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 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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- - c rx o authors: singh, manmeet; khan, reas s.; dine, kimberly; das sarma, jayasri; shindler, kenneth s. title: intracranial inoculation is more potent than intranasal inoculation for inducing optic neuritis in the mouse hepatitis virus-induced model of multiple sclerosis date: - - journal: front cell infect microbiol doi: . /fcimb. . sha: doc_id: cord_uid: c rx o neurotropic strains of mouse hepatitis virus (mhv) induce acute inflammation and chronic demyelination in the spinal cord and optic nerves mediated by axonal spread following intracranial inoculation in mice, with pathologic features similar to the human demyelinating disease multiple sclerosis. spinal cord demyelination is also induced following intranasal inoculation with neurotropic mhv strains, however much higher viral doses are required as compared to intracranial inoculation. recently, it was shown that intranasal administration of low concentrations of proteins leads to significant, rapid accumulation of protein in the optic nerve and in the eye, with only low levels reaching spinal cord and other brain regions. thus, we examined whether intranasal inoculation with mhv at doses equivalent to those given intracranially could induce optic neuritis—inflammation, demyelination and loss of retinal ganglion cells (rgcs) in the optic nerve with or without inducing spinal cord demyelination. four week old male c bl/ j mice were inoculated intracranially with the recombinant demyelinating strain rsa , or intranasally with rsa or the non-demyelinating strain rsmhv as control. one month post-inoculation, mice inoculated intracranially with rsa had significant myelin loss in both spinal cord and optic nerves, with significant loss of rgcs as well, consistent with prior studies. as expected, intranasal inoculation with rsa failed to induce demyelination in spinal cord; however, it also did not induce optic nerve demyelination. no acute inflammation was found, and no viral antigen was detected, in the optic nerve or retina day after inoculation. results confirm the neurotropic effects of rsa following intracranial inoculation, and suggest that direct infection with axonal transport of virus from brain to spinal cord and optic nerve is required to induce demyelinating disease. these studies suggest that mhv does not selectively concentrate in optic nerve and retina to sufficient levels to induce demyelination following intranasal inoculation. intracranial inoculation should continue to be considered a preferred method for studies of mhv-induced optic neuritis and central nervous system (cns) demyelinating disease. neurotropic strains of mouse hepatitis virus (mhv) induce acute inflammation and chronic demyelination in the spinal cord and optic nerves mediated by axonal spread following intracranial inoculation in mice, with pathologic features similar to the human demyelinating disease multiple sclerosis. spinal cord demyelination is also induced following intranasal inoculation with neurotropic mhv strains, however much higher viral doses are required as compared to intracranial inoculation. recently, it was shown that intranasal administration of low concentrations of proteins leads to significant, rapid accumulation of protein in the optic nerve and in the eye, with only low levels reaching spinal cord and other brain regions. thus, we examined whether intranasal inoculation with mhv at doses equivalent to those given intracranially could induce optic neuritis-inflammation, demyelination and loss of retinal ganglion cells (rgcs) in the optic nerve with or without inducing spinal cord demyelination. four week old male c bl/ j mice were inoculated intracranially with the recombinant demyelinating strain rsa , or intranasally with rsa or the non-demyelinating strain rsmhv as control. one month post-inoculation, mice inoculated intracranially with rsa had significant myelin loss in both spinal cord and optic nerves, with significant loss of rgcs as well, consistent with prior studies. as expected, intranasal inoculation with rsa failed to induce demyelination in spinal cord; however, it also did not induce optic nerve demyelination. no acute inflammation was found, and no viral antigen was detected, in the optic nerve or retina day after inoculation. results confirm the neurotropic effects of rsa following intracranial inoculation, and suggest that direct infection with axonal transport of virus from brain to spinal cord and optic nerve is required to induce demyelinating disease. these studies suggest that mhv does not selectively concentrate in optic nerve and retina to sufficient levels to induce demyelination following intranasal inoculation. intracranial inoculation should continue to be considered a preferred method for studies of mhv-induced optic neuritis and central nervous system (cns) demyelinating disease. neurotropic strains of mhv have been extensively used to induce neuroinflammation mediated acute and chronic demyelinating disease of cns. depending upon route of inoculation and strain of mhv, different regions of the cns are affected. inoculation with experimental neurotropic strains jhm and mhv-a induces a biphasic disease with acute meningoencephalitis (in first - days post inoculation) followed by subacute and chronic inflammatory demyelinating disease (stohlman and weiner, ; lavi et al., ; das sarma et al., ) . both jhm and mhv-a strains of mhv cause some subacute and chronic inflammatory demyelination in the brain, but a much larger disease burden in the spinal cord. translocation of virus from initial site of inoculation in brain to spinal cord occurs by trafficking of virus particle in neural and glial cells sun and perlman, ) . intranasal as well as intracranial inoculation of jhm has been shown to induce similar symptoms in balb/c mice (robbins et al., ) . similarly, intracranial inoculation has been well used as a method for mhv-a to cause the biphasic disease (das sarma et al., , , while a higher dose of mhv-a is required to reach the same level of inflammation in ceacam-/-mice when inoculated through the nasal route (blau et al., ; hemmila et al., ) . with intracranial inoculation, the inflammation is not limited to brain and spinal cord. mhv-a and recombinant strain rsa cause inflammation in optic nerve with subsequent demyelination of optic nerve and rgc loss (shindler et al., (shindler et al., , . studies of isogenic recombinant strains rsa and rsmhv of demyelinating strain (mhv-a ) and non-demyelinating strain (mhv ), respectively, containing enhanced green fluorescent protein (egfp) have elaborated the mechanisms of demyelination and axonal loss and have helped in tracking and tracing of virus in vitro as well as in vivo (das sarma et al., ) . rsa can cause demyelination, but rsmhv cannot, which makes it a suitable control to determine the cellular and molecular basis of demyelination in mice. following intranasal inoculation of mice, mhv accesses the cns through the olfactory nerve and spreads from the olfactory system (jacobsen and perlman, ; perlman et al., ) into structures of the limbic system and their brainstem connections. this has led investigators to suggest that interneuronal transport is one mechanism of viral spread during acute encephalitis (barthold, ; lavi et al., ; , and studies showing spread of virus sequentially from cerebral hemispheres to brainstem to spinal cord provide further support for this interneuronal transport mechanism. similar axonal transport of virus from brain to spinal cord (das sarma et al., ) , as well as from brain to optic nerve (shindler et al., (shindler et al., , , has been reported following intracranial inoculation with mhv-a or rsa and may serve as one mechanism for virus to avoid immune surveillance; however, axonal spread to optic nerve has not been well examined following intranasal inoculation. different intra-and extracellular pathways may help facilitate viral transport across olfactory or respiratory epithelial barriers. endocytosis into olfactory sensory neurons followed by intraneuronal transport to the olfactory bulb, or transcellular transport to the lamina propria via sustentacular cells, have been suggested as potential intracellular pathways (kristensson and olsson, ; broadwell and balin, ; thorne et al., ; doty, ; kristensson, ) . delivery of large molecular weight biological therapies (e.g., stem cells, gene therapy vectors, and large proteins) to the cns via intranasal administration has been explored as a potential method to treat multiple cns diseases/disorders including parkinson's and alzheimer's diseases, multiple sclerosis, seizures, strokes, and psychiatric disorders (costantino et al., ; neuwelt et al., ; dhuria et al., ) . spread of smaller peptides through rodent brain following intranasal administration occurs rapidly, with diffuse brain distribution and greatest levels found in olfactory bulbs and trigeminal nerves, just h after treatment. igf- (mw = . kda) is one of the most studied proteins using intranasal delivery to the cns (thorne et al., ) . even entry of some high molecular weight proteins such as vegf (mw = . kda) to the cns has been shown following intranasal administration (yang et al., ) . recently, it has been shown that proteins in a complex biologic therapy, st , administered via the intranasal route in rats reached the cns within min, and st proteins were detected in the vitreous and the optic nerve at markedly higher concentrations than in the brain (khan et al., ) , suggesting a rapid, direct nose-to-optic nerve delivery method for proteins. whether viruses can follow similar pathways to preferentially spread to optic nerve at low inoculation titers has not been reported, but if such pathways are present, the rapid spread of virus could provide an additional mechanism for immune evasion and therefore promote viral infection at lower inoculation titers. we hypothesized that rsa can be used to induce optic neuritis when inoculated intranasally at lower doses than required to induce brain and spinal cord disease due to enhanced viral spread to optic nerve. mice were inoculated with rsa and rsmhv as control, both intranasally as well as intracranially at equivalent concentrations to compare if both routes of administration result in the same optic nerve pathology. four-week-old virus-free c bl/ j mice were purchased from the jackson laboratory (barharbor, me, usa). all animal procedures and care were conducted in accordance with ethical guidelines approved by the institutional animal care and use committee at the university of pennsylvania. rsa and rsmhv , the isogenic recombinant strain of mhv-a and mhv , respectively, were used as previously described (das sarma et al., ) . rsa and rsmhv are each engineered to express enhanced green fluorescence protein (egfp), thus allowing viral antigen detection by fluorescence without immunohistochemical staining (das sarma et al., ) . mice were monitored daily for signs of mhv induced neurologic up to days (chronic stage) post-infection. % ld doses of strains rsa ( , pfu), and rsmhv ( pfu) were used to inoculate -week-old, mhv-free, c bl/ j mice (jackson laboratory). desired pfu of viruses were prepared in a total volume of µl in pbs and were pipeted as intranasal drops noninvasively every min to alternating nares until all µl were delivered, with simultaneous occlusion of the opposite naris. drops were placed at the opening of the nares, allowing them to be snorted into the nasal cavity. the mice for day studies were also inoculated intracranially as a positive control for disease pathogenesis with rsa , as in prior studies (das sarma et al., ) . control mice mock-infected with pbs were inoculated in parallel. animals were euthanized ( mice per group) at day post-inoculation (p.i.) and day p.i. at and days p.i., tissues, including whole eyes, optic nerves, brains, spinal cords, and livers, were isolated from both mock-and virus-infected mice. for paraffin sectioning, eyes and optic nerves were fixed for min after dissecton in % paraformaldehyde (pfa) while brains, spinal cords, and livers were fixed in % pfa overnight. five micrometer sections were cut for routine cns pathology staining following fixation and tissue processing. sections were stained with luxol fast blue (lfb) to detect myelin loss in spinal cord and optic nerve as in prior studies (shindler et al., ) . demyelination was scored based on detection of focal white matter areas lacking lfb staining using a relative three-point scale. areas of demyelination were quantified using a - point scale, where -no demyelination; -rare foci of demyelination; a few foci of demyelination; and -large (confluent) areas of demyelination. all slides were coded and read in a blinded manner. serial sections from eye, optic nerve, and brain were stained by the avidin-biotin-immunoperoxidase technique (vector laboratories) using , -diaminobenzidine as substrate, and a : dilution of anti-iba (wako, richmond, va, usa), and : dilution of antiviral nucleocapsid antiserum (mouse monoclonal anti-n; nucleocapsid protein of mhv-jhm, monoclonal clone - - , kindly provided by julian lebowitz, texas a&m, college station, tx) as primary antibodies. control slides from mock-infected mice were incubated in parallel. rgc immunolabeling and counting was performed as in prior studies (khan et al., ) . briefly, eyes removed at the time of sacrifice were fixed with % pfa. retinas were isolated and whole-mounted on glass slides, washed several times in pbs, permeabilized at − • c in . % triton x solution, then thawed and washed again in . % triton x . retinas were incubated overnight with goat anti-brn a (rgc marker) antibody (santa cruz biotechnology) diluted : in blocking buffer (pbs containing % bovine serum albumin and % triton x ). after washing in pbs, retinas were incubated for h with alexa fluor- anti-goat secondary antibody diluted : . retinas were then washed and mounted with vectashield mounting medium for fluorescence. photographs of rgcs were taken in standardized fields at / , / , and / of the retinal radius from the center of the retina in four quadrants at x magnification. rgcs were counted in each field by a blinded investigator using image-pro plus . (media cybernetics, silver spring, md) software. four week old c bl /j mice were inoculated with % ld doses of rsa or rsmhv by intranasal administration or by intracranial injection, or mock transfected by intranasal administration of solution without virus. pathology was assessed from lfb stained cross-sections of spinal cord isolated from mice at day (peak of demyelination) p.i. rsa , when injected intracranially, induced significant myelin loss within formed demyelinating plaques, [average demyelination score . ± . ; (mean ± se); n = mice ( sections/group); p < . vs. control] as in prior studies (figures j-l) . as expected, mice infected intracranially with rsmhv did not show any significant myelin loss (data not shown). interestingly, no demyelination plaques were observed in any level of spinal cord sections of rsa infected mice when given via the intranasal route (figures d-f) . similarly, as expected, no myelin loss was observed in intranasally mockinfected (figures a-c) or rsmhv -infected mouse spinal cord (figures g-i) . intracranial, but not intranasal, inoculation with rsa induces optic neuritis mice inoculated intracranially with rsa have been found to exhibit retrograde axonal transport of virus from the lateral geniculate nuclei along the optic nerve into the retina, and can cause optic nerve inflammation and demyelination (shindler et al., ) , whereas rsmhv does not. to investigate whether intranasal rsa administration can induce optic nerve demyelination similar to intracranial inoculation, µm thick serial optic nerve sections from the same mice shown in figure were stained with lfb. rsa , when infected intracranially, induced significant myelin loss with notable demyelinating plaques in optic nerves, (figure ) as previously observed (shindler et al., ) . similar to spinal cord, little or no demyelinating plaques were observed in optic nerve sections of rsa infected mice when injected intranasally, which was comparable to rsmhv and mock infected mice. demyelinating optic neuritis induced by intracranial inoculation with rsa has been shown previously to lead to neuronal damage with loss of rgcs (khan et al., ) . to examine whether intranasal infection with demyelinating strains of mhv figure | comparative demyelination study between intracranially inoculated and intranasally infected mouse spinal cords. serial thoracic (left column), cervical and lumbar (center and right columns) cross sections of spinal cord from post-inoculation day intranasally mock-infected (a-c), rsa -infected (d-f), and rsmhv -infected (g-i) mice, and intracranially rsa -inoculated mice (j-l) were stained with lfb (scale bar = µm). marked area indicate typical demyelinating plaques found in spinal cord white matter. average demyelination score is . ± . ; (mean ± se) (m); n = /group; data comparisons were done by one-way anova and tukey's multiple comparison post-hoc testing with graphpad prism . software. ****p < . . results in neuronal loss, retinas were isolated from the same mice shown in figure , and rgcs were labeled and counted in a blinded fashion. intracranially rsa -infected mice had significantly fewer surviving rgcs compared to mock-infected mice, whereas mice infected intranasally with either rsa or rsmhv did not show rgc loss (figure ) . proteins rapidly accumulate at high concentrations in optic nerve and in the eye following intranasal administration (khan et al., ) . thus, the potential for mhv viruses to similarly spread figure | comparative optic nerve demyelination study between intracranially-and intranasally-infected mice. representative optic nerve sections from chronic stage (day p.i.) mock-infected (n = ) (a), rsa intrancranially-infected (n = ) (b), rsa intranasally-infected (n = ) (c), and rsmhv intranasally-infected (n = ) (d) mice stained with lfb show demyelination only in rsa intracranially-infected mice (scale bar = µm). the relative level of demyelination scored by a blinded investigator showed significant demyelination in optic nerves of mice inoculated intracranially with rsa , but not in mock-infected (control) mice nor in mice infected intranasally with either rsa or rsmhv (*p < . vs. all other groups) (e). data comparisons were done by one-way anova and tukey's multiple comparison post-hoc testing with graphpad prism . software. to optic nerve and retina was assessed day following intranasal inoculation. four-week-old, mhv-free, c bl/ j mice were inoculated intranasally with % of the ld dose of rsa or rsmhv , and mice were euthanized day later. retinas and optic nerves were isolated, sectioned, and immunostained with anti-viral nucleocapsid antisera to detect viral spread. no significant staining was observed in any of the retinas or optic nerves from mock-infected (n = ), rsa -infected (n = ), or rsmhv -infected (n = ) mice (figure ) . as shown in prior studies (shindler et al., ) , viral antigen does not reach the retina within day following intracranial inoculation with rsa (data not shown). viral antigen is found in the retina days after intrancranial inoculation (figure ) , while no antigen is detectable in retina following intranasal inoculation at day (figure ) or any later time points (data not shown). to further confirm that intranasal rsa administration fails to induce optic neuritis, acutely, optic nerve sections were immunostained for the microglial/macrophage marker iba . previously, it has been observed that intracranial inoculation with rsa induces acute optic nerve inflammation containing almost entirely activated microglia/macrophages (shindler et al., ) - days post-inoculation. to study whether intranasal figure | rsa infection induces rgc loss. representative photos illustrate the decreased rgc numbers in eyes of mice inoculated intracranially with rsa (b) compared to mock-infected control mice (a). mice inoculated intranasally with rsa (c) or rsmhv did not show rgc loss (d) (scale bar = µm). the total number of labeled rgcs present in standardized retinal fields was counted. the average number of surviving rgcs/eye (n = /group) shows intracranial rsa induced a significant decrease in rgc numbers compared to control mice (**p < . ). neither rsa nor rsmhv induced rgc loss compared to control mice when administered intranasally. rgc numbers in rsmhv -infected mice were significantly higher than in mice intracranially inoculated with rsa (*p < . ) (e). data comparisons were done by one-way anova and tukey's multiple comparison post-hoc testing with graphpad prism . software. inoculation rapidly induces similar optic nerve inflammation, optic nerve sections were stained with anti-iba antibody. sections from mock-infected mice were used to demonstrate resting levels of microglial staining. iba- staining did not reveal any increased numbers of microglia/macrophages in optic nerve sections following intranasal viral inoculation as compared to mock-infected (figure ) . to confirm that lack of viral antigen detection in the optic nerve represents a failure of the virus to spread to optic nerve and retina, and not a failure to detect viral antigen, viral antigen was also examined in olfactory bulb sections from the same mice by autofluoresence of egfp (figures j,k) as well as immunostaining with anti-viral nucleocapsid antisera (figures l,m) . the current studies compared effects of rsa infection via two different routes of inoculation on the development of demyelinating disease in the optic nerve and spinal cord. results suggest intracranial inoculation is the best method to induce neuroinflammation. in the current study, intracranial infection of rsa lead to chronic stage inflammation and demyelination in both the optic nerve and spinal cord. this result is consistent with earlier studies where intracranial inoculation of rsa or its parental strain mhv-a led to demyelination and axonal loss in spinal cord ) and optic nerve (shindler et al., (shindler et al., , . induction of optic neuritis by intracranial inoculation of rsa is dependent on retrograde transport of viral antigen along rgc axons that occurs over several days and results in late stage demyelination (shindler et al., ) as seen again in the current study. together, spinal cord and optic nerve pathology observed after intracranial inoculation of rsa in the current study confirm that the viral titer used retains its previously identified ability to induce cns demyelinating disease. while intrancranial inoculation was shown previously to induce optic neuritis (shindler et al., (shindler et al., , , effects of intranasal inoculation with rsa on optic nerve pathology were not reported. in the current study, intranasal infection by rsa at the same dose used for intracranial inoculation was not able to induce any neuropathogenesis, either in optic nerve or spinal cord. earlier studies with several other strains showed that intranasal inoculation of mhv can cause cns disease, but much higher concentrations of virus were used than in intracranial inoculation studies. mhv-jhm enters the central nervous system (cns) via the olfactory after intranasal inoculation . the intranasal inoculation of jhm strain can lead to encephalitis and demyelination. the oblv strain of mhv can infect the main olfactory bulb (schwob et al., ) . intranasal inoculation of mhv-a can lead to hepatitis with measurable viral load in brain as well (hemmila et al., ) . these studies did not report optic nerve pathogenesis following intranasal inoculation although it is not known if that was examined. there are several possible reasons that we did not see retinal infection or optic nerve inflammation and demyelination lesions after intranasal inoculation. most likely, the dose of virus may have been too low to cause the pathogenesis. this finding was not unexpected in the spinal cord, where previous studies showed higher doses were necessary. however, based on the high levels of protein accumulation in optic nerve and eye following intranasal inoculation (khan et al., ) , it was anticipated that rsa would also preferentially accumulate in the eye, but results suggest that higher doses are likely required. alternatively, the intranasal inoculation may be less effective for inducing optic neuritis and retinal lesions than the intracerebral route because of the longer distance required for the virus to travel to the eye if it travels via axonal transport and neuronneuron spread similar to what has been observed following intracranial inoculation (das sarma et al., ; shindler et al., ) . the precise mechanisms mediating spread of a virus or a drug from the nose to various cns regions are not fully elucidated. at least three steps are necessary following intranasal administration ( ) crossing the epithelial barrier in the nasal cavity, ( ) transport from nasal mucosa to site of brain entry, likely across the cribiform plate, ( ) transport from the site of brain entry to other anatomical regions. alternatively, they may be absorbed into the systemic circulation and gain secondary access to the cns through the blood brain barrier. the speed at which proteins were reported to reach the eye and optic nerve, min after intranasal administration (khan et al., ) , suggests hematogenous spread is very unlikely, and that intraaxonal transport is even more unlikely. it was hypothesized that perhaps some pathway of local diffusion or lymphatic pathway may allow rapid protein diffusion over the relatively short distance from absorption through the cribiform plate to optic nerve (khan et al., ) . for rsa , prior intracranial studies demonstrate that the virus can use axonal transport machinery to spread intraneuronally (das sarma et al., ) , thus it is likely that a similar mechanism would be used after intranasal administration and entry into the olfactory nerve. the path from there to optic nerve is not direct, and thus may require much higher titers of virus to occur at a pathologic level, or may not be possible at all. these hypothesized mechanisms may explain why we were not able to see viral staining day post infection whereas the drug st and other small molecules can be found in optic nerve as early as min post intranasal inoculation (dhuria et al., ; khan et al., ) . intranasal administration provides a potential non-invasive method for delivering material to the cns. interestingly, complex mixtures containing physiologic concentrations of multiple proteins show that protein can rapidly accumulate in the eye and optic nerve following intranasal delivery, suggesting a direct nose to eye diffusion pathway that remains to be fully elucidated (khan et al., ) . the current results show that rsa does not follow a similar pattern of accumulation in the optic nerve, suggesting that viral particle may be too large or complex to follow the same pathway, or may actively enter neurons locally and restrict their movement to intraneuronal axonal transport. nonetheless, the ability of neurotropic mhv viruses to infect different cells, translocate throughout the cns, and induce inflammatory demyelination, continues to provide a reproducible model to study optic nerve and spinal cord demyelinating disease following intracranial inoculation. thus, intracranial inoculation should continue to be considered a preferred method for studies of mhv-induced optic neuritis and cns demyelinating disease. ms, rk and kd performed the wet lab experiments and wrote their contributions. ks and jd designed the studies and wrote the paper. delhi and dupre fellowship, multiple sclerosis society international federation (msif), uk for a fellowship. this work was supported by the department of biotechnology bt/pr /med/ / / to jd. ks thanks research to prevent blindness, nih grant ey , and the f. m. kirby foundation. the authors thank iiser kolkata and university of pennsylvania, philadelphia, for support. the funders had no role in the study design, data collection, and 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the rat brain and spinal cord along olfactory and trigeminal pathways following intranasal administration direct transport of vegf from the nasal cavity to brain the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.the reviewer sm and handling editor declared their shared affiliation at time of review.copyright © singh, khan, dine, das sarma and shindler. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -egy d x authors: shindler, kenneth s.; chatterjee, dhriti; biswas, kaushiki; goyal, ashish; dutt, mahasweta; nassrallah, mayssa; khan, reas s.; sarma, jayasri das title: macrophage-mediated optic neuritis induced by retrograde axonal transport of spike gene recombinant mouse hepatitis virus date: - - journal: journal of neuropathology & experimental neurology doi: . /nen. b e da sha: doc_id: cord_uid: egy d x following intracranial inoculation, neurovirulent mouse hepatitis virus (mhv) strains induce acute inflammation, demyelination and axonal loss in the cns. prior studies using recombinant mhv strains that differ only in the spike gene, which encodes a glycoprotein involved in virus-host cell attachment, demonstrated that spike mediates anterograde axonal transport of virus to the spinal cord. a demyelinating mhv strain induces optic neuritis, but whether this is due to retrograde axonal transport of viral particles to the retina, or if it is due to traumatic disruption of retinal ganglion cell axons during intracranial inoculation is not known. using recombinant isogenic mhv strains, we examined the ability of recombinant mhv to induce optic neuritis by retrograde spread from the brain through the optic nerve into the eye following intracranial inoculation. recombinant demyelinating mhv induced macrophage infiltration of optic nerves, demyelination and axonal loss whereas optic neuritis and axonal injury were minimal in mice infected with the non-demyelinating mhv strain that differs in the spike gene. thus, optic neuritis was dependent on a spike glycoprotein-mediated mechanism of viral antigen transport along retinal ganglion cell axons. these data indicate that mhv spreads by retrograde axonal transport to the eye and that targeting spike protein interactions with axonal transport machinery is a potential therapeutic strategy for cns viral infections and associated diseases. neurotropic mouse hepatitis virus (mhv) infection in mice causes meningoencephalitis, myelitis, and demyelination, with relative axonal preservation. recent studies have additionally demonstrated that neurotropic mhv strains can also induce axonal loss ( , ) ; direct virus-mediated axonal damage can occur concurrently with and independently of demyelination ( ) . thus, neurovirulent mhv strains provide useful tools for studying the neuroinflammation, demyelination, and axonal loss and as a virus-induced model of multiple sclerosis. recombinant mhv strains rsa (demyelinating strain; dm) and rsmhv (nondemyelinating strain; ndm) are isogenic except for the spike gene, which encodes the host attachment spike glycoprotein. studies of these strains have elucidated mechanisms of axonal loss and demyelination ( ) . rsa and rsmhv both cause hepatitis, encephalitis, and meningitis after intracranial inoculation. however, they differ in their ability to induce macrophage infiltration and subsequent demyelination and axonal loss in spinal cord ( ) . there is a lack of viral antigen spread and subsequent inflammation extending into spinal cord white matter after intracranial infection with the ndm strain, whereas there is extensive macrophage-mediated white matter pathology secondary to dm strain infection. thus, the spike protein plays a critical role in anterograde axonal transport of viral particles, an important mechanism mediating axonal damage and demyelination ( , ) . because both strains cause encephalitis after transcranial inoculation, the differences in spike protein between dm and ndm strains do not impair viral entry; however, differential neural cell tropism may contribute to the mechanism of demyelination ( , , ) . infection of brain neurons and oligodendrocytes occurs on inoculation with either strain, whereas in the spinal cord, oligodendrocyte infection is only seen with the dm strain. this is likely due to the route by which the virus gains access to white matter, that is, spinal cord infection does not occur as a result of direct trauma, whereas transcranial inoculation results in traumatic disruption of the brain gray-white matter interface. viral particles that would require anterograde axonal transport from infected neurons to reach myelin are able to gain direct access to the myelin sheath and spread proximally to oligodendrocyte cytoplasm. anterograde axonal transport and spread of virus from neurons to oligodendrocytes have been documented, but retrograde axonal spread of virus from nerve ending to neural cell body also needs to be considered. earlier studies suggested that mhv strains may spread via retrograde axonal transport ( , ) , but the molecular mechanisms mediating such transport are not well defined. in the optic nerve, the parental demyelinating strain mhv-a causes inflammation, demyelination, and axonal loss (ie, optic neuritis), in contrast to the nondemyelinating mhv- strain ( ) . whether mhvinduced optic neuritis is dependent on retrograde axonal transport of viral particles or is due to local traumatic disruption of the intracranial portion of retinal ganglion cell (rgc) axons during inoculation is not known. moreover, the immune response in mhv recombinant strain optic neuritis has not been well characterized. here, we compared the incidence and phenotype of optic neuritis after inoculation with rsa and rsmhv and assessed the ability of spike protein to facilitate retrograde axonal transport and induce optic neuritis. recombinant isogenic dm strain of mhv (rsa ) and ndm strain (rsmhv ) have been described in previous studies ( , ) . rsa and rsmhv strains of mhv are isogenic except for the spike gene, which encodes an envelope glycoprotein that mediates many biological properties of mhv including viral attachment to host cells and virus-cell and cell-cell fusion ( ) . these recombinant strains also express enhanced green fluorescence protein (egfp) ( , ) . mhv-free, c bl/ (b ) mice (jackson laboratory, bar harbor, me) were inoculated intracranially at weeks of age with % ld dose of rsa strain ( , plaque forming units) or rsmhv ( plaque forming units), as described previously ( ) . mice were monitored daily for signs of disease. mock-infected controls were inoculated similarly but with an uninfected cell lysate at a comparable dilution. animals were killed ( y mice per group) at day , days to , and day post inoculation (pi). all experimental procedures adhered to guidelines of, and were approved by, the institutional animal care and use committee. mice were killed at days to pi (peak of inflammation) or at day pi (during chronic demyelination) and were perfused transcardially with phosphate-buffered saline followed by phosphate-buffered saline containing % paraformaldehyde. brain, spinal cord, eyes, and optic nerve tissues were collected, postfixed in % paraformaldehyde overnight, and embedded in paraffin; sections were then stained with hematoxylin and eosin (h&e) to evaluate inflammation and luxol fast blue to evaluate demyelination. experiments were repeated at least times with to mice. areas of demyelination and inflammation were quantified as previously described ( , ) . all slides were coded and read in a blinded fashion. to confirm expected virulence of the strains used, livers from the infected mice were embedded in paraffin, sectioned at km, and stained with h&e ( , ) . the degree of optic nerve inflammation was scored by blinded investigators on a -(no inflammation) to -point (severe inflammation) scale, as described ( , ) , during the time of peak inflammation (days y pi). any amount of inflammation (score of y ) was considered positive for optic neuritis. serial sections from optic nerves were stained by the avidin-biotin-immunoperoxidase technique (vector laboratories, burlingame, ca) using , ¶ diaminobenzidine as substrate, and antibodies against lymphocytic cell markers, antiviral nucleocapsid antiserum, or the axonal marker antineurofilament antiserum as primary immunoglobulin g antibodies. the sources and dilution of primary antibodies are listed in the table. control slides from mock-infected mice were incubated in parallel. fixed sections of optic nerve, brain, and eyes from mice at or days pi were stained by the previously described immunohistochemical methods using antiviral nucleocapsid antiserum. to visualize viral antigen by egfp expression directly, frozen sections from infected mice were examined by fluorescence microscopy. viral antigen was also assayed by western blotting using antiyhepatitis virus nonstructural protein monoclonal antibody (rockland, inc., gilbertsville, pa), according to the manufacturer's instructions. briefly, protein was extracted from the optic nerve and retina using ripa buffer (sigma, st. louis, mo) in the presence of a complete protease inhibitor cocktail (pierce, rockford, il) at -c. protein concentration was determined with a micro bca protein assay kit (pierce). protein ( kg) was electrophoresed on % to % sds-page, transferred to cellulose membrane, blocked, and probed with : dilution of primary antibody. expression of glyceraldehyde -phosphate dehydrogenase (gapdh) was determined as a loading control using anti-gapdh (sigma) diluted : , . after incubation with horseradish peroxidaseyconjugated secondary antibodies, signals were developed with enhanced chemiluminescence agent (ge healthcare, buckinghamshire, uk), and intensity was determined using the nih image j program. longitudinal optic nerve sections were stained with antineurofilament antibody, and areas of axonal staining were quantified as described ( ) . briefly, photographs were taken at  magnification of each stained nerve at predefined locations (one each of the proximal, central, and distal portion of the nerve) covering a total area of , km of each nerve. the amount of tissue within this area that stained positively for neurofilament was calculated using imagepro plus . (media cybernetics, silver spring, md) software. data shown represent the cumulative area of positive staining/nerve. comparisons of optic neuritis incidence, demyelination, and axonal density were analyzed by one-way analysis of variance followed by tukey multiple comparison test using graphpad prism . (graphpad software, san diego, ca). data represent mean (sd) percentage of eyes that developed optic nerve inflammation or demyelination or the mean (sd) density of axonal staining. as in prior studies, rsa -infected mice showed meningitis, encephalitis, myelitis, and concurrent axonal loss and demyelination as early as day pi with an increase at day , whereas rsmhv showed only meningitis, encephalitis, and myelitis with no significant demyelination or axonal loss (data not shown) ( ) . the livers of both strains showed moderate to severe hepatitis (data not shown), thereby confirming virulence. as expected, mice infected with parental demyelinating strain mhv-a at days to pi had optic nerve inflammation ( fig. c) , whereas optic nerves from mice infected with parental nondemyelinating strain mhv- did not (fig. b) ; their optic nerves appeared similar to those of mock-infected mice (fig. a) ( ) . the inability of mhv- to induce optic neuritis was expected because mhv- does not infect the brain parenchyma and is unable to induce encephalitis ( , ) . by contrast, the rsmhv- strain induces encephalitis ( , , ) . rsa induced optic neuritis similar to the parental mhv-a strain at days to pi (fig. f) , whereas most eyes of rsmhv -infected mice exhibited no optic nerve inflamma-tion (fig. d) ; however, there was mild inflammation in a few optic nerves (fig. e) . mhv-a induced optic neuritis in a mean of . % of optic nerves in experiments, whereas mhv- induced optic neuritis in only . % of optic nerves (fig. g) ; this result is similar to the incidence in prior studies ( ) . rsa infected mice developed optic neuritis with a high incidence ( . %) similar to that in mhv-a -infected mice, and significantly higher than the incidence in rsmhv- infected mice ( . %) (fig. h) . serial sections from rsa -and rsmhv -infected mice were stained with anti-cd (leukocyte common antigen; lca), antiyiba- (microglia/macrophage marker), anti-cd (t-cell marker), or anti-cd (b-cell marker) (table) . lca staining confirmed the presence of infiltrating inflammatory cells in optic nerves from rsa -infected mice, whereas few lca-positive cells were found in those of rsmhv -infected mice (not shown). among the lca-positive inflammatory cells, the majority in the rsa -infected mice were iba- + microglia/macrophages (fig. c) . the optic nerves of some control mice (fig. ) and rsmhv -infected mice (fig. b ) also had scattered iba + cells that likely represent resident microglia, but there were far fewer than in the rsa -infected samples. this result was confirmed by blinded investigators. significantly more optic nerves from rsa -infected mice had increased iba + cells versus nerves from rsmhv -infected mice ( / total nerves from experiments versus / , respectively; p = . by fisher exact test). few or no cd -positive t cells were present in optic nerves of rsa -infected mice (fig. d ) and no cd stained b cells were observed (fig. e ). spleen sections from mock-infected mice stained with either anti-cd (fig. f ) or anti-cd antibodies (not shown) served as positive controls. thus, there was an increase of iba- ypositive cells and few cd -positive cells in optic nerves of rsa -infected mice. optic nerves from rsa -infected mice had areas of demyelination detected by luxol fast blue staining both at day and day pi (fig. ) , whereas no demyelinating plaque was observed in day pi rsmhv mouse optic nerve and little or no demyelination was observed at day pi. these data are consistent with the previous result obtained from parental demyelinating strain mhv-a and nondemyelinating strain mhv- ( ). no axonal loss was identified at to days pi in optic nerves of rsa -or rsmhv -infected mice (figs. ayc). at day pi, rsmhv -infected mice continued to show no axonal loss (fig. d) , whereas optic nerves from rsa infected mice showed regions of mildly reduced axonal staining, with focal areas of axon loss intermixed with areas of normal axons (figs. e, f) . quantification of the area of axonal staining across all sections of optic nerves of rsa infected mice demonstrated a small but significant decrease compared with optic nerves of either rsmhv -infected or mock-infected control mice (p = . ) (fig. g ). the optic nerve inflammation, demyelination, and axonal loss observed after rsa infection, but not after rsmhv infection, demonstrate that the mhv-a spike protein is required for the induction of optic neuritis. this suggests that the spike protein may mediate retrograde axonal transport of the virus, allowing it to travel from brain regions containing rgc axonal projections along the optic nerve. viral antigen was consistently detected within thalamic neurons in rsa -infected mouse brains (figs. c, d) , as well as in some rsmhv -infected brains (fig. b) . rsa viral antigen was also detected in the superior colliculi in some animals (data not shown). light diffuse staining detected in optic nerves from rsa -infected mice (fig. g ), but not rsmhv -infected mice (fig. f ), suggests that only the rsa viral antigen is transported to the optic nerves. to further investigate this, the optic nerves were isolated or days pi and frozen sections were examined for the presence of egfp. viral antigeny positive egfp signal was observed in optic nerves from rsa -infected mice (figs. i, j) but not rsmhv -infected mice (fig. h) . the punctate egfp signal in optic nerve sections observed by fluorescent microscopy in rsa -infected mice (fig. j ) further suggests axonal transport through the optic nerve and was seen as early as days pi. to determine whether viral antigen is transported all the way to the rgc cell bodies, whole eyes were isolated and sectioned. viral antigen immunostaining of retinal sections demonstrated no viral antigen in cells of the rgc layer of rsmhv -infected mice (fig. a) , whereas rsa viral antigen was detected in some cells within the rgc layer (figs. b, c) at day pi. overall, viral antigen staining was detected in % of eyes from rsa -infected mice, with serial cross sections examined from each of eyes. the timing of viral antigen spread and relative levels of antigen in retina and optic nerve were determined in protein extracts isolated at days and pi. western blot analysis demonstrated presence of viral antigen in retinal tissue from rsa infected mice at day pi but not at day and no viral antigen was detected in rsmhv -infected mice (fig. d ). viral antigen was detected in protein extracts from optic nerves of rsa -infected mice earlier than it was detected in retina. antigen was detected on western blots at both days and pi (fig. e) . these results suggest that the viral antigen was able to enter the rgc axons and was transported retrograde to the cell bodies within the eye. rsa induces optic neuritis at comparable levels of severity and incidence as that seen in mice infected with its parental strain mhv-a ( ), whereas rsmhv has a limited ability to induce optic nerve inflammation. accordingly, nerves from rsa -infected mice at the chronic stage showed significantly decreased axonal density compared with nerves from rsmhv -infected mice. this differential ability of mhv strains to induce optic neuritis and axonal injury is dependent on spike glycoprotein mediated retrograde transport of viral antigen along rgc axons. prior studies demonstrated that rsa and rsmhv both can cause meningitis and encephalitis, but rsmhv did not induce subsequent demyelination and axonal loss in spinal cord. our current results demonstrate that in addition to demyelination and axonal loss, rsmhv also has limited ability to cause significant optic nerve inflammation and rgc infection. however, rsmhv did induce some optic nerve inflammation, unlike the parental strain mhv . this difference may be due to the ability of rsmhv to enter neurons in the brain parenchyma and replicate, as demonstrated previously ( , , ) and shown again here, leading to a higher viral load that may allow some diffusion of the virus at an undetectable level. alternatively, the higher viral load might trigger a more diffuse central nervous system (cns) inflammatory response associated with mild, nonspecific inflammation in some optic nerves. mhv , on the other hand, does not enter the brain parenchyma, and infection with it results in meningitis without inducing encephalitis ( ). prior studies demonstrated that one mechanism limiting the ability of the ndm strain to induce demyelination and axonal loss in the spinal cord involves impaired interneuronal spread of viral particles and defective translocation of viral antigen from gray matter to white matter ( ) . evaluation of axonal loss and demyelination in the spinal cord demonstrated that dm mhv infection begins in the neuronal cell body, propagates to the axon, and subsequently induces axonal degeneration and demyelination. the propagation of viral antigen from gray to white matter is dependent on anterograde axonal transport of virus particle mediated by the spike protein ( , ) . spike proteinymediated axonal transport as an underlying mechanism is further supported by the current studies and demonstrates that it also plays a role in mediating retrograde axonal transport. it is especially intriguing that mhv uses similar molecular mechanisms to interact with cellular axonal transport machinery, suggesting that targeted disruption of this protein function might prevent all pathogenic spread of the virus. although it has been reported previously that neurotropic mhv strains can spread within neurons in a retrograde direction, the molecular interactions mediating this spread have not been fully examined ( , ) . in cultured neurons, viral interaction with the microtubule network in neuronal processes has been shown to be important; based on cross-interaction of antimicrotubule antibodies and the nucleocapsid protein (n), it is possible that such interactions might be involved in axonal transport mechanisms ( ) . however, in view of the fact that the dm and ndm strains used in the present study differ only in spike protein and contain identical n proteins, our data suggest more of a role for the spike protein. how the dm strain of mhv initially infects neurons requires further study. one possible explanation could be that traumatic disruption to the nerve endings in the brain allows access of the virus through the damaged axolemma; or the virus may be capable of directly infecting intact axons either in the brain, or after diffusing into the optic nerve in the cerebrospinal fluid. alternatively, virus may be engulfed into the axons at synaptic terminals. although direct infection after diffusion in cerebrospinal fluid could potentially explain the presence of viral antigen in optic nerves, the fact that antigen is detected in rgc bodies in the retina demonstrates that retrograde transport does occur. although the presence of viral antigen in rgcs alone cannot exclude hematogenous spread as an alternate mechanism from retrograde transport, the timing of its spread (ie, detected only at day pi in the retina versus day pi within optic nerves and within liver via hematogenous spread), the lack of hematogenous spread to the cns after systemic infection with equivalent doses of mhv-a ( ) , and the presence of viral antigen in areas of the brain containing rgc projections all suggest that retrograde transport is the more likely mechanism of viral spread. retrograde transport may follow fusion of the spike protein with axonal membranes followed by loss of most of the viral structural proteins and then binding of the nucleocapsid via one or more viral proteins to the retrograde molecular motors. such a mechanism has been seen in other neurovirulent viruses that follow a retrograde direction of axonal transport ( , ) . although it is not common that a single virus can use both directional transport mechanisms, this is not the first virus to demonstrate such properties. for example, herpesviruses can be transported rapidly along microtubules in the retrograde direction from the axon terminus to the dorsal root ganglion and then anterograde in the opposite direction ( , ) . axonal transport is an important strategy used by several neurovirulent viruses, including herpes simplex, rabies, polio, influenza, and borna disease viruses, which can be transported in axons either in anterograde or in retrograde direction ( y ). in some instances, viruses can be transported within axons for a long distance, yet this journey occurs within the cell. therefore, the virus cannot be inactivated by neutralizing antibody during its transit and may spread in the cns without inducing an antivirus immune response while it is within the cell. when viruses can spread only within axons and/or via direct cell-tocell contact, they could potentially escape the attack of antiviral drugs or neutralizing antibodies. in the current studies, optic nerve inflammation induced by dm strain rsa consisted of predominantly macrophages/microglia, similar to immune responses in spinal cord ( ) and without the marked t-cell infiltration seen in other demyelinating disease models ( y ). iba- staining was diffuse and was observed in cell bodies as well as cell processes, as has been seen previously with this macrophage/microglia marker ( , ) . although iba- cannot distinguish between macrophages and microglia, we suspect most labeled cells represent infiltrating macrophages based on the overall increase in the number of cells observed. macrophages have been shown to play both proinflammatory and anti-inflammatory roles in optic nerves ( ) , and we suggest that the dm strain viral antigen might be moving within axons to avoid being cleared by infiltrating macrophages. it is known that some viruses make use of the microtubules and/or the actin cytoskeleton for axonal transport ( y ). our studies have shown that a major mechanism of both retrograde and anterograde axonal transport of neurovirulent mhv is mediated by spike protein and further experiments will focus on identifying the molecular mechanisms by which the dm virus interacts with the axonal transport system and whether specific interventions targeting the transport system can delay or prevent the dm strain-induced axonal loss and demyelination. analyzing the underlying principles of mhv axonal transport will be helpful in the design of viral vectors to be used in research, in human gene therapy, and in the identification of new antiviral therapies. such therapies may also have the potential to prevent cns demyelinating diseases that might be triggered initially by viral infection. axonal damage is t cell mediated and occurs concomitantly with demyelination in mice infected with a neurotropic coronavirus mechanisms of primary axonal damage in a viral model of multiple sclerosis a mechanism of virus-induced demyelination demyelinating and nondemyelinating strains of mouse hepatitis virus differ in their neural cell 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containing a green fluorescent protein-b r membrane protein chimera the authors thank the du pre foundation for support of ag. 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- - qfhltg authors: chatterjee, dhriti; biswas, kaushiki; nag, soma; ramachandra, s. g.; das sarma, jayasri title: microglia play a major role in direct viral-induced demyelination date: - - journal: clin dev immunol doi: . / / sha: doc_id: cord_uid: qfhltg microglia are the resident macrophage-like populations in the central nervous system (cns). microglia remain quiescent, unable to perform effector and antigen presentation (apc) functions until activated by injury or infection, and have been suggested to represent the first line of defence for the cns. previous studies demonstrated that microglia can be persistently infected by neurotropic mouse hepatitis virus (mhv) which causes meningoencephalitis, myelitis with subsequent axonal loss, and demyelination and serve as a virus-induced model of human neurological disease multiple sclerosis (ms). current studies revealed that mhv infection is associated with the pronounced activation of microglia during acute inflammation, as evidenced by characteristic changes in cellular morphology and increased expression of microglia-specific proteins, iba (ionized calcium-binding adaptor molecule ), which is a macrophage/microglia-specific novel calcium-binding protein and involved in membrane ruffling and phagocytosis. during chronic inflammation (day postinfection), microglia were still present within areas of demyelination. experiments performed in ex vivo spinal cord slice culture and in vitro neonatal microglial culture confirmed direct microglial infection. our results suggest that mhv can directly infect and activate microglia during acute inflammation, which in turn during chronic inflammation stage causes phagocytosis of myelin sheath leading to chronic inflammatory demyelination. microglia are specialized macrophages of the cns that constitute - % of total glial cells in rodents, depending on the specific neuroanatomical region of the cns. microglia are distinguished from neuron as well as glial cells, such as astrocytes and oligodendrocytes, by their origin, morphology, gene expression pattern, and function. while neuron and conventional glial cells are neuroectodermal in origin, microglia are of haematopoietic origin and act as primary responding cells for pathogen infection and injury like monocytes/macrophages in peripheral tissues. microglia exhibit several features that distinguish them from other populations of macrophages, such as their "ramified" branches that emerge from the cell body and communicate with surrounding neurons and other glial cells. microglia can rapidly respond to infectious and traumatic stimuli and adopt a "phagocytotic" nature. activated microglia are known to produce many proinflammatory mediators including cytokines, chemokines, reactive oxygen species (ros), and nitric oxide which mainly contribute to the clearance of pathogens or infections. however, prolonged or unwarranted microglial cell activation may result in pathological forms of inflammation which can lead to several neuroinflammatory conditions of the nervous system. microglia-mediated innate immune response in the cns is now considered to be potentially one of the major pathogenic factors in a number of cns neuroinflammatory diseases that lack clinical and developmental immunology the prominent leukocytic infiltrates of adaptive immune responses [ ] . neuroinflammation is associated with many neurodegenerative diseases, including alzheimer's disease (ad), parkinson's disease (pd), amyotrophic lateral sclerosis (als), and multiple sclerosis (ms) [ ] . while ad, pd, and als are commonly known to be neurodegenerative disease with underlying neuroinflammatory mechanisms, ms is one of the major chronic inflammatory cns diseases in humans with heterogeneous (chronic/remitting) clinical presentations and course [ , ] . ms is believed to be an autoimmune inflammatory demyelinating disease in which exposure of genetically predisposed people to environmental factors triggers a breakdown in t-cell tolerance to myelin antigens. demyelination is a complex process, and while the precise mechanisms of this pathology are unclear, inflammatory demyelination is thought to be the result of adaptive immune-mediated responses to myelin antigens in the myelin sheaths of axons and/or in the myelin-forming oligodendrocytes. most studies have focused on the pathogenic role of myelin-specific cd + t cells because of the relatively strong association of susceptibility to ms with major histocompatibility complex (mhc) class ii alleles [ , ] . there is also increasing recognition of the potential importance of cd + t cells in the pathogenesis of demyelination [ , ] . however, the contribution of innate immune cells in mediating ms pathogenesis has been recently gained attention, as several studies demonstrated the role of various innate immune cells in mediating ms pathogenesis, in particular, the potential anti-inflammatory or proinflammatory function of microglial cells along with its physical interaction with myelin [ ] [ ] [ ] . for long time, microglia were known to be present in the chronic inflammatory demyelinating plaque to remove myelin from the dead sick neuron in ms patients but the emerging recognition of microglia as cns resident immune cells and their role in cns health and diseases stimulated substantial efforts to redefine the role and function of microglia in the regulatory mechanisms of demyelination. ms is best studied in some experimental models such as experimental autoimmune encephalitis (eae), theiler's murine encephalomyelitis (tmev), and mouse hepatitis virus-(mhv-) induced neuroinflammation. virtually, all types of adaptive immune response have been proposed to play important roles in the pathogenesis of eae [ , ] , tmev [ ] , and a neurotropic strain of mouse hepatitis virus (mhv); mhv-jhm [ , ] , mimicking the pathogenesis of the ms. upon intracranial (i.c.) infection of neurotropic mhvs, acute meningoencephalitis (with or without hepatitis) is the major pathologic process (see supplementary figure available online at http://dx.doi.org/ . / / ) [ ] . natural and genetically constructed recombinant mhv strains (generated by targeted rna recombination) with differential pathological properties were used in several studies to understand the mechanisms of demyelination and concomitant axonal loss [ ] [ ] [ ] [ ] . the outcome and degree of mhv-induced disease are dependent on several factors, including the age and strain of the mouse, the strain of mhv, and the route of virus inoculation. even very closely related strains of mhv differ in pathogenic properties. some strains of mhv are purely hepatotropic (e.g., mhv- ) [ ] ; some are primarily neurotropic (e.g., jhm, mhv- , an isolate of jhm) [ , ] ; while others (e.g., mhv-a and mhv ) [ , ] are both hepatotropic and neurotropic. viral titer reaches its peak at days and postinfection (p.i.) [ ] . infectious virus is cleared within the first - days; however, at this time mice begin to develop demyelination, either clinical or accompanied by chronic hind limb paralysis [ , ] . both mhv-jhm and mhv-a cause inflammatory demyelination in the brain and spinal cord whereas mhv only causes vasculitis [ , ] . it was formerly believed that in primary mhv-induced demyelination neuronal axons remain relatively preserved. recently, it has been shown that axonal damage is, in large part, immune mediated in mhvinfected mice and occurs concomitantly with demyelination. concurrent axonal loss and demyelination have recently also been observed with s protein recombinant demyelinating strain-infected mouse spinal cord [ ] . evidence from highly neurovirulent jhm strains of mhv suggests that mhv-induced demyelination is primarily immune mediated [ , ] . clearance of infectious virus is mediated by both cytolytic and cytokine-mediated mechanisms and microglia, and t cells modulate pathologic changes. demyelination can be prevented in jhminfected lymphocyte-deficient (rag −/− ) mice [ ] . however, demyelination will occur upon transfer of splenocytes from immunocompetent mice to rag −/− mice [ ] . it has also been shown by depletion and transfer studies in the jhm model that cd + t cells can induce demyelination. these studies suggest that an intact adaptive immune system is required to promote demyelination in jhm-mhv infection. contrary to these findings, demyelination, induced by mhv-a , has been shown to develop in adult immunocompromised mice lacking b and t cells [ ] . it has also been demonstrated that the depletion of cd + or cd + t cells after the acute stage of infection does not reduce demyelination [ , ] . indeed, mhv-a or its isogenic spike protein (hostattachment protein) recombinant strain, rsa [ , , ] , induces a ms-like disease in mice mediated by microglia, along with a small population of t cells. the mechanism of demyelination is at least, in part, due to macrophagemediated myelin stripping, with some direct axonal injury as well as without involving the conventional t cells. in our current studies, we have used rsa infection in vivo, in vitro, and ex vivo as a model to understand whether mhv can directly infect cns resident microglia and the mechanism of microglial activation in the induction of chronic demyelination. use of animals and all experimental procedures were reviewed and approved by the institutional animal care and use committee at the indian institute of science education and research kolkata and indian institute of science, bangalore india. animal protocols adhered to the guidelines of the cpcsea, india. rsa an isogenic recombinant demyelinating strain of mhv-a , where the spike gene (encodes virus host-attachment protein), was exchanged by mhv-a spike gene only in the background of mhv-a gene by targeted rna recombination as described in our previous studies [ , ] . this recombinant strain also expresses enhanced green fluorescence protein (egfp) [ ] for easy detection of viral particle by egfp fluorescence. to engineer the targeted recombinant strains, molecularly cloned vector pmh [ , ] , which contains the entire end of the genome from mhv-a , was used for construction of the recombinant viruses. rsa and rsmhv are isogenic except the spike protein. rsa strain is expressing the mhv-a spike in the mhv-a background, whereas rsmhv strain is expressing the mhv- spike in the mhv-a background [ ] . furthermore, egfp gene was inserted into the mhv genome in place of the nonessential gene in both rsa and rsmhv [ ] . in order to replace gene with the egfp gene, pmh was modified by the introduction of a sali site nucleotides downstream of the intergenic sequence for gene a and a noti site bp upstream of the stop codon for gene b, using the quick change site-directed mutagenesis kit (stratagene, la jolla, ca, usa). (these are coding-silent nucleotide changes.) the coding sequence of egfp was cleaved from the pegfp-n vector (clontech, palo alto, ca, usa) using sali and noti and inserted in the place of the sali/noti fragment of pmh . the resulting plasmid contains bp of non-mhv sequence, including the -bp egfp open-reading frame, replacing the entire gene a and the rest bp of gene b. previous studies reported with jhm strain revealed that the interruption of the orf did not alter the neurovirulence neither in vivo nor the replication in vitro [ ] . targeted recombination was used to select mhv isolates with stable and efficient expression of the gene encoding egfp to facilitate the in vivo detection of virus in the mouse cns as well as to trace the viral entry and spread in tissue culture. the viruses replicated with similar kinetics as wild-type virus both in tissue culture and in the mouse cns. they caused similar encephalitis and demyelination in animals as the wildtype virus or their recombinant strains; however, they were somewhat attenuated in virulence [ ] . four-week-old, ten mhv-free, c bl/ (b ) mice (jackson laboratory, obtained from iisc, bangalore, india) were inoculated intracranially with % ld dose of rsa strain ( , pfu) as described previously [ , ] . mice were monitored daily for signs of disease. three mock-infected controls were inoculated similarly but with an uninfected cell lysate at a comparable dilution. three mice were sacrificed in between days , , or (period for peak of inflammation postinfection for routine paraffinbased histopathological analysis), and the other three were used for frozen sections. the rest of the infected mice were sacrificed at day postinfection for routine paraffin-based histopathological analysis. cervical, thoracic, and lumbar regions of spinal cord were successively processed, and quadrants (dorsal/posterior column, anterior column, and two anterior horns) from two separate sections of each spinal cord level were examined. histopathology. at , , or and days postinfection, brain and spinal cord tissues were harvested from both mock infected and rsa -infected mice. for routine paraffin sectioning, brain and spinal cord tissues were postfixed in % pfa for overnight. fixed tissues were processed and micron thin sections were prepared for routine cns pathology, whereas frozen sections tissues were postfixed with % pfa for − hours and then transferred in % sucrose solution for - hours and in % sucrose solution for - hours and mounted in cryomatrix (thermo shandon). ten micron thin sections were prepared for frozen tissue immunofluorescence. the paraffin-embedded tissue sections were stained with hematoxylin and eosin (h&e) to determine the inflammation, whereas luxol fast blue (lfb) staining was used to detect the loss of myelin sheath. all slides are coded and read in blind manner. analysis. serial sections from brain and spinal cord were stained by the avidin-biotinimmunoperoxidase technique (vector laboratories) using , -diaminobenzidine as substrate and a : dilution of anti-iba (wako, richmond, va, usa), : dilution of anti-cd (lca; leukocyte common antigen, ly- , bd pharmingen), anti-iba (wako, richmond, va, usa), or cd (dako; carpinteria, ca, usa), and : dilution of monoclonal antibody directed against the nucleocapsid protein (n) of mhv-jhm (monoclonal antibody clone - - (kindly provided by julian leibowitz)) as primary antibodies. control slides from mock-infected mice were incubated in parallel. cryosections from the spinal cord tissues were washed with pbs at room temperature in a humidified chamber, incubated for min at room temperature with mg/ml nabh in pbs to reduce autofluorescence, washed, incubated for h at room temperature with m glycine in pbs to reduce nonspecific cross-linking, and then washed subsequently with pbs, pbs with . % triton x- (tx), and pbs with tx and % goat serum (gs). the sections were incubated overnight at ∘ c with a : dilution of a rabbit anti-iba antibody diluted in pbs with tx and gs, washed, and then incubated with a secondary antiserum diluted into pbs with gs for hrs at ∘ c. all incubations were carried out in a humidified chamber. viral antigen was detected by egfp in a fluorescein isothiocyanate channel [ ] . control slides were incubated in parallel with preimmune rabbit sera, and sections from mock-infected mice were incubated with secondary antibodies only. tissue sections were sequentially washed with pbs plus tx and with pbs and mounted and visualized by ix- fluorescence microscopy with a x uplanapo objective, with the iris diaphragm partially closed to limit the contribution of out-of-plane fluorescence, and with filter packs suitable for green fluorescence and red fluorescence. images were acquired with a hamamatsu orca- charge-coupled device camera and image-pro image analysis software (media cybernetics, silver spring, md, usa). chronic inflammatory stage of rsa infection. to further characterize the presence of microglia in the chronic demyelinating plaque at the ultrastructural level, mice were anesthetized, perfused with % pfa, and spinal cords from mock-infected and rsa -infected were harvested and fixed overnight in % glutaraldehyde as described earlier [ ] . samples for transmission electron microscopy (tem) were postfixed with % osmium tetroxide, dehydrated, and flatembedded in poly-bed epoxy resin (polysciences). half micrometer thick sections were cut from the lesional epicenter, stained with toluidine blue, and examined by light microscopy. ultrathin tem sections ( Å) were cut from representative foci of demyelination from the toluidine bluestained semithin sections and mounted on mesh copper grids, stained with uranyl acetate and bismuth subnitrate, and viewed under a jeol jem . culture. four-week-old, mhv-free, c bl/ were perfused transcardially with sterile pbs. spinal cord was harvested and washed with pbs containing % penicillin/streptomycin (pen/strep). the spinal cord was then embedded in % agarose mould, and micron thick crosssections were prepared by vibratome (leica vibrating blade microtome; vt s). the slices were washed twice with pbs containing % pen/strep. the slices were then transferred to a -well plate with one slice in each well. l of dmem containing % fbs, % pen/strep, and % l-glutamine were added in each well and incubated overnight with % co . after hrs of explantation, slices were infected with rsa at , pfu (half of the ld dose) in low serum ( %) containing medium for hr and then washed with pbs to remove the unbound viruses, and % serum containing medium were added to the infected culture and the cultures were maintained for hrs. at hrs, hrs, and hrs of postinfection, slices were processed for immunostaining with anti-iba antibody, and egfp fluorescence was used to detect viral antigen. briefly, at different times postinfection slices were washed gently with pbs and fixed with % pfa for hours. postfixed slices were washed with pbs, permeabilized with . % triton x- for mins, and blocked with % goat serum for hr at rt followed by overnight incubation with anti-iba antibody. for better staining, next day antibody solution was replaced with fresh antibody and incubated at ∘ c for additional - hrs. slices were washed to remove any unbound antibody and then labelled with tritc conjugated goat anti-rabbit igg for hrs. labelled slices were then washed to remove any unbound fluorescent tagged antibody and then mounted in vectashield (vector laboratories, ca, usa with dapi and observed in zeiss confocal microscope (lsm ). images were acquired and processed by using zen software (carl zeiss). brain. primary cultures of mixed glia from day to day newborn mice were prepared as described previously [ ] . briefly, following the removal of meninges, brain tissues were minced and incubated in a rocking water bath at ∘ c for min in hanks balanced salt solution (hbss, gibco) in the presence of g/ml of dnasei (sigma) and . % trypsin (sigma). enzyme-digested-dissociated cells were triturated with . % of fetal calf serum (fcs), followed by a wash and centrifugation ( ×g for min). the pellet was resuspended in hbss, passed through a micron nylon mesh, followed by a second wash and centrifugation ( ×g for min). following dilutions with astrocytespecific medium (dulbecco's essential medium containing % penicillin-streptomycin, . mm l-glutamine, and % fcs), cells were plated and allowed to adhere for day in a humidified co incubator at ∘ c. after hrs, any nonadherent cells were removed and fresh astrocyte-specific medium was added. adherent cells were maintained in astrocyte-specific medium for days. culture. after establishment of the mixed glia culture, feeding was stopped for days to allow for significant microglial growth on top of the astrocyte monolayer. the microglia population peaked at - days in these cultures. to remove any cells adherent to the astrocyte monolayer, microgliaenriched cultures were thoroughly agitated in an orbital incubator shaker ( rpm for min at ∘ c). immediately following agitation, all cells suspended in the culture medium were collected and centrifuged at ×g for min at ∘ c. the cell pellet was resuspended and diluted with fresh astrocytespecific medium bringing the cells to a final concentration of × cells/ml; ml was added to each well of a two-well cc -treated chamber slide (specifically made for primary cell culture; nunc) or ml/well of a six-well plate. after min, any non-adherent cells were discarded and adherent cells were maintained in fresh astrocyte-specific medium until infected with a medium change every - days. to examine different cell types in a given culture, primary antibodies directed against cell-specific antigens were used to determine the presence and/or purity of each of the major glial cell types as described previously. microglia were labelled with biotinylated anti-mouse cd b (chemicon, diluted : in f- nutrient medium) followed by cy streptavidin (jackson immunoresearch, diluted : in f- ). astrocytes were labelled with polyclonal rabbit antiglial fibrillary acidic protein (anti-gfap; dako) followed by either goat anti-rabbit alexa (molecular probes), cy , or fitc (jackson immunoresearch) secondary antibodies. before processing for double-label immunofluorescence microscopy, cells were washed in f- nutrient medium. cells were incubated with primary antibodies to the surface clinical and developmental immunology markers cd b at room temperature followed by three min washes with f- . cells were then incubated with fluorescently coupled secondary antibodies for min followed by three washes with pbs containing ca ++ /mg ++ . surfacelabelled cells were fixed for min in % paraformaldehyde followed by pbs washes, permeabilized with pbs/tx (pbs with ca ++ /mg ++ , . % triton-x) for min, and successively washed with pbs/tx/gs (pbs with ca ++ /mg ++ , . % triton-x, . % normal goat serum) three times for min each. cells were incubated for min with the astrocytic marker gfap, washed three times with pbs/tx, labelled with an appropriate secondary antibody, and stained with dapi ( : diluted in pbs without ca ++ /mg ++ from g/ml stock solutions) for min. cells were then washed, mounted using vectashield (vector laboratories), and visualized by fluorescence microscopy (olympus i x- ) with a planapo objective ( . numerical aperture). images were acquired with a hamamatsu orca ccd camera and data were analyzed by using image-pro software. on day after seeding, neonatal microglial cultures were infected at a multiplicity of infection (moi) of : with rsa or mock-infected with noninfected cell lysate. after allowing viral adsorption for hr, cells were washed and placed in fresh media without virus. at , , and hrs after infection, cultures were examined by microscopy for egfp fluorescence. . . rsa growth curve. confluent monolayers of l cells were infected with undiluted and : diluted culture supernatant collected from the in vitro infected microglia and incubated for hr at ∘ c. following adsorption, the cells were washed with tris-buffer saline times and then fed with dmem with % fbs mixed with . % agarose for overlaying. hours postinfection, culture was subjected for plaque count [ ] . to confirm the rsa -induced cns inflammation, brain and spinal cord sections from day (peak of inflammation) and day (peak of demyelination) postinfected mice were stained with h&e or lfb and examined. rsa -induced meningitis (supplementary figure (a) ), and encephalomyelitis (accumulation of inflammatory cell and perivascular cuffing) ( supplementary figures (b) and (c) ) were observed as shown previously [ , ] (supplementary figure ; these data are partly published but for the ready information compiled in one figure. ). to characterize inflammatory cell types, brain sections from day postinfection were stained immunohistochemically with anti-cd (leukocyte common antigen (lca)), anti-cd b and/or anti-iba (macrophage/microglial marker), or anti-cd (pan t-cell marker) (data not shown). the majority of inflammatory cells in rsa -infected brains were immunoreactive for both lca (supplementary figure (d) ) and cd b (supplementary figure (e) ) and iba (supplementary figure (f) ). some cd -stained infiltrating t cells were also found (data not shown), although nonspecific background staining of neurons with available anti-cd antibodies made quantification difficult. no cd -and cd positive cells but few cd -positive cells were observed in the inflamed brain and spinal cord sections in rsa -infected mice (data not shown). demyelination was observed by lfb staining as early as day as examined (supplementary figure (h) ) and it reaches its peak at day postinfection (supplementary figure (i) ) as observed earlier [ , ] . lfb-stained spinal cord section showed no myelin loss (supplementary figure (g) ). together, the data indicate that rsa causes meningoencephalitis and demyelination. cns inflammation consists of a mixed population of inflammatory cells, predominantly macrophages/microglia as well as a smaller population of t lymphocytes as shown previously [ , , ] . infection during acute inflammation. previously, it has been demonstrated that neurotropic strains of mhv can directly infect different neural cell types [ , , , ] but there is no evidence whether neurotropic strain can directly infect microglia or only acquire activity indirectly due to the infection of other neural cell types. in order to determine the tropism of rsa in cns resident microglia, fourweek-old, mhv-free, c bl/ (b ) mice (jackson laboratory) were inoculated intracranially with rsa . mice were sacrificed at the peak of inflammation (day ), and the spinal cord sections were prepared for cryostat sectioning. since rsa expresses egfp, viral antigen was viewed directly by fluorescence microscopy. identification of cns resident microglia was performed by using iba as a specific marker for microglia/macrophages [ ] . while iba immunofluorescence was observed in both gray and white matter, double fluorescence/immunofluorescence demonstrated dual labelling of egfp (viral antigen) positive iba positive microglia/macrophages were present only in the white matter of rsa infected mice (figure ). in the white matter, all the microglia (iba -positive) were not infected as shown by arrowheads (figures (e) and (f) ). in the control mock infected spinal cord section, no double fluorescent labelled cells were observed as expected (data not shown). previous studies demonstrated that with time of postinfection viral antigen spread from gray matter to white matter [ ] in the infected mice. this phenomenon is more prevalent in the spinal cord of infected mice as gray and white matter is clearly separated from white matter. immunostained section demonstrated that the viral antigen is localized both in gray and white matter at day postinfection (figure (a) ). at day viral antigen is below the detection level, more specifically after day postinfection (as observed) viral antigen is below the detection limit (data not shown) as discussed previously [ , ] . to determine whether microglia also follow the trajectory of the viral spread at days and postinfection, debris in demyelinating plaque. previously microglial accumulation was observed in the demyelinating plaque of rsa with an emphasis on the stripping of the myelin sheath [ ] . to reemphasize on the accumulation of microglia in the demyelination plaque during chronic phase of the inflammation at ultrastructural level, semithin sections were cut at micron intervals from five infected mice at day post infection. semithin sections were stained with toluidine blue. control mock-infected mouse spinal cord was used to evaluate for background fixation and/or postfixation artefacts ( supplementary figure (a) ). rsa -infected spinal cords showed significant myelin loss and accumulation of phagocytotic microglia within plaques as observed earlier ( supplementary figures (b) and (c)). representative foci of demyelination were selected from semithin sections, and Å ultrathin sections from poly-bed embedded blocks were processed for tem. high-resolution tem images show accumulation of large number of microglia with no basement membrane which is the characteristic features of microglia/macrophages (supplementary figure (e) ). multiple vacuoles with myelin fragments were seen within the cytoplasm of the microglia in the plaque (supplementary figure (f) ). no such microglial accumulation was observed in the control mock infected mice at high-resolution tem images (supple figure (d) ). cells. in vivo colocalization of iba with egfp-(viral antigen) positive cells demonstrated that rsa can directly infect microglia but that does not confirm that infected microglia were resident microglia because in intracranial (ic) inoculation blood brain barrier can be disrupted and blood monocytes/macrophages can migrate and acquire infection. (figures (g) and (k)) which demonstrated that cns resident microglia can directly acquire infection and become activated (by morphological analysis as number of processes increased and enlarged). arrowheads in figures (b) , (c), (d), (f), (g), (h), (j), (k), and (l) showed that some of the resident microglia did not get infection. control noninfected explant cultures were also immunolabeled with anti-iba antibody (figures (a) , (e), and (i)) as microglia in vitro in culture system behave like activated macrophages due to perturbation of the culture system. figures (d) , (h), and (l) show a merged image of egfp (viral antigen; green), iba (microglia; red), and dapi (nucleus; blue) and demonstrate the presence of viral antigen in the cell cytoplasm of microglia. due to the thickness of the slices, clarity of the images was slightly compromised. rsa infection in ex vivo explant cultures demonstrated that in the absence of peripheral immune cells cns resident microglia can directly be infected. syncytia. in order to determine whether rsa can infect microglial cells in vitro in absence of any neural cells, -dayold neonatal microglial cultures were infected at a multiplicity of infection (moi) of : with rsa or mock infected with noninfected cell lysate. microglia harvested in the cell suspension by the conventional shake-off method as described earlier [ ] were ± . % positive for cd b staining (figure (a) ). very few gfap (astrocyte marker) positive cells were observed in the isolated microglia culture (data not shown). at , , , and hrs after infection, cultures were examined by microscopy for egfp fluorescence. at and hrs, no fluorescence was observed in the infected culture but at hrs bright fluorescence started to appear denoting the presence of viral antigen in the microglia. at hrs postinfection, infected microglia demonstrated stressed morphology and started to fuse with the neighbouring cells, and at hrs postinfection, most of the infected cells were involved into large syncytia formation (figure (c) ) which is a characteristic of some enveloped rna viruses and more specifically characteristic of mhv-a (parental strain of rsa ), an enveloped demyelinating strain of mhv [ ] . nucleus of the syncytia was very obvious as shown in figure staining. in vitro experiment demonstrated that rsa can infect primary microglia in isolated culture and can also induce syncytia in primary microglia. to demonstrate that the virus is replicating in the microglia, culture supernatant of infected microglia was assessed by routine plaque assay [ ] . routine plaque assay found very few plaques which were below the detection limit. but there, discrete syncytia was observed in the infected plates which denoted that the titer could be - pfu/ml. to understand the cellular mechanism of demyelination of neurotropic strain of mhv, prior studies in our laboratory have analyzed the detailed pathogenesis of recombinant mhv strain, rsa (demyelinating strain (dm)) and compared it with rsmhv (nondemyelinating strain (ndm)) that is isogenic except for the spike gene that encodes the virus-host-attachment spike glycoprotein [ , ] . both strains are capable of causing hepatitis, encephalitis, and meningitis. however, the two strains differ in their ability to induce subsequent demyelination and axonal loss [ ] . seven days post infection, rsa produces demyelination that is best observed in the spinal cord at day postinfection (peak of inflammation). in contrast, rsmhv does not produce demyelination and only rarely demonstrates axonopathic changes in spinal cord white matter [ ] . the inability of rsmhv to induce demyelination is due in part to a lack of transport of viral antigen (and the subsequent inflammatory reaction) to the white matter. furthermore, in vivo and in vitro experiments demonstrate deficits in the ability of rsmhv to spread between neurons when compared to interneuronal spread by rsa [ ] . rsa -induced demyelination occurs in the setting of both axonal degeneration and macrophage mediated myelin stripping along intact axons [ ] . while spike glycoprotein mediates spread of viral antigen to white matter through axonal transport, specific mechanisms leading to subsequent demyelination are not known. one plausible explanation is that mhv spreads intra-axonally within gray matter and when it reaches the white matter, viral particles may need to spread directly into oligodendrocytes, astrocyte, and microglia, using the spike protein, and can induce local oligodendroglial dystrophy and inflammation. viral antigen in white matter axons may be sufficient to trigger an inflammatory response via microglial activation. infected and activated microglia due to its intrinsic nature of chemotaxis can recruit more microglia to the site of inflammation and strip myelin from the damaged axon and successively cause demyelination. our current in vivo studies support this hypothesis that rsa can infect cns resident microglia. the migration and activation of numerous microglia to the white matter during acute inflammation and the retention of microglia in the chronic inflammatory plaque reinforce the hypothesis that cns resident microglia can be recruited to the region of local cns injury. ultrastructural morphology of microglia containing multiple vacuoles with myelin fragments in the cytoplasm in the demyelinating plaque further substantiate that cns resident activated microglia can mediate myelin stripping and can successively mediate demyelination. as rsa spread intra-axonally, no colocalization was observed within the gray matter cns resident microglia. if haematogenous propagation of peripheral monocytes/macrophages occurred to the cns, one would expect more widespread distribution of activated microglia throughout the spinal cord which may not discriminate gray/white matter track. furthermore, the delay in complete development of demyelination following partial resolution of encephalitis (up to days after peak inflammation) documented in previous studies would not be expected [ , , ] . moreover, ex vivo colocalization of egfp-positive cells with microglia confirmed that rsa can directly infect cns resident microglia in absence of peripheral immune cells. in vitro infections of neonatal microglia demonstrate that rsa not only infects, but microglia can also forms syncytia which suggests that microglia supports rsa infection via cell-to-cell contact. current combined in vivo, in vitro, and ex vivo explants culture studies established that the recruitment of microglia occurred from the cns resident microglial pool rather than peripheral monocyte/macrophages. our current studies are focused on the understanding of the innate immune mechanism of cns resident microglia activation and maturation to perform phagocytotic activity. affymetrix microarray analyses for mrna expression have revealed that expression of inflammatory mediators by mhv infected microglia, including chemokine and inflammatory cytokines. mhv infection of the mouse spinal cord was also associated with increased expression of genes involved in ifn signalling compared to mock-infected controls in the cns. during chronic infection (day postinfection), microglia are still present within areas of demyelination and microgliaassociated inflammatory mediators are still produced which indicates that microglia are still active. our results suggest that putative activated microglia and inflammatory mediators contribute to a local cns microenvironment that eventually regulates viral replication and ifn-gamma production during acute phase of infection. sequentially, ifn-can activate microglia by promoting phagolysosomes maturation and activation (engulfment of the myelin sheath) leading to demyelination. affymetrix microarray data warrants further confirmation. viral infection in the cns is classically recognized as inflammatory in nature, with meningeal perivascular and parenchymal infiltrates of peripheral leukocytes but rsa infection could be an exception where inflammation can proceed with cns resident glial activation without involving the peripheral immune responses like rabies virus infection [ ] , hiv infection [ ] , and prion diseases [ , ] . in this perspective, it is tempting to speculate that the underlying mechanism of chronic myelin loss in ms could be a combination of persistence of myelin-related autoimmunogens that has escaped self-tolerance with persistence of activated cns resident microglia which can mediate demyelination by phagocytised myelin. microglia are known for their innate immune function for long time but the role of microglia in chronic inflammation opens a new episode in the field of glial biology of neuroinflammatory diseases. the concept of chronic inflammation as opposed to acute inflammation is more relevant in the context of understanding other cns diseases, more specifically neurodegenerative diseases like alzheimer's disease, amyotrophic lateral sclerosis, parkinson's disease, and huntington's disease. these neurodegenerative diseases lack the prominent infiltrates of mononuclear cells but the underlying mechanism of inflammation could be through activation of cns resident microglia. activation of cns resident microglia in the context of chronic neuroinflammation as one of the underlying mechanism of neurodegeneration warrants further study. microglia as the prime components of an intrinsic cns resident immune system become a major focus in cellular neuroimmunology and, therefore, in neuroinflammation. it has been known for long time that in absence of conventional t cells microglia play a major role in neurotropic mhv-induced demyelination but the mechanism of infection and route of infection were not very clearly known for long time. our current microglial tropism studies revealed that rsa , an isogenic demyelinating strain of mhv, can infect and activate cns resident microglia, and microglia can help to mediate demyelination by engulfing myelin debris. rsa -induced neuroinflammatory models are helpful in understanding direct cns cellular injury and demyelination that does not require an intact adaptive immune system. understanding the role of direct cns resident microglial infection and activation will shed some light on the pathogenesis of cns inflammatory disease, not only infectious diseases but also chronic cns disorders. the vision of cnsresident-microglia-driven neuroinflammatory responses in rsa with neuropathological consequences has extended the avenue to explore the contribution of microglia in chronic neuroinflammatory cns diseases. microglia and neuroinflammation: a pathological perspective mechanisms underlying inflammation in neurodegeneration multiple sclerosis immunology of multiple sclerosis a full genome search in multiple sclerosis a complete genomic screen for multiple sclerosis underscores a role for the major histocompatibility complex a pathogenic role for myelin-specific cd + t cells in a model for multiple sclerosis autoreactive cd + tcell responses to human myelin protein-derived peptides role of the innate immune system in the pathogenesis of multiple sclerosis t cells-innate immune lymphocytes? multiple sclerosis: a complicated picture of autoimmunity myelin-specific cd t cells in the pathogenesis of experimental allergic encephalitis and multiple sclerosis persistent infection with theiler's virus leads to cns autoimmunity via epitope spreading pathogenesis of mouse hepatitis virus-induced demyelination chronic central nervous system demyelination in mice after jhm virus infection experimental demyelination produced by the a strain of mouse hepatitis virus a mechanism of virus-induced demyelination demyelination determinants map to the spike glycoprotein gene of coronavirus mouse hepatitis virus demyelinating and nondemyelinating strains of mouse hepatitis virus differ in their neural cell tropism mechanisms of primary axonal damage in a viral model of multiple sclerosis mouse hepatitis virus type- infection in mice: an experimental model system of acute meningitis and hepatitis pathogenesis of virusinduced demyelination selective tropism of a neurotropic coronavirus for ependymal cells, neurons, and meningeal cells limbic encephalitis after inhalation of a murine coronavirus ependymitis, leukoencephalitis, hydrocephalus, and thrombotic vasculitis following chronic infection by mouse hepatitis virus (mhv ) bystander cd t cell-mediated demyelination after viral infection of the central nervous system demyelination induced by murine hepatitis virus jhm strian (mhv- ) is immunologically mediated cd and cd t cells have redundant but not identical roles in virusinduced demyelination neither b cells nor t cells are required for cns demyelination in mice clinical and developmental immunology persistently infected with mhv-a cd + and cd + t cells are not major effectors of mouse hepatitis virus a -induced demyelinating disease experimental optic neuritis induced by a demyelinating strain of mouse hepatitis virus enhanced green fluorescent protein expression may be used to monitor murine coronavirus spread in vitro and in the mouse central nervous system the internal open reading frame within the nucleocapsid gene of mouse hepatitis virus encodes a structural protein that is not essential for viral replication analysis of a recombinant mouse hepatitis virus expressing a foreign gene reveals a novel aspect of coronavirus transcription inactivation of expression of gene of mouse hepatitis virus strain jhm does not affect virulence in the murine cns magnetic cell sorting: a fast and effective method of concurrent isolation of high purity viable astrocytes and microglia from neonatal mouse brain tissue macrophagemediated optic neuritis induced by retrograde axonal transport of spike gene recombinant mouse hepatitis virus selective localization of wild type and mutant mouse hepatitis virus (jhm strain) antigens in cns tissue by fluorescence, light and electron microscopy viral infections and demyelinating diseases neurofibromatosis- heterozygosity increases microglia in a spatially and temporally restricted pattern relevant to mouse optic glioma formation and growth syncytia formation induced by coronavirus infection is associated with fragmentation and rearrangement of the golgi apparatus rabies virusinduced activation of mitogen-activated protein kinase and nf-b signaling pathways regulates expression of cxc and cc chemokine ligands in microglia microglia in human immunodeficiency virusassociated neurodegeneration neuroinflammation in alzheimer's disease and prion disease atypical inflammation in the central nervous system in prion disease key: cord- -rjl dpa authors: taguchi, f.; yamada, a.; fujiwara, k. title: asymptomatic infection of mouse hepatitis virus in the rat date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: rjl dpa after intranasal inoculation of suckling rats mouse hepatitis virus multiplied mostly in the nasal epithelium; though there were no symptoms, antibodies were produced. antibodies were also demonstrated in adult rats. these findings suggest that the rat may be a natural host for the virus. , mhv-s multiplied in the "anterior part of the head", which includes the nasal bones as well as the nasal mucosa, and reached a maximum titer of about pfu/ . g on day . virus grew to much lower titres in the brain and low titres (less than pfu/ . g) were also detected in the lung, liver and spleen in some animals examined to days after virus inoculation. other tissues, such as salivary glands or blood, contained no infectious virus. limited to the nasal mueosa and brain (manuscript in preparation). two other strains of mhv, namely jhm and mhv-nu ( ), were also inoculated into -day-old rats and were found to multiply, but to a lesser extent than mhv-s. figs. a and b. necrotic changes (fig. a) and cytoplasmic immunofluorescenee (fig. b) in epithelial cells of the nasal mueosa. tissue from -day-old rats days after i.n. inoculation with pfu of mhv-s. bar represents . mm two-, -, -and t -week-old rats were also inoculated i.n. with t pfu of mhv-s and infectious viruses were assayed in the lung, brain, salivary glands and liver. in less than one-third of the total, were infectious viruses detected in the lung and salivary glands and the titers were less than t pfu/ . g. no infectious virus was demonstrable in the liver. sera were collected days after inoculation and examined for neutralizing antibody. table shows that almost all contained neutralizing antibody to a titre of > . the two rats inoculated at weeks and seronegative when tested at : were nevertheless positive at i : . so far only mice have been considered to be natural hosts of mhv and rat; has been excluded because highly virulent mhv inoculated by various routes did not cause any sy~lptomatic infection like that produced in the mouse ( , ) . however, this does not imply that mtiv does not infect the rat. in the present study, we clearly demonstrated that by the intranasal route mhv-s infects rats of various ages from the following criteria: . mhv replicated in the "anterior part of the head" and several other organs; . viral antigen was detected by immunoftuoreseenee in the epithelial cells of nasal mneosa where histopathological changes were also found ; . rats of all ages produced neutralizing antibodies after virus inoculation. i n some cases, neither infectious virus nor histopathologieal changes were demonstrable in infected rats, however, neutralizing antibodies were constantly detected in the sera of inoculated rats, revealing that mhv replication occurs in every cases, but m a y be below the detection level in some cases. two eoronaviruses, namely rat eoronavirus (rcv) ( ) and sialodaeryoadenitis virus (sdav) ( ) , are known to infect and cause disease in the rat. these viruses multiply first in the epithelial cells of the nasal cavity and thereafter, they manifest their organ tropism i.e., rcv goes to the respiratory system and woduees interstitial pneumonia ( , ) and sdav affects the salivary and lacrymal glands, and produces adenitis ( ) . the pathogenesis of nhv-s infection in rats resembles, in part, that of sdav or i~cv, in that these viruses cause the necrotic changes in the epithelial cells of the nasal mucosa at early stage of infection ( ) . however mhv is confined there, or affects slightly the central nervous system as it does in infections of mice (manuscript in preparation). b~tatt and his eoworkers showed that sdav multiplied in mice producing some histopathological changes in the respiratory system after i.n. inoculation ( ) and we demonstrated in the w e s e n t paper that {hv infected rats of any ages. these facts indicate that sdav and mitv are infectious for both mice and rats, so that both m a y play an important part in the survival of these viruses. a murine virus (jhm) causing disseminated encephalitis with extensive destruction of myelin. ii experimental infection of adult axenic rats with parker's coronavirus respiratory infection in mice with sialodacryoattenitis virus, a coronavirus of rats lethal enteritis in infant mice caused by mouse hepatitis virus a rnurine virus (jhm) causing disseminated encephalitis with extensive destruction of myelin. i. isolation and biological properties of the virus problems in checking inapparent infections in laboratory mouse colonies. an attempt at serological checking by anamnestic response mouse hepatitis virus and its pathogenic action antibodies to mouse hepatitis virus in human sera pathogenicity of mouse hepatitis virus for mice depending upon host age and route of infection isolation of low-virulent mouse hepatitis virus from nude mice with wasting syndrome and hepatitis pathogenesis of sialodaeryoadenitis in gnotobiotic rats rat eoronavirus ( %cv): a prevalent, naturally occurring pneumotie virus of rats experimental viral hepatitis mouse hepatitis virus infection as a. highly contagious, prevment, enteric infection of mice difference in response to mouse hepatitis virus a, mong suseeptible mouse strmns age-dependent response of mice to a mouse hepatitis virus, mttv-s. japan we th~nk dr. t. sato (chugai pharmaceutical co., ltd) and dr. h. ikeda of the same institute for their many helpful discussions of the research.this work was supported partly by grant-in-aid for scientific research from ministry of education, science and culture, japan. key: cord- -a pkh q authors: tardieu, m.; goffinet, a.; harmant-van rijckevorsel, g.; lyon, g. title: ependymitis, leukoencephalitis, hydrocephalus, and thrombotic vasculitis following chronic infection by mouse hepatitis virus (mhv ) date: journal: acta neuropathol doi: . /bf sha: doc_id: cord_uid: a pkh q mouse hepatitis virus (mhv ) is either avirulent (resistant mice), hepatotropic (susceptible mice). or neurotropic (semisusceptible mice), depending on the strain of mice infected. in semisusceptible mice, infection led first to a transient meningitis, ependymitis, and leukoencephalitis, followed by a permanent communicating hydrocephalus and, later on, to a chronic thrombotic vasculitis affecting meningeal and parenchymal vessels at the brain stem level. small foci of ischemic necrosis related to vascular occlusions were seen in the dorsal brain stem. cyclophosphamide treatment of semisusceptible mice significantly reduced the meningeal infiltrates but did not prevent the development of hydrocephalus and other neuropathologic changes. identical lesions occurred in fully susceptible mice infected with a low dose of virus, but no neurologic disorder could be induced in genetically resistant mice even following immunosuppression or intracranial inoculation. the leukoencephalitis differed from the demyelinating lesions observed with mhv . vascular lesions were of particular interest. more attention should be given to the possibifity of virus induced chronic cerebral vasculitis in man. differences in susceptibility to virus infection depend on characteristics of injected virus (weiner et al. wolinsky and stroop ) , the route of infection (johnson ) , and on host genetic factors and immune function (allison ; hirsch et al. ; levy-leblond et al. ). mouse hepatitis virus (mhv ), a member of the corona virus group, is either avirulent, hepatotropic, or neurotropic, depending on the strain of mice infected. adult a/j mice are fully resistant to the virus. susceptible strains (e.g., dba/ or balb/c) develop an acute hepatic necrosis leading to death within a few days. some mouse strains (e.g., c h/he) as well as f hybrids between resistant and susceptible strains exhibit a "semisusceptibility" resulting either in early death or, in surviving animals, in the development of a chronic disease with neurologic manifestations and virus persistence (dupuy et al. ; le prevost et al. a, b) . ependymitis and vasculitis have been reported . susceptibility or resistance to mhv infection has been shown to be under the influence of at least two major gene complexes, one for the acute disease and the other, h- linked, for the chronic disease (levy-leblond et al. ). in addition to genetic factors, one of us has observed that host defense against mhv infection requires at least three types of immunocompetent cells: t-lymphocytes, splenic macrophages, and nk-like lymphoid cells (tardieu et al. ) . the aim of the present neuropathologic study was to better delineate the interaction of mhv with the different cells of the central nervous system (cns), as influenced by the genetic background and immunologic status of the host. of particular interest were an early transient leukoencephalitis and a chronic thrombotic vasculitis in the later stage of the disease. x a/j) fi hybrids (thereafter designed as baf mice) were bred in our mouse colony as previously described (le prevost et al. a) . passage, assay, and recovery of mhv were performed in susceptible dba/ mice as reported previously (le prevost et al. a) . virus titer was expressed in ld /g of tissue. the virus was injected intraperitoneally (i.p.) at a dose of ld in . ml of saline buffer unless otherwise specified. in a group of a/j strain mice, ld of viral suspension were injected intracerebrally through a burhole in the parietal bone (total injected volume: i gl). a total of mice were injected. ninety-one survived and were available for study. at various times after infection [on days , , , , , , , , , , , , , and post infection (p.i.) ], mice were anesthetized with ether and perfused through the left ventricle with either formaldehyde ( % w/v in phosphate buffer) for opto-microscopic studies, or formaldehyde ( % w/v in phosphate buffer) plus glutaraldehyde ( . % v/v) for electron-microscopic studies. the whole cranium was fixed overnight. the brain was then removed, embedded in paraffin, and sliced coronally on a serial basis for the preparation of paraffin sections which were processed by conventional techniques. in a series of mice prepared for electron-microscopic studies, selected fragments of ependyma, white matter, cortex and brain stem were obtained, postfixed in % oso , and embedded in epoxy resin. semithin sections were stained with toluidine blue or with the pas method. thin sections were stained with uranyl acetate and lead citrate and examined using a philips em electron microscope. cyclophosphamide treatment. cyclophosphamide was reconstituted with saline buffer and a freshly prepared solution was injected i.p. at a dose of mg/kg. cyclophosphamide treatment, in semisusceptible baf mice, given either before or just after mhv infection, leads to % mortality (willenborg et al. ) . to avoid acute mortality, cyclophosphamide was injected on days , , p.i. or and p.i. in some experiments, a/j strain mice were injected with cyclophosphamide , , and/or days before infection. a strontium treatment. cu of strontium ( sr) were injected i.p. in baf mice on two separate occasions, weeks apart. six weeks after the last injection, animals were considered " rtreated", as described previously (tardieu et al. ) . mhv was injected weeks after the second injection. after infection, ( %) of the semisusceptible mice (baf , ch ) survived and developed a progressive neurologic disease characterized by incoordination, paresis of the hind limbs, enlargement of the head, and progressive neurologic deterioration leading to death within - months. in this group of animals, no neuropathologic changes could be detected when examined on days and p.i. the first abnormalities, at days p.i., consisted of an important meningeal in-filtration of polymorphonuclear and mononuclear cells. at days p.i., the infiltrate consisted of small lymphocytes, plasmocytes, and macrophages. after days p.i., the diffuse meningeal infiltrate diminished progressively and was minimal after days p.i. during the same period inflammatory cuffing of meningeal vessels became progressively more marked. perivascular inflammatory cells consisted of small lymphocytes, plasmocytes, and macrophages. between and days p.i. perivascular infiltrations of mononuclear cells and microglial nodules appeared in the hemispheric white matter (fig. l a) . in rare instances, a microglial nodule was observed in the adjacent cortex. at days the inflammatory lesions in the white matter had disappeared, leaving widespread destruction and cavitation (fig. i b) . at days p.i., the first signs of an ependymitis appeared. between and days p.i. the granular ependymitis was particularly marked. inflammatory lesions predominated in the fourth ventricle and aqueduct ( fig. a) . focal aggregates of inflammatory cells consisting of mononuclear cells were formed beneath the ependyma and the ependymal cell line was focally disrupted. some of these granulomas bulged into the ventricular lumen and became even detached to form rosettes which were found free in the csf or were attached to the cilia of the ependymal cells. under the electron microscope, these granulomatous buds were seen to consist of lymphocytes and histio-monocytes together with ependymocytes. no viral particles could be seen within the affected ependymal cells. the cells of the subependymal aggregates did not stain with an antiserum against gfap, suggesting that they did not contain proliferating astrocytes. no changes were detected in the chroid plexus. after days p.i., the ependymal lesions became progressively less active and demonstrated residual scarring. the ependymal cell line was focally disrupted, and in the posterior part of the fourth ventricle it was completely destroyed. proliferation of subependymal atrocytes was not conspicuous (fig. b) . the first evidence of ventricle enlargement was noted at days p.i. hydrocephalus progressed thoughout the life span of the animals. on serial sectioning of the aqueduct no stenosis was observed at and days p.i. in mice killed after days p.i. vascular lesions were observed. numerous meningeal arterioles showed severe alterations : hyaline necrosis of the media, fragmentation of the elastic lamina and intimal proliferation (fig. ) . some arterioles and capillaries were occluded by an inflammatory thrombus (figs. , ) consisting of plasmocytes, a few polymorphonuclear leukocytes, and necrotic cells of uncertain origin (fig. ) . between - days p.i. vasculitis and thrombosis were seen to affect intraparenchymal yes- fig. a , b. inflammatory nodules in hemispheric white matter. neocortex is situated below and subiculum above the central white matter. fragments of ependyma are seen amidst the inflammatory cells. a days p.i. nissl, x . b widespread destruction of cerebral white matter, no inflammation; days p.i. he, x sels beneath the iv ventricle and in the dorsolateral part of the brain stem (fig. ) . a t some points, the nervous tissue surrounding these vessels showed distinct signs of ischemic necrosis, characterized by the presence of macrophages and proliferating astrocytes. aggregates of inflammatory cells were seen around vessels and in the surrounding parenchyma. n o microglial nodules and no intranuclear inclusions were observed. the infected semisusceptible mice which died acutely during week showed lesions similar to those observed in fully susceptible mice, i.e., acute hepatitis with a normal appearance of the cns. cyclophosphamide treatment. a group of baf mice were injected i.p. with mhv ( ld ). two died acutely and the others were treated with cyclophosphamide as described in material and methods. among the infected and cyclophosphamide-treated mice, survived and two died at and days p.i. pathologically, the ependymal lesions and ventricular dilatation were identical to non-treated mice. hyaline necrosis of the walls of meningeal arteries and thrombosis existed as in controls. in contrast, there was a marked diminution of the meningeal and perivascular inflammatory cell infiltration as compared to control mice which were infected by mhv but were not treated with cyclophosphamide. there was no evidence of leukoencephalitis. strontium treatment. treatment with sr resulted in acute liver necrosis with death between day and p.i. in the five semisusceptible mice tested. these mice showed a pattern of lesions similar to that observed in susceptible mice. when six susceptible balb/c mice were injected i.p. with mhv ( ld ), they died of an acute hepatic necrosis - d a y s after m h v infection, and no neuropatholigic lesion was observed except a slight degree of meningeal infiltration. a group of balb/c mice was then infected with a low dose of virus ( ld ). eight of these animals died acutely with in - days, the other two survived. the neuropathologic lesions were identical to those observed in semisusceptible mice, i.e., meningitis, leukoencephalitis, ependymitis, ventricular dilatation, and vasculitis. if the dose of inoculated virus was further lowered ( ld ), neither acute nor chronic lesions developed in five injected animals. to determine whether resistant mice could develop a chronic disease, three experiments were performed. first, three resistant a/j mice were injected in the same way as the semisusceptible animals. no clinical manifestations or neuropathologic abnormalities were observed. secondly, a/j strain mice were immunosuppressed with cyclophosphamide. after infection with mhv , the most profoundly immunosuppressed animals (two or three cyclophosphamide injections before mhv infection) died acutely as previously shown (willenborg et al. ) ; in the two surviving animals a slight meningeal infiltration and a perivascular infiltrate were observed on day p.i. without any ependymal damage, in a third experimental group mhv was injected directly into the brain of nine mice. only minimal neuropathologic lesions were observed (a light microglial infiltration at the point of injection) without any ependymal or vascular change. systemic infection of semisusceptible strains of mice with mhv leads to a remarkable sequence of brain lesions, such as meningitis and leukoencephalitis to mention those appearing first. the diffuse meningeal infiltrate tends to disappear progressively within a few weeks without fibrotic changes. however, perivascular infiltrates remain, especially at the level of the brain stem. the acute leukoencephalitis (fig. a) , which is more marked in c h, is restricted to the central white matter of the cerebral hemisphere and is visible only during a limited period of time between days and p.i., leaving behind severe destructive lesions (fig. b) . this type of leukoencephalitis is different from white matter lesions produced by mhv , which consist of noninflammatory patchy demyelination (waksman and adams ; lampert et al. ; weiner ; herndon et al. ; haspel et al. ; weiner and stohlman ; stohlman and weiner ) . the presence of glial nodules in the white matter and occasionally in the adjacent cortex, may be considered as an indication of a direct action of the virus on glial cells. inflammatory changes in the ependyma appeared approximately at day and persisted for about month (fig. a) . they resulted in widespread destruction of the ependymal lining (fig. b) . viral particles were not detected with the electron microscope in the ependymal cells, in contrast with the findings in other viral ependymitis (nielsen and baringer ; wolinsky ) . progressive hydrocephalus starting at about weeks p.i. was probably a major factor in the death of the animals. virus-induced experimental hydrocephalus has been attributed either to stenosis of the aqueduct produced by an ependymitis (johnson et al. ; johnson and johnson ; johnson ) or to a post infectious fibrosis of the meninges (masters et al. ) . in our animals aqueductal stenosis was not observed (at least until the th day p.i.), and there was no meningeal fibrosis (arachnoid villi were not examined). immunosuppression of semisusceptible animals with cyclophosphamide did not prevent the appearance of hydrocephalus; its most remarkable effect was the nearly complete suppression of the meningeal infiltrate. fibrosis of the meninges can, therefore, probably be discounted as the cause of intraventricutar hypertension and the mechanism of hydrocephalus remains unsettled. however, the parallelism between the intensity of ependymitis and hydrocephalus should be noted: both abnormalities were more important in baf mice than in c h mice. in contrast to the early, self-limiting, inflammatory lesions observed in the cerebral white matter and ependyma, vasculitis appeared late, after ependymitis had subsided, and increased progressively thoughout the life of the animals. these remarkable lesions consisted in hyaline necrosis of vessel walls and thromboses (figs. , ) . plasmocytes were remarkably numerous in the inflammatory thrombi (fig. ). perivascular cuffings with lymphocytes and plasmocytes were even more abundant than in the early phases of the disease. arterioles, capillaries, and venules were affected, essentially in the meninges surrounding the pons and medulla, deep in the subependymal region of the ivth ventricle, and within the dorsolateral parts of the brain stem, where small loci of ischemic necrosis related to vascular occlusions were observed (fig. ) . it is not impossible that some of the parenchymal lesions could be ascribed to an encephalitic process; however, the presence of vascular occlusions and ischemic necrosis in these areas is unquestionable. in man, particularly in the fetus, the possibility for a chronic viral infection to induce ischemic brain lesions after a long delay, through a slowly progressive thrombotic vasculitis, has not been sufficiently considered. such a sequence of events is known, however, in congenital rubella, where degeneration of vessel walls leads to multifocal ischemic necrosis of the brain (rorke and spiro ; singer et al. ; rorke ) and similar lesions have been shown in experimental animals (rorke et al. ) . a rare condition, granulomatous angiitis has been related to varicella-zoster infection (kolodny et al. ; rosenblum and hodfield ; linnemann and alvira ) and to other viral infections (reyes et al. ) in man. also, it is known that inflammatory perivascular infiltrates perist for many months in human poliomyelitis (esiri ) . at the time vascular lesions develop, a low titer of virus can still be detected in the brain, as late as months p.i. (le prevost et al. b), and dupuy et al. ( ) have demonstrated at that stage a severe non specific immunodepression (dupuy et al. ) . to investigate possible immune mediation in the generation of vascular lesions, we studied the effects of two immunomodulating regimen in semisusceptible mice: cyclophosphamide, which depresses t-and b-cell activities (stockman et al. ) and s sr, which abolishes selectively nk cell activity (kumar et al. ; tardieu et al. ). cyclophosphamide did not prevent necrosis of vessel walls and thrombosis. sr treatment changed the pattern from semisusceptible to susceptible mice, dying of acute hepatic necrosis. to study further the effect of r, preliminary experiments were performed in which mice were injected with mhv day after the second injection of sr, to avoid acute hepatic necrosis. the intensity of vasculitis in these mice and in controls was identical. these experiments suggest that t, b, and nk-cells do not play a prominent role in the induction of vascular lesions. however, there is as yet no clear evidence in favor of a direct action of the virus on the vessels, and further research is needed into the pathogenesis of the thrombotic vasculitis following mhv infection. the same neurologic disease can occur in fully susceptible mice infected with a low dose of virus, but attempts to induce this disease in genetically resistant mice have been unsuccessful, even following immunosuppression or intracranial inoculation. these findings suggest that host cells in the brain from fully susceptible and semisusceptible strains are equally able to support viral replication and are equally susceptible to give rise to destructive lesions. the absence of disease in resistant mice may be related to an inability of host cells to either bind or replicate mhv . it is also possible that other means of immunosuppression may alter this genetic resistance. genetic factors in resistance against virus infection persistent virus infection with neurological involvement in mice infected with mhv c ( ) 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nervous system lesions in the congenital rubella syndrome granulomatous angiitis of the nervous system in cases of herpes zoster and lymphosarcoma pathology of the congenital rubella syndrome differential effects of cyclophosphamide on the b-and t-cell compartments of adult mice chronic central nervous system demyelination in mice after jhm virus infection neonatal susceptibility to mhv infection in mice. ii. role of natural effector marrow cells in transfer of resistance neuropathological effects of persistent infection of mice by mouse hepatitis virus infectious leukoencephalitis. a critical comparison of certain experimental and naturallyoccuring viral leukoencephalitis with experimental allergic encephalomyelitis pathogenesis of demyelination induced by a mouse hepatitis virus (jhm virus) molecular basis of reovirus virulence: role of the s gene viral models of demyelination effect of cyclophosphamide on the genetic resistance of c h mice to mouse hepatitis virus mumps virus-induced hydrocephalus in hamsters. ultrastructure of the chronic infection virulence and persistence of three prototype strains of mumps virus in newborn hamster key: cord- - ozhye q authors: yu, haijing; liu, yang; wang, hongwu; wan, xiaoyang; huang, jiaquan; yan, weiming; xi, dong; luo, xiaoping; shen, guanxin; ning, qin title: clara cell kda protein alleviates murine hepatitis virus strain -induced fulminant hepatitis by inhibiting fibrinogen-like protein expression date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: ozhye q background: fulminant hepatitis (fh) is a serious threat to human life, accompanied by massive and rapid necroinflammation. kupffer cells, the major immune cell population involved in innate immune responses, are considered to be central for fh. fibrinogen-like protein (fgl ) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and fgl depletion represses murine hepatitis virus strain (mhv- ) infection. clara cell kda (cc ) protein is a secretory protein with anti-inflammatory properties in allergic rhinitis and asthma. however, its mechanisms of action and pathogenic roles in other disease are still unclear. in this study, we aimed to determine the role of cc in fh and the regulation of fgl by cc . methods: a mouse fh model was established by peritoneal injection of mhv- . the mice received cc protein through tail vein injection before viral infection. survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined. the regulatory effect of cc on fgl expression was investigated using thp- cells and mouse peritoneal macrophages in vitro. results: in the mouse fh model induced by mhv- , the survival rate increased from to . % in the cc group compared to that in the saline-only control group. meanwhile, the levels of alt and ast in serum were significantly decreased and liver damage was reduced. furthermore, hepatic fgl , tnf-α, and il- β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of cc protein. in vitro, cc was found to significantly inhibit the expression of fgl in ifn-γ-treated thp- cells and mhv- -infected mouse peritoneal macrophages by western blot and real-time pcr. however, there was no direct interaction between cc and fgl as shown by co-immunoprecipitation. microarray investigations suggested that hmg-box transcription factor (hbp ) was significantly low in cc -treated and ifn-γ-primed thp- cells. hbp -sirna treatment abrogated the inhibitory effect of cc on fgl expression in human umbilical vein endothelial cells (huvecs). conclusion:cc protects against mhv- -induced fh via suppression of fgl expression in macrophages. such effects may be mediated by the transcription factor hbp . fulminant hepatitis (fh) is a serious life-threatening disease characterized by massive hepatocyte necrosis, severe liver damage, and high mortality. the underlying mechanisms and the pathogenesis of fh are not clear. however, accumulating evidence suggests that, regardless of the pathogenesis of fh, the host's inflammatory responses contribute to liver microcirculatory disorders and injuries. accordingly, it has been shown that immune cell activation and inflammatory cytokines play an important role in fh ( ) . in recent years, our laboratory has conducted extensive research on the pathogenesis of fh and found that immune cells play a key role in it. kupffer cells, natural killer (nk) cells ( , ) , cytotoxic t-lymphocytes (ctls), and double negative t-cells (dnt) ( ) ( ) ( ) in liver and the cytokines that are produced by these cells cause liver damage. prothrombinase fgl belongs to the fibrinogen superfamily and is produced by activated macrophages or endothelial cells, transforming prothrombin directly into thrombin, so as to quickly initiate the process of coagulation. this promotes the conversion of fibrinogen into fibrin, resulting in thrombosis ( ) ( ) ( ) ( ) ( ) ( ) . our study found that fgl was highly expressed in peripheral blood mononuclear cells (pbmcs) and in liver tissue of humans or mice with severe viral hepatitis, and was positively related to the severity of the disease ( , ) . gene therapy targeting fgl silencing showed that the survival rate of fulminant hepatitis mice increased from to . % ( ) . thus far, the discovery and related research involving fgl have provided new insights into the molecular mechanism of hepatocyte necrosis in fh. in view of the important role of fgl in severe viral hepatitis, investigations concerning the regulation of fgl will be beneficial in the search for new strategies for treatment of severe hepatitis. clara cell kda protein (cc ), also considered to be uteroglobin, clara cell secretory protein, is one of members of secretoglobin superfamily. expressed in mucosal epithelial cells of organs (including lungs and nose) that communicated with the outside world ( ) . cc has immunomodulatory and anti-inflammatory effects. compared to wild-type mice, cc -knockout mice exhibited excessive airway inflammation abbreviations: fh, fulminant hepatitis; mhv- , murine hepatitis virus strain ; fgl , fibrinogen-like protein ; cc , clara cell kda protein; alf, acute liver failure; pfu, plaque-forming units; pbs, phosphate-buffered saline; alt, alanine aminotransferase; ast, aspartate aminotransferase; pca, pro-coagulant activity; hrp, horseradish peroxidase; tunel, terminal deoxynucleotidyl transferase dutp nick end labeling. caused by allergic reaction and bacterial and viral infections ( ) . reduced levels of cc are associated with inflammatory and allergic airway diseases, including sinusitis, asthma and allergic rhinitis ( ) ( ) ( ) ( ) . previous studies and published articles show that cc protein can not only inhibit th cell responses by inhibiting expression of related molecules of dendritic cells and cytokines in mice with allergic rhinitis, but also can inhibit chitosan- like protein ( , ) . moreover, cc inhibits the expression of an important immune regulator, osteopontin (opn), in models of allergic rhinitis ( ) . in this study, we investigated the role of cc in hepatitis virus strain (mhv- )-induced fh in mice and explored whether cc protein could regulate fgl in the disease process. female balb/cj mice (shanghai shilaike animal seed center, shanghai, china), - weeks of age, with a body weight of . - . g, were kept in tongji hospital with food and water. mice were divided into two groups: cc group (experimental group) and phosphate-buffered saline (pbs) group (control group). this study was carried out in accordance with the recommendations of the guidelines of the national institutes of health and the animal experiment committee of tongji hospital. this study was reviewed and approved by the animal experiment committee of tongji hospital. the human monocyte cell line thp- was purchased from the cell institute of the chinese academy of sciences (shanghai, china). human umbilical vein endothelial cells (huvecs) were obtained from the biology treasure center of wuhan university, china. the chinese hamster ovary (cho) cell line was acquired from the typical culture preservation commission cell bank, the chinese academy of sciences (shanghai, china). human umbilical vein endothelial cells (huvecs) and cho cells were cultured in dulbecco's modified eagle's medium (dmem), and thp- cells were maintained in rpmi , containing % heat inactivated fetal bovine serum (fbs, gibco life technologies, usa), u/ml penicillin, and mg/ml streptomycin and cultured at • c, ml/l co and % humidity. peritoneal exudative macrophages (pems) were obtained from balb/cj mice. cells were resuspended in rpmi , supplemented with % fbs at - × cells/ml in a -well plate and incubated for h. they were then washed with rpmi medium and non-adherent cells discarded. the adherent cells were macrophages and were incubated for a further h. peritoneal exudative macrophages (pems) were divided into two groups. one group was supplemented with cc protein ( ng/ml) and in the other group, pbs was added. after h of stimulation, , plaque forming units (pfus) of mhv- was added to the cells, which were then cultured for h. peritoneal exudative macrophages (pems) were harvested and lysed for real-time pcr and western blotting analysis. cell apoptosis was detected by the terminal deoxynucleotidyl transferase dutp nick end labeling (tunel) method with a tunel apoptosis detection kit (roche, switzerland). briefly, µm sections were deparaffinized, dehydrated through an alcohol series and incubated with proteinase k for min at • c. after stopping the proteinase k digestion reaction with pbs, the samples were incubated with terminal deoxynucleotidyl transferase end-labeling cocktail (a mixture of terminal deoxynucleotidyl transferase and dutp at a ratio of : , respectively), for h at • c in an immunohistochemistry wet box. following washing and blocking, each section was supplemented with reagent (converter-pod) to cover the tissues and incubated for min at • c in a wet box. then, the liver tissue sections were washed with pbs, and colored with diaminobenzidine (dab) subsequently. hepatocytes with nucleus stained brownish yellow were considered to be apoptotic cells. the expression of fgl on thp- cells was measured by flow cytometry (bd facs canto ii, usa). briefly, cells ( × per tube) were incubated with human trustrain fcx (fc receptor blocking solution, biolegend, usa) for min at room temperature and then incubated in the dark with mouse anti-fgl antibody ( : , abnova,) or normal goat serum (an isotype control) at • c for min. cells were washed with pbs and incubated in the dark with pe-conjugated goat anti-mouse igg antibody ( : , biolegend, usa) at • c for min. cells were then washed with pbs and resuspended in µl pbs for study. liver slices were fixed in % paraformaldehyde and then embedded in paraffin. immunohistochemistry of liver tissues was performed using sp- splink detection kits (biotin-streptavidin hrp detection systems) (zsgb-bio, beijing, china) according to the manufacturer's instructions. for immunohistochemistry staining, the expression of fgl , fibrinogen, fas and tnf-receptor in mouse liver tissues was detected with polyclonal rabbit anti-mouse fgl antibody ( : , proteintech, usa), polyclonal rabbit anti-mouse fibrinogen antibody ( : , , abcam, england), polyclonal rabbit antimouse fas antibody ( : , abcam, england), and polyclonal rabbit anti-mouse tnf-receptor antibody ( : , abcam, england), respectively. after incubation with an horseradish peroxidase (hrp)-labeled goat igg fraction to rabbit igg fc, the target protein was detected using a dab kit (zsgb-bio, beijing, china). the slides were then counterstained with hematoxylin and visualized under a microscope (olympus, tokyo, japan). liver tissue and cells were homogenized in ripa lysis buffer with phenyl methane sulfonyl fluoride (pmsf) protease inhibitor. protein lysates were separated by sds-page, and western blotting was performed using a monoclonal mouse antihuman/mouse fgl ( : , abnova), a monoclonal mouse antihuman hbp ( : , santa cruz, usa), and a monoclonal rabbit anti-human/mouse β-actin ( : , , cell signaling technology, usa). liver tissues were collected from mhv- -infected balb/cj mice at h, and total rna was extracted using trizol reagent (invitrogen, usa) and then reverse transcribed into cdna by using revertra ace qpcr rt kit (toyobo, japan). the cdna was then amplified by rt-pcr by using dream taq green pcr master mix ( ×) (thermo scientific, usa). realtime quantitative pcr (qpcr) with sybr green real-time pcr master mix (toyobo, japan) was performed using a cfx real-time pcr detection system (bio-rad, usa) and mrna levels were normalized with reference to those of the house keeping gene gapdh. primer sequences for qpcr amplification were as follows: mtnf-α forward, ′ -ttt gag atc cat gcc gtt gg- ′ ; mtnf-α reverse, ′ -gcca cca cgc tct tct gt- ′ ; mil- β forward, ′ -tgt aat gaa aga cgg cac acc- ′ ; mil- β reverse, ′ -tct tct ttg ggt att gct tgg- ′ . mfgl forward, ′ -gcc aaa tgt gag tcc ctg gaa- ′ ; mfgl reverse, ′ -ttc cac cca aga gca cgt tta ag- ′ ; hfgl forward ′ -aca gtt cag gct ggt ggt- ′ ; hfgl reverse, ′ -ggc tta aag tgc ttg ggt- ′ ; hbp forward, ′ -tga agc aga agc tgg gagt- ′ ; hbp reverse, thp- cells were treated with ng/ml phorbol -myristate -acetate (pma) (sigma, usa) for h to induce differentiation toward adherent macrophage-like cells as reported previously ( ) . the cc group was supplemented with cc protein ( ng/ml). after h of stimulation, ifn-γ ( ng/ml) was added to these cells, which were then cultured for h before they were collected for western blotting and real-time pcr studies. the chinese hamster ovary (cho) cells were cultured in cm cell culture dishes with dmem supplemented with % fbs until - % confluence. next, µg pcdna . -hfgl (constructed in our lab) was mixed with µg pcdna . -hcc in serumfree dmem. the mixture was then combined with lipofectamine , (invitrogen, usa) and mixed gently. after incubation at • c for min, the solution was added to cho cells and incubated at • c in % co . four to six hour after transfection, the medium was removed and fresh medium containing % fbs was added. at h after transfection, the cells were collected for co-immunoprecipitation analysis to evaluate the interaction of cc with fgl . both huvec and thp- cells express fgl . however, in the transfection experiments, it is difficult to transfect the thp- cells with sirna, so we use huvec instead of thp- . human umbilical vein endothelial cells (huvecs) were cultured in figure | cc protein increased survival rate and reduced liver damage in mice. (a) the survival rate of cc group is higher than the control group comprised of mhv- -infected balb/cj mice treated with saline. cc protein ( µg) or saline were injected into mice by tail vein. balb/cj mice then received pfu of mhv- intraperitoneally h later to develop fulminant viral hepatitis. then, cc protein ( µg) or saline were injected into mice by tail vein following mhv- infection h later. the survival rate was observed for days (n = /group). representative data from three independent experiments are shown. the survival curve was analyzed by using the log-rank test. ***p < . compared with saline group. (b) histopathology of liver tissues (h&e staining; original magnification, × , n = /group) at h post-mhv- infection was evaluated in the two groups of mhv- -infected balb/cj mice. livers were collected from saline-treated (a) and cc -treated (b) balb/cj mice at h after mhv- infection. arrows point to inflammatory cell infiltration areas or necrotic regions with inflammation. (c) effect of cc on serum alt and ast levels (n = - /group). values represent means and standard error of three independent experiments performed in triplicate. **p < . compared with the saline group. six-well plates with dmem supplemented with % fbs until - % confluence. pmol hbp -sirna was mixed with µl serum-free dmem. two microliter lipofectamine , was gently mixed with serum-free dmem. after incubation at • c for min, the solution was added to huvecs and incubated at • c. four hour after transfection, the medium was removed and fresh medium containing % fbs was added. at h after transfection, cells were collected for real-time pcr and western blot analysis to evaluate the effects of hbp on fgl . at h after transfection, the cc group was supplemented with the cc protein ( ng/ml). after h of stimulation, ifn-γ ( ng/ml) was added to these cells. these cells were then cultured for h before they were harvested for real-time pcr studies to evaluate the effects of cc on fgl by hbp . negative control was used as a control. to detect whether there was a potential interaction between cc protein and fgl , cho cells were transfected with pcdna . -hcc and pcdna . -hfgl for h. cells transfected with empty plasmid pcdna . (mock) were used as negative controls for cc gene transfection. immunoprecipitation and immunoblotting were performed by using pierce co-immunoprecipitation kit (pierce, usa). total cell proteins were extracted as previously described ( ) . the proteins were immunoprecipitated by mouse anti-human fgl antibody ( : , abnova). for co-immunoprecipitation experiments, western blotting was performed using both rat anti-human uteroglobin/scgb a antibody ( : , r&d, usa) frontiers in immunology | www.frontiersin.org and mouse anti-human fgl antibody ( : , abnova). control isotype rat igg was used as a negative control for primary antibodies. the human cc coding region gene, including a bp sequence, was amplified from homogenized human turbinate tissue by rt-pcr. in this study, the sequences of pcr primers for cc were as follows: hcc -forward, ′ -ccc tcc acc atg aaa ctcg- ′ ; hcc -reverse, ′ -tga gat gct tgt ggt tta ttg aag- ′ . the pcr products were cloned into peasy-t cloning vector (transgen, beijing, china) and then subcloned into hindiii/xbai site of pcdna . vector (invitrogen, usa) to form eukaryotic expression plasmids pcdna . -hcc . microarray analysis was used to screen changes in genome-wide gene expression patterns in thp- cells with or without cc protein. the changes in over , human gene expression patterns were assessed using affymetrix gene microarrays (human genome u plus . ) (capitalbio co.,ltd., beijing, china). three replicates were used for microarrays analysis. data obtained from the experiments are expressed as means ± sem. survival curve comparisons were performed with the log rank test. multiple group analyses for data were evaluated by one-way analyses of variance. analyses of two group results were performed using student's t-test to evaluate the statistical significance of differences. values of p < . indicated significance. to establish an animal model of mouse fh, mhv- was injected intraperitoneally to balb/cj mice ( mice/group). to further study the role of cc in fh, recombinant mouse cc protein ( µg/mouse) or saline was administrated into the tail vein h prior to mhv- infection. the same dose of cc protein or saline was then administered h later. the survival rate of the cc and saline groups was observed for days. the results showed that mice in the two groups began to die at h after injection of mhv- and exhibited symptoms of horripilation, slow activity, and reduced food consumption. in the cc group mice were alive on day after infection, mice alive on day , and of ( . %) mice recovered from fulminant viral hepatitis. at the same time, in saline treated group, there were mice alive on day , mice alive on day after infection, and no mice survived to day . that is to say, the mice in the saline group died within or days. three of ( . %) mice of the cc group recovered from fulminant viral hepatitis ( figure a) . to better understand the mechanisms underlying the biological effects of the cc protein, liver function (alt and ast levels in serum) and liver histology in mice of mhv- -infected was performed. liver tissues were harvested h following mhv- infection, and liver histology was detected by h&e staining. these results showed that there was substantial inflammatory cell infiltration and widespread necrosis of hepatocytes in the liver tissue of the saline group mice (figure ba ). there were rare or no infiltrating inflammatory cells, and few or no hepatocyte necrosis in the livers of mice in the cc group h after mhv- infection (figure bb) . serum alt and ast levels in mice were observed h after mhv- infection. the results showed that serum alt and ast levels in the saline group reached a peak h after mhv- infection, but there was no significant increase in the cc group compared to the levels in the control group (p < . , figure c) . these results suggested that cc protein has a role in protection against mhv- -induced liver injury in mice. to further elucidate the mechanisms of reduced liver injury following cc protein injection, we investigated the cytokines tnf-α and il- β expression. because these two cytokines play a crucial role in the liver damage of fh. they are characterized by an increase in apoptosis. levels of tnf-α and il- β in liver tissues were markedly reduced in the cc group (as shown in figure a) . hepatic apoptosis (figure b ) was significantly reduced in the cc group. we and collaborators have a long standing interest in studying the role of fgl in viral hepatitis. fgl has been verified to play an essential role in the progression of fulminant viral hepatitis as we appreciate from previous reports. we have provided liver pathology figures and liver function for mhv- infected mice with a fgl gene knockout as shown in supplementary figure . the data was comparable with previous reports from our center and collaborators. from this current study we shown that cc plays a protective role in liver damage.to study the related molecules of cc in mhv- -induced fh mice, we evaluated whether there was crosstalk between fgl and cc . we found that the expression of fgl in the liver of mice was reduced h after mhv- infection and treatment with cc protein (figures a,b) . furthermore, fibrin deposition, an indicator of liver injury associated with fgl expression in fh, was also decreased in the livers of cc -treated mice compared to that in controls (figure c ). this indicates that cc treatment reduced liver injury after viral infection by inhibiting fgl expression. we examined the effect of increasing doses of cc protein ( , , , and ng/ml) on ifn-γ-induced fgl expression in thp- cells. cc treatment showed a . % decrease in thp- cells compared to that in control after stimulation with ng/ml ifn-γ for h. cc protein inhibited fgl expression between doses of ng/ml and ng/ml (figure a ). in particular, ng/ml cc protein had the strongest inhibitory effect on fgl expression among the doses, and we chose this dose for the following experiments. we explored the effect of different time points of stimulation with a concentration of ng/ml cc protein. after stimulation with cc protein for , , and h compared to the pbs control, the strongest inhibitory effect on fgl expression was noted at h; hence, we chose this time point for the following studies ( figure b ). an increasing number of studies suggest that macrophages are the primary source of fgl . in order to ascertain that cc has a direct effect on macrophages, we treated thp- cells with recombinant cc and assessed the expression of fgl . unlike in controls, ifn-γ induced a significant increase in fgl expression. this effect was attenuated when cells were treated with cc protein (figures c,d) , revealing that cc directly reduces the levels of fgl in macrophages. to further explore the possibility that cc protein directly acts on macrophages, we infected murine pems with mhv- in the presence of recombinant cc and determined fgl expression. compared to levels in the controls, mhv- infected macrophages exhibited a significant increase in fgl production, and this effect was abolished by using cc protein (figures a,b) , indicating that cc directly modulates fgl production in macrophages. in order to determine genes that were downregulated after stimulation by cc protein, we used dna microarray analysis to screen for differentially expressed genes. thp- cells were cultured and pma was added to induce differentiation into macrophages. the production of fgl was stimulated by ifnγ. the experimental group was treated with cc protein for microarray detection of differentially expressed genes. the results showed that the most obviously downregulated genes were ube w, hectd , mir , atrx, sox , hbp , and fgl (supplementary table ) . and then these genes were tested by qpcr. however, ube w, hectd , mir , atrx, and sox was not differentially expressed by qpcr, while hbp and fgl were still down-regulated genes. dna microarray analysis identified hbp as a down-regulated gene involved in the pathological processes of the regulation of cc . recently, very limited studies have explored the role of hbp in fh. nevertheless, the mechanistic functions of hbp in fh remain largely unexplored. therefore, we selected this gene for further study. qpcr analysis confirmed that mrna levels of hbp were significantly decreased in thp- cells after cc protein stimulation compared to that in the pbs control group (figure a ). we knocked down hbp using hbp -sirna. then, transfection of hbp -sirna into huvecs was detected by qpcr and western-blotting methods. as expected, hbp knockdown led to significantly decreased expression of hbp (figures b,c) . furthermore, hbp knockdown impaired expression of fgl (figure d ), suggesting that hbp was able to activate fgl . hbp -sirna was used to transfect huvecs. then, ifn-γ was added to induce the expression of fgl followed by stimulation with cc protein ( ng/ml) after h. finally, we explored the expression of fgl by qpcr. the results showed that hbp -sirna treatment abrogated the inhibitory effect of cc on fgl expression in huvecs (figure ) . that is to say, cc could suppress fgl expression in macrophages. such an effect may be mediated by the transcription factor hbp . it is well-known that cc protein can suppress the immune response. in animal models of allergic diseases of the respiratory tract, most of evidences confirm this inhibition ( ) . its function in fh has not been investigated yet. here, we used a murine fh model established by mhv- infection to explore the effects of cc in this disease process. to determine the role of cc in the pathogenesis of fh, cc protein was injected into a mouse fh model established by mhv- infection. mhv- -induced liver injury in cc -treated mice occurred rarely and the areas of lesions were much fewer than those in saline-treated control mice. in summary, these results suggested that cc could reduce pathological liver damage in this fh model together with lower mortality rates followed by mhv- infection. mhv- induced fulminant viral hepatitis progresses rapidly and infected mice die within - days. previous studies suggested fgl played a vital role in this process with a - % increase of survival when fgl was deleted ( , , , ) . multiple inflammatory factors or mediators including tnf-α and ifn-γ, il- β and c ar have been demonstrated to promote fh progression with significant discrepancies between liver damage and survival rate ( ) ( ) ( ) ( ) , which is accordant with our observation that cc substantially alleviated liver injury though survival rate improved mildly. the survival rate based on hours may be more accurate to examine the effect of cc on fh. it is speculated that fgl can mediate lethality in mhv- -induced fh. this is due to the fact that fgl induces the deposition of fibrinogen, which leads to activation of the coagulation cascade and induction of procoagulant activity ( ) . to determine whether the tissue necrosis was mediated by fgl in cc -treated mice following infection, fgl expression was observed. results suggested that the expression of fgl was significantly increased in mhv- -induced fh mice and cc treatment significantly reduced the production of fgl in the infected liver and serum. in addition, decreased fibrinogen deposition was also observed in the livers of cc -treated mice. therefore, our research results strongly clarify that the lower mortality of cc -treated mice after mhv- infection is due to the lower levels of fgl and decreased fibrinogen deposition. indeed, it has been reported that fgl is expressed on macrophages, and the expression of fgl is believed to be induced by ifn-γ and tnf-α ( ) . cultured thp- cells activated by ifn-γ or il- have been demonstrated, with induction of fgl expression and enhanced activation of human prothrombin ( ) . therefore, in this study, we explored this cell line to investigate the modulation of cc on fgl . surprisingly, we found that cc directly inhibited ifn-γ-induced fgl expression in thp- cells. as we know, ifn-γ has proved to be the main cytokine that leads to the development and progression of fh. also, it was shown that ifn-γ might exert its own proinflammatory biological function through enhancing fgl expression. therefore, in our study, cc might counter the effect of ifn-γ in the setting of fh, which substantiates its role in fh. these results demonstrated that cc regulates the expression of fgl in macrophages. in the current study, we used co-immunoprecipitation to analyze binding between cc and fgl . in this study, we investigated possible protein-protein interactions between cc and fgl in vitro. the chinese hamster ovary (cho) cells transfected with pcdna . -hcc and pcdna . -hfgl . cellular proteins were immunoprecipitated with anti-cc antibody or anti-fgl antibody. immunoblotting was performed with anti-fgl and anti-cc antibodies. immunoprecipitation of protein extracts from pcdna . -cc and pcdna . -fgl co-transfected cho cells with anti-fgl or anti-cc antibody followed by western blotting with fgl and cc antibodies indicated that cc did not co-immunoprecipitate with fgl , showing that there is no direct relationship between cc and fgl (data not shown). the results showed that cc has no direct interaction with fgl . from our previous study the gene of fgl contributed profoundly in mhv- induced fulminant hepatitis and is extensively expressed in macrophages and endothelium ( , ) . our microarray indicated a cc down-regulated fgl expression and this is further confirmed by qpcr and western blotting in vivo (peritoneal macrophages) and in vitro (thp- , macrophage cell line). therefore, it is reasonable to focus on macrophages to display the effect of cc on fgl expression and eventually mice survival. we entirely agree there may be other possibilities for a protective effect of cc to contribute to the disease process. this is worth further studies. the potential receptor of cc has not been revealed yet. our previous study have demonstrated that cc have effect of dendritic cells in allergic rhinitis ( ) . in this research, we evaluated the effect of cc on macrophages functions and found fgl was substantially down-regulated upon cc treatment, therefore, we speculate that potential cc receptor may be also expressed on macrophages. the potential target of cc on other immune cells cannot be excluded. dna microarray analysis is one of the most powerful approaches for the potential identification of unexpected genes involved in pathogenic processes. by using this approach, hmgbox transcription factor (hbp ) was found to be one of the most downregulated genes after cc treatment of thp- cells. hbp is a well-described transcriptional repressor that modulates expression of genes involved in cell cycle progression. in a recent study, it was found that hbp is a direct target of mir- and confirmed that hbp modulates the inhibitory function of mir- -aso in hepatosteatosis and carcinogenesis simultaneously ( ) . hbp is an endogenous inhibitor of the wnt signaling pathway in both normal and cancer cells. the tumor suppressor role of hbp has been reported in some malignancies, such as oral cancer and glioma ( ) . however, an association between hbp and fgl has not been investigated yet. the current study clearly demonstrated that cc protects against mhv- induced fh via suppression of fgl expression. such effects might be mediated by hbp . however, the functional status of hbp in the cc pathway requires further research, and such studies are conducting in our laboratory. in conclusion, we demonstrated that cc could limit the immunopathological damage in mhv- -induced fh mice. our results suggest that enhancing cc expression by an immunotherapeutic approach might be an effective treatment for fh. hy performed all the described experiments and wrote the manuscript. yl assisted with some experiments, analyzed experimental results, and edited the manuscript. hw analyzed experimental results. xw reviewed and 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can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material table | list of down-regulated genes by dna microarray. in order to determine genes that were downregulated after stimulation by cc protein, we used dna microarray analysis to screen for differentially expressed genes. briefly microarray analysis was used to screen changes in genome-wide gene expression patterns in thp- cells with or without cc protein. the changes in over , human gene expression patterns were assessed using affymetrix gene microarrays (human genome u plus . ) (capitalbio co. ltd., beijing, china). three replicates were used for microarrays analysis. thp- cells were cultured and pma was added to induce differentiation into macrophages. the production of fgl was stimulated by ifn-γ. the experimental group was treated with cc protein for microarray detection of differentially expressed genes. the results showed that the most obviously downregulated genes were ube w, hectd , mir , atrx, sox , hbp , and fgl . key: cord- -t bz kmk authors: macphee, peggy j.; dindzans, vincent j.; fung, lai‐sum; levy, gary a. title: acute and chronic changes in the microcirculation of the liver in inbred strains of mice following infection with mouse hepatitis virus type date: - - journal: hepatology doi: . /hep. sha: doc_id: cord_uid: t bz kmk the acute and chronic effects of mouse hepatitis virus type on the microcirculation of the liver in both semisusceptible c heb/fej and fully resistant a/j mice were studied. in the c heb/fej mice, abnormalities of microcirculatory flow were noted as early as hr after infection and by hr, localized avascular foci appeared. disturbances were characterized by granular blood flow, sinusoidal microthrombi, distortion of sinusoids by edematous hepatocytes and necrotic lesions. following the acute infection, day , two patterns of chronic disease were observed. eighty percent of the mice developed chronic granulomatous hepatitis whereas in the remaining % a more severe chronic aggressive hepatitis was observed which was characterized by ongoing hepatocellular necrosis and a marked mononuclear cell infiltrate. in both cases, in vivo microcirculatory abnormalities were found predominantly around visible lesions. onset of the microcirculatory abnormalities was found to be concomitant with a rise in monocyte related procoagulant activity. procoagulant activity rose acutely and remained elevated throughout the chronic phase but was higher in animals with severe disease. in contrast to the above, normal blood flow and histology were seen in the resistant a/j mice at all times following infection, and procoagulant activity remained at basal levels despite active viral replication as demonstrated by immunofluorescence studies and recovery of infectious virus. these observations suggest a role for monocyte procoagulant activity in the development of microcirculatory abnormalities following mouse hepatitis virus type infection which may be important in the pathogenesis of the disease. liver ( ) . the sinusoids of the simple liver acini along with the nutrient portal triad and draining terminal hepatic venule comprise the basic microvascular unit of the liver. oxygen levels, blood composition, flow and pressure in the microcirculation contribute to a local environment which is in dynamic balance with the functionally heterogeneous hepatocytes across the simple liver acini; this is reflected by significant gradients in oxygen tension, redox state and cytoplasmic enzyme activities across zones to ( , ). selective zonal injury to the hepatic parenchymal plate by toxic agents in turn results in localized zonal abnormalities of the microcirculation ( , ) . infection with mouse hepatitis virus type (mhv- ), a member of the single-stranded, positive polarity, rnacontaining coronaviruses, causes a strain-dependent spectrum of liver disease in inbred mice ( ) . mice of fully susceptible strains (balb/cj, c b / j and dba) die of fulminant hepatitis; mice of semisusceptible strains (c h/st, c heb/fej) develop acute hepatitis which ultimately results in chronic liver disease while resistant adult mice (a/j) develop no liver disease. previous experiments showed that viral replication occurs to comparable levels in resistant as well as susceptible mice, hence it is apparent that viral replication alone contributes only in part to the pathogenesis of viral hepatitis ( ) . experimental evidence has suggested that host-immune defects may have a major role in determining strain-dependent susceptibility to mhv- infection ( ) . we have previously shown that strain-dependent susceptibility to mhv- correlates directly with the spontaneous t lymphocyte controlled expression of a procoagulant monokine which exhibits prothrombin cleaving activity [procoagulant activity (pca)] ( ) . a biological role for this activity in the pathogenesis of liver disease has been suggested by in uiuo microscopic observations during acute mhv- infection in the fully susceptible balb/cj strain ( ) . in this system, abnormalities of the microcirculation consisting of granular blood flow and sinusoidal microthrombi preceded in uiuo viral replication by hr. subsequently, focal thrombotic and/or ischemic lesions formed and finally resulted in confluent liver necrosis. the present study was designed to examine the hepatic microcirculation during both the acute and chronic phases of mhv- infection in mice of the semisusceptible c heb/fej strain ( , ) . the in uiuo hepatic microcirculation was studied and compared with both the spontaneous expression of monocyte/macrophage pca and histological parameters of liver disease. the origin and growth of mhv- has been previously described ( ) . mhv- , obtained from the american type culture collection, rockville, md. (attcc vr ), was plaque-purified on monolayers of dbt cells. stock virus was grown to a titer of . x lo plaque-forming units per ml in cl cells. the virus was harvested by one cycle of freeze thawing and clarified by centrifugation at , x g for hr at °c. virus was assayed on monolayers of l cells in a standard plaque assay ( ) . viral purification was accomplished by centrifugation through polyethylene glycol and sequential potassium tartrate gradients ( ) . purified virus at a t,iter of . x lo plaque-forming units per ml was finally resuspended in normal saline. a/j and c heb/fej mice, to weeks of age, were obtained from jackson laboratories, bar harbor, maine. the presence of serum antibody to mouse hepatitis virus was ruled out by a standard radioimmunoassay as described previously ( ) . mice were i.p. injected with lo plaque-forming units of stock mhv- in . ml. of dulbecco's modified eagle's medium (dmem). they were followed for up to months and sacrificed at various time intervals. blood was obtained by axillary bleeding; samples of liver tissue were processed for immunofluorescence, viral titers and routine h and e histology. a heterologous antibody to mhv- was produced in new zealand white male rabbits by hyperimmunization with purified mhv- in complete freund's adjuvant ( ) . the antiserum was found to be suitable for use a t / dilution. immunofluorescence was studied by an indirect method using a fluorescein-labeled goat antirabbit igg as the probe. by radioimmunoprecipitation, the antibody demonstrated predominant specificity for the nucleocapsid protein with weak e l and e envelope glycoprotein reactivity. liver tissue was homogenized in dmem as a % homogenate (w/v) at °c. viral titers were then determined on monolayers of l cells in a standard plaque assay ( ) . isolation of peripheral blood mononuclear cells (pbms) heparinized blood from mice for each time point was pooled and suspended in an equal volume of dmem. mononuclear cells were isolated over ficoll-hypaque gradients by centrifugation at °c at , x g for min. cells at the interface were collected and found to be greater than % mononuclear cells by cytologic examination using giemsa stain. viability was greater than % by trypan blue exclusion. cells were washed twice and resuspended in dmem at a concentration of x lo mononuclear cells per ml. spleens were removed aseptically from c heb/fej mice, and cells were suspended in dmem. mononuclear cells were isolated by centrifugation over ficoll-hypaque at °c for min at , x g. cells from the interface were greater than % mononuclear by cytologic examination. cells were washed twice and resuspended in dmem at a concentration of x lo mononuclear cells per ml. samples of viable cells or cells which were subjected to three cycles of freeze thawing and sonication were assayed for the capacity to shorten the spontaneous clotting time of normal citrated human plasma in a onestage clotting assay ( ) . t o . ml of cellular homogenate or viable cells at "c, . ml of citrated normal human platelet-poor plasma or factor-deficient plasma (helena laboratories, beaumont, tex.) was added, followed by . ml of mm cac to start the reaction. the time for the appearance of a fibrin gel was recorded. clotting times were converted to units of pca by comparison to a rabbit brain thromboplastin standard (dade division, american hospital supply, miami, fla.) where mg dry weight per ml were assigned a value , mu pca. the assay was utilized over the range of to , mu or ' to ' cells, and it was linear over this range with normal plasma substrate. media with or without % fetal calf serum and buffers were all without activity. mice were injected i.p. with , pfu of stock mhv- in . ml dmem and examined at various time intervals. control mice were injected with . ml of dmem. anesthesia was obtained with nembutal ( . mg per gm body weight) by i.p. injection. the animals were immobilized on a heated surgical table with temperature control at °c. mice were intubated (pe- intramedic tubing, clay adams, ny) by tracheostomy and ventilated with room air by a volume ventilator at cycles per min and ml per breath. laparotomy by midline incision was performed, and the peritoneal cavity was irrigated with ringer's lactate solution warmed to °c. tubocurare ( . ml of mg per ml solution) was injected intramuscularly as required. the liver margin was transilluminated with a quartz rod connected to a fiberoptic light source (intralux h volpi, zurich, switzerland). the microcirculation was observed through an e. leitz microscope as previously described ( ) . no abnormalities in liver histology could be found in a/j mice for up to days following infection ( figure a ). in contrast, at hr, the livers from c hebifej mice had focal necrotic lesions consisting of acidophilic degenerating hepatocytes and nuclear debris with a sparse inflammatory infiltrate ( figure a ). by days postinfection, these early lesions became larger, more numerous and were associated with an infiltrate of pyknotic polymorphonuclear leukocytes ( figure c ). at days, confluent necrosis was evident in some sections ( figure a ). by to days, the lesions were densely infiltrated with intact mononuclear cells, and the adjoining liver parenchyma underwent intense hepatocyte regeneration as evidenced by many mitotic figures ( figure a ). following the acute phase of the disease (day lo), two patterns of chronic disease were observed. the first was characterized by the appearance of epithelioid cells with large pale nuclei and pale granular cytoplasm. these appeared within the foci of necrosis by days postinfection, and by day , most remaining lesions were organized into granulomas consisting of centralized epit helioid cells surrounded by mononuclear cells ( of disease was observed in approximately % of mice. these animals developed an aggressive form of hepatitis characterized by persistent necrotic foci with a mixed inflammatory cell infiltrate, largely mononuclear, a severe terminal portal venule vasculitis and focal hepatocellular dropout ( figure a ). both of these groups of animals died within to months of the infection, a marked reduction from the normal to months. by indirect immunofluorescence, viral antigens could be detected at hr in both the fully resistant a/j mice and the semiresistant c hebfej mice. in the a/j mice, large viral deposits were found within hepatocytes, endothelial cells and kuppfer cells ( figure a ). by to hr, antigens were predominantly localized to the cytoplasm of the hepatocytes but some membrane deposition could be seen ( figure b ). viral antigens were detected up to days following infection but were undetectable at day ( figure c ). in livers from c heb/fej mice, viral antigens were present in the cytoplasm and at the cell surface of hepatocytes at hr postinfection ( figure a ). by to hr, viral antigens were found in areas of necrosis as well as in apparently normal hepatocytes ( figure b ). at days, viral antigens were seen in areas of inflammation, which were characterized by a mononuclear cell infiltrate ( figure c ). for up to months postinfection, viral antigens were detected in both areas of inflammation and morphologically normal hepatocytes ( figure d ). for hr following infection, virus could not be recovered from the livers of either a/j mice or c heb/ fej mice. however, by hr, high titers were detected in both strains of mice ( figure ). the viral titers peaked of disease was observed in approximately % of mice. these animals developed an aggressive form of hepatitis characterized by persistent necrotic foci with a mixed inflammatory cell infiltrate, largely mononuclear, a severe terminal portal venule vasculitis and focal hepatocellular dropout ( figure a ). both of these groups of animals died within to months of the infection, a marked reduction from the normal to months. by indirect immunofluorescence, viral antigens could be detected at hr in both the fully resistant a/j mice and the semiresistant c hebfej mice. in the a/j mice, large viral deposits were found within hepatocytes, endothelial cells and kuppfer cells ( figure a ). by to hr, antigens were predominantly localized to the cytoplasm of the hepatocytes but some membrane deposition could be seen ( figure b ). viral antigens were detected up to days following infection but were undetectable at day ( figure c ). in livers from c heb/fej mice, viral antigens were present in the cytoplasm and at the cell surface of hepatocytes at hr postinfection ( figure a ). by to hr, viral antigens were found in areas of necrosis as well as in apparently normal hepatocytes ( figure b ). at days, viral antigens were seen in areas of inflammation, which were characterized by a mononuclear cell infiltrate ( figure c ). for up to months postinfection, viral antigens were detected in both areas of inflammation and morphologically normal hepatocytes (figure d ). for hr following infection, virus could not be recovered from the livers of either a/j mice or c heb/ fej mice. however, by hr, high titers were detected in both strains of mice ( figure ). the viral titers peaked by to days postinfection and were no longer detected by day in a/j mice and by day in c heb/fej mice (figure ). previously, we have shown that pbm cells, isolated from the blood of uninfected mice and stimulated in uitro with mhv- responded with an increase in pca which directly correlated with the in i h susceptibility to hepatic injury in that strain ( ). to determine whether a similar response occurs in uiuo, pbms from a/j and c heb/fej mice were assayed directly following isolation from the blood for both viable (cell surface) and total content pca ( figure ). each pca determination was made on pbms from the pooled blood of mice. mononuclear cells from a/j and c hebfej mice which had been injected with pl of dmem as controls had a basal surface pca of mu per " pbms. following infection, there was no increase in pca in pbms from fully resistant a/j mice. in c: heb/fej mice, there was a greater than -fold increase in viable pca by hr which reached a maximum -fold increase over baseline at day . this elevated pca persisted for the subsequent to months with minor fluctuations in activity. the values of total pca (obtained from disrupted cellular homogenates) were approximately twice those expressed at the cell surface (viable), and both activities followed parallel patterns. in order to examine the pca response of individual mice, splenic mononuclear cells were harvested from c heb/fej mice and assayed for total content pca ( table ) . early in the course of the infection, all animals had markedly elevated pca corresponding with the severe histologic and microcirculatory disturbances. at days to , two divergent patterns of pca were observed. although in both strains, the pca remained elevated, animals that developed severe hepatitis (type ) maintained the high levels of pca found during the acute phase of the disease whereas animals that developed chronic granulomatous hepatitis (type ) had pca elevations at to times background but only to % of the pca observed in type animals. no increase in total pca was found in pbms or splenic macrophages from a/j mice over initial basal values. i n uiuo microscopy of normal control a/j and c heb/ fej mice that had received pl i.p. of dmem revealed normal streamlined blood flow of the terminal hepatic vessels and the sinusoids ( figure b ). blood flowed from the terminal portal venules, with occasional bursts of arteriolar flow, into the proximal sinusoids. the blood could be followed as it drained into terminal hepatic venules. liver cell cords to cells wide separated the sinusoids. at hr postinfection in c hebfej mice, the velocity of blood flow in the terminal hepatic vessels was slowed, however, the architecture of the parenchyma was normal. small avascular, pale-colored parenchymal lesions, spanning to sinusoids appeared by hr ( figure b ). localized granular blood flow, caused by reduced velocity of clumped erythrocytes, was prominent in dilated sinusoids which shunted blood around the lesions. most of the lesions were found in zone (periportal) of the simple liver acinus with normal intervening parenchyma. at hr, diffuse granular flow occurred in the microcirculation, and sinusoidal microthrombi could be seen ( figure d) . by to days, the parenchymal lesions were more numerous and larger, spanning as much as a full acinus. sinusoidal flow was disrupted by microthrombi and edematous hepatocytes, and many sinusoids were difated with large pools of stagnant blood. at days, parenchymal lesions had coalesced with widespread hepatic necrosis and severe parenchymal edema ( figure b ). by days to , the diffuse flow abnormality had resolved and many lesions demonstrated dark hemorrhagic centers ( figure b ). at day , small pale and more sharply circumscribed lesions were noted ( figure d ). by day , the lesions were further condensed and contained occasional patches of a yellow-green stained pigment. from day to months, avascular foci of heaped-up tissue were scattered throughout the liver while the intervening parenchyma exhibited normal architecture and sinusoidal blood flow. histological examination of serial sections showed these lesions to be giant cell granulomas ( figure b ). in the animals that developed granulomatous hepatitis, in uiuo microcirculatory flow abnormalities were confined to the sinusoids surrounding the granulomatous lesions. this consisted largely of granular blood flow at the periphery of the granuloma, and no blood flow was observed through the granuloma itself. histologic examination demonstrated large numbers of mononuclear cells in the sinusoids adjacent to the lesions. in approximately % of c h/ ebfej mice, marked localized abnormalities of the liver microcirculation correlated histologically with foci of aggressive hepatitis ( figure b ). these foci were composed of a central avascular core which was surrounded by pale edematous hepatocytes containing dilated and disorganized sinusoids with slow or absent blood flow. subsequent histologic analysis of these lesions which had been studied in uiuo demonstrated large areas of necrosis with a marked predominantly mononuclear inflammatory cell infiltrate ( figure a ). the outcome of viral infection in a host is the result of a complex set of interactions between the elements of the immune system, host cells and the virus ( , ) . a number of immune effector networks have been studied in the model of murine hepatitis virus infection; however, to date, none of these have shown good correlation with the strain dependence of disease susceptibility ( , ) . in this paper, we present data that associate the spontaneous expression of monocyte pca with acute and chronic liver inflammation in mhv- -induced hepatitis. this relationship emphasizes the possible role of the coagulation cascade in the disturbances of the microcirculation that enhance liver cell necrosis. activation of the coagulation system is a common feature of inflammation ( ) ( ) ( ) ( ) . activated coagulation proteins serve as mediators of the inflammatory response by in turn recruiting the complement, kallikrein-kinin and fibrinolytic systems ( ) . furthermore thrombin and its activation fragment . directly elicit chemotaxis in monocytes ( ) . it has been known for several years that leukocytes express a number of pcas following stimulation both in uitro and in uiuo ( , ) . the cellular source of this activity is the monocyte/macrophage but t lymphocytes and/or their products are necessary for the induction of these procoagulant monokines ( ) . the coagulation cascade may be activated at local sites of inflammation by the procoagulant products of infiltrating leukocytes resulting in amplification of the inflammatory response with increased tissue injury. the pattern of spontaneous monocyte pca observed in these experiments in the a/j and c hebfej mice is in agreement with the previously described direct correlation of monocyte pca with susceptibility to liver disease following mhv- infection of pbm with mhv- in uitro ( , ) . in the c heb/fej mice, a constant proportion ( %) of pca was expressed at the cell surface, consistent with the physiologic importance of pca in situ. during the early stage of the infection, splenic macrophage pca did not discriminate between those mice that develop chronic aggressive hepatitis or chronic granulomatous hepatitis. only by day could distinct patterns of pca response be identified, and these correlated with the severity of the histologic lesion ( table ). as the mice studied are inbred strains, it is apparent that factors other than genetic are involved in the evolution of the immune-inflammatory process. other investigators have shown that diet, age, sex, stress and temperature affect immune responses to infectious processes ( ). however, in studies using recombinant inbred strains of mice, we have shown that pca and susceptibility/resistance to mhv- infection are genetically linked and are controlled by two independent non-h- linked genetic loci (dindzans, v. et al., hepatology ; : , abstract). determinations of p-glucuronidase, 'nucleotidase and elastase in macrophages from mhv- infected and control animals did not correlate with susceptibility to infection and resultant hepatitis ( ) and thus, monocyte pca is not simply a nonspecific marker of inflammation. the immunofluorescence studies and viral titers in the resistant a/j mice demonstrate that permissiveness to viral replication alone cannot explain the disease observed. in addition, the abnormalities in the microcirculation following mhv- infection were detected prior to viral replication. evidence for a role of the microcirculation in the pathogenesis of acute hepatitis has been generated by in uiuo microscopic studies of the liver during toxic hepatitis. severe flow abnormalities, consisting of sinusoidal constriction and dilation, granular blood flow, microhemorrhages and microthrombi, are prominent features of toxic hepatitis ( ) . during hepatitis caused by toxic agents, microcirculatory abnormalities begin in a zonal distribution with the most severe changes occurring in areas of greatest hepatocellular damage ( , ) . the earliest abnormality seen following mhv- infection in uiuo was granular blood flow, a relatively nonspecific finding whose mechanism has been attributed to several factors ( , ) . these include: hypotension, neurally mediated vasoconstriction and vasodilation, humoral mediators (histamine, bradykinin) of vasodilation, hemoconcentration due to capillary permeability in-creases and changes in the surface charge of erythrocytes due to fibrin adsorption. studies using fluorescent-labeled proteins during the acute phase of ally formate hepatitis revealed increased sinusoidal permeability within min of administration of the toxin, coincident with the appearance of granular flow ( ) . similar abnormalities were detected in mhv- -infected c heb/fej mice, suggesting similar pathophysiological mechanisms may be operative. in the acute phase of mhv- infection, viral antigens were diffusely deposited in the liver, and extensive microcirculatory disturbances were observed. in contrast, during the chronic phase, viral antigens were localized in focal deposits. thus, continued specific stimulation of pca in the chronic phase may result in localized microcirculatory disturbances confined to the areas of antigen deposition. since it is known that pca directly correlates with in vitro and in uivo measures of delayed cutaneous h-ypersensitivity ( , it is conceivable that pca exerts its influence through both cellular and humoral immune mechanisms. this could occur by the production of lymphokines and/or the recruitment of activated t cells macrophages and nk cells into the chronically affected area. finally, since pca is a potent serine protease, it may have effects through its proteolytic action on substrates other than prothrombin. as an example, one of the earliest pathologic features of experimental allergic hepatitis is the deposition of immune complexes within the sinusoids ( ). this is followed by thrombotic occlusion of sinusoids with resultant focal coagulative necrosis ( ). a similar pathologic mechanism has been proposed for fulminant hepatitis b ( ). we have previously reported that immune complexes stimulate the production of pca, thus asso-ciating the production of immune coagulants with liver injury ( , ) . in this paper, we present further evidence suggesting a link between viral induction of monocyte/macrophage pca and disturbances in the microcirculation in liver disease. hepatic vascular bed the microcirculatory acinar concept of normal and pathological hepatic structure functional implications of liver cell heterogeneity hepatocytes of zones and conjugated sulfobromopthalein with 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macrophages blood coagulation and the inflammatory response the pathobiology of viral hepatitis and immunologic activation of the coagulation protease network a quantitative evaluation of anti-coagulants in experimental nephrotic nephritis relationships among the complement, kinin, coagulation and fibrinolytic systems in the inflammatory reaction monocyte chemotaxis: stimulation by specific exosite region in thrombin cellular pathways and signals for the induction, biosynthesis of initiators of the coagulation protease cascade and of plasminogen activators in cells of the monocyte lineage. monocytes, macrophages and inflammation the role of lymphokines in delayed type hypersensitivity reactions the immune response to mouse hepatitis virus: expression of monocyte procoagulant activity and plasminogen activator during infection in uiuo physioanatomical basis of toxic liver injury study of the effects of acetaminophen (paracetamol) on the hepatic microcirculation the circulatory manifestations of bacterial endotoxemia microscopic observations of the circulating blood in the bulbar conjunctiva in man in health and disease. ergebenisse der anatomie und entwicklungs geshichte electron microscopy of the hepatocellular and kupffer cell lesion of mouse hepatitis with particular reference to the effect of cortisone in uiuo studies of the responses of the liver to endotoxin the inflammatory response to endotoxin electron microscopy of hypersensitivity reactions: allergic hepatic necrosis mechanisms of liver cell injury in acute and chronic hepatitis b immune complex induced human monocyte procoagulant activity. . cellular kinetics and metabolic requirements key: cord- -o cijp a authors: knobler, r.l.; linthicum, d.s.; cohn, m. title: host genetic regulation of acute mhv- viral encephalomyelitis and acute experimental autoimmune encephalomyelitis in (balb/cke × sjl/j) recombinant-inbred mice() date: - - journal: j neuroimmunol doi: . /s - ( ) - sha: doc_id: cord_uid: o cijp a in the present report we provide the strain distribution patterns of susceptibility to acute mouse hepatitis virus type- (mhv- ) encephalomyelitis, acute experimental allergic encephalomyelitis (eae) and vasoactive amine sensitivity (vaas) for (cxj) recombinant-inbred strains between balb/cke (c) and sjl/j (j) mice. we confirm that susceptibility to mhv- is not linked to the h- complex, and that all strains susceptible to acute eae have both a responder h- haplotype (h- (s) or h- (d)) and induced (b. pertussis) vaas. in addition, we provide evidence that susceptibility to acute eae induction is controlled by an additional presently unmapped locus and that an eae-like histopathological disease does not usually follow mhv- infection intracerebrally in animals susceptible to mhv- , acute eae and induced vaas. genetic factors regulate susceptibility to viral infection and specific immune responses to a variety of antigens. viral susceptibility genes are usually located outside of the major histocompatibility complex (mhc), the h- region on murine chromosome (brinton and nathanson ) , while many immune response genes are located within the mhc (benacerraf and mcdevitt ) . genetically regulated virus-induced and immune-mediated models of demyelination in the mouse illustrate the role of specific host genes. susceptibility to acute mhv- encephalomyelitis is controlled by a single autosomal dominant gene, unlinked to the h- complex (knobler et al. ) . acute infection in susceptible strains is frequently characterized by a fatal necrotizing encephalomyelitits, but, survivors develop demyelinating disease due to direct infection of oligodendrocytes without perivascular infiltration of mononuclear cells (bailey et al. ; waksman and adams ; lampert et al. ; weiner ) . the incidence of demyelination can be enhanced by infection with an attenuated temperature-sensitive mutant of mhv- , designated ts (haspel et al. ) . mhv- induced disease has been extensively studied in balb/c mice (knobler et al. a, b) , but does not usually occur in sjl/j mice which lack the gene for susceptibility (stohlman and frelinger ; knobler et al. knobler et al. , a . susceptibility to acute experimental autoimmune encephalomyelitis (eae), an acute perivascular inflammatory demyelinating disease, is controlled by at least two genes, one, h- linked [responder haplotype (h- s, h- q or h- d)]; the other associated with natural vasoactive amine sensitivity (vaas) or that induced by bordetella pertussis (linthicum and frelinger ) . acute eae is easily induced in sjl/j mice, but rarely in balb/c mice (levine and sowinski ; bernard ; lando et al. ; raine et al. ) which are genetically resistant to vaas and the histamine sensitizing factor (hsf) of b. pertussis vaccine, although they have an h- d responder haplotype for eae (linthicum and frelinger ) . the development of recombinant-inbred (ri) strains allows determination of genetic linkage, characterization of multiple gene inheritance and investigation of genetic reassortments. ri strains are derived by systematic inbreeding of progenitors differing at identifiable loci, providing a set of strains containing a fixed recombination of linked alleles characteristic of either progenitor strain (bailey ; taylor ) . the more gene loci mapped the more useful the ri strains become in detecting linkage. once the strain distribution pattern (sdp) is established for a large enough set of markers, the sdp of any new one can be compared to establish linkage. nine ri strains between balb/cke and sjl/j (cxj) have been tested to provide the sdp of susceptibility to acute mhv- encephalomyelitis, acute eae and vaas. a seed of mhv- initially provided by dr. l. weiner (departments of neurology and microbiology, university of southern california) was plaque-purified times and grown as a stock in l- - cells. the handling of virus, culture and plaque assay conditions for mhv- and ts have been reported elsewhere (haspel et al. ; knobler et al. knobler et al. , a . the (cxj) ri lines were initially derived in (in the laboratory of m.c., salk institute for biological studies) from the successive breeding of f z offspring obtained by mating f hybrid mice ( sjl/j male x balb/cke females and balb/cke male x sjl/j females), f offspring were brother-sister-mated for successive generations and at least experimental colonies were thus established. progenitor strains balb/cke and sjl/j, and sja (congenic with s j l / j except at igh locus; gift of roy riblet) were also maintained in the vivarium of the salk institute. mice of both sexes at - wk of age were used for these studies. these were determined by pvp microhemagglutination (takasugi and hildemann ) , with h- s or h- d specific antisera (from dr. r. hyman) and primary mlr of each parent against each ri strain, which could rule out intra-h- recombinants. macrophages were obtained from the peritoneal cavities of no fewer than mice, of each strain tested, inoculated intraperitoneally with ml of . % thioglycollate broth (brewer's modified thioglycollate, becton, dickinson and co, cockeysville, md). five days later peritoneal exudate cells were harvested by lavage with - ml of sterile eagle's minimal essential media which contained units/ml heparin, % glutamine, /~g/ml penicillin and streptomycin, while the animal was under ether anesthesia. cells were spun down, resuspended in fresh media and counted. sufficient numbers of cells were plated to provide x adherent cells/dish. nonadherent cells were removed - h after plating by vigorous washing, as previously reported (brautigam et al. ; knobler et al. ) . adherent cells were identified as macrophages by their ability to ingest zymosan particles, and by their morphology (van furth et al. ) . homogeneity was usually > % with a range of - %. macrophage cultures were infected at a multiplicity of infection (moi) of . , by adsorbing virus for h at °c and then washing the cells times in phosphatebuffered saline (ph . ) before replacing fresh media. the cells were incubated at °c in a % co atmosphere and observed for the development of cytopathic effect - h after infection. mhv- permissive macrophages fuse to form multinucleated giant cells (syncytia) within h after infection. nonpermissive cells do not fuse. in addition, the rd wash after infection and the supernatant culture fluids at h after infection were assayed to quantitate virus replication. a minimum of but usually more mice of each strain were inoculated intracerebrally with . ml of mhv- or ts while under ether anesthesia. this inoculum contained pfu mhv- or pfu ts . mice were observed daily for signs of illness which included ruffled fur, irritability, lethargy, limb paralysis and death. surviving animals were killed at and days while under ether anesthesia, by exsanguination followed by intracardiac perfusion with % formalin. after fixation, the brain was embedded transversely and the spinal cord was embedded longitudinally in paraffin, sectioned and stained with hematoxylin and eosin (h & e). mice were immunized with mouse spinal cord homogenate (msch), mg dry weight, suspended in saline and emulsified in an equal volume of freund's complete adjuvant supplemented with mg/ml mycobacterium tuberculosis (difco, detroit, mi, h ra). all four footpads were injected, the total inoculum volume being . ml. immediately thereafter and h later, b. pertussis vaccine, x organisms, was given i.v. this immunization procedure produces - % incidence of eae in (sjl x balb/c) f~ hybrids (linthicum and frelinger ) . mice were examined daily for clinical signs of eae, scored on a scale of - : (no disease), (tail atonia, slight hind limb weakness), (hind limb paralysis, incontinence of bladder), and (moribund state or death due to eae). histological assessment of eae was made on mid-sagittal paraffin sections of brain and spinal cord stained with h&e, without knowledge of the specific cxj strain. the size and frequency of perivascular mononuclear infiltrates in the white matter of the cns were graded on a scale of - , as (no lesions), (few lesions, mainly leptomeningial and ependymal), (numerous infiltrates in the white matter of the brain stem, cerebellum, and spinal cord), and (florid lesions throughout the brain and spinal cord white matter). a minimum of mice but usually more of each strain were tested. body temperature was measured using a bailey bat- thermistor probe. the rectal temperature of mice immunized for eae was measured each day in the afternoon. body weights were determined by weighing mice on an ohaus b d digital balance with + . g sensitivity. b. pertussis has been demonstrated to induce a 'hypersensitivity' to both vaa serotonin and histamine (bergman and munoz ) which is characterized by the onset of hypotensive and hypovolemic shock following vaa challenge. for the sake of simplicity, we chose to test 'natural' and 'induced' hypersensitivity to vaa with only histamine. to determine 'natural' histamine sensitivity, naive mice were challenged with mg histamine i.p. and the deaths recorded h later. 'induced' histamine hypersensitivity due to b. pertussis administration was determined by intraperitoneal injection of mg histamine free base (denoted as 'challenge') in . ml neutral buffered saline days after the initial inoculation of x b. pertussis whole cells. deaths (due to hypotensive and hypovolemic shock) were recorded over the next h, and the results reexpressed as number of deaths divided by the total number of animals challenged. mice challenged with mg histamine without pretreatment with b. pertussis served as negative controls. following mhv- infection of permissive macrophages (balb/cke, cxj- , - , - , - , - ) at an moi of . , - pfu/ml of virus was detected in the supernatant culture fluid at h after infection (table ) . this was not due to the carryover of virus inoculum, since viral content of the rd pbs wash after infection was below the level of detection (< pfu/ml). cytopathic effects (cpe) in permissive macrophages was fusion to form multi-nucleated giant cells (syncytia). in contrast, at h after infection the viral content of the supernatant culture fluid of non-permissive macrophages (sjl/j, sja , cxj- , - , - , - ) was below the level of detection, and there was no evidence of cpe. further, there was neither cpe nor evidence of viral replication in the non-permissive macrophage cultures at h after infection and later. aging the non-permissive macrophages up to weeks prior to infection did not alter their response to mhv- . the intracerebral inoculation of pfu of mhv- represents an infection of approximately lds for susceptible strains of mice, such as balb/c. all mice of each strain with mhv- permissive macrophages (balb/cke, cxj- , - , , - , - ) were dead at days after infection (table ). in contrast, all mice of each strain with macrophages non-permissive for mhv- (sjl, sja , cxj- , - , - , - ) survived intracerebral challenge with balb/c lds (table ) , except cxj- . the cxj- strain had fatalities ( and days after infection) and the appearance of clinical symptoms in a rd mouse, which persisted until killed at weeks after infection. five cxj- mice challenged with -fold less virus ( balb lds ) showed no clinical symptomatology and survived until killed at weeks. in the mhv- susceptible strains (balb/cke, cxj- , - , - , - , - ) symptoms included ruffled fur, irritability which progressed to lethargy and a moribund state. histopathologically these mice showed evidence of a severe necrotizing encephalomyelitis, enlarged ventricles, with infiltration of polymorphonuclear and mononuclear cells in the meninges and choroid plexus, hippocampus, diencephalon, pons extending into the cerebellum and the spinal cord (knobler et al. a) . similar findings were observed in the two cxj- animals that died. in the symptomatic surviving cxj- mouse symptoms included ruffled fur, lethargy and bilateral hind limb paralysis. histopathologically this mouse showed evidence of demyelinating lesions, however, infiltrating mononuclear cells were not in a perivascular distribution. similar histopathological findings were apparent at and days after i.c. infection with ts in balb/cke, cxj- , - , - , - and - mice. the asymptomatic cxj- mice and the other asymptomatic survivors (sjl/j, sja , cxj- , - , - ), of the ri strains examined, strains (cxj- , - , - , - , - ) developed clinical and histopathological signs of eae and strains (cxj- , - , - , - ) were not susceptible (table ) . parental sjl/j, congenic sja and ( s j l × b a l b / c ) f hybrid mice were all susceptible to eae, while balb/c mice were not. susceptibility to eae did not require 'natural' histamine sensitivity, but all strains which developed eae were susceptible to b. pertussis-induced vaa hypersensitivity ( table ). using body weight and rectal thermometer probe temperature readings we followed several strains (fig. ) of the ri strains of mice tested only (cxj- and - ) revealed a 'natural' sensitivity to histamine (table ) hybrids are in the present report we have provided the strain distribution pattern (sdp) of susceptibility to acute mhv- encephalomyelitis, acute e a e and vaas for (cxj) ri strains between b a l b / c k e and s j l / j mice. we confirm that susceptibility to m h v - is unlinked to the h- complex and is consistent with control by a single gene locus (k_nobler et al. ) . we demonstrate that despite all cxj strains having both a required responder h- haplotype (h- s or h- d) and induced (b. (linthicum and frelinger ) , strains (cxj- , - , - and - ) are resistant, indicating control by at least an additional presently un~mapped, genetic locus. finally, although some (cxj) ri strains are susceptible to acute eae, induced vaas and acute m h v - encephalomyelitis eae-like histopathological disease does not occur in these animals following mhv- intracerebral infection. ri strains are derived by systematic inbreeding of pre-existing inbred progenitor strains that are unrelated (bailey ; taylor ) . for a trait that differentiates the progenitor strains, one or the other progenitor phenotypes will be recovered in association with each derived ri strain if it is controlled by a single locus. the appearance of novel phenotypes indicates a more complex pattern of inheritance. the probability of control by one or more loci may be calculated from the distribution of responses amongst the ri strains tested (taylor ; bailey ) . the difference between b a l b / c and s j l / j for susceptibility to m h v - (table ) is (table ) , marginally compatible with two ( p = . ) and incompatible with three ( p = . ). this gene is unlinked to the other markers analyzed here, i.e., eae and vaas, and unlinked to other markers established in the (cxj) ri strains on chromosomes , , , and (data not shown). recent work maps this gene to the proximal end of murine chromosome where it is linked to the sop- locus (knobler et al., in press ). control of resistance to acute mhv infection by a single recessive gene has also been reported for mhv- (weiser et al. ) and mhv- (levy-leblond et al. ) . our results indicating a single gene controlling mhv- differ, however, from a proposed two-gene model (stohlman and frelinger ) . the cause for these differences is not apparent. the difference between balb/c and s j l / j for susceptibility (sensitivity) to eae (table ) is compatible with a one-gene difference (table ) and incompatible with two or more genes ( p = < . ); for histamine sensitivity ( table ) the data is marginally compatible with one gene ( p = . ), compatible with two or three genes (table ) and marginally ~compatible with four genes ( p = . ); for b. pertussis enhancement of histamine sensitivity (table ) , the data is incompatible with one gene ( p = . ), marginally compatible with two genes (p--- . ) and compatible with three or more genes (table ). we will discuss the difference between b a l b / c and s j l / j for susceptibility to eae in terms of one gene locus; to histamine, two-gene loci; and to b. pertussis enhancement, three loci. sensitivity to acute eae had previously been demonstrated to be inherited in a dominant fashion and dependent upon a background of both an h- responder haplotype and sensitivity to histamine inherited in a recessive fashion or dominantly inherited responsiveness to b. pertussis enhancement of histamine sensitivity (linthicum and frelinger ) . in the present report, these relevant background genes were present in all (cxj) strains, which thus unmasked the newly found locus. the distribution of sensitives and resistants based .upon a single gene difference would predict a ratio of sensitives to resistants of . . five sensitives and resistants were found, giving a ratio of . , which is compatible with a single gene difference. possibly this eae locus is an ir-gene present in the non-h- - inked background which controls responsiveness to myelin basic protein (mbp) or another encephalitogenic component of the inoculum used to induce acute eae. alternatively, this locus controls a suppressor response to the relevant antigen(s). these hypotheses may be tested by determining immune-responsiveness to mbp or other components in the (cxj) ri strains to determine if they have a correlation with sensitivity to eae induction. the difference between balb/c (r) and s j l / j (s) as regards histamine sensitivity and its enhancement by b. pertussis is a quantitative not qualitative property. histamine sensitivity was assayed at rag/animal under conditions where the lds for balb/c is mg/animal and s j l / j is . mg/animal (linthicum and frelinger ) . the proposed two-gene difference between the parents and the dominance of the resistant phenotype in the (sjl/j × b a l b / c ) f hybrid, might be explained as follows: one gene (hd) determines a histamine destroying activity, e.g., histaminase, the activity of which is presumed higher in b a l b / c (hi) than in s j l / j ( o). the other gene (hr) controls the histamine receptor which is proesumed to be of higher affinity in s j l / j (hi) than in b a l b / c (lo). the ( b a l b / c × sjl/j)f , under the conditions of our assay ( m g / a n i m a l ) would score as resistant (r), protected by the histaminase activity. the four projected phenotypes would be b a l b / c (hdhr)hi io resistant, s j l / j k, hi hi hi (hdh r) sensitive, (cxj) ri resistant, ( h d h r ) (cxj)ri ( h~h~) resistant. the expected proportion of sensitives in the ri strains would be / ( . ) and / ( . ) were found. the b. pertussis enhancement of histamine sensitivity was assayed at mg/animal. under these conditions, the expected proportion of ri strains scoring as resistant under this assumption would be / , whereas . were found. this difference is marginally compatible with two genes ( p = . ). if it is assumed that b a l b / c and s j l / j differ by a rd gene (e) controlling degree of enhancibility [balb/c(ei°), sjl/j(ehi)] then the expected proportion of strains scoring as resistant would become / versus / (i.e., _< / ) found, a better fit (table ) . furthermore, this hypothesis suggests that the ri strains would differ quantitatively in response to b. pertussis, which is testable by titration of the enhancement of sensitivity. acute e a e is considered by many to be an immune reaction to myelin basic protein (mbp) and other myelin antigens, and has been studied as a model system of multiple sclerosis (hashim ) . it has been suggested that a prior viral encounter may lead to development of an eae-like response in man (maugh ) . recent evidence demonstrates cellular sensitization to mbp may follow acute measles infection in man (johnson et al. ) or mhv- infection in rats (watanabe et al. h o = histamine destroying activity; hg= histamine receptor; e= enhancing effect of histaminesensitizing factor from b. pertussis; hi = high activity; io = low activity; r = resistance, while s sensitivity, both relative to the quantity of histamine challenge. ), where clinical disease is associated with central nervous system perivascular infiltration of mononuclear cells. in the present study, ri strains (cxj- , - , - and - ) sensitive to both acute mhv- encephalomyelitis and acute eae did not develop perivascular infiltration of mononuclear cells following mhv- infection i.c., possibly because death from necrotizing encephalomyelitis occurred prior to the time for such lesions to develop. however, these same strains did not develop perivascular infiltration of mononuclear cells following ts infection i.c., although surviving with histopathological evidence of demyelination at and days after infection (data not shown). the lack of such 'chronic' lesions following mhv- infection in these mice may reflect absence of natural vaas characteristic of s j l / j mice (linthicum and frelinger ) , which can develop chronic eae (lublin et al. ), or a direct or indirect effect of mhv- or ts infection on cells of the murine immune systems (knobler et al., unpublished observations) limiting their usefulness as effectors. these possibilities may now be addressed by cell transfer experiments between (cxj) ri strains. finally, these (cxj) ri s~trains highlight the usefulness and value for determining the sdp and pattern of gene inheritance for other neurological disorders which differ between balb and sjl mice such as sensitivity to theiler's murine encephalomyelitis virus (lipton and dal canto ) and measles encephalomyelitis (rammohan et al. ) amongst others. recombinant-inbred strains --an aid to finding identity, linkage and function of histocompatibility and other genes recombinant-inbred strains and bilineal congenic strains a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin, part (pathology) histocompatibility-linked immune response genes action of histamine sensitizing factor from bordetella pertussis on inbred and random bred strains of mice pathogenesis of murine cytomegalovirus infection-the macrophage as a permissive cell for cytomegalovirus infection, replication and latency genetic determinants of virus suscepibility-epidemiologic implications of murine models myelin basic protein --structure, function, and antigenic determinants temperature-sensitive mutants of mouse hepatitis virus produce a high incidence of demyelination measles encephalomyelitis-clinical and immunologic studies mouse hepatitis virus type (jhm strain)-induced fatal central nervous system disease, part (genetic control and the murine neuron as the susceptible site of disease) selected mutants of mouse hepatitis virus type (jhm strain) induce different cns diseases --pathobiology of disease induced by wild type and mutants ts and tsl in balb/c and sjl/j mice virus persistence and recurring demyelination produced by a temperature sensitive mutant of mhv- host genetic control of mouse hepatitis virus type- (jhm strain) replication, part (the gene locus foi" susceptibility is linked to the syp- locus on murine chromosome ) mechanism of demyelination of jhm virus encephalomyelitis genetic control of susceptibility to experimental allergic encephalomyelitis in inbred strains of mice experimental allergic encephalomyelitis in inbred and outbred mice genetic study of mouse sensitivity to mhv infection --influence of the h- complex acute autoimmune encephalomyelitis in mice, part (susceptibility is controlled by the combination of h- and histamine sensitization genes) susceptibility of inbred mice to chronic central nervous system infection by theiler's murine encephalomyelitis virus delayed, relapsing experimental allergic encephalomyelitis in mice the eae model --a tentative connection to multiple sclerosis neuropathology of experimental allergic encephalomyelitis in inbred strains of mice chronic measles encephalitis in mice resistance to fatal central nervous system disease by mouse hepatitis virus, strain jhm regulation of immunity toward allogenic tumors in mice, part (effect of antiserum fractions on tumor growth) genetic analysis of susceptibility to isoniazid-induced seizures in mice genetic variants and strains of the laboratory mouse in vitro determination of phagocytosis and intracellular killing by polymorphonuclear and mononuclear phagocytes infectious leukoencephalitis-a critical comparison of certain experimental and naturally-occurring viral leukoencephalitides with experimental allergic encephalomyelitis adoptive transfer of eae-like lesions from rats with coronavirus-induced demyelinating encephalomyelitis pathogenesis of demyelination induced by mouse hepatitis virus (jhm virus) congenic strains of mice susceptible and resistant to mouse hepatitis virus the authors thank ms. munira sheikh for her careful breeding and maintenance of the ri strains. they also thank ms. linda tunison, mr. anthony russo and ms. caroline mcniel for technical assistance and ms. gay wilkins for manuscript preparation. key: cord- -go cmgpo authors: masihi, k.noel; kröger, hans; lange, werner; chedid, louis title: muramyl peptides confer hepatoprotection against murine viral hepatitis date: - - journal: int j immunopharmacol doi: . / - ( ) - sha: doc_id: cord_uid: go cmgpo the hepatoprotection induced by synthetic muramyl peptides was investigated using a model of lethal murine mouse hepatitis mhv- virus infection. mdp and a nonpyrogenic analog, murametide, inhibited the steep elevation of serum transaminases induced by mhv- irrespective of whether the immunomodulators were administered before or after the infection. a significant proportion of mdp or murametide-treated animals, in contrast to controls, survived the mhv- infection. the histopathological examination of the liver revealed marked necrosis of the hepatic parenchymal cells and infiltration of the inflammatory cells in controls but not in mdp-treated animals. due to hepatitis viruses and mycobacterium tuberculosis are endemic in southeast asia and amongst refugees from that region. a study of immune responses to both infections in indochinese refugees showed a significant association in the reactivity to purified tuberculin protein derivative (ppd) and the presence of hepatitis be antigen (mcglynn, lustbader & london, ) . persons having a positive ppd skin test tended to be hbeag negative suggesting that mycobacterium tuberculosis infection may affect the outcome of viral hepatitis. mouse hepatitis virus type (mhv- ) belongs to the group of coronaviruses. parenteral administration of mhv- to susceptible mice causes fatal hepatic necrosis culminating in death within a matter of few days. hepatic necrosis liberates several enzymes that are usually present intracellularly within the liver into the blood circulation. the elevation of serum transaminases is an important biochemical manifestation of human and murine viral hepatitis and can be used diagnostically as a marker of liver damage. measurements of serum alanine aminotransferase in indochinese refugees showed normal transaminase levels in ppd-positive persons compared with ppd-negative individuals (mcglynn et al., ) . it would be of considerable interest if the protection against virus-mediated liver damage associated with tuberculosis infection could be duplicated by immunomodulators of mycobacterial origin. mycobacteria contain on their cell walls, n-acetylmuramyl-l-alanyl-d-isoglutamine (mdp), a small glycopeptide which represents the minimal structure essential for bacterial adjuvanticity. synthetic mdp and its analogs are endowed with multifarious properties including the stimulation of nonspecific resistance against viral pathogens (chedid, ) . already in it was reported that mdp, in combination with trehalose dimycolate, could induce resistance against influenza virus infection (masihi, brehmer, lange & ribi, , . several mdp analogs like -o-acyl, ubiquinone (masihi, brehmer, azuma, lange & miiller, a) , seryl and aminobutyryl (masihi, brehmer, lange, ribi & schwartzman, b ) conferred long-term resistance against aerogenic influenza virus in combination with trehalose dimycolate. subsequently, mdp and analogs were shown to induce protection against various strains of influenza (dietrich, hochkeppel & lukas, ) , herpes simplex virus (dietrich et al., ; koff, showalter, hampar & fidler, ) , vaccinia virus (ikeda, negishi & nishimura, ) , sendai virus (yamamura, ishihara, hamada, yamamoto & azuma, ) and in combination with an interferon-inducer, against semliki forest virus (george, jain, gupta & anand, ) . mdp can also protect rat hepatocytes against the in vitro toxic effects of acrolein, chloroform and carbon tetrachloride and decrease serum transaminases (farghali, machkovfi, kameinikov~, jank~ & mgek, ) . in the present study, the effect of mdp and a nonpyrogenic analog, murametide, on biochemical and other parameters was investigated using a model of lethal routine mhv- virus infection. k, noel masihi el al. nuclei from liver cells were isolated using the technique previously described (blobel & potter, ) . the adpr transferase activity was measured in the presence of dnase (kidwell & burdette, ) . five to six-week old nmri mice were purchased from zentralinstitut fur versuchstiere, hannover, f.r.g. for histological studies, mice were given saline or mg of mdp h, h, and h after the mhv- infection. livers were removed on day after the viral infection and fixed in % formalin. histological sections were stained with hematoxylin and eosin. mdp and its analog, murametide, were synthesized by p. lefrancier, institut choay, paris, france (lefrancier, derrien, jamet, choay, lederer, audibert, parant, parant & chedid, ) . desired amounts of muramyl peptides were dissolved in pyrogen-free physiological saline. all substances were administered by the intraperitoneal (i.p.) route. mhv- was passaged i.p. in young nmri mice. livers were removed days after the infection and homogenized in ml of medium/liver using a tissue grinder. supernatant obtained after centrifugation was diluted and further passaged in mouse l-cells. marked cytopathic effects could be observed in tissue cultures of l-cells using supernatant dilutions of ~ to . three-day old cultures infected with : dilution of the supernate were frozen and thawed three times. the supernate obtained after centrifugation was stored in liquid nitrogen. various dilutions were injected i.p. into mice for the determination of lethal dose. half a milliliter of : dilution injected i.p. consistently gave ld,o o in -week old nmri mice and was used for all experiments. the got and gpt enzyme activities present in nonhemolytic sera collected at different intervals were determined using the standard method (bergmeyer, ) . reagents for the test were purchased from boehringer mannheim, f.r.g. the enzyme activity is presented in mu/ml. thirty animals were administered a single dose of /~g of mdp by the i.p. route. the effect of mdp itself on liver enzymes was determined in a group of pretreated mice. twenty mice from the mdp-pretreated group and normal mice injected with saline were infected i.p. with mhv- h after the mdp administration. sera were collected everyday for four days. the results of enzyme activities are presented in fig. . serum got and gpt activities were not induced after the administration of mdp alone. in contrast, the mhv- infection induced increased got and gpt activities on day and the enzyme levels were elevated even further on day , a time period when many of the animals were dying. pretreatment with mdp greatly reduced the rise in got and gpt levels observed after the mhv- infection. sixteen mice were given /~g of mdp h, h, h and h after the mhv- infection. another group of mice was similarly treated with mdp but did not receive the viral infection. a third group of mice received mhv- infection only. sera were collected every day for days after the infection. results presented in fig. show that the multiple administration of mdp alone did not affect the liver transaminases. mdp given after the mhv- infection could inhibit the induction of serum got and gpt (fig. ) . the effect of mdp or its potent nonpyrogenic analog murametide was investigated at a higher dosage. twenty-eight animals each were given mg of mdp or mg of murametide h, h, and h after the mhv- infection. eight animals were each the virus controls (fig. ) . nonpyrogenic murametide was active and inhibited the got and the gpt induction (fig. ) . neither mdp alone nor murametide alone induced liver enzyme activities at this dosage. the activity of adpr transferase in the nuclei of liver cells increases after infection with mhv- (table ). in contrast, treatment with mg of mdp h after infection resulted in a very small reduction of adpr transferase activity. additional treatments with mdp at h and h post-infection did not after the adpr transferase activity at h or h. one milligram of mdp or murametide administered three times after the mhv- infection conferred significant protection to respectively % and ° o of treated mice compared to only % survivors in the control group (<p . ) (fig. ) . lower doses of mdp given prior to or after the mhv- infection did not significantly affect the mortality (data not shown). mice infected with mhv- developed extensive necrotic lesions around the central artery on day (fig. a) . marked necrosis of the hepatic parenchymal cells was accompanied by infiltration of the inflammatory cells. in contrast, mdp-treated animals exhibited a strong suppression of the hepatic necrosis and a lack of cellular infiltration on day after the mhv- infection (fig. b) . the results of the present study demonstrate the hepatoprotective activity of synthetic muramyl peptides in viral hepatitis. mdp and murametide k. noel masih el al. could prevent the steep elevation of serum got and gpt transaminase levels induced by mhv- infection. the inhibition of both got and gpt enzyme activities was observed up to at least h after the mhv- infection irrespective of whether mdp was given before or after the infection. the histopathological examination of the liver revealed the marked protection conferred by mdp treatment in contrast to the extensive necrosis of hepatic parenchymal cells in the control animals. certain chemicals can cause profound liver damage. the ability of mdp to protect rat hepatocytes against the in vitro toxic effects of acrolein, chloroform and carbon tetrachloride has been described. serum aspartate and alanine transaminases detected h after i.p. carbon tetrachloride administration to rats could also be decreased by intravenous mdp pretreatment (farghali et al., ) . the ability to protect the liver against damage by viral or chemical agents may constitute an important property of immunomodulators like mdp. nicotinamide adenine dinucleotide (nad) functions as an important catalytic coenzyme in the oxidation reduction reactions. in addition, nad also participates in certain biological processes like adenoribosylation as a substrate. the enzyme adpr transferase cleaves nad with subsequent adenoribosylation of specific proteins. the nad-adenoribosylation metabolism is involved in quite a number of processes like dna repair differentiation, enzyme regulation etc. the activity of adpr transferase in the early stages of mhv- infection was somewhat lower in mdp-treated animals than in the controls. further experiments are currently in progress to define the role of nad metabolism in the damaged liver. the mechanisms involved in the protection conferred by mdp are not fully elucidated as yet. mdp administration increases the incorporation of radiolabeled palmitic acid into the hepatocyte phospholipids suggesting a stabilizing effect on hepatocytes (farghali, machkovfi, julis, buchar, jank~ & mgek, ). in addition, stimulation of nonspecific resistance mechanisms by mdp may have been responsible, in part, for the protection against mhv- infection. the clearance of colloidal carbon is impaired by mhv- (gledhill, bilbey & niven, ) . since mdp and several of its analogs can enhance the clearance of colloidal carbon particles from the blood by the reticuloendothelial system (fraser-smith, waters & matthews, ) and activate a variety of macrophage functions (leclere & chedid, ) , the mhv- induced dysfunction may be balanced by mdp. a protective role for endogenous interferon is suggested by interferon induction in mhv- infection in both the resistant and susceptible strains of mice and the finding that prior administration of anti-interferon serum causes accelerated mortality (virelizier, ) . muramyl peptides can augment viral interferon production (sakuma, azuma & yoshida, ) , against murine hepatitis stimulate the nk cell activity in the liver (talmadge, schneider, collins, phillips, herberman & wiltrout, ) and spleen (masihi, lange & rohde-schulz, ) , and activate the cytotoxic properties of murine kupfer cells (xu & fidler, ) . nuclei from rat liver: isolation method that combines purity with high yield stimulation of nonspecific host defence mechanisms against infections enhancement of host resistance against virus infections by mtp-pe, a synthetic lipophilic muramyl peptide- . increased survival in mice and guinea pigs after single drug administration prior to infection, and the effect of mtp-pe on interferon levels in sera and lungs possible effects of muramyl dipeptide on liver cell membranes the protection from hepatotoxicity of some compounds by the synthetic immunomodulator muramyl dipeptide (mdp) in rat hepatocytes and in vivo correlation between in vivo anti-pseudomonas and anti-candida activities and clearance of carbon by the reticuloendothelial system for various muramyl dipeptide analogs, using normal and immunosuppressed mice enhancement in anti-semliki forest virus activity of ds rna by muramyl dipeptide effect of certain murine pathogens on phagocytic activity enhancement of nonspeciflc resistance to viral infection by muramyldipeptide and its analogs poly (adp-ribose) synthesis and cell division protection of mice against fatal herpes simplex type infection by liposomes containing muramyl tripeptide macrophage activation by synthetic muramyl peptides apyrogenic adjuvant-active n-acetyl-muramyl dipeptides stimulation of chemiluminescence and resistance against aerogenic influenza virus infection by synthetic muramyl dipeptide combined with trehalose dimycolate effects of bcg, mycobacterial fractions and synthetic adjuvant (mdp) on the resistance of mice to aerogenic influenza virus infection effects of mycobacterial fractions and muramyl dipeptide on the resistance of mice to aerogenic influenza virus infection protective effect of muramyl dipeptide analogs in combination with trehalose dimycolate against aerogenic influenza and mycobacterium tuberculosis infections in mice modulation of natural killer cytotoxicity by muramyl dipeptide and trehalose dimycolate incorporated in squalane droplets immune responses to hepatitis b virus and tuberculosis infections in southeast asian refugees effect of n-acetylmuramyl-l-alanyl-d-isoglutamine on interferon production in mice by newcastle disease virus augmentation of nk cell activity to tissue specific sites by liposomes incorporating mtp-pe role of macrophages and interferon in natural resistance to mouse hepatitis virus infection the in situ activation of cytotoxic properties in routine kupfer cells by the systemic administration of whole ma,cobacterium boris organisms or muramyl tripeptide experimental and clinical studies on the effects of synthetic muramyl dipeptide derivatives on viral and opportunistic infections acknowledgements --we thank beate rohde-schulz and monika klewer for their expert technical assistance. key: cord- - e yu do authors: homberger, f. r. title: maternally-derived passive immunity to enterotropic mouse hepatitis virus date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: e yu do maternally-derived antibody to enterotropic mouse hepatitis virus (mhv) strain y was transferred to pups by both intrauterine (igg) and lactogenic (iga and igg) routes. antibody present in the gastric whey of pups suckling immune dams dropped to undetectable levels by weaning age ( days post partum). mhv-specific igg was found in the serum of passively immune pups up to weeks of age. immune dams transferred equal levels of antibody to consecutive litters of pups, without evidence of decline. immunoblots showed that iga and igg in whey and serum were directed against nucleoprotein n and glycoprotein s. mhv-specific igm was not detected in any sample. coronaviruses are important pathogens in many species of birds and mammals, including man. they are usually associated with either respiratory or enteric disease. mouse hepatitis virus (mhv), family coronaviridae, genus coronavirus, is a common, highly contagious virus of laboratory mice. the mhv group contains numerous, closely related and highly mutable viruses with either respiratory or enteric tropism [ ] . mhv is important both as a natural pathogen of laboratory mice and as a model of coronaviral infection, particularly enteritis in the neonate. as with other enterotropic coronaviruses, outbreaks of enterotropic mhv in naive mouse populations cause enteritis with high mortality among neonates. older pups and adult mice show minimal or no apparent disease [ , ] . observations in enzootically infected mouse populations give reason to suspect that maternally-derived passive immunity is very significant in protecting newborn mice against apparent disease [ , , ] . the passive transfer of immunoglobulins from dam to young in mice differs from that of most other mammals. igg is absorbed in utero by means of specific receptors in the yolk sac wall, and is selectively absorbed postnatally for up to f.r. homberger days via igg receptors in the intestine [ ] . pilot studies with mhv-y, an enterotropic strain of mhv [t] , have shown that pups born to immune dams and transferred to naive dams at week of age, then challenged with mhv-y, all developed enteritis. in contrast pups born to naive dams and fosternursed by immune dams were protected against disease. this finding suggested that ingestion of immune milk is essential for passive immunity to enterotropic coronavirus infection and that locally active, intraluminal antibody such as iga, might play an important role in protecting pups from enteritis. the purpose of the present study was to evaluate the route of transfer and the role of the different immunoglobulins in passive immunity against enterotropic mhv and to determine which viral proteins are recognized by these immunoglobulins. mhv-y was isolated in nctc- cells during a natural outbreak of enteritis in infant mice [ ] , passaged in infant cd mice and used in the form of a clarified intestinal homogenate. mhv-s was obtained from the american type culture collection, bethesda, md and passaged twice in nctc- cells. nctc-l cell-adapted mhv- was obtained from the institut f/ir labortierkunde, universitfit ziirich, switzerland and passaged in nctc-l cells. outbread cd (crl-cd br) albino mice were obtained from charles river breeding laboratories, portage, mi in filtered containers. all mice were kept in autoclaved microisolator cages (lab products, maywood, nj) containing pine shavings. cages were changed aseptically in a laminar flow hood as previously described [ ] . all animals were seronegative to mhv. dams were orally inoculated with a single dose of mhv-y containing median neonatal enteritis doses of virus or with sterile tissue culture fluid (controls) and bred days later. pilot studies have shown that oral inoculation results in higher mhv antibody titers than parenteral inoculation and mimics more closely immunity in naturally infected animals. the day interval between immunization and breeding ensured that the dams were not shedding virus when the pups were born (approximately days after inoculation). adult mice were killed with carbon dioxide gas and neonates by decapitation. serial blood samples were taken with capillary tubes by periorbital puncture, under methoxyflurane (metofane, pitman-moore inc., washington crossing, n j) anesthesia. terminal blood samples were obtained from pups by decapitation and from dams by cardiac puncture. whey was collected by removing the stomach curds from pups, suspending them in phosphate buffered saline ( % w/v) and centrifuging the homogenate at , × g for min. the supernate was stored at - °c. serology sera and whey were tested for mhv-specific igg by an enzyme immunoassay (eia) using mhv-s-infected formalin-fixed nctc- cells as antigen [ ] . mhv-s is the prototype strain to which mhv-y, which grows poorly in cell culture, is most closely related eli. mhv-specific iga was best detected with an emzyme-linked immunosorbent assay (elisa) using purified mhv- as antigen. mhv- grown in nctc-l cells was pelleted at , x g for h. the resulting pellet was resuspended in ste buffer ( ram nac , passive immunity to enterotropic mhv mm tris, mm edta), layered on a continuous sucrose gradient ( %/ % w/v) and centrifuged for h at , x g. the visible band was collected, stored at - °c and diluted in bicarbonate buffer (ph . ) for coating wells of -well microtiter plates (dynatech laboratories, alexandria, va). a peroxidase-labeled goat-anti-mouse iga conjugate and abts substrate (both kirkegaard and perry laboratories, gaithersburg, md) were used in the elisa [ ] . sera of sentinel mice and of dams of replacement litters were screened for mhv antibody by indirect immunofluorescence assay [ ] . attempts to detect mhvspecific igm used all of the assays described above. immunoblots were performed on nitrocellulose paper using mhv-s as antigen. mhv-s was propagated in nctc- cells, and clarified culture fluid was treated overnight at °c with polyethylene glycol- ( g/ ml) in the presence of nac ( . g/ ml). the resulting precipitate was centrifuged at , x g for min. the pellet was resuspended in ste buffer, and layered on a discontinuous gradient ( %/ % sucrose in ste buffer) and centrifuged at , x g for rain. the visible band was collected and stored in aliquots at - °c. purified mhv-s was boiled for rain in sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page) sample buffer and electrophoretically separated on a . % acrylamide gel at ma for h. the proteins from the gel were then electrobtotted onto nitrocellulose paper at v for h [ ] . the nitrocellulose paper was stained with ponceau-s, cut into strips along the visible lanes and saturated in blocking buffer ( % fetal calf serum) overnight. nitrocellulose strips were twice washed for min in tris-buffered saline, covered with test serum or whey at different dilutions and incubated for h at °c. after two more wash cycles, phosphatase-labeled goat anti-mouse igg, iga, or igm conjugate was added and incubated for another h at °c followed by two rain washes. the strips were then stained with bcip/nbt ( -bromo- -chloro- -indolylphosphate/nitroblue tetrazolium) phosphatase substrate system (kirkegaard and perry laboratories) and air dried. the isotype and titer of mhv immtmoglobulins transferred in utero and in milk, as well as the duration of secretion during lactation were first examined. immune and naive adult females were bred and allowed to whelp. serum was collected from half of each litter immediately after birth before suckling to assess intrauterine transfer of passive immunity. the rest of the litter was sacrificed h later to collect serum and whey, then replaced by a litter from a naive dam to maintain the nursing stimulus. on days , , and post partum, the pups were removed for h and replaced by naive pups which had previously fasted for h. after feeding, serum and whey were collected from these pups. sera and whey were tested for mhv-specific iga, igg, and igm by eia or elisa. mhv-specific iga and igg but not igm could be found in the milk of immunized dams throughout the days of lactation (table ) . during this period, iga titers remained relatively constant, whereas igg titers peaked at to days and declined by days post partum. sera of pups sacrificed immediately after birth (day ) contained mhv-specific igg, indicating in utero transfer of antibody (table ) . virus-specific igm could not be detected at any time. in of the replacement litters used to collect whey on days , , and , igg antibody could be found in the serum (data not shown). this showed that igg can appear in the serum of naive pups after only h of suckling on an immune dam. toward the end of lactation, suckling activity declined and pups started to eat solid food. to find out how this change affected passive immunity, adult females were mhv-y-or sham-inoculated and bred days later. at , , , and weeks of age, two pups from each litter were sacrificed and serum and stomach contents were collected and tested. mhv-igg, but not iga or igm, could be found in the serum of pups suckling immune dams. mhv serum igg titers peaked at days (table ), when they reached levels higher than the ones found in their dams' sera collected immediately post partum. serum igg titers dropped markedly after weaning at days (table ) . mhv-specific igm could not be detected in sera from either pups or dams. stomach contents of pups suckling immune dams yielded constant igg titers and gradually declining iga titers up to the age of days, but neither isotype of mhv antibody was detected at or beyond days (table ). this coincided with the macroscopically obvious change of the stomach contents from milk curd to solid food mass. in a final experiment, the titer and duration of passively acquired mhv igg in serum were evaluated in first, second, and third litter pups weaned from immune vs. naive dams to determine the duration of detectable passive antibody and if the concentration of passively transferred antibody declined with sequential litters. pups from three consecutive litters born to immune and naive dams were exsanguated at , , , , and weeks of age and sera were tested for mhv-specific igg. naive males used for breeding also served as sentinels to verify that dams were not re-exposed to mhv, thereby causing booster infections. passively acquired mhv antibodies persisted in the sera of pups born and nursed by immunized females for to weeks. the mhv serum igg titers of two week old pups correlated closely with those of their corresponding dam, but dropped substantially during the next two weeks (table ). antibodies persisted for weeks in pups born to dams with low mhv igg serum titers and then declined to undetectable levels. in other animals, serum antibodies could be found through weeks. the duration of passive immunity did not decrease with the second and third litter of individual dams. to ensure that the persisting titers were not due to an inadvertant mhv infection, all sera were tested for iga, which could be found in the immunized dams later than five months after inoculation but not in any of the offspring (data not shown). selected whey and serum samples were also examined by western blot to determine the viral protein specificity of the immunoglobulin isotypes. serum of mice experimentally infected with other prototype strains of mhv crossreacted with mhv-s. mhv-jhm igg reacted with all three structural proteins of mhv-s, including glycoprotein s (fig. , lane ) . mhv-y serum igg reacted with the nucleoprotein n and glycoprotein s (fig. , lane ) . passively acquired mhv-y serum igg in pups (fig. , lane ) , as well as whey igg and iga (fig. , lanes and ) also reacted with n and s. reactivity to the m protein of mhv- -- --- i - ...... rdfitter nd e -- --- - - - - ....... "reciprocal mhv igg titer b reciprocal geometric mean of mhv igg titer, replicates c uninfected controls d<l: e dam died at weaning s was not detected in sera or whey of mhv-y-immune mice (fig. , lanes - and ). since igm against mhv could not be detected in any samples, sera from mice infected for , , , , or days with mhv-y were tested. all were igm-negative using elisa, eia, and western blot. the goat-anti-mouse igm conjugates that were used yielded positive results when tested by elisa and immunoblots with a different antigen (borrelia burgdorferi [ ] ). the mouse selectively transfers igg to fetuses in utero through yolk sac receptors and to pups for about two weeks via specific igg receptors on intestinal epi- this study demonstrated that igg to enterotropic mhv-y was transferred in utero as well as through the milk. the same results were found previously with dams immunized intraperitoneally with killed mhv strain dvim, another enterotropic strain of mhv [ ] . intrauterine antibody transfer could not be detected from dams inoculated oronasally with respiratory mhv-jhm [ ] . the present study also demonstrated lactogenic transfer of iga, but absorption from the intestine into the blood of pups did not occur. intraluminal iga and igg in the gastro-intestinal tracts of immune pups could be detected until the suckling activity declined and pups started to eat solid food. serum antibody in pups nursed by mhv-y-immune dams persisted much longer than expected. earlier studies with mice inoculated either oronasally or intraperitoneally with non-enterotropic mhv-jhm had shown that igg in the serum of passively immune pups persisted for about two weeks after the receptormediated absorption of igg in the intestine ceased at approximately two weeks of age [ , ] . in contrast, passively acquired igg to mhv-y persisted at low titer for more than weeks in some mice. this finding suggests that results of serologic monitoring should be interpreted cautiously among animals of this age group. the possibility must be considered that low positive results are due to maternally-derived passive immunity. the current study has also shown that an iga assay could be used to differentiate between passive and active immunity, since serum iga was only found in mice that were actively immune. compared with a similar study on respiratory strains of mhv [ ] , serum igg titers of dams inoculated with enterotropic mhv-y did not so obviously decline over the course of their first three litters. mhv igg titers passed on to the second or third litter were not substantially reduced. serum igg titers of pups dropped to the same level in all three consecutive litters after weaning and duration of passive immunity did not decrease in later litters. this was not due to repeated infections of the dams, since sentinel mice remained seronegative. mhv-specific igm was not detected in these experiments using three independent assays. literature reports of mhv igm could not be found, implying that no specific igm response is generated by mhv infection. in vitro studies showing that plasma cells in culture cease to produce igm when infected with mhv may support this contention [ ] . among the three major structural proteins of mhv, nucleoprotein n is moderately and membrane glycoprotein m highly conserved among mhv strains, whereas spike glycoprotein s is very variable. a fourth protein, hemagglutinin esterase he, appears in only a few mhv strains [ , , , ] . mhv-s virions possessed n, m, and s bands on immunoblots, but not he when stained with mhv-jhm antiserum. additional bands at kda are virusrelated proteins of cell origin antigenetically related to n protein and vary in expression dependent on culture conditions of the virus [ ] . in this study, mhv-y-specific igg and iga were directed against structural proteins n and s of mhv-s. that no specific reaction to the m protein was found is most likely due to insufficient antibody titer of the samples. epizootic coronaviral typhlocolitis in suckling mice mouse hepatitis virus s in weanling swiss mice following intranasal inoculation mouse hepatitis virus biology and epizootiology new ae (eds) viral and mycoplasmal infection of laboratory rodents: effects on biomedical research host age and genotypic effects on enterotropic mouse hepatitis virus infection mouse hepatitis virus and host determinants of vertical transmission and maternally derived passive immunity in mice the transmission of antibodies from mother to fetus. in: villee ca (ed) the placenta and fetal membranes suppression of immune response induction in peyer's patch lymphoid cells from mice infected with mouse hepatitis virus monoclonal antibodies to murine hepatitis virus- (strain jhm) define the viral glycoprotein responsible for attachment and cell-cell fusion antigenetic relationships of murine coronaviruses: analysis using monoclonal antibodies to jhm (mhv- ) virus new strain of mouse hepatitis virus as cause of lethal enteritis in infant mice mouse hepatitis virus: molecular biology and implications for pathogenesis etablierung des serologischen nachweises yon infektionen mit dem m/iusehepatitisvirus und dem pneumonievirus der maus mittels elisa. dissertation maternally derived immune resistance to fatal diarrhea in infant mice to mouse hepatitis virus experimental chronic lyme borreliosis in lewis rats ter meulen v ( ) influence of maternal immunity on the outcome of murine coronavirus jhm infection in suckling mice coronavirus jhm: tryptic peptide fingerprinting of virion proteins and intracellutar polypeptides an immunofluorescence test for the detection of serum antibody to rodent coronaviruses two enzyme immunoassays for the detection of antibody to rodent coronaviruses structural polypeptides of the murine coronavirus dvim western and dot immunoblotting analysis of viral antigens and antibodies: application to murine hepatitis virus this work was supported by grant rr from the national center for research resources, national institutes of health, bethesda, md. the author was supported by a fellowship from the schweizerische nationalfond, bern, switzerland. i thank drs. stephen, w. barthold, and abigail l. smith for their invaluable help and debby beck and deborah winograd for their technical assistance. received march , key: cord- -sn p iw authors: taguchi, fumihiro; goto, yoshitaka; aiuchi, masamine; hayashi, toshiharu; fujiwara, kôsaku title: pathogenesis of mouse hepatitis virus infection: the role of nasal epithelial cells as a primary target of low‐virulence virus, mhv‐s date: - - journal: microbiol immunol doi: . /j. - . .tb .x sha: doc_id: cord_uid: sn p iw the pathogenesis of mouse hepatitis virus (mhv‐s) infection in suckling and weanling mice was comparatively studied after intranasal inoculation. in sucklings, infectious virus as well as specific antigen was first detected in the nasal mucosa at hr, then in the nerve cells of the olfactory bulbs. at this stage viral particles were demonstrated both in the supporting cells and olfactory cells of the nasal mucosa. in the posterior part of the brain and spinal cord, virus was detected on days to postinoculation when viral growth was clearly demonstrable in the liver, spleen and intestines. in weanlings too, infection was first established in the nasal mucosa, shedding infectious virus in the nasal washing until day postinoculation, and later infection spread to the brain and spinal cord. in weanling mice, however, neither infectious virus nor viral antigen was detected in the liver or other visceral organs, while serum neutralizing antibody became detectable on day postinoculation, increasing in titer thereafter. histopathologically degenerative and necrotic changes were observed in the nasal mucosa and central nervous system of both age groups of animals coincidentally with the presence of viral specific antigen, while inflammatory response was much less prominent in sucklings. in the liver, spleen and intestines, however, some lesions were observed only in sucklings. it has been known that there is a wide variance in virulence and organ tropism among many established strains or fresh isolates of mouse hepatitis virus (mhv) ( ) . among these strains mhv- and mhv- are of high-virulence for mice inoculated by the intraperitoneal (i.p.) or intravenous route, multiplying not only in the liver but also in other organs including the nervous system ( , , ) irrespective of age of the host ( ) , although some differences in susceptibility among mouse strains have been reported ( , ) . on the other hand, mhv- or jhm, a highly neurotropic strain, is not fully virulent when inoculated by the i.p. or other routes, but is capable of causing a prominent demyelination resulting in systemic paralysis when inoculated intracerebrally or intranasally (i.n.) ( ) . in contrast to these extensive studies on virulent mhv, the pathogenesis of low-virulence mhv infection remains unclear. such types of mhv are usually pathogenic only for suckling ( , ) or athymic ( , ) mice but not for weanling or adult mice. the natural infection of low-virulence mhv was suggested to occur through nasal infection ( ) , but it is still unknown how the virus is transmitted from mouse to mouse after multiplying in the host. in the previous paper ( ) we reported that icr mice gained an age-dependent resistance against low-virulence mhv-s irrespective of the route of inoculation and that the nasal cavity is the most probable route of natural infection ( ) . the present paper describes comparative studies on the response of susceptible suckling and resistant weanling mice to low-virulence mhv-s. mice assay of neutralizing antibody. sera were collected from infected weanling mice at an interval of or days after virus inoculation. they were diluted in fold steps with mem supplemented with % tryptose phosphate broth (tpb) (difco, u.s.a.) after heating at c for min. to . ml of the serum dilutions were mixed equal volumes of a virus suspension containing approximately pfu/ . ml. after incubation at c for min, . ml of the mixture was inoculated in triplicate on confluent dbt cells grown in -mm petri dishes. after hr incubation for virus adsorption, the cells were overlayed with mem supplemented with % tpb, % calf serum and . % bacto agar (difco, u.s.a.). two days later, cells were stained with neutral red ( : , ) and developed plaques were counted hr after staining. neutralizing titer of the serum was expressed as reciprocal of the serum dilution giving % plaque reduction as compared to the control. as for suckling mice, sera were collected on day from animals, pooled, heated at c for min and assayed in the same manner as in the cases of -week-old mice. no infectious virus was detectable in the sera after inactivation. immunofluorescence. three liters of mhv-s propagated on dbt cells was subjected to concentration with ammonium sulfate followed by partial purification by discontinuous sucrose gradient centrifugation ( ) . a pool of ml fractions showing a distinct virus band was diluted : with pbs, and an equal volume of freund's complete adjuvant (difco, u.s.a.) was added. the mixture was injected in the amount of ml into the footpads of a rabbit weighing kg, and weeks later, ml of the virus material without adjuvant was injected intravenously as a booster. the rabbit was bled week after the booster injection. the antiserum showed a neutralizing titer of : , by the % plaque reduction method using dbt cells. labeling of the antiserum with fluorescein isothiocyanate was carried out according to the method of kawamura ( ) . histopathology. at or hr intervals, to weanling mice were sacrificed. tissues from the main organs were sampled and fixed with % formalin, and sections were made and stained with hematoxylin and eosin. as for -to -day-old suckling mice, sampling was performed every or hr; some materials were fixed with bouin's solution immediately after killing for microscopic examination. electon microscopy. three to suckling mice were sacrificed at , and hr after viral inoculation and tissues of the nasal mucosa were sampled and fixed in . sodium cacodylate (ph . ) containing . % glutaraldehyde. they were postfixed with % osmium tetroxide, dehydrated in ethanol, transferred to propylene oxide, and embedded in epon . sections nm in thickness were made and stained with uranyl acetate and lead citrate, and then examined under electron microscopes, jel s and hitachi hu- . the titers in the brain and spinal cord as well as the head without brain are shown in fig. a . during an early stage of infection the head without brain was found to have titers to logs higher than that of the brain with a peak of about pfu/ . g at to hr postinoculation, which decreased thereafter. but eventually about pfu/ . g were recovered at hr when most mice were moribund. on the other hand, the titer in the brain increased gradually and attained pfu/ . g at the time of death. the pattern of viral growth in the spinal cord resembled that in the brain, although being to logs lower throughout the experiment. in some sucklings which received the same inoculation as above, the anterior and posterior parts of the brain were examined separately for virus. as presented in fig. b , until day postinoculation, the virus titer was significantly higher in the anterior part than in the posterior one. on day , however, the titer in the latter exceeded slightly that in the former. and then, the titer of both parts attained pfu/ . g on day of infection. figure c shows the virus titers in the liver, blood and intestines of infected sucklings. in the liver, virus was first detected at hr postinoculation and the titer increased with time attaining pfu/ . g when moribund. viral growth patterns in the blood and intestines were similar to that in the liver, though to logs lower. viral growth in the small and large intestines is shown in fig. d which indicates that the virus titer was to logs higher in the small intestine. virus assay was performed also in -week-old weanlings at , or hr intervals after inoculation of the same dose of virus. as shown in figs. a and b , significant viral growth was observed in the brain, spinal cord ( fig. a) and head without brain (fig. b) , whereas no virus was demonstrated in the spleen or liver of the infected mice with a few exceptions (not included in the figures). virus was first detectable from the head without brain and then to hr later in the brain. peaks were attained on days and in the head without brain and brain, respectively. in the spinal cord, very low-titered virus was demonstrated with a delay by a few days. on day postinoculation, virus was detectable in neither the brain nor spinal cord, but in one case it was detected from the head without brain. infectious virus was detected in the nasal washings until day , although there were some variations in positivity of virus recovery and titer (fig. b) . none of the other visceral organs examined included infectious virus throughout the experimental period. sera collected from individual weanlings were assayed for neutralizing antibody titers. since the circulating blood of the mice showed no infectious virus during the course of the experiment, the sera were subjected to antibody assay without any virus-killing treatment. as shown in fig. a , neutralizing antibody appeared on day postinoculation which increased gradually in titer until the end of the experimental period. no antibody was detected in pooled sera of infected sucklings. fig. a histopathology at to hr postinoculation, degeneration and necrosis appeared in epithelial cells of the nasal mucosa and some necrotized cells were desquamated to fall into the nasal cavity (fig. a ) in sucklings. at to hr some proliferation of nasal epithelial cells was demonstrable, and thereafter a regenerating process was clear, resulting in structural recovery on days to postinoculation. at this stage of infection, the olfactory bulb underwent degeneration and necrosis of nerve cells with hyperemia in the arachnoid. on days to , these necrotized cells increased in number with proliferation of glia cells. later, the other parts of the brain and spinal cord were involved. in the liver, a few small necrotic foci were seen on day , which increased in size and number on days to (fig. b) . inflammatory response at the necrotic sites was rather poor while a few monocytes were detected in some cases. at the same stage or later, some neutrophils were found in the red pulp of the spleen, and necrosis occurred in exocrine cells of the pancreas. degeneration of the epithelium with accumulation of neutrophils was seen in the small and large intestines (fig. c) . also in infected weanling mice there were changes in the nasal mucosa and central nervous system but not in other organs such as the liver. on day postinoculation, necrosis and desquamation of epithelial cells of the nasal mucosa as well as some meningitis and chorioependymitis were demonstrated in all cases examined. on days to , however, the nasal mucosa turned to be recovered rapidly with replacement by proliferated epithelial cells. while necrosis appeared in neurons of the olfactory bulb on days to (fig. a) , recovery was rapid, leaving only a slight demyelination in the white matter of the midbrain on day (fig. b) . electron microscopy of infected sucklings revealed necrotic changes with proliferation of epithelial cells of the nasal mucosa at hr postinoculation. viral particles were present in a space between the microvilli of supporting cells (fig. a and also in the cytoplasm of the same cells. the particles were also numerous in the olfactory cells (fig. b) . most of the particles were seen in the cytoplasmic vesicles, being morphologically circular and to nm in diameter; they contained a donut-shaped nucleoid and some were covered with fuzzy surface materials . it was shown in a previous paper ( ) that mhv-s was highly virulent for suckling mice aged week or less while being practically non-virulent for -week-old mice. the age-dependent resistance may be due to the ability of mice to suppress viral growth in the liver after i.n. infection despite the lack of such a suppression in the brain. it was also suggested that natural infection of mhv in mice might presumably occur via the i.n. route ( ) . then, the present investigation was designed to see the comparative pathogenesis of mhv-s infection in suckling and weanling mice. as early as hr after virus inoculation in -to -day-old mice, a high virus titer in the head without brain as well as viral antigen in the nasal mucosa was demonstrated, suggesting that the inoculated virus grew first within the epithelial cells of the nasal mucosa. the virus titer increased logarithmically in this site, reaching a maximum at hr postinoculation when viral antigen was densely distributed within the nasal epithelial cells as well as in the tunica propria. numerous viral particles were observed in the epithelial cells of the nasal mucosa as well as in a space between microvilli of the epithelium, suggesting that mhv-s grown in the nasal mucosa and shed into the nasal cavity might be a source of transmission to other individuals. concomitantly with viral growth, there were degeneration and necrosis in the nasal mucosa followed by a regenerating process. at this stage of nasal infection there was no significant viral growth in any other part of the body. also, in -week-old mice, an immunofluorescent study and virus titration revealed that virus multiplied first in the nasal epithelial cells. thus, the present study revealed that mhv did multiply and produce some lesions in the nasal mucosa in both susceptible suckling mice and resistant weanling ones, as suggested in our previous studies using mhv-jhm ( ), although it had not been considered as yet that mhv can multiply and cause some histopathological changes in the nasal mucosa ( ) . it is really of importance that the nasal epithelial cells serve as an initial target in natural infection of mhv. in both suckling and weanling mice, neither infectious virus nor viral antigen was detected in the bronchi or lungs. also in rat coronavirus (rcv) and sialodacryoadenitis virus (sdav) infections, the epithelium of the nasal mucosa of the rat seems to serve as the initial target ( , ) , as has been observed at the early stage of mhv-s infection in the mouse. however, rcv has a tropism to the respiratory system producing histopathological changes and sdav to the salivary and lacrimal glands rather than the respiratory system. the murine coronaviruses, mhv-s, rcv and sdav, can be said to have a similarity in the tropism at the early stage of infection ( ) . the phenomenon observed in mhv -s infection is also compatible with human coronavirus infection which involves only the upper respiratory tract ( ) . the viral growth in the brain seems to follow that occurring in the nasal mucosa of both sucklings and weanlings, and virus was first detected at the anterior portion of the brain, mainly in nerve cells of the olfactory bulb. the mitral cells of the bulb were shown to have viral antigen at an early stage of infection, indicating that the virus grown in olfactory epithelial cells gains access to mitral cells through or along the olfactory fiber, as mentioned by johnson ( ) in herpes simplex virus infection and by goto et al ( ) in jhm infection. this concept is also supported by the electron microscopic observation that viral particles were found in the olfactory cells of the nasal epithelium as early as hr postinoculation. in suckling mice, viral antigens became to be distributed widely in the posterior part of the brain to days postinoculation and thereafter also in the spinal cord, mainly in nerve cells and sometimes in the ependyma or meninges. this suggests that infection in the anterior part of the brain is transmitted to the posterior part through nerve cells, cerebrospinal fluid and/or blood stream, while there appeared no or little tissue damage in the meninges. since infectious virus appeared in the liver on day , it is likely that the infection of the liver and other visceral organs is due to virus disseminated after multiplication in the nasal mucosa, although viremia failed to be detected. the virus grown in the liver may be liberated into bile and then into the intestinal lumen, where it may have a chance to infect the epithelial cells of the mucosa. at the last stage of infection, infectious virus was found also in the large intestine with some virus specific antigen or histopathological changes, although the titer was much lower than in the small intestine. from these findings, virus excreted in feces is supposed to be mostly derived from the liver or the small intestine. transmission of mhv-s from the liver to the lumen of intestine has a similarity to that of human hepatitis a virus infection ( , ) , although no viral multiplication in the intestines has been evidenced in the latter ( ) . the excretion of mhv in feces of mice was reported by rowe et al ( ) and we also experienced that naturally infected but apparently healthy adult mice were shedding infectious virus in the feces (unpublished observation). but we could not detect mhv-s in the feces or intestines of weanling mice. in suckling mice, however, infectious virus and viral antigens were demonstrated in the intestines and it can be considered that the transmission may occur also through feces. thus, the mode of mhv transmission resembles that of other members of coronavirus, human coronavirus ( ) and swine transmissible gastroenteritis virus ( ) . it can be concluded that in mhv-s infection in suckling mice, virus first grows in the nasal mucosa of mice and the infection spreads later in two directions, either to the olfactory bulb probably through nerve fibers and then to the posterior parts of the brain and spinal cord, or to the liver and spleen via blood stream resulting in excretion of the virus in feces. in -week-old mice, however, no or little infectious virus was detected in the liver or other visceral organs, although high titered virus demonstrable in the brain was probably disseminated from the nasal mucosa as was observed in suckling mice. mhv-s infection was reported to be characterized by demyelinating changes in the central nervous system in -to -week-old mice as shown in jhm ( ) . in the present experiments, demyelination was frequently encountered in -week-old mice on days to , being severe on day postinoculation. in -to -day-old suckling mice, however, no demyelinating changes were observed, probably because of too acute course of the infection. pathogenesis on such demyelinating process in mhv-s infection is now being studied and the results will be published later. 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hepatitis virus response of nude mice to a mouse hepatitis virus isolated from a wasting nude mouse neuropathological effects of persistent infection of mice by mouse hepatitis virus key: cord- -k om prn authors: barthold, s. w. title: olfactory neural pathway in mouse hepatitis virus nasoencephalitis date: journal: acta neuropathol doi: . /bf sha: doc_id: cord_uid: k om prn the mechanism of brain infection with mouse hepatitis virus-jhm was studied in balb/cbyj mice following intranasal inoculation, and found to be a consequence of direct viral spread along olfactory nerves into olfactory bulbs of the brain. infection was followed sequentially from nose to brain, using microscopy, immunohistochemistry and virus quantification. lesions, antigen and virus were observed in the olfactory bulb and anterior brain as early as days and posterior brain by days after inoculation. viral antigen extended through nasal mucosa into submucosa, then coursed along the olfactory nerve perineurium and fibers, through the cribriform plate into the olfactory bulbs. on days and , viral antigen was found in the antero-ventral brain, along ventral meninges, olfactory tracts and anterior ramifications of the lateral ventricles. virus was cleared from nose by days and anterior brain by days, but persisted in posterior brain for days after inoculation. mice also developed disseminated infection, with viremia and hepatitis. infection of brain did not correlate with presence of viremia. in contrast to intranasally inoculated mice, orally-inoculated mice did not develop encephalitis, despite evidence of disseminated infection. for direct extension of mhv from the nose into the brain, it has not been conclusively proven. the purpose of this study was to determine the route of virus entry into brain by examination of the sequential progression of infection with neurotropic mhv-jhm following i.n. inoculation of mice. three-week-old balb/cbyj mice were purchased from the jackson laboratory (bar harbor, me) and crl:cd br mice were purchased from charles river laboratories (portage, mich). both sources were mhv free. mice were shipped in filtered boxes and transferred on arrival into sterile micro-isolator (lab products) cages containing pine shavings, food (purina laboratory chow) and water. mice were inoculated i. n. or per os (p.o.) with gl of cell-free culture fluid containing median tissue culture infectious doses of mhv-jhm. mhv-jhm was obtained from the american type culture collection (bethesda, md), passaged twice in nctc- cells, once in balb/cbyj brain and once in c cells [ ] . infectious virus was quantified in tissues by infant mouse infectivity assay. frozen tissues were thawed, weighed and diluted % (w/v) in dulbecco's minimal essential medium containing % fetal bovine serum. they were homogenized, then clarified in a refrigerated centrifuge at , rpm for rain. serial tenfold dilutions of supernates in . ml were inoculated intracerebrally into -day-old crl: cd br mice in groups of four dilution. mortality was established at h and the logloldso/gram of tissue was determined [ ] . tissues were immersion fixed in neutral buffered formalin, ph . , and paraffin embedded. skulls were demineralized in a solution containing . g tetrasodium ethylenediaminetetraacetic acid, . g sodium tartrate, ml hydrochloric acid and ml distilled water. tissues were sectioned at gm and stained with hematoxylin and eosin. selected tissues were stained for mhv antigen using a streptavidin-biotin-horseradish peroxidase (bethesda research laboratories) method and tissue treatment as previously described [ ] . primary antibody was hyperimmune mouse ascitic fluid prepared in multiparous crl:cd br mice by -weekly intraperitoneal injections of mhv-jhm-infected infant mouse brain emulsified in freund's complete adjuvant. groups of five balb mice were killed at intervals ( and h; , , , , , , and days) after i. n. mhv-jhm inoculation. mice were randomly selected for necropsy and killed with carbon dioxide gas followed by cardiac exsanguination. following removal of the lower jaw and skin, the head was sagitally hemisected. one hemisection was placed in formalin. the nasal turbinates and brain were removed for virus recovery from the other hemisection. the brain was then cut coronally into anterior and posterior segments. nose, olfactory bulb, brain, and liver were collected for histology and immunohistochemistry. nasal turbinate, anterior brain, posterior brain and whole blood were collected and frozen at - ~ for virus determination. in addition, groups of five balb mice were inoculated i.n. or p.o. and the subsequent patterns of infection in nose, brain and liver were compared microscopically on day after inoculation. the distribution of lesions suggested a sequential progression of infection from nose to posterior brain ( fig. ) . necrosis of nasal respiratory and olfactory epithelium was observed as early as h after mhv inoculation, with extension into submucosa by h. by day , necrotizing lesions were visible in the anterior olfactory bulb. at subsequent intervals, necrotizing inflammation extended posteriorly along the meninges and olfactory tracts of the anteroventral brain, as well as into the rostral ramifications of the lateral ventricles and hippocampus. involvement of the posterior brain was first evident on day . on days and , necrotizing changes were absent, but mild giiosis and nonsuppurative meningitis were present in areas lytically affected at previous intervals. livers from all mice on days and had multifocal necrotizing hepatitis. the presence of mhv antigen correlated with microscopic lesions in target tissues in the early phase of infection (fig. ). in the anterior nose, mhv antigen was present in ciliated respiratory epithelial cells as early as h after inoculation ( fig. a) . in the posterior nose, mhv antigen was present in olfactory epithelial cells, as well as within submucosal tissues by h (fig. b) . antigen appeared to extend along olfactory nerve perineurium (fig. c) and to a lesser extent, within nerve fibers (fig. d) , often in areas without involvement of overlying mucosa. antigen was present around nerve fibers as they penetrated the ethmoid cribriform plate (fig. e) and appeared within the olfactory bulb meninges and cells of the glomerular layer within h (fig. f ). antigen also appeared in anterior brain at this interval. on days and , mhv antigen was evident in anteroventral (fig. a) , ependyma of the rostra lateral ventricles (fig. b ) and brain (fig. c ). brain appeared to be infected by spread from meninges and ventricles, as well as by extension along olfactory tracts. antigen was seen nonselectively in meningeal, neuronal, gtial and ependymal cells. virus detection confirmed morphological impressions of an anterior to posterior progression of mhv from nose to posterior brain, as well as anterior to posterior regression of infection ( fig. ) . at h after i. n. inoculation, only one of five mice had detectable virus in nasal tissue, which probably represented residual inoculum. by h, all mice had demonstrable virus in nasal tissue, which rose in titer through day , then declined. virus was first detected in anterior brain on day after inoculation and cleared by day . in contrast, virus was not detected in posterior brain until day and was still present in brains of all infected mice at day . viremia was confirmed in four of five infected mice at h and all mice at , , and days after i.n. inoculation. to confirm that brain infection resulted from direct extension of virus along olfactory nerves and not due to viremia, groups of five mice were inoculated either i.n. or p.o. with mhv-jhm. at days after inoculation, the distribution of mhv lesions was compared between the two groups (table ). both groups developed the same prevalence and severity of hepatitis, indicative of viremic dissemination. however, only i.n.-inoculated mice developed brain lesions. interestingly, orally inoculated mice had mhv lesions in the anterior nose involving respiratory epithelium, this study provides insight into one mechanism of central nervous system infection by viruses with respiratory tropism. mouse hepatitis virus, similar to coronaviruses of other species, utilizes upper respiratory mucosa as its primary target [ , , , , ] . a number of studies have demonstrated olfactory bulb or tract lesions following i.n. inoculation of different mhv strains [ - , , , , , ] . results of the present study provide evidence that virus enters the brain di-rectly by extension along olfactory nerves. sequential studies of lesion, antigen and virus distribution in nose and brain indicated anterior to posterior progression of infection. furthermore, mice inoculated orally with mhv did not develop brain infection, although they showed evidence of viremic dissemination to liver. this suggests that there is an effective blood-brain barrier to mhv in mature mice. in contrast, the pattern of brain infection is decidedly different in neonatal mice. in these animals, mhv lesions are diffusely distributed throughout the brain, suggesting blood-borne infection [ , ] . the mechanism of this age-dependent blood-brain mhv barrier is worthy of further investigation. orally inoculated mice developed mfiv lesions in their anterior nasal tissues, presumably by entry of virus through the incisive foramina, patent openings between the mouth and anterior nose [ ] . the fact that none of these mice developed brain infections despite anterior nasal infection suggests that olfactory neural extension of virus is inefficient and requires infection of the posterior nasal tissues, where olfactory epithelium and nerves prevail. other studies have indicated that among several prototype mhv strains tested, all strains infected anterior olfactory pathways of brain following i.n. inoculation of adult mice. however, only those vi~rus strains with relative neurotropism were capable of progressing into the posterior brain [ ] . thus, progression of infection within brain is mhv strain specific. the sequential distribution of lesions within the brain up to days after i.n. inoculation of neurotropic mhv-jhm has been well described [ ] , and resembled the posterior progression seen in our studies. the present study examined infection only for days, but showed a sequential anterior-posterior regression of lesions and virus within the brain. persistence of low levels of virus in the posterior brain stem corresponded with the location of persistent spongiform and demyelinating lesions in the brain stem following i.n. inoculation of several different strains of mhv [ , , , ] . although mhv-jhm is usually cleared from brain by days after i.n. inoculation of susceptible mice [ ] , temperature-sensitive mutants of mhv-jhm have been shown to persist for up to one year after intracerebral inoculation [ , ] . a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin. ii. pathology mouse hepatitis virus s in weanling swiss mice following intranasal inoculation mouse hepatitis virus strainrelated patterns of tissue tropism in suckling mice response of genetically susceptible and resistant mice to intranasal inoculation with mouse hepatitis virus jhm enterotropic mouse hepatitis virus infection in nude mice mouse hepatitis virus nasoencephalopathy is dependent upon virus strain and host genotype experimental models of virus-induced demyelination of the central nervous system nasoencephalopathy of mice infected intranasally with a mouse hepatitis virus, jhm strain anatomy. in: green el (ed) biology of the laboratory mouse pathology of diarrhea due to mouse hepatitis virus in the infant mouse virus persistence and recurring demyelination produced by a temperature sensitive mutant of mhv- selected mutants of mouse hepatitis virus type ohm strain) induce different cns diseases. pathobiology of disease induced by wild type and mutants ts and ts in balb/c and sjl mice temperature-sensitive mutants of mouse hepatitis virus strain a : isolation, characterization and neuropathogenic properties the organ tropism of mouse hepatitis virus a in mice is dependent on dose and route of inoculation a simple method of estimating fifty percent endpoints enhanced growth of a murine coronavirus in transformed mouse cells pathogenesis of mouse hepatitis virus infection. the role of nasal epithelial cells as a primary target of low virulence virus virus infection in demyelinating diseases the biology and pathogenesis of coronaviruses viral models of demyelination acknowledgement. the technical assistance old. s. beck is gratefully acknowledged. key: cord- - qu j authors: ulferts, rachel; imbert, isabelle; canard, bruno; ziebuhr, john title: expression and functions of sars coronavirus replicative proteins date: - - journal: molecular biology of the sars-coronavirus doi: . / - - - - _ sha: doc_id: cord_uid: qu j the discovery of a previously unknown coronavirus as the causative agent of the sars epidemic in / stimulated a large number of studies into the molecular biology of sars coronavirus (sars-cov) and related viruses. this research has provided significant new insight into the functions and activities of the coronavirus replicase–transcriptase complex, a multiprotein complex that directs coordinated processes of both continuous and discontinuous rna synthesis to replicate and transcribe the large coronavirus genome, a single-stranded, positive-sense rna of ~ kb. in this chapter, we review our current understanding of the expression and functions of key replicative enzymes, such as rna polymerases, helicase, ribonucleases, ribose- ′-o-methyltransferase and other replicase gene-encoded proteins involved in genome expression, virus–host interactions and other processes. collectively, these recent studies reveal fascinating details of an enzymatic machinery that, in the rna virus world, is unparalleled in terms of the number and nature of virally encoded activities involved in virus replication and host interactions. coronaviruses and their closest relatives from the order nidovirales have exceptionally large rna genomes of about kb and synthesize an extensive set of -leader-containing, subgenome-length rnas. the synthesis of these rnas is mediated by the viral replicase-transcriptase complex (rtc), a large multisubunit complex that is comprised of more than a dozen proteins encoded by the viral replicase gene and other proteins. the rtc includes the key replicative proteins of the virus, such as rna-dependent rna polymerase (rdrp) and helicase activities, as well as enzymes that are thought to be involved in the processing and modification of viral and/or cellular rnas, such as primase, endoribonuclease, exoribonuclease and ribose- -o-methyltransferase activities (for recent reviews, see masters ; ziebuhr ziebuhr , . the rtc is anchored through three replicase-gene encoded integral membrane proteins to intracellular membranes derived from the endoplasmic reticulum masters ; ziebuhr and snijder ) . besides proteins encoded by the replicase gene, the rtc contains the nucleocapsid (n) protein (almazan et al. ; schelle et al. ) and several cellular factors, which have not been characterized in great detail (shi and lai ; van hemert et al. ) . it is becoming increasingly clear that the coronavirus replicase gene also encodes proteins that are not essential for viral rna synthesis but are involved in viral pathogenicity and specific virus-host interactions (see below). the single-stranded, positive-sense rna genome of coronaviruses fulfills a double role, serving as an mrna for the expression of the replicase gene and a template for the synthesis of genome-and subgenome-length minus-strand rnas. a large part of the genome (about two-thirds) is occupied by the replicase gene, which consists of two (slightly) overlapping open reading frames (orf) located in the -proximal region of the viral genome rna. the remaining -terminal third of the genome is dedicated to encoding the four major structural proteins of the virus (s, e, m, and n) and a varying number of so-called accessory proteins ( fig. . ). compared to other coronaviruses, sars coronavirus (sars-cov) encodes an unusually large number of accessory proteins, some of which are involved in counteracting antiviral host responses (chap. ). the orfs located downstream of the sars-cov replicase gene are expressed from eight subgenomic (sg) rnas (fig. . ) . a peculiarity of the sgrnas produced by coronaviruses and some other nidoviruses is that both the plus-strand genome rna and all plus-strand sgrnas share a short, so-called leader sequence at their ends, whose functional relevance is currently unknown (spaan et al. ). it has been speculated that the minus-strand complement of the leader sequence (present at the ends of all minus-strand rnas) might facilitate initiation of plus-strand rna synthesis. alternatively, the leader might promote the translation of sgrnas, for example through the presence of sequence elements required for efficient recognition by the viral capping apparatus or other mechanisms enhancing translation initiation. the precise mechanism used to join the "leader" and "body" sequences of sgrnas (which are located more than kb apart in the genome) is unknown but is generally accepted to involve a discontinuous step during sg minus-strand rna synthesis and be guided by complementary base-pairing between transcriptionregulating sequences (for recent reviews, see pasternak et al. ; sawicki et al. ). the specific requirements for maintaining genome rnas of an unparalleled size and synthesizing sg minus-strand rnas in a discontinuous manner are reflected by an exceptional complexity of the protein machinery involved in viral rna synthesis, which will be the topic of this review. with few exceptions, the proteins and mechanisms involved in genome replication and transcription are conserved among sars-cov and other coronaviruses. in this chapter, we will review recent work on the sars-cov rtc and refer to related work on other coronaviruses as appropriate. the sars-cov replicase gene contains more than , nucleotides and is comprised of two orfs called orf a and orf b ( fig. . ) (marra et al. ; rota et al. ; thiel et al. ) . upon infection of the cell, orfs a and b are translated into two large polyproteins, pp a and pp ab, of approximately and kda, respectively. polyprotein ab is a c-terminally extended version of pp a; pp ab expression requires a programmed (- ) ribosomal frameshift prior to termination of orf a translation, resulting in continuation of translation in the orf b reading frame . frameshifting depends on two cis-acting rna elements, a "slippery" heptanucleotide sequence and an rna pseudoknot structure (see chap. for details). based on in vitro translation experiments, it has been estimated that frameshifting occurs in roughly % of translation events (brierley ; dos ramos et al. ; herold and siddell ; namy et al. ) . this results in a considerably higher amount of orf a-encoded proteins compared to orf b-encoded proteins, which is likely to be of biological relevance as the frameshifting mechanism is conserved in coronaviruses and other nidoviruses. sars-cov pp a and pp ab are co-and post-translationally processed by two proteases, a papain-like protease (pl pro ) and the main protease (m pro , nsp ), resulting in mature products called nonstructural proteins (nsps) - the -terminal orf(s) expressed from specific rnas is/are shown as boxes. the small black box indicates the -leader sequence present on each of the viral rnas. the replicase gene (orfs a and b) are shown in black. to the right, sars-cov genomic and subgenomic rnas as detected by northern blotting using a end-specific probe are shown together with information on sizes (in kilobases) and names of proteins expressed from these rnas. (c) overview of the domain organization and proteolytic processing of the sars-cov replicase polyproteins, pp a ( kda) and pp ab ( kda). for comparison, the mouse hepatitis virus (mhv)-a replicase polyproteins are shown. the processing end-products of pp a are designated nonstructural proteins (nsps) to nsp and those of pp ab are designated nsp to nsp and nsp to nsp . cleavage sites that are processed by the viral main protease, m pro , are indicated by grey arrowheads; sites that are processed by the papain-like proteases and (pl and pl ), are indicated by white and black arrowheads, respectively. ac, acidic domain; a, adrp (adp-ribose- -phosphatase); sud, sars-cov unique domain; nab, nucleic acid-binding domain; rp, noncanonical rna polymerase (putative primase); rdrp, rna-dependent rna polymerase; tm , tm , tm , transmembrane domains , , and , respectively; hel, helicase; exon, -to- exoribonuclease; neu, nendou (nidoviral uridylate-specific endoribonuclease); mt, ribose- -o-methyltransferase < (harcourt et al. ; prentice et al. ; snijder et al. ; thiel et al. ) . the pl pro domain is part of nsp and processes the nsp | , nsp | and nsp | cleavage sites. the m pro cleaves the c-terminal part of pp a/ ab at the remaining sites ( fig. . ), releasing the most conserved proteins of the coronavirus rtc. the individual cleavage sites are thought to be processed with varying efficiencies, resulting in the presence of some reasonably stable intermediates. this has been studied in some detail for the proteins released by the pl pro activity (harcourt et al. ) . while the nsp | and nsp | sites are rapidly processed, the nsp | site was found to be cleaved at a lower rate, giving rise to a relatively long-lived nsp - intermediate in infected cells. the rapid cleavage of the nsp | site results in fast separation of the domains processed by pl pro and those processed by m pro . even though an initial study of the nonstructural proteins in sars-cov-infected cells failed to detect processing intermediates (prentice et al. ) , the cleavage of interdomain junctions by m pro is also thought to occur at varying rates (fan et al. ). this is supported by data obtained for mouse hepatitis virus (mhv), showing that nsp - is a relatively stable processing intermediate (schiller et al. ). in addition to the sequence of the individual cleavage sites, the efficiency of processing appears to be influenced by the secondary structure of the substrate (fan et al. (fan et al. , . thus, cleavage of peptides with a high propensity to form b-sheets is enhanced, consistent with the substrate binding mode observed in crystal structures of m pro in complex with inhibitors yang et al. yang et al. , . it is generally accepted that processing intermediates fulfil important functions that, in some cases, differ from those of the fully processed proteins. this strategy is commonly employed by plus-strand rna viruses to expand the functional repertoire of the relatively small number of protein domains present in viral polyproteins (dougherty and semler ; palmenberg ) . the idea of different functions being associated with precursors and final processing products, respectively, is supported by the fact that coronavirus rna synthesis requires ongoing protein synthesis, with minus-strand synthesis declining more rapidly than positive-strand synthesis after inhibition of translation by cycloheximide (sawicki and sawicki ). nonstructural proteins the proteins processed from the n-terminal regions of the coronavirus replicative polyproteins a/ ab are highly divergent among coronaviruses. nsp proteins from group and group coronaviruses, respectively, are not evidently related to each other, whereas group coronaviruses lack an nsp -counterpart altogether, making nsp the most n-terminal processing product in this case. the significant sequence diversity is also evident when nsp proteins from the same group ( or ) are compared with each other connor and roper ; snijder et al. ) . a nuclear magnetic resonance structure was reported for sars-cov nsp . the protein has a complex b-barrel fold flanked by disordered n-and c-terminal domains (see chap. for details) and lacks statistically significant structural similarity to other cellular or viral proteins. although the protein is not required for replication of sars-cov and mhv in cell culture wathelet et al. ; züst et al. ) , there is increasing evidence to suggest that the protein has important functions in vivo. sars-cov nsp was suggested to counteract cellular innate immune responses by inhibiting ifn signaling pathways, and recombinant sars-cov with an attenuating mutation in nsp was found to decrease the ability of the virus to replicate in cells with an intact ifn response (wathelet et al. ). narayanan and co-workers (narayanan et al. ) reported that another sars-cov nsp mutant, but not the wild-type virus, induced high levels of inf-b, suggesting that nsp may also have a role in type i ifn induction, which contradicts findings reported earlier by others (wathelet et al. ; züst et al. ). characterization of an mhv mutant expressing a c-terminally truncated form of nsp showed that the protein is a major pathogenicity factor of this virus (züst et al. ). there is evidence to suggest that mhv nsp is involved in counteracting type i ifn signaling and/or the antiviral activities of ifn-induced proteins, whereas a role in suppression of ifn induction seems less likely. furthermore, sars-cov nsp was shown to ( ) promote host mrna degradation, ( ) inhibit cellular translation (kamitani et al. ; narayanan et al. ) and ( ) affect cell cycling (wathelet et al. ). mhv nsp was reported to colocalize (and interact) with other subunits of the viral replicase-transcriptase at the site of viral rna synthesis but appears to migrate to virion assembly sites at a later stage of infection (brockway et al. ) . it remains to be seen if this relocalization is linked to distinct functions of nsp at different stages of the replication cycle of mhv and, possibly, other coronaviruses. for nsp , no specific function has yet been identified. it has been suggested that nsp is a component of sars-cov virions (neuman et al. ). the protein is not essential for replication in vitro as deletion of the entire nsp coding sequence was tolerated in both sars-cov and mhv (graham et al. ) . nsp deletion mutants grew to slightly reduced titers and rna synthesis was diminished by about %, with all rna species being equally affected. more recently, it was shown that the replication defects observed in mhv nsp deletion mutants could not be compensated by nsp expressed from either a subgenomic rna or from a c-proximal position in the replicase polyprotein, when inserted between nsp and nsp (gadlage et al. ) . nsp is a large, membrane-anchored multidomain protein. despite significant sequence diversity among coronavirus nsp proteins, up to putative functional domains, including ubiquitin-like, metal-binding, nucleic acid-binding, rna chaperone-like, poly(adp)-ribose-binding, protease, transmembrane (tm) and other conserved domains have been identified in nsp (neuman et al. ; ziebuhr et al. ). nsp has been proposed to be a major "hub" for protein-protein and protein-rna interactions between viral and (possibly) cellular macromolecules neuman et al. ). many of the domains are common to all coronaviruses while some are group-or species-specific (neuman et al. ; snijder et al. ; ziebuhr et al. ) . among the conserved domains are papain-like proteases (pl pro s) that cleave the n-terminal part of the polyproteins at up to three sites ( fig. . ). while most coronaviruses possess two pl pro s (pl pro and pl pro ), sars-cov and ibv encode only one active pl pro , which are orthologues of the pl pro of other coronaviruses thiel et al. ; ziebuhr et al. ). the sars-cov pl pro employs a cys-his-asp catalytic triad and exhibits a narrow substrate specificity, with all three processing sites conforming to the consensus sequence lxgg (harcourt et al. ; thiel et al. ) . besides its important role in pp a/pp ab processing, the sars-cov pl pro has deubiquitinating activity (barretto et al. ; lindner et al. ) and shares structural features with cellular ubiquitin-specific proteases (see chap. for details) (ratia et al. ; sulea et al. ) . the pl pro homolog of hcov-nl , a group b coronavirus, also has ubiquitin-specific protease activity (chen et al. b) , suggesting that this activity may be of general biological relevance to the coronavirus life cycle. sars-cov pl pro removes ubiquitin and ubiquitin-like modifiers (ubl) from fusion proteins and debranches polyubiquitin chains, with a marked preference for lys- -over lys- -conjugated chains (lindner et al. ). the c-terminal residues of ubiquitin and the ubiquitinlike modifier isg , lrlrgg, match the consensus sequence of sars-cov pl pro cleavage sites in pp a/pp ab very well (harcourt et al. ; thiel et al. ) . sars-cov pl pro is able to distinguish between isg and ubiquitin and preferentially cleaves isg -modified proteins. the exact mode of recognition of ubl modifiers by pl pro is not known but might involve interactions between the zn + ribbon domain connecting the a and b subdomains of the pl pro and the b-grasp fold of ubiquitin and additional, currently unknown, interactions. the deubiquitinating and deisgylating activities of pl pro have been speculated to be involved in ( ) protecting viral and/or cellular proteins from degradation and ( ) counteracting innate immune responses (lindner et al. ; sulea et al. ) . recently, pl pro was shown to inhibit ifn signaling by binding to irf- and interfering with its hyperphosphorylation, dimerization and nuclear translocation (devaraj et al. ) . surprisingly, the inhibition of ifn response was not abrogated by substitution of pl pro active-site residues, suggesting that the protease/deubiquitylase activity of pl pro was not involved. slightly contrasting data were reported by zheng and coworkers (zheng et al. ) . in this case, the reduction of the type i inf response induced by vesicular stomatitis virus was dependent on the proteolytic activity of the sars-cov pl pro . another nsp domain is the adp-ribose phosphatase (adrp) domain (also called x domain or macro domain) (fig. . ) . the domain is conserved across members of the genera coronavirus, torovirus and bafinivirus (draker et al. ; schütze et al. ) and several other plus-strand rna viruses (gorbalenya et al. ) . viral adrps are related to a large family of macro domain proteins found in many cellular organisms. the protein family is named after the nonhistone domain of macroh a histones. macro domain proteins share a conserved fold and bind to (and, in some cases, process) a range of substrates related to adp-ribose (karras et al. ) . several members of the macro domain family have been shown to ( ) bind poly(adp)-ribose and/or poly(a) rna and ( ) hydrolyze adp-ribose- -phosphate karras et al. ; neuvonen and ahola ; putics et al. putics et al. , saikatendu et al. ; xu et al. ). the adrp domains of hcov- e, sars-cov and transmissible gastroenteritis virus (tgev) have been shown to hydrolyze adp-ribose- phosphate to yield adp-ribose and inorganic phosphate karras et al. ; neuvonen and ahola ; putics et al. putics et al. , saikatendu et al. ; xu et al. ). the molecular mechanisms of the dephosphorylation reaction have not been elucidated but are thought to differ from other phosphatases as there is no sequence and/or structural similarity between the respective enzymes (allen et al. ; egloff et al. ) . based on the structure of coronavirus adrps in complex with adp-ribose, two conserved residues (asn and his) were proposed to be important for activity saikatendu et al. ; xu et al. ). substitutions of these residues by alanine abolished adrp activity activity in an in vitro assay, supporting their critical role putics et al. ) . the biological role of the adrp in the coronavirus life cycle has not been established. characterization of an hcov- e mutant (hcov- e-n a) expressing an inactive adrp revealed no apparent defects in virus reproduction and rna synthesis in a cell culture system (putics et al. ) . similar observations were made for the corresponding mhv-a mutant (mhv-n a) (eriksson et al. ) , confirming that adrp activity is dispensable for coronavirus replication in vitro. however, when characterized in vivo, the mhv-n a mutant was found to be attenuated. at high infection doses, the mutant and wild-type viruses replicated to similar titers in the liver of c bl/ mice but, in contrast to wild-type virus, the n a mutant failed to cause severe liver pathology. similar observations were made in c bl/ ifnar -/mice, arguing against a major role of the adrp activity in counteracting ifn-a host responses. the underlying mechanisms for the observed low pathogenicity of the mhv-n a mutant in the liver have not been identified conclusively but may be linked to a reduced expression of proinflammatory cytokines, in particular il- . the data support the biological relevance of the catalytic activity of coronavirus adrp domains which, on the basis of in vitro observations, has previously been questioned by others neuvonen and ahola ) . the biologically relevant substrate(s) of the adrp domains of sars-cov and other coronaviruses remain(s) to be identified. there is increasing evidence to suggest that macro domains have evolved different functionalities using a conserved fold and may have more than one activity which might not necessarily be mutually exclusive in any one domain. a striking example to support this idea is the recent identification of another macro domain within the sars-cov nsp , located downstream of the adrp (chatterjee et al. ) and representing the central domain (sud-m) of the sars-cov unique domain (sud). on the basis of the nmr structure of sud-m the adrp was identified as the closest structural homolog of sud-m, even though the sequence similarity between the two domains is very low ( % identical residues). it is tempting to speculate that the sud-m domain may have evolved from adrp through gene duplication, similar to what was previously proposed for the two paralogous pl pro domains found in many coronaviruses (ziebuhr et al. ). sud-m was found to bind to single-stranded poly(a)-rna while others (tan et al. ) had previously reported a poly(dg)/poly(g)-binding activity for sud. the reasons for these differences are currently unclear but might be due to the different domain boundaries of the proteins characterized in the two studies. the full-length sud has also been shown to have metal-binding activity (neuman et al. ) which likely resides in the n-terminal part of the protein and involves some of the six conserved cysteine residues (sars-cov nsp positions , , , , , and ) and two conserved histidine residues (positions and ). additional domains with nucleic acid-binding activity are present in sars-cov nsp and probably other coronaviruses. the bacterially-expressed n-terminal domain of nsp consistently copurified with nucleic acids (serrano et al. ) . the n-terminal region of nsp is particularly rich in acidic residues and is therefore also referred to as acidic (ac) domain (ziebuhr et al. ). the domain is conserved in all coronaviruses but exhibits a low degree of sequence identity (serrano et al. ). the nmr structure of the ac domain revealed an n-terminal ubiquitin-like fold (residues - ) followed by a disordered tail particularly rich in glutamic acid residues (serrano et al. ) . the n-terminal globular domain differs from the ubiquitin-like fold by an elongated a helix and the presence of two additional helices, a and , which are important for rna binding. the protein was found to preferentially bind (g)au(a) sequences. a further domain linked to rna binding was identified in sars-cov nsp just downstream of the pl pro (neuman et al. ). this domain, named nucleic acidbinding domain (nab), is conserved in group and (but not in group ) coronaviruses. bacterially-expressed nab exhibited atp-independent doublestranded nucleic acid-unwinding activity, consistent with a possible chaperone function. coronavirus replication takes place at virus-induced double membrane vesicles (dmvs) (gosert et al. ; knoops et al. ) . the tm domain present in nsp (neuman et al. ; snijder et al. ; ziebuhr et al. ) , along with tm domains in nsp and nsp , is thought to be involved in tethering the viral replication-transcription complex to intracellular membranes. the topology of the sars-cov nsp tm domain was recently determined (oostra et al. ) . the data suggest that the n-and c-termini are located in the cytoplasm, thus placing the pl pro at the same face of the membrane as all its cleavage sites in pp a/pp ab. the domain traverses the membrane twice, while the third, central, predicted transmembrane helix does not appear to function as such. by analogy with the tm domains located in the equine arteritis virus nsp and nsp proteins (snijder et al. ) , whose role in dmv formation has been established earlier, oostra and co-workers (oostra et al. (oostra et al. , suggest that dmv formation in sars-cov infected cells is mediated by nsp and nsp . they further hypothesize that the central non-tm hydrophobic domain might play an important role in this process by dipping into the membrane and inducing curvature of the membranes. nsp is a tetra-spanning membrane protein (oostra et al. ) . both termini are located in the cytoplasm (oostra et al. ). tm helices and are connected by a long lumenal loop that is n-glycosylated (oostra et al. ). the presumed critical role of nsp in dmv formation is supported by studies using a temperaturesensitive mutant of mhv-a in which substitution of the nsp asn- residue with thr led to impaired dmv formation, while polyprotein processing was not affected (clementz et al. ) . the critical role of nsp , including its transmembrane-spanning regions - , in coronavirus replication has been established in a recent reverse genetics study using mhv-a (sparks et al. ). the study also showed that the c-terminal nsp residues lys- to thr- are dispensable for mhv replication. nsp is the viral main protease (m pro ). it cleaves at sites in the central and cterminal pp a/pp ab regions ziebuhr et al. ; ziebuhr et al. ) . because of its prominent role in pp a/pp ab processing and the large body of structural and biochemical information available for this enzyme, the m pro is considered an attractive target for the development of antivirals. the enzyme is distantly related to the c proteases of picornaviruses (hence its traditional name " c-like protease," cl pro ) but diverged significantly from these viral homologs (gorbalenya et al. b ). for example, in place of the typical cys-his-asp/glu catalytic triad of picornaviral c proteases, the coronavirus m pro employs a cys-his catalytic dyad (anand et al. , tan et al. ; xue et al. ; yang et al. yang et al. , zhao et al. ) . a water molecule was found to occupy the position of the third member of the catalytic triad in coronavirus m pro structures and it has been suggested that this water molecule might stabilize the protonated his during catalysis. in common with many cellular and viral chymotrypsin-like proteases, coronavirus m pro has a two-b-barrel fold which, in the coronavirus enzymes, is linked to a unique a-helical domain at the c-terminus (anand et al. ) . m pro forms homodimers (anand et al. , yang et al. ) . dimerization mainly occurs through interactions between the c-terminal domains of the two protomers in the dimer as well as a stretch of n-terminal residues (n-finger) (see chap. for details). dimerization is generally believed to be a prerequisite for trans-processing activity. the dimeric structure is another feature that sets the coronavirus m pro apart from c proteases which function as monomers. over the past few years a large number of studies have provided significant insight into the structural details and dynamics of m pro dimerization and their functional consequences. for a review of this work, the reader is referred to chap. . nsp is another tm protein. both the n-and c-terminus are located cytoplasmically (oostra et al. ) , indicating an even number of tm helices and thus placing the m pro on the same face of the membrane as all its substrates. the protein has seven putative tm helices. to satisfy the observed n endo -c endo localization of the protein, oostra and co-workers ( ) proposed that only six of the predicted tm helices function as such in the context of the full-length protein and helix or possibly helix might not traverse the membrane. further, it has been suggested that the nonmembrane-spanning helix may act as interaction platform for other replicase components or aid in the formation of dmvs. the function of nsp remains to be characterized. nsp was recently reported to be a second "noncanonical" rna-dependent rna polymerase (imbert et al. ) (see chap. ). the protein synthesizes short oligonucleotides of up to nts and requires an rna template and metal ions for activity. rna synthesis was sequence specific, with a preference for the internal -(g/u)cc- trinucleotide sequence as site of initiation, but exhibited a relatively low fidelity and processivity. nsp is well conserved among coronaviruses. the noncanonical rdrp is therefore suggested to be an essential enzymatic activity involved in rna synthesis in all coronaviruses and, possibly, other related nidoviruses. imbert and co-workers (imbert et al. ) hypothesized that nsp could act as a primase that produces rna primers which are then extended by the main rdrp, nsp , in a mechanism reminiscent of dna replication. nsp was shown to interact with nsp and together these proteins form a hexadecameric ring structure consisting of eight copies of each protein as shown by x-ray crystallography (zhai et al. ) . the complex encircles a channel with a diameter of~ Å that is lined with positively-charged residues (for details, see chap. ). addition of nsp to the primase activity assay did not increase the primase activity. however, nsp exhibits a low thermostability and nsp might therefore act as a stabilizing mortar (imbert et al. ; zhai et al. ) . the structure of sars-cov nsp was solved by two groups (egloff et al. ; sutton et al. ) . a distant structural relationship with domain ii of the m pro was noted, suggesting that both proteins may be evolutionary related and thus represent another example of domain duplication in the coronavirus replicase, similar to what was discussed above for coronavirus pl pro and macro domains. furthermore, nsp is structurally related to proteins containing an oligosaccharide/oligonucleotidebinding (ob) fold, although the connectivity of the individual secondary structural elements differs in nsp . a large proportion of ob-fold proteins bind nucleic acids and sars-cov nsp was confirmed to also bind ssrna. binding of ssrna by nsp was unspecific but specificity might possibly be attained by interaction with other viral or cellular proteins. sutton and co-workers ( ) showed that sars-cov nsp interacts with nsp in an analytical ultracentrifugation analysis. the experiment further suggested that this interaction might help stabilize an otherwise disordered domain of nsp . nsp forms dimers through the interaction of parallel a-helices that contain a gxxxg protein-protein interaction motif (miknis et al. ). substitutions of either of the gly residues with glu disrupted dimer formation while rna binding was only marginally affected. viable sars-cov mutants carrying either mutation could not be recovered, suggesting that nsp dimerization is critical for virus replication. nsp is another small replicase protein with rna-binding activity. two studies analyzing the structure of nsp described a single-domain protein that contains two zn + -fingers (joseph et al. ; su et al. ) . nsp was shown to bind single-and double-stranded rna and dna with low affinity and no apparent specificity. nsp interacts with nsp as shown by cross-linking (joseph et al. ) and in a gst-pulldown assay . as nsp , in turn, interacts with nsp and nsp forms a complex with nsp and all these proteins are involved in homotypic interactions (see above and chap. ), it is tempting to believe that these proteins form a multiprotein complex that, in the course of infection, undergoes structural rearrangements (possibly due to m pro -mediated cleavages), thereby activating, modulating or inactivating specific rtc function(s) as required at the various steps of viral rna synthesis. consistent with the presumed key role of m pro processing in the formation of a functional rtc, viable mhv mutants could not be recovered if cleavage at the nsp | , nsp | or nsp | ( ) sites was abolished (deming et al. ). disruption of proteolytic processing at the nsp | site gave rise to an attenuated, but viable phenotype. the mhv nsp | cleavage site mutant restored near wild-type growth kinetics after serial passaging which was not caused by restoration of processing at the nsp | site but by a number of compensatory mutations at distant positions in the viral genome. this suggests that nsp - can function as a fusion protein, though efficient replication requires adaptation of the virus. the functional role of the specific changes identified in some of the recovered viruses remain to be characterized. nsp forms the c-terminal part of pp a. sars-cov nsp is an oligopeptide of residues. it is produced if no programmed frameshift occurs at the "slippery sequence" (see chap. ), leading to translation termination at the orf a stop codon. nsp shares its n-terminal amino acids (upstream of the frameshift site) with nsp . most of the nsp coding sequence overlaps with the rna sequence involved in frameshifting and the nsp coding sequence, posing severe constraints on the nsp sequence and arguing against a functional role of nsp . the protein has also not been detected in infected cells. the rna-dependent rna polymerase (rdrp, nsp ) is the most conserved protein of the coronavirus replicase-transcriptase. expression of nsp (and all other orf b-encoded proteins) requires ribosomal frameshifting, implying that nsp - are produced at significantly lower levels compared to orf a-encoded functions. sars-cov nsp is a protein of residues. the actual catalytic domain containing the conserved rdrp motifs (gorbalenya et al. b; koonin ) occupies the c-terminal region (c-terminal domain, ctd) of nsp while the n-terminal domain (ntd) spanning the first amino acids has no known counterpart in other rdrps (xu et al. ) . xu and co-workers ( ) proposed a three-dimensional ( d) homology model for the sars-cov nsp ctd. this model showed the characteristic cupped right hand palm-finger-thumb structure encircling a nucleic acid-binding tunnel. the rdrp activity of nsp was recently confirmed by showing that bacterially-expressed nsp is able to extend an oligo (u) primer hybridized to a poly(a)-template (cheng et al. ) . sars-cov nsp is a multidomain protein of amino acid residues. the n-terminal region contains a zinc-binding domain (zbd) while a helicase domain featuring the typical conserved morifs of superfamily helicases is present in the c-terminal half (gorbalenya et al. a (gorbalenya et al. , b . the zbd contains conserved cysteine and histidine residues that are predicted to form a binuclear zn + -binding cluster (seybert et al. ; van dinten et al. ) . it is conserved in all nidoviruses and is critical for helicase activity in vitro (seybert et al. ) and rna synthesis of hcov- e in cell culture (hertzig and ziebuhr, unpublished) . coronavirus helicases (including sars-cov nsp ) were shown to unwind rna and dna duplexes in a -to- direction with respect to the single-stranded rna they initially bind to (ivanov and ziebuhr ; ivanov et al. a; seybert et al. ; tanner et al. ) . translocation of nsp along rna (and concomitant duplex unwinding) is fueled by ntp or dntp hydrolysis. consistent with many other helicases, the nsp -associated ntpase/dntpase activity is stimulated by nucleic acids (heusipp et al. ) . additionally, nsp exhibits rna -triphosphatase activity which was proposed to catalyze the first step of the -capping reaction of viral rnas (ivanov and ziebuhr ; ivanov et al. a) . the n-terminal part of nsp contains a -to- exoribonuclease (exon) domain. exon is related to the dedd superfamily of exonucleases (zuo and deutscher ) but carries an additional putative zn + -finger structure that is inserted between the conserved motifs ii and iii . the exon activity of sars-cov nsp was demonstrated in vitro and shown to be specific for single-stranded and double-stranded rna (minskaia et al. ) . the protein was shown to require metal ions for activity and isothermic titration calorimetry data suggest that nsp binds two mg + ions per molecule (chen et al. a) . this data, together with the profound reduction of activity upon substitution of putative metal ion-coordinating residues (minskaia et al. ) , suggests that catalysis occurs through a two-metal-ion mechanism similar to that used by many cellular enzymes mediating phosphoryltransfer reactions (beese and steitz ) . it has been proposed that coronaviruses and other nidoviruses with genome sizes of about kb have evolved specific mechanisms to replicate their large rna genomes. the argument is that, in the absence of such mechanisms, the intrinsically error-prone rdrps of rna viruses would cross a (postulated) threshold of nucleotide misincorporations above which the survival of a given virus population becomes impossible. the question of how coronaviruses are able to maintain their exceptionally large rna genomes has not been resolved but a number of recent observations suggest that nsp was critically involved in the evolution of these large genomes. first, coronaviruses were shown to encode a -to- exon that is related to cellular enzymes involved in proof-reading mechanisms during cellular dna replication. second, the exon activity is not conserved in nidoviruses with much smaller genomes (i.e., arteriviridae), even though the pp a/pp ab domain organization is otherwise very well conserved among small and large nidoviruses snijder et al. ) . third, exon activity was found to be essential for hcov- e replication in cell culture (minskaia et al. ) . transfection of genome-length rnas containing substitutions of exon active-site residues failed to produce viable virus. northern blot analysis of viral rna in transfected cells revealed a severe reduction in genome replication, an altered molar ratio of sgrnas and aberrant migration of two sgrnas in some of the mutants (minskaia et al. ) . consistent with these data, deletion of the exon domain from a sars-cov replicon was reported to abolish rna synthesis and substitution of a putative exon active-site residue reduced genome replication and transcription about -fold in this system (almazan et al. ) . fourth, the characterization of mhv-a exon active-site mutants revealed defects in viral rna synthesis and rapid accumulation of mutations across the genome (eckerle et al. ). the authors calculated that, during passage (and under strong selection pressure), mhv-a exon mutants accumulated approximately -fold more substitutions than the wild-type virus. the data obtained in these studies are consistent with the proposed role of exon in increasing the fidelity of the coronavirus rdrp, although there is still no direct proof for this specific function. coronaviruses encode a second conserved ribonuclease, nendou (nidoviral endoribonuclease, specific for u), which is located within nsp . the domain is conserved not only in coronaviruses but also in all other members of the order nidovirales. nendou homologs could not be identified in any other rna virus, which makes the domain a genetic marker of nidoviruses (ivanov et al. b ). sars-cov nsp was shown to be an endoribonuclease that cleaves preferentially of uridylates and generates - cyclic phosphate ends (bhardwaj et al. ; ivanov et al. b ). the activity is significantly enhanced by mn + while addition of mg + or ca + only had minor effects on activity. the structure of sars-cov nsp revealed a novel fold (see chap. ). in spite of unrelated structures and lack of sequence similarity, nsp and rnasea were proposed to use the same catalytic mechanism (ricagno et al. ) . residues forming the catalytic triad of bovine rnase a (his- , lys- , his- ) could be superimposed with his/lys residues known to be critical for activity of sars-cov nsp (ivanov et al. b ) and both rnasea and sars-cov nendou generate - cyclic phosphate ends, suggesting that endoribonucleolytic cleavage by nendou proceeds through the same catalytic mechanism (ricagno et al. ). the proposed rnasea-like catalytic mechanism does not involve metal ions and therefore the observed stimulatory effects of mn + on nendou activities of several (but not all) coronaviruses (bhardwaj et al. ; cao et al. ; ivanov et al. b; xu et al. ) cannot readily be reconciled with a role in catalysis. based on intrinsic tryptophan fluorescence data, bhardwaj and co-workers ( ) suggested that mn + ions induce specific conformational changes in the protein that might promote its nuclease activity. however, the available crystal structure information does not provide evidence for the presence of mn + ion-binding sites in any of the coronavirus nendous studied (ricagno et al. ; xu et al. ). more recently, it was suggested that mn + ions increase the rna-binding activity of nendou (bhardwaj et al. ) . coronavirus nendous form hexamers consisting of a dimer of trimers in crystals and in solution (see chap. ) . amino acid substitutions that interfere with hexamerization were reported to reduce the nucleolytic activity and rna affinity of sars-cov nendou, suggesting that hexamerization is critically involved in activity (guarino et al. ) . however, the presence of six independent active sites in the nendou crystal structure, together with relatively minor differences in the k m and k cat values determined for monomeric and hexameric mhv nendou (xu et al. ) and the fact that maltose-binding protein-nendou fusion proteins that are unable to form hexamers possess endoribonucleolytic activity (ivanov et al. b) suggest that hexamerization may not be essential for activity. it therefore remains possible that nendou may be active prior to its proteolytic release from pp ab, for example in the context of nendou-containing processing intermediates or the fulllength polyprotein. the role of nendou in the coronavirus life cycle is not well understood. reverse genetics data obtained for hcov- e (ivanov et al. b ) and mhv (kang et al. ) suggest that nendou activity is not essential for viral replication. substitutions of nendou active-site his and lys residues in mhv were shown to cause subtle defects in sgrna accumulation and reduce virus titers by about -fold. substitutions of a conserved asp residue abolished viral rna synthesis, both in hcov- e and mhv (ivanov et al. b; kang et al. ). both structural information and biochemical data suggest that this particular residue may have an important structural role, suggesting that the observed defects in viral replication may be due to misfolding of nsp or other pp a/pp ab subunits rather than caused by the lack of nendou activity. more studies are needed to understand the function of the conserved nendou activity in nidoviral replication. nendou belongs to a family of proteins that is prototyped by a cellular endoribonuclease, called xendou, from xenopus laevis (laneve et al. ) . xendou is a poly(u)-specific endoribonuclease that, together with exon and methyltransferase (mt) activities, is involved in the processing of intron-encoded small nucleolar (sno) rnas and site-specific rna methylation pathways in x. laevis oocytes (laneve et al. ) . it has been suggested (but not yet explored experimentally) that a similar set of activities associated with coronavirus nsp (exon), nsp (nendou) and nsp (mt), which are coexpressed in the c-proximal pp ab region, may be involved in related rna-processing and methylation pathways . the most c-terminal processing product of pp ab, nsp , has been proposed to be related to the rrmj/ftsj family of s-adenosyl-methionine-dependent ribose- -o-methyltransferases (feder et al. ; snijder et al. ) . the predicted mt activity was recently confirmed for nsp of feline coronavirus (fcov) (decroly et al. ) . fcov nsp was shown to methylate me gpppac n at the ribose- -o moiety of the adenosine, converting a cap- to a cap- structure. the domain is critically involved in coronavirus replication as deletion or ablation of expression of nsp abolished rna synthesis in sars-cov (almazan et al. ) and hcov- e (hertzig, schelle and ziebuhr, unpublished data) while substitution of one of the residues forming the catalytic tetrad reduced sgrna synthesis about -fold in a sars-cov replicon system (almazan et al. ). the -cap structure is known to be critically important for the stability of cellular mrnas and translation initiation (reviewed in shuman ) . the cellular capping apparatus is located in the nucleus, implying that viruses that replicate in the cytoplasm need to either provide all the enzymes required to produce rna cap structures or employ alternative mechanisms, such as cap snatching (plotch et al. ) . in eukaryotic cells, the production of me gpppg ome n ( ome) cap- (and cap- ) structures is achieved through four consecutive enzymatic reactions: ( ) removal of the rna g-phosphate by an rna -triphosphatase, ( ) transfer of gmp to the remaining diphosphate end by a guanylyltransferase, ( ) methylation of the guanine at the n position by a guanine-n -methyltransferase, resulting in a cap- structure, ( ) methylation of ribose- -o-moieties of the first (and second) nucleotide of the mrna by a ribose- -o-methyltransferase, resulting in cap- and cap- structures, respectively (langberg and moss ; shuman ) . coronaviral rnas are modified at the -end, probably with a cap structure (lai et al. ) , and it seems reasonable to suggest that the nsp -associated ribose- -o-methyltransferase activity catalyzes the conversion of cap- into cap- structures whereas the nsp -associated rna -triphosphatase activity might catalyze the first step of -cap formation (ivanov and ziebuhr ; ivanov et al. a ). homologs of cellular guanylyltransferase and guanine-n -methyltransferases have not been identified in coronaviruses and it remains to be seen what viral or cellular proteins/activities mediate the two remaining reactions, namely gmp transfer and guanine-n methylation, to synthesize rna cap structures on coronavirus rnas. while some + rna viruses, such as alphaviruses, encode all four required activities (reviewed in salonen et al. ) , other viruses use one and the same domain to perform two different reactions. for example, the west nile virus mt methylates at both the guanine n position and the ribose- -o moiety using an interesting substrate-repositioning mechanism and a single s-adenosylmethionine-binding site (dong et al. ). while fcov nsp had no guanine-n -methyltransferase activity and did not bind unmethylated gpppac n (decroly et al. ) the authors point out that recognition of unmethylated, guanylylated rna might depend on regulatory rna elements located further downstream in the rna, similar to what was described for flaviviruses (dong et al. ; ray et al. ). the emergence of sars-cov led to a renewed interest in coronavirology and recent years saw a significant increase in research involving this family of viruses. many of the previously predicted coronavirus replicase gene-encoded enzyme activities were characterized by biochemical and structural approaches using viral proteins expressed in heterologous systems (ziebuhr ) . in a few cases, structural studies informed subsequent biochemical studies, resulting in the identification of rna-binding domains and other functions (egloff et al. ; joseph et al. ; zhai et al. ) . most of the biochemical and structural studies reported over the past years involved isolated nsps or subdomains of these proteins rather than multidomain complexes. although these studies provided invaluable new insight into structurefunction relationships of many of these proteins, there is hardly any information regarding the quarternary structure(s) and subunit compositions of replicase and/or transcriptase complexes catalyzing specific reactions during viral rna synthesis. for example, very little is known about the special factors and macromolecular interactions involved in discontinuous minus-strand rna synthesis, a unique feature of coronaviruses and several other nidoviruses. similarly, the replication and maintenance of the exceptionally large coronavirus genome is likely to involve the concerted action of replicase gene-encoded nsps, possibly including processing precursors and intermediates as pointed out above. the physical and functional interactions between the various components of the replicase-transcriptase probably undergo significant changes in the course of the viral replication cycle, further complicating the characterization of these complexes. despite these major technical challenges, recent studies increasingly try to elucidate the functions and structures of complexes involving two or more proteins (zhai et al. ) . also, the availability of reverse genetics systems for several coronaviruses has greatly facilitated the characterization of specific protein functions and critical interactions between domains encoded by very different regions of the replicase gene (donaldson et al. ). the recently established method for purification of entire functional (membranebound) rtcs (van hemert et al. ) presents exciting new possibilities to study coronavirus rna synthesis. advanced imaging techniques including cryo-electron microscopy have provided fascinating new insight into key structures involved in virus replication. for example, cryo-electron microscopy of ribosomes stalled at a specific stage of frameshifting provided a glimpse of how the structure of the mrna template affects ribosome function to mediate this frameshift (namy et al. ) , and electron tomography of sars-cov-infected cells provided a d view of the unique continuous reticulovesicular network, the formation of which is induced by the virus . however, much remains to be studied to understand how the virus coaxes the cell to produce these structures. the precise role of these membrane structures in virus replication and, possibly, immune evasion need to be characterized in more detail. further progress has been made in our understanding of the interactions of the virus with the host cell, especially with regard to innate immune responses to coronavirus infections and the role of specific viral proteins in counteracting these antiviral host responses. the understanding of these pathways in combination with 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how to make sense a unique cap(m g pppxm)-dependent influenza virion endonuclease cleaves capped rnas to generate the primers that initiate viral rna transcription identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins adp-ribose- -monophosphatase: a conserved coronavirus enzyme that is dispensable for viral replication in tissue culture identification of protease and adp-ribose -monophosphatase activities associated with transmissible gastroenteritis virus non-structural protein severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme west nile virus -cap structure is formed by sequential guanine n- and ribose '-o methylations by nonstructural protein crystal structure and mechanistic determinants of sars coronavirus nonstructural protein define an endoribonuclease family characterization of a novel coronavirus associated with severe acute respiratory syndrome structural basis of severe acute respiratory syndrome coronavirus adp-ribose- -phosphate dephosphorylation by a conserved domain of nsp viral rna replication in association with cellular membranes coronavirus minus-strand rna synthesis and effect of cycloheximide on coronavirus rna synthesis a contemporary view of coronavirus transcription selective replication of coronavirus genomes that express nucleocapsid protein processing of the coronavirus mhv-jhm polymerase polyprotein: identification of precursors and proteolytic products spanning kilodaltons of orf a characterization of white bream virus reveals a novel genetic cluster of nidoviruses nuclear magnetic resonance structure of the n-terminal domain of nonstructural protein from the severe acute respiratory syndrome coronavirus the human coronavirus e superfamily helicase has rna and dna duplex-unwinding activities with -to- polarity a complex zinc finger controls the enzymatic activities of nidovirus helicases viral and cellular proteins involved in coronavirus replication structure, mechanism, and evolution of the mrna capping apparatus non-structural proteins and interact to modify host cell membranes during the formation of the arterivirus replication complex unique and conserved features of genome and proteome of sarscoronavirus, an early split-off from the coronavirus group lineage coronavirus mrna synthesis involves fusion of non-contiguous sequences genetic analysis of murine hepatitis virus nsp in virus replication dodecamer structure of severe acute respiratory syndrome coronavirus nonstructural protein nsp deubiquitination, a new function of the severe acute respiratory syndrome coronavirus papain-like protease? the nsp replicase protein of sars-coronavirus ph-dependent conformational flexibility of the sars-cov main proteinase (m(pro)) dimer: molecular dynamics simulations and multiple x-ray structure analyses the "sars-unique domain" (sud) of sars coronavirus is an oligo(g)-binding protein the severe acute respiratory 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implications for substrate binding and antiviral drug design the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor design of widespectrum inhibitors targeting coronavirus main proteases insights into sars-cov transcription and replication from the structure of the nsp -nsp hexadecamer structure of the main protease from a global infectious human coronavirus, hcov-hku plp , a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production the coronavirus replicase coronavirus replicative proteins the coronavirus replicase gene: special enzymes for special viruses characterization of a human coronavirus (strain e) c-like proteinase activity virus-encoded proteinases and proteolytic processing in the nidovirales the autocatalytic release of a putative rna virus transcription factor from its polyprotein precursor involves two paralogous papain-like proteases that cleave the same peptide bond exoribonuclease superfamilies: structural analysis and phylogenetic distribution coronavirus non-structural protein is a major pathogenicity factor: implications for the rational design of coronavirus vaccines key: cord- -va ft we authors: taguchi, fumihiro title: coronavirus receptors date: journal: experimental models of multiple sclerosis doi: . / - - - _ sha: doc_id: cord_uid: va ft we the major receptor for murine coronavirus, mouse hepatitis virus (mhv), is identified as a protein, cell-adhesion molecule in the carcinoembryonic antigen family (ceacam ), which is classified in the immunoglobulin superfamily. there are four ceacam isoforms, with either four or two ectodomains, resulting from an alternative splicing mechanism. ceacam is expressed on the epithelium and in endothelial cells of a variety of tissues and hemopoietic cells, and functions as a homophilic and heterophilic adhesion molecule. it is used as a receptor for some bacteria as well. the n terminal domain participates in mediating homophilic adhesion. this domain is also responsible for binding to the mhv spike (s) protein; the cc’ face protruding in this domain interacts with an n terminal region of the s protein composed of amino acids (called s n ). the binding of ceacam with mhv s protein induces s protein conformational changes and converts fusion-negative s protein to a fusion-positive form. the allelic forms of ceacam found among mouse strains are thought to be an important determinant for mouse susceptibility to mhv. the coronavirus family includes a number of different viruses that infect a variety of animal species, causing numerous diseases, mainly in organs of the enteric, respiratory and central nervous systems. they are chapter ci o classified into three distinct groups in terms of serological cross-reactivity and sequence homology. group consists of porcine transmissible gastroenteritis virus, human coronavirus (hcov) e, feline infectious peritonitis virus and so on. group ii includes mouse hepatitis virus (mhv), hcov-oc , bovine coronavirus and some others. group iii is comprised of avian coronaviruses; infectious bronchitis virus and turkey coronavirus. all of these viruses infect animals in a highly species-specific fashion, although some of them can experimentally infect animals different from their natural hosts. the receptor protein for group i viruses has been revealed to be an aminopeptidase n, while the receptor for mhv in group ii is a protein classified in the immunoglobulin (ig) superfamily. the receptors for other viruses in group ii, as well as for viruses in group iii, have not yet been identified. in this chapter, i describe the receptor for mhv as well as the interaction of mhv receptor and the virus spike (s) protein. the receptors of other coronaviruses can be found in a review article ( ). . discovery of mhv receptor proteins mhv infects mice, but few other species. the major target organs are the liver, intestine and central nervous system. this host-species specificity or organ tropism of mhv has been thought to be determined mainly by the cellular receptor for mhv. a series of studies carried out by kay holmes and her colleagues, which began with analysis of differential susceptibility to mhv infection among mouse strains, has led to the finding of a major mhv receptor molecule. boyle et al. found that the plasma membranes isolated from mhv-susceptible balb/c mouse hepatocytes or enterocytes contained a to -kda protein that binds to mhv particles, but those derived from mhv-resistant sjl mice lacked such a protein ( ). this finding suggested that the difference in mhv susceptibility among mouse strains is determined by this protein, presumably the mhv receptor. by using monoclonal antibody (mab) cc- specific to - kda protein from balb/c, they purified a protein of ca. kda and determined the amino acid sequence in its n terminal region, from which the ll -kda protein was postulated to be a glycoprotein classified in the carcinoembryonic antigen (cea) family ( , ). finally, they isolated a gene encoding this protein, which was revealed to be cell adhesion molecule in the cea family of the ig superfamily [formerly called biliary glycoproteinl (bgpl) and currently termed ceacam ] ( ). mhv non-permissive bhk cells transfected with this gene were converted to mhv-susceptible cells, indicating that this molecule is the receptor for mhv. it was also found that mhv-resistant sjl mice express a homologous protein ( , ). two other species of glycoprotein, bgp ( ) and pregnancy-specific glycoprotein ( ), both of which belong to cea family members, were thereafter found to serve as the mhv receptor in mouse species. however, none of these are as highly efficient as ceacam in terms of receptor functionality or receptor utility by mhv strains. human cea glycoprotein works as an mhv receptor as well ( ). ceacam is a member of the ig superfamily and its prototypical -kda glycoprotein consists of four ectodomains (in the order of n, a , b and a from the n terminus), a transmembrane region (tm) and a cytoplasmic tail (cy) (fig. , ). the n domain is similar to an igvariable domain, and the three other domains resemble a c ig-constant domain. four different isoforms of ceacam are known to exist, and have been produced by alternative splicing (fig. ) . two of the isoforms have ectodomains and the other two have domains, consisting of an n terminal and a domains, one of which has either a short or long cy. the two-domain protein is to kda in size. ceacam has two allelic forms, ceacam a and ceacam b (fig. ) . the former is expressed in most laboratory mouse strains, while the latter, insofar as is currently known, is expressed only in mhv-resistant sjl mice ( ) . in wild mice, however, both of those forms are widely distributed ( ) . the major structural differences between ceacam a and ceacam b lie in the n domain, which differs in of its amino acids ( , ). ceacam a is -to -fold higher than ceacam b in terms of receptor function ( , ) . there is no apparent difference in virus-binding activity as examined by a neutralization test between the -ectodomain isoform and the domain ceacam a ( ) . however, mice deleting the -domain ceacam a and expressing the -domain isoform alone are more resistant to mhv than those expressing both of the -and -domain isoforms ( ) . thus, there could be a difference between them in terms of mhv receptor function in mice. on the contrary, ceacam b isoform containing domains neutralizes mhv-a strain more efficiently than does the isoform containing domains (n and a domains), while both of these isoforms showed similar neutralization activity to mhv-jhm strain ( ), suggesting a virus-strain specificity in the interaction with ceacam . the n domain is responsible for receptor function ( ) . since the ceacam splice variant deleting the a and b domains is functional, then it is evident that these domains are not necessary for receptor function. the ceacam isoform containing the n and second a domains is also • )))))))))))))))))))~)i functional, indicating that the fourth a domain is not absolutely critical. although ceacam consisting of n domain alone bound mhv, it did not work as a functional receptor when expressed on ceacaml-negative cells ( ) . however, the chimeric ceacam having the n domain linked to the mouse poliovirus receptor homolog deleting n domain that has a binding specificity to poliovirus served as a functional receptor for mhv ( ) . as a result of these reports, it is believed that the n domain alone is sufficient for receptor function; however, when expressed on the cell surface, ceacam containing the n domain alone was buried among the various molecules expressed on the cell surface because of its shortness and hence it failed to bind to viruses ( ) . by using a soluble form of ceacam , we have recently found that ceacam with n domain alone converted mhv s protein from a fusion-negative to fusion-positive form ( ) . collectively, the n domain is sufficient for the mhv receptor function. detailed analysis using n domain deletion mutants of ceacam showed that a stretch composed of amino acids (aa to ) in the n domain is particularly important for receptor function ( ) . the stretch is located in the cc' loop in the n domain composed by -strands and is supposed to protrude from the n domain ( , ) . the difference in receptor function observed between ceacam a and ceacam b results from a -amino-acid difference in the above-described amino acids in the n domain ( ) . ceacam is reported to be distributed in various cells in different organs, not only in the target organs of mhv, but also in those in which mhv infection has not been detected. a high level of ceacam expression is reported on epithelium and endothelial cells of a variety of tissues, and in hemopoietic cells, such as monocytes, macrophages, granulocytes and their precursors, the b cells, activated t cells and thymic stromal cells ( , ) . it is also demonstrated on both apical membranes of epithelial cells as well as on sites of cell-cell contact (e.g., between hepatocytes, stratified epithelia, junctional epithelium that forms a transition zone between gingival epithelium and teeth, and between pericytes and endothelial cells of blood vessel walls). furthermore, during early mouse embryonic development, ceacam is abundantly expressed in endodermal and mesenchymal derivatives ( ), but is not detected by immune histochemistry in any type of cells in the brain ( ). however, mhv infection in the brain was blocked by anti-ceacam mab cc- ( ). also, a ceacam isoform with domains was detected by rt-pcr in the brain ( ). these findings suggest that the ceacam molecule is expressed, albeit in very small amounts, in some cell populations of the brain. the major biological function of ceacam is cell adhesion. it serves as both a homophilic and heterophilic adhesion molecule ( ) . homophilic adhesion, confirmed by in vitro studies of rodent and human ceacam , is thought to be important in the embryonic organization of the intestinal epithelium and liver hepatocytes, in placental trophoblasts, during muscle and tooth development and vascularization of the central nervous system ( ). ceacam also plays an important role in neutrophil activation and adhesion during inflammatory responses ( ), lymphoregulation and immunosurveillance ( ), angiogenesis ( ), and the negative regulation of cell proliferation ( , ) . heterophilic adhesion of ceacam to other ceacam family members has been shown ( ). also, heterophilic adhesion to opa proteins of neisseria gonorrhoeae, neisseria meningitis and haemophilus influenzae mediates their infections ( , ), indicating that ceacam is a receptor for those bacteria. this also facilitates bacterial colonization of the gut and bacterial phagocytosis by neutrophils and is involved in the initial tethering of granulocytes to e-selectin on the endothelium prior to their transendothelial migration during inflammatory responses. it was recently shown that homophilic adhesion of ceacam involves n-terminal domain interactions. the gfcc'c" face of the n domain, which includes the mhv binding site cc' region, is responsible for homophilic interaction ( ) . and mhv s the mhv s protein comprising a petal-like projection on virion surface is the ligand for the ceacam molecule. the projection is composed of two or three molecules of the s -$ heterodimer derived from s protein. s protein is a type i glycoprotein. it is synthesized and cotranslationally glycosylated as a -kda, protein, becomes as - kda protein after modification of glycans and cleaved by a host-derived proteinase into two subunits, n terminal s and c terminal $ ( ). s comprises an outer knob-like structure of the spike, and $ consists of the stem-like part beneath the knob ( ). s and $ units are associated by non-covalent linkage, and they can be easily dissociated from each other by denaturing reagents or even during a purification process. alpha-helices constructed by the heptad repeats in the $ play an important role for oligomerization of s -$ heterodimers, though there is another determinant in the s for oligomerization ( ). following its synthesis, the s protein is incorporated into the envelope of viral particles after interaction with viral integral membrane protein in the internal compartments from endoplasmic reticulum to the golgi apparatus. the s protein is also transported to the plasma membrane. the n terminal region in the s composed of amino acids (s n ) is responsible for binding to ceacam ( ) . among s proteins of a variety of mhv strains, there are three conserved regions in s n (s n - , -ii and -iii) composed of or more identical aminoacid stretches ( ) . site-directed mutagenesis analysis suggested that two of these regions, s n -i and -ii, located far from one another, are involved in receptor binding ( ) . studies using mhv variants containing mutations in s n -i confirmed the significance of this region in receptor binding ( ) . s n -iii was recently suggested to be responsible for virus entry into the cell in combination with a region in the $ ( ). denaturing of s n abolished the receptor-binding activity, indicating that the tertiary structure composed of different regions in the s n or/and the dimerization of s which takes place within s n ( ) is important. the interaction of mhv s protein and ceacam leads to the s protein functional conversion from a fusion-negative to a fusion-positive form ( ) . recently, it was also shown that this functional activation is accompanied with conformational changes in the s protein; receptor-bound s protein has a fraction resistant to proteinase digestion, while receptorunbound s protein is susceptible ( ) . these functional and structural changes of the s protein greatly resemble those of the envelope protein of retroviruses that take place after they bind to their receptors ( , ) , suggesting that mhv enters cells in a fashion similar to that of retroviruses. a number of investigators have reported that balb/c, c bl and most other mouse strains, later revealed to have a ceacaml ~ ( ~) gene, are susceptible, while sjl mice with a ceacaml t' ( b) gene are resistant ( , ) . genetic analyses indicated that a single dominant gene located on chromosome is responsible for susceptibility to mhv ( ) . the ceacaml could be a gene determining susceptibility, since ) the a makes mice susceptible and ) ceacaml is also mapped on chromosome ( ). the expression of either i a or b in ceacaml-negative cells converted them to mhv susceptible, suggesting that allelic differences in receptor proteins were not sufficient to explain the differences in mouse susceptibility to mhv ( , ). in detailed studies, however, cells transiently expressing a were to times more sensitive to mhv than were cells expressing b ( , ) , indicating a small, but significant difference between a and b. this also suggested that the mhv receptor expressed in sjl is still functional. if the receptor allele controls mhv susceptibility, then sjl should be relatively, but not completely, resistant to mhv. sjl mice are, in fact, resistant to mhv when challenged with a low dose of virus, but susceptible when inoculated with a high dose of virus ( , ) . these observations suggest that ceacaml is a gene controlling mhv susceptibility. of mice of (balb/c x sjl) f and backcrossed mice to sjl, all mice with ~/ ~ and u/ b genotypes were susceptible, while all mice with lb/ b genotype were resistant after infection with a low dose of virus ( ) . this implies the mhv receptor gene and mhv-susceptibility gene are identical, and if not, they are located within . cm on chromosome . to finally examine whether the receptor gene is identical to the gene that controls mhv susceptibility, gene replacement is a useful strategy. the mhv susceptibility of balb/c mice in which la/ a is replaced by lb/ b and sjl mice in which lb/ b is replaced with i"/u will conclusively establish whether the mhv receptor gene is the gene which controls the mhv susceptibility of mice. redefined nomenclature for members of the carcinoembryo-nic antigen family mouse susceptibility to mouse hepatitis virus infection is linked to viral receptor genotype a study on mouse hepatitis virus receptor genotype in the wild mouse identification of a contiguous -residue determinant in the mhv receptor that controls the level of virion binding to cells difference in virus-binding activity of two distinct receptor proteins for mouse hepatitis virus purified, soluble recombinant mouse hepatitis virus receptor, bgpl(b), and bgp murine coronavirus receptors differ in mouse hepatitis virus binding and neutralizing activities targeted disruption of the ceacaml (mhvr) gene leads to reduced susceptibility of mice to mouse hepatitis virus infection mouse hepatitis virus strain a and blocking antireceptor monoclonal antibody bind to the n-terminal domain of cellular receptor mouse hepatitis virus receptor activities of an mhvr/mph chimera and mhvr mutants lacking nlinked glycosylation of the n-terminal domain mutational analysis of the virus and monoclonal antibody binding sites in mhvr, the cellular receptor of the murine coronavirus mouse hepatitis virus strain a homophilic adhesion of human ceacam involves n-terminal domain interactions: structural analysis of the binding site crystal structure of murine sceacamla [ , ]: a coronavirus receptor in the cea family localization of neutralizing epitopes and the receptor-binding site within the amino-terminal amino acids of the murine coronavirus spike protein analysis of the receptor-binding site of murine coronavirus spike protein identification of spike protein residues of murine coronavirus responsible for receptor-binding activity by use of soluble receptorresistant mutants communication between s n and a region in $ of murine coronavirus spike protein is important for virus entry into cells expressing ceacam i b receptor soluble receptor potentiates receptor-independent infection by murine coronavirus receptor-induced conformational changes of murine coronavirus spike protein soluble receptor -induced retraviral infection of receptordeficient cells hiv entry and its inhibition resistance to fatal central nervous system disease by mouse hepatitis virus, strain jhm. . genetic analysis control of mouse hepatitis virus replication in macrophages by a recessive gene on chromosome mouse hepatitis virus type (jhm strain)-induced fatal central nervous system disease. . genetic control and the murine neuron as the susceptible site of disease key: cord- -qmx umk authors: barthold, stephen w.; smith, abigail l. title: response of genetically susceptible and resistant mice to intranasal inoculation with mouse hepatitis virus jhm date: - - journal: virus research doi: . / - ( ) -x sha: doc_id: cord_uid: qmx umk abstract mouse hepatitis virus (mhv)-jhm infection was studied in genetically susceptible (balb/cbyj) and resistant (sjl/j) mice following intranasal inoculation at , , or wk of age. markers of infection included histology, immunohistochemistry, virus quantification and virus serology. all bale mice developed severe disseminated disease with high mortality due to encephalitis and hepatitis. peak mhv titers appeared in brain, liver, spleen and intestine on days or . age at inoculation did not influence virus titers in brain, spleen or intestine, but virus titers in liver were inversely proportional to age at inoculation. in -wk-old bale mice, virus was cleared from spleen, intestine and liver by day and from brain by day . in intestine, mhv was localized to lymphoid tissue, without fecal excretion. sjl mice of all ages developed remarkably milder disease with low mortality occurring only among mice inoculated at wk of age. sjl mice inoculated at wk had disseminated infection at day , but lesions and antigen were cleared from most organs by day . mice inoculated at and wk of age had minimal or no involvement of peripheral organs, and mice inoculated at wk of age had infections restricted to the nose. at day , mhv titers in brain, liver, spleen and intestine were significantly lower or undetectable in sjl mice of all ages compared to age-matched balb mice. in -wk-old mice, mhv was cleared from all organs by day . serum antibody titers to mhv were many-fold higher in balb mice, compared to sjl mice, which mounted only a modest response. mouse hepatitis virus (mh~-jh~ infection was studied in genetically susceptible (balb/cby~ and resistant (sjl/j) mice follo~ng intranasal inoculation at , , or wk of age. markers of infection included histology, immuno~stochemistry, virus quantification and virus serology. all balb mice developed severe disseminated disease with high mortality due to encephalitis and hepatitis. peak mhv titers appeared in brain, liver, spleen and intestine on days or . age at inoculation did not influence virus titers in brain, spleen or intestine, but virus titers in liver were inversely proportional to age at inoculation. in -wk-old balb mice, virus was cleared from spleen, intestine and liver by day and from brain by day . in intestine, mhv was localized to lymphoid tissue, without fecal excretion. sjl mice of all ages developed remarkably milder disease with low mortality occurring only among mice inoculated at wk of age. sjl mice inoculated at wk had disseminated infection at day , but lesions and antigen were cleared from most organs by day . mice inoculated at and wk of age had minimal or no involvement of peripheral organs, and mice inoculated at wk of age had infections restricted to the nose. at day , mhv titers in brain, liver, spleen and intestine were significantly lower or undetectable in sjl mice of all ages compared to age-matched balb mice. in -wk-old mice, mhv was cleared from all organs by day . serum antibody titers to mhv were many-fold higher in balb mice, compared to sjl mice, which mounted only a modest response. since its initial isolation from the brains of mice with posterior paresis (cheever et al., ) , the jhm strain of mouse hepatitis virus (mhv) has received considerable attention as a neurotropic mhv strain that experimentally induced encephalitis and demyelination in mice and rats (bailey et al., ; goto et al., ; knobler et al., knobler et al., , sorenson et al., ; stohlman et al., ; wege et al., wege et al., , weiner et al., ) . although its neurotropic properties have been emphasized, mhv-jhm is similar to several other mhv strains and isolates, with tropism for not only brain, but also many other tissues in susceptible hosts (barthold and smith, ; barthold ) . strains such as mhv-s and mhv-a also produce brain lesions similar to mhv-jhm (barthold et al., ; barthold and smith, ; koolen et al., ; taguchi et al., ; woyciechowska et al., ) . it is now becoming apparent that different mhv strains, like coronaviruses of other species (wege et al., ) , are either primary upper-respiratory or enteric pathogens in mice. in hosts that are more susceptible by virtue of immaturity, genotype, or impaired lymphoreticular function, respiratory mhv strains are likely to disseminate secondarily to multiple organs, but enterotropic mhv strains tend to be more restricted to enteric mucosa (barthold, ; barthold et al., ) . commonly studied, protot~e mhv strains ( , , a , jhm, s), are primary respiratory viruses in the mouse. since most studies with mhv-jhm have utilized artificial, usually intracerebral (i.c.), routes of inoculation with emphasis on neurotropism, this study was initiated to investigate the full spectrum of effects induced by this virus following a natural (intranasal) route of inoculation. genotype and age are important determinants in mhv disease (wege et al., ; barthold, ; bang, ) and were thus incorporated into this investigation. these results will provide a clearer understanding of the pathogenesis of this frequently studied mhv strain, as well as of respiratory mhv strains in general. mouse strains were selected for known susceptibility (balb/cbyj) or resistance (sjl/j) to i.c. inoculation with mhv-jhm (knobler et al., b (knobler et al., ,c, stohlman and frelinger, ) . pilot studies confirmed this dichotomy in susceptibility following intranasal (in.) inoculation. resistance of various mouse genotypes to different mhv strains, including mhv-jhm, has been shown to evolve between and wk of age (bang, ; gallily et al., ; pickel et al., ; taguchi et al., taguchi et al., , b . resistance of sjl mice to i.c. inoculation with mhv-jhm further evolves between and wk of age (stohlman et al., (stohlman et al., , . thus, mice were inoculated with mhv-jhm at , , and wk of age. sufficient numbers of mice were inoculated to obtain groups of - mice of each genotype and of each age group at days , , , and after inoculation. additional groups of balb mice exposed at wk of age were collected at days and after inoculation. within age groups, mice were selected for necropsy on these days using a table of random numbers. day was chosen as a peak interval for mhv infection (barthold and smith, ) , and other intervals were selected to examine the early phase (day ) and recovery or chronic phases (days - ) of infection. markers of infection included immunohistochemistry on tissues from all major organs, virus quantification in selected target tissues and serum antibody to mhv-jhm. in open cages in the animal room during the course of these studies. they were tested periodically for serum mhv antibody to ensure that proper containment of experimental mhv was effected and that adventitious mhv had not been introduced to the animal room. mice from both commercial sources were mhv-free. mice were killed with carbon dioxide gas and exsanguinated by cardiac puncture. tissues were frozen at - o c until tested for mhv infectivity, or were placed in % neutral buffered formahn (ph . ) for immunohistochemistry. mhv-jhm was obtained from the american type culture collection, bethesda, maryland, passaged twice in nctc cells, once in adult balb/cbyj brain and once in cl cells (sturman and takemoto, ) and frozen in aliquots at - o c until used. all mice were inoculated twice i.n. with ~ of cell-free culture fluid containing tcids of mhv-jhm. unlike other routes of inoculation, disease severity is not affected by higher i.n. doses of mhv beyond the infectious dose (barthold et al., ) . because of the relative insensitivity of cell culture for mhv detection, infant mouse infectivity assays were used for mhv quantification in target tissues. the logi ld,, per g of tissue was determined for brain, liver, spleen and intestine at day after inoculation in mice of both genotypes and all age groups and at days , , , , and after inoculation in mice of both genotypes inoculated at wk of age. in addition, virus in brains of balb mice exposed at wk of age and collected on days and after inoculation was titrated. tissues were thawed, weighed and diluted % (w/v) in dulbecco's minimal essential medium containing % fetal bovine serum. they were homogenized and clarified in a refrigerated centrifuge at rpm for min. serial lo-fold dilutions of supernates in . ml vol were inoculated i.c. into -day-old suckling swiss pups. four pups were inoculated per dilution and endpoint mortality was established at h after inoculation. the log,, ld,, per g of tissue was calculated using the method of reed and muench ( ) . means were calculated as geometric means. virus titers were compared between organs or groups using the student's paired t-test (same animal) or unpaired t-test. linear regression was utilized to analyze the relationship of virus titers in a target organ with age (steel and torrie, ) . formalin-fixed tissues were paraffin embedded, sectioned at - pm and examined for histopathology. mhv antigen was detected using an avidin-biotin peroxidase complex method, counterstained with hematoxylin, as previously described (barthold, ) . hyperimmune mouse ascitic fluid was prepared in multiparous female swiss mice by once-weekly intraperitoneal injections of mhv-jhm infected infant mouse brain emulsified in freund's complete adjuvant. the following tissues were specifically examined for mhv antigen and lesions in mice of both genotypes, all ages and all intervals: nose, eye, brain, spinal cord, lung, liver, spleen, submaxillary and mesenteric lymph nodes, salivary glands, bone and bone marrow, small intestine, cecum, colon, kidney, urinary bladder and gonad. immunohis-tochem&ry was performed in batches, each of which included positive and negative antiserum and antigen controls. sera collected from mice inoculated at wk of age were tested at two-fold dilutions beginning at : (sjl) or : (balb) in an enzyme immunoassay (eia) to determine antibody titers (smith and winograd, ) . the antigen in the eia was formalin fixed, mhv-jhm-infected cl cells, and the detecting antibody was horseradish peroxidase-conjugated goat anti-mouse igg (biorad, richmond, ca) diluted : . after addition of abts substrate (kirkegaard and perry, gaithersburg, md), plates were read spectrophotometrically at nm. wells were considered positive if the mean od,,a for infected cells exceeded by three standard deviations the mean oddi for uninfected cells treated with the same serum dilution. /pathology balb/cbyj mice. mice of all age groups developed disseminated mhv infections on days and , with mortality rate of approximately % by day among mice inoculated at or more wk of age and % among mice inoculated at wk of age. encephalitic signs became pronounced on day after inoculation. viral antigen was accompanied by necrotizing inflammation and viral syncytium formation in nose, olfactory bulb, brain, spinal cord, bone marrow, spleen, lymph nodes, liver and gut-associated lymphoid tissue (fig. ) . the time of appearance, organ distribution and duration after inoculation of mhv antigen correlated identically with virus detection in tissues. in lung, viral antigen was sparse and associated with vascular endothelium. antigen was found sporadically in the eye, but generally not in genitourinary organs, bone, salivary glands or intestinal mucosa. in nose, lung and lymphoid tissues, mhv antigen was most pronounced on day , with diminution on day , and most pronounced on day in brain, liver and bone marrow. mortality was associated with encephalitis and hepatitis, since brain and liver were the most severely affected organs. with the exception of brain in mice inoculated at wk of age, no differences in lesion severity and/or distribution of mhv antigen were apparent among age groups. in mice inoculated at - wk of age, lesions and antigen extended from the nose to the olfactory bulb, then along the meninges and parenchyma of the anteroventral brain to the hippocampus and posterior brain stem. olfactory bulbs were infected at day , and encephalitis was most severe and widespread at day . neurons, glia and their cell processes contained mhv antigen (fig. ) . in other organs, lesions and antigen were more randomly distributed, suggesting hematogenous infection. lesions were often found adjacent to blood vessels. in liver, groups of hepatocytes, and to a lesser extent kupffer cells, contained antigen, often with necrosis and leukocytic infiltration (fig. ) . in spleen, antigen was most prevalent in lymphoid regions (fig. ) . mice inoculated at wk of age had a similar pattern of disseminated infection. however, lesions and antigen in brain were diffusely distributed, rather than restricted to olfactory pathways as seen in older mice. this suggested hematogenous infection of brain in the neonate. in surviving balb mice, lesions and antigen had largely disappeared from nose, lymphoid tissue, bone marrow, spinal cord, olfactory bulb and lung by day after inoculation. encephalitis and hepatitis in the presence of mhv antigen were less severe and associated with lymphocytic, and in brain, glial infiltrates. early spongiosis was present in the brain stem of some mice. by day , livers had residual mineralized scars without mhv antigen. encephalitis had also largely disappeared, but there was a high prevalance of spongiosis with gliosis and demyelination localized to brain stem. in these spongiform areas, antigen was present in a few glial cells (fig. ) . brains of most mice exposed at wk and examined days later had mild resolving spongiform lesions, and brains at days had no visible lesions. sjl mice of all ages had remarkably milder disease than balb mice. mice inoculated at wk of age experienced moderate mortality, which was heralded by encephalitic signs. lesions and antigen of -wk-old sjl mice resembled balb mice, and were found in nose, olfactory bulb, brain, liver, spleen, lymph nodes, bone marrow and gut associated lymphoid tissue (fig. ). lesions and antigen had cleared from most organs, with the exception of nose and brain, by day . no mortality occurred among sjl mice inoculated at or more wk of age. they had minimal visible lesions, with mild nasal involvement and sporadic, mild inflammation of olfactory bulbs, liver and lymphoid tissues on days and . on day , livers contained multiple microgranulomas, consisting of small nodular accumulations of macrophages and lymphocytes. viral antigen was restricted to a few kupffer cells at day . complete recovery was apparent by day in sjl mice of all age groups, including the surviving mice inoculated at wk of age. age and genotypic differences at day after inoculation virus titers in major target organs at day after inoculation revealed age and genotype-related differences (figs. , ) . among balb mice, virus reached equally high titers in brain and liver, and lower titers were detected in spleen and intestine. no statistically significant differences in virus titer could be detected in any organ between balb age groups. however, there was an inverse correlation between virus titers in liver and age at inoculation (p . , r = - . ). among sjl mice, age appeared to influence involvement of different organs. sjl pups infected at wk of age had highly variable virus titers in brain. sjl mice infected at wk of age had no or low titers of brain virus, and older groups were negative. these data suggested that wk of age represented a pivotal point in age-related resistance of brain to mhv-jhm infection. therefore, an additional group of -day-old sjl pups was inoculated. virus titers in their brains were uniformly high at days (range . to . , mean . k . log,, ld,,/g). livers were uniformly negative for detectable virus at day in all sjl age groups and only of livers from sjl pups inoculated at days of age had a low level of virus activity. virus was present in spleen and intestine in l-and -wk-old mice, intestine of -wk-old mice, but neither spleen nor intestine contained virus in -wk-old mice. since all organs were negative at day in -wk-old mice, they were tested for virus at days after inoculation and also found to be negative. nevertheless, viral antigen was present in the nasal mucosa of sjl mice from all age groups, including all of the -wk-old mice examined on day after inoculation. comparison of virus titers between genotypes at day in different organs revealed marked differences (fig. ) . with the exception of virus titers in brain of mice infected at wk of age, balb mice had higher titers of virus in brain and liver than sjl mice of corresponding age (p i . ). virus titers in spleen were consistently higher in balb compared to sjl mice in all age groups (p '< . for , wk; p i . for , wk). although intestinal virus titers were higher in land -wk balb mice compared to sjl mice, they were not statistically different. significant differences were found in intestinal virus titers between genotypes in the -and -wk age groups ( i . , < . , respectively). titers of mhv-jhm were determined in brain, liver, spleen and intestine at , , , , and days after inoculation of gwk-old balb and sjl mice (fig. ) . no virus was detected on day (within h after inoculation). in balb mice, virus was detectable in all organs by day . virus activity peaks at this interval in spleen and intestine. virus titers in brain and liver continued to rise through day , when mortality reached its zenith. virus was cleared from spleen and intestine between days and and from liver between days and . brains, however, had uniform but low virus activity through day . additional groups of balb mice examined at and days revealed no detectable virus in brain among mice at days or among mice at days. in sjl mice, low titers of virus were transiently detectable in liver and spleen on day and in intestine on day , but not other intervals. brain was not infected. virus titers on day in liver and spleen were lower in sjl mice than in balb mice (p < . liver, . spleen). mparison of mhv titers (log,, lds,/g) in selected tissues on day after mhv-jhm inoculation of l-, -, -or -wk-old balb/cbyj and sjl mice. because virus was frequently detected in intestine, but antigen was found only in gut associated lymphoid tissue, an attempt was made to confirm the location of virus activity in the intestine. an additional group of five -wk-old balb/cbyj mice was inoculated i.n. with mhv-jhm. on day after inoculation, the small intestine was dissected free of mesenteric tissue and its lumen flushed free of digesta with sterile saline. mesenteric lymph node, ileal wall with and without peyer's patches, and cecal feces were collected separately and assayed for virus. virus activity was localized to lymphoid tissue (mesenteric lymph node and peyer's patches), but was not detected in non-lymphoid ileal wall or feces (table ) . there was a marked difference in antibody response between balb and sjl mice that were inoculated at wk of age ( table ) . mice of both genotypes had detectable antibody at , but not days after inoculation. antibody titers were significantly higher among balb mice and all had seroconverted by day and beyond. in contrast, not all sjl mice seroconverted, despite confirmed infection in all wk sjl mice at days after inoculation, based on mhv antigen in nasal mucosa. antibody titers never exceeded : (one mouse on day ). based on mortality, resistance of several mouse genotypes to different mhv strains has been shown to evolve between and wk of age (bang, ; gallily et al., ; taguchi et al., ; b) . among susceptible balb mice in the present study, mortality was highest in the youngest age group, but virus titers in most target organs did not reflect age-related differences. we did find, however, decreasing virus titers in liver with increasing age at inoculation in balb mice. mice of the sjl genotype are exceptionally resistant to mortality following i.c. inoculation with mhv-jhm, particularly after wk of age (knobler et al., b (knobler et al., , c, stohlman and frelinger, ; stohlman et al., stohlman et al., , . we found that resistance of sjl mice to i.n.-inoculated mhv-jhm occurs early in life and may evolve at different rates in different organs. brain resistance to i.n. inoculation appeared to begin in mice inoculated at wk of age, was relatively strong in mice inoculated at wk of age, and absolute in older mice. mice infected at days of age were highly susceptible to brain infection. liver was remarkably resistant to infection in all sjl age groups, including mice infected as young as days of age. lymphoid tissue was a target in sjl mice inoculated at or less wk of age, but not older mice. it is difficult to assess if these differences were due to reduced access of virus to target tissues or tissue resistance per se, since mice were inoculated i.n. with virus. tissue resistance is likely, since it has also been shown that direct i.c. inoculation of -wk-old sjl mice with mhv-jhm resulted in low levels of virus replication in brain, liver and spleen at days, but virus had cleared by - days after inoculation (knobler et al., ) . age-related differences do not appear to be due to acquired immunity to mhv, since our studies precluded natural exposure. in balb mice infected at wk of age or older, antigen distribution patterns suggested that virus reached brain primarily by direct naso-olfactory extension. other studies have also suggested this mechanism of brain infection, following i.n. inoculation with mhv-jhm, as well as with mhv- , - , -a , and -s ( barthold et al., ; barthold and smith, ; goto et al., ; koolen et al., ; taguchi et al., a) . mice infected at wk of age developed diffuse brain infection, with a vascular distribution, as seen in suckling mice infected oronasally with several mhv strains (barthold and smith, ) . regardless of age, there was vascular dissemination of mhv-jhm to other organs, particularly liver and lymphoid tissues. nasal mucosa seems to be the major mucosal target for mhv-jhm excretion, since other potential excretory organs were not significantly involved. the nose has been shown to be an important target organ of mhv-jhm and mhv-s ( barthold and smith ; goto et al., ; taguchi et al., a) . a number of mhv strains, including mhv-jhm, have been shown to infect lung following i.n. inoculation, however, viral antigen is restricted to alveolar septal cells, usually endothelium, rather than airway epithelium (barthold and smith, smith, , carthew and sparrow, ) . intestine was infected in mice of the present study, but virus activity was localized to gut-associated lymphoid tissue, with no appreciable virus in feces. in contrast, enterotropic mhv strains replicate nearly exclusively in enteric mucosal epithelium, regardless of age or genotype (barthold, ; barthold and smith, ; barthold et al., ) . our current data support other studies (barthold and smith, ) showing that urinary organs do not play a significant role in mhv replication and excretion. reproductive tract transmission has not been fully explored. vertical trans~ssion in utero has been shown experimentally following intravenous mhv-jhm inoculation (katami et al., ) . furthermore, me-iv-like particles have been observed in endometrium of naturally infected athymic nude mice (consmith et al., ) . in. inoculation of adult balb mice induced a uniform pattern of brain infection, resulting in encephalitis at days after inoculation and development of localized brainstem spongiform lesions with demyelination in surviving mice, as previously described with mhv-jhm (bailey et al., ; barthold et al., ; goto et al., ) . lesions were present through day , but were resolving at day and had disappeared by day . infectious virus was detectable through day , but not at day . central nervous system infection is far more variable following i.e. insulation, since outcome is deter~ned by virus strain, virus dose, host age and host genotype (haspel et al., ; weiner, ) . persistent brain infection has been demonstrated in some mice that survive i.e. inoculation with mhv-jhm and mhv-a . with mhv-jhm, active demyelination has been observed in one study for up to months (herndon et al., ) . foci of recurrent demyelination and infectious virus have been shown in brain at days after i.c. inoculation with a temperature-sensitive mutant of mhv-jhm (knobler et al., b, c) . demyelination has also been observed for up to months after i.c. inoculation of mhv-a , and low levels of mhv-a rna can be detected for up to months (lavi et al., a, b) . others (robb et al., ) have found that mhv-jhm, mhv-a and their temperature-sensitive mutants induce a high rate of demyelination, but few mice have persistent demye~nation beyond - months. most mhv studies, including our current data, have demonstrated a transient rather than persistent course of mhv infection in all tissues, including brain (reviewed in barthold, ) . virus persistence and recurring demyelination produced by a temperature sensitive mutant of mhv- selected mutants of mouse hepatitis virus type (jhm strain) induce different cns diseases. pathobiology of disease induced by. wild type and mutants ts and ts in balb/c and sjl mice temperature-sensitive mutants of mouse hepatitis virus strain a : isolation, characterization and neuropathogenic properties experimental demyelination produced by the a strain of mouse hepatitis virus persistence of mouse hepatitis virus a rna in a slow virus demyelinating infection in mice as detected by in situ hybridization analysis of age-dependent resistance to murine coronavirus jhm infection in mice a simple method of estimating fifty percent endpoints pathogenic murine coronaviruses. iii. biological and biochemical characterization of temperature-sensitive mutants of jhmv two enzyme immunoassays for the detection of antibody to rodent coronaviruses in viva and in vitro models of demyelinating diseases: iii. jhm virus infection of rats resistance to fatal central nervous system disease by mouse hepatitis virus resistance to fatal central nervous system disease by mouse hepatitis virus, strain jhm. ii. adherent cell mediated protection chronic central nervous system demyelination in mice after jhm infection age-dependent response of mice to a mouse hepatitis virus, mhv-s pathogenesis of mouse hepatitis virus infection. the role of nasal epithelial cells as a primary target of low-virulence virus factors involved in the age-dependent resistance of mice infected with low virulence mouse hepatitis virus neutrovirulence of murine coronavirus jhm temperature-sensitive mutants in rats jhm infections in rats as a model for acute and subacute demyelinating disease pathogenesis of demyelination induced by a mouse hepatitis virus (jhm virus) arch acute and subacute demyelination induced by mouse hepatitis virus strain a in c h mice the technical assistance of d.s. beck and s.d. moore is gratefully acknowledged. this work was supported by phs grant rr- from the division of research resources. bailey, o.t., pappenheimer, a.m., cheever, f.s. and daniels, j.b. ( ) a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin: ii. pathology. j. exp. med. , - . bang, f.b. ( ) key: cord- -bb lyvhy authors: nan title: monoclonal antiprothrombinase ( d . ) prevents mortality from murine hepatitis virus (mhv- ) infection date: - - journal: j exp med doi: nan sha: doc_id: cord_uid: bb lyvhy the induction of monocyte/macrophage procoagulant activity (pca) has been implicated in the pathogenesis of murine hepatitis virus strain (mhv- ) infection and disease. previously, we have shown that induction of pca by mhv- correlated with resistance/susceptibility to infection in different mouse strains. in this study, all balb/cj mice that were infected with ( ) plaque-forming units of mhv- developed severe liver disease and died within - h. examination of the livers of these animals showed marked hepatic necrosis, deposition of fibrin, and cellular expression of pca by direct immunofluorescence staining in areas of necrosis as well as in hepatic sinusoids. splenic mononuclear cells recovered from these mice expressed high concentrations of pca with time after infection. infusion into mice of a high-titered monoclonal antibody that neutralized pca ( d . ) attenuated the development of hepatic necrosis and enhanced survival in a dose- dependent manner. all of the animals receiving micrograms, and % and % of the animals that received and micrograms per day, respectively, survived for d and made a full recovery. administration of the antibody resulted in a dose-dependent reduction in fibrin deposition, pca expression as detected by direct immunofluorescence staining and by a functional assay. in animals treated with high concentrations of antibody, titers of antibody to pca fell from +/- micrograms/ml to +/- ng/ml during the active phase of the disease, consistent with sequestration due to binding of the immunoglobulin to cells expressing pca. surviving animals, when rechallenged with mhv- , had a % mortality, consistent with the known rates of metabolism of immunoglobulin. this further suggested that protection was by a passive mechanism. the results reported here demonstrate that a neutralizing antibody to pca protects animals from fulminant hepatitis and death associated with mhv- infection, and supports the notion that pca is a potent inflammatory mediator that plays a pivotal role in the pathogenesis of liver injury resulting from mhv- infection. . furthermore, using recombinant inbred strains of mice, we showed that genetic linkage between resistance/susceptibility to mhv- infection and induction of pca was controlled by two non-h - inked recessive genes ( ) . after infection with mhv- , disturbances of the hepatic microcirculation associated with sinusoidal thrombosis occurred coincident with the rise in pca ( , ) . intravenous infusion of macrophages, induced to express high amounts of pca by mhv- , resulted in rapid ( - min) death from disseminated intravascular coagulation in both susceptible and resistant mice (our unpublished observations). together, these observations suggest that coagulative necrosis occurring as a result of induction of pca may be a crucial feature of mhv- -induced hepatic injury. we have recently produced a panel of mabs to mhvinduced macrophage pca ( ) . the antibodies did not react with purified viral proteins nor did they inhibit viral replication. one of these mabs ( d . ) , an igg ak, strongly inhibited pca expression in a one-stage dotting assay and inhibited conversion of prothrombin to thrombin ( ) . this antibody had no reactivity with murine, rabbit, or human tissue factor. the monoclonal bound to a -kd protein that is distinct from murine and human tissue factor ( kd) ( , ) . the purpose of this study was to determine whether treatment with this specific murine antiprothrombinase would modify the morbidity and/or mortality associated with murine hepatitis virus infection. virus. the origin and growth of mhv- have been previously described ( ) . mhv- , obtained from the american type culture collection (kockville, md) (atcc-vr ), was plaque purified on monolayers of dbt cells. stock virus was grown to a titer of . x pfu/ml in cl cells. the virus was harvested by one cycle of freeze-thawing and clarified by centrifugation at , g for h at ~ virus was then assayed on monolayers of l cells in a standard plaque assay as previously described ( ) . to induce mhv- infection, each mouse received pfu of mhv- by intraperitoneal injection. mice. balb/cj mice - wk of age, were obtained from charles river laboratories, (st. constant, quebec). animals were housed in the d class facility at the university of toronto fed a diet of standard chow and water ad libitum. mice were killed on days , , , , , and after infection. blood was obtained by axillary bleeding. splenic mononuclear cells were harvested and assayed for pca both by immunofluorescence staining and in a onestage clotting assay. livers were harvested for viral titers, histopathology, and pca by immunofluorescence staining as described below. all mice used were screened by an elisa for exposure to mhv, and were found to be negative ( ) . anti-pca mat~ the preparation of mab d . has been previously described ( ) . after injection of hybridoma cells into pristane-primed caf mice, ascites was harvested. animals were treated with ascitic fluid containing , , or #g of mab daily for d preinfection and d postinfection (p.i.) by intraperitoneal injection. as a standard control, ~ of ascites from animals that were infected with sp myeloma cells was injected into six separate mice. all ascitic fluid whether from sp -or d . injected mice had no reactivity with mhv- either in the elisa or plaque reduction assays ( ) . to ensure that the protective effect of ascites containing mab to pca was specific, mab to pca ( d . ) was isolated from hybridoma supematants by affinity chromatography using goat anti-mouse igg immobilized on sepharose b (pharmacia, montreal, quebec). immunoblot analysis of the column eluate confirmed that igg was the only ig present. subclass analysis by elisa, as previously described ( ) , confirmed that igg ak was the only subclass present. #g of the purified mab was injected into mice for d preinfection and for d p.i. as described above. histology. histology was assessed by a blinded observer as previously described ( ) . briefly, li~r were cut into . x . -cm blocks and fixed by immersion into % formalin in . molar phosphate buffer, ph . . after fixation, the tissue was dehydrated in graded alcohols and xylene, then embedded in paraffin. -#m sections were cut, stained with harris' hematoxylin for min, and counterstained with eosin y for s. the sections were then washed with distilled water, dehydrated in graded alcohols and xylene, and mounted with parmount. for each group five animals were used. to quantitate the effect of the mab on liver histology, a digitalized image analysis system (hp- ; hewlett packard co., ltd., mississauga, ontario) with customized software was used. this constitutes a modification of a technique described previously ( ) . the areas of necrosis were encircled as well as the entire section yielding a percentage figure representing the proportion of diseased liver present in that particular section. for each animal, three random sections were assayed in this fashion, and the mean +_ sd was calcuhted. hbrin deposition was assessed by the morris-lendrum hcro-mallory stain as previously described ( ) . viral titers. livers that had been snap frozen at - ~ were homogenized in dmem supplemented with % fcs and mm glutamine as a % homogenate at ~ as previously described ( ) . viral titers of liver homogenates were then determined on monolayers of l cells in a standard plaque assay ( , ) . spleens were harvested aseptically and cells teased from splenic tissue and suspended in ml of dmem as previously described ( ) . smnc were isolated over ficoll-hypaque gradients (density, . ) (pharmcia) by centrifugation at , g for rain at ~ cells at the interface were collected. viability was > % as assessed by trypan blue exclusion. cells were washed three times and resuspended in dmem at a concentration of x smnc/ml. procoagutant activity. samples of frozen thawed smnc were assayed for the capacity to shorten the spontaneous clotting time of human citrated platelet-poor plasma in a one-stage clotting assay as previously described ( ) . equal volumes ( # ) of the cellular homogenate were admixed with citrated normal human plateletpoor plasma, and then # of mm caclz was added at ~ to start the reaction. the time in seconds for the appearance of a fibrin gel was then recorded. to establish arbitrary units, a rabbit brain thromboplastin standard at mg dry mass/ml (dade division, american hospital supply, miami, fl) was assigned a value of , mu. the assay was used over the range of - , mu, and the results were linear with normal human plasma substrate. data were converted to pca per splenic macrophages and expressed as the mean and standard deviation from six mice done in triplicate. media and buffers were all without activity in this assay. inhibition of viral replication. mab purified from hybridoma cell cultures containing d . and ascites were assayed for their ability to inhibit replication of mhv- in a standard plaque reduction assay as previously described ( ) . briefly, pfu of virus was admixed with dilutions of purified antibody or ascites, media as a negative control, or a high titered anti-mhv- -neutralizing antibody as a positive control, for min at ~ the mixture was then added to a monolayer of l cells in culture medium, overlayed with % agarose, and incubated at ~ in a % co environment for an additional h. the effect of antibody on viral replication was assessed by reduction of viral plaques ( ) . immunofluorescence. blocks of liver tissue were snap frozen in liquid nitrogen. cryostat sections ('~ #m thick) were fixed for rain in acetone and air dried for h as previously described ( ) . unoccupied sites were then blocked with % horse serum in pbs, ph . , for h. mab d . was conjugated with fitc (sigma chemical co., st. louis, mo) according to the method of thi and feltkamp ( ) . the fluoresceinated antibody did not react with normal liver or uninduced normal peritoneal macrophages. tissues were then stained with fitc-conjugated mab d . for h at room temperature, washed three times, mounted in % glycerol in pbs, and viewed on a phase-epifluorecence microscope equipped with a x fluotar objective (e. leitz, inc., rockleigh, nj). elisa for anti-pca antibody. titres of antibody to pca were determined in a standard elisa as previously described ( ) . well enzyme immunoabsorbant assay (eia) plates (dynatech, mclean, va) were coated with /~l/well of mhv- -stimulated pca-positive macrophage membranes or unstimuhted pcanegative membranes ( x ~ macrophage/ml) at ~ overnight. the plates were then washed three times with pbs, ph . , conraining . % tween (washing buffer), and the unoccupied sites were blocked with /zl of % ig-free horse serum (flow laboratories, mississauga, ontario) that had been dissolved in washing buffer for h at room temperature. -/~l/well dilutions of sera from treated animals were then added to the plates and incubated for h at ~ after washing three times, #l/well of alkaline phosphatase-conjugated goat anti-mouse ig in pbs containing . % bsa and . % tween was added for i h at ~ after three washings, p-nitrophenyl phosphate in . m -amino- -methyl- , -proponediol buffer, ph . (zymed laboratories, san francisco, ca), substrate was then added. the plates were then incubated at ~ for h and read at nm with a plate reader (titertek mcc/ ; icn/flow, mississauga, ontario). antibody levels were expressed (/~g/ml) by comparison to a standard curve. statisticalanalysis. statistical analysis was carried out using analysis of variance and the wilcoxon ranked sum test. a p value of . % or less was considered statistically significant. suwival. mice infected with , pfu ofmhv- (n = ) all succumbed to the infection within d (fig. ) . this was consistent with previous data reported by our group as well as others ( , ) . in contrast, there was survival in some animals that were treated with ascites containing antibody to pca (fig. ). animals treated with ascites containing /~g/d had a % survival; % of those treated with ascites containing #g/d and % of animals treated with /~g/d survived. all animals survived when treated with /~g of monodonal anti-pca (igg ak), which had been purified from hybridoma superuatant, confirming that the beneficial effect of ascites was due to the monoclonal anti-pca. mice immunized with /~ of ascites from sp injected animals and infected with mhv- all died within d (data not shown). liver histology. mhv- -infected mice that did not receive antibody developed histologic evidence of severe liver disease. by h p.i., small, discrete loci of necrosis with a sparse pmn infiltrate could be seen. at h, these lesions became both more pronounced and more numerous (fig. a) , and by - h, confluent liver necrosis was apparent (fig. b) . in contrast, mice infected with mhv- but treated with antibody to pca showed a marked reduction in liver disease in all groups ( , , and /.r ( were a few small loci of inflammatory cells with no necrosis (fig. d) . morphometric image analysis showed that the proportion of the liver that was necrotic was significantly different between the mhv- -infected, anti-pca-treated, and untreated groups at , and h (fig. ) . the histolog of livers from all survivors at and d postinfection appeared normal. fibrin deposits were seen in hepatic sinusoids as well as in areas of necrosis of untreated and mhv- -infected mice (fig. ) . in animals treated with and /~g of anti-pca, a marked reduction in fibrin was noted. no fibrin was seen in the livers of infected mice treated with /~g of mab. treatment with anti-pca alone resulted in no detectable histological evidence of liver disease in nonirlf~ed animals. viral titers. by h p.i., large amounts of infectious virus were recovered from liver homogenates of mhv- -infected and untreated animals, and these persisted until the death of the animals (fig. ) . in animals treated with and /~g of antibody to pca, there was no significant difference in viral titers from those observed in untreated mice at days and . viral titers remained high at day , however, viral titers decreased by day and no virus could be detected after day . in animals treated with the highest dose of antibody to pca ( #g/d), viral titers were markedly reduced (p < . ) and approached those seen in resistant a/j mice ( , ) . using the method of reed and meunch, the mhv- recovered from infected and antibody-treated animals was as pathogenic as stock mhv- or virus recovered from infected and untreated mice (data not shown) ( ) . pca. in untreated, but mhv- -infected animals, a sharp increase in splenic macrophage pca was noted at h p.i. (fig. ) . maximal pca was seen at h and pca remained elevated until the animals' death on day . animals treated with low concentrations of antibody to pca ( and /zg/d) expressed high amounts of pca at early time points, but by day , pca levels fell and only basal levels of pca were detected in smnc by day . in contrast, no expression of pca could be detected in animals treated with /zg of antibody to pca during the course of infection (fig. ). immunofluorescence. by direct immunofluorescence staining, pca could be detected at h in livers from mhv- - infected and untreated mice. pca was seen in areas of inflammation and necrosis, and also in hepatic sinusoids, localized primarily in endothelial cells and kupffer cells, but not expressed by hepatocytes (fig. ) . livers from animals treated with antibody to pca had significantly less pca expression, although small amounts of pca could be seen in macrophages and endothelial cells in hepatic sinusoids in animals treated with high-dose antibody to pca ( ~tg/d), even as late as day (fig. ) . antibody to pca. sera were collected from animals at all time points and analyzed in an elisa for the presence of antibody to pca as previously described ( ) . no antibody to pca could be detected in sera from normal control animals or in animals infected with mhv- who had not received antibody to pca (data not shown). sera from animals treated with ascites containing - /zg of antibody to pca contained large amounts of anti-pca before mhv- infection. after mhv- infection, the concentration of circulating antibody fell, but remained at > ng/ml in animals treated with /~g/d. in animals that were treated with and /zg of antibody, by day , antibody was undetectable (< ng/ml) (fig. ) . antibody to pca either in ascites or purified from supernatants did not neutralize mhv- in a standard plaque reduction assay as previously described ( ) to determine whether treated animals that had survived the acute infection developed long-term resistance to mhv- infection, mice previously infected with mhv- and treated with /~g of antibody (n = ) were infected with , pfu of mhv- , d after their last exposure to virus. pca antibody titers in this group were ~ % of that present during antibody therapy, consistent with known rates of disappearance of igg. the mhv-rechallenged mice experienced a % mortality rate, suggesting that protection was by passive immunization of antibody, not an acquired, active immune process (fig. ). in addition, no antibody to mhv- was detected in these mice either before or after rechallenge with virus. mhv- infection produces fulminant hepatic failure and death in balb/cj mice ( , ) . the availability of a mab to pca that neutralized acceleration of coagulation in vitro ( ) provided us an opportunity directly to examine the role of pca in vivo in murine hepatitis virus strain infection. all balb/cj mice which were infected with pfu of mhv- developed severe liver disease and died within - h. administration of the mab to pca attenuated the hepatic necrosis, fibrin deposition and enhanced survival in a dosedependent manner. all of the animals receiving /~g, and % and % of the animals that received and /~g/d, respectively, survived for d and made a full recovery. furthermore, the same protective effect was seen in mice treated with igg ak purified from hybridoma culture supernatants ( d . ). the increased survival of the treated animals was specific for anti-pca, since ascites from mice injected with sp cells alone, which contained no antibody to pca, failed to protect mice from mhv- infection. although antibodytreated, mhv- -infected animals demonstrated clinical evidence of viral hepatitis early in the course of the infection, by - d their behavior appeared normal and liver sections showed little or no apparent disease. examination of the livers of antibody-treated animals showed marked reduction in hepatic necrosis and inflammatory cells (neutrophils and mononuclear cells), each of which are prominent features of mhv- infection ( , , ) . pca expression is a feature of mhv- infection in this mouse strain ( ) ( ) ( ) , and in untreated mhv- -infected mice, pca was detected by immunofluorescence staining in areas of necrosis as well as in hepatic sinusoids. both macrophages and endothelial cells expressed detectable pca, but hepatocytes did not. it is likely that the hepatic necrosis is secondary to ischemic changes resulting from induction of pca, leading to the deposition of fibrin. administration of mab resulted in a dose-dependent reduction in the deposition of fibrin and in expression of pca as detected by immunofluorescence staining in the liver and by a direct functional assay in smnc. previously, we have shown that mhv- infection of peritoneal macrophages in vitro results in the production of pca, tnf, leukotriene b ( ) , and il- ( ) . thus, expression of pca by endothelial ceils might either be due to direct induction by mhv- or due to induction by il. and/or tnf, which have previously been shown to induce pca in endothelial cells in vitro ( , ) . treatment of mice with /~g of mab to pca not only increased survival and reduced hepatic necrosis, but also resulted one possible explanation for the decrease in viral replication observed in mice treated with antibody d . is that this antibody reacts with the mhv receptor recently described by holmes and coworkers ( , ) . we regard this explanation as unlikely, since d . does not show any neutraliza- tion of viral infectivity in a plaque reduction assay, whereas antibody to the mhv receptor does inhibit infectivity. furthermore, the mhv receptor has a molecular mass of kd, considerably different than that of the pca molecule ( kd). a second possible explanation for the effect of anti-pca antibody on mhv- growth in mice is related to the normal cleavage of the mhv s protein. it has been shown that cleavage of s by proteases is necessary to activate the membrane-fusing properties of the s protein ( ) . this fusion property facilitates the spread of virus to uninfected cells by cell-cell fusion and also increases the specific infectivity of mhv when compared with virus in which s has not been cleaved ( ) . it is possible that in infected macrophages, pca, a serine protease, mediates at least in part the proteolytic cleavage of s into $ and $ , and thereby activates the fusion properties of this molecule. consistent with this idea is the observation that infection of a/j macrophages, which do not produce pca in response to mhv infection, does not result in the appearance of syncytial giant cells (our unpublished observations). thus, antibody to pca could inhibit the spread of virus to uninfected cells by decreasing the activation of the fusion properties of s. although the mechanism by which anti-pca treatment protects the susceptible animals is not clear, the use of antibody to pca neutralizes pca, thereby preventing activation of the coagulation system and inhibiting fibrin formation. the antibody could also result in complement-mediated destruction of mhv- -infected cells that express membranebound pca or promote macrophage activation with restriction of viral growth. in animals treated with #g of antibody for d before infection and d p.i., concentrations of antibody to pca in sera fell from /~g/ml before mhv- infection to ng/ml during the active phase of disease (day ), consistent with sequestration perhaps due to binding of the ig to cells expressing pca. furthermore, in these mice, levels of pca in smnc remained at basal levels. in animals treated with lower amounts of antibody ( or #g/d) to pca, hepatic necrosis and survival were only partially attenuated, and antibody was not detected (< ng/ml) after day p.i. together, the data strongly support the notion that anti-pca antibody neutralizes pca in vivo during the infection and that this may be the basis for its protective effect. in a previous report we have demonstrated that dimethyl prostaglandin e inhibited procoagulant activity and prevented fulminant viral hepatitis, yet all animals still succumbed to the infection ( ) . in pge-treated mice, viral replication proceeded at a rate similar to that in untreated animals. recently, we have demonstrated that although pge inhibited functional pca, antigenic expression of pca was not altered as determined by western immunoblotting ( ) . we have proposed that pca may exert its effect through activation of the coagulation system with microvascular and macrovascular thrombosis ( , ), but the present results, in concert with the inhibition studies of pca by pge , suggest that pca has other sites of action as well. the protective effects of the mab to pca occurred even though the balb/cj mice failed to mount an antiviral humoral response. our ob-servations are consistent with a recent report by korner et al. ( ) and support the notion that the murine antiviral antibody response may not be required for protection from acute viral infection. cytokines can play a potent role in the course of inflammatory injuries in vivo, and interference with their action can alter the course of certain inflammatory diseases ( , ) . treatment of rats with recombinant antibody to tnf has been shown to protect animals from the hypotension, hypothermia, and mortality of gram-negative sepsis ( ) , and treatment of rabbits with an il- r antagonist reduced the mortality associated with endotoxin shock ( , ) . our studies support the notion that induction of pca during mhv- infection in mice is an integral and potentially central step in the disease. a previous report by taylor et al. ( ) demonstrated that lethal escherichia coli septic shock can be prevented by blocking tissue factor (a distinct procoagulant) with mab, demonstrating the importance of cellular coagulants in the pathophysiology of other infectious diseases. we conclude that pca is a potent inflammatory mediator that plays a pivotal role in hepatic injury resulting from mhv- infection. the biology and pathogenesis of coronaviruses induction of monocyte procoagulant activity by murine hepatitis virus type parallels disease susceptibility in mice the pathobiology of viral hepatitis and immunologic activation of the coagulation protease network activation of the immune coagulation system by murine hepatitis virus strain susceptibility/resistance to murine hepatitis virus (mhv- ) and monocyte procoagulant activity (mpca) are genetically linked and controlled by non-h- linked genes acute and chronic changes in the microcirculation of the liver in inbred strains of mice following infection with mouse hepatitis virus type the effects of mouse hepatitis virus type on the microcirulation of the liver in inbred strains of mice monoclonal antibody analysis of a unique macrophage procoagulant activity induced by mufine hepatitis virus strain infection complete sequence of the human tissue factor gene, a highly regulated cellular receptor that initiates the coagulation protease cascade molecular cloning of the cdna for tissue factor, the cellular receptor for the initiation of the coagulation protease cascade a sensitive radioimmunoassay for the detection of antibodies to mhv- induction of resistant hepatocytes as a new principle for a possible short-term in vivo test for carcinogens morris-lendrum picro-mallory stain for fibrin dimethyl prostaglandin e prevents the development of fulminant hepatitis and blocks the induction of monocyte/macrophage procoagulant activity after murine hepatitis virus strain infection lymphocyte cooperation is required for amplification of macrophage procoagulant activity, f ex f med conjugation of fluorescein isothiopropriate to antibodies. i. experiments in the conditions of conjugation a sensitive radioimmunoassay for the detection of antibody to mvh- mechanism of protective effect of prostaglandin e in murine hepatitis virus strain infection: effects on macrophage production of tumor necrosis factor, procoagulant activity and leukotriene b susceptibility to mouse hepatitis virus strain in balb/cj mice: failure of immune cell proliferation and interleukin production interleukin- activation of vascular endothelium. effects on procoagulant activity and leukocyte adhesion the role of endothelial cells in inflammation monoclonal antibody to the receptor for murine coronavirus mhv-a inhibits viral replication in vivo purification of the -kilodalton glycoprotein receptor for mouse hepatitis virus (mhv)-a from mouse liver and identification of a nonfunctional, homologous protein in mhv- resistant sjl/j mice. f virol proteolytic cleavage of the e glycoprotein of murine coronavirus: host dependent differences in proteolytic cleavage and ceu fusion effect of eicosanoids on induction of procoagulant activity by murine virus strain in vitro nucleocapsid or spike protein-specific cd + t lymphocytes protect against coronavirus-induced encephalomyelitis in the absence of cd + t cells, f immunol shcokd and tissue injury induced by recombinant human cachectin cachectin/tumor necrosis factor induces lethal shock and stress hormone responses in the dog development of partial tolerance to the gastrointestinal effects of high doses of recombinant tumor necrosis factor-alpha in rodents treatment with recombinant human tumor necrosis factor-alpha protects rats against the lethality, hypotension and hypothermia of gram-negative sepsis interleukin- receptor antagonist reduces mortality from endotoxin shock lethal e. coli septic shock is prevented by blocking tissue factor with monodonal antibody key: cord- - wc f rl authors: sengupta, sourodip; addya, sankar; biswas, diptomit; sarma, jayasri das title: matrix metalloproteinases and tissue inhibitors of metalloproteinases in murine coronavirus-induced neuroinflammation date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: wc f rl mouse hepatitis virus (mhv) belongs to the same beta-coronavirus family as sars-cov- , mers-cov, and sars-cov. studies have shown the requirement of host cellular proteases for priming the surface spike protein during viral entry and transmission in coronaviruses. the metzincin family of metal-dependent endopeptidases called matrix metalloproteinases (mmps) is involved in virus encephalitis, enhanced blood-brain barrier permeability, or cell-to-cell fusion upon viral infection. here we show the role of mmps as mediators of virus-induced host neuroinflammatory response in the mhv model. infection of mice with wild-type mhv-a or its isogenic recombinant strains, rsa or rsmhv significantly upregulated mmp- , mmp- , and mmp- transcript levels. functional network assessment with ingenuity pathway analysis revealed a direct involvement of these mmps in disrupting junctional assembly between endothelial cells via interaction with junctional adhesion molecules and thereby facilitating transmigration of peripheral lymphocytes. our findings also suggest mrna upregulation of park , which is involved in nadph oxidase-dependent ros production, following rsa infection. rsa infection resulted in elevated mrna levels of rela, a subunit of nf-κb. infection with mhv-a is known to generate ros, and oxidative stress can activate nf-κb. thus, our findings indicate the existence of a possible nexus between ros, nf-κb, and mmps in rsa -induced neuroinflammation. we also assessed the expression of endogenously produced regulators of mmp activities. elevated mrna and protein levels of tissue inhibitors of metalloproteinases (timp- ) in mhv-a infection are suggestive of a timp- mediated host antiviral response. importance the newly emergent coronavirus has brought the world to a near standstill. in the past, studies have focused on the function of host proteases in virus attachment and entry. our research indicates the involvement of a group of metal-dependent host proteases in inflammation associated with coronavirus infection. inflammation is the first response of the host to virus infection. while it helps in restricting the spread and clearance of viral particles, uncontrolled inflammation results in several inflammatory consequences. therefore, it becomes vital to limit unchecked host immune response. the inhibition of specific metalloproteases represents a potential new therapeutic approach in coronavirus infection and disease outcome. junctional adhesion molecules and thereby facilitating transmigration of peripheral lymphocytes. our findings also suggest mrna upregulation of park , which is involved in nadph oxidase-dependent ros production, following rsa infection. as the number of plaques times dilution factor (df) per ml per gram of tissue per ml [pfu= (no. of plaques*df per ml)/ (tissue weight in gram per ml)]. gene expression analysis. total rna was extracted from brain tissues of mhv-a , rsa or rsmhv infected as well as mock-infected mice using trizol reagent (invitrogen) following the manufacturer's instructions. rna concentration was measured using a nanodrop / c spectrophotometer (thermo fisher scientific), and cdna was prepared with µg of total rna using a cdna reverse transcription kit. quantitative real-time pcr (rt-qpcr) was performed using sybr green dye-based assay in a quantstudio real-time pcr system (thermo fisher scientific) with the following reaction conditions: initial denaturation at °c for min, cycles of °c for s and °c for s, and melting curve analysis at °c for s. reactions were performed in triplicates (n= ). primer sequences are provided in table . the comparative threshold (ΔΔct) method was used for relative quantification. the mrna levels of target genes were normalized with the housekeeping gapdh gene and represented as the relative fold change values compared to their respective mock-infected controls. western blotting. brain tissues ( mg) were harvested from mice following transcardial pbs perfusion and flash-frozen in liquid n . tissues were homogenized (using qiagen homogenizer) and lysed in ul of ripa buffer containing protease-cocktail inhibitor and phosphatase inhibitors ( mm navo and mm naf) for hr mins with intermittent vortex every mins. the samples were kept on ice during the entire process. samples were then centrifuged for mins at , rpm at °c to separate the supernatant. the total protein content in the supernatant was estimated with a bca protein assay kit. for immunoblotting, µg of total protein per sample was resolved by sds-page on a % polyacrylamide gel followed by transfer onto pvdf membranes using transfer buffer ( mm tris, mm glycine, and % methanol) . membranes were blocked for hr at room temperature in % v/v goat serum prepared in tbst (tris-buffered saline containing . % v/v tween ) and subsequently incubated for overnight at °c in polyclonal anti-mouse timp- antibody at : dilution in blocking solution. the membranes were washed in tbst and incubated for hr at room temperature with hrp-conjugated donkey anti-goat secondary igg antibody. as an internal loading control, g-actin was used, and membranes were blocked separately in % w/v non-fat skimmed milk in tbst. polyclonal anti-mouse g-actin antibody ( : dilution) and hrp-conjugated goat anti-rabbit secondary igg antibody ( : , dilution) were used. the blots were washed in tbst, and the immunoreactive bands visualized using the chemiluminescent hrp substrate. non-saturated bands were visualized with syngene g: box chemidoc system using gensys software. to the median of all samples used as the baseline option. data were filtered by percentile, and a lower cut off was set at . a fold change of ≥ . -fold was considered for differential expression of a gene. statistical analysis using unpaired student t-tests was performed to compare two groups, with p-values ≤ . considered significant. the list of mmp and timp genes from the microarray data was loaded into qiagen's ingenuity pathway analysis software (ipa®, qiagen, usa) to perform biological network and functional analyses. statistical analysis. data shown are mean ± standard error mean (sem) for all graphs. unpaired student t-test with welch's correction, assuming unequal standard deviations, was performed to examine significant differences between two groups. multiple comparisons were achieved using ordinary one-way anova, followed by dunnett's multiple comparison test. a p-value < . was considered statistically significant. availability of data. all the data sets used and analyzed in the current study are available from the corresponding author on request. four weeks old, male c bl/ mice inoculated with mhv-a ( pfu) or mock- infected were sacrificed at day - (acute), (acute-chronic), and (chronic) post-infection (p.i), and brains were harvested. routine plaque assay was performed with serially diluted brain homogenates to estimate viral replication. mhv-a titer was significant between day - p.i (fig. , a; p< . ) and viral particles were below the detection limit at later time points (data not shown). total rna was isolated from mock and virus-infected brain tissues for expression analysis of viral nucleocapsid and mmp genes through rt-qpcr. primer sequences are given in table . levels of viral nucleocapsid mrna ( fig. , b ; p< . ) coincided with viral replication reaching its peak between day - p.i, which also marks the acute phase of inflammation. mhv-a infected mice exhibited neuroinflammation reaching its peak by - days p.i, which is associated with meningitis, encephalitis, perivascular cuffing, and macrophage/microglia nodule formation ( ). we found similar results in paraffin-embedded brain sections stained by hematoxylin-eosin and immunohistochemistry (data not shown). transcript levels of mmp , mmp , mmp , and mmp were significantly upregulated at day - p.i ( tissue inhibitors of metalloproteinases or timps are endogenous protein regulators of mmps. to understand the regulation of mmps upon mhv-a infection, we also considered the gene expression of timps. as described above, total rna from brain samples of mock and mhv-a infected mice were subjected to rt-qpcr using specific primers (table ) to determine the transcript levels of timp , timp , timp , and timp . mhv-a infection resulted in significant upregulation of timp mrna at day - p.i (fig. , a; p< . ), while mrna levels of timp , timp , and timp remained significantly downregulated (fig. , c-e; p-values varies as < . to < . ). while timp mrna followed a similar expression pattern as the mmps following mhv-a infection-induced inflammation, its protein levels remained high throughout post-infection, as shown in the representative figure (fig. , b) . overall, mhv-a resulted in elevated timp- levels in the brain of infected mice. to determine whether the spike (s) protein has any role in inducing mmp cord. to validate the findings from microarray data, we performed rt-qpcr from brain samples of rsa , rsmhv , and mock-infected mice sacrificed at day - , , and p.i. brain samples were also harvested for titer assay to estimate viral replication. like the parental mhv-a strain, viral titer and nucleocapsid mrna levels peaked by day - p.i in both the recombinant strains as demonstrated in the representative graphs (fig. , a-c) . although we detected no nucleocapsid mrnas between - days p.i in rsmhv , its presence was observed at day p.i in rsa (data not shown). this data corroborates with previous findings that demyelinating rsa persists in the brain while rsmhv does not persist or, if present, nucleocapsid level is significantly low compared with rsa ( ). biological and functional network analysis were performed for mmp , mmp , and mmp genes using qiagen's ingenuity pathway analysis (ipa) software. ipa analysis identified that these mmps could influence several canonical pathways associated with an immune response such as leukocyte extravasation signaling, granulocyte and agranulocyte adhesion and diapedesis (fig. , a) . also, the top disease pathways involved both inflammatory response and immune cell trafficking (fig. , b) . furthermore, ipa revealed that mmps facilitate the transmigration of firmly adhered granulocytes (fig. , a) and agranulocytes (fig. , b) pathway genes brain samples from mice infected with rsa and sacrificed at day - and p.i, were harvested for total rna isolation followed by cdna synthesis. mock-infected samples were kept in parallel. we performed rt-qpcr using primers (table ) in the oxidative and anti-oxidative pathways. transcript levels of parkinson's disease (park ) gene were significantly upregulated following rsa infection and remained elevated p.i compared to mock-infected samples (fig. , a; p< . ). rela, a subunit of nf- b, also showed elevated mrna levels during the acute infection, i.e., - days p.i (fig. , b; p< . ). on the contrary, mrna levels of nfb , a negative regulatory subunit of nf- b, remained unchanged p.i (fig. , c) . similar to another study of our lab (unpublished data), we detected significantly high mrna levels of nuclear factor erythroid -related factor (nrf ) and heme oxygenase- (hmox ) genes during the acute disease phase (fig. levels. in our current study, rsa infection increased transcript levels of park , which is involved in oxidative stress. park has a double-sword effect. in lower ros concentration, it can affect nadph oxidase by phosphorylating its p phox subunit during nadph oxidase activation, which is crucial for nadph oxidase-dependant ros production ( ). in one of our studies (unpublished data), we show that park also induces the anti-oxidative pathway via nrf and hmox activation during higher cellular ros concentration. activation of metalloproteases via oxidative pathways has been demonstrated in the past ( - ). ros-induced oxidative stress can also activate nf-b signaling ( ). the nuclear factor kappa-light-chain-enhancer of activated b cells (nf-b) acts as a transcription factor and is known to induce inflammation-related genes. rela, a subunit of nf-b, which gets activated in the canonical pathway via toll-like receptors that recognize pathogenic patterns ( ), showed increased mrna levels following rsa infection. on the other hand, nfb , which acts as both a precursor and suppressor of nf-b ( ), demonstrated unchanged mrna levels upon infection. previously, it has been documented that nf-b can induce mmp genes ( , ). therefore, our result indicates that park mediated ros generation leads to the induction of mmp genes via nf-b signaling during mhv-induced acute disease (fig. ) . we also found that rsa infection-induced upregulation of nrf and hmox genes. the anti-oxidative pathway mediated by nuclear factor erythroid -related factor (nrf ) and its dependant heme oxygenase- (hmox ) ( ), could therefore play an essential role in restoring homeostasis through inhibition of ros overproduction. one limitation of this study that will be addressed in our future experiments is that the interplay between ros and mmps has not been validated using inhibitors of ros as positive controls. in previous studies ( - ) involving mhv-a , it has been shown that virus infection reduced expression of connexins (cxs) that form intercellular gap junctional channels and thereby disrupt functional communications between cns glial cells and fibroblasts. however, the mechanism through which cx trafficking is altered is not well understood. the ( ) infected mice at different days post-infection (p.i) were harvested, and viral replication was estimated by routine plaque assay. rna isolated from brain tissues was subjected to cdna synthesis. an equal amount of cdna template was used for rt-qpcr. gene expression was normalized to gapdh and fold-change values obtained using ∆∆ct method. a: mhv-a titer peaked between day - p.i. b: viral nucleocapsid mrna levels peaked at day - p.i like viral replication. c-f: mmp , mmp , mmp , and mmp mrna levels elevated between - days p.i. and coincided with viral replication peak. g: membrane-associated mmp- mrna levels peaked only at later stages p.i. data shown are mean ± sem from two independent biological experiments with nine technical replicates. a significant difference between the two groups was compared with the student t-test. multiple group comparison was made with ordinary one-way anova followed by dunnet's test. a p-value of < . was considered statistically significant (**, p< . ; ***, p< . ; ****, p< . ). of rt-qpcr revealed a significant upregulation in timp- mrna levels at day - p.i in mhv-a compared with mock-infected samples (a). representative immunoblot assay from two independent experiments showed elevated protein levels of timp- at all examined days following mhv-a infection (b). in contrast, timp- , - , and - mrnas remained downregulated throughout p.i (c-e). graphs show mean ± sem values from two independent experiments with nine technical replicates. ordinary one-way anova followed by dunnet's test was performed for multiple group comparisons. statistical significance was considered for p values < . (*, p< . ; ***, p< . ; ****, p< . ). strains of the wild-type mhv-a and differs only in the spike gene. brain samples from mice infected with rsa ( pfu) or rsmhv ( pfu) were harvested at different days p.i for routine plaque assay and total rna extraction. a: both rsa and rsmhv showed peak viral replication between day - p.i. no detectable viral particles were observed at later time points in a routine plaque assay (data not shown). rt-qpcr data showed significantly elevated mrna levels of viral nucleocapsid gene in both rsa (b) and rsmhv (c) at early days p.i, coinciding with peak levels of viral particles as detected in a plaque assay. data shown are mean ± sem from three independent experiments having three technical replicates each. multiple group comparison was made with ordinary one-way anova followed by dunnet's test. a p-value of < . was considered statistically significant (***, p< . ; ****, p< . ). china novel coronavirus i, research t. . a novel coronavirus from patients 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and - production by vascular smooth muscle cells role of nrf /ho- system in development, oxidative stress response and diseases: an evolutionarily conserved mechanism mouse hepatitis virus infection remodels gap junction intercellular communication in vitro and in vivo microtubule-assisted altered trafficking of astrocytic gap junction protein connexin is associated with depletion of connexin during mouse hepatitis virus infection loss of cx -mediated functional gap junction communication in meningeal fibroblasts following mouse hepatitis virus infection we thank the ministry of education, india, and the department of mice infected with rsa ( pfu) or mock-infected was subjected to cdna synthesis, and subsequently, rt-qpcr was performed. a: data analysis revealed elevated mrna levels of park during acute ( - days p.i) and acute-chronic (day p.i) disease phase. b: mrna upregulation was detected for rela, a subunit of the nf-b transcription factor. c: in contrast, no change was observed in the mrna level of nfb , a negative regulator of nf-b. d & e: moreover, rsa infection also induced increased transcription of nrf and hmox genes. data shown are mean ± sem from two independent experiments. a significant difference between multiple groups was compared with ordinary one-way anova, followed by dunnet's test. a p-value of < . was considered statistically significant (*, p< . ; ***, p< . ; ****, p< . ). key: cord- - dxy e authors: van berlo, m.f.; warringa, r.; wolswijk, g.; lopes‐cardozo, m. title: vulnerability of rat and mouse brain cells to murine hepatitis virus (jhm‐strain): studies in vivo and in vitro date: - - journal: glia doi: . /glia. sha: doc_id: cord_uid: dxy e the pathogenicity and cell tropism of mouse hepatitis virus (mhv‐jhm‐strain) in the developing mouse (balb/c) and rat (wistar and lewis) brain were analysed. intracranial infection of balb/c mice at postnatal day induced a lethal encephalitis in all animals. of wistar rats infected at day or after birth, to %, respectively, survived. the distribution of viral antigen was studied in frozen brain sections of animals that died after infection; astrocytes were found to be the major virus‐infected cell type throughout the central nervous system. more than % of the surviving rat pups developed paralysis, but viral antigen was detected in only few brain cells and not in astrocytes. the cell tropism of mhv‐jhm was examined further in virus‐infected glial cell cultures derived from brains of rats or mice. in the glial cultures derived from wistar rats, only oligodendrocytes were infected, whereas in cultures derived from mouse or lewis rat brain viral antigen was detected in both astrocytes and oligodendrocytes. infection of astrocytes led to the formation of syncytia and degradation of the cytoskeleton. infected rat oligodendrocytes gradually disappeared from the cultures because of cell death. these phenomena indicate that, besides an indirect autoimmune response triggered by infected astrocytes, direct virus‐induced injury to astrocytes or to oligodendrocytes can have a dominant role in the neuropathogenicity of mouse hepatitis virus. the present results underscore the importance of species and developmental stage of experimental animals in the neurotropism and pathogenicity of mhv‐jhm. mouse hepatitis virus (mhv), a coronavirus, is widespread in mouse colonies. although mhv causes no apparent illness in most (fujiwara et al., ), a n acute or chronic state of disease may result after experimental infection. the consequences of the infection depend on the pathogenic potential of the mhv strain (for review, see wege et al., ) and on the mouse strain (stohlman and frelinger, ) . rats and hamsters are not natural hosts for mhv but are susceptible alan r. liss, inc. to experimental infection (bailey et al., ; cheever et al., ) . the neurotropic jhm strain of mhv can cause a n acute encephalitis after intracranial (i.c.) inoculation of suckling rats. postmortem analysis has shown that virus particles are present in oligodendroglial cells and in neurons . in contrast, a chronic demyelinating condition develops when weanling rats are infected. in this case, virions are found only in degenerated oligodendroglial cells nagashima et al., ) . this shift in cellular tropism of mhv-jhm has also been described for infections of mice (knobler et al., a,b) . it has been reported that a temperature-sensitive (ts) mutant of mhv is rarely lethal for mice but frequently results in chronic demyelination (haspel et al., ) . virions were found mainly in oligodendrocytes (knobler et al., ) , suggesting that infection of this cell type by the mutant is the primary cause of demyelination. although replication of mhv-jhm in brain cells is well documented, cell tropism especially in rat brain, remains controversial. for example, in wistar-furth rats, only oligodendrocytes are target cells (beushausen and dales, ) , whereas in lewis rats all glial cells can be infected (massa et al., ) . in both cases the infection induces a pathological condition of the central nervous system (cns). an age-related disease develops in infected wistar-furth rats (sorensen et al., ) , whereas a n "experimental allergic encephalitis (eae) type" mechanism of myelin destruction has been described in lewis rats (watanabe et al., ) . the present study examined the effects of mhv-jhm on cultured brain cells derived from balb/c mice and from wistar and from lewis rats. the results obtained from studies in vitro were correlated with those of in vivo infections. the present observations indicate that sensitivity to mhv-jhm infection depends not only on the species of the animal, but is also affected by the differentiation of both oligodendrocytes and astrocytes. mouse l-cells, obtained from dr. e. lehmann-grube, hamburg, west germany, and sac(-) cells, a moloney sarcoma virus-transformed cell line defective in virus production (weiland et al., ) were grown in dulbec-co's modified eagle's medium containing % fetal calf serum (fcs), supplemented with unitdm penicillin and pg/ml streptomycin (dmem- % fcs). cells from the cerebral hemispheres of newborn rats and mice were isolated as described previously (van berlo et al., ) and grown in dmem- % fcs supplemented with % heat-inactivated horse serum and uaiter insulin. cultures were grown for days in a coa incubator at % humidity and at °c and then were used for the infection experiments. at that stage, the cultures consisted predominantly of astrocytes as shown before (van berlo et al., ) . animals were obtained from the central institute for the breeding of laboratory animals, tno, zeist, the netherlands, and were tested serologically for the absence of anti-mhv antibodies by a plaque-reduction assay. glial cell cultures enriched in oligodendrocytes were prepared as described previously (koper et al., ) . briefly, forebrains of -day-old rat pups were minced and incubated with trypsin at °c. the tissue was then gently disrupted by trituration in the presence of soybean trypsin inhibitor. the cell suspension was sieved through a fine nylon screen to remove tissue debris. dissociated cells were suspended in dmem containing % newborn-calf serum and plated onto poly-l-lysine-coated tissue culture dishes. the following day, the culture medium was replaced by a serum-free, chemically defined medium (koper et al., ) . the virus stock of mhv-jhm (weiner, ) was plaque-purified twice and used to infect sac(-) cells at a multiplicity of infection (moi) of . . culture medium of infected cells was collectec and centrifuged at lowspeed centrifugation to remove cell debris. to precipitate the virus, g/liter polyei hylene glycol (bdh, poole, england) and . g naclaiter at ph . was added followed by incubation a t °c for hours and centrifugation ( , g, minutes). the pellet was resuspended in a small volume of dmem- % fcs, and the suspension was then clarified by short centrifugation. to determine the yield of infectious virus, virus stocks were plaque-titrated on l-cells using a n overlay of . % bacto-agar (difco, detroit, mi, usa) in dmem- % fcs. the plaques were wad days postinfection. virus stocks usually had titers of about lo plaqueforming units (pfu) per ml. wistar rats ( and days old) and balb/c mice ( days old) were injected i.c. with approximately x lo pfu of mhv-jhm in a volume of pl. control animals received pl of the dilution medium. kinetics of virus g r o w t h sac(-) cells and primary brain cells were infected as described previously (van berlo et al., ) . culture supernatants of infected sac:(-) cells and brain cells were collected and hours postinfection (pi.), respectively, and infectious virus was assayed by plaque titration. a n t i s e r a and indirect immunofluorescence a monoclonal antibody against the nucleocapsid protein of mhv-jhm (a gift from m. buchmeier, la jolla, ca, usa) and a rabbit anti-mhv-a serum (rottier et al., ) were used to recognize the viral proteins of mhv-jhm. rabbit antiglial fibrillary acidic protein (gfap) serum (obtained from dakopatts, copenhagen, denmark) and a monoclonal antigalactocerebroside (galc) antibody (ranscht et al., ) were used as markers for astrocytes (bignami et al., ) and oligodendrocytes (raff et al., ) , respectively. monoclonal antibody a b was used to distinguish between type astrocytes, which are a b -and gfap+, and type astrocytes (a b +, gfap+) (raff et al., ) and was also used to identify oligodendrocytetype astrocyte ( - a) progenitor cells, which are a b +, galc-, gfap- (raff et al., ) . goat antimouse igg or igm, conjugated to rhodamine (tritc), were used to visualize the binding of the anti-galc antibody or the binding of the a b antibody, respectively; and swine antirabbit immunoglobulin g (igg) conjugated to fluorescein (fitc) was used to detect the binding of the anti-gfap and anti-mhv antibodies. double-immunolabelling procedures were carried out a s described previously (noble and murray, ) . brains were quickly frozen in semisolid isopentane a t - °c. sections ( pm) were cut, fixed in methanol at - °c for minutes, and immunolabelled as described above. sac(-) and brain cells grown in -mm tissue culture dishes were infected and labelled from to hours p.i. (sac[-]) or from to hours p i (brain cells) using ml methionine-deficient minimal essential medium (mem), supplemented with % fcs, and pci smethionine, ci/mmol (the radiochemical centre, amersham, england). after the labelling period, cells were rinsed with cold pbs and lysed for minutes in . ml lysis buffer (koolen et al., ) . lysates were centrifuged for minutes a t , g and then processed directly or stored at - ooc. virus-specific proteins were immunoprecipitated from p of portions of the lysates with p rabbit anti-mhv-jhm serum (a gift from s. siddell, wurtzburg, west germany) following the methods previously described (koolen et al., ) . samples were electrophoresed in % acrylamide- . % bisacrylamide gels (rottier et al., ) , and proteins were visualized by fluorography on preflashed fuji rx film at - ooc. the mortality of rat pups after viral infection dropped rapidly with the age of the animals. whereas % ( / ) of the rat pups died of acute encephalitis when infected at postnatal day , only % ( / ) died when infected days after birth. in addition, most ( / ) of the rat pups inoculated i.c. with mhv-jhm a t postnatal day that survived developed tremors and paralysis of the hind legs. in contrast to the effects of mhv-jhm inoculation in rats, mice infected with mhv-jhm at days after birth all died (table ) . to determine the presence of viral antigen in the cns of acutely or lethally afflicted and chronically paralysed rats, frozen brain sections were immunolabelled with viral-specific antibodies and anti-gfap antibodies and examined by fluorescence microscopy. in the acute disease, high concentrations of viral antigen were found throughout the brain. double immunolabelling showed that viral antigen could be traced in astrocytes (fig. a,b) but was also present in other cells. in contrast, the very low levels of viral antigen present in brain sections of paralysed animals were not present in astrocytes (fig. ic,d) . no antigen was detected in the brains of uninfected animals (fig. e,f) . to study the infection of astrocytes with mhv-jhm in vitro, cultures of brain cells derived from either newborn mice or rats were prepared and infected with virus. twenty hours after infection, cultures, consisting predominantly of type astrocytes (van berlo et al., ) , were fixed and immunolabelled with anti-mhv and anti-gfap antibodies. many multinucleated cells (syncytia) were observed in cultures of mouse brain cells, and these syncytia were positive both for viral antigens and gfap (data not shown). syncytium formation was associated with a breakdown of glial filaments, as judged by their diffuse distribution within the infected cells ( fig. a) . in contrast, no mhvinfected astrocytes and no multinucleated gfap-positive cells were observed in cultures derived from the brains of newborn rats (fig. b) . to analyse the expression of viral proteins, infected sac(-) and mouse or rat astrocytes were labelled with "s-methionine. viral proteins in the lysates of these infected cells were immunoprecipitated with a serum, directed against purified virions, and separated by polyacrylamide gel electrophoresis (page). in mhv-jhm infected sac(-) cells, eight virus-specific smethionine labelled polypeptides were detected (fig. ) . the four low-molecular-weight proteins ( , k, , k, , k, and k) are molecular species of the e l protein brain cells from wistar rats after growth in culture for days. cells were inoculated with mhv-jhm (moi ) and hours later fixed and stained for gfap using a rabbit antiserum and a fitc-conjugated swine antirabbit igg. note that syncytium formation with breakdown of glial filaments is observed only in the mouse preparation (a). x . (siddell et al., ) . the high-molecular-weight protein bands of k and k are the membrane protein (e ) and its precursor, respectively (siddell et al., ) . in addition, two prominent bands were observed with molecular weights ( k and k) in the range of the nucleocapsid protein (n). the lower band might be a degradation or not-phosphorylated product of the upper band. the induced polypeptides in mhv-jhm infected mouse brain cells were comparable to those in infected sac(-) cells. in rat astrocytes, no viral-induced proteins were observed (fig. ) . additional experiments were carried out whether or not the cultures produced infectious virus. culture supernatants were plaque-titrated on l-cells, and the amounts of infectious virus particles in the mouse and rat brain cell cultures were compared with those in infected sac(-) cells. the results showed that the jhm strain of mhv was produced by sac(-) cells ( pfu/ cell at hours p.i.) and by cultures of mouse brain cells ( pfu/cell a t hours p.i.), but not by cell cultures derived from rat brain. fir (astrocytes). cells were harvested and viral proteins immunoprecipi-in a serum-free, chemically defined medium that tated from cell lysates using a n anti-mhv-jhm serum. immunoprecip-favours the development of oligodendrocytes (koper et itates were analysed by page in % polyacrylamide gels. molecular weight of standards ( -a) and mhv proteins ~ , ~ , and n are al., ) . one day after plating, most of these cells indicated. were positive for a b and expressed neither galc nor gfap. this antigenic phenotype is characteristic of the oligodendrocyte type astrocyte progenitor cells described by raff et al. ( ) . as expected, after days of growth in a chemically defined medium, most a b positive cells had differentiated into galc-positive oligodendrocytes, and virtually all a b -positive cells had disappeared from the cultures ( van der pal et al., ) . infection of these oligodendrocyte-enriched cultures with mhv-jhm showed that day after infection only a few galc-positive (fig. a,b) and no a b -positive cells (results not shown) contained viral proteins. after hours infected oligodendrocytes lost their complex network of brahches and appeared to be dying (fig. c,d) . similar experiments were performed with oligodendrocyte-enriched cultures derived from lewis rats or balb/c mice. double-label immunofluorescence revealed that oligodendrocytes of both species stained strongly with the anti-mhv antibody (fig. a,c, respectively) . cultures derived from lewis rats also showed foci of fig. . rat oligodendrocytes in culture can be infected with mhv-jhm. oligodendrocyte-enriched cultures derived from cerebra of week-old wistar rats were infected with mhv-jhm on day in culture. one day (a,b) or hours (c,d) later, the preparation was incubated with a monoclonal antibody against galc, followed by a tritc-conjugated goat antimouse igg. after fixation the preparation was incubated with a rabbit anti-mhv antiserum coupled with fitc. a,c: fitc fluorescence (mhv+); b,d: tri'rc fluorescence(galc+). note the necrotic appearance of a n infected oligodendrocyte in panels c and d. virus-positive galc-negative multinucleated cells, probably infected astrocytes (fig. ) . in primary brain cell cultures from lewis rats consisting mainly of astrocytes, many multinucleated cells were observed that were positive for viral antigen (results not shown). no viral antigen was detected in mock-infected cells (fig. e ,f). it should be emphasized, however, that in cultures from both rat species, few galc-positive cells were infected. the number of infected oligodendrocytes increased with the virus dose when cultures were inoculated at higher multiplicities of infection (moi) (results not shown). however, even after a n infection with a n moi of fpu/cell, only a small fraction (less than %) of the galc+ oligodendrocytes was infected compared with mouse cultures (table ). in addition, the number of infected oligodendrocytes in wistar rat cultures decreased significantly after hours, suggesting that the infection was lethal for these cells. in lewis rats the number of infected cells remained constant, whereas in balb/c mice there was a n increase in the number of viral positive cells, suggesting that the produced virus infected other cells. the effects of mhv-jhm infection in the cns depend on the species of the experimental animal. mice developed a n acute, lethal encephalitis within to days; in contrast, rats that survived a n infection by mhv-jhm often exhibited a chronic disease (table ) . how can we explain these differences between related animal species? an answer might be that the susceptibility to infection with mhv of the various cns cell types is species dependent. the present experiments support this hypothesis and agree with previous observations made in situ (powell and lampert, ; nagashima et al., ; sorensen et al., ) and in vitro (dubois-dalcq et al., ; beushausen and dales, ; wilson et al., ) . when mhv-jhm infection caused an acute encephalitis in young rat pups, antigen could be found throughout the brain, mainly in astrocytes (fig. a,b) . however, when chronic paralysis followed infection, unidentified cells were found to be positive for the viral antigen. these results suggest that a susceptibility of immature brain cells to infection by mhv-jhm may result in a n acute disease in young rat pups. acute encephalitis in mice, after infection with mhv- number of virus-uositive cellsb jhm, is always associated with infection of astrocytes (wilson et al., ) . this susceptibility to mhv-jhm infection is found also in cultures of mouse astrocytes and lewis rats (massa et al., ) . astrocytes from wistar rats are not susceptible to mhv-jhm infection, suggesting that they lack distinct binding sites for mhv-jhm. in this context, it is noteworthy that boyle et al. ( ) reported that target cells from susceptible animals have a mhv-binding molecule on their membranes. a specific receptor, therefore, must be present on astrocytes from lewis rats, a s viral replication occurs in these cells. in cultures of wistar rat glid cells, only oligodendrocytes (galc-positive) were infected (fig. ) , whereas a b -positive cells (progenitor cells and type- astrocytes) were resistant to virus infection. beushausen and dales ( ) showed that mhv-jhm replication in oligodendrocytes, derived from newborn wistar-furth rats, is maximal between and days after explantation. replication is suppressed between day and , which fits with the report of the same group (sorensen et al., ) that their rats became highly resistant to mhv-jhm infection weeks after birth. it appears, therefore, that mhv-jhm can induce paralysis in wistar rats only during a short time span of the differentiation phase of oligodendrocytes. oligodendrocytes (walker et al., ) and astrocytes (schachner, ) in culture develop according to a fixed time schedule. future studies are necessary to sort out which differentiation phase and surface property is essential for infection of oligodendrocytes by mhv-jhm. the present results underscore the notion that pathogenesis of mhv-jhm coronavirus-induced demyelination may involve two completely different mechanisms. first, astrocytes of lewis rats infected with mhv-jhm support a persistent infection in which the rate of virus production is low (massa et al. ). such infections could initiate processes having indirect long-term effects on oligodendrocytes and myelin. second, a n infection by mhv-jhm in astrocytes in mice (wilson et al., ) and in wistar rat pups by mhv-jhm causes a fatal acilte disease. older wistar rat pups survive but sometimtts become paralysed; lytic infection of oligodendrocytee may cause foci of viral induced demyelination. the authors thank martin van eijk for his skillful technical assistance and dr. mark noble for his advice and for providing the antisera against the neural cell markers. these investigations were supported by the prinses beatrix fonds. a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin. in vivo and in vitro models of demyelinating disease. xi. tropism and differentiation regulate the infectious process of coronaviruses in primary explants of the rat cns localization of the glial fibrillary acidic protein in astrocytes by immunofluorescence genetic resistance to mouse hepatitis virus correlates with absence of virusbinding activity on target tissue a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin. i. isolation and biological properties of the virus cell tropism and expression of mouse hepatitis viruses (mhv) in mouse spinal cord cultures carrier state of antibody and virus in a mouse breeding colony persistently infected with sendai and mouse hepatitis viruses temperature-sensitive mutants of mouse hepatitis virus produce a high incidence of demyelination mouse hepatitis virus type (jhm strain)-induced fatal central nervous system disease. i. genetic control and the murine neuron a s the susceptible site of disease selective localization of wild type and mutant mouse hepatitis virus (jhm strain) antigens in cns tissue by fluorescence, light and electron microscopy virus persistence and recurring demyelination produced by a temperaturesensitive mutant of mhv- temperaturesensitive mutants of mouse hepatitis virus strain a : isolation, characterization and neuropathogenic properties culture of rat cerebral oligodendrocytes in a serum-free, chemically defined medium effects of triodothyronine on the synthesis of sulfolipids by oligodendrocytes enriched glial cell cultures analysis of murine hepatitis virus (jhm strain) tropism toward lewis rat glia cells in vitro: type astrocytes and brain macrophages (microglia) a s primary glial cell targets early and late cns-effects of coronavirus infection in rats purified astrocytes promote the in vitro division of a potential glial progenitor cell oligodendrocytes and their myelin-plasma membrane connections in jhm mouse hepatitis virus encephalomyelitis gacactocerebrogide is a specific cell-surface antigenic marker for oligodendrocytes in cultures a glial progenitor cell that develops in vitro into a n astrocyte or a n oligodendrocyte depending on culture medium development of oligodendrocytes and schwann cells studied with a monoclonal antibody against galactocerebroside translation of three mouse hepatitis virus strain a subgenomic rnas in xenopus laeuis oocytes glial antigens and the expression of neuroglial phenotypes coronavirus jhm: intracellular protein synthesis in vivo and in vitro models of demyelinating diseases. . jhm virus infection in rats resistance of fatal central nervous disease by mouse heuatitis virus strain jhm. i. genetic analysis restricted replication of coronavirus mhv-a in primary mouse brain astrocytes correlates with reduced pathogenicity effects of insulin and insulin-like growth factor (igf- ) on oligodendrocyte-enriched glial cultures immunocytochemical characterization of cell cultures grown from dissociated - -day post-natal rat cerebral tissue. a developmental study adoptive transfer of eae-like lesions from rats with coronavirus-induced demyelinating encephalomyelitis jhm infections in rats a s a model for acute and subacute demyelinating disease non-producer malignant tumor cells with rescuable sarcoma virus genome isolated from a recurrent moloney sarcoma pathogenesis of demyelination induced by a mouse hepatitis virus (jhm virus) in vivo and in vitro models of demyelinating diseases. xv. differentiation influences the regulation of coronavirus infection in primary explants of mouse cns key: cord- - qhoqa authors: hingley, susan t.; leparc-goffart, isabelle; seo, su-hun; tsai, jean c.; weiss, susan r. title: the virulence of mouse hepatitis virus strain a is not dependent on efficient spike protein cleavage and cell-to-cell fusion date: journal: j neurovirol doi: . / sha: doc_id: cord_uid: qhoqa the cleavage and fusion properties of recombinant murine hepatitis viruses (mhv) were examined to assess the role of the cleavage signal in determining the extent of s protein cleavage, and the correlation between cleavage and induction of cell-to-cell fusion. targeted recombination was used to introduce amino acid substitutions into the cleavage signal of the fusion glycoprotein (spike or s protein) of mhv strain a . the recombinants were then used to address the question of the importance of s protein cleavage and viral-mediated cell-to-cell fusion on pathogenicity. our data indicate that cleavage of spike is not solely determined by the amino acid sequence at the cleavage site, but may also depend on sequences removed from the cleavage site. in addition, efficient cell-to-cell fusion is not necessary for virulence. murinehepatitis viruses (mhv) are coronaviruses that can be hepatotropic and/or neurotropic (hirano et al, ) . for example, strains mhv-a and mhv- are both neurotropic, and can produce meningitis, encephalitis, and demyelinating disease. in addition, mhv-a , along with another strain, mhv- , can cause severe hepatitis. mhv- , although considerably more neurovirulent than mhv-a , causes minimal hepatitis, whereas mhv- causes only minimal disease in the central nervous system (cns) . an important determinant of pathogenicity for mhv is the spike, or s, glycoprotein. this protein is responsible for both viral attachment to the host cell receptor and virus-induced membrane fusion. the s protein of all mhv strains mediates fusion between the viral envelop and the eukaryotic cell membrane, an event that is necessary for entry into a host cell. the s protein of some strains of mhv can also induce cell-to-cel l fusion, producing syncytia. in this report, we consider viruses capable of syncytia formation to have a fusion-positive phenotype, whereas those that do not produce syncytia are referred to as nonfusogenic. the s protein is a -kda glycoprotein that is cleaved into two -kda subunits, the aminoterminal s and carboxyterminal s subunits (luytjes et al, ) . although cleavage of the s protein correlates with more ef cient syncytia formation in vitro (bos et al, ; gombold et al, ; stauber et al, ; taguchi et al, ) , the relationship between fusogenicity in vitro and pathogenicity in vivo has not been clearly established for the different strains of mhv. this is in contrast to several other virus systems, where cleavage of the fusion glycoprotein is required for fusion activity of this protein, and cleavage has a direct affect on virulence (maisner et al, ; rott et al, ; stadler et al, ; volchkov et al, ) . cleavageof mhv s proteins occurs following a basic-x-basic-basi c amino acid sequence motif that is recognized by furin or furin-like proteases (barr, ; nakayama, ) . the furin-like proteases are cellular enzymes that are responsible for processing hormone precursors into mature, active hormones. these proteases have also been shown to cleave a variety of viral fusion glycoprotein s (bolt and pedersen, ; bolt et al, ; hallenberger et al, ; vey et al, ; volchkov et al, ) . the fusogenic strains mhv-a and mhv- both have a cleaved s protein and have cleavage signals of rrahr and rrarr, respectively. mhv- , on the other hand, is nonfusogenic, and expresses an uncleaved s protein having the sequence hrars at the region homologous to the mhv cleavage signal. in order to evaluate the role of the cleavage signal in determining fusion phenotype, and potentially pathogenicity, we wanted to generate viruses with different mutations in the cleavage signal of their s proteins. our previous studies using mutants of mhv-a derived from persistently infected glial cells indicate that a wild-type fusion phenotype is not required for replication in the cns (hingley et al, ) . these mutants, however, encode two amino acid substitutions in the s protein. one mutation lies within a putative receptor binding region of s (kubo et al, ) , and the second, at amino acid codon (h d), is within the cleavage signal of s (luytjes et al, ) . previous data suggested that the h d substitution prevents cleavage of the s protein, which would account for the delayed fusion phenotype of these mutants, because several cleavage and fusion revertant viruses replaced the aspartic acid residue (gombold et al, ) . the mutant cleavage and fusion phenotypes were seen only when the h d substitution was present (hingley et al, ; leparc-goffart et al, ; however, none of the glial cell mutants had the h d mutation alone, so it was necessary to isolate recombinant viruses expressing the h d mutation alone in order to assess the potential affect of this particular mutation on cleavage of s, fusogenicity, and virulence. targeted recombination has been used to isolate murine hepatitis viruses with a de ned spike gene placed into an mhv-a background (fischer et al, ; koetzner et al, ) . we have used this technology to select viruses that contain either the h d point mutation, or the corresponding sequence from the mhv- s gene, in the cleavage signal of the mhv-a spike protein. these cleavage site recombinants indicate that the cleavage signal alone is not the sole determinant of s protein cleavage and viral fusion phenotype. to assess the in vivo and in vitro phenotypes of the recombinants, it was important to include as a control wild-type recombinants in which the wild-type s genes of mhv-a or mhv- are (re)introduced into the parent mhv-a background. these recombinants, described in previous studies (das sarma et al, ; leparc-goffar t et al, ; phillips et al, ) , would control for possible effects of mutations outside of the s gene, because all recombinants have the same background genes and therefore differ only by the de ned mutation near the cleavage site. this is the rst time that pairs of viruses have been available for study that differ only by de ned mutations known to disrupt cleavage and fusion properties of the s protein. several laboratories have shown that lack of cleavage of s correlates with less ef cient induction of cellto-cell fusion (bos et al, ; gombold et al, ; stauber et al, ; taguchi et al, ) . previous studies from this laboratory using mutants isolated from persistently infected glial cells (gombold et al, ) have suggested that an h d mutation, which changes the rrahr cleavage signal to an rradr sequence, is associated with an uncleaved s protein, but did not directly assess the affect of that mutation on virulence. as one would predict, and as seen in figure , the h d mutation did dramatically reduce the amount of s protein cleavage compared to cleavage of the wild-type protein (s h d r and s h d r versus s a r ). however, upon examining multiple blots of s h d r and s h d r lysate and virion samples, it was also evident that, although cleavage was inhibited, it rarely appeared to be completely prevented. cleavage of s protein in recombinant viruses. virions derived from infected l cells were analyzed by western blot as described in materials and methods. on the left is shown the migration of molecular weight markers, in kda, while on the right is shown the migration of cleaved and uncleaved forms of s protein (arrowheads). image analysis of either viral pellets or infected cell lysates offered a semiquantitative measure of the relative amounts of cleaved and uncleaved s protein. the measure of s cleavage was based on the relative intensity of protein bands migrating at either kda (uncleaved s) or kda (cleaved s). it does not take into account oligomeric, unglycosylated , or alternatively processed forms of s. these data indicate that for s protein derived from s h d recombinants, less than % of the material in the two bands migrated as cleaved s, whereas approximately % of the s a r s protein migrated as cleaved s (table ) . to further examine the relationship between cleavage of the s protein and the sequence of the cleavage signal, we isolated a second pair of recombinant viruses (s cs recombinants) that contain the mhv- cleavage signal (tshrars) in an mhv-a spike background. because the s protein of mhv- is not cleaved, it was anticipated that this change would create a variant of mhv-a with an uncleaved s protein. however, cleavage did occur in the two s cs recombinants, s cs r and s cs r , albeit at reduced levels relative to wild-type mhv-a s protein. the amount of cleaved s, or -kda protein, observed with the s cs recombinants ( % to %) was typically slightly greater than that of the s h d recombinants, but markedly less than that seen with s a r (table ). in contrast, s protein expressed by penn- - , a recombinant virus with the entire mhv- s gene placed into the mhv-a background, appeared uncleaved in western blots ( figure ). the calculated % cleaved s of penn- - , based on image analysis, was < % (table ), indicating that the optical density of the portion of the lane corresponding to the migration of a -kda protein was minimally above background levels. previous studies indicated that the h d mutation in s was necessary for a delayed fusion phenotype, but did not prove that the mutation alone figure fusion phenotype of recombinant viruses. con uent l monolayers were infected with virus at an moi of . the percentage of nuclei involved in syncytia was quanti ed as described in materials and methods. the results shown are representative of three separate fusion assays. the mean viral titers of duplicate infected wells at or hours post infection are listed in table . was responsible for this phenotype (gombold et al, ) . figure compares the fusion phenotype in l cells of s h d recombinants and that of a wildtype recombinant (s a r ) that contains the wildtype mhv-a s sequence. as shown in figure , the fusion phenotype of the s h d recombinants was delayed relative to the wild-type control, and therefore this mutation was suf cient to inhibit cell-to-cell fusion. because the mhv- s protein is known to be nonfusogenic, it was predictable that penn- - , which expresses the mhv- s protein, would have a similar cleavage and fusion phenotype, as indeed was the case (figure ). we had originally expected the s cs recombinants, s cs r and s cs r , to be nonfusogenic, but instead they displayed a delayed fusion phenotype similar to that of the s h d recombinants, and consistent with the low level of cleavage observed with these viruses. any observed differences in the amount of cell-to-cel l fusion produced by the various recombinant viruses were not due to inhibition of viral replication in cell culture. as seen in table , all viruses had similar titers at and h post infection. . £ . £ sh dr . £ . £ scs r . £ . £ scs r . £ . £ penn- - . £ . £ a pfu/ml indicates the mean viral titer of supernates from duplicate wells infected for the fusion experiment presented in figure . in some viral systems, cleavage and subsequent activation of the fusion protein correlates with viral virulence and tropism (maisner et al, ; mccune et al, ; rott et al, ; stadler et al, ) . the cleavage site recombinants provide a means to examine the importance of viral-induced cell-to-cell fusion on pathogenicity for mhv. the virulence of these viruses, as well as their ability to replicate in vivo, was assessed following intracerebral inoculation of c bi/ mice with s a r , s h d r , s h d r , s cs r , and s cs r . the ld of the wild-type recombinant is approximately £ pfu following intracerebral (ic) inoculation, similar to that of wild-type mhv-a (hingley et al, ; leparc-goffar t et al, ) (table ). the ld values of s h d r and s h d r are four-to vefold higher, at : £ and : £ , respectively, indicating that these viruses are only slightly attenuated relative to wild-type virus. on the other hand, the ld values of the s cs recombinants (> £ ; table ) are considerably higher than both wild type and s h d recombinants. as seen in figure , the s h d recombinants replicated in the brains of infected animals as well as or better than wild-type recombinants, although liver titers tended to be more variable, and were more likely to be reduced in mice infected with the s h d recombinants. in contrast, replication of the s cs recombinants was signi cantly impaired, in both liver and brain, compared to that of s a r (figure ). brain titers in mice infected with the s cs recombinants peaked anywhere from to days post infection, at which point they were typically more than -fold lower than those in mice infected with s a r or the s h d recombinants. liver titers were often undetectable in mice infected with the s cs recombinants, though low titers were seen occasionally, especially at days post infection. immunohistochemistry was performed to examine the distribution of viral antigen in brain and liver sections from infected mice. positive staining for viral antigen was observed in similar regions of the brains of mice infected with s a r , s h d recombinants, or s cs recombinants ( figure ) . however, consistent with the low titers detected in tissue from animals infected with s cs recombinants, viral antigen staining was much less prevalent in these mice. positive staining for viral antigen was often observed in the following regions: ( ) at the level of the hippocampus and habenular nucleus; (figure ; a, c, e) ( ) in the frontal cortex just off the midline, at the level of the accumbuns nucleus, and including the cingulate and infralimbic cortex; (figure ; b, d, f) and ( ) in the vicinity of the hippocampus and along the lateral ventricle. viral antigen was associated predominately with cells that morphologically resembled glial cells, neurons, and choroid plexus epithelia. in ammation was often observed in regions staining positive for viral antigen. extensive labeling for viral antigen was observed in livers of mice infected with the three types of recombinant viruses ( figure ), even though viral titers were often undetectable in mice infected with s cs recombinants. however, necrotic foci were more prevalent and larger in animals infected with a a r or s h d recombinants than in mice infected with the s cs recombinants. our initial studies on the pathogenesis of mhv-a focused on the role of the s protein in determining pathogenicity, because this protein mediates both viral attachment and cell-to-cel l fusion. a mutation located near the cleavage site of s, h d, correlated with lack of cleavage of the s protein and a delayed fusion phenotype (gombold et al, ) . however, it was not established that this mutation alone was suf cient to inhibit cleavage and fusion. to assess the relationship between cleavage of the s protein, fusogenicity and virulence, targeted recombination was used to generate recombinant mhv viruses whose spike proteins contained altered cleavage signals, including the h d amino acid substitution. a pair of recombinant viruses that replaced the mhv-a cleavage signal with the corresponding mhv- sequence (s cs recombinants) was also isolated. mhv- has a noncleaved, nonfusogenic s protein, so it was thought that the s cs recombinants would have similar cleavage and fusion phenotypes. interestingly, cleavage was only inhibited in the s cs recombinants, not abolished. the low level of s cleavage observed in both the s h d and s cs recombinants could account for the delayed fusion phenotype of these viruses. the mhv-a s protein is cleaved following a basic-x-basic-basi c motif (rahr). by substituting the mhv- sequence (tshrars) for the mhv-a sequence (ksrrahr) at the cleavage site, we disrupt two consecutive basic-x-basic-basi c motifs (rahr and ksrr). we do not know if the ksrr sequence is used as an alternate cleavage site in s; however, eliminating both potential cleavage motifs would prevent the possible use of the ksrr sequence as a cleavage site in the absence of the rahr signal. as shown in this study, disruption of the rahr motif inhibits, but does not abolish, both cleavage of s and s-mediated cell-to-cel l fusion. this observation figure replication of wild-type and recombinant viruses in the brains and livers of infected animals. c bi/ mice were infected ic with pfu of virus. animals were sacri ced at the indicated times, and virus present in brain (a) or liver (b) homogenates was titered by plaque assay on l cells. data are expressed as the log pfu/g of tissue; shown is the mean § standard deviation of the titers from to animals/virus. titers that are signi cantly different from sa r are indicated ( ). the limit of detection was . log pfu/g. is consistent with the results from several laboratories that use a vaccinia expression system to demonstrate that mutating the basic amino acid residues at the cleavage site disrupts both cleavage and fusion properties of s, but does not completely prevent cellto-cell fusion (bos et al, ; stauber et al, ; taguchi et al, ) . however, the levels of s protein present on the cell surface by the vaccinia expression system may be arti cially high, and not accurately represent the relationship between cleavage of s and fusogenicity for intact virus. therefore, the level of cell-to-cel l fusion observed following infection with the s h d and s cs recombinant viruses would be a more precise measure of the importance of cleavage of s for induction of cell-to-cel l fusion. these data con rm, using intact virus instead of a vaccinia expression system, that mutations altering the basic-xbasic-basic motif of the cleavage signal of s are sufcient to disrupt both cleavage of s and viral-mediated cell-to-cel l fusion. sequences outside of the cleavage signal must in uence both cleavage and fusogenicity. previous studies showed that fusogenicity is in uenced by sequences away from the cleavage site, in viruses in which spike protein is cleaved. for example tsai et al ( ) hypervariable region of s that converts a cleaved, fusogenic s protein into an s protein that is still cleaved, but nonfusogenic. there are additional examples of mutations either in the hypervariable region of s (phillips et al, ) , or in the heptad repeat region of s (gallagher et al, ; luo and weiss, ) , that disrupt s-mediated cell-to-cel l fusion independently of cleavage. the data reported here are consistent with the observation that cleavage of s correlates with more ef cient induction of cell-to-cel l fusion. however, our data differ from these other studies in that they are the rst observation of viruses with the same cleavage signal sequence (penn- - versus s cs recombinants), yet different levels of cleavage and fusion. thus, the cleavage signal alone does not determine cleavage of s. krueger et al ( ) suggest that s-mediated cell-tocell fusion correlates with a dissociation of the s and s subunits of the mhv spike protein. consequently, a more stable interaction between s and s would be associated with a delayed fusion phenotype. inhibition of s protein cleavage would be associated with a more stable interaction between s and s and correlate with a loss of fusogenicity. this could account for the delayed fusion phenotype observed with the s h d and s cs recombinants. for coronaviruses, the relationship between induction of cell-to-cel l fusion and virulence is complex. the data on the s h d and s cs recombinants suggest that the ability to produce syncytia in vitro is not a predictor of pathogenicity. the s cs recombinants were considerably more attenuated than the s h d recombinants, even though both sets of recombinants had the same cleavage and fusion phenotypes. similarly, the s cs recombinants did not replicate in vivo as well as the s h d recombinants, although the regions of the brain that were infected by the different recombinants was similar. for the viruses examined in this study, there was no correlation between ability to induce cell-to-cel l fusion in vitro and ability to cause disease in vivo. the only difference between the s h d and s cs recombinants is the amino acid sequence at the carboxy-terminus of the s subunit. it is surprising that these two different cleavage signals, which produce the same fusion/cleavage phenotypes in vitro, produce virus with a greater than log difference in ld , and a signi cant difference in ability to replicate in vivo. the different in vivo properties of these two sets of recombinants indicate that disrupting the cleavage signal of s affects more than just cleavage and fusion phenotypes. this observation, along with evidence indicating that mutations removed from the cleavage signal disrupt cleavage and fusogenicity of the s protein, implies that the conformation of s is an important determinant of function. mutations in one region of s may alter the conformation of this protein as a whole, and subsequently disrupt protein functions mediated by other regions of s. given the role of the s protein in viral attachment and induction of immune response, protein functions that might be altered by mutations in s include viral af nity for host cell receptors, as well as induction of neutralizing antibody and t cells. it is possible that the s cs recombinants have lower af nity for cellular receptors in the brain and/or liver, which could account for their impaired ability to replicate in these tissues. alternatively, conformational differences between the s h d and the s cs recombinants could produce differences in the epitopes recognized by the immune response, which could in turn in uence viral clearance. thus, the mutations introduced into the cleavage signal of the s protein, by altering the conformation of s, might indirectly affect the in vivo phenotype of the recombinant viruses, even though their in vitro fusion phenotypes are similar. virus and cells mhv-a was obtained from lawrence sturman (albany, ny). alb , obtained from paul masters, is a temperature-sensitiv e variant of mhv-a that contains an -nucleotide deletion (resulting in a -amino acid in-frame deletion) in a nonessential spacer region in the n gene (koetzner et al, ) . the recombinant viruses s a r and penn- - have been described previously (das sarma et al, ; leparc-goffart et al, ) . s a r and penn- - contain the mhv-a and mhv- s gene, respectively, in the mhv-a background. the recombinant viruses s cs r and s cs r contain an mhv-a s gene with the mhv- cleavage site (tshrars replaces ksrrahr). the s h d recombinant viruses, s h d r and s h d r , contain a single point mutation (d replaces h) at amino acid codon . this mutation results in a cleavage signal of rradr, mimicking the mutation observed in the fusion-delayed glial cell mutants previously charac- terized (gombold et al, ; hingley et al, ) . the isolation of the s h d recombinants and the s cs recombinants is described below. murine l cells and cl- cells were maintained on plastic tissue culture dishes in dulbecco's minimal essential medium (mem) with % fetal bovine serum (fbs). spinner cultures of l cells were maintained in joklik's mem with % fbs at densities between £ and £ cells per milliliter. the construction of pfv (obtained from paul masters) was described previously (fischer et al, ) . this plasmid contains the entire mhv-a s gene (gene ), and all the structural genes of s, including a wild-type n gene sequence. the sequence of the s gene in pfv is the same as that of our wildtype mhv-a strain, except for silent changes in codons and , resulting in the loss of a hindiii site and the concomitant addition of an asei site. as described previously (leparc-goffart et al, ) , these changes enabled a hindiii site following the polya tail to be used for linearization of the plasmid for rna transcription, as well as providing a way to distinguish the s gene of the parent virus from that derived from pfv in the recombinant viruses. polymerase chain reaction (pcr) mutagenesis, with vent polymerase, was used to introduce the h d mutation into the s gene of pfv . a fragment containing the h d mutation was generated by ampli cation of two shorter fragments. the end fragment was generated using primers fs and rsh d, and the end using primers wzl and rs (table ) . primers rsh d and wzl introduced a histidine to aspartic acid nucleotide substitution in amino acid codon . the -bp and -bp pcr fragments were gel puri ed and used as templates for a third pcr reaction with primers fs and rs . the resulting -bp fragment was gel puri ed, digested with restriction enzymes draiii and xhoi, and then cloned into the corresponding sites in pfv . this clone was designed pfv -h d. the entire draiii/xhoi fragment of pfv -h d was sequenced to verify the presence of the h d mutation, as well as the absence of other mutations. pcr mutagenesis was similarly used to alter the cleavage signal of mhv-a s gene so that it resembled that of mhv- (cs mutation). primers fs and wzl a were used to amplify a -bp fragment from two smaller overlapping templates generated using primers fs and csr, and csf and wzl b (table ). the -bp fragment was digested with draiii and xhoi and cloned into the corresponding sites in the plasmid pmh , creating the plasmid pmh -cs . the vector pmh is similar to pfv , but contains a extension of the s gene that includes the end of the he gene (fischer et al, ) . the draiii/xhoi fragment of pmh -cs was sequenced to con rm the presence of the cleavage signal mutations and the absence of additional mutations. targeted rna recombination targeted rna recombination was carried out between parent virus having an mhv-a background, and rnas transcribed from either wild-type pfv or pfv -h d. infection of l cells with parent virus, transfection with the synthetic rna, and isolation and plaque puri cation of recombinant virus were carried out as described previously (leparc-goffart et al, ; masters et al, ) . targeted recombination using the pmh -cs plasmid was performed in a similar manner, except the parent virus contained the feline s protein necessitating a different screening protocol for recombinant virus. this protocol has also been previously described (kuo et al, ; phillips et al, ) . the presence of the desired mutation, h d or cs , was con rmed by sequencing. templates for sequencing were derived by reverse transcriptase-polymerase chain reaction ampli cation (rt-pcr) of cytoplasmic rna extracted from virus-infected l cell monolayers. pcr products were puri ed (wizard prep; promega) and analyzed by automated sequencing using the taq dye terminator procedure according to the manufacturer's protocol (taq dyedeoxy terminator cycle sequencing kit; applied biosystems, foster city, ca). each fragment was sequenced in both directions. viral fusion assay l cell monolayers were prepared in -well plates in dubelcco's mem with % fbs. con uent monolayers were infected with each virus at a multiplicity infection (moi) of and incubated for h at ± c. following adsorption, the cells were washed with hanks balanced salt solution (gibco) two times and then fed with ml of dmem- % fbs. at the times indicated, the supernatants were removed and titered by plaque assay on l cells as previously described (gombold et al, ) . the infected cells were xed with % paraformaldehyde, stained with hematoxylin, and fusion quanti ed by counting the number of nuclei involved in syncytia, and expressing this as a percent of total nuclei in a eld. two elds of approximately nuclei each were counted, in duplicate wells, for each virus. the mean § sd of percent fusion was then calculated from the four elds (approximately nuclei) examined. western blot analysis of s l cell monolayers in -well plates or t asks were infected with or ¹l virus, respectively, at an moi of to pfu/cell. after the virus was allowed to attach to the cells for h, additional media was added and the infection continued for to h. virions present in culture supernates were obtained by ultracentrifugatio n at , £ g for h. viral lysates were prepared by lysing infected cells with ¹l lysis buffer ( mm tris ph . , mm nacl, % np- ). viral pellets and lysates were electrophoresed into % sodium dodecyl sulfate (sds)polyacrylamide gels in a tris acetate buffer system (novex) and transferred to nitrocellulose or pvdf membrane. mhv s protein was detected using a polyclonal goat anti-s antibody (ao ; provided by k holmes, university of colorado hsc, denver, co) and a chemiluminescence detection system (amersham). image analysis of western blots was performed using gel-pro analyzer software from media cybernetics. protein bands migrating at a molecular weight of and kda were selected, and the analysis program generated an optical density measurement for these bands after ltering out background. the relative amount of material migrating at kda, corresponding to cleaved s, compared to protein migrating at kda, or uncleaved s, was calculated using the following formula: od kda /(od kda c od kda ), and expressing the results as a percentage. this number is de ned as the percent cleaved s. a limitation of this type of quanti cation is that it does not take into account oligomeric, alternatively processed, or immature forms of s. thus, the numbers obtained in this manner must be considered to be a semiquantitative measure of s cleavage. all animal experiments used four week old mhvfree c bi/ male mice (jackson laboratories, bar harbor, me). viruses were diluted into phosphatebuffered saline containing . % bovine serum albumin (pbs/bsa). mice were anesthetized with methoxy urane (metofane, pittman-moore, mundelein, il), and inoculated intracerebrall y with ¹l of diluted virus ( pfu) injected into the right cerebral hemisphere. for measurement of virus replication in the brain and the liver, at selected times post infection, mice were sacri ced and brains and livers removed. brains were placed directly into ml of isotonic saline with . % gelatin (gel saline), whereas livers were rst rinsed with pbs and then placed in ml of gel saline. all organs were weighed and stored frozen at ¡ ± c until titered for virus. organs were homogenized and virus titered by plaque assay on l cell monolayers all as previously described (hingley et al, ) . in vivo replication of the different viruses was compared using wilcoxon's rank sum test. mice were inoculated intracerebrall y with either -fold or -fold serial dilutions of wild-type or recombinant virus, ve mice per dilution. mice were examined for signs of disease or death on a daily basis up to days post infection. ld values were mammalian subtilisins: the long-sought dibasic processing endoproteases the role of subtilisin-like proprotein convertases for cleavage of the measles virus fusion glycoprotein in different cell types cleavage of the respiratory syncytial virus fusion protein is required for its surface expression: role of furin mutational analysis of the murine coronavirus spike protein: effect on cell to cell fusion demyelination determinants map to the spike glycoprotein gene of coronavirus mouse hepatitis virus analysis of a recombinant mouse hepatitis virus expressing a foreign gene reveals a novel aspect of coronavirus transcription alteration of ph dependence of coronavirus-induced cell fusion: effect of mutations in the spike glycoprotein fusion-defective mutants of mouse hepatitis virus a contain a mutation in the spike protein cleavage signal the role of eukaryotic subtilisin-like endoproteases for the activation of human immunode ciency virus glycoproteins in natural host cells mhv-a fusion mutants are attenuated and display altered hepatotropism hepatitis mutants of mouse hepatitis virus strain a comparison of mouse hepatitis virus strains for pathogenicity in weanling mice infected by various routes repair and mutagenesis of the genome of a calculated by the reed-meunch method (lavi et al mice were perfused with % paraformaldehyde, and tissue was processed and embedded in paraf n. immunohistochemistry was performed using a rabbit anti-mhv-a polyclonal primary antibody (uv ) and horseradish peroxidase (hrp)-conjugated secondary antibody. diaminobenzadin e tetrahydrochloride was used as a substrate, and slides were conterstained with hematoxylin. deletion mutant of the murine coronavirus mouse hepatitis virus by targeted rna recombination variations in disparate regions of the murine coronavirus spike protein impact the initiation of membrane fusion localization of neutralizing epitopes and the receptor binding site within the amino-terminal amino acids of the murine coronavirus spike protein retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier experimental demyelination produced by the a strain of mouse hepatitis virus altered pathogenesis of a mutant of the murine coronavirus mhv-a is associated with a q l amino acid substitution in the spike protein targeted recombination within the spike gene of murine coronavirus mouse hepatitis virus-a : q is a determinant of hepatotropism roles in cell-to-cell fusion of two conserved hydrophobic regions in the murine coronavirus spike protein primary structure of the glycoprotein e of coronavirus mhv-a and identi cation of the trypsin cleavage site recombinant measles virus requiring an exogenous protease for activation of infectivity optimization of targeted rna recombination and mapping of a novel nucleocapsid gene mutation in the coronavirus mouse hepatitis virus endoproteolytic cleavage of gp is required for the activation of human immunode ciency virus furin: a mammalian subtilisin/ kex p-like endoprotease involved in processing of a wide variety of precursor proteins pathogenesis of chimeric mhv /mhv-a recombinant viruses: the murine coronavirus spike protein is a major determinant of neurovirulence multiple regions of the murine coronavirus spike glycoprotein in uence neurovirulence a simple method of estimating fty per cent points in uenza viruses, cell enzymes, and pathogenicity proteolytic activation of tick-borne encephalitis virus by furin proteolytic cleavage of the murine coronavirus surface glycoprotein is not required for infectivity molecular cloning and expression of a spike protein of neurovirulent murine coronavirus jhmv variant cl- a -amino acid stretch in the hypervariable region of the spike protein s subunit is critical for cell fusion activity of mouse hepatitis virus proteolytic processing of human cytomegalovirus glycoprotein b (gpul ) is mediated by the human endoprotease furin processing of the ebola virus glycoprotein by the proprotein convertase furin proteolytic processing of marburg virus glycoprotein key: cord- -btjq q authors: duhalde-vega, maite; loureiro, maría e.; mathieu, patricia a.; retegui, lilia a. title: the peptide specificities of the autoantibodies elicited by mouse hepatitis virus a date: - - journal: j autoimmun doi: . /j.jaut. . . sha: doc_id: cord_uid: btjq q synthetic decapeptides (n = ) covering the entire sequence of mouse liver fumarylacetoacetate hydrolase (fah) were used to analyze the specificities of the autoantibodies (autoab) elicited towards this enzyme in mice infected with mouse hepatitis virus (mhv). these autoab bound mainly to n- and c-terminal fah peptides, the most reactive sequences being – and – , respectively. surprisingly, although fah sequence – shares a high degree of homology with various mhv proteins, the c-terminal portion does not. moreover, whereas the autoab reacted with homologous peptides surrounding residues , and , non-similar sequences around residues , , , , and were also recognized, indicating that autoab were not restricted to epitopes with sequence homologies. there was also a lack of correlation between the amount of anti-mhv or anti-fah antibodies produced and the reactivity towards the peptides. moreover, the spectrum of peptides recognized by the autoab of a given mouse did not change significantly with time, which suggests that the mhv-elicited autoimmune response does not induce an epitope recognition spreading. finally, anti-fah ab produced after immunization with rat liver fah recognized essentially the same mouse fah regions than autoab from mhv-infected mice. results indicated that the induction of the autoab is not only related to molecular or structural mimicry, but rather supports the danger model, in which any aggression, in this case the mhv infection, is susceptible to trigger the production of autoab. mouse hepatitis virus strain a (mhv-a ) is a coronavirus that triggers various pathologies in susceptible mice, including hepatitis and thymus involution, igg a-restricted hypergammaglobulinaemia and transient demyelination [ , ] . in a previous paper we reported the presence of autoantibodies (autoab) in sera from various mouse strains after mhv infection [ ] . the autoab were directed to a -kda protein present in mouse liver and kidney, later identified as fumarylacetoacetate hydrolase (fah), a soluble cytosolic enzyme that mediates the hydrolytic formation of fumarate and acetoacetate [ ] . since molecular mimicry of viral antigens with self determinants could be the mechanism involved in the mhv induction of autoab to liver fah, the putative cross-reaction between the enzyme and mhv proteins was afterward examined [ ] . elisa and western blot competition assays indicated that the autoab could recognize either cryptic or native fah epitopes, the response being different between individuals [ ] . furthermore, to analyze the ab repertoire to sequential fah epitopes in mhv-infected mice, a set of decapeptides displaying at least % of identity between the sequence of mouse fah and viral proteins e , nucleocapside, e and rna polymerase was used. the results suggested that the fah sequence e was one of the epitopes recognized by the mhv-elicited autoab [ ] . herein we examined whether the mhv-elicited autoimmune response was based on molecular mimicry and whether the epitope spreading, an immune diversification originated from only a single autoreactive determinant frequently associated with autoimmune disorders [ e ] , occurs in the present anti-fah autoimmune response. overlapping decapeptides corresponding to the entire mouse fah sequence were prepared using the pepscan method and their reactivities with sera from mhv-infected mice at different times was determined by elisa. results indicated that various regions of the enzyme, including sequence e , are recognized as soon as days after infection and that the autoimmune response is not restricted to peptides homologous to viral proteins. besides, the determinant spreading phenomenon was discarded because individual mouse sera did not display the corresponding pattern of response. female pathogen free balb/c mice from the university of la plata, argentina, were bred in isolators and used for experiments at the age of e weeks. several mice were inoculated intraperitoneally with % tissue culture infectious doses (tcid ) of mhv a , grown in nctc cells [ ] and bled at different times. the nctc adherent cell line derived from normal mouse liver was purchased from the american type culture collection. cells growing in t- bottles were inoculated with mhv a virus at a multiplicity of e tcid /cell. after an adsorption period of h at c, ml of nctc medium with % fetal calf serum was added to each bottle and incubated at c. several cycles of freezing and thawing were used to release the virus h after inoculation. the harvested virus was centrifuged at g for min to remove debris and the supernatant was frozen at À c for storage. virus titration by endpoint method was performed by inoculating serial dilutions of the mhv stock onto cell monolayers in multiwell. after h, wells with viral cytopathic effect were counted for each dilution and titer was expressed as % tissue infectious doses (tcid ). before using in elisa assays the virus was inactivated by incubating the mhv stock h at c [ ] . protein concentration in both mhv and nctc stocks was determined by lowry et al. [ ] . to test anti-mhvab, elisa plates were coated with ml of uv-inactivated mhv-a ,  pfu/well, diluted in . m glycine, . m nacl, ph . . after overnight incubation at room temperature and washing with phosphate buffer saline containing . ml of tween per liter (pbsetween), the plates were blocked h at c with . m tris, . m nacl, ph . , containing % of fetal calf serum (tms-fcs), which minimizes non-specific binding. the plates were then incubated h at room temperature with mouse serum diluted in tms-fcs and after washing with pbsetween, the bound ab were revealed with peroxidase labeled donkey igg anti-mouse igg (jackson immunoresearch laboratories, inc., west grove, pa) diluted : , in tms-fcs. as a substrate, orthophenylene-diamine-dihydrochloride (opd, sigma chemical co, st. louis, mo) with freshly added h o was used. the reaction was stopped after min by addition of m h so . the absorption was measured by elisa reader (metertech inc., taipei, taiwan) at nm. non-specific values of optical density were obtained in the absence of mouse serum. essentially the same procedure was used to test anti-fah ab, except that elisa microplates were coated with ml of . m nahco , ph . , containing mg of rat liver fah prepared as indicated before [ ] . as reported previously [ ] , -week-old balb/c mice were immunized subcutaneously on day with mg of purified rat fah in ml of saline, emulsified in an equal volume of complete freund's adjuvant (difco laboratories, usa). the animals were boosted on day with the same amount of fah in incomplete freund's adjuvant (difco laboratories, usa) and bled days after the last inoculation. lalign program (http://www.ch.embnet.org/software/ lalign_form.html) using two different algorithms or matrices (pam .mat, blosum .mat) was utilized to locate multiple matching sub-segments in two protein sequences. sequences of mhv a surface glycoprotein (e ), membrane glycoprotein (e ), nucleocapside (n), rna-direct rna polymerase (rna), hemagglutinin-esterase and kda non-structural protein were aligned with the mouse liver fah amino acid sequence. our minimum criterion for homology was the existence of at least % of sequence identity between fah and each viral protein. a set of overlapping peptides ( mers) representing the entire sequence of mouse liver fah were synthesized according to the method of geysen et al. [ ] onto activated polyethylene pins, in a standard -well microtiter plate format (mimotopes, san diego, ca). each consecutive peptide was offset by two residues from the preceding one (i.e. e , e ,., e ). serum reactivity with synthetic peptides was determined by elisa as follows: immobilized pins were blocked for h at room temperature with pbs, ph . , containing % bsa and . % tween . after washing with pbs, ph . , for min at room temperature, pins were incubated overnight at c in ml of each serum, diluted : in the above-described blocking buffer. pins were then washed four times with pbs, ph . , and incubated for h at room temperature with peroxidase labeled donkey igg anti mouse igg diluted : in pbs, ph . , containing % fcs and . % tween . after several washes, the bound ab were detected by incubating the pins for min at room temperature in ml of . mg/ml , -azino-bis( -ethylbenthiazoline- -sulfonic acid) (abts) dissolved in . m na hpo , . m citric acid, ph . , containing . % h o . the absorption was measured by elisa reader at nm and control values obtained with non-immune serum were subtracted in each experiment. to follow the production of ab to viral proteins and liver fah, both kinds of ab were assayed by elisa in individual mice at different times after viral infection. representative results obtained with four infected mice showed that low but significant titers of ab to mhv were present and days after viral infection, the amount of ab sharply increasing after days post-infection and persisting at least days in mice # , # and # (fig. ). by contrast, serum from mouse # contained significant ab to viral proteins only days after infection (fig. ) . since mouse fah was not available, rat liver fah was used to assay by elisa the amount of autoab produced by mhvinfected mice. using rat fah rather than mouse fah should not change the results markedly since rat and mouse fah share % of sequence identity [ ] . the titers of anti-fah ab were significantly lower than those of anti-mhv ab and the kinetics of the production of these two ab were quite different (fig. ) . large variations were also observed between each mouse. autoab were detectable only at and days post-infection in mouse # , after and days in mouse # whereas, in mice # and # , anti-fah ab appeared at various times after mhv inoculation (fig. ). overlapping decapeptides (n ¼ ) corresponding to the mouse liver fah sequence were prepared using the pepscan method, and their reactions with sera from various mhvinfected mice were tested by elisa. sera were collected at , , , , and days after viral infection. representative results showed that no correlation was found between the binding of the ab to the peptides and to the viral proteins ( figs. and ) . sera from mice # and # ( fig. a and b ) reacted more strongly with peptides than did sera from mice # and # (fig. c and d) , whereas sera from mice # , # and # displayed the highest anti-mhv titers (fig. ) . similar discrepancies were found when the bindings of ab to peptides were compared to the amount of autoab detected by elisa ( figs. and ) . sera from mouse # displayed more autoab than sera # , # and # , whereas ab from mouse # displayed the most potent binding to peptides (figs. and ) . ab titers to viral proteins were low and days postinfection, compared with values for , , and days in mice # , # and # (fig. ) . in contrast, the sera collected later did not react with peptides more strongly than the sera obtained earlier (fig. a, b and d) . similar observation could be ascribed to serum from mouse # , since peptide reactivity did not raise days post-infection, when the highest amount of anti-mhv ab was detected (figs. and c ). data obtained with mouse serum # and # showed that ab would recognize essentially the same fah regions. in fact, both sera reacted mainly with n-terminal (residues e ) and c-terminal (residues e ) portions of the enzyme, whereas sequences around residues , , , , , and were recognized at diverse extents ( fig. a and b) . moreover, although ab from mouse serum # displayed lower reactivity than the former, essentially the same fah regions were recognized (fig. c) , and the scarce reaction of mouse serum # with peptides was limited to n-and c-terminal fah sequences (fig. d) . various mouse fah portions exhibiting e % of identity with peptides from different mhv proteins (fig. ) were placed as solid bars below the mouse fah sequence (fig. ) . it was observed that although homologous sequences e , e , and e did react with ab, others did not, i.e., sequences e , e , and e ( fig. a, b and c) . a horizontal bars indicate sequence homology ( % or more) between mouse liver fah and mhv proteins according to data presented in fig. . thus, homologous sequence e correspond to overlapping fah peptides , , , , , , , and , sequence e to peptides , , , and , sequence e to peptides , and , sequence e to peptides , , , , and , sequence e to peptide and sequence e to peptides and (see fig. ) . surprisingly, the highly reactive c-terminal mouse fah sequence (residues e ) does not display significant homology with any viral protein ( fig. a, b and c) . besides, results obtained with serum # , even if very low, followed basically the same pattern of reactivity than sera # e (fig. ) . same results as those described in the last two paragraphs could be distinguish when the sum of optical density values at all time points for each peptide was collected for the four representative mice shown in fig. plus the addition of values displayed by sera from other three animals (fig. a ). furthermore, optical density values exhibited by different pooled serum samples obtained after and days of mhv infection also indicated that autoab reacted with the fah sequence portions listed before, even though residues around position as well as the homologous sequence e were also reactive ( fig. b and c) . finally, sera reacted similarly with peptides from the au-toag at the various times after mhv infection, i.e., there was no evidence of a major sequence being the first target of the autoimmune response, suggesting the lack of spreading of the immune response (fig. ) . various mice were immunized with purified rat liver fah [ ] and the ab tested for their binding to the mouse fah synthetic peptides. results indicated that anti-rat fah ab reacted like the autoab from mhv-infected mice. n-and c-terminal peptides were recognized, as well as regions surrounded residues , , , , , and (fig. d) . the only remarkable difference was that anti-rat fah ab also bound to peptides corresponding to mouse fah sequence e , whereas sera from mhv-infected mice did not (fig. ) . we have reported that mice infected with mhv produced autoab to mouse liver and kidney fah [ ] . competition assays indicated that the autoab detected both conformational and cryptic fah epitopes in elisa but only cryptic determinants in western blot assays, whereas anti-mhv ab were directed to native epitopes of the viral proteins [ ] . such results suggested that mhv infection could trigger a crossreaction of either sequential or conformational epitopes between the viral proteins and the autoag and that mhv-infected mice produce at least three different ab populations: ab specifically directed either to viral proteins or to the autoag, and cross-reacting ab [ ] . molecular mimicry between viral proteins and self-ag is one of the most probable mechanisms that explain autoimmune responses induced by viral infections [ , ] coxsackie virus and cytomegalovirus have been found to mimic physiologically important host proteins [ ] . we previously observed that most autoab from mhvinfected mice reacted with the fah n-terminal sequence (residues e ) [ ] . however, it could be expected that other sequences were also recognized. thus, in the present work, we extended the study of the autoab specificities using peptides spanning the entire sequence of mouse liver fah. moreover, to examine the possible occurrence of determinant spreading [ e ], we tested the sera from individual animals at various times post-infection. representative results from four different mice showed that, in spite of individual disparity, the autoab recognized mainly n-and c-terminal peptides, the most reactive sequences being e and e , respectively. strikingly, whereas the nterminal portion of fah shares a high degree of homology with various mhv proteins, sequence e does not display any similarity with viral proteins. furthermore, the au-toab reacted with homologous peptides surrounding residues , and , but non-homologous sequences around residues , , , and were also recognized. thus, autoab were not restricted to similar sequences, suggesting that structural patterns other than linear epitopes were involved in the autoimmune response. accordingly, it has been proposed that viral regions that are able to initiate autoimmune responses do not need to have sequences analogous to the autoantigen [ ] and, since the only requirement should be structural similarity, some authors propose that ''molecular mimicry'' should be changed to ''structural mimicry'' [ ] . the present data also show a lack of correlation between the amount of autoab and ab to mhv proteins determined by elisa, and ab binding to the peptides. these facts could be explained by the production of three kinds of ab, as mentioned above: virus-specific ab, fah-specific autoab, and cross-reacting ab, together with previous observation indicating than mainly conformational epitopes are recognized by elisa assays [ , ] . moreover, no immune diversification originated from a single autoreactive determinant was observed, indicating that the autoimmune response induced by mhv is not associated with determinant spreading as described in other autoimmune processes [ e ]. when the specificities of the mhv-elicited autoab were compared with those of ab induced by injections of rat liver fah, no significant differences were observed. the same mouse fah regions were recognized, with the exception of sequence e , recognized only by the anti-rat fah ab. the danger model proposes that the immune system is more concerned with damage than with foreignness, and is called into action by alarm signals from injured tissues rather than by the recognition of non-self [ ] . thus, it was suggested that structural features of autoantigens, their locations, and catabolism during cell death and their translocation to cells that can present antigens to the immune system could contribute to selection of the autoimmune repertoire [ , ] . mhv is known to be lymphotropic and to induce diverse alterations of immune responses that depend on the mouse genetic background [ e , , ] , but why liver fah was chosen as autoantigen among the large variety of liver proteins? as said by p. h plotz [ ] : ''the repertoire of target autoantigens is a wunderkammer e a collection of curiosities e of molecules with no obvious linking principle''. in the present model of mhv-induced anti-fah autoab, it seems that antigen mimicry e fah n-terminal sequence is about % homologous with a mhv protein e together with the alarm signals released by the mhv-injured tissues leads to the autoimmune response. morphological analysis of mouse hepatitis virus a -induced pathology with regard to viral receptor expression polyclonal b lymphocyte activation induced by mouse hepatitis virus a infection identification of two liver proteins recognized by autoantibodies elicited in mice infected with mouse hepatitis virus a sequence similarity and structural homologies are involved in the autoimmune response elicited by mouse hepatitis virus a epitope mimics and determinant spreading: pathways to autoimmunity epitope spreading and molecular mimicry as triggers of autoimmunity in the theiler's virus induced demyelinating disease model of multiple sclerosis molecular mimicry and the role of b lymphocytes in the processing of autoantigens the coronavirus protein measurements with the folin phenol reagent detection of mouse hepatitis virus infection by assay of anti-liver autoantibodies strategies for epitope analysis using peptide synthesis the viral-autoimmunity relationship evidence for mimicry by viral antigens in animal models of autoimmune disease including myocarditis mimicking the way to autoimmunity: an evolving theory of sequence and structural homology molecular mimicry or structural mimicry? the danger model: a renewed sense of self the autoantibody repertoire: searching for order in vivo polyclonal b-lymphocyte activation elicited by murine viruses role of virus receptor-bearing endothelial cells of the bloodebrain barrier in preventing the spread of mouse hepatitis virus-a into the central nervous system the authors are indebted to drs. pierre l. masson (icp, brussels, belgium) and leonor p. roguin (iquifib, buenos aires, argentina) for helpful discussions and critical revision of the manuscript. this work was supported by grants from conicet, foncyt and universidad de buenos aires, argentina. key: cord- -v r idg authors: nan title: induction of monocyte procoagulant activity by murine hepatitis virus type parallels disease susceptibility in mice date: - - journal: j exp med doi: nan sha: doc_id: cord_uid: v r idg the in vitro induction of procoagulant activity (pca) in murine peripheral blood mononuclear cells (pbm) by murine hepatitis virus type (mhv- ) correlates with the disease susceptibility in three strains of mice. pbm from balb/c mice, a strain in which mhv- infection results in fatal acute fulminant hepatitis, responds to the virus with a robust pca response, whereas pbm from c h/st mice, a strain which develops mild acute hepatitis followed by chronic hepatitis, only exhibit a modest pca response. in contrast, pbm from a/j mice, a strain fully resistant to mhv- , generate no increase in pca above control levels. the induction phase of mhv- pca is rapid, with an increase within - . h, with maximum activity at h, and it precedes mhv- replication in either cl cells, a fully permissive cell line, or in monocytes from these strains of mice. the pca response of balb/c pbm exceeds the response to any other known stimulus. no induction occurs upon direct stimulation of monocytes by mhv- , but in the presence of lymphocyte collaboration, the pca response is observed first at a lymphocyte:monocyte ratio of : and reaches a maximum as the lymphocyte:monocyte ratio approaches : . this response appears to provide a functional marker for susceptibility to mhv- infection in inbred strains of mice and could be important in the pathogenesis of mhv- -induced disease. induced by a variety of stimuli, including lipopolysaccharides (lps), antigen-antibody complexes, and lectins ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . we have recently established ( , ) that the monocyte and macrophage represent the cellular sources of lps and antigen:antibody induced pca and that lymphocyte/macrophage collaboration is a necessary requirement for the full induction of pca response. current evidence suggests this is an immune response that functions via unidirectional lymphocyte induction of procoagulant monokines. the murine hepatitis viruses (mhv) are a group of serologically related pathogenic coronaviruses ( , ) . infection of mice by mhv may produce a broad spectrum of diseases, including encephalitis, hepatitis, interstitial pneumonitis, nephritis, and enteritis, depending on the serotype of mhv, the age and strain of the mouse, and the route of infection ( ) . murine hepatitis virus type (mhv- ) is a primarily hepatotropic serotype and causes hepatitis when administered intraperitoneally, intracerebrally, intravenously, or orally in susceptible strains of mice ( , ) . balb/c and dba/ mice develop acute fulminant hepatitis, c h mice develop a mild chronic disease, and mice of the a strain develop no evidence of hepatitis ( ) . the mechanism of resistance to mhv infection in animals is both complex and poorly understood. it has been demonstrated that resistance to fatal mhv infection may be associated with a resistance gene that inhibits mhv replication in macrophages and is also influenced by a susceptibility factor that is related to lymphocytes ( ) ( ) ( ) ( ) ( ) . in this study, we demonstrate that the induction by lymphocytes of monocyte pca in response to mhv- infection reflects the susceptibility of the host to infection by mhv- . we further indicate that there exists a genetic restriction in the mhv- induced pathway of lymphoreticular pca. this is characterized by the lack of generation of pca in the resistant animal, modest induction in mice that develop mild and chronic disease, and a profound pca response by the fully susceptible animal who develops fatal hepatitis. the induction of lymphoreticular pca antedates viral replication and appearance of histologic lesions in susceptible organs. furthermore, we demonstrate that the monocyte is the cellular source of this activity but cellular collaboration between lymphocytes and monocytes is required for the generation of the pca response. cells. the origin and growth of cl , dbt, and l cells has been described previously ( ) ( ) ( ) . cells were routinely propagated in dulbecco's modified eagle's medium (dmem) (flow laboratories inc., rockville, md.) supplemented with % newborn calf serum (biocell laboratories, carson city, calif.) and #g/ml chlortetracycline hydrochloride grade ii (sigma chemical co., st. louis, mo.) and buffered with mm hepes, -[n-morpholino]propanesulfonic acid, n-tris [hydroxymethyl] methyl- aminoethane sulfonic acid, and mm glutamine (sigma chemical co.). peripheral blood mononuclear cells (pbm) were isolated from mice as previously described ( ) . the cells were separated over ficoll-hypaque (density, . g/ml) at °c for rain at , g and were recovered at the interface. the cells were adjusted to x pbm/ml. lymphocytes and monocytes were separated by adherence to plastic, as previously described ( ) . the recovery of cells was > % and viability was > %, as evidenced by trypan blue exclusion. lymphocytes were defined by failure of adherence, morphology, and failure of uptake of neutral red and were < % esterase positive. monocytes were defined by morphology and uptake of neutral red and were > % esterase positive, as previously described ( ) . mice. c h/st, a/j, and balb/c mice (research institute of scripps clinic breeding colony) were used at - wk of age. after infection with mhv- , they were maintained in strict isolation. virus. mhv- was obtained from the american type culture collection, rockville, md. (attcc vr- ), and the lyophilized infected liver homogenate was reconstituted with ml sterile phosphate-buffered saline (pbs), ph . . the virus was plaque purified twice on monolayers of dbt cells. the virus was subsequently passaged once in dbt cells to prepare a seed stock. a working stock of virus was grown in cl cells infected at a multiplicity of infection (moi) of - plaque-forming units (pfu)/eeli. this stock virus had a titer of . × v pfu/ml and was used for all subsequent experiments. virus was assayed on monolayers of l cells in a standard plaque assay, as previously described ( ) ( ) ( ) ( ) . lethal dose-- % (ldso) was determined in balb/c, c h/st, and a/j mice and was calculated according to the method of reed and mueneh ( ) , using an endpoint of d. pca assay. pca was determined in a one-stage recalcified clotting assay, as previously described ( ) . samples of frozen-thawed and sonicated cells in rpmi were assayed for the capacity to shorten the spontaneous clotting of human plasma. the assay consisted of # of test sample, #l of mm cac , and # of citrated normal-fasting platelet-poor human plasma. units were assigned from a log-log plot and standardized with dilutions of rabbit brain thromboplastin (dade div., american hospital supply corp., miami, fla.), which at /~g/ml was assigned a value of , mu. there was no activity present in dmem, rpmi , pbs, complete media, or the virus preparations. endotoxin contamination. dmem, rpmi , pbs, and virus preparations were assayed for endotoxin contamination by standard limulus assay (e. toxate; sigma chemical co.) and contained < . ng/ml of endotoxin in the lower limits of this test ( , ) . resistance of mice to lethal infection with mhv- . balb/c mice were infected in groups of seven with , , , or , pfu of mhv- by intraperitoneal injection and were observed for d (table i) . using the method of reed and muench ( ) , an lds for balb/c of < - pfu was established. in similar experiments, the lds for c h/st was ~ pfu, and a/j mice were resistant to > pfu. mhv- replication in vitro. the one-step growth curve of mhv- in cl cells was determined after innoculation of cultures at an moi of . pfu/cell, and the virus titers in replicate cultures were determined at various subsequent time intervals (fig. ). mhv- had an eclipse phase of -to h, reaching a maximum virus titer at h postinfection. one-step growth curves were then determined in monocytes harvested from the peripheral blood of a/j, balb/c and c h/st mice. for these experiments mice were killed, and pbm were isolated asceptically over ficoll-hypaque. monocytes were separated from lymphocytes by adherence to plastic for h and were > % esterase positive ( ) . monocytes at × were seeded in -well cluster plates (costar data fzc. . one-step growth curve of mhv- in x | cli cells. cells were infected in suspension with mhv- at an moi of . ( ) . after virus adsorption for rain at °c, the cells were collected by low-speed centrifugation and resuspended in media containing % fcs, and replicate cultures were seeded at × e cells per x -ram culture dish. at appropriate intervals, cultures were frozen, and the cells were harvested by freeze-thawing, scraped with a rubber policeman, and disrupted by sonication. the homogenates were then clarified by centrifugation, and virus titer was determined by plaque assay at serial -fold dilutions. each data point represents the mean of duplicate cultures, and each line represents individual experiments. packaging, cambridge, mass.) in ml dmem fortified with % fetal calf serum and were infected at an moi of . with mhv- . virus was adsorbed to the cells for min at °c. unadsorbed virus was removed by washing with dmem, the media were replaced, and the cells were further incubated at °c. cultures were frozen at appropriate intervals, treated as described above, and assayed for virus by plaque assay at serial -fold dilutions (fig. ) . in contrast to the growth of mhv- in a totally permissive cell line ( cli) , in balb/c monocytes, a prolonged period of h was observed postinfection, during which time no virus replication was evident. however, subsequent to the -h lag period, replication was progressive, with peak yields h after infection. in contrast, no virus replication was detected in a/j or c h/st monocytes within h postinfection. these observations parallel the observations of acute fulminant hepatitis in balb/c mice and resistance of a/j mice to overt hepatitis or death. basal cellular pca. the total cellular content of pca immediately after isolation of cells and before any form of in vitro culture was determined for a/j, balb/c, and c h/st mice and was remarkably consistent at - mu/ pbm. this activity rises spontaneously to a maximum stable concentration of mu/ pbm within h of culture in serum-free medium in the absence of added stimulus (table ii) . when the control cultured cells were fractionated by adherence into lymphocytes and mhv- induction of pca. pbm were isolated from each of the mice strains, and x pbm were incubated with pfu of mhv- for h at °c in ml of rpmi supplemented with % heat-inactivated fcs at °c. the cells were washed three times with rpmi , disrupted, and assayed for total pca. as a positive control, × pbm from each strain was stimulated with escherichia coli l:b lps at #g/ml, as previously described ( ) . cells from all three strains responded to lps with a six-to eightfold increase in pca (table iii) , in accord with previous observations ( ) . the pca response of balb/c pbm to pfu mhv- was greater than previously observed for any stimulus, reaching levels of > , mu/ pbm (table iii) . in contrast to the profound pca response of balb/c pbm, no response to mhv- was observed in pbm from a/j mice, even though a normal pga response was observed to lps stimulation. a moderate pca response was observed for pbm from c h/st mice stimulated with mhv- , but the response was only % of the response observed for lps stimulation (table iii) . thus, the pca response of pbm to mhv- stimulation exhibited profound strain differences. the production of the pga by pbm correlates with the susceptibility of the strain to hepatic disease. furthermore, the pca response of balb/c mice was of a magnitude - times greater than that observed for the response to lps or antigen-antibody complexes ( ) ( ) ( ) . no increase in pca above basal levels was observed when x pbm from balb/c, a/j, or c h/st mice were cultured with an aliquot from mockinfected dbt, l , or gl cells (data not shown). dose-response titrations were examined for mhv- -induced pca in which x pbm from balb/c mice were incubated for h with mhv- . when pfu of mhv- was used, a fourfold increase in pca as compared with unstimulated control cultures was observed (fig. ) . a dose-dependent increase of pca achieved a -fold maximum increase of pca with pfu of mhv- (fig. ) . comparable experiments were conducted with pbm from c h/st and a/j mice. when x pbm from c h/st mice were incubated for h with increasing amounts of mhv- , no pga response was observed until concentrations of pfu of mhv- were added; and the maximum pca response was observed at pfu of mhv- , with a . -fold maximum increase in pca observed over control cultures. in contrast, no pca response was observed for pbm from a/j mice over a dose titration of - pfu of mhv- . to determine the kinetics of mhv- -induced amplification of pca, x pbm from balb/c, g h/st, and a/j mice were each incubated with pfu of mhv- for various time intervals and assayed for total cellular pca. a -fold pca response was observed for balb/c pbm that were cultured for h with mhv- (fig. ) . there was a progressive increase of pga with time of exposure, which reached a maximum of , mu at h. this level of pga was maintained for h, which was the maximum interval of observation. in contrast, the pca response of c h/st mice did not rise until h after exposure to mhv- and was maximum at h (fig. ) at , mu, which was a . -fold increase over control. finally, there was no increase in pca from a/j mice above a background level of mu at any time interval of cultivation (fig. ) . from either c h/st or balb/c mice were directly stimulated with mhv- , no increase of cellular pca was observed (table iv) . however, when pbm were first stimulated by mhv- for h and then subsequently fractionated into lymphocytes and monocytes by adherence to plastic for h, the increase in pca observed for the whole pbm was localized to the monocyte population. this is similar to the increase demonstrated for lps stimulation ( ) . the pca response of pbm from balb/c mice was -fold, increasing from to , mu. % of the increase in pca could be accounted for in the monocyte fraction, which demonstrated an increase in total cellular pca from , (control) to , mu/ ~ monocytes (table v) . were prepared from c h/st, a/j, and balb/c mice. they were stimulated either directly by mhv- alone attached in the -mm plastic wells or in the presence of variable numbers of lymphocytes, i.e., with lymphocyte:monocyte ratios from : to : . monocytes were then isolated from lymphocytes by vigorous washing and assayed for total cellular pca. monocytes ( × ) exposed to pfu of mhv- in the absence of lymphoeytes exhibited no pca response, nor was the response at lymphocyte:monocyte ratios of less than : particularly notable (fig. ) . however, at lymphocyte'monocyte ratios of . : or greater, a significant pca response was observed and it reached maximum levels as the ratio approached : . no further increase in pca was noted as the ratio was increased to : . these data indicate that monocytes are the cellular source of the mhv- -induced pca, but the presence of lymphocytes is necessary for induction of the pca response in monocytes. this study further characterizes the generation of monocyte procoagulant activity in response to stimuli, a response mediated by the coagulation pathways. in previous reports, we have demonstrated that monocytes from a wide variety of mouse strains, including the a/j, balb/c, and c h/st used in this study, produce an equivalent pca response to such agents as lps ( , ) and antigen-antibody complexes ( ) . others ( , ( ) ( ) ( ) ( ) have observed this response in a variety of species, including human and rabbit, using lectins, lps, and antigen-antibody complexes. in the present study, a genetically restricted pathogenic agent, mhv- , stimulates the pca response of cells only from strains of mice susceptible to the pathologic effects of the virus. this, to our knowledge, is the first example of a normal genetic restriction of the pca response and is characterized by maximum output of pca by balb/c mice that are fully susceptible to mhv- , with a fatal outcome within - d, a moderate pca response of c h/st mice who develop enzymatic and histologic lesions of hepatitis (g. a. levy, j. l. leibowitz, and t. s. edgington, manuscript in preparation), and no pca response of mice fully resistant to mhv- , as exemplified by the a/j strain. we have shown that these strains are all functionally capable of producing a pca response to lps and also to antigen:antibody complexes (unpublished data) and that the magnitude of the pca response is equivalent, a therefore, the lack of response by the a/j strain to mhv- is specific for the stimulus. the response of cells from c h/st mice is comparable to that observed for other unrestricted stimuli, such as lps or antigen:antibody complexes ( ) and before this study would have been considered a maximum response. of great interest is the unprecedented response of cells from balb/c mice. it represents - times the response observed for any other previously examined stimulus, including those reported by other investigators ( ) ( ) ( ) ( ) . whether or not the increased magnitude of the response represents increased synthesis of procoagulant molecules, a new and as yet uncharacterized initiator of the coagulation pathways or conformation realignment of either tissue factor or the monocyte prothrombinase ( ) and amplification of activity by a cofactor such as factor va ( ) remains to be investigated. mhv- -induction of the pca response is also more rapid than was previously observed, with a significant increase of pca within - . h in vitro and with maximum activity at - h. this contrasts to the - -h lag period before monocyte pca increases in response to lps-triggered lymphocytes. virus growth in a fully susceptible cloned cell line ( cl ) was not detectable until h after infection, and in monocytes from balb/c mice infectious virus was not produced until h postinfection. thus, the induction of the pca response in pbm antedates production of infectious virus and cannot be ascribed to it per se. furthermore, little or no viral replication occurred in monocytes from c h/st mice, and yet a robust generation of monocyte pca followed. thus, these initial observations suggest that the induction of the pca response by pbm does not depend on virus replication. the induction of this activity temporally precedes induction by other stimuli, such as lps or antigen-antibody complexes, as reported previously. at h, although only % of total virally induced pca is seen in the susceptible balb/c strain, the quantity of pca exceeds a maximum response by other agents, such as lps. we have recently described ( ) the kinetic characteristics and metabolic requirements for the induction of lymphoreticular pca. we demonstrated, using lps as a model stimulus, that lymphocyte triggering by this stimulus was a brief event, requiring only - min for detectable effect and min for maximum triggering. lymphocyte contact and induction of monocytes required min for complete effect, but monocytes then required an additional h for production of maximum pca. in the viral induction of pca, it appears there are differences in the kinetics of induction of pca, with an initial response in just . h, although maximum pca induction requires - h. this is not simply a matter of recruiting more monocytes or macrophages because we have demonstrated ( ) in cytologic assays that virtually all monocytes/macrophages are recruited in the previously described responses. thus, it is possible that different cellular mechanisms or products are operative for the induction of pca by different stimuli. we have extended these observations by demonstrating that lymphocyte collaboration is necessary for the induction of monocyte pca. when highly purified monocytes from c h/st or balb/c strains of mice are directly stimulated by the virus, no induction of pca follows. these results parallel the pca response to other stimuli ( ) ( ) ( ) . in studies involving human pbm ( , ) , we have observed that both helper and suppressor lymphocytes exist that appear central to the induction and regulation of the pca response to lps. indeed, the induction of monocyte pca by helper t cells can be markedly attenuated by ty cells. this finding may be significant in explaining the response to mhv- . it is possible that the failure of response by pbm from a/j mice could reflect an excess of suppressor cells rather than a lack of lymphocyte recognition and generation of triggered t helper cells. furthermore, the different responses observed in c h/st and balb/c mice could reflect similar disproportions in the suppressor and helper cell participants. another possible explanation of the differences in pca induction by mhv- could reflect differences in the capacity of monocytes to receive signals from mhv- -triggered lymphocytes or otherwise accept lymphocyte collaboration for this specific stimulus. the lack of a monocyte subpopulation (which is inducible) by mhv- within a/j mice might explain the failure of induction of pca in this strain. application of the cytologic pca assay previously described ( ) , in which we can visualize individual cells and the presence or absence of pca, promises to provide additional information. a number of investigators ( , ) have demonstrated that the susceptibility of adult mice of different strains to mhv, using mortality as the index, parallels the cytopathic effects that the virus has in mouse peritoneal macrophages in vitro. the cellular basis for the resistance of a strain macrophages is not known; however, it has been observed ( ) that resistance can be overcome by increasing the infecting dose, suggesting that resistant strains do not entirely lack receptors for mhv- . it has also been demonstrated ( ) ( ) ( ) that young or immunologically depressed a strain mice develop disease similar to that occurring in susceptible strains, in contrast to the fully resistant adult a mouse. further observations suggest that the state of susceptibility is under the influence of t cells ( ) ( ) ( ) , humoral factors ( ) , and splenic adherent cells ( ) . it has also been demonstrated ( , ) that nude mice and mice treated with cyclophosphamide ( ) develop full susceptibility to mhv- infection. thus, it appears that a complex series of interactions between lymphocytes, monocytes, and humoral factors may be required to explain the balance between susceptibility or resistance to this virus. the present study reports that pbm from susceptible strains of mice respond to mhv- with a rapid and robust pca response. in contrast, there is a failure of induction of pca in the fully resistant a/j strain. although most mechanistic details remain to be resolved, this system provides a biochemical marker for susceptibility to mhv- -induced disease and perhaps may play a role in the pathogenesis of disease induced by this virus. the in vitro induction of procoagulant activity (pca) in murine peripheral blood mononuclear cells (pbm) by murine hepatitis virus type (mhv- ) correlates with the disease susceptibility in three strains of mice. pbm from balb/c mice, a strain in which mhv- infection results in fatal acute fulminant hepatitis, responds to the virus with a robust pca response, whereas pbm from c h/st mice, a strain which develops mild acute hepatitis followed by chronic hepatitis, only exhibit a modest pca response. in contrast, pbm from a/j mice, a strain fully resistant to mhv- , generate no increase in pca above control levels. the induction phase of mhv- pca is rapid, with an increase within - . h, with maximum activity at h, and it precedes mhv- replication in either cl cells, a fully permissive cell line, or in monocytes from these strains of mice. the pca response of balb/c pbm exceeds the response to any other known stimulus. no induction occurs upon direct stimulation of monocytes by mhv- , but in the presence of lymphocyte collaboration, the pca response is observed first at a lymphocyte:monocyte ratio of : and reaches a maximum as the lymphocyte:monocyte ratio approaches : . this response appears to provide a functional marker for susceptibility to mhv- infection in inbred strains of mice and could be important in the pathogenesis of mhv- -induced disease. the macrophage. in future trends in inflammation induction of cytocidal macrophages after in vitro interactions between listeria-immune t cells and macrophages--role of h- macrophage t cell interactions involving listeria monocytogenes--role of the h- gene complex secretion of macrophage enzymes in relation to the pathogenesis of chronic inflammation the pathogenetie role of the coagulation process in glomerular diseases of immunologic origin anaphylactoid pupura. ii. immunofluorescent and electron microscopic studies of glomerular lesions clotting changes including disseminated intravascular coagulation during rapid renal homograft rejection participation of intravascular coagulation in the pathogenesis of glomerular and vascular lesions fibrinopeptide a in plasma of normal subjects and patients with disseminated intravascular coagulation and systemic lupus erythematosus role of leukoeytes in blood coagulation and the generalized schwartzman reaction immunohistochemical study of allergic encephalomyelitis the immunopathology of joint inflammation in rheumatoid arthritis stimulation of human leukocyte thromboplastic activity by endotoxin lymphocyte cooperation is required for amplification of macrophage procoagulant activity lymphocyte collaboration is required for the induction of murine monocyte procoagulant activity by immune complexes the kinetics and metabolic requirements for direct lymphocyte induction of human procoagulant monokines by bacterial lipopolysaccharide tissue thromboplastin activity of isolated human monocytes tissue factor activity of normal and leukemic cells increased production and expression of tissue thromboplastin-like procoagulant activity in vitro by auogeneically stimulated human leukocytes the role of human t cells and t cell products for monocyte tissue factor generation goronaviridae experimental viral hepatitis immunopathology of mouse hepatitis virus type infection. i. role of humoral and cell mediated immunity in resistance mechanisms immunopathology of mouse hepatitis virus type infection. iii. clinical and virologic observation of a persistant viral infection correlation of persistent mouse hepatitis virus (mhv- ) infection with its effect on mouse macrophage cultures mouse macrophages as host cells for the mouse hepatitis virus and the genetic basis of their susceptibility macrophages genet~ally resistant to mouse hepatitis virus converted in vitro to susceptible macrophages blocking of in vitro and in vivo susceptibility to mouse hepatitis virus resistance to fatal central nervous system disease by mouse hepatitis virus strain jhm. i. genetic analysis enhanced growth of a murine coronavirus in a transformed mouse cells replication and plaque formation of mouse hepatitis virus (mhv- ) in mouse cell line dbt culture. archives fur die ges virusforsch pathogeneic murine coronaviruses. ii. characterization of virus specific proteins of murine coronaviruses jhmv and a v in vivo and in vitro models of demyelinating diseases: tropism of the jhm strain of murine hepatitis virus for cell of glial origin a simple method of estimating fifty percent endpoints detection of endotoxin in the blood of patients with sepsis due to gram-negative bacteria gram negative sepsis detection of endotoxemia with the limulus test esterases in human leukocytes plasma lipoprotein induction and suppression of the generation of cellular procoagulant activity in vitro. i. two procoagulant activities are produced by peripheral blood mononuclear cells interaction of coagulation factor xa with human platelets t cell collaboration is required for amplification of monocyte procoagulant activity by endotoxin the existence oft helper (t/z) and t suppressor (t),) cells for the generation of monocyte thromboplastin activity by lipopolysaccharide lps generation of a murine procoagulant monokine is an unidirectional lymphocyte to macrophage instructed event requiring ia compatibility thymus-dependent maturation ofmacrophages neonatal susceptibility to mhv- infection in mice. i. transfer of resistance hepatitis and brain lesions due to mouse hepatitis virus accompanied by wasting in nude mice response of nude mice to a mouse hepatitis virus isolated from a wasting nude mouse t lymphocyte-dependent difference in susceptibility between ddd and c h mice to mouse hepatitis virus mhv- effect of cyclophosphamide on the genetic resistance of c h mice to mouse hepatitis virus the authors are grateful to james de vries, eric paavola, and maryellen pizzolato for their dedicated technical assistance, and to sandy thompson for preparation of this manuscript.received for publication june . key: cord- -mfptcw t authors: lu, yiqi; denison, mark r. title: determinants of mouse hepatitis virus c-like proteinase activity date: - - journal: virology doi: . /viro. . sha: doc_id: cord_uid: mfptcw t abstract the coronavirus, mouse hepatitis virus strain a (mhv), expresses a chymotrypsin-like cysteine proteinase ( clpro) within the gene polyprotein. the mhv clpro is similar to the picornavirus c proteinases in the relative location of confirmed catalytic histidine and cysteine residues and in the predicted use of q/(s, a, g) dipeptide cleavage sites. however, less is known concerning the participation of aspartic acid or glutamic acid residues in catalysis by the coronavirus c-like proteinases or of the precise coding sequence of clpro within the gene polyprotein. in this study, aspartic acid residues in mhv clpro were mutated and the mutant proteinases were tested for activity in anin vitro transcleavage assay. mhv clpro was not inactivated by substitutions at asp (d ) or asp (d ), demonstrating that they were not catalytic residues. mhv clpro was able to cleave at a glutamine–glycine (qg - ) dipeptide within the clpro domain upstream from the predicted carboxy-terminal qs - cleavage site of clpro. the predicted full-length clpro (s to q ) had an apparent mass of kda, identical to the p clpro in cells, whereas the truncated proteinase (s to q ) had an apparent mass of kda. this -amino-acid carboxy-terminal truncation of clpro rendered it inactive in atranscleavage assay. thus, mhv clpro was able to cleave at a site within the putative full-length proteinase, but the entire predicted clpro domain was required for activity. these studies suggest that the coronavirus cl-proteinases may have a substantially different structure and catalytic mechanism than other c-like proteinases. the role of aspartic acid or glutamic acid residues in clpro activity is less well understood. mutagenesis of the coronavirus, mouse hepatitis virus, strain a asp/glu residues of c and c-like proteinases suggests (mhv-a ), contains a chymotrypsin-like proteinase that they might not participate as catalytic residues in all within the -kda gene polyprotein ( fig. ) (lu et al., cases. aspartic/glutamic acid residues have been shown ). the c-like proteinases of the coronaviruses to be essential for proteinase activity of tobacco etch mhv-a , infectious bronchitis virus (ibv), and the huvirus (tev), poliovirus, and human rhinovirus (hrvman coronavirus e (hcv- e) are encoded in a con- ) (gorbalenya and koonin, ) . the positioning of served region of orf a (boursnell et al., ; gorba- his, cys, and glu residues in the hrv- cpro has been lenya et al., ; lee et al., ; herold et al., ). shown to be very similar to that of cellular trypsin by mhv, hcv- e, and ibv encode clpro molecules with analysis of the hrv crystal structure (matthews et al., apparent masses of , , and kda, respectively, ) . in contrast, analysis of the crystal structure of as determined by sds-page analysis (lu et al., ; hepatitis a virus cpro indicates that asp most likely ziebuhr et al., ; tibbles et al., ) . the classificadoes not participate directly in catalysis (allaire et al., tion of the coronavirus proteinases as '' c-like'' is sup- ) . more directly relevant to mhv, it has been shown ported by mutagenesis studies of predicted catalytic cysthat mutagenesis at glu residues in ibv clpro does teine or histidine residues. we have demonstrated that not abolish activity of the expressed proteinase (liu and mutations at his or cys of mhv clpro abolish pro- brown, ) . teolytic activity. similar results have been obtained for several clpro cleavage sites within the gene clpro of ibv and hcv- e, confirming the essential polyprotein recently have been defined for mhv, ibv, nature of these residues and demonstrating that his and and e. the experimentally confirmed coronavirus cys residues are in positions similar to those of the picor- clpro cleavage sites have a leucine, isoleucine, or navirus c proteinases (lu et al., ; liu and brown, valine at position p , glutamine at position p , and ; ziebuhr et al., ; tibbles et al., ) . serine or alanine at position p (liu and brown, ; lu et al., lu et al., , ziebuhr et al., ; tibbles et the amino acid sequences of the coronaviruses were obtained from genbank; mhv (x ) (bonilla et al., ) , ibv (m ) (boursnell et al., ) , hcv- e (x ) (herold et al., ) , and tgev (z ) (eleouet et al., ) . deduced amino acid sequences of coronavirus clpro domains were compared using a pam scoring matrix and a word size of (macvector . . , ibi-kodak) (fig. ) . numbering of mhv amino acid sequences was from the beginning of orf a. numbering of amino acid residues within mhv clpro is based the linear schematic of the mhv genome shows the organization of ser (lu et al., ) site-directed mutagenesis of pgpro with bovine chymotrypsin (gorbalenya and koonin, ) , cpro of human rhinovirus (matthews et al., ) , and hepatitis a virus asp and asp were mutagenized by the chame- cpro (allaire et al., ) . black bars reflect the number of amino acid leon double-stranded, site-directed mutagenesis kit per residues in the proteinase. the proteins are aligned at the confirmed the manufacturer's instruction (stratagene). two primers cys or ser catalytic residues to show the carboxy-terminal extent of the protein. catalytic asp residues of hrv- and chymotrypsin have were simultaneously annealed to the template. one sebeen confirmed by crystallography. asp and asp of mhv are prelection primer changed one nonessential unique restricdicted catalytic residues. lqs indicates cleavage sites at amino termition site alwni on the pgpro vector to a new restriction nus (confirmed) and carboxy terminus (putative) of clpro. site. the other primer encoded for a specific mutation. after annealing and extension, all new plasmid dna was incubated with the restriction enzyme alwni. the dialanine at p and glycine at p (lee et al., ) . the gested mixtures were then transformed into repair-defiprecise determinants of clpro cleavage site seleccient xlmuts cells, and the resultant colonies were isotion remain to be determined. finally, comparison of lated. the mutant plasmids were then purified and the coronavirus clpro sequences with those of other digested with alwni again, and the resultant dna digesproteinases suggests that they may differ from other tion was transformed into xl -blue cells. all specific muviral and cellular proteinases in their size and structations were confirmed by bidirectional sequencing ture. the coronavirus clpro domains contain signifi-(sequenase ii, u.s. biochemicals, per the manufacturer's cantly more amino acids downstream from the putative instructions). substrate binding site than other c or c-like proteinases ( fig. ) (gorbalenya and koonin, ) . the role constructs expressing full-length and truncated of this additional region of polypeptide in structure or versions of clpro activity of the coronavirus proteinases is not known. in this study, we demonstrate that aspartic acid resi-the ser to gln (pg-s/fq) and ser to dues in mhv clpro are not required for catalytic activity. gln (pg-s/lq) fragments were obtained by pcr am-in addition, we show that the mhv clpro is able to plification of the region between nt and and cleave at a gln-gly cleavage site upstream from the prethat between nt and , respectively (fig. ). dicted carboxy terminal gln-ser cleavage site and within the pg-s/fq left primer with an added on xbai restricthe proteinase itself. the clpro extending from the contion site ( -attctagatgtctggtatagtgaagatgg firmed amino-terminal serine to the internal glutamine/ tgtcg- ) and pg-s/fq right primer with an added on glycine cleavage site is inactive in vitro. in contrast, the hindiii restriction site ( -taaaataagctt tcactggaa-''full-length'' protein extending to the predicted glutamine/ tccagaatgcagcct- ) were used to prime dna synserine cleavage site is identical in size to clpro exthesis from the pgpro construct. the pcr products were pressed in vitro and in virus-infected cells and is an digested by xbai and hindiii for hr and then run on an active proteinase. thus, it appears that the entire pre- . % low-melting-point agarose gel. the product band was excised and ligated into the ecori and hindiii sites dicted coding region is required for clpro activity. . with a pam scoring matrix. the mhv-a his and cys residues, and the corresponding residues of ibv, tgev, and e are shown in boldface letters. the locations of aspartic acid residues (asp and asp ) of mhv-a are shown by asterisks. other conserved asparagine (n ) and aspartic/glutamic acid residues (d ) are indicated by a dot. residues predicted to be involved in substrate binding (thr and his ) are indicated by a diamond. the solid arrowhead indicates the experimentally confirmed amino-terminal cleavage site of the mhv and e clpro (lu et al., ; ziebuhr et al., ) . the open arrowhead indicates the predicted carboxyl terminal lq_s/a cleavage sites of the proteinases (gorbalenya et al., ; lee et al., ; gorbalenya and koonin, ) . the location of the fq_g sequence in mhv is indicated as an underlined arrowhead. numbering of mhv his , cys , asp , asp , asn , and glu residues is based on identifying ser of the orf a polyprotein as ser of clpro. mhv-a amino acid numbers were derived from the submitted nucleotide sequence of bonilla, et al. ( ) . ''iisvkes'' is a seven-amino-acid region present only in the ibv sequence. of pgem- zf( ) (promega) behind the t promoter which in vitro transcription and translation constituted pg-s/fq. pgopt-s/fq was similarly con-recombinant plasmids were transcribed and transstructed using a left primer with an optimal atg ( lated using a coupled in vitro transcription/translation gggcgaattcgccaccatgagtggtatagtgaagat rabbit reticulocyte lysate system (tnt, promega), as pre-ggtgtcg- ). pc-s/fq was constructed by using a left viously described (lu et al., (lu et al., , . approximately primer with an added ncoi restriction site ( -tcatcc- . mg of plasmid dna was incubated at Њ with . atggcctctggtatagtgaagat g- ) and a right ml tnt lysate, ml tnt reaction buffer, . ml t rna primer with an added ecori restriction site ( -aatttpolymerase, units rnasin, . ml mm methionine-gaattcactggaatccagaatgcagcct- ). the fragfree amino acid mixture, and mci [ s]methionine in ment was then subcloned into pcite (novagen). pc-s/ a final volume of ml. samples were taken at various lq was similarly constructed by using a left primer with time points and electrophoresed on an sds - % gradian added ncoi restriction site ( -tcatccatggcctctent polyacrylamide gel (sds-page). ggtatagtgaagatg- ) and a right primer with an added ecori restriction site ( -tgtgcg aattcactg-trans cleavage assay tagcttgacaccagcta- ). the fragment was then subcloned into the ncoi and ecori sites of pcite (nova-inactive site-directed mutants of pgpro (pgproh g or pgproh q) were translated in the presence of [ s]-gen) behind the t promoter. methionine. the parental pgpro construct, plasmids en-asp (d ) was conserved as either asp or glu among the four viruses. glu (e ) of mhv was conserved coding the predicted full-length clpro (pc-s/lq), and plasmids encoding the truncated forms of clpro (pg-as glu or asp, and asn (n ) of mhv was identical in all four coronavirus sequences; however, the location s/fq, pgopt-s/fq, and pc-s/fq) were transcribed and translated in the presence of nonradiolabeled l-methio-of these residues relative to the essential his and cys residues makes them less appealing as potential cata-nine. after min, transcription and translation were terminated by the addition of rnase ( mg/ml) and cyclo-lytic residues. comparison of the coronavirus clpro amino acid se-heximide ( mg/ml) for min. following termination of transcription and translation, labeled mutant and unla-quences with chymotrypsin confirmed the additional amino acids between the putative substrate binding resi-beled clpro reaction lysates were mixed : and incubated for an additional min. the reaction mixtures due his (h ) and the probable carboxy-terminal qs - cleavage site of clpro. the comparison of the were checked for residual expression and processing from the pgpro construct by the addition of [ s]-four coronavirus clpro sequences revealed two potential cleavage sites present only in mhv, qs - , and methionine to an aliquot of the unlabeled reaction mixture after treatment with rnase and cycloheximide and qg - . overall, the comparison of the coronavirus sequences indicated that there was variation among the incubation for an additional min. all products were analyzed by electrophoresis by sds gradient page, fol-proteinases in the location of potential catalytic residues and cleavage sites. lowed by fluorography. mutagenesis of aspartic acid residues in vitro transcription and translation were performed based on the analysis of the protein alignments, we in a total volume of ml with . mg pgpro dna in the chose asp and asp residues for mutagenesis studpresence of mci [ s]methionine (dupont nen), ies. asp has been considered the most likely candi-mci [ h]valine (amersham), or mci [ h]leucine (amerdate for a third residue to be involved in catalysis. asp sham) for min at Њ. the products were separated was in a less favorable position relative to the his, but on - % gradient polyacrylamide gels, transferred to a was conserved among the coronaviruses and provided polyvinylidene difluoride (pvdf) membrane at v at Њ a good control. in addition, studies of other viruses have for hr in transfer buffer containing mm tris-base, demonstrated that deviation from predictions of active mm glycine, and % (v/v) methanol. after transfer, residues is not uncommon. the construct used for these the pvdf membrane was air dried and exposed to x-ray studies (pgpro) encoded amino acids - of film. radiolabeled proteins were identified by autoradiog-mhv gene , including clpro ( - ) and portions raphy, and the corresponding bands were excised from of the flanking domains (fig. a) . we have previously the pvdf membrane and subjected to amino-terminal shown that translation of pgpro in vitro results in a presequencing on an abi sequencer. the amino acid cursor polypeptide from which active clpro is autoprofraction from each cycle was quantitated in a beckman teolytically cleaved and that clpro has an apparent scintillation counter. mass of - kda (p ) following sds-page (lu et al., ) . liberation of p clpro was therefore used results as a marker of proteolytic activity of proteins expressed from different constructs in vitro. the wild-type proteinase construct (pgpro) and mutant proteinase domains proteinase constructs were transcribed and translated in a rabbit reticulocyte lysate (fig. b) . the proteinase predictions of catalytic residues of the coronavirus clike proteinases have not strongly predicted aspartic or expressed from pgpro was able to process p clpro (fig. b, lane ) , whereas the proteinase with the his glutamic acid residues. comparison of the deduced amino acid sequences of clpro from the coronaviruses to gln mutation (h q) did not cleave p (fig. b , lane ). mutation of asp to pro or ala (d p and d a) mhv-a , ibv, hcv- e, and tgev revealed no completely conserved asp or glu residues at positions analo-resulted in a proteinase with activity comparable to that expressed from wild-type pgpro (fig. b, lanes and ) . gous to catalytic asp or glu residues of other c or clike proteinases (fig. ) . there was relative conservation substitution of asp by glu (d e) did not affect clpro activity (fig. b, lane ) , whereas the substitution of of asp, glu, or asn among the coronaviruses at the residue analogous to asp (d ) of mhv. it has been asp by pro (d p) impaired processing of p approximately % relative to pgpro (fig. b, lane ) . the d p shown that the analogous residues within the ibv clpro at asp or asp (d or d ) are not required for change might be expected to cause a change in the proteinase structure with a concomitant alteration of ac-proteolytic activity (liu and brown, ). the mhv pgpro in the presence of leupeptin blocked cleavage of p and also completely blocked processing of the small polypeptide fragment (fig. , lanes - ) . the small cleavage fragment indicated by the arrow was consistently seen when pgpro or proteolytically active mutants were translated, but not when proteolytically inactive mutants were expressed (fig. b) . the cleavage fragment was detected following translation of pgpro, h q, h m, and c r (fig. b, lanes , , , and , respectively) , all of which also processed p . in contrast, no small fragment was seen after translation of c g or h q (fig. b, lanes and ) , both of which are inactive in p processing. together these results indicated that this cleavage fragment was processed by products expressed from the proteinase constructs in vitro, rather than by proteinases in the reticulocyte lysate. the smallest proteolytic fragment was used for amino terminus radiosequencing since it was the most discrete and abundant. the pgpro construct was transcribed and translated in the presence of [ h]leucine, the peaks of radioactivity were consistent with leucine and asp were expressed in a combined transcription and translation at residues and , cysteine at residue , and valine lysate as previously described (lu et al., ) . samples were taken at residue . the only cleavage site within the pgpro at min for analysis by - % sds gradient page. the wild-type pgpro construct and the his to gln mutant (h q) were used as expression product that could result in a product with controls. d a refers to an ala substitution at asp ; other constructs this pattern was gln-gly - . are similarly labeled. mass markers are to the right of the gel and the the qg - was not conserved in any of the other location of p is shown to the left of the gel. processing of p coronaviruses and previously had not been predicted as by clpro expressed from pgpro was considered as %, and the a cleavage site for clpro. radiosequencing with three percentage of proteinase activity of each expressed protein is shown beneath the lane markers. different amino acids confirmed specific cleavage between glutamine and glycine by the in vitro translated proteinase. we could not define the presumed cartivity; however, the d p substitution diminished but did boxy-terminal fragment containing the predicted qs - not abolish activity, indicating that d was not an indiscleavage site, possibly due to the compression of propensable residue. it was interesting that the d p teins in this region ( . kda) of the gel by the nonlabeled change did not diminish proteolytic activity, suggesting globin protein from the lysate. additionally, the protein that even a major change at this location was inconsefrom the construct may have been targeted for rapid degquential for liberation of p clpro in the in vitro system. radation. we also did not detect any prominent alterna-deletion of asp (d del) resulted in complete loss of tive form of p clpro. since we do know the order of proteinase activity (lane ). this was not surprising since cleavages or pattern of precursors expressed from pgpro such a change might be expected to significantly alter it has not been possible to determine when the fq/g the structure of the proteinase. these experiments demsite is cleaved. direct comparison of these cleavage sites onstrated that neither asp nor asp was directly inwill require constructs expressing single cleavage sites volved in catalysis with his or cys . to determine specificity. identification of a clpro cleavage site truncation of clpro and trans cleavage activity during in vitro translation of pgpro several proteins in vitro with apparent masses of less than . kda were seen along with p (fig. ) . the pulse-label expression (fig. since clpro was able to cleave upstream of the predicted qs - cleavage site, we determined whether a) showed that the smallest of these polypeptides appeared concurrently with p clpro but then decreased ser to gln was the entire coding region for the active p clpro protein detected in virus-infected cells over a -hr period (fig. , lanes - ) . translation of and during in vitro translation of pgpro (fig. ) . the amino proteins by incubating the nonradiolabeled translation products of these constructs with radiolabeled sub-acid sequence extending from ser to gln would predict a protein of with a calculated mass of kda, strate expressed from the inactive proteinase mutant pgproh g (his to gly) (fig. b, lane ) . the ''full-whereas cleavage at gln would predict a protein of kda in mass, somewhat closer in size to the apparent length'' -kda clpro expressed from the ser -gln construct was able to cleave the pgproh g mass of p (fig. a ). we constructed a panel of plasmids containing cdnas encoding amino acids from expressed protein in trans (fig. b, lane ) , whereas the -kda truncated clpro expressed from the s -gln or s -gln (fig. a) . the cdnas were expressed in a variety of plasmids, using either the first ser -gln constructs did not process the mutant protein (fig. b , lanes , , and ). this result demon-natural aug (pg-s/fq) or an optimized aug before ser (pgopt-s/fq). we also used vectors containing strated that a -amino-acid carboxy terminal truncation of clpro abolished proteolytic activity. emcv ires elements to ensure that translation initiated before the ser (pc-s/fq and pc-s/lq). the constructs were used to direct translation in vitro discussion and the proteins either were radiolabled or were translated in nonlabeled medium and used in a trans cleavage mhv clpro is postulated to mediate the majority of assay of the inactive mutant pgproh g (fig. b ). transcleavages in the gene polyprotein during virus replicalation of pc-s/lq, encoding ser to gln , resulted in tion. we have shown that aspartic acid residues of mhv a single -kda protein, the same migration pattern as clpro in locations analogous to essential asp/glu resi-p clpro detected after expression of pgpro (fig. b , dues of other c and c-like proteinases are not neceslanes and ). in contrast, translation of three different sary for processing of substrate by clpro in vitro. reconstructs encoding the truncated clpro domain from sults similar to ours have been reported for infectious ser to gln resulted in a single -kda protein (fig. bronchitis virus (ibv) (liu and brown, ) . our study b, lanes , , and ). these data indicated that proteins demonstrates that conservation of asp/glu in this region expressed from the clpro domain differed in their calof the coronavirus c-like proteinases is not due to an culated and apparent masses by to kda. the results indispensable catalytic role. the mechanism of the mhv also supported the conclusion that the active clpro in clpro may be more similar to that of hepatitis a virus, mhv-infected cells and from in vitro translation products in which the asp is on an external motif and not directly incorporated ser to gln . involved in the catalytic unit (allaire et al., ) . it is we assessed the in vitro cleavage activity of the possible that this variation in the use of a third residue may have coevolved with the specificity for cleavage -kda ser -gln and the -kda ser -gln sites. there is a precedent in other virus systems for a consensus substrate binding residues, whereas the mhv sequence extends an additional residues to its contribution of asp residues to proteinase specificity even though they may not be involved in catalysis. for carboxy terminus (lee et al., ) . our study demonstrates that a small deletion of this part of the proteinase example, poliovirus contains an frd (d ) sequence that was initially thought to be involved in catalytic activity abolishes its ability to cleave new molecules of p clpro in trans, demonstrating that the entire carboxy-but subsequently was found to be in a flanking turn domain and to be involved in autocatalytic cleavage of cd terminal region is essential for clpro activity. analysis of confirmed and predicted cleavage sites in (hammerle et al., ) . despite the lack of use of an asp residue, the mhv clpro should still be classified the gene polyproteins of mhv-a , hcv- e, ibv, and tgev has revealed a preference for a gln at p and leu, as a chymotrypsin-like enzyme because of the localization of histidine and cysteine residues as well as flanking ile, val, or less often phe or met at p (boursnell et al., ; breedenbeek et al., ; lee et al., ; herold residues considered to be important in protein structure and substrate binding (gorbalenya and koonin, ). et al., ; bonilla et al., ; eleouet et al., ) . although the phe-gln-gly (fq/g) clpro cleavage site analysis of the full-length clpro domain (ser to gln ) reveals several possible differences between the we identified within clpro has similarities to other predicted clpro sites from p to p , it is not present in mhv clpro and other viral proteinases in the group of cysteine-containing enzymes (fig. ) . first, most of these the other sequenced coronaviruses. we have not determined if the fq/g site can be cleaved in virus-infected enzymes terminate within amino acids following the intracellular and in vitro translated -kda proteins contain the c-like proteinase activity of the picornaviral c cysteine proteinases have a fold similar to chymocoronavirus mhv-a identification and characterizatrypsin-like serine proteinases mouse hepatitis tion of a serine-like proteinase of the murine coronavirus mhv-a effect of expression of the aphthovirus protease c on viral infection and gene expression completion of the sequence of the virology genome of the coronavirus avian infectious bronchitis virus the primary structure and ase reveals a trypsin-like polypeptide fold, rna-binding site, and means for cleaving precursor polyprotein characterization of a human coronavirus (strain e) c-like proteinase activity eleouet, j. f., rasschaert, d., lambert, p., levy, l., vende, p., and laude, cells. it is possible that the fragment of gene used in h. ( ) . complete sequence ( kilobases) of the polyprotein-enthese studies allows presentation of this site in a manner g cleavage site is used by the proteinase in cells, it gorbalenya, a., and koonin, e. ( ) . comparative analysis of aminoacid sequences of key enzymes of replication and expression of might represent a pathway for regulation of the protein- in conclusion, we have identified several unique fea- ase gene polyprotein processing and virus replication. lawson, m. r., and semler, b. l. ( ) . alternate poliovirus nonstructural protein processing cascades generated by primary sites of c key: cord- -q ogrem authors: barthold, s. w.; smith, abigail l. title: viremic dissemination of mouse hepatitis virus-jhm following intranasal inoculation of mice date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: q ogrem using a sensitive infant mouse bioassay to detect infectious virus, the pattern of mouse hepatitis virus (mhv) jhm dissemination in blood and other tissues was examined during the first days following intranasal inoculation. mhv replicated in nasal turbinates of both susceptible balb and resistant sjl mice from days through , but balb mice had higher titers on days and . viremia was detectable on days through in balb mice, but only on days and in sjl mice. transient virus replication occurred in the lungs of both mouse genotypes at and days, then ceased. this correlated with more consistently demonstrable virus in blood collected from the left atrium of the heart, compared to jugular vein, portal vein and right atrial blood. virus was associated equally with the plasma and cellular fractions of blood on day , but was primarily in the buffy coat of the cellular fraction on day . interferon-α/β was detected in serum and spleen, but not liver or brain of balb mice or in any tissue of sjl mice. balb serum and spleen interferon was first detected at h, peaked between and h, and was undetectable by h. the distribution of virus in nose, cervical, axillary and mesenteric lymph nodes, spleen, peyer's patch, thymus, bone marrow and liver was examined at , , and days. the resulting pattern suggested lymphatic spread of virus to cervical lymph node and mesenteric lymph node as pathways of dissemination in addition to viremia. mouse hepatitis virus (mhv) is a highly contagious and prevalent coronavirus of laboratory mice, with numerous related strains that partially differ antigenically, genetically and biologically [ , ] . like coronaviruses of other species, mhv strains display primary tropism for upper respiratory or enteric mucosa [ , ] . in susceptible mice inoculated with respiratory-type mhv strains by the s.w. barthold and abigail l. smith intranasal (i.n.) route, virus spreads by direct extension from the nose to the brain [ , ] and to other target organs such as liver and lymphoid tissue by a presumed viremic course. viremia is suspected, since lesions and antigen are distributed in a vascular pattern [ , , , ] . mhv viremia has been very difficult to demonstrate, since cell culture systems are relatively insensitive for detection of infectious virus in tissues. despite evidence that mhv disseminates in a vascular distribution and infects blood-associated tissues such as bone marrow, lyrnphoid organs and vascular endothelium, viremia following i.n. mhv inoculation of immunocompetent mice has only recently been demonstrated by using an infant mouse infectivity assay. under these circumstances, infectious virus could be detected in blood as early as h after i.n. inoculation [ ] . the purpose of the present study was to examine the early kinetics of viremia as the mechanism of mhv dissemination in genetically susceptible and resistant mice inoculated with a moderately virulent, respiratory-type strain of mhv. the sequential appearance of mhv in respiratory tissues and blood was initially examined in genetically susceptible balb/cbyj (balb) and resistant sjl/j (sjl) mice following i.n. inoculation with moderately virulent mhv strain jhm. previous studies have shown that both of these mouse strains develop disseminated infections between days and after i.n. inoculation, but virus titers and disease are significantly greater in balb mice compared to sjl mice [ ] . groups of randomly selected mice of each genotype were killed on days , , , , and after inoculation. infectious virus in nasal turbinates and lung on clays , , , and , and blood on days , , , , and was titrated. sera were tested for detectable mhv antibody. to determine the relative rate of infection and mhv titers in each blood compartment, sets of blood samples were obtained from jugular vein, portal vein, left cardiac atrium and right cardiac atrium in a group of balb mice at h after i.n. inoculation. viremic blood samples from additional balb mice on days and were pooled and differentially separated into cellular (erythrocyte and buffy coat) and plasma fractions. mhv titers were determined in whole blood, cellular and plasma fractions. the association of interferon-a/~ with viremia was explored initially by analyzing pooled samples of nasal turbinate, blood, liver, brain, or spleen obtained from mice of each genotype on days (controls), , , , and after i.n. inoculation. based on findings from this experiment, interferon was assayed in individual samples of serum and spleen from balb mice per interval at , , , , , , , , and h after i.n. inoculation. finally, as another means of examining the routes of mhv dissemination, the sequential appearance of mhv in nose, cervical lymph nodes, axillary lymph nodes, ruesenteric lymph nodes, spleen, peyer's patches, thymus, bone marrow, and liver was examined in groups of balb mice at , , and clays after i.n. inoculation. certified virus-free, - week old balb and sjl mice were purchased from the jackson laboratory, bar harbor, me and pregnant outbred cri:cd br (cd ) mice were purchased from charles river laboratories, portage, mi, shipped in filtered boxes, then transferred upon arrival into autoclaved micro-isolator containment cages (lab products, mouse hepatitis virus viremia maywood, nj) containing wood shavings, food (prolab animal diet, agway, syracuse, ny) and water. cages and mice were manipulated within a class ii biological containment cabinet to preclude inadvertant exposure to adventitious murine viruses. randomly selected mice were killed with carbon dioxide gas at specific intervals, and tissues were collected aseptically and frozen at - °c until tested for virus. nasal turbinates were collected with forceps after removal of the dorsal nasal bones with a razor blade. blood was collected in non-heparinized glass syringes and placed in vials containing edta as an anticoagulant. peyer's patches consisted of intestinal mucosa containing gut associated lymphoid tissue. blood was fractionated by centrifugation. plasma was drawn off from the cellular fraction in separate aliquots. in additional samples, buffy coat was removed from erythrocytes, then erythrocytes were washed and centrifuged twice in saline: virus mhv-jhm was obtained and maintained as previously described [ ] . mice were inoculated i.n. with gl of cell-free culture fluid containing approximately ~ tcids of mhv-jhm. virus was detected in tissues by intracerebral inoculation of neonatal cd mice with % (w/v) tissue homogenates or whole blood in . ml volume. virus titers were determined by similar inoculation of infant mice with serial -fold dilutions of tissue homogenates, and expressed as log lds /g, as previously described [ ] . interferon-a/ was assayed in tissues by a cytopathic effect reduction assay, using mouse l cell monolayers challenged with median infectious doses of vesicular stomatitis virus (indiana serotype), as previously described [ , ] . international units (iu) were determined with reference to a niaid, who international reference standard (g - - ). test tissues were triturated as % (w/v) homogenates, acidified to ph . overnight and neutralized prior to assay. antibody to mhv-jhm was determined by enzyme immunoassay, using formalin-fixed mhv-jhm-infected c - cells as antigen and horseradish peroxidase-conjugated goat anti-mouse igg (biorad, richmond, ca) as described [ ] . differences in proportions were determind by ~ analysis and virus titers were compared with student's paired or unpaired t tests. mhv was detected in nasal turbinates of both balb and sjl mice during the first days after i.n. inoculation, but significantly higher titers were found in balb mice compared to sjl mice on days (p ~< . ) and (p ~< . ) (fig. t) . viremia was detectable in balb mice on days , , , , and , but only on days and in sjl mice (fig. ) . remarkably, mhv titers in blood on days and were equivalent between genotypes. mhv was detected in of balb (table ) . mhv lung titers at days were significantly higher (p ~< . ) in balb compared to sjl mice. viremia was most consistently detected in blood samples taken from the left atrium compared to jugular vein, portal vein and right atrium at days after i.n. inoculation (table ). cellular and plasma fractions of balb blood contained equivalent titers of mhv on day , but titers in plasma dropped significantly on day relative to day (p ~< . ) ( table ) . on day , the cellular fraction contained significantly more virus as the plasma (p ~< . ). in of the blood samples tested on day , virus could only be detected in the cellular table . mhv-jhm titers (log intracerebral lds /g) and rate of lung infection at intervais after intranasal inoculation of balb and sjl mice genotype interval after inoculation (days) interferon-a/ was detected in pooled serum and spleen, but not nasal turbinate, liver or brain, of balb mice and was not detected in any tissues of sjl mice. levels in serum and spleen of balb mice peaked at the day interval. based on these findings, interferon levels were assayed in serum and spleen of individual balb mice (table ). interferon titers were much higher in spleen compared to blood. in both tissues, interferon was first detectable at h and peaked between and h. none was detectable by h. mhv antibody was not detectable in sera of balb or sjl mice through day . at day after i.n. inoculation, infectious virus was detectable in nasal turbinate of all mice, in cervical lymph node in of mice, in spleen and peyer's patch of a few others, but not other sites (table ) . thus, cervical lymph node sustained significantly higher rate of early infection compared to more distant axillary lymph node (p <~ . ), suggesting lymphatic spread of virus. by days, mhv was present in nose, cervical lymph node, mesenteric lymph node and spleen of all mice; axillary lymph node, peyer's patch and liver of over half of the mice; bone marrow of a few mice and not in thymus. both cervical lymph node and mesenteric lymph node had higher rates of infection compared to axillary a number positive/number tested lymph node (p ~< . ), suggesting lymphatic spread through both the head and gut. by days, most tissues were infected in high prevalence, except thymus, which was positive in only of mice. hepatitis and inflammation of other internal organs are important features of respiratory mhv infection in laboratory mice and are responsible for the name "hepatitis virus" that has been given to this group of murine agents. we now know that some mhv strains are strictly entertropic and seldom cause hepatitis regardless of the age or immune status of the host, while others, which replicate initially in nasal mucosa, can cause hepatitis if the virus is sufficiently virulent or the host is susceptible [- ]. the polytropic, generalized nature of disease caused by these latter mhv strains in susceptible mice following i.n. inoculation is suggestive of viremic dissemination, but demonstration of mhv viremia has been inconsistent and usually following artificial routes of inoculation of immune-impaired mice with highly virulent strains of mhv. viremia was detected with c - cell culture in c bl/ mice on day and after intracerebral (i.c.) inoculation with mhv-a , but not following intraperitoneal (i.p.), i.n., or intragastric inoculation [- ] . viremia was demonstrable with dbt cell culture in athymic balb mice on day after i.n. inoculation with wild-type mhv [- ] . swiss mice inoculated i.n. with mhv-s at days of age had viremia detected in dbt cells on day , but not days , , or . viremia could not be detected in older mice [ ] . much greater success was achieved in detecting and quantifying viremia as early as h and through days after i.p. inoculation of ddd and cdf mice with virulent mhv- , using dbt cell culture [ ] . highly virulent mhv- could be detected and quantified in the serum of day old a mice on days , , and after i.p. inoculation, using a mouse infectivity bioassay [ ] . likewise, virus titers in blood were monitored at several intervals up to hours after intravenous inoculation of adult swiss mice with mhv- , using a mouse bioassay [ ] . on the other hand, we and others [ , ] have failed to detect viremia with cell culture assays following i.n. inoculation of adult mice with mhv-jhm. these inconsistencies are no doubt due to difficulties of growing nonadapted mhv in cell culture systems and their relative insensitivity for detecting infectious virus. we have found that the most sensitive means of detecting infectious mhv in tissues is the infant mouse bioassay and we have previously shown viremia following i.n. inoculation of young adult, immunocompetent mice with moderately virulent mhv [ using the infant mouse bioassay that was employed in the current study. the current results suggest that viremia is a very early event following i.n. inoculation, and can be detected within h. furthermore, lung appears to be an important early target for virus replication, and probably contributes to secondary viremia. mhv has been shown to replicate in pulmonary capillary endothelium and interstitium, with minimal infection of airway epithelium [ , , ] . virus titers in cardiac blood samples were highest in the left atrial samples, compared to the right atrial, jugular or portal samples, supporting the pulmonary origin of some of the virus in the circulating blood. fractionation studies of cardiac blood samples demonstrated infectious virus in both the plasma and cellular fractions on day , but virus had largely cleared from plasma by day , when it was associated with the buffy coat cells (leukocytes). mhv has a well known tropism for lymphoid tissue, as well as hematopoietic elements in bone marrow and spleen. the mechanism of plasma clearance by day was not determined, but it was preceded by a drop in virus titer on day , which was apparent in both balb and sjl mice. this biphasic pattern would suggest the influence of antibody or interferon, but antibody was not detected and serum interferon was present at low concentrations only in balb mice. in a previous study, antibody was detectable on day , but not on day , in balb mice and only of sjl mice tested on day had detectable antibody [ ] . the sequential distribution of mhv in parenchymal tissues suggests that virus dissemination also occurs through lymphatic drainage of the head, since cervical lymph nodes became infected earlier than other lymphoid tissues. this is not a significant event in mhv dissemination, since generalized involvement of virtually all target tissues has taken place by days. the higher rate of mesenteric lymph node infection relative to axillary lymph node at days suggests enteric entry as well. mhv-jhm, like most respiratory mhv strains, does not replicate to a very great extent in intestinal mucosa, but has a marked tropism for gut associated lymphoid tissue (galt) [ ] . the current study suggests early infection of galt from the intestinal lumen, then mesenteric lymph nodes following i.n. inoculation. as previously demonstrated, this study confirms that lymphoid tissue in general is an important target for mhv. involvement of the thymus in mhv-jhm infection has been reported in mice inoculated intracerebrally [ ] , but appears to be relatively rare following i.n. inoculation. interferon has been detected in serum, liver, spleen, and peritoneal exudate cells of mice infected with various mhv strains [ , , , , ] . correlation of interferon levels with susceptibility has been variable. serum interferon concentrations were low in mhv-s infected, immature mice and elevated in adult mice [ ] . others [ , ] have shown that resistant mouse genotypes (ddd, a/j) produce less serum and tissue interferon than susceptible genotypes (cdf , c bl) when infected with mhv- or mhv- . no differences in serum interferon concentrations were detected between susceptible (c h, balb) and resistant (ddd, cf ) mice infected with mhv- or mhv-jhm [ , ] , although peak interferon titers occurred later in susceptible balb compared to resistant cf mice [ ] . in the current study, interferon was found in spleen and serum of mhv-jhm-infected balb mice, but not nose, liver or brain and was not detectable in any tissue of sjl mice. since all of these tissues were infected in both genotypes, as demonstrated previously [ ] , interferon had no correlation with presence, clearance or absence of infectious mhv. these data, and those of others, indicate that serum or tissue interferon responses of mice to mhv vary greatly with mouse genotype. furthermore, mhv strains vary in their sensitivity to interferon in vitro [- , ] . sjl mice are well known to possess remarkable resistance to mhv disease. they have been shown to lack a functional cellular receptor for mhv-a in at least some target tissues [ , ] . we have previously demonstrated that sjl mice develop very mild infections of liver, spleen and lymphoid tissue compared to balb mice, when inoculated by the i.n, route [ ] . the current study demonstrates that sjl mice support virus replication in their nasal turbinates and also develop a demonstrable viremia, albeit intermittent. thus, their resistance to mhv-jhm-induced disease is not a reflection of their absolute resistance to infection or ability to support virus replication. mouse hepatitis virus biology and epizootiology olfactory neural pathway in mouse hepatitis virus nasoencephalitis susceptibility of laboratory mice to intranasal and contact infection with coronaviruses of other species mouse hepatitis virus strain-related patterns of tissue tropism in suckling mice response of genetically susceptible and resistant mice to intranasat inoculation with mouse hepatitis viurs jhm genetic resistance of mhv correlates with absence of virus-binding activity on target tissues pulmonary vascular lesions in nude mice persistently infected with mouse hepatitis virus responses of mice susceptible or resistant to lethal infection with mouse hepatitis virus, strain jhm, after exposure by a natural route nasoencephalopathy of mice infected intranasally with a mouse hepatitis virus, jhm strain infection and involution of mouse thymus by mhv- effects of cortisone on interferon response of germfree mice to mouse hepatitis virus mouse hepatitis virus viremia mhv-a pathogenesis in mice immunopathology of mouse hepatitis virus type infection. i. role of humoral and cell-mediated immunity in resistance mechanisms the fate of murine hepatitis virus (mhv- ) after intravenous injection into susceptible mice convenient assay for interferons activation of natural killer cells and induction of interferon after injection of mouse hepatitis virus type in mice two enzyme immunoassays for the detection of antibody to rodent coronoaviruses pathogenesis of mouse hepatitis virus infection. the role of nasal epithelial cells as a primary target of lowvirulence virus difference in response to mouse hepatitis virus among susceptible mouse strains difference in sensitivity to interferon among mouse hepatitis viruses with high and low virulence for mice factors involved in the age-dependent resistance of mice infected with low virulence mouse hepatitis virus the role of circulating interferon in the modifications of immune responsiveness by mouse hepatitis virus (mhv- ) the biology and pathogenesis of coronaviruses purification of the -kilodalton glycoprotein receptor for mhv (mhv)-a from mouse liver and identification of a nonfunctional, homologous protein in mhv-resistant sjl/j mice t-lymphocyte.dependent difference in susceptibility between ddd and c h mice to mouse hepatitis virus, mhv- this work was supported by grant rr from the national center for research resources, national institutes of health, bethesda, maryland. the assistance of d. s. beck and d. winograd is gratefully acknowledged. received april , key: cord- -lodjcj c authors: zhang, xuming; hinton, david r.; cua, daniel j.; stohlman, stephen a.; lai, michael m.c. title: expression of interferon-γ by a coronavirus defective-interfering rna vector and its effect on viral replication, spread, and pathogenicity date: - - journal: virology doi: . /viro. . sha: doc_id: cord_uid: lodjcj c abstract a defective-interfering (di) rna of the murine coronavirus mouse hepatitis virus (mhv) was developed as a vector for expressing interferon-γ (ifn-γ). the murine ifn-γ gene was cloned into the di vector under the control of an mhv transcriptional promoter and transfected into mhv-infected cells. ifn-γ was secreted into culture medium as early as hr posttransfection and reached a peak level (up to u/ml) at hr posttransfection. the di-expressed ifn-γ (de-ifn-γ) exhibited an antiviral activity comparable to that of recombinant ifn-γ and was blocked by a neutralizing monoclonal antibody against ifn-γ. treatment of macrophages with de-ifn-γ selectively induced the expression of the cellular inducible nitric oxide synthase and the ifn-γ-inducing factor (igif) but did not affect the amounts of the mhv receptor mrna. antiviral activity was detected only when cells were pretreated with ifn-γ for hr prior to infection; no inhibition of virus replication was detected when cells were treated with ifn-γ during or after infection. furthermore, addition of ifn-γ together with mhv did not prevent infection, but appeared to prevent subsequent viral spread. mhv variants with different degrees of neurovirulence in mice had correspondingly different levels of sensitivities to ifn-γ treatmentin vitro,with the most virulent strain being most resistant to ifn-γ treatment. infection of susceptible mice with de-ifn-γ-containing virus caused significantly milder disease, accompanied by more pronounced mononuclear cell infiltrates into the cns and less virus replication, than that caused by virus containing a control di vector. this study thus demonstrates the feasibility and usefulness of this mhv di vector for expressing cytokines and may provide a model for studying the role of cytokines in mhv pathogenesis. ). resistance to ifn-g may lead to incomplete viral clearance and contribute to the establishment of persis-interferon-g (ifn-g) is a pleiotropic cytokine produced tent infection (moskophidis et al., ) . by contrast, ifnby activated cd / and cd / t cells and natural killer g is also involved in inflammatory processes. ifn-g incells (trinchieri and perussia, ; pestka and langer, duces the expression of many other inflammatory cyto- ; ijzermans and marquet, ) , which exerts both kines, such as interleukin- (il- ) and tumor necrosis antiviral and immunomodulatory effects. these include factor (tnf), and acts synergistically with these cytokines the activation of mononuclear phagocytes, enhancement (wong and goeddel, ) . the multitude of immunoof the generation of oxygen-free radicals, modulation of modulatory effects of ifn-g makes it a particularly interclass i and ii major histocompatability complex (mhc) esting cytokine for studying viral pathogenesis. in the antigen expression, and promotion of differentiation of central nervous system (cns), no cells constitutively exboth t and b cells (for reviews, see references by pestka press ifn-g. during encephalomyelitis, for example as and langer, ; benveniste, ) . it plays an ima result of mouse hepatitis virus (mhv) infection, actiportant role in the early phase of many viral infections vated nk cells and t cells which pass through the blood- (wheelock, ; wong and goeddel, ; leist et al., brain barrier into the cns express ifn-g (bukowski et ; klavinskis et al., ; feducchi and carrasco, al., ; pearce et al., ) . in addition to its effects on ; ramsey et al., ; heise and virgin iv, ; mononuclear cells, ifn-g acts upon cells of the cns, rodriguez et al., ) , inhibiting the replication of a varisuch as astrocytes, microglia, and macrophages (benety of viruses prior to activation of antiviral effector cytoveniste, ) . toxic t lymphocyte (ctl) or antibodies. because of its mhv, a murine coronavirus, causes a variety of disantiviral activity, ifn-g has been implicated in virus cleareases in rodents, such as hepatitis, enteritis, and neuroance and resolution of viral infection (ramshaw et al., logical diseases, depending on the viral strain (cheever et al., ; gledhill and niven, ; ishida et al., ) . lination (stohlman et al., ; lai and stohlman, ) . may allow studies of the interaction between mhv and the host's immune system by expressing immunoregula-the dl variant derived from the parental jhmv causes an acute, fulminant, necrotizing encephalomyelitis with tory proteins at the foci of viral infection. minimal or no demyelination. by contrast, the neuroattenuated variant . -v- derived from dl produces a nonfa-materials and methods tal encephalomyelitis with extensive demyelination virus and cells (fleming et al., (fleming et al., , wang et al., ) . disease outcome also depends on the genetic background, the the following virus strains were used in this study: the developmental stage, and the immunological status of neuropathogenic mhv strain jhm isolate (dl), which is the host. previous studies have shown that immunocoma large plaque variant derived from the parental jhm petent mice infected with mhv exhibited increased exstrain (stohlman et al., ) ; the small plaque variant pression of a number of cytokines, including il- , il- , ds (stohlman et al., ) ; the neutralization-escape mu-tnf-a, and ifn-g, in the cns at the time of viral cleartant . -v- (fleming et al., ; wang et al., ) , and ance (pearce et al., ) . however, the role of these strain a , which is both neurotropic and hepatotropic. cytokines in mhv pathogenesis is not fully understood. the murine astrocytoma cell line (dbt) (hirano et al., for example, it has been suggested that ifn-g may not ) and j . macrophage cell line (obtained from be necessary for induction of the mhc class i molecules the american type culture collection) were used for in on neural cells in vivo (pearce et al., ) , a prerequisite vitro experiments. dbt cells were also used for plaque to ctl-mediated clearance (stohlman et al., ) . howassay. ever, ifn-g treatment ameliorates mhv-induced disease (smith et al., ) , suggesting that either the antiviral plasmid construction role or the immunomodulatory role of ifn-g is a critical a previously constructed plasmid p cat (liao and component of mhv infection. lai, ) , which contains the plasmid bluescript (pro-mhv contains a single-strand, positive-sense rna gemega) sequence with a cat gene inserted behind an ig nome of kb (lee et al., ) . it undergoes rapid recombisequence in the disse cdna (makino et al., a) , was nation, probably due to its large rna genome and the used as the basic di vector. for cloning the murine ifnspecial properties of its rna-dependent rna polymerase g gene into the di vector, a cdna fragment containing . similarly, defective interfering (di) rnas are the complete ifn-g gene (kindly provided by dr. j. a. frequently generated in mhv-infected cells. recently, re-frelinger, university of rochester) was generated by combinant di rnas have been developed which can replipolymerase chain reaction (pcr) using a pair of primers. cate in the presence of a helper mhv (makino et al., a, the sense primer ( -taactagtaatctaatctaa- ; van der most et al., ) . we have modified an mhv actttaaggaatgaacgctacacact- ) contains a re-di rna and developed an expression vector. this di rna striction enzyme spei site (underlined), the coronavirus contains both the -and the -ends, an internal region of intergenic sequence (in boldface), and the first nucleothe parental mhv genome (makino et al., b) , and an tides of the ifn-g open reading frame (orf). the intergenic (ig) sequence, which is a recognition signal for antisense primer ( -tcagaattcaatcagcagcgasubgenomic mrna transcription, followed by an exoge-ctcct- ) contains the last nucleotides of the ifn-g nous gene. upon transfection of this di rna into mhv-orf and a restriction enzyme ecori site (underlined). infected cells, a subgenomic mrna is synthesized and the after restriction enzyme digestion of the pcr products inserted gene expressed. this system has been used to with spei and ecori, a . -kb cdna fragment was puriexpress the chloramphenicol acetyltransferase (cat) profied by low-melting-point agarose gel electrophoresis tein and the coronavirus structural protein hemagglutinin/ and directionally cloned into the spei and ecori sites of esterase (he) in mhv-infected cells (liao and lai, ; p cat, resulting in pde-ifn-g (fig. a) . the resulting liao et al., ) . these proteins are expressed only in construct contains the ifn-g gene placed behind the ig infected cells during virus replication, thus providing some sequence between genes and (ig ) of mhv. degree of targeted gene expression. furthermore, the expressed he protein can be incorporated into virus particles, rna transcription and transfection and the expression can be detected in serial virus passages (liao et al., ) . thus, this di rna expression plasmid dna (pde-ifn-g) was linearized with xbai, and rna was transcribed in vitro using t rna polymer-system provides an alternative to an infectious full-length cdna clone, which is still not available, for studying the ase according to the manufacturer's recommended procedure (promega). rna transfection was carried out molecular biology and pathogenesis of coronaviruses. in the present study, we have used this di rna system using the dotap method (boehringer-mannheim) as described previously (zhang et al., ) . briefly, mono-to express the murine ifn-g gene. the expressed ifng exhibited antiviral activity, prevented virus spread in layers of dbt cells grown at approximately % confluence in -mm petri dishes were infected with mhv at vitro, and altered viral pathogenesis in mice. this system de-ifn-g rna. following centrifugation at g for min, supernatants were tested for ifn-g using a sand-cells were washed with phosphate-buffered saline (pbs) and covered with ml of prewarmed eagle's minimum wich elisa as previously described (cua et al., ) . r - a (anti-ifn-g) (american type culture collection) essential medium (mem) containing % newborn calf serum (intragen). five to ten micrograms of in vitro tran-serum-free hybridoma supernatant was used to coat well plates. biotinylated xmg- . (anti-ifn-g) was ob-scribed rnas were mixed slowly with ml of dotap (boehringer-mannheim) in hbs buffer ( mm hepes; tained from pharmingen. avidin-peroxidase and o-phenylenediamine (opd) were obtained from sigma chemical mm nacl; ph . ), and incubated at room temperature for min. the mixture was then added to the cell co. recombinant ifn-g (rifn-g) (zymogen) was used as elisa standard, and the concentration of ifn-g is re-culture. the final concentration of dotap was mg/ml. ported in international units per milliliter (u/ml). enzyme-linked immunosorbent assay (elisa) for ifn-g mhv replication in the presence of ifn-g to quantitate expression of ifn-g, medium was collected at , , , , , and hr posttransfection from dbt cells were seeded at a concentration of cells per well into -well plates and incubated for hr dbt cells infected with jhm or a and transfected with at Њ in mem containing % newborn calf serum. j . extension. pcr products were analyzed by agarose gel electrophoresis. cells were seeded at a concentration of cells per well into -well plates and incubated for hr at Њ in dulbecco's modified mem (dmem) containing % dot blot analysis fetal calf serum. cells were treated with various concen-rt-pcr products were quantitated using the dot blot trations of the di-expressed ifn-g (de-ifn-g) or rifn-g method previously described (murphy et al., ; cua and infected with viruses at an m.o.i. of , . , . , or et al., ) . briefly, pcr-amplified cdna ( ml) was . . after virus adsorption for hr, the respective medenatured in ml of denaturing solution ( . n naoh dium with or without ifn-g was added and the cells were and mm edta) for min and neutralized by the incubated for the indicated periods of time. addition of an equal volume of m tris-hcl, ph . . samples were transferred to a nylon membrane via a isolation and detection of intracellular mrnas minifold i dot blot apparatus (schleichel and schuell), to study the effects of ifn-g treatment on the expresand the wells were washed with ssc ( . % sodium sion of cellular genes [inducible nitric oxide synthase chloride, . % sodium citrate). membranes were air-dried (inos), interferon-g-inducing factor (igif), and mhv reand the cdna was fixed using a stratalinker uv oven ceptor (mhvr)], macrophage cells (j . ) were grown (stratagene). following prehybridization [ % ssc, to % confluence in -mm petri dishes and then treated . % sodium dodecyl sulfate (sds), . mg/ml salmon with medium from cells expressing de-ifn-g or de-cat, sperm dna] at room temperature for min, p-labeled both of which had been irradiated with uv to inactivate specific probes (table ) were added. following hybridhelper virus. at and hr after treatment, cells were ization at Њ, the membranes were washed three times collected and intracellular rna was isolated as dewith ssc containing . % sds for min, air dried, scribed previously (zhang et al., ) . to determine the and scanned on an ambis radioanalytic imaging system effects of mhv infection on the expression of cellular (ambis systems). total counts of each duplicate sample genes, j . cells were infected with mhv-jhm virus at for inos, igif, and mhvr at each time point were noran m.o.i. of . at hr after ifn-g treatment. rna was malized to the control hprt. the blots were further autoisolated at hr postinfection. the rna samples were radiographed. used for synthesis of cdnas by reverse transcription (rt) with random priming hexamers (boehringer-mannheim). mice to detect individual genes, cdna pools were subjected to pcr amplification using gene-specific primers (table c bl / mice were purchased at weeks of age from the jackson laboratory. mice were infected with ). the gene encoding the housekeeping enzyme hypoxanthine phosphoribosyltransferase (hprt) was used as pfu of a expressing de-ifn-g or de-cat. preliminary experiments showed no difference in virus replication in an internal control. the pcr was performed for cycles under the following condition: Њ for min for denatur-the cns comparing parental a and a virus containing the de-cat vector. ation, Њ for min for annealing, and Њ for min for virus titers in the cns were determined by homogenization of half of the brain in pbs followed by plaque assay on monolayers of dbt cells as previously described (stohlman et al., ) . the remaining half of the brains were fixed in clark's solution ( % ethanol, % glacial acetic acid), embedded in paraffin, and stained with hematoxylin and eosin to examine the extent of encephalitis or with the immunoperoxidase method (vectastain abc kit; vector laboratories, burlingame, ca) using the anti-nucleocapsid monoclonal antibody j. . . (fleming et al., ) to determine the percentage of cuture medium from dbt cells infected with jhm virus and transfected virus-infected cells. with either de-ifn-g or de-cat rna was harvested at various time points posttransfection, and virus titers were determined by plaque assays. expression of ifn-g using an mhv di rna vector cell metabolism prior to infection or it may be that interferon acts at an early stage of viral replication. the murine ifn-g gene was cloned into the mhv di to distinguish these possibilities, the culture medium rna vector (liao et al., ) under the control of the harvested from jhm-infected and de-ifn-g-transfected mhv ig sequence. the resulting rna, de-ifn-g rna, cells late in infection was used to infect dbt cells. this was transfected into mhv-infected cells, and the producmedium contained not only jhm virus but also ifn-g tion of ifn-g in the culture medium was detected by ( u/ml) (fig. ) . therefore, ifn-g was present through-elisa. as shown in fig. b , when mhv-jhm was used out the infection, beginning with the initiation of viral as helper virus, ifn-g was secreted into the medium ( infection. no significant differences in virus titer released u/ml) as early as hr posttransfection and increased from the de-ifn-g-and de-cat-infected cells were dewith time. at hr posttransfection, when cell monotected (both yielded approximately pfu/ml) (data not layers were completely lysed, the amount of ifn-g shown). thus, ifn-g has little antiviral effect even when reached approximately u/ml. when a was used present at the initiation of viral infection. as helper virus, the production of ifn-g was detected at in view of the known mechanisms of action of ifn-a u/ml at hr posttransfection and reached a maximum and -b, whose antiviral activities require preadsorption (approximately u/ml) earlier (at hr posttransfecto cells prior to viral infection (bianzani and autonelli, tion) (fig. c) , consistent with the observation that a ), we examined the effects of pretreatment of cells replicates faster than jhm. these results indicated that with ifn-g prior to infection. for this study, the culture mhv di vector can be used for the production of a semedium from jhm-infected and de-ifn-g-transfected creted cytokine during mhv infection in vitro. cells was uv-irradiated to inactivate infectious virus and then used as a source of ifn-g to pretreat dbt cells. twenty-four hours later, cells were infected with jhm or replication in vitro a virus at m.o.i.'s ranging from . to . in the continual presence of de-ifn-g. virus titers were deter-ifn-g exerts multiple biological functions both in vitro and in vivo (trinchieri and perussia, ; pestka and mined at hr postinfection. as shown in fig. a , de-ifng exhibited a slight inhibitory effect on jhm replication langer, ), but its effects on coronavirus infections have not been extensively examined. we first determined (approximately log reducation in virus titer), when an m.o.i. of . was used; similar results were obtained whether di-expressed ifn-g had antiviral effects on helper viral replication. virus titers in the medium of dbt with a virus (fig. a) , suggesting that pretreatment of cells with ifn-g prior to viral infection induces an antiviral cells infected with jhm and transfected with de-ifn-g rna were determined at various time points after infec-state. this inhibitory effect was less pronounced when higher m.o.i.'s were used (data not shown), suggesting tion and compared to de-cat rna-transfected cells. figure shows that the virus titers in the presence of de-that the observed antiviral activity was weak and could be overcome by a higher virus titer. ifn-g were lower by approximately half a log compared to cultures transfected with the de-cat rna. this differ-to further establish that the antiviral effect was due to the specific effects of ifn-g, the uv-inactivated de-ifn-ence was small but reproducible, suggesting that ifn-g exerts at most a weak antiviral effect. the absence of g preparation was preincubated for hr with a hamster neutralizing monoclonal antibody specific for rifn-g. significant anti-viral effect of ifn-g in this system could be due to the requirement for interferon to modify host antiviral effects were completely blocked by this treat- the uv-irradiated supernatants were used either as a source of ifn-g or as a control (cat) to pretreat cells for hr, and the cells were then infected with either jhm or a at an m.o.i. of . . after virus adsorption, cells were incubated with the same supernatants for hr, and the virus titers in culture medium at hr postinfection were determined by a standard plaque assay. (b) neutralization assay of ifn-g. both uv-irradiated supernatants (ifn-g and cat) were incubated with mg/ml of a hamster anti-ifn-g neutralizing monoclonal antibody for hr at room temperature prior to being used for pretreatment of cells. subsequent procedures were the same as in (a). ment (fig. b ), demonstrating that ifn-g, but not the repli-log , similar to the data obtained with dbt cells. thus, the absence of strong antiviral effects of ifn-g is not cation of the di vector itself, was responsible for the antiviral activity. these combined results suggest that due to nonresponsiveness of cells to ifn-g. ifn-g has a weak antiviral effect, which was evident only di rna-expressed ifn-g prevents virus spread when cells were pretreated with ifn-g prior to infection. the relatively weak antiviral effects of ifn-g also could the results described above indicated that antiviral be due to the possibility that dbt cells do not respond effects of ifn-g could be demonstrated only when cells well to ifn-g. since it is known that macrophages are were pretreated with ifn-g before viral infection and particularly sensitive to ifn-g treatment (ijzermans and when a low m.o.i. was used. they suggested the possibil-marquet, ), we further determined the inhibitory efity that ifn-g could prevent virus spread, if virus initially fects of ifn-g on mhv replication in an mhv-susceptible infects only a small number of cells. to establish an in macrophage cell line (j . ). j . cells were previtro model for studying the potential effects of ifn-g in treated with various concentrations of rifn-g for hr preventing virus spread, uv-irradiated culture medium before and throughout virus infection. as shown in fig. from de-ifn-g-transfected cells, which contained ifn-g , both a and jhm were inhibited by rifn-g by to at u/ml, was mixed with a very low titer of jhm virus at approximately one infectious particle in each well of a -well plate. cells were observed for cytopathic effects daily for days and the number of fusion plaques was counted. results of these experiments are presented in table . the number of plaques increased more slowly when the de-ifn-g was present (for example, from plaque on day to plaques on day ), as compared to those in the control wells, in which diexpressed cat preparation was used (i.e., from plaque on day to plaques on day and too numerous to count by day ) (table ) . initially, the plaque sizes in the presence of ifn-g were indistinguishable from those of the control wells (data not shown); however, by day or postinfection, while all plaques in the ifn-g-treated cultures remained of uniform size, plaques in the absence of ifn-g became numerous and heterogeneous it has been suggested that ifn-g induces a number no. of plaques c on of cellular proteins and enzymes which either act as t cells (okamura et al., ) . mhvr is a member of virus. one milliliter of each culture medium was then mixed with jhm the biliary glycoprotein (bgp)/carcinoembryonic antigen virus and added to the cell monolayers, so that an average of pfu per well was present. (cea) family and serves as a receptor for mhv infection b each sample was quadruplicated in wells of a -well plate. (williams et al., ) . treatment of cells with di-exc plaques were counted in the liquid medium using a light micropressed ifn-g for hr increased the expression of inos scope. and igif mrnas. mhv infection did not affect the expresd uc, uncountable due to extensive cytopathic effects and detachment of cells. due to the rapid spread of progeny virus before ifn-g exhibited its antiviral effect (data not shown). similar results were obtained when various concentrations of rifng ( , , and u/ml) were used, suggesting that u/ml rifn-g is sufficient to prevent virus spread in vitro (data not shown). sensitivity of different jhm variants to ifn-g treatment in vitro was assessed in an effort to determine whether the ifn-g sensitivity correlates with the pathogenicity of the virus in vivo. three jhm variants with different degrees of neurovirulence were used: dl (ld - pfu), ds (ld - pfu), and . -v- (ld - , pfu) (stohlman et al., (stohlman et al., , fleming et al., fleming et al., , . dl causes little demyelination and infects predominantly neurons whereas variant . -v- causes extensive demyelination and infects predominantly glial cells with a particular tropism for oligodendrocytes. variant ds causes less demyelination than variant . -v- . dbt cells pretreated with ifn-g ( u/ml) for hr were infected, and the same concentrations of ifn-g were maintained throughout the infection. at hr postinfection, culture medium was collected and virus titer determined by plaque assay. as shown in fig. , a reduction of approximately . log in pathogenicity in vivo, groups of c bl/ mice were infected with pfu of a virus containing either de-ifn-g small numbers of perivascular and subarachnoid mononuor de-cat. preliminary experiments showed no difference clear cells, the brains of the de-ifn-g-expressing group in virus replication in cns between mice infected with pashowed widespread meningomyeloencephalitis with promrental a virus and those infected with a -de-cat (data inent perivascular cuffs, infiltration of mononuclear cells not shown). at days postinfection, four mice in each group into the parenchyma, and subarachnoid infiltrates (fig. ) . were sacrificed and the brains were examined for mhv this result supports the immunostimulatory effects of ifntiter and histological changes. the remaining mice in each g. although this experiment used only a small number of group were monitored daily for survival. table shows that mice, the data suggest that expression of immunomodulathere was approximately . log less virus in the cns of tory molecules from the di vector can alter the pathogenemice infected with a expressing de-ifn-g vector comsis of mhv-induced disease. pared to the mice infected with a expressing de-cat vector. correspondingly, all the mice infected with de-ifng-expressing a survived the entire -day observation the molecular basis for the relative ifn resistance of different mhv strains is not yet known. previous studies this study demonstrates that the mhv di rna system have shown that the neutralization-escape mutant . -vcan be utilized as a vector to express the ifn-g gene of jhm strain has a single nucleotide mutation at posiand that the ifn-g protein is translated and secreted tion of the s gene, which results in a leucine to from infected cells as a biologically active molecule. phenylalanine substitution (wang et al., ) . whether these data represent the first successful attempt to exthis single mutation affects the sensitivity of the virus to press a mammalian cellular gene product using a coro-ifn-g remains unclear. in lymphocytic choriomeningitis navirus di rna vector. thus far, we have demonstrated virus, resistance of various virus strains to ifn-a/b or the feasibility of this di rna system for expressing a ifn-g in vitro correlates with their ability to establish prokaryotic bacterial gene cat (liao and lai, ) , a persistent infections in adult immunocompetent mice viral structural protein gene he (liao et al., ) , and (moskophidis et al., ) . one possibility is that ifn the mammalian cellular gene ifn-g (this report). these resistance allows enhanced viral replication and spread, studies showed a broad range of usage of this di rna facilitating exhaustion of antiviral ctl, thereby resulting system for expressing various genes of interest. in virus persistence. whether mhv utilizes a similar currently, an infectious, full-length cdna clone of mhv mechanism to modulate its infection in mice is an inter-rna is not available; therefore, it is difficult to unequivoesting issue. correlation between ifn resistance and cally elucidate the mechanism of pathogenesis of mhv viral pathogenicity has also been documented for meaat the molecular level. the development of a di rna sles virus, adenovirus, and herpes simplex virus type i expression system thus provides an alternative ap- (carrigan and kehl-knox, ; su et al., ; kalvakoproach, allowing the expression of both viral and cellular lanu et al., ) . genes to be manipulated. further, this system allows the in vitro experiments showed that the di-expressed expression of heterologous gene products at the site of ifn-g had inhibitory effects on virus spread from initially viral replication. this system has an advantage over the infected cells to neighboring uninfected cells. the inhibipassive administration of cytokines for studying viral tory effect was more pronouced at a lower m.o.i., which pathogenesis, since cytokines usually have a short halfapparently allowed sufficient time for ifn-g to activate life, making it difficult to maintain high local concentraan antiviral state in adjacent uninfected cells. pretreattions at the site of infection. one drawback of the di ment of cells (astrocytoma and macrophages) with ifnsystem, however, is its limited expression. the di rna g is required to induce an antiviral state (figs. and ) , cannot be packaged beyond the fourth passage in vitro consistent with previous findings from studies of primary (data not shown). we have attempted to increase retenmouse macrophages (lucchiari et al., ) and other tion of the di rna via incorporation of a packaging signal. target cells (lewis, ) . expression of both inos and however, the expression level of the gene product was igif mrna in macrophages was induced by ifn-g. howreduced; no significant retention was found (lin and lai, ever, whether these molecules mediate the antiviral ef- ). nevertheless, our data indicated that, during the fects of ifn-g is not clear. recently, it was demonstrated first several passages, the expression level of ifn-g was that inos expression did not play a significant role in such that a sufficiently high level of ifn-g can be mainthe pathogenesis of the mhv oblv strain (lane et al., tained locally at the beginning of viral infection. ). nevertheless, we can conclude from our study the virulence of several mhv variants correlates with that the antiviral effects of ifn-g are not mediated by their resistance to ifn-g treatment, suggesting that ifndown-regulation of mhvr. the precise mechanism of the g may play a role in the pathogenesis of mhv. an earlier antiviral effects of ifn-g will require additional studies, study analyzed the effects of ifn-g during jhm infection as there appears to be discordance between the antiviral using passive transfer of an anti-ifn-g-antibody (smith effects of no in vivo and its effects in vitro (lane et al., et al., ) . this treatment significantly enhanced virus ). replication and resulted in a higher mortality with de- the alteration of a neuropathogenesis by de-ifn-g creased survival times. ifn-g treatment of macrophages provides further support for the significance of ifn-g from a/j mice rendered them partially resistant to mhv in mhv infection. inhibition of ifn-g action by passive infection, whereas the macrophages from susceptible transfer of antibody (smith et al., ) enhanced virus balb/c mice did not respond to ifn-g, suggesting that replication and increased mortality, suggesting that local the resistance of mice to mhv infection involves the production of ifn-g by infiltrating leukocytes is a critical sensitivity of macrophages to ifn-g (lucchiari et al., component of the host response to mhv infection. in ; vassao et al., a,b) . ifn-g was also shown to our experiments, the production of ifn-g by de-ifn-g be more effective than ifn-a/b in inducing an antiviral resulted in an exaggeration of the host response with state in macrophages infected with mhv (vassao et al., more prominent encephalitis, improved viral clearance, a). these reports support the notion that ifn-g may and decreased mortality. the increased encephalitis may, in turn, induce local cytokine production and ctl play a role in mhv infection. the complete , - . sequence ( kilobases) of murine coronavirus gene encoding the -sequence as an upstream cis-acting element for coronavirus sub- . genomic mrna transcription a murine virus (jhm) causing disseminated encephalomyelitive-interfering rna as an expression vector: the generation of a tis with extensive destruction of myelin. i. isolation and biological pseudorecombinant mouse hepatitis virus expressing hemagglutiproperties of the virus deletion mapping of a mouse hepatiduced th responses in experimental allergic encephalomyelitis tis virus defective interfering rna reveals the requirement of an (eae)-resistant mice: th -mediated suppression of autoimmune disinternal and discontiguous sequence for replication acquired interferon and tumor necrosis factor exert a synergistic blockade on immunity of a/j mice to mouse hepatitis virus infection: dependence the replication of herpes simplex virus defectiveruses: analysis using monoclonal antibodies to jhm (mhv- ) virus. interfering particles of murine coronavirus: mechanism of synthesis virology primary structure and translation of a defective-interfering navirus jhm selected with monoclonal antibodies experimental demyelination induced by coronavidefective-interfering rna results from intergenic site insertion isolation and characterization of two plaque morphology variants of the jhm neurotropic strain mouse hepatitis virus-specific cytotoxic t lymphocytes protect from lethal infection without eliminating virus from the central ingitis virus to alpha/beta interferon and to gamma interferon detection of in vivo expression of interleukin- using a semi-quantitative polymerherpes simplex virus type i strain is associated with heightened sensitivity to alpha/beta interferon immune interferon: a pleiotropic lymphokine with multiple effects cloning of a new cytokine that induces ifn-g production by dation of coronavirus defective interfering rnas a genetic analysis of macrophage activation and specific antibodies in relation cytokine induction during t-cell-mediated clearance of mouse hepatitis virus from neurons in vivo the astrocyte is a target cell in mice persistently infected with mouse hepatitis virus, strain interferons and their actions. annu. of genetic heterogeneous mouse populations to mouse hepatitis virus infection sequence analysis of the spike protein gene of murine coronavirus variants: study of as effector molecules in the resolution of virus infection expression of cytokines by recombinant vaccinia viruses: a model leukocytes by phytohemagglutinin hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins tumour necrosis factor a and b inhibit virus replication and synergize with interferons coronavirus leader the role of gamma interferon in infection of susceptible mice with murine coronavirus, mhv-jhm gledhill, a. w., and niven, j. s. f. ( ) . latent virus as exemplified by activity. altogether, these data demonstrated that ifn-g mouse hepatitis virus (mhv). vet. rev. annotat. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] plays a critical role at least early in a infection. the haller, o. ( ) . inborn resistance of mice to orthomyxoviruses. curr.longer-term consequences of ever, cannot be definitively determined from this study heise, m. t., and virgin, iv, h. w. ( ) . key: cord- -fn pkec authors: shi, xiaodong; yu, lijia; zhang, yinglin; liu, zequan; zhang, huawei; zhang, yansong; liu, ping; du, peishuang title: glycyrrhetinic acid alleviates hepatic inflammation injury in viral hepatitis disease via a hmgb -tlr signaling pathway date: - - journal: int immunopharmacol doi: . /j.intimp. . sha: doc_id: cord_uid: fn pkec various human disorders are cured by the use of licorice, a key ingredient of herbal remedies. glycyrrhizic acid (gl), a triterpenoid glycoside, is the aqueous extract from licorice root. glycyrrhetinic acid (ga) has been reported to be a major bioactive hydrolysis product of gl and has been regarded as an anti-inflammatory agent for the treatment of a variety of inflammatory diseases, including hepatitis. however, the mechanism by which ga inhibits viral hepatic inflammatory injury is not completely understood. in this study, we found that, by consecutively treating mice with a traditional herbal recipe, licorice plays an important role in the detoxification of mice. we also employed a murine hepatitis virus (mhv) infection model to illustrate that ga treatment inhibited activation of hepatic inflammatory responses by blocking high-mobility group box (hmgb ) cytokine activity. furthermore, decreased hmgb levels and downstream signaling triggered by injection of a neutralizing hmgb antibody or tlr gene deficiency, also significantly protected against mhv-induced severe hepatic injury. thus, our findings characterize ga as a hepatoprotective therapy agent in hepatic infectious disease not only by suppressing hmgb release and blocking hmgb cytokine activity, but also via an underlying viral-induced hmgb -tlr immunological regulation axis that occurs during the cytokine storm. the present study provides a new therapy strategy for the treatment of acute viral hepatitis in the clinical setting. in contrast to western medicine which normally consists of a single compound designed for specific cellular targets, traditional chinese medicine (tcm) incorporates a mixture of herbal remedies that have been developed through experience over thousand years and accepted conventionally in eastern medicine. however, the therapeutic basis and underlying molecular mechanism for the majority of the ingredients in tcm have yet to be elucidated [ , ] . interestingly, a few herbs, including licorice, are present in the majority of conventional tcm recipes [ ] . in addition, the major bioactive component of licorice, glycyrrhizic acid (gl), has been used to treat viral hepatitis and inflammatory disease [ , ] . the gl compound consists of two molecules glucuronic acid and one molecule glycyrrhetinic acid (ga) [ ] . ga is also a key hydrolysis product of gl and is a triterpenoid saponins with a molecular weight of . kda ( fig. ) . ga has been shown to have anti-inflammatory effects in different systems through multiple mechanisms, such as inhibitory effects on reactive oxygen species (ros) overproduction and nf-κb pathway activation [ , ] . however, little is known about the role of ga in the immunological regulation during viral infection. high-mobility group box (hmgb ) is a nuclear dna-binding protein with highly conserved sequence, and plays an important role acting as an endogenous damage-associated molecular patterns (damps) molecular [ ] . hmgb also functions as an extracellular signaling molecule during inflammation, cell differentiation, cell migration, and tumor metastasis [ ] . gl has been reported to interact with a motif within hmgb and inhibit its chemoattractant and mitogenic activities [ , ] . additionally, ga can also bind selectively to hmgb protein and inhibit its cytokine activities by preventing accumulation of hmgb protein [ ] . some recent studies regarding the anti-inflammatory function of ga on hepatic injury have been performed. transgenic mice model can be alleviated by treatment with ga, which can also inhibit the downstream hmgb signaling pathway in acetaminophen-induced acute liver injury [ , ] . hmgb is passively released from damaged or necrotic tissue cells and is actively secreted by inflammatory cells in response to stress, which can activate innate immune receptors, such as toll-like receptors (i.e., tlr and tlr ), as well as the receptor for advanced glycation endproducts (rage) [ ] . therefore, hmgb also acts as a therapeutic target for both sterile inflammation and infection [ ] . however, the underlying mechanism for the effectiveness of ga on viral-induced liver injury is largely unclear. in asia, viral infections play an important role in acute and chronic liver failure with hbv being an important cause of acute-on-chronic liver failure (aclf) in china [ , ] . recently it has been reported that targeting the inhibition of nk cell activity can ameliorate liver damage in viral fulminant hepatitis [ ] . mouse hepatitis virus (mhv), a coronavirus with single-stranded rna, causes several murine pathological illnesses, including enteritis, hepatitis, and encephalitis [ ] . the outcome of viral infection depends on the virus strain and genetic background of the mouse. the mhv-a strain is a moderately hepatotropic virus, which may also cause moderate acute encephalitis and chronic demyelination [ , ] . in mhv-a infection animal models, adaptive immune cells have an unexpected role in tempering initial innate responses [ ] . although gl can exert anti-viral activity in sars-associated coronavirus infection, the effect of its major metabolite, ga, on hepatic virus-induced tissue injury is not very clear [ ] . therefore, it was hypothesized that in a hepatotropic coronavirus model, ga may have different effects on virus replication and its protective function in tissue injury. to test this hypothesis, we examined whether the presence of licorice serves as a hepatoprotective agent against potentially toxic bioactive herbal ingredients. then we characterized the effectiveness of ga in inhibiting hmgb cytokine activity and suppressing the induction of inflammatory genes known to be involved in hepatic injury while assessing the involvement of hmgb /tlr signaling in mediating tissue injury during viral hepatitis. there is a pressing need for new approaches to prevent and manage viral hepatitis, and we hope that a better understanding of ga function will allow for emergence of new therapeutic approaches in western medicine. the recipe for xiaoyao powder was first recorded in "taiping huimin heji jufang", one of the earlier pharmacopoeia from song dynasty in china [ ] . in this tcm study, our modified recipe consists of radix bupleuri, radix angelicae sinensis, radix paeoniae alba, rhizoma atractylodis macrocephalae, poria, rhizoma zingiberis recens, radix glycyrrhizae preparata and herba menthae in a ratio of : : : : : : : . ga crystalline powder, in the form of β-glycyrrhetinic acid ( % purity) was kindly supplied by huzhou r&d center, institute for nutritional sciences, shanghai institute for biological sciences, cas (huzhou, china). the powder was dissolved in a pbs-buffered solution of % ethanol. after adding . % dimethyl sulfoxide (dmso), the solution was then heated to ℃and stirred evenly and continuously. the composition of the modified xiaoyao powder with licorice (tcm+l) included licorice ( . % of total weight), while licorice was excluded from tcm-l mixtures. we divided the mice into three groups, which received a daily intragastric (i.g.) injection of pbs (control group), tcm+l pbs solution, or tcm-l pbs solution ( mg/ml, g/kg,) for days. all mice were monitored for weight loss or mortality. serum samples were collected on , , and days after the first injection. subsequently, animals were sacrificed, and liver tissue and serum samples were collected days after the first injection. in the mhv infection model, mice were infected by intraperitoneal (i.p.) injection of × plaque forming unit (pfu)/mouse (lethal) or × pfu/mouse (sublethal) of mhv-a in . ml dulbecco's modified eagle's medium (dmem) (gibco, usa ). mice were monitored daily for weight loss or mortality. animals were sacrificed and liver tissue and serum samples were collected days post-infection (dpi). murine fibroblast cl- cells, murine dendritic d sc cells, and mouse embryonic hepatic two hours after viral infection, mice were administered ga solution ( mg/kg, i.p.) or an equivalent volume of pbs as a control. mice were then i.p. injected every other day for total of three times. purified anti-hmgb monoclonal neutralizing antibody e was prepared as previous described [ ] . two hours after mhv viral infection, mice were administered a diluted antibody solution ( . mg/kg, i.p.) or an equivalent volume of pbs as a control. mice were then i.p. injected every other day for total of three times. to assess the effects of ga on hmgb cytokine activity, d sc cells ( x cells/well) were co-incubated with hmgb ( μg/ml hmgb -flag protein) (sigma-aldrich, usa) and different amounts ( , , μg/ml) of ga for hours. the tnf-α and il- levels in the supernatant were then assayed using an elisa kit (ebioscience, usa). serum harvested from mhv-a -infected mice was used for detecting the circulating levels of murine hmgb according to the manufacturer's instructions of elisa kit (chondrex systems, usa). serum alt levels were measured with a kit according to the manufacturer's instructions (biosino bio, beijing, china) analysis. liver specimens were fixed in % neutral-buffered formalin. then paraffin-embedded liver sections ( μm thick) were stained with hematoxylin and eosin (h&e) using standard techniques [ ] . were incubated with the same volume of supernatant containing hmgb for hour at °c, and then the mixture was added to the medium of raw . cells. eight hours after incubation, the total rna of stimulated cells was isolated and ip- gene expression was assayed using real-time pcr. the tnf-α cytokine levels in the supernatants were also measured after stimulation. serum harvested from mhv-a -infected mice was used for detecting the circulating levels of cytokine, including il- β, ip- , il- , il- , il- a, and il- , according to the instructions of the elisa kits (ebioscience, usa). total rna was isolated from liver tissue or cultured cells using trizol (invitrogen, usa) according to the manufacturer's instructions. the cdna synthesized as described previously [ ] . the reverse-transcribed mrna expression of ip- , il- , tnf-α, il- β, tlr , and glyceraldehyde- -phosphate dehydrogenase (gapdh) genes were determined by qrt-pcr using sybr green master mix kit (applied biosystems, foster city, ca, usa). the primers for the following genes are listed in table . table list of primers used for qrt-pcr all results were expressed as the mean ± standard error of the mean. the difference between the two groups was examined using a student's t test after analyzing the variance. statistics were performed using graphpad prism (graphpad software, la jolla, ca, usa). a p-value of < . was considered statistically significant (*p < . , **p< . , and ***p < . ). as a well-known herb formulation, xiaoyao powder contains licorice as one of the ingredients of the recipe. this recipe is widely used to treat liver damage and mental disorders [ , ] . this xiaoyao powder solution was subjected to ultra-performance liquid chromatography coupled with mass spectrometry (uplc-ms), and common fingerprint peaks were defined and identified. fourteen target compounds, such as saikosaponin c, albiflorin, paeoniflorin, quercetin, and ferulic acid, were characterized distinctly in the plasma from rats orally administrated the xiaoyao powder solution rat as determined by uplc-ms/ms [ , ] . in this study, chronic hepatic toxicity investigation of a modified recipe solution was carried out by injecting the solution to mice. the difference in the formulation between mixtures with licorice ( . %) and mixtures without licorice (including seven herbal ingredients) was tested. interestingly, long-term daily administration of tcm (i.g., g/kg) without licorice (tcm-l) led to body weight loss as compared to mice that received tcm+l ( fig. a) . furthermore, serum alt and ast levels increased significantly days after the first injection in the tcm-l group as compared to control mice and the tcm+l group ( fig. b-c) . additionally, we examined the induction of ip- and il- β, two inflammatory cytokines induced during liver injury. removing licorice from the tcm formula resulted in increased ip- and il- β cytokine release (fig. d -e) as well as the induction of other inflammatory genes, such as ip- , tnf-α and il- ( fig. f-h) . these findings suggest that licorice could be a hepatoprotective agent in xiaoyao powder that alleviates the long-term toxic effects of the other bioactive ingredients in this formulation. therefore, as the key metabolite of licorice, ga has the potential to alleviate viral-induced hepatic injury. in the following experiment, we set out to evaluate the effectiveness of ga in alleviating hepatic injury in a viral infection model. mhv causes hepatic and central nervous system diseases of varying severity, depending on the strain and is therefore used as a model for hepatitis, viral encephalitis, and demyelination. in our lab, we have established acute lethal and sub-lethal mhv infection models using the mhv-a virus strain, which is a moderately hepatotropic and neurotropic virus (fig. s ). murine survival studies suggested that administration of ga ( mg/kg, i.p.) on the day of infection and every other day after infection leads to an increased survival rate in mhv-infected mice as compared to the control group (fig. a ). in addition, the virus titer assay showed that ga treatment significantly inhibited the viral proliferation in the liver ( fig. b ). as the marker of hepatotoxicity, alt exhibited a decreasing trend until the level of iu/ml (fig. c) . furthermore, ga treatment decreased mhv-induced hepatic injury and exhibited fewer necrotic foci upon liver histological analysis (fig. d ). in some sterile hepatic injury diseases, ga attenuates hmgb -mediated inflammatory by inhibiting tnf-α, il- β and il- production [ , , ] . pro-inflammatory chemokine ip- is a predictive marker of hepatitis c virus (hcv) and hbv infection [ , ] . we then determined the gene expression of these four pro-inflammatory cytokines, which have been shown to be key triggers in the innate immune response in hepatic injury in mouse models (fig. e ). our qpcr data demonstrated that ga treatment during viral infection in mice can suppress the induction of several pro-inflammatory cytokine genes, except il- β (fig. e) , which has some different expression in sterile inflammation disease [ ] . in line with previous reports about sterile hepatic injury, it was found that ga treatment also significantly inhibited hmgb levels in the serum of infected mice as compared to control mice ( fig. f ) [ ] . surprisingly, the high level of tlr gene expression in hepatic tissue from mhv-infected mice was not affected by ga treatment, which is in accordance with sterile hepatic injury ( fig. g ) [ , ] . thus, studies regarding hmgb cytokine activity induced by ga needs to be explored further. it has been reported that gl has a variety of pharmacological properties, including anti-inflammatory and anti-viral activities [ , ] . a and fig. s ). in order to determine whether ga is able to inhibit the hmgb -induced expression of inflammatory genes, d sc cells were co-incubated with hmgb solution and different doses of ga for hours. analysis of the culture supernatants suggested that hmgb stimulation leads to the release of il- and tnf-α，which were inhibited by ga treatment in a dose-dependent manner (fig. b-c) . the supernatant solution from infected bnl.cl cells was also exposed to uv radiation for virus inactivation and then added to raw . cells, after incubating with anti-hmgb antibody or ga for hours, qpcr analysis showed that ip- expression in macrophages was able to be inhibited by both hmgb antibody and ga treatment (fig. d) . tnf-α cytokine levels released from raw . cells, which was triggered by mhv-infected hepatocyte supernatant, showed the same trend as ip- (fig. e ). similar to our in vivo data, ga treatment in vitro was able to inhibit the induction of ip- in mhv-infected bnl.cl cells (fig. f) . therefore, the above results suggested that ga exerts regulation functions in hepatic cells and immune cells. interestingly, ga and hmb antibody treatment did not affect the viral proliferation in bnl.cl cell (fig. g) . this was not in accordance with the data from other murine cells. this difference may be due to indirect and immunological viral inhibition mechanisms of ga in hepatic infection model. mhv-infected mice showed high levels of hmgb ( ng/ml) in their serum at dpi, suggesting that mhv infection causes release of hmgb into the serum either through active release by inflammatory cells or due to hepatocellular injury caused by viral replication (fig. a) . in order to examine whether hmgb mediates the pathogenesis of mhv-induced lethality, mice were injected with a hmgb neutralizing antibody after infection and then every other day. during mhv infection with a lethal dose ( × pfu/mouse), injection of the blocking antibody completely rescued mice as compared to the pbs control (fig. b) . interestingly, the virus titers on dpi in the infected livers of the antibody-treated group and control group were not significantly different (fig. c) , suggesting that hmgb antibody treatment does not affect viral replication in the liver. however, the neutralizing antibody-treated group expressed fewer indicators of liver injury, such as decreased release of alt and lack of histological necrotic foci and inflammatory cell infiltration (fig. d-e) . these results indicate that hmgb plays an important role in mediating liver injury in a mhv infection model. furthermore, inflammatory cytokine expression data illustrated that a hmgb neutralizing antibody significantly suppresses the transcriptional induction of ip- , tnf-α and il- genes in infected liver tissue ( fig. f-h) , which is similar to the effect in the ga treatment group. tlr signaling has been reported to contribute to tissue damage in both pathogenic hepatic injury and sterile inflammatory hepatitis [ ] [ ] [ ] . as a ligand of tlr , hmgb plays an important role in many viral infection models [ ] [ ] [ ] . therefore, we examined whether tlr signaling is also critical in a mhv infection model. survival curve data showed that tlr ko mice are protected against lethal mhv infection as compared to infected wt mice (fig. a) . interestingly, there was no distinct difference in virus titer between tlr ko and wt infected-mice, which suggests that virus inhibition is not the major cause underlying the protective role during lethal dose infection in tlr ko mice (fig. b) . furthermore, tlr ko mice exhibited decreased signs of liver injury as compared to control mice, such as lower alt levels in serum and fewer of necrotic foci in histological analysis (fig. c-d) . additionally, ip- , tnf-α and il- gene were not strongly induced in the tlr ko mice infected with mhv, further suggesting that tlr is an important immune receptor in mediating inflammation during mhv infection ( fig. e-g) . although tlr ko mice may not affect mhv proliferation in vivo, the tlr gene could play a key role in determining the level of hmgb release in infected mouse serum, as well as the ga treatment effect (fig. h ). tlr is one of downstream receptors of the hmgb , but tlr mediates hmgb release in viral infection similar to its regulation in hepatic sterile inflammation disease [ , ] . in accordance with our survival curve data, body weight loss in mhv-infected tlr ko and ga treated mice groups was rescued at dpi (fig. a) . assay results of il- β, ip- , il- suggested that tlr deficiency significantly inhibits the production of pro-inflammatory cytokines in the serum of infected mice, which is similar to ga treatment effect (fig. b-d) . viral-induced fulminant hepatitis can cause acute liver failure involved with il- and ifn-γ release and th cell responses [ ] . viral infection triggers early production of il- from γδt cells in the liver, and il- plays an important role in host immunity and tissue homeostasis induced by infectious and inflammatory diseases [ ] . our findings illustrated that il- a and associated cytokine il- in infected tlr ko mice decreased significantly, similar to mice treated with ga ( fig. e-f) . the production of these cytokines are usually from the liver, spleen, and thymus stimulated by viral infection. unlike above pro-inflammatory cytokines, il- was stimulated and reached the concentration of pg/ml in the serum of ga-treated mice, which suggests that ga may function as positive stimulator to il- cellular secretion. furthermore, il- production in mhv-infected mice depended, to a great extent, on the expression of the tlr gene (fig. g ). although derived from the same interleukin family, il- and il- exhibited differing trends in ga-treated mice (fig. f-g) . therefore, the detailed molecular and cellular mechanisms of the secretion of these cytokines need to be explored further. in this study, we first demonstrated that licorice, a common component in the majority of herbal remedies, may be included as hepatoprotective agent to alleviate potential toxic adverse effects of ingredients in tcm. next, we investigated if ga, as the active metabolic ingredient of licorice, could influence viral-induced hepatic injury. our data demonstrated that ga inhibits mhv-a -induced hepatitis by suppressing hmgb release and cytokine activity in vivo and in vitro, which was consistent with the results from using hmgb neutralizing antibody. additionally, our findings further demonstrated that the hmgb -tlr axes is also involved in mediating tissue injury during hepatic viral infections, as blocking this pathway can effectively hinder the inflammatory injury in mhv-infected mice. licorice root extract contains various bioactive compounds and has potent hepatoprotective activity, which makes it a key detoxification ingredient in a wide variety of traditional herbal formulations [ ] . however, it is unclear what molecular actions make this herb an effective ingredient. xiaoyao powder usage has been proven to be safe and is commonly used to treat hepatic stagnation and splenic deficiency without any reports of significant hepatotoxicity. however, the formula without licorice led to body weight loss and increased serum aminotransferase levels in mice, which are both indicators of hepatotoxicity. therefore, our studies have suggested that the inclusion of licorice protects against hepatotoxicity induced by other ingredients (such as radix bupleuri ) in the herbal formula. ga is a hydrolysate of glycyrrhizic acid in the aqueous extract of licorice root, which is known to have various immune-modulating and antivirus activities [ ] . for example, ga can attenuate lps-induced fulminant hepatic failure through the deactivation of mapks and nf-κb pathway, resulting in the inhibition of tnf-α production [ ] . ga also inhibits viral proliferation directly and indirectly in vitro and in vivo [ , ] . our ga treatment results demonstrated that ga can not only rescue mortality of virus-infected mice and alleviate liver injury, but ga injection can also significantly inhibit viral proliferation in the liver. gl and ga both have been reported to exert antiviral effects, but there is a difference between the antiviral profiles of gl and ga [ , ] . although gl exerts antiviral activity in sars-associated coronavirus infection in vitro, solid evidence of the antiviral activity of ga in the mhv infection animal model is absent [ ] . in this study, we first demonstrated the antiviral activity of ga in a murine coronavirus infection model in vivo. to further investigate the mechanism of ga, we performed a cellular assay in vitro and found that it was involved with hmgb release and activity. interestingly, in both hepatic and inflammatory cells, ga treatment or hmgb neutralizing antibody treatment significantly inhibited hmgb cytokine-inducing activity. surprisingly, ga did not affect viral proliferation in vitro, which was different from previous reports [ ] . this may be because we used mouse embryonic liver cells in our study, whereas previous studies have used vero cells. the response of mouse embryonic liver cells to mhv infection may differ from vero cells, and as such, a distinct cellular signaling pathway may be activated in mouse embryonic liver cells in vitro. furthermore, it is well known that the virus titer in infected mice is determined directly by drug inhibition and indirectly by host immune-regulation [ , ] . therefore, the antiviral activity of ga in mhv infection may depend on the activation of cytotoxic t lymphocytes, which protect against infected hepatic cells. many viruses cause cytopathologic effects indirectly by inducing host cells to release inflammatory factors and cause an inflammatory response [ ] . when the amount of inflammatory factor is moderate, the host can resist viral invasion. if an excessive amount is produced, it will cause serious pathological damage [ ] . during respiratory viral infection, serum hmgb acts as a biomarker and a therapeutic target [ ] . however, in viral infection disease, the effect of hmgb on virus replication is multifactorial. for example, it has been reported that hmgb protein is the functional domain that interacts with hcv rna and enhances viral replication [ , ] . to reduce viral inflammatory tissue injury, hmgb neutralizing antibody combined with a conventional anti-proliferation drug might be more useful against severe influenza a virus infection [ ] . based on our neutralization experiment, hmgb antibody exerted therapeutic effect during mhv-a infection, but blocking hmgb did not significantly affect viral replication in vivo, which is consistent with the previous study on influenza virus [ ] . tlrs are sensors for pathogen-associated molecular patterns (pamps) and play important roles in immune responses. it has been reported that tlr is key receptor of innate immune signaling responses to influenza virus and other respiratory viruses [ ] . although host hmgb and rage interaction is also a major mechanism driving serious liver injury, the tlr pathway has recently been demonstrated to be more involved in respiratory syncytial virus and human papilloma virus (hpv) infection [ ] [ ] [ ] . previously, the expression of tlr , tlr , tlr , tlr and tlr genes from hepatic tissue was shown to be significantly upregulated in some viral infection models [ , ] . although ga did not significantly affect tlr gene expression during viral infection, expression of tlr gene mediated the mhv-induced hepatic inflammation damage and determined hmgb release levels in the serum (fig. g and h ). in fact, pretreatment with a tlr blocking agent decreased the hmgb levels from virus-infected cells via the tlr -nfκb pathway, and the inactivation of nfκb resulted in a decreased release of various pro-inflammatory cytokines, including il- β, il- , tnf-α and hmgb [ , ] . furthermore, reduced hmgb levels led to the inhibition of nfκb pathway in various immune cells, and the inflammation response was alleviated significantly. however, the involvement of tlr signaling in virus-induced hmgb secretion remains elusive and needs to be investigated further. interestingly, tlr gene deficiency did not lead to the down-regulation of virus titer in the liver, which suggests that in mhv-a infection, the hmgb -tlr axis exerts pro-inflammatory function without directly affecting virus replication. this result was not consistent with hcv and human adenovirus infection, which may due to the different viral pathological and replication mechanism [ , , ] . acute viral hepatitis caused by mhv is characterized by acute necrosis of hepatocytes, inflammation and production of tnf-α, ifn-γ, il- , and il- a [ , ] . in acute hepatitis b virus infection, the anti-inflammatory cytokine il- can suppress liver inflammation by reducing il- and il- production, which results in regulating hbv peptides-inducing th cells [ ] . in this study, we show for the first time that il- a, il- , and il- cytokines were released into the serum in response to mhv-a infection and are inhibited by ga treatment or tlr deficiency. however, there were different variations of il- levels under the two intervening pathways. although the anti-inflammatory cytokine il- has been shown to have efficacy in reducing inflammatory injury in the liver, its effect on viral titers in some types of hepatitis remains unclear [ ] . based on our mhv infection data, il- was up-regulated by ga treatment or mediated in a tlr -dependent manner, which might be a new ga regulatory mechanism of cytokine storms resulting from viral infection, excluding the hmgb -tlr -il- axis. in summary, we demonstrated here for the first time that ga significantly ameliorates mhv-induced hepatic inflammatory damage via hmgb -tlr signaling pathway, which is similar to treatment with a hmgb neutralizing antibody. furthermore, this protective effect of ga was specifically associated with impaired il- and il- levels, and was not due to the direct inhibition of intracellular viral replication. future studies are warranted to investigate the molecular mechanisms underlying how ga inactivates the hmgb -tlr axis and which immune cell plays a major role in the cytokine storm induced by hepatic virus infection. conclusively, this present study provides a new strategy for the immunological therapy of acute viral hepatitis in the clinical setting. all authors have no conflict of interest to disclose. using an elisa method. naturally derived anti-inflammatory compounds from chinese medicinal plants 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tnf-alpha-dependent pathway elevated interleukin- suppresses liver inflammation by regulation of t helper cells in acute hepatitis b virus infection potential role of high mobility group box in viral infectious diseases key: cord- -grw s pf authors: lai, michael m.c.; cavanagh, david title: the molecular biology of coronaviruses date: - - journal: advances in virus research doi: . /s - ( ) - sha: doc_id: cord_uid: grw s pf publisher summary this chapter discusses the manipulation of clones of coronavirus and of complementary dnas (cdnas) of defective-interfering (di) rnas to study coronavirus rna replication, transcription, recombination, processing and transport of proteins, virion assembly, identification of cell receptors for coronaviruses, and processing of the polymerase. the nature of the coronavirus genome is nonsegmented, single-stranded, and positive-sense rna. its size ranges from to kb, which is significantly larger when compared with other rna viruses. the gene encoding the large surface glycoprotein is up to . kb, encoding an imposing trimeric, highly glycosylated protein. this soars some nm above the virion envelope, giving the virus the appearance-with a little imagination-of a crown or coronet. coronavirus research has contributed to the understanding of many aspects of molecular biology in general, such as the mechanism of rna synthesis, translational control, and protein transport and processing. it remains a treasure capable of generating unexpected insights. viruses. the coronavirus polymerase gene alone ( - kb) is about the same size as the whole of the picornavirus (- kb) and vesicular stomatitis virus (- kb) genomes added together. the gene encoding the large surface glycoprotein is up to . kb, encoding an imposing trimeric, highly glycosylated protein. this soars some nm above the virion envelope, giving the virus the appearance-with a little imagination-of a crown or coronet (latin corona, hence the name of the genus). coronaviruses are responsible for a number of economically important diseases. avian infectious bronchitis virus (ibv) was the first coronavirus to be isolated, from the domestic fowl, and propagated in the s. in addition to respiratory disease, which can predispose chickens to possibly lethal secondary bacterial infections, some strains also cause nephritis (king and cavanagh, ; cook and mockett, ) . porcine transmissible gastroenteritis virus (tgev) causes devastating disease in newborn pigs, with mortality often approaching % (enjuanes and van der zeijst, ) . intriguingly, there are also naturally occurring mutants [i.e., porcine respiratory coronavirus (prcv)] of tgev which cause only mild respiratory disease and no enteritis. several other coronaviruses also cause enteritis: bovine coronavirus (bcv), turkey coronavirus (tcv; bluecomb virus), feline coronavirus (fcv), canine coronavirus (ccv) and porcine epidemic diarrhea virus (pedv). fcv may also cause feline infectious peritonitis. an fcv has been isolated from a cheetah and bcvs from wild sambar deer and waterbuck (tsunemitsu et al., ) . these bcvs caused enteritis when inoculated into domestic calves. humans are known to suffer from two very different coronaviruses, human coronavirus (hcv) oc and hcv , both of which are a cause of the common cold. there is evidence for the presence of coronaviruses in tissues taken from multiple sclerosis (ms) patients (reviewed by cavanagh and macnaughton, ) . this inflammatory, demyelinating neurological disease is associated with autoreactive t lymphocytes sensitized to myelin components of the central nervous system. recently, talbot and colleagues ( ) have demonstrated that many cd ' t-cell lines derived from ms patients showed a human leukocyte antigen-(hla)-dr-restricted, crossreactive pattern of antigen activation after in vitro selection of either myelin basic protein or hcv- e proteins, suggesting that molecular mimicry between hcv and myelin may be a n immunopathological mechanism in ms. other coronaviruses [some strains of murine hepatitis virus (mhv) and porcine hemagglutinating encephalomyelitis virus (hev)] are well-known causes of neurological diseases, and mhv has been studied for many years in this context (dales and anderson, , although many mhv strains cause primarily hepatitis. the s and early s was the period in which coronavirus virion proteins and nested-set arrangements of mrnas were identified and the discontinuous nature of coronavirus transcription was initially demonstrated. the first published sequence of a coronavirus gene appeared in , starting an era in which the whole of the genomes of four coronaviruses were clonedin piecesand sequenced. this decade has seen the manipulation of these clones, and of complementary dnas (cdnas) of defective-interfering (di) rnas, to study coronavirus rna replication, transcription, recombination, processing and transport of proteins, virion assembly, identification of cell receptors for coronaviruses, and processing of the polymerase. this review is largely concerned with these areas. some topics are notable by their absence, space not permitting their inclusion. for example, the elucidation of the molecular basis of the antigenic properties of the large surface (spike) glycoprotein and its role in tissue tropism has been omitted. for these topics and all others both within and without the compass of our review for which a concurrently comprehensive and in-depth treatise is desired, the reader is referred to the book edited by siddell ( a) . individual chapters in that book will be referenced a t the appropriate places in this review. all coronaviruses belong to one genus, coronavirus, within the family coronaviridae . initially, serological analysis was used to differentiate coronavirus species and showed that they could be divided into four antigenic groups (holmes, ) . the species and group divisions were subsequently refined by monoclonal antibody analysis and nucleotide sequencing, which revealed the close relatedness between tcv and bcv, resulting in the current classification of three antigenic groups (table i) . the same groupings emerge regardless of which structural protein sequences are compared (siddell, b) . within group , tgev, fcv, and ccv are particularly closely related, all the members of group being tightly clustered. the sole member of group , ibv, not only differs extensively from all other coronaviruses but also exhibits extensive variation within the species. the coronaviridae had remained a monogeneric family for a quarter of a century, until an accumulation of observations which showed that many of the features thought to be characteristic of the coronaviridae applied equally well to the genus torovirus, which had not been officially assigned to a family (figs. and , table ). therefore, in , the international committee for the taxonomy of viruses (ictv) formally expanded the coronaviridae to include torovirus . the bringing together of coronavirus and torovirus was not the end of the taxonomic story; another family, arteriviridae, shared important characteristics in relation to the genome, structure, and strategies of transcription and translation (table i ) (plagemann and moennig, ; snijder and spaan, ) . however, the distinct morphology of the arteriviruses (fig. , and their underlying differences from the coronaviruses in the size of the genome (fig. ) and structural proteins (table ii) , precluded their inclusion in the coronaviridae. the common features uniting the two families (table ) are at the heart of a proposal that an order be created to contain coronaviridae and arteriviridae to reflect their common features and, probably, their evolutionary relationships. the name nidovirales, from the latin nidus, meaning nest, has been designated for the order, as all members produce mrnas in an extensive nested-set arrangement. the remainder of this review is restricted largely to the coronaviruses. coronaviruses are enveloped, more or less spherical, approximately nm in diameter, with a prominent fringe of -nm-long petalshaped surface projections (spikes) composed of a heavily glycosylated type i glycoprotein, spike protein ( s ) (fig. ) . a subset of the coronaviruses (table i ) has an additional layer of short spikes (caul and egglestone, ; dea and tijssen, ) , which consist of hemagglutininesterase (he) protein, also a type i glycoprotein. these small spikes are not essential for viral infectivity. both the large and small spikes are anchored in the envelope, which is a lipid bilayer formed by virus budding from intracellular membranes. the envelope is associated with, in addition to the s and he proteins, a smaller type i integral membrane protein (m), which spans the envelope three times. an even smaller protein [envelope (e) or small membrane (sm) protein] has recently been shown to be an integral membrane protein of the viral envelope. inside the envelope is a ribonucleoprotein (rnp) core, which comprises the rna genome and a single species of nucleocapsid protein n. electron microscopic observation of viral rnp showed a long helix of to nm (macnaughton et al., ; sturman and holmes, ) . a very recent study of intact and detergent-treated tgev virions (risco et al., ) by negative-staining, ultrathin sectioning, freezefracture, immunogold mapping and cryoelectron microscopy showed a surprising new feature of coronavirus particles, namely, a spherical, probably icosahedral, core inside the virion (fig. ) . these internal cores comprise not only the n protein and rna but also the m protein, m being the major core shell component. disruption of the cores released helical nucleocapsids. the presence of an icosahedral core in the coronavirus virion had heretofore been unsuspected. this core structure was also detected with mhv virion (risco et al., ) . this surprising new finding gives us cause to reconsider our view of coronavirus architecture. thus, the precise structure of the core and rnp inside the virion is not certain. toroviruses and coronaviruses have a similar morphology and virion composition ( fig. , table ) but are distinguishable in a number of ways (table ) (weiss and horzinek, ; horzinek, , ; koopmans and horzinek, ) , necessitating their inclusion in separate genera. the morphology of the arteriviruses is substantially different from that of coronaviruses and toroviruses, particularly in having an icosahedral rnp core ( fig. ) ; hence, a separate family is maintained for arteriviruses. however, the recent discovery of the icosahedral core for coronavirus (risco et al., ) may have blurred this distinction. the s glycoprotein is the outermost component of the virion, and is responsible for the attachment of the virus to cells (collins et al., ; godet et al., ; kubo et al., ) and for instigating the fusion of the virus envelope with cell membranes. it is the primary target for the host's immune responses; neutralizing antibodies are induced mainly by s (collins et al., , and immunization in animals with s alone can induce protection from some coronaviruses (ignjatovic and galli, ; torres et al., ) . within a coronavirus species, sequence variation is usually exhibited more by s than by any other structural proteins; the variation of the s protein sequence probably confers a selective advantage in immune animals. these and other aspects have recently been reviewed in detail . the s protein is large, ranging from some (ibv) to amino acids (fcv). there are many potential n-linked glycosylation sites ( to , most of which have glycans attached. the s preproprotein has a n-terminal signal sequence and a membrane-anchoring sequence near the c terminus (fig. ) . the s protein may be cleaved into s and s subunits; the extent of its cleavage varies greatly among the species (cavanagh, ) . a high proportion, up to loo%, of the s protein is cleaved in some coronaviruses (ibv, mhv, bcv, tcv, pedv) (cavanagh, a) ; none is cleaved in others (tgev, fcv, ccv) (garwes and pocock, ) ; and very little of the s protein of hcv- and hcv-oc is cleaved, although the s of oc is completely fig . models of the virions of a coronavirus, a torovirus, and an arterivirus. the he protein is present only in antigenic group coronaviruses (see table i ). reproduced with permission from cavanagh et al. ( ) . present in only a subset of coronaviruses (table i) . may have an isometric core in addition (risco et al., ) . no such protein described. table i ) and with respect to the number and position of nonstructural protein genes. the polymerase genes encode two orfs, l a and lb, which overlap. l, leader sequence; he, hemagglutinin-esterase; s, spike; e, small membrane protein; m, integral membrane protein; n, nucleocapsid protein; an, poly(a) tail; g, and gl, small and large glycoproteins, respectively. risco et al. ( ) for tgev. this model illustrates the observation that internal cores (ic), possibly icosahedral, were observed inside virions of tgev. the cores comprise the helical ribonucleoprotein (nc) (genome rna + n protein) and the m protein. reproduced with permission from risco et al. ( ). cleaved if trypsin is present (hogue and brian, ) . the extent of s cleavage depends on the cell type (frana et al., ) . cleavage generates two glycopolypeptides, n-terminal s and c-terminal s , the latter being acylated . s is probably linked to the s subunits by noncovalent linkage: trypsin treatment of mhv virions caused cleavage of all s proteins without disrupting the spikes ; however, s can be released from virion by either urea or mild alkali treatment (cavanagh and davis, ; weismiller et al., ) . among the coronavirus genus as a whole, the s polypeptide is much more conserved than s . regions of up to % amino acid identity (particularly in the transmembrane domain) exist between the s polypeptides of coronaviruses in the different antigenic groups, whereas and (b) (schmidtet al., ) . the amino acid numbering has been normalized with respect to that of the longest known mhv s protein, that of mhv (jhm) ( s . e. . (a) the protein has an amino-terminal signal peptide (sp) and a transmembrane (tm) sequence near the c terminus. the glycosylated propolypeptide is cleaved a t a basic connecting peptide (cp) to yield glycopolypeptides s and s . the locations shown are those of three mutations present in mutants of mhv recovered from a persistently infected neural cell line, the mutants requiring a ph of . - . for membrane fusion (gallagher et al., ) . (b) s of another mhv-jhm (schmidt et al., ) , which has a -amino acid deletion with respect to (a). bacterial expression products containing residues - and - bound mab f and g, respectively, both of which neutralize virus infectivity and inhibit membrane fusion. the arrow indicates the positions of amino acid substitutions in jhm mab f-resistant mutants (grosse and siddell, ) . a peptide comprising residues - bound another mab that neutralized virus and inhibited fusion (luytjes et al., ). there is almost no conservation of the s sequence. furthermore, comparison of s sequences among strains of a given species, or between species of a given group, reveals hypervariable regions, which include frequent deletions, mutations, or recombination (cavanagh et al., ; s. e. parker et al., ; banner et al., ; gallagher et al., , suggesting that this region is externally exposed and not essential for the structure. the s polypeptide has two regions with a seven-residue periodicity, forming heptad repeats (fig. ) indicative of a coiled-coil structure (de groot et al., ) . indeed, current evidence suggests that the mature s protein forms an oligomer; for tgev, it is probably a trimer (delmas and laude, ). however, a dimer structure has been proposed for ibv s protein (cavanagh, ~) . therefore, the oligomeric s protein is envisaged as being anchored in the membrane by a n a-helical region near to the c terminus of s . just beyond the outer membrane surface is the shorter (minor) repeat structure predicted to be an a! helix of - nm. the major repeat indicates a helix of - nm, which may form the narrow stalk of the spikes (de groot et al., ) . all coronavirus s proteins have a highly conserved eight-residue sequence kwpww/yvwl, the last five residues of which probably form the beginning of the membrane-spanning domain. terminating residues upstream of kwp is a leucine-zipper motif, the length varying from three to five heptad repeats (britton, ) . the highly conserved sequences of s may play a role in forming the stalk, which has a more rigid structure. in contrast, the s domain is predicted to form the globular portion of the spikes, consistent with its highly variable nature. the s protein has two important biological activities for the virus: a. induction of membrane fusion. this activity may be required for viral entry into cells or for cytopathic effects. expression of the recombinant s gene has provided unequivocal evidence that the s protein alone is sufficient to cause membrane fusion, as shown by syncytium formation (de groot et al., ; pfleiderer et al., ; yo et al., ; taguchi, ) . several regions of the s protein, widely separated in a linear sense, have been implicated in the membrane fusion process by the following observations: ( ) s of bcv expressed in insect cells caused fusion (yo et al., ) . ( ) a monoclonal antibody that inhibited cell fusion was shown to bind to the s domain of mhv ( fig. ) (luytjes et al., ) . ( ) changes at three s residues ( , , and in the mhv s protein; fig. ) were associated with a change from a requirement for a neutral ph to an acidic ph for fusion (gallagher et al., ) . ( ) two bacterial expression products containing residues - ) and - ) ofthe jhm strain ofmhvinducedmonoclonal antibodies f and g, respectively, both of which inhibited fusion ( fig. ) . ( ) chemical modifications of the cysteine residues, specifically residue in the ectodomain of s , reduced the fusion activity of the jhm strain of mhv (gallagher, ) . this result also suggests strain-specific differences in the conformation of the s protein, since the fusion activity of the a strain of mhv was not affected by this modification. ( ) some mutations to cysteine residues within the transmembrane domain of s adversely affected fusion, suggesting that the transmembrane domain is involved in conformational changes that are associated with fusion activity (bos et al., ) . these results combined suggest that the s ectodomain contains the major determinants for membrane fusion; however, s does not contain hydrophobic domains typical of fusion proteins. thus, several disparate regions, including some in the s , may contribute to the fusion activity, probably because some of these regions are juxtaposed in the threedimensional structure or can affect the overall conformation of the spikes. interestingly, monoclonal antibody-resistant mutants of the jhm strain ofmhvselected with antibody f had mutations not a t the antibody-binding site (residues - of s l ) , but at a distant site, i.e., residues - in the s domain (grosse and siddell, ) (fig. ) , suggesting that s is folded such that regions which are widely separated in the linear sense are juxtaposed to form functional domains. early studies of coronvirus-induced cell-cell fusion suggested that only cleaved s was able to promote cell fusion . more recent studies in which mhv s proteins with mutated s connecting peptides were expressed have shown that cleavage is not essential for fusogenic activity, although cell-cell fusion is more efficient when the s protein is cleaved (stauber et al., ; taguchi, ; bos et al., ) . furthermore, naturally occurring mutants of mhv, derived from persistently infected mouse cells, which are defective in s cleavage, have delayed fusion activity (gombold et al., ) . expression of the feline infectious peritonitis virus (fipv) s protein, which is not cleaved a t all, also resulted in syncytia formation (de groot et al., ) . these results indicate that s protein cleavage is not required for but can enhance membrane fusion. whether membrane fusion activity, as manifested by syncytia formation, is required for viral infectivity has not been established. there are mhv strains (e.g., mhv- ) that do not cause syncytia formation in cultured cells; however, these viruses may be able to cause virus-cell membrane fusion within the infected cells. b. receptor binding. monoclonal antibodies (mab) against the s protein of most coronaviruses can neutralize viral infectivity; thus, it is assumed that the s protein mediates virus binding to the receptors on target cells. indeed, the s protein or a portion of it can bind to the viral receptor molecules in uitro. this has been demonstrated for mhv and tgev s proteins (godet et al., ; kubo et al., ) . the binding domain has been mapped to the n-terminal amino acids of mhv s protein. site-directed mutagenesis of this region showed that mutations of the residues at position and positions , , and abolished the binding of the protein to the receptor (suzuki and taguchi, , suggesting that the receptor-binding site might comprise discontiguous regions in the linear sense. the s subunit is not involved in receptor binding (taguchi, ) . the receptor-binding sites of tgev s protein have been mapped to a -residue region (aa - ) of the s (godet et al., , which overlaps with a n epitope for a neutralizing mab. this neutralizing mab was able to block the binding of the -residue polypeptide to the receptor; conversely, the receptor did not block the binding of the mab to this polypeptide, suggesting that the receptor-binding determinants and the neutralizing epitopes are distinct and are part of a domain of s whose configuration is independent of the remainder of the s protein. s proteins of bcv and hcv-oc bind to -o-acetylneuraminic acid (schultze et al., a) ; this binding is required for viral infection. the significance of this binding will be discussed in section v,b on virus attachment. intriguingly, several coronavirus s proteins share some sequence identity with the receptor for the fc fragment of mammalian immunoglobulins (fcy receptor). thus, mab to the fcy receptor could immunoprecipitate s protein from the mhv-infected cells, and s could bind to the fc fragment of immunoglobulin. this molecular mimicry was first demonstrated for mhv and, more recently, for bcv and tgev as well (oleszak and leibowitz, ; oleszaket al., oleszaket al., , . it may play a role in modulating viral pathogenicity. this potential function is significant because expression of the s protein in the infected cells induces not only humoral antibodies but cellular immunity as well (welsh et al., ) ; the potential binding of s to the fc fragment of immunoglobulin may modulate these immune responses. the m protein is one of only two of the structural proteins [the other being the e protein (see below)] that are essential for the production of coronavirus-like particles. the sequence of the m protein reveals that the m polypeptides comprise - amino acids, except for some members of the tgev group, which have an additional or so residues a t the amino terminus, forming a cleavable membrane insertion signal. the amino-terminal or so residues of the mature m protein of all the coronaviruses are hydrophilic, exposed at the virion surface, and have a small number of glycosylation sites. glycans are of the n-linked type for ibv and the tgev group and o-linked for the mhv group (rottier, ) . the remainder of the n-terminal half of the molecule forms three helical membrane-spanning domains, although a mutant m protein which lacked all three of the membrane-spanning domains did associate with membranes in uitro (mayer et al., ) . the structure of the c-terminal half is uncertain, but it is believed to be largely situated on the inside of the viral envelope, based on protease susceptibility cavanagh et al., b) and sequence-based predictions (armstronget al., ; rottier et al., ) . however, some m molecules of tgev virions have the c terminus exposed a t the virion surface (laviada et al., ; risco et al., ) . moreover, mab specific for the c-terminal amino acids of m neutralized tgev virions in the presence of complement and caused antibody-mediated, complementdependent cytolysis of tgev-infected cells (risco et al., ) . studies with mutant mhv m proteins expressed from vaccinia virus recombinants had shown that some had the n terminus and others the c terminus a t the luminal side of the endoplasmic reticulum, equivalent to the outer surface of virions (locker et al., ) . some molecules of one mutant m protein had both termini at the luminal surface, and other molecules had both termini at the cytoplasmic surface (locker et al., , ) . thus, the precise topology and the structural role of the m protein are still not certain. recent studies have shown that some m proteins are also associated with the rnp core of tgev and constitute the outer shell of the internal core (risco et al., ) . this core-associated m can be clearly separated from the viral envelope. therefore, m may play a dual structural role in forming both the envelope and the internal core of the virion. several properties of the m protein suggest that it is involved in virus particle assembly: ( ) the m protein binds to the purified nucleocapsid in uitro (sturman et al., ) . ( ) when the m protein was expressed alone, it was localized in the golgi complex, near the location where virus particles bud (tooze et al., ; tooze and tooze, ) . however, recent studies showed that the site of m protein retention in the golgi was slightly different from that for viral particle budding (klumperman et al., , suggesting that additional factors are involved in virus particle assembly. this will be discussed in section v,h on virus assembly. the m protein of tgev has a n additional biological activity: induction of a-interferon (charley and laude, ; laude et al., ) . thus, it may play a role in viral pathogenesis. monoclonal antibodies against the m protein do not neutralize viral infectivity, suggesting that m is not involved in receptor binding. however, some of these antibodies can neutralize viral infectivity in the presence of complement (collins et al., ; laviada et al., ) , indicating that part of the m protein is exposed on the virion surface. the he glycoprotein-or perhaps one should say the he gene-of coronaviruses is something of a n enigma. only coronaviruses belonging to the mhv group possess the he gene (table i) . even there, not all virus strains within a species express the he protein (luytjes et al., ; . as with many of the so-called nonstructural protein genes of coronaviruses, the product of the he gene is not essential for viral replication, certainly not in the cell types used in the laboratory. the he protein was first detected in bcv (king et al., ) and some mhv strains; however, acceptance of it as a legitimate virus-encoded protein was delayed because in one of the mhv strains studied most thoroughly, a , virions lacked he. the he gene of a was later shown to lack the initiation codon of the he open reading frame (orf); thus, the he gene is a pseudogene in this (luytjes et az., ) and several other mhv strains . a complete, functional he gene was subsequently identified in the jhm strain and several others . the he glycoprotein, of approximately kda ( amino acids in bcv), has been detected in virions of hev, mhv, hcv-oc , bcv, and tcv. when analyzed under nonreducing conditions, the he protein migrates as a dimer of approximately kda (king et al., ) . the mature protein is believed to exist in the virion as a dimer, anchored by the c terminus, forming a fringe of short spikes visualized by electron microscopy (caul and egglestone, ; dea and tijssen, ) . it is not known whether each spike consists of more than one he dimer. those coronaviruses which contain he in their virions cause hemagglutination much more efficiently than those that do not. similar to the s protein, he alone can mediate hemagglutination and hemadsorption (king et al., ; hogue and brian, ; vlasak et al., b; deregt et al., ; pfleiderer et al., ; schultze et al., a) ; however, he seems to have weaker activity than s (schultze et al., a) . he binds to -o-acetylated neuraminic acid (vlasak et al., ; schultze et al., a) , which is also a target for s binding. some he-specific mab can neutralize bcv infectivity (deregt and babiuk, ; deregt et al., ) . thus, he protein of bcv may participate in virus binding to the receptor. the relative importance of he and s in hemagglutination and tissue tropism of bcv is not known. as its name implies, the he protein also has esterase activity; specifically, it is a neuraminate-o-acetylesterase. it hydrolyzes the - acetylated sialic acid on erythrocytes, thereby reversing hemagglutination induced by the he or s protein; thus, he is considered a receptordestroying enzyme (vlasaket az., a,b; yokomori et az., ; . the putative esterase active site is fgds, encoded by amino acids - of the mature he polypeptide of bcv (m. d. parker et al., ; kienzle et al., ) . in these respects, it resembles the hemagglutinin-esterase-fusion (hef) glycoprotein of influenza c viruses, which also has hemagglutinating and -o-acetylated sialic acidhydrolyzing esterase activities. moreover, the he protein of coronavi-ruses shares some % amino acid identity with the hef of influenza c virus, including conservation of the position of the putative esteraseactive site fgds and many cysteine residues (luytjes et al., ; s. e. parkeret al., ; kienzle et al., ; zhanget al., ) . unlike the hef protein of influenza c virus, which is cleaved into two subunits (nakada et al., , the coronavirus he protein is not cleaved and lacks most of the c-terminal subunit of the hef of influenza c virus. because of the close relatedness between the coronavirus he protein and the influenza c virus hef protein, and because the he gene is present in only one coronavirus group, it was proposed that the he gene was acquired by a coronavirus as a result of recombination between an ancestral coronavirus and influenza c virus (luytjes et al., ) . interestingly, the torovirus berne virus also has an he pseudogene (gene ; fig. ) (snijder and horzinek, ) , the amino acid sequence of which has approximately % identity with the c-terminal part of the coronavirus he. the functional significance of he for coronaviruses is not known. among coronaviruses, only bcv requires he for infectivity; however, the presence of he may affect the pathogenicity of some coronaviruses, as evidenced by the findings that passive administration of he-specific mab in mice altered mhv pathogenicity and that mhvs with an he have different neuropathogenicity from those without he . conceivably, the presence of he in an mhv may allow the virus to utilize an alternative receptor independently of the s protein. however, this is not the case, as evidenced by the finding that an mab specific for the murine biliary glycoprotein molecule, which is the major mhv receptor recognized by s, inhibited the infectivity of an he-containing mhv (gagneten et al., ) . thus, the he protein does not enable a virus to bypass the primary mhv receptor and may provide only an auxiliary function for virus binding to target cells. until recently it was thought that coronaviruses possessed three (s, n, m) or four (including he) structural proteins. it is now clear that coronaviruses, but not toroviruses, possess an additional virion protein, the e protein. it plays an essential role in virion assembly. it has been shown that the e and m proteins are the only two viral proteins absolutely required for virion assembly (bos et al., ; vennema et al., ) . this protein has been demonstrated for ibv (smith et al., ; liu et al., , tgev (godet et al., ) and mhv (yu et al., ) . when the deduced e proteins of the other coronaviruses are taken into account, it transpires that the e proteins vary from t o amino acids, corresponding to molecular weights of to , (siddell, ~) . siddell has highlighted a number of features common to all the e proteins, namely, a hydrophobic region of some two dozen residues, starting near the n terminus; a cysteine-rich region immediately downstream from this; a conserved proline residue in the middle of the molecule, and otherwise very low amino acid identity in the genus as a whole; and an abundance of charged residues in the cterminal half of the protein (siddell, ~) . it is now well established that this protein is associated with highly purified virion preparations (liu et al., ; godet et al., ; yu et al., ) . liu and inglis calculated the ratio of s:n:m:e proteins in virions of ibv-beaudette strain to be : : : , indicating an amount of e protein similar to that of s protein (liu et al., ) . in contrast, godet et al. estimated that the s:m:e protein ratio in virions of tgev was : : (godet et al., , and vennema et al. ( ) have suggested an m:e ratio of approximately oo:l for virions of mhv. it is not clear why there is such a wide range of variations. the e protein in the cells is localized in the perinuclear region, with some migrating to the cell surface (godet et al., ; yu et az., ) . experimental evidence suggests that the e protein is anchored in the membrane by sequence in the n-terminal half of the molecule. thus antibodies specific for epitopes in the c-terminal half of the tgev e protein produced cell-surface fluorescence in paraformaldehyde-fixed, tgev-infected cells (godet et al., ) , but the precise topology of the protein has not been elucidated. the role of the e protein in virion assembly will be discussed in section v,h on virus assembly and release. the e proteins of ibv and mhv are translated from the third and second orfs, respectively, of mrnas and of the respective viruses. both of these are polycistronic mrnas (see figs. and and section v,g, ). in contrast, in all other viruses, the e protein is derived from a monocistronic mrna. the mechanism of translation of the ibv and mhv e proteins is discussed in section v,g. the n protein is a -to -kda phosphoprotein which, together with the genomic rna, forms a helical nucleocapsid (rnp). the rnp of coronaviruses have been reported variously as being from - to - nm in diameter (see laude and masters, , for references) . the n protein in rnp provides only limited protection to the rna genome against ribonucleases. the n proteins vary from to amino acids in length, are highly basic, and have a high ( - %) serine content, potential targets for phosphorylation. sequence conservation within the genus is low. thus, the n proteins of ibv and tgev have only % identity with that of bcv. even within the mhv group, the n proteins of mhv and bcv share only % identity, whereas the m proteins of these two viruses have % identity (lapps et al., ) . based on sequence comparison, three structural domains in the n protein have been identified (parker and masters, ) . the middle domain is an rna-binding domain (masters, ; nelson and stohlman, ) which binds to both coronaviral and nonviral rna sequences in uitro (robbins et al., ; stohlman et al., ; masters, ) ; however, it does not contain any motifs characteristic of other rna-binding proteins. under specific binding conditions, the mhv n protein binds to the leader rna sequence, particularly nucleotides - . furthermore, an anti-n mab immunoprecipitated all of the mhv rna molecules which had the leader sequence . the n protein of ibv also bound to the ' untranslated region of the ibv rna in uitro (zhou et al., ) . these rna-binding properties are consistent with the fact that the n protein interacts with the viral genomic rna to form nucleocapsid. this interaction is necessary for the formation of virus particles, as n alone cannot be incorporated into virus particles, whereas the n-rna complex can (bos et al., ; vennema et az., ) . however, the specificity of the rna-n protein interaction required for nucleocapsid formation has not been elucidated. the n protein also binds to membranes and phospholipid (anderson and wong, ) . this may be another property which facilitates the formation of virus particles. the finding that the n protein binds to the ' and ' ends of viral rna suggests that the n protein may also modulate viral rna synthesis because the ends of the rna are likely involved in the regulation of rna synthesis. in an in uitro rna replication system, the addition of mhv n-specific antibodies inhibited viral rna synthesis (compton et al., ) , suggesting that the n protein is a component of the rnasynthesizing machinery. the ability of n to bind to the membrane (anderson and wong, ) may enable the formation of the rna replication or transcription complex, in view of the fact that viral rna synthesis occurs in the membrane fraction of infected cells dennis and brian, ) . the three structural domains of the n protein are separated by spacer regions, which are not conserved (masters, ) . the functions of the n-and c-terminal conserved domains are not yet clear. using a targeted recombination approach (koetzner et al., ; masters et al., ) to generate recombinant viruses that have a chimeric n gene containing parts of bcv and mhv sequences, peng et al. ( a) have shown that there is strict sequence specificity within the conserved structural domains for viable recombinants. since the n protein constitutes the nucleocapsid, mutations within the n protein will likely affect the stability or viability of the virus. indeed, several temperaturesensitive and thermolabile mutants of mhy have deletions or mutations within the n protein (koetzner et al., ; peng et al., b) . viruses with site-specific mutations of the n gene have been generated by targeted recombination techniques; interestingly, revertants of these mutants often have second-site mutations located a t different domains, suggesting that there are interactions between different domains of the n protein (peng et al., ) . the role of phosphorylation in the n protein has not been elucidated. the coronavirus contains a positive-sense, single-stranded rna genome, which is the largest viral rna genome known, ranging from . to kb. the large size of the viral rna requires the virus to develop special mechanisms of rna synthesis to counter the deleterious effects of the possible errors during rna synthesis. the virion rna functions as an mrna and is infectious. it contains approximately - functional genes, or of which encode structural proteins. the genes are arranged in the order '-polymerase-(he)-s-e-m-n- ', with a variable number of other, mostly nonstructural and largely nonessential, genes interspersed among them (fig. ) . this gene arrangement also applies to toroviruses and arteriviruses (fig. ) . the ' terminus of the coronavirus genome is capped, and the rna starts with a leader sequence of - nucleotides, which is also present a t the ' end of mrnas, followed by a -to -nucleotide untranslated region (utr). at the other end of the genome is a ' utr of - nucleotides followed by a poly(a) tail. almost two-thirds of the entire rna is occupied by the polymerase gene, which comprises two overlapping orfs, la and lb. at the overlap region is a specific seven-nucleotide "slippery" sequence and a pseudoknot structure, characteristic of the ribosomal frameshifting signal (brierley et al., (brierley et al., , lee et al., ; , which is required for the translation of orf lb. the architecture of the nonstructural protein genes interspersed between the known structural protein genes varies significantly among different coronavirus species (fig. ) . for example, in hcv- e, gene contains two orfs, whereas in the related virus pedv these two orfs are fused (duarte et al., ) . in hcv-oc , gene is missing altogether (mounir and talbot, ) . finally, in ibv, two orfs are inserted between m and n genes. the variability of gene structure indicates the plasticity of coronavirus rna and the frequent occurrence of recombination and also suggests that there is no strong conservation pressure on these nonstructural proteins. there is a stretch of consensus sequence, ucuaaac (for mhv), or a related bcv (hcv-oc ) fig . comparative genome structure of the different coronaviruses. the complete sequences are available for mhv, ibv, tgev, and hcv- e. the gene sequences of the remaining viruses have not been completed. gene sequences are interrupted and shortened to highlight the remaining genes. the vertical lines represent mrna start sites; thus, each region between two vertical lines represents a separate gene ("transcription unit"). the structural protein genes are marked by various symbols, and nonstructural protein genes are represented by unfilled boxes. the gene arrangements of ns protein genes and e protein gene are very heterogeneous in terms of transcription unit and the relative size and position among different strains of the same virus species; only the representative one is presented. the numbering system for the genes of hcv- e deviates from the published one to be consistent with the other viruses. hcv-oc does not have a gene . sequence, a t sites immediately upstream of most of the genes. these sequences represent signals for transcription of subgenomic mrnas (see section v,e). finally, a pseudoknot structure has been shown to be present a t the ' end of the coronaviral rna (williams et al., ) . a characteristic feature of the coronaviridae, and of the arteriviridae as well, is that all known member species generate a '-coterminal nested set of five or more mrnas (see fig. ). each coronavirus and arterivirus subgenomic mrna has the leader sequence a t its ' end. curiously, no leader rna sequence is present in the torovirus rnas (fig. ) . in , the coronavirus study group published its recommendations for the nomenclature of coronavirus genes, mrnas, and proteins (cavanagh et al., ) . at that time it was reluctant to apply the term "nonstructural" t o the potential products of genes which were suspected of not being structural proteins. this caution was a consequence of our lack of knowledge of those gene products, a situation which has improved greatly in the last years or so. this has resulted in the term "nonstructural (nsl" being applied more widely to several gene products. every gene that encodes the ns proteins has been deleted in a t least some naturally occurring virus isolates; thus, most of the ns genes are not essential for viral replication. however, some of the ns proteins may play a role in viral tissue tropism or pathogenicity. the polymerase is encoded by gene , which accounts for approximately two-thirds of the genome (fig. ) . the complete polymerase gene of four coronaviruses (ibv, mhv, hcv- e, and tgev) covering each of the three coronavirus groups has been sequenced lee et al., ; bonilla et al., ; eleouet et al., ) . although the polymerase genes vary in size from approximately to kb, the encoded proteins have many structural features in common. the degree of amino acid identity for this gene product is greater than is observed for any other coronavirus gene product. the polymerase gene is predicted to encode a protein of approximately - kda. proteins of this size have not been detected in coronavirus-infected cells, in part because of co-translational polypro-tein processing. the pol gene encodes two orfs, l a and lb, which overlap by a few dozen nucleotides (figs. and ). the second, orf lb, is in the - reading frame with respect to the upstream orf l a and is translated following ribosomal frameshifting in the overlap region. this will be examined in more detail in section v,g. there is greater amino acid identity among the l b than the l a orfs. for example, l a and l b of ibv, the least typical coronavirus in terms of protein sequences, have amino acid identityhimilarity of approximately / % and / %, respectively, compared with those of mhv, hcv, and tgev. it is the l a orf which accounts for the mhv polymerase gene being approximately - kb longer than those of ibv, hcv, and tgev. a number of functional domains within pol have been predicted following computer-based motif analyses hodgman, ; gorbalenya et al., a,b; lee et al., ) ; some of these functional domains have been confirmed by experimental analysis. the location of these motifs is illustrated in fig. . three motifs have been identified in orf l a , indicating the presence of one or two papain-like cysteine proteases (plp): a chymotrypsinlpicornaviral c-like protease (lee et al., ) . (a) the polymerase gene comprises two orfs, l a and lb, which overlap, the l b orf being translated after ribosomal frameshifting. (b) the positions of motifs: plp and , papain-like protease; x domain, highly conserved between ibv and mhv; clp, picornavirus- c-like protease; md, membrane-associated domain; gfl, growth factorlike; pol, rna-dependent rna polymerase; mb, metal-binding motif; hel, helicase. (c) genetic complementation groups fu and baric, ) . (d) processing scheme for part of the l a orf (denison et al., ( clp) and a cysteine-rich growth factor-related protein (gfl). mhv, hcv- e, and tgev have two plp domains ( and , with plpb corresponding to the single plp domain of ibv. sequence corresponding to a cysteine protease of streptococcus pneumoniae has been identified in l a of ibv. upsteam of plpb is a region termed the x domain, a region of particularly high conservation between ibv and mhv and similar to one near the thiol protease of alpha-and rubiviruses . there is no functional evidence so far to link the gfl with known growth factors, but the predictions of most of the protease domains have been confirmed by experimental analysis. the first plp domain of mhv is responsible for the cleavage of p /p and p from the n terminus of the mhv orf l a polyprotein ( fig. ) (baker et al., (baker et al., , bonilla et al., bonilla et al., , . this plp was inhibited by zinc chloride but not by leupeptin (baker et al., ; denison et al., ) . deletion analysis defined this proteinase domain to be within the sequence encoded by the . - . -kb region from the ' end of the genome. site-directed mutagenesis showed that residues cys- and his- were essential for protease activity (baker et al., ) . some amino acid sequences between the p cleavage site and the plp domain were also essential for the cis cleavage that generates p (baker et al., ; bonilla et al., ) . the function of plp has not been demonstrated. the clp domain extends for approximately amino acids and is homologous to proteases encoded by picornaviruses and several other virus genera. the putative clp domain of hcv- e has been expressed as a /?-galactosidase fusion protein in escherichia coli and shown to have autocatalytic proteolytic activity, releasing an active clp protein (ziebuhr et al., ) . an antiserum against this fusion protein immunoprecipitated a -kda protein from hcv- e-infected cells. similar activity has been demonstrated for the clps of mhv and ibv (tibbles et al., ) . this protease cleaves not only its own boundaries but also several downstream sites within orf l a and orf lb, probably both in cis and in trans. computer analysis predicted that the catalytic center of the ibv clp would include cys- , his- , and glu- (gorbalenya et al., a,b) . site-directed mutagenesis confirmed the role of the cys and his residues but showed that the glu residue was not essential (liu and brown, ) . the same approach confirmed that the predicted qs(g) dipeptide bonds in the l b orf are the targets for the protease activity of the clp of ibv (discussed further in section v,g). similar conclusions were reached for clp activity of mhv and hcv- e grotzinger et al., ) . the importance of cys- in the clp of mhv has been demonstrated (seybert et al., ) . in uitro transcription and translation of a cdna containing the putative clp of mhv produced polypeptides of and kda, which were subsequently processed to products of and kda . the -kda protein possesses the c-like protease activity (lu et al., ) . the clp domain is flanked by predicted membrane-spanning domains, which may be important for the proteolytic activity (tibbles et al., ) (fig. ) . poor expression of the ibv clp protein in vitro led to the discovery that this protease was ubiquinated and subsequently degraded by an adenosine triphosphate (atp)-dependent protease present in reticulocyte lysate (tibbles et al., ) . this is the third example of a viral protein subject to turnover in this manner and involves a different virus class from the previously reported examples, in a picornavirus (oberst et al., ) and an alphavirus . the ubiquitin-mediated, atp-dependent proteolytic pathway is a major cellular, nonlysosomal, protein degradation system, which may cause rapid turnover of the coronaviral polymerase. the functional domains associated with rna synthesis are located within the more conserved l b orf. these include domains for an rnadependent rna polymerase, a nucleoside triphosphate (ntp)-binding/ helicase domain, and a zinc-finger nucleic acid-binding domain (metal binding domain) (fig. ) . computer analyses identified the polymerase domain h o d p a n , ; gorbalenya et al., a,b) . unlike the gdd motif present in many viruses, the corresponding sequence in coronaviruses is sdd. whether the polymerase gene products contain activities other than proteases and polymerases is not known. the coronaviruses exhibit great heterogeneity with respect to the number and genome location of ns protein genes and in regard to the number of orfs within a gene (fig. ) . the functions of these ns proteins are still unknown. there are two genes located between the polymerase and s genes of these viruses (fig. ) . gene - encodes the he protein, while gene encodes an ns protein of unknown function. the gene protein com-prises approximately amino acids ( kda) (luytjes et al., ; shieh et al., ; labonte et al., ) . the bcv and mhv homologs share % amino acid identity, while the homolog of hcv-oc has % identity with that of bcv. this gene product has been detected in the cytoplasm of mhv-, bcv-, and hcv-oc -infected cells and may be phosphorylated (bredenbeek et al., ; zoltick et al., ; coxet al., ; labonte et al., ) . computer analysis of its sequence suggested the presence of a nucleotide-binding site (luytjes et al., ) . however, no function has been assigned to this protein, and it is not required for virus replication in culture (schwarz et al., ) . interestingly, the c terminus of the torovirus orf l a product (polymerase) has - % sequence identity with the gene product of mhv (snijder et al., ) . this evolutionary relationship between coronavirus and torovirus suggests that the gene product is probably involved in viral rna synthesis, since it is expressed as part of the torovirus polymerases. there are two to three orfs in this region, and their structure and the mechanism of expression of gene products vary markedly among different coronavirus species. they can be expressed as two different genes, i.e., expressed from two separate mrnas (e.g., mrnas and of mhv and bcv and mrnas and - of tgev) or localized in one gene, thus requiring internal initiation of translation from a single polycistronic mrna (e.g., mrna of the ibv and fcv groups). in ibv, it contains three orfs ( a, b, and c); orf c encodes the e protein, which is a viral structural protein, while a and b encode ns proteins. the gene products of both orfs a and b (approximately kda) have been detected in small quantities in virus-infected cells (liu et al., ) . in tgev, this region contains two orfs, being separated from the e protein gene. these two orfs are encoded by mrnas and - , respectively, the predicted protein products being approximately and kda, respectively. in a related nonenterogenic strain, prcv, however, there are multiple deletions in this region, essentially inactivating one or both of the orfs (rasschaert et al., ; wesley et al., ) . it has been suggested that the absence of the a product, in addition to a shorter s protein, might be associated with their lack of enteropathogenicity. however, vaughn et al. ( ) have recently described two prcv strains which have an intact a gene (vaughn et al., ) . canine coronavirus has gene orfs equivalent to those of tgev, exhibiting high amino acid identity (> %), although the second orf is truncated by a stop codon (horsburgh et al., ) . two other members of the tgev group exhibit a variation on the same theme. pedv and hcv- e lack a homolg of orf a of tgev and ccv. pedv has an orf corresponding to b of tgev, while hcv- e has two orfs corresponding to the single orf of pedv (duarte et al., ) . members of the group i coronaviruses also exhibit great heterogeneity in this region. mhv-jhm produces mrna , which encodes a -kda protein. this protein has been detected in virus-infected cells (ebner et al., ) . in contrast, hcv-oc contains only amino acids in this region (mounir and talbot, ) . gene of mhv has two orfs, a and b. the latter encodes the structural e protein and is the predominant product made from mrna . it is not clear whether orf a is translated at all. at least one strain of mhv lacks the a orf ; also, hcv-oc has the a orf but is unable to produce a corresponding mrna (mounir and talbot, ) . in summary, there is great heterogeneity with respect to the number, size, and mechanism of expression of orfs between the s and e genes. these ns proteins probably are not required for viral replication. the lack of necessary function may account for the heterogeneity which arose during evolution. ibv is unique in that it has two orfs ( a and b), which encode proteins of . and . kda, respectively. these proteins have been detected in very small amounts in virus-infected cells (liu and inglis, a) . the function of these orfs is not clear. tgev has an additional gene , which encodes a . -kda protein (garwes et al., ; tung et al., ) , in the region corresponding to the ' end untranslated region of other viruses (fig. ) . this protein is hydrophobic and is associated with the endoplasmic reticulum and cell surface membranes (tung et al., ) , but its nuclear localization has also been reported (garwes et al., ) . fcvs and ccv have two orfs in the same region, the first being analogous to the single orf of tgev. the second ( b) orf encodes a -kda soluble protein containing the sequence ktel (vennema et al., , which is similar to the endoplasmic reticulum retention signal, kdel. the protein is partially retained in the endoplasmic reticulum but is also slowly secreted out of the cells. the functions of these proteins are not known. coronaviruses have relatively restricted host ranges, infecting only their natural hosts and closely related animal species. occasionally, cross-species infection of coronaviruses occurs, such as the experimental infection of monkey by mhv, which causes central nervous system demyelination (murray et al., ; cabirac et al., ) , and the occasional infection of humans by bcv, which causes diarrhea. bcv also infects turkeys and tgev infects dogs, suggesting some flexibility in their host range. the expansion of viral host range can be achieved by passing the coronavirus in a heterologous cell line, as demonstrated by the emergence of an mhy variant with the ability to infect originally nonpermissive cell lines, such as human cells, after serial passages (baric et al., ) . in animals, coronaviruses have restricted tissue tropism; for example, most hcv strains cause only respiratory infections. different strains of a coronavirus may have distinct tissue specificity; for example, tgev infects both the gastrointestinal tract, causing fatal diarrhea, and respiratory tract tissues without causing primary respiratory symptoms, whereas prcv, which is closely related to tgev, infects the respiratory tract of pigs but replicates poorly in the intestinal tract (cox et al., ) . the species and tissue specificity of a coronavirus infection is a t least partially dictated by the nature and distribution of cellular receptors and other related molecules that regulate virus entry, as evidenced by the viral replication that results when viral rna is directly introduced into cell types of other animal species. thus, coronaviruses have the potential to replicate in many cell types. the complete coronavirus replication cycle takes place in the cytoplasm. it has been shown that mhv can replicate in enucleated cells and in the presence of actinomycin d and a-amanitin, suggesting that nuclear functions are not required for viral replication wilhelmsen et al., ) . there are, however, reports of the inhibition of replication by actinomycin d of some coronaviruses, including feline enteric coronavirus (lewis et al., , ibv (evans and simpson, , hcv- e (kennedy and johnson-lussenberg, , and mhv in some cell lines (dupuy and lamontagne, ) . thus, nuclear functions may be required for viral replication under certain conditions. this issue has not been resolved. the first step in viral infection is the binding of the virus to target cells. hemagglutination and hemadsorption have been used as assays for studying virus-cell interaction, although the erythrocyte itself is not a target cell for coronavirus infection. several coronaviruses, including hev, ibv, bcv, and some strains of mhv and hcv, can cause hemagglutination (sugiyama and amano, ; schultze et al., ; zhang et al., a) . the binding residue on the cell surface is a -o-acetylated neuraminic acid of glycoproteins or glycolipids (schultze et al., , although different coronaviruses may prefer different structural isoforms of -o-acetylated neuraminic acid. for bcv, the virus binding to erythrocytes is mediated through either the s or he protein, both of which have hemagglutinating activities, the s protein having the stronger activity (king et al., ; schultze et al., a,b) . the he protein of bcv and hev also recognizes -o-acetylated neuraminic acid, and its esterase activity is also specific for this molecule; thus, he protein has both receptor-binding and receptor-destroying activities (vlasak et al., a,b; schultze et al., b) . expression of the he protein of mhv on the cell surface conferred a hemadsorption activity ; however, even viruses that lack he protein (e.g., ibv) can cause hemagglutination, suggesting the role of s protein in hemagglutination. thus, the he and s proteins of various coronaviruses may have comparable functions, enabling the virus to bind the sialic acid residues; however, only the he protein confers the receptordestroying activity. the residue necessary for hemagglutination by ibv is a , -linked n-acetylneuraminic acid . curiously, the hemagglutinating activity of ibv is not evident until the virus particle is treated with neuraminidase, suggesting that the s protein itself is covered by sialic acid. although virus binding to erythrocytes provides a good model system for studying virus-cell interactions, it may not necessarily reflect the actual mechanism of virus attachment to target cells. the classical study of virus attachment to target cells involved the in uitro binding of mhv to macrophages from genetically susceptible m i c w l m. c. lai and david cavanagh and resistant mouse strains (shif and bang, ) . this study showed that mhv bound equally well to cells from resistant and susceptible mice, even though macrophages from resistant mice were resistant to virus infection. similar observations have been made on splenic lymphocytes, thymocytes (krzystyniak and dupuy, , and glial cells (wilson and dales, ) ; thus, it appears that genetic resistance is not exerted at the level of virus binding in these cases. similarly, established tissue culture cell lines, including murine and primate cells, irrespective of their degree of susceptibility or resistance to mhv, bound mhv to the same extent (van dinter and flintoff, ; kooi et al., kooi et al., , . thus, virus may bind to a ubiquitous molecule on the cell surface, which, however, may not lead to virus infection. whether this ubiquitous molecule is a sialic acid-containing glycoprotein has not been established. the binding of bcv to its target cells, such as mdck cells, is mediated by -o-acetylneuraminic acid residues similar to those on erythrocytes. removal of the sialic acid by neuraminidase abolished virus attachment, while resialization restored it . hcv-oc binds to a similar sialic acid residue but prefers a form slightly different from that for bcv (kunkel and herrler, ) . the he protein of bcv can also mediate virus binding to target cells, and this binding may be required for viral infection, as suggested by the finding that mab against he inhibited bcv infectivity (deregt and babiuk, ; deregt et al., ) . one inhibitor of the esterase activity of he protein, diisopropylfluorophosphate, also inhibited bcv infection (vlasak et al., a) . the s protein of bcv probably also participates in virus binding to target cells, as suggested by the finding that the mab against s protein can neutralize bcv infectivity . the relative importance of s and he proteins is not clear. in contrast, none of the mab against the he protein of mhv inhibited mhv infection . despite the finding that the binding of he and s proteins to target cells is necessary for bcv infection, the binding of bcv or hcv-oc to n-acetylneuraminic acid in itself is not likely the basis of viral cell tropism because sialic acid is a common cell surface carbohydrate residue; thus, an additional, more cell type-specific molecule is probably required for viral infection. the finding that mhv and other coronaviruses bound to resistant as well as susceptible cells indicates that this binding may represent an initial step in the virus attachment process, which is not sufficient for viral infection. it is likely that a more specific binding between virus and cells is required for the establishment of viral infection. this binding involves a specific virus receptor molecule on the cell surface. a. mhv receptor. the mhv receptor was the first coronavirus receptor to be identified. it is the murine homolog of a member of the carcinoembryonic antigen (cea) family (dveksler et al., ; williams et al., ) and belongs to the biliary glycoprotein (bgp) subfamily. the terminology of mhv receptors in the literature is somewhat controversial, the following terms being used interchangeably: mmcgm , mhvr- , and bgpa. it has an immunoglobulin-like structure, consisting of four immunoglubulin-like loops, the n-terminal loop being the virus-binding domain (dveksler et al., b) . the sequence of the c terminus (cytoplasmic domain) of the receptor is not essential. glycosylation of the protein also is not necessary for its receptor function in uiuo (dveksler et al., ) . the functional significance of the receptor in viral infection in uiuo was demonstrated by the finding that an mab against the mhv receptor inhibited viral infection in mice (smith et al., ) . subsequently, several additional members of cea family were found to serve as mhv receptors, including an mmcgm -like protein (also termed mhvr- and bgpb), which is the product of an alternatively spliced form of mmcgml rna and is expressed in both the liver and brain, in contrast to the liver-specific expression of mmcgml (yokomori and dveksler et al., a) ; an allelic gene product of the bgp gene in sjl mice, a mouse strain resistant to mhv infection (yokomori and lai, ; dveksler et al., a) ; bgp- , which is the product of a new member of the murine bgp gene (nedellec et al., ) ; and a novel pregnancy-specific glycoprotein (psg)-like protein, which is expressed in the mouse brain, in contrast to placenta-specific expression of other psg molecules . all these molecules contain a consensus motif in the virus-binding domain (n-terminal loop). thus, several different cea family members, which are differentially expressed in different cells and tissues, can potentially serve as an mhv receptor. different strains of mhv may use different cearelated molecules as receptors at different efficiencies (compton, ; . the prototype mhv receptors (mhvr- ) are expressed in the liver, gastrointestinal tract, b cells, macrophages, and endothelial cells but not in t cells (coutelier et al., ; godfraind et al., ) , consistent with the target cell specificity of mhv. however, the mhv receptor is also expressed in other tissues, e.g., kidney, which are not targets for mhv infection. also, sjl mice express a functional mhv receptor (yokomori and lai, ; dveksler et al., a) but are resistant to mhv infection (knobler et al., ) . thus, receptor expression is not sufficient for viral infection. it is not yet clear which molecules are used by mhv as receptors in cross-species infection (e.g., rats and monkeys) (murray et al., ; cabirac et al., ) . recently it was shown that bgp and cea molecules of human origin could serve as receptors for some mhv strains . the expression of the receptor molecules on the cell surface is necessary for virus infection, and the expression level of the receptor may determine the relative susceptibility or resistance to viral infection in some cells. during persistent viral infection of cultured murine cells, the expression level of the receptor is offen reduced, resulting in the relative resistance of the cells to viral superinfection, which could be overcome by the expression of an exogenous receptor (sawicki et al., ; chen and baric, ) . thus, there is a rough correlation between receptor expression and the susceptibility of a cell type to virus infection. under certain circumstances, virus may infect cells by a receptorindependent mechanism; for example, mhv-infected murine cells may fuse with human cells, which do not have mhv receptors, and cause the latter cells to become infected (gallagher et al., ) . it has been shown that mhv infects polarized epithelial cells through the apical, but not the basolateral, surface (rossen et al., a (rossen et al., , . it is not clear whether the virus receptor is differentially expressed on the two different surfaces. b. receptors for tgevand hcv- e. the receptors for tgev and hcv- e have been identified as aminopeptidase n (apn) of the porcine and human species, respectively (delmas et al., ; yeager et al., ) . prcv also uses porcine apn as a receptor; thus, virus binding to the receptor is not sufficient to explain the differences in tissue tropism between tgev and prcv. apn is a member of the membrane-bound metallopeptidase family and is widely distributed on diverse cell types; it is highly expressed on the brush border membrane of enterocytes. some of the antibodies against human apn can block hcv- e binding (yeager et al., ) ; however, the catalytic site of the protease activity of apn is not required for receptor function, and the inhibitors of apn do not block viral infection . similar to mhv, tgev infects polarized cells through the apical, but not the basolateral, surface (rossen et al., ) . again, it is not clear whether this is restricted by the differential expression of apn on the different sides of the cells. tgev has also been shown to bind to a -kda protein on the surface of the enterocytes on the villi of the small intestine (weingartl and derbyshire, ) . pcrv does not bind to this molecule. both the temporal expression (mainly in the newborn) and spatial distribution patterns (on the villi of the gastrointestinal tract) of the -kda protein correspond to the pattern of susceptibility of piglets to tgev infection. thus, the expression pattern of this molecule appears to have a better correlation than the porcine a€" with the tissue tropism of tgev. this -kda molecule may be an alternative receptor used by tgev. the relative functional significance of this molecule and a€" as tgev receptors is not yet clear. the fipv strains of fcv and canine coronavirus apparently utilize the a€" of feline and canine species, respectively, as receptors (benbacer et al., ) . cross-species utilization of feline a€" by coronaviruses of different species (canine, feline, and human) has also been reported (tresnan et al., ) . fipv, however, is unique among coronaviruses in that it causes an antibody-dependent enhancement (ade) phenomenon (weiss and scott, , which is the result of the binding of the virus-antibody complex to fc receptors on the surface of macrophages, leading to enhanced virus uptake and spread. this ade phenomenon has been attributed to the s protein-antibody complex (vennema et al., b; corapi et al., ; olsen et al., ) . the fc receptor may be a co-factor or an alternative receptor for fipv entry into macrophages. in this regard, the s protein of mhv has been shown to have limited sequence homology with the murine fc receptor and to have the ability to bind to the fc fragment of immunoglobulin oleszak et al., ) . whether the fc receptor plays a role in mhv infection is not clear. however, mhv does not exhibit ade. c. receptors for other coronaviruses. sialic acid (n-acetyl- - acetylneuraminic acid)-containing glycoproteins are probably a component of the cell surface molecules required for bcv and hcv-oc infection because the removal of sialic acids inhibits bcv infection and resialylation restores virus infectivity ; however, it is unlikely that it is the primary receptor molecule used by these viruses since the distribution of these molecules is more widespread than the susceptible target cells. the identity of the specific receptor for these viruses has not been determined. for hcv-oc , it has been shown that the virus binds to a major histocompatibility complex class i molecule (collins, ) . however, the receptor function of this molecule has not been established. the mechanism of coronavirus entry into target cells has been controversial. early electron microscopic studies visualized virus (mhv and ibv) particles inside lysosome-like vesicles near plasma membranes, suggesting that virus enters cells by endocytosis ("viropexis") (david-ferreira and manaker, ) ; however, other studies suggested that virus enters cells by direct fusion between virions and the plasma membrane (doughri et al., ) . lysosomotropic drugs, such as ammonium chloride and chloroquine, inhibited mhv- virus entry (krzystyniak and dupuy, ) . also, mhv-specific antibodies did not lyse virusinfected cells during the virus-entry process, as would be the case if the virus fused with the cell membrane (krzystyniak and dupuy, ) . these results suggested that mhv- enters cells by an endocytotic pathway. similar studies using the a strain of mhv, however, showed that ammonium chloride delayed, but did not inhibit, the viral infection of l- cells (mizzen et al., ) . the effects of ammonium chloride were interpreted to be inhibiting virus uncoating in this case. recent studies by the same group have further shown that only a small proportion of adsorbed virus enters cells by the endocytotic pathway since ammonium chloride, chloroquine, and dansylcadaverine, all of which inhibit receptor-mediated endocytosis, did not have significant effects on mhv entry (kooi et al., ) . the majority of mhv particles enter cells by virus-cell fusion at the plasma membrane. this interpretation is consistent with the finding that the optimum ph for mhvinduced cell fusion is . (weismiller et al., ; kooi et al., , rather than the acidic ph expected for a virus that enters cells by an endocytotic pathway (e.g., vesicular stomatitis virus). the optimum ph for bcv-and ibv-induced cell fusion is also neutral (payne and storz, ; li and cavanagh, ) . these findings suggest that coronavirus enters cells by virus-cell fusion at the plasma membrane. on the other hand, virus internalization by endocytosis may be a nonproductive mechanism which does not depend on virus-receptor interaction, since some mhv-resistant cell lines can internalize mhv particles as efficiently as susceptible cell lines (kooi et al., ) . most surprisingly, even vero cells, an african monkey kidney cell line which presumably does not have an mhv receptor, can internalize virus (kooi et al., ) . therefore, it is likely that mhv enters cells by both acidic-phdependent (endocytosis) and -nondependent pathways (kooi et al., ) . the exact mechanism of virus entry may depend on cell types and virus strains. interestingly, an mhv variant which has mutations in the s protein has an acidic optimum ph of . - . , in contrast to the ph of . for the parental virus (gallagher et al., ) . this virus variant probably enters cells by an endocytic pathway, a fact supported by the finding that infection of this variant virus is inhibited by ammonium chloride or chloroquine. what triggers virus internalization after virus-receptor binding is not clear. it has been shown that a conformational change in the s protein could be induced at ph . and incubation at °c . whether this represents the expected conformational change following virus-receptor binding is not clear. irrespective of the mechanism of virus internalization, fusion between the viral envelope and cell membrane must occur, either at the cell surface or in the endosome, for viral infection to take place. virus-induced cell-cell fusion has been used to investigate the ability of a virus to induce fusion. early studies with mhv indicated that virus-induced fusion from without (caused by virions at the cell surface) or fusion from within (caused by de ~o u o synthesized s protein on the cell surface) required cleavage of the s protein (frana et al., ; sturman et al., ) . work on bcv supported this view (payne and storz, ; storz et al., ) . however, more recent experiments involving the expression of s protein (de groot et al., ; stauber et al., ; taguchi, ) and studies of mhv fusion mutants (gombold et al., ) have indicated that uncleaved s can cause syncytium formation, though less efficiently than the cleaved s. of course, coronaviruses such as tgev, which have no cleaved s protein, are infectious, in fact, highly so. since fusion of the virion envelope with a cell membrane is an essential part of the infection process, these results suggest that tgev must be able to cause viruscell fusion. thus, virus-cell fusion and cell-cell fusion may have different requirements, and, for at least some coronaviruses, s cleavage is not required for the fusion of a virion with a cell membrane. nevertheless, cleaved s may be more efficient at inducing fusion for some coronaviruses. the concentration of s at the surface of a virion may be higher than at the cell surface, such that even the uncleaved s can induce virion-cell fusion, even though it cannot cause cell-cell fusion. virusreceptor interaction may also trigger a signal transduction pathway to facilitate the internalization of the virus-receptor complex. one study showed that tyrosine kinase is activated in macrophages immediately following mhv- infection (dackiw et al., ) . it is not yet known whether this is required for virus entry. the mechanism of virus uncoating, i.e., the release of virion rna from the nucleocapsid, after the virus has been internalized remains unclear. one study suggested that virus uncoating may involve an endosomal neutral phosphatase, which preferentially dephosphorylates the nucleocapsid protein (mohandas and dales, ) . furthermore, while immature oligodendrocytes were sensitive to jhm virus infection, differentiated oligodendrocytes were resistant, probably due to a block in virion uncoating (beushausen et al., ) . the factors responsible for the differences in these two types of cells may involve protein kinases (wilson et al., ) . additional cellular factors may be required for viral penetration and uncoating. various murine cell lines, all of which express virus receptors, show different degrees of susceptibility to infection by different mhv strains (kooi et al., ; asanaka and lai, ; yokomori et al., ) . cell-cell and virus-cell fusion studies indicated that virus infection is blocked at different stages of virus entry, including penetration and uncoating, in different cell lines (van dinter and flintoff, ; asanaka and lai, ) . these cell lines could be grouped into at least three complementation groups with respect to the virus entry process (flintoff, ; asanaka and lai, ) . thus, virus penetration and uncoating appear to require separate cellular factors. it has been suggested from the studies using recombinant viruses between the a and jhm strains of mhv that viral s protein may interact with these cellular factors . the nature of these factors is not yet clear. following virus uncoating, the first macromolecular synthetic event is predicted to be the synthesis of an rna-dependent rna polymerase(s) from the incoming viral genomic rna, as is the case for all positive-strand rna viruses. the polymerase is translated from gene at the ' end of the genomic rna, most likely directly from the incoming genomic rna. the process of primary translation has not been observed experimentally. however, inhibitors of protein synthesis applied early in the infection blocked rna transcription (mahy et al., ; perlman et al., ; sawicki and sawicki, , indicating that protein synthesis, most likely the translation of a viral polymerase, is necessary for viral rna synthesis. this virus-specific polymerase is responsible for the synthesis of negative-strand rna from the incoming genomic rna and subsequent transcription of mrnas from the negative-strand template. the nature of polymerase is discussed in section iv,a. since the genomic-sized rna is used for both packaging into virus particles to become virion rna and as an mrna for protein translation, the distinction between rna transcription and rna replication is often blurred. in this review, we will use the term "transcription" to describe the synthesis of subgenomic mrnas as well as genomic rna used for translation; the term "replication" will be used to describe the synthesis of the genomic rna destined to be packaged into virions. coronavirus rna synthesis occurs via an rna-dependent rna transcription process; thus, rna synthesis can occur in the presence of actinomycin d (with the exception of some coronaviruses, as discussed in section v,a). the majority of the virus-specific rnas in the cells are mrnas, which are transcribed from a negative-strand rna template. for clarity of discussion, the structure of the mrnas will be discussed first. coronavirus mrnas consist of six to eight species of different sizes, depending on the coronavirus species and strains (lai, ) . the largest mrna is equivalent to the genomic rna, and the remainder are subgenomic in size. these rnas are designated mrnas through , in order of decreasing size, according to the recommendations of the coronavirus study group in (cavanagh et al., ) . some mrnas have been given a hyphenated name, e.g., mrna - , because they were discovered after the original set of mrnas was named. they have a nested-set structure, and all of them contain sequences starting at the ' terminus and extending to various distances toward the ' end (stern and kennedy, b; lai et al., ; leibowitz et al., ) . the smallest mrna contains only the ' terminal orf, while each next larger mrna contains one additional orf. the structure of the mrnas in relation to the genome structure is shown in fig. . thus, except for the smallest mrna, all of the mrnas are structurally poly- cistronic. in general, each orf in the genome is represented by an mrna, whose sequence starts from a consensus signal upstream of the orf, and only the ' most orf of each mrna can be translated; thus, each mrna is functionally monocistronic. however, there are exceptions: some mrnas, e.g., mrna of mhv and mrna of ibv, are translated into two or three proteins by different mechanisms (see section v,g). several additional minor mrna species have been detected, some of which could only be detected by reverse transcription-polymerase chain reaction (la monica et al., ; schaad and baric, ) . these minor rnas probably represent rna transcripts from weak or atypical mrna start signals (see below). most do not contain a complete orf at the ' end; thus, they are probably not functional. furthermore, in mhv, several mrnas, e.g., mrnas - , - , and - , are transcribed only in some virus strains la monica et al., ) . the syntheses of these mrnas appear to be differentially regulated by the sequence at the ' end of the viral genome la monica et al., ) . coronavirus mrnas have another unique structural feature: their ' ends have a leader sequence of approximately - nucleotides, which is derived from the ' end of the genomic rna (lai et al., , spaan et al., ) . the leader sequences of all the mrnas are identical for a given strain of virus, except for slight variations at some of the leader-mrna fusion sites, and are identical to the sequence present at the ' end of the genomic rna. at the mrna start sites on the viral genomic rna, there is a short stretch of sequence that is nearly homologous to the ' end of the leader rna (budzilowicz et az., ) . this sequence constitutes part of the signal for subgenomic mrna transcription (makino et al., ) . sequence comparison of viral genomic and mrnas suggests that subgenomic mrnas are derived by fusion of the ' end genomic rna sequence (leader) to the mrna start sites on the viral genomic rna. the mrna start sites are usually located between the genes; hence, they are termed intergenic (ig) sequences. however, some of the igs may overlap the coding region of the upstream gene. the core sequence of the ig for mhv is ucuaaac or a slightly variant form of this sequence at various ig sites (joo and makino, ) . other virus species also have similar ig sequences. the leader sequence of mhv ranges in length from to nucleotides, the variation resulting from the heterogeneity of the ' end sequence, which contains two to four copies of a pentanucleotide (ucuaa) repeat. the homologous nucleotides (ucuaa) at the ' end of the leader and ig sites serve as fusion sites for the leader and mrnas. some of the mhv mrnas are heterogeneous, consisting of several subspecies, each containing different copy numbers of the ucuaa repeat . this fact suggests that the fusion between the leader rna and the mrnas is not very precise. the length and sequence of the leader rna in other coronaviruses vary. however, the ' end of the leader sequence always contains a pentanucleotide ucuaa or a closely related sequence. mrnas of coronaviruses other than mhv are usually homogeneous in their structure, probably a reflection of the fact that leader rna at the ' end of the genome and ig sites in these viruses contain only a single copy of the the ucuaa-like sequence (hofmann et al., a) . the copy number of this pentanucleotide repeat apparently plays an important role in the regulation of mrna transcription. coronavirus rna synthesis is mediated by rna-dependent rna synthesis via a negative-strand rna intermediate (complementary to the genomic rna). coronavirus negative-strand rna represents no more than - % of the total intracellular virus-specific rna sawicki and sawicki, ) . bothgenome-sized and subgenomic negative-strand rnas, which correspond in number of species and size to those of the virus-specific mrnas, have been detected (sethna et al., ; hofmann et al., ) . the relative molar ratios of the various subgenomic negative-strand rna species are comparable to those of the positive-strand subgenomic mrnas. the ' end of the negativestrand rna contains poly(u) sequences, which are shorter than the poly(a) sequences present on the positive-strand rnas . at the ' end of the negative-strand rna is the complementary sequence of the leader rna (anti-leader) (sethna et al., ) . structurally speaking, the subgenomic negative-strand rnas appear to be mirror images of the positive-strand subgenomic mrnas. all of the negative-strand rnas in the infected cells are present in the form of double-stranded rna, no free negative-strand rna is detected . in virus-infected cells, virus-specific mrna synthesis can usually be detected a few hours after infection and throughout most of the viral replication cycle (stern and kennedy, a; leibowitz et al., ; keck et al., a) . the molar amounts of the different mrna species vary; smaller mrnas are generally more abundant than larger ones, but this rule does not always hold true. nevertheless, the relative ratio of different subgenomic mrna species remains constant throughout, suggesting that the synthesis of the various subgenomic mrna species is regulated coordinately. some viruses may show slight variations in the amounts of individual mrna species present during infection (hiscox et al., a) . later in infection, there appears to be an enhanced synthesis of the genomic-sized rna (keck et al., a) . the kinetics of negative-strand rna synthesis follows a pattern similar to that of positive-strand mrna synthesis; however, the peak of negative-strand rna synthesis appears to occur earlier than for positive-strand rna sawicki and sawicki, ) . thereafter, negative-strand rna synthesis drops significantly, in contrast to that of positive-strand rna synthesis, and negative-strand rna appears to be stable sawicki and sawicki, ) . a similar pattern of kinetics of negative-strand rna synthesis is also seen in the accumulation of the negative-strand rna of a di rna, which very rapidly reaches a steady-state level after transfection . therefore, the negative-strand rna probably functions as a template for multiple rounds of positive-strand rna synthesis. this conclusion is supported by the study of a ts mutant defective in negative-strand rna synthesis (schaad and baric, ) . however, the ability to synthesize negative-strand rna seems to be maintained throughout the viral life cycle, as evidenced by the finding that a transfected di rna can replicate even when transfected late in the infection (jeong and makino, ) . since all subgenomic rnas consist of a leader rna derived from the ' end of the genome and a body sequence derived from various downstream sequences, they must be synthesized by fusion of two discontiguous sequences either during or after transcription. an early study showed that the leader sequence of each mrna can be exchanged freely between two coinfecting viruses, suggesting that the leader rna and mrnas are transcribed independently and can conjoin in a random fashion (makino et al., b) . more recent studies using di rna constructs that contain an inserted mrna start signal (see below) established that the leader rna and mrnas are usually derived from two separate rna molecules (jeong and makino, ; zhang et al., b) . these studies unequivocally showed that coronaviral mrna synthesis is carried out by either a discontinuous transcription or transsplicing process, which fuses sequences from two different rna molecules. several transcription models have been proposed, each of which is consistent with some of the experimental data. these models are not mutually exclusive, as components of each model may operate at different stages of the viral replication cycle. before presenting these models, we will discuss several findings pertinent to coronaviral rna transcription. . coronavirus replication takes place entirely in the cytoplasm. nuclear functions are believed not to be required for rna synthesis wilhelmsen et al., ) ; thus, viral rna transcription does not involve the conventional rna splicing machinery present in the nucleus. . early ultraviolet (w) transcriptional mapping studies indicated that in the late stage of viral replication, the w target size of each subgenomic and genomic mrna is approximately equivalent to the physical size of the respective mrna (jacobs et al., ; stern and sefton, a) ; thus, each mrna is transcribed independently rather than derived by the processing of a large precursor rna. however, early in infection, the w target sizes of the subgenomic mrnas were found to be equivalent to that of the genomic rna (yokomori et al., ) ; thus, at least early in infection, the synthesis of a genomic-length rna is required for subgenomic mrna synthesis, although it is not clear whether this requirement is for a positive-or a negative-stranded, full-length rna. a more recent analysis of the uv target sizes of subgenomic mrnas of mhv suggested that, even late in the infection, the w target sizes of some subgenomic mrnas are slightly larger than their physical lengths but smaller than genomic size (den boon et al., ) . similar observations were made for equine arteritis virus (an arterivirus). this recent result is consistent with either of two interpretations: (a) the subgenomic mrnas are derived from a slightly longer rna template or (b) they are derived from a mixture of templates of different sizes (genomic as well as subgenomic). the difference in w target size between the early and late stages of viral rna replication suggests that different mechanisms of rna synthesis may operate at the different stages of the viral replication cycle. . the molar ratios of different subgenomic mrna species and those of subgenomic negative-strand rnas are similar (sethna et al., ; hofmann et al., ) , suggesting that subgenomic mrnas and subgenomic negative-strand rnas are derived from each other or under the same transcriptional regulation. . the leader rna at the ' end of each mrna is identical in each mrna and to the leader rna at the ' end of genomic rna. furthermore, there is sequence homology between the ' end of the leader rna and the mrna start sites on the genomic rna (budzilowicz et al., , where the leader sequence is fused to the mrnas. there is some sequence divergence between the leader rna and some of the mrna start sites; in these cases, the leader rna of the resulting mrnas usually mimics the sequence of the mrna start site rather than the leader at the ' end of the genome. this finding was used to suggest the possible presence of rna proofreading activity during coronavirus transcription (lai, (lai, , ; van der most et al., ). the following transcriptional models (fig. ) address the possible mechanism of fusion between the leader sequence and mrnas. most of the experimental evidence came from mhv studies. the exceptions will be noted. a. leader-primed transcription. this model proposes that the virion genomic rna is first transcribed into a genomic-length, negativestrand rna, which, in turn, becomes the template for subsequent subgenomic mrna synthesis. the leader rna is transcribed from the ' end of the negative-strand rna and dissociated from the template. the free rna subsequently associates with the template rna at various mrna start sites and serves as a primer for transcription of mrnas. it is proposed that the discontinuous transcription step takes place during positive-strand rna synthesis. several pieces of evidence are compatible with this model: detected in the cytoplasm of mhv-infected cells (baric et al., ) . some of these are dissociated from the template rna and, thus, may serve as a potential source of primers in this transcription model. these rnas have distinct sizes which are reproducible from cell to cell (baric et al., ) ; however, they are not exactly the same size as the leader sequence present in the subgenomic mrnas. thus, these free leader rnas must be processed before they are incorporated into mrnas. a temperature-sensitive mutant of mhv, which synthesizes leader rna but not mrnas at the nonpermissive temperature, has been isolated . the isolation of this mutant suggests that mhv mrna synthesis is discontinuous, requiring different viral proteins for the synthesis of leader rna and mrnas. thus, a distinction can be made between leader rna synthesis and mrna synthesis. of the viruses is derived from the other coinfecting virus (makino et al., b) . this result suggests that the leader sequence and body sequence of each mrna are derived from two separate pools. this phenomenon is reminiscent of the rna reassortment that occurs in rna viruses with segmented rna genomes. this result is best explained by the possibility that free leader rnas participate in viral rna synthesis. in an in vitro transcription system utilizing cytoplasmic extracts from mhv-infected cells, exogenous leader rnas can be utilized for mrna synthesis (baker and lai, ) . the exogenous leader rna was incorporated into the subgenomic mrnas at a site that matched precisely that of the endogenous leader rna present in the viral subgenomic mrnas, regardless of the length of the exogenous leader rna used, suggesting that the exogenous leader rna sequence was processed before being incorporated into mrnas. furthermore, the truncated leader rna which lacked the ' end ucuaa sequence could not be incorporated into mrnas, suggesting the importance of this sequence in transcription (baker and lai, ) . . the leader rna sequence, specifically the copy number of the ucuaa repeats at the ' end of the leader rna, can affect the transcription of some viral subgenomic mrnas. for example, whereas an mhv strain containing two ucuaa repeats transcribes mrna - , a strain with three ucuaa repeats does not, despite identical sequences in the mrna start sites of these two viruses la monica et al., ) . this finding suggests that the leader rna plays an essential role in the regulation of mrna transcription. according to this model, the free leader rna binds to the mrna start site (ig) of the full-length negative-strand template via the complementary sequences between the ' end of the leader (positive-strand) and the ig site of the template rna (negative-strand) and serves as the primer for rna transcription. the free leader rna (primer) may be longer than the leader sequence in the subgenomic mrnas. there are certain mismatched nucleotides between the leader and template at some mrna start sites; in the latter case, sequences in the mature mrnas usually match those of the template instead of the leader. therefore, the free leader rna probably undergoes ' end cleavage before transcription starts to remove the leader nucleotides that are not complementary to the template rna (lai, (lai, , van der most et al., ) . transcription is then initiated from the ' end of the processed leader rna. this model is consistent with most of the sequence data of mrnas. it also explains the curious finding that some mrnas of mhv are heterogeneous in the copy number (from two to four) of the pentanucleotide (ucuaa) repeats at the leader-mrna fusion site . this heterogeneity is best explained by the imprecise binding between the leader rna and template rna due to the presence of multiple copies of ucuaa (lai, ) . indeed, bcv, which contains only one copy of ucuaa in both the ' leader and ig sites, does not show this type of heterogeneity in its mrnas (hofmann et al., a) . some recent data, however, cannot be explained by this rna sequence-homology-driven transcription model. a particular mhv strain (mhv-bc), which has four copies of the ucuaa in the leader rna, synthesizes some subgenomic mrnas that are very heterogeneous in length and in leader-mrna fusion sites . the sequence data of its mrnas showed that the leader rna of this virus is randomly fused to sites where no sequence homology exists between the leader and fusion sites (zhang et al., ) . a similar though less conspicuous heterogeneity in the leader-mrna fusion sites has also been observed in another mhv strain in a di rna-based transcription system (see section v,e, ) (van der most et al., ) . these findings suggest that the sequence complementarity between the leader rna and ig sites may not be the driving force for mrna transcription. thus, a modified version of the leader-primed transcription model proposes that the ucuaaac sequence provides a recognition signal for viral polymerases and viral or cellular transcription factors. these proteins bind to the leader and ig sites of the template rna, and the subsequent rna-protein and protein-protein interactions result in the formation of a transcription complex to initiate mrna transcription and effect leader-mrna fusion zhang and lai, ) . the salient feature of this model is that the discontinuous transcription step occurs during positive-strand rna synthesis; thus, transcriptional regulation is exerted mainly during positive-strand rna synthesis. this is consistent with current knowledge of the regulation of mhv rna synthesis. it has been shown that mhv mrna transcription requires multiple cis-acting rna sequences (see section v,e, ). in contrast, the initiation of negative-strand rna synthesis requires only the ' end -nt plus poly(a) . thus, most of the regulatory elements appear to regulate positive-strand rna synthesis. since the free leader rna is the centerpiece of this transcription model, it readily explains why the leader rna from a different virus can be utilized freely in trans during mixed infections (makino et al., b) . however, this model does not explain the finding that subgenomic replicative-intermediates (ri) and replicative-form (rf) rnas were detected and were functional during viral rna synthesis (sawicki and sawicki, ; schaad and baric, ) (see section v,e, ,b). it is possible that the subgenomic mrnas synthesized can be transcribed into subgenomic negative-strand rnas, which, in turn, become the templates for mrna transcription at a later stage in the viral replication cycle. this would explain why the uv target sizes for mrnas are nearly equivalent to the physical sizes of mrnas late in the infection and yet are equivalent to the genomic-sized rna early in the infection (yokomori et al., b) . in contrast to the leader-primed transcription model, this model proposes that the discontinuous transcription step occurs during negative-strand rna synthesis, generating subgenomic negativestrand rnas, which then serve as templates for subgenomic mrnas in uninterrupted transcription. this model was proposed to account for the detection of subgenomic negative-strand rnas (sethna et al., ; hofmann et al., ) and subgenomic ris (sawicki and sawicki, ) in virus-infected cells. in this model, ig (mrna start site) sequences on the genomic rna serve as termination or pausing signals for negative-strand synthesis (konings et al., , and the nascent subgenomic negative-strand rna then jumps to the leader rna sequence at the ' end of the genomic rna by an unknown mechanism to continue rna synthesis. as a result, the nascent negative-strand subgenomic rna fuses with the negative-strand leader sequence, generating a subgenomic negative-strand rna that contains an anti-leader sequence at its ' end and a poly(u) sequence at its ' end sethna et al., ) . structurally, these negativestrand rnas are mirror images of the subgenomic mrnas and, thus, can potentially serve as a template for uninterrupted transcription of subgenomic mrnas. in this model, the regulation of subgenomic mrna transcription would be exerted on negative-strand instead of positive-strand rna synthesis. this model is consistent with the following observations: . subgenomic negative-strand rnas have been detected in virusinfected cells (sethna et al., ; hofmannet al., ) . these rnas have structures that are mirror images of those of the completed subgenomic mrnas. the relative molar ratios of the different subgenomic negative-strand rnas are similar to those of the corresponding viral mrnas (sethna et al., ; hofmann et al., ). in the infection (sawicki and sawicki, ) . the smaller ris were precursors of the smaller mrnas and the larger ris generated the larger mrnas, suggesting that each subgenomic mrna was transcribed from the corresponding subgenomic-sized negative-strand template (sawicki and sawicki, ) . another study, which analyzed the subgenomic rfs of a temperature-sensitive mutant of m w , also suggested that subgenomic negative-strand rnas are functional (schaad and baric, ) ; although, in this study, ris were not directly examined. . the uv targets for subgenomic mrna synthesis at the later stage of viral replication are subgenomic in length (jacobs et al., ; stern and sefton, a; yokomori et al., b) , roughly corresponding to the physical lengths of each subgenomic mrna, suggesting that the templates for these mrnas are subgenomic. . in di rna systems (see section v,e, ), when multiple ig sequences were present, the sequences in the ' end often had a higher transcription efficiency than those at the ' end, consistent with the proposal that igs serve as transcriptional termination sites, which impede the elongation of the negative-strand rnas (van marle et al., ; krishnan et al., ) . however, in some cases, the higher transcription efficiency of the ' proximal ig was observed only when the neighboring igs were very close together, suggesting a spatial constraint rather than sequential interference (joo and makino, ) . this model, however, cannot explain why the w targets for subgenomic mrna synthesis early in infection are of genomic size (yokomori et al., b) and why, later in the infection, the targets for these same mrnas are still larger than the respective subgenomic mrnas but not longer than genomic size (den boon et al., ) . it also cannot explain why the nature of the leader sequence can regulate differential transcription of various mrna species, such as mrna - of mhv, inasmuch as the leader sequence on the template rna is localized downstream of the transcription termination site for negative-strand rna synthesis. finally, it is difficult to explain why the leader rnas are derived in trans. c. trans-splicing of nascent rna transcripts. this model proposes that the fdl-length positive-or negative-strand rnas are spliced posttranscriptionally to generate subgenomic rnas. it was initially considered unlikely because of the findings that coronavirus replicates in the cytoplasm rather than in the nucleus wilhelmsen et al., ) , where the splicing machinery is present, and that uv target sizes of subgenomic mrnas are equivalent to the physical sizes of subgenomic mrnas (jacobs et al., ) . furthermore, there are no consensus splicing donor and acceptor sequences in the coronavirus genomic rnas. however, the trans-splicing model is compatible with recent findings that early in infection, the w targets for subgenomic mrna synthesis are of genomic length (yokomori et al., b) , and that both the leader rna and ig sequence of mhv negative-strand rna bind to a cellular factor, heterogeneous nuclear rnp (hnrnp) al, which is involved in alternative rna splicing h.-p. li and m. m. c. lai, unpublished observation) . a modified splicing model thus can be proposed as follows: a full-length negative-strand rna is first synthesized. components of the splicing machinery derived from the nucleus or cytoplasm then bind to the leader sequence and ig sites on the negative-strand rna and form a splicing complex. the leader and ig can be derived from different rna molecules. splicing between the leader and ig generates a subgenomic negative-strand rna. once the spliced subgenomic negative-strand rnas are generated, they are used as templates for subsequent mrna synthesis. later in infection, even the subgenomic negative-strand rnas may be able to participate in rna splicing to generate smaller subgenomic negativestrand rnas because they themselves also contain the leader and ig sequences. this model may thus explain why the w target for mrna transcription is of genomic length early in infection (yokomori et al., b) and may shed light on the recent puzzling finding that later in infection, the w target sizes are still larger than the actual sizes of the subgenomic mrnas (den boon et al., ) . it also explains the functional roles of subgenomic ris (sawicki and sawicki, ) . this potential splicing, however, must be different from conventional rna splicing because it occurs in the cytoplasm, and the splicing donor and acceptor sequences must also be different from the conventional ones. since some of the splicing factors are probably derived from the nucleus, this model predicts that nuclear functions are involved in mhv rna transcription. based on the findings that some coronaviruses, including bcv, tgev, and ibv (sethna et al., ; hofmann et al., ; zhao et al., ) , contain subgenomic mrnas in the virion, probably as a result of nonspecific rna packaging, it was proposed that these virion-associated subgenomic mrnas can be used directly as templates for the synthesis of subgenomic negative-strand rnas, which, in turn, serve as templates for the synthesis of additional subgenomic mrnas (sethna et al., ) . this model may explain the presence of subgenomic negative-strand rnas and ris in the infected cells, but it cannot explain the genomiclength nature of the w target sizes for mrna synthesis early in infection (yokomori et al., b) , nor can it explain how leader rnas from different virus strains can be randomly incorporated into mrnas of a different virus. furthermore, the virion-associated subgenomic mrnas have not been detected in all coronavirus species. the available data cannot unequivocally rule out any of the proposed transcription models. the primary difficulty in experimental analysis is that once the subgenomic mrnas are synthesized, by whatever mechanism, they are transcribed into negative-strand rnas because the cis-acting signal for negative-strand rna synthesis in mhv resides in the nucleotides at the ' end plus poly(a) (lin et al., ) , which is present in every subgenomic rna. thus, it is difficult to separate the primary and secondary events of transcription. it is possible that these transcription models are not mutually exclusive. for example, early in infection, a leader-primed transcription or trans-splicing mechanism may operate, generating subgenomic mrnas, which are then amplified into subgenomic negative-strand rnas; the latter serve as templates for further amplification of subgenomic mrnas thereafter. the subgenomic negative-strand rna can be used for either uninterrupted transcription or leader-primed transcription to generate positive-strand subgenomic rnas. a combination of these models would be consistent with most of the experimental data. this twostep model of primary and secondary transcription (jeong and makino, ) may explain the apparent differences in the possible mechanism of transcription between early and late stages of viral infection. because of the large size of coronavirus rna, no infectious cdna or rna clones are now available for reverse genetics studies. this difficulty has hampered progress in the study of the molecular biology of coronaviruses. di rnas of several coronaviruses (see section vi,e) have been molecularly cloned and used as a substitute for the genomic rna to study the cis-and trans-acting signals involved in viral rna synthesis. although natural di rnas do not contain an mrna start signal and, consequently, cannot transcribe an mrna, the insertion of such a signal into the di rna allows an mrna to be transcribed from the transfected di rna in the virus-infected cells, thus enabling studies of the regulatory sequences for transcription. following is a summary of information that has been obtained using this approach. it should be cautioned, however, that regulation of rna transcription probably depends on overall rna conformation and that the cis-acting sequence required for rna synthesis very often varies with the di rna vector used; therefore, the results obtained from di rna studies may not be directly applicable to the viral genome. a full understanding of the regulation of viral rna synthesis still awaits the development of an infectious cdna clone. the following cis-acting signals for coronavirus rna transcription have been determined primarily from mhv di rna studies (with some from bcv di) (fig. ) . a. ig sequence. the ig sequence can be considered to be the promoter element for transcription. it also serves as the mrna start site and the site of fusion between the leader rna and body sequence of mrnas. a seven-nucleotide core sequence, ucuamc, is sufficient to initiate mrna synthesis (makino et al., ) . extensive site-specific mutagenesis studies have shown that most of the single-nucleotide mutations within this core sequence could be tolerated, although the transcription efficiency of some of these mutants was lower (joo and makino, ; van der most et al., ) . these seven nucleotides represent the minimum promoter; deletion of a nucleotide results in complete ablation of mrna transcription. the effects of the sequences near the promoter on transcription are contradictory: in certain situa- tions, the nature of the neighboring sequences did not affect transcription (makino and joo, , but under other circumstances, it did (jeong et al., ) . thus, the strength of the promoter appears to depend on the context of the overall rna sequence and structure. the relative flexibility of sequence requirement of the promoter sequence in the di rna system appears to differ significantly from that seen in the viral genomic rna. in the mhv genome, there are more than stretches of sequence resembling the ucuaaac sequence, in addition to the six promoters for the known subgenomic mrnas (joo and makino, ). yet, most of these did not promote mrna synthesis from the viral rna genome to any appreciable extent, in contrast to their ability to promote transcription in the di rna vector system (joo and makino, ) . in the viral genome, the single-nucleotide substitution of a g residue in the core promoter sequence completely abolished mrna synthesis (shieh et al., , whereas this is tolerated in the di rna (joo and makino, ) . thus, there appear to be significant differences between the sequence requirement for mrna synthesis in the di rna and in the natural viral genomic rna. when there are multiple ig sequences in the di rna, the order of the ig sequences may influence transcriptional efficiency. an ig located at the ' end generally has an advantage in initiating mrna synthesis (van marle et al., ; krishnan et al., ) . the sequences near the igs may suppress transcription (jeong et al., ) . b. the leader sequence at the ' end of the dz rna. the leader sequence at the ' end of the viral genomic rna becomes the leader sequence of subgenomic mrnas; thus, it fills a structural role for mrna synthesis. however, the leader rna of the subgenomic mrnas is not derived exclusively from the leader rna of the same (di) rna; in fact, most are derived in trans from a separate rna molecule, such as helper virus rna (jeong and makino, ; liao and lai, ; zhang et al., ) . nevertheless, mrna transcription from an ig site in the di rna still requires the presence of a leader rna sequence at the ' end of the di rna as a cis-acting sequence . deletion of this cis-acting leader abolished transcription. furthermore, the sequence of this leader rna, particularly its ' end sequence, can affect the efficiency of transcription from certain ig sequences on the di rna (zhang et al., ) . for example, the leader rna containing two pentanucleotide (ucuaa) repeats transcribes an mrna from the ig - site more efficiently than the leader rna with three ucuaa repeats. thus, the cis-acting leader rna plays a role similar to that of an enhancer. these findings suggest that the leader rna serves two functions : ( ) it supplies the leader rna to the subgenomic mrnas, and ( ) it serves as an enhancer-like sequence to regulate transcription. this finding also suggests that there is either a direct or an indirect interaction between the leader and ig sequences. some additional sequences downstream of the leader may also enhance transcription from an ig site in the di rna ; however, the precise sequence requirement is not known. this sequence requirement shows some virus sequence specificity, since it cannot be replaced with other viral rna sequences . it may be needed to maintain overall rna conformation for the recognition of the ig sequence. c. the ' utr. in an mhv di rna construct, partial deletion of the ' utr completely abolished transcription from an upstream ig site in the di rna (lin et al., ) . this stretch of ' utr is probably involved in positive-strand rna synthesis, since the length of this required sequence ( nt) is significantly longer than that required for negative-strand rna synthesis ( nt). the ' utr requirement for mrna transcription is surprising, since positive-strand rna synthesis starts from the ' end; thus, the ' end sequence is the last to be transcribed. this ' utr sequence requirement is similar to that for rna replication (kim et al., b; lin and lai, ) (see section v,f). this finding suggests that the ' end may interact with the ' end and possibly with ig sequences during transcription. a nine-nucleotide sequence, uuuauaaac. this sequence, located immediately downstream from the ucuaa repeats at the ' end of the leader rna in the viral genome, plays a significant role in rna transcription. it is deleted from the genome of one of the mhv strains and is often deleted in naturally occurring di rnas . in this particular mhv strain (mhv- , the leader-mrna fusion sites are very heterogeneous and do not always occur at the usual ucuaaac sites (zhang et al., ) . this nine-nucleotide sequence can serve as an mrna start signal, allowing transcription of an almost genomic-length mrna . in the di rnas, the presence or absence of this nine-nucleotide sequence influences transcription efficiency from the downstream ig site and, most importantly, affects the source of the leader rna incorporated into subgenomic mrnas (zhang et al., b) . when this nine-nucleotide sequence is present, the leader sequence in the subgenomic mrnas is contributed both from the di rna in cis and from helper virus rna in trans. when this sequence is missing, the leader rna is derived exclusively from the helper virus rna (zhang et al., b) . thus, this nine-nucleotide sequence appears to regulate the mechanism by which the leader rna is fused t o the subgenomic mrnas. these results combined suggest that multiple rna regions are involved in the regulation of mrna transcription. however, a recent study appears to contradict the need of cis-acting sequences other than the igs for mrna transcription. when a negative-strand rna containing only an ig sequence of tgev and a reporter gene was transcribed in situ from a transfected cdna by using a recombinant vaccinia virus-t rna polymerase expression system, this rna was transcribed in the presence of tgev, generating an mrna with a correctly fused tgev leader sequence (hiscox et al., b) . the leadercontaining mrna could have been generated by either of the transcription mechanisms described (section v,e, ,a or section v,e, ,b) above. this study suggests that this negative-strand ig site is sufficient for transcription. however, it is possible that this activity represents a basal level of transcription and that other cis-acting sequences may enhance the efficiency of transcription. the application of inhibitors of protein synthesis at any time during the viral life cycle inhibits viral rna synthesis, suggesting that continuous protein synthesis is required for rna synthesis sawicki and sawicki, ) . a similar observation has been made using an inhibitor of cysteine proteases, which inhibits a specific step of the processing of gene l a products of mhv (see section v,g, ), suggesting that continuous production of polymerase is required for viral rna synthesis. the precise nature of the viral proteins involved has yet to be determined. temperature-sensitive mutants of mhv that are defective in rna synthesis at the nonpermissive temperature have been divided into at least five complementation groups, indicating that at least five proteins are involved in viral rna synthesis (leibowitz et al., a; baric et al., ) (see fig. ). all of these complementation groups are mapped within the gene region (including both l a and lb). sequence analysis showed that gene l b contains an rna polymerase motif (gorbalenya et al., ; lee et al., ) . polymerase activities have been demonstrated in membrane fractions of bcv-and mhv-infected cells dennis and brian, ) , and several in vitro rna synthesis systems have been reported (compton et al., ; baker and lai, ) ; however, the nature of polymerases in these systems has not been identified. in one study, it was demonstrated that the antibodies against the n protein could inhibit rna synthesis, suggesting that n protein may be involved in rna synthesis (compton et al., ) . in addition to viral proteins, cellular factors may also be involved in rna synthesis. several cellular proteins have been shown to bind to the regulatory elements of mhv rna, including the ' and ' ends of the genomic rna and the ' end of the negative-strand rna and ig sites yu and leibowitz, a,b; zhang and lai, ) . the binding sites for the cellular proteins at the ' end of genomic rna and the ' end of negative-strand rna are complementary . the protein p , which binds to the negative-strand leader sequence and the ig site, is particularly interesting. site-specific mutations of the ig site affected the binding of this protein and the efficiency of rna transcription to the same extent, suggesting that the binding of this protein is required for rna transcription . this protein recently has been identified as hnrnp a (h.-p. li and m. m. c. lai, unpublished observation) . the mutations at the ' end of the viral genomic rna that abolished the binding of cellular proteins also inhibited both negative-strand and positive-strand rna synthesis, although the correlation between protein binding and rna replication was not absolute (yu and leibowitz, a) . thus, cellular proteins probably play a significant role in viral rna replication and transcription. curiously, viral proteins in the infected cell extract could not be cross-linked to the viral rna in vitro, suggesting that viral proteins may interact with viral rna only indirectly through cellular proteins. this is in contrast to the finding that the purified n protein can bind to the leader rna sequence in vitro stohlman et al., ) . the reason for this discrepancy is not clear. the genomic-sized rna in coronavirus-infected cells theoretically consists of two populations: the messenger rna (mrna l), which is translated to yield gene l a and l b products, and the genomic rna, which is destined to be packaged into virion. early studies demonstrated that, late in the infection, most ( %) of the genomic-sized rna in the cells was associated with the viral nucleocapsid, while the remainder ( %) was present in polysomes perlman et al., ) . presumably, early in infection, most of the genomic-sized rna would be associated with polysomes to serve as mrnas for the synthesis of polymerase; however, this has not been demonstrated. it is not clear whether there is any difference in structure and mechanism of synthesis between these two rna populations. since genomic rna requires uninterrupted synthesis from the fulllength template, whereas mrnas involve discontinuous transcription, the two types of genomic-sized rna (mrna and virion genome rna) may be synthesized by two different mechanisms. a recent study suggests that at least some of the mhv genomic-sized rnas are indeed synthesized by a discontinuous transcription, using the u c u m repeat in the leader rna and the nine-nucleotide uuuauaaac immediately downstream of the leader rna as the transcription start site . this raised the possibility that mrna and virion genomic rna are distinguishable. however, it cannot be inferred from this study that the fate of the genomic-sized rna products derived from discontinuous transcription is different from the fate of those derived from uninterrupted rna synthesis. the possible involvement of discontinuous transcription in generating genomic-sized rna may explain several interesting findings regarding mhv genomic rna . the copy number of the ucuaa repeat in the leader sequence of the genomic rna, which ranges from two to four copies in different mhv strains, rapidly evolves during virus passage (makino and lai, a; la monica et al., ) . starting with a pure virus population, the copy number in the viral genomic rna rapidly becomes heterogeneous during serial passages in tissue culture, and a new virus population with a different copy number of ucuaa repeats emerges (makino and lai, a) . since this sequence variation is seen in the leader region but not in the ig regions, where uninterrupted rna synthesis probably occurs, this finding is best explained by the discontinuous transcription mechanism involving the ' leader region. the imprecise fusion of the leader rna to the mrna start sites would result in heterogeneity of the copy number of the ucuaa repeats lai, ) . such heterogeneity is not observed when the virus, e.g., bcv, contains only one ucuaa copy in the leader rna (hofmann et al., a) . the ucuaa region at the ' end of the genomic rna is a hot spot of rna recombination during mixed infection of mhvs, resulting in recombinant mhvs with a crossover site at the ' end of the leader rna sequence . this result is best explained by the discontinuous rna synthesis at the ' end of the genomic rna. . if the generation of di rnas is viewed as an anomaly of rna replication, the structure of naturally occurring di rnas reveals an insight into the mechanism of rna replication. most of the naturally occurring mhv di rnas have a copy number of the ucuaa repeat different from that of the parental virus, and most lack a ninenucleotide sequence downstream of the ucuaa repeats . as discussed above, this is a reflection of discontinuous transcription in the region. the understanding of the mechanism of rna replication has been aided by the use of in uitro-transcribed di rna generated from cloned cdna. when di rna was transfected into virus-infected cells, the leader rna was rapidly replaced by that of the helper virus (makino and lai, b; . this leader exchange is dependent on the presence of the nine-nucleotide sequence (uuuauaaac) in the di rna (makino and lai, b) , consistent with the finding that this sequence serves as an mrna start signal for discontinuous transcription . the use of the cloned di rna also allowed the determination of the cis-acting signals for rna replication (kim et al., ; lin and lai, ) . it was shown that more than nucleotides at both the ' and ' ends of the di rna are required for rna replication, and that some mhv di rnas also required a stretch ( nt) of internal sequence in the gene region for rna replication; however, the requirement for the internal sequence was not observed in other mhv or bcv di rna constructs (chang et al., ; luytjes et al., ) . thus, this internal sequence probably plays a role in maintaining the overall rna conformation for some di rnas (y. n. . again, the requirement of a ' end sequence ( nt) that is longer than that required for negative-strand rna synthesis ( nt) is a surprise. these ' end sequences are probably required for positive-strand rna synthesis during rna replication. this finding is reminiscent of the sequence requirement for rna transcription discussed above and suggests that there is a direct or indirect rna-rna interaction between the ' and ' ends during rna replication. these di rna studies also showed that replication of di rna is inhibited when an mrna is transcribed from an ig site within the same di rna, and that the mechanism of inhibition is due not to competition for the same transcription machinery (jeong and makino, , but most likely to the overlap of the cis-acting signals for these two different processes. however, the sequence requirements for replication and transcription are different, indicating that these two processes are distinguishable. the mrna transcription and genomic rna replication may be regulated by the same mechanism throughout most of the viral replication cycle. however, the ratio between the genomic rna and subgenomic rnas, as detected by radioactive uridine incorporation, increases during the late stages of the bcv replication cycle (keck et al., a) , suggesting a possible switching mechanism from transcription to replication. it has been shown that genomic rna replication is coupled to encapsidation, since no free genomic rna is found . since the encapsidation of rna requires the n protein, this protein may participate in the regulation of switching between transcription and replication. the sequences of coronavirus mrnas usually start from a site immediately upstream of a gene. these mrnas, except for the smallest mrna, are structurally polycistronic, containing multiple orfs. only the ' most orf in the mrnas is translatable; the remaining orfs are usually functionally silent. thus, most of these mrnas are functionally monocistronic (see fig. ) . the s, he, m, and n proteins, and in most coronaviruses the e protein, are translated from separate mrnas by this mechanism; initiation of their translation is unremarkable, utilizing a cap-dependent translation mechanism. many ns proteins, however, are translated from truly polycistronic mrnas, i.e., two or three proteins are translated from the same mrna. for these mrnas, the first orf, e.g., a of ibv or a of mhv, is probably also translated by the same mechanism as the structural protein genes. for internal orfs, e.g., e protein of ibv and mhv, an alternative mechanism must be employed to initiate translation internally. one characteristic of coronavirus mrnas is the presence of the leader rna sequence at the ' end, which not only participates in rna transcription, but also regulates the efficiency of translation. it has been shown that the presence of the mhv leader sequence on a heterologous mrna in a chimeric rna construct can enhance its translation in virus-infected cell lysates but not in uninfected cell lysates (tahara et al., ) . this effect conceivably will enable the efficient translation of viral mrnas in the face of shutoff of translation of cellular mrnas in the infected cells (siddell et al., ; hilton et al., ) . the mechanism of translational enhancement by the leader rna has not been determined. it has been shown that during persistent infection of bcv, the leader rna sequence underwent frequent mutations (hofmann et al., b) . one of these mutants had an intraleader short orf and a lower translation efficiency, indicating that the leader sequence in-deed can modulate translation. another region which can potentially regulate the translation of coronavirus mrnas is the ' utr (other than the leader sequence) of mrnas. the genomic rna (mrna ) has a particularly long ' . an mhv with a specific point mutation within the ' utr was selected during persistent infection in uitro (chen and baric, ) . this mutant had a significantly higher translation efficiency than the wild-type virus. different subgenomic mrnas had ' utr of various lengths, which may also affect their translation. for the translation of internal orfs, several different mechanisms are used by coronaviruses: a. ribosomal frameshifing within the polymerase gene. all of the coronavirus genes (polymerase) sequenced so far contain two overlapping orfs. several features of the ibv polymerase gene sequence , coupled with the absence of a distinct mrna for orf lb, suggested that translation of orf l b involved ribosomal frameshifting from orf la, thus synthesizing a large polyprotein containing both l a and l b sequences. subsequently, a highly efficient ( % frequency) - frameshift was demonstrated experimentally in uitro (brierley et al., ; somogyi et al., ) and in uiuo (brierly et al., ) . this mechanism has been shown to operate in gene of mhv, hcv- e, and tgev as well (bredenbeek et al., ; lee et al., ; eleouet et al., ) . in all cases, the mechanism involves two essential elements: a slippery site followed by an rna pseudoknot (brierley et al., ) . the site at which the ribosome slips backward has the sequence uuuaaac. the pseudoknots of ibv and mhv are similar, comprising two base-paired regions stacked coaxially in a quasi-continuous manner and connected by two singlestrand loop regions. the hcv- e pseudoknot is more complex . it is the overall shape and stability of the pseudoknot that are important, not the nucleotide sequence per se. two reasons have been put forward to explain why coronaviruses should employ ribosomal frameshifting to translate orf l b (brown and brierly, ) . one reason is that this is done primarily to control the relative amounts of the l a and l b products. that could be achieved in other ways, of course, e.g., by translating orf l b from a separate mrna, this will require that the transcription of l a and l b mrnas is tightly regulated. the other reason may be to avoid making a l b mrna. such an mrna might be packaged into virions in competition with genomic rna, as the rna region corresponding to the l b orf of mhv contains a sequence that is essential for packaging into virions (fosmire et al., ) (see section vi,e,l). mrna. the e proteins of ibv and mhv are encoded by the third and second orf, respectively, of the corresponding genes and (fig, ) . cells infected with ibv contain the products of all three of the gene orfs (liu et al., ) . both of the mhv gene orfs are translated in vitro (budzilowicz and weiss, , but only the b orf product has been detected in virus-infected cells . experiments have shown that the e protein orf of both ibv and mhv mrnas is translated by a cap-independent, internal ribosomal entry mechanism (liu and inglis, b; thiel and siddell, ) . furthermore, if the a and b orfs were eliminated from the ibv mrna, translation of the c (e) orf did not occur (liu and inglis, b) . this suggested that the d b region contains an internal ribosome entry site (ires) for the e protein orf. le and colleagues have predicted the existence of secondary structures in the d b region of ibv which resemble the ires elements of picornaviruses (le et al., ) . they predicted a -nucleotide sequence in d b which would fold into five stem-loops, forming a compact structure by the interaction of two pseudoknots. c. translation of nonstructural proteins. in addition to the ns proteins encoded from the '-most orfs of mrnas, several other ns proteins are encoded from an internal orf of some viral mrnas, e.g., b of ibv and hcv- e, b of bcv, and b of fcv (fig. ) . most of these products have been detected in virus-infected cells; however, the mechanism of the internal initiation of translation has not been elucidated. bcv and mhv rna contains an additional internal orf within the n protein gene. this orf (termed i) is in a different reading frame from that of n protein and encodes a hydrophobic protein (senanayake et al., ; fischer et al., ) . this protein is translated in virusinfected cells by a leaky ribosomal scanning mechanism from the bicistronic mrna of n gene (senanayake et al., ) . it is a nonessential gene. the mechanism of its regulation is not yet clear. the gene product is predicted to be nearly - kda. it is probably processed into multiple proteins posttranslationally by its own proteases. the processing pathway has just begun to be explored. remarkably, the protease domains and potential cleavage sites predicted by computer analysis (gorbalenya et al., ; lee et al., ) have largely been confirmed by experimental data. initially, in vitro translation of virion rna of mhv revealed several polypeptides of more than kda (leibowitz et al., b; denison and perlman, ) . in addition, a -kda product was detected and shown to have originated by cleavage from the n terminus of a precursor (denison and perlman ; soe et al., ) , now known to be the beginning of the orf l a polyprotein (fig. ) . the cleavage which generates p is carried out by plp (fig. ) . it cleaves between residues gly- and val- , mutation of either residue resulting in almost no cleavage (dong and baker, ; hughes et al., ) . in addition to p , the mhv orf l a encodes a protein of more than kda, which is cleaved to a -kda product, which, in turn, is cleaved to produce a -kda and a -kda product (denison et al., ) (fig. ) . another protein of kda is derived from sequence immediately downstream of the p -encoding region, thus representing the n-terminal part of the large polyprotein initially found in in uitro translation (probably more than kda) (fig. ) . the cleavage of p from the polyprotein was also carried out by plpl (bonilla et al., , . inhibition of the c-terminal cleavage of p by e d, an irreversible inhibitor of cysteine (thio) proteinases, inhibited mhv replication . in addition, the clp domain is cleaved from the polyprotein by the autocatalytic cleavage activity of clp itself to generate a - kda protein, which contains both the trans-and cis-acting proteolytic activities (lu et al., , liu and brown, ; ziebuhr et al., ) . e d also inhibited the clp protease activity. the processing pathway of the l b protein sequence is less clear. there is experimental evidence with ibv and hcv- e that the l b polyprotein is cleaved in trans by the clp encoded by orf l a (liu et al., ; ziebuhr et al., ; grotzinger et al., ) . a polypeptide of approximately kda, representing the extreme c terminus of orf l a and the n terminus of the frame shifted orf lb, was immunoprecipitated from ibv-infected cells. the cleavage sites of the -kda protein appear to be at the q/s sites, as predicted from the computer analysis and consistent with the known substrate specificity of the picornavirus c protease. a similar observation was recently made with hcv- e (grotzinger et al., ) . this -kda protein contains the putative rna polymerase motif and thus may represent the coronavirus polymerase. the coding region for this protein belongs to complementation group d, which has been shown to effect mrna transcription (fig. ) . b. processing of the structural proteins the s protein is co-translationally glycosylated with nlinked glycans. conversion of the high mannose (simple) glycans of the s protein to complex ones is a slow process, the half-life being one to several hours (vennema et al., a) . the s protein undergoes multiple disulfide linkages to fold into a more complex structure (opstelten et al., ) and oligomerize into a trimer in the golgi complex (delmas and laude, ) . the s prepropolypeptide is converted to a propolypeptide by removal of the n-terminal signal peptide. whether the propolypeptide is cleaved to generate s and s depends on the virus species and strain and, to some extent, on the cell type in which the virus is grown (frana et al., ) . the sl-s cleavage site in ibv and mhv is adjacent to several basic residues (cavanagh et al., a; luytjes et al., ) . those coronaviruses whose s protein is not cleaved, e.g., fcv, tgev, and ccv, have no such pairs of basic residues. cleavage of the mhv s protein occurred after conversion of the glycans from simple to complex forms (vennema et al., a) . after cleavage, the s and s subunits are held together by noncovalent linkages (cavanagh et al., b; sturman et al., ) . the s protein of mhv is acylated, possibly involving some of the many cysteine residues in the c-terminal, hydrophilic tail of s (schmidt, ; sturman et al., ; van berlo et al., ) . the processing of s proteins is reviewed in greater detail by cavanagh . modification of the m protein depends greatly on the virus species. the major modification is glycosylation. the oligosaccharides of ibv and the tgev group are of the co-translationally added n-linked glycans (stern and sefton, b) . the conversion of the high mannose to complex glycans is not very efficient. in contrast, viruses of the mhv group have o-linked glycans which are added posttranslationally (holmes et al., ; niemann et al., ; ; tooze et al., ; locker et al., a; krijnse-locker et al., ) . the m protein of tgev is also sulfated (garwes et al., , but whether this is linked directly to the polypeptide or to glycans is unknown. unlike the m proteins of ibv and the mhv group, which have an internal membrane insertion sequence, those of the tgev group have an n-terminal membrane insertion sequence that is absent from the mature m protein (laude et al., ) . this signal sequence, however, is not an essential requirement for the membrane insertion of the m protein (kapke et al., ; vennema et al., ) . . he protein. the he glycoprotein has n-linked glycans which are converted to complex ones in the golgi complex. the n-terminal signal sequence is cleaved from the mature protein, which then forms dimers by disulfide bonds (king et al., ; hogue et al., ; kienzle et al., ; yo et al., ) . of mhv e protein (yu et al., ) . however, this was not observed for the e protein of tgev when expressed in insect cells (godet et al., ) . . nprotein. the n protein is phosphorylated, the phosphate linkage being exclusively to serine residues (stohlman and lai, ) . the role of phosphorylation is unknown. in virus-infected cells, the assembly of virus particles presumably starts with the formation of rnp, which interacts with the components of viral envelope proteins to form enveloped virus particles and bud into the endoplasmic reticulum (er) and golgi complex. several recent advances shed light on this process: early studies have shown that the s proteins are not necessary for virus particle formation; thus, denuded virus particles without spikes can be formed in the virus-infected cells treated with tunicamycin, which inhibits n-glycosylation and transport of the s and he proteins (holmes et al., ) . further, recent studies have shown that the minimum requirement for the formation of viruslike particles (vlp), i.e., empty virus particles, is the m and e proteins (bos et al., ; vennema et al., ) ; . the sites of virus budding are in the er and golgi, near the sites of accumulation of the m protein (dubois-dalcq et al., ; tooze et al., ; tooze and tooze, ; klumperman et al., ) ; thus, the interaction between the m and e proteins appears to be the key event for virus particle assembly. the incorporation of the nucleocapsids and s and he proteins into virus particles may involve subsequent interactions of these components with the m-e complex. the virus assembly and release process has been studied in most detail for mhv (j. tooze et al., tooze et al., , tooze and tooze, ; s. a. tooze et al., ; krijnse-locker et al., ) , and the gross features have recently been confirmed for ibv, tgev, and fipv . recently, an ultrastructural study of the replication of ibv in renal ductotubular epithelial cells of infected chicks has also been very informative (chen and itakura, ) . the first virions form in the perinuclear region, in small, smooth vesicles/ tubules between the rough er and the cis side of the golgi stack. later, the rough er becomes the major site of virion assembly, extending beyond the perinuclear region. virions then proceed through the golgi complex, at the trans side of which they are collected into vesicles of the constitutive exocytic pathway and subsequently released from the cell. the major determining factor for the site of virus assembly appears to be the site of localization of the m protein, which is in the golgi complex. there are some points of difference among the coronaviruses. when the m protein of mhv was expressed, it accumulated in the trans-golgi membranes, consistent with its o-linked glycosylation, which occurs efficiently (locker et al., a; klumperman et al., ) . in contrast, expression of the ibv m protein from cdna resulted in its accumulation in cis-golgi membranes; consequently the highmannose n-linked glycans of the m protein were not efficiently converted to complex ones (machamer et al., ; klumperman et al., ) , in agreement with the properties of the m protein in the ibv virions. glycosylation of the coronavirus m protein is not essential for its translocation or for virus particle formation. the m protein exists as monomers in the er, but it oligomerizes to form variously sized complexes during transport through the golgi and trans-golgi network (locker et al., ) . it is likely that the m molecules in the virus particles are in complexed form. the sequence requirements for insertion of the nascent m polypeptide into the rough er have not been precisely defined. with the exception of the tgev group, the coronavirus m proteins do not have an amino-terminal signal peptide. even in the case of the tgev group, the signal peptide is not essential for membrane insertion of the m protein (kapke et al., ; vennema et al., ) . rather, one of the three transmembrane sequences of the m protein is responsible for the insertion of m into the er and its final localization in the golgi complex (machamer and rose, ; mayer et al., ; armstrong et al., ; locker et al., ) . different domains of the m protein of ibv and mhv have been identified as the sequences responsible for the final localization of the protein. the first membrane-spanning domain of the ibv m protein performs this function, the m protein being concentrated in the cis-golgi membranes (machamer and rose, ; machamer et al., swift and machamer, ) . in contrast, the carboxyterminal domain of the mhv m protein, probably in combination with a middle domain, directs the protein to the trans-golgi (armstrong and patel, ; weisz et al., ; krijnse-locker et al., ) . it should be borne in mind, however, that the major site ofvirus particle formation is proximal to either of the golgi compartments, namely, in an intermediate compartment between the er and the golgi complex . thus, it is proximal to the major site of m accumulation. what is responsible for that? the answer would appear to be that the s glycoprotein and the nucleocapsid interact with the m protein molecules before the m proteins have migrated to the golgi, precipitating virus particle formation. it has been shown that the coronavirus m protein can interact with nucleocapsids (sturman et al., ) . this interaction requires the presence of viral rna, since the n protein alone cannot be incorporated into the vlps (bos et al., ; vennema et al., , suggesting either that m interacts with viral rna, or that rna-n protein binding induces a conformational change in the n protein, enabling it to interact with m. interaction between the m and s proteins has also been demonstrated. the m and s proteins co-sediment under certain ionic conditions after dissolution of virions with mild detergents (cavanagh, b) , and cell-associated complexes containing m and s have been detected (opsteltenet al., ) . the s protein undergoes certain conformational changes induced by disulfide linkage before it is able to interact with m (opstelten et al., . inhibition of correct oligomerization of s by dithiothreitol prevented interaction of s with m and, as a result, the rate of transport of the m protein to the trans-golgi increased (opstelten et al., ) . this result suggests that s-m interaction can retard the transport of the m protein. the ability of the s or he protein to interact with the m protein appears to be a prerequisite for their incorporation into virus particles. in this regard, it is interesting to note that mhv ts mutants with a deletion in the ectodomain of the s protein or those with defects in oligomerization of the s protein do not incorporate the s protein (ricard et al., ; luytjes et al., ) . also, partial deletions in the ectodomain of the he protein prevent its incorporation into virus particles . these results suggest that the interaction of s or he with m occurs through the ectodomain or requires the correct protein conformation in the ectodomain. the formation of the s-m complex occurs in the pre-golgi complex, whereas the s-m complex progresses until the golgi complex, indicating that this interaction is not sufficient to localize it in the pre-golgi complex, the ultimate site of virion budding . thus, m-nucleocapsid interaction may also contribute to the determination of the site of virus assembly. in this regard, it is important to note that the recent discovery that m is present in the viral rnp core, as well as in the envelope (risco et al., ) may further indicate the crucial role of the m protein in the virus assembly process. only the m and e proteins are required for the production of vlps (bos et al., ; vennema et al., ) . these particles were formed when the m and e proteins were expressed from transfected plasmids. s protein was incorporated into the vlps if expressed. in the absence of viral rna, the n protein also was not incorporated. when all the structural proteins were expressed from plasmids in the presence of an mhv di rna, which contains a packaging signal, and in the absence of helper virus, the vlps contained the di rna (bos et al., ) . moreover, these vlp were "infectious," i.e., on transfer of the released vlps to a new cell culture, they were able to infect the cells, as revealed by the rescue of the di rna by helper virus. these results show that n is dispensable for the formation of vlps but the packaging of rna into virion requires an interaction between m and the n-containing ribonucleoprotein, as previously demonstrated (sturman et al., ) . the expression of the m protein alone in the cells did not lead to vlp formation or induction of curvature in the m-containing intracellular membranes. the presence of the e protein together with the m protein triggered both events, but the ratio of m:e in virions was as high as oo:l (vennema et al., ) . this has led to the suggestion that e does not have frequent, regular positions in the lattice formed by m but rather occupies strategic positions within the lattice to cause membrane curvature. alternatively, its role may be to close the neck of the virus particle as it pinches off from the membrane in the final stage of budding. what determines the site of virion budding? it is possible that the e protein dictates the site of budding, since this protein is also localized in the perinuclear region and associated with membrane (godet et al., ; yu et al., ) . alternatively, it may be the interaction of the rnp-nucleocapsid with the s-m complexes which halts the migration of the latter and promotes budding. relevant to this notion is the observation that the nucleocapsids and free n protein have affinity for membranes (anderson and wong, ) . it should be remembered, however, that in the absence of s, he, and nucleocapsids, the e and m proteins alone can induce budding to form vlps (bos et al., ; vennema et al., ) . it is not yet clear whether the budding site of vlp containing only m and e is the same as that for the complete virion. empty virus particles have previously been isolated from ibv, which were grown in embryonated fowl eggs (macnaughton and davies, ) . this supports the view that even during natural infection, virus budding can be induced without involvement of the viral nucleocapsid. parallels have been drawn between the e protein of coronaviruses, the m protein of orthomyxoviruses, and the k protein of alphaviruses. all are minor envelope proteins that play a role in virus assembly. once the virus particles bud into the pre-golgi compartment, they are transported through the golgi complex. whether the golgiassociated posttranslational modifications occur before or after incorporation of the proteins into virus particles is not known. retrograde transport of the proteins may be required for some steps of the virus assembly process. finally, the release of virus particles from the cells appears to be restricted to certain areas of cells. tgev grown in polarized llc-pk cells both enter and exit by the apical surface (rossen et al., ) , whereas mhv-a enters polarized murine kidney cells (mtal) by the apical surface but is released via the basolateral surface (rossen et al., a) . however, the site of virus release varies with different cell lines (rossen et al., ) . the factors governing this process are not known (rossen et al., b) . probably because of the large size of their rna genomes, coronaviruses have developed a variety of genetic mechanisms, among which are rna recombination and generation of di rna, to maintain their genetic stability and, as a side product, generate diversity. coronaviruses also readily undergo genetic mutation, a characteristic common to all rna viruses. thus, they evolve rapidly and are heterogeneous. these genetic phenomena provide virologists with useful tools for understanding coronavirus biology, particularly because reverse genetics studies for coronaviruses are not yet feasible. using a variety of chemical mutagens, several laboratories have isolated mhv temperature-sensitive (ts) mutants which cannot produce infectious virus particles or cause different plaque morphology at the nonpermissive temperature (haspel et al., ; robb et al., ; wege et al., ; koolen et al., ; schaad et al., ) . some of these mutants have been characterized with respect to their ability to synthesize rna and have been grouped into at least seven complementation groups (leibowitz et al., a , five of which have the rna (- phenotype (i.e., cannot synthesize rna at the nonpermissive temperature) (leibowitz et al., a; schaad et al., ) (see fig. ). with the use of recombination analysis (see below), the possible genetic defects of the mutants were mapped on the rna genome fu and baric, ) . it appears that all of the rna (-) mutants have genetic defects within gene , suggesting that gene encodes rna polymerase and other proteins involved directly or indirectly in viral rna synthesis. the genetic defects of some of these mutants have been confirmed by rna sequence analysis of the mutants and their revertants . these five different complementation groups have been demonstrated to affect different steps of rna synthesis, including the synthesis of leader rna, negative-strand rna, and positive-strand rna (fig. ) , suggesting that different steps of rna synthesis require different viral proteins . it is still not possible, however, to correlate the genetic defects definitively with the known processed products of the gene polyprotein. among the rna (+) mutants, two complementation groups have been assigned to the gene encoding the s protein fu and baric, , but the phenotype of these mutants has not been well characterized. another rna (+) mutant, alb , has a single amino acid substitution in the n-terminal domain of s protein and cannot incorporate s protein into the virus particles (ricard et al., ) . still another group of rna (+) mutants have a defective n protein (koetzner et al., ; masters et al., ; peng et al., a) and produce smaller plaques at the nonpermissive temperature; several of these mutants have a deletion in the n gene (masters, ) and are defective in rna-binding activity (peng et al., a) . most wildtype revertants have a second-site mutation in the n protein and restored rna-binding activity (peng et al., a) . another class of viral mutants was obtained by a specific selection scheme, e.g., by treating viruses with neutralizing mab and selecting mutant viruses resistant to neutralization. since neutralizing antibodies are usually directed against the s protein, all of the neutralizationescape mutants were presumed to have defects in the s gene. this was indeed the case (reviewed by . depending on the neutralizing mab used for selection, the mutants obtained had either deletions or point mutations in the neutralization epitopes of the s protein (gallagher et al., ; wang et al., ) . these mutants generally retain growth properties very similar to those of the parental virus but often have significantly different pathogenic properties with altered tissue tropism (dalziel et al., ; fleming et al., fleming et al., , wege et al., ) . during serial virus passages in tissue culture or in animals, coronaviruses often undergo various deletions or substitutions even in the absence of experimentally applied selection pressure. these genetic changes probably provide the emerging virus variants with evolutionary advantages under experimental conditions or in natural infection. the deletions occur most frequently within the s gene, particularly within a hypervariable region encoding the s subunit (s. e. wang et al., ) . in fact, some natural isolates of mhv have a deletion of - nucleotides in this region (fig. ) . similar deletions have been detected in virus variants during central nervous system (cns) infections of rats . in persistent infections of cultured cells of cns origin, viruses with point mutations or deletions in the gene encoding s protein are frequently selected (gallagher et al., ; gombold et al., ; rowe et al., ) . these viruses often have altered cell fusion and pathogenic properties. the most striking effect of deletions during natural virus infection is illustrated by the emergence of prcv from tgev. tgev causes epizootic enteric infection in pigs, resulting in a very high mortality rate in newborn pigs. an attenuated virus strain that is related t o tgev but infects only respiratory tissues was isolated in western europe in the early s (pensaertet al., ). an independent isolate of prcv was subsequently obtained in the united states (wesley et al., ) . both of these prcv isolates have similar extents of deletion in the n terminus of the s protein, in addition to smaller deletions in gene , which eliminates its expression (rasschaert et al., ; wesley et al., ; laude, ) . although it is not yet possible to link the changes in viral pathogenicity to the deletions in the s gene or gene , the tgev-prcv evolution illustrates the power of deletions in coronavirus evolution. different ts mutants with defects in different coronaviral genes have been demonstrated to complement each other. the available ts mutants of mhv have been divided into at least seven complementation groups, five of which have an rna (-phenotype (leibowitz et al., a) (fig. ) . it is worth noting that these five rna (-) complementation groups have been mapped in gene , which is translated into a polyprotein. the existence of five complementation groups within this gene indicates that this polyprotein is processed into at least five different proteins that function independently. it is not possible, however, to complement the genetic defects of a virus by expressing a wildtype viral protein from an exogenous vector. mixed infection with mhv and murine leukemia virus in tissue culture cells yielded a pseudotype mhv which contained a murine leukemia virus envelope protein and was neutralized by antibodies against both murine leukemia virus and mhv (yoshikura and taguchi, ) . this phenotypic mixing of viral proteins suggests the lack of a stringent requirement for a virus-specific spike protein for the formation of coronavirus particles. pseudotype formation of virus particles has also been achieved by expressing a viral protein, e.g., he protein, from a di rna vector (see section vi, e), which was incorporated into virus particles . one unique genetic feature of coronaviruses is their ability to undergo rna recombination at a very high frequency; this is particularly true of mhv, in which recombinant viruses containing parts of the genomic sequences of both parental viruses could be isolated at high frequency when two strains of mhv with defined genetic markers were co-infected into culture cells or animals. this genetic phenomenon was first discovered using two ts mutants of mhv . subsequently, many different recombinant mhvs were isolated (keck et al., , b makino et al., ) using a combination of selection markers, such as ts markers, resistance to neutralizing antibodies, and cytopathic effects (the ability of the virus to cause fusion). based on the distribution of the crossover sites on the viral rna genome, it appears that recombination can occur practically anywhere on the viral genome, although some combinations of virus strains favor selection of viruses with certain recombination sites (lai, ) . for example, between the mhv a and jhm strains, recombination occurs mostly at the ' end of the genome and rarely at the ' end. in contrast, recombination between the mhv- and jhm strains occurs readily a t the ' end . the most surprising finding with regard to mhv recombination is the extremely high frequency of recombination, which has been calculated to be nearly % for the entire mhv genome . this high frequency of recombination is reminiscent of the reassortment of segmented rna genomes in viruses such as influenza virus and reovirus. the recombination map for mhv is nearly linear, suggesting the random occurrence of recombination ; however, more careful analysis of the recombination frequency showed that there is an increasing gradient of recombination frequency (in the direction of '+ ') across the genome, suggesting that subgenomic mrnas, which represent preferentially the ' end sequences, may participate in rna recombination baric, , ) . recombination has now been demonstrated experimentally for ibv (kotier et al., ) and tgev (ballesteros et al., ) in embryonated eggs or tissue culture; however, the recombination frequency for these viruses has not been determined. recombination can provide a powerful tool for virus evolution. for example, in a study in which ts mutants of the a strain of mhv were co-infected with a wild-type jhm strain, the majority of the progeny viruses after a single passage were recombinants which contained the ' end of the a genome (makino et al., a) , suggesting that this recombinant virus has evolutionary advantages. recombination has also been demonstrated during virus infection in animals (keck et al., ) . similar to the situation in other rna viruses, coronavirus recombination probably occurs by a copy-choice mechanism (lai, ) . it has been shown that mhv rna synthesis normally pauses at certain sites on the rna genome (baric et al., ) . the nascent, incomplete rna transcripts may dissociate from the template rna and then rebind to the template to resume rna synthesis. when the nascent rna binds to a different template, the resumed rna synthesis will result in a recombinant rna. whether coronavirus recombination occurs more frequently at certain rna sites with more complex secondary structure is not yet known. when rna recombination was examined under nonselective conditions (by reverse transcription-polymerase chain reaction detection of the intracellular rna from virus-infected cells), recombination sites appeared to be random; only after serial passages did "hot spots" of rna recombination become apparent . this finding indicates that the recombination hot spots may be the result of selection. recombination has been detected during natural infections of coronaviruses, most notably ibv. sequence analysis of natural ibv strains has provided convincing evidence that some ibv strains are recombinants between different ibv strains; recombination sites have been detected so far in the ' half of the s gene and at the ' end of viral rna (kusters et al., ; cavanagh and davis, ; wang et al., wang et al., , jia et al., ) . thus, recombination is a natural evolutionary strategy for coronaviruses. rna recombination may also explain the difference in genome structure among different coronaviruses. for example, ibv contains an additional gene, gene (a nonstructural protein gene) inserted between gene m and gene n (fig. ) . this insertion could be the result of a recombination mechanism involving the consensus ig sequence, which provides a favored recombination site. since all of the coronavirus genes are flanked by consensus ig sequences, each gene can be considered a gene "cassette," which can be rearranged by homologous recombination involving the consensus ig sequence. a nonhomologous recombination event between coronavirus rnas and other virus or cellular rnas may also explain the gene insertions in some coronaviruses. for example, mhv and bcv contain an additional gene, he, which is similar in sequence to the he gene of influenza c virus (luytjes et al., ) . this gene may have been derived by recombination between a coronavirus and influenza c virus. comparison between genome structures of coronavirus and torovirus also suggests that several recombination events may have been involved in rearranging the order of several genes during the evolution of these viruses (snijder et al., ) . recombination has been demonstrated to occur between viral rna and a transfected rna fragment derived from the viral genome (koetzner et al., ; liao and lai, ) . since transfection of both the positive-and negative-strand rna fragments led to recombination, these results suggested that recombination can occur during both positive-and negative-strand rna synthesis (liao and lai, ) . recombination can also take place between di rnas and viral rna reciprocally, i.e., the viral rna sequence can be incorporated into di rna, and vice versa, during viral rna replication. the incorporation of a helper viral rna sequence into di rna accounts at least partially for the continuous evolution of mhv di rna species during serial passages in cultured cells (see the next section). this phenomenon also explains why some genetic markers in the di rna were rapidly replaced by the helper viral rna sequences during di rna replication kim et al., a) . on the other hand, the incorporation of di rna sequences into viral rna by recombination provides an important tool to introduce desired sequences into the viral genome. for example, when an mrna or di rna containing the n gene of mhv was transfected into cells infected with an mhv ts mutant containing a defective n protein, recombination occurred between the di rna and the wild-type viral rna, resulting in recombinant viruses which had a wild-type rna sequence derived from the transfected rna in place of the defective n gene (koetzner et al., ; van der most et al., ; masters et al., ; peng et al., a ). an mhv recombinant containing a chimeric n protein of bcv and mhv has also been derived by this rna recombination strategy (peng et al., ) . this targeted rna recombination promises to be a powerful tool. recombination is thus one of the most unique aspects of coronavirus biology. it can potentially provide a genetic mechanism by which coronaviruses maintain their sequence integrity. in view of the large size of the coronavirus rna, it is predictable that most of the viral rna molecules would contain mutations due to the high error frequencies of rna polymerases; recombination may provide a repair mechanism for the virus (lai, ). similar to most rna viruses, coronaviruses can readily generate di particles when viruses are passaged in tissue culture at a high multiplicity of infection. this has been demonstrated for mhv, ibv, and tgev. when mhv was serially passaged, different types of di rna appeared at different passage levels, suggesting that di rnas continue to evolve and that new dis have a selective advantage under the evolving cellular conditions . however, the ibv and tgev dis appear to be more stable (penzes et al., ; mendez et al., ) . the generation of di rnas is probably caused by polymerase jumping during rna replication or nonhomologous rna recombination. although no sequence homology exists at the fusion sites of different rna regions within the di rna, a high degree of potential secondary structure does exist at some of its rna fusion sites (makino et al., b) , which may have facilitated the pausing and template switching of rna polymerase during synthesis. if nonhomologous recombination is involved in generating di rna, it probably occurs between two different rna molecules because di rnas are generated only at high multiplicity of infection. recombination between an existing di rna and helper virus rna has been shown to contribute to the evolution of mhv di rnas during virus passages . the coronavirus di rnas can be grouped into three types. the first type is of nearly genomic size and is typified by dissa rna of mhv . this di rna is efficiently packaged into virus particles and contains several deletions in the viral genome, but it contains a functional gene , which encodes rna polymerase, and a functional gene , which encodes n protein. these two functional gene products are sufficient to support di rna replication (k. h. ; thus, this type of di rna can replicate without a helper virus (makino et al., a; k. h. kim and makino, ) . by definition, it is not a di rna, inasmuch as it is not defective in replication; however, because it is smaller than the genomic rna and is produced at a high multiplicity of infection, it is classified as a di rna. this type of di is unique to coronavirus. a -kb di rna has been described for tgev (mendez et al., , but whether it can replicate in the absence of a helper virus has not been examined. the second type di rna is typified by disse of mhv (makino et al., ) . this di rna is truly defective and can replicate only in the presence of helper viruses. it replicates very efficiently, but is poorly packaged into virus particles because it lacks a specific rna-packaging signal. this type of di rna typically contains both the ' and ' ends of the wild-type viral rna and one or several discontiguous regions of the wild-type rnas. because of the high efficiency of replication, this type of di can still be serially passaged in tissue culture for at least several passages, probably because a small amount of di rna can be nonspecifically packaged into the virion. the third type of di rna is represented by dissf of mhv-jhm (makino et al., ) and di-a of mhv a (van der most et al., ). it is similar to the second type but contains an rna-packaging signal and is thus packaged efficiently into virus particles. this type of di rna has been detected in ibv (penzes et al., ) and tgev (mendez et al., ) . a small di rna ( . kb) of bcv may also belong to this type , but whether this di rna can be specifically packaged into virion is not certain. all three types of di rnas contain an orf, which encodes a protein fused from two different viral proteins. this orf is not required for the replication of mhv di rna (liao and lai, ' ) ; nevertheless, mhv di rnas with a functional orf usually have an evolutionary advantage over those without one or with a smaller orf kim et al., a) . therefore, a di rna containing a short orf was often rapidly replaced by di rnas containing a longer orf that had been generated by recombination or mutation kim et al., a) . the translatability of the orf may be more important than the nature of the actual protein translated from this orf (van der most et al., ) , suggesting that translation of rna may facilitate rna replication. reduction of the orf of a n ibv di rna to just amino acids did not diminish its capacity to be replicated or packaged (penzes et al., ) . however, it has been shown for a bcv di rna that a bcv-specific n protein translated from the di orf (a cis-acting protein) is required for efficient di rna replication . the variation in the sequence requirement for rna replication of these di rnas may be related to their overall rna conformation. the significance of di rna in the biology and natural evolution of coronaviruses is not known. di rnas provide useful tools for studying the sequence and structural requirements for various functions of viral genomic rna. as they contain cis-acting signals for rna replication, they are mini-versions of the viral genomic rna. however, it should be cautioned again that because of the small size of the di rna compared to the genomic rna, the structural requirements for various rna functions, as determined from the use of di rna constructs, may be different from those of the whole viral genome. the following cis-acting signals for various rna functions have been determined using various di rnas: . rna-packaging signal. in a comparison of mhv di rnas that are efficiently and inefficiently packaged, it was determined that the packaging signal for mhv di rna is localized near the ' end of gene (in the l b region, approximately kb from the ' end) (makino et al., ; van der most et al., ; fosmire et al., ) . this packaging signal forms a stem-loop structure which may be required for the rna-packaging activity (fosmire et al., ) . it is necessary and sufficient for the packaging of di rna or a heterologous rna into the virions (woo et al., ) . the fact that this packaging signal is localized in gene , which is present in genomic but not subgenomic rnas, is consistent with the packaging of genomic but not subgenomic rnas in virus particles. the packaging signal for di rnas of other coronaviruses has not been determined. however, some coronaviruses have been shown to package subgenomic mrnas at low efficiency (sethna et al., ; hofmann et al., ; zhao et al., ) . these are probably packaged nonspecifically; however, the possibility that these viruses may have a different rna packaging signal cannot yet be ruled out. similarly, di rnas that do not contain this packaging signal, such as disse rna of mhv (makino et al., ) and di rna of bcv chang et al., , can be packaged at low efficiency, thereby maintaining themselves for at least several passages in tissue culture. for mhv di rna, it has been shown that only nucleotides at the ' end plus a stretch of poly(a) sequence are required for negative-strand rna synthesis ; no specific upstream rna sequences are required. however, when an mrna is transcribed from an ig site in the same di rna, the negative-strand rna synthesis from this di rna is inhibited, suggesting a common element involved in mrna transcription and negative-strand rna synthesis . one unanswered question is whether or not the sequence requirements for the synthesis of genomic and subgenomic negative-strand rna are identical. . replication signal. sequential deletion analysis has shown that the replication (i.e., complete cycles of negative-and positive-strand rna synthesis) of mhv disse or dissf rnas requires approximately - nucleotides from both the ' and ' ends. the minimum sequence requirement for rna replication may vary with different di rnas. these issues have been discussed in section v,f. . transcriptional signal. di rnas normally do not transcribe subgenomic mrnas because they do not have ig sequences. thus, natural di rnas can synthesize only the full-sized di rna. however, by introduction of the consensus ig sequences into di rna (makino et al., , it has been possible to use di rna as a vector for determining the sequence requirement for subgenomic rna transcription. the cis-and trans-acting signals for transcription have been described in section v,e. . recombination. di rnas of mhv have been demonstrated to undergo a high frequency of recombination with helper virus rna. as discussed above, this accounts for the evolution of mhv di rna species during serial passages of viruses . furthermore, mhv di rnas with a smaller orf are frequently replaced by a di rna with a larger orf by recombination with the helper virus rna (de groot et al., ; kim et al., a) , suggesting that recombination between di rnas and helper virus rnas occurs readily. the reciprocal recombination between di rna and helper virus rna, i.e., the transfer of di rna sequences to the helper virus rna, also has been observed. as a result, the genetic markers on the di rna can be incorporated into the helper virus rna (koetzner et al., ) . recombination between two di rnas, however, has not been described. sequence requirements for rna recombination also have not been studied. bcv di rnas also undergo frequent recombination . however, di rnas of ibv and tgev appear to be more stable. coronavirus research has made tremendous progress in the last decade. the virus family has grown in size, and many of the features thought to be unique to coronaviruses have now been found to be shared by some other viruses. since the last time this serial publication published the first comprehensive review of the molecular biology of coronaviruses (sturman and holmes, ) , the literature on this virus has grown to exceed anyone's ability to do a comprehensive review of every topic relating to coronaviruses. in this review, we have concentrated on areas which have shown the most progress and which present the most challenges. our choice of literature was meant to be representative but is by no means comprehensive. notably missing from this review are the molecular studies related to viral pathogenesis and the interactions between the virus and cells. coronavirus research has contributed to the understanding of many aspects of molecular biology in general, such as the mechanism of rna synthesis, translational control, and protein transport and processing. it remains a treasure capable of generating unexpected insights. despite two decades of studies on the molecular biology of this virus, there are still many problems to be solved: . with regard to the mechanism of rna transcription, many conflicting data remain. coronavirus undoubtedly utilizes a unique, discontinuous transcription mechanism, but how it acts is a subject of debate. an in uitro rna transcription system, so necessary for an understanding of rna synthesis, is still in its infancy. related to this question is the nature of rna polymerase. the sheer size of the polymerase gene presents a daunting task. the availability of the cdna clones and expression vectors for this gene has just begun to allow this black box to be cracked open. this will undoubtedly be a fruitful area of future research. . the last two years have seen the unraveling of the mechanism of coronavirus assembly, which, as it turns out, involves a littlecharacterized e protein. how the various viral structural proteins interact with each other in the various subcellular compartments to form a complete virus particle is an exciting frontier. . after more than years since the first coronavirus was seen under electron microscope, an unexpected new feature of the virus, namely, an icosahedral core with a helical nucleoprotein, was recently uncovered. this structure places coronavirus in a unique position among rna viruses because it takes on the characteristics of positive-, negative-and double-strand rna viruses in morphology. this recent finding challenges us to reevaluate the structure of coronaviruses. . the ability to perform reverse genetic studies of coronavirus is still very limited. expression of individual viral genes and targeted recombination of very limited rna regions are the only available genetic means for examining the structure and function of the coronavirus genome. perhaps it is an unrealistic dream, but progress in polymerase chain reaction technology may one day allow an infectious cdna for coronavirus rna to be made. the early events of viral replication have so far been largely ignored. identification of the cellular receptors for the viruses may finally provide penetrating molecular tools to allow these issues to be examined. it will not be a surprise to discover that virus penetration and uncoating play defining roles in the cellular tropism of viruses. . are nonstructural protein genes really unnecessary? even if they are auxiliary genes, they may prove to play significant roles in the biology of the virus. . finally, what of the potential interaction between the virus and host, which has been one of the major themes of virology in recent years? it may be a little premature to conclude that cellular factors play major roles in coronavirus replication, but there is little doubt that cells are playing more active roles than was previously suspected. is the nucleus contributing to the coronavirus replication? this may require reexamination. these are but some of the exciting challenges for the coronavirologists to tackle. the next decade should bring us an even better understanding of the various aspects of the molecular biology of coronaviruses. membrane and phospholipid binding by murine the golgi sorting domain of coronavirus e l protein. coronaviral nucleocapsid n protein sequence and topology of a model intracellular membrane protein, e l glycoprotein, from a coronavirus lysosomal sorting mutants of coronavirus e l protein, a golgi membrane protein cell fusion studies identified multiple cellular factors involved in mouse hepatitis virus entry identification of a domain required for the autoproteolytic cleavage of murine coronavirus gene a polyprotein identification of the catalytic sites of a papain-like cystein proteinase of murine coronavirus two amino acid changes at the n-terminus of transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism random nature ofcoronavirus rnarecombination in the absence of selection pressure a clustering of rna recombination sites adjacent to a hypervariable region of the peplomer gene of murine coronavirus characterization of leader-related small rnas in coronavirus-infected cells: further evidence for leaderprimed mechanism of transcription analysis of intracellular small rnas of mouse hepatitis virus: evidence for discontinuous transcription interactions between coronavirus nucleocapsid protein and viral rnas: implications for viral transcription establishing a genetic recombination map for murine coronavirus strain a complementation groups episodic evolution mediates interspecies transfer of a murine coronavirus interspecies aminopeptidase-n chimeras reveal species-specific receptor recognition by canine coronavirus, feline infectious peritonitis virus in vivo and in vitro models of demyelinating disease: activation of the adenylate cyclase system influences jhm virus expression in explanted rat oligodendrocytes mouse hepatitis virus strain a rna polymerase gene orf la: heterogeneity among mhv strains characterization of the leader papain-like proteinase of mhv-a : identification of a new in vitro cleavage site characterization of a second cleavage site and demonstration of activity in trans by the papain-like proteinase of the murine coronavirus mouse hepatitis virus strain a mutational analysis of the murine coronavirus spike protein: effect on cell-to-cell fusion the production of recombinant infectious di-particles of a murine coronavirus in the absence of helper virus completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus host cell nuclear function and murine hepatitis virus replication characterization of two rna polymerase activities induced by mouse hepatitis virus further characterization of mouse hepatitis virus rna dependent rna polymerases 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 an efficient ribosomal frame-shifting signal in the polymeraseencoding region of the coronavirus ibv characterization of an efficient coronavirus ribosomal frame-shifting signal: requirement for an rna pseudoknot. cell (cumbridge products of the polymerase-encoding region of the coronavirus ibv the coronavirus nonstructural proteins in vitro synthesis of two polypeptides from a nonstructural gene of coronavirus mouse hepatitis virus strain a 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 entry of coronavirus into primate cns following peripheral infection further studies on human enteric coronavirus coronavirus ibv glycopolypeptides: size of their polypeptide moieties and nature of their oligosaccharides coronavirus ibv further evidence that the surface projections are associated with two glycopolypeptides coronavirus ibv: structural characterization of the spike protein the coronavirus surface glycoprotein coronavirus ibv removal of spike glycopolypeptide s by urea abolishes infectivity and haemagglutination but not attachment to cells sequence analysis of strains of avian infectious bronchitis coronavirus isolated during the s in the uk coronaviruses. in "principles and practice of clinical virology coronavirus ibv partial amino terminal sequencing of the spike polypeptide s identifies the sequence argarg-phe-arg-arg at the cleavage site of the spike precursor propolypeptide of ibv strains beaudette and m amino acids within hypervariable region of avian coronarvirus ibv (massachusetts serotype) spike glycoprotein are associated with neutralization epitopes recommendations of the coronavirus study group for the nomenclature of the structural proteins, mrnas and genes of coronaviruses revision of the taxonomy of the coronavirus, torovirus and arterivirus genera coronaviridae. in "virus taxonomy. sixth report ofthe international committee on taxonomy ofviruses cis requirement for n-specific protein sequence in bovine coronavirus defective interfering rna replication a cis-acting function for the coronavirus leader in defective-interfering rna replication the ucuaaac promoter motif is not required for high-frequency leader recombination in bovine coronavirus defective interfering rna induction of alpha interferon by transmissible gastroenteritis coronavirus: role of transmembrane glycoprotein el cytopathology of chick renal epithelial cells experimentally infected with avian infectious bronchitis virus a pregnancy-specific glycoprotein is expressed in the brain and serves as a receptor for mouse hepatitis virus human carcinoembryonic antigen and biliary glycoprotein can serve as mouse hepatitis virus receptors function ofa '-end genomic rnamutation that evolves during persistent mouse hepatitis virus infection in vitro molecular anatomy of mouse hepatitis virus persistence: coevolution of increased host cell resistance and virus virulence human coronavirus oc interacts with major histocompatibility complex class i molecules at the cell surface to establish infection monoclonal antibodies to murine hepatitis virus- (strain jhm) define the viral glycoprotein responsible for attachment and cell-cell fusion enterotropic strains of mouse coronavirus differ in their use of murine carcinoembryonic antigen-related glycoprotein receptors in vitro replication of mouse hepatitis virus strain a epidemiology of infectious bronchitis virus monoclonal antibody analysis of neutralization and antibody-dependent enhancement of feline infectious peritonitis virus b lymphocyte and macrophage expression of carcinoembryonic antigen-related adhesion molecules that serve as receptors for murine coronavirus intestinal replication of a respiratory coronavirus closely related antigenically to the enteric transmissible gastroenteritis virus bovine coronavirus nonstructural protein ns is a phosphoprotein induction of macrophage procoagulant activity by murine hepatitis virus strain : role of tyrosine phosphorylation pathogenesis and diseases of the central nervous system caused by murine coronaviruses site-specific alteration of murine hepatitis virus type- (mhv- ) peplomer glycoprotein e results in reduced neurovirulence an electron microscope study of the development of a mouse hepatitis virus in tissue culture cells identification of the structural proteins of turkey enteric coronavirus evidence for a coiled-coil structure in the spike proteins of coronavirus stably expressed fipv peplomer protein induces cell fusion and elicits neutralizing antibodies in mice sindbis virus rna polymerase is degraded by the n-end rule pathway the fitness of defective interfering murine coronavirus di-a and its derivatives is decreased by nonsense and frameshift mutations assembly of coronavirus spike protein into trimers and its role in epitope expression aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev determinants essential for the transmissible gastroenteritis virus-receptor interaction reside within a domain of aminopeptidase-n that is distinct from the enzymatic site equine arteritis virus subgenomic rna transcription: w inactivation and translation inhibition studies translation and processing of mouse hepatitis virus virion rna in a cell-free system intracellular processing of the n-terminal orf l a proteins of the coronavirus mhv-a requires multiple proteolytic events identification and characterization of a -kda protein processed from the gene polyprotein of the murine coronavirus mhv-a rna-dependent rna polymerase activity in coronavirus-infected cells monoclonal antibodies to bovine coronavirus: characteristics and topographical mapping of neutralizing epitopes on the e and e glycoproteins monoclonal antibodies to bovine coronavirus glycoproteins e and e : demonstration of in vivo neutralizing activity determinants of the p cleavage site recognized by the first papain-like cysteine proteinase of murine coronavirus morphology and morphogenesis of a coronavirus infecting intestinal epithelial cells of newborn calves sequence analysis of the porcine epidemic diarrhea virus genome between the nucleocapsid and spike protein genes reveals a polymorphic orf cell tropism and expression of mouse hepatitis viruses (mhv) in mouse spinal cord cultures genetically-determined sensitivity to mhv infections is expressed in vitro in lymphoid cells and macrophages cloning of the mouse hepatitis virus (mhv) receptor: expression in human and hamster cell lines confers susceptibility to mhv several members of the mouse carcinoembryonic antigen-related glycoprotein family are functional receptors for the coronavirus mouse hepatitis virus-a mouse hepatitis virus strain a and blocking antireceptor monoclonal antibody bind to the n-terminal domain of cellular receptor mouse hepatitis virus receptor activities of an mhvwmph chimera and mhvr mutants lacking n-linked glycosylation of the n-terminal domain identification of the coronavirus mhv-jhm mrna product complete sequence ( kilobases) of the polyprotein-encoding gene of transmissible gastroenteritis virus molecular basis of transmissible gastroenteritis virus epidemiology the coronavirus avian infectious bronchitis virus requires the cell nucleus and host transcriptional factors the internal open reading frame within the nucleocapsid gene of mouse hepatitis virus encodes a structural protein that is not essential for viral replication pathogenicity of antigenic variants of murine coronavirus jhm selected with monoclonal antibodies experimental demyelination induced by coronavirus jhm (mhv- ): molecular identification of a viral determinant of paralytic disease replication of murine coronaviruses in somatic cell hybrids between murine fibroblasts and rat schwannoma cells identification and characterization of a coronavirus packaging signal proteolytic cleavage of the e glycoprotein of murine coronavirus: host-dependent differences in proteolytic cleavage and cell fusion evidence for variable rates of recombination in the mhv genome map locations of mouse hepatitis virus temperaturesensitive mutants: confirmation of variable rates of recombination three different cellular proteins bind to the complementary sites on the '-end positive-and '-end negative-strands of mouse hepatitis virus rna natural evolution of coronavirus defective-interfering rna involves rna recombination interaction of mouse hepatitis virus (mhv) spike glycoprotein with receptor glycoprotein mhvr is required for infection with a n mhv strain that expresses the hemagglutinin-esterase glycoprotein murine coronavirus membrane fusion is blocked by modification of thiols buried within the spike protein neutralization-resistant variants of a neurotropic coronavirus are generated by deletions within the aminoterminal half of the spike glycoprotein alteration of the ph dependence of coronavirus-induced cell fusion: effect of mutations in the spike glycoprotein cell receptor-independent infection by a neurotropic murine coronavirus the polypeptide structure of transmissible gastroenteritis virus isolation of subviral components from transmissible gastroenteritis virus the polypeptide of m, , of porcine transmissible gastroenteritis virus: gene assignment and intracellular location tgev coronavirus orf encodes a membrane protein that is incorporated into virions major receptor-binding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein tissue and cellular distribution of an adhesion molecule in the carcinoembryonic antigen family that serves as a receptor for mouse hepatitis virus fusion-defective mutants of mouse hepatitis virus a contain a mutation in the spike protein cleavage signal an ntpbinding motif is the most conserved sequence in a highly diverged monophyletic group of proteins involved in positive strand rnaviral replication coronavirus genome: prediction of putative functional domains in the nonstructural polyprotein by comparative amino acid sequence analysis putative papain-related thiol proteases of positive-strand rna viruses single amino acid changes in the s subunit of the mhv surface glycoprotein confer resistance to neutralization by s subunit-specific monoclonal antibody characterization of a -kda polypeptide encoded in gene of the human coronavirus hcv temperature-sensitive mutants of mouse hepatitis virus produce a high incidence of demyelination an "elaborated" pseudoknot is required for high frequency frameshifting during translation of hcv polymerase mrna nucleotide sequence of the human coronavirus rna polymerase locus translational control in murine hepatitis virus infection quantification of individual subgenomic mrna species during replication of the coronavirus transmissible gastroenteritis virus investigation of the control of coronavirus subgenomic mrna transcription by using t -generated negative-sense rna transcripts anew superfamily ofreplicative proteins the '-end of coronavirus minus-strand rnas contains a short poly(u) tract bovine coronavirus mrna replication continues throughout persistent infection in cell culture leader-mrna junction sequences are unique for each subgenomic mrna species in the bovine coronavirus and remain so throughout persistent infection a translation-attenuating intraleader open reading frame is selected on coronavirus mrnas during persistent infection structural proteins of human respiratory coronavirus oc synthesis and processing of the bovine enteric coronavirus hemagglutinin protein coronaviridae and their replication tunicamycin-resistant glycosylation of coronavirus glycoprotein: demonstration of a novel type of viral glycoprotein analysis of a . kb sequence from the ' end of canine coronavirus genomic rna identification ofthe murine coronavirus p cleavage site the s glycoprotein but not the n or m proteins of avian infectious bronchitis virus induces protection in vaccinated chickens synthesis of subgenomic mrna's of mouse hepatitis virus is initiated independently: evidence from uv transcription mapping mechanism of coronavirus transcription: duration of primary transcription initiation activity and effects of subgenomic rna transcription on rna replication evidence for coronavirus discontinuous transcription coronavirus transcription mediated by sequences flanking the transcription consensus sequence a novel variant of avian infectious bronchitis virus resulting from recombination among three different strains mutagenic analysis of the coronavirus intergenic consensus sequence the effect of two closely inserted transcription consensus sequences on coronavirus transcription the amino-terminal signal peptide on the porcine transmissible gastroenteritis coronavirus matrix protein is not an absolute requirement for membrane translocation and glycosylation multiple recombination sites at the '-end of murine coronavirus rna temporal regulation of bovine coronavirus rna synthesis in vivo rna-rna recombination of coronavirus in mouse brain rna recombination of murine coronaviruses: recombination between fusion-positive mhv-a and fusion-negative mhv- inhibition of coronavirus replication by actinomycin d structure and orientation of expressed bovine coronavirus hemagglutinin-esterase protein coronavirus protein processing and rna synthesis is inhibited by the cysteine proteinase inhibitor e d two murine coronavirus genes suffice for viral rna synthesis characterization of a murine coronavirus defective interfering rna internal cis-acting replication signal generation and selection of coronavirus defective interfering rna with large open reading frame by rna recombination and possible editing analysis of cis-actingsequences essential for coronavirus defective interfering rna replication bovine coronavirus hemagglutinin protein infectious bronchitis coronavirus m proteins accumulate in the golgi complex beyond the site of virion budding host genetic control of mouse hepatitis virus type- (jhm strain) replication. . the gene locus for susceptibility is linked to the svp- locus on mouse chromosome repair and mutagenesis of the genome of a deletion mutant of the coronavirus mouse hepatitis virus by targeted rna recombination differential premature termination of transcription as a proposed mechanism for the regulation of coronavirus gene expression early events of importance in determining host cell permissiveness to mouse hepatitis virus infection differentiation of acid-ph-dependent and -nondependent entry pathways for mouse hepatitis virus temperature-sensitive mutants of mouse hepatitis virus strain a : isolation, characterization and neuropathogenic properties toroviruses of animals and humans: a review experimental evidence of recombination in coronavirus infectious bronchitis virus 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 tandem placement of a coronavirus promoter results in enhanced mrna synthesis from the downstream-most initiation site early interaction between mouse hepatitis virus and cells entry of mouse hepatitis virus into cells localization of neutralizing epitopes and the receptor-binding site within the amino-terminal amino acids of the murine coronavirus spike protein structural and fimctional analysis of the surface protein of human coronavirus oc phylogeny of antigenic variants of avian coronavirus ibv. virology sequence and expression of the ns coronavirus leader rna-primed transcription: an alternative coronavirus: organization, replication and expression of genome rna recombination in animal and plant viruses mouse hepatitis virus a : messenger rna structure and genetic localization of the sequence divergence from the hepatotropic strain mhv the presence of leader sequences in the mrna of mouse hepatitis virus characterization of leader rna sequences on the virion and mrnas of mouse hepatitis virus-a cytoplasmic rna virus recombination between nonsegmented rna genome of murine coronaviruses coronavirus: a jumping rna transcription coronavirus: how a large rna viral genome is replicated and transcribed localization of extensive deletions in the structural genes of two neurotropic variants of murine coronavirus jhm coronavirus mrna synthesis: identification of novel transcription initiation signals which are differentially regulated by different leader sequences sequence analysis of the bovine coronavirus nucleocapsid and matrix protein genes porcine respiratory coronavirus: molecular features and virus-host interactions the coronavirus nucleocapsid protein single amino acid changes in the viral glycoprotein m affect induction of alpha interferon by the coronavirus transmissible gastroenteritis virus expression of swine transmissible gastroenteritis virus envelope antigens on the surface of infected cells: epitopes externally exposed distinct structural elements and internal entry of ribosomes in mrna encoded by infectious bronchitis virus the complete sequence ( kilobases) of murine coronavirus gene encoding the putative proteases and rna polymerase synthesis of virus-specific rna in permeabilized murine coronavirus-infected cells thevirus specific intracellular rna species of two murine coronaviruses: mhv a and mhv-jhm genetic analysis of murine hepatitis virus strain jhm cell-free translation of murine coronavirus rna detection of a murine coronavirus nonstructural protein encoded in a downstream open reading frame differential in vitro inhibition of feline enteric coronavirus and feline infectious peritonitis virus by actinomycin d coronavirus ibv-induced membrane fusion occurs at near-neutral ph rnarecombination in a coronavirus: recombination between viral genomic rna and transfected rna fragments requirement of the '-end genomic sequence as an upstream cis-acting element for coronavirus subgenomic mrna transcription a cis-acting viral protein is not required for the replication of a coronavirus defective-interfering rna coronavirus defective-interfering rna as an expression vector: the generation of a pseudorecombinant mouse hepatitis virus expressing hemagglutinin-esterase deletion mapping of a mouse hepatitis virus defectiveinterfering rna reveals the requirement of an internal and discontiguous sequence for replication identification of the cis-acting signal for minus-strand rna synthesis of a murine coronavirus: implications for the role of minus-strand rna in rna replication and transcription the ' untranslated region of the coronaviral rna is required for subgenomic mrna transcription from a defectiveinterfering rna characterisation and mutational analysis of an orf la-encoding proteinase domain responsible for proteolytic processing of the infectious bronchitis virus l d l b polyprotein identification of two new polypeptides encoded by mrna of the coronavirus infectious bronchitis virus internal entry of ribosomes on a tricistronic mrna encoded by infectious bronchitis virus a polycistronic mrna specified by the coronavirus infectious bronchitis virus a -kilodalton polypeptide encoded by open reading frame (orf) l b of the coronavirus infectious bronchitis virus is processed by orf la products -glycosylation of the coronavirus m protein. differential localization of sialyltransferases in n-and -linked glycosylation membrane assembly of the triple-spanning coronavirus m protein. individual transmembrane domains show preferred orientation the cytoplasmic tail of mouse hepatitis virus m protein is essential but not sufficient for its retention in the golgi complex oligomerization of a trans-golgvtrans-golgi network retained protein occurs in the golgi complex and may be part of its retention intracellular and in vitro-translated -kda proteins contain the c-like proteinase activity of the coronavirus mhv-ass identification and characterization of a serine-like proteinase of the murine coronavirus mhv-a primary structure of the glycoprotein e of coronavirus mhv-a and identification of the trypsin cleavage site. virology sequence of mouse hepatitis virus a mrna : indications for rna-recombination between coronavirus and influenza c virus amino acid sequence of a conserved neutralizing epitope of murine coronaviruses replication of synthetic defective interfering rnas derived from coronavirus mouse hepatitis virus-a characterization of two temperature-sensitive mutants of coronavirus mouse hepatitis virus strain a with maturation defects in the spike protein a specific transmembrane domain of a coronavirus e l glycoprotein is required for its retention in the golgi region the e l glycoprotein of an avian coronavirus is targeted to the cis golgi complex retention of a cis golgi protein requires polar residues on one face of a predicted alpha-helix in the transmembrane domain two particle types of avian infectious bronchitis virus ribonucleoprotein-like structures from coronavirus particles rna-dependent rna polymerase activity in murine coronavirus-infected cells effect ofintergenic consensus sequence flanking sequences on coronavirus transcription evolution of the '-end of genomic rna of murine coronaviruses during passages in vitro high-frequency leader sequence switching during coronavirus defective interfering rna replication structure of the intracellular defective viral rnas of defective interfering particles of mouse hepatitis virus high-frequency rna recombination of murine coronaviruses leader sequences of murine coronavirus mrnas can be freely reassorted: evidence for the role of free leader rna in transcription rna recombination of coronaviruses: localization of neutralizing epitopes and neuropathogenic determinants on the carboxyl terminus of peplomers defective interfering particles of murine coronavirus: mechanism of synthesis of defective viral rnas primary structure and translation of a defective-interfering rna of murine coronavirus discontinuous transcription generates heterogeneity a t the leader fusion sites of coronavirus mrnas analysis of efficiently packaged defective-interfering rnas of murine coronavirus: localization of a possible rnapackaging signal a system for study of coronavirus mrna synthesis: a regulated, expressed subgenomic defective-interfering rna results from intergenic site insertion localization of an rna-binding domain in the nucleocapsid protein of the coronavirus mouse hepatitis virus optimization of targeted rna recombination and mapping of a novel nucleocapsid gene mutation in the coronavirus mouse hepatitis virus membrane integration and intracellular transport of the coronavirus glycoprotein e l , a class i membrane glycoprotein molecular characterization of transmissible gastroenteritis coronavirus defective interfering genomes: packaging and heterogeneity attenuation ofmurine coronavirus infection by ammonium chloride endosomal association of a protein phosphatase with high dephosphorylating activity against a coronavirus nucleocapsid protein human coronavirus oc rna lacks two open reading frames located downstream of the s gene of bovine coronavirus coronavirus infects and causes demyelination in primate central nervous system influenza c virus hemagglutinin: comparison with influenza a and b virus hemagglutinins bgp , a new member of the carcinoembryonic antigen-related gene family, encodes an alternative receptor for mouse hepatitis virus localization of the rna-binding domain of mhv nucleocapsid protein post-translational glycosylation of coronavirus glycoprotein el: inhibition by monensin the carbohydrates of mouse hepatitis virus (mhv) a : structures of the -glycosidically linked oligosaccharides of glycoprotein el the encephalomyocarditis virus c protease is rapidly degraded by an atp-dependent proteolytic system in reticulocyte lysate immunoglobulin fc binding activity is associated with the mouse hepatitis virus e peplomer protein mhv s peplomer protein expressed by a recombinant vaccinia virus vector exhibits igg fc-receptor activity molecular mimicry between fc receptor and s peplomer protein of mouse hepatitis virus, bovine coronavirus, and transmissible gastroenteritis virus monoclonal antibodies to the spike protein of feline infectious peritonitis virus mediate antibody-dependent enhancement of infection of feline macrophages disulfide bonds in folding and transport of mouse hepatitis coronavirus glycoproteins envelope glycoprotein interactions in coronavirus assembly cloning and in vitro expression of the gene for the e haemagglutinin glycoprotein of bovine coronavirus expression and secretion of the bovine coronavirus hemagglutinin-esterase glycoprotein by insect cells infected with recombinant baculoviruses sequence comparison of the n genes of five strains of the coronavirus mouse hepatitis virus suggests a three domain structure for the nucleocapsid protein sequence analysis reveals extensive polymorphism and evidence of deletions within the e glycoprotein gene of several strains of murine hepatitis virus analysis of cell fusion induced by bovine coronavirus infection analysis of second-site revertants of a murine coronavirus nucleocapsid protein deletion mutant and construction of nucleocapsid protein mutants by targeted rnarecombination construction of murine coronavirus mutants containing interspecies chimeric nucleocapsid proteins isolation of a porcine respiratory, non-enteric coronavirus related to transmissible gastroenteritis characterization of a replicating and packaged defective rna of avian coronavirus infectious bronchitis virus replication and packaging of coronavirus infectious bronchitis virus defective rnas lacking a long open reading frame mhvnucleocapsid synthesis in the presence of cyclohexamide and accumulation of negative-strand mhv rna functional analysis ofthe coronavirus mhv-jhm surface glycoproteins in vaccinia virus recombinants lactate dehydrogenase-elevating virus, equine arteritis virus and simian haemorrhagic fever virus, a new group of positive strand rna viruses porcine respiratory coronavirus differs from transmissible gastroenteritis virus by a few genomic deletions a conditionallethal murine coronavirus mutant that fails to incorporate the spike glycoprotein into assembled virions membrane protein molecules of transmissible gastroenteritis coronavirus also expose the carboxy-terminal region on the external surface of the virion the transmissible gastroenteritis coronavirus contains a spherical core shell consisting of m and n proteins pathogenic murine coronaviruses. . biological and biochemical characterization of temperature-sensitive mutants of jhmv rna-binding proteins of coronavirus mhv: detection of monomeric and multimeric n protein with an rna overlay-protein blot assay entry and release of transmissible gastroenteritis coronavirus are restricted to apical surfaces of polarized epithelial cells mhv-a enters polarized murine epithelial cells through the apical surface but is released basolaterally coronavirus infection of polarised epithelial cells mouse hepatitis virus strain a is released from opposite sides of different epithelial cell types assembly in vitro of a spanning membrane protein of the endoplasmic reticulum: the e l glycoprotein of coronavirus mouse hepatitis virus a the coronavirus membrane glycoprotein analysis of murine coronavirus surface glycoprotein functions by using monoclonal antibodies evolution of mouse hepatitis virus: detection and characterization of spike deletion variants during persistent infection 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 persistent infection of cultured cells with mouse hepatitis virus (mhv) results from epigenetic expression of the mhv receptor evidence for new transcriptional units encoded at the ' end of the mouse hepatitis virus genome genetics of mouse hepatitis virus transcription: identification of cistrons which may function in positive and negative strand rna synthesis nucleotide sequence ofthe gene encoding the surface projection glycoprotein of coronavirus mhv-jhm acylation of viral spike glycoproteins: a feature of enveloped rna viruses bovine coronavirus uses n-acetyl- - -acetylneuraminic acid as a receptor determinant to initiate the infection of cultured cells hemagglutinating encephalomyelitis virus attaches to n-acetyl- - -acetylneuraminic acidcontaining receptors on erythrocytes: comparison with bovine coronavirus and influenza c virus the s protein of bovine coronavirus is a hemagglutinin recognizing - -acetylated sialic acid as a receptor determinant isolated he-protein from hemagglutinating encephalomyelitis virus and bovine coronavirus has receptordestroying and receptor-binding activity neuraminidase treatment of avian infectious bronchitis coronavirus reveals a hemagglutinating activity that is dependent on sialic acid-containing receptors on erythrocytes murine coronavirus nonstructural protein ns is not essential for virus replication in transformed cells the nucleocapsid protein gene of bovine coronavirus is bicistronic coronavirus subgenomic minusstrand rnas and the potential for mrna replicons minus-strand copies of replicating coronavirus mrnas contain antileaders expression and characterization of a recombinant murine coronavirus c-like proteinase identification of a new transcriptional initiation site and the corresponding functional gene b in the murine coronavirus rna genome in vitro interaction of mouse hepatitis virus and macrophages from gneetically resistant mice. i. adsorption of virus and growth curves the coronaviridae the coronaviridae: an introduction the small membrane protein coronavirus jhm: intracellular protein synthesis monoclonal antibody to the receptor for murine coronavirus mhv-a inhibits viral replication in vivo identification of a new membrane-associated polypeptide specified by the coronavirus infectious bronchitis virus toroviruses: replication, evolution and comparison with other members of the coronavirus-like superfamily the molecular biology of toroviruses the coronavirus superfamily comparison of the genome organization of toro-and coronaviruses: evidence for two nonhomologous rna recombination events during berne virus evolution sequence and translation of the murine coronavirus '-end genomic rna reveals the n-terminal structure of the putative rna polymerase ribosomal pausing during translation of an rna pseudoknot isolation and identification of virus-specific mrna in cells infected with mouth hepatitis virus (mhv-ass) coronavirus mrna synthesis involves fusion of non-contiguous sequences proteolytic cleavage of the murine coronavirus surface glycoprotein is not required for fusion activity coronavirus multiplication strategy identification and characterization of virus-specified rna coronavirus multiplication strategy. . mapping the avian infectious bronchitis virus intracellular rna species to the genome synthesis of coronavirus mrnas: kinetics of inactivation of infectious bronchitis virus rna synthesis by uv light coronavirus proteins: structure and function of the oligosaccharides of the avian infectious bronchitis virus glycoproteins phosphoproteins of murine hepatitis viruses specific interaction between coronavirus leader rna and nucleocapsid protein monoclonal antibodies differentiate between the haemagglutinating and the receptordestroying activities of bovine coronavirus the molecular biology of coronaviruses isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid proteolytic cleavage of the e glycoprotein of murine coronavirus: activation of cell fusing activity of virions by trypsin and separation of two different k cleavage fragments conformational change of the coronavirus peplomer glycoprotein at ph . and degrees c correlates with virus aggregation and virus-induced cell fusion hemagglutination and structural polypeptides of a new coronavirus associated with diarrhea in infant mice analysis ofreceptor-binding site ofmurine coronavirus spike protein a golgi retention signal in a membranespanning domain of coronavirus e l protein fusion formation by the uncleaved spike protein of murine coronavirus jhmv variant cl- the s subunit of the murine coronavirus spike protein is not involved in receptor binding coronavirus translational regulation: leader affects mrna efficiency myelin basic protein and human coronavirus cross-reactive t cells in multiple sclerosis internal ribosome entry in the coding region of murine hepatitis virus mrna a region of the coronavirus infectious bronchitis virus l a polyprotein encoding the c-like protease domain is subject to rapid turnover when expressed in rabbit reticulocyte lysate characterization in vitro of an autocatalytic processing activity associated with the predicted c-like proteinase domain of the coronavirus avian infectious bronchitis virus infection of att murine pituitary tumour cells by mouse hepatitis virus strain a : virus budding is restricted to the golgi region replication of coronavirus mhv-a in sac-cells: determination of the first site of budding of progeny virions sorting of progeny coronavirus from condensed secretory proteins at the exit from the trans-golgi network of att cells site of addition of n-acetyl-galactosamine to the e l glycoprotein of mouse hepatitis virus-a induction of antibodies protecting against transmissible gastroenteritis coronavirus (tgev) by recombinant adenovirus expressing tgev spike protein feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i isolation of coronaviruses antigenically indistinguishable from bovine coronavirus from wild ruminants with diarrhea the -kda hydrophobic protein encoded at the '-end ofthe porcine transmissible gastroenteritis coronavirus genome is membrane-associated a domain a t the '-end of the polymerase gene is essential for encapsidation of coronavirus defective interfering rnas homologous rna recombination allows efficient introduction of site-specific mutations into the genome of coronavirus mhv-a via synthetic co-replicating rnas subgenomic rna synthesis directed by a synthetic defective interfering rna of mouse hepatitis virus: a study of coronavirus transcription initiation translation but not the encoded sequence is essential for the efficient propagation of the defective interfering rnas of the coronavirus mouse hepatitis virus rat glial c cells are defective in murine coronavirus internalization regulation of coronavirus mrna transcription sequence comparison of porcine respiratory coronavirus isolates reveals heterogeneity in the s intracellular transport of recombinant coronavirus spike proteins: implications for virus assembly early death after feline infectious peritonitis virus challenge due to recombinant vaccinia virus immunization primary structure of the membrane and nucleocapsid protein genes of feline infectious peritonitis virus and immunogenicity of recombinant vaccinia viruses in kittens a novel glycoprotein of feline infectious peritonitis coronavirus contains a kdel-like endoplasmic reticulum retention signal nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes the e protein of bovine coronavirus is a receptor-destroying enzyme with acetyltransferase activity human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza c viruses sequence analysis of the spike protein gene of murine coronavirus variants: study of genetic sites affecting neuropathogenicity evidence of natural recombination within the s gene of the infectious bronchitis virus evolutionary implications of genetic variations in the s gene of infectious bronchitis virus genetic variation of neurotropic and non-neurotropic murine coronaviruses the peplomer protein e of coronavirus jhm as a determinant of neurovirulence: definition of critical epitopes by variant analysis evidence for a putative second receptor for porcine transmissible gastroenteritis virus on the villous enterocytes of newborn pigs monoclonal antibodies to the peplomer glycoprotein of coronavirus mouse hepatitis virus identify two subunits and detect a conformational change in the subunit released under mild alkaline conditions the proposed family toroviridae: agents of enteric infections. brief review pathogenesis of feline infectious peritonitis: nature and development of viremia oligomerization of a membrane protein correlates with its retention in the golgi complex natural cytotoxicity against mouse hepatitis virus-infected cells. . a cytotoxic effector cell with a b lymphocyte phenotype evidence for a porcine respiratory coronavirus, antigenically similar to transmissible gastroenteritis virus, in the united states genetic analysis of porcine respiratory coronavirus, an attenuated variant of transmissible gastroenteritis virus the replication of murine coronaviruses in enucleated cells evidence for a pseudoknot in the '-untranslated region of the bovine coronavirus genome receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins in vivo and in vitro models of demyelinating disease: efficiency of virus spread and formation of infectious centers among glial cells is genetically determined by the murine host in vivo and in vitro models of demyelinating disease. possible relationship between induction of regulatory subunit from camp dependent protein kinases and inhibition of jhmv replication in cultured oligodendrocytes murine coronavirus packaging signal confers packaging to nonviral rna human aminopeptidase n is a receptor for human coronavirus mouse hepatitis virus s rna sequence reveals that nonstructural proteins ns and ns a are not essential for murine coronavirus replication mouse hepatitis virus utilizes two carcinoembryonic antigens as alternative receptors the receptor for mouse hepatitis virus in the resistant mouse strain sjl is functional: implications for the requirement of a second factor for viral infection biosynthesis, structure, and biological activities of envelope protein gp of murine coronavirus heterogeneity of gene expression of hemagglutinin-esterase (he) protein of murine coronaviruses hemagglutininesterase (he)-specific monoclonal antibodies alter the neuropathogenicity of mouse hepatitis virus coronavirus mrna transcription: uv light transcriptional mapping studies suggest an early requirement for a genomiclength template a spike proteindependent cellular factor other than the viral receptor is required for mouse hepatitis virus entry synthesis and processing of the haemagglutinin-esterase glycoprotein of bovine coronavirus encoded in the e region of adenovirus the subunit of the spike glycoprotein of bovine coronavirus mediates membrane fusion in insect cells mouse hepatitis virus strain mhv-s: formation of pseudotypes with a murine leukemia virus envelope a conserved motif a t the ' end of mouse hepatitis virus genomic rna required for host protein binding and viral rna replication specific binding of host cellular proteins to multiple sites within the '-end of mouse hepatitis virus genomic rna mouse hepatitis virus gene b protein is a new virion envelope protein unusual heterogeneity of leader-mrna fusion in a murine coronavirus: implications for the mechanism of rna transcription and recombination interactions between the cytoplasmic proteins and the intergenic (promoter) sequence of mouse hepatitis virus r n a correlation with the amounts of subgenomic mrna transcribed a '-proximal rna sequence of murine coronavirus as a potential initiation site forgenomic-lengthmrna transcription the hemagglutinidesterase glycoprotein of bovine coronaviruses: sequence and functional comparisons between virulent and avirulent strains biological and genetic characterization of a hemagglutinating coronavirus isolated from a diarrhoeic child coronavirus leader rna regulates and initiates subgenomic mrna transcription, both in trans and in cis presence of subgenomic mrnas in virions of coronavirus ibv the infectious bronchitis virus nucleocapsid protein binds rna sequences in the ' terminus of the genome characterization of a human coronavirus (strain ) c-like proteinase activity mouse hepatitis virus orf a is expressed in the cytosol of infected mouse fibroblasts we would like to thank the following individuals for making data available prior to publication: luis enjuanes, hubert laude, peter rottier, and pierre talbot. we would also like to thank daphne shimoda for her tremendous help with editorial revisions, preparation of the figures, and the typing of the manuscript. m.m.c.l. is investigator of the howard hughes medical institute. key: cord- -b ea u authors: fu, kaisong; baric, ralph s. title: evidence for variable rates of recombination in the mhv genome date: - - journal: virology doi: . / - ( ) -h sha: doc_id: cord_uid: b ea u abstract mouse hepatitis virus has been shown to undergo rna recombination at high frequency during mixed infection. temperature-sensitive mutants were isolated using -fluorouracil and -azacytidine as mutagen. six rna+ mutants that reside within a single complementation group mapping within the s glycoprotein gene of mhv-a were isolated which did not cause syncytium at the restrictive temperature. using standard genetic techniques, a recombination map was established that indicated that these mutants mapped into two distinct domains designated f and f . these genetic domains may correspond to mutations mapping within the s and s glycoproteins, respectively, and suggest that both the s and s domains are important in eliciting the fusogenic activity of the s glycoprotein gene. in addition, assuming that most distal is alleles map roughly . kb apart, a recombination frequency of % per – by was predicted through the s glycoprotein gene. interestingly, this represents a threefold increase in the recombination frequency as compared to rates predicted through the polymerase region. the increase in the recombination rate was probably not due to recombination events resulting in large deletions or insertions (> bp), but rather was probably due to a combination of homologous and nonhomologous recombination. a variety of explanations could account for the increased rates of recombination in the s gene. mouse hepatitis virus (mhv), a member of coronaviridae, contains a single-stranded nonsegmented plus-polarity rna of about kb in length (lee et a/., ; pachuk et a/., ) . the genomic rna is arranged into seven or eight coding regions and is encapsidated within multiple copies of a -kda nucleocapsid protein (n) (lai, ; siddell, ) . the n protein forms a helical nucleocapsid structure that is probably associated with the transcriptional complex compton et a/., ; stohlman and lai, ; stohlman eta/., ; sturman eta/., ) . the nucleocapsid structure is surrounded by a lipid envelope and contains two or three virus-specific glycoproteins. the s glycoprotein has a molecular weight of kda and is frequently cleaved into two -kda glycoproteins designated sl and s (frana et a/., ; sturman et a/., . the n-terminal signal sequence is contained within the sl domain while the c-terminus is contained within the s region (boireau et al., ; luytjes eta/., ; rasschaert et al., ; schmidt et al., ) . the sl domain can undergo significant amino acid alteration without losing its function while the s domain is more highly conserved and contains a hydrophobic heptad repeat element that probably forms a complex ' to whom correspondence should be addressed. coiled coil (luytjes et a/., ; schmidt et a/., ) . the s glycoprotein is responsible for virus binding to the specific cellular receptor, induction of cell fusion, and elicitation of neutralizing antibody and cell-mediated immunity (collins et al., ; fleming et a/., ; sturman et a/., . in addition to s, a -kda m glycoprotein is present in the virion that probably functions in assembly and release (sturman et a/., . in some strains of mhv, a -kda hemagglutinin (he) that shares considerable sequence homology with the influenza c virus hemagglutinin is also present (luytjes et al., ; shieh et al., ; yokomori et al., ) . upon entry into susceptible cells, the genomic rna is translated into one or more polyproteins that act as an rna dependent rna polymerase (mahy et a/., ; brayton et al., brayton et al., , . while the exact mechanism for mhv transcription is still under study, the preponderance of data suggest that the genome is initially transcribed into a full-length minus strand rna, which acts as template for the synthesis of a genomelength rna and six subgenomic mrnas by "leaderprimed" transcription (baker et a/., ; baric et al., baric et al., , makino et a/., a makino et a/., , . in turn, the subgenomic mrna then act as template for the synthesis of subgenomic minus strands, which participate in successive rounds of mrna amplification (sawicki and sawicki, ; sethna et a/., sethna et a/., , . homologous recombination among viruses with nonsegmented rna genomes has been reported among picornaviruses (cooper, (cooper, , cooper et al., ; king et al., king et al., , king et al., , lake et al., ) , brome mosaic virus (bujarski and kaesberg, ) alphaviruses (hahn et al., ; weiss and schlesinger, ) , cowpea chlorotic mottle virus a (allison et al., ) , and coronaviruses (keck et al., a,b; lai et a/., ; makino et al., b) . during mhv replication, the rna recombination frequency is unusually high and approaches % or more during mixed infection . recently, biochemical analysis has revealed extensive polymorphisms and deletions in the sequence of the s glycoprotein gene of different mhv variants. the variant viruses were probably derived from nonhomologous recombination because no consensus or conserved sequences flanked the deletions (banner et al., ; parker et a/., ) . sequence analysis of rna recombinant viruses also suggests the presence of a recombination "hot spot" in the mhv s glycoprotein gene (banner el al., ) . however, in the absence of selection pressure, rna recombination sites in this region are random, suggesting that this preferred site reflects the selection for biologically more efficient recombinants . recombination analysis provides a powerful tool for mapping genetic loci and determining the recombination frequency between individual temperature sensitive (ts) mutations cooper, cooper, , lake et al., ) . in this study, several group f rna+ ts mutants were used to establish a genetic recombination map in the s glycoprotein gene of mhv-a . temperature-sensitive mutants representing the group f rna+ mutants (la , la , nc , nc , nc , nc ) and group e rna-mutants (la ) of mhva were used throughout the course of this study. all virus stocks were propagated at " in -cm flasks containing dbt cells as described previously . plaque assays were performed at either the permissive ( ") or nonpermissive ( . ") temperatures in dbt cells in dulbecco's modified essential medium (dmem) (sigma) containing % nu-serum (collaborative research, inc.), % gentamycin/kanamycin (gibco), and . % agarose (brl). all plaque assays were stained - hr postinfection by the addition of neutral red for hr. cloned ci- cells were kindly provided by dr. sue baker (loyola university, chicago) and maintained at " in dmem containing % newborn calf serum (gibco), % gentamycin/kanamycin, % glucose (sigma), and % ttyptose-phosphate broth (sigma) . most of the mutants used in this study have been characterized previously (la , la , la , nc ) . to increase the accuracy of our recombination mapping data, additional mutants residing within complementation group f were isolated. briefly, cultures of mhv-a -infected cells were mutagenized with either pug/ml -azacytidine or pg/ ml -fluorouracil for hr. supernatant fluids containing mutagenized virus were plaqued at permissive temperature and individual plaques were isolated. each plaque was diluted into . ml ice-cold pbs, screened at " and . " by plaque assay, and isolates displaying ts phenotypes were repurified two to three times at ". virus stocks were assayed at the permissive and restrictive temperatures and only those isolates displaying reversion frequencies of less than om were used for future studies. complementation and rna phenotype analysis were performed as described previously (leibowitz et al., ; martin et al., ; , and three additional group f rna+ mutants were isolated and designated nc , nc , and nc . nc and nc were isolated using -fluorouracil, while nc was isolated using -azacytidine. to select for revertants from the group f rna+ ts mutants, virus stocks were plaque assayed at . ", and individual plaques were isolated and repurified by plague assay at the restrictive temperature. individual stocks of revertant virus were grown in -cm flasks at " and the reversion frequency of each was determined by plague assay at and . ". at least three independent revertants from each rna+ mutant were isolated. to isolate rna recombinant virus, dbt cells were coinfected with two different ts mutants at an m.o.i. of each at ". the medium was harvested at hr postinfection and stored at - ". recombinant viruses were isolated at the restrictive temperature, repurified at . ", and grown in dbt cells at ". culture of cl- cells ( x ) in -mm six-well dishes (costar) were infected with various group f ts mutants, revertants, or recombinant viruses at a multiplicity of infection of for hr at room temperature. following adsorption, the inoculum was removed, and the cultures were rinsed with pbs and incubated in complete dmem for hr at ". actinomycin d ( pgi ml) was added for hr and one-half the cultures shifted to the restrictive temperature at hr postinfection by the addition of prewarmed media. viral progeny were harvested at varying intervals and analyzed by plaque assay. to determine whether these mutants could transcribe viral mrna, cultures of cells were infected with various ts mutants and maintained at the permissive or restrictive temperature for hr. the media were removed and the cultures washed with ml of iso-tkm ( m/w tris-hci, ph . , mni kci, and . mm mgci,). the rna was isolated as described by sawicki and sawicki, , and extensively purified by successive rounds of phenol (u.s. biochemical company), phenol/chloroform/isoamy alcohol, and chloroform extraction. the rna was bound to nitrocellulose filters and probed with radiolabeled rna probes specific for the mhv n gene . the blots were washed and exposed to xar- film for hr. recombination analysis and establishing a genetic recombination map of the group f rna+ mutants recombination analyses were performed as previously described and the recombination frequencies calculated as the percentage of ts+ virus present in the progeny from the following formula: rf = vw,,., -ta + b) ~.s x , ooo,o, . . -* bw (ab),,., was the titer of the cross at nonpermissive temperature while (ab),, represented the titer of the same cross at permissive temperature. (a + b) . was the sum of the revertants of each parent strain assayed at the nonpermissive temperature. the formula only measured single or odd-number cross-over events resulting in the ts+ phenotype and did not account for recombination events that resulted in the doublets mutant phenotype. all recombination frequencies were standardized to a standard cross [la x nc ; recombination frequency . f . as previously described . the mutants were aligned according to their standardized recombination frequencies and positioned from la , la , and nc . cultures of dbt cells in loo-mm dishes were infected at a m.o.i. of with different ts mutants, revertant viruses, or recombinant viruses, and intercellular rna was harvested at -l hr postinfection. the precipitate was resuspended in te buffer ( mni tris-hci, ph . , mm edta) and used as template for reverse transcription and taq amplification reactions. cdna synthesis was carried out in a -~ reaction mixture containing pg intercellular rna, mm mgci,, m/l/l tris-hci ph . , mm kci, . % triton x-l (sigma), mm dntp each (promega), . pg random hexamer (perkin elmer cetus), u/pi rnasin (promega), u amv reverse transcriptase (promega) and incubated at " for hr. the products were extracted and precipitated in ethanol and resuspended in ~ h,o prior to successive rounds of polymerase chain amplification (pcr). one-tenth of the cdna was amplified in a ~ reaction mixture containing mm tris-hci (ph . ) mm kci, mm mgci,, . o/o triton x-l , . u taq dna polymerase (promega), . mm dntp mixture and the appropriate primer pairs ( ng of each). pcr amplification was performed for successive cycles at . min at " to denature the dna, min at " for primer annealing, and min at " for primer extension. the products were loaded onto % agarose gels in tae ( mltltris, mm naci, mm edta, (ph . )) separated electrophoretically, stained with ethidium bromide, and visualized under uv light. the dna products were transferred to nitrocellulose filters, and confirmed by hybridization with p-labeled oligomer probes specific for internal sequences in each gene fragment amplified. three sets of overlapping primer pairs were obtained from highly conserved sequences in the mhv-a and jhm s glycoprotein genes (banner et a/., ; luytjes et al., ; parker et a/., ) . set a was derived from sequences spanning nucleotides - to (cat gct ggt cgt gtt t) and - (aac gta gta gcg gag g), respectively, and should result in a dna fragment of . kb in length. set b was derived from nucleotides - (gcg tac tat tcg gat aaa cc) and - (ccc acg acc gaa tac g) and were used to amplify a - . -kb fragment encompassing the hypervariable region in the sl domain (banner et a/., ; parker et a/., ) . set c was derived from nucleotides - (acg gat gag gcg ctt c) and - (gtc llt cca gga gag g) and were used to amplify a - . -kb fragment spanning the c terminus of the s glycoprotein gene. internal oligodeoxynucleotides were used as probe to demonstrate the specificity of the pcr reaction. six complementation group f rna+ mutants and one group e rna-mutant were used during the course of this study. three of the rna' mutants (la , la , . x lo . x . x o- . x lo* . x lo . x - . x . x lo* . x o- . x lo . x lo . x - . x ' . x lo . x o- . x ' . x lo . x o- . x ' . x ' . x loo . x lo . x ' . x loo . x ' . x lo . x loo . x lo . x ' . x -l . x lo* . x " . x lo-' . x lo . x " . x -z . x ' . x lo . x loo . x ' . x lo . x - . x ' . x ' . x -l . x lo . x lo . x lo . x lo . x . x loo . x ' . x . x loo . x lo . x lo . x lo . x . x lo . x -l . x ' . x ' . x -l . x ' . x ' . x loo . x lo . x ' . x -j . x ' . x ' . x -l . x ' . x ' . x -l + + + + + + + + "calculated as . " nc ) and one rna-mutant (la ) have been characterized previously (schaad el al., ) . to increase the accuracy of the recombination map and obtain more representative mutants in the s glycoprotein gene, three additional rna+ mutants were isolated by mutagenesis as previously described . two mutants were isolated following mutagenesis with pg/ml -fluorouracil (nc , nc ) and one mutant with pg/ml -azacytidine (nc ). all mutants had reversion frequencies of less than e , and did not complement each others defect at the restrictive temperature (table , data not shown). all of the rna+ mutants were capable of transcribing viral mrna when maintained at the restrictive temperature (data not shown, table ). while nc produced a small amount syncytium, the remainder of the mutants were not capable of giant cell formation at the restrictive temperature (fig. ). to assist in precisely defining the location of these mutations and examining the mechanism for mhv rna recombination within the s glycoprotein gene, a panel of revertants were obtained from six of the rna+ mutants (table ) . revertant viruses had similar titers, produced syncytium, and were of the rna+ phenotype when assayed at both permissive and restrictive temperatures. to conclusively document the revertant phenotype and determine if growth characteristics were similar to parental controls, growth curves were compared at the permissive and restrictive temperatures. cultures of cells were infected with nc or its revertant, nc -rl, at " and shifted to . " at . hr postinfection. viral progeny were harvested at different times after infection and assayed by plaque assay at the permissive temperature. similar growth curves were evident between ts nc and its revertant nc -rl in cultures maintained at the permissive temperature (fig. ) . however, following temperature shift, growth of ts nc was inhibited significantly under conditions in which the revertant replicated normally. similar results have also been obtained with other mutants and their revertants (data not shown). to assess the ability of these mutants to be used in recombination studies, we performed a series of crosses between different group f ts mutants and examined the differences between the recombination and reversion frequencies. cultures of cells were infected at a m.o.i. of each with two different ts mutants and maintained at " for hr postinfection. the progeny were harvested and titered at the permissive and restrictive temperatures. recombination frequencies ranged from as little as . % to > . / suggesting that the mutants map in different locations in the gene. the ratio of recombinants to revertants ranged from to times higher than the sum of the spontaneous reversion frequencies of each individual ts mutant used in the cross and indicated that recombination mapping was feasible throughout this region ( table ). the extent of the difference in the ratio of recombinants to revertants probably reflected the stability of the mutants used in the cross and the distance between the individual ts lesions. establishing a recombination map for the group f rna+ mutants in contrast to poliovirus and apthovirus ts mutants, mhva ts mutants were amenable to complementa- tion analysis, providing strong evidence that all of the rna+ ts mutants used in this study contain defects in a single gene or a noncomplementable function (koolen et al., ; leibowitz et al., ; martin et al., ; schaad et al., ) . all mutants were originally crossed three to five times with the reference mutants la , nc , or a group e rna-mutant la and standardized to the reference cross la x nc . to obtain a more detailed map of the group f mutants, crosses were also performed among each of the viruses used in this study and standardized to the reference cross. the results of these experiments are shown in table . the distances between different ts mutants were within statistical limits and permitted the construction of a genetic map (fig. ) . on the basis of their genetic recombination frequencies, the group f rna+ ts mutants appeared to map into two distinct domains, designated fl (la , la , nc ) and f (nc , nc , nc , la ). ts la mapped closely to representative mu-tants within each subgroup suggesting that it may be a double mutant. assuming that crossover events occur in both directions and that nc /la and nc /nc map at different ends of the . -kb s glycoprotein gene, a % recombination frequency occurred over - nucleotide pairs of rna. since these mutants do not produce syncytium at the restrictive temperature ( fig. ) and sequence analysis of rna recombinant viruses suggested that the defect in la mapped within the first .l kb of the s glycoprotein gene (banner et al., ; keck eta/., keck eta/., , a makino eta/., b makino eta/., , , these data provided an anchor for mapping the location of the remaining group f rna+ mutants. these data predicted that the fl mutants map in the sl glycoprotein while the f mutants map within the s glycoprotein sequences (fig. ) . comparisons between the recombination frequencies in the polymerase and s glycoprotein genes using well characterized ts mutants of mhv-a , we have predicted that the recombination frequency between the group f rna' mutant la , and the group a rna-mutants la /la to be approximately l%/ -l nucleotides of dsrna ( fig. ) . to determine if recombination frequencies vary in different portions of the genome, we compared the maximum recombination frequencies between mutants spanning the polymerase genes of mhv (group a (la /la ) x group e (la )) across gene b encoding the p and he genes (group e (la ) x group f (la /la )) or through the s glycoprotein gene (group f (la x nc inc )). all mutants were crossed three to five times and average recombination frequencies calculated as previously described (fig. ) . interestingly, the maximum recombination frequency predicted between the two most distant ts mutants in complementation groups a (la , la ) and e (la ) in the -kb polymerase region ranged from about %/l (la x la ) to %i (la x la ) nucleotides of ds rna. the maximum recombination frequency predicted between the group e and f mutants that spanned the p and he genes ranged from about %/l (la x la ) to %/ (la x la ) nucleotides of dsrna. since the maximum predicted recombination frequency across the . -kb s glycoprotein gene ranged from about o/ / (la x nc ) to o/o/ (la x nc ) nucleotides of dsrna, these data suggested that variable rates of recombination occurwithin different portions of the mhv genome ( figure ). are deletions contributing to the increased rates of recombination among the group f rna+ mutants? extensive amounts of polymorphism and deletion have been detected in both the he and the s glycoprotein gene sequences of different mhv strains and in the sequence of other coronaviruses (banner et al., ; boireau et a/., parker et al., ; rasschaert et a/., ) . it is possible that the increased rate of recombination within the s glycoprotein gene may represent the sum of homologous recombination and recombination (homologous or nonhomologous) resulting in deletions. to test this possibility, cultures of cells were infected with various combinations of ts mutants as shown in fig. and rna recombinant viruses isolated hr postinfection. since the ratio of recombinants/revertants ranged from to in these crosses (table ) these data suggested that the majority of ts+ isolates represented true rna recombinants and not revertants. based on the recombination rate in the s glycoprotein gene, the predicted distance between the ts alleles used in these crosses ranged from - bp to >l nucleotides (fig. ) . to determine if deletions were present within the s gene of rna recombinant viruses, a series of overlapping primers were synthesized that span the entire s glycoprotein gene of mhv-a . the location of these primers as well as the size of predicted pcr products are shown in fig. . cultures of cells were infected with various rna recombinant viruses and intercellular rna was harvested at - hr postinfection. following cdna synthesis and rounds of amplification with the taq polymerase and specific oligonucleotide primer pairs, the dna products were separated on % agarose gels and visualized by uv light or southern blotting techniques (fig. ) . in fig. a , pcr products spanning the hypervariable region of mhv-a , jhm, and mhv clearly demonstrate the amino acid deletion that is present in the jhm s glycoprotein gene (banner eta/., ; parker eta/., ) . southern blotting techniques using an internal oligomer probe demonstrate that the - . -kb fragment is specific for the mhv s glycoprotein sequences spanning nucleotides - and is not present in uninfected cells (fig. b) . no obvious deletions were detected through the hypervariable region (fig. c) , n-terminus, or c terminus in the s glycoprotein gene of over different rna recombinant viruses tested (fig. d) . since the distance between the group fl and f mutants would have required sizable deletions (>loo bp) to result in ts+ virus (fig. ) these data suggested that large deletions were rare events in these crosses, and did not contribute to an increase in the recombination frequency within the s glycoprotein gene. these studies cannot rule out the possibility of small deletions in one ts parent contributing to an increase in the ts+ progeny. however, no obvious deletions were detected in the s glycoprotein gene of different revertant viruses. in fig. e , pcr products spanning the hypervariable region of nc and its four revertants are presented. while these data cannot conclusively rule out the possibility of small deletions (~ bp) that would not be detected under these conditions, the data suggested that most revertants probably contain a single nucleotide reversion. although homologous recombination occurs during the replication strategy of several nonsegmented rna viruses (bujarski and kaesberg, ; cooper, ; hahn et al., ; king el al., ; weiss and schlesinger, ) , high frequency rna recombination is probably a unique phenomenon associated with mhv and perhaps the replication strategies of other coronavirus keck et al., keck et al., , a makino et a/., b makino et a/., , lai et a/., ) . using highly characterized ts mutants of mhv-a , we established a genetic recombination map in the polymer- predrcted nucleotide domarns of the group f rna' mutants. assumrng a recombination frequency of lo/ bp of dsrna and that la maps roughly . kb from the ' end of the s glycoprotern gene, the tentatrve nucleotide domains of the mutants mapping in the fl and f subgroups were predrcted. the hatched boxes represent the locatron of the region of polymorphism and putative hot spot of recombination in the mhv genome (banner et a/., ; parker et al., ) . arrows represent the approxrmate locatron of neutralizing epitopes in the s glycoprotein (routledge et al., ; weissmiller et a/., ) . to determine if recombinatron events resulting in deletions were contributing to the increased recombinatron rate in the s glycoprotern gene, a series of crosses were obtained between different rna+ mutants as shown at the top of the figure. pcr products spannrng different portions of the mhv s glycoprotein gene were obtained using specific oligomer products and the taq polymerase. the locatron of these primer pairs and their predicted products are shown in the bottom of the figure. to predict the maximum recombination frequency at the 'end of the genome, we calculated the recombination frequency between the three most distant rna-mutants (group a rna-mutants: la . la ; group f rna-mutant: la ) assuming that these mutants map at the most distant ends of the .kb polymerase region (- kb apart). to map the maximum recombination rate across the he and p genes, recombination rates were determined between mutants mapping approximately . kb apart at the ' end of the polymerase gene (la ) and the ' end of the s glycoprotein gene (group f rna+ mutants: la , la ). recombination rates were calculated through the s glycoprotein gene, assuming the group f rna+ mutants la and nc /nc map roughly . kb apart. ase genes of mhv. assuming that the recombination frequency was equivalent throughout the entire genome, these data suggested that the recombination frequency approached % or more . in this study, we have demonstrated that the ts mutants from a rna+ complementation group of mhv-a can also be arranged into an additive, linear, genetic map. several lines of evidence strongly suggest that the group f rna+ mutants map in the s glycoprotein gene of mhv-a . first, the group f mutants do not produce syncythium when maintained at the restrictive temperature. induction of cell fusion has been mapped to either the s domain of the bovine coronavirus s glycoprotein gene (yoo eta/., ) or the mhv sl and s glycoprotein domains (gallagher et al., ; routledge et al., ; weismiller eta/., ) . second, tl fingerprint analysis of rna recombinant viruses derived from crosses between ts la or la and mhv-jhm demonstrated that the mhv-a sl domain was always replaced by heterologous mhv-jhm sequences. since no other region in the mhv-a genome was uniformly replaced in recombinant viruses, these data strongly suggested that the mutations in these ts viruses reside within the sl coding sequences (keck et al., (keck et al., , a makino et al., b makino et al., , . finally, sequence analysis of rna recombinant viruses derived from these mutants place the ts allele in la within the ' most . kb of the sl glycoprotein gene (banner et a/., ) . while these data do not definitively prove that the group f mutants map in the s glycoprotein gene sequence, these data have strongly supported the localization of these mutations within this coding region. on the basis of recombination mapping data, the group f rna+ mutants appear to map into two discrete domains in the s glycoprotein gene sequence (fig. ) . data from our laboratory and others suggest that the fl mutants probably map within the sl glycoprotein while the f mutants map within the glycoprotein sequences (banner et a/., ; keck et al., keck et al., , a makino et al,, makino et al,, , . sequence analysis of the group f rna+ mutants and revertant viruses will be required to definitively map the location of these alleles. however, since mutants from both subgroups are incapable of producing syncytium at the restrictive temperature, these data suggest that both domains are important in eliciting cell fusion. currently, it is unclear whether the alterations in the fl and f conditional lethal mutants result in a temperature-sensitive fusogenic domain or alter the synthesis, transport, and surface expression of the s glycoprotein gene. the recombination frequency for the entire mhv genome has been estimated to approach % or more . these estimates were based on the assumption that the recombination frequency was uniform and approached / /l - nucleotides of dsrna throughout the entire -kb mhv genome. however, recombination rates measured within the polymerase region and s glycoprotein gene were estimated to occur at frequencies of about %/l - and %/ - bp of dsrna, respectively (fig. ) . recombination rates across the p /he genes were estimated to occur at about /o/l -l bp of dsrna. currently, our best estimate for an mhv recombination frequency was measured within the s glycoprotein gene because these mutations must have resided within a . -kb stretch of rna. moreover, the frequency of recombination was internally consistent between several independent crosses within a single complementation group (table ) . while definitive proof will require the identification of the exact location of the ts alleles used in these studies, these data suggest that the recombination frequency varies in different portions of the genome and is roughly threefold higher in the s glycoprotein gene. several explanations could account for an increase in the recombination frequency within the s glycoprotein gene. first, the recombination rate through the fig. . pcr ampliflcatron and size analysrs of the s glycoprotein gene of wildtype, ts, recombmant, and revertant viruses. cultures of cells were infected with different strains of mhv ts mutants, recombinant viruses, or revertant viruses. the intercellular rna was extracted and used as template for reverse transcription and rounds of pcr amplification usrng the taq polymerase and different primer parrs. (a) pcr products spanning the hypervariable region of mhva , jhm, and mhv- ( - ) (lanes - , respectively; lane , uninfected control); (b) southern blot analysis of the pcr products shown in (a) using an internal ollgomer probe; (c) pcr products spanning the hypervariable region in the s glycoprotein gene of mhv-a (lane ) and rna recombinant viruses derived from nc x nc [lanes - ) nc x la (lanes - ) and nc x la (lanes o-l ); (d) pcr products spanning the c (lanes - ) and n (lanes - ) terminl of mhv-a (lanes , ) and rna recombinant viruses derived from nc x nc (lanes - and -l ). nc x la (lanes - and -l ) and nc x la (lanes -l and ); (e) pcr products spanning the hypervariable region of nc (lane ) and four different revertant viruses (lanes - ). lane contains dna markers in all panels. polymerase region of mhv-a may be grossly under-la ) was unclear. this seems unlikely because a uniestimated because the exact location of the ts allele in form recombination frequency of lo/o/ nucleotides the group e mutant (la ) and group a mutants (la / of dsrna throughout the genome would: ( ) predict the recombination frequency for the mi-iv genome to approach - % or more; ( ) place rna-complementation groups a, b, and c in orf b and rna-complementation groups d and e in the p /he nonstructural proteins, and ( ) suggest that conditional lethal mutants were not isolated within orfla at the 'most three-fifths (- kb) of the genome. this is highly unlikely since several proteolytic, hydrophobic membrane-anchoring and cysteine-rich domains have been identified in orf a, which should be amenable to mutagenesis (baker et a/., ; lee et a/., ) . more importantly, tl fingerprint analysis of rna recombinant viruses derived from ts mutants in complementation groups c, d, and e clearly place each genetic function in the polymerase gene (keck et a/., ; lai et al., ) . since rna-complementation groups c, d, and e have also been demonstrated to function in mrna synthesis and map within orf b, which contains polymerase, helicase, and metal-binding sequence motifs, it is extremely unlikely that these mutants map in the he or p subgenomic orfs bredenbeck et a/., ; lee et al., ; schaad et al., ) . in contrast, the recombination rate predicted through the polymerase region was not inordinately high ( %/ - bp of dsrna), but rather closely approximated the recombination frequencies estimated to occur in apthovirus and poliovirus infections (cooper, ; king, ) . thus, it seems likely that the frequencies measured between the polymerase and s glycoprotein genes actually represent true differences in the recombination rate. since the enhanced recombination frequencies in mhv were observed within a physically smaller ( . -kb) region as compared to the -kb polymerase region, it is possible that a mechanism similar to high negative interference in dna viruses may account for the increased rates of recombination (chase and doermann, ) . this seems unlikely since high negative interference is probably mediated by dna polymerase repair mechanisms, which have not been demonstrated in the mhv rna polymerase (glickman and radman, ) . the increase in the recombination rate in the s glycoprotein gene could also not be attributed to a combination of true homologous recombination and recombination resulting in large deletions. nonhomologous recombination probably explains the appearance of defective interfering rnas of sindbis virus, vesicular stomatitis virus, and mhv (holland, ; makino et a/., monroe and schlesinger, ) . since deletions have been demonstrated in the mhv s and he glycoprotein genes as well as in the nonstructural genes encoded in mrna and and in the tgev s glycoprotein gene and other subgenomic orfs (ban-ner el al., ; la monica et al., ; luytjes et al., ; parker et al., ; rasschaert et al., i ; schwartz et al., ; yokomori and lai, ) , these data have suggested that the subgenomic orfs may be very amenable to frequent deletions and insertions. insertions in the mhv s glycoprotein gene have also been reported previously (taguchi et al., ) . analysis of rna recombinant viruses derived from different crosses between the group f mutants suggested that large deletions or insertions were rare and occurred at <&the rate of true homologous recombination. while these data could not conclusively rule out the presence of very small deletions, this seems unlikely since the predicted distances between the ts alleles used in these crosses should have resulted in deletions ranging from - bp to > bp in length. it is also unlikely that the ts+ virus isolated from these crosses were revertants containing small deletions (~ bp) in a singlets parent, since the ratio of recombinants: revertants was at least : or greater in each of the crosses and none of the revertants analyzed from each parental ts mutant had evidence of deletions. since revertants of sb ts mutants usually contain single nucleotide reversions at the site of mutation (hahn et al., a,b) , the most likely interpretation from these data is that the increase in the rate of recombination in the s glycoprotein gene probably reflects an increase in the rate of true homologous rna recombination. if the mechanism for homologous recombination and recombinations resulting in deletions are similar as suggested by banner et al., , these data suggest that the frequency of these two recombination events is very different. alternatively, the regions flanked by the ts mutations used in these crosses may be critical for s glycoprotein gene function or plaque formation and could not be deleted. two possible mechanisms could explain variable rates of recombination in the mhv genome. the first possibility is a preferred recombination site in the mhv s glycoprotein gene. a clustering of rna recombination sites adjacent to the hypervariable region in the s glycoprotein region suggest the presence of a preferred site of recombination (banner eta/., ) . however, in the absence of selection, crossover sites spanning the hypervariable region of the mhv s glycoprotein gene were random, suggesting that the preferred site of recombination reflects in vitro selection for certain types of recombinants . although the majority of data support non-site-specific homologous recombination throughout the entire poliovirus genome (kirkegaard and baltimore, ; sarnow et al,, ) brian, , and sawickr and sawicki, , have clearly demonstrated the presence of subgenomic minus strands in mhv and tgev infected cells. if the subgenomic minus strands partrcipate in template switching with full-length genomrc rnas, recombrnation rates should increase from the ' to the ' end of the genome. panel a demonstrates how template switching could occur during negative-strand synthesis between a full-length and subgenomic-length plus strand rna and result in a full-length negative-stranded rna recombinant molecule. panel b demonstrates how template switching between full-length and subgenomic negative strands during positrve-strand synthesis could result in recombinant genome-length molecules, switching occur after the synthesis of uu in sites chosen to minimize the adverse free energy change involved in switching to a heterotypic template (king, ) . in addition, a nonrandom distribution of recombination sites has also been reported among intertypic poliovirus recombinants (tolskaya et al., ) . if mhv rna recombination is mediated by freely segregating rna segments that are generated by transcriptional pausing during rna synthesis, preferred sites of recombination may also exist in au-rich regions and/or in regions of secondary structure in the mhv genome (baric et al., ) . however, it is difficult to envision how these types of preferred sites of recombination would result in higher intratypic recombination frequencies between mhv-a ts mutants since extensive secondary structure is found throughout the mhv genome and the g:c:a:u and aa/uu dimer ratios are roughly equivalent in the polymerase and s glycoprotein regions (banner et al., ; baric et al., ; soe et al., ; fu et al., unpublished) . a more likely mechanism to explain the increase in the recombination rate in the s glycoprotein gene is based on the basic replication strategy of coronaviruses (fig. ) . recently, subgenomic minus-strand and replicative intermediate rnas have been demonstrated during tgev and mhv infection (sethna et a/., (sethna et a/., , sawicki and sawicki, ) . these data indicate that the amount of negative-strand template rna is unequal and increases from the ' to ' end of the genome. because of the availability of more negative strand template, recombination rates should increase proportionately from the ' to ' end of the genome and be highest in the n gene coding region contained within mrna . for example, recombination events in the polymerase region can only involve template switching between full-length negative-or positive-strand rna templates. however, recombination events in the s glycoprotein gene or other subgenomic orfs could not only occur between full-length rna templates but also involve subgenomic mrna and subgenomic negative-strand rna templates as well. several findings support this hypothesis. first, recombination frequencies between the polymerase gene and s glycoprotein genes of mhv may vary threefold. second, in vitro transcribed subgenomic-length di rnas rapidly undergo rna recombination with fulllength (or subgenomic) rnas supporting the notion that recombination events can occur between different-sized template rnas . third, analysis of sepharose b-cl column purified full-length ri rna has demonstrated the presence of mrna subgenomic nascent-plus strands bound to the fulllength negative-strand rna (baric et al., ) . while the original interpretation of these data supported "leader-primed" transcription, an alternative explanation for these findings is that these subgenomic nascent-plus strands have disassociated from subgenomic ri rna template and recombined with the fulllength negative-strand rna. finally, a large number of rna recombinant viruses selected with markers in the s glycoprotein gene of mhv contain additional crossover sites in the m and n structural genes encoded at the ' end of the genome (keck et al., b) . these findings are consistent with the idea that subgenomic minus strands function in rna recombination and suggest that recombination events may increase in frequency toward the ' end of the genome. high-frequency rna recombination is a unique property associated with mhv replication. our data suggest that the frequency of rna recombination is mediated in part by the large size of the mhv genome ( kb) and its novel mechanism for rna synthesis involving "leader primed" discontinuous transcription and mrna replication via subgenomic minus strands (baric et al., (baric et al., , sethna et a/,, sethna et a/,, , sawicki and sawicki, ) . the possibility that the mhv recombination rate increases proportionately from the ' to ' 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recombination between sindbis virus rnas biosynthesis, structure, and biological activities of envelope protein gp of murine coronavirus mouse hepatitis virus s rna sequence revealed that nonstructural proteins ns and ns a are not essential for murine coronavirus replication. . viral the s subunit of the spike glycoprotein of bovine coronavirus mediates membrane fusion in insect cells we thank sheila peel, lorraine alexander and mary schaad for helpful comments and criticisms. this work was supported by american heart association grant (aha -l , aha - ) and a grant from the national institutes of health (a ). this work was done during the tenure of an established investigator of the american heart association (aha - ) (r.s.b.). key: cord- - lvn f authors: shi, stephanie t.; huang, peiyong; li, hsin-pai; lai, michael m.c. title: heterogeneous nuclear ribonucleoprotein a regulates rna synthesis of a cytoplasmic virus date: - - journal: the embo journal doi: . /emboj/ . . sha: doc_id: cord_uid: lvn f heterogeneous nuclear ribonucleoprotein (hnrnp a ) is involved in pre-mrna splicing in the nucleus and translational regulation in the cytoplasm. in the present study, we demonstrate that hnrnp a also participates in the transcription and replication of a cytoplasmic rna virus, mouse hepatitis virus (mhv). overexpression of hnrnp a accelerated the kinetics of viral rna synthesis, whereas the expression in the cytoplasm of a dominant-negative hnrnp a mutant that lacks the nuclear transport domain significantly delayed it. the hnrnp a mutant caused a global inhibition of viral mrna transcription and genomic replication, and also a preferential inhibition of the replication of defective-interfering rnas. similar to the wild-type hnrnp a , the hnrnp a mutant complexed with an mhv polymerase gene product, the nucleocapsid protein and the viral rna. however, in contrast to the wild-type hnrnp a , the mutant protein failed to bind a kda cellular protein, suggesting that the recruitment of cellular proteins by hnrnp a is important for mhv rna synthesis. our findings establish the importance of cellular factors in viral rna-dependent rna synthesis. introduction hnrnp a is an rna-binding protein that contains two rna-binding domains (rbds) and a glycine-rich domain responsible for protein±protein interaction. it is involved in pre-mrna splicing and transport of cellular rnas (reviewed by dreyfuss et al., ) . it is predominantly located in the nucleus, but also shuttles between the nucleus and the cytoplasm (pin Äol-roma and dreyfuss, ) . the signal that mediates shuttling has been identi®ed as a amino acid sequence, termed m , located near the c-terminus of hnrnp a between amino acids and (michael et al., ; siomi and dreyfuss, ; weighardt et al., ) . yeast two-hybrid screening with m as bait resulted in the discovery of a novel transportin-mediated pathway for nuclear import of hnrnp a (pollard et al., ; fridell et al., ; siomi et al., ) . the function of the cytoplasmic hnrnp a has not been well de®ned. studies have shown that cytoplasmic and nuclear hnrnp a exhibit different rna-binding pro®les. cytoplasmic hnrnp a is capable of high-af®nity binding to au-rich elements that modulate mrna turnover and translation (hamilton et al., (hamilton et al., , henics et al., ) . it has also been shown to promote ribosome binding to mrnas by a cap-mediated mechanism, and prevent spurious initiation at aberrant translation start sites (svitkin et al., ) . mhv belongs to the coronaviridae family of positivesense, single-stranded rna viruses. mhv replication and transcription occur exclusively in the cytoplasm of infected cells via the viral rna-dependent rna polymerase (rdrp) (reviewed by lai and cavanagh, ) . initially, the ¢-most gene of the viral genome is translated into the viral rdrp, which then replicates the viral genomic rnas into negative-strand rnas. subsequently, the negative-strand rnas are used as templates to transcribe mrnas, which include a genomic-sized rna and a nested set of subgenomic mrna transcripts, all with an identical ¢ non-translated leader sequence of ± nucleotides and ¢ co-terminal polyadenylated ends. the subgenomic mrna transcription of mhv utilizes a unique discontinuous mechanism in which the leader sequence, often derived from a different molecule, is fused to rnas at the intergenic (ig) sites (i.e. transcription initiation site) to generate subgenomic mrnas (jeong and makino, ; liao and lai, ; zhang et al., ) . the exact mechanism of how these mrnas are made is still controversial. however, it has been shown that the process of discontinuous rna transcription is regulated by several viral rna elements, including the cis-and trans-acting leader rna zhang et al., ) , ig sequence (makino et al., ) and ¢-end untranslated sequence (lin et al., ) . there is considerable biochemical evidence suggesting possible direct or indirect interactions between the various rna regulatory elements. hnrnp a binds mhv negative (±)-strand leader and ig sequences (furuya and lai, ; li et al., ) . site-directed mutagenesis of the ig sequences demonstrated that the extent of binding of hnrnp a to the ig sequences correlated with the ef®ciency of transcription from the ig site (zhang and lai, ; li et al., ) . immunostaining of hnrnp a showed that hnrnp a relocated to the cytoplasm of mhv-infected cells, where viral rna synthesis occurs (li et al., ) . hnrnp a also mediates the formation of a ribonucleoprotein complex containing the mhv (±)-strand leader and ig sequences . these results suggest that hnrnp a may serve as a protein mediator for distant rna regions to interact with each other. heterogeneous nuclear ribonucleoprotein a regulates rna synthesis of a cytoplasmic virus the embo journal vol. no. pp. ± , ã european molecular biology organization many cellular proteins, including calreticulin (singh et al., ) , polypyrimidine tract-binding protein (ptb) (hellen et al., ; wu-baer et al., ) , la protein (pardigon and strauss, ), sam (mcbride et al., ) , poly(rc)-binding protein (parsley et al., ) and nucleolin (waggoner and sarnow, ) , have been implicated to be involved in viral rna transcription or replication. in addition to mhv, hnrnp a has also been reported to interact with human cytomegalovirus immediate-early gene protein, which plays an important role in the regulation of virus replication (wang et al., ) . furthermore, a yeast protein related to human core rna splicing factors, lsm p, has been shown to be required for the ef®cient replication of brome mosaic virus rna (diez et al., ) . recently, reddy and colleagues demonstrated an inhibition of hiv replication by dominant-negative mutants of sam (reddy et al., ) . however, none of these cellular proteins has been shown experimentally to participate directly in rna-dependent rna synthesis. in order to demonstrate the involvement of hnrnp a in mhv rna replication and transcription, we established several dbt cell lines stably expressing either the wildtype (wt) hnrnp a or a c-terminus-truncated mutant lacking the m sequence and part of the glycine-rich domain. we showed that the mutant hnrnp a , which was localized predominantly in the cytoplasm, exhibited dominant-negative effects on viral genomic rna replication and subgenomic mrna transcription. in contrast, overexpression of the wt hnrnp a accelerated the synthesis of all viral rnas. our results provide strong evidence that hnrnp a is directly or indirectly involved in mhv rna synthesis in the cytoplasm and that the c-terminal part of the protein is important for its function. this ®nding thus reveals a novel function for hnrnp a in the cytoplasm. characterization of stable cell lines expressing the wt and a c-terminus-truncated hnrnp a to explore a potential role for hnrnp a in mhv rna synthesis, we established murine dbt cell lines stably expressing the flag-tagged wt hnrnp a (dbt-a ) or a mutant hnrnp a , which has a amino acid deletion from the c-terminus (dbt-a dc) ( figure a ). this mutant lacks part of the glycine-rich domain and the m sequence responsible for shuttling hnrnp a between the nucleus and the cytoplasm. immunoblot of the whole-cell lysates with an anti-flag antibody detected a kda protein in dbt-a cells and a kda protein in three independent clones of dbt-a dc cells ( figure b ), whereas no protein was cross-reactive to the anti-flag antibody in the control cell line stably transfected with the pcdna . vector (dbt-vec). the amounts of the flagtagged wt and truncated hnrnp a were comparable in these cell lines. a chicken polyclonal antibody against hnrnp a detected two endogenous hnrnp a isoforms or hnrnp a -related proteins in the whole-cell lysates of all of the cell lines. the bottom band ( kda) overlaps the flag-tagged wt hnrnp a in dbt-a cells. there was only a slight increase in the overall amount of hnrnp a in dbt-a cells as compared with dbt-vec cells, indicating that the exogenous hnrnp a constituted a small fraction of the total hnrnp a in the cells. in dbt-a dc cells, an additional band of smaller size ( kda) corresponding to the mutant hnrnp a was detected. the overall expression levels of the exogenous hnrnp a and hnrnp a dc were~ -fold lower than that of the endogenous hnrnp a in whole-cell lysates ( figure b ). similar to the endogenous hnrnp a protein (pin Äol-roma and dreyfuss, ) , the flag-tagged wt hnrnp a was localized almost exclusively in the nucleus ( figure c ). the mutant hnrnp a , however, was localized predominantly in the cytoplasm ( figure c) , consistent with the previous ®nding that the m nuclear localization signal is necessary to localize hnrnp a to the nucleus weighardt et al., ) . thus, hnrnp a dc was much more abundant than the endogenous hnrnp a in the cytoplasm. the expression levels of the wt or mutant hnrnp a varied among individual cells based on immuno¯uorescent staining ( figure c ). the growth rate ( figure d ) and cell morphology (data not shown) were similar among the different cell lines. the effects of overexpression of the wt and mutant hnrnp a on syncytium formation and virus production we ®rst assessed the effects of hnrnp a overexpression on the morphological changes induced by mhv-a infection using several different clones of dbt cell lines. virus infection was performed at a multiplicity of infection (m.o.i.) of . to detect the subtle morphological differences among the different cell lines. syncytia appeared at~ h post-infection (p.i.) in dbt-vec cells and~ h earlier in dbt-a cells. at both and h p.i., syncytia were signi®cantly larger and more spread out in dbt-a cells than those in dbt-vec cells ( figure a ). similar differences were observed with two additional clones of dbt-a cells (data not shown). in contrast, no syncytium was observed in three different clones of dbt-a dc cells, even at h p.i. at h p.i., almost all dbt-a cells detached from the plate, but~ ± % of dbt-vec cells still remained on the plate (data not shown). remarkably, there was no sign of syncytium formation in dbt-a dc cells until h after virus infection, when the overall morphology of the cells was similar to that of dbt-vec cells at h p.i. (data not shown). all of the dbt-a dc cells were eventually killed at~ h p.i., suggesting that the inhibition of viral replication was not a result of the disruption of the mhv receptor. correspondingly, virus production from these cell lines was signi®cantly different. between and h p.i., virus production from dbt-a dc cells was -to -fold less than that from dbt-vec and dbt-a cells ( figure b ). dbt-a cells produced twice as many viruses as those from dbt-vec cells during that time period. relocalization of hnrnp a during mhv infection mhv rna synthesis occurs exclusively in the cytoplasm of infected cells. in order for hnrnp a to participate directly in viral transcription, it has to be recruited to the site of rna synthesis. although hnrnp a shuttles between the nucleus and the cytoplasm in normal cells (pin Äol-roma and dreyfuss, ) , the level of cytoplasmic hnrnp a is very low. we have demonstrated previously that hnrnp a relocates from the nucleus to the cytoplasm of mhv-infected cells (li et al., ) . to determine whether the overexpressed hnrnp a may participate in mhv rna synthesis, we performed immunostaining experiments using an anti-flag antibody to localize flag-tagged hnrnp a . in dbt-a cells, a signi®cant increase in the cytoplasmic level of hnrnp a and a corresponding decrease of nuclear hnrnp a were observed in virus-infected cell syncytia at h p.i. ( figure b ); these cells express the mhv nucleocapsid (n) protein in the cytoplasm ( figure a ). by comparison, in the uninfected cells, which did not have n protein staining, hnrnp a was predominantly localized to the nucleus (arrow in figure b ). in dbt-a dc cells, very few cells were stained positive for the mhv n protein at h p.i. ( figure c ). signi®cantly, the viral n protein was detected only in the cells that were stained weakly or not at all for flag-hnrnp a ( figure d ), suggesting that the expression of a high level of hnrnp a dc interfered with viral replication. the effects of wt and mutant hnrnp a on mhv protein production we further investigated the effects of the wt and mutant hnrnp a on the production of mhv structural and nonstructural proteins. cytoplasmic protein was extracted from infected cell lines at different time points after infection for immunoblot analysis to detect an open reading frame (orf) a product, p (lu et al., ) and the n protein. p expression in dbt-vec cells was clearly detected at h p.i. and peaked at~ h p.i. ( figure a ). in dbt-a cells, p appeared at h p.i. and peaked at~ h p.i. in dbt-a dc cells, no p protein was detected until h p.i. similar patterns of differences were observed for the n protein in these three cell lines. actin levels in different cell lines remained relatively constant throughout the infection, except that, in dbt-a cells, actin was not detected at and h p.i. due to the loss of the dead cells ( figure a ). these results clearly demonstrated that overexpression of the wt hnrnp a accelerated viral protein production, whereas expression of the mutant hnrnp a delayed it. we also performed immuno¯uorescent staining of the n protein at h p.i. to further con®rm the western blot results. as represented by images shown in figure b , there were more dbt-a cells stained positive for the n protein than dbt-vec cells. very few cells were found to express the n protein in dbt-a dc cells. the p and n proteins appeared as doublets in some of the lanes of figure a , but the results varied from experiment to experiment. the n protein is known to be phosphorylated (stohlman and lai, ) . whether p is post-translationally modi®ed is not known. figure a ). dbt-a cells showed a signi®cantly higher level of [ h]uridine incorporation, which peaked at~ h p.i. dbt-a dc cells did not show any detectable level of incorporation of the radioactivity. these results suggest that hnrnp a regulates mhv rna synthesis. we further assessed the production of genomic and subgenomic mhv rnas in these cell lines by northern blot analysis. the genomic and the six subgenomic rna species were detected at h p.i. in both dbt-vec and dbt-a cells; there were signi®cantly higher steady-state levels of all of the rna species in dbt-a cells ( figure b ). in contrast, no viral rna was detected in dbt-a dc cells at that time point. at h p.i., mhv rna levels in dbt-vec and dbt-a cells decreased generally because of the loss of the dead cells, while the smaller subgenomic rnas became detectable in dbt-a dc cells. by h p.i., most viral rna species became detectable in dbt-a dc cells ( figure b , lane ), while most of the dbt-a cells were dead (lane ). these results con®rmed that the synthesis of all of the viral rna species is accelerated by overexpression of the wt hnrnp a and delayed by a dominant-negative mutant of hnrnp a . in this analysis, we also detected an additional rna species (arrow in figure b ), which was determined to be a defective-interfering (di) rna by northern blot analysis using a probe representing the ¢-untranslated region (without the leader), which is present only in genomic and di rnas (data not shown). interestingly, this di rna was inhibited to a greater extent than other rna species in dbt-a dc cells. this result suggests that the replication of di rnas is more sensitive to the dominant-negative inhibition by cytoplasmic hnrnp a . to demonstrate further that mhv rna transcription machinery is defective in cells expressing the mutant hnrnp a , we studied transcription of an mhv di rna, cat, which contains a transcription promoter (derived from the ig sequence for mrna , ig ) and a chloramphenicol acetyltransferase (cat) reporter gene . cat activity can be expressed from at h p.i., serum-free medium was replaced by virus growth medium containing % ncs and mg/ml actinomycin d. [ h]uridine ( mci/ml) was added to the infected cells at , , , , , , , , and h p.i. after h labeling, cytoplasmic extracts were prepared and precipitated with % tca. the tca-precipitable counts were measured in a scintillation counter. (b) northern blot analysis of mhv genomic and subgenomic rna synthesis in dbt cells. cytoplasmic rna was extracted from mhv-a -infected cells at , and h p.i. for northern blot analysis. the naturally occurring di rna of mhv-a is indicated by an arrow. this di rna only if a subgenomic mrna containing cat sequences is produced . the cat rna was transfected into mhv-a -infected cells h after infection. at h p.i., cat activity in dbt-a cells was signi®cantly higher than that in dbt-vec cells ( figure a ). on the other hand, cat activity was very low in dbt-a dc cells. at h p.i., cat activity in dbt-a cells became slightly lower than that in dbt-vec cells because of the loss of the dead dbt-a cells. the cat activity in dbt-a dc was still signi®cantly lower than that in dbt-vec or dbt-a cells. these results established that mrna transcription from the di rna was also inhibited by hnrnp a dc. the results shown above ( figure b ) also suggest that di rna replication is more sensitive to the inhibitory effects of the hnrnp a mutant. to con®rm this result, we further studied replication of another di rna during serial virus passages. dbt cells were infected with mhv-a and transfected with disse rna derived from jhm virus (makino and lai, ) ; the virus released (p ) was passaged twice in dbt cells to generate p and p viruses. dbt cells were infected with these viruses, and cytoplasmic rna was extracted for northern blot analysis using glyoxalated rna for a better resolution of smaller rnas. for dbt-a dc cells, rna was extracted at h p.i. since viral rna synthesis was delayed in this cell line. cells infected with p viruses did not yield detectable amounts of disse, but contained the naturally occurring a di rna, whose replication was inhibited more strongly than the synthesis of mhv genomic and subgenomic rnas in dbt-a dc cells ( figures b, lanes ± and b, lanes ± ). however, this a di rna was not detectable in cells infected with p and p viruses ( figure b , lanes ± ). in contrast, disse appeared in cells infected with p viruses and further increased in cells infected with p viruses, indicating that the replication of the smaller disse may have an inhibitory effect on the replication of the larger a di rna (jeong and makino, ) . similar to the a di rna, the replication of disse rna was much more strongly inhibited than that of mhv genomic and subgenomic rnas in dbt-a dc cells ( figure b , lanes and ). our results thus suggest that mhv di rna replication is more dependent on the function of cytoplasmic hnrnp a . the mechanism of dominant-negative inhibition by the c-terminal deletion mutant of hnrnp a to understand the underlying mechanism of the inhibition of mhv rna transcription by the c-terminal-deletion mutant of hnrnp a , we ®rst examined the rna-and protein-binding properties of this mutant protein. electrophoretic mobility shift assay demonstrated that hnrnp a dc retained the ability to bind the mhv (±)strand leader rna and to form multimers with itself, similar to the wt hnrnp a (data not shown); this is consistent with the fact that both of its rbds are intact ( figure a) . furthermore, uv-crosslinking experiments showed that increasing amounts of puri®ed glutathione s-transferase (gst)±hnrnp a dc ef®ciently competed with the endogenous hnrnp a for the binding of the mhv (±)-strand leader rna ( figure a ), indicating that the binding of hnrnp a dc to rna was not affected. these results suggest that the rna-binding properties of hnrnp a dc were intact. we next examined the protein-binding properties of hnrnp a dc. since hnrnp a has been shown to interact with the n protein, which also participates in mhv rna synthesis (compton et al., ; wang and zhang, ) , we ®rst determined whether the dominantnegative mutant of hnrnp a retained the ability to interact with the n protein in vitro. gst pull-down assay using various truncation mutants of hnrnp a showed that the n protein bound the n-terminal domain (aa ± ) of hnrnp a ( figure b) ; thus, the binding of hnrnp a dc [equivalent to hnrnp a ( ± )] to the n protein was not affected. we next examined the in vivo interaction of the wt and mutant hnrnp a with an mhv orf a product, p , which has been shown to co-localize with the de novo synthesized viral rna (s.t.shi and the viruses were passaged twice in wt dbt cells to obtain p and p viruses. cytoplasmic rna was extracted from the dbt cells infected with p , p and p viruses and treated with glyoxal before electrophoresis and northern blot analysis using a p-labeled (±)-strand mrna as a probe. the a di rna and disse rna are indicated by arrows. m.m.c.lai, unpublished results) and associate with the viral replicase complex (gibson bost et al., ) . cytoplasmic extracts from mhv-a -infected cells were immunoprecipitated with anti-flag antibody-conjugated beads, followed by western blotting with a rabbit polyclonal antibody against p . at h p.i., p was co-precipitated with the flag-tagged hnrnp a from dbt-a cells, whereas no precipitation of p was observed in dbt-vec cells ( figure c ). for dbt-a dc cells, co-immunoprecipitation was performed at h p.i., when abundant mhv proteins were synthesized. p was shown to co-precipitate with hnrnp a dc, indicating that hnrnp a dc still formed a complex with the viral polymerase gene product. these results suggest that the ability of hnrnp a dc to interact with the n and polymerase proteins was not altered. we next investigated whether the mutant hnrnp a is de®cient in the interaction with any other cellular proteins in this rna±protein complex. we labeled proteins in mhv-infected cells or mock-infected cells at different time points after infection and immunoprecipitated with the anti-flag antibody. signi®cantly, a cellular protein of kda was shown to be associated only with the wt hnrnp a , but not the mutant hnrnp a ( figure d ), suggesting that hnrnp a binds to this protein through its c-terminal domain. we propose that this cellular protein is another important component of the mhv rna transcription/replication complex. there is an accumulating body of evidence signifying the importance of cellular factors in rna synthesis of rna viruses (reviewed by lai, ) . previous studies have shown that hnrnp a binds to the cis-acting sequences of mhv template rna and that this interaction correlates with the transcription ef®ciency of viral rna in vivo (zhang and lai, ; li et al., ) . in addition, hnrnp a is also implicated in viral rna replication by the recent ®nding that hnrnp a interacts with the ¢-ends of both positive-and negative-strand mhv rna (p.huang and m.m.c.lai, unpublished results). however, hnrnp a modulates cytoplasmic viral rna synthesis the functional importance of hnrnp a in viral rna synthesis has so far not been directly demonstrated. in the present study, we established that mhv rna transcription and replication were enhanced by overexpression of the wt hnrnp a protein, but inhibited by expression of a dominant-negative hnrnp a mutant in dbt cell lines. our results suggest that hnrnp a is a host protein involved in the formation of a cytoplasmic transcription/ replication complex for viral rna synthesis. this represents a novel function for hnrnp a in the cytoplasm. our results indicate that the inhibitory effects on mhv replication exhibited by the dominant-negative mutant of hnrnp a were relatively more prominent than the enhancement effects by overexpression of the wt hnrnp a . this is consistent with the subcellular localization patterns of the wt and mutant hnrnp a proteins. the overexpressed exogenous wt hnrnp a in dbt-a cells was predominantly localized in the nucleus, similar to the endogenous hnrnp a ( figure c ). the c-terminal-deletion mutant, however, was localized mainly in the cytoplasm. thus, the level of hnrnp a dc was much higher than the endogenous wt hnrnp a in the cytoplasm of dbt-a dc cells, where mhv replication occurs. this result explains why hnrnp a dc could have a strong dominant-negative inhibitory effect, despite the fact that it was expressed at a lower level than the endogenous hnrnp a ( figure b) . the effects of the expression of the wt and mutant hnrnp a on virus production ( figure b ), viral protein synthesis ( figure a ) and viral rna synthesis ( figure a ) correlated with each other. furthermore, hnrnp a dc caused not only a global inhibition of genomic rna replication and subgenomic mrna transcription, but also a preferential inhibition of at least two di rna species. these results suggest that the inhibition of mhv replication by the hnrnp a mutant was most likely a direct effect on viral rna synthesis rather than an indirect effect on other aspects of cellular or viral functions. since hnrnp a binds directly to the cis-acting mhv rna sequences critical for mhv rna transcription (li et al., ) and replication (p. huang and m.m.c.lai, unpublished results) , it is most likely that hnrnp a may participate in the formation of the transcription/replication complex. indeed, our data show that hnrnp a interacts directly or indirectly with the n protein and a gene product, p , both of which are probably associated with the viral transcription/replication complex (compton et al., ; wang and zhang, ; gibson bost et al., ) . hnrnp a may participate directly in viral rna synthesis in a similar role to that of transcription factors in dnadependent rna synthesis, e.g. by maintaining favorable rna conformation for rna synthesis. alternatively, hnrnp a may modulate mhv rna transcription or replication by participating in the processing, transport and controlling the stability of viral rnas. it has been reported that rna processing of retroviruses, human t-cell leukemia virus type (black et al., ) and hiv- (black et al., ) , is altered by the binding of hnrnp a to the viral rna regulatory elements. it is also possible that hnrnp a may participate in mhv rna synthesis indirectly by affecting the production of other host cell proteins, which may, in turn, regulate mhv rna synthesis. since hnrnp a is a dose-dependent altern-ative splicing factor (caceres et al., ) , even small changes in the intracellular level of hnrnp a can alter the splicing of other cellular proteins. regardless of the mechanism, our study established the importance of cellular factors in viral rna-dependent rna synthesis. the transcription from cat rna was strongly inhibited by the dominant-negative mutant of hnrnp a , as shown by cat assays ( figure a ). in addition, the replication of the naturally occurring a di rna and the arti®cial disse rna was completely abolished ( figure b) . surprisingly, the replication of mhv di rnas suffered a stronger inhibition by the dominantnegative mutant of hnrnp a than the synthesis of mhv genomic and subgenomic rnas, suggesting that di rna replication may be more dependent on hnrnp a . although di rnas contain all of the cis-acting replication signals that are essential for their replication in normal cells (kim and makino, ) , the small size of di rna may cause it to require more hnrnp a to maintain a critical rna structure. it has been shown that different di rnas require different cis-acting signals for rna replication (kim and makino, ) . our results demonstrate that the c-terminal domain of hnrnp a , including the m sequence and the glycinerich region, is important for mhv rna transcription and replication, but the mechanism of the dominant-negative effects of hnrnp a dc is still not clear. hnrnp a dc retains the rna-binding and self-association ability and is capable of binding the viral proteins n and p , which are associated with the transcription/replication complex. it is possible that hnrnp a dc is not productive due to its inability to interact with other viral or cellular proteins that are involved in mhv rna synthesis. we have found a protein of~ kda that binds only the wt, but not the mutant hnrnp a ( figure d ). it remains to be shown whether this cellular protein is involved in mhv rna synthesis. in our preliminary study, we found that mhv could replicate in an erythroleukemia cell line, cb , which was reported to lack detectable hnrnp a expression as a result of a retrovirus integration in one allele and loss of the other allele (ben-david et al., ) . since hnrnp a protein is involved in a variety of important cellular functions, including rna splicing, transport, turnover and translation, it is conceivable that other redundant gene products may substitute for the function of hnrnp a in cb cells. indeed, uv-crosslinking assays using cb cell extracts detected two proteins comparable to hnrnp a in size that could interact with the mhv negative-strand leader rna (data not shown). these proteins may represent hnrnp a -related proteins, since many of such hnrnps exist in the cells (buvoli et al., ; burd et al., ) . therefore, multiple cellular proteins may have the capacity to be involved in mhv rna synthesis. based on previous ®ndings (kim and makino, ; zhang and lai, ; li et al., ) and the results from this study, we propose a model for the regulation of transcription/replication of mhv rna by hnrnp a . we hypothesize that hnrnp a is one of the components of the mhv rna transcription or replication complex, and the crosstalk between hnrnp a and another viral or cellular rna-binding protein (designated x in figure ) is essential for mhv replication and transcription. the x protein binds to the c-terminus of hnrnp a and cooperates with hnrnp a to recruit more proteins to form the transcription or replication complex. the c-terminaldeletion mutant of hnrnp a loses the ability to interact with the x protein and to bring it into the initiation complex, resulting in an inhibition of mhv rna transcription and replication. the residual replication and transcription activities of mhv rna in the absence of functional hnrnp a may be due to a limited af®nity of the x protein to a cis-acting signal that is only present in mhv genomic rna (site b). on the other hand, di rnas may lack this cis-acting signal. when the crosstalk between the x protein and hnrnp a is abolished by the dominant-negative mutant of hnrnp a , the x protein can no longer participate in the formation of the initiation complex, resulting in a complete loss of di rna replication. in summary, our data provide direct experimental evidence that hnrnp a is involved directly or indirectly in mhv rna synthesis, probably by participating in the formation of an rna transcription/replication complex. this ®nding reveals a novel cytoplasmic function for hnrnp a . cells and viruses dbt cells, a mouse astrocytoma cell line (hirano et al., ) , were cultured in eagle's minimal essential medium (mem) supplemented with % newborn calf serum (ncs) and % tryptone phosphate broth. mhv strain a (robb and bond, ) was propagated in dbt cells and maintained in virus growth medium containing % ncs. plasmid construction and establishment of dbt stable cell lines the cdna of the murine hnrnp a gene was ampli®ed by rt±pcr using rna extracted from dbt cells and a set of primers representing the ¢-and ¢-ends of hnrnp a -coding region, and cloned into pcdna . (invitrogen, carlsbad, ca). the amino acid flag tag was attached to the n-terminus of hnrnp a by including the flag tag in the forward pcr primer. the truncated hnrnp a dc was similarly constructed using a pcr-ampli®ed fragment that represents hnrnp a (aa ± ). for the establishment of permanent dbt cell lines, pcdna . alone or the plasmid containing the flag-tagged hnrnp a or hnrnp a dc was transfected into % con¯uent dbt cells using dotap according to the manufacturer's instructions (boehringer mannheim, indianapolis, in) . after h, the transfected cells were selected in dbt cell medium containing . mg/ml geneticin (g ) (omega scienti®c, tarzana, ca) for days. single colonies were then collected and cultured individually for additional days before screening for the expression of flag-tagged proteins. the polyclonal rabbit antibody against p was a gift from dr susan c.baker at loyola university, il. the chicken polyclonal antibody against hnrnp a was produced by aves labs, inc. (tigard, or) by immunizing chickens with the puri®ed mouse hnrnp a protein expressed in bacteria. the polyclonal anti-flag antibody was purchased from af®nity bioreagents (golden, co). the goat polyclonal antibody against actin was obtained from santa cruz biotechnology (santa cruz, ca). the mouse monoclonal antibody against the n protein has been described previously (fleming et al., ) . examination of growth rate of permanent dbt cells equal numbers ( ) of dbt-vec, dbt-a and dbt-a dc cells were plated in -cm culture plates and maintained in culture medium for days. cells were trypsinized, stained with trypan blue (gibco-brl, grand island, ny) and counted at -h intervals with a hemacytometer (hausser scienti®c, horsham, pa). plaque assay dbt cells in -cm plates were infected with mhv-a at an m.o.i. of . after h for virus adsorption, the cells were washed three times with serum-free mem, which was then replaced with virus growth medium containing % serum. at , , , , and h p.i., ml of medium was taken from each plate for plaque assay. [ h]uridine labeling of mhv rna cells plated in -well plates were infected with mhv-a at an m.o.i. of . at h p.i., mg/ml actinomycin d was added to the virus growth medium to inhibit cellular rna synthesis. to label newly synthesized mhv rna, mci/ml of [ h]uridine (nen, boston, ma) were added to the medium at hourly intervals. after h of labeling, the cells were washed twice in ice-cold pbs and scraped off the plates in ml of pbs. the cells were then collected by centrifugation and incubated in ml of nte buffer ( mm nacl, mm tris ph . , mm edta) containing . % np- , . mm dithiothreitol (dtt) and u/ml of rnasin on ice for min. after centrifugation, ml of the cytoplasmic extract were spotted on a piece of mm paper and incubated with % trichloroacetic acid (tca). the radioactivity remaining on the mm paper was measured in a scintillation counter. northern blot analysis dbt cells were infected with mhv-a at an m.o.i. of . at , and h p.i., cytoplasmic extract was prepared as described above and subjected to phenol/chloroform extraction and ethanol precipitation to purify cytoplasmic rna. approximately mg of rna were separated by electrophoresis on a . % formaldehyde-containing agarose gel and transferred to a nitrocellulose membrane. for a better resolution of the disse rna ( figure b ), rna was glyoxalated before being electrophoresed on a % agarose gel. an in vitro transcribed, p-labeled negative-strand mrna of mhv-jhm was used as a probe to detect mhv genomic and subgenomic rnas. for detecting di rna species, rna blots were probed with an rna representing a sequence complementary to the sequence of the ¢-untranslated region of mhv-jhm rna, but excluding the leader sequence. western blot analysis dbt cells in -well plates were infected with mhv-a and cytoplasmic extracts were prepared as described previously (li et al., ) at various hnrnp a modulates cytoplasmic viral rna synthesis time points p.i. the extracts were electrophoresed on a % polyacrylamide gel and transferred to a nitrocellulose membrane for western blotting. immuno¯uorescence staining cells were washed in phosphate-buffered saline (pbs) and ®xed in % formaldehyde for min at room temperature, followed by min in ± °c acetone. primary antibodies were diluted in % bovine serum albumin and incubated with cells for h at room temperature. after three washes in pbs,¯uorescein-conjugated secondary antibodies were added to cells at : dilution for h at room temperature. fitc-or tritcconjugated secondary antibodies were used to generate green or red uorescence. cells were then washed in pbs and mounted in vectashield (vector laboratories, burlingame, ca). uv-crosslinking assay uv-crosslinking assay was performed as described previously (huang and lai, ) . in brief, dbt cell extracts ( mg protein), mg/ml trna and c.p.m. of an in vitro transcribed, p-labeled negativestrand mhv ¢-end rna ( bp) were incubated for min at °c. increasing amounts of puri®ed gst ( , . , . and ng) or recombinant gst±hnrnp a fusion protein ( , , and ng) were included in the reaction to compete with the endogenous hnrnp a for binding. the reaction mixture was placed on ice and uv-irradiated in a uv stratalinker (stratagene) for min, followed by digestion with mg/ml rnase a for min at °c. the protein±rna complexes were then separated on a % sds±polyacrylamide gel and visualized by autoradiography. gst pull-down assay gst pull-down was performed as described previously (tu et al., ) . in brief, gst±hnrnp a fusion proteins on glutathione beads (pierce, rockford, il) were incubated with the in vitro translated, s-labeled n protein in . ml of gst-binding buffer containing . % np- for h at °c. the beads were washed ®ve times with the gst-binding buffer containing . % np- . proteins bound to beads were eluted by boiling in laemmli buffer for min and separated on a % polyacrylamide gel. [ s]methionine labeling and immunoprecipitation dbt cells were infected with mhv-a at an m.o.i. of . the cells were incubated with methionine-free medium for min before labeling and were labeled in mci/ml [ s]methionine starting at . , or h p.i. after labeling for h at each time point, the cells were harvested for protein extraction as described previously (li et al., ) . the protein extracts were immunoprecipitated with anti-flag antibody-conjugated beads (sigma, st louis, mo) in tm buffer ( mm tris±hcl ph . , . m kcl, . mm mgcl , mm edta, % glycerol, mm dtt, . % np- , mm phenylmethylsulfonyl¯uoride) at °c for h. the immunoprecipitates were washed and separated on a ± % gradient sds±polyacrylamide gel and visualized by autoradiography. plasmid cat was linearized by xbai and in vitro transcribed with t rna polymerase to produce the di rna . the di rna was transfected into mhv-a -infected dbt cells using dotap as described previously (huang and lai, ) . in brief,~ % con¯uent dbt cells were infected by mhv-a at an m.o.i. of . at h p.i., the cells were transfected with mg of in vitro transcribed di rna and incubated at °c for the desired lengths of time. to amplify the di rna, viruses (p ) were passaged twice in wt dbt cells to generate p and p viruses. cells were harvested at or h p.i. and lysed by freezing and thawing for three times. after centrifugation at r.p.m. for min, the supernatant was used in a cat assay as described previously (lin et al., ) . retroviral insertions downstream of the heterogeneous nuclear ribonucleoprotein a gene in erythroleukemia cells: evidence that a is not essential for cell growth speci®c binding of polypyrimidine tract binding protein and hnrnp a to hiv- crs elements primary structure of the heterogeneous nuclear ribonucleoprotein a , b and c proteins: a diversity of rna binding proteins is generated by small peptide inserts cdna cloning of human hnrnp protein a reveals the existence of multiple mrna isoforms regulation of alternative splicing in vivo by overexpression of antagonistic splicing factors in vitro replication of mouse hepatitis virus strain a identi®cation and characterization of a host protein required for ef®cient template selection in viral rna replication ) hnrnp proteins and the biogenesis of mrna antigenic relationships of murine coronaviruses: analysis using monoclonal antibodies to jhm (mhv- ) virus nuclear import of hnrnp a is mediated by a novel cellular cofactor related to karyopherin-b three different cellular proteins bind to the complementary sites on the ¢-end positive-and ¢-end negative-strands of mouse hepatitis virus rna four proteins processed from the replicase gene polyprotein of mouse hepatitis virus colocalize in the cell periphery and adjacent to sites of virion assembly association of heterogeneous nuclear ribonucleoprotein a and c proteins with reiterated auuua sequences modulation of auuua response element binding by heterogeneous nuclear ribonucleoprotein a in 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nucleolar±cytoplasmic relocalization of nucleolin in poliovirus-infected cells the nucleocapsid protein of coronavirus mouse hepatitis virus interacts with the cellular heterogeneous nuclear ribonucleoprotein a in vitro and in vivo the interaction between human cytomegalovirus immediate-early gene (ie ) protein and heterogeneous ribonucleoprotein a nucleo-cytoplasmic distribution of human hnrnp proteins: a search for the targeting domains in hnrnp a identi®cation of a group of cellular cofactors that stimulate the binding of rna polymerase ii and trp- to human immunode®ciency virus tar rna interactions between the cytoplasmic proteins and the intergenic (promoter) sequence of mouse hepatitis virus rna: correlation with the amounts of subgenomic mrna transcribed coronavirus leader rna regulates and initiates subgenomic mrna transcription both in trans and in cis formation of a ribonucleoprotein complex of mouse hepatitis virus involving heterogeneous nuclear ribonucleoprotein a and transcriptionregulatory elements of viral rna. virology, , ± hnrnp a modulates cytoplasmic viral rna synthesis we thank dr susan c.baker at loyola university, il, for generously providing the polyclonal antibody against p . we also thank drs jong-won oh, guang yang, deborah r.taylor and peter koetters for their helpful discussions. this work was partially supported by a national institutes of health research grant. s.t.s. is supported by a postdoctoral fellowship from the national institute for allergy and infectious diseases, national institutes of health. m.m.c.l. is an investigator of the howard hughes medical institute. key: cord- - lntii authors: talbot, pierre j.; buchmeier, michael j. title: antigenic variation among murine coronaviruses: evidence for polymorphism on the peplomer glycoprotein, e date: - - journal: virus research doi: . / - ( ) - sha: doc_id: cord_uid: lntii abstract a panel of monoclonal antibodies (mab) against the structural proteins of murine hepatitis virus- , strain jhm (mhv- ) was used in three antigen binding assays to determine the extent of antigenic homology among six strains of murine coronaviruses. the antigenic determinants studied were highly conserved on the e glycoproteins and nucleocapsid (n) proteins of all strains tested. in contrast, antigenic polymorphism was observed among the e glycoproteins. of three previously described antigenic determinants against which neutralizing antibodies are directed, only one, termed a(e ), was conserved on all strains. antigenic site b(e ) was found only on the strongly neurotropic mhv- and site c(e ) was present on the virulent mhv- and mhv- (hepatotropic) strains, but absent on the weakly pathogenic mhv-a , mhv- and mhv-s strains. four non-neutralizing antibodies against at least one topographically distinct antigenic determinant, which we previously designated d(e ), gave binding patterns consistent with two distinct sites. one of these was present on all mhv strains tested and the other was present on all strains except mhv-s. these non-neutralizing antigenic sites were redesignated e(e ) and d(e ) respectively. murine hepatitis viruses (mhv) are coronaviruses which cause a variety of diseases such as hepatitis, gastroenteritis and encephalomyelitis (siddell et al., a (siddell et al., , b wege et al., ) in the natural host. despite these variations in pathogenesis, the viruses are closely related in structure and in their mode of replication. viral rna is complexed with n protein (& = - ~) and the envelope is studded with petal-shaped peplomers composed of a major glycoprotein, e (m, - ) made up of two nonidentical dalton subunits. a second glycoprotein, el (m, = - ), spans the membrane and interacts internally with the viral rna. el may determine the intracellular site of virus maturation (siddell et al., ; sturman and holmes, ) . rna homologies between strains of mhv with different virulence characteristics and organ tropisms have been analyzed. oligonucleotide fingerprinting analysis of the highly virulent hepatotropic strain mhv- and the weakly pathogenic mhv-a strain revealed structural differences in the genes coding for the rna polymerase and the e glycoprotein (lai et al., ) . in similar analyses with other strains, each mhv isolate yielded a unique oligonucleotide pattern. the neurotropic mhv- strain showed the highest degree of difference among the strains tested, which also included mhv- , and a (wege et al., ; weiss and leibowitz, ) . hybridization studies with cdna clones of the viral rnas revealed extensive nucleic acid homologies between mhv strains (cheley et al., ; weiss and leibowitz, ) . more recently sequence analyses of the n gene indicated an overall % homology between the strongly neurotropic mhv- and the weakly neurotropic mhv-a (armstrong et al., ; skinner and siddell, ) . antigen& homologies were initially probed in cross-neutralization assays (hierholzer et al., ; taguchi et al., ; wege et al., ) and neutralization kinetics (childs et al., ) . these studies showed that mhv strains could be distinguished, but that significant levels of cross-reactivity occurred. strains mhv- and were closely related, as were strains mhv- , , s and a . there was no correlation between serological typing and virulence. these neutralization assays likely measured antigenic homologies at the level of the e glycoprotein, which is the target for neutralizing antibodies (collins et al., ; fleming et al., ) . tryptic maps of the n proteins from five mhv strains also showed a strong level of similarity (cheley et al., ) . recently, antigen&c relationships among eleven mhv isolates were probed in radioi~unoassays with monoclonal antibodies to mhv (fleming et al., ) . extensive antigenic variation of the e glycoprotein was observed but a correlation was drawn between less extensive variations in the n protein and pathogenicity. el glycoprotein was markedly conserved among the mhv isolates tested. mhv- is a useful animal model for virus-induced primary demyelination (haspel et al., ; knobler et al., a knobler et al., , lampert et al., ; nagashima et al., ; sorensen et al., ; stohlman and weiner, ; weiner, ) . rapidly fatal encephalitis normally masks the more slowly developing demyelinating disease in mice (knobler et al., b; stohlman and weiner, ; weiner, ) . how-ever, infection with mutants or variants of mhv- results in survival with accompanying primary demyelination, due to infection of the oligodendrocyte (haspel et al., ; stohlman et al., ; knobler et al., ) . monoclonal antibodies (mab) to mhv structural proteins were raised and characterized as described previously (collins et al., ) . a minimum of four topographically distinct antigenic determinants on e and two on el were mapped by competition binding assays (talbot et al., a) . at least two distinct antigenic determinants were mapped on the n protein (talbot et al., ) . antibodies to sites a, b and c(e ) were capable of neutralizing viral infectivity in vitro and passive antibody transfer experiments showed that neutralizing antibodies to sites a and b(e ) but not c(e ) could protect mice from a lethal intracerebral virus challenge. such antibodies modulated disease from fatal encephalitis to sublethal demyelination (buchmeier et al., ) . in the present study, we have used a panel of eighteen mab to mhv generated in our laboratory and an independently derived panel of ten additional mab to e raised by fleming et al. ( ) to analyze antigenic relationships among six mhv strains differing in virulence and organ tropism. these were mhv (jhm), both the wild-type (wt) strain (cheever et al., ) and the ts mutant (haspel et al., ) ; mhv- (dick et al., ) ; mhv-a (manaker et al., ) ; mhv- (gledhill and andrewes, ); and mhv-s (rowe et al., ) . murine hepatitis virus strains a , and s were purchased from the american type culture collection. dr. julian leibowitz kindly provided strain mhv- , as well as dbt cells. the origin and cultivation of the wt and ts strains of mhv was described previously (haspel et al., ) . all viruses were plaque purified two or three times, propagated and assayed on l- . or dbt cells. the generation and characterization of mab to the structural proteins of mhv- was described previously (collins et al., ; buchmeier et al., ; talbot et al., a) . in addition to our panel we obtained an independently generated panel of ten mab to the e glycoprotein of mhv-jhm kindly supplied by dr. john fleming, usc medical school, los angeles (fleming et al., ) . for indirect immunofluorescence, l- . cells grown on coverslips were infected with each mhv strain at a mo of . - . and fixed in cold acetone when extensive syncytia were observed ( - h after infection). antigenic preparations for dot immunoblotting and enzyme immunoassays (eia) consisted of microsomal fractions from mhv infected or uninfected control cells, as described previously (talbot et al., a) . indirect immunofluorescence was performed as described previously (collins et al., ) using monoclonal hybridoma cell culture fluids undiluted or after a lo-fold concentration in a macrosolute concentrator, type b- (amicon corp., danvers, ma). the degree of reactivity was scored qualitatively on a scale of to by comparison with positive and negative controls. enzyme immunoassays were as described previously (talbot et al., a) except that -fold concentrates of tissue culture fluids were used ( -fold initial concentration by precipitation with % (w/w) saturated ammonium sulfate followed by -fold additional concentration as described above). viral antigens were in the form of subcellular fractions, hence it was necessary to calibrate the amount of protein used for each virus strain. this was accomplished by ~determining, in preliminary titrations, the quantity of antigen necessary to saturate the binding of an optimal dilution of a mixture of five mab to the highly conserved n protein as previously described (talbot et al., a) . dot immunoblotting assays were performed as previously detailed (talbot et al., b ) in a bio-dot microfiltration apparatus (bio-rad laboratories, richmond, calif. ). an excess of viral antigen (as determined by enzyme immunoassay) was bound to nitrocellulose and reacted with six serial -fold dilutions of each mab culture fluid. bound immunoglobulin was detected by incubation of the well nitrocellulose replica in a mm petri dish with ' -labeled, affinity-purified goat antibody to mouse immunoglobulin (h + l chain specificity). after autoradiography, radioactivity bound on each dot was quantitated by gamma counting, and comparisons were made in the linear portion of the antibody binding curves. as with enzyme immunoassays, a positive reaction was scored as greater than twice background binding, a plus/minus reaction as twice background binding and a negative reaction as less than twice background binding. indirect immunofluorescence on acetone-fixed cells was initially used to screen hybridoma antibodies and proved to be a helpful tool to delineate antigenic relationships among mhv strains (fig. ) . intense fluorescence staining was seen in multinucleated giant cells infected by strains of mhv in which a particular antigenic site was conserved. in this example, the variation of the neutralization sites, previously assigned to the e glycoprotein (talbot et al., a) , is shown for three mhv strains. epitope a(e ), recognized by mab b . was conserved among these and all other mhv strains tested. in contrast, epitope b(e ), recognized by mab a . , was unique to mhv- . epitope c(e ) recognized by mab b . was observed on mhv and mhv- but not on the other strains tested. staining of control uninfected cells with these mab was always at background negative levels. a second antigen-binding assay was developed to confirm antigenic relationships initially determined by indirect immunofluorescence. we have previously used the dot blotting assay to analyze detergent sensitivities of different viral epitopes (talbot et al., b) . in the present study, this assay was modified into a solid-phase radioimmunoassay using nitrocellulose as an efficient antigen-binding matrix. the is x assay was very sensitive and quantitative (jahn et al., ; talbot et al., b) , and also provided a visual representation of results. determined by enzyme immunoassay to be in excess of that required for maximal antibody binding was applied on the nitrocellulose solid support. serial dilutions of mab culture fluids were then reacted and detected with a radiolabeled second antibody. after autoradiography, each dot was cut out and counted for *"i radioactivity. comparisons between antibody binding on viral antigens from different mhv strains were established in the linear portion of the binding curves and the results obtained are shown in fig. . binding of each mab to control antigen dots was negligible ( - cpm), whereas specific binding on viral antigen dots reached levels of up to cpm. background binding on mhv- was often higher than on the other mhv strains (fig. , mab a . and b . ) and this background binding was not diluted out with decreasing concentration of antibody. in agreement with the results of fleming et al. ( ) , extensive conservation of the el glycoprotein was evident and similar levels of antibody binding were seen on each mhv strain, with the exception of the ts mutant of mhv- . antigenic variation was observed in the n protein, particularly with mhv-a and mhv-s. however, the most extensive polymorphism was seen on the e glycoprotein. as was shown by indirect immunofluorescence, site a(e ) was conserved among all mhv strains tested, whereas site b(e ) was specific for mhv- and site c(e ) was specific for mhv- and mhv- . four non-neutralizing mab directed against at least one antigenic site provisionally designated d(e ) (talbot et al., a) gave two distinct binding patterns. mab b . and - b . reacted with all strains tested except mhv-s, whereas mab b . and b . reacted with all of the strains we tested. thus, these two pairs of antibodies likely recognize at least two different structures on the e glycoprotein, which we have redefined d(e ) and e(e ), respectively. in order to confirm the antigenic polymorphism observed on the e glycoprotein, we obtained a panel of mab against e generated independently by fleming et al. ( ) , and assayed these antibodies for strain-specific binding in the dot immunoblotting assay described above. results of such an experiment are shown in fig. . polymorphism similar in extent to that described above was also observed with these antibodies. moreover, similarities in binding patterns emerged between these. two panels of mab. mab . . , . . and . . were similar to our neutralizing mab b . and . . was similar to our a . . all of these antibodies, except . . neutralized virus in vitro (fleming et al., ; collins et al., ) . non-neutralizing antibodies . . and . . showed a similar binding pattern to that of antibodies against nonneutralizing site e ( b . and b . ). the binding pattern of antibodies to antigenic site d was not observed with the fleming panel of antibodies. conversely, the specificity patterns of antibodies . . and . . , as well as that of . . and . . were unique to the panel obtained from fleming. a third antigen-binding assay was also used to delineate antigenic relationships. an enzyme immunoassay (eia) which we have previously described (talbot et al., a) was applied to the present study. we found eia to be less sensitive than either the immunofluorescence or dot blotting assays when mab culture fluids were used. thus, it was necessary to concentrate these fluids to be used in eia. nevertheless, only ten of eighteen mab could reliably be used in the assay. the cumulative results of the eia are combined with results of the two other antigen-binding assays and presented in table . in dot immunoblotting assays and eia, a positive reaction was scored as greater than twice background binding. a plus or minus reaction corresponded to approximately twice background binding and' a negative reaction to less than twice background binding. the results of all three antigen binding assays generally agreed, with a few exceptions. for example, eia results were sometimes negative but dot blotting and immunofluorescence results scored as positive, such as mab b . and b . with mhv- and s, mab b . and b . for mhv- and mab b . for mhv ts . similarly, dot blotting results could be negative while immunofluorescence was positive, such as mab b . and b . for mhv rs , mab b . for mhv-a and s and mab b . for mhv-a . in the latter case, eia results were also positive. such discrepancies likely result from differences in assay sensitivity and/or antigen stability. it is apparent from table that nearly complete conservation of el glycoprotein antigens exists among the six mhv strains tested. some variation was shown on the n protein in agreement with fleming et al. ( ) . antigenic site a(n) is apparently lost on mhv-s and a distinct determinant recognized by mab b . is lost on mhv-a . extensive antigenic variation is evident on the e glycoprotein. the three different antigen binding assays confirm conservation of sites a and e(e ) but strain variation was observed in sites b, c and d(e ). site b(e ) was only found on the neurotropic strain mhv , whereas site c(e ) was present on the two virulent (fleming et al., ) . the experiment was performed as described in the legend to fig. . the hybridoma designation is on the left. all these mab were specific to e , except . . which is directed to el. strains mhv- and - but lost on the weakly pathogenic mhv strains a , and s. finally, site d(e ) was found on all mhv strains tested, with the exception of mhv-s. monoclonal antibodies against the jhm strain of murine hepatitis virus (mhv- ) were used in three different antigen binding assays to delineate antigenic relationships among the structural proteins of six mhv strains. in agreement with studies of fleming and colleagues (fleming et al., ) , we found extensive conservation of antigenic determinants on the internal viral protein n, and particularly on the transmembrane glycoprotein, el. using eighteen mab against the peplomer glycoprotein, e and raised in two laboratories, we found significant antigenic polymorphism on this glycoprotein. in contrast to the report of fleming and colleagues (fleming et al., ) , we found that antigenic variation of the e glycoprotein was a better correlate of pathogenicity of a given virus strain than was variation in the n protein. variation on e has been correlated with previously mapped antigenic determinants (talbot et al., a) . neutralizing antibodies a . and b . are directed against two topographically distinct antigenic determinants designated b and c(e ), respectively (talbot et al., a) . in separate studies, detection of these two determinants was found to be dependent on the native conformation of the molecule (talbot et al., b) . in the present study, we found minimal conservation of these two antigenic sites in the mhv strains tested: site b(e ) was only recognized on the mhv- strain, which is known to be strongly neurotropic (siddell et al., ; wege et al., ) and site c(e ) was preserved on the two virulent strains mhv and mhv- but absent on the weakly pathogenic strains mhv-a , mhv- and mhv-s. a third neutralization site on the mhv e glycoprotein, designated a(e ), was conserved among all mhv strains tested. separate studies have shown that this site is stable to sodium dodecyl sulfate (sds) denaturation suggesting that it may consist of a primary sequence of amino acids (talbot et al., b) and that the frequency of mutation at this site reflected as antibody resistance is very low (data not shown). conservation of this determinant and its resistance to antibody selection may indicate a structure that is essential for productive infection. conservation of site a(e ) may account for the reported cross-neutralization between different mhv strains (childs et al., ; hierholzer et al., ; taguchi et al., ; wege et al., ) . in other studies (talbot et al., ) we have shown that mice infected with mhv- , a- , mhv- and mhv-s all respond by producing antibody to site a(e ). finally, non-neutralizing sites d and e(e ), which are also stable to sds denaturation (talbot et al., b) , are extensively conserved among mhv strains, with the exception of mhv-s which has lost site d(e ). in contrast to the small plaque variant of mhv- generated by stohlman and colleagues (stohlman et al., ) , the ts mutant generated by haspel and colleagues (haspel et al., ) appears to be antigenically indistinguishable from the wild-type mhv- strain. in our study using three different antigen binding assays, we assumed that a positive reaction in any one of the assays was an indication of antigenic conservation and that only in those instances where a negative reaction was obtained in all assays could we conclude a loss of antigenic determinants. in several instances, the importance of using more than one assay is stressed since often a positive reaction was found in only one or two assays. our studies use two independently derived panels totaling mab against the structural proteins of mhv to analyze antigenic relationships among murine hepatitis viruses. in part these studies confirm and extend the studies of fleming et al. ( ) which showed extensive conservation of antigenic determinants on mhv el and n proteins and minimal conservation of epitopes on the e glycoprotein. this report differs from the previously reported studies in that we found a better correlation between antigenic variation, organ tropism and virulence at the level of the e glycoprotein rather than the previously reported correlation in n. this may reflect the larger number of e antibodies employed in the present study. we have also confirmed the identity of each mhv as a separate strain with a distinctive mosaic of antigenic markers. more detailed structural studies on e , its functional properties, and interaction(s) with cellular receptors will be helpful to gain a better understanding of the molecular basis for coronavirus tropism and virulence. coronavirus induced subacute demyelinating encephalitis in rats: a morphological analysis mouse hepatitis virus infection as a highly contagious, prevalent, enteric infection of mice the structure and replication of coronaviruses the biology of coronaviruses coronavirus jhm: nucleotide sequence of the mrna that encodes nucleocapsid protein in vivo and in vitro models of demyelinating disease. iii. jhm virus infection of rats chronic central nervous system demyelination in mice after jhm virus infection key: cord- -bh xgl authors: snijder, e.j.; decroly, e.; ziebuhr, j. title: the nonstructural proteins directing coronavirus rna synthesis and processing date: - - journal: adv virus res doi: . /bs.aivir. . . sha: doc_id: cord_uid: bh xgl coronaviruses are animal and human pathogens that can cause lethal zoonotic infections like sars and mers. they have polycistronic plus-stranded rna genomes and belong to the order nidovirales, a diverse group of viruses for which common ancestry was inferred from the common principles underlying their genome organization and expression, and from the conservation of an array of core replicase domains, including key rna-synthesizing enzymes. coronavirus genomes (~ – kilobases) are the largest rna genomes known to date and their expansion was likely enabled by acquiring enzyme functions that counter the commonly high error frequency of viral rna polymerases. the primary functions that direct coronavirus rna synthesis and processing reside in nonstructural protein (nsp) to nsp , which are cleavage products of two large replicase polyproteins translated from the coronavirus genome. significant progress has now been made regarding their structural and functional characterization, stimulated by technical advances like improved methods for bioinformatics and structural biology, in vitro enzyme characterization, and site-directed mutagenesis of coronavirus genomes. coronavirus replicase functions include more or less universal activities of plus-stranded rna viruses, like an rna polymerase (nsp ) and helicase (nsp ), but also a number of rare or even unique domains involved in mrna capping (nsp , nsp ) and fidelity control (nsp ). several smaller subunits (nsp –nsp ) act as crucial cofactors of these enzymes and contribute to the emerging “nsp interactome.” understanding the structure, function, and interactions of the rna-synthesizing machinery of coronaviruses will be key to rationalizing their evolutionary success and the development of improved control strategies. coronaviruses (covs) are the best-known and best-studied clade of the order nidovirales, which is comprised of enveloped plus-stranded (+rna) viruses and currently also comprises the arteriviridae, roniviridae, and mesoniviridae families (de groot et al., a,b; lauber et al., ) . in addition to including various highly pathogenic covs of livestock (saif, ) and four "established" human covs causing a large number of common colds (pyrc et al., ) , covs have attracted abundant attention due to their potential to cause lethal zoonotic infections (graham et al., ) . this was exemplified by the outbreak of severe acute respiratory syndrome-coronavirus (sars-cov) in southeast asia and the ongoing transmission-since -of the middle east respiratory syndromecoronavirus (mers-cov), which causes $ % mortality among patients seeking medical attention. both these viruses are closely related to covs that are circulating in bats (ge et al., ; menachery et al., ) and other potential reservoir species. they may be transmitted to humans either directly or through intermediate hosts, like civet cats for sars-cov (song et al., ) and dromedary camels for mers-cov (reusken et al., ) . formally, the family coronaviridae now includes about species, divided into the subfamilies torovirinae and coronavirinae, the latter being further subdivided in the genera alpha-, beta-, gamma-, and deltacoronavirus. sars-cov and mers-cov are betacoronaviruses, and the same holds true for one of the best-characterized animal cov models, murine hepatitis virus (mhv). this explains why the bulk of our current knowledge of cov molecular biology is betacoronavirus based, even more so for the replicative proteins that are the central theme of this review, which will mainly summarize data obtained studying sars-cov proteins. despite their unification in the same virus order, nidoviruses cover an unusually broad range of genome sizes, ranging from $ - kilobases (kb) for arteriviruses, via $ kb for mesoniviruses, to $ - kb for covs (nga et al., ) . together with the genomes of roniviruses, which infect invertebrate hosts, cov genomes are the largest rna genomes known to date . the common ancestry of these extremely diverse virus lineages was inferred from their polycistronic genome structure, the common principles underlying the expression of these genomes, and-most importantly-the conservation of an array of "core replicase domains," including key enzymes required for rna synthesis. while retaining this conserved genomic and proteomic blueprint, nidovirus genomes are thought to have expanded gradually by gene duplication and acquisition of novel genes (lauber et al., ) , most likely by rna recombination. in addition to the high mutation rate that characterizes all rna viruses, these genomic innovations appear to have enabled nidoviruses to explore an unprecedented evolutionary space and adapt to a wide variety of host organisms, including mammals, birds, reptiles, fish, crustaceans, and insects. whereas the poor replication fidelity generally restricts rna virus genome sizes, it has been postulated that nidovirus genome expansion was enabled by the acquisition of specific replicative functions that counter the error rate of the rna polymerase (deng et al., ; eckerle et al., ; snijder et al., ) (discussed in more detail later). as in all nidoviruses, at least two-thirds of the cov genome capacity is occupied by the two large open reading frames (orfs) that together constitute the replicase gene, orf a and orf b (fig. ). these orfs overlap by a few dozen nucleotides and are both translated from the viral genome, with expression of orf b requiring a - ribosomal frameshift to occur just fig. outline of the cov genome organization and expression strategy, based on sars-cov. the top panel depicts the sars-cov genome, including various regulatory rna elements, and the -and -coterminal nested set of subgenomic mrnas used to express the genes downstream of the replicase gene. utr, untranslated region; trs, transcription-regulatory sequence. below the rnas, the open reading frames in the genome are indicated, i.e., the replicase orfs a and b, the four common cov structural protein genes (s, e, m, and n) and the orfs encoding "accessory proteins." the bottom panel explains the organization and proteolytic processing of the pp a and pp ab replicase polyproteins, the latter being produced by - ribosomal frameshifting. the nsp (pl pro ) and nsp ( cl pro ) proteases and their cleavage sites are indicated in matching colors. the resulting cleavage products (nonstructural proteins (nsps)) are indicated, as are the conserved replicase domains that are relevant for this review. domain abbreviations and corresponding nsp numbers: pl pro , papain-like proteinase (nsp ); cl pro , c-like proteinase (nsp ); tm, transmembrane domain (nsp , nsp , and nsp ); niran, nidovirus rdrp-associated nucleotidyl transferase (nsp ); rdrp, rna-dependent rna polymerase (nsp ); zbd, zinc-binding domain (nsp ); hel , superfamily helicase (nsp ); exon, exoribonuclease (nsp ); n -mt, n -methyl transferase (nsp ); endou, uridylate-specific endoribonuclease (nsp ); -o-mt, -omethyl transferase (nsp ). upstream of the orf a termination codon (brierley et al., ) . the efficiency of this highly conserved frameshift event, which may approach % in the case of covs (irigoyen et al., ) , is promoted by specific primary and higher-order rna structures. as a result, in cov-infected cells, the replicase subunits encoded in orf a are overexpressed in a fixed ratio relative to the proteins encoded in orf b. the primary translation products of the cov replicase are two huge polyproteins, the orf a-encoded pp a and the c-terminally extended pp ab frameshift product (fig. ) . the former is roughly - amino acids long, depending on the cov species analyzed. the size of the orf b-encoded extension is more conserved (around residues), resulting in pp ab sizes in the range of - amino acids. probably already during their synthesis, either two or three orf aencoded proteases initiate the proteolytic cleavage of pp a and pp ab to release (sometimes) or (mostly) functional nonstructural proteins (nsps; fig. ). the highly conserved nsp protease has a chymotrypsin-like fold ( clike protease, cl pro ) (anand et al., (anand et al., , gorbalenya et al., ) and is the viral "main protease" (therefore sometimes also referred to as m pro ). the cl pro cleaves the nsp -nsp part of pp a and the nsp -nsp part of pp ab at and conserved sites, respectively. these sites can be summarized with the p -p consensus motif (small)-x-(l/i/v/f/m)-q#(s/a/g), where x is any amino acid and # represents the cleavage. the processing of three sites in the nsp -nsp region is performed by one or two papain-like proteases (pl pro ) residing in the very large nsp subunit (mielech et al., ) . whereas alphacoronaviruses and most betacoronaviruses (though not sars-cov and mers-cov) have two pl pro domains in their nsp , presumably the result of an ancient duplication event, gamma-and deltacoronaviruses have only a single pl pro . the cleavage sites (lxgg# or similar) resemble the c-terminal lrgg# motif of ubiquitin, which explains why cov pl pro domains were found capable to also act as deubiquitinases (ratia et al., ) . this secondary function has been implicated in the disruption of host innate immune signaling by removing ubiquitin from certain cellular substrates. more than any other cov-encoded enzyme, the cov cl pro and pl pro domains have been characterized in exquisite structural and biochemical detail, both in their capacity of critical regulators of nsp synthesis and as two of the primary drug targets for this virus family. space limitations unfortunately prevent us from summarizing these studies in more detail, but a variety of excellent reviews is available to compensate for this omission (baez-santos et al., ; hilgenfeld, ; mielech et al., ; steuber and hilgenfeld, ) . once released from pp a and pp ab, most covs nsps studied thus far assemble into a membrane-bound ribonucleoprotein complex that drives the synthesis of different forms of viral rna (see later) and is sometimes referred to as the replication and transcription complex (rtc) . while viral rna production takes off, peculiar convoluted membrane structures, spherules tethered to zippered endoplasmic reticulum, and doublemembrane vesicles begin to accumulate in cov-infected cells (gosert et al., ; knoops et al., ; maier et al., ) . as for other +rna viruses, they have been postulated to serve as scaffolds, or perhaps even suitable microenvironments, for viral rna synthesis. nevertheless, many questions on their biogenesis and function remain to be answered, and the exact location of the metabolically active rtc still has to be pinpointed "beyond reasonable doubt" for covs and other nidoviruses (hagemeijer et al., ; neuman et al., a; van der hoeven et al., ) . three orf a-encoded replicase subunits containing transmembrane domains (nsp , nsp , and nsp ; fig. ) have been implicated in the formation of the membrane structures that are induced upon cov infection and with which the rtc is thought to be associated (angelini et al., ; hagemeijer et al., ) . in addition to actively engaging in host membrane remodeling, they may serve as membrane anchors for the rtc by binding the nsps that lack hydrophobic domains, like all of the orf b-encoded enzymes. for more details, the reader is referred to the numerous recent reviews of the "replication organelles" of covs and other +rna viruses (den boon and ahlquist, ; hagemeijer et al., ; neuman et al., a; romero-brey and bartenschlager, ; van der hoeven et al., ; xu and nagy, ) . the common ancestry of nidovirus replicases is not only reflected in their conserved core replicase domains but also in the synthesis of subgenomic (sg) mrnas that are used to express the genes located downstream of orf b ( fig. ) . although some nidoviruses (e.g., roniand mesoniviruses) have only a few of these genes, they are much more numerous in arteriviruses and covs, their number going up to about a dozen orfs for some covs. in addition to the standard set of four cov structural protein genes (encoding the spike (s), envelope (e), membrane (m), and nucleocapsid (n) protein), genomes in different cov clusters contain varying numbers of orfs encoding so-called "accessory proteins" narayanan et al., ) . the proteins they encode are often dispensable for the basic replicative cycle in cultured cells, but highly relevant for cov viability and pathogenesis in vivo, for example, because they enable the virus to interfere with the host's immune response. most of the genes downstream of orf b are made accessible to ribosomes by positioning them at the end of their own sg transcript. occasionally, two or even three genes are expressed from the same sg mrna, usually by employing ribosomal "leaky scanning" during translation initiation. nidoviral sg mrnas are -coterminal with the viral genome, but in most nidovirus taxa, including covs, the sg transcripts also carry common leader sequences ($ - nucleotides in covs), which are identical to the -terminal sequence of the viral genome ( fig. ) (pasternak et al., ; sawicki et al., ; sola et al., ) . the joining of common leader and different sg rna "body" sequences occurs during minus-strand rna synthesis (sawicki and sawicki, ; sethna et al., ) . this step can be either continuous, to produce the full-length minus strand required for genome replication, or interrupted (discontinuous) to produce a subgenome-length minus-strand rna that can subsequently serve as the template for the synthesis of one of the sg mrnas. the polymerase jumping that is the basis for leader-to-body joining occurs at specific "transcriptionregulatory sequences" (trss). these conserved sequence motifs are comprised of up to a dozen nucleotides, and are found in the genome at the end of the leader sequence and at the end of each of the sg mrna bodies. quite likely, also higher-order rna structure and transcription-specific protein factors play a role in the interruption of minus-strand rna synthesis at a body trs, after which the nascent minus strand (with a body trs complement at its end) is translocated to the -proximal part of the genomic template. guided by a base-pairing interaction with the leader trs, the synthesis of the subgenome-length minus-strand rna is resumed and completed with the addition of the complement of the genomic leader sequence. in this manner, a nested set of subgenome-length templates for sg mrna synthesis is produced, providing a mechanism to regulate the abundance of the different viral proteins by fine-tuning the level at which the corresponding sg mrna is generated (nedialkova et al., ) . the cov transcription strategy allows the rtc to use the same -terminal recognition/initiation signals in both full-and subgenome-length templates of either polarity. moreover, the presence of the common leader sequence may be important for mrna capping or other translation-related features. during the past two decades, studies on the cov enzyme complex that controls this elegant replication and transcription mechanism have been accelerated by four important developments. first, using bioinformatics, expression systems, and virus-infected cells, the replicase polyprotein processing scheme and the proteases involved were elucidated, thus defining the boundaries of the mature nsps (fig. ) that are working together during cov replication (ziebuhr et al., ) . second, using this information and promoted by rapidly advancing methods in structural biology, x-ray or nmr structures were obtained for numerous (recombinant) full-length cov nsps or domains thereof, in particular for sars-cov (neuman et al., b) . third, multiple techniques for the targeted mutagenesis of cov genomes were developed and refined, which was a specific technical challenge due to the exceptionally large size of the cov rna genome (almazan et al., ) . by launching engineered mutant genomes in susceptible cells, the rna and protein players in the cov replication cycle can now be interrogated directly, to reveal their importance, function(s) and/or interactions in vivo. finally, in vitro biochemical assays were developed for a variety of cov replicative enzymes, including many of those involved in rna synthesis and processing. for the purpose of this review, we have chosen to focus on these latter functions, as performed by the cov nsp to nsp products nga et al., ; sevajol et al., ; subissi et al., a) . these subunits include several replicative enzymes that are more or less universal among + rna viruses, such as rna polymerase (nsp ) and helicase (nsp ), but also a number of rare or even unique domains involved in, e.g., mrna capping, cap modification, and promoting the fidelity of cov rna synthesis. several smaller subunits, in particular nsp to nsp , have been identified as crucial cofactors of these enzymes and contribute to the emerging cov "nsp interactome," which will likely need to be advanced considerably to achieve a more complete understanding of the intricacies of cov rna synthesis. making that step will obviously be key to understanding the evolutionary success of covs, and nidoviruses at large. moreover, this knowledge will lay the foundation for the development of improved strategies to combat current and future emerging covs, including targeted antiviral drug development. . coronavirus nsp - : small but critical regulatory subunits? the -terminal part of orf a, the approximately . kb separating the nsp -coding sequence and the orf a/ b ribosomal frameshift site, encodes a set of four small replicase subunits, named nsp to nsp (fig. ) . although highly conserved among coronavirinae, these proteins seem to lack enzymatic functions. instead, they have emerged as (putative) interaction partners and modulators of orf b-encoded core enzymes like nsp (rna-dependent rna polymerase, rdrp), nsp (exoribonuclease, exon), and nsp (ribose -o-methyl transferase, -o-mtase). furthermore, several of them have been predicted or shown to interact with rna. additionally, a fifth, very small cleavage product is assumed to be released from this region of pp a: the nsp peptide resulting from cleavage of pp a at the nsp / junction (fig. ) . in the pp ab frameshift product, the n-terminal sequence of nsp (encoded between the nsp / junction and orf a/ b frameshift site) equals the n-terminal part of the nsp subunit. depending on the cov species, nsp consists of - residues and its actual release, function (if any), or fate in cov-infected cells have not been established. in cell culture models, for some (infectious bronchitis virus (ibv)) but not other (mhv) covs, the nsp / and nsp / cleavages were found to be dispensable for virus replication (deming et al., ; fang et al., ) , even though the conservation of this cleavage site suggests that it is generally required for full replicase functionality. processing of the nsp -nsp region of pp a/pp ab has been studied in some detail for mhv (bost et al., ; deming et al., ) , human cov e (hcov- e) (ziebuhr and siddell, ) , and ibv (ng et al., ) , confirming the release of these subunits in infected cells and the use of the predicted cl pro cleavage sites. processing at these sites was found to be critical for mhv replication, the exception being inactivation of the nsp / cleavage site, which yielded a crippled mutant virus. depending on antibody availability, the subcellular localization of nsp to nsp has been studied for several covs using immunofluorescence microscopy. without exception, and in line with their role as interaction partner of key replicative enzymes, these subunits localize to the perinuclear region of infected cells (bost et al., ) , where the membranous replication organelles of covs accumulate (gosert et al., ; knoops et al., ; maier et al., ) . it should be noted, however, that these labeling techniques cannot distinguish between fully processed nsps and polyprotein precursors or processing intermediates. the structure of the -amino acid sars-cov nsp was determined using both nmr (peti et al., ) and x-ray crystallography (zhai et al., ) , with the latter study resolving the structure of a hexadecameric supercomplex consisting of recombinant nsp and nsp (see later; fig. ). in both structures, the nsp -fold includes four helices, but their position and spatial orientation is quite different, suggesting that the protein's conformation is strongly affected by the interaction with nsp , in particular, where it concerns helix α (johnson et al., ) . reverse-genetics studies targeting specific residues in sars-cov nsp confirmed the protein's importance for virus replication (subissi et al., b) , although the impact of single point mutations was smaller than anticipated on the basis of the biochemical characterization of the rna-binding properties of nsp -containing protein complexes in vitro (see later). the $ -amino-acid-long nsp subunit initially took center stage due to two studies, the first describing a fascinating hexadecameric structure consisting of eight copies each of nsp and nsp (fig. ) (zhai et al., ) , and the second reporting an nsp -specific "secondary" rna polymerase fig. crystal structure of the sars-cov nsp -nsp hexadecamer (pdb ahm) (zhai et al., ) . purified recombinant sars-cov nsp and nsp were found to self-assemble into a supercomplex of which the structure was determined at . Å resolution. (a) the complex forms a doughnut-shaped hollow structure of which the central channel is lined with positively charged side chains (in blue) and was postulated to mediate doublestranded rna binding. the outside of the structure is predominantly negatively charged (red) surface shading). (b and c) sars-cov nsp resembles a "golf club"-like shape that can adopt two conformations, as presented here in orange and green. these nsp conformations are integrated into a much larger, hexadecameric structure that is composed of eight nsp subunits and eight nsp subunits, of which one is shaded pink. in (b), the hexadecamer is depicted against the background of the surface plot presented in (a). activity (imbert et al., ) that was implicated in the mechanism of initiation of cov rna synthesis. this template-dependent activity was reported to depend on the presence of mn + or mg + and to typically generate products of up to six nucleotides (for more details, see section . ). around the same time, purified recombinant sars-cov nsp and nsp were found to self-assemble into the hexadecameric supercomplex of which the structure was determined at . Å resolution (zhai et al., ) . the complex was described, and also visualized by electron microscopy, as a doughnut-shaped hollow structure of which the central channel is lined with positively charged side chains ( fig. a) . a combination of structural modeling, rna-binding studies, and site-directed mutagenesis led to the hypothesis that the complex may slide along the replicating viral rna together with other viral proteins, possibly as a processivity factor for the rdrp (nsp ; see later). within the nsp -nsp hexadecamer, sars-cov nsp was found to adopt two different conformations ( fig. b and c). these were named "golf club" and "golf club with a bent shaft" (zhai et al., ) , with the globular head of the golf club being considered a new fold. although the structures of feline coronavirus (fcov) nsp and nsp were found to resemble their sars-cov equivalents, they were found to assemble into a quite different higher-order complex, with two copies of nsp and a single copy of nsp forming a heterotrimer (xiao et al., ) . biochemical and reverse-genetics studies pointed toward an important role in rna synthesis for sars-cov nsp residues k , p , and r , whose replacement was lethal to sars-cov. of these residues, p and r were postulated to be involved in interactions with nsp , whereas k may be critical for nsp -rna interactions (subissi et al., b) . reverse-genetics studies targeting the -proximal rna replication signals in the mhv genome provided strong evidence for an interaction between nsp and these rna structures (a so-called "bulged stemloop" and rna pseudoknot). when making a particular -nucleotide insertion in the rna pseudoknot, which strongly affected mhv replication, multiple suppressor mutations evolved, of which several mapped to the genomic region encoding nsp and nsp (z€ ust et al., ) . these interactions were postulated to be part of a molecular switch that controls minus-strand rna synthesis, or its initiation from the end of the viral genome (te velthuis et al., ; z€ ust et al., ) . using screening approaches based on yeast two-hybrid and glutathione s-transferase (gst) pull-down assays, sars-cov nsp was reported to be an interaction partner of many other viral proteins (including nsp , nsp , and nsp to nsp ), although most of these interactions remain to be verified in the infected cell (von brunn et al., ) . the cov nsp subunit is about amino acids long and was the second replicase cleavage product, after nsp , for which crystal structures were obtained (egloff et al., ; sutton et al., ) . the biologically active form of the protein is believed to be a dimer that is capable of binding nucleic acids in a nonsequence-specific manner, with an apparent preference for single-stranded rna (egloff et al., ; ponnusamy et al., ; sutton et al., ) . several nsp point mutations that block cov replication have now been described (chen et al., a; miknis et al., ), but the protein's exact function has remained enigmatic thus far. the nsp monomer consists of a β-barrel, composed of seven β-strands, and a c-terminal domain formed by a single α-helix. the latter domain plays a key role in the formation of the parallel helix-helix dimer conformation that-based on sequence conservation, structural considerations, and experimental data (miknis et al., )-is thought to be the biologically most relevant state of sars-cov nsp . nevertheless, multiple alternative structures were described, including a sars-cov form that is stabilized by β-sheet interactions (sutton et al., ) and, for hcov- e nsp , an antiparallel helix-helix dimer that is stabilized by a disulfide bond (ponnusamy et al., ) . replacement of the hcov- e cys residue involved in dimerization (cys- ) resulted in conversion to the parallel helix-helix dimer described for sars-cov nsp . whereas wild-type hcov- e nsp is organized as a trimer of dimers, the cys- ! ala mutant and sars-cov nsp both form rod-like polymers (ponnusamy et al., ) . disulfide bonding of the latter protein could not be detected (miknis et al., ) . although sars-cov and other betacoronaviruses do contain an equivalent cys residue, the feature is not conserved in alphacoronaviruses that are much more closely related to hcov- e. thus, it cannot be excluded that the disulfide-bonded form of hcov- e nsp is an artifact of recombinant protein purification and crystallization, although it was suggested that oxidative stress due to viral infection may favor its formation in cov-infected cells (ponnusamy et al., ) . we are not aware of experiments directly addressing the existence of such a disulfide-linked nsp dimer in covinfected cells. the importance of nsp dimerization for sars-cov and ibv viability was demonstrated in reverse-genetics studies (chen et al., a; miknis et al., ) that also independently confirmed the importance of dimerization of the α-helical domain and in particular a putative gxxxg proteinprotein interaction motif. although rna binding in vitro was not disrupted in dimerization-incompetent sars-cov nsp variants, their affinity for ssrna -mers was reduced by -to -fold compared to the wild-type protein (miknis et al., ) . replacement of some of the basic residues (e.g., lys- , lys- , and lys- ) in the β-barrel domain of ibv nsp also significantly reduced the protein's capability to bind rna in vitro, but these mutations only modestly affected virus replication upon reverse engineering (chen et al., a) . it remains to be studied how nsp dimerization and mutagenesis may affect interactions with other replicase subunits, like nsp and nsp -rdrp. these proteins were identified as nsp interaction partners using different technical approaches (brockway et al., ; sutton et al., ; von brunn et al., ) and colocalize with nsp on the membranous replication organelles (bost et al., ) . at present, the available data suggest that, for efficient cov replication, nsp homodimerization is a more critical feature than the protein's affinity for rna per se. alternatively, the correct positioning of rna on larger protein complexes consisting of (or containing) nsp may be important for the protein's correct functioning in viral rna synthesis (miknis et al., ) . currently, the fact that suppressor mutations arose in mhv nsp (and nsp ) after mutagenesis of -proximal mhv replication signals (see earlier) is the most compelling evidence for the involvement of nsp -rna interactions in a critical step of cov replication. the protein may be part of a molecular switch (z€ ust et al., ) and/or possess features that are relevant to viral pathogenesis, as mutations in nsp were found to contribute to increased sars-cov pathogenesis in an animal model employing young mice infected with a mouse-adapted virus strain (ma- ) (frieman et al., ) . the small nsp subunit ( residues in the case of sars-cov) is among the more conserved cov proteins and is thought to serve as an important multifunctional cofactor in replication. using yeast two-hybrid assays, nsp was shown to interact with itself, as well as with nsp , nsp , nsp , and nsp . these interactions were confirmed by coimmunoprecipitation and/or gst pull-down assays (brockway et al., ; imbert et al., ; pan et al., ; von brunn et al., ) . the important role of nsp in replication was first inferred from the phenotype of temperature-sensitive mutants of mhv in which an nsp mutation was responsible for a defect in minus-strand rna synthesis (sawicki et al., ) . in addition, the protein was implicated in the regulation of polyprotein processing since an engineered mhv nsp double mutant (asp- and his- to ala) was partially impaired in the processing of the nsp -nsp region (donaldson et al., ) . when nsp was characterized in biochemical and structural studies, the protein was found to bind two zn + ions with high affinity, suggesting the presence of two zinc-finger motifs (matthes et al., ) . additionally, in in vitro assays, nsp displayed a weak affinity for single-and doublestranded rna and dna, although no obvious sequence specificity could be established, suggesting that the protein may function as part of a larger rna-binding complex. crystal structures of monomeric and dodecameric forms of sars-cov nsp were solved by different laboratories, but obvious structural rearrangements between the two forms were not detected (joseph et al., ; su et al., ) . the structures revealed a new fold in which the zn + ions are coordinated in a unique conformation and in which a cluster of basic residues on the protein's surface probably contributes to the rna-binding properties of nsp . more recent biochemical studies revealed that nsp interacts with nsp and nsp and regulates their respective exon and ribose- -o-mtase ( -o-mtase) activities (bouvet et al., (bouvet et al., , . both these cofactor functions will be discussed in more detail later, in section . although a virus-encoded rdrp is at the hub of the replication of all rna viruses, special properties have long been attributed to the cov rdrp. these ideas find their origin in a combination of cov features, like the exceptionally long rna genome , the complex mechanism underlying subgenomic rna synthesis pasternak et al., ; sawicki et al., ; sola et al., ) , the reported high rna recombination frequency (graham and baric, ; lai and cavanagh, ) , and the size and positioning of the rdrpcontaining subunit, nsp , within the replicase polyprotein. it remains to be elucidated to which extent features like polymerase processivity, fidelity, and template switching (during either genomic recombination or subgenome-length negative-strand rna synthesis) are determined by the properties of the nsp -rdrp subunit itself or by some of its protein cofactors, such as nsp and nsp (see earlier). in fact, some cofactors have been studied more extensively than nsp itself, and the same holds true for some of the specific rna signals employed by the rdrp during, e.g., replication and subgenomic mrna synthesis. protein subunits of the larger rnasynthesizing complex, like nsp -nsp , the nsp -helicase, and the nsp -exon, likely exert a strong influence on rdrp behavior and performance. on the other hand, a recent study employing homology modeling and reverse genetics of the mhv rdrp domain described the first two nsp mutations that can induce resistance to a mutagen and reduce the mhv rdrp error rate during virus passaging (sexton et al., ) . so, not unexpectedly, also features within nsp itself contribute to properties like nucleotide selectivity and fidelity regulation. all of the currently identified nsp cofactors, and most other cov nsps, assemble into membrane-associated enzyme complexes (see earlier). the large number of viral subunits in these complexes (subissi et al., a) , the likely requirement for host factors (van hemert et al., ) , and the concept of rna synthesis occurring in a dedicated microenvironment in the infected cell (knoops et al., ; v'kovski et al., ) complicate the straightforward characterization of the cov rdrp. to reconstitute the enzyme's activities in vitro, purified recombinant nsp is a key reagent but, for many years, such studies were hampered by poor nsp expression in escherichia coli. the first in vitro activity assays have only been developed recently (subissi et al., b; te velthuis et al., ) , and the same technical issues with protein production explain the current lack of an nsp crystal structure. consequently, structural information is restricted to sequence comparisons and some homology-based structure models of the c-terminal rdrp domain of the $ -residue-long nsp (xu et al., ) . moreover, most of what we have learned so far is based on the characterization of a single nsp homolog only, that of the sars-cov. the nsp -coding sequence includes the orf a/ b ribosomal frameshift site and a programmed - frameshifting event directs orf b translation to yield the pp ab polyprotein that includes nsp . the cl pro -driven cleavage required to release the n-terminus of nsp is the same that separates nsp and nsp . about - amino acids downstream ( in the case of sars-cov), the nsp /nsp cleavage site separates the cov rdrp subunit from the helicase-containing cleavage product, whichuniquely among +rna viruses-resides downstream of the rdrp domain for reasons that are poorly understood thus far . nsp consists of at least two domains, the recently described n-terminal "nidovirus-wide conserved domain with nucleotidyl transferase activity" (nidovirus rdrp-associated nucleotidyltransferase (niran); see later) (lehmann et al., a ) and the c-terminal canonical rdrp domain (gorbalenya et al., ) . the latter possesses the common motifs and structural features found in other rna polymerases, which are often summarized as a "cupped right hand" with subdomains called fingers, palm, and thumb each playing specific roles in binding of templates and ntps, initiation, and elongation (te velthuis, ; xu et al., ) . in simplified form, the reaction catalyzed by the rdrp comes down to selecting the appropriate ntp to match with the template and the formation of a phosphodiester bond to extend the end of the nascent rna chain with this incoming nucleotide (ng et al., ; van dijk et al., ) . reconstituting these activities in vitro using a purified rdrp preparation can be relatively straightforward, but sometimes is a huge technical challenge depending-among other factors-on the efficiency of recombinant rdrp expression and purification, the existence of specific template requirements (e.g., recognition signals), and the need for protein cofactors. the initiation mechanism of the cov rdrp, primer dependent or de novo, continues to be a much-debated issue, with important implications for the question of how covs maintain the integrity of the crucial terminal sequences of their genome. compared to a de novo-initiating rdrp, the enzyme's active site, which is enclosed by the thumb and fingers domains, needs to be more accessible when a primer-template duplex has to be accommodated. de novo initiation, on the other hand, requires specific structural elements (so-called "priming loops") that serve to properly position the initiating ntps for catalysis, thus creating an initiation platform for rna synthesis. bioinformatics analyses grouped the cov rdrp with primer-dependent rdrps, as found in, e.g., picornaviruses and caliciviruses, in part based on the identification of a specific sequence motif (motif g) that is thought to mediate primer recognition ( fig. a ) (beerens et al., ; fig. comparison of coronavirus nsp and arterivirus nsp , containing the highly conserved niran and rdrp domains. (a) similarity density plot derived from a multiple sequence alignment including rdrp subunits from all nidovirus lineages. to highlight local deviations from the average, areas displaying conservation above and below the mean similarity are shaded in black and gray, respectively. conserved sequence motifs of niran (subscript n; see also b) and rdrp (subscript r) are labeled. domain boundaries used for bioinformatics analyses and uncertainty with respect to the niran/rdrp domain boundary are indicated with vertical and by dashed horizontal lines, respectively. below each plot, the predicted secondary structure elements are presented in gray for α-helices and black for β-strands. gorbalenya et al., ; xu et al., ) . this prediction appeared to be further supported by the identification of sars-cov nsp as a de novoinitiating second rna polymerase (see earlier), capable of synthesizing products of up to six nucleotides in length that could serve to prime rna synthesis by the nsp -rdrp (imbert et al., ) . support for a direct interaction between nsp and nsp was obtained using different technical approaches subissi et al., b; von brunn et al., ) . however, although a similar primer-independent rdrp activity was reported for the fcov nsp (xiao et al., ) , other studies have called into question this concept of a primase-main rdrp (i.e., nsp /nsp ) tandem working in concert to achieve initiation of processive cov rna synthesis (see later). using recombinant sars-cov nsp , preliminary evidence for primer-dependent rdrp activity on poly(a) templates was first obtained using a gst-nsp fusion protein, although these efforts were hampered by protein instability, which also led to the conclusion that the n-terminal domain of nsp is required for activity (cheng et al., ) . subsequently, a c-terminally his -tagged sars-cov nsp was found to mediate homopolymeric rna synthesis in a primer-dependent manner (te velthuis et al., ) . both these activities must probably be considered relatively weak and nonprocessive compared to the activity observed when a sars-cov nsp rdrp assay was supplemented with nsp and nsp (subissi et al., b) . however, at the same time, this study reinvigorated the debate on the initiation mechanism of the coronavirus rdrp, as the nsp - - tripartite complex displayed both primer-dependent and de novo initiation of rna synthesis, whereas no de novo-initiating rdrp activity could be detected for nsp or the nsp -nsp complex alone (subissi et al., b) . to add to the confusion, other studies reported de novo initiation by sars-cov nsp alone (ahn et al., ) and primer-dependent rdrp activity of sars-cov nsp , when expressed without affinity tags commonly used to facilitate purification (te velthuis et al., ). technical differences between these studies and those summarized earlier (e.g., regarding expression constructs and templates used) may have contributed to the contradictory results obtained on the rdrp activities of nsp and nsp . thus far, five different laboratories addressed the two (putative) coronavirus rdrps in seven independent studies, none of which succeeded in exactly reproducing the results of any of the other studies (ahn et al., ; cheng et al., ; imbert et al., ; subissi et al., b; te velthuis et al., te velthuis et al., , xiao et al., ) . nidovirus rdrps appear to be technically challenging and sensitive proteins that may respond to minute changes in purification protocols or assay conditions. clearly, both the role of nsp (primase or processivity factor?) and the initiation mechanism employed by the nsp -rdrp require further study. although the bioinformatics-based prediction that nsp uses a primer-dependent initiation mechanism is compelling, it lacks the direct support of an nsp crystal structure. at the same time, the question of the nature and source of the primer that would be used by nsp seems to be wide open again. as for other rna viruses, the nsp -rdrp of covs is a primary drug target that may, in principle, be inhibited without major toxic side effects for the host cell. nucleoside analogs constitute an important class of antiviral drug candidates that can target viral rdrps, but efforts to use them to inhibit cov replication were not very successful thus far (chu et al., ; ikejiri et al., ) . moreover, it remains to be established that their target in the infected cell is indeed the nsp -rdrp. the mismatch repair capabilities attributed to the nsp -exon domain (see later) (bouvet et al., ) may pose an additional hurdle, as the efficacy of a nucleoside analogue with anticoronavirus activity may be determined by the balance between its propensity to be incorporated by the nsp -rdrp and its tendency to resist excision by the mismatch repair mechanism mediated by nsp -exon. similar considerations apply to ribavirin, a guanosine analog with broadspectrum antiviral activity that is used to treat patients infected with a variety of rna viruses. its mechanism of action appears to differ on a case-by-case basis, but may include the induction of lethal mutagenesis by increasing the rdrp error rate, inhibition of viral mrna capping, and reduction of viral rna synthesis by inhibition of the cellular enzyme inosine monophosphate dehydrogenase (impdh), which decreases the availability of intracellular gtp (crotty et al., (crotty et al., , smith et al., smith et al., , . although ribavirin was used to treat small numbers of sars and mers patients, high doses were used and the benefits of the treatment remained essentially unclear (zumla et al., ) . experiments with different covs in animal models falzarano et al., ) and infected cell cultures (ikejiri et al., ; pyrc et al., ) also established its poor activity and strongly suggested that ribavirin does not target the cov rdrp directly or is targeted (itself ) by the nsp -exon activity (smith et al., ) . innovative nucleoside inhibitors continue to be identified or developed (peters et al., ; warren et al., ) and the recently described in vitro rdrp assay (subissi et al., b) may prove very useful for establishing their mechanism of inhibition more precisely. a better understanding of nsp -rdrp structure and function will also be required to design strategies that minimize the impact of drug resistance-inducing mutations, which are a common problem when targeting enzymes of rapidly evolving rna viruses. since the delineation of the borders of the cov rdrp-containing replicase cleavage product (boursnell et al., ; gorbalenya et al., ) , which is now known as nsp , it had been clear that the protein must be a multidomain subunit, with the canonical rdrp domain roughly occupying its c-terminal half (fig. a ). only recently, first clues to some of the properties and possible functions of the n-terminal part of nsp were obtained (lehmann et al., a) . a renewed bioinformatics analysis across the (still expanding) order nidovirales revealed that the nidoviral rdrp-containing replicase subunit contains a conserved n-terminal domain of - residues ($ residues in cov nsp ; fig. b ). in cov nsp , about residues separate the niran and rdrp domains, leaving space for the presence of an additional domain between the two. based mainly on biochemical data obtained with the arterivirus homolog (see later), the n-terminal domain was concluded to possess an essential nucleotidylation activity and hence it was coined nidovirus rdrp-associated nucleotidyltransferase (niran) (lehmann et al., a) . niran conservation was found to be lower than that of the downstream rdrp domain (fig. a) , but the analysis suggested that the evolutionary constraints on niran have been similar in different nidovirus lineages, which would be in line with a conserved function. gorbalenya and colleagues identified three key niran motifs (a-b-c) containing seven invariant residues (fig. b) , with domains b and c being most conserved (lehmann et al., a) . the identification of the niran domain was further supported by the conservation of its predicted secondary structure elements in different nidovirus families (fig. a) . extensive database searches did not reveal potential niran homologs in either the viral or the cellular world, although it cannot be excluded that the domain has diverged from cellular ancestors to a level that prevents their identification with the currently available sequences and tools. nevertheless, its unique presence in nidoviruses and its association with the important rdrp domain suggest that niran may be a crucial regulator or interaction partner of the downstream rdrp domain that must have been acquired before the currently known nidovirus lineages diverged. niran and the zinc-binding domain (zbd) that is associated with the nsp -helicase protein (see later) are the only unique genetic markers of the order nidovirales identified thus far. mainly due to the lack of sufficient amounts of recombinant cov nsp , the preliminary biochemical characterization of niran was restricted to its arterivirus homolog, using recombinant nsp of equine arteritis virus (eav) (lehmann et al., a) . for both eav and sars-cov, it could be shown that replacement of conserved niran residues can cripple or completely block virus replication in cultured cells. a combination of biochemical assays revealed that in vitro the niran domain exhibits a specific, mn + -dependent enzymatic activity that results in the self-nucleotidylation of eav nsp . the activity was abolished upon mutagenesis of conserved key residues in niran motifs a, b, and c. although utp was found to be the preferred substrate for niran's in vitro nucleotidylation activity, also gtp could be used, albeit less efficiently. the conserved lysine residue in motif a (the eav equivalent of lys- in sars-cov nsp ) was concluded to be the most likely target residue for nucleotidylation via formation of a phosphoamide bond. although the importance of the niran domains of arterivirus nsp and coronavirus nps was supported by the outcome of reverse-genetics studies (lehmann et al., a) , the role of the produced protein-nucleoside adducts in viral replication remains unclear at present. in fact, the unique dual specificity for utp and gtp seems to argue against two initially considered potential niran functions (lehmann et al., a) . the first of these was a role as an rna ligase, a type of activity however that commonly is atp dependent. the second was its involvement in synthesizing mrna cap structures. one of the four enzymes required for this process, the crucial guanylyl transferase (gtase), still remains to be identified for covs (see later). however, niran's substrate preference for utp over gtp is difficult to reconcile with this hypothesis and has not been observed for other gtases involved in mrna capping. the third hypothesis that was put forward links back to the open question of the initiation of coronavirus rna synthesis, which presumably is a primer-dependent step (see earlier). nsp nucleotidylation could be envisioned to play a role in protein-primed rna synthesis, a strategy used by, e.g., picornaviruses and their relatives, which covalently attach an oligonucleotide to a viral protein (called vpg in the case of picornaviruses) that subsequently mediates the initiation of rna synthesis (paul et al., ) . the first step in the synthesis of the "protein primer" is a nucleotidylation step during which a nucleotide monophosphate is covalently attached to the vpg. niran could be involved in a similar mechanism either directly or indirectly, by transferring the bound nucleotide to another protein player. although such a mechanism would definitely revolutionize the concept of the initiation of cov rna synthesis, it is clearly not very compatible with some of the currently available data, such as the reported presence of a cap structure (rather than a vpg-like molecule) on cov mrnas. evidently, the further in-depth characterization of niran is needed to fill the current knowledge gaps, starting with the biochemical characterization of a cov niran domain, which may confirm and extend the features now deduced from the analysis of its distantly related arterivirus homolog. helicases are versatile ntp-dependent motor proteins that play a role in cellular nucleic acid metabolism in the broadest possible sense, including processes like dna replication, recombination and repair, transcription, translation, as well as rna processing. helicases are also encoded by all +rna viruses with a genome size exceeding kb, suggesting they are required for the efficient replication of +rna viral genomes above this size threshold. given the large size of the genomes of covs and related nidoviruses, they may depend on the function(s) of a replicative helicase even more than other +rna virus taxa. however, despite their abundance and conservation, the specific role of helicases in +rna virus replication remains poorly understood. for an extensive recent review of nidovirus helicases, the reader is referred to lehmann et al. ( c) . currently, helicases are classified into six superfamilies (sfs) (singleton et al., ) , with +rna viral helicases belonging to sf (e.g., alphaviruses and nidoviruses), sf (e.g., flaviviruses), or sf (e.g., picornaviruses). the presence of a sf helicase (hel ) domain in the cov replicase polyprotein was discovered upon the early in-depth analysis of the first full-length cov genome sequence that became available (ibv) (gorbalenya et al., ) . the hel domain maps to the c-terminal part of the replicase cleavage product that is now known as nsp , which is about residues long. the cov hel domain contains all characteristic sequence motifs of the sf superfamily. the n-terminal part of nsp is formed by a multinuclear zbd, one of the most conserved domains across the order nidovirales (gorbalenya, ; nga et al., ) . this qualification also applies to the helicase-containing subunit as a whole, despite considerable size differences between, e.g., cov nsp and its arterivirus homolog (designated nsp ) (lehmann et al., c) . the zbd and hel domains occupy a conserved position downstream of the rdrp domain in all nidovirus replicase polyproteins studied so far. sf helicases contain at least a dozen conserved motifs that direct the binding of ntps and nucleic acids. of these, motifs i and ii (also known as the walker a and b boxes) are common to helicases of all sfs as well as ntpases. structurally, the catalytic core of sf helicases like the cov hel domain is formed by two reca-like domains, designated a and a (fig. ) , that bind to nucleic acids through stacking interactions of aromatic residues with the bases of their nucleic acid substrates (velankar et al., ) . cyclic conformational changes of the reca-like domains mediate the conversion of the energy from hydrolysis of the phosphodiester bonds of ntps into directional movement along the nucleic acid substrate, with the so-called "inchworm" model now widely being considered as best supported by the available experimental data (lehmann et al., c; velankar et al., ; yarranton and gefter, ) . additional domains, located upor downstream of a and a, or inserted internally, can mediate supplemental protein-protein and protein-nucleic acid interactions or enzymatic activities, thus contributing to the functional versatility and specificity of the enzyme (lehmann et al., c; singleton et al., ) . within helicase sf , the cov hel domain belongs to the upf -like family (sf b) which is characterized by moving in the -to- direction along the nucleic acid strand to which they bind. upf -like helicases may unwind either dna or rna and, in some cases, also both substrates without a clear preference, as was readily observed during the in vitro characterization of different nidovirus helicases. the cov hel activity was first demonstrated in vitro using recombinant hcov- e nsp (seybert et al., a) . bacterially expressed nsp from hcov- e and sars-cov, and also the homologous helicase (nsp ) of the arterivirus eav, displayed -to- unwinding activity on double-stranded rna or dna substrates containing single-stranded overhangs (ivanov and ziebuhr, ; ivanov et al., b; seybert et al., a,b; tanner et al., ) . following the biochemical characterization of sars-cov nsp , it was calculated that unwinding occurs in discrete steps of . base pairs each, with a catalytic rate of steps per second (adedeji et al., a) . the nsp ntpase activity can use all four natural ribonucleotides and nucleotides as substrate, with atp, datp, and gtp being hydrolyzed most efficiently, and utp being the least preferred substrate (ivanov and ziebuhr, ; ivanov et al., b; tanner et al., ) . replacement of a conserved lys in motif i, the walker a box (walker et al., ) , kills the in vitro ntpase activity of all nidovirus helicases tested thus far and, when introduced by reverse genetics, this mutation also abolished replication of the arterivirus eav (seybert et al., b) . the substrate preferences summarized earlier support a threedimensional model of the sars-cov hel core domains ( a and a) that was based on structural information available for multiple cellular helicases (hoffmann et al., ) . the model predicts both the existence of multiple hydrogen bonding interactions with the βand γ-phosphates of the ntp and a lack of specific interactions with the nucleobase. thus, the mere presence of a triphosphate group appears to be the main determinant for ntp/dntp binding. since nidovirus helicases are presumed to unwind double-stranded rna intermediates that are formed during viral replication, considerable attention was given to the in vitro characterization of their nucleic acid substrate preferences (seybert et al., a,b) . the hcov- e and eav helicases could not unwind substrates with single-stranded tails or blunt-ended substrates. in contrast, rna and dna substrates with one or two single-stranded regions were unwound efficiently, suggesting that the nidovirus helicase must bind to a single-stranded region before initiating unwinding in the -to- direction. however, the in vitro assays did not yield any clear indications for the preferred recognition of specific sequences or higher-order structures in the substrate (lehmann et al., c) . also a more in-depth biochemical characterization, performed with sars-cov nsp , confirmed that the cov helicase does not discriminate between rna and dna substrates (adedeji et al., a) . consequently, it cannot be excluded that the enzyme, in addition to being engaged in viral rna synthesis, may also target host dna. nuclear translocation of nidovirus helicases has not been reported thus far, but the light microscopy techniques used to study the protein's subcellular distribution would not suffice to detect the nuclear import of only a small fraction of the protein. as a final caveat it should be stressed that the biochemical properties summarized earlier are all derived from in vitro studies using recombinant helicases, expressed in different systems and sometimes containing substantial foreign sequences. the in situ characterization of the helicase as one of the key enzymes of the nidovirus rna-synthesizing machinery remains to be addressed. in that context, sequence specificity, for example, could be conveyed by other subunits of the replicase complex, which may target the helicase to, e.g., the initiation sites for viral genome or antigenome synthesis, or to signals controlling the production of subgenomic mrnas. as summarized by lehmann et al. ( c) , other important helicase features that could be dramatically different in the setting of the infected cell are (the need for) helicase oligomerization, cooperativity between multiple helicase molecules binding to the same substrate, and-consequentlythe overall processivity of the enzyme, which in vitro appeared to be quite low given the large cov genome size (adedeji et al., a) . the nidovirus helicase subunit domain is unique among its +rna virus homologs in having a conserved n-terminal domain of - residues that contains or conserved cys/his residues (den boon et al., ; van dinten et al., ) . the domain was recognized as a potential zbd (gorbalenya et al., ) and early in vitro studies with the recombinant hcov- e and eav helicases confirmed that zn + ions are essential for retaining the protein's enzymatic activities, suggesting that zbd modulates nidovirus helicase function (seybert et al., ) . a recent structural study of the arterivirus nsp -helicase (deng et al., ) will be discussed in more detail later. this first nidovirus helicase structure confirmed the binding of three zinc ions by the zbd, which adopts a unique fold that combines a ring-like module with a so-called "treble-clef" zinc finger. zbd and hel interact extensively (deng et al., ) but, in the helicase primary structure, they are separated by a variable and uncharacterized domain that essentially explains the size difference of about residues between cov and arterivirus helicase subunits (seybert et al., ) . using the arterivirus prototype eav, the functional importance of zbd was probed extensively by combining biochemistry and reverse genetics (seybert et al., ; van dinten et al., ) . this yielded a variety of phenotypes for nsp -zbd mutants, the most striking being mutants deficient in subgenomic mrna synthesis while remaining capable of (and even enhancing) viral genome replication (van dinten et al., (van dinten et al., , (see later). most replacements of conserved zbd cys and his residues profoundly impacted the helicase activity of eav nsp , even when performed in a semiconservative manner that could preserve zinc binding. in reverse-genetics studies, most of these zbd mutations rendered the virus nonviable. recently, the impact of these mutations on zbd integrity and zbd-hel interactions could be rationalized with the help of the nsp crystal structure (deng et al., ) . despite its importance as a potential drug target, a cov nsp or hel crystal structure has not been obtained thus far due to technical complications with recombinant protein production and crystallization. instead, several cov helicase models have been described, mainly based on cellular helicase structures (bernini et al., ; hoffmann et al., ; lehmann et al., c) . given this limitation, and despite the large evolutionary distance between the two enzymes, it is interesting to have a closer look at the recently published eav nsp -helicase structure (deng et al., ) . the overall structure of eav nsp (fig. ) consists of the n-terminal zbd, a new domain designated b, the two reca-like hel domains ( a and a), and a short c-terminal domain, which is not conserved among nidoviruses and needed to be deleted to allow nsp crystallization. this -amino-acid c-terminal truncation did not affect the helicase core domains and only modestly influenced levels of atpase and helicase activity compared to the full-length protein (deng et al., ) . compared to cellular representatives of the sf helicase superfamily, nsp is most similar to upf and its close homolog ighmbp , which also contain an n-terminal zbd. moreover, the location and orientation of the newly discovered b domain of nsp (residues - ) resembles that of the domain with the same name found in the upf -like helicase subfamily. the nsp zbd uses conserved cys and his residues to coordinate three zinc ions and folds into two zinc-binding modules that are connected by a disordered region. the n-terminal ring-like structure of nsp coordinates two zinc ions and the closest similarity that was found for this module was with the n-terminal zinc-binding ch-domain of upf . both proteins have a second zinc-binding module downstream (a so-called treble-clef zinc finger in the case of nsp ), but these are structurally different, suggesting that the nidoviral zbd represents a new kind of complex zinc-binding element. the previous suggestion of zbd codetermining hel function was strengthened by the presence of an extensive interface of Å that was proposed to be involved in intramolecular signaling (deng et al., ) . a second crystal structure was obtained for nsp in complex with a partially double-stranded dna substrate, revealing possible nucleic acid-binding clefts at the protein's surface that are formed by the zbd + b and zbd + a domains. although the exact path of the nucleic acid strands could not yet be determined, it became clear that the positively charged zbd, and in particular its n-terminal ring-like module, must be involved in nucleic acid binding. in line with the biochemical data summarized earlier, most of the nsp -substrate contacts identified are not base-specific and occur with the nucleic acid backbone. whereas the hel core domains were found to be quite similar in the absence or presence of bound substrate, a remarkable degree rotation was observed for domain b, enlarging the dimensions of the nucleic acid substrate channel formed by domains a and b, but not allowing it to accommodate a duplex substrate. consequently, it was postulated that an element near the entrance of the substrate channel may destabilize the duplex and facilitate the entrance of one of the strands into the channel. since the double-stranded region of the duplex could not be modeled, additional studies are needed to verify the existence and molecular details of this proposed unwinding mechanism (deng et al., ; lehmann et al., c) . likewise, direct structural information on cov nsp is needed to be able to assess to which extent the structural observations made for arterivirus nsp can be translated to distantly related (and larger) nidovirus helicases (fig. ) . in general, however, the analysis of nsp provided a clear basis for a model in which the function of the common reca-like core domains of nidovirus helicases is modulated by specific extension domains, presumably to facilitate the involvement of the nidovirus helicase in multiple processes in the infected cell (see later). as outlined earlier, the biochemical characterization of purified recombinant nidovirus helicases, the functional probing of (in particular) the eav nsp -helicase using reverse genetics, the eav nsp structure, and advanced bioinformatics analyses together have painted a picture of an enzyme that is involved in multiple critical steps of the viral replicative cycle. space limitations do not allow an in-depth discussion of all of these (putative) functions, which-based on eav nsp studies-may include a poorly characterized role in virion biogenesis (van dinten et al., ) , not unlike what was uncovered for, e.g., the helicase-containing ns protein of flaviviruses (liu et al., ; ma et al., ) . likewise, we will not discuss the first reports on possible interactions with host proteins, such as the ddx helicase (for sars-cov nsp ; chen et al., b) and polymerase δ (for ibv nsp ; xu et al., ) . instead, we will focus on the most significant findings related to nidovirus helicase functions in rna synthesis and processing, specifically (i) genome replication, (ii) transcription of subgenomic mrnas, (iii) mrna capping, and (iv) posttranscriptional mrna quality control. the presumed "default" function of +rna viral helicases is to cooperate with the viral rdrp to achieve the efficient amplification of the genome. in this context, helicases are presumed to promote rdrp processivity by opening up the double-stranded rna intermediates of viral replication, and possibly also by removing rna secondary structures in single-stranded template strands (kadare and haenni, ) . in this light, reports on molecular interactions between the cov rdrp (nsp ) and helicase (nsp ) were not unexpected von brunn et al., ) . the same holds true for the observation that sars-cov nsp can stimulate the in vitro helicase activity of nsp (adedeji et al., a) and for the fact that both nsp and nsp (like most other cov replicase cleavage products) associate with the membranous replication organelles in nidovirus-infected cells (denison et al., ; ivanov et al., b; knoops et al., ) . in spite of all these indications for rdrp-helicase interplay, the polarity of nidovirus helicase-mediated unwinding ( -to- ) remains a major conundrum, as it is opposite to the polarities of the rdrp and many other +rna helicases, which move in a -to- direction on the rna strand they initially bind to (seybert et al., a) . this strongly suggests that the two enzymes cannot simply operate as a tandem that moves along the same template strand while copying it, a consideration that also applies to the sf b helicase employed by alphaviruses. this problem could be resolved if rdrp and helicase would move along different strands of the same rna duplex, which might allow the helicase to separate the two strands and provide a single-stranded template to be copied by the rdrp (lehmann et al., c) . also, it is tempting to speculate that the helicase, by trailing along the nascent strand (following the rdrp at a certain and, possibly, somewhat flexible distance), provides (i.e., leaves behind) a single-stranded template, thus facilitating initiation and elongation of rna synthesis by the next rtc. this, for example, could occur in cases when multiple rtcs act simultaneously/consecutively on the same template to produce multiple plus-strand rnas from the same minus-strand template, a process that is generally thought to add to the large excess of plus-over minus-strand rnas in nidovirus-infected cells. clearly, significantly more work is needed to explore this possibility. nidovirus sg mrnas are each produced from their own subgenomelength minus-strand template (see section ). in the case of arteri-and coronaviruses, these derive from a process of discontinuous minus-strand rna synthesis and this unique mechanism likely requires specific functional interactions between (among others) rdrp and helicase. these interactions may contribute to maintaining a proper balance between continuous and discontinuous minus-strand rna synthesis, and thus between the production of new genomes and sg mrnas. the serendipitous identification of an arterivirus nsp -zbd mutation (ser- ! pro) that essentially inactivated transcription while leaving replication unaffected was an early indication for the involvement of the nidovirus helicase in the control of sg mrna synthesis (van dinten et al., ) . such control functions could also be related to the recognition of trss (fig. ) , the frequency with which each of the trss is used to produce a subgenome-length minus strand, or mechanistic aspects of the stalling and reinitiation of rna synthesis or the transfer of the nascent strand to an upstream position on the template (see earlier), which must occur during the discontinuous step in sg rna synthesis (lehmann et al., c) . recently, the eav nsp ser- ! pro mutation, which selectively reduces transcription of all subgenomic mrnas to below % of their normal levels, was reanalyzed in the context of the nsp crystal structure. as postulated when this virus mutant was first described, its phenotype appears to be based on the special structural properties of the pro residue in combination with the position of residue in a "hinge" region that connects zbd to the rest of the protein (deng et al., ) . although residue , located just downstream of the second zinc-binding module of the zbd, is fairly distant from the rna-binding surface, it resides in a region that connects the zbd treble-clef zinc finger to a helix that interacts with domains a and b and with the nucleic acid. thus, specific mutations affecting the flexibility of this hinge region may drastically influence the long-distance signaling within nsp , apparently preventing the rnasynthesizing machinery to work in "transcription mode" and dedicating it exclusively to full-length minus-strand rna synthesis and genome amplification. since this kind of mutations barely affected nsp s in vitro ntpase and helicase activities (seybert et al., ) , it may well be that changed interactions with specific protein partners will turn out to be the key to explaining the transcription-negative phenotype of the corresponding virus mutants (lehmann et al., c) . for the coronavirus ibv, a point mutation in a somewhat comparable position of nsp (arg- ! pro; just downstream of zbd) was reported to cause a similar block of sg mrna synthesis (fang et al., ) but, thus far, this observation has not been followed up in more detail for ibv or confirmed for nsp of another cov. in addition to its role in rna synthesis, the nidovirus helicase is also assumed to be involved in the capping pathway of viral mrnas by exhibiting an rna -triphosphatase (rtpase) activity that can remove the -terminal triphosphate from the rna substrate. for sars-cov and hcov- e nsp , this first step in viral cap synthesis was shown to rely on the same ntpase active site that provides the energy for the protein's helicase activity (ivanov and ziebuhr, ; ivanov et al., b) . the cov capping pathway is discussed in section of this review. finally, the remarkable similarities between eav nsp and the cellular helicase upf (deng et al., ) have given rise to the intriguing but still speculative hypothesis that the nidovirus helicase may be involved in the posttranscriptional quality control of viral rna. common features of the two helicases include their -to- polarity of unwinding, their lack of substrate specificity and striking similarities in terms of domain organization and fold (fig. ) (lehmann et al., c) . using several pathways, including nonsense-mediated mrna decay, upf mediates rna quality control in eukaryotic cells (cheng et al., ; clerici et al., ) , while its activity can be modulated through interactions of its n-terminal zbd. it was postulated that a similar function in nidovirus replication, e.g., detection and elimination of defective viral mrnas (including the genome), could explain the conservation of the unique zbd across the nidovirus order, which stands out for containing members with very large + rna genomes. such a function would prevent the synthesis of defective viral polypeptides, which might interfere with the proper functioning of full-length viral proteins. in this manner, not unlike the nsp -exon domain (see earlier), the nidovirus helicase may have contributed to genome expansion by providing a form of compensation for the relatively poor fidelity of genome replication by the nidoviral rdrp (deng et al., ) . clearly, this is just one of the scenarios for the involvement of the helicase in the posttranscriptional fate of viral rna products. further experimental work will be needed to explore these possibilities in more detail, as they are compatible with the much broader realization that the functions of rna helicases can extend far beyond merely the unwinding of rna structure. due to its multifunctionality and involvement in several key processes in viral rna synthesis and processing, the nidovirus helicase is an important target for antiviral drug development, which was mainly explored for covs following the sars-cov outbreak. the highly conserved nature of the helicase offers the interesting perspective of developing inhibitors with a potential broad-spectrum activity. on the other hand, avoiding toxicity resulting from inhibition of the abundant cellular ntpases/helicases poses a serious challenge, which is why-as in the case of the rdrp-obtaining a crystal structure for a cov helicase should be considered a research priority. in theory, a variety of helicase properties may be targeted with specific inhibitors, ranging from the active and nucleic acid-binding sites of the enzyme to interaction surfaces for multimerization and modulation by protein cofactors (kwong et al., ) . several compound families were found to target the atpase of nsp , and thus also its helicase activity. these include naturally occurring flavonoids (yu et al., ) , chromones (kim et al., ) , and bananins (kesel, ; tanner et al., ) , all exhibiting in vitro ic values in the low-micromolar range. other compounds appear to target the helicase of nsp activity more specifically, like the triazole ssya - , which was found to inhibit the replication of multiple beta-covs (sars-cov, mers-cov, and mhv) in cell culture-based infection models (adedeji et al., b (adedeji et al., , . the ic value in in vitro helicase assays was about . μm, whereas ec values in cell cultures assays were in the range of - μm, depending on the cov analyzed, suggesting that broad-spectrum activity may indeed be achieved. to our knowledge, the antiviral potential and toxicity of ssya - in animal models remain to be tested. another interesting group of helicase-directed antiviral hits are bismuth complexes, which were postulated to inhibit the ntpase and helicase functions by competing for zinc ions with the zbd. in sars-cov-infected cell cultures the determined ec and cc values were μm and mm, respectively (yang et al., ) . nsp - - - cap structures consists of a -methylguanosine ( m g) linked to the first nucleotide of the rna transcript through a - triphosphate bridge (for a review, see decroly et al., ) . in eukaryotic cells, the synthesis of the cap structure is a multistep process that occurs in the nucleus and is coupled to rna pol ii-driven transcription (shatkin, ) . capping begins with the hydrolysis of the γ-phosphate of the nascent rna transcript by an rna -triphosphatase (rtpase). subsequently, a gmp molecule is transferred to the -diphosphate of the rna by a gtase, leading to the formation of gpppn-rna. the cap structure is methylated at the n position of the guanosine by an (adomet)-dependent n -mtase, yielding cap- ( m gpppn). the cap- structure is then converted into cap- ( m gpppn -om ) or cap- by an adomet-dependent -o-mtase that methylates the -o position of the ribose of the first or first and second rna nucleotide, respectively. due to the cytoplasmic localization of their mrna synthesis, nidoviruses, and all other +rna viruses of eukaryotes, cannot rely on the standard capping pathway outlined earlier, which is executed by host cell enzymes in the nucleus. cap structures can protect viral mrnas from degradation by the cellular -to- exoribonuclases involved in rna turnover (liu and kiledjian, ) . cap methylation is critical for mrna recognition by translation initiation factor eif e (filipowicz et al., ; ohlmann et al., ) , and thus for viral translation and replication as a whole . in addition to promoting mrna stability and securing their recognition by host cell ribosomes, capping of viral mrnas also promotes escape from certain antiviral responses of the host cell. the retinoic acidinducible gene and melanoma differentiation-associated protein (mda ) were shown to detect either uncapped triphosphorylated rna or cap- -containing rna (devarkar et al., ; hyde and diamond, ; hyde et al., ; schuberth-wagner et al., ; z€ ust et al., ) , resulting in the expression of antiviral interferon-stimulated genes (isgs) in infected and neighboring cells. interferon-induced proteins with a tetratricopeptide repeat (ifit / ) and ifit / (ifit ) have been shown to recognize miscapped rnas, in order to restrict viral translation (daffis et al., ; pichlmair et al., ) . a subsequent study identified ifit as the only interferon-induced protein whose rna-binding affinity was affected by the ribose- -o methylation state of the cap structure (cap- /cap- ). the data support a model in which ifit efficiently binds and sequesters capped mrna that lacks a ribose- -o-methyl group. consistent with this, viral mrna translation was shown to be reduced in cells infected with -o-mtase-deficient covs (habjan et al., ) . other studies suggested that -o methylation of cap structures prevents or delays the mda -dependent recognition of viral mrnas as "nonself." this mrna cap modification thus limits the antiviral response launched upon infection, thereby affecting viral pathogenesis (daffis et al., ; schuberth-wagner et al., ; z€ ust et al., ) . the genomic and sg mrnas of nidoviruses are presumed to be capped at their end and polyadenylated at their end (fig. ) . the presence of a cap structure was first suggested based on studies using p-labeled mhv rna that was digested with rnase t and t and subjected to deaecellulose chromatography (lai and stohlman, ) . the presence of a cap was further substantiated by immunoprecipitation experiments using a cap-specific monoclonal antibody that was shown to specifically bind to equine torovirus mrnas (van vliet et al., ) . although the (presumed) capping machinery of arteriviruses has remained essentially uncharacterized thus far (lehmann et al., b) , three conserved putative capping enzymes were identified in the conserved orf b-encoded part of the replicase of coronaviridae, roniviridae, and mesoniviridae, which all have substantially larger genomes. these enzymatic activities, which were proposed to participate in the synthesis of a cap- structure ( n m gpppn om ), are the following: (i) the nsp helicase/rtpase (ivanov and ziebuhr, ) , (ii) the nsp n -mtase (chen et al., c; ma et al., ) , and (iii) the nsp -o-mtase (bouvet et al., ; decroly et al., ; snijder et al., ; von grotthuss et al., ; zeng et al., ) . at present, structural and functional studies, which were mainly performed with purified recombinant sars-cov nsps, suggest that covs follow a capping pathway that is very similar to that of eukaryotic cells. cap synthesis is presumed to start by hydrolysis of the end of a nascent rna by the rtpase function of nsp to yield pp-rna (ivanov and ziebuhr, ) . then, a still elusive gtase must transfer a gmp molecule onto the pp-rna to yield gppp-rna. the cap structure is then methylated at the n position by the n -mtase domain of the bifunctional nsp (bouvet et al., ; chen et al., c) . cap modification is completed by the conversion of the cap- into a cap- structure, which involves the nsp /nsp complex (bouvet et al., ; chen et al., c) in which nsp possesses -o-mtase activity. in the following paragraphs, we will describe the different cov enzymes involved in mrna capping in more detail. as described earlier, the nsp helicase domain is thought to unwind double-stranded rna in a -to- direction, an activity that energetically depends on the nucleotide triphosphatase (ntpase) function of the enzyme (ivanov and ziebuhr, ; ivanov et al., b; seybert et al., a) . additionally, using the same active site, the protein was found to exhibit rtpase activity (ivanov and ziebuhr, ) , which was found to be abolished if the conserved active-site lys residue of the walker a box motif was replaced with ala (ivanov and ziebuhr, ; ivanov et al., b) . specific rtpase activity on viral mrna species would require specific recruitment of nsp to the end of viral mrnas, which has not been demonstrated for cov helicases but may involve yet other factors. in this context, it remains to be studied if the common leader sequence present on cov mrnas contributes to the recruitment of nsp and/or other proteins involved in capping reactions. several other +rna virus helicases were shown to possess an activity that can target the phosphodiester bond between the β and γ phosphate groups of the -terminal ntp of diverse substrate rnas, suggesting that this dual function of helicase and capping rtpase is a common feature in this group of viruses ivanov and ziebuhr, ) . even though experimental evidence has been obtained to suggest an nsp -associated rtpase activity, coronavirus nsp homologs proved to be unable to transcomplement the yeast strain ybs , which lacks the cet locus encoding the yeast rtpase involved in mrna capping (chen et al., c) . the lack of trans-complementation by nsp could be due to technical reasons, such as inappropriate subcellular localization of nsp , misfolding of the protein, or a functional mismatch with other players of the distantly related yeast capping machinery. alternatively, it could indicate specific substrate requirements for coronavirus nsp -associated rtpase activities. in any case, a possible involvement of nsp in the first step of cov mrna capping remains to be corroborated in further studies, for example, by in vitro reconstitution experiments of the entire cov capping pathway. the cov nsp involved in the second step of rna capping, gtase, remains to be identified. bioinformatics analysis of cov genome sequences failed to identify replicase gene-encoded domains that may perform this activity. eukaryotic rna capping enzymes belong to the ligase family and have been shown to form a gtase-gmp adduct upon incubation with gtp . a substantial number of sars-cov nsps were expressed and purified (nsp , nsp , nsp , and nsp - ), but covalent linkage of gmp to any these proteins could not be demonstrated (jin et al., ) . in addition, nsps were screened for gtase activity in a yeast trans-complementation system using the ybs strain lacking the gene (ceg ) encoding the yeast gtase (chen et al., c) , but also this powerful approach failed to identify the cov gtase. consequently, several hypotheses remain to be explored. first, it is possible that the n-terminal niran domain of nsp (see earlier) forms a covalent adduct with gtp, as observed for its arterivirus homolog nsp (lehmann et al., a) . another possibility is that the cov capping pathway is unconventional and, for example, resembles that of alphaviruses in which the gtp molecule needs to be methylated at its n position before the gtase-m gmp adduct can be formed (ahola and ahlquist, ) . interestingly, this second possibility might explain nsp s capability to convert gtp into m gtp (see later) (jin et al., ) . finally, the involvement of a host gtase remains an interesting possibility, in particular since cytoplasmic forms of cellular capping enzymes have been described recently (mukherjee et al., ; schoenberg and maquat, ). further work is needed to explore these various hypotheses and resolve this important question. coronavirus nsp is a bifunctional protein that plays a crucial role in viral rna synthesis. its n-terminal exonuclease (exon) domain (minskaia et al., ; snijder et al., ) , which is thought to promote the fidelity of cov rna synthesis, will be discussed in more detail in section . the c-terminal part of nsp carries an adomet-dependent guanosine n -mtase activity. interestingly, as in the case of the gtase (see earlier), bioinformatics analyses of cov genome sequences failed to identify proteins or protein domains related to cellular and/or viral n -mtases, again illustrating the significant divergence of nidoviruses from other viral and cellular systems. however, using a trans-complementation assay and a yeast strain lacking the abd (n -mtase) gene, guo et al. discovered a sars-cov nsp -associated n -mtase activity (chen et al., c) by demonstrating that the protein was able to restore the growth of the △abd yeast mutant. they also showed that a range of alphacoronavirus nsp homologs were able to complement the defect of the △abd yeast strain. the n -mtase activity of nsp was subsequently confirmed and characterized using purified recombinant enzymes (bouvet et al., ; chen et al., c) . the sars-cov n -mtase was shown to methylate cap structures in a sequenceindependent manner using a range of rnas and it also proved to be active on cap analogues and gtp (jin et al., ) , corroborating the trans-complementation experiments in yeast in which rescue required efficient methylation of a wide range of cellular rnas. in contrast to the exon activity, the in vitro n -mtase activity was not found to be affected by interactions with nsp (bouvet et al., ) . the n -mtase domain was further characterized by alanine scanning mutagenesis and key residues for enzymatic activity were identified (chen et al., c) including crucial residues distributed throughout the domain and two clusters of residues essential for mtase activity (fig. ). the first cluster (nsp residues - ) corresponds to the dxgxpxa motif of the adomet-binding site. in cross-linking experiments, mutations in this motif strongly decreased the binding of h-labeled adomet. the role of the second cluster, between residues and , was revealed by x-ray structure analysis of a sars-cov nsp /nsp complex expressed in e. coli (fig. ) (ma et al., ) . these residues form a constricted pocket that holds the cap structure (gpppa) between two β-strands (β and β ) and helix , placing the n position of the guanine in close proximity of adomet and ready for methyl transfer using an in-line mechanism. , and ibv (genus gammacoronavirus). the alignment was generated using clustal omega (sievers et al., ) and rendered using espript version . (robert and gouet, ) . conserved exon motifs i, ii, and iii and clusters of residues involved in sam binding and n -mtase activity ( and ) are highlighted in gray. catalytic residues of exon and residues involved in the formation of zinc fingers are indicated by asterisks and arrowheads, respectively. also shown are the secondary structure elements of sars-cov nsp (pdb c s) and the border between the n-terminal exon and c-terminal n -mt (nmt) domains. the structure also revealed that the nsp n -mtase domain exhibits a noncanonical mtase fold. whereas the canonical fold contains a -strand β-sheet that is commonly present in the rossman fold, nsp contains only a -strand β-sheet and an insertion of a -strand antiparallel β-sheet between β and β . in line with previous mutagenesis data (chen et al., c) , the fig. surface representation of the three-dimensional structure of the nsp /nsp complex (pdb c s). the nsp ribbon structure is shown with conserved residues colored in blue (using a scale from dark to light blue). the coloring of the nsp surface is based on the conservation of the respective residues among covs (using a scale from dark to light red). the upper panel shows the surface containing the exon catalytic site with one mg + ion bound in the active site (green sphere). the lower panel shows the opposite side of the structure with the n -mtase active site. the cap analog gpppa and sah are shown in stick representation. the figures were generated using ucsf chimera (pettersen et al., ) . the degree of conservation of specific residues was determined using an alignment of nsp and nsp sequences of eight coronaviruses representing the four genera of the coronavirinae subfamily (sars-cov, mers-cov, mhv, tgev, fcov, hcov- e, ibv, and bulbul coronavirus hku ). nsp x-ray structure revealed functionally relevant interactions between the n-terminal (exon) and c-terminal (n -mtase) domains, with three α-helices of exon stabilizing the base of the n -mtase substrate-binding pocket. the specific role of n -mtase activity in virus replication was supported by reverse genetics. nsp mutations were introduced in sars-cov rna replicons expressing a luciferase reporter gene. a d a mutation in the adomet-binding site, which blocks the n -mtase activity of nsp , was shown to reduce the luciferase expression (by %), indicating that viral rna replication and/or transcription were impaired in this in vitro system (chen et al., c) . the nsp n -mtase is an attractive target for antiviral strategies, especially because it exhibits a range of features that are distinct from host cell mtases . the druggability of the enzyme was explored using a small set of previously documented mtase inhibitors. these in vitro assays revealed that adohcy (the coproduct of the methylation reaction), sinefungin (another adomet analog), and ata efficiently inhibited nsp n -mtase activity with ic values of μm, . nm, and . μm, respectively (bouvet et al., ) . ata was also shown to limit sars-cov replication in infected cells (he et al., ) . in the yeast-△mtase trans-complementation assay mentioned earlier, micromolar concentrations of sinefungin were reported to effectively suppress the nsp n -mtase activity of sars-cov, mhv, transmissible gastroenteritis virus (tgev), and ibv (sun et al., ) . however, other compounds, such as ata and adohcy, did not exert an inhibitory effect in the context of yeast cells. these discrepancies may reflect differences in cell penetration of the compounds between yeast and (virus-infected) mammalian cells. the yeast system was also applied to screen a library of natural product extracts, and three hits were obtained displaying potent inhibitory effects on the cov n -mtase (sun et al., ) . further work is needed to optimize these hits and test their inhibitory activities in assays using cov-infected cells. the presence of a -o-methyl transferase ( -o-mtase) domain in cov nsp was first inferred using bioinformatics (snijder et al., ; von grotthuss et al., ) . computational threading produced a model containing a conserved k-d-k-e catalytic tetrad that is characteristic of adomet-dependent -o-mtases and a conserved adomet-binding site ( fig. ) (von grotthuss et al., ) . the -o-mtase activity was then confirmed by in vitro biochemical assays using purified fcov nsp (decroly et al., ) . the recombinant protein was shown to specifically recognize short, cap- -containing rnas and to transfer a methyl group from adomet to the -o position of the first nucleotide of the n -methylated substrate. in contrast, a recombinant form of the sars-cov nsp homolog was inactive under similar experimental conditions. since yeast two-hybrid experiments and gst pull-down assays had revealed that sars-cov nsp strongly interacts with nsp pan et al., ) , a possible involvement of the latter in regulating or supporting the -o-mtase activity was tested. it was shown that purified nsp interacts with nsp in vitro and thereby triggers a robust -o-mtase activity (bouvet et al., ) . effective methyl transfer was demonstrated using synthetic capped n -methylated rna and longer rna mimicking the end of the sars-cov genome (bouvet et al., ) . in contrast, rna with a nonmethylated cap structure (gppp-rna) was not bound by the nsp / nsp complex and, consequently, could not serve as a substrate. these observations suggest that sars-cov mrna capping follows a strict order of reaction steps: after gtp transfer by the still elusive gtase, the cap is methylated by the nsp n -mtase at the guanosine-n position to produce a cap- structure that, in a subsequent reaction, is bound by the nsp / nsp complex and converted to a cap- structure employing the -o-mtase activity of nsp . mutagenesis of sars-cov nsp and nsp confirmed the importance of the catalytic tetrad of the nsp -o-mtase (decroly et al., ) and showed that the nsp -nsp interaction is absolutely required for activity (decroly et al., ; lugari et al., ) . crystallographic studies of the nsp /nsp complex revealed the molecular basis for the stimulation of the nsp -o-mtase activity by nsp (fig. ) (chen et al., ; decroly et al., ) . the cov -o-mtase belongs to the rrmj/fibrillarin superfamily of ribose -o-methyl transferases (feder et al., ) which have a number of viral orthologs in flaviviruses, alphaviruses, and other nidoviruses. as mentioned earlier, this family contains a conserved k-d-k-e catalytic tetrad (fig. ) that is located in close proximity to the substrate-binding pocket accommodating the rna substrate. the structure revealed that nsp is an allosteric regulator that stabilizes nsp . moreover, structural and biochemical analyses indicated that nsp binding extends and narrows the rna-binding groove that accommodates the rna substrate, thereby promoting the rnaand adomet-binding capabilities of nsp (fig. ) . ) , and ibv (genus gammacoronavirus). the alignment was generated using clustal omega (sievers et al., ) and rendered using espript version . (robert and gouet, ) . residues of the catalytic tetrad k-d-k-e are indicated by asterisks and secondary structure elements of sars-cov nsp (pdb xyv) are shown. (b) surface representation of the three-dimensional structure of the nsp /nsp (continued) the functional relevance of -o-mtase regulation by nsp for virus replication is not yet understood. the nsp -associated -o-mtase activity (itself ) is highly conserved across the coronaviridae family and its functional relevance has been supported by reverse-genetics studies using genetically engineered alpha-and betacoronavirus mutants that lack -o-mtase activity (devarkar et al., ; hyde and diamond, ; schuberth-wagner et al., ; z€ ust et al., ) . furthermore, the phenotype of temperature-sensitive mhv mutants suggested nsp to play a role in rna synthesis or in the stability of plus-strand rna (sawicki et al., ). an early sars-cov study was able to show that the insertion of a stop codon immediately upstream of the nsp -coding sequence blocked viral rna synthesis (almazan et al., ) . subsequently, for hcov- e, mhv, and sars-cov, several mutants with reduced or ablated -o-mtase activity were described that generally retained robust viral replication in cell culture (menachery et al., ; z€ ust et al., ) . the studies also revealed that the impact of nsp mutations may depend on the cell types used and that the lack of -o-mtase activity causes more profound effects in primary cells and immune cells. the sars-cov nsp mutants were further characterized in infected mice and showed a robust attenuation as judged by viral titers, weight loss, lung histology, and respiratory function. the nsp mutants also displayed increased sensitivity to treatment with type i interferon. this was also observed for the corresponding mhv and hcov- e nsp mutants (z€ ust et al., ) . however, in contrast to the latter study, the sars-cov nsp mutant was not found to induce type i interferons, either in vitro or in vivo (menachery et al., ) . this observation suggests that sars-cov may have a larger repertoire of functions for preventing induction of type i interferons following cellular sensing of "nonself" rnas, such as viral rnas with incompletely methylated cap structures. together, these data established that the highly conserved nsp -o-mtase plays an important role in limiting the detection of viral fig. -cont'd complex (pdb xyv). nsp is shown in ribbon representation with conserved residues colored in dark to light blue according to their conservation among covs. zinc molecules are shown as spheres and zinc-coordinating residues are shown in stick representation. the surface of nsp is colored in dark to light red according to the conservation of the respective residues among coronaviruses. sah is depicted in a stick model. the figure was generated using ucsf chimera (pettersen et al., ) . the degree of conservation of specific residues was determined using an alignment of nsp and nsp sequences of eight coronaviruses (see fig. ). rna by the host's antiviral sensors, but that the specific role of this activity in escaping host innate immune responses may differ to some extent among covs. the mechanism underlying the induction of the antiviral immune response following infection with -o-mtase knockout mutants was studied using specific knockout mice in which viral replication was found to be restored. the absence of mda , a cap- sensor, restored mhv replication, whereas the nuclear translocation of irf and interferon induction were reduced (z€ ust et al., ) . this suggested mda to function in the primary recognition of the rna produced by cov -o-mtase mutants and to initiate the isg cascade that restricts viral replication. among the isg products, the ifit family of proteins was shown to be critically involved in reducing the replication of cov nsp mutants. the replication of mhv and hcov- e nsp mutants and wild-type controls was similar in ifit À/À knockout mice (habjan et al., ; z€ ust et al., ) . likewise, replication of a sars-cov Δnsp mutant was increased in both ifit and ifit knockdown mice (menachery et al., ) , suggesting that ifit family proteins mediate the primary attenuation of sars-cov -o-mtase knockout mutants. the earlier results indicate that the nsp -o-mtase constitutes a new and attractive target for the development of antiviral drugs against sars-cov and hcov- e, as well as newly emerging covs like mers-cov and porcine epidemic diarrhea virus (pedv). for example, the nsp -nsp interface may be targeted to limit viral -o-mtase activity and thus restore the antiviral responses mediated by mda and ifit (menachery and baric, ) . interestingly, the nsp residues involved in the nsp /nsp interaction are quite conserved within the cov family and it was recently demonstrated that nsp of different covs (fcov, mhv, sars-cov, mers-cov) is functionally interchangeable in the stimulation of nsp -o-mtase activity . thus, molecules or peptides blocking this interface may have broad-spectrum anti-cov effects, a concept that was explored and supported using synthetic peptides that mimic the nsp interface and suppress nsp -o-mtase activity in vitro (ke et al., ; wang et al., ) . the antiviral effect of the mhv tp peptide, for example, was first demonstrated in mhv-infected cells and was subsequently confirmed to limit mhv replication in mice and to enhance the type i interferon response . the same peptide was also effective in blocking the replication of a sars-cov replicon. the cov exon domain was identified on the basis of comparative sequence analyses (snijder et al., ) that suggested a distant relationship of the nsp n-terminal domain (and equivalent polyprotein regions of other nidoviruses) with cellular dedd exonucleases, a large protein superfamily containing rna and dna exonucleases from all kingdoms of life (zuo and deutscher, ) . the designation "dedd" alludes to four invariant asp/glu residues that are part of three sequence motifs, i-iii, that are conserved in members of this superfamily (fig. ) . the dedd superfamily is also referred to as dnaq-like family because it includes dnaq, the ε subunit of e. coli dna polymerase iii, a well-characterized proofreading enzyme scheuermann et al., ) . conservation of a fifth residue, his, located four positions upstream of the conserved asp in motif iii identifies exon as a member of the deddh subgroup, while members of the deddy exonucleases contain tyr at the equivalent position. the acidic residues are required to form two metal-binding sites. based on catalytic models initially developed for cellular exonucleases and catalytic rna (beese and steitz, ; steitz and steitz, ) , the conserved his and the site a metal ion are thought to activate a water molecule that launches a nucleophilic attack on the phosphorus group of the -terminal phosphodiester, while the site b metal ion is thought to stabilize the transition state (derbyshire et al., ) . exon is conserved in covs and all other known nidoviruses with genome sizes of > kb minskaia et al., ; nga et al., ; snijder et al., ; zirkel et al., ) . the correlation between genome size and exon conservation suggests that, in nidoviruses with medium-and large-size genomes, the correct nucleotide selection and recognition of properly formed base pairs by the rdrp is not enough to accomplish the necessary replication fidelity and, therefore, requires additional functions suitable to detect and remove misincorporated nucleotides. recently, biochemical evidence has been provided to suggest that exon may have exactly this function (bouvet et al., ) . cov mutants that lack exon activity provided additional evidence for exon being involved in mechanisms that keep the cov mutation rate at a relatively low level (< À mutations per site per round of replication for mhv and sars-cov) (eckerle et al., (eckerle et al., , , while other rna viruses have much higher mutation rates, ranging from À to À mutations per site per round of replication (drake and holland, ; sanjuan et al., ) . exon knockout mutants of sars-cov and mhv were shown to display a mutator phenotype with significantly increased mutation frequencies approaching those of other rna viruses (eckerle et al., (eckerle et al., , . considering that these studies were performed under selection pressure favoring genotypes with high replication efficiency, the total number of mutations in rnas produced by exon-deficient viruses may be even higher than calculated in that study, especially in genome regions that are not subject to selection in in vitro cell culture systems. while inactivation of exon activity was tolerated by mhv and sars-cov (albeit with reductions in replication efficiency), stable exon-deficient mutants of the alphacoronaviruses hcov- e and tgev could not be recovered (becares et al., ; minskaia et al., ) , supporting the critical role of this activity in cov replication. taken together, the available information provides compelling evidence for exon playing a key role in high-fidelity replication of covs. consistent with this hypothesis, genetically engineered exon knockout mutants were shown to be significantly more sensitive to rna mutagens such as ribavirin and -fluorouracil (up to -fold). furthermore, compared to wild-type virus, the exon knockout mutants were shown to accumulate a much higher number of mutations when propagated in the presence of mutagens (smith et al., ) . the lack of exon activity was also shown to have profound effects on viral replication and pathogenesis in vivo (graham et al., ) . exon-negative mutants displayed a stable mutator phenotype in a number of mouse models of human sars, providing a promising approach for the stable attenuation of highly pathogenic covs with important implications for vaccine development (graham et al., ) . coronavirus nsp is a bifunctional protein comprised of an n-terminal exon domain and a c-terminal n -mtase domain. surprisingly, the latter domain is not conserved in the torovirinae (genera torovirus and bafinivirus), representing the other subfamily of the coronaviridae, but in other (more distantly related) nidovirus branches (lauber et al., ; nga et al., ) . this conservation pattern suggests that a common ancestor of the corona-, mesoni-, and roniviridae contained the two-domain exon/n -mtase structure while some lineages lost the n -mtase domain at a later stage. although nsp does not require other proteins for activity minskaia et al., ) , its ribonucleolytic (but not the n -mtase) activity was shown to be stimulated significantly in the presence nsp . in line with this, nsp variants carrying amino acid substitutions that prevent the interaction with nsp failed to stimulate exon activity (bouvet et al., (bouvet et al., , . to date, there is no evidence to suggest a direct role for nsp in catalysis. most likely, interactions between the nsp and the n-terminal domain of nsp stabilize the exon active site in a catalytically competent conformation. mutagenesis data and a recent x-ray structure analysis of a sars-cov nsp /nsp complex (see fig. ) revealed that the nsp surface required for interaction with nsp overlaps with the surface involved in the interaction and activation of the nsp -o-mtase activity (see earlier) (bouvet et al., ; ma et al., ) . coronavirus exon activities were first demonstrated using recombinant forms of sars-cov nsp expressed in e. coli (minskaia et al., ) . the protein was shown to require mg + or mn + ions for activity and to degrade a range of single-stranded (ss) synthetic rnas with -to- directionality to yield reaction products of about - nucleotides. the data further suggested that rna secondary structure affects exon activity (minskaia et al., ) . mutational analysis of predicted active-site (asp/glu) residues confirmed their critical involvement in catalysis (minskaia et al., ) . in a subsequent study, using nsp /nsp complexes, the substrate specificity was characterized in more detail and revealed dsrna with a terminal mismatch to be the preferred substrate for exon activity. excision efficiencies using different mismatched base pairs (a:g, a:a, a:c, u:g, u:c, u:u) were found to be similar, suggesting that the mismatch rather than the nature of the nucleotide misincorporated at the end determines exon activity (bouvet et al., ) . in a recent study, the structures of unliganded, sam-bound, and sah-gpppa-bound sars-cov nsp -nsp complexes were determined by x-ray crystallography (ma et al., ) . the structures provide important insight into the two-subdomain structure of nsp , the two catalytic sites of exon and n -mtase, critical substrate-binding residues, the contribution of nsp to enhancing exon activity, and the roles of as many as three zinc fingers present in nsp (fig. ) . in the structure, one molecule of nsp was found to bind one molecule of nsp . given that nsp tends to form multimers , it is tempting to speculate that, in infected cells, the nsp -nsp complex may form even larger complexes, for example, by interacting with nsp -nsp complexes that might be stabilized by nsp -nsp interactions. in this way, consecutive methylation reactions of the cap structure could be spatially coordinated. comparison of the nsp surfaces that interact with nsp and nsp , respectively, revealed a significant overlap (bouvet et al., ) , with a substantially larger surface being involved in interactions between nsp and nsp . buried solvent accessible areas for interactions with nsp and nsp were determined to be and Å , respectively (decroly et al., ; ma et al., ) . the structure of the nsp -nsp complex also helps to explain the observed stimulation of exon activity by nsp (see earlier). two regions of nsp interact extensively with different structural elements of the nsp n-terminal domain, most likely to maintain the structural integrity of the exon domain. interestingly, the observed interaction of n-terminal residues of nsp with nsp led to interpretable electron density for these residues that had not been observed in previous structures of nsp (joseph et al., ; su et al., ) or the nsp -nsp complex (chen et al., ; decroly et al., ) , consistent with the proposed role of the n-terminal loop of nsp in stabilizing interactions with nsp . the structure of the exon domain is essentially comprised of a twisted β-sheet that is formed by five β-strands and flanked by α-helices on either side. it resembles that of other dedd superfamily exonucleases, such as the ε subunit of e. coli dna polymerase iii, but also has unique features. these include two segments (residues - and - ) that are involved in the interaction with nsp and two zinc fingers in the exon domain (see fig. ). the second zinc finger was found in close proximity to the catalytic site. both its position in the structure and mutagenesis data support a role for this zinc finger in catalysis (ma et al., ) . the other zinc finger appears to be required to maintain the structural integrity of nsp . consistent with this, nsp variants containing substitutions in zinc finger proved to be insoluble when expressed in e. coli. five residues predicted to coordinate two mg + ions, , were found in the catalytic site, with one mg + ion being coordinated by asp- (exon motif i) and glu- (motif ii) (fig. ) . the second mg + ion expected to be involved in the two-metalion-assisted catalytic mechanism of exon (beese and steitz, ; chen et al., ; ulferts and ziebuhr, ) was not identified, presumably due to the lack of an rna substrate and/or product in this structure. with one exception, metal ion-coordinating residues identified in the active site corresponded well to those identified in related cellular proofreading exonucleases (beese et al., ; hamdan et al., ) and previous predictions for nidovirus homologs (snijder et al., ) . the structure clearly revealed to be involved in catalysis, thus revising previous predictions on the identity of exon motif ii in the cov nsp primary structure and making nsp a "deed outlier" in the dedd superfamily of exonucleases. the combined structural and functional information obtained for nsp including its subdomains and the nsp cofactor provides an excellent basis for studies using even larger multisubunit complexes to obtain insight into the coordinated action of key replicative enzymes involved in rna synthesis, quality control, capping, methylation, and other functions (subissi et al., a) . the nsp -associated endou domain is one of the most conserved proteins among covs and related viruses (fig. ) , suggesting important functions in the viral replicative cycle. already in the first sequence analyses of torovirus and arterivirus replicase genes published more than years ago (den boon et al., ; snijder et al., ) , the identification of a conserved sequence in the -terminal orf b region, including the (at the time unknown) endou domain, was key to establishing phylogenetic relationships between corona-and toroviruses and, subsequently, also arteriviruses (cavanagh and horzinek, ; snijder et al., ) . outside the nidovirales, no viral homologs of endou have been identified to date. together with the helicase-associated zbd (see earlier), the nidoviral endou has therefore been proposed to be a unique and universally conserved genetic marker common to all nidoviruses (ivanov et al., a; snijder et al., ) . only recently, with the identification of the first nidoviruses in insects (now classified in the family mesoniviridae) and reanalysis of the ronivirus replicase gene, it was found that endou is not conserved in those nidovirus branches that replicate in invertebrate hosts (mesoniviridae, roniviridae) (lauber et al., (lauber et al., , nga et al., ; zirkel et al., ) , suggesting specific roles in vertebrate hosts. to date, these functions and, more specifically, the biologically relevant substrates of endou have not been identified. characterization of mhv endou knockout mutants revealed only minor effects on viral rna synthesis in infected cells, with all rna species being equally affected, and caused a slight reduction in virus titers, which was most evident at later time points post infection (kang et al., ) . for the arterivirus eav, substitutions of several conserved residues in the endou domain were found to cause more profound effects, both in virus reproduction and viral rna accumulation, with sg rna being more affected than genome replication in several mutants. in some cases, reduction in virus titers by up to log was observed (posthuma et al., ) . the (limited) information obtained in these studies suggests that endou activity is not strictly required for nidovirus rna synthesis, at least in cell culture. however, the strong conservation clearly suggests an important in vivo function that remains to be identified in suitable model systems. initial evidence for specific functions of nidovirus endou domains in infected cells was obtained in studies showing that sars-cov nsp , but surprisingly not the homologous proteins from hcov-nl and hcov-hku , counteracts mavs-induced apoptosis (lei et al., ) . further experiments will be necessary to confirm and assess the significance of these functions for virus replication and/or virus-host interactions. nidovirus-encoded endou activities have been characterized using recombinant forms of cov nsp (sars-cov, hcov- e, mhv-a , ibv) and arterivirus nsp (eav, prrsv) (bhardwaj et al., ; ivanov et al., a; kang et al., ; nedialkova et al., ) . in a number of studies, recombinant nidovirus endous were shown (i) to have endonucleolytic activity, (ii) to cleave of pyrimidines, preferring uridine over cytidine, and (iii) to release reaction products with , -cyclic phosphate and -oh ends. using suitable test substrates, rna structural features were shown to affect endou cleavage efficiency, with unpaired pyrimidines being processed more efficiently (bhardwaj et al., ; nedialkova et al., ). the role of metal ions in endou activity is not entirely clear, with somewhat contradictory data being reported for different homologs. while the activities of cellular and cov homologs were shown to require (or to be significantly stimulated by) mn + ions (bhardwaj et al., ; ivanov et al., a; laneve et al., laneve et al., , , arterivirus endou activities proved to be less dependent on metal ions. low concentrations of mn + were found to stimulate only marginally the arterivirus endou activities, whereas higher concentrations (previously shown to be required for optimal nucleolytic activities in cov and cellular homologs) inhibited the activities of eav and prrsv endous (nedialkova et al., ) . metal ion requirements are commonly used to distinguish between the two basic catalytic mechanisms employed by ribonucleases: the metal-independent mechanism that, for example, is employed by rnase a and results in products with , cyclic phosphate ends (as described earlier for endou), and the metaldependent mechanism in which catalysis is aided by two divalent cations coordinated by conserved acidic residues and generates products with -oh and -phosphate ends (as described earlier for exon). the critical (or supportive) role of metal ions observed for several cellular and viral endou homologs is inconsistent with an rnase a-like (metal-independent) reaction mechanism. also, metal ions were not detected in any of the structures determined for coronavirus nsp s or xendou, arguing against a direct role of metal ions in catalysis (renzi et al., ; ricagno et al., ) . however, mn + ions were found to change the intrinsic tryptophan fluorescence of sars-cov nsp , suggesting conformational changes that, potentially, may affect activity and were shown to be unrelated to protein multimerization (bhardwaj et al., ; guarino et al., ) . also, regarding the role of mn + in rna binding, contradicting data have been reported, with rna binding by sars-cov nsp being enhanced in the presence of mn + or not affected by metal ions in the case of xendou (bhardwaj et al., ; gioia et al., ) . structural information has been obtained by x-ray crystallography and cryoelectron microscopy studies for several cov and cellular endou homologs (bhardwaj et al., ; renzi et al., ; ricagno et al., ; xu et al., ) . sars-cov and mhv nsp s were shown to form homohexamers comprised of a dimer of trimers. the nsp monomers have an α + β structure with three subdomains, a small n-terminal, an intermediate-sized middle, and a large c-terminal domain, the latter basically representing the "conserved domain" of the cov-like superfamily (see earlier). one side of this domain contains two β-sheets that line the positively charged active-site groove, while the other side is formed by five α-helices. the structures of the catalytic domains are largely conserved among cov endous and xendou, supporting the proposed common phylogeny of viral cellular members of this large endoribonuclease family (bhardwaj et al., ; renzi et al., ; snijder et al., ) . the endou hexamer reported for sars-cov nsp (ricagno et al., ) has dimensions of -  Š, forming a three-petal-shaped surface that surrounds a small, predominantly negatively charged central channel with an inner diameter of $ Å . the hexamer has six-independent active sites located on the surface of the molecule. interactions between individual protomers are predominantly mediated by residues located in the n-terminal and middle domains. database searches failed to identify closely related structural homologs, suggesting that endous diverged profoundly from other ribonucleases (renzi et al., ; ricagno et al., ) . nevertheless, the presumed endou catalytic residues (his/his/ lys, fig. ) could be superimposed with the catalytic his/his/lys residues of bovine rnase a, the prototype of a large superfamily of pyrimidinespecific ribonucleases (ricagno et al., ; ulferts and ziebuhr, ) . the superposition also includes a number of conserved substrate-binding residues. a comparison of viral and cellular endou structures with that of rnase a and related nucleases using the pdbefold server revealed similarities between the structural cores of these enzymes that may be described as "interrelated by topological permutation," providing initial evidence for a common ancestry of the two endonuclease families (ulferts and ziebuhr, ) . further studies are required to substantiate this hypothesis (see later). the hexameric form is thought to be the fully active form of cov nsp . this is supported by the exponential increase of activity with increased protein concentrations, the reduced activities determined for protein variants that do not multimerize and the increased rna-binding activities observed for hexameric forms of endou (bhardwaj et al., ; guarino et al., ; xu et al., ) . consistent with this, hexamerization has been confirmed for different cov endou homologs and residues confirmed to be involved in intersubunit interactions are highly conserved among covs. in the structure of a truncated, monomeric form of sars-cov nsp that lacks n-terminal and c-terminal residues, two loops of the catalytic domain were found to be displaced compared to their location in the hexamer, resulting in the destruction of the active site (joseph et al., ) . in hexameric structures, the two loops pack against each other and are stabilized by intermonomer interactions, suggesting that hexamerization may induce an allosteric switch. furthermore, cross-linking and cryoelectron microscopy studies support a specific role of hexamerization in rna binding (bhardwaj et al., (bhardwaj et al., , . in the structure model, the proposed active-site his and lys residues (fig. ) identified by comparative sequence analysis and site-directed mutagenesis (bhardwaj et al., ; gioia et al., ; guarino et al., ; ivanov et al., a; kang et al., ; snijder et al., ) were found to be embedded in a positively charged groove of the catalytic domain. other residues identified in the active site and proposed to be involved in binding to the substrate phosphate include the side chain of a conserved thr (fig. ) and the main chain amide of a conserved gly (fig. ) (bhardwaj et al., ; ricagno et al., ; xu et al., ) . the proposed functional role of the latter residues has also been corroborated by mutagenesis data for several endou homologs (kang et al., ; renzi et al., ; ricagno et al., ) . as mentioned earlier, endou and rnase a share a number of features in their active sites. in rnase a, pyrimidine binding primarily involves thr- and phe- . while thr- forms hydrogen bonds with the pyrimidine base, phe- interacts with the base through stacking interactions (raines, ) . the ser- /tyr- residues of sars-cov nsp and the thr- /phe- residues of rnase a occupy similar positions in the active-site clefts of the two enzymes (ulferts and ziebuhr, ) . the ser/thr residue is conserved in viral and cellular domains, while tyr- is conserved in viral endou homologs while conservative substitutions (phe, trp) are occasionally found in cellular endou homologs (renzi et al., ; ricagno et al., ) . the role of sars-cov endou ser- and tyr- (and equivalent residues in related enzymes) in substrate binding received strong support by molecular modeling and site-directed mutagenesis data, with the conserved ser/thr residue being confirmed to have a critical role in the differential cleavage of uridine-and cytidinecontaining substrates, respectively (bhardwaj et al., ; nedialkova et al., ; ricagno et al., ) . similarities in their active-site structures and reaction products containing , -cyclic phosphate ends suggest that endous and rnase a-like endoribonucleases employ similar catalytic mechanisms (nedialkova et al., ; ricagno et al., ) . for rnase a, it has been established that two his residues in the active site act as general base and acid, respectively. the his residue that acts as a general base attracts a hydrogen from the ribose -hydroxyl group that subsequently attacks the p-o bond. the second his donates a hydrogen to the -o, thus facilitating displacement of this group and subsequent product release (raines, ) . in a second step, the , -cyclic phosphate is hydrolyzed, resulting in a -phosphomonoester product and recovery of the enzyme. the latter is essentially a reverse reaction of the transphosphorylation reaction, with the protonated his now acting as an acid and the other his acting as a base. in both reaction steps, lys interacts with the phosphate to stabilize the pentavalent reaction intermediate. although the reaction mechanisms employed by endous have not been studied in detail, the enzymes are thought to use an rnase a-like catalytic mechanism. this is supported by several lines of evidence, including (i) the conserved spatial positions in the structure and the critical functional role of two his and one lys residue(s) (ricagno et al., ) , and (ii) the release of , -cyclic phosphate-containing reaction products (bhardwaj et al., ; ivanov et al., a) that are converted to products with -hydroxyl ends (nedialkova et al., ). since the first in-depth analysis of a cov replicase in (gorbalenya et al., ) , significant progress has been made in terms of its structural and functional characterization. a multitude of enzymatic functions has been identified and characterized in vitro, although mainly using artificial substrates so far. protein structures were obtained for most of the subunits from the nsp - region (neuman et al., b) , but unfortunately two prominent remaining "blank spots" on this map concern two key enzymes in cov rna synthesis, rdrp and helicase. filling those gaps would constitute an important step forward, to address basic questions like the priming mechanism employed by the rdrp and the function of the niran domain, and to accelerate targeted drug discovery, for example, in the area of nucleoside inhibitors of cov rna synthesis, which has received little attention thus far. clearly, where cov mrna capping is concerned, identification of the elusive gtase remains a research priority (subissi et al., a) . for other nsps, potential functions (nsp , the n-terminal domain of nsp ) or substrates (nsp -endou) remain to be found. as highlighted by several nsp-wide interaction screening studies pan et al., ; von brunn et al., ) and more specifically by the in vitro data on the interplay between nsp - - - (subissi et al., b) and nsp - - (bouvet et al., ) , cov nsps need to work together in many ways. the further characterization of the "nsp interactome," now also inside the cov-infected cell, will undoubtedly provide more clues as to how specific functions are switched on and off or modulated. likewise, attention should be given to defining the interactions of cov nsps with the specific rna signals for genome replication (madhugiri et al., ; yang and leibowitz, ) , discontinuous minus-strand rna synthesis (attenuation at body trss, nascent minus-strand transfer, and reinitiation; pasternak et al., ; sawicki et al., ; sola et al., ) , and the transcription, capping, and polyadenylation of sg mrnas (fig. ) . these rna sequences, several of which may be cis-acting, could provide starting points for improved biochemical assays, ultimately paving the way for the complete in vitro reconstitution of some of these multi-nsp-driven processes. the analysis of cov rtc structure and function in the living infected cell remains an enormous technical challenge, requiring continued toolbox development. it is likely that several functional riddles can only be solved by studying infected cells. for example, the endoribonuclease activity of the nsp endou domain, a potential "suicide enzyme" for an rna virus, must be controlled tightly in the infected cell. whereas the enzyme is highly active and displays only very limited substrate specificity in vitro (ivanov et al., a; nedialkova et al., ) , it may be confined to a specific compartment in the infected cell and/or its activity may be modulated by interactions with other nsps or host factors. such differences between in vitro and in vivo activities will surely emerge for other nsps as well, and they may be better understood following the further characterization (including their lipid composition) of the membranous replication organelles with which the metabolically active cov rtc presumably is associated (hagemeijer et al., ; neuman et al., a; van der hoeven et al., ) . these studies should also answer the question of how both nsps and viral rna substrates are targeted to or recruited by the membrane-bound cov rtc, in particular also during the earlier stages of infection when viral rna synthesis appears to be taking off in the absence of the prominent membrane rearrangements observed later in infection. specific mutations in cov genomes can now be reverse engineered, but many of the functions encoded by nsp -nsp are so basic that their inactivation will merely result in dead virus mutants that do not provide many deeper insights into nsp function. this in part explains why most progress thus far has been made for some of the functions that can-fortunatelybe inactivated without such lethal consequences, like the nsp exon and nsp -o-mtase enzymes (eckerle et al., (eckerle et al., , graham et al., ; menachery et al., ; smith et al., ; z€ ust et al., ) . for this reason, the field should continue to also employ "traditional" (forward) genetic methods to characterize (and produce more) conditionally defective covs, like temperature-sensitive mutants (sawicki et al., ) . thanks to the advent of next-generation sequencing technologies, tracing the evolution of crippled virus mutants and (pseudo) revertants has become much more straightforward than before, and this approach (letting the virus do the work) likely is among the most economical ones in uncovering previously unknown interactions between the protein and rna players in cov replication (z€ ust et al., ) . furthermore, it may be possible to develop cell-based assays for the analysis of cov nsp functions that do not rely on having a replication-competent virus to start with. unraveling the molecular mechanisms underlying the presumed mismatch excision function (bouvet et al., ) of the nsp -exon, which is uniquely encoded by rna virus genomes larger than kb (nga et al., ) , connects to the mechanisms driving the evolution of nidoviruses at large (lauber et al., ) . also, replicases from other nidovirus branches will need to be studied to fully understand the basic principles governing the profound divergence and genome expansion of this exceptional order of +rna viruses. the error rate and genomic plasticity of rna viruses are among their most fascinating features, and also form the basis for the many problems caused by rna virus mutation and adaptation, including successful zoonotic transfer. as exemplified by the viable cov mutants lacking the exon or -o-mtase functions (graham et al., ; habjan et al., ; menachery et al., ; z€ ust et al., ) , the functional characterization of cov replicative enzymes can be key to the development of conceptually new live attenuated vaccine prototypes. likewise, it will contribute to the development of broad-spectrum and highly effective antiviral drugs targeting essential enzyme functions, critical interactions with nsp cofactors, or "nonessential" nsp functions that promote efficient viral replication and/or pathogenesis. as highlighted by the sars and mers outbreaks of the past years, having such compounds available would definitely strengthen our first line of defense against cov infections in humans. with great respect, we acknowledge the many ground-breaking contributions of our longterm collaborator dr. alexander gorbalenya, founding father of the functional characterization of the replicase of coronaviruses and nidoviruses at large. we are also grateful for the scientific and technical contributions over many years by numerous past and present lab members and colleagues in leiden (e.j.s.), marseille (e.d.), and giessen/ belfast/w€ urzburg (j.z.). 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the sars-coronavirus nsp +nsp complex is a unique multimeric rna polymerase capable of both de novo initiation and primer extension nidovirus ribonucleases: structures and functions in viral replication biogenesis and architecture of arterivirus replication organelles initiation of viral rna-dependent rna polymerization an infectious arterivirus cdna clone: identification of a replicase point mutation that abolishes discontinuous mrna transcription the predicted metalbinding region of the arterivirus helicase protein is involved in subgenomic mrna synthesis, genome replication, and virion biogenesis the in vitro rna synthesizing activity of the isolated arterivirus replication/transcription complex is dependent on a host factor discontinuous and nondiscontinuous subgenomic rna transcription in a nidovirus crystal structures of complexes of pcra dna helicase with a dna substrate indicate an inchworm mechanism new insights on the role of paired membrane structures in coronavirus replication analysis of intraviral protein-protein interactions of the sars coronavirus orfeome mrna cap- methyltransferase in the sars genome distantly related sequences in the alpha-and beta-subunits of atp synthase, myosin, kinases and other atp-requiring enzymes and a common nucleotide binding fold coronavirus nsp / nsp methyltransferase can be targeted by nsp -derived peptide in vitro and in vivo to reduce replication and pathogenesis protection against filovirus diseases by a novel broad-spectrum nucleoside analogue bcx nonstructural proteins and of feline coronavirus form a : heterotrimer that exhibits primer-independent rna polymerase activity expanding use of multi-origin subcellular membranes by positivestrand rna viruses during replication molecular model of sars coronavirus polymerase: implications for biochemical functions and drug design new antiviral target revealed by the hexameric structure of mouse hepatitis virus nonstructural protein nsp coronavirus infection induces dna replication stress partly through interaction of its nonstructural protein with the p subunit of dna polymerase delta the structure and functions of coronavirus genomic and ends inhibition of sars coronavirus helicase by bismuth complexes enzyme-catalyzed dna unwinding: studies on escherichia coli rep protein identification of myricetin and scutellarein as novel chemical inhibitors of the sars coronavirus helicase, nsp identification and characterization of a ribose -o-methyltransferase encoded by the ronivirus branch of nidovirales insights into sars-cov transcription and replication from the structure of the nsp -nsp hexadecamer processing of the human coronavirus e replicase polyproteins by the virus-encoded c-like proteinase: identification of proteolytic products and cleavage sites common to pp a and pp ab virus-encoded proteinases and proteolytic processing in the nidovirales an insect nidovirus emerging from a primary tropical rainforest identification and characterization of genetically divergent members of the newly established family mesoniviridae coronaviruses-drug discovery and therapeutic options exoribonuclease superfamilies: structural analysis and phylogenetic distribution genetic interactions between an essential cis-acting rna pseudoknot, replicase gene products, and the extreme end of the mouse coronavirus genome ribose -o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda key: cord- -woktjl h authors: holmes, kathryn v.; boyle, john f.; frana, mark f. title: mouse hepatitis virus: molecular biology and implications for pathogenesis date: - - journal: viral and mycoplasmal of laboratory rodents doi: . /b - - - - . - sha: doc_id: cord_uid: woktjl h nan it has long been recognized that host factors play an important role in coronavirus-host interactions ( , ). age is an important determinant of coronavirus virulence. infections with most enterotropic coronaviruses cause much more severe disease in neonatal animals than in older animals ( ). bang and his colleagues ( , ) showed nearly years ago that certain host genes could determine the outcome of infection with mhv. the murine genes for susceptibility or resistance to mhv which they identified determine both susceptibility of the mouse to death from mhv infection and permissiveness for mhv replication of cultured peritoneal macrophages from susceptible or resistant animals. in later studies, genetically determined host resistance to mhv was also found in cultured oligodendroglial cells or hepatocytes ( , ). the molecular mechanisms for host genetic resistance to coronavirus replication have not yet been elucidated. the integrity of the host's immunological system is another important determinant of the outcome of coronavirus infection. immunosuppression enhances susceptibility to some coronaviruses, such as mhv ( ). mice given transplanted tumors or immunosuppressive drugs, or infected with other viruses, bacteria or parasites may develop fulminating hepatitis and die from what would normally have been a mild or inapparent infection with mhv ( ). nude mice, which lack functional t lymphocytes, show greater morbidity and mortality from mhv infection than do immunologically intact animals. in an mhv epidemic in a mouse colony, the spectrum of disease ranges from inapparent infection to overwhelming infection and death. host factors undoubtedly play important roles in this variation in virulence of virus infection. animals with inapparent mhv infections are carriers which can transmit infection to susceptibles. in such carriers, the sites and extent of virus replication and the duration of virus shedding have not yet been clearly defined. there is widespread belief that coronaviruses can cause asymptomatic persistent infection with virus shedding over a period of months. mhv can cause persistent infection in vitro ( ) and in vivo ( ) , but many infections in vivo appear to resolve rather quickly without virus persistence ( , ) . however, a temperature-sensitive mutant of mhv-jhm was observed to persist for at least one year in balb/c mice ( ) . the host and virus factors which permit persistence of mhv have not been defined. mhv epidemics in laboratory animal colonies have been implicated as a possible cause of variability in diverse experimental protocols. the time has come to progress from anecdotes about the effects of mhv on murine research to documentation of specific effects of mhv infection on well defined experimental protocols and to characterization of the pathogenic mechanisms responsible. in this paper we will summarize current concepts of coronavirus structure and replication ( ) ( ) ( ) , and then evaluate the critical role of the host in determining the outcome of mhv infection. mhv, like all coronaviruses, is an enveloped rna virus with a helical nucleocapsid. the genome is single stranded, non-segmented rna of positive or message sense ( ) . it interacts with a k phosphoprotein, n, to form the long flexible, helical nucleocapsid. figure shows the characteristic long, petal-shaped peplomers or spikes on the viral envelope which give the coronaviruses their name. the viral envelope is composed of two viral glycoproteins in a lipid bilayer derived from intracellular membranes. the peplomers are composed of a glycoprotein, e , in both the intact form ( k) and its proteolytically cleaved products ( a and b) ( ) . the e glycoprotein is responsible for attachment to receptors on susceptible cells and for virusinduced cell fusion ( , [ ] [ ] [ ] . the other glycoprotein of the coronavirus envelope, el, is an unusual transmembrane model of coronavirus replication. following ads genomic rna serum as mrna, and rna polymerase ( k) is made. th length, negative strand rna template, six subgenomic mrnas and translated to yield one protein, except that mrna # yields two virus assembles at intracellular membranes, and virions are vesicles by cellular secretion. adapted from sturman and h academic press. translation of each of the viral mrnas yields a single protein encoded by the gene at the f end of the mrna. an exception is mrna , which encodes two nonstructural proteins whose amino acid sequences have been deduced ( ) . the functions of these and the other non-structural proteins are not yet known. the nucleocapsid (n) protein is synthesized on free ribosomes and is then phosphorylated ( ) ( ) ( ) . phosphorylated n interacts with viral genomic rna to form helical nucleocapsids which are either assembled into virions or accumulated in cytoplasmic inclusions. the majority of the n protein synthesized in infected cells is not released in virions, but remains cell-associated ( ) . n is processed to form several faster migrating species of unknown functional significance ( ) . the two envelope glycoproteins of mhv are synthesized, transported and processed differently in infected cells ( , ) . e , the peplomeric glycoprotein, is synthesized on the rough endoplasmic reticulum (rer). mannose-rich core sugars are added co-translationally, and e is transported to the golgi apparatus where n-linked oligosaccharides are trimmed and terminal sugars such as fucose and sialic acid are added by cellular enyzmes ( ). also in the golgi vesicles, palmitic acid is covalently bonded to e ( ) . glycosylation of e is inhibited by tunicamycin ( ) . e is incorporated into virions which bud at the rer and golgi membranes, and excess e is carried by a host glycoprotein transport mechanism to the plasma membrane ( ) . there it may participate in virus-induced cell fusion and make the infected cell a target for an antiviral immune response. in vitro translation studies have demonstrated that the el glycoprotein can be synthesized on free ribo somes. but in the presence of microsomes, it can insert through the lipid bilayer by means of an internal signal sequence ( , , ). glycosylation of serine and threonine residues in the external domain of el occurs only after the molecule has reached golgi membranes and is not inhibited by tunicamycin ( , ). in infected cells, el accumulates in vesicles of the golgi apparatus but is not transported to the plasma membrane ( figure ) . we have postulated that the restruction of el to intracellular membranes determines the site of coronavirus budding in infected cells ( ) . a cl cell was fixed with acetone hours after infection with mhv-a , and stained with monoclonal antibody to the el glycoprotein. el is found in small amounts in the rer, accumulates in large amounts in a perinuclear region which is the golgi apparatus, and does not migrate to the plasma membrane. in contrast, e is found in the rer and is readily transported to the plasma membrane (data not shown). assembly of virions occurs in the rer or golgi apparatus where viral nucleocapsids interact with regions of membrane containing viral glycoproteins el and e (figure ). virions released into smooth-walled vesicles apparently escape from intact cells by cellular secretion. released virions are frequently observed adsorbed to the plasma membrane of infected cells ( ) . released virions may adsorb to the plasma membrane, but coronaviruses do not bud from the plasma membrane. magnification: x , . the preceding discussion shows that coronavirus replication depends upon a large number of host cell processes (table ) . it is therefore not surprising that the ability of different cell types to support coronavirus replication varies markedly. many coronaviruses can only be isolated in cells from mice genetically resistant to mhv, virus replication appears to be arrested at a very early step, since no virus specific antigens can be detected ( ). it is not yet clear whether this inhibition is at the level of virus adsorption and penetration or early rna synthesis. complementation studies indicate that at least genes are required for coronavirus rna synthesis ( , ). if some host cell functions are also necessary for transcription of viral rna, this could provide another mechanism for host control of coronavirus replication. the fate of the genomic rna of the input virus in an abortive infection is not known. if this rna could survive within an abortively infected cell, it might later be reactivated, providing a mechanism for persistence of coronaviruses jln_ vitro and in vivo. possible persistence of unexpressed viral rna could be explored using cell cultures persistently infected with mhv. in such cultures although viral antigens are not detectable in the majority of cells, cloning of the cells leads to recovery of infectious virus ( ). when different strains of mhv are used to infect the same cell line, the relative abundance of different mrna species varies markedly ( ) . the host or viral factors which determine the relative abundance of these mrna species are not understood, but the resulting differences in abundance of viral specific polypeptides in the infected cells may affect the outcome of infection. the cytopathic effects of coronaviruses are host cell dependent, and may include either cell lysis, cell fusion, or no morphological change. certain bovine cell lines infected with bcv normally show no cpe, but fuse extensively if trypsin is added to the medium ( , ) . this suggests that proteolytic cleavage of a viral glycoprotein might be required to activate coronavirus cell fusing, as has been demonstrated with paramyxoviruses and orthomyxoviruses ( , )· trypsin treatment of mhv virions released from the cl line of balb/c t cells results in quantitative conversion of the k form of e into k species and also renders concentrated virions able to cause fusion from without (ffwo), that is immediate fusion of uninfected cells ( ) . the extent and time course of mhv-a induced fusion caused by virus replication (fusion from within, ffwi) vary considerably among different permissive cell types. infection of l , dbt and sac-cells results in extensive fusion and death by to hours post inoculation. in contrast, fusion of the cl cell line involves only a small proportion of the cells until to hours after infection. comparison of the proteins of virions released from these four cell lines revealed significant differences in the proportion of the k e which had been cleaved to k ( figure ) . the more slowly fusing cl cells released virions containing e that was only approximately % cleaved, whereas virions released from sac-cells, which fused rapidly, contained e completely cleaved to k. this suggests that cleavage of e by a host cell protease during intracellular transport of the glycoprotein might be a virulence factor influencing the extent of virus-induced cpe. however, other cell lines which fuse extensively, such as l and dbt cells, show incomplete cleavage of e . in repeated experiments the e cleavage products from virions from the sac-cells differed slightly in molecular weight from those on virions produced by the other three cell types. this suggests that the protease cleavage site on the e molecule may differ from one host cell to another and that the specificity of the host protease which cleaves e may also be an important determinant of virulence. alternatively, host dependent differences in glycosylation could explain the observed differences in molecular weights of the e cleavage products. incorporation of e into the plasma membrane depends upon cellular transport mechanisms. thus, depending upon the relative amount of cleaved e carried to the plasma membrane, different mhv-infected cell lines show more or less fusion. the amount of e on the plasma membrane may also determine susceptibility of infected cells to attack by anti-viral antibody or immune lymphocytes. proteolytic cleavage glycoprotein is required virus infectivity ( , ) . can significantly enhance of a paramyxovirus envelope not only for fusion, but also for trypsin treatment of bcv virions virus infectivity ( , ) , but protease treatment of other coronaviruses results in only minor changes in infectivity ( ) . possibly the e on these viruses is already sufficiently cleaved by enzymes of the cells in which the virus was made to permit fusion of the viral envelope with the membrane of another cell. such fusion may be required for infectivity. no cell line which yields mhv with completely uncleaved e has yet been identified. the availability of a host cellular protease which can cleave the coronavirus e glycoprotein and activate virus infectivity may be another host determinant of coronavirus virulence. for example, human enteric coronaviruses (hecv) may be difficult to propagate for more than one cycle even in human fetal intestinal organ cultures due to inability of the cells to activate viral infectivity ( ). (figure ) . the slight strain-specific differences in the mobility of the bands labeled n reflect those seen in n proteins from virion. two faster migrating intracellular species of n were observed in infected cl cells, and their mobilities and relative abundance were also strain dependent. infection of the macrophage-derived cell line j a. with the same three strains of mhv resulted in detection of up to five intracellular species of n which showed strain-specific differences in mobility (figure ). the strain-specific differences in the mobilities of virionassociated n and the intracellular n species must be due to differences in the amino acid sequence of n proteins of different strains. the n proteins of mhv-a and mhv-jhm share % amino acid homology ( , ), but specific amino acid changes which account for the different electrophoretic mobility of n proteins of different strains have not yet been identified. the host cell dependent differences in the intracellular forms of n may be due to differences in mrna transcription, translation, phosphorylation or proteolytic cleavage. the biological significance of these multiple forms is unknown. mhv-a is shown in lanes and ; mhv-s, in lanes and ; and mhv-yale, in lanes and . mhv specific proteins are labeled. arrows indicate faster migrating intracellular species antigenically related to n. another factor which may affect the yield of infectious virus from different cell types is the rate of secretion from smooth-walled, post-golgi vesicles. thus, cells which secrete cellular products rather slowly may accumulate virions in intracellular vesicles and release relatively little infectious virus. the preceding discussion indicates that coronavirus replication may be influenced by many cellular processes that differ from one cell type to another (table i) . the outcome of coronavirus infection jln_ vivo depends not only upon virus interactions with individual cells as described above, but also upon interactions between virus antigens and the immune system. the virulence of mhv infection may be enhanced by immunosuppression, and mhv infection may change the immunologie responsiveness of the host. in this section we will discuss recent studies on the interactions of mhv with cells of the immune system and consider their implications for pathogenesis of mhv. in mice infected with some strains of mhv, viral antigens are observed in the spleen, and occasionally splenolysis results ( ). thus, mhv can infect lymphoid cells. we have demonstrated the availability of cell surface receptors for mhv-a on splenic lymphocytes. interaction of viral e glycoprotein on the surface of an infected cell with receptors on splenocytes resulted in an unusual form of natural cell-mediated cytotoxicity ( ) . the effector cell for this rapid, h independent natural cytotoxicity for mhv-infected targets was a b lymphocyte ( ) . antibody to e prevented cytotoxicity. thus, the unique ability of the e glycoprotein of mhv to bind to a receptor on b lymphocytes has revealed a previously unsuspected cytotoxic activity of these cells. levy and his co-workers ( ) demonstrated that intravenous inoculation of mhv- virions rapidly induces monocyte procoagulent activity (mpc) which results in alteration of the hemodynamics in the livers of infected mice. microthrombus formation and swelling of hepatocytes were detected within a few hours after virus inoculation. the susceptibility of different mouse strains to death from mhv- was correlated with the ability of mhv- to induce mpc activity in the strains. thus mpc may be an important determinant of genetic susceptibility of different mouse strains to mhv. it will be of considerable interest to identify the viral component which stimulates mpc activity. the extensive use of mice for production of hybridoma antibodies in ascites fluid may present a new opportunity for dissemination of mhv in animal colonies. we have found that mhv-a can readily infect hybridoma cells generated by fusion of murine splenic lymphocytes with myeloma cells. viral antigens were detected in the cytoplasm by immunofluoresence, numerous coronavirions were observed in the rer and golgi and adsorbed to the plasma membrane (figure ) , and infectious virus was released from infected hybridoma cultures. it is therefore possible that hybridomas may become infected when, during preparation of hyperimmune ascites fluids, they are passaged in the peritoneal cavity of mice with inapparent mhv infections. the contaminated hybridoma cells might then transmit mhv to other mice, thus becoming a source for spread of infection within a mouse colony. inapparent mhv infection in a mouse colony can also cause another problem for hybridoma research. we have prepared many hybridomas which produce antibody against one or another of the three structural proteins of mhv-a . although antibody in the supernatant medium from each hybridoma clone reacted with only one viral protein, ascites fluids induced by some of the hybridoma clones contained antibody to several mhv antigens. we found that the mice in which these ascites fluids had been prepared had been infected asymptomatically with an enterotropic strain of mhv. the ascites fluids thus contained both monoclonal anti-mhv a produced by the hybridoma cells and polyclonal antibodies to the enterotropic strain of mhv produced by resident b cells. we have also found anti-mhv antibody in ascites fluids elicited by hybridomas producing antibodies to non-viral antigens, when the mice had been previously exposed to mhv endemic in the animal colony. unless immunoglobulins prepared from ascites fluids from mhv-immune mice are purified by immunoaffinity chromatography with the specific epitope, contamination of the monoclonal antibody with anti-mhv antibodies could complicate interpretation of experimental results. we have discussed some of the ways in which coronavirus replication may depend upon host cell processes. this marked dependence of virus replication on the host cell may explain in part why coronaviruses show such pronounced species and tissue tropisms. differences in the yields of virus and the extent of viral cytopathic effects may be correlated with the ability of the cells to secrete virus and to transport appropriately processed and cleaved viral e glycoprotein to the host cell membrane. further studies on the functions of the coronavirus non-structural proteins and analysis of temperature-sensitive virus mutants ( , ) should elucidate additional virus-host interactions which may affect coronavirus virulence. perhaps due to special properties of coronavirus proteins, mhv infection can have unusual effects on the immune system, resulting in monocyte procoagulant activity or b cell-mediated cytotoxicity. inapparent mhv infection of a mouse colony can result in appearance of mhv antibodies in ascites fluids prepared by i.p. inoculation of hybridomas producing antibody to unrelated antigens. accidental infection of hybridoma cells with mhv could result in further spread of the virus through mouse colonies. these studies indicate new insights into viral immunology which can result from studies on mhv and illustrate the importance of eliminating mhv from mouse colonies in order to prevent compromise of research data. . rottier, p., brandenburg, d., armstrong, j., van der zeijst, b. and warren, g. ( ) . assembly in vitro of a spanning membrane protein of the endoplasmic reticulum: the el glycoprotein of coronavirus mouse hepatitis virus a . proc the use of a genetically incompatible combination of host and virus (mhv) for the study of mechanisms of host resistance coronavirus muturation mouse macrophages as host cells for the mouse hepatitis virus and the genetic basis of their susceptibility host and virus factors associated with cns cellular tropism leading to encephalomyelitis or demyelination induced by the jhm strain of mouse hepatitis virus evolution of a coronavirus during persistent infection in vitro immunopathology of mouse hepatitis virus type infection clinical and virologie observations of a persistent viral infection experimental demyelination produced by a temperature-sensitive mutant of mhv- acute and subacute demyelination induced by mouse hepatitis virus strain a in c h mice mouse hepatitis virus type (jhm strain) induced fetal nervous system disease. part i. genetic control and the murine neuron as the susceptible site of disease the structure and replication of coronaviruses the molecular biology of coronaviruses proteolytic cleavage of peplomeric glycoprotein e of mhv yields two k subunits and activates cell fusion analysis of the functions of coronavirus glycoproteins by differential inhibition of synthesis with tunicamycin monoclonal antibodies to murine hepatitis virus- (strain jhm) define the viral glycoprotein responsible for attachment and cell-cell fusion sequence and topology of a model intracellular membrane protein, el glycoprotein, from a coronavirus transcription strategy of coronaviruses: fusion of non-contiguous sequences during mrna syntheses studies on the mechanism of rna synthesis of a murine coronavirus coronavirus jhm. a virion-associated protein kinase intracellular protein synthesis posttranslational glycosylation of coronavirus glycoprotein el: inhibition by monensin cellular synthesis and modification of murine hepatitis virus polypeptides tunicamycin resistant glycosylation of a coronavirus glycoprotein: demonstration of a novel type of viral glycoprotein acylation of viral spike glycoproteins, a feature of enveloped rna viruses cultivation of a novel type of common-cold virus in organ culture cell tropism and expression of mouse hepatitis virus (mhv) in mouse spinal cord cultures comparison of the rnas of murine and human coronaviruses enhancement of plaque formation and cell fusion of an enteropathogenic coronavirus by trypsin treatment trypsin-enhanced replication of neonatal calf diarrhea coronavirus the role of viral glycoproteins in adsorption, penetration, and pathogenicity of viruses cotranslational and posttranslational processing of viral glycoproteins further studies on human enteric coronaviruses nucleotide sequencing of mouse hepatitis virus strain jhm messenger rna natural cytotoxicity against mouse hepatitis virus-infected target cells. i. correlation of cytotoxicity with virus binding to leukocytes natural cytoxicity against mouse hepatitis virus infected cells. ii. a cytotoxic effector cell with a b lymphocyte phenotype induction of monocyte procoagulant activity by murine hepatitis virus type parallels disease susceptibility in mice a genetic analysis of murine hepatitis virus, strain jhm temperature-sensitive mutants of mouse hepatitis virus strain a : isolation, characterization and neuropathogenic properties this work was supported in part by usuhs grant r and nih grant r . we are grateful for the outstanding technical assistance of margaret kerchief, barbara f neill, eileen bauer and cynthia dúchala.the opinions expressed are the private views of the authors and should not be construed as official or necessarily reflecting the views of the uniformed services university of the health sciences or the department of defense. key: cord- -xu elmak authors: collins, arlene r.; knobler, robert l.; powell, harry; buchmeier, michael j. title: monoclonal antibodies to murine hepatitis virus- (strain jhm) define the viral glycoprotein responsible for attachment and cell-cell fusion date: - - journal: virology doi: . / - ( ) - sha: doc_id: cord_uid: xu elmak abstract hybridoma cell lines producing monoclonal antibodies to the jhm strain of mouse hepatitis virus- (mhv- ) were established. by indirect immunofluorescence and immune precipitation, monoclonal antibodies of three viral polypeptide specificities were characterized. monoclonal antibodies to nucleocapsid reacted in the cytoplasm of infected cells and precipitated the , d nucleocapsid polypeptide (vp- ) of mhv- . other monoclonal antibodies reacted both in the cytoplasm and on the surface of infected cells and were found to precipitate the , d viral glycoprotein (gp- ). a third set of monoclonal antibodies reacted both in the cytoplasm and on the surface of infected cells and precipitated the , d viral glycoprotein (gp- ) and its precursor vp- ( , d). antigp- alone had direct neutralizing activity for mhv- virus, while in the presence of complement both anti-gp- and anti-gp- neutralized virus. only anti-gp- had the ability to inhibit the spread of infection due to fusion in l cells. thus, the viral glycoprotein gp- likely contains both the attachment and fusion activities of mhv- . at least four murine coronaviruses are associated with hepatitis and/or encephalitis in mice (gledhill and andrewes, ; nelson, ; dick et al., ; manaker et al., ; bailey et al., ) , and these viruses are collectively referred to as murine hepatitis viruses (mhv). strains of mhv exhibit distinct organ tropisms, being predominantly either hepatotropic or encephalitogenic, and may cause persistent infections in mouse colonies. mhv- (jhm strain) is an encephalitogenic strain which causes fatal acute encephalomyelitis following intracerebral inoculation in its natural host (lampert et al., ; weiner, destruction of oligodendrocytes (haspel et al., ) . in viva studies with jhmv and its mutants may provide valuable insights into the pathogenesis of human demyelinating diseases; however, a careful analysis of the pathogenesis of primary demyelination has been hampered by the lack of sensitive and specific probes for viral products as well as a firm understanding of coronavirus structure, replication, and mechanism(s) of persistence. serologic evidence of infection and detection of virus antigens in target organs has been difficult to demonstrate due to the poor quality of serum antibodies elicited in mice. antigens responsible for cross-reactivity among murine coronaviruses have not been well defined. the importance of making accurate comparisons is underscored by a recent report of serologic cross-reactivity between murine coronaviruses and two human coronavirus isolates from brains of multiple sclerosis patients (burks et al., ) . of mhv- and other murine coronaviruses has not been well defined. the virion contains positivestranded rna and infected cells reportedly contain seven major and two minor mrna species, the largest of which is the same size as the genome . the mode of synthesis and processing of these mrnas has not been elaborated. six polypeptides, four of which are glycosylated, have been reported in infected sac cells by siddell et al. ( ) . of these six polypeptides, the two largest are the glycopeptides gp-l( ' ,oood) and gp- ( ,oood) which appear to represent dimerit and monomeric forms, respectively, of the same glycopeptide as shown by tryptic peptide mapping. a ,oood nonglycosylated polypeptide, vp- , is associated with the viral nucleocapsid. the two smallest polypeptides, oood) and vp- ( ,oood), found in the infected cells, are identical to those found in the virion envelope (wege et al., ) . pulse-chase experiments have shown that vp- is processed to gp- by the addition of polysaccharide. a minor glycopeptide gp- ( ,-oood) appeared in gels when dithiothreitol was used as reducing agent, and its dimerit form ( ,oood) was found in gels when / -mercaptoethanol was used (wege et al., ) . sturman et al. ( ) have recently proposed a structural model for the membrane proteins of the mouse hepatitis virion based on experimental data obtained with the a- strain. in this proposed model, the largest virion glycopeptides gp- ( ,oood) and gp- ( ,oood) are associated with the virion peplomers and have been designated e- protein. d) and vp- ( , d) are deeply embedded in and may transsect the viral envelope, interacting internally with the nucleocapsid protein. in the proposed model, these polypeptides form a structure designated the e-l protein which plays an essential role in virus maturation intracellularly (holmes et al., ) . the viral glycoproteins bearing receptor sites for virus adsorption and cell-cell fusion during mhv infection have not been defined. in this report we describe the generation and characterization of monoclonal anti-bodies to mhv- and their use in initial studies to define biological activities associated with the virion glycopeptides. v+~.g and cezz culture. the jhm strain of mhv- was provided by l. weiner (university of southern california). it was plaque purified in nctc- cells and then propagated and plaque assayed in l- cells obtained from l. sturman ( production of nacmochaz antibadies to jhm'v. balb/c mice - weeks of age were primed by ip inoculation of to lo plaque-forming units (pfu) of mhv- grown in l- cells. after weeks, boosting doses consisting of . ml of a % (v/v) extract of virus-infected cells were inoculated ip for consecutive days prior to fusion. fusion with p x ag clone nonsecretor plasmacytoma cells was done essentially by the method of kohler and milstein ( ) as we have described elsewhere . briefly, lo* spleen cells were fused with lo plasmacytoma cells using peg at % concentration. cells were diluted in hat medium and plated out in eight -well plates. wells were observed for - weeks for hybrid colonies and these colonies were screened for production of antibody to mhv- . antibody producing cultures were immediately subcloned by limiting dilution and rechecked. screening of culture fluids for antiviral antibody was usually done by indirect immunofluorescence on both acetone-fixed and paraformaldehyde-fixed cells. as controls, uninfected l- cells and culture supernatant from parental myeloma cells were included in each assay. fluorescein isothiocyanate-conjugated goat anti-mouse igg was prepared in this laboratory . clones of each culture producing the highest titers of antibody in vitro were selected for ascites production in pristane ( , , , ,-tetramethylpentadecane)-primed balb/c mice. spetifiicities of monoclonal immunoglub ulins. virus-specific antibody concentrations in cell culture and ascites fluids were estimated by indirect immunofluorescence endpoint titration on acetone-fixed target cells. igg concentrations were estimated by radial immunodiffusion using sheep anti-mouse igg. determination of immunoglobulin isotype was determined by agar gel diffusion using subclass-specific antisera (meloy laboratories, springfield, va.). immune precipitation. mhv- polypeptide specificities of monoclonal antibodies were determined by immune precipitation of [ s]methionine-labeled viral proteins from infected l- cells. previously described methods (schauffhausen et al., ; were adapted as follows. radiolabeled cytosol extracts of infected and control l- cells were prepared by solubilizing . to . x ' infected cells in . to ml of lysis buffer ( mm tris-hcl, ph . ; mm nacl; mm cacl,; . mm mgcib; % aprotinin; % nonidet p ; and % (v/v) glycerol), then centrifuging at , g for min to remove nuclear debris. a volume of ~ of cytosol was mixed with ~ of hybridoma culture fluid or ~ of ascites fluid and incubated at " for hr. heat-killed, formalin-fixed staphylococcus aureus bacteria (sa) in sac buffer ( . mphosphatebuffered saline, ph . ; . % nonidet p- ; mm methionine; . % sodium azide; mg/ml ovalbumin) were added to each mixture in volumes of ~ of a % (v/ v) suspension, and incubation was continued for min at ". pellets were collected by centrifugation at g for min and washed three times in tris-hcl ( . m, ph ) containing . m licl. the final pellet was resuspended in ~ of % sds, % mercaptoethanol sample preparation buffer and heated at " for min. bacteria were removed by centrifugation and samples were analyzed by sds-page on . % gels. controls included precipitation of polypeptides from uninfected cytosol and inability to precipitate mhv- proteins by monoclonal antibodies directed against an unrelated virus (lcmv). virus neutralization. virus neutralizing capacity of monoclonal antibodies was quantitated by a semimicro plaque reduction assay. ascites fluids were first centrifuged at , g heat inactivated at " for min, then preadsorbed with l- cells ( x lo cells per ~ of ascites) for min at ". virus was diluted to contain - pfu in ~ and mixed with ~ of diluted antibody. virus-antibody mixtures were incubated for hr at ", then plated onto l- cell monolayers in -well plates (flow laboratories, mc-lean, va.). the mixtures were adsorbed for hr and then removed and replaced with culture medium. agar overlay was not necessary due to the short (~ hr) incubation period and cell-associated nature of jhmv. neutralization endpoint titers were expressed as the reciprocal of the highest antibody dilution which gave a % reduction in plaque number (prd %). controls included ascites fluids containing neutralizing monoclonal antibodies to an unrelated virus. inhibition of spread of virus in cell cdture. the growth of mhv- in l- cells is characterized by rapid formation of large syncytia and resultant intercellular virus spread. effect of virus-specific hybridoma antibodies on spread of virus infection in cell cultures was determined by incubation of infected monolayers with monoclonal antibodies in the culture medium. l- cell monolayers in -well linbro plates were infected at - pfu per well and incubated at " for hr. inoculum was removed and culture medium containing serially diluted monoclonal antibody prepared as described above was added at the concentrations indicated and incubation continued for - hr at which time plates were fixed and stained with . % crystal violet. the inhibition titer of monoclonal antibody was expressed as the highest dilution which inhibited the number of visible plaques and syncytia by %. inhibitory effect of virus-specific monoclonal antibody on syncytium formation was also visualized microscopically by indirect immunofluorescence staining. after , , and hr of incubation in the presence of monoclonal antibody, coverslip cultures were washed three times in pbs and fixed in acetone. fluorescent staining for detection of syncytia was performed using monoclonal antibody to mhv- nucleocapsid protein ( b- . ). surface labeling of mcmoclunal antibody bound to in&ected cells. cell surface binding of monoclonal antibodies was assessed both by indirect immunofluorescence and by electron microscopic examination of immunoferritin-labeled infected cells. l- cells were infected for hr at " with mhv- in suspension at an m.o.i of . , washed, then incubated at " for hr. cells were then transferred to an ice bath, washed in cold medium, and viable cells enumerated by trypan blue exclusion. aliquots of x lo viable cells were mixed with ~ of monoclonal ascites fluid at :loo dilution for min at ". cells were then washed three times with cold medium and reacted for hr on ice with ~ of rabbit anti-mouse igg conjugated to ferritin (cappel laboratory, dorrington, pa.). after incubation, cells for electron microscopy were washed four times with cold medium, once with pbs, and fixed with . % glutaraldehyde in . m cacodylate buffer and examined as described elsewhere (knobler et al., ) . for surface immunofluorescence cells were reacted with monoclonal antibody as described above, then with sheep anti-mouse igg coupled to biotin. after min at " cells were washed then reacted with fitc coupled avidin (e-y laboratories, san mateo, calif.) for min. cells were then washed and examined using a zeiss fluorescence microscope equipped for incident illumination. antibodies to mhv- fifty-two cell lines making monoclonal antibodies to mhv- were established from cultures. initial screening by immunofluorescence segregated these into those which reacted with antigens at the surface of infected cells, and those which reacted with antigens expressed only in the cytoplasm. to determine the polypeptide specificity of these antibodies, we immunoprecipitated radiolabeled viral polypeptides from cytosol extracts of infected cells. under the conditions which we employed, the monoclonal antibodies fell into three groups: those which precipitated gp- , those reacting with vp- , and those reacting with gp- and vp- . figure illustrates these results for selected antibodies. correlating these data with immunofluorescence observations, we found that antibodies which stained cell surface viral antigens at , , or hr reacted with either gp- or gp- , vp- whereas antibodies which stained antigens only in the cytoplasm immunoprecipitated vp- . under the conditions employed with [ s]methionine label, we observed little gp- ( ,oood) surface glycopeptide; however, preliminary experiments using glucosamine label (data not shown) suggest that anti-gp- antibodies also react with gp- . forty-four of the fifty-two monoclonal antibodies were analyzed for their immunoglobulin isotype using monospecific typing reagents. of these, eight were igg- , were igg-za, two were igg-zb, six were igg- , three were iga, four were igm and nine were undetermined. the replication of murine coronaviruses is characterized by synthesis of viral rna and proteins in the cytoplasm, envelopment of the nucleocapsid, and maturation by budding into intracellular vesicles. cell fusion and resultant syncytium formation are not essential for maturation (robb and bond, ) ; however, cell-cell fusion between infected cells and uninfected cells may contribute to intercellular spread of infection. viral antigens at the cell surface can also serve as targets for immune attack. presence of viral giycoproteins gp- and gp- on the surfaces of infected cells was examined using two monoclonal antibodies, b- . (anti-gp- ) and - . (anti-gp- ). l- cells were examined hr after infection with mhv- by indirect immunofluorescence and by immunoferritin labeling for surface antigen. six hours was selected for detailed study because this preceded both release of mature virus into the supernatant medium and cell death. both anti-gp- (fig. b ) and anti-gp- (fig. c ) stained infected cells at hr; however, anti-vp- did not ( fig. a) . the binding of anti-gp- was visualized by immune electron microscopy using ferritin-conjugated rabbit anti-mouse igg (fig. ) . antigen at the cell surface was observed in the absence of virions. the mhv- antigen responsible for adsorption to target cells has not been identified. in order to address this point, we selected four monoclonal antibodies demonstrated by immunoprecipitation to react with the gp- polypeptide, three which precipitated gp- , and as controls, two which precipitated vp- . globulin class, igg concentration, and immunofluorescence endpoint titer were also determined. the ability of these monoclonal antibodies to neutralize mhv infectivity is illustrated in fig. . neutralization was mediated only by those monoclonal antibodies which precipitated gp- . one anti-gp- ( b- . ) which did not neutralize was of the iga class. monoclonal antibodies to the gp- glycoprotein and to np showed no significant neutralizing activity in the absence of complement; however, we found that the addition of guinea pig complement to the reaction mixture substantially increased the neutralizing activity of the gp- antibodies. in one such experiment with antibody a- . (anti-g ) the titer of , pfu of jhmv was reduced by only % without complement, but by % with the addition of l/ fresh guinea pig complement. in contrast, antibody b- . (anti-gp- ) neutralized and % of the infectivity without and with complement, respectively. neutralizing antibody titers did not always correlate with igg concentration, and this may reflect differences in avidity. other controls consisting of monoclonal antibodies to jhmv vp- and neutralizing antibodies to an unrelated virus (lcmv) showed no significant neutralizing activity against mhv- . an interesting biological property of mhv- is its ability to spread infection to adjacent uninfected cells by fusion. this property is especially evident in certain cells such as l- , primary macrophage, primary brain, and embryonic fibroblast cultures from susceptible mouse strains such as balb/c. other cell lines such as the -cl-l line of mouse fibroblasts do not demonstrate extensive fusion after infection with collins, unpublished observation) . the viral glycopeptide associated with fusion activity has not been identified. monoclonal antibodies with specificity for gp- and gp- were tested for ability to inhibit spread of infection in l- cells. inhibition of spread was quantitated by incubating previously infected cells in medium containing monoclonal antibodies and scoring the number of visible plaques and foci of syncytia - hr after infection. only incubation with monoclonal antibodies to gp- resulted in inhibition in plaque and syncytium number (fig. ) , indicating involvement of that polypeptide in cell to cell spread of infection by fusion. titers observed were unrelated to the neutralizing antibody titer but did correlate with indirect immunofluorescence antibody titers. for example, considering the data summarized in table , the relative titers of three different anti-gp- antibodies ( b- . , b- . , and b- . ) in neutralization assays were less than, equal to, and greater than their respective titers in inhibition of spread of infection. measurement of antibody activity by inhibition of spread of infection was approximately lo-fold less sensitive than indirect immunofluorescence. the inhibitory effects of monoclonal antibody to gp- upon development of syncytia was also observed morphologically by indirect immunofluorescence on mhv- -infected l- cells. monoclonal antibodies at a final dilution of l/ were added to the medium of l- cultures on coverslips one hour after infection with mhv. as early as hr after infection, a marked inhibition both in the numbers and size of syncytia was observed in cultures incubated with monoclonal antibody to gp- . inhibition of development of syncytia was even more evident in such cultures hr after infection (fig. ) . monoclonal antibodies to gp- , vp- , and an unrelated virus (lcmv, not shown) had no effect on the development of syncytia. discussion we have prepared a library of monoclonal antibodies to mhv- and have identified their polypeptide specificity toward the viral proteins gp- , vp- , gp- , and vp- . these antibodies have been used in and functional assays to strated that despite this intracellular mode probe mhv infection. of maturation both viral gp- . and gp- coronaviruses mature by budding into are expressed at the surfaces of infected intracytoplasmic vesicles (reviewed in cells. surface expression occurred by hr, robb and bond, shown by immunoelectron microscopy to occur in areas free of morphologically distinguishable virions. this observation has several implications with respect to hostvirus interactions. first, expression of virus-specific surface components during eclipse may render the infected cell susceptible to specific antibody-dependent or cellular host defense mechanisms. thus the infected cell can potentially be eliminated prior to release of new virus. conversely, spread of mhv infection in culture and presumably in v&o is largely cell to cell, and is facilitated by the ability of viral polypeptides to mediate cell fusion resulting in syncytia formation. presence of these viral polypeptides on the cell sur-face suggests that cell fusion may begin prior to virus release. we have observed cell fusion in culture as early as hr after infection, coincident with the appearance of gp- and gp- at the cell surface. sturman and co-workers ( ) have proposed that gp- and vp- are structurally associated with a transmembrane protein complex which they have termed e-l. this protein is thought to be involved in virus maturation along the endoplasmic reticulum. the ability of anti-gp- to bind to the infected cell surface and to the intact virion indicates that this putative transmembrane polypeptide of the coronaviruses is expressed at the cell surface independent of virus maturation and differs to had no effect at a l/ or l/ dilution. in this regard from the membrane (m) proteins of the orthomyxoviruses and rhabdoviruses which apparently are not exposed to the external environment (gerhard et al., ; compans and klenk, ) . we investigated the biological roles of the gp- and gp- polypeptides in initiation and spread of infection using monoclonal antibodies. neutralization tests employing several anti-gp- , anti-gp- , and anti-vp- monoclonals demonstrated that only anti-gp- possessed strong virusneutralizing activity. antibodies to gp- did not neutralize virus infectivity unless complement was added to the r,eaction. thus the virion polypeptide gp- probably contains the site of attachment of virion to host cell. one anti-gp- ( b- . ) did not neutralize infectivity despite a binding titer of l/ , . it is likely that this antibody recognizes an epitope on gp- that is unrelated to the attachment site. blocking experiments utilizing b- . to attempt to competitively inhibit binding to known neutralizing antibodies are currently under way to clarify this question. despite their failure to neutralize, antibodies to gp- clearly bind to virions. the addition of fresh guinea pig complement to anti-gp- antibody + virus reaction mixtures resulted in virus neutralization comparable to that seen with anti-gp- antibody and this may reflect a lytic effect of complement on the virus. since both complement and antibody-dependent cellmediated cytotoxocity (adcc) mechanisms are operative in the whole animal, the ability of these antibodies to modify the course of disease in viva must be considered. virus-specific membrane antigens clearly also play a role in the intercellular spread of infection through the mechanism of cell fusion. fusion activity has been described for many enveloped viruses. among the paramyxoviruses this activity is the function of a distinct fusion glycoprotein (f,) (homma and ohuchi, ; scheid and choppin, ) . in the orthomyxoviruses a hydrophobic region of the hemagglutinin peplomer has fusion activity (lazarowitz and choppin, ; klenk et al., ) . when we examined our collection of monoclonal antibodies for the ability to inhibit spread of infection, only anti-gp- was effective, suggesting that the virion peplomer contains the active site for cell-cell co % fusion. the mechanism by which anti-gp- inhibits spread is not clear. anti-gp- may directly inhibit the molecular events of membrane fusion or may bind to a site which sterically hinders membrane penetration. although our experiments suggest that gp- is not directly involved in cell fusion the possibility exists that gp- participates in events occurring after initiation of fusion by gp- . alternatively, expression of gp- at the surface of the infected cell may be merely a fortuitous occurrence. recent data (holmes et al., ) suggest that the gp- , vp- complex e-l plays an essential role in the intracellular maturation of mhv. thus surface expression of this polypeptide may reflect a breakdown in the normal pathways of glycoprotein expression in the infected cell. clearly, monoclonal reagents will be valuable tools to probe the precise mechanisms of infection, cell-cell spread, and cell fusion by coronaviruses. a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin monoclonal antibodies to lymphocytic choriomeningitis and pichinde viruses: generation, characterization and crossreactivity with other arenaviruses protein structure of lymphocytic choriomeningitis virus: evidence for a cell-associated precursor of the virion glycopeptides monoclonal antibodies to lymphocytic choriomeningitis and pichinde viruses: generation, characterization, and crossreactivity with other arenaviruses two coronaviruses isolated from central nervous system tissue of two multiple sclerosis patients viral membranes a virus related to that causing hepatitis in mice (mhv) monoclonal antibody hybridomas: a new dimension in biological analyses a hepatitis virus of mice. &-it temperature-sensitive mutuants of mouse hepatitis virus produce a high incidence of demyelination tunicamycin resistant glycosylation of a coronavirus glycoprotein: demonstration of a novel type of viral glycoprotein trypsin action on the growth of sendai virus in tissue culture cells. iii. structural difference of sendai viruses grown in eggs and in tissue culture cells activation of influenza a viruses by trypsin treatment selective localization of wild type and mutant mouse hepatitis virus (jhm strain) antigens in cns tissue by fluorescence, light and electron microscopy continuous cultures of fused cells secreting antibody of pre-defined specificity mechanism of demyelination in jhm virus encephalomyelitis enhancement of infectivity of influenza a and b viruses by proteolytic cleavage of the hemagglutinin polypeptide a hepatitis virus complicating studies with mouse leukemia acute hepatitis associated with mouse leukemia . pathological features and transmission of the disease tumor antigen(s) in cells productively infected by wild type polyoma virus and mutant ng- identification of the biological activities of paramyxovius glycoproteins. activation of cell fusion, hemolysis and infectivity by proteolytic cleavage of an inactive precursor protein of sendai virus coronavirus jhm: intracellular protein synthesis isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid coronavirus jhm: characterization of intracellular viral rna structural polypeptides of the murine coronavirus jhm pathogenesis of demyelination induced by a mouse hepatitis virus (jhm) virus this is publication no. from scripps clinic and research foundation, la jolla, california. we thank michael b. a. oldstone for helpful discussion; ricarda defries, hannah lewicki, and linda tunison for excellent technical assistance, and susan edwards for manuscript preparation.the work described here was supported by nih grants ns , ns , and ai , and was performed during the tenure of an american heart association established investigatorship granted to m.j.b. r.l.k. is the recipient of a national multiple sclerosis society postdoctoral fellowship award. key: cord- -up vftj authors: brayton, cory; mähler, michael; nicklas, werner title: viral infections date: - - journal: the laboratory mouse doi: . /b - - / - sha: doc_id: cord_uid: up vftj nan in interpreting the microbiological status of laboratory animals, it must be understood that infection and disease are not synonymous. infection refers to the invasion and multiplication of microorganisms in body tissues and may occur with or without apparent disease. disease refers to interruption or deviation from normal structure and function of any tissue, organ, or system. many of the infections with which we are concerned may not cause discernable disease in many strains of mice. however, they may cause inapparent or subclinical changes that can interfere with research. such interference often remains undetected, and therefore modified results may be obtained and published. the types of interference of an agent with experimental results may be diverse. there is no doubt that research complications due to overt infectious disease are significant and that animals with clinical signs of disease should not be used for scientific experiments. but also clinically inapparent infections may have severe effects on animal experiments. there are numerous examples of influences of microorganisms on host physiology and hence of the interference of inapparent infections with the results of animal experiments. many microorganisms have the potential to induce activation or suppression of the immune system or both at the same time but on different parts of the immune system, regardless of the level of pathogenicity. all infections, apparent or inapparent, are likely to increase inter-individual variability and hence result in increased numbers of animals necessary to obtain reliable results. microorganisms, in particular viruses, present in an animal may contaminate biological materials such as sera, cells, or tumours (collins and parker, ; nicklas et al., ) . this may interfere with in vitro experiments conducted with such materials and may also lead to contamination of animals (lipman et al., polymerase chain reaction (pcr) testing of biologics to be inoculated into mice is an important component of a disease prevention programme. finally, latent infections may be activated by environmental factors, by experimental procedures, or by the combination and interaction between various microorganisms. for all these reasons, prevention of infection, not merely prevention of clinical disease, is essential. unfortunately, research complications due to infectious agents are usually considered artefacts and published only exceptionally. information on influences of microorganisms on experiments is scattered in diverse scientific journals, and many articles are difficult to detect. to address this problem, several congresses were held on viral complications on research. the knowledge available was summarized in conference proceedings (melby and balk, ; bhatt et al., b; hamm, ) and has later repeatedly been reviewed (lussier, ; national research council, ; baker, ; nicklas et al., ) . this chapter covers only viral infections of laboratory mice. viral infections of mice have been studied in detail, and comprehensive information on their pathogenic potential, their impact on research, and the influence of host factors such as age, genotype, and immune status on the response to infection is available. bacterial agents may be similarly important, but with few exceptions (e.g. helicobacter species) little is known about their potential to influence host physiology and experiments. even less is known about most parasites in this regard. among fungal agents, only pneumocystis carinii can be expected to play a significant role in contemporary mouse colonies. the nomenclature and taxonomy of viruses described are based on recent nomenclature rules by the international union of microbiological societies ( ) and the universal virus database of the international committee on the taxonomy of viruses (http://www.ictvdb.iacr.ac.uk). retroviruses are not covered in this chapter because they are not included in routine health surveillance programmes and cannot be eradicated with presently available methods. this is because most of them are incorporated in the mouse genome as proviruses and thus are transmitted via germline. the ability to accurately determine whether or not laboratory animals or animal populations have been infected with virus depends on the specificity and sensitivity of the detection methods used. most viral infections in immunocompetent mice are acute or short-term, and lesions are often subtle or subclinical. the absence of clinical disease and pathological changes has therefore only limited diagnostic value. however, clinical signs, altered behaviour, or lesions may be the first indicator of an infection and often provide clues for further investigations. serology is the primary means of testing mouse colonies for exposure to viruses, largely because serological tests are sensitive and specific, are relatively inexpensive, and allow screening for a multitude of agents with one serum sample. they are also employed to monitor biological materials for viral contamination using the map test. serological tests detect specific antibodies, usually immunoglobulin g (igg), produced by the host against the virus and do not actually test for the presence of virus. an animal may have been infected, mounted an effective antibody response, and cleared the virus, but remains seropositive for weeks or months or forever, even though it is no longer infected or shedding the agent. active infection can only be detected by using direct diagnostic methods such as virus isolation, electron microscopy, or pcr. meanwhile, pcr assays have been established for the detection of almost every agent of interest. they are highly sensitive and depending on the demands, they can be designed to broadly detect all members of a genus or only one species. however, good timing and selection of the appropriate specimen is critical for establishing the diagnosis. in practice, combinations of diagnostic tests are often necessary including the use of sentinel animals or immunosuppression to get clear aetiological results or to avoid consequences from false-positive results. reports on the prevalence of viral infections in laboratory mice throughout the world have been published frequently. in general, the microbiological quality of laboratory mice has constantly improved during the last decades, and several agents (e.g. herpes-and polyomaviruses) have been essentially eliminated from contemporary colonies due to advances in diagnostic methodologies and modern husbandry and rederivation practices (jacoby and lindsey, ; zenner and regnault, ; livingston and riley, ) . they may, however, reappear, since most have been retained or are still being used experimentally. furthermore, the general trend towards better microbiological quality is challenged by the increasing reliance of biomedical research on genetically modified and immunodeficient mice, whose responses to infection and disease can be unpredictable. increasing numbers of scientists are creating genetically modified mice, with minimal or no awareness of infectious disease issues. as a consequence, they are more frequently infected than 'standard' strains of mice coming from commercial breeders, and available information on their health status is often insufficient. frequently, they are exchanged between laboratories, which amplifies the risk of introducing infections from a range of animal facilities. breeding cessation strategies that have been reported to eliminate viruses from immunocompetent mouse colonies may prove to be costly and ineffective in genetically modified colonies of uncertain or incompetent immune status. it must also be expected that new agents will be detected, although only occasionally. infections therefore remain a threat to biomedical research, and users of laboratory mice must be cognizant of infectious agents and the complications they can cause. two members of the family herpesviridae can infect mice (mus musculus). mouse cytomegalovirus (mcmv- ) or murid herpesvirus (muhv- ) belongs to the subfamily betaherpesvirinae, genus muromegalovirus. murid herpesvirus (muhv- ) or mouse thymic virus (mtv) has not yet been assigned to a genus within the family herpesviridae. both viruses are enveloped, doublestranded dna viruses that are highly host-specific and relatively unstable to environmental conditions such as heat and acidic ph. both agents are antigenically distinct and do not cross-react in serological tests, but their epidemiology is similar (cross et al., ) . seropositivity to mcmv- was reported in less than % of specified pathogen-free (spf) mouse colonies in the usa in (jacoby and lindsey, ) , and some institutions reported to have mice 'on campus' that were positive for mtv. in a more recent study, a low rate ( . %) of samples was found to be positive for mcmv- whereas no sample tested positive for mtv (livingston and riley, ) . the data available suggest that the prevalence of both viruses in contemporary colonies and thus their importance for laboratory mice is negligible. however, both mcmv- and mtv are frequently found in wild mice, which may be coinfected with both viruses (national research council, ; singleton et al., ) . natural infection with mcmv- causes subclinical salivary gland infection in mice. the virus persists in the salivary glands (particularly in the submaxillary glands) and also in other organs (osborn, ; kercher and mitchell, ; lenzo et al., ) . most information concerning the pathogenesis of mcmv- infection is based on experimental infection studies. these results are very difficult to summarize because the outcome of experimental infection in laboratory mice depends on various factors such as mouse strain and age, virus strain and passage history, virus dose, and route of inoculation (osborn, ) . in general, newborn mice are most susceptible to clinical disease and to lethal infection. virus replication is observed in newborn mice in many tissues (for details, see osborn, ) and appears in the salivary glands towards the end of the first week of infection when virus concentrations in liver and spleen have already declined. resistance develops rapidly after weaning between days and of age. experimental infection of adult mice results in mortality only in susceptible strains and only if high doses are administered. not even intravenous or intraperitoneal injections of adult mice usually produce signs of illness in resistant strains (shanley et al., ) . mice of the h- b (e.g. c bl/ ) and h- d (e.g. balb/c) haplotype are more sensitive to experimental infection than are mice of the h- k haplotype (e.g. c h), which are approximately -fold more resistant to mortality than are those of the b or d haplotype (osborn, ) . subclinical or latent infections can be activated by immunosuppression (e.g. with cyclophosphamide or cortisone). reactivation of mcmv- occurs also after implantation of latently infected salivary glands into prkdc scid mice (schmader et al., ) . immunodeficient mice lacking functional t cells or natural killer (nk) cells, such as foxn nu and lyst bg mice, are more susceptible than are immunocompetent animals. experimental infection in prkdc scid mice causes severe disease or is lethal, with necrosis in spleen, liver, and other organs, and multinucleate syncytia with inclusion bodies in the liver (reynolds et al., ) . similar to aids patients infected with human cytomegalovirus, athymic foxn nu mice experimentally infected with mcmv also develop adrenal necrosis (shanley and pesanti, ) . the virus also replicates in the lungs leading to pneumonitis whereas in heterozygous (foxn nu / ϩ ) littermates replication and disease are not seen (shanley et al., ) . the most prominent histological finding of cytomegaloviruses is enlarged cells (cytomegaly) of salivary gland epithelium with eosinophilic nuclear and cytoplasmic inclusion bodies. the inclusion bodies contain viral material and occur in other organs such as liver, spleen, ovary, and pancreas (osborn, ) . depending on inoculation route, dose, strain, and age of mice, experimental infections may result in inflammation or cytomegaly with inclusion bodies in a variety of tissues, pneumonitis, myocarditis, meningoencephalitis, or splenic necrosis in susceptible strains (national research council, ; osborn, ; percy and barthold, ) . virus is transmitted oronasally by direct contact and is excreted in saliva, tears, and urine for several months. wild mice serve as a natural reservoir for infection. the virus is most frequently transmitted horizontally through mouse-to-mouse contact but does not easily spread between cages. it is generally assumed that mcmv- has a very low prevalence in contemporary colonies of laboratory mice. the risk of introduction into facilities housing laboratory mice is very low if wild mice are strictly excluded. monitoring is necessary if populations of laboratory mice may have been contaminated by contact with wild mice. as for other viruses, enzyme-linked immunosorbent assay (elisa) and indirect immunofluorescence assay (ifa) are the most appropriate tests for detecting antibodies. as the virus persists, direct demonstration of mcmv- in infected mice is possible by pcr (palmon et al., ) or by virus isolation using mouse embryo fibroblasts ( t cells). although mcmv- does not play a significant role as a natural pathogen of laboratory mice, it is frequently used as a model for human cytomegalovirus infection (bolger et al., ) . however, the virus is known to influence immune reactions in infected mice and may therefore have impact on immunological research (osborn, ; national research council, ; baker, ) . mouse thymic virus was detected during studies in which samples from mice were passaged in newborn mice. unlike other herpesviruses, the virus can not yet be cultured in vitro and is propagated by intraperitoneal infection of newborn mice. the thymus is removed - days later, and thymus suspensions serve as virus material for further studies. the prevalence of mtv is believed to be low in laboratory mice, and for this reason and also due to the difficulties in virus production for serological assays, it is not included in many standard diagnostic or surveillance testing protocols. limited data are available indicating that it is common in wild mice, and it is also found in laboratory mice (osborn, ; morse, ; national research council, ) . further, mtv obviously represents a significant source of contamination of mcmv- (and vice versa) if virus is prepared from salivary glands since both viruses cause chronic or persistent salivary gland infections and can coinfect the same host. all mouse strains are susceptible to infection, but natural or experimental infection of adult mice is subclinical. gross lesions appear only in the thymus and only if experimental infection occurs at an age of less than about days. virus is present in the thymus but may also be found in the blood and in salivary glands of surviving animals. salivary glands are the only site yielding positive virus isolations if animals are infected as adults. mouse thymic virus also establishes a persistent infection in athymic foxn nu mice, but virus shedding is reduced compared to euthymic mice and virus recovery is possible only in a lower percentage of mice (morse, ) . pathological changes caused by mtv occur in the thymus, and reduced thymus mass due to necrosis in suckling mice is the most characteristic gross lesion (percy and barthold, ) . lymphoid necrosis also may occur in lymph nodes and spleen (wood et al., ) , with necrosis and recovery similar to that in the thymus. in mice infected during the first days after birth, necrosis of thymus becomes evident within - days, and its size and weight are markedly reduced at day - . intranuclear inclusions may be present in thymocytes between days - post infection. the thymus and the affected peripheral tissues regenerate within weeks after infection. regardless of the age of mice at infection, a persistent infection is established in the salivary glands, and infected animals shed virus for life. several alterations of immune responses are associated with neonatal mtv infection. there is transient immunosuppression, attributable to lytic infection of t lymphocytes, but activity (e.g. response of spleen cells to t cell mitogens) returns to normal as the histological repair progresses (wood et al., ) . selective depletion of cd ϩ t cells by mtv results in autoimmune disease (morse and valinsky, ; morse et al., ) . information about additional influences on the immune system is given by osborn ( ), national research council ( ), and baker ( . in experimentally infected newborn mice, oral and intraperitoneal infections similarly result in thymus necrosis, seroconversion, and virus shedding suggesting that the oral-nasal route is likely to be involved in natural transmission (morse, ) . the virus spreads to cage mates after long periods of contact. it is transmitted between mice kept in close contact, and transmissibility from cage to cage seems to be low. mouse thymic virus is not transmitted to foetuses by the transplacental route, and intravenous infection of pregnant mice does not lead to congenital damage, impairment in size or development, or abortion (st-pierre et al., ) . mouse thymic virus and mcmv- do not crossreact serologically (cross et al., ) . serological monitoring of mouse populations for antibodies to mtv is possible by ifa testing, which is commercially available; elisa tests have also been established (morse, b) . elisa and complement fixation yield similar results . it must be noted that the immune response depends on the age at infection. antibody responses are not detectable in mice infected as newborns whereas adult mice develop high titres that are detectable by serological testing. if neonatal infection is suspected, homogenates of salivary glands or other materials can be inoculated into pathogen-free newborn mice followed by gross and histological examination of thymus, lymph nodes, and spleens for lymphoid necrosis (morse, ) . alternatives to the in vivo infectivity assay for detecting mtv in infected tissues include a competition elisa (prattis and morse, ) and map testing, although this is slightly less sensitive than infectivity assays (morse, a) . very little experience exists on eradication methods for mtv due to its low prevalence in contemporary mouse colonies. methods that eliminate other herpesviruses likely will eliminate mtv. procurement of animals of known negative mtv status is an appropriate strategy to prevent infection. strict separation of laboratory mice from wild rodents is essential to avoid introduction into laboratory animal facilities. mousepox (ectromelia) virus (ectv) is a member of the genus orthopoxvirus belonging to the family poxviridae. it is antigenically and morphologically very similar to vaccinia virus and other orthopoxviruses. poxviruses are the largest and most complex of all viruses with a diameter of nm and a length of - nm. mousepox (ectromelia) virus contains one molecule of double-stranded dna with a total genome length of , nucleotides. it is the causative agent of mousepox, a generalized disease in mice. experimental transmission to young rats (up to days of age) is possible (jandasek, ; buller et al., ) . the virus is resistant to desiccation, dry heat, and many disinfectants. it is not consistently inactivated in serum heated min at Њc (lipman et al., b) and persists for weeks when maintained at Њc in foetal bovine serum (bhatt and jacoby, a) . effective disinfectants include vapour-phase formaldehyde, sodium hypochlorite, and iodophores (small and new, ; national research council, ) . historically, ectv has been an extremely important natural pathogen of laboratory mice. the virus was widespread in mouse colonies worldwide and can still be found in several countries. between and almost individual ectromelia outbreaks were reported in the usa. the last major epizootic in the usa occurred in - and has been described in great detail (e.g. wagner and daynes, ) . severe outbreaks were also described in various european countries (deerberg et al., ; owen et al., ; osterhaus et al., ) . a more recent outbreak in the usa, which resulted in the eradication of almost mice in one institution, was described by dick et al. ( ) . the most recent well-documented case of mousepox was published by lipman et al. ( b) . few additional but unpublished cases of ectromelia have been observed thereafter. in a recent survey conducted in the usa, one population was reported to be seropositive for mousepox (jacoby and lindsey, ) . natural infections manifest differently depending on many factors. mousepox may occur as a rapidly spreading outbreak with acute disease and deaths, or may be inconspicuous with slow spreading and mild clinical signs. the mortality rate can be very low in populations in which the virus has been present for long periods. the infection usually takes one of three clinical courses: acute asymptomatic infection, acute lethal infection (systemic form), or subacute to chronic infection (cutaneous form ; fenner, fenner, , manning and frisk, ; national research council, ; dick et al., ) . the systemic or visceral form is characterized clinically by facial oedema, conjunctivitis, multisystemic necrosis, and usually high mortality. this form is less contagious than the cutaneous form because the animals die before there is virus shedding. the cutaneous form is characterized by typical dermal lesions and variable mortality. the outcome of infection depends on many factors including strain and dose of virus; route of viral entry; strain, age, and sex of mouse; husbandry methods; and duration of infection in the colony. while all mouse strains seem to be susceptible to infection with ectv, clinical signs and mortality are strain-dependent (fenner, ; wallace and buller, , ; brownstein et al., b) . acute lethal (systemic) infection occurs in highly susceptible inbred strains such as dba/ , dba/ , balb/c, a, and c h/hej. immunodeficient mice may also be very susceptible (allen et al., ) . outbreaks among susceptible mice can be explosive, with variable morbidity and high mortality (Ͼ %). clinical disease may not be evident in resistant strains such as c bl/ and akr, and the virus can be endemic in a population for long periods before being recognized. furthermore, females seem to be more resistant to disease than males, at least in certain strains of mice brownstein et al., b) . the mechanisms determining resistance versus susceptibility are not fully understood but appear to reflect the action of multiple genes. the genetic loci considered to be important include h- d b (termed rmp- , resistance to mousepox, on chromosome ; o'neill et al., ) , the c genes (rmp- , on chromosome ), rmp- , localized to a region on chromosome encoding the nk cell receptor nkr-p alloantigens (brownstein and gras, ) , the nitric oxide synthase locus on chromosome (karupiah et al., ) , and the signal transducer and activator of transcription locus on chromosome (mahalingam et al., ) . clearance of the virus by the immune system is absolutely dependent upon the effector functions of cd ϩ t cells while nk cells, cd ϩ t cells, and macrophages are necessary for the generation of an optimal response (niemialtowski et al., ; delano and brownstein, ; karupiah et al., ) . mousepox (ectromelia) virus usually enters the host through the skin with local replication and extension to regional lymph nodes (fenner, (fenner, , wallace and buller, ; national research council, ) . it escapes into the blood (primary viraemia) and infects splenic and hepatic macrophages resulting in necrosis of these organs and a massive secondary viraemia. this sequence takes approximately week. many animals die at the end of this stage without premonitory signs of illness; others develop varying clinical signs including ruffled fur, hunched posture, swelling of the face or extremities, conjunctivitis, and skin lesions (papules, erosions, or encrustations mainly on ears, feet, and tail; figure . ). necrotic amputation of limbs and tails can sometimes be seen in mice that survive the acute phase, hence the original name of the disease 'ectromelia' (meaning absent or short limbs; figure . ). common gross lesions of acute mousepox include enlarged lymph nodes, peyer's patches, spleen, and liver; multifocal to semiconfluent white foci of necrosis in the spleen and liver; and haemorrhage into the small intestinal lumen (allen et al., ; fenner, ; dick et al., ; percy and barthold, ) . in animals that survive, necrosis and scarring of the spleen can produce a mosaic pattern of white and red-brown areas that is a striking gross finding. the most consistent histological lesions of acute mousepox are necroses of the spleen (figure lymph nodes, peyer's patches, thymus, and liver (allen et al., ; fenner, ; dick et al., ; lipman et al., b; percy and barthold, ) . occasionally, necrosis may also be observed in other organs such as ovaries, uterus, vagina, intestine, and lungs. the primary skin lesion, which occurs about a week after exposure at the site of inoculation (frequently on the head), is a localized swelling that enlarges from inflammatory oedema. necrosis of dermal epithelium provokes a surface scab and heals as a deep, hairless scar. secondary skin lesions (rash) develop - days later as the result of viraemia (figure . ). they are often multiple and widespread and can be associated with conjunctivitis. the skin lesions also can ulcerate and scab before scarring. mucosal and dermal epithelial cells may have characteristic intracytoplasmic eosinophilic (cowdry type a) inclusion bodies ( figure . ). basophilic (cowdry type b) inclusions may be found in the cytoplasm of all infected cells, especially in hepatocytes. natural transmission of ectv mainly occurs by direct contact and fomites (fenner, ; wallace and buller, ; national research council, ) . the primary route of infection is through skin abrasions. faecal-oral and aerosol routes may also be involved (werner, ) . in addition, the common practice of cannibalism by mice may contribute to the oral route of infection (bhatt and jacoby, b) . intrauterine transmission is possible at least under experimental conditions (schwanzer et al., ) . virus particles are shed from infected mice (mainly via scabs and/or faeces) for about - weeks, even though the virus can persist for months in the spleen of an occasional mouse (bhatt and jacoby, b; national research council, ) . cage-to-cage transmission of ectv and transmission between rooms or units is usually low and largely depends on husbandry practices (e.g. mixing mice from different cages). importantly, the virus may not be transmitted effectively to sentinel mice exposed to dirty bedding (lipman et al., b) . various tests have been applied for the diagnosis of ectromelia. previous epidemics were difficult to deal with because of limited published data and information on the biology of the virus and the lack of specific and sensitive assays (wallace, ) . in the s, diagnosis relied on clinical signs, histopathology, and animal passages of tissues from moribund and dead animals. culture of the virus on the chorioallantoic membrane of embryonated eggs was also applied. serology is currently the primary means of testing mouse colonies for exposure to ectv. the methods of choice are elisa and ifa; they are more sensitive and specific than the previously used haemagglutination inhibition (hi) assay (collins et al., ; buller et al., ; aclad, ) . both tests detect antibodies to orthopoxviruses and do not distinguish between ectv and vaccinia virus. vaccinia virus is commonly used as antigen for serological testing to avoid the risk of infection for mice. thus, false-positive serological reactions may be found after experimental administration of replication-competent vaccinia virus. it has been shown that even cage contact sentinels may develop antibodies, and vaccinia virus leading to seroconversion may even be transmitted by dirty bedding (gaertner et al., ) . confirmation of positive serological results is important before action is taken because vaccinia virus is increasingly prevalent in animal facilities as a research tool (e.g. for vaccination or gene therapy). as observed in different outbreaks, serological testing is of little value in the initial stages of the disease. for example, in the outbreak described by dick et al. ( ) depopulation was nearly completed before serological confirmation was possible. for this reason, negative serological results should be confirmed by direct detection methods (pcr, immunohistochemistry, virus isolation) or by histopathology, especially when clinical cases suggestive of mousepox are observed. polymerase chain reaction assays to detect different genes of poxviruses in infected tissues have been described by dick et al. ( ) , neubauer et al. ( ) , and lipman et al. ( b) . the key to prevention and control of mousepox is early detection of infected mice and contaminated biological materials. all institutions that must introduce mice from other than commercial barrier facilities should have a health surveillance programme and test incoming mice. perhaps even more important than living animals are samples from mice (tumours, sera, tissues). the virus replicates in lymphoma and hybridoma cell lines (buller et al., ) , and such cells or material derived from them may therefore be a vehicle for inadvertent transfer between laboratories. the last two published outbreaks of ectromelia were both introduced into the facilities by mouse serum (dick et al., ; lipman et al., b) . lipman et al. ( b) found that the contaminated serum originated from a pooled lot of l that had been imported from china. because mouse serum commonly is sold to the end user in small aliquots (few millilitres), it has to be expected that aliquots of the contaminated lot are still stored in numerous freezers. both cases provide excellent examples of why map or pcr testing should be performed on all biological materials to be inoculated into mice. eradication of mousepox usually has been accomplished by elimination of the affected colonies, disinfection of rooms and equipment, and disposal of all infected tissues and sera. while culling of entire mouse colonies is the safest method for eradication of mousepox, it is not a satisfactory method due to the uniqueness of numerous lines of genetically modified animals housed in many facilities. several studies indicate that mousepox is not highly contagious bhatt and jacoby a,b ) and that it may be self-limiting when adequate husbandry methods are applied. therefore, strict quarantine procedures along with cessation of breeding (to permit resolution of infection) and frequent monitoring with removal of clinically sick and seropositive animals are a potential alternative. the period from the last births before the break until the first matings after the break should be at least weeks (bhatt and jacoby, b) . sequential testing of immunocompetent contact sentinels for seroconversion should be employed with this option. in the past, immunization with live vaccinia virus was used to suppress clinical expression of mousepox. vaccination may substantially reduce the mortality rate, but it does not prevent virus transmission or eradicate the agent from a population bhatt and jacoby, c) . after vaccination, typical pocks develop at the vaccination site, and infectious vaccinia virus is detectable in spleen, liver, lungs, and thymus (jacoby et al., ) . vaccination also causes seroconversion so that serological tests are not applicable for health surveillance in vaccinated populations. it is therefore more prudent to control mousepox by quarantine and serological surveillance than by relying on vaccination. mortality and clinical disease are the major factors by which ectv interferes with research. severe disruption of research can also occur when drastic measures are taken to control the infection. the loss of time, animals, and financial resources can be substantial. murine adenoviruses (madv) are non-enveloped, double-stranded dna viruses of the family adenoviridae, genus mastadenovirus. two distinct strains have been isolated from mice. the fl strain (madv- ) was first isolated in the usa as a contaminant of a friend leukaemia (hartley and rowe, ) ; the k strain (madv- ) was first isolated in japan from the faeces of a healthy mouse (hashimoto et al., ) . both strains are now considered to represent different species (hamelin and lussier, ; jacques et al., a,b) . in laboratory mice, seropositivity to adenoviruses was reported in % of spf colonies and in % of non-spf colonies in the usa (jacoby and lindsey, ) . antibodies were also detected at a low prevalence rate in french colonies (zenner and regnault, ) , but the virus strain used as antigen is not mentioned. a similar range of positive samples was reported by livingston and riley ( ) . antibodies to madv were also found in wild mice and in rats (otten and tennant, ; smith et al., ) . both viruses are not known to cause clinical disease in naturally infected, immunocompetent mice. however, madv- can cause a fatal systemic disease in suckling mice after experimental inoculation (hartley and rowe, ; heck et al., ; wigand, ) . disease is characterized by scruffiness, lethargy, stunted growth, and often death within days. experimental infection of adult mice with madv- is most often subclinical and persistent (richter, ) but can cause fatal haemorrhagic encephalomyelitis with neurological symptoms, including tremors, seizures, ataxia, and paralysis, in susceptible c bl/ and dba/ j mice (guida et al., ) . balb/c mice are relatively resistant to this condition. athymic foxn nu mice experimentally infected with madv- develop a lethal wasting disease (winters and brown, ) . similarly, prkdc scid mice succumb to experimental infection with madv- (pirofski et al., ) . gross lesions in response to natural madv infections are not detectable. occasional lesions observed after experimental infection with madv- include small surface haemorrhages in the brain and spinal cord of c bl/ and dba/ j mice (guida et al., ) , duodenal haemorrhage in foxn nu mice (winters and brown, ) , and pale yellow livers in prkdc scid mice (pirofski et al., ) . histologically, experimental madv- infection of suckling mice is characterized by multifocal necrosis and large basophilic intranuclear inclusion bodies in liver, adrenal gland, heart, kidney, salivary glands, spleen, brain, pancreas, and brown fat (heck et al., ; margolis et al., ; national research council, ; percy and barthold, ) . in experimentally induced haemorrhagic encephalomyelitis, multifocal petechial haemorrhages occur throughout the brain and spinal cord, predominantly in the white matter, and are attributed to infection and damage to the vascular epithelium of the central nervous system (cns; guida et al., ) . histopathological manifestations in madv- -infected prkdc scid mice are marked by microvesicular fatty degeneration of hepatocytes (pirofski et al., ) . in contrast to madv- , the tissue tropism of madv- is limited to the intestinal epithelium. naturally or experimentally infected mice develop intranuclear inclusions in enterocytes, especially in the ileum and caecum (takeuchi and hashimoto, ; otten and tennant, ; national research council, ; percy and barthold, ) . transmission of madv primarily occurs by ingestion. madv- is excreted in the urine and may be shed for up to years (van der veen and mes, ). murine adenovirus- infects the intestinal tract and is shed in faeces for only a few weeks in immunocompetent mice (hashimoto et al., ) ; immunodeficient mice may shed the virus for longer periods (umehara et al., ) . murine adenovirus infections are routinely diagnosed by serological tests. however, there is a one-sided cross reactivity of madv- with madv- (wigand et al., ) . serum from mice experimentally infected with madv- yielded positive reactions in serological tests with both viruses while serum from mice infected with madv- reacted only with the homologous antigen . smith et al. ( ) reported that sera may react with madv- or madv- or both antigens. occasional reports of mice with lesions suggestive of adenovirus infections and negative serology (with madv- ) indicate that the infection may not be detected if only one virus is used as antigen (luethans and wagner, ) . it has therefore become standard practice to test sera for antibodies to both madv- and madv- . the common methods are ifa and elisa, and both are more sensitive than the previously used complement fixation test. the low prevalence in colonies of laboratory mice indicate that madv can easily be eliminated (e.g. by hysterectomy derivation or embryo transfer) and that barrier maintenance has been very effective in preventing infection. the low pathogenicity and the low prevalence in contemporary mouse populations are the main reasons why adenoviruses are considered to be of little importance. however, immunodeficient mice are increasingly used and candidates for natural infections and wasting disease (richter, ) , and the viruses might easily be spread by the exchange of genetically modified mice and therefore re-emerge. only few influences on research attributable to madv have been published. for example, it has been shown that madv- significantly aggravates the clinical course of scrapie disease in mice (ehresmann and hogan, ) . natural infections with madv could also interfere with studies using adenovirus as a gene vector. polyomaviridae are enveloped, double-stranded dna viruses. two different agents of this family exclusively infect mice (mus musculus), and both belong to the genus polyomavirus. murine pneumotropic virus (mptv) has formerly been known as 'newborn mouse pneumonitis virus' or 'k virus' (named after l. kilham who first described the virus). the second is murine polyomavirus (mpyv). both are related but antigenically distinct from each other (bond et al., ) . they are enzootic in many populations of wild mice but are very uncommon in laboratory mice. even older reports indicate that both have been eradicated from the vast majority of contemporary mouse colonies, and their importance is negligible (national research council, ) . seropositivity to these viruses was not reported in a survey conducted in the usa (jacoby and lindsey, ) . in a retrospective study in french facilities, antibodies to mpyv were found in of colonies, and all samples tested for mptv were negative (zenner and regnault, ) . comparable data were reported by livingston and riley ( ) . due to their low prevalence, both viruses are not included in the list of agents for which testing is recommended on a regular basis by felasa (nicklas et al., ) . although polyomavirus genes, especially those of sv are used widely in gene constructs for insertional mutagenesis, very few reports have been published on spontaneous or experimental disease due to mpyv or mptv in the last - years. the reader is therefore referred to previous review articles for details (eddy ; parker and richter, ; richter, ; shah and christian, ; national research council, ; orcutt, ; porterfield and richter, ) . natural infections with mptv are subclinical. the prevalence of infection is usually low in an infected population. the virus may persist in infected animals for months and perhaps for life depending on the age at infection and is reactivated under conditions of immunosuppression. virus replicates primarily in endothelial cells, but renal tubular epithelial cells are the major site of viral persistence (greenlee et al., (greenlee et al., , . clinical signs are observed only after infection of infant mice less than - days of age. infected pups suddenly develop respiratory symptoms after an incubation period of approximately week, and many die within a few hours of onset of symptoms with an interstitial pneumonia caused by productive infection of and damage to pulmonary endothelium. endothelial cells in other organs are involved in virus replication also (ikeda et al., ; greenlee et al., ) . in older suckling mice, mptv produces a more protracted infection, and the virus or viral antigen can be detected for as long as months. in adult animals, the virus produces a transient asymptomatic infection. even in immunodeficient foxn nu mice, experimental infection of adults is clinically asymptomatic although virus is detectable for a period of several months (greenlee, ) . in vitro cultivation of mptv is difficult. no susceptible permanent cell line is known to support growth. it can be cultured in primary mouse embryonic cells, but viral titres are not sufficient for use in serological assays (greenlee and dodd, ) . for this reason, the hi test using homogenates of livers and lungs of infected newborn mice is still frequently used, but ifa and elisa tests are also available (groen et al., ) . furthermore, a pcr test for demonstration of mptv in biological samples has been published (carty et al., ) . murine polyomavirus was first detected as a contaminant of murine leukaemia virus (mulv) when sarcomas developed in mice after experimental inoculation of contaminated samples. it has later been shown to be a frequent contaminant of transplantable tumours (collins and parker, ) . natural infection of mice is subclinical, and gross lesions including tumours are usually not found. tumour formation occurs if mice are experimentally infected at a young age or if they are inoculated with high virus doses. development of tumours may be preceded by multifocal necrosis and mortality during the viraemic stage (percy and barthold, ) . parotid, salivary gland, and mammary tumours are common, and sarcomas or carcinomas of kidney, subcutis, adrenal glands, bone, cartilage, teeth, blood vessels, and thyroid occur also. virus strains vary with regard to the tumour types or lesions that they induce, and mouse strains vary in their susceptibility to different tumour types. those of c bl and c br/cd lineage are considered to be the most resistant strains; athymic foxn nu mice are considered to be most susceptible; c h mice are particularly susceptible to adrenal tumours and a mice tend to develop bone tumours. immunosuppression or inoculation into immunodeficient strains (e.g. foxn nu ) also support the growth of tumours. on the other hand, experimental infection of adult immunocompetent mice does not result in tumour formation because the immune response suppresses tumour growth, and newborn immunocompetent mice develop runting only if inoculated with high virus doses (atencio et al., ) . after experimental intranasal infection, mpyv initially infects the respiratory tract followed by a systemic phase in which liver, spleen, kidney, and the colon become infected (dubensky et al., ) . the virus is shed in faeces and in all body fluids, and transmission occurs rapidly by direct contact between animals, but also between cages in a room. further, intrauterine transmission has been documented after experimental infection (mccance and mims, ) . murine polyomavirus persists in all organs in prkdc scid mice while viral dna is detectable in immunocompetent mice after experimental infection for only a limited period of about weeks (berke et al., ) . however, virus may persist and can be reactivated by prolonged immunosuppression (rubino and walker, ) or during pregnancy, at least in young mice (mccance and mims, ) . biological materials of mouse origin are likely to be the most common source of contamination of laboratory mice emphasizing the importance of map or pcr screening of biological materials to be inoculated into mice. the most frequently used tests for health surveillance of mouse colonies are elisa and ifa (aclad, ) ; in addition, the hi test is still used. latent infections can be detected by intracerebral inoculation of neonate mice or by map testing, but direct demonstration of virus in biological samples is also possible by pcr testing (porterfield and richter, ; carty et al., ) . parvoviruses are non-enveloped small viruses (approximately nm in diameter) with a single-stranded dna genome of approximately nucleotides. murine parvoviruses are members of the family parvoviridae, genus parvovirus. they are remarkably resistant to environmental conditions like heat, desiccation, acidic and basic ph-values. two distinct serotypes infect laboratory mice: the mice minute virus (mmv) and the mouse parvovirus (mpv). nonstructural proteins (ns- and ns- ) are highly conserved among both viruses whereas the capsid proteins (vp- , vp- , vp- ) are more divergent and determine the serogroup (ball-goodrich and johnson, ) . both viruses require mitotically active cells for replication. severe infections are therefore not found in mature animals due to the lack of a sufficient number of susceptible cells in tissues. general aspects of rodent parvovirus infections and their potential effects on research results have been reviewed (tattersall and cotmore, ; national research council, ; jacoby et al., ) . already in the mid- s mouse colonies were identified that gave positive reactions for mmv by ifa but not by hi tests. it was subsequently shown that these colonies were infected with a novel parvovirus, initially referred to as 'mouse orphan parvovirus'. the first isolate of mpv was detected as a contaminant of cultivated t-cell clones interfering with in vitro immune responses (mckisic et al., ) and was named 'mouse parvovirus'. it does not replicate well in currently available cell cultures, and sufficient quantities of virus for serological tests are difficult to generate. hitherto, only very few isolates of mpv have been cultured and characterized on a molecular basis (ball-goodrich and johnson, ; besselsen et al., ) . at present, mpv is among the most common viruses in colonies of laboratory mice. the prevalence of sera positive for parvoviruses was nearly % in a study from livingston et al. ( ) , with the majority of sera being positive for mpv. this is consistent with a recent survey conducted in the usa showing that almost % of non-spf colonies were seropositive (jacoby and lindsey, ) . similar results were obtained for genetically modified mice in japan (yamamoto et al., ) , in contrast to earlier studies indicating that the infection was rare in japan (ueno et al., ) . clinical disease and gross or histological lesions have not been reported for mice naturally or experimentally infected with mpv. infections are subclinical even in newborn and immunocompromised animals . in contrast to many other viruses infecting mice, viral replication and excretion is not terminated by the onset of host immunity. tissue necrosis has not been observed at any stage of infection in infected infant or adult mice . humoral immunity to mpv does not protect against mmv infections and vice versa (hansen et al., ) . serological surveys have indicated that mpv naturally infects only mice. differences in mouse strain susceptibility to clinical mpv infection do not exist. however, seroconversion seems to be strain-dependent. after experimental infection, seroconversion occurred in all c h/hen mice, fewer balb/c, dba/ , and icr mice, and seroconversion could not be detected in c bl/ mice (besselsen et al., ) . diagnosis of mpv infection by pcr testing of small intestine and mesenteric lymph nodes also depended on the mouse strain. mpv dna was detected in all mouse strains evaluated except dba/ even though seroconversion was detected in these mice. after oral infection, the intestine is the primary site of viral entry and replication. the virus spreads to the mesenteric lymph nodes and other lymphoid tissues, where it persists for more than months , and seems to be excreted via the intestinal and the urinary tract. after experimental inoculation of weanling mice, mpv is transmitted to cagemates by direct contact for - weeks , and transmission by dirty bedding is also possible. these results implicate a role for urinary, faecal, and perhaps respiratory excretion of virus. another study showed that naturally infected mice may not transmit the virus under similar experimental conditions (shek et al., ) . serology is a useful tool to identify mpv infections in immunocompetent hosts, but reaching a diagnosis based on serological assays may be difficult and requires a good knowledge of the available techniques. neither the virion elisa nor hi are practical screening tests for mpv because they require large quantities of purified mpv which is difficult to obtain. diagnosis of mpv infections has long been made on the basis of an mmv hi-negative result coupled with an mmv ifapositive result. a generic rodent parvovirus elisa using a recombinant ns- protein as antigen has been developed , but mpv ifa and mpv hi assays are more sensitive techniques than the ns- elisa and the mmv ifa (besselsen et al., ) . recently, elisa tests have been described that use recombinant vp- and provide sensitive and serogroupspecific assays for the diagnosis of mpv infections in mice (ball-goodrich et al., ; livingston et al., ) . in immunodeficient mice that do not generate a humoral immune response, pcr assays can be used to detect mpv (besselsen et al., ; redig and besselsen, ) and other parvoviruses. mpv has been shown to persist for at least weeks in the mesenteric lymph nodes . this tissue is considered the best suited for pcr analysis, but spleen and small intestine can also be used with good success (besselsen et al., ) . the virus persists sufficiently long in mesenteric lymph nodes so that pcr assays may also be used as a primary screening tool for laboratories that do not have access to specific mpv antigenbased serological assays. polymerase chain reaction is further a good confirmatory method for serological assays and has also been described for the detection of parvoviruses in cell lines and tumours (yagami et al., ) . in addition, the map test has been reported as a sensitive tool to detect mpv (shek et al., ) . given the high environmental stability of the virus and the potential fomite transmission together with the long virus persistence in infected animals, spontaneous disappearance from a mouse population (e.g. by cessation of breeding) is very unlikely. eradication of infection is possible by elimination of infected animals and subsequent replacement with uninfected mice, and the agent can be eliminated from breeding populations only by embryo transfer or by hysterectomy. although there are few published reports of confounding effects of mpv on research, it is lymphocytotropic and may perturb immune responses in vitro and in vivo. infections with mpv have been shown to influence rejection of skin and tumour grafts (mckisic et al., , . mice minute virus is the type species of the genus parvovirus. the virus was formerly called 'minute virus of mice' (mvm) and was renamed recently (international union of microbiological societies, ) . it was originally isolated by crawford ( ) from a stock of mouse adenovirus, and this prototype isolate was later designated mvmp. its allotropic variant was detected as a contaminant of a transplantable mouse lymphoma (bonnard et al., ) and designated mvmi because it exhibits immunosuppressive properties in vitro. both variants have distinct cell tropisms in vivo and in vitro. the mmvp infects fibroblast cell lines and does not cause clinical disease (kimsey et al., , brownstein et al., . the mmvi grows lytically in t cells and inhibits various functions mediated by these cells in vitro. both strains are apathogenic for adult mice, but the immunosuppressive variant is more pathogenic for neonatal mice than is mmvp. serological surveys show that the mouse is the primary natural host (parker et al., ; smith et al., b; singleton et al., ) , but the virus is also infective for rats, hamsters (garant et al., ; ward and tattersall, ) , and mastomys (haag et al., ) during foetal development or after parenteral inoculation. natural infections are usually asymptomatic in adults and infants, and the most common sign of infection is seroconversion. kilham and margolis ( ) observed mild growth retardation a few days after experimental infection of neonatal mice with mmvp. studies of transplacental infection yielded no pathological findings in mice (kilham and margolis, ). the immunosuppressive variant but not the prototype strain is able to produce a runting syndrome after experimental infection of newborn mice (kimsey et al., ) . depending on the host genotype, experimental infections of foetal and neonatal mice with mmvi produce various clinical presentations and lesions. infection in c bl/ mice is asymptomatic, but the virus causes lethal infections with intestinal haemorrhage in dba/ mice. infection of strains such as balb/c, cba, c h/he, and sjl is also lethal and mice have renal papillary haemorrhage (brownstein et al., ) . the mmvi also infects haematopoietic stem cells and mediates an acute myelosuppression (segovia et al., (segovia et al., , . due to their dependency on mitotically active tissues, the foetus is at particular risk for damage by parvoviruses. mice minute virus and other parvoviruses may have severe teratogenic effects and cause foetal and neonatal abnormalities by destroying rapidly dividing cell populations, often resulting in foetal death. adult prkdc scid mice develop an acute leukopenia month after experimental infection with mmvi and die within months. the virus persists lifelong in the bone marrow of these mice (segovia et al., ) . mice minute virus is shed in faeces and urine. contaminated food and bedding are important factors in viral transmission because the virus is very resistant to environmental conditions. direct contact is also important and the virus does not easily spread between cages. routine health surveillance is usually conducted by serological methods. unlike mpv, mmv can easily be cultured in cell lines so that antigen production for hi and elisa (using whole purified virions) is easy. haemagglutination inhibition is a highly specific diagnostic test whereas ifa always exhibits some degree of cross reactivity with mpv and other closely related parvoviruses. enzyme-linked immunosorbent assay is probably the most frequently used test, but depending on the purity of the antigen preparation, cross reactions with mpv may occur due to contamination with nonstructural proteins that are common to both viruses. this problem can be avoided by the use of recombinant vp- antigen (livingston et al., ) . viral detection is also possible by pcr in biological materials and in organs (intestines, kidney, spleen) from infected animals (yagami et al., ; chang et al., ; redig and besselsen, ) . in contrast to mpv, pcr is not appropriate as a confirmatory method for serology because mmv has not been shown to persist in immunocompetent animals for sufficiently long periods. the virus can be eliminated from infected breeding populations by caesarean derivation or by embryo transfer. in experimental colonies, elimination of infected animals and subsequent replacement with uninfected mice is practical if careful environmental sanitation is conducted by appropriate disinfection procedures. it is important that reintroduction is avoided by exclusion of wild mice and by strict separation from other infected populations and potentially contaminated materials in the same facility. admission of biological materials must be restricted to samples that have been tested and found free from viral contamination. both allotropic variants of mmv have been used as models for molecular virology, and their small size and simple structure have facilitated examination of their molecular biology and expedited understanding of cell tropism, viral genetics, and structure. the significance for laboratory mouse populations was considered low or uncertain because natural infections are inapparent. however, various effects on mouse-based research have been published (tattersall and cotmore, ; jacoby et al., ; baker, ; nicklas et al., ) . due to their predilection for replicating in mitotically active cells, they are frequently associated with tumour cells and have a marked oncosuppressive effect (rommelaere and cornelis, ) . special attention is also necessary for immunological research and other studies involving rapidly dividing cells (embryology, teratology). in addition, mmv is a common contaminant of transplantable tumours, murine leukaemias, and other cell lines (collins and parker, ; nicklas et al., ; garnick, ) . lactate dehydrogenase-elevating virus (ldv) is a single-stranded rna virus of the genus arterivirus belonging to the family arteriviridae. lactate dehydrogenase-elevating virus has repeatedly been detected in feral mice (mus musculus), which are considered to be a virus reservoir (rowson and mahy, ; li et al., ) . only mice and primary mouse cells are susceptible to infection with ldv. after infection, virus titres of - particles per ml serum are found within - h after infection. the virus titre drops to particles per ml within - weeks and remains constant at this level for life. lactate dehydrogenase-elevating virus replicates in a subpopulation of macrophages in almost all tissues and persists in lymph nodes, spleen, liver, and testes tissues (anderson et al., a) . the virus can be stored in undiluted mouse plasma at Ϫ Њc without loss of infectivity, but it is not stable at room temperature and is very sensitive to environmental conditions. lactate dehydrogenase-elevating virus was first detected during a study of methods that could be used in the early diagnosis of tumours (riley et al., ) . it produces a persistent infection with continuous virus production and a lifelong viraemia despite ldv-specific immune reactions of the host ( van den broek et al., ) . lactate dehydrogenase-elevating virus has been found in numerous biological materials that are serially passaged in mice such as transplantable tumours including human tumours (nicklas et al., ; ohnishi et al., ) , monoclonal antibodies or ascitic fluids (nicklas et al., ) , or infectious agents (e.g. haemoprotozoans, k virus, clostridium piliforme). these materials are contaminated after passage in an infected and viraemic animal. contamination with ldv leads to the infection of each sequential host and to transmission of the virus by the next passage and remains associated with the specimen. it is therefore the most frequently detected contaminant in biological materials (collins and parker, ; nicklas et al., ) . infection with ldv is usually asymptomatic, and there are no gross lesions in immunocompetent as well as in immunodeficient mice. the only exception is polyomyelitis with flaccid paralysis of hind limbs developing in c and akr mice when they are immunosuppressed either naturally with aging or experimentally (anderson et al., b; monteyne et al., ) . it has been shown that only mice harbouring cells in the cns that express a specific endogenous mulv are susceptible to poliomyelitis (anderson et al., c) . the characteristic feature of ldv infection is the increased activity of lactate dehydrogenase (ldh) and other plasma enzymes (brinton, ; national research council, ) , which is due to the continuous destruction of permissive macrophages that are responsible for the clearance of ldh from the circulation. as a consequence, the activity of plasma ldh begins to rise by only h after infection and peaks - days after infection at - -fold normal levels, or even be up to -fold in sjl/j mice. the enzyme activity declines during the next weeks but remains elevated throughout life. antigen-antibody complexes produced during infection circulate in the blood and are deposited in the glomeruli (brinton, ; national research council, ) . in contrast to other persistent virus infections (e.g. lymphocytic choriomeningitis virus lcmv), these complexes do not lead to immune complex disease and produce only a very mild glomerulopathy. the only gross finding associated with ldv infection is mild splenomegaly. microscopically, necrosis of lymphoid tissues is visible during the first days of infection. in mouse strains that are susceptible to poliomyelitis, ldv induces lesions in the grey matter of the spinal cord and the brain stem (brinton, ) . lactate dehydrogenase-elevating virus is not easily transmitted between mice, even in animals housed in the same cage. fighting and cannibalism increase transmission between cage mates most likely via blood and saliva. infected females transmit the virus to their foetuses if they have been infected few days prior to birth and before igg anti-ldv antibodies are produced, but developmental and immunological factors (e.g. gestational age, timing of maternal infection with ldv, placental barrier) are important in the regulation of transplacental ldv infection (haven et al., ; zitterkopf et al., ) . maternal immunity protects foetuses from intrauterine infection. immunodeficient prkdc scid mice transmit virus to their offspring also during chronic infection (broen et al., ). an important means of transmission is provided by experimental procedures such as mouse-to-mouse passage of contaminated biological materials or the use of the same needle for sequential inoculation of multiple mice. in principal, serological methods such as ifa may be used for detecting ldv infection (hayashi et al., ) but they are not of practical importance. circulating virus-antibody complexes interfere with serological tests (aclad, ) , and sufficient quantities of virus for serological tests are difficult to generate because ldv replicates only in specific subpopulations of primary cultures of murine macrophages and monocytes for one cell cycle (brinton, ) . therefore, diagnosis of ldv infection is primarily based on increased ldh activity in serum or plasma of mice. lactate dehydrogenase-elevating virus activity in serum or plasma can be measured directly, or samples (e.g. plasma, cell or organ homogenates) are inoculated into pathogen-free mice and the increase in ldh activity within - days is measured. an - -fold increase is indicative of ldv infection. detection of infectivity of a plasma sample by the induction of increased ldh activity in the recipient animal is the most reliable means of identifying an infected animal. however, it is important to use clear nonhaemolysed samples because haemolysis will (falsely) elevate activities of multiple serum or plasma enzymes, including ldh. while this assay may be included in a commercial 'map test', it does not involve antibody detection. persistent infection makes ldv an ideal candidate for pcr detection in plasma or in organ homogenates (van der logt et al., ; chen and plagemann, ) . however, reports exist that pcr may produce false-negative results and should be used cautiously (lipman et al., a) . similarly important as detecting ldv in animals is its detection in biological materials. this may be done by assay for increased ldh activity after inoculation of suspect material into pathogen-free mice (collins and parker, ; nicklas et al., ) or by pcr (goto et al., ; bootz and sieber, ) . lactate dehydrogenase-elevating virus spreads slowly in a population because direct contact is necessary. therefore ldv-negative breeding populations can easily be established by selecting animals with normal plasma ldh activity. embryo transfer and hysterectomy derivation are also efficient. the presence of ldv in experimental populations is indicative of contaminated biological materials. in such cases, it is essential that the virus is also eliminated from these samples. this is easily achieved by maintenance of cells by in vitro culture instead of by animal-to-animal passages (plagemann and swim, ) . due to the extreme host specificity of the virus, contaminated tumour samples can also be sanitized by passages in nude rats or other animal species. lactate dehydrogenase-elevating virus is a potential confounder of any research using biological materials that are passaged in mice. once present in an animal, the virus persists lifelong. the most obvious signs are increased levels of plasma ldh and several other enzymes. lactate dehydrogenase-elevating virus may also exhibit numerous effects on the immune system (thymus involution, depression of cellular immunity, enhanced or diminished humoral responses, nk cell activation, development of autoimmunity, and suppression of development of diabetes in nod mice; cafruny and hovinen, ; nicklas et al., ; takei et al., ; markine-goriaynoff et al., ; gomez et al., ) and enhance or suppress tumour growth (brinton, ; baker, ; nicklas et al., ) . lymphocytic choriomeningitis virus is an enveloped, segmented single-stranded rna virus of the genus arenavirus, family arenaviridae. its name refers to the condition that results from experimental intracerebral inoculation of the virus into adult mice and is not considered to be a feature of natural infections. mice (mus musculus) serve as the natural virus reservoir (salazar-bravo et al., ) , but syrian hamsters are also important hosts (ackermann, ) . additional species such as rabbits, guinea pigs, squirrels, monkeys, and humans are susceptible to natural or experimental infection. infection in hamsters is considered to be asymptomatic (national research council, ) . natural infection of callitrichid primates (marmosets and tamarins) leads to a progressive hepatic disease that is known as 'callitrichid hepatitis' (montali et al., ; asper et al., ; lukashevich et al., ) . antibodies to lcmv have been found in wild mice in europe (ackermann et al., ) , africa (el karamany and imam, ), asia (morita et al., (morita et al., , , australia , and america (childs et al., ) . thus, it is the only arenavirus with worldwide distribution. infection with lcmv is rarely found in laboratory mice (smith et al., ) . seropositivity to lcmv was reported in approximately % of non-spf mouse colonies in the usa in (jacoby and lindsey, ) and in % of french colonies in - (zenner and regnault, ) . recent studies confirm that only a small percentage of mice tested are positive for lcmv (livingston and riley, ) . in addition to laboratory mice and other vertebrate hosts, the virus has frequently been found in transplantable tumours and tissue culture cell lines from mice and hamsters (bhatt et al., a; nicklas et al., ) . despite the low prevalence in laboratory mice, seropositivity to this zoonotic agent should raise serious concern for human health. lymphocytic choriomeningitis virus is frequently transmitted to humans from wild mice (childs et al., ) and is also endemic to a varying degree in the human population (childs et al., ; marrie and saron, ; lledo et al., ) due to contact with wild mice. lymphocytic choriomeningitis virus is further transmitted to humans by domestic syrian hamsters rousseau et al., ) . in addition, infected laboratory mice (dykewicz et al., ) and contaminated biological materials are important sources of infections for humans, and several outbreaks of lcm among laboratory personnel have been traced to transplantable tumours biggar et al., ; mahy et al., ) . in mice, clinical signs of lcmv infection vary with strain and age of mouse, strain and dose of virus, and route of inoculation (lehmann-grube, ; national research council, ) . two forms of natural lcmv infection are generally recognized: a persistent tolerant and an (acute) nontolerant form. the persistent form results from infection of mice that are immunotolerant. this is the case if mice are infected in utero or during the first days after birth. this form is characterized by lifelong viraemia and shedding. mice may show growth retardation, especially during the first - weeks, but appear otherwise normal. infectious virus is bound to specific antibodies and complement, and these complexes accumulate in the renal glomeruli, the choroid plexus, and sometimes also in synovial membranes and blood vessel walls. at - months of age, immune complex nephritis develops with ruffled fur, hunched posture, ascites, and occasional deaths. this immunopathological phenomenon is called 'late onset disease' or 'chronic immune complex disease'. the incidence of this type of disease varies between mouse strains. gross lesions include enlarged spleen and lymph nodes due to lymphoid hyperplasia. kidneys affected with glomerulonephritis may be enlarged with a granular surface texture or may be shrunken in later stages of the disease process. microscopically, there is generalized lymphoid hyperplasia and immune complex deposition in glomeruli and vessel walls, resulting in glomerulonephritis and plasmacytic, lymphocytic perivascular cuffs in all visceral organs (percy and barthold, ) . the nontolerant acute form occurs when infection is acquired after the development of immunocompetence (in mice older than week). these animals become viraemic but do not shed virus and may die within a few days or weeks. natural infections of adults are usually asymptomatic. surviving mice are seropositive and in most cases clear the virus to below detection levels of conventional methods. however, virus may persist at low levels in tissues (particularly spleen, lung, and kidney) of mice for at least weeks after infection as determined by sensitive assays such as nested reverse transcriptasepolymerase chain reaction (rt-pcr) or immunohistochemisty (ciurea et al., ) . such nonlethal infection leads to protection against otherwise lethal intracerebral challenge. protection from lethal challenge is also achieved by maternally derived anti-lcmv antibodies through nursing or by the administration of anti-ldv monoclonal igg a antibodies (baldridge and buchmeier, ) . in experimentally infected animals, the route of inoculation (subcutaneous, intraperitoneal, intravenous, intracerebral) also influences the type and degree of disease (lehmann-grube, ; national research council, ) . intracerebral inoculation of adult immunocompetent mice typically results in tremors, convulsions, and death due to meningoencephalitis and hepatitis. neurological signs usually appear on day postinoculation, and animals die within - days after the onset of symptoms or recover within several days. the classic histological picture is of dense perivascular accumulations of lymphocytes and plasma cells in meninges and choroid plexus. while infection following subcutaneous inoculation usually remains inapparent, reaction of mice to intraperitoneal or intravenous inoculation depends on the virus strain and on the mouse strain. infection by these routes primarily causes multifocal hepatic necrosis and necrosis of lymphoid cells. athymic foxn nu mice and other immunodeficient mice do not develop disease but become persistently viraemic and shed virus. as a general rule, all pathological alterations following lcmv infection are immune-mediated; and mice can be protected from lcmv-induced disease by immunosuppression (gossmann et al., ) . lymphocytic choriomeningitis virus disease is a prototype for virus-induced t-lymphocyte-mediated immune injury and for immune complex disease. for detailed information on the pathogenesis of lcmv infection, the reader is referred to a recent review article by oldstone ( ) . extensive information on the clinical and pathological features of lcmv infection in mice has been assembled by lehmann-grube ( ) . in nature, carrier mice with persistent infection serve as the principal source of virus. intrauterine transmission is very efficient, and with few exceptions all pups born from carrier mice are infected. furthermore, persistently infected mice and hamsters can shed large numbers of infectious virions primarily in urine, but also in saliva and milk. the virus can replicate in the gastric mucosa after intragastric infection (rai et al., (rai et al., , . gastric inoculation elicits antibody responses of comparable magnitudes as intravenous inoculation and leads to active infection with lcmv indicating that oral infection is possible, e.g., by ingestion of contaminated food or cannibalism. a self-limiting infection frequently results from infection of adult mice. the virus does not spread rapidly after introduction in populations of adult mice, and the infectious chain usually ends. however, if the virus infects a pregnant dam or a newborn mouse, a lifelong infection results, and soon a whole breeding colony of mice may become infected if the mice live in close proximity (which is the case under laboratory conditions). lymphocytic choriomeningitis virus is most commonly diagnosed by serological methods. methods of choice are ifa and elisa, which have replaced the relatively insensitive complement fixation test. it is important that bleeding of mice is done carefully because of a potential risk due to viraemic animals. historically, direct viral detection was performed by inoculating body fluids or tissue homogenates into the brain of lcmv-free mice or by subcutaneous injection into mice and subsequent serological testing (map test). more recently, pcr assays have been developed for the direct detection of viral rna in clinical samples or animals (park et al., . both map test and pcr can also be used to detect contamination of biological materials (bootz and sieber, ) . vertical transmission of lcmv by transuterine infection is efficient so this virus cannot reliably be eliminated by caesarean rederivation. caesarean derivation may be effective if dams acquired infection after the development of immunocompetence (nontolerant acute infection) and subsequently eliminated the virus, but such a strategy is difficult to justify in light of lcmv's zoonotic potential. in breeding colonies of great value, virus elimination might be possible soon after introduction into the colony by selecting nonviraemic breeders. this procedure is expensive and time consuming and requires special safety precautions. fortunately, infections of laboratory mice with lcmv are very uncommon. however, once lcmv has been detected in animals or in biological materials, immediate destruction of all contaminated animals and materials is advisable to avoid risk of human infection. foxn nu and prkdc scid mice may pose a special risk because infections are silent and chronic (mahy et al., ) . cages and equipment should be autoclaved, and animal rooms should be fumigated with disinfectants such as formaldehyde, vaporized paraformaldehyde, and hydrogen peroxide. appropriate precautions are necessary for experiments involving lcmv, or lcmv-infected animals or materials. biological safety level (bsl) will be considered to be sufficient in most cases. biological safety level practices may be considered when working with infected animals owing to the increased risk of virus transmission by bite wounds, scratching, or aerosol formation from the bedding. animal biosafety level (absl) practices and facilities are generally recommended for work with infected hamsters. appropriate precautions have been defined for different bsls or animal biology safety levels by cdc ( ) . lymphocytic choriomeningitis virus is an important zoonotic agent. it has been transmitted to humans working with infected animals or with contaminated biological materials and can cause mild to serious or fatal disease in humans (dykewicz et al., ; barton et al., ; barton and hyndman, ) . congenital infection in humans may result in hydrocephalus, or foetal or neonatal death (barton et al., ) . lymphocytic choriomeningitis virus is also frequently utilized as a model organism to study virus-host interactions, immunological tolerance, virus-induced immune complex disease, and a number of immunological mechanisms in vivo and in vitro (slifka, ; zinkernagel, ) . accidental transmission may have a severe impact on various kinds of experiments (for details, see lehmann-grube, ; bhatt et al., b; national research council, ; baker, ; nicklas et al., ) . mammalian orthoreoviruses (mrv) are nonenveloped, segmented double-stranded rna viruses of the family reoviridae, genus orthoreovirus. they have a wide host range and are ubiquitous throughout the world. the designation reo stands for respiratory enteric orphan and reflects the original isolation of these viruses from human respiratory and intestinal tract without apparent disease. the term 'orphan' virus refers to a virus in search of a disease. mammalian orthoreovirus can be grouped into three serotypes ( , , ). mammalian orthoreovirus- (synonyms: hepatoencephalomyelitis virus; echo virus) infection remains prevalent in contemporary mouse colonies and has been reported in wild mice barthold, a) . seropositivity to mrv- was found in less than % of spf colonies and in approximately % of non-spf mouse colonies in the usa in (jacoby and lindsey, ) . a study in france reported antibodies to mrv- in % of mouse colonies examined (zenner and regnault, ) . more recently, a study in north america found a low rate ( . %) of mouse sera to be positive for antibodies against this virus (livingston and riley, ) . in addition, contamination of mouse origin tumours and cell lines by mrv- has been reported many times (national research council, ; nicklas et al., ; barthold, a) . experimentally, mrv- infection of infant mice has been used to model human hepatobiliary disease, pancreatitis, diabetes mellitus, and lymphoma (kraft, ; national research council, ; fenner et al., ) . the literature on mrv- infections in mice is dominated by studies on experimentally infected animals. the virus can cause severe pantropic infection in infant mice (kraft, ; tyler and fields, ; barthold, a) . after parenteral inoculation, virus can be recovered from the liver, brain, heart, pancreas, spleen, lymph nodes, and blood vessels. following oral inoculation, reoviruses gain entry by infecting specialized epithelial cells (m cells) that overlie peyer's patches. the virus then becomes accessible to leukocytes and spreads to other organs by way of the lymphatic system and the bloodstream. neural spread to the cns has also been well documented (morrison et al., ) . the mechanisms of viral pathogenesis and their interactions with the host cell are reviewed in detail by and . natural infection by mrv- in a mouse colony usually is subclinical although diarrhoea or steatorrhoea and oily hair effect in suckling mice may be noted (kraft, ; tyler and fields, ; national research council, ; barthold, a; percy and barthold, ) . the latter term has been used to describe the matted, unkempt appearance of the hair coat that results from steatorrhoea due to pancreatitis, maldigestion, and biliary atresia. in addition, runting (attributed to immune-mediated destruction of cells in the pituitary gland that produce growth hormone), transient alopecia, jaundice (due to excessive bilirubin in the blood, which is attributed to the liver pathology, especially biliary atresia), and neurological signs such as incoordination, tremors, or paralysis may develop. when present in natural infections, clinical signs and lesions are similar to but milder than in experimental neonatal infections. early descriptions of naturally occurring disease may have been complicated by concurrent infections such as mhv or murine rotavirus a (murv-a)/epizootic diarrhoea of infant mice (edim) virus that contributed to the severity of the lesions especially in liver, pancreas, cns, and intestine. the outcome of mrv- infection depends on age and immunological status of mouse, dose of virus, and route of inoculation. adult immunocompetent mice typically show no clinical signs and have no discernible lesions even in experimental infections. mucosal and maternally conferred immunity are considered to be important in protection from or resolution of disease (cuff et al., ; barthold et al., b) . experimental infection of adult prkdc scid mice is lethal (george et al., ) . depending on the route of inoculation, experimental infection of adult foxn nu mice is subclinical or results in liver disease (carthew, ; george et al., ) . histological findings reported to occur after experimental mrv- infection of neonatal mice include inflammation and necrosis in liver, pancreas, heart, adrenal, brain, and spinal cord; lymphoid depletion in thymus, spleen, and lymph nodes; and hepatic fibrosis with biliary atresia (papadimitriou and robertson, ; tyler and fields, ; barthold et al., b; barthold, a; percy and barthold, ) . transmission of reoviruses probably involves the aerosol as well as the faecal-oral route (national research council, ) . fomites may play an important role as passive vectors because reoviruses resist environmental conditions moderately well. serological screening with elisa or ifa is in widespread use for detection of antibodies to mrv- in diagnostic and health surveillance programmes. both elisa and ifa detect cross-reacting antibodies to heterologous mrv serotypes that can infect mice (aclad, ) . the hi test does not detect such cross-reacting antibodies but is prone to give false positive results due to nonspecific inhibitors of haemagglutination (kraft and meyer, ; van der logt, ; aclad, ) . reverse transcriptase-polymerase chain reaction methods for the detection of mrv- rna (steele et al., ) or mrv rna (leary et al., ) are also available. reports on contamination of mouse origin tumours and cell lines by mrv- and its interference with transplantable tumour studies (bennette, ; nelson and tarnowski, ) emphasize the importance of screening of biological materials to be inoculated into mice by map test or pcr. natural seroconversion to mrv- without clinical disease is also observed in laboratory rats, hamsters, and guinea pigs (national research council, ; barthold, a) . caesarean derivation and barrier maintenance have proven effective in the control and prevention of mrv- infection (kraft, ; national research council, ) . the virus may interfere with research involving transplantable tumours and cell lines of mouse origin. it has the potential to alter intestinal studies and multiple immune response functions in mice. in enzootically infected colonies, protection of neonates by maternal antibody could complicate or prevent experimental infections with reoviruses. it could further complicate experiments that require evaluation of liver, pancreas, cns, heart, lymphoid organs, and other tissues affected by the virus. the term murine hepatitis virus (mhv; commonly referred to as 'mouse hepatitis virus') designates a large group of antigenically and genetically related, singlestranded rna viruses belonging to the family coronaviridae, genus coronavirus. they are surrounded by an envelope with a corona of surface projections (spikes). murine hepatitis virus is antigenically related to rat coronaviruses and other coronaviruses of pigs, cattle, and humans. numerous different strains or isolates of mhv have been described. they can be distinguished by neutralization tests that detect strain-specific spike (s) antigens. the best studied strains are the prototype strains mhv- , mhv- , mhv- , jhm (mhv- ), a , and s, of which mhv- is regarded as the most virulent. murine hepatitis virus, like other coronaviruses, mutates rapidly, and strains readily form recombinants, so that new (sub)strains are constantly evolving. strains vary in their virulence, organotropism, and cell tropism (homberger, ) . based on their primary organotropism, mhv strains can be grouped into two biotypes: respiratory (or polytropic) and enterotropic. however, intermediate forms (enterotropic strains with tropism to other organs) exist. murine hepatitis virus is relatively resistant to repeated freezing and thawing, heating ( Њc for min), and acid ph but is sensitive to drying and disinfectants, especially those with detergent activity (national research council, ) . mus musculus is the natural host of mhv. it can be found in wild and laboratory mice throughout the world and is one of the most common viral pathogens in contemporary mouse colonies. while polytropic strains have historically been considered more common, this situation is thought to have reversed. a survey conducted in the usa in reported antibodies to mhv in more than % of spf mouse colonies and more than % of non-spf colonies (jacoby and lindsey, ) , though very recent monitoring results for research institutions across north america indicate that the prevalence of mhv has decreased during the past few years (livingston and riley, ) . a retrospective study in france covering the period from to reported antibodies to mhv in % of mouse colonies examined (zenner and regnault, ) . suckling rats inoculated experimentally with mhv had transient virus replication in the nasal mucosa and seroconversion but no clinical disease . similarly, deer mice seroconverted but showed no clinical disease after experimental infection (silverman et al., ) . murine hepatitis virus is also a common contaminant of transplantable tumours (collins and parker, ; nicklas et al., ) and cell lines (sabesin, ; yoshikura and taguchi, ) . the pathogenesis and outcome of mhv infections depend on interactions among numerous factors related to the virus (e.g. virulence and organotropism) and the host (e.g. age, genotype, immune status, and microbiological status; kraft, ; barthold, ; national research council, ; compton et al., ; homberger, ; percy and barthold, ) . murine hepatitis virus strains appear to possess a primary tropism for the upper respiratory or enteric mucosa. those strains with respiratory tropism initiate infection in the nasal mucosa and then may disseminate via blood and lymphatics to a variety of other organs because of their polytropic nature. respiratory (polytropic) strains include mhv- , mhv- , mhv- , a , s, and jhm. infection of mice with virulent polytropic mhv strains, infection of mice less than weeks of age, infection of genetically susceptible strains of mice, or infection of immunocompromised mice favour virus dissemination. virus then secondarily replicates in vascular endothelium and parenchymal tissues, causing disease of brain, liver, lymphoid organs, bone marrow, and other sites. infection of the brain by viraemic dissemination occurs primarily in immunocompromised or neonatal mice. additionally, infection of adult mouse brain can occur by extension of virus along olfactory neural pathways, even in the absence of dissemination to other organs. in contrast, enterotropic mhv strains (e.g. livim, mhv-d, and mhv-y) tend to selectively infect intestinal mucosal epithelium, with no or minimal dissemination to other organs such as mesenteric lymph nodes or liver. all ages and strains are susceptible to active infection, but disease is largely age-related. infection of neonatal mice results in severe necrotizing enterocolitis with high mortality within h. mortality and lesion severity diminish rapidly with advancing age at infection. adult mice develop minimal lesions although replication of equal or higher titres of virus occurs compared with neonates. the age-dependent decrease in severity of enterotropic mhv disease is probably related to the higher mucosal epithelium turnover in older mice, allowing more rapid replacement of damaged mucosa. another factor that is of considerable importance to the outcome of mhv infections is host genotype. for example, balb/c mice are highly susceptible to enterotropic mhv disease while sjl mice, at the other end of the spectrum, are highly resistant (barthold et al., a) . unlike in polytropic mhv infection where resistance is correlated with reduced virus replication in target cells (barthold and smith, ) , enterotropic mhv grows to comparable titres in sjl and balb/c mice at all ages (barthold et al., a) . therefore, the resistance of the sjl mouse to disease caused by enterotropic mhv seems to be mediated through an entirely different mechanism than resistance to polytropic mhv. furthermore, mouse genotypes that are susceptible to disease caused by one mhv strain may be resistant to disease caused by another strain (barthold, ) . it is therefore not possible to strictly categorize mouse strains as susceptible or resistant. the genetic factors determining susceptibility versus resistance in mhv infections are as yet poorly understood. both polytropic and enterotropic mhv infections are selflimiting in immunocompetent mice. immune-mediated clearance of virus usually begins about a week after infection, and most mice eliminate the virus within - weeks (barthold, ; barthold and smith, ; barthold et al., a) . humoral and cellular immunity appear to participate in host defences to infection, and functional t cells are an absolute requirement (williamson and stohlman, ; kyuwa et al., ; lin et al., ; haring and perlman, ) . therefore, immunodeficient mice such as foxn nu and prkdc scid mice cannot clear the virus (barthold et al., ; compton et al., ) . similarly, some genetically modified strains of mice may have deficits in antiviral responses or other alterations that allow the development of persistent mhv infection (rehg et al., ) . recovered immune mice are resistant to reinfection with the same mhv strain but remain susceptible to repeated infections with different strains of mhv (barthold and smith, a,b; . similarly, maternal immunity protects suckling mice against homologous mhv strains but not necessarily against other strains . however, maternal immunity, even to homologous strains, depends on the presence of maternally acquired antibody in the lumen of the intestine . therefore, the susceptibility of young mice to infection significantly increases at weaning. most mhv infections are subclinical and follow one of two epidemiological patterns in immunocompetent mice (national research council, ; homberger, ) . enzootic (subclinical) infection, commonly seen in breeding colonies, occurs when a population has been in contact with the virus for a longer period (e.g. several weeks). adults are immune (due to prior infection), sucklings are passively protected, and infection is perpetuated in weanlings. epizootic (clinical) infection occurs when the virus is introduced into a naive population (housed in open cages). the infection rapidly spreads through the entire colony. clinical signs depend upon the virus and mouse strains and are most evident in infant mice. typically, they include diarrhoea, poor growth, lassitude, and death. in infections due to virulent enterotropic strains, mortality can reach % in infant mice. some strains may also cause neurological signs such as flaccid paralysis of hind limbs, convulsions, and circling. adult infections are again usually asymptomatic. as the infection becomes established in the colony, the epizootic pattern is replaced by the enzootic pattern. in immunodeficient (e.g. foxn nu and prkdc scid ) mice, infection with virulent polytropic mhv strains often is rapidly fatal while less virulent strains cause chronic wasting disease (compton et al., ) . in contrast, adult immunodeficient mice can tolerate chronic infection by enterotropic mhv, with slow emaciation and diarrhoea, or minimal clinical disease (barthold et al., ; barthold, ) . subclinical mhv infections can be activated by a variety of experimental procedures (e.g. thymectomy, whole body irradiation, treatment with chemotherapeutic agents, halothane anaesthesia) or by co-infections with other pathogens (e.g. eperythrozoon coccoides, k virus; reviewed by kraft, ; national research council, ) . in most natural infections, gross lesions are not present or are transient and not observed. gross findings in neonates with clinical signs include dehydration, emaciation, and in contrast to edim, an empty stomach (ishida et al., ; barthold et al., ; kraft, ) . the intestine is distended and filled with watery to mucoid yellowish, sometimes gaseous contents. haemorrhage or rupture of the intestine can occur. depending on the virus strain, necrotic foci on the liver (ishida et al., ; kraft, ; percy and barthold, ) and thymus involution godfraind et al., ) may also be seen in susceptible mice. liver involvement may be accompanied by jaundice and haemorrhagic peritoneal exudate. splenomegaly may occur as a result of compensatory haematopoiesis (fox et al., ) . histopathological changes in susceptible mice infected with polytropic mhv strains include acute necrosis with syncytia in liver, spleen, lymph nodes, gut-associated lymphoid tissue, and bone marrow (kraft, ; barthold, ; national research council, ; percy and barthold, ) . neonatally infected mice can have vascular-oriented necrotizing (meningo)encephalitis with demyelination in the brain stem and peri-ependymal areas. lesions in peritoneum, bone marrow, thymus, and other tissues can be variably present. mice can develop nasoencephalitis due to extension of infection from the nasal mucosa along olfactory pathways to the brain, with meningoencephalitis and demyelination, the latter of which is thought to be largely t cell-mediated (haring and perlman, ) . this pattern of infection regularly occurs after intranasal inoculation of many mhv strains but is a relatively rare event after natural exposure. syncytia arising from endothelium, parenchyma, or leukocytes is a hallmark of infection in many tissues including intestine, lung, liver, lymph nodes, spleen, thymus, brain, and bone marrow. lesions are transient and seldom fully developed in adult immunocompetent mice, but they are manifest in immunocompromised mice. highly unusual presentations can occur in mice with specific gene defects. for example, granulomatous peritonitis and pleuritis were found in interferon-␥-deficient mice infected with mhv (france et al., ) . histopathological changes caused by enterotropic strains of mhv are mainly confined to the intestinal tract and associated lymphoid tissues (kraft, ; barthold, ; national research council, ; percy and barthold, ) . the most common sites are terminal ileum, caecum, and proximal colon. the severity of disease is primarily age-dependent, with neonatal mice being most severely affected. these mice show segmentally distributed areas of villus attenuation, enterocytic syncytia (balloon cells), and mucosal necrosis accompanied by leukocytic infiltration. intracytoplasmic inclusions are present in enterocytes. erosions, ulceration, and haemorrhage may be seen in more severe cases. lesions can be fully developed within - h, but are usually more severe at - days after infection. surviving mice may develop compensatory mucosal hyperplasia. mesenteric lymph nodes usually contain lymphocytic syncytia, and mesenteric vessels may contain endothelial syncytia. pathological changes in older mice are generally much more subtle and may only consist of transient syncytia. an occasional exception seems to occur in immunodeficient animals such as foxn nu mice, which can develop chronic hyperplastic typhlocolitis of varying severity (barthold et al., ) , but other agents such as helicobacter species may have been involved. in general, enterotropic mhv strains do not disseminate, but hepatitis and encephalitis can occur with some virus strains in certain mouse genotypes. murine hepatitis virus is highly contagious. it is shed in faeces and nasopharyngeal secretions and appears to be transmitted via direct contact, aerosol, and fomites (kraft, ; national research council, ) . vertical (in utero) transmission has been demonstrated in experimental infections (katami et al., ) but does not seem to be of practical importance under natural conditions. diagnosis during the acute stage of infection can be made by histological demonstration of characteristic lesions with syncytia in target tissues, but clinical signs and lesions can be highly variable and may not be prominent. suckling, genetically susceptible or immunocompromised mice are the best candidates for evaluation. active infection can be confirmed by immunohistochemistry (brownstein and barthold, ) or by virus isolation. virus recovery from infected tissues is difficult but can be accomplished using primary macrophage cultures or a number of established cell lines such as nctc or dbt (aclad, ) . these cells, however, may not be successful substrates for some enterotropic mhv strains. virus in suspect tissue can also be confirmed by bioassays such as map testing or infant or foxn nu mouse inoculation (de souza and smith, ; aclad, ) . amplification by passage in these mice increases the likelihood of detection of lesions and antigen, or virus recovery. other direct diagnostic methods that have been successfully utilized to detect mhv in faeces or tissue of infected mice include monoclonal antibody solution hybridization assay (casebolt and stephensen, ) and a number of rt-pcr assays (homberger et al., ; kunita et al., ; yamada et al., ; besselsen et al., ) . because of the transient nature of mhv infection in immunocompetent mice, serology is the most appropriate diagnostic tool for routine monitoring. enzyme-linked immunosorbent assay and ifa are well established and sensitive, and all known mhv strains cross-react in both tests (smith, ; aclad, ) . the magnitude of antibody response depends on mhv strain and mouse genotype (nakanaga et al., ; barthold and smith, ) . dba/ mice are poor antibody responders whereas c bl/ mice produce a high antibody titre and are therefore good sentinels. antibody titres remain high over a period of at least months (barthold and smith, b; . infected mice may not develop detectable antibodies for up to days after initial exposure (smith, ). in such cases, a direct diagnostic method as discussed above may be useful. another drawback of serology is that mice weaned from immune dams can have maternal antibodies until they are weeks of age (homberger, ) . this may impact serological monitoring because the possibility must be considered that low positive results are due to maternally-derived passive immunity. because the virus can be transmitted by transplantable tumours and other biological materials from mice, including hybridomas (holmes et al., ) and embryonic stem cells (okumura et al., ; kyuwa, ) , these materials should also be routinely screened for mhv contamination. mouse inoculation bioassay, map test, and rt-pcr can be used for this purpose. the best means of mhv control is to prevent its entry into a facility. this can be accomplished by purchase of mice from virus-free sources and maintenance under effective barrier conditions monitored by a welldesigned quality assurance programme. control of wild mouse populations, proper husbandry and sanitation, and strict monitoring of biological materials that may harbour virus are also important measures to prevent infection. if infection occurs, the most effective elimination strategy is to cull the affected colony and obtain clean replacement stock. however, this is not always a feasible option when working with valuable mice (e.g. genetically modified lines, breeding stocks). caesarean derivation or embryo transfer can be used to produce virus-free offspring, and foster-nursing also has been reported to be effective (lipman et al., ) . quarantine of an affected colony with no breeding and no introduction of new animals for approximately months has been effective in immunocompetent mice (weir et al., ) . the infection is likely to be terminated because mhv requires a constant supply of susceptible animals. this method works best when working with small numbers of mice. large populations favour the development of new mhv strains that may result in repeated infections with slightly different strains (adami et al., ) . it may be practical to select a few future breeders from the infected population and quarantine them for approximately weeks (compton et al., ) . this can be achieved in isolators, or in individually ventilated cages if proper handling is guaranteed. after this interval, breeding can resume. the -week interval should permit recovery from active infection, and the additional -week gestation period effectively extends the total quarantine to weeks. it is advisible to select seropositive breeders because the possibility of active infection is lower in such animals. the breeding cessation strategy may not be successful if immunodeficient mice are used because they are susceptible to chronic infection and viral excretion (barthold et al., ) . genetically engineered mice of unclear, unknown or deficient immune status pose a special challenge because they may develop unusual manifestations of infection or may be unable to clear virus. rederivation likely is the most cost effective strategy in such situations. along with the measures described, proper sanitation and disinfection of caging and animal quarters as well as stringent personal sanitation are essential to eliminate infection. careful testing with sentinel mice should be applied to evaluate the effectiveness of rederivation. if transplantable tumours are contaminated with mhv, virus elimination can be achieved by passage of tumours in athymic whn rnu rats (rülicke et al., ) . murine hepatitis virus is one of the most important viral pathogens of laboratory mice and has been intensively studied from a number of research perspectives (e.g. as a model organism for studying coronavirus molecular biology or the pathogenesis of viral-induced demyelinating disease). numerous reports document the effects of natural and experimental infections with mhv on host physiology and research, especially in the fields of immunology and tumour biology (reviewed by barthold, ; national research council, ; compton et al., ; homberger, ; baker, ; nicklas et al., ) . murine pneumonia virus, commonly referred to as 'pneumonia virus of mice' (pvm), is an enveloped, singlestranded rna virus of the family paramyxoviridae, genus pneumovirus. it is closely related to human respiratory syncytial virus (hrsv). the virus name is officially abbreviated as 'mpv' according to the international union of microbiological societies ( ); however, the former designation 'pvm' will be used in this chapter to avoid confusion with the official abbreviation of mouse parvovirus (mpv). 'pneumonia virus of mice' infection is relatively common in colonies of mice and rats throughout the world. seropositivity to pvm was reported in less than % of spf mouse colonies and in approximately % of non-spf mouse colonies in the usa (jacoby and lindsey, ) . a serological survey in france demonstrated antibodies to pvm in % of mouse colonies examined (zenner and regnault, ) . in a more recent study in north america, such antibodies were found in only . % of mice monitored (livingston and riley, ) . antibodies to pvm have also been detected in hamsters, gerbils, cotton rats, guinea pigs, and rabbits (parker and richter, ; richter, ; national research council, ) . experimentally, pvm infection of mice is used as a model for hrsv infection (domachowske et al., ) . in immunocompetent mice, natural infection with pvm is transient and usually not associated with clinical disease or pathological findings (parker and richter, ; national research council, ; brownstein, b) . however, natural disease and persistent infection may occur in immunodeficient mice (carthew and sparrow, ; richter et al., ; weir et al., ) . in particular, athymic foxn nu mice seem to be susceptible to pvm infection, which can result in dyspnoea, cyanosis, emaciation, and death due to pneumonia (richter et al., ; weir et al., ) . similar clinical signs have been reported for experimentally infected, immunocompetent mice (cook et al., ) . necropsy findings in naturally infected foxn nu mice include cachexia and diffuse pulmonary oedema or lobar consolidation (weir et al., ) . pulmonary consolidation (dark red or grey in colour) also has been found after experimental infection of immunocompetent mice (brownstein, b) . histologically, natural infection of foxn nu mice with pvm presents as interstitial pneumonia (richter et al., ; weir et al., ) . experimental intranasal inoculation of immunocompetent mice can result in rhinitis, erosive bronchiolitis, and interstitial pneumonia with prominent early pulmonary eosinophilia and neutrophilia (brownstein, b; domachowske et al., ) . hydrocephalus may result from intracerebral inoculation of neonatal mice (lagace-simard et al., ) . susceptibility to infection is influenced by age of mouse, dose of virus, and a variety of local and systemic stressors (parker and richter, ; national research council, ) . pneumonia virus of mice is labile in the environment and rapidly inactivated at room temperature (parker and richter, ; national research council, ) . the virus is tropic for the respiratory epithelium (carthew and sparrow, ; cook et al., ) , and transmission is exclusively horizontal via the respiratory tract, mainly by direct contact and aerosol (parker and richter, ; national research council, ) . therefore, transmissibility in mouse colonies is low, and infections tend to be focal enzootics. serology (elisa, ifa, or hi) is the primary means of testing mouse colonies for exposure to pvm. immunohistochemistry has been applied to detect viral antigen in lung sections (carthew and sparrow, ; weir et al., ) , however, proper sampling (see chapter on health monitoring) is critical for establishing the diagnosis due to the focal nature of the infection. an rt-pcr assay to detect viral rna in respiratory tract tissues has also been reported . however, the use of direct methods requires good timing because the virus is present for only up to about days in immunocompetent mice (brownstein, b) . embryo transfer or caesarean derivation followed by barrier maintenance can be used to rear mice that are free of pvm. because active infection is present in the individual immunocompetent mouse for only a short period, strict isolation of a few (preferably seropositive) mice with the temporary cessation of breeding might also be successful in eliminating the virus (richter, ; national research council, ) . pneumonia virus of mice could interfere with studies involving the respiratory tract or immunological measurements in mice. in addition, pvm can have devastating effects on research using immunodeficient mice because they are particularly prone to develop fatal disease (richter et al., ; weir et al., ) or become more susceptible to the deleterious effects of other agents such as pneumocystis carinii (roths et al., ) . murine rotavirus-a/edim (commonly referred to as 'mouse rotavirus' or 'epizootic diarrhoea of infant mice virus') is a nonenveloped, segmented double-stranded rna virus of the family reoviridae, genus rotavirus. it is antigenically classified as a group a rotavirus, similar to rotaviruses of many other species that cause neonatal and infantile gastroenteritis (fenner et al., ) . murine rotavirus-a/edim infection remains prevalent in contemporary mouse colonies and appears to occur worldwide. seropositivity to murv-a/edim was reported in approximately % of spf colonies and in almost % of non-spf mouse colonies in the usa in (jacoby and lindsey, ) . more recently, livingston and riley ( ) found a low rate ( %) of mouse sera to be positive for antibodies against murv-a/edim. experimentally, murv-a/edim infection in mice is used as a model for human rotavirus infection, especially in investigations on the mechanisms of rotavirus immunity and in the development of vaccination strategies (ward and mcneal, ) . clinical symptoms following murv-a/edim infection range from inapparent or mild to severe, sometimes fatal, diarrhoea. 'epizootic diarrhoea of infant mice' describes the clinical syndrome associated with natural or experimental infection by murv-a/edim during the first weeks of life (kraft, ; sheridan and vonderfecht, ; national research council, ; barthold, b; percy and barthold, ) . diarrhoea usually begins around h after infection and persists for about week. affected suckling mice have soft, yellow faeces that wet and stain the perianal region. in severe instances, the mice may be stunted, have dry scaly skin, or are virtually covered with faecal material. morbidity is very high but mortality is usually low. gross lesions in affected mice are confined to the intestinal tract. the caecum and colon may be distended with gas and watery to paste-like contents that are frequently bright yellow. the stomach of diarrheic mice is almost always filled with milk, and this feature has been reported to be a reliable means to differentiate diarrhoea caused by rotavirus from the diarrhoea caused by mhv infection. histopathological changes may be subtle even in animals with significant diarrhoea. they are confined to the small intestine and are most prominent at the apices of villi, where rotaviruses infect and replicate within epithelial cells. hydropic change of villous epithelial cells is the hallmark finding of acute disease. the villi become shortened, and the cells that initially replace the damaged cells are less differentiated, typically cuboidal instead of columnar, and lack a full complement of enzymes for digestion and absorption, resulting in diarrhoea due to maldigestion and malabsorption. undigested milk in the small intestine promotes bacterial growth and exerts an osmotic effect, exacerbating damage to the villi. intestinal fluid and electrolyte secretion is further enhanced by activation of the enteric nervous system (lundgren et al., ) and through the effects of a viral enterotoxin called nsp (for nonstructural protein ; ball et al., ) . it is hypothesized that nsp is released from virusinfected cells and then triggers a signal transduction pathway that alters epithelial cell permeability and chloride secretion. susceptibility to edim depends on the age of the host and peaks between and days of age (kraft, ; sheridan and vonderfecht, ; national research council, ; barthold, b; percy and barthold, ) . mice older than about weeks can still be infected with murv-a/edim, but small numbers of enterocytes become infected, there is little replication of virus, and diarrhoea does not occur. the exact reason for this age-related resistance to disease is unknown. pups suckling immune dams are protected against edim during their period of disease susceptibility (rosé et al., ) . in general, the infection is selflimiting and resolves within days. successful viral clearance is promoted by an intact immune response (feng et al., ; mcneal et al., ; rosé et al., ) , and some immunodeficient mice (e.g. prkdc scid and rag tm fwa mice) may shed virus for extended periods or become persistently infected (riepenhoff-talty et al., ; franco and greenberg, ) . protection against murv-a/edim reinfection is primarily mediated by antibodies (feng et al., ; rosé et al., ) . murine rotavirus-a/edim is highly contagious and transmitted by the faecal-oral route (kraft, ; sheridan and vonderfecht, ; national research council, ) . dissemination of the virus occurs through direct contact or contaminated fomites and aerosols. murv-a/edim is stable at Ϫ Њc but otherwise tends to be susceptible to extreme environmental conditions, detergents, and disinfectants. enzyme-linked immunosorbent assay and ifa are in widespread use for detection of serum antibodies to murv-a/edim in diagnostic and health surveillance programmes; other assay systems such as those using latex agglutination are also utilized (ferner et al., ) . rotazyme ii is a commercially available elisa for detection of rotavirus antigen in faeces; however, great care must be used in interpreting the results because some feeds have been reported to cause false positive reactions (jure et al., ) . electron microscopy of faeces of diarrheic pups should reveal typical wheelshaped rotavirus particles, - nm in diameter. reverse transcriptase-polymerase chain reaction also can be used to detect rotavirus rna in faecal samples (wilde et al., ) . good timing is critical for establishing the diagnosis from faeces because virus is shed for only a few days in immunocompetent mice. embryo transfer or caesarean derivation followed by barrier maintenance is recommended for rederivation of breeding stocks (kraft, ; national research council, ) . in immunocompetent mice in which infection is effectively cleared, a breeding suspension strategy combined with excellent sanitation, filter tops, and conscientious serological testing of offspring may also be effective. murine rotavirus-a/edim has the potential to interfere with any research utilizing suckling mice. it may have a significant impact on studies where the intestinal tract of neonatal or infant mice is the target organ. the infection also poses a problem for infectious disease and immune response studies, particularly those involving enteropathogens in infant mice (newsome and coney, ) . in addition, runting could be interpreted erroneously as the effect of genetic manipulation or other experimental manipulation. sendai virus (sev) is an enveloped, single-stranded rna virus of the family paramyxoviridae, genus respirovirus. it is antigenically related to human parainfluenza virus . the virus was named for sendai, japan, where it was first isolated from mice. infections of mice and rats are relatively common and occur worldwide. in addition, there is evidence that hamsters, guinea pigs, and rabbits are susceptible to infection with sev (machii et al., ; aclad, ; national research council, ; percy and palmer, ) ; however, some apparently seropositive guinea pigs may in fact be seropositive to other parainfluenza viruses instead of sev. seropositivity to sev was reported to be absent from spf mouse colonies and to be approximately % in non-spf mouse colonies in the usa (jacoby and lindsey, ) . a study in france reported antibodies to sev in % of mouse colonies examined (zenner and regnault, ) . a low rate of seropositive mice ( . %) was found in a recent survey in north america (livingston and riley, ) . furthermore, sev can contaminate biological materials (collins and parker, ) . sendai virus is pneumotropic and the leading cause of viral respiratory disease in mice. the pneumotropism is partially a consequence of the action of respiratory serine proteases such as tryptase clara, which activate viral infectivity by specific cleavage of the viral fusion glycoprotein (tashiro et al., ) . in addition, the apical budding behaviour of sev may hinder the spread of virus into subepithelial tissues and subsequently to distant organs via the blood. two epidemiologic patterns of sev infection have been recognized, an enzootic (subclinical) and epizootic (clinically apparent) type (parker and richter, ; national research council, ; brownstein, a) . enzootic infections commonly occur in breeding or open colonies, where the constant supply of susceptible animals perpetuates the infection. in breeding colonies, mice are infected shortly after weaning as maternal antibody levels wane. normally, the infection is subclinical, with virus persisting for approximately weeks, accompanied by seroconversion that persists for a year or longer. epizootic infections occur upon first introduction of the virus to a colony and either die out (self-cure) after - months or become enzootic depending on colony conditions. the epizootic form is generally acute, and morbidity is very high resulting in nearly all susceptible animals becoming infected within a short time. clinical signs vary and include rough hair coat, hunched posture, chattering, respiratory distress, prolonged gestation, death of neonates and sucklings, and runting in young mice. breeding colonies may return to normal productivity in months and thereafter maintain the enzootic pattern of infection. factors such as strain susceptibility, age, husbandry, transport, and copathogens are important in precipitating overt disease. dba and /j strains of mice are very susceptible to sev pneumonia whereas sjl/j and c bl/ /j strains and several outbred stocks are relatively resistant. a/j, balb/c, and swr/j are among the strains that show intermediate susceptibility. there is no evidence for persistent infection in immunocompetent mice, but persistent or prolonged infection may occur in immunodeficient mice and can result in wasting and death due to progressive pneumonia (ward et al., ; iwai et al., ; percy et al., ) . clearance of a primary sev infection is mediated by cd ϩ and cd ϩ t cell mechanisms (kast et al., ; hou et al., ) . heavier than normal, consolidated, plum-coloured or grey lungs are a characteristic gross finding in severe sev pneumonia (parker and richter, ; national research council, ; brownstein, a; percy and barthold, ) . lymphadenopathy and splenomegaly reflect the vigorous immune response to infection. histologically, three phases of disease can be recognized in susceptible immunocompetent mice: acute, reparative, and resolution phases (brownstein, a; percy and barthold, ) . lesions of the acute phase, which lasts - days, are primarily attributed to the cell-mediated immune response that destroys infected respiratory epithelial cells and include necrotizing rhinitis, tracheitis, bronch(iol)itis, and alveolitis. epithelial syncytiae and cytoplasmic inclusion bodies in infected cells may be seen early in this phase. alveoli contain sloughed necrotic epithelium, fibrin, neutrophils, and mononuclear cells. atelectasis, bronchiectasis, and emphysema may occur as a result of damage and obstruction of airways. the reparative phase, which may overlap the acute phase but continues through about the third week post infection, is indicated by regeneration of airway lining epithelium. adenomatous hyperplasia and squamous metaplasia (with multilayered flat epithelial cells instead of normal columnar cells) in the terminal bronchioles and alveoli are considered to be a hallmark of sev pneumonia. mixed inflammatory cell infiltrates in this phase tend to be primarily interstitial rather than alveolar as they are in the acute phase. the resolution phase may be complete by the fourth week post infection and lesions may be difficult to identify subsequently. residual, persistent lesions that may occur include organizing alveolitis and bronchiolitis fibrosa obliterans. alveoli and bronchioles are replaced by collagen and fibroblasts, foamy macrophages, and lymphoid infiltrates, often with foci of emphysema, cholesterol crystals, and other debris, which represent attempts to organize and wall off residual necrotic debris and fibrin. lesions are more severe and variable when additional pathogens such as mycoplasma pulmonis are present (national research council, ) . otitis media has also been reported in natural infections with sev although some of these studies have been complicated by the presence of other pathogens (ward, ) . sendai virus has been detected in the inner ear after experimental intracerebral inoculation of neonatal mice (shimokata et al., ) . sendai virus is extremely contagious. infectious virus is shed during the first weeks of infection and appears to be transmitted by direct contact, contaminated fomites, and respiratory aerosol (parker and reynolds, ; parker and richter, ; national research council, ) . serology (elisa, ifa, or hi) is the approach of choice for routine monitoring because serum antibodies to sev are detectable soon after infection and persist at high levels for many months, although active infection lasts only - weeks in immunocompetent mice. the short period of active infection limits the utility of direct methods such as immunohistochemistry (carthew and sparrow, ) and rt-pcr (hayase et al., ; wagner et al., ) . although sev is considered to be highly contagious, studies have shown that dirty bedding sentinel systems do not reliably detect the infection and that outbred stocks may not seroconvert consistently (dillehay et al., ; artwohl et al., ) . mouse antibody production test and rt-pcr can be used to detect sev in contaminated biological materials. sendai virus infection in mouse colonies has proven to be one of the most difficult virus infections to control because the virus is highly infectious and easily disseminated. depopulation of infected colonies probably is the most appropriate means to eliminate the virus in most situations. embryo transfer followed by barrier maintenance has also been used successfully in eliminating the virus (national research council, ) . a less effective alternative is to place the infected animals under strict quarantine, remove all young and pregnant mice, suspend all breeding, and prevent addition of other susceptible animals for approximately months until the infection is extinguished and then breeding and other normal acitivities are resumed (parker and richter, ; national research council, ) . vaccines against the virus have been developed (brownstein, ; national research council, ) , but these probably do not represent a practical means to achieve or maintain the seronegative status of colonies that is in demand today. sendai virus has the potential to interfere with a wide variety of research involving mice. reported effects include interference with early embryonic development and foetal growth; alterations of macrophage, nk cell, and t and b cell function; altered responses to transplantable tumours and respiratory carcinogens; altered isograft rejection; and delayed wound healing (reviewed by national research council, ; baker, ; nicklas et al., ) . pulmonary changes during sev infection can compromise interpretation of experimentally induced lesions and may lead to opportunistic infections by other agents. they could also affect the response to anaesthetics. in addition, natural sev infection would interfere with studies using sev as a gene vector. theiler's murine encephalomyelitis virus (tmev) or murine poliovirus is a member of the genus cardiovirus in the family picornaviridae. members of this genus are nonenveloped viruses with singlestranded rna. the virus is rapidly destroyed at temperatures above Њc. it is considered to be a primary pathogen of the cns of mice and can cause clinical disease resembling that due to poliomyelitis virus infections in humans. antibodies to tmev have been identified in mouse colonies and feral populations worldwide, and mus musculus is considered to be the natural host of tmev (lipton et al., ) . the most well-known and most frequently mentioned tmev strain is gdvii, which is virulent for mice. infant or young hamsters and laboratory rats are also susceptible to intracerebral infection. the original isolate is designated to (theiler's original) and represents a group of tmev strains with low virulence for mice. many additional virus strains have been isolated and studied, and they all fall in the broad grouping of to and gdvii. a similar virus strain has also been isolated from rats, but in contrast to mouse isolates this virus is not pathogenic for rats and mice after intracerebral inoculation (hemelt et al., ) . recently, another rat isolate has been characterized and shown to be most closely related to but quite distinct from other tmev viruses (ohsawa et al., ) . antibodies to tmev (strain gdvii) have been detected in guinea pigs and are considered to indicate infection with another closely related cardiovirus (hansen et al., ) . seropositivity to tmev was reported in approximately % of spf mouse colonies and approximately % of non-spf mouse colonies in the usa (jacoby and lindsey, ) . zenner and regnault ( ) reported a prevalence rate of % in french mouse colonies in a retrospective study, and it has been one of the most common virus infections in rodent colonies. in a recent study, antibodies were found in . % of mice monitored (livingston and riley, ) indicating that tmev, like most viruses, has meanwhile been eliminated from the majority of mouse colonies. theiler's murine encephalomyelitis virus is primarily an enteric pathogen, and virus strains are enterotropic. in natural infections, virus can be detected in intestinal mucosa and faecal matter, and in some cases it is also found in the mesenteric lymph nodes. however, histological lesions in the intestine are not discerned. virus may be shed via intestinal contents for up to weeks, sometimes intermittently (brownstein et al., a) , and transmission under natural conditions is via the faecal-oral route by direct contact between mice as well as by indirect contact (e.g. dirty bedding). the host immune response limits virus spread, but it does not immediately terminate virus replication in the intestines. virus is cleared from extraneural tissues, but it persists in the cns for at least a year. clinical disease due to natural tmev infection is rare, with a rate of only in - , infected immunocompetent animals (percy and barthold, ) . in immunodeficient mice, especially in weanlings, clinical signs may be more common and mortality may be higher (rozengurt and sanchez, ) . this group of viruses usually causes asymptomatic infections of the intestinal tract. they may spread to the cns as a rare event where they cause different neurological disease manifestations. the most typical clinical sign of tmev infection is flaccid paralysis of hind legs. the animals appear otherwise healthy, and there is no mortality. experimental infection in mice provides models of poliomyelitis-like infection and virus-induced demyelinating disease including multiple sclerosis (mcgavern et al., ) . after experimental infection, tmev causes a biphasic disease in susceptible strains of mice. the acute phase is characterized by early infection of neurons in the grey matter. encephalomyelitis may develop during this phase and may be fatal, but most animals survive and enter the second phase of the disease at - months after the acute phase. this phase is characterized by viral persistence in the spinal cord white matter, mainly in macrophages, and leads to white matter demyelination. persistence and demyelination occur only in genetically susceptible mouse strains while resistant strains clear the infection after early grey matter encephalomyelitis through a cytotoxic t lymphocyte response. for this reason, the nude mutation (foxn nu ) confers susceptibility on mice with an otherwise resistant background. the severity and nature of disease depend on virus strain, route of inoculation, host genotype and age (downs, ; lipton and rozhon, ; national research council, ; percy and barthold, ) . in general, virus isolates with low virulence produce persistent cns infection in mice whereas virulent strains are unable to cause persistent infection. intracerebral inoculation results in the most severe infections, but the intranasal route is effective also. experimental intracerebral infections with virulent fa and gdvii strains of tmev are more likely to cause acute encephalomyelitis and death in weanling mice - days after inoculation ('early disease'). death may be preceded by neurological manifestations of encephalitis such as hyperexcitability, convulsions, tremors, circling and rolling, and weakness. animals may develop typical flaccid paralysis of hind limbs, and locomotion is possible only by use of the forelimbs. interestingly, the tail is not paralysed. experimental infections with low virulence virus strains (e.g. to, da, ww) are more likely to cause persistent infection with development of mild encephalomyelitis followed by a chronic demyelinating disease after a few months ('late disease'). these virus strains infect neurons in the grey matter of the brain and spinal cord during the acute phase of viral growth, followed by virus persistence in macrophages and glial cells in the spinal cord white matter. sjl, swr, and dba/ strains are most susceptible to this chronic demyelinating disease. cba and c h/he are less susceptible strains, and strains a, c bl/ , c bl/ , and dba/ are relatively resistant (lipton and dal canto, ) . differences in humoral immune responses play a role in resistance to tmev infection (pena rossi et al., a) , but genetic factors are also important. several genetic loci implicated in susceptibility to virus persistence, demyelination, or clinical disease have been identified, including the h- d region of the major histocompatibility complex (brahic and bureau, ) . furthermore, the age at infection influences the severity of clinical disease. in infant mice, intracerebral infection with low virulence virus strains (e.g. to) is often lethal. young mice develop paralysis after an incubation period of - weeks while adult mice often show no clinical signs of infection (downs, ) . the only gross lesions are secondary to the posterior paralysis and may include urine scald or dermatitis due to incontinence of urine and trauma to paralysed limbs, or wasting or atrophy of the hind limbs in long term survivors. theiler's murine encephalomyelitis virus infects neurons and glial cells, and histological changes in the cns include nonsuppurative meningitis, perivasculitis, and poliomyelitis with neuronolysis, neuronophagia, and microgliosis in the brainstem and ventral horns of the spinal cord (percy and barthold, ) . demyelination in immunocompetent mice is considered to be immune-mediated. susceptible strains develop a specific delayed-type hypersensitivity response which is the basis for inflammation and demyelination. this reaction is mediated by cytotoxic t lymphocytes (lindsley et al., ; pena rossi et al., b) and by the activation of cytokines as a consequence of infection of macrophages and other cells of the cns (rubio and capa, ; sierra and rubio, ; palma et al., ) . protection from chronic demyelinating disease is possible by vaccination with live virus given previously by subcutaneous or intraperitoneal inoculation (crane et al., ; kurtz et al., ) . early immunosuppression at the time of infection, e.g. by treatment with cyclophosphamide or antithymocyte serum, inhibits or diminishes demyelination. immunosuppression in mice chronically infected with tmev leads to remyelination of oligodendrocytes (rodriguez and lindsley, ) . further details related to the pathogenesis of tmev infections and the role of immune mechanisms have been reviewed by yamada et al. ( ) . experimental infection of foxn nu mice results in acute encephalitis and demyelination. demyelination associated with minimal inflammation and neurological signs including the typical hind limb paresis develop weeks after inoculation, and most animals die within weeks. in foxn nu mice, demyelination is caused by a direct lytic effect of the virus on oligodendrocytes (rosenthal et al., ) . demyelination and lethality are reduced after administration of neutralizing antibodies (fujinami et al., ) . histopathological changes in prkdc scid mice are very similar to those in foxn nu mice (rozengurt and sanchez, ) . young mice born in infected populations usually acquire infection shortly after weaning and are almost all infected by days of age. intrauterine transmission to foetuses is possible during the early gestation period, but a placental barrier develops during gestation and later prevents intrauterine infection (miyamae, ; abzug et al., ) . all tmev isolates are closely related antigenically and form a single serogroup as determined by complement fixation and hi (lipton and rozhon, ) . hemelt et al. ( ) demonstrated cross reactions among four strains used in experimental infections, but differences were evident in homologous and heterologous titres. the viral strain most commonly used as antigen for serological testing is gdvii. this strain agglutinates human type erythrocytes at Њc, and hi has been the standard test for routine screening of mouse populations. meanwhile, hi has been replaced by elisa or ifa, both of which are more sensitive and specific. virus isolation is possible from brains or spinal cords of mice with clinical disease or from the intestinal contents of asymptomatic mice. pcr techniques also are available to test for virus-specific nucleotide sequences in biological samples (trottier et al., ) . mice that have been shown to be free from tmev by serological testing can be selected for breeding populations. if the virus is introduced into a mouse population, depopulation of infected colonies may be the most appropriate means to eliminate tmev. embryo transfer or caesarean derivation are the methods of choice for eliminating virus from valuable breeding populations. foster-nursing has been reported to be effective in generating virus-free offspring (lipman et al., ) although transplacental transmission has been demonstrated with experimental infection early in gestation. lesions of demyelination in cns of mice with clinically inapparent chronic infection may interfere with investigations that require evaluation of the cns (krinke and zurbriggen, ) . conceivably, such lesions also could affect neuromuscular responses or coordination, and affect neurological and behavioural evaluations. viral and mycoplasmal infections of laboratory rodents: effects on biomedical research monographs on pathology of laboratory animals: digestive system monographs on pathology of laboratory animals: digestive system viral and mycoplasmal infections of laboratory rodents: effects on biomedical research the mouse in biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research monographs on pathology of laboratory animals: respiratory system monographs on pathology of laboratory animals: respiratory system biosafety in microbiological and biomedical laboratories (bmbl), th edn. u.s. department of health and human services proc. natl. acad. sci. usa the mouse in biomedical research the mouse in biomedical research the mouse in biomedical research veterinary virology complications of viral and mycoplasmal infections in rodents to toxicology research and testing viral and mycoplasmal infections of laboratory rodents: effects on biomedical research virus taxonomy. seventh report of the international committee on taxonomy of viruses the mouse in biomedical research the mouse in biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research proc. natl. acad. sci. usa the importance of laboratory animal genetics, health, and the environment in biomedical research proc. natl. acad. sci. usa national research council, committee on infectious diseases of mice and rats manual of microbiologic monitoring of laboratory animals the mouse in biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research the mouse in biomedical research the mouse in biomedical research pathology of laboratory rodents and rabbits proc. natl. acad. sci. usa manual of microbiologic monitoring of laboratory animals viral and mycoplasmal infections of laboratory rodents: effects on biomedical research lactic dehydrogenase virus viral and mycoplasmal infections of laboratory rodents: effects on biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research viral and mycoplasma infections of laboratory rodents: effects on biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research handbook of animal models of infection the mouse in biomedical research key: cord- -tcltmhi authors: barthold, stephen w. title: mouse hepatitis virus biology and epizootiology date: - - journal: viral and mycoplasmal of laboratory rodents doi: . /b - - - - . - sha: doc_id: cord_uid: tcltmhi nan mouse hepatitis virus (mhv) is a highly contagious and ubiquitous coronavirus of laboratory mice. it is present among mice in many commercial breeding colonies and most biomédical research institutions and has been documented in laboratory mice throughout the world. recognition of the ubiquity of mhv is often not realized because of unfamiliarity with its biology, reliance on insensitive diagnostic methods and the subtle effects of the virus. its subtle effects should not be underestimated, however, since mhv has been associated with a panoply of effects on mice and research data derived from them. considering the large financial investment in biomédical research and that mice represent the majority of animals used in such research, the potential scientific and economic impact of this single virus is enormous. since the original isolation of mhv in ( ), a body of literature representing nearly citations has been developed on the subject of mhv. the voluminous literature on mhv would not have been generated if mhv were simply a pathogen of mice. interest in mhv has evolved from a number of different perspectives, including emphasis on mhv as a model of viral hepatitis, viral encephalitis and demyelinating disease, genetic mechanisms of host resistance to viral infection, and the molecular biology of coronaviruses, using mhv as a prototype. these have been the subjects of several reviews ( - ). on the one hand, the literature clearly illustrates the complexity of mhv pathogenesis. virus strain, passage history, dose, route of inoculation, host genotype, age, immune function and co-infection with other agents all interact to determine the ultimate expression of mhv disease. on the other hand, this complexity interferes with a clear view of the biology of mhv as a natural pathogen of mice. many of these studies have emphasized different aspects of mhv-host interactions, using artificial routes of inoculation and analysis of only one or a few target organs or effects. as a result, still relatively little is known about the natural pathobiology of mhv in mice. without such information, the true significance of this agent cannot be assessed and its effect under diverse experimental conditions cannot be predicted. this review will discuss recent updates in mhv pathobiology and incorporate past literature in that discussion wherever possible. coronaviruses, including mhv, are enveloped viruses containing single-stranded, non-segmented, infectious rna with positive (messenger) polarity. the molecular biology of mhv will be discussed in dr. holmes' chapter and has been reviewed elsewhere ( , ). physical resistance properties have also been reviewed ( ). coronaviruses infect many species of birds and mammals, causing respiratory or enteric infections in their hosts (table ). the relationship among viruses of this family is complex. they do not share a common group antigen, yet many are antigenically and genetically related ( , , ). although "mhv" is a singular term, it refers to many named and unnamed strains of mouse coronavirus (table ) . certain prototype strains have served as standards for study of the mhv group, including mhv-l,- ,- ,-a ,-jhm and -s. these strains have been used in most experimental studies and as antigenic or genetic standards for mhv serodiagnosis and comparison to new mhv all mhv strains or isolates share cross-reacting antigens, but each strain also possesses strain-specific antigenicity, with considerable overlap ( , - )· similarly, while there is rna and polypeptide homology between mhv strains, there is also strain-related heterogeneity ( , ). this suggests ongoing "drift" or mutation as a factor in the biology of mhv. relatedness of one mhv strain to another is not a useful means of predicting biological behavior. for example, mhv-s and mhv-jhm are relatively unrelated genetically, yet induce similar patterns of disease. related strains such as mhv-s and mhv-s/cdc produce different patterns of disease ( , - ). furthermore, the behavior of a defined or named mhv strain depends on its passage history and thus varies from laboratory to laboratory. mhv also cross-reacts antigenically with coronaviruses of the rat and some strains of coronavirus of other species, including man ( , - ). the biological significance of these relationships has not been thoroughly explored. mhv produces disease in mice by lytic infection of cells, although viral replication and exocytosis can take place in the absence of cytolysis. in addition to cytolysis, cell fusion is a hallmark of mhv, both in vivo and in vitro, although it is not necessary for viral replication ( , in contrast to respiratory mhv strains, very little is known about host resistance factors to enteric mhv. most of the mhv strains listed in table have not been characterized as to their primary pattern of infection, but it is clear that both respiratory and enteric strains exist in contempory mouse populations ( ). selective organotropisra is often stressed as an mhv strain-related phenomenon, such as neurotropism of mhv-jhm ( ), hepatotropism of mhv-a ( ), lymphocytotropism and bone marrow tropism of mhv- ( , ). such differences are only relative, with considerable overlap between mhv strains ( ). many of these differences are simply a reflection of the emphasis an author places on a particular target organ. nevertheless, recent work is suggesting that selective cell tropism can take place with specific mhv strains and that host cell type can influence the outcome of mhv infection. in primary central nervous system cell cultures, mhv strains exhibit different cell tropism, cytopathic effects and viral assembly, which may account for different patterns of disease ( , - ). understanding the pathogenesis of mhv is best approached by catagorizing infections into the respiratory and enteric patterns, which are determined by virus strain. relative differences in órgano-and cell tropism exist between virus strains in each of these catagories. furthermore, host factors play a significant role in severity of infection within these basic themes. the following discussion will detail what is known about the pathogenesis of respiratory and enteric patterns of mhv infection with supporting references for each pattern. these mhv strains rely on upper respiratory mucosa as a site of primary replication and excretion. in weanling or adult outbred mice infected with low-virulence mhv (mhv-s), infection is largely restricted to upper respiratory mucosa ( , since brain is often infected with several strains of mhv in susceptible hosts, neurotropism is not a particularly unique feature of any single mhv strain. however, some mhv strains (s and jhm) seem to possess the relatively unique ability to extend directly from the nasal lining into the olfactory tracts of the brain, even in older, resistant mice ( - , , ). within hours after intranasal inoculation, mhv-jhm causes meningitis and necrosis of mitral cells in the olfactory bulbs, with extension into the olfactory tracts, meninges, piriform cortex, septum pelucidum, anterior commissure, and hippocampus within hours. during this phase, both neurons and glia are infected. at hours, virus is present in the pons, medulla and spinal cord and by hours, severe demyelination is present. at this time, titers are diminishing ( ). a similar course of events probably occurs with mhv-s ( , ). the demyelinating component has been shown to be due to selective destruction of oligodendroglia ( - ) and reportedly does not seem to occur with other encephalitogenic strains of mhv, such as mhv- ( , , ). virus strain tropism for different nervous system cell types in vitro correlates with disease phenotype ( - ). the pattern of nervous system disease that occurs is also dependent on host age, genotype as well as dose and route of virus inoculation ( , - ). duration of mhv infection is a subject of considerable contention. the behavior of the virus under natural conditions has created the impression that infections are chronic and latent, since many experimental manipulations such as immunosuppression, tumor transplantation or co-infection with other agents will precipitate acute disease ( ). it appears, however, that this impression is created by the generally subclinical (but not latent) nature of mhv infection and that experimental manipulations that exacerbate disease are coincidentally applied during active infection. an overwhelming body of literature with a number of mhv strains clearly supports the concept of short-term infection, from which the host fully recovers within - weeks of infection ( , , , , , - ). aggressive immunosuppression after the acute phase of mhv infection will not reactivate the virus, even when residual mhv antigen and lesions remain in the brain ( , ). a smaller number of reports exist, however, that uphold the hypothesis of persistence. mice of the semisusceptible c h genotype that survive intraperitoneal inoculation of virulent mhv- can have persistent infections, in which virus can be recovered in low titer from liver, brain and lymphoid organs for up to days ( ). chronic brain infections have also been established in mice following intracerebral inoculation of mhv- , intracerebral or intranasal inoculation of brainpassaged mhv-jhm and selected temperature sensitive jhm mutants ( , - ). however, others have shown neurotropic mhv is eventually cleared from the brain ( , , , , ). these studies suggest that mhv can potentially cause chronic persistent infections, particularly under artificial experimental conditions, but limited infections, without a chronic carrier state, are the norm. an obvious exception is the athymic nude mouse, which suffers from a wasting syndrome due to chronic, persistent mhv infections. in contrast to their heterozygous euthymic counterparts, which recover within - weeks, homozygous athymic mice develop increasingly high titers of virus in multiple organs, ultimately dying from hepatitis or encephalitis. during the early acute phase of infection, euthymic and athymic mice have parallel titers and distribution of virus ( , , , ). persistent mhv infections incontestably take place in vitro in cultured cells in the absence of immune surveillance in the live mouse. this has been demonstrated repeatedly with a variety of mouse and rat cell types ( , [ ] [ ] [ ] [ ] [ ] [ ] . the addition of a low concentration of antiviral antibody to the culture medium was found to modulate infection, producing a carrier state with intracytoplasmic antigen, but no virus production or expression of viral antigens on the cell surface. the carrier state continued in the absence of antibody, and virus could not be rescued by co-cultivation with susceptible cells. virus could only be recovered following fusion of persistently infected cells to permissive cells with polyethylene glycol ( , ). the significance of these observations for the mouse remain to be determined. host-related factors in mhv pathogenesis have been elucidated using several respiratory prototype mhv strains. mice less than - weeks of age are susceptible, regardless of genotype ( , , - ). other workers have suggested that resistance to intracerebral jhm infection further evolves at - weeks of age ( , mhv-jhm productively infects both neuronal and non-neuronal cells in vitro, while laboratory-created temperature-sensitive mutants of jhm cause non-productive infection of non-neuronal cells only, with expression of antigen without virus replication. on the other hand, mhv-a selectively infects non-neuronal cells in a productive fashion ( ). differences among various mhv strains have been observed in their ability to productively infect adherent cells (macrophages) from liver expiant cultures ( ). intrinsic susceptibility of cells in vitro can be modified with lymphokines ( , ) and interferon ( , , ). lymphoreticular function in the whole animal also influences the course of mhv infection. agents that interfere with or stimulate macrophage activity alter the course of mhv infections ( , , , - ). interferon (if) plays a disputed role in mhv pathogenesis. serum if levels are low in mhv-infected immature mice and elevated in adults ( ), but others have found that resistant mouse genotypes may produce lower levels of if than susceptible genotypes in response to mhv infection ( , ). others have found the same degree of if levels ( ). tissue if levels have also been variably correlated with resistance ( , ). it is apparent that if plays a role in the initial response of mice to mhv ( ), and becomes elevated in adult, susceptible genotypes because of permissive infections, also resulting in increased natural killer (nk) cell activity in some mice. in resistant adults, virus titers, if and nk cells may all remain low ( ). immunosuppression by a variety of means, including x-irradiation, neonatal thymectomy, antilymphocyte serum, graft vs host reaction, cyclophosphamide and corticosteroid treatments, renders resistant mice susceptible to mhv. immunosuppression, however, does not modify intrinsic genetically determined resistance of cells cultured ±n_ vitro or abrogate a secondary immune response to virus challenge ( , , , , ). cell-mediated immunity is crucial in recovery from mhv infections. this fact is most evident in t cell deficient, athymic nude mice, in which mhv titers initially parallel their heterozygous euthymic counterparts, but continue to rise as hétérozygotes recover ( ) only a single study has demonstrated delayed-type hypersensitivity in mhv immunized mice ( ). it becomes apparent from this overview that mhv strains interact at different levels with mice of different ages and genotypes. expression of susceptibility or resistance is mediated first through the intrinsic ability of the target cell to support or restrict mhv replication. this can be further modified by if and nk cells, among other factors. secondly, infection is restricted by a specific immune response to the virus, resulting in susceptibility, attenuated disease or recovery. this is influenced by immunosuppressive regimens or impaired immune responsiveness. both intrinsic resistance and development of an effective immune response are age and genotype dependent ( , , , , , , , ). this is why susceptibility to mhv can be predicted in genotypes that allow high-titer virus replication early in infection, prior to mounting an immune response ( , , , ). it also explains the confusion over whether genetic susceptibility or resistance are inherited as one or two genes, recessive or dominant, h- haplotype linked or unlinked with different mhv strains, different routes of inoculation and different mouse genotypes ( , , , , , ). the course of mhv infection is notoriously sensitive to modification of lymphoreticular function. normally resistant mice are rendered susceptible by x-irradiâtion, cyclophosphamide, cortisone, neonatal thymectomy, antilymphocyte serum and graft vs host reaction, among other manipulations ( ). co-infection with other infectious agents also potentiates mhv disease. eperythrozoon coccoides and k virus are normally minimally pathogenic, but they exacerbate mhv in resistant hosts ( , ). leukoviruses and even schistosoma mansoni also enhance mhv disease. these diversely related infectious agents have been suggested to have a common mechanism by blocking if responsiveness of the host to mhv ( - ). several enterotropic strains of mhv have been identified. like other mhv strains, they can be differentiated antigenically, but also share antigenic and genetic homology with other members of the mhv group ( , , , - ). the upper respiratory mucosa is often a target of enterotropic mhv ( ), but intestinal mucosa is the major target for virus replication and excretion. unlike respiratory mhv strains that infect intestine only in highly susceptible hosts, enterotropic mhv universally infects intestine, regardless of host age. in neonatal mice, epithelial degeneration, necrosis and syncytia predominate, resulting in erosions, ulcération and villus attenuation. virusinduced syncytia, or "balloon cells," are diagnostic. intracytoplasmic inclusions have also been described. lesions can be fully developed within - hours ( ), but are usually most severe at three to five days ( , , ). in surviving mice or mice exposed at older ages, there tends to be less necrosis and more compensatory mucosal proliferation, or hyperplasia. adult mice are also susceptible, but usually have mild infections with only minor lesions ( , , , , , ). lesions can be found anywhere from pylorus to anus, but stomach is not involved. distal small intestine, cecum and ascending colon are preferential sites ( , , , , - ). dissemination beyond the intestine can also occur. some mhv strains are highly restricted to intestinal mucosa and mesenteric lymph nodes, while others spread to liver and other organs ( , , , , , ). mhv-d, like mhv-jhm and -s, appears to share the ability of infecting the olfactory bulbs of the brain ( ). these differences in behavior have not been carefully examined and may be either virus strain or host related. the influence of host genotype and immune response have not been well studied with enterotropic mhv. however, clinical symptoms and lesions associated with intestinal protozoal infestations are exacerbated by enterotropic mhv ( , ) and x-irradiation exacerbates enterotropic mhv infection. duration of intestinal mhv infections is limited, with no known carrier state beyond two weeks after exposure ( , , , , ). an exception is the athymic nude mouse, which develops persistent infections of the intestinal and nasal mucosa. enteric mucosa tends to be segmentally hyperplastic with syncytia. involvement of other organs is minimal or absent. these mice suffer from progressive emaciation, but lesions and distribution of lesions differ markedly from the typical wasting syndrome caused by respiratory mhv strains ( , , ). clinical disease is usually absent or mild and lesions are often restricted to the primary target organ (nose or intestine), except in highly susceptible hosts. disease and lesions are most apt to be seen in naive mouse populations experiencing an initial epizootic. suckling, genetically susceptible or immunologically compromised mice are the best candidates for diagnostic evaluation. suckling mice naturally infected with respiratory mhv suffer moderate to high mortality between - days of age. they may have signs of encephalitis, but more often signs are nonspecific. gross necropsy findings include multiple white spots on the liver ( , ). flaccid paralysis was described in adult swiss mice naturally infected with mhv-jhm ( ), but this is uncommon. a variety of experimental manipulations will exacerbate otherwise mild infections in resistant mice ( ). enterotropic mhv strains tend to produce explosive epizootics of diarrhea and high mortality among infants but these signs may be absent in enzootically-infected colonies. necropsy findings include enteritis, usually in the absence of liver lesions. surviviors are often runted and pot-bellied ( , , , - ) . histopathology is a useful means of confirming a diagnosis of mhv, but only in susceptible mice during the active phase of infection. in susceptible hosts, particularly neonates and athymic nude mice, mhv induces multinucleate syncytia in multiple tissues, which are pathognomonic for mhv. syncytia are especially obvious in enteric mucosa of mice infected with enterotropic mhv. histopathology details are given elsewhere ( , , ) . a more definitive diagnosis can be made with the use of immunohistochemical techniques such as immunofluorescence and immunoperoxidase. formalin-fixed, paraffin-embedded tissues can be ultilized for this purpose if subjected to prior trypsinization. antigen is very stable in formalin-fixed tissue, allowing retrospective analysis of archival cases ( , , , ) . electron microscopy can also be utilized. mhv has characteristic coronavirus morphology and buds into cisternae of endoplasmic reticulum. it can also produce intracytoplasmic aggregates of viral products ( , , ) . virus recovery from actively infected tissues is difficult but can be accomplished using a variety of effective cell lines, such as nctc , dbt, c - or l cells. these cell lines, however, may not be successful substrates for some enterotropic mhv strains. syncytium formation is the hallmark mhv cytopathic effect. identification of a suspect agent as an mhv strain can be accomplished by immunocytochemistry. reciprocal serum neutralization is used as a means of characterizing the antigenic relatedness of a new isolate to prototype mhv strains. however, because of extensive and complex antigenic inter-relationships, this method is not definitive for strain classification ( - , , ) . several serological assays can be used for detection of antibody in mhv-recovered mice. the complement fixation test was used extensively at one time, but is usually too insensitive to detect seroconversion following natural exposure ( , - ). as mentioned above, serum neutralization against prototype mhv strains grown ±n_ vitro can be used, but it tends to be highly strain-specific. the neutralizing profiles of sera from different mouse populations or from different mhv outbreaks within a population can be compared to determine if the same or unrelated mhv strains are involved ( - ). hemagglutination inhibition is used for detecting antibody to coronaviruses of other species, but only one mhv isolate (dvim) has been found to possess a hemagglutinin ( , ). two very sensitive and specific assays for mhv antibody are an indirect immunofluorescence assay (ifa), using mhv-infected cells and an enzyme-linked immunosorbent assay (elisa) ( - ). both offer the advantage of using multiple mhv strains simultaneously as antigens in order to cover a broad range of reactivity to mhv serotypes. under experimental conditions, these two tests are nearly equally sensitive and both are more sensitive than serum neutralization ( ). nude mice are not good candidates for serology. although they can produce neutralizing antibody to mhv, titers are widely variable and sometimes absent ( , ). morphological, immunohistochemical, virus recovery and serological methods can be embellished with the use of susceptible mhv hosts. for example, if mhv is suspected but cannot be confirmed in a mouse population, athymic nude mice can be placed in the animal room as sentinels. when these mice become infected, lesions and antigen are easily confirmed and virus is easier to isolate. infant mouse brain inoculation will amplify virus titers from suspect tissues, allowing easier antigen detection or virus recovery. similarly, inoculation of cortisonized mice will serve the same purpose. the mouse antibody production test can also be effectively utilized. inoculation of adult, virus antibody free mice with suspect material will result in seroconversion if mhv is present. under natural conditions, mhv is highly contagious and is transmitted by the respiratory or oro-fecal routes. expression of overt disease requires immature, immunocompromised or genetically susceptible mice. under experimental conditions, mice seroconvert by ifa or elisa between to days after inoculation ( ) . under natural conditions, seroconversion occurs in % of mice within two weeks after introduction to an mhv-infected population, underscoring the rapidity of exposure that takes place (unpublished observations). outbreaks of clinical disease among susceptible neonatal mice are also very rapid with high morbidity ( , ). as already discussed, infections last less than two weeks with seroconversion at the time of recovery ( , ). maintenance of infection in a mouse population (enzootic infection) requires continual exposure of new, susceptible mice, either through newly introduced mice or breeding populations. in the absence of susceptible mice, mhv will die out of a population. frequently, mhv-free mice are received into enzootically infected mouse rooms. if they are neonatal mice, they suffer high mortality ( ). however, extramural mice are usually weanlings or older, developing only subclinical infections upon exposure, but perpetuating the infectious cycle. use of these newly acquired exposed mice within the first one to two weeks after arrival creates a high potential for exacerbation of overt disease from subclinical infection when experimentally immunosuppressed or otherwise stressed. in enzootically infected populations, disease or lesions are most apt to be encountered in weanlings, rather than neonates, because neonates are protected by passive immunity. colostral igg from immune dams is partially protective under experimental conditions and seems to occur naturally ( , ). therefore, when mhv initially infects a naive mouse population, neonatal mice suffer high mortality, particularly with enterotropic mhv strains ( , ). once infection is enzootic, adult mice confer protection to neonates, so the infection cycle is perpetuated among older mice, when their passive immunity wanes. vertical transmission of mhv is possible, but unlikely in the short time span of an acute infection relative to the breeding life of a female mouse. transplacental transmission to fetuses has been shown in mice inoculated with mhv-jhm intravenously, but depended upon stage of gestation at the time of inoculation ( ) . neither transplacental transmission to fetuses nor vaginal transmission to newborns could be demonstrated in dams inoculated intraperitoneally with mhv- ( ) . thus, the potential is there, but it is not a likely consideration. one aspect of mhv epizootiology that is not well understood is whether repeated infections can occur with the same mhv strain or different mhv strains. challenge resistence to mhv has been accomplished by vaccination of mice with denatured virus or surface peplomers, but not with virus membrane or ribonucleoprotein subcomponents, suggesting that the most immunogenic components of the virion are virus-strain specific ( , ). the host range of mhv has not been defined. under natural conditions, rats and mice develop serum antibody to coronaviruses of the heterologous species but this is not surprising because of the antigenic cross-activity of rat and mouse coronaviruses ( , ). mice are experimentally susceptible to rat sialodacryoadenitits virus (sdav) ( ). suckling mice infected with sdav develop encephalitis, but not hepatitis or enteritis. weanling mice develop interstitial pneumonia, with antigen in alveolar lining cells (type pneumocytes). in contrast, mhv infects alveolar septal cells, but not lining cells in the mouse ( ). suckling rats are susceptible to mhv by intranasal inoculation. lesions are restricted to the nasal mucosa and antigen is present for only - days ( ) . intracereberal inoculation of rats with mhv-jhm causes encephalitis and demyelination ( ). Ιτ± vitro growth characteristics of viruses from these two species also differ. sdav will grow in primary rat kidney but not mouse cells and mhv will grow in mouse but not rat cells ( , ). sdav passaged through infant mouse brain remains unable to grow in mouse cells in vitro ( ) . it therefore appears that cross-species infections are laboratory, rather than natural, phenomena. the antigenic relatedness of mhv to coronaviruses of other species, including man, has unexplored biological significance. mhv has a long history of interference with research. most mhv strains were isolated and identified as inadvertant contaminants that were exacerbated by experimental protocol. subclinical mhv infections are potentiated by a variety of experimental manipulations that modify immune or observed ( , , - ) . the ubiquity of mhv among laboratory mice creates a high potential for contamination of other agents by mhv or confusion with mhv when passed in mice. examples are not hard to find. tettnang virus was an unclassified virus isolated from ticks and man in germany, egypt and czechoslovakia. the agent was unrelated to a variety of other tick-born arboviruses, and was eventually found to be mhv. retrospective study revealed that there was a relationship among other examples include isolation of mhv when human encephalitis brain biopsy material was passed in infant mice ( ), confusion over the human or mouse origin of coronaviruses isolated from multiple sclerosis material passed in mice or mouse tissue ( ) and confusion over avian or mouse origin of coronavirus isolated from manx shearwaters suffering from puffinosis ( ). mhv has a number of effects on immunologie and nonspecific host responses to antigens. following intraperitoneal injection of mhv- , mice had a modified immune response to sheep red blood cells. during acute infections, if mice were infected prior to antigen exposure, immunodepression occurred, while simultaneous injection with mhv and antigen resulted in immunostimulation. mice inoculated oronasally with mhv-jhm and an enterotropic mhv isolate display transient but significant functional disturbances in t and b lymphocyte populations, in mitogenesis assays with splenocytes from susceptible (balb/cbyj) but not resistant (sjl) genotypes ( ). it remains to be determined if these effects are due to direct or indirect action of the virus on lymphoid tissue. mhv certainly has the potential of direct action on lymphoid organs ( , ) . pre-infection of mice with mhv, either naturally or experimentally, alters the ability of normally permissive respiratory tract tissues to support replication of pneumonia virus of mice and also reduces susceptibility of dba/ j mice to sendai virus ( ). regardless of the mechanisms involved, these observations have broad implications for experimental virology and laboratory animal science, since mhv-infected mice respond abnormally to respiratory and possibly other viruses. mhv has been shown to activate nk cells and alter the if responsiveness of infected mice ( , ) . -recovered mice ( , , , ) . athymic nude mice infected with mhv develop a number of immunological peculiarities. responses to sheep red blood cells, including a secondary immune response ( , ) . in addition, they are able to reject tumor xenografts ( , ) . since mhv causes acute infections and because its contagiousness results in almost simultaneous infection of an entire population, it can be controlled like coronaviruses of the rat by simply breaking the infectious cycle ( ). cessation of breeding or quarantine without introduction of new mice will achieve this objective. selection of seropositive breeding stock for re-establishment of a breeding colony will ensure that mice are recovered from infection. judgement on the success of this approach must be made only under conditions that prevent re-infection of the population. prevention of infection is extremely difficult in facilities receiving mice from outside sources at various intervals. such conditions can allow introduction of subclinically infected animals that can infect resident stock or, conversely, perpetuation of the infectious cycle by introduction of naive mice following exposure to infected populations. furthermore, mhv is so highly contagious that it is very likely to re-contaminate mouse populations that have become rid of the agent by re-derivation, re-population or quarantine, particularly if identical husbandry practices prevail. the best means of mhv control is preventing its entry into a facility. this can be achieved by purchase of mice from mhv-free sources and shipment in filtered boxes. also, transplantable tumors and other mouse biological products must be mhv-free. since most institutions are unable to achieve this goal, mhv-free mice can be maintained in properly controlled barrier facilities, plastic film isolators or filtered cage systems. filter-top cages offer a relatively inexpensive means of preventing mhv infections, but handling and service of the cages must be strictly aseptic ( , , , ) . vaccination has been shown to effectively reduce severity of challenge mhv infection, but not totally prevent it ( , ). since vaccination immunity is produced against virus peplomeric antigens, it is likely to be strainspecific ( ). the number of antigenic mhv biotypes therefore makes vaccination impractical. furthermore, mhv usually causes subclinical infections, particularly when the virus is enzootic in a colony. it is likely that vaccination will cause greater effects on the host than natural infection· if exacerbation of mhv disease is a problem in an experimental protocol, newly arrived mice can be allowed to become infected and recover prior to use, or virus-free mice under appropriate preventive husbandry conditions can be used. a number of aspects of mhv biology are worthy of future research pursuit. rather than generating a list of such topics, the author has attempted to indicate throughout the text where deficiencies in knowledge exist. since scientific knowledge evolves best from a multi-disciplinary approach, it is hoped that scientists in various fields will seek ways to better understand this agent from their own perspectives. effect of corticosteroids on mouse hepatitis virus infection chronic central nervous system demyelination in mice after jhm infection immunopathology of mouse hepatitis virus type infection. iii. clinical and virologie observation of a persistent viral infection mouse hepatitis virus-induced recurrent demyelination. a preliminary report virus persistence and recurrent demyelination produced by a temperature-sensitive mutant of mouse hepatitis virus selected mutants of mouse hepatitis virus type (jhm strain) induce different cns diseases. pathobiology of disease induced by wild type and mutants ts and ts in balb/c and sjl/j mice hepatosplenic myelosis in the nude mouse naturally infected with mouse hepatitis virus persistent infection with mouse hepatitis virus of low virulence in nude mice persistent infection with mouse hepatitis virus in vivo and ±n_ vitro models of demyelinating diseases. ii. persistence and host-regulated thermosensitivity in cells of neural derivation infected with mouse hepatitis and measles viruses fusion resistance and decreased infectability as major host cell determinants of coronaviruses persistence enterotropic mouse hepatitis virus infection in nude mice mouse hepatitis immunofluorescence in formalin-or bouin'sfixed tissues using trypsin digestion complications of viral and mycoplasmal infections in rodents to toxicology research and testing peritoneal and macrophage alterations caused by naturally occurring mouse hepatitis virus the enzyme-linked immunosorbent assay for mouse hepatitis and rat coronaviruses enzyme-linked immunosorbent assay for detection of antibodies to murine hepatitis virus an immunofluoresence test for detection of serum antibody to rodent coronaviruses igm and igg response 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liver cells. am. j. gastroenterol. : - . . stohlman, s.a., sakaguchi, a.y., weiner, l.p. ( ) .characterization of the cold-sensitive murine hepatitis virus mutants rescued from latently infected cells by cell fusion. virol key: cord- - ht xm authors: kraft, lisbeth m. title: viral diseases of the digestive system date: - - journal: diseases doi: . /b - - - - . -x sha: doc_id: cord_uid: ht xm this chapter discusses three virus infections affecting the digestive system of mice and their properties: ( ) epizootic diarrhea of infant mice (edim), ( ) reovirus infection, and ( ) murine hepatitis virus infection (mhv). all three infections may cause serious, debilitating, and sometimes fatal diarrheal disease in nursling and weanling mice. mice of all ages can be infected by the edim virus but overt disease is restricted to animals up to about – days of age at the time of first exposure. the edim virus is worldwide in distribution. its prevalence is difficult to estimate because serologic tests have not been readily available, and it is not customary to sacrifice animals for the purpose of examining the appearance of their intestinal tract or for electron microscopic visualization of fecal contents. the acute disease of reovirus infection affects mainly sucklings and weanlings, whereas the chronic disease is encountered in animals over days of age. the mhv virus, on the other hand, has been found to affect cotton rats, rats, and hamsters. epidemic diarrhea of infant mice, it is common knowledge that virtually every colony of conventional mice suffered that infec tion to a greater or lesser extent. in recent years, with the advent of measures such as cesa rean derivation, barrier-sustained breeding and maintenance procedures, routine serologic surveillance, laminar flow hoods or rooms, and filter-top cages, the problems associated with these diseases have become somewhat less critical. neverthe less, they are at times and under certain circumstances still troublesome. all three infections may cause serious, debilitating, and sometimes fatal diarrheal disease in nursling and weanling mice. thus, the economic impact on commercial mouse col onies can be severe. furthermore, newborn mice, pooled from many dams and then redistributed to them at random, are used for the isolation and identification of certain viruses in diagnos tic and epidemiological studies, for example arboviruses (shope, ) , coronaviruses , and reoviruses (stanley, ) . clearly, indigenous infection with related or identical agents in only a few such infants could confound and compromise the validity of observations follow ing the inoculation of test materials. it should be pointed out, too, that collins and parker ( ) demonstrated both reovirus and murine hepatitis virus as contaminants in some murine leukemia and transplantable tumor specimens. whereas it is mainly from these standpoints that the diseases in question derive importance, they should not be dismissed without considering their intrinsic value as models for elucidat ing the pathogenesis and control of related infections in man and other animals as well as their utility for the molecular biologist. cheever and mueller ( , ) , pappenheimer and en ders ( ) , and pappenheimer and cheever ( ) were the first to describe the pathological changes and epidemiology of edim. runner and palm ( ) and cheever ( ) also con tributed to knowledge concerning the epidemiology and etiol ogy of the disease. thereafter kraft ( kraft ( , kraft ( , kraft ( , b kraft ( , reported on studies regarding the etiology, mode of transmission, carrier state, immune response, pathogenesis, and control of the disease. serologic studies were also under taken by blackwell et al ( ) . kraft ( , ) first demonstrated the agent in electron micrographs of infected infant mouse intestinal epithelium. banfield et al ( ) enlarged on those findings, comparing the virions to those of the reoviruses. particles of similar morphology were subsequently observed in the diarrheal feces of many species, primarily in the young: cattle (mebus et al, ) , man (flewett et al, a) , horse (flewett et al, ) , pig (rodger et al, ) , sheep , rabbit , deer (tzipori et al, ) , goat (scott et al, ) , and dog (england and poston, ) . in addition, one virus, sa- , was isolated from a nondiarrheal monkey, and another, oa (offal agent), was recovered from intestinal washings of sheep in an abattoir (els and lecatsas, ) . much and zajac ( ) purified edim virus from diarrheal infant mice and further characterized it. based on its moφhology and other known characters, edim virus has been placed into the genus rotavirus in the family reoviridae (matthews, ) . during the past decade, knowl edge of the rotaviruses as agents of diarrheal disease of young mammals has burgeoned, especially with regard to those af fecting children, calves, and piglets. reviews concerning the genus have been published by wyatt et al ( ), mcnulty ( , flewett and woode ( ) , andrewes et al ( ) , and holmes ( ) . additional comments may be found in the american veterinary medical association panel the young (anonymous, ) . the classification of edim virus in the genus rotavirus, family roeviridae, derives from its moφhology and mode of replication as seen in electron micrographs (adams and kraft, ; banfield et al, ) , which were later shown to re semble those observed in ultrathin sections in human intestinal material containing ''reovirus-like" particles (bishop et al, ) . the term rotavirus (l. rota, wheel) was proposed by flewett et al ( ) because of its moφhology as viewed in negative-contrast electron micrographs. it has become widely used in preference to duovirus, which is synonymous (david son et al, ) . there is no evidence that antigenic or pathogenetic variants of edim virus exist. analysis of rna segment and struc tural polypeptide variation, however, as has been accom plished for other rotaviruses derbyshire and woode, ; rodger and holmes, ) , may reveal differences among strains. further, several serotypes have been described a m o n g the h u m a n rotaviruses by m e a n s of the serum neutralization test (beards et al., ) . this technique may also prove useful in future studies of e d i m virus strains. a. morphology, size, and composition. kraft ( b) , using millipore filters, determined the size of the infective e d i m virion to be between and n m . in electron micro graphs, a d a m s and kraft ( ) mined the m e a n diameter of virions of purified e d i m virus to be . ± n m . o n the other h a n d , melnick ( ) gives about % is lost at x for hr or at t for hr. although some infectivity (> . %) remains at either °c or °c for min, it is abolished at t for min (cheever and muel ler, ; kraft, kraft, , b . much and zajac ( ) found that the purified virus is unstable at both ° and - ''c for weeks but found that at - °c infectivity is retained for at least weeks. c. ejfect of chemicals. in an intestinal filtrate, edim virus titer is not significantly reduced when held in ether or . % sodium deoxycholate at °c for hr (kraft, b) . according to much and zajac ( ) , the purified virus is stable in % ether, % chloroform, or . % sodium deoxycholate at °c for hr and, as is true for other rotaviruses, is resistant to pancreatin. ward and ashley ( a,b) examined the effects of the anionic detergent sodium dodecyl sulfate and of the chelating agent ethylenediaminetetraacetate on purified simian virus and determined that low concentrations and mild temper ature conditions readily inactivated the agent. both chemicals modified the viral capsid to prevent adsoφtion of the inacti vated virions to cells. indeed, this study was the outgrowth of a need to determine the survival of enteric viruses in wastewater. it had been determined that wastewater sludge reduced the heat necessary for simian rotavirus inactivation. ionic detergents in the sludge were identified as the active components. nonionic detergents did not destabilize the virus; further, these com pounds protected the virus from the destabilizing effect of sodium dodecyl sulfate. destabilization by both cationic and anionic detergents was found to be dependent on the ph of the medium. a systematic study of the stability of edim virus to extremes of ph has not been reported. much and zajac ( ) kraft ( ) replication of edim virus occurs in epithelial cells of the villi of the small intestine. in electron micrographs, kraft ( ), banfield etal. ( ) , and holmes etal. ( ) demonstrated replication by budding in distended cistemae of the endoplasmic reticulum. the significance of intracytoplas mic and intranuclear tubular structures encountered is not clear. using direct immunofluorescence, wilsnack et al. ( ) found that edim virus antigen in infant mice is limited to the cytoplasm of the villous epithelium from the duodenum to the colon and is detectable within hr after peroral inoculation. stainable virus was also seen in the intestines of contact infant mice without necessarily causing clinical signs to appear. the virus could not be stained in the stomach or liver of mice infected either naturally or experimentally. kraft ( ) studied the distribution of infectious edim virus in -day-old mice following peroral inoculation. at hr, virus was detected in the stomach and small and large intes tines (the cecum was not tested) and could not be recovered from the lungs, liver, spleen, kidney, or blood. at hr, all those tissues were positive except for the kidney. the bladder and urine, as well as the brain, were also devoid of the agent, but at hr, these too were positive. at hr, the liver, spleen, kidney, and intestines yielded virus, but the brain was negative. the blood, stomach, lungs, bladder, and urine were not tested at that interval. at days, blood, liver, and intestines, the only tissues examined, still contained virus. ingested virus from a diarrheal litter brings about an active infection in the nursing dam that previously had been nondiarrheal herself and had had only nondiarrheal litters (kraft, ) . blood, liver, spleen, and feces all contain infectious virus in the dam week after her litter is given virus perorally as an intestinal filtrate. later, kraft ( ) determined that adult male mice can also be intestinal carriers for at least days after a single peroral exposure. with regard to the mechanism of cell penetration. holmes et al. ( ) proposed that lactase of the villous brush border of the intestine may be the receptor that uncoats the rotavirus virion by attacking the glycolysated polypeptides of the outer capsid. in addition, pancreatic enzymes within the lumen may also be instrumental in the infectious process (see section ii,b, ). the question of cofactors that might enhance pathogenicity or of interfering substances, in addition to antibodies, that might inhibit edim virus replication is open. kraft ( ) found increased numbers of clostridium tertium in the intes tine of diarrheal animals, and pappenheimer and enders ( ) remarked on the persistent presence of ''coccoid bodies" in the intestines of diarrheal animals as seen by light microscopy. in both instances, these may be considered opportunistic or ganisms. their effect on the severity of the clinical disease is unknown, however. one report (labonnardiere and devaurieux, ) the use of standard methods to grow edim virus in tissue culture has resulted for the most part in failure. habermann ( ) (thiel et al., ) . further, babiuk et al. ( ) and al meida et al. ( ) state that calf rotavirus production is mark edly enhanced when passaged in the presence of trypsin. clark et al. ( ) produced high-titer calf rotavirus in a variety of continuous cell lines, also by utilizing trypsin, and matsuno et al. ( ) were successful in producing plaques with bovine rotavirus in monkey kidney cell monolayers when trypsin was incorporated in the overlay medium. estes and graham ( ) found that simian virus titers were enhanced by both trypsin and elastase in vero as well as ma cells. ramia and sattar ( ) studied additional proteolytic enzymes utilizing plaque formation by simian virus sa- in ma cells as the endpoint. elastase, a-chymotrypsin, subtilase, pronase, and pan creatin were as effective as trypsin, whereas pepsin, papain, and thermolysin were ineffective. another approach with human rotavirus has been to incoφorate the agent into cells by means of low-speed centrifugation of the two together (banatvala et al., ; bryden et al., ; schoub et al., ; wyatt et al., ) . it remains to be seen if similar treatments will lead to suc cessful cultivation of edim virus in tissue culture. chick embryos appeared unaffected when the chorioallantois was inoculated. the agent did survive up to days, but an increase in infectious titer could not be demonstrated (kraft, ) . moon ( ) of each disease appears to be reflected not only in the particular group of cells but also in the numbers of host cells involved. lucid descriptions of the natural clinical disease are given by cheever and mueller( ) , seamer ( ) , and mcclure et al. ( ) . the experimental disease does not differ signifi cantly (kraft, (kraft, , b . overt illness is confined to pre- typical appearance of infant mice infected with enterotropic mhv (pair of mice at left) or rotavirus (pair at right) compared with normal mice of the same age (center pair). note the evidence of inanition and dehydration in the mhv-infected animals, whereas those infected with rotavirus show mainly a somewhat prominent abdomen and only a slightly smaller size than the normal control mice. weanling mice. t h e first signs usually appear at - days of age. in mild cases diarrhea is manifested by a minimal amount of pasty fecal material about the perineum. in severe cases the amount is copious, the entire infant becoming soiled. rectal impaction may occur at about - days of a g e , and death can ensue if the impacted mass is not removed spontaneously or deliberately. death does not seem to be the result of the virus infection per se, but rather as a consequence of protracted obstipation. in pure e d i m virus infections, mice continue to nurse throughout their illness (in the absence of impaction). they may be slightly stunted (fig. ), but those with mild cases soon attain the weight of their nondiarrheal peers. especially when diarrhea is mild, morbidity in the natural disease m a y be difficult to ascertain, but one could quite cor rectly assume that, if diarrheal signs are observed in a few litters, it is likely that all infants in a colony will sooner or later be affected. concerning mortality, cheever and mueller ( ) state that there was both % recovery and % lethality in their outbreaks. based on present k n o w l e d g e , the first is suggestive of e d i m virus infection and the latter of another disease, perhaps murine (mouse) hepatitis. at no time do a d u h mice exhibit signs of illness ascribable to e d i m virus infection. in the experimentally induced dis ease, the absence of external clinical signs cannot be relied upon for a negative diagnosis (kraft, ) . necropsy of each animal is essential. in this w a y , the appearance of the colonic contents can be observed, which in normal infant mice are burnt orange in color and semisolid but formed in consistency. t h e colon is not distended. in diarrheal m i c e , however, even in those with no external soiling, the colonic contents are fluid or m u c o i d , bright lemon yellow to amber, or gray-green, with no formed feces in evi dence. g a s is often seen in the colon and c e c u m , which are distended. t h e stomach, t o o , is distended with curdled milk (except in terminal cases with anal impaction). all other or gans appear normal. gross change is not seen in the intestines of adults exposed perorally. it is noteworthy that in germ-free infant m i c e , ex perimental e d i m virus infection appears in the gross as it does in conventional mice (kraft, ) . the histologic picture has been confirmed by others (adams and kraft, ; mcclure et al, ) . there is no inflam matory reaction in the intestines. inclusions may indeed be found in enterocytes near the tips of villi, especially in the jejunum, and infrequently in the sloughed cells seen in the lumen. epithelial cells near and at the tips of villi are fre quently vacuolated. acres and babiuk ( ) have pointed out that in all species studied, the rotaviruses cause diarrhea by attacking and de stroying the columnar epithelium of the small intestine. middleton ( ) considers that cell migration from the crypts is speeded in response to diarrhea and that the infected cells are relatively immature. enzyme levels, high thymidine kinase and low sucrase, are similar in such cells to those of normal crypt cells and thus support this view. the microscopic appearance belies the severity of the gross and clinical findings of edim. in this regard, d-xylose absorp tion has been studied in calves in which - % reduction occurred during the acute illness with the calf rotavirus . aberrations in sodium transport have also been investigated in other animals and in humans (middleton, ) , but such studies have not been carried out in mice using the mouse agent. cheever and mueller ( ) and kraft ( ) demonstrated that the agent is transmissible perorally. kraft ( ) showed further that dissemination in a mouse colony is mediated prin cipally by the airborne route. kraft ( ) examined the neutralization test in edim and found that antibodies were not always formed following infec tion, and when they were present, they tended to be low in titer. sera from adult mice having had lifelong contact with the agent (in the form of consistently diarrheal litters) were more apt to neutralize the virus than those of mice exposed for the first time as adults. on the other hand, hyperimmunization of mice has resulted in the production of significant serum titers of both complement-fixing and neutralizing antibodies (blackwell etal, ) . antibody response measured by other serologic tests has not been investigated for edim, nor have immune substances in lacteal secretions been measured directly. suggestive of their presence, however, are data showing that infants of primiparae who themselves had been diarrheal in infancy resisted about id more of edim virus than did offspring of previously nondiarrheal dams (kraft, ) . antirotaviral immunoglobu lins have been found in both colostrum and milk in man (yolken et al, c; simhon and mata, ; thouless et al, a) , bovines (acres and babiuk, ) , and lambs (snodgrass and wells, ) . indeed, local immunity afforded by lacteal immune substances is regarded as crucial for protection against any intestinal infection in the young (welliver and ogra, ; snodgrass and wells, as indicated, mice of all ages can be infected, but overt disease is restricted to animals up to about - days of age at the time of first exposure. there seems to be no predilection for a particular sex. the observation that first litters are more apt to show copious and protracted soiling than those of later parity has been reported by cheever and mueller ( , ) and by runner and palm ( edim virus is probably worldwide in distribution. whether it occurs in wild mouse populations is unknown. its prevalence is difficult to estimate, since serologic tests have not been readily available, and it is not customary to sacrifice animals for the purpose of examining the appearance of their intestinal tract or for electron microscopic visualization of fecal contents. as has been shown experimentally, latent carriers can exist. the carrier rate in a diarrheal colony is not known, nor is the frequency of viral shedding under colony conditions. cheever and mueller ( ) examined seasonal variations in the weaning percentage in their mouse strains and found that there was a significant effect in only the cfw mice. they experienced the lowest weaning rate in the late fall and winter. runner and palm ( ) , studying c h mice, indicated that there was a higher incidence of diarrhea in december/january (kraft, ; blackwell et al., ) , complement fixation (wilsnack et al., ; kapikian et al, ; thouless et al., b) , direct immunofluorescent staining or precipitin (wilsnack et al., ; spence et al., ; foster α/., ; peterson α/., ) , immune electron microscopy (kapikian et al., ; bridger and woode, ) , immunoelectroosmophoresis (tufvesson and johnsson, ; middleton et al., ) , enzyme-linked im munosorbent assay (elisa) (scherrer and bernard, ; el lens etal., ; yolken etal., a,b,c) , radioimmunoas say (acres and babiuk, ; kalica et al., ; middleton et al., ) , immunodiffusion (woode et al., ) , hemagglutination inhibition (fauvel et al., ) , enzymelinked fluorescence assay (elisa) (yolken and stopa, ) , an unlabeled soluble enzyme peroxidase-antiperoxidase method , plaque reduction test (estes and graham, ) , serologic trapping on antibody-coated electron microscope grids (nicolaieff et al., ) , a solid phase system (space, solid phase aggregation of coupled erythrocytes) for detection of rotaviruses in feces (bradbume et al., ) , and immune electron microscopy with serum in agar diffusion (lamontagne et al., ) . more recently, sheridan and aurelian ( ) have described an elisa test for edim virus which should prove beneficial for both practical (serologic) purposes and for investigations of the antigenic structure of the virion. in the absence of a reliable tissue culture system, edim virus isolation is generally impractical. bacteria-free filtrates of intestinal suspensions can, of course, be given to diarrheafree animals (gnotobiotes or axenics), but this is expensive and inefficient. on the other hand, such filtrates may be concen trated by ultracentrifugation and examined in the electron mi croscope for characteristic virus particles flewett, ) . the particles may also be identified by immune electron microscopy. a practical approach to a presumptive diagnosis would be to kill selected animals in order to examine the appearance of the colonic contents. the inclusion of sentinel dams with litters in a breeding colony should be considered. these might be sac rificed at intervals to determine the presence of edim in the colony. together with the clinical history of the mouse colony, this practice may provide a fairly reliable, although not pathognomonic, indication of the presence of edim. his topathologic examination could also be of value. based on experimental results, kraft et al. ( ) proposed the use of air-filter devices, essentially dust caps for each cage, for the practical control of airborne transmission of edim in a commercial mouse colony. it was subsequently shown (kraft, ) that % of first litters and % of all other litter parities were weaned from cages without filters, whereas in cages pro vided with filters, weaning percentages were and %, respectively, during the same observation period. it should be pointed out that both edim and mouse hepatitis virus (livim) were present simultaneously in that colony. although the filter devices did not eliminate the disease(s), they probably reduced the pathogenic microbial load in the immediate environment of the susceptible animals, but the precise mechanism by which this control method succeeds to the extent that it does is obscure. since that time ( ), various devices based on a filter cage or filter-top design have been utilized. some of these are de scribed by simmons and brick ( ) . woods et al. ( ) evaluated them from the viewpoint of environmental factors within the cages. they found that dry bulb temperature dif ferentials, comparing environments inside and outside the cage, were not significantly different (about °c) between fil tered and unfiltered cages, but that dew point differentials were significantly greater in the filtered cages (about °c) than in unfiltered cages (about °c). however, they concluded that a suitable cage size for a particular species and number of ani mals could compensate for the higher wet bulb readings under filters to maintain acceptable conditions for the animals. cur rently, several types of filter covers or bonnets are available commercially. vaccination as a method of control has not been attempted. judging from the lack of reports to the contrary, caesarian derivation together with barrier maintenance apparently elimi nates and controls the infection. kunstyf ( ) attempted to control edim by decontamina tion of air by the use of triethylene glycol but was unable to do so. the use of antibiotics, too, is without value, although there seems to be amelioration of signs for a short period as secon dary organisms are temporarily reduced in number. as indicated by kraft ( ) , it may be relatively easy to establish a colony of mice free from edim virus infection. this may be accomplished by eliminating those breeding pairs whose first litter is diarrheal. filter devices are required for this, and they and the animals must be handled with the aid of a transfer or laminar flow hood using sterile techniques during observation and handling of the animals. the method is suita ble for small colonies, but it is impractical for commerce where caesarian derivation and barrier maintenance are the methods of choice. reovirus was first isolated from the feces of an australian child manifesting a cough, fever, vomiting, hypertrophic tonsils, and bilateral bronchopneumonia. it was named hepatoencephalomyelitis virus by stanley et al. ( ) , who originally recovered the agent. sabin ( ) proposed the name reovirus for a group of agents associated with the respi ratory and enteric tracts of humans. they were found to be ether resistant, about nm in size by membrane filtration but of unknown shape, and caused distinctive cytopathic effects in monkey kidney tissue cultures. one of the echo group of viruses, echo , now synonymous with reovirus, became a member of the new group on that basis. (echo is the acronym for enteric cytopathic /zuman >φhan, agents isolated in tissue culture from asymptomatic humans-so-called viruses in search of disease.) stanley ( ) then demonstrated that the hepatoencephalomyelitis virus was serologically identical to reovirus . additional strains have since been recovered from humans, other mammals, marsupials, birds, insects, and reptiles (for review, see stanley, ) , and from mouusks (meyers, ; meyers and hirai, ) . reoviruses have been divided into three serotypes on the basis of hemagglutination inhibition and neutralization tests (sabin, ; rosen, ) . reovirus was established as an indigenous murine virus by hartley et al. ( ) and by cook ( ) . reviews concerning the biological and clinical aspects of disease caused by this agent have been prepared (stanley, (stanley, , . when gomatos et al. ( ) and discovered that reoviruses possess double-stranded rna, a unique characteristic among viruses, molecular biologists were inspired to study them in minute detail. currently, knowledge of reovirus replication on biochemical and biophysical planes is as extensive as that available for any other virus and is, for the most part, outside the scope of this chapter. interested readers are therefore referred to reviews by shatkin ( ) and joklik ( ) for extended literature coverage and detailed dis cussion and to andrewes et al. ( ) for a condensed over view. or reovirus, in the family reoviridae (melnick, ) . wild-type strains of reovirus include: dearing, the pro totype strain (sabin, ) , isolated from a child with diarrhea; abney (rosen, ) , isolated from a child with a febrile upper respiratory infection; can , from a case of burkitt's lymphoma (bell et al., ) ; and several strains obtained from naturally infected cattle (rosen, ) . mutant, temperature-sensitive (ts) strains have been developed in the laboratory (fields and joklik, ) and have been used for studying the synthesis of viral rna and peptides (cross and fields, ; as well as for examining problems of pathogenesis. a neurotropic strain was also de-rived from the m o r e hepatotropic original isolate (stanley et al, ) . t h e reovirus virion (fig. ) has a m e a n diameter of about - n m and is icosahedral in shape, with : : symmetry. particles have a core, an inner layer or shell containing a n u m b e r of capsomers formalin at °c. ether treatment was ineffective. rozee and leers ( ) determined that although cholorform does not affect infectivity, it does destroy the hemagglutinin. wallis et al. ( ) found that mg^^ enhances the titer of reovirus at °c. the infective titer increased four to eight times by heat ing for - min in Μ mgcl . other divalent cations and nacl were ineffective. hemagglutinin was not affected. it is thought that the high temperature and mg^+ caused activation of reovirus particles that were inactive in the original prepa rations. as with rotavirus. ward and ashley ( ) found that reovirus was sensitive to anionic detergents in wastewater sludge, i.e., these chemicals decreased the temperature needed to inactivate the virus. cationic detergents were more active than anionic, and nonionic detergents were inactive in decreas ing reovirus thermal stability. mutagens (nitrous acid, nitrosoguanidine, and proflavine) have been applied to reovirus (fields and joklik, ) . the resulting mutants are of interest not only to the molecular biologist but to the clinical virologist as well, since some of them produce altered disease pictures (fields and raine, ) . for example, when inoculated into rats, wild-type virus produced a necrotizing encephalitis, whereas a mutant gave rise to a slowly progressive communicating hydrocephalus. the effects of enzymes are described below (see section iii,b, ). in phosphate-citrate buffers, stanley et al. ( ) ascertained that the virus is stable between ph . and . . e. antigenic determinants. sabin ( ) demonstrated that the mammalian reoviruses known at that time could be divided into three serologic groups by neutralization tests. they could also be differentiated by hemagglutination inhibi tion (rosen, ), hull et al. ( having discovered that echo virus possessed a hemagglutinin for human o eryth rocytes. later, reported that reovirus , but not reovirus or , agglutinated ox erythro cytes. the reovirus hemagglutinin was inhibited by nonspecific substances such as normal mouse, rabbit, or rat serum, and by vibrio cholerae filtrate. weiner et al. ( ) were able to show that the rna segment, which is associated with type specificity, encodes the polypeptide that determines the hemagglutinating properties of the virion. complement-fixing antigens were prepared by stanley et al. ( , ) and by j. c. parker et al. ( , ) . these are group specific. leers et al. ( ) determined that reovirions display at least one type-specific and one to two group-specific antigens when studied by immunodiffusion. with the more sensitive im munoelectrophoresis technique, however, these authors en countered two type-specific and four group-specific precipitin lines. the agent is regarded as pantropic in mice. in neonates, stanley et al. ( ) (wolf et al., ) . papadimitriou ( papadimitriou ( , papadimitriou ( , also studied viral replica tion by electron microscopy in the mucosa of the common bile duct and in the liver. reovirus has been reported to be oncolytic for a mouse ascites tumor by bennette ( ) , bennette et al. ( ,a,b) , and nelson and tamowski ( ) . most of the studies dealing with replication of the reoviruses have been accomplished in tissue culture, frequently in plaque assays in l-cell monolayers. other cells that have been suc cessfully employed are primary kidney monolayers of rhesus, patas, and capuchin monkeys, as well as those of pigs, cats, and dogs. continuous lines, such as fl human amnion, bs-c- , and kb, have also been used (hsiung, ; cook, ; mcclain etal., ; rhim and melnick, ; harford et al., ) . harford et al. ( ) ( a,b,c, , ) . stanley et al. ( , ) reported the development of pocks on the chorioallantois of -day-old chick embryos in oculated with infectious brain and liver. the embryos appeared unaffected, and with succeeding passages, the pocks could no longer be observed, although oral inoculation of suckling mice with chorioallantoic suspensions resulted in active disease. essentially the same results were found following amniotic inoculation. the experimental and natural diseases appear identical ex cept for variations in intensity of signs, perhaps due to dif ferences in infectious dose. up to days after intraperitoneal inoculation, the mice appear emaciated and uncoordinated. the hair is oily and matted-the so-called oily hair effect (ohe)-an effect that can be demonstrated in contact animáis as well. however, in these mice it disappears as soon as the diseased animal is removed from the healthy ones. the effect was ultimately traced to a high proportion of fat in the intesti nal contents (steatorrhea): . % in infected animals as com pared with . % in normal mice. feces may contain as much as % fat in infected mice (stanley et al., (stanley et al., , . the thymus and other organs appeared normal. in the central nervous system, neuronal degeneration began about the ninth day and was most prominent in the brain stem and cerebral hemispheres. by the tenth day, perivascular cuf fing as well as neuronal satellitosis was evident. the meninges were infiltrated with round cells and netrophilic leukocytes. by the fourteenth day encephalitis was severe and widespread, with small hemorrhages occurring in necrotic regions. in suckling rats inoculated intracerebrally with reovirus types , , or , viral cytoplasmic inclusions and intranuclear bodies corresponding to cowdry type Β inclusions have been ob served (margolis et al., ) . the latter were seen in cells that were free from intracytoplasmic inclusions; they were readily found in weanlings, were unassociated with inflam and their microvilli swollen. the common bile duct is dilated, and the ampullary region becomes obstructed with debris. it is this obstruction that leads to both hepatic and pancreatic dys function, for after studying the pancreatic lesions further, papadimitriou and walters ( ) concluded that, even though virions were seen in pancreatic acinar cells, the principal cause of acinar degeneration is ductal obstruction. based on the neutralization test, determined that the si genome segment is linked to type speci ficity, and finberg et al ( ) found that the same genome segment is also responsible for the production of cytolytic Τ lymphocytes after reovirus infection. tytell et al ( ) , working with reovirus rna, found that it was highly active in inducing interferon in rabbits and tissue culture, and lai and joklik ( ) showed that coreless virions as well as those lacking the outer capsid shell induce no interferon. the question of the role of interferon in protection of mice from either the acute or chronic infection remains an intriguing problem. in an effort to permit a precise definition of the host cellular immune response to viral antigens, weiner et al ( b) and greene and weiner ( ) which also determined serotype-specific humoral (neutraliz ing) and cytolytic t-cell responses. it is clear that the agent can be transmitted by the oral route as well as by parenteral inoculation. mice respond to the natural infection with neutralizing, hemagglutination-inhibiting, and complement-fixing an tibodies. as indicated earlier (section iii,b, ,^), precipitating antibodies can also be demonstrated. there is no evi dence that any mouse strain is more or less susceptible to reovirus infection than any other, provided the animals come from a colony that is free of the infection. the host range is broad. stanley ( ) cited at least species that may be infected with reoviruses, and, as men tioned above, it is thought that the prevalence of antibodies in otherwise normal mammals is related to this fact and that mosquitoes or other insects may be operational in the spread of the infection. the acute disease affects mainly sucklings and weanlings, whereas the chronic disease is encountered in animals over days of age. there is no indica tion that either sex is more or less susceptible than the other. in an epizootic described by cook ( ) , of first litters were affected. later-parity litters were involved hardly at all. in view of the absent or low complement-fixing antibody titers in the presence of significant hemagglutination-inhibiting titers that follow natural infection, prevalence estimation by immunologic means may be difficult to assess. the data cited by parker et al. ( ) , % of colonies positive, and by descoteaux et al. ( ) , % of five colonies posifive, may be typical incidences for conventional mouse colonies. as already indicated, reovirus infection is regarded as worldwide in distribution. this aspect of reovirus infection has been covered above (section iii,b,l, a,¿?). munoperoxidase method (enzyme-labeled antibody) has been employed for reovirus (ubertini et al., ) and may find application for reovirus diagnosis as well. although ohe may not be absolutely pathognomonic for reovirus infection, it seems distinctive enough so that a provisional diagnosis may be made when it is seen. stronger evidence is afforded if the animals are also jaundiced and wasted. necropsy coupled with histopathologic examination is never a mistake and is to be encouraged. the placement of sentinel animals at strategic locations in an animal colony should be considered. such animals may be regarded as expendable for sacrific, necropsy, and virus isolation as well as for the acquisition of serum for antibody determinations. there is no evidence that epizootics occur preferentially at a particular time of the year. j. c. parker et al. ( , ) discussed the serologic diagnosis of reovirus infection, concluding that for these puφoses the hemagglutination inhibition test was the most reliable. in preparing type-specific antisera for standardizafion and controls, behbehani et al. ( ) found that the ubiquity of inhibitory substances (antibodies included) in most mamma lian species precluded accurate work. they therefore used domestic geese, in which virtually no hemagglutinafion inhibi tion or neutralizing antibodies could be detected prior to im munization. in any event, for routine surveillance, the hemagglutination inhibition test is currently utilized. comments similar to those expressed for edim virus recov ery and visualization apply. although it is possible to perform these procedures, they are inefficient for routine diagnostic puφoses. stanley ( ) has outlined methods for this pur pose. in brief, infectious material can be inoculated into tissue cultures (primary rhesus monkey or human kidney) or newbom mice (from reovirus free colonies!), or the material may be subjected to immunofluorescent methods. an im- cesarean derivation and barrier maintenance are believed to be suitable techniques for control and prevention of reovirus infection. although no experimental evidence has been found, it is possible that the use of filter devices in conventional colonies might also be helpful in preventing the spread of infection. in the absence of information on the vertical trans mission of the agent, it is impossible to evaluate the influence of that route on successful control of the endemic disease. although therapy is impractical from the standpoint of con trolling epizootics of reovirus infection, it is nonetheless of considerable interest that willey and ushijima ( ) found that thymosin given intraperitoneally to -day-old mice that had been neonatally infected with reovirus ( ld ) signifi cantly increased their mean survival time, provided it was ad ministered at hr. when given at hr, significantly increased survival time was not observed, but when inoculated at hr, there was an apparent decrease in mean survival time. reviews on the subject include those by piazza ( ) , mcintosh ( ) , kapikian ( ) , andrewes et al ( ) , holmes ( ) , garwes ( ) , and robb and bond ( ) . based on their own electron microscopic studies and on those of david-ferreira and manaker ( ) , becker et al ( ) considered that mhv might be an 'tbv-like" (infecti ous bronchitis viruslike) agent. virions of similar moφhology seen in electron micrographs and also ether sensitive, as is ibv, were being isolated at that time from human cases of colds (almeida and tyrell, ) . the following year, the term coronavirus was proposed for the group (tyrell et al, ) , and in the coronaviridae became an official fam ily with a single genus, coronavirus (tyrell etal, additional agents may be added to this group, e.g., ''runde" virus (traavik et al, ) , a coronavirus causing car diomyopathy in rabbits (small et al, ) . as noted, many strains of mhv have been recovered from mice under various circumstances. in addition to jhm, these include: mhvl, from "white mice" (p or parkes strain), dur ing attempts to adapt human hepatitis virus to animals (the strain was originally isolated as a dual agent consisting of the virus and a protozoon parasite, eperythrozoon coccoides (gledhill and andrewes, ) ; mhv , from mice used to propagate murine leukemia virus (nelson, a,b) ; mhv , also found during studies on adaptation of human hepatitis virus to mice (dick et al, ) ; mhv-b (ehf- ), from mice used for human epidemic hemorrhagic fever (hehf) adaptation attempts (buescher, ) ; an unnamed strain from mice undergoing murine leukemia chemotherapy trials (braunsteiner and friend, ) ; h , following intracere bral inoculation of suckling mice with hehf materials (morris, ) ; mhv-a , during transfer of moloney leukemia virus in mice (manaker et al, ) ; mhv-s, from cesareanderived mice that had been barrier-maintained before exposure to conventional mice ; mhv-c (mhv-balb/c), isolated during passage of spleens from leukemic mice (nelson, ) ; four addiiional strains from spleens of leukemic mice or from natural outbreaks (mhv-srl, -sr , -sr , -sr ) (nelson, (nelson, , ; lethal intestinal virus of infant mice (livim), from infant mice dying of a spontaneous infection (kraft, a) , identified as an mhv strain by broderson et al ( ) and hierholzer et al ( ) ; and nuu, nua, nu , from nude mice with hepatitis and wasting syndrome . other isolates have also been derived from nude mice (sebesteny and hill, ; ward et al, ) , and fox et al ( ) have described a strain that appeared during passage of an ascites myeloma cell line in balb/c mice. *hev may also denote the hepatoencephalomyelitis virus of stanley et al. ( ) , which is identical to reovirus . a s reviewed by mcintosh ( ) , coronaviruses are p l e o m o φ h i c , enveloped, and variable in size, measuring about - n m in diameter. peplomers are - n m in length (fig. ) . using various techniques, a n u m b e r of workers have con firmed the diameter of m h v virions to fall within the range of the coronaviruses (gledhill et al., ; miyazaki et al., ; kraft, a; starr etal., ) . svoboda etal. ( ) further described the virion as consisting of a nucleoid sepa rated from an outer m e m b r a n e by an electron-lucent space. david-ferreira and manaker ( ) , working with m h v -a in tissue culture, found that the virions had a m e a n diameter of nm with an electron-dense inner shell, nm in diameter, separated from the outer double m e m b r a n e by an electronlucent space nm wide. peplomers were . - . n m long. not only was the virion significantly smaller than that of i b v , but the peplomers dif fered from those of both i b v and h c v e , being conerather than club-shaped. mallucci ( ) in general, coronaviruses are inacti vated at °c in - min, at °c in several days, and at °c in several months (tyrell etal., ; kapikian, ) . wildtype mhv isolates also fall into this range of sensitivity gledhill and andrewes, ; gledhill et al., ; kraft, a) . hirano et al. ( ) , however, indicated that mhv is not completely destroyed at °c for min and is stable at t for min in Μ mgclg or mgs but not in water. freezing and thawing or sonication at kc for min does not affect the virus titer. c. effect of chemicals. all coronaviruses are sensitive to ether when exposed overnight at °- °c. chloroform also de stroys or reduces infectivity (mcintosh, ) . fifty percent glycerol inactivates mhvl after weeks at °c (gledhill and andrewes ( ) . sodium deoxycholate reduces the titer of livim significantly (kraft, a) , but calisher and rowe ( ) regard mhv virus as moderately resistant. according to hirano et al. ( ) , mhv is completely inactivated by ether, chloroform, sodium deoxycholate, and / -propiolactone, but it is completely resistant to trypsin. mutagenesis has been reported by means of nmethyl-/v'-nitroguanidine or -fluorouridine and by -azacytidine or -fluorouracil (haspel et al., ) . the ph stability of all known murine hepatitis viruses has not been reported. for coronaviruses in general, acid sensitivity is regarded as variable (mcintosh, ) . hirano et al. ( ) found that mhv is stable be tween ph and at °c for min. calisher and rowe ( ) although they have been sought, hemagglutinins have not been demonstrated for mhv strains (see, e.g., hirano et al., ; kraft, a; bradbume, ; miyazaki etal., ) , but two human coronavirus strains do agglutinate human erythrocytes (kaye and dowdle, cheever et al. ( ) found jhm virus to be unrelated to other neurotropic viruses, including gd vii, pseudorabies, lansing poliomyelitis, and mengo virus. kraft ( b) found no relationship between livim, edim, and reovirus . with regard to other coronaviruses, the picture is somewhat different, for as a group, coronaviruses display complex serologic variability (bradburne, ; mcintosh et al., ) . mhv is serologically closely related to rcv and sdav in complement fixation tests and distantly related to rcv in cross-neutralization tests (parker et al., ; bhatt et al., ) . several strains of mhv are closely related to human coronaviruses oc and oc , and mhv is related to hcv- e (bradbume, ) . antibody to mhv strains commonly found in human sera is probably present because of endemic human infection with related coronavimses (hartley et al., ) . electron micrographs and studies using fluorochrome stains indicate that coronaviruses develop exclusively in the cyto plasm of infected cells, that the virions collect in cytoplasmic vesicles of diverse size, that particles may also be seen in the matrix outside of the endoplasmic reticulum as well as in the golgi apparatus, and that they are not observed in the nucleus. in the main, replication involves budding into cytoplasmic cis temae, but tubular structures have also been seen within the cytoplasm during virus formation (ruebner et al., ; stan di α/., ; watanabe, a,b) . wilsnack ( ) , confirming the work of boss and jones ( ) , elicited immunofluorescent antigen staining in sinusoidal lining cells in necrotic liver foci of weanling mice within hr after intraperitoneal inoculation of the a strain. stainable antigen in intestinal impression smears of mice in fected by cage contact was also demonstrated. piazza et al. ( ) examined the fate of mhv after in travenous inoculation. the agent was not demonstrable be tween min and . hr, when it appeared first in the spleen, then in the liver ( hr) and blood ( . hr). at hr it was recoverable from brain and kidney. high titers were then reached in all organs. barinsky and dementiev ( ) thereafter, the cells of the olfactory bulb and other brain re gions are affected. a number of cell systems have been successfully employed for in vitro growth of mouse hepatitis viruses: mhv-c in mouse embryo explants (mosley, ) ; mhvl in newborn mouse kidney explants (starr and pollard, ) ; mhv-s in mouse embryo explants (compels, ) and in liver (gallily et al, ) ; mhv-b in liver cell monolayers (paradisi and piccinino, ) ; mhv in liver explants (vainio, ) ; mhv-b in liver cells (miyazaki et al, ) ; mhv and mhv in dbt cells (hirano et al, ; takayama and kim, ) ; and various strains in nctc cells (david-ferreira and manaker, ; wilsnack etal, ; hartley and rowe, ) . mallucci ( ) , seamer ( ) , and lewis and starr ( ) described syncytium formation by mhv in mouse macrophage cultures, and a plaque assay for mhv in primary peritoneal macrophage cultures was described by shif and bang ( ) . laufs ( ) also described multinucleated giant cells with as many as nuclei per cell in macrophage cultures infected with mhv . using autoradiography, he ascertained that there was no dna synthesis in them and that they originated from cell fusion. macrophages derived from either liver or peritoneal wash ings are of enormous interest for the question of host cell-vims interactions. bang and warwick ( , ) macrophages from the mice mirror these changes in resistance as the animals age. kantoch et al. ( ) determined that temporary susceptibility could be induced in resistant cells in culture if they were exposed to homogenates of susceptible cells, and gallily et al. ( ) showed that macrophages from genetically resistant mice treated with cortisone to enhance susceptibility behave in culture as if they were from susceptible animals. macrophages from mice susceptible to mhv virus can be converted to resistance by the intraperitoneal inoculation of concanavalin a in the donor mice. this enhanced resistance is also expressed in vivo (weiser and bang, ) . cheever etal. ( ) , nelson ( b) , and kraft ( a) in suckling mice, rapid wasting with or without neurologic signs may take place, accompanied in some cases by diarrhea, inanition, and dehydration (fig. ) . mortality and morbidity are variable, ranging between al most and % depending on factors like those affecting clinical signs. of importance to users and breeders of mice alike is the fact that a number of agents and procedures are known to modify the reactivity (and therefore the clinical signs) of mice to both spontaneous and experimental infection. examples of these, together with pertinent references, are presented in table i . leprévost et al. ( a,b) have taken the view that there are three types of sensitivity to mhv infection in mice: resis tance, full susceptibility, and semisusceptibility. these are re flected in the susceptibility of their macrophages response of nuinu mice to sheep erythrocytes dba/ mice, on the other hand, are regarded as fully suscep tible since deaths begin - days after infection, even when the mice are days old, whereas the a/j strain is resistant, being susceptible to the acute disease only up to - weeks of age. although c h mice are partially susceptible to the acute phase of the disease, they are fully susceptible to the chronic stage. in nude (nu/nu) mice, mhv takes on special significance. indeed, perhaps the best description of the clinical signs may be found in the original report describing this mutant mouse (flanagan, ) , published before runt disease, as the wast ing syndrome was called, came to be recognized as something other than a genetic effect. mhv-infected nude mice lose weight slowly or rapidly. they move stiffly with a stilted gait, and their faces assume a pointed, anxious appearance. partial paralysis may develop first in the hindlimbs and then in the forelimbs, resulting in almost total immobility. nu/+ heterozygotes are not affected in this way. flanagan ( ) found that at weaning, the nude animals were much smaller than controls (heterozygotes), that % died within weeks of birth, and that % were dead by weeks of age, whereas only % of controls died in the same period. it was not until sebesteny and hill ( ) uv-inactivated virus did not elicit the effect. the authors be lieve that fixed macrophages in athymic mice may be acti-vated, as was indicated by nickol and bonventre ( ) and by cheers and waller ( ) in certain bacterial infections in nude mice. t a m u r a et al ( ) (see, e . g . , r u e b n e r and bramhall, ; gledhill etal., ; nelson, ) . in liver regeneration m a y take place as early as - days after infection (ruebner and bramhall, ) , ranging from complete healing to chronic scarring with intermediate gradations. kupffer cells were examined by r u e b n e r and miyai ( ) and r u e b n e r et al. ( ) . t h e y m a y undergo nuclear pyknosis and karyorrhexis hr after intravenous inoculation of m h v . in infection with neurotropic variants, such as j h m , the principal lesions appear in the central nervous system . meningitis is present but varies in degree and loca tion. in the brain, lesions m a y be found in all regions, but the hippocampus and its connections, the olfactory lobes, the periependymal tissues, and the brain stem seem to b e affected most often. necrotizing lesions predominate in the olfactory lobes and hippocampal regions, whereas demyelination is the major change in the brain stem. s o m e exudate m a y be found around blood vessels associated with lesions, and at about days after infection, proliferating pericytes and scant lym phocytic cuffing can be seen. peripheral nerves show n o change. in sucklings, j h m virus produces extensive lesions in the brain and cord at - days. meningitis is present, and large regions of necrosis with m a n y giant cells occur throughout the brain. in the cord, the lesions consist of spongy necrosis of the central gray matter. ganglion cells appear unaffected. powell and lampert ( ) resolved into small foci of fibrillary gliosis with an increased size and n u m b e r of astrocytic processes. t h e importance of this finding for investigations into the cause of multiple sclerosis and other demyelinating diseases in m a n is obvious and has been addressed b y , a m o n g others, lucas et al. ( ) and lampert ( ) . in sucklings infected with enterotropic strains (kraft, a; broderson et al., ; ishida et al., ; ishida and fuji wara, ; r o w e et al., ; hierholzer et al., ) strains (dick et al., ; r u e b n e r and bramhall, ; hirano and ruebner, ) , and biggart and ruebner ( ) attribute the change to virus replication in lymphocytes. in other organs, minute superficial necrotic foci m a y be found in the stomach. n o changes are seen in the heart, lungs, pancreas, kidney, adrenals, voluntary m u s c l e , femoral o r ver tebral bone m a r r o w , or pituitary gland, although virus m a y be isolated from some of those organs. occasional giant cells are found in peripancreatic lymph nodes and in p e y e r ' s patches. flanagan ( ) islands of hepatocytes, markedly hypertrophied, remained. changes in other organs were not described. the liver lesions observed in other nude mice infected with mhv were similar. sebesteny and hill ( ) noted central nervous system lesions in their nude mice. ward et al. ( ) also encountered central nervous system lesions in addition to vascular changes, giant cell peritonitis, ascites, and giant cells in the villous epithelium of the intestines. since virus can be transmitted perorally and intranasally, it may be assumed that these are the principal routes of natural infection. transmission may be mediated by both the airborne and contact modes. feces, nasopharyngeal exudates, and perhaps urine would be sources of infection. evidence of vertical transmission is at hand, but reports are conflicting. piccinino et al. ( ) with or without booster, showed no antibody in the same colony. in a conventional colony of ddd mice, furthermore, seropositivity increased from to % and in ddy mice from . to % as a result of the booster technique. in barrier-maintained animals, the booster did not elicit antibodies where there had been none before. presumably, these animals were free from mhv infection. from the foregoing, it is evident that the classic humoral immune response to mhv seems to be weak, and that a colony in contrast to the findings of bang and warwick ( ) , who concluded that one gene (or factor) was responsible for host susceptibility in mhv infection, stohlmann and freiinger ( ) showed that two genes are required for resistance of the central nervous system in sjl mice to fatal disease due to mhv-jhm. further, stohlmann et al. ( ) reported that there is an age-related change in resistant mice that protects them from acute central nervous system disease. they iden tified this change as due to a maturing adherent spleen or peritoneal exudate cell population. in extensive genetic studies, levy-leblond et al. ( ) found no correlation between the h- locus and either the acute or chronic disease in c bl (susceptible) or a/j (resistant) animals. using congenie c h lines, however, they were able to show that the h- ^ allele enables both heterozygous and homozygous animals to resist the development of the chronic disease. they believe, therefore, that mhv sensitivity appears to be influenced by at least two major genes: one for the acute disease, and the other, linked to the h- gene complex, for the chronic disease. mice also produce interferon as a result of mhv infection virelizier and gresser, ) , and taguchi et al ( a) ascribe the greater susceptibility of suckling c h/hejms mice to serum levels of interferon that are considerably lower than in weanling and aduh mice, as well as to greater macrophage sensitivity in the neonates. mhv-induced interferon may be regarded as the principal mechanism by which the virus modifies the immune respon siveness of mice to sheep red blood cells, for example. (see also ( ) infected cotton rats, rats, and hamsters intracerebrally, but rabbits and guinea pigs failed to respond. sebesteny and hill ( ) at tempted to infect infant wistar rats and hamsters using virus recovered from nude mice. all survived at least days with out signs of illness. of considerable importance is a report by taguchi et al ( c) concerning asymptomatic mhv-s infection in suckl ing rats following intranasal inoculation. the agent multiplied mainly in the nasal epithelium without any clinical signs. neu tralizing antibodies were produced, however, and could also be demonstrated in adult rats following infection. necrotic changes took place in the nasal mucosa, and cytoplasmic im munofluorescence was demonstrated in the nasal epithelium days after intranasal inoculation of -day-old rats. age susceptibility has been amply addressed above in the consideration of virus growth in mice and tissue culture and in the discussion of the clinical picture. there appears to be no difference in susceptibility between the sexes (taguchi et al., ) . parker et al. ( ) found that the incidence of complement-fixing antibodies was greater in females than in males under colony conditions, as cribing this difference to continual exposure to virus-infected litters. there appears to be no evidence that first litters are significantly more susceptible than later ones. as noted above (section iv,Β, and c,l), the susceptibility of various mouse strains depends on the age of the host at the time of infection, the virus strain, and the host genotype. a completely resistant mouse strain has not been reported. prevalence of mhv infection in an animal colony is often difficult to ascertain. in addition to examples given earlier, descoteaux et al. ( ) found a low prevalence of hepatitis antibodies in three of five colonies studied in canada. complement-fixing antibodies ranged in titer from to , being considered positive. fewer than % of the animals, which were - months old at the time of testing, were posi tive. in distribution, mhv is regarded as occurring worldwide. these have been defined as occurring more than months after intracerebral, intraperitoneal, intranasal, or intravenous inoculation (as reviewed in robb and bond, ) . the chronic clinical manifestations range from none to porenceph aly, paralysis, hepatitis, immunodeficiency manifestations, encephalitis, lymph node adenopathy, and vasculitis. virus may or may not be isolated. cells stainable by immunofluores cence may be found. demyelination with or without remyelination may occur. occasional scattered mononuclear cell infil trates may be evident. there is no evidence that seasonal changes influence the oc currence of epizootics of mhv infection. as indicated above, serologic testing is routinely carried out by means of the complement fixation test. cross-reactions with other coronaviruses must be taken into account when inteφreting the results, however. neutralization tests performed in tis sue culture systems are also possible. employing virus strain a as antigen in the elisa, peters et al. ( ) found a high prevalence of mhv antibodies in colonies with a low incidence of both complement-fixing and neutralizing antibodies. hierholzer and tannock ( ) have used the single radial hemolysis test for human coronavirus serodiagnosis. they then applied it to some mhv strains (hierholzer et al, ) . these techniques are helpful under certain circumstances, e.g., experimental investigations, but they are inefficient for field conditions. necropsy of dead or sick animals is always useful, although a definitive diagnosis in the absence of serologic evidence cannot be made. nevertheless, wherever possible, gross and microscopic pathologic examination should be undertaken. sentinel animals, especially gnotobiotes, could be Ιηοοφοrated into a program of colony health surveillance. these ani mals can then be checked at predetermined intervals for clini cal, serologic, and histopathologic evidence of endemic dis ease in the colony. control of mhv is difficult in mouse colonies unless caesa rian derivation coupled with barrier maintenance is under taken. with the finding that vertical transmission is possible, however, barrier maintenance alone may not be adequate if, for example, such transmission is frequent. using small number of animals for experimental puφoses, kraft ( a) tamura et al ( ) found that nu/nu mice could resist mhv infection when they previously received thymocytes from weanling nu/+ littermates. they were then not only able to produce antibody but survived a challenge infection as well. studies on rotaviral antibody in bovine serum and lacteal secretions, using radioimmunoassay epidemic diarrhea of infant mice. identification of the etiologic agent electron microscopic study of the intestinal epithelium of mice infected with the agent of epizootic diarrhea of infant mice (edim virus) the morphology of three pre viously uncharacterized human respiratory viruses that grow in organ culture the effect of trypsin on the growth of rotavirus viruses of verte brates panel report of the colloquium on selected diarrheal diseases of the young rotavirus isolation and cultivation in the presence of trypsin a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin. ii in vitro detection of human rotaviruses further observa tions on the virus of epizootic diarrhea of infant mice. an electron microscopic study genetics of resistance of animals to viruses: i. introduc tion and studies in mice macrophages and mouse hepatitis mouse macrophages as host cells for the mouse hepatitis virus and the genetic basis of their susceptibility virological and cytogenetic studies on the involvement of bone marrow of mice in some hepatoencephalotropic viral infections rotavirus serotypes by serum neutralisation moφhogenesis of avian infectious bronchitis virus and a related human virus (strain e) preparation of type-specific antisera to reoviruses isolation of a reovirus from a case of burkitt's lymphoma isolation of a non-pathogenic tumour-destroying virus from mouse ascites characteristics of a newborn runt disease induced by neonatal infection with an oncolytic strain of reovirus type (reo mh). ii. immunological aspects of the disease in mice characteristics of a newborn runt disease induced by neonatal infection with an oncolytic strain of reovirus type (reo mh). i. pathological investigations in rats and mice characterization of the virus of sialodacryoadenitis of rats: a member of the coronavirus group lymphoid necrosis in the mouse spleen produced by mouse hepatitis virus (mhv ): an electronmicroscopic study lethal intestinal virus infection of mice (livim). an important new model for study of the response of the intestinal mucosa to injury virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis serological studies with an agent of epizootic diarrhea of infant mice pathogenic murine coronaviruses. ii. characterization of virus-specific proteins of murine coronaviruses jhmv and a v specific monovalent cation effects on modification of reovirus infectivity by chymotrypsin digestion in vitro ex traordinary effects of specific monovalent cations on activation of reovirus transcriptase by chymotrypsin in vitro new intermediate subviral particles in the in vitro uncoating of reovirus virions by chymotrypsin reovirus transcriptase activation in vitro: involvement of an endogenous uncoating activity in the second stage of the process two modes of entry of reovirus particles into l cells hepatic localization of infectious agent in murine viral hepatitis antigenic relationships amongst coronaviruses a solid-phase system (space) for the detection and quantification of rotavirus in faeces small intestinal epithelial brush border enzymatic changes in suckling mice infected with reovirus type reovirus type infection in a suckling mouse: the effects on pancreatic structure and enzyme content viral hepatitis associated with trans plantable mouse leukemia. i. acute hepatic manifestations following treatment with urethane or methylformamide neonatal calf diarrhoea: identifica tion of reovirus-like (rotavirus) agent in faeces by immunofluorescence and immune electron microscopy lethal en teritis in infant mice caused by mouse hepatitis virus the laboratory diagnosis of epizoo tic diarrhoea of infant mice a rabbit rotavirus. vet. ree. diagnosis of rotavirus infection by cell culture a hepatitis virus of mice two coronaviruses isolated from central nervous system tissue of two multi ple sclerosis patients mouse hepatitis, reo- , and the theiler viruses activated macrophages in congenitally athymic "nude" mice and in lethally irradiated mice epidemic diarrheal disease of suckling mice epidemic diarrheal disease of suckling mice. i. manifestations, epidemiology, and attempts to trans mit the disease epidemic diarrheal disease of suckling mice. iii. the effect of strain, litter, and season upon the incidence of the disease a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin. i. isolation and biological prop erties of the virus serological inter relationships of murine hepatitis viruses production of high-titer bovine rotavirus with trypsin murine virus contaminants of leukemia viruses and transplantable tumors reovirus type infection in laboratory mice temperature-sensitive mutants of reovirus type : studies on the synthesis of viral rna effect of corticosteroids on mouse hepatitis virus infection an electron microscope study of the development of a mouse hepatitis virus in tissue culture cells importance of a new virus in acute sporadic enteritis in children comparison of the morphology of three coronaviruses classification of rotaviruses: report from the worid health organization/food and agriculture or ganization comparative virology program serologic study on the prevalence of murine viruses in five canadian mouse colonies a virus related to that causing hepatitis in mice (mhv) immunopathology of mouse hepatitis virus type . ii. effect of im munosuppression in resistant mice the appearance of a hepatotrophic virus in mice thymectomized at birth comparison of five diagnostic methods for the detection of rotavirus antigens in calf faeces morphological studies on simian virus sa and the "related" agent electron microscopic identification and subsequent isolation of a rotavirus from a dog with fatal neonatal diarrhea enhancement of rotavirus infectivity by trypsin and elastase identification of rotaviruses of different origins by the plaque-reduction test hemagglutination and hemagglutination-inhibition studies with a strain of nebraska calf diarrhea virus (bovine rotavirus) isolation and preliminary genetic and biochemical characterization of temperature-sensitive mutants of reovirus altered disease in rats due to mutants of reovirus type temperature-sensitive mutants of reovirus type : studies on the synthesis of viral peptides generation of cytolytic Τ lymphocytes after reovirus infection: role of the si gene nude", a new hairless gene with pleiotropic effects in the mouse electron microscopy in the diagnosis of infectious diarrhea the rotaviruses relation between viruses from acute gastroen teritis of children and newborn calves virus diarrhoea in foals and other animals. vet. ree. fluorescent virus precipitin test adverse effects of mouse hepatitis virus on ascites myeloma passage in the balb/cj mouse problems in checking inapparent infections in laboratory mouse colonies: an attempt at serological checking by anamnestic re sponse implication pancréatique chez la souris infectée avec le virus de l'hépatite murine immunisation de la souris "nude" contre le virus de l'hépatite murine par transfert de lymphocytes sensibilisés carrier state of anti body and viruses in a mouse breeding colony persistently infected with sendai and mouse hepatitis viruses effect of cortisone on genetic resistance to mouse hepatitis virus in vivo and in vitro ontogeny of macrophage resistance to mouse hepatitis in vivo and in vitro structure and physicochemical properties of coronaviruses comparison of an enzyme-linked immunosorbent assay for quantitation of rotavirus an tibodies with complement fixation in an epidemiological survey enhancement of the pathogenicity of mouse hepatitis virus (mhvl) by prior infection of mice with certain leukaemia agents a hepatitis virus of mice production of hepatitis in mice by the combined action of two filterable agents mouse hepatitis virus and its pathogenic action reactive sites of reovirus type and their interaction with receptor substances reovirus type : physical characteristics and interaction with l cells the propagation of s virus of mouse hepatitis in tissue culture comparison of methods for immunocytochemical detection of rotavirus infections delayed hypersensitivity in mice infected with reovirus. ii. induction of tolerance and suppressor Τ cells to viral specific gene products spontaneous diseases and their control in laboratory animals electron microscopic examination of cells infected with reovirus tissue culture cytopathic and plaque assays for mouse hepatitis viruses recovery of reoviruses from wild and laboratory mice antibodies to mouse hepatitis viruses in human sera temperature-sensitive mutants of mouse hepatitis virus produce a high incidence of demyelination mouse hepatitis virus-induced recurrent demyelination quantitation of antibody to non-hemagglutinating viruses by single radial hemolysis: serological test for human coronaviruses new strain of mouse hepatitis virus as the cause of lethal enteritis in infant mice isolation of low-virulent mouse hepatitis virus from nude mice with wasting syndrome and hepatitis studies on the mechanism of destruc tion of lymphoid tissue in murine hepatitis virus (mhv ) infection. i. selective prevention of lymphoid necrosis by cortisone and puromycin physico-chemical properties of mouse hepatitis virus (mhv- ) grown on dbt cell culture viral gastroenteritis infantile enteritis viruses: morphogenesis and morphology is lactase the receptor and uncoating enzyme for infantile enteritis (rota) viruses? some distinctive biological characteristics of echo- virus new viral agents recovered from tissue cultures of monkey kidney cells. i. origin and properties of cytopathogenic agents svj, sv , sv , sv , svg, svn, sv , and sv pathology of diarrhea due to mouse hepatitis virus in the infant mouse isolation of mouse hepatitis virus from infant mice with fatal diarrhea some aspects on the transmission of hepatitis Β antigen; model experiments by mosquitoes with murine hepatitis virus the molecular biology of reovirus studies on the effect of chymotrypsin on reovirions reproduction of reoviridae the effect of a murine hepatitis virus on the liver the fine structure of reovirus, a new member of the icosahedral series a microtiter solid phase radioimmunoassay for detection of the human reovirus-like agent in stools detection of differences among human and animal rotaviruses using analysis of viral rna the cellular nature of genetic susceptibility to a virus the coronaviruses reoviruslike agent in stools: association with infantile diarrhea and development of serologic tests antigentic relationships among five reovirus-like agents by complement fixation vertical transmission of mouse hepatitis virus infection in mice some characteristics of hemaggluti nation of certain strains of "ibv-like" virus studies on the etiology and transmission of epidemic diarrhea of infant mice observations on the control and natural history of epidemic diarrhea of infant mice (edim) responses of the mouse to the virus of epidemic diarrhea of infant mice. neutralizing antibodies and carrier state two viruses causing diarrhea in infant mice an apparently new lethal virus disease of infant mice epizootic diarrhea of infant mice and lethal intestinal virus infection of infant mice practical control of diarrheal disease in a commercial mouse colony reovirus infection in suckl ing mice: immunofluorescent and infectivity studies discussion of kraft, l. m. two viruses causing diarrhea in infant mice in vivo interference between heterologous rotaviruses the induction of interferon by temperature-sensitive mutants of reovirus, uv-irradiated reovirus, and subviral reovirus particles diagnosis of rotavirus, adenovirus, and heφes virus infections by immune electron microscopy using a serum-in-agar diffusion method autoimmune and virus-induced demyelinating dis eases mechanism of demyelination of jhm virus encephalomyelitis untersuchungen über die entstehung von riesenzellen in mäusemakrophagenkulturen nach infektion mit dem mäusehepatitisvirus (mhv- ) differential growth of mhv(pri) and mhv(c h) in genetically resistant c h rendered susceptible by eperythrozoon coccoides relationship of phagocytic activity to pathogenicitiy of mouse hepatitis virus as affected by triolein and cor tisone immunodiffusion and immunoelectrophoretic studies of reovirus antigens immunopathology of mouse hepatitis virus type infection. iii. clinical and virologic observation of a persistent viral infection immunopathology of mouse hepatitis virus type infection. i. role of humoral and cell-mediated immunity in resistance mechanisms neonatal susceptibility to mhv infection in mice. i. transfer of resistance genetic study of mouse sensitivity to mhv infection: influence of the h- complex polykaryocytosis and replication of mouse hepatitis virus in peritoneal macrophages in vivo and in vitro models of demyelinating diseases: tropism of the jhm strain of murine hepatitis virus for cells of glial origin an ultrastructural study of virions and cores of reovirus type infectivity assay of reoviruses: comparison of immunofluorescent cell count and plaque methods the digestive system the nature of the polypeptide encoded by each of the double-stranded segments of reovirus type trans-stadial maintenance of reovirus type in the mosquito culex pipiens fatigans weidmann and its implications coronaviruses: a comparative review coronaviruses as causes of diseases: clinical observa tions and diagnosis growth in suckling-mouse brain of "ibv-like" viruses from patients with upper respiratory tract disease antigenic relationships among the coronaviruses of man and between human and animal coronaviruses the polypeptides of human and mouse coronaviruses rotaviruses-a review moφhology and chemical composition of rotaviruses observations on the growth of mouse hepatitis virus (mhv- ) in mouse macrophages a. hepatitis virus complicating studies with mouse leukemia identity of cowdry type Β inclusions and nuclear bodies: observations in reovirus encephalitis an antigenic subunit pre sent in rotavirus infected faeces plaque assay of neonatal calf diarrhoea and the neutralising antibody in human sera the classification and nomenclature of viruses calf diarrhea (scours) reproduced with a virus from a field outbreak taxonomy of viruses a reo-like virus isolated from juvenile american oys ters (crassostrea virginica) moφhology of a reo-like virus isolated from juvenile american oysters (crassostrea virginica) pathogenesis of rotaviral infection counter-immunoelectro-osmophoresis for the detection of infantile gastroenteritis virus (orbi-group) antigen and antibody solid phase radioimmunoassay for the detection of rotavirus experimental studies on hepatitis virus of mice in tissue culture mechanisms in the pathogenesis of diarrhea: a review a new member of hepatoencephalitis group of murine viruses multiplication and cytopathogenicity of mouse hepatitis virus in mouse cell cultures purification and characterization of epi zootic diarrhea of infant mice virus acute hepatitis associated with mouse leukemia. i. etiology and host range of the causal agent in mice acute hepatitis associated with mouse leukemia. i. pathological features and transmission of the disease acute hepatitis associated with mouse leukemia. iv. the relationship of eperythrozoon coccoides to the hepatitis virus of prince ton mice acute hepatitis associated with mouse leukemia. v. the neurotropic properties of the causal virus recovery and behavior of hepatitis virus from swiss mice injected with ascites tumor pathogenicity of murine hepatitis virus recovered from infant swiss mice an oncolytic virus recovered from swiss mice during passage of an ascites tumour anomalous high native resistance of athymic mice to bacterial pathogens detection of rotavirus by serological trapping on antibody-coated electron micro scope grids further light on mouse hepatitis defective virions of reovirus virusinduced diabetes mellitus: reovirus infection of pancreatic / -cells in mice electron micrographic features of acute murine reovirus hepatitis ultrastructural features of chronic murine hepatitis after reovirus type infection an electron microscopic study of murine reovirus- encephalitis the biliary tract in acute murine reovirus infection studies on the exocrine pancreas. Π. ultrastructural investigation of reovirus pancreatitis epidemic diarrheal disease of suckling mice. iv. cytoplasmic inclusion bodies in intestinal epithelium in relation to the disease an epidemic diarrheal dis ease of suckling mice. ii. inclusions in the intestinal epithelial cells propagation of mouse hepatitis virus (mhv- ) in monolayer cell cultures from liver of newborn mice virus studies with germfree mice. i. preparation of serologic diagnostic rea gents and survey of germfree and monocontaminated mice for indige nous murine viruses prevalence of viruses in mouse colonies rat coronavirus (rcv): a prevalent, naturally occurring pneumotropic virus of rats the isolation of reovirus type from mosquitoes and a sentinel infant mouse enzyme-linked immunosorbent assay for detection of antibodies to murine hepatitis virus detection of neonatal calf diarrhea virus, infant reovirus-like diarrhea virus, and a coronavirus using the fluorescent virus precipitin test chronic obstructive jaundice induced by reovirus type in weanling mice experimental viral hepatitis the fate of murine hepatitis virus (mhv- ) after intravenous injection into susceptible mice lack of transplacental transmissibility of mhv- virus oligodendrocytes and their myelin-plasma membrane connections in jhm mouse hepatitis virus encephalomyelitis proteolytic enzymes and rotavirus sa- plaque formation a genetic map of reovirus. ii. assignment of the double-stranded rnanegative mutant groups c, d, and Ε to genome segments plaque formation by reoviruses cytochemical, fluorescent-antibody and electron microscopic studies on the growth of reovirus (echo ) in tissue culture pathogenic murine coronaviruses. i. characterization of biological behavior in vitro and virus-specific in tracellular rna of strongly neurotropic jhmv and weakly neurotropic a v viruses pathogenic murine coronaviruses. iii. biological and biochemical characterization of temperature-sensitive mutants of jhmv demonstration of reovirus-like particles in intestinal contents of piglets with diarrhoea comparison of the genomes of simian, bovine, and human rotaviruses by gel electrophoresis and detec tion of genomic variation among bovine isolates serologic grouping of reoviruses by hemagglutinationinhibition mouse hepatitis virus infection as a highly contagious, prevalent, enteric infection of mice chloroform inactivation of reovirus hemagglutinins the growth of the virus of epidemic diarrhea of infant mice (edim) in organ cultures of intestinal epithelium pathology of experimental hepatitis in mice the kupffer cell reaction in murine and human viral hepatitis with particular reference to the origin of acidophilic bodies electron microscopy of the hepatocellular and kupffer-cell lesions of mouse hepatitis, with par ticular reference to the effect of cortisone factors associated with the incidence of infantile diarrhea in mice reoviruses. a new group of respiratory and enteric viruses formerly classified as 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of mouse hepatitis virus (gledhill) in tissue culture electron and fluorescence microscopy of mouse hepatitis virus resistance to fatal central nervous system disease by mouse hepatitis virus, strain jhm. i. genetic analysis resistance to fatal central nervous system disease by mouse hepatitis virus, strain jhm. ii. adherent cell-mediated protection postsplenectomy viral hepatitis characterization of a coronavirus. i. structural pro teins: effects of preparative conditions on the migration of protein in polyacrylamide gels an electron microscopic study of viral hepatitis in mice difference in response to mouse hepatitis virus among susceptible mouse strains factors involved in the age-dependent resistance of mice infected with low virulence mouse hepatitis virus pathogenesis of mouse hepatitis infection. the role of nasal epithelial cells as a primary target of low virulence virus asymptomatic infection of mouse hepatitis virus in the rat in vitro growth characteristics and heterogeneity of mouse hepatitis virus type igm and igg response to sheep red blood cells in mouse hepatitis virus-infected nude mice response of nude mice to a mouse hepatitis virus isolated from a wasting nude mouse persistent infection with mouse hepatitis virus of low virulence in nude mice the role of macrophages in the early resistance to mouse hepatitis virus infection in nude mice enhanced phagocytic activity of macrophages in mouse hepatitis virus-infected nude mice neonatal susceptibility to mhv infection in mice. ii. role of natural effector marrow cells in transfer of resistance techniques for rotaviral propagation rotavirus neutralization by human milk serological relation ships between rotaviruses from different species as studied by comple ment fixation and neutralization potentiating effect of k-virus on mouse hepatitis virus (mhv-s) in weanling mice runde" virus, a coronavirus-like agent associated with seabirds and ticks immunoelectroosmophoresis for de tection of 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enzyme-linked immunosorbent assay key: cord- -m q roay authors: agostini, maria l.; pruijssers, andrea j.; chappell, james d.; gribble, jennifer; lu, xiaotao; andres, erica l.; bluemling, gregory r.; lockwood, mark a.; sheahan, timothy p.; sims, amy c.; natchus, michael g.; saindane, manohar; kolykhalov, alexander a.; painter, george r.; baric, ralph s.; denison, mark r. title: small-molecule antiviral β-d-n( )-hydroxycytidine inhibits a proofreading-intact coronavirus with a high genetic barrier to resistance date: - - journal: j virol doi: . /jvi. - sha: doc_id: cord_uid: m q roay coronaviruses (covs) have emerged from animal reservoirs to cause severe and lethal disease in humans, but there are currently no fda-approved antivirals to treat the infections. one class of antiviral compounds, nucleoside analogues, mimics naturally occurring nucleosides to inhibit viral replication. while these compounds have been successful therapeutics for several viral infections, mutagenic nucleoside analogues, such as ribavirin and -fluorouracil, have been ineffective at inhibiting covs. this has been attributed to the proofreading activity of the viral ′- ′ exoribonuclease (exon). β-d-n( )-hydroxycytidine (nhc) (eidd- ; emory institute for drug development) has recently been reported to inhibit multiple viruses. here, we demonstrate that nhc inhibits both murine hepatitis virus (mhv) ( % effective concentration [ec( )] = . μm) and middle east respiratory syndrome cov (mers-cov) (ec( ) = . μm) with minimal cytotoxicity. nhc inhibited mhv lacking exon proofreading activity similarly to wild-type (wt) mhv, suggesting an ability to evade or overcome exon activity. nhc inhibited mhv only when added early during infection, decreased viral specific infectivity, and increased the number and proportion of g:a and c:u transition mutations present after a single infection. low-level nhc resistance was difficult to achieve and was associated with multiple transition mutations across the genome in both mhv and mers-cov. these results point to a virus-mutagenic mechanism of nhc inhibition in covs and indicate a high genetic barrier to nhc resistance. together, the data support further development of nhc for treatment of covs and suggest a novel mechanism of nhc interaction with the cov replication complex that may shed light on critical aspects of replication. importance the emergence of coronaviruses (covs) into human populations from animal reservoirs has demonstrated their epidemic capability, pandemic potential, and ability to cause severe disease. however, no antivirals have been approved to treat these infections. here, we demonstrate the potent antiviral activity of a broad-spectrum ribonucleoside analogue, β-d-n( )-hydroxycytidine (nhc), against two divergent covs. viral proofreading activity does not markedly impact sensitivity to nhc inhibition, suggesting a novel interaction between a nucleoside analogue inhibitor and the cov replicase. further, passage in the presence of nhc generates only low-level resistance, likely due to the accumulation of multiple potentially deleterious transition mutations. together, these data support a mutagenic mechanism of inhibition by nhc and further support the development of nhc for treatment of cov infections. keywords coronavirus, nucleoside analogue, rdrp, rna-dependent rna polymerase, sars-cov, mers-cov, pandemic, antiviral resistance t he emergence of severe acute respiratory syndrome (sars) in and middle east respiratory syndrome (mers) in has underscored the ability of coronaviruses (covs) to cause lethal disease in humans ( , ) . mers-cov continues to infect humans in the middle east, and four additional human covs (hcovs), hcov- e, hcov-nl , hcov-oc , and hcov-hku , continue to circulate globally and cause respiratory disease ( ) ( ) ( ) ( ) . the continued circulation in bat populations of sars-and mers-like covs that can replicate efficiently in primary human airway cells further demonstrates the potential for covs to emerge and cause severe disease in the future ( ) ( ) ( ) ( ) . while sars-cov and mers-cov outbreaks have been controlled, largely through public health measures ( ) ( ) ( ) , the potential for future outbreaks highlights the need for safe and effective therapeutics to combat cov infections. there are currently no approved therapeutics or vaccines for any human cov infection. previous efforts to treat cov infections with existing antivirals did not conclusively benefit clinical outcomes; thus, the current standard of care remains mostly supportive ( ) ( ) ( ) . several targets for direct-acting antivirals are being investigated to treat cov infections ( ) ( ) ( ) . because the viral replication machinery performs an essential role in genome replication, therapeutics approved to treat multiple different viral infections are aimed at this target ( ) . many approved antivirals are classified as nucleoside analogues, compounds that mimic natural nucleosides to inhibit viral replication ( ) . inhibition by nucleoside analogues can be accomplished through a variety of mechanisms. common mechanisms of action include incorporation of the analogue by the viral polymerase to induce premature termination of strand synthesis and loss of essential genetic information through mutagenesis ( ) ( ) ( ) ( ) . a previous study reported that the nucleoside analogues ribavirin (rbv) and -fluorouracil ( -fu) did not potently inhibit covs, and this finding was attributed to the proofreading capabilities of the viral =- = exoribonuclease (exon) ( ) . recent reports have demonstrated the inhibition of wild-type (wt) covs by nucleoside analogues such as galidesivir (bcx ) and remdesivir (gs- ) ( ) ( ) ( ) . while these compounds have shown efficacy against covs, administration of multiple compounds simultaneously may be required to effectively treat cov infections and control the emergence of drug resistance, as has been demonstrated for other viral infections ( ) . ␤-d-n -hydroxycytidine (nhc) (eidd- ; emory institute for drug development), a cytidine analogue, has recently been shown to inhibit multiple viruses, including chikungunya virus, venezuelan equine encephalitis virus (veev), respiratory syncytial virus (rsv), hepatitis c virus, norovirus, influenza a (iav) and b viruses, and ebola virus ( ) ( ) ( ) ( ) ( ) ( ) . previous reports have demonstrated increased introduction of transition mutations in viral genomes after treatment, as well as a high genetic barrier to resistance ( , ) . antiviral activity of nhc has also been reported against the human ␣-cov hcov-nl , as well as the ␤-cov sars-cov ( , ) . neither the nhc mechanism of action nor nhc resistance has been described for any cov to date. in this study, we investigated nhc inhibition and resistance in two divergent ␤-covs, murine hepatitis virus (mhv) and mers-cov. we show that nhc potently inhibits wt mhv and mers-cov with minimal cytotoxicity. we also demonstrate that mhv exon proofreading activity has a limited but measurable effect on sensitivity to nhc. we observed an nhc inhibition profile consistent with a mutagenic mechanism of action featuring an accumulation of transition mutations, indicative of a high genetic barrier to resistance. experiment with two divergent ␤-covs: the model cov mhv and the epidemically circulating zoonotic cov mers-cov. nhc treatment resulted in a dose-dependent reduction in viral titers for mhv ( fig. a ) and mers-cov (fig. b ). this inhibition resulted in % effective concentrations (ec s) of . m for mhv (fig. c ) and . m for mers-cov (fig. d) . we detected negligible changes in dbt- cell viability out to m (fig. e ) and % cytotoxic concentration (cc ) values above m in vero cells (fig. f ). the antiviral activity was not due to cytotoxicity, as the selectivity indexes were Ͼ , for mhv and Ͼ for mers-cov. together, these results confirm potent inhibition of ␤-covs by nhc. the nhc inhibition profile in covs is consistent with mutagenesis. to better understand the mechanism through which nhc inhibits cov replication, we performed a time of drug addition assay to determine at what point in the viral replication cycle nhc acts ( ) . we added m (ϳ times the ec ) nhc at the indicated times preor postinfection (p.i.) of cells with wt mhv at a multiplicity of infection (moi) of pfu/cell and quantified viral replication after a single infectious cycle. compared to the vehicle (dimethyl sulfoxide [dmso]) control, nhc significantly inhibited mhv replication when added at or before h postinfection (fig. a) , suggesting that nhc acts at early stages of the viral replication cycle. we next determined the effect of nhc on mhv rna levels and compared it to the effect on the infectious-virus titer. rna levels were reduced by approximately -fold at the highest tested concentration of nhc in both mhv-infected cell monolayers (fig. b ) and supernatants (fig. c ). in contrast, the viral titer was reduced up to , -fold at this concentration. we therefore calculated the ratio of infectious virus per viral rna genome copy number normalized to the untreated control (specific infectivity) after nhc treatment and found that the specific infectivity of wt mhv was reduced in a dose-dependent manner after treatment with increasing concentrations of nhc (fig. d ). together, these data are consistent with a mutagenic mechanism of nhc anti-cov activity. nhc treatment increases transition mutations present across the mhv genome. to directly test the effect of nhc treatment on the mutational burden, we treated wt mhv with increasing concentrations of nhc and performed full-genome next-generation sequencing (ngs) on viral populations released after a single round of infection. our data demonstrated a dose-dependent increase in mutations present at low frequencies (Ͻ % of the viral population) across the genome after treatment with increasing concentrations of nhc ( fig. a to c). further analysis of the types of mutations introduced by nhc revealed an increase in the total number of transition mutations with increasing nhc concentrations ( fig. d to f). the relative proportions of g:a and c:u transitions among all observed mutations were increased by to % in the presence of m nhc and to % in the presence of m nhc compared to the vehicle control ( fig. g and h) . conversely, the relative proportions of a:g and u:c transitions decreased with increasing nhc concentrations compared to the vehicle control ( fig. g and h) . together, these results demonstrate that nhc treatment during a single round of wt mhv infection causes predominantly g:a and c:u transition mutations that are detectable at low frequencies across the genome. these data further support a mutagenic mechanism of action for nhc inhibition of wt mhv. nhc inhibition is modestly enhanced in the absence of exon proofreading. mutagenic nucleoside analogues, such as rbv and -fu, have been ineffective at potently inhibiting wt covs due to the exon proofreading activity ( ) . a proofreadingdeficient [exon(Ϫ)] mhv mutant displays increased sensitivity to previously tested nucleoside analogues, indicating that proofreading dampens inhibition by these compounds ( , , ) . therefore, we tested the sensitivity of exon(Ϫ) mhv to nhc inhibition. our results indicate that nhc decreases the titers of both wt and exon(Ϫ) mhv in a dose-dependent manner but that exon(Ϫ) mhv demonstrates a statistically passage in the presence of nhc yields low-level resistance associated with multiple transition mutations. to better understand the development and impact of nhc resistance in covs, we passaged two lineages of wt mhv times in the presence of increasing concentrations of nhc and tested the sensitivity of passage (p ) mhv populations to nhc inhibition. we found that the lineage (mhv p . ) viral population showed no change in sensitivity to nhc compared to wt mhv (fig. a ). however, lineage (mhv p . ) showed a decrease in sensitivity to nhc inhibition in a titer reduction assay, especially at higher concentrations of compound. we observed a modest (approximately -fold) increase in ec values for mhv nhc passage viruses (wt mhv, ec ϭ . m; mhv p . , ec ϭ . m; mhv p . , ec ϭ . m) (fig. b ). this suggests that mhv passage resulted in minimal resistance to nhc. we next sought to determine if passaging wt mhv in the presence of nhc altered the replication capacities of these viruses. we found that both lineages showed a delay in replication but ultimately reached peak titers similar to that of wt mhv (fig. c ). this delay in replication suggests that mhv p is less fit than wt mhv. to identify mutations associated with these phenotypes after passage, we sequenced complete genomes of mhv p . and mhv p . . both lineages passaged in the presence nhc had accumulated over consensus mutations distributed across the genomes ( fig. d and e; see table s in the supplemental material). in comparison, a previous study reported that wt mhv accumulated only total mutations after passages in the absence of drug ( ) . further analysis of the p mhv mutational profile demonstrated that slightly more of the total mutations in both lineages were synonymous changes that did not result in an amino acid change as opposed to nonsynonymous changes, which did alter the amino acid sequence ( fig. f ; see table s ). additionally, the vast majority of mutations in both lineages were transition mutations resulting in a purine-to-purine or pyrimidine-to-pyrimidine change (fig. g ). both lineages contained only two transversion mutations resulting in a purine-topyrimidine or pyrimidine-to-purine change. though all possible transition mutation types were detected in both viral-lineage populations, the majority in both passage lineages were g:a transitions (fig. h) , which is consistent with the mhv ngs data (fig. ) . to determine if the mutational profile at p was consistent with an earlier passage, we analyzed the whole genomes of both lineages and at p . both lineages demonstrated fewer mutations at p than at p , but the profiles of synonymous versus nonsynonymous changes and the transition mutations were similar (see fig. s and table s in the supplemental material). to determine whether the lack of robust resistance to nhc was broadly applicable across ␤-covs, we assessed the capacity of mers-cov to evolve resistance to nhc. as (fig. b ), corresponding to approximately -fold resistance. similar to mhv, we observed no substantial shift in the dose-response curve for mers-cov, indicating minimal acquired resistance. nhc p viruses replicated similarly to wt p mers-cov (fig. c) . we sequenced both lineages of the mers-cov p population and detected consensus mutations in mers-cov nhc p . (fig. d ; see table s in the supplemental material) and consensus mutations in mers-cov nhc p . ( fig. e ; see table s ) that were randomly distributed across the genome. both mers-cov nhc p . and mers-cov nhc p . accumulated nonsynonymous and synonymous mu- tations in roughly equal proportions (fig. f) . as in mhv, the mutations detected in mers-cov p lineages were predominantly transition mutations (fig. g ). further analysis of these mutations revealed that the predominant type of transition was lineage dependent. the majority of transition mutations in mers-cov nhc p . were g:a transitions, as was observed in both p mhv lineages, whereas mers-cov nhc p . contained similar numbers of each type (fig. h) . these results indicate that mers-cov can achieve low-level resistance to nhc and that development of resistance is associated with the accumulation of multiple transition mutations. together, our data suggest nhc acts as a mutagen and that it poses a high genetic barrier to resistance for ␤-covs. in this study, we demonstrate that nhc potently inhibits the divergent ␤-covs mhv and mers-cov. our data are consistent with a virus-mutagenic mechanism of action, as evidenced by a decrease in specific infectivity and an increase in g:a and c:u transition mutations present at low frequencies across the genome after treatment with nhc. we also demonstrate that robust resistance to nhc is difficult to achieve in both mhv and mers-cov. both wt mhv and exon(Ϫ) mhv are sensitive to nhc inhibition, suggesting that nhc is able to overcome exon-mediated proofreading to inhibit wt covs and that it interacts with covs differently than other previously tested nucleoside analogues. utility of the broad-spectrum antiviral nhc as a pan-cov therapeutic. early work with nhc focused on the mutagenic effects of the compound in multiple bacterial systems ( , , ) . more recently, the antiviral properties of the compound have been reported for multiple rna viruses, including chikungunya virus, venezuelan equine encephalitis virus, respiratory syncytial virus, hepatitis c virus, norovirus, influenza a and b viruses, and ebola virus ( ) ( ) ( ) ( ) ( ) ( ) . nhc has also been shown to potently inhibit sars-cov and hcov-nl ( , ) , suggesting potential utility in treating cov infections ( ) . based on previous studies, nhc appears to primarily inhibit viral replication by mutagenesis ( , ) . serial passaging in the presence of nhc led to low-level resistance for veev, but no detectable resistance for rsv, iav, or bovine viral diarrhea virus, indicating a high barrier to resistance ( , , ) . consistent with the previous studies, we demonstrated that nhc is mutagenic in covs and that serial passaging yields low-level, approximately -fold resistance. low-level resistance has also been observed for remdesivir, another nucleoside analogue that potently inhibits covs. approximately -fold resistance to remdesivir is conferred by two mutations in the cov rna-dependent rna polymerase (rdrp) ( ) . this study further expands the known antiviral spectrum of nhc to include mhv and mers-cov, two genetically divergent ␤-covs, and supports nhc development as a broad-spectrum cov antiviral. nhc inhibition may circumvent exon-mediated proofreading. nhc is the first mutagenic nucleoside analogue demonstrated to potently inhibit proofreading-intact covs. previous studies have demonstrated that viruses lacking exon proofreading activity [exon(Ϫ) viruses] are more sensitive to inhibition by nucleoside analogues, especially rbv and -fu ( , , , ) . this increased sensitivity has been attributed to the inability of exon(Ϫ) viruses to efficiently remove incorrect nucleosides ( ) . however, we observed a minimal change in nhc sensitivity between wt mhv and exon(Ϫ) mhv, especially by ec . this suggests that nhc interacts with the cov replicase differently than other previously tested nucleoside analogues. one explanation is that nhc may evade removal by the proofreading exon. studies investigating nucleosides that inhibit dna viruses have suggested an inability of the viral exonuclease to efficiently excise some nucleoside analogues ( , ) . further, a previous study suggested that the t dna exonuclease activity was incapable of removing nhc ( ) . while the sars-cov exon efficiently removes =-terminal mismatches regardless of type ( , ) , the effect of nhc on this activity has not been investigated. interestingly, mismatches readily observed during single-nucleotide elongation by the sars-cov polymerase in the absence of drugs correspond to mismatches that would lead to the g:a and c:u transitions observed after nhc treatment ( ) . this suggests that the cov polymerase could be naturally more prone to make these types of errors, which are then magnified by nhc. this could lead to a scenario where exon cannot prevent dipping below the error threshold, ultimately resulting in lethal mutagenesis and similar inhibition of both wt mhv and exon(Ϫ) mhv ( ) . several nucleosides, including the mutagenic rbv, have multiple demonstrated mechanisms other than direct incorporation into the genome ( , ) . thus, another explanation for the unique potency of nhc in the presence of an active proofreading exon is that it may inhibit viral replication by additional mechanisms beyond mutagenesis. indeed, previous reports have suggested that nhc may also interfere with the rna secondary structure or virion release to cause inhibition ( , ) . further, exogenous c or u in the presence of nhc could rescue viral replication in hcv, chikungunya virus, rsv, and influenza a virus ( , , ) , indicating that nhc competes with exogenous nucleosides at some stage prior to viral inhibition. these results raise the possibility that nhc could inhibit a process that results in similar inhibition of these viruses by a mechanism unrelated to exon. thus, future studies will be important to investigate the role of proofreading in nhc inhibition of covs to shed light on the intricacies of nhc inhibition of the cov replication complex. nhc mutagenesis may hinder emergence of robust resistance to nhc. the decrease in specific infectivity, along with the accumulation of transitions across the cov genome, supports a mutagenic mechanism of action for nhc in covs. nhc resistance in covs was modest and difficult to achieve, as we obtained approximately -fold resistance after passages. resistance was associated with multiple mutations. interestingly, mers-cov accumulated fewer mutations over passages than mhv. while differences in viral mutation rates could be the driver of this difference, previous studies have suggested that mhv does not have a higher mutation rate than mers-cov ( ) ( ) ( ) . the differences in mutation accumulation between mhv and mers-cov may be a product of different passage conditions. while mhv was passaged with a consistent transfer volume, mers-cov passage volumes were adjusted over time to sustain viral replication under escalating selection for drug resistance. the constant-volume passaging conditions may have more severely bottlenecked mhv populations and fixed more mutations in the genome than the variable-volume passaging conditions applied to mers-cov ( ) . alternatively, this difference could also reflect a difference in mutational robustness of the mhv and mers-cov genomes, though this proposition needs to be investigated further ( , ) . while a portion of the mutations that accumulated during passage likely contribute to nhc resistance, mutations in proteins dispensable for viral replication in cell culture, such as ns and nsp , may be merely tolerated because of their limited effect on viral fitness in the context of our passage conditions ( ) ( ) ( ) . few common mutations arose in both mhv and mers-cov passage series (see tables s to s ), suggesting that multiple pathways to low-level nhc resistance exist in covs. interestingly, for both mhv and mers-cov, the p lineage that demonstrated a greater change in sensitivity to nhc was the lineage that had fewer overall mutations (fig. and ) . both mhv passage lineages replicated less well than wt mhv, suggesting that the accumulation of mutations during passage may negatively impact viral fitness and the ability of mhv to evolve robust resistance to nhc. further, the mhv lineage that did not result in changed sensitivity to nhc by p (mhv p . ) had fewer mutations present at consensus by p than the other lineage (see fig. s ). thus, it is possible that the accumulation of deleterious mutations counteracts the potential benefits of resistance mutations ( ) . if this is the case, mutations promoting nhc resistance would need to arise early during passage to help mitigate the accumulation of excess deleterious mutations. alternatively, the inability to evade inhibition by nhc may lead to the accumulation of a greater number of nhc-associated transitions and ultimately a higher mutational burden that may impact viral fitness ( , ) . together, our results support the hypothesis that establishment of resistance to nhc in covs requires a delicate balance of resistance-promoting mutations, viral fitness, and accumulation of deleterious mutations. thus, defining the roles of individ-ual nhc resistance-associated mutations will be an important goal for future studies. overall, our results support further development of nhc as a broad-spectrum antiviral for treatment of cov infections and contribute new insights into important aspects of cov replication. cell culture. murine astrocytoma delayed brain tumor (dbt) ( ) and vero (atcc ccl- ) cells were maintained at °c in dulbecco's modified eagle medium (dmem) (gibco) supplemented with % fetal bovine serum (fbs) (invitrogen), % penicillin and streptomycin (gibco), and . % amphotericin b (corning). viruses. all work with mhv was performed using the recombinant wt strain mhv-a (genbank accession number ay [ ] ). mers-cov stocks were generated from cdna clones (genbank accession number jx [ ] ). compounds and cell viability studies. nhc was synthesized at the emory institute for drug development and prepared as a mm stock solution in dmso. cell viability was assessed using celltiter-glo (promega) in -well plates according to the manufacturer's instructions. dbt and vero cells were incubated with the indicated concentrations of compound at °c for h (dbt) or h (vero). cell viability was determined using a veritas microplate luminometer (promega) or glomax (promega), with values normalized to those of vehicle-treated cells. nucleoside analogue sensitivity studies and generation of ec curves. subconfluent monolayers of dbt cells were infected with mhv at an moi of . pfu per cell for h at °c. the inoculum was removed and replaced with medium containing the indicated compound concentration. cell supernatants were harvested h postinfection. titers were determined by plaque assay as described previously ( ) . subconfluent monolayers of vero cells were infected at an moi of . pfu/cell of mers-cov. after virus adsorption for min at °c, the inoculum was removed. the cells were washed with pbs and incubated with medium containing the indicated concentrations of nhc or dmso (vehicle control). after h, the supernatant was collected and titers were determined by plaque assay as described previously ( ) . ec and ec values and curves were generated using the nonlinear regression curve fit in graphpad (la jolla, ca) prism software. time of drug addition assay. subconfluent monolayers of dbt cells were treated with medium containing dmso or m nhc (ϳ times the ec ) at the indicated times pre-or postinfection. the cells were infected with wt mhv at an moi of pfu/cell for h at °c. the virus inoculum was removed and replaced with fresh medium. culture supernatant was harvested h postinfection, and the viral titer was determined by plaque assay. quantification of viral genomic rna. subconfluent dbt cells were infected with wt mhv at an moi of . pfu/cell. the inoculum was removed after h of incubation at °c, and medium containing the indicated concentration of nhc was added. total rna from cells and supernatant rna were harvested using trizol reagent (invitrogen) after h. both total rna and supernatant rna were extracted by phase separation. total rna was purified by ethanol precipitation, and supernatant rna was purified using a purelink rna minikit (invitrogen) according to the manufacturer's protocol. total rna was reverse transcribed using superscript iii (invitrogen) to generate cdna, which was quantified by quantitative pcr (qpcr) as previously described ( ) . data are presented as ϪΔΔct , where ΔΔc t denotes the change in the threshold cycle for the viral target (nsp ) normalized to the control (glyceraldehyde- -phosphate dehydrogenase [gapdh]) before and after drug treatment. the supernatant rna was quantified using one-step quantitative reverse transcriptase pcr (qrt-pcr) as previously described ( ) . the data are presented as the fold change in genome rna copies normalized to vehicle control. determination of specific infectivity. subconfluent dbt cells were infected with wt mhv at an moi of . pfu/cell. the inoculum was removed after h of incubation at °c, and medium containing the indicated concentration of nhc was added. supernatant rna was harvested using the trizol reagent (invitrogen) after h, followed by extraction and quantification as described above. the viral titer was determined by plaque assay. the specific infectivity was calculated as the number of pfu divided by the supernatant genome rna copy number. this ratio was then normalized to that of the vehicle control. ngs studies. subconfluent dbt cells were infected with wt mhv at an moi of . pfu/cell and treated with the indicated concentrations of nhc. the supernatant was collected h postinfection. purified viral rna was submitted to genewiz (south plainfield, nj) for library preparation and sequencing. briefly, after quality controls, viral rnas were randomly fragmented using heat. libraries were prepared and sequenced on the illumina hiseq platform. genewiz performed base calling and read demultiplexing. trimmomatic was used to trim adapter contaminants and reads shorter than bp and to filter low-quality bases (q score Ͻ ) ( ). the paired-end fastq reads were then aligned with the mhv genome using bowtie to generate a sam file ( ) . samtools was used to process the resultant alignment file and to calculate the coverage depth at each nucleotide, generating a sorted and indexed bam file. lofreq was used to call substitution variants, including low-frequency variants, and to generate a variant file ( ) . the bash shell and excel were used to further process and analyze the resultant vcf file. a frequency of . was used as a cutoff for variants, consistent with previous reports ( ) . absolute numbers of mutations are reported for each nhc treatment. the percentage of the total mutations for each specific mutation type was calculated using these numbers. the difference in percentage for each class of mutation after treatment compared with vehicle control is referred to as the relative proportion of these mutations. mhv population passage in the presence of nhc. wt mhv was passaged in triplicate in increasing concentrations of nhc from m to a maximum of m. infection was initiated for passage at an moi of . pfu/cell. viral supernatants were harvested from each viral lineage and frozen when the cell monolayer demonstrated % cytopathic effect (cpe) or after h. a constant volume of l was used to initiate subsequent passages. all three lineages were maintained until passage , when lineage demonstrated no visible cpe upon multiple attempts at varying concentrations. lineages and were maintained until passage . after each passage, total rna was harvested from infected cell monolayers using the trizol reagent. viral rna was extracted from passage and passage samples and reverse transcribed using superscript iii, followed by generation of pcr amplicons to cover the whole genome. dideoxy amplicon sequencing was performed by genewiz and analyzed to identify mutations present at greater than % of the total using macvector. viral mutation maps depicting the identified mutations were generated using macvector. mers-cov population passage in the presence of nhc. three parallel independent passage series of wt mers-cov were performed on vero cells in the presence of gradually increasing concentrations of nhc up to a maximum concentration of . m to select for drug-resistant mutant viruses. virus adaptation to nhc-supplemented complete culture medium was assessed by monitoring the progression of characteristic mers-cov cpe. the volumes of transferred culture supernatants were adjusted empirically to balance continuous selective pressure against culture extinction. each of triplicate lineages in the mers-cov passage experiment was sustained through passage . however, the third lineage was severely impaired in replication and was excluded from further analysis. total infected-cell mers-cov rna purified from monolayers infected with terminal-passage (p ) culture supernatant was used to generate rt-pcr products for consensus sanger sequencing of the complete viral genome (genewiz). changes in passaged virus nucleotide and deduced amino acid sequences were identified via alignment with the wt parental virus genomic sequence using macvector. virus replication assays. subconfluent monolayers of dbt (mhv) or vero (mers-cov) cells were infected with wt or nhc-passaged viral populations at an moi of . pfu/cell for h (mhv) or min (mers-cov). inocula were removed, and the cells were washed with pbs before addition of prewarmed medium. supernatants were harvested at the indicated times postinfection, and titers were determined by plaque assay. statistics. statistical tests were performed using graphpad (la jolla, ca) prism software as described in the respective figure legends. supplemental material for this article may be found at https://doi. a novel coronavirus associated with severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia global seasonal occurrence of middle east respiratory syndrome coronavirus (mers-cov) infection seroepidemiologic studies of coronavirus infection in adults and children epidemiology and clinical presentations of the four human coronaviruses e, hku , nl , and oc detected over years using a novel multiplex real-time pcr method clinical impact of human coronaviruses e and oc infection in diverse adult populations a sars-like cluster of circulating bat coronaviruses shows potential for human emergence sars-like wiv -cov poised for human emer further evidence for bats as the evolutionary source of middle east respiratory syndrome coronavirus receptor usage and cell entry of bat coronavirus hku provide insight into bat-to-human transmission of mers coronavirus control measures for severe acute respiratory syndrome (sars) in taiwan description of a hospital outbreak of middle east respiratory syndrome in a large tertiary care hospital in saudi arabia control of an outbreak of middle east respiratory syndrome in a tertiary hospital in korea clinical management and infection control of sars: lessons learned sars: systematic review of treatment effects ribavirin and interferon therapy for critically ill patients with the middle east respiratory syndrome: a multicenter observational study potential antivirals and antiviral strategies against sars coronavirus infections coronavirusesdrug discovery and therapeutic options antiviral drugs specific for coronaviruses in preclinical development antivirals and antiviral strategies approved antiviral drugs over the past years advances in the development of nucleoside and nucleotide analogues for cancer and viral diseases antimicrobial strategies antiviral nucleoside and nucleotide analogs: a review inhibitors of the hepatitis c virus polymerase; mode of action and resistance coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics protection against filovirus diseases by a novel broad-spectrum nucleoside analogue bcx therapeutic efficacy of the small molecule gs- against ebola virus in rhesus monkeys broad-spectrum antiviral gs- inhibits both epidemic and zoonotic coronaviruses antiviral combination therapy for treatment of chronic hepatitis b, hepatitis c, and human immunodeficiency virus infection ␤-d-n( )-hydroxycytidine is a potent antialphavirus compound that induces high level of mutations in viral genome characterization of ␤-d-n -hydroxycytidine as a novel inhibitor of chikungunya virus antiviral activity of nucleoside analogues against norovirus orally efficacious broadspectrum ribonucleoside analog inhibitor of influenza and respiratory syncytial viruses identification of a new ribonucleoside inhibitor of ebola virus replication ribonucleoside analogue that blocks replication of bovine viral diarrhea 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correction and ribavirin excision from coronavirus rna inhibition of purified human and herpes simplex virus-induced dna polymerases by cidofovir diphosphate inhibits adenovirus dna polymerase via both nonobligate chain termination and direct inhibition, and polymerase mutations confer cidofovir resistance on intact virus effect of proofreading and dam-instructed mismatch repair systems on n -hydroxycytidineinduced mutagenesis rna =-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp /nsp exoribonuclease complex theories of lethal mutagenesis: from error catastrophe to lethal defection demethylation therapy as a targeted treatment for human papillomavirus-associated head and neck cancer the predominant mechanism by which ribavirin exerts its antiviral activity in vitro against flaviviruses and paramyxoviruses is mediated by inhibition of imp dehydrogenase viral mutation rates mers coronavirus in dromedary camel herd, saudi arabia spread, circulation, and evolution of the middle east respiratory syndrome coronavirus viral quasispecies evolution evolution favors protein mutational robustness in sufficiently large populations the origins of mutational robustness murine coronavirus nonstructural protein ns is not essential for virus replication in transformed cells cell-typespecific type i interferon antagonism influences organ tropism of murine coronavirus the nsp replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for viral replication pathways to extinction: beyond the error threshold the contribution of epistasis to the architecture of fitness in an rna virus mutation and epistasis in influenza virus evolution molecular anatomy of mouse hepatitis virus persistence: coevolution of increased host cell resistance and virus virulence systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a engineering infectious cdnas of coronavirus as bacterial artificial chromosomes high fidelity of murine hepatitis virus replication is decreased in nsp exoribonuclease mutants growth and quantification of mers-cov infection trimmomatic: a flexible trimmer for illumina sequence data scaling read aligners to hundreds of threads on general-purpose processors lofreq: a sequence-quality aware, ultra-sensitive variant caller for uncovering cell-population heterogeneity from high-throughput sequencing datasets sequence-specific error profile of illumina sequencers we thank members of the denison laboratory for thoughtful discussions regarding this work. key: cord- - ufgwxxg authors: lai, m. m. c.; fleming, j. o.; stohlman, st. a.; fujiwara, k. title: genetic heterogeneity of murine coronaviruses date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: ufgwxxg several mouse hepatitis viruses (mhv) with different pathogenicity were studied by oligonucleotide fingerprinting. two strains, mhv-k and mhv-d, which were isolated in japan and, which cause anaplasia and necrosis of bone marrow and diarrhea, respectively, were found to be closely related to mhv-a , the prototype mhv. two other mhv strains, isolated from nude mice, were found to have diverged extensively from the known mhv strains. the mhvs isolated from separate cloned neuroblastoma cell lines persistently infected with jhm strain were also found to have diverged more markedly than the corresponding virus maintained under the conditions of lytic infection. genetic divergence during persistent infection may be one of the mechanisms by which the mhv diverges. mouse hepatitis virus (mhv), a member of the coronaviridae, is an p~na virus containing a single-stranded rna genome of . × a molecular weight ( ) . it has been isolated from mice in many parts of the world and has been associated mainly with hepatitis and encephalitis ( , ) . it repiieates in a variety of tissue culture cells of rodent origin, including fibroblasts and neuronal cells ( ) . mhv infection in vitro generally results in the lysis of infected cells, but can also lead to persistent infection ( , , ) . in nature, the virus survives by establishing latent infections in the gastrointestinal tract of mice ( ) . viruses of this group have also been shown to cause enteric infections and wasting diseases in nude mice ( ) . thus, the various strains of mouse hepatitis virus exhibit a wide range of pathogenic potentials. we have previously studied several mhv strains by oligonucleotide fingerprinting, and found that they were widely divergent (t ). however, some strains are much more closely related, e.g. mhv-a and mhv- , suggesting a close genetic origins (t ). this approach is useful in determining the evohltionary relationship among various mhv strains. in this study, we have further examined several mouse hepatitis virus strains isolated in japan. two of these viruses revealed extensive genetic homology with the prototype mitv-a strain. thus, these strains appear to have remained genetically stable in nature. we have also compared the jhm strain of mhv (mhv-jhm) passaged under the conditions of lyric infection with those maintained as persistent infections in vitro. the viruses maintained under the latter conditions exhibit much more variability, suggesting that persistent infections may be a mechanism contributing to the genetic heterogeneity of the current mhv strains. the mhv-d strain was isolated from an infant mouse with fatal diarrhea, and has been shown to cause hepatitis and diarrhea in suckling mice ( , , ) . mi-iv-k was isolated from all asymptomatie adult nude mouse (nu/nu, balb/c). this strain causes bone marrow anaplasia and necrosis as well as hepatosplenic hemopoiesis ( ). mttv-i~tuu was isolated from a nude mouse with wasting syndrome and hepatitis ( ) , and causes fatal wasting disease in nude mice but is not pathogenic in euthymic mice ( ) . these three strains were all isolated in japan. mhv-i~ was isolated from a nude mouse and was a generous gift of dr. michael collins, microbiological associates, bethesda, maryland. the two mhv-a strains studied were originally obtained from dr. c. bond of the university of california at san diego and from dr. k. ]~lolmes of the uniformed services university, bethesda, maryland. all viruses were grown in dbt cells as previously described ( ) . the in vitro persistent infection was established by growing mhv-jhm in a mouse neuroblastolna cell line ( ) . single cell clones were obtained in the presence or absence of anti-jhm hyperimmune aseitic fluid. two of the viruses were rescued from the nonprodueer cell clones by fusing with dbt cells in the presence of polyethylene glycol ( ) . these two viruses, designated cs i and cs , were found to be cold-sensitive mutants ( ) . two other virus strains, a and a , were obtained from the virusproducing persistently infected cell clones. the same procedures as described by lai and sto~il~a~-( ) were followed. the ~ p-]abeled virus was extracted with phenol in the presence of per cent si)s, and the rna was prepared by separation on -- per cent sucrose gradients in an sw rotor at , rpm for hour. the s l%l~a peak was collected and used for oligonucleotide fingerprinting. the analysis of ~ °-labeled s rna by two-dimensional polyacrylamide gel eleotrophoresis followed essentially the same procedures as described previously (]i, ). to study the genetic relationship of different mhv strains causing various diseases in mice, we first examined the oligonucleotide fingerprints of two of the ikhvs isolated in japan. one, mhv-d, was isolated from a suckling mouse with fatal diarrhea ( ) and could cause a similar disease in experimentally infected. infant mice ( ) . the other, mhv-k, causes anaplastie and necrotic lesions in bone marrow of mice (t ). the ~ p-labeled s rnas of these two viruses were digested with rnase tt, and separated by two-dimensional polyaerylamide gel electrophoresis. as shown in fig. , these two viruses have very similar oligonueleotide fingerprints, suggesting a close genetic origin. n{ost surprisingly, the fingerprints of mhv-d (fig. b) is indistinguishable from that of mhv-a (fig. c) , the prototype mhv, which we have studied previously ( ) . these data suggest that the mhv-d might have been derived from the mhv-a strain quite recently. the oligonucleotide fingerprint of mhv-k (fig. t a) was also similar to that of mhv-a , but, contained six additional oligonueleotides which were not present in mhv-d or nhv-a . the very high extent of homology between mhv-k and mhv-a suggests that mhv-k is also a recent derivative of mitv-a . the finding that ~hv-k contained six more large tl-o]igonucleotides than mhv-a suggests either that mtiv-k has greater genetic complexity than mhv-a or that mitv-k is heterogeneous. to distinguish between these two possibilities, several subelones of mttv-k were plaquepurified and analyzed by oligonuclcotide fingerprinting. such an analysis showed that all of the mhv subclones are similar but not identical, suggesting that the virus is indeed heterogeneous; however, all of them are very similar to mhv-a (data not shown). the nihv-a strain used here for comparison is the same strain we have examined previously ( ) . it has relatively weak pathogenicity ( ) . no clinically apparent symptoms were induced by the intracranial injection of plaqueforming units of mhv-a into mice (data not shown). however, the nhv-a strains maintained in other laboratories have much higher hepatotropic properties, inducing very severe hepatitis (k. holmes, personal communication). we therefore compared the mhv-a strains obtained from different laboratories. we found that their oligonueleotide fingerprints are indistinguishable (data not shown). therefore, these two mhv-a strains are essentially identical, or have only very minor differences that could not be detected by this technique. several mhvs have been isolated from latently infected nude mice which had developed wasting syndrome. these virus strains are of relatively low virulence in euthymic mice but produced fatal wasting diseases in nude mice ( ) . in order to assess the genetic origin and the pathogenic potentims of these viruses peculiar to nude mice, we examined the oligonucleotide fingerprints of two of these isolates : one, designated nnu, was isolated in japan ( ) comparison with other known [hv isolates ( ) revealed that they were also very distinct from any other mhv strains studied so far. thus, these viruses represent quite unique mhv isolates. to assess the genetic stability and variability of mitv and to study the possible mechanism of viral divergence observed as above, we examined the heterogeneity of viral genomic sequences in viruses isolated from persistently and latently infected cells. the persistent infection was established by infection of a mouse neuroblastoma cell line with the j h m strain of mhv ( ) . the viruses were rescued from separate cell lines derived from single cell clones by polyethylene glycol-mediated fusion with dbt or cl cells ( ) . two of these rescued viruses, cs and cs , have been shown to be cold-sensitive ( ) . as shown in fig. : the oligonucleotide fingerprints of both cs t (fig. b) and cs (fig. c) are very similar to that of the small-plaque strain of the parental j h m (jhiw-ds) (fig. a) ( ) . however, they both contain some oligonucleotides which are unique for each strain. in addition, cs does not contain three oligonucleotides present in the jh /lds. this result suggests that these two viruses represent the variants of jhm-ds, which were selected during persistent infection. two other viruses, a (fig. e ) and as (:pig. f), were produced spontaneously from two different persistently infected neuroblastoma cell clones. these two viruses have oligonucleotide fingerprints similar to that of a large-plaque variant of jht { (jhm-dl), i.e. they contain a unique spot z, but lack the spot. x, the latter of which is characteristic of jhm-ds ( fig. a; ) . however, they also contain at least one unique oligonueleotide spot not seen in the oligonueleotide fingerprints of either jhm-dl or jhm-ds, suggesting that these two viruses are derivatives of jhm-dl. the isolation of jhm-ds and jhm-dl variants h'om the persistently infected cells was in sharp contrast with the viruses isolated under the conditions of lytic infection. as we have shown previously, mhv-jtim produced plaques of varying size on i)bt cells ( ) . we have randomly picked at least plaques on dbt cells and analyzed them by oligonucleotide fingerprinting. only two fingerprint patterns were observed, one with the ds-type fingerprint and the other with the dl-type ( ) . the jhm-ds was subsequently serially passaged for at least passages on dbt cells via lytic infection. the cloned viruses were studied at passages and . the oligonucleotide fingerprints of the viruses passaged times were exactly identical to that of jhm-ds (data not shown). no viruses of other genotypes were isolated, suggesting that mhv viruses are relatively stable during lyric infection, whereas persistent or latent infection in the-~euroblastoma cell line tends to select for various types of variants. we have previously studied six strains of mouse hepatitis virus isolated at different geographical locations and found them to be genetically heterogeneous ( ) . in this report, we have analyzed additional mhv strains, some of which were isolated in japan. one surp ~sing finding is the genetic similarity of those strains isolated in japan to the mhv-a strain, the miiv prototype. these strains might, therefore, represent, very recent derivatives of mhv-a or vice versa. this finding also suggests that these strains have remained relatively stable during passages through animals. it is interesting to note that these genetically highly related viruses have different pathogenicity: the mtiv-a used in our laboratory is weakly pathogenic ( ) , n[hv-d causes diarrhea ( ), mhv-k causes bone marrow diseases ( ), while another closely related strain mhv- causes severe hepatitis ( ) . some of the genetic differences observed among these strains might be associated with the pathogenicity of the respective viruses. however, it is equally possible that these differences are not related to viral pathogenicity. in this regard, it is worth noting that several strains of mhv-a have different pathogenieities and yet they could not be distinguished by oligonueleotide fingerprinting. it is possible that there are minor genetic differences among these viruses or that the different pathogenicity was contributed by host factors. this issue requires complete nueleotide sequence analysis of these and additional miiv strains. the finding that the viruses rescued from the persistently in~ected cells were more heterogeneous than those passaged under the conditions of lyric infection is similar to the observation with vesicular stomatitis virus ( ) . in that case, the vsv underwent progressive genetic changes as the cells were passaged in vitro ( ) . similar long-term culturing of cells infected with other mhv strains has not been carried out; nevertheless, it is remarkable that the viruses rescued by cell fusion or produced spontaneously from these persistently infected cells are different from the parental viruses, while the randomly selected viruses maintained under the conditions of lyric infection are genetically stable. it is unclear why these cell lines would select for these unusual virus variants. it is noteworthy, however, that these variants produce lytic infection upon reinfection of the neuroblastoma cell line, and preliminary data indicated that. these viruses do not produce diseases different from those induced by the parental viruses (s. a. s~ohlma~¢. unpublished results). whether the selection of such variants in vivo contributes to the pathogenicity of the viruses is not clear at the present time. the propensity of the persistently infected cells to select for viral mutants might be one of the mechanisms by which the mhv diverges. it is also noteworthy that the two viruses rescued by fusion of ]atently infected cell clones with the permissive cells were the derivatives of jhm-ds, while the viruses spontaneously produced by the persistently :infected cells were jhm-dl derivatives. these two viruses, i.e. ds and dl, differ in their ability to cause c:hronic demyelination ( ) . there might be a correlation between the dl ~nd the ds viruses and the ability to yield virus progeny under these conditions. such a possibility is being studied in our laboratories. mouse hepatitis, reo- and theiler viruses latent virus as exemplified by mouse hepatitis virus (mhv) persistent infection with mouse hepatitis virus jhm strain in dbt ceil culture isolation of low-virulent mouse hepatitis virus from feces in infected mouse breeding colony isolation of low-virulent mouse hepatitis virus from nude mice with wasting syndrome and hepatitis evolution of multiple genome mutations during long-term persistent infection by vesicular stomatitis virus evolution of a coronavirus during persistent infection in vitro pathology of diarrhea due to mouse hepatitis virus in the infant mouse isolation of mouse hepatitis virus from infant mice with fatal diarrhea hepatosplenic myelosis in naturally occurring mouse hepatitis virus infection in the nude mouse mouse hepatitis virus a : mrna structure and genetic localization of the sequence divergence from hepatotropic strai~ ?¢ii-tv- the i%na of mouse hepatitis virus comparative analysis of rna genomes of mouse hepatitis viruses pathogenic murine coronaviruses. i, characterization of the biological behavior in vitro and virus-specific intraeellular rna of strongly neurotropic jhmv and weakly neurotropic a v viruses coronaviridae. in: comprehensive virology murine eoronaviruses: isolation and characterization of two plaque morphology variants of the jh v~ neurotropic strain characterization of the coldsensitive murine hepatitis virus mutants rescued from latently infected cells by fusion rescue of a positive-stranded i%na virus form antigen-negative neuroblastoma cells stability of neurotropic mouse hepatitis virus (jhm strain) during chronic infection of neuroblastoma cells persistent infection with mouse hepatitis virus of low virulenee in nude mice the biology and pathogenesis of coronaviruses we thank the excellent technical a~sistance of todd lasman, todd kennell, and chris jatton. we also wish to thank claudia troesch, alisa young, and raymond mitche]i for excellent editorial assistance. this work was supported by a research grant -a from the national multiple sclerosis society. key: cord- -dlmzcl g authors: mcintosh, kenneth; kapikian, albert z.; turner, horace c.; hartley, janet w.; parrott, robert h.; chanock, robert m. title: seroepidemiologic studies of coronavirus infection in adults and children( ) date: - - journal: am j epidemiol doi: . /oxfordjournals.aje.a sha: doc_id: cord_uid: dlmzcl g mcintosh, k. a. z. kapikian, h. c turner, j. w. hartley, r. h. parrott and r. m. chanock. (lab. of infectious diseases, niaid, nih, bethesda, md. ) sero-epidemiologic studies of coronavirus infection in adults and children. amer. j. epid., , : – -a seroepidemiologic study of infection by coronavirus strains e, oc , oc , and mouse hepatitis virus (mhv) strain a- , is described. in adults with upper respiratory disease, two “outbreaks” of coronavirus infection occurred, one during the winter of – associated with complement fixing (cf) antibody responses to oc , oc and mhv, and the other during the following winter associated with cf antibody responses to e. in hospitalized children, infection with e was rare; infection with oc , oc , and mhv occurred less often in hospitalized children with lower respiratory tract disease ( . %) than in a control group with non-respiratory tract disease ( . %). the limitations of the cf test using available coronavirus antigens are discussed. several investigators have recently reported the recovery from adults with upper respiratory infection of ether-labile viruses morphologically indistinguishable from mouse hepatitis virus (mhv) and avian infectious bronchitis virus (ebv) ( ) ( ) ( ) ( ) ( ) ( ) . the distinctive appearance of these viruses by electron microscopy suggested that they be designated as a new group, the "coronaviruses" ( ) . members of the group are medium-sued, rna-containing, and etherlabile, and bear characteristic club-shaped surface projections. nine of the strains recovered from the human respiratory tract were originally isolated in human embryonic tracheal organ culture (hetoc) ( , , , ) . three of the nine were subsequently adapted to growth in monolayer tissue cultures and two others (oc and oc ) were successfully adapted to growth in suckling mice and shown to be antigenically related to several strains of mhv by cf tests and fluorescence staining ( , , ) . the remaining human strains were originally recovered in tissue culture monolayers and appeared antigenically similar or identical to the prototype tissue culture strain, e ( , ) . little is known of the epidemiology of human infection with coronaviruses. cf and neutralizing antibodies to several strains of mhv were found in military recruits before any coronavirus strains had been recovered from man ( ) . epidemiologic analysis of these findings indicated that, although fourfold rises in antibody were frequent, there was no discernible association with disease. both strain e and strain b have been shown to cause colds in adult volunteers ( , ) . preliminary seroepidemiologic surveys performed in this laboratory suggested that e, oc , and oc infection occurred with sporadic frequency in adults with upper respiratory tract disease ( , ) . this report describes a more extensive seroepidemiologic study using the available cf antigens from human strains and an antigen of mhv, strain a- , shown by hartley and others to detect cf antibody in human sera ( ) . adults. nasopharyngeal washings and acute phase sera were obtained from employees of the national institutes of health with upper respiratory tract disease on or before the fourth day of illness, and convalescent phase sera three weeks later. there were patients admitted to the study, some of them on multiple occasions, so that serum pairs were available for study. the periods covered were october, through may, , and september, through september , . some aspects of the study have been published ( , ) . infants and children. throat or nasal swabs and acute phase sera were obtained from pediatric patients on admission to children's hospital, washington, d.c., and convalescent phase sera about three weeks later. a diagnosis of lower respiratory tract disease (lrtd) indicated croup (laryngotracheobronchitis), bronchitis, pneumonia, or bronchiolitis. control sera were obtained at similar intervals from hospitalized patients with non-respiratory tract disease. individuals with incidental respiratory tract disease at the time of selection were excluded from the group. there was difficulty in finding sufficient control infants under months of age (see table ). members of the control group were not matched with respect to their length of hospital stay. the sera chosen for study were obtained from october, through april, , and from october, through april, ( ) . the preparation and maintenance of human embryonic intestine (hei) diploid cell cultures, and their use in the recovery of coronaviruses have been described ( ) . human embryonic tracheal organ cultures were prepared and maintained as previously reported ( ) . the methods used for recovery of coronaviruses in organ culture have also been described ( ). dr. hamre kindly supplied coronavirus strain e which had been purified by the terminal dilution technique. it was passaged several times in human diploid cell strain wi in this laboratory. cf antigen was prepared as a single lot of infected wi cells ( ) . eight units of antigen as measured with a standard convalescent human serum were used in all tests. cf antigen of mhv, strain a- , was prepared from infected mouse liver nctc tissue culture as previously described ( ) . a single lot of antigen was used for the study reported here. four to eight units of antigen as measured with hyperimmune mouse serum were used. the adaptation of organ culture grown coronavirus strains oc and oc to swiss mice and the preparation of cf antigens from infected mouse brain have been described ( ) . the antigens used in this study were from a single lot for each virus and were free of contamination with mycoplasma and cytopathic agents. moreover, screening cf tests were performed for possible contamination with theiler's agent, lymphocytic coriomeningitis virus, sendai virus, mouse leukemia, reoviruses, rubella, mumps, measles, respiratory syncytial (es) virus, parainfluenza virus types , , , and , and influenza virus types a, b, and c; all such tests were negative. the techniques of insuring that the antigens were also free of mhv strains have been previously described ( ) . eight to units of antigen as measured with hyperimmune mouse serum were used. control uninfected wi cell, nctc cell and mouse brain antigens were prepared in parallel to infected antigen lots and were included in each test. sera reacting with control antigens were not included in • the data reported. these omissions account for the differences in the total number of serum pairs analyzed for each antigen (see table ). cf tests were performed by the microtiter technique using overnight fixation at c and . - . units of complement as previously described ( ) . figure is a diagram representing the number of and per cent antibody responses to the various coronavirus antigens tested in mhv, and because of their reported close antigenic relationships ( ), in certain analyses these agents are grouped together. table shows the proportion of infants and children with and without lrtd showing fourfold or greater antibody responses to the three related coronavirus antigens oc , oc , and mhv, strain a- . when all age groups were combined, a positive correlation between coronavirus infection and lrtd did not exist. however, the youngest age group (under one year) with lrtd tended to have more coronavirus infections than the control group, but the difference was not statistically significant (p > . ). in all age groups taken together there was a significant negative correlation with lrtd (x* = . , p <. ). this negative correlation was particularly striking in the children over the age of one (x* = . , p <. ). in table is shown the proportion of individuals tested who had measurable ( : or greater) cf antibody to coronavirus antigens. during the period - cf antibody to strain e was rare in children ( . per cent) and quite common in adults ( per cent). cf antibody to oc and oc was increasingly common in children up to the age of three years, when it was measurable in approximately per cent of those tested. in adults, per cent of those tested had measurable cf antibody to one or both strains during - , and per cent during - . antibody to mhv, strain a- , was of somewhat lower prevalence (approximately per cent in children and per cent in adults during the periods noted above). information obtained from serologic, tissue culture, and organ culture studies is summarized in figures and . among adults (figure ) coronaviruses appeared to be rarely associated with upper respiratory tract disease during and . the frozen nasopharyngeal washings of a small group of patients, who in preliminary studies had shown cf antibody responses to strain e, were examined in hei tissue culture. three of these yielded coronaviruses serologically similar to strain e. sporadic frozen specimens were examined in tracheal organ in the second "outbreak" antibody responses to oc , oc , and mhv antigens were rare, and no coronaviruses were recovered in organ culture. in children, on the other hand, (figure ) there appeared to be no meaningful temporal pattern of coronavirus cf antibody responses, and no strains were recovered in organ culture. the children under study were examined for evidence of infection by other respiratory viruses, and a certain number of dual infections with myxo-or paramyxoviruses and coronaviruses were found. there was no evidence that coronavirus infection potentiated the pathogenic effect of infection by either parainfluenza virus or rs virus, the two viruses found most often in conjunction with coronaviruses (table ) . discussion these studies were undertaken to define more clearly the seroepidemiology of coronavirus infection in adult and pediatric popu-lations. serum samples and nasopharyngeal washings were studied from adults with colds occurring in two two-year periods. during the first period ( - ) detectable coronavirus infection was uncommon. however, in the winter of - , members of the oc -oc -mhv group were prevalent and detectable both by serologic and organ culture techniques. one year later members of the e group were prevalent and again detectable by both serologic responses and virus isolation. it is of interest that among adults all the coronavirus recoveries and most of the coronavirus serologic responses occurred in the four winter months of december, january, february, and march. in several surveys of colds in adults, respira-tory disease was common during these winter months but rhinovirus infection, though frequent in the fall and spring, was rare ( , , ) . this pattern of rhinovirus infection prevailed in this study during the period - . thus, during these winter months coronaviruses although still accounting for less than per cent of colds, became the predominant identifiable organisms associated with adult upper respiratory disease. it is evident from the studies of hospitalized children that infection with coronaviruses was not significantly associated in this survey with pediatric lower respiratory tract disease. indeed, a negative correlation of coronavirus infection with lrtd was found. it may be that the presence of severe lrtd requiring hospitalization interfered in some way with infection of the respiratory epithelium by coronaviruses. on the other hand, it seems more likely that this negative correlation reflects the fact that it was not possible to select a completely comparable control group. control children were drawn from different pediatric wards, and their lengths of stay in the hospital frequently differed from those of the test group. in any case, the data reported suggest that the coronaviruses tested were not an important cause of lrtd in infants and children during the period covered by this study. our studies did not provide information on the etiologic association of coronavirus infection with respiratory tract disease in adults. two coronavirus strains, e and b , were administered to adult volunteers and in both instances a significant number of common colds occurred ( , ) . data are still lacking, however, to show that members of the oc -oc serologic type cause disease under natural conditions. it is of interest that in the seroepidemiologic studies of hartley and others, where several strains of mhv were tested with sera from military recruits, no epidemiologic association of mhv or mhv-like virus infection with respiratory tract disease was made ( ) . the infrequency of detectable cf antibody to e in the pediatric population studied here was surprising. although e virus infection may indeed be rare in children, several other explanations for this anomalous finding are possible. the e antigen, in contrast to the other coronavirus antigens, may have reacted with cf antibody to a narrow range of coronavirus serologic types, owing either to the type of tissue in which it was made, or to some characteristic of the virus itself. possibly serum cf antibody (as opposed to serum neutralizing, or secretory antibody) did not develop in children in response to e virus infection. studies of e neutralizing antibody in different age groups would contribute to the clarification of this finding. antigenic studies of human coronaviruses have shown that those members of the group which were originally recovered in tissue culture are all closely related to the prototype virus strain e ( , ) . in contrast, the strains isolated in organ culture include at least two and probably several more anti-genie types ( ) . one type, exemplified by strains oc and oc , is clearly related to the mhv group. the other type or types have an indefinite relationship with mhv and with the oc -oc group. in particular, patients yielding coronaviruses in organ culture varied markedly in their antibody responses to the cf antigens used in this study. one such patient showed in multiple tests no rise to any of the coronavirus antigens. two others showed responses in some tests and not in others. infection with strain b was likewise difficult to detect serologically, using oc , oc , mhv and e antigens ( ) . the heterogeneity of antibody response in subjects with known or presumed coronavirus infection, and the probable insensitivity of presently available serologic tests in the detection of coronavirus infection indicate that the serologic studies reported here do not describe a complete picture of coronavirus infection. it is probable that undetected coronavirus infections occurred in these populations during the study periods. cf antigens from the other known coronavirus strains, and further efforts to isolate and characterize new strains will contribute to further definition of the epidemiology of coronavirus infection. cultivation of a novel type of common-cold virus in organ culture a new virus isolated from the human respiratory tract recovery in tracheal organ cultures of novel viruses from patients with respiratory disease the morphology of three previously uncharacterized human respiratory viruses that grow in organ culture isolation from man of "avian infectious bronchitis viruslike" viruses (coronaviruses) similar to e virus, and some epidemiologic observations sensitivity of l cells to some "new" respiratory viruses cultivation of difficult viruses from patients with common colds growth in suckling-mouse brain of "ibv-like" viruses from patients with upper respiratory tract disease antigenic relationships among the coronaviruses of man and between human and animal coronaviruses antibodies to mouse hepatitis virus in human sera effects of a "new" human respiratory virus in volunteers etiology of upper-respiratory-tract illness among civilian adults respiratory syncytial virus. ii. serologic studies over a -month period of children with bronchiolitis pneumonia, and minor respiratory diseases application of a microtechnique to viral serological investigations virologic studies of acute respiratory disease in young adults. iv. virus isolations during four years of surveillance rhinovirus infections in an industrial population. i. the occurrence of illness key: cord- - de oaq authors: madhugiri, ramakanth; fricke, markus; marz, manja; ziebuhr, john title: rna structure analysis of alphacoronavirus terminal genome regions date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: de oaq coronavirus genome replication is mediated by a multi-subunit protein complex that is comprised of more than a dozen virally encoded and several cellular proteins. interactions of the viral replicase complex with cis-acting rna elements located in the ′ and ′-terminal genome regions ensure the specific replication of viral rna. over the past years, boundaries and structures of cis-acting rna elements required for coronavirus genome replication have been extensively characterized in betacoronaviruses and, to a lesser extent, other coronavirus genera. here, we review our current understanding of coronavirus cis-acting elements located in the terminal genome regions and use a combination of bioinformatic and rna structure probing studies to identify and characterize putative cis-acting rna elements in alphacoronaviruses. the study suggests significant rna structure conservation among members of the genus alphacoronavirus but also across genus boundaries. overall, the conservation pattern identified for ′ and ′-terminal rna structural elements in the genomes of alpha- and betacoronaviruses is in agreement with the widely used replicase polyprotein-based classification of the coronavirinae, suggesting co-evolution of the coronavirus replication machinery with cognate cis-acting rna elements. coronaviruses are enveloped, positive-strand rna viruses with exceptionally large genomes of approximately kb. they have been assigned to the subfamily coronavirinae within the family coronaviridae (de groot et al., a; masters and perlman, ) . together with the families arteriviridae, roniviridae, and mesoniviridae, the coronaviridae form the order nidovirales (de groot et al., b) . the family coronaviridae is currently comprised of four genera called alpha-, beta-, gammaand deltacoronavirus. closely related virus species in these genera are grouped together in specific lineages. coronaviruses infect mammals and birds and include pathogens of major medical, veterinary and economic interest (de groot et al., a) , with severe acute respiratory syndrome (sars) coronavirus (sars-cov) and middle east respiratory syndrome (mers) coronavirus (mers-cov) providing two prominent examples of newly emerging, highly pathogenic coronaviruses in humans (drosten et al., ; ksiazek et al., ; zaki et al., ) . besides their large genome sizes, coronaviruses and related nidoviruses stick out from other plus-strand rna viruses by the large number of virally encoded nonstructural proteins that are either involved in viral rna synthesis or interact with host cell functions (reviewed in masters and perlman, ; ziebuhr, ) . most of the nonstructural proteins (nsp) are expressed as subdomains of two large replicase gene-encoded polyproteins called pp a (∼ kda) and pp ab (∼ kda). co-and posttranslational cleavage of pp a/ ab by two types of virus-encoded proteases gives rise to a total of - mature proteins that (together with the nucleocapsid protein and cellular proteins) form the viral replication-transcription complex (rtc) (almazán et al., ; schelle et al., ; ziebuhr, ; ziebuhr et al., ) . this multi-protein complex replicates the viral genome and produces an extensive set of -coterminal subgenomic messenger rnas (sg mrnas), the latter representing a hallmark of corona-and other nidoviruses (pasternak et al., ; sawicki et al., ; ziebuhr and snijder, ) . the sg mrnas are used to express open reading frames located in the -proximal third of the genome. they essentially encode the viral structural proteins, such as the nucleocapsid (n), membrane (m), spike (s) and envelope (e) proteins, and a varying number of accessory proteins, the latter often involved in functions that counteract antiviral host responses (masters and perlman, ; narayanan et al., b) . coronavirus sg mrnas contain a common leader sequence (approximately - nt) that is identical to the end of the genome. the complement of this sequence is attached to the end of nascent negative strands in a complex process called "discontinuous extension of negative strands" sawicki, , ; sawicki et al., ; sethna et al., ) . this process involves attenuation of negativestrand rna synthesis at transcription-regulating sequences (trs) located upstream of the individual orfs in the -proximal genome region ("body trss", trs-b). guided by basepairing interactions between the negative-strand complement of a trs-b and the trs located downstream of the leader on the viral genome ("leader trs", trs-l), the nascent minus strand may be transferred from its downstream position on the template (at the trs-b) to the trs-l, where negative-strand rna synthesis is then resumed and completed by copying the leader sequence. the set of antileader-containing sg minus-strand rnas is subsequently used as templates for the production of the characteristic nested set of capped, leader-containing and -polyadenylated sg mrnas in coronavirus-infected cells (lai et al., ; sawicki et al., ; sawicki and sawicki, ; sethna et al., ; spaan et al., ) . sg minus-strand rnas contain a u-stretch at their end, thus providing a template for polyadenylation of sg mrnas wu et al., ) . similar to other rna viruses, coronavirus genomes contain important rna signals in their and -terminal genome regions, mainly (but not exclusively) in the untranslated regions (utr). these signals are required for viral rna synthesis (replication and/or transcription) and are collectively referred to as cis-acting rna elements (chang et al., ; dalton et al., ; izeta et al., ; kim et al., ; liao and lai, ; lin et al., lin et al., , zhang et al., ) . as indicated above, coronaviruses also contain cis-acting elements at internal positions in the genome, the best documented ones being the leader and body trss which play a vital role in the transfer of the nascent minus-strand rna to an upstream position on the template (see above). other internal cis-acting elements include specific rna signals required for genome packing, which have been characterized in a small number of coronaviruses escors et al., ; morales et al., ; penzes et al., ) , and a complex rna pseudoknot structure located in the orf a-orf b overlap region that mediates a (− ) ribosomal frameshift event and thus controls the expression of the second large orf on the coronavirus genome rna (orf b) (brierley et al., (brierley et al., , de haan et al., ; namy et al., ) . both viral and cellular proteins, including the viral n protein, heterogeneous ribonucleoprotein (hnrnp) family members, polypyrimidine tract-binding protein, and poly(a)-binding protein (pabp), have been shown to bind to specific coronavirus cis-acting elements and there is evidence to support the biological significance of some of these protein-rna interactions (reviewed in shi and lai, ; sola et al., b) . the coronavirus rtc is a multi-subunit assembly comprised of more than a dozen viral and an unknown number of cellular proteins. the complex is anchored through transmembrane domains present in nsps , and to intracellular membranous structures that provide a specialized membrane-shielded compartment in (or at) which viral rna synthesis takes place (den boon and ahlquist, ; gosert et al., ; kanjanahaluethai et al., ; knoops et al., ; oostra et al., ; oostra et al., ; snijder et al., ; van hemert et al., ) . over the past years, viral components of the coronavirus rtc have been characterized in considerable detail, providing a wealth of functional and structural information (reviewed in imbert et al., ; masters, ; ulferts et al., ; ulferts and ziebuhr, ; ziebuhr, ) . a large number of virally encoded enzymes, including protease, adp-ribose- -phosphatase, ntpase, -to- helicase, rna -triphosphatase, rna polymerase, guanosine-n and ribose -o methyltransferases, -to- exoribonuclease and uridylatespecific endoribonuclease, have been identified (baker et al., ; bhardwaj et al., ; chen et al., chen et al., , decroly et al., decroly et al., , eckerle et al., ; ivanov et al., ; kanjanahaluethai and baker, ; minskaia et al., ; putics et al., ; saikatendu et al., ; seybert et al., ; te velthuis et al., ; ziebuhr et al., ) . in some cases, these activities could be linked to specific steps of viral rna synthesis and/or rna processing or were shown to interfere with cellular functions (reviewed in masters and perlman, ; ziebuhr, ) . there are multiple interactions between the individual replicase gene-encoded nsps and the structural basis and functional implications of these interactions have been studied in a few cases. for example, it has been shown that the exoribonuclease and ribose o-methyltransferase activities associated with nsp and nsp , respectively, are each stimulated by specific interactions with nsp (bouvet et al., ; decroly et al., ) . also, there is evidence that a heteromultimeric complex formed by nsp and nsp interacts with (and serves as a processivity factor for) the rna-dependent rna polymerase (rdrp, nsp ) (zhai et al., ) . additional interactions between individual subunits of the rtc have been suggested on the basis of two-hybrid screening data (pan et al., ; von brunn et al., ) and there is evidence that a substantial number of coronavirus nsps form homo-and/or heterooligomeric complexes (anand et al., (anand et al., , bouvet et al., ; chen et al., ; ricagno et al., ; su et al., ; xiao et al., ; zhai et al., ) . despite major progress in the characterization of proteins and cis-acting rna elements involved in coronavirus rna synthesis, the molecular mechanisms that mediate specific steps of coronavirus rna replication and transcription are far from being understood. important information on cis-acting rna elements has been obtained from studies using defective interfering (di) rnas and, more recently, genetically engineered coronavirus mutants (reviewed in brian and baric, ; masters, ; sola et al., b) . in this article, we will summarize previous work on (beta)coronavirus genomic cis-acting rna elements and will then move on to present conclusions from our recent bioinformatic and rna structure probing studies on cis-acting rna elements conserved in alphacoronaviruses. historically, cis-acting rna elements required for coronavirus rna synthesis have mainly been studied by using naturally occurring and genetically engineered defective interfering rnas (di rnas) (reviewed in brian and baric, ; brian and spaan, ; masters, ; sola et al., b) . di rnas are replicationcompetent genome-derived rna molecules that contain extensive internal deletions but retained all the cis-acting rna signals required for replication by functional replicase complexes provided by a helper virus that replicates in the same cell (levis et al., ; weiss et al., ) . essentially, these cis-acting elements comprise the untranslated -and -terminal genome regions but, in some cases, may also extend into coding regions. in some cases, they also contain noncontiguous sequences derived from internal genome regions. coronavirus di rnas were first described for the betacoronaviruses mhv and bcov (chang et al., ; de groot et al., ; hofmann et al., ; luytjes et al., ; makino et al., makino et al., , a makino et al., , b makino et al., , . subsequently, these studies were extended to alpha-and gammacoronaviruses (izeta et al., ; mendez et al., ; penzes et al., penzes et al., , . over the years, studies of defective genomes proved to be very useful for identifying coronavirus rna elements required for replication (and packaging). however, di rnas also have disadvantages, a major one being homologous recombination between the rna replicon and the helper virus genome. thus, for example, artificial di rnas containing mutant leader sequences rapidly acquire the leader sequence of the helper virus, a process commonly referred to as "leader switching" (chang et al., (chang et al., , . the latter occurs very often if poorly replicating mutant di rnas are to be characterized which generally require amplification steps by serial passaging to determine their phenotype. with the development of reversegenetic systems suitable to produce and manipulate full-length coronavirus cdna copies, an attractive alternative for studying cisacting rna elements at the genome level (including long range rna-rna interactions) is now available that overcomes some of the limitations of di rna-based systems (almazán et al., ; casais et al., ; scobey et al., ; tekes et al., ; thiel et al., ; van den worm et al., ; yount et al., yount et al., , . di rnas studies on representative betacoronaviruses revealed that less then nt from the end of the genome ( nt in mhv and nt in bcov) are required for replication (chang et al., ; kim et al., ; luytjes et al., ) . in subsequent studies on alphaand gammacoronaviruses, minimal cis-acting signals of and nt were determined for tgev (escors et al., ) and ibv, respectively (dalton et al., ) . the -terminal genome regions of nt (mhv) to nt (tgev) comprise the entire utr ranging in size from nt (mhv, bcov and hcov-oc ) to nt (tgev) and thus extend into the nsp -coding region of orf a (see below). by contrast, the gammacoronavirus ibv features a much larger utr ( nt) and lacks a counterpart of nsp (ziebuhr et al., ) . it therefore appears that the gammacoronavirus utr (alone) contains all the rna signals required for genome replication. cis-acting rna structures in the -terminal region of the coronavirus genome have first been studied for bcov using di rnabased systems (brown et al., ; chang et al., chang et al., , gustin et al., ; raman et al., ; raman and brian, ) . in the terminal nts of the bcov genome, four stem-loops (designated i [comprised of ia and ib], ii, iii, and iv) were defined. enzymatic probing and mutational analysis of both naturally occurring and genetically engineered di rnas were used to (i) corroborate the predicted rna secondary structures and (ii) determine their functional significance in di rna replication. more recently, two further stem-loops (called sl-v and sl-vi) were identified in the bcov nsp -coding region of which sl-vi was confirmed to be essential for di rna replication (brown et al., ) . subsequent studies suggested (a varying degree of) conservation of cis-acting elements among betacoronaviruses and even the more distantly related alpha-and gammacoronaviruses (chen and olsthoorn, ; kang et al., ) . to facilitate the discussion of data obtained in studies of different viruses by different laboratories, we will use in this article a uniform nomenclature for the main rna structural elements in the -proximal genome region (sl , sl , [sl if present], sl and sl ). the nomenclature is based on sl designations used by the leibowitz and giedroc laboratories and in predictions of genus-and lineage-specific conservation patterns of cis-acting elements in alpha-, beta-and gammacoronaviruses (chen and olsthoorn, ; kang et al., ; liu et al., liu et al., , . the proposed functional and structural conservation of cis-acting elements among betacoronaviruses received strong support by reverse-genetic data demonstrating that sars-cov sl , sl , and sl can functionally replace their counterparts in the mhv genome when introduced individually (kang et al., ) . by contrast, replacement of the entire mhv utr with that of sars-cov did not produce a viable mhv mutant, suggesting a requirement for additional stable or transient long-range rna-rna interactions of the utr with other genome regions. evidence to support this hypothesis was obtained in subsequent studies. for example, the energetically unstable lower part of mhv sl was found to be involved in long-range rna interactions with the utr ) (see below). also, in a study using mhv/bcov chimera, a region downstream of sl was revealed to be engaged in long-range interactions with the nsp -coding region, thus possibly forming an extensive higher-order rna structure (guan et al., ) . a subsequent bcov di rna mutagenesis study further suggested that this multipartite rna structure may involve several stem-loop (sub)structures identified in earlier studies (gustin et al., ; raman and brian, ) but require refolding of other rna structures suggested earlier to be essential for di rna replication (brown et al., ) . the study by su et al. ( ) also identified an intriguing requirement in cis of an oligopeptide sequence in the nproximal part of nsp , suggesting that nsp may be an essential cis-acting protein factor in betacoronavirus replication, in addition to its multiple other functions (brockway and denison, ; huang et al., a,b; kamitani et al., kamitani et al., , lei et al., ; lokugamage et al., ; narayanan et al., a; tanaka et al., ; tohya et al., ; wathelet et al., ; züst et al., ) . possible interacting partners for nsp remain to be identified. the -proximal sl and sl are predicted to be conserved across all genera of the coronavirinae (chen and olsthoorn, ; liu et al., ) . nuclear magnetic resonance (nmr) spectroscopy studies of mhv and hcov-oc sl rnas supported the predicted stem-loop and revealed - noncanonical base pairs in the middle of the stem. the fully base-paired sl was proposed to exist in equilibrium with higher-energy (partially unfolded) conformers. characterization of mhv mutants containing specific replacements in sl and sequence analysis of second-site revertants supports a "dynamic sl " model in which sl is structurally and functionally bipartite . while the upper part of sl is (required to be) stable, the lower part is (required to be) unstable, possibly indicating the requirement for an optimized stability to permit or fine-tune transient long-range (rna-or protein-mediated) interactions between the and utrs required for sgrna transcription and genome replication, respectively. sl is the most conserved structure in the coronavirus utr (chen and olsthoorn, ; liu et al., ) . it is comprised of a -bp stem and a highly conserved loop sequence, -cuugy- , that was shown to adopt a -uynmg(u)a or ucuyg(u)a-like tetraloop structure (lee et al., ; liu et al., ) . reverse genetics data confirmed that sl is required for mhv replication and may have a specific role in sgrna synthesis. within certain structural constraints, nucleotide replacements were found to be tolerated or could be rescued by increasing the stem stability, suggesting a limited plasticity of this important cis-acting rna element . the predicted sl (named sl-ii in bcov di rna studies) appears to be conserved only in a subset of beta-and gammacoronaviruses (chen and olsthoorn, ) . for bcov and closely related viruses, the trs-l core sequence (cs) has been proposed to be exposed in the sl loop region, a structure similar to the trs-l hairpin structure reported for the related arterivirus equine arteritis virus (eav) (chang et al., ; van den born et al., van den born et al., , . by contrast, in most other coronaviruses, the trs-l cs and flanking regions were suggested to be located in nonstructured regions (chen and olsthoorn, ; stirrups et al., ; wang and zhang, ) . sl is a long hairpin located downstream of the trs-l cs and suggested to be conserved in all coronavirus genera (chen and olsthoorn, ; raman et al., ; raman and brian, ) . in the vast majority of coronaviruses, sl contains a short orf comprised of only a few codons. because of its position in the colors are used to indicate conserved base pairs: from red (conservation of only one base-pair type) to purple (all six base-pair types are found); from dark (all sequences contain this base pair) to light colors ( or sequences are unable to form this base pair). the gray bars below the alignment indicate the extent of sequence conservation. gray shadows are used to link rna structures with the corresponding dot-bracket notations above the alignment. to refine the alignment, an anchor at the highly conserved apical loop of sl was used. genome upstream of orf a it is generally referred to as the uorf. recent reverse genetics work in the mhv system yang et al., ) showed that disruption of the uorf yields viable mutants that, however, acquire alternate uorfs upon serial passaging in cell culture. in vitro, uorf-disrupted rnas showed enhanced translation of the downstream orf. the available data suggest that the uorf represses orf a/ b translation and has a beneficial but non-essential role in coronavirus replication in cell culture. sl may be further subdivided into sl a and sl b. interestingly, despite its conservation in coronaviruses, sl tolerates extensive mutations. thus, for mhv, it was shown that base pairing in sl a is not required for replication and separate deletions of sl a and sl b are tolerated. by contrast, complete deletion of sl and a -nt deletion immediately downstream of sl abolished or profoundly impaired viral rna synthesis. the characterization of second-site mutations and a viable mhv mutant in which sl was replaced with a shorter sequence-unrelated stem-loop led to a model in which sl was proposed to function as a spacer element that controls the orientation of sl , sl , and trs-l and thereby directs subgenomic rna synthesis . the sl sequence overlaps with the 'hotspot' of the -proximal genomic acceptor required for bcov discontinuous transcription (wu et al., ) , thus further supporting a role of the region immediately downstream of trs-l in subgenomic rna synthesis. chen & osthoorn ( ) predicted that variations of another rna structure called sl may be conserved in specific coronaviruses genera and/or lineages. in alpha-and betacoronaviruses, sl extends into orf a. depending on the lineage studied, conserved loop sequences could be identified in substructural hairpins of sl . sequence conservation was found to be more pronounced in alpha-compared to betacoronaviruses. thus, for example, in alphacoronaviruses, three hairpins, called sl a, b, and c, each containing a -uuccgu- loop sequence, were identified. similar structures were only partly conserved in betacoronaviruses and significant lineage-specific variations in the substructural hairpins and their loop sequences were identified. a possible sl equivalent in gammacoronaviruses was predicted to adopt a rod-like structure that lacks conserved loop sequences (chen and olsthoorn, ). as outlined above, possible betacoronavirus sl substructures located within (or extending into) the nsp -coding region (termed sls iv, v, vi, and vii) have been characterized structurally and functionally using bcov di rna and mhv reverse genetics systems (brown et al., ; guan et al., guan et al., , raman and brian, ) . to a large extent, previous work on coronavirus cis-acting elements has focused on only two species of closely related lineage a betacoronaviruses (represented by bcov and mhv), while there is limited information on functionally and structurally related elements in other coronaviruses. we therefore decided to embark on a more detailed characterization of putative alphacoronavirus cis-acting elements located in the and genome regions. as a starting point, we used coronavirus genomes representing all the currently approved species from the four genera of the coronavirinae and the newly identified mers-cov (de groot et al., ) and calculated alignment-based secondary structures using locarna (v. . . ) . although locarna considers both sequence and secondary structure to calculate these multiple alignments, we failed to detect rna secondary structures conserved across all coronavirus genera when using these highly diverse sequences. we therefore resorted to producing separate genus-wide alignment-based secondary structure predictions for coronaviruses. to do this, we used complete utr regions and nts from the orf a end to calculate consensus structures with rnaalifold -r -p --color --nolp --mea (v. . . ) (lorenz et al., ) and -c, respectively, if constraints were used. the subgroups (lineages) obtained in these sequence alignments were consistent with the previously recognized subgroups of alpha-and betacoronaviruses (de groot et al., a, ) (not shown). as shown in fig. , all the functionally relevant rna secondary structure elements identified previously in the betacoronavirus genome regions, sl , sl and sl , could be identified. in line with previous predictions and despite the pronounced sequence diversity in the -terminal genome regions, these rna structures are suggested to be conserved among all currently approved betacoronavirus lineages and species. this conclusion is also supported by the large number of covariations, suggesting a strong selection pressure to retain these base-pairing interactions. fig. also shows the lack of conservation of a stable hairpin structure containing the trs-l. it should be noted that, for several betacoronaviruses, it is possible to force a stem-loop containing the trs-l, but this stem-loop is only supported by two conserved base pairs. in line with previous reports, bovine coronavirus (and other viruses belonging to the species betacoronavirus ) appear(s) to be an exception in that a more stable sl containing the -ucuaaac- sequence in the loop region can be predicted in this case. overall, the structure prediction shown in fig. turned out to be in perfect agreement with previous studies (see chapter . ). we therefore used this approach in subsequent studies of conserved rna structure elements located in the genome regions of alphacoronaviruses. predictions were verified and refined by rna structure probing analyses (ehresmann et al., ; qu et al., ) using in vitro-transcribed rnas with sequences corresponding to the -terminal genome regions of hcov- e and hcov-nl , respectively (to be published elsewhere). for the calculation of secondary structures of single sequences, we used rnafold --nolp (v. . . ) (lorenz et al., ) and -c, respectively, if constraints were used. using multiple alignments calculated with locarna, we were able to identify rna secondary structures conserved among (all) alpha-and betacoronaviruses, thus confirming and extending earlier studies (figs. and ) (chen and olsthoorn, ; raman et al., ) . these include (i) sl , (ii) sl (with its short stem and highly conserved loop region [ -uuugu- in alphacoronaviruses]), (iii) a poorly structured region containing the trs-l cs and some flanking sequence, (iv) sl (containing the uorf) and (v) sl , the latter being conserved very well among all alphacoronavirus species (fig. ) and significantly more diverse in betacoronaviruses (chen and olsthoorn, ) . the large number of covariant base pairs in the alphacoronavirus sl (fig. ) suggest significant constraints and a major functional role for this structure in alphacoronavirus (and, possibly, betacoronavirus) replication and there is indeed some experimental evidence to support this hypothesis (brown et al., ; su et al., ) . using rna structure probing information obtained for the -terminal nts of hcov- e and hcov-nl , we confirmed and refined our rna secondary structure predictions for two b-lineage alphacoronaviruses (figs. and ) . the data obtained in these studies (details to be published elsewhere) support a model in which the ∼ -nt genome regions consistently fold into major rna structures called sl , sl , sl , and sl . the latter contains hairpin substructures, sl a, b, and c, featuring highly conserved ( )-nt loop sequences. the consensus secondary structure predicted for alphacoronaviruses ( fig. ) was found to fit very well the individual structure predictions for hcov- e and hcov-nl ( fig. a and a ) and the inclusion of structure probing information as additional constraints required only very few minor adjustments in our structure predictions (figs. b and b). most importantly, the basal part of the predicted sl was now predicted to be unpaired, thereby extending the single-stranded region downstream of the trs-l and also affecting the spacing between sl and sl . the (predicted) basal stem of sl contains the most conserved sequence within the alphacoronavirus -terminal rna structural elements (fig. , see the red base pairs). it is therefore reasonable to think that this structurally flexible region is involved in long-range rna-rna interactions. in line with this idea, a previous tgev reverse genetic study showed that mutants permitting additional base-pairing interactions of the copy trs-b upstream of a reporter sgrna with the -gaaa- sequence immediately downstream of the tgev trs-l cs ( -acuaaac- ) (see also fig. ) enhance the production of this specific reporter sgrna (zúñiga et al., ) . these functional data and our structural analyses of alphacoronavirus -terminal genome regions lead us to suggest that the basal part of sl exists in a flexible state, thereby possibly facilitating strand transfer during sg minus-strand rna synthesis. both this rna structural flexibility and the role of proteins that bind in this region and thereby likely affect the sl structure remain to be further investigated. of note, hnrnp family members along with the viral n protein have been shown to bind in this region and the n protein has been suggested to have chaperone and trs-l/trs-b unwinding activities (galán et al., ; grossoehme et al., ; huang and lai, ; keane et al., ; li et al., li et al., , shi and lai, ; sola et al., a,b; zúñiga et al., zúñiga et al., , . it is therefore tempting to speculate that cellular and/or viral proteins bind and unwind the energetically labile sl substructure to facilitate the strand transfer during sg minus-strand rna synthesis. initial information on cis-acting elements required for rna replication was (again) derived from betacoronavirus di rna studies (kim et al., ; lin and lai, ; luytjes et al., ) . deletion mutagenesis data suggested that cis-acting elements encompass the entire -nt utr plus poly(a) tail, along with a portion of the nucleocapsid (n) protein gene. however, subsequent studies showed that the structural protein genes (including the n protein coding region) tolerate major changes including combinations of single-site mutations and rearrangements of entire genes, suggesting that the -proximal coding regions do not form part of the cis-acting element (de haan et al., ; goebel et al., b; lorenz et al., ) . similarly, for members of the species alphacoronavirus (tgev, fcov), it was shown that the n gene was not required for replication (izeta et al., ) and even deletions of the accessory protein genes a and b were tolerated in fcov, suggesting that the cis-acting replication signals do not exceed nts plus poly(a) tail (haijema et al., ) . for the gammacoronavirus ibv, a minimal -terminal sequence of nts was reported to be required for di rna replication (dalton et al., ) , supporting the idea that, across all coronavirus genera, the utr contains all the cis-acting elements required for replication. for mhv, it was shown that a significantly smaller fragment of no more than nts suffices for the initiation of negative-strand rna synthesis (lin et al., ) , suggesting differential requirements for plus-versus minus-strand rna synthesis. furthermore, using betacoronavirus di rna systems, a short poly(a) tract of at least - nts was found to be required for replication (spagnolo and hogue, ) . cis-acting rna elements present in the -terminal genome regions have been studied most extensively in betacoronaviruses. the first essential rna structural element, called bulged stemloop (bsl), was discovered in mhv (hsue and masters, ) . it comprises nts immediately downstream of the mhv n gene stop codon and was discovered as a result of attempts to replace the mhv utr with that of bcov (hsue and masters, ) . despite limited sequence identity in the utrs of the two viruses, replacements of the entire utr (and specific parts of it) were tolerated, suggesting the presence of conserved structures (rather than sequences) that perform cis-acting functions in betacoronavirus replication (hsue and masters, ) . the conservation of functionally equivalent elements in the (and ) utr(s) among betacoronavirus genomes was further supported by a study showing that a bcov-derived reporter di rna was efficiently replicated by a range of bcov-and mhv-related betacoronaviruses (wu et al., ) . interestingly, a possible bsl equivalent was also identified in ibv and other gammacoronaviruses and its functional significance was demonstrated using ibv di rna constructs (dalton et al., ) . the nearly perfect stem-loop structure in ibv comprises nts and ((((... ) )))))).. )))))))...........))))))).. (((....(((((((((..... .) ))))))) )....))))).....))). (......)))))..)....)))))).))..)) ))))))))) )...))))))......... fig. . alignment-based secondary structure prediction of genome regions of alphacoronaviruses. the viruses included in this analysis represent all currently recognized species in the genus alphacoronavirus. the alignment was calculated by locarna, the structure by rnaalifold. the consensus sequence is represented using the iupac code. colors are used to indicate conserved base pairs: from red (conservation of only one base-pair type) to purple (all six base-pair types are found); from dark (all sequences contain this base pair) to light colors ( or sequences are unable to form this base pair). the gray bars below the alignment indicate the extent of sequence conservation at a given position. gray shadows are used to link rna structures with the corresponding dot-bracket notations above the alignment. to refine the alignment, an anchor at the highly conserved core trs-l was used. is located at the upstream end of region ii, a conserved region in the gammacoronavirus utr. the second essential rna element within the (betacoronavirus) utr is a classical hairpin-type rna pseudoknot (pk) structure that was first discovered in bcov and shown to be required for di rna replication (williams et al., ) . in bcov, the pk comprises nts and is located immediately downstream of the bsl. equivalent pk structures were predicted to be conserved in beta-and alphacoronaviruses while gammacoronaviruses were proposed to retain only a few features of this pk or to lack this structure altogether (williams et al., ) . in a subsequent study using a reverse genetics approach, the functional significance of the pk in genome replication was demonstrated for mhv (goebel et al., a) . also the more distantly related betacoronaviruses hcov-hku (woo et al., ) and sars-cov were confirmed to contain this pk structure (goebel et al., b) . the utr regions of minimum free ene rgy = - . kcal/ mol mhv and sars-cov, which only share % sequence identity, were shown to be interchangeable, again supporting the conservation of functionally equivalent structures among different betacoronavirus lineages. apparently, this conservation does not extend to alpha-and gammacoronaviruses because replacements of the mhv utr with that of tgev and ibv, respectively, did not give rise to viable mhv mutants (goebel et al., b) . together, these data suggest that coronaviruses evolved several genus-specific cisacting rna elements. for example, the presence of a bsl followed by a pk structure is limited to betacoronaviruses, while other genera appear to contain only one of these elements, with the pk being conserved in alphacoronaviruses and the bsl in gammacoronaviruses (dalton et al., ; hsue and masters, ; williams et al., ) (see section . ). the structures and functionally important substructures of both the bsl and pk have been characterized in significant detail for bcov and mhv (goebel et al., a; hsue et al., ; williams et al., ) . in the primary structure, the bsl and pk regions overlap by several nucleotides. formation of the first stem of the pk structure requires base-pairing interactions with the downstream segment f of the bsl, thereby destabilizing the latter structure. in an extensive mhv mutagenesis study, the functional significance of both structures was demonstrated conclusively. because the two structures cannot exist simultaneously and, yet, each of them is essential for viral replication, it was proposed that the two elements may adopt alternate structures that act as a 'molecular switch' controlling the transition between different steps of the viral replication cycle (goebel et al., a) . initial mechanistic insight into how this 'molecular switch' might work was obtained in a subsequent study that provided evidence for a direct interaction between loop of the pk with the extreme end of the mhv genome (züst et al., ) . the characterization of second-site revertants arising from mhv mutants with genetically engineered oligonucleotide insertions in loop revealed distinct replacements at the extreme end, thereby retaining specific base-pairing interactions with the loop region and thus precluding the formation of stem of the pk. another set of mutants contained second-site mutations that suggested specific interactions of the pk region with nsp and nsp . based on this study, a model was proposed in which the formation and disruption of the pk by differential base-pairing interactions with the bsl and -terminal genome sequences, respectively, may lead to alternate structures that govern different steps of the minimum free energy = - . kcal/mol initiation and continuation of negative-strand rna synthesis (züst et al., ) . further evidence to support this model was obtained in a subsequent mhv reverse genetics study by liu et al. (liu et al., ) . thermodynamic investigations revealed a limited stability of the pk structure (stammler et al., ) , further supporting the structural flexibility of this cis-acting element and, thus, its proposed role as a 'molecular switch'. the region downstream of the pk is less well conserved among betacoronaviruses. it is generally referred to as the "hypervariable region (hvr)" and is not identical to the hvr identified at the end of the utr in ibv (dalton et al., ; williams et al., ) . the betacoronavirus hvr was predicted to contain a complex rna structure whose existence and functional significance was supported by enzymatic probing and mhv di rna mutagenesis studies (liu et al., ) . by contrast, more recent studies showed that large parts or even the entire hvr region can be deleted without causing major defects in mhv replication, arguing against an important role of this genome region in viral replication (goebel et al., ; züst et al., ) . interestingly however, mhv hvr mutants proved to be highly attenuated in vivo, suggesting a possible role in pathogenesis (goebel et al., ) . about - nts from the end of the coronavirus genome, there is a conserved octanucleotide sequence, -ggaagagc- , which was identified in early coronavirus sequence analyses performed in the late s (boursnell et al., ; lapps et al., ; schreiber et al., ) and subsequently found to be universally conserved across all coronavirus genera, with only very few viruses containing single-site replacements in this sequence (goebel et al., ) . this strict conservation suggests an important functional role for the octanucleotide sequence. to date, however, the function of the sequence has not been identified. as mentioned above, the entire hvr including the octanucleotide sequence can be deleted from the mhv genome without causing major defects in viral replication in vitro (goebel et al., ) . in line with this, replacements of single nucleotides within the octanucleotide motif were tolerated although, in most cases, the octanucleotide mutants exhibited small-plaque phenotypes and/or delayed single-step growth kinetics. in both high-and low-multiplicity-of-infection experiments, octanucleotide and hvr deletion mutants lagged behind the wildtype virus but reached near-wildtype titers at later time points and had no detectable defect in grna or sgrna synthesis (goebel et al., ) . mhv and bcov di rna studies provided evidence that the poly(a) tail present at the end of coronavirus genomes is another essential component of the coronavirus cis-acting signals, with a minimum of to adenylate residues being required for di rna replication (spagnolo and hogue, ) . this requirement corresponds well to the minimal binding site of the poly(a)-binding protein (pabp) on di rnas poly(a) sequences (spagnolo and hogue, ) . recent studies further suggest that, in the course of bcov infection, poly(a) tail lengths vary between and nts (wu et al., ) . this poly(a) tail length variation was confirmed to occur in beta-and gammacoronavirus infections and in a range of cell types, both in vitro and in vivo (shien et al., ) . the biological significance of these observations remains to be determined. to identify putative cis-acting elements in the alphacoronavirus utr, we used a range of rna folding algorithms to identify rna structural elements in the hcov- e and hcov-nl -terminal genome regions encompassing the last nts from the n gene and the entire -utr. because of the length of these sequences ( nts), many local secondary structures with similar free energies and base pair probabilities were identified and, thus, it proved to be difficult to make reliable predictions on stable rna secondary structures in this region. as described above for the utr, we therefore decided to use a combination of sequence and structural alignments of all currently recognized alphacoronavirus species to identify conserved rna structures. the predictions were then further refined using structure probing data obtained for hcov- e and hcov-nl . the validity of the approach was first tested by analyzing conserved rna structural elements in the betacoronavirus utr for which a large body of information has been obtained in previous structural and functional studies (see above). as shown in fig. , we were able to detect conserved rna structural elements in the betacoronavirus utr, including the bsl and the two sl structures that form the pk immediately downstream of the bsl. consistent with previous studies (goebel et al., a) , our predictions suggest that the formation of the pk requires structural rearrangements at the base of the bsl to permit the base-pairing interactions required to form pk stem , the latter involving the pk-sl loop sequence and the bsl -terminal sequence ( fig. a and b) . interestingly, our analyses also revealed another conserved structural element, a short hairpin, immediately upstream of the pk-sl . formation of this hairpin would compete with base-pairing interactions required to form the basal part of the bsl and the pk stem , respectively (fig. b) . furthermore, the hairpin overlaps partly with the pk loop region that, in a previous study, was suggested to interact with the extreme end of the genome (züst et al., ) . the conservation of both structure and sequence of this hairpin suggest a biological function for this element. in this context, it may be worth mentioning that the hairpin structure is predicted to be disrupted by the -nt insertion in loop that was reported previously to cause a poorly replicating and unstable phenotype in mhv (goebel et al., a) . it remains to be seen if the small hairpin represents yet another element in the intricate network of basepairing interactions between the bsl, the pk, and the end that together constitute the complex molecular switch proposed by the masters laboratory (goebel et al., a) . consistent with previous studies, we identified only one conserved rna structural element in the hvr downstream of pk-sl . this stem-loop contained the conserved octanucleotide sequence in a single-stranded region. as pointed out above, the role of this element is currently unclear as both the hvr and octanucleotide sequence proved to dispensable for betacoronavirus (mhv) replication in vitro (goebel et al., ; liu et al., ) . overall, the alignment-based structure prediction algorithms used in our analysis led to conclusions that were consistent with results obtained in previous studies on betacoronavirus utrs, suggesting that the approach might also be suitable to make reliable predictions on conserved rna structural elements in the highly variable alphacoronavirus utrs. our alignment-based secondary structure predictions using representative viruses from all currently recognized alphacoronavirus species revealed the conservation of several rna structural elements in the alphacoronavirus utr. a counterpart of the betacoronavirus bsl structure (goebel et al., a; hsue and masters, ) could not be identified in the alphacoronavirus utr while structural elements suitable to form a pk structure could be identified in all alphacoronaviruses (fig. a) . interestingly, despite the absence of an upstream bsl in alphacoronaviruses, the formation of this putative pk structure is predicted to require the disruption of a short hairpin immediately upstream of pk-sl , a scenario that is similar but less complex compared to the situation described above for betacoronaviruses. it remains to be investigated in further studies whether or not alphacoronaviruses employ a molecular switch mechanism similar to what has been described for betacoronaviruses (goebel et al., a) . the predicted sl structure (fig. ) could be confirmed by structure probing data obtained for hcov- e and hcov-nl (((.....) )).)))....... (((((((((((...........) )))))))))).. ... ((((((..(((((((((((..((((...((.....) )...))))....))))))))))))))))).. ' pk-sl bulged stem-loop hvr octanucleotide a b c fig. . alignment-based secondary structure prediction of betacoronavirus genome regions. the viruses included in this analysis represent all currently recognized lineages and species in the genus betacoronavirus. the alignment was generated using locarna and the structure was calculated using rnaalifold. the consensus sequence is represented using the iupac code. colors are used to indicate conserved base pairs: from red (conservation of only one base-pair type) to purple (all six base-pair types are found); from dark (all sequences contain this base pair) to light colors ( or sequences are unable to form this base pair). gray bars below the alignment indicate the extent of sequence conservation. gray shadows are used to link rna structures with the corresponding dot-bracket notations above the alignment. (a) alignment-based secondary structure prediction of the bulged stem-loop (bsl) in the utr. (b) alignment-based secondary structure prediction of the pseudoknot (pk) region in the utr. note that pk-sl is an alternate structure that requires base-pairing interactions between the loop region of pk-sl and the basal part of the bsl shown in (a). formation of the bsl basal part and pk structure, respectively, are mutually exclusive (see text for details). (c) alignment-based secondary structure prediction of the hypervariable region (hvr) in the utr. to refine the alignment, an anchor at the highly conserved octanucleotide sequence was used. ( fig. , detailed structure probing data to be published elsewhere). furthermore, our structure probing data indicated base-pairing interactions upstream of sl in hcov-nl , supporting the formation of the predicted small hairpin in this region ( fig. a and b). by contrast, no such interactions were seen in hcov- e. also, the structure probing data did not support the formation of a stable pk structure, possibly reflecting a similarly low thermodynamic stability as determined for the equivalent pk in betacoronaviruses (stammler et al., ) . further studies including reverse genetics experiments are required to confirm the existence and biological significance of the predicted alphacoronavirus pk structure. with respect to the hvr downstream of pk-sl , an extensive stem-loop structure was predicted in our analyses of alphacoronavirus utrs (fig. b) . the structure is supported by a large number of covariant base pairs and contains the conserved octanucleotide sequence in a single-stranded region. the large distal part of the stem-loop structure was further corroborated by structure probing data (fig. , details to be published elsewhere). in both hcov- e and hcov-nl , the octanucleotide sequence was found to be located in the loop region. of note, the cell cultureadapted hcov-nl isolate used for our structure probing analysis contained a short deletion apparently acquired upon serial passaging in cell culture, resulting in a significantly smaller loop but retaining the octanucleotide sequence (with one g-to-a replacement) in an identical position in the loop when compared to hcov- e (see fig. a and b ). this serendipitous deletion shows that the distal part of the extended stem-loop structure is not essential for hcov-nl replication in cell culture. the data also indicate that, despite the deletion, the octanucleotide sequence retains a position in a loop region of the stem-loop structure and tolerates (.((...) ))))))... (((((((((((...........) ))))))))))... ' pk-sl ..) ))) ......)) ).....))) )).)))). ..))))).. . )))))..))...... )))...... )))..))) ))...)))) .. fig. . alignment-based secondary structure prediction of alphacoronavirus -terminal genome regions. the viruses included in this analysis represent all currently recognized species in the genus alphacoronavirus. the alignment was calculated by locarna, the structure by rnaalifold. the consensus sequence is represented using the iupac code. colors are used to indicate conserved base pairs: from red (conservation of only one base-pair type) to purple (all six base-pair types are found); from dark (all sequences contain this base pair) to light colors ( or sequences are unable to form this base pair). gray bars below the alignment indicate the extent of sequence conservation at a given position. gray shadows are used to link rna structures with the corresponding dot-bracket notations above the alignment. (a) alignment-based secondary structure prediction of the pseudoknot (pk) region in the utr. formation of the stem of pk-sl requires base-pairing interactions with the loop region of sl . formation of the pk and the two sl structures shown above the alignment are mutually exclusive (see text for details). (b) alignment-based secondary structure prediction of the hypervariable region (hvr) in the utr. to refine the alignment, an anchor at the highly conserved octanucleotide sequence was used. pk-s fig. . rna secondary structure predictions of -terminal genome regions of hcov- e (a) and hcov-nl (b). predictions were generated using rnafold --nolp -c. as constraints, structure probing data were used. formation of the predicted pseudoknot (pk) requires base-pairing interactions between the loop region of sl and an upstream sequence (and, possibly, structural rearrangements), resulting in the formation of pk stem (pk-s ) as indicated. also shown is the octanucleotide sequence that is conserved across all genera of the coronavirinae. minimal changes, the latter being consistent with mhv reverse genetics data obtained for the hvr/octanucleotide region (goebel et al., ) . based on numerous studies on betacoronaviruses including structural, biochemical, and reverse genetic work (di rna and replication-competent virus), a picture of putative cis-acting elements is beginning to emerge (reviewed in masters, ; sola et al., b) . previous work also identified a growing number of cellular and viral proteins that bind to these structures and likely have important functions at different steps of genomic and/or subgenomic rna synthesis, genome packaging, genome expression or intracellular targeting of structures engaged in viral rna synthesis (reviewed in narayanan and makino, ; sola et al., b) . using a combination of bioinformatic and biochemical methods, the present study confirms and extends this previous work. our study suggests that rna secondary structure elements may be more conserved than previously thought, both within individual coronavirus genera and across different genera as shown here for the genera alphacoronavirus and betacoronavirus. although significantly more work is needed to further characterize the structures identified in this and previous studies and understand their functional role, the available data suggest a cross-genus conservation of a number of rna structural elements among alpha-and betacoronaviruses. the conservation pattern is consistent with the replicase gene-based classification of genera within the subfamily coronavirinae (de groot et al., a) . conserved elements include stem-loops , , , and, possibly, in the -terminal genome region and a putative pk in the utr. the data further suggest that, in both alpha-and betacoronaviruses, the formation of the pk may require structural rearrangements in other regions (upstream of the sl ) and it remains an attractive idea to suggest specific functions for these alternative structures whose mechanistic and functional details remain to be investigated (goebel et al., a; züst et al., ) . finally, in line with previous observations (brian and baric, ; masters, ; sola et al., b) , the study confirms a significant degree of variation in the -terminal region of the utr, with the conserved octanucleotide sequence being consistently located in a single-stranded region of a stem-loop structure. interestingly, specific lineages of beta-, gamma-and deltacoronaviruses (as well as other plus-strand rna viruses) may contain another structural element, called s m, in the utr, thus further adding to the variability of this genome region in coronaviruses (jonassen et al., ; robertson et al., ; tengs et al., ) . the conservation pattern of the various lineage-specific structural elements argues against a conserved function in viral rna synthesis but rather suggests that the utr may contain elements that are involved in specific virushost interactions and/or pathogenesis as has been shown for the mhv hvr (goebel et al., ) . the nucleoprotein is required for efficient coronavirus genome replication engineering the largest rna virus genome as an infectious bacterial artificial chromosome structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain coronavirus main proteinase ( cl pro ) structure: basis for design of anti-sars drugs identification of a domain required for autoproteolytic cleavage of murine coronavirus gene a polyprotein the severe acute respiratory syndrome coronavirus nsp protein is an endoribonuclease that prefers manganese as a cofactor sequences of the nucleocapsid genes from two strains of avian infectious bronchitis virus completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus coronavirus nsp : a critical co-factor for activation of multiple replicative enzymes coronavirus genome structure and replication recombination and coronavirus defective interfering rnas an efficient ribosomal frame-shifting signal in the polymeraseencoding region of the coronavirus ibv characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot mutagenesis of the murine hepatitis virus nsp -coding region identifies 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defective-rna replication and packaging family coronaviridae middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group order nidovirales the fitness of defective interfering murine coronavirus di-a and its derivatives is decreased by nonsense and frameshift mutations coronaviruses maintain viability despite dramatic rearrangements of the strictly conserved genome organization crystal structure and functional analysis of the sars-coronavirus rna cap -o-methyltransferase nsp /nsp complex coronavirus nonstructural protein is a cap- binding enzyme possessing (nucleoside- o)-methyltransferase activity organelle-like membrane compartmentalization of positive-strand rna virus replication factories identification of a novel coronavirus in patients with severe acute respiratory syndrome infidelity of sars-cov nsp -exonuclease mutant virus replication is revealed by complete genome sequencing probing the structure of rnas in solution transmissible gastroenteritis coronavirus packaging signal is located at the end of the virus genome host cell proteins interacting with the end of tgev coronavirus genome influence virus replication characterization of the rna components of a putative molecular switch in the untranslated region of the murine coronavirus genome a hypervariable region within the cis-acting element of the murine coronavirus genome is nonessential for rna synthesis but affects pathogenesis the cis-acting genomic replication element of the severe acute respiratory syndrome coronavirus can function in the murine coronavirus genome rna replication of mouse hepatitis virus takes place at double-membrane vesicles coronavirus n protein n-terminal domain (ntd) specifically binds the transcriptional regulatory sequence (trs) and melts trs-ctrs rna duplexes genetic evidence of a long-range rna-rna interaction between the genomic untranslated region and the nonstructural protein coding region in murine and bovine coronaviruses 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form a : heterotrimer that exhibits primer-independent rna polymerase activity mouse hepatitis virus stem-loop functions as a spacer element required to drive subgenomic rna synthesis strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia insights into sars-cov transcription and replication from the structure of the nsp -nsp hexadecamer coronavirus leader rna regulates and initiates subgenomic mrna transcription both in trans and in cis coronavirus replicative proteins characterization of a human coronavirus (strain e) c-like proteinase activity the coronavirus replicase gene: special enzymes for special viruses virus-encoded proteinases and proteolytic processing in the nidovirales the autocatalytic release of a putative rna virus transcription factor from its polyprotein precursor involves two paralogous papain-like proteases that cleave the same peptide bond coronavirus nucleocapsid protein facilitates template switching and is required for efficient transcription sequence motifs involved in the regulation of discontinuous coronavirus subgenomic rna synthesis coronavirus nucleocapsid protein is an rna chaperone coronavirus non-structural protein is a major pathogenicity factor: implications for the rational design of coronavirus vaccines genetic interactions between an essential cis-acting rna pseudoknot, replicase gene products, and the extreme end of the mouse coronavirus genome the work of r.m. and j.z. is supported by the deutsche forschungsgemeinschaft (sfb , a ). key: cord- -ck eto authors: baric, ralph s.; shieh, chien-kou; stohlman, stephen a.; lai, michael m.c. title: analysis of intracellular small rnas of mouse hepatitis virus: evidence for discontinuous transcription date: - - journal: virology doi: . / - ( ) - sha: doc_id: cord_uid: ck eto abstract we have previously shown the presence of multiple small leader-containing rna species in mouse hepatitis virus (mhv)-infected cells. in this paper, we have analyzed the origin, structure, and mechanism of synthesis of these small rnas. using cdna probes specific for leader rna and genes a, d, and f, we demonstrate that subsets of these small rnas were derived from the various viral genes. these subsets have discrete and reproducible sizes, varying with the gene from which they are derived. the size of each subset correlates with regions of secondary structure, whose free energy ranges from − . to − . kcal/mol, in each of the mrnas examined. in addition, identical subsets were detected on the replicative intermediate (ri) rna, suggesting that they represent functional transcriptional intermediates. the biological significance of these small rnas is further supported by the detection of leader-containing rnas of , , and nucleotides in length, which correspond to the crossover sites in two mhv recombinant viruses. these data, coupled with the high frequency of rna recombination during mhv infection, suggest that the viral polymerase may pause in or around regions of secondary structure, thereby generating pools of free leader-containing rna intermediates which can reassociate with the template, acting as primers for the synthesis of full-length or recombinant rnas. these data suggest that mhv transcription uses a discontinuous and nonprocessive mechanism in which rna polymerase allows the partial rna products to be dissociated from the template temporarily during the process of transcription. we have previously shown the presence of multiple small leader-containing rna species in mouse hepatitis virus (mhv)-infected cells. in this paper, we have analyzed the origin, structure, and mechanism of synthesis of these small rnas. using cdna probes specific for leader rna and genes a, d, and f, we demonstrate that subsets of these small rnas were derived from the various viral genes. these subsets have discrete and reproducible sizes, varying with the gene from which they are derived. the size of each subset correlates with regions of secondary structure, whose free energy ranges from - . to - . mouse hepatitis virus (mhv), a member of coronaviridae, contains a linear single-stranded and positivesense rna of . x o da (lai and stohlman, ) . the genomic rna is enclosed in a helical nucleocapsid structure constructed from multiple copies of nucleocapsid protein (n) (sturman, ) . virions are enveloped and contain two virus-specific glycoproteins of kda (e ) and kda (el) (sturman et al., ) . upon entry into the host cell, the genomic rna is translated into an early polymerase which directs the synthesis of a full-length negative-sense viral rna (brayton et a/., ; lai et al., ) . in turn, the negative-sense rna is transcribed by a late or altered early polymerase into the genomic rna and six subgenomic mrnas (bray-ton eta/., ) . these mrnas range from . to . x lo da in length and are arranged in the form of a nested set from the '-end of the genome (lai et a/., ) . the '-ends of each mrna and the genomic rna appear to contain an identical leader sequence of about nucleotides, which are encoded only at the y-end of the genome (lai eta/., (lai eta/., , spaan et al., ) . the uv transcriptional mapping studies suggest that the mrnas are not derived from cleavage of a large precursor rna (jacobs era/., ) nor is there any evidence indicating that nuclear factors are required for mhv replication (brayton et a/., ; wilhelmsen et al., ) . these data suggest that conventional eukaryotic rna splicing is not involved in mhv transcription. analysis of mhv replicative intermediate and replicative form rnas suggests that a free leader rna may be involved in subgenomic mrna synthesis (baric eta/,, ). in addition, discrete small leader-related rnas of various sizes have been detected in mhv-infected cells (baric et a/., ) . we have also isolated a ts mutant of mhv which synthesizes only small leader rnas, but not mrnas at the nonpermissive temperature (baric et a/., ) . these data suggest that the mhv mrnas are synthesized discontinuously and may utilize a single or multiple free leader rnas as primers for the transcription of different subgenomic mrnas. additional proof that free transacting leader rna(s) function in mhv transcription came from the demonstration of reassortment of leader rnas between two different strains of mhv (makino et a/., b) . however, the exact leader rna species which function in mhv transcription has not been identified. rna recombination occurs at very high frequencies during mixed infection with two heterologous strains of mhv (lai et a/., ; makino et a/., a) . these data, coupled with the presence of discrete large leader-containing rnas which range from to nucleotides in length in mhv-infected cells (baric et a/., ) suggest that discontinuous rna intermediates may be dissociated and reassert between viral rna templates to generate recombinant viruses by a copy-choice mechanism (makino eta/., a). therefore, the larger leader-containing rnas present in mhvinfected cells may represent functional intermediates of rna transcription and recombination. in this paper, we have analyzed the origin, structure, and probable mechanism of synthesis of these rnas. the data suggest that mhv rna transcription may pause at sites corresponding to hairpin loops in the rna template, and they support a mechanism of discontinuous rna transcription in which rna intermediates can be dissociated and reassociated with the rna template intermittently during the course of transcription. the a strain of mhv was propagated in either dbt or l cells at " in dulbecco's modified mem medium supplemented with % fetal calf serum containing pg/ml penicillin and pg/ml streptomycin. infection was performed at a m.o.i. of to , as previously described (baric er a/., ). preparation of mhv intracellular rna rna was extracted from infected cells by the phenol/ chloroform method between and hr postinfection ( % cpe) (baric et al., ) . following ethanol precipitation, the rna was washed once in % ethanol to remove excess salts and analyzed by electrophoresis on polyacrylamide gels as previously described (baric et al., ) . intracellular rna was extracted from infected cells as described above and applied to - % sucrose gradients made in nte buffer ( . ili naci, . m tris-hci, ph . , and . \/ edta) containing . % sds. rna was sedimented at , rpm in an sw ti rotor for hr at " and . -ml fractions were collected. in duplicate gradients, , , and s rna were included as size markers. to isolate mhv ri rnas, the - s rna fractions were pooled and precipitated with ethanol. it has previously been shown that the ri rnas are contained within this size fraction (bark et al., (bark et al., , sawicki and sawicki, ) . the - s rna fractions were collected as the free small rnas. the cdna clones of mhv genomic rna used in this study are summarized in fig. . clones f and c represent overlapping regions in the '-end of the genomic rna (shieh et a/., ). f is a . -kb clone representing the very 'end of the genome including the leader rna sequences, while clone c represents internal sequences in gene a. two other clones representing the internal sequences of mhv genome were also used: clone d contains entire sequences of genes d and e and part of genes c and f (shieh, unpublished), and clone phn spans the entire gene f (kindly provided by dr. heiner niemann, giessen, germany). an internal xbal fragment ( . kb), designated d x, of clone d , was used as the probe for gene d. this fragment includes nucleotides - in the mrna coding sequence as reported (skinner and siddell, ) . the construction of subclones derived from phn followed the procedures described by maniatis er al. ( ) . subclone el- was derived by blunt-end ligating a nucleotide ddel fragment, located between nucleotides and in the gene f sequence (armstrong et al., ) into plasmid pt - at the smal site (pt - was purchased from amersham). subclone el- was derived from a -nucleotide fragment, located between nucleotides and in the el rna (armstrong et a/., ) and was inserted into plasmid pt - at the /-/indllllsmal site. el-l was excised from phn by an ecoriiscal restriction digestion. it contains the first nucleotides of the mrna , including the -nucleotide leader rna sequence. this fragment was used directly for nick translation without subcloning. nick translations of cdna clones ( . -l .o pg) were performed in mlll tris-hci (ph . ) mm mgc , mlll dlt, pg/ml bovine serum albumin (bsa), mm each of datp, dgtp, and l'tp, j&i [a- p]dctp ( ci/mmol), ob glycerol, fig/ml dnase i (worthington), and u of dna polymerase i (boehringer mannheim biochem) for hr at ". unincorporated nucleotides were removed by several precipitations with % ethanol. hybridizations were performed at " for hr in % formamide, mm na phosphate (ph . ) x ssc ( x ssc = . a# naci, . \/ na citrate, ph . ) ox denhardt's solution ( x denhardt's solution: . % each of ficoll, polyvinylpyrrolidone, and bsa) and pg/ml salmon sperm dna (baric et a/., ) . approximately - x lo cpm/pg of dna were hybridized to each blot. after hybridization, the filters were washed, dried, and exposed to xar films at - " in the presence of an intensifying screen. equivalent amounts of intracellular rna ( or rg) were separated by electrophoresis on % polyacrylamide gels in . m tris-borate buffer (ph . ) containing mm edta and m urea. for analysis of small rnas ranging between and nucleotides in length, the samples were electrophoresed at v ( - ma) until the bromphenol blue dye marker had migrated approximately cm from the origin. to analyze larger rnas ranging between and nucleotides in length, electrophoresis was at v until the xylene cyanol dye marker had migrated - cm from the origin. following electrophoresis, urea was removed by twice washing the gel in ice-cold tae buffer ( . m tris-hci, ph . , . m na acetate, and . m edta) for min each, and the rna was electroblotted to zeta-probe paper (bio-rad) in cold tae. electroblotting was performed at " in a circulating chamber at ma for - hr and an additional hr at a to ensure the transfer of larger rnas above nucleotides in length (baric et a/., ; stellwag and dahlberg, ) . following electrotransfer, the paper was gently washed in tae and baked at " for hr. prehybridization was performed for hr at " in % formamide, mm na phosphate (ph . ) x ssc, ox denhardt's solution and pg/ml salmon sperm dna. precise sizes of the small rnas were determined by electrophoresis of ml mp sequencing t-ladders in lanes adjacent to intracellular rna preparations. since electrotransfer oftentimes decreases the resolution of individual bands, complete sets of ml sequencing a, t, c, g ladders were also electrophoresed with each set of rna preparations, fixed, dried, and separately exposed to xar- film. the ml -t ladders electrotransferred with rna preparations were aligned with the a, t, c, g ladders to determine the exact sizes of small rnas. computing stability of mhv rna secondary structure predictions for regions of secondary structure in mhv rna were made using the zucker rna folding program through bionet (zucker and stiegler, ) . stability of individual hairpin loops within the mhv sequence was calculated from the thermodynamics of adding a base pair to a double-stranded helix at " as reported by tinoco et a/. ( ) and modified by salser ( ) . more specifically, the energies for stacking of internal gu pairs were computed as follows: gu next to gc, - . kcal/mol; gu next to au or gu, - . kcal/mol. in addition, - .o kcai/mol was subtracted from loop structures containing u nucleotides (tinoco et al., ) . to determine the structure and origin of the small rnas present in mhv-infected cells, we constructed several cdna probes specific for different regions of the mhv genome. we chose three genes for this study, namely, genes a, d, and f. gene a probably encodes an rna polymerase, while genes d and f encode a nonstructural protein p and the el protein, respectively. since gene a represents the '-most region of the genome, the small rnas originating from this gene represent products of rna replication. the other two genes represent two internal genes which have been well characterized (armstrong eta/,, ; skinner and siddell, ) . the map positions of the probes used in this study are depicted in fig. . the probe specific for leader region was derived from a gene f-specific clone (phn ) which contains the entire leader sequence. the fragment (el-l) containing the first nucleotides of mhv mrna was used as the leader-specific probe in subsequent experiments (see fig. b) . the clones el - and el - were used to serve as the probes for the '-and '-ends of the gene f, respectively (fig. b) . the el - clone would not detect any leader-containing rnas smaller than nucleotides. clones specific for genes a and d were derived from cdna clones as described under materials and methods. to determine the structure and origin of the small rnas present in mhv-infected cells, we first used a leader-specific cdna probe to identify small rnas terminated within or around the leader rna sequence. these small rnas should represent rna species derived from the '-end of genomic or subgenomic mrnas. these rna species have been detected previously (baric et a/., ) but their size and structure have not been precisely determined. intracellular rna was extracted from mhv-infected cells and separated by electrophoresis on polyacrylamide gels. following transfer to zeta-probe paper, the blot was probed with a leader-specific cdna probe (el-l) (fig. b) . as shown in fig. , several distinct small rnas ranging from to nucleotides were detected, similar to our previous a cbb\~~ d pt p fig. . the structure of the mhv cdna probes. clone f contains the complete '-end of the genome and overlaps with clone c . these clones were used to detect small rnas originating during rna replication (a). clones d x, el - , and el - were used to detect small rnas originating during transcription of mrna (gene d) and mrna (gene f) (b). probe e -l was used to detect small leader rnas containing the leader sequence. the exact sizes of these clones are described under materials and methods. abbreviations used for cdna fragments: b, bamhi; d, ddel; e, ecori; h, hindlll; p. pstl; s, seal; x, xbal. report (baric et a/., ) . the sizes of these rna spe-larger rna species were also reproducibly detected cies were precisely determined from the accompanying by the leader-specific probe, consistent with previously t-ladder of ml sequences. their sizes were repro-published results (baric et a/., ); however, under ducible, as evident from the rna patterns of two prep-the conditions used, they were not well resolved. these arations derived from different cell lines, although the leader-containing rnas range from to more than relative amounts of the various rna species varied. nucleotides in length. to determine the structure the four smallest rna species of , , , and of these larger leader-containing rna species, the rna nucleotides correspond in size to the lengths between blots were hybridized with cdna clone f , reprethe '-end of the leader rna and a region of two weak senting . kb of the sequences at the '-end of the overlapping hairpin loops around nucleotides - genomic rna (shieh et al., ) (fig. a) . several disand - ( fig. ; table ) (shieh et a/., ) . thus, tinct rna species ranging from to larger than these rna species are likely to represent rna tran-nucleotides were resolved (fig. a) . it is notable that scriptional products terminating within the leader se-the two independently isolated preparations of intraquences. the larger leader-containing rnas of and cellular rna gave identical rna bands. when the clone nucleotides, and occasionally an rna of nu-c , representing the internal region of gene a (apcleotides, also correspond to the region of a hairpin proximately . kb from the '-end) (fig. a) , was used, loop at nucleotides -l from the '-end of the ge-no distinct small rna bands were detected (fig. b ). nome and a postulated transcriptional termination sig- the hybridization with c was generally weaker than nal (uuuauaaa) for the mhv leader rna synthesis that with f , since the c probe would not detect (shieh et a/., ) (fig. ) ""g%h c u g'- (fig. ) . the free energy of these hairpin loops is shown in table . data suggest that these rnas represent the transcriptional intermediates derived from the '-end of the genomic rna, and that transcription from the '-end of the genome terminates or pauses at distinct sites, the length of these rnas correspond to the lengths between the '-end and the sites of potential hairpins (table ; see discussion). the leader-containing rnas larger than nucleotides in length detected by the leader-specific cdna probe were more heterogeneous (fig. ) (baric et al., ) suggesting that multiple rna species in this size range were present. however, the probe representing the '-end sequences of gene a detected discrete species (fig. a) , suggesting that the larger leader-containing rnas may also represent rna species derived from other regions of the genome during subgenomic rna synthesis. therefore, cdna probes specific for the '-coding sequences of genes d and f were tested. the probe representing the '-end of the gene f (el- ) detected rna species of , , , /l , , , , , and two groups of poorly resolved rnas ranging between - and - nucleotides in length (fig. a ). these rna species were different from those detected with the gene a probe (fig. a ). in addition, they were not detected with a probe (el- ) representing the '-end of the gene between nucleotides and (fig. c ). the gene d-specific cdna probe (d x) (fig. a) also detected a different set of small rna species of , , , , , and nucleotides in length (fig. ) . smaller amounts of rna of and nucleotides in length were also detected. these data suggest that transcription of subgenomic mrnas also terminates or pauses at many sites. computer models of rna folding suggest the presence of hairpin loops in regions of nucleotides - , - , - , - , and - within the sequence of mrna (gene f) (fig. b) and in the regions of nucleotides -l , -l , and - and l- within mrna (gene d) (fig. a) . the predicted stability of these hairpin structures is shown in table . these data show that most of the small rnas detected in mhv-infected cells have termination sites within the hairpin loop structures, suggesting a possible correlation between the generation of small rnas and the presence of secondary structure in the template or product rnas. if the small rna species detected in mhv-infected cells represent specific products released from the template rna during transcriptional pausing, rather than degradation products of the mrnas, then the small rna species would be expected to be present in the ri structure which is involved in rna transcription. conversely, if rna transcription proceeds in a continuous manner, the rna species on the ri structure should be heterogeneous, without predominant rna species. to distinguish between these two possibilities, - s ri rna (baric et a/., ; sawicki and sawicki, ) was isolated and analyzed for the presence of small leader-containing rna using two probes, f and el - , representing genes a and f, respectively. as shown in fig. , the small rna species present on the ri rna are the same as those present in the whole cell lysates. furthermore, the s rna fractions also contain an identical set of small rnas, indicating that some of the small rnas were dissociated from the ri rna. these results suggest that the small rna species are true transcriptional intermediates. previous findings in our laboratory demonstrate that rna recombination could occur at a very high frequency during a mixed infection with two strains of mhv (makino et a/., a). this high frequency is reminiscent of rna reassortment described for viruses with segmented rnas (fields, ) and suggests that mhv replication might involve the generation of free and discontinuous rna intermediates. these intermediates could participate in rna recombination by a copy-choice mechanism. the detection of multiple discrete species of leader-containing rnas, which range from to more than nucleotides in length, in mhv-infected cells further supports this model (baric et al,, ) . in this report, we examined the origin, structure, and mechanism of synthesis of these rnas. the data suggest that these rna species are likely to represent transcriptional pausing products of mhv rna synthesis. the pausing sites correspond, in general, to the potential hairpin loops present in the template or product rnas. this transcriptional pausing mech-anism is reminiscent of the transcriptional pausing of q/ phage rna, in which rna polymerase pauses in regions of hairpin loop structures (mills et a/., ) . pausing also occurs during dna-directed dna synthesis (huang and hearst, ; sherman and gefter, ) , dna-directed rna synthesis (maizels, ; rosenberg et al., ) and in reverse transcriptase reactions (efstratiadis et al., ; haseltine et al., ) . similar to the mechanism of qj rna transcrip- tion, the majority of mhv small rnas terminate at the '-side of hairpin loop structures in the product or template strands, which have free energies ranging from - . to - . kcal/mol ( fig. and table ). the stabilities of these hairpin loops are comparable to those of q/ (- . to - . kcal) and trna (- . to - . kcal) hairpin loops, whose existence in the rna has been demonstrated by various physical and biochem-ical methods (auron et al., ; gehrke et al., ; kramer and mills, ) . whether the pausing rna intermediates of qp or other pausing transcriptional model systems are dissociated from the rna template is not known; however, the data presented in this paper clearly show that at least some of the mhv rna intermediates are separated from the template rna strand. it has previously been shown that the nascent rna chains of g&i and ms phages are probably bound to the template strand only by short duplex regions (weissman, ) . it is also possible that the mhv rna polymerase is a nonprocessive enzyme, thus releasing some of the rna intermediates after pausing. currently, there is no direct evidence to prove that these free small rnas actually rebind to the rna template and participate in continuing transcription; however, the high frequency of rna recombination (makino et a/., a) suggests that such rejoining does occur. the recent isolation of several mhv recombinants with multiple crossovers further strengthened this probability (keck et a/., ) . most interestingly, the crossover sites in the two recently isolated recombinants, a-l and a- , have been mapped within a region of the leader sequences (nucleotides - from the '-end) (keck eta/., ) , where three small rna species were detected (fig. ) . the isolation of these recombinants strongly suggests the functional roles of the pausing rna intermediates. it is noteworthy that, during the transduction of the proto-oncogene c-fps by a retrovirus, right-handed recombination occurred near or in stable hairpin loop structures (huang et al., ) . these data, coupled with the fact that reverse transcriptase "pauses" in regions of secondary structure (haseltine et al., ) suggest a common mechanism of recombination for retroviruses and coronaviruses, involving paused transcriptional intermediates. in addition, it has previously been shown that short oligonucleotides can act as primers for rna synthesis in vitro (minkley and pribnow, ; niyogi and stevens, ) . thus, it seems likely that the free rnas in mhvinfected cells could also be used for chain elongation. recently, our laboratory has shown that the mhv leader rna can act in vans to participate in rna transcription (makino et al., b) . the four leader-containing rna species of - nucleotides in length are the most likely candidates for the primers of subgenomic mrna transcription, since they fall within the size range of the leader sequences present in the mhv subgenomic mrnas (lai et a/., ; spaan et a/., ) . it is not clear, however, whether multiple leader rnas are involved in priming mrna transcription. according to our present model of leader-primed transcription, these leader rnas would have to be cleaved before serving as primer (shieh et a/., ) . these rnas are likely generated due to the hairpin loop structures in nucleotides - and -l , and the intervening au-rich sequences at the '-end of the genome (fig. ) (shieh eta/., ) . it should be noted that the pausing or termination sites of these small rnas were determined by assuming that they were generated from correct transcriptional initiation sites. although the extremely small amount of these rnas makes it impossible to resolve this issue definitively, several reasons argue for the interpretation that they were initiated from the same site: first, the leader-primed transcription mechanism of mhv ( baric et al., ; shieh et a/., ) indicates that the same primer is used for the transcription of various mrnas; second, these rna species are very reproducible in different rna preparations and are present on replicative intermediate rna; third, rna recombinants have been isolated whose recombination sites may be in an area corresponding to the termination sites of these small rnas (keck et a/., ) suggesting that these rnas have biological functions; and finally, correct initiation was noted in a similar study with qb phages (mills et a/., ) . the biochemical and biological data presented here and elsewhere (baric eta/., ; makino eta/., a) are, thus, consistent with a mechanism of mhv transcription which is discontinuous and nonprocessive (fig. ). in this model, the viral polymerase pauses in molecular cloning-a laboratory manual optimal computer folding of large rna sequence using thermodynamics and auxillaty information. nucleic acids res. , -l . key: cord- -z utwbk authors: fu, li; gonzales, donna m.; das sarma, jayasri; lavi, ehud title: a combination of mutations in the s part of the spike glycoprotein gene of coronavirus mhv-a abolishes demyelination date: journal: j neurovirol doi: . / sha: doc_id: cord_uid: z utwbk the a strain of coronavirus, mouse hepatitis virus (mhv), produces acute hepatitis, meningoencephalitis, and chronic demyelination. the authors have previously shown that the spike (s) glycoprotein gene of mhv contains determinants of virulence, hepatitis, and demyelination. they then identified viruses containing mutations in the s gene that exhibit alterations in viral pathogenesis. in the present study, the authors produced new recombinant viruses with each one of these s gene mutations by site-directed mutagenesis and targeted recombination and studied the effect of each individual mutation on the pathogenesis of the virus. they identified a combination of mutations in the s gene (i m and l i) that abolishes demyelination. individual mutation and other combinations of mutations in the s gene only interfere with virulence and hepatitis and only reduce demyelination (i m), but do not abolish demyelination completely. thus, demyelination determinants exist within genomic regions on both sides of the hypervariable region, downstream from the receptor-binding domain in the s part of the mhv spike glycoprotein gene. the structure and precise function of these regions awaits further investigation. mouse hepatitis virus (mhv) is a member of the coronavirus genus, which belongs to the coronaviridae family of the nidovirales order (cavanagh, ; lai and cavanagh, ) . nidoviruses pro-duce a variety of hepatic, enteric, and neurologic diseases in animals, and upper respiratory and enteric diseases in humans (lai and cavanagh, ; lavi et al, ; lavi and weiss, ) . infection of mhv-a in -week-old c bl/ (b ) mice produces a biphasic disease with acute hepatitis and meningoencephalitis followed by chronic inflammatory demyelinating disease (lavi et al, (lavi et al, , . mhv infection in mice (with both a and jhm strains) serves as an excellent laboratory model for virus-induced demyelination, similar to multiple sclerosis (ms) in humans (houtman and fleming, ; knobler et al, ; lavi et al, b; perlman et al, ; sorensen et al, ; wege et al, ; weiner, ) . because viral infection is considered as a potential trigger for demyelination in ms (allen and brankin, ) , understanding the mechanism of mhv-induced demyelination and the upstream events that lead to the autoimmune phenomenon may provide important insight into the mechanism of interaction between viruses and the nervous system, especially with respect to to understand the molecular basis of mhv neurotropism, we investigated a strain of mhv (mhv- ) that is closely related to mhv-a but is only weakly neurotropic (hirano et al, (hirano et al, , wege et al, ; yamada et al, ; yokomori et al, ) . we sequenced the entire genome of mhv- and studied its detailed pathologic properties (das sarma et al, b) . we found that mhv- produces acute hepatitis and meningitis, without encephalitis, without demyelination, and without persistence of virus in the central nervous system (cns) (das sarma et al, b) . we then replaced the s gene of a with that of mhv- by targeted recombination and produced recombinant viruses that have a persistence-positive, demyelination-negative phenotype. we concluded that demyelination determinants, independent of viral persistence, map to the s gene of mhv (das sarma et al, ) . additional studies identified molecular determinants of pathogenesis in the s gene of mhv (leparc-goffart et al, ; navas et al, ; phillips et al, phillips et al, , . to further explore which parts of the s gene are directly responsible for the demyelinating phenotype, we studied a set of recombinant viruses that were produced by infection with both mhv- and la- , a temperature-sensitive (ts) mutant of mhv-a (keck et al, ) . these recombinant viruses were all found to be nondemyelinating. sequencing the s genes of these recombinant viruses revealed that the recombinants' s gene was either derived from mhv- or from mhv-a . all the recombinant viruses with an s gene derived from mhv-a had three identical amino acid substitutions within the s gene (das sarma et al, a) . because these recombinant viruses were produced by dual infection of cultures with mhv- and la- , we could not rule out that they also contained other mutations in addition to the mutations in the s gene. we therefore used site-directed mutagenesis and targeted recombination to produce new recombinant viruses, with selective s gene mutations that were associated with the demyelinationnegative phenotype. we have previously identified three amino acid substitutions in the s gene ( figure ) that are potentially associated with demyelination (i m, l i, and t n). by using the site-directed mutagenesis kit (see materials and methods), we mutated three different sites individually or jointly. targeted recombination was carried out between synthetic rnas transcribed from recombinant vectors and recipient virus alb (penn - , ) or fmhv (all other recombinants) according to previously described methods (peng et al, ; das sarma et al, ) . alb is thermolabile and displays small plaque morphology at • c; thus putative recombinants were selected as large plaque viruses after treatment for h at • c and plating on l cell at • c. the use of the virus with feline spike protein (fmhv) also provided a coronavirus s gene demyelination determinants l fu et al useful method to select recombinant viruses against the background of parental viruses. because fmhv does not grow in mouse cells, the recombination process with the transfected plasmid could be achieved in feline cells (fcwf). recombinant viruses with s genes derived from mutated mouse s gene regained the ability to grow in mouse cl- cells. by switching from feline cells to mouse cells that did not support the growth of parental fmhv, we were able to select the recombinant viruses because only recombinant viruses with a mouse s gene were present in these cultures. the potential recombinants were plaque purified twice and screened by using polymerase chain reaction (pcr) sequencing to detect the expected mutations made in the s gene. to verify that no secondary site mutations had been introduced into the s gene, we sequenced the entire s gene of the new recombinant viruses. we analyzed the ability of the new recombinant viruses to grow in l cells by growth-curve kinetics analysis, and compared it with the mhv-a kinetics. we also performed growth analysis of the new viruses in different temperatures ( • c, • c, • c) to rule out a ts phenotype. the growth kinetics of the viruses at • c is shown in figure . it demonstrates that all viruses have similar growth properties. in addition, none of the viruses was ts. we analyzed the virulence and pathologic properties of the new recombinant viruses and compared it to that of wtmhv-a and the control recombinant virus wtr-a . the virulence of the viruses is shown in table . the t n mutation in s reduced viral virulence by fivefold. however, all other individual and combination of mutations in the s gene reduced ). reduced virulence correlated with reduction in the amount of hepatitis, mostly seen in the viruses that had mutations in the s gene. the effect of the three mutations on the pathologic picture of encephalitis and hepatitis is shown in figures and . generally, none of the mutations or combination of mutations affected the extent of encephalitis. hepatitis was abolished in all the viruses that contained the i m mutation individually or with l i (penn - , ; penn k- , ; and penn k- , ). hepatitis was reduced in penn - , (l i individual mutation), but interestingly, hepatitis was not significantly changed in combinations of the t n mutation with each one of the other s mutations (penn - , and penn - , ). the effect of the three mutations on viral replication in the infected organs (brain and liver) is shown in figure . viral titers in the brain and liver were consistent with the pathologic picture. because viruses were injected into the brain, the brain titers on day generally represent residual injected virus. the effect of the three mutations on demyelination is shown in table . demyelination was not affected by the reduced virulence and most nonlethal viruses were still able to cause demyelination. only viruses containing both i m and l i mutations in the s gene were nondemyelinating. therefore, the two combinations that abolished demyelination were the one that included the i m and l i mutations (penn k- and penn k- ) and the one that included all three mutations i m, l i, and t n (penn k- and penn k- ). neither one of these individual mutations was able to abolish demyelination completely. the i m individual mutation and the combination of l i and t n partially reduced demyelination, especially at the lower doses of viral injections. interestingly, the combination of i m and t n mutations did not reduce demyelination at the same level as the individual i m mutation. viral rna persistence of all recombinant and parental viruses in mouse organs (brain, spinal cord and liver) was analyzed by reverse transcriptase l fu et al figure pathologic analysis of recombinant viruses during acute infection. mice were injected ic with pfu per mouse with each virus. only one recombinant virus from each set of duplicate viruses is shown; however, similar findings were found for both viruses. each time point represents the pathologic analysis of two to three mice. dark bars represent encephalitis whereas white bars represent hepatitis. the intensity of the pathologic damage of encephalitis was based on a -level scale: = no inflammatory infiltrates in the brain; = focal inflammatory infiltrate; = multifocal inflammatory infiltrates; = diffuse inflammatory infiltrates in the entire brain (panencephalitis). the intensity of the hepatitis was evaluated based on a -level scale: = no hepatitis; = one to two foci of necrosis in each section of liver; = three to six necrotic foci in each liver section; = more than six foci of nonbridging necrosis in each liver section; = diffuse bridging necrosis of the liver with islands of preserved parenchyma. (rt)-pcr. the results of rna persistence in the spinal cord are shown in figure . both mhv-a and recombinant virus wtr-a showed a prominent band consistent with mhv rna. similar bands were detected following infections with penn - , penn - , penn - , and penn - , all of which are demyelinating viruses. the demyelinatingnegative virus penn k- showed no rna persistence in the cord; however, the other demyelinatingnegative virus penn k- showed a band similar to demyelinating-positive viruses. the demyelinationpositive virus penn - showed a weaker positive band. thus, although some degree of rna persistence may be required for demyelination, the presence of persistence is insufficient for the development of demyelination. rna persistence in the brain was similar to the spinal cords but with a slightly weaker signal. rna persistence in the liver was minimal and was only detected in mhv-a and wtr-a infections (data not shown). mice were injected ic with pfu per mouse with each virus. each time point represents the mean titer analysis of two to three mice. data present log pfu/ml of w/v organ homogenates. only one recombinant virus from each set of duplicate viruses is shown; however, similar findings were found for both viruses. samples of liver and brain from infected mice at different time points post infection were tested by plaque assay on l cells. the mhv genome is the largest among rna viruses. although the generation of an infectious mhv clone has been recently reported (yount et al, ) , the new construct appears to be attenuated in vivo. thus, molecular pathogenesis studies continue to require a complex manipulation of the mhv genome in order to introduce targeted changes into specific locations. to date, the best technique available for this purpose is targeted end heterologous recombination, originally introduced by paul masters (peng et al, ) . we used this technique in our previous studies, in which we identified the demyelination determinants in the s gene (das sarma et al, ) . we continued our investigation in the present study to better define the regions of genomic control of demyelination within the s gene. although incidental mutations during coronavirus replication are not uncommon, we used strict precautions to reduce the chance that incidental mutations might interfere with molecular pathogenesis studies. we sequenced the entire s gene of each new recombinant virus immediately before it was used in pathogenesis studies to confirm the presence of the desired mutations and to exclude the possibility of incidental undesired mutations. the pathogenesis of new recombinant viruses was tested immediately after the generation of the viruses to avoid unnecessary passages of the viruses in culture, which tend to increase the chance for mutations. we generated two identical recombinant viruses for each mutation in two independent recombination procedures. both viruses were tested in mice to ensure an identical in vivo phenotype. these precautions reduced the chance for incidental mutations to a negligible level. using these principles, we found that a combination of mutations in the s gene abolishes demyelination only when introduced simultaneously. the i m mutation, which appears to be the dominant and the more important one, reduces the amount of demyelination but does not abolish it completely. however, i m in combination with l i completely abolishes demyelination. thus, although this may not be the only genomic site that controls demyelination, the region in the s gene downstream from the receptor-binding domain contains important determinants of demyelination (figure ). the coronavirus spike protein is a type i membrane protein with approximately a -residue ectodomain, an -residue transmembrane portion, and a -residue cytoplasmic tail (schmidt et al, ) . the full-length spike glycoprotein oligomerizes rapidly after synthesis and then cleaves by a furin-like protease into two similar-sized fragments: a peripheral s and a membrane-anchored s (sturman et al, ) . s and s are associated by noncovalent interactions. s is responsible for binding to cellular receptors whereas s has the primary responsibility for membrane fusion. the receptors for mhv are members of the family of carcinoembryonic antigen cell adhesion molecules (ceacam), of the immunoglobulin superfamily (dveksler et al, (dveksler et al, , a (dveksler et al, , b . dimers of s bind to a single molecule of ceacam. the binding sites on s are presumably juxtaposed, causing a steric hindrance or global conformational changes that preclude additional interactions (lewicki and gallagher, ) . after binding to receptor, the spike protein undergoes conformational changes, including alternative disulfide linkages and separation of s from s , which may increase the propensity of s to fuse membranes (taguchi and matsuyama, ) . we hypothesize that the i m and l i mutations mark potential s genomic sites that control demyelination. these mutations are in the middle of two regions of yet undesignated name or function on either side of the hypervariable region of s . as shown in figure , we provisionally labeled these regions as s demyelination region (sdr ) and s demyelination region (sdr ). there are no known t-or b-cell epitopes in these regions; however, these s regions may influence spike protein functions in several ways. binding of s to receptor is unlikely to be significantly affected by sites outside the first amino acids because the ceacam binding site can exist wholly in the amino terminal residues of s . however, sdr and sdr may still have influence on s -ceacam binding kinetics, which may affect pathogenesis. similarly, the sdr and sdr regions may alter the kinetics and/or extent of the fusion activity of the spike protein, or the kinetics and/or extent of separation of s from s . either one of these changes may have an effect on the pathogenesis of the virus. the fact that two mutations are necessary for the abolishment of demyelination may rely on the three-dimensional spatial structure and folding of the s part of the molecule and on potential chemical interactions between the two sites. both i m and l i mutations also reduce hepatitis and therefore reduce the overall virulence of the viruses resulting in a nonlethal phenotype. however, the reduced virulence per se is not enough to abolish demyelination. we have shown here and in a previous publications (das sarma et al, sarma et al, , a sarma et al, , b that viruses that lack the ability to produce demyelination can be either less virulent (penn k- , and penn k- ) or significantly more vir-ulent (penn - and penn - , and mhv- ) than mhv-a . moreover, the individual l i mutation has a nonlethal phenotype but no effect on demyelination. in addition, ability of the virus to produce encephalitis may be a prerequisite, but is insufficient for the development of subsequent demyelination. for example, penn k- and penn k- produce encephalitis similar to demyelinating viruses but do not produce demyelination. this study, therefore, provides further evidence that the ability of a virus to produce chronic demyelination is not in direct correlation with the virulence of the virus. similarly, viral persistence may be a prerequisite but is insufficient for the development of demyelination. the property of demyelination is therefore a unique additional neurotropic property, which appears to be at least in part encoded by the s gene of the mhv genome. the following plaque-purified viruses were used in this study: mhv-a (budzilowicz et al, ; lavi et al, a lavi et al, , b , alb (obtained from paul masters, albany, ny), and fmhv (kuo et al, ) (obtained from paul masters, albany, ny). stock viruses had titers of to pfu/ml. viruses were propagated and titered on murine l cells, cl- cells, and feline fcwf cells in dulbecco's minimum essential medium (dmem) with % fetal bovine serum (fbs). the pmh plasmid (obtained from paul masters) contains the . -kb, end of mhv-a genome from the he gene downstream (das sarma et al, ; kuo et al, ) . the sequence of the s gene in pmh is identical to that of mhv-a except for the introduction of avrii and sbfi restriction sites, which allowed the insertion of different s genes into the background of pmh , and enabled the identification of recombinant viruses containing full length recombinant s gene. site-directed mutagenesis experiments were performed using the stratagene quickchange kit (cedar creek, tx). targeted rna recombinations were carried out between alb (for penn - , ), fmhv (for all other recombinant viruses), and synthetic rnas. alb is thermolabile and displays small plaque morphology at • c. fmhv contains an s gene from fipv on an mhv-a background (kuo et al, ) and can only grow in feline cells fcwf. the synthetic rnas were supplied by the pmh plasmid containing the entire end of the mhv genome from gene (he) to (n). for recombination, l cells spinner culture (for penn - , only) or fcwf (all other recombinants) coronavirus s gene demyelination determinants l fu et al was harvested and infected at multiplicity of infection (m.o.i) = of alb (at • c), or fmhv (at • c), respectively. cells were then transfected by electroporation with the synthetic rna by using two consecutive pulses from bio-rad gene pulser apparatus set at . kv and μf. infected and transfected cells were plated onto a monolayer of cl- cells, and the released viruses were harvested at approximately to h post infection. for selection of the recombinant viruses, a two-step plaque purification was performed and pcr sequencing of the s gene was used to confirm the presence of the desired mutations. for each mutation, we produced two identical viruses in two independent recombination events. this strategy was designed to reduce the possibility that random unrecognized mutations would affect the phenotype. the new recombinant viruses were designated as the following (also in table ): ( ) penn k and penn k contain an mhv-a s gene with the i m, l i, and t n -point mutations; ( ) penn k and penn k contain an mhv-a s gene with the i m and l i -point mutations; ( ) penn - and penn - contain an mhv-a s gene with the t n single-point mutation (das sarma et al, c) ; ( ) penn - and penn - contain an mhv-a s gene with the i m single-point mutation; ( ) penn - and penn - contain an mhv-a s gene with the l i single-point mutation; ( ) penn - and penn - contain an mhv-a s gene with the i m and t n two-point mutations; ( ) penn - and penn - contain an mhv-a s gene with the l i and t n -point mutations. finally, a control virus, wtr-a , was generated by targeted recombination in which the s gene of mhv-a was recombined back into the mhv-a background, to eliminate the possibility that any changes in pathogenesis might derive from the recombination process itself. for sequencing of the s gene, rt-pcr was carried out on templates of cytoplasmic rna extracted from virus-infected l cells. double-stranded pcr products were analyzed by automated sequencing using the taq dye terminator procedure, with previously described primers, according to the manufacturer's protocol (das sarma et al, , a , b . mice (c bl/ ) were infected intracerebrally with pfu of each of the recombinant viruses along with same dose mhv-a and wtr-a . two mice per virus were analyzed independently days post infection. briefly, mice were sacrificed and perfused intracardially with ml phosphate-buffered saline (pbs). liver, brain and spinal cord were removed, cleaned with pbs, then snap-frozen with liquid nitrogen. tissue rna was isolated with rneasy mini kit (qiagen). after quantification, μg of each rna sample was re-verse transcribed with superscript rt-pcr system (invitrogen) to synthesis first strand cdna. pcr was performed with the oligonucleotide primers izj ( -gctccaacagttggtgcc- ) and izj ( -acgtaggaccttgctaacttc- ). the primers izj and izj were designed to detect the most conserved region of mhv-a n gene and the untranslated region. a -bp pcr product was analyzed by agarose gel electrophoresis. viral growth curves l cell cultures were prepared in -well plates in dmem with % fbs. confluent monolayers were infected with each virus (m.o.i. = ) in duplicate wells and incubated for h at • c. after adsorption, the cells were washed with pbs three times and then fed with . ml medium. at the indicated times, the cells were lysed by three cycles of freeze-thawing, and the supernatants were removed and titered by plaque assay on l cells as previously described (lavi et al, b) . all animal experiments used -week-old virus-free c bl/ mice (nci; bethesda, md). mice were anesthetized with methofane. the amount of virus inoculated by in each experiment was diluted in pbs containing . % bovine serum albumin (bsa) and a total volume of μl was injected into the left cerebral hemisphere (ic). virulence assays (ld dose) were performed as previously described by ic inoculation of mice per dilution with -fold serial dilutions of wild-type or recombinant viruses (lavi et al, b) . mice were examined for signs of disease or death on a daily basis for up to days post infection. for organ analysis, mice were sacrificed at various time points post infection. after perfusion with pbs, brains and livers were removed. half of the brain and a portion of liver were fixed in % buffered formalin, paraffin-embedded, and stained with hematoxylin and eosin (h&e) for a blinded histologic analysis. samples of brain and liver were frozen in − • c, to be used later for assessment of viral titers by plaque assays. for assessment of demyelination, mice were sacrificed at days post infection. mice were perfused with pbs followed by % buffered formalin. spinal cords were removed, formalin-fixed, and paraffin embedded. four to six transverse sections of cervical, thoracic, and lumbar levels of the cord were stained with luxol fast blue (lfb) and the number of quadrants of cord containing demyelination were counted as a fraction of the total number of cord quadrants evaluated in each individual mouse (lavi et al, b (lavi et al, , (lavi et al, , (lavi et al, , . pathogenesis of multiple sclerosis-the immune diathesis and the role of viruses three intergenic regions of mouse hepatitis virus strain a genome rna contain a common nucleotide sequence that is homologous to the end of the viral mrna leader sequence nidovirales: a new order comprising coronaviridae and arteriviridae sequence analysis of the s gene of recombinant mhv- /a coronaviruses reveals three candidate mutations associated with demyelination and hepatitis mouse hepatitis virus type- infection in mice: an experimental model system of acute meningitis and hepatitis the effect of the t n s gene mutation on mhv-a pathogenesis demyelination determinants map to the spike glycoprotein gene of coronavirus mouse hepatitis virus several members of the mouse carcinoembryonic antigen-related glycoprotein 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spike protein determines the ability of the virus to replicate in the liver and cause hepatitis construction of murine coronavirus mutants containing interspecies chimeric nucleocapsid proteins identification of the spinal cord as a major site of persistence during chronic infection with a murine coronavirus multiple regions of the murine coronavirus spike glycoprotein influence neurovirulence pathogenesis of chimeric mhv- /mhv-a recombinant viruses: the murine coronavirus spike protein is a major determinant of neurovirulence nucleotide sequence of the gene encoding the surface projection coronavirus mhv-jhm in vivo and in vitro models of demyelinating disease. ix. progression of jhm virus in the central nervous system of rat during overt and asymptomatic phases proteolytic cleavage of the e glycoprotein of murine coronavirus: activation of cell-fusing activity of virions by trypsin and separation of two different k cleavage fragments soluble receptor potentiates receptor-independent infection by murine coronavirus the biology and pathogenesis of coronaviruses genetic variation of neurotropic and nonneurotropic murine coronaviruses pathogenesis of demyelination induced by a mouse hepatitis virus (jhm virus) acquired fusion activity of a murine coronavirus mhv- variant with mutations in the proteolytic cleavage site and the siganal sequence of the s protein heteroginity of gene expression of the hemagglutinin-esterase (he) protein of murine coronaviruses systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a key: cord- -wl jk authors: totoiu, minodora o.; nistor, gabriel i.; lane, thomas e.; keirstead, hans s. title: remyelination, axonal sparing, and locomotor recovery following transplantation of glial-committed progenitor cells into the mhv model of multiple sclerosis date: - - journal: exp neurol doi: . /j.expneurol. . . sha: doc_id: cord_uid: wl jk the behavior and myelinogenic properties of glial cells have been well documented following transplantation into regions of focal experimental demyelination in animal models. however, the ability of glial cell preparations to remyelinate in such models does not necessarily indicate that their transplantation into demyelinated lesions in clinical disease will be successful. one of the precluding factors in this regard is a greater understanding of the environmental conditions that will support transplant-mediated remyelination. in this study, we determined whether the complex and reactive cns environment of the mouse hepatitis virus (mhv) model of multiple sclerosis (ms) could support transplant-mediated remyelination. striatal neural precursors derived from postnatal day mice were committed to a glial cell lineage and labeled. immunohistochemical staining indicated that this population generated > % glial cells following differentiation in vitro. transplantation of glial-committed progenitor cells into the t spinal cord of mhv-infected mice demonstrating complete hindlimb paralysis resulted in migration of cells up to mm from the implantation site and remyelination of up to % of axons. transplanted-remyelinated animals contained approximately × the number of axons within sampled regions of the ventral and lateral columns as compared to non-transplanted animals, suggesting that remyelination is associated with axonal sparing. furthermore, transplantation resulted in behavioral improvement. this study demonstrates for the first time that transplant-mediated remyelination is possible in the pathogenic environment of the mhv demyelination model and that it is associated with locomotor improvement. remyelination is a successful regenerative event within the adult central nervous system (cns), as it can restore saltatory conduction (smith et al., ) and facilitate functional recovery (jeffery and blakemore, ) . remyelination has been demonstrated in a variety of experimental demyelination/mutant models (blakemore, (blakemore, , (blakemore, , (blakemore, , duncan et al., ; gumpel et al., ; herndon et al., ; blakemore, , ; jeffery et al., ; moore et al., ; rodriguez et al., ; sasaki and ide, ; yajima and suzuki, ) and occurs spontaneously in naturally occurring demyelinating diseases such as ms (prineas et al., a) . however, remyelination failure is more prevalent than its success when considering the spectrum of pathologies that affect the cns. although remyelination is usually successful in most animal models, it is often incomplete in the theiler's model of demyelination (murray et al., ) . furthermore, remyelination is less efficient in old animals than in young animals (shields et al., ) . the failure of remyelination is illustrated within foci of chronic demyelination within the later stages of ms (prineas et al., b) . chronic ms lesions are characterized by oligodendrocyte loss (ozawa et al., ) , with remyelination limited to the borders of inactive plaques (suzuki et al., ) . repairing persistent demyelination may ameliorate clinical deterioration. in addition to restoring normal impulse conduction (utzschneider et al., ) , remyelination may decrease axonal degeneration/transection. this is suggested by the demonstration that chronically demyelinated axons are vulnerable to degeneration/transection and that axonal loss in the later stages ms contributes to clinical deterioration (bjartmar et al., ; de stefano et al., ; . these findings underscore the importance of developing therapeutic strategies to enhance remyelination. one approach is to activate or enhance the response of endogenous mechanisms for repair, as has been demonstrated following growth factor administration in the experimental autoimmune encephalomyelitis (eae) demyelination model (yao et al., ) , or passive transfer of antiserum (rodriguez et al., ) or purified immunoglobulin (rodriguez and lennon, ) from mice immunized with spinal cord homogenate into the theiler's demyelination model (miller and rodriguez, ) . an alternative approach to remyelinating areas of demyelination is the transplantation of remyelination-competent cells. stem cells and neural precursors represent attractive sources for the generation of remyelination-competent cells, as they can be readily amplified and differentiated to the oligodendrocyte lineage (ben-hur et al., ; brustle et al., ) . stem cell-derived glial precursors have been shown to myelinate following transplantation into the myelindeficient rat (brustle et al., ) , and neural precursorderived glial-committed progenitors (ben-hur et al., ; have been shown to myelinate following transplantation into regions of acute experimental demyelination . more recently, intracerebroventricular or intrathecal implantation of neural precursors into the eae demyelination model resulted in migration of transplanted cells into white matter and their differentiation to astrocytes and oligodendrocytes, although no assessment of their ability to myelinate was performed (ben-hur et al., ) . to determine whether transplantation represents a viable strategy for treating demyelination, it is necessary to better understand the range of environmental conditions that support transplantation-mediated remyelination. in this study, we investigated the ability of the complex and reactive disease state of the chronic demyelinating mhv model of ms to support transplant-mediated remyelination. intracerebral injection of the j . v- strain of mhv results in acute encephalomyelitis with demyelination, followed - days later by an immunemediated demyelinating encephalomyelitis with hindlimb paralysis and progressive cns destruction, including the initiation of new demyelination foci probably for the life of the mouse (fleming et al., ; haring and perlman, ; lane et al., ; stohlman and hinton, ; stohlman et al., ) . this model provides an environment of ongoing demyelinating pathogenesis, and is thus distinct from gliotoxin lesions. intraspinal transplantation of glial-committed progenitors into the mhv model of ms resulted in extensive migration of transplanted cells, robust remyelination, axonal sparing, and behavioral improvement. these results show that transplant-mediated remyelination is possible following intraspinal transplantation into an environment of ongoing pathogenesis resembling ms. striata from postnatal day c bl/ mice were dissected, triturated, and maintained as previously described (ben-hur et al., ) . trypan blue was used to determine cell viability. cells were grown for days as floating clusters in -well low-adherent plastic dishes (corning life sciences acton, ma) at an initial density of  cells/ ml of dmem:f , b supplement (gibco-invitrogen, carlsbad, ca) with insulin, sodium selenite, transferrin, putrescin, progesterone, t , detailed in ben-hur et al. ( ) , and . ag/ml epidermal growth factor (egf; sigma-aldrich, st. louis, mo). on day , culture supernatant containing the floating clusters was removed and centrifuged at  g for min. clusters were then resuspended in fresh media and added to new culture dishes. on day , . ag/ml egf was added to the culture dishes. on day , clusters were washed, resuspended in fresh media, and added to new culture dishes. on day , clusters were incubated with mm brdu (sigma-aldrich) in the culture medium overnight. one culture dish was not labeled with brdu to compare viability and differentiation in the presence and absence of brdu. on day , cultures were prepared for transplantation. clusters were dissociated with . % trypsin-edta (invitrogen canada inc., burlington, on) for min, triturated, centrifuged for min at  g, resuspended three times in calcium-and magnesium-free dmem (gibco-invitrogen), and concentrated to a final density of  cells/ al in the same media. the cell preparation was kept on ice for a maximum of h before transplantation. on the day of transplantation, some cells were prepared for immunocytochemistry. clusters were centrifuged for min at  g and resuspended three times in fresh media without egf, and were plated on mg/ml poly-l-lysine (sigma-aldrich) and ag/ml laminin (sigma-aldrich) coated four chamber, imaging slides (nalgene-nunc international, rochester, ny). to assess differentiation potential, cells were grown on imaging slides on adherent substrate for days, then fixed in % paraformaldehyde (fisher scientific, pittsburgh, pa) in pbs for min and immunocytochemical staining was performed using standard protocols. imaging chambers were blocked with % normal goat serum (ngs) (chemicon, temecula, ca) for min at room temperature. primary antibodies (polyclonal rabbit anti-galc, chemicon, : dilution in % ngs; monoclonal mouse anti-neun, chemicon, : dilution in % ngs; polyclonal rabbit anti-gfap, dako, denmark, : dilution in % ngs; monoclonal mouse anti-cd b, serotec, uk, : dilution in % ngs; polyclonal rat anti-brdu, accurate chemical and scientific corporation, westbury, ny, : dilution in % ngs) were applied to imaging chambers overnight at jc. imaging chambers were rinsed three times with pbs, incubated for min in % ngs, and fluorescent-conjugated secondary antibodies (alexa or , goat anti-rabbit, goat anti-rat, or goat antimouse igg h+l, : dilution in % ngs; vector laboratories, burlingame, ca) was applied and incubated for h at room temperature. chambers were rinsed three times in pbs, and nuclear staining was conducted by exposing cultures to bis-benzimide (hoechst , molecular probes, eugene, or) for min. cell quantification was conducted using an olympus ax- light microscope with a  objective. the percentage of immunopositive cells was determined by dividing the total number of immunopositive cells by the total number of hoechst-positive cells in each imaging chamber, and averaging the results from three different imaging chambers per marker. a total of hoechst-positive cells were counted for these analyses. each -chamber imaging slide had one no-primary control chamber and three stained chambers for each of the markers mentioned above. only immunopositive cells with clearly hoechstpositive nucleus were counted. age-matched, weight-matched ( - g) male c bl/ mice (h- b background; national cancer institute, bethesda, md; n = ) were anesthetized by methoxyflurane inhalation (pitman-moore inc., washington crossing, nj). mice received intracerebral injections of plaque forming units of the neurotropic corona virus mhv strain j . v- (kindly provided by j. fleming, university of wisconsin, madison, wi), suspended in al of sterile saline (lane et al., ) . intracerebral injection of mhv results in a biphasic disease: acute encephalomyelitis with myelin loss, followed - days later by an immunemediated demyelinating encephalomyelitis with hindlimb paralysis and progressive destruction of the cns (fleming et al., ; haring and perlman, ; lane et al., ; stohlman et al., ) . two of the mhv injected animals died during the first week post injection; there is an - % survival rate of animals injected with this viral strain, and the animals usually die during the first days of acute infection. if the animals survive the acute stage of disease there is > % chance of survival. control (sham) animals (n = ) were injected with al of sterile saline alone and did not develop any behavioral or histological deficits. twelve days after mhv intracerebral injection, each animal (n = ) received a single injection of , cells in al of calcium-and magnesium-free dmem (gibco-invitrogen) at t of the spinal cord, using the protocol described in blakemore and crang ( ) . briefly, animals were anesthetized with avertin and received a laminectomy at t . the spinal process cranial to the laminectomy was immobilized using a micromanipulator and the transplant needle, a -al hamilton syringe (hamilton company, reno, nv) with a silicon-coated pulled glass tip, was lowered into the spinal cord using a stereotactic manipulator arm. cell suspensions were injected into one site at the midline of the spinal cord. the needle was removed from the cord min after injection of the cells was complete. in addition, six control animals that received an mhv intracerebral injection received no transplant. one animal died during transplantation/anesthesia. animals were randomly selected for inclusion in either group and all received an incision to the skin overlying t to allow blinded analyses for the duration of the experiment. behavioral testing of all animals was conducted by a blinded observer, every other day using the -point clinical scoring scale (houtman and fleming, ) where = normal, = limp tail, = waddling gait and partial hindlimb weakness, = complete hindlimb paralysis, = death animal. this -point scale was supplemented with a single increment between each point, such that . = limp tail and partial waddling gait and no hindlimb weakness, . = hindlimb weakness and partial hindlimb paralysis, . = complete hindlimb paralysis and moribund disposition. animals were acclimated and behaviorally tested day before mhv injection. after mhv injection, animals were tested every day. after transplantation, animals were tested every other day for weeks and then every rd day until the end of the experiment. statistical significance was determined using the mann -whitney u test. animals were euthanized under chloral hydrate anesthesia (fisher scientific) days following transplantation or days after mhv infection and fixed by cardiac perfusion with % paraformaldehyde (fisher scientific) in . m pbs, ph . . the length of spinal cord extending mm cranial and mm caudal to the site of implantation was cut into -mm transverse blocks and processed so as to preserve the craniocaudal sequence and orientation. alternating tissue blocks were processed for resin and cryostat sectioning and analyzed in a blinded fashion. resin sections were used to determine the area of the ventral and lateral columns, and the number of demyelinated, remyelinated, and normally myelinated axons. cryostat sections were used to determine spread of brdu-prelabeled transplanted cells and their differentiation profile. for resin sectioning, odd numbered blocks were post fixed in % glutaraldehyde (fisher scientific), then exposed to % oso (electron microscopy sciences, fort washington, pa), dehydrated in ascending alcohols, and embedded in spurr resin (electron microscopy sciences) according to standard protocols. transverse semi-thin ( am) sections were cut from the cranial face of each block, stained with alkaline toluidine blue, cover slipped, and examined by light microscopy on an olympus ax- microscope using olympus microsuite b sv software. total areas of the ventral and lateral columns were measured with the  objective and averaged. a t test was performed to compare the areas of the ventral and lateral columns in transplanted and non-transplanted animals. the state of myelination was determined by assessing the thickness of the myelin sheath in relation to the axon diameter (guy et al., ; hildebrand and hahn, ) . demyelinated, remyelinated, and normally myelinated axons were counted within  am areas, totaling am , on each tissue section using the  objective with  optical zoom. am represents approximately % of the total area of remyelination within transplanted animals, which was determined by measuring the total area of remyelination in tissue sections from each block in each animal using the  objective, and averaging areas from all animals in each group. these quantitative assessments were conducted throughout the region extending mm cranial and mm caudal to the implantation site. the number of demyelinated axons, remyelinated axons, the total number of axons (normally myelinated plus demyelinated plus remyelinated axons), and the percentage of remyelinated axons (number remyelinated/number total axons) were determined for each of the four regions on each tissue block, averaged, then averaged across animals within each group for each tissue block. a t test was performed to compare these values for transplanted and non-transplanted groups. to determine whether the number of remyelinated axons in each animal correlated with the total number of axons in each counting area, remyelinated and total axons counts were each averaged among all four counting areas for all blocks for each animal, and the correlation coefficient between these two values was determined for both transplanted and non-transplanted groups. statistical analyses were conducted using spss software. for cryostat sectioning, even numbered blocks were cryoprotected in % sucrose solution in pbs, embedded in oct (fisher scientific) and frozen sectioned in the transverse plane at am on a jung cm leica cryostat for anti-brdu staining. for brdu staining, sections were washed in pbs, exposed to % formamide (sigma-aldrich) and n hydrochloric acid (fisher scientific) for min at jc for dna denaturation, followed by a -min exposure to . % h o in pbs, then incubated overnight at room temperature with rat anti-brdu polyclonal antibody (accurate chemical and scientific corporation) at a : dilution in % ngs. secondary antibody was biotinylated goat anti-rat heavy and light chain igg (sigma-aldrich). vectastain abc (vector laboratories) and , v-diaminobenzidine (vector laboratories) were used for signal visualization. the number of brdu-positive cells was counted on three sections -am apart from each tissue block for each animal using olympus microsuite b sv software, and averaged. the numbers of brdu-positive cells within corresponding blocks from animals within a group were then averaged. for brdu, apc-cc double staining, sections were washed in pbs, exposed to n hydrochloric acid (fisher scientific) for min at jc for dna denaturation, and primary antibodies (rat anti-brdu, : , accurate chemical and scientific corporation; mouse anti-apc-cc , : , oncogene research products, san diego, ca) were diluted in % bovine serum albumin and applied to slides overnight at room temperature. slides were rinsed three times with pbs, incubated for min in % bovine serum albumin, and secondary antibodies (alexa or , goat anti-rat or goat anti-mouse igg h+l, : dilution in % bovine serum albumin; vector laboratories) were applied and incubated for h at room temperature. trypan blue analysis indicated that approximately . - .  viable cells were obtained from each postnatal day mouse. floating cell clusters reached approximately am in diameter after days of culture (fig. a) . on day , clusters were either ( ) dissociated and transplanted into animals or ( ) transferred to an adherent substrate in the absence of growth factors, grown for an additional days and assayed for differentiation potential. as early as h after transfer of cell clusters to an adherent substrate and concomitant growth factor withdrawal, cells started to spread out from the adherent clusters and by day, displayed complex cellular morphologies (fig. b) . after days of growth on adherent substrate, oligodendrocytes and astrocytes could be identified by their morphology and immunolabeling. oligodendrocytes displayed a multipolar morphology with membranous extensions and galc immunoreactivity (fig. c) , whereas astrocytes displayed a flat or stellate morphology and gfap immunoreactivity (fig. d) . few neun immunoreactive neurons (fig. e ) and cd b immunoreactive microglia (results not shown) were detected. no-primary antibody control staining chambers had no positive staining. different cell types often occupied discrete regions of the culture dishes. a total of hoechst-positive cells were counted for the following analyses. quantification of immunolabeled cells indicated that f . % (range = - , median = ) of the total cell population was brdu immunoreactive following an overnight pulse (fig. f) , and that the differentiation protocol yielded . f . % oligodendrocytes (range = - , median = ), f . % astrocytes (range = - , median = ) and . f . % other cell types (range = - , median = ), which included neun + neurons, cd b + microglia, and other hoechst-positive cells not identified by the immunostains tested (fig. g) . no difference was observed in the viability or differentiation of brdu-labeled or -unlabeled cells. transplanted glial-committed progenitors, labeled with brdu before implantation, survived and colonized long lengths of the spinal cord during the -day survival period (fig. ) , and differentiated into mature oligodendrocytes (fig. ) . brdu-labeled cells were detected mm cranial and mm caudal to the site of implantation (the extent of tissue examined), and extended throughout the transverse plane of the spinal cord, primarily within white matter tracts. the number of brdu-labeled cells was greatest around the site of implantation, and fell off sharply - mm either side of the site of implantation. brdu-labeled cells were absent in non-transplanted animals (fig. c) and in no-primary antibody control stains of transplanted animals. non-transplanted animals infected with mhv develop a demyelinating disease accompanied by mononuclear cell infiltration, widespread myelin destruction, and progressive degeneration of the cns in the survivors (fleming et al., ; haring and perlman, ; liu et al., ; stohlman et al., ) . systematic random analyses (fig. a) indicated that numerous demyelinated axons were present (figs. b, e, g) among vacuoles, myelin debris, activated macrophages, lymphocytes, and necrotic cells throughout the region of spinal cord examined, indicative of ongoing pathogenesis. the number of demyelinated axons was not significantly different ( p > . ) in transplanted and non-transplanted animals (fig. a) , likely because mhv is an ongoing demyelinating disease generating a similar number of newly demyelinated axons in both non-transplanted and transplanted animals. the number (fig. b) and percentage (g) quantification of cell types after days of growth on adherent substrate in the absence of growth factors indicated that the differentiation protocol yielded . f . % oligodendrocytes, f . % astrocytes, and . f . % other cell types, which included neun+ neurons, cd b + microglia, and other hoechst-positive cells not identified by the immunostains tested. error bars represent standard deviation.  magnification for a and b,  for magnification for c and d,  magnification for e,  magnification for f. (fig. c ) of remyelinated axons in non-transplanted animals was significantly lower ( p < . ) than in transplanted animals, and composed less than % of the total number of axons counted (fig. c ). normally myelinated axons were exceedingly rare at this time point. control animals injected with saline only did not develop clinical or histopathological deficit. transplantation of glial-committed progenitors resulted in widespread remyelination (figs. and ). remyelinated axons were identified by their characteristically thin my- elin sheaths (figs. c, f, h) and appeared in aggregates distributed throughout the dorsal, ventral, and lateral columns among demyelinated axons. remyelination extended mm cranial and mm caudal to the implantation site in six of seven transplanted animals. counts of remyelinated axons throughout the region extending mm cranial and mm caudal to the implantation site (the extent of our quantification; fig. b ) indicated that regions of remyelination contained % to % remyelinated axons (fig. c) . normally myelinated axons were exceedingly rare at this time point; note that for each tissue block, the number of remyelinated axons (fig. b) plus the number of demyelinated axons (fig. a) approximates the total number of axons (fig. d) . one transplanted animal contained few remyelinated axons, similar to non-transplanted animals ( p > . ); immunohistological analysis of this animal revealed that it contained no brdu-positive cells. we did not observe tumor, teratoma, or non-neuronal tissue formation in transplant recipients, nor did we observe a qualitative difference in schwann cell presence or remyelination between transplanted and non-transplanted animals. counts of the total number of axons (normally myelinated, demyelinated, and remyelinated) within the region extending mm cranial and mm caudal to the transplantation site (the extent of spinal cord examined) suggest that transplanted animals had significantly more axons ( p < . ) within the ventral and lateral columns as compared to non-transplanted animals (figs. g, h and d). area measurements of the ventral and lateral columns revealed no significant difference between animals within and between the transplanted and non-transplanted groups ( p > . , t test), however, a two-sample t test power analysis indicated that an experimental n of per group was insufficient to conclude statistical significance of the differences between the groups. nonetheless, these data suggest that the higher number of total axons within transplanted animals was not a result of tissue shrinkage in the transplanted animals. comparison of the total number of axons with the number of remyelinated axons for each animal within the transplanted group indicated a statistically significant correlation between these two values (r = . ; p < . ), indicating that a greater degree of axonal sparing correlated with a greater degree of remyelination. all transplanted animals survived for the duration of the experiment. non-transplanted animals developed clinical disease typically characterized by a waddling gait and partial hindlimb weakness by - days post-infection, which progressed to complete hindlimb paralysis by days post-infection, persisting for the duration of the experiment (fig. ) . control saline-injected animals displayed normal locomotor behavior throughout the duration of the experiment (all animals scored ). transplantation of glial-committed progenitors resulted in a significant improvement in locomotor abilities (fig. ) . remyelination extended mm cranial and mm caudal to the implantation site (arrow) in transplanted animals (the extent of tissue examined) and was significantly greater than the degree of remyelination in non-transplanted animals at every point examined ( p < . ). (c) throughout this region, % to % of the total number of axons in the ventral and lateral columns of transplanted animals were remyelinated. remyelination in non-transplanted animals was significantly less at every point examined ( p < . ). (d) the total number of axons within am of white matter in transplanted animals was approximately  the total number of axons within similar regions in non-transplanted animals ( p < . for all points), suggesting that remyelination is associated with axonal sparing. error bars represent standard deviation. fig. . transplantation of glial-committed progenitor cells resulted in an improvement in locomotor abilities. transplanted animals demonstrated a significant improvement ( p < . ) in locomotor abilities beginning days after transplantation (arrow). from days after induction of disease, transplanted animals walked with a waddling gait and partial hindlimb weakness, whereas non-transplanted animals demonstrated complete hindlimb paralysis. error bars represent standard deviation. all transplanted animals had complete hindlimb paralysis at the time of transplantation. six of seven transplanted animals demonstrated a significant ( p < . ) improvement in locomotor abilities beginning days after transplantation, which progressed to weight-supported waddling locomotion with partial hindlimb weakness. one transplanted animal demonstrated no improvement in locomotor abilities; postmortem analysis of this animal revealed that it contained no brdu-positive transplanted cells and a degree of remyelination that was not significantly different ( p > . ) from non-transplanted animals. cell replacement strategies must contend with the fact that the differentiation of transplanted cells is strongly influenced by the environmental signals and cellular deficiencies operating at the site of implantation. for example, stem cells isolated from the adult hippocampus generate new hippocampal neurons and glia following transplantation into the hippocampi of other animals, while these same cells generate olfactory bulb neurons expressing appropriate neurotransmitter phenotypes following transplantation into the rostral migratory stream, or glial cells following transplantation into regions of the cns which do not normally generate neurons in the adult (gage et al., ; suhonen et al., ) . these influences present formidable challenges to cell replacement strategies aimed at promoting repair of neurodegenerative disease or injury due to the complex and reactive environment of such lesions, and point towards lineage commitment of multipotent cells before transplantation (keirstead, ) . our differentiation protocol resulted in the generation of oligodendrocytes and astrocytes in high yield ( . % and %, respectively) from postnatal day derived striatal neural precursors (fig. ) , supporting previous studies using a similar differentiation protocol (ben-hur et al., ) . cellular migration in vitro was evident as early as h, and cellular differentiation in vitro was evident as early as day, after transfer of cell clusters to an adherent substrate and concomitant growth factor withdrawal. to assess migration and differentiation in vivo, undifferentiated prelabeled glial-committed progenitors were transplanted into regions of demyelination in the mhv model of ms. intracerebral injection of the j . v- strain of mhv results in initial viral replication in the ependymal cells lining the ventricles, which subsequently extends to the central canal, gray and white matter of the spinal cord. infectious virus is usually eliminated by days - post infection, but is not completely cleared. acute encephalomyelitis with myelin loss is present by day and extends rapidly in the anterior funiculi. an immune-mediated demyelinating encephalomyelitis with hindlimb paralysis and progressive destruction of the cns then follows; although some remyelination takes place, the initiation of new foci of demyelination likely continues for the entire life of the mouse (fleming et al., ; haring and perlman, ; lane et al., ; liu et al., ; stohlman and hinton, ; stohlman et al., ) . in the current study, undifferentiated glial-committed progenitors were transplanted into the thoracic spinal cords of mice with fulminate demyelinating pathology and complete hindlimb paralysis as a result of mhv infection. transplanted cells survived for the duration of the -day post-transplantation period and spread throughout the mediolateral extent of the spinal cord as well as mm cranial and mm caudal to the site of implantation, the extent of the tissue being examined (fig. ) . these findings demonstrate that transplanted progenitor cells are capable of extensive migration throughout regions of ongoing pathogenesis. survival and extensive migration of transplanted gliogenic cell populations has been demonstrated in the developing normal cns (jacque et al., ) , myelin-deficient mutant cns (brustle et al., ; gansmuller et al., ; liu et al., ; tontsch et al., ) , chemically demyelinated cns (groves et al., ; , and in inflamed white matter tracts in the eae model of ms (ben-hur et al., ; pluchino et al., ) . however, the ability of gliogenic cell populations to survive and migrate in these environments does not indicate that they will do so in the intact normal adult cns. when glial progenitors (franklin et al., ; o'leary and blakemore, ) are transplanted into the intact normal adult cns, they fail to survive or migrate; only when the cells are placed in the immediate vicinity of active regions of demyelination, where survival factors are elevated (hinks and franklin, ) , do they enter the lesion. these findings suggest that the survival and extensive migration of cells in our study was due in part to the continuity of pathology throughout the region of migration. transplantation of glial-committed progenitors resulted in extensive remyelination, ranging from % to % of the axons present within randomly selected regions of the spinal cord (figs. and ) . the in vitro (fig. ) and in vivo (fig. ) differentiation profile of these cells, the presence of extensive remyelination only in transplanted animals (figs. b, c) , and the presence of remyelination throughout the region over which transplanted cells migrated (figs. d and b) suggest that remyelination was conducted by the transplanted cell population. nonetheless, we cannot rule out the possibility that remyelination in transplanted animals was carried out by endogenous cells that may have been activated as a result of trophic support by the transplanted population. cumulative axonal loss has recently been demonstrated in several human (bitsch et al., ; ferguson et al., ; trapp et al., ) and rodent (bjartmar et al., ; griffiths et al., ; yin et al., ) demyelinating pathologies, using in vivo magnetic resonance imaging (stevenson and miller, ; van waesberghe et al., ) , in vivo magnetic resonance spectroscopy (arnold et al., ; matthews et al., ) , morphological analysis of brain sections (ferguson et al., ; trapp et al., ) , and biochemical methods (bjartmar et al., ) . chronic demyelination is a probable cause of such axonal loss (bjartmar and trapp, ) . to determine whether remyelination was associated with axonal sparing in the current study, total axon numbers were determined within the ventral and lateral columns of transplanted and non-transplanted animal groups days after induction of disease. although the area of the ventral and lateral columns were not significantly different between the transplanted and nontransplanted groups ( p > . , t test), the total number of axons within the ventral and lateral columns of transplanted remyelinated animals was approximately  the number of axons within the ventral and lateral columns of non-transplanted animals (fig. d) . furthermore, comparison of the total number of axons with the number of remyelinated axons for each animal within the transplanted group indicated a statistically significant correlation between these two values (r = . ). these findings suggest that a greater degree of axonal sparing correlated with a greater degree of remyelination. reduced axonal loss has also been shown after intravenous or intrathecal neurosphere transplantation in the eae mouse model of ms (pluchino et al., ) . these data support the view that chronic demyelination is a cause of axonal loss, and suggest that transplant-mediated remyelination protects axons from degeneration/transection. further studies are required to determine the molecular mechanisms underlying demyelination-associated axonal degeneration/transection. transplantation of glial-committed progenitors resulted in a significant improvement in locomotor abilities beginning days after transplantation, which progressed to weightsupported waddling locomotion with partial hindlimb weakness (fig. ) . non-transplanted animals remained completely paralyzed throughout this time period (fig. ). in regions of experimental demyelination induced by lysolecithin (smith et al., (smith et al., , yezierski et al., ) or ethidium bromide (felts and smith, ) , return of secure axonal conduction occurs at the same time as spontaneous remyelination and transplanted glial cells have been shown to improve conduction velocity to near-normal values in myelin-deficient rats (utzschneider et al., ) and ethidium bromide lesions (honmou et al., ) . transplantation of clonal neural cells into the intracerebroventricular system of the shiverer mice at birth resulted in myelination of up to % of host neuronal processes and reduced the symptomatic tremor in some animals (yandava et al., ) . thus, it is intriguing that the time course of behavioral improvement following transplantation in the current study approximates the time required for remyelination (pender et al., ; smith et al., ) . our findings demonstrate that transplant-mediated remyelination is possible in the complex and reactive environment of the mhv model of multiple sclerosis. transplant-mediated remyelination has been demonstrated in regions of the cns subjected to experimental demyelination or in myelin mutants, environments that are characterized by a clear cellular deficit, a largely non-reactive cns environment, and a lack of inflammation. thus, transplantation of embryonic stem cell-derived precursors (brustle et al., ) or oligodendrocyte progenitors (zhang et al., ) into postnatal myelin-deficient rats resulted in differentiation of transplanted cells into myelinating oligodendrocytes and astrocytes. multipotential psa-ncam + neural precursors isolated from the postnatal rat brain have been shown to differentiate into oligodendrocytes, schwann cells, and astrocytes following transplantation, to completely remyelinate regions of acute demyelination in the adult rat induced by ethidium bromide injection into x-irradiated dorsal column white matter . similarly, transplantation of embryonic stem cells into regions of ethidium bromide induced demyelination in the adult rat spinal cord or into myelin-deficient shiverer mutant mice, resulted in the generation of stem cell-derived oligodendrocytes that were capable of myelinating axons (liu et al., ) . more recently, transplant-mediated remyelination has been illustrated in the eae model of ms, following intravenous or intrathecal injection of adult neural precursor cells (pluchino et al., ) . our findings confirm and extend those outlined above, in demonstrating that intraspinal transplantmediated remyelination can occur in the fulminate neurodegenerative environment of the mhv model of ms and is associated with locomotor improvement. the ability of glial cell preparations to remyelinate in such models does not necessarily indicate that their transplantation into demyelinated lesions in clinical disease will be successful (stangel, ) . firstly, transplanted cells may be subject to the same demyelinating process that led to the development of the initial pathology. notably, the viral 'trigger' of pathology in the mhv model of ms is largely cleared by days post-infection (lane et al., ; stohlman and hinton, ) , before the time of implantation in the current study. secondly, the inability of glial progenitors to migrate through normal tissue (o'leary and renders them less than ideal for remyelinating disparate foci of demyelination as seen in clinical disease (prineas et al., a) . thirdly, human transplantation necessitates a human progenitor or stem cell population that is capable of amplification and is myelinogenic. although robust amplification of human embryonic stem cells has been achieved (carpenter et al., ; reubinoff et al., ) , the conditions needed to differentiate them into a remyelinationcompetent transplant population have not yet been defined. and fourthly, the gliotic environment of clinical lesions, which may contribute to the failure of endogenous remyelination, may prohibit the transplant population from effecting repair. clearly, several key issues must be addressed before a cellular replacement strategy can be considered for the treatment of human demyelinating pathologies such as ms. use of proton magnetic resonance spectroscopy for monitoring disease progression in multiple sclerosis growth and fate of psa-ncam+ precursors of the postnatal brain transplanted multipotential neural precursor cells migrate into the inflamed white matter in response to experimental autoimmune encephalomyelitis acute axonal injury in multiple sclerosis. correlation with demyelination and inflammation axonal and neuronal degeneration in multiple sclerosis: mechanisms and functional consequences axonal pathology in myelin disorders neurological disability correlates with spinal cord axonal loss and reduced nacetyl aspartate in chronic multiple sclerosis patients remyelination of the superior cerebellar peduncle in the mouse following demyelination induced by feeding cuprizone remyelination of the superior cerebellar peduncle in old mice following demyelination induced by cuprizone remyelination by schwann cells of axons demyelinated by intraspinal injection of -aminonicotinamide in the rat ethidium bromide induced demyelination in the spinal cord of the cat transplantation of glial cells into areas of demyelination in the adult rat spinal cord embryonic stem cellderived glial precursors: a source of myelinating transplants enrichment of neurons and neural precursors from human embryonic stem cells axonal damage correlates with disability in patients with relapsing -remitting multiple sclerosis. results of a longitudinal magnetic resonance spectroscopy study transplantation of oligodendrocytes and schwann cells into the spinal cord of the myelin-deficient rat conduction properties of central nerve fibers remyelinated by schwann cells axonal damage in acute multiple sclerosis lesions experimental demyelination induced by coronavirus jhm (mhv- ): molecular identification of a viral determinant of paralytic disease transplanted cg cells (an oligodendrocyte progenitor cell line) survive, migrate, and contribute to repair of areas of demyelination in x-irradiated and damaged spinal cord but not in normal spinal cord survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain tracing transplanted oligodendrocytes during migration and maturation in the shiverer mouse brain axonal swellings and degeneration in mice lacking the major proteolipid of myelin repair of demyelinated lesions by transplantation of purified o- a progenitor cells myelination and remyelination in the central nervous system by transplanted oligodendrocytes using the shiverer model. discussion on the remyelinating cell population in adult mammals spectra of g ratio, myelin sheath thickness, and axon and fiber diameter in the guinea pig optic nerve mouse hepatitis virus regeneration of oligodendroglia during recovery from demyelinating disease relation between myelin sheath thickness and axon size in spinal cord white matter of some vertebrate species distinctive patterns of pdgf-a, fgf- , igf-i, and tgf-beta gene expression during remyelination of experimentally-induced spinal cord demyelination restoration of normal conduction properties in demyelinated spinal cord axons in the adult rat by transplantation of exogenous schwann cells pathogenesis of mouse hepatitis virusinduced demyelination comparative migration and development of astroglial and oligodendroglial cell populations from a brain xenograft remyelination of mouse spinal cord axons demyelinated by local injection of lysolecithin locomotor deficits induced by experimental spinal cord demyelination are abolished by spontaneous remyelination behavioural consequences of oligodendrocyte progenitor cell transplantation into experimental demyelinating lesions in the rat spinal cord stem cell transplantation into the central nervous system and the control of differentiation the role of oligodendrocytes and oligodendrocyte progenitors in cns remyelination polysialylated neural cell adhesion molecule-positive cns precursors generate both oligodendrocytes and schwann cells to remyelinate the cns after transplantation dynamic regulation of alpha-and beta-chemokine expression in the central nervous system during mouse hepatitis virusinduced demyelinating disease embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and after spinal cord transplantation neutralization of the chemokine cxcl reduces inflammatory cell invasion and demyelination and improves neurological function in a viral model of multiple sclerosis putting magnetic resonance spectroscopy studies in context: axonal damage and disability in multiple sclerosis spontaneous and induced remyelination in multiple sclerosis and the theiler's virus model of central nervous system demyelination dose-dependency of mbp-induced demyelination in the guinea pig spontaneous remyelination following extensive demyelination is associated with improved neurological function in a viral model of multiple sclerosis oligodendrocyte precursors survive poorly and do not migrate following transplantation into the normal adult central nervous system patterns of oligodendroglia pathology in multiple sclerosis demyelination and early remyelination in experimental allergic encephalomyelitis passively transferred with myelin basic protein-sensitized lymphocytes in the lewis rat injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis multiple sclerosis: remyelination of nascent lesions multiple sclerosis. pathology of recurrent lesions effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method immunoglobulins promote remyelination in the central nervous system remyelination by oligodendrocytes stimulated by antiserum to spinal cord demyelination and remyelination in the dorsal funiculus of the rat spinal cord after heat injury remyelination occurs as extensively but more slowly in old rats compared to young rats following gliotoxin-induced cns demyelination central remyelination restores secure conduction the restoration of conduction by central remyelination transplantation of myelinating cells as regenerative therapy for multiple sclerosis-experimental basis and present state of clinical studies magnetic resonance imaging in the monitoring of disease progression in multiple sclerosis viral induced demyelination the art of survival during viral persistence differentiation of adult hippocampus-derived progenitors into olfactory neurons in vivo ultrastructural studies of multiple sclerosis transplantation of an oligodendrocyte cell line leading to extensive myelination axonal transection in the lesions of multiple sclerosis transplantation of glial cells enhances action potential conduction of amyelinated spinal cord axons in the myelin-deficient rat axonal loss in multiple sclerosis lesions: magnetic resonance imaging insights into substrates of disability demyelination and remyelination in the rat central nervous system following ethidium bromide injection. lab global'' cell replacement is feasible via neural stem cell transplantation: evidence from the dysmyelinated shiverer mouse brain insulin-like growth factor i treatment reduces demyelination and up-regulates gene expression of myelin-related proteins in experimental autoimmune encephalomyelitis effects of dorsal column demyelination on evoked potentials in nucleus gracilis myelin-associated glycoprotein is a myelin signal that modulates the caliber of myelinated axons adult brain retains the potential to generate oligodendroglial progenitors with extensive myelination capacity we thank oswald steward for discussion and advice. we thank michael liu for assistance with viral injections, rafael gonzalez and giovanna bernal for assistance with animal surgeries, and josh kunellis for assistance with tissue processing. this project was supported by the national multiple sclerosis society, and individual donations to the reeve-irvine research center. key: cord- -yibbiiij authors: wege, helmut title: immunopathological aspects of coronavirus infections date: journal: springer semin immunopathol doi: . /bf sha: doc_id: cord_uid: yibbiiij nan coronaviruses are widespread pathogens that cause a number of important diseases in mammalian and avian species. their pathogenic potential ranges from respiratory and gastrointestinal diseases to hepatitis, encephalomyelitis, vasculitis and coagulopathies. the following diseases are of major economical importance: transmissible gastroenteritis and porcine respiratory coronavirus infections in pigs, diarrhea in calves, infectious bronchitis in chickens, feline infectious peritonitis in cats [ , ] . about % of common colds in human beings are attributed to these viruses. furthermore, they cause a broad range of diseases in mice and rats. the majority of coronaviruses induce acute, self-limiting diseases. the major exceptions are infections caused by feline and murine coronaviruses. feline infectious peritonitis (fip) represents a debilitating condition based on infection of the monocyte/macrophage lineage and is associated with immune-mediated damage [ , , ] . murine coronaviruses (mouse hepatitis virus, mhv) can spread inapparently or may hide as persistent infections that modulate the immune response [ , ] . therefore, mhv infections may severely impair results of experimental studies and are of major concern in livestock breeding. on the other hand, these infections are interesting for analysing disease processes. mhv-jhm and mhv-a are mainly employed for studies of virus-induced demyelination in the central nervous system, whereas mhv- is a versatile model for the study of diseases of the liver and the lymphoreticular system. these aspects constitute the major focus of the following sections. typical coronaviruses are pleomorphic to rounded particles with a lipid envelope surrounded by a fringe of surface projections termed spikes or peplomers [ , , ] . their genome consists of a large, single-stranded rna molecule comprising about - kilobases of nucleotides. this is the largest known rna genome. the rna is h. wege of positive polarity, with a cap structure at the ' end and polyadenyl sequences at the ' end. the ' end codes for the polymerase, followed by genes for envelope proteins and the nucleocapsid protein. expression of the coronavirus genome involves a series of subgenomic mrna, each containing one or more open reading frames. in the case of structural proteins, only the ' end of the gene is translated. a similar gene organisation and expression strategy is employed by arteriviruses (e.g. equine arteritis virus) and toroviruses (e.g. berne virus). therefore, despite considerable morphological differences, these families may derive from a common ancestor of the "coronavirus-like superfamily" [ ] . they each have a similar polymerase unit coupled to different sets of structural genes (modular evolution). about two thirds of the genome is required to code for the polymerase functions, corresponding to about kilodaltons of amino acids. the genomic rna and the nucleocapsid protein n form a helical structure, which is surrounded by a lipid bilayer. attached to n is the matrix glycoprotein m, which protrudes into the lipid envelope. the major surface glycoprotein is the spike protein s. this protein is important for cell fusion, attachment to receptors on the cell surface and induction of protective immunity. the s gene displays a considerable genetic heterogeneity involving mutations, deletions and recombination events, which has a strong impact on virulence and tissue tropism. the virus receptors on the cell surface mirror this heterogeneity. murine coronaviruses employ carcinoembryonic antigens as receptors; these are related to the igg superfamily and consist of a large number of variants expressed in different tissues [ ] . other coronaviruses, such as the porcine transmissible gastroenteritis virus or the human coronavirus e, bind to a completely different type of molecule, the aminopeptidases [ ] . a further surface glycoprotein, the haemagglutinin esterase (he), is not expressed by all coronaviruses. this protein promotes binding to neuraminic acid and functions as a second receptor system. depending on the coronavirus strain, the haemagglutinating activity is either associated with he or can be a property of the s protein [ , ] . in several virus diseases such as dengue haemorrhagic fever, yellow fever or some stages of hiv infections, a cytokine-mediated dysregulation of the immune system results in an augmentation of pathology by the antibody response through deposition of immune complexes or antibody-mediated enhancement of infectivity [ , , ] . chronic inflammations elicited by feline coronavirus infections provide an interesting virus-host system in which such mechanisms can be studied. many organ systems [ , ] . the disease is predominant in young cats (of less than year of age). it is debatable whether the greater incidence among purebred cats is the result of a certain genetic predisposition or a management problem of catteries. the disease is transmitted by contact (ingestion, inhalation, bites) and shed via feces and secretions. the manifestation of clinical disease occurs after an incubation period of a few weeks to months and years. asymptomatic carders may be involved. two different manifestations can be diagnosed, either an effusive (wet) form or a non-effusive (dry) form [ , ] . furthermore, combinations of these phenotypes complicate the clinical definition of fip. characteristic of the effusive form is a fibrin-rich fluid accumulating in the peritoneal, pericardial or renal, subcapsular spaces. the severity and type of signs depends on the site of effusion, in most classical cases the cats develop an enlargement of the abdomen. general symptoms are anorexia, weight loss, dehydration, deafness and fever. depending on the site of inflammation, liver functions or the pancreas can be involved. the disease process leads, within weeks to months, to the death of the animal. in the effusive form multiple granulomas can affect different organ systems. neurological signs such as paresis, ataxia, behavioural changes and seizures are often observed. these clinical signs can fluctuate in phases over many months. infection by the oronasal route is followed by replication in the pharyngeal, respiratory or intestinal epithelial cells. monocytes are the predominant target, whereby the viremic phase remains predominantly cell associated [ , ] . in the next step, virus spreads to the macrophages of the reticuloendothelial organs or into perivascular areas. this results in necrotising pyogranulomas with phlebitis and thrombosis. the most puzzling phenomenon observed during fip is the acceleration of disease if seropositive cats are challenged with fipv. this more fulminant accelerated fip is primarily associated with the existence of serum antibodies. experimental transmission of immune serum or purified anti-fipv igg to seronegative kittens before challenge also resulted in an acceleration of disease [ , [ ] [ ] [ ] . two major mechanisms promote and drive the pathological changes. the first mechanism involves immune complexes consisting of antibodies, complement (c t) and fipv proteins [ , , ] . activation of c' appears to be the major factor eliciting inflammations and the blood-coagulation cascade [ ] . under experimental conditions, subclinical disseminated intravascular coagulation (dic) can be elicited and the inflammations are associated with an array of mediators such as interleukin (il)-i, il- , leukotriene b or prostaglandin e . the second mechanism is triggered by antibodies that enhance the infection instead of blocking or neutralising the virus. such an antibody-dependent enhancement (ade) can be emulated by experiments with macrophages in vitro [ ] . the detailed mechanism of ade appears to be quite variable. basically, the efficiency of infection of monocytes or macrophages is much higher with virus-antibody complexes than with virus alone [ , ] . this phenomenon involves a receptor-mediated endocytosis employing fc receptors or c ~ receptors and cd receptors. it is not yet clear whether fc receptor-mediated enhancement of fip infections occurs in absence or presence of c ~. the viral structures that are involved in the induction of enhancing antibodies have been defined in more detail [ ] [ ] [ ] . in the case of fipv, certain epitopes on the s protein are associated with this phenomenon, whereby the same epitope can mediate virus neutralisation by binding of antibodies in a dose-dependent manner [ ] . a smaller amount of antibodies is required for a maximal enhancement effect than for neutralisation. furthermore, antibodies binding to the matrix protein m can also result in an enhancement. in addition, the igg subclass plays a role. whereas igg a-specific monoclonal antibodies (mab) can be responsible for either enhancement or neutralisation, igg mab are strictly associated with neutralisation. the enhancing antibody may promote the infection by targeting the virus to the surface of cells bearing fc receptors for igg, thus resulting in phagocytosis or a more intense interaction of the virus proteins with the specific receptor on the cell surface. however, experiments were presented which demonstrate a correlation between the number of infected macrophages and the presence of enhancing antibodies. it was also speculated that the enhancing antibodies promote the uncoating of virus within the cell. the implications for ade in vaccination strategies are obvious. trials to protect cats by vaccination with recombinant vaccinia virus expressing the fipv s protein were, however, a failure. challenge of vaccinated cats displaying s-specific antibodies resulted in a significantly accelerated infectious peritonitis instead of protection [ ] . furthermore, depending on the challenge schedule, a correlation between accelerated fip and enhancing antibodies in the serum was demonstrated. by contrast, no such acceleration was encountered if cats were vaccinated with recombinants expressing either n or m protein of fipv. many diseases of the central nervous system (cns) are accompanied by inflammatory demyelination. diseases such as visna in sheep, canine distemper in dogs or a number of parainfectious encephalomyelitides in humans are typical examples [ ] . furthermore, an involvement of virus infections in multiple sclerosis (ms) is a widely discussed and attractive hypothesis. this enigmatic disease process is driven by a combination of immunological, genetic and environmental factors [ , ] . coronavirus infections in mice and rats have been employed as a model to analyse such pathogenic mechanisms. the murine coronaviruses (or mhv) comprise a large number of strains and biotypes [ , , ] . most studies have been performed with mhv-jhm and mhv-a in rats and mice. the outcome of infection depends on the properties of the virus, route of inoculation and host factors such as genetics, age and immune status. mice are in general more susceptible and a variety of organ systems can be involved. rats must be infected intracerebrally, but the virus remains more restricted to the cns. the major target cells in the cns are neurons and oligodendroglia, whereas during persistent infection the astrocytes are the sites of predilection which harbour and spread the virus [ ] . in both mice and rats, the immune response has a marked influence on the outcome of infection. on the one hand, a strong cd § t cell response is elicited, whereby the n protein represents a dominant antigen [ , , ] . for protection and virus elimination in the early stages of infection, both cd § and cd § t cells are important [ , , , , , , ] . in the mouse system, non-immune b cells have been described which are capable of inducing apoptosis of mhv-a -infected cells [ ] . this mechanism differs from the classical cytotoxic t lymphocyte function and may be triggered by cell fusion activity of the viral s protein. on the other hand, if the immune response fails to eliminate the virus, a persistent infection may be established. depending on the experimental conditions, an immune-mediated demyelination can be induced [ , , , , , ] . infection of several inbred rat strains with mhv-jhm results in a demyelinating cns disease [ , ] . in lewis rats, following intracerebral infection with mhv-jhm different forms of encephalomyelitis were observed. the acute encephalomyelitis (ae) develops within a short incubation time and leads rapidly to the death of the animal. the lesions are located mainly in the grey matter of the cns. in contrast, the subacute demyelinating encephalomyelits (sde) is a paralytic disease, which develops after an incubation time of several weeks to months and is based on a persistent infection [ , , ] . inflammatory demyelinating lesions are restricted to the cerebral white matter including the optic chiasm, brain stem and spinal cord. the disease develops after an incubation time of several weeks to months and in a number of cases relapse occurs [ ] . lewis rats and bn rats react very differently to infection with mhv-jhm [ ] . lewis rats are highly susceptible and display a strong inflammatory response in the cns driven by cd § t calls. bn rats are quite resistant to mhv-jhm infection and react with a strong intrathecal antibody response [ , , ] . the virus remains restricted to the periventricular regions, whereby a heavy infiltration of virus-specific antibody-secreting plasma cells occurs in the brain parenchyma. in lewis rats, macrophages and cd § t cells are the major cell type in the infiltrates [ , ] . the cytokines released by these infiltrates may contribute to the up-regulation of major histocompatibility complex (mhc) class ii antigens including microglia cells and astrocytes [ ] . results from in vitro studies have indicated that interferon (ifn)--y induces mhc class ii expression in astrocytes, which are then capable of functioning as antigen-presenting cells [ , ] . furthermore, mhv-jhm virions have the capacity to elicit an up-regulation of mhc class ii in astrocyte cultures [ ] . this effect is rat strain dependent; astrocytes derived from bn rats do not display a virus-dependent up-regulation of mhc class ii expression [ , ] . astrocytes can prime cd § t cells and can perpetuate cd § t cell functions [ ] . however, in vivo the priming and proliferation of antigen-specific cd § t lymphocytes occurs probably in cervical lymph nodes outside the cns. the t lymphocytes which home to the infected areas of the cns are unresponsive to proliferation signals, but continue to produce cytokines and function as effector cells [ , ] . therefore, it is conceivable that it is the role of activated astrocytes or microglia to mitigate the inflammatory response within the cns. most of the inflammatory t cells in older lesions are eliminated by apoptosis. the influence of immunity on the outcome of infection was investigated by employing for immunisation recombinant vaccinia viruses, which express structural proteins of mhv-jhm. protective immunity was induced against mhv-jhm if the challenge infection of rats was performed within days following vaccination against s protein [ ] . as shown by depletion experiments, the protective effect depends on the presence of s protein-specific cd § t cells. however, a subclinical and protracted course of disease associated with a persistent infection was induced if the mhv-jhm challenge was performed weeks after vaccination [ ] . employing this immunisation schedule, the cd § t cells appear to be of less importance. it is conceivable that the presence of antiviral antibodies shields the neurons against a cytolytic virus infection and thus prevents lethal disease [ ] . the conclusion that the humoral immune response may modulate the disease is supported by data from immune-histological analysis of rats displaying inflammatory demyelinating lesions [ ] . within the lesions, binding of antiviral antibodies, complement c and an increased amount of b cells are detectable, whereas the amount of s protein expressed is drastically reduced in comparison to the nucleocapsid protein. as shown by tissue culture experiments with measles virus-infected cells, during persistent infection in the presence of antibodies viral glycoproteins disappear from the cell surface and their expression can be down-regulated. this mechanism of antigenic modulation may help the virus escape from immmune surveillance [ ] . most fascinating was the finding that, during sde, lymphocytes are sensitised against myelin basic protein (mbp) [ ] . to test the pathogenetic potential of these cells, such lymphocytes were restimulated in vitro to enhance their number and then transferred to healthy syngeneic rats. these animals developed inflammatory lesions and clinical signs characteristic of experimental allergic encephalitis (eae). it is noteworthy that no autoimmune response was observed in bn rats (a strain relatively resistant to induction of eae and other autoimmune diseases [ ] ). these results support the hypothesis that autoimmunity specific for brain antigens can be triggered by a virus infection and have implications for pathogenesis. the susceptibility of neuronal cells and macrophages is a key element in determining the outcome of infection and may involve only one single dominant gene. by contrast, the development of chronic mhv-induced cns disease is probably under the control of several genes [ ] . a chronic demyelinating encephalomyelitis was observed in a few mice that survived the acute disease [ , ] . whereas demyelination during the acute stage might be a direct consequence of a cytolytic infection affecting oligodendrocytes, astrocytes are the predominant ceils that maintain the virus as a smoldering persistent infection throughout the chronic stage [ ] . some evidence has been obtained that mhv-jhm also triggers also autoimmune responses in mice. however, it is not known whether this phenomenon plays a pathogenic role leading to inflammatory demyelination. in mhv-jhm-infected balb/c mice, the frequency of self-reactive t cells was significantly elevated [ ] . in this context, it is interesting that a murine coronavirus infection strongly influenced the pathology of an autoimmune relapsing encephalomyelitis in a mouse model [ ] . in pl/j mice, eae was elicited by transfer of mbp-specific lymph node cells. only mice containing antibodies against mhv displayed large demyelinating lesions in the spinal cord. in addition, mhv-jhm induces a biphasic retinal disease in balb/c mice [ ] . in the early phase, the virus induces a retinal vasculitis. later, when both viral particles and inflammatory ceils are no longer detectable, these animals suf-fer from a degeneration of the retina associated with the presence of autoantibodies against retina and retinal pigment epithelial cells. the major factor leading to primary demyelination and inflammation appears to be provoked by the antiviral immune response. a persistent virus infection and demyelination can be induced if c b / mice that are nursed by already immune mothers are infected [ ] . this chronic disease may involve humoral antibodies, which prevent virus spread and damage of neurons during the acute phase. a similar modulation of the disease was observed after infection of mice infused with mab against the s protein [ , ] . the pathology of virus-induced cns infections depends on changes caused directly by the virus and the effects mediated by proinflammatory cytokines [ , ] . during the acute stage, the infection of mhv-jhm results in an increase of mhc class i expression on oligodendrocytes and astrocytes [ , ] . this induction of mhc class i expression in astrocytes is dependent on the virus strain employed for infection [ ] . in addition, a virus-mediated induction of il- was demonstrated on endothelial cells and astrocytes [ ] . these virus-specific immune modulatory effects are thought to involve soluble factors released from brain cells. furthermore, besides neural cells, infiltrating monocytes and macrophages are a target for infection. surviving the acute disease depends strongly on an efficient clearance of virus involving both cd § and cd § t lymphocytes, which act through the release of cytokines [ , ] . at the time of clearance, an increase of mrna for il-lc~, il- / , il- , tumor necrosis factor (tnf)-c~ and ifn- was demonstrated [ ] . in irradiated mice, the clearance of virus from the cns is severely impaired. however, although the amount of infectious virus is significantly enhanced, demyelination is abrogated. passive transfer of splenocytes from immune animals again resulted in inflammatory demyelination. virus-specific thy- + cells are, therefore, considered to be of central importance in mediating demyelination [ , , ] . the process of demyelination, the involvement of autoimmunity and the clinical course of experimental coronavirus infections emulate some aspects of ms. eae is investigated as the classical model to illustrate the involvement of autoimmunity in cns diseases [ , ] . to induce eae, animals are immunised with mixtures of mbp or myelin extracts with adjuvants.the essential pathogenic element consists of cd § t cells sensitised against "encephalitogenic" epitopes of mbp. passive transfer of activated mbp-specific t cell lines also results in eae. however, the lesions associated with this type of eae model only consist of perivascular infiltrations of t cells and monocytes, whereas inflammatory demyelination is a hallmark of ms. to emulate such pathological changes, a number of experimental designs were evaluated. one of the most successful modifications of the eae model was achieved by a combined transfer of t cells specific for brain antigens and antibodies against surface proteins on oligodendrocytes or myelin. the intensity of inflammation and the degree of demyelination depend on the balance between encephalitogenic t cells and antibodies. the neuropathological changes observed in these eae models and in coronavirus-induced demyelinating disease are similar to demyelinating lesions associated with ms. in both systems a spectrum of lesions can be analysed, which represent different stages of plaque development. the pathogenic mechanism driving the different types of lesions appear to vary even within the same rat. in general, a persisting virus infection may provide a first antigen stimulus and disturb the balance of the immune system [ ] . viruses that replicate in neural tissue can incorporate host material, including myelin proteins, and thus provoke a normally irrelevant immune response [ ] . furthermore, epitopes of the virus may cross-react with potential neuroantigens, thus provoking autoimmunity by molecular mimicry [ ] . many direct or indirect disturbances of oligodendrocyte functions can impair myelin production, expose new antigens and progress to phases of increasing pathology. oligodendrocytes are sensitive to exposure to tnf or il- [ ] . other factors are released by activated macrophages such as nitric oxide or proteases. finally, oligodendrocytes are sensitive to c ~ or c'-mediated antibody reactions. therefore, demyelination may also be the consequence of an "innocent bystander" effect related to an antiviral immune response. astrocytes are coupled through gap junctions to oligodendrocytes and secrete trophic factors. in addition, the viability of oligodendrocytes depends on the integrity of the associated axons. furthermore, the immunomodulatory functions of astrocytes, microglia and endothelial cells can be affected by a viral infection. in particular, the virus-induced up-regulation of mhc or of cytokines can be implicated in the disruption of immune regulation. an immunopathological process may, thus, prevail even when the eliciting infectious agent has already been eliminated. despite the fascinating observations in such model systems, a direct etiological involvement of coronavirus infections in ms remains unlikely. the reports on coronaviruses isolated from cns tissue of ms patients using mice or murine cell cultures are debatable [ ] . the risk of a hidden murine coronavirus infection during prolonged passages is obvious. on the other hand, there are reports of coronavirus rna or protein being detected in brain tissue in some ms cases [ , ] . furthermore, murine coronaviruses can induce encephalomyelitis in primates [ , ] . there have been numerous attempts to incriminate viruses as an agent which triggers ms. no epidemiological evidence indicates that coronaviruses are involved in this disease. a number of low-virulent mhv viruses have been isolated from mouse colonies, which display a highly variable tropism for macrophages and t or b cell lineages [ ] . natural mhv infections can impair the function of splenic cells or influence the release of immunomodulatory neurotransmitters [ , , , ] . oral infection of balb/cbyj mice with mhv-jhm resulted in a transient but marked depression of t cell functions [ , ] . within the first weeks of infection, the spleen cell response to polyclonal stimuli was decreased. on the other hand, spleen cells from mice infected with mhv-jhm proliferated spontaneously and produced elevated levels of il- and il- . this spontaneous lymphokine production was measured at a time point when infectious virus had already been eliminated [ ] . moreover, the frequency of self-reactive t cells was significantly elevated [ ] . following intranasal infection of balb/c mice with mhv-a , a transient acute phase was observed accompanied by a markedly decreased number of cells in lymphoid organs [ ] . later, when infectious virus was no longer detectable, these mice had an impaired ability to reject skin grafts. this long-term dysfunction of t cells was not detectable by in vitro assays. mhv-a does not infect t or b cells. therefore, it is conceivable that this dysfunction is caused by an impairment of accessory cell functions induced during the phase of acute infection. this murine coronavirus displays a broad spectrum of organ tropisms and can affect liver, lymphoid organs and the cns [ , , ] . the outcome of disease strongly depends on the genetic background, the route of infection and the mhv- biotype. a/j mice are resistant to intraperitoneal infection, the virus being cleared within days. c bl/ mice by contrast die within a few days post infection (p.i.) from an acute hepatitis. this genetic restriction is based on the interaction between the virus, macrophages and lymphoid cells. the lymphoid organs of animals with acute disease are severely affected. the virus induces a lytic infection of cells belonging to the b cell lineage in spleen and bone marrow, which correlates with the pathogenicity of mhv- [ , ] . in addition, the splenic and thymic t cell population is depleted. in these cells, a lytic viral infection is maintained through contact with stromal cells of the thymus [ ] . a completely different outcome of infection was observed, when f hybrids derived from cross-breeding between those strains; (c b / xa/j) f were employed [ ] . these "semi-susceptible" animals are resistant to acute disease, but develop chronic diseases based on persistent infections, which can be associated with immunosuppression. major signs consist of a wasting syndrome, hind limb paralysis and incoordination. these mice occasionally die within months. similar manifestations of chronic disease are also inducible by mhv- infection of c h, akr or a g mice. the chronic disease is under the control of several h- - inked genes, which influence the degree of hepatic damage. the acute phase depends on another gene complex. the development of neurological disease depends on the level of virus replication involving meningeal cells, ependymal cells and neurons [ , ] . although antibodies against mhv- are continuously detectable, a strong impairment of both primary and secondary antibody responses occurs. furthermore, the number of thymocytes, splenic cells and macrophages decreases significantly. the number of spleen cells decreases already within days p.i. and remains low for up to months. infectious virus can be isolated from different organs of chronically infected mice. the major targets are mature b cells and thymic stromal cells. viruses isolated from cns tissue are less pathogenic and have lost the tropism for thymocytes [ ] . it is noteworthy that neurological signs develop at a time when the spleen cellularity and number of peritoneal macrophages have reached normal levels. the paralysis is thought to result from an immune-mediated lysis of infected neural cells [ ] . as a consequence of the t and b cell depletion during the acute phase, the elimination of infectious virus may be inefficient and, thus, establishment of a persistent infection could be promoted. macrophage function determines to a significant extent the outcome of infections with mhv- . the restriction of mhv- replication is strongly associated with macrophage activation by ifn-' [ ] . furthermore, the activation of the blood coagulation system by infection with mhv- is a major pathogenic mechanism related to susceptibility and resistance [ ] . activation of the coagulation system can result in disseminated intravascular deposition of fibrin and, thus, lead to severe liver damage. a low basal procoagulant activity (pca), a prothrombinase, is an intrinsic property of lymphoreticular cells. this enzymatic activity can be up-regulated by lipopolysaccharides, immune complexes or lectins. monocytes and macrophages play a pivotal role in the induction of the pca response. the manner in which infection of macrophages by mhv- stimulates pca is strictly dependent on the genetic background: high activity is induced in fully susceptible balb/c mice, moderate responses are measurable in c h/st mice and no pca activity occurs in the fully resistant a/j strain. the pca response develops within - . h p.i., before the virus replicates. infusion of infected balb/c mice with an mab that neutralises pca activity has a protective effect and limited hepatic necrosis [ ] . therefore, the mechanism by which macrophages contribute to genetic restriction is via induction of the blood coagulation cascade by the virus and is not directly dependent on the virus replication. in addition, mhv- infection of macrophages elicits the production of tnf, leukotriene b and il- . these mediators may in turn elicit the expression of pca in endothelial cells. the induction of pca in macrophages is not possible without t cell cooperation. t lymphocytes from resistant, immunised a/j mice are capable of inhibiting expression of pca in susceptible, h- -compatible recombinant mice. a virus-specific cd § thl line has been established, which blocks the induction of pca in macrophages [ ] . furthermore, this t cell line is capable of conferring resistance to otherwise susceptible mice. results from several studies imply that ifn play a central role in susceptibility or resistance [ ] . these cytokines display both antiviral and immunomodulatory activities. a major mechanism mediating an antiviral effect is elicited through - adenylate synthetase, which activates rnase l. this endoribonuclease activity can impair the synthesis of viral rna by degradation of single-stranded rna. compounds that stimulate rnase l interfere with virus replication in macrophages [ ] . however, mhv- -induced pca activity is not diminished and, thus, these substances did not prevent necrotic hepatitis. the beneficial role of ifn- , appears to operate through stimulation of the thl lymphocyte response and may not be a consequence of its antiviral effect. a number of coronavirus-induced disease processes are driven or accompanied by immunopathological mechanisms. of major clinical importance is feline infectious peritonitis, which is a management problem in catteries. this antibody-and c'-mediated disease process displays interesting mechanistic parallels to important virus diseases of human, involving haemorrhagic shock syndromes and damage to the lymphoreticular system [ , ] . murine coronavirus infections do not appear to be of clinical importance. however, these viruses provide interesting experimental models by emulating complex disease processes and may represent as-yet-unrecognized threats or underestimated problems in biomedical studies. infections with mhv strains affect about - % of mouse colonies and are often not recognised because of their tendency to induce subclinical and inapparent infections. therefore, the long-term effects of such infections on immune functions and the neuroendocrine system can severely impair the reliability of results of experiments employing mice and rats. in particular, studies with genetically manipulated animals may be invalidated if such colonies 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pathogenesis of feline infectious peritonitis: nature and development of viremia pathogenesis of feline infectious peritonitis: pathologic changes and immunofluorescence disseminated intravascular coagulation in experimentally induced feline infectious peritonitis local cytokine responses during acute and chronic viral infections of the central nervous system effective clearance of mouse hepatitis virus from the central nervous system requires both cd § and cd § t cells coronavirus induced primary demyelination: indications for the involvement of a humoral immune response immune protection vs. immunpathology vs. autoimmunity: a question of balance and of knowledge acknowledgements. the author acknowledges financial support from the deutsche forschungsgemeinschaft and hertie stiftung. key: cord- - bhnlsgy authors: trifilo, matthew j.; lane, thomas e. title: the cc chemokine ligand regulates cd c(+)cd b(+)cd α(−) dendritic cell maturation and activation following viral infection of the central nervous system: implications for a role in t cell activation date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: bhnlsgy the role of cc chemokine ligand (ccl ) in activation of dendritic cells (dcs) following mouse hepatitis virus (mhv) infection of the central nervous system (cns) was examined. the results indicate that ccl participates in an effective host response to mhv infection by contributing to cd c(+)cd b(+)cd α(−) dc maturation, activation, and migration to cervical lymph nodes (cln). diminished cd α(−) dc activation correlated with reduced ifn-γ expression by virus-specific t cells accompanied by increased il- production suggesting that ccl contributes to an effective host response to viral infection by enhancing the t cell activation potential of dc. the cc chemokine ligand (ccl -macrophage inflammatory protein- a) is capable of activating monocytes and lymphocytes and serves an important role in the initial recruitment of these cells to tissues following microbial infection (cook et al., ; domachowske et al., ) . in support of the importance for ccl in imparting functional signals to t cells are data from our laboratory demonstrating that instillation of mouse hepatitis virus (mhv) into the brains of ccl À/À mice results in an inability to clear virus from the central nervous system (cns) (trifilo et al., ) . mhv-infected ccl À/À mice exhibited a significant reduction in the numbers of infiltrating virus-specific cd + t cells present within the brain indicating that trafficking was impaired. moreover, the ability to produce ifn-g as well as the cytolytic activity of virus-specific cd + t cells was dramatically reduced in the absence of ccl signaling. taken together, these data indicate that ccl signaling significantly enhances the differentiation of primed cd + t cells into effector cells that allows their release from secondary lymphoid organs into circulation and effective migration to the cns. the present study was undertaken to characterize potential mechanisms by which ccl signaling imparts effector function to antigen-specific t cells following mhv infection of the cns. to further understand the relationship between ccl signaling and t cell activation, ccl +/+ and ccl À/À mice were infected with mhv and the presence and activation state of dc-like cells within the brain and draining cervical lymph nodes (cln) determined. our results delineate a ccl -dependent pathway of t cell activation that involves the maturation and activation of a subpopulation of (dendritic cells) dcs (cd c + cd b + cd a À ) within the cns as well as influencing the accumulation of these cells within the cln following mhv infection of the cns. characterization of cd c + cells within the cns following mhv infection of ccl +/+ and ccl À/À mice to characterize the populations of cells present within the cns of mhv-infected mice, cells were harvested at days , , and post-infection (p.i.) and immunophenotyped by flow cytometry. we chose to focus our attention on markers that are associated with professional antigen presenting cells such as dc as recent studies have indicated dc-like cells can be detected within the brains under inflammatory conditions (fisher and reichmann, ; fischer et al., ) . furthermore, we have previously determined that ccl mrna expression is detected within the cns early (b days) and therefore may participate in the appearance of cd c + cells within the brain following mhv infection (trifilo et al., ) . therefore, we sought to characterize the populations of cd c + cells within the brain following mhv infection of ccl +/+ and ccl À/À mice. results in fig. a indicate the frequency of cd c + cells present within the cns of narve ccl +/+ and ccl À/À mice is b %. however, within days following intracranial infection with mhv, there is a marked increase in the frequency of cd c + cells within the brains of both strains of mice (fig. a) . analysis of cd b expression revealed that approximately % of cd c + cells in both ccl +/+ and ccl À/À mice were also cd b + (fig. a) . cd c + cd b + cells isolated from the cns of either ccl +/+ or ccl À/À mice expressed little to no cd a or dec suggesting a phenotype similar to myeloid derived dc (cd c + , cd b + , cd a À , dec À ) ( fig. b ) (anjuere et al., ; henri et al., ) . increased cd a and dec expression on cd c + cd b À cells indicated that the majority of the remaining cd c + cells present within the brain were similar phenotypically to lymphoid derived dc (cd c + , cd b À , cd a + ) (fig. b) (anjuere et al., ; henri et al., ) . although the fig. . analysis of dendritic cells (dcs) within the brain following mhv infection. (a) cells were isolated from the brains of uninfected (narve) or infected (day p.i.) ccl +/+ and ccl À/À mice and stained for cd c + and cd b + expression. gated populations represent cd c + cd b + (upper-right quadrant) or cd c + cd b À (lower-right quadrant) and numbers indicate frequencies of gated cells within the isolated population. (b) cd a and dec expression on cd c + cells. cd c + cd b + cells from either mhv-infected ccl +/+ or ccl À/À mice at day p.i. did not express detectable levels of either cd a or dec while expression of both cd a and dec was readily detectable on cd c + cd b À cells present within the brains of both populations of mice at day p.i. (c) total numbers of cd a + and cd a À dcs within the brains of mhv or sham-infected ccl +/+ and ccl À/À mice at days and p.i. data presented represent an average cell number derived from two separate experiments with a minimum of mice analyzed per experimental group. (d) cd a À cells isolated from brains of ccl +/+ or ccl À/À mice at day p.i. were gated upon and cd , cd , and cd expression was determined by flow cytometry. the mean fluorescence intensity (mfi) for cells obtained from either ccl +/+ or ccl À/À mice is indicated. flow data shown in panels a, b, and d are representative of two separate experiments with a total of mice for each experimental condition. remaining cd c + cd b À cd a À population was not further characterized, it is likely that these cells may be plasmacytoid in origin (cd c + cd b À cd a À b + ) or consist of a yet to be defined population of dc. comparison of the total numbers of cd a + cells within the brains of mhv-infected ccl +/+ and ccl À/À mice revealed no dramatic differences between the two populations of mice at either or days p.i. (fig. c) . in contrast, numbers of cd a À cells were increased by approximately % within the brains of mhv-infected ccl +/+ mice as compared to ccl À/À mice at day p.i. however, by day p.i., there were increased numbers of cd a À cells present in the brains of ccl À/À mice when compared to ccl +/+ mice. in attempt to better evaluate the activation state of cd c + cells within the brains of mhv-infected mice, we next determined the expression levels of co-stimulatory molecules cd (b- . ), cd (b- . ), and cd on cd a À cells within the cns of ccl +/+ and ccl À/À mice. we chose to focus on this subpopulation of dcs in more detail as this clearly was the predominant dc population within the brains of infected mice suggesting a potentially more important role in defense. analysis of cd a À cells isolated from the brains of ccl +/+ mice at day p.i. revealed these cells expressed detectable levels cd , cd , and cd as determined by measuring the mean fluorescence intensity (mfi) (fig. d ). although mhv infection of ccl À/À mice also resulted in enhanced expression of cd on the surface of cd a À cells, the mfi for both cd and cd was dramatically reduced as compared to cd a À cells within the brain of ccl +/+ mice at day p.i. (fig. d ). together, these results indicate that although ccl signaling is not required for the appearance of dc-like cells within the brain, expression of the co-stimulatory molecules cd and cd is muted in the absence of ccl signaling. characterization of cd c + cells within the cln of mhv-infected ccl +/+ and ccl À/À mice following mhv infection of the cns, virus-specific t cells are present within the cln suggesting that the bulk of virus-specific t cells are generated in the periphery (marten et al., ) . therefore, the accumulation of cd c + cells within the cln of infected ccl +/+ and ccl À/À mice was determined. before infection, cd c + cells expressing a myeloid dc phenotype (cd c + cd b + ) and lymphoid dc phenotype (cd c + cd b À ) were present within the cln of both ccl +/+ and ccl À/À mice at an approximate : ratio, respectively ( fig. a) . however, within days of mhv infection of the cns, the frequency of cd c +-cd b + dcs, but not cd c + cd b À dcs, dramatically increased within the cln of ccl +/+ mice ( fig. a) . further analysis revealed that the cd c + cd b + cell population was cd a À dec À while the cd c +-cd b À population was cd a + dec + (fig. b) . although the total number of cd a À cells increased within the cln of ccl À/À mice, there was an approximate -fold reduction in total numbers of cd a À cells as compared to ccl +/+ mice at days and p.i. (fig. c ). similar to the brain, no difference in numbers of cd a + cells within the cln was detected at either days or p.i. (fig. c ). examination of co-stimulatory molecule expression on cd a À dcs present within the cln of ccl +/+ mice correlated with the increased expression of co-stimulatory molecules cd , cd , cd as well as increased mhc i and ii expression when compared to sham-infected mice (figs. d and e) . these data suggest that this population of activated cd a À cells within the cln is able to present antigen and induce t cell differentiation within the cln following mhv infection of the cns. although cd a À dcs isolated from the cln of ccl À/À mice expressed similar levels of cd and cd as compared to ccl +/+ (determined by mfi), expression of cd as well as mhc i and ii were reduced as compared to cd a À cells isolated from mhv-infected ccl +/+ mice (figs. d and e). one mechanism by which dcs influence the t cell response to infection is through the secretion of cytokines that can subsequently polarize the immune response towards either a th or th phenotype depending on the antigenic challenge. to determine if cd a À dcs present within the cln of infected ccl +/+ and ccl À/À mice were capable of secreting either chemokines or cytokines following mhv infection of the cns, these cells were isolated and production determined by elisa. ccl was readily detectable from cd a À dcs obtained from ccl +/+ mice while ccl was not detected in supernatants collected from ccl À/À cd a À dcs (fig. ) . the ccl +/+ cd a À dc population secreted il- p with low-level production of il- ( fig. ) . in contrast, the cd c + cd b + cd a À cells isolated from ccl À/À mice secreted approximately -fold less il- p while il- secretion was increased by -fold as compared to cells from ccl +/+ mice (fig. ) . altered cytokine production in ccl À/À t cells we next evaluated the ability of t cells obtained from the cln of either mhv-infected ccl +/+ or ccl À/À mice to synthesize cytokines following exposure to defined viral antigens. t cells were isolated from the cln of ccl +/+ and ccl À/À mice at days and p.i. following intracranial infection with mhv and stimulated with peptides corresponding to either the immunodominant cd epitope present within the matrix (m) glycoprotein at residues - (m - ) or the immunodominant cd epitope in the surface (s) glycoprotein spanning residues - (s - ) and cytokine production by t cells determined by intracellular cytokine staining (castro and perlman, ; xue et al., ) . the results presented in table indicate similar frequencies of cd + and cd + t cells from ccl +/+ mice produced ifn-g at days and p.i. both cd + and cd + ccl +/+ t cells also secreted il- following specific peptide exposure at day , although expression was limited to the acute stage of mhv infection as the frequency of il- producing cells was reduced at day p.i. (table ). in contrast, the frequency of cd + and cd + t cells isolated from ccl À/À mice secreting ifn-g following peptide stimulation was dramatically reduced. expression of il- by ccl À/À t cells was comparable with ccl +/+ mice at day p.i. however, by day p.i., the frequency of ccl À/À t cells expressing il- remained elevated as compared to ccl +/+ t cells (table ). in addition, only limited frequencies of ccl +/+ cd + and cd + t cells produced il- following mhv infection whereas ccl À/À cd + and cd + t cells displayed an overall increase in the frequency of il- ( table ) . the major findings of this study are (i) mhv infection of the cns results in the appearance of two distinct populations of cd c + cells each expressing markers characteristic of lymphoid (cd c + cd b À cd a + dec + ) and myeloid dendritic cells (cd c + cd b + cd a À dec À ), (ii) the accumulation of cd a À dcs within the draining cln is reduced in the absence of ccl signaling, (iii) expression of co-stimulatory molecules such as cd by cd a À dcs within either the brain and cln of mhv-infected ccl À/À mice is diminished suggesting that ccl signaling enhances expression of these molecules, and (iv) absence of ccl signaling results in the re-direction of the t cell response to viral antigens as determined by cytokine production. these data support and extend recent studies from our laboratory demonstrating an important role for ccl in generating effector anti-viral t cells capable of migrating to the brain in response to viral infection (trifilo et al., ) . i.) ccl +/+ and ccl À/À mice and stained for cd c + and cd b + expression. gated populations represent cd c + cd b + (upper-right quadrant) or cd c + cd b À (lower-right quadrant) and numbers indicate frequencies of gated cells within the isolated population. (b) cd a and dec expression on cd c + cells. cd c + cd b + cells from either ccl +/+ or ccl À/À at day p.i. did not express detectable levels of either cd a or dec while expression of both cd a and dec was readily detectable on cd c + cd b À cells present within the brains of both ccl +/+ and ccl À/À mice at day p.i. (c) total numbers of cd a + and cd a À dcs within the clns of mhv or sham-infected ccl +/+ and ccl À/À mice at days and p.i. data presented represent an average cell number derived from two separate experiments with a minimum of mice analyzed per experimental group. (d) cd a À cells obtained from mhv-infected (day p.i.) or sham mice were gated and the level of cd , cd , and cd expression was determined by flow cytometry. the mfi for cells obtained from either ccl +/+ or ccl À/À mice is indicated. (e) cd a À cells obtained from mhv-infected or sham mice were evaluated for expression of mhc i and ii. the mfi for staining of either mhc i or ii is indicated in the histogram. flow data shown in panels a, b, d, and e are representative of two separate experiments with a total of mice for each experimental condition. the activation of dc and their mobilization to secondary lymphoid organs is thought to be a key step in the initiation of an adaptive immune response (banchereau and steinman, ). recent studies have indicated that following infection of the cns with toxoplasma gondii, cd c + cells are present within the brain and these cells were able to stimulate the proliferation of narve t cells (fisher and reichmann, ) . similarly, our results also indicate an increase in cd c + cells within the cns following viral infection, suggesting that these cells are likely critical for successful t cell priming following migration to draining lymph nodes. whether these cells are present within the cns by differentiation of local antigen presenting cells, or through migration of immature dc has not been determined and is currently under investigation. regardless, our data imply that ccl expression and signaling contributes to the migration of cd a À cd c + cells to secondary lymphoid tissue where they participate in priming of t cells. in support of this, we have shown that ccl is important in arming these cells with the capability to optimally stimulate antigen-specific t cells with the ability to fully differentiate into effector cells (trifilo et al., ) . the data presented in this study support and extend these observations and indicate that these results may be the result of a combination of diminished expression of both mhc class i and ii, reduced expression of cd , as well as a shift in cytokine production by cd a À cells. indeed, cd l:cd mediated interactions between t cells and apc can enhance il- production by dc and blockade of this interaction has been shown to result in reduced autoimmunity by down-regulating th differentiation (cella et al., ; macatonia et al., ) . accumulating evidence indicates that in addition to driving virus-specific t cell proliferation, the activation state of dcs can also directly influence the effector function of t cells through the secretion of proinflammatory cytokines (fischer et al., ; maldonado-lopez et al., pulendran et al., ) . for example, following several viral and bacterial infections, cd a + dcs have been shown to be able to secrete large amounts of the proinflammatory cytokine il- both in vitro and in vivo and this results in a preferential expression of th -associated cytokines, such as ifn-g by responding t cells (aliberti et al., ; maldonado-lopez et al., pulendran et al., ; reis e sousa et al., ) . until recently, the prevailing thought was that cd a + dcs were primarily responsible for production of il- and contributing to a th response. however, recent studies have indicated that cd a À dcs also have the potential for secreting il- and influencing the t cell response (doxsee et al., ) . our studies clearly indicate that cd a À dcs isolated from the draining cln of mhv-infected ccl +/+ mice secrete il- suggesting that these cells help influence a protective th -mediated immune response characterized by the majority of antigen-specific t cells expressing ifn-g rather than il- (table ). in stark contrast is the data indicating that cd a À dcs present in the cln of infected ccl À/À mice predominantly secrete il- and this correlates with limited ifn-g expression and enhanced expression of the th -associated cytokine il- (table ) . therefore, the data indicate that cytokine production, rather than the type of cd c + cell, may control the predominant t cell immune response within the cln. taken together, these data point to an important role in ccl expression in linking innate and adaptive immune responses following viral infection of the cns by contributing to the activation fig. . cytokine and chemokine secretion by cd c + cd b + cd a À cells isolated from mononuclear cells pooled from the cln of mhv-infected ccl +/+ and ccl À/À mice at day p.i. supernatants were analyzed for the production of il- p , il- , and ccl by elisa h following culture. a minimum of three to six mice per group were used for isolation of cells and data presented indicate the average f sd. *p v . . table frequency of cytokine-producing t cells present within draining cervical lymph nodes following mhv infection a day p.i. ifn-g il- il- cd + t cells ccl +/+ f f f data represent two separate experiments with at least three mice per group, n = . data are presented as average f sd. a cytokine expression determined by pooling cells from draining cln of mhv-infected mice at defined times p.i. and pulsing with defined cd + epitope (m - ) and cd + epitope (s - ). b p v . . decreased frequency of ifn-g secreting t cells and increased frequency of il- producing t cells following mhv infection of ccl À/À mice as compared to ccl +/+ mice. c p v . . increased frequency of il- producing t cells in ccl À/À mice as compared to ccl +/+ mice following mhv infection. of dcs through regulating the migration of cells from the cns to lymphoid tissues as well as the expression of both co-stimulatory molecules and cytokine production. however, it is important to note that there is the possibility of other chemokines and chemokine receptors participating in dc responses following viral infection. indeed, the cc chemokine receptor (ccr ) is expressed on professional apc including macrophages and dcs and is thought to contribute to defense following microbial challenge by enhancing recruitment as well as production of antimicrobial products such as tnf-a and no by these cells (luster, ; mccoll, ; serbina et al., ) . in addition, the absence of ccr signaling results in diminished trafficking and accumulation of dendritic cells within secondary lymphoid tissues following antigenic challenge (peters et al., (peters et al., , sato et al., ) . studies are currently in progress to evaluate the contributions of additional chemokine signaling pathways that may also participate in dc activation and migration following coronavirus infection. in conclusion, the studies presented support and extend previous work from our laboratory indicating an important role for chemokines in the migration of t cells into the cns following mhv infection glass et al., ; liu et al., ) . here, we have demonstrated a novel role for the chemokine ccl in enhancing the accumulation and activation of cd a À dcs within secondary lymphoid tissue and this correlates with altered t cell activation and differentiation following viral infection. these results indicate that cd a À dcs likely function in an apc-like role within the cln following mhv infection and that these cells rely upon chemokine instruction to activate t cells. at a more fundamental level, the results presented demonstrate that chemokines serve as critical upstream signals in the innate immune response that later is important with regards to the initiation of a protective adaptive immune responses to viral infection. mhv j . v- was kindly provided by j. fleming (u. wisconsin). ccl À/À and ccl +/+ mice (c bl/ , h- b ) were purchased from jackson laboratories (bar harbor, me). mice were anesthetized by inhalation of methoxyflurane (pitman-moor inc., washington crossing, nj) and injected intracranially (i.c.) with pfu mhvj . v- suspended in al sterile pbs. control (sham) mice were injected with al sterile pbs alone. mononuclear cells were obtained from the brains and cervical lymph nodes [two draining cervical lymph nodes (cln) per mouse] of either ccl +/+ or ccl À/À mice at defined times post-infection (p.i.) using a previously described protocol (trifilo et al., ) . cell surface expression of phenotypic markers was examined using the following reagents for flow cytometric analysis: apcconjugated rat anti-mouse cd ; percp-conjugated rat anti mouse cd (pharmingen, san diego, ca). pe conjugated d b /s - mhc class i tetramer (beckman coulter, san diego, ca) was utilized for identification of cd + t cells specific for viral spike protein antigen (trifilo et al., ) . to determine the presence of dendritic-like cells within the cns and lymph nodes, cells were stained using fitcconjugated rat anti-mouse cd c (serotec, oxford, england) in combination with percp-conjugated rat anti-mouse cd a (pharmingen), rat anti-mouse apc-conjugated cd b (pharmingen), and rat anti-mouse dec (pharmingen). the maturation and activation state of dc were determined using fitc-conjugated rat anti-mouse cd , cd , cd , mhc i, and mhc ii (pharmingen). isotypematched antibodies were used as controls for all staining conditions described. isolation of cd c + cells from the cln ccl +/+ and ccl À/À mice were infected intracranially with pfu of mhv and brains and cln were removed at defined times post-infection for analysis. brain samples were minced and homogenized into a single cell suspension followed by fractionation on a / % percoll gradient at  g for min. for isolation of mononuclear cells from the cln, lymph nodes were homogenized using frosted glass slides and the resulting single cell suspension was treated with sterile h o to lyse red blood cells. due to the low frequency and numbers of cd c + cells within the brain and cln, samples from three to six mice were pooled for each experiment. to enrich for cd c + cd b + cd a À dcs, both brain and cln samples were separately magnetically sorted by negative selection against cd a using macs microbeads coated with anti-cd a (miltenyi biotec, auburn, ca) according to the manufacturer's instructions. cd c + cells were then magnetically selected from the cd a À fraction using macs microbeads coated with anti-cd c (miltenyi). the resulting population was n % pure for cd c + cells that were subsequently determined to be cd b + and cd a À by flow cytometry (data not shown). macs enriched cells were then resuspended in dmem supplemented with % fbs. cytokine and chemokine expression from cd c + cd a À cells freshly prepared cd c + cd a À cells were isolated from the cln from ccl +/+ and ccl À/À mice at day p.i. and seeded into -well plate at a cell density of  cells/ al in dmem containing % fetal bovine serum (fbs, biowhittaker, walkersville, md). following isola-tion, cell viability was n %. after h, supernatants from the samples were collected, and the level of il- , il- , and ccl were determined using quantikine m mouse immunoassays kits (r&d systems, minneapolis, mn) according to manufacturer's specifications. assays had a minimum sensitivity of pg/ml (il- and il- p ) and pg/ml (ccl ). mononuclear cells were obtained from the clns of mhv-infected mice at defined times post-infection and cytokine production by t cells determined by intracellular cytokine staining to defined viral antigens using a previously described protocol (trifilo et al., ) . statistically significant differences between experimental groups was determined by the mann-whitney rank sum test, and p values of v . were considered significant. ccr provides a signal for microbial induced production of il- by cd a+ dendritic cells definition of dendritic cell subpopulations present in the spleen, peyer's patches, lymph nodes, and skin of the mouse dendritic cells and the control of immunity cd + t-cell epitopes within the surface glycoprotein of a neurotropic coronavirus and correlation with pathogenicity ligation of cd on dendritic cells triggers production of high levels of interleukin- and enhances t cell stimulatory capacity: t-t help via apc activation lack of ccr results in increased mortality and impaired leukocyte activation and trafficking following infection of the central system with a neurotropic coronavirus requirement of mip- alpha for an inflammatory response to viral infection the chemokine macrophage-inflammatory protein- alpha and its receptor ccr control pulmonary inflammation and antiviral host defense in paramyxovirus infection the immune response modifier and toll-like receptor agonist s- selectively induces il- and tnf-alpha production in cd c+cd b+cd -dendritic cells brain dendritic cells and macrophages/ microglia in central nervous system inflammation phenotype and functions of brain dendritic cells emerging during chronic infection of mice with toxoplasma gondii reduced macrophage infiltration and demyelination in mice lacking the chemokine receptor ccr following infection with a neurotropic coronavirus the dendritic cell populations of mouse lymph nodes the t cell chemoattractant ifninducible protein is essential in host defense against viral-induced neurologic disease the role of chemokines in linking innate and adaptive immunity dendritic cells produce il- and direct the development of th cells from narve cd + t cells cd alpha(+) and cd alpha(À) subclasses of dendritic cells direct the development of distinct t helper cells in vivo cytokines regulate the capacity of cd alpha(+) and cd alpha(À) dendritic cells to prime th /th cells in vivo kinetics of virus-specific cd +-t-cell expansion and trafficking following central nervous system infection chemokines and dendritic cells: a crucial alliance a mechanism for the impaired ifn-gamma production in c-c chemokine receptor (ccr ) knockout mice: role of ccr in linking the innate and adaptive immune responses chemokine receptor serves an early and essential role in resistance to mycobacterium tuberculosis distinct dendritic cell subsets differentially regulate the class of immune response in vivo in vivo microbial stimulation induces rapid cd ligand-independent production of interleukin by dendritic cells and their redistribution to t cell areas cc chemokine receptor (ccr) is required for langerhans cell migration and localization of t helper cell type (th )-inducing dendritic cells. absence of ccr shifts the leishmania major-resistant phenotype to a susceptible state dominated by th cytokines, b cell outgrowth, and sustained neutrophilic inflammation tnf/inos-producing dendritic cells mediate innate immune defense against bacterial infection the cc chemokine ligand (ccl ) regulates cd + t cell effector function and migration following viral infection identification of a cd + t cell epitope within the m protein of a neurotropic coronavirus this work was supported by national institutes of health grant and national multiple sclerosis society grant -a- to t.e.l. key: cord- - hw qx authors: grunewald, matthew e.; fehr, anthony r.; athmer, jeremiah; perlman, stanley title: the coronavirus nucleocapsid protein is adp-ribosylated date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: hw qx adp-ribosylation is a common post-translational modification, although how it modulates rna virus infection is not well understood. while screening for adp-ribosylated proteins during coronavirus (cov) infection, we detected a ~ kda adp-ribosylated protein in mouse hepatitis virus (mhv)-infected cells and in virions, which we identified as the viral nucleocapsid (n) protein. the n proteins of porcine epidemic diarrhea virus (pedv), severe acute respiratory syndrome (sars)-cov and middle east respiratory syndrome (mers)-cov were also adp-ribosylated. adp-ribosylation of n protein was also observed in cells exogenously expressing n protein by transduction using venezuelan equine encephalitis virus replicon particles (vrps). however, plasmid-derived n protein was not adp-ribosylated following transient transfection but was adp-ribosylated after mhv infection, indicating that this modification requires virus infection. in conclusion, we have identified a novel post-translation modification of the cov n protein that may play a regulatory role for this important structural protein. adp-ribosylation is the covalent attachment of adp-ribose (adpr) moieties to a protein, resulting in either mono-adpr (mar) or poly-adpr (par). adp-ribosylation is catalyzed by poly-adpr polymerases (parps), also known as adp-ribosyltransferases (artds). the parp family consists of proteins in humans and in mice, which utilize nad as the adpr donor [reviewed in (bock and chang, ) ]. removal of adpr from proteins (de-adp-ribosylation) is catalyzed by different cellular proteins including par glycohydrolase (parg) and macrodomain proteins (bernardi et al., ; jankevicius et al., ; rosenthal et al., ; sharifi et al., ) . detection of adp-ribosylated proteins on a proteomic level is difficult due to the reactive nature and short half-life of the modification in cells (cervantes-laurean et al., ; wielckens et al., ) . despite this, studies of individual parps and adp-ribosylated proteins have elucidated several physiological roles for adp-ribosylation, including dna damage and repair, regulation of rna transcription, cellular stress response, inflammation, differentiation, and apoptosis (bock and chang, ) . parps are well-established to have both proviral and antiviral properties. parp has been shown to facilitate epstein-barr virus replication and latency, simian virus induction of cellular necrosis, and hiv integration (gordon-shaag et al., ; ha et al., ; lupey-green et al., ; tempera et al., ) . tiparp has been shown to inhibit interferon (ifn) production by adp-ribosylation of tbk- , leading to enhanced replication of several viruses (yamada et al., ) . finally, adp-ribosylation of the adenovirus core protein has been implicated in aiding viral replication and modulating stability of viral chromatin-like structures (dery et al., ) . other data suggest that parps can also be antiviral. many parps are expressed following ifn stimulation and several parps show evidence of rapid evolution, suggesting a microbial "arms race" between parps and cellular pathogens (atasheva et al., ; daugherty et al., ; macdonald et al., ) . for example, parp , parp , and parp restrict venezuelan equine encephalitis virus (veev) replication and can block cellular translation when overexpressed (atasheva et al., (atasheva et al., , . one notable parp, the zinc-finger antiviral protein (zap) or parp , has been demonstrated to inhibit replication of several different viruses, potentially by binding to viral rna and directing it to be degraded by the rna exosome (bick et al., ; gao et al., ; guo et al., guo et al., , liu et al., ; muller et al., ; zhu et al., ) . parp is enzymatically inactive, and thus its antiviral activity is independent of adp-ribosylation. in addition, liu et al. have described a novel mechanism in which an unknown parp adp-ribosylates two subunits of the influenza rna polymerase, allowing subsequent binding to zap and degradation of these subunits by the proteasome (liu et al., ) . three different virus families, hepeviridae, togaviridae, and coronaviridae, encode for a viral macrodomain, which has been shown to de-adp-ribosylate proteins in vitro (li et al., ; rosenthal et al., ) . these macrodomains have been proposed to counter the antiviral effects of adp-ribosylation during infection. our previous work has focused on the coronavirus (covs) macrodomain. covs are large, positive-sense, single-stranded rna viruses which include human pathogens such as the severe acute respiratory syndrome (sars)-cov and middle east respiratory syndrome (mers)-cov as well as important veterinary pathogens such as bovine cov and porcine epidemic disease virus (pedv). all covs encode a macrodomain within non-structural protein (nsp ) that can remove both mar and par from proteins (li et al., ) . covs lacking this enzymatic activity generally replicate normally in vitro but are highly attenuated in vivo and elicit an enhanced innate immune response (eriksson et al., ; fehr et al., fehr et al., , kuri et al., ) . to identify potential targets of the cov macrodomain, we analyzed infected cells for changes in adp-ribosylation patterns utilizing antibodies specific for adpr. we focused on cells infected with a murine cov, mouse hepatitis virus (mhv). mhv causes acute and chronic encephalomyelitis, hepatitis and gastroenteritis (bailey et al., ) . surprisingly, we found that the cov nucleocapsid (n) protein was adpribosylated in cells during infection with mhv as well as several other covs. . . cell culture, plasmids and reagents delayed brain tumor (dbt) cells, cl- cells, vero cells, and hela cells expressing the mhv receptor carcinoembryonic antigen-related cell adhesion molecule (ceacam ) (hela-mhvr) were grown in dulbecco's modified eagle medium with % fetal bovine serum as previously described (zhou and perlman, ) . codon-optimized mhv-a n protein was synthesized and cloned directly into pcdna (genscript). a tagged construct was synthesized by inserting a x-flag sequence to the c terminus of the n protein using overlapping primers and recombination by in-fusion (clontech). control plasmid pcdna -gfp was described previously (fehr et al., ) . recombinant mouse hepatitis virus (mhv) strains a (yount et al., ) and jhmv (wild-type and n a) (fehr et al., ) were propagated on cl- cells, and titers were determined on hela-mhvr cells. sars-cov (ma ) was propagated and titered on vero e cells, and mers-cov (emc ) and pedv (isu - e, a gift from dr. kyoung-jin yoon, iowa state university) were propagated on vero- cells. for virus infections, cl- , dbt, calu- , vero e , or vero cells were infected with virus at the indicated multiplicity of infection (moi) and collected at the indicated hours post-infection (hpi). all work with sars-cov or mers-cov infectious virus was performed in a biosafety level laboratory according to the guidelines set forth by the university of iowa. dbt cells were infected with mhv-a at an moi of . pfu/cell, and supernatant was collected and filtered at hpi. the filtrate was subjected to ultracentrifugation at , rpm for h over a % sucrose cushion as described previously. pellets were resuspended in mm nacl and mm tris-cl (ph . ) and treated with or without proteinase k (new england biolabs) in the presence or absence of sds. the reaction was stopped by incubation at °c for min. cells were transfected with polyjet in vitro transfection reagent (signagen labs) as per the manufacturer's instructions. h after transfection, cells were either treated with or without u/ml of ifn-β (pbl) for h or were infected with mhv-a at an moi of pfu/cell for h before collection. veev replicon particles (vrps) encoding either gfp or mers nucleocapsid protein were created and titered as previously reported (zhao et al., . the vrps were transduced into vero cells at indicated mois and collected at h post-transduction. sample buffer containing sds, β-mercaptoethanol, protease/phosphatase inhibitor cocktails (roche), pmsf, parp inhibitor -aminobenzamide ( -ab, tocris bioscience), parg inhibitor adenosine ʹ-diphosphate (hydroxymethyl)pyrrolidinediol (adp-hpd, calbiochem) and universal nuclease (thermofisher scientific) was used to collect cell lysates. proteins were resolved on an sds polyacrylamide gel and transferred to a polyvinylidene difluoride (pvdf) membrane. following binding with a primary antibody, blots were then visualized by using a peroxidase-conjugated secondary antibody (thermo fisher scientific) detected with a chemiluminescent substrate (thermo fisher scientific) or by using an infrared (ir) dye-conjugated secondary antibody detected with a li-cor odyssey imager (li-cor, lincoln, ne) . ir secondary antibodies of different wavelengths were used to obtain different signals for antibody bound proteins. images of α-adpror α-nstained immunoblots were merged using image studio software. primary antibodies used for immunoblotting and immunoprecipitation included polyclonal (pab) α-mhv rabbit serum (perlman et al., ) , monoclonal (mab) α-mhv n (collins et al., ) (mab b . , a kind gift from dr. m. buchmeier, university of california, irvine), pab α-sars-cov n (novus biologicals), pab α-nsp (gift from mark denison, vanderbilt university), mab α-pedv n (gift from dr. kyoung-jin yoon, iowa state university), pab α-mers-cov n , mouse mab α-adpr ( h, millipore sigma), rabbit pab α-adpr (trevigen), chicken pab α-adpr (tulip biolabs inc.), α-flag (sigma), α-gapdh (poly , biolegend), and α-actin (ac ; abcam, inc.) antibodies. secondary antibodies used included horseradish peroxidase-conjugated α-rabbit or α-mouse (sigma #a / a ) antibodies or ir-conjugated α-rabbit, α-mouse, or α-chicken (li-cor, # - / - / - ) antibodies. dbt cells infected with mhv-a at an moi of pfu/cell were collected at hpi and pelleted by low-speed centrifugation. cell pellets were lysed with immunoprecipitation (ip) buffer ( . % np- , mm nacl, % glycerol, and mm tris ph . ) containing protease/phosphatase inhibitor cocktails, pmsf, parp inhibitor -ab, parg inhibitor adp-hpd, and a universal nuclease for h at °c. nuclei were pelleted by centrifugation ( , g for min at °c). one aliquot of cell lysate was saved as the input control and boiled in sds sample buffer described above. protein g magnetic beads were conjugated to α-adpr or α-n antibodies (described above) as per manufacturer's instructions (thermofisher scientific). protein g antibody-conjugated were mixed with cell lysates overnight at °c. beads were washed with pbs-tween before elution by boiling in sds sample buffer. to screen for changes in protein adp-ribosylation during cov infection, we infected dbt cells, an astrocytoma cell line, with the a strain of mhv. cells were collected throughout the infection, and cell lysates were immunoblotted with a mouse mab antibody to adpr (mab h). the h antibody has been described to bind preferentially to linear +-mers of par with no binding activity to dna, rna, or adenosine-monophosphate. however, more recent reports have demonstrated that mab h also binds to auto-marylated proteins (eckei et al., ; goenka et al., ; kawamitsu et al., ; kleine et al., ) . while the adp-ribosylation status of most proteins did not change over the course of infection, we noted the appearance of a ~ kda band at h post-infection (hpi) which increased in abundance up to hpi (fig. a) . immunoblotting with two other commercial α-adpr antibodies raised in two other species also detected a similar protein band (fig. a) . a negative control antibody (α-ha) did not bind to this protein, suggesting a specific interaction of the kda protein with adpr antibodies. based on to the size and abundance of this protein, we hypothesized that it was the nucleocapsid (n) protein. staining with monoclonal α-n antibody produced a signal that completely overlapped the signal from the adpr antibody (fig. b, left) . to confirm that this overlap was consistent in other cell lines and mhv strains, we infected cl- cells (a fibroblast cell line) with mhv-a or dbt cells with mhv-jhm (jhmv), a neurotropic strain of mhv, and immunoblotted with α-mhv serum or α-adpr antibody. the results showed that in all cases the n protein completely overlapped with thẽ kda adp-ribosylated protein, demonstrating that n protein adpribosylation is not mhv strain or cell type specific (fig. b, middle and right) . to confirm the identity of this protein as the n protein, we collected mhv-infected dbt cells at hpi and immunoprecipitated proteins with either monoclonal α-adpr or α-n antibodies and immunoblotted with the reciprocal antibodies. as expected, this~ kda protein could be stained with the α-adpr antibody after immunoprecipitation with α-n and stained with α-n after immunoprecipitation with α-adpr (fig. c) , confirming that the n protein is adp-ribosylated. previous reports have demonstrated that the nsp macrodomain removes both mono-and poly-adpr from auto-adp-ribosylated substrates in vitro (fehr et al., ; li et al., ) . because the n protein is known to associate with nsp , we speculated that the nsp macrodomain may de-adp-ribosylate the n protein (hurst et al., ) . to test this, we infected dbt cells with mhv-a or mhv-jhm encoding either wild-type or a catalytically-deficient (jhmv-n a, a -n a) nsp macrodomain and immunoblotted for n protein adpribosylation. if the macrodomain was indeed removing the adpr from the n protein, we would expect to see increased n protein adp-ribosylation in cells infected with mutant virus compared to wild-type infected cells. however, we found that the level of the adp-ribosylated n protein was the same in cells infected with either mhv strain (fig. ) . these findings suggest that the nsp macrodomain does not affect n protein adp-ribosylation under these conditions. next, we tested whether n protein adp-ribosylation was conserved in cov genera and lineages other than mhv. we infected vero cells with pedv (⍺-cov), sars-cov (lineage b β-cov), or mers-cov (lineage c β-cov) and then collected the cells and analyzed whether the n proteins from these viruses were adp-ribosylated (fig. ) . in all cases, adpr antibody staining overlapped with the n protein of each virus, lysates were collected at and hpi, respectively, and immunoblotted with indicated antibodies. (c) immunoprecipitation with α-adpr antibody confirms n protein is adp-ribosylated. dbt cells were mock infected (m) or infected with mhv-a at moi of pfu/cell. at hpi, cells were collected and the lysates were subjected to immunoprecipitation with mouse α-adpr or α-n (mab) bound to protein g beads. cell lysates and eluted proteins were analyzed by immunoblotting with indicated antibodies. asterisk indicates cellular adp-ribosylated protein that is not immunoprecipitated with α-n antibody. m.e. grunewald et al. virology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] demonstrating that this modification is conserved across multiple genera and lineages of covs. it has previously been shown that the n protein is phosphorylated and that some phosphorylated residues depend on whether the protein is intracellular or virion-associated (white et al., ) . specifically, mhv n protein at amino acid s is phosphorylated in infected cells, but this modification is absent on n protein in virions (wu et al., ) . to determine if n protein adp-ribosylation is maintained within the virion, we purified virions and analyzed whether n protein from the cov virion was adp-ribosylated by immunoblotting. to rule out the possibility of detecting of any residual n protein from non-virion sources, pelleted virions were treated with proteinase k with or without virion lysis by sds. both the n protein as well as the spike (s) protein were detectable with α-mhv serum in both cells and virions (fig. ) . although the s protein was not completely eliminated upon proteinase k treatment, the abundance of the s protein was reduced, consistent with the protein being exposed on the surface of the virus. a co-sedimented nonspecific band of~ kda was also degraded by proteinase k treatment. in contrast, n protein, located in the interior of the virion, was protected from proteinase k treatment unless sds was also added to lyse the viral envelope. importantly, immunoblotting with α-adpr antibody demonstrated that the n protein maintained the adp-ribose modification in virions. to determine if n protein expressed in the absence of cov infection could be adp-ribosylated in cell culture, we transduced vero cells with veev replicon particles (vrps) encoding the mers-cov n protein or control gfp at different mois . transduced cells were collected at hpi, and immunoblotting of cell lysates showed that n protein expressed from an alphavirus replicon was adp-ribosylated ( fig. a) . because the vrp platform utilizes a virus infection to express exogenous proteins, we hypothesized that the adp-ribosylation of the n protein may require a virus infection. to examine this possibility, we transfected dbt cells with a plasmid encoding codon-optimized mhv-n protein or gfp and then tested whether the n protein was adp-ribosylated. because several parps are ifn-stimulated genes, we also treated cells with or without ifn-β (atasheva et al., ; macdonald et al., ) . importantly, we were unable to detect any adp-ribosylation of the exogenous transfected n protein (fig. b) . in contrast, a positive control of mhv-a -infected cell lysate, normalized to total n protein, stained with α-adpr antibody. furthermore, ifn-β treatment did not rescue the modification, suggesting that other factors present during infection drove adp-ribosylation of n protein. to confirm that n protein requires an infection to be adp-ribosylated, we added a x-flag tag to our plasmid-expressed n (n-flag) protein. we then . the n protein is adp-ribosylated within the mhv-a virion. supernatant from dbt cells infected with mhv-a at moi of . was collected at h. whole virions were purified by filtration and ultracentrifugation with a % sucrose cushion. virions were then treated with proteinase k to degrade extramembranous protein or proteinase k in the presence of sds to degrade all viral proteins. proteinase k was then inactivated at °c, and proteins were blotted with indicated antibodies. the nucleocapsid (n), spike (s), and a nonspecific (ns) proteins are indicated with arrows. m.e. grunewald et al. virology ( ) - transfected plasmid expressing gfp or n-flag into dbt cells and then mock infected or infected cells with mhv-a . at hpi, we collected cells and immunoblotted with α-adpr antibody. the n-flag protein was detectable with α-adpr antibody following infection with mhv-a but not in mock infected cells (fig. c ). this suggests that n protein is only adp-ribosylated within the context of virus infection. the n protein was initially identified as a major structural protein, binding directly to viral rna, providing stability to the bound rna, and self-oligomerizing into the virus nucleocapsid. n protein also binds to the viral membrane (m) protein to facilitate genome loading and viral assembly (narayanan et al., ) . it plays a prominent role in transcription and replication of the viral genome (hurst et al., ) . in fact, the addition of n protein from an exogenous plasmid is often utilized to initiate cellular infection from recombinant cov cdna (yount et al., ) . the n protein provides additional accessory functions, including the ability to promote cell cycle arrest, inhibit host translation, and block the ifn response during infection [reviewed in (mcbride et al., ) ]. many of these functions are regulated by posttranslational modifications, most notably phosphorylation by host proteins. for example, sars-cov n protein phosphorylation modulates n protein oligomerization, translation suppression, and localization to stress granules (peng et al., ) . furthermore, phosphorylation of the mhv n protein is implicated to function as a cellular switch to control transcription of either genomic or subgenomic rna (wu et al., ) . in this paper, we have identified an additional post-translational modification of the n protein, adp-ribosylation, which could also play a role in the regulation of n protein functions. we have found that the n protein of multiple covs was apd-ribosylated in vitro, using multiple antibodies to adpr (figs. and ) . the h α-adpr antibody primarily used in this study binds preferentially to par but can be used to detect marylated proteins as well (eckei et al., ; goenka et al., ; kawamitsu et al., ; kleine et al., ) . because the n protein band stained with either α-adpr or α-n antibodies did not appear as a smear, which is seen with long-chain parylated proteins, it is likely that the n protein is either marylated or parylated with only a few monomers of adpr. this is further supported by the fact that adp-ribosylation did not alter migration of the n protein to a detectable level ( fig. b and c) . furthermore, our data indicate that the n protein adp-ribosylation was detectable during infection ( fig. , fig. a, fig. c ) but not following transfection alone (fig. b ). this could be due to a number of factors including, but not limited to, virus infection-dependent expression of an adp-ribosylating enzyme or the localization of the n protein to a distinct cellular compartment during infection. n protein in infected cells has been shown to localize both in the cytoplasm as well as the nucleus, and the sars n protein has been shown to localize to stress granules under stress conditions (hiscox et al., ; peng et al., ) . several parps are known to colocalize with stress granules, which could potential sites of n protein modification (leung et al., ) . future experiments are required to parse out the number of adpr monomers that are attached to the n protein, to identify the amino acid residue(s) that is modified, and to determine where this modification occurs. the fact that adp-ribosylation of n protein was conserved across multiple cov lineages (fig. ) suggests that it is important either for viral replication or pathogenesis or as a host defense mechanism. individual parps have been identified to be either proviral or antiviral [reviewed in (kuny and sullivan, ) ]. because the n protein is the major structural protein of the cov nucleocapsid, it is tempting to speculate that adp-ribosylation is providing a regulatory role for the structure of the genome, similar to adp-ribosylation of histones or of the adenovirus core protein (dery et al., ; strickfaden et al., ) . it is also possible that adp-ribosylation of n protein could regulate one or more of the other functions of the n protein such as inhibition of cellular translation or of ifn expression (kopecky-bromberg et al., ) . on the other hand, adp-ribosylation of the n protein may be an antiviral defense mechanism, similar to adp-ribosylation of influenza a virus polymerase subunits (liu et al., ) . if this were the case, the virus must have evolved methods to combat this modification, possibly including removal of the adp-ribose moieties from the n protein to mitigate its antiviral effects. however, we found that the nsp macrodomain, a likely candidate to catalyze this de-adp-ribosylation, unexpectedly does not de-adp-ribosylate the n protein based on our assays ( fig. ) (fehr et al., ) . however it is possible that a small, localized subset of the very abundant n protein is de-adp-ribosylated, which would not be detectable in our assay. future experiments will focus on identifying the function of n protein adp-ribosylation and the specific targets of the cov macrodomain. here we have showed that the n proteins of multiple covs are adpribosylated, representing a previously undescribed modification of a rna virus structural protein. this modification only occurred during virus infection and was maintained both in the cell and in virions. future experiments will work to identify the amino acids that are adpribosylated and to demonstrate the effect of this novel modification on virus infection. new parp gene with an antialphavirus function interferon-stimulated poly(adp-ribose) polymerases are potent inhibitors of cellular translation and virus replication a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin: ii. pathology analysis of poly(adp-ribose) glycohydrolase activity in nuclear extracts from mammalian cells expression of the zinc-finger antiviral protein inhibits alphavirus replication new directions in parp biology preparation of low molecular weight model conjugates for adp-ribose linkages to protein monoclonal antibodies to murine hepatitis virus- (strain jhm) define the viral glycoprotein responsible for attachment and cell-cell fusion rapid evolution of parp genes suggests a broad role for adp-ribosylation in host-virus conflicts possible role of adp-ribosylation of adenovirus core proteins in virus infection the conserved macrodomains of the non-structural proteins of chikungunya virus and other pathogenic positive strand rna viruses function as mono-adp-ribosylhydrolases mouse hepatitis virus liver pathology is dependent on adp-ribose- ''-phosphatase, a viral function conserved in the alpha-like supergroup the nsp macrodomain promotes virulence in mice with coronavirus-induced encephalitis the conserved coronavirus macrodomain promotes virulence and suppresses the innate immune response during severe acute respiratory syndrome coronavirus infection inhibition of retroviral rna production by zap, a ccch-type zinc finger protein collaborator of stat (coast )-associated poly (adp-ribose) polymerase activity modulates stat -dependent gene transcription the abundant nuclear enzyme parp participates in the life cycle of simian virus and is stimulated by minor capsid protein vp the zinc finger antiviral protein directly binds to specific viral mrnas through the ccch zinc finger motifs the zinc-finger antiviral protein recruits the rna processing exosome to degrade the target mrna poly(adpribose) polymerase- is required for efficient hiv- integration the coronavirus infectious bronchitis virus nucleoprotein localizes to the nucleolus characterization of a critical interaction between the coronavirus nucleocapsid protein and nonstructural protein of the viral replicase-transcriptase complex an interaction between the nucleocapsid protein and a component of the replicase-transcriptase complex is crucial for the infectivity of coronavirus genomic rna a family of macrodomain proteins reverses cellular mono-adp-ribosylation monoclonal antibodies to poly(adenosine diphosphate ribose) recognize different structures substrate-assisted catalysis by parp limits its activity to mono-adp-ribosylation severe acute respiratory syndrome coronavirus open reading frame (orf) b, orf , and nucleocapsid proteins function as interferon antagonists virus-host interactions and the artd/parp family of enzymes the adp-ribose- ''-monophosphatase domains of severe acute respiratory syndrome coronavirus and human coronavirus e mediate resistance to antiviral interferon responses poly(adpribose) regulates stress responses and microrna activity in the cytoplasm viral macro domains reverse protein adp-ribosylation battle between influenza a virus and a newly identified antiviral activity of the parp-containing zapl protein parp restricts epstein barr virus m lytic reactivation by binding the bzlf promoter the zinc finger antiviral protein acts synergistically with an interferon-induced factor for maximal activity against alphaviruses the coronavirus nucleocapsid is a multifunctional protein inhibition of filovirus replication by the zinc finger antiviral protein characterization of the coronavirus m protein and nucleocapsid interaction in infected cells phosphorylation of the arginine/serine dipeptiderich motif of the severe acute respiratory syndrome coronavirus nucleocapsid protein modulates its multimerization, translation inhibitory activity and cellular localization late onset, symptomatic, demyelinating encephalomyelitis in mice infected with mhv-jhm in the presence of maternal antibody macrodomain-containing proteins are new mono-adp-ribosylhydrolases deficiency of terminal adp-ribose protein glycohydrolase targ /c orf in neurodegenerative disease poly(adp-ribosyl)ation-dependent transient chromatin decondensation and histone displacement following laser microirradiation regulation of epstein-barr virus orip replication by poly(adp-ribose) polymerase identification of mouse hepatitis coronavirus a nucleocapsid protein phosphorylation sites dna fragmentation and nad depletion. their relation to the turnover of endogenous mono(adp-ribosyl) and poly(adp-ribosyl) proteins nucleocapsid phosphorylation and rna helicase ddx recruitment enables coronavirus transition from discontinuous to continuous transcription constitutive aryl hydrocarbon receptor signaling constrains type i interferon-mediated antiviral innate defense systematic assembly of a fulllength infectious cdna of mouse hepatitis virus strain a rapid generation of a mouse model for middle east respiratory syndrome airway memory cd (+) t cells mediate protective immunity against emerging respiratory coronaviruses mouse hepatitis virus does not induce beta interferon synthesis and does not inhibit its induction by double-stranded rna zinc-finger antiviral protein inhibits hiv- infection by selectively targeting multiply spliced viral mrnas for degradation we thank the members of the perlman, wendy maury, and pat sinn laboratories for valuable discussion, kyoung-jin yoon for pedv virus and antibodies, and rudragouda channappanavar for critical reading of the manuscript.this study was supported by national institute of health grants to s.p. (p ai and r ns ). m.g. and j.a. were supported by institutional predoctoral nrsa training grants (t ai and t ai , respectively). a.r.f was supported by institutional (t -ai ) and individual (f -ai ) nih nrsa grants. none. key: cord- -tgpsu qz authors: brockway, sarah m.; denison, mark r. title: mutagenesis of the murine hepatitis virus nsp -coding region identifies residues important for protein processing, viral rna synthesis, and viral replication date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: tgpsu qz despite ongoing research investigating mechanisms of coronavirus replication, functions of many viral nonstructural proteins (nsps) remain unknown. in the current study, a reverse genetic approach was used to define the role of the -kda amino-terminal product (nsp ) of the gene polyprotein during replication of the coronavirus murine hepatitis virus (mhv) in cell culture. to determine whether nsp is required for mhv replication and to identify residues critical for protein function, mutant viruses that contained deletions or point mutations within the nsp -coding region were generated and assayed for defects in viral replication, viral protein expression, protein localization, and rna synthesis. the results demonstrated that the carboxy-terminal half of nsp (residues k( ) through l( )) was dispensable for virus replication in culture but was required for efficient proteolytic cleavage of nsp from the gene polyprotein and for optimal viral replication. furthermore, whereas deletion of nsp residues amino-terminal to k( ) failed to produce infectious virus, point mutagenesis of the nsp amino-terminus allowed recovery of several mutants with altered replication and rna synthesis. this study identifies nsp residues important for protein processing, viral rna synthesis, and viral replication. mutagenesis of the murine hepatitis virus nsp -coding region identifies residues important for protein processing, viral rna synthesis, and viral replication coronaviruses belong to a family of enveloped positivestrand rna viruses that are responsible for devastating illnesses in livestock and domestic animals. the identification of a novel human coronavirus as the etiological agent of severe acute respiratory syndrome (sars) in highlighted the potential of this virus family to also cause severe human disease (kuiken et al., ) . even with continuing research addressing how coronaviruses replicate and cause disease, the functions of many viral proteins remain to be elucidated. for example, - nonstructural proteins (nsps) are expressed from coronavirus gene polyproteins, but at least seven of these nsps have no known roles in viral replication. in the past, gene has been referred to as the ''replicase gene'' and gene nsps as ''replicase proteins'' named by their molecular weight in kilodaltons. more recently, nsps have been named based on their order in gene and are numbered consecutively beginning at the amino-terminus of the polyprotein (i.e., nsp through nsp ) (harcourt et al., ; prentice et al., b; snijder et al., ) . the gene nsps of the coronavirus murine hepatitis virus (mhv) are similar in number, size, and organization to those of sars coronavirus (sars-cov) (marra et al., ; snijder et al., ) . this resemblance suggests that mhv is an excellent model for studies of coronavirus nsp function and may - /$ -see front matter d elsevier inc. all rights reserved. doi: . /j.virol. . . increase our understanding of sars-cov replication and pathogenesis. following entry of mhv into a host cell, the first event in the virus life cycle is translation of two polyproteins from gene of the input rna genome. gene is comprised of two overlapping open reading frames (orf a and orf b) that are connected by a À ribosomal frameshift (fig. a ) (bonilla et al., ; bredenbeek et al., ; brierley et al., ; lee et al., ; pachuk et al., ) . translation of either orf a or the orf ab fusion results in possible -kda or -kda polyproteins, respectively. these co-aminoterminal polyproteins are proteolytically processed by three virus-encoded proteinases, including two papain-like proteinases (plp and plp within nsp ) and a picoronavirus c-like proteinase (nsp ), to yield at least mature gene nsps as well as intermediate precursors. to date, all mhv gene nsps tested co-localize with sites of active viral rna synthesis at cytoplasmic viral replication complexes on intracellular double-membrane vesicles (bost et al., (bost et al., , brockway et al., brockway et al., , gosert et al., ; prentice et al., a; shi et al., ; van der meer et al., ) . nsps are thought to mediate replication of the mhv rna genome and subgenomic rna synthesis at these membrane-bound complexes. however, the viral proteinases are the only mhv nsps with experimentally confirmed functions (baker et al., ; bonilla et al., ; lu et al., ; lu et al., ) . based on homology to proteins with known functions, roles in viral rna synthesis have been predicted for several other mhv gene nsps. these proteins include two trans-membrane scaffolding proteins (nsp and nsp ) (gosert et al., ) , an rna-dependent rna polymerase (nsp ) (cheng et al., ; gorbalenya et al., ; lee et al., ) , an rna helicase (nsp ) (seybert and ziebuhr, ; seybert et al., ) , and several rna processing enzymes (nsp , nsp , and nsp ) (bhardwaj et al., ; ivanov et al., ; thiel et al., ; ziebuhr, ) . currently, there are no known or envisaged functions in replication for at least seven mhv gene proteins (nsp , nsp , and nsp - ) . previous studies have provided intriguing evidence about the potential functions of the -kda amino-terminal protein nsp (previously referred to as p ) in mhv replication. the kinetics of nsp expression suggest that it might have an early regulatory role during the viral life cycle. nsp is the first mature protein processed from the gene polyprotein and is likely cleaved quickly following trans- orf a -orf b fusion polyprotein is illustrated with mature nonstructural protein (nsps) represented as numbered boxes. the gray box represents the aminoterminal cleavage product (nsp ). nsps with confirmed or predicted functions include: two papain-like proteinases (plp and plp within nsp ), the c-like proteinase ( cl pro ; nsp ), two trans-membrane proteins (mp and mp ; nsp and nsp , respectively), the rna-dependent rna polymerase (pol; nsp ), the rna helicase (hel; nsp ), the v-to- v exonuclease (exo; nsp ), the endoribonuclease (endo; nsp ), and the rna methyltransferase (mt; nsp ). (b) nsp mutant proteins. the schematics illustrate the engineered deletions and point mutations within nsp . nsp amino acid numbers are listed below each protein, and the predicted protein size (in kilodaltons) is listed to the right of each. the amino-terminal charge-to-alanine mutations for each vusb mutants are listed below the bottom nsp protein. the asterisks (*) indicate mutants that did not establish productive infections as determined by lack of recovered virus from electroporated cells. lation of plp within nsp (baker et al., (baker et al., , denison and perlman, ; denison and perlman, ; denison et al., denison et al., , . mhv mutants that are incapable of liberating nsp from the nascent polyprotein exhibit delayed replication, diminished peak titers, small plaques, and reduced rna synthesis compared to wild-type controls . these results emphasize the importance of nsp cleavage for optimal viral rna synthesis and suggest that nsp might play an important role at mhv replication complexes. in support of this notion, nsp localizes to replication complexes in the infected cell cytoplasm during times of peak viral rna synthesis, and biochemical experiments demonstrate interactions between nsp and two other replication complexassociated proteins (nsp and nsp ) . however, later in infection, nsp is distinct from replication complexes and instead co-localizes with mhv structural proteins at virion assembly sites . it has also been reported that exogenous mhv nsp expression induces cell-cycle arrest (chen et al., ) . together, these results led to the hypothesis that nsp may participate in multiple stages of the mhv life cycle. still, the function of this protein remains a mystery as analysis of nsp primary amino acid sequence does not reveal common motifs or protein homologs, and the nsp crystal structure has not been solved. the goal of the current study was to determine the role of nsp during mhv replication in cell culture and to identify regions of the protein that are critical for its function. using the reverse genetic system for mhv developed by yount et al. ( ) , genomic rna molecules that contained deletions or point mutations within the nsp -coding region were generated and tested for their capacity to produce infectious virus following transfection into permissive cells. the results demonstrate that the carboxy-terminal half of nsp is dispensable for replication in culture but is important for efficient proteolytic cleavage of the protein and optimal viral replication. in contrast, deletion of the entire nsp protein or a middle region of the protein resulted in the lack of virus recovered from electroporated cells. analysis of nsp point mutants identified residues important for viral replication and rna synthesis. the results of this study provide valuable information regarding the possible functions of the amino-terminal nsp protein in coronavirus replication. the carboxy-terminal half of nsp is dispensable for mhv replication to determine whether nsp is required for mhv replication, viruses were engineered to lack nsp residues a through l (nsp dfl) or to express truncated forms of the protein lacking either q through n (nsp dmid) or k through l (nsp dc) (fig. b) . all deletion constructs were designed to maintain the start codon, the translational reading frame, and the minimal residues thought to be required for cleavage of nsp from the polyprotein by plp (nsp residues k through g ) . mutations were engineered into the nsp -coding region within the mhv infectious cdna (icmhv) fragment a plasmid. the mutagenized plasmids were then used to assemble full-length mhv cdna, which was transcribed in vitro to yield mhv genomic rna (yount et al., ) . bhk-mhvr cells were electroporated with either assembled wild-type (icwt) or nsp mutant genomic rna transcripts. cells were incubated at -c and were monitored for syncytia formation, a cytopathic effect (cpe) of mhv replication. virus-induced syncytia were detected at h postelectroporation (p.e.) in cells transfected with icwt genomic rna and at h p.e. in cells transfected with genomic rna for the carboxy-terminal truncation mutant (nsp dc). clarified cell culture media harvested from cells electroporated with either icwt or nsp dc rna were capable of initiating a productive infection in freshly inoculated dbt- cells, demonstrating the presence of viable virus. in contrast, cells electroporated with nsp dfl or nsp dmid rna showed foci containing - cell nuclei at - h p.e., but these multi-nucleated foci did not progress or spread over time like wild-type mhv syncytia. the clarified media from cells electroporated with nsp dfl or nsp dmid genomes were not capable of producing cpe in freshly inoculated dbt- cells. this result indicated that nsp dfl and nsp dmid were not capable of completing a productive life cycle, defined by the lack of infectious virus particles in the cell media supernatant following electroporation. nsp dfl and nsp dmid rna molecules were tested for their capacity to produce viruses in three independent experiments, all of which yielded this same result. the nsp dc virus was purified by three rounds of plaque isolation, and rt-pcr was used to sequence across the entire nsp -coding region. the mutant virus maintained the engineered deletion and had no other mutations within nsp . these results demonstrate that the carboxy-terminal half of nsp is dispensable for mhv replication and suggest that deletion of residues in the amino-terminal half is not tolerated for a productive infection. charge-to-alanine mutagenesis of the nsp amino-terminus the fact that nsp dfl and nsp dmid did not generate productive infections suggests that this region of the protein has a critical function in mhv replication. however, it is also possible that the deletions removed required rna elements within the v end of the nsp coding sequence. to investigate the function of the nsp amino-terminus with minimal disruption of the correspond-ing rna sequence, viruses were engineered to contain point mutations within this region. mutations were introduced into the icmhv fragment a plasmid using sitedirected mutagenesis. single or paired charged residues (d, e, r, k, and h) within the amino-terminal half of mhv nsp that are conserved across all group coronaviruses were substituted with alanine (fig. b) . mutated fragment a cdnas were used as before to generate full-length genomic viral rna. virus-induced syncytia were detected within h in bhk-mhvr cells electroporated with either icwt rna or with rna for nsp mutants vusb , vusb , vusb , or vusb . the timing and extent of cpe following transfection with these mutants were indistinguishable from icwt. cells transfected with rna for vusb exhibited cpe at h p.e., similar to the kinetics observed with nsp dc. the clarified media supernatants from cells transfected with vusb , vusb , vusb , vusb , or vusb rna were capable of infecting dbt- cells, indicating that they contained nsp mutant viruses. as with nsp dc, the nsp point mutants were purified by three rounds of plaque isolation, and rt-pcr was used to sequence across the entire nsp -coding region. the mutant viruses maintained the engineered changes and lacked other mutations within nsp . similar to the results with nsp dfl and nsp dmid, cells transfected with vusb or vusb rna produced foci containing - cell nuclei at - h p.e, but the extent of these multi-nucleated foci did not increase over time. moreover, clarified media supernatant harvested from cells transfected with vusb or vusb rna did not generate a productive infection following incubation with dbt- cells, indicating that vusb and vusb were blocked at some stage in the viral life cycle. vusb and vusb rna molecule were assayed for their capacity to produce viruses in two independent experiments, both of which showed the same result. it has been reported that charge-to-alanine mutagenesis of viral proteins may generate temperaturesensitive mutants (hanley et al., ; hassett and condit, ; tang et al., ) . in the current study, nsp mutants vusb , vusb , and nsp dmid did not exhibit temperature-sensitive phenotypes as no infectious virus was recovered at either -c or -c. the nsp dfl virus was not tested at -c. experiments were next performed to determine if viral gene expression occurred in cells electroporated with rna for mutants that did not produce infectious virus (vusb , vusb , nsp dfl, and nsp dmid). using antisera against mhv virions (a-mhv) or against nsp (a-nsp ), no viral proteins could be detected by immunofluorescence or immunoprecipitation (data not shown). furthermore, no specific products were amplified from electroporated cells using rt-pcr and a primer that detects subgenomic rnas (data not shown). these results suggest that, if viral protein or rna expression occurred, it was below the level of detection using these assays. to determine whether mutagenesis of nsp results in viral replication defects, single-cycle replication assays were performed with the nsp mutants (fig. ) . dbt- cells were infected with icwt or nsp mutants (vusb , vusb , vusb , vusb , vusb , or nsp dc). samples of infected cell media supernatant were taken at various times from - h post-infection (p.i.), and viral titers in each sample were determined by plaque assays. wild-type (icwt) or nsp mutant viruses were used to infect dbt- cells at an moi of . cells were rinsed three times with pbs, incubated under medium at -c, and samples of medium were obtained at the indicated times post-infection. viral titers in each sample were determined using plaque assays on dbt- cells at -c. the graph shows the results of a representative experiment. values are the averages obtained from duplicate media samples. (b) nsp mutant viral yield. to determine viral yield, viral titer at h p.i. was subtracted from peak titer for each virus. bars represent average viral yield calculated from the experiments (n = ). lines represent standard error from the experiments. statistical analysis software was used to determine p values using a two-sample t test. asterisks (*) indicate p values . . (c) relative plaque size of icwt, vusb , and vusb ( h p.i.). images were obtained at the same resolution ( Â) on a nikon eclipse te -e microscope. white circles were drawn to facilitate visualization of the plaque boundary. the nsp mutants exhibited viral replication kinetics similar to icwt with peak virus release occurring between - h p.i. (fig. a) . however, several of the nsp mutants (vusb , vusb , and nsp dc) showed a reduction in viral yield compared with icwt ( fig. b ). whereas icwt gave an average yield of approximately .  pfu/ml, vusb , vusb , nsp dc viruses exhibited viral yields of .  , .  , .  pfu/ml, respectively. all of the nsp mutants exhibited decreased yields; however, only the vusb , vusb , and nsp dc yields were statistically significant ( p . using a twosample t test). vusb and vusb also demonstrated small plaque phenotypes (fig. c ). these results indicate that nsp mutants have replication defects in cultured dbt- cells. nsp mutant viruses were evaluated to determine whether they are defective in the expression or processing of viral proteins (fig. ) . immunoprecipitation experiments were performed to analyze processing of nsp and nsp or clpro-mediated processing of nsp . to confirm expression of the structural proteins, antisera raised against mhv virions (a-mhv) were used. dbt- cells were either mockinfected or infected with icwt or nsp mutants, radiolabeled from - h p.i. with [ s]met/cys, and used to prepare cytoplasmic lysates for immunoprecipitations. antisera against nsp (a-nsp ) immunoprecipitated a -kda protein (nsp ) from cells infected with icwt or the nsp point mutants (vusb , vusb , vusb , vusb , or vusb ) (fig. a) . migration of vusb -nsp differed from icwt and all other point mutants, suggesting differences in protein folding or charge. no -kda proteins were immunoprecipitated from mock-infected cell lysates or from lysates generated from cells infected with the nsp dc virus ( fig. a) . rather, from nsp dc-infected cell lysate, a-nsp immunoprecipitated a unique -kda protein, the predicted size of nsp dc, as well as a previously undescribed -kda protein (fig. a) . in both vusb -and nsp dc-infected cells, several additional proteins ( to kda) of unknown identity were also precipitated using a-nsp (fig. a ). antisera against nsp (a-nsp ) immunoprecipitated a -kda protein (nsp ) from all infected cell lysates, but not from mock-infected lysates (fig. b ). similar to the result using a-nsp , an -kda protein was also detected by a-nsp in nsp dc-infected cells (fig. b ). antisera against nsp (a-nsp ) immunoprecipitated a -kda protein (nsp ) from all infected cell lysates, but not from mock-infected cell lysates (fig. c ). the viral structural protein s, n, and m were detected in all infected cell lysates using antisera against virions (a-mhv) (fig. d ). the nsp mutants showed band intensities slightly increased compared with icwt. the reason for this difference in band intensity is unknown but might be a reflection of more protein synthesis or processing with the mutants at the time of label. still, although the amount of protein detected differed slightly, the results show that amino-terminal nsp point mutants do not have defects in their capacities to express or process viral proteins. nonetheless, the detection of an -kda protein from nsp dc-infected cells using either a-nsp or a-nsp suggests that cleavage of nsp dc from nsp may be inefficient. fig. . nsp mutant viral protein expression and processing. cytoplasmic lysates were generated from radiolabeled dbt- cells that were either mock-infected (m) or infected with icwt or nsp mutant viruses. labeled proteins were immunoprecipitated from cytoplasmic lysates with the indicated polyclonal antisera. proteins were resolved by sds-page in - % polyacrylamide gradient gels and visualized following fluorography. images were obtained following -day film exposure. bands corresponding to unique or predicted proteins are indicated on the right of the fluorograms, and molecular weight standards (in kilodaltons) are shown on the left. the mhv structural proteins are designated as follows: spike (s), nucleocapsid (n), and membrane (m). the antisera used for immunoprecipitation are indicated: the carboxy-terminal half of nsp is required for efficient cleavage at cs previous studies have shown that nsp is processed from the gene polyprotein very rapidly, and nsp -containing precursors have not been detected (denison et al., ) . in contrast, nsp is processed with slower kinetics likely from a -kda nsp -nsp precursor protein (denison et al., harcourt et al., ; schiller et al., ) . to determine the expression kinetics of the -kda protein in nsp dc virus-infected cells and to determine whether this protein is capable of being processed into -kda (nsp dc) and -kda (nsp ) proteins, pulse-chase translation experiments were performed (fig. ) . dbt- cells were infected with either icwt or the nsp dc virus, and proteins were radiolabeled from - . h p.i. with [ s]met/cys. infected cells were then chased in medium without radiolabel but containing cyclohexamide to inhibit new protein synthesis. cells were harvested at various times post-chase (p.c.), and cytoplasmic lysates were generated for immunoprecipitations using a-nsp and a-nsp . in the current study, the kinetics of nsp and nsp expression and processing in icwt-infected cells were similar to published reports (fig. a ). nsp was detected as a mature -kda protein at min p.c., suggesting that this protein was processed during the min radiolabeling period. although nsp was detected as a mature -kda protein at min p.c., nsp detection was increased at - min p.c. the result that nsp and nsp remained detectable even at min p.c. suggested that these proteins were stable. in these experiments, the -kda nsp -nsp precursor was not detected by a-nsp . like nsp from icwt, nsp dc ( kda) was detectable at min p.c., indicating that processing at cs occurred within fig. . pulse-chase translation in nsp dc virus-infected cells. proteins in infected dbt- cells were radiolabeled for min with [ s]met/cys at h p.i. and then incubated in medium containing cyclohexamide for to min as described in materials and methods. cells were lysed at the indicated times (min) post-chase (p.c.), and cytoplasmic lysates were generated for immunoprecipitation studies using a-nsp and a-nsp . proteins were analyzed as in fig. . the identities of proteins are indicated to the right of the fluorograms. the number of days the gels were exposed to film (d exp) to generate the image is listed next to the antisera used for immunoprecipitation. pulse-chase translation in cells infected with (a) icwt or (b) nsp dc virus. . intracellular localization of mutant nsp proteins. dbt- cells grown on glass coverslips were infected with icwt or nsp mutant viruses for h, fixed and permeablized with % methanol, and incubated with antibodies against nsp (red), nsp (green), and n (purple). cells were imaged using a zeiss lsm confocal microscope at nm (red), nm (green), and nm (purple). images are single confocal slices obtained using a  objective. co-localization of green and purple for vusb is shown in the merged image as light green pixels. co-localization of all three colors is shown in the merged images as white pixels. multi-nucleated cells are a cytopathic effect of mhv replication. the min period of radiolabel. however, in contrast to icwt nsp , nsp dc was not detectable after min p.c., suggesting that once processed the protein may be subject to degradation. the kinetics of nsp expression in nsp dc virus-infected cells were similar to those of nsp from icwt (fig. b) ; nsp ( kda) was first detected at - min p.c., but detection was increased at - min p.c. using either a-nsp or a-nsp , the -kda protein was detectable by min p.c. and remained stable or increasing until min p.c. at min p.c., the -kda band was slightly less intense; however, at this time point, there was no concurrent increase in nsp dc or nsp detection. this result suggests that the -kda protein may be degraded at late times of chase rather than processed. as with icwt, the high molecular weight nsp dc viral precursor proteins were not detected by the antisera. during early times of infection ( - h p.i.), nsp colocalizes with nsp and the viral nucleocapsid protein (n) at viral replication complexes in the cytoplasm . at these times, nsp is mostly distinct from the virion membrane protein (m), which localizes to sites of particle assembly . to determine the intracellular localization of viral proteins from the nsp mutants, immunofluorescence confocal microscopy was performed. dbt- cells on glass coverslips were infected for h, fixed and permeabilized with methanol, and incubated with antisera against nsp , nsp , and either n or m (fig. ) . the a-nsp staining pattern in cells infected with vusb , vusb , vusb , vusb , or nsp dc was similar to icwt. nsp from these mutant viruses co-localized with nsp and n in punctate cytoplasmic replication complexes (fig. ) . at this time point, the nsp proteins from vusb , vusb , vusb , vusb , nsp dc, or icwt viruses were also distinct from sites of virion assembly as determined by lack of predominant nsp co-localization with m (data not shown). using the a-nsp antisera, no specific staining above background levels was detected in vusb -infected cells, whereas nsp , n, and m staining was indistinguishable from icwt ( fig. and data not shown) . the nsp antiserum is capable of detecting vusb -nsp by immunoprecipitation (fig. a) , demonstrating that the engineered substitutions did not abolish the epitope(s). therefore, the lack of staining during immunofluorescence assays might reflect alternative folding of vusb -nsp . nonetheless, these results suggest that nsp mutant viruses do not have defects in intracellular protein localization or replication complex formation. to investigate whether the nsp mutant viruses exhibit changes in the timing or levels of viral rna synthesis, metabolic labeling assays were performed (fig. ). dbt- fig. . nsp mutant viral rna levels. (a) nsp mutant viral rna levels represented as percent of icwt. dbt- cells were infected at an moi of with icwt or nsp mutant viruses. actinomycin d was added to a final concentration of ag/ml min prior to the addition of [ h]uridine. viral rna was radiolabeled from - h p.i. and then precipitated from equal volumes of cytoplasmic lysates in replicate using trichloracetic acid. to quantitate [ h]uridine incorporation as counts per minute (cpm), liquid scintillation was used. for each experiment (n = ), labeled viral rna levels (cpm) for icwt were set to %, and nsp mutant viral rna was calculated as a percentage of the icwt value. the bars represent the average percent viral rna from all experiments, and lines indicate standard error. statistical analysis software was used to determine p values using a one-sample t test. asterisks (*) indicate p values . . (b) gel analysis of viral rna. dbt- cells were mock-infected or infected with icwt or the indicated nsp mutants. rna was labeled as in panel (a) above, and cells were lysed using trizol. rna was isolated from cell lysates and normalized so as to electrophorese the same amount of radiolabeled viral rna for each mutant (approximately , cpm from a maximum of cells). for the mock-infected control (m), rna from cells was used. the rna was separated in an . % formaldehyde/agarose gel, and individual rna species were visualized following fluorography. the image is from a -day exposure of the gel to film. individual viral rna species are numbered to the right of the fluorogram (rna = genome and rna - = subgenomic rnas). the asterisk (*) indicates a unique rna band detected in nsp dc virus-infected cells. cells were infected with icwt or nsp mutants (vusb , vusb , vusb , vusb , vusb , or nsp dc). viral rnas were radiolabeled from - h p.i. at intervals using aci/ml of [ h]uridine in the presence of actinomycin d, a drug that inhibits dna-dependent rna synthesis. rna was precipitated from cytoplasmic lysates using trichloroacetic acid, and h levels were quantitated using liquid scintillation. the timing of rna synthesis for the nsp mutants was indistinguishable from icwt, with all viruses exhibiting maximal [ h]uridine incorporation when cells were labeled between - h p.i. (data not shown). however, the total levels of radiolabeled viral rna differed among the nsp mutants when compared with icwt. vusb and vusb rna levels were only % and % of icwt, respectively, whereas vusb rna levels were % if icwt (fig. a) . these data suggest that nsp may function at the replication complex in the synthesis of viral rna. to determine whether nsp mutants have specific defects in genome replication or subgenomic rna synthesis, radiolabeled viral rna was analyzed by gel electrophoresis (fig. b ). dbt- cells were either mock-infected or infected with icwt or nsp mutant viruses, and rna was labeled in the presence of actinomycin d from - h p.i. using [ h]uridine. total viral and cellular rna was extracted from cell lysates, and radioactivity in each sample was quantitated using liquid scintillation. equal amounts of radiolabeled viral rna (equal cpm) were separated by electrophoresis in formaldehyde/agarose gels and then visualized following fluorography. all seven species of mhv viral rna were detected with the nsp mutant viruses and icwt. interestingly, with the nsp dc virus, an additional band that migrated above rna was consistently detected. the identity of this band is not known, but it might represent a more stable replicative intermediate or a new subgenomic rna species. still, the approximate ratio of rna (genome) to rna for each nsp mutant was similar to that of icwt, suggesting that nsp mutants do not have specific defects in either genome replication or subgenomic rna synthesis as detected by this assay. coronavirus gene nsps are predicted to function in the synthesis of viral rna at cytoplasmic replication complexes; however, many of these proteins have no known roles during the viral life cycle. in the current study, a reverse genetic approach was used to investigate the role of nsp during mhv replication in cell culture and to identify residues critical for its function. it was shown that viruses containing deletions within nsp amino-terminal to k are not capable of establishing productive infections, suggesting that residues essential for mhv viability reside within the amino-terminal half of the protein. several such residues were identified using point mutagenesis, and the importance of the nsp for viral replication and rna synthesis was characterized. moreover, the results show that the carboxyterminal half of nsp is not required for mhv to complete its life cycle but is necessary for efficient cleavage of nsp from the gene polyprotein and for optimal viral replication. together, these data are consistent with the hypothesis that mhv nsp contains at least two domains important for virus replication: ( ) an essential aminoterminal domain involved in viral rna synthesis and ( ) a non-essential carboxy-terminal domain that influences cs cleavage efficiency. the analyses of mhv nsp deletion and point mutants emphasize the significance of the amino-terminus for virus viability, replication, and rna synthesis. deletion of different portions of the nsp amino-terminal half, as was done with nsp dfl and nsp dmid, was not tolerated for virus viability. this result is consistent with the idea that critical replication determinants reside within the region spanning residues m through p . charge-to-alanine mutagenesis identified four candidate residues (r and e with vusb ; r and d with vusb ), all or any of which may be vital for some stage of the mhv life cycle. in cells transfected with rna genomes for these nonproductive mutants (nsp dfl, nsp dmid, vusb , or vusb ), foci containing - cell nuclei were seen. how these foci form in electroporated cell monolayers is unknown, but the presence of this cpe suggests that transient viral gene expression may have occurred. however, no viral protein was detected in transfected cell monolayers by immunoprecipitation or immunofluorescence assays, and rt-pcr did not amplify subgenomic rna, indicating that, if gene expression occurred, it was below the limits of detection using these assays. consequently, the exact stage of the mhv life cycle at which these mutant viruses are blocked could not be determined. still, the result that nsp dfl, nsp dmid, vusb , or vusb did not establish productive infections underscores the importance of the nsp amino-terminus and suggests that this protein has a critical function during viral replication. in support of nsp having a central role in the mhv life cycle, it was demonstrated that all of the viable nsp point mutants exhibited reduced viral yields during singlecycle replication assays. vusb and vusb were the most defective and resulted in viral titers approximately . -to . -log reduced compared with icwt. these two viruses also exhibited small plaque phenotypes, suggesting defects in cell-to-cell spread. the reduced replication and small plaques seen with vusb and vusb are most likely a reflection of reduced viral gene expression as both of these nsp point mutants showed significantly lower levels of viral rna. interestingly, compared with icwt, vusb had increased levels of viral rna associated with a slight reduction in viral replication, suggesting that any deviation from wild-type viral rna levels may negatively impact viral replication. there were no detectable differences in the ratios of genome rna versus subgenomic rnas for any of the viable nsp mutants. these results suggest that nsp might play a role in the regulation of total viral rna amounts rather than in species-specific viral rna synthesis. it has been previously shown that mhv mutants unable to liberate nsp from the gene polyprotein (dcs mutants) exhibit phenotypes very similar to the nsp mutant viruses described in this study . because the dcs mutations resulted in a noncleaved nsp -nsp fusion protein, it could not be concluded that the viral phenotypes were solely related to defects in nsp function. this study corroborates and extends these previous findings and directly correlates mutations in the mhv nsp -coding region with changes in viral replication and rna synthesis. the results of this study raise important questions regarding the mechanistic basis for the described replication and rna synthesis defects with nsp mutants. why is the nsp amino-terminus required for mhv replication? what function does this domain have during mhv rna synthesis? do the deletion and point mutations change the nature of nsp -protein interactions? yeast two-hybrid and co-immunoprecipitation assays previously demonstrated that wild-type nsp directly binds to nsp and nsp , two other gene proteins . the interactions between nsp , nsp , and nsp do not require the nsp carboxy-terminus (f through g ) but do require residues amino-terminal to f (i.e., residues e through r ). because the nsp dmid virus was engineered to lack this putative interaction domain, it is interesting to speculate whether the lack of viability for this mutant correlates with a loss of nsp binding to either nsp and/or nsp . furthermore, it is thought that charge-to-alanine mutagenesis alters a protein's capacity to participate in interand intra-molecular interactions (cunningham and wells, ) . this mutagenesis approach is supposed to minimize disruption of protein secondary structure; however, it is possible that some of the charge-to-alanine mutations caused nsp to misfold. in support of this possibility, vusb -nsp was detected by a-nsp antisera during immunoprecipitation assays but not during immunofluorescence assays, suggesting that this mutant protein may have a different structural conformation. regardless, the introduced mutations may have altered the capacity of nsp to bind to nsp , nsp , or other proteins resulting in viral rna synthesis and viral replication defects. during wild-type mhv infection, nsp , nsp , and nsp are processed in an ordered manner (fig. a) . immediately following translation of plp within nsp , cs is rapidly cleaved, releasing nsp as polyprotein synthesis continues (denison et al., harcourt et al., ; schiller et al., ) . next, cleavage site (cs ) is cleaved (likely by plp ) liberating an nsp -nsp precursor, which is finally processed at cleavage site (cs ) to release mature nsp and nsp products (denison et al., harcourt et al., ; schiller et al., ) . in nsp dc virus-infected cells, nsp dc ( -kda) and nsp are present, demonstrating that the carboxy-terminal half of nsp is not absolutely essential for cleavage at either cs or cs . however, the identification of an -kda nsp dc -nsp polypeptide in nsp dc virus-infected cells reveals that this region of the protein (residues k through l ) is important for efficient cs processing. fig. . model of icwt and nsp dc virus protein processing. shown are schematics of wild-type and nsp dc virus amino-terminal gene nsps (boxes). plp and plp are shown as gray boxes within nsp . the individual cleavage sites are labeled, and the order of processing is indicated above the closed arrowheads. lines below the protein schematics illustrate the size of mature proteins following cleavage at individual sites. (a) processing of wild-type nsp , nsp , and nsp . during infection, nsp ( kda) is cleaved rapidly at cleavage site (cs ) as the polyprotein is translated (indicated by forward slashes). next, cleavage site (cs ) is processed to yield a -kda nsp -nsp precursor. finally, cleavage site (cs ) is cleaved to liberate nsp ( kda) and nsp ( kda). (b) wildtype pattern of processing for nsp dc mutant. the order of cleavage for nsp dc mutant polyprotein is identical to that of icwt. nsp dc protein (dc; kda) is liberated by cs cleavage. next, cs is processed to yield a -kda nsp -nsp precursor. finally, cs is cleaved to liberate nsp ( kda) and nsp ( kda). (c) alternative pattern of processing for nsp dc mutant. if cs is not initially cleaved, a -kda nsp dc -nsp -nsp precursor is made following cs processing. this precursor is then cleaved at cs to yield an -kda protein (nsp dc -nsp ) and nsp . the results described in this report are consistent with a model in which the nsp dc gene polyprotein exists in two different conformations: a cs cleavable form and a cs non-cleavable from. if the mutant polyprotein is folded in such a manner that cs is accessible by the proteinase, then this site is cleaved normally and the mutant polyprotein follows a wild-type pattern of processing (fig. b) . alternatively, if the mutant polyprotein is folded in a way that masks cs , this site is not processed and nsp dc remains fused to nsp (fig. c ). in this model, an nsp dc-nsp -nsp precursor is the first processed protein following cs cleavage. this polypeptide is then processed at cs to yield the -kda protein (nsp dc -nsp ) and nsp . although the high molecular weight precursors were not detected in these studies, the kinetics of appearance for nsp dc, nsp , and the -kda protein are compatible with the proposed model. during pulse-chase experiments, nsp dc and nsp are first detected in nsp dc virus-infected cells with the same timing as the corresponding nsp and nsp proteins in icwt-infected cells. however, the expression kinetics for the -kda protein are unlike either nsp or nsp , suggesting that the timing and order of polyprotein processing are different under circumstances where cs is not immediately cleaved. whether the -kda protein is capable of being subsequently processed into nsp dc and nsp could not be determined from these studies. at extended times p.c. ( min), the -kda protein is still detectable, albeit at slightly reduced levels, and there is no new accumulation of nsp dc at this same time. these data are most consistent with the idea that the -kda protein is not further processed once it is generated. in addition to cs processing defects, the nsp dc virus also exhibits subtle replication defects, but normal viral rna synthesis levels, compared with icwt. a unique rna species migrating above rna was detected in nsp dcinfected cells. the identity of this rna band remains to be determined, but it might represent a stable replicative intermediate and implies that this mutant virus may have minor defects in discontinuous transcription. another interesting possibility is that this band might represent a new species of subgenomic rna. because some molecules of nsp dc are fused to nsp , defects in nsp function may contribute to the mutant viral phenotype. alternatively, the reduced replication of the nsp dc virus might suggest that nsp plays an important role downstream of viral rna synthesis, such as in virion assembly. at late times of infection, wild-type nsp co-localizes with the mhv structural protein m at virion assembly sites . because the a-nsp antisera used in this study also detects the -kda nsp dc -nsp protein, we were unable to precisely determine whether the -kda nsp dc protein is capable of localizing to virion assembly sites late in infection. nonetheless, it will be valuable to determine whether nsp dc, or any of the mutant nsp proteins, exhibit differences in protein localization during late times of infection. in summary, the phenotypes of these mhv mutant viruses reveal new information about the possible roles of nsp during viral replication. however, it is important to note the limitations of the reverse genetic approach used in this study and to explore alternative explanations of the available data. for example, it has been reported that exogenous mhv nsp expression induces cell-cycle arrest (chen et al., ) . therefore, it is also possible that the nsp mutations have an indirect effect on mhv replication as a result of cell biological changes during infection. moreover, while it is clear that some of the nsp mutants have differences in nsp confirmation and interactions, it is possible that uncharacterized rna structural elements have also been disrupted as a result of nsp mutagenesis resulting in changes in viral rna synthesis and replication. furthermore, in the absence of sequencing the entire -kb genomes of the nsp mutant viruses, the possibility that other mutations contribute to the described phenotypes cannot be excluded. to rule out the contributions of other mutations or rna elements, wild-type nsp expressed in cells must complement the defects of these viruses. while ideal, trans-complementation using protein expression has not been successful for coronaviruses. in fact, efficient complementation for other positive-strand rna viruses has required the development of replicon systems to facilitate viral replication complex formation (appel et al., ; grassmann et al., ; khromykh et al., ; lindenbach and rice, ; liu et al., ) . despite these limitations, this is the first report of detailed mutagenesis of an mhv gene nsp-coding region within the context of a virus. these results will enhance our knowledge of mhv replication determinants, and future analysis of nsp mutant viruses will provide greater insight into the function of rna and protein elements within the v end of gene . even more, studies with these engineered mutant viruses are expected to contribute to understanding the replication strategies of other coronaviruses, such as sars-cov. delayed brain tumor cells selected for high-level expression of the mhv receptor carcino-embryonic antigen cell adhesion molecule- (dbt- ) (chen et al., ; hirano et al., ; yount et al., ) and baby hamster kidney- cells expressing the mhv receptor (bhk-mhvr) (chen et al., ; yount et al., ) were grown in dulbecco's modified eagle medium (dmem) (gibco) that was supplemented with % heat-inactivated fetal calf serum (fcs) (sigma) for all experiments. medium for bhk-mhvr cells was supplemented with g ( ag/ ml) to select for cells expressing the mhv receptor. polyclonal antisera used for biochemical and immunofluorescence experiments have been previously described. these include guinea pig a-nsp antisera (gp ) ; rabbit a-nsp antisera (vu ) (sims et al., ) ; rabbit a-nsp antisera (p a- ) (bost et al., ) ; and rabbit a-mhv antisera generated against intact virions . j. fleming (university of wisconsin, madison) kindly provided murine monoclonal antibodies specific for the structural proteins nucleocapsid (a-n, j. . ) and membrane protein (a-m, j. . ). to delete portions of the nsp -coding sequence, pcr was performed using the icmhv-a fragment a plasmid (pcr-xl-topoa) as template (yount et al., ) . to generate nsp dfl, deleting nearly the entire nsp -coding sequence (nsp amino acids a through l ), a pcr product was amplified using oligodeoxynucleotide primers (sense) v-gtt taa acg aga cat aat acg- v and (antisense) v-ctt aag cat tat gca acc tat- v. to generate nsp dmid, deleting the middle portion of nsp (q through n ), two separate pcr products were amplified. the first pcr reaction used primers (sense) v-ccg ccg gcc tgg tct tgt- v and (antisense) v-gga tcc tca tct aca aa- v to amplify sequences corresponding to the vutr and the amino-terminal residues (m through p ) of nsp . the second reaction used primers (sense) v-gat ccc ggc cgt ttt ata ggc- v and (antisense) v-ctt aag aag agc ata- v to amplify sequences corresponding to the carboxy-terminal residues (g through l ). to generate nsp dc, deleting the carboxy-terminus of nsp (k through l ), a pcr product was amplified using primers (sense) v-ccg ccg gcc tgg tct tgt- v and (antisense) v-ctt aag ggg aag cac acc caa- v. all pcr products were ligated into pgem-t-easy (promega) and sequenced to ensure the fidelity of pcr. nsp sequences were then subcloned into pcr-xl-topoa in place of wild-type nsp using primer-generated restriction sites: nsp dfl ( vpmei to vaflii), nsp dmid (first ligated at the bamhi site then subcloned into pcr-xl-topoa using v sacii and vaflii), and nsp dc ( v sacii to vaflii). all fragment a plasmids were sequenced across the nsp coding region to ensure proper ligation and maintenance of the translational reading frame. the mhv-a nucleotides (nt) to (restriction sites v sacii to vaflii) were pcr amplified using pcr-xl-topoa as template and cloned into pgem-t-easy to generate pgem-nsp . charge-to-alanine substitutions were introduced into pgem-nsp using complementary pairs of mutagenic primers and the quikchange site-directed mutagenesis kit (stratagene). mutagenized pgem-nsp plasmids were sequenced to verify the incorporation of the appropriate substitutions and the fidelity of pcr. restriction enzyme sites v sacii and vaflii were then used to subclone mutant nsp sequences into pcr-xl-topoa in place of the wild-type nsp sequence. viruses containing nsp mutations were produced using the infectious clone strategy for mhv-a (icmhv) described by yount et al. ( ) and modified by denison et al. ( ) . all assembled viruses, including the wildtype control (icwt), were generated using the icmhv fragment f plasmid corresponding to the virulent vuss strain (sperry et al., ) . plasmids containing the cdna cassettes of the mhv genome were digested using mlui and bsmbi for fragment a, bgli and bsmbi for fragments b and c, ncii and bsmbi for fragments d and e, bsmbi for f, and sfii and bsmbi for fragment g. gel purified restriction fragments were ligated together using t dna ligase (new england biolabs) in a total reaction volume of al at -c overnight. following chloroform extraction and isopropanol precipitation of ligated cdna, full-length transcripts of icmhv rna were generated in vitro using the mmessage mmachine t transcription kit (ambion) according to the manufacturer's protocol with the following modifications. fifty microliter reactions were supplemented with . al of mm gtp, and transcription was performed at . -c for min, . -c for min, and . -c for min. in parallel, rna transcripts encoding the mhv nucleocapsid protein (n) were generated from n cdna. n transcripts and icmhv genomic rna were then mixed and electroporated into bhk-mhvr cells. briefly, bhk-mhvr cells were grown to sub-confluence, trypsinized, then washed twice with phosphate-buffered saline (pbs) and resuspended in pbs at cells/ml. six hundred microliters cells were then added to rna transcripts in a -mm gap electroporation cuvette, and three electrical pulses of v at af were delivered with a gene pulser ii electroporator (bio-rad). transfected cells were then seeded on a monolayer of uninfected dbt- cells in a cm flask and incubated at either -c or -c for to h. virus viability was determined by syncytia formation in the electroporated cell culture and by the capacity of the clarified transfected cell supernatant to induce syncytia in a monolayer of fresh dbt- cells. plaque purification, rt-pcr, and sequencing mutant viruses were subjected to three rounds of plaque purification, and reverse transcription (rt)-pcr was used to amplify the v end of the viral genome for sequence analysis. rna was harvested from infected dbt- cells using trizol (invitrogen) according to the manufacturer's protocol and used as template for rt-pcr. to generate viral cdna, reverse transcription was performed using superscript ii rt (invitrogen) and an antisense primer complimentary to nt - of the mhv-a genome. the nsp -coding region was then amplified using pcr with primers corresponding to nt - (sense) and nt - (antisense). the resulting amplicons were sequenced across the nsp -coding region to confirm the retention of the introduced nsp mutations and the absence of second-site mutations. viable nsp mutant viruses were analyzed for replication using single-cycle replication assays as previously described . briefly, dbt- cells (approximately cells) were infected at an moi of pfu/cell with icwt or nsp mutant viruses. virus inoculum was removed following min adsorption at -c, and cells were washed three times with pbs. cells were then incubated at -c in fresh pre-warmed dmem with % fcs, and samples of medium were collected at various times up to h post-infection (p.i.). viral titers in the medium were determined by plaque assay (hirano et al., ) . to determine viral yield, titer at h p.i. was subtracted from peak titer for each virus, and an average was calculated using data from three separate experiments. data analysis was performed using smith's statistical package-version . . a two-sample t test was performed for each virus ( p value . ). for imaging relative plaque size, confluent dbt- cells in -mm culture dishes were infected with serial dilutions of icwt, vusb , or vusb virus and then were overlaid with % agar -dmem supplemented with % fcs. at h p.i., the agar medium was removed, and the cells were fixed for min using À -c % methanol. cells were incubated in pbs for min, and then images of plaques were obtained on a nikon eclipse te -e microscope using a  objective. images were prepared using adobe photoshop . (adobe). dbt- cells (approximately  cells) were either infected at an moi of or mock-infected using dmem with % fcs. at . h p.i., the medium was replaced with fresh dmem lacking methionine and cysteine and supplemented with % fcs and actinomycin d ( ag/ml). proteins were radiolabeled from - h p.i. using aci/ml of [ s]met/ cys (translabel; icn). for pulse-chase analysis, proteins were radiolabeled from - . h p.i. and then chased for various times with dmem supplemented with % fcs and cyclohexamide ( ag/ml). cells were washed using al of pbs and then lysed in al of lysis buffer containing mm nacl, % np , . % doc, mm tris ph . . lysates were subjected to centrifugation at  g to remove cell nuclei. immunoprecipitations were performed in a final volume of al using protein a -sepharose beads (sigma), al of radiolabeled cytoplasmic lysate (derived from approximately cells) which was boiled for min in % sds, and - al of polyclonal antisera in immunoprecipitation buffer containing mm nacl, . % sds, . % tx- , mm edta, . % dtt, and mm tris ph . . immunoprecipitation reactions were incubated at -c for h with rotation. protein-bead conjugates were washed three times in immunoprecipitation buffer, and proteins were eluted from beads by boiling for min in  protein loading buffer ( mm dtt, mm tris ph . , . % bromophenol blue, % glycerol). proteins were resolved by sds-page in - % polyacrylamide gradient gels and analyzed by fluorography. the [ c] high molecular weight standard (gibco) and full-range rainbow marker (invitrogen) were used as molecular weight standards. images were prepared using adobe photoshop . (adobe). dbt- cells cultured on glass coverslips were either mock-infected or infected at an moi of at -c for min. following virus adsorption, infected medium was replaced with pre-warmed dmem with % fcs, and cells were incubated at -c. at h p.i., cells were fixed and permeablized with À -c % methanol for a minimum of min. indirect immunofluorescence assays were performed as previously described (bost et al., ) . gp was used at : dilution, and all rabbit polyclonal antisera were used at : dilution. murine monoclonal antibodies were used at : dilution. secondary antibodies conjugated to fluorophores (molecular probes) were used at : and included a-guinea pig-alexa , arabbit-alexa , and a-murine-alexa . immunofluorescence was detected using a zeiss lsm laser scanning confocal microscope with a  oil immersion objective. image analysis and merging were performed using adobe photoshop . (adobe). dbt- cells (approximately cells) were infected at an moi of with icwt or nsp mutant viruses. virus inoculum was removed following min adsorption at -c, and cells were washed three times with pbs. cells were then incubated for various times in fresh pre-warmed dmem with % fcs at -c. at min prior to addition of radiolabel, cell medium was supplemented with actinomycin d ( ag/ml). viral rna was metabolically labeled in the presence of actinomycin d using aci/ml [ h]uridine. to harvest viral rna, cells were washed twice with pbs and then lysed with al of cell lysis buffer ( mm nacl, % np , . % doc and mm tris ph . ). lysates were centrifuged at  g to remove nuclei, and rna in al of cytoplasmic extract was precipitated in replicate using % trichloroacetic acid. precipitated rna was dried onto glass microfiber filters (whatman) using vacuum filtration, and radioactivity was measured as counts per minute (cpm) using a liquid scintillation counter (beckman). statistical analysis software (smith's statistical package-version . ) was used for data analysis. for each experiment (n = ), icwt values (cpm) from the - h time intervals were set to equal %. and, nsp mutant values were calculated as percent of icwt. one-sample t tests were performed using a p value . . analysis of [ h] viral rna by gel electrophoresis was performed as described previously (kim et al., ) with a few modifications. viral rna was radiolabeled from - h p.i. as before and was isolated using trizol (invitrogen) according to the manufacturer's protocol. the amount of [ h] incorporation was quantitated using liquid scintillation. equal amounts of radiolabeled viral rna (approximately , cpm from a maximum of cells) were denatured in formamide gel loading buffer at -c for min and then electrophoresed in a . % formaldehyde/ agarose gel at v for h. for mock-infected samples, rna from cells was used. following electrophoresis, the gel was incubated in % methanol for h then in % diphenyloxazole/methanol for h prior to overnight incubation in distilled water. the gel was dried at -c using vacuum filtration before exposing it to film. images were prepared using adobe photoshop . (adobe). efficient rescue of hepatitis c virus rna replication by trans-complementation with nonstructural protein a identification of a domain required for autoproteolytic cleavage of murine coronavirus gene a polyprotein identification of the catalytic sites of a papain-like cysteine proteinase of murine coronavirus the severe acute respiratory syndrome coronavirus nsp protein is an endoribonuclease that prefers manganese as a cofactor mouse hepatitis virus strain a rna polymerase gene orf a: heterogeneity among mhv strains characterization of the leader papain-like proteinase of mhv-a : identification of a new in vitro cleavage site four proteins processed from the replicase gene polyprotein of mouse hepatitis virus colocalize in the cell periphery and adjacent to sites of virion assembly mouse hepatitis virus replicase protein complexes are translocated to sites of m protein accumulation in the ergic at late times of infection 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 an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot characterization of the expression, intracellular localization, and replication complex association of the putative mouse hepatitis virus rnadependent rna polymerase intracellular localization and protein interactions of the gene protein p during mouse hepatitis virus replication human carcinoembryonic antigen and biliary glycoprotein can serve as mouse hepatitis virus receptors murine coronavirus nonstructural protein p arrests cell cycle in g /g phase expression, purification, and characterization of sars coronavirus rna polymerase high-resolution epitope mapping of hgh -receptor interactions by alanine-scanning mutagenesis translation and processing of mouse hepatitis virus virion rna in a cell-free system identification of putative polymerase gene product in cells infected with murine coronavirus a intracellular processing of the n-terminal orf a proteins of the coronavirus mhv-a requires multiple proteolytic events identification and characterization of a -kda protein processed from the gene polyprotein of the murine coronavirus mhv-a 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 cleavage between replicase proteins p and p of mouse hepatitis virus is not required for virus replication coronavirus genome: prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis rna replication of mouse hepatitis virus takes place at doublemembrane vesicles genetic analysis of the pestivirus nonstructural coding region: defects in the ns a unit can be complemented in trans paired charge-to-alanine mutagenesis of dengue virus type ns generates mutants with temperature-sensitive, host range, and mouse attenuation phenotypes identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity targeted construction of temperaturesensitive mutations in vaccinia virus by replacing clustered charged residues with alanine mouse hepatitis virus (mhv- ). plaque assay and propagation in mouse cell line dbt cells identification of the murine coronavirus p cleavage site major genetic marker of nidoviruses encodes a replicative endoribonuclease trans-complementation of flavivirus rna polymerase gene ns by using kunjin virus replicon-expressing bhk cells coronavirus protein processing and rna synthesis is inhibited by the cysteine proteinase inhibitor e d newly discovered coronavirus as the primary cause of severe acute respiratory syndrome the complete sequence ( kilobases) of murine coronavirus gene encoding the putative proteases and rna polymerase trans-complementation of yellow fever virus ns reveals a role in early rna replication complementation analysis of the flavivirus kunjin ns and ns proteins defines the minimal regions essential for formation of a replication complex and shows a requirement of ns in cis for virus assembly identification and characterization of a serine-like proteinase of the murine coronavirus mhv-a intracellular and in vitro-translated -kda proteins contain the c-like proteinase activity of the coronavirus mhv-a molecular cloning of the gene encoding the putative polymerase of mouse hepatitis coronavirus, strain a coronavirus replication complex formation utilizes components of cellular autophagy identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins processing of the coronavirus mhv-jhm polymerase polyprotein: identification of precursors and proteolytic products spanning kilodaltons of orf a guanosine triphosphatase activity of the human coronavirus helicase the human coronavirus e superfamily helicase has rna and dna duplexunwinding activities with v-to- v polarity colocalization and membrane association of murine hepatitis virus gene products and de novo-synthesized viral rna in infected cells mouse hepatitis virus replicase proteins associate with two distinct populations of intracellular membranes unique and conserved features of genome and proteome of sarscoronavirus, an early split-off from the coronavirus group lineage single-amino-acid substitutions in open reading frame (orf) b-nsp and orf a proteins of the coronavirus mouse hepatitis virus are attenuating in mice clustered charge-toalanine mutagenesis of human respiratory syncytial virus l polymerase generates temperature-sensitive viruses mechanisms and enzymes involved in sars coronavirus genome expression localization of mouse hepatitis virus nonstructural proteins and rna synthesis indicates a role for late endosomes in viral replication systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a the coronavirus replicase we express our appreciation to xiao tao lu key: cord- - nfqusv authors: molenkamp, richard; spaan, willy j.m. title: identification of a specific interaction between the coronavirus mouse hepatitis virus a nucleocapsid protein and packaging signal date: - - journal: virology doi: . /viro. . sha: doc_id: cord_uid: nfqusv abstract the coronavirus mouse hepatitis virus (mhv) is an enveloped positive stranded rna virus. in infected cells mhv produces a ′ coterminal nested set of subgenomic messenger rnas. only the genomic rna, however, is encapsidated by the nucleocapsid protein and incorporated in infectious mhv virions. it is believed that an rna packaging signal (ps), present only in the genomic rna, is responsible for this selectivity. earlier studies mapped this signal to a -nt stem–loop structure positioned in the ′ end of orf b. the selective encapsidation mechanism probably initiates by specific interaction of the packaging signal with the nucleocapsid protein. in this study we demonstrate thein vitrointeraction of the mhv-a nucleocapsid protein with the packaging signal of mhv using gel retardation and uv cross-linking assays. this interaction was observed not only with the nucleocapsid protein from infected cells but also with that from purified virions and from cells expressing a recombinant nucleocapsid protein. the specificity of the interaction was demonstrated by competition experiments with nonlabeled ps containing rnas, trna, and total cytoplasmic rna. the results indicated that no virus specific modification of the n-protein or the presence of other viral proteins are required for thisin vitrointeraction. the assays described in this report provide us with a powerful tool for studying encapsidation (initiation) in more detail. gion a domain (from here on called ps) of nt could be identified that is probably required for the encapsidation the murine coronavirus mouse hepatitis virus (mhv) of defective genomes (fosmire et al., ) . this signal is an enveloped virus containing a positive stranded rna is present in genomic rna, but not in sgrnas and it is genome of about kb (holmes, ; likely that it has also a similar function in the encapsida- ). the virion envelope is composed of a lipid bilayer tion of genomic rna. recently, it was demonstrated that derived from an internal compartment of the host cell the encapsidation of a heterologous rna by mhv was and three or four virus-encoded structural membrane fully dependent on the presence of this ps (woo et al., proteins (luytjes, ) : the spike protein (s), the mem- ). furthermore, it was shown by bos et al. ( ) that brane protein (m), the small membrane protein (e), and transferring the ps to a sgrna resulted in the specific the optional hemagglutinin-esterase protein (he). the viencapsidation of this sgrna, though with reduced effiral envelope surrounds a nucleocapsid with helical symciency. metry composed of the genomic rna and multiple copies the nucleocapsid protein of mhv is a basic phosphoof the nucleocapsid protein (n). evidence for the presprotein of amino acids and has an apparent molecuence of a fifth structural envelope protein translated from lar weight of approximately kda (armstrong et al. , an internal open reading frame (orf) within the n gene ; parker and masters, ; laude and masters, has been published recently (fischer et al., (fischer et al., ). . it is phosphorylated exclusively on serine residues in infected cells mhv produces a coterminal nested (stohlman and lai, ) . the n protein contains of set of subgenomic mrnas (sgrnas) which possess an these potential phosphorylation sites, but the exact numidentical leader sequence derived from the end of ber and location of phosphoserines have not been identithe genome (lai et al., ; spaan et al., spaan et al., , . fied yet. the basic amino acids are not clustered in only genomic length rna is packaged into virus partistrings, but local densities of positive charge can be cles; however, trace amounts of sg rnas are sometimes found, particularly in two regions in the middle of the n detected in purified virus (makino et al., ) . earlier protein (laude and masters, ) . in contrast, the cstudies (fosmire et al., ; most et al., ) have terminus is quite acidic. the mhv n protein does not mapped a region in the end of orf b that is essential contain known rna binding motifs, like the arginine-rich for encapsidation of defective genomes. within this remotif (arm) or zinc fingers (draper, ; holmes and behnke, ; burd and dreyfuss, ) . leader rna has been reported although there is some analysis. all enzyme incubations and biochemical reactions were performed according to the instructions of the discrepancy about the specificity of this interaction (stohlman et al., ; bredenbeek, ) . a leader-rna manufacturers. binding domain in the n protein was mapped and com-construction of plasmids prises the two basic regions mentioned above (nelson and stohlman, ; masters, ) . furthermore, spe-(i) pps . a -nt fragment containing ps was obcific interaction of the coronavirus infectious bronchitis tained by polymerase chain reaction (pcr) using pmidivirus (ibv) nucleocapsid protein with the terminus of c as a template (most et al., ) and oligonucleotide the genome has recently been reported (zhou et al., primers c and c (table ) . to obtain pps this ). fragment was cloned in pcrii using the ta cloning kit the -nt ps is able to form a stable secondary struc-(invitrogen) according to the instructions of the manufacture and the integrity of this structure is essential for turer. the encapsidation of defective genomes (fosmire et al., (ii) pemcv-n. a ncoi restriction site was created at ). it has been postulated (fosmire et al., ) that the position of the aug start codon of the n gene by the ps functions as an encapsidation initiation site, prob-pcr mutagenesis using oligonucleotide primers c ably by interacting specifically with the n protein. initiaand c (table ). the pcr fragment was digested with tion of encapsidation by packaging signal/(nucleo)capsid ncoi and apai and fused to the remaining sequences protein interactions has been observed for several other of the n gene. a consequence of this procedure was rna viruses, including alphaviruses, retroviruses, and that the second amino acid of the n protein was changed escherichia coli bacteriophages. (owen and kuhn, ; from a ser to an ala. the reconstituted n gene was berkowitz et al., ; zhang and barklis, ; schle- then exchanged with the ncoi-bamhi fragment of pl a singer et dupraz and spahr, ; witherell et (snijder et al., ) and the sequence of the ncoi-apai al., ; aldovini and young, ; weis et al., ) . fragment was confirmed by sequence analysis. the final however, a specific interaction of the mhv n protein expression vector, pemcv-n, contained a t promoter with the ps has not been demonstrated yet. and emcv-ntr, followed by the entire n gene and a t in this report we have used gel retardation and uv terminator sequence. cross-linking assays to study the in vitro interaction of mhv-a nucleocapsid protein and a small rna con-preparation of riboprobe taining the ps domain. we observed specific interaction in order to serve as template dna, pps was linearbetween in vitro transcripts containing the ps and n proized with bamhi, extracted with phenol/chloroform, and tein isolated from infected cells, but also with n protein precipitated with ethanol. alternatively, templates for the extracted from purified viruses. furthermore, we were production of ps and psdhp rna were produced by able to identify a similar interaction with recombinant pcr using a oligonucleotide containing the t pronucleocapsid protein expressed in the vaccinia t exmoter sequence in addition to mhv specific sequences pression system. these experiments underline the possi- (table ) . radiolabeled rna was synthesized by t tranbility of studying the encapsidation of mhv-a rna at scription for . h at Њ. reactions contained mg of a molecular basis and allow us to map important dolinearized plasmid dna, mm (each) atp, ctp, and mains in both the nucleocapsid protein as well as the gtp, mm utp, mci of [a- p]utp ( ci/mmol), rna packaging signal. and units of t polymerase in a final volume of ml transcription buffer (gibco brl). the reaction products materials and methods were extracted with phenol/chloroform, purified on a sephadex g collumn and precipitated by adding / cells and viruses volume of m nh ac (ph . ) and vol of ethanol. the mouse l cells were grown in dulbecco's modified eaamount of incorporated label was determined by tca gle's medium (dmem; gibco) supplemented with % fetal precipitation. calf serum. mhv-a stocks were grown as described (spaan et al., ) phenylmethylsulfonyl fluoride, . mm dithiothreitol ml binding buffer ( mm hepes (ph . ), mm kac, . mm mgac , . mm edta, . mm dtt, mm atp, (dtt)) (dignam et al., ) . subsequently, the cells were disrupted by freezing ( Њ) and thawing ( Њ) once or . mm gtp). where necessary specific or nonspecific competitor rna was added as indicated in the figure by strokes of a dounce homogenizer. cellular debris and nuclei were removed by centrifugation for min at legends. in supershift experiments, ml of the n-specific monoclonal antibody b . (talbot and buchmeier, , rpm at Њ. the supernatant was aliquoted and stored at Њ. the protein concentration was deter- ) or b-galactosidase monoclonal antibody (boehringer mannheim) and units of rnaguard (pharmacia) mined using the bicinchoninic acid protein assay kit (sigma). were added. the mixtures were incubated at room temperature for min, after which ml of % glycerol (ii) protein lysates from purified viruses. virus was purified as described before (luytjes et al., ) . briefly, was added to each reaction. the mixtures were then separated by electrophoresis on a % polyacrylamide/ viruses harvested from cells were precipitated with polyethylene glycol and loaded on top of a to % % glycerol gel (mono:bis Å . : ) in . tbe ( mm tris-cl (ph . )), mm boric acid, mm edta) for linear sucrose gradient. the gradient was centrifuged for h at , rpm at Њ in a sw ti rotor. subsequently h at ma (fixed). subsequently, the gel was dried and exposed to x-ray film with an intensifying screen at Њ. the gradient was fractioned into fractions. all fractions were assayed for viral proteins by western blot using uv cross-linking the rabbit polyclonal mhv-a antiserum k . the two fractions corresponding to the virus peak and two bottom rna binding reactions were performed as described fractions (control) were combined, and their volumes above. subsequently the samples were irradiated at a were adjusted to ml with tesv ( mm tris (ph . ), -cm distance with . j/cm of -nm uv light in a mm edta, mm nacl). virus was pelleted by centrif-stratalinker (stratagene). the complexes were then ugation in a sw ti rotor for h at , rpm at Њ. incubated with a mixture of ng rnase a (pharmacia) the resulting virus pellet was lysed in ml buffer c and . units rnase t (gibco brl) for min at Њ. the supplemented with . % np- . complexes were analyzed by sodium dodecyl sulfate-(iii) recombinant n protein lysates. vaccinia virus inpolyacrylamide gel electrophoresis on % gels. fections and dna transfections were performed as de-alternatively, cross-linked and rnase treated samples scribed previously . briefly, rk cells were immunoprecipitated prior to electrophoresis as dewere grown to subconfluency in -cm-diameter petri scribed earlier with monoclonal antidishes ( cells per dish) and infected with the t body b . (talbot and buchmeier, ) or rabbit rna polymerase expressing vaccinia virus recombinant polyclonal mhv-a antiserum k . (vtf . ) at a m.o.i. of . at h postinfection the cells were transfected with ml lipofectin (gibco brl) containing results mg of pemcv-n. after an incubation of h protein ly-ps rna binds specifically to proteins from mhvsates were made as described above. infected and mock-infected cells gel mobility shift assays in the studies described here we have analyzed the interaction between the mhv-a n protein and rnas rna binding reactions contained ng of radiolabeled riboprobe and mg of protein lysate in a final volume of containing the -nt ps signal. in order to identify this ). these observations indicate that there is a specific interaction between ps rna and proteins from mhvinfected cells as well as with proteins from mock-infected cells. the mhv-a nucleocapsid protein interacts specifically with ps rna to investigate whether the n protein is part of the protein-ps rna complex, a supershift assay using a n-specific antibody was performed. if the n protein is indeed part of the complex, binding to n of a n-specific antibody should result in the formation of a large complex composed of ps rna, n protein, and n-specific antibody. this complex is expected to migrate slower in the gel as compared to the protein-ps rna complex and meier, ) was used to analyze the protein-ps rna complex and the monoclonal b-galactosidase-specific antibody was used as a control. when n-specific antiinteraction we have first used gel mobility shift assays body b . was added to the complex formed between using protein lysates from both mhv-infected and mock-ps rna and proteins from the i-lysate, a second cominfected cells and an in vitro transcript containing the ps plex, migrating slightly slower than the protein-ps (fig. ) . from pilot experiments using ng of labeled rna complex was readily observed (fig. , lane ) . in rna we determined that approximately mg of protein contrast, addition of b . to the complex formed belysate was required to observe a distinct retarded band tween ps rna and proteins from the mi-lysate did (data not shown). we therefore used these amounts as not result in a supershift (fig. , lane ) . this demonstandard in all binding experiments. strates the presence of the mhv-a n protein in the an interaction between ps rna and proteins from complex formed between ps rna and proteins from infected cells (i-lysate), as well as from mock-infected the i-lysate. cells (mi-lysate) was readily observed (fig. , lanes the b-galactosidase-specific antibody was not able to and ). however, the complex formed between ps shift the complex formed between ps rna and pro-rna and proteins from the mi-lysate migrated slightly teins from the i-lysate or from the mi-lysate (fig. , lanes slower in the gel and appeared to be more diffuse, indi- and ). furthermore, incubation of ps rna and cating that there are some differences in binding activity antibody b . or b-galactosidase-specific antibody between i-and mi-lysates. without protein lysate, did not result in the formation of in order to determine the specificity of the interaction, competition experiments were performed. competition of the interaction between ps rna and proteins from the i-lysate with a -fold molar excess of nonlabeled ps rna resulted already in a decrease of intensity of the retarded band (fig. , lane ) . at a level of fold molar excess of nonlabeled ps rna the retarded band was no longer visible and all of the labeled rna migrated at the position of the unbound rna probe (fig. , lane ) . competition with a -fold molar excess of yeast trna did not affect the intensity or the mobility of the retarded band, indicating that the observed interaction between ps rna and proteins from the i-lysate is specific for the ps rna (fig. , lane ) . competition of the interaction between ps rna and proteins from the mi-lysate with non-labeled ps rna or yeast trna beled ps rna competed entirely for protein binding formed using a -to -fold molar excess of unlabeled ps rna or (fig. , lane ) , whereas a -fold molar excess of a -to -fold molar excess of yeast trna. the positions of the unbound rna (probe) and complexed rna (complex) are indicated. yeast trna had no effect on the interaction (fig. , lane tor rna. cross-linked and rnase treated samples were immunoprecipitated with the monoclonal n-specific antibody b . or the rabbit polyclonal mhv-a antiserum k as indicated in the figure legends. an interaction between the n protein and ps rna was readily observed (fig. , lane ) , but no interaction was observed between the n-protein and psdhp rna (fig. , lane ) . complexes formed between proteins from the mi-lysate and ps rna or psdhp rna could not be immunoprecipitated with the n-specific antibody (fig. , lanes and ) . when the ps and psdhp rnas were used in a competitive gelretardation experiment with radiolabeled ps and the i-lysate, complete competition was observed with the ps rna, whereas no immunoprecipitation and that this interaction is dependent on the presence of the -nt ps hairpin. to determine the specificity of the nucleocapsid pro-rna-protein complexes (fig. , lanes - and ) , tein-ps rna interaction in uv cross-linking assays, clearly indicating that the observed supershift was not competition experiments were performed. addition of a the result of an interaction between the rna probe and -fold molar excess of nonlabeled ps rna resulted the n-specific antibody. in the complete inhibition of n protein binding (fig. , addition of a -fold molar excess of nonlabeled lane ), whereas, a -fold molar excess of trna had ps competitor rna resulted in the complete inhibition no effect on the interaction between the n protein and of protein binding to the rna probe (fig. , lane ) . in ps rna (fig. , lane ) . this clearly demonstrates the contrast, a -fold molar excess of trna did not affect specificity of the interaction between the n protein and the formation of rna-protein and rna-protein-anti-ps rna. body complexes (fig. , lane ) , which demonstrates the specificity of the interaction between the n protein and ps rna. nucleocapsid protein from purified viruses and recombinant expressed nucleocapsid protein interact specifically with ps rna uv cross-linking demonstrates specific nucleocapsid protein ps -rna interaction during rna encapsidation or particle formation, the n protein might undergo structural changes or other modifi-another assay for studying the interaction between the cations. it would therefore be interesting to see if the n n protein and ps containing rnas is uv cross-linking. protein incorporated in virus particles is still able to inter-to exclude the possibility that the non-mhv-specific sequences present in ps rna are involved in the interaction between the nucleocapsid-protein and ps rna, an rna probe (ps ) containing only mhv-specific sequences was constructed. furthermore, in order to investigate whether the -nt hairpin of ps (and ps ) rna is responsible for specific nucleocapsid protein binding, psdhp rna was constructed. this rna is identical to ps rna except that the largest part of the -nt ps hairpin has been removed. the u contents of ps and psdhp are similar (approx. %). pilot experiments were performed to estimate the optimal uv dose. from these experiments it was determined that an uv dose of . j/cm gave the most distinct bands with the least background (data not shown). from uninfected cells was added as nonspecific competi-observed using the vtf -lysate (fig. c, lane ) . these experiments clearly demonstrate that no virus specific modifications of the n protein or any other viral proteins are required for the specific interaction with ps . initiation of encapsidation by a specific interaction between the (nucleo-) capsid protein and a rna packaging signal is a common mechanism used by positive stranded rna viruses. such an interaction has already weis et al., ) , retroviruses (zhang were immunoprecipitated with n-specific monoclonal antibody and barklis, ; berkowitz et al., ; dupraz and b . . competition was performed with unlabeled ps rna or yeast trna as indicated above the lanes. the position of the n-protein spahr, ; aldovini and young, ) but also in e. coli specific band is indicated. bacteriophages like r and ms (witherell et al., ) . in this report we describe the specific in vitro interaction of the mouse hepatitis virus a n protein with tranact specifically with ps . in order to address this quesscripts containing the rna packaging signal of mhv. tion, virus particles were purified on a sucrose gradient. this interaction was studied by gel retardation and uv lysates were prepared from the virus peak fractions (pcross-linking assays using an rna probe containing the lysate) and from the gradient bottom fractions (b-lysate; -nt ps and the n protein from mhv-a infected cell negative control). uv cross-linking was performed using lysates. a similar interaction was identified between the both lysates, radiolabeled ps rna, and competitor n protein from purified virus and recombinant n-protein rna ( mg of total cytoplasmic rna extracted from uninexpressed by the vtf expression system. fected cells; fig. b ). a specific interaction was observed in addition to the n-ps rna complex we also observed between the n-protein from the p-lysate and ps rna that cellular proteins bind to ps containing rnas. the (fig. b, lanes and ) . in contrast, no complex could nature of these cellular proteins and the significance of be immunoprecipitated with n-specific antibodies when this interaction, however, remains unclear. the b-lysate was used (fig. b, lanes and ) . this indicates that any structural change of the n protein which might occur during virus assembly does not affect the ability of the n protein to interact with ps rna. in mhv-infected cells, the n protein might also undergo virus-specific or virus-dependent posttranslational modifications. these modifications might play a role in the specific interaction with the ps. furthermore, it is not yet known if the interaction between the n protein and the ps involves other viral proteins as well. in order to investigate this, recombinant n protein was expressed by the vtf . expression system. a n protein expression vector (pemcv-n) was transfected in vtf . -infected rk cells and protein lysates were made h posttransfection (vtf pn lysate). as a control protein lysates from mock-transfected but vtf . -infected rk cells were made (vtf -lysate). uv cross-linking experiments, followed by immunoprecipitation with n-specific anti- fig. c ). using the vtf pn lysate, a clear band was fraction lysate and p denotes the peak-fraction lysate. immunoprecipitaobserved migrating at the expected position of the nution with b . and k were performed as indicated above the lanes. the position of the n-protein-specific band is indicated. cleocapsid protein (fig. c, lane ) . this band was not the nature of the nucleocapsid protein-ps rna cells (woo et al., ) . although it is not known whether this encapsidation was efficient, it might be possible that interaction the heterologous flanking sequences have increased the the n-protein of mhv does not posess any well known flexibility of the rna molecule, allowing it to adapt the rna binding domains, such as the arm (lazinski et al., proper structure for encapsidation. ; talbot and buchmeier, ) or zinc fingers (draper, ) . it interacts, although, specifically with significance of the nucleocapsid protein-ps rna leader-rna (stohlman et al., ; baric et al., ) and interaction an rna binding domain has been identified (nelson and stohlman, ) . this domain comprises the two major since mhv virions contain only mhv genomic rna, it hydrophobic basic regions in the middle of the protein. is evident that rna encapsidation is a highly specific since this central part of the n protein contains a high process. the location of the encapsidation signal in degree of basic amino acids, it is possible that the ps orf b ensures the specific encapsidation of only genorecognition domain might also be positioned somewhere mic rna. it is unclear if the mhv ps is by itself sufficient within this part of the n protein. for efficient encapsidation. it is known that rous sarcoma in infected cells the n protein of mhv is very rapidly virus has multiple encapsidation sites, which are rephosphorylated on serine residues (stohlman and lai, quired to interact for efficient packaging of the genome ) and part of it concomitantly becomes associated (sorge et al., ; pugatsch and stacey, ) . recently, with a cell membrane fraction (stohlman et al., ; it was shown that a subgenomic rna of mhv containing anderson and wong, ) . it is still unknown if phosthe ps can be encapsidated specifically but the efficiency phorylation is carried out by a host cell or viral encoded was much lower as compared to the encapsidation effiprotein kinase and its (biological) role remains unclear. ciency of a defective interfering rna (bos et al., ) . it has been suggested that it might govern the tightness preassembled nucleocapsids have never been obof the interaction between n and rna (laude and masserved in coronavirus infected cells, but an electronters, ). it will be interesting to compare the phosphordense structure, which may correspond to the nucleoylation of the n protein from infected cells and the recomcapsid, can be found at the cytoplasmic face of the binant n protein and to investigate the possible role of budding site (dubois-dalcq et al., ; holmes, ) . phosphorylation in rna binding. phosphorylation and nucleocapsid incorporation in budding virions is exlikewise dephosphorylation could also induce a major pected to be mediated by m-n protein interactions. asconformational change of the n protein. it has been sugsociation of the m protein to the nucleocapsid in np- gested that dephosphorylation of the nucleocapsid after disrupted virions has been reported (sturman et al., virus entry is involved in uncoating of the viral rna (ma- ) ; however, the same study demonstrated that the hondas and dales, ) . m protein was able to bind rna in the absence of n. it in general the recognition of an rna signal by a protein is still unclear if the interaction between m and n is a involves secondary structure elements in addition to the prerequisite for rna encapsidation in vivo. this interacprimary sequence of the rna signal (draper, ) . tion could position the n protein in a favorable way to there is accumulating evidence for an induced-fit mechinteract with the genomic rna. this must then be folanism in rna-protein interactions affecting both the lowed by interaction with additional n proteins, forming rna and the protein (beck and nassal, ; allain et the helical nucleocapsid. involvement of other viral proal., ) . sufficient flexibility in the rna molecule could teins in ps rna binding, however, was not observed in be a major determinant in protein binding. from that our in vitro studies. recombinant nucleocapsid protein, perspective, the recognition of the ps by the n protein expressed in the vtf expression system, interacts also could be highly dependent on the secondary structure specifically with the ps rna (fig. c) , although the effiof the ps domain. when a short rna probe consisting ciency of this interaction was not determined. a striking of only the -nt ps was used in our studies, no gel feature of coronaviruses is that the nucleocapsid has a retardation or uv cross-linking to the n protein was obhelical symmetry (macnaughton et al., ; holmes and served (unpublished observations). computer prediction behnke, ) , in contrast to the nucleocapsids of all of the secondary structure of this small -nt rna moleother positive stranded rna viruses, which are icosaecule showed that the secondary structure was entirely dral or spherical (murphy et al., ) . however, electron different from that of the ps in the orf b context. this microscopy studies on the transmissible gastroenteritis suggests that the flanking orf b sequences are necescoronavirus and mhv (risco et al., ) have revealed sary to force the ps in its specific structure or that the recently a spherical core structure inside the virion (intersmall rna probe lacks the flexibility to form the specific nal core) and this structure reacted with m-and n-spestructure. recently, it has been shown that the -nt ps, cific antibodies. a structural model for coronaviruses was flanked by non-mhv sequences could confer specific proposed, in which a spherical core, composed of a combination of n and m proteins, was present in addition to encapsidation to a heterologous rna in mhv infected interaction between nucleocapsid protein and packaging signal of a murine coronavirus in the absence of helper virus. virology , a helical nucleocapsid. it will be of interest to study the - . possible relationship between the morphology of the nu-bos, e. c. w., dobbe, j., luytjes, w., and spaan, w. j. m. ( ) . a subcleocapsid or internal core and the mechanism of encapgenomic mrna transcript of the coronavirus mouse hepatitis virus sidation initiation. strain a defective interfering (di) rna is packaged when it contains it has been shown that, as opposed to alphavirus asthe di packaging signal. j. virol. , - . bredenbeek, p. j. ( ) . ''nucleic acid domains and proteins involved sembly, nucleocapsid formation and rna encapsidation in the replication of coronaviruses.'' ph.d. thesis, university of are not strictly required for virion formation (vennema et utrecht, utrecht, the netherlands. al., ; strauss and strauss, ) . burd, c. g., and dreyfuss, g. ( ) . conserved structures and diversity coexpression of m and e protein was sufficient for partiof functions of rna-binding proteins. science , - . cle formation. however, incorporation of nucleocapsids dignam, j. d., lebowitz, r. m., and roeder, r. g. ( ) . acurate transcription initiation by rna polymerase ii in a soluble extract from during the budding process could greatly increase the isolated mammalian nuclei. nucleic acids res. , res. , - efficiency of virion formation. draper, d. e. ( ) . protein-rna recognition. annu. rev. biochem. , the study of the encapsidation of coronaviruses is - . greatly hampered by the absence of an infectious clone. dubois-dalcq, m. e., doller, e. w., haspel, m. v., and holmes, k. v. the in vitro binding assay, described in this report, can ( ) . cell tropism and expression of mouse hepatitis viruses (mhv) greatly enhance our understanding of the encapsidation in mouse spinal cord cultures. virology , - . dupraz, p., and spahr, p. f. ( ) . specificity of rous sarcoma virus mechanism. we have shown that recombinant nucleonucleocapsid protein in genomic rna packaging. j. virol. , capsid protein expressed in the vtf expression system . is able to interact with the ps in a similar fashion as the fischer, f., peng, d., hingley, s. t., weiss, s. r., and masters, p. s. nucleocapsid protein produced in infected cells. muta- ( ) . the internal open reading frame within the nucleocapsid gene tional analysis of both the nucleocapsid protein and the of mouse hepatitis virus encodes a structural protein that is not essential for viral replication. j. virol. , - . ps should provide us with information that allows us to fosmire, j. a., hwang, k., and makino, s. ( ) . identification and understand the encapsidation mechanism in more detail. characterization of a coronavirus packaging signal. j. virol. , - . holmes, k. v. ( ) . in ''fundamental virology'' (b. n. fields and d. m. sequence specific sequences involved in human immunodeficiency virus type packagrecognition of rna hairpins by bacteriophage antiterminators reing result in production of noninfectious virus -characterisation of two temperature-sensitive mutants of coronavi- ribo- . nucleoprotein-like structures from coronavirus particles sequence of endosomal association of a acids res cascoronavirus nucleocapsid protein nucleocapsid protein and viral rnas: implications for viral transcrip-primary structure and translation of a defective interfering rna of tion localization of an rna-binding domain in the minantsin the interaction between the rna encapsidation signal and nucleocapsid protein of the coronavirus mouse hepatitis virus. arch. reverse transcriptase of avian hepatitis b viruses retrovimain at the end of the polymerase gene is essential for encapsidaral nucleocapsid domains mediate the specific recognition of genotion of coronavirus defective interfering rnas virus taxonomy, classification and nomenclature of viruses binding domain of mouse hepatitis virus nucleocapsid protein. j. synthesis and subcellular localization of the murine coronavirus nu-gen and sindbis virus nucleocapsid protein that is involved in specificity of deans phosphoproteins of murine n genes of five strains of the coronavirus mouse hepatitis virus hepatitis viruses relikely to be required for avian retroviral packaging the navirus envelope glycoproteins and interaction with the viral nucleotransmissible gastroenteritis coronavirus contains a spherical core capsid antigenic variation among molecular cloning: murine coronaviruses: evidence for polymorphism on the peplomer a laboratory manual nucleocapsidelements and trans-acting proteins involved in the assembly of rna independent assembly of coronavirus-like particles by co-expression viruses the arterivirus nsp protease is the protofor specificity in the encapsidation of sindbis virus rnas cis-acting rna packaging action between rna phage coat proteins and rna. prog. nucleic locus in the -nucleotide direct repeat of rous sarcoma virus nucleocapsid protein effects on the - . specificity of retrovirus rna encapsidation the infectious bronchitis virus nucleocapsid protein binds navirus mrna synthesis involves fusion of non-contiguous se-rna sequences in the terminus of the genome key: cord- -ipfs hcb authors: mathieu, patricia a.; gómez, karina a.; coutelier, jean-paul; retegui, lilia a. title: sequence similarity and structural homologies are involved in the autoimmune response elicited by mouse hepatitis virus a date: - - journal: j autoimmun doi: . /j.jaut. . . sha: doc_id: cord_uid: ipfs hcb the features of autoantibodies (autoab) to liver fumarylacetoacetate hydrolase (fah) elicited in mice infected with mouse hepatitis virus (mhv) were studied by elisa and western-blot competition assays. all sera tested contained ab to cryptic fah epitopes according with results from western-blot tests, whereas elisa data indicated that some of these same sera did recognize native epitopes of the autoantigen (autoag). such differences were detected in individual sera from various mouse strains, and were ascribed to the fact that proteins insolubilized on solid supports expose a variety of conformational and cryptic antigenic determinants. on the other hand, whereas results from both experimental protocols showed that anti-mhv ab did not cross-react with the soluble autoag, the opposite situation did not show analogous results. thus, binding of autoab to insolubilized fah could be inhibited by mhv depending on the mouse serum or the experimental protocol used. additionally, a set of synthetic homologous peptides from mouse fah and various viral proteins was employed to analyze the ab repertoire of mhv-infected mice. results indicated that two homologous peptides were recognized by most ab: the n-terminal sequences ( – ) from fah and the nucleocapside, both sharing % of identity, and sequence – of the rna polymerase, a peptide showing % of identity with fah – . results indicated that mhv-infection triggers at least three distinct ab populations: anti-mhv, anti-fah and cross-reacting ab. this cross-reaction implies either sequential or conformational epitopes from both the viral proteins and the autoag and may differ between individuals. viruses have been implicated in the generation of autoimmune disorders over the last years [ e ]. it has been proposed that these infectious agents trigger an autoimmune humoral response by diverse mechanisms, including polyclonal b-lymphocyte activation, release of sequestered autoantigens (autoag), antigenic mimicry, modification of self-antigen, epitope spreading of the anti-viral immune response or enhancement of major histocompatibility complex molecule expression [ , , ] . mouse hepatitis viruses (mhv) are known to be lymphotropic and, depending on the viral strain and also on the mouse genetic background, they induce diverse alterations of the immunologic system [ e ]. the mhv strain a (mhv-a ) is a coronavirus that triggers various pathologies in susceptible mice, including hepatitis and thymus involution, igg a-restricted hypergammaglobulinaemia and transient demyelination [ , ] . in a previous paper we reported the presence of autoantibodies (autoab) in sera from various mouse strains after mhv-infection [ ] . the autoab were directed to a -kda protein present in mouse liver and kidney. the autoag was isolated and identified as fumarylacetoacetate hydrolase (fah), a soluble cytosolic enzyme that mediates the hydrolytic formation of fumarate and acetoacetate [ ] . furthermore, during fah purification we found that the autoab detected more weakly another liver protein, which turned to be the enzyme alcohol dehydrogenase (adh). there was no correlation between the igg titers and the presence of autoab to liver fah in cba/ht mice, suggesting that the autoab production was not related to the non-specific polyclonal activation of b-lymphocytes produced after viral infection. additionally, mice immunized with extracts of mouse liver did not develop autoab, thus discarding the release of a self-antigen as the mechanism involved in the autoimmune response elicited by mhv-infection [ ] . since molecular mimicry of viral antigens with self determinants seemed to be the mechanism involved in the mhv-induction of autoab to liver fah, we explored the putative cross-reaction between the enzyme and mhv proteins. elisa and western-blot competition assays, as well as ab reactivity with synthetic peptides from both fah and viral proteins, indicated that the autoab recognized a wide range of cryptic and conformational epitopes of the antigen and that the cross-reaction showed by the anti-mhv ab could be different between individuals. female cba/ht and balb/c mice were bred in isolators at the ludwig institute for cancer research (brussels branch) by dr g. warnier and used for experiments at the age of e weeks. their microbiological status was described previously [ ] . mice were inoculated intraperitoneally with % tissue culture infectious doses (tcid ) of mhv a , grown in nctc cells [ ] . efficiency of mhv infection was checked by testing anti-viral ab by elisa [ ] . the enzyme was prepared as previously described [ ] . briefly, livers from -day-old wistar rats were homogenized in vol (v/w) of chilled . m sucrose, mm tris/hcl buffer containing . mm cacl , u/ml of trypsin inhibitor and mm pmsf, ph . . after centrifugation at , g for min and then at , g for h, ethyl alcohol was added to the supernatant as to obtain a final concentration of % ethanol. this mixture was allowed to stand overnight at (c and then centrifuged at , g for min. the supernatant was mixed with % ethyl alcohol to yield a final alcohol concentration of %. after incubating overnight at (c the precipitated enzyme was packed by centrifugation at , g for min. the pellet was resuspended in mm phosphate buffer ph . and stirred for min. the supernatant fluid was recovered after centrifugation of the suspension at , g for min and solid ammonium sulfate was added so as to obtain a final salt concentration of %. after h at (c and centrifugation at , g for min the pellet was resuspended in mm tris/hcl ph . and dialyzed against the same buffer. an aliquot of this solution was chromatographied in a monoq hr / column (pharmacia lkb biotechnology) equilibrated with mm tris/hcl ph . . proteins were eluted at a flow rate of . ml/min by using a continuous e . m nacl gradient. effluent fractions containing the enzyme were concentrated and then applied to a sephadex g- column ( . ! cm) equilibrated with mm tris/hcl, . m nacl ph . . elution was carried out at a flow rate of ml/h with the buffer cited before and the protein concentration in the effluent fractions was determined at nm. fractions were analysed by western-blot as indicated before [ ] . the nctc adherent cell line derived from normal mouse liver was purchased from the american type culture collection. cells growing in t- bottles were inoculated with mhv a virus at a multiplicity of to tcid /cell. after an adsorption period of h at (c, ml of nctc medium with % fetal calf serum was added to each bottle and incubated at (c. several cycles of freezing and thawing were used to release the virus h after inoculation. the harvested virus was centrifuged at g for min to remove debris and the supernatant was frozen at ÿ (c for storage (mhv stock). the same procedure, but without viral inoculation, was carried out to prepare a control cellular stock (nctc stock). virus titration by endpoint method was performed by inoculating serial dilutions of the mhv stock onto cell monolayers in -multiwell. after h wells with viral cytopathic effect were counted for each dilution and titer was expressed as % tissue infectious doses (tcid ). before using in western-blot and elisa assays the virus was inactivated by incubating the mhv stock h at (c [ ] . protein concentration in both mhv and nctc stocks was determined by lowry et al. [ ] . viral and cellular stocks ( e mg) or purified fah ( e mg) were subjected to % sds-page [ ] and then transferred onto nitrocellulose sheets (amersham, buckingghamshire, uk). after reversible staining with ponceau s to check satisfactory transfer, non-specific ab-binding sites were blocked by incubating the sheets with % non-fat milk in mm tris, . m nacl, . % (v/v) tween , ph . (tbs-m-t) for h at room temperature with shaking. the strips were then incubated overnight at (c with an ab dilution in tbs-m-t. after several washings with tbs containing . % tween , bound ab were revealed with peroxidaselabeled donkey anti-mouse igg diluted : in tbs-m-t (jackson immunoresearch laboratories, inc, west grove, pa, usa) and ecl reagents (amersham, buckingghamshire, uk). the apparent molecular mass (kda) of the detected bands was determined using a wide range protein standard (bdh laboratory supplies poole bh td, uk). to perform competition experiments the diluted ab was incubated overnight at (c with the strips in the presence of different concentrations of competitors, i.e., mhv or nctc stocks, or the purified fah. the intensity of the bands was quantified by densitometric scan of the autoradiograms and the results expressed as percent of control, i.e. band intensity in the absence of competitor. elisa microplates (nunc maxi-sorb) were coated with ml of purified fah ( mg/ml) in . m nahco , ph . , or mhv stock ( mg/ml) in mm glycine, mm nacl, ph . . after overnight incubation at room temperature, the plates were washed with phosphate buffer saline (pbs) containing . % tween (pbs-t) and blocked for h at (c with . m tris, . m nacl, ph . (tms) containing % fetal calf serum (tms-fcs). the plates were then incubated h at (c with the ab diluted in tms-fcs and different concentrations of the competitors. after washing with pbs-t, the bound ab were revealed with peroxidase-labeled donkey igg anti-mouse igg (jackson immunoresearch laboratories, inc., west grove, pa) diluted : in tms-fcs. as a substrate, orthophenylene-diamine-dihydrochloride (opd, sigma chemical co, st. louis, mo, usa) with freshly added h o was used. the reaction was stopped after min by addition of m h so . the absorption was measured by elisa reader (metertech inc., taipei, taiwan) at nm. ten-week-old balb/c mice were immunized subcutaneously on day with mg of fah in ml of saline, emulsified in an equal volume of complete freund's adjuvant (difco laboratories, usa). the animals were boosted on day with the same amount of fah in incomplete freund's adjuvant (difco laboratories, usa) and bled days after the last inoculation. lalign program (http://www.ch.embnet.org/ software/lalign_form.html) using two different algorithms or matrices ( pam .mat, blosum .mat) was utilized to locate multiple matching sub-segments in two protein sequences. sequences of mhv a surface glycoprotein (e ), membrane glycoprotein (e ), nucleocapside (n), rna-direct rna polymerase (rna), hemagglutinin-esterase and kda non-structural protein were aligned with the mouse liver fah amino acid sequence. our minimum criterion for homology was the existence of at least % of sequence identity between fah and each viral protein. homologous peptides ( mers) from fah and viral proteins were synthesized according to the method of geysen et al. [ ] onto activated polyethylene pins, in a standard -well microtiter plate format (mimotopes, san diego, ca, usa). serum reactivity with synthetic peptides was determined by elisa as follows: immobilized pins were blocked for h at room temperature with pbs, ph . , containing % bsa and . % tween . after washing with pbs, ph . , for min at room temperature, pins were incubated overnight at (c in ml of each serum, diluted : in the above-described blocking buffer. pins were then washed four times with pbs, ph . , and incubated for h at room temperature with peroxidase-labeled donkey igg anti-mouse igg diluted : in pbs, ph . , containing % fcs and . % tween . after several washes, the bound ab were detected by incubating the pins for min at room temperature in ml of . mg/ml , #-azino-bis( ethylbenthiazoline- -sulfonic acid) (abts) dissolved in . m na hpo , . m citric acid, ph . , containing . % h o . the absorption was measured by elisa reader at nm. as described previously [ ] , because mouse and rat liver fah share % of sequence identity, we were able to use the purified rat liver enzyme as a substitute of the murine protein. additionally, trace amounts of rat adh co-purified with fah, accounting by about % of total proteins [ ] . thus, for the sake of simplicity hereafter we are going to refer only to ab recognizing rat liver fah as autoag. it was shown that the autoab appeared as soon as days after mhv-infection and persisted up to days post-infection [ , ] . accordingly, ab directed to the different viral proteins could be detected in sera from mhv-infected mice [ ] . under our experimental conditions, after days of mhv-infection only a band corresponding to the mw of the nucleocapside protein (n) was detected in western-blot assays. ab to proteins of mw analogous to surface glycoprotein e (also called spike glycoprotein, peplomer protein or protein s) and membrane glycoprotein e (also termed matrix glycoprotein or protein m) appeared days post-infection and continued to be secreted up to days after the virus inoculation (data not shown). in a previous work we communicated that the autoab occurring in mhv-infected mice were directed to cryptic fah epitopes, since western-blot experiments indicated that simultaneous incubation of sera with either mouse liver extracts or purified rat liver fah did not affect the autoab binding to the insolubilized autoag [ ] . in the present paper we further explored this fact adding competition elisa experiments and testing individual sera from balb/c and cba/ht mouse strains. results from fig. illustrate the different behaviors of the autoab when tested either by competition elisa or by western-blot assays. thus, data from both experimental protocols obtained with a serum from a balb/c mouse indicated that soluble rat liver fah did not alter the binding of autoab to the insolubilized enzyme, corroborating that the autoab were mainly directed to cryptic epitopes of the autoag ( fig. a and b) . however, the soluble enzyme behaved as a good competitor in elisa when a serum from a cba/ht mouse was tested, suggesting the presence of some autoab to native fah epitopes (fig. a) . in contrast, results from western-blot competition experiments performed with this same mouse serum showed that incubation with fah in solution failed to inhibit autoab binding to the insolubilized autoag (fig. b) . results similar to those presented in fig. for the balb/c serum were obtained with three balb/c and one cba/ht sera tested, whereas autoab from one balb/c and two cba/ht sera behaved as that of the cba/ht mouse presented in the same figure. mhv was insolubilized on elisa microplates or nitrocellulose sheets and allowed to react with sera from mhv-infected mice in the presence of different concentrations of soluble rat liver fah. results obtained with sera from five balb/c and three cba/ht mice showed that the enzyme did not compete for ab binding to mhv neither in elisa nor in western-blot competition assays, suggesting that the anti-mhv ab did not recognize the native epitopes exposed in the soluble autoag (see representative results in fig. ) . when the binding of the autoab to insolubilized rat liver fah was tested in the presence of mhv as competitor, three different patterns of autoab reactivity could be distinguished (fig. ) . for instance, when sera from three different cba/ht mice were tested, results indicated that the virus did not inhibit autoab binding to the insolubilized enzyme neither in elisa nor in western-blot experiments (see representative results in fig. a) . conversely, the mhv did compete for autoab binding to the ag in both procedures when a serum from a balb/c mouse was used (fig. b) . lastly, results obtained with four sera from balb/c mice indicated that the virus could behave as a poor competitor in elisa but a very good one in westernblot experiments (see representative results in fig. c ). in no case nctc stock, used as a control, produced any effect (data not shown). various decapeptides displaying at least % identity between the mouse liver fah sequence and the viral surface glycoprotein e , the nucleocapside, the membrane glycoprotein e or rna polymerase were prepared using the pepscan method (fig. ) . as indicated in section , ab binding to the insolubilized peptides was determined by elisa. . . reactivity of ab from mice immunized with rat liver fah sera from balb/c mice immunized with rat liver fah exhibited strong reactivity towards the insolubilized enzyme when tested by elisa, but the anti-fah ab did not recognize mhv proteins neither in elisa nor in western-blot assays (data not shown). results from elisa competition assays showed that soluble rat liver fah strongly inhibited the binding of anti-fah ab to the insolubilized ag, whereas only % of competition was observed in western-blot experiments (fig. ) . on the other hand, the presence of mhv did not affect the ab binding in any procedure (fig. ) . in order to investigate the fine specificity of the anti-fah ab, sera from six mice immunized with the enzyme were tested for their reactivity towards the homologous fah/mhv peptides displayed in fig. . representative results from three different experiments shown in fig. indicated that the individual responses were diverse. however, although a unique pattern of ab reactivity could not be found, anti-fah ab consistently recognized sequences e and e of the enzyme and, depending on the serum tested, bound to various mhv peptides, mainly sequences from e and rna polymerase proteins (fig. ) . as stated before, mice infected with mhv developed autoab to mouse liver and kidney fumarylacetoacetate hydrolase (fah). these ab bound to liver fah from various origins, i.e., the rat, sheep and human enzyme, and also recognized rat liver alcohol dehydrogenase (adh) [ ] . in addition, western-blot competition experiments suggested that the autoab elicited in mhv-infected mice were directed to cryptic epitopes of the ag [ ] . since hypergammaglobulinaemia or tissue damage as mechanisms triggering the mhv-elicited autoimmune process appeared less likely than molecular mimicry, we undertook competition experiments. because individual sera were tested, it could be found that results using elisa and western-blot competition assays were sometimes different, and that such differences did not depend on the mouse strain utilized. thus, whereas western-blot assays indicated that autoab were directed to cryptic fah epitopes in all sera tested, elisa results indicated that some of these same sera did recognize native epitopes of the autoag (fig. ) . these results could be explained taking into consideration that insolubilized proteins expose cryptic epitopes while still retaining some conformational features [ , ] . since proteins submitted to sds-page and then transferred to nitrocellulose should be more denatured than those insolubilized on plastic surface, it is possible that the autoab detected both conformational and cryptic epitopes in elisa and only cryptic determinants in western-blot assays. so, sera showing lack of competition by the soluble autoag in both protocols should have autoab directed mostly to cryptic epitopes of the fah, whereas those displaying dissimilar reactivity may have a mixture of autoab directed to both hidden and conformational antigenic determinants of the enzyme. on the other hand, results from elisa as well as from western-blot competition assays showed that ab to mhv did not cross-react with the soluble autoag (fig. ) . however, results from the opposite situation were not the same. in fact, when mhv stock was used as competitor for binding of autoab to insolubilized fah different patterns of reactivity were obtained. elisa as well as western-blot competition tests showed lack of effect of mhv in solution or, depending on the mouse serum used, both procedures did show an inhibitory activity of the virus (fig. ) . furthermore, some sera displayed different behavior in both procedures, mhv being inhibitory in western-blot experiments but not in elisa assays (fig. ) . optical density ( nm) it is noteworthy to mention than anti-mhv ab elicited in all mice tested were directed to native epitopes of the viral proteins, because mhv strongly inhibited ab binding to the insolubilized virus in elisa as well as in western-blot competition experiments (data not shown). molecular mimicry between viral proteins and self-ag is one of the most probable mechanisms that explain autoimmune responses induced by viral infections [ , ] . murine adenovirus, semliki forest virus, lactate dehydrogenase-elevating virus, herpes simplex virus type- , hepatitis b virus, encephalomyocarditis virus, theiler's murine encephalomyelitis virus, coxsackievirus and cytomegalovirus have been found to mimic physiologically important host proteins [ ] . thus, in order to analyze the ab repertoire of mhv-infected mice a set of mers homologous peptides corresponding to the sequence of mouse fah and viral proteins e , nucleocapside, e and rna polymerase was employed (fig. ) . most ab recognized the n-terminal sequence ( e ) of both fah and the nucleocapside, two peptides that share % of identity. in addition, ab from and days post-infection mice bound to the sequence e of the rna polymerase, a peptide showing % of identity with fah e . thus, recognition of the fah sequence e by ab elicited by mhv infection could be at least partially responsible of the autoimmune response described herein. this observation should also explain the lack of reactivity of anti-fah ab with viral stock. in fact, sera from fahimmunized mice did not react with the n-terminal sequence e of the enzyme, and only showed binding to sequence e or e (figs. and ) . the crystal structure of fah showed that the protein folds into a -residue n-terminal domain of unknown function and a -residue c-terminal domain defined by a novel b-sandwich roll structure [ ] . in that structure, sequence e appears as a coil exposed to the solvent, suggesting that autoab could recognize this structure in the enzyme either insolubilized or in solution. we cannot discard that sequences other than fah e were recognized by the autoab elicited in mhvinfected mice because we did not test peptides spanning the entire fah sequence. moreover, it was reported that similarity of sequences is sometimes not sufficient to mimic epitopes and that structural considerations contribute significantly to the underlying mechanisms of molecular mimicry [ ] . accordingly, results presented in this work indicate that mhv-infection triggers a cross-reaction of either sequential or conformational epitopes from both the viral proteins and the autoag. data also suggest that mhv-infected mice could develop at least three different ab populations: ab directed either to viral proteins or to the autoag, and cross-reacting ab. the observation that immune responses undergo determinant spreading is a major finding shaping current theories regarding autoimmunity and molecular mimicry [ ] . therefore, immune diversification originated from only a single autoreactive determinant, i.e., a common sequence, could probably provide a pathway for the generation of the multifaceted autoimmune response described in this paper. the absence of autoimmune disease in our model remains to be explained. in fact, mice infected with mhv-a develop acute hepatitis but liver regeneration may take place as early as e days after infection [ , ] . additionally, other mhv-a effects, such as thymus involution, the enlargement of the spleen and production of great amounts of igg a, are also transient [ , , ] . thus, in spite of the presence of the autoab reported in this work, no signs of autoimmune hepatitis were evident. our findings remind the response to allo-hppd (allotype of the hepatocyte enzyme -hydroxy-phenyl-pyruvate dioxigenase, alias f liver protein) that has been long studied [ e ]. the mouse f antigen is expressed mainly in the liver ( ÿ m) but leaks into body fluids at a concentration of z ÿ m. it has been proposed that this protein concentration is just sufficient to induce complete tolerance of t cells, whereas b cells are not tolerized [ , ] . although the concentration of liver fah in serum is not known, its similarity in mw and localization with hppd [ , , ] enabled us to speculate that a t cell effect similar to that proposed by n.a. mitchinson and colleagues [ e ] may take place in our model. reactivity of synthetic peptides with sera from mice immunized with rat liver fah. ab binding to the homologous decapeptides showed in fig. was determined by elisa as indicated in section . results are expressed as specific optical density values for ab binding to sequences corresponding to rat liver fah (black bars) or the indicated different mhv proteins (white bars). peptides synthesized on pins were numbered according to fig. . results presented in a, b and c were obtained with individual sera from three different balb/c mice immunized with rat liver fah as indicated in section . the viraleautoimmunity relationship evidence for mimicry by viral antigens in animal models of autoimmune disease including myocarditis mimicking the way to autoimmunity: an evolving theory of sequence and structural homology in vivo polyclonal b-lymphocyte activation elicited by murine viruses the role of infection in the pathogenesis of autoimmune disease viral diseases of the digestive system role of virus receptor-bearing endothelial cells of the bloodebrain barrier in preventing the spread of mouse hepatitis virus-a into the central nervous system morphological analysis of mouse hepatitis virus a -induced pathology with regard to viral receptor expression polyclonal b lymphocyte activation induced by mouse hepatitis virus a infection identification of two liver proteins recognized by autoantibodies elicited in mice infected with mouse hepatitis virus a igg a restriction of murine antibodies elicited by viral infections the coronavirus protein measurements with the folin phenol reagent cleavage of structural proteins during the assembly of the head of bacteriophage t strategies for epitope analysis using peptide synthesis detection of mouse hepatitis virus infection by assay of anti-liver autoantibodies autoantibodies to cryptic epitopes elicited by infection with lactate dehydrogenase-elevating virus binding properties of antibodies to prothrombin and b -glycoprotein i (b -gpi) assayed by elisa and dot blot crystal structure and mechanism of a carbonecarbon bond hydrolase epitope mimics and determinant spreading: pathways to autoimmunity thymus involution induced by mouse hepatitis virus a in balb/c mice self-and allo-specific suppressor t cells evoked by intravenous injection of f protein self-reactive t cell hybridomas and tolerance regulation by non-major histocompatibility complex genes of the allo- -hydroxy-phenylpyruvate dioxygenase (f liver protein) response selection of anti-f protein b-cell repertoires in normal mice the authors are indebted to drs. pierre l. masson (icp, brussels, belgium) and leonor p. roguin (iqui-fib, buenos aires, argentina) for helpful discussions and critical revision of the manuscript. this work was supported by grants from conicet, foncyt and universidad de buenos aires, argentina, and fonds national de la recherche scientifique (fnrs), fonds key: cord- -ydkigome authors: villarreal, luis p. title: the widespread evolutionary significance of viruses date: - - journal: origin and evolution of viruses doi: . /b - - - - . - sha: doc_id: cord_uid: ydkigome in the last years, the study of virus evolution has undergone a transformation. originally concerned with disease and its emergence, virus evolution had not been well integrated into the general study of evolution. this chapter reviews the developments that have brought us to this new appreciation for the general significance of virus evolution to all life. we now know that viruses numerically dominate all habitats of life, especially the oceans. theoretical developments in the s regarding quasispecies, error rates, and error thresholds have yielded many practical insights into virus–host dynamics. the human diseases of hiv- and hepatitis c virus cannot be understood without this evolutionary framework. yet recent developments with poliovirus demonstrate that viral fitness can be the result of a consortia, not one fittest type, a basic darwinian concept in evolutionary biology. darwinian principles do apply to viruses, such as with fisher population genetics, but other features, such as reticulated and quasispecies-based evolution distinguish virus evolution from classical studies. the available phylogenetic tools have greatly aided our analysis of virus evolution, but these methods struggle to characterize the role of virus populations. missing from many of these considerations has been the major role played by persisting viruses in stable virus evolution and disease emergence. in many cases, extreme stability is seen with persisting rna viruses. indeed, examples are known in which it is the persistently infected host that has better survival. we have also recently come to appreciate the vast diversity of phage (dna viruses) of prokaryotes as a system that evolves by genetic exchanges across vast populations (chapter ). this has been proposed to be the “big bang” of biological evolution. in the large dna viruses of aquatic microbes we see surprisingly large, complex and diverse viruses. with both prokaryotic and eukaryotic dna viruses, recombination is the main engine of virus evolution, and virus host co-evolution is common, although not uniform. viral emergence appears to be an unending phenomenon and we can currently witness a selective sweep by retroviruses that infect and become endogenized in koala bears. phylogenetic tools have greatly aided our analysis of virus evolution, but these methods struggle to characterize the role of virus populations. missing from many of these considerations has been the major role played by persisting viruses in stable virus evolution and disease emergence. in many cases, extreme stability is seen with persisting rna viruses. indeed, examples are known in which it is the persistently infected host that has better survival. we have also recently come to appreciate the vast diversity of phage (dna viruses) of prokaryotes as a system that evolves by genetic exchanges across vast populations (chapter ). this has been proposed to be the " big bang " of biological evolution. in the large dna viruses of aquatic microbes we see surprisingly large, complex and diverse viruses. with both prokaryotic and eukaryotic dna viruses, recombination is the main engine of virus evolution, and virus host co-evolution is common, although not uniform. viral emergence appears to be an unending phenomenon and we can currently witness a selective sweep by retroviruses that infect and become endogenized in koala bears. our understanding of virus evolution has reached a threshold in that it now appears to provide a much broader vista regarding its general signifi cance and infl uence on host evolution. several developments have brought us to this point. one has been the realization that viruses often evolve by processes involving the collective action of a consortia, or quasispecies. and the resulting adaptability and power of such evolution is unmatched by any other genetic entity. much of this volume is dedicated to this issue. in such consortia, the concept of a " wild-type " virus is no longer considered to be the fi ttest type, as the quasispecies itself provides fi tness (see chapters and ). the quasispecies model resembles population genetics in some ways, but it has led to some signifi cant departures from population genetics, and these departures are very well supported by experiments. another development that has recalibrated our view of the overall signifi cance of viruses is information on the scale and diversity of viruses. viruses are present at a previously unappreciated global level and appear to have affected the evolution of all life on earth. much of this realization has been brought about by the development of metagenomic methods as applied to various habitats. measurements of major habitats (the oceans, soil, extreme environments) have established that our biological world is predominantly viral, in terms of both numbers and diversity ( paul et al. , ; breitbart et al. , ; rohwer, a, b ; edwards and rohwer, ; comeau et al. , ) . these two developments would seem reason enough to consider virus evolution in a new light. however, there have also been numerous theoretical proposals suggesting viral involvement in some of the very earliest events and major transitions in the evolution of life. we no longer think of viruses as recent agents that escaped from the host chromosomes as run away replicons. viruses now appear very old to us and they relate to and trace all branches of life. the last years have been very active regarding virus evolution. major developments in theory, technology, medicine, and the study of human disease with respect to virus evolution have all occurred. and as we seek to grow and manage various life forms for human use, virus evolution has also had major impact on such efforts. as a science, virus evolution has benefi tted greatly from traditional evolutionary biology. however, since viruses are molecular genetic parasites that are inscrutable by casual observation, our understanding of virus evolution has been dependent upon measuring sequence variation and sequence diversity in a large number of virus genomes. because of this, these small genetic parasites have been the last domain of life to yield their secrets of evolution. and viruses harbor some clearly distinct evolutionary abilities. for one, they are polyphyletic. all major viral lineages have their own distinct origins. they are also difficult if not impossible to defi ne as species and are able to exchange genetic information across normal boundaries. even " dead " and defective viruses can participate in such exchanges, which confuses the defi nitions of fi tness. we now know that viruses can evolve by a consortia process and also exchange information by recombination across vast genetic pools to assemble new mosaic combinations of genes. we thus no longer think of a specifi c genetic lineage in understanding virus evolution, but instead think of a cloud, matrix, or a population as the basis of virus evolution. viruses are inherently fuzzy entities that can differ from their relatives in any specifi c feature. yet even with such fuzziness, it is clear that common themes also link them. patterns of evolution have become clear. diversity and variation are often (but not always) observed. stability and host congruence can also be observed. nevertheless, the evolutionary power of viruses has been learned at a human cost. the application of numerous analytical and phylogenetic tools have provided crucial insights into virus origin and evolution. yet these methods struggle to incorporate the fuzzy nature of viruses and have clear limits, especially regarding quasispecies and high recombination rates. structural biology now also adds tools that extend our vision of virus evolution beyond what can be seen in the genetic sequence. for example, common structural motifs from phage to eukaryotic dna viruses (t and herpesvirus) suggest very ancient links in virus evolution that span all domains of life (see below). nevertheless, our analytical methods are currently lacking as we struggle to understand complex genetic mixtures that provide fi tness, reticulated relationships, polyphyletic origins, and virus-host congruence. virus evolution has, for the most part, been considered to be a specifi c, esoteric part of broader evolutionary biology and has been given limited attention in reference works on evolutionary biology (see pagel, ) . historically, the focus has been on various rna viruses and some dna viruses that cause disease in humans and domesticated animals and plants . however, i have asserted that all life forms must be examined from the perspective of virus evolution ( villarreal, ) , not just those pathogens that impact on us. how viruses evolve in a more general sense informs us of evolutionary paradigms that have not been previously well understood (especially the evolution of consortia or the dynamics of vast reticulated gene pools). this volume now extends these traditional topics of virus evolution to include the vast virology of the prokaryotic world. in so doing, it illuminates the global consequences viruses have had on all life forms. prior to the s we saw some stunning successes in vaccine control for major viral human diseases, such as polio, measles, mumps, infl uenza, and especially smallpox virus. due to this historic success, american health agencies and educators considered virus disease as a thing of the past, no longer a serious threat. the era of infectious disease that they represented was now one for the historical record, an unfortunate part of human history, or so we thought. in what now seems to have been a clear case of hubris and naivety, we have been humbled by the evolutionary power of viruses, which was woefully underappreciated, even by most virologists. by the end of that decade, the evolution and emergence of hiv- permanently changed our views (see chapters and ) . this has also been followed by a seemingly never-ending series of viral threats as newly emerging viral diseases have come to our attention. hiv- provides the only example of a public health situation that has reversed centuries of progress for extending human health and lifespan. it now limits human life expectancy in many parts of the world, especially sub-saharian africa. this development could not have been imagined in the s. we are much less confi dent now about predicting the future of virus evolution and its potential impact on human health. diseases of domesticated microbiological, plant, and animal species have also experienced the trauma of the consequences of emerging viral diseases along with huge losses. however, the human hiv- story may not be a fl uke of virology but may be telling us something basic about human and primate evolution. as we sought to understand the origin of this new virus, we have come to appreciate a much broader virus-host story which involves simian immunodefi ciency virus (siv), foamy viruses, and the speciation of old world primates. we have also come to learn that the genomes of these primates show much evidence of past viral interaction and ongoing endogenous retrovirus colonization. the evolution of retroviral endogenization has taken on a much greater signifi cance in basic evolutionary biology. thus it is with great interest that we now study the ongoing endogenization of retroviruses in the koala bear genome (see below). historically, we are compelled to study viruses because they can cause serious disease. new viruses come to our attention also mainly because of disease. it is therefore understandable that most evolutionary biologists mainly think of viruses strictly as agents of disease. these are the products of run away replicons that provide negative selection to host survival. in this light, the application of predator-prey based mathematical models has seemed most appropriate. with such viral disease, variation has long been observed and was initially used for the generation of most vaccines. however, this disease-centric view has also occluded another more prevalent virus-host relationship. for example the emergence of hiv- has led us conclude that it likely evolved from various versions of siv. but siv is not pathogenic in its native african primate host. nor does it show the genetic diversifi cation of hiv- in these native primate hosts. it is a silent, asymptomatic infection. genomic and metagenomic analysis now allows us to identify many more silent, asymptomatic viruses that would not have previously been observed. we now know of many such viruses that are prevalent in a specifi c host. evolutionary biology must escape the confi nes of disease-centric thinking and seek to understand these relationships as well. in the last ten years i have attempted to provide another view concerning virus-host evolution. i have argued that viruses often attain evolutionary stability by species-specifi c persistence and that such states apply to all domains of life, including prokaryotes. on an evolutionary time-scale, the majority of viral lineages tend to exist as species-specifi c persistent (aka temperate, latent, and chronic) infections in which individual hosts will be colonized by mostly silent (asymptomatic) viruses for the duration of their life . such persistence can have major consequences to the evolution of both virus and host, which also leads us to more directly link virus evolution to broader issues of host evolution. it is from this perspective that we start to clearly see that viruses indeed belong on the tree of life as major participants ( villarreal, ( villarreal, , . persisting viruses are not simply agents responsible for destruction of life, but are also agents that create genetic novelty on a vast scale that infl uences all life and promotes symbiosis ( marquez et al. , ; ryan, ; villarreal, ) . the persistent lifestyle of such symbiotic virus-host relationships is not simply a less effi cient, acute infection; nor is it simply a " reservoir " for acute virus (as epidemiologists are prone to assert). neither can it be attributable to concepts of selfi sh dna. persistence represents a major virus life strategy that is both fundamental and highly adapted. it has distinct genetic, fi tness, and evolutionary characteristics that require intimate, host (tissue)-specifi c viral strategies and precise gene functions to attain stable maintenance in the presence of immunity and to allow biologically controlled reactivation. persistence also must resist displacement by similar viruses and competitors. it is virus-host persistence that provides the thread that allows us to link these polyphyletic viral lineages (and their clouds) with the entire tree of life. in turn, this link identifi es a much more fundamental role for viruses in the evolution of host, visible from the very earliest to the most recent events in host evolution. it is from such species-specifi c persistent states that the large majority of acute diseases evolve and emerge by various mechanisms. we know much about virus replication and disease. however, our understanding of the specifi c mechanisms of persistence is generally poor. persistence is a generally silent and inscrutable state, it does not lend itself to in vitro or cell culture experimental models. we are left with but a few examples from which to attempt to extrapolate the possible existence of general relationships. the study of virus evolution thus struggles to incorporate concepts of persistence. another recent and major development in virus evolution is the arrival of various proposals suggesting that viruses have been involved in some major innovation and transition in the evolution of life. in all these proposals, however, it is necessary that the virus in question has attained a stable genomic persistence with its host. these evolutionary events thus seem to be the products of viral-mediated symbiogenesis of host ( ryan, ; villarreal, ) . proposals include the possibility that viruses may have originated the dna replication system of all three cellular domains (archaea, bacteria, eukarya) of life ( forterre, ( forterre, , ( forterre, , a ( forterre, , b filee et al. , ; forterre et al. , ) . the discovery and analysis of the largest dna virus ( orf mimivirus), a lytic cytoplasmic virus of amebae (a distant relative of phycodnavirus and poxviruses), has also led to proposals that this virus lineage may represent an ancient fourth domain of life ( raoult et al. , ; desjardins et al. , ; claverie et al. , ) . it is interesting that in an initial structural analysis, the large complex replication centers for mimivirus were confused with the host nucleus ( suzan-monti et al. , ) . thus, it seems relevant that others have proposed that a distant relative of phycodnaviruses and poxviruses may have originated the eukaryotic nucleus ( villarreal, ; villarreal and defilippis, ; bell, bell, , takemura, ) . such proposals, although consistent with various observations, however, remain outside of the consensus of most evolutionary biologists. nevertheless, numerous other observations continue to suggest viral involvement in other major host innovations, such as a viral origin of rag / of the adaptive immune system ( dreyfus et al. , kapitonov and jurka, ; fugmann et al. , ) or the role of endogenous retroviruses (ervs) in the evolution of the placenta ( villarreal and villareal, ; harris, ; blond et al. , ; mi et al. , ; dupressoir et al. , ; caceres and thomas, ) . such possible roles for viruses in host evolution are at odds with accepted views of virus-host relationships, but might be the products of viral symbiogenesis. the metagenomic viral measurements mentioned above for prokaryotic dna viruses, along with the increasing realization that viruses and host can co-evolve, has led to various calls that a viral tree of life needs to be considered and developed ( forterre, ; villarreal, ; filee et al. , b ) . a virosphere clearly exists but its nature and boundaries are not so clear. multiple viral origins, their diversity and numerical dominance in distinct and sometimes harsh environments as well as their presence in host genomes suggest that any viral tree of life will be huge, multidimensional, and connected to the host tree of life. as discussed below regarding double-stranded (ds)dna viruses of prokaryotes, they have all the above characteristics. such viruses may represent the big bang of biological novelty. with their unmatched capacity to generate diversity they can function as the mass creators of biological novelty as well as destroyers of most species. surely, such capacity must have had big infl uences on the evolution of life. symbiosis, simply defi ned, is the stable coexistence of two previously separate lineages of organisms. there can be little doubt that many temperate phage can stably colonize a bacterial cell resulting in a stable descendent from two lineages. this is clearly symbiotic. endogenous retroviruses can similarly be found to persist in vertebrate genomes and also appear symbiotic. yet studies of symbiosis seldom consider a role for virus ( villarreal, ) . how important are viruses in general to evolutionary biology? the core concepts of evolutionary biology were developed well before we had a modern understanding and defi nition of viruses ( luria, ) . after all, the basic lysogenic model of phage integration was only clarifi ed in when developed by campbell (see campbell, ) . that cryptic and defective phage are ubiquitous in the genomes of all prokaryotes is generally considered uninteresting by many in the fi eld of evolutionary biology. i suggest that the seemingly applicable concepts of selfi sh dna effectively derailed any thinking that persisting genetic parasites might have a more germinal role in the evolution of life ( doolittle and sapienza, ; orgel and crick, ). yet as outlined above, virus footprints in major evolutionary transitions are clear and a direct role in such events now seems much more plausible. we therefore must seek to defi ning the nature of the virosphere and how its evolution relates to the tree of life. this book represents the fi rst integration of the entire fi eld of virus evolution, including both prokaryotic and eukaryotic life forms. however, because our understanding of virushost relationships remains uneven, the chapters necessarily focus on well-studied models (exemplars). these exemplars also tend to refl ect a historic disease focus (i.e., e. coli , fl owering plants, mouse, humans). it is unfortunate that the silent species-specifi c viruses that tend to exist in stable states with long evolutionary histories seldom provide our examplars. we understand these infections poorly and lack basic defi nitions concerning fi tness or selective advantages. only metagenomics tools now seem able to inform us of their presence, but not their biology. self-organization and the evolution of rna molecules as an origin of biological information are discussed in chapter . autocatalytic chemical reactions, such as replication of rna, presents issues such as how to optimize a rugged fi tness landscape yet allow the study of evolution in vitro with rna. rna in vitro reduces genotype-phenotype issues to rna secondary structure and minimal free energy states. this allows both continuity and discontinuity to be measured. these same issues are crucial for the study of rna viruses, whose sites of secondary structure often defi ne replicator identity. these models currently offer the best system to evaluate an early and simple biological world for evolutionary principles (see chapter ). viruses and viroids with their rna genomes may be the only extant survivors of this pre-dna world. since it was the consideration of error-prone replication that led to the development of the concept of quasispecies (see chapter ), such models have provided a conceptual foundation which led to several basic concepts. chapter discussed the foundations and various aspects of quasispecies theory. viruses appear to operate close to the error threshold, thus allowing maximum evolutionary exploration ( biebricher and eigen, ) . however, as presented below, the loss of the " fi ttest " type concept has also led to clear experimental evaluations of consortia-based evolutionary behaviors. such behaviors were not predicted by classical darwinian models. although virologists were initially attracted to quasispecies models, many evolutionary biologists were initially hostile to the application of the quasispecies concept to evolution. it was thought that the classical mathematical models of population biology, as originally developed by wright and fisher, and later applied by kimura and maruyama to asexual haploid populations at the mutationselection balance, had already fully developed the needed models and precluded additional need for the quasispecies concept. the classical models were thus argued to provide adequate mathematical coverage for viruses, including quasispecies and error threshold ( wilke, ) . however, these two approaches differ fundamentally with regard to the signifi cance of error-prone replication and it was the quasispecies approach that led to the clear experimental establishment that quasispecies selection, per se , is important for viral pathology and fi tness (see below). the development of quasispecies theory to virology does indeed demonstrate distinct differences with population genetics. various phenomena, such as complementation, cooperation, competition, and even defective mediated extinction ( domingo et al. , , domingo grande-perez et al. , ) have been observed, all of which fall outside of the parameters of classical population genetics. viral fi tness has indeed been shown to be due to interaction within a diverse population, and not to the fi ttest or master type. and with rna viruses, error threshold has become a central issue ( biebricher and eigen, ) . the collective experience has thus made clear the value of theory to biology. many working biologists understand that life seems overly complex and defi es most generalizations. thus they do not always appreciate attempts at general theory. although in reality, biology may indeed often be too complex for accurate theoretical predictions, these theories have nevertheless clearly stimulated crucial concepts and experimental evaluation, and biologists should be encouraged by them. by providing new ways of thinking, entirely new experimental approaches can be developed. the existence of error-prone replication and quasispecies also raises the issue of the conservation of information. how is information stability and higher fi tness attained with such errors? how can genetic complexity be created in such a circumstance? can cooperation (or consortia behavior) result from any of these models? these issues have yet to be resolved. some interesting suggestions, however, have been proposed. one involved cooperative evolution that results from ligated genomes. a model was proposed in by stadler and schuster in which they considered the dynamics of replicator networks resulting from higher order reactions involving the templated ligation of smaller genomes ( stadler et al. , ) . although this was based on concepts such as triple-stranded nucleic acids, this clearly has some elements that also resemble the ligation of recombinational processes for dna phage (presented below). a most interesting outcome of these models is that, depending on initial replicator concentrations, permanent coexistence of replicators could result in a cooperative network. such cooperation is a rare outcome for most models and given the conclusion that early dna based life was a " horizontal " consortium, such models are of special interest. the issue of consortia will come up often. consortia selection directly implies cooperation, but cooperation of selfi sh replicators presents dilemmas. replicator networks with interaction functions that give highly non-linear dynamics can result in complex mixtures, with behaviors ranging from survival of the fi ttest to also including attainment of globally stable equilibrium tantamount to permanent coexistence. the fi tness of populations, however, is inherent to current quasispecies concepts in virology. there may also be other ways to explain the genetic origin of cooperation (such as stable persistence involving addiction strategies). the stable persistence of a genetic parasite can compel cooperation and promote the conservation of information (see below). the defi nitions of fi tness with respect to a virus in a natural habitat are far from clear. although the concept of relative replicative fi tness is often applied to lab experiments of virus growth, we know many situations in which virus replication is not maximized in natural settings and many viruses can exist in relatively non-replicative states for long periods. even in the context of an acutely replicating virus in a host organism, the concept of fi tness is clearly conditional, as the virus must replicate through various in vivo habitats that can have opposing selection. as presented below, in vivo models that study fi tness and viral diversity have clearly indicated that diversity per se is important and fi tness is the result of consortia. how do we defi ne such fi tness since the mixture clearly matters? also, how do we defi ne information content or integrity of a consortia? currently, we cannot. in the lab, the viability of a virus is usually measured by the ability to produce plaques. this has been a crucial and main assay for many experimental systems that study virus population. here, the defi nition of fi tness seems direct: plaque formation equals fi tness. various highly useful models have thus been defi ned and developed that depend on these plaque assays (see chapter ). with this, populations and population growth are defi ned as relative growth of plaque-forming units. however, the concept has always been problematic when considered from a natural virology perspective. plaque formation is clearly not equal to fi tness in natural habitats. there are many examples of highly successful viruses that either plaque poorly or not at all. consider the roughly types of human papillomavirus (hpv), a simple small circular dna virus of epithelia; this does not form plaques in any known system (chapter ). hpv is clearly fi t, well adapted to, and stable in its human host. in addition, hpv evolution is phylogenetically congruent with their primate host, as are most persistent viral infections. we have yet to understand the definition of fi tness in this situation. in some cases, it seems selection for plaque propagation has clearly resulted in loss of highly conserved genes; such as with the plaque-adapted laboratory strains of cytomegalovirus (cmv). the problem posed by viruses with ineffi cient plaque formation is not limited to dna viruses. many persisting rna viruses also do not plaque well or at all, such as most rna viruses of plants or many insect picorna-like viruses, such as those found in drosophila and bees (which also conserve an extra orf). nor do most persistent infections make lots of virus. low-level persistence, such as hantavirus in rodents, for example, is common ( hart and bennett, ) . clearly, our simplifying assumptions of viral fi tness and population dynamics cannot apply to these stable evolutionary states. however, if we limit our defi nition of viral fi tness to relative replication or plaque formation we can perform some clear and quantitative evaluations. experimental evaluation forces us to study fi tness by only those defi nitions that we can currently measure. as fi tness appears to be a relativistic and transient concept, depending very much on the tissue, time, place, extant adaptive and innate immunity, and competition, it is likely that we can only measure with any accuracy one aspect of fi tness at any one time. hiv infection of humans shows evidence of this in that the r virus is more fi t for transmission and early disease whereas the x virus is fi t later during the aids disease phase. clearly conditional, time-dependent issues relate to fi tness defi nitions. however, much more problematic is that we have no theory for viral persistence or its fi tness. we lack specifi c or measurable parameters other than the simple maintenance of genetic material. yet it seems clear that some distinguishing features of persistence can already be recognized. for example, the possible participation of viral defectives (normally considered unfi t), which in numerous circumstances can modify or mediate persistence, would need to be included. clearly, a defective role in persistence would also preclude them from being considered as genetic " junk, " or selfish elements, since they would then matter in measurable ways to the biological outcome of virus persistent infection. persistence also requires an extended duration of infection, not simply maximized replication. in fact, persistence generally requires mechanisms to limit the replication of at least the same virus for at least some time. thus, limited replication must be an essential element for this life strategy. in my judgment, and much like the quasispecies concept, the concept of persistence will eventually be recognized for the fundamental (symbiotic) force it represents in virus evolution. the experimental work of domingo and holland spans the modern assessment of quasispecies theory that occurred in the s and s. these investigators were the chief proponents of this theory, bringing it to the attention of the broader virology community (chapter ). this work has transformed our thinking and laid the experimental foundations that we now build upon. this current volume is an extension from an earlier book on quasispecies and now encompasses both prokaryotic and eukaryotic viruses . since early experimental phage studies provided the foundations for quasispecies theory ( eigen et al. , ) , using mathematical descriptions (differential equations) of mutation rates in t-even phage ( luria and delbruck, ) , this inclusion is appropriate. interestingly, a second early paper measuring replication rates by these same authors also noted the problems of viral interference and defectives ( delbruck, ) . other early experiments of phage rna polymerase ( batschelet et al. , ) , especially with rna phage qβ ( domingo et al. , ) , helped set the stage for the subsequent experiments of the s and s. from the test tube to mouse models to the study of human disease, the work of domingo and colleagues has spanned the entire history of viral quasispecies ( domingo and gomez, ; chapter ) . quasispecies deals with the products of error-prone replication. however, it is worth repeating that products of error-prone replication are not behaving in a simple " selfish dna " capacity and are not devoid of biological relevance and phenotype. in their complex populations, they create clear and varied affects on viral adaptability, competition, and fi tness. since quasispecies necessarily involve defective and mutant virus, it is easy (and common) to think of these entities simply as genetic junk ( villarreal, ( villarreal, , . defective and even lethal or interfering variation in viral genomes can contribute to adaptability. thus, viruses can clearly adapt as a cloud with a mutant spectra. in addition, unending competition and exclusion, consistent with the red queen hypothesis, has also been observed ( clarke et al. , ) . the poliovirus-mouse model (see below) in particular has provided a solid experimental system for evaluating the adaptive consequence of quasispecies. it is thus ironic that these same experiments have also made clear that the original simplifying assumptions of the quasispecies ordinary differential as presented by eigen are violated by the resulting quasispecies. these products of error-prone replication do indeed strongly interact with each other in both positive and negative ways and such interactions contribute signifi cantly to the observed fi tness of the population. errors and interaction are important for fi tness. for example, defectives have been reported to mediate extermination of a competing wild-type virus ( grande-perez et al. , ). complementation has also been observed ( garcia-arriaza et al. , ) , as has trans -dominant inhibition ( crowder and kirkegaard, ) . genetic memory of past selection has been shown to be maintained in a minority of the population ( ruiz-jarabo et al. , ; briones et al. , ) . such cooperative (consortia) behavior, which can also depend on unfi t or defective members, is at odds with classical darwinian notions regarding survival of the fi ttest. consider, for example, the fi tness of a defective or mutant outside of its role in a quasispecies. such a consideration ignores the very nature of a quasispecies yet it is an issue that has often been posed and experimentally evaluated. we should refrain from thinking of viruses simply as fi t or non-fi t individual types since they clearly exist in populations that provide population-based adaptability. the selection of viral consortia or population raises some fundamental issues for evolutionary biology. this is essentially group selection in which a population, not the fi ttest individual, is selected. this view makes cooperation or interaction of individual genomes a signifi cant component of selection, which is not commonly thought to be a general or accepted process in evolution. yet population selection is no longer a contestable issue in rna virus evolution (see below). i expect that many classical evolutionary biologists might interpret this as evidence that viruses really are an oddity in this feature and are not representative of broader processes of evolutionary biology. furthermore, viral-based group selection may not be limited to quasispecies-based evolution. as presented below, persistent viral infections may also provide population-based selective advantage (see below for the p and mouse hepatitis virus (mhv) persistence exemplars; villarreal, villarreal, , . since viruses are ancient, numerically dominant, and the most diverse biological entities on earth, no life form can escape exposure to them. all extant life forms have evolved in a viral habitat. thus we should expect that the viral footprints (including defectives) that we now fi nd in all genomes have likely played an active role in their evolution; a role, i would argue, that is fundamental, dynamic, and unending. if we can accept this assertion, we may start to see and appreciate the vast evolutionary power that viruses can bring to bear onto host evolution. we can start to attain a global perspective and appreciation for their ability to assemble genetic function from enormous, complex mixtures of genomes, and select gene sets needed to solve multivariant, temporally dynamic evolutionary problems. we can then seek evidence for the role of viral elements in fundamental host innovation and be open to evaluating the occurrence of viral entities from a constructive perspective and not instinctively dismiss such observations as due to coincidence, " junk, " or selfi sh dna. the advantage of such a perspective is that it will promote the specifi c experimental evaluations that can better assess any constructive role genetic parasites might have played in host evolution. for example, there is much reason to think ervs have played an active role in human evolution (for references see ryan, ) . the quasispecies concept has provided the foundation for us to understand virus evolution and informed us of the evolutionary power viruses possess. if that power also links to host evolution, then the tree of life becomes enriched by virus, much larger and more dynamic. the recent experimental studies from andino and colleagues using poliovirus in the mouse model should, in my judgment, provide the keystone exemplar regarding the in vivo fi tness of quasispecies (see chapters and ; vignuzzi et al. , ) . these studies make clear the importance of quasispecies and error-prone replication. such detailed in vivo experiments were made possible by a long and detailed history of poliovirus studies that has identifi ed the nature of rna polymerase fi delity as well as developed mouse models for the study of pathogenesis. few other virus-host systems could have provided such potential for high resolution. these results also provide the experimental observations that distinguish quasispecies-based evolution from the classical fisher-based population genetics. the general importance of this story for understanding virus evolution thus deserves special emphasis. the very origins of modern animal virology stem from poliovirus studies with the need to develop in vitro cell culture technology in order to grow and evaluate poliovirus and generate variants. the live poliovirus vaccine is of special interest with regards to virus evolution and adaptability. the " live " oral sabin vaccine can be considered to have been a miracle of the practical approach to virology developed in the s ( horaud, ) in that it was used well before our understanding of the relevant evolutionary theory. the sabin vaccine strain was the result of rodentadapted virus and differs from the neurovirulent mahoney strain by point mutations (in the consensus sequence), although only a small number of these mutations were needed for neurovirulence ( christodoulou et al. , ) . one of the important neurovirulent mutations was within the rna polymerase gene ( tardy-panit et al. , ) . however, the signifi cance of this observation took many years to unravel and exploit. in time it became apparent that dpol mutants could affect replication fi delity. one poliovirus point mutant, dg s, was shown to have enhanced highfi delity replication and that selective pressure could be designed to increase fi delity in rna polymerase ( pfeiffer and kirkegaard, ) . another major development was the molecular identifi cation of the poliovirus receptor and the subsequent creation of transgenic mice expressing this receptor, making them susceptible to poliovirus infection. one of these transgenic lines allowed mouse brain infections with neurovirulent versions of poliovirus , and has provided a very useful animal model that allowed the evaluation of viral fi tness in the context of in vivo pathogenesis. although dg s replicates well in culture (with lowered error rate), it was less pathogenic in this mouse model and competed poorly with d wild-type virus. it seemed that the decreased viral diversity was less able to generate the variation needed to get past bottlenecks due to multiple selective differences presented in vivo in tissues in the host, such as brain infection ( pfeiffer and kirkegaard, ). this experimental system also makes clear the greater complexity of fi tness in vivo relative to that typically measured in culture. thus it seems that in vivo there may not be one fi tness but several that cannot be distinguished or individually measured. it is likely that various in vivo barriers require distinct fi tness solutions that tend to create bottlenecks and that the diversity per se is essential to get past such bottlenecks. a population, not a clone or a consensus, appeared more fi t as higher titer infections of dg s also failed to be pathogenic. thus, higher levels of a consensus virus are not equivalent to higher diversity. the relationship between rna polymerase structure, error rates, and ribavirin action is discussed by cameron in chapter and has been the subject of numerous studies ( crotty et al. , ; vignuzzi et al. , ) . knowledge of the structure and catalytic mechanism of rna polymerase function has allowed a greatly enhanced level of detail to be considered into what affects error rate (see castro et al. , ; korneeva and cameron, ; marcotte et al. , ) . this has provided insight into the likely action of ribavarin on product fi delity ( harki et al. , ) . thus, it appeared that even a mutant of rna polymerase with increased fi delity could still generate elevated diversity by various methods. such control of fi delity allowed for the design of control experiments in which the same consensus virus genome could be forced to generate either less or more diverse progeny populations. in no other virus-host system have we attained such detailed insight into issues of error rate as those that were put to such excellent use in the poliovirus-mouse system. how generally important is this poliovirus in vivo quasispecies result? although the poliovirus-mouse system provides us with a fi rm experimental result, it seems likely that the generality of this relationship will be questioned by evolutionary biologists for several reasons. for one, this was observed in a lab constructed model system, which, it could be argued, is not an accurate representation of in vivo virus-host fi tness. also, as mentioned above, group selection is a process that will not readily be accepted as representative by the broader community. is there any evidence that this result with poliovirus indeed represents a general virus-host evolutionary relationship in natural settings? as presented in chapters - , retroviruses and also human hepatitis virus c clearly exist as quasispecies populations that affect disease outcome. in the case of the retroviruses, viral populations show diversity that far exceeds that seen for other rna viruses. in both hiv- and hcv there is clear circumstantial evidence for the importance of quasispecies for in vivo disease outcome, drug resistance, and fi tness. in addition, with hcv, cns infection may sometimes result, and such brain infections appear to be mediated by distinct quasispecies ( forton et al. , ; forton et al. , ) , reminiscent of the polio virus mouse model. quasispecies memory, as mentioned above, also seems to be an important issue with regard to failure of antiretroviral therapy ( kijak et al. , ) and it appears that pol gene mutations could also be involved in this ( carobene et al. , ) . measurements of hiv quasispecies in individual patients indicates that multiple evolutionary patterns can be found in typical individual patients ( casado et al. , ) , thus mixtures of hiv exist in patients ( bello et al. , ( bello et al. , , . and hiv- recombination is clearly contributing to diversity ( kijak and mccutchan, ) . thus, with both hiv- and hcv, their capacity to cause human disease is clearly associated with quasispecies compositions that affect fi tness in complex ways. the poliovirus mouse system therefore appears to refl ect quasispecies issues as observed in natural virus-host situations. consideration of retrovirus-host evolution introduces another large issue in evolution: genomic viruses. unlike poliovirus and most rna viruses, retroviruses (e.g. non-lentivirus) have colonized the genomes of animal species in large numbers and represent a large fraction of these genomes. genomic retroviruses are present in vast numbers, most of which are defective and mutant copies. in this genomic colonization they resemble the dsdna viruses of prokaryotes (discussed below) that also colonize all prokaryotes although at a much lower numbers. the human genome has fewer than genes, but appears to have retroviral-related ltr elements. some of these elements are intact and conserved (human ervs (hervs)) and this genomic population has some clear characteristics of a viral quasispecies. such large amounts of genetic material have previously been dismissed simply as selfi sh or junk dna of no fi tness consequences to the host. however, given the importance of quasispecies mutant genomes for viral fi tness and persistence, we might need to re-evaluate this dismissal. retroviruses are clearly part of the human ancestry thus we should seek to understand, not dismiss their role in human evolution. in contrast to the story above in which polio infection of mouse brain was dependent on the quasispecies resulting from lowered fi delity replication, a different relationship has been proposed for the nidoviruses. these are also positive single-stranded polycistronic rna viruses ( gorbalenya et al. , ) . this group of virus includes the coronaviruses (e.g. mouse hepatitis virus and sars-associated coronavirus), which are the largest rna viruses known ( - kb). it has been proposed that such large genomes have required the adaptation of a high-fi delity rna polymerase in order to increase the error threshold and accommodate large rna genomes. based on the phylogenetics of this polymerase and other rna-processing enzymes, this group of viruses appears to be monophyletic and it is thought that the acquisition of a high-fi delity rna replicase was central to the origin of this lineage. this type of replicase is unique to rna viruses. the monophyletic view stems from an analysis of a small set of conserved genes. overall, however, these larger genomes have many other genes that show no similarities to related viruses. the origins and evolution of these more diverse and numerous genes cannot be currently traced. this is an inherent problem in the analysis of virus evolution: a small selected set of hallmark genes with some similarity are assumed to trace an apparently linear (tree-based) viral lineage whereas the larger number of genes are not included and cannot be traced. if most of rna virus evolution is indeed mediated by a mixed cloud of genomes, any role for mutant mixtures thus becomes obscure. but perhaps there is little else we can currently do given the lack of information. how might we explain the increased fi delity and genome size of the nidoviruses? was there some change in viral adaptation in which quasispecies and generation of mixtures was no longer as important for adaptation? did the need and selection for a larger genome override the use of error to generate adaptability as seen in poliovirus and hiv- ? if so, what selective pressures might have changed this seemingly basic feature? what do we know about the natural biology of these viruses, which might provide some insight into this? unfortunately, the natural distribution and gene functions of the nidoviruses are generally poorly understood. in terms of coronaviruses, numerous mammal and avian species can be infected and the virus will cause acute disease. in several of these acute infections, the virus involved seems to have recently been adapted to the new host from other, often unknown sources. with the recent emergence of the sars virus and human infections, however, much greater attention has been focussed on trying to understand the origin and evolution of this virus. it has recently become clear that there indeed appears to exist an evolutionary stable source of this virus from which adaptation to humans was possible. various bat species have been found to support persistent asymptomatic infections by specifi c versions of sars viruses ( tang et al. , ; vijaykrishna et al. , ) . these studies also indicate that there appear to be three different and independent groups of sars viruses in bats. in fact six novel coronaviruses were isolated from six different bat species showing an astonishing diversity in bats. furthermore, phylogenetic analysis indicates that all bat coronaviruses appear to have descended from a common ancestor. only one of these bat groups includes sars and sars-like coronaviruses that adapted to acute human infections. thus, a prevalent and speciesspecifi c persistence of sars viruses is found in particular geographical populations. why is this relationship stable? could the adaptation to a host-specifi c persistence-based basal life strategy provide some explanations for the evolution of the higher fi delity rna replicase of these coronaviruses? as i have argued, persistent viral infection represents the majority of evolutionary stable viral lineages ( villarreal, ) . however, we have almost no knowledge regarding how these bat sars viruses persist and escape elimination by innate and adaptive immunity and what, if any, role the high-fi delity replicase (or other genes) have in this life strategy. although we cannot yet evaluate natural sars virus persistence in native bat hosts, another coronavirus may be more informative regarding the effects of persistence on host populations. mouse hepatitis virus (mhv) may provide our best exemplar of virus-host relationships and show how the concept of virus addiction relates to population persistence. mhv is the best-studied coronavirus. as a natural and prevalent virus of rodents, mhv is our best natural model of persistent rna virus-host relationships for any mammal. in general, rodents are the most studied non-domestic mammals with regard to natural virus distribution. overall, we know that wild-caught rodents seldom show signs of acute virus infection ( kashuba et al. , ) . however, asymptomatic virus persistence is ubiquitous in wild rodents ( descoteaux et al. , ; gannon and carthew, ; schoondermark-van de ven et al. , ) , including voles ( descoteaux and mihok, ) . some fi eld studies have evaluated broader patterns of virus persistence in mice ( singleton et al. , ; becker et al. , ) which indicated that wild house mice are highly colonized with mhv ( - % prevalence). in addition to mhv, mouse cytomegalovirus, mouse parvovirus, mouse thymic virus, and mouse adenovirus are also prevalent. other well-studied mouse viruses, such as lymphocytic choriomeningitis virus (lcmv) and polyomavirus (pyv), were at low natural prevalence. interestingly, some non-native house mice that have colonized isolated islands may lack mhv ( moro et al. , ) , although most other isolated island populations retain mhv ( moro et al. , ) . other small mammals have yet to show any viral disease whatsoever (hedgehogs, chinchillas, prairie dogs, gerbils, sugar gliders) ( kashuba et al. , ) . thus, asymptomatic persistent viral infection is clearly the norm in rodents. yet, in spite of this usual asymptomatic viral persistence, historically, some zoonotic viral disease outbreaks have occasionally been documented in natural populations. one such early outbreak was an epizootic diarrhea that occurred in infant mice ( adams and kraft, ) . later, it was established that one such infection was due to mouse hepatitis virus ( carthew, ; ishida et al. , ) . in spite of this disease outbreak, with mhv, it has since become clear that asymptomatic persistent infections are the norm and are highly stable. yet mhv disease outbreaks, especially in virus-free mouse facilities, are also common and severe. how does mhv attain such stable and prevalent persistence in natural population yet retain the ability to cause disease in naive populations? what maintains the mhv fi tness of natural persistence? it is well known that once mhv is established in a mouse or rat colony it can be very diffi cult to eliminate ( gannon and carthew, ; lussier and descoteaux, ) , clearly indicating that stability is rapidly attained and likely genetically programmed by the virus. i propose that these stable evolutionary states of viral persistence are due to a strategy we can call virus addiction ( villarreal, ) and that mhv can provide the exemplar of such a state. with mhv, only persistently infected mice colonies are protected from the disease that is otherwise caused by the virus. in wild asymptomatic mice, mhv is found mostly as an enteric infection. the cns demyelinated disease that mhv can induce is most observed in newborn pups ( homberger, ; nash et al. , ) and once in the brain, mhv can persist in cns with recurring disease ( marten et al. , ) . this recurring cns disease is also associated with quasispecies (in the s gene) and recombination ( rowe et al. , ) . the most serious cns disease is in s-gene variant of mhv- (jhm), thus as with the poliomouse model, pathogenic fi tness with mhv is also associated with quasispecies. such mhv disease is the bane of all mouse colonies ( knobler et al. , ) . however, once mhv persistence is attained, the problem to a mouse facility is not due to acute disease, but because immunological measurements are signifi cantly affected by mhv persistence. thus mhv alters mouse molecular identity regarding immunological (t-cell) reactions ( wilberz et al. , ) . to establish stable asymptomatic persistence, however, mhv needs to infect newborns ( weir et al. , ) , in which acute disease is prevented due to maternal passive immune antibody transfer ( gustafsson et al. , ) . being born to immune mothers thus protects against cns disease and promotes enteric (not brain) virus colonization. in addition, it appears that persistence also promotes cross-species transfer ( baric et al. , ) . mhv persistence may involve genome stability and result in a distinct evolutionary dynamic. asymptomatic persisting infections in a lewis rat, for example, showed no variation in mhv s gene sequence, and no quasispecies as seen in brain infections ( stuhler et al. , ) . the need to establish stable persistence could then be providing a strong selection for increased genome complexity and stability and might better explain the selection for the enhanced rna polymerase fi delity in nidoviruses. how might such selection operate in natural populations? evolutionary biologists often consider what might differentiate one group from another very similar group in a way that leads to two isolated and distinct populations. consider two hypothetical adjacent hay stacks harboring two mus musculus colonies, one of which is persistently infected with mhv the other which is not. what is the fi tness consequence to the colony harboring mhv relative to its uninfected neighbor? our experience with mhv in mouse breeding colony provides a clear answer. the colony that is persistently infected with mhv will have a distinct advantage over its neighbor as mhv introduced into this uninfected colony will have severe effects on the offspring. eventually, we can expect only the mhv-harboring colony will prevail in both hay stacks. this is a state i have called virus addiction. only mice harboring persistent mhv are protected against the potential pathogenic consequence of acute mhv (or related virus) infection. the population is addicted to the virus. such a state, however, is clearly affecting colonies (or groups) of host, not individuals. an individual either quickly succumbs to the virus infection or, if infected, transmits it to others in the colony. a colony is thus under selection by mhv. to generalize this state, we expect that the persistence of sars in specifi c bat populations would be expected to also affect the fi tness of the corresponding specifi c bat populations. persistence is a more demanding phenotype than acute replication. it requires greater gene complexity to counter host immunity and also to promote self-regulation. thus the enhanced fi delity of rna replication is selected in order to conserve this greater genetic complexity and stability. we know that the high-fi delity rna replication system (including rna pol, helicase, endoribonuclease, and other activities) is also present in an ancient nidovirus relative of coronaviruses, such as fi sh-isolated white bream virus ( kb rna). i suggest there will also likely be species-specifi c persistent infections with this virus that require this enhanced replication fi delity and maintain this virus in its natural habitat. thus, i suggest, an ancient persistent life strategy could more easily explain the monophyletic character of the nidovirus virus lineage. it is particularly interesting that one of these unique and conserved replication proteins (adp ribose- -monophosphate) is dispensable for culture growth ( putics et al. , ) . i suggest it will not be dispensable for persistence. the hiv- pandemic is an unfi nished story. hiv- represents a real-time biological event in human evolution that confi rms for us the importance of quasispecies and retroviruses to human biology. however, even though its human toll is huge, modern medicine and culture has responded rapidly enough to limit the impact of hiv- to the point at which it will not likely be the cause of a selective evolutionary sweep that could have altered human genetic makeup (in contrast to the koala bear endogenization presented below). as described earlier, its amazing adaptability via quasispecies along with extensive recombination contribute directly to hiv- ' s diversity and makes it the most dynamic genetic entity ever studied. many studies track the dominant hiv population and fail to examine minority populations. yet it is precisely these minority populations, which evolve independently of the majority population, that can determine drug resistance phenotype and biological outcome ( charpentier et al. , ; briones et al. , ; morand-joubert et al. , ) . clearly, the specifi c makeup of a complex hiv population matters. furthermore, hiv defectives and variants can also have major consequences. in some cases, long-term non-progressors of hiv- have shown mixed populations and unusual polymorphism in the early phase of hiv infection, sometimes contributing to long-term non-progression (ltnp) ( alexander et al. , ) . one population of ltnps was reported to have been colonized by an hiv variant that showed low virus replication and slow or arrested evolution ( bello et al. , ) . in another case, a stable non-progressor was colonized by a replication incompetent version of hiv- ( wang et al. , ) . some of these non-progressors also appear to resist super-infection . it seems clear that at least in these exceptional situations, non-majority hivs are crucial to the outcome. there is also reason to think that other retroviruses have had a major infl uence on recent primate and human evolution, such as apathogenic persisting foamy virus in primates ( switzer et al. , ; murray and linial, ) . human antiretroviral genes seem to have undergone recent adaptations, such as apobec , which can interfere with exogenous retroviruses (such as mlv and siv) and underwent an expansion in the hominid lineage ( esnault et al. , ) . it thus seems clear that human and primate evolution has been signifi cantly affected by earlier, prevalent primate retroviruses. another important human-virus quasispecies story that has long been recognized is with hepatitis c virus (hcv), (see chapter ; domingo and gomez, ) . hcv seems to have adapted to humans in the recent past, possibly from asymptomatic enteric primate viruses currently found in africa ( smith et al. , ) . as hcv remains an infection predominantly transmitted by blood, it does not appear to have fully adapted to the tissues of and transmission within its human host. however, like hiv- , hcv has long been recognized to generate quasispecies in chronically infected people ( martell et al. , ) and it soon became apparent that the viral quasispecies are affected by and affects the outcome of antiviral therapy hohne et al. , ; kurosaki et al. , ; okamoto and mishiro, ) . thus, successful antiviral therapy is directly correlated with an initial dramatic reduction in genetic diversity. unfortunately, it has become clear that only a minority of hcv-infected individuals will respond favorably to a combination of interferon and ribivarin. thus it seems to be diversity per se and the resulting structure of an hcv quasispecies that has a direct consequence to human health. however, since hcv is less well-adapted to humans compared with hiv- , it does not pose the same threat to potentially provoke an evolutionary event in human evolution. vsv is a negative-stranded rna virus that has been a very important experimental model and has provided many laboratory measurements regarding quasispecies theory (see chapter ). using vsv, evidence supporting the red queen hypothesis, involving unending adaptation to greater competition and mueller ' s ratchet has been presented ( clarke et al. , ; novella et al. , ; elena et al. , ) . when vsv was evaluated as an arbovirus, requiring adaptation to alternating and opposing fi tness of insect and mammalian host, it was also apparent that minority quasispecies populations were responsible for maintaining the apparently antagonistic phenotypes ( novella et al. , ) . thus here too, the consortia character of a quasispecies is clear. yet in natural settings several very different virus-host relationships can be seen with rhabdoviruses. a distant relative of vsv (vhsv) is also known to be responsible for mass die-off of commercially important fi sh ( marty et al. , ) . this virus infects many teleost species and has shown % mortality in many experiments (i.e. with i.p. inoculation). in natural outbreaks, however, it has also shown surprising genetic stability ( einer-jensen et al. , ) . clearly error-prone rhabdovirus replication must be kept in check by purifying selection in this situation. in contrast, another rhabdovirus, sigma virus of drosophila , is associated with no mortality but is a vertically transmitted persisting virus in specifi c drosophila populations ( fleuriet, ) . yet in some recent population measurements, sigma virus infected drosophila are expanding for unknown reasons ( fleuriet, ) . clearly this particular virus-host persistent relationship has some undefi ned selective advantage that operates beyond the lab-based concepts as measured above. other rhabdoviruses also have peculiar host-specifi c relationships, such as bats that tend to support many persistent infections ( badrane and tordo, ; li et al. , ) , or birds that seem to be free of almost all rhabdoviruses. clearly, although vsv lab results have been highly informative, we still have much to learn regarding natural settings that affect rhabdovirus adaptation and evolution. another major paradigm for the high rates of negative-strand virus evolution is found with infl uenza virus. due to its history and potential for initiating great human epidemics, it has long held the special interest of evolutionary virologists (see chapter ; nelson and holmes, ) . however, this research has not much emphasized the quasispecies character of infl uenza virus evolution. instead, it concentrates on the evolution of the master template or clades of template for the purposes of vaccine development ( webster and govorkova, ) . the views stemming from this type of evolution have lent themselves well to master template-based phylogenetic analysis and have dominated how many researchers think of virus-host evolution. thus it is curious, given the above emphasis, that the quasispecies character of infl uenza populations often seems of low relevance to issues of acute disease and vaccination, other then to provide a source of diversity. in some situations, viral competitive interference may contribute to drift variation and displacement in antigenic epitopes ( levin et al. , ). yet outcomes of individual human and bird infections do not seem much affected by specifi c quasispecies structures, as we saw with hiv- and hcv. with infl uenza, we are mainly concerned with epidemic human disease. however, by shear numbers of infections and deaths worldwide, it must be admitted that infl uenza virus is really a virus that affects mostly birds. for example, during the outbreak in china, only humans died whereas million domestic birds died ( smith et al. , ) . although our concern on the large potential for human disease is understandable, these numbers should inform us of a more basic virushost biology. in this case, infl uenza shows a high affi nity for various birds; migratory water birds in particular can have high prevalence ( wallensten et al. , ) . some waterfowl, such as wild mallard ducks, have been called the stealth (asymptomatic) carriers of infl uenza h n and free grazing ducks seem to introduce virus into domestic bird populations ( gilbert et al. , ) . thus waterfowl represent the well-accepted epidemiological concept of a reservoir species ( louz et al. , ) . but these wild waterfowl, shorebirds, and gulls that are a natural host for avian infl uenza also seem to show a much slower rate of evolution ( spackman et al. , ) . in contrast, the much higher rate of evolution as seen in chickens and turkeys indicates that these hosts should not be considered as natural reservoirs ( suarez, ) . in waterfowl, infl uenza infections show several distinctions, such as virus co-infection or virus interference ( sharp et al. , ) as well as phylogenetically distinguishable waterfowl dendograms, including specifi c m lineages ( makarova et al. , ; widjaja et al. , ) . the diverse and stable avian pool of infl uenza virus appears to be ancestral to the infl uenza viruses that infected human populations. the phylogenetic methods that have been adapted from evolutionary biology have been tremendously helpful and have allowed us to trace the seemingly untraceable, virus evolution (see chapter ). thus, we have often been able to make informed judgments concerning broader patterns of virus evolution and this has become the major tool for the current study of virus evolution, such as infl uenza virus ( nelson and holmes, ) . infl uenza a, for example can be seen to show extended periods of stasis followed by periods of rapid adaptation that necessitates adaptations in vaccine strategy ( wolf et al. , ) . however, the evolutionary variation between seemingly similar viruses can be surprisingly large (see above vsv section). for example, the very different phylogenetic behaviors between infl uenza a and measles virus, both acute human respiratory infections due to membrane-bound negative-stranded rna viruses, are striking. the reasons for the maintained genetic stability of measles virus remain poorly understood, but may well involve more complex fi tness associated with systemic infections. phylogenetic methods can also be highly informative regarding the likely origins of viral lineages and possible sources of emergence. for example, the studies of dengue virus by holmes and colleagues suggest that this virus fi rst entered its human host about years ago, and that sylvatic (african jungle) asymptomatic infection of primates may have provided the origin of this virus that later became a human pathogen ( holmes and twiddy, ; holmes, ) . such insight provides valuable clues concerning the likely selective pressures that may lead to the emergence of dengue virus. phylogenetic methods are also highly informative regarding classifi cation and taxonomy relationships and have allowed us to understand viral relationships across broad species defi nitions ( zanotto et al. , ) . however, phylogenetic approaches necessarily assume the master template is the fi ttest type and that mutations or variants in the rna populations are a source of genetic load that are deleterious and limiting to virus adaptation ( pybus et al. , ) . such variation is mostly due to " unfi t " mutations, which indicates that a viral cloud is mostly and unfi t consortia. it would seem that such conclusions go against the concept of quasispecies as being fi t per se as described above. in this consideration we see a major weakness of extant phylogenetic methods. they were not developed to access the evolutionary relationship and fi tness of interacting mixtures. nor were they designed to follow the evolution of systems with high rates of recombination between numerous parental templates. we currently lack the analytical tools for such a population analysis. without such tools, however, it seems we can only evaluate those parameters we can defi ne and will remain confused by those we cannot. evolution of a consortia thus provides a new directions for theoretical and laboratory research. we should seek to investigate the mixture, not just its average. another major virus-host system that has been highly studied is the viruses of agricultural plants. our understanding of plant viruses has also been highly infl uenced by disease associated with agricultural domestic species, thus natural virus-plant relationships are much less understood, although some recent fi eld studies are starting to change this situation (see chapter ). we currently have a rather uneven understanding of broader virus-host relationships and evolution in plants. for example, viruses of the more ancient ferns, if they exist, are essentially unknown. the prevalence and diversity of positivestranded rna viruses in plants is striking. in addition, we are starting to appreciate that virus-virus interactions are also frequently involved, although this issue remains poorly studied. one well-studied family of plant virus are the tobamoviruses of angiosperms (see chapter ; gibbs, ) . progenitors of this virus family appear to also be found in algae and fungi consistent with a very long evolutionary history. both high transmission between host and virus-host congruence are observed with these viruses. virus-virus interactions also seem to be important. for example, tobacco mosaic virus (tmv) and tomato golden mosaic virus (tmgv) appear to have shown interactions in australia which have apparently led to the extinction of tmv, but the retention of tmgv with no increase in genetic diversity ( fraile et al. , ) . plant viruses have also been seen as quasispecies in some but not all settings (see chapter ; roossinck, ; roossinck and schneider, ) . besides the interactions expected for typical viral quasispecies, plants often show evidence of more extensive mixed virus infections. there are, for instance, many examples of satellite viruses that must necessarily interact with other rna viruses of plants. it is also clear that the subviral elements of even a single viral lineage can greatly affect the virus-host relationship. such subviral elements (dis) have been observed to both reduce and intensify disease, and also interact with satellite viruses ( qiu and scholthof, ) , thus virus-virus interactions are clearly crucial in many situations ( simon et al. , ) and viral interactions and synergism appear to have led to signifi cant events in plant virus emergence ( fargette et al. , ) . virus-virus interactions are not limited to plant rna viruses. the ssdna plant geminiviruses also display complex interactions with satellites as well as high diversity in fi eld isolates of east africa ( ndunguru et al. , ) . thus, plant viruses seem particularly prone to interactions. more recently, virus-mediated symbiosis with respect to host survival has been reported ( roossinck, ) (discussed below). phylogenetic methods also struggle to address the occurrence of high rates of recombination in viral lineages. such a situation complicates the analysis, creating hardto-defi ne, reticulated trees, although these limitations can be partially overcome by using sliding windows for the analysis. such approaches have allowed surveys of recombination in some viral lineages, such as with the plant potyviruses ( chare and holmes, ) . however, the rampant recombination and quasispecies generation of hiv- makes a quantitative assessment of the virus population problematic. one proposed solution is to use a composition vector method ( gao and qi, ) . the issue of measuring recombination and tracing evolution in large populations is especially a problem that applies to the dna viruses (phage) of prokaryotes (see below). cells. our perception regarding the overall importance of dna viruses of prokaryotes to the evolution of life on earth has undergone a major shift in recent years. the main realization is that dna phage are the numerically dominant genetic entity in most habitats on earth (mentioned above). in addition, as discussed in chapter , it is now clear that some of these viruses are surprisingly complex and that essentially the entire pool of dsdna viruses of prokaryotes may be exchanging dna via recombination at high rates. this would constitute by far the largest common gene pool on earth. historically, the evolution of the dna viruses of prokaryotes has seldom been considered in the broader context of virus evolution or evolutionary biology. although it has long been realized that there are many basic similarities between viruses of bacteria and eukaryotes ( luria et al. , ) , not until structural studies solved the capsid genes of prokaryotic and eukaryotic viruses did the evolutionary relationships between these viruses become clear. in addition, there have been a number of striking proposals that suggest that dna viruses of prokaryotes may be involved in the origin of several major systems used by cells and that viruses appear to be involved in several major transitions during host evolution. thus we now consider the possibility that these dna phage were fundamental to the origin and evolution of life on earth. it now seems likely that some large dna viruses infecting eubacteria, archaea, and eukaryotes share some common evolutionary histories. it also seems clear that such viruses can link all three domains of life. this realization was not apparent based on phylogenetic sequence conservation, which is absent. it stems from the structure and assembly of virion capsids in which t phage, halophage, and the herpesviruses all show clear similarity as well as similarity in replication strategies. in addition, phage prd and adenoviruses show similar broad structural and strategic conservation. some biochemical (dna pol family) and genetic similarities (gene order, gene programming) are also apparent, which taken together supports the common origins of these viruses ( hendrix, ( hendrix, , hendrix et al. , hendrix et al. , , . t -like viruses in particular seem to represent a major source of global genetic diversity. this giant genetic pool represents a huge potential to affect life ( filee et al. , ) and the viral genetic creativity represented by this pool would also be vast ( nolan et al. , ) . since t -like phage that infect cyanobacteria also encode virus-specifi c type ii photosynthetic core genes, viruses appear able to create the most complex of genes as well ( clokie et al. , ; sullivan et al. , ) . as presented in chapter , phage are now thought to evolve by distinct and highly mosaic " horizontal " processes of rampant recombination ( hendrix, ( hendrix, , . large dna phage appear to be ancient, present before the split of the three main branches of cellular life: bacteria, archaea, and eukarya ( benson et al. , ) . luca, the last universal common ancestor, would represent the putative cell ancestor prior to this split. however, phyogenetic analysis of common or conserved genes of luca identifi es only about or fewer genes in extant cellular genomes ( mushegian, ; koonin et al. , ; mirkin et al. , ) . ironically, the genes needed for dna replication are not part of this conserved set, calling into question the nature of the fi rst dna-based cell. large-scale " horizontal " transfer seems to have clearly prevailed early in the evolution of dna-based cellular life and it has recently been asserted that luca existed in a highly horizontal " consortia " of cooperative genes that developed the common genetic code ( vetsigian et al. , ) . since the dna replication proteins in the extant three domains of life have distinct compositions, it has been proposed by forterre that dna viruses and retroviruses were directly involved in the invention of the three extant cellular dna replication systems . according to this view, early cellular life was completely entangled with viral (phage) lineages; hence cells must have evolved from an ancestral " virus " -mediated population not a single genetic lineage. thus the evolution of early life would have clear similarity to the quasispecies (consortia) state of genetic information as seen in rna viruses above. thus the huge creative and adaptive potential of virus would have been directly involved in the very earliest evolution of life. clearly, such conjectures regarding the most ancient events in the evolution of life are hard to substantiate. but, these theories are as viable as any other and deserve serious consideration. in spite of this seemingly unending mosaic exchange in dsdna phage, some phage isolates show surprisingly stable genetic makeup. we now accept that t -related phage are an important source of the larger global phage genetic diversity and that most such viral genes are novel ( filee et al. , b ; nolan et al. , ). yet even with t -like viruses, there can be clear barriers to horizontal gene transfer which promote the evolution of stable viral lineages ( filee et al. , a ) . in t -type phage, similar core genes could be seen in all genomes, which seem to be inherited in gene blocks that preclude recombination. however, these blocks were not seen in the broader t-even and pseudo t-even genomes. other phage also show surprising genetic stability when repeatedly isolated from similar habitats, such as soil phages of burkholderia ( summer et al. , ) and bam ( saren et al. , ) as well as some hot spring isolates ( khayat et al. , ) . this bam capsid also identifi es another structural motif mentioned above that is broadly conserved in evolution and shows clear similarity to that capsids found in prd and pbcv- (discussed below). sh also has a clear prd related capsid, membrane, and genome; thus this halophilic euryarchaeon virus, although showing no sequence similarity to prd or any other bacterial phage, is clearly structurally related ( bamford et al. , a ) . it is interesting that overall the viruses of hyperthermophilic crenarchaeota generally show no sequence relationship to phage of bacteria. in addition, the use of the term phage for these viruses can also be questioned as most establish non-lytic chronic infections. many of these crenarchaeota viruses have unique morphologies not found in any other domain of life ( prangishvili et al. , a ( prangishvili et al. , , b ortmann et al. , ) . some, however, have clear structural and genetic similarity to specifi c phage (i.e. t ). considerations of phage evolution and rampant recombination (especially with t and t-even phage) often emphasize the viral lytic lifestyle and host death. in fact this lytic relationship was argued by many early phage researchers to be the fundamental and only character of phage-host relationships in general. we now know, however, that persisting (temperate) phage are also common, some of which have no independent lytic phase. the fundamental model of phage persistence by unique integration into host chromosomes (temperate lysogeny) marks a major development in our understanding of molecular virology and virus-host relationships which was fi rst clarifi ed by campbell in (see campbell, . all free-living prokaryotes show the presence of colonized phage in their genomes. both complete and defective genomes of dsdna viruses have been observed in the sequenced dna of all free living prokaryotic genomes ( gelfand and koonin, ) (exceptions are some intracellular parasites and plastids). thus, the massive genetic diversity and novelty of phage evolution as presented above has a direct conduit into the genetic composition of all prokaryotes via lysogeny. the fi tness and evolutionary consequences of such colonization to the evolution of the host and its virus should be considerable but is in need of theoretical development. fitness of temperate phage, however, is more complicated then that of a lytic virus and, like fi tness of persistence discussed above, cannot be simply described by relative replication or effi cient virion production. here too, successful phage colonization must inherently limit the replication of the same virus. thus, a temperate lifestyle also requires an autoinhibitory capacity. this generally involves an immunity gene set that not only limits self-replication but can also affect replication of other temperate and lytic viruses, i.e. lambda (even as a defective) precludes t and other t-even phage. uncolonized hosts are thus susceptible to lysis by highly prevalent acute tailed phage. host fi tness is thus strongly affected by a temperate phage due to its ability to preclude and survive other competing phage. i suggest this situation is similar to the mhv-mouse exemplar above, in that virus-colonized hosts are in a state of " virus addiction " in which persistence is needed to provide protection from the same or similar virus ( villarreal, ( villarreal, , . it is well established that most natural populations of bacteria have specifi c patterns of phage colonization, hence the utility of phage typing for strain identifi cation. from this, we can infer that virus-virus competition is a prevalent and major issue regarding the prokaryotic fi tness resulting from a symbiotic temperate phage-host combination. in addition, such virus-host symbiosis can also affect competition with other bacteria. this would be very much like the virus addiction concept outlined above for the mhv examplar. the original observation of a lysogenic process and coining of this term occurred in the s when two pure cultures of bacteria were grown together. it was observed that in some combinations, one strain would lyse the other strain (was lysogenic). later, it became clear that such lysis was mediated by reactivation of temperate phage present in the lysogenic strain, but absent from the non-lysogenic susceptible strain. in this relationship, we see another example of group selection operating on bacterial populations harboring a persistent virus. thus, what host is fi t depends very much on the prevalent viruses it will encounter as well as the viruses that colonize it. bacterial populations that are colonized by the same or similar phage express the appropriate immunity functions and are protected from lysis by the same or similar phage. such a situation has signifi cant implication for the evolution of immunity and group identity for cells. host stability becomes a major fi tness issue for a persistent virus life strategy. it is generally thought that a temperate virus attains a stable colonization of its host by simply integrating into and become one with the host genome. however, there are also clear examples of stable phage persistence that does not integrate and uses other strategies to attain host stability (similar to eukaryotic dna viruses; see below for the p phage exemplar of this). like a temperate phage, a host that is colonized by episomal persisting viruses has also been much affected in its evolutionary potential. it is clear that phage can have complex effects on host populations, but these phage themselves often exist in complex and mixed states that can be diffi cult to unravel ( harcombe and bull, ) . it has been known for some time that the presence of otherwise silent phage can greatly affect the growth of other virus and susceptibility of host. one such silent and common phage that has long been studied is p . p was initially discovered due to its effect on t and lambda. however, p has been a very interesting model, not because it causes disease or offers potential therapy against bacterial pathogens, but simply because it persists effi ciently as an episome and competes effectively with many other phage ( yarmolinsky, ) . since it does so without integrating, p provides us with one of the only well-studied models that can inform us regarding the molecular strategies and details of how stability in non-genomic persistence is attained. curiously, a main strategy by which p attains this stability was inapparent and not suspected after several decades of study. it became apparent only after replication mutations were made that induced self-destruction and uncovered the existence of what came to be called " addiction modules " ( lehnherr et al. , ) . p encodes several gene pairs (toxins/ antitoxins, such as the phd/doc pair) that protect bacteria harboring p , but kill daughter bacteria that have lost the p genome ( gazit and sauer, ) . this strategy compels colonized e. coli to maintain p or die (doc, death on curing). however, these very same addiction systems are also involved in protecting a p -colonized colony from t and lambda infection and will also induce self-destruction when cells are infected by those viruses, protecting the colony (population). p also provides an exquisite level of molecular self-identifi cation in that it will recognize a single second copy of its own genome ( yarmolinsky, ) . what then is the fi tness and evolutionary consequence to e. coli harboring p ? clearly it is major, but mostly host fi tness is affected relative to other viruses. accordingly, when contemplating the amazing complexity of the p immunity and how it evolved, yarmolinsky posed the question; " could the byzantine complexity of the controls at immi be the outcome, not of successive host-parasite accommodations, but of competition among related phages? " ( yarmolinsky, ) . if we answer yes to this question, then we would also conclude that virus-virus interactions and competition in general are major forces in the adaptability and evolution of persisting phage and surviving colonized host. in this light, viral persistence takes on a major role in virus and host evolution. the p exemplar has thus provided us the concept of viral addiction that also promotes host group selection. historically, we are biased to think of viruses (and phage) as agents that simply kill their host. some have proposed that the prokaryotic global biomass is phage partitioned into those populations that live and those that die due to viral lysis. from such a perspective, viral novelity would seen of little relevance to host evolution. metagenomic projects as noted above, have sequenced nearly million phage genomes and report that most of these phage genes are unique, not in the database, and likely not derived from host ( edwards and rohwer, ) . the protein repertoire of sequenced phage indicates that % of conserved phage genes are specifi c to phage and show an evolutionary independence from genes of host . this identifi es a massive genetic novelty from virus, which is especially apparent in large dna phage. as just discussed above, however, those hosts that live are also products of phage selection, and persisting temperate phage play a major role in this. such phage colonization allows this massive phage novelty to fi nd its way into host genomes, which allows viral complex gene sets to be applied to novel problems of host adaptation. host novelty can thus be introduced by phage ( comeau and krisch, ) . that persistence is a major life strategy of phage is confi rmed by the large numbers of genes associated with persistence (i.e. integrases, immunity) observed in metagenomic screens. there is also much practical experience that supports the crucial role of prophage in host evolution. one particularly well-studied system that has been studied for over years is the ongoing evaluation of phage evolution as observed in the dairy industry ( canchaya et al. , brussow et al. , ) . the temperate phage analysis of these bacteria follows a long tradition of lambda and e. coli studies ( campbell et al. , ; canchaya et al. , canchaya et al. , , . since lytic phage can severely disrupt dairy fermentation, it was of particular interest to understand and trace their evolution. these studies have led brussow to conclude that much of the more recent dairy bacteria evolution can be considered to have resulted from the action of temperate phage. a similar view applies to e. coli and cyanobacteria. in addition, the ecor collection of sequenced e. coli genomes of medical interest shows that they differ from each other mainly due to patterns of genetic colonization, mostly by prophage, but they also show the presence trna-adjacent defective prophage and plasmid elements that differentiate these strains ( hurtado and rodriguez-valera, ; mazel et al. , ; nilsson et al. , ) . cyanobacteria ( prochlorococcus ) is major model for the study of the origin of the type ii (plant-like) photosynthetic system. since such genes show much evidence of recent and massive horizontal movement, it seem quite likely that prophage are mediators of such transfers, especially as these phage encode their own version of these photosynthetic genes ( lindell et al. , ; sullivan et al. , ) . very similar prochlorococcus strains exist in distinct oceanic populations in various habitats known as ecotypes. some think that such ecotypes represent the initial type of genetic variation that leads to speciation. the sequencing of six ecotypes has shown that they are % similar to one another, but the genetic variation that distinguishes them is mostly due to patterns of prophage colonization (called phage islands) ( bouman et al. , ; coleman et al. , ) . thus in all these prokaryotic models, persisting viruses play a fundamental role in host evolution and host genetic novelty is mostly phage derived. such observations have led some to propose that " war is peace " regarding virus-host evolution ( comeau and krisch, ) . massive and complex innovation by phage appears to be a major force in the prokaryotic world. prokaryotes are the most adaptable of all cells. if we can accept the above conclusion concerning the role for viruses in the evolution of prokaryotes, we must then ask why such a successful evolutionary strategy was not apparently maintained in eukaryotes? in eukaryotes we see little evidence that largescale integration by dna viruses is an important evolutionary process (although the story with retroviruses is different). why should prokaryotes and eukaryotes differ is such a fundamental way? nevertheless, as noted at the start of this section, we do see good evidence that links the evolution of large dna viruses of prokaryotes to the large dna viruses of eukaryotes. in case we were becoming comfortable with the apparently clear distinctions between rna and dna virus evolution as outlined above (quasispecies vs. domain recombination respectively), the evolution of the parvoviruses informs us that dna viruses can also evolve by a quasispecies process. parvovirus evolution (see chapter ) can show a sharp contrast to the evolutionary pattern displayed by other small dsdna viruses above (hpv, py). with the emergence of an acute pandemic in domestic dogs and cats (as well as other wild carnivore species), we see what is essentially evolution driven by single point mutations, mostly affecting the capsid genes and host cell receptor binding. this system provides us with one of the better studied examples of the evolutionary dynamics of an emergent viral disease. in addition, in vivo mouse studies with minute virus of mouse (mvm) now make it clear that parvoviruses can behave much like rna viruses, generating quasispecies of diverse progeny that allow a high adaptability for the generation of fi tness and disease in vivo ( lopez-bueno et al. , ) . this story is very reminiscent of the study of poliovirus in mice mentioned above. human studies with b parvovirus are also consistent with high mutation rates ( parsyan et al. , ; shackelton and holmes, ) . although not specifi cally addressed in this volume, the viruses of eukaryotic unicellular green algae are of special interest from the perspective of dna virus evolution. these large, complex dsdna membrane-containing icosahedral viruses are abundant in some water habitats ( van etten, ; ghedin and claverie, ) . the reason they deserve special attention is that they clearly have many features that are characteristic of both prokaryotic and eukaryotic viruses. they resemble prokaryotic viruses in that their life cycle is clearly phagelike, such as external virion attachment, injection of dna and no pinocytosis. in addition, they also encode many phage-like genes, such as restriction-modifi cation enzymes and homing endonucleases ( filee et al. , c ) . they also resemble eukaryotic viruses in that they have eukaryotic dna replication proteins (dna polymerase beta and pcna; chen and suttle, ; nagasaki et al. , ; villarreal and defilippis, ) as well as many genes associated with eukaryotic signal transduction ( van etten et al. , ) . thus they represent a clear link between prokaryotic and eukaryotic dna viruses. for example, the dna polymerase of paramecium bursaria chlorella virus (pbcv- ) is the most conserved gene and most closely resembles that found in human herpesvirus and is distantly related to the similar family dna pol encoded by t . this polymerase is distinct from that of the poxviruses or prd /adenoviruses (associated with protein-primed dna replication). however, numerous other genes of the phycodnaviruses are similar to some genes found in the mimiviruses (giant dna virus of ameba), including the presence of conserved intenes in the dna pol gene ( ogata et al. , ) . in view of this it is most curious that in structural similarity, polydnavirus capsids clearly resemble prd capsid ( khayat et al. , ; nandhagopal et al. , ) . prd contains the double-barrel trimer capsid structure that was fi rst observed in adenovirus (for references see saren et al. , ) . adenovirus also closely resembles prd in dna replication strategy (i.e. linear dna with covalently closed ends ( benson et al. , ; khayat et al. , ) . the lineage of adenovirus-like dna viruses, however, is thought to be distinct from that herpes and poxviruses and its dna polymerase is clearly distinct from polyndavirus. it is clear that related elements of all these viruses can be found in phycodnaviruses. overall, the phycodnaviruses, like phage, also appear to be creating genes in large numbers and they encode many genes unrelated to their host. what then is the evolutionary relationship that links all of these seemingly distinct viruses? as outlined above, the pattern of evolution of dsdna phage involves lots of exchange by recombination from a vast gene pool. this pool resembles a cloud from which various mosaic subelements and substrategies are assembled to allow viral gene acquisition and novelty ( blum et al. , ; benson et al. , ) . does such a distributed pattern of evolution and gene novelty also apply to the phycodnaviruses? recently, another distinct phycodnavirus has been sequenced: coccolithovirus (ehv- ) ( allen et al. , a ( allen et al. , , b conserves only core genes in common with pbcv- and is unique to the phycodnaviruses in that it has acquired six dnadep rna polymerase subunit genes, which are absent in all other phycodnaviruses. as rna polymerase is considered a core viral gene function, it is clear that phycodnaviruses can alter some very basic molecular functions during their evolution. oceanic phycodnaviruses are thought to have large infl uence on the free-living populations of eukaryotic algae, such as the termination of algal blooms reported for emilian huxley virus ( martinez et al. , ; schroeder et al. , ) . however, not all phycodnaviruses are lytic. another lineage of phycodnaviruses is represented by two viruses of fi lamentous brown algae, esv- and firrv- ( delaroque et al. , ) . unlike the lytic phycodnaviruses noted above, these two viruses are " temperate phage " like. that is they exist as silent viruses whose dna is integrated into the germlines of their host. in this, they are unique to all known eukaryotic dna viruses; host chromosome integration is a normal part of their persistent life strategy. esv- has a -bp genome and encodes likely genes ( delaroque et al. , ) . these genes are mostly unique and only are clearly related to pbcv- genes. the gene differences include many replication genes and their gene order is completely different. like the temperate phage-host evolutionary relationship outlined above, it would be most interesting to understand how the integration of these large dna viruses has affected host evolution. thus, the phycodnaviruses appear to represent a basal but diverse viral lineage that has both acute and persistent lifestyle and have some clear relationships to most large eukaryotic dna viruses and many phage. the phycodnavirus exemplar above should leave us with several impressions regarding the nature and evolution of these large and ubiquitous dna viruses of algae, an early eukaryotic host. they show clear linkages by structure and function to both phage and various eukaryotic dna viruses. they also show major variation and novelty in their own genetic composition, including their core genes. in addition, they show clear relationships to distinct and seemingly separate viral lineages (adenoviruses, herpesviruses, poxviruses, iridoviruses). the picture we are left with is that they seem to resemble phage evolution in that they appear to have evolved from a diverse pool that has exchanged many basic viral features and created many new genes. this view, however, contrasts sharply with the work of iyer et al. ( iyer et al. ( , . by considering the small number of conserved genes in four families of eukaryotic dna viruses (poxviruses, asfarviruses, iridoviruses, phycodnaviruses), they suggest that these viruses are monophyletic, evolving from a common nucleo-cytoplasmic large dna virus (ncldv) with an icosahedral capsid. given the above information, i fi nd this view unhelpful and possibly confusing. it has numerous problems. the main problem is that it fails to acknowledge the clear link between prokaryotic and eukaryotic viruses. furthermore, by focussing on a small set of related genes, it represents a traditional perspective as found in evolutionary biology that assumes a common (fi ttest) linear lineage, not a cloud, cooperative, or mosaic pool as the main source of novelty resulting in the matrix pattern of virus evolution. the virosphere is clearly not disconnected from itself, but it is also clearly not a linear or tree-like evolutionary system as suggested above. we must learn to think of virus evolution in its own terms; fuzzy, mixed, reticulated, and cloud-like. as mentioned in the phage section, there have been various publications that suggest a deep evolutionary relationship between the herpesviruses and dsdna viruses of prokaryotes ( rice et al. , ; khayat et al. , ; duda et al. , ; akita et al. , ) . such enormously distant relationships, however, cannot now be measured by any reliable metric. although herpes-like viruses are found in invertebrates (such as ostreid herpesvirus (oshv- )) in both lytic and asymptomatic states ( barbosa-solomieu et al. , ) , our interest in their evolution has been mainly focussed on the vertebrate herpesviruses. vertebrate herpesvirus do tend to show clear sequence conservation that suggests broad patterns of evolution. one interesting feature of this evolution is the apparent link between the biology of the virus and its evolution. a common, but not universal pattern is that of virus and host co-evolution ( mcgeoch et al. , ( mcgeoch et al. , , mcgeoch and gatherer, ) . this trend has maintained several biological characteristics, such as highly species host-and tissue-specifi c persistence (i.e. neuronal and lymphoid persistence). the discovery of hhv- has further stimulated studies of herpesvirus evolution in that hhv- appears to have undergone much recombination with herpesviruses of related primate lineages ( mcgeoch and davison, ) . thus recombination seems prevalent in herpesviruses. the apparent link between herpesvirus evolution and recent human evolution, as well as an apparent link to primate retroviral evolution, is fascinating, but of unknown signifi cance ( kung and wood, ; lacoste et al. , ) . the herpesviruses lineages will often show the presence of lineage-specifi c genes. many of these genes affect innate and adaptive host functions, whereas others affect host metabolism. when the source of such genes has been contemplated, in contrast to phage, phycodnaviruses, or baculoviruses ( herniou et al. , ) , it is often proposed that most such herpes genes originate from the host. it is well accepted that the three major lineages of herpesviruses descended from a common ancestor in vertebrates ( mcgeoch et al. , ) . there have been numerous proposals that most new lineage-specifi c herpesvirus genes have originated from host (see becker and darai, ) . this includes herpesvirus dutpase ( davison and stow, ) , and viral chemokines and viral bcl- ( nicholas et al. , ) . in my evaluation of such claims, however, it seems clear that the possibility that there was an ancient viral source of such genes was not considered and cannot now be dismissed. we currently believe that ancient herpesvirus ancestors can be traced to tailed phage ( hendrix, ; bamford, ; baker et al. , ; duda et al. , ; mcgeoch et al. , ) . other phage lineages also appear to trace to eukaryotic viruses ( bamford et al. , b ) . within the herpesviruses, the same t- icosahedral structure, as well as invertable dna regions are also present in the very distant but much more recognizable oceanic ostreid herpesvirus . given the highly diverse and mosaic nature of large dna virus evolution in prokaryotes and lower eukaryotes described above, it seem quite possible that many other viral genes might also trace far back in virus evolution. consider the example of dutpase in avian and mammalian herpesvirus ( davison and stow, ; mcgeehan et al. , ) . the current view requires very complicated gene rearrangements to account for the viral source of this gene from its host. yet we know that diverse dutpases are found in many ancient viral lineages. for example, the ervs present in all vertebrate genomes also conserve dut-pase ( jern et al. , ) , as do exogenous retroviruses (i.e. lentiviruses) ( mcintosh and haynes, ) . in fact, since the herpesviruses genes are especially poor in introns, it would seem likely that any herpesviral gene acquisition would necessarily involve a retrovirus via a cdna. the oceans are especially fi lled with large complex dna viruses (such as mimivirus and phycodnavirus, plus numerous relatives of oshv- ) thought to be ancient ancestors of herpesvirus. the phycodnavirus (chlorella virus, pbcv- ) provides a clear bridge between phage and eukaryotic dna viruses. pbcv- also encodes a dutpase that has the highly conserved motif iii ( zhang et al. , ) . many phage are also known to encode dutpases of diverse types, such as b. subtilis (spbeta) ( persson et al. , ) , and a phage of thermus thermophilus ( naryshkina et al. , ) . this thermus phage (phiys ) is of special note since its dutpase gene is clearly related to the dutpases of eukaryotic viruses and has a version that has undergone multiple events of recombination from apparently distinct phage, exactly as expected for mosaic phage genes. thus, the origin of new herpesvirus genes might not be so different than that seen in other large dna viruses and a potential ancient source of new genes from these ancestral viruses remains plausible. similar considerations apply to other possible examples of herpesvirus gene capture. for example, the herpes thymidylate synthase (ts) has also been considered to have originated by host gene capture ). yet distinct versions of these genes are also found in different herpesviral lineages, which would necessitate multiple independent " capture " events of different version of host ts genes. ts genes are present in ancient virus sources. for example, bacillus phage beta encodes ts, which also has a self-splicing intron ( bechhofer et al. , ) . also, phage phikz has a highly conserved ts ( mesyanzhinov et al. , ), yet this virus lacks a dna polymerase or other replication proteins, clearly indicating that the viral ts genes has a basic viral role. similarly, the cytokines-like genes (such as il- ) as found in poxviruses and herpesviruses appear to have originated in at least three independent events prior to the divergence of mammalian eutherian orders. yet it is still presupposed that they are necessarily the products of host gene capture ( hughes, ) . comparative genomics supports the idea that the herpesviruse lineages are originating viral genes. a broader phylogenetic analysis of all herpesvirus genomes identifi ed only genes in common to all taxa of herpesvirus . thus only genes appear to be in common to all the herpesviruses. in this analysis, only a few genes of recent origin could be identifi ed as possibly having been transferred between virus and host (e.g. new genes found at tips of phylogenetic dendograms). thus, gene gain in the herpesviruses (as in dna phage and phycodnavirus) is prevalent but the origination of such genes from the host is not prevalent. i suggest that our tendency to assume that new viral genes are usually " stolen " from the host should be revised ( moreira and lopez-garcia, ). in contrast to the herpesviruses, the poxviruses evolution tend to have little congruence to host evolution (see chapter ). yet, they too show evidence of ancient linkages to other viruses. the replication of poxvirus dna is distinct in that it involves a linear genome with inverted ends that have covalently closed " snapback " dna. the resulting replication structures involve head-to-tail and tail-to-tail intermediates. this replication strategy is very different from that used by the host (and most other dna viruses), but is clearly related to that found in other eukaryotic and prokaryotic viruses. similar replication mechanisms are seen in all poxviruses, as well as african swine fever virus and phycodnaviruses (pbcv- ). this exact replication strategy is also present in archaeal lipothrixviruses (sirv and sirv ) which has been proposed to be ancestral to phycodnaviruses and poxviruses ( persson et al. , ) . a similar replication strategy is also seen with n ( lobocka et al. , ) , an unusual phage of e. coli that persists as a linear dna ( casjens et al. , ) . conservation of such replication similarities clearly suggests ancestral relationships, but no sequence similarity can be seen between these viruses. the similarity between poxvirus and pbcv- dna replication deserves some additional comment. pbvc- and herpesvirus have very similar dna polymerase genes, yet differ fundamentally in replication strategy. furthermore, the poxviral dna polymerase gene is very different from that found in the herpesviruses. yet, the pbcv- capsid was clearly similar to that of adenoviruses and prd phage (and iridovirus capsids). how then do we link poxvirus evolution to other more ancient dna viruses, such as pbcv- which has the same dna replication mechanism, but distinct replication proteins? such observations might seem confusing, but they are clearly consistent with mosaic, reticulated evolution of dna viruses. various distinct phage lineages can link in multiple ways to various distinct eukaryotic dna viruses. the concept of a net or matrix rather than a tree is thus a better way to describe the broad topology of dna virus evolution. the issue of gene gain and gene loss is also of central interest to orthopoxvirus evolution. typically, we seek to understand poxviruses evolution from the perspective of pathogenesis, such as the origin of human-specifi c smallpox virus. with the comparative genomics of several orthopoxviruses now possible, we see curious overall patterns of gene loss in their evolution ( randall et al. , ) . for example, comparing human smallpox to cowpox dna (a rodent virus that is phylogenetically basal to smallpox), we observe an overall diminution of gene content in smallpox virus. several poxviruses seem to have also lost genes relative to cowpoxvirus, especially genes that appear to affect immunity ( hughes and friedman, ) . i suggest that this evolutionary tendency for gene reduction is associated with a switch from a more demanding species-specifi c persistent life strategy to a less demanding, acute life strategy in a new host. cowpox is a naturally persistent infection in rodents (bank voles) ( feore et al. , ; chantrey et al. , ) , which has been called a natural virus reservoir ( hazel et al. , ) . smallpox is a strictly acute and human-specifi c disease. such gene loss in association with lost persistence could be a general situation and might also explain why clinical isolates of human cytomegalovirus isolates show a strong tendency to delete genes with passage in culture ( davison et al. , ) . most orthopoxviruses are not phylogenetically congruent with their vertebrate host. host switching and acute replication seem to be relatively common but recent occurrences in their evolution ( babkin and shchelkunov, ) . the avian poxviruses are not as well studied in this context, but curiously have signifi cantly more complex genomes than the orthopoxviruses ( jarmin et al. , ) . the entomopoxviruses are even less well understood from both a biological and molecular perspective, although they do conserve genes found in all poxvirus family members ( gubser et al. , ) . clearly these poxviruses share some degree of evolutionary history. it is most curious that entomopoxviruses have even larger, more diverse and complex genomes than the other poxviruses. why? as insects lack an adaptive immune system (the target of many orthopoxvirus genes), they would seem to present a simpler host for virus adaptation. this group appears to be the most basal phylogenetically, but evolutionary relationships between entomopoxvirus and insect evolution have not been studied. the enotomopoxviruses are particularly prevalent in grasshopper and locust species, often in unapparent states. interestingly, within these viruses we can fi nd examples of major shifts in core replication genes, such as the family of dna pol gene that is used (a shift from dna pol x to dna pol b in two entomopoxvirus lineages). we can recall that the dna pol b gene closely resembles that found in phycodnaviruses (and herpesvirus), but is distinct from that in orthopoxvirus ( zhu, ) . we also see in the entomopoxviruses some clear links to phage genes, such as t -like rna ligase found in all entomopoxviruses ( ho and shuman, ) as well as a lambdalike integrase seen in d epv ( hashimoto and lawrence, ) . this integrase in d epv implies possible integration and persistence, thus it is most signifi cant that d epv also shows a clear persistent host infection as well as symbiosis and apparent phylogenetic congruence between virus and host. this virus is symbiotic in its parasitoid wasp host in that virus is injected into larval host along with the wasp egg (and also along with a second d rhv rhabdovirus) and virus is needed for successful host parasitization. this symbiosis is clearly very reminiscent of the genomic polydnaviruses of other parasitoid wasp species. diepv is also expressed in the male poison gland. however, it is unknown if diepv is integrating into the host dna. clearly, d epv it is part of a complex virus-virus-host symbiotic interaction. the overall evolution of orthopoxviruses contrasts sharply with that of the papillomaviruses as presented by bernard in chapter . here, highly species-specifi c and tissuespecifi c host infection are the norm and the viral evolution is typically highly congruent with the host (with some exceptions). the resolution between virus and host can be high, in that human racial and geographical populations, for example, can often be differentiated based on the type of hpv they harbor. yet here too there is evidence of signifi cant shifts in core gene usage early during papillomavirus evolution. in the human and rodent viruses, a highly conserved gene function associated with replication and cell control are the e and e early genes. in particular, the prb-binding domain of the e gene is thought to be central to the biological strategy of the virus. thus, it is most curious that the papillomaviruses of lagomorphs, such as bovine and reindeer papillomavirus, lack an e rb-binding domain and instead appear to use e or e genes for this regulatory function ( narechania et al. , ) . it seems an early but signifi cant and bifurcating shift occurred in the molecular strategy during the virus-host evolution of this group of viruses for unknown reasons. other small dna viruses (jcv, bkv, py) can also show similar high-resolution host congruence ( shadan and villarreal, ) . as well as similar curious shifts in basic molecular strategies. for example, the presence of a middle tantigen in mouse virus (a third early gene), but its absence from primate viruses ( gottlieb and villarreal, ) , clearly differentiates these viral lineages. although the origins of these entire small dna viruses are obscure, and any links to prokaryotic viruses are unknown, it does appear they have tended to retain their overall biological strategy and show a strong tendency for tissue-specifi c (especially kidney) persistence and virus-host congruence. since persistence requires the stable coexistence of a virus and its host, it also fi ts the simple defi nition of symbiosis (the stable living together of two distinct lineages of organisms). viral involvement in symbiosis is a foreign idea to many and possibly presents a fundamentally different view of the role viruses may have in host evolution. a major role for persisting (temperate, cryptic) viruses in the evolution of prokaryotes is no longer a controversial idea. thus, at least in the prokaryotic world, virus persistence can be accepted as adaptive. in eukaryotes, however, viral persistence is seldom considered adaptive. the mhv-mouse exemplar as presented above has suggested how persistence can directly affect host survival. can this be considered an example of symbiosis in the accepted sense? a crowning achievement in the fi eld of symbiosis has been to explain the origin of plastids (chloroplasts, mitochondria) from symbiotic prokaryotes in eukaryotic cytoplasm ( margulis and bermudes, ) . this idea involves the high adaptability of prokaryotes to provide innovation but would seem not to involve virus in any way. yet here too we can fi nd viral footprints that suggest some involvement. for example, various plastid-specifi c rna and dna polymerases clearly resemble polymerases from t /t -like phage ( cermakian et al. , ; shutt and gray, ) . other models of symbiosis also show evidence of a viral role, such as the sexual isolation of buchnera ( moran et al. , ) . another very popular topic in the fi eld of symbiosis is the symbiotic origin of the photosynthetic sea slug, elysa chlorotica . what could be more fascinating than a green sea slug-an animal that can use light for photosynthesis? e. chlorotica eats photosynthetic eukaryotic algae ( vaucheria litorea ) and retains the functional chloroplast from algae for months. here too, however, there lies a viral footprint. this slug harbors an unusual endogenous retrovirus which is expressed in large numbers during sexual reproduction, following which all slugs die via synchronized apoptosis and in which the chloroplasts have accumulated numerous viral particles ( pierce et al. , ; mondy and pierce, ) . since there is reason to think gene movement from the algae to the slug genome is involved in this symbiosis, the presence of this retrovirus is a strong candidate to also be involved in symbiogenesis. clearly we should thus investigate retroviral elements as possible symbiotic participants and not dismiss them beforehand as irrelevant or " junk dna " (as is automatically done in many database screens). if viral persistence is a kind of symbiosis, viruses may also mediate the establishment of other symbiotic relationships ( villarreal, ) . the recent studies by roossinck and colleagues (see chapter ), in which a persisting virus, a plant, and a fungus were all symbiotically involved in altering the thermal tolerance of the plant could be an example of this ( marquez et al. , ) . many other virus-host relationships should also be examined for possible symbiosis. for example, placental vertebrate evolution has involved various endogenous retroviruses (i.e. herv-w, herv-frd). intact herv genomes, including env orfs, are important for placental trophoblast fusion (for references see ryan, ) . some will dismiss this situation as the quirky usurping of a viral gene for host function which is of little general signifi cance. the specifi c erv involved is simply selfi sh and mostly defective genetic material of no general consequence. if so, why is it that in sheep a distinctly different lineage of retrovirus (enjsrv) was also selected to provide a related placental function to a another mammal with signifi cantly different placental reproductive biology? it has been experimentally well established the enjsrv env is essential for sheep embryo implantation ( dunlap et al. , a ( dunlap et al. , , b . enjsrv is the endogenous version of jsrv, a problematic sheep-specifi c retrovirus that induces lung tumors (responsible for the death of dolly, the famous fi rst cloned sheep). the endogenous virus (enjsrv) is present in copies in the sheep genome and all sheep have this virus. sheep genomes also encoded a trans -dominant enjrsv gag that is inhibitory to exogenous jsrv ( mura et al. , ; oliveira et al. , ; murcia et al. , ) . it seems clear that this situation can also be considered from the perspective of viral symbiosis and/or virus addiction in host evolution. we should thus seek to understand why colonization by an erv population might generally provide a good solution to the evolutionary demands of placental biology. there are many other opportunities to examine the potential role of persistent and symbiotic viruses in the evolution of viruses, animals, primates, and humans. for example, as we seek to understand the origins of the adaptive immune system we should pay attention to viral footprints. we can ask, for example, why the major histocompatibility complex (mhc) locus, the most polymorphic, diverse, and rapidly evolving gene set in our chromosome, is so densely colonized with retroviral elements ( andersson et al. , ) . why is a retrovirus also the basic element of the duplication unit that was thought to have been the progenitor for the expansion of the mhc class i (and ii) genes ( gaudieri et al. , ; kulski et al. , kulski et al. , , ? why do similar herv element (l and ) also differentiate between human and chimpanzee mhc i ( watkins, ; kulski et al. , ) ? what was the role for siv in the evolution of primate mhc ( vogel et al. , ) ? humans and primates appear to have undergone some signifi cant and relatively recent evolution with regard to their endogenous and exogeneous retroviruses. along these lines, apobec-like genes are basic component of the adaptive immune response but they are also antiretroviral genes that act on retroviral cdna and gag ( ohainle et al. , ) . the apobec antiviral system has expanded recently in humans, but not chimpanzees ( sawyer et al. , ; ohainle et al. , ) . why? all african primates support unapparent foamy viruses (and also siv co-infection), but not humans ( murray and linial, ) . apobec c is active against foamy viruses ( delebecque et al. , ) . old world primates also underwent an expansion of ervl colonization (a clear relative of foamy virus) ( sawyer et al. , ) . was this ervl colonization of relevance to the ancient co-speciation of simian foamy virus and their primate host ( switzer et al. , ) ? what exactly was the relevance of herv endogenization to human survival and adaptations? curiously, human brain (neocortex) specifically expresses many of these more recent ervs as transcripts ( nakamura et al. , ; yi et al. , ) . if we consider these situations as possible examples of virus-mediated symbiosis in human evolution, perhaps they may make more sense of the otherwise confusing role or hervs. as noted, all primates, but especially humans show much evidence of recent endogenization by retroviruses. but these events mostly occurred in our extinct ancestors and we do not see ongoing evidence that any hervs remain active. however, we are currently witnessing a related virus-host evolutionary event of considerable interest. koala bears, native marsupials of australia, are currently experiencing a major epidemic caused by a leukemiainducing retrovirus. as a consequence, they are undergoing massive endogenization by a gammaretrovirus (mlv-related). this virus is similar to gibbon ape leukemia virus, but most likely originated from rodent ancestry ( tarlinton et al. , ; fiebig et al. , ) . the expectation is that extinction awaits those koalas that do not adapt or endogenize the retrovirus successfully ( stoye, ) . this event has the appearances of a retroviral-driven addiction that will result in a genetic variant of koala bear that has acquired a new antiretroviral state. this seems equivalent to the expansion of human apobec ; or perhaps a closer analogy is the endogenization of a suppressive gag as occurred with enjsrv. the surviving koala bears will likely tolerate or be persistently infected with this retrovirus pool. the genome of the species will have undergone considerable (but unpredictable) genetic perturbations and likely contain a large pool of variant and defective retrovirus. however, in so doing, the descendent koalas will likely present a biological hazard to any koala species that remain virus-free (as in virus addiction). currently, one island colony of koalas is suffi ciently isolated to have remained virus-free. this population will henceforth be under persistent threat from populations of endogenized koalas, now favored by group selection. from the very earliest events in evolution of prebiotic replicators to very recent events in human evolution, including the emergence of human-specifi c hiv, we expect viral evolution to show profound effects on the evolution of all life. unlike accepted host evolution, viruses also employ consortia and mixed populations to evolve, sometimes at unprecedented rates. thus viruses have informed us of quasispecies, group dynamics, and group selection in evolution. virus evolution should now be considered as basic science, not just a medical concern. we must acknowledge that the tree of life cannot be properly understood without virus evolution. this book helps to lay the foundation for such understanding. epizootic diarrhea of infant mice: indentifi cation of the etiologic agent the crystal structure of a virus-like particle from the hyperthermophilic archaeon pyrococcus furiosus provides insight into the evolution of viruses unusual polymorphisms in human immunodefi ciency virus type associated with nonprogressive infection a ) genome comparison of two coccolithoviruses b ) evolutionary history of the coccolithoviridae retroelements in the human mhc class ii region host switching in lyssavirus history from the chiroptera to the carnivora orders common ancestry of herpesviruses and tailed dna bacteriophages do viruses form lineages across different domains of life? a ) what does structure tell us about virus evolution? b ) constituents of sh , a novel lipid-containing virus infecting the halophilic euryarchaeon haloarcula hispanica ostreid herpesvirus (oshv- ) detection among three successive generations of pacifi c oysters ( crassostrea gigas ) persistent infection promotes cross-species transmissibility of mouse hepatitis virus the proportion of revertant and mutant phage in a growing population, as a function of mutation and growth rate an intron in the thymidylate synthase gene of bacillus bacteriophage beta : evidence for independent evolution of a gene, its group i intron, and the intron open reading frame serological survey of virus infection among wild house mice ( mus domesticus ) in the uk molecular evolution of viruses, past and present: evolution of viruses by acquisition of cellular rna and dna viral eukaryogenesis: was the ancestor of the nucleus a complex dna virus? sex and the eukaryotic cell cycle is consistent with a viral ancestry for the eukaryotic nucleus coexistence of recent and ancestral nucleotide sequences in viral quasispecies of human immunodefi ciency virus type patients a subset of human immunodefi ciency virus type long-term nonprogressors is characterized by the unique presence of ancestral sequences in the viral population does common architecture reveal a viral lineage spanning all three domains of life? the error threshold what is a quasispecies an envelope glycoprotein of the human endogenous retrovirus herv-w is expressed in the human placenta and fuses cells expressing the type d mammalian retrovirus receptor the genome of the archaeal virus sirv has features in common with genomes of eukaryal viruses oceanographic basis of the global surface distribution of prochlorococcus ecotypes a ) here a virus, there a virus, everywhere the same virus? b ) method for discovering novel dna viruses in 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population evolutionary insights into the ecology of coronaviruses dna virus contribution to host evolution viruses and the evolution of life how viruses shape the tree of life virus-host symbiosis mediated by persistence a hypothesis for dna viruses as the origin of eukaryotic replication proteins on viruses, sex and motherhood acute and persistent viral life strategies and their relationship to emerging diseases major histocompatibility complex class i genes in primates: co-evolution with pathogens high prevalence of infl uenza a virus in ducks caught during spring migration through sweden first demonstration of a lack of viral sequence evolution in a nonprogressor, defi ning replication-incompetent hiv- infection phylogenetic analysis, genome evolution and the rate of gene gain in the herpesviridae the evolution of major histocompatibility class i genes in primates h n infl uenza-continuing evolution and spread elimination of mouse hepatitis virus from a breeding colony by temporary cessation of breeding matrix gene of infl uenza a viruses isolated from wild aquatic birds: ecology and emergence of infl uenza a viruses persistent mhv (mouse hepatitis virus) infection reduces the incidence of diabetes mellitus in non-obese diabetic mice long intervals of stasis punctuated by bursts of positive selection in the seasonal evolution of infl uenza a virus bacteriophage p in retrospect and in prospect human endogenous retroviral elements belonging to the herv-s family from human tissues, cancer cells and primates: expression, structure, phylogeny and evolution a reevaluation of the higher taxonomy of viruses based on rna polymerases chlorella virus-encoded deoxyuridine triphosphatases exhibit different temperature optima persistence of extraordinarily low levels of genetically homogeneous human immunodefi ciency virus type in exposed seronegative individuals key: cord- - p rsf authors: de haan, cornelis a.m.; rottier, peter j.m. title: molecular interactions in the assembly of coronaviruses date: - - journal: adv virus res doi: . /s - ( ) - sha: doc_id: cord_uid: p rsf this chapter describes the interactions between the different structural components of the viruses and discusses their relevance for the process of virion formation. two key factors determine the efficiency of the assembly process: intracellular transport and molecular interactions. many viruses have evolved elaborate strategies to ensure the swift and accurate delivery of the virion components to the cellular compartment(s) where they must meet and form (sub) structures. assembly of viruses starts in the nucleus by the encapsidation of viral dna, using cytoplasmically synthesized capsid proteins; nucleocapsids then migrate to the cytosol, by budding at the inner nuclear membrane followed by deenvelopment, to pick up the tegument proteins. viruses are multimolecular assemblies that range from small, regular, and simple to large, pleiomorphic, and complex. they consist of virus-specified proteins and nucleic acids and, in the case of enveloped viruses, of host-derived lipids. in infected cells the assembly of these different components into virions occurs with high precision amidst a huge background of tens of thousands of host compounds. two key factors determine the efficiency of the assembly process: intracellular transport and molecular interactions. directional transport ensures the swift and accurate delivery of the virion components to the cellular compartment(s) where they must meet and form (sub)structures. some viruses achieve this goal relatively simply when genome production occurs in close proximity to the virion assembly site (e.g., picornaviruses). many viruses, however, have evolved more elaborate strategies. this is illustrated, for instance, by the a-herpesviruses. assembly of these viruses starts in the nucleus by the encapsidation of viral dna, using cytoplasmically synthesized capsid proteins; nucleocapsids then migrate to the cytosol, by budding at the inner nuclear membrane followed by deenvelopment, to pick up the tegument proteins. subsequently, the tegumented capsids obtain their final envelope by budding into vesicles of the trans-golgi network (tgn), where the viral envelope proteins have congregated after their synthesis in the endoplasmic reticulum; the assembled viral particles are finally released by fusion of the virion-containing vesicles with the plasma membrane. to achieve their transport goals viruses provide their components with address labels that can be read by the transport machinery of the cell. once brought together, formation of the viral (sub)structures is governed and driven by their interactions. whereas the assembly of nonenveloped viruses is generally restricted to the cell cytoplasm, although often in association with membranes, that of enveloped viruses involves multiple cellular compartments, as exemplified already for herpesviruses. this review deals with the assembly of coronaviruses. we first describe what is known about the structure of the coronavirion and about the relevant properties of the structural components. we summarize the limited ultrastructural information about coronavirus assembly and budding. the main body of the review describes the interactions between the different structural components of the viruses and discusses their relevance for the process of virion formation. this review has a limited scope; for further information about other aspects of coronavirus biology the reader is referred to other reviews (de vries et al., ; enjuanes et al., ; gallagher and buchmeier, ; holmes, ; holmes et al., ; lai, ; lai and cavanagh, ; lai et al., ; masters, ; perlman, ; rossen et al., ; sawicki and sawicki, ; siddell, ; ziebuhr et al., ) . coronaviruses are a group of enveloped, plus-stranded rna viruses presently classified as a genus, which, together with the genus torovirus, constitutes the family coronaviridae. these viruses are grouped with two other families, the arteriviridae and the roniviridae, into the order nidovirales. this classification is not based on structural similarities-in fact, structure and composition of the viruses from the different families differ significantly-but on common features of genome organization and gene expression (de vries et al., ; lai and cavanagh, ) . coronaviruses infect a wide variety of mammals as well as avian species (table i ). in general they cause respiratory or intestinal infections, but some coronaviruses can also infect other organs (liver, kidney, and brain). until recently, these viruses were mainly of veterinary importance. this situation has changed quite dramatically because of the emergence of severe acute respiratory syndrome- (sars-cov) in late , which emphasized the potential relevance of coronaviruses for humans. on the basis of antigenic and genetic relationships the coronaviruses have been subdivided into three groups (table i) ; the taxonomic position of sars-cov has not been formally assigned. coronavirus particles have a typical appearance under the electron microscope. by the characteristic, approximately -nm-long spikes that emanate from their envelope the viruses acquire the solar image to which they owe their name (fig. ) . the -to -nm virions have a pleiomorphic appearance that, whether artifact or real, reflects a pliable constellation, a feature that has severely hampered the ultrastructural analysis of these viruses. hence, our knowledge about the structure of coronaviruses is still rudimentary. the schematic representation of the current model of the coronavirion drawn in fig. is based on morphological and biochemical fig . electron micrographs of mouse hepatitis virus strain a (mhv-a ) virions without (a) and with (b) the hemagglutinin-esterase (he) envelope protein (viruses kindly provided by r. de groot, virology division, utrecht university, the netherlands; image courtesy of j. lepault, vms-cnrs, gif-sur-yvette, france). large, club-shaped protrusions consisting of spike (s) protein trimers give the viruses their corona solis-like appearance. viruses containing the he protein display a second, shorter fringe of surface projections in addition to the spikes. (c) schematic representation of the coronavirion. the viral rna is encapsidated by the nucleocapsid (n) protein forming a helical ribonucleoprotein (rnp), which is in turn part of a structure with spherical, probably icosahedral, configuration. the nucleocapsid is surrounded by a lipid bilayer in which the s protein, the membrane glycoprotein (m), and the envelope protein (e) are anchored. in addition, some group coronaviruses contain the he protein in their lipid envelope as illustrated on the right side of the particle. observations. as this picture illustrates, the particle consists of a nucleocapsid or core structure that is surrounded by a lipid envelope. anchored in this envelope are the three canonical coronavirus membrane proteins: the membrane (m) protein, the envelope (e) protein, and the spike (s) protein. viruses from group have an additional, fourth membrane protein, the hemagglutinin-esterase (he) protein. as a consequence these viruses display a second, shorter ( nm) fringe of surface projections in addition to the spikes (fig. b) (bridger et al., ; king et al., ; sugiyama and amano, ) . the ribonucleoprotein (rnp) core contains one copy of the viral genomic rna. this rna is packaged into a helical structure by multiple copies of nucleocapsid protein (n). size estimations of the flexible cylindrical structures varied quite considerably, ranging between and nm in diameter and up to . mm in length (see laude and masters, ) . the ribonucleoprotein helix appears in turn to be contained within a spherical, probably icosahedral, configuration as indicated by various ultrastructural approaches using purified transmissible gastroenteritis virus (tgev) and mouse hepatitis virus (mhv) (risco et al., (risco et al., , . the molar ratio of the major structural proteins, s:n:m, has been variously estimated to be approximately : : (sturman et al., ) , : : (cavanagh, a) , : : (hogue and brian, ) , and : : (liu and inglis, ) , although an m:n molar ratio of has also been reported (escors et al., a) . the s:he molar ratio was estimated to be (hogue and brian, ) . the e protein is only a minor virion component and was calculated to occur in infectious bronchitis virus (ibv), tgev, and mhv virions at a rate of approximately , , and molecules per particle, respectively (godet et al., ; liu and inglis, ; vennema et al., ) . the lipid composition of coronaviral envelopes has been studied only to a limited extent. comparison of the phospholipid composition of mhv with that of its host cell showed increased levels of sphingomyelin, phosphatidylserine, and phosphatidylinositol and a decrease in the level of phosphatidylethanolamine (van genderen et al., ) . whether the lipid composition of mhv is an accurate reflection of its budding compartment or whether certain lipids become enriched in the virus during budding is not known. what follows is a general description of the individual virion components and their properties. this description is by no means complete as it is restricted to the information that is of relevance to the main topic of this review. for a schematic representation of the coronavirus life cycle see fig. . the coronavirus life cycle. the replication cycle starts with attachment of the virion by its s protein, that is, through the s subunit thereof, to the receptors on the host cell. this interaction leads to fusion of the virus envelope with a cellular membrane, coronaviruses contain a single-stranded positive-sense rna genome of some to kilobases, the largest nonsegmented viral rna genomes known. the rna has a -terminal cap and a terminal poly(a) tract. both genomic termini contain untranslated regions (utrs) of some - nucleotides that harbor several cis-acting sequences and structural elements functioning in viral replication and transcription. coronaviruses have a typical genome organization characterized by the occurrence of a distinctive set of genes that are essential for viability and occur in a fixed order: -polymerase ( pol)-s-e-m-n- (fig. ) . the pol gene comprises approximately twothirds of the genome, from which it is translated directly. it encodes two large precursors (pol a and pol ab), the many functional cleavage products of which are collectively responsible for rna replication and transcription (for reviews on coronavirus transcription and replication see de vries et al., ; lai, ; lai and cavanagh, ; lai et al., ; sawicki and sawicki, ; ziebuhr et al., ) . the more downstream pol b gene is translated by translational readthrough, for which the s subunit is responsible. from the genomic rna that is released by disassembly of the incoming particle the pol a and pol b genes are translated, resulting in the production of two large precursors (pol a and pol ab), the many cleavage products of which collectively constitute the functional replication-transcription complex. genes located downstream of the pol b gene are expressed from a -coterminal nested set of subgenomic (sg) mrnas, each of which additionally contains a short leader sequence derived from the end of the genome (shown in red). transcription regulatory sequences (trss) located upstream of each gene serve as signals for the transcription of the sgrnas. the leader sequence is joined at a trs to all genomic sequence distal to that trs by discontinuous transcription, most likely during the synthesis of negative-strand sgrnas. in most cases, only the -most gene of each sgrna is translated. multiple copies of the n protein package the genomic rna into a helical structure in the cytoplasm. the structural proteins s, m, and e are inserted into the membrane of the rough endoplasmic reticulum (rer), from where they are transported to the er-to-golgi intermediate compartment (ergic) to meet the nucleocapsid and assemble into particles by budding. the m protein plays a central role in this process through interactions with all viral assembly partners. it gives rise to the formation of the basic matrix of the viral envelope generated by homotypic, lateral interactions between m molecules, and it interacts with the envelope proteins e, s, and he (if present), as well as with the nucleocapsid, thereby directing the assembly of the virion. virions are transported through the constitutive secretory pathway out of the cell-the glycoproteins on their way being modified in their sugar moieties, whereas the s proteins of some but not all coronaviruses are cleaved into two subunits by furin-like enzymes (see text for references). using a ribosomal frameshift mechanism for which a "slippery" sequence and a pseudoknot structure are required. the genes located downstream of pol b are expressed from a -coterminal nested set of subgenomic (sg) rnas, each of which additionally contains a short leader sequence derived from the end of the genome. transcription regulatory sequences (trss) located upstream of each gene serve as signals for transcription of the sgrnas. the leader sequence is joined at a trs to all genomic sequence distal to that trs by discontinuous transcription, most likely during the synthesis of negative-strand sgrnas (sawicki and sawicki, ) . besides the characteristic genes encoding the replicative and structural functions, coronaviruses have a more variable collection of additional genes that are located in two clusters in the -terminal onethird of the genome. the genes differ distinctly in their nature and genomic position among the coronavirus groups, but they are specific for each group. these so-called group-specific genes appear not to be essential as shown by the occurrence of natural mutants defective in some of them (brown and brierley, ; herrewegh et al., ; kennedy et al., ; luytjes, ; shen et al., ; vennema, ; vennema et al., ; woods, ) and by the observed viability of engineered deletion mutants lacking some or all of these genes (de haan et al., b; fischer et al., ; haijema et al., ; ortego et al., ; sola et al., ) . except for the group -specific he protein and, possibly, the poorly characterized i protein (fischer et al., ; senanayake et al., ) , the latter encoded by an open reading frame completely contained within the n gene, the group- the single-stranded, positive-sense rna genome contains -and -terminal untranslated regions (utrs) with a -terminal cap and a -terminal poly(a) tract. the leader sequence (l) in the utr is indicated. all coronaviruses have their essential genes in the order -pol-s-e-m-n- . the pol a and pol b genes comprise approximately twothirds of the genome. the more downstream pol b gene is translated by translational readthrough, using a ribosomal frameshift mechanism. transcription regulatory sequences (trss) located upstream of each gene, which serve as signals for the transcription of the subgenomic (sg) rnas, are indicated by circles. the genes encoding the structural proteins he, s, e, m, and n are specified. gray boxes indicate the accessory, group-specific genes, in the case of group coronaviruses genes a, he, , a, and i. specific proteins do not appear to occur in virions. although their functions have not yet been resolved, mutant studies indicate that they play important roles in the interaction of coronaviruses with their host (de haan et al., b; fischer et al., ; haijema et al., ; ortego et al., ) . the n protein is the most abundantly expressed viral protein in infected cells (for a review, see laude and masters, ) . its size varies considerably between viruses from different groups ( - amino acids, i.e., molecular masses ranging between and kda), n proteins from group coronaviruses (table i) being the largest. whereas the amino acid sequences of n proteins are quite similar within the groups, the homology between proteins from different coronavirus groups is rather limited ( - %). an exception is a region spanning about residues within the amino-terminal one-third of the n molecule, where high sequence identity has been conserved across the different groups. despite the overall sequence variation the n proteins have a number of common characteristics. consistent with their role as nucleic acidbinding proteins they are all highly basic because of the abundance of arginine and lysine residues. these are clustered mainly in two nearby regions in the middle of the molecules. the abundance of basic residues is reflected in the calculated overall isoelectric points of the n proteins, the values of which are in the range of . - . . these numbers are the more significant in view of the acidic nature of the very carboxyterminal domain; pi values ranging from . to . were calculated for the terminal residues (parker and masters, ) . another general characteristic of the n proteins is their high content ( - %) of serine residues, which are potential targets for phosphorylation. although these residues occur all over the n molecule, their relative abundance within the first of the two basic regions is notable. little is known about the three-dimensional structure of the n protein. of the sars-cov n protein the amino-terminal domain (residues - ) was analyzed by nuclear magnetic resonance spectroscopy. it appeared to consist of a five-stranded b sheet with a folding distinct from that of other rna-binding proteins (huang et al., ) . in coronavirus-infected cells the n protein can often be detected as one major and several minor forms, the latter polypeptides having a slightly lower molecular weight. the major species appeared to comigrate in gels with the n protein observed in virions, indicating that only the full-len gth n spec ies is incorp orated in to partic les. how th e minor n spec ies ar ise and whet her they are of partic ular significan ce for infection is unc lear. they are most likely derived by proteoly tic proc essing from the major n species. this is supp orted by stud ies from eleo uet et al. ( ) , who show ed the tge v n prot ein to be cleav ed by casp ases. cas pase cleava ge sites wer e also predicted in the carboxy terminus of several other coronavirus n proteins (eleouet et al., ; ying et al., ) . these features are in agreement with observations showing that antibodies directed against the carboxy terminus of the mhv and tgev n proteins were not reactive with the faster migrating electrophoretic forms. furthermore, these smaller n protein forms appeared to be derived from the major species as judged from pulse-chase analyses (for a review see laude and masters, ) . the n protein is the only coronavirus structural protein known to become phosphorylated (for references see laude and masters, ) . both the major and minor n species appear to be phosphorylated as shown for mhv-a in sac(À) cells (rottier et al., b) and for tgev in llc-pk cells (garwes et al., ) . of the many potential target serines only a few are actually modified in the case of mhv (stohlman and lai, ; wilbur et al., ) . n protein phosphorylation does not seem to play a critical role in the regulation of virus assembly. in contrast, it has been hypothesized that dephosphorylation of the protein might facilitate disassembly during mhv cell entry (kalicharran et al., ; mohandas and dales, ) . immunofluorescence microscopy has shown the n protein to be localized in a particulate manner throughout the cytoplasm of coronavirus-infected cells. although the protein lacks a membrane-spanning domain it was found in association with membranes (anderson and wong, ; sims et al., ; stohlman et al., ) . for mhv, the n protein was found to colocalize partly with the membrane-associated viral replication complexes van der meer et al., ) . in addition to its cytoplasmic localization, the n proteins of ibv, mhv, and tgev have also been demonstrated to localize to the nucleolus both in coronavirus-infected cells and when expressed independently wurm et al., ) . putative nuclear localization signals were identified in these proteins. the ibv n protein was found to interact with nucleolar antigens, which appeared to occur more efficiently when the n protein was phosphorylated, and to affect the cell cycle (chen et al., ) . however, because mhv is able to replicate in enucleated cells (brayton et al., ; wilhelmsen et al., ) the nucleolar localization of the n protein does not appear an essential step during infection. although the primary function of the n protein is the formation of the viral ribonucleoprotein complex, several studies indicate the protein to be multifunctional. as indicated by its intracellular localization, the n protein is a likely component of the coronavirus replication and transcription complex. its presence is not an absolute requirement for replication and transcription because a human coronavirus (hcov) rna vector containing the complete pol ab gene appeared to be functional in the absence of the n protein (thiel et al., ) . however, the efficiency of the system was much enhanced when the protein was present. furthermore, using an in vitro system, it was demonstrated that antibodies to the n protein, but not those against the s and m proteins, inhibited viral rna synthesis by % (compton et al., ) . interactions that have been observed between the n protein and leader/trs sequences nelson et al., ; stohlman et al., ) and between n protein and the utr (zhou et al., ) suggest a role for the n protein in the discontinuous transcription process. furthermore, the n protein was also shown to interact with cellular proteins that play a role in coronavirus rna replication and transcription (choi et al., ; shi et al., ) . in addition, the n protein was reported to function as a translational enhancer of mhv sgrnas (tahara et al., ) . the m protein (previously known as e protein) is the most abundant envelope protein. it is the "building block" of the coronavirion and has been shown to interact with virtually every other virion component, as detailed in section iv. the m protein is - residues in length, except for the group m proteins, of which the amino terminus is about residues longer. despite large differences in primary sequences between m proteins from different antigenic groups, their hydropathicity profiles are remarkably similar. the m protein is highly hydrophobic. it has three hydrophobic domains alternating with short hydrophilic regions in the amino-terminal half of the protein, with the exception of the aforementioned group m proteins, which have at their amino terminus a fourth hydrophobic domain that functions as a cleavable signal peptide. the carboxy-terminal half of the protein is amphipathic, with a short hydrophilic domain at the carboxy-terminal end (fig. ) . in the center of the protein, directly adjacent to the third hydrophobic domain, is a stretch of eight amino acids that is well conserved (swwsfnpe). the conservation of the overall chemical features suggests that there are rigid structural constraints on the m protein as a result of functional requirements (for a review on the m protein, see rottier, ) . biochemical and theoretical studies led to a topological model for the mhv m protein rottier et al., rottier et al., , , in which the polypeptide spans the lipid bilayer three times, leaving a small amino-terminal domain ( - residues) in the lumen of intracellular organelles (or outside the virus), whereas the carboxyterminal half of the protein is located on the cytoplasmic side of the membrane (or inside the virion). the lumenal domain and the hydrophilic carboxy terminus are susceptible to protease digestion and are thus exposed. the bulk of the carboxy-terminal half of the m protein is protease resistant, indicating that the amphipathic part of the protein is either folded tightly or embedded in the polar surface of the membrane. indeed, a mutant lacking all three transmembrane domains was found to be associated with membranes (mayer et al., ) . the model for the disposition of the m protein in the membrane was confirm ed for ibv (cavanag h et al., ) . interest ingly, the fig . membrane topology of the coronavirus envelope proteins. the he and s proteins are both type i membrane proteins, with short carboxy-terminal cytoplasmic tails. the he protein forms disulfide-linked homodimers, whereas the s protein forms noncovalently linked homotrimers. the s subunits presumably constitute the globular head, whereas the s subunits form the stalk-like region of the spike. the m protein spans the lipid bilayer three times, leaving a small amino-terminal domain in the lumen of intracellular organelles (or on the outside of the virion), whereas the carboxy-terminal half of the protein is located on the cytoplasmic side of the membrane (or inside the virion). in tgev virions some of the m proteins have their cytoplasmic tail exposed on the outside (not shown). the m protein is glycosylated at its amino terminus (indicated by a diamond). the amphipathic domain of the m protein is represented by an oval. the hydrophilic carboxy terminus of the e protein is exposed on the cytoplasmic side of cellular membranes or on the inside of the virion. the e protein may span the bilayer once (b) or twice (a). m protein of tgev was shown to adopt an additional conformation. in virions about one-third of the m molecules have their carboxy terminus exposed on the virus surface rather than buried inside the particle (escors et al., a; risco et al., ) . this appears to have immunological consequences (risco et al., ) but the real significance of the dual topology is unclear. in mhv the m protein was found to assume only one defined membrane topology . the coronavirus m protein is almost invariably glycosylated in its exposed amino-terminal domain. this provides the virion with a diffuse, hydrophilic cover on its outer surface. whereas the group and coronaviruses and sars-cov all contain m proteins with only n-linked sugars, the m proteins of group coronaviruses are o-glycosylated (for a review see rottier, ). an exception is mhv- , the m protein of which carries both o-and n-linked sugars (yamada et al., ) . n-glycosylation is initiated in the endoplasmic reticulum by the cotranslational linkage of a large oligosaccharide structure to the polypeptide at asparagine residues within the consensus sequence nxs/t (where x is any amino acid). in contrast, mucin-type o-glycosylation starts posttranslationally with the addition of an n-acetylgalactosamine (galnac) monosaccharide to a hydroxylamino acid. o-glycosylation is subsequently completed by stepwise addition of other monosaccharides such as galactose, n-acetylglucosamine, fucose, and sialic acid. mhv m proteins carry a well-conserved ss(x)ttxxp sequence at their extreme amino terminus. despite the apparent presence of multiple hydroxylamino acids as potential oligosaccharide acceptor sites the m protein of mhv-a was found to be modified by the addition of only a single oligosaccharide side chain (de haan et al., b) . this side chain, when studied in ost - cells, appeared to be attached to the threonine at position . mutation studies, however, revealed that alternative acceptor sites can also be used. no unique sequence motifs for o-glycosylation of mhv m could be identified, which is probably related to the occurrence in cells of multiple galnac transferases (de haan et al., b) . as the expression of these enzymes varies in cells, conservation of the ss(x)ttxxp motif in mhv m protein may serve to increase opportunities for the protein to become glycosylated in different cell types. the distinct conservation of n-and o-glycosylation among the m proteins of the different groups of coronaviruses suggests that the presence and the particular type of carbohydrates are somehow beneficial to the virus, most likely in its interaction with the host. glycosylation of the m protein appeared not to be required for envelope assembly (de haan et al., a,) or for interaction with the s protein (de haan et al., ) , nor did it influence virus replication in vitro (de haan et al., a laude et al., ) . coronaviruses are able to induce interferon a (ifn-a) by their glycoproteins (baudoux et al., a,b) . for tgev (charley and laude, ; laude et al., ) and mhv (de haan et al., ) , the oligosaccharides linked to the m protein were demonstrated to be important for efficient ifn induction in vitro. the glycosylation status of the mhv m protein was found to influence the ability of the virus to replicate in the liver but not in the brain . thus, viruses with n-glycosylated m proteins replicated to a significantly higher extent in liver than otherwise identical viruses carrying o-glycosylated m proteins. mhv with unglycosylated m proteins replicated to the lowest extent. the mechanism behind these observations remains to be elucidated. when expressed in cells independently from the other viral proteins, the m proteins of mhv, ibv, tgev, and feline coronavirus (fcov) accumulate in the golgi compartment, that is, beyond the site of virus budding locker et al., ; machamer and rose, ; machamer et al., ; rottier and rose, ) , which is the intermediate compartment between the er and the golgi (ergic). the fine localization of the different proteins is, however, not the same. for instance, whereas the mhv m protein is concentrated in the trans-most golgi compartments, the ibv m protein localizes to the cis side of the golgi complex. signals for localization appear to reside in the hydrophilic part of the cytoplasmic tail and in the transmembrane domains. the extreme carboxy-terminal tail of mhv m was shown to be necessary, although not sufficient, for golgi localization (armstrong and patel, ; locker et al., ) . mutant proteins lacking this domain were transported to the plasma membrane. also, mutation of a single tyrosine in this domain, which occurs in the context of a potential internalization signal, resulted in plasma membrane localization (c. a. m. de haan and p. j. m. rottier, unpublished results). the first transmembrane domain of the ibv m protein was shown to be required and sufficient for localization to the cis-golgi region (machamer and rose, ; machamer et al., machamer et al., , swift and machamer, ) . this is not the case for the mhv m protein of which mutants with only the first transmembrane domain did not leave the er (armstrong et al., ; locker et al., ; rottier et al., ) . moreover, insertion of the first transmembrane domain of mhv m into a reporter protein resulted in a chimeric protein that was transported to the cell surface (armstrong and patel, ; machamer et al., ) , unlike a similar chimeric protein containing the first transmembrane domain of ibv that was retained in the golgi compartment . other mhv m mutant proteins lacking the first and second transmembrane domains were also not efficiently retained in the golgi compartment and were diverted to endosomal structures (armstrong et al., ; locker et al., ) . the mechanism by which golgi retention of m proteins is regulated has not yet been resolved. however, oligomerization of the proteins, mediated by the transmembrane domains, seems to play an important role, perhaps in combination with retrieval mechanisms maceyka and machamer, ) . formation of oligomeric complexes has been demonstrated to correlate with golgi retention of a reporter protein containing the first transmembrane domain of ibv m (weisz et al., ) while also the golgi-resident mhv m protein was found to occur in large, homomeric complexes (locker et al., ) . the lumenal domain of the m protein does not appear to contribute to localization; its deletion from mhv m did not affect the intracellular destination of the protein (mayer et al., ; rottier et al., ) . in infected cells the m proteins of ibv and mhv were observed to occur in the membranes of the budding compartment as well as in the golgi compartment. under these conditions their cis-trans distribution in the golgi compartment was the same as when these proteins were expressed independently machamer et al., ) . the e protein (previously known as sm protein) is a small protein ( - residues) and a minor component of the coronaviral envelope. although the primary structures of e proteins are quite conserved within the different coronavirus groups, they share little homology between the groups. however, the proteins have several structural features in common. the e protein contains a relatively large hydrophobic region in its amino-terminal half, followed by a cysteine-rich region, an absolutely conserved proline residue, and a hydrophilic tail. e is an integral membrane protein, which is assembled in membranes without the involvement of a cleaved signal peptide . its membrane topology has not been firmly established. although the opposite was proposed initially for the tgev e protein (godet et al., ) , there seems to be consensus about the hydrophilic carboxy terminus being exposed on the cytoplasmic side in cells or on the inside of the virion (corse and machamer, ; raamsman et al., ) . the amino terminus of the mhv e protein was not detectably present on the virion outside but appeared to be exposed cytoplasmically when it was extended with an amino-terminal epitope tag (maeda et al., ) , consistent with a topological model in which the hydrophobic domain spans the bilayer twice. for the ibv e protein evidence was provided indicating that the amino terminus is exposed lumenally in cells, consistent with a single spanning topology (corse and machamer, ) (fig. ) . the e protein is not glycosylated but appears to become palmitoylated. this was shown most convincingly for the ibv e protein by labeling with [ h]palmitate (corse and machamer, ) , both in ibv-infected cells and when the protein was expressed. mutagenesis revealed that one or both of the two conserved cysteines became modified. the result is consistent with the observed increase in electrophoretic mobility of the mhv e protein after treatment with hydroxylamine, an agent that cleaves thioester-linked acyl chains (yu et al., ) . others, however, were not able to confirm this posttranslational modification (godet et al., ; raamsman et al., ) . in coronavirus-infected cells the e protein has been observed by immunofluorescence studies to occur at intracellular membranes as well as at the cell surface (godet et al., ; smith et al., ; tung et al., ; yu et al., ) . when expressed exogenously from cdna, the e protein was detected only in intracellular organelles, although at different locations. the mhv e protein localized to pre-golgi membrane compartments, as was demonstrated by its colocalization with rab- , a marker for the endoplasmic reticulum and the ergic, by electron microscopy . the ibv e protein, tagged at its amino terminus with an epitope, was also localized to pre-golgi compartments (lim and liu, ) . in another study, however, the ibv e protein was shown to accumulate in the golgi apparatus, being distributed throughout the complex machamer, , ) . while the former study identified an ertargeting signal in the extreme carboxy terminus of the e protein (lim and liu, ) , the latter studies, using carboxy-terminal truncations, mapped the golgi-targeting information to a region between tail residues and (corse and machamer, ) . in addition, these authors showed the ibv e cytoplasmic tail to be necessary and sufficient for golgi targeting. the e protein was identified as a virion component relatively late, due to its low abundance and its small size (godet et al., ; liu and inglis, ; yu et al., ) . it was estimated to occur in ibv, tgev, and mhv virions at a rate of about , , and molecules per particle, respectively (godet et al., ; liu and inglis, ; vennema et al., ) . because of its low abundance the e protein may not have a genuine structural function in the virion envelope. rather, it may have a morphogenetic function by taking strategic positions within the m protein lattice to generate the required membrane curvature. alternatively, it may serve to close the neck of the budding particle as it pinches off the membrane (vennema et al., ) . expression of the e protein alone induced the formation of characteristic membrane structures also observed in infected cells, which apparently consist of masses of tubular, smooth convoluted membranes (david-ferreira and manaker, ; raamsman et al., ) . in addition, it resulted in the formation of vesicles containing the e protein, shown to be released from the cells (corse and machamer, ; maeda et al., ) . mhv infection induces caspase-dependent apoptosis in some, but not all, cells. by expressing the viral structural proteins separately in cells, the activity could be attributed to the e protein . apoptosis induction has not been reported for e proteins from other coronaviruses. coronavirus e proteins share structural similarities with small hydrophobic membrane proteins found in other enveloped viruses. examples are the vpu protein of hiv- , the k protein of alphaviruses, and the m protein of influenza virus. these proteins, also known as viroporins (gonzalez and carrasco, ) , were demonstrated to modify membrane permeability and to help the efficient release of progeny virus. the s protein (previously known as e ) constitutes the spikes, the hallmark of coronaviruses under the electron microscope. it is the major determinant of host range, tissue tropism, pathogenesis, and virulence. it is a relatively large, -to -amino acid-long type i glycoprotein with a cleavable n-terminal signal sequence and a membrane-anchoring sequence followed by a short hydrophilic carboxy-terminal tail of about residues (fig. ) . when comparing primary sequences, the s protein shows two faces: an amino-terminal half with hardly any sequence similarities and a carboxy-terminal half in which regions with significant conservation can be observed (de groot et al., a,b ; for a review see cavanagh, ) , consistent with the distinctive functions of these domains (see later). the s protein is synthesized as a heavily glycosylated polypeptide as demonstrated by the susceptibility of the glycans to endoglycosidases and by the dramatic effect of the n-glycosylation inhibitor tunicamycin. the number of potential n-glycosylation sites ranges from (mhv) to [feline infectious peritonitis virus (fipv)]. the s protein has not been reported to contain o-linked sugars. cotranslational n-glycosylation is an essential requirement for proper folding, oligomerization, and transport of the s protein, as has also been shown for other (viral) glycoproteins (doms et al., ) . growth of coronaviruses in the presence of tunicamycin resulted in the production of spikeless, noninfectious particles mounir and talbot, ; rottier et al., a; stern and sefton, ) . these particles were devoid of s protein, which was found to aggregate in the endoplasmic reticulum when glycosylation was inhibited (delmas and laude, ) . folding of the s protein is a relatively slow process. besides the addition of oligosaccharides it involves the formation and rearrangement of many intramolecular disulfide bonds. for the s protein of mhv-a , the lumenal domain of which contains cysteine residues, the major conformational events appear to take about min during which the protein passes through a continuous spectrum of folding intermediates (opstelten et al., a) . folding of s is probably the rate-limiting step in the process of oligomerization. sufficiently folded s protein monomers associate in the endoplasmic reticulum to form trimers (delmas and laude, ; lin et al., ) , with a half-time of approximately h (delmas and laude, ; vennema et al., a,b) . trimerization is likely to be required for export out of the endoplasmic reticulum. in infected cells s protein trimers interact with m protein and perhaps also with e protein, and migrate to the virus assembly site. a fraction of the s protein is transported to the plasma membrane where it can cause cell-cell fusion, a feature formally attributed to the s protein by its individual expression in cells (de groot et al., ; pfleiderer et al., ) . under such expression conditions the bulk of the s protein remains intracellularly (vennema et al., a) in the endoplasmic reticulum . retrieval signals have been identified in the cytoplasmic tail of the s proteins from coronavirus groups and as well as in the tail of the sars-cov s protein, but not in the group mhv s protein (lontok et al., ) . during its transport to the cell surface, either alone or as part of virions, the s protein undergoes further modifications. the n-linked sugars are modified and become mature during passage through the golgi complex. the mhv s protein was shown to become palmitoylated, a modification that may already take place in the endoplasmic reticulum (van berlo et al., ) . as a late step the s protein can be cleaved. a basic amino acid sequence resembling the furin consensus sequence motif (rxr/kr) occurs approximately in the middle of the protein and was shown to be the target of a furin-like enzyme in the case of mhv-a (de haan et al., ) . cleavage has been demonstrated for s proteins from coronavirus groups and , but not for s proteins from group viruses (cavanagh, ) or from sars-cov (bisht et al., ) . the resulting amino-terminal s subunit and the membrane-anchored s subunit remain noncovalently linked. it has been suggested that the s subunit constitutes the globular head, whereas the s subunit forms the stalk-like region of the spike (cavanagh, b; de groot et al., a,b) . the coronavirus s protein has two functions, which appear to be spatially separated. the s subunit (or the equivalent part in viruses with uncleaved s protein) is responsible for receptor binding, and the s subunit is responsible for membrane fusion. for several coronaviruses the receptor-binding site in s has been mapped. for mhv strain jhm (mhv-jhm), for instance, it was located in the domain composed of the amino-terminal residues of the s molecule (kubo et al., ) , residues - and - being particularly important (saeki et al., ; suzuki and taguchi, ) . this amino-terminal domain also determined ceacam receptor specificity of various mhv strains (tsai et al., ) . for tgev (godet et al., ) , hcov- e breslin et al., ) , and sars-cov (babcock et al., ; wong et al., ) the receptor-binding domains have also been mapped to the s subunit, although in different regions. in several cases neutralizing antibodies were demonstrated to bind the receptor-binding domains and to prevent the interaction with the receptor (godet et al., ; kubo et al., ; sui et al., ) . the interaction between the s protein and its receptor is the major determinant for virus entry and host range restriction. nonpermissive cell lines can be rendered susceptible by making them express the receptor (see later references). coronaviruses can also be retargeted to specific cells by exchanging the ectodomain of the s protein for that of an appropriate other coronavirus, as was demonstrated for mhv (kuo et al., ) and fipv (haijema et al., ) . receptors have so far been identified for the group coronavirus mhv (ceacam; dveksler et al., dveksler et al., , williams et al., ) ; the group coronaviruses tgev and porcine respiratory coronavirus (prcov) (papn; delmas et al., t resnan et al., ) , and hcov- e (hapn; yeager et al., ) ; and for sars-cov (ace ; li et al., ) . the s proteins of the group coronaviruses have been observed to exhibit hemagglutinating activities. although for bovine coronavirus (bcov), hcov-oc , and hemagglutinating encephalomyelitis virus (hev) -o-acetylated sialic acids were identified as a receptor determinant (krempl et al., ; kunkel and herrler, ; schultze and herrler, ; schultze et al., a; vlasak et al., b) , specific receptors for these viruses have not been identified. also, the mhv s protein appears to bind sialic acid derivatives in addition to its specific receptor ceacam, which may suggest that sialic acids function as an additional receptor determinant for mhv-like coronaviruses (wurzer et al., ) . the ectodomain of the s subunit, which is involved in the fusion process, contains two heptad repeat (hr) regions (de groot et al., a,b) , a sequence motif characteristic of coiled coils. mutations in the first (i.e., membrane-distal) hr region of the mhv s protein resulted in fusion-negative phenotypes (luo and weiss, ) or in a low-ph dependence for fusion (gallagher et al., ) , whereas mutations in the second hr region caused defects in s protein oligomerization and fusion ability (luo et al., ) . a fusion peptide has not yet been identified in any of the coronavirus spike proteins, but is predicted to be located at (bosch et al., b; chambers et al., ) or within (luo and weiss, ) the amino terminus of the first hr region. binding of the s subunit to the (soluble) receptor, or exposure to c and an elevated ph, has been shown to trigger conformational changes that are supposed to facilitate virus entry by activation of the fusion function of the s subunit (breslin et al., ; gallagher, ; lewicki and gallagher, ; matsuyama and taguchi, ; miura et al., ; sturman et al., ; taguchi and matsuyama, ; zelus et al., ) . this conformational change is thought to lead to exposure of the fusion peptide and its interaction with the target membrane, further changes resulting in the formation of a heterotrimeric six-helix bundle, characteristic of class i viral fusion proteins, during the membrane fusion process. indeed, peptides corresponding to the hr regions of mhv (bosch et al., ; xu et al., ) and sars-cov (bosch et al., b; ingallinella et al., ; liu et al., ; tripet et al., ; zhu et al., ) were found to assemble into stable oligomeric complexes in an antiparallel manner, which in the natural situation would result in the close colocation of the fusion peptide and the transmembrane domain. these peptides were further shown to be inhibitors for viral entry (bosch et al., (bosch et al., , b liu et al., ; yuan et al., ; zhu et al., ) . besides the hr regions, other parts of the s protein are also likely to be important for the fusion process. all coronavirus s proteins contain a highly conserved region (de groot et al., a) , rich in aromatic residues, downstream of the second hr region, part of which may form the start of the transmembrane domain. the function of this domain is unknown, but a similar region in the hiv- env protein was demonstrated to be important for viral fusion and env incorporation into virions (salzwedel et al., ) . immediately downstream of the transmembrane domain all s proteins contain a cysteine-rich region (de groot et al., a) . using a mutational approach including deletions, insertions, and substitutions, both the transmembrane domain and the cysteine-rich region immediately downstream thereof, but not the carboxy-terminal part of the cytoplasmic tail, were shown to be important for mhv s protein-induced cell-cell fusion (bos et al., ; chang and gombold, ; chang et al., ) the cleavage requirements of the s proteins for the biological activities of the coronavirus spike remain enigmatic. whereas the s proteins of group coronaviruses, such as fipv (vennema et al., a) , are not cleaved, those of other coronaviruses, particularly of groups and , are cleaved to variable extents, depending on the viral strain and the cell type in which the viruses are grown (frana et al., ; reviewed by cavanagh, ) . cleavage of the s proteins is not required to expose the internal fusion peptide. whereas cleavage of the mhv s protein generally correlates strongly with cell-cell fusion (cavanagh, ) , virus-cell fusion appeared not to be affected by preventing s protein cleavage, indicating that these fusion events have different requirements (de haan et al., ) . similarly, whereas trypsin activation of sars-cov s protein was required for cell-cell fusion, it did not enhance the infectivity of cell-free pseudovirions (simmons et al., ) . for mhv- , the spikes of which are able to initiate fusion without prior interaction with the primary mhv receptor (gallagher et al., ) , the stability of the s -s heterodimers after s protein cleavage is low, allowing receptor-independent fusion. during cell culture adaptation, however, selected mutant viruses carried deletions in the s subunit, downstream of the receptor-binding domain, which resulted in stabilized s -s heterodimers and receptor-dependent fusion activity (krueger et al., ) . virions of group coronaviruses generally contain a fringe of shorter surface projections in addition to the characteristic spikes (bridger et al., ; king et al., ; sugiyama and amano, ) . these viruses express and incorporate into their particles an additional membrane protein, he (for a review see brian et al., ) . although all group viruses contain an he gene, the protein is not expressed by all mhv strains (luytjes et al., ; yokomori et al., ) , indicating that he is a nonessential protein also in these viruses. the he gene encodes a type i membrane protein of - residues that contains a cleavable signal peptide at its amino terminus (hogue et al., ; kienzle et al., ) and a transmembrane domain close to its carboxy terminus, leaving a short cytoplasmic tail of about residues (fig. ) . the ectodomain contains - putative n-linked glycosylation sites. the putative esterase active site (fgds) is located near the (signal-cleaved) he amino terminus. the coronavirus he protein has % amino acid identity with the he- subunit of the he fusion protein of influenza c virus and the he protein of torovirus (cornelissen et al., ) . it has been suggested that coronaviruses have captured their he module from influenza c virus or a related virus (luytjes et al., ) . however, influenza c virus, toroviruses, and coronaviruses may well have acquired their he sequences independently, not from each other but from yet another source (cornelissen et al., ) . the he protein becomes cotranslationally n-glycosylated when expressed in cells, giving rise to a polypeptide of approximately - kda that rapidly forms disulfide-linked dimers (hogue et al., ; kienzle et al., ; king et al., ; parker et al., ; yokomori et al., ; yoo et al., ) . the he dimers (or a higher order structure thereof) become incorporated into virions, while a proportion is transported to the cell surface (kienzle et al., ; pfleiderer et al., ) . little is still known about the function(s) of the coronavirus he protein. the protein contains hemagglutinin and acetyl esterase activities (brian et al., ) . while the he proteins of bcov, hev, and hcov-oc hydrolyze the -o-acetyl group of sialic acid and therefore appear to function as receptor-destroying enzymes (schultze et al., b; vlasak et al., a) , the he proteins of mhvlike coronaviruses function as sialate- -o-acetylesterases (klausegger et al., ; regl et al., ; wurzer et al., ) . although inhibition of the esterase activity of bcov resulted in a -to -fold reduction in viral infectivity (vlasak et al., a) , it was shown both for bcov and for an mhv strain expressing an he gene that the s protein is required and sufficient for infection (gagneten et al., ; popova and zhang, ) . in view of these results it has been proposed that the he protein might play a role at an even earlier step and may mediate viral adherence to the intestinal wall through the specific yet reversible binding to mucopolysaccharides. the process of binding to sialic acid receptors followed by cleavage and rebinding to intact receptors could theoretically result in virus motility and even allow migration through the mucus layer covering the epithelial target cells in the respiratory and enteric tracts (cornelissen et al., ) . several studies have indicated the he protein to play a role in pathogenicity. the he protein of bcov (deregt and babiuk, ) , but not that of mhv, was able to induce neutralizing antibodies. however, passive immunization of mice with nonneutralizing, mhv he-specific antibodies protected the animals against a lethal mhv infection (yokomori et al., ) . furthermore, intracerebral expression of the he protein in mice was found to affect the neuropathogenicity of mhv (yokomori et al., ; zhang et al., ) . strikingly, he protein-defective mhv mutants were rapidly selected during viral infection in the mouse brain (yokomori et al., ) , which may suggest that the he protein plays a more critical role during the infection of other tissues. early electron microscopic studies demonstrated that coronavirus morphogenesis takes place at intracellular membranes and identified the cisternae of the endoplasmic reticulum as the site of budding of ibv and hcov- e (becker et al., ; chasey and alexander, ; hamre et al., ; oshiro et al., ) . later studies revealed that early in infection particle formation occurs predominantly at smoothwalled, tubulovesicular membranes located intermediately between the rough endoplasmic reticulum and the golgi complex (ergic). this so-called intermediate compartment was shown to be used as the early budding compartment by mhv, ibv, fipv, tgev, and sars-cov (goldsmith et al., ; klumperman et al., ; tooze et al., ) . at later times during infection the rough endoplasmic reticulum was seen to gradually become the major site of mhv budding in fibroblasts (tooze et al., ) . as already mentioned, ultrastructural studies localized the mhv and ibv m proteins in the budding compartment(s) but also in the golgi complex, that is, beyond the site of budding tooze et al., ) . apparently, accumulation of m protein alone is not sufficient to determine the site of budding; other viral and/or cellular factors are required as well. for mhv the e protein, which was found to localize to the intermediate compartment by immunoelectron microscopy (immuno-em), was suggested to be such a candidate , but more players are likely to be involved. whether the helical nucleocapsids, visible as electron-dense cytoplasmic elements adjacent to budding profiles (david-ferreira and manaker, ; dubois-dalcq et al., ; massalski et al., ; risco et al., ; and references given previously), are a determining factor is unclear. in this respect, knowledge about the budding location of coronavirus-like particles (see later) might be informative. coronavirions are subject to an intracellular postbudding maturation process that occurs while they are on their way through the constitutive exocytic pathway by which they are exported out of the cell (risco et al., ; salanueva et al., ; tooze et al., ) . indications of this had already been noticed in early morphological studies with hcov- e (becker et al., ; chasey and alexander, ; hamre et al., ; oshiro et al., ) and mhv (holmes and behnke, ; , but were described in somewhat more detail for mhv by tooze and coworkers ( ) . the pictures show "immature" virions in pre-golgi compartments and golgi cisternae that appear as spherical structures with the ribonucleoprotein core immediately below the viral envelope and with an "empty" center. by contrast, virions in the trans-golgi network and beyond have the mature morphology showing a fairly uniform, high internal electron density. an extensive analysis of the structural maturation of coronavirions was reported for tgev (risco et al., ; salanueva et al., ) . budding was shown to yield relatively large virions with an annular, electron-dense internal periphery and a clear central area. smaller particles, with the characteristic morphology of extracellular virions, that is, having a compact, dense inner core with polygonal contours, were seen to accumulate in secretory vesicles in the periphery of the infected cell. both types of particles appeared to coexist in the golgi complex (fig. ) . obviously, the larger particles are the precursors of the smaller mature virions (salanueva et al., ) and probably undergo their morphological maturation during their transport through the golgi complex. the reorganization of the particle gives rise to the supposedly icosahedral core shell and is accompanied by a dramatic, approximately % reduction of the particle volume. it is presently unknown what triggers the morphological reorganization in the golgi complex. application of drugs affecting the state of the organelle did not give clues. as virions encounter an increasingly acidic ph on passage through the golgi stack, studies addressing this parameter were done with lysosomotropic agents. thus, chloroquine and nh cl were applied to mhv-infected cells to elevate the ph at the trans side of the golgi complex, but no effect on the maturation of mhv was observed (tooze et al., ) . monensin, a drug that reversibly disorganizes the golgi complex and blocks transport along the exocytic pathway, led to the accumulation of the large, annular tgev virions; after reversal of the blockade formation of the small, compact particles was again restored (salanueva et al., ) . these observations confirm that the golgi complex is necessary for tgev structural transformation. nocodazole treatment of cells causes a reversible fragmentation of the golgi complex. under these conditions tgev virions were still able to undergo normal structural maturation. in contrast, still another golgi-disrupting compound, brefeldin a, prevented their maturation (risco et al., ) . this compound leads (risco et al., ) . (b) for a direct comparison of size and morphology a small, dense particle and a large particle are shown (salanueva et al., ) . pictures were kindly provided by c. risco. to a redistribution of golgi membranes to the er, leaving no definable golgi system. interestingly, mhv particles accumulated in infected cells under these conditions appeared to be infectious when liberated by sonication (j. meertens and p. j. m. rottier, unpublished results), leaving us with an intriguing question about the function of the maturation process. a. nucleocapsid assembly helical nucleocapsids are assembled in the cytoplasm of coronavirus-infected cells. they have been recognized by their tubular appearance in electron microscopy studies with several viruses including ibv, hcov- e, tgev, and mhv (becker et al., ; chasey and alexander, ; david-ferreira and manaker, ; dubois-dalcq et al., ; hamre et al., ; massalski et al., ; oshiro et al., ; risco et al., ) . large inclusions of nucleocapsids were seen to accumulate late in the infection of cells with hcov (caul and egglestone, ) and mhv-jhm (dubois- dalcq et al., ) . the structure of the nucleocapsid as it occurs in infected cells has not been studied in any detail. ribonucleoprotein particles supposed to represent nucleocapsids have been isolated from mhv-infected cells and were shown to consist of genomic rna and n protein (perlman et al., ; robb and bond, ; spaan et al., ) . the particles sedimented as edta-resistant structures of - s in sucrose gradients. during the active phase of viral replication the majority ( %) of the intracellular genome-size rna was found in these structures (spaan et al., ) . ultrastructural studies of nucleocapsids derived from purified virion preparations have shown quite a variety of helical structures, depending on the virus and the experimental conditions used. the overall feature, however, was that of a thread-like coil, sometimes appearing to be hollow, with a diameter varying between and nm and a length ranging from . mm up to mm (caul et al., ; davies et al., ; kennedy and johnson-lussenburg, ; macnaughton and davies, ) biochemical analysis of nucleocapsids prepared by detergent disruption of purified coronaviruses revealed the presence of genomic rna and n protein. interestingly, however, particles obtained by treatment of virions with nonidet p- appeared to be spherical when viewed under the electron microscope and, in addition, to contain m protein, as was observed with tgev (garwes et al., ) , hev (pocock and garwes, ) , and mhv-jhm (wege et al., ) . the presence of the m protein was found for mhv-a to depend on the preparation conditions. whereas the protein was absent when the virions had been disrupted with nonidet p- at c, solubilization at c resulted in copurification of m protein with the nucleocapsid (sturman et al., ) . the higher temperature was found to cause a conformational change in the m protein, leading to its aggregation and association with the viral rna in the nucleocapsid. similarly, nucleocapsid structures essentially lacking the m protein were also reported for ibv when virions were treated with detergent at low temperature (davies et al., ) . although there is no direct evidence yet, it seems reasonable to assume that the helical nucleocapsids seen accumulating in the cytosol of infected cells constitute the reservoir that feeds into the viral budding system. the location where these nucleocapsids are assembled has not been defined. their production may take place either free in the cytoplasm, where the n protein is synthesized, or, alternatively, in association with the membrane-bound structures where genomic rna is produced. the observed colocalization of n protein with the replication complexes (bost et al., (bost et al., , denison et al., ; van der meer et al., ) is consistent with the latter possibility. coronavirus replication appears to occur on double-membrane vesicles (gosert et al., ) , which utilize components of the cellular autophagy pathway (prentice et al., ) . whereas early in infection the replication complexes were shown to be almost entirely discrete from sites of m protein accumulation, at later times of infection helicase and n proteins appeared to colocalize with the m protein (bost et al., (bost et al., , . it was proposed that the translocation of helicase-n protein complexes to sites of virus assembly may serve as a mechanism to deliver the newly synthesized rna and nucleocapsids and to facilitate the retention of the m protein in the intermediate compartment. encapsidation of genomic rna into a nucleocapsid is presumably initiated by an interaction of the n protein with a specific nucleotide sequence, the packaging signal, which is subsequently followed by the polymerization of n proteins around the rna molecule in a nonsequence-specific manner. the selective incorporation of genomic rna into virions would predict the packaging signal to be located in sequences unique to this rna, that is, within the approximately -kb region comprising the utr and open reading frame (orf ) with the exception of the leader sequence. although the data obtained so far support this prediction, no consistent picture has emerged yet. the approach generally used to map the packaging signal involved the study of helper virus-assisted encapsidation of natural and artificially obtained defective rna genomes. thus, a -nucleotide region located at the end of the pol b gene was initially identified for mhv (van der most et al., ) , which was subsequently narrowed to an area of nucleotides (fosmire et al., ) . within this area a stable stem-loop of nucleotides was predicted. mutation studies revealed that the integrity of this secondary structure was important and that the sequence of the packaging signal could be trimmed further to a minimum stretch of nucleotides (fosmire et al., ) . the signal appeared to be sufficient for rna packaging as its inclusion allowed a synthetic subgenomic mrna of mhv-a to be packaged specifically; the encapsidation efficiency of the mrna was, however, significantly lower than that of the defective genomic rna from which it was transcribed . even a nonviral rna was found to be packaged into mhv particles when provided with the packaging signal (woo et al., ) . buoyant density analysis of the particles revealed that the rna was not assembled separately but copackaged with helper virus rna. studies of the corresponding pol b region of another group coronavirus, bcov, indicated that within this group the packaging signal is structurally and functionally conserved. a -nucleotide sequence with significant homology ( %) to that of mhv was identified within a cloned -nucleotide segment sharing % homology overall . when this segment was fused to a noncoronavirus reporter gene sequence, the resulting rna appeared to be packaged not only by the homologous helper virus bcov but also by mhv. conversely, when the mhv packaging signal was fused to the reporter gene sequence, the rna was found to be encapsidated also in the context of a bcov-infected cell . mapping studies of packaging signals in the genomes of group and group coronaviruses have yielded quite different results. for ibv, deletion mutagenesis of a defective rna led to the conclusion that only the sequences in the utr and/or a region of the utr were specifically required for packaging, although parts of the pol b sequence, but not any part in particular, also enhanced the efficiency (dalton et al., ) . somewhat similar conclusions could be drawn from a study with tgev (izeta et al., ) . by comparing packaging efficiencies of different defective genomes it was inferred that information for packaging was present both at the genomic end (about . kb) and in parts of pol b. a packaging signal was subsequently mapped to a fragment representing the terminal nucleotides of the genome by inserting a series of overlapping segments covering a stretch of about nucleotides from the end of the genome into an mrna reporter expression construct contained within a defective genome (escors et al., ) ; only the terminal sequence conferred to the mrna the ability to become packaged by helper virus. it is too early to conclude that the apparently contrasting results reflect true, fundamental differences in encapsidation strategies between the different (groups of ) coronaviruses. although the overview may suggest the existence of different cis-acting signals, the data still allow a scenario in which multiple domains in the genome are involved cooperatively, each one contributing differently to (the efficiency of) the encapsidation process. such contributions would not necessarily concern n protein binding only; the exceptional complexity of the coronaviral genome might call for additional provisions, related perhaps to the structuring of the encapsidation complex. several observations indeed imply the involvement of multiple domains. the efficient rescue, for instance, of a bcov defective genome (drep) that completely lacks the putative -nucleotide packaging signal entails the participation of additional sequence(s) (chang and brian, ; . another example is the strongly increased rescue of an otherwise poorly packaged defective tgev genome (m ) due to the presence of about . kb of sequences derived from the pol b gene (m ) (izeta et al., ) . there is no direct evidence yet for the actual functioning of the presumed packaging signals in the initiation of nucleocapsid assembly. binding of n protein to these signals, the first step in the process, has so far been addressed only for the -nucleotide sequence of mhv. specific binding to rna transcripts containing this sequence was indeed demonstrated biochemically with mhv n protein derived from infected cells, from virions, and from cells expressing the protein (molenkamp and spaan, ) . the binding efficiency, however, appeared to be relatively weak as was shown by comparing n protein binding to different parts of a packageable defective genome (midi-c) . the highest binding efficiency was observed with an rna transcript representing about kb from the end of pol a. remarkably, not even removal of the packaging signal from midi-c rna affected binding to the n protein as measured in the filter-binding assay used. the observations indicate that the domain containing the -nucleotide sequence does not function as a packaging signal in the conventional sense, adding further support to the notion that the intricacies of coronaviral nucleocapsid assembly are complex. apart from the studies mentioned, the occurrence of n-rna interactions has been amply documented. this is not surprising as the n protein has been implicated in several other processes that involve interaction with rna, such as replication, transcription, and translation (lai and cavanagh, ; see also section ii.c). as the relevance of these interactions for viral assembly is generally unclear, a brief survey of the available information is included here. in addition, an overview of data on the mapping of rna interactions on the n polypeptide is schematically presented in fig. . a high-affinity interaction between the n protein and the leader was demonstrated for mhv-a . using an rna overlay protein blot assay and various in vitro rna transcripts, the binding of n protein was localized to a stretch of nucleotides (nucleotides - ) at the end of the leader . the stretch included the pentanucleotide repeat ucuaa now known to be critical for transcription. biochemical analyses of the interaction measured a dissociation constant (k d ) of nm for bacterially expressed mhv n protein to the leader rna (nelson et al., ) . consistent with the presence of a leader at the end of all viral rnas, an n protein-specific monoclonal antibody coimmunoprecipitated genomic rna as well as the subgenomic rnas from mhv-infected cells . similar observations were made for bcov . packaging of subgenomic rnas has been reported for tgev (sethna et al., ) , bcov (hofmann et al., ) , and ibv (zhao et al., ) but not for mhv, suggesting that their incorporation is not mediated by n protein-leader interaction. whereas for tgev and ibv the relative packaging of subgenomic rnas was found to be inefficient, genomic rna appearing in virions at a more than -fold molar excess over any subgenomic rna species, the bcov subgenomic n and m mrnas appeared to be packaged as abundantly as the genome (hofmann et al., ) . a reevaluation for tgev revealed that the detection of subgenomic rnas in virions was related to the purity of virus preparations, indicating that mrnas were not specifically encapsidated (escors et al., ) . besides the leader, the n protein has been found to bind to other parts of the coronaviral genome. in addition to the binding site in the mhv fig . structural organization of the coronavirus n protein. common features and their distribution along the polypeptide chain are shown schematically. the hatched box indicates the most conserved part of the n protein, with a high proportion of aromatic residues. the n protein contains many basic residues throughout the polypeptide, but with particular clustering in two regions (þþþ). the upstream cluster contains a serine/arginine-rich region (s/r). the carboxy terminus, which contains a high proportion of acidic residues, is also indicated (ÀÀÀ). the bars indicate parts of the n protein that have been implicated in n-n and n-rna interaction. furthermore, the location of the deletion in the mhv-a mutant alb (d) is indicated as well as the domain where second-site mutations in revertant viruses of alb are mapped. finally, the parts of the n protein that could not be transferred from bcv into the mhv genome are marked. references are included in the figure. pol a gene mentioned previously, a high-efficiency binding site was identified by the same authors in the half of the n gene of both mhv and bcov . the ibv n protein was shown to bind sequences in the terminal utr of the genome (zhou et al., ) . coronavirus n proteins do not contain sequence motifs typically found in other rna-binding proteins. they appear to bind rna both nonspecifically (masters, ; robbins et al., ) and in a sequencespecific way nelson and stohlman, ; nelson et al., ; stohlman et al., ) . non-sequence-specific rna binding has been mapped to a large central domain of the mhv n molecule (fig. ) (masters, ) . also, the leader-binding property was assigned to this domain; this activity was initially mapped to the area containing the two highly basic regions (nelson and stohlman, ) , but was later narrowed to a -residue segment containing the serine/ arginine-rich basic region (nelson et al., ) . interestingly, this particular region could not be interchanged with its bcov counterpart in a study on the functional equivalence of the n proteins from these related viruses (peng et al., b) . another domain in the mhv n protein implicated in viral rna binding was mapped to an area that partly overlaps with the second basic region. the assignment was based on an analysis of second-site revertants of mhv mutant alb , the virions of which are extremely thermolabile because of a -residue deletion located between the central and carboxy-terminal domain of the n protein (koetzner et al., ) . the reverting mutations correlated with restoration of the disturbed rna-binding capacity of the mhv n protein and were found clustered close to the basic region some residues on the amino side of the deletion (fig. ) (peng et al., a) . although all these studies consistently attribute a major role in rna binding to the central portion of the coronaviral n protein, the interaction of the ibv n protein with the utr of ibv rna mentioned previously was mapped to the amino-and carboxy-terminal domains of the molecule (zhou and collisson, ) . utr rna-binding activity was also assigned to the amino-terminal domain of the sars-cov n protein on the basis of studies using nuclear magnetic resonance spectroscopy (huang et al., ) . it is obvious that the wrapping of the -kb coronaviral genome into the compact helical nucleocapsid is largely driven by n protein interactions. as there are no indications for packaging of the rna into a preformed capsid, these interactions can be described by the following model. packaging is initiated by binding of the n protein, either as a monomer or in a multimeric form, to the rna. by analogy to other rna viruses, this sequence-specific interaction may induce a conformational change in the n protein, thereby creating a nucleation site for the cooperative stacking of n protein units along the entire length of the rna, now in a non-sequence-specific way. these n units can again be monomeric or consist of defined multimers. finally, helix formation is driven by interactions between n molecules separated along the ribonucleoprotein chain but that become adjacent in neighboring helices. this model predicts multiple nonequivalent interactions between n molecules. n-n interactions have been experimentally demonstrated for mhv, bcov, and hcov-oc . high molecular weight species of the n protein, possibly trimers, were detected by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page) of virion preparations under nonreducing (but not under reducing) conditions, which is indicative of intermolecular disulfide bonds between the n subunits (hogue et al., ; narayanan et al., b; robbins et al., ) . these complexes are likely to be additionally stabilized by noncovalent interactions as coronavirus n protein cysteines are not well conserved, the sars-cov n protein even lacking any cysteine residues. both monomeric and oligomeric n species were able to bind rna (robbins et al., ) . multimeric forms of the n protein were also found in association with intracellular genomic rna in mhv-infected cells as shown after the selective isolation of this ribonucleoprotein through coimmunoprecipitation with the m protein (narayanan et al., b) . high molecular weight forms of the n protein corresponding to dimers and trimers were also demonstrated in vitro after ultraviolet (uv) cross-linking of bcov n protein to rnas . few studies have addressed the identification of the n-n interaction domains. the results so far are inconsistent (fig. ), but this might as well reflect the predicted occurrence of nonequivalent interactions. interaction sites were mapped to the amino-terminal part of the mhv n protein (wang and zhang, ) . using an in vitro binding assay in which the full-length n protein was incubated with bacterially expressed fusion proteins containing different segments of the n protein, interaction was observed with a polypeptide derived from the amino-terminal one-third (residues - ) of the protein and with a polypeptide representing the central part (residues - ). the latter domain contains the serine/arginine-rich region implicated in the binding to the leader/trs-specific sequences (nelson et al., ) . this domain could not be replaced by the corresponding domain from bcov without loss of viral viability, from which the authors indeed inferred an involvement in protein-protein interactions (peng et al., b) . the same domain was also shown to be essential for the homotypic association of the sars-cov n protein . using a mammalian two-hybrid approach, the n-n interaction appeared to be abolished completely when the serine/arginine-rich region had been deleted. however, in another study using the yeast two-hybrid system this interaction was not confirmed. a polypeptide consisting of the amino-terminal two-thirds of the sars-cov n protein, that contains the serine/arginine-rich region, exhibited no association with the full-length protein (surjit et al., ) . rather, self-association was attributed to the carboxy-terminal residues of the molecule, which lacks the motif. unlike most other enveloped viruses, coronaviruses have the remarkable feature of being able to independently assemble their envelope. indications for this were already noticed in early electron microscopy studies of viral preparations and infected cells showing the occurrence of apparently "empty" particles (afzelius, ; chasey and alexander, ; macnaughton and davies, ) . incomplete virions with the typical coronavirus morphology but lacking the n protein and the genome could indeed be separated from normal ibv particles by their lower density in sucrose gradients (macnaughton and davies, ) . the definition of virus-like particles (vlps) and the requirements for their formation were established by the coexpression of the coronaviral structural proteins in mammalian cells (vennema et al., ) . membranous particles were assembled when the mhv envelope proteins m, e, and s were coexpressed, without the need for an n protein or genomic rna. the particles were released from the cells and, when examined under the electron microscope, appeared to be morphologically indistinguishable from authentic virions, that is, they had the characteristic shape and dimensions of normal virions. also, their membrane protein composition was similar to that of mhv, with a high abundance of m protein and only trace amounts of e protein. quite surprisingly, only the m and e proteins were required for particle assembly. both s and n proteins were dispensable for particle formation but, whereas the s protein became incorporated when present, this was not the case for the n protein (vennema et al., ) , except in combination with (defective) genomic rna, in which case a nucleocapsid was coassembled (bos et al., ; kim et al., ) . individual expression of the m or the e protein in cells did not give rise to formation of vlps although e protein synthesis by itself led to the secretion of e-containing vesicles (corse and machamer, ; maeda et al., ) . the nature of these particles has not been characterized in much detail; the vesicles induced by mhv e sedimented slightly slower than virions (maeda et al., ) whereas the particles obtained with ibv e had about the same density as virions (corse and machamer, ) . besides for mhv and ibv, vlps have so far been described for bcov (baudoux et al., b) , fipv , and tgev (baudoux et al., b) . the observations demonstrate the unique budding mechanism of coronaviruses, which is dependent solely on the envelope proteins m and e but independent of a nucleocapsid. somewhat similar observations have been described for the flavivirus tick-borne encephalitis virus and for hepatitis b virus, which also produce proteolipid particles on expression of their envelope proteins prem and e (allison et al., ; mason et al., ) and s (patzer et al., ; simon et al., ) , respectively, but these particles are much smaller than the corresponding virions. in contrast, particles with the typical, large size of coronaviruses are acquired by the concerted action of just the proteins m and e (baudoux et al., b; vennema et al., ) . budding of enveloped viruses generally requires a nucleocapsid (for a review see garoff et al., ) . for retroviruses the gag protein, the precursor to the nucleocapsid, is all that is needed to obtain particles resembling immature virions; the env protein is dispensable. budding of alphaviruses, on the other hand, requires both the envelope proteins and the nucleocapsid. interestingly, the same appears to hold true for arteriviruses (r. wieringa, a. a. f. de vries, and p. j. m. rottier, unpublished observations), which are closely related to the coronaviruses, share with them a triple-spanning envelope protein, and bud into early membranes of the secretory pathway, like coronaviruses but unlike alphaviruses. it is unknown how the coronavirus m and e proteins cooperate in budding. as the extensive electron microscopy work with m proteins from various coronaviruses gave no indications that this protein causes membrane bulging by itself, it is believed that the function of the e protein in coronavirus budding is in the induction of curvature in the m protein lattice (see later) and the subsequent budding of the membrane (vennema et al., ) . by its low abundance in the virion, the e protein does not seem to serve a genuine structural function in that it occupies frequent, regular positions in the m protein framework. consistent with an important role of the e protein in particle morphogenesis, mutations in its hydrophilic carboxy-terminal part, introduced by targeted recombination into the mhv genome, yielded thermolabile viruses one of which showed aberrant virion morphology with pinched and elongated shapes when viewed in the electron microscope (fischer et al., ) . revertant analyses revealed that a single second-site amino acid change within the e protein was able to reverse the phenotypic effect of the original mutations, providing support for possible interactions between e protein monomers during budding (fischer et al., ) . unexpectedly, complete deletion of the e gene from the coronaviral genome does not abolish virion formation, demonstrating that the protein is not essential for budding. whereas this deletion dramatically (at least -fold) reduced the release of infectivity from infected cells in the case of mhv-a , knockout of the tgev e gene resulted in a lethal phenotype (curtis et al., ; ortego et al., ) . remarkably, however, in the latter case virions still assembled but these appeared to be unable to leave the cells (j. ortego and l. enjuanes, personal communication). the vlp system offers a convenient assay to study many aspects of coronavirus envelope assembly. it was thus used to analyze the primary structure requirements of the m and e proteins for particle formation. for the m protein such studies demonstrated each of its different domains to be important. in general, mutations (deletions, insertions, and point mutations) in the lumenal domain, the transmembrane domains, the amphiphilic domain, or the carboxy-terminal domain of the mhv m protein strongly affected its ability to form vlps (de haan et al., a) . the assembly process was particularly sensitive to changes in the carboxy terminus of the protein. truncation by only one residue reduced the efficiency severely whereas removal of two residues fully abolished particle formation. these effects appeared to be less severe in the context of a normal coronaviral infection, probably because additional interactions can compensate. the single-residue deletion, when introduced into the mhv genome, was without measurable phenotype and also a mutant virus with a truncation of two residues could be obtained, although with difficulty, as it was severely affected in its growth (de haan et al., a; kuo and masters, ) . the importance of the m protein cytoplasmic and transmembrane domains was confirmed by vlp studies in the ibv system; mutant proteins lacking portions of either of these domains were unable to support particle assembly (corse and machamer, ) . studies of the primary structure requirements of the e protein for vlp formation revealed that the sequence of its hydrophobic domain was not critical. the assembly capacity of the protein was maintained when its transmembrane region was partly or completely replaced by the corresponding domain of the vesicular stomatitis virus (vsv) g protein (corse and machamer, ) . however, when its small aminoterminal ectodomain was additionally replaced by the large vsv g protein counterpart, the chimeric protein became nonfunctional. deletion of the transmembrane, but not the amino-terminal, domain rendered the e protein essentially assembly incompetent (lim and liu, ) . deletions in the cytoplasmic carboxy-terminal half of the e protein mapped the cysteine-rich region as the most important part for vlp assembly (lim and liu, ) . although the interaction between m and e proteins is amply demonstrated by their interdependence for vlp formation, direct evidence for their interaction has actually been provided only for the ibv proteins (corse and machamer, ; lim and liu, ) . the two proteins could be cross-linked to each other in ibv-infected cells and in cells coexpressing the m and e genes (corse and machamer, ) . it appeared that the cytoplasmic tails of both proteins were required, suggesting they are involved in the interaction. in another study m-e interaction was demonstrated by a coimmunoprecipitation assay. also in this assay the cytoplasmic domain of the e protein, comprising the cysteine-rich region, was found to be important as its deletion affected m-e interaction to the greatest extent when compared with other deletion mutant e proteins (lim and liu, ). the results from both studies also showed that the ability of mutant e or m proteins to interact did not correlate with their assembly competence. apparently, other requirements such as homotypic e or m interactions or interactions with host cell components must be met. the specificity of the interaction between the m and e proteins during particle assembly was further demonstrated by the poorly successful attempts to generate chimeric vlps. no particles were observed when heterologous combinations of tgev and bcov m and e proteins were coexpressed (baudoux et al., b) and the same was true for heterologous combinations of fipv and mhv m and e proteins (h. vennema and p. j. m. rottier, unpublished results) . in both studies chimeric m and e proteins were also tested, demonstrating that, except in one case, exchanges between corresponding domains rendered the proteins assembly incompetent. only when the tgev m protein amino-terminal ectodomain was replaced with that of bcov did the chimeric polypeptide support vlp formation in combination with the tgev e protein; vlp formations was also supported, but to different extents, with tgev/bcov chimeric e proteins and-poorly, however-with the bcov e protein (baudoux et al., b) . the reciprocal m construct, the bcov m protein carrying the tgev ectodomain, was nonfunctional, even in combination with tgev e protein. consistently, replacement of the ectodomain with that of fipv m also abolished the productive partnership of mhv m protein with mhv e (de haan et al., ) . as the disproportionate amounts of m and e proteins in vlps already imply, homotypic interactions between m molecules must constitute the energetic basis underlying the formation of the coronaviral envelope. in mhv-based vlps generated by coexpression of m and e proteins, for instance, the sheer excess of m protein-the relative molar presence of e in the particles is less than %-is evidence for the strong interactive forces between m molecules. hence, envelope assembly is thought to be driven primarily by laterally interacting m molecules that form a two-dimensional lattice in intracellular membranes (opstelten et al., b . large multimeric complexes of m protein have indeed been demonstrated biochemically after individual expression of the mhv protein in cells. when the association of the m molecules was maintained by the careful selection of cell lysis conditions, sucrose gradient analysis revealed the existence of large heterogeneous (up to about molecules) complexes, which accumulated in the golgi compartment (locker et al., ) . somewhat smaller complexes were obtained when the cytoplasmic tail of the protein was removed; these complexes were no longer retained in the golgi apparatus but transported to the cell surface. apparently, the tail domain is not essential for the lateral interactions between m proteins, but it is critically required for budding (de haan et al., a . similar higher order complexes of the m protein have also been demonstrated in mhv-infected cells as well as in mhv virions (opstelten et al., b . further support for the existence of homotypic m protein interactions and additional insight into the domains involved in these interactions came from work with mutant m proteins that are unable to assemble into vlps. in these studies mhv m proteins with deletions in either the transmembrane regions, the amphipathic domain, or the extreme carboxy terminus or with substitutions of the lumenal domain were tested for their ability to associate with other m proteins and to be rescued into vlps formed by assembly-competent m proteins (de haan et al., a . it appeared that the mutant proteins maintained these biological activities despite the often severe alterations; actually, the only mutant protein that had lost these abilities was one in which all three transmembrane domains had been replaced by a heterologous transmembrane domain. it was concluded that m protein molecules interact with each other through multiple contact sites, particularly at the transmembrane level. it was furthermore hypothesized that the full complement of interactions between the m molecules is required for efficient particle formation; possibly, all these interactions are required to provide the free energy to generate and stabilize the budding envelope. the failure of m protein mutants capable of associating with assembly-competent m protein to assemble into vlps by themselves (de haan et al., a indicates that additional interactions with viral (e) and/or host proteins is required. in this respect it is of note that the ifn-inducing capacity of the m protein, demonstrated for tgev and bcov, also requires the presence of the e protein, which suggests that the induction of ifn is dependent on a specific, probably regularly organized structure of the m protein (baudoux et al., a,b) . it is unclear how the presence of the e protein alters the m protein lattice to achieve this effect. coronavirus envelope assembly is not dependent on the s protein or the he protein. this is obvious from work with vlps as well as with viruses showing that bona fide particles were produced when these proteins were either simply absent or unavailable for assembly. availability can be compromised under conditions in which proper folding of the proteins is affected. inhibition of n-glycosylation by the drug tunicamycin, for instance, can lead to aggregation and retention of membrane proteins in the endoplasmic reticulum and has been shown to prevent the incorporation into virions of both the s protein mounir and talbot, ; rottier et al., a; stern and sefton, ) and the he protein (mounir and talbot, ) . the same effect has been observed with temperature-sensitive mhv mutants carrying defects in their s gene, which, when grown at the restrictive temperature, gave rise to spikeless particles ricard et al., ) . both s and he proteins are assembled into the coronaviral envelope through interactions with the m protein. such interactions have been demonstrated for mhv and bcov m and s proteins and for bcov m and he proteins, in infected cells, in cells coexpressing the proteins, and in virions (nguyen and hogue, ; opstelten et al., ) . complexes of the proteins were shown by coimmunoprecipitation and cosedimentation analyses as well as by immunofluorescence studies in which the intracellular transport of s and he proteins to the plasma membrane was found to be inhibited by coexpressed m protein, the proteins being retained in the golgi apparatus, the natural residence of the m protein. the kinetics with which the proteins engage in heteromeric complex formation appeared to be different for the different proteins. this effect is due to their different rates of folding and oligomerization. for the s protein these rates are low, involving the formation of multiple intramolecular disulfide bonds and the addition of numerous oligosaccharide side chains (delmas and laude, ; opstelten et al., a; vennema et al., a,b) . in contrast, folding of the mhv and bcov m proteins is independent of disulfide bonds and glycosylation, as a result of which they are, for instance, swiftly transported out of the endoplasmic reticulum (opstelten et al., a) . as a consequence, m molecules enter into m-s and m-he complexes immediately after their synthesis whereas for newly synthesized s and he molecules it took - min before they started to appear in these heterocomplexes (nguyen and hogue, ; opstelten et al., ) . the importance of folding as a major rate-limiting step was illustrated by the inability of the s protein to interact with m protein when its folding had been inhibited by in vivo reduction; only completely oxidized s molecules were association competent (opstelten et al., a . whether the m protein interacts with s and he proteins while they are still in their monomeric form or only after their oligomerization remains to be elucidated. it is, however, clear that the proteins engage in interaction with each other in early compartments, most likely the endoplasmic reticulum, as judged from the oligosaccharide maturation states of freshly formed protein complexes (de haan et al., ; nguyen and hogue, ; opstelten et al., ) . only dimers of he were associated with he-m-s complexes that were observed in bcov-infected cells; because the appearance of he in these complexes correlated with the kinetics of he dimerization it was concluded that proper oligomerization is most likely a requirement for its association (nguyen and hogue, ) . interestingly, such heterotrimeric complexes were not observed on coexpression of the three proteins in cells. under these conditions only the heterodimeric m-s and m-he associations were detected. the structural domains of m and s proteins that are involved in the formation and stabilization of their complex have been identified. using the coimmunoprecipitation and colocalization assays referred to previously, the essential domains in the mhv m protein were mapped by a mutag enetic appro ach (de ha an et al ., ) . it appear ed tha t m-s complex formation was sensitive to changes in all membrane-associated parts of the m molecule. interactions between m and s proteins were found to occur at the level of the transmembrane domains and of the amphipathic domain, which is located on the cytoplasmic face of cellular membranes. in contrast, neither the lumenally exposed amino terminus nor the hydrophilic cytoplasmic tail of the m protein was required; even the deletion of these parts-known to abrogate the ability of the protein to form vlps-did not prevent association with the s protein. chimeric s proteins were used to show that the large ectodomains of the spikes are not involved in interaction with m proteins. such chimeric proteins were constructed from the mhv and fipv s proteins and consisted of the ecto-or lumenal domain from the one and the transmembrane plus endodomain from the other. these proteins, which seemed biologically fit as they were still fusion active, were initially tested in coexpression studies with the m and e proteins from either virus for their ability to be incorporated into vlps. they were found to assemble only into viral particles of the species from which their carboxy-terminal domain originated . the chimeric s genes were subsequently incorporated into the proper coronavirus genomic background, creating the chimeric viruses fmhv and mfipv, the spike ectodomains of which are from the feline and murine coronavirus, respectively; these studies provided the basis for the development of a novel targeted recombination system for reverse genetics of coronaviruses (haijema et al., ; kuo et al., ) . further fine mapping of the carboxy-terminal parts of the s protein involved in m-s protein interaction revealed the importance of the cytoplasmic tail. again using coimmunoprecipitation and vlp incorporation assays, it appeared that increasing truncations gradually abolished the association with the m protein (b. j. bosch, c. a. m. de haan, and p. j. m. rottier, unpublished results) . the significance of the tail domain was demonstrated most convincingly by showing the coimmunoprecipitation and vlp assembly of a chimeric vsv g protein the cytoplasmic tail of which had been replaced by that of mhv s. tail truncations were tolerated in the context of the coronavirus; recombinant mhvs were generated that lacked or (but not ) residues from the s protein carboxy terminus, but their growth was impaired by about -and -fold, respectively. also, tail extensions were tolerated, allowing the construction of a recombinant mhv with a spike protein extended at its carboxy terminus by the green fluorescent protein (gfp), yielding green fluorescent virions (bosch et al., a) . the extension was, however, lost quite rapidly on serial passaging of the virus. molecular details of the interaction of m and he proteins and the requirements of he for incorporation into viral particles have not been described. one study reported that he protein mutants lacking part of their ectodomain were not assembled into particles (liao et al., ) . most likely, however, this observation was due to folding or maturation defects of the mutant proteins. in another study the bcov s and he proteins were shown to be incorporated into mhv particles when coexpressed in mhv-infected cells. apparently, homology between the proteins of these related group coronaviruses is sufficiently high for heterologous m-s and m-he interactions to occur (popova and zhang, ) . numerous electron microscopy studies have pictured the process of virion assembly in the coronavirus-infected cell. they show the close apposition of-presumably preassembled-tubular nucleocapsids to intracellular membranes, the appearance of membrane curvature at the contact sites, the "growth" of these buds into particle-sized vesicles, and the ultimate detachment of virions from the membranes by pinching off. it has become clear that the m protein is the central player, which, through its interactions with every known component of the virion, orchestrates the entire assembly process (see fig. ). in the process two levels of interaction can be distinguished. one is the level of the membrane where, as detailed in the previous section, the m protein interacts ( ) with itself, to generate the basic molecular framework of the viral envelope, ( ) with the e protein, to induce curving and budding of the m protein-modified membrane, and ( ) with s and he, to coassemble these spikes into the viral envelope. the other level at which the m protein operates involves the incorporation of the nucleocapsid into the virion. here, two types of interactions have been described: interactions of the m protein with the n protein and with the viral genome. an instrumental role of the m protein in drawing the nucleocapsid into the budding particle is indicated by their demonstrated interaction in studies with virion preparations. the m protein has been shown to remain associated with subviral particles obtained after treatment of virions with detergent that removes the spikes (escors et al., a,b; garwes et al., ; lancer and howard, ; wege et al., ) . the association was shown for mhv to be temperature dependent (sturman et al., ) . in the case of tgev the association of m with the spherical structure, termed the core, was stabilized by basic ph and divalent cations but lost at high salt concentration, resulting in disruption of the core structure and release of the helical nucleocapsid (escors et al., a,b; risco et al., ) . in an elegant study of the binding of in vitro-translated m polypeptides to purified nucleocapsids the ionic interaction was mapped to a -residue sequence in the hydrophilic carboxy-terminal tail domain (escors et al., b) . also in infected cells, interaction of the m protein with ribonucleoprotein structures, presumably nucleocapsids, has been demonstrated. using m-specific antibodies, structures containing both n protein and genomic rna were coimmunoprecipitated with m protein from mhv-infected cell lysates (narayanan et al., ) . conversely, m protein was coprecipitated when an n-specific antibody was used, while in this case all viral mrnas copurified because of their known leader-mediated affinity for the n protein. these interactions did not require an s or an e protein (narayanan et al., ) . although interactions between the m and n proteins might intuitively be expected to drive the process of attachment of the nucleocapsid to the intracellular target membrane, direct experimental evidence for this interaction is strikingly lacking. significantly, mhv m and n proteins coexpressed in cells were found not to interact (narayanan et al., ) nor did purified tgev n protein interact with in vitrosynthesized m protein (escors et al., b) . because the n protein occurs in coronavirus-infected cells in various configurations-as a free protein and in association with an array of partners including the viral genome-it is obvious that a selection mechanism must act to ensure that only nucleocapsids are assembled into particles. consistently, coexpressed n protein is not incorporated into vlps, but its inclusion depends on the presence of viral rna (bos et al., ; vennema et al., ) . thus, unless the selection process is performed by a mechanism not involving the n protein, its association with genomic rna to form a nucleocapsid seems required to generate the unique conformation that enables it to interact with the m protein. the only evidence to date for an interaction between m and n proteins is indirect and comes from genetic studies. analysis of second-site revertants of a constructed mhv-a mutant virus lacking the two carboxyterminal residues of its m protein revealed that the highly defective growth phenotype of this virus could be restored, among others, by mutations in the carboxy-terminal domain of the n protein (kuo and masters, ) . in two independently obtained revertants the n protein had lost residues of this-among different strains of mhv highly conserved-domain because of a frameshifting -nucleotide deletion. the results argue strongly for a direct cooperation of the carboxy-terminal regions of the m and n proteins during virion formation. other indications supporting the occurrence of m-n interactions come from studies of complexes of m protein with ribonucleoprotein from mhv-infected cells and with tgev cores (escors et al., b; narayanan et al., ) . when such complexes were treated with rnase the association of m and n proteins was not destroyed, suggesting a direct interaction. however, the presence of short rnas inaccessible to the rnase but sufficient to bridge the m-n interaction could not be excluded. the most unusual interaction that the coronaviral m protein seems to engage in involves genomic rna. this interaction has so far been reported only for mhv, by makino and co-workers. these workers had shown earlier that the -nucleotide packaging signal located in the pol b gene could mediate the incorporation into virions of rnas of even noncoronaviral origin (woo et al., ) . they subsequently showed that this incorporation is most likely effected by a direct and specific interaction of the signal with the m protein. when defective genomic rnas or nonviral rnas were introduced into helper mhv-infected cells, they could subsequently be isolated as ribonucleoproteins from lysates of the cells by immunoprecipitation with an m-specific antibody, but only if the rnas contained the packaging signal (narayanan and makino, ) . coexpression experiments using noncoronaviral vectors showed the interaction to be independent of the n protein. a reporter gene transcript generated in cells expressing the m protein could be coimmunoprecipitated with an anti-m monoclonal antibody provided that the rna carried the packaging signal (narayanan et al., a) . moreover, when the e protein was additionally coexpressed, the signal-containing rna-but not an identical rna lacking this sequence-was found to be coincorporated into vlps, irrespective of the presence of the n protein. altogether these observations reveal a hitherto unknown type of interaction between a viral envelope protein and genomic rna. although its significance remains to be further established the m-rna interaction seems to provide additional selectivity to the assembly of the coronaviral nucleocapsid. assembly of viruses is a process of generally high specificity. directed by specific targeting signals, the viral structural components colocalize to distinct places in the cell where unique and complex molecular interactions control their assembly. these rules hold particularly for naked viruses and many of the smaller enveloped viruses; there are, however, many examples where the process is considerably less selective and where "nonself " (host or viral) components are coassembled (see, e.g., garoff et al., ) . interestingly, formation of the large, pleiomorphic coronaviruses appears to combine aspects of both great selectivity and extreme flexibility. with the m and e proteins as the fixed minimal requirement, coronaviral particles appear to tolerate the presence of all other viral components in practically every possible combination. a nucleocapsid is not required but, if available, it can take almost any length as defective (including chimeric) genomes of largely varying sizes have been accommodated. rnas need not necessarily be packaged into a nucleocapsid; whether of viral or nonviral origin, if provided with the proper packaging signal they can be taken in even in the absence of an n protein. also in the composition of their viral envelope these viruses are highly flexible. spikes seem to be incorporated in variable numbers depending on availability. they tolerate severe manipulation, both of their ectodomain and of their endodomain. thus, swapping of ectodomains between unrelated coronaviruses (i.e., from different groups) creates viable chimeric viruses whereas a foreign protein such as gfp appended to the s protein endodomain is accommodated in the particle, although reluctantly. some coronaviruses have an he protein but continue growing well if for any reason the gene is not (properly) expressed as happens among different mhvs. consistently, s and he proteins are incorporated independent from each other. direct interactions between s and he were not observed when the proteins were coexpressed in cells (nguyen and hogue, ) . this result is consistent with the idea that these proteins are separately drawn into the m protein lattice by their distinctive interactions with m molecules. it is also consistent with the concept that these proteins assume different positions within this lattice, a hypothesis based on the presumed different geometric requirements for the incorporation of trimeric and di-or tetrameric s and he complexes, respectively. how, in the face of this enormous flexibility in accommodating all these various numbers and combinations of viral components, do coronaviruses manage to maintain specificity? host proteins have not been noticed to occur in virions, although this may simply not have been looked at carefully enough. by probing the specificity, using viral and nonviral membrane proteins, it appeared that foreign proteins are effectively excluded from coronaviral particles . however, some missorting was found to occur, consistent with earlier observations (yoshikura and taguchi, ) . the picture of coronaviral envelope formation is one that is directed entirely by lateral interactions between the envelope proteins. in infected cells, membrane proteins-viral and cellular-are sampled for fit into the lattice formed by m molecules. the specificity of the molecular interactions acts as a quality control system to warrant the formation of the two-dimensional assemblies that contain the full complement of viral membrane proteins but from which cellular proteins are segregated. for each cellular protein the efficiency of this exclusion process is determined by its lack of interaction with the m protein, its lack of fit in the m protein framework, and its success in competing with the s and he oligomers for the (geometrically different) vacancies within this framework. the precise location of coronavirus budding and the factors that govern it have not been established. although it is clear that particle formation occurs at membranes early in the secretory pathway, up to the cis-golgi compartment, the precise site has not been identified for any coronavirus. several considerations may explain this lack of knowledge. one is that these early compartments are themselves rather complex and highly dynamic and have hence been difficult to define structurally. another is the possible alteration of the structural integrity of these compartments by infection; studies of these effects have not been described. a third complication may be that coronaviruses do not behave uniformly, different viruses possibly preferring different membranes for budding. in this respect it may be of note that differences have been observed, for instance, in the intrinsic localization of the m proteins from ibv and mhv; they appeared to accumulate on the cis and trans side of the golgi apparatus, respectively machamer et al., ) . it has long been assumed that the m protein determines the site of coronavirus budding. when, however, this protein appeared to localize beyond this site, the idea became attractive that the association of the envelope proteins may create the novel targeting signals that direct these multimeric complexes to the budding site. in support of such a notion is the fact that the s and he proteins, when coexpressed with the m protein, are retained in the golgi apparatus rather than being transported to the plasma membrane. the critical question now is whether and how the e protein affects the localization of the m protein. as the e protein by itself does not seem to localize to the virion budding site it will be of great interest to determine the membranes at which vlps assemble. as the envelope proteins can direct particle formation by themselves, it may seem that the nucleocapsid is not leading the assembly process. still, besides giving rise to virions rather than vlps, its involvement in assembly might have important consequences. first of all, nucleocapsids may enhance the efficiency of the budding process. the physical yields of vlps obtained by coexpression of the envelope proteins in cells are generally poor. although there may be many reasons for this, in infected cells the availability of nucleocapsids is likely to facilitate particle production. empty particles, considered to be vlps, have nevertheless been observed during natural infection (afzelius, ; macnaughton and davies, ) . their formation might simply serve as a means to dispose of excess viral membrane proteins from infected cells if required. another effect of the nucleocapsid could involve the localization of budding. it is conceivable that, unless a defined budding station is created by a specific interplay between viral and host proteins (for which no indications yet exist), preassembled nucleocapsids dock at those intracellular membrane sites where sufficiently sized patches of m protein-based envelope structure have accumulated. early in infection such patches might start to form only after the envelope proteins have left the endoplasmic reticulum and become concentrated in intermediate membranes on their way to the golgi complex. later, when viral protein synthesis increases, this density might be reached earlier, perhaps explaining the observed late budding in the endoplasmic reticulum (goldsmith et al., ; klumperman et al., ; tooze et al., ) . it will again be interesting to learn where vlps independently bud and how this relates to the local density of the envelope proteins because this information will shed light on the role of the nucleocapsid in the localization of virion assembly. the picture of coronavirus assembly that the available literature allows us to draw in this review is still a rough draft. we know the identity and some characteristics of the key elements of the picture, we know the relative positions and orientations of most of them, but we are unable to fit them all into a sensible composition. although this may seem like a discontented retrospective, it certainly is not. in the years that the senior author has been in coronavirology research, enormous progress has been made in practically all its aspects including virion assembly. however, the rewarding act of compiling and ordering the available information and trying to abstract from it actual knowledge was at the same time a sharp and recurrent confrontation with the unknown. we want to conclude this work by summarizing what in our opinion will be the main issues for the near future. with the obstacle of reverse genetics technology solved, "structure" will be the dominating issue of the next decade. biology has taught that molecular insight into processes will eventually depend critically on detailed structural information. for coronavirus assembly this means data on the individual structural components and, particularly, on the virion. with respect to the former, this will be most challenging for the membrane proteins, notably m and e. virions, by their apparent elasticity, have eluded structural analysis. here, despite the still limited resolution to be expected, cryoelectron microscopy should provide the urgently required insight into the structural organization of the particle. another issue will be the cell biology of assembly. this actually refers to a number of poignant problems at every stage of the process. starting with nucleocapsid formation we must admit that we know practically nothing. by which interactions and where on the genome the packaging is initiated, how the wrapping of the rna proceeds, how the condensation of the ribonucleoprotein structure takes place, and where in the cell these activities take place are all unresolved questions. although we seem to know more about the budding process, several fundamental issues are still unresolved. obvious issues are the site of budding and the determinants of its location, and the inclusion of the nucleocapsid into the budding particle. an intriguing issue is the budding mechanism itself: how is membrane curvature generated and, particularly, how is the directionality determined. coronaviruses, like other intracellularly budding viruses, direct their particles out of the cytoplasm into the organelles, that is, opposite to the natural direction of cellular vesicle budding. once again, simply nothing is known about the governing principles. a third field of research that has yet to open is the contribution of host cellular factors to the assembly process. work has so far been concentrating on the viral components and their interplay. although there have been incidental indications, studies on the specific involvement of host proteins apparently had to await the development of appropriate technologies and these are now becoming available. although the serious health threat caused by the - epidemic of sars apparently has waned, 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virus-infected cells and e protein-expressing cells membrane topology of coronavirus e protein japanese encephalitis virus-vaccinia recombinants produce particulate forms of the structural membrane proteins and induce high levels of protection against lethal jev infection in vivo and in vitro models of demyelinating diseases. v. comparison of the assembly of mouse hepatitis virus, strain jhm, in two murine cell lines localization of an rna-binding domain in the nucleocapsid protein of the coronavirus mouse hepatitis virus reverse genetics of the largest rna viruses receptor-induced conformational changes of murine coronavirus spike protein membrane integration and intracellular transport of the coronavirus glycoprotein e , a class iii membrane glycoprotein n-terminal domain of the murine coronavirus receptor ceacam is responsible for fusogenic activation and conformational changes of the spike protein endosomal association of a protein phosphatase with high dephosphorylating activity 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determinant bovine coronavirus uses n-acetyl- -o-acetylneuraminic acid as a receptor determinant to initiate the infection of cultured cells isolated he-protein from hemagglutinating encephalomyelitis virus and bovine coronavirus has receptordestroying and receptor-binding activity the nucleocapsid protein gene of bovine coronavirus is bicistronic minus-strand copies of replicating coronavirus mrnas contain antileaders emergence of a coronavirus infectious bronchitis virus mutant with a truncated b gene: functional characterization of the b protein in pathogenesis and replication heterogeneous nuclear ribonucleoprotein a regulates rna synthesis of a cytoplasmic virus the coronaviridae characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry secreted hepatitis b surface antigen polypeptides are derived from a transmembrane precursor mouse hepatitis virus replicase proteins associate with two distinct populations of intracellular membranes identification of a new membrane-associated polypeptide specified by the coronavirus infectious bronchitis virus expression of transcriptional units using transmissible gastroenteritis coronavirus derived minigenomes and full-length cdna clones isolation and identification of virus-specific mrnas in cells infected with mouse hepatitis virus (mhv-a ) coronavirus proteins: structure and function of the oligosaccharides of the avian infectious bronchitis virus glycoproteins specific interaction between coronavirus leader rna and nucleocapsid protein synthesis and subcellular localization of the murine coronavirus nucleocapsid protein mouse hepatitis virus nucleocapsid protein-specific cytotoxic t lymphocytes are ld restricted and specific for the carboxy terminus phosphoproteins of murine hepatitis viruses isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid conformational change of the coronavirus peplomer glycoprotein at ph 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coronaviruses in enucleated cells receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins a -amino acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme murine coronavirus packaging signal confers packaging to nonviral rna efficacy of a transmissible gastroenteritis coronavirus with an altered orf- gene localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the protein may disrupt host cell division the sialate- -o-acetylesterases of coronaviruses related to mouse hepatitis virus: a proposal to reorganize group coronaviridae structural basis for coronavirus-mediated membrane fusion: crystal structure of mhv spike protein fusion core unique n-linked glycosylation of murine coronavirus mhv- membrane protein at the conserved o-linked glycosylation site human aminopeptidase n is a receptor for human coronavirus e neuropathogenicity of mouse hepatitis virus jhm isolates differing in hemagglutinin-esterase protein expression hemagglutininesterase specific monoclonalantibodies alter the neuropathogenicity of mouse hepatitis virus heterogeneity of gene expression of the hemagglutinin-esterase (he) protein of murine coronaviruses biosynthesis, structure, and biological activities of envelope protein gp of murine coronavirus the detection and characterization of multiple hemagglutinin-esterase (he)-defective viruses in the mouse brain during subacute demyelination induced by mouse hepatitis virus synthesis and processing of the haemagglutinin-esterase glycoprotein of bovine coronavirus encoded in the e region of adenovirus mouse hepatitis virus strain mhv-s: formation of pseudotypes with a murine leukemia virus envelope mouse hepatitis virus gene b protein is a new virion envelope protein suppression of sars-cov entry by peptides corresponding to heptad regions on spike glycoprotein conformational changes in the spike glycoprotein of murine coronavirus are induced at c either by soluble murine ceacam receptors or by ph expression of hemagglutinin/esterase by a mouse hepatitis virus coronavirus defective-interfering rna alters viral pathogenesis presence of subgenomic mrnas in virions of coronavirus ibv the amino and carboxyl domains of the infectious bronchitis virus nucleocapsid protein interact with genomic rna the infectious bronchitis virus nucleocapsid protein binds rna sequences in the terminus of the genome following the rule: formation of the -helix bundle of the fusion core from severe acute respiratory syndrome coronavirus spike protein and identification of potent peptide inhibitors virus-encoded proteinases and proteolytic processing in the nidovirales we thank henk halsema for making most of the drawings. we are grateful to cristina risco for providing the electron micrographs demonstrating the structural maturation of virions; to raoul de groot, berend jan bosch, and jean lepault for their combined efforts in providing the electron micrographs of the purified virions; and to raoul de groot for critical reading of the manuscript. key: cord- -krw zmr authors: rao, pasupuleti v.; kumari, suman; gallagher, thomas m. title: identification of a contiguous -residue determinant in the mhv receptor that controls the level of virion binding to cells date: - - journal: virology doi: . /viro. . sha: doc_id: cord_uid: krw zmr abstract murine carcinoembryonic antigens serve as receptors for the binding and entry of the enveloped coronavirus mouse hepatitis virus (mhv) into cells. numerous receptor isoforms are now known, and each has extensive differences in its amino terminal immunoglobulin-like domain (ntd) to which mhv binds via its protruding spike proteins. some of these receptor alterations may affect the ability to bind viral spikes. to identify individual residues controlling virus binding differences, we have used plasmid and vaccinia virus vectors to express two forms of mhv receptor differing only in their ntd. the two receptors, designated biliary glycoproteins (bgp) aand b ntd, varied by residues in the amino acid ntd. when expressed from cdnas in receptor-negative hela cells, these two bgp molecules were displayed on cell surfaces to equivalent levels, as both were equally modified by a membrane-impermeant biotinylation reagent. infectious center assays revealed that the aisoform was to times more effective than b ntdin its ability to confer sensitivity to mhv (strain a ) infection. bgp awas also more effective than bgp b ntdin comparative virus adsorption assays, binding times more mhv (strain a ) and . times more mhv (strain jhmx). bgp awas similarly more effective in promoting the capacity of viral spikes to mediate intercellular membrane fusion as judged by quantitation of syncytia following cocultivation of spike and receptor-bearing cells. to identify residues influencing these differences, we inserted varying numbers of bresidues into the bgp abackground via restriction fragment exchange and site-directed mutagenesis. analysis of the resulting chimeric receptors showed that residues to of the ntd were key determinants of the binding and fusion differences between the two receptors. these residues map to an exposed loop (c-c′ loop) in a structural model of the closely related human carcinoembryonic antigen. nized mhv strains (barthold et al., ; barthold, ) . these receptors are type i single-pass transmembrane coronaviruses are attractive objects for studies of aniglycoproteins containing an ectodomain structure made mal virus entry into cells because there exists a remarkup of four immunoglobulin-like domains (dveksler et al. , able level of variation in the ligands that engage virions ). the variability between a and b lies primarily in with cells. several strains of mhv are now recognized the most membrane-distal domain (yokomori et al., (siddell, ) and several receptors are also known b, dveksler et al., a) . this amino terminal do- (dveksler et al., ; yokomori and lai, b; nedellec main (ntd) is essential for virus binding in vitro (dveksler et al., ; chen et al., ) . this abundant natural et al., b) and is similarly required to render cells variation provides the opportunity to compare different susceptible to infection by mhv (dveksler et al., ) . receptors for their ability to interact with different virus initial studies on the coronavirus-binding functions of strains. such comparative analyses will allow one to bgp a and b indicated that they differ substantially in identify the determinants responsible for differences in virion adsorptive capacity -when the two receptors virus:cell interactions. were immobilized on nitrocellulose filters, only the a in this study, we have focussed attention on two allelic isoform supported detectable virus-binding (dveksler et isoforms of the mhv receptor, biliary glycoprotein (bgp) al., a) . this finding suggested that one or more of isoforms a (dveksler et al., ; the amino acid differences between the a and b a) and b (yokomori and lai, b; ntds (yokomori and lai, b ) are responsible for a; mccuaig et al., ) , and we have examined controlling virion adsorptive capacity, either because their capacity to interact with purified enveloped corothey provide direct receptor:virion contact points or benavirions. the a isoform is expressed in mhv suscepticause they control the overall conformation of the recepble mice, while mouse strains homozygous for the b tors. if the ntd differences were indeed critical, then isoform are generally resistant to many currently recogidentification of the relevant residues could be achieved by constructing a / b hybrid cdnas and then measuring virus binding to the protein products. we have used duce chimeras that have been useful in identifying resi-changed. b - and b - were made by exchange of acci -psti and bamhi-psti fragments, respec-dues controlling the virus-adsorptive capacity of the mhv receptor. tively (see fig. ). additional exchanges were performed after creation of a ndei site at codon using the megaprimer mutagenesis procedure (aiyar and leis, ) . ndei site creation altered codon (n h) in bgp a and cells and viruses codons and in bgp b ntd (n h, k m). b - and b - were made by exchange of ndei -ndei baby hamster kidney (bhk), rabbit kidney (rk ), and and ncoi -ndei fragments, respectively (see fig. ). rehuman carcinoma (hela) cells were grown in dulbecco combinant b - was made by oligonucleotide-dimodified eagle medium (dmem) supplemented with % rected pcr mutagenesis using a primer spanning coheat-inactivated fetal bovine serum (dfbs). murine dons - and the ndei site (scharf, ) . nucleotide cl cells (sturman and takemoto, ) were grown in sequencing methods (sanger et al., ) were employed dmem containing % tryptose phosphate broth (tpb; to confirm that bgp a and b clones matched those re-difco laboratories, detroit, mi) and % dfbs. murine ported for these genes (mccuaig et al., ) and to astrocytoma (dbt) cells (hirano et al., ) were grown verify the isolation of all of the mutant receptors dein minimal essential medium (mem) containing % tpb scribed herein. and % dfbs; these cells were used to measure mhv infectivities by plaque assay. murine coronavirus strains generation of vaccinia virus recombinants. a and jhmx (makino et al., ) (felgner et al., (icn radiochemicals, irvine, ca) . clarified supernatants ) . at days postinfection, cultures were frozen at were overlaid above % (w/w) sucrose cushions in pbs Њ. after three freeze -thaw cycles, debris was recontaining . % bsa and subjected to ultracentrifugamoved by centrifugation and virus in the clarified matetion (beckman-spinco sw rotor for hr at , rpm). rial was plated on rk cells. plaques arising from spe-virion-containing pellets were resuspended in ice-cold cific amplification of recombinant virus were revealed pbs containing . % bsa and stored in silanized glass by overlay of cells with mycophenolic acid-containing vials at Њ. medium, as described by falkner and moss ( ) . virus from well isolated plaques was grown in rk cells synthesis and cloning of mhv receptor cdna to generate stocks of recombinant virus (vbgp a , constructs vbgp d, vbgp b ntd , vbgp a q g,v g , and additional a / b methods for construction of vaccinia virus insertion/ chimeras). expression plasmids (ptm ; moss et al., ) encoding mhv receptor isoform bgp a or bgp a lacking codons immunoblot analysis of bgp proteins - (bgp d) have been described previously (gal-cell monolayers were infected with the respective lagher, ). these plasmid vectors were further modi-vbgp recombinants (m.o.i. Å ) and coinfected with fied by reinsertion of bgp ntd sequences into bgp d. vtf . (m.o.i. Å ). at hr postinfection, lysis buffer ( to this end, the bgp a and b ntd sequences were mm tris -hcl (ph . ), mm nacl, mm edta, . % amplified from outbred cd mouse liver rna by rt-pcr np- , . mm pmsf, and . u/ml aprotinin) was added, (kawasaki, ) using primers based on the reported and -ml aliquots (equivalent to cells) were bgp a sequence (mccuaig et al., ) and products subjected to western immunoblotting. receptor was encoding for the a and b ntd were identified by identified using antireceptor antibody (a gift of dr. restriction mapping and by sequencing (sambrook et m. j. buchmeier, scripps research institute, la jolla, ca) al., ) . b ntd sequences were cloned in-frame into which was directed against conserved carboxy-terminal the unique xmai site of bgp d to generate the chimeric residues - . bgp b ntd . two undesired codon alterations that resulted at the junctions of the insertion (q g and v g) quantitation of relative bgp cell surface levels were introduced into a control bgp a construct by performing a parallel insertion of the bgp a ntd into ptm -at hr postvaccinia infection, hela cells expressing the bgp molecules were washed three times with ice-bgp d. the control recombinant was designated as bgp a q g,v g . cold pbs and incubated for hr at Њ with pbs containing mg/ml nhs-lc biotin (sulfosuccinimidyl- -(biotina-to construct additional bgp chimeras for use in identifying virus binding determinant(s), restriction fragments mido) hexanoate; pierce co., rockford, il). unreacted reagent was then quenched by the addition of mm between ptm -bgp a and ptm -bgp b ntd were ex-tris-hcl (ph . ) for min at Њ. monolayers were virus binding assays washed thoroughly with pbs and lysed ( cells/ hela cells overexpressing bgp receptors from vacml in lysis buffer). clarified cytoplasmic extracts ( ml) cinia vectors were washed twice with ice-cold sfm at were then mixed with antipeptide antibody ( . ml) hpi, then radiolabeled mhv particles were diluted in and left overnight at Њ. twenty microliters of gammabind sfm and added at varying multiplicities. after incuba-g-sepharose (pharmacia biotech) was then added for tions at Њ, unadsorbed virions were removed and cells hr at Њ. sepharose beads were collected by centrifugawere rinsed five times with ice-cold pbs containing . % tion, washed twice in ripa buffer ( mm tris-hcl (ph bsa and . % tween . ripa buffer was added, and . ) mm nacl, % sodium deoxycholate, . % sds, radioactivities associated with the cell lysates and superand % triton x- ), then once in mm tris -hcl (ph natant media were quantified by scintillation counting. . ) containing . % v/v nonidet p- . immunoprecipitated receptors were then subjected to western blotting, intercellular fusion assay and the biotinylated fraction of each sample was identi- the cell fusion-dependent reporter gene (b-galactosified by incubation of blots for hr with . % v/v streptavidase) activation assay of nussbaum et al. ( ) was din-horseradish peroxidase (hrp) conjugate (pierce adapted for studies of mhv fusion as described pre-co.) in pbs/ % bsa. immobilized hrp was detected by viously (gallagher, ) , with minor modifications. in incubation in pbs containing . mg/ml -chloro- -naphbrief, stably transfected hela-bgp a cells were infected thol, % v/v methanol, and . % hydrogen peroxide. with mhv-a (m.o.i. Å ) and with vaccinia virus strain blots were photographed (polaroid film) and signal wr (m.o.i. Å ). after hr at Њ, these cells were further intensities were quantified with an lkb ultrascan xl dentransfected by lipofection with the reporter gene consitometer. struct pg nt b-gal (kindly provided by dr. richard a. morgan, national center for human genome research, bethesda, md). five hours later (before the onset of mhv vaccinia-infected bhk cells were challenged with a -induced syncytium formation yet after pg nt b-gal mhv-a (m.o.i. Å ) at hr postvaccinia infection. dna amplification via vaccinia-encoded polymerases) fourteen hours later, cells were washed with pbs, fixed the cells were trypsinized, suspended in dmem % in acetone, and incubated for hr at Њ with a : dfbs, and . -ml ( cell) aliquots were overlaid dilution of antispike mab b . (collins et al., ) on to confluent -cm monolayers of hela cells that were in pbs/ % bsa. bound mab was detected with fitccoinfected hr earlier with vtf . and vbgp recombiconjugated goat ig directed against mouse ig (cappell, nants. after hr at Њ, the mixed monolayers were fixed durham, nc) and cells were photographed using a leitz and stained with x-gal for in situ localization of b-gal fluorescence microscope. activity (macgregor et al., ) . alternatively, the monolayers were lysed by addition of . % np- in pbs and infectious center assays the quantity of b-galactosidase in each lysate was calculated using a colorimetric enzyme assay involving hydro-cdna constructs encoding bgp a and bgp b ntd were lysis of chlorophenyl red b-galactopyranoside (nussinserted into plasmid expression vector puhd- - baum et al., ). od values were normalized by (gossen and bujard, ) and the plasmids ( . mg) comparing the hydrolysis rates for each sample with that were transfected by lipofection into aliquots of obtained for a standard preparation of e. coli b-galactosi-hela-tta cells (gossen and bujard, ) . to determine dase (calbiochem, la jolla, ca) and were expressed as transfection efficiencies, parallel cultures were cotransnanograms/well. fected with each puhd-bgp construct in conjunction with . mg of the b-galactosidase-encoding pcmv-b (clon-results tech labs, inc.). in situ x-gal staining (macgregor et al., ) at hr posttransfection revealed . and . % synthesis of chimeric mhv receptors from vaccinia b-gal-positive cells for the a and b ntd transfectants, revirus vectors spectively. to assess susceptibilities to mhv infection, cells were washed at hr posttransfection with cold a method for receptor overexpression on cell surfaces was necessary to reproducibly measure the binding of serum-free dmem (sfm) and inoculated with mhv-a at Њ for hr. unbound virus was removed by washing mhv particles to a series of different receptors. hela cell lines in which the mhv receptor gene was stably and with pbs containing . % bsa and . % tween and cells were then incubated in dmem % dfbs for hr constitutively expressed (hela-bgp a cells) have been developed (gallagher, ) ; however, we found that at Њ. cells were then trypsinized, washed twice with dmem % dfbs, and serial dilutions were plated on clones varied dramatically in receptor levels (data not shown). therefore we anticipated difficulty in identifying dbt indicator monolayers. plaques were visualized after a -day incubation period. a series of stable hela cell transfectants in which differ- the carboxy-terminal amino acids are identical for to increase the likelihood of equivalent receptor producall four receptors; and an antipeptide antibody ( ) tion, different receptor cdnas were expressed from vacraised against these cytoplasmic residues was available cinia virus vectors. such vectors have historically produced to us for use in monitoring receptor levels. initial tests high levels of surface glycoprotein (broder et al., ; nus- using this anti-c-terminal antibody were performed by sbaum et al., ) and their preparation is known to be immunoblotting bhk and hela cell lysates collected relatively straightforward (falkner and moss, ) . hr after coinfection with vtf . and vtm -bgp. the re-a set of cdnas capable of encoding four distinct sults (fig. ) revealed identical patterns of immunoreacforms of mhv receptor were each recombined into tive protein in lysates expected to contain bgp b ntd , the vaccinia virus genome using insertion vector ptm a q g,v g , and a (lanes - , respectively). a sharp band (elroy-stein and moss et al., ) and at c. kda was detected, which likely represent underrecombinant viruses (designated vtm -bgp) were seglycosylated form(s) of the amino acid proteins. the lected as described under materials and methods. the series of bands ranging from to kda indicated use of the ptm vector places the cdnas under the that equivalent levels of the various glycoforms of receptranscriptional control of a bacteriophage t promoter; tor were present in each infected culture. lysates exthus expression of mhv receptors required coinfection pected to contain bgp d had a similar, slightly more of hela cells with vtm -bgp and vtf . , which encodes bacteriophage t rna polymerase (fuerst et al., ) . the four translation products predicted from this expression scheme are depicted in fig. . the mature bgp a , after removal of its signal peptide, has an ectodomain composed of four immunoglobulin-like domains, designated d to d (dveksler et al., ) . a deletion mutant of bgp a (bgp d) was prepared by excision of nucleotides encoding the bulk of the virus-binding d domain as well as amino acids of the d domain. corresponding sequences from the allelic bgp b (mccuaig et al., ) were then placed back into the truncated cdna to generate a hybrid capable of encoding the chi- to test the contribution that these two changes might an antipeptide antibody directed against conserved carboxy-terminal have on interaction with virions, cdna for a bgp a con- ( ) taining the mutations (designated bgp a q g,v g ) was bgp d, ( ) vtf . alone, ( ) molecular weight markers, kilodaltons. prepared by ligating the ntd sequences of bgp a into identical profiles of immunoreactive protein were observed in similarly infected hela cell lysates. the deletion mutant. sion vector puhd- - and transfected into hela-tta cells (gossen and bujard, ) tantly, the results of fig. showed that bgp only to log higher. moreover, these differences in receptor effectiveness could not be eliminated by increasing input multiplicities during virus challenge. fiintense immunoreactive protein profile, with the apparent nally, we observed a straightforward correlation between molecular weights of the bands lower by approximately infectious centers and virus yields; hr after challenge kda (lane ). of the bgp transfectants with mhv a at pfu per to determine whether the four different mhv receptors cell, yields were , , and pfu/ml for the a , were displayed on the cell surface at similar levels, we b ntd , and control transfectants, respectively. thus, when first made the assumption that each receptor contains expressed in the hela-tta cells, bgp a was utilized as similar numbers of exposed amines available for conjuan mhv-a receptor much more effectively than gation with the membrane-impermeant biotinylation re-bgp b ntd . agent sulfo-nhs-lc biotin. with this in mind, we derivatized the proteins on intact cells at hr postvaccinia infection with the reagent and then we lysed cells and virus binding capacities of the chimeric mhv immunoprecipitated the receptor proteins. the relative receptors proportion of biotinylated receptor among the immuno-with the hela cell monolayers each expressing a difprecipitates was then assessed by western blotting usferent mhv receptor on the cell surface, straightforward ing streptavidin-horseradish peroxidase as a detection virus adsorption assays became feasible. thus we colreagent. by this method, the relative abundance of biotinlected particles from supernatants of mhv-infected cl ylated receptors were similar in all cultures; densitometcells that had been incubated for hr in the presence ric scans of the blots revealed less than % variation of trans[ s] label, and we concentrated the radiolabeled in signal intensities ( percentage of added radioactivity remaining bound to the monolayers was determined. the results (fig. ) re-spike antigen (fig. , panels , , and ) while the truncated receptor failed to support mhv-a infection vealed a gradual increase in the levels of nonspecific adsorption of virions to cells displaying bgp d, from . % (panel ), in concert with the results of dveksler et al. ( b) . after min, to . % by hr, and . % by hr. mhv-a bound far more avidly to cells with bgp a or b ntd ; . additional examination of the various receptors for their ability to confer susceptibility to infection involved and %, respectively, after hr. subtraction of nonspecific adsorption values revealed that the bgp b ntd was -fold infectious center assays. to this end, cdnas encoding bgp a or bgp b ntd were introduced into plasmid expres-less effective than bgp a in virion adsorptive capacity. this difference in efficiency of binding was maintained identify the residues involved in virus binding by systematically exchanging a and b residues. this was accomthroughout the -hr, Њ incubation period. moreover, this plished through a series of restriction fragment ex-pattern whereby bgp a bound more virus than b ntd rechanges to produce recombinant receptors containing mained constant over a -fold range of virus input multivarying numbers of b residues within a bgp a backplicities (data not shown). ground (fig. ) . these receptors were expressed in in all subsequent assays, we chose to interact [ s] hela cells from vaccinia vectors and then tested for virions with receptor-bearing cells for hr at Њ, as bindtheir ability to bind [ s] labeled mhv particles. chimeric ing differences between the two receptor isoforms were receptors containing b residues from positions to readily discernable under these conditions. our next were as effective as a in binding, while the reciprocal assay involved testing the related strain jhmx for bindchimeras containing b residues to were ineffective ing. for jhmx, a similar pattern of virus adsorption (table ) . emerged (fig. ) . the amount of virion adsorption, how-to further narrow down residues controlling receptor ever, was lower for jhmx and the difference between binding efficiency, we focused on codons to , as bgp a and bgp b ntd in jhmx adsorptive capacity was only this region is the most variable among the presently se- . -fold, comparatively lower than the -fold difference quenced bgps (see fig. ; boxed region). additionally observed when strain a was used. this stretch of six residues is predicted to form a protruding loop connecting two b strands that form the frame-mapping the mhv receptor determinants that control work of the immunoglobulin-like cea domain (bates et virus adsorption al., ) . thus the bgp a residues to were the two receptors contain a limited number of amino changed to b residues by oligonucleotide-directed mutaacid differences and show measurable differences in genesis. the resulting chimera ( b - ) exhibited a weak binding efficiency that was equivalent to that of virus binding capacity. this provided an opportunity to (table ) . thus a key determinant of binding differences between the isoforms was present within enhance intercellular fusion with s-expressing cells. to these six residues. do this, we employed a cell fusion-dependent reporter gene activation assay (nussbaum et al., ) . in brief, in performing these experiments comparing the efficiency of bgp a and bgp b ntd receptors, we readily identified a correlation between virus binding capacity and syncytium formation. upon infection by mhv-a , stable hela-bgp a transfectants fused rapidly into polykaryons, while the corresponding hela-bgp b ntd cells were much less susceptible. this was readily evident by simple microscopic examination of infected cell monolayers anywhere from to hr postinfection. however, previously published information suggested that measurements of additional receptor mutants for virus binding and fusion promotion capacities might not reveal a direct relationship between these two properties. first, evidence for the uncoupling of virus binding and fusion activation functions has been demonstrated in studies of mutant retrovirus receptors (james et al., ; lifson et al., ; truneh et al., ) . second, two reports focusing on the the results obtained from a subset of these tests is shown in fig. . when cells were fixed and stained with transfectants with vaccinia virus (strain wr) and mhv strain a and then loading a fraction of the cells with x-gal (macgregor et al., ) , intense blue clusters were abundantly evident in monolayers expressing bgp a (fig. a transcriptionally silent b-galactosidase gene under the control of the phage t promoter. these cells, which a, panel a) and the bgp a q g,v g mutant. fewer blue syncytia were seen in the bgp b ntd -expressing mono-bear s proteins on their surface, were then overlaid at subconfluent densities onto monolayers of hela cells layers (fig. a, panel c) , while blue cells were rare in the cultures lacking the bgp ntd altogether (fig. a , coinfected with vtf . and the respective vtm -bgp recombinants. transfer of the t polymerase to the s-ex-panel b). quantitation of the b-galactosidase activity in detergent lysates prepared from each monolayer at vari-pressing cells causes b-galactosidase expression, which can be measured and taken as an indicator of the ous times after cell mixing revealed the potency of each receptor in promoting fusion (fig. b) . from to hr after amount of fusion between receptor and spike-bearing cells. cocultivation, specific b-galactosidase expression in cultures harboring bgp a was - times the level found in bgp b ntd -containing cultures, in concert with the sixfold previous qualitative assays in which virions were al-bound to bgp b ntd . when identical assays were performed using [ s] mhv strain jhmx, . % bound specifi-lowed to interact with denatured and immobilized mhv receptors showed that allelic receptor isoforms termed cally to bgp a , while . % bound bgp b ntd (fig. ). these differences between the two receptors in virus binding bgp a and b do indeed differ in virion adsorptive capacity (boyle et al., ; dveksler et al., a) , thereby capacity impact the outcome of mhv infection. we found that transfection of either bgp a or bgp b ntd genes into providing the opportunity for determining which nonhomologous region(s) control the differences. our initial hela cells conferred sensitivity to mhv a infection, but bgp b ntd was inefficiently utilized as revealed by goal was to establish assays in which we could accurately measure the relative virus binding capacities of quantitation of infectious centers (fig. ) . this latter finding was generally consistent with a variety of previous these two receptors. we anticipated that the success of these assays might require abundant receptor levels observations. chen and baric ( ) reported that challenge of stable bhk-bgp a transfectants with mhv a because previous attempts to measure virion binding to murine cells endogenously expressing bgps were not led to titers of pfu/ml after day, while parallel challenge of bhk-mmcgm (bgp b ) transfectants sensitive enough to reveal any specific adsorption (yokomori et al., ; wilson and dales, ) . our binding yielded only pfu/ml. in similar tests, compton ( ) found analagous (but less dramatic) differences between assays therefore involved interaction of radiolabeled virions to native receptors that were overexpressed on the the two stably transfected bhk cell lines in their support of mhv a infection. finally, yokomori and lai ( b) surface of hela cells from vaccinia vectors. by this method, specific binding measurements were obtained, found that cos cells transiently transfected with bgp a or b supported very low levels of mhv a ; the a even though the proportions of cell-associated [ s] virions were relatively low (fig. ) . adsorption might be lim-transfectants were marginally superior as virus hosts. we have exchanged portions of bgp a cdna with ited in part by slow diffusion to cell monolayers, as prolonged incubation periods gradually increased binding those from bgp b and have assessed the resulting hybrid gene products for virus binding capacity. our most infor- (fig. ) . ligand densities also likely play a role-recombinant bgp a that is immobilized onto sepharose at ex-mative recombinant receptor was a bgp a in which only residues - were specific to b . this chimera was traordinarily high densities will bind the majority of [ s] virions in our recently developed in vitro receptor binding indistinguishable from the complete bgp b ntd in its support of virus binding (table ; see data highlighted by assays (gallagher, ) . we established conditions in which parallel hela cell bold type), thus strongly suggesting that these six residues are the critical determinants controlling virus ad-monolayers displayed equivalent amounts of either bgp a or a chimeric bgp b ntd on the cell surface. these sorption levels. despite this compelling data, it must be fig. . quantitation of intercellular fusion between spike-and receptor-bearing hela cells. stable hela-bgp a transfectants were infected with mhv strain a (m.o.i. Å ) and concomitantly transfected by lipofection with pg nt -bgal, which produces the b-gal product only in the presence of t rna polymerase. hr later, the spike-bearing cells were trypsinized and overlaid onto hela cell monolayers that had been infected hr earlier with vtf . and the indicated vtm -bgp recombinants. the resulting intercellular fusion levels were quantitated by measuring b-gal enzyme activities produced by the mixing of t rna polymerase (in vtf . /vtm -bgp cytosol) with pg nt -bgal (in mhv-a cytosol). (a) cocultivated cells were fixed after a -hr incubation and incubated with x-gal substrate to reveal the b-gal product in situ (a) bgp a , (b) bgp d, (c) bgp b ntd , (d) bgp a q g,v g . (b) at the indicated times after overlay, cell monolayers lysed with pbs containing . % np- . b-gal activity in the lysates was measured by a colorimetric assay and the od nm values were normalized by comparison with od nm produced by a preparation of purified bgalactosidase. remembered that a tremendous number of possible re-the binding capacities of the receptors. in fact we have come to similar conclusions using mhv jhm in place of ceptor chimeras can be generated, each with a unique combination of a and b residues. it therefore remains a -while receptor independent fusion was observed with jhm, enhancement of this endogenous activity was possible that investigations of additional a / b combinations will reveal a role for residues outside the - most pronounced in the presence of receptors with a residues - . at present we have no evidence of sepa-stretch in binding, perhaps because some patterns will impact overall receptor conformation. rate domains on the bgp molecule that are individually responsible for binding and induction of membrane fu-exchange of bgp a residues - with those from b also eliminates a potential n-linked glycan addition site sion. however, such separate domains may eventually be identified, as studies of both the hiv and poliovirus at residue . however it has been established from thorough mutagenesis studies in the holmes laboratory receptors have revealed specific regions that induce changes in virion conformation required for membrane that a carbohydrate at this position does not contribute to virus binding (dveksler et al., ) . identification of penetration (james et al., ; morrison et al., ) . finally, the results from these quantitative binding these critical amino acid residues led us to attempt blockade of binding with synthetic peptides. to date we assays may shed light on the determinants of murine coronavirus tropism in vivo. jhmx can infect murine brain have been unable to interfere with the virus:receptor interaction using synthetic bgp a peptide - , implying tissue more readily than a (lavi et al., ; robb and bond, ) , and the overall abundance of bgp a and b a requirement for the immunoglobulin-like framework region in presenting a defined conformation of the resi-in brain is very low lai, a, b) . given that jhmx is actually less avid than a in the dues. according to a predicted three-dimensional model for bgp binding properties, it appears likely that the different in vivo tropisms of these strains are not dictated simply the corresponding human cea ntd (bates et al., ) , residues to would represent a ''loop'' that is held by their capacity to bind these bgps but rather by utilization of alternative receptors or by additional events oc-in place by two of the beta strands (strands c and c) that comprise a portion of the framework region (fig. ) . curring subsequent to binding. this c-c loop is further predicted to protrude from the internal framework and thus it is reasonable to infer its choe and sodroski, ; harrison, ) . po- gossen and herman bujard for the hela-tta cells. this work was liovirus binding is influenced by mutations in the c-cЉ, supported by nih grant r -ns- and by a grant from the cold spring harbor using recombinant vaccinia virus vectors a pregnancy-specific glycoprotein is expressed in the brain and serves as a receptor for mouse hepatitis fusion mutants are attenuated and display altered hepatotropism utility of mouse cell line dbt for propagation and assay of mouse hepatitis virus persistence: coevolution of increased host cell resistance and virus virulence contribution of charged amino acids in the cdr region of cd to hiv- gp binding pcr protocols'' (m. a. monoclonal antibodies to murine hepatitis virus- (strain jhm) define the viral glycoprotein responsible for attachment and cell-cell fusion the organ tropism of mouse hepatitis virus a in mice is dependent on dose in their use of murine carcinoembryonic antigen-related glycoprotein receptors synthetic cd peptide derivatives that inhibit hiv infection and cytopathicity. science , - . the mouse hepatitis virus (mhv) receptor: expression in human and hamster cell lines confers susceptibility to mhv expression in vivo analysis of genomic and intracellular viral rnas of small plaque mutants of related glycoprotein family are functional receptors for the coronavirus mouse hepatitis virus-a virus strain a and blocking antireceptor monoclonal antibody bind to the n-terminal domain of cellular receptor quantitative equilibrium and kinetic binding studies of mutants in conjunction with a high resolution cd atomic structure homolog-scanning mutagenesis reveals poliovirus receptor residues important for virus binding and replication escherichia coli gpt gene provides dominant selection for vaccinia virus open reading frame expression lipofection: a highly efficient, lipid-mediated dna-transfection procedure. mouse hepatitis virus infection proc. natl. acad. sci. usa bgp , a new member of the carcinoembryonic antigen-related gene family, encodes an transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage t rna polymerase fusogenic mechanisms of enveloped-virus glycoproteins analyzed by a novel recomindependent infection by a neurotropic murine coronavirus murine coronavirus membrane fusion is blocked by modification of thiols buried within the spike protein a role for naturally occurring variation of the measles virus hemagglutinin and cd pathogenic murine coronaviruses. murine coronavirus spike protein in stabilizing association with the cellular receptor characterization of biological behavior in vitro and virus-specific intracellular rna of strongly neurotropic jhmv and weakly neuro-gombold molecular cloning: protein cleavage signal tight control of gene expression in a laboratory manual in vivo and in vitro models of demyelinating disease: efficiency of virus spread and formation of cloning with pcr. in ''pcr protocols'' (m. a. innis, infectious centers among glial cells is genetically determined by the murine host mouse hepatitis virus utilizes two carcinoembryonic antigens as alternative receptors tis virus in the resistant mouse strain sjl is functional: implications for the requirement of a second factor for viral infection -spike protein-dependent cellular factor other than the viral receptor is required for mouse hepatitis virus entry cЉ-d, and d-e loops (aoki et al., ; morrison et al., schweppe foundation of chicago. ). additional comparisons of receptor structure and function will require definitive resolution of the bgp a references receptor through crystallographic methods. this is fast becoming a realistic possibility as a system for produc- aiyar, a., and leis, j. ( ) . modification of the megaprimer method tion and purification of reasonably large quantities of of pcr mutagenesis: improved amplification of the final product. , - . that the relative fusion levels corresponded directly with key: cord- - tok mqk authors: nanda, s. k.; johnson, r. f.; liu, q.; leibowitz, j. l. title: mitochondrial hsp , hsp , and hsp bind to the ′ untranslated region of the murine hepatitis virus genome date: - - journal: arch virol doi: . /s - - - sha: doc_id: cord_uid: tok mqk we have previously shown that mitochondrial-aconitase binds specifically to the ′ terminal nucleotides of the murine hepatitis virus (mhv) rna along with three additional proteins of , and kda to form a stable rna-protein complex. supershift and western blot assays have identified these three proteins as mitochondrial hsp (mthsp ), hsp , and hsp . a series of co-immunoprecipitation assays have established that these four mhv rna binding proteins are associated, even in the absence of mhv rna. however, the presence of a synthetic rna containing the sequence bound by these four proteins does increase the amount of co-precipitated protein, in particular the amount of hsp which is brought down with antibodies directed against hsp and mthsp . we have provided evidence for the interaction of these four proteins with the ′ end region of mhv rna in infected cells by a series of immunoprecipitation rt-pcr assays. we believe it is likely that mhv rna interacts with m-aconitase prior to its import into mitochondria in cooperation with extra-mitochondrial mthsp , hsp , and hsp . murine hepatitis virus (mhv) is a prototypic member of the family coronaviridae, and contains a single-stranded, positive-sense rna genome approximately kb in length [ ] . viral proteins are translated from six to seven subgenomic mrnas as well as from the genome. the virus-specific subgenomic and genomic rnas make up a -coterminal nested-set [ , ] and contain a leader sequence of approximately nucleotides (nt) at the end [ , ] . coronaviruses perform their entire replication program in the cytoplasm of infected cells. following uncoating, coronaviruses express the largest known replicase polyproteins, which in turn are proteolytically processed to yield a large number of mature proteins including rna-dependent rna polymerase (rdrp) [ , ] . the rdrp, perhaps in association with host proteins, directs the synthesis of negative-sense rnas from the end of the viral genome. analysis of the structure of defective interfering (di) rnas indicated that approximately nucleotides (nt) at the terminus, and nt at the terminus are required for di rna replication in mhv-infected cells [ , ] . the cis-acting signals for the synthesis of negative-strand rna are contained within the last nucleotides plus the poly (a) tail at the end of the mhv genome [ ] . specific binding of host cellular proteins to two distinct sites within the -utr of mhv-jhm genomic rna has been reported [ ] . one site, the (+) protein binding element [ (+) ] , was mapped within the most nt of the genomic rna [ ] , the other site was mapped to nucleotides - upstream from the end of the viral genome [ ] . cytosolic protein extracts from murine cl- cells assayed using a probe containing the (+) element formed three rna-protein complexes, with the slowest migrating and presumably largest complex, complex , being the most abundant [ , ] . recently, we established that mitochondrial aconitase (m-apo-aconitase) binds specifically to the last nucleotides of the utr of mhv rna [ ] . however, the interaction of purified m-apo-aconitase by itself was not stable under electrophoretic conditions. four different proteins with molecular weights of kda (m-apo-aconitase), kda, kda and kda were required to form a complex with the last nucleotides of the mhv utr which was stable enough to survive electrophoresis. in this work we have identified the kda protein bound to the mhv protein binding element as mitochondrial hsp (mthsp ), the kda protein as hsp , and the kda protein as hsp . murine cl- cells were cultured in dulbecco's minimal essential medium supplemented with % fetal bovine serum at • c in a % co atmosphere [ ] . the origin and growth of the jhm strain of mhv (mhv-jhm) virus used in this study have been described previously [ ] . infected cell extracts were prepared from confluent cl- monolayers infected with mhv-jhm at a multiplicity of infection (m.o.i.) of pfu/cell. theatpase activity of the post-mitochondrial lysate was measured by determining the amount of free radioactive p i liberated by [γ- p]atp hydrolysis. the assays were done essentially as previously described [ ] except that the partially purified post-mitochondrial lysates (approximately µg total protein) were incubated at • c with purified m-apo-aconitase ( - µg, graciously supplied by m.c. kennedy, gannon university, erie, pa) and mhv-jhm utr rna in atpase buffer ( mm hepes, ph . , mm kcl, mm mgcl , . % np- , mm dtt, mm atp containing µci [γ- p]atp plus cocktails of protease and phosphatase inhibitors). at the times indicated, the reaction was stopped by the addition of µl of acid washed charcoal (sigma) ( . % in mm hcl and mm h po ) to bind free nucleotides. the samples were centrifuged at , rpm for min, and the radioactivity mthsp , hsp and hsp bind to the mhv -utr contained in the supernatant was determined by liquid scintillation counting. control reactions were carried out in the absence of purified m-apo-aconitase or by replacing the enzyme with bsa. all assays were performed in triplicate and mean values and standard deviations calculated. mouse monoclonal antibodies directed against mthsp , hsp and hsp were obtained from stressgen biotechnologies corp (victoria, b.c., canada) and affinity bioreagents inc. (golden, colorado). a rabbit antiserum against m-aconitase was provided by dr. richard s. eisenstein, university of wisconsin, madison [ ] . the monoclonal antibody directed against the mhv nucleocapsid gene, antibody - - , has been described [ ] . monoclonal antibodies directed against a t -tag and anti-sv t ag (ab- ) were purchased from novagen (madison, wi) and oncogene science (cambridge, ma) respectively. a mouse monoclonal antibody directed against phosphotyrosine (py ) was purchased from bd transduction laboratories (lexington, ky). in vitro transcription reactions were carried out with [α- p]utp to generate radiolabeled probes [ ] . after transcription, the radiolabeled rnas were purified through sep-pak light c cartridges (waters, milford, ma). the concentration of radiolabeled rna was measured spectrophotometrically. crude cytoplasmic lysates and post-mitochondrial lysates were prepared from cl- cells as previously described [ ] . rna-protein binding reactions were performed in a volume of µl as described previously [ ] . briefly, extracts containing - µg of total protein, to ng of one of the p-labeled mhv (+) rna probes, µg heparin (sigma), ng of poly(i)-poly(c) (sigma) and % glycerol were incubated at • c for min in binding buffer ( mm tris ph . , mm mgcl , mm dtt, mm kcl) and then digested at • c for min with rnase t (calbiochem). rna-protein complexes were subjected to nondenaturing polyacrylamide gel electrophoresis, dried, and autoradiographed as described [ ] . for supershift assays approximately µg of protein in the post-mitochondrial lysate was incubated either with . or µg of monoclonal antibody, or with buffer ( mm tris/hcl, ph . , and . % np- ) alone in a final volume of µl at • c for h. after the incubation the mixture was used for the standard rna binding assay. a biotinylated synthetic rna ( -aguaaaugaa ugaaguugaucauggccaauugg aaga- ) corresponding to nucleotides - at the end of the mhv genome (counting the first nucleotide upstream from the poly(a) tail as position ) was purchased from dharmacon research (boulder, co). aliquots of biotinylated rna were cleaved and deprotected as per the manufacturer's guidelines and bound to magnetic strepavidin beads (perseptive biosystems, farmingham, ma) as described previously [ ] . the beads were washed free of unbound rna with buffer b ( mm tris, ph . , mm mgcl , mm edta, mm dtt, % glycerol, mm pmsf, µg/ml leupeptin, µg/ml aprotinin . µg/ml pepstatin) and a preparation of mhv (+) rna binding proteins, partially purified by sequential ion exchange and heparin agarose binding steps [ ] , was added to the beads in the presence of heparin (sigma), poly(i)-poly(c) (sigma), and trna, at concentrations of µg/µl, ng/µl, and µg/µl, respectively. the binding reaction was incubated for h at • c and washed four times with buffer b. the bound proteins were eluted by boiling the beads in × sds loading buffer. s. k. nanda et al. samples (either immunoprecipitates or rna affinity purified proteins) were added to sdslysis buffer preheated to • c and boiled for min. proteins were separated on either % standard or - % gradient (w/v) sds-polyacrylamide gels and transferred to nitrocellulose membranes (biorad) using a semi-dry blotting procedure [ ] . membranes were blocked overnight in tbst ( mm tris-hcl, ph , mm nacl, . % (v/v) tween ), % (w/v) dried milk powder, incubated for h with primary antibodies in tbst, % (w/v) dried milk powder, washed in tbst, and incubated with a second antibody conjugated to horseradish peroxidase for h. membranes were developed using an ecl (enhanced chemiluminescence) detection kit (amersham pharmacia biotech, piscataway, nj) and visualized by exposure to biomax light film (kodak, rochester, ny). uv cross-linking was performed as described previously [ ] with the modification that after uv crosslinking and digestion with rnase a samples were immunoprecipitated and the labeled proteins resolved by sds-page. uninfected and infected cl- cells were lysed in ml of ice-cold lysis buffer containing mm tris-cl ph . , mm nacl, mm edta, mm sodium pyrophosphate, mm orthovandate, % np- , mm naf, µg/ml leupeptin, µg/ml pepstatin and mm phenylmethylsulphonyl fluoride. lysates were clarified by centrifugation at , × g for min. immunoprecipitation was carried out using dynabeads protein a (dynal biotech, lake success, ny) as per the manufacturer's recommendations. antibodies were bound to protein a immobilized on the magnetic beads and cross-linked using dmp (dimethyl pimelimidate) in . m triethanolamine ph . . antibodies cross-linked to the protein a magnetic beads were used to capture the antigens from the cl- cell lysates. immunoprecipitates were washed several times with pbs, extracted in × sds-buffer and then separated by sds-polyacrylamide gel electrophoresis and analyzed by western blotting. fifty microliters of dynabeads-protein a suspension per reaction were washed twice with . ml of rnase-free . m na-phosphate (pbs, ph . ) and resuspended in µl of pbs. approximately µg of each monoclonal antibody tested [ - - , directed against jhm strain n protein ( ) , anti-mitochondrial hsp , anti-hsp , anti-hsp , µl of anti-t -tag, anti-sv t ag] was added to magnetic beads, and rotated slowly for min at room temperature. the test tubes were then placed in a magnetight separation stand (novagen) for min and supernatants were removed. the beads were washed twice with . ml of rnasefree . m na-phosphate (ph . ) containing . % bsa. five hundred microliters of binding buffer [ mm tris (ph . ), mm kcl, mm mgcl , and mm dtt] used for rnase protection/gel mobility shift assays was used for the last wash and subsequently discarded. two hundred micrograms of mock-infected and mhv-jhm infected post-mitochondrial fractions were resuspended in binding buffer and added to the beads.antigen-antibody binding reactions were rotated for h at room temperature. the immunocomplexes bound to the beads were washed three times with binding buffer. proteins and protein-rna complexes binding to the beads were eluted in µl of mm tris (ph . ), % sds, and mm edta at • c for min. the eluted material was treated with µg of proteinase k ( mg/ml) at • c for min, extracted twice by phenol-chloroform and nucleic acids precipitated by ethanol in the presence of µg mthsp , hsp and hsp bind to the mhv -utr glycogen carrier. rna was pelleted by centrifugation and used as template for reverse transcription (rt). rt was performed using a mhv-jhm specific primer, spanning nt to at the end of jhm genome ( gtagtgccagatgggtta ), and superscript ii (invitrogen) at • c for h. synthesized cdna was used as a template for pcr. the positive sense primer for pcr was the same primer as used in rt reactions. the negative sense primer corresponds to the complementary sequence from nt to at the end of jhm genome ( gtgattcttccaattggc ). amplification was performed at • c for sec, • c for sec, • c for sec for cycles; at • c for sec, • c for sec, • c for sec for the next cycles, at • c for sec, • c for sec for the last cycles; followed by a min extension at • c. ten nanograms of the plasmid de was used as a positive control in pcr reactions. rt-pcr was also carried out to detect the abundant cellular gapdh mrna. the sequence of the rt primer for the synthesis of gapdh cdna is: -gccaaaagggtcatcatctc- . the rt primer also serves as the positive sense primer for pcr. the sequence of the negative sense primer for gapdh is: -gtagaggcagggatgatgttc- (primers were provided by dr. george davis, tamus-hsc). pcr reactions underwent cycles of amplification (each cycle consists of at • c for sec, • c for sec), followed by a min extension at • c. pcr products were resolved by agarose gel electrophoresis. we have recently demonstrated that m-apo-aconitase is one of the four proteins which bind to the last nucleotides upstream of the poly(a) tail of the mhv genome [ (+) protein binding element] as an rnp complex using gel shift/rnase protection and uv-cross-linking assays. in these assays the addition of a rnase t digestion step prior to electrophoresis increases both the resolution and sensitivity of gel shift assays by digesting unbound rna and rna nonspecifically bound to protein, and by decreasing the size and apparent heterogeneity of the rna-protein complexes [ ] . the digested rna runs as a smear with the dye front at the bottom of the gel, with the majority of the digested probe running out of the gel. three rna-protein complexes are detected by this assay. the largest complex, complex , is typically the most abundant complex detected, with the two faster migrating complexes being produced in variable amounts [ , ] . in the course of experiments investigating the binding of m-apo-aconitase to the (+) protein binding element, we determined that the addition of atp and purified m-apoaconitase to cytoplasmic lysates increased rna binding activity (fig. a , compare lanes and ), in gel shift/rnase protection assays. the formation of the largest complex, complex , was most dramatically affected (black arrow, fig. a ). it should be noted that the lysates used in these experiments contain a large amount of m-aconitase, only a small fraction of which is the apo-enzyme which binds mhv rna [ ] . the addition of atp to the binding reactions in the absence of added m-apo-aconitase produced only a small increase in rna binding activity (fig. a , lane ). when atp was omitted from the binding reaction (fig. a , lane ) no increase in rna binding activity was detected in the presence of m-apo-aconitase. these effects were consistently observed in multiple experiments. substituting bsa for m-apo-aconitase in these binding reactions resulted in no increase in rna binding activity (data not shown). these finding led us to generate a working hypothesis that the lysate might possess anatpase activity and thatatp hydrolysis generated energy for a conformational change in m-apo-aconitase which resulted in more efficient rna binding. we therefore analyzed rna binding activity in the presence of atp-γs, a non-cleavable analogue of atp. the substitution of atp-γs for atp did not stimulate rna binding activity (fig. a, to further investigate the role of atp in the rna binding reaction, we measured the ability of rna binding reactions containing m-apo-aconitase, atp, and cytoplasmic lysate to hydrolyze [γ- p]atp, as described in materials and methods. as shown in fig. b , atp was hydrolyzed in the complete binding reaction. only minor amounts of atpase activity were observed when purified m-apo-aconitase was omitted from the reaction or when it was replaced with bsa. similar results were obtained in multiple experiments. thus stimulation of rna binding activity and atpase activity were both dependent on the presence of cytoplasmic lysate, m-apo-aconitase, and atp. atpase activity was strictly dependent on the presence of mgcl in the reaction buffer, since no activity was detected in the absence of mgcl or in the presence of mm edta (not shown). thus cytoplasmic lysate exhibits a cation dependent atpase activity characteristic of members of the hsp family of chaperones [ ] . we have previously shown by northwestern blotting and affinity chromatography that in addition to m-apo-aconitase, three other proteins with molecular masses of approximately , , and kda bind to rna corresponding to the last nucleotides of the mhv genome [ ] . the presence of an atpase activity in cytoplasmic lysates which was stimulated by the addition of m-apo-aconitase and rna containing the (+) protein binding element, plus the association of hsp family members with proteins destined to be imported into mitochondria [ ] , suggested to us that the kda protein might be a member of the hsp family. to investigate this possibility we performed a "supershift" assay with antibodies to mthsp and hsp/hsc . these two monoclonal antibodies to related hsp family members do not cross-react. the antibody specific for mthsp produced a supershifted rnp complex ( fig. a, lane , gray arrow) . although the amount of the supershifted rna-protein complex was relatively small, it was quite specific; antibody to the closely related hsp/hsc protein failed to produce a supershifted band ( fig. a, lane ) as did cytoplasmic lysate in the absence of antibody ( fig. a, lane ) , although both contained rnaprotein complex (indicated by the black arrow). the relatively small fraction of rna-protein complexes which were supershifted suggests that an excess of mthsp was present in the binding reaction over that which bound to mhv rna in complex . alternatively, complex might be formed by a heterogeneous population of proteins and only those containing mthsp are supershifted, or the antibody used had a relatively low affinity for mthsp . labeled rna incubated with antibody to mthsp in the absence of cytoplasmic lysate did not produce any rna-protein complexes, only small rnase t digestion products were detected (fig. a, lane ) . similar experiments were attempted with an antisera to mitochondrial aconitase (kindly provided by dr. richard s. eisenstein, university of wisconsin, madison) but were unsuccessful due to large amounts of ribonuclease present in this rabbit polyclonal antisera (data not shown). identical experiments using a labeled rna containing an nucleotide mutation known to interfere with formation of the rna-protein complex [ ] , as expected, failed to contain either the shifted or supershifted complexes (not shown). mthsp undergoes tyrosine phosphorylation under stress conditions [ ] . it has been reported previously that infection with mhv- induces the tyrosine phosphorylation of cellular proteins [ ] ; thus it seemed logical to investigate the phosphorylation status of the mthsp proteins in the rnp complex. preincubation of the cytoplasmic lysate with anti-phosphotyrosine antibodies led to the presence of two supershifted rnp complexes with a corresponding decrease in the intensity of the unshifted rnp complex (fig. b, lane ) . these data suggest that at least one of the proteins binding to the mhv (+) protein binding element is tyrosine phosphorylated, possibly mthsp . hsp potentiates the function of the hsp family of protein both in vitro and in vivo [ ] . this suggested to us the possibility that the kda mhv (+) binding protein could be hsp . to investigate this hypothesis we performed a "supershift" assay with an antibody to hsp . as shown in fig. c , preincubation of crude post-mitochondrial lysate with anti-hsp antibody prior to our standard rnase protection/gel mobility shift assay resulted in the presence of two additional slowly migrating protected rnp complexes (lane ) compared to our standard assay (lane ). the result of the supershift assay suggested that the kda protein we had observed upon uv-crosslinking was hsp . the finding that two of three rna binding proteins which we had identified were hsp family members suggested to us that the kda protein might be hsp . a supershift assay with anti-hsp produced a supershifted band, as shown in fig. c , lane , confirming our suspicion that the kda protein was hsp . the finding that antibodies to hsp and hsp only supershifted a relatively small portion of rna-protein complex suggests that for these two chaperones, only a small fraction of these molecules participated in the formation of mhv rna-protein complexes. alternatively, complex might be formed by a heterogeneous population of proteins and only those containing hsp or hsp are supershifted, or the antibodies used had relatively low affinities. to confirm the results of our gel shift analyses, we employed two additional sets of immunologic assays. previously we showed that four proteins with molecular weights of kda (m-apo-aconitase), kda, kda and kda bound to an rna affinity reagent corresponding to the mhv (+) protein binding element and were eluted with sds-page sample buffer [ ] . mhv (+) rna binding proteins were affinity purified in the presence of trna, heparin and poly(i)-poly(c) to block non-specific binding to the affinity matrix as described in materials and methods, resolved by sds-page and electrophoretically transferred to membranes. immunoblot analysis of the material eluted from the specific rna affinity matrix with anti-mthsp (fig. a, lane ) or anti-hsp (lane ) antibodies demonstrated immunoreactive species with molecular masses of approximately kda and kda, respectively. these experiments confirmed the identity of the kda protein as mthsp and the kda protein as hsp . the second immunologic assay we employed was an immunoprecipitation assay after transferring label from a p-labeled rna probe containing the mhv (+) protein binding element to its cognate protein binding partners by uvcrosslinking. immunoprecipitation with anti-hsp pulled down four proteins protein binding element were partially purified and then subjected to affinity purification with a biotinylated rna immobilized on magnetic beads as described in materials and methods. bound proteins were eluted by boiling the beads in × sds-page loading buffer, resolved by sds-page ( - % gradient gel), and transferred to a nitrocellulose membrane. the membrane was probed with either anti-mthsp antibody ( ) or with anti-hsp antibody ( ) . the positions of molecular weight markers are indicated. b post-mitochondrial lysates were incubated with ng of p-labeled mhv (+) rna for min at • c and digested with limiting amounts of rnase t as described previously [ ] . the rna-protein complexes formed were irradiated with uv light for min. the samples were digested with rnase a ( µg/µl) and directly immunoprecipitated with anti-mthsp ( ) or irrelevant antibody ( ) (fig. b, lane ) . a parallel immunoprecipitation reaction with anti-mthsp antibody produced the identical result (fig. b, lane ). an irrelevant control polyclonal rabbit antibody (fig. b, lane ) failed to precipitate any labeled proteins.a control immunoprecipitation reaction with a monoclonal antibody to the hsp/hsc mthsp , hsp and hsp bind to the mhv -utr (cytoplasmic) protein also failed to precipitate any labeled proteins (not shown). immunoprecipitation of uv-cross linked rnp complexes with anti-hsp also pulled down all four proteins, although the signals were weak even after days of exposure (fig. b, lane ) as opposed to a one day exposure for the other immunoprecipitation experiments. the immunoprecipitation reactions were all performed after treating the rna-protein complex with rnase. the finding that each of these antibodies pulled down the same four proteins after extensive rnase a digestion suggests that these four proteins bound to the mhv (+) containing rna as a complex rather than as four proteins binding to different regions on this rna. this data is consistent with our previous glutaraldehyde crosslinking experiments which suggested that there were protein:protein interactions amongst all four proteins ( , , and kda) which made up rnp complex [ ] . a multi-protein rnp complex can form either by binding of a pre-formed multiprotein complex to an rna, or multiple proteins may recognize the rna separately, and only establish protein:protein contacts after they have bound to the rna. in order to clarify the situation we immunoprecipitated rnp complexes formed in uv-crosslinking reactions using unlabeled rna containing the mhv (+) protein binding element. these immunoprecipitates were compared to immunoprecipitates prepared in parallel from partially purified cytoplasmic lysate in the absence of rna. both sets of immunoprecipitates were subjected to immunoblot analysis. when we used anti-mthsp or anti-hsp to pull down the rnp complex or lysate in the absence of added rna, and blotted the immunoprecipitated material separated by sds-page with anti-hsp , hsp could be detected in both the cases, whereas samples immunoprecipitated with non-specific antibody did not contain hsp (fig. a) . however, the signal for hsp in immunoprecipitates with anti-hsp was considerably augmented by the addition of rna. when the immunoprecipitation experiments were carried out with anti-mthsp , anti-hsp , or anti-hsp , and the immunoprecipitated materials were blotted and probed with anti-m-aconitase, m-aconitase was detected irrespective of the presence of rna, but was not detected in immunoprecipitates with non-specific antibody (fig. b) . however, the signals for m-aconitase were greatly increased by the presence of rna in the immunoprecipitation reactions with anti-hsp and anti-hsp . thus the association of hsp with hsp and with m-aconitase is stimulated by the presence of mhv (+) containing rna. these data suggest that these proteins are recruited to an rna-protein complex. to search for the presence of specific mhv rna-protein complexes in cells rather than formed after the in vitro addition of synthetic rna probes we performed a series of immunoprecipitation-rt-pcr assays. mhv rna-protein complexes ( , , ) or with primers for the abundant mrna for gapdh ( , , ) . pcr products were resolved by electrophoresis in a . % agarose gel mthsp , hsp and hsp bind to the mhv -utr were immunoprecipitated from post-mitochondrial lysates prepared from mock infected and mhv-jhm infected cl- cells with various monoclonal antibodies and amplified by rt-pcr with primers directed against the untranslated region of the mhv genome. a monoclonal antibody to the mhv nucleocapsid (n) protein, a protein which is known to bind to mhv rnas, was used as a positive control. antibodies to mthsp , hsp , and hsp all co-precipitated mhv rna which could be amplified by rt-pcr (fig. a, lanes - ) . the assay appeared to be specific in that monoclonal antibodies to irrelevant antigens (lanes and ) did not bring down complexes containing mhv rna. to further investigate the specificity of this assay we performed a second set of immunoprecipitation reactions and attempted to detect mrna for the highly expressed house keeping gene gapdh as well as for mhv specific rnas (fig. b ). antibodies to mthsp , hsp , and hsp captured mhv rna (lanes , , ) , but failed to precipitate gapdh mrna (lanes , , ) . the relative amount of mhv rna captured by the three anti-hsp antibodies in this experiment correlates well with the immunoprecipitation uv-crosslinking experiment shown in fig. b . thus we have now demonstrated that mthsp , hsp , and hsp bind to mhv rnas present in infected cells. the untranslated regions of most positive stranded rna viruses interact with host factors during their replication. we have previously shown that m-aconitase binds specifically to the last nucleotides of the utr of mhv rna along with three other proteins with molecular weights of approximately kda, kda and kda to form an rnp complex [ ] . in this study, we have used supershift assays and western blot assays of rna affinity purified protein to identify the three additional components of this rna-protein complex as mthsp , hsp , and hsp . we have also demonstrated this four protein -rna complex in postmitochondrial extracts of mhv infected murine cells by immunoprecipitation of rna-protein complexes followed by rt-pcr assays. both m-aconitase and mthsp are nuclear encoded, posses mitochondrial targeting sequences, and are thought to translocate into mitochondria shortly after synthesis. mthsp plays an important role in importation of proteins into the mitochondrial matrix [ ] . however mthsp has been documented to be present in extra-mitochondrial locations by biochemical fractionation, immunofluorescent labeling and confocal microscopy, and immunoelectron microscopy [ , , ] . this protein is also known as a glucose response protein (grp ); a senescencerelated gene product, mortalin; and as a peptide binding protein, pbp . this compendium of names reflects the different intracellular compartments and functions which have been described for this protein in addition to its role in refolding and import of mitochondrial proteins. mammalian hsp s have a predominantly cytoplasmic distribution in the cell [ ] . although the functional hsp complex is predominantly localized to mitochondria in eukaryotes [ ] , detailed immunoelectron microscopy studies in a wide variety of cells and tissues show that - % of hsp immunoreactivity is present at discrete extra-mitochondrial sites, including unidentified cytoplasmic vesicles [ , , ] and secretory granules [ ] . mhv replicates in the cytoplasm, making it unlikely that mhv interacts with these proteins inside mitochondria. we believe that it is most likely that mhv rna interacts with m-aconitase prior to its import into mitochondria in cooperation with extra-mitochondrial mthsp , hsp , and hsp . the alternative scenario which would allow this interaction to occur, requires export of at least m-aconitase from mitochondria. hsp , hsp , and mthsp are all molecular chaperones. the folding of most newly synthesized proteins in the cell requires the interaction of a variety mthsp , hsp and hsp bind to the mhv -utr of protein cofactors known as molecular chaperones. these molecules have been identified in most cellular compartments of different organisms, and recognize and bind to nascent polypeptide chains and partially folded intermediates of proteins, preventing their aggregation and misfolding [reviewed in [ ] ]. there are several families of chaperones; those most involved in protein folding are the -kda heat shock protein (hsp ; dnaj), -kda heat shock protein (hsp ; groel), and -kda heat shock protein (hsp ; dnak) families. hsp family members are regulated by hsp (dnaj or its homologs). these essential and ubiquitous partner proteins make up the dnaj family, with hsp (hdj- ) [ ] and hsdj (hdj- ) [ ] being the best studied human homologs. they function to increase atp turnover, facilitate chaperone action, and promote a more stable interaction with protein substrates [ , ] . the atpase activity we detected in partially purified lysates during rna binding reactions is consistent with the known properties of hsp 's atpase activity. it also suggests the possibility that mthsp is interacting with a dnaj domain containing partner, such as hsp during rna binding reactions. gel supershift assays with anti-phosphotyrosine antibodies (fig. b ) demonstrate that at least one of the proteins in the complex formed with mhv (+) containing rnas and m-aconitase, mthsp , hsp , and hsp is tyrosine phosphorylated. although m-aconitase, hsp , and mthsp contain predicted potential tyrosine phosphorylation sites, only mthsp has been experimentally demonstrated to undergo tyrosine phosphorylation [ , ] . thus mthsp is the protein most likely to be responsible for the supershift we observed with antiphosphotyrosine antibody. immunoprecipitation/western blot assays with anti-mthsp and anti-phosphotyrosine antibodies indicate that a portion of the mthsp is tyrosine phosphorylated, at least in mouse fibroblasts which contain mhv (+) rna binding activity (nanda and leibowitz, unpublished) . the phosphorylation/dephosphorylation status of many rna binding proteins plays a major role in terms of their interaction with rna. enzymatic treatment of crude lysates with alkaline phosphatases greatly inhibits their rna binding activity (nanda and leibowitz, unpublished) . this marked decrease in rna binding activity after dephosphorylation of the lysate argues in favor of an important role for mthsp tyrosine phosphorylation in the formation of the rnp complex. previously published northwestern blot data [ ] suggest that all four of the proteins are individually able to interact with mhv (+) containing rna. a series of co-immunoprecipitation assays (fig. ) have established that these four proteins are associated, even in the absence of rna. however, the data clearly indicated that the presence of rna enhanced the association of hsp with hsp and with m-aconitase. the addition of rna also enhanced the association of m-aconitase with hsp , but did not affect the association of mthsp with hsp or m-aconitase. one possible interpretation of these results is that those associations which we observed to be rna independent are due to the known associations of these proteins with each other (mthsp with m-aconitase, hsp s with hsp ), whereas the remainder are dependent on rna and the formation of an rna-protein complex. our data do not distinguish between two possibilities, formation of this four protein rnp complex from the separate proteins as opposed to rna binding to a pre-existing four protein complex. in either case our results clearly indicate that these four proteins form a complex with mhv-jhm (+) containing rna. we have established here that m-aconitase, mthsp , hsp and hsp interact with each other as well as with mhv rna. the nature of the interaction between hsp and hsp is complex. the interaction between the j domain and the hsp atpase domain is well established. an interaction between hsp and the peptide-binding domain of hsp also occurs in vitro. other hsp family members have been shown to bind to rna substrates and may guide the appropriate folding of these rnas and affect subsequent regulatory processes such as mrna stability and/or translation [ , , ] . thus it is quite possible that hsp potentiates the activity of mthsp and hsp , and thus stabilizes the interaction of m-aconitase with the rna. it is quite interesting that tomita and colleagues have recently reported that a yeast hsp family member, ydj p, is involved in forming brome mosaic virus replication complexes active in negative-strand rna synthesis, and suggested that a chaperone system involving ydj p participates in viral replicase folding or assembly into the active replication complex [ ] . it has recently been demonstrated that groel, the e. coli hsp homolog, can associate with lipid membranes while remaining functional as a protein folding chaperone [ ] . replicating mhv rna has been localized to early endosomal vesicles [ ] . it is possible that the association of hsp with the viral rna may contribute to its intracellular localization. this could occur if the interaction of hsp (likely in association with the other three proteins which make up the rna-protein complex we have identified) with the rna assists the intracellular translocation of rna-protein complexes [ ] . groel has also been suggested to be part of a protein complex that protects bacterial transcripts from rnase e-mediated degradation [ , ] . molecular chaperones rarely function alone; rather they function together in complex pathways [ ] . in mammalian cells, components of the mitochondria, namely grp (mthsp ) and hsp , a homolog of bacterial groel, were found to associate with each other and transiently interact with newly synthesized mitochondrial proteins [ ] . only a few partnerships between various hsp s and hsp s have been elucidated. the most well-studied chaperone partnership is one found in e. coli between dnak, a chaperone of the hsp class, and dnaj, an hsp . both genetic and biochemical evidence indicates a functional partnership between these two chaperones [ , , ] . however, a chaperone complex containing mthsp , hsp and hsp along with m-aconitase has not been reported previously. we have provided evidence for the existence of such a complex and for its interaction with the end region of mhv rna. the precise functional significance of this interaction for mhv replication is still unclear, although our previous work is consistent with its having an enhancing effect on viral rna stability [ ] . it is clear that a single hsp protein may interact with more than one hsp family member [ ] . our knowledge of the interactions between the various chaperones themselves, as well as with newly synthesized mthsp , hsp and hsp bind to the mhv -utr proteins or other chaperone target proteins is incomplete. it will be interesting to see how modulating the expression of these hsps affects mhv replication. the hsp and hsp chaperone machines dietary iron intake modulates the activity of iron regulatory proteins and the abundance of ferritin and mitochondrial aconitase in rat liver regulation of hsp function by a eukaryotic dnaj homolog intracellular processing of the n-terminal orf a proteins of the coronavirus mhv-a requires multiple proteolytic events identification of groel as a constituent of an mrna-protection complex in escherichia coli ) p , a member of the heat shock protein family, undergoes tyrosine phosphorylation in response to oxidative stress mammalian hsp and hsp proteins bind to rna motifs involved in mrna stability mammalian -kda stress protein (chaperonin homolog). identification, biochemical properties, and localization protein folding in vivo: unraveling complex pathways requirement for hsp in the mitochondrial matrix for translocation and folding of precursor proteins analysis of cis-acting sequences essential for coronavirus defective interfering rna replication the molecular biology of coronaviruses further characterization of mranas of mouse hepatitis virus: presence of common -end nucleotides cytoplasmic protein binds in vitro to a highly conserved sequence in the untranslated region of ferritin heavy-and light-subunit mrnas increased hepatotropism of mutants of mhv, strain jhm, selected with monoclonal antibodies the virus-specific intracellular rna species of two murine coronaviruses: mhv-a and mhv-jhm escherichia coli dnaj and grpe heat shock proteins jointly stimulate atpase activity of dnak identification of the cis-acting signal for minus-strand rna synthesis of a murine coronavirus: implications for the role of minus-strand rna in rna replication and transcription deletion mapping of a mouse hepatitis virus defective interfering rna reveals the requirement of an internal and discontinous sequence for replication mitochondrial hsp ssc : role in protein folding a specific host cellular protein binding element near the end of mouse hepatitis virus genomic rna the bovine papilloma virus e protein has atpase activity essential to viral dna replication and efficient transformation in cells murine hepatitis virus strain induces the macrophage prothrombinase fgl- through p mitogen-activated protein kinase activation subcellular localization of the hsp -homolog encoded by beet yellows closterovirus regulation of the heat-shock protein reaction cycle by the mammalian dnaj homolog, hsp cell-cycle dependent tyrosine phosphorylation on mortalin regulates its interaction with fibroblast growth factor- the two mammalian mitochondrial stress proteins, grp and hsp , transiently interact with newly synthesized mitochondrial proteins mitochondrial aconitase binds to the -untranslated region of the mouse hepatitis virus genome human cdna encoding dnaj protein homologue cloning of a cdna for heat-shock protein hsp , a human homologue of bacterial dnaj mammalian hsp /dnaj homologs: cloning of novel cdnas and a proposal for their classification and nomenclature extramitochondrial localization of mortalin/mthsp /pbp /grp interaction of erythropoietin rna binding protein with erythropoietin rna requires an association with heat shock protein mthsp , hsp and hsp bind to the mhv maturation of the polymerase polyprotein of the coronavirus mhv strain jhm involves a cascade of proteolytic processing events cloning and some novel characteristics of mitochondrial hsp from chinese hamster cells functional interaction of heat shock protein groel with an rnase e-like activity in escherichia coli immunoelectron microscopic localization of the -kda heat shock chaperonin protein (hsp ) in mammalian cells mitochondrial-matrix proteins at unexpected locations: are they exported? coronavirus mrna synthesis involves fusion of non-contiguous sequences sequence relationships between the genome and the intracellular rna species , , , and of mouse hepatitis virus strain a mutation of host dnaj homolog inhibits brome mosaic virus negative-strand rna synthesis evidence for a lipochaperonin: association of active protein-folding groesl oligomers with lipids can stabilize membranes under heat shock conditions electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications localization of mouse hepatitis virus nonstructural proteins and rna synthesis indicates a role for late endosomes in viral replication molecular chaperones in pancreatic tissue: the presence of cpn , cpn and hsp in distinct compartments along the secretory pathway of the acinar cells the nh -terminal amino acids of the escherichia coli dnaj protein stimulate the atpase activity of dnak and are sufficient for lambda replication a conserved motif at the end of mouse hepatits virus genomic rna required for host protein binding and viral rna replication specific binding of host cellular proteins to multiple sites within the end of mouse hepatitis virus genomic rna analysis of sequence-specific binding of rna to hsp and its various homologs indicates the involvement of n-and c-terminal interactions this work was supported by in part by national multiple sclerosis society grant rg -b- and a generous gift from the stearman family. we gratefully acknowledge dr. claire kennedy for providing us with purified m-aconitase and richard eisenstein for providing us with antim-aconitase antibody. the authors would like to thank dr. lori bernstein and dr. van wilson for their helpful comments and reading of the manuscript. we also thank elena belyavskaya for her help and encouragement. key: cord- - qiwui u authors: torrecilhas, a c t; faquim-mauro, e; da silva, a v; abrahamsohn, i a title: interference of natural mouse hepatitis virus infection with cytokine production and susceptibility to trypanosoma cruzi date: - - journal: immunology doi: . /j. - . . .x sha: doc_id: cord_uid: qiwui u mouse hepatitis virus (mhv) infection can have a pronounced impact on several investigation areas. reports on natural mhv outbreaks are rare and most studies have been conducted by deliberately infecting mice with mhv laboratory strains that cause moderate to severe disturbances to the immune system. we have investigated the effects of a natural acute outbreak of mhv in our otherwise specific-pathogen-free (spf) inbred mouse colonies, and of enzootic chronic mhv infection on cytokine production and resistance to the intracellular pathogen trypanosoma cruzi. we found that balb/c and/or c bl/ spf mice that had been injected with t. cruzi blood trypomastigotes from recently mhv-contaminated (mhv(+)) mice developed significantly higher parasite blood counts, accelerated death, and showed higher il- production by spleen cells than their counterparts whose t. cruzi inoculum was derived from mhv-negative (mhv(−)) donors. interferon-γ (ifn-γ) production by mhv(+) and mhv(−) mice was not significantly different. in contrast, t. cruzi infection of chronically mhv-infected mice did not result in major changes in the course of infection when compared with that observed in mice from mhv(−) colonies, although a trend to higher parasitaemia levels was observed in balb/c mhv(+) mice. nevertheless, both balb/c and c bl/ t. cruzi-infected mhv(+) mice had diminished ifn-γ production to parasite-antigen stimulation in comparison with similarly infected mhv(−) mice. interleukin- (il- ) production levels by spleen cells did not differ between chronic mhv(+) and mhv(−) mice, but ifn-γ neutralization by monoclonal antibody treatment of anti-cd -stimulated spleen cell cultures showed higher levels of il- synthesis in mhv(+) balb/c mice. immune responses and on their interference with experimental models of infection. most reports deal with virulent laboratory mouse hepatitis virus (mhv ) collectively designates corona strains, although more recently, attenuated mhv laboratory viruses of a wide range of virulence. the strains that are strains have been used in an attempt to mimic the prevalent endemic in most mouse colonies over the world show relatively strains. the objective of this study was to investigate the low virulence and animals from infected colonies do not effects of a natural acute outbreak of mhv due to accidental present overt signs of illness. nevertheless, although tolerated exposure, in our otherwise specific-pathogen-free (spf ) inbred by many researchers, evidence has accumulated over the years mouse colonies, and of enzootic chronic mhv infection on that results from several investigation areas can be comprocytokine production and resistance to the intracellular pathomised by concomitant mhv infections. in particular, the gen trypanosoma cruzi. study of immunological parameters that determine resistance trypanosoma cruzi is a dygenetic protozoan that infects to infections can be seriously affected by mhv infection several kinds of mammals and is the aetiological agent of (reviewed in refs , ) . there are few studies on the effects of chagas' disease in man. the parasite replicates in the cytoinfection by natural-low-virulence enzootic mhv strains on plasm of virtually any nucleated cell type including macrophages; non-dividing forms of the parasite are found free in more resistant. susceptible mice have higher parasite numbers ), pneumonia virus of mice (pvm ), minute virus of mice (mvm ), lymphocytic chorio-meningitis virus (lcmv ), in the blood and tissues and most animals die within weeks of inoculation of as few as parasites; in contrast, resistant sendai virus, ectromelia virus, mouse polio virus (gd ), and on elisa for the bacteria mycoplasma pulmonis. all pro-mice have lower parasite loads and survive infection with parasites. both t. cruzi-resistant and -susceptible cedures with the animals were in accordance with the principles of the 'brazilian code of laboratory animals use'. mouse strains show elevated production of interferon-c (ifn-c) during infection and very low interleukin- (il- ) and il- production. - ifn-c and tumour necrosis factor-a trypanosoma cruzi infection, parasitaemia counting and experimental design (tnf-a), triggered by il- , are essential to control of parasitism by innate and acquired cellular immune systems in infective blood trypomastigotes were obtained from y strain t. cruzi-infected anaesthetized mice by drawing cardiac blood; mice. - il- exerts an important in vivo regulatory role on ifn-c and nitric oxide production , and its absence in motile blood forms were counted and the desired number of parasites was injected intraperitoneally (i.p.). infection was il- -gene-deprived mice , or neutralization by monoclonal antibody (mab) treatment results in lower parasitism, maintained by weekly i.p. infection of balb/c mice. in the experiments designated 'acute' mhv infection, parasites were whereas increased parasitism occurs when mice are treated with recombinant il- (ril- ) or receive il- -and il-maintained in mice coming from either recently infected mhv+ or from mhv− colonies and or forms were -producing t cells. il- , depending on the parasite strain, is also involved in negative regulation of parasitism. inoculated in recipient balb/c or c bl/ mice from mhv− colonies. in later experiments, designated 'chronic' mhv we found that balb/c and c bl/ mice that had been injected with blood trypomastigotes from recently mhv-infection, the t. cruzi strain was started anew from tissueculture-grown trypomastigotes and maintained in mhv− contaminated mice became much more susceptible to t. cruzi infection and produced higher il- levels than their counter-mice, whose blood was used as a source of t. cruzi to infect recipients derived from mhv+ colonies that had been infected parts whose t. cruzi inoculum was derived from mhvnegative (mhv−) donors. comparison between mice coming for more than months or from mhv− colonies. in these experiments the infective t. cruzi dose was , , or from chronically mhv-infected and from mhv− colonies, showed higher parasitaemia levels in balb/c mhv-positive blood forms in balb/c mice and , (not shown), , or blood forms in c bl/ mice. as c bl/ (mhv+) mice but otherwise no major significant differences in susceptibility to t. cruzi. nevertheless, quantitative differ-mice are much more resistant to t. cruzi infection, the high inocula would allow comparison between mhv− and mhv+ ences in ifn-c, il- and nitric oxide production were found between mhv-infected and uninfected mice. c bl/ colonies submitted to moderate to severe t. cruzi infection. parasitaemia determination was performed by direct microscope (× ) counting of motile parasites in a ml fresh materials and methods blood sample, obtained from the lateral tail veins. mice and mhv infection c bl/ and balb/c female mice ( - -week-old), acutely spleen cell cultures spleen cell suspensions were prepared from t. cruzi-infected or chronically infected with mhv were obtained from biotério de camundongos isogênicos do departamento de imunologia and uninfected mice, mhv+ or mhv−, depleted of erythrocytes by hypotonic lysis with distilled water and resuspended do icb/usp (são paulo, sp, brazil ). mice with negative serological tests for mhv, from both these strains, were in rpmi- (cultilab, campinas, sp, brazil ) complete medium containing % fetal calf serum (fcs; cultilab) and obtained from biotério de camundongos isogênicos da universidade estadual de campinas (campinas, sp, brazil ). supplemented with glutamine, -mercaptoethanol ( -me ) and antibiotics as described. spleen cell suspensions were pooled mhv− mice were housed and handled separate from and before those coming from mhv-contaminated animal facili-from three mice and were cultured in duplicate or triplicate in -well flat-bottomed plates at /ml or at × /well ties. the mice were fed autoclaved food and water, and were handled using disposable gloves. mhv infection was diagnosed and stimulated with t-ag ( × frozen-thawed tissue culture trypomastigotes parasites) prepared as described. by antibody testing of the sera. the mhv enzyme-linked immunosorbent assay ( elisa) diagnostic kits sold by charles concanavalin a (con a at · m/ml ) or plate-bound anti-cd [mab - c , americal type culture collection (atcc ) river laboratories ( wilmington, ma) were used according to the manufacturer's instructions. when the outbreak of crl ], coated at mg/ml, ml/well ) were also used as t-cell stimulants. . supernatants from the cultures were mhv was detected, levels of anti-mhv antibodies were very high with corrected optical density (od) values for sera harvested after hr from the higher cell-density cultures and after hr from the lower-density cultures. the following ranging from · to · (positive test values > ). these were calculated (as indicated by the manufacturer) by the following neutralizing rat mab anti-mouse cytokines were incorporated to the cultures in some experiments: jes - a anti-il- , and formula: [(od obtained for the test serum diluted at / incubated with the cells containing the virus) -(od obtained xmg . anti-ifn-c; both were used at final concentrations of mg/ml. the anti-cd mab gk . was added to cultures for the same serum and dilution incubated with uninfected cells)]/ · . fluorescence antibody testing was also performed at mg/ml. as an additional control and ranked + + + + for serum incubated with mhv-infected cells. the colonies were serologi-cytokine assays cytokine levels in the culture supernatants were measured by cally negative on elisa and immunofluorescence testing for the following viruses: respiratory-enteric orphan virus (reo two-site sandwich elisa using the following mab pairs of which the second cited was biotinylated. ifn-c, xmg . and when the inoculum came from mhv− donor mice. the an ; il- , jes- a and sxc- ; il- , b and differences in parasitaemia could be observed with inocula of bvd g . minimum levels of detection for the assays or blood trypomastigotes (fig. a,b) . moreover, for were:. ifn-c, · ng/ml; il- , · units/ml; and il- , balb/c mice injected with mhv+ inocula, mortality reached · ng/ml. the rat anti-mouse cytokines producing % by day ( t. cruzi forms) and % by day hybridomas were a generous gift from dr r. l. coffman, ( t. cruzi forms) whereas all recipients of mhv− inocula dnax research institute of molecular and cellular biology, survived to days after infection. all c bl/ mice palo alto, ca. standard curves were obtained with recombisurvived after infection whether inoculated with t. cruzi from nant mouse cytokines. the supernatants were tested in serial mhv+ or mhv− donor mice. twofold dilutions and the results were expressed as the arith-serological tests for mhv became positive in t. cruzimetic mean of duplicate determinations. the sd did not infected (from mhv+ donors) c bl/ mice by day after exceed % of the mean. infection and in % of a group of balb/c mice that survived to days after being infected with t. cruzi statistical analysis forms. recipients of inocula from mhv− mice remained the significance of differences in parasitaemia between distinct serologically negative for mhv. looking into possible causes experimental groups was examined by analysis of variance with of the observed increase in susceptibility to t. cruzi of mice repeated measurements followed by tukey's test for multiple infected with mhv+-derived inocula. we investigated whether comparisons. differences of · log or over were significant cytokine production was altered concomitant to the viral at least at p< · . differences in cytokine production levels infection. spleen cells (stimulated with con a or t-ag) from were tested by bonferroni's multiple comparison test. mice that received an mhv+ inoculum of t. cruzi produced detectable and much higher levels of il- during the first weeks of infection than spleen cells from mice that results received an identical inoculum of t. cruzi derived from t. cruzi infection and cytokine production in mhv− mice mhv− mice. in fact, il- production in this last situation inoculated with t. cruzi blood forms obtained from donor-mice was very low and often below · units/ml (table ). il- that were serologically mhv+ or mhv− production, on day after infection, in balb/c (but not in c bl/ ) mice was cd -activation dependent as treatment our preliminary observation, suggestive of an infection with gk . mab suppressed most of its synthesis outbreak in the mouse colonies, was increased susceptibility ( units/ml in untreated versus units/ml in gk . treated to t. cruzi infection first detected in balb/c mice. as the cultures in con a-stimulated and units/ml versus diagnosis of mhv infection was confirmed, we first investi- units/ml, respectively, in t-ag-stimulated balb/c spleen gated how a t. cruzi inoculum derived from donor mice that cell cultures). in c bl/ mice, the values were units/ml had concomitant mhv infection would compare with a in untreated versus units/ml in gk . -treated con similar inoculum originated in mhv− mice in regard to their a-stimulated cultures and versus units/ml, respectively, course of infection in a t. cruzi-susceptible (balb/c) and in after t-ag stimulation. in spite of the higher il- proa resistant (c bl/ ) mouse strain. as shown in fig. , duction by spleen cells from mice that had received the parasitaemia levels during the first days of infection were mhv+ inoculum, ifn-c levels were not significantly different significantly higher for both strains of mice when infected with t. cruzi blood forms derived from mhv+ donors than from those secreted by spleen cells from mhv− t. cruzi recipients and il- production was below detection levels in resulted in significant increases in ifn-c production of the order of - % (fig. ) . we next investigated whether all groups (data not shown). infecting mhv− or mice that were chronically infected mhv with t. cruzi would affect cytokine production and/or alter cytokine production by spleen cells from mice derived from the course of infection. spleen cell cultures from balb/c and c bl/ mice coming in contrast with the marked increase in parasitaemia and (data not shown). production of ifn-c by spleen cells from susceptibility to t. cruzi determined by the situation of acute mhv+ c bl/ and balb/c mice cells to con a stimulation simultaneous infection with blood parasites derived from was not significantly higher than in spleen cell cultures from acutely infected mhv+ donors as described above, no statis-mhv− animals (fig. ) . production of ifn-c to con a was tically significant differences of parasitaemia levels or mortunder control by il- both in mhv− and in mhv+ mice ality were observed between chronically infected mhv+ and because the addition of anti-il- mab a to cultures mhv− balb/c or c bl/ mice infected with t. cruzi. however, peak t. cruzi parasitaemia levels were attained earlier and were - % higher in balb/c mhv+ mice as compared to mhv-free mice (data not shown). differences in cytokine production between t. cruziinfected mhv+ and mhv-free groups of mice were only observed in the first week of infection. balb/c mice spleen cell cultures from mhv+ mice infected with t. cruzi produced lower ifn-c levels to con a and t-ag stimulation than cultures from mhv− mice (fig. ) . although the potential to produce ifn-c to polyclonal con a stimulation was preserved in mhv+c bl/ mice, ifn-c production was markedly suppressed on parasite-specific stimulation (fig. ) . the comparison of il- production levels among balb/c and c bl/ mice, mhv− or mhv+, infected with t. cruzi yielded no significant differences (data not shown). nevertheless, when balb/c (but not c bl/ ) spleen cell uninfected mhv+ mice (fig. , normal group) , suggesting mice, suppression of ifn-c responses is limited to the antigenspecific compartment whereas mhv infection affects more that the potential to increased il- production stimulated by mhv infection was, in these mice, maintained in check by severely balb/c mice by suppression of both polyclonal and specific ifn-c responses. ifn-c. taken together, these results indicate that in c bl/ immune response have been performed in situations of deliberate infection of mice with mhv laboratory strains of varying enzootic mhv strains are of low virulence, mostly entervirulence that cause moderate to severe disturbances to the otropic and notoriously difficult to isolate and to grow in immune system. mice infected with the pantropic strain of vitro. when the outbreak was detected in the mouse colony, medium-virulence, jhm, show suppression of con a-induced we unsuccessfully tried to isolate infective viral particles from proliferation, decreased il- and il- synthesis and a delay the plasma and liver of seropositive mice. immunosuppressing in ifn-c production in the first week of infection, whereas, the animals with cyclophosphamide also failed to promote later in infection, large amounts of ifn-c are produced by viral isolation. the difficulty in isolating low virulence enzootic balb/c mice. - macrophage function was impaired in mice enterotropic mhv, as opposed to polytropic laboratory mhv infected with this strain or with naturally occurring mhv strains, has been frequently reported. , , we had (as many strains. , increased message levels for ifn-c, il- , il- , other authors) to rely on serum antibody screening and tnf-a and inducible nitric-oxide synthase (inos ) were found serological conversion as a criterion to identify occurrence of in the brain of mice infected with a neurotropic jhm variant mhv infection. serological testing, although highly specific, strain. mhv a- is yet another laboratory strain, but does not distinguish between acute and chronic mhv infection of relatively low virulence that, although subclinical, is and thus we will discuss the immunological alterations found accompanied by increased production of ifn-c and suppresin the group of mice that had recently become seropositive for sion of con a spleen responses. , mhv (recent mhv-testers) in comparison with those found there have been few reports on immunological alterations in mice originating from colonies with longstanding record of resulting from natural mhv outbreaks. however, established mhv seropositivity. both groups of mice were serologically 'chronic' natural infection with mhv was reported to affect negative on testing for several other mouse pathogenic viruses mostly splenic t lymphocytes, with a - % decrease in and for mycoplasma pulmonis (see the materials and methods). proliferative responses to con a and resistance to the effects we have found a much more severe course of t. cruzi of nor-adrenalin or dibutyryl-camp. we found that lymphoinfection in mhv− mice infected with blood forms obtained proliferative responses of spleen cells from balb/c and from mouse colonies that had recently become seropositive c bl/ mice, mhv+ or mhv− recipient mice to con a, for mhv, as compared with mice inoculated with parasites t-ag, or anti-cd stimulation were not significantly different. derived from mhv− donors; increased susceptibility to infec-suppressed lymphoproliferative responses to these stimuli, tion was accompanied by increased synthesis of il- by commonly seen in the course of t. cruzi infection, were spleen cell cultures. interleukin- down-regulates il- and observed from day of infection, with minimal levels of ifn-c production, besides antagonizing ifn-c-and tnf-asuppression seen on day and recovery by day (data not dependent macrophage activation and intracellular t. cruzi shown). thus, in the course of t. cruzi infection, we did not killing. , , , - in spite of the enhanced il- secretion by observe, neither in recently seropositive recipient mice nor in spleen cells from mhv+ recipients, no decrease of ifn-c mice from chronically mhv-infected colonies, suppression of levels, in comparison to recipients of mhv− donors could be lymphoproliferation to con a, to parasite antigens or to antidetected in these same cultures. however, the data on enhanced cd stimulation that could be ascribed to the viral infection. ifn-c synthesis by anti-il- mab treatment, showed that however, its occurrence could have been masked by the intense endogenous il- was down-regulating ifn-c production in suppression characteristic of acute t. cruzi infection. this situation of probable acute concomitant infection with our results on the aggravation of t. cruzi infection in the parasite and the virus. the maintenance of in vitro ifn-c balb/c and c bl/ mice by using parasite inocula mainsecretion rates in the presence of increased il- concentained in mice that had positive serology for mhv are in trations has been described upon treatment of t. cruzi-infected agreement with a previous report on cba/j mice infected with mice with high doses of ril- that aggravate infection. t. cruzi derived from corona virus-positive mice. , these both mouse strains, balb/c and c bl/ , respectively, authors were able to demonstrate, in the plasma, infective susceptible and resistant to t. cruzi, showed increased parasitavirus particles that could be neutralized by anti-mhv antiemia and augmented il- production, but increased mortality serum. nevertheless, the underlying mechanisms leading to as a consequence of mhv infection was not observed for increased susceptibility to the parasite were not explored. c bl/ mice and they maintained their resistant phenotype we further investigated the influence of a longstanding to t. cruzi infection. although mouse strain resistance to ( year) established endemic mhv infection on the course of mhv infection is highly dependent on the mhv strain, studies t. cruzi infection and immune response. in contrast with the with laboratory mhv-strains have concluded that indifferently marked effects on the immune response observed in mhv− to the degree of mhv-strain virulence, replication in macrohosts infected with t. cruzi derived from recently mhv+ mice, phages does always occur. viral replication inside macrothe disturbances of immune response found in mice from phages may directly interfere with t. cruzi killing ability by chronically mhv-infected colonies were milder. production these cells and stimulate a number of macrophage functions of il- was similar between mhv+ and mhv− mice, while including il- synthesis. in this regard, c bl/ mice are parasite-antigen-stimulated ifn-c production was lower in t. semisusceptible to most mhv strains; , as production of cruzi-infected balb/c and c bl/ mice. however, when il- by mhv-infected c bl/ mice (but not by balb/c neutralization of ifn-c in the cultures unmasked the full mice) was cd +-activation independent, virus-infected or potential of il- secretion, mhv+ balb/c mice (but not virus-stimulated macrophages could be the main source of c bl/ ), showed higher il- production levels. this result this cytokine. is duration of mouse hepatitis virus infection: studies in immunocompetent and chemically enterotropic mouse hepatitis virus. lab immunosuppressed mice the cellular synergism between tumor necrosis factor-alpha and interferonand molecular pathogenesis of coronaviruses. lab animal sci gamma on macrophage activation for the killing of intracellular , . trypanosoma cruzi through a nitric oxide-dependent mechanism the microbicidal activity of interferon-gamma-treated san diego cytokines in innate and acquired dependent, nitrogen oxide-mediated mechanism inhibitable by immunity to trypanosoma cruzi infection tumor necrosis factor alpha mediates resistance to trypanosoma cruzi: early parasite pro-infect immun , . liferation and host resistance in inbred strains of mice trypanosoma cruzi: serum sci , . antibody reactivity to the parasite antigens in susceptible and ifn-gamma and il- production during trypanosoma cruzi infec- l failure of immune cell proliferation and interleukin production trypanosoma cruzi: mainten-adv exp med biol , . ance of parasite-specific t cell responses in lymph nodes during trypanosoma cruzi infection alters in vitro splenic t cell proliferation and cytokine production. suppresses nuclear factors that bind to specific sites on the lab animal sci , . interleukin- enhancer characterization of accessory cell function during acute infection of balb/cbyj mice with lactate dehydrogenase-elevating virus-permissive macrophages and t lymphocyte alterations. virus res , . mouse hepatitis virus (mhv ), strain jhm effect of d.a. ( ) mouse hepatitis virus suppresses modulation of inapparent murine hepatitis virus infections on macrophages and mouse spleen t cell activation murine virus contaminant of trypanosoma ( ) kinetics of cytokine mrna expression in the central cruzi experimental infection. rev inst med trop são paulo , nervous system following lethal and non-lethal coronavirus- . induced acute encephalomyelitis trypanosoma cruzi: murine virus contaminant of the experimental infection key: cord- -psjua authors: v’kovski, philip; al-mulla, hawaa; thiel, volker; neuman, benjamin w. title: new insights on the role of paired membrane structures in coronavirus replication date: - - journal: virus res doi: . /j.virusres. . . sha: doc_id: cord_uid: psjua the replication of coronaviruses, as in other positive-strand rna viruses, is closely tied to the formation of membrane-bound replicative organelles inside infected cells. the proteins responsible for rearranging cellular membranes to form the organelles are conserved not just among the coronaviridae family members, but across the order nidovirales. taken together, these observations suggest that the coronavirus replicative organelle plays an important role in viral replication, perhaps facilitating the production or protection of viral rna. however, the exact nature of this role, and the specific contexts under which it is important have not been fully elucidated. here, we collect and interpret the recent experimental evidence about the role and importance of membrane-bound organelles in coronavirus replication. organelles, this has been difficult to test experimentally. however, there are several lines of experimental and genetic evidence that suggest that rna synthesis is tied to the formation of replicative organelles. viral rna accumulates in the coronavirus organelles, suggesting that the organelles may be a site of rna synthesis (knoops et al., (knoops et al., , gosert et al., ; hagemeijer et al., ) . furthermore, viral organelles are not formed when rna synthesis is stopped (stokes et al., ; verheije et al., ) . while it is clear that rna synthesis is linked with the organelles, it has proved difficult to directly test whether or to what extent the process of organelle formation is necessary for the process of rna synthesis, because of the practical difficulty in separating the two processes in an experimental setting. electron tomography studies have revealed that the replicative organelles of different nidoviruses are drawn from a repertoire of paired-membrane structures, including (paired) convoluted membranes, pouch-like double-membrane spherules, long paired membranes and double-membrane vesicles (knoops et al., (knoops et al., , maier et al., a) , though studies of the more recently discovered mesoniviruses and roniviruses remain poorly characterized (zirkel et al., ; spann et al., ) . the nidoviruses that have been studied to date all induced a combination http://dx.doi.org/ . /j.virusres. . . - /© elsevier b.v. all rights reserved. posthuma et al. ( ) , wood et al. ( ) , pedersen et al. ( ) , pol et al. ( ) a hsu et al. ( ) , richards et al. ( ) , teterina et al. ( ) , suhy et al. ( ) secoviridae p er v? nr roberts and harrison ( ) betaflexiviridae p er v nr edwardson and christie ( ) , rudzinska-langwald ( ) guix et al. ( ) , mendez et al. ( ) bromoviridae p er z,s a+ a pol moreira et al. ( ) , schwartz et al. ( ) , schwartz et al. ( ) fig. . the common element in nidovirus-like membrane rearrangement is that the membranes are paired, usually maintaining a consistent-sized gap between the two membranes (reviewed here, angelini et al., ) . since protein-induced membrane fig. . conservation and functional organization of the carboxyl-terminal region of nidovirus polyprotein a. domains that are homologous at the amino acid level are shown at left in solid colors. more distantly related potential homologs identified by genome position and comparison of predicted secondary structures are marked with stripes. positions of transmembrane regions (black bars) and hydrophobic non-transmembrane regions (striped bars) were predicted by tmhmm . (krogh et al., ) and amended to reflect known topologies (kanjanahaluethai et al., ; oostra et al., oostra et al., , wherever possible. clusters of conserved cysteine and histidine residues that may bind metal ions are marked with white ovals. a jagged line denotes the uncertain position of the amino terminus. regions that induce membrane pairing, proliferation or vesiculation in betacoronavirus sars-cov and arterivirus eav are shown above and below the domain annotation, respectively, and all annotations come from the references listed for table . double-membrane organelles observed (x) or uncertainly observed (?) in infected cells are marked at right. virus names are abbreviated as follows: white bream virus (wbv), fathead minnow nidovirus (fhmnv), equine arteritis virus (eav), lactate dehydrogenase elevating virus (ldv), porcine reproductive and respiratory syndrome virus (prrsv), simian hemorrhagic fever virus (shfv) and wobbly possum nidovirus (wpnv). pairing appears to be a consistent feature associated with nidovirus replication, and in the absence of data carefully dissecting the relationship between the shape and function of these different paired membrane structures, it makes sense to refer to the resulting structures collectively as double-membrane organelles (dmo). despite a relative wealth of structural data, it has proved difficult to test hypotheses about the role of dmos in viral replication and fitness directly because dmo formation is linked so closely to replication and expression of replicase proteins. here, we will discuss the implications of two recent studies that address questions about the role of dmos in nidovirus replication (al-mulla et al., ) , and characterize the effects of a new dmo-blocking drug against a variety of coronaviruses (lundin et al., ) . further evidence of the probable importance of nidovirus replicative organelles for viral rna replication comes in the form of genetic conservation. nidoviruses, and most particularly coronaviruses, are highly genetically variable and contain several genus-specific or even species-specific genes (lauber et al., ) . however, there are two clusters of genes that are conserved in all known nidoviruses lauber et al., ) . the first is a highly conserved cluster of genes homologous to the severe acute respiratory syndrome coronavirus (sars-cov) nsp - ( fig. ) . expression of the membrane-anchored proteins nsp , nsp and nsp is sufficient to induce the formation of sars-like paired-membrane replicative organelles (angelini et al., ) . the second conserved gene cluster encodes the viral rna polymerase and superfamily helicase (deng et al., ) . the conservation of membrane-pairing genes in the context of an otherwise hypervariable group of viruses is a strong argument in favor of the importance of at least the membrane-pairing genes for rna synthesis the proteins that form sars-cov replicative organelles have several features in common with distant homologs found throughout the nidovirales. we will refer to the transmembrane proteins homologous to sars-cov nsp , nsp and nsp a as tm , tm , and tm , respectively. the relative genomic positions and functions attributed to tm - in nidoviruses are shown in fig. . of the three proteins involved in sars-cov replicative organelle formation, the least conserved is tm , which has a multidomain architecture (neuman et al., ) . many nidovirus and all coronavirus tm proteins contain one or more ubiquitin-like domains which may help to anchor the viral rna to the membranes where replication takes place (hurst et al., ) . potentially rna-binding macrodomains (serrano et al., ; xu et al., a; wojdyla et al., ; saikatendu et al., ; tan et al., ; chatterjee et al., ; johnson et al., ) , papain-like proteinases (ratia et al., ; wojdyla et al., ; van kasteren et al., ) , other rna binding domains ) and a well conserved but poorly understood region known only as the y domain (neuman et al., ) are also commonly but not ubiquitously found in nidovirus tm proteins. all putative tm proteins are predicted to contain one or more transmembrane domains, as shown in fig. . the c-terminal region of tm , from the first transmembrane region to the end of the y domain induces membrane proliferation, which in some ways resembles an autophagy response (angelini et al., ) . tm and tm are recognizable because they contain four or more predicted transmembrane regions, and are encoded immediately before and after the viral main protease (m pro ). bioinformatics generally predicts an even number of transmembrane spans in these proteins, which would be necessary to localize m pro on the same side of the membrane as all of its predicted upstream and downstream cleavage sites. however there are additional hydrophobic regions that are strongly predicted to span the membrane, but which do not for several viruses, including most coronaviruses (kanjanahaluethai et al., ; oostra et al., oostra et al., , . tm contains two potential conserved domains located between the first and second transmembrane domains in coronavirus, and after the final transmembrane domain in most nidoviruses. mutations in the first non-hydrophobic domain of tm , which is the largest part of the coronavirus replicase to localize on the luminal face of the membrane, have been demonstrated to disrupt rna replication and may cause defects in membrane pairing (gadlage et al., ) . deletion of the latter conserved domain of tm , which has been structurally solved xu et al., b) , was surprisingly well tolerated sparks et al., ) . tm localizes to membranes, but does not induce any recognizable change to intracellular membranes in the absence of other viral proteins (angelini et al., ) . however, co-expression of tm with full-length tm results in extensive pairing of perinuclear membranes in both coronavirus (angelini et al., ) and arterivirus (snijder et al., ; posthuma et al., ) . additionally, it has recently been shown that co-expression of a fragment of mhv tm including the transmembrane region and the c-terminus with tm induced er membrane zippering and curvature similar to the phenotype observed after sars-cov tm and tm co-expression (hagemeijer et al., ) . in that report tm and tm were demonstrated to interact via protein loops on the luminal face of the membrane. the maze-like paired-membrane structures that resulted from coexpression of sars-cov tm and tm have not ever been reported in coronavirus-infected cells, suggesting that this should be interpreted as a conditional, or perhaps partial phenotype, that is not observed when the full viral replicase polyprotein is expressed. this suggests that membrane pairing is caused by heterotypic interactions between tm and tm on opposing membranes, but that the final architecture of the paired membranes is dependent on additional viral proteins. tm largely consists of transmembrane regions, without the hallmarks of amino acid conservation or predicted structural conservation that would be expected for an enzyme. overexpression of tm alone disturbs intracellular membrane trafficking (cottam et al., (cottam et al., , , resulting in an accumulation of singlemembrane vesicles around the microtubule organization complex (angelini et al., ) . however, quantitative electron microscopy revealed that expression of tm with tm prevents the membrane disruption seen with tm expression alone (angelini et al., ) . when sars-cov tm , tm and tm are coexpressed, membranecontaining bodies which resembled authentic sars-cov replicative organelles were formed. however, in each of the cell sections where dmv-like membranes were observed, the membrane proliferation phenotype of tm , the paired membrane phenotype of tm + tm and the single membrane vesicle accumulation from tm were each visible, suggesting that these proteins do not always colocalize efficiently when expressed from plasmids in different parts of the cell instead of being expressed in the natural form as a polyprotein (bwn, personal communication) . this suggests that while tm is not necessary for membrane pairing, tm may be necessary to induce the formation of the double-membrane vesicles (dmvs) that are characteristic of coronavirus replicative organelles. the formation of large intracellular structures such as the mazelike tm + tm bodies and dmv-like tm + tm + tm bodies suggests that nsp , nsp and nsp may interact both homotypically and heterotypically. sars-cov nsp -nsp interactions have been detected in cells by yeast two-hybridization (pan et al., ) and gst pulldown (imbert et al., ) , and in purified protein by perfluorooctanoic acid polyacrylamide gel electrophoresis (neuman et al., ) . while sars-cov nsp -nsp interactions were not found in yeast-two hybrid or mammalian two-hybrid screens (pan et al., ; von brunn et al., ) studies with another coronavirus did detect nsp -nsp interactions by venus reporter fluorescence (hagemeijer et al., ) . to date, homotypic interactions have not been demonstrated for nsp despite several attempts (pan et al., ; imbert et al., ; von brunn et al., ) . heterotypic interactions between coronavirus tm - proteins have been demonstrated biochemically: a tm -tm interaction was detected by mammalian two-hybridization (pan et al., ) and weakly detected by venus reporter fluorescence (hagemeijer et al., ) . a tm - interaction has been demonstrated by venus reporter fluorescence (hagemeijer et al., ) , though it did not appear in other hybridization studies. a one-way interaction between the amino-terminal amino acid domain of tm and tm detected by yeast two-hybridization (imbert et al., ) has also been reported. however, the apparent independence of tm and tm phenotypes after coexpression, coupled with the abrupt change in both phenotypes in the presence of tm suggests that interactions between these proteins may be largely mediated by tm (angelini et al., ) . molecular interactions between host and viral factors are observed in virtually every step of the viral life cycle. viruses rely on and manipulate established cellular pathways to accommodate their needs during replication and to counteract host innate immune signaling. replication of coronaviruses is no exception; while some host factors have been described in the context of viral rna replication and transcription (zhong et al., ) , few studies have looked closely at the complex interplay of host pathways in the establishment of virus-induced membrane-bound replication complexes. the best available evidence suggests that most coronavirus dmo structures derive from er membranes (knoops et al., (knoops et al., , maier et al., a) , but the precise mechanism of membrane rearrangement remains elusive. dmo membranes were initially suggested to derive from the early secretory pathway, although the absence of conventional er, ergic and golgi protein markers on viral replicative membranes argues against this hypothesis (verheije et al., ; knoops et al., ) . since dmvs are reminiscent of the double-membranes of autophagosomes, several lines of controversial evidence hypothesized a diversion of atg (autophagyrelated) proteins and autophagosome function during coronavirus replication, as it is the case for other +rna viruses (prentice et al., ; snijder et al., ; zhao et al., ; maier and britton, ; richards and jackson, ) . the involvement of autophagy was recently investigated in the context of the avian cov infectious bronchitis virus (ibv) infections (cottam et al., ) . the authors conclude that the presence of exogenous, individually expressed ibv nsp , which localizes to the er, induces the formation of autophagosomes in contrast to other ibv replicase proteins. additionally, although autophagosomes induced by ibv nsp or ibv infection appeared smaller than conventional autophagosomes observed after starvation of cells, they were similar in size to dmvs (cottam et al., ) . however, the data reported here do not appear to support the assumption that there is a functional link between ibv nsp and autophagosomes, and a role of the autophagy in the formation of ibv replicative structures can hereby not be demonstrated. moreover, neither induction nor inhibition of autophagy seems to affect ibv replication (maier et al., b) . it also has to be noted that the induction of autophagy by virus infection is cell type and possibly species dependent, as exemplified by the absence of lc puncta accumulation in ibv-infected primary chick kidney cells (maier et al., b) . new evidence concerning the source of membranes for covinduced dmos was proposed, in which mouse hepatitis virus (mhv) probably co-opts a cellular degradation pathway of erassociated degradation (erad) regulators, known as the erad tuning pathway . the erad pathway is responsible for the turnover of folding-defective polypeptides in the er and is modulated by stress-inducible positive regulators of erad-mediated protein disposal such as edem (er degradationenhancing alpha mannosidase-like ) and os- (osteosarcoma amplified ). the latter assist in transporting misfolded proteins into the cytosol for subsequent degradation by the proteasomal system. under physiological conditions, however, low concentrations of edem and os- are maintained in the er lumen in order to avoid premature degradation of proteins that are undergoing folding programs (calì et al., ) . in this case, edem and os- are selectively confined by interacting with the transmembraneanchored cargo receptor sel l (suppressor of lin- -like protein ) and later released from the er lumen in small short-lived vesicles, called edemosomes, which rapidly fuse with the endolysosomal compartments (bernasconi et al., ) . this steady-state disposal of edem and os- is known as erad tuning pathway. while not relying on the coat protein complex ii (copii) or atg , it critically depends on the non-lipidated form of lc (lc -i), which is recruited to edemosomes. however, the specific autophagosomal marker gfp-lc does not associate with edemosomes, which are therefore distinct structures (noack et al., ) . the coronavirus mhv is hypothesized to divert the erad tuning machinery for the generation of dmos. similarly to edemosomes, colocalization of edem , os- , sel l, lc -i and double-stranded (ds) rna is observed upon mhv infection. moreover, replication of mhv, which does not require an intact autophagy pathway, is impaired upon knockdown of lc or sel l (bernasconi et al., ) . dmos furthermore lack conventional er markers and do not associate with gfp-lc . altogether, the evidence from this study may suggest that mhv exploits the erad-tuning machinery to establish dmos for replication. in order to learn whether this mechanism might be common to other nidoviruses, other viruses that use a similar replication strategy to mhv were examined. one of these, the arterivirus equine arteritis virus (eav) has been shown to require the same subset of erad tuning factors as mhv to ensure replication (monastyrska et al., ) . recently, investigations of the even more distantlyrelated japanese encephalitis virus (jev), which belongs to the flaviviridae family, revealed that it may usurp the same components of the erad-tuning pathway as well (sharma et al., ) . consistent with this hypothesis, both viruses were shown to replicate independently of a functional autophagy pathway. the non-lipidated lc marker protein, which is essential for the replication of eav and jev, associated with their replication complexes together with edem whereas gfp-lc did not label these structures. these observations parallel the ones seen for mhv but raise further questions whether this feature is even more widespread amongst +rna viruses. despite the resemblance of mhv, eav and jev in the requirement of host factors for efficient replication, diversion of the erad tuning pathway cannot be considered as a generic way of inducing replicative membranes by these viral families. probable variations within families have to be kept in mind as exemplified by the comparison of dmos from two different coronavirus genus members. indeed, ibv's recently described spherules derived from paired er membranes significantly differ from the dmo structures observed upon alpha-and beta-coronaviruses infections (maier et al., a; neuman, ) and their generation might require a different set of factors. furthermore, the morphology of dmos induced by flaviviruses such as hepatitis c virus, dengue virus or west nile virus is highly heterogeneous and the identification of a common, conserved membrane diversion strategy seems unlikely (romero-brey and bartenschlager, ). however, it is possible that the diversion of one pathway could lead to the generation of the different arrangements of membrane that we collectively refer to as the dmo. importantly, it has been shown that, in contrast to what is observed during eav infection, endogenous lc does not colocalize with membrane puncta induced by expression of eav nsp and nsp , and the membrane modifications induced by the latter are not affected by lc knockdown (monastyrska et al., ) . similarly, lc and edem were not recruited to rearranged membranes induced by co-expression of mhv tm and tm (hagemeijer et al., ) . while this still has to be proven in the context of cov tm , tm and tm expression, it raises the questions whether lc participates to the biological function of dmvs rather than its generation. a novel hypothesis has been recently suggested for poliovirus, according to which the virus might not only co-opt a host pathway, but also divert the functional network of individual proteins (belov and sztul, ) . host factors could therefore have a proviral function during infection, distinct from the function for which they have been initially described. accordingly, this is reminiscent with novel functions attributed to lc during cellular homeostasis, cytoprotection against invading pathogens or during chlamydia trachomatis' intracellular life cycle (bestebroer et al., ) . the dmos of the model coronavirus mhv take the form of perinuclear dmvs which appear either singly, or grouped around and interconnected with a region of paired, convoluted membrane (cm;). a recent study examined dmv formation by wild-type mhvinf- (wt) and five temperature-sensitive (ts) mhv mutants, each of which differed from wt by a single amino acid substitution. the panel of ts viruses chosen contained mutations in an interdomain linker of nsp (tm ), m pro , the viral rna polymerase, cap n-methyltransferase and cap o-methyltransferase, respectively (stokes et al., ; al-mulla et al., ; sawicki et al., ) . with the exception of the polymerase mutant, which was attenuated tenfold, these viruses produced the same amount of infectious progeny as wt (al-mulla et al., ) . all of the mutants produced significantly smaller dmvs than wt virus, varying from almost wt size to % smaller (table ). in two of the mutants that produced normal amounts of infectious progeny, not only were the dmvs smaller, there were only about half as many dmvs per visibly infected cell compared to wt (table ) . examination of the size and number of intracellular virus particles from the same samples did not reveal corresponding changes, suggesting that the observed dmv phenotypes were not an artifact of sample preparation. the number of cms remained in a constant ratio to the number of dmvs present, suggesting that the mutations affected production of the entire dmo. the dmos of human coronavirus e (hcov- e) include dmvs similar to those observed after mhv infection (lundin et al., ) . in testing a new antiviral called k , it was observed that infectivity, viral rna, and dmv formation were all blocked by treatment with m k . a time of addition study revealed that k did not block viral entry, and had the greatest antiviral effects after virus entry during the first few hours of infection, leading to the interpretation that k inhibits a cellular or viral component involved in a post-entry, early stage of viral replication. after serial passage of the virus in the presence of k , resistant mutants were selected. surprisingly, two independently isolated resistance mutations mapped to opposite ends of transmembrane helices in tm (nsp ) at positions h l and m v. the resistant viruses released similar amounts of new progeny compared to wt, but produced only about half as many dmvs per infected cell. in addition, the dmvs induced by resistance mutants appeared structurally impaired. similarly to mhv nsp mutants (gadlage et al., ) , k escape mutants induced dmv with partially collapsed inner membranes, even when k was not present. however, it is important to bear in mind that the fixation, staining and imaging conditions may have an influence on the appearance of membrane structures (knoops et al., ) . moreover, the specific infectivity of those newly released virions was about tenfold lower for tm mutants than for wt. this suggested that the mutations in nsp conferred resistance to k at a cost of impairing an early intracellular step in the establishment of infection. from these experiments it was clear that hcov- e viruses with k resistance mutations in tm incurred a steep fitness cost, in the form of decreased specific infectivity. there were also indications of a similar decrease in efficiency in the mhv nsp mutant brts , which produced significantly more intracellular rna than wt, but without a corresponding increase in infectious progeny. to find out if the mhv mutants also incurred a fitness cost associated with producing smaller and fewer dmvs, competitive fitness assays were carried out. to do this, equal infectivities of two viruses were added to the same flask at a temperature where both viruses could grow normally. after h in direct competition, the amount of each virus was quantified either by sequencing to look for the ts mutation, or by phenotypically screening for ts and non-ts virus. none of the mhv mutants tested was significantly less fit than wt in continuous or primary fibroblasts, and two mutants were significantly fitter than wt under the assay conditions. one of the viruses with increased fitness compared to wild-type was the n-methyltransferase mutant brts , which produced only half as many dmvs as wt. these results demonstrated that at least under these experimental conditions, producing larger or more numerous dmvs did not confer a corresponding fitness advantage. when interpreting these findings, it is important to consider that none of the hcov- e or mhv mutants tested to date has been able to replicate entirely without dmos. and while some of these tests were carried out in primary cells, work in animal models was not possible because of the lack of a small animal model for hcov- e, and because the mutations restricted the growth of mhv mutants at physiological temperatures. these two studies do not disprove the fundamental connectedness between coronavirus rna replication and dmo formation, but together, they reveal an unexpected plasticity in the size and number of dmvs that are needed to carry out wild-type amounts of rna synthesis. for these reasons, along with the observation that rna replication is detectable before the first appearance of organelles , we favor an interpretation in which the organelles are a late manifestation of accumulated viral proteins resulting from abundant rna expression. in this interpretation, dmos could still play an obligate role in viral replication under specific conditions or in specific cell types, but the primary role for dmos would be to increase the efficiency of either rna production, delivery of newly synthesized rna to sites where it could be translated or packaged, and/or shielding abundantly synthesized viral rna from host cell innate immune sensing pathways. these studies also suggest that at least half of the dmvs present in infected cells may be in excess of what is strictly needed to sustain normal levels of rna synthesis, given that both mhv and hcov- e mutants replicated normally despite producing only half the normal complement of dmvs. before these studies, very little was known about the potential for natural and induced variation in intracellular membrane rearrangement. the viruses described in these studies all produced normal amounts of progeny virus particles, and were all selected for analysis for reasons unrelated to dmo formation. these represent only a handful of the available nidovirus replicase mutants that have been published. from this work we can hypothesize that other mhv ts mutants, or k -resistant hcov- e mutants with replicase defects would probably make either smaller or fewer dmvs, and a larger collection of such mutants will like be highly informative to further our understanding on the pivotal role(s) of dmos in the coronavirus life cycle. hopefully the unique insight provided by these results, together with the relative ease of analysis will make quantitative electron microscopy a routine part of the characterization of new virus mutants. in addition, the accumulated knowledge on the nature of coronavirus dmos and the possibility to experimentally interfere with dmo formation by using small compound inhibitors, such as k , will allow us to dissect similarities and differences between viral dmos and related cellular organelles. the study of viral replicative organelles remains an area of active research where there are more questions than answers. for the coronavirus dmo, there are still fundamental questions that need to be answered including the roles of specific parts of the dmo in replication, the cellular pathways involved in dmo formation, and whether there are host antiviral defences that work by specifically targeting dmo formation. some of these questions could be addressed by developing methods to study active replicase complexes and dmo membranes in vitro. there are also larger questions about the 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viral nucleic acids on hospital surfaces suggested surfaces could play a role in spread in health care environments. surface disinfection may interrupt transmission, but few data exist on the effectiveness of health care germicides against coronaviruses on surfaces. methods the efficacy of health care germicides against surrogate coronaviruses, mouse hepatitis virus (mhv) and transmissible gastroenteritis virus (tgev), was tested using the quantitative carrier method on stainless steel surfaces. germicides were o-phenylphenol/p-tertiary amylphenol) (a phenolic), % ethanol, : sodium hypochlorite, ortho-phthalaldehyde (opa), instant hand sanitizer ( % ethanol), and hand sanitizing spray ( % ethanol). results after -minute contact time, for tgev, there was a log reduction factor of . for % ethanol, . for phenolic, . for opa, . for : hypochlorite, . for % ethanol, and . for % ethanol. for mhv, log reduction factors were . for % ethanol, . for phenolic, . for opa, . for : hypochlorite, . for % ethanol, and . for % ethanol. conclusion only ethanol reduced infectivity of the coronaviruses by > -log after minute. germicides must be chosen carefully to ensure they are effective against viruses such as severe acute respiratory syndrome coronavirus. health care-associated infections are responsible for thousands of deaths worldwide each year. approximately % of all nosocomial infections are because of viral exposure, and, in pediatric wards, viruses account for at least % of health care-associated infections. studies have shown viruses to be common in health care environments and capable of surviving for extended periods of time on environmental surfaces. in these settings, health care workers, medical devices, and environmental surfaces can act as both a reservoir for infection and a mode of transmission of infection to patients and staff. , in , the nosocomial transmission of viral disease proved to be a major contributor to a worldwide outbreak of severe acute respiratory syndrome (sars), caused by a novel human coronavirus (cov) (sars-cov). outbreaks of sars occurred in multiple health care facilities, infecting patients, staff, visitors, and volunteers. sars-cov was also found on environmental surfaces in hospitals where outbreaks occurred, and studies demonstrated that it could survive on surfaces for to hours. airborne transmission was the main route of spread; however, rabenau et al observed that ''there are a number of instances when transmission occurred through other means that are often still not well defined, '' and other studies of outbreak settings showed that providing handwashing facilities reduced transmission in hospitals, suggesting that hands and surfaces could have played a role in transmission. the outbreak highlighted the need for effective and quick evaluation of means for controlling the spread of nosocomial infection. disinfection of hospital surfaces is an effective measure for reducing the risk of exposure for health care workers and patients; appropriate disinfection of contaminated surfaces and equipment is crucial in interrupting the spread of viruses such as sars-cov. , [ ] [ ] [ ] however, to assist in the selection of appropriate germicidal agents for use against coronaviruses on hospital surfaces and equipment, data are needed on the effectiveness of commonly used hospital germicides against coronaviruses. these data must accurately reflect disinfectant efficacy against viruses under the conditions in which they occur on surfaces, such as desiccation and embedding in proteinaceous matrices. many previous disinfection studies have used liquid suspension methods for testing germicide efficacy. [ ] [ ] [ ] these studies report greater efficacy against viruses than studies performed with carrier methods. viruses may be more resistant on surfaces than in suspension because they can adsorb to the surface or become embedded in organic material and may be more difficult to inactivate with chemical germicides than viruses suspended in liquid. thus, it is possible that suspension tests overestimate the level of antimicrobial activity of germicides against viruses on surfaces. carrier-based methods may more closely resemble real environmental conditions in which viruses contaminate surfaces and provide a more conservative estimate of germicide activity against viruses that are dried onto environmental surfaces. this study was undertaken using the carrier method to evaluate chemical germicides commonly used in health care settings for their efficacy in reducing infectivity of coronaviruses on environmental surfaces. the germicides selected were surface germicides and hand sanitizers. although hand sanitizers are not used for surface disinfection, the quantitative carrier test can help determine whether or not the active ingredients are effective against coronaviruses. germicide evaluation was done using non-human coronaviruses as surrogates for the coronaviridae family and pathogenic human coronavirus such as sars-cov. the family coronaviridae is divided into groups. groups i and ii include human, mammalian, and avian coronaviruses, and group iii consists of avian coronaviruses. although sars is thought to be related to the group coronaviruses, and phylogenetic analyses have indicated it may be closely related to mouse hepatitis virus (mhv), there is still disagreement about the exact placement of sars-cov within the coronavirus family. based on this uncertainty, representative of each group of mammalian coronaviruses was included in the study to determine whether there was any difference in their survival and persistence in water. the viruses included in the study were transmissible gastroenteritis virus (tgev), a diarrheal pathogen of swine and a member of the group i coronaviruses, and mouse hepatitis virus (mhv), a pathogen of laboratory mice and a member of the group ii coronaviruses. mhv and tgev were kindly provided by r. baric, university of north carolina, chapel hill. tgev was grown in swine testicular cell cultures. mhv was grown in delayed brain tumor cell cultures. viral stocks were propagated by infecting confluent layers of host cell cultures in flasks, harvesting cell lysates, clarifying by centrifugation ( , g, minutes, c), and storing resulting supernatants as virus stock at c. viral titers were determined by the plaque assay method on confluent host cell layers in -mm petri dishes with overlay medium consisting of % agarose, eagle's minimum essential medium, % bovine serum replacement (fetal clone ii; hyclone, logan, ut), % lactalbumin hydrolysate, and gentamicin ( . mg/ml)/ kanamycin ( . mg/ml). cell layers were stained with a second overlay containing % neutral red at hours postinfection, and plaques were visualized at hours postinfection. hard water was prepared according to the usepa opp microbiology laboratory standard operating procedure for disinfectant sample preparation for hard water preparation ml of solution a and ml of solution b were added to a volumetric flask and brought up to l with sterile deionized water. this solution was diluted with additional liters of sterile deionized water. final solution was adjusted to ph . to . by drop wise addition of sodium hydroxide or citric acid. a hardness testing kit (hach model -ep mg/l no. - ; hach corp, loveland, co) was used to confirm that hardness of the prepared water was to mg/l caco . six hospital-grade germicides were tested. the germicide types, active ingredients, and use-dilutions are summarized in table . germicides requiring dilution were prepared on the day of the experiment, using hard water as the diluent. all germicides were used by the manufacturer's expiration date. neutralizing solutions were used to inactivate germicide activity after the experimental contact time. vesphene iise (steris corp, mentor, oh), % ethanol, clorox anywhere spray (clorox co, oakland, ca), and purell sanitizing hand gel (johnson & johnson inc, new brunswick, nj) were neutralized using % glycine. chlorine bleach was neutralized with . % thiosulfate and cidex-opa (johnson & johnson inc, new brunswick, nj) with . % sodium bisulfite. to prevent cytotoxicity in cell culture assays, each neutralizing solution, with the exception of % glycine, was prepared as a stock solution and diluted to the use concentration with cell culture medium. glycine was prepared by adding . ml cell culture medium to . ml of a % glycine solution. sodium thiosulfate was prepared as a % (wt/vol) stock solution and diluted with cell culture medium to a use concentration of . %. sodium bisulfite was prepared as a % stock solution and diluted in cell culture medium to a use concentration of . %. test surfaces were -cm stainless steel carriers with a no. polish. the quantitative carrier test method used was adapted from sattar et al. each germicide experiment used control carriers (no germicide applied) and test carriers (germicide applied). each experiment was performed in duplicate. each carrier was placed in a -well plate, and ml of virus suspension was applied. the virus was allowed to dry for hours. after drying, ml of use-dilution germicide was placed on the dried virus suspension on test carriers, and ml of cell culture medium was placed on control carriers. after -minute contact time, ml of neutralizing solution was added to the test carriers to halt virucidal activity, and ml of cell culture medium was added to the control carriers. to elute viruses from carriers, ml of % beef extract (ph . ) was then added to all carriers. carriers were agitated on a shaking platform ( rpm) for minutes. the liquid from each well was then recovered, diluted, and assayed for virus infectivity as previously described. to determine the reduction in virus infectivity, the concentration of virus per -ml sample volume was calculated. reduction in viral titer was calculated using difference in virus concentration between test carriers and control carriers. log reductions were calculated for each germicide based on independent exposure trials. statistical analysis was performed using sas . ( ; sas institute inc, cary, nc). a -way analysis of variance (anova) was used to compare the log reduction among germicides. additionally, a -way anova was performed to compare the efficacy of all surface germicides between the virus types. reductions of mhv and tgev infectivity on surfaces by hospital germicides are shown in table . for mhv, only % ethanol and purell hand gel ( % ethanol) produced a log infectious virus titer reduction factor . . after -minute contact time. hypochlorite ( : use dilution) was least effective, producing a log reduction factor , . hypochlorite, vesphene iise, cidex-opa, and clorox anywhere spray each produced a log reduction factor of , . , with log reduction factors of . , . , . , and . , respectively. statistical analysis using -way anova showed that the mean log reduction factors for the germicides differed significantly (p , . ). for tgev, infectivity reduction factors of . -log were observed for % ethanol ( . ) , purell hand gel ( . ), and clorox anywhere spray ( . ). vesphene and cidex against tgev produced intermediate log infectivity reduction factors of . and . , respectively. as seen with mhv, hypochlorite exposure resulted in an infectious tgev titer log reduction factor , ( . ). statistical analysis using -way anova showed that the mean log reduction factors were significantly different (p , . ) among the germicides. analysis of the mean log reductions of mhv and tgev by germicides was done using the tukey multiple comparison test to determine whether reductions by individual germicides significantly differed from one another (p , . ) ( table ). in addition, -way anova was used to compare independent variables, germicide and virus type, and their influence on the log virus reduction factor. this analysis aids in determining how much of the variability in reduction is explained by each of these variables, as well as potential interaction between them. using -way anova, infectivity reduction results are statistically significant (p , . ). a type iii sum of squares test was used to examine variation among mean reduction results. germicide had a greater influence on log viral reduction (p , . ) than did the virus type (tgev vs mhv) (p . ). additionally, when interaction between germicide and virus type was evaluated using the anova test, it was determined that there is a statistically significant interaction between virus type and germicide type (p . ). health care-associated transmission can play an important role in the spread of coronavirus infection, and coronavirus nucleic acids have been found on hospital surfaces in outbreak settings. data from surrogate coronaviruses suggest that these viruses can survive for long periods on hard surfaces, potentially posing a continued risk of infection if health care surfaces are not adequately disinfected. the efficacy of hospital surface germicides was tested against coronaviruses, mhv and tgev, used as surrogates for sars-cov. these findings expand the available data on disinfection beyond what has been previously studied using other surrogates such as human coronavirus e. although e shares some tissue tropism with sars, there are important differences. studies of sars patients and outbreaks demonstrated that sars-cov is also a fecally shed virus, with intestinal tissue tropism; e lacks this. this may indicate important differences in resistance to environmental stressors because fecally shed viruses must be able to survive the conditions in the gastrointestinal tract, including extremes of ph, abundance of other microbes, and bile salts. this gastrointestinal tropism has played an important role in at least major outbreak; this suggests that surrogates such as mhv (a virus with multiple tropisms) and tgev (an enteric virus) that reflect this diversity in tissue tropism are necessary. a log viral reduction factor of . has been previously suggested as a benchmark for effective virucidal activity against coronaviruses and other viruses on surfaces. , , , the results of this study show that, of the commonly used hospital germicides tested, only the ethanol-based germicides were able to achieve this level of reduction of infectious virus after minute of contact time. for mhv, the ethanol-based germicides ( %, %, and % ethanol) gave the greatest reduction in infectivity, with log reduction factors ranging from . to . . this was greater than the reductions observed with hypochlorite, phenolic, and orthophthalaldehyde based germicides. these same performance trends are evident in tests of these germicides against tgev. the ethanol-based germicides gave log infectivity reduction factors ranging from . to . , greater than those observed for hypochlorite, phenolic, and orthophthalaldehyde germicides. the phenolic and orthophthalaldehyde germicides had greater virucidal activity against tgev than against mhv. however, only the reductions in infectivity by orthophthalaldehyde were significantly different between mhv and tgev. statistical analysis indicates that mean log viral reductions differed significantly based on both the type of germicide and the type of virus tested and that there are interaction effects between germicide and virus type. hence, both the selection of germicide and the type of virus will influence the resultant magnitude of reduction of virus infectivity titer. several previous studies have determined that surface disinfection is an important method for preventing viral transmission from surfaces to humans. the risk of acquiring infection decreases proportionately to the amount of viral agent present on surfaces. this study shows that ethanol-based germicides achieve the greatest reduction in viral titer on surfaces. these findings are consistent with previous studies of coronavirus disinfection, but this study provides more precise estimates of inactivation on surfaces than have been previously observed with other human coronaviruses, such as e. some previous studies of chemical disinfection of e have been limited by cytotoxicity problems that limited the ability to measure infectious virus reduction. sattar et al found that % ethanol reduced human coronavirus e dried onto a stainless steel surface by . . %. this study shows that the actual reduction is slightly greater than -log ( . for tgev and . for mhv). the available data on disinfection of sars-cov itself are not extensive, partly because of the challenges of working with sars. much of the available data focuses on alcohols. seventy percent ethanol was found in study to inactivate sars by . log . rabenau et al reported a log reduction factor of . after -second contact time using % ethanol and % propoanol, a reduction that was actually greater than what they observed with other germicides classified as chemical steriliants. these results support the findings of this study that ethanol-based disinfectants are efficacious against coronaviruses. however, flammability limits the use of ethanol for spills or contamination events involving large surface areas. other studies show that disinfectant efficacy can vary by virus type, with nonenveloped viruses such as adenovirus differing from enveloped viruses such as the coronaviruses. studies of hospital germicide efficacy against adenovirus using the same carrier-based method with -minute contact times found greater log reduction factors by opa ( . ) than were observed in this study for tgev ( . ) and mhv ( . ). in contrast, vesphene iise reduced adenovirus by a log reduction factor of only . , compared with . for mhv and . for tgev in this study. reduction of adenovirus by % ethanol was similar to that observed for coronavirus in this study, but . % hypochlorite reduced adenovirus approximately -log greater than reductions observed for coronaviruses with . % hypochlorite. this suggests that disinfection efficacy may differ greatly by virus type and that nonenveloped viruses may not be appropriate surrogates for predicting the effects of disinfectants on enveloped viruses such as coronavirus and influenza. hypochlorite demonstrated a log reduction factor , after minute for both tgev and mhv when applied at the : ( . %) use-dilution prescribed by the manufacturer. previous studies of coronavirus disinfection have found higher reductions with concentrations of hypochlorite greater than the recommended usedilution, suggesting these results are consistent with a concentration-dependent effect. sattar et al reported . % reduction of viral titer for human coronavirus e when . % and . % sodium hypochlorite solutions were tested with -minute contact times. commercial marketers of sodium hypochlorite recommend contact time of minutes. in actual use, it is likely that contact times are shorter than this, and increases in concentration may be necessary to offset the use of shorter contact times. the results of this study suggest that the : use-dilution should not be recommended for use on surfaces with suspected contamination by coronaviruses. hypochlorite is an important environmental surface disinfectant in health care; without the flammability and rapid evaporation of ethanol, it is suitable for large surface area spills. increases in both sodium hypochlorite concentration and contact time should be evaluated to determine whether these factors would improve virucidal activity of hypochlorite on hard surfaces to achieve a . -log reduction performance target. organic matter may play an important role in the poor performance of hypochlorite on surfaces observed in this study. a concentration of mg/l on a surface produced a log reduction factor , of mhv and tgev in this study. the poor performance of hypochlorite against viruses dried into surfaces may be due to the high oxidant demand exerted by the proteinaceous cell culture medium matrix in which the viruses were suspended. this results in consumption of available hypochlorite by the proteins and other organic compounds (eg, amino acids) present in the matrix, rendering it unavailable for disinfection. use of viruses suspended in a proteinaceous matrix simulates the real world conditions under which viruses shed by human hosts occur in health care environments. viruses are not shed by an infected host as single purified particles; they occur as aggregates, surrounded by membranes and embedded in feces, mucus, and other proteinaceous matrices. together, these results suggest that changing the recommended use-dilution of hypochlorite may address the problem of oxidant demand exerted by proteinaceous material such as body fluids and result in effective inactivation of coronaviruses shed from human hosts. hypochlorite, phenolic, and opa disinfectants tested reduced infectious viral titer by , log after -minute contact time. opa is used as a high-level disinfectant for semicritical equipment such as endoscopes; viruses can be deposited on the surfaces of semicritical equipment items during patient care. these results suggest that sufficient contact time is crucial to ensure that inactivation of viruses on the surfaces of semicritical equipment items takes place. according to the manufacturer, the phenolic disinfectant tested demonstrated a -to -log reduction in viral titer after minutes contact time when tested against another surrogate www.ajicjournal.org vol. no. coronavirus, avian infectious bronchitis virus. the results from this study, using -minute contact time, suggest that virucidal efficacy is greatly compromised if germicides are not used according to label directions and contact time is shorter than manufacturer recommended contact times. enveloped viruses of great nosocomial importance and pandemic potential, such as sars cov and avian influenza, are extremely challenging to work with in the laboratory. in the event of re-emergence of sars-cov, and in the context of pandemic influenza control, data are needed to guide decisions on appropriate disinfection of health care surfaces for control of viral transmission. tgev and mhv are nonpathogenic to humans and easily propagated and assayed in cell culture by plaque and quantal mpn assays. , this study shows that these types of surrogate viruses can help expand our knowledge of practical aspects of virus control, such as inactivation by disinfectants, for viruses of public health importance. it also increases the available data both for disinfectants that have previously been studied with sars co-v and disinfectants that have not. previous studies using sars have evaluated benzalkonium chloride and magnesium monoperphthalate-based products, povidone iodine, formalin, and glutaraldehyde. this current study included sodium hypochlorite and a phenolic, which have not been evaluated in previous studies. previous investigators have tested several types and concentrations of alcohols, including isopropanol, propanol, and ethanol, for which varying results have been observed in studies of sars. the data from this study can add to existing knowledge to help clarify how effective alcohols are against coronaviruses on surfaces. four of the germicides tested showed greater reductions of tgev compared with mhv. this suggests that mhv is potentially a more conservative surrogate for evaluating disinfectant efficacy against sars-cov. these studies should be replicated using sars-cov to determine which surrogate virus is a more suitable model for the response of sars to these germicides. there is still an important role to be played by surrogate viruses, especially for the evaluation of new disinfectants or the re-evaluation of use of current disinfectants (such as changes in dose and contact time); the available data suggest that both tgev and mhv may serve as conservative surrogates for modeling control of sars-cov by health care germicides in worst case scenarios. learning from sars: preparing for the next disease outbreak nosocomial spread of viral disease microbicides and the environmental control of nosocomial viral infections disinfection and prevention of infectious disease efficacy of hospital germicides against adenovirus , a common cause of epidemic keratoconjunctivitis in health care facilities nosocomial transmission of sars severe acute respiratory syndrome coronavirus on hospital surfaces contamination, disinfection, and crosscolonization: are hospital surfaces reservoirs for nosocomial infection? stability and inactivation of sars coronavirus why did outbreaks of severe acute respiratory syndrome occur in some hospital wards but not in others? severe acute respiratory syndrome (sars): paradigm of an emerging viral infection importance of environmental decontamination: a critical view potential role of hands in the spread of respiratory viral infections: studies with human parainfluenza virus and rhinovirus hierarchy of susceptibility of viruses to environmental surface disinfectants: a predictor of activity against new and emerging viral pathogens guidelines for environmental infection control in health-care facilities efficacy of various disinfectants against sars coronavirus antimicrobial activity of home disinfectants and natural products against potential human pathogens study on the resistance of severe acute respiratory syndrome-associated coronavirus a disc-based quantitative carrier test method to assess the virucidal activity of chemical germicides the relationship of severe acute respiratory syndrome coronavirus with avian and other coronaviruses phylogenomics and bioinformatics of sars-cov severe acute respiratory syndrome coronavirus phylogeny: toward consensus opp microbiology laboratory standard operating procedure for disinfectant sample preparation disinfection of human enteric viruses on fomites survival of human coronaviruses e and oc in suspension and after drying onsurfaces: a possible source of hospital-acquired infections chemical disinfection of non-porous inanimate surfaces experimentally contaminated with four human pathogenic viruses inactivation of sars coronavirus by means of povidone-iodine, physical conditions and chemical reagents apic guideline for selection and use of disinfectants replication and plaque formation of mouse hepatitis virus (mhv- ) in mouse cell line dbt culture studies on transmissible gastroenteritis of swine: ii. selected characteristics of a cytopathogenic virus common to five isolates from transmissible gastroenteritis inactivation of the coronavirus that induces severe acute respiratory syndrome, sars-cov key: cord- -cual qv authors: abraham, sushma; kienzle, thomas e.; lapps, william; brian, david a. title: deduced sequence of the bovine coronavirus spike protein and identification of the internal proteolytic cleavage site date: - - journal: virology doi: . / - ( ) -r sha: doc_id: cord_uid: cual qv abstract the sequence of the spike (also called peplomer or e ) protein gene of the mebus strain of bovine coronavirus (bcv) was obtained from cdna clones of genomic rna. the gene sequence predicts a , mol wt apoprotein of amino acids having an n-terminal hydrophobic signal sequence of amino acids, potential n-linked glycosylation sites, a hydrophobic anchor sequence of approximately amino acids near the c terminus, and a hydrophilic cysteinerich c terminus of amino acids. an internal lysargargserargarg sequence predicts a protease cleavage site between amino acids and that would separate the s apoprotein into s and s segments of and mol wt, respectively. amino terminal amino acid sequencing of the virion-derived gp spike subunit confirmed the location of the predicted cleavage site, and established that gp and gp are the glycosylated virion forms of the s and s subunits, respectively. sequence comparisons between bcv and the antigenically related mouse hepatitis coronavirus revealed more sequence divergence in the putative knob region of the spike protein (s ) than in the stem region (s ). the bovine coronavirus (bcv) is an important cause of neonatal calf diarrhea ( , ) and may also be the cause of winter dysentery in adult cattle ( ). the mechanisms by which bcv causes disease and persistent infection are not understood, nor are current vaccines universally regarded as effective. toward these ends, we have begun a detailed study of the bcv protein and genome structure. bcv is comprised of four major structural proteins ( ). these are (i) a -kda spike (peplomer) glycoprotein (s), that exists on the virion as cleaved subunits of approximately and kda, (ii) a -kda glycoprotein (he) that has both hemagglutinating ( ) and esterase ( ) activities, and which is comprised of two identical, disulfide-linked -kda subunits ( , , , ) , (iii) a -kda integral membrane glycoprotein (m) ( ), and , (iv) an internal phosphorylated nucleocapsid protein (n) ( ). of these, the s protein is presumed to ' sequence data from this article have been deposited with the embugenbank data libraries under accession no. m . ' present address: department of molecular biology and microbiology, case western reserve university, cleveland, oh . present address: dnx corporation, one president street, athens, oh - . to whom requests for reprints should be addressed. be the major structure by which coronaviruses attach to cells and initiate infection (reviewed by spaan et a/. ( )). the he protein, however, may also bind to cells to initiate infection, and for bcv, the relative importance of these two proteins in initiating infection is not known. both s and he are probably important in inducing immunity since antibodies to each are known to neutralize virus infectivity in cell culture and in calves ( , ) . s and he, therefore, may both be useful in developing effective engineered vaccines against bcv. cdna cloning of bcv genomic rna was accomplished essentially as previously described ( , ) except that random mer oligodeoxynucleotides (pharmacia) and -mer oligodeoxynucleotides of specific sequences were used as primers for first-strand synthesis. clones were mapped relative to one another and to the ' end of the genome using a matrix spot hybridization technique. some clones were sequenced by the chemical method of maxam and gilbert ( ) and some by the dideoxynucleotide-induced chain termination method of sanger ( ) as described by kraft et a/. ( ) using sequenase enzyme (united states biochemicals). for much of the sequencing, restriction endonuclease fragments were subcloned into the pgem z vector (promega) and forward and reverse sequencing kilobases , ' i i i i i i primers for the pgem vectors were used. sequencespecific oligodeoxynucleotides were also synthesized and used for sequencing within certain regions of the large clones. the amino-terminal ends of purified gp and gplo subunits were subjected to sequencing by the method of matsudaira ( ) . unlabeled bcv was purified by isopycnic sedimentation in sucrose gradients and the proteins were electrophoretically separated after reduction in -mercaptoethanol ( ) and electroblotted ( ) onto polyvinylidene difluoride membrane ( ). proteins were visualized by staining with coomassie brilliant blue and the gpl and gplo bands were excised and shipped to dr. matsudaira for analysis. complete sequencing of clone ma which extends . kilobases from the ' end of the genome (fig. ) revealed a continuous open reading frame located on the ' side of the orf for a potential . -kda protein (abraham et al., to be published elsewhere). the deduced amino acid sequence of the extended orf demonstrated high sequence similarity to the c-terminal end of the antigenically related mhv-a ( ) and mhv-jhm ( ) s proteins, both antigenic homologs of the bcv s protein ( ). these data suggested that the s protein gene of bcv lies in the same relative position on the genome as does the spike protein gene of mhv. to complete the sequencing of the s gene, both strands of three clones, ii, hpa , and g , generated by random priming, and three clones, lk , lp and , generated by specific priming, were sequenced ( fig. ) . the total sequence for the putative s orf extended to a position . kb from the ' end of the genome and contained bases (fig. ) . we conclude this orf to be the s gene since it potentially encodes a amino acid protein of , da, the approximate size of the unglycosylated spike precursor (io), and be-cause its deduced amino acid sequence shows extensive sequence similarity throughout with the s proteins of both strains of mhv. five other open reading frames ranging in size from to amino acids were also found within the s gene sequence in the plus one reading frame, but their significance is not known at this time. the putative s orf is preceded immediately upstream (beginning at base in fig. ) by the consensus cyaaac sequence thought to play a role in leader priming of coronavirus transcription. the sequence is also found three times within the s orf, beginning at positions , , and , but it is not established that transcripts initiate at any of these sites. five features of the deduced bcv s protein reflect the properties of four other coronavirus spike proteins that have been characterized to date from nucleotide sequence data (i, , , , , , , ) . (i) there is an n-terminal hydrophobic stretch of amino acids which predicts a signal peptide with a cleavage site between amino acids and ( ). (ii) there are potential asparagine-linked glycosylation sites that could give rise to the only kind of glycosylation demonstrated for this protein (hogue and brian, unpublished data; ) . (iii) there is a hydrophobic stretch of amino acids near the c terminus that could serve as a stoptransfer and anchor sequence. (iv) there is a stretch of amino acids on the immediate n-terminal side of the predicted anchor sequence (-k-w-p-w-y-v-w-l-, beginning with amino acid ) that is identical in all coronavirus s proteins sequenced to date. (v) there is a cysteine-rich hydrophilic c-terminus of amino acids that is probably the intravirion domain. in common with mhv-( , ) and ibv ( , , , ) , but not in common with tgev ( , ; tung and brian, unpublished) and fipv (i!?), is also an internal sequence of basic amino acids that, in the case of mhv and ibv, lies on the immediate n-terminal side of the protease cleavage site ( , ) . in bcv the sequence is k-r-r-s- r-r beginning tagaccataa~tgtmtgatact~~~tccttaccaatggct~gctg~ataggaga~~gtgtactacgg~cca~mtgatg~acaccggtgctcc~ -mflillislpmafavigdlkcttvsindvdtgap ctattagcactgatattgtcgatgttactaatgg~aggtac~attatg~agatcgtgtgta~~tactacg~g~gc~~t~~a~accctac~cagg~ctacat sistdivdvtnglgtyyvldrvylbttlll ngyyptsgst b atcgtaatatggcactgaagggaacmactattgagcagactattgagcagactatggttt~ccacc~~ct~ctga~a~~tggta~gct~~tc~taccaagg~a~~ yrnmalkgtlllsrlwfkppflsdfingifakvkntkvik b i advyrripnlpdcnieawlndksvpsplnwerktfs attgtaattttaatatgag~agcctgatgtcttttattcaggcagactca~acttgt~taata~gatgctgct~gatatatggtatgtg~ccagcat~ctatagat~gt ncnfmmsslmsfiqad sftcnnidaakiygmcfssitidk q ttgctatacccaatggtaggaaggttgacctacaattgggcaat~gggctatttgcagtc~tt~ctatag~ttgatactactgctacaagttgtcag~gta~ataat~acctg faipngrkvdlqlgnlgylqs fnyridttatscqlyynlp ctgctaatgmctgttagcaggtttaatccttctacttggaataggagat~ggt~acag~caa~gt~~aagcctc~cctgtaggtg~actcatcatgatg~g~ aabvsvsrfnpstwnrrfgfteq fvfkpqpvgvfthhdvv ls atgcacaacattgttttaaagctccctc~at~ctgtccgtgtaaa~ggatgggtct~gtgtgtaggtaatggtcctggtatagatgctgg~at~~tagt~tataggcac~ yaqhcfkapsnfcpckldgslc vgngpgidagyknsgigt ---- t;t b tagttggcataggtgagcactgttcggggtc~gcta~aa~gtga~attgtggaggtaatcc~gtac~gcc~ccacaagca~~gggctggtctg~gactc~g~ac~g lvgigehcsclaiksdyc~cnpctcppqaflgwsvdselq -_--_-_------ b gggataggtgtaatamttgctaatmatmgcatgatgatgttaatagtggtactacttg~ctactga~ac~~atc~acacagacat~~c~ggtg~gtg~aattatg gdrcnifanfilhdvnsgttcstdlqksntdi ilgvcvny atctttatggta~acaggcc~ggta~~g~gaffi~aatgcgac~attat~tag~ggcagaacc~tatatga~ct~tggt~tctctatgg~tagagactac~~ dlygitgq gifvevbatyynswqnllyd sngnlygfrdyl caaacagaactmatgattcgtagttgctatagcggtcgtgtttcagcggcctttcatgct~ctc~ccg~ccagcattgcta~cggaata~~tgc~~acg~~~ta tbrtfmirscysgrvsaafhaes b sepallfrnircnyvfb atactcmcacgacagctgcaacctattaactamtgata~gatagttatc~gg~gtg~gtc~tgctgat~tagtac~ctagtg~g~c~catgtgat~cacagtaggtagtg ntlsrqlqpinyfd sylgcvvnadbs tssvvqtcdltvgs gttactgtgtggattactctacaaaaagacgaagtagcctgtag gyc"dystkrrsrraittgyrfttfepftvnsv,?jdslepv gtggtttgtatgaaattcaaataccttcagagag~actataggt~tatggaggag~a~c~acmgctctcct~g~acta~ga~g~ctgc~tctgtggtga~atg gglyeiqi pseftignmeefiqts spkvtidcsafvcgdy cagcatgtaaatcacagttgg~g~tatggtagcttctgtgacaatattaatgctatactcacagaagtaaatgaactacttgacactacacagttgcaagtagct~ta~t~~~ga aacksqlvey gsfcdninailtevne lldttqlqvan atggtgtcactcttagcactaagcttaaagatggcgtt~~tc~tgtagacgacatcaatt~cccctgta~aggttg~aggaagcgattgtaat~gt~ccagcagatctg ngvtlstklkdgvnfnvddiwfs pvlgclgsdcnkvssrs sei ctatagaggatttacttmtctaaagtapiagtaaag~atctgatgtcggt~cgttgaggcttataat~ttgtactggaggtgccgaaattagggacctca~gtgtgc~gttataatg aiedllfskvklsdvgfveaynac tggaeirdlicvqsyn ttpcaacacccaacctccatgattttaaggaaggaagagttggatcaatggttt~aa pictcagtggcaccagatptgtcacttgattatatp~cttggacctacaag istpnlhdfkeeldqwfk&svapdlsldyi,ii,vtfldlq atgaaatgaataggttacaggaggcaataaaagttttaag~tt~atcagagctacatcaatctcaaggaca~ggtacatatgagta~atgt~aatggccttggtatgtatggc~aa~g demnrlqeaikvloq syinlkdigtyeyyvkwpwyvwlli sb gctttgctggtgtagctatgcttgmpactattctattc~catatgctg~gtacaggatgtgggactag~gt~taagatatgtggtgg~gttgtgatgattatactggacaccaggagt gfagvamlvllfficcctgcgtscfkicggccddytghqe tagtaattaaaacatcacatgacgactaa lviktshdd m t t fig. . nucleotide sequence of the s gene and its deduced amino acid sequence. the nucleotide sequence shown begins with the tag termination codon of the he gene (underlined) bases upstream of the presumed s start site ( bases from the poly(a) tail), and ends with the taa termination codon of the s protern. the first three amino acids of the putative . .kda protein are shown beginning at base position . consensus cyaaac sequences are boxed. the presumed amino-terminal signal peptide and carboxy-terminal anchor sequences are underlined. potential n-linked glycosylation sites (nxs or nxt. where x # p) are boxed. the proteolytic cleavage site separating sl and is identified with an arrow. the extended sequence of amino acids missing in mhv jhm is identified by individually underlined amino acids, and that missing in mhv a , by asterisks. with amino acid , and, on the basis of the pattern in mhv and ibv, predicts a cleavage between amino acids and (note arrow in fig. ). cleavage at this point would divide the unglycosylated s protein into an n-terminal segment of , da (sl) and a cterminal segment of , da (s ), from amino acid sequencing studies, no n-terminal sequence could be obtained from the virion-derived -kda subunit, possibly because of n-terminal blockage. the n-terminal sequence of the loo-kda subunit could be obtained, however, and was determined to be x-l-t-t-g-y-x-f-, identifying the first amino acids downstream from the predicted internal cleavage site. these results confirmed the predicted internal cleavage site and established that the -kda subunit is sl and the -kda subunit is s . the bcv and mhv s proteins show remarkable sequence homology suggesting that these viruses are re-a ' i i i ii iii i bcvq ' j ' i i illll i i i i iiiii ii i i ii i ill i ii i ii ii i i i i i ii* iii i ii ii ii i i i cently diverged. after aligning sequences for maximal homology, the following points emerge. (i) relative to bcv, a large deletion appears in the mhv sl subunits. for jhm it is a contiguous gap of amino acids, and for a it is a discontiguous gap of amino acids (figs. and ). the function of the additional sequence in the bcv sl subunit is not known, but it is possibly a structure that interacts in some way with the he glycoprotein, a structural protein not found on mhv ( , ) except under certain rare conditions ( ). no electron micrographic or chemical data exist, however, to suggest that s and he do physically interact ( , , ) . it is interesting to note that the entire region in the bcv s protein corresponding to the gap region of the jhm s protein is especially rich in cysteine residues and contains ( %) of the total cysteines in the bcv s protein (figs. and ). this suggests that this part of the molecule may be important for intramolecular or intermolecular disulfide linkages. (ii) exclusive of the large gap in the mhv sequences, the sl subunits of jhm and a show and % identity, respectively, with bcv, and the s subunits show and % respectively. throughout the s protein, of cysteine positions and of potential n-linked glycosylation sites are conserved. the internal proteolytic cleavage position (not yet confirmed for jhm) is also conserved. the pattern of greater amino acid sequence divergence in the sl subunit is consistent with the model of cavanagh ( ) and de groot et a/. ( ) which proposes that the sl subunit comprises the exposed bulbous structure of the spike and probably contains most ( ) but not all ( , ) of the neutralizable antigenic sites. it is the structure most likely to undergo changes as a result of immunologic selective pressures. fusion of cells in culture is one biological activity associated with cleavage of the mhv s protein ( ). despite its extensive sequence similarity with the mhv s protein, however, the bcv s protein shows little fusion activity. in fact, fusion is a behavior we have not observed with the mebus strain of bcv even though the s protein is primarily in the cleaved form on the virion ( , ). it is not clear why bcv and mhv behave so differently in their fusogenic properties, but functional evaluation of sequence differences near the cleavage sites of these two viruses may aid in clarifying the mechanisms of fusion by mhv. this is especially interesting since hydrophobic regions, common at the cleavage sites on fusion proteins of paramyxoviruses and myxoviruses, are absent in the mhv s protein ( ) and different mechanisms of fusion may be employed. biotech-niques proc. /vat/. acad. sc;. usa proc. nat/. acad. sc;. usa this work was supported by grant al- from the national institute of allergy and infectious diseases, and by grant -crsr-z- from the united states department of agriculture. t.e.k. and w.l. were predoctoral trainees on grant t -al from the national institutes of health. key: cord- -zfjy m i authors: alsaadi, entedar a. j.; neuman, benjamin w.; jones, ian m. title: identification of a membrane binding peptide in the envelope protein of mhv coronavirus date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: zfjy m i coronaviruses (covs) are enveloped, positive sense, single strand rna viruses that cause respiratory, intestinal and neurological diseases in mammals and birds. following replication, covs assemble on intracellular membranes including the endoplasmic reticulum golgi intermediate compartment (ergic) where the envelope protein (e) functions in virus assembly and release. in consequence, e potentially contains membrane-modifying peptides. to search for such peptides, the e coding sequence of mouse hepatitis virus (mhv) was inspected for its amino acid conservation, proximity to the membrane and/or predicted amphipathic helices. peptides identified in silico were synthesized and tested for membrane-modifying activity in the presence of giant unilamellar vesicles (guvs) consisting of , -dipalmitoyl-sn-glycero- -phosphocholine (dppc), sphingomyelin and cholesterol. to confirm the presence of membrane binding peptides identified in the context of a full-length e protein, the wild type and a number of mutants in the putative membrane binding peptide were expressed in lenti-x- t mammalian and insect cells, and the distribution of e antigen within the expressing cell was assessed. our data identify a role for the post-transmembrane region of mhv e in membrane binding. the coronavirus envelope protein (e) is a small hydrophobic protein ranging from - amino acids [ , ] . it has an n-terminal domain, a long alpha helical transmembrane domain and a c-terminal hydrophilic domain, and is found incorporated into the virus particles of all coronavirus groups at low levels [ ] [ ] [ ] [ ] [ ] . two membrane topologies have been suggested for the e protein: hairpin or transmembrane [ , ] . e also interacts with the m protein, and mutants of m that are unable to bud from cells can be complemented by forms of e [ , ] . the membrane curving properties of e are such that the coexpression of m and e is adequate for the efficient formation of virus-like particles (vlp) [ , ] , and these can also incorporate the s protein if it is coexpressed [ ] . for many coronaviruses, including mhv, e protein also functions as an ion channel, a viroporin [ , ] , affecting the trafficking of virions in the secretory pathways and membrane permeability, both of which are essential for virus growth [ , , ] . while e function is critical for virus assembly, its viroporin activity in mobilizing calcium ions and its interactions with host cell tight junction proteins have also been implicated in the pathologies of some coronavirus infections [ , ] . an additional role for e in viral pathogenesis is an anti-apoptotic effect on host cells during virus replication [ ] . the relative significance of the e encoded ion channel activity to virus morphogenesis, as opposed to its structural role in binding m, is uncertain. however, its role as a virulence factor has been demonstrated by many studies on sars-cov e protein which have shown altered pathogenesis in a mouse model, either by an effect of virus production and by effects on expressing cells [ , , ] . stimulated by the pandemic of sars-cov- [ ] , a recent protein interaction map of virus infected hek cells identified host proteins of the vesicular sorting pathway and bromodomain proteins as interacting with e, the latter of which may have therapeutic potential [ ] . notwithstanding its multifunctional nature, the interaction of e with membranes is central to its biological role, and peptides with direct membrane binding properties are present within the coding region. the mapping of such peptides may help to interpret e mechanisms of action and offer the potential for inhibitor development. here, we test e-derived peptides for membrane binding activity in vitro and confirm those identified as positive in the context of the full length protein expressed in two different cell types. all chemical reagents were purchased from thermofisher scientific unless otherwise stated, and were used following the vendor's recommendations. lipids were obtained from avanti polar lipids. peptides used for the in vitro analysis (table ) were derived from the e protein of mhv coronavirus (accession number aau ), synthesized and supplied as a lyophilized product with a stated mean purity of ≥ % by cambridge research biochemicals, uk. peptide stock solutions were prepared in a buffer consisting of . mm sucrose, . mm glucose, mm dtt and . % (v/v) dmso. a positive control peptide, the m -influenza peptide, a well characterized amphipathic helix sufficient for budding into guvs and the formation of large luminal vesicles (luvs) [ ] , was used to validate the guv assay. negative controls were provided by buffer only in order to match test samples in all but the test peptide. guvs were generated by electroformation using a vesicle prep pro (nanion technologies gmbh, munich, germany) with a mix of mm , -dipalmitoyl-sn-glycero- -phosphocholine (dppc), mm egg sphingomyelin and . mol% cholesterol, which were first dissolved in chloroform. to visualize the guvs, . mol% of naphtha [ -alpha] pyrene (tokyo chemical industry uk ltd, oxford, uk) was added. lipids were mixed in an amber vial to a final total lipid concentration of mm before preparation by electroformation. briefly, µl of lipid stock was spread on the conductive side of an indium tin oxide slide (ito-slide), air dried and put into a vacuum desiccator for h to remove all solvent. a -mm o-ring was coated on one side with silicon grease and placed around the dried lipid film, which was then hydrated with µl of . mm sucrose solution in deionized water. a second ito slide was placed on top of the o-ring with the conductive sides facing, and a voltage of v peak to peak at a frequency of hz was applied to the ito slides over a period of h at • c [ ] . guvs were imaged using an evos-fl digital fluorescence microscope (evos, usa). following electroformation, the chamber was disassembled and the sucrose solution removed to leave the guvs attached to one ito slide. diluted peptide was added immediately at the desired concentration and the field was imaged , , and min thereafter. imagej was used to measure the shape and the relative size of the guvs. experiments were performed in triplicate and the average and standard deviation were calculated. buffer only and m -influenza peptide controls were treated in the same way. guv-peptide incubations were at room temperature. statistical significance was calculated using spss version , using linear mixed model (lmm) (p• . ). results were expressed as mean ± sem. figures were generated using graphpad prism . b software. lenti-x t cells were cultured and maintained in dulbecco's modified eagle medium (dmem) (sigma aldrich) supplemented with % fetal bovine serum (fbs) (ge healthcare) and . % penicillin-streptomycin solution (gibco/invitrogen, paisley, uk) at • c with % co . spodoptera frugiperda (sf ) cells (invitrogen) were maintained in ex-cell medium (sigma, gillingham, uk) supplemented with % fetal bovine serum and % penicillin/streptomycin solution at • c with shaking. the coding sequence of mhv e (genbank accession: ay . ) was amplified by pcr from cdna kindly supplied by dr. volker thiel using the primers hsv-e-fw ( 'gcgccatggcaca gccagaactcgccccggaagaccccgaggattttaatttattccttacagacacagtatggtat gtgggg- ') and hsv-e-rv ( '-gcgctcgaggatatcatccacctctaatag ggg- ') and the amplicon cloned into ptriex . (merck millipore, darmstadt, germany) between the ncoi and xhoi restriction sites. to provide markers for the detection of expression by western blot, the wt e coding region was cloned in frame with an upstream hsv and downstream his tag, both provided by the vector. following initial expression experiments, detection of the hsv tag was found to be poor. as a result, mutant e sequences were synthesized de novo (integrated dna technologies, dresden, germany) and cloned similarly, except they were in frame only with the downstream his tag. all constructs were confirmed by dna sequencing prior to use. one day before transfection, . × lenti-x t cells (takara, saint-germain-en-laye, france) were seeded into well plates containing a glass coverslip in each well and incubated for h at • c/ % co . the following day, the cells were transfected using lipofectamine transfection reagent (invitrogen) with plasmids encoding wildtype or mutant mhv-e and incubated for h at • c/ % co . a control vector ptriex . -gfp expressing green fluorescent protein (gfp) gene was used to visualize the efficiency of transfection, typically - %. twenty four hours after transfection, the media was removed and the cells were washed twice with cold pbs for min, then incubated in fix and permeabilization buffers for h at room temperature (ebioscience, san diego, usa). fixed and permeabilized cells were incubated with anti-his-alexa fluor conjugate ab ( e d hh /e , thermofisher scientific) for h at room temperature diluted : in × permeabilization buffer, washed twice with tbs for min each at room temperature in the dark, and then counterstained with dapi. the fixed cells were mounted with a drop of slowfade™ gold reagent before being imaged using an evos-fl digital fluorescence microscope. typical images were captured at × magnification and further manipulated, if required, using imagej software [ ] . the flashbac™ gold (fbg) baculovirus expression system (oxford expression technologies, oxford, uk) was used to produce recombinant baculoviruses. small-scale protein expression was performed by infection in a -well plate seeded with × sf cells per well using µl of a high titre stock of the recombinant baculovirus, typically passage , and incubated for days at • c. after incubation, cells were harvested and used for western blot with an anti-his antibody. proteins were separated by sds-page using - % precast tris-glycine sds polyacrylamide gels (invitrogen) for min at v. after electrophoresis, gels were either stained by coomassie blue r or transferred to pvdf membranes for western blot analysis. following transfer to pvdf membranes (whatman, chalfont st. giles, uk) using a semidry western blotting apparatus, membranes were incubated in blocking buffer ( % skimmed milk powder in tbst ( . % tween- , × tbs)) for h. membranes were incubated with the primary antibody at : , in × tbst buffer for h, followed by three washes for min each in tbst buffer, and subsequently, with a secondary horseradish-peroxidase (hrp) antibody conjugate (dako, glostrup, denmark) diluted : , in × tbst for h followed by three washes for min each with tbst buffer. the membrane was finally washed with tbs and the bound hrp activity was detected using chemiluminescence imagery on a syngene g: box. to allow reprobing of a pvdf membrane with a different antibody, the membrane was stripped using a stripping buffer ( mm β-mercaptoethanol, % sds, . mm tris-hcl ph . ) for min at • c. the membrane was then washed three times with tbst buffer, min each, and then reblocked and probed as before. a total of . × sf cells were seeded in t flasks as monolayers, infected with recombinant baculoviruses at an moi = and incubated for h at • c. the infected cells were harvested by loosening the monolayer into the media and collected by centrifugation at × g at • c for min. cell pellets were resuspended in µl of cold pbs and lysed by sonication for m with on/off pulses at -s intervals (sonics, vibracell tm ). the cell lysates were briefly clarified ( × g for min) and then centrifuged at low speed, i.e., , × g, at • c for min to collect large membrane debris. the pelleted material was kept as a low-speed pellet (lsp). the collected supernatants were then centrifuged at high speed, i.e., , × g, for min at • c in a beckman tl- ultracentrifuge and the pellets were retained as high-speed pellets (hsp). low-and high-speed pellets were tested for the presence of mhv e by western blot using an anti-his antibody, as before. several viral proteins encode amphipathic helices that modulate membrane curvature, e.g., the m protein of the influenza virus and the nonstructural protein b of the hepatitis c virus [ , , ] . the program amphipaseek [ ] was used to assess local amphipathy and putative membrane-binding regions for a phylogenetically diverse set of e proteins representing covs, and the output was visualized using jalview v . . b [ ] . cov e proteins have related topologies but otherwise limited direct homology outside of the clearly predicted tm domain. however, amphipathicity occurred in a region immediately downstream of the tm domain, a region which contains the previously mapped cysteine region [ ] at the end of the predicted transmembrane domain ( figure ) and which is also palmitoylated intracellularly [ , ] to facilitate contact with the lipid bilayer [ ] . in addition, two conserved proline residues occur in this region, plausibly providing conformational flexibility that may be important for e-e interactions or between e and other proteins [ , ] . spanning the first conserved proline, a residue sequence, i.e., - in mhv a e, including a number of hydrophobic residues, was evidently relatively well conserved among most α and β covs, and was also shared with the more distantly related γ and δ covs, with the exception that insertions containing further multiple hydrophobic residues were also present ( figure ). based on these criteria this peptide, the e post tm peptide (eptm), was considered to have potential for direct membrane interaction. ; green represents polar amino acids (n, q, s, t); pink represents cysteines (c); orange represents glycines (g); yellow represents prolines (p); cyan represents aromatic amino acids (h, y); white represents any unconserved/gap. the sars-cov- e protein is % identical to sars e, so was not included as a distinct entry. to address its potential as a membrane active peptide, the residue wild type peptide was synthesised and reconstituted with giant unilamellar membranes (guvs), and the effect on size and morphology were measured. in addition, peptides etm, representing the e protein tm domain itself, and the m influenza peptide were tested (table ) . guvs, composed of mm dppc, mm egg sm and . mol% cholesterol were reconstituted with the peptides at a concentration of µm and the guvs were measured at , and min thereafter by fluorescence microscopy, as described elsewhere [ ] . shape was measured as the ratio between the longest and shortest radii, while relative guv size was estimated by ramanujan's first approximation, taking an average of guvs per experiment for three separate experiments. peptide eptm changed the size but not the shape of the guvs membrane, leading to membrane deformation that increased with time, while guvs treated with buffer only showed no deformation ( figure ) . interestingly, peptide etm showed no effect on either the shape or size of the guvs. interaction of mhv-eptm peptide with the guv membranes made the guvs smaller in size and more rugged in appearance, which was possibly indicative of fragmentation of the lipid bilayers after peptide insertion. the lack of effect by the etm peptide suggested that this peptide alone is not membrane active and may need to be presented in the context of the complete e protein for biological activity. the assay was validated by use of the m -influenza peptide which showed a statistically significant change in guv shape with the formation of intraluminal-vesicles (ilvs) as described [ ] , although no significant change in size was apparent ( figure ). to validate the eptm peptide sequence as membrane active in the context of the full length e protein, e was expressed in both mammalian and insect cells through the use of a dual promoter vector, ptriex . , which permits expression from the same vector in both systems. mhv e protein was expressed as the wild type protein and also as a series of alanine scanning mutants in which conserved and hydrophobic residues were exchanged for alanine ( table ). in addition, the entire region was deleted within the e coding sequence. mammalian expression was done by transfection of lenti-x t cells where expression is driven by the vector resident cag promoter, and the expressed e protein was detected by immunofluorescence imaging with an anti his tag antibody h post-transfection. the wt mhv e protein was found to be distributed evenly throughout the cytoplasm with a slight concentration near the nucleus, plausibly the golgi body. in all the alanine scanning mutants of e, expression levels were similar with little diminution of the overall signal, but in most cases, the pattern of staining was altered to a more granular punctate appearance (figure ). this was particularly notable for mutation l a, where almost all of the fluorescence signal was associated with a punctate distribution. this data suggests that the eptm sequence, identified as causing membrane association in an isolated peptide, also influences the behaviour of the complete protein in a physiologically relevant environment. del ---------- to provide a more quantifiable measure of membrane association, a more productive protein expression system, i.e., expression in insect cells via the construction of recombinant baculoviruses, was investigated with the same panel of e variants. following the generation of recombinant viruses, insect cells were infected at high moi and their ability to express e protein assessed by western blot at days postinfection when synthesis from the vector encoded p promoter was maximal. to facilitate detection, the wt e protein was expressed with both hsv and his tags, at the n-and c-termini respectively, a predicted molecular mass of~ kda. of these two tags, however, only probing with the his antibody detected the expressed product consistently. as a result, all mutant e proteins were expressed with only the c-terminal his tag, a predicted molecular mass of~ kda. all e proteins were expressed at the molecular weight expected, but the expression level varied with mutation ( figure a ). half of the mutants expressed at levels similar to the wild type, but mutations l a, v a, l a and y a were present at reduced levels, consistent with a potential role of these residues in protein folding and stability. to ensure the levels of infection were equivalent, the blot was stripped and reprobed with a monoclonal antibody to the major baculovirus glycoprotein, gp , a marker for infection level, which showed near equivalent infection in all cases ( figure b ). to investigate whether the mutations introduced into mhv-e resulted in an effect on cellular localization in more detail, plausibly by altered membrane association, infection of insect cells with each recombinant virus was repeated on a larger scale and the infected cells harvested at days postinfection. the cells were lysed by sonication in the absence of detergent, clarified and subjected to differential membrane fractionation to produce low-speed (lsp) and high-speed membrane pellet (hsp) fractions. to confirm the fractionation, lsp and hsp samples were probed with an antibody to calnexin, an er marker, which was largely found in the lsp fraction ( figure ). the expression of the wt mhv e was found in both fractions, but was more strongly associated with the hsp fraction ( figure a ), consistent with its known primary localization in mhv-cov infected cells [ ] . similarly, mutations l a and v a partitioned mainly into the hsp fraction, although expression levels overall were reduced. strikingly, the "all" mutant, in which all targeted residues were mutated to alanine, and the "del" mutant, in which the target peptide was deleted from mhv e, were found almost exclusively in the lsp fraction, despite high levels of expression. the remaining mutants carrying mutations l a, p a, y a and y a also associated preferentially with the lsp, although, as before, expression levels were lower in some cases, notably l a and y a. relative densitometry of the hsp and lsp bands revealed significant differences among the mutants with regard to their localisation to the different membrane fractions of e expressing insect cells ( figure b ) and confirmed a role for the amphipathic mhv cov e - peptide in membrane interaction. the cov e proteins consist from a short hydrophilic amino terminal region, a hydrophobic transmembrane region and a carboxy-terminal region that encompasses the majority of the protein [ ] . the e proteins are multifunctional proteins involved in virus assembly and release, and have been located in the ergic and golgi of infected cells but do not traffic to the infected cell surface [ ] . as studies of e have often involved different coronaviruses and different experimental systems, the unambiguous basis of this localisation has not been described. an amphipathic helix, eptm, detected in the post-tm region of e, was suggested by bioinformatics analysis and assessed for direct membrane interaction in vitro by binding to guvs. for comparison, the predicted e protein tm domain, etm, and an established membrane active peptide from the influenza m protein were also included. eptm, but not etm, caused a change in guv size and appearance, consistent with direct membrane binding. following expression of the complete e protein with mutations in the same identified peptide, altered membrane binding in two distinct cell types, mammalian and insect, was apparent. these data support the hypothesis that the membrane binding observed for the eptm peptide in vitro occurs also in the context of the complete e protein expressed in a physiologically relevant environment. cov e protein is a known integral membrane protein, and it has been proposed that the basis of membrane interaction is by one of a number of possible topologies, e.g., as a type iii membrane protein, as a membrane hairpin or as a glycosylated type ii membrane protein [ ] . the expression of e in insect cells resulted in a single band detected by western blot that did not have the appearance typical of a glycosylated protein. moreover, in the latter topology, the eptm region is predicted to be free on the luminal side of the membrane and not to interact with it. the data here are inconsistent with this prediction and are more explicable by assuming that either the type iii membrane protein or the membrane hairpin topology is more likely. in both cases, it is postulated that the post-tm region folds back against the membrane, where it may be additionally anchored by cysteine palmitoylation, although the precise mechanism of interaction remains unknown. assuming that the predominant punctate staining in mammalian cells represents misfolded e protein that induced stress granules, then mutants l a, y a and y a formed a group with a shared phenotype. the "all" mutant, which necessarily included these mutations, was also punctate in appearance. by contrast, l a and v a were more similar to the wt straining pattern. biochemical fractionation of membrane from insect cells following the construction and validation of recombinant baculoviruses broadly recapitulated this division. the relative level of e protein following differential centrifugation of the cell membranes showed that wt, l a and v a mutants were predominantly associated with the hsp fraction with a minority in the lsp fraction ( . %, . % and . % respectively), while the remaining mutants were predominantly associated with the lsp fraction. some mutants, notably l a and y a, were expressed at lower levels but were hardly present in the hsp faction. as the wt exhibited an hsp pattern, we interpret this to be authentic partitioning into membranes, whilst the lsp pattern is likely to be aggregated protein trapped in the er by virtue of its inability to associate with membranes correctly. despite its conservation, the role of the p a mutant was equivocal, i.e., some way between the two extremes but tending towards non-wt-like mutants. these data suggest a possible model for eptm interaction with the membrane in which the c-terminal hydrophobic residues within the residue region are the most relevant. interestingly, these fall broadly on one side of the helix prediction for eptm, while those broadly tolerated fall on the other ( figure ). plausibly, this distinction relates to membrane binding verses the requirement for e to also bind other ligands. further work will be required to address the level of membrane curvature afforded by this interaction and the extent to which it relates to the various functions reported for cov e, such as reorientation of the plasma membrane within the golgi [ ] , interaction with the m protein [ , , ] and viral particle scission [ , ] . in sars cov, e residues which reportedly bind to the membrane include valine- , tyrosine- and lys- [ ] . these residues are fully conserved in sars-cov- within an overall e similarity of %, and are also equivalent in the mhv e peptide identified here. membrane binding by these residues in sars e is thus supported by the observations made here for v a and y a, although lys was not examined. e has been considered a therapeutic target for sars in relation to its viroporin function which is linked to inflammation [ ] , and more recently, for sars-cov- as a result of its mapped interactions with bromodomain proteins, for which drugs already exist [ ] . a more detailed mapping of 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the terms and conditions of the creative commons attribution (cc by) license we thank fellow members of the virology laboratory for their help during the course of this study. the authors declare no conflict of interest. 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- - vx aq authors: leibowitz, julian l.; devries, james r. title: synthesis of virus-specific rna in permeabilized murine coronavirus-infected cells date: - - journal: virology doi: . / - ( ) -x sha: doc_id: cord_uid: vx aq abstract we have developed a permeabilized cell system for assaying mouse hepatitis virus-specific rna polymerase activity. this activity was characterized as to its requirements for mono- and divalent cations, requirements for an exogenous energy source, and ph optimum. this system faithfully reflects mhv-specific rna synthesis in the intact cell, with regard to both its time of appearance during the course of infection and the products synthesized. the system is efficient and the rna products were identical to those observed in intact mhv-infected cells as judged by agarose gel electrophoresis and hybridization. permeabilized cells appear to be an ideal system for studying coronavirus rna synthesis since they closely mimic in vivo conditions while allowing much of the experimental flexibility of truly cell-free systems. the coronaviruses comprise a group of large enveloped positive-strand viruses with a unique replication scheme. the genomic rna is about kb in size (boursnell et al., ) and shares many structural features with the genomes of other positive-strand viruses, i.e., it is capped at the 'terminus (lai et al., a) , is polyadenylated at the ' end (lomniczi, ; yogo et al., ) and can function as messenger rna in vitro (leibowitz et al,, ; denison and perlman, ) and presumably in vivo as well (denison and perlman, ) . the translation product(s) of the virion rna is hypothesized to include the coronavirus-specific rnadependent rna polymerase, although this has never been rigorously demonstrated. in addition to the virion rna, infected cells contain several other classes of coronavirus-specific rna (stern and kennedy, a; lai et al., ; leibowitz et a/., ; spaan et a/., ) . lai and co-workers have demonstrated that mhv-infected cells contain a single rna species of negative polarity which is genome length ( ) . this negative-strand rna is thought to serve as the template for positive-strand mrna synthesis. for mouse hepatitis virus (mhv), one of the most extensively studied coronaviruses, there are seven species of mhv-specific mrna present in infected cells, the largest of which is indistinguishable from virion rna (leibowitz et a/., ; lai et a/., ; spaan et a/., ) . structural analyses of these rnas have shown them to make up a "nested set" with coterminal ' ends (leibowitz et al., ; stern and kennedy, a,b; lai et a/., ; cheley et a/., ; ' to whom requests for reprints should be addressed. weiss and leibowitz, ; spaan et al., ) . a unique feature of coronavirus replication is the presence of a common leader sequence of about bases at the ' end of each message which is present only once (at the ' end) in the virion rna (spaan et a/., ; lai et al., lai et al., , baric et a/., ) . the mechanism of synthesis of all species of coronavirusspecific rnas is largely unknown. progress in studying the details of the synthesis of the mhv mrnas has been hampered somewhat by the lack of an efficient and easily reproducible in vitro transcription system which faithfully reproduces the events which occur in intact cells. several groups of workers have demonstrated actinomycin d-resistant rna polymerase activity in extracts of coronavirus-infected cells. dennis and brian ( ) have reported the presence of a membrane-associated polymerase activity in cytoplasmic extracts of tgev-infected cells. a similar activity has been demonstrated to be present in extracts of mhv-infected cells (brayton et al., (brayton et al., , mahey et a/., ) . recently, compton et al. ( ) have described a system based on an extract from lysolecithin-treated cells. these systems have either been difficult to work with due to their relatively low efficiencies, or they have been hard to reproduce and maintain on a daily basis, or they do not faithfully reflect the pattern of mhv rna synthesis observed in infected cells. to circumvent the short-comings of the existing in vitro mhv polymerase systems and to overcome the relative experimental limitations of intact cells, we have taken an approach similar to that used by condra and lazzarini ( ) for studying vsv replication. in this paper we report the characteristics of a permeabilized cell system and demonstrate that it incorporates ribonucle-otide triphosphates into rna molecules which appear identical to the virus-specific mrnas synthesized in mhv-infected cells. monolayer cultures of cl- cells were grown as described previously (sturman and takemoto, ; leibowitz et a/., ) . the origin and growth of the a (mhv-a ) and jhm (mhv-jhm) strains of mouse hepatitis virus have been described (robb and bond, ) . monolayer cultures of ci- cells were trypsinized and infected in suspension as described previously (robb and bond, ) at a multiplicity of infection equal to pfu per cell. infected or mock-infected cells were plated in -mm -well cluster dishes (costar) at x o cells/well in dulbecco's modified eagle's medium (dme) containing % fetal bovine serum and incubated at ". at hr postinfection actinomycin d (sigma) was added to the cultures at pg/ml to inhibit host dna-dependent rna synthesis. at the times indicated for each individual experiment, the dishes were placed on ice and washed twice with serum-free dme. buffera [ mmtris, ph . , . mm mgac,, mm kci, mm naci, mll/l rnase-free sucrose (swartz/ mann biotech)] was then added to the cultures. for our standard permeabilization conditions, synthetic lysolecithin (l-d-lysophosphatidylcholine, palmitoyl, sigma) was added to a concentration of pg/ml and the cells were held on ice for sec. lysolecithin was removed from the cultures by aspirating the buffer followed by one wash with buffer a without lysolecithin. buffer b was then added to the cultures and the cells were incubated at ". the make up of buffer b varied, as described under results, over the course of the work reported here. mm dithiothreitol, mm sucrose, pg/ml actinomycin d, . tiu/ml aprotinin (sigma), rig/ml ouabain octahydrate (sigma). [ h]ctp (icn) was added at . &i/ml yielding a final ctp concentration of . fm. following incubation at " for the indicated times ( min for most experiments), the reaction was stopped by the addition of an equal volume of % sodium dodecyl sulfate (sigma) to the cultures. polymerase activity was assayed by precipitating labeled rna from replicate cultures by the addition of trichloroacetic acid (tca) containing yo sodium pyrophosphate (sigma) to a final concentration of %. tcainsoluble precipitates were collected on glass fiber filters and extensively washed with % tca, and the tca-precipitable radioactivity was quantitated by liquid scintillation counting. extraction and electrophoresis of rna rna was extracted from cell cultures and permeabilized cells as described (wittek et al., ; cabirac eta/., ) . the cells were dissolved in m guanidium hydrochloride, . m -mercaptoethanol, and . m sodium acetate, ph . , and the dna was sheared by passage through a hypodermic needle. the rna was selectively precipitated overnight by the addition of ethanol to a concentration of %. the precipitated rna was collected by centrifugation and dissolved in mm sodium acetate, ph . , mm edta, yo sds, mg/ml proteinase k and digested at " for hr. after phenol extraction the rna was ethanol precipitated and collected by centrifugation prior to further analysis. rna to be analyzed by gel electrophoresis was dissolved in a buffer containing % formamide, % formaldehyde, mm mops [ -(iv-morpholino)propane sulfonic acid), mm sodium acetate, mm edta, ph . , and electrophoresed in . % agarose gels containing formaldehyde (lerach et a/., ) . the plasmids used in this work include (kindly provided by dr. susan weiss, university of pennsylvania), a cdna clone of mhv-a which encompasses a portion of gene all of genes and , and the 'pottion of gene (budzilowicz et al., ) . previously undescribed molecular clones of mhv-jhm used in this work are - a, a cdna clone which extends from the ' poly(a) of the genome to position in gene , a distance of bases (spaan et al., ) ; - , a cdna clone extending from the pvull site at position of gene (spaan et al. ) to the pstl site at position of gene (schmidt et a/., ) , a distance of almost . kbp; - a, which extends from nucleotide to nucleotide in gene (schmidt et a/., ) ; and - a, a clone which extends from the ddel site at position in gene into mhv gene for an additional . kbp. a molecular clone representing the barnhi fragment k ( . kbp) of the leporipoxvirus malignant rabbit fibroma virus (strayer et a/., a,b) was used as a control for some experiments. plasmids were digested with the appropriate restriction enzyme according to the manufacturer's suggested conditions. the resulting digests were electrophoresed in a o/o agarose gel and transferred to nitrocellulose as described previously (southern, ) . nitrocellulose filters were probed either with randomprimed cdna prepared with [ p]dctp using purified mhv-a virion rna as template (weiss and leibowitz, ) or with the permeabilized cell reaction products. hybridization was performed at " in /o formamide, x sspe, x denhardt's. the filters were washed twice in . x sspe, % sds at " and then washed four additional times in the same buffer at ". our initial experiments were geared toward determining the optimal conditions for permeabilizing mhvinfected cl- cells. cells were infected with mhv-a or mock-infected and incubated until approximately % of the cells were involved in syncytia. for the experiments reported here this was usually between . and . hr postinfection, a time when mhvspecific rna synthesis was maximal. at this time the cells were washed as described under materials and methods and permeabilized in buffera containing lysolecithin which was varied in concentration from to pg/ml. after permeabilization the cells were stained with trypan blue to determine the percentage of cells which had been made permeable to the dye at each lysolecithin concentration. these preliminary experiments demonstrated that a lysolecithin concentration of pg/ml permeabilized virtually all of the mhv-infected cells and greater than % of the uninfected cells without making the cells too fragile to withstand the subsequent incubations. higher concentrations of lysolecithin impaired our ability to subsequently maintain the cells for the polymerase reaction (data not shown). our standard permeabilization conditions were therefore set at pg/ml lysolecithin. once the conditions for permeabilization were es-tablished, we investigated the ability of permeabilized cells, in the presence of actinomycin d, to incorporate labeled precursors into tca-precipitable material. these initial experiments were pet-formed at a ph of . , . mh/l mg +, mm na+, mm k+, and mll/l nh,+. these conditions were based upon those used by brayton et a/. ( ) in a cell-free mhv polymerase system. permeabilized cells were incubated with [ h]utp or [ h]ctp in the presence of the three other unlabeled ribonucleotide triphosphafes, actinomycin d, and an energy regenerating system, in buffer b. the amount of tca-precipitable radioactivity was severalfold greater in mhv-infected cells than in mock-infected controls (data not shown). the synthesis of radioactivity labeled material from labeled ribonucleotide triphosphates required permeabilization; cells in which the lysolecithin treatment was omitted did not incorporate any radioactivity (table ). the tca-precipitable material synthesized in permeabilized cells was demonstrated to be rna in subsequent experiments on the basis of it being completely sensitive to rnase digestion and completely resistant to digestion with dnase ( table ) . the ability of permeabilized, mhv-infected cells to incorporate [c~-~~p]ctp into acid-precipitable material percentage activity present under control reaction condition@ -permeabilization -spermidine -creatine phosphokinase and creatine phosphate -mg'+ -mg*+, +mn*+ ( mm) -mg'+, +ca*+ ( , , or mm) -gtp -utp -utp, -gtp + rm pmsfb +aprotininb + units/ml rnasin* +ouabain, rig/ml" +ouabain, ng/mp was dependent upon adding an excess of atp as compared to the three other ribonucleotide triphosphates. an atp concentration of .o mm achieved the best results. higher levels of atp made the permeabilized cells extremely fragile and inhibited incorporation (data not shown). previous investigators using cell-free systems had demonstrated ph optima for mhv-specific rna-dependent rna polymerase activity at . , . , or . , depending upon the system used. to determine the optimum ph for measuring mhv-specific polymerase activity in permeabilized cells, infected and mock-infected cells were permeabilized and assayed for polymerase activity as described above, with the exception that the ph of buffers a and b was varied between . and . among replicate cultures. in this assay, incorporation of labeled substrate in the presence of actinomycin d into tca-precipitable material increased as the ph was raised from . to . (fig. ) . the increase of polymerase activity as the ph was raised proceeded in a step-wise fashion, with the greatest increment in activity occurring as the ph was increased from . to . . further increases in the ph from . to . had a relatively small effect upon the activity, with the greatest portion of that increase occurring as the ph was changed from . to . . the ability of permeabilized cells to synthesize actinomycin d-resistant rna remained almost constant as the ph was varied from . to . . therefore we adopted ph . for our standard reaction conditions. the magnesium requirement of mhv rna synthesis in permeabilized cells was then determined. infected and mock-infected cells were permeabilized at ph . and the magnesium concentration was varied from to mm in replicate cultures. as can be seen in fig. and in table , there was a strict requirement for mg*+ in this system. in the absence of mg*+ the polymerase activity was reduced to % of the amount observed at . mm mg*+. although mg*+ was needed for measuring the mhv polymerase activity in permeabilized cells the optimum was rather broad. the requirement for magnesium cannot be replaced by either mn*+ or ca*+ (table l) , both of which resulted in less activity than that obtained when magnesium was simply omitted from the reaction mix. a magnesium concentration of . mm was chosen for our standard reaction conditions. to further optimize the system we next investigated the monovalent cation requirements of the mhv polymerase/permeabilized cell system. infected and mockinfected cells were permeabilized as described above, except that the ph and magnesium concentration were held constant at . and . mm, respectively, and the naf, k+, and nh,+ concentrations were varied as described below. initial experiments determined that k+ and nh + seemed to be interchangeable in this sys- fig. . determination of ph optimum for mhv rna polymerase activity in permeabilized cells. cells were infected with mhva at a m.o.i. of , or mock-infected, and incubated until . hr postinfection. the cells were permeabilized as described under materials and methods. replicate cultures were assayed for mhv rna polymerase activity using the original formulation of buffer b ( mm nh&i, mm naci, mm kci, . mm mgcip, as described under materials and methods with the exception that the ph was varied between . and . among the replicate cultures. after min of incubation the assay was terminated and the amount of radioactivity incorporated into tca-precipitable material was determined. all data points represent the mean of duplicate samples. the results were calculated by subtracting the amount of radioactivity in mock-infected samples from that incorporated into mhv-infected samples under identical conditions. the results are expressed in arbitrary units with the maximum activity being set at . the cells were permeabilized and incubated in a formulation of buffer b which contained rnm tris, mm nh&i, mm naci, mm kci, ph . , as described under materials and methods with the exception that the magnesium concentration was varied between and mm among replicate cultures. at min incubation the assay was terminated and the amount of radioactivity incorporated into tca-precipitable material was determined. all data points represent the mean of duplicate samples. the results were calculated by subtracting the amount of radioactivity in mock-infected samples from that incorporated into mhv-infected samples under identical conditions. tern, na+ was required for activity, and a total monovalent cation concentration greater than rnm resulted in a decrease in polymerase activity (data not shown). the mhv polymerase activity present in permeabilized cells was relatively insensitive to monovalent cation concentrations, as long as the total monovalent cation remained below mn/l. k+ was not required; the omission of k+ from buffer b decreased activity by -l /o. at concentrations above mm na+ + k+ the polymerase activity decreased somewhat. we adopted final concentrations of mm na+ and mm k+ in buffer b for our standard reaction conditions. these concentrations were convenient to use and approximately in the center of the broad optimum concentrations of monovalent cations. the requirements of the mhv polymerase/permeabilized cell system for various cofactors were determined. as shown in table , the omission of spermidine decreased the activity to /o of that observed with the complete system. there was a requirement for an energy regenerating system; the omission of cpk and creatine phosphate decreased activity to % of control values. the system also required all four ribonucleotide triphosphates. the omission of either gtp or utp decreased incorporation of labeled ctp by and %, respectively. the omission of both utp and gtp decreased synthesis by % of that observed in the complete system. these results suggested that the activity we were detecting was not a polynucleotide terminal transferase-like activity. similarly, the ability of permeabilized cells to incorporate radiolabeled ctp as well as utp into tca-precipitable material suggests that the polymerase activity we are detecting is not due to the polyuridylate polymerase present in the cytoplasm of mammalian cells (hayashi and mcfarlane, ) . protease inhibitors and rnase inhibitors have both been reported to increase the rna-dependent rna polymerase activity present in extracts of west nile virus-infected cells (grun and brinton, ) . we therefore determined the effect of adding pmsf or aprotinin, two protease inhibitors, on the ability of permeabilized mhv-infected cells to direct the synthesis of actinomytin d-resistant rna. as shown in table , &i pmsf decreased incorporation of ctp into tca-precipitable rna by about %. however, aprotinin increased activity by about %. we attribute the different effects of these compounds to the much broader spectrum of activity of pmsf, a drug which inhibits most serine esterases (fahrney and gold, ; laskowski and sealock, ) . surprisingly, the addition of placental rnase inhibitor had little effect on rna synthesis by permeabilized cells. the effect of ouabain on the mhv polymerase/permeabilized cell system was investigated because of the dependence of the system on an exogenous energy source. ouabain is an inhibitor of the na+/k+-dependent atpase present in the plasma membrane (ruoho and kyte, ) . we reasoned that after permeabilization this enzyme might be competing with the mhv polymerase complex for atp. if this hypothesis is true we felt that the addition on an inhibitor of the atpase to the system might stimulate the mhv polymerase activity. this did appear to be the case. ouabain at concentrations of and rig/ml increased the polymerase activity to and % of that observed in controls. we could not increase the concentration of atp above mm to directly test the idea that ouabain exerted its stimulator-y effect on mhv polymerase activity by increasing the biologically effective atp concentration in our reaction since concentrations of atp greater than mn/l caused the permeabilized cells to detach from the substrate and subsequently disintegrate. we therefore included ouabain at rig/ml and aprotinin at . tiu/ml in all subsequent experiments. the tca-precipitable material synthesized in mhvinfected permeabilized cells was identified as rna by its sensitivity to rnase. it was not sensitive to dnase (table ) . to further characterize the rna synthesized in our system, we extracted rna from permeabilized cells labeled with [a- p]ctp and from parallel cultures of intact mhv-infected cells labeled with [ 'p]orthophosphate in the presence of actinomycin d. these samples were then analyzed by electrophoresis on a formaldehyde gel. the autoradiograph shown in fig. illustrates that the relative amounts and sizes of the rna species synthesized in the permeabilized cells is very similar to the mhv-specific rnas observed in intact cells. further evidence of the virus-specific nature of these rnas was obtained by southern blot hybridization. mhv-specific plasmid clones and a plasmid clone derived from the unrelated malignant rabbit fibroma virus were digested with the appropriate restriction enzyme to excise the cloned insert and resolved by agarose gel electrophoresis. the band at approximately . kbp (fig. a, lane a) represents the cloning vector pgem- . the band at approximately . kbp (fig. a, lanes be) represents pbr . the band at approximately . kbp (fig. a , lane f) represents puc . replicate filters of molecular clones representing the most ' kb of the mhv genome were hybridized with either randomprimed cdna synthesized from a purified virion rna template (fig. b) , rna extracted from mhv-infected permeabilized cells labeled with [a- p]utp after permeabilization (fig. c) , or rna prepared from mockinfected permeabilized cells. as expected, the randomprimed cdna probe hybridized to all of the mhv-specific clones (lanes a-e) and did not recognize the plasmid containing the malignant rabbit fibroma virus barnhi fragment k (lane f). the labeled rna synthesized after permeabilization of infected cells also hybridized specifically with the mhv inserts, although it did not give as strong a signal as random-primed cdna probe (fig. c) . the apparent band at about . kbp in panel c, lane e, as artifactual since no dna is present at that position in the ethidium bromide stained gel. the signal with clones - a and - a was considerably weaker than the signal obtained with the other mhv-specific inserts. we attribute these differences in signal, at least in part, to the lower amount of these two inserts present in the gel (fig. a ). this is also reflected in the relative signals obtained with the random-primed probe. the specificity of the hybridization reaction was confirmed by the lack of hybridization of mhv-infected permeabilized reaction product with an irrelevant plasmid insert (fig. c , lane g) and the failure of permeabilized cell reaction products from mock-infected cells to hybridize with these mhv clones (data not shown). additionally, the extent of hybridization of the reaction products from permeabilized mhv-infected cells to mhv cdna clones bound to nitrocellulose circles was similar to that of rna prepared by labeling intact mhvinfected cells with [ p]orthophosphate in the presence of actinomycin d (data not shown). the time course of incorporation of label in perme- cultures of mhv-infected and mock-infected cells, incubating them for . hr, permeabilizing them using the standard conditions we had developed, and labeling them for the times indicated in fig. . the accumulation of radioactivity in tca-precipitable products increases, although not in a linear fashion, over the first min of labeling. after that time the amount of tcaprecipitable radioactive product in the cells decreases dramatically. the kinetics of the development of the mhv-specific rna polymerase activity over the course of infection was determined in permeabilized cells. as shown in fig. , the accumulation of mhv rna polymerase activity in infected cells (panel a), as detected by our assay, faithfully mirrored the kinetics of actinomycin dresistant, [ h]uridine incorporation into tca-precipitable material (panel b) during a series of -hr pulses. polymerase activity is first detectable at hr postinfection in these experiments. subsequent experiments showed that the peak level of polymerase activity in permeabilized cells occurred at hr postinfection (data not shown). similar experiments with mhv-jhm yielded similar results, although polymerase activity appeared hr later than during mhv-a infection. it should be noted that the infection proceeded somewhat slower in the experiments reported here than in our previously reported work (leibowitz et a/., ) . the reasons for this discrepancy are not known at this time. in this work we report the development and characterization of a permeabilized cell system for assaying mhv-specific rna polymerase activity. this activity was characterized as to its requirements for mono-and divalent cations, requirements for an exogenous energy source, ph optimum, and its time of appearance during the course of infection. the rna products synthesized in permeabilized cells were demonstrated to be mhv-specific by agarose gel electrophoresis. the purpose of the present work was to develop and characterize a system for studying the mhv-specific rna polymerase that was more amenable to experimental manipulations than intact cells. to avoid difficulties in reproducing in vitro the intracellular environment which evolves during mhv infection we elected to pursue a path which would leave as much of the cell machinery in place as possible. the system we have developed has several advantages when compared to the cell-free systems developed by other workers. although it is difficult to compare the relative efficiencies of different systems due to the different ways in which the experimental results have been presented, we can calculate the amount of rna synthesized in our system. using the optimized reaction conditions, one mm well of mhv-infected permeabilized cells incorporated about fmol of ump into mhv-specific rna. this is estimated to be about fivefold more rna synthesis on a per cell basis than that obtained from a cell-free extract prepared from permeabilized cells (compton et a/., ) . other workers using cell-free systems have reported yields on the basis of femtomoles of ump/h/ mg protein (dennis and brian, ; mahey et a/., ; brayton et a/., brayton et a/., , . these have been in the range of - fmol/hr/mg protein. one -mm well of mhv-infected cl- cells contains about pg of protein, providing a yield on a per milligram basis which is approximately fmol of ump/mg protein/ min, a figure which makes it at least four times more efficient than the previously described cell-free systems. there are no data at this time to suggest that our system, or any other mhv polymerase assay, is capable of initiating the synthesis of new strands of rna. a second advantage of permeabilized cells for studying mhv rna synthesis is that the products synthe-sized accurately reflect the rna species synthesized in intact mhv-infected cells. all seven of the mhv mrna species are made in approximately the same ratios as they are in vivo. this contrasts with truly cell-free systems in which the rna products synthesized were not characterized as to the precise molecular species of rna synthesized (brayton et al., (brayton et al., , mahey et a/., ) or those in which the major product was genome length (compton et al., ) . although the reasons for this difference in the rna products synthesized are unknown, a possible explanation for this observation is the loss of a soluble factor responsible for regulating mhv transcription during preparation of cellfree extracts. other explanations are possible as well, and additional work is needed to identify putative factors needed for the appropriate regulation of mhv rna synthesis. the kinetics of the accumulation of polymerase activitywe observed parallels the increase of actinomycin d-resistant uridine incorporation which occurs during mhv infection. no early peak of polymerase activity or uridine incorporation in intact cells was detected. in this regard our results are similar to those of compton et al. ( ) and sawicki and sawicki ( ) . these results differ from those of earlier workers (brayton et a/., (brayton et a/., , who detected a peak of polymerase activity at hr postinfection followed by a fall in activity prior to a subsequent increase to maximal levels. the reasons for these differences is not known. it could relate to the different cell lines used by different laboratories ham post hktbl ham post hfectii fig. . the accumulation of mhv-specific rna polymerase activity during infection. replicate cultures of ci- cells were infected with mhva (m) or mock-infected (a) and incubated for hr. at that time, and at hourly intervals thereafter, duplicate sets of cultures were either permeabilized and assayed forthe incorporation of radioactive ctp into tca-precipitable material under standard reaction conditions (a) or exposed to actinomycin d ( pg/ml) for min and labeled with &i of [ h]uridine for hr and then solublized with sds and assayed for incorporation of label into tca-precipitable material (b). or be related to the vastly different reaction conditions that are employed by the different methods of assaying mhv polymerase activity. the system we have described for assaying the mhv-induced rna-dependent rna polymerase activity should prove useful for other workers. it is simple to set up, provides a system which should be amenable to pulse-chase-type experiments, and furnishes a systern where macromolecules such as rna templates or purified proteins can be added and their effect on mhv synthesis observed. it may also serve as a point of departure for developing a completely cell-free system which better reflects the intracellular synthesis of mhv rnas than the currently available systems. characterization of leader-related small rnas in coronavirus-infected cells: further evidence for leader-primed mechanism of transcription completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus characterization of two rna 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rna the virus-specific intracellular rna species of two murine coronaviruses: mhv-a and mhv-jhm rna molecularweight determinations of gel electrophoresis under denaturing conditions, a critical reexamination biological properties of avian coronavirus rna rna-dependent rna polymerase activity in murine coronavirusinfected cells pathogenic murine coronaviruses. i. characterization of biologic behavior in vitro and virusspecific intracellular rnaof strongly neurotropic jhmv and weakly neurotropic a v viruses photoaffinity labeling of the ouabainbinding site on (na+ + k+) adenosine triphosphatase. proc. nat coronavirus minus-strand rna synthesis and effect of cycloheximide on coronavirus rna synthesis nucleotide sequence of the gene encoding the surface projection glycoprotein of coronavirus mhv-jhm. detection of specific sequences among dna fragments separated by gel electrophoresis coronavirus mrna synthesis involves fusion of non-contiguous sequences isolation and identification of virus-specific mrnas in cells infected with mouse hepatitis virus (mhv-a ) sequence relationships between the genome and the intracellular rna species , , , and of mouse hepatitis virus strain a coronavirus multiplication strategy. i. identification and characterization of virus-specified rna coronavirus multiplication strategy. ii. mapping the avian infectious bronchitis virus intracellular rna species to the genome malignant rabbit fibroma virus: observations on the culture and histopathologic characteristics of a new virus-induced rabbit tumor malignant rabbit fibroma virus causes secondary immunosupression in rabbits enhanced growth of a murine coronavirus in transformed mouse cells characterization of murine coronavirus rna by hybridization with virus-specific cdna probes mapping of a gene coding for a major late structural polypeptide on the vaccinia virus genome polyadenylate in the virion rna of mouse hepatitis virus acknowledgments merase gene product in cells infected with murine coronavirus a . virology , - . dennis, d. e., and brian, d. a. ( ) . rna-dependent rna polymerase activity in coronavirus-infected cells. j. viral. , -l . fahrney, d. e., and gold, a. m. ( ) . sulfonylfluorides.i. rates of reaction with acetylcholinesterases, a-chymotrypsin and trypsin. . amer. chem. sot. , -l . grun, . b., and brinton, m. a. ( ) key: cord- -bq u d z authors: phillips, joanna j.; chua, mingming; seo, su-hun; weiss, susan r. title: multiple regions of the murine coronavirus spike glycoprotein influence neurovirulence date: journal: j neurovirol doi: . / sha: doc_id: cord_uid: bq u d z the spike (s) glycoprotein of mouse hepatitis virus (mhv) is a major determinant of neurovirulence. using targeted recombination we previously demonstrated that the s gene of the highly neurovirulent mhv- conferred a dramatic increase in neurovirulence to the mildly neurovirulent mhv-a . to identify the genetic determinants of neurovirulence within the mhv- spike, we generated isogenic recombinant viruses containing various mhv- /mhv-a chimeric spike genes, and studied their phenotypes in vivo. the mhv- /mhv-a chimeric spike genes consisted of either reciprocal exchanges between the s and s spike subunits, or smaller exchanges specifically in the hyper-variable region (hvr) of s . the chimeric spike gene containing recombinants all exhibited efficient replication in vitro, yet many were severely attenuated for virulence in vivo. furthermore, these attenuated recombinants exhibited decreased titers of infectious virus in the brain relative to the parental recombinant viruses containing the full-length mhv- or mhv-a spike genes. this is the first report that compares the neurovirulence and pathogenesis of isogenic viruses with defined alterations in the mhv spike protein. from these studies, it appears that the interactions of multiple regions of the mhv spike, including the hvr, act in concert to allow for efficient infection of and virulence in the murine central nervous system. murine coronaviruses, or mouse hepatitis viruses (mhv) , are enveloped positive-stranded rnaviruses that can induce a variety of respiratory, gastrointestinal, and neurologic diseases in rodents. as with a number of viral pathogens, the severity and organ tropism of the disease depend, in part, on the viral strain. infection with the highly neurovirulent mhv type (mhv- , a strain jhm isolate) results in a potentially fatal acute encephalomyelitis (dalziel et al, ) . infection with a similar dose of the mildly neurovirulent mhv type a (mhv-a ) results in amuch less severe disease with only mild encephalomyelitis and virtually no mortality (lavi et al, ; lavi et , ) . recently, using targeted recombination, we have demonstrated that the spike gene ofmhv- is sufficient to confer this highly neurovirulent phenotype to mhv-a (phillips et al, ) . the mhv spike or s glycoprotein, expressed on the virion envelope and on the plasma membrane of infected cells, plays a vital role in viral entry, viral spread, and in the immune response to infection (collins et , ; buchmeier et al, ; dveksler etal, ; castro and perlman, ; bergmann etal, ) . depending both on the viral strain and the host cell the spike is posttranslationally cleaved dividing the spike into an amino-terminal, s , subunit and a carboxy-terminal, s , subunit (sturman et , ) . sl, thought to form the globular head of the spike, mediates binding to the viral receptors (collins et , ; degroot et al, ; kubo et , ) that are members of the ceacam(previously bgp (beauchemin et al, ) ) subclass of the jj f)hillips et ill figure i schemutic diagram of the spike gene from the recombinant viruses and their corresponding virulence following intracranial inoculation. ldc,o assays were conducted as descnhcd previously (hingley at , t ( ). the recombinant viruses are represented as indicated. ,\ ,r and "r contain the mhv-i\ spike and the mhv- spike, respectively, r and contain the of the 'v l- v- spike with the of the mhv-t\ spike, and ,r and contains the oflvlhv-t\ and the of mhv- . .; .hvri go and gi represent recombinants that have a z-amin o acid deletion in the !\-lhv· hypervariuble region (hvrj. while s hv-!\ r :j and r contain an exchange of the ivmv- i-ive for the corresponding mhv'f\ region. asohv- r and r contain the mhv-t\ ! spike with the i- vr of mi-iv- . the amino acid residues at the exchange sites are indicated by their position in the mhv- spike. recombinant .,r contained a single coding mutation lgg q. and recombinants ,hv-a r and r each contained a single coding mutation and n y, respectively. grey bars imlicalc the lvlhv-i\ sequence, and closed bill's inrlicate thu mhv- seqnonr.e. the -amino acid deletion in the ivii-lv·a i!vr is indicated. to begin to identify specific regions of the mi-i)' spike which playa role in neurovirulence, we generated recombinant viruses containing exchanges between the s and s subunits of the highly neurovirulent mhv- spike and the much less ncurovirulent lvihv-a spike, the recombinants were generated using a modification of the targeted recombination strategy as recently described by kuo ei ( ) (see materials and methods). to reduce the risk of secondary site rnutations influencing the viral phenotype, two independently selentcd recombinants (r), as indicated by their numbering, were isolated and characterized for each desired recombinant virus. as shown in figure , the recombinants containing the st of mhv- and the s of mi-iv-a were named s r ancl s r , and the recombinants containing the s of lvihv-i\ ~j and the s of mhv- were called r and s r . the s / junction was located just ' to the cleavage site at amino acid (see materials and castro and perlman, ; borgmann et , ) . despite the dramatic differences in ncurovirulenco conferred by spike proteins of lvii-iv- and lvihv-a they are highly homologous proteins, sharing % amino acid identity in and % identity in s . within the s subunit of the loss nourovirulent mhv-a spike, however, there is deletion of amino acids in a region termed tho hypervariable region (hvr) (luytjes ei ol, ) . the spike hvr, loosely defined between amino acids and , exhibits a great deal of variation in length from strain to strain (parker et al, ; banner et , ) . the highly neurovirulenl lvii-iva has the longest known hvr, but deletions in this region of up to amino acids have been reported (parker et , ) . the specific role of the hvr in pathogenesis appears to be complex and multifunctional. deletions or mutatiuns in this region have been found to alter viral fusion, eliminate neutralizing monoclonal antibody epitopes, and abrogate cd + t-cell epitopes (dalziel or , ; fleming et , ; wege et ol, ; parker et ol, ; taguchi and fleming, ; gallagher ei al, ; pewe et , ; tsai et , ; wang et al, ) . in addition, deletions or mutations in the hvr have been associated with neuroattenuation. in one such study, a -arriino acid deletion in the i-ivr was associated with decreased neurovirulence and decreased viral spread in the cns (fazakorloy et ai, ) . with the recent development of targeted rna recombination the structural gones of lvii-iv have become accessible, for the first time, to genetic manipulation (fischer et , ; leparc-goffart et , ) . using this technique we have definitively demonstrated that the s gene is a major determinant ofmhv neurovirulence (phillips et , ( ) . to further identify genetic determinants of neuroviru lence within the lvihv spike, we generated a series of recombinants containing mhv- /mi-iv-i\ chimeric spike genes, and examinod the pathogenesis of these isogenic viruses that differed exclusively in spike. from these studies, we conclude that multiple regions of the mhv- spike, including the hypervariable region, are required for efficient infection and virulence in the mouse cns. methods). as controls, recombinants containing the full-length spike gene ofmhv- , s r , or mhv-a , sa r , were isolated and characterized in parallel. to further decrease the impact of secondary site mutations in the recombinant viruses, we sequenced the entire s gene from at least one of each pair of recombinants. s r , sa r , and s r contained no secondary site mutations in spike. s r contained a single point mutation in the si subunit that resulted in a codon change l q. an independently isolated recombinant virus, s r , contained no secondary site mutations in s. to examine the efficiency of replication of the chimeric spike gene recombinants we performed growth curves on l cell monolayers with s r , s r , s r , and sa r . the results are shown in figure a . consistent with previous reports, recombinants containing the mhv-as spike, sa r, replicated to higher titer in cell culture than recombinants containing the mhv- spike, s r (phillips et al, ) . both chimeric spike gene recombinants, s r and s r , replicated in cell culture, and exhibited similar kinetics as the two parental recombinants, sa r and s r.the titers of infectious virus, however, were intermediate between sa yr and s r. the plaque morphology of the chimeric spike gene recombinants was also intermediate between sa yr and s r. at h after infection of l cell monolayers, the s recombinants and the s recombinants exhibited a medium-plaque phenotype ( . mm and . mm, respectively) as compared to the large-plaque phenotype produced by sa r ( . mm) and the small-plaque phenotype produced by s r ( . mm), thus, the sl/s chimeric spike gene recombinants replicated efficiently in cell culture, but exhibited a slightly different in vitro phenotype from either of the parental mhv- or mhv-a spike containing recombinants. o determine if either the sl or the s subunit of the mhv- spike was sufficient to confer an increase in neurovirulence, we infected mice intracranially with s r , s r , s r , s r , s r . and sas r , monitored for lethality, and calculated ldso values. the neurovirulence of each recombinant is shown in figure . the ldsos for the s and sas recombinants were similar to those reported previously (phillips et ol, ) . the virulence of the chimeric s gene recombinants, however, was very different from either of the parental viruses. both s and s recombinants were highly attenuated for virulence. consistent with this decrease in virulence, mice exhibited clinical signs such as hunched posture, ruffled fur, arid abnormal gait only at high doses of virus [» pfu) . to determine if the reduced virulence of the chimeric spike gene recombinants was attributable to decreased viral .raplication in the brain, we titered brain homogenates for infectious, virus at various days after intracranial inoculation with pfu of virus (see materials and methods). the results are shown in figure . as we have shown previously, the titers of infectious virus in the brain were similar following inoculation with s r and sa r despite the dramatic difference in virulence exhibited by the two recombinant viruses (phillips et al, ) . the two chimeric s gene recombinants, however. exhibited markedly reduced virus titers. s r exhibited low levels of virus while the amount of infectious s r virus was at or below the level of detection ( pfu/g). thus, despite the high amino acid identity between the mhv- and mhv-a spike proteins they could not substitute functionally for one another in an in vivo infection. this result suggests that the high neurovirulence conferred by the mhv- spike protein requires specific homotypic interactions between regions in and , and that disruption of these interactions has a major impact on the pathogenesis of the virus, within the subunit there is a region, termed the hypervariable region (hvr), which can tolerate extraordinary variation particularly with respect to its length (banner et al, ; parker et al, ) . it has been proposed that the hvr has a vital function in mhv pathogenesis. numerous variant viruses have been identified in which mutations or deletions in this region have been associated with alterations in neuropathogenesis (dalziel et al, ; fleming et al, ; wege et ol, ; parker et al, ; taguchi and fleming, ; gallagher et al, ; wang et al, ) . to definitively assess the role of the hypervariable region (hvr) in neurovirulence, we used targeted recombination to generate a series of recombinant viruses with small exchanges in the hvr between mhv- and mhv-a . to determine if the hvr of the highly neurovirulent mhv- spike was necessary to confer an increase in neurovirulence, we generated recombinants in which the mhv- hvr was deleted or replaced with the mhv-a hvr (see figure ). deletion of the mhv- hvr was based on a previously identified neutralizing monoclonal antibody escape mutant with a deletion in the hvr, and the two independently isolated recombinants containing this amino acid deletion were named ahvr and s ahvr (dalziel et al, ; parker et al, ) . recombinants s hv-a r and s hv-a r contained the mhv- gene with replacement of a l z-amino acid region in the hvr with the corresponding -amino acid region from the mhv-a s gene. in addition, to examine if the mhv- hvr was sufficient to confer some degree of increased neurovirulence we replaced themhv-a hvrwith the mhv- hvr (see figure ). thus, recombinants a hv- r and sa hv- r contained the mhv-a spike with replacement of a -amino acid region in the hvr with the corresponding -amino acid region from the mhv- spike. once again, we sequenced the entire gene from at least one recombinant of each pair of recombinants. recombinants s ahvr and sa hv- r contained no secondary site mutations. sequencing of recombinant hv-as r , containing the mhv- spike with the mhv-a spike hvr, revealed a single co~ing mutation s . interestingly we were unable to select a hv-a r recombinant without a secondary site mutation. s hv-a r , an independently isolated recombinant, contained a single coding mutation n y, and two additional recombinants, s hv-as r and r , were partially sequenced and found to contain single coding mutations (l f and l q, respectively). these two recombinants were not further characterized. the inability to select s hv-as recombinants without secondary site mutations in s suggests that these mutations were selected for during replication in cell culture. the replication efficiency of these recombinants on l cell monolayers is shown in figure b . the recombinants all exhibited similar kinetics of viral replication; however, the extent to which they replicated differed. in general, the growth phenotype of the recombinants segregated with the spike gene. recombinants with primarily mhv- spike sequence, ahvr and s hv-a r, replicated to a similar extent as s r, and the hvr recombinant with primarily mhv-a spike sequence, sa hv r, replicated to a similar extent as s a r. the plaque morphology of the recombinants was also dependent on the spike gene. hpj. of l cell monolayers, sa r, and sa hv- r exhibited a large-plaque morphology ( . rom). the recombinants with primarily the mhv- spike exhibited smaller plaque-phenotypes. s hv-as r exhibited an intermediate-plaque size ( . mm), and s ahvrexhibited a delayed plaque" phenotype as plaques were not yet visible at h p.i, the delay in plaque-formation observed with s ahvr was consistent with previous reports associating deletions in the mhv- hvr with delayed fusion and decreased cytopathicity (gallagher et al, ) . in general, recombinants with alterations in the hypervariable region exhibited similar properties in cell culture as the parental spike from which they were derived. to determine the effect of mutations in the hvr on virulence, we inoculated animals intracranially with serial dilutions of the recombinant viruses, observed them for lethality, and then calculated ld doses. the results are shown in figure . removal of the mhv- hvr, either by deletion, s ahvr, or by replacement with the mhv-a hvr, hv-as r, resulted in recombinant viruses with attenuated virulence. thus the mhv- hvr was necessary for the highly neurovirulent phenotype conferred by the mhv- spike. in contrast, insertion of the mhv- hvrinto the mhv-a spike, sa hv- , did not alter the virulence of the recombinant viruses indicating that the mhv- hvr was not sufficient to confer an increase in virulence to the mhv-as spike. in addition, the similar virulence exhibited by sa r and the two recombinants sa hv- r and r indicate that an insertion of amino acids is well tolerated in the mhv-a hvr. to determine the effect of alterations in the hvr on the efficiency of viral replication in the brain, we inoculated mice intracranially with the hvr recombinants, and determined the titers of infectious virus in the brain at various times p.i. (figure ). consis-. tent with their poor virulence, the mhv- gene containing recombinants with deletions or exchanges in the hvr'exhibited dramatically reduced virus titers in the brain which were at or below the level of rao and gall<lglwr, ' mj ). to examine tho fusion phenotype of t!ie recombinant viruses following virus infection \'\' pnrl'llrmccl c ~cell fusion assays on l cell monolayers (sec materials and methods). by h p.i., all of the recombinant viruses had induced syncytia formation, bul at early times postinfection, there were clearly differences among the recornbinanl viruses with respect to their ability to induce cell-cell fusion. the percent fusion relative ( s h. was found to he similar at and h p.i.: the results at hours are shown in figure . similar levels of cell-cell fusion were observed in ., cells infected with the l\vo parental recombinants, \ and si\s r, and the severely attenuated recombinant \, expressing the s ' of mhv-a and the of me\!- . in contrast, recombinants expressing spikes wi th alterations in the hvr, s l'-.hvr, s hv-a r, and sasghv- r, or with pairing of the s subunit ofmhv- with the sz subunit of mhv-a , r, all exhibited some degree) of delay in cell-cell fusion. deletion of the mhv- i-ivrresulted in the most dramalic decrease in fusion, while replacement of the mhv-a hvr with the mhv- hvr resulted in only a moderate decfcase in fusion. the delayed fusion formation observed with s l'-.hvr is consistent with the observation by gallagher et al ( ) that deletions in the hvr can result in decreased fusion. thus, the neuruauenuaicd virulence observed v"ith some but virus figure (al cell-cell fusion assay on lz cell monolayers. cells were infected at a multiplicity of infection of . or . pfu/cell with s..rz!), s""grig, s r , s ,r , sa nhv- r . s hv- \ r , and s.,t.hvr . at h p.i., cells were fixed. using phase-contrast microscopy and a zox-objective the total nurnbel' of nuclei in svncvtiu per field was determined for at least random fields per g-well plate. the percent fusion relative to that observed with s rz\] w[ls calculated for each experiment and the means (and standard deviations) are shown. figure , and is shown here for comparison. the limit of dptection was zoo pfu/g of tissue. on day pi., the mean titers of infectious virus in ,hz!) inffjctccl mice was signifir.anlly gruahlr than in s hv-a r -or s cl.]-lvr -infecter\ mice \wo-taiibli r-test, p> . for both). detection ( pfu/g). on days and p.i. with s ~hvr or s -iv-a r the titers of infectious virus were significantly different from the titers following infection with s r (two-tailed r-test, p > . ). in contrast, replacement of the mhv-a hvr with the corresponding region from mhv- did not ' su [ in a dramatic reduction in the amount of infectious virus recovered from the brain. although sa ~hv- r exhibited similar average liters of infectious virus as s,wjr, there was a large degree of animal to animal variation as indicated in figure . on clay p.i., some :animals exhibited relatively high titers of infectious virus. to demonstrate that these high tilers late in infection were not attributable to the replication of variant viruses with secondary site mutations in the spike, we amplified virus from the brain al days p.i., and sequenced the spike gene to generate a coni sensus sequence. no secondary site mutations were founel in the spike gene. despite efficient replication and growth in cell culture most of the chimeric spike containing recombinant viruses exhibited attenuated virulence in vivo. due to the numerous selective pressures present during an in vivo infection it is possible that small alterations jn viral entry, spread, or replication, which were not readily observable in cell culture, had a profound impact on virulence. previous studies have found an association between alterations in the abilty of the mhv spike to induce fusion and a terations iii pathogenesis (frana et ol, ; gallagher et al, ; gallagher et al, ; gallagher ei al, ; multiple regionsof spike influenceneurovirulence jj phillips et al not all of the recombinants may be associated with decreased cell-cell fusion in cell culture. with the development of targeted recombination the structural genes of mhv have become amenable to genetic manipulation. using this technique we previously demonstrated that the spike gene of the highly neurovirulent mhv- virus can confer a dramatic increase in virulence to the much less virulent mhv-a virus (phillips et , ) . here we report on studies designed to localize this phenotype to a specific region of the spike, and to examine the effect of specific alterations in the spike on pathogenesis. using targeted recombination, we generated a series of recombinants containing mhv- /mhv-a chimeric spike genes. we first characterized their in vitro phenotype to ensure that the chimeric spike genes were functional for infection and replication in tissue culture. we then examined the in vivo phenotype of the recombinants. the two parental recombinants, r (containing the mhv- spike gene) and as r (containing the mhv-a spike gene), exhibit similar kinetics of replication in cell culture, but as r achieves higher peak titers than r. in cell culture, all of the chimeric spike gene recombinants replicated efficiently. they exhibited similar kinetics of replication as r and as r, and their peak titers were similar to or intermediate between the high titers observed with as r and the lower titers observed with r. when we infected mice with the recombinant viruses, however, we observed that many of the recombinants displayed a different phenotype from either of the two parental recombinants. recombinants containing exchanges between the and subunits ( r or r) or deletion or replacement of the mhv- spike hypervariable region ( ~hvr or hv-a r, respectively) exhibited a profound decrease in viral replication in the brain as demonstrated by the titers of infectious virus. furthermore, these recombinants were highly attenuated for neurovirulence as demonstrated by their high intracranial ld so values. the high titers of infectious virus in the brains of mice infected with recombinant a hv- r, containing the mhv-a spike with the mhv- spike hvr, demonstrated that not all of the chimeric spike gene recombinants exhibited attenuated replication. in addition, the virulence of as hv- r was not attenuated as it was similar to the parental sa r rec~mbinant. as illustrated by the two parental recombinants decreased neurovirulence is not always associated with decreased titers of infectious virus in the brain. s r and sas r exhibit a iog difference in intracranial ld so, yet when mice are infected with the same dose of virus, s rand as r exhibit similar titers of infectious virus in the brain. the chimeric spike gene recombinants, however, demonstrate that alterations in both sl and s can result in reduced viral titers in the brain, and that this decrease in viral replication in the brain is associated with decreased neurovirulence. the neuroattenuated phenotype of recombinants containing /s exchanges between the mhv- and mhv-a genes suggest that under the stringent conditions of an in vivo infection homotypic /s interactions are required for efficient replication in the ens. moreover, in combination with previous studies associating mutations in si and with neuroattenuation, these data suggest that regions within both subunits of the mhv- spike are required to confer the highly neurovirulent phenotype (parker et al, ; wang et al, ; grosse and siddell, ; aeki et , ) . these regions of spike, which when mutated, deleted, or replaced influence neuropathogenesis, may interact structurally in the tertiary conformation of the protein, or cooperate functionally for efficient functioning of spike. there is experimental evidence to support the idea that regions of and s closely interact. variant viruses have been identified in which mutations in s'l, responsible for receptor binding, alter fusion, and mutations in s , responsible for viral fusion, alter the conformation of sl or confer resistance to neutralization by soluble receptor (gallagher et al, ; grosse and siddell, ; saeki et al, ) . although the spike proteins of mhv- and mhv-a share a high degree of amino acid homology, the severely attenuated phenotype of the recombinants with chimeric spike genes indicate that there are critical differences between the two spike genes in both sl and s . as has been demonstrated with theiler's virus (pritchard et al, ; zhang et al, ) , the in vivo attenuation observed with viruses containing chimeric viral proteins between similar strains demonstrates the complexity and sequence specificity of viral protein interactions, and attests to the stringent constraints imposed on a virus in vivo. additional recombinants containing alterations exclusively in the mhv- hypervariable region (hvr) demonstrated that this region of sl is required for the high neurovirulence of the mhv- spike. deletion or replacement of the mhv- hvr resulted in neuroattenuation of the recombinant viruses. these results definitively confirm the data from previous studies suggesting an association between mutations or deletions in the mhv- hvr and neuroattenuation (dalziel et al, ; fleming et al, ; wege et al, ; parker et al, ; taguchi and fleming, ; gallagher et al, ; wang et al, ) . although no single function has been attributed to the hvr, studies of variant viruses suggest that alterations in this region can influence - interactions and fusion (gallagher et al, ; gallagher, ) . in ad-'dition, the mhv- spike hvr contains neutralizing antibody epitopes and t-cell epitopes (dalziel et ai, ; taguchi and fleming, ; gallagher et al, ; castro and perlman, ) , and thus alterations in this region could also influence the immune response to infection. due to the severely attenuated in vivo replication and virulence exhibited by recombinants with deletion or replacement of the mhv- spike hvr, however, it appears that this region is critical for the in vivo functioning of the mhv- spike. in contrast, replacement of the mhv-a spike hvr with the corresponding region from the mhv· spike demonstrated that the hvr of mhv- was not sufficient to confer an increase in neurovirulence. interestingly, the presence of the mhv- hvr in the mhv-as spike did not.appear to alter the ld so of the resulting recombinant virus. thus, not only could the mhv- hvr grossly substitute for the corresponding region ofmhv-a , but the mhv-a spike hvr was able to tolerate an insertion of s amino acids with no obvious effect on neurovirulence. to identify more subtle alterations or defects in the mhv- /mhv-as chimeric spike proteins we tested a number of their in vitro properties. previous studies have suggested that the fusogenicity of the mhv spike is an important factor in both viral spread in the host and in virus-induced cytotoxicity (gallagher et al, ; gallagher et ol, ; rao and gallagher, ) . to determine if the spike proteins of the attenuated recombinants exhibited an altered ability to induce cell-cell fusion we performed cell-cell fusion assays. four of the recombinants (s r , s ~hvr . s hv-as r , and sa hv- r ) exhibited some degree of delayed fusion. although not all of the attenuated recombinants exhibited decreased cell-cell fusion, it appeared that a severe reduction in cellcell fusion was correlated with neuroattenuation. interestingly, the slight decrease in cell-cell fusion observed with the recombinants containing a replacement of the mhv-as hvr with the mhv- hvr did not result in a corresponding decrease in virulence. this result suggests that mhv infection and replication in the ens may require a certain threshold level offusion. although not all viruses with fusion defects exhibit neuroattenuation ( st hingley, unpublished data) , decreased fusogenicity of the mhv spike has previously been found to associate with decreased spread in the cns (gallagher et al, ; fazakerley et al, ) . thus for some ofthe recombinant viruses decreased fusion resulting in decreased spread and dissemination, may contribute to the decreased replication observed in the cns. in additio,n to cell-cell fusion we also examined the temperature sensitivity and the thermostability of the recombinant viruses. the temperature sensitivity of tj:le recombinants, as determined by the plating effi-c~ency at . °c compared to at . °c, revealed no differences between the highly virulent and highly atteuuated recombinant viruses (data not shown). the thermo stability of the recombinant viruses was also examined by determining the decrease in viral stock titers over time at °c, ph . (data not shown). the neuroattenuated recombinants fell into a range of thermostabilities, however, they were bounded by the highly thermostabile sa r and much less fuermostabile r (data not shown). thus. there was no clear correlation between thermostability and neurovirulence. due to the many selective pressures inherent in an in vivo infection. small differences in the kinetics of an infection can dramatically affect pathogenesis. such differences may explain why many of the recombinants with alterations in the spike displayed efficient replication in cell culture. yet highly inefficient replication in vivo. by creating recombinant viruses with heterotypic sl/s interactions we may have inadvertently altered either structural or functional interactions between the and subunits. these perturbations in spike may not have been apparent in cell culture due to differences between in vitro and in vivo infection such as receptor density on the host cell, the availability of free virus, the virion density required for an efficient infection, and the fusogenicity of the host cell. it is also possible that there were slight alterations in the processing or expression of the chimeric spike proteins that were not apparent in vitro. the lack of a correlation between replication in cell culture and replication in vivo is not unique to the chimeric spike gene recombinants as it has been observed previously for other mhv strains and for other viruses (gallagher et ol, ; pritchard et al, ) . the targeted recombination technique is a powerful tool for studying mhv pathogenesis as defined alterations in spike can be introduced into isogenic viruses. as with all strains of mhv, point mutations can arise during viral replication. to decrease the impact of such secondary site mutations in the recombinant viruses we selected and characterized two independent recombinants for each desired recombinant, and sequenced the entire s gene from at least one recombinant. interestingly, s hv-a r was the only recombinant for which multiple. independent recombinants were found to contain single coding mutations in s after minimal passage in cell culture. the location of one of these compensatory mutations identified in s (ll f) is striking as numerous studies have identified alterations in this exact residue. the codon has been identified by gallagher et al ( ) (l r) as one of three amino acids responsible for conferring ph-dependent fusion on mhv- , by saeki et aj ( ) (l f) in the spike gene of soluble-receptor resistant mutants, and by wang et a ( ) (ll f) in a neuroattenuated monoclonal antibody escape mutant. it is interesting to speculate that this region of s plays a prominent role in the conformation of spike and perhaps in the dynamic interaction that occurs between s and s . the identification of compensatory mutations that arise both in vivo and in vitro as a result of various alterations in spike may provide valuable clues as to which regions of spike undergo functional and/or structural interactions. the spike of mhv is a major determinant of neurovirulence. this is the first report using targeted recombination to examine the role of specific regions of the mhv spike in neuropathogenesis. despite efficient replication in cell culture many of the mhv- /mhv-a chimeric s gene recombinants displayed severely attenuated replication in vivo. thus, the degree of amino acid homology between the mhv- and mhv-a spike glycoproteins was high enough for the chimeric spike gene recombinants to replicate efficiently in vitro, yet not high enough for efficient replication in the murine ens. in addition, recombinants with alterations in the spike hvr have confirmed the importance of this region in the highly neurovirulent phenotype conferred by mhv- spike. thus the pronounced neurovirulence conferred by the mhv- spike appears to depend on the structural and/or functional interaction of multiple regions of spike rather than on discreet independent functional domains within spike. feline fcwf cells, fmhv, and aim were obtained from paul s masters (albany, ny). the fmhv virus is a recombinant mhv that contains the ectodomain of the s protein of feline infectious peritonitis virus, the' alb replicase, and the mhv-a '-end (kuo et al, ) . aim, derived from mhv-a , is a temperature-sensitive n gene deletion mutant that produces small plaques at the non-permissive temperature ( °c) and is thermolabile (koetzner et al, ) . viruses were propagated on either murine ci-l cells or feline fcwf cells, and plaque assays and purifications were carried out on murine l cells. cells were maintained on plastic tissue culture flasks in dulbecco's minimal essential medium (mem) with % fetal bovine serum (fbs). spinner cultures of l cells were maintained in [oklik's mem with % fbs at densities of between x and x cells per ml. the pmh plasmid (obtained from paul s masters) includes from codon of the he pseudogene through to the ' end of the mhv-a genome as described .by kuo et a ( ) . the pgem z plasmid containing the mhv- gene, p wt, was obtained from michael j buchmeier (lalolla, cal (gallagher et al, ) . the pgem-s 'and pgem-a plasmids were derived from pswt and contained the mhv- and the mhv-a spike genes, respectively (phillips et al, ) . these plasmids also contained silent mutations at codons and of the s gene to create an avril site, and at nt past the s gene stop codon to create a sbfl site. these restriction sites were used for shuttling the various spike genes into the pmh vector. to generate the various exchanges in thels gene, coding-silent changes were introduced into the mhv· and mhv-a spike genes in pgem-s or pgem-a , respectively, that created unique andlor common restriction sites. dna manipulations were carried out using standard methods (maniatis et al, ) . all cloning sites and regions generated by per mutagenesis were verified by sequencing. and the composition of each construct was checked by restriction analysis. to exchange the mhv- and mhv-a and s subunits an ecorv site was introduced at codon (mhv- sequence). avrll/ecorv or ecorv/sbfi segments were then exchanged between the mhv- and mhv-a spikes. the resulting plasmids, pg-s , containing the sl of mhv- and the s of mhv-a , and pg- , containing the sl of mhv-a and the of mhv- , were digested with avrll/sbfi and the chimeric s genes were shuttled into the pmh plasmid creating pmh - and pmh , , a s gene with the mhv-a spike and the mhv- hvr was generated by introducing silent mutations into the mhv- spike creating a pstl site at codon and a kpni site at codon . using these two sites the resulting -nt mhv- fragment was cloned into the mhv-a spike creating plasmid pg-s a hv- . the avrll/sbfi segment from this' plasmid was then cloned into pmh creating plas mid pmh -sa hv- . the mhv-a hvr was in troduced into the mhv- spike by introducing silenm utations in the mhv-a spike at codons aug! creating an ecor ill site and a bglll site, respeca tively. for cloning purposes a silent mutation creati! ing a bglll site was also introduced into the mhv.{ } s gene at codon . using the ec ill and bgin! restriction sites the mhv-a hvr was cloned intg: the corresponding region of the mhv- spike create ing plasmid pg-s hv-a . the avrll/sbfl segment from this plasmid was then cloned into pmh creã ting pmh -s hv-a . deletion of the mhv- h\lb was based on a previously identified deletion in wy. mhv spike (dalziel et ol, ; parker et al, ): the -amino acid deletion between codons jl: and and codon substitution at k n was crã ted using pcr mutagenesis in pgem-s . the rer' sulting plasmid, pg-s ~hv, was used to shuttle thà vrll/sbfl region of s into pmh creating pmh a for rna transcription the designated pmh pla~' mids were linearized just ' to the poly(a) tail by di· gestion with pacl, targeted rna recombination and selection of recombinants , targeted recombination was carried out, as describe, by kuo ( ) , between the recipient virus fmhvan· synthetic capped rnas transcribed from. pmh - , pmh , pmh -s , pmh -s , pmh -s hv-a , and pmh -s . . . recombination with rna transcribed from pmh -sas hv- was carried out with the alb recipient virus as described previously (masters et al, ; leparc-goffart et ai, ; phillips et al, ) . the potential recombinants were plaque-purified and screened, using pcr amplification and gel electrophoresis, for the presence of the ' portion of the recombinant s gene (fischer et ai, ; leparc-goffart et ol, ; phillips et al, ; kuo et al, ) . the selected recombinants were then plaque-purified once again, and viral stocks were grown on ci- cells for further analysis (fischer et al, ) . for each desired recombinant virus at least two recombinants derived from independent recombination events were characterized. s gene sequencing for sequencing of the gene, cytoplasmic rna extracted from virus-infected l cells was used as a template for reverse transcriptase-mediated pcr (rt-pcr) amplification. the oligonucleotides described previously (leparc-goffart et , ; phillips et al, ) were used for amplification of the designated regions. double-stranded per products were analyzed by automated sequencing by the taq dye terminator procedure according to the manufacturer's protocol (taq dyedeoxy terminator cycle sequencing kit; applied biosystems). the entire s gene was sequenced from at least one member of each pair of recombinant viruses. the sequence of each s gene was identical to the previously published sequences for the mhv- and mhv-a s genes parker et , ; gallagher et al, ; phillips et al, ) except for recombinants containing the ofmhv- , s r, and recombinants containing the mhv- spike with the mhv-a hypervariable region, s hv-a r. s r contained a single point mutation in the subunit that resulted in a codon change l q. s r , an independently isolated recombinant virus, contained no secondary site mutations in s. s hv-a r contained a single coding mutation s . an independently isolated recombinant, s hv-a r , also contained a single coding mutation n y. two additional recombinants, s hv-a r and r , were partially sequenced and found to contain single coding mutations (l f and l q, respectively). virus growth curves onfluent l cell monolayers in -well plates werẽ fected with each virus at the indicated multiplic-ity of infection. infections were carried out in duplicate. following adsorption for h at °c, the cells were washed with tris-buffered saline three times and then fed with . ml ofdmem-l % fbs. at the specified times, the cells were lysed by three cycles of freeze-thawing, and the supernatants were removed and titered by plaque assay on l cells as previously described (gombold et al, ) . animals qj bli (b ) mice were purchased from the national cancer institute (frederick, md). four-week-old, mhv-free male mice were used in all experiments. mice were anesthetizedwith isoflurane (isoflo; abbott laboratories, north chicago, il). the amount of virus designated in each experiment was diluted in pbs containing . % bovine serum albumin, and a total volume of ill was injected into the left cerebral hemisphere. mock-infected controls were inoculated similarly but with an uninfected ci-l cell lysate at a comparable dilution. fifty percent lethal dose (ldso) ..assays were carried out as described previously (hingley at al, ) . mice were inoculated intracranially with -or -fold serial dilutions of recombinant viruses. a total of animals per dilution per virus were analyzed. mice were examined for signs of disease or death on a daily basis up to days p.i, ld so values were calculated by the reed-muench method (reed and muench, ; smith and barthold, ) . the levels of infectious virus in the cns as a function of time following infection with the recombinant viruses was determined in mice inoculated intracranially with pfu of virus. on days , , , and postlnfsction, mice were sacrificed, perfused with ml of pbs, and the brains were removed. the left-half of the brain was placed directly into ml of isotonic saline with . % gelatin (gel saline) (ramig, ) . all organs were weighed and stored frozen at -bo°c until titered for virus. brains were homogenized, and virus titers were determined by plaque-assay on l cell monolayers (ringley et al, ) . the titers of infectious virus in the brain were determined for all of the recombinant viruses at once in three independent experiments. to quantitate cell-cell fusion induced by the recombinant viruses, l cell monolayers ,in -well plates were infected with virus in duplicate (mol . or . pfu/cell) and incubated for h at °c. following adsorption, the cells were washed with 'iris-buffered saline twice and then fed with ml of dmem-l % fbs. at and h p.i., the cells were washed with pbs and fixed with % paraformaldehyde (sigma chemical co, st. louis, mo). for each x-objective field under phase-contrast microscopy the total number of nuclei in syncytia was counted. at least random fields were counted per well, and the mean number of nuclei in syncytia per field was calculated. the percent fusion relative to that observed with s r was calculated for each experiment. to demonstrate that each x-objective field had a similar number of cells, the total number of cells per field was counted at random. the results. shown in figure . represent the means (and standard deviations) of at least three independent experiments except s hvr . which is from two independent experiments. a clustering of rna recombination sites adjacent to a hypervariable region of the peplomer gene of murine coronavirus redefined nomenclature for members of the carcinoembryonic antigen family the jhm strain of mouse hepatitis virus induces a spike proteinspecific db.restricted cytotoxic t cell response murine hepatitis virus-a (strain jhm)-induced neurologic disease is modulated in vivo by monoclonal antibody cd + t-cell epitopes within the surface glycoprotein of a neurotropic coronavirus and correlation with pathogenicity monoclonal antibodies to murine hepatitis virus-a (strain jhm) define the viral glycoprotein responsible for attachment and cell-cell fusion site-specific alteration of murine hepatitis virus type peplomer glycoprotein e results in reduced neurovirulence evidence for a coiledcoil structure in the spike proteins of coronaviruses cloning of the mouse hepatitis virus (mhv)receptor: expression jn human and hampster cell lines confers susceptibility to mhv envelope glycoprotein deletion mutant of mouse hepatitis virus type- is neuroattenuated by its reduced rate of spread in the central nervous system analysis of a recombinant mouse hepatitis virus expressing a foreign gene reveals a novel aspect of coronavirus transcription antigenic relationships of murtna coronaviruses: analysis using monoclonal antibodies to jhm (mhv- ) virus pathogenicity of antigenic variants of murine coronavirus jhm selected with monoclonal antibodies proteolytic cleavage of the e glycoprotein of murine coronavirus: host-dependent differences in proteolytic cleavage and cell fusion a role for naturally occurring variation of the murine coronavirus spike protein in stabilizing association with the cellular receptor cell receptor-independent infection by a neurotropic murine coronavirus alteration of ph dependence of coronavirus-induced cell fusion effect of mutations in the spike glycoprotein neutralization-resistant variants of a neurotropic coro navirus are generated by deletions within the aminoter minal half ofthe spike glycoprotein fusion-defective mutants of mouse hepatitis virus a contain a mutation in the spike protein cleavage signal single amino acid changes in the sz subunit of the mhv surface glycoprotein confer resistance to neutralization by sl subunit-specific man oelonal antibody mhv a fusion mutants are attenuated and display altered hepatotropism repair and mutagenesis of the genome of a deletion mutant of the murine coronavirus mouse hap atitis virus by targeted rna recombination localization ofneu tralizing epitopes and the receptor-binding site withij the amino-terminal amino acids of the murine coro: navirus spike protein retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier limbic encephalitis after inhalation of a murine coronavirus the organ tropism of mouse hepatitis virus strain a is dependent on dose and route of inoculation targeted recombination within the spike gene of murine coronavirus mouse hepatitis virus-a : q is a determinant of hepatotropism primary structure of the glycoprotein e of coronavirus mhv-a and identification ofthe trypsin cleavage site molecular cloning, a laboratory manual optimization of targeted rna recombination and mapping of a novel nucleocapsid gene mutation in the corona virus mouse hepatitis virus sequence analysis reveals extensive polymorphism and evidence of deletions within the e glycoprotein gene of several strains of murine hepatitis virus cytotoxic t cell-resistant variants are selected in a virus-induced demyelinating disease pathogenesis of chimeric mhv /mhv-a recombinant viruses: the murine coronavirus spike protein is a major determinant of neurovirulence assembly of theiler's virus recombinants used in mapping determinants ofneurovirulence isolation and genetic characterization of temperature sensitive mutants of simian rotavirus sall intracellular complexes of viral spike and cellular receptor accumulate during cytopathic murine coronavirus infections a simple method of estilflating fifty per cent points identification of spike protein residues of murine coronavirus responsible for receptor-binding activity by use of soluble receptor-resisitant mutants methods in viral pathogenesis conformational change of the coronavirus peplomer glycoprotein at ph . and °c correlates with virus aggregation and virusinduced cell fusion comparison of six different murine coronavirus jhm variants by monoclonal antibodies against the e glycoprotein a -amino acid stretch in the hypervariable region of the spike protein subunit is critical for cell fusion activity of mouse hepatitis virus sequence analysis of the spike protein gene of murine coronavirus variants: study ofgenetic sites affecting neuropathogenicity the peplomer protein e of coronavirus jhm as a determinant of neurovirulence: definition of critical epitopes by variant analysis monoclonal antibodies to the peplomer glycoprotein of coronavirus mouse hepatitis virus identify two subunits and detect a conformational change in the subunit released under mild alkaline conditions receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins chimeric cdna studies of theiler's murine encephalomyelitis virus neurovirulence this work was supported by public health service grants ns- and ns- . j.j.p. was supported in part by training grant gm- z .we thank paul s masters, lili kuo, and peter m 'rottier for pmh , alb , and fmhv. we thank jean tsai for providing us with sa sr , danielle linn letting for her help, and paul bates. amy matthews, and jean tsai for critical reading of the manuscript. key: cord- - kgy o authors: de vries, antoine a.f.; horzinek, marian c.; rottier, peter j.m.; de groot, raoul j. title: the genome organization of the nidovirales: similarities and differences between arteri-, toro-, and coronaviruses date: - - journal: seminars in virology doi: . /smvy. . sha: doc_id: cord_uid: kgy o abstract viruses in the families arteriviridae and coronaviridae have enveloped virions which contain nonsegmented, positive-stranded rna, but the constituent genera differ markedly in genetic complexity and virion structure. nevertheless, there are striking resemblances among the viruses in the organization and expression of their genomes, and sequence conservation among the polymerase polyproteins strongly suggests that they have a common ancestry. on this basis, the international committee on taxonomy of viruses recently established a new order, nidovirales, to contain the two families. here, the common traits and distinguishing features of the nidovirales are reviewed. the nidovirales (summarized in table ) is a newly established order comprising the families arteriviridae (genus arterivirus) and coronaviridae (genera coronavirus and torovirus). species in the genus coronavirus can be grouped into three clusters on the basis of serological and genetic properties ( ) . two torovirus species have been recognized: the equine and bovine toroviruses (etv, berne virus; and botv, breda virus). in addition, a human torovirus is thought to exist ( ) and we have recently identified a porcine torovirus (potv) (kroneman et al., unpublished) . the genus arterivirus presently contains four species. despite considerable differences in genetic complexity and virion architecture, coronaviruses, toroviruses, and arteriviruses are strikingly similar in genome organization and replication strategy ( ) (fig. ) . the name nidovirales (from the latin nidus, nest) refers to the coterminal nested set of subgenomic (sg) viral mrnas that is produced during infection. sequence similarities, although mostly restricted to the lb poly-protein (pol b) from which the replicase-associated proteins are derived, suggest that the nidovirales have evolved from a common ancestor. apparently their divergence has been accompanied by extensive genome rearrangements through heterologous rna recombination. here, we review the common traits and distinguishing features of the genome organization, gene expression, and evolution of the nidovirales. other reviews are references to and the different models proposed for sg mrna synthesis are discussed in references to . the phylogenetic relationship among arteriviruses, toroviruses, and coronaviruses is not apparent from their morphology. coronavirions are roughly spherical, - nm in diameter, with a fringe of c. -nm-long petal-shaped spikes. some group ii coronaviruses exhibit a second fringe of smaller surface projections about nm in length. torovirus particles are pleiomorphic, measuring to nm in their largest axis; spherical, oval, elongated, and kidneyshaped virions have been described. the surface projections on torovirus virions closely resemble coronavirus peplomers ( ) . arterivirions are only - nm in diameter and lack large surface projections. instead, cup-like structures with a diameter of to nm have been observed ( ) . the difference in virion architecture become even more apparent when comparing the nucleocapsid structures. that of coronaviruses is a loosely wound helix ( ) , that of toroviruses is a compact tubular structure ( ) , and that of arteriviruses is isometric, about - nm in diameter, and possibly icosahedral ( ) . the nucleocapsid proteins (n) differ considerably in size (c. , , and kda for corona-, toro-, and arteriviruses, respectively) and amino acid sequence. the compositions of the viral envelopes also differ. coronavirus membranes contain: (i) -to -kda spike protein (s), (ii) - -kda triple-spanning membrane protein m, and (iii) c. -kda transmembrane protein e, a minor virion component but essential for virus assembly ( , ) . the small surface projections of group ii coronaviruses are dimers of a -kda class i membrane protein, the hemagglutinin-esterase (he), possibly acquired by heterologous rna recombination ( , ) . toroviruses also specify m and s proteins of and kda, respectively. although different in sequence, the m and s proteins of toro-and coronaviruses are alike in size, structure, and function. the m proteins have a similar triple-spanning membrane topology ( ) , and the heptad repeats, indicative of a coiled-coil structure in the spike proteins of coronaviruses ( ) , are also present in the torovirus peplomer ( ) . thus, the s and m genes of these viruses may well be phylogenetically related ( , , ) . puzzlingly, toroviruses seem to lack a homologue for the e protein, which could indicate a difference in assembly. we have found recently that botv virions contain a third membrane protein, the -kda hemagglutinin-esterase ( ) . the structural proteins of arteriviruses are unrelated to those of the coronaviridae. there is a basic set of three envelope proteins ( ) ( ) ( ) ( ) . (i) a -to -kda nonglycosylated membrane protein (m) which traverses the membrane three times and thus structurally resembles the m protein of corona-and toroviruses, (ii) a heterogeneously n-glycosylated triple-spanning protein (designated g l for eav) of variable size, and (iii) a class i glycoprotein of - kda (designated g s for eav) which is a minor virion component. the g l and m proteins associate into disulphide-linked heterodimers and probably form the cup-like structures on the virion surface ( ) ( ) ( ) . nidoviral genome rna is single-stranded, infectious, polyadenylated ( ) ( ) ( ) , and, at least for arteri-and coronaviruses, capped ( , ). nucleotide sequences are known for the complete rna of coronaviruses mhv, ibv, tgev, and hcv e and arteriviruses eav, ldv, and prrsv ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) and for parts of rna of several other nidovirales, including etv strain berne ( , ) and shfv (godeny et al., in press ). the size of the arterivirus genome is from to kb. the genomes of toroviruses and coronaviruses are considerably larger (up to kb) and include the largest known rna genomes. despite the differences in genetic complexity and gene composition, the genome organizations of arteri-, toro-, and coronaviruses are remarkably similar. more than two-thirds of each genome are taken up by two huge overlapping open reading frames (orfs), designated orf a and b. the more downstream, orf b, is only expressed after translational read-through via a - frameshift mediated by a pseudoknot structure ( ) . the polypeptides encoded by these orfs are proteolytically cleaved by virus-encoded proteinases to yield the proteins involved in viral rna synthesis. downstream of orf b, there are four to nine genes that encode the structural proteins and, at least for coronaviruses, a number of nonstructural proteins. these genes are expressed from a coterminal nested set of sg mrnas ( , , , ) . although these mrnas are structurally polycistronic, translation is restricted to the unique sequences not present in the next smaller rna of the set. cells infected by arteriviruses or coronaviruses contain negative-stranded rnas which correspond to each mrna and which may serve as templates for transcription ( ) ( ) ( ) ( ) ( ) . each transcription unit (comprising one or more genes expressed from a single mrna species) is preceded by a short consensus sequence, the complement of which is thought to function as a promoter: the transcription-associated sequence (tas) ( , , ) . the relative strength of coronavirus promoters is influenced by the primary structure of the tas ( , , ) and the presence downstream of other tass. in general, downstream tass have a negative effect on transcription levels from upstream sites ( ) ( ) ( ) . for mhv, host proteins of and kda have been identified that specifically bind to the tas and may serve as transcription factors ( , , ) . the sg mrnas of corona-and arteriviruses carry a leader sequence of - and about nt, respectively, which are derived from the ends of the viral genomes. the mrna synthesis thus requires, at least at one point, a discontinuous transcription event ( , ) . the fusion of ''leader'' and ''body'' sequences occurs within or in close proximity to the tas ( , , , ) . puzzlingly, the torovirus mrnas seem to lack an extensive leader sequence ( , ) . thus if the use of a leader sequence evolved before the divergence of the nidovirales, toroviruses must have lost their leader relatively recently. the close evolutionary relationship between toro-and coronaviruses suggests that this event took place after the coronaviridae and arteriviridae diverged. alternatively, the common ancestor of the nidovirales may have used a leader-independent transcription mechanism and arteri-and coronaviruses acquired a leader independently. in either view, the addition of noncontiguous leader sequences would not be a mechanistically important aspect of mrna synthesis (as suggested by the ''leader-primed'' transcription model) ( ) but rather a modification of a common transcription scheme, based primarily on transcriptase-promoter recognition ( , ) . what then is the function of the leader sequence? perhaps the discontinuous transcription seen in arteri-and coronaviruses has evolved merely to provide each viral mrna with a translational enhancer, allowing efficient competition with host mrnas for the cellular translational machinery. indeed, there is evidence that the coronavirus leader sequence stimulates viral translation in cis, possibly in conjunction with a virusspecified or virus-induced factor ( ) . for a complete understanding of nidovirales transcription-initiation, studies on torovirus mrna synthesis will be pivotal. in fact, the existence of a small torovirus leader rna cannot entirely be excluded. sequence analysis of etv defective interfering rnas, combined with results of primer extension studies, suggest that a tas is present at the extreme end of the viral genome which could give rise to a leader of approximately nt ( ). the promoters required for genome replication are commonly found at the and ends of the genome. coronaviruses have nontranslated regions (ntrs) ranging from . to . kb ( ) and from . to . kb ( ) . their primary structure is poorly conserved among the different subgroups. deletion mapping studies using synthetic di rnas suggest that for the group ii coronaviruses, about . kb of each end of the genome is required for replication, implying that promoter elements may extend into orf a and the n gene ( , ( ) ( ) ( ) ( ) ( ) . all coronavirus genome rnas have the sequence u/gggaagagc about nt upstream of the poly(a) tail ( , ) . the strict conservation of this sequence element suggests that it has a role in replication. surprisingly, however, the most nt of the ntr of mhv appear to be sufficient to drive minus-strand synthesis ( ) . the ntrs of toroviruses are about . kb. the ntr of etv strain berne is . kb ( ) but the lengths of ntrs of other toroviruses are unknown. the ntrs of arteriviruses are about . kb and, unlike those of coronaviruses, consist almost entirely of the leader ( ) ( ) ( ) ) . the ntrs of arteriviruses are also short, ranging from to nt, and conserved sequence elements have not been found. the overlapping orfs a and b found at the end of the nidoviral genome are frequently referred to as the ''polymerase gene.'' however, there is little doubt that the processing of the encoded polyproteins yields proteins required for rna synthesis as well as a number of products involved in other aspects of virus replication. the a and b polyproteins of coronaviruses are to and to residues long, respectively. pol b of etv strain berne consists of residues; only limited sequence data are available for torovirus orf a. the polyproteins of arteriviruses are much smaller, with lengths of - (pol a) and - (pol b) residues. amino acid sequence comparisons show that the b polyproteins of corona-, toro-, and arteriviruses are basically colinear ( , ) (fig. b) . the sequence conservation between the more closely related corona-and toroviruses is clustered in six domains, four of which are also found in the arterivirus pol b: the ''classical'' rna-dependent rna polymerase (rdrp) and helicase (h) domains, which are also present in the polymerases of most other viruses, a zinc finger motif (zf), and a short region of - residues, which has not yet been identified in other viral polymerases and was called the ''coronavirus-like'' (cvl) domain ( ) (motif in fig. b) . there is little sequence conservation among the n-termini of the pol a polyproteins of the three coronavirus subgroups. size differences can mostly be attributed to these regions (fig. ) and sequence similarities are limited to papain-like cysteine proteinase (pcp) domains ( , , , ) . pol a of hcv e, tgev, fipv (subgroup i), and mhv (subgroup ii) have two pcp domains, whereas that of ibv (subgroup iii) contains a single pcp domain. these pcp seem to be involved in the processing of the n-termini of the a polyproteins. the proteolytic cleavage of the n-terminus of the coronavirus a polyprotein has been studied in most detail for mhv. in vitro translation of genomic rna gave products of and kda and the production of p was sensitive to proteinase inhibitors, suggesting that it arose by a proteolytic cleavage ( ) . p was also detected in mhv-infected cells ( ) . partial peptide mapping revealed that p is derived from the n-terminus of pol a ( ). baker et al. ( ) subsequently showed that the proteolytic activity responsible for the production of p mapped to residues - of pol a which contains the n-terminal-most pcp domain (pcp ) ( ) . mutagenesis showed that any change of either cys or his (cys and his of mhv-a ) ( , ) resulted in the loss of proteinase activity, suggesting that these residues form the catalytic dyad ( ) . cleavage to give p was at an rgv motif at the g /v dipeptide bond ( , ) , and presumbably occurred in cis ( ) . reactions of specific antisera raised against different regions of mhv pol a with potential cleavage products with apparent molecular weights of , , , and kda in mhv-infected cells ( , ) showed that processing of the n-terminus of pol a involves multiple cleavage events. p is thought to be immediately adjacent to p ( , ) . gao et al. ( ) reported that p of mhv strain jhm is generated from a p precursor, but this precursor has not been observed by others studying mhv strain a ( ) . kinetic analysis suggests that p is a precursor to p and p . a provisional map of the pol a region of mhv is shown in fig. . the proteinases involved in the release of p , p , and p have not yet been identified. although some authors have implicated pcp in the cleavage of p ( ) this is disputed by others ( ) . only limited data are available on the processing of the n-terminus of pol a of ibv. using monospecific antisera raised against residues - or - , liu et al. ( ) detected a -kda product in ibv-infected cells. it is not known if ibv p represents the n-terminal cleavage product or if an additional smaller product is released from the n-terminus of pol a. p was also found upon in vivo expression of the n-terminal residues of ibv pol a ( ) , which include the pcp domain ( , ) . interestingly, p was not detected after in vivo expression of a shorter n-terminal polypeptide of residues that lacked pcp, strongly suggesting that pcp is involved in the release of this product. because p did not appear when the -residue polypeptide was produced by in vitro translation, cellular factors may also be involved in this cleavage event. however, in vivo processing of this polypeptide was also inefficient, possibly because the pcp is located at the c-terminus of the -residue expression prod-uct and sequences downstream of this domain are required for optimal proteolytic activity. in our laboratory, a monospecific antiserum, raised against the n-terminal residues of the a polyprotein of fipv, specifically recognized products of , , and kda in fipv-infected cells. these products were also found upon in vivo expression of the n-terminal residues of fipv pol a containing the pcp domain. kinetic analysis suggested that p and p are mature products with p as their precursor. p reacted with antiserum raised against the n-terminal in contrast to the n-termini, the c-terminal third of coronavirus pol a polyproteins are well conserved. all contain a proteinase domain flanked by hydrophobic regions, designated mp and mp (fig. ) . this proteinase is related to the chymotrypsin-like serine proteases, but with a cysteine rather than a serine residue as the active site nucleophile ( , , , , ) . a similar situation exists in the c proteinases of picornaviruses and c-like proteinases of plant viruses ( ) . the c-like proteinases ( clp) of coronaviruses are involved in the processing of the c-terminus of pol a and of pol ab. the results obtained for ibv, mhv, and hcv e differ only in details. the clp mediates at least four cleavage events. it autocatalytically excises itself from the polyprotein precursor, yielding products of , , and kda for ibv, mhv, and hcv e, respectively ( - ) (fig. ) . the release of ibv clp (but not that of mhv) from a synthetic precursor in vitro was dependent on the presence of microsomal membranes and apparently required membraneassociation of the flanking lipophilic domains ( , ). lu et al. ( ) proposed that because production of the mhv p in vitro was sensitive to dilution, the autocatalytic release of clp occurs mainly in trans. protein sequence analysis identified q /s and q /a as the respective n-terminal cleavage sites of mhv p and hcv p with the gln residues in the p position ( , ) . p of ibv is generated by cleavage of qs dipeptides at positions - and - ( ) . the cleavage sites flanking clp are well conserved among the different coronaviruses. processing of the pol ab polyprotein by clp also resulted in the production of a polypeptide of c. kda, containing the rdrp domain ( ) ( ) ( ) . the cleavage sites for ibv and hcv e were at the positionally conserved dipeptides q /s and q /s or q /s and q /a , respectively, the n-terminal most of which are located in pol a (fig. ) . processing leading to the release of the rdrp can occur in trans, both in vitro and in vivo ( , ) . gorbalenya et al. ( ) predicted that the catalytic site of the ibv clp consists of a triad formed by his , glu , and cys . the cys and his residues are conserved in the clp domain of the other coronaviruses and their involvement in proteolysis has been confirmed by site-directed mutagenesis ( , , , ) . glu is not part of the catalytic site. this residue is not conserved in other clp and substitution by asn, asp, or gln did not affect proteolytic activity ( ) . in agreement with the assumed evolutionary relationship with cellular trypsin-like serine proteases, the coronavirus clp are sensitive to both serine and cysteine protease inhibitors ( , ) . moreover, substitution of the active site cys by ser yielded an ibv clp which was still partially active ( ) . the cleavage sites of the coronavirus clp conform to the consensus xqz, with x being a hydrophobic residue (l, v, i, m or f) and z a small uncharged residue (s, a, g or c). these data provide experimental support to earlier predictions ( , ) . alignment of pol ab sequences suggests that clp may cleave at seven additional conserved sites (fig. ) . cleavage at the sites in mhv pol a would produce four extra polypeptides with predicted molecular weights of , , , and kda. the -kda product would contain the hydrophobic domain mp , whereas the -kda product would be a cysteine-rich polypeptide resembling murine epidermal growth factor in sequence ( ) . processing of pol b would yield the rdrp and four other products. the zinc finger and helicase motifs would be in a product of about kda and the conserved motif would be in a polypeptide of kda, whereas motifs (the cvl domain) and would be in products of and kda, respectively (figs. and ). the latter may correspond to a -kda protein in lysates of mhv-infected cells which reacted with antiserum against the c-terminal amino acids of pol b ( ) . most of what is known about arterivirus polyprotein processing stems from the work of snijder and colleagues on eav; only limited information is available for prrsv and ldv. as for coronaviruses, most sequence variation occurs in pol a. processing of the n-terminus of pol a is mediated by papain-like cysteine proteinases, whereas the c-terminus of pol a and the conserved b polyprotein is probably processed by a c-like proteinase which is located at the c-terminus of pol a and flanked by hydrophobic domains (fig. ) . for both prrsv and ldv ( , ) , the n-terminus of pol a contains two papain-like proteinase domains, pcpa and pcpb, which mediate their own release by cleavage in cis at c-terminal cleavage sites, giving rise to products nsp a and nsp b (fig. ) ( ) . the prrsv and ldv leader proteinases share % sequence identity. for prrsv, cys and his are crucial for pcpa activity ( ) , whereas cleavage by pcpb was dependent on cys and his . for ldv, cys and cys were identified as active site cysteines. the cleavage sites in pol a have not been mapped but from the sizes of nsp a and nsp b, and from the results of deletion analyses, are predicted to be around position for pcpa and between tyr and gly for prrsv pcpb, and between tyr and gly for ldv pcpb. eav is thought to have a single leader proteinase ( ), corresponding to pcpb of ldv and prrsv. however, relicts of nsp a are still present in the n-terminus of eav pol a ( ) . the eav pcpb homologue releases a -kda protein, nsp (fig. ) , apparently exclusively by cleavage in cis at g /g . the results of site-directed mutagenesis suggested that cys and his form the catalytic dyad ( ) . four additional mature cleavage products were identified in lysates of eav-infected cells ( ) and were designated nsp to (fig. ) . the -kda nsp protein is released by cleavage between gly and gly and the catalytic activity responsible is within the n-terminal residues of nsp as this domain can induce cleavage at the / site in trans ( ) . sequence comparisons suggested that the catalytic residues in the cysteine proteinase domain were cys and his . substitutions of these residues completely abolished proteolytic activity, but so did replacement of three other conserved cysteine residues (positions , , and ). the n-and c-terminal sequences of nsp are highly ( ) . also shown are the apparent molecular weights of the cleavage products. the papain-like proteinase domains (pcp) and the nsp cysteine (cp) and the nsp serine proteinases (sp) are indicated by shading as are the rna-dependent rna polymerase (rdrp), zinc finger (zf), and helicase domains (h). also shown are the hydrophobic domains, mp and mp , that flank nsp . cleavage sits that have been identified experimentally are indicated by black arrows. white arrows indicate cleavages for which the exact cleavage site has not yet been determined. cleavages performed by the serine proteinase are given. arched arrows depict cleavages performed by the leader proteinases. open arrowheads indicate predicted sp cleavage sites, black arrowheads mark cleavages possibly performed by a cellular proteinase. conserved among eav, ldv, and prrsv. in contrast, the middle portions differ markedly in size ( - residues) and sequence ( ) ( ) ( ) (fig. ) , suggesting that nsp has species-specific rather than genus-specific functions ( ) . multiple sequence alignments suggest that the nsp /nsp cleavage sites for ldv and prrsv are gly-gly at positions / and / , respectively. inhibition of cleavage at the nsp / junction abolishes downstream proteolytic events, which are probably all mediated by a c-like serine protease (sp) ( ) located within nsp . site-directed mutagenesis results suggest that the catalytic triad of the nsp protease comprises his , asp , and ser , while thr and his may be involved in substrate recognition. snijder et al. ( ) further identified three cleavage sites within pol a (e /g , e /s , and e /g ) and two additional cleavage sites were proposed in the c-terminus of pol a ( ) . the corresponding cleavage sites in ldv and prrsv in fig. are inferred. three putative recognition sequences for the nsp protease were predicted in pol b. proteolytic cleavage at these sites would separate the rdrp motif from the putative metal binding and helicase domains. reaction with specific antisera detected four possible cleavage products designated p , p , p , and p , respectively (fig. ) , and a number of putative precursor proteins in lysates of eav-infected cells ( ) . the most n-terminal cleavage product, p , contains the rdrp domain, and the putative zinc finger and helicase motifs are in the adjacent p . the cvl domain (motif ; fig. b) is in p . no information is available on the processing of pol b of toroviruses, although the sequence contains a number of potential clp-cleavage sites. because the pol b sequences of toro-and coronaviruses are colinear (fig. b) , the processing of torovirus pol b is likely to be very similar to that of coronaviruses. there are some marked differences between coronaviridae and arteriviridae. the latter lack a cleavage product containing motif (figs b and c) . moreover, it remains to be seen whether the c-terminal pol b cleavage products of the arteri-and coronaviridae are functionally equivalent. for the arteri-and coronaviruses, pol b processing would yield a product containing both the helicase domain and the zinc finger motif. such a combination is rare, but not unprecedented as it has also been seen in glh- , a putative rna helicase from caenorhabditis elegans ( ) , and the (putative) yeast rna helicases yer w ( ) and nam ( , ) . most helicases lack zinc finger motifs, and it is therefore unlikely that the zinc fingers are required for helicase activity ( ) . perhaps, they may confer sequence specificity, for example, in promoter recognition. the arteriviruses prrsv, ldv, and eav each possess six genes, numbered - from the end, that are expressed from subgenomic mrnas ( ) ( ) ( ) ) . these orfs usually overlap (fig. a) . orfs , , , and are conserved among all arteriviruses and, using eav terminology, code for g s , g l , m, and n, respectively ( , , , , ) . sequence similarity can be detected only at the amino acid level; the conservation is generally low and, especially in the eav proteins, restricted to short domains. orfs and are conserved among prrsv, ldv, and shfv and code for membrane glycoproteins, which in the case of prrsv, are present in purified virions ( , ) . the orf product of eav shares no obvious sequence similarity with that of the other arteriviruses and has not been detected in virus preparations. surprisingly, shfv possesses three additional orfs. from the limited sequence similarities and the apparent positional conservation of cysteine residues it appears that these orfs have arisen from a heterologous rna recombination event by which orfs - were duplicated (e. godeny, personal communication). toroviruses apparently express only four genes from subgenomic mrnas, all of which encode structural proteins. etv and botv are genetically and serologically closely related and share % sequence identity in the -most kb of their genomes ( ) . potv is more distant as judged from the sequence of its nucleocapsid protein, which is only % identical to those of the other two viruses (kroneman et al., unpublished). snijder et al. ( ) noted the presence of a small orf completely contained within the n gene of etv. this orf, which would encode a hydrophobic polypeptide of approximately kda, is conserved in botv but abrogated by a termination codon in potv. coronaviruses possess up to nine orfs that are expressed from sg mrnas. of these, the genes for only the main structural proteins are conserved among the three subgroups (sequence identities of approximately %) as is their relative position in the genome ( s-e-m- n ) . apparently, as coronaviruses diverged, subgroup-specific sets of accessory genes were acquired ( , , ) . for instance, the he gene and orf a, which encodes a cytoplasmic nonstructural phosphoprotein of about kda ( , , ) (fig. ) , are only found in group ii viruses. differences in gene composition occur even among viruses of the same subgroup. in ccv and fcov, orfs a and b are at the end of the genome ( , ) , but tgev, which is serologically and genetically very closely related to ccv and fcov, lacks b ( ) . hcv e lacks both orfs ( ) . all accessory genes tested thus far are dispensible for replication in vitro and in vivo ( , ( ) ( ) ( ) ( ) ( ) ( ) . the functions of the encoded proteins are poorly understood, but at least some may be involved in virus-host interactions and thus contribute to viral fitness. for example, the b gene of fcov codes for a nonstructural -kda secretory glycoprotein ( ) . fcov variants that lack orf b readily arise in tissue culture, but among naturally occurring fcov strains, the gene is strictly maintained and its loss correlates with reduced virulence ( ) . in contrast to the other nidovirales, a number of coronaviruses have polycistronic mrnas which contain up to three orfs clustered in a single transcription unit. downstream orfs are usually translated by leaky scanning but the synthesis of the e proteins of ibv (orf c) and mhv (orf b) may involve internal intiation of translation mediated by a ribosomal landing pad ( , ( ) ( ) ( ) . the n gene of some group ii coronaviruses contains a small internal orf in the reading frame (fig. ) that is expressed in infected cells ( , ) . it encodes a hitherto unrecognized structural protein that is not essential for virus replication in vitro and in vivo ( ) . the variation in coronavirus gene composition is probably the result of heterologous rna recombination events during which gene modules ( ) were obtained either from nonrelated viruses or from the host. the most compelling example is the he gene, the product of which is % identical to the n-terminal subunit of the hemagglutinin-esterase fusion protein (hef) of influenza c virus (icv) ( ) . heterologous rna recombination events must also have taken place during torovirus evolution. a . -kb remnant of an he gene was found in the etv genome ( ) and an intact, functional he gene of . kb is present in the genome of botv ( fig. ; ) . the torovirus he protein shares % sequence identity with both the influenza c virus hef and the coronavirus he. in addition, sequences related to orf a of group ii coronaviruses were found at the end of etv orf a ( ) (fig. ) . the he and the orf a-related sequences found in corona-and toroviruses were probably not inherited from a common ancestor, but acquired through separate heterologous rna recombination events ( , ) because (i) the genes are in different positions in the two virus genomes (fig. ) and (ii) it is highly unlikely that genes retained during the considerable evolutionary divergence between corona-and toroviruses would have been lost from the genomes of coronavirus subgroups i and iii. the differences among the main structural proteins of the nidovirales could also be explained by heterologous recombination ( ). a switch from an arteriviruslike isometric nucleocapsid structure to the extended helical nucleocapsid structures of the coronaviridae may have been a determining step in the divergence of the nidovirales ( ) . removal of constraints on genome size would have allowed toro-and coronavirus ancestors to acquire large genomes and thus develop the variation in gene composition seen today. a relatively recent replacement of the n gene may subsequently have led to the divergence of the toro-and coronaviruses. homologous rna recombination ( , ) may also be an important force in nidovirales evolution. high frequency recombination of coronavirus genomes has been observed in tissue culture ( , ) , in experimentally infected animals ( ) and in embryonated eggs ( ) . homologous recombination allows the rapid exchange of beneficial mutations and also serves as a correction mechanism counteracting muller's ratchet ( ) . there is evidence that homologous recombination occurs in ibv genomes in the field ( , , ) and a genetic exchange between ccv and fcov serotype i strains may have resulted in the emergence of a new fcov serotype ( , , ) . the nidoviral replicase module has given rise to viruses that utilize similar replication strategies and yet differ markedly in genetic complexity. common to the nidovirales is the use of a nested set of mrnas. this property, often regarded as ''unique,'' is shared with the phylogenetically unrelated closteroviruses, a genus of filamentous rna viruses of plants ( , ) . closteroviruses have genomes of up to kb in length, thus approaching the coronaviridae in genetic complexity. they also resemble the nidovirales in genome organization and expression, including the use of large polymerase polyproteins, encoded by two overlapping orfs located at the end of the genome, and down-regulation of rdrp synthesis by ribosomal frameshifting. these recent findings underscore the power of convergent evolution and indicate that similarities in genomic organization and common mechanisms of gene expression and regulation are not reliable taxonomic criteria by themselves. even the results of comparative sequence analysis should be regarded with caution. alignments of rdrp domains have been presented to illustrate the evolutionary relationship between the nidovirales, but the phylogenetic signal in this domain is not sufficient to support a common ancestry of corona-and arteriviruses ( ) . here, the toroviruses provide the ''missing link'' and thus justify a phylogenetic grouping of corona-, toro-, and arteriviruses ( ) (p. zanotto, personal communication). the analyses of nidovirales genomes and the studies on polyprotein processing have led to the identification of many viral proteins, some of which are conserved and some of which are genus-or even species-specific. the next formidable task will be to determine the function of each of these products. what is the added value of the nonconserved pol aderived cleavage products? are they antagonists of the intracellular antiviral response or involved in host shut off? what are the functions of the proteins derived from pol b? why are proteins containing motifs and lacking in arteriviruses and what are the consequences for replication and transcription? are replication and transcription distinct processes? is there a developmental shift from replication to transcription and if so, how is this regulated? what is the function of the various accessory genes of coronaviruses and how do they contribute to viral fitness? many of these questions may well be solved in the near future. both in leiden ( ) and in utrecht (glaser et al., in preparation), full-length cdna clones of the eav genome have been constructed, from which infectious transcripts can be derived. for coronaviruses, no such clones are yet available. however, homologous rna recombination can be exploited to introduce sitespecific mutations into the viral genome using syn-thetic (di) rnas as donor sequence ( , , , ) . targeted rna recombination provides an attractive strategy to characterize the various ts-mutants of mhv ( ) . undoubtedly, the recent development of methods to study arteri-and coronaviruses by reverse genetics heralds a new era in nidovirales 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by using t -generated negative-sense rna transcripts coronavirus transcription mediated by sequences flanking the transcription consensus sequence the effect of two closely inserted transcription consensus sequences on coronavirus transcription regulation of coronavirus mrna transcription tandem placement of a coronavirus promoter results in enhanced mrna synthesis from the downstream-most initiation site three different cellular proteins bind to complementary sites on the -end-positive and -end-negative strands of mouse hepatitis virus rna interactions between the cytoplasmic proteins and the intergenic (promoter) sequence of mouse hepatitis virus rna: correlation with the amounts of subgenomic mrna transcribed sequences of end of genome and of end of open reading frame la of lactate dehydrogenaseelevating virus and common junction motifs between leader and bodies of seven subgenomic mrnas subgenomic rnas of lelystad virus contain a conserved leader-body junction sequence 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proteolytic processing of the replicase orf a protein of equine arteritis virus the arterivirus nsp protease: an unusual cysteine protease with primary structure similarities to both papain-like and chymotrypsin-like proteases the arterivirus nsp protease is the prototype of a novel group of chymotrypsin-like enzymes, the c-like serine proteases processing of the equine arteritis virus replicase orf b protein: identification of cleavage products containing the putative viral polymerase and helicase domains glh- , a germ-line putative rna helicase from caenorhabditis, has four zinc fingers gene products that promote mrna turnover in saccharomyces cerevisiae nam nuclear gene encodes a novel member of a family of helicases with a zn-ligand motif and is involved in mitochondrial functions in saccharomyces cerevisiae characterization of proteins encoded by orfs to of lelystad virus identification and characterization of a sixth structural protein of lelystad virus: the glycoprotein gp 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about the relationships between feline coronaviruses and their evolution molecular biology and evolution of closteroviruses: sophisticated build-up of large rna genomes principles of molecular organization, expression, and evolution of closteroviruses: over the barriers a reevaluation of the higher taxonomy of viruses based on rna polymerases repair and mutagenesis of the genome of a deletion mutant of the coronavirus mouse hepatitis virus by targeted rna recombination homologous rna recombination allows efficient introduction of site-specific mutations into the genome of coronavirus mhv-a via synthetic co-replicating rnas 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 hemagglutinin-esterase, a novel structural protein of torovirus sequence evidence for rna recombination in field isolates of avian coronavirus infectious bronchitis virus an infectious arterivirus cdna clone: identification of a replicase point mutation that abolishes discontinuous mrna transcription the authors thank drs. e. godeny for sharing unpublished results and dr. p. zanotto for advice and stimulating discussions. we are grateful to katharina schick and jolanda mijnes for their help in the preparation of the manuscript. r. j. de groot was supported by a fellowship of the royal netherlands academy for sciences and arts. key: cord- -ys n authors: whary, mark t.; baumgarth, nicole; fox, james g.; barthold, stephen w. title: biology and diseases of mice date: - - journal: laboratory animal medicine doi: . /b - - - - . - sha: doc_id: cord_uid: ys n today’s laboratory mouse, mus musculus, has its origins as the ‘house mouse’ of north america and europe. beginning with mice bred by mouse fanciers, laboratory stocks (outbred) derived from m. musculus musculus from eastern europe and m. m. domesticus from western europe were developed into inbred strains. since the mid- s, additional strains have been developed from asian mice (m. m. castaneus from thailand and m. m. molossinus from japan) and from m. spretus which originated from the western mediterranean region. laboratory animal medicine development of the 'modern' laboratory mouse. research use of mice has grown exponentially during the past and current century with the recognition of the power of the mouse for gene and comparative mapping and have made the laboratory mouse, in genetic terms, the most thoroughly characterized mammal on earth (silver, ; lyon et al., ; morse, a) . the current ability to create highly sophisticated, genetically engineered mice by inserting transgenes or targeted mutations into endogenous genes has also made the laboratory mouse the most widely and heavily used experimental animal. historical reviews have documented the origins of the laboratory mouse, which extend thousands of years into antiquity (keeler, ; morse, ; silver, ) . the laboratory mouse belongs within the genus mus, subfamily murinae, family muridae, superfamily muroidea, order rodentia, and within the m. musculus clade collectively called the 'house mouse' (lundrigan et al., ) . anatomic features of molar teeth and cranial bones were traditionally used by zoologists to identify over different species within the genus, and to differentiate them from other murids. because of considerable phenotypic variation within a single mus species, this approach has proven to be inaccurate, and given way to contemporary genetic analysis. the native range of the genus mus is eurasia and north africa. members of this genus are generally classified as aboriginal, consisting of species that live independent of humans, or commensal, which includes taxa that have coevolved and geographically radiated with human civilization since the dawn of agriculture , years before present (bp). this close association with human agrarian society gave rise to the genus name, derived from sanskrit, mush: to steal. the commensal group is known as the 'house mouse' clade, consisting of several subspecies of mus musculus, including m. m. domesticus, m. m. musculus, m. m. castaneus, m. m. bactrianus , and a lesser known lineage, m. m. gentilulus (prager et al., ) . the japanese house mouse, m. m. molossinus, is a natural hybrid of m. m. musculus and m. m. castaneus. the progenitor of the m. musculus clade arose in the northern indian subcontinent and diverged into genetically isolated and distinct species or subspecies due to geographic barriers (mountain ranges). there is debate whether these taxa are species or subspecies, and some have referred to them as 'incipient species,' but their genetic divergence is now blurring as they colonize the world and hybridize. the native ranges of these taxa are important for understanding the origins of various laboratory mice, whose genomes are mosaics derived from m.m. domesticus (~ %), m.m. musculus (~ %), and m.m. castaneus (~ %) (wade and daly, ; wade et al., ) . it is now apparent that the m.m. musculus and m.m. castaneus contributions to the laboratory mouse genome were primarily derived from m.m. molossinus japanese fancy mice (takada et al., ) . mus m. domesticus is indigenous to western europe and southwest asia, m.m. musculus to eastern europe and northern asia, m.m. castaneus to southeast asia, and m.m. molossinus to japan and the korean peninsula. the cohabitation of humans with commensal mice gave rise to captive breeding for coat color and behavioral variants in china over years bp. by the s, mouse 'fanciers' in asia had created many varieties of fancy mice, as did european fanciers, who subsequently acquired asian stocks, particularly japanese fancy mice (m.m. molossinus) , to mix with european (m.m. domesticus) fancy mouse varieties. this genetic mixing for fancy variants was also occurring in the united states, and these mouse lines contributed to many of the major laboratory mice used today. meanwhile, the european colonial expansion era contributed to the worldwide dissemination of m.m. domesticus, which now occupies every continent of the world. it is well documented that wild-caught m.m. domesticus also contributed to the genetic composition of fancy and laboratory mice on multiple occasions. despite their diverse genetic origins and phenotypic differences, most laboratory mouse strains are closely related, since many were derived from a genetically mixed but small number of fancy mice from a single mouse breeder (abbie lathrop's granby mouse farm, massachusetts) at the beginning of the th century. most inbred laboratory mice share a common maternal mitochondrial genome derived from m.m. domesticus (ferris et al., ; yu et al., ) , and a common y chromosome contributed by m.m. musculus (bishop et al., ) through its contribution to the genome of m.m. molossinus (nagamine et al., ) . thus, the most inclusive name that can be assigned to the genetically mosaic laboratory mouse is m. musculus, the over-arching name for the entire commensal clade. there are exceptions, however. c bl/ mice contain minor genetic elements derived from m. spretus (hardies et al., ) , and a number of wild aboriginal species that are not members of the m. musculus clade, including m. spretus, m. caroli, and others, have been established as inbred lines of mice. genetic mapping in mice began in the early s with a focus on inheritance of coat color. the first autosomal genes, albino and pink-eyed dilution, were linked in (haldane et al., ) . extensive linkage maps and an impressive array of inbred strains are now available to expedite genetic research (table . ) (lyon et al., ) . mice have pairs of telocentric chromosomes that are differentiated by their size and patterns of transverse bands. the chromosomes are designated by arabic numbers in order of decreasing size. during the s, chromosome rearrangements were used to assign known genetic linkage groups -identified by roman numerals -to specific chromosomes and for determining locus order with respect to the centromere. genes can be located physically on chromosomes by fluorescent in situ hybridization (fish). development of quantitative trait loci (qtl) methodology for mapping genes and the similarity between mouse and human genomes have made the mouse invaluable for identifying genes and underlying complex traits that are inherent to the most common human genetic diseases (moore and nagle, ) . for more information on comparative genomics, see chapter animal models in biomedical research, subsection c. one of the most thoroughly studied genetic systems of the mouse is the histocompatibility complex. histocompatibility (h) loci control expression of cell surface molecules that modulate critical immune responses, such as the recognition of foreign tissue. for example, the time, onset, and speed of skin graft rejection are controlled by two groups of h loci. the major group is located in the major histocompatibility complex (mhc, h ) on chromosome . the h complex contains several loci, including k, d, l, i-a, and i-e. inbred strains of mice, being homozygous, each have unique sets of h alleles, termed h haplotypes. for example, the balb h haplotype is h d and the c bl h haplotype is h b . the international immunogenetics (imgt) information system provides details on h haplotypes for various inbred mice (www.imgt.org/imgtrepertoiremhc/polymorphism/ haplotypes/mouse/mhc/mu_haplotypes.html). minor h loci groups are scattered throughout the genome and are responsible for delayed graft rejection. genes associated with the h complex also control other immunological functions, such as cell-cell interactions in primary immune responses and the level of response to a given antigen. immune-mediated responses to infectious agents such as viruses and complement activity are influenced directly or indirectly by the h complex (stuart, ) . non-mhc or minor histocompatibility systems also are under active study (roopenian et al., ) . mouse genomics have accelerated tremendously in the last two decades, heralded by the development of a robust physical map and high-quality genome sequence of the c bl/ j mouse in by the international mouse genome sequencing consortium (waterston et al., ) . the mouse genomes project/wellcome trust sanger institute is extending this effort to include the genomic sequences of key mouse strains. completed and evolving sequence data are available through the european nucleotide archive (www.ebi.ac.uk/ena/home). the burgeoning numbers of inbred mouse strains, natural mutants, induced mutants, transgenic lines, and targeted mutant lines of mice are cataloged in the mouse genome informatics (mgi) database: http://www.informatics.jax.org/mgihome). the growing number of mutant mice has fostered the development of a number of mouse repositories, from which specific mice can be located and acquired. in the united states, there are four regional national institutes of health (nih)-supported mutant mouse regional resource centers (http://www.mmrrc.org), which link to international repositories in europe, japan, china, australia, and canada, as well as additional resource programs in the united states through the international mouse strain resource (imsr; http://www.informatics.jax.org/ imsr/index.jsp) for depositing, archiving, and distributing mutant mouse and embryonic stem cell lines to the scientific community. in addition to numerous mutant mice produced independently by scientists in various academic institutions, three major targeted gene knockout programs, all utilizing c bl/ n embryonic stem cells, are under way internationally, and funded by the nih, the european community, and genome canada (collins et al., ; skarnes et al., ) . these include the knock out mouse project (komp; http://www.knockoutmouse.org), the european conditional mouse mutagenesis program (eucomm; http://www.eucomm.org), and the north american conditional mouse mutagenesis project (norcomm; http://norcomm.phenogenomics.ca/index. htm). these mouse lines will be available through laboratory animal medicine three distribution centers: the german resource center for genome research (rzpd; http://www.rzpd.de), the komp repository (https://komp.org), and the canadian mouse consortium (cmc; http://www.mousecanada. ca/index.htm). the repositories are all linked to the imsr, and provide access to mice, germplasm, genomic detail, and phenotypic data. genetic, genomic, and biological data are also available through the international mouse phenotyping consortium (impc; www.mousephenotype. org) and the mouse genome database (mgd; http://www. informatics.jax.org) (eppig et al., ) . inbreeding is a fundamental genetic tool applied to the laboratory mouse and detailed information is available on the web (table . ). the first inbred strain (dba) was developed by c.c. little in , with the subsequent creation of over inbred strains and stocks of mice (festing, ) . genetic origins, basic characteristics, references, and breeding performance of inbred strains of mice are available through michael festing's online version of inbred strain characteristics (http:// www.informatics.jax.org/external/festing/mouse/ strains.shtml). overviews of genetic manipulation for the creation of different types of mice are available (lyon et al., ; silver, ) . inbred mouse lines are termed strains, and are achieved by or more brother × sister (filial; f) generations (table . ). mice within an inbred strain, for practical purposes, are genetically identical (syngeneic or isogenic) to other mice of the same strain and sex. because of residual heterozygosity, a strain is not fully inbred until after f generations. most commonly used inbred mouse strains represent or more f generations, providing a high degree of experimental reproducibility. the mouse genome is not static, so when branches of an inbred strain are separated, spontaneous mutations, residual heterozygosity, and retroelement integrations result in genetic differences. therefore, if branches of an inbred strain are separated before f , if branches have separated for generations, or if genetic differences arise, the different branches become substrains. the same holds true if branches of a substrain diverge, resulting in substrains of the inbred substrain. when two inbred mouse strains are crossed, the f hybrids are genetically identical to one another (isogenic), but maximally heterozygous (with chromosomes of each chromosomal pair separately contributed by each parental strain), whereas f hybrids are maximally genetically diverse from one another (with chromosomes of both chromosomal pairs containing a mixture of contributions from each parental strain). with each subsequent f generation, mice once again approach inbred status. this technique is used for creating recombinant inbred (ri) strains. ri strains are sets of inbred strains of mice derived from crossing two inbred strains, and developed by singlepair random matings of sibling mice from the f generation, thereby creating separate breeding lines. each line created is maintained separately, and then propagated by brother-sister matings for generations, with each line becoming a separate inbred strain, but belonging to a set of ri strains. ri mice are useful for mapping phenotypic or quantitative traits that differ between the progenitor strains (bailey, ) . ri sets are generally limited to two parental strains. an ongoing international effort has been undertaken to increase allelic diversity among ri strains by creating the collaborative cross (cc) in which a panel of ri strains are being generated mixing the genomes from eight disparately related inbred (octo-parental) mouse strains, including a/j, c bl/ j, s /svimj, nonobese diabetic (nod)/shiltj, nzo/ hlltj, cast/eij, pwk/phj, and wsb/eij. these eight strains capture nearly % of the known genetic variation present among laboratory mice. future applications of the cc will utilize ri intercrosses of pairs of ri cc lines (threadgill and churchill, ; welsh et al., ) . recombinant congenic strains are sets of inbred strains derived in a manner similar to that for ri sets, except that one or more backcrosses to one parental strain (designated the background strain) are made after the f generation, before inbreeding is begun. the other parental strain is designated as the donor strain. the proportion of background and donor genomes is determined by the number of backcrosses preceding inbreeding (demant and hart, ) . advanced intercross lines (ails) are another type of ri lines. they are made by producing an f generation between two inbred strains and then, in each subsequent generation, intercrossing mice but avoiding sibling matings. the purpose is to increase the possibility of recombination between tightly linked genes. when a mutation arises spontaneously or is induced within an inbred strain, that mutant mouse becomes co-isogenic with the parental inbred strain, being virtually identical except for the single mutant allele. frequently, a mutation that arose in one inbred strain may be desired within the genetic background of another inbred strain. this can be accomplished by backcrossing, in which an f hybrid is created by mating the donor mutant strain to the desired background strain, with subsequent matings to the background strain while retaining the mutant locus. after backcross generations (n generations), the mutant mouse line is now congenic to the background inbred strain. backcrossing to create congenic strains of mice has been used extensively when targeted mutations have been induced in embryonic stem cells, with backcrossing onto c bl/ inbred mice. congenic mice are never co-isogenic, as the preserved locus in a congenic mouse is invariably surrounded by flanking dna, which may significantly influence phenotype (linder, ) . in contrast to inbred mice, outbred mice are genetically heterogeneous and are maintained by breeding systems that intentionally minimize inbreeding. outbred mice are called stocks, which are defined as a closed population (for at least four generations) of genetically variable mice that are bred to maintain maximal heterozygosity. outbred mice may be used when high genetic heterogeneity is desired or for experiments requiring large numbers of mice. outbreeding can be achieved only in a large breeding population using a systematic breeding scheme, or randomized selection of breeders from the population. a small breeding population or passage through the genetic 'bottleneck' of rederivation to improve health status will reduce genetic heterogeneity and lead eventually to some degree of inbreeding. in a population of breeding pairs, e.g., heterozygosity will decrease at % per generation with standard randomization techniques. random breeding involves the statistically random selection of breeders by using a random numbers table or computer program. an outbreeding program that is easy to manage is the circular pair mating system, in which each pair is mated only once. conceptually, cages are visualized in a circle, and each cage contains one breeding pair in the nth generation. another 'circular' set of cages serves as the breeding nucleus for the n + generation. each mated pair in the nth generation contributes one female and one male to the n + generation. outbreeding is accomplished by assigning the female and male derived from each nth generation cage to different cages in the n + generation. most outbred mouse stocks are of 'swiss' origin, derived from nine mice imported to the united states in , and are therefore quite homogeneous genetically (chia et al., ) . various lines of these mice have been maintained at different institutions, giving rise to numerous closely related stocks. although considered outbred, they have a high degree of homozygosity, exemplified by the fact that many swiss mouse stocks are blind due to the homozygous recessive rd allele (serfilippi et al., b) . it is preferable to ensure genetic heterogeneity by intercrossing multiple inbred strains to achieve heterogeneity with known genetic input. in that regard, the diversity outbred mouse has been developed, which is a heterogeneous stock derived from the same eight founder inbred strains of the cc . additional types of inbred mice are utilized in research, including consomic and conplastic strains. consomic strains, also known as chromosome substitution strains, are inbred mice that are congenic for entire chromosomes, and are useful for studying polygenic traits (singer et al., ) . conplastic mice are inbred mice that are congenic for different mitochondrial genomes (mtdna) contributed by other inbred strains, other subspecies, or other species of mus (yu et al., ). in addition to spontaneously occurring mutations that are maintained as co-isogenic strains (such as the c bl/ beige mouse), mutant lines of mice have been created by radiation mutagenesis, chemical mutagenesis, or transgenesis. radiation was one of the earlier methods for in vivo mutagenesis (silver, ) , but in vitro radiation of embryonic stem (es) cells is also performed (thomas et al., ) . chemical mutagenesis involves in vivo treatment of male mice or in vitro treatment of es cells with mutagenic chemicals such as ethylmethanesulphonate (ems) or n-ethyl-n-nitrosourea (enu) , which induce point mutations in dna (o'brien and frankel, ; justice et al., justice et al., , . technically, a transgenic mouse is any mouse in which foreign dna has been integrated into its genome, regardless of method. however, the term transgenic commonly refers to mice that are genetically altered by additive transgenesis through microinjection of foreign dna into the pronucleus of a fertilized egg. each ensuing embryo results in a genetically different founder mouse, since the transgene is integrated in random sites of the genome of each founder mouse. since the injected dna is not homologous to the mouse genome and is not an allele, transgenic founders are hemizygous (rather than heterozygous) for the transgene until the mice carrying the transgene are bred into homozygosity for the transgene. transgenes typically integrate as tandem repeats, copy numbers affect phenotype of each founder, and may be lost in subsequent generations, thereby changing the phenotype of the mouse line (tinkle and jay, ) . transgenes are often constructed with an upstream promoter, which confers widespread (ubiquitous) or tissuespecific expression of the cdna, so that the transgene expression pattern reflects the expression pattern of the promoter. transcriptional regulation of the transgene can be inducible by drug-dependent regulatory control, such as the widely used tetracycline (tet) regulatory system, in which treatment of mice with tetracycline or doxycycline induces up-or down-regulation of the transgene (jaisser, ) . es cells are used for the less efficient integration of genetic material by homologous dna recombination, but allow large-scale screening of es cell clones for transformation. integration can be achieved in a random fashion by gene trapping, or by targeted mutation. both methods involve homologous dna recombination. gene trapping is a high-throughput approach that randomly introduces insertional mutations within the genome. vectors contain a gene trapping cassette with a promoter-less reporter gene and/or selectable genetic marker flanked by an upstream ′ splice site and a downstream termination sequence. when inserted into an laboratory animal medicine intron of an expressed gene, the gene trap is transcribed from the endogenous promoter of that gene. gene traps simultaneously inactivate and report the expression of the trapped gene at the insertion site, and provide a dna tag for the rapid identification of the disrupted gene (skarnes et al., ) . targeted gene mutations are achieved by homologous recombination of specific sites within the genome of es cells. homologous sequences flank the upstream and downstream regions of the targeted gene, and the construct between the flanking sequences may inactivate (knock out) or replace (knock in) a gene, and typically contains a reporter gene to track the integration. a variation on this approach is site-specific recombinase (ssr) technology. two of the most common recombinases are cre from the coliphage p and flp from saccharomyces cerevisiae. cre and flp mediate recombination between target sites, termed loxp and frt, respectively. for example, cre loxp target sites are engineered to flank the gene target, which can be used in different ways to achieve different outcomes (conditional mutations), depending upon the orientation and location of the flanking loxp sites. if the loxp sites are oriented in opposite directions, cre recombinase mediates inversion of the floxed segment. if the loxp sites are on different chromosomes (trans), cre recombinase mediates a chromosomal translocation. if the loxp sites are oriented in the same direction on the same chromosome (cis), cre recombinase mediates deletion of the floxed segment. once the floxed mutation is created in es cells, the transformed es cells are developed into a mouse with the conditional mutation. the conditional mutant mouse is then genetically crossed with a cre transgenic mouse, in which cre recombinase is under the control of a ubiquitous or tissue-specific promoter. wherever and whenever cre is expressed, cre recombinase will recognize and recombine the loxp sites. this approach can include insertion of reporter genes and selectable markers, and can be under the control of inducible gene expression systems (http:// www.eucomm.org/docs/protocols/mouse_protocol_ _ sanger) (nagy, ) . es cells are pluripotent with the full genetic capacity to develop into mice when implanted into the blastocyst of a developing embryo. interest in 'embryonal carcinomas' (teratomas) that arose in relatively high frequency in the testes of mice and early gene transfer experiments in the late s and early s led to the development of es cell lines derived from several different strains. this early emphasis on teratomas prompted creation of 'better' mouse lines that were more prone to development of testicular teratomas, resulting in genetic corruption of the mouse (simpson et al., ; threadgill et al., ) . this realization gave rise to the need to revise mouse nomenclature (festing et al., ) . this was necessary because genetic variation significantly impacts homologous recombination in order to match genome sequence of the es cell line with the mouse from which it was derived. es cells can be created from any mouse strain or hybrid, but es cell lines have been commonly used. recent international knockout mouse program efforts use c bl/ n es cells. transformed es cells are microinjected into the inner cell mass of recipient blastocysts, which are then implanted into the uteri of pseudopregnant surrogate mothers. the pups that are born are composed of a mixture of cells derived from recipient blastocysts and the transformed es cells (chimeras). the goal is for male chimeric progeny to produce spermatozoa of es cell origin (containing the mutation), in order to create f progeny by mating the chimera with the desired background strain (http://www.eucomm.org/docs/protocols/mouse_protocol_ _sanger). for this reason, most es cell lines are xy, which favors male chimerism. if the es cells are of (or other) strain origin, the chimeras are often bred to a desired background mouse strain (commonly c bl/ ) and backcrossed for n generations, thereby creating congenic inbred mouse lines. recent international knockout mouse efforts utilize c bl/ n es cells, so that chimeric males are bred directly with c bl/ mice, thereby creating co-isogenic lines. the latter approach saves time and money, and creates a more genetically refined mutant mouse. an alternate approach is to allow es cells to aggregate with a developing embryo to form blastocysts in culture (aggregation chimera), then implant the chimeric blastocysts (tanaka et al., ) . rna interference (rnai), which functions through short double-stranded rna (dsrna), has also been utilized to produce transgenic mice, known as gene knockdown mice (gao and zhang, ; peng et al., ) . the dsrna is enzymatically processed into small molecules, termed small interfering rna (sirna), which find homologous target mrnas, resulting in interference. this phenomenon is believed to be a self-defense mechanism against viral infection. in order to adapt this approach to generation of transgenic mice, small hairpin rna (shrna) can be expressed in the same way as other transgenes in mice, resulting in processing of the shrna into sirna with gene-silencing effects. constructs are introduced into mouse es cells by electroporation or lentiviral infection. this method can be embellished conditionally, as with other transgenes. although rnai knockdown mice are genetically stable, rnai-mediated transgenesis is never complete, has variable tissue expression, and cannot induce point mutations (peng et al., ) . recent advances in engineered endonuclease (ee) technology, including zinc finger nucleases (zfns), laboratory animal medicine transcription activator-like effector nucleases (talens), and rna-guided endonucleases (rgens), have revolutionized the field of transgenics (sung et al., ; wijshake et al., ; gaj et al., ) . zfns and talens consist of engineered proteins that target dna fused to the nonspecific endonuclease, fok (cathomen and joung, ; joung and sander, ) . zfns are comprised of three to six tandem zinc finger proteins, each of which targets a specific bp nucleotide sequence. paired zfns are generated, with each half of the pair targeting opposite dna strands, allowing dimerization of fok which is required for introduction of double-stranded breaks (dsbs) in the dna of interest (cathomen and joung, ) . talens function similarly, but are composed of tandem repeats of - amino acids, each with nucleotide specificity occurring in two hypervariable amino acids, the 'repeat variable di-residue (rvd)', at positions and (joung and sander, ) . in contrast to zfns and talens, clustered regularly interspaced short palindromic repeats (crisprs) paired with crispr-associated (crispr/cas) systems are rgen systems that target specific dna sequences. cas proteins, rather than fok , produce dsb (hsu et al., ) . dsb generated by ee are repaired by host cells by either nonhomologous end joining (nhej) or, less commonly, by homologous recombination (hr). nhej is an error-prone repair system and results in insertions or deletions (indels) with a relatively high frequency, which can result in gene disruption. hr is a less common repair pathway, but certain manipulations of the engineered nucleases can increase hr efficiency. for example, nucleases can be engineered to generate a break in a single strand of dna rather than inducing dsb, and the resulting nickases increase the incidence of hr with high fidelity (gaj et al., ; wijshake et al., ) . hr allows for the introduction of donor dna to generate knock-ins, specific point mutations, or for the generation of larger modifications such as insertions of loxp sites (brown et al., ; wijshake et al., ) . vectors encoding the ee can be injected into mouse embryos by pronuclear injection of dna, intracytoplasmic injection of rna, or transfection of mouse es cells (sung et al., ; wijshake et al., ) . one advantage of ee technologies over more traditional transgenic methods is the ability to target dna and induce mutations in any background strain of mouse negating the need to backcross onto the desired strain. multiple genes can be targeted with crisprs simultaneously, thus avoiding the need to cross single knockout animals (zhou et al., ) . in addition, it is possible to obtain bi-allelic mutations in some cases, allowing for the generation of functional gene knockout animals in a single generation (zhou et al., ; wijshake et al., ) . vectors for generating ee are available through plasmid repositories; websites are available to assist in identifying appropriate dna sequences to target; and multiple websites post protocols for generating the various types of engineered endonucleases (xie et al., ; sander et al., ; bae et al., ; reyon et al., ; herscovitch et al., ; wolfson, ) . crisprs tend to be particularly cost effective and easy to design, with minimal restrictions for targeting specific dna sequences. there are currently more than separate outbred stocks and traditional inbred strains, often with multiple substrains (table . ). in addition, there are thousands of induced mutant strains. therefore, it is critical that strain or stock designations be complete and accurate to avoid semantic and genetic confusion, and to ensure reproducibility of research results. as an example of substrain variation that makes precise nomenclature important, cba/j mice are homozygous for the retinal degeneration allele (rd ), whereas cba/caj mice do not carry this allele. the international committee on standardized genetic nomenclature for mice and rats, established in the early s, is responsible for genetic nomenclature rules. the rules are available online at the mgi website (http:// www.informatics.jax.org/mgihome/nomen). inbred mouse strains are designated by a series of capital letters and/or numbers, which often provide a shorthand description of the origin and history of the strain. the c bl/ j mouse serves as an example. the inbred strain c bl originated from abbie lathrop's female (and male ) at the cold spring harbor laboratory (c), and was the black (bl) line from this female. early in their history, inbred c bl mice split into major substrains, e.g., c bl/ and c bl/ . substrains are identified by appending a forward slash (/) after the inbred strain name. since , uniform international nomenclature has been built upon these historical names, so that substrains of an inbred strain are now designated using lab codes that are registered in the international laboratory code registry maintained at the institute for laboratory animal research (ilar) of the national academies (dels. nas.edu/global/ilar/lab-codes). laboratory codes are composed of one to five letters that identify an institute, laboratory, or investigator. each lab code starts with an uppercase letter, followed by lowercase letters if more than one letter is used (such as n, j, jci, crl, and tac). the j in c bl/ j means it is a substrain maintained at the jackson laboratory (j). another common substrain of c bl/ mice is c bl/ n, which is maintained at nih (n). substrains can be cumulative, reflecting the genetic history of the mouse strain. for example, there are a number of c bl/ j substrains (such as c bl/ jjci and c bl/ jjmsslc), and a number of c bl/ n substrains (such as c bl/ njci, c bl/ ncrlcrlj, dba/ j inbred strain named for its characteristic coat color genes (using their original gene symbols), dilute (d), brown (b), and nonagouti (a); it is the second of two sublines separated before generations of brother × sister breeding and is the subline maintained at the jackson laboratory (j) c h/hesn-ash/+ co-isogenic segregating inbred mutant strain carrying the ashen (ash) mutation, which arose on c h/hesn c bl/ j-tyrc- j /+ co-isogenic segregating inbred mutant strain carrying the albino j mutant allele of the cloned tyrosinase gene (tyr) aej/gnj-a e /a w-j inbred strain segregating for two alleles at the agouti gene congenic inbred strain in which the b haplotype at the h complex was transferred from c bl/ j (b ) to the akr background b .cba-d mit -d mit congenic strain in which the chromosomal segment between d mit and d mit was transferred from cba to b b .cg m lepr db /++ congenic inbred strain in which the linked mutant genes misty (m) and diabetes (lepr db ) were transferred from multiple, mixed, or unknown genetic backgrounds to b and are carried in coupling, i.e., on the same chromosome b .cg-m +/+ lepr db congenic inbred strain in which the m and lepr db mutations are carried in repulsion bxd- /ty recombinant inbred (ri) strain number in a set of ri strains derived from a c bl/ j (b) female mated to a dba/ j (d) male and made by taylor (ty) recombinant congenic (rc) strain number in a set made by crossing the balb/c (c) and sts (s) strains, backcrossing one or two times to balb/c and then inbreeding as with ri strains and c bl/ ntac). significant differences may exist among these substrains (mekada et al., ). thus, a string of substrain designations indicate the genetic progression of the substrain, which can be identified when reading the entire strain name. this nomenclature is highly nuanced, as c bl/ ncrlcrlj mice, whose last letter is a lowercase j, are not a substrain maintained at the jackson laboratory (j), but rather at charles river japan (crlj), underscoring the importance of upper-and lowercase lettering in rodent nomenclature. balb/c mice are another popular inbred strain with numerous substrains. like the ' ' in c bl/ , the 'c' that follows laboratory animal medicine of the mutational event as a superscript. for example, cftr tm unc is a targeted mutation (tm), first line ( ) congenic mice are often derived from es cells, backcrossed onto a background strain, such as c bl/ . under such circumstances, when the backcross generation is at n , the '.' symbol is used between the background inbred strain and the donor strain (e.g., c bl/ n. p /olahsd-abc tm zzz , abbreviated as b . -abc tm zzz . when backcrossing is incomplete but at the n generation, the mouse is an incipient congenic, designated with a ';' in lieu of a '.': b ; -abc tm zzz . if the background strain is mixed genetic origin, it is designated stock. -abc tm zzz . if the donor strain is mixed origin, it is designated 'cg'. for example, b .cg-abc tm zzz outbred stock that meets specific criteria is designated by placing the lab code before the stock symbol, separated by a full colon (':'). for example, hsd:icr designates an icr (swiss) outbred stock maintained by harlan sprague dawley (hsd). the above overview covers the nomenclature of commonly encountered types of mice. there are numerous additional specifications for nomenclature of mice. details are available at the mgi website (http://www. informatics.jax.org/mgihome/nomen). optimum housing conditions and husbandry practices for research mice should be guided by program requirements to ensure biosecurity, occupational health, efficient use of equipment, labor and financial resources, behavioral needs of mice, and investigator needs for consistent colony maintenance, including standardized husbandry practices and nutrition. the emerging interest in the mouse microbiome in combination with the immune competency of diverse genetically engineered mouse strains demands high standards of mouse care. mouse colonies are optimally maintained as specificpathogen-free (spf) which obligates veterinary and facility management to exclude specific organisms. housing options for spf immunocompetent mice typically include static or individually ventilated microisolator cages, which differ significantly in cost and labor required to maintain. severely immunodeficient strains such as nod.cg-prkdcscid il rgtm wjl/szj (nsg) mice require staff training, caging systems and husbandry practices that minimize risk for opportunistic infections the '/' in balb/c is a lowercase letter because of historical precedent. subsequent substrains follow accepted nomenclature, e.g., balb/cbyj and balb/cann. hybrids of two inbred strains are often used in research, and are particularly common with engineered mutations that are created in -derived es cells, followed by intercrossing the chimeric mice with c bl/ or other background strains of mouse. when an f hybrid is created, the female partner is listed first, e.g., a c bl/ j × s /svpas hybrid would be designated: c bl/ j s /svpasf . ri strain sets that are derived from two parental inbred strains are identified by an x between the two parental strains followed by a hyphen designating the specific ri line, e.g., c bl/ jxdba/ j- , c bl/ jxdba/ j- , etc. cc ri strains do not use the x between the parental strains because they are derived from eight parental strains, so they are designated cc- , cc- , etc. in order to simplify the complexity of this nomenclature, abbreviations are used for common inbred strains and substrains of mice (table . ), but it is important to include the full genetic nomenclature in publications. using the abbreviated nomenclature, c bl/ j s /svpasf mice would be b f and c bl/ jxdba/ j- ri mice would be bxd- . parental order is an important consideration in nomenclature, as a b mouse is genetically different from a b mouse due to mitochondrial dna (from the female) and y chromosome (from the male) differences. mutant genes are designated by a brief abbreviation for the mutation (e.g., bg for beige which arose at the jackson laboratory, j). the symbol for the parent gene is noted in italics, starting with an uppercase letter (e.g., lyst) and the mutant allele is designated in superscript (e.g., lyst bgj ). thus, the beige mutation arose in c bl/ j mice, so that c bl/ j beige mice, which are co-isogenic with c bl/ j mice, are designated c bl/ j-lyst bgj . a transgenic strain is designated by the strain and substrain name, followed by a symbol for the transgene. transgene symbols take the form tg(yyy)#zzz, where 'tg' indicates transgenic, yyy defines the transgene as a brief description of the inserted dna (such as a gene symbol), '#' is the assigned number in the series of events generated using a given construct, and 'zzz' is the lab code. for example, fvb/n-tg(mmtv-erb ) led mice are inbred fvb/n mice in which the rat erb gene was introduced under control of the mouse mammary tumor virus (mmtv) ltr promoter (mmtv-erb ), the first line ( ) created in the laboratory of phil leder (lab code led). when a transgene causes an insertional mutation in an identified endogenous gene, the mutant allele of the gene is designated by using the gene symbol and an abbreviation for the transgene as a superscript (-abc tg zzz ). a targeted mutation, or knockout, is designated by the mutated gene with the identification laboratory animal medicine (foreman et al., ) . barrier practices and microisolator techniques may include autoclaved or irradiated feed and bedding, autoclaved or acidified water, cage-tocage transfer of mice using disinfected forceps, positive displacement change hoods, and verified sanitation of caging and equipment through tunnel or rack washers to prevent fomite transmission of infectious agents (compton et al., ) . in addition to husbandry staff, it is critical to maintenance of colony health status that investigators who handle cages are also trained in these techniques. the microenvironment for mice is the cage which will vary in design, size, and composition. vendors often successfully house production colonies in open-top cages to expedite detection of pathogen transmission should a break occur. end-users usually prefer filter-top microisolator cages which prevent (at least) gross contamination between cages by fecal contamination and aerosolized debris. the objective is to keep mice in an uncrowded, socially compatible, low-odor, dry and clean environment. ambient temperature should minimize any confounding impact on the animal model and energy expenditure for the mice, while also being suitable for staff and investigators. shoebox static cages made of polycarbonate, polypropylene, or polystyrene plastic (in order of decreasing cost and durability) with filtered microisolator tops continue to be used for housing and breeding mice. older cage designs are being rapidly supplanted by individually ventilated caging systems that promote the advantages of increasing housing capacity, decreasing labor costs, and mitigating exposure of mice to noxious gases such as ammonia and exposure of humans to allergens. as more advanced caging systems are developed, the level of biosecurity may be increased but at the cost of increased health surveillance efforts to detect the source of an infectious outbreak (shek, ) . disposable, recyclable polyethylene caging is a recent innovation, particularly for facilities not equipped with a cage wash facility. animal care programs should carefully consider the necessity for housing mice on wire-mesh flooring because of injury risk to limbs and thermoregulation issues in neonates and hairless mice which are more difficult to maintain without nesting material. solidbottom cages should contain sanitary bedding, such as hardwood chips, paper products, or ground corn cob. criteria for selecting bedding vary with experimental and husbandry needs. it may be preferable to irradiate or autoclave bedding, but if this is not done, the bedding should be used only after its origin and microbial content have been evaluated (table . ). germfree and gnotobiotic mice require positive pressure isolators, most usually flexible film, with additional protection provided by sterile air through high-efficiency particulate air (hepa) filters. this equipment can be negatively pressurized when the objective is to contain known or unknown pathogens. animal care programs should establish enrichment policies which for mice should include social housing when mice are compatible and experiments do not require single housing. species-specific behaviors are encouraged by nesting material and hiding places such as tubes or shacks. nutrient requirements for the mouse are influenced by genetic background, disease status, growth rate, pregnancy, lactation, and environmental factors such as ambient temperature. the best current estimate of nutritional requirements is shown in table . . nutritional requirements for laboratory mice are also published periodically by the national research council and have been reviewed by knapka and coworkers (knapka et al., ; knapka, ) . feed intake and weight gain data are used to estimate the nutritional needs of a particular stock or strain. mice consume about - g of feed per day after weaning, and maintain this intake throughout life. outbred mice tend to gain weight faster than inbred mice and are heavier at maturity (figs. . and . ). diet is often neglected as a variable in animal-related research. diet can influence responses to drugs, chemicals, or other factors and lead to biased research results. therefore, diet must provide a balance of essential kraft ( ) . knapka ( ) . b linoleic acid: . % is adequate. c john and bell ( ) . d theuer ( ) . e knapka et al. ( ). f nutrition ( . g hurley and bell ( ) . h pleasants et al. ( ) . nutrients, and contaminants must be kept to a minimum (see also chapter ). natural-product commercial diets for mice are usually satisfactory for breeding and maintenance. animal care programs should avoid using fresh produce, grains, fish meal, or other supplements to minimize exposure of colonies to pathogens or harmful chemicals such as pesticide residues or phytoestrogens (guerrero-bosagna et al., ) . mouse diets can be purchased as open-formula, fixedformula, constant nutrition, and closed-formula which laboratory animal medicine are designed to reduce variation in experimental data attributable to diet (reviewed in barnard et al. ( ) ). diets are supplied in standard, irradiated, or autoclavable formulations. irradiated diets will be virtually free of live microorganisms but have the risk of residual, radio-resistant bacteria. autoclavable diets are higher in heat-labile nutrient content. many programs use sterilized mouse chow exclusively to minimize risk of opportunistic infections. because commercial diets vary in nutrient content, diets should be selected for optimal maintenance of adult mice or for growth and reproduction in breeding colonies. mice should have continuous access to potable water even if a high-moisture diet is fed. water is needed for lubrication of dry food and for hydration. adult mice drink - ml of water per day. decreased water intake will decrease food consumption. water imbalance may occur immediately post weaning and weanlings on automatic watering systems need extra attention. water intake will decrease in sick mice. therefore, dosing mice with medicated water requires careful assessment of hydration and clinical or experimental efficacy of the compound administered. the main reference used to update this section of the rd edition is volume iii; normative biology, husbandry and models in the mouse in biomedical research, nd edition, aclam series published by academic press. normative data on the mouse are presented in table . , and clinical chemistry reference ranges are summarized in table . . mice have a relatively large surface area per gram of body weight. this results in dramatic physiologic changes in response to fluctuations in the ambient temperature (t a ). the mouse responds to cold exposure, e.g., by nonshivering thermogenesis. a resting mouse acclimated to cold can generate heat equivalent to triple the basal metabolic rate, a change that is greater than for any other animal. a mouse must generate about kcal/m per h to maintain body temperature for each °c drop in t a below the thermoneutral zone. mice cannot tolerate nocturnal cooling as well as larger animals that have a greater heat sink. therefore, it is not advisable to conserve energy in animal quarters at night by lowering t a . because of the ratio of evaporative surface to body mass, the mouse has a greater sensitivity than most mammals to water loss. its biological half-time for turnover of water ( . days) is more rapid than for larger mammals. water conservation is enhanced by cooling of expired air in the nasal passages and by highly efficient concentration of urine. the conservation of water can preempt thermal stability. if the mouse had to depend on the evaporation of body water to prevent elevations of body temperature, it would go into shock from dehydration. the mouse has no sweat glands, it cannot pant, and its ability to salivate is severely limited. mice can partially compensate for changes in t a increases from °c to °c. it adapts to moderate but persistent increases in environmental temperature by a persistent increase in body temperature, a persistent decrease in metabolic rate, and increased blood flow to the ears to increase heat loss. its primary means of cooling in the wild is behavioral -retreat into a burrow. in the confinement of a cage, truck, or plane, mice do not survive well in heat and begin to die at an ambient temperature of °c or higher. thus, the mouse is not a true endotherm. in fact, the neonatal mouse is ectothermic and does not have well-developed temperature control before days of age. the thermoneutral zone for mice varies with strain and with conditioning but is about . - . °c, narrower than that of any other mammal measured thus far. thermoneutrality should not be equated with comfort or physiological economy. recent data have suggested that mice housed under routine vivarium conditions are chronically cold-stressed. mice maintained at °c were shown to expend more energy compared with mice housed at intermediate ( °c) and a higher temperature ( °c) with an increase in glucose utilization and activation of brown adipose tissue (david et al., ) . in contrast, other studies report that mice in a t a range of - °c grow faster, have larger litters, and have more viable pups than those maintained in the thermoneutral zone. the respiratory tract has three main portions: the anterior respiratory tract consists of nostrils, nasal cavities, and nasopharnyx; the intermediate section consists of larynx, trachea, and bronchi, all of which have cartilaginous support; and the posterior portion of the respiratory tract consists of the lungs. the left lung is a single lobe. the right lung is divided into four lobes: superior, middle, inferior, and postcaval (cook, ) (fig. . loeb and quimby ( ) . a mouse at rest uses about . ml o /g/h, which is about times more o /g/h than is used by an elephant. to accommodate for this high metabolic rate, the mouse has a high alveolar p o ; a rapid respiratory rate; a short air passage; a moderately high erythrocyte (rbc) concentration; high rbc hemoglobin and carbonic anhydrase concentrations; a high blood o capacity; a slight shift in the o -dissociation curve, enabling o to be unloaded in the tissue capillaries at a high p o ; a more pronounced bohr effect, i.e., the hemoglobin affinity for o with changes in ph is more pronounced; a high capillary density; and a high blood sugar concentration. the kidneys, ureters, urinary bladder, and urethra form the urinary system. the paired kidneys lie against the dorsal body wall of the abdomen on either side of the midline. the right kidney is normally located anterior to the left kidney. kidneys from males of many inbred strains are consistently heavier than kidneys from females. the glomeruli of mice are small, about μm in diameter, or about half the size of glomeruli in rats. there are, however, . times as many glomeruli in the mouse, and the filtering surface per gram of tissue is twice that of the rat. mice excrete only a drop or two of urine at a time, and it is highly concentrated (table . ). the high concentration is made possible by long loops of henle and by the organization of giant vascular bundles (vasa recta) associated with the loops of henle in the medulla. the mouse can concentrate urine to mosm/l, whereas humans can concentrate to a maximum of mosm/l. mice normally excrete large amounts of protein in the urine. taurine is always present in mouse urine, whereas tryptophan is always absent. creatinine is also excreted in mouse urine, a trait in which mice differ from other mammals. the creatinine/creatine ratio for fasting mice is about : . . mice excrete much more allantoin than uric acid. the submaxillary salivary gland, a mixed gland in most animals, secretes only one type of saliva (seromucoid) in the mouse. the tubular portion of the gastrointestinal (gi) tract consists of esophagus, stomach, small intestine, cecum, and colon. the esophagus of the mouse is lined by a thick cornified squamous epithelium, making gavage a relatively simple procedure. the proximal portion of the stomach is also keratinized, whereas the distal part of the stomach is glandular. gastric secretion continues whether or not food is present. the gastrointestinal flora consists of (at least) species of bacteria that begin to colonize the alimentary canal selectively shortly after birth. the ceca of normal mice contain up to bacteria/g of feces. the bacteria throughout the gastrointestinal tract form a complex ecosystem that provides beneficial effects, such as an increase in resistance to certain intestinal pathogens, production of essential vitamins, and homeostasis of important physiological functions. gnotobiotic animals colonized with known microbiota have been used to great advantage as models for biomedical research (see chapter ). for certain studies, it is desirable to colonize germfree mice with a defined microbiota. in the mid- s, schaedler was the first to colonize germfree mice with selected bacteria isolated from normal mice (schaedler and orcutt, ) . he subsequently supplied animal breeders with this group of microorganisms. these defined bacteria included aerobic bacteria and some less oxygen-sensitive anaerobic organisms. the so-called extremely oxygen-sensitive (eos) fusiform bacteria, which make up the majority of the normal microbiota of rodents, were not included, because of technical difficulties in isolation and cultivation. of the defined microbiotas later used for gnotobiotic studies, the one known as the 'schaedler flora' was the most popular. in , the national cancer institute (nci) decided to revise the schaedler flora, or 'cocktail' consisting of eight bacteria, in order to standardize the microbiota used to colonize germfree rodents. the new defined microbiota, now known as the 'altered schaedler flora' (asf), consisted of four members of the original schaedler flora (two lactobacilli, bacteroides distasonis, and the eos fusiform bacterium), a spiral-shaped bacterium, and three new fusiform eos bacteria. studies have quantified the regional colonization of the asf strains along the gastrointestinal tract (sarma-rupavtarm et al., ) (fig. . ) . individual strain abundance was dependent on oxygen sensitivity, with microaerotolerant lactobacillus murinus asf present at - cells/g of tissue in the upper gastrointestinal tract and obligate anaerobic asf strains being predominant in the cecal and colonic flora at - cells/g of tissue. it is difficult to monitor a gnotobiotic mouse colony with a defined microbiota. it is necessary to demonstrate that microorganisms of the specified microbiota are present and that adventitious microorganisms are absent. in the past, monitoring relied on bacterial morphology, limited evaluation of biochemical traits, and growth characteristics. with the advent of polymerase chain reaction (pcr) technology, the eight asf strains were identified taxonomically by s rrna sequence analysis . three strains were previously identified as lactobacillus acidophilus (strain asf ), l. salivarius (strain asf ), and bacteroides distasonis (strain asf ), based on phenotypic criteria. s rrna analysis and genome sequencing indicated that each of the strains differed from its presumptive identity (wannemuehler et al., ) . the s rrna sequence of strain asf is essentially identical to the s rrna sequences of the type strains of l. murinus and l. animalis (both isolated laboratory animal medicine from mice), and all of these strains probably belong to a single species. strain asf is a novel lactobacillus that clusters with l. acidophilus and l. lactis. strain asf is a parabacteroides sp. the spiral-shaped strain, strain asf , is in the flexistipes phylum, exhibits sequence identity with rodent isolates of robertson, and has been formally named, mucispirillum schaedleri (robertson et al., ) . the remaining four asf strains, which are eos fusiform bacteria, group phylogenetically with the low-g + c content gram-positive bacteria (firmicutes, bacillus-clostridium group) (asf -eubacterium plexicaudatium; asf -firmicutes bacterium; asf and asf -clostridium sp.) (fig. . ) . the s rrna sequence information was determined by dewhirst et al. ( ) and draft genome sequences for each member of asf were recently published (wannemuehler et al., ) . this genetic data will permit detailed analysis of the interactions of asf organisms during development of intestinal disease in mice that are coinfected with a variety of pathogenic microorganisms. the lymphatic system consists of lymph vessels, thymus, lymph nodes, spleen, solitary peripheral nodes ( fig. . ) , and intestinal peyer's patches. mouse lymph nodes are numerous but typically are small, reaching only a few millimeters. the typical lymph node is beanshaped and consists of a cortex and a medulla. the cortex is divided into b lymphocyte domains, called primary follicles, and t lymphocyte domains, known s i i i i i i c c l l l e s s i i i i i i c c l l l e s s i i i i i i c c l l l e s s i i i i i i c c l l section of the gi tract bacteroides sp. asf the sections are taken from the esophagus (esop.) (section e ), stomach (sections s and s ), small intestine (sections i to i ), ileocecal junction and apical cecum (sections c and c , respectively), and colon (sections l to l ). from sarma-rupavtarm et al. ( ). as the diffuse cortex. the mouse does not have palatine or pharyngeal tonsils. the spleen lies adjacent to the greater curvature of the stomach. different strains of mice have varying degrees of accessory splenic tissue. age, strain, sex, and health status can affect the size, shape, and appearance of the spleen. male spleens, e.g., may be % larger than those of females. most lymphocytes enter and leave the spleen in the bloodstream. the so-called white pulp of the spleen is organized along the central arteriole and is subdivided into t-and b-cell zones. the periarteriolar sheath is composed mainly of cd + and cd + t cells, and lymph follicles, which often contain germinal centers, are located at the periphery. the red pulp consists of sinusoids and hemoreticular tissue. cellular and humoral components of immunity are distributed to the bloodstream and tissues by efferent lymphatic vessels and lymphatic ducts, which empty into the venous system. the thymus is a bilobed lymphoid organ lying in the anterior mediastinum. it reaches maximum size around the time of sexual maturity and involutes between and days of age. the thymus plays a major role in maturation and differentiation of t lymphocytes. this function is not complete in newborn mice. thymectomy is routinely performed in immunological research for experimental manipulation of the immune system. thymectomy of newborn mice causes a decrease in circulating lymphocytes and marked impairment of certain immune responses, particularly cellular immune responses. thymectomy in adult mice produces no immediate effect, but several months later mice may develop a progressive decline of circulating lymphocytes and impaired cellular immune responses. the mutant athymic nude mouse is a powerful experimental tool in the study of the thymus in immune regulation (fogh, ) . the mucosa-associated lymph tissue (malt) contains more lymphoid cells and produces greater amounts of immunoglobulin than both the spleen and the lymph nodes. the term malt designates all peripheral lymphoid tissues connecting to cavities communicating with the external milieu. they include the peyer's patches, the cecal lymphoid tissue, and the lymphoid tissue in upper and lower respiratory tract, as well as the respiratory and genitourinary system. lymphatics drain these lymphoid-rich areas, thus providing a direct link with lymph nodes and the bloodstream. bone marrow and splenic red pulp produce erythrocytic, granulocytic, and megakaryocytic precursors over the life of the mouse. bone marrow is located in the protected matrix of cancellous bone and is sustained by reticular tissue rich in blood vessels and adipose cells (pastoret et al., ) . normal hematologic values are listed in table . . bone marrow-derived mononuclear phagocytes remove particulate antigens and act as antigen-presenting cells for lymphocytes. tissue macrophages, which often function in a similar way, are found in many tissues, including peripheral lymphoid tissues, lung, liver, intestine, and skin. the cardiovascular system of mice is reviewed extensively by hoyt et al. in the nd edition of volume iii; normative biology, husbandry and models in the aclam series the mouse in biomedical research (hoyt, ) . the heart consists of four chambers, the thin-walled atria and the thick-walled ventricles ( fig. . ) . mice conditioned to a recording apparatus have mean systolic blood pressures ranging from to mmhg. an increase in body temperature does not lead to an increase in blood pressure. heart rate, cardiac output, and the width of cardiac myofibers are related to the size of the animal. heart rates from to /min have been recorded for mice, and there are wide variations in rates and blood pressure among strains. the skeleton is composed of two parts: the axial skeleton, which consists of the skull, vertebrae, ribs, and sternum, and the appendicular skeleton, which consists of the pectoral and pelvic girdles and the paired limbs. the normal vertebral formula for the mouse is c t l s c , with some variations among strains, especially in the thoracic and lumbar regions. normal mouse dentition consists of an incisor and three molars in each quadrant. these develop and erupt in sequence from front to rear. the third molar is the smallest tooth in both jaws; the upper and lower third molar may be missing in wild mice and in some inbred strains. the incisors grow continuously and are worn down during mastication. the mouse brain has a typical mammalian structure as documented by a detailed study of the neuroanatomy of the c bl/ j mouse (sidman et al., ) . more recently, gene expression patterns have been used to study the functional anatomy of the mouse brain (bohland et al., ) . use of wild-type and genetically modified mice in behavior, learning, and memory paradigms has exponentially increased over the last decade. the male reproductive organs consist of paired testes, urethra, penis, prostate and associated ducts and glands ( fig. . ) . the female reproductive organs consist of paired ovaries and oviducts, uterus, cervix, vagina, clitoris, and paired clitoral glands ( fig. . ). the clitoral glands are homologous to the male preputial glands and secrete a sebaceous substance through ducts entering the lateral wall of the clitoral fossa. the female mouse normally has five pairs of mammary glands, three in the cervicothoracic region and two in the inguinoabdominal region ( fig. . ). the mammary glands are often not appreciated for how far they extend over the cervical, axillary, and inguinoabdominal flank regions which become the following section summarizes normal reproduction in the mouse. the reader is referred to a more comprehensive text in the aclam series (pritchett and taft, ) and online resources such as the jackson laboratories publication of the biology of the laboratory mouse (http://jaxmice.jax.org/jaxnotes/ / j.html). external influences, such as noise, vibration, diet, light cycle, and cage density, and intrinsic factors, such as health status, genetics, and parity impact reproductive success by directly or indirectly influencing the hypothalamic-pituitary axis for hormonal control of ovarian and testicular function. genotype also dramatically affects the reproductive performance of the mouse. coincident with the explosion in the number of mouse strains, each with unique induced or spontaneous mutations, a sound breeding program must include training of care staff to recognize anticipated and unanticipated breeding performance and strain or stock characteristics. in the new age of genomics, older methods of confirming genetic purity of mouse lines are being replaced with formal genetic monitoring by comparing strain-specific panels of single-nucleotide polymorphisms (snps). follicle-stimulating hormone promotes gametogenesis in both sexes. luteinizing hormone promotes the secretion of estrogen and progesterone in the female and androgen in the male. prolactin promotes lactation and development of the ovary during pregnancy. these gonadal hormones also ensure proper maintenance of the reproductive tract and modulate behavior to promote successful mating. the hypophysis is usually responsive to hormonal influence by day in the male and day in the female. ovarian follicle development begins at weeks of age and matures by days. rising levels of gonadotropins evoke signs of sexual maturity at about the same age. in the female, estrogen-dependent changes such as cornification of vaginal epithelium at the vaginal opening can occur as early as - days. puberty is slightly later in the male (up to weeks). sexual maturation varies among strains and stocks of mice and is subject to seasonal and environmental influences. mating behavior and the ability to conceive and carry fetuses to parturition are under complex hormonal control mediated by the anterior pituitary. the mouse is polyestrous and cycles every - days. in the first two phases (proestrus and estrus), active epithelial growth in the genital tract culminates in ovulation. degenerative epithelial changes occur during the third phase, followed by diestrus, a period of quiescence or slow cell growth. the cycle can be followed by changes in the vaginal epithelium that are often used to determine optimum receptivity of the female for mating and fertilization (table . ). patency of the vaginal orifice and swelling of the vulva are useful signs of proestrus and estrus ( fig. . ) . irregularities of the estrous cycle occur during aging. seasonal and dietary factors, such as estrogenic substances found in a variety of feeds, and genetic backgrounds also influence estrous cycles. estrus is routinely observed in mice at about - h after parturition (postpartum estrus). however, cornification of the vagina is not complete, and fertile matings are not as frequent compared with normal estrus. mice are spontaneous ovulators. ovulation does not accompany every estrus, and estrus may not coincide with every ovulation, because estrus is dependent on gonadal hormones, whereas ovulation is responsive to gonadotropin. the cyclicity of estrus and ovulation is controlled by the diurnal rhythm of the photoperiod. mating, estrus, and ovulation most often occur during the dark phase of the photoperiod. reversing the timing cook ( ) . laboratory animal medicine of the light-dark cycle reverses the time of estrus, ovulation, and mating. pheromones (table . ) and social environment also affect the estrous cycle. for example, estrus may be suppressed in group-housed female mice and reentry into estrus can be synchronized by exposure to pheromones in male mouse urine ('whitten effect'). once exposed to male urine, most female mice will be in estrus within days with a second estrus in about days. hence, estrus can be synchronized by group-housing females prior to pairing with males. in contrast, pheromones from a strange male mouse, particularly of a different strain, may prevent implantation or pseudopregnancy in recently bred females and is known as the 'bruce effect'. see section ii.c on behavior for more detail on the effect of pheromones on mouse reproductive behavior. mating is normally detected by formation of a vaginal plug (a mixture of the secretions of the vesicular and coagulating glands of the male) whose prevalence is highly strain dependent. the plug usually fills the vagina from cervix to vulva (fig. . ). plug detection is often coupled with vaginal cytology to evaluate fertility and conception. when the cervix and vagina are stimulated physically during estrus, prolactin is released from the anterior pituitary to enable the corpus luteum to secrete progesterone. secretion continues for about days. if fertilization has occurred, the placenta takes over progesterone production. if fertilization does not occur, a pseudopregnant period ensues, during which estrus and ovulation do not occur. fertilization usually takes place ( ) testis, ( ) head of epididymitis, ( ) caudal epididymitis, ( ) vas deferens, ( ) testicular vein, ( ) ampullary gland, ( ) seminal vesicle, ( ) anterior prostate, ( ) ureter, ( ) bladder, ( ) ventral prostate, ( ') dorsal prostate, ( ) urethra, ( ) bulbourethral muscle, ( ) ischiocavernosus, ( ) bulbourethral gland, ( ) diverticulum of bulbourethral gland, ( ) penis, ( ) preputial gland, ( ) glans penis, ( ) prepuce, ( ) testicular artery, and ( ) vas deferens artery. adapted from (komarek, ) . fallopian tube, ( ) uterine horn, ( ) endometrium, ( ) cervix, ( ) vagina, ( ) vaginal vestibulum, ( ) clitoris, ( ) clitoral gland, ( ) urethra, ( ) bladder, ( ) medial ligament of bladder, ( ) lateral ligament of bladder, ( ) left ureter, ( ') right ureter, ( ) mesovarium, ( ) mesometrium, ( ) ovarian artery, ( ) uterine horn artery, and ( ) ovarian artery and vein. adapted from (komarek, ) . adapted from komarek ( ) . in the ampulla or the upper portion of the oviduct. ova can be fertilized to produce normal embryos for - h after ovulation. gestation is usually - days. because of postpartum estrus, lactation and gestation can occur simultaneously. lactation can delay gestation because of delayed implantation. this may cause prolongation of gestation for up to - days in certain inbred strains. the effective reproductive life of some inbred strains approaches years where optimum environmental conditions are maintained, but litter size usually decreases as the female ages. therefore, females are usually retired by months of age. average litter size is strain dependent and commonly ranges from to pups. maternal care can account for about % of the variation in body weight of neonatal mice. nursing females usually lactate for weeks. milk production increases up to days postpartum and then declines until weaning at days. interestingly, oxytocin is required for nursing but is not essential for parturition or reproductive behavior (nishimori et al., ) . some transmission of humoral immunity from dam to progeny occurs in utero, but the majority of antibody is transferred through colostrum. transmission of passive immunity by colostral antibodies has been demonstrated to a wide variety of antigens, including viruses, bacteria, and parasites. antibodies continue to be secreted in the milk throughout lactation. decay of maternally acquired immunity occurs within several months after weaning. loss of maternal immunity increases susceptibility to infection and warrants continued care of weaned mice under barrier conditions. mice are socially gregarious animals with strong family bonds who communicate through complex olfactory, auditory, tactile, and visual signals. wild mice aggregate into groups called demes with low exchange of individuals between different groups. each deme consists of kinrelated members with a high degree of natural inbreeding, higher mutation rates compared to other mammals, and a wide range of developmental flexibility based on early life experience, which all contribute to their remarkably successful environmental adaptability. the deme is composed of a dominant breeding male, a hierarchy of females, subordinate males, and juveniles. wild mice occupy territories measuring just a few square meters when food is abundant to several square kilometers. mice are crepuscular (active during the twilight hours of dawn and dusk), strongly territorial, and omnivorous. coprophagy contributes to approximately one-third of their ingesta as an essential nutritional activity. aside from territoriality, social interactions, breeding, burrowing (when conducive substrates are available), and nest building are major activities. in managing laboratory mice, it is important to understand the complex behavioral biology of their free-living counterparts (latham and mason, ) . chemo-olfactory communication is mediated through extremely diverse chemical factors that trigger innate (non-learned) social responses among conspecifics, known as pheromones (table . ). pheromones have been traditionally divided into two broad categories: releaser pheromones, which elicit an immediate behavioral response, and primer pheromones, which mediate a slowly developing and longer-lasting endocrine response. this original definition of pheromone categories has been expanded to another category, termed signaler pheromones, which convey individual or group identity, as well as mediating parent-offspring recognition and mate choice. the biology and genetics of pheromone signaling is being extensively studied in the mouse as a model of mammalian pheromone communication (brennan and zufall, ; rodriguez and boehm, ) . mouse pheromones are excreted in the urine, as well as plantar, salivary, lacrimal, preputial, and mammary glands. in the urine, major urinary proteins (mups), small peptides, mhc class i peptides, volatile chemicals, and sex hormones all contribute to chemosignals that communicate dominance, kinship, diversity, and gender. wild mice possess a great deal of individual variations of roberts et al. ( ) . c ferrero et al. ( ) . laboratory animal medicine these elements, providing a 'bar code' that distinguishes individuals. inbreeding of laboratory mice has reduced individual variation, but each inbred strain possesses a characteristic array of signals, and to a certain extent, unique signals exist among individuals within a strain (sharrow et al., ; sturm et al., ) . pheromones are detected by sensory neurons in the vomeronasal organ, the olfactory epithelium, and the lesser known septal organ of masera within the olfactory epithelium, and the gruenberg ganglion, which is located at the anterior end of the nasal cavity (breer et al., ; chamero et al., ; liberles and buck, ; restrepo et al., ) . neuronal signals are transmitted to the ganglion layer of the olfactory bulb, and thence to the brain. mups are important components of chemosensory communication in mice, and also an important occupational hazard to human handlers. chromosome contains a cluster of mup genes, plus a number of pseudogenes. mups are small soluble proteins known as lipocalins, which bind small organic chemicals (pheromones) with high affinity, and function as pheromone transporters and stabilizers (thereby contributing to slow release), but also act as protein pheromones themselves. they are synthesized in the liver and excreted in the urine, as well as nasal mucosa, lacrimal glands, and salivary glands. their endogenous role on metabolic activity is not yet understood. male mice excrete significantly more mups in the urine than females. one wellcharacterized mup is 'darcin', named after fitzwilliam darcy, the romantic hero in pride and prejudice. as its name implies, it is a female attractant. mups also act as kairomones, which function as chemical signals between species. for example, cat and rat mups invoke fear in mice. mups are important in the laboratory animal management context, as they are excreted in copious amounts ( - mg/ml in urine) and are potent allergens for humans, particularly mus m (ag or ma ), which is encoded by the mup gene (sharrow et al., ) . chemosensory communication has numerous behavioral effects that influence mouse social interactions. one of the most studied behavioral effects is the bruce effect, or pregnancy block, which is a complex physiologic response in which recently conceived females resorb fetuses during early pregnancy in the presence of an unrelated male, particularly a dominant male. the continued presence of the original mate protects the female from this effect (bruce, ) . the vandenbergh effect results in acceleration of puberty of juvenile females in response to male urine (vandenbergh, ) . the lee-boot effect occurs among group-housed females that are isolated from males, in which there is suppression of estrus cyclicity (van der lee and boot, ) . the whitten effect results in synchronization of estrus among a group of females in response to a male (whitten et al., ) . the lee-boot and whitten effects are utilized in the laboratory to assist in induction of synchronized timed pregnancy, but the bruce effect can have deleterious consequences on breeding colonies when foreign males are introduced to a breeding colony, as pheromone communication can occur in the absence of direct contact. the above effects are well-defined pheromone-driven behavioral responses, but chemosensory communication has a myriad of other effects. estrus, pregnant, or lactating females also accelerate puberty among juvenile females. females use odor cues to avoid parasite-laden males, males prefer odors of estrus females, and estrus females prefer odors of dominant males. mice have strong mating and social preferences based upon mhc proteins, which indicate genetic relatedness. maternal recognition of young is also mhc-related, and pups prefer nest odors of maternal and sibling pups based upon mhc relatedness. male aggression against unrelated males is also a strong mhc-related phenomenon. mhc haplotypes determine not only mhc proteins in the urine, and mhc-specific olfactory receptors, but also the composition of volatile chemicals in the urine (kelliher and wersinger, ) . the complexity of social communication extends to auditory stimuli as well. male mice utilize ultrasonic 'birdsong' to vocally communicate and attract females. mouse vocalization patterns are largely genetically innate and unique to each strain of mouse, but they can also be modified, or learned, to a limited extent (arriaga et al., ) . the behavioral biology of the mouse is highly complex, and depends upon genetic, physiologic, social, and environmental variables, which all impact on how laboratory mice can best be managed in captivity. it is clear that this rich complexity cannot be fully addressed under laboratory conditions, but that does not mean that basic needs, such as nest building, burrowing, foraging, and olfactory environments, cannot be provided. for example, intermale aggression, which is particularly apparent in some strains of mice such as balb/c and swiss-origin stocks and strains, can be minimized by maintaining males from infancy as sibling groups, since adult siblings tend not be aggressive to one another. this sibling bond, however, can be easily broken by short-term separation. environmental enrichment often features provision of plastic houses, which may make vivarium managers feel good, but maximal enrichment can be provided by provision of nesting material, which includes structural scaffolding, such as crinkled cardboard, which facilitates construction of three-dimensional nests. mouse nests are replete with 'appeasement' pheromones, thereby contributing to harmony within the cage, whereas introduction of dirty bedding has the opposite effect. frequent cage changing, including removal of established nests, is highly stressful and disruptive to social harmony within a cage. provision of appropriate and adequate amounts of bedding material that is conducive to burrowing is desirable. it is important to remember that mice are socially gregarious, and that mouse welfare is optimally enriched by other mice within a socially harmonious deme (latham and mason, ; van loo et al., ) . a laboratory mouse ethogram, defined as an operationalized list of mouse behaviors, arranged by their adaptive meaning to the animal, is available on the web: www. mousebehavior.org. behavioral phenotyping, particularly of transgenic mice, is used extensively in genomic research. a wide variety of standardized test batteries and approaches are used, depending upon the focus of research (reviewed in crawley ) . initial behavioral evaluations include general health, body weight, body temperature, appearance of the fur and whiskers, and neurological reflexes assessment. specific tests include observations of home cage behaviors, righting reflex, acoustic startle, eye blink, pupil constriction, vibrissae reflex, pinna reflex, digiscan open field locomotion, rotarod motor coordination, hanging wire, footprint pathway, visual cliff, auditory threshold, pain threshold, and olfactory acuity. novel and complex environmental enrichment in animal housing conditions facilitates enhanced sensory and cognitive stimulation as well as physical activity. environmental enrichment and exercise have beneficial effects such as cognitive enhancement, delayed disease onset, enhanced cellular plasticity, and associated molecular processes in animal models of brain disorders (pang and hannan, ) . the immune system of the mouse is very similar to that of humans. the availability of inbred mouse strains, in which each individual animal expresses identical mhc alleles so that tissues and cells can be transplanted without tissue rejection, greatly simplifies and indeed enables functional analyses of immune system components not possible with any other outbred mammalian species. in addition, the ability to genetically manipulate the mouse genome, adding to, altering, and deleting existing genes, enables unprecedented in vivo analysis of immune cell functions. it is for these reasons that the mouse is the primary animal model for immunology research. the immune system is an unusual organ system in that it consists of both solid tissues and various migrating cell populations. the bone marrow and thymus are considered primary lymphoid organs, as sites of hematopoiesis and b-and t-lymphocyte development, respectively. lymph nodes, spleen, and intestinal peyer's patches are considered secondary lymphoid tissues, as sites of immune response initiation. lymph nodes and spleen are analyzed frequently for studies of immune responses and as organs for immune cell isolation. tertiary lymphoid tissue sites are those that form in other solid organs in response to an insult or microbial exposure. among them are the lymphoid cell aggregates of the gastrointestinal and respiratory tract, also called 'gut-associated lymphoid tissue' (galt) and bronchusassociated lymphoid tissues (balt). leukocytes are classified as belonging to the innate or adaptive immune system. the innate immune system responds rapidly to an antigen insult via recognition of pathogen-associated molecular patterns (pamps), such as lipopolysaccharide, bacterial flagellin, single (s)-and double-stranded (ds) rna, and non-methylated dna, via extracellular or intracellular pattern recognition receptors (prrs). receptors include the toll-like receptors (tlrs), such as tlr (recognizing lps), tlr / (ss and dsrna) and tlr (dna), nod-like receptors (nod / ), and rig-like receptors (rig-i, mda- ) among others (takeuchi and akira, ) . cells of the innate immune system are monocytes/macrophages, granulocytes and dendritic cells as well as innate-like lymphocyte populations (ilc) , and , which include natural killer (nk) cells (spits et al., ) . cells of the adaptive immune system (t and b lymphocytes) express a highly antigen-specific receptor that has arisen through gene rearrangement (t-cell and b-cell receptors, respectively). b cells of the b- lineage and γδ t cells are regarded as innate-like cells, as they express a rearranged antigen receptor but seem to respond in an innate-like manner. leukocytes are identified and classified by sets of monoclonal antibodies (mab) against uniquely expressed surface receptors, typically measured by flow cytometry. identification of a unique receptor by one or more mab of the same specificity leads to the assignment of a receptor name, as a 'cluster of differentiation (cd)'. for example, t cells are differentiated into two subsets based on their expression of either cd or cd . cd + t cells (t helper cells) recognize peptides presented in mhc class ii and promote b-lymphocyte activation and activate and regulate cellular immune responses via secretion of differing cytokines (see below). cd + t cells recognize antigenic peptides presented in mhc class i and serve as cytotoxic cells during the cell-mediated immune response where they can destroy infected cells (e.g., against cells containing infectious agents). the major function of b cells is to respond to an encounter with an antigen/pathogen with the production of highly antigen-specific immunoglobulins (ig; antibodies), which can bind to and inactivate pathogens and toxins. activation of b cells can lead to their differentiation to plasma cells, which produce large amounts of ig. five laboratory animal medicine classes or ig 'isotypes' can be distinguished, which differ in effector function: igm, igg, iga, ige, and igd. the latter is expressed only on the surface of b cells in mice. the igg class, the most abundant antibody class in the serum, is further divided into subtypes: igg , igg a/c , igg b , and igg . polymorphisms exist on the ig locus such that some strains of mice produce the igg a subtype (e.g., balb/c), whereas others produce igg c (e.g., c bl/ ) (zhang et al., ) . additional allelic polymorphisms of the locus also exist. for example, balb/c and sv mice express the igh-a allotype, whereas c bl/ mice express the igh-b allotype. recombinant inbred strains of mice exist for both balb/c and c bl/ , which harbor the reciprocal igh locus (i.e., igh-b for balb/c and igh-a for c bl/ mice). these mice are useful tools for tracking b cells following adaptive cell transfer via allotype-specific mab (see below). immunoglobulin isotype production varies according to the type of immunogen used to evoke the response. igm is secreted short term after initial exposure to an antigen, followed by the other ig isotypes. in viral and intracellular bacterial infections, igg a/c is dominant, whereas in extracellular bacterial infections igg dominates the response. igg b and igg are usually induced to carbohydrate or lipid antigens. ige is linked to parasitic infections and to allergy. serum antibodies specific for an immunogen can often be measured for the life of the animal. while serum iga levels are low, iga is the highest produced ig in mice. iga production, however, occurs in plasma cells lodged in the lamina propria of mucosal tissues, from where the iga is actively transported in dimeric form onto the luminal surface of mucosal tissues as 'secretory' iga (brandtzaeg, ). cytokines are secreted signaling molecules involved in cell-cell communication in a complex biological system (table . ). these include the large family of interleukins (ils, currently il- to il- ), tumor necrosis factors (tnfs), interferons (type i, ii, and iii) and growth factors such as granulocyte-macrophage colonystimulating factor (gm-csf) and stem cell factor (scf). cytokine secretion often occurs in response to recognition of antigen via prr or tcr. because of their importance in modulating immunity to antigenic stimuli, mice with specific deletions or overexpression of individual cytokines have been made and have contributed to a detailed understanding of many of their often pleotropic functions (akdis et al., ) . chemokines are a similarly large group of small, secreted molecules that regulate cell trafficking to sites of antigen encounter but also facilitate cell-cell contact by acting as chemoattractants. chemokines are grouped according to the number of cysteines and disulfide bonds in the molecule into c-x-c-, c-c, c, and cx cl chemokine ligands (l) and receptors (r) and designated accordingly as cxcr - /cxcl - and ccr - /ccl - (allen et al., ) . immune responses must be coordinated to provide the most appropriate effector functions for the type of pathogen/antigen encountered. immune effector responses differ depending on the life cycle (facultative or obligate intracellular, extracellular, localized, systemic, etc.) and antigen types displayed by the encountered antigen/ pathogen, because this affects the type of prr engaged and activated. prr engagement leads to cytokine and chemokine responses by the first responders, i.e., epithelial cells, local macrophage populations and other innate cells. the type of cytokines and chemokines produced then dictates the types of cells recruited to the site of infection and their subsequent differentiation and functions. the prr engagement also leads to antigen uptake, activation and migration of dendritic cells (dcs) from the site of insult to the regional lymph nodes, where dcs present antigen peptides on mhc molecules to t cells. in addition, the dcs secrete cytokines induced by the initial prr activation, which cause the differentiation of cd t cells towards a particular effector response. for example, secretion of il- in response to activation of tlr or will result in the induction of interferongamma (ifn-γ) production by cd t cells, whereas il- and tgf-β production by dc will induce cd t cells to secrete il- (kara et al., ) . because the dc translates signals from prr at the site of infection into differentiation signals for t cells in the lymph tissues, these cells are regarded as a 'bridge' between the innate and adaptive immune systems. the specific ig isotype secreted in response to a pathogen depends to a large degree on the type of cytokine produced by cd t cells that provide 't-cell help' for b cells. t cells that interact with b cells are identified as a discrete subset termed 't follicular helper cells (t fh )' and it is their cytokine profile that directs b cells to secrete a particular ig isotype (kara et al., ) . the classic t h /t h dichotomy outlined above was in part shaped by the observation that ifn-γ production will lead to switching of b cells to secrete igg a/c, whereas production of il- leads to the secretion of igg . interestingly, it appears that the cytokine profile induced by the effector t-cell population is mirrored by the innate immune response. innate-like lymphocytes also have effector phenotypes that correspond to those of cd t cells and are induced by the same signals and transcriptional regulators (spits et al., ) and the same appears to be true also for macrophages and other innate immune cells (sica and mantovani, ) . while initial studies identified two particular antagonistic effector response types (termed t h and t h and classified by t-cell production of ifn-γ and il- , respectively), more recent studies now demonstrate a much wider array of effector responses in which innate and adaptive immunity acts together to reinforce an immune response phenotype as well as modulate its size by induction of t regulatory cells (t regs ) that generate inhibitory cytokines (kara et al., ; sica and mantovani, ; spits et al., ) . the use of cytokine-deficient and reporter mice that enabled the identification of cytokine-producing cells via expression of a fluorescent reporter was particularly valuable for the development of this more nuanced view of the quality of immune responses. spontaneous mouse models of immune deficiencies have been used extensively in research. their use, plus the expanding number of knockout, transgenic, and dominant negative mouse mutants, has advanced understanding of human immune deficiency diseases as well as basic understanding of the immune system (table . ). interbreeding of multiple immune-deficient mice has allowed the development of 'humanized' mice in which immune cells of the mouse are replaced with those of humans. while many challenges remain to fully replenish mice with components of the human immune system, the use of immune-deficient nod/severe combined immunodeficient (scid)/il- rγ -/recipients for transfer of human peripheral blood lymphocytes, cordblood or bone marrow-derived cd + stem cells with human liver and thymus (blt-mice) is yielding promising results (akkina, ) . investigators using genetically engineered mice are constantly reminded that phenotypic analysis of these animals must be done cautiously because the immune system may be profoundly affected and in ways that are not always anticipated. this may make it difficult to determine whether a given gene product is directly involved or may be secondary to a more global dysregulation of the immune system. as with other biological systems, compensation mechanisms also may mask the phenotype. experimental approaches are being increasingly used to refine the knockout technology by restricting a specific genetic deficiency to a particular tissue of interest using the cre-lox system, in which tissue-specific or temporal restricted expression of the cre recombinase induces the deletion of a 'floxed' gene (mak et al., ) . transgenic mice are available that restrict cre expression to various hematopoietic cells or tissue or drive cre recombination following injection of tamoxifen. other approaches are the generation of 'bone marrow irradiation' chimeras. here, inbred wild-type mice or mice deficient in certain immune cells (table . ) are lethally irradiated by exposure to a gamma-irradiation source to deplete the hematopoietic stem cells. these are then replaced by transfer of bone marrow cells to the irradiated mice. reconstitution of the hematopoietic system is usually achieved within about weeks, during which time mice are provided with antibiotic-containing drinking water to avoid infections of these temporarily immune-compromised animals. transfer of bone marrow from a congenic knockout restricts the genetic defect to the hematopoietic system. a mix of bone marrow from two sources is also often used to generate tissue-specific knockouts. for example, mixing a bone marrow from t-cell-deficient mice ( %) with that of a gene knockout ( %) generates 'mixed bone marrow chimeras' in which all t cells only develop from the knockout, thus lack the gene of interest, whereas most of the other cells t from the wild-type source, effectively constraining the genetic defect to the t-cell population. sets of congenic mice with defined allotypic differences are often used to confirm the source of individual cells. such markers include the gene locus cd . /cdc . or cd . /cd . (thy . /thy . ). alternatively, cells may express a fluorescent transgene, such as green-fluorescent protein (gfp). identification is usually performed by flow cytometry, or less commonly by immunofluorescence or immunohistochemistry. generation of bone marrow chimeras circumvents the time-consuming breeding of cre recombinase-expressing flx/flx mice. however, numerous controls are needed to exclude off-target effects due to irradiation damage. repeat injection of antibodies targeting specific cell populations is another rapid approach that avoids the potential for irradiation damage and allows short-term depletion of individual cell subsets. its main disadvantage is the need to identify mab that bind to surface receptors uniquely expressed by a cell subset of interest and the verification of the efficacy of the depletion. frequently used is antibody treatment for the short-term depletion of t-cell subsets using mab against cd or cd as well as individual cytokines. contemporary knowledge about diseases of laboratory mice has developed primarily from examining the effects of disease on traditional strains and stocks. the widespread use of genetically engineered mice is likely to modify current concepts because of novel or unpredictable interactions among genetic alterations, the genetic backgrounds on which they are expressed, and exogenous factors, such as infectious agents. because the number of combinations is extraordinarily high, clinical and laboratory diagnosticians should be alert to the potential for altered disease expression in genetically engineered mice and not be misled by unexpected signs, lesions, and epizootiology. many microbial agents have the potential to cause disease in mice or interfere with mouse-based research. housing and husbandry in microbiologically sheltered environments are designed to reduce the risks of disruptive infection, especially among immunologically dysfunctional mice, but must be accompanied by effective microbiological surveillance. surveillance should encompass resident mice and mouse products (serum, cell lines, transplantable tumors) procured from external sources. because surveillance strategies will vary with research needs and operating conditions, it is prudent to consult a number of sources, such as the federation of european laboratory animal science associations (felasa) (nicklas et al., ) and commercial laboratories, for guidance. detailed discussion of microbial quality control is provided in chapter . there are also recommendations regarding specific agents in following sections. diagnostic methods involve gross and microscopic pathology, parasitology, microbial isolation and culture, serology, and pcr. serology is particularly important for viral surveillance, and now relies principally on enzyme-linked immunosorbent assay (elisa), multiplex fluorescent immunoassay (mfi) for simultaneous detection of antibodies to multiple agents (hsu et al., ) , indirect fluorescent antibody (ifa) assay, or hemagglutination inhibition (hai), with the latter two methods generally used for confirmation (livingston and riley, ; pritchett-corning et al., ) . mouse antibody production (map) testing has been historically used for testing biological materials for contamination by infectious agents. pcr panels for murine infectious agents are now commercially available and have cost and time-saving advantages as well as improved assay sensitivity and specificity. beyond the classic bacterial and viral murine infections, pcr assays are now available for endo-and ectoparasites (see chapter ). etiology mousepox is caused by ectromelia virus (ectv), an orthopoxvirus that is antigenically and genetically closely related to a number of other poxviruses, including vaccinia, variola, and cowpox viruses. the original isolate of ectv, known as the hampstead strain, was discovered by j. marchal in (marchal, as the cause of epizootic disease among laboratory mice in england. the disease featured amputation of extremities, which marchal termed ectromelia (from the greek, ectro, amputation and melia, limb). other strains of the virus include moscow, nih- , washington university, st. louis , beijing , ishibahsi i-iii, and naval (nav) strains, which vary in virulence, but are essentially indistinguishable genetically and serologically, suggesting a common origin. virus can be isolated from infected tissues by inoculation of cell cultures (bs-c- , hela, l cells) or embryonated eggs. the natural host (and original source of infection of laboratory mice) of ectv remains unknown. clinical signs the expression of clinical signs reflects an interplay among virus-related factors, including virus strain, dose and portal of entry, and host-related factors, including age, genotype, immunological competence, and gender (brownstein et al., a) . during natural epizootics, it was observed that a, bc, dba/ , dba/ , and cba strains developed acute fatal infections, whereas c bl/ mice were resistant to severe disease (briody, ) . experimental studies have shown that all strains of mice are susceptible to infection, but balb/c, a, dba/ , and c h/he mice were highly susceptible, akr and sjl mice were moderately susceptible, and c bl/ mice were highly resistant to lethal infection (bhatt and jacoby, c; wallace and buller, ) . the mechanisms of genetic resistance are not fully understood but appear to reflect multiple genes, some of which appear to be expressed through lymphoreticular cells, including nk cells (brownstein et al., a; jacoby et al., ) . the nuances of cytokine and cellular immune responses to ectv infection have received recent attention (reviewed in buller and fenner ( ) and esteban and buller ( ) ). outbreaks among susceptible mice are often volatile, with variable morbidity and high mortality in susceptible strains of mice. clinical signs such as ruffled fur or prostration may occur for only a few hours before death. mice that survive acute infection may develop chronic disease characterized by a focal or generalized rash anywhere on the body ( fig. . ). conjunctivitis also may occur. skin lesions usually recede within several weeks, but hairless scars may remain. additionally, severe viral infection of the feet and tail during the rash syndrome can lead to necrosis and amputation. epizootiology mousepox is not a common disease. outbreaks occur sporadically and recent outbreaks have been traced to the importation of contaminated mice or mouse products. for example, contaminated mouse serum was responsible for recent outbreaks in the united states (dick et al., ; . natural exposure is thought to occur through direct contact and skin abrasions. cage-to-cage transmission is low and can be virtually nil if filter-topped cages are used (bhatt and jacoby, b) . ectromelia virus is highly stable at room temperature, especially under dry conditions, leading to the potential for prolonged environmental contamination in infected colonies (bhatt and jacoby, d) . aerogenic exposure is not a major factor in natural outbreaks, and arthropod-borne transmission does not appear to occur. virus-free progeny can be obtained laboratory animal medicine from immune dams (bhatt and jacoby, b) . however, intrauterine infection and fetal deaths, albeit rare, have been reported. natural transmission is facilitated by intermediately resistant mice, which survive long enough to develop skin lesions that can shed virus for relatively long periods of time. the risks for transmission are further increased by persistence of infectious virus in excreta and exfoliated scabs. although virus excretion typically lasts for about weeks, virus has been found in scabs and/or feces for up to weeks. resistant mouse strains also are dangerous because they can shed virus during subclinical infections. however, infections in resistant mice tend to be short-lived. highly susceptible mice are a relatively small hazard for dissemination of infection, if properly discarded, because they die before virus shedding becomes prominent. thus, juxtaposition of resistant or intermediately resistant infected mice with highly susceptible mice can provoke explosive outbreaks. infant and aged mice are usually more susceptible to lethal infection than young adult mice. maternal immunity among enzootically infected breeding mice may perpetuate infection by protecting young mice from death, but not from infection. such mice may subsequently transmit infection by contact exposure. pathology the classic descriptions of ectv pathogenesis by fenner remain timely, including the frequently cited and reproduced figure summarizing the pathogenesis of infection ( fig. . ) (fenner, b) . interest in smallpox has renewed the interest in ectv as a model of host response to infection (esteban and buller, ) . ectv multiplies in the cell cytoplasm and produces two types of inclusion bodies. the a type (marchal body) is well demarcated and acidophilic in histological sections. it is found primarily in epithelial cells of skin ( fig. . ) or mucous membranes and can also be found in intestinal mucosa. the b type of inclusion is basophilic and can be found in all ectromelia-infected cells. however, it is difficult to visualize unless cells are stained intensely with hematoxylin. ectv antigen can be readily visualized by immunohistochemistry on formalin-fixed, paraffin-embedded tissue sections (esteban and buller, ; jacoby and bhatt, ) . following skin invasion, viral multiplication occurs in the draining lymph node and a primary viremia ensues. splenic and hepatic involvement begin within - days, whereupon larger quantities of virus are disseminated in blood to the skin. this sequence takes approximately week and, unless mice die of acute hepatosplenic infection, ends with the development of a primary skin lesion at the original site of viral entry. the primary lesion is due to the development of antiviral cellular immunity. severe hepatocellular necrosis occurs in susceptible mice during acute stages of mousepox. white spots indicative of necrosis develop throughout the liver ( fig. . ). in nonfatal cases, regeneration begins at the margins of necrotic areas, but inflammation is variable. splenic necrosis in acute disease commonly precedes hepatic necrosis but is equally or more severe. necrosis and scarring of red and white pulp can produce a macroscopic 'mosaic' pattern of white and red-brown the primary skin lesion, which occurs - days after exposure, is a localized swelling that enlarges from inflammatory edema. necrosis of dermal epithelium provokes a surface scab and heals as a deep, hairless scar. secondary skin lesions (rash) develop - days later as the result of viremia. they are often multiple and widespread and can be associated with conjunctivitis, with blepharitis, and, in severe cases, with buccal and lingual ulcers. the skin lesions also can ulcerate and scab before scarring. diagnosis mousepox can be diagnosed from clinical signs, lesions, serological tests, and demonstration of virus or viral antigen in tissues. observation of characteristic intracytoplasmic eosinophilic inclusions aids detection of infection. several serological tests are available to detect mousepox. historically, the standard test was hai, using vaccinia antigen as a source of hemagglutinin. elisa is more sensitive and specific and has replaced hai for serological monitoring among nonvaccinated mice (buller et al., ) . ectv infection also can be detected by ifa (buller et al., ) and pcr. serological differentiation of mousepox from vaccinia infection in vaccinated mice is based on the lack of hemagglutinin in the vaccine strain of virus. thus, serum from vaccinated mice may react by elisa but should not react by hai. differential diagnosis mousepox must be differentiated from other infectious diseases associated with high morbidity and high mortality. these include sendai pneumonia, mouse hepatitis, and tyzzer's disease. the latter two can be expressed by acute necrosis in parenchymal organs, but they can be differentiated by morphological, serological, and virological criteria. the skin lesions of chronic mousepox must be differentiated from other skin diseases caused by opportunistic or pathogenic bacteria, ascariasis, and bite wounds. prevention and control mousepox is a dangerous disease because of its virulence for susceptible mice. therefore, infected colonies should be quarantined immediately. depopulation has been used as a primary means for control, but confirmation of infection should be obtained before exposed mice are destroyed. tissues, supplies, instruments, or other items that have had potential contact with infected mice should be disinfected by heat or chemicals such as formalin, sodium hypochlorite, or chlorine dioxide. materials should be autoclaved or, preferably, incinerated. disinfected rooms should be challenged with susceptible sentinel animals that are observed for clinical signs and tested for seroconversion after several weeks. depopulation and disinfection must be carried out vigorously. because modern housing and husbandry methods based on the use of microbarrier caging are effective for containing infection, testing and culling properly isolated mice is a potential alternative, especially for irreplaceable breeding mice. such mice can be quarantined along with cessation of breeding to permit resolution of infection (bhatt and jacoby, b) . sequential testing with contact-exposed sentinels should be employed with this option. additionally, maternal immunity from fully recovered dams can protect mice from infection, thereby enhancing opportunities to derive virus-free mice from previously infected dams, with the caveat that progeny will be transiently seropositive with maternally derived antibody. vaccination can control or prevent clinically apparent mousepox. the hemagglutinin-deficient strain of vaccinia virus (ihd-t) is used to scarify skin on the dorsum of the tail. 'takes' should occur in previously uninfected mice by - days, but not in infected mice (bhatt and jacoby, a) (fig. . ). infected mice should be quarantined separately or eliminated. vaccination may not prevent infection, although infection in vaccinated mice is often transient. furthermore, vaccinia virus can be shed from scarification sites for at least several days. therefore, other preventive measures, such as strict controls on the entry of mice or mouse products, combined with periodic serological monitoring, should not be relaxed until diagnostic testing has confirmed the elimination of vaccinia and ectromelia virus. additionally, seroconversion evoked by vaccination must be taken into account in serological monitoring of vaccinated colonies. finally, vaccinia virus is a human pathogen, so vaccination procedures should include personnel protective measures to prevent exposure. research complications the primary threat from mousepox is mortality in susceptible mice. the loss of time, animals, and financial resources can be substantial. (shellam, , ) mice are naturally susceptible to two herpesviruses from the subfamily betaherpesvirinae and in the genus muromegalovirus, the two species murid herpesvirus (of which one of the members is mouse cytomegalovirus (mcmv)) and murid herpesvirus (of which one of the members is mouse thymic virus (mtv)). they are species-specific viruses and distinct from each other and from other rodent herpesviruses. mctv has received considerable attention as a model of human cmv infection. etiology mouse cytomegalovirus (mcmv) is a mouse-specific betaherpesvirus. it can, however, replicate in cell cultures from several species, including mouse (fibroblasts and t cells), hamster, rabbit, sheep, and nonhuman primate. cocultivation may be required to rescue latent virus. clinical signs mcmv causes subclinical infection in adult immunocompetent mice, but experimental inoculation of neonates can cause lethal disease due to multisystemic necrosis and inflammation. epizootiology the prevalence of mcmv in laboratory mice is probably uncommon but undefined, since infection is clinically silent and serological surveillance is not widely practiced. wild mice are commonly infected and serve as a natural reservoir for infection, which implies that the entry of virus into a modern vivarium is most likely to occur from contaminated animal products. persistence is a central feature of nonlethal infection. persistently infected mice excrete virus in saliva, urine, and tears for many months, resulting in horizontal transmission through mouse-to-mouse contact. virus also can infect prostate, testicle, and pancreas, implicating other modes of excretion. vertical transmission does not appear to be a common factor in natural infection. further, maternal immunity protects sucklings from infection. pathology mouse cytomegalovirus can replicate in many tissues, and viremia commonly occurs. lesions are not remarkable during natural infection and may be limited to occasional enlarged cells (megalocytosis) containing eosinophilic intranuclear and/or cytoplasmic inclusions associated with lymphoplasmacytic interstitial inflammation, especially in the cervical salivary glands. susceptibility to experimental infection varies with age, dose, route, virus strain, and host genotype. infection can occur in young and adult mice. however, the pathogenicity of mcmv for mice decreases with age. neonates are highly susceptible to lethal infection, but resistance to disease develops by the time mice are weaned. immunodeficient mice, however, remain susceptible to pathogenic infection as adults. persistent infection often affects the salivary glands and pancreas. the persistence of salivary gland infection appears to be dose dependent. there is experimental evidence that mcmv can produce latent infection of b cells, probably t cells as well as aforementioned tissues. persistent infection may lead to immune complex glomerulonephritis. latent persistent infection can be reactivated by lymphoproliferative stimuli and by immunosuppression. diagnosis mcmv antigens appear to be weak stimuli for humoral antibody production, which is consistent with the fact that cellular immunity is critical for protection against infection. neutralizing antibody titers are low during acute infection and difficult to find during chronic infection. serology and pcr-based diagnosis are available, but neither is widely used because of assumptions that infection has a very low prevalence. detection of enlarged cells with intranuclear inclusions, especially in salivary glands, is diagnostic, if they are present. in situ hybridization can be used as an adjunct to routine histopathology. differential diagnosis mcmv infection must be differentiated from infection with mtv. the latter virus can produce necrosis of thymic and peripheral lymphoid tissue when infant mice are experimentally inoculated. lytic lesions of lymphoid tissues are not a hallmark of mcmv. the viruses can also be distinguished from each other serologically. sialoadenitis with inclusions can occur during infection with mouse polyoma virus. like mcmv, mtv infects the salivary gland as its primary target organ. prevention and control control measures for mcmv have not been established, because it has not been considered an important infection of laboratory mice. cage-to-cage transmission has not been demonstrated, but horizontal infection from contaminated saliva must be considered. the exclusion of wild mice is essential. research complications mcmv can suppress immune responses. apart from the potential for interfering with immunology research, it can exacerbate the pathogenicity of opportunistic organisms such as pseudomonas aeruginosa. etiology mouse thymic virus (mtv) is a herpesvirus (murid herpesvirus ) that is antigenically distinct from mcmv. no suitable in vitro method for cultivation has been developed; therefore, viral propagation depends on mouse inoculation. clinical signs natural infections are subclinical. epizootiology the prevalence of mtv is thought to be low. mice can be infected at any age, although lesions develop only in mice infected perinatally. mice infected as infants or adults can develop persistent infection of the salivary glands lasting several months or more. excretion of virus in saliva is considered the primary factor in transmission. seroconversion occurs in adults but does not eliminate infection. infection in neonates may not elicit seroconversion, rendering such mice serologically negative carriers. the mode of infection is obscure, but virus is excreted in saliva, suggesting that transmission from infected dams to neonatal mice occurs by ingestion. mtv also has been isolated from the mammary tissue of a lactating mouse, suggesting the potential for transmission during nursing. prenatal transmission has not been found. pathology mtv causes severe, diffuse necrosis of the thymus and lymphoid tissue with tropism for cd + t cells in mice inoculated within approximately week after birth. the severity of thymic and lymph node necrosis can be mouse strain-dependent. grossly, the thymus is smaller than normal. infected thymocytes display mtv-positive intranuclear inclusions. necrosis is followed by granulomatous inflammation and syncytium formation. reconstitution of lymphoid organs takes - weeks. diagnosis thymic necrosis associated with intranuclear viral inclusions is the hallmark lesion. viral antigen can be detected by immunohistochemistry. serologic detection is effective, but generally not utilized, and is potentially negative in neonatally exposed mice. suspicion of infection in seronegative mice can be tested by inoculation of virus-free neonatal mice with homogenates of salivary gland or with saliva. inoculated mice should be examined for thymic necrosis - days later. pcr or the mouse antibody production (map) test can also be used to detect infection. differential diagnosis reduction of thymus mass can occur in severe mouse coronavirus infection, during epizootic diarrhea of infant mice, or following stress. prevention and control because mtv induces persistent salivary infection, rederivation or restocking should be considered if infection cannot be tolerated as a research variable. research complications mtv transiently suppresses cellular and humoral immune responses because of its destructive effects on neonatal t lymphocytes. parvoviruses are among the most common viral infections in contemporary laboratory mouse populations (livingston et al., ) (pritchett-corning et al., ) , and pose major challenges to both detection and control. the mouse parvoviruses are composed of two antigenically and genetically distinct but related groups, including minute virus of mice (mvm) and mouse parvovirus (mpv), with each group containing a number of strains. the international committee on taxonomy of viruses classifies mpv, mvm, and several other rodent parvoviruses into one genus, protoparvovirus, and species, rodent protoparvovirus , but these viruses will be treated separately herein. etiology minute virus of mice (mvm) is a small ( -kb) single-stranded dna virus. the prototypic strain is designated mvmp. an allotropic variant with immunosuppressive properties in vitro is named mvmi, and additional laboratory animal medicine named strains include mvmc and mvmm. the genome encodes two nonstructural proteins, ns- and ns- , which are highly conserved among the rodent parvoviruses and account for prominent cross-reactivity in serological assays that utilize whole virus antigen. the viral capsid proteins, vp- and vp- , are virus-specific and form the basis for serological differentiation of mvm from mpv. mvm has a broad in vitro host range. it replicates in monolayer cultures of mouse fibroblasts (a cells), c rat glial cells, sv (simian virus )-transformed human newborn kidney ( k cells), t-cell lymphomas (el ), and rat or mouse embryo cells, producing cytopathic effects that can include the development of intranuclear inclusions. clinical signs natural mvm infections are subclinical. neonatal mice of some inbred strains are experimentally susceptible to lethal renal and/or intestinal hemorrhage during mvmi infection, but this syndrome has not been reported in natural outbreaks. experimental inoculation of adult c.b- -prkdc scid (scid) mice with mvmi results in lethal infection (lamana et al., ; segovia et al., ) , and similar severe illness has been noted in naturally infected b-cell-deficient nod. cg-h h -igh null mice (naugler et al., ) . epizootiology mvm is a common virus that naturally infects laboratory mice, but appears to be less common than mpv (besselsen et al., ; livingston et al., ) . mvm is moderately contagious for mice, its only known natural host. virus can infect the gastrointestinal tract and is excreted in feces and urine. the resistance of rodent parvoviruses to environmental inactivation increases the risks of transmission after virus is excreted. therefore, contamination of caging, bedding, food, and clothing must be considered a risk for the spread of infection. transmission occurs by oronasal exposure, but viral contamination of biologicals used for experimental inoculation, such as transplantable tumors, also can be a source of infection. continuous contact exposure to infected animals or soiled bedding usually induces a humoral immune response within weeks, but limited exposure may delay seroconversion. young mice in enzootically infected colonies are protected by maternal antibody, but actively acquired immunity develops from infection sustained after the decay of maternal immunity. mvm, in contrast to mpv, is not thought to cause persistent infection; infection in immunocompetent adult mice usually lasts less than weeks (smith, ; smith and paturzo, ). infection appears to last less than month, even in oronasally inoculated neonatal mice, but immunodeficient mice may be persistently infected. there is no evidence that mvm is transmitted in utero. pathology natural infections or experimental inoculation of adult mice appears to be nonpathogenic. contact-exposed neonates have been reported to develop cerebellar lesions, but these are very rare. experimental infection of neonatal balb/c, swr, sjl, cba, and c h mice with mvmi can cause renal hemorrhage and infarction (brownstein et al., b) . dba/ mice also developed intestinal hemorrhages and accelerated involution of hepatic hematopoiesis. c bl/ neonates are resistant to vascular disease. this lesion has been attributed to viral infection of endothelium. infection of immunodeficient mice, including scid and b-cell-deficient mice, results in lethal damage to granulomacrophagic, megakaryocytic, and erythrocytic hematopoietic tissue with severe leukopenia (lamana et al., ; naugler et al., ; segovia et al., ) . intranuclear viral inclusions and viral antigen have been observed in splenic mononuclear cells of b-cell deficient mice (naugler et al., ) . diagnosis serology is the primary method of detecting infection, which utilizes recombinant mvm and mpv major capsid viral proteins (vp ) as antigens, which discriminate between the two groups of mouse parvoviruses. in contrast, the conserved nonstructural protein, ns can be used to detect antibody to both groups, but is less sensitive than vp assays (livingston et al., ) . mvm infection also can be detected by pcr, in situ hybridization, and immunohistochemistry. pcr assays can be used to detect mvm-or mpv-specific vp or all rodent parvovirus group specific ns exons (besselsen, ; besselsen et al., ) . mvm can be isolated from the spleen, kidney, intestine, and other tissues by inoculation of the c rat glial cell line. it also can be detected by the mouse antibody production test. prevention and control because mvm does not persist in immunocompetent mice, control and elimination should exploit quarantine combined with thorough disinfection of the environment, because parvoviruses are resistant to environmental inactivation. mpv has been shown to be successfully eliminated by a cage-bycage test (serology and fecal pcr) and cull approach, although there are no published reports confirming the success of this strategy for eliminating mvm (macy et al., ) . cesarean rederivation or embryo transfer may also be used to rederive virus-free progeny. prevention of mvm infection depends on strict barrier husbandry and regular surveillance of mice and mouse products destined for use in vivo. research complications mvm contamination of transplantable neoplasms can occur; therefore, infection can be introduced to a colony through inoculation of contaminated cell lines. failure to establish long-term cell cultures from infected mice or a low incidence of tumor 'takes' should alert researchers to the possibility of mvm contamination. mvmi has the potential to inhibit the generation of cytotoxic t cells in mixed lymphocyte cultures. etiology mouse parvovirus (mpv) is among the more common viruses detected within contemporary laboratory animal medicine mouse colonies, and is more common than mvm (livingston et al., ; livingston and riley, ; pritchett-corning et al., ) . mpv was initially isolated following its detection as a lymphocytotropic contaminant in in vitro assays for cellular immunity. the virus grew lytically in a cd + t-cell clone designated l and inhibited the proliferation of cloned t cells stimulated with antigen or interleukin (il- ) (mckisic et al., ) . molecular analysis of mpv indicates that regions encoding the ns proteins are similar to those of mvm (and other rodent parvoviruses). however, they differ significantly in regions encoding the capsid proteins, accounting for their antigenic specificity. the prototype isolate was first called an 'orphan' parvovirus of mice because its biology and significance were obscure, but it has subsequently been named mouse parvovirus (mpv). immortalized t cells (l ) are the only cells found thus far to support replication of mpv. there are three genetically distinct variants of mpv, including mpv- , mpv- , and mpv- . mpv- includes a number of closely related variants, including mpv- a, mpv- b, and mpv- c. in addition, a hamster parvovirus isolate is closely related to mpv- , which is infectious to mice and likely to be of mouse origin (besselsen et al., ; christie et al., ) . clinical signs mpv infection is clinically silent in infant mice and adult immunocompetent or immunodeficient mice (besselsen et al., ) . immunologic perturbations are the most likely signs of infection (mckisic et al., ) . epizootiology mpv causes persistent infection in infant and adult mice, a property that differentiates it from mvm. in situ hybridization has identified the small intestine as a site of viral entry and early replication, but respiratory infection cannot be excluded. experimental studies following inoculation of neonatal balb/c and c.b- -prkdc scid (scid) mice revealed that balb/c mice shed high levels of virus for weeks, with transmission to sentinels exposed during the first weeks of infection. thereafter, balb/c mice shed extremely low virus intermittently. in contrast, scid mice shed high levels of virus until weaning, but lower levels at weeks of age, yet they effectively transmitted infection to sentinels at all stages of infection (besselsen et al., ) . others have shown that transmission of mpv by sencar mice inoculated as infants was intermittent up to weeks, whereas transmission by mice inoculated as weanlings occurred during the first weeks of infection . transmission to balb/c progeny from infected dams was shown to occur, but embryo transfer rederivation was found to be successful in experimentally infected scid mice (besselsen et al., ) . humoral (e.g., passively or maternally acquired) immunity can protect against mpv infection. however, immunity to mvm may not confer cross-immunity to mpv (hansen et al., ) . pathology mpv appears to enter through the intestinal mucosa, which is a site of early virus replication ( fig. . ). acute infection is widespread but mild, involving the lung, kidney, liver, and lymphoid organs. histological lesions are not discernible. lymphocytotropism is a characteristic of acute and persistent mpv infection in infant and adult mice. during acute infection, virus is dispersed within lymph nodes, but during persistent infection virus localizes in germinal centers (fig. . ) . diagnosis because infected mice do not manifest signs or lesions and the virus is very difficult to propagate in cell culture, detection and diagnosis rely on serology and molecular methods. serology that utilizes mpv vp as antigen is a sensitive and specific assay that differentiates mpv from mvm (livingston et al., ) . the map test also can be used to detect parvovirus infections but is relatively time-consuming and expensive. as noted for mvm, pcr for murine parvoviruses, using nucleoprotein gene sequences that are conserved among murine parvoviruses, can be used as a screening test. pcr also can be used to detect mpv-specific sequences in the vp gene. although diagnostic pcr is sensitive and specific, it is effective only in actively infected animals. it can be used on feces to detect virus shedding, or applied to tissues, such as mesenteric lymph nodes, obtained at necropsy. differential diagnosis mpv infection must be differentiated from mvm infection. because both viruses are enterotropic and lymphocytotropic, serology and pcr must be used to distinguish between them. prevention and control the persistence of mpv in individual mice, its potential for provoking immune dysfunction, and the resistance of murine parvoviruses to environmental inactivation favor active control and prevention of mpv infection. quarantine of infected rooms is appropriate. elimination (depopulation) of infected mice should be considered if they are an immediate threat to experimental or breeding colonies and can be replaced, but a cage-by-cage test and cull approach has been shown to be successful under natural conditions (macy et al., ) . for mice that are not easily replaced, virus persistence in the absence of transplacental transmission favors cesarean rederivation or embryo transfer as relatively rapid options to eliminate infection. control of infection also should include environmental decontamination. chemical disinfection of suspect animal rooms and heat sterilization of caging and other housing equipment are prudent steps. prevention is based on sound serological monitoring of mice and surveillance of biologicals destined for inoculation of mice. with the increasing use of mouse germplasm, it is important to note that mouse sperm, oocytes, ovarian tissue, and preimplantation embryos from enzootically mpvinfected mouse colonies may have a high prevalence of mpv contamination, based upon pcr (agca et al., ) . research complications murine parvoviruses can distort biological responses that depend on cell proliferation. for mpv, such effects are seen on immune function and include augmentation or suppression of humoral and cellular immune responses. etiology adenoviruses are nonenveloped dna viruses that produce intranuclear inclusions in vitro and in vivo. two adenovirus species in the genus mastadenovirus have been associated with mice: murine mastadenovirus a (with the representative strain being mav- or fl) and murine mastadenovirus b (with the representative strain being mav- or k ). both strains replicate in mouse kidney tissue culture but are antigenically distinct. clinical signs mav- can cause severe clinical disease after experimental inoculation of infant mice. signs include scruffiness, lethargy, stunted growth, and often death within days. mav- virus is enterotropic and is responsible for virtually all naturally occurring infections in contemporary mouse populations. infection is usually subclinical in immunocompetent mice, with the possible exception of transient runting among infant mice. wasting disease can occur in athymic mice infected with mav- . epizootiology the prevalence of adenovirus infection in mouse colonies is low, particularly mav- riley, , pritchett-corning et al., ) . transmission occurs by ingestion. adult mice experimentally infected with mav- may remain persistently infected and excrete virus in the urine for prolonged periods. adult mice experimentally infected with mav- excrete virus in feces for at least weeks but eventually recover. athymic mice can shed mav- for at least weeks and episodically for at least months. pathology infection with mav- causes multisystemic disease characterized by necrosis. infant mice are especially susceptible to rapidly fatal infection characterized by necrosis of brown fat, myocardium, adrenal cortex, salivary gland, and kidney, with the development of intranuclear inclusions. more mature mice usually develop subclinical infection leading to seroconversion; however, athymic and scid mice can develop intestinal hemorrhage and wasting, with fatal disseminated infection (lenaerts et al., ) . infection with mav- produces amphophilic, intranuclear inclusions in intestinal epithelium, especially in the distal small intestine (fig. . ) . inclusions are easier to detect in infant mice than in adults. infection of c.b- -prkdc scid mice with mav- results in enteric infection, but also hepatic lesions resembling reye's syndrome (pirofski et al., ) . diagnosis although mav strains can be isolated in tissue culture, routine diagnosis depends on detection of infection by serological assay and/or demonstration of adenoviral inclusions, most commonly in the intestinal mucosa. cross-neutralization tests have revealed that antiserum to mav- neutralizes both strains, but antiserum to mav- neutralizes mav- weakly at best. therefore, mav- antigen should be used for the serological detection of adenovirus infection irrespective of the assay employed. mav also can be detected by pcr. differential diagnosis intranuclear adenoviral inclusions in intestinal epithelium are pathognomonic laboratory animal medicine and differentiate mav- infection from other known viral infections of mice. infection may resemble rotavirus infection, with runting and abdominal bloating in infant mice. prevention and control prevention requires serological monitoring of mice and examination for contamination of animal products such as transplantable tumors. because mav- infection appears to be transient in individual mice, segregation of infected colonies may be effective for control. however, rederivation coupled with subsequent barrier housing is a more conservative approach. research complications mav infection is unlikely to affect research using immunocompetent mice. however, it has the potential for pathogenicity in immunodeficient mice. mice can incur natural infection with two polyomaviruses: polyoma virus (pyv) and k virus. these viruses belong to the family polyomaviridae. k virus belongs to the genus polyomavirus and the species murine pneumotropic virus, while the classical polyoma virus belongs to the species murine polyomavirus. etiology polyoma virus (pyv) is a small dna virus that derives its name 'polyoma' (many tumors) from its ability to experimentally induce multiple types of tumors in mice experimentally infected as neonates. its primary importance stems from use in murine models of experimental oncogenesis, with natural infection being rare. the transformative activity is mediated by 't' (tumor) antigens, encoded by large t, middle t, and small t genes, with middle t (mt) being considered the major viral oncogene, and as a result has been used extensively in transgenic constructs. clinical signs natural infections in immunocompetent mice are usually subclinical. however, tumor induction, neurological disease, and wasting can occur in naturally exposed immunodeficient mice (mccance et al., ; sebesteny et al., ) . epizootiology modern husbandry and health care have essentially eliminated natural exposure in laboratory mice. pyv is used for experimental studies and thus can inadvertently be introduced to mouse colonies. inoculation of mice with contaminated biologicals or cell cultures is a potential source of entry and spread. natural transmission occurs via the respiratory route. exposure of neonatal mice results in persistent infection and shedding of the virus in urine, feces, and saliva, thereby contaminating the environment for spread to other mice. infection of adult mice is transient, with minimal virus shedding, although pcr has revealed infection lasting up to months in cba mice inoculated with virus as adults (berke and dalianis, ) . maternal antibody is highly effective at preventing infection of newborn mice, but as maternal antibody wanes, mice are partially susceptible, with transient virus shedding. thus, the natural cycle of transmission in enzootically infected populations requires contamination of bedding and nesting material in order to infect and be inefficiently transmitted, which is readily precluded by modern husbandry. intrauterine infection also can occur, and persistent renal infection, contracted neonatally, can be reactivated during pregnancy. as in immunologically immature neonatal mice, pyv infection can persist in adult immunodeficient mice. pathology pyv-induced tumors are essentially a laboratory phenomenon, optimized by virus strain and mouse strain, with akr, c h, c , cba, swr, and others being most susceptible, and c bl/ being among the most resistant to pyv oncogenesis. intranasal inoculation of neonatal mice results in initial replication in pulmonary respiratory epithelium (gottlieb and villarreal, ) followed by viremic dissemination and acute, lethal disease. tumors appear - months after inoculation of surviving mice. tumors of both epithelial and mesenchymal origin arise in multiple organs, particularly mammary carcinomas, basal cell tumors of the skin, carcinomas of salivary glands, thymomas, and various types of sarcomas. athymic mice can develop cytolytic and inflammatory lesions, followed by multisystemic tumor formation. intranuclear inclusions may be present in cytolytic lesions. demyelinating disease and skeletal tumors have been reported in experimentally . laboratory animal medicine inoculated and naturally exposed athymic mice, and myeloproliferative disease has been reported in experimentally inoculated c bl/ -scid mice (szomolanyi-tsuda et al., ) . diagnosis pyv can be isolated in mouse fibroblast cell lines, but infection is ordinarily detected serologically. additionally, pcr and immunohistochemistry can be used. differential diagnosis wasting in athymic mice can be caused by other infectious agents, including coronaviruses, sendai virus (sv), and pneumocystis. intranuclear inclusions can occur in infections caused by mouse adenovirus, mouse cytomegalovirus, and k virus. prevention and control control depends on elimination of infected mice and material, together with prevention of airborne spread. biological material destined for mouse inoculation should be tested for pyv by the map test or molecular diagnostics. research complications pyv infection can affect experiments by inadvertent contamination of cell lines or transplantable tumors, leading to infection of inoculated mice and the potential for epizootic spread. k virus infection k virus has historical importance, and is apparently absent from contemporary mouse populations (livingston and riley, ; pritchett-corning et al., ) , but it continues to be tested for, adding to the expense of infectious disease surveillance. oral inoculation of neonatal mice results in initial infection of capillary endothelium in the intestine, followed by viremic spread. vascular endothelium is the primary target in affected tissues, which often include the lung, liver, spleen, and adrenal glands. dyspnea occurs from pulmonary infection because of edema and hemorrhage. infection of immunocompetent adult mice is subclinical and results in a vigorous immune response. however, both adults and infant mice develop persistent infection. the primary organ for persistence is the kidney, with shedding of virus from tubular epithelium, and shedding can be reactivated by immunosuppression (greenlee et al., ) . additionally, infection of athymic mice can lead to clinical signs and lesions akin to those described for neonatally inoculated mice. gross lesions are limited to pulmonary hemorrhage and edema. histologically, intranuclear inclusions, which are visualized more easily using immunohistochemistry, are present in vascular endothelium of infected tissues. mild hepatitis with hepatocyte degeneration also may develop. infection can be detected by serology or pcr. prevention and control measures, if ever to be found within a mouse population, are similar to those described for pyv. etiology lactate dehydrogenase-elevating virus (ldv) is a mouse-specific small enveloped rna virus belonging to the family arteriviridae. infected mice are persistently viremic, resulting in increased concentration of several serum enzymes, most notably lactate dehydrogenase (ldh). infection is common among wild mice, but is now rare in contemporary laboratory mouse populations. however, surveys of biologic material indicate that ldv may be a common contaminant of biologic materials (nicklas et al., ) . clinical signs infection is subclinical. however, poliomyelitis has occurred in immunosuppressed c and akr mice inoculated with ldv, and has recently been observed in icr-scid mice following inoculation with contaminated biologic material (carlson-scholz et al., ) . epizootiology the primary mode of mouse-tomouse transmission is mechanical transfer from aggressive behavior (e.g., bite wounds). inoculation of mice with contaminated animal products such as cell lines, transplantable tumors, or serum is probably the most common source of induced infection. it is important to note, with respect to mechanical transmission, that infection induces lifelong viremia. natural transmission between cagemates or between mother and young is rare even though infected mice may excrete virus in feces, urine, milk, and probably saliva. pathology viremia peaks within day after inoculation, then persists at a diminished level. the elevation of enzyme levels in blood is thought to result primarily from viral interference with clearance functions of the reticuloendothelial system. ldv selectively targets mature f /f -positive macrophages, which are continually produced by uninfected progenitor cell populations, thereby maintaining persistent infection. virus also escapes immune clearance by evolution of neutralizing antibody-resistant quasi-species. no lesions are seen in naturally infected mice. the only significant lesion that can arise from experimental infection is poliomyelitis. this syndrome requires a combination of immunosuppression (due to age, genetics or induced means), mouse strain (c , akr, c h/fg, and pl), neurotropic ldv strains, and endogenous ecotropic murine leukemia virus. the mouse strain-dependent element is homozygosity for the fv- n allele, which permits replication of endogenous n-tropic ectotropic murine leukemia virus. mice develop spongiosis, neuronal necrosis, and astrocytosis of the ventral spinal cord and brain stem, with axonal degeneration of ventral roots. lesions contain both ldv and retrovirus. although this syndrome is largely experimentally induced, a natural outbreak of poliomyelitis has been reported in fv- homozygous icr-scid mice following inoculation with contaminated biologic material (carlson-scholz et al., ) . diagnosis plasma ldh levels are elevated, a response that is used to detect and titrate ldv infectivity. of the five isoenzymes of ldh in mouse plasma, only laboratory animal medicine ldh-v is elevated. sjl/j mice in particular show spectacular increases in ldh levels ( - times normal), a response controlled by a recessive somatic gene. ldv is detected by measuring ldh levels in mouse plasma before and days after inoculation of specific pathogenfree (spf) mice with suspect material. it is important to use nonhemolyzed samples because hemolysis will produce falsely elevated readings. plasma enzyme levels are measured in conventional units/ml, conventional unit being equivalent to . international units (iu). normal plasma levels are - iu, whereas in ldv infection, levels as high as iu can occur. ldv also interferes with the clearance of other serum enzymes and results in their elevation in serum. in a recent survey, serum samples were tested by serum ldh enzyme assays, among which % were deemed potentially positive. however, pcr revealed that all were false-positives (pritchett-corning et al., ) , emphasizing the inaccuracy of traditional enzyme assays. infection provokes a modest humoral antibody response, but it is difficult to detect because of formation of virus-antibody immune complexes. molecular diagnostics also can be used to diagnose infection in mouse tissues and serum and biologic materials. however, inhibitory factors in cells and serum may cause falsenegative results in pcr testing, so appropriate quality control measures are essential if this method is used (lipman and henderson, ) . prevention and control transplantable tumors have been a common source of ldv historically. therefore, tumors or cell lines destined for mouse inoculation should be monitored for ldv contamination. although ldv can contaminate tumor cell lines, it does not replicate in the tumor cells. therefore, one can attempt to free tumors of virus by passaging them through athymic nude rats, which are not nonpermissive to ldv but are permissive to xenografts. research complications ldv has numerous potential effects on immunological function. it may reduce autoantibody production, cause transient thymic necrosis and lymphopenia, suppress cell-mediated immune responses, and enhance or suppress tumor growth. etiology the house mouse is the natural host for lymphocytic choriomeningitis virus (lcmv), an old world member of the arenaviridae family that has spread worldwide along with m. musculus. lcmv virions are pleomorphic, containing single-stranded rna, and bud from the cell membrane. disease associated with infection is due to host immune response to the otherwise non-cytolytic virus. its name is derived from the immune-mediated inflammation resulting from the intracerebral inoculation of virus into immunologically competent mice. lcmv is a zoonotic virus that may cause a variety of clinical manifestations in humans, including meningitis. it has been extensively studied as an experimental model of virus-induced immune injury, using a number of closely related strains, including ones that have been selected for their relative neurotropism or viscerotropism. lcmv can be propagated in a variety of mammalian, avian, and even tick cell lines, with minimal cytopathic effect. these characteristics favor its propensity to persistently and silently contaminate biologic products, such as tumor cell lines. clinical signs natural infection in immunocompetent adult mice is usually self-limiting and subclinical. during enzootic infection of a mouse population, lcmv is transmitted in utero from persistently infected dams to their fetuses or to neonates, which are persistently infected and immunologically tolerant to lcmv. since lcmv is non-cytolytic in and of itself is minimally pathogenic, congenitally infected mice grow into adulthood, reproduce, and therefore transmit infection to the next generation. however, with age, immune tolerance breaks down, and mice develop a syndrome known as 'late disease' in which mice will progressively lose weight and die. in utero infection results in a low level of fetal mortality and maternal cannibalism of infected pups. the immune tolerance to lcmv is virus-specific, with the mice capable of eliciting effective immune responses against other agents. clinical signs following experimental inoculation of lcmv vary with age and strain of mouse, route of inoculation, and strain of virus. when virus is inoculated intracerebrally into immunocompetent adult mice, mice develop immune-mediated lymphocytic choriomeningitis, characterized by illness beginning - days after inoculation. sudden death may result or subacute illness associated with one or more of the following signs may develop: ruffled fur, hunched posture, motionlessness, and neurological deficits. mice suspended by the tail display coarse tremors of the head and extremities, culminating in clonic convulsions and tonic extension of the rear legs. spontaneous convulsions also can occur. animals usually die or recover in several days. a visceral form of infection can occur in adult mice inoculated by peripheral routes with 'viscerotropic' strains. it can be subclinical or lead to clinical signs, including ruffled fur, conjunctivitis, ascites, somnolescence, and death. if mice survive, recovery may take several weeks. surviving mice may have immune exhaustion due to consumption of lymphoid tissue, in contrast to immune tolerance that occurs when mice are infected in utero or as neonates. runting and death from lcmv infection may occur in neonatally infected mice and can lead to transient illness or to death. clinical signs are nonspecific, recovery is slow, and survivors may remain runted. this early form of disease is attributed to endocrine dysfunction caused laboratory animal medicine by lcmv infection. late-onset disease can occur in previously subclinical carrier mice that develop immune complex glomerulonephritis. it is usually the result of prenatal or neonatal infection and occurs in persistently infected mice when they are - months old. clinical signs are nonspecific and include ruffled fur, hunched posture, weight loss, proteinuria, and ascites. epizootiology lcmv is distributed widely in wild m. musculus throughout the world. among common laboratory species, mice, hamsters, guinea pigs, and nonhuman primates are susceptible to infection, but only the mouse and the hamster are known to transmit virus. lcmv infection is rare in laboratory mice produced and maintained in modern quarters (livingston and riley, ; pritchett-corning et al., ) . infection is usually introduced through inoculation of virusinfected biologicals, such as transplantable tumors, or by feral mice. wild mice are a natural reservoir of infection and a potential threat to research colonies if they gain entry inadvertently. naturally infected carrier mice can have persistently high concentrations of virus in many organs, thereby facilitating virus excretion in saliva, nasal secretions, and urine. persistently infected neonates usually reach breeding age and can perpetuate infection in a breeding colony. thus introduction of a single lcmv carrier mouse to a breeding colony can eventually result in a high prevalence of persistently infected mice. infection in adult mice, in contrast, is often acute because of the onset of effective immunity, and the spread of virus is halted. horizontal spread of infection is enhanced by close contact, but rapid horizontal spread is not characteristic. mice can transmit lcmv to hamsters, which can remain viremic and viruric for many months, even if they contract infection as adults. infected hamsters can transmit virus to other hamsters and mice and are the primary source of human lcmv infection. persistent infection in immunodeficient mice may carry greater risks for viral excretion and zoonotic transmission. pathology lcmv disease is a prototype for virusinduced, t-lymphocyte-mediated immune injury, noncytolytic endocrine dysfunction, and immune complex disease. however, lesions comparable to experimentally induced disease are rare during natural infection. intracerebral inoculation of virus into immunocompetent adult mice induces nonsuppurative leptomeningitis, choroiditis, and focal perivascular lymphocytic infiltrates. host tissues are damaged during the course of the cellular immune response to the virus. the character of visceral lesions depends on virus strain and mouse strain; the ratio of cytolytic to proliferative responses in lymphoid organs is mouse strain-dependent. in severe infection, nonsuppurative inflammation can occur in many tissues. the severity of accompanying cytolytic lesions seems to parallel the intensity of cellular immunity. liver lesions can include hepatocyte necrosis accompanied by nodular infiltrates of lymphoid cells and kupffer cells, activated sinusoidal endothelium, an occasional granulocyte or megakaryocyte, and fatty metamorphosis. cytolysis, cell proliferation, and fibrinoid necrosis can develop in lymphoid organs. necrosis of cortical thymocytes can lead to thymic involution. lesions of late-onset disease are characterized by formation of immune complexes and associated inflammation. renal glomeruli and the choroid plexus are most severely affected, but complexes may also be trapped in synovial membranes, blood vessel walls, and skin. lymphoid nodules can form in various organs. lesions associated with early deaths in neonatally infected mice have not been thoroughly described but include hepatic necrosis. the lesions of acute and persistent lcmv infection reflect separate immunopathologic processes. in adult mice with acute lcmv infection, virus multiplies in dcs, b cells, and macrophages, whereas t cells are resistant. internal viral epitopes induce humoral immune responses, but surface epitopes elicit cell-mediated immunity and neutralizing antibodies. thus, elimination of virus and virus-associated immunological injury are both t-cell-mediated. this apparent paradox has been explained by the view that prompt cellular immunity limits viral replication and leads to host survival, whereas slower cellular immune responses permit viral spread and increase the number of virus-infected target cells subject to attack once immunity is fully developed. antibody can be detected by week after infection but does not play a significant role in eliciting acute disease. lesions of lcmv infection appear to develop from direct t-cell-mediated damage to virus-infected cells and may involve humoral factors released from immune effector t cells. lcmv also can suppress humoral and cellular immunity in acutely infected mice. persistent infection commonly evolves from exposure early in pregnancy, and virus has been demonstrated in the ovaries of carrier mice. prenatal or neonatal infection induces immunological tolerance to lcmv, which can then replicate to high titer in many tissues. nevertheless, persistently infected mice develop humoral antibody to lcmv. antibody can complex with persistent virus to elicit complement-dependent inflammation in small vessels. immune complex glomerulonephritis exemplifies this process, as noted above. diagnosis lcmv infection can be diagnosed serologically. whereas immunocompetent adult mice will normally seroconvert after exposure, carrier mice may develop poor humoral immune responses. therefore, testing must avoid false-negative results. employment of adult contact sentinel mice is a useful strategy for detecting lcmv infection by seroconversion. tissues, including biologic products and cell lines, can be tested laboratory animal medicine by pcr. a traditional method for detection involved collection of small blood samples from persistently infected live suspects, which are often viremic, and using them to inoculate cultured cells or adult and neonatal mice. intracerebral inoculation of lcmv-positive tissues should elicit neurological signs in adult mice within days, whereas infant mice should remain subclinical. histological examination of brains from affected adults may reveal nonsuppurative inflammation, but lesions may be minimal in mice infected with viscerotropic isolates. immunohistochemistry can be used to detect viral antigen in brains of suckling and adult mice. intraperitoneal inoculation of adult mice may yield short-lived infection with seroconversion, i.e., the map test. virus can be grown and quantified in several continuous cell lines, including mouse neuroblastoma (n- ) cells, bhk- cells, and l cells. application of immunofluorescence staining to detect lcmv antigen in inoculated cultured cells yields results more quickly than animal inoculation. of course, all diagnostic procedures involving potential contact with live virus should be carried out under strict containment conditions to avoid infection of laboratory personnel (see chapter ). the use of in vitro detection has the added advantage, in this regard, of reducing biohazardous exposure and the use of live animals for testing. differential diagnosis neurological signs must be differentiated from those due to mouse hepatitis virus, mouse encephalomyelitis virus, and meningoencephalitis from bacterial infection. trauma, neoplasia, and toxicities also must be ruled out in neurological disease with low prevalence. late-onset disease is associated with characteristic renal lesions, including deposition of viral antigen in tissues. early-onset disease must be differentiated from other causes of early mortality, such as mouse hepatitis virus, ectromelia virus, reovirus infection, tyzzer's disease, or husbandry-related insults. prevention and control adequate safeguards for procurement and testing of animals and animal products are essential to prevent entry. because mouse-to-mouse spread is slow, selective testing and culling for seropositive or carrier mice is possible. if mice are easily replaced, however, depopulation is a safer and more reliable option. valuable stock can be rederived, but progeny must be tested to preclude in utero transmission. because infected hamsters can excrete large quantities of virus, exposed hamsters should be destroyed and hamsters should not be housed with mice. infection of immunodeficient mice poses similar risk. lcmv can be transmitted to human beings, who can contract flu-like illness or severe cns disease. more frequently, human infection is subclinical. the zoonotic potential of lcmv infection makes it especially important to detect and eliminate carrier animals and other potentially contaminated sources, such as cell cultures, transplantable neoplasms, and vaccines to prevent human exposure. serum banking and periodic serological testing of highrisk human populations, such as those working with lcmv experimentally, are recommended. research complications lcmv may stimulate or suppress immunological responses in vivo and in vitro, and it can replicate in cells used as targets or effectors for immunological studies. introduction of immune cells to a carrier animal may elicit an immunopathological response. immune complex disease can complicate longterm experiments and morphological interpretations. illness and death in mice and zoonotic risk to humans are obvious research-related hazards. etiology sv is a paramyxovirus that is antigenically related to human parainfluenza virus . viral particles are pleomorphic, contain single-stranded rna, and have a lipid solvent-sensitive envelope that contains glycoproteins with hemagglutinating, neuraminidase, and cell fusion properties. sv grows well on embryonated hens' eggs and in several mammalian cell lines (e.g., monkey kidney, baby hamster kidney , and mouse fibroblast [l]). virus replicates in the cytoplasm and by budding through cell outer membranes. once common in laboratory rodent populations, sv is now rare or absent (livingston and riley, ; pritchett-corning et al., ) . clinical signs clinically affected adult mice often assume a hunched position and have an erect hair coat. rapid weight loss and dyspnea occur, and there may be chattering sounds and crusting of the eyes. although highly susceptible adults may die, lethal infection is more common in suckling mice. sex differences in susceptibility have not been found. genetically resistant mice usually have subclinical infection. athymic mice and immunodeficient mice are at high risk for development of a wasting syndrome. they develop illness later than their immunocompetent counterparts, since clinical signs in immunocompetent mice are related to immune-mediated destruction of respiratory epithelium. opportunistic infections can complicate the clinical presentation. for example, secondary bacterial infections of the ear can cause vestibular signs. epizootiology sv is transmitted by aerosol and is highly contagious. morbidity in infected colonies is commonly %, and mortality can vary from % to %, partly because strains of mice vary greatly in their susceptibility to lethal sv infection. for example, c bl/ mice are highly resistant to clinically apparent infection, whereas dba/ mice are highly susceptible. aerogenic infection is promoted by high relative humidity and by low air turnover. prenatal infection does not occur. enzootic infection is commonly detected in postweaned mice ( - weeks old) and is associated with seroconversion within - days and the termination of infection. therefore, entrenched infection is perpetuated by the introduction of susceptible animals. there is no evidence for persistent infection in immunocompetent mice, but prolonged infection is common in immunodeficient mice. maternally acquired immunity protects young mice from infection, and actively acquired immunity is thought to be long-lived. rats, hamsters, and guinea pigs also are susceptible to sv infection. therefore, bidirectional cross-infection is a risk during outbreaks. pathology viral replication is nominally restricted to the respiratory tract and peaks by the first week after infection. gross lesions feature partial to complete consolidation of the lungs (fig. . ) . individual lobes are meaty and plum-colored, and the cut surface may exude a frothy serosanguinous fluid. pleural adhesions or lung abscesses caused by secondary bacterial infection are seen occasionally, and fluid may accumulate in the pleural and pericardial cavities. sv targets airway epithelium and type ii pneumocytes. type i pneumocytes are less severely affected. histologically, the pattern of pneumonia is influenced by mouse genotype. susceptible mice usually have significant bronchopneumonia and interstitial pneumonia, whereas the interstitial component may be less prominent in resistant mice. typical changes begin with inflammatory edema of bronchiolar lamina propria, which may extend to alveolar ducts, alveoli, and perivascular spaces. necrosis and exfoliation of bronchiolar epithelium ensue, frequently in a segmental pattern (fig. . ). alveolar epithelium also may desquamate, especially in severe disease, and necrotic cell debris and inflammatory cells can accumulate in airways and alveolar spaces. alveolar septae are usually infiltrated by leukocytes to produce interstitial pneumonia (fig. . ) . lymphoid cells also invade peribronchiolar and perivascular spaces. the lymphocytic response to sv infection reflects the fact that cellular immunity contributes both to lesions and to recovery. local immunoglobulin synthesis by infiltrating cells also occurs. the extent of inflammatory cell infiltration corresponds to the level of genetic resistance expressed by the infected host, with clinically susceptible hosts mounting a more florid immune response than resistant hosts. additionally, strain-related differences in the severity of infection may reflect differences in airway mucociliary transport. multinucleated syncytia are occasionally seen in affected sucklings and scid mice, and inclusion bodies have been reported in infected athymic mice. regeneration and repair begin shortly after the lytic phase and are characterized by hyperplasia and squamous metaplasia of bronchial epithelium, which may extend into alveolar septae. proliferation of cuboidal laboratory animal medicine epithelium may give terminal bronchioles an adenomatoid appearance. repair of damaged lungs is relatively complete in surviving mice, but lymphocytic infiltrates, foci of atypical epithelium, and mild scarring can persist. acute phase lesions are prolonged in immunodeficient mice, which can lead to wasting and death. aged mice also have a prolonged recovery phase accompanied by focal pulmonary fibrosis (jacoby et al., ) . diagnosis sv is notable for its ability to cause epizootics of acute respiratory distress in adult genetically susceptible strains. serology is an effective means to detect infection in all strains of immunocompetent mice. antibody can be detected by days postinfection and coincides with development of clinical signs related to the immune-mediated necrotizing bronchiolitis and alveolitis. repeated serologic sampling over several weeks can help stage infection within a population. alternatively, sentinel animals can be added to seropositive colonies to detect active infection. irrespective of serologic results, histopathology, immunohistochemistry (which can be performed on formalin-fixed, paraffin-embedded sections), and, where possible, virus isolation should be used to confirm infection. virus can be isolated from the respiratory tract for up to weeks, with peak titers occurring at about days postinfection. nasopharyngeal washings or lung tissue homogenates are most reliable and should be inoculated into embryonated hens' eggs or bhk- cell monolayer cultures. sv infection of cultured cells is non-cytolytic, so erythrocyte agglutination or antigen detection methods must be used. rt-pcr also can be used to detect virus in infected lungs. differential diagnosis respiratory infection caused by pneumonia virus of mice (pvm) is generally milder or subclinical. histologically, necrosis of airway epithelium is less severe. bacterial pneumonias of mice, including murine respiratory mycoplasmosis, are sporadic and can be differentiated morphologically and by isolation of causative organisms. because sv pneumonia may predispose the lung to opportunistic bacterial infections, the presence of bacteria should not deter evaluation for a primary viral insult. control and prevention sv infection is self-limiting in surviving immunocompetent mice. suckling mice from immune dams are protected from infection by maternal antibody until after weaning. control and eradication measures must eliminate exposure of susceptible animals, so that infection can 'burn out.' this is most easily accomplished by a quarantine period of - weeks wherein no new animals are introduced either as adults or through breeding. control also is aided by the fact that sv is highly labile. barrier housing is preferred for prevention and for control of transmission. vaccination with formalin-killed virus can provide short-term protection of valuable mice but is not commonly used for prevention. research complications sv can cause immunosuppression and can inhibit growth of transplantable tumors. this effect has been attributed to virus-induced modification of tumor cell surface membranes. pulmonary changes during sv pneumonia can compromise interpretation of experimentally induced lesions and may lead to opportunistic infections by other bacteria. they also have been associated with breeding difficulties in mice. this sign is thought to be an indirect effect due to stress, fever, or related changes during acute infection. clinical signs natural pvm infection in mice is subclinical. therefore, its name is clinically misleading, being derived from pneumonic illness that occurred after serial passage of the agent in mice. however, dyspnea, listlessness, and wasting may develop in immunodeficient mice infected with pvm (weir et al., ) . pvm is used experimentally as a model to study acute respiratory infection, using highly pathogenic strains of the virus (dyer et al., ) . epizootiology pvm causes natural infections of mice, rats, hamsters, and probably other rodents and may be infectious for rabbits. serological data indicate that pvm was once common, but is now relatively uncommon (livingston and riley, ; pritchett-corning et al., ). pvm appears to spread less rapidly than sv. intimate contact between mice is probably required for effective transmission. this characteristic may reflect the fact that environmental inactivation of virus occurs rapidly. infection is acute and self-limiting in immunocompetent mice but may persist in immunodeficient mice. pathology pvm replicates exclusively in the respiratory tract and reaches peak titers in the lung - days after infection. although pulmonary consolidation can occur in experimentally infected mice, gross lesions are rare during natural infection. histological lesions can occur in the upper and lower respiratory tract. they consist of mild necrotizing rhinitis, necrotizing bronchiolitis, and interstitial pneumonia, which usually occur within weeks after exposure to virus and are largely resolved by weeks. the predominant inflammatory infiltrate is comprised of mononuclear cells, but some neutrophils laboratory animal medicine are usually present. immunohistochemistry on paraffinembedded tissues can be used to detect viral antigen in bronchiolar epithelium, alveolar macrophages, and alveolar epithelium during acute infection. residual lesions include nonsuppurative perivasculitis, which can persist for several weeks after acute infection has ceased. severe progressive pneumonia, with wasting, can occur in immunodeficient mice. it is characterized by generalized pulmonary consolidation that reflects severe interstitial pneumonia with desquamated alveolar pneumocytes and leukocytes filling alveolar spaces (fig. . ) . diagnosis diagnosis is based primarily on serological detection that can be supplemented by histopathology, immunohistochemistry, in situ hybridization, and virus isolation. virus replication in bhk- cells is detected by immunofluorescence or other antigen detection methods. virus also can be detected in tissues by rt-pcr. differential diagnosis because pvm is antigenically distinct from other murine viruses, serology is the most useful method to separate pvm infection from other respiratory infections of mice. however, in immunodeficient mice, where clinical signs and lesions are typical, it must be differentiated from other pneumonias, especially those due to sv and pneumocystis. additionally, pvm can coexist with and exacerbate pneumocystis infection in immunodeficient mice (bray et al., ) . prevention and control pvm infection is acute and self-limiting in immunocompetent mice, but persistent in immunodeficient mice. seropositive mice should be viewed as either immune or in the final stages of acute infection. therefore, control and prevention follows guidelines applicable to sv infection. research complications pvm can exacerbate pneumocystosis, as noted above. (ward et al., ) two members of the family reoviridae infect laboratory mice: reovirus per se (species: mammalian orthoreovirus) and murine rotavirus (species: rotavirus a), also known as epizootic diarrhea of infant mice (edim) virus. etiology reoviruses of mammals, although taxonomically considered one type species, have been divided into three cross-reacting prototypic serotypes: reovirus , and , which can be differentiated by cross-serum neutralization. mice can be infected with any serotype, but reovirus is emphasized because it has been associated with naturally occurring disease. natural infections in mice are usually not caused by pure serotypes, because reoviruses actively recombine. a number of wild-type and laboratory strains have been characterized, and related viruses have been recovered from virtually every mammal tested, as well as birds, reptiles, and insects. the virion contains segmented, double-stranded rna and is relatively heat stable. reoviruses replicate well in bhk- cells and other continuous cell lines, as well as in primary monolayer cultures from several mammals. clinical signs clinical disease is rare and age dependent. acute disease affects sucklings at about weeks of age, whereas adults have subclinical infection. signs in sucklings include emaciation, abdominal distension, and oily, matted hair due to steatorrhea. icterus may develop and is most easily discerned as discoloration in the feet, tail, and nose. incoordination, tremors, and paralysis occur just before death. convalescent mice are often partially alopecic and are typically runted. alopecia, runting, and icterus may persist for several weeks, even though infectious virus can no longer be recovered. infants born to immune dams are protected from disease by maternal immunity. epizootiology the prevalence of reovirus infection in contemporary mouse colonies is rare (livingston and riley, ; pritchett-corning et al., ). reoviruses are highly contagious among infant mice and can be transmitted by the oral-fecal or aerosol routes, but mechanical transmission by arthropods has also been documented. additionally, virus may be carried by transplantable neoplasms and transmitted inadvertently by injection. transmission is inefficient among adult mice. there is no evidence that vertical transmission is important or that genetic resistance or gender influence expression of disease. infection in immunocompetent mice appears to be self-limiting, lasting up to several weeks but terminating with the development of host immunity. the course of infection in immunodeficient mice should be considered prolonged, but the duration has not been determined. pathology reovirus can cause severe pantropic infection in infant mice. after parenteral inoculation, virus can be recovered from the liver, brain, heart, pancreas, spleen, lymph nodes, and blood vessels. following ingestion, reoviruses gain entry by infecting intestinal epithelial cells (m cells) that cover peyer's patches. virus can be carried to the liver in leukocytes, where it is taken up by kupffer cells prior to infecting hepatocytes. in acute disease, livers may be large and dark, with yellow foci of necrosis. the intestine may be red and distended, and, in infants, intestinal contents may be bright yellow. myocardial necrosis and pulmonary hemorrhages have been reported. myocardial edema and necrosis are especially prominent in papillary muscles of the left ventricle. the brain may be swollen and congested. central nervous system lesions have a vascular distribution, and are most prevalent in the brain stem and cerebral hemispheres. neuronal degeneration and necrosis are followed quickly by meningoencephalitis and satellitosis. severe encephalitis may evoke focal hemorrhage. in the chronic phase, wasting, alopecia, icterus, and hepatosplenomegaly may persist. orally infected suckling mice can develop multifocal hepatocyte necrosis, which may include the accumulation of dense eosinophilic structures resembling councilman bodies. hepatocytomegaly, kupffer cell hyperplasia, and intrasinusoidal infiltrates of mononuclear cells and neutrophilic leukocytes also can develop. in experimentally inoculated mice, necrotic foci can persist in the liver for at least weeks. chronic active hepatitis may develop after acute infection and result in biliary obstruction. acinar cells of the pancreas and salivary glands can undergo degeneration and necrosis. because pancreatic duct epithelium is susceptible to infection, parenchymal lesions in the pancreas may be caused by obstruction rather than by viral invasion of parenchyma. pulmonary hemorrhage and degeneration of skeletal muscles also have been observed. both humoral and cellular immunity seem to participate in host defenses, but it is unclear how host immunity may influence the course of chronic infection. oronasal inoculation of infant mice with reovirus results in a similar distribution but significantly milder lesions compared to reovirus . in contrast, reovirus is highly enterotropic, inducing mild enteritis without lesions in other tissues, similar to epizootic diarrhea of infant mice (edim) . diagnosis serology uses reovirus as antigen, which detects seroconversion to all serotypes, and viral rna can be detected by rt-pcr. a presumptive diagnosis of reovirus infection is aided clinically by detection of the oily hair effect, accompanied by jaundice and wasting. the presence histologically of multisystemic necrosis is consistent with severe reovirus infection but should be confirmed by immunohistochemistry or virus isolation. differential diagnosis reovirus infection must be differentiated from other diarrheal diseases of infant mice, including those caused by mouse coronaviruses, edim virus, salmonella spp., or clostridium piliforme. prevention and control although surviving mice appear to recover completely from infection, the potential for a carrier state is unresolved. therefore, it may be necessary, after adequate testing for the continued presence of virus by the use of sentinels, map testing, or other appropriate means, to rederive or replace infected stock. prevention depends on adequate barrier husbandry coupled with adequate serological monitoring. research complications reovirus infection can interfere with research in several ways. infections in breeding colonies can result in high mortality among sucklings from nonimmune dams. virus has been commonly recovered from transplantable neoplasms and is suspected of being oncolytic. the potential exists for interference with hepatic, pancreatic, cardiovascular, or neurological research. etiology rotaviruses are double-stranded, segmented rna viruses that have a wheel-like ultrastructural appearance. edim virus is a group a rotavirus that replicates in differentiated epithelial cells of the small intestine by budding into cisternae of endoplasmic reticulum. currently, only a single antigenic strain is recognized, but antigenically distinct variants may exist. edim virus shares an inner capsid antigen with rotaviruses of rabbits, fowl, nonhuman primates, human beings, and domestic and companion animals. these agents tend to be species-specific under natural conditions and can be differentiated by serum neutralization tests. cultivation of edim virus requires the presence of proteolytic enzymes to cleave an outer capsid polypeptide. clinical signs clinical signs occur in infant mice less than weeks old. this age-related susceptibility also applies to infection in immunodeficient mice. furthermore, clinical signs occur only in offspring of nonimmune dams, because maternal immunity protects infants until they have outgrown susceptibility to clinical disease. the cardinal signs are bloated abdomens with fecal soiling of the perineum, which may extend to the entire pelage in severe cases. despite high morbidity, mortality is low because affected mice continue to nurse. transient weight loss does occur, and there may be a delay in reaching adult weight. recovery from infection usually occurs in about weeks and, once weight is regained, is clinically complete. epizootiology edim virus appears to be infectious only for mice and occurs episodically in mouse colonies, and infection is probably widespread geographically (livingston and riley, ; pritchett-corning laboratory animal medicine et al., ) . all ages and both sexes can be infected, but genetic resistance and susceptibility have not been determined. the virus is highly contagious and is transmitted by the oral-fecal route. subclinically infected adult mice can shed virus in feces for at least days, an interval that may be extended in immunodeficient mice. after oral inoculation, virus is essentially restricted to the gastrointestinal tract, although small amounts of virus may be present in the liver, spleen, kidney, and blood. nursing dams can contract infection from their litters. transplacental transmission has not been demonstrated. pathology gross lesions occur primarily in the gastrointestinal tract, but thymic involution can result from infection-related stress. the intestine is often distended, flaccid, and filled with gray-green gaseous liquid or mucoid fecal material that soils the pelage. the stomach contains curdled milk, except in terminal cases with anal impaction due to caking of dried feces. virus preferentially infects terminally differentiated enterocytes in the small and large intestines, which accounts for the agerelated susceptibility to disease; the number of such cells decreases as the intestinal tract matures. characteristic histological lesions are often very subtle, but are most easily discerned in the small intestine in mice less than weeks old. they consist of vacuolation of villar epithelial cells with cytoplasmic swelling, which give villi a clubbed appearance (fig. . ) . the vacuoles must be differentiated from normal absorption vacuoles in nursing mice. the lamina propria may be edematous, but necrosis and inflammation are not prevalent. diagnosis edim virus infection is readily detected serologically. clinical disease is diagnosed from signs and typical histological lesions in the intestine, which can be confirmed by immunohistochemical or ultrastructural demonstration of virus in the intestine or in intestinal filtrates or smears. rotavirus antigen can be detected in feces by elisa, but certain dietary ingredients can cause false-positive reactions. infection can also be diagnosed by rt-pcr. differential diagnosis edim virus infection must be differentiated from other diarrheal diseases of suckling mice such as intestinal coronavirus (mouse hepatitis virus) infection, reovirus infection, tyzzer's disease, and salmonellosis. the presence of milk in the stomach can be helpful in differentiating edim virus infection from more severe enteric infections, such as those caused by pathogenic coronaviruses, during which cessation of nursing often occurs. the possibility of dual infections must also be considered. thymic necrosis in edim virus-infected mice, although nonspecific, must be differentiated from that due to mouse thymic virus (mtv) infection or other stressors. prevention and control the spread of edim can be controlled effectively by the use of microbarrier cages and good sanitation. because infection appears to be acute and self-limiting, cessation of breeding for - weeks to allow immunity to build in adults while preventing access to susceptible neonates also is recommended. alternatively, litters with diarrhea can be culled, in combination with the use of microbarrier cages. the duration of infection in immunodeficient mice has not been determined, but it is reasonable to assume that chronic infection occurs. therefore, such animals should be eliminated. litters from immune dams are more resistant to infection. if edim virus is allowed to become enzootic within a colony, clinical signs will disappear within the population, which may be an appropriate management approach in conventional colonies. prevention of edim virus infection depends on maintenance of sanitary barrier housing with adequate serological surveillance. research complications the research complications of edim infection pertain to clinical illness with diarrhea and retarded growth. transient thymic necrosis may perturb immunological responses. infection (barthold, a,b) etiology coronaviruses are large, pleomorphic, enveloped rna viruses with radially arranged peplomers (spikes). in mice, early clinical and laboratory investigations emphasized their potential to induce hepatitis, so their original designation, which is still used actively, is mouse hepatitis virus (mhv). during that time, enteritis in infant mice was recognized as a separate entity caused by an uncharacterized virus, known as lethal intestinal virus of infant mice (livim). subsequent studies revealed that hepatitis-causing mhv and enteritis-causing livim were closely related coronaviruses, now collectively termed mhv. mhv isolates differ in biologic behavior according to their organ tropism into two biotypes: enterotropic strains, which infect primarily the intestinal tract, and polytropic strains, which initially infect the respiratory tract but may progress to multisystemic dissemination, including the liver and brain. these differences are often reflected in their cell tropism in vitro. however, natural isolates may contain features of both biotypes. several prototype polytropic strains have been extensively studied as experimental models of hepatitis and encephalitis. they include jhm (mhv ), mhv- , mhv- , mhv-s, and mhv-a . numerous additional strains have been identified that differ in virulence, tissue tropism, and antigenicity. differentiation by strain, particularly under natural conditions, is irrelevant, since mutation is common among coronaviruses, and even named prototype strains differ significantly depending upon passage history. although mhv isolates and strains share internal antigens (m and n), they can be distinguished by neutralization tests that detect strainspecific spike (s) antigens. mhv shares antigens with the coronaviruses of rats, a finding that has been exploited to develop heterologous antigens for serological tests. mhv also is related to human coronavirus oc . a number of established cell lines may be used for propagating polytropic mhv strains in vitro. however, field isolates are difficult to maintain in vitro. nctc mouse liver cells are useful for growing many polytropic strains. mhv can also be grown in mouse macrophages, cells that have been used for genetic studies of resistance and susceptibility to infection. enterotropic strains, because of their tendency to be strictly enterotropic, have been grown in cmt- cells derived from a rectal carcinoma in a c bl mouse, but are generally difficult to propagate in cell culture. irrespective of cellular substrate used for isolation or propagation, syncytium formation is emblematic of mhv infection (fig. . ) . clinical signs clinical signs depend primarily on the age, strain, and immunological status of infected mouse and strain and tropism of virus. as with many murine viruses, infection is often clinically silent among immunologically competent mature mice. clinical morbidity is most often associated with suckling mice less than weeks old or with immunodeficient mice. suckling mice infected with enterotropic mhv develop inappetence, diarrhea, and dehydration, often terminating in death (fig. . ) . epizootics of enterotropic mhv have been known to result in % mortality among neonatal mice in a breeding colony. older mice ( - weeks of age) may have ruffled pelage and runting. neurotropic strains such as mhv-jhm may induce flaccid paralysis of the hindlimb, but this sign is rarely encountered alone during natural infection. conjunctivitis, convulsions, and circling may be seen occasionally. enterotropic strains may not cause acute disease in athymic mice when exposed as adults, whereas mildly pathogenic polytropic strains can cause a progressive wasting syndrome that may be accompanied by progressive paralysis. epizootiology mhv infection, despite constant surveillance and preventive programs, continues to be a common threat to laboratory mouse populations (livingston and riley, ; pritchett-corning et al., ). there are no reports of natural transmission from mice to other species, but suckling rats have been found to develop necrotizing rhinitis after intranasal inoculation with mhv-s. mhv is highly contagious, with natural transmission occurring by respiratory or oral routes. mouse appears normal and has a milk-filled stomach. lower mouse is runted and dehydrated and has an empty stomach. from barthold et al. ( ) . enterotropic biotypes predominate in natural infections in contemporary laboratory animal facilities, since they tend to be the most contagious due to copious excretion of virus in feces, whereas polytropic strains generally spread by direct respiratory contact. natural vertical transmission has not been demonstrated. introduction of mhv through injection of contaminated biologicals can be an important factor in epizootics, especially because some isolates infect b lymphocytes and, by implication, hybridomas nonlytically. infection in immunocompetent mice is self-limiting. immune-mediated clearance of virus associated with seroconversion usually begins about a week after infection, and mice recover fully within - weeks. humoral and cellular immunity participate in host defenses to infection, and t-cell-dependent immunity is an absolute requirement. thus, age-related resistance to mhv correlates with maturation of lymphoreticular tissues, but intestinal proliferative kinetics are critical determinants of disease susceptibility with enterotropic mhv. enzootic infection had been construed to include persistent infection in individual mice. current evidence suggests, however, that enzootic infection results either from the fresh and continuous introduction of immunologically naive or deficient mice or from the recurrent infection of immune mice with mhv variants that arise by natural mutation. mutation is favored by immune pressure in enzootically infected colonies as well as missteps during natural replication, which include copying errors and recombination. thus, mice that have developed immunity to one strain of mhv can remain susceptible to one or more genetically and antigenically divergent strains, resulting in reinfection. this caveat has practical importance for breeding colonies. maternal immunity protects suckling mice against homologous mhv strains but not against antigenically variant strains. however, maternal immunity, even to homologous strains, depends on the presence of maternally acquired antibody in the lumen of the intestine. therefore, the susceptibility of young mice to infection increases significantly at weaning. strain differences in resistance and susceptibility to polytropic mhv can be inherited as an autosomal dominant trait. for example, dba/ mice are highly susceptible to mhv- and die acutely even as adults, whereas a/j mice develop resistance to lethal infection shortly after weaning. however, genetic resistance is also virus strain-dependent. therefore, mice resistant to one strain of mhv may be susceptible to another strain. it also is worth noting that the expanded use of genetically altered mice with novel or unanticipated deficits in antiviral responses may alter the outcome of virus-host interactions unpredictably. this pertains to mhv as well as other agents. for example, mhv infection has presented as granulomatous peritonitis and pleuritis in interferongamma (ifn-γ) knockout mice (france et al., ) . pathology polytropic strains replicate initially in the nasal mucosa, where necrotizing rhinitis may occur. viremic dissemination can follow if virus gains access to regional blood vessels and lymphatics. thus, viremia leads to secondary infection of vascular endothelium and parenchymal tissues in multiple organs including liver, brain, lymphoid organs, and other sites. mice also may develop central nervous system disease by direct extension of infection from the olfactory mucosa along olfactory tracts. at necropsy, yellow-white foci indicative of necrosis can occur in multiple tissues, with the involvement of the liver as the classical lesion. liver involvement may be accompanied by icterus and peritonitis. histologically, necrosis can be focal or confluent and may be infiltrated by inflammatory cells (fig. . ) . syncytia commonly form at the margin of necrotic areas and, in mild infections, may develop in the absence of frank necrosis. syncytia formation is a hallmark of infection in many tissues, including the intestine (fig. . ) , lung, liver, lymph nodes, spleen, thymus, brain, and bone marrow and in vascular endothelium in general. although syncytia are transient in immunocompetent mice, they are a persistent feature in chronically infected, immunodeficient mice ( fig. . ) . neurotropic variants cause acute necrotizing encephalitis or meningoencephalitis in suckling mice, with demyelination in the brain stem and in peri-ependymal areas secondary to viral invasion of oligodendroglia. convalescent mice may have residual mononuclear cell infiltrates around vessels or as focal lesions in the liver. immunodeficient mice can develop progressive necrotic lesions in the liver and elsewhere. compensatory splenomegaly may occur because of expansion of hematopoietic tissue. enterotropic strains infect primarily the intestine and associated lymphoid tissues, although some may also figure . necrosis, inflammation, and syncytium in the liver of a mouse infected with mhv. courtesy of s.w. barthold. cause systemic lesions, especially in the liver and brain. the most common sites are terminal ileum, cecum, and proximal colon. the severity of disease is age-related, and dependent upon intestinal proliferative kinetics, similar to edim, with young infants being at highest risk for lethal infection. pathogenic strains can cause lesions ranging from villus attenuation to fulminant necrotizing enterotyphlocolitis, which can kill suckling mice within a few days (fig. . ) . the stomach is often empty, and the intestine is filled with watery to mucoid yellowish, sometimes gaseous contents. syncytia are a consistent feature in viable mucosa (fig. . ) and not only are formed in intestine but also may be present in mesenteric lymph nodes and endothelium of mesenteric vessels. enterocytes may contain intracytoplasmic inclusions, but they are not diagnostic. surviving mice develop compensatory mucosal hyperplasia, which eventually recedes, but may contribute to clinical signs due to osmotic, secretory, and malabsorptive diarrhea. older mice are equally susceptible to infection, but are resistant to severe disease due to their mature (more rapid) intestinal proliferative kinetics. pathology may be subtle, consisting of transient syncytia without necrotic lesions. in adult mice, syncytia can be found most often in the surface mucosal epithelium of the ascending colon. the exception occurs in immunodeficient mice, such as athymic and scid mice, which can develop chronic proliferative bowel disease of varying severity with mhv antigen in mucosal epithelium (figs. . and . ). this may not always be present, as athymic nude mice exposed as adults may only manifest a few enterocytic syncytia without hyperplasia. diagnosis because mhv infection is often subclinical, serological testing is the most reliable diagnostic tool. many animal resources rely on sentinel mouse protocols for continuous serological surveillance. serology is well established, sensitive, and reliable. neutralization tests are used to differentiate individual virus strains in the research laboratory but are inappropriate for routine use, because of cost, technical complexity, and serologic identification per se does not predict biological behavior, including virulence or tissue tropism. serology also can be used in the context of map testing in which adult mice are inoculated with suspect tissues to elicit seroconversion. rt-pcr protocols to detect virus in tissues or excreta are available. the detection of syncytia augmented, when possible, by immunohistochemistry to laboratory animal medicine detect mhv antigen is a useful and practical means to confirm infection. this strategy should attempt to select mice that are in early stages of infection, because necrosis in infant mice or seroconversion in older mice may reduce the chances of detecting syncytia or viral antigens. the option of using immunodeficient mice as sentinels can be considered, because they sustain prolonged infection. however, they should be securely confined because they also amplify virus loads. if properly controlled, amplification in immunodeficient mice can, however, facilitate subsequent virus isolation in tissue culture. differential diagnosis mhv infection must be differentiated from other infectious diseases that cause diarrheal illness, runting, or death in suckling mice and wasting disease in immunodeficient mice. these include edim, mousepox, reovirus infection, tyzzer's disease, and salmonellosis. neurological signs or demyelinating lesions must be differentiated from mouse encephalomyelitis virus infection or noninfectious cns lesions, such as neoplasms, including polyoma virus-induced tumors in athymic mice. prevention and control control and prevention of mhv infection can be difficult because of the numerous variables that influence its expression. perhaps the most important factor is the duration of infection in individual mice and in mouse colonies. there is evidence that infection in an individual immunocompetent mouse is acute and self-limiting. such mice can be expected to develop immunity and eliminate virus within days. therefore, selective quarantine at the cage (not room) level with the temporary cessation of breeding can be used effectively to eliminate infection. quarantine at the room basis is likely to fail, since mutations arise and continually reinfect the mouse population. additionally, maternally derived immunity can protect infant mice from infection until they are weaned and moved to uncontaminated quarters. careful testing with sentinel mice should be used to assess the effectiveness of quarantine or 'natural rederivation,' as just described. immunodeficient mice, in contrast, are susceptible to chronic infection and viral excretion. mice with unrecognized or unanticipated immune dysfunction or with selective immune dysfunction may impact on mhv infection and its control. such colonies, which may contain highly valuable or irreplaceable mice, may be rescued by cesarean rederivation or embryo transfer if vertical transmission of mhv infection is subsequently ruled out. although rodent coronaviruses are not viable for extended periods in the environment, excreted virus may remain infectious for up to several days, so proper sanitation and disinfection of caging and animal quarters as well as stringent personal sanitation are essential to eliminate infection. the prevention of mhv requires procurement of animals from virus-free sources and maintenance under effective barrier conditions monitored by a well-designed quality assurance program. control of feral mouse populations, proper husbandry and sanitation, and strict monitoring of biological materials that may harbor virus (e.g., transplantable neoplasms, cell lines) are also important strategies to prevent adventitious infection. research complications numerous research complications have been attributed to mhv, and the unpredictable outcome of infection in genetically altered mice is likely to lengthen the list. for example, apart from its clinical impact, mhv may stimulate or suppress immune responses, contaminate transplantable neoplasms, and be reactivated by treatment of subclinically infected animals with several classes of drugs, including immunosuppressive agents, and by intercurrent infections. it also can alter tissue enzyme levels. additionally, the ubiquitous threat of mhv infection and uncertainty about its potential effects on a given research project provoke concerns that may exceed its true impact. for example, transient infection with a mild enterotropic strain is unlikely to disrupt systemic immune responses, whereas infection with a polytropic strain may be highly disruptive. this is not to say that subclinical or strictly enterotropic infection should be taken lightly but simply to caution against overreaction in assessing the impact of an outbreak. etiology mice are susceptible to infection by two members of the cardiovirus genus within the theilovirus. emcv has a less selective host range and can infect wild mice, but is not known to infect laboratory mice. tmev is a small, nonenveloped, rna virus that was discovered by max theiler during experimental studies of yellow fever virus in mice. established prototype strains include to (theiler's original), fa, da, and gd vii, the last of which is named after george martine (george's disease), an assistant in theiler's laboratory. tmev is rapidly destroyed by temperatures over °c and by alcohol but not by ether. it can be cultivated in vitro in several continuous cell lines, but bhk- cells are routinely used for isolation and propagation. tmev is antigenically related to emcv. as with other nonenveloped viruses, tmev is resistant to environmental inactivation, a factor that must be considered in control and prevention of infection. clinical signs the development of clinical disease depends on virus strain, mouse strain, and route of exposure, but natural disease is exceedingly rare (estimated at . - . % of infected mice). when clinical signs occur, they are expressed as neurological disease. the characteristic sign is flaccid posterior paralysis, which may be preceded by weakness in the forelimbs or hindlimbs, but in mice that are otherwise alert (fig. . ) . some mice may recover, but death frequently ensues, often because of failure to obtain food or water. furthermore, mice that recover from the paralytic syndrome are disposed to a chronic demyelinating phase, which is expressed as a gait disturbance. epizootiology infection occurs primarily in laboratory mice with the exception of the mgh strain, which has been isolated from laboratory rats and is pathogenic in mice and rats after experimental inoculation. the prevalence of tmev in mouse colonies is low, a reflection of the slow rate at which virus is transmitted from mouse to mouse, but it continues to be among the more common viral contaminants of mouse colonies (livingston and riley, ; pritchett-corning et al., ) . tmev infection is acquired by ingestion and replicates primarily in the intestinal mucosa. enteric infection can persist after the development of host immunity and can result in chronic or intermittent excretion of virus in feces over several months . mice often become infected shortly after weaning, but virus is seldom recovered in mice over months of age. however, neurologic infection can persist in the brain and spinal cord for at least year. immunity to one strain of tmev provides cross-protection to other strains. there are no reports of differences in mice with respect to susceptibility to infection under natural conditions. prenatal transmission has not been found. pathology intestinal tmev infection does not cause lesions, but virus can be detected in enterocytes by immunohistochemistry or in situ hybridization. poliomyelitis-like disease, the syndrome that may be encountered during natural infections, is characterized by acute necrosis of ganglion cells and neurons, neuronophagia, and perivascular inflammation, which occur particularly in the ventral horn of the spinal cord gray matter but also can involve higher centers such as the hippocampus, thalamus, and brain stem. during the subsequent demyelinating phase, mononuclear cell inflammation develops in the leptomeninges and white matter of the spinal cord, accompanied by patchy demyelination. the white-matter lesions are due to immune injury. spontaneous demyelinating myelopathy, affecting the thoracic spinal cord and associated with mev infection, has also been reported in aged mice. virulent strains may cause acute encephalitis after experimental inoculation, whereas less virulent isolates produce acute poliomyelitis followed by chronic demyelinating disease. diagnosis infection is usually detected serologically or by pcr of feces, but virus shedding from infected mice may be intermittent. clinical signs are striking, if they occur, but are too rare to rely on for routine diagnosis. histological lesions in the cns and especially the spinal cord are characteristic when present. differential diagnosis neurotropic variants of mhv may, on occasion, cause similar neurological signs. injury or neoplasia affecting the spinal cord can also produce posterior paralysis. polyoma virus infection in athymic mice can induce tumors or demyelination in the cns, which may result in clinical signs resembling those of tmev infection. prevention and control disease-free stocks were originally developed by foster-nursing infant mice. this technique, cesarean rederivation, or embryo transfer can be used successfully to eliminate infection. in either case, foster mothers should be surveyed in advance to ensure their mev-free status. selective culling can be considered as an option to eliminate infection, because laboratory animal medicine infection spreads slowly. however, the virus is hardy in the environment and resists chemical inactivation, so it may be prudent to depopulate and disinfect rooms if the presence of infection is unacceptable. research complications the principal hazard from tmev for research relates to its potential effects on the cns. noroviruses are nonenveloped rna viruses that belong to the family caliciviridae. they are notoriously resistant to environmental inactivation, and cause significant gastrointestinal morbidity in humans. noroviruses are species-specific, including mnv, which exclusively infects mice. until the discovery of mnv, replication of noroviruses in vitro has not been possible. for this reason, mnv has emerged as an important small animal model of norovirus pathogenesis. mnv was relatively recently discovered in , and subsequent surveillance has revealed that it is the most common adventitious virus infection in laboratory mice (hsu et al., ; pritchett-corning et al., ) . over mnv isolates have been found in mouse research colonies around the world, which display nearly % genetic identity, comprising a single genetic cluster. although genetically homogeneous, significant biological differences exist among mnv strains (thackray et al., ) . mnv effectively replicates in macrophages and dendritic cells, including the mouse macrophage-like raw . cell line, as well as a microglial cell line (wobus et al., ) . clinical signs clinical signs of infection in immunocompetent mice are usually absent, but infection leads to systemic disease with high mortality in interferon αβγ receptor and stat null mice. affected mice have loss of body weight, ruffled fur, and hunched posture (ward et al., ) . experimental infection of and c h mice with mnv- caused mild diarrhea (kahan et al., ) . epizootiology mnv is transmitted by the fecaloral route, and contaminates the environment as an environmentally resistant virus. for this reason, it can efficiently infect sentinel mice with soiled bedding (manuel et al., ) . duration of infection varies with mnv strain, mouse immunocompetence and mouse genotype. experimental studies have revealed that several mnv strains persist in various tissues of c bl/ j, hsd:icr, and jcl:icr and c.b- -prkdc scid mice, with fecal shedding for at least - days (goto et al., ; hsu et al., ; thackray et al., ) . although not clinically ill, rag null mice are unable to clear infection . comparative studies with mnv- and mnv- have shown differences in virus replication and shedding (kahan et al., ) . mnv has a tropism for macrophages and dendritic cells, and virus can be detected in the intestine, intestinal lymphoid tissue, liver, and spleen (hsu et al., ; kahan et al., ; wobus et al., ) . pathology naturally and experimentally infected stat or ifnγr null mice may develop splenomegaly and multifocal pale spots on the liver. microscopic findings include varying degrees of hepatitis, focal interstitial pneumonia, vasculitis, peritonitis, and pleuritis (karst et al., ; ward et al., ) . encephalitis, cerebral vasculitis, pneumonia, and hepatitis have also been described in intracerebrally infected stat null mice (karst et al., ) . infection of immunocompetent mice may be associated with mild inflammation of the intestine, splenic hypertrophy, and lymphoid hyperplasia of spleen and lymph nodes (mumphrey et al., ) . diagnosis mnv infection can be detected by serology or rt-pcr. sentinel mouse surveillance, using soiled bedding, is an effective strategy for detecting mnv (manuel et al., ) differential diagnosis the mild change in fecal consistency associated with mnv in adult mice may mimic rotavirus, coronavirus, helicobacter spp., citrobacter rodentium, or other enteric diseases. disseminated lesions in stat or ifnγr null mice must be differentiated from other polytropic viral diseases in immunodeficient mice, including mhv. prevention and control depopulation and decontamination has been shown to be effective at eliminating mnv from an enzootically infected colony, whereas testand-removal of positive mice was found to be ineffective (kastenmayer et al., ) . embryo transfer and cesarean rederivation are also effective (goto et al., ; perdue et al., ) . neonatal mice are resistant to infection, so that cross-fostering neonates onto uninfected dams is another effective means of rederivation mnv-free mice (artwohl et al., ; compton, ) . research complications the tropism of mnv for macrophages and dendritic cells is likely to modify immune responses, and mnv infection may interfere with studies involving enteric disease. hantaviruses are rna viruses belonging to the very large bunyaviridae family. they differ from other members of this family by not being arthropod-borne. each hantavirus is antigenically distinct and maintained within single or at most a few rodent or insectivore hosts, but are infectious for other hosts. infection is lifelong, and virus is transmitted by shedding of virus in urine, feces, and saliva. several hantaviruses are zoonotic and may cause severe disease in humans. although there is overlap, hantaviruses in asia and europe cause hemorrhagic fever with renal syndrome (hfrs) in humans, a multisystem disease with significant renal involvement, and hantaviruses that are endemic in the americas cause laboratory animal medicine hantavirus pulmonary syndrome (hps) in humans, which is a multisystem disease with pulmonary involvement. among the better-known old world hfrs hantaviruses are hantaan, seoul, puumala, and dobrava-belgrade viruses. sin nombre virus is the best known new world phs hantavirus, among many others. most notably from the perspective of laboratory animal medicine, the norway rat, rattus norvegicus, serves as a reservoir host for hantavirus in the wild, but infection has also been associated with laboratory rats. in addition to being endemic in wild rats in asia, it has been found to be endemic in wild rats in the eastern united states and associated with human cases of hfrs (childs et al., ; leduc et al., ; tsai et al., ) . over cases of hantavirus infection have been transmitted to humans from laboratory rats in japan, belgium, and the united kingdom (desmyter et al., ; kawamata et al., ; lloyd et al., ; umenai et al., ) . m. musculus is not considered to be a primary reservoir host, but hantavirus infection has been documented serologically in conventional and barrier-maintained laboratory mice and rats in korea (won et al., ) , infection of wild m. musculus has been documented in the united states (baek et al., ) , and infection of wild mice in europe has been associated with human exposure (diglisic et al., ) . hantaviruses are difficult to culture in vitro. infection in rodents is subclinical and is detected by serology or rt-pcr. the main research complication from natural infection is the zoonotic risk and potentially subclinical effects on the immune response associated with viral defenses such as cd + t cell (taruishi et al., ) and nk function as demonstrated in human studies (braun et al., ) . the mouse is host to a number of enveloped rna viruses of the family retroviridae, subfamily orthovirinae, including the two type species (and their variants) mouse mammary tumor virus (mmtv) and murine leukemia virus (mlv). these viruses belong to a diverse assemblage of related mobile dna elements that are integrated into the host genome, and collectively termed 'retroelements', which include retrovirus-related elements and nonviral elements. during cell division, retroelements are transcribed into rna, and subsequently reverse-transcribed into dna copies that become integrated into a new location within the genome. this process utilizes reverse transcriptase, which is encoded by the retroelement. over millennia, retroelements have been repeatedly integrated within the genome in large numbers, comprising approximately % of the mammalian genome. various families of mouse retroelements share sequence similarity, despite their random distribution throughout the mouse genome, and the majority of them are truncated, mutated, and methylated to become incapable of infectivity. nevertheless, many of them continue to be mobile within the genome. noninfectious retrovirus-related retroelements include iap, vl , musd, and etn elements. replication-competent retroviruses represent the pinnacle of the retroelement constellation and are best considered as the most evolutionarily recent members. these include mmtv and mlv. mtv and mlv share similar genetic structure, except that the long terminal repeat (ltr) region of the mmtv genome encodes an additional superantigen (sag). both mmtv and mlv include exogenous viruses, which are horizontally transmitted, replication-competent viruses, and endogenous viruses, which are closely related to exogenous viruses, encoded within the mouse genome, and transmitted by mendelian inheritance. exogenous mmtv and mlv exist in wild mouse populations, but have been eliminated from contemporary laboratory mice. however, they may continue to be used experimentally, including bittner mmtv, and gross, friend, moloney, and rauscher mlvs. in particular, mouse colonies may be purposely infected with mmtv for mammary cancer research and are termed 'mmtv-positive', reflecting their exogenous virus status, even though the mice may also carry endogenous mmtv. the genomes of all inbred strains of mice encode one or more (over in some mouse strains) endogenous mmtv loci, the distribution of which is unique to each inbred strain of mouse. most mmtv genomic loci do not encode infectious virus or are transcriptionally inactive, except for mouse strains (dba, c h, grs) that carry mtv or mtv loci. these loci encode infectious virus, which can be visualized as b-type particles by electron microscopy. likewise, all mouse strains carry endogenous mlv loci within their genome, but not all mice carry replication-competent mlv sequences. some endogenous mlvs encode infectious virus, which can be visualized as c-type particles by electron microscopy. mice have often evolved mechanisms to counter the deleterious effects of retroviruses by preventing reentry or replication of virus into other cells. if an endogenous retrovirus is still infectious to other mouse cell targets, it is termed ecotropic, whereas if it is no longer infectious for mouse cells, but can infect cells of other species, it is termed xenotropic. viruses capable of infecting cells of mice as well as other species are termed polytropic. the combinations of endogenous replication-competent mlvs and cell tropism factors are a reflection of selective breeding of mouse strains for susceptibility to various types of cancer. clinical signs mice were originally inbred for specific phenotypes, including mammary tumors and lymphomas. thus, some strains of mice were genetically selected for unique combinations of endogenous mmtv and mlv in concert with susceptibility factors laboratory animal medicine that favored their expression and disease manifestations. in addition, noninfectious retroelements continue to reintegrate randomly within the genome during cell division as retro transposons. these ongoing integrations contribute to genetic drift, spontaneous mutations, and well-recognized mouse strain phenotypes, including the athymic nude allele, the hairless allele, and the rodless retina allele, among others. epizootiology exogenous mmtv and mlv are horizontally transmissible, primarily through the milk of lactating females. endogenous retroviruses and retroelements are inherited through the genome. replicationcompetent endogenous mmtvs and mlvs are also transmissible like their exogenous counterparts, but differ by being integrated within the genome of the mouse. pathology replication-competent mmtv and mlv, regardless of their exogenous or endogenous origin, are usually clinically silent. their ability to cause neoplasia is a reflection of genetic selection for susceptibility factors that are genetically encoded within individual mouse strain genomes. mmtv derives its name from its association with induction of mammary carcinomas in mammary cancer-susceptible strains of mice. mlv is associated with lymphomas, the pattern of which is mouse strain specific. for example, akr mice develop % prevalence of thymic lymphoma between and months of age, whereas aging balb/c mice commonly develop multicentric lymphoma. in these strains of mice, multiple endogenous mlvs are coexpressed in tissues and undergo recombination events that allow them to target and transform cells into neoplasia. despite its name, mmtv can induce lymphomas in some strains of mice, such as sjl mice which develop lymphomas arising from enteric lymphoid tissue and mesenteric lymph nodes. diagnosis exogenous retroviruses have been eliminated from contemporary mouse populations, unless purposely introduced for experimental purposes. because endogenous retroviruses and retroelements are encoded within the genome, and reflect the unique genetic composition of each strain of mouse, they are not targets of diagnostic pursuit. differential diagnosis patterns of some types of neoplasia within individual inbred strains of mice are a reflection of their endogenous retroviral integration. prevention and control exogenous retroviruses have been eliminated from laboratory mice by cesarean rederivation and foster nursing. mmtv-s, the 'bittner agent', continues to be purposely maintained in some mouse breeding populations, but can be eliminated by foster-nursing or other means. caution is advised when re-deriving such mouse colonies for other purposes, as elimination of exogenous mmtv will be an unintended consequence. research complications endogenous retroviruses and retroelements influence the life span of individual strains of mice, and random integrations during cell division can give rise to spontaneous mutations and genetic drift. it is estimated that significant mutations may arise due to mobile retroelement integrations every generations. astroviruses are small, nonenveloped, singlestranded rna viruses that have been associated with human gastroenteritis and detected in association with other enteric pathogens. the viral family astroviridae is split into two genuses: avastrovirus for those astroviruses infecting avians and mamastrovirus for those infecting mammals. astrovirus infection has been detected in research mice (muastv) using metagenomic analyses and appears to have a wide geographical, institutional, and host strain distribution. clinical signs none reported. epizootiology pcr screening has found muastv infection in up to % of a variety of mouse strains housed in vendor and academic facilities in the united states and japan. the virus has been detected most commonly in immunocompromised mice (nsg, nod-scid, nsg- gs, c bl -timp- −/− , and upa-nog), but also in immuncompetent strains (b j, icr, bash , and balb/c). both immunodeficient and immunocompetent mice are susceptible to muastv, but adaptive immunity is required to clear the virus. based on human epidemiology indicating children are at highest risk for infection, the virus may preferentially infect young mice. pathology immunodeficient mice showed no sign of pathology based on histopathology. diagnosis pcr data has indicated that muastv causes a systemic, chronic infection in immunocompromised mice, indicating samples from most tissues will be pcr positive. yokoyama et al. ( ) detected high viral load (up to genome copies) per fecal pellet from immunocompetent mice. differential diagnosis none, in the absence of lesions and clinical disease. prevention and control because immunocompetent mice clear the infection, quarantine may be successful but lack of routine screening for muastv in laboratory mice will allow for uncontrolled spread of the infection. research complications based on limited surveys, muastv may have a high prevalence in laboratory mice. the impact of infection on both innate and adaptive immune responses warrants further investigation to assess the potential for confounding research data. this section briefly describes the etiology, clinical signs, epizootiology, pathology, diagnosis, differential diagnoses, prevention and control, and research complications of the most common bacterial diseases encountered in research colonies of mice. as sequencing technology becomes more available, the number and genus/species classification of bacteria potentially responsible for infections, in particular, opportunistic infections, will grow (benga et al., ) . potential candidates include members of pasteurellacaeae, bordetella hinzii, streptococcus danielae, acinetobacter spp., and others, for which little is currently known about their pathogenic potential. etiology lawsonia intracellularis, an obligate intracellular bacterium and the causative agent of proliferative enteropathy, is not a pathogen encountered in research colonies of mice but has been reported to infect wild mice and rats in close contact with infected livestock (collins et al., ) . clinical signs none reported but should consider lawsonia as a differential in necropsy cases with gross or histologic evidence of proliferative lower bowel lesions. epizootiology although mice are experimentally susceptible to infection and develop classic lesions of hyperplastic ileitis and typhlocolitis (murakata et al., ) , susceptibility varied with mouse strain and source of inoculum from rabbits or swine, suggesting important differences in l. intracellularis strains. pathology lawsonia infection may result in hyperplastic ileitis, typhlitis and/or colitis, and hemorrhagic intestines may be noted (percy and barthold, ) . diagnosis lawsonia spp. has been diagnosed using a variety of techniques, including pcr, immunohistochemistry, in situ hybridization, and warthin-starry silver stains. differential diagnosis bacterial infections associated with hyperplastic intestinal epithelium, including c. rodentium and enterohepatic helicobacter species in susceptible (typically immunodeficient) mouse strains. prevention and control species separation from hosts more commonly associated with natural infection (hamsters, ferrets, pigs). research complications none reported. the following section describes infection due to mycoplasma pulmonis and summarizes infections associated with other murine mycoplasmas including m. arthriditis, m. neurolyticum, m. collis, and m. muris. antigenic cross-reactivity among these species, and especially between m. pulmonis and m. arthriditis, mandates that reliable diagnostic strategies incremental to serology (elisa, ifa, mfia) such as culture (often false negative) and pcr be employed to distinguish potentially pathogenic infections. when screening cell lines for opportunistic pathogens, pcr is the most efficient method to discriminate between m. pulmonis and mycplasma contaminants associated with cell culture. etiology m. pulmonis is a pleomorphic, gram-negative bacterium that lacks a cell wall and has a single outer limiting membrane. it causes murine respiratory mycoplasmosis (mrm). clinical signs mice are relatively resistant to florid mrm; thus, subclinical infection is more common. when clinical signs occur, they reflect suppurative rhinitis, otitis media, and chronic pneumonia. affected mice may display inactivity, weight loss, and ruffled hair coat, but the most prominent signs are 'chattering' and dyspnea, due to rhinitis and purulent exudate in nasal passages. otitis media may cause a head tilt, whereas suppurative inflammation in the brain and spinal cord, although rare, can cause flaccid paralysis. experimental infection of the genital tract can cause oophoritis, salpingitis, and metritis, which may lead to infertility or fetal deaths. experimental inoculation of scid mice has caused systemic infection accompanied by severe arthritis (evengard et al., ) . epizootiology mrm historically was a common infectious disease of mice, but improved housing, husbandry, and health surveillance have reduced its prevalence dramatically. serologic data from a large diagnostic laboratory indicated m. pulmonis infection affects about . % of conventionally housed mouse colonies in the united states and . % in europe (pritchett-corning et al., ) . m. pulmonis infection is contracted by inhalation and can occur in suckling and adult mice. therefore, infection should be considered highly contagious. mice injected with cells harvested from m. pulmonis contaminated cell cultures may develop disease. m. pulmonis can also be transmitted venerally; in utero infection has been demonstrated in rats but not in mice. because transplacental infection occurs in rats, the same route may be possible in mice, particularly immunocompromised strains. concomitant viral pneumonia (sv, mouse coronavirus) or elevated environmental ammonia concentrations may increase susceptibility to mrm. m. pulmonis also infects rats, hamsters, guinea pigs, and rabbits. among these species, only rats are significant reservoirs of infection for mice. pathology m. pulmonis is an extracellular organism that colonizes the apical cell membranes of respiratory epithelium. attachment occurs anywhere from the anterior nasal passages to the alveoli and may be mediated by surface glycoproteins. the organism may injure host cells through competition for metabolites such as carbohydrates and nucleic acids or by release of toxic substances such as peroxides. ciliostasis, reduction in the number of cilia, and ultrastructural changes leading to laboratory animal medicine cell death have also been described. detrimental effects on ciliated epithelium can lead to disrupted mucociliary transport, which exacerbates pulmonary disease. experimental infection of mrm is dose dependent. doses of colony-forming units (cfus) or less cause mild, transient disease involving the upper respiratory tract and middle ears, whereas higher doses often lead to acute, lethal pneumonia. additionally, m. pulmonis strains can differ in virulence. survivors of severe infection may develop chronic bronchopneumonia with bronchiectasis and spread infection to other mice. intravenous inoculation of m. pulmonis can cause arthritis in mice, but arthritis is not a significant feature of natural infection. host genotype also is a major factor in the outcome of infection, with resistance being expressed phenotypically through the bactericidal efficiency of alveolar macrophages. strains derived from a c bl background appear to be resistant to pathogenic infection, whereas balb/c, c h, dba/ , swr, akr, cba, sjl, and other strains have varying degrees of increased susceptibility (cartner et al., ; lai et al., ) . the initial lesion of mrm is suppurative rhinitis, which may involve the trachea and major airways. early inflammatory lesions, if not quickly resolved, progress to prominent squamous metaplasia. transient hyperplasia of submucosal glands may occur, and lymphoid infiltration of the submucosa can persist for weeks. syncytia can sometimes be found in nasal passages, in association with purulent exudate (fig. . ) . affected mice also develop suppurative otitis media and chronic laryngotracheitis with mucosal hyperplasia and lymphoid cell infiltrates. pulmonary lesions are typified by bronchopneumonia, which spreads from the hilus. lymphoid cells and plasma cells accumulate around bronchi which often contain neutrophils in their lumen. chronic lung disease features suppurative bronchitis, bronchiolitis, and alveolitis (fig. . ) . chronicity also increases the prevalence of bronchiectasis and abcessation. diagnosis accurate diagnosis should exploit the complementary use of clinical, serological, microbiological, molecular, and morphological methods. clinical signs are variable but can be characteristic when they occur. serology is sensitive but although antibodies do not clear the infection, seroconversion may be weak or take months and may not accurately differentiate between m. pulmonis infection and m. arthriditis infection (cassell et al., ) . therefore samples for culture and pcr of the upper respiratory tract should be obtained to confirm diagnosis. buffered saline or mycoplasma broth should be used to lavage the trachea, larynx, pharynx, and nasal passages. specimens for culture from the genital tract are warranted if this site is suspected. mycoplasma spp. may be difficult to grow, so it is prudent to confirm that the relevant expertise and quality control exist in the diagnostic laboratory. speciation can be accomplished by immunofluorescence or immunoperoxidase staining or by growth inhibition. immunohistochemistry should be considered to supplement basic histopathologic examination. immunofluorescence and immunoperoxidase techniques are available to identify mycoplasma antigens in tissue sections or in cytological preparations of tracheobronchial or genital tract lavages (brunnert et al., ) . pcr assays for m. pulmonis at veterinary diagnostic laboratories and pcr kits to screen cell culures for mycoplasma are readily available. differential diagnosis mrm must be differentiated from bronchopneumonia associated with ciliaassociated respiratory (car) bacillus. silver stains may reveal car bacilli adherent to the respiratory epithelium. sv also can cause bronchopneumonia in mice but can be detected by serology and immunohistochemistry. other causes of respiratory infection include pvm, corynebacteriosis and, in immunodeficient mice, pneumocystis murina infection. combined infections with known pathogens or secondary opportunists also must be considered. prevention and control mice mount an effective immune response to m. pulmonis, as measured by their recovery from mild infection and their resistance to infection after active or passive immunization (cartner et al., ) . antibodies of various classes are produced locally and systemically, but clearance of the infection has been attributed to innate immune responses (love et al., ; sun et al., ) . there is some evidence that antibody may facilitate phagocytosis of m. pulmonis. t-cell responses, however, appear to exacerbate m. pulmonis in mice, because immunity cannot be transferred with immune cells. in addition, athymic and neonatally thymectomized mice are not more susceptible than immunocompetent mice to m. pulmonis pneumonia. nude and scid mice develop less severe respiratory disease than immunocompetent mice but infection becomes systemic and they may develop suppurative disease in multiple organs and joints (arthritis). host immunity aside, effective control and prevention of mrm depend primarily on maintenance of mycoplasma-free colonies under barrier conditions supported by careful surveillance for infection by serology, microbiology, pcr, and histopathology. cesarean or embryo rederivation may eliminate infection, although vertical transmission may occur in immunocompromised mice. treatment with tetracycline suppresses clinical disease but does not eliminate infection. earlier interest in developing dna-based vaccines against m. pulmonis has not achieved clinical application (lai et al., ) . research complications m. pulmonis can interfere with research by causing clinical disease or death. experiments involving the respiratory tract, such as inhalation toxicology, can be compromised by chronic progressive infection. additionally, affected mice are at greater risk during general anesthesia. m. pulmonis may alter immunological responsiveness. for example, it is mitogenic for t and b lymphocytes and can increase nk cell activity. perhaps one of the most important complications of mycoplasma infection is contamination of cell lines and transplantable tumors. other murine mycoplasmas cell lines are often contaminated with mycoplasma species such as m. arginini, m. hyorhinis, m. orale, or m. fermentans that can distort the results of in vitro assays (garner et al., ) . initial evidence of a contamination is often by pcr evidence of mycoplasma at the genus level when cell lines are pcr screened for opportunistic murine pathogens prior to use in mice. other than m. pulmonis, these mycoplasmas are not normally considered mouse pathogens in immunocompetent mice. in contrast, injection of mycoplasma contaminated cells into immunodeficient mice (e.g., xenografts) may result in clinical disease or confounding effects on immune responses (peterson, ) . mycoplasma contamination of murine embryonic stem cells has adversely affected germline transmission and postnatal health of chimeric progeny (markoullis et al., ) . mycoplasma arthritidis is antigenically related to m. pulmonis. therefore, serological evidence of mycoplasma infection must be supplemented by other diagnostic tests, as outlined above, to differentiate between these agents. differentiation is important because m. arthritidis, though arthritogenic in mice after intravenous inoculation, is nonpathogenic during natural infection. mycoplasma collis has been isolated from the genital tract of mice but does not appear to cause natural disease. mycoplasma neurolyticum is the etiological agent of rolling disease, a rare syndrome which occurs within hours after intravenous inoculation of m. neurolyticum exotoxin. characteristic clinical signs include spasmodic hyperextension of the head and the raising of one foreleg followed by intermittent rolling on the long axis of the body. the rolling becomes more constant, but mice occasionally leap or move rapidly. after - h of rolling, animals become comatose and usually die within h. all published reports of rolling disease are associated with experimental inoculation of m. neurolyticum or exotoxin. large numbers of organisms are needed to produce disease, and there is no indication that, under natural conditions, organisms replicate in the brain to concentrations required for the induction of these signs. because animals are frequently inoculated with biological materials by parenteral routes, contamination with m. neurolyticum may induce rolling disease inadvertently. diagnosis can be made from the appearance of typical clinical signs, astrocytic swelling, and isolation of the causative organism. clinical signs must be differentiated from rolling associated with pseudomonas-and p. pneumotropica-caused otitis. m. pulmonis has been recovered from the brain of mice but does not seem to cause overt neurological disease. hemotropic mycoplasmas ribosomal rna sequencing has reclassified hemobartonella muris and eperythrozoon coccoides as mycoplasma hemomuris and mycoplasma coccoides, respectively (neimark et al., ; percy and barthold, ) . distinct from the mycoplasmas just discussed, these agents are trophic for red blood cells and cause anemia and hemolytic disease. these laboratory animal medicine infections could be encountered in wild mice but are rarely found in research mice. diagnosis is by morphologic assessment of blood smears and pcr. clinical signs mice infected with m. coccoides may remain clinically normal or develop febrile, hemolytic anemia and splenomegaly, which can be fatal. hepatocellular degeneration and multifocal necrosis have been recorded in acute infections. hemotropic mycoplasma infections are long-lived and are expressed clinically in one of two ways: acute febrile anemia and latent or subclinical infection that can be reactivated by splenectomy. the carrier state may be lifelong. epizootiology the primary natural vector of m. coccoides, historically, is the mouse louse, polyplax serrata. infection was associated with primitive housing and husbandry conditions that no longer occur in modern vivaria. although the risks for infection have been reduced substantially by modern animal care procedures, m. coccoides can be transmitted to mice from contaminated biological products such as transplantable tumors or blood plasma. diagnosis splenectomy or inoculation of test material into splenectomized mice is the most sensitive means of detecting m. coccoides infection. these procedures provoke mycoplasmemia, usually within - days. because mycoplasmemia may be transient, blood smears stained by the romanowsky or indirect immunofluorescence procedures of the blood should be prepared every h, beginning at h after splenectomy of index animals or inoculation of test specimens into splenectomized animals to ensure that mycoplasmemia is not missed. prevention and control treatment of m. coccoides infection is not practical. control is based on culling or rederivation of infected stock. if replacement animals are readily available, euthanasia is a more prudent course. suspect biological materials destined for animal inoculation should be screened for mycoplasma contamination by inoculation of splenectomized mice. research complications subclinical infection can be reactivated by irradiation, immunosuppressive therapy, or intercurrent disease. conversely, m. coccoides may potentiate coincident viral infections in mice. this effect has been clearly demonstrated for mouse coronavirus and has been suspected for lymphocytic choriomeningitis virus and ldv. active infection also may suppress interferon production. etiology car bacillus is a slender, gram-negative, non-spore-forming bacillus, which, in rats, produces clinical disease and lesions that closely resemble those of mrm (see chapter ). clinical signs chronic respiratory disease has been produced in mice by experimental inoculation, but natural clinical disease is rare (griffith et al., ; pritchett-corning et al., ) . furthermore, putative natural cases were reported in mice that were seropositive for sv and pneumonia virus of mice. therefore, car bacillus may exacerbate respiratory disease as an opportunist rather than as a primary pathogen. on balance, it is assumed that mice contract natural infection, but attributing severe chronic respiratory disease in mice solely to car bacillus should be supported by screening for other respiratory pathogens. epizootiology car bacillus is transmitted by direct contact; dirty bedding transfer to sentinel mice may not reflect colony infection status. pathology lung lesions are typically mild in mice and are similar to respiratory mycoplasmosis. uncomplicated car bacillus infection results in peribronchiole cuffing with lymphocytes and plasma cells. severe bronchiolitis and pneumonia are possible (fig. . ) . fatal bronchopneumonia was reported in ob/ob mice (griffith et al., ) . diagnosis an elisa for serological screening is routinely used; pcr and histology are used for definitive diagnosis. in active infection, histologic assessment using warthin-starry or similar stains will reveal argyrophilic bacilli adherent to the apical membranes of bronchial respiratory epithelium along with the presence of peribronchial lymphocytes (fig. . ) . alternatively, immunohistochemistry assays have also been used successfully to detect infection. recovery of car bacillus requires cell culture or culture in embryonated eggs. differential diagnosis respiratory mycoplasmosis, bordetella (avium, hinzii). prevention and control given car bacillus does not form spores, disinfection of the environment should be effective. treatment using sulfamerazine ( mg/l) in drinking water may eradicate infection (matsushita and suzuki, ) but culling or embryo rederivation is recommended. research complications infection is most often subclinical, but like other infectious agents for mice, may confound studies particularly when mice are immunocompromised (griffith et al., ) . etiology the causative agent of transmissible murine colonic hyperplasia, c. rodentium (formerly citrobacter freundii strain ), is a nonmotile, gramnegative rod that ferments lactose but does not utilize citrate or does so marginally (barthold, ; schauer et al., ) . clinical signs c. rodentium infection can be a selflimiting colitis with sterilizing immunity or lead to severe colitis with life-threatening dehydration. clinically apparent infection is characterized by retarded growth, ruffled fur, soft feces or diarrhea, rectal prolapse, and moderate mortality in older suckling or recently weaned mice (barthold et al., ) . epizootiology c. rodentium is not detected in the gastrointestinal flora of normal mice, and therefore, there is not a carrier state. it is thought to be introduced by contaminated mice, food, or bedding, from which it spreads by contact or additional fecal contamination. c. rodentium shares several pathogenic mechanisms, such as attaching and effacing lesions mediated by the intimin receptor, with select escherichia coli (reviewed in collins et al. ( ) ). c. rodentium is used experimentally to model colitis caused by enteropathogenic (epec) and enterohemorrhagic e. coli (ehec) in humans (mallick et al., ; collins et al., ) . host genotype can influence the course and severity of disease (barthold et al., ) . for example, dba, nih swiss, and c bl mice are relatively resistant to mortality, whereas c h/hej mice are relatively susceptible both as sucklings and as adults. interestingly, c bl mice obtained from different commercial sources have varying susceptibility to c. rodentium (ostensibly due to the presence or absence of segmented filamentous bacteria). diet also can modulate infection, but specific dietary factors responsible for this effect have not been identified. pathology c. rodentium attaches to the mucosa of the descending colon and displaces the normal flora. attachment is accompanied by effacement of the microvillus border and formation of pedestal-like structures (attaching and effacing lesions) (schauer and falkow, ; newman et al., ) . colonization results in prominent mucosal hyperplasia, by unknown mechanisms. the characteristic gross finding is severe thickening of the descending colon, which may extend to the transverse colon and lasts for - weeks in surviving animals ( fig. . ) . affected colon segments are rigid and either are empty or contain semiformed feces. histologically, accelerated mitotic activity results in a markedly hyperplastic mucosa, which may be associated with secondary inflammation and ulceration (fig. . ). lesions subside after several weeks. intestinal repair is rapid and complete in adults but slower in sucklings. diagnosis diagnosis depends on clinical signs, characteristic gross and histological lesions, and isolation of c. rodentium from the gastrointestinal tract or feces. the organism can be cultured on macconkey's agar during early phases of infection, whereas the intestine may be free of c. rodentium during later stages of the disease. c. rodentium also can be detected by molecular hybridization (schauer et al., ) . barthold et al. ( ) . diagnosis transmissible murine colonic hyperplasia must be differentiated from other diarrheal diseases of mice, including infections caused by coronavirus, rotavirus, adenovirus, reovirus, salmonella, c. piliforme, and helicobacter spp. prevention and control some success in curtailing epizootics has been achieved by adding antimicrobials to the drinking water (barthold, ; silverman et al., ) . because c. rodentium may contaminate food, bedding, or water, proper disinfection of such materials is prudent before they are used for susceptible animals. additionally, the employment of microbarrier caging can reduce transmission. surveillance for c. rodentium should be incorporated into quality-assurance programs, and the organism screened for during quarantine of incoming mice from atypical sources. research complications the potential effects on research of colonic hyperplasia as a clinically severe disease are obvious. colonic hyperplasia has been shown to increase the sensitivity of colonic mucosa to chemical carcinogens and to decrease the latent period between administration of carcinogen and the appearance of focal atypical cell growth (barthold and beck, ) . c. rodentium infection has been incriminated in immune dysfunction, poor reproductive performance, and failure to thrive in t-cell receptor transgenic mice (maggio-price et al., ) . immunocompromised mice infected with c. rodentium will die from sepsis. etiology pseudomonas aeruginosa is a motile, gramnegative rod. clinical signs p. aeruginosa infections are almost always silent, but immunologically compromised animals are prone to septicemia (brownstein, ) . p. aeruginosa can, e.g., cause severe or lethal infections in athymic and scid mice. sick mice may have equilibrium disturbances, conjunctivitis, serosanguinous nasal discharge, edema of the head, weight loss, and skin infections. immunosuppressed mice may also develop gastrointestinal ulcers. generalized infection is associated with severe leukopenia, especially neutropenia. neurologic signs are rare, but there are reports of central nervous system infection. chronic proliferative inflammation in the cochlea and vestibular apparatus with dissolution of surrounding bone may cause torticollis. epizootiology p. aeruginosa is not considered a component of the normal flora. however, it is an opportunist that inhabits moist, warm environments such as water and skin. once established in a host, it may be found chronically in the nasopharynx, oropharynx, and gastrointestinal tract, all sites from which additional environmental contamination or direct transmission to susceptible mice can occur. pathology pathogenic infection is most common in immunodeficient mice. organisms enter at the squamocolumnar junction of the upper respiratory tract and, in some cases, the periodontal gingiva. bacteremia is followed by necrosis or abscess formation in the liver, spleen, or other tissues. if otitis media occurs, the tympanic bullae may contain green suppurative exudate. the bowel may be distended with fluid, and gastrointestinal ulceration has been reported. diagnosis infection is diagnosed on the basis of history (e.g., immune dysfunction or recent immunosuppression), clinical signs, lesions, and isolation of p. aeruginosa from affected mice. carrier mice can be detected either by nasal culture or by placing bottles of sterile, nonacidified, nonchlorinated water on cages for - h and then culturing the sipper tubes. p. aeruginosa can also be cultured from feces. differential diagnosis pseudomoniasis must be differentiated from other bacterial septicemias that may occur in immunodeficient mice. these include, but are not limited to, corynebacteriosis, salmonellosis, colibacillosis, staphylococcosis, and tyzzer's disease. prevention and control infection can be prevented by acidification or hyperchlorination of the drinking water (homberger et al., ) . these procedures will not, however, eliminate established infections. entry of infected animals can be prevented by surveillance of commercially procured colonies. maintenance of pseudomonas-free animals usually requires barrierquality housing and husbandry. p. aeruginosa has a long history in the literature of antibiotic resistance and resistance to quaternary amine disinfectants. research complications p. aeruginosa infection is not a substantial threat to immunocompetent mice but can complicate experimental studies by causing fatal septicemia in immunodeficient mice. viral infections that alter host defense mechanisms, such as mcmv may enhance susceptibility to pseudomoniasis. (lindsey et al., a; percy and barthold, ) etiology pasteurella pneumotropica is a short, gramnegative rod. clinical signs many early observations concerning the pathogenicity of p. pneumotropica are questionable because they were made on colonies of mice with varying levels of bacterial and viral contamination. infection is usually subclinical. therefore, p. pneumotropica is most properly viewed as an opportunistic pathogen. studies of experimental p. pneumotropica suggest that it may complicate pneumonias due to mycoplasma pulmonis or sv. it has also been associated with suppurative or exudative lesions of the eye, conjunctiva, skin, mammary glands, and other tissues, especially in immunodeficient mice or in mice with a predisposing primary infection. epizootiology p. pneumotropica is a ubiquitous inhabitant of the skin, upper respiratory tract, and gastrointestinal tract of mice. litters from infected dams can become infected during the first week after birth. pathology infections can cause suppurative inflammation, which may include abcessation. dermatitis, conjunctivitis, dacryoadenitis, panophthalmitis, mastitis, and infections of the bulbourethral glands have been attributed to p. pneumotropica. preputial and orbital abscesses also occur, especially in athymic mice (fig. . ). its role in metritis is unclear, but it has been cultured from the uterus, and there is some evidence that it may cause abortion or infertility. cutaneous lesions can occur without systemic disease. they include suppurative lesions of the skin and subcutaneous tissues of the shoulders and trunk. diagnosis diagnosis requires isolation of the organism on standard bacteriological media. although infection can be detected serologically by elisa (wullenweber-schmidt et al., ; boot et al., a, b) , subclinical carriers often do not seroconvert. pcr assays also are available (dole et al., ) and have shown that p. pneumotropica did not transmit from infected mice to contact or dirty bedding sentinels (ouellet et al., ; dole et al., ) . differential diagnosis suppurative lesions in mice may be caused by other bacteria, including staphylococcus, streptococcus, corynebacterium, klebsiella, and mycoplasma. treatment antibiotic sensitivity testing in vitro indicated p. pneumotropica was significantly more sensitive than p. aeruginosa to enrofloxacin (sasaki et al., ) . enrofloxacin in the drinking water at mg/kg daily for days eliminated clinical signs and infection in a closed breeding colonic of transgenic mice and after days of treatment there were no detectable carriers when the colony was screened weeks later (matsumiya and lavoie, ) . prevention and control because p. pneumotropica is an opportunistic organism, it should be excluded from colonies containing immunodeficient mice and from breeding colonies. achieving this goal will normally require barrier housing supported by sound microbiological monitoring. rederivation should be considered to eliminate infection in circumstances where infection presents a potential threat to animal health or experimentation. research complications clinically severe infection in immunodeficient mice is the major complication. although clinically silent, experimental evidence has shown that p. pneumotropica infection in immunocompetent mice (c bl/ ) stimulated transcription of multiple proinflammatory cytokines for at least days with residual elevation detectable days later (patten et al., ) . pioneering studies conducted in the s first linked a novel microaerobic bacterium, helicobacter hepaticus, with chronic active hepatitis and hepatic tumors in a/jcr mice (fox et al., , ward et al., ) . the organism could be visualized by electron microscopy in the bile canaliculi of the liver in susceptible mouse strains (fig. . ). subsequently, it was associated with inflammatory bowel disease in several murine models (table . ) which were further developed to examine the role of immune cell subsets, such as t regulatory cells, in the pathogenesis of inflammatory bowel disease (ibd) and colon cancer (fig. . ). helicobacteriosis is laboratory animal medicine now appreciated to be a common infection of laboratory mice. it is caused by a growing list of helicobacter spp. that vary in clinical, pathologic, and epidemiologic significance (whary and fox, ; fox et al., ) . because recognition and investigation of helicobacteriosis continues to evolve, many important questions about the impact of this infection on mice remain unresolved. h. hepaticus infection is emphasized here, because it is among the most prevalent causes of helicobacteriosis and has been studied more extensively than other murine enterohepatic helicobacter spp. (ehs) (fox et al., , ward et al., ; suerbaum et al., ) . however, current information about other murine helicobacters is summarized in the concluding section. etiology helicobacter spp. are gram-negative, microaerophilic, curved to spiral-shaped organisms that have been isolated from the gastrointestinal mucosa of many mammals, including humans and mice whary and fox, ) . to date, the genus includes formally named helicobacter spp. assigned on the basis of s rrna analysis, complemented by biochemical, molecular, and morphological characteristics. the organisms can be grown on freshly prepared antibiotic impregnated blood agar or in broth supplemented with fetal bovine serum in a microaerobic atmosphere ( % co , % n , % h ). there are currently formally named helicobacter species have been isolated from laboratory mice, as well as several other novel helicobacter spp. awaiting formal naming. species isolated from mice include h. hepaticus, h. bilis (which also infects rats), h. muridarum, h. rappini, and h. rodentium, h. ganmani, h. mastomyrinus, h. magdeburgensis, and h. typhlonius , each of which cahill et al. ( ) tcrα, β mutants defective t-receptors typhlocolitis chin et al. ( ) scid icr-defined flora b t-and b-cell deficient typhlocolitis shomer et al. ( ), shomer et al. ( c bl/il- −/−c lacks il- typhlocolitis burich et al. ( ) , kullberg et al. ( ) , kullberg et al. ( ) , kullberg et al. ( ) c blrag mice infected with h. bilis also developed ibd (shomer et al., ) . c ibd also developed in c bl/il- −/− mice experimentally infected with a novel urease-negative helicobacter spp. now named h. typhlonius (franklin et al., ) ; also ibd produced with h. trogontum (whary et al., ) and h. cinaedi (shen et al., ) . d h. bilis produces ibd (maggio-price et al., and colon cancer (maggio-price et al., ) . have been formally named (except for h. rappini) (fox and lee, ; franklin et al., ; whary and fox, ) . most recently, helicobacter pullorum, a human pathogen, has been isolated from commercial, barriermaintained mice (boutin et al., ) . these ehs are most commonly urease-, catalase-, and oxidase-positive. however, h. rodentium, h. typhlonicus, and another novel helicobacter sp. are urease-negative. clinical signs helicobacteriosis in adult immunocompetent mice is usually asymptomatic. liver enzymes are elevated in h. hepaticus-infected a/jcr mice (fox et al., a) . infection of immune-dysregulated mice with h. hepaticus can cause inflammatory bowel disease, which may present as rectal prolapse and/or diarrhea (miller et al., ) . epizootiology recent surveys and anecdotal evidence suggest that helicobacteriosis is widespread among conventional and barrier-maintained mouse colonies (shames et al., ; fox et al., b; taylor et al., ; lofgren et al., ) . furthermore, h. hepaticus (and probably other helicobacters) can persist in the gastrointestinal tract, particularly the cecum and colon, and is readily detected in feces. these results indicate that transmission occurs primarily by the fecal-oral route and imply that carrier mice can spread infection chronically in enzootically infected colonies. pathology helicobacter spp. colonize the crypts of the lower bowel, where, depending on host genotype, the organisms can be pathogenic or nonpathogenic. h. hepaticus and h. bilis, e.g., can cause inflammation in the gastrointestinal tract, which is expressed as ibd and colon cancer in immunodeficient mice or typhlitis in a/jcr mice infected with h. hepaticus (ward et al., ; knutson et al., ; shomer et al., ; erdman et al., b; nguyen et al., ) . thickening of the cecum and large bowel develops because of proliferative typhlitis, colitis, proctitis, and lower bowel carcinoma. these lesions can occur without coincident hepatitis. indeed, helicobacter spp. induced ibd and colon carcinoma are increasingly popular models to study pathogenesis of the disease in humans (table . ). helicobacter spp. also can cause liver disease. bacterial translocation is thought to occur and results in colonization of the liver and progressive hepatitis. it is characterized by angiocentric nonsuppurative hepatitis and hepatic necrosis (fig. . ) . inflammation originates in portal triads and spreads to adjacent hepatic parenchyma. hepatic necrosis also may occur adjacent to intralobular venules, which can contain microthrombi. additionally, phlebitis may affect central veins. this lesion has been linked to the presence of organisms in bile canaliculi by silver stains and electron microscopy. age-related hepatocytic proliferation can develop in infected livers, a response that is more pronounced in male mice than in female mice (fox et al., a) . this lesion may laboratory animal medicine increase susceptibility to hepatomas and hepatocellular carcinomas among aged male a/jcr and b c f mice from infected colonies. an increased incidence of hepatic hemangiosarcoma also has been noted in h. hepaticusinfected male b c f mice. in this context, a/jcr, c h/ hencr, and sjl/ncr mice are susceptible to hepatitis, whereas c bl/ mice are resistant (ward et al., ) . the finding of severe liver disease and tumor induction in b c f mice infected with h. hepaticus infers that genetic susceptibility to h. hepaticus-induced neoplasia has a dominant pattern of inheritance. studies with h. hepaticus in recombinant inbred mice also indicate that disease susceptibility has multigenetic properties (hailey et al., ; fox and lee, ; ihrig et al., ; franklin, ; hillhouse et al., ) . diagnosis rapid generic diagnosis can be accomplished by pcr detection of the highly conserved s rrna region of the helicobacter genome in feces or tissues, using suitable oligonucleotide primers (fox et al., a; shames et al., ; beckwith et al., ) . however, genus-specific pcr does not differentiate among different helicobacter spp. molecular speciation can be accomplished by s rrna sequencing, restriction fragment length polymorphism analysis of the pcr product or use of species-specific pcr assays. this procedure requires suitable skill and experience to avoid technological pitfalls and should be performed by qualified laboratories. an igg elisa using the outer membrane protein as the antigen has been proposed for serological diagnosis, but shared antigens among ehs create lack of specificity for the assay. as noted above, helicobacters can be isolated on antibiotic-impregnated blood agar under microaerobic conditions and can then be speciated biochemically, and by helicobacter species-specific pcr. isolation of h. hepaticus and from other helicobacter spp. with spiral to curved morphology from feces should be preceded by passing slurried samples through a . -μm filter before plating. if infection with larger fusiform helicobacters (h. bilis, h. rappini) is suspected, filtration at . μm is preferred. helicobacters grow slowly and require prolonged incubation of cultures (up to weeks) before they can be deemed negative. signs (rectal prolapse) and lesions (hepatitis, typhlocolitis), depending on host genotype, can be suggestive of infection. histopathological examination should include silver stains, especially of liver, to attempt to visualize spiral or curved organisms (whary and fox, ) . differential diagnosis clinically apparent helicobacteriosis must be differentiated from other gastrointestinal or hepatic infections of mice. coronavirus infection, clostridium piliforme, and salmonella spp. can cause enterocolitis and/or hepatitis. c. rodentium also causes colonic hyperplasia, which can present as rectal prolapse. infections caused by other helicobacters of mice h. bilis has been isolated from the livers and intestines of aged mice and experimentally induces ibd in scid mice as does h. hepaticus. h. bilis also experimentally produces lower bowel cancer in immunocompromised mice (nguyen et al., ) . helicobacter muridarum colonizes the ileum, cecum, and colon. it appears to be nonpathogenic, although it can colonize the stomach of mice and induce gastritis under certain circumstances. h. 'rappini' has been isolated from the feces of mice without clinical signs. h. rodentium also colonizes the intestine and may be a component of normal flora. a dual infection of h. bilis and h. rodentium was noted in a natural outbreak of ibd in immunocompromised mice (shomer et al., ) . a novel urease negative helicobacter, which has been named h. typhlonius, causes ibd in il- −/− and scid mice (franklin et al., (franklin et al., , fox et al., ) . decreased reproductive efficiency has been reported in il knockout mice infected with h. rodentium and/or h. typhlonius (sharp et al., ) . prevention and control eradication of infection from small numbers of mice, such as quarantine groups, can be achieved by standard rederivation or intensive antibiotic therapy. the best results have been obtained by triple therapy with amoxicillin, metronidazole, and bismuth given for weeks (del carmen martino-cardona et al., ) . this strategy requires repeated daily gavage rather than administration in drinking water, but it has successfully eliminated h. hepaticus from naturally infected mice. antibiotic impregnated wafers have been used to eradicate helicobacter spp. in mouse colonies (kerton and warden, ) . wide-scale, eradication of enzootic helicobacteriosis can be expensive and time-consuming, without guarantee of success. careful husbandry procedures can limit infection within a colony (whary et al., ) . therefore, strategies have to be weighed carefully against risks of enzootic infection for the health and use of mice. in contrast, infection should be avoided in immunodeficient mice, including genetically engineered mice with targeted or serendipitous immune dysfunction. lastly, the outcome of opportunistic helicobacteriosis has not been thoroughly examined. this condition could occur during simultaneous infection with two or more helicobacter species or during combined infection with an intestinal virus (e.g., coronavirus) and helicobacter spp. if highly valuable animals are exposed, antibiotic therapy or rederivation may be warranted. research complications chronic inflammation of the liver and or gastrointestinal tract may be injurious to health. additionally, it may impede the development and assessment of noninfectious disease models, such as ibd models in mice with targeted deletions in t-lymphocyte receptors (fox et al., ) . h. hepaticus infections provoke a strong th proinflammatory response, which may perturb other immunological responses. h. hepaticus infection also has been incriminated as a cofactor or promoter in the development of hepatic neoplasia in a/ jcr, b c f , ab f , b af , and carko mice (hailey et al., ; fox et al., a; garcia et al., garcia et al., , h. salmonellosis (ganaway, ; lindsey et al., etiology the genus salmonella contains two species, s. bongori which infects mainly poikilotherms and rarely, humans, and s. enterica which includes approximately serovars and are a major cause of food-borne illness in humans (fookes et al., ) . the salmonella of historical importance in mice that are now rare include s. enterica subsp. enterica serovar typhimurium (aka s. typhimurium) and serovar enteritidis (s. enteritidis). s. enteritidis is a motile, gram-negative rod that rarely ferments lactose. the genomes of many strains have been sequenced. virulence factors carried on pathogenicity islands and plasmids include antimicrobial resistance genes, type iii secretion systems, vi antigen, lipopolysaccharide and other surface polysaccharides, flagella, and factors essential for a intracellular life cycle in macrophages (de jong et al., ) . pathogenassociated molecular patterns (pamps) unique to salmonella interact with tlrs and nod-like receptors (nlrs) which recruit neutrophils and macrophages leading to inflammasome formation and release of pro-inflammatory il- , il- β, tnf-α, and ifn-γ. clinical signs acute infection is especially severe in young mice (casebolt and schoeb, ) . it is characterized by anorexia, weight loss, lethargy, dull coat, humped posture, and occasionally conjunctivitis. gastroenteritis is a common sign, but feces may remain formed. subacute infection can produce distended abdomens from hepatomegaly and splenomegaly. chronic disease is expressed as anorexia and weight loss. enzootic salmonellosis in a breeding colony can produce episodic disease with alternating periods of quiescence and high mortality. the latter can be associated with diarrhea, anorexia, weight loss, roughened hair coat, and reduced production. epizootiology s. typhimurium is commonly used experimentally and cross-contamination in a mouse facility is a risk. modern production and husbandry methods have reduced the importance of salmonellosis as a natural infection of mice. however, the organisms are widespread in nature. therefore, cross-infection from other species or from feral mice remains a potential hazard. salmonellas are primarily intestinal microorganisms that can contaminate food and water supplies. infection occurs primarily by ingestion. salmonella have a broad host range and vermin, birds, feral rodents, and human carriers are potential sources of infection. other common laboratory species such as nonhuman primates, dogs, and cats also can serve as carriers. conversely, murine salmonellosis presents a zoonotic hazard to humans. the induction and course of infection are influenced by the virulence and dose of the organism, route of infection, host sex and genetic factors, nutrition, and intercurrent disease. suckling and weanling mice are more susceptible to disease than mature mice. immune deficiency, exposure to heavy metals, and environmental factors such as abnormal ambient temperatures can increase the severity of disease. nutritional iron deficiency has an attenuating effect on salmonella infection in mice, whereas iron overload appears to promote bacterial growth and enhance virulence. resistance to natural infection is increased by the presence of normal gastrointestinal microflora. resistance to infection also can be an inherited trait among inbred strains. among the most important considerations is that mice that recover from acute infection can become subclinical carriers and a chronic source of contamination from fecal shedding. pathology the virulence of s. enteritidis depends on its ability to penetrate intestinal walls, enter lymphatic tissue, multiply, and disseminate. organisms reach peyer's patches within h after inoculation and spread quickly to the mesenteric lymph nodes. bacteremia results in spread to other lymph nodes, spleen, and liver within several days. in chronic infections, organisms persist in the spleen and lymph nodes as well as in the liver and gallbladder and from the latter are discharged into the intestinal contents. bacteria reaching the intestine can reinvade the mucosa and can be shed intermittently in the feces for months. s. enteritidis infection also has been associated with chronic arthritis. acute deaths may occur without gross lesions, but visceral hyperemia, pale livers, and catarrhal enteritis are more common. if mice survive for up to several laboratory animal medicine weeks, the intestine may be distended and reddened, whereas the liver and spleen are enlarged and contain yellow-gray foci of necrosis. affected lymph nodes are also enlarged, red, and focally necrotic. focal inflammation can develop in many organs, including the myocardium (percy and barthold, ) . histologic lesions reflect the course of disease and the number of bacteria in affected tissues. during acute infection, necrotic foci are found in the intestine, mesenteric lymph nodes, liver, and spleen. neutrophilic leukocytes and histiocytes accumulate in lymphoid tissues. thrombosis from septic venous embolism may occur, especially in the liver. granulomatous lesions are particularly characteristic of chronic salmonellosis, especially in the liver. diagnosis diagnosis is based on isolation of salmonellas together with documentation of compatible clinical signs and lesions. in mice with systemic disease, bacteria may persist in the liver and spleen for weeks. during acute stages, bacteria can also be isolated from the blood. subclinically infected animals can be detected by fecal culture using selective enrichment media (selenite f broth plus cystine followed by streaking on brilliant green agar). culture of the mesenteric lymph nodes may be more reliable, because fecal shedding can be intermittent. isolates can be speciated with commercial serotyping reagents. alternatively, isolates can be sent to a reference laboratory for confirmation. antibodies to salmonellas can be detected in the serum of infected mice by an agglutination test. however, this method is not entirely reliable, because serological crossreactivity is common even among bacteria of different genera. pcr-based assays are also available. differential diagnosis salmonellosis must be differentiated from other bacterial diseases, including tyzzer's disease, helicobacter spp., pseudomoniasis, corynebacteriosis, c. rodentium, and pasteurellosis. viral infections that cause enteritis or hepatitis must also be considered, especially infections caused by coronavirus, ectromelia virus, and reoviruses. among noninfectious conditions, mesenteric lymphadenopathy is an agingassociated lesion in mice and is not indicative of chronic salmonellosis. prevention and control salmonellosis can be prevented by proper husbandry and sanitation. contact between mice and potential carriers, such as nonhuman primates, dogs, and cats, should be prevented. diets should be cultured periodically to check for inadvertent contamination. contaminated colonies should be replaced to eliminate infection and its zoonotic potential. research complications apart from the clinical manifestations, the zoonotic potential for salmonellosis is a major concern. this includes transmission among laboratory species, but especially between mice and the personnel working with them. i. streptobacillosis (lindsey et al., e; percy and barthold, ) etiology streptobacillus moniliformis is a nonmotile, gram-negative, pleomorphic rod that can exist as a nonpathogenic l-phase variant in vivo. however, it can revert to the virulent bacillus form. clinical signs streptobacillosis generally has an acute phase with high mortality, followed by a subacute phase and finally a chronic phase that may persist for months. signs of acute disease include a dull, damp hair coat and keratoconjunctivitis. variable signs include anemia, diarrhea, hemoglobinuria, cyanosis, and emaciation. cutaneous ulceration, arthritis, and gangrenous amputation may occur during chronic infection. the arthritis can leave joints deformed and ankylosed. hindlimb paralysis with urinary bladder distention, incontinence, kyphosis, and priapism may occur if vertebral lesions impinge on motor nerves. breeding mice may have stillbirths or abortions. epizootiology streptobacillosis has historical importance as a disease of rats and mice, but modern husbandry, production, and health surveillance strategies have reduced its impact dramatically (wullenweber, ) . subclinical, persistently infected rats are the most likely source of dissemination to mice, but mouse-tomouse transmission then ensues. transmission may occur from aerogenic exposure, bite wounds, or contaminated equipment, feed, or bedding. s. moniliformis is also pathogenic for humans, causing rat bite fever (haverhill fever). pathology during acute disease, necrotic lesions develop in thoracic and abdominal viscera, especially in the liver, spleen, and lymph nodes. histological lesions include necrosis, septic thrombosis of small vessels, acute inflammation, fibrin deposition, and abscesses. chronically infected mice may develop purulent polyarthritis because of the organism's affinity for joints. diagnosis diagnosis depends on clinical and pathological evidence of septicemia and isolation of the organism on blood agar. the organism has been recovered from joint fluid as long as months after infection. isolation from chronic lesions requires serumenriched medium. s. moniliformis as a cause of septic joints in humans has been diagnosed using pcr and electrospray-ionization followed by mass spectrometry (mackey et al., ) . differential diagnosis clinical signs must be differentiated from septicemic conditions, including mousepox, tyzzer's disease, corynebacteriosis, salmonellosis, mycoplasmosis, pseudomoniasis, and traumatic lesions. prevention and control control is based on exclusion of wild rodents or carrier animals such as latently infected laboratory rats. bacterins and antibiotic therapy are not adequately effective. the potential laboratory animal medicine for cross-infection is a reason not to house rats and mice in the same room. research complications infection can be disabling or lethal in mice and has zoonotic potential for humans. j. corynebacteriosis (lindsey et al., ; weisbroth, ; percy and barthold, ) etiology corynebacteria are short gram-positive rods. corynebacterium kutscheri is the cause of pseudotuberculosis in mice and rats. corynebacterium bovis has been associated with hyperkeratosis, especially in immunodeficient mice (clifford et al., ; scanziani et al., ; dole et al., ) . clinical signs c. kutscheri infection is often subclinical in otherwise healthy mice. active disease is precipitated by immunosuppression or environmental stresses and is expressed as an acute illness with high mortality or a chronic syndrome with low mortality. clinical signs include inappetence, emaciation, rough hair coat, hunched posture, hyperpnea, nasal and ocular discharge, cutaneous ulceration, and arthritis. c. bovis infection causes hyperkeratotic dermatitis characterized by scaly skin, which is accompanied by alopecia in haired mice. severe infection may cause death. corynebacterial keratoconjunctivitis has been reported in aged c bl/ mice (mcwilliams et al., ) . epizootiology subclinically infected animals harbor c. kutscheri in the upper alimentary tract, colon, respiratory tract, regional lymph nodes, middle ear, and preputial gland. c. bovis colonizes skin and is shed in feces. therefore, transmission is by direct contact, fecaloral contact, and aerosol. resistance to infection appears to be under genetic control in some mouse strains. rats are susceptible to c. kutscheri, so cross-infection to mice may occur. pathology lesions caused by c. kutscheri develop from hematogenous spread to various internal organs and appear as gray-white nodules in the kidney, liver, lung, and other sites. cervical lymphadenopathy and arthritis of the carpometacarpal and tarsometatarsal joints also may occur. septic, necrotic lesions often contain caseous material or liquefied exudate. histologic lesions are characterized by coagulative or caseous necrosis bordered by intense neutrophilic infiltration. colonies of gram-positive organisms with 'chinese letter' configurations can usually be demonstrated using tissue gram stains of caseous lesions. mucopurulent arthritis of carpal, metacarpal, tarsal, and metatarsal joints are related to bacterial colonization of synovium accompanied by necrosis, cartilage erosion, ulceration, and eventually ankylosing pan arthritis. c. kutscheri is not a primary skin pathogen, but skin ulcers or fistulas follow bacterial embolization and infarction of dermal vessels. subcutaneous abscesses have also been reported. hyperkeratotic dermatitis caused by c. bovis is characterized grossly by skin scaliness and alopecia. microscopically, skin lesions consist of prominent acanthosis and moderate hyperkeratosis accompanied by mild nonsuppurative inflammation (fig. . ) . hyperkeratosis is typically more severe in glabrous athymic mice than in haired mice. organisms can be demonstrated in hyperkeratotic layers by gram stain. diagnosis c. kutscheri is usually diagnosed by culture and tissue gram stains on lesions from clinically apparent cases. agglutination serology is available, and immunofluorescence, immunodiffusion, and elisa tests have been reported (boot et al., a) . pcr of skin swabs or feces is a sensitive and specific method for the detection of c. bovis infection in mice (dole et al., ) . differential diagnosis the caseous nature of c. kutscheri-induced lesions helps separate them from necrotic changes or abscesses caused by other infectious agents of mice. thus, they can be differentiated from streptococcosis, mycoplasmosis, and other septicemic bacterial infections in which caseous necrosis does not occur. because mice can sustain natural infections with mycobacterium avium, histochemical techniques for acidfast bacilli and appropriate culture methods for mycobacteria should be considered if nodular inflammatory lesions of the lung are detected. diffuse scaling dermatitis in athymic nude mice is classic for c. bovis infection; however, in one case report staphylococcus xylosus was instead isolated in high numbers from the skin lesions (russo et al., ) . hyperkeratotic dermatitis caused by laboratory animal medicine c. bovis must be differentiated from scaly skin caused by low humidity in glabrous mice. prevention and control c. kutscheri infection occurs sporadically and infected colonies should be culled or rederived into an spf facility as treatment is not curative and control is difficult. c. bovis can be endemic in athymic nude mouse colonies. prevention and control are difficult because both immunocompetent and athymic mice as well as humans can carry c. bovis on the skin and in the upper respiratory system, respectively. c. bovis readily contaminates the environment as aerosolization within a class ii biosafety cabinet was shown to spread the bacterium during cage-change procedures (burr et al., ) . antibiotic treatment has been unrewarding (burr et al., ) research complications corynebacteriosis can cause morbidity and mortality, especially among immunodeficient mice. dermatologic disease in suckling mice can be fatal but is less severe and transient in weanling mice. etiology staphylococci are gram-positive organisms that commonly infect skin and mucous membranes of mice and other animals. the two most frequently encountered species are staphylococcus aureus, which can be highly pathogenic, and s. epidermidis, which is generally nonpathogenic. species subtypes are identified by phage typing and biochemistry profiles. pathogenic staphylococci are typically coagulase-positive, although s. xylosus has caused serious infections and is coagulasenegative (gozalo et al., ) . clinical signs staphylococcosis causes suppurative conjunctivitis, periorbital and retroorbital abscesses, preputial adenitis, and pyoderma in mice, particularly in immunocompromised strains such as nude mice. some evidence suggests that staphylococci can produce primary cutaneous infections, but they are more likely opportunistic organisms that induce lesions after contamination of skin wounds. eczematous dermatitis develops primarily on the face, ears, neck, shoulders, and forelegs and can progress to ulcerative dermatitis, abscessation (including botryomycotic granulomas), and cellulitis. because lesions are often pruritic, scratching causes additional trauma and autoinoculation. staphylococcal infection in the genital mucosa of males may produce preputial gland abscesses. these occur as firm, raised nodules in the inguinal region or at the base of the penis and may rupture to spread infection to surrounding tissues. male mice also may develop septic balanoposthitis secondary to penile self-mutilation. retrobulbar abscesses caused by s. aureus are frequently noted in athymic mice. sjl mice, which are nk cell deficient, are prone to necrotic dermatitis on the tail secondary to s. xylosus infection. epizootiology staphylococci are ubiquitous and can be carried on the skin and in the nasopharnyx and gastrointestinal tract. they also can be cultured from cages, room surfaces, and personnel. the prevalence of staphylococcal dermatitis appears to be influenced by host genotype, the overall health of the animal, and the degree of environmental contamination with staphylococcus spp. c bl/ , c h, dba, and balb/c mice are among the most susceptible strains. age may also influence susceptibility, with young mice being more susceptible than adults. immunodeficient mice (e.g., athymic mice) contaminated with staphylococci often develop abscesses or furunculosis (fig. . ). as noted above, behavioral dysfunction resulting in selfmutilation, including scratching and trichotillomania, is a likely predisposing factor. once virulent staphylococci contaminate the environment, colonization of the gastrointestinal tract can occur and produce a carrier state. phage typing can help to determine the source of infection. human phage types of staphylococci can infect mice, but the zoonotic importance of this connection is not clear. pathology gross lesions are typified by suppurative, ulcerative and necrotic dermatitis involving the head and neck but may extend to the shoulders and forelegs (percy and barthold, ) . superficial or deep abscesses may occur in conjunction with dermatitis or separately, as, e.g., in the external male genitalia. histologically, acute skin infections result in ulceration with neutrophils in the dermis and subcutis. chronic lesions contain lymphocytes, macrophages, and fibroblasts. deep infections appear as coalescing botryomycotic pyogranulomas with necrotic centers containing bacterial colonies. infected athymic mice may develop laboratory animal medicine furunculosis of the muzzle and face accompanied by regional lymphadenitis. diagnosis diagnosis is made by documenting gross and histological lesions, including gram staining of suspect tissues, complemented by isolation of grampositive, coagulase-positive (s. aureus), or coagulasenegative staphylococcus species. differential diagnosis staphylococcosis must be differentiated from other suppurative infections of mice, including pasteurellosis, streptococcosis, corynebacteriosis, and pseudomoniasis. ectoparasitism, fight wounds, and self-mutilation per se should also be considered. prevention, control, and treatment removal of affected animals, sterilization of food and bedding, and frequent changing of bedding may limit or reduce transmission. in affected animals, nail trimming can reduce self-inflicted trauma. conditions that facilitate aggressive or self-mutilating behavior should be avoided. research complications staphylococcosis can cause illness and disfigurement in mice. immunodeficient mice are at increased risk. etiology streptococci are ubiquitous commensal gram-positive organisms and in some cases, primary pathogens. pathogenic streptococcal infections in laboratory mice are caused by β-hemolytic organisms in lancefield's group c, but epizootics caused by group a streptococci have occurred, and group g organisms have been isolated occasionally. group d has been reclassified as an enterococcus. alpha-hemolytic streptococci can cause systemic disease in scid mice, and group b streptococcus sp. infection has been reported to cause meningoencephalitis in athymic mice (schenkman et al., ) . additionally, streptococcus dysgalactiae subsp. equisimilis has lancefield group g or c antigens and was isolated from visceral abscesses of immunocompetent mice (greenstein et al., ) . clinical signs cutaneous infections can cause ulcerative dermatitis over the trunk, which may appear gangrenous, whereas systemic infections may be expressed as conjunctivitis, rough hair coat, hyperpnea, somnolescence, and emaciation. epizootiology mice can carry streptococci subclinically in their upper respiratory tracts. lethal epizootics can occur, but factors leading to clinical disease are unknown, although some infections may be secondary to wound contamination. pathology systemic lesions reflect hematogenous dissemination and include abscessation, endocarditis, splenomegaly, and lymphadenopathy (percy and barthold, ) . streptococcal cervical lymphadenitis can lead to fistulous drainage to the neck complicated by ulcerative dermatitis. infection with α-hemolytic streptococci can cause inflammatory lesions affecting kidney and heart. diagnosis diagnosis and differential diagnosis depend on isolation of organisms from infected tissues, combined with histopathologic confirmation. differential diagnosis streptococcosis must be differentiated from other suppurative infections of mice, including staphylococcosis, pasteurellosis, corynebacteriosis, and pseudomoniasis. prevention and control removal of affected animals, sterilization of food and bedding, and frequent changing of bedding may limit or reduce transmission. research complications immunodeficient mice are at increased risk for streptococcosis. etiology e. coli is a small gram-negative rod that is a normal inhabitant of the mouse intestine. epizootiology infection is considered nonpathogenic in immunocompetent mice. however, hyperplastic typhlocolitis resembling transmissible murine colonic hyperplasia has been reported in scid mice infected with a non-lactose-fermenting e. coli (waggie et al., ; arthur et al., ) . clinical signs affected mice develop lethargy and fecal staining. pathology gross lesions consist of segmental thickening of the colon or cecum, which may contain blood-tinged feces. microscopically, affected mucosa is hyperplastic and may be inflamed and eroded. diagnosis diagnosis depends on demonstrating lesions and isolating non-lactose-fermenting e. coli. differential diagnosis this condition must be differentiated from proliferative and inflammatory intestinal disease caused by lawsonia intracellularis, c. rodentium, or enterotropic mouse hepatitis virus, especially in immunodeficient mice. colibacillosis provides an example of the morbidity associated with a nominally innocuous organism when it affects an immunocompromised host. prevention and control removal of affected animals and disinfection of caging and equipment will limit or reduce transmission. research complications clinical illness may develop in immunodeficient mice. historically, klebsiella pneumoniae is a ubiquitous gram-negative organism that is a natural inhabitant of the mouse alimentary tract. most commercial vendors have excluded it from their barriers. it can be pathogenic for the respiratory and urinary tract of mice after experimental inoculation but is not a significant cause of naturally occurring disease. etiology klebsiella oxytoca is an opportunistic pathogen implicated in various clinical diseases in animals and humans. epizootiology k. oxytoca also is purported to be an etiological agent of antibiotic-associated hemorrhagic colitis (aahc) in adult humans and adolescents. in animals, k. oxytoca has been isolated from apparently healthy sentinel rodents being monitored for pathogens in health surveillance programs and from utero-ovarian infections including suppurative endometritis, salpingitis, perioophoritis, and peritonitis in aged b c f mice (davis et al., ; rao et al., ) . a model of aahc has been developed in rats by administering amoxicillinclavulanate followed by orally infecting rats with a strain of k. oxytoca cultured from a patient with aahc. studies in humans suggest that k. oxytoca exerts its pathogenicity in part through a cytotoxin. recently, authors have showed that several animal isolates of k. oxytoca, including clinical isolates, produced secreted products in bacterial culture supernatant that display cytotoxicity on hep- and hela cells, indicating the ability to produce cytotoxin. using mass spectroscopy techniques, they also confirmed tilivalline as the cytotoxin present in animal k. oxytoca strains. tilivalline may serve as a biomarker for k. oxytoca-induced cytotoxicity (darby et al., ) . clinical signs k. oxytoca has been cultured from cases of suppurative otitis media, urogenital tract infections, and pneumonia in c h/hej and nmri-foxn (nu) mice (bleich et al., ) . additionally, k. oxytoca was recently cultured from three breeding colonies of nod. cg-prkdc scid il rg tm wjl /szj (nsg) mice with chronic renal inflammation and ascending urinary tract infections (foreman et al., ) . differential diagnosis other bacterial infections capable of causing suppurative lesions, including staphylococci, streptococci, pasteurella sp., and e. coli, among others are considered a differential diagnosis. research complications morbidity and mortality from spontaneous infections can affect ongoing research. etiology clostridium difficile was identified as the etiology of antimicrobial-associated pseudomembranous colitis in humans and currently a considerable cause of morbidity in hospitalized patients who acquire nosocomial infections. in the early s, an increased interest in c. difficile infection (cdi) resulted from the emergence of a hyper-virulent strain (nap /bi/ ) associated with frequent recurrences and more severe clinical disease (abou chakra et al., ; mcfarland, ; kuijper et al., ) . c. difficile has also been implicated in antibioticassociated colitis in syrian hamsters (bartlett et al., ) , guinea pigs (lowe et al., ) , rabbits (thilsted et al., ; ryden et al., ) , prairie dogs (muller et al., ) , ostriches (frazier et al., ) , and horses (diab et al., ) . c. difficile is a rod-shaped strict anaerobe. cycloserinecefoxitin-fructose agar (ccfa) is a commonly used selective medium for c. difficile. cultures are incubated under anaerobic conditions at - °c. when grown on blood agar, c. difficile colonies are nonhemolytic and gray, and have a slightly raised umbonate profile with filamentous edges and a ground-glass appearance. colonies grown on blood agar have fluorescence under ultraviolet light. c. difficile forms acid from glucose and fructose, but is negative on lactose, maltose, and sucrose. two closely related exotoxins, toxin a and toxin b, are produced by c. difficile. recent taxonomic classification support placement of c. difficile and its close relatives within the family peptostreptococcaceae. the authors suggested renaming it peptoclostridium difficile (yutin and galperin, ) . epizootiology it is estimated that c. difficile spores germinate and establish infection less than h after ingestion. spores rapidly transit through the upper gastrointestinal tract and colonize the colon and cecum. spore shedding begins less than h postingestion. when c bl mice were challenged with cfu of c. difficile spores, severe cdi signs developed and all mice were clinically affected by h postchallenge (chen et al., ) . specific methods to control and prevent c. difficile infections in mice have not been described. given the method of transmission of c. difficile and c. perfringens are via ingestion or spores, these clostridia can probably be excluded from mouse colonies by maintaining strict husbandry practices, robust sanitation, and use of autoclaved feed, bedding, cages, and cage accessories. sudden dietary changes should be avoided and antibiotics should be used judiciously to minimize disruption of the normal gut microbiota of mice. diagnosis of c. difficile-associated disease is generally based on detection of cytotoxin using a tissue culture cytotoxicity assay. pcr assays for detection of both c. difficile and its cytotoxins have been developed (eastwood et al., ) . there are no published regimens specifically for the treatment of natural c. difficile infections in mice. oral doses given twice daily of mg vancomycin for days to experimentally infected gnotobiotic mice caused a -to -log decrease in vegetative bacterial cell count and no detectable cytotoxin. bacterial counts and cytotoxin levels returned to previous levels after treatment was discontinued. clinical signs untreated mice are relatively resistant to infection with c. difficile and do not develop fatal infections, although these mice can become asymptomatic carriers that persistently shed low numbers of spores (lawley et al., ) . susceptibility of mice to infection must be induced by disrupting the microbiota through antibiotic treatment. brief exposure to environmental laboratory animal medicine spore contamination is sufficient for transmission of c. difficile to naïve but susceptible mice. the cdi transmission model has been used to demonstrate that clindamycin treatment of asymptomatic carriers of c. difficile can inadvertently trigger the excretion of high levels of spores (lawley et al., ) . a c bl mouse model of recurrence/relapse cdi has been reported (sun et al., ) . the primary bout of cdi induced little or no protective antibody response against c. difficile toxins and mice continued shedding c. difficile spores. antibiotic treatment of surviving mice induced a second episode of diarrhea. a simultaneous reexposure of mice to c. difficile bacteria or spores elicited a full clinical spectrum of cdi similar to that of the primary infection. immunosuppressive agents resulted in more severe and fulminant recurrent disease. vancomycin treatment only delayed disease recurrence; however, neutralizing polysera against both tcda and tcdb completely protected mice against cdi relapse (sun et al., ) . a recent study in c bl mice demonstrated that antibiotic-mediated alteration of the gut microbiome favors a global metabolic profile, and therefore increases susceptibility to c. difficile clinical diseases (theriot et al., ) . c. difficile is not tissue invasive and only toxigenic strains are associated with disease. experimental c. difficile infections include diarrhea, cecitis, polymorphonuclear cell infiltration of the lamina propria, inflammation, pseudomembrane formation, and death. differential diagnosis c. difficile-induced diarrhea is most often associated with antibiotic treatment. other clostridial diseases in mice must be ruled out as well as other enteric pathogens in mice causing diarrhea and mortality. salmonella spp. and c. rodentium should be considered in the differential diagnosis. etiology clostridium perfringens is associated with a number of diseases in domestic animals and humans. c. perfringens is a nonmotile, rod-shaped, encapsulated, anaerobic bacterium measuring - µm in length and . - . µm in diameter (murray et al., ) . c. perfringens grow rapidly on blood agar, and colonies are smooth, round, and grayish in color, and are surrounded by a double zone of hemolysis. c. perfringens is grouped into five types based on the production and secretion of four major toxins. c. perfringens produces a number of other virulence-enhancing toxins and hydrolytic enzymes. the most significant of these is probably enterotoxin, released with the bacterial spore after cell lysis. epizootiology c. perfringens is most likely acquired by the ingestion of spores that originated in the soil or in the intestinal tract of a carrier animal. the organism can be a member of the normal microbiota in human and domestic animals. factors that have been associated with the proliferation of the organism of these species include poor husbandry and sudden dietary changes (quinn et al., ) . methods to control and prevent c. perfringens infections have not been evaluated in mice. because the bacterium is most likely acquired by the ingestion of spores, it can probably be excluded from mouse colonies by maintaining good sanitation and sterilizing feed, bedding, cages, and cage accessories. sudden dietary changes have also been associated with proliferation of the organism and should be avoided if possible (quinn et al., ) . clinical signs only a few reports in the literature exist describing clinical disease associated with c. perfringens infection in mice (matsushita and matsumoto, ; rozengurt and sanchez) . disease has been observed in mice of both sexes, from to days old, and in female mice of breeding age. clinical signs have included hunched posture, ruffled hair coat, enlarged painful abdomen, soft or impacted feces, hindquarter paralysis, and dyspnea. sudden death without premonitory signs has also been reported. the toxin types of c. perfringens isolated from these cases were reported to be non-type a (matsushita and matsumoto, ) , type b (rozengurt and sanchez, ) , and type d (clapp and graham, ) . mucosal necrosis in both the large and small intestine is a consistent finding on microscopic examination of tissues from mice with clinically apparent c. perfringens infections. differential diagnosis c. perfringens produces a number of major and minor toxins. different types of the bacterium produce different toxins which account for different disease outcomes. c. perfringens type a is a constituent of the normal microbiota of the intestine of humans and other animal species. bacterial culture should be obtained from live or recently dead animals, and placed in anaerobic transfer medium for transport to a microbiology laboratory and should be cultured soon after their arrival. a presumptive diagnosis for c. perfringens can be based on the presence of large grampositive rods in fecal smears or in histologic sections of intestines (quinn et al., ) . definitive diagnosis is based on toxin identification. mice treated with chlortetracycline hydrochloride in drinking water at a level of mg/l for weeks have eliminated c. perfringens-associated disease (matsushita and matsumoto, ) . penicillin g in the diet or changing the diet has also been reported to be effective in disease remission. c. perfringens treatments in domestic species include ampicillin, amoxicillin-clavulanate, tylosin, clindamycin, metronidazole, and bacitracin (marks, ; mcgorum et al., ) . commercially available bacterins for use in mice were not effective in controlling the disease (clapp and graham, ) . research complications clostridia are large, rodshaped, gram-positive anaerobic bacteria. naturally occurring clostridial infection in mice is rare. epizootics of c. perfringens type d infection with high mortality laboratory animal medicine have been reported in a barrier colony where heavy mortality occurred in -to -week-old suckling mice. clinical signs included scruffy hair coats, paralysis of the hindquarters, and diarrhea or fecal impaction. however, attempts to reproduce the disease experimentally with clostridia isolated from naturally infected animals were unsuccessful. c. perfringens also has been isolated from sporadic cases of necrotizing enteritis in recently weaned mice. clostridium piliforme -tyzzer's disease (fujiwara and ganaway, ; ganaway, ; ganaway et al., ; percy and barthold, ) etiology tyzzer's disease is named for ernest tyzzer, who first described it in a colony of japanese waltzing mice. the causative organism, c. piliforme (formerly bacillus piliformis), is a long, thin, gram-negative spore-forming bacterium that appears to require living cells for in vitro growth. it has not been grown successfully on cell-free media, but it can be propagated by inoculation of susceptible vertebrates, in select cell lines, the yolk sac of embryonated eggs, or hepatocyte cell cultures obtained from mice (ganaway et al., ; kawamura et al., ) . clinical signs clinical disease occurs as unexpected deaths that may be preceded by diarrhea and inactivity. although outbreaks can be explosive and mortality is usually high, morbidity varies. additionally, subclinical infections can occur, accompanied by the development of antibodies to c. piliforme. stresses, such as overcrowding, high temperature and humidity, moist food, and immunosuppression, and young age, may predispose mice to tyzzer's disease. susceptibility and resistance also are influenced by host genotype. it has been shown, e.g., that c bl/ mice are more resistant than dba/ mice to tyzzer's disease (waggie et al., ) . resistance to severe infection appears to be due, in part, to b-lymphocyte function. the role of t cells in resistance is not clear, because susceptibility among athymic mice appears to vary (livingston et al., ) . however, the involvement of t cells can be inferred by the fact that several interleukins modulate resistance and susceptibility. depletion of neutrophils or nk cells also increases susceptibility to infection. epizootiology current prevalence rates, reservoirs of infection, carrier states, and the mechanism of spread remain speculative. tyzzer's disease occurs in many species of laboratory animals and in domestic and free-living species. some strains appear capable of cross-infecting mice, rats, and hamsters, whereas others have a more restricted host range (franklin et al., ) . therefore, the risks for cross-infection depend on the strain causing a given outbreak. although the vegetative form of c. piliforme is unstable, spores can retain infectivity at room temperature for at least year and should be viewed as the primary means of spread. natural infection is probably due to ingestion of organisms, which are subsequently shed in feces. feces-contaminated food and soiled bedding are the most likely sources of environmental contamination. prenatal infection can be induced by intravenous inoculation of pregnant mice, but its importance in the natural transmission of infection has not been determined. pathology infection begins in the gastrointestinal tract, followed by bacteremic spread to the liver and, to a smaller extent, the heart. the lesions are characterized by necrosis in these tissues and in the mesenteric lymph nodes. grossly, segments of the ileum, cecum, and colon may be red and dilated, with watery, fetid contents, whereas the liver, mesenteric lymph nodes, and heart often contain gray-white foci. histologically, intestinal lesions include necrosis of mucosal epithelium, which may be accompanied by acute inflammation and hemorrhage. in the liver, foci of coagulation necrosis are generally distributed along branches of the portal vein, a finding compatible with embolic infection from the intestine. peracute lesions are largely free of inflammation, but neutrophils and lymphocytes may infiltrate less fulminant lesions. myocardial necrosis is sporadic in natural infection. diagnosis tyzzer's disease is diagnosed most directly by the demonstration of characteristic intracellular organisms in tissue sections of liver and intestine. bundles of long, slender rods occur in the cytoplasm of viable cells bordering necrotic foci, especially in the liver (fig. . ) and intestine. they are found more easily during early stages of infection. organisms in tissue sections do not stain well with hematoxylin-eosin stain. silver stains, giemsa stains, or periodic acid-schiff stains are usually required for visualization of the organism. pcr and serologic assays are readily available at diagnostic laboratories. older supplemental procedures included inoculation of cortisonized mice or embryonated eggs laboratory animal medicine with suspect material, followed by histological or immunocytochemical demonstration of organisms in tissues. differential diagnosis the histological detection of organisms is essential for differentiating tyzzer's disease from other infections that can produce similar signs and lesions, especially mousepox, coronaviral hepatitis, reoviral hepatitis, helicobacteriosis, and salmonellosis. it also is important not to misconstrue extracellular rods as c. piliforme. prevention and control barrier housing and husbandry that incorporate sanitation measures to avoid the introduction or buildup of spores in the environment are the bases for control or prevention of tyzzer's disease. if infection occurs, spore formation will make control or elimination by antibiotic therapy problematic. therefore, strict quarantine, followed by replacement of affected or exposed stock, must be considered. rederivation by embryo transfer or cesarean section should take the potential for prenatal transmission of infection into account in housing and testing offspring. thorough decontamination of the environment with an oxidizing disinfectant must be included in any control program. additionally, procurement of food and bedding from suppliers with thorough quality assurance and vermin control programs is essential for both prevention and control. husbandry supplies should be stored in vermin-proof quarters, and the option of heat sterilization of food and bedding should be considered. research complications research complications stem from clinical morbidity and mortality. mice with immune dysfunction are at increased risk. there is recent evidence that infection causes elevations in selected cytokines (van andel et al., ) . etiology two mycobacteria are known to be pathogenic for laboratory mice: mycobacterium avium-intracellulare and m. lepraemurium. both are acid-fast, obligate intracellular bacteria. epizootiology mycobacteria are widespread in water and soil. their presence in laboratory mice would indicate a significant break in husbandry practices. infection with m. avium-intracellulare should be considered extremely rare, with the only published report describing an episode in a breeding colony of c bl/ mice . the source of the outbreak was presumed to be drinking water. mycobacterium lepraemurium has been isolated from healthy laboratory mice and can persist as a latent infection, but its significance is primarily historical, as a model for human leprosy. it is highly unlikely to encounter this infection in a modern, well-managed mouse colony. clinical signs m. avium-intracellulare infection is typically subclinical but mice have developed granulomatous pneumonia . pathology lesions are classically a chronic granulomatous disease with granulomas, langhans giant cells, and concurrent presence of acid-fast bacteria in various organs including the lungs, liver, spleen and lymph nodes. m. lepraemurium may cause alopecia, thickening of skin, subcutaneous swellings, and ulceration of the skin. disease can lead to death or clinical recovery. gross lesions are characterized by nodules in subcutaneous tissues and in reticuloendothelial tissues and organs (lung, spleen, bone marrow, thymus, and lymph nodes). lesions can also occur in the lung, skeletal muscle, myocardium, kidneys, nerves, and adrenal glands. the histologic hallmark is perivascular granulomatosis with accumulation of large, foamy epitheloid macrophages (lepra cells) packed with acid-fast bacilli. diagnosis acid-fast bacilli in lesions are the hallmark of presumptive diagnosis of mycobacteriosis. definitive diagnosis results from positive culture which takes days to weeks to rule out or positive pcr assays which are more time-efficient but require associated expertise. differential diagnosis other bacterial species that cause granulomatous lesions in mice. research complications natural infection is very rare. etiology proteus mirabilis is a ubiquitous gram-negative organism that can remain latent in the respiratory and intestinal tracts of normal mice (percy and barthold, ) . epizootiology proteus mirabilis colonizes the intestinal tract of most humans and is commonly found in research mice unless specifically excluded. clinical signs clinical disease can occur following stress or induced immunosuppression. immunodeficient mice have a heightened susceptibility to pathogenic infection. pathology proteus has been associated with ulcerative lesions in the gastrointestinal tract of immunodeficient mice. infected animals lose weight, develop diarrhea, and die within several weeks. if septicemia develops, suppurative or necrotic lesions, including septic thrombi, may be found in many organs, but the kidney is commonly affected. proteus pyelonephritis is characterized by abscessation and scarring. ascending lesions may occur following urinary stasis, but hematogenous spread cannot be ruled out. proteus mirabilis and pseudomonas aeruginosa have been isolated concomitantly from cases of suppurative nephritis or pyelonephritis. infection in immunodeficient mice is typified by splenomegaly and focal necrotizing hepatitis. pulmonary lesions include edema and macrophage activation. septic thrombi can occur, however, in many tissues. diagnosis culture recovery of proteus mirabilis as a predominant or single isolate confirms an opportunistic local or systemic infection. differential diagnosis gram-negative bacterial infections. research complications natural infections are typically isolated cases. etiology leptospirosis remains one of the most common zoonoses transmissible from rodents (desvars et al., ) but is exceedingly rare in laboratory mice. infection with leptospira interrogans serovar ballum has been reported on several occasions (see chapter ). epizootiology leptospira are gram-negative organisms that, after a septicemic phase, establish persistent infection in the renal tubules and are periodically excreted in the urine. clinical signs natural infection is subclinical and causes no significant lesions. experimental infections can result in severe vascular, hepatic and renal lesions dependent on serovar, mouse strain and immunocompetency. diagnosis diagnosis requires isolation of organisms in kidney culture. serological testing should be used with caution because neonatal exposure can lead to persistent infection without seroconversion. histologic examination of kidney using silver stains can also be attempted. pcr assays are reliable for preliminary diagnosis. differential diagnosis not applicable in research colonies. research complications persistent murine infections associated with active shedding present a zoonotic hazard for humans; therefore, infected mice should be culled. elimination of infection from highly valuable mice requires rederivation. (percy and barthold, ) etiology chlamydia trachomatis is an intracellular organism that produces glycogen-positive intracytoplasmic inclusions (elementary bodies). c. trachomatis causes ocular and urogenital disease in humans. however, at least one strain historically referred to as the 'nigg agent' after clara nigg, is most recently classified as chlamydia muridarum and is used experimentally to model human chlamydia infection. epizootiology mice are susceptible to natural infection and experimental infection with c. trachomatis and chlamydophila psittaci, especially immunodeficient mouse strains. clinical signs natural infections are typically subclinical but persistent. pathology c. muridarum is also known as the 'mouse pneumonitis agent' due to severe acute infection which is characterized by ruffled fur, hunched posture, and labored respiration due to interstitial pneumonitis and death in h. mice with more chronic infections may develop progressive emaciation and cyanosis of the ears and tail. experimental infections to model human venereal chlamydia infections will develop hydrosalpinx, cervical, and vaginal infections in female mice and urethritis in male mice. diagnosis chlamydia can be diagnosed by impression smears stained with giemsa or macchiavello stains, cell culture, or inoculation of embryonated eggs. pcr and sequencing can be used to speciate the type of chlamydia. differential diagnosis c. muridarum, c. trachomatis, and c. psittaci are included in the differential diagnosis. research complications chlamydia is a rare spontaneous infection in research mice; its potential significance is low. etiology pneumocystis murina (pm) is a common opportunistic organism of laboratory mice and other mammals. when first described by chagas in , p. carinii was misidentified as trypanosoma cruzi and was considered a protozoan (chagas, ) . it was renamed as a new species, p. carinii, when observed in a rat in (delanoë, p. and delanoë, m. ) . p. carinii, however, has now been grouped taxonomically with the fungi based on dna analysis and the homology of p. murina housekeeping genes with those found in fungi (edman et al., ; stringer et al., ; wakefield et al., ) . these dna studies and apparent differences of host susceptibility prompted a new name, p. jiroveci, for pneumocystis isolated from humans (stringer et al., ; frenkel, ) . p. carinii is now used to name the organism in rats and p. murina, the organism in mice. clinical signs pm infection is subclinical in immunocompetent mice. however, it can be clinically severe in immunodeficient mice, because an adequate complement of functional t lymphocytes is required to suppress infection (roths et al., ; shultz and sidman, ; walzer et al., ; weir et al., ) . b cells have also been shown to be critical to clearance of infection and the mechanism appears only partially related to igg and has a more important role in promoting activation and expansion of t cells (lund et al., ) . b cells may also protect early hematopoietic progenitor activity during systemic responses to pneumocystis infection (hoyt et al., ) . infection proceeds slowly, but relentlessly in immunodeficient mice leading to clinical signs of pneumonia, usually within several months. primary signs include dyspnea and hunched posture, which may laboratory animal medicine be accompanied by wasting and scaly skin. severe cases, such as those that occur in advanced disease in scid mice, may be fatal. epizootiology pm is known to infect a number of mammalian hosts, including ferrets, rats, mice, and humans. pm is a ubiquitous organism that is often present as a latent infection. although firm prevalence data are not available, because detection methods are not simple to apply, infection is assumed to be present in mouse colonies unless ruled out by extensive surveillance. although these organisms appear morphologically similar, there are antigenic and genetic differences among p. murina isolated from different hosts (weinberg and durant, ; cushion, ) . furthermore, studies indicate that p. carinii isolated from one host species is unable to survive and replicate after inoculation into a different immunodeficient host species (gigliotti et al., b) . pm infection also occurs in human beings, but transmission between rodents and human beings has not been documented. pm is transmitted by aerosol and establishes persistent, quiescent infection in the lungs of immunocompetent mice. prenatal infection has not been demonstrated. pathology pm is normally not pathogenic but can be activated by intercurrent immunosuppression. activation fills the lung with trophic and cystic forms. gross lesions occur in the lungs, which are often rubbery and fail to deflate (fig. . ). histopathological changes are characterized by interstitial alveolitis with thickening of alveolar septa from proteinaceous exudate and infiltration with mononuclear cells (fig. . ) (roths et al., ) . alveolar spaces may contain vacuolated eosinophilic material and macrophages. special stains are required to visualize pm. silver-based stains reveal round or partially flattened -to -mm cysts in affected parenchyma (fig. . ) . in florid cases, alveolar spaces may be filled with cysts, but cysts may be sparse in mild cases. disease can be especially severe when subclinically infected immunodeficient mice are reconstituted with competent immune cells that subsequently promote pneumonitis. diagnosis respiratory distress in immunodeficient mice should elicit consideration of pneumocystosis. pathologic examination of the lung, including silver laboratory animal medicine methenamine staining, is essential to confirm a presumptive clinical diagnosis. past infections of immunocompetent mice also can be detected by elisa (furuta et al., ) . pcr can be used to detect active infection (gigliotti et al., a; reddy et al., ) and is particularly useful for screening immunodeficient mice. differential diagnosis pneumocystosis must be differentiated from viral pneumonias of mice. it is worth noting, in this regard, that pneumonia virus of mice has been shown to accelerate the development of pneumocystosis in scid mice (bray et al., ; roths et al., ) . prevention and control pm infection is a significant disease threat to immunodeficient mice. its widespread distribution strongly suggests that susceptible mice should be protected by microbarrier combined, where possible, with macrobarrier housing. husbandry procedures should include proper sterilization of food, water, and housing equipment and the use of hepa-filtered change stations. infected colonies can be rederived by embryo transfer or cesarean methods, because infection does not appear to be transmitted in utero. research complications pneumonia in immunodeficient mice is the major complication of pm infection. trichophyton mentagrophytes is the most common fungal agent of mice. however, infection rarely causes clinical disease. clinical signs include sparse hair coats or well-demarcated crusty lesions, with a chalky surface on the head, tail, and legs (favus or ringworm). skin lesions are composed of exfoliated debris, exudate, mycelia, and arthrospores with underlying dermatitis. invasion of hair shafts is not characteristic. diagnosis depends on effective specimen collection. hairs should be selected from the periphery of the lesion, and hairless skin should be scraped deeply to obtain diagnostic specimens. t. mentagrophytes rarely fluoresces under ultraviolet light, and hyphae must be differentiated from bedding fibers, food particles, and epidermal debris. histological sections should be stained with a silver stain or schiff's reagent to reveal organisms. trichophyton also can be cultured on sabouraud agar. plates are incubated at room temperature ( - °c), and growth is observed at - days. ringworm is not easily eradicated from laboratory mice. the use of antifungal agents to treat individual mice is time-consuming, expensive, and variably effective. rederivation is a more prudent course. cages and equipment should be sterilized before reuse. concurrent infection with ectoparasites also must be considered during eradication steps. candida albicans and other systemic mycoses are not important causes of disease in mice, but they can be opportunistic pathogens in immunodeficient mice. etiology giardia muris is a pear-shaped, flagellated organism with an anterior sucking disk. it inhabits the duodenum of young and adult mice, rats, and hamsters. clinical signs infection is often subclinical, unless organisms proliferate extensively, and can cause weight loss, a rough hair coat, sluggish movement, and abdominal distension, usually without diarrhea. additionally, immunodeficient mice may die during heavy infestation. epizootiology the contemporary prevalence of affected mouse colonies is not well documented, but surveys during the s found the rates exceeding %. transmission occurs by the fecal-oral route. crossinfection between mice and hamsters after experimental inoculation of organisms has been demonstrated, whereas rats were resistant to isolates from mice and hamsters (kunstyr et al., ) . c h/he mice are particularly susceptible to giardiasis, whereas balb/c and c bl/ mice are more resistant. additionally, female mice appear to be more resistant to infection than male mice (daniels and belosevic, ) . c bl/ females, e.g., have lower trophozoite burdens and for a shorter interval than male mice. females also shed cysts later than male mice. these differences may be related to a more potent humoral immune response to giardia in female mice. pathology gross lesions are limited to the small intestine, which may contain yellow or white watery fluid. histopathology reveals organisms in the lumen that often adhere to microvilli of enterocytes or reside in mucosal crevices or mucus. the crypt/villus ratio may be reduced, and the lamina propria may have elevated numbers of inflammatory cells. diagnosis diagnosis is based on detection of trophozoites in the small intestine or in wet mounts of fecal material. organisms can be recognized in wet preparations by their characteristic rolling and tumbling movements. ellipsoidal cysts with four nuclei also may be detected in feces. infection also can be detected by serology (daniels and belosevic, ) and by pcr (mahbubani et al., ) . treatment, prevention, and control murine giardiasis can be treated by the addition of . % dimetridazole to drinking water for days. prevention and control depend on proper sanitation and management, including adequate disinfection of contaminated rooms. research complications accelerated cryptal cell turnover and suppression of the immune response to sheep erythrocytes have been observed in infected mice. the potential for severe or lethal infection in immunodeficient mice was noted previously. etiology spironucleus muris is an elongated, pearshaped, bilaterally symmetrical flagellated protozoan that commonly inhabits the duodenum, usually in the crypts of lieberkühn. it is smaller than giardia muris and lacks an anterior sucking disk. clinical signs s. muris infection is usually subclinical in normal adult mice. it is more pathogenic, however, for young, stressed, or immunocompromised mice (kunstyr et al., ) . additionally, clinical morbidity may indicate an underlying primary infection with an unrelated organism. clinically affected mice can have a poor hair coat, sluggish behavior, and weight loss. mice at - weeks of age are at notably higher risk for clinically evident infection. they can develop dehydration, hunched posture, abdominal distension, and diarrhea. severe infections can be lethal. epizootiology transmission occurs by the fecaloral route and can occur between hamsters and mice as well as between mice. it does not appear to be transmitted between mice and rats (schagemann et al., ) . the most recent surveys, which are somewhat dated, indicated that prevalence rates exceeded % among domestic mouse colonies in the mid- s. there is some evidence that inbred strains vary in their susceptibility to infection and their rate of recovery (baker et al., ; brett and cox, ) . pathology gross findings associated with infection include watery, red-brown, gaseous intestinal contents. however, it is essential to rule out primary or coinfection by other organisms before attributing these lesions to spironucleosis. microscopically, acute disease is associated with distension of crypts and intervillous spaces by pear-shaped trophozoites and inflammatory edema of the lamina propria. organisms can be visualized more easily with periodic acid-schiff staining, which may reveal invasion of organisms between enterocytes and in the lamina propria. chronic infection is associated with lymphoplasmacytic infiltration of the lamina propria and occasional intracryptal inflammatory exudate. diagnosis diagnosis is based on identification of trophozoites in the intestinal tract. they can be distinguished from giardia muris and tritrichomonas muris by their small size, horizontal or zigzag movements, and the absence of a sucking disk or undulating membrane. pcr-based detection also is available (rozario et al., ) . it is not clear whether duodenitis is a primary pathogenic effect of s. muris or represents opportunism secondary to a primary bacterial or viral enteritis. therefore, it is prudent to search for underlying or predisposing infections. treatment, prevention, and control treatment consists of adding . % dimetridazole to drinking water for days, as described for giardiasis. prevention and control require good husbandry and sanitation. research complications as with giardiasis, infection can accelerate enterocytic turnover in the small intestine. there is some evidence that infected mice may have activated macrophages that kill tumor cells nonspecifically and that infection can diminish responses to soluble and particulate antigens. additionally, infected mice also have increased sensitivity to irradiation. such effects should, however, be interpreted cautiously in order to rule out intercurrent viral infections. tritrichomoniasis t. muris is a nonpathogenic protozoan that occurs in the cecum, colon, and small intestine of mice, rats, and hamsters. no cysts are formed, and transmission is by ingestion of trophozoites passed in the feces. it can be detected by microscopy or by pcr (viscogliosi et al., ) . coccidiosis eimeria falciformis is a pathogenic coccidian that occurs in epithelial cells of the large intestines of mice. it was common in european mice historically but is seldom observed in the united states. heavy infection may cause diarrhea and catarrhal enteritis. klosiella muris causes renal coccidioisis in wild mice but is rare in laboratory mice. mice are infected by ingestion of sporulated sporocysts. sporozoites released from the sporocysts enter the bloodstream and infect endothelial cells lining renal arterioles and glomerular capillaries, where schizogony occurs. mature schizonts rupture into bowman's capsule to release merozoites into the lumen of renal tubules. merozoites can enter epithelial cells lining convoluted tubules, where the sexual phase of the life cycle is completed. sporocysts form in renal tubular epithelium and eventually rupture host cells and are excreted in the urine, but oocysts are not formed. infection is usually nonpathogenic and subclinical. gray spots may occur in heavily affected kidneys and are the result of necrosis, granulomatous inflammation, and focal hyperplasia. destruction of tubular epithelium may impair renal physiology. diagnosis is based on detection of organisms in tissues. prevention and control require proper sanitation and management techniques. there is no effective treatment. cryptosporidiosis cryptosporidium muris is a sporozoan that adheres to the gastric mucosa. it is uncommon in laboratory mice and is only slightly pathogenic. cryptosporidium parvum inhabits the small intestine and is usually nonpathogenic in immunocompetent and athymic mice (ozkul and aydin, ; taylor et al., ) . athymic mice may develop cholangitis and hepatitis, however, if organisms gain access to the biliary tract. entamoebiasis entamoeba muris is found in the cecum and colon of mice, rats, and hamsters throughout the world. organisms live in the lumen, where they feed on particles of food and bacteria. they are considered nonpathogenic. encephalitozoonosis encephalitozoon cuniculi is a gram-positive microsporidian that infects rabbits, mice, rats, guinea pigs, dogs, nonhuman primates, humans, and other mammals. infection is extremely rare among laboratory mice. the life cycle of the organism is direct, and animals are infected by ingesting spores or by cannibalism. spore cells are disseminated in the blood to the brain and other sites. infection can last more than year, and spores shed in the urine serve as a source of infection. vertical transmission has not been confirmed in mice. e. cuniculi is an obligate intracellular parasite, but infection usually elicits no clinical signs of disease. organisms proliferate in peritoneal macrophages by asexual binary fission. they have a capsule that accepts giemsa and goodpasture stains but is poorly stained by hematoxylin. fulminating infection can cause lymphocytic meningoencephalitis and focal granulomatous hepatitis. in contrast to encephalitozoonosis in rabbits, affected mice do not develop interstitial nephritis. infection is diagnosed by cytological examination of ascitic fluid smears, histopathologic examination of brain tissues stained with goodpasture stain, and elisa serology. no effective treatment has been reported. prevention and control require rigid testing and elimination of infected colonies and cell lines. pcr-based assays may also be useful. toxoplasmosis toxoplasma gondii is a ubiquitous gram-negative coccidian parasite for which the mouse serves as a principal intermediate host. however, the prevalence of natural infection is negligible because laboratory mice no longer have access to sporulated cysts shed by infected cats, which were historically the major source for cross-infection. toxoplasmosis can cause necrosis and granulomatous inflammation in the intestine, mesenteric lymph nodes, eyes, heart, adrenals, spleen, brain, lung, liver, placenta, and muscles. diagnosis is based on elisa serology, histopathology, and pcr. control and prevention depend largely on precluding access of mice to cat feces or to materials contaminated with cat feces. oocytes are very resistant to adverse temperatures, drying, and chemical disinfectants; therefore, thorough cleaning of infected environments is required. b. cestodiasis baker, ) etiology hymenolepis (rodentolepis) nana, the dwarf tapeworm, infects mice, rats, and humans although the zoonotic risk has been questioned (macnish et al., ) . adults are extremely small ( - mm) and have eggs with prominent polar filaments and rostellar hooks (fig. . ) . clinical signs young adult mice are most frequently infected. signs and lesions include weight loss and focal enteritis, but clinical disease is rare unless infestation is severe. epizootiology the life cycle may be direct or indirect (r. nana is the only cestode known that does not require an intermediate host). the indirect cycle utilizes arthropods as intermediate hosts. liberated oncospheres penetrate intestinal villi and develop into a cercocystis stage before reemerging into the intestinal lumen - days later. the scolex attaches to the intestinal mucosa, where the worm grows to adult size in weeks. the cycle from ingestion to patency takes - days. pathology cysticerci are found in the lamina propria of the small intestine and sporadically in the mesenteric lymph nodes, whereas adults, which have a serrated profile, are found in the lumen. inflammation is not a feature of infection. diagnosis infection can be diagnosed by demonstrating eggs in fecal flotation preparations or by opening the intestine in petri dishes containing warm tap water to facilitate detection of adults. r. nana can be differentiated from another species of rodent tapeworm, h. diminuta, by the fact that r. nana has rostellar hooks and eggs with polar filaments. however, h. diminuta requires an intermediate arthropod host, so it is rarely found in contemporary mouse colonies. treatment, prevention, and control drugs recommended for treatment and elimination include praziquantel ( . % in the diet for days), albendazole, mebendazole, and thiabendazole. although the benzimidazoles have an excellent activity against cestodes and nematodes in rats, they have not been tested extensively in mice. the potential for successful treatment is high, however, because eggs do not survive well outside the host and because the prevalence of infestation is low in caged mice kept in properly sanitized facilities. because r. nana can directly infect humans, proper precautions should be taken to avoid oral contamination during handling of rodents (see chapter ). hymenolepis microstoma is found in the bile ducts of rodents and could be confused with r. nana in the mouse. however, the location of the adult as well as the large size of h. microstoma eggs compared with those of r. nana make differential diagnosis relatively simple. the mouse and the rat are intermediate hosts of the cestode taenia taeniaformis. the definitive host is the cat. this parasite should not be found in laboratory mice housed separately from cats. c. nematodiasis (wescott, ) syphacia obvelata (mouse pinworm) infestation etiology syphacia obvelata, the common mouse pinworm, is a ubiquitous parasite of wild and laboratory mice. the rat, gerbil, and hamster are also occasionally infected. female worms range from . to . mm in length, and male worms are smaller ( . - . mm). eggs are flattened on one side and have pointed ends (fig. . ). the nucleus fills the shell and is frequently at a larval stage when eggs are laid. clinical signs infestation is usually subclinical, although heavily infested mice can occasionally sustain intestinal lesions, including rectal prolapse, intussusception, enteritis, and fecal impaction. epizootiology pinworm infestation is one of the most commonly encountered problems in laboratory mice. a national survey revealed that more than % of barrier colonies and about % of conventional colonies were affected (jacoby and lindsey, ; carty, ) . syphacia obvelata infestation can occur unexpectedly in commercial barrier murine colonies, resulting in widespread dissemination of the parasite into academic mouse colonies. the epizootiological impact of pinworm infestation is increased by the airborne dissemination of eggs, which can remain infectious even after drying. the life-cycle is direct and completed in - days. females deposit their eggs on the skin and hairs of the perianal region. ingested eggs liberate larvae in the small intestine and they migrate to the cecum within h. worms remain in the cecum for - days, where they mature and mate. the females then migrate to the large intestine to deposit their eggs as they leave the host. there is unconfirmed speculation that larvae may reenter the rectum. infestation usually begins in young mice and can recur, but adult mice tend to be more resistant. syphacia infestation often occurs in combination with aspiculuris tetraptera. because the life cycle of syphacia is much shorter than that of aspiculuris, the number of mice that are apt to be infected with s. obvelata is correspondingly greater. there is evidence that resistance to infestation may be mouse strain-specific (derothe et al., ) . pathology gross lesions are not prevalent, aside from the presence of adults in the lumen of the intestine. diagnosis infestation is diagnosed by demonstrating reniform-shaped eggs in the perianal area or adult worms in the cecum or large intestine. four-to -weekold mice should be examined because the prevalence is higher in this age group than in older mice. because most eggs are deposited outside the gastrointestinal tract, fecal examination is not reliable. eggs are usually detected by pressing cellophane tape to the perineal area and then to a glass slide that is examined by microscopy. aspiculuris tetraptera eggs are not ordinarily found in tape preparations and are easily differentiated from eggs of s. obvelata (see below). adult worms can be found in cecal or colonic contents diluted in a petri dish of warm tap water. they are readily observed with the naked eye or with a dissecting microscope. an elisa also is available to detect serum antibodies to s. obvelata somatic antigens (sato et al., ) . pcr assays are increasingly being used to augment traditional diagnostic methods and to discriminate between pinworm species (dole et al., ) . pcr panels for pinworm detection using fecal pellets are available from commercial diagnostic laboratories. treatment, prevention, and control pinworm infestation can be treated effectively by a number of regimens, which include the use of anthelmintics such as piperazine, ivermectin, and benzimidazole compounds alone or in combination (klement et al., ; le blanc et al., ; lipman et al., ; flynn et al., ; wescott, ; zenner, ) . because some of the recommended therapies have the potential for toxicity, it is prudent to keep mice under close clinical observation during treatment (davis et al., ; skopets et al., ; toth et al., ) . fenbendazole diets can be fed with week on/ week off rotation with normal chow although the potential impact on experimental data must be considered (duan et al., ; gadad et al., ; landin et al., ) . prevention of reinfestation requires strict isolation because syphacia eggs become infective as soon as h after they are laid, and they survive for weeks, even in dry conditions. strict sanitation, sterilization of feed and bedding, and periodic anthelmintic treatment are required to control infestation. the use of microbarrier cages can reduce the spread of infective eggs. syphacia muris is the common rat pinworm. it can potentially infest mice but is not found in well-managed colonies. it can be differentiated from s. obvelata because s. muris eggs are smaller. treatment is the same as for pinworms of mice. etiology aspiculuris tetraptera is the other major oxyurid of the mouse and may coinfect mice carrying s. obvelata. females are . - . mm long, and males are slightly smaller. the eggs are ellipsoidal (fig. . ) . clinical signs ingested eggs hatch, and larvae reach the middle colon, where they enter crypts and remain for - days. they move to the proximal colon about weeks after infection of the host. because the life cycle is - days longer than in s. obvelata, infestations appear in somewhat older mice; heaviest infestation is expected in - weeks after initial exposure. infection is usually subclinical, but heavy loads can produce signs similar to those discussed for s. obvelata. light to moderate loads do not produce clinical disease. epizootiology as noted under s. obvelata, pinworm infestation is highly prevalent and contagious in laboratory mice. the life cycle is direct and takes approximately - days. mature females inhabit the large intestine, where they survive from to days and lay their eggs. the eggs are deposited at night and are excreted in a mucous layer, covering fecal pellets. they require - days at °c to become infective and can survive for weeks outside the host. pathology see s. obvelata (section iii, a, ,c). diagnosis aspiculuris tetraptera eggs can be detected in the feces, and adult worms are found in the large intestine. eggs are not deposited in the perianal area; therefore, cellophane tape techniques are not useful. measures for treatment, prevention, and control are similar to those described for s. obvelata. because a. tetraptera takes longer to mature and because eggs are deposited in feces rather than on the host, adult parasites are more amenable to treatment by frequent cage rotations. immune expulsion of parasites and resistance to reinfection are hallmarks of a. tetraptera infection. research complications see s. obvelata (section iii, a, ,c). several species of mites infest laboratory mice. they include myobia musculi, radfordia affinis, myocoptes musculinus, and, less commonly, psorergates simplex. the common murine mites are described below, while less frequently encountered species are listed in table . . these include the mouse mite trichoecius romboutsi, which resembles myocoptes and ornithonyssus bacoti, the tropical rat mite, which can infect laboratory mice. characteristics of specific infestations are described after a general introductory section. clinical signs mites generally favor the dorsal anterior regions of the body, particularly the top of (jacoby and lindsey, ; carty, ) reported mite infestations in % of colonies. acarids spend their entire lives on the host. populations are limited by factors such as self-grooming, mutual grooming, the presence of hair, and immunological responses, which tend to produce hypersensitivity dermatitis. inherited resistance and susceptibility also affect clinical expression of acariasis. mite populations, e.g., vary widely among different stocks and strains of mice housed under similar conditions. pathology gross lesions include scaly skin, regional hair loss, abrasions, and ulcerations. histologically, hyperkeratosis, acanthosis, and chronic dermatitis may occur. long-standing infestation provokes chronic inflammation, fibrosis, and proliferation of granulation tissue. ulcerative dermatitis associated with acariasis may have an allergic pathogenesis but often results in secondary bacterial infections. lesions resemble allergic acariasis in other species and are associated with mast cell accumulations in the dermis. diagnosis classic methods of detection include direct observation of the hair and skin of dead or anesthetized mice. hairs are parted with pins or sticks and examined with a dissecting microscope. examination of young mice, prior to the onset of immune-mediated equilibrium, is likely to be more productive. alternatively, recently euthanized mice can be placed on a black paper, and double-sided cellophane tape can be used to line the perimeter to contain the parasites. as the carcass cools, parasites will vacate the pelage and crawl onto the paper. sealed petri dishes can also be used. cellophane tape also can be pressed against areas of the pelt of freshly euthanatized mice and examined microscopically. skin scrapings made with a scalpel blade can be macerated in % koh/glycerin or immersion oil and examined microscopically. this method has the disadvantage of missing highly motile species and low-level populations of slower moving immature forms. it is important to remember that mite infestations may be mixed, so the identification of one species does not rule out the presence of others. detecting mites in sentinels exposed to dirty bedding from colony animals has been reported to be unreliable (lindstrom et al., ) . thus, pcr assays offered by commercial diagnostic laboratories are increasingly being used to augment traditional diagnostic methods and to test individual animals or equipment using a swabbing technique; samples can be pooled to decrease cost (jensen et al., ) . gross anatomical features facilitate differentiation of intact mites. myocoptes has an oval profile with heavily chitinized body, pigmented third and fourth legs, and tarsal suckers (fig. . ) . myobia and radfordia have a similar elongated profile, with bulges between the legs. myobia has a single tarsal claw on the second pair of legs (fig. . ) , whereas radfordia has two claws of unequal size on the terminal tarsal structure of its second pair of legs (fig. . ) . histopathological examination of skin is helpful for diagnosing unique forms of acariasis, such as the keratotic cysts associated with psorergates simplex infestation. treatment, prevention, and control ivermectin can be used topically, in drinking water or as a medicated feed and often is the first-choice approach for attempting eradication although cost and potential toxicity are concerns. because of potential differences in laboratory animal medicine blood-brain barrier permeability to ivermectin, pilot treatments should be evaluated. for large facilities, ivermectin medicated feed may be an attractive option (ricart arbona et al., ) . for valuable lines of mice, rederivation may be cost-and time-effective. control and prevention programs should be carried out on a colony-wide basis, which includes thorough sanitation of housing space and equipment to remove residual eggs. research complications hypersensitivity dermatitis has the potential to confound immunological studies (jungmann et al., ) , especially those involving skin, and has been shown to elevate serum ige (morita et al., ) . heavy mite infestations can cause severe skin lesions and have been associated with weight loss, infertility, and premature deaths. chronic acariasis also may provoke secondary amyloidosis due to long-standing dermatitis. myocoptes musculinus this is the most common ectoparasite of the laboratory mouse but frequently occurs in conjunction with myobia musculi. the life cycle includes egg, larva, protonymph, tridonymph, and adult stages. eggs hatch in days and are usually attached to the middle third of the hair shaft. the life cycle may range from to days. transmission requires direct contact, for mice separated by wire screens do not contract infestations from infested hosts. bedding does not seem to serve as a vector. neonates may become infested within - days of birth, and parasites may live for - days on dead hosts. myocoptes appears to inhabit larger areas of the body than myobia and tends to displace myobia during heavy infestations. it has some predilection for the skin of the inguinal region, abdominal skin, and back, but it will also infest the head and neck. it is a surface dweller that feeds on superficial epidermis. infestation can cause patchy thinning of the hair, alopecia, or erythema. lesions can be pruritic, but ulceration has not been reported. chronic infestations induce epidermal hyperplasia and nonsuppurative dermatitis. myobia musculi this is a common mite of laboratory mice. the life cycle of myobia can be completed in days and includes an egg stage, first and second larval stages, protonymph, deutonymph, and adult. eggs attach at the base of hair shafts and hatch in - days. larval forms last about days, followed by nymphal forms on day . adults appear by day and lay eggs within h. myobia are thought to feed on skin secretions and interstitial fluid but not on blood. they are transmitted primarily by contact. mite populations increase during new infestations, followed by a decrease to equilibrium in - weeks. the equilibrated population can be carried in colonies for long periods (up to years). population fluctuations may represent waves of egg hatchings. because mites are thermotactic, they crawl to the end of hair shafts on dead hosts, where they may live for up to days. infestation may result in hypersensitivity dermatitis, to which c bl mice are highly susceptible. clinical signs vary from ruffled fur and alopecia to pruritic ulcerative dermatitis. therefore, lesions can be exacerbated by self-inflicted trauma. radfordia affinis radfordia is thought to be common in laboratory mice, but it closely resembles myobia and may occur as a mixed infestation. therefore, its true prevalence is conjectural. additionally, its life cycle has not been described. it does not appear to cause clinical morbidity. psorergates simplex this species has not been reported as a naturally occurring infection in well-managed colonies for several decades, but it is unique in that it inhabits hair follicles. its life cycle is unknown, but developmental stages from egg to adult may be found in a single dermal nodule. transmission is by direct contact. invasion of hair follicles leads to development of cyst-like nodules, which appear as small white nodules in the subcutis. histologically, they are invaginated sacs of squamous epithelium, excretory products, and keratinaceous debris. there is usually no inflammatory reaction, but healing may be accompanied by granulomatous inflammation. diagnosis is made by examining the subcuticular surface of the pelt grossly or by histological examination. sac contents also can be expressed by pressure with a scalpel blade or scraped and mounted for microscopic exam. mesostigmoid mites rarely, blood-sucking ornithonyssus bacoti and laelaps echidnina, normally limited to wild rodents, can also infect laboratory rodent colonies (watson, ; fox, ) . these mites may also transiently bite humans and can transmit zoonotic infections (see chapter ). unlike the more common rodent fur mites, mesostigmoid mites live off the host and can travel a long distance in search of a blood meal. they access research colonies via contaminated supplies or wild rats and mice gaining access to the facility. polyplax serrata, the mouse louse, is encountered in wild mice but no longer is a significant issue in research colonies. eggs are deposited at the base of hair shafts and nymph stages and adults can be found principally on the dorsum. p. serrata causes pruritus with associated dermatitis, anemia and debilitation and historically is the vector for mycoplasma coccoides. amyloidosis is caused by the deposition of insoluble (polymerized), mis-folded amyloid protein fibrils in organs and/or tissues. primary amyloidosis is a naturally occurring disease in mice, associated with the deposition of amyloid proteins consisting primarily of immunoglobulin light chains. secondary amyloidosis is associated with antecedent and often chronic inflammation. it results from a complex cascade of reactions involving release of multiple cytokines that stimulate amyloid synthesis in the liver (falk and skinner, ) . primary amyloidosis is common among aging mice (lipman et al., ) but also may occur in young mice of highly susceptible strains such as a and sjl or somewhat older c bl mice. other strains, such as balb/c and c h are highly resistant to amyloidosis (percy and barthold, ) . secondary amyloidosis is usually associated with chronic inflammatory lesions, including dermatitis resulting from prolonged acariasis. it can be induced experimentally, however, by injection of casein and may occur locally in association with neoplasia or in ovarian corpora lutea in the absence of other disease. in reactive amyloid a (aa) amyloidosis, serum aa (saa) protein forms deposits in mice, domestic and wild animals, and humans that experience chronic inflammation. aa amyloid fibrils are abnormal β-sheet-rich forms of the serum precursor saa, with conformational changes that promote fibril formation. similar to prion diseases, recent findings suggest that aa amyloidosis could be transmissible in mice and other species (murakami et al., ) . amyloid fibrils induce a seeding-nucleation process that may lead to development of aa amyloidosis. amyloidosis can shorten the life span of mice and can be accelerated by stress from intercurrent disease. amyloid appears histologically as interstitial deposition of a lightly eosinophilic, acellular material in tissues stained with hematoxylin and eosin. however, it is birefringent after staining with congo red when viewed with polarized light. deposition patterns vary with mouse strain and amyloid type. although virtually any tissue may be affected, the following sites are common: hepatic portal triads, periarteriolar lymphoid sheaths in spleen, renal glomeruli and interstitium (which can lead to papillary necrosis), intestinal lamina propria, myocardium (and in association with atrial thrombosis), nasal submucosa, pulmonary alveolar septa, gonads, endocrine tissues, and great vessels (fig. . ) . naturally occurring mineralization of the myocardium and epicardium and other soft tissues is a common finding at necropsy in some inbred strains of mice. although this condition is usually an incidental finding at necropsy, interference with organ function such as the heart cannot be ruled out if lesions are severe. it occurs in balb/c, c h, and especially dba mice (eaton et al., ; brownstein, ; brunnert et al., ) . it is found in the myocardium of the left ventricle ( fig. . ) , in the intraventricular systems, and in skeletal muscle, kidneys, arteries, and lung and may be accompanied by fibrosis and mononuclear inflammatory infiltrates. dba mice also can develop mineralization in the tongue and cornea. dietary, environmental, disease-related, and endocrine-related factors are thought to influence the prevalence of this lesion. ectopic mineralization is associated clinically with skin and vascular connective tissue conditions in humans and mouse models have been developed to study metastatic and dystrophic tissue mineralization (li and uitto, ) . pseudoxanthoma elasticum (pxe), a heritable ectopic mineralization disorder in humans, is caused by mutations in the abcc gene. knockout abcc −/− mice model the histopathologic and ultrastructural features of pxe, notably with mineralization of the vibrissae dermal sheath, serving as a biomarker of tissue mineralization (benga et al., ) . other inbred mouse strains, including kk/hlj and s /svimj, also develop vibrissae dermal mineralization and have an snp (rs ) in the abcc gene associated with low levels of abcc protein expression in the liver. dba/ j and c h/hej mice have the same polymorphism and low abcc protein levels; however, these mice only develop tissue mineralization when fed an experimental diet enriched in phosphate and low in magnesium. a reye's-like syndrome has been reported in balb/ cbyj mice (brownstein et al., ) . the etiology is unknown; however, antecedent viral infection may be involved. affected mice rapidly become lethargic and then comatose. they also tend to hyperventilate. high mortality ensues within - h, but some mice may recover. lesions are characterized grossly by swollen, pale liver and kidneys. the major histopathological findings include swollen hepatocytes with fatty change and nuclear swelling among astrocytes in the brain. hepatic lesions resembling changes in reye's syndrome have been reported in scid mice infected with madv- (pirofski et al., ) . deficiencies (tobin et al., ) vitamin deficiencies in mice have not been thoroughly described. unfortunately, much of the information that does exist reflects work carried out - years ago; thus, the reliability and specificity of some of these syndromes is questionable. vitamin a deficiency may produce tremors, diarrhea, rough hair coat, keratitis, poor growth, abscesses, hemorrhages, and sterility or abortion. vitamin a is recognized for its importance in development of the immune system (ross, ) and knockout mouse models have been used to demonstrate genetic polymorphisms in humans that negatively regulate intestinal β-carotene absorption and conversion to retinoids in response to vitamin a requirements for growth and reproduction (von lintig, ) . vitamin e deficiency can cause convulsions and heart failure, as well as muscular dystrophy and hyaline degeneration of muscles. two knockout mouse models of severe vitamin e deficiency were independently developed and lack α-tocopherol transfer protein (α-ttp), a gene that controls plasma and tissue α-tocopherol concentrations by exporting α-tocopherol from the liver. ttpa −/− mice have very low to undetectable levels of α-tocopherol and are infertile. the phenotype includes neuronal degeneration associated with progressive ataxia and age-related behavioral defects (yu and schellhorn, ) . deficiency of b complex vitamins produces nonspecific signs such as alopecia, decreased feed consumption, poor growth, poor reproduction and lactation, as well as a variety of neurological abnormalities. choline deficiency produces fatty livers and nodular hepatic hyperplasia, as well as myocardial lesions, decreased conception, and decreased viability of litters. folic acid-deficient diets cause marked decreases in red and white cell blood counts and the disappearance of megakaryocytes and nucleated cells from the spleen. pantothenic acid deficiency is characterized by nonspecific signs, such as weight loss, alopecia, achromotrichia, and posterior paralysis, as well as other neurological abnormalities. thiamin deficiency is associated with neurological signs, such as violent convulsions, cartwheel movements, and decreased food consumption. dietary requirements for ascorbic acid have not been shown in mice, and mouse diets are generally not fortified with ascorbic acid. the gulonolactone oxidase knockout mouse (gulo −/− ) on the c bl/ background requires vitamin c supplementation although the plasma ascorbate concentration of gulo −/− mice fed a vitamin c-deficient diet is maintained at % of wild-type concentrations, suggesting an uncharacterized pathway to generate a small amount of ascorbate (yu and schellhorn, ) . the gulo −/− mouse has become the model of choice in studying the role of vitamin c in complex diseases. vitamin c production has been successfully restored in gulo −/− mice using adenovirus vectors, making it possible to robustly manipulate physiological ascorbate concentrations in an inbred mouse. mineral deficiencies have been described only for several elements, and the consequences of the deficiencies are similar to those observed for other species. for example, iodine-deficient diets produce thyroid goiters; magnesium-deficient diets may cause fatal convulsions; manganese deficiency may cause congenital ataxia from abnormal development of the inner ear; and zinc deficiency may cause hair loss on the shoulders and neck, emaciation, decreased liver and kidney catalase activity, and immunosuppression. chronic essential fatty acid deficiency may cause hair loss, dermatitis with scaling and crusting of the skin, and occasional diarrhea. infertility has also been associated with this syndrome. mice have an absolute requirement for a dietary source of linoleic and/or arachidonic acid. (sundberg, ; ward, ) the significant syndrome of ulcerative dermatitis (ud) is a common idiopathic skin lesion that causes morbidity and early euthanasia losses in c bl/ and related lines of mice. significant pruritus leads to skin trauma associated with opportunistic bacterial infection and deep dermal ulcerations. initial signs include alopecia and papular dermatitis, which usually occur over the dorsal trunk (fig. . ) . progressive inflammation can be halted, sometimes reversed, by nail trimming and therapy with a wide spectrum of topical or systemic antibiotics, steroids, and other drugs such as vitamin e and aloe, all of which speak to the frustrating search for a primary etiology. treatment should be based on microbiological culture and sensitivity and screening for ectoparasites as hypersensitivity to acariasis has been proposed. seasonal fluctuation in the incidence of disease suggests that environmental factors may play a role. the incidence appears to increase during periods of significant seasonal changes in temperature and humidity, i.e., the onset of winter and early spring. there is some evidence that incidence is related to dietary fat with mice on high fat or ad libitum diets being more susceptible than those on restricted diets (neuhaus et al., ) . ileus associated with high mortality has been reported to occur in primiparous female mice during the second week of lactation (kunstyr, ) . this disorder has been described as acute intestinal pseudo-obstruction (ipo) in c bl/ mice free of known pathogens (feinstein et al., ) . lactating mice are either found dead or becoming moribund. segments of the small intestine become distended with fluid contents and histologically there is apoptosis of the villus epithelium of the small intestine and superficial epithelial cells of the large intestine. the enteric nervous system appears morphologically normal but necrotic enterocytes, mucosal erosions, and acute mucosal inflammation are commonly observed. there is no strong evidence for metabolic issues such as hypocalcemia or low blood glucose. the direct cause is unknown but death probably results from sepsis secondary to loss of barrier function reflected in apoptosis of the gut epithelium during peak lactation. environmental variables can affect responses of mice in experimental situations. changes in respiratory epithelial physiology and function from elevated levels of ammonia, effects of temperature and humidity on metabolism, effects of light on eye lesions and retinal function, and effects of noise on neurophysiology are examples of complications that can vary with the form of insult and the strain of mouse employed. mice do not easily acclimatize to sudden and dramatic changes in temperature. therefore, they are susceptible to both hypothermia and hyperthermia. mice also are susceptible to dehydration. poorly functioning automatic watering system valves or water bottles, resulting in spills (hypothermia) or obstructed sipper tubes (dehydration), are a significant cause of husbandry-related morbidity. shipping mice between facilities, irrespective of distance, warrants institutional guidelines to minimize exposure to temperature extremes. reheat coils should be designed to fail in the closed position to avoid overheating holding rooms. ringtail is a condition associated with low relative humidity. clinical signs include annular constriction of the tail and occasionally of the feet or digits, resulting in localized edema that can progress to dry gangrene ( fig. . ). it should be differentiated from dryness and gangrene that may occur in hairless mice exposed to low temperatures and perhaps other environmental or nutritional imbalances. necrosis of legs, feet, or digits also can occur in suckling mice because of disruption of circulation by wraps of stringy nesting material such as cotton wool. corneal opacities can result from acute or chronic keratitis, injury (unilateral) and developmental defects; the latter may occur in combination with inherited microphthalmia in c black mice (koch and gowen, ) . there is some evidence that the buildup of ammonia in mouse cages may contribute to inflammatory keratitis, because it can be controlled by increasing the frequency of cage cleaning. corneal opacities and anterior polar cataracts are a developmental defect in inbred c black mice (pierro and spiggle, ) . corneal opacity may be associated with keratolenticular adhesions involving a persistent epithelial stalk of the lens vesicle, which normally disappears around day of gestation (koch and gowen, ) . typically noted in runted or cachectic mice soon after weaning, malocclusion of the open-rooted, continually growing incisor teeth is an inherited trait expressed as poorly aligned incisors, especially of the lower incisors causing osteomyelitis, soft tissue abscesses, or necrosis in the lips or oral cavity. the incidence of inherited malocclusion varies with mouse strain (petznek et al., ) . malocclusion in older mice may be the result of trauma or oral neoplasia. overgrown molar teeth have been associated with trauma to developing tooth buds. skin lesions can be caused by fighting, tail biting, and overgrooming such as whisker chewing. barbering of facial hair and whiskers in subordinate mice by a dominant cagemate is common and may be solved by removing the dominant, normally haired mouse. hair or whisker chewing (barbering) has long been interpreted to be a manifestation of social dominance. apparent dominant animals retain whiskers, whereas cagemates have 'shaved faces' (fig. . ). chronic hair chewing can produce histological abnormalities such as poorly formed or pigmented club hairs. once chewing has ceased, many mice regrow previously lost hair in several weeks. both sexes may engage in this activity, and sometimes females may be dominant. barbering of whiskers and fur-plucking behavior in mice has been suggested to model human trichotillomania (compulsive hair plucking) because of similarities including elevated serotonin levels (dufour et al., ) , 'barbers' predominately pluck hair from the scalp and around the eyes and the genitals; the behavior is female biased, and begins during puberty and is impacted by genetic background (garner et al., ) . fighting is more common in male mice and more aggressive in some strains (sjl, fvb, balb/c) with bite wounds typically located on the head, neck, shoulders, perineal area, and tail. often one mouse in the cage is free of lesions and is the likely aggressor. removal of the unaffected male may end the fighting or simply reorder the dominance order. removing males for breeding and then regrouping them often results in fighting. for programs that produce sentinel mice in-house, castration is an option to reduce aggression in group-housed male sentinels (lofgren et al., ) . regional alopecia, especially around the muzzle, may result from abrasion against cage surfaces. improperly diluted disinfectants may also cause regional hair loss. ear tags used for identification may cause pruritis and self-induced trauma. hair removal products or clipping prior to imaging or application of experimental compounds to the skin may cause pruritus and can augment lesions that interfere with test results. dermatophytosis, ectoparasitism, or idiopathic hair loss must be considered in the differential diagnoses for muzzle or body alopecia. (burek et al., ; percy and barthold, ) common idiopathic lesions in aging mice include cardiomyopathy (with or without mineralization or arteritis), chronic nephropathy (frequently with mineralization), myelofibrosis (fibrotic change in the bone marrow) especially in female mice, melanosis in the meninges, ovarian atrophy (with or without hyaline material), pigment (ceroid-lipofuscin), tubular or stromal hyperplasia, cystic endometrial hyperplasia, testicular tubular degeneration or mineralization, prostate atypical epithelial hyperplasia, gastric glandular epithelial hyperplasia, pancreatic islet cell hyperplasia, dental dysplasia of incisor teeth, pituitary hyperplasia of pars intermedia and pars distalis, cataracts, increased extramedullary hematopoiesis in spleen, and lymphocytic infiltrates or other inflammatory changes in various tissues, including harderian gland, salivary gland, kidney, liver, gall bladder, nasal, trachea, thyroid, periovarian fat, epididymis, and urinary bladder. lymphoma is also very common . spontaneous atrial thrombosis is rare in mice (< % in -year-old mice) and appears to be strain-related, with a high prevalence in rfm mice. it also is more common in aged mice affected by kidney disease and amyloidosis. organizing thrombi will be found usually in an enlarged, hyperemic left atrium and auricle and may be accompanied by amyloidosis. affected mice may display signs of heart failure, particularly severe dyspnea. induction of atrial thrombosis in b c f mice has been used to assess cardiovascular risk of chemical exposures (yoshizawa et al., ) . myocardial and epicardial mineralization is described above (section iii,b, ). periarteritis, also known as arteritis, polyarteritis, or systemic arteritis, impacts older mice and lesions may be observed in multiple tissues, including the spleen, heart, tongue, uterus, testes, kidney, and urinary bladder. the media of the affected vessels is homogenous and intensely eosinophilic with hematoxylin and eosin stain. fibrosis and mononuclear cells infiltrate the vessel wall. experimental coronary arteritis with cardiac hypertrophy has been model in dba/ and other strains by intraperitoneal administration of mannoprotein-beta-glucan complex isolated from c. albicans (nagi-miura et al., ) . hyperplasia of alveolar or bronchial epithelium occurs in old mice and must be differentiated from pulmonary tumors. pulmonary histiocytosis, acidophilic macrophage pneumonia, and acidophilic crystalline pneumonia are synonymous morphologic descriptions of an idiopathic lung lesion that can be incidental or the cause of significant morbidity. incidence varies with mouse strain or stock, with c bl, s /svjae and swiss mice and older mice in general particularly susceptible. histologically, alveoli and bronchioles are filled with varying quantities of macrophages containing eosinophilic crystalline material . the crystalline material consists of ym and/or ym chitanases and can be found in other tissues including the upper respiratory tract, stomach, gall bladder, and bone marrow where it is described as hyalinosis (nio et al., ) . gastric lesions include crypt dilatation, submucosal fibrosis, adenomatous gastric hyperplasia, mineralization, and erosion or ulceration. gastric ulcers have been induced by cold stress, food restriction (rehm et al., ) , chemical injury (yadav et al., ) , and gastritis and gastric tumors by helicobacter infection (fox et al., ) . germfree mice have reduced muscle tone in the intestinal tract. cecal volvulus is a common cause of death in germfree mice and is caused by rotation of the large, thin-walled cecum. age-associated lesions are common in the livers of mice. cellular and nuclear pleomorphism, including binucleated and multinucleated cells, are detectable by months. mild focal necrosis occurs with or without inflammation, but an association of mild focal hepatitis with a specific infectious disease is often hard to confirm. other geriatric hepatic lesions include biliary hyperplasia with varying degrees of portal hepatitis, hepatocellular vacuolization, amyloid deposition (especially in periportal areas), strangulated or herniated lobes, hemosiderosis, lipofuscinosis, and fibrosis. extramedullary hematopoiesis occurs in young mice and in response to anemia. exocrine pancreatic insufficiency has been reported in cba/j mice. acinar cell atrophy is common but is strain-and sex-dependent. blood-filled mesenteric lymph nodes may occur in aged mice, especially c h mice. this condition is an incidental finding and should not be confused with infectious lymphadenopathy such as that associated with salmonellosis. aggregates, or nodules of mononuclear cells, are found in many tissues of aged mice, including the salivary gland, thymus, ovary, uterus, mesentery and mediastinum, urinary bladder, and gastrointestinal tract. these nodules should not be mistaken for lymphosarcomas. grossly observable black pigmentation in the spleen of c bl/ is normal and is melanosis caused by melanin deposition (weissman, ) . the spleen is subject to amyloidosis and hemosiderin deposition. lipofuscin deposition is common, especially in older mice. the thymus undergoes age-associated atrophy. a variety of genetic immunodeficiencies have been described in mice, many of which increase susceptibility to infectious diseases. perhaps the most widely known of these is the athymic nude mouse that lacks a significant hair coat and, more importantly, fails to develop a thymus and thus has a severe deficit of t-cellmediated immune function. additionally, scid mice, which lack both t and b lymphocytes, are used widely and are highly susceptible to opportunistic agents such as pneumocystis murina. specific immune deficits have become excellent models for studying the ontogeny and mechanisms of immune responsiveness (table . ). age-associated osteoporosis or senile osteodystrophy can occur in some mice. it is not associated with severe renal disease or parathyroid hyperplasia. nearly all strains of mice develop some form of osteoarthrosis. it is generally noninflammatory, affects articulating surfaces, and results in secondary bone degeneration. glomerulonephritis is a common kidney lesion of mice. it is more often associated with persistent viral infections or immune disorders rather than with bacterial infections. its prevalence in some strains approaches %. nzb and nzb × nzw f hybrid mice, e.g., develop immune complex glomerulonephritis as an autoimmune disease resembling human lupus erythematosus, whereas glomerular disease is relatively mild in nzb mice (nzb mice have a high incidence of autoimmune hemolytic anemia). renal changes occur as early as months of age, but clinical signs and severe disease are not present until - months. the disease is associated with wasting and proteinuria, and lesions progress until death intervenes. histologically, glomeruli have proteinaceous deposits in the capillaries and mesangium. later, tubular atrophy and proteinaceous casts occur throughout the kidney. immunofluorescence studies show deposits of immunoglobulin and the third component of complement, which lodge as immune complexes with nuclear antigens and antigens of murine leukemia virus in glomerular capillary loops. mice infected with lcmv or with retroviruses can also develop immune complex glomerulonephritis. mice also can develop chronic glomerulopathy characterized by progressive thickening of glomerular basement membrane by pas-positive material that does not stain for amyloid. this lesion can be accompanied by proliferation of mesangial cells; local, regional, or diffuse mononuclear cell infiltration; and fibrosis. advanced cases may lead to renal insufficiency or failure. interstitial nephritis can be caused by bacterial or viral infections but may also be idiopathic. typical lesions include focal, regional, or diffuse interstitial infiltration of tubular parenchyma by mononuclear cells, but glomerular regions also may be involved. severe lesions can be accompanied by fibrosis, distortion of renal parenchyma, and intratubular casts, but not by mineralization. if renal insufficiency or failure ensues, it can lead to ascites. some strains of mice, such as balb/c, can develop polycystic kidney disease, which, if severe, can compromise normal renal function. urinary tract obstruction occurs as an acute or chronic condition in male mice. clinical signs usually include wetting of the perineum from incontinence. in severe or chronic cases, wetting predisposes to cellulitis and ulceration. at necropsy, the bladder is distended, and proteinaceous plugs are often found in the neck of the bladder and proximal urethra. in chronic cases the urine may be cloudy, and calculi may develop in the bladder. additionally, cystitis, urethritis, prostatitis, laboratory animal medicine balanoposthitis, and hydronephrosis may develop. this condition must be differentiated from infectious cystitis or pyelonephritis and from the agonal release of secretions from accessory sex glands, which is not associated with an inflammatory response. hydronephrosis also may occur without urinary tract obstruction. ascending pyelitis occurs in mice secondary to urinary tract infection. parvovarian cysts are observed frequently and may be related to the fact that mouse ovaries are enclosed in membranous pouches. amyloidosis is also common in the ovaries of old mice. cystic endometrial hyperplasia may develop unilaterally or bilaterally and may be segmental. in some strains, the prevalence in mice older than months is %. endometrial hyperplasia is often associated with ovarian atrophy. mucometra is relatively common in adult female mice. the primary clinical sign is abdominal distension resembling pregnancy among mice that do not whelp. testicular atrophy, sperm granulomas, and tubular mineralization occur with varying incidence. preputial glands, especially of immunodeficient mice, can become infected with opportunistic or pathogenic bacteria. spontaneous lesions in prostate, coagulating gland (anterior prostatic lobe), seminal vesicles, and ampullary glands were described in control b c f mice from national toxicology program -year carcinogenicity and toxicity studies conducted in one of four different laboratories (suwa et al., ) . lymphocytic infiltration, inflammation, edema, epithelial hyperplasia, mucinous cyst, mucinous metaplasia, adenoma, adenocarcinoma, granular cell tumor, and glandular atrophy were variously observed in accessory sex glands. accessory adrenal cortical nodules are found in periadrenal and perirenal fat, especially in females. these nodules have little functional significance other than their potential effect on failures of surgical adrenalectomy. lipofuscinosis, subcapsular spindle cell hyperplasia, and cystic dilatation of cortical sinusoids are found in the adrenal cortices of aged mice. some inbred strains have deficiencies of thyrotropic hormone, resulting in thyroid atrophy. thyroid cysts lined by stratified squamous epithelium and generally of ultimo-branchial origin may be seen in old mice. amyloid can be deposited in the thyroid and parathyroid glands as well as in the adrenal glands. spontaneous diabetes mellitus occurs in outbred swiss mice and genetic variants of several strains such as nod mice (lemke et al., ) . high levels of estrogen in pregnancy may influence postpartum hair shedding. various endocrine effects on hair growth have also been described. abdominal and thoracic alopecia have been reported in b c f mice. symmetrical mineral deposits commonly occur in the thalamus of aged mice. they may also be found in the midbrain, cerebellum, and cerebrum and are particularly common in a/j mice. lipofuscin accumulates in the neurons of old mice. age-associated peripheral neuropathy with demyelination can be found in the nerves of the hindlimbs in c bl/ mice. deposits of melanin pigment occur in heavily pigmented strains, especially in the frontal lobe. a number of neurologically mutant mice have been described. they commonly have correlative anatomical malformations or inborn errors of metabolism. a seizure syndrome in fvb mice has been described (goelz et al., ) and can be spontaneous or associated with tail tattooing, fur clipping, and fire alarms. mice are most often female with a mean age of . months (range, - months) and can exhibit facial grimace, chewing automatism, ptyalism with matting of the fur of the ventral aspect of the neck and/or forelimbs, and clonic convulsions that may progress to tonic convulsions and death. ischemic neuronal necrosis was consistently observed in these mice and is consistent with status epilepticus in humans. unilateral and bilateral microphthalmia and anophthalmia are frequent (as high as %) developmental defects in inbred and congenic strains of c bl mice, especially impacting the right eye and female mice. these conditions may first be recognized due to ocular infections, secondary to inadequate tear drainage. other common findings include central corneal opacities, iridocorneal and corneal-lenticular adhesions, abnormal formation of the iris and ciliary body, cataracts, extrusion of lens cortical material with dispersion throughout the eye, failure of vitreous development, and retinal folding. these syndromes can be reproduced by exposure to alcohol at critical stages of embryogenesis when the optic cup and lens vesicle are developing and impacting normal development of other ocular structures, including the iris, ciliary body, vitreous, and retina . retinal degeneration can occur as either an environmental or a genetic disorder (chang et al., ) in mice. nonpigmented mice, both inbred and outbred, can develop retinal degeneration from exposure to light, with the progression of blindness being related to light intensity and duration of exposure. mouse genetics have laboratory animal medicine been shown to be more important than potential light associated tissue injury (serfilippi et al., a) . other strains such as c h, cba, and fvb are genetically predisposed to retinal degeneration because they carry the rd gene, which leads to retinal degeneration within the first few weeks of life and has been used extensively as a model for retinitis pigmentosa (farber and danciger, ) . presence of the rd gene in some mouse strains highlights that impaired vision must be a consideration when selecting strains for behavioral assays that rely on visual clues (garcia et al., ) . blindness does not interfere with health or reproduction and blind mice cannot be distinguished from non-blind mice housed in standard caging. cataracts can occur in old mice and have a higher prevalence in certain mutant strains. vestibular syndrome associated with head tilt, circling, or imbalance can result from infectious otitis or from necrotizing vasculitis of unknown etiology affecting small and medium-sized arteries in the vicinity of the middle and inner ears. (jones et al., (jones et al., - maronpot et al., ; percy and barthold, ) neoplasms of lymphoid and hematopoietic tissues are estimated to have a spontaneous prevalence of - %. there are, however, some strains of mice that have been specifically inbred and selected for susceptibility to spontaneous tumors. leukemogenesis in mice may involve viruses and chemical or physical agents. viruses associated with lymphopoietic and hematopoietic neoplasia belong to the family retroviridae (type c oncornaviruses) and contain rna-dependent dna polymerase (reverse transcriptase). these viruses are generally noncytopathogenic for infected cells, and mice appear to harbor them as normal components of their genome. although they may be involved in spontaneous leukemia, they are not consistently expressed in this disease. recombinant viruses have recently been discovered that can infect mouse cells and heterologous cells and are associated with spontaneous leukemia development in high leukemia strains such as akr mice. their phenotypic expression is controlled by mouse genotype. endogenous retroviruses are transmitted vertically through the germ line. horizontal transmission is inefficient but can occur by intrauterine infection or through saliva, sputum, urine, feces, or milk. the leukemia induced by a given endogenous virus is usually of a single histopathological type. loss of function in nucleic acid-recognizing, tlr , tlr , and tlr can result in spontaneous retroviral viremia and acute t-cell lymphoblastic leukemia (yu et al., ) . chemical carcinogens, such as polycyclic hydrocarbons, nitrosoureas, and nitrosamines, and physical agents such as x-irradiation can also induce hematological malignancies in mice. the most common hematopoietic malignancy in the mouse is lymphocytic leukemia that originates in the thymus. disease begins with unilateral atrophy and then enlargement of one lobe of thymus as tumor cells proliferate. cells can spread to the other lobe and then to other hematopoietic organs, such as the spleen, bone marrow, liver, and peripheral lymph nodes. clinical signs include dyspnea and ocular protrusion. the latter sign is due to compression of venous blood returning from the head. tumor cells spill into the circulation late in disease. most of these tumors originate from t lymphocytes or lymphoblasts, but there are leukemias of b-lymphocyte or null cell lineage. in the last two syndromes, the lymph nodes and spleen are often involved, but the thymus is generally normal. reticulum cell sarcomas are common in older mice, especially in inbred strains such as c bl/ and sjl. primary tumor cell types have been divided into several categories based on morphological and immunohistochemical features. histiocytic sarcomas correspond to the older dunn classification as type a sarcomas and are composed primarily of reticulum cells. the tumor typically causes splenomegaly and nodular lesions in other organs, including the liver, lung, kidney, and the female reproductive tract. follicular center cell lymphomas correspond to dunn type b sarcomas. they originate from b-cell regions (germinal centers) of peripheral lymphoid tissues, including the spleen, lymph nodes, and peyer's patches. typical tumor cells have large vesiculated, folded, or cleaved nuclei and ill-defined cytoplasmic borders. tumors also often contain small lymphocytes. type c reticulum cell tumors often involve one or several lymph nodes rather than assuming a wide distribution. they consist of reticulum cells with a prominent component of well-differentiated lymphocytes. myelogenous leukemia is uncommon in mice and is associated with retrovirus infection. disease begins in the spleen, resulting in marked splenomegaly, but leukemic spread results in involvement of many tissues including the liver, lung, and bone marrow. leukemic cells in various stages of differentiation can be found in peripheral blood. in older animals, affected organs may appear green because of myeloperoxidase activity, giving rise to the term chloroleukemia. the green hue fades on contact with air. affected mice are often clinically anemic and dyspneic. erythroleukemia is rare in mice. the major lesion is massive splenomegaly, which is accompanied by anemia and polycythemia. hepatomegaly can follow, but there is little change in the thymus or lymph nodes. erythroleukemia can be experimentally induced in mice by friend spleen focus-forming virus (sffv) which initially activates the erythropoietin (epo) receptor and the receptor tyrosine kinase sf-stk in erythroid cells, resulting in proliferation, differentiation, and survival. in a second stage, sffv activates the myeloid transcription factor pu. , blocking erythroid cell differentiation, and in conjunction with the loss of p tumor suppressor activity, results in the outgrowth of malignant cells (cmarik and ruscetti, ). mast cell tumors are also very rare in mice. they are found almost exclusively in old mice and grow slowly. they should not be confused with mast cell hyperplasia observed in the skin following painting with carcinogens or x-irradiation. natural plasma cell tumors are infrequent in the mouse. they can, however, be induced by intraperitoneal inoculation of granulomatogenic agents such as plastic filters, plastic shavings, or a variety of oils, particularly in balb/c mice. mineral oil-induced plasmacytomas in balb/c mice produce large amounts of endogenous retroelements such as ecotropic and polytropic murine leukemia virus and intracisternal a particles. associated inflammation may promote retroelement insertion into cancer genes, thereby promoting tumors (knittel et al., ) . similar to other spontaneous cancers, plasmacytoma development in mice is inhibited by innate immune responses of nk cells which when activated by viruses will release γinf (thirion et al., ) . mammary tumors can be induced or modulated by a variety of factors, including viruses, chemical carcinogens, radiation, hormones, genetic background, diet, and immune status. certain inbred strains of mice, such as c h, a, and dba/ , have a high natural prevalence of mammary tumors. other strains, such as balb/c, c bl, and akr, have a low prevalence. among the most important factors contributing to the development of mammary tumors are mammary tumor viruses. several major variants are known. the primary tumor virus mmtv-s (bittner virus) is highly oncogenic and is transmitted through the milk of nursing females. infected mice typically develop a precursor lesion, the hyperplastic alveolar nodule, which can be serially transplanted. spontaneous mammary tumors metastasize with high frequency, but this property is somewhat mouse strain dependent. metastases go primarily to the lung. some mammary tumors are hormone dependent, some are ovary dependent, and others are pregnancy dependent. ovary-dependent tumors contain estrogen and progesterone receptors, whereas pregnancy-dependent tumors have prolactin receptors. ovariectomy will dramatically reduce the incidence of mammary tumors in c h mice. if surgery is done in adult mice - months of age, mammary tumors will develop, but at a later age than normal. grossly, mammary tumors may occur anywhere in the mammary chain. they present as one or more firm, welldelineated masses, which are often lobular and maybe cystic (fig. . ) . histologically, mammary tumors have been categorized into three major groups: carcinomas, carcinomas with squamous cell differentiation, and carcinosarcomas. the carcinomas are divided into adenocarcinoma types a, b, c, y, l, and p. most tumors are type a or b. type a consists of adenomas, tubular carcinomas, and alveolar carcinomas. type b tumors have a variable pattern with both well-differentiated and poorly differentiated regions. they may consist of regular cords or sheets of cells or papillomatous areas. these two types are locally invasive and may metastasize to the lungs. type c tumors are rare and are characterized by multiple cysts lined by low cuboidal to squamous epithelial cells, and they have abundant stroma. type y tumors, which are also rare, are characterized by tubular branching of cuboidal epithelium and abundant stroma. adenocarcinomas with a lacelike morphology (types l and p) are hormone dependent and have a branching tubular structure. the control or prevention of mammary neoplasms depends on the fact that some strains of mammary tumor virus are transmitted horizontally, whereas others are transmitted vertically. although horizontally transmitted virus such as mmtv-s can be determined by cesarean rederivation or by foster nursing, endogenous strains of tumor virus may remain. fortunately, these latter tumor viruses have generally low oncogenicity relative to the bittner virus. mammary tumors are increased in frequency in c bl apc +/− female mice infected with h. hepaticus (rao et al., ) . mice develop an assortment of liver changes as they age, including proliferative lesions which can progress from hyperplastic foci to hepatomas to hepatocellular carcinomas. almost all strains of mice have a significant prevalence of hepatic tumors, some of which appear to result from dietary contamination or deficiency and h. hepaticus infections in susceptible strains of mice such as the a/jcr male mouse ward et al., ) . the prevalence of spontaneous liver tumors in b c f hybrids is increased by feeding choline-deficient diets or when infected with h. hepaticus (hailey et al., ) . tumors also can develop in mice exposed to environmental chemicals, many of which are carcinogenic or potentially carcinogenic (hoenerhoff et al., ) . spontaneous liver tumors in mice occur grossly as gray to tan nodules or large, poorly demarcated darkred masses. they are usually derived from hepatocytes, whereas cholangiocellular tumors are rare. hepatomas are well circumscribed and well differentiated, but they compress adjacent liver tissue as they develop. hepatocellular carcinomas are usually invasive and display histopathological patterns ranging from medullary to trabecular. large carcinomas also may contain hemorrhage and necrosis. carcinomas also may metastasize to the lungs. primary respiratory tumors of mice occur in relatively high frequency. it has been estimated that more than % of these tumors are pulmonary adenomas that arise either from type pneumocytes or from clara cells lining terminal bronchioles. pulmonary adenomas usually appear as distinct whitish nodules that are easily detected by examination of the lung surface. malignant alveologenic tumors are infrequent and consist of adenocarcinomas and squamous cell carcinomas. they invade pulmonary parenchyma and are prone to metastasize. the prevalence of spontaneous respiratory tumors is mouse straindependent. for example, the prevalence is high in aging a strain mice but low in aging c bl mice. the number of tumors per lung is also higher in susceptible mice. pulmonary tumors often occur as well-defined gray nodules. microscopically, adenomas of alveolar origin consist of dense ribbons of cuboidal to columnar cells with sparse stroma. adenomas of clara cell origin are usually associated with bronchioles. they have a tubular to papillary architecture consisting of columnar cells with basal nuclei. pulmonary adenocarcinomas, though comparatively rare, are locally invasive. they often form papillary structures and have considerable cellular pleomorphism. given the rapid development of mouse strains genetically predisposed to neoplasia, the mouse tumor biology database maintained by jackson laboratory is a valuable centralized resource for the most current tumor descriptions. the database contains information on more than strains and substrains, tissues and organs, over , tumor frequency records, and nearly histopathological images and descriptions. risk factors for recurrence, complications and mortality in clostridium difficile infection: a systematic review detection of 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astroviruses chemical-induced atrial thrombosis in ntp rodent studies nucleic acid-sensing toll-like receptors are essential for the control of endogenous retrovirus viremia and erv-induced tumors recent applications of engineered animal antioxidant deficiency models in human nutrition and chronic disease dissecting the effects of mtdna variations on complex traits using mouse conplastic strains a genomic update on clostridial phylogeny: gram-negative spore formers and other misplaced clostridia effective eradication of pinworms (syphacia muris, syphacia obvelata and aspiculuris tetraptera) from a rodent breeding colony by oral anthelmintic therapy possible allelic structure of igg a and igg c in mice one-step generation of different immunodeficient mice with multiple gene modifications by crispr/cas mediated genome engineering key: cord- -rfw njpo authors: olsen, christopher w. title: a review of feline infectious peritonitis virus: molecular biology, immunopathogenesis, clinical aspects, and vaccination date: - - journal: veterinary microbiology doi: . / - ( ) -r sha: doc_id: cord_uid: rfw njpo abstract feline infectious peritontis (fip) has been an elusive and frustrating problem for veterinary practitioners and cat breeders for many years. over the last several years, reports have begun to elucidate aspects of the molecular biology of the causal virus (fipv). these papers complement a rapidly growing base of knowledge concerning the molecular organization and replication of coronaviruses in general. the fascinating immunopathogenesis of fipv infection and the virus' interaction with macrophages has also been the subject of several recent papers. it is now clear that fipv may be of interest to scientists other than veterinary virologists since its pathogenesis may provide a useful model system for other viruses whose infectivity is enhanced in the presence of virus-specific antibody. with these advances and the recent release of the first commercially-available fipv vaccine, it is appropriate to review what is known about the organization and replication of coronaviruses and the pathogenesis of fipv infection. list of abbreviations: ade=antibody-dependent enhancement; bcv=bovine coronavirus; c' =complement; c'-ade=complement-mediated antibody-dependent enhancement; ccv=canine coronavirus; cns=central nervous system; cr=complement receptor; cvlp=coronavirus-like particle; ds=double-stranded; dth=delayed-type hypersensitivity; eav=equine arteritis virus; fcr = fc receptor; fecv = feline enteric coronavirus; felv = feline leukemia virus; fip = feline infectious peritonitis; fipv = feline infectious peritonitis virus; hcv- e = human coronavirus e; hcv-oc =human coronavirus oc ; he=hemagglutinating esterase; hev=hemagglutinating encephalomyelitis virus; hiv=human immunodeficiency virus; hrsv=human respiratory syncytial virus; ibv = infectious bronchitis virus; kb = kilobases; kda = kilodaltons; ldhv = lactate dehydrogenase virus; m = membrane (protein); mab = monoclonal antibody; mhc = major histocompatability; mhv=mouse hepatitis virus; mrna=messenger rna; n=nucleocapsid (protein); nlinked = asparagine-linked (glycosylation); ns = nonstructural (protein); o-linked = serine-or th reonine-linked (glycosylation); orf--open reading frame; pol = polymerase (protein); prcv = porcine respiratory coronavirus; rcv = rat coronavirus; recv = rabbit enteric coronaivirus; ri = replicative intermediate; rhuifn~ =recombinant human interferon alpha; s= spike (protein); sdav = sialodacryoadenitis virus; siv = simian immunodeficiency virus; spf = specific-pathogen-free; tcids =tissue culture infectious dose %; tcv=turkey coronavirus; tgev=transmissible gastroenteritis virus; ts=temperature-sensitive; vn=virus neutralization (-izing). history feline infectious peritonitis was first described as a disease entity in the s (holzworth, ; wolfe and griesemer, ) . the etiology of this disease was uncertain until , when virus particles were described from of cats with fip (ward, ) . this early paper by ward is notable because it outlined several salient features regarding the viral etiology of fip which have been confirmed by more recent studies. these include the tropism of the virus for macrophages, the presence of virions in vesicles and cysternae of the golgi body, the lack of plasma membrane budding by the virus, and the presence of club-like projections from the surface of the virions. ward and colleagues (ward, ; ward et al., ) suggested that the virus responsible for fip might be a member of the coronaviridae. evidence for this characterization included structural similarities between mouse hepatitis virus (mhv) and the putative fipv, disease similarities between fip and mhvrelated disease, and the possible association of a coronavirus with a retrovirus (feline leukemia virus (felv)/fipv and murine leukemia virus/mhv, respectively) in the etiology of both diseases. confirmation of the viral etiology and elucidation of the pathogenesis of fip was hampered for a number of years because of difficulties encountered in isolating fipv from clinical cases and growing the virus in vitro. the first cultivation of the virus in vitro was accomplished using peritoneal exudate cell cultures (pedersen, a) . in so doing, pedersen confirmed the subcellular localizations, virion features, and macrophage tropism first described by ward. the virus was subsequently grown in feline small intestinal organ cultures (hoshino and scott, ) . finally, the growth and serial passage, in a continuous cell line of feline origin, of a virus which produced fip upon experimental inoculation of cats was demonstrated in . this virus was also characterized as a coronavirus (o'reilly et al., ) . coronaviridae the family coronaviridae includes pathogens of several mammalian and avian species. this family has been defined morphologically as a group of spherical to pleomorphic, large ( - nm), enveloped viruses with helical nucleocapsid cores (wege, et al., ) . projecting from the envelope of coronavirus virions is a fringe of club- (wege, et al., ) or petal-shaped (holmes, ) spikes or peplomers. these spikes have given rise to the name coronavirus by analogy to either the corona of the sun or the crowns of thorns (corona spinarum) used in medieval artworks (holmes, ) . coronaviruses have historically been arranged antigenically into five groups (wege, et al., ) . one group includes fipv and the related feline enteric coronavirus (fecv), canine coronavirus (ccv), transmissible gastroenter-itis virus of swine (tgev), and human coronavirus e (hcv- e). a second group includes human coronavirus oc (hcv-oc ) and related isolates, mhv, rat coronavirus (rcv) and sialodacryoadenitis virus (sdav), bovine coronavirus (bcv), and hemagglutinating encephalomyelitis virus of swine (hev). the avian coronaviruses have been classified into two separate groups typified by infectious bronchitis virus (ibv) of chickens and turkey coronavirus (tcv). there are also several unclassified coronaviruses including an enteric coronavirus of rabbits (recv) (descoteaux and lussier, ) , a coronavirus which induces myocarditis after experimental inoculation of rabbits (edwards, et al., ) , and a coronavirus isolated from a horse (mair et al., ) . within this classification framework, recent work suggests that the barriers between antigenic groups are not absolute. tcv has been shown to be related antigenically to ibv as well as two mammalian coronaviruses, mhv and bcv (dea, et al., ) . furthermore, an evolutionary relationship between tcv, bcv, mhv, and hcv-oc is supported by genetic sequence analysis of the ' ends of these viruses (verbeek and tijssen, ) . there is also evidence from a recent monoclonal antibody (mab) analysis that hcv- e should be moved out of the tgev group (sanchez, et al., ) . beyond these changes within the coronavirus family, the coronaviridae is now seen to represent a larger rna virus superfamily. toroviruses have been isolated from horses (snijder, et al., b) , cattle (koopmans, et al., ) , cats (muir, et al., ) , and humans (beards, et al., ) . though morphologically different (toroviruses have a tubular nucleocapsid), the genomic organization (snijder, et al., b; snijder, et al., ) and protein structure (snijder, et al., a) of toroviruses and coronaviruses are very similar and there is evidence for recombination between toroviruses and coronaviruses during their co-evolution . equine arteritis virus (eav) contains an icosahedral-core and has been classified historically as a togavirus but is now included in the coronavirus superfamily because of its similar genetic organization . another traditional member of the togaviridae, lactate dehydrogenase-elevating virus (ldhv), is also similar genetically to coronaviruses kuo, et al., ) . coronaviruses appear to enter cells via plasma membrane fusion without significant involvement of phagocytic or endosomal pathways (kooi, et al., ; payne, et al., ; stoddart, ; sturman, et al., ; weismiller, et al., ) . neither cytochalasin b (which disrupts cellular microfilaments and phagocytosis) nor lysosomotropic bases (which elevate endosomal ph) affected infection of macrophages by fipv (stoddart, ) . bcv infection of human rectal tumor cells was likewise unaffected by lysosomotropic bases and an electron microscopic analysis demonstrated bcv fusion only with the plasmalemma, not with intracellular membranes (payne, et al., ) . mhv infection of fibroblasts has also been found to be largely independent of endocytotic pathways (kooi, et al., ) . the ability of a given coronavirus to infect specific hosts and/or tissues is thought to be dependent upon the presence of a specific cellular receptor (lai, ) . a receptor for mhv has recently been identified on murine enterocyte and hepatocyte membranes and cloned (dvelsler, et al., ) . the receptor is a kilodalton (kda) glycoprotein of the immunoglobulin superfamily, with amino acid sequence and antigenic homologies to members of the carcinoembryonic antigen family of proteins . transfection of fibroblasts with the putative mhv receptor gene conferred susceptibility to infection whereas mab to the receptor blocked infection (dvelsler, et al., ) . in addition, the presence of a functional form of the receptor correlated with tissue and mouse strain susceptibility to infection with mhv . to date, no work has been published concerning a receptor for fipv. a number of reviews have outlined the molecular biology of coronaviruses (holmes, ; lai, ; spaan, et al., ; sturman and holmes, ). these will be drawn upon to create a model into which recent advances in the molecular biology of fipv can be integrated. replication of coronaviruses is accomplished entirely in the cytoplasm of infected cells. the genome of coronaviruses is single-stranded rna of positive polarity. upon initial infection of a cell, the '-capped and '-polyadenylated genomic rna can function as messenger rna (mrna). coronaviruses are among the largest rna viruses; the genome of ibv is over kilobases (kb) . viral replication and transcription occur via the formation of double-stranded (ds) rna replicative intermediates (ris). a stretch of highly conserved nucleotides about bases from the ' end of the genome has been identified as a negative-strand initiation signal (lai, ) . traditionally it was thought that new positive-sense, genomiclength rna as well as positive-sense, subgenomic mrnas were all transcribed off a genomic-length ri (lai, et al., ) . however, results from recent studies of tgev (sethna, et al., ; sethna, et al., ) , mhv (sawicki and sawicki, ) , and bcv (liu, et al., ) have demonstrated transcription via subgenomic ris as well. coronavirus mrnas form a '-nested set, i.e. all of the mrnas share the same sequences at their ' ends. the number ofmrnas varies among different coronaviruses. fipv and ibv produce mrnas, tgev produces mrnas, and bcv produces mrnas, including the genomic-length mrna in each case. despite this variation in number, there is a consensus coding pattern among all coronaviruses. from ' to ' the gene order is polymerase (pol) polyprotein, spike (s) protein, membrane (m) protein, and nucleocapsid (n) protein, with nonstructural (ns) protein genes variably interspersed (lai, ) . the coding assignments (where known ) and sizes of the fipv (strain - ) mrnas are as follows: mrna , kb-genomic rna (de groot, et al., d) ; mrna , . kb-s protein (de groot, et al., c; de groot, et al., d) ; mrna , . kb (de groot, et al., d) ; mrna , . kb-m protein (de groot, et al., d; vennema, et al., ); mrna , . kb-n protein (de groot, et al., d; vennema, et al., ) ; and, mrna , . kb-open reading frames (orfs) and (de groot, et al., ) . each of the fecv strain mrnas has been found to be . kb shorter than the corresponding fipv - mrnas, suggesting a deletion of nucleotides at the ' end of the fecv genome (baines, ) . although all but the smallest coronavirus mrnas are structurally polycistronic, most are functionally monocistronic since only the unique '-most orf of each mrna is translated (lai, ; spaan, et al., ) . exceptions to this include the mrnas which encode the polymerase proteins (see below), mrnas and of mhv and mrna of tgev spaan, et al., ) , mrnas and oflbv (liu, et al., ) , and mrna of fipv (de groot, et al., ) . fipv mrna encodes polypeptides, an kda protein from orf- and a kda protein from orf- . orf- is homologous to orf-x from tgev, which is also encoded by the '-most mrna of tgev, mrna . the kda protein from orf- has structural similarity to membrane proteins (de groot, et al., ) . tgev mrna lacks a homolog to orf- (de groot, et al., ) . thus, the deletion at the ' end of the fecv genome compared to the fipv genome (baines, ) may be very interesting since both tgev and fecv are largely restricted to replication in enterocytes, whereas fipv infects macrophages in a variety of tissues systemically. the genomic-length mrnas of coronaviruses encode the rna-dependent rna polymerase proteins (lai, ) . research with mhv has demonstrated two "temporally and enzymatically distinct...polymerase activities" in mhv transcription . in addition, in vitro translation of the genomic-length mrna of mhv-a resulted in the detection of two predominant protein products, p and p . the p product was shown to be the n-terminal portion of a larger precursor protein (p ) and could be detected by two-dimensional gel electrophoresis of mhv-infected cells only at late times ( - hours ) after infection (denison and perlman, ) . cloning, sequencing, and in vitro translation of the ibv brieirly, et al., ; brieirly, et al., ) and mhv (lee, et al., ) pol mrnas has revealed that in both cases two orfs are translated via a - frameshifting-pseudoknot to produce the pol polyprotein. nucleotide (lee, et al., ) and protein (denison, et al., ) sequence analysis of the mhv pol region has also delineated a number of putative functional domains. these include membrane anchor, cysteine-rich, and protease domains in orf-la (lee, et al., ) , as well as polymerase, helicase, and zinc-finger domains and protease cleavage sites in orf- b (denison, et al., ; lee, et al., ) . similar domains have also been identified in the ibv (gorbalenya, et al., ), torovirus, and eav (denboon, et al., ) polymerases. but despite identification of these domains by sequence analysis, it is still unclear what the exact relationship is between the component proteins identified and the early and late polymerase activities which have been functionally defined. however, a group of temperature-sensitive (ts) mutants of mhv have been identified. these mutations fell into complementation groups relative to positive-and negative-sense rna transcription (schaad, et al., ) . sequence analysis of such mutations may allow assignment of distinct polymerase functions to specific polymerase protein components. for a given coronavirus, genomic-length and subgenomic mrnas all contain an identical ' leader sequence, the length of which varies among different coronaviruses . lai and colleagues, working with mhv, have pioneered research into the role of leader rna in coronavirus transcription. in , the leader segments of several mhv a mrna species were sequenced and found to be identical . the same sequence was also found at the ' end of the genomic-length rna, suggesting that the '-genomic rna coded for the mrna leaders via the complementary ' end of the negative-sense strand of the ri. lai and colleagues reasoned that since coronavirus replication is an entirely cytoplasmic process, conventional eukaryotic splicing, a nuclear process, could not explain their findings. they also found a sequence complementary to a region near the ' end of the leader at the initiation site for mrna , suggesting a role for the leader in transcription initiation . in , makino and colleagues demonstrated that during a mixed infection with two different mhv strains (bi and ca ), the respective leader sequences could freely reassort (makino, et al., b) . they have further demonstrated that such reassortment/ recombination occurs at a high frequency (makino, et al., a; makino et al., b) . it also appears that recombination occurs randomly with respect to the site of crossover (banner and lai, ) . (a previous report had suggested that recombination crossover sites were clustered near a hypervariable region in the s gene (banner et al., ) . ) the ability to study such recombinants may play a critical role in elucidating questions of coronavirus pathogenesis (baines, ) . recombinants have already been used to localize functionally important epitopes on the s protein of mhv (makino, et al., ) . coronavirus transcription is thus proposed to occur via a discontinuous, leader-primed mechanism (lai, ) . the leader is transcribed from the ' end of the ri's negative-sense strand, dissociates from this template, then reassociates with the template at intergenic sites and primes downstream transcription. the sites of leader reassociation with the template are determined by homologous binding between sequences near the ' end of the leader sequence and a consensus sequence in the intergenic regions. (such a consensus intergenic sequence has been identified preceeding the orfs for the fipv s (degroot, et al., c ) , m, and n (vennema, et al., ) proteins. ) the ' end of the mhv leader contains repeats of a ucuaa sequence which is also imperfectly repeated at intergenic sites (lai, ) . the number of repeats in the leader not only varies among strains of mhv but can also change during passage in vitro (lai, ; makino, et al., a) . these pentanucleotide repeats are thought to play an important role in the binding of leader to intergenic sequence rna (makino, et al., a) . coronavirus mrnas are not all synthesized in equal amounts during transcription, but their individual, relative rates of synthesis are constant throughout transcription . the relative abundance of the mrna species progressively increases from ' to ' along the genome (konings, et al., ) . two factors may be responsible for governing this gradient in mrna levels: the relative degree of homology, and thus binding efficiency, of leader rna to the intergenic sequences makino, et al., a) ; or, the internal initiation events during leader-primed transcription, which may occur slowly enough to block the elongation of passing transcripts (konings, et al., ) . the later theory proposes that upstream transcripts are forced to pause at new sites of initiation and thus prematurely terminate and dissociate from the template. such incomplete transcripts had previously been detected (makino et al., a) . ultimately, both mechanisms may be acting together to govern mrna levels. in addition, with the recognition of subgenomic-mrna replication (hoffman, et al., ; sawicki and sawicki, ; sethna, et al., ; sethna, et al., ) , it is possible that differing efficiencies of subgenomic-mrna replication may also affect mrna levels. all coronaviruses encode at least three structural proteins: s (formerly e ), m (formerly e ), and n (holmes, ; spaan, et al., ; sturman and holmes, ) . the s protein forms the peplomers which project from the surface of virions and are responsible for receptor binding, induction of cellto-cell fusion and fusion of the viral envelope with target cell membranes, induction of neutralizing ab, and induction of cell-mediated immune (cmi) responses. in addition, alterations in the s protein have been shown to affect the virulence ofmhv (dalziel, et al., ) and bcv . the mhv s protein also has fc receptor-like activity (oleszak, et al., ) ; binding of nonspecific ab to cell surface-exposed s proteins may protect the infected cell from virus-specific ab attachment and antibody-dependent cellular cytoxicity (oleszak, et al., ) . coronavirus s proteins are - kda glycoproteins with asparaginelinked (n-linked) oligosaccharide attachment. they contain an n-terminal signal sequence, a large domain exterior to the virus envelope, a transmembrane domain, and a c-terminal hydrophilic tail interior to the virus envelope . each peplomer consists of molecules of s protein arranged in a coiled-coil structure (de groot, et al., a; de groot, et al., b) . the sequence of each s polypeptide can be divided into two regions, s and $ . s is the n-terminal portion and $ is the region between s and the transmembrane domain . studies of fipv have confirmed that its s protein is also n-linked glycosylated (olsen, et al., c; vennema, et al., b) , structurally similar to the generalized coronavirus s protein (de groot, et al., c) , and responsible for mediating cell-to-cell fusion and neutralizing ab induction (de groot, et al., ) . there has been substantial interest in the extent to which s /$ proteolytic cleavage occurs among coronaviruses. results appear to depend upon the virus in question as well as the cell type involved (frana, et al., ; . the s proteins of ibv and bcv appear to be cleaved consistently (binns, et al., ; cavanaugh, et al., ) . cleavage of the mhv s proteins varies among different mhv strains and the s proteins of fipv, ccv, and tgev were not thought to be cleaved at all . however, a trypsin-sensitive cleavage site has recently been demonstrated in the s protein of fipv and fecv (baines, ) . cleavage at this site did not occur during normal processing of the virus in cell culture and cleavage with exogenous trypsin did not increase virus infectivity for a or crandell feline kidney cells (crfkc). cleavage did, however, increase the ability of attenuated strains of fipv to infect feline macrophages (baines, ) . a latent cleavage site may also exist in the tgev s protein (rasschaert and laude, ) . the degree of amino acid sequence homology among the s proteins of various coronaviruses differs for the s and $ portions of the polypeptides. a comparison between fipv, mhv, and ibv revealed - % homology in $ , but little homology in s (degroot, et al., b) . within the fipv antigenic group of viruses, however, there is much greater homology throughout the s protein. a significant degree of antigenic homology has been noted among the s proteins of tgev, porcine respiratory coronavirus (prcv), fipv, fecv, and ccv (sanchez, et al., ) . comparison of the s gene sequences of tgev and fipv has revealed % homology over the first amino acids and % homology for residues - (jacobs, et al., ) . extensive efforts have gone into mapping the dominant antigenic sites on the s proteins oftgev correa, et al., ; delmas, et al., ; garwas, et al., ; gebauer, et al., ; laviada, et al., ; posthumus, et al., ) , bcv (yoo, et al., ) , ibv (koch, et al., ; kusters, et al., ) , mhv (makino, et al., ; routledge, et al., ; stuhler, et al., ; takese-yoden, et al., ) , and fipv olsen, et al., a) . in all cases, multiple neutralizing domains have been delineated, with immunodominant domains generally located in the s portion of the protein. neutralization domains have, however, been identified in $ as well (koch, et al., ; kusters, et al., ; makino, et al., ; posthumus, et al., ; routledge, et al., ) . on the fipv s protein, a number of epitopes have been defined which appear to mediate both virus neutralization and antibody-dependent enhancement (ade) of fipv infectivity olsen, et al, a; olsen, et al., b) . coronavirus s proteins are co-translationally glycosylated, with the majority of s protein incorporated into budding virions and a smaller portion transported to the plasma membrane (sturman and holmes, ) . s protein which was incorporated into virions was transported substantially faster than recombinant-expressed s protein (degroot, et al., ; vennema, et al., b) , suggesting the existence of a retention signal in the cytoplasmic tail of s (vennema, et al., b) . a second study, however, would appear to refute the presence of such a retention signal since c-terminally-truncated s protein expressed by a recombinant vaccinia virus was completely retained in the endoplasmic reticulum (er) of infected cells (pulford and britton, ) . trimerization of the tgev s protein has been shown to occur primarily at a stage when the carbohydrate moieties are only partially trimmed, with terminal glycosylation occurring mainly on trimeric s protein (delmas and luade, ). in addition, oligomerization appears to be the rate-limiting step in transport oftgev s protein from the er to the golgi body (delmas and luade, ) . antigenic sites on the tgev s protein may be structurally dependent upon or independent of trimerization . however, the majority of antigenic sites on both the s and m proteins of tgev appear to require at least core-glycosylation (delmas and laude, ). this appears not to be the case for the s protein of fipv (olsen et al., c ) , despite the high degree of antigenic homology between the s proteins of fipv and tgev (jacobs, et al., ) . the m or membrane proteins of coronaviruses are smaller surface glycoproteins, approximately - kda in size . as opposed to the substantial external portion of coronavirus s proteins, only about % of the n-terminal portion of the m proteins protrudes from the viral envelope . the form of glycosylation (n-linked or o-linked) and the presence or absence of a signal peptide on the m protein varies among different coronaviruses . the m proteins oftgev (pulford and britton, ) , ibv (lai, ) , and fipv and fecv (baines, ) are n-linked glycoproteins, whereas glycosylation of the m protein of mhv is o-linked (lai, ) . for those coronaviruses lacking an n-terminal signal sequence on the m protein, one of the three alpha helices which span the developing virus membrane may function as a signal sequence . the first membrane spanning domain may also be responsible for targetting m proteins to the golgi (machamer, et al., ) . m protein is necessary for virus maturation and its insertion into golgi membranes determines, via interaction with the n protein, the sites of virus assembly and budding (holmes, ; lai, ; spaan, et al., ) . though the s proteins have traditionally been associated with the induction of protective immune responses to coronaviruses, mab to the m protein of mhv has been shown to protect mice against acute encephalitis (fleming, et al., ) . in addition, a recombinant vaccinia virus expressing the m protein of fipv - induced protection against challenge with virulent fipv for of vaccinated cats (vennema, et al., ) . the third structural protein common to all coronaviruses is the n protein. the n proteins are highly basic proteins of - kda which are phosphorylated on serine residues ). like many viral nucleocapsid proteins, the coronavirus n proteins encase the genomic rna and play an integral part in formation of the helical nucleocapsids ). in addition, n protein appears to be critical for viral transcription. in vitro studies have demonstrated that antibody to n protein inhibited transcription of mhv . subsequent studies confirmed a specific interaction of n protein with mhv leader rna at bases to (from the ' end of the leader) and ruled out direct binding of n protein to negative-sense rna in ris stohlman, et al., ) . these authors suggested that n protein may be an integral part of the transcription complex, possibly functioning to open secondary structure constraints known to occur at the region of n binding, targeting the transcription complex of protein and leader rna during the discontinuous transcription process, or serving to prevent rna-rna base pairing after transcription. a sequence comparison of the n genes from strains of mhv has demonstrated that each n protein contains highly homologous domains separated by more variable "spacer regions". the central domain appears to be responsible for rna binding ( parker and masters, ). the level of n protein may also act as the switch between transcription/mrna synthesis and replication/genomic rna synthesis for coronaviruses (lai, ) . involvement of n proteins in the transcription of helical rna viruses is by no means limited to coronaviruses. n protein functions similarly during the transcription and replication of rhabdoviruses (banerjee, ) . a fourth structural protein, he, is present in certain strains of mhv as well as bcv and hcv- c . it is a glycoprotein of approximately kda which functionally exists as a dimer (hogue, et al., ; spaan, et al., ) . this protein from bcv has been shown to have hemagglutinating activity and to be a "receptor-destroying enzyme with acetylesterase activity" (hogue, et al., ; spaan, et al., ; storz, et al., ; vlasak, et al., ) . recent work, however, indicates that the s protein of bcv is actually a more efficient hemagglutinin than the he protein (schultze, et al., ) . no such he protein has been identified for fipv. completion of the coronavirus replication cycle requires encapsidation of viral rna and release of virions from infected cells. recent studies with mhv have demonstrated a -nucleotide encapsidation sequence at the ' end of orf-lb (makino, et al., ; van der most, et al., ) . this location would explain the fact that only genomic-length mhv rna is packaged (van der most, et al., ) . however, bcv (hoffman, et al., ) and tgev (sethna, et al., ) subgenomic rnas are also packaged, suggesting that this encapsidation sequence site may not be consistent among all coronaviruses. subsequent to passage through the golgi, mature virions may be released from post-golgi vesicles by either fusion with the plasma membrane or cell lysis (holmes, ) . coronaviruses produce a broad range of disease manifestations in their respective hosts (mclntosh, ; wege, et al., ) . many coronaviruses induce diseases limited to a single organ system. for instance, tcv, bcv, ccv, recv, fecv, and tgev produce primarily enteric disease while ibv, rcv, and the hcvs are mainly respiratory tract pathogens. a respiratory coronavirus has also been identified recently in swine. it was initially sought out because of a high incidence of tgev ab in populations of pigs without evidence of tgev-related enteric disease (pensaert, et al., ; van nieuwstadt and pol, ) . experimentally, the severity of clinical disease associated with porcine respiratory coronavirus (prcv) infection has varied from mild (o'toole, et al., ) to fatal (van nieuwstadt and pol, ) and may depend upon the strain and dose of prcv and the age of piglets at challenge. though prcv primarily infects epithelial cells and macrophages in the respiratory tract, it is also capable of limited replication in the gastrointestinal tract (cox, et al., a; cox, et al., b; o'toole, et al., ) . like fipv and fecv, tgev and prcv are very similar antigenically across the s, m, and n proteins, though mab analysis of their s proteins appears to offer a method for differentiating these two viruses (callebaut, et al., ; callebaut, et al., ) . genetic comparisons of prcv and tgev have revealed deletions in the prcv s gene and the first ns gene downstream of s (page, et al., ; rasschaert, et al., ; wesley, et al., ) . interestingly, analysis of a small plaque variant of tgev also revealed alterations in the ns genes downstream of s (wesley, et al., ) . this variant virus also exhibited a different cell tropism within the small intestine. as previously noted, the fecv genome also appears to be deleted in comparison to fipv. as opposed to these coronaviruses which induce disease of primarily a single organ system, disease manifestations of mhv and fipv infections involve a variety of organs (wege, et al., ) . in mhv infections, the exact nature of the disease produced (hepatic, neurologic, or enteric) depends upon the strain of virus and the route and dose of inoculation, as well as host factors such as age and genetic make-up (levy, et al., ; wege, et al., ) . the pathogenicity of mhv- for different strains of mice has also been correlated with the ability of the virus to either replicate in or lyse macrophages or lymphocytes or mediate t cell-induced expression of a procoagulant monokine (chung, et al., ; lamontagne, et al., a; lamontagne, et al., b; lamontagne and jolicoeur, ; levy and abecassis, ) . similar correlations between host resistance to virus infection in vivo and intrinsic resistance (morahan, et al., ) ofmacrophages to infection in vitro have been documented for several other virus families (mogensen, ) . as will be seen, the nature of fip is also multifactorial with regard to virus strain, host parameters, and virus-immune cell interactions. fipv is only one of the coronaviruses which can infect cats. most notably, a distinction must be drawn between fipv and fecv (pedersen, a; pedersen, et al., b; pedersen, et al., ; stoddart and gaskell, ; tupper, et al., ) . fecv infections are generally inapparent or induce only mild enteritis, though more severe enteric disease has been seen in young, specific-pathogen-free (spf) kittens (pedersen, a; pedersen, et al., b) . clinical signs may include a low-grade fever, mucoid diarrhea, sometimes with hematochezia, occasional vomiting, anorexia, and lethargy. a transient leukopenia may accompany the onset of diarrhea. fecv targets the epithelium of the small intestine. histologically, villous atrophy may develop (most prominently in the jejunum and ileum) in severe cases. serum antibodies appear - days after infection (pedersen, a) . fecvs must also be differentiated from "coronavirus-like particles" (cvlps). cvlps have been detected in the feces of a variety of species of animals, both with and without diarrhea. these include rodents, dogs, cats, pigs, cows, poultry species, man, and non-human primates (hoshino and scott, ) . cvlps detected in the feces of clinically normal cats were morphologically similar to cvlps from other species (hoshino and scott, ) . cvlps can be distinguished from enteric coronaviruses based upon the ultrastructural characteristics of their surface projections. coronavirus peplomers are typically petal-shaped with a short stalk whereas the projections from the surface of cvlps are spherical or teardrop-shaped and attached via a long stalk (hoshino and scott, ) . (based upon this distinction, one report (dea, et al., ) of feline cvlps may have been describing a true enteric coronavirus.) the role of cvlps as pathogenic agents or their identity as viruses is yet to be conclusively determined (mcintosh, ) . it is vitally important to realize that there are multiple strains of both fipv and fecv which vary substantially in pathogenicity (fiscus, et al., ; fiscus and teramoto, b; pedersen and floyd, ; stoddart, ; stoddart and scott, ). among fipv strains, for instance, ucd is relatively avirulent, ucd and are intermediate in virulence, and ucd and - are extremely virulent (pedersen and floyd, ) . feline coronaviruses have also been separated into two groups based upon reactivity with s protein-specific mabs. group i viruses are typified by fipv strains ucd and and nw , while group ii consistently includes - , df /nor , and fecv (fiscus and teramoto, a; fiscus and teramoto, b; hohdatsu, et al., lb) . the classification of fipv strains ucd l and and tn /black varies between studies. this type of antigenic classification may also reflect biological characteristics such as rate of internalization and cellto-cell spread (fiscus and teramoto, b) . certain strains of feline coronaviruses (e.g., the yayoi strain) can apparently produce both enteritis with diarrhea as well as fatal fip (hayashi, et al., ) . the existence of such intermediate strains and the antigenic similarity among fipv and fecv isolates may indicate that all feline coronaviruses are simply different biotypes of a single prototypical virus (scott, ). it has even been suggested that that fipv strains are directly derived from fecvs by mutation or recombination in the gastrointestinal tract of an infected cat (evermann, et al., ; pedersen, et al., b) . finally, cats can also be infected with heterologous coronaviruses from other species. cats which were infected experimentally with tgev shed the virus in their feces and mounted homologous and heterologous humoral immune responses (reynolds and garwes, ) . conversely, pigs infected orally with virulent fipv demonstrated clinical signs, histologic lesions, and coronaviral antigen localization typical of tgev infection (woods, et al., ) . cats infected with ccv remained clinically normal and demonstrated homologous as well as heterologous humoral immune responses, but virus was not detectable in their stools (barlough, et al., ) . infection of cats with hcv- e resulted in only an homologous antibody response, with "little or no replication of the virus in inoculated animals" (barlough, et al., ) . given this potential for cross-species infection and the close antigenic relationship between fipv, fecv, tgev, and ccv pedersen, et al., ; sanchez, et al., ) , all of these viruses may be host-range mutants of one another . the clinical characteristics of fip in domestic cats have been well outlined (barlough and scott, ; hoskins, ; pedersen, b; pedersen and floyd, ; scott, ; stoddart and gaskell, ) . although a cat of any age may be affected, disease occurs most often in cats from months to years of age, with the majority of cases occurring in cats ~< year of age (addie and jarrett, ; scott, ) . it has been suggested that the incidence of fip is greater among purebred cats (pedersen, b ) , though it is unclear whether this reflects an actual genetic predisposition to infection or only increased fipv exposure and transmission in cattery situations (scott, ) . although the attack rate for fip is greater in catteries and multi-cat households, it is still a disease which usually occurs only sporadically (pedersen, b) . transmission of fipv is thought to occur via ingestion or inhalation subsequent to cat-to-cat contact. fipv is shed from infected cats in their feces and oronasal secretions. virus shedding has only been documented to occur for - days after experimental infection. thus, most cats are probably not shedding the virus at the time of clinical illness (scott, ; stoddart, et al., a,b,c) . there have been reports of transplacental transmission and reproductive tract disease manifestations (scott, ) , but this route of transmission and form of disease are yet to be conclusively proven experimentally. anecdotal reports, however, indicate that queens, both seropositive and possibly seronegative, may repeatedly give birth to kittens which develop fip over the first few months of life (scott, ) . survival of fipv in the environment has traditionally been assumed to be quite limited, given the enveloped nature of coronaviruses. a recent report, however, suggests that after drying onto a surface, infectious virus may persist for up to weeks (scott, ) . nonetheless, fipv is readily inactivated by commonly used disinfectants (scott, ) . the incubation period for fip varies greatly. it may be as short as two weeks or clinical disease may not be evident for months to years following infection (scott, ) . the existence of an fipv latent carrier state remains an assumption, but is supported by such circumstantial evidence as the prolonged incubation periods and the apparent activation of clinical disease upon subsequent infection with felv (pedersen, b) or corticosteroid treatment (pedersen and floyd, ) . in addition, a recent study of multicat households in the uk also supports the existence of asymptomatic cartier cats, their importance in fipv transmission, and the potential for fipv shedding subsequent to the first days after infection (addie and jarrett, ). there are two major forms of fip, the effusive or "wet" form and the noneffusive or "dry" form. in addition, there may be a combination of these forms with multiple granulomas but only limited, localized fluid accumulation. in the effusive form of the disease, fibrin-rich fluid accumulates in the peritoneal, pleural, pericardial, and/or renal subcapsular spaces. the specific signs exhibited by each cat depend upon the site of the effusion. the abdominal enlargement seen with peritoneal fluid accumulation is generally nonpainful. dyspnea may accompany significant pleural effusion and signs ofcor pulmonale may accompany pericardial effusion. renal subcapsular fluid accumulation is sometimes palpable upon physical examination. regardless of the site(s) of fluid extravasation, there are certain general signs which are typically present in all cases: anorexia, weight loss, listlessness, dehydration, and fever. the fever may fluctuate over a diurnal or longer pattern. other specific signs may occur with extension of the inflammatory process in the peritoneum to specific organs, e.g., jaundice with hepatic involvement. likewise, exocrine or endocrine pancreatic insufficiency may become evident with pancreatic involvement, but this is rare. signs associated with noneffusive fip are more unpredictable because of the more discrete nature of organ involvement by the pyogranulomatous lesions. fever, however, is particularly consistent with this form of fip and may be the only initially-evident clinical sign. consequently, fip should be a prominent rule-out for cats which present with a fever-of-unknown-origin. central nervous system (cns) and ocular involvement are more common with the non-effusive form of fip. cns signs may reflect spinal cord involvement with upper-motor neuron paresis and ataxia, or cerebral or cerebellomedullary lesions with ataxia, nystagamus, behavioral changes, or seizures. signs of ocular disease may accompany other manifestations of fip or may be the only clinically apparent evidence of fip. most commonly the uveal tract is involved, resulting in iritis, uveitis, or chorioretinitis. retinal hemorrhage and detachment or panophthalmitis may also occur. the course of disease tends to be different for the various forms of fip. cats with effusive disease generally progress along a more fulminant course, with death in weeks to several months. young kittens tend to survive for the shortest periods of time. the noneffusive form of the disease may wax and wane over a period of many months, particularly in cases with only ocular involvement. our understanding of the pathogenesis of fip is still incomplete and evolving. it is clear that exposure to fipv does not consistently correlate with the development of clinical disease (pedersen, b) . other disease determinants may include the strain of fipv involved (pedersen and floyd, ) , the dose and route of infection (pedersen, et al., la) , nonspecific stress factors, and the age (stoddart and gaskell, ) , immune status/felv status (pedersen, b ) , and genetic makeup of the cat (pedersen, b ) . support for the role of genetic factors comes from the possible increased incidence of fip in purebred cats and from the nature of fip in cheetahs. fipv has been documented to cause clinical disease in a variety of exotic felid species including cheetahs, sand cats, caracals, lynx, cougars, jaguars, leopards, pallas cats, and lions (barlough and scott, ; pedersen, b) . cheetahs, however, appear to be exceptionally sensitive to fipv infection. fipv infection in a captive breeding colony of cheetahs in oregon in produced a "disease storm" with high morbidity and mortality. investigations of this epizootic (evermann, et al., ; o'brien, et al., ) demonstrated that cheetahs are genetically homogeneous at their major histocompatability (mhc) loci. it has been hypothesized that because of a lack of mhc variability, cheetahs may be deficient in their ability to present fipv antigen and mount protective immune responses (o'brien, et al., ) . the recent characterization of the domestic cat mhc (winkler, et al., ) should allow further investigation into the possible genetic basis for any increased incidence of fip in purebred domestic cats. following oronasal infection, fipv first replicates in pharyngeal, respiratory (hayashi, et al., b; scott, ) , or intestinal epithelial cells (pedersen, b) . a primarily cell-associated viremia subsequently occurs in which monocytes are the predominant cell type infected (weiss and scott, l b) . monocytes likely serve to distribute fipv to target organs throughout the body. the virus localizes in macrophages in the reticuloendothelial organs and perivascularly in many organs (weiss and scott, c) . the histologic lesions of fip are characterized as necrotizing pyogranulomas with phlebitis and thrombosis (weiss and scott, c) . one of the most perplexing aspects of the pathogenesis of fip is the frequent occurrence of accelerated, more fulminant disease upon fipv challenge of seropositive as compared with seronegative cats. accelerated fip was first documented when it was shown that the onset of clinical disease among experimentally infected kittens correlated with the appearance of serum antibodies (pedersen and boyle, ) . confirmation of these results came with the demonstration that the onset of viremia, clinical signs, thrombocytopenia, lymphopenia, and the appearance of viral antigen and necrotizing lesions in affected tissues all occurred earlier in seropositive kittens than in seronegative kittens (weiss and scott, l b; weiss and scott, l c) . survival times were also significantly shorter for seropositive kittens (pedersen and boyle, ; weiss and scott, lb) . in addition, seronegative kittens given immune serum (pedersen and boyle, ; weiss and scott, a) or anti-fipv igg (pedersen and boyle, ) before challenge developed clinical disease in the same manner and over the same time course as seropositive kittens. jacobse-geels and colleagues (jacobse-geels, et al., ; demonstrated immune complex and complement (c') deposition in fip. both host proteins as well as fipv proteins have been demonstrated in the immune complexes purified from sera and ascites fluid of cats with fip (horzinek, et al., ) , though the potential significance of an autoimmune component in the pathogenesis of fip is yet to be evaluated. with activation of c', other inflamatory mediator cascades as well as the blood coagulation cascade may be initiated. subclinical disseminated intravascular coagulation (dic) has been demonstrated during experimental fip (weiss, et al., ) . other inflamatory mediators which have been implicated in the pathogenesis of fip include ill (goitsuka, et al., ) , il (goitsuka, et al., ) , and leukotriene b and prostaglandin e (weiss and vaughn, ) . the demonstration of immune complex deposition in fip initially seemed to explain both the pathologic changes of fip and the phenomenon of accelerated fip. however, a new consideration has been introduced with the demonstration of antibody-dependent enhancement (ade) of fipv infection of macrophages in vitro hohdatsu, et al., a; olsen, et al., b olsen, et al., , stoddart, ). antibody-dependent enhancement of virus infection occurs when monocytes or macrophages are more efficiently infected by complexes of virus plus ab, via receptor-mediated endocytosis, than by virus alone (porterfield, ) . since ade of virus infection in vitro was first documented in (hawkes, ) , it has been demonstrated for a wide range of viruses. a partial list includes flaviviruses such as west nile virus and murray valley encephalitis virus (cardosa, et al., ; pieris and porterfield, ) , alphaviruses such as semliki forest virus, sindbis virus, and western equine encephalitis virus (chanas, et al., ; pieris and porterfield, ) , bunyaviruses (lewis, et al., ; pieris and porterfield, ) , human respiratory syncytial virus (hrsv) (gimenez, et al., ; krilov, et al., ) , influenza a virus (ochiai, et al., ; ; ) , ldhv (inada and mims, ) , rabies virus (king, et al., ) , murine cytomegalovirus , reoviruses (burstin, et al., ) , and arenaviruses (lewis, et al., ) . however, the most informative work regarding ade of virus infection has come from studies of dengue virus (dv), human immunodeficiency virus (hiv), and most recently fipv (see below). despite all of the descriptions of ade of virus infectivity, numerous questions remain regarding the actual basis of the enhancement. for instance, which receptors mediate ade of virus infectivity? traditionally the process has been attributed to fc receptors (fcrs) for igg, both fcri and fcrii (littaua, et al., ; porterfield, ) . work with hiv and simian immunodeficiency virus (siv) has, however, demonstrated enhancement of virus infectivity via fcrs alone (homsy, et al., ; takeda, et al., ) ; via c' receptors (crs) and cd in the presence of both c' and ab (c'-ade) (montefiori, et al., a,b; robinson, et al., a robinson, et al., , b or in the presence of c' alone (robinson, et al., b) ; via fcr and cd (connor, et al., ; takeda, et al., ) ; and, in the presence of soluble cd (allan, et al., ; werner, et al., ) or synthetic peptides to hiv (derossi, et al., ) in the absence of serum and c'. crs have been implicated in ade of flaviviruses as well (cardosa, et al., ) . to date, reports of ade of fipv infectivity hohdatsu, et al., a; olsen, et al., b; stoddart, ) have reported only fcr-mediated enhancement in the absence of c'. given the involvement of c' activation in the pathogenesis of fip (jacobse-geels, et al., ; jacobse-geels, et al., ) , the potential for c'-ade of fipv needs to be addressed as well. a second question is what are the specific viral components which mediate ade of infectivity for each virus? mabs have been used to delineate viral proteins and specific epitopes involved in ade ofdv , hiv (robinson, et al., a,b; , and fipv hohdatsu, et al., a; olsen, et al., b) . for fipv, studies olsen, et al., b) indicate that enhancement is strictly mediated by epitopes on the s protein, while a third paper found that m protein-specific mabs could also mediate ade of fipv infectivity (hohdatsu, et al., a ) . an associated fact is that individual epitopes are capable of mediating both virus neutralization (vn) and ade of virus infectivity. this has been clearly shown for dv (halstead, et al., ; morens and halstead, ) and hiv (robinson, et al., ) , as well as for fipv hohdatsu, et al., a; olsen, et al., b) . does the subclass of a given ab determine whether it is able to mediate both neutralization and ade of virus infectivity? results with fipv suggest that igg a mabs can mediate both processes while igg mabs can only mediate vn . a report of ade of hiv infectivity found no dependence upon ig subclass, but since this was a report of c'-ade, the results don't refute the conclusions from the fipv report . work with ade of fipv infectivity has demonstrated a bell-shaped distri-bution of enhancement relative to ab concentration, with maximal enhancement occurring at subneutralizing concentrations of ab (olsen, et al., b) . similar results have been obtained with dv (morens and halstead, ) . mechanistically, how does the binding of enhancing ab to a particular epitope on a virus induce an increase in virus infectivity? it has been suggested that enhancing abs increase the binding of virus to cell surfaces, thereby potentiating a productive interaction between virus and its normal cell surface receptor (connor, et al., ; mady, et al., ; porterfield, ) . in support of this, bispecific abs to dv or hiv and a variety of cell surface molecules in addition to fcrs were found to enhance virus infectivity (connor, et al., ; mady, et al., ) . however, results of an in situ hybridization analysis of ade of fipv infectivity found no difference in the initial uptake of virus by macrophages infected in the presence or absence of enhancing ab . in other virus systems it has also been suggested that enhancing ab may facilitate uncoating (jolly, ; porterfield, ; robinson, et al., a) . the ultimate question is what bearing ade of virus infection, as described in vitro, has upon the pathogenesis of disease in vivo and the development of virus vaccines. this question has fueled an ongoing debate among researchers ofdv (halstead, ; morens and halstead, ; rosen, ) and hiv (bolognesi, ; homsy, et al., ; robinson, et al., b) . there is a substantial body of epidemiological information to suggest that ade of dv infection of macrophages underlies the development of a more severe form of dengue called dengue hemorrhagic fever or dengue shock syndrome. experimental data has, however, been largely restricted to measurements of viremia levels or clinical pathology parameters in nonhuman primates (halstead, ; halstead, et al., ; ). an attempt has been made to correlate the presence of enhancing abs in the serum of hiv-infected patients to the progression of aquired immunodeficiency syndrome. however, the in vitro enhancement levels demonstrated were fairly low and the number of patients was limited (homsy, et al., ) . with hrsv, ab may not only function to enhance infectivity, but also to enhance leukotriene production in infected cells (ananaba and anderson, ) . recent results of fipv candidate vaccine testing seem to provide the strongest support to date for a direct relationship between enhanced infectivity in vitro and enhanced disease in vivo. inoculation of cats with a recombinant vaccinia virus expressing the s protein of fipv - sensitized the cats and led to accelerated disease after fipv challenge (vennema, et al., a) . inoculation with recombinant vaccinia viruses expressing the m or n proteins of fipv - did not predispose the cats to accelerated disease (vennema, et al., ) . (these results in vivo also support the in vitro-defined localization of enhancing epitopes to the s protein of fipv olsen, et al., b) ). in addition, recent experiments among groups of cats used to study the efficacy of a candidate fip vaccine demonstrated a statistically significant association (under certain challenge conditions) between the ability of a cat's serum to mediate enhancement of fipv infectivity in vitro and the development of accelerated fip in vivo . it is very difficult to make a definitive diagnosis of fip. biopsy and subsequent histopathologic examination is the only absolutely conclusive method for antemortem diagnosis (barlough and scott, ). short of this, a variety of factors must be taken together to support a diagnosis of fip (barlough and scott, ; pedersen, b; scott, ; stoddart and gaskell, ; sparkes, et al., ) . clinicopathologically, patients may demonstrate any of the following, depending upon the particular organ system affected: elevated serum liver enzyme and bilirubin levels, elevated serum urea nitrogen and creatinine levels, elevated fibrinogen levels, decreased packed cell volumes, neutrophilia, lymphopenia, or proteinuria. analysis of blood protein levels may be very helpful. while albumin levels may be normal or decreased, globulin levels are often increased. serum protein electrophoresis commonly demonstrates a polyclonal gammopathy. in cases of effusive fip, analysis of fluid obtained by abdomino-or thoracocentesis may be helpful. the fluid is generally yellow (though there may be various degrees of blood-tinging) and viscous with visible strands of fibrin. fluid analysis should reveal a high specific gravity and elevated protein level, with variable numbers of inflammatory cells. the fluid may clot upon standing. cerebrospinal fluid may reveal elevated protein levels and increased cellularity when fip affects the cns. the ability to utilize serologic testing in the diagnosis of fipv infection and clinical fip is limited. an early serologic study revealed that % of a local general cat population and % of cats in fip "problem catteries" were seropositive, but very few of the cats developed clinical disease (pedersen, b) . these results suggested that there was a mild primary form of the disease (pedersen, b) and that more severe, classical fip was an uncommon secondary sequela (pedersen, b) . subsequent to the discovery of the antigenically-related fecvs, it has been suggested that the vast majority of seropositive test results may indicate exposure to fecvs rather than fipvs (pedersen, b) . at this point, "the presence of serum coronavirus antibody in any cat, whether healthy or diseased, is indicative only of prior exposure to a coronavirus in the fipv antigenic group" (barlough and scott, ) , and has "little predicitive or diagnostic value" (scott, ) . in addition, not all cats with fip will have elevated coronavirus ab titers. in a recent study, of cats with fip had coronavirus titers of ~< , and of the cats tested negative for coronavirus ab (sparkes, et al., ) . early attempts to serologically discriminate between cats infected with fipv versus fecv were uniformly unsuccessful (ingersoll and wylie, a,b) . however, mabs which distinguish fipv - and fecv have recently been identified hohdatsu, et al., c) . such mabs may provide the basis for a competitive enzyme-linked immunosorbent assay to differentiate between a cat's exposure to fipv and fecv (fiscus and teramato, a) . one final complication in feline coronavirus serologic testing is that cats may produce ab directed against bovine serum components, possibly as a result of their presence in routine vaccine preparations (barlough and scott, ) . these abs can produce false positive coronavirus titers if bovine serum is also used to propagate the target viruses used for coronavirus ab testing. such cross-reacting abs decrease over a period of - months after vaccination (barlough and scott, ). treatment of cats suffering from fip is largely symptomatic, e.g. fluid replacement and nutritional support. due to the immunologic nature of the disease, immunosuppressive doses of corticosteroids ( - mg of prednisolone per kg per day) are often prescribed, along with broad spectrum antibiotics (barlough and scott, ) . less commonly, immunosuppressive drugs such as cyclophosphamide or melphalan are used (barlough and scott, ) . ribavirin, human recombinant alpha interferon (rhulfna), and feline fibroblastic beta ifn have been shown to inhibit fipv replication in vitro (weiss and oostrom-ram, ; weiss and toivio-kinnucan, ) . in vivo, high-dose therapy with rhulfna + an immunomodulating drug derived from propionibacterium acnes was successful in suppressing clinical signs of disease and prolonging survival times in cats, but was unable to significantly protect against fatal disease (weiss, et al., ) . with the potentially long and unpredictable incubation period for fip, the difficulty inherent in identifying cats which are shedding the virus and distinguishing fipv from fecv serologic responses, and the survivability of fipv in the environment, it is not surprising that feline coronaviruses have been notoriously difficult to control in multicat environments. it is becoming increasingly clear that controlling contact between kittens and adult carrier cats may be the critical factor in controlling enzootic fip in catteries. addie and jarret have shown that isolation of kittens and their queen from the rest of the cattery population reduced seroconversion among the kittens, and that isolation of kittens from their queens as well (from to weeks of age ) completely eliminated seroconversion among the kittens (addie and jarrett, jarrett, , . however, the role of queens is more complicated. a special form of immunity may be conferred by infected queens to their offspring. pedersen and floyd demonstrated that kittens born to fipv-infected queens could resist challenge with virulent fipv. they suggested that "natural" transmission of fipv from queens to kittens at an early age may provide a form of premunition immunity (pedersen and floyd, ) . given the immune-mediated pathogenesis of fip, what is the basis for protective immunity to fipv infection? clearly, humoral immunity is not protective (pedersen, b) . some form of immunity would seem to exist, however, since there are reports (though rare) of spontaneous remissions, and the rate of seropositivity in cats far exceeds the incidence of clinical disease (pedersen, b) . it has been suggested that cmi responses may be important in immunity (pedersen and floyd, ; scott, ). weiss and cox evaluated delayed-type hypersensitivity (dth) skin reactions to fipv antigen in cats. they initially found a strong dth response in an fip-resistant cat, but only "minimal" response in a susceptible cat (weiss and cox, ) . subsequent work with more subjects documented similar results; after lethal challenge with fipv, of cats with positive dth responses to fipv antigen had increased survival times compared to dth ( -) cats (weiss and cox, ) . further support for the importance of cmi responses to fipv resistance comes from work in nude mice. following intracerebral challenge with fipv, mortality rates were significantly higher for homozygous (nu/nu) than heterozygous (nu/+ ) nude mice (takenouchi, et al., ) . more severe intestinal lesions have also been noted after fipv challenge in thymectomized kittens as opposed to normal kittens (hayashi, et al., a) . however, cmi responses may also contribute to the granulomatous lesions seen in noneffusive fip when cmi responses are only partially protective (pedersen, b; weiss and cox, ) . considering the lack of understanding as to what constitutes protective immunity to fipv infection and the problem of ade of infection and disease, the development of a safe and effective vaccine against fipv has been very problematic. a variety of approaches has been unsuccessful (olsen and scott, ) including the administration of inactivated fipv (scott, ) , avirulent fipv (fipv ucd , , and ) or sublethal doses of virulent fipv (pedersen, ; pedersen and black, ) , heterologous (ccv, hcv- e, tgev) live virus vaccines (barlough, et al., (barlough, et al., , woods and pedersen, ) , and a recombinant vaccinia virus expressing fipv s protein (vennema, et al., a) . most recently, a temperature-sensitive mutant of fipv df has been developed as an fipv vaccine (christianson, et al., ) . this vaccine strain was derived after passages of parental fipv df in cell culture (passages - at ° c ) followed by ultraviolet irradiation. intranasal vaccination of cats induces strong mucosal iga and lymphocyte blastogenesis responses (gerber, ; gerber, et al., ) . the vaccine's safety has been demonstrated after parenteral administration, administration to corticosteroid-and felv-immunosuppressed cats, and administration to cats previously exposed to fecv (gerber, ; gerber, et al., ) . the mean vaccine efficacy (calculated by the author from published data as preventable fraction) for experimental challenge studies conducted by the manufacturer (gerber, ) was %. however, independent testing of this vaccine which has just been completed partially refutes these initially reported (gerber, ) efficacy and safety results. the data presented by scott et al. indicate that the ability of the vaccine to protect cats against experimental challenge is dependent upon the strain and particularly the dose of challenge virus. low dose exposure ( tcidso) resulted in protection of >t % of vaccinated cats, while higher dose exposure ( >/ tcidso) resulted in virtually no protection and even induced accelerated fip in many cases. to date, accelerated fip has not been reported under field conditions. however, field studies conducted by the manufacturer in multicat facilities also failed to demonstrate a significant difference in the incidence of fip or kitten mortality between vaccinated and unvaccinated cats (fanton, ) . summary coronaviruses are now seen as representative of a larger rna virus superfamily whose viruses employ a unique and interesting replication scheme. in addition, traditional antigenically-defined boundaries within the coronaviridae are falling and the emerging relationships between prcv and tgev and fipv and fecv should provide for a fruitful area of viral pathogenesis research. while our knowledge of the molecular biology of feline coronaviruses is limited compared to that of mhv and ibv, the cloning and characterization of the fipv s, m, n, and ' orfs has provided very useful information. the recent characterization of the mhv receptor should provide an impetus for the identification of an fipv receptor. development of the ts df vaccine also represents a significant achievement. but while this vaccine has provided encouragement for the control of fipv infections, there are still questions concerning its safety under experimental conditions and its efficacy under field conditions. in this regard, the immunopathogenesis of fip has become even more complex with recent advances in our understanding of ade of fipv infectivity. the identification of discrete epitopes which mediate ade may allow for the development of a recombinant-engineered fipv vaccine from which enhancing epitopes have been deleted. in addition, the work of vennema and colleagues ( ) with a recombinant vaccinia virus expressing the m protein of fipv deserves further consideration. as regards the treatment of fip, weiss and colleagues' work ( - ) with specific antiviral drugs will hopefully be expanded, perhaps evaluating the combination of ribavirin and interferon in vivo. in addition, addle and jarrett's ( , ) data regarding control of enzootic fip in catteries is very encouraging. however, the lack of a diagnostic test which is specific for fipv exposure remains as a significant hurdle to the routine control of fipv infections. control of feline coronavirus infection in kittens 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gastroenteritis virus genetic analysis of porcine respiratory coroanvirus, an attenuated variant of transmissible gastroenteritis virus receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins genetic characterization of fla, the cat major histocompatability complex feline infectious peritonitis cross-protection studies between feline infectious peritonitis and porcine transmissible gastroenteritis viruses lesions in the small intestine of newborn pigs inoculated with porcine, feline, and canine coronaviruses structural analysis of the conformational domains involved in neutralization of bovine coronavirus using deletion mutants of the spike glycoprotein s subunit expressed by recombinant baculoviruses comparison of the nucleotide and deduced amino acid sequences of the s genes specified by virulent and avirulent strains of bovine coronaviruses key: cord- -jwflooop authors: clementz, mark a.; kanjanahaluethai, amornrat; o’brien, timothy e.; baker, susan c. title: mutation in murine coronavirus replication protein nsp alters assembly of double membrane vesicles date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: jwflooop coronaviruses are positive-strand rna viruses that replicate in the cytoplasm of infected cells by generating a membrane-associated replicase complex. the replicase complex assembles on double membrane vesicles (dmvs). here, we studied the role of a putative replicase anchor, nonstructural protein (nsp ), in the assembly of murine coronavirus dmvs. we used reverse genetics to generate infectious clone viruses (icv) with an alanine substitution at nsp glycosylation site n or n , or an asparagine to threonine substitution (nsp -n t), which is proposed to confer a temperature sensitive phenotype. we found that nsp -n a is lethal and nsp -n t generated a virus (designated alb ts icv) that is temperature sensitive for viral replication. analysis of alb ts icv-infected cells revealed that there was a dramatic reduction in dmvs and that both nsp and nsp partially localized to mitochondria when cells were incubated at the non-permissive temperature. these results reveal a critical role of nsp in directing coronavirus dmv assembly. all positive-stranded rna viruses that infect mammalian and plant hosts form membrane-associated replication complexes in the cytoplasm of infected cells (salonen et al., ) . coronaviruses, such as mouse hepatitis virus (mhv) and severe acute respiratory syndrome coronavirus (sars-cov) that causes severe respiratory illness in humans (peiris et al., ; stadler et al., ) , generate double membrane vesicles (dmvs), which are the sites of viral rna synthesis (baker and denison, ; goldsmith et al., ; gosert et al., ; snijder et al., ) . the dmvs are generated by the association of coronavirus nonstructural proteins (nsps) with host intracellular membranes (gosert et al., ; harcourt et al., ; prentice et al., ; shi et al., ; snijder et al., ) . however, the role of each of the coronavirus nonstructural proteins in the assembly of dmvs is not yet clear. the coronavirus nonstructural proteins are translated from the ′-most gene of the genome, gene . gene contains two open reading frames (orfs) that produce two large polyproteins, pp a and pp ab (fig. a) . polyprotein a is processed by viral proteinases to generate intermediates and mature products nsp -nsp . polyprotein ab is generated by a ribosomal frameshift between orf a and orf b and contains all nsps (masters, ; ziebuhr and snijder, ) . for mhv, these large polyproteins undergo extensive proteolytic processing by three virally encoded proteinases, papain-like proteinase (plp) , plp , and c-like proteinase ( clpro) to produce intermediate and mature replicase proteins (baker et al., ; bonilla et al., ; bost et al., ; denison et al., ; graham and denison, ; kanjanahaluethai and baker, ; kanjanahaluethai et al., ; lu et al., lu et al., , schiller et al., ) . the replicase intermediate p contains the mature products nsp -nsp and has been proposed to be functional in the synthesis of negative strand rna and/or as a scaffold for the assembly of the transcriptase/ replicase complex (kanjanahaluethai and baker, ; sawicki et al., sawicki et al., , . bioinformatic analysis indicates that the replicase products nsp , nsp , and nsp have membrane spanning helices (ziebuhr et al., ) . biochemical fractionation studies have shown that nsp and nsp are integral membrane glycoproteins (gosert et al., ; kanjanahaluethai et al., ; oostra et al., ) . recently, sparks et al. ( ) showed that the amino terminal region of nsp is essential for viral replication as deletions in this region are lethal. furthermore, sawicki et al. ( ) sequenced a large panel of temperature sensitive (ts) mutants of mhv and identified one ts mutant, alb ts , that contained a substitution of asparagine to threonine at amino acid position within the amino terminal region of nsp . this mutation was predicted to confer the rna minus ts phenotype, but how this mutation affects mhv rna synthesis is not yet understood. to investigate the role of specific asparagine residues within the amino terminal domain of nsp in mhv replication, we engineered amino acid substitutions at putative glycosylation sites nsp -n and nsp -n and the putative ts lesion nsp -n and isolated infectious clone virus (icv). we found that nsp -n and nsp -n are glycosylated and that substitution of nsp -n to alanine was lethal for virus replication, whereas substitution of nsp -n to alanine had no effect on virus replication. our results show that the asparagine to threonine substitution at nsp - was sufficient to bestow a ts phenotype, and this virus was designated alb ts icv. we found that proteolytic processing of p is unaffected in the alb ts icv at either temperature. however, at the non-permissive temperature, dmv assembly and mitochondria morphology are disrupted in alb ts icv-infected cells and viral replicase proteins partially localize with mitochondria. these data demonstrate that nsp is an important factor in dmv assembly and are consistent with the the first two-thirds of the mhv genome (orf a and orf b) encode the viral replicase proteins. the nonstructural proteins (nsps) are synthesized as polyproteins processed into precursors then mature replicase products. (b) analysis of p and nsp after tunicamycin and endo h treatments. hela-mhvr cells were infected with mhv-jhm, untreated (u) or treated with µg/ml of tunicamycin (t) for h prior to and during labeling. endo h treatment (e) was performed after immunoprecipitation with α-nsp . (c) hela-mhvr cells were infected with vtf . , and co-transfected with plasmids encoding plp and substrate encoding either wt or mutant nsp . proteins were radiolabeled ( s-trans-label) and immunoprecipitated with α-nsp antibodies. ip products were untreated or treated with endo h, separated on - % gradient sds-page gel, and visualized by autoradiography. (d) predicted topology of nsp indicating the location of two glycosylation sites and a ts lesion within the luminal loop. hypothesis that nsp is an anchor or scaffold for the replication complex. mhv replicase precursor p and product nsp are modified by n-linked glycosylation previous studies indicated that mhv nsp is a glycoprotein (oostra et al., ) , but it was unclear if the p precursor was also modified. to determine if both the precursor p and the mature product nsp are modified by n-linked glycosylation, we analyzed these proteins for sensitivity to tunicamycin and endoglycosidase h (endo h). mhv-infected cells were radiolabeled with s-trans-label and either mock-treated or treated with tunicamycin and lysates and were subjected to immunoprecipitation with anti-nsp and then treated with endo h. we found that both the precursor p and the mature product nsp were modified by n-linked glycosylation and that these modifications were sensitive to endo h, indicating that these proteins did not progress past the endoplasmic reticulum (er) (fig. b) . bioinformatic analysis indicated that nsp residues n and n are consensus sites for n-linked glycosylation (consensus sequence: nxs/t). to determine if nsp -n and nsp -n are specifically modified by n-linked glycosylation, a cdna clone [mhv-cen-nsp (kanjanahaluethai and baker, ) ] expressing the entire nsp region and the cleavage site recognized by plp , was subjected to site-directed mutagenesis to generate alanine substitutions at these sites, and the mobility of wt and mutant forms of nsp was assessed by sds-page. cells were transfected with constructs expressing pplp (proteinase) and either wt or mutant forms of pcen-nsp dna (substrate) and newly synthesized proteins were radiolabeled with s-trans-label. cell lysates were prepared and subjected to immunoprecipitation with an α-nsp antibody to detect the mature form of nsp generated by cleavage of cen-nsp by plp . the immunoprecipitated nsp was either mocktreated or endo h treated and electrophoretic mobility of protein was assessed. as seen in fig. c , nsp encoding either the n a or n a substitutions exhibited faster migration as compared to wt nsp . the nsp -n a/n a double mutant migrated faster than either single mutant. for products treated with endo h, which cleaves n-linked oligosaccharides, the mobility of all four proteins was similar to that of the nsp -n a/n a double mutant, as expected. these data indicate that nsp -n and nsp -n are in fact subjected to n-linked glycosylation. recent studies have suggested that an asparagine residue in the luminal domain of nsp is important for mhv rna synthesis. sawicki et al. ( ) analyzed a series of mhv temperature sensitive mutants that do not make viral rna at the non-permissive temperature. they identified one virus, designated as alb ts , with an asparagine to threonine substitution at nsp residue . this residue was implicated as the site responsible for temperature sensitive defect. however, the mechanism by which this substitution in nsp causes the defect in rna synthesis in alb ts is not known. a schematic diagram of nsp topology indicating the position of the two asparagine residues modified by n-linked glycosylation, and an asparagine to threonine change predicted to be responsible for the temperature sensitive phenotype are depicted in fig. d . to determine if nsp -n , nsp -n , or nsp -n is important for nsp function, we generated virus encoding each specific substitution. each substitution was introduced into the mhv-a genome using a reverse genetics approach pioneered by yount et al. ( ) as described in the materials and methods. briefly, pcr based site-directed mutagenesis was performed on the plasmid dna containing the region of nsp to be mutated (clone b) using specific primers (table ) . each mutant clone b dna fragment was ligated with the remaining six wt fragments to produce full-length mhv cdna which was then in vitro transcribed using t rna polymerase. infectious rna was electroporated into bhk-mhvr cells and cells were laid over a semi-confluent monolayer of dbt cells. cells were incubated at °c and scored for cytopathic effect. supernatant from cells showing syncytia formation was collected and passaged over a fresh monolayer of dbt cells to generate a stock of infectious clone virus. rna was isolated from mock and icv-infected dbt cells and rt-pcr was performed to amplify the region containing the mutation of interest. amplicons were sequenced to verify the presence of the engineered mutation (fig. ) . infectious viruses were successfully obtained for position n a (referred to as nsp -n a icv) and n t (designated alb ts icv). however, we were unable to generate the nsp -n a single or nsp -n a/n a double mutant virus, which indicates that mutation of n may be lethal for nsp function. to determine if either alb ts icv or nsp -n a icv is temperature sensitive, we measured the amount of infectious particles produced by virus-infected cells incubated at the permissive ( °c) or non-permissive ( . °c) temperature and titrated at the permissive temperature. two sets of dbt cells were infected with wt-a icv, alb ts icv, or nsp -n a icv at an moi of . . one set of infected cells was maintained at the fig. . sequence analysis of mutant infectious clone virus. dbt cells were infected with wt-a icv, nsp -n a icv, or alb ts icv and at h.p.i. rna was isolated. rt-pcr was performed on viral rna using primers listed in table , and pcr products were sequenced across the nsp region. permissive temperature of °c, while the other was incubated at the non-permissive temperature of . °c. at h.p.i., cellfree supernatant was collected. ten-fold serial dilutions (in triplicate) of isolated supernatant were used to infect dbt cells incubated at the permissive temperature. after h, plaques were counted and viral titer was determined. wt-a icv replicated to high titers of . × plaque forming units (pfu)/ ml at °c and . × pfu/ml at . °c. nsp -n a icv also produced a similar size of plaques and reached titers of . × pfu/ml at °c and . × pfu/ml at . °c. the alb ts icv replicated efficiently at °c ( . × pfu/ ml), but replication was dramatically reduced at . °c ( . × pfu/ml) (fig. a) . one-step growth curve analysis was then performed on wt-a icv, alb ts icv, and nsp -n a icv. dbt cells were infected with icv at a multiplicity of infection of . , incubated at the permissive temperature, and production of infectious virus was monitored by plaque assay. as shown in fig. b , when grown at °c, wt-a icv, alb ts icv, and nsp -n a icv all replicated with indistinguishable kinetics. temperature shift experiments were then performed to further assess the ts phenotype. two sets of dbt cells were infected with wt-a icv, alb ts icv, or nsp -n a icv at an moi of . and incubated at °c. at h.p.i., one set of infected cells was shifted to . °c. supernatant was harvested at two hour intervals and virus production was measured by plaque assay. as depicted in fig. , wt-a icv, nsp -n a icv, and alb ts icv at °c grew to similar titers and statistical analysis revealed that all icvs exhibited a common growth curve ( p = . ). at . °c however, alb ts icv titers fell over logs by - h.p. i. as compared to wt-a icv or nsp -n a icv, and alb ts icv exhibited distinct growth kinetics ( p b . ). taken together, these data indicate that the n t mutation in nsp is sufficient to confer a ts phenotype to the alb ts icv. analysis of proteolytic processing of p in the alb ts icv and nsp -n a icv one possible explanation for the ts phenotype of the alb ts icv is that nsp processing is altered at the non-permissive temperature. to address this issue, dbt cells were infected with wt-a icv, alb ts icv, and nsp -n a icv at an moi of . and at h.p.i. radiolabeled with s-trans-label for h. at h.p.i., cell lysates were prepared and subjected to immunoprecipitation with nsp-specific antibodies. as seen in figs. a-d, a large precursor of kda is detected from which the mature nsp -nsp / products are generated (kanjanahaluethai and baker, ) . the liberation of nsp was detected in cells infected with wt-a icv, alb ts icv, or nsp -n a icv virus at both the permissive and non-permissive temperature. the mobility of nsp in the nsp -n a icv was increased, which is consistent with the absence of glycosylation at amino acid (fig. a) . the processing of nsp , nsp , nsp , and nsp was also unaffected in cells infected with wt-a icv, alb ts icv, or nsp -n a icv at both temperatures (figs. b-d). overall, these results indicate that the ts phenotype is not due to any alteration in the processing of nsp or other p -derived replicase products. we hypothesize that nsp is a key anchor for dmv assembly and therefore asked if the formation of dmvs was altered fig. . temperature shift assay on infectious clone virus. two sets of dbt cells were infected with wt-a icv, alb ts icv, or nsp -n a icv at an moi of . and incubated at °c. at h.p.i., one set of infected cells was shifted to . °c. supernatant was harvested at two hour intervals and virus production was measured by plaque assay. arrow indicates time of temperature shift. in cells infected with the alb ts icv incubated at the nonpermissive temperature. we assessed dmv formation by transmission electron microscopy (tem) analysis to determine if the reported defect in rna synthesis is due to a defect in dmv assembly. two sets of dbt cells were infected with wt-a icv or alb ts icv at an moi of . and incubated at °c. at . h.p.i., one set of infected cells was shifted to . °c. at . h.p.i., cells were harvested and processed for tem analysis. dmvs can be visualized by tem as darkly ringed vesicles in the cytoplasm of mhv-infected cells (gosert et al., ) . as shown in fig. , dmv formation induced by wt-a icv was similar at both permissive and non-permissive temperatures. at °c, the alb ts icv induced dmv formation comparable to wt-a icv; however, at the non-permissive temperature of . °c, the alb ts icv produced fewer dmvs. the most striking feature we observed was that the morphology of the mitochondria was altered in cells infected with the alb ts icv incubated at . °c. as seen in fig. d , the mitochondria were larger and extensively vacuolated. the overall reduction in dmvs and the striking change in mitochondrial morphology lead us to hypothesize that the mutation in nsp resulted in altered localization of nsp and potentially other mhv replicase products, resulting in a block in viral rna synthesis. the abnormalities observed in the mitochondria of cells infected with alb ts icv incubated at the non-permissive temperature led us to explore whether mhv replicase proteins, which normally co-localize with er in dbt cells (shi et al., ) , were co-localizing with mitochondria. two sets of dbt cells were infected with wt-a icv or alb ts icv at an moi of . and incubated at °c. at . h.p.i., one set of infected cells was shifted to . °c. at h.p.i., cells were labeled with mitotracker red fluorescent dye, which is concentrated by active mitochondria. at . h.p.i., cells were harvested, fixed, and permeabilized for immunofluorescence assays. permeabilized cells were then incubated with antibodies to either nsp or nsp . as shown in fig. a , staining for nsp (green) and mitochondria (red) was non-overlapping in cells infected with wt-a icv at either temperature. at the permissive temperature, alb ts icv nsp and mitochondria displayed a very slight increase in co-localization versus wt-a icv. at . °c however, co-localization of nsp and mitochondria was substantially increased. the intensity of the red signal was also increased in cells infected with alb ts icv at the nonpermissive temperature, which may reflect the increased in size of the mitochondria that we observed by tem. similar results were obtained in three independent experiments, with extensive co-localization detected only in the alb ts icv-infected cells incubated at the non-permissive temperature. to extend these findings, we performed similar experiments using hela cells stably transfected with the mhv receptor (mhvr). two sets of hela-mhvr cells were infected with wt-a icv or alb ts icv at an moi of . and incubated at °c. at . h.p.i., one set of infected cells was shifted to . °c. at h.p.i., mitochondria were labeled with mitotracker red fluorescent dye or following fixation with an antibody to pyruvate dehydrogenase (pdh), which is a mitochondrial matrix protein. antibodies against nsp and nsp were again used to detect replicase products. as seen in fig. , extensive overlap between replicase proteins nsp and nsp and mitochondria was detected only in the alb ts icv-infected cells incubated at the non-permissive temperature ( . °c). similar results were obtained with mitotracker stained hela-mhvr cells and mitotracker and pdh were found to completely overlap (data not shown). these results are consistent with the tem studies and reveal that the mutant form of nsp is partially localized to mitochondria at the non-permissive temperature. furthermore, we also detected co-localization with mitochondria using the α-nsp antibody in alb ts icv-infected cells incubated at . °c (figs. b and b) . importantly, nsp , and perhaps other replicase products, are misdirected due to nsp mis-localization and are likely unable to efficiently assemble into functional dmvs in the alb ts icv-infected cells. this inability to generate functional dmvs and/or replication complexes would lead to an inability to synthesize viral rna, which is the reported phenotype of alb ts (sawicki et al., ) . positive-strand rna viruses express viral replicase proteins that must interact with host intracellular membranes to create an environment for optimal viral rna synthesis. coronaviruses express replicase proteins that assemble to generate dmvs in the cytoplasm of infected cells (goldsmith et al., ; gosert et al., ; snijder et al., ) . in this study, we investigated the role of one coronavirus replicase product, nsp , in mhv replication. because nsp is a transmembrane protein, we hypothesize that nsp is critical for assembly of the replication complex on dmvs. to test this hypothesis, we generated viruses with specific amino acid substitutions in nsp and assessed the effect of these substitutions on viral replication. first, we investigated the role of two putative n-linked glycosylation sites in mhv replication. using endo h assays, we found that both the nsp - precursor (p ) and the nsp product are modified by n-linked glycosylation (fig. b) . we engineered alanine substitutions (nsp -n a and nsp -n a) into wt-a virus and found that nsp -n a icv behaved identically to wt-a icv suggesting that glycosylation at this site is not required for mhv replication. however, we were unable to generate either nsp -n a icv or nsp -n a/n a (double glycosylation knockout) icv suggesting that glycosylation of nsp -n a, or specific folding of nsp in this luminal domain, is required for mhv replication. next, we investigated the role of nsp -n in mhv replication. sawicki et al. ( ) analyzed the sequence of mhv ts mutants and reported that one of these mutant viruses, alb ts , encoded a substitution of asparagine for threonine at nsp - . they hypothesized that this mutation alone was sufficient to confer the ts and rna synthesis-negative phenotype to mhv-a . via reverse genetics, we engineered the nsp -n t mutation into mhv and designated this virus as alb ts icv. we found that the nsp -n t substitution was indeed sufficient to induce temperature sensitivity. alb ts icv titers were reduced approximately orders of magnitude when incubated at the nonpermissive temperature; however, alb ts icv titers at °c were comparable to wt-a icv (fig. a) . growth kinetics, assayed by a one-step growth curve at the permissive temperature, were indistinguishable between alb ts icv and wt-a icv. however, temperature shift experiments revealed that upon incubation at the non-permissive temperature, alb ts icv titers fell fold (fig. ) . using a similar reverse genetics approach, donaldson et al. ( ) found that a single amino acid substitution in nsp conferred temperature sensitivity to the icts-la virus. this analysis revealed that nsp is a necessary cofactor for clpro activity as proteolytic processing of the replicase intermediate p was defective in icts-la -infected cells incubated at the non-permissive temperature. in contrast, we found that alb ts icv had no defects in proteolytic processing when virus-infected cells were incubated at the non-permissive temperature (fig. ). an alternative explanation for the rna minus ts phenotype of alb ts is that a mutation in nsp affects assembly of dmvs. to test this hypothesis, we performed tem analysis of alb ts icvinfected cells. this analysis revealed that dmv assembly is severely impaired in the alb ts icv-infected cells incubated at the non-permissive temperature (fig. d) . the failure to assemble dmvs, which are necessary for viral rna synthesis, is consistent with the rna minus phenotype observed by sawicki et al. ( ) . our results demonstrate that nsp plays a critical role in the formation and/or maintenance of dmvs. also, tem analysis of alb ts icv-infected cells incubated at the non-permissive temperature showed a disruption of mitochondrial morphology; the mitochondria were enlarged and extensively vacuolated (fig. d ). using confocal microscopy, we assessed whether nsp -n t was localized to the mitochondria. we found that nsp -n t partially co-localized with mitochondria in virus-infected cells incubated at the nonpermissive temperature (figs. a and a) . interestingly, we found that replicase product nsp also co-localized with mitochondria, suggesting that nsp -n t may direct the localization of other replicase components (figs. b and b). currently, it is unclear if a replicase precursor or only the final replicase products are directed to specific membrane sites or if nsp is actually penetrating the mitochondrial membrane. since nsp is an integral membrane protein originally derived from the er, the co-localization detected may be due to membrane reorganization. dmvs are likely diffusible in the cytoplasm and perhaps nsp -n t is directing the localization of dmvs to mitochondria where they are sequestered or fused with mitochondrial membranes. further experiments will be required to address this important issue. the aberrant mitochondrial morphology and partial colocalization with nsp and nsp raises questions about the role for mitochondria in mhv replication. could nsp -n t be localizing to mitochondria in error resulting in reduced dmv assembly? or is there a mitochondrial phase in mhv replication whose progression is inhibited by the nsp -n t substitution? previous studies demonstrate that for some viruses, the replicase complex can be directed to use different membrane sources for efficient virus replication. for example, flock house virus (fhv) normally induces spherules within the outer membrane of the mitochondria providing precedence for the use of mitochondrial membranes as the site of membrane-bound replication complex assembly (kopek et al., ; miller and ahlquist, ; miller et al., ) . to determine if mitochondrial membranes were required for replication, miller et al. ( ) replaced the mitochondrial outer membrane targeting signal of fhv protein-a with that of an er targeting signal and measured viral replication. they found that the er-targeted replication complex functioned as efficiently, if not more efficiently, than the normal mitochondria-targeted replication complex. there-fore, a specific source of membranes for replication complex assembly is not required for fhv. for mhv, it is unclear if the replication complex could be appropriately targeted to mitochondria, or if cytoplasmic dmvs are critical for mhv replication. in addition, it will be interesting to determine if wt nsp or nsp -n t expressed in trans can direct mhv replication complexes to specific membrane sites. complementation studies are useful for identifying products which can act in trans to provide a functional protein for a defective gene product. complementation analyses have been done with a large panel of ts mutants within the mhv replicase and have provided insights into the functions of intermediate and fully processed replicase proteins donaldson et al., ; fu and baric, ; sawicki et al., ; schaad et al., ; siddell et al., ; younker and sawicki, ) . interestingly, although mhv orf a encodes eleven mature nsps, mutants within orf a do not complement each other. there are at least two possible explanations for these results: ) a polyprotein precursor, such as p , may function itself, or function in cis and therefore cannot be complemented by mature nsp products (deming et al., ; sawicki et al., ) ; and ) mutations in one nsp may affect the production, function or localization of multiple products and therefore cannot be complemented by a trans-acting factor. for example, virus with a mutation in nsp (ts-la ) crossed with a nsp mutant (alb ts ) do not complement each other (sawicki et al., ) . donaldson et al. ( ) suggest that the icts-la , which exhibits a processing defect, fails to complement due to the inability to generate mature forms of nsp -nsp . thus, a mutation in a single nsp (nsp ) affects the production of several nsps (nsp -nsp ). likewise, the mutation analyzed in this study, nsp -n t, which results in defects in dmv assembly and localization, also affects the localization of other nsps, such as nsp . therefore, like the icts-la , the defect in alb ts icv induces an overarching defect in mhv replication. these observations highlight the complex nature of coronavirus replication complex assembly and maturation and indicate that interplay among partially and fully processed replicase products ultimately leads to competent replication complexes. the results presented in this study indicate that nsp is a key component in dmv assembly and are consistent with nsp serving as an anchor or scaffold for the replication complex. analysis of cis and trans-acting viral and host factors will further elucidate the processes required for assembly of the coronavirus transcription/replication complex. wt-a icv, alb ts icv and nsp -n a icv were generated using the mhv-a reverse genetics system developed by yount et al. ( ) . wt and mutagenized clone b plasmids were transformed into chemically competent mds (scarab genomics) cells. the remaining clones were transformed into chemically competent xl- blue cells. competent cells were heat shocked for s at °c and plated on luria-bertani (lb) plates containing appropriate selection antibiotics. single colonies were picked and grown in selection media (lb + antibiotic) overnight at °c. subcultures of wt and mutant clone b were grown at °c in ml of lb + antibiotic until culture density reached an o.d. of . - . at nm. the remaining clones were treated similarly, but grown at °c. delayed brain tumor (dbt) and baby hamster kidney (bhk) expressing the mhv receptor (bhk-mhvr) cells were incubated at °c in minimal essential medium, mem, (gibco) containing % fetal calf serum (fcs), % tryptose phosphate broth, % penicillin/streptomycin, and % l-glutamine. hela-mhvr cells were propagated in dmem (gibco) containing % fcs, . m sodium n- -hydroxyethylpiperazine-n′- ethanesulfonic acid, ph . , % penicillin/streptomycin and % l-glutamine. the coronavirus reverse genetics system described by yount et al. ( ) was used to generate virus encoding a single amino acid substitution compared to mhv-a . primers with two nucleotide changes designed to generate amino acid substitutions at nsp positions n and n to alanine, and n to threonine were incorporated into the mhv b plasmid dna via pcr based site-directed mutagenesis (quikchange kit, stratagene, primers listed in table ). plasmid dnas containing the specific mutations of interest were isolated and sequenced across the entire b region (sequencing primers shown in table ). the b plasmid dna region of interest was excised and ligated with the mhv a, c, d, e, and f isolated dna fragments to produce full-length viral cdna, which was in vitro transcribed using mmessage mmachine t kit (ambion) according to the manufacturer's instructions. infectious rna was electroporated into × bhk-mhvr cells and laid over . × dbt cells in mm dishes in duplicate. cells were incubated at °c for - h and monitored for the characteristic cytopathic effect (cpe) of mhv, which is syncytia formation. supernatant from cultures with cpe was passaged over a fresh monolayer of confluent dbt cells to generate a stock of icv. rna was isolated from virus-infected cells and subjected to reverse transcriptase (rt)-pcr using primers that flanked the region of interest. pcr amplicons were sequenced to verify the presence of the mutation in infectious clone virus rna (fig. ) . the region of viral rna containing the mutation of interest was rt-pcr amplified using the improm-ii rt system (promega) followed by the advantage cdna pcr kit (clonetech) according to the manufacturer's instructions. specific primers are listed in table . hela-mhvr cells were infected with a recombinant vaccinia virus expressing the bacteriophage t polymerase (vtf . ) at a multiplicity of infection of for h. then, cells were co-transfected with pplp -cen dna and either pcen-nsp wild type or pcen-nsp mutant dna (n a, n a, or n a/n a) using lipofectamine (gibco) according to the manufacturer's instruction as previously described (fuerst et al., ; kanjanahaluethai and baker, ) . proteins were radiolabeled with μci of s-trans-label from . to . h. p.i. cells were harvested and lysed with lysis buffer a containing % sds, % dtt, % glycerol and . m tris at ph . . cell lysates were subjected to immunoprecipitation assays as described previously (schiller et al., ) . briefly, radiolabeled cell lysates was diluted in ripa buffer ( . % triton x- , . % sds, mm nacl, mm edta and mm tris-hcl, ph . ) and immunoprecipitated with α-nsp rabbit antiserum and protein-a sepharose beads (amersham biosciences, piscataway, nj). for endoglycosidase h (endo h) treatment, protein-a sepharose-antibody-antigen complexes were washed once in ripa buffer. the endo h treatment was performed according to the manufacturer's instruction (roche). briefly, the complexes were resuspended in µl of mm sodium phosphate buffer, ph . , and incubated in the presence and absence of a final concentration of u/μl of endo h for h at °c. the complex-bound sepharose beads were pelleted by centrifugation. the products were eluted from the beads by incubating with × laemmli sample buffer at °c for min. protein products were separated via electrophoresis on - % gradient sds-page gels and were visualized by autoradiography. in tunicamycin treatment experiments, mhv-infected hela-mhvr cells were treated with µg/ml tunicamycin (boehringer mannheim) for h prior to addition of s-trans-label, and the drug was present during the h labeling period. whole cell lysates were prepared and subjected to immunoprecipitation as described above. viral titer of the wt-a icv, alb ts icv, and nsp -n a icv was determined via plaque assay. two sets of dbt cells were infected with wt-a icv, alb ts icv, or nsp -n a icv at an moi of . . one set of infected cells was maintained at the permissive temperature of °c, while the other was incubated at the non-permissive temperature of . °c. at h.p.i. cellfree supernatant was collected. ten-fold serial dilutions (in triplicate) of isolated supernatant were used to infect dbt cells seeded to % confluency in well plates. following a h absorption period, a ml mixture of . % noble agar (difco, detroit, mi) and mem with % fcs and % penicillin/ streptomycin was added to each well. infection was maintained for h at the permissive temperature ( °c) and plates were stained with . % crystal violet solution for min at room temperature to visualize and count plaques. one-step growth curves were generated by infecting dbt cells at an moi of . in -well plates. cells were washed three times with phosphate-buffered saline (pbs) following a h absorption phase. three milliliters of fresh medium were added and cells were incubated at °c. aliquots of supernatants were collected , , , , , and h.p.i. and the viral titer was determined by plaque assay in dbt cells maintained at °c. temperature shift growth kinetics were assessed by infecting two sets of dbt cells with wt-a icv, alb ts icv, or nsp -n a icv at an moi of . and incubated at °c. at h.p.i., one set of infected cells was shifted to . °c. supernatant was harvested at two hour intervals from - h.p.i. and virus production was measured by plaque assay in dbt cells maintained at °c. the logarithm of the titer of each virus (y) was analyzed using nonlinear regression modeling and the sas® software package. since these data clearly exhibit an asymptotic growth pattern, the two-parameter exponential model, y = θ ( − exp{− θ x}) + ε, was fit to the data using h post infection as the baseline. separate curves were fitted to each virus and temperature combination; parameter estimates were obtained using maximum likelihood methods, and subsequent tests were performed using likelihood-based f tests (ratkowsky, ) . two sets of dbt cells were infected with wt-a icv, alb ts icv and nsp -n a icv at an moi of . and incubated at °c. one hour post infection, actinomycin d (sigma, st. louis, mo) was added. at . h.p.i., cells were grown in media lacking methionine for min. cells were radiolabeled with s-trans-label for h at h.p.i. at h.p.i., one set of infected cells was shifted to . °c. cell lysates were prepared h.p.i. and subjected to immunoprecipitation with nsp-specific antibodies as described above. two sets of dbt cells were infected with wt-a icv or alb ts icv at an moi of . and incubated at °c. at . h.p.i., one set of infected cells was shifted to . °c. at . h.p.i., cells were harvested and processed for tem analysis as previously described (gosert et al., ) . two sets of dbt or hela-mhvr cells were grown to semiconfluence in well chamber slides coated with permanox. cells were infected with wt-a icv or alb ts icv at an moi of . and incubated at °c for a h absorption period. at . h.p.i., one set of infected cells was shifted to . °c. at h.p.i., cells were labeled with nm mitotracker red fluorescent dye (invitrogen). at . h.p.i., cells were washed times with pbs and fixed for min at room temperature with . % formaldehyde in pbs. cells were then permeabilized for min at room temperature with . % triton x- in pbs. following permeabilization, cells were incubated with either α-nsp or α-nsp and/or α-pdh antibodies overnight at °c. cells were then washed times for min in pbs. after washing, cells were incubated with alexafluor conjugated chicken α-rabbit igg (invitrogen) and/or alexa fluor goat α-mouse igg (invitrogen) secondary antibody for min at room temperature. cells were again washed times for min in pbs. cells were imaged on the zeiss confocal microscope. identification of a domain required for autoproteolytic cleavage of murine coronavirus gene a polyprotein cell biology of nidovirus replication complexes establishing a genetic recombination map for murine coronavirus strain a complementation groups characterization of a second cleavage site and demonstration of activity in trans by the papain-like proteinase of the murine coronavirus mouse hepatitis virus 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identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity identification of mouse hepatitis virus papain-like proteinase activity processing of the replicase of murine coronavirus: papain-like proteinase (plp ) acts to generate p and p identification of the murine coronavirus mp cleavage site recognized by papain-like proteinase membrane topology of murine coronavirus replicase nonstructural protein three-dimensional analysis of a viral rna replication complex reveals a virus-induced mini-organelle identification and characterization of a serine-like proteinase of the murine coronavirus mhv-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 molecular biology of coronaviruses flock house virus rna polymerase is a transmembrane protein with amino-terminal sequences sufficient for mitochondrial localization and membrane insertion flock house virus rna replicates on outer mitochondrial membranes in drosophila cells engineered retargeting of viral rna replication complexes to an alternative intracellular membrane localization and membrane topology of the coronavirus nonstructural protein : involvement of the early secretory pathway in replication severe acute respiratory syndrome coronavirus replication complex formation utilizes components of cellular autophagy handbook of nonlinear regression models viral rna replication in association with cellular membranes functional and genetic analysis of coronavirus replicasetranscriptase proteins a contemporary view of coronavirus transcription genetics of mouse hepatitis virus transcription: identification of cistrons which may function in positive and negative strand rna synthesis processing of the coronavirus mhv-jhm polymerase polyprotein: identification of precursors and proteolytic products spanning kilodaltons of orf a colocalization and membrane association of murine hepatitis virus gene products and de novo-synthesized viral rna in infected cells identification of the mutations responsible for the phenotype of three mhv rna-negative ts mutants ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex genetic analysis of murine hepatitis virus nsp in virus replication sars-beginning to understand a new virus negative strand rna synthesis by temperature-sensitive mutants of mouse hepatitis virus systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a the coronavirus replicase gene: special enzymes for special viruses virus-encoded proteinases and proteolytic processing in the nidovirales we thank ralph baric for the generous donation of clones for the reverse genetics system. we also thank linda fox of the loyola core imaging facility for her help with imaging studies, and katrina sleeman, surendranath baliji, naina barretto, dalia jukneliene, and other members of the baker lab for their technical assistance and suggestions. this research was supported by public health service research grant ai . key: cord- -cdg im h authors: van beurden, steven j.; berends, alinda j.; krämer-kühl, annika; spekreijse, dieuwertje; chénard, gilles; philipp, hans-christian; mundt, egbert; rottier, peter j. m.; verheije, m. hélène title: a reverse genetics system for avian coronavirus infectious bronchitis virus based on targeted rna recombination date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: cdg im h background: avian coronavirus infectious bronchitis virus (ibv) is a respiratory pathogen of chickens that causes severe economic losses in the poultry industry worldwide. major advances in the study of the molecular biology of ibv have resulted from the development of reverse genetics systems for the highly attenuated, cell culture-adapted, ibv strain beaudette. however, most ibv strains, amongst them virulent field isolates, can only be propagated in embryonated chicken eggs, and not in continuous cell lines. methods: we established a reverse genetics system for the ibv strain h , based on targeted rna recombination in a two-step process. first, a genomic and a chimeric synthetic, modified ibv rna were co-transfected into non-susceptible cells to generate a recombinant chimeric murinized (m) ibv intermediate (mibv). herein, the genomic part coding for the spike glycoprotein ectodomain was replaced by that of the coronavirus mouse hepatitis virus (mhv), allowing for the selection and propagation of recombinant mibv in murine cells. in the second step, mibv was used as the recipient. to this end a recombination with synthetic rna comprising the ′-end of the ibv genome was performed by introducing the complete ibv spike gene, allowing for the rescue and selection of candidate recombinants in embryonated chicken eggs. results: targeted rna recombination allowed for the modification of the ′-end of the ibv genome, encoding all structural and accessory genes. a wild-type recombinant ibv was constructed, containing several synonymous marker mutations. the in ovo growth kinetics and in vivo characteristics of the recombinant virus were similar to those of the parental ibv strain h . conclusions: targeted rna recombination allows for the generation of recombinant ibv strains that are not able to infect and propagate in continuous cell lines. the ability to introduce specific mutations holds promise for the development of rationally designed live-attenuated ibv vaccines and for studies into the biology of ibv in general. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. avian coronavirus infectious bronchitis virus (ibv) primarily infects the upper respiratory epithelium of chickens, causing a respiratory disease that is frequently complicated by secondary bacterial pathogens [ ] . in addition, some ibv strains affect the renal tubuli, oviduct and parts of the gastrointestinal tract, leading to pathological lesions in these organ systems, with subsequent reduced weight gain and a drop in egg production. the virus has a worldwide presence in both commercial and backyard chickens, appearing in a wide variety of geno-, sero-and protectotypes [ ] . ibv is currently regarded as one of the economically most relevant viral pathogens in the poultry industry. infectious bronchitis virus is the prototype gammacoronavirus in the family coronaviridae, order nidovirales [ ] . the enveloped virus particles have a positive-sense rna genome of . kb (fig. a ) [ ] . the ′ two-third of the viral genome comprises gene , divided into two large open reading frames a and b, which code for nonstructural proteins primarily involved in rna replication and transcription. the ′ one-third of the viral genome codes for structural proteins: spike protein (s, encoded by gene ), envelope protein (e, encoded by gene c), and membrane protein (m, encoded by gene ), each located in the viral envelope. the nucleocapsid protein (n, encoded by gene ) occurs in the ribonucleoprotein core [ ] . interspersed between the structural genes, coronaviruses carry a variable number of genus specific accessory genes [ ] . most of their gene products are nonstructural, and their expression is not essential for virus replication in vitro [ ] [ ] [ ] [ ] [ ] [ ] . the ibv genome contains the accessory genes and , encoding the proteins a and b, and proteins a and b, respectively [ ] . in addition, an open reading frame located in the intergenic region was identified between genes and [ ] . the typical coronavirus spikes are formed by trimers of the type membrane protein s, which is often proteolytically cleaved into two subunits, s and s [ , ] . the glycosylated s domain forms the 'head' of the spike and contains the receptor-binding domain [ ] . avian gammacoronaviruses typically interact with glycans on the host cell surface. ibv in particular requires α , -linked sialic acids for attachment and entry [ ] [ ] [ ] . the s domain builds the remaining part of the ectodomain (the 'stalk'), the transmembrane domain and the internally located endodomain. the s protein is the main determinant of the coronaviral host species tropism [ ] . many mammalian coronaviruses of the genera alphacoronavirus and betacoronaviruses can be propagated in cultured cells, unlike most avian coronaviruses of the genus gammacoronavirus. ibv can, however, readily be propagated in, and isolated from, embryonated fowl eggs. during passaging in embryonated eggs adaptation occurs, often leading to attenuation. for example, ibv strain h represents the nd serial passage of a massachusetts-like ibv strain isolated in the netherlands [ ] , which causes embryonic death within h post infection (hpi), and still has a residual virulence in young chickens. another passages resulted in the ibv strain h , which is more attenuated and has a lower pathogenicity in young chicks. similar serial passaging of another ibv strain of the massachusetts serotype isolated in the usa resulted in the generation of the non-pathogenic and cell-culture adapted ibv strain beaudette [ ] . in order to study its characteristics, several research groups have independently developed a reverse genetics system (rgs) for ibv which allow the manipulation of its genome [ ] [ ] [ ] [ ] [ ] . all these systems are based on the nonpathogenic cell-culture adapted ibv strain beaudette, or the highly attenuated ibv vaccine strain h . a major drawback of the use of the non-virulent ibv strains beaudette and h [ , ] is the inability to provide insights in the infection process in chickens, as these strains no longer cause a clinically relevant phenotype in vivo. yet, the rgs has provided significant insight in the fundamentals of avian gammacoronavirus replication. key findings include that ibv cell tropism is determined by the spike gene [ , ] , that the low virulence of ibv beaudette is caused by changes in the replicase gene [ ] , and that one or more of the ibv accessory gene products interfere with the hosts' interferon response [ ] [ ] [ ] . targeted rna recombination is another reverse genetics approach, so far only developed for mammalian coronaviruses from the genera alphacoronavirus and betacoronavirus [ , , ] . this system is based on the exchange of the spike gene by that of a coronavirus with a different host tropism, which enables subsequent selection on cells susceptible to the heterologous species [ ] . as a consequence, manipulation is limited to the last third of the coronavirus genome, covering all genes encoded that are located ′ of gene , starting with the spike gene. targeted rna recombination has been shown to be easy in use and to allow the rescue of highly defective mutants [ , , ] . however, the system is based on the ability to propagate both the donor and the recipient coronavirus in cell culture, and is hence not implementable for pathogenic ibv. this problem was solved by transfecting ibv genomic rna into otherwise non-susceptible cells, exchanging the ibv spike gene by that of the mouse hepatitis virus (mhv) provided as part of a synthetic rna, and by subsequently rescuing recombinant ibv from infected/ transfected cells in embryonated eggs (fig. ) . this system was successfully established to introduce marker mutations in the last one-third of the genome of ibv. the resulting recombinant viruses demonstrated growth kinetics in ovo and the in vivo phenotypic characteristics in one-day-old chickens similar to ibv wild type. the results presented here demonstrate for the first time a host species switch for an avian gammacoronavirus by exchanging the spike gene with that of the highly divergent betacoronavirus mhv. this rgs enables the manipulation of the structural and accessory protein genes from the genome of virulent ibv. baby hamster kidney (bhk- ) cells (atcc ccl- ) and murine lr cells kuo were cultured in dulbecco's modified eagle medium (dmem) (biowhittaker), supplemented with mm l-glutamine (lonza, basel, switzerland), % fetal bovine serum (fbs) (biowhittaker) and . mg/ml gentamicin (gibco invitrogen), at °c and % co . virus titers in cells were obtained by determining the % tissue culture infective dose (tcid ) per ml at days post inoculation (p.i.) according to the spearman-kärber method [ ] . fertilized specific pathogen free (spf) white leghorn eggs (animal health service, deventer, the netherlands) were incubated at . °c and - % relative humidity. a b c d fig. coronavirus genome organization and schematic overview of targeted rna recombination. a schematic genome representations of ibv (blue) and mhv (red). the first two-third of the genome is truncated, the structural and accessory genes are drawn to scale. the lengths of the first two-thirds and last one-third of the ibv genome are indicated at the top. the different domains of the spike gene are indicated: ss = signal sequence; ec = ectodomain; tm = transmembrane domain; en = endodomain. pcr amplicons are depicted as black bars drawn to scale above the genomes, with encircled letters referring to the primer sets in table and fig. . b stage in targeted rna recombination: an interspecies chimeric murinized ibv with a mhv spike ectodomain (mibv) is generated by a single recombination event of ibv genomic rna with synthetic rna transcribed from donor plasmid p-mibv in the ′-end region of the b gene (indicated by a black curved line). murinized ibv is selected on murine lr cells. plasmid inserts are indicated above p-mibv, with numbers in black circles referring to the plasmid junctions. c stage in targeted rna recombination: a recombinant ibv with the ibv spike gene (ribv) is recreated by a single recombination event of mibv with synthetic rna transcribed from donor plasmid p-ibv. recombinant ibv is selected on embryonated chicken eggs. d nucleotide sequences of the plasmid junctions, marked with corresponding numbers in the schematic donor plasmid drawings. nucleotide sequences are indicated for wild-type ibv and donor plasmids p-ibv and p-mibv, with restriction enzyme sites in italics, mhv spike gene sequences in lower case, and spike domains (i.e. ss, ec, tm and en) separated by vertical dashes. stop codons are highlighted in red. open reading frames (orfs) are underlined, overlapping orfs are double-underlined, and orf translations are indicated as amino acids below the nucleotide sequences if applicable eight-day-embryonated chicken eggs (ece) were inoculated via the allantoic cavity unless stated otherwise, and candled twice daily. upon embryonic death or no later than days p.i., eggs were transferred to °c for - h prior to allantoic fluid (af) and chorio-allantoic membrane (cam) collection. virus titration in ovo was based on the determination of the % embryonic infectious dose (eid ) per ml, as determined at day p.i. according to reed and muench [ ] . for the production of a virus stock, ten -day-old ece were inoculated with eid , incubated for h, and subsequently cooled for - h before the af was harvested and pooled. ibv strain h (boehringer ingelheim (bi), ingelheim, germany) was propagated in embryonated spf eggs and titrated. ibv strain beaudette (animal health service, deventer, the netherlands) was propagated and titrated in bhk- cells. mouse hepatitis virus (mhv) strain a was propagated and titrated in lr cells. monoclonal antibody (mab) ch/ibv . against the ibv s protein was obtained from prionics (thermo fisher scientific, waltham, ma, usa) [ , ] . the production of rabbit polyclonal antiserum k against mhv was described previously [ ] . chicken polyclonal antiserum was derived from a spf chicken vaccinated with ibv strain h (bi, ingelheim, germany). secondary fluorescently-labeled antibodies alexa fluor goat anti-chicken igy, alexa fluor goat antirabbit igg, and alexa fluor goat anti-mouse igg (invitrogen by thermo fisher scientific) were stored in % glycerol at − °c. cams were collected from eces, washed in pbs, fixed in neutral buffered % formalin in pbs for h, stored in % ethanol and finally paraffin-embedded. four micrometer sections of cam were mounted on glass slides and subsequently deparaffinized and rehydrated in alcohol series. next, the sections were subjected to endogenous peroxidase inactivation and antigen retrieval as described before [ ] . sections were washed in phosphate buffered normal antibody diluent (nad, scytek laboratories, logan, usa) containing . % tween- , and after primary antibody incubation with pbs . % tween- . sections were incubated for min at room temperature with mab ch/ibv . diluted : in nad. antibody binding was detected by dako envision hrpo labeled polymer anti-mouse (dako, by agilent technologies, santa clara, usa) diluted : in nad, and visualized by -amino- -ethylcarbazole (aec, dako). slides were counterstained with hematoxylin, mounted with aquatex (merck, darmstadt, germany), and viral antigen presence was assessed by light microscopy (bx , olympus, tokyo, japan). rna was isolated from harvested af using the qiaamp viral rna mini kit (qiagen, hilden, germany) according to manufacturer's protocol. reverse transcription (rt) was performed using the transcriptor first strand cdna synthesis kit (roche, basel, switzerland) according to manufacturer's protocol, with random hexamers for standard pcr, or with specific primers for sequencing and cloning purposes. pcr was performed with recombinant taq dna polymerase (thermo fisher scientific) for plasmid characterization or with phusion hot start ii high-fidelity dna polymerase (thermo fisher scientific) for sequencing and cloning purposes. one-step rt-qpcr was used to semi-quantitatively assess virus load in af. forward primer ibv.rdrp.f ( ′-catgcagtttgttggagatcct- ′) and reverse primer ibv.rdrp.r ( ′-gtgacctggttttaccgt ttga- ′) targeting the conserved region of gene b (nucleotide position , to , in ibv beaudette genbank accession number m . ) coding for the rna-dependent rna polymerase protein. primers were obtained from biolegio (nijmegen, the netherlands) and used at a final concentration of nm each with the itaq universal sybr green one-step kit (bio-rad laboratories, hercules, california, usa). the rt-qpcr reaction was carried out in a bio-rad cfx connect realtime pcr system, starting with min at °c and min at °c, followed by cycles of s at °c and s at °c, and ending with a dissociation step for the determination of the melting point of the obtained pcr fragment. the complete genome sequence of ibv h bi was determined by sanger sequencing using primers as described by zhou et al. [ ] . the ′-and ′-utr sequences were identified using the nd generation ′/ ′ race kit (roche, basel, switzerland). the ibv h bi genome sequence was , nucleotides (nt) in length, including an annotated nt polya tail. the design of the donor plasmids principally followed the strategy previously described by kuo et al. [ ] . the final donor plasmid p-ibv was constructed from the stepwise ligation of fragments derived from five plasmids (fig. b and table , and described below in detail): plasmid (p)ibv- comprises a t rna polymerase promotor, g nucleotides, and the near full-length ′-untranslated region (utr), with an unintended a to c substitution at position . plasmid ibv- b comprises the last nt of the pol b gene, including the nt overlap with the spike gene, and the first nt of the spike gene, including the signal sequence. plasmid ibv-s contains the near full length ectodomain of the spike gene, nt in length. plasmid ibv-sir comprises the last nt of the spike gene (the transmembrane and the endodomain), the accessory gene , the envelope gene, the membrane gene and half of the intergenic region. plasmid ibv- t comprises the ′-terminal region of the ibv genome, including the second half of the ir, the accessory gene , the nucleocapsid gene, the ′-utr and a nt poly-a sequence. all plasmids were generated by gen-script (piscataway, nj, usa) and provided in the plasmid puc -simple, a standard cloning plasmid with the polylinker removed. in order to allow cloning of the fragments in a stepwise approach, naturally occurring restriction enzyme sites (res) located in the viral cdna were used, except for the bstbi site between p-ibv- and p-ibv- b, which is only partly present in the ′-utr, and the xhoi site between p-ibv- b and p-ibv-s, which was introduced without changing the amino acid sequence (silent mutation). restriction enzyme sites were made unique by silently removing these res from other parts of the genome included in the donor plasmid (additional file : table s ). in addition, semi-unique res were introduced by silent mutations within nt up-and downstream of the accessory genes and . finally, unique res mssi and paci were included after the poly-a sequence, allowing linearization of the plasmid by a single restriction enzyme digest. all genome fragments were ligated step-by-step into p-ibv- using the restriction enzymes specified in table . each ligation mixture was subsequently transfected into hb competent cells and plasmid dna was isolated by performing midiprep dna isolation (qiagen, hilden, germany). the final plasmid consisted of p-ibv- - b-s-sir- t, now called p-ibv (fig. c) . the composition of each plasmid was confirmed after each cloning step by pcr, restriction enzyme digestion and sequencing of each of the inserts (macrogen, amsterdam, the netherlands). the ectodomain of the mhv a spike gene was amplified from ptug [ ] by pcr using primers with an xhoi overhang (table ) and ligated into pjet . resulting in p-mhv-s. site directed mutagenesis (sdm) with the q sdm kit (new england biolabs, ipswich, usa) was used to silently remove an ecori and an xhoi res interfering with subsequent cloning steps ( table ). the ectodomain of mhv spike was ligated into p-ibv- - b, followed by subsequent cloning steps using the ibv fragments sir and t. this resulted in the plasmid p-ibv- - b-mhvs-sir- t, now called p-mibv (fig. b) . a plasmid comprising the nucleocapsid gene and ′-utr sequence of ibv h bi was generated by pcr amplifying the respective region using primers ibv-h .n.atg.fw and ibv-m # .ir.rv ( table ). the amplicon was ligated into pjet . downstream of the t promotor sequence, resulting in p-ibv-n, and the correctness of the insert was verified by sequencing. capped, run-off donor transcripts were synthesized from p-ibv, p-mibv and p-ibv-n using the mmessage mmachine t kit (ambion by thermo fisher scientific). in brief, p-mibv was paci-linearized, and p-ibv and p-ibv-n were mssi-linearized. linearized plasmid dna was ethanol precipitated. transcription reactions were prepared according to the manufacturer's instructions, using . and . μg linearized dna per ul reaction for p-(m)ibv and p-ibv-n, respectively. after h of incubation at °c, production of rna was verified by analyzing μl of the reaction volume by gel electrophoreses. after an incubation of h the reaction was stopped by transferring the reaction tubes to ice. targeted rna recombination and rescue of mibv the ibv spike gene was replaced by a chimeric mhv-ibv spike gene in the ibv genome by targeted rna recombination between p-mibv generated donor rna and recipient virus (ibv) rna, as described before [ ] . ibv h viral rna was transfected into bhk- cells, a cell line known to support replication of ibv [ ] , but not infection with ibv h (data not shown). thus, μl of ibv h bi rna obtained from allantoic fluid, mixed with μl transcript reaction mixtures of p-mibv and p-ibv-n each were transfected into bhk- cells by electroporation using two pulses at v and μf in a gene pulser electroporation apparatus (bio-rad). transfected bhk- cells were seeded onto monolayers of lr cells having an approximate confluence of - % and incubated at °c. two days after seeding, when syncytia in the lr monolayer were observed, the cell culture supernatant was harvested and rescued viruses were purified by two rounds of plaque purification on lr cells. characterization of the last one-third of the genome of candidate recombinants was performed by rt-pcr and subsequent sanger sequencing of the obtained cdna-fragments, using the primer sets specified in table . murinized ibv (mibv) strain # b -iia was selected based on sequence analysis, and virus stocks were propagated and stored at − °c. aliquots were titrated on lr cells. recombinant ibv (ribv) was generated by substituting the ibv spike ectodomain back into the mibv genome by targeted rna recombination between p-ibv-generated donor rna and recipient virus mibv. lr cells were infected with mibv at a multiplicity of infection (moi) of . for h. capped, run-off donor transcripts from p-ibv were transfected into the mibv-infected lr cells by electroporation with two pulses at v and μf. electroporated lr cells were resuspended in ml dmem (at °c) and tenfold dilutions (up to − ) were prepared. two hundred microliters of lr cell suspensions were inoculated into the allantoic cavity of -day-old eces, using eggs per dilution. the eggs were candled twice daily and scored for embryonic death. upon death, or at days p.i., the eggs were transferred to °c. sixteen to twenty-four hrs later the af was collected aseptically for rt-qpcr, and the cams were fixed in % formalin for immunohistochemistry (ihc). the af from eggs inoculated with the highest dilution of electroporated lr cells, in which virus was detected by rt-qpcr and ihc, was subjected to two additional rounds of end-point dilution in -day-old ece. genetic characterization of candidate recombinants was performed by rt-pcr and subsequent sanger sequencing of the region encoding the structural and accessory genes using the primer sets specified in table . biological characterization of the chimeric nature of mibv was performed by immunofluorescence (if) double staining for ibv and mhv. bhk- and lr cells were grown on coverslips and inoculated with ibv beaudette, and mhv a and mibv # b -iia, respectively, at an moi of . . cells were fixed with pbs % paraformaldehyde (aurion, wageningen, the netherlands) for min at room temperature after ¼, , and hpi for mhv, ibv, and mibv, respectively. subsequently, cells were permeabilized with pbs containing . % triton x- , blocked with goat serum (gibco by life technologies), and incubated for - min with a combination of two primary antibodies in ngs; rabbit anti-mhv polyserum k diluted : and chicken anti-ibv-h serum diluted : , or rabbit anti-mhv polyserum k diluted : and mouse mab ch/ibv . animals were housed in separate groups and inoculated via eye-drop with eid in . ml of ibv h bi (n = ), ribv-wt (n = ), or not inoculated (n = , negative control). clinical symptoms monitored included ruffled feathers, decreased consciousness, depression, gasping, coughing, tracheal rales, and nasal discharge. seven days p.i. animals were euthanized, and evaluated for their tracheal ciliary activity. to this end, the trachea was sliced into transversal sections: from the upper part, from the middle part, and from the lower part. ciliary activity was examined by low-magnification microscopy within h after sampling. ciliostasis of each tracheal section was scored on a scale from ( % ciliary activity) to (no ciliary activity, i.e. complete ciliostasis), with the maximum score for each trachea being . finally, the mean ciliostasis score for each group of animals was calculated. a unpaired t-test was performed to analyse whether there were differences in ciliostasis scores between ibv h bi and ribv-wt. generation and antigenic characterization of mibv and ribv-wt viral rna of ibv h bi and rnas transcribed from plasmids p-mibv and p-ibv-n were co-transfected into bhk- cells and seeded onto monolayers of lr cells. at days post transfection, syncytia were observed in the lr monolayers, suggesting the successful generation of recombinant mibv. after two rounds of plaque purification on lr cells, candidate recombinants were characterized antigenically and genetically. if staining of lr cells infected with mibv showed positive staining with both anti-ibv and anti-mhv sera, indicating the chimeric nature of mibv (fig. a) . if staining with an anti-ibv-s mab was positive for ibv beaudetteinfected bhk- cells (taken along as positive control for if), but not for lr cells infected with mibv, indicating the absence of ibv s protein in mibv (fig. b) . lr cells infected with mibv and subsequently transfected with rna transcribed from plasmid p-ibv were inoculated in tenfold dilution series into the allantoic cavity of -day-old eces. no embryonic death was observed up to days p.i., but embryos in the lowest dilutions showed signs of stunting and curling typical for embryos infected with ibv. the presence of replicating recombinant ibv was demonstrated by rt-qpcr on viral rna extracted from the af (data not shown), and by ihc on cam tissue (fig. c) . in contrast, cams of eggs inoculated with mibv-infected lr cells (not transfected) did not show any positive signal for ibv in ihc. during the first and second passage of ribv-wt in eight-day-old eces for endpoint dilution purposes, infected embryos died between and days p.i. using specifically located primers ( fig. and table ) , the intended genome structure and sequence of mibv and recombinant ibv wild-type (ribv-wt) were verified by rt-pcr (fig. ) . the most important findings were that ( ) murinized ibv contained the correct ′ s gene sequence (primer set [f] -with a forward primer located in the ibv b gene at a position upstream of the b sequence present in p-mibv and a reverse primer located in the mhv spike gene -resulted in a detectable pcr product for mibv, but not for ibv or p-mibv); ( ) murizined ibv contained the mhv spike gene at the location of the ibv spike gene (as both primer set [f] and [h] -each with a primer in the mhv spike gene and one in ibv gene b [f] or in the m gene [h] -resulted in a detectable pcr product for mibv, but not for ibv or mhv); ( ) the ibv spike gene was absent from the mibv genome (as primer set [d] targeting the ibv spike gene resulted in a detectable pcr product for ibv, but not for mibv or mhv( ); recombinant ibv was the result of recombination between genomic rna from mibv and rna transcribed from p-ibv (as primer set [c] -with a forward primer in ibv gene b located upstream of the b sequence present in p-mibv and a reverse primer located in the ibv spike gene -resulted in a detectable pcr product for ribv-wt, but not for mibv or p-ibv); ( ) recombinant ibv contained the ibv spike gene at the location of the mhv-derived spike in mibv (as both primer set [c] and primer set [e] -each with one primer in the ibv spike gene and the other in ibv orf b [c] or the m gene [e] -resulted in a detectable pcr product for ribv-wt, but not for mibv or mhv); ( ) the mhv spike gene was absent from the ribv-wt genome (as primer set [g] targeting the mhv spike gene resulted in a detectable pcr product for mhv and mibv, but not for ribv-wt). sequence analysis of the ′ kb of the mibv genome (starting kb upstream of the bstbi res in gene b, which marks the start of p-mibv) confirmed the expected genetic identity of mibv as observed after rt-pcr analysis (additional file : figure s ). the ′ kb of mibv and ribv-wt were exactly as designed, including the deliberate synonymous mutations listed in additional file : table s . a single spontaneous silent mutation (t to c) was observed in the spike of ribv-wt at position , . in ovo growth kinetics of ibv and ribv-wt were assessed after inoculating eces with eid per egg by determination of the relative viral load in the af of five eggs per virus at , , , and hpi by rt-qpcr. at hpi, the parental wild-type ibv showed somewhat higher viral loads as compared to ribv-wt, while from hpi onwards, viral loads were comparable for both viruses (fig. a) . the virus titers remained at the same level until embryos started to die between and hpi. no differences in embryonic death between ribv-wt and ibv h bi groups was observed ( fig. b ; p > . ). the pathogenicity of ribv-wt was compared to that of the parental ibv h bi strain by inoculating one-dayold spf chickens. during the course of the infection, no clinical symptoms were observed in any of the groups (data not shown). at days p.i. the animals were euthanized and the mean ciliostasis scores were determined as a readout for the ability of the respective viruses to infect and cause lesions in the primary target organ, the trachea. as expected, the negative control animals scored very low (fig. ) , while ibv h bi infection resulted in a score of . ribv-wt had a mean score of (p > . ), indicating that ribv maintained the ability to a b c fig. antigenic characterization of mibv and ribv-wt. a immunofluorescence analyses of ibv beaudette, mhv a and mibv # b -iia infected cells. lr cells infected with mibv were fixed and double-immunolabeled with a polyclonal against ibv (green) and a polyclonal antibody against mhv (red). ibv beaudette-infected bhk- cells and mhv-infected lr cells were taken along for comparison. nuclei are visualized with dapi (blue). overlay pictures (merge) are shown on the right. b similar to (a), except that a monoclonal antibody against ibv s was used instead of a polyclonal against ibv, indicating the absence of ibv s protein in mibv infected cells. c immunohistochemistry of ibv h bi and ribv-wt infected cam tissues. ten-day-old embryonated chicken eggs were inoculated with ibv h bi (positive control), mibv-infected and p-ibv transcript electroporated lr cells (resulting in generation of ribv-wt), mibv infected and p-ibv transcript, but not electroporated, lr cells (mibv + p-ibv mock) or pbs (mock). formalin-fixed and paraffin-embedded cam tissues were immunohistochemically stained using a monoclonal antibody against ibv s . replication of (r)ibv in the epithelial cells of the cam is indicated by red cytoplasmic staining, which is absent in eggs inoculated with mibvinfected non-transfected lr cells infect one-day-old chickens and induced lesions to a similar extent as the parental virus strain. here we developed a novel rgs based on targeted rna recombination, which allows manipulation of the genome of virulent ibv. the resulting recombinant virus has the same characteristics as the wild type ibv h both in embryonated eggs and in one-day-old chickens. by adapting the classical targeted rna recombination approach [ , , ] to ibv, the inability to culture ibv strains like h on continuous cell lines has been overcome. for this, a cell-line known to support the replication, but not the entry of, ibv was used. this observation was used to create, by co-transfection of ibv viral rna and a transcript of chimeric ibv carrying the mhv spike gene, the recombinant mibv virus. upon transfection of synthetic ribv donor rna into mibv-infected murine lr cells, subsequent infectious ibv virus particles could be rescued in ece. the feasibility of the approach was demonstrated by the generation of recombinant ribv virus carrying silent marker mutations. the inability to select for individual ibv recombinants by plaque purification was circumvented by a combination of three approaches. first, the mibv-infected and ribv donor rna-transfected lr cells were inoculated into eces by end-point dilution. second, early rt-pcr and sequencing based screening of the genetic make-up of recombinants helped to identify and discard erroneous recombinants. third, two subsequent end-point dilution series were executed in eces, each leading to the selection of ribv-wt. finally, the genetic identity of the ′ kb of ribv-wt, i.e. the part of the ibv genome had been subject to manipulation, was confirmed by sequence analysis. the replication and pathogenicity of ribv-wt in ece was comparable to that of the parental ibv h bi. viral loads in the af were similar with respect to maximum virus titers (fig. ) and embryonic death induced by both viruses did not differ (not shown). the pathogenicity of ribv-wt in one-day-old chickens was also comparable to that of parental ibv h bi, as demonstrated by comparable mean ciliostasis scores at days p.i. (fig. ) . taken together, ribv-wt has the same properties as ibv h both in ovo and in vivo and can thus be used to provide insights in the infection process in chickens. previously described rgs based on non-pathogenic ibv [ ] [ ] [ ] [ ] [ ] can be used for in vivo studies only upon introduction of virulence factors including spikes from other ibv serotypes [ ] [ ] [ ] . our newly developed rgs for ibv-h directly allows the elucidation of factors that determine the pathogenicity of ibv, as well as studying its protective immunity in vivo. fig. genetic characterization of wild type viruses, recombinant viruses and donor plasmids. pcr was performed on cdna templates of viral rna extracted from infected lr cell culture supernatants (mhv and mibv) and allantoic fluid of inoculated embryonated eggs (ibv and ribv-wt), plasmid dna (p-mibv and p-ibv), or no template control (−).primer set letters a to h correspond with letters depicted in fig. ; detailed information on the primers is given in table here, for the first time host species switching of an avian gammacoronavirus to mammalian cells was demonstrated, by exchanging the spike gene with that of the betacoronavirus mhv. manipulation of coronavirus genomes by targeted rna recombination using an interspecies chimeric coronavirus has been demonstrated for alpha-and betacoronaviruses, thereby switching species tropism between mammalian hosts. our observation confirms the spike gene as the principal determinant of host species tropism of both avian and mammalian coronaviruses. in summary, a novel reverse genetics system (rgs) based on targeted rna recombination that allowed manipulation of the genome of virulent ibv was developed. this system makes use of an interspecies chimeric coronavirus, which is created by replacing the ectodomain of the ibv spike protein by that of mhv. the spike ectodomain exchange results in a host species tropism switch, which enables replication of mibv in cell culture. upon recombination of mibv with synthetic donor rna carrying the ibv spike gene, ribv a b fig. in ovo characteristics of ibv and ribv-wt. a growth kinetics of ibv and ribv-wt were assessed by quantitative rt-qpcr analysis of rna extracted from allantoic fluid of inoculated embryonated eggs collected at , , , and hpi. data points represent means and standard deviations of eggs per condition, with all samples run and analyzed in triplicates, using a tenfold dilution series of ibv h bi as reference for quantification. b embryonic death is indicated as a percentage of all remaining animals at each time point could be rescued in eces. in ovo growth kinetics and in vivo characteristics were comparable for ribv-wt and parental ibv h bi, suggesting no attenuating effect of the recombination process or of the introduced synonymous marker mutations. this system will allow the introduction of mutations in the ′ one-third of the ibv genome, allowing the manipulation of the structural and accessory genes. the use of this system for both fundamental and applied research is promising, and potentially enables the development of a new generation of rationally designed live-attenuated ibv vaccines. additional file : table s . silent mutations introduced in ribv. nucleotide positions and sequences refer to the ibv h bi genome (see additional file : figure s ). modified nucleotides in recombinant ibv wt are depicted in lower case; n.a. = not applicable. purpose of introduction of restriction enzyme site are indicated for each site; in case of enzyme site removal the purpose was to create unique restriction enzyme sites for cloning; n.a. = not applicable in this study. (docx kb) additional file : figure s . alignment of ′ kb of mibv and ribv-wt with ibv h bi. alignment of the ′ kb of mibv b iia p (excluding the mhv derived spike ectodomain sequence) and recombinant (r)ibv wild-type (wt) p with ibv h bi. numbers refer to nucleotide positions in the ibv h bi genome. restriction enzyme sites are highlighted in yellow, with the corresponding enzyme indicated above the sequences. an additional thymidine residue to keep the mhv spike gene ectodomain sequence in frame with the ibv spike gene signal sequence at position , is highlighted in green and marked with a # above the sequence. the long view: years of infectious bronchitis research infectious bronchitis virus variants: a review of the history, current situation and control measures family coronaviridae in: ninth report of the international committee on taxonomy of viruses coronavirus avian infectious bronchitis virus coronaviridae accessory proteins of sars-cov and other coronaviruses gene of the avian coronavirus infectious bronchitis virus is not essential for replication live, attenuated coronavirus vaccines through the directed deletion of group-specific genes provide protection against feline infectious peritonitis neither the rna nor the proteins of open reading frames a and b of the coronavirus infectious bronchitis virus are essential for replication manipulation of the porcine epidemic diarrhea virus genome using targeted rna recombination severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice identification of a noncanonically transcribed subgenomic mrna of infectious bronchitis virus and other gammacoronaviruses the avian coronavirus spike protein mapping of the receptor-binding domain and amino acids critical for attachment in the spike protein of avian coronavirus infectious bronchitis virus sialic acid is a receptor determinant for infection of cells by avian infectious bronchitis virus binding of avian coronavirus spike proteins to host factors reflects virus tropism and pathogenicity novel receptor specificity of avian gammacoronaviruses that cause enteritis retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier development and use of the h strain of avian infectious bronchitis virus from the netherlands as a vaccine: a review the pathogenesis of virulent and avirulent avian infectious bronchitis virus generation of a recombinant avian coronavirus infectious bronchitis virus using transient dominant selection reverse genetics system for the avian coronavirus infectious bronchitis virus an arginine-to-proline mutation in a domain with undefined functions within the helicase protein (nsp ) is lethal to the coronavirus infectious bronchitis virus in cultured cells in vitro assembled, recombinant infectious bronchitis viruses demonstrate that the a open reading frame is not essential for replication establishment of reverse genetics system for infectious bronchitis virus attenuated vaccine strain h recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism a recombinant avian infectious bronchitis virus expressing a heterologous spike gene belonging to the / serotype the replicase gene of avian coronavirus infectious bronchitis virus is a determinant of pathogenicity activation of the chicken type i interferon response by infectious bronchitis coronavirus infectious bronchitis coronavirus inhibits stat signaling and requires accessory proteins for resistance to type i interferon activity infectious bronchitis coronavirus limits interferon production by inducing a host shutoff that requires accessory protein b switching species tropism: an effective way to manipulate the feline coronavirus genome coronavirus reverse genetics by targeted rna recombination the method of "right and wrong cases" (constant stimuli) without gauss's formula a simple method of estimating fifty per cent endpoints antigenic domains on the peplomer protein of avian infectious bronchitis virus: correlation with biological functions detection by immunofluorescent assay of serotype-specific and group-specific antigens of infectious bronchitis virus in tracheas of broilers with respiratory problems viral protein synthesis in mouse hepatitis virus strain a -infected cells: effect of tunicamycin contributions of the s spike ectodomain to attachment and host range of infectious bronchitis virus nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes development and evaluation of a real-time taqman rt-pcr assay for the detection of infectious bronchitis virus from infected chickens 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 geert de vrieze, maartje woelders, maloeke de jong, and alexandra negatsch are acknowledged for excellent technical support. this research was financially supported by boehringer ingelheim, ingelheim, germany. data generated or analyzed during this study and described in this manuscript are included in this manuscript and its supplementary information files. authors' contributions sjvb, em, pjmr and mhv designed the studies. sjvb, ajb, akk, gc, ds, and hcp carried out the experiments. sjvb wrote the manuscript. mhv, em, and pr edited the manuscript. all authors read and approved the final manuscript. submit your next manuscript to biomed central and we will help you at every step: key: cord- -dap zo m authors: bose, abhishek; basu, rahul; maulik, mahua; das sarma, jayasri title: loss of cx -mediated functional gap junction communication in meningeal fibroblasts following mouse hepatitis virus infection date: - - journal: mol neurobiol doi: . /s - - - sha: doc_id: cord_uid: dap zo m mouse hepatitis virus (mhv) infection causes meningoencephalitis by disrupting the neuro-glial and glial-pial homeostasis. recent studies suggest that mhv infection alters gap junction protein connexin (cx )-mediated intercellular communication in brain and primary cultured astrocytes. in addition to astrocytes, meningeal fibroblasts also express high levels of cx . fibroblasts in the meninges together with the basal lamina and the astrocyte endfeet forms the glial limitans superficialis as part of the blood–brain barrier (bbb). alteration of glial-pial gap junction intercellular communication (gjic) in mhv infection has the potential to affect the integrity of bbb. till date, it is not known if viral infection can modulate cx expression and function in cells of the brain meninges and thus affect bbb permeability. in the present study, we have investigated the effect of mhv infection on cx localization and function in mouse brain meningeal cells and primary meningeal fibroblasts. our results show that mhv infection reduces total cx levels and causes its intracellular retention in the perinuclear compartments reducing its surface expression. reduced trafficking of cx to the cell surface in mhv-infected cells is associated with loss functional gjic. together, these data suggest that mhv infection can directly affect expression and cellular distribution of cx resulting in loss of cx -mediated gjic in meningeal fibroblasts, which may be associated with altered bbb function observed in acute infection. the meninges are formed by three tissue membranes that primarily provide protective covering to the central nervous system (cns). they comprise of the outer dura matter, middle arachnoid matter, and inner pia matter that consist of different cell populations including the fibroblasts, mast cells, endodermal cells, and smooth muscle cells [ ] . the arachnoid and the pia matter, together called as leptomeninges, harbor a large population of fibroblasts [ ] . meningeal fibroblasts are known to have important role in blood-brain barrier (bbb) formation, glial scar formation during injury response, angiogenesis, and early astrocyte activation [ , ] . in adult cns, the meninges cover and penetrate the neural tissue deeply at every level of its organization: as large projections between the major brain structures, as stroma of the choroid plexus, and as sheaths of blood vessels forming the perivascular space. they also form the roof of the lateral ventricles, the third and the fourth ventricles. their widespread distribution and complex organization suggest a more important role of the meninges as modulator of brain function in addition to being a mere protective covering of the brain. a further indication that the meninges are functionally linked to the neural tissue is the presence of gap junction proteins called connexins (cxs). gap junctions (gjs) are intercellular channels that allow exchange of small molecules (less than kda) including small glucose derivatives [ ] , and second messengers such as atp, cyclic amp, inositol , , -trisphosphate, and ca + [ ] [ ] [ ] between coupled cells. gjs are composed of two hemichannels each contributed by the two opposing cells. the hemichannels or connexons are formed by six cx protein subunits which after synthesis and oligomerization in endoplasmic reticulum/endoplasmic reticulum golgi intermediate complex are trafficked to the cell surface where they are mostly assembled into gap junction plaques. cx proteins, mainly cx , cx , and cx , have been found in the meninges and in their projections into the brain, including meningeal sheaths of blood vessels and stroma of the choroid plexus [ ] [ ] [ ] [ ] [ ] . the distribution of these proteins suggests the existence of anatomical and functional interactions between meningeal cells, meningeal perivascular cells, ependymocytes, and astrocytes, thus providing a rapid means to spread signals in physiological and pathological events. cx , the most abundantly expressed cx in the cns, is also highly expressed by the meningeal fibroblasts [ ] . recent findings suggest that cx -mediated functional coupling between the astrocytes and in the panglial system is altered in various neurodegenerative conditions like alzheimer's disease, parkinson's disease, ischemia, and multiple sclerosis (ms) [ ] [ ] [ ] [ ] . additionally, neuroinfections caused by borna virus, bovine papillomavirus, rous sarcoma virus, and human influenza virus can alter cx-mediated gap junction communication [ ] [ ] [ ] [ ] [ ] . our previous studies have demonstrated that mouse hepatitis virus (mhv) infection significantly reduces cx expression and functional gap junction formation in mice brain and in cultured astrocytes [ ] . mhv-a , a neurotropic strain of the coronavirus, causes meningitis and encephalitis in acute infection followed by demyelination and concurrent axonal loss in chronic stage of infection, serving as a model to study certain pathology of virusinduced meningitis and the human cns-demyelinating disease ms [ , ] . although earlier reports suggest that mhv infection causes loss of cx expression and negatively affects gap junction communication in the brain, particularly in astrocytes, at present, very little is known about virus-induced alterations of cx channels in meningeal fibroblasts. this is particularly important in injuryinduced brain parenchymal reaction, where gjs in the astrocyte-fibroblast interface play an important role in reforming glial limitans that forms a part of the bbb. moreover, cx proteins of bbb have been shown to regulate bbb permeability in inflammatory conditions [ ] . in the present study, we investigated the expression and cellular distribution of cx in mhv-a -infected mouse brain meningeal cells. in parallel, we have established an in vitro model of viral meningitis by infecting fibroblast-enriched primary meningeal cultures with mhv to understand the effect of viral infection on cx -mediated gap junction communication in meningeal fibroblasts. our findings provide important insights in understanding the role of glial-pial gjic in altered barrier function of the inflamed brain. virus a neurotropic demyelinating strain of mouse hepatitis virus, mhv-a , was used from our previous studies [ ] , for infecting mice and primary meningeal cultures to study the effect on gap junction protein cx . four-week old c bl/ mice were intracranially inoculated with % ld dose of a neurotrophic demyelinating strain of mouse hepatitis virus, mhv-a ( pfu) as described previously [ ] . mice were monitored daily for symptoms of disease and mortality. mock-infected controls were inoculated similarly with the vehicle containing phosphate-buffered saline (pbs) containing . % bovine serum albumin (bsa) and maintained in parallel. all animals were euthanized at day post-infection (p.i.) and brain tissues were harvested as described below. all experimental procedures were approved by the institutional animal ethical committee and committee for the purpose of control and supervision on experiments on animals (cpcsea, india). mice were perfused transcardially with pbs followed by pbs containing % paraformaldehyde (pfa) at day p.i. brain tissue was collected, post-fixed in % pfa overnight, and embedded in paraffin [ ] . brains were sectioned at μm and stained with hematoxylin and eosin (h&e) to evaluate inflammation or meningitis. the paraffin-embedded slides were first deparaffinized on a hot plate followed by xylene treatment for mins. sections were then rehydrated gradually through different alcohol grades from to %. sections were then treated with hematoxylin for min and excess stain was washed under running tap water. subsequently, sections were dehydrated by passing through to % ethanol. sections were then stained with eosin stain for s and washed with % ethanol twice. further dehydration was carried out with % ethanol and xylene. sections were mounted with refrax mounting medium (anatech ltd., mi, usa) and observed under the upright light microscope (nikon eclipse i) and analyzed with nikon imaging software (nis, nikon corp. tokyo, japan). mock and mhv-infected mice at day p.i. were transcardially perfused and fixed with % pfa. brain tissues were further post-fixed in % pfa overnight and isotonically equilibrated in % pbs sucrose. tissues were embedded in oct cryomatrix (tissue tek, hatfield, pa) and sagittal sections ( μm) were done on cryostat (thermo scientific). tissue sections were mounted on charged slides and processed as described earlier [ ] . briefly, sections were incubated with m glycine in pbs for h at room temperature (rt) to reduce non-specific cross-linking and subsequently treated with mg/ml nabh in pbs for min at rt to reduce autofluorescence. sections were washed in pbs and incubated with blocking serum containing pbs with . % triton x- and % normal goat serum. sections were incubated overnight at °c with anti-cx in combination with either anti-vimentin or anti-viral nucleocapsid (n) antisera prepared in blocking serum at dilutions listed in table . the following day, the sections were washed and incubated in appropriate fitc or texas red conjugated fluorescent secondary antibody ( : ; jackson immunoresearch inc., pa, usa) for h at rt. all incubations were done in humid chambers. slides were washed and mounted in vectashield® mounting media containing dapi (vector laboratories, ca, usa). sections were visualized and imaged using a zeiss® confocal microscope (lsm ; carl zeiss ag, germany). primary meningeal cultures from day - c bl/ mouse pup brains were prepared as described earlier, with minor modifications [ ] . briefly, the meninges were carefully removed, homogenized in hank's balanced salt solution (hbss), and passed through a -μm cell strainer. the resulting flow-through were collected and centrifuged at rpm for mins to pellet the meningeal cells. the cell pellet was washed twice with hbss and re-suspended in growth medium containing dulbecco's minimal essential medium (dmem) supplemented with % fetal bovine serum (fbs), % l-glutamine, and % penicillin-streptomycin. the cells were plated and allowed to grow at °c in a humidified co incubator. after h, all non-adherent cells were removed and fresh medium was added. adherent cells were maintained as meningeal fibroblast culture in growth medium till confluency with a media change every - days. primary cultures of mixed glia from newborn (day - ) c bl/ mouse pup brains were prepared as described earlier [ ] . briefly, the meninges were removed and brain tissue was minced and incubated in a shaking water bath at °c for min in hbss containing mg/ml dnase i (sigma®) and . % trypsin (sigma®). enzyme dissociated cells were triturated with . % of fbs, washed and pelleted at ×g for min. the pellet was resuspended in hbss and passed through a -μm strainer and centrifuged at ×g for min. the pellet was resuspended in astrocyte-specific medium (dmem containing % l-glutamine, % fbs, and % penicillin-streptomycin), plated in t flasks, and allowed to grow at °c in a humidified co incubator. after h, all non-adherent cells were removed and fresh astrocyte-specific medium was added. adherent cells were allowed to grow to confluence with a media change every to days. confluent cultures were trypsinized and washed twice in flow buffer (pbs containing % fbs). following centrifugation ( ×g, min), the cell pellet was resuspended in flow buffer and approximately × cells were distributed per polystyrene round-bottom -ml flow tubes. cells were fixed in μl cyto-fix buffer (bd biosciences, san jose, ca) for min at rt. following a wash in flow buffer, cells were incubated with anti-vimentin and anti-glial fibrillary acidic protein (gfap) antibody diluted ( : ) in × bd perm/wash buffer for mins at rt. cells were washed, centrifuged, and labeled with goat anti-mouse tritc and goat anti-rabbit fitc diluted ( : ) in bd perm/wash buffer and incubated for mins at rt. after a final wash, all tubes were resuspended in ml of flow buffer and subjected to flow cytometry in a bd facs verse™ flow cytometer and analyzed using bd facsuite™ software [ ] . primary meningeal fibroblasts were infected with mhv-a for investigating the effect of virus infection on meningeal gap junction protein cx , following previously published protocols [ ] . in brief, meningeal cultures were infected with mhv-a diluted in inoculation medium (dmem containing % fbs and % penicillin-streptomycin) at a multiplicity of infection of and incubated for h at °c in a humidified co chamber. after initial viral adsorption for h, medium was changed and infected cells were maintained in normal growth medium for h. for mock infection, parallel cultures were initially incubated in the inoculation medium for h followed by normal growth medium. all cells were collected h p.i. and used for immunolabeling and biochemical assays. immunofluorescence staining of primary meningeal fibroblasts was performed as described previously [ ] , with minor modifications. in brief, cells fixed with % pfa for mins at rt were permeabilized in pbs containing . %triton x- and incubated in blocking serum (pbs containing . %tritonx- and . % goat serum) for h. cells were incubated for h at rt with a combination of anti-cx , antivimentin, anti-n anti-gfap, and anti-calnexin antisera at dilutions listed in table membranes were subsequently re-probed with γactin antibody and densitometric analysis of immunoreactive bands was carried out using imagej software. to evaluate gap junction coupling, meningeal fibroblasts were grown on -mm culture dishes to % confluency. permeability mediated by gap junctions under control and mhv-a -infected conditions ( h p.i.) was determined by gap junction permeable lucifer yellow (ly) dye transfer experiment, using a protocol described previously [ ] , with minor modifications. ly dye (sigma, saint louis, mo, usa) dissolved in pbs ( mg/ml) was scrape loaded onto confluent monolayer of meningeal fibroblasts. after min of incubation, ly was thoroughly washed with pbs, fresh % dmem was added, and dye spreading from scrape line was imaged using an olympus ix- microscope system appended with a hamamatsu orca- ccd camera. images were processed and analyzed using imagepro (media cybernetics) and imagej software and the distance of dye spread from the scrape-loading line was measured. cells plated on coverslips were washed with pbs and treated with %triton x- at °c for mins to solubilize the cx molecules not involved in gap junction plaques while the cx molecules involved in cell surface gap junction plaques remain insoluble. upon thorough washing and extraction of solubilized cx molecules, cells were fixed and immunofluorosence labelling was performed as described previously [ ] . cells were visualized in axio observer microscope with apoptome module (carl zeiss, germany) and images acquired and processed with zen (carl zeiss ag, germany) software. data are presented as mean ± sem. significance of the difference between two experiment groups for the ly dye transfer assay and western blotting was determined by unpaired two-tailed student's t test. the data from cell enrichment experiment were analyzed by one-way analysis of variance, and pairwise comparisons were made using the post hoc tukey method for multiple testing. statistical significance was set at p < . . all statistical analysis were carried out using graphpad prism (ver. ) software (graphpad software, inc). four-week old c bl/ mice were intracranially inoculated with mhv-a or mock-infected with pbs-bsa as controls. mice were sacrificed on day p.i., the time point previously reported to result in peak cns inflammation [ ] . paraffinembedded brain sections were stained with hematoxylin and eosin to visualize inflammation in different brain regions (fig. a-f ). the mock-infected mouse brain sections showed no signs of inflammation and exhibited intact meningeal layer lining the cortex and choroid plexus in the ventricular spaces ( fig. a-e) . in contrast, acute meningitis and inflammatory tissue damage was evident in the corresponding brain regions from mhv-a -infected mice (fig. b-f ). previous studies suggested that the meningeal cells express high levels of cx and are tightly coupled by gap junctions both in developing and adult brain [ ] . moreover, cxmediated gap junction communications are known to be affected in several cns-associated injuries and pathologies. therefore, to investigate the effect of viral infection on cx expression and distribution in the infected meningeal cells of mhv-a -infected mouse brains, we performed doubleimmunolabeling with a combination of anti-cx and antiviral nucleocapsid (n; fig. a-l) or anti-vimentin antibody (fig. a-l) . our results clearly showed that mhv-a infection altered the expression pattern of cx in infected meningeal cells. interestingly, we observed a marked downregulation in the overall cx staining in infected and vimentinpositive cells of the meninges (fig. a-f, fig. a-f ), choroid plexus ( fig. g-l) , and perivascular region (fig. g-l) in infected brains compared to controls. additionally, while the control brain sections showed a characteristic punctate distribution of cx (fig. b, h) , the infected and vimentin-positive meningeal fibroblasts in the infected mouse brains showed loss of its characteristic punctate staining (fig. e, k) . to determine the mechanisms by which viral infection can modulate cx -mediated gap junction communication particularly in the meningeal cells, we established a complimentary in vitro model of primary meningeal cultures infected with mhv-a . purity of the primary cultures was first determined by flow cytometry, using vimentin as fibroblast marker [ ] and gfap as astrocyte marker (fig. a) . viable cell population (p ) was normalized for each marker expression with isotype controls. as expected, majority of the cells ( . %) in our established cultures were vimentin-positive meningeal fibroblasts. in addition, a small population ( . %) of the cells were found to be gfap-positive, and a few ( . %) were positive for both gfap and vimentin and probably represents stem/progenitor cells with a potential to differentiate to either astrocytes or fibroblasts at a later stage in culture. supporting this, our double-immunostaining of primary meningeal cultures with anti-gfap and antivimentin antibodies also showed that most of the cells in cultures expressed vimentin only (fig. b, arrows) . however, a few gfap-positive and gfap and vimentin dual positive cells were also observed. visual counting of the cells from three independent cultures (with different fields from each set) demonstrated that about . % cells were positive for vimentin only, while . % cells expressed gfap only and . % stained for both markers (fig. c) . additionally, we also quantified the vimentin expression in our primary meningeal cultures. our immunoblotting experiments clearly showed that the primary meningeal cultures expressed a significantly higher level of the fibroblast marker vimentin compared to mixed glial cultures demonstrating the enrichment for meningeal fibroblasts in the primary cultures (fig. d) . to determine if viral infection can alter cellular expression/ distribution of the cx in primary meningeal fibroblasts, we infected the primary meningeal cultures with mhv-a , a neurotropic demyelinating strain of mouse hepatitis virus [ ] . at h p.i., cells were double-labeled with a combination of anti-cx and anti-n or anti-vimentin antibodies (fig. a-l) . in the mock-infected meningeal fibroblast cultures, vimentin positive cells expressed profuse cx at the cell surface, demonstrated by its characteristic punctate staining (fig. a -c, g-i; arrow and inset). interestingly, in mhv-a -infected cultures, vimentin-positive fibroblasts displayed cx staining predominantly localized in the intracellular compartments with a typical perinuclear distribution ( fig. d -f, j-l; arrow and inset). interestingly, most of these intracellular cx puncta in mhv-infected cultures was found to co-localize with the endoplasmic reticulum marker, calnexin ( fig. a-f ). earlier studies have shown that mhv-a infection can reduce cx protein levels in the cns and mouse primary astrocytes [ ] . moreover, our immunofluorescence studies also suggest a possible reduction in total cx levels in vimentinlabeled cells of virus-infected mouse brains and altered cellular distribution in primary meningeal fibroblasts. to quantify the observed change, we measured total cx levels in primary meningeal cultures infected with mhv-a . cx levels were found to be significantly downregulated in the infected cultures compared to control (− . ± . %; p < . ) ( fig. a; lower panel) . to determine if reduced cx expression together with its increased intracellular retention affected functional gap junction communication in infected meningeal cells, we performed ly dye transfer in a scrape-loading assay. cx channels are permeable to ly, a small molecular dye that moves from cells to the neighboring cells via gap junctions [ ] . primary meningeal fibroblasts were either mockinfected or infected with mhv-a and gap junction activity was assayed by scrape-loading dye transfer h p.i. mhv-a infection prevented cultured meningeal fibroblasts from transferring the dye to neighboring cells, as the distance of dye spread beyond the injured cells at the scratch boundary was significantly less compared to the control cultures (fig. b) . control meningeal fibroblasts exhibited active gap junction communication, while infected fibroblasts displayed less gap junction communication, as evident from shorter dye travel distance (control: . ± . μm vs. infected: . ± . μm; p < . ) (fig. b; lower panel) . musil and goodenough demonstrated that cx assembled into the gap junction plaques are resistant to solubilization by % triton x- at °c, while cx monomers are mostly solubilized under such conditions [ ] . in order to understand the assembly of cx into plaques, mock-and mhv-a -infected cells were treated in situ with % triton x- at °c, h p.i., and double immunofluorescence labelling was performed for anti-cx and anti-vimentin (fig. a-i) or anti-n (fig. j-l) . interestingly, triton x- solubilization resulted in a marked decrease in cx staining in mock- infected cultures compared to non-triton x- -treated experimental controls (fig. a-f ). this might be because intracellular soluble monomers of cx were extracted by triton x- treatment leaving behind the cx puncta that were assembled in gap junction plaques and resistant to triton x- solubilization. in contrast, mhv-infected meningeal fibroblasts showed mostly intracellular accumulation of cx in the perinuclear region (fig. g-l; arrows in h, i, k, and l). this might be due to aggregation of misfolded protein or increased amount of prematurely oligomerized and triton x- -resistant cx that failed to traffic to the cell surface to form gap junction plaques. using mouse brain slices and cultured primary meningeal fibroblasts, we show that mhv-a can directly infect meningeal cells in vivo in mice as well as in culture conditions. our results reveal that (i) mhv infection reduces total cx protein levels in primary meningeal fibroblasts, (ii) majority of cx in infected, viral antigen-positive fibroblasts show intracellular perinuclear staining, both in vivo and in vitro, (iii) mhv infection causes retention of triton x- insoluble intracellular aggregates of cx which may not be able to assemble into gap junctions, and (iv) the observed alterations in cx expression and cellular distribution is associated with loss of functional gap junction communication between fibroblasts in primary culture as demonstrated by ly scrapeloading and dye transfer assay. taken together, our results suggest that mhv-a infection can directly affect expression and intracellular distribution of cx in meningeal fibroblasts which might play an important role in altered cns homeostasis during acute infection. earlier studies suggest that mhv hijacks host translational machinery to produce its own proteins during viral multiplication thereby arresting the synthesis of large number of host cellular proteins [ ] . virus-induced host translational shutdown and mrna decay can affect molecules particularly with shorter half-life such as cx [ , ] resulting in reduced cx levels. in addition, it has been shown that er stressmediated unfolded protein response can result in downregulation of cx expression at both mrna and protein levels [ ] . thus, it is also possible that mhv-induced er stress can lead to accumulation of misfolded cx in the perinuclear compartments, subsequently leading to downregulation of fig. confocal images showing reduced cx punctate staining in vimentin-positive fibroblasts in the meninges and perivascular region (a-l). confocal images of -μm thin brain cryosections double-immunolabeled with antivimentin (red) and anti-cx (green) showing reduced cx staining in the inflamed cortical meningeal layer and perivascular region of mhv-a -infected (df, j-l) mouse brains compared to mock-infected controls (a-c, g-i). sections were counterstained with dapi (blue) to visualize nuclear localisation. meningeal fibroblasts in the cortical and perivascular region of mhvinfected brains (d-f, j-l: arrow) depict reduction in cx immunostaining compared to respective mock-infected brain regions (a-c, g-i: arrow). insets in f and l demonstrate reduced cx immunoreactivity in a magnified view of fibroblast. note the higher intensity of cx staining is noted in vimentin-positive fibroblasts in representative brain images from the mock-infected cortical meningeal layer and perivascular region (a, c and g, i) cx expression and reduced trafficking to cell surface. this is further supported by our triton x- solubilization experiment showing that infected cells have increased intracellular accumulation of cx in the perinuclear region that was not extracted by triton x- treatment. in contrast, the control cells showed cx puncta only at the cell surface present in gap junction plaques and no intracellular monomers that were supposedly removed by the triton x- . neurovirulent strains of mhv are preferentially transported by microtubules in neurons [ ] . interestingly, cx is also known to directly bind to microtubules [ ] and transported to the cell surface along microtubules as conduit [ ] . thus, in infected cultures (d-f), cx puncta are mostly seen in the perinuclear region which colocalize with er marker calnexin (f; arrows). in control cells (a-c), most of the cx staining is observed towards the periphery (c; arrowheads) with only a few cx puncta seen to colocalize with calnexin (c; arrow). cells were counterstained with dapi (blue) to visualize nuclear localization in merged images virus transport using the cellular microtubule network might possibly hinder microtubule-dependent cx trafficking to the cell surface. in this regard, our previous studies have shown that depolymerization of the microtubule network by colchicine in primary astrocytes can lead to similar retention of cx in the perinuclear compartments as in virus-infected cells [ ] . thus, it is possible that virus-specific utilization of the microtubules in the infected meningeal fibroblasts resulted in reduced trafficking and assembly of cx subunits into gj plaques causing loss of gap junctional communication. nevertheless, further studies are needed to understand the underlying regulatory mechanisms of cx association with the microtubule network in infected meningeal cells and its oligomerization into gap junction plaques. impaired gap junction communication in meningeal fibroblasts can contribute to disruption of functional link to the neural tissue. it is reported that meningeal cells can communicate with astrocytes via ca + signaling [ ] . interestingly, ablation of astrocytic cx has been shown to cause nearly complete depletion of cx immunoreactivity in leptomeninges [ ] . moreover, the meninges are important in maintaining the functional coupling among the glia limitans astrocytes, which are more coupled than the astrocytes in the molecular layer [ ] . during injuryinduced parenchymal reaction, reactive astrocytes and the meningeal cells form a glia-fibroblast interface that forms new basal lamina which along with astrocytic endfeet reforms the glial limitans [ , ] . this process is crucial for restoring the bbb function and re-establishing cns homeostasis. thus, cx channels play an important role in maintaining bbb function, and loss of these junctional communications might affect bbb permeability. the effect of cx on bbb may be mediated via tight junction proteins such as claudin, occludin, and zona occludens (zo- ). there are reports of cx interacting with tight junction protein zo- through its carboxyl terminus [ ] . the pdz domain of zo- serves to recruit several signaling molecules and also provides anchorage to several cytoskeletal elements. moreover, the dynamic association of zo- with cx also modulates the stability of gap junction channel and regulates its internalization and endosomal turnover [ ] . zo- and other tight junction proteins are reported to be downregulated in bbb leakage during different pathological conditions [ ] . hence, the virus-induced retention of cx might alter the stability of membrane resident tight junction proteins and disrupt the bbb integrity. increased permeability of the bbb is a pathological hallmark in several neurological disorders such as multiple sclerosis, bacterial meningitis, and neurotropic virus infections including human immunodeficiency virus, measles virus, and japanese encephalitis virus [ ] [ ] [ ] . experimental intracranial inoculation with mhv might increase bbb permeability, leading to rapid viral dissemination and enhanced transmigration of activated immune cells into the brain. although the homing mechanism of immune cells and molecular mechanisms of bbb disruption in mhv infection are not well understood, viral infection-associated loss of cx-mediated intercellular communication might play an important role in altered bbb permeability and altered cns homeostasis during neuroinflammation. in summary, we have shown that mhv infection in brain can reduce cx expression in meningeal fibroblasts and induce intracellular retention of cx , which might contribute to reduced gap junction communication. such loss of junctional communication might have a potential role in modulating coupling between fibroblasts of the meningeal layer and at fibroblast-astrocyte interface of glial limitans superficialis subsequently leading to altered bbb function. council for scientific and industrial research (csir), india. mm is a recipient of young scientist award from science and engineering research board (serb), india. compliance with ethical standards all animal experiments were performed following the approved guidelines of the institutional animal ethical committeee and committee for the purpose of control and supervision on experiments on animals (cpcsea, india). the authors declare that they have no conflict of interest. control cells not subjected to triton x- solubilization (−triton x- ; a-c), exhibited characteristic punctate cx immunostaining (b, c; arrow). upon triton x- solubilization (+tritonx- ; d-f), mockinfected control cells displayed a few cx puncta representative of triton x- -insoluble gap junction plaques at the cell surface (e, f; arrow). in contrast, vimentin-positive primary fibroblasts in mhv-a infected cultures (g-i) displayed increased immunostaining for cx mostly in the perinuclear regions following triton x- solubilization (+triton x- ) suggesting intracellular cytosolic aggregates of cx (h, i; arrow). anti-n staining illustrating that most of these intracellular cytosolic aggregates of cx noted in +triton x- condition are in mhv-infected cells (j-l). cells were counterstained with dapi (blue) to visualize nuclear localization meninges: from protective membrane to stem cell niche microscopic morphology and histology of the human meninges the astrocyte/meningeal cell interface is a barrier to neurite outgrowth which can be overcome by manipulation of inhibitory molecules or axonal signalling pathways a new in vitro model of the glial scar inhibits axon growth endothelin- regulates glucose utilization in cultured astrocytes by controlling intercellular communication through gap junctions intercellular calcium signaling via gap junctions in glioma cells calcium waves in astrocytes-filling in the gaps hepatocyte gap junctions are permeable to the second messenger, inositol , , -trisphosphate, and to calcium ions immunohistochemical 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astroglial coupling in mptplesioned, fgf- -treated parkinsonian mice phosphorylation of connexin gap junction protein in uninfected and rous sarcoma virus-transformed mammalian fibroblasts the bovine papillomavirus type e protein binds to ductin and causes loss of gap junctional intercellular communication in primary fibroblasts viral regulation of aquaporin , connexin , microcephalin and nucleolin persistent borna disease virus infection changes expression and function of astroglial gap junctions in vivo and in vitro hiv increases the release of dickkopf- protein from human astrocytes by a cx hemichannel-dependent mechanism mouse hepatitis virus infection remodels connexin -mediated gap junction intercellular communication in vitro and in vivo experimental demyelination produced by the a strain of mouse hepatitis virus a mechanism of virus-induced demyelination a new angle on blood-cns interfaces: a role for connexins? demyelination determinants map to the spike glycoprotein gene of coronavirus mouse hepatitis virus microglia play a major role in direct viral-induced demyelination cocultures of meningeal and astrocytic cellsa model for the formation of the glial-limiting membrane magnetic cell sorting: a fast and effective method of concurrent isolation of high purity viable astrocytes and microglia from neonatal mouse brain tissue multimeric connexin interactions prior to the trans-golgi network formation of a distinct connexin phosphoisoform in mitotic cells is dependent upon p cdc kinase regulation of connexin -protein binding in astrocytes in response to chemical ischemia/hypoxia lucifer yellow-an angel rather than the devil biochemical analysis of connexin intracellular transport, phosphorylation, and assembly into gap junctional plaques mouse hepatitis coronavirus replication induces host translational shutoff and mrna decay, with concomitant formation of stress granules and processing bodies hys- , a novel analogue of combretastatin a- , enhances connexin expression and gap junction intercellular communication in rat astrocytes two tyrosine-based sorting signals in the cx c-terminus cooperate to mediate gap junction endocytosis downregulation of gap junction expression and function by endoplasmic reticulum stress effect of microtubule disruption on neuronal spread and replication of demyelinating and nondemyelinating strains of mouse hepatitis virus in vitro gap junction protein connexin- interacts directly with microtubules connexin modulates cell polarity and directional cell migration by regulating microtubule dynamics meningeal cells can communicate with astrocytes by calcium signaling meningeal cells increase in vitro astrocytic gap junctional communication as measured by fluorescence recovery after laser photobleaching interactions between meningeal cells and astrocytes in vivo and in vitro ephrin-b and ephb regulation of astrocyte-meningeal fibroblast interactions in response to spinal cord lesions in adult rats the gap junction protein connexin interacts with the second pdz domain of the zona occludens- protein connexin gap junction plaque endocytosis implies molecular remodelling of zo- and c-src partners enhancement of blood-brain barrier permeability and reduction of tight junction protein expression are modulated by chemokines/cytokines induced by rabies virus infection blood-brain barrier disruption in multiple sclerosis viral disruption of the blood-brain barrier defense at the border: the bloodbrain barrier versus bacterial foreigners acknowledgements authors thank iiser-k confocal facility and mr. ritabrata ghosh for his technical assistance with microscopy. key: cord- -vft v authors: thackray, larissa b.; turner, brian c.; holmes, kathryn v. title: substitutions of conserved amino acids in the receptor-binding domain of the spike glycoprotein affect utilization of murine ceacam a by the murine coronavirus mhv-a date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: vft v the host range of the murine coronavirus (mhv) is limited to susceptible mice and murine cell lines by interactions of the spike glycoprotein (s) with its receptor, mceacam a. we identified five residues in s (s , l , t , y and k ) that are conserved in the receptor-binding domain of mhv strains, but not in related coronaviruses. we used targeted rna recombination to generate isogenic viruses that differ from mhv-a by amino acid substitutions in s. viruses with s r and k r substitutions had wild type growth, while l a/t a viruses formed small plaques. viruses with s g, l m/t m or k g substitutions could only be recovered from cells that over-expressed a mutant mceacam a. viruses with y h or y q substitutions were never recovered, while y a viruses formed minute plaques. however, viruses with y f substitutions had wild type growth, suggesting that y may comprise part of a hydrophobic domain that contacts the mhv-binding site of mceacam a. viruses of the coronaviridae family form three distinct antigenic and phylogenetic groups (lai and holmes, ) . coronaviruses in group ii infect mice, rats, cattle or humans, as well as several other host species. only one cellular glycoprotein has been identified as a receptor for a group ii coronavirus. the murine coronavirus [murine hepatitis virus (mhv)] utilizes as receptors murine carcinoembryonic antigen cell adhesion molecule a (mceacam a) and related murine glycoproteins in the cea family of glycoproteins in the immunoglobulin (ig) superfamily (dveksler et al., a (dveksler et al., , b yokomori and lai, ) . the envelope of most group ii coronaviruses contains a hemagglutinin esterase (he) glycoprotein that binds to -oacetylated neuraminic acid, but the a strain of mhv (mhv-a ) does not express he (yokomori et al., ) . thus, mhv-a infection of susceptible mice and murine cell lines depends solely on interactions of the viral spike glycoprotein (s) with mceacam a and related murine glycoproteins (hemmila et al., ) . although the variety of hosts infected by coronaviruses illustrates that these viruses can emerge in a new host species, the mechanisms required for the introduction of a coronavirus into a new host are not well understood. the interaction of the viral spike glycoprotein with a specific cellular glycoprotein receptor is a major determinant of coronavirus host range. cell lines from host species that are normally resistant to mhv, porcine coronavirus [transmissible gastroenteritis virus (tgev)] or human coronavirus strain e (hcov- e) are rendered susceptible to infection by transfection with cdna encoding the specific coronavirus receptors mceacam a, porcine aminopeptidase n (papn) (delmas et al., ; dveksler et al., ; yeager et al., ) . the -kda s of mhv is a type i viral fusion protein that mediates both receptor binding and membrane fusion activities (bosch et al., ; gallagher and buchmeier, ) . s of mhv-a is post-translationally cleaved by a cellular protease into kda s and s proteins that remain non-covalently associated on the viral envelope. variations in the amino acid (aa) sequence of s of mhv strains are associated with differences in tissue tropism and pathogenesis (leparc-goffart et al., ; phillips et al., ) . the membrane-anchored s contains a coiled-coil domain that is likely associated with membrane fusion and is more highly conserved among mhv strains than s (bosch et al., ) . mhv and the blocking anti-mceacam a monoclonal antibody, mab-cc , bind to the amino (n)-terminal ig-like domain of mceacam a (dveksler et al., a (dveksler et al., , b . binding of the n-terminal domain of soluble mceacam a to s on mhv virions at c induces conformational changes in s and s proteins, and neutralizes virus (gallagher, ; lewicki and gallagher, ; matsuyama and taguchi, ; miura et al., ; zelus et al., ) . incubation of purified mhv-a virions with soluble mceacam a at neutral ph, or without receptor at ph . , triggers major conformational changes in s . these conformational changes may expose a hydrophobic domain in s that allows virus to bind liposomes and presumably initiates fusion of the viral envelope with host cell membranes, as well as cell-to-cell fusion. mutational analyses of determinants in mcea-cam a that alter mhv binding and entry implicated residues in the predicted ccv loop and the cv h sheet in the n-terminal domain of mceacam a (rao et al., ; wessner et al., ) . the predicted ccv loop was also shown by mutational analyses to be critical for recognition of human ceacam by bacterial pathogens such as neisseria, as well as for homophilic cell adhesion (bos et al., ; virji et al., ; watt et al., ) . the crystal structure of mceacam a [ , ] showed that the ccv loop has a convoluted conformation unlike other ig superfamily glycoproteins (tan et al., ) . the side chain of an isoleucine at residue (i ) projects upwards away from the membrane and was predicted to play an important role in mceacam a recognition by mhv s proteins. the structural characterization of s is limited to fusion cores of s proteins of mhv and severe acute respiratory syndrome coronavirus (sars-cov) (bosch et al., ; liu et al., ; tripet et al., ; xu et al., ) . domains of s that are responsible for receptor binding have been identified for several coronaviruses. aa - of s of hcov- e comprise a minimal receptor binding domain (rbd) for hapn expressed on cell membranes breslin et al., ) , while aa - of s of mhv (s ) comprise the minimal domain for binding to soluble mceacam a in vitro and initiating infection via anchored mceacam a (kubo et al., ; tsai et al., ) . aa - of s of sars-cov comprise a minimal domain for binding to angiotensin converting enzyme (ace ) in vitro (babcock et al., ; li et al., ; wong et al., ; xiao et al., ) . recently, we demonstrated that the n-terminal region of s containing aa substitutions and a -aa insert derived from mhv/bhk, a virus variant generated during persistent mhv-a infection of murine cells, is sufficient to extend the host range of mhv-a in vitro (sawicki et al., ; schickli et al., schickli et al., , thackray and holmes, ) . residues critical for receptor utilization and host specificity have not been identified for most coronaviruses, although several residues that play a role in the interactions of the mhv s protein with mceacam a or of the sars-cov s protein with ace have been identified (saeki et al., ; suzuki and taguchi, ; wong et al., ) . with the emergence of sars-cov in humans and the search for effective drugbased interventions for coronavirus infection, the need to identify residues critical for the interactions of coronaviruses with their cellular receptors is greater than ever. we compared the n-terminal aa of s proteins of seven mhv strains with the n-termini of s proteins of the extended host range variant mhv/bhk, as well as related group ii coronaviruses of rats, cattle and humans. we identified five residues that are highly conserved in s of all mhv strains and mhv/bhk, but are not found in s of rat, bovine or human coronaviruses of group ii. to examine the roles of these residues in the receptor specificity of s, we used targeted rna recombination (trr) to generate isogenic viruses that differ from mhv-a at one or two aa in the rbd of s. we showed that y is critical for the recovery of mhv-a from cells expressing mceacam a. we also found that certain aa substitutions at s , l , t or k can inhibit the recovery of mhv-a from murine cells. to identify residues that are involved in the receptor specificity of the s glycoprotein of mhv, we compared the n-terminal aa of s proteins of seven mhv strains with the n-termini of s proteins of related coronaviruses of rats, cattle and humans. we identified four residues, s , t , y and k , that are conserved in s of all mhv strains, but not in the corresponding domains of rat, bovine and human coronaviruses in group ii (fig. a) . another residue, l , is conserved in s of all mhv strains, except mhv , but not in s of rat, bovine or human group ii coronaviruses. these five residues are also conserved in the extended host range variant mhv/bhk that infects a wide range of non-murine cell lines while maintaining the ability to infect murine cells (schickli et al., ; thackray and holmes, ) . to examine the roles of these residues in the receptor specificity of s, we used trr to introduce single aa substitutions at s , y or k and double aa substitutions at l and t into the genome of mhv-a . the aa substitutions were chosen to reflect residues found in rat, bovine or human group ii coronaviruses, a conservative y to f substitution, or neutral alanine substitutions (fig. a) . although aa substitutions at t or l in s of mhv-jhm were previously associated with reduced mceacam a binding (saeki et al., ; suzuki and taguchi, ) , we observed that t and l were conserved in s of all mhv strains, as well as s of related rat, bovine and human coronaviruses (fig. a) . to examine the roles of these residues in the receptor specificity of s in the context of infectious isogenic viruses, we used targeted rna recombination (trr) to introduce aa substitutions at t or l into the genome of mhv-a . the aa substitutions were chosen to reflect substitutions made previously, s and h for t and l , respectively, (saeki et al., ; suzuki and taguchi, ) or neutral alanine substitutions. donor rnas, transcribed in vitro from pmh constructs, were transfected into feline (fcwf) cells that had been inoculated with the chimeric helper virus fmhv (kuo et al., ) . rna recombination occurs between fmhv, which contains a chimeric s gene with the ectodomain of feline infectious peritonitis virus and the rest of the mhv-a genome, and the pmh donor rna containing the vmost . kb of the mhv-a genome (kuo et al., ) . the infected and transfected fcwf cells were immediately overlaid onto monolayers of murine cl cells to select for isogenic recombinant viruses that had gained the ability to infect murine cells. for each mutant pmh construct, three recombinant viruses (a, b and c) were independently recovered and plaque-purified to control for adventitious mutations that might arise during trr. in addition, in every experiment, pmh rna encoding wild-type mhv-a s protein was used to reconstitute wild-type mhv-a virus (sa ) in triplicate. extensive cytopathic effects (cpe) were seen at h in cl cell monolayers overlaid with fmhv-inoculated fcwf cells transfected with pmh rna or pmh rnas encoding s r, t s, t a, y f or k r substitutions. in contrast, cl cell monolayers overlaid with fmhv-inoculated fcwf cells transfected with pmh rnas encoding l h, l a or l a/t a substitutions exhibited less cpe at - h, while cl cell monolayers overlaid with fmhv-inoculated fcwf cells mock-transfected or transfected with pmh rnas encoding s g, l m/t m, y h, y q, y a or k g substitutions exhibited no detectable cpe even after h. most of the recombinant viruses formed uniform plaques on cl monolayers, although recombinant viruses derived from the l h or l a constructs formed clear and opaque plaques (data not shown). several mutant viruses were never recovered from cl cell monolayers (table ) , either because the mutant s proteins interacted inefficiently with mceacam a or because the mutant s proteins were inefficiently incorporated into mhv virions. trr using fmhv-inoculated fcwf cells transfected with pmh rnas encoding s g, l m/t m, y h, y q, y a or k g substitutions was repeated several times, but none of these mutant viruses could be recovered from cl cell monolayers. in order to isolate these crippled viruses with mutant s proteins, we used hamster (bhk) cells stably expressing mceacam a [ , ] containing an i r substitution [bhk + mceacam a(i r)]. these cells expressed -fold more receptor than either cl cells or bhk cells stably expressing wild type mceacam a[ , ], as measured by flow cytometry using anti-mceacam a mab cc (data not shown). bhk + mceacam a(i r) cell monolayers overlaid with fmhv-inoculated fcwf cells transfected with pmh or pmh rnas encoding s r, l m/t m or k g substitutions exhibited extensive cpe at h, while no cpe was detected even at h in cells transfected with pmh rnas encoding y h, y q or y a substitutions. all three recombinant viruses derived from each of the sa , s g, l m/t m or k g constructs formed uniform plaques on bhk + mceacam a(i r) cell monolayers, as well as on cl cells (data not shown). we do not know whether the recovery of the s g, l m/ t m or k r viruses from bhk + mceacam a(i r) cells was facilitated by the high level of receptor expression in these cells or the i r substitution in the n-terminal domain of mceacam a. however, the recovery phenotypes and plaque morphologies shared by the three replicates of each mutant virus suggest that the engineered mutations in s were responsible for the observed phenotypes of these viruses. we further characterized the s g, l m/t m and k g viruses that were subsequently plaque-purified and propagated in cl cells. viruses derived from the y h, y q and y a constructs were not recovered from either cl or bhk + mceacam a(i r) cell monolayers (table ) . trr using fmhv-inoculated fcwf cells transfected with pmh rnas encoding y h, y q or y a substitutions was repeated several times, but these mutant viruses were not recovered from bhk + mceaca-m a(i r) cell monolayers. to examine the infectivity and spread of these crippled viruses with mutant s proteins immediately after trr, we used a pmh -egfp plasmid that contains the enhanced green fluorescent protein (egfp) gene in place of gene (das sarma et al., ) . gene is not essential for replication of mhv-a , since mhv-a egfp has wild type growth in vitro and in vivo. donor rnas, transcribed in vitro from mutant pmh -egfp constructs, were transfected into fcwf cells that had been inoculated with the chimeric helper virus fmhv (kuo et al., ) . the infected and transfected fcwf cells were immediately overlaid in duplicate onto monolayers of cl or bhk + mceacam a(i r) cells. for each mutant pmh -egfp construct, two recombinant viruses (a and b) were independently recovered. in addition, wild-type pmh -egfp rna was used to reconstitute wild type mhv-a egfp virus (sa -egfp) in duplicate. nearly all of the cl or bhk + mceacam a(i r) cells overlaid with fmhv-inoculated fcwf cells transfected with pmh -egfp or pmh -egfp encoding a y f substitution exhibited extensive cpe and strong egfp expression at h (fig. ) . in contrast, less than . % of the cl or bhk + mceacam a(i r) cells overlaid with fmhv-inoculated fcwf cells transfected with pmh -egfp encoding y h, y q or y a substitutions exhibited cpe and egfp expression at h. although y a-egfp viruses induced several large syncytia in bhk + mceacam a(i r) cells, the number of cells infected by y a-egfp viruses decreased between and h (fig. ) . these results suggested that h, q or a substitutions for y that reflect residues found at corresponding residues in s of rat, bovine or human coronaviruses in group ii altered the interaction of mhv-a virions with mceacam a. the detection of the mutant egfp viruses in cl cells prompted us to examine the plaque forming ability of the y h, y q and y a-egfp viruses on cl cell monolayers. both replicates of the sa , y a and y f-egfp viruses formed plaques that exhibited egfp expression in cl cells monolayers (data not shown). both of the y f-egfp viruses formed large, - mm plaques on cl cell monolayers like mhv-a (fig. ) . however, both of the y a-egfp viruses formed minute, . mm plaques that were difficult to visualize with neutral red staining, but were readily apparent using immunoper- oxidase labeling of viral nucleocapsid (n) protein in cl cell monolayers (fig. ) . the yields of the y a-egfp viruses from cl cells were , -fold lower than those of the sa and y f-egfp viruses. consequently, the egfp viruses were not studied further. to identify mutant viruses that were free of adventitious mutations in the s gene, we sequenced the s genes of plaque-purified recombinant viruses derived from mutant pmh constructs. point mutations were found in the s genes of the sa a, s g a and b, l h a and b, l a a and b and l a/t a a viruses ( table ). the s genes of the sa b, s r a, s g c, t s a, t a a, l h c, l a c, l m/t m a, l a/ t a b and k g a viruses were free of adventitious mutations, while the s genes of the y f a and k r a viruses had only non-coding adventitious mutations ( table ). the lack of adventitious mutations in the s genes of the s g c, l m/t m a, b, and c, and k g a, b, and c viruses suggests that second-site mutations in the s gene were not responsible for the observed ability of these viruses to form plaques on cl cells after their recovery from bhk + mceaca-m a(i r) cell monolayers. most of the plaque-purified recombinant viruses released between  and  pfu/ml into the tissue culture medium by h p.i., except for all three replicates of the l a/t a virus that released only  pfu/ml, as determined by plaque assay on cl cell monolayers. to determine whether aa substitutions in the rbd of s altered the growth of mhv-a in murine cells, we examined the plaque morphologies of the plaque-purified recombinant viruses on cl cell monolayers. when examined using neutral red stained cells, the sa , s r, l m/t m, y f and k r viruses formed clear plaques like mhv-a , whereas the s g, t s, t a and k g viruses formed turbid plaques (fig. ) . the plaque-purified l h and l a viruses formed opaque plaques that were difficult to visualize with neutral red staining, but were readily apparent using immunoperoxidase labeling of viral n protein in cl cell monolayers. most of the recombinant viruses formed large, - mm plaques at h like mhv-a , whereas the three l a/t a viruses formed small, . mm plaques (fig. ) . the plaque morphologies shared by the three replicates of each mutant virus indicate that the engineered mutations in s were responsible for the observed phenotypes of these viruses. differences in plaque morphology could be caused by alterations in receptor binding or membrane fusion activities of mutant s proteins on virions. to investigate whether aa substitutions in the rbd of s altered the susceptibility of mhv-a virions to neutralization by soluble mcea-cam a, we incubated the sa b, s r a, s g c, t s a, t a a, l h c, l a c, l m/t m a, y f a, k r a and k g a viruses at neutral ph and c with nm of purified, anchorless mceacam a [ , ] . this concentration of mceacam a[ , ] was -fold higher than the nd for mhv-a , but did not neutralize the extended host range variant mhv/bhk (schickli et al., ; zelus et al., ) . like mhv-a , all of the recombinant viruses were neutralized by mceacam a [ , ] at neutral ph and c (fig. ) .the ability of soluble mceacam a to neutralize the l a/t a b virus was not measured due to the low titer of this virus. the neutralization of the mutant viruses by soluble mceacam a [ , ] at neutral ph and c suggested that the soluble receptor bound to mutant s proteins on the recombinant virions and induced a conformational change in s like that observed for mhv-a (gallagher, ; matsuyama and taguchi, ; zelus et al., zelus et al., , . to further characterize the mceacam a utilization of the mutant viruses, we measured the yields of the sa b, s r a, s g c, t s a, t a a, l h c, l a c, l m/t m a, l a/t a b, y f a, k r a and k g a viruses from cl cells treated with anti-mceacam a mab-cc . mab-cc binds to the nterminal domain of mceacam a using an epitope that overlaps, but is not identical to, the virus-binding site (dveksler et al., a (dveksler et al., , b wessner et al., ) . like mhv-a , none of the recombinant viruses infected cl cells in the presence of mab-cc (fig. ) . thus, aa substitutions at s , t , l , l /t , or k in s did not prevent binding of mhv-a virions to soluble or anchored mceacam a. since some of the aa substitutions in the rbd of s were chosen to reflect residues found in s of related rat, bovine or human coronaviruses in group ii (fig. a) , we examined the ability of the sa b, s r a, s g c, t s a, t a a, l h c, l a c, l m/t m a, l a/ t a b, y f a, k r a and k g a viruses to infect non-murine cells lines that are normally resistant to mhv-a infection. as determined by immunofluorescence labeling of viral n protein and plaque formation, none of the mutant viruses infected rat (rie), human (hrt- g) or hamster (bhk) cells. rie cells were susceptible to infection by rat coronavirus (rcov), hrt- g cells were susceptible to infection by bovine coronavirus and human coronavirus strain oc , while all three cell lines were susceptible to infection by the extended host range variant mhv/bhk (schickli et al., ) . in addition, viruses derived from the y h, y q and y a constructs did not infect rie, hrt- or bhk cell lines. thus, aa substitutions at s , t , l , l /t , y or k did not extend the host range of mhv-a . we used comparative analysis of s proteins from coronaviruses in group ii to identify residues in the receptor binding domain, s , that may be important for the mceacam a specificity of mhv virions. to study the biological significance of aa substitutions in the rbd of s, we used targeted rna recombination (trr) to generate isogenic recombinant viruses that differ from mhv-a at one or two residues in s . aa substitutions were selected by identification of five residues, s , l , t , y and k , that are highly conserved in s of seven mhv strains and the extended host range variant mhv/bhk, but not in s of related group ii coronaviruses of rats, cattle and humans. since mhv-a does not bind or infect rat, bovine or human cell lines and rcov does not bind to or utilize mceacam a (compton et al., ; gagneten et al., ) , we reasoned that these residues might be important for the interactions of mhv-a with mceacam a. in this study, we found that y in the rbd of s is critical for the recovery of mhv-a from cells expressing mceacam a. fcwf cells inoculated with fmhv and transfected with pmh -egfp or pmh -egfp rnas encoding y h, y q, y a or y f substitutions showed limited egfp expression when plated alone (data not shown). however, fmhv-inoculated fcwf cells transfected with pmh -egfp or pmh -egfp rnas encoding y h, y q, y a or y f substitutions that were overlaid onto cl or bhk + mceacam a(i r) cell monolayers showed increased cpe and egfp expression (fig. ) . these data strongly suggested that the majority of cpe and egfp expression observed was due to infection of cl or bhk + mceacam a cells. recombinant viruses with y h and y q substitutions inefficiently infected cl and bhk + mceacam a(i r) cell monolayers (fig. ) and these viruses were not recovered from either cl or bhk + mceacam a(i r) cells (table ) . although viruses with y a substitutions were recovered from cl and bhk + mceacam a(i r) cells, the y a viruses formed minute plaques on cl cell monolayers (fig. ) and had markedly lower yields than mhv-a . in contrast, f substitutions for y were well tolerated by the s proteins of mhv-a , since the y f viruses infected both cl and mceacam a(i r) cells as well as mhv-a (figs. - and ) . these data suggest that the phenyl ring of y may form an essential hydrophobic contact with the mhv-binding domain of mceacam a, and that this hydrophobic contact is disrupted in s proteins with h, q or a substitutions at y . alternatively, it is possible that h, q or a substitutions at y may alter the local conformation or stability of s proteins, since a s g substitution in the s of mhv-jhm is associated with decreased s -s stability (ontiveros et al., ) and a q l substitution in the s of infectious bronchitis virus has been shown to inhibit maturation and incorporation of s proteins into virions (shen et al., ) . additional studies will be needed to determine whether y participates directly in mcea-cam a binding, triggers the fusion activity of s and/or maintains the conformation of the rbd of the mhv s protein. in this study, we found that viruses with s r, l a/ t a or k r substitutions in the rbd of s were readily recovered from cl cells. the s r and k r viruses had wild-type growth in cl cells, while the l a/t a viruses formed small plaques on murine cell monolayers (fig. ) and had reduced viral yields compared to mhv-a . viruses with s g, l m/t m or k g substitutions in s were never recovered from cl cells. however, these viruses could initially be recovered from bhk + mceacam a(i r) cells that express -fold more receptor than cl cells or bhk cells stably expressing wild type mceacam a. the s r, s g, l m/t m, k r and k g viruses were neutralized like mhv-a by soluble mceacam a at neutral ph and c (fig. ). in addition, just like mhv-a , the s r, s g, l m/t m, l a/t a, k r and k g viruses bound mcea-cam a using an epitope that overlaps that of the blocking anti-mceacam a mab-cc (fig. ) . the recovery, growth and plaque phenotypes shared by each independently generated replicate of each mutant virus strongly suggests that the engineered mutations in s , rather than adventitious mutations outside of the s gene, were responsible for the observed phenotypes of these viruses. the failure of the s g, l m/t m and k g viruses to be recovered from cl cells may have been due to impaired interactions between mutant s proteins and mceacam a. during trr, feline fcwf cells infected with fmhv and transfected with mutant mhv-a rnas may generate virions whose envelopes contain both chimeric fmhv s proteins and mutant mhv-a s proteins due to phenotypic mixing. the phenotypically heterogeneous s g, l m/t m and k g virions may have impaired interactions with mceacam a. however, phenotypically homogenous s g, l m/t m and k g virions, generated in bhk + mceacam a cells, may regain the ability to interact with mceacam a on cl cells. alternatively, the high level of receptor expression on bhk + mceacam a(i r) cells may facilitate the recovery of these crippled viruses by increasing the number of receptors available on cell membranes. in a similar manner, increased expression of its cellular receptor partially restores the infectivity of a friend murine leukemia virus with aa substitutions in its receptor binding pocket (davey et al., ) . in addition, the i r substitution in the nterminal domain of mceacam a may facilitate the recovery of crippled viruses with mutant s proteins. several residues in s that may be important for binding to mceacam a have previously been identified. a single t s substitution or a triple t s, y s, y s substitution in soluble s of mhv-jhm reduced binding to soluble mceacam a in vitro (suzuki and taguchi, ) . soluble receptor resistant (srr) mutants of mhv-jhm with l h substitutions in s also had reduced binding to soluble mceacam a in vitro (saeki et al., ) . however, these srr mutants had wild-type interactions with anchored mceacam a expressed in hamster cells (matsuyama and taguchi, ) . in this study, we found that isogenic recombinant mhv-a viruses with t s, t a, l h or l a substitutions had wild-type growth in cl cells and were neutralized, like mhv-a , by soluble mceacam a at neutral ph and c (figs. and ) . the t s and t a viruses formed turbid plaques on cl cell monolayers, while the l h and l a viruses formed opaque plaques (fig. ) . thus, although t and l are not essential for mceacam a utilization by mhv-a , these residues may nevertheless influence spike-receptor interactions (rao and gallagher, ) . we noted that t and l are conserved in s of murine, rat, bovine and human coronaviruses in group ii (fig. a) . since mhv-a does not bind rat, bovine or human tissues or infect rat, bovine or human cell lines (compton et al., ) , t and l , individually, most likely do not determine the receptor specificity of mhv. in this study, we demonstrated that single aa substitutions at s , t , l , y or k and double aa substitutions at l and t in the rbd of s of mhv-a did not allow the virus to interact with alternative receptors on murine or non-murine cell lines. like wild type mhv-a , infection of murine cells by viruses with substitutions at s , t , l , l /t , y or k was blocked by anti-mceacam a mab-cc (fig. ) . none of the viruses with substitutions at s , t , l , l /t , y or k infected rat, hamster or human cell lines. perhaps multiple aa substitutions are needed to change the host range of mhv-a . we recently showed that aa substitutions and a -aa insert in the n-terminal region of s of mhv-a are sufficient to extend the viral host range (schickli et al., ; thackray and holmes, ) . isogenic recombinant viruses that differ from mhv-a by the aa substitutions and the -aa insert in s not only utilize mceacam a, but also as yet unidentified receptors on murine and non-murine cells . alternatively, residues in the s proteins of rat, bovine and human coronaviruses in group ii critical for receptor binding may not be found in s . an aspartic acid at residue in the rbd of the distantly related sars-cov s protein is essential for binding to ace in vitro (wong et al., ) . the mceacam a-binding site in the mhv s glycoprotein has previously been proposed to be conformationdependent based on the inhibition of mhv infection by conformation-dependent anti-s mabs (suzuki and taguchi, ) . we postulate that y may comprise part of a hydrophobic pocket or patch in the rbd of s that interacts with hydrophobic residues in the mhv-binding site of mceacam a. hydrophobic residues such as y, f and w are known to play important roles in spike-receptor interactions of other enveloped viruses such as herpes simplex virus, human immunodeficiency virus and murine retroviruses (carfi et al., ; connolly et al., ; kwong et al., ; wu et al., ; zhang et al., ) . residues s , t , l , l , t and k are most likely located peripheral to this hydrophobic binding site, since binding hot spots in spike-receptor interactions are often surrounded by energetically less critical residues, predominantly charged in nature (bogan and thorn, ; wang, ) . residues that extend the host range of mhv-a may also be located peripheral to the hydrophobic binding site. during persistent mhv infection in murine cells, the important mceacam a binding residues in s are retained (baric et al., ; schickli et al., ) , while additional aa substitutions may be selected that enhance the affinity or avidity of s for mceacam a and allow mhv to utilize as yet unidentified alternative receptors on murine and nonmurine cells. a mouse monoclonal antibody (mab) to the mhv nucleocapsid protein (n) (anti-n mab) was provided by julian leibowitz (department of pathology and laboratory medicine, texas a and m university, college station, tx). mouse anti-mceacam a mab-cc blocks binding of mhv to mceacam a and infection of murine cells that express mceacam a, such as cl cells (dveksler et al., a (dveksler et al., , b williams et al., ) . a mouse mab directed against the h subunit of cholera toxin (mab-ctrl) was used as an isotype matched control for anti-n mab and mab-cc . the cl cell line of spontaneously transformed balb/c t fibroblasts, rat intestinal epithelial (rie) cells, baby hamster kidney bhk- (bhk) cells, felis catus whole fetus (fcwf) cells, felis catus lung epithelial (ak-d) cells and african green monkey kidney (vero ) cells were propagated as previously described (schickli et al., ; thackray and holmes, ) . human rectal tumor clone g cells (hrt- g) were kindly provided by johannes storz (department of veterinary microbiology and parasitology, louisiana state university school of veterinary medicine, baton rouge, lo) and propagated in dulbecco's modified eagle medium (dmem; gibco, invitrogen corporation, grand island, ny) supplemented with % heat-inactivated fetal bovine serum (fbs; hyclone laboratories, inc., logan, ut), % antibiotic-antimycotic (psf; gibco) and . g/l sodium bicarbonate. to generate bhk cells stably transfected with a murine ceacam a cdna encoding an i r substitution [bhk + mceacam a(i r)], site-directed mutagenesis of mcea-cam a [ , ] in pci-neo (invitrogen) was performed with the mutagenic forward primer vctacggctagagac-aaagaaattg, and reverse primer vcaatttctttg-tctctagccgtag. bhk cells were transfected with cdna encoding the mceacam a(i r) or wild type mcea-cam a construct using lipofectamine (invitrogen), as specified by the manufacturer, and selected using ag/ml of geneticin (gibco). stably transfected bhk cells were sorted twice on a cytomation moflo cell sorter (ft. collins, co) for high levels of mceacam a expression using anti-ceacam a mab-cc followed by phycoerythrin (pe)conjugated goat anti-mouse igg (jackson immunoresearch laboratories, inc., west grove, pa). mhv-a and fmhv were propagated in cl and fcwf cells, respectively, as previously described (kuo et al., ; schickli et al., ) . virus titers were measured by plaque assay on cl , fcwf, ak-d, rie or bhk cells as previously described (gagneten et al., ; kuo et al., ) . virus-inoculated hrt- g cells were incubated under . % seakem agarose (biowhittaker molecular applications, rockland, me) and mem (gibco) with % fbs and % psf. virus inoculation of cells grown on coverslips and detection of newly synthesized viral nucleocapsid (n) protein by immunofluorescence were performed as previously described . virus-inoculated cell monolayers were incubated under agar, and plaques were visualized by neutral red staining or immunolabeling of viral n protein in cell monolayers. cell monolayers were washed with isotonic phosphate buffered saline and fixed in methanol/acetic acid at À c for min. expression of n protein was detected using anti-n mab followed by biotinylated anti-mouse igg and avidin dh/biotinylated horseradish peroxidase (hrp) h complexes (vectastain elite abc kit; vector laboratories inc., burlingame, ca). avidin-hrp complexes were visualized by deposition of , v-diaminobenzidine (dab; vector laboratories inc., burlingame, ca). s sequences of mhv strains and the corresponding domains of other group ii coronaviruses were obtained using the following genbank accession numbers: mhv-a (ay ), mhv-jhm (x ), mhv- (d ), mhv- (d ), mhv- (d ), mhv-s (d ), mhv-u (d ), mhv/bhk (ay ), rcov (aaf ), bcov strain l (p ) and hcov-oc (z ). deduced aa sequences were aligned and five residues that were highly conserved in s of all mhv strains, but not in s of other group ii coronaviruses were identified (fig. a) . the s constructs used in this paper were assembled in pbc sk+ (stratagene, la jolla, ca) (fig. b) and used to replace the s gene of pmh (kuo et al., ) or pmh -egfp (das sarma et al., ) . the transcription vector pmh -egfp, containing the v-most . kb of the mhv-a genome and the gene for enhanced green fluorescent protein (egfp) in place of gene , was kindly provided by susan weiss (department of microbiology, university of pennsylvania school of medicine, philadelphia, pa). s gene sequences in this paper were numbered according to genbank accession number ay (schickli et al., ) . site-directed mutagenesis was used to introduce point mutations into a cdna encoding the s protein, the s/pbc sk+ construct containing an avrii site at nt . mutant s constructs were amplified with cloned pfu dna polymerase (pfu; stratagene, la jolla, ca) using various primer pairs (table ) essentially as previously described (wentworth and holmes, ) . mutant s constructs were screened by restriction enzyme digestion and/or sequence analysis. all s constructs were subcloned into the s gene of pmh using avrii and a draiii site at nt . y h, q, a and f mutant s constructs were also subcloned into pmh -egfp. the v ends of each construct were sequenced by the university of colorado cancer center dna sequencing and analysis core facility using abi prism kits (applied biosystems, foster city, ca) as previously described (wentworth and holmes, ) . six primers: sstart, g d-, a . , a . , a . and a . (schickli et al., ; thackray and holmes, ) were used to generate overlapping sequences for each construct. mutations were introduced into the genome of mhv-a by targeted rna recombination (trr) as previously described . briefly, fcwf cells were inoculated with the chimeric helper virus fmhv, transfected with pmh or pmh -egfp derived donor rnas containing engineered mutations in the s gene, and immediately overlaid onto cl or bhk + mceaca-m a(i r) cell monolayers in individual -cm flasks. after h, culture media were collected, clarified by centrifugation, and flash frozen. independently recovered replicates were plaque-purified and propagated in cl cells. total cellular rna from cl cells inoculated with each of the recombinant viruses was extracted and reverse transcribed as previously described . the cdna was amplified with primers sstart and a . or a . and a(igs) as previously described . amplification products were sequenced by the university of colorado cancer center dna sequencing and analysis core facility as described above. eleven primers: , a . , a . , a .c , s(xbai)+, a . , s(noti)+, a . , a . , a . , and bz [table and (schickli et al., ; thackray and holmes, ) ] generated overlapping sequences for each s gene. virions were incubated with purified, anchorless mcea-cam a[ , ] essentially as described previously (zelus et al., ) . briefly, al of virus ( pfu) were incubated with al of soluble mceacam a diluted in trisbuffered saline with % glycerol (tbs-g) and . mg/ml bovine serum albumin fraction v (bsa) or tbs-g with . mg/ml bsa alone. after incubation for h at c, virus survival was determined by plaque assay on cl cell monolayers. percent virus neutralization was calculated as: À [(number of plaques from virus incubated with mceacam a/number of plaques from virus incubated with buffer alone)  ]. virus inoculation of murine cells in the presence of . mg/ml anti-ceacam a mab-cc or isotype matched mab-ctrl was performed as previously described (schickli et al., ; thackray and holmes, ) . amino acids to of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor persistent infection promotes cross-species transmissibility of mouse hepatitis virus anatomy of hot spots in protein interfaces identification of a receptor-binding domain of the spike glycoprotein of human coronavirus hcov- e homologue scanning mutagenesis reveals cd receptor residues required for neisserial opa protein binding the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex human coronavirus e: receptor binding domain and neutralization by soluble receptor at degrees c herpes simplex virus glycoprotein d bound to the human receptor hvea coronavirus species specificity-murine coronavirus binds to a mouse-specific epitope on its carcinoembryonic antigen-related receptor glycoprotein structure-based mutagenesis of herpes simplex virus glycoprotein d defines three critical regions at the gd-hvea/ hvem binding interface enhanced green fluorescent protein expression may be used to monitor murine coronavirus spread in vitro and in the mouse central nervous system identification of a receptorbinding pocket on the envelope protein of friend murine leukemia virus aminopeptidase-n is a major receptor for the enteropathogenic coronavirus tgev cloning of the mouse hepatitis-virus (mhv) receptor-expression in human and hamster-cell lines confers susceptibility to mhv several members of the mouse carcinoembryonic antigen-related glycoprotein family are functional receptors for the coronavirus mouse hepatitis virus-a mouse hepatitis-virus strain-a and blocking antireceptor monoclonal-antibody bind to the n-terminal domain of cellular receptor interaction of mouse hepatitis-virus (mhv) spike glycoprotein with receptor glycoprotein mhvr is required for infection with an mhv strain that expresses the hemagglutinin-esterase glycoprotein attachment glycoproteins and receptor specificity of rat coronaviruses a role for naturally occurring variation of the murine coronavirus spike protein in stabilizing association with the cellular receptor coronavirus spike proteins in viral entry and pathogenesis ceacam a(À/À) mice are completely resistant to infection by murine coronavirus mouse hepatitis virus a localization of neutralizing epitopes and the receptor-binding site within the amino-terminal amino-acids of the murine coronavirus spike protein retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier structure of an hiv gp envelope glycoprotein in complex with the cd receptor and a neutralizing human antibody coronaviridae and their replication targeted recombination within the spike gene of murine coronavirus mouse hepatitis virus-a : q is a determinant of hepatotropism quaternary structure of coronavirus spikes in complex with carcinoembryonic antigen-related cell adhesion molecule cellular receptors angiotensin-converting enzyme is a functional receptor for the sars coronavirus interaction between heptad repeat and regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors impaired entry of soluble receptorresistant mutants of mouse hepatitis virus into cells expressing mhvr receptor receptor-induced conformational changes of murine coronavirus spike protein n-terminal domain of the murine coronavirus receptor ceacam is responsible for fusogenic activation and conformational changes of the spike protein enhanced virulence mediated by the murine coronavirus, mouse hepatitis virus strain jhm, is associated with a glycine at residue of the spike glycoprotein multiple regions of the murine coronavirus spike glycoprotein influence neurovirulence intracellular complexes of viral spike and cellular receptor accumulate during cytopathic murine coronavirus infections identification of a contiguous -residue determinant in the mhv receptor that controls the level of virion binding to cells identification of spike protein residues of murine coronavirus responsible for receptor-binding activity by use of soluble receptor-resistant mutants persistent infection of cultured-cells with mouse hepatitis-virus (mhv) results from the epigenetic expression of the mhv receptor the murine coronavirus mouse hepatitis virus strain a from persistently infected murine cells exhibits an extended host range the n-terminal region of the murine coronavirus spike glycoprotein is associated with the extended host range of viruses from persistently infected murine cells a single amino acid mutation in the spike protein of coronavirus infectious bronchitis virus hampers its maturation and incorporation into virions at the nonpermissive temperature analysis of receptor-binding site of murine coronavirus spike protein crystal structure of murine sceacam a[ , ]: a coronavirus receptor in the cea family amino acid substitutions and an insertion in the spike glycoprotein extend the host range of the murine coronavirus mhv-a structural characterization of the sars-coronavirus spike s fusion protein core the n-terminal domain of the murine coronavirus spike glycoprotein determines the ceacam receptor specificity of the virus strain critical determinants of host receptor targeting by neisseria meningitides and neisseria gonorrhoeae: identification of opa adhesiotopes on the n-domain of cd molecules protein recognition by cell surface receptors: physiological receptors versus virus interactions homophilic adhesion of human ceacam involves n-terminal domain interactions: structural analysis of the binding site molecular determinants of species specificity in the coronavirus receptor aminopeptidase n (cd ): influence of n-linked glycosylation mutational analysis of the virus and monoclonal antibody binding sites in mhvr, the cellular receptor of the murine coronavirus mouse hepatitis virus strain a purification of the -kilodalton glycoprotein receptor for mouse hepatitis-virus (mhv)-a from mouse-liver and identification of a nonfunctional, homologous protein in mhv-resistant sjl/j mice a -amino acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme kinetic and structural analysis of mutant cd receptors that are defective in hiv gp binding the sars-cov s glycoprotein: expression and functional characterization structural basis for coronavirus-mediated membrane fusion-crystal structure of mouse hepatitis virus spike protein fusion core human aminopeptidase-n is a receptor for human coronavirus- e mouse hepatitis-virus utilizes carcinoembryonic antigens as alternative receptors heterogeneity of geneexpression of the hemagglutinin-esterase (he) protein of murine coronaviruses purified, soluble recombinant mouse hepatitis virus receptor, bgp (b), and bgp murine coronavirus receptors differ in mouse hepatitis virus binding and neutralizing activities conformational changes in the spike glycoprotein of murine coronavirus are induced at degrees c either by soluble murine ceacam receptors or by ph identification of the receptor binding domain of the mouse mammary tumor virus envelope protein the authors are grateful for technical assistance from jason bartsch, jacinta cooper, sonia tusell and mark young. we also thank mk smith and sonia tusell for many helpful discussions.this work was supported by nih grant r -ai- . sequencing of dna samples at the university of colorado cancer dna sequencing and analysis core facility and facs analysis at the university of colorado cancer center flow cytometry core were supported by a nih/nci cancer core support grant (p ca ). key: cord- -bd u udl authors: joseph, jeymohan; grun, james l.; lublin, fred d.; knobler, robert l. title: interleukin- induction in vitro in mouse brain endothelial cells and astrocytes by exposure to mouse hepatitis virus (mhv- , jhm) date: - - journal: journal of neuroimmunology doi: . / - ( ) -g sha: doc_id: cord_uid: bd u udl abstract interleukin- (il- ) induction, as detected by bioassay and northern analysis, was examined in vitro in endothelial cells or astrocytes derived from balb/c (susceptible) or sjl (resistant) mice following exposure to mouse hepatitis virus (mhv- ) or uv inactivated mhv- (uv-mhv- ). in balb/c endothelial cells, up to -fold more il- (> u/ml) was induced, compared to sjl cells which showed a minimal response ( u/ml), relative to basal levels (< u/ml). in contrast, both balb/c and sjl astrocytes showed a substantial il- response to mhv- and uv-mhv- exposure, although a strain difference persisted. despite strain and cell specific differences in released il- , equivalent levels of il- mrna were induced in all cell types following exposure to mhv- or uv-mhv- . mouse hepatitis virus (mhv- , jhm) is a neurotropic coronavirus which causes a spectrum of disease ranging from fatal encephalomyelitis to demyelination in susceptible murine hosts (bailey et al., ) . both direct infection of oligodendrocytes and immune mediated events have been reported to play a role in the pathologic events in the central nervous system (cns) following mhv- infection (knobler et al., (knobler et al., , wang et al., ; williamson et al., ) . the identification of cd ÷ t-cells, nk cells, b-cells and pmn in the cns of mhv-jhm infected mice suggests that immune mechanisms may be playing a role in the virus induced disease process (knobler et al., ; stohlman et al., ; welsh et al., : natuck et al., williamson et al., ; zimprich et al., ) . the role of locally released cytokines in the regulation of immune and inflammatory events in the cns following mouse hepatitis virus infection has not yet been systematically studied. cytokines play a critical role in the modulation of immune and inflammatory events, important in both anti-viral immunity, and the virus induced pathology observed in the cns. endothelial cells and astrocytes in the cns are potential sources of cytokines and have been demonstrated to synthesize interleukin- (il- ) in response to treatment with tumor necrosis factor (tnf), interleukin- (il- ) and lipopolysaccharide (frei et al., ; jirik et al., ; lieberman et al., ; shalaby et al., ; sironi et al., ; beneveniste et al., ) . endothelial ceils and astrocytes in the cns are also readily infected by mhv- and the products released by these cells may be important in regulating immune mediated events in the cns (joseph et al., (joseph et al., , (joseph et al., , . il- was chosen for this study because its release following infection has been demonstrated in other virus model systems (frei et al., (frei et al., , houssiau et al.. ; breen et al., ; nishimoto ct al., . additionally, il- is a multifunctional cytokine with immunoregulatod, effects on b-cells, t-cclls and neutrophil functions (cernetti et al., j : takai et al., ; borish et al., ; lc and vilcck, ) . we demonstrate that il- induction in mhv- exposed endothelial cells or astrocytes is strain dependent. balb/c (mhv- susceptible) derived endothelial cells can produce up to -fold higher levels of il- , and release this earlier than sjl (mhv- resistant), as determined both by bioassay and northern analysis. active infection is not necessary since exposure to uv inactivated mhv- (uv-mhv- ) can also induce il- in these cells. cerebral endothelial cells were isolated from the brains of balb/c (mhv susceptible) and sjl (mhv resistant) mice as previously described (debault et al., ) . endothelial cell lines obtained were maintained in medium with ( % fetal bovine serum and additional supplements that included basal medium eagle (bme) amino acids, bme vitamins, glutamine, bacto-peptone and penicillin-streptomycin. endothelial identity was established by studying the uptake of dii-ac-ldl (biomedical technologies, stoughton, ma, usa) and specific binding of fluorescein labeled griffonia simplicifolia agglutinin (voyta et at., : sahagun et al., . the purity of endothelial cell cultures was greater than %, as determined by these methods. astrocyte cultures were established from balb/c and sjl mice using the previously described method (knobler et al., adapted from mccarthy and devellis ( ) . brain cortices derived from -day mouse embryos were mechanically dissociated through nitex mesh bags ( p.m). astrocytes were cultured in dmem and ham's f- nutrient medium, : , containing % fetal bovine serum. astrocytic identity of these cells was established by their positive reactivity to an antibody directed against glial fibrillary acidic protein. the purity of the astrocyte cultures was greater than %, as determined by this method. endothelial cells or astrocytes grown in t- flasks ( x <' cells/flask) were infected with mhv- or uv-mhv- for h at °c using a multiplicity of infection (moi) of . . this moi reflects the working titer of virus that elicits a cellular response without extensive cytolytic effects based on previous studies (joseph ct al., l c (), ~ ) . mhv- ~as grown to a stock containing x ( ~ pfli/'ml as prcviously described (knobler et al.. ) . uv inactivation of virus (uv-mhv- ) was carried out for rain ( p.w,/cm:) under a spcctrolinc uv-illuminator (fisher scientific, springfield, n j, usa). virus infectivity was tested by plaque assays on l- cclls (knoblcr ct al., to determine if the inactivation was complctc following uv treatment. after a i-h incubation of the cells with mhv- nr uv-mhv- the cultures were washed three times with phosphate buffered saline (pbs). after washcs the cultures were fed with endothelial cell or astrocytc culture medium. supernatants wcre collected on day - aftcr virus exposurc r~r testing in an !t.- bioassay. the proliferation bioassay for il- was performed using an il- dependent b-cell hybridoma (tl tc) (jayaraman et al., . the tl c cells werc cultured at a density of × ( cells/well in the presence of endothelial or astrocyte supernatants collected on day i- after mhv- or uv-mhv- treatment. thc supernatants from mhv- infected cells were uv-inactivated prior to testing for il- in order to prevent virus mediated killing of the il- dependent cell line. on day cells were pulsed with [ h]thymidine for h and then counted in triplicate in a beta counter as a measure of il- induced proliferation. in order to test the specificity of the proliferative response to il- , a neutralizing rat anti-mouse il- monoclonal antibody (genzyme corporation, cambridge, ma, usa) was used. the anti-ll- antibody was used at a concentration of /.tg/m[. four hours after exposure of endothelial cells and astrocytes to mhv- , uv-mhv- or tnf ( u/ml, genzyme, boston, ma, usa), total cellular rna was isolated by immediate solubilization of cells in guanidine hydrochloride by standard procedures (maniatis et al., . the solution was sonicated and centrifuged to remove cellular debris. the rna was ethanol precipitated, phenol extracted and then reprecipitated with ethanol the rna was quantified by absorbance at nm and by visual estimation of ribosomal rna content by ethidium bromide staining following electrophoresis in denaturing formaldehyde gels. rna was transferred to nylon membranes (hybond-n, amersham, arlington heights, il, usa) and fixed by uv irradiation. the rna was prehybridized in m naci, . .% sds, . mg/ml salmon sperm dna, and ~ dextran for h at °c. the rna was then hybridized overnight at °c with a radiolabeled routine il- cdna probe (obtained from dr. frank lee, dnax, palo alto, ca, usa). the probe was radiolabeled ( p) in low melting agarose by random hexamer priming. the blots were washed under high stringency conditions ( . × ssc, . % sds, mm edta (ph ), mm sodium phosphate (ph . ) at °c) and analyzed by autoradiography with intensifying screens (dupont, hoffman estates, il, usa). densitometry of the autoradiographs was carried out using a macbeth densitometer (model td- , macbeth process measurements, newburg, ny, usa). tables and demonstrate that il- is induced in both balb/c and sjl derived endothelial ceils following exposure to mhv- as determined by bioassays, albeit at very different levels. in the mhv- susceptible balb/c cells, the peak of il- induction occurred on day of infection, and the levels of il- were -fold higher (> u/ml) than those obtained from the mhv- resistant sjl cells ( table ). the level of il- release from sjl endothelial cells ( u/ml), following exposure to mhv- , was not substantially different from the basal levels ( < u/ml) detected in control cultures. active infection of the cells was not apparently necessary since uv inactivated virus also induced il- in a strain dependent fashion. in table the drop of il- levels on day and the subsequent increase on day following uv-mhv- treatment of balb/c endothelial cells was an isolated observation in the representative experiment presented. the general trend was toward a gradual decline in il- levels from day onward. tables and demonstrate that il- was induced in both balb/c and sjl astrocytes by mhv- , as determined by bioassay of the culture supernatant. levels of il- were maximal on day (> u/ml) after mhv- infection of balb/c astrocytes. the levels of il- continued to remain high even days post infection. in contrast, il- activity in sjl astrocytes peaked later, on day ( u/mi), but were not sustained, declining from day onward. as seen with the endothelial cell cultures, uv inactivated virus was also able to induce il- ( table ) . to confirm that il- in the supernatants being tested was the inductive signal for proliferation of the il- dependent cell line a neutralizing monoclonal anti-mouse il- antibody was used. as shown in tables - , the proliferative response in all cases was blocked by the antibody to il- . this confirms that il- activity was present in supernatants collected after exposure of endothelial ceils or astrocytes to mhv- and uv-mhv- . the mhv- preparation used in these studies was obtained by infection and sonication of l-cells. it is il- units were determined by the dilution of supernatants yielding half maximal proliferation. the data presented in rows b and d show the effect of rat anti-mouse il- monoclonal antibody ( /zg/ml) in neutralizing the il- activity in the supernatants. il- levels in cell culture supernatants on day were << u/ml. possible that tnf was produced by the l-cells during mhv- production. tnf can induce il- in various cell types, including endothelial cells and astrocytes (shalaby et al., : beneveniste et al., . therefore a bioassay was employed to rule out the presence of tnf in the virus preparation used in our studies. this bioassay is based on the ability of tnf to inhibit proliferation of a mouse fibrosarcoma cell line, wehi . . . uv-mhv- l-cell lysates (virus stock) did not inhibit the proliferation of the wehi . . cells. this documented the absence of detectable tnf activity in the i,-cell derived virus preparation used for the il- induction studies. uv-mhv- was used in these studies to avoid the cytolytic effects of mhv- on the wehi . . cells. a positive control using recombinant murine tnf (genzyme, cambridge. ma. usa) at doses as low as u/ml caused a -fold decrease in proliferation of the wehi . . cells. northern analysis was performed to look for differences in the induction of _- mrna between balb/c and sjl derived astrocytes (fig. i) . the il- edna probe (moil- cdna, dnax, palo alto, ca. usa), used in our studies, hybridizes to a . -kb species (frci et al,, ). the rna from untreated cells (lanes a and e) showed no evidence for induction of il- mrna. in contrast, il- mrna induction was noted in lanes b-d and f-g. il- mrna was induced to a comparablc degree in both balb/c (susceptible) and sjl (resistant) derived astrocytes following exposure to either mhv- (lanes b and f), uv-mhv- (lanes c and g) or tnf at u/ml (lanes d and h) for h. tnf exposure was included as a positive control for il- induction. densitometric analysis did not indicate major differences in il- mrna induction in balb/c astrocytcs relative to sjl cells (table ) . a similar pattern of mhv- induction was obtained when evaluating il- mrna of endothelial cells (data not shown). these results show that il- mrna is induced at comparable levels in the different strains and cell types, although there are dramatic differences in the quantity of il- released. this suggests that these differences may bc accounted tor by altered translation or posttranslational processing. the study of the immune and inflammatory events in the cns following infection with mhv- is important in understanding the pathologic events and mechanisms of virus induced demyelination, lmmunoregulatory cytokines can profoundly affect the cascades of both humoral and/or cell mediated immune events in the cns. however, there has not yet been a systematic analysis of cytokine induction following mhv infection of cns derived cells. the cytokine il- was studied because of ( ) demonstration of its release following viral infections with lcmv (lymphocytic choriomeningitis virus), vsv (vesicular stomatitis virus), hiv (human immunodeficiency virus) and htlv- (human t-cell leukemia virus), and ( ) its multifunctional immunoregulatory role in modulating t, b and neutrophil functions (cernetti et al., ; frei et al., frei et al., , houssiau et al., ; takai et al., ; borish et al., : le and vilcek, ; breen et al., ; nishimoto et al., ) . we report on the induction of il- in cultures of cerebral endothelial cells or astrocytes following infection with mhv- . the mhv susceptible balb/c derived endothelial cells and astrocytes produce substantially higher levels of il- (> u/ml), and at earlier time points, than resistant sjl derived cells. the sjl endothelial cells yield barely more ( u/ml) than basal levels (< u/ml) of il- in response to mhv- . in contrast, sjl astrocytes are induced to release a significant quantity of l- ( u/ml), although not as great as in the balb/c astrocytes (> u/ml). therefore, the strain differences in il- induction in response to mhv- are more striking between the endothelial cells than with the astrocytes. the mechanisms regulating this strain dependent induction of il- are under further study. one possible explanation may reflect the differences in mhv receptor expression on endothelial cells and astrocytes. mhv binding receptors have been demonstrated on balb/c but not sjl derived intestinal brush border and liver cells (williams et al., ) . the binding of the virus to its receptor on the cell surface may activate a signal transduction pathway for il- production. the results obtained with uv-mhv- suggest that infection is not required and that the binding of the viral particles to its receptor on the cell surface may be sufficient to trigger the release of il- . this ability of uv-mhv- to exert a biological effect is not unique to the induction of ii- . previous studies in our laboratory have demonstrated that uv-mhv- can block y-interferon induced mhc class ii antigen expression on endothelial cells to the same degree as infectious virus (joseph et al., ) . the greater degree of difference in il- induction noted in cerebral endothelial cells compared to astrocytes is intriguing in the face of the potential role of this cytokine in triggering a variety of immune mediated inflammatory mechanisms. since the mhv- replication in balb/c mouse brain is two to three logs greater than in sjl mice (knobler et al., ) , and there is a correspondingly greater inflammatory response to mhv- infection in balb/c than sjl mice, this observation deserves further investigation. current studies are directed at elucidating these mechanisms. a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin. part (pathology) induction and regulation of interleukin- gene expression in rat astrocytes activation of neutrophils by recombinant interleukin- genetic resistance to mouse hepatitis virus correlates with absence of virus binding activity in target tissues infection with hiv is associated with elevated levels of il- production identification of a kd cytokine that is required for the development of cytolytic t lymphocytes cerebral microvessel and derived cells in tissue culture. ii. establishment, identification and preliminary characterization of an endothelial cell line production of b-cell stimulator),' factor- and interferon gamma in the central nervous system during viral meningitis and encephalitis on the cellular source and function tit [[llcrletlkill-++ prtv~ltlccd ir the cetltld[ ilel~.otl: -, s\~,icill ill enhanccmcnt ot m ','p,'o cell mcdmlcd imnlune rc~,ponsc b_,' three dimincl c}-tokincs. ,i. h+nmunol. t lgs£) bacterial lipopolysaccharide and it+lllatnnlato~ mediators augment l[.-h secretion by human endothelial culls f.l). and ttarl. m.n lt)stl) l)iftelcntial nlodttlation of mt|(" class j arlltgen e',q)rc,sion on lllouqc brain cndolhehal cell', i+,y mhv- infection regulation of mh(" class i and antigens on cerebral endothelial cells and astroc},tes following mhv- infection mouse hepatitis virus (mhv . jhm) blocks y interferon-induced major hi>tocompatibility complex class ii antigen expression on murine cerebral endothelial cells mou>,e hepatitis virus type (jhm strain) induced fatal central nervous s}melll dneasc. i. genetic control and the nlurine neuron as the su,ceptiblc site of di,ease it) "p selected tnutants of mouse hepatitis virus type (,ihm ,tt'ain) induce diflerent cns disea,e. pathobiology of disease induced b, v, im type and rnutant ts and tsl in balb/c and sjl mice differential effects of mhv- infection of astrocytes and oligodendrocytes in vitro biology ol disease: interleukin- a mtdtifunctional cytokine regulating ~mmunc reactions and the acute phase protein response l ( ) production of tumor necrosis factor and other cytokines by astroc,vtes ~timulaled ,aith lipopolvsaccharidc or a neurotropic virus molecular cloning, a kaboratod manual preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue accumulation and chemotaxis of natural killer,'large granular lymphocytes at sites of virus replication s of intcrieukin ,' > in ,crum and celcbro-,pu]ul lhlld of l ii i_v+ i associated myclopathy 'tmrncal { t}) purification ol nlullnc endothelial cell cuhurc, , tim', cyt~mlctd' usnlg fluorescctn-labeled (;nlt;,m+ ~#tqqt<'z/+dm ,tgghl train..\ill ~;) ('~,tokinc regulation ol interleukm-t+ production tr:,' huttlan endt~theliul cell il-i ,timulalc', il- production b~, cndolhelialcells iq ) natural killer cell ;.ictivit~, during nlouhc hepatitis xirus infection: le,ponse in the absence ol interferon si production oi b celimtmulalod factor- interlcukinm activity by human endothelial cells < ' ,~ + t-cell gro,.,th and diflcrentiation indt.ced by interlcukm-i, the murmc h_~-bridoma plasmacytoma growth factor i~js )specific labeling of endothelial cells using fluorescent acutylatcd io < ) l)cnb:ehnation induced by murine hepatitis ,fit.s jhm strain cmiiv- '~ i, im munologicall} mediated ) natural c.vlotoxlclty against mouse hepatitis virus-mlected cells. . a cytotoxic effector cell ,aith at b b'tnphocyte phenotypc ) purification of the i io-kd gb'coprotein receptor for mifv-a from mouse liver and identification ul a non-functional homologous protein in mhv-resistant sjl j mice ) ('haractcrization ol brain-infiltrating nlononuclear cells during infection with mouse hepatitis virus qrain jhm coronavirus induced primary demyelination: indications for tile involvement of a humoral immune response this study was supported by research grants rg- and rg- from the national multiple sclerosis society and ns and ns from the national institutes of health. the advice and facilities provided by dr. terry hieman-patterson and dr. rebecca hoffman for performing the northern analysis are gratefully acknowledged. the excellent technical assistance of ms. concetta d'imperio and ms. claire leighton is also acknowledged. key: cord- -frkb iso authors: gao, hong-qiang; schiller, jennifer j.; baker, susan c. title: identification of the polymerase polyprotein products p and p of the murine coronavirus mhv-jhm date: - - journal: virus research doi: . /s - ( ) - sha: doc_id: cord_uid: frkb iso abstract the rna polymerase gene of murine coronavirus mhv-jhm encodes a polyprotein of greater than kda. this polyprotein is proposed to be processed by two papain-like cysteine proteinases, pcp- and pcp- , and a poliovirus c-like proteinase domain, c-pro, to generate protein products. the amino-terminal product of the mhv polymerase polyprotein, p , is generated by cleavage of the polyprotein by pcp- . to identify the viral products downstream of p , we generated a fusion-protein specific antiserum directed against the region adjacent to p and used the antiserum to detect virus-specific proteins from mhv-jhm infected cells. when this antiserum was used to immunoprecipitate radiolabeled proteins from mhv-jhm infected cell lysates, virus-specific proteins of and kda were detected. furthermore, pulse and chase experiments demonstrated that p is likely a precursor to the mature protein product, p . to investigate which viral proteinase may be responsible for generating p and p , we expressed the ′-region of the mhv-jhm rna polymerase gene including the two papain-like cysteine proteinase domains in an in vitro transcription/translation system and analyzed the translation products for proteolytic processing. we also cloned and expressed the kda region immediately downstream from p , and tested the ability of in vitro translated pcp- and pcp- to cleave p to p in trans. our results indicate that neither viral proteinase domain pcp- nor pcp- is capable of cleavage of p to produce p in vitro. the role of mhv proteinases in the processing of p and p is discussed. the mhv genomic rna is surrounded by a phosphoprotein to form a helical nucleocapsid structure within a lipid envelope. upon infection, the mhv genomic rna is translated to produce a viral rna-dependent rna polymerase which mediates the synthesis of negative strand rna and subgenomic mrnas. the subgenomic mrnas form a '-coterminal nested set and are synthesized by a process that involves discontinuous transcription (baric et al., ; spaan et al., ) . the discontinuous transcription of mhv rna is thought to contribute to the high frequency of rna recombination that is also observed in mhv-infected cells (makino et al., ) . the novel transcription mechanism proposed to generate mhv mrnas and the high frequency of mhv rna recombination implicate a polymerase enzyme with unusual discontinuous properties; however, that polymerase must also be capable of accurately replicating the kb genomic rna to produce infectious virus (reviewed by lai et al., ; van der most and spaan, ) . to understand the unique process of mhv rna synthesis, we aim to identify the components of the rna-dependent rna polymerase, to determine how these proteins are generated and to elucidate their function in the complex mhv replication strategy. the mhv rna-dependent rna polymerase is encoded by the '-most gene, gene . gene is kb and contains two long open reading frames (orfs) designated orf la and orf lb (pachuk et al., ; lee et al., ; bonilla et al., ) . orf la and orf lb overlap by nucleotides and may be translated to produce a single polyprotein of approximately kda by a ribosomal frameshift mechanism (brierly et al., (brierly et al., , bredenbeek et al., ; lee et al., ) . this large polymerase polyprotein is proposed to be processed into mature protein products by viral proteinases encoded within the polyprotein, and perhaps by cellular proteinases as well. three viral proteinase domains, including two papainlike cysteine proteinases (pcp- and pcp- ) and a poliovirus c-like proteinase domain ( c-pro), have been predicted by amino acid sequence comparison (gorbalenya et al., . the first direct evidence for proteolytic processing of the polymerase polyprotein came from in vitro translation studies of mhv genomic rna. denison and perlman showed that in vitro translation of viral genomic rna produced p and p , and that production of p was inhibited by the addition of a proteinase inhibitor, zinc chloride (denison and perlman, ) . using an antiserum generated against a synthetic peptide representing a portion of p , the p protein was detected in mhv-infected cells and shown to be the amino-terminal protein of the polymerase polyprotein (denison and perlman, ; soe et al., ) . translation of rnas representing the ' end of the viral genome revealed that the pcp- domain was required for the autoproteolytic cleavage of p (baker et al., ) . the cleavage of p by pcp- was inhibited by leupeptin in both in vivo and in vitro reactions (denison and perlman, ; baker et al., ) . furthermore, the catalytic cysteine and histidine residues of the pcp- domain have been identified (baker et al., ) . the cleavage site recognized by pcp- for the cleavage of p has been determined to be the gly- /val- dipeptide bond (dong and baker, ; hughes et al., ) . a number of potential pcp- cleavage sites are predicted, but only the p cleavage site has been experimentally confirmed. a second, independent cleavage event has recently been implicated in the release of a kda product in mhv-a infected cells . using an antiserum which was directed against a fusion protein encoding the entire p domain and the region adjacent to the p cleavage site, denison and co-workers immunoprecipitated a kda protein from mhv-a infected cells. additional in vitro experiments using cloned cdnas of mhv-a suggest that p may be cleaved by the '-most proteinase pcp- . however, kim et al. ( ) have shown that e d, an irreversible inhibitor of cysteine proteinases, specifically blocks the in vivo cleavage of p but surprisingly has very little effect on the cleavage of p . this suggests that a proteinase other than pcp- , which cleaves p , is responsible for the liberation of p . in this study, we aimed to identify the proteolytic product ad-jacent to p in the mhv-jhm polymerase polyprotein and to determine how that protein is processed. to examine the synthesis and processing of the mhv-jhm polymerase polyprotein, we used antisera directed against the amino-terminal domains of the polymerase, anti-p and anti- , to immunoprecipitate [ s]methionine-labeled proteins from mhv-jhm infected cells. the anti-p serum was generated using a synthetic peptide representing amino acids - of mhv-jhm and has been previously described (baker et al., ) . for this study, the anti- serum was generated against a bacterial gst-mhv fusion protein which encodes a basepair domain that is adjacent to p (see fig. ). the bacterial expression vector, pgex-kg, which contains the glutathione s-transferase (gst) gene under the control of a tac promoter, was kindly provided by dr. steven broyles (purdue university, west lafayette, in). to generate a gst-mhv fusion protvin, a basepair xhoi fragment (nt - of mhv jhm-x), was isolated from pt -nbgl plasmid dna (baker et at., ) and ligated into the xhoi site of pgex-kg. the resulting expression plasmid which encodes a gst-mhv fusion protein with a predicted molec- ular mass of kda, was designated pgex- . the fusion protein was induced and isolated following the method of guan (guan and dixon, ) , and injected into rabbits to generate polyclonal antiserum. to identify mhv gene products, a mouse fibroblast cell line, c - , was infected with mhv-jhm at a multiplicity of infection (m.o.i.) of and labeled at . h post-infection (p.i.) with [ s]methionine for min. whole cell lysates were prepared and immunoprecipitated as previously described (baker et al., ) using protein a-sepharose beads and anti-p , anti- or pre-immune serum, and analyzed by electrophoresis in a % sds-polyacrylamide gel (fig. ) . the amino-terminal product of the polymerase polyprotein, p , was detected in mhv-jhm infected cells using anti-p (lane ), demonstrating that translation and processing of the polymerase polyprotein was occurring during the labeling period. the anti- serum specifically precipitated a protein with an apparent molecular mass of kda (lane ), which was not detected in mock infected cells (lane ) or from mock or mhv-jhm infected cell lysates immunoprecipitated with pre-immune serum (lanes and ). two additional mhv-jhm specific proteins of kda and greater than kda were also detected using anti- serum. to determine if the p and/or kda proteins were precursors to p , we performed pulse and chase experiments. mhv-jhm infected cells were pulse-labeled with [ s]methionine from . to . h p.i. following the pulse, the radiolabeled proteins were chased by replacing the labeling media with media containing excess unlabeled methionine. whole cell lysates were prepared at various times during the chase and immunoprecipitated with anti-p and anti- , and products were analyzed by electrophoresis in % sdspolyacrylamide gels (fig. (a,b) ). the p protein is detected immediately after the min pulse and no precursor protein is observed (fig. (a) ). the p protein also appears to be stable throughout the min chase. this result is consistent with previous data demonstrating that p is cleaved in cis and is stable in mhv-infected cells (denison and perlman, ; baker et al., ) . immunoprecipitation of the mhv-jhm infected lysates with anti- reveals a precursorproduct relationship of p and p (fig. (b) ). the p protein is the predominant viral protein precipitated from the pulse-labeled cells. the p protein is detected by min of chase and gradually accumulates with a concomitant decrease in p . these results indicate that the p is likely the precursor to the p protein. the p and p proteins may share either amino-or carboxy-terminal regions and be recognized by the anti- serum. the high molecular weight protein (greater than kda) was also detected at very low levels in the pulse-chase experiment. this large protein likely represents a primary translation product of orf la. this precursor polyprotein may be cleaved to produce p /p and the p which was detected by denison and co-workers (denison et al., ) . analysis of the mhv strain a polymerase gene products has also identified a kda protein as the mature proteolytic product adjacent to p . the mhv-a p protein was identified using a fusion protein antiserum (up ) that was generated against the entire p coding region and approximately kb of adjacent sequence. consistent with our results, denison and co-workers demonstrated that p is detected immediately after a min pulse, and that p is detected after approximately min of chase. however, no precursor protein of kda was detected from mhv-a infected cells using antiserum up . there are several possible explanations for this difference. first, because the up and antisera were generated against different virus strains and in different fusion protein constructs, the antisera may recognize different epitopes. the p precursor protein may be folded in a conformation that is not efficiently recognized by the up antiserum. a second possible explanation is that the proteolytic processing of the mhv-a strain and the mhv-jhm strain may differ in this region. to determine how p and p are generated from the polymerase polyprotein, we expressed cdna clones encoding the '-end of orf l a, including the papain-like cysteine proteinase domains, and analyzed the translation reactions for the presence of proteolytic products. the cdna clones pt -na and pt -nbgl, representing the '-end . and . kb, respectively (see fig. ; baker et al., ; lee et al., ) , were expressed in vitro in the presence of [ s]methionine in a coupled transcription/translation system according to the manufacturer's instructions (tnt system, promega, madison, wi). the translation products were analyzed by sdspolyacrylamide gel etectrophoresis (sds-page) directly (fig. , lanes and ) or after immunoprecipitation with anti- (lanes and ) or anti-p (lanes and ). as expected, p is detected from the primary translation products of both pt -na and pt -nbgl, since both encode pcp- , and it has been shown that pcp- is responsible for the cleavage of p (baker et al., ) . clone pt -na contains an extension of the '-end of the polymerase gene which includes the putative pcp- domain. analysis of pt -na in vitro translation products reveals protein products of approximately and kda. no additional proteolytic products were specifically detected either in the primary translation products (fig. , lane ) or after immunoprecipitation with anti- (lane ). these results indicate that although pcp- can cleave p from the polyprotein representing the ' . kb of orf-la, neither pcp- nor pcp- can cleave p or p from the precursor polyprotein in vitro. although the viral proteinase domains pcp- and pcp- do not cleave p or p from the precursor polyproteins described above, they may still be responsible for the cleavage of p if the appropriate precursor, p , is presented. alternatively, p may be processed by mhv proteinase c-pro or some other unidentified viral proteinase, to generate p . to address this possibility we asked the following question: if p is presented independently, will a viral proteinase domain recognize and cleave it to produce p ? for this purpose, we reverse-transcribed and polymerase chain reaction (pcr) amplified the kda coding region adjacent to p starting at nucleotide , encoding the valine in the pi' position of the p cleavage site, and ending precisely at a potential c-pro e/s cleavage site, at nucleotide . this region was cloned into the expression vector pet- ld (novagen, madison, wi), generating plasmid pet-lid/p (fig. ) . in vitro transcription/translation of this plasmid dna yields a protein which, when analyzed by sds-page, is approximately kda (fig. (a) , lane ). to determine if mhv proteinase domains pcp- or pcp- act in trans to cleave p , we generated radiolabeled p protein and unlabeled pt -nbgl and pt -na the translation products were analyzed by % sds-page directly (lanes and ) or following immunoprecipitation with anti- (lanes and ) or anti-p (lanes and ). dots highlite higher molecular weight immunoprecipitated products. lane contains t c-labeled molecular marker polypeptides. molecular mass standards are given in kilodaltons. translation products, mixed the proteins and incubated the mixtures for or min. the proteins were then analyzed by electrophoresis on an sds-polyacrylamide gel and visualized by autoradiography (fig. ) . in samples where fullytranslated proteins are mixed and incubated together for an additional min, p is not cleaved into p by pcp- and pcp- -containing proteins (translated from pt -nbgl and pt -na , respectively) (fig. (a) , lanes - ). similarly, when p is included in the pt -nbgl and pt -na translation mixtures to test the possibility of a co-translational activity of pcp- and pcp- ( fig. (a) , lanes - ), p also remains uncleaved. incubation of the p protein with rabbit reticulocyte lysates (fig. (a) , lanes and ), a major component in the transcription/translation reactions, serves as a negative control and reflects p stability throughout the incubation period. results from this experiment' indicate that neither pcp- nor pcp- can mediate the trans-cleavage of p as a substrate to generate p in vitro. in summary, we have identified two proteolytic products, p and p of the mhv-jhm polymerase polyprotein, from mhv-jhm infected cells. a kda protein has also been identified for mhv-a by denison et al. ( ) , but the p protein has not previously been reported for any coronavirus. our pulse-chase experiments indicate that the kda protein is likely a precursor to the p protein (fig. ) . using both cis and trans-cleavage assays, we showed that neither the pcp- nor pcp- domain mediates the cleavage of p or p in vitro (figs. and ). these results suggest that a proteolytic pathway distinct from that used to generate p is used to generate p and p in mhv-jhm infected cells. it is likely that a number of distinct proteolytic events are required to generate the functional mhv rna polymerase. to date, a number of proteins have been identified from mhv infected cells, including p , p , p , p , p and p (denison and perlman, ; denison et al., denison et al., , this report), but only the proteolytic pathway for generating p has been elucidated (baker et al., ; dong and baker, ; hughes et al., ) . there has been no report of the cleavage activity of the pcp- domain. re- methionine for min. the p translation product was then incubated with unlabeled transcription/translation products generated from: rabbit reticulocyte lysates alone (lanes and ), pt -nbgl plasmid dna (lanes and ) or pt -na plasmid dna (lanes and ). to test the potential co-translational activity of pt -nbgl and pt -na translation products to cleave p in trans, plasmid dnas were transcribed and translated in the absence of [ s]methionine, but in the presence of [ s]methionine radiolabeled p protein (lanes - ). the trans-cleavage assay products were analyzed by electrophoresis on a % sds polyacrylamide gel and visualized by autoradiography. molecular weight markers are indicated in kilodaltons on the left of the gel. (b) in vitro transcription/translation products generated from plasmid dna pt -na (lane ) and pt -nbgl (lane ) in the presence of [ ss]methionine as a control for parallel unlabeled translation reactions. products were analyzed on a % sds-polyacrylamide gel and visualized by autoradiography. the position of the p c/s-cleavage product is indicated by the arrow on the right. cently, the activity of the coronavirus c-pro domain has been identified for mhv-a , human coronavirus -e (ziebuhr et al., ) and the avian coronavirus infectious bronchitis virus tibbles et al., ) . using bacterial expression vectors, it has been shown that the coronavirus c-like proteinases recognize predicted cleavage sites, q/s for mhv-a and ibv, and q/a for e, to release the c domain from the polyprotein precursor. these cleavage sites are consistent with the consensus recognition sequence of q, e/g, s, a for c proteinases (dougherty and semler, ; krausslich and wimmer, ; palmenberg, ) . the picornavirus c proteinases recognize multiple sites in their polyprotein and cleave both in cis and in trans. the mhv-jhm c-pro may also have multiple cleavage sites, including a putative e/s cleavage site in the region predicted for cleavage of p . we are currently investigating whether the c-pro domain is in fact responsible for cleavage of p or p , or if an as yet unidentified proteinase domain is involved in these processing events. identification of a domain required for autoproteolytic cleavage of murine coronavirus gene a polyprotein identification of the catalytic sites of a papain-like cysteine proteinase of murine coronavirus characterization of replicative intermediate rna of mouse hepatitis virus: presence of leader rna sequences on nascent chains mouse hepatitis virus a rna polymerase gene orf a: heterogeneity among mhv strains characterization of the leader papain-like proteinase of mhv-a : identification of a new in vitro cleavage site 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 an efficient ribosomal frame-shifting signal in the polymeraseencoding region of the coronavirus ibv characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot identification and characterization of a kda protein processed from the gene polyprotein of the murine coronavirus mhv-a translation and processing of mouse hepatitis virus virion rna in a cell-free system identification of a putative polymerase gene product in cells infected with murine coronavirus a intracellular processing of the n-terminal orf la proteins of the coronavirus mhv-a requires multiple proteolytic events expression of virusencoded proteinases: functional and structural similarities with cellular enzymes determinants of the p cleavage site recognized by the first papain-like cysteine proteinase of murine coronavirus coronavirus genome: prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis putative papain-related thiol proteases of positive-strand rna viruses eukaryotic proteins expressed in escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione s-transferase identification of the murine coronavirus p cleavage site coronavirus protein processing and rna synthesis is inhibited by the cysteine proteinase inhibitor e d viral proteinases coronavirus: how a large rna viral genome is replicated and transcribed the complete sequence ( kilobases) of murine coronavirus gene i encoding the putative proteases and rna polymerase characterization and mutational analysis of an orf la-encoding proteinase domain responsible for proteolytic processing of the infectious bronchitis virus la/lb polyprotein identification, expression, and processing of an -kda polypeptide encoded by orf la of the coronavirus infectious bronchitis virus identification and characterization of a serine-like proteinase of the murine coronavirus mhv-a high frequency rna recombination of murine coronaviruses proteolytic processing of picornaviral polyprotein molecular cloning of the gene encoding the putative polymerase of mouse hepatitis coronavirus, strain a sequence and translation of the murine coronavirus '-end genomic rna reveals the n-terminal structure of the putative rna polymerase coronavirus mrna synthesis involves fusion of non-contiguous sequences characterization in vitro of an autocatalytic processing activity associated with the predicted c-like proteinase domain of the coronavirus avian infectious bronchitis virus characterization of a human coronavirus (strain e) c-like proteinase activity this research was supported by public health service research grant ai and a junior faculty research award (to s.c.b.) from the american cancer society. 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 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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- -np xdag authors: barnett, e. m.; cassell, m. d.; perlman, s. title: two neurotropic viruses, herpes simplex virus type and mouse hepatitis virus, spread along different neural pathways from the main olfactory bulb date: - - journal: neuroscience doi: . / - ( ) -h sha: doc_id: cord_uid: np xdag abstract several neurotropic viruses enter the brain after peripheral inoculation and spread transneuronally along pathways known to be connected to the initial site of entry. in this study, the pathways utilized by two such viruses, herpes simplex virus type and mouse hepatitis virus strain jhm, were compared using in situ hybridization following inoculation into either the nasal cavity or the main olfactory bulb of the mouse. the results indicate that both viruses spread to infect a unique and only partially overlapping set of connections of the main olfactory bulb. both quantitative and qualitative differences were observed in the patterns of infection of known primary and secondary main olfactory bulb connections. using immunohistochemistry for tyrosine hydroxylase combined with in situ hybridization, it was shown that only herpes simplex virus infected noradrenergic neurons in the locus coeruleus. in contrast, both viruses infected dopaminergic neurons in the ventral tegmental area, although mouse hepatitis virus produced a more widespread infection in the a group, as well as infecting a and a . the results suggest that differential virus uptake in specific neurotransmitter systems contributes to the pattern of viral spread, although other factors, such as differences in access to particular synapses on infected cells and differences in the distribution of the cellular receptor for the two viruses, are also likely to be important. the data show that neural tracing with different viruses may define unique neural pathways from a site of inoculation. the data also demonstrate that two viruses can enter the brain via the olfactory system and localize to different structures, suggesting that neurological diseases involving disparate regions of the brain could be caused by different viruses, even if entry occurred at a common site. to be important. the data show that neural tracing with different viruses may define unique neural pathways from a site of inoculation. the data also demonstrate that two viruses can enter the brain via the olfactory system and localize to different structures, suggesting that neurological diseases involving disparate regions of the brain could be caused by different viruses, even if entry occurred at a common a number of viruses have been shown to spread transneuronally into and throughout the rodent cns following intranasal inoculation, including herpes simplex virus type (hsv-i)," vesicular stomatitis virus, borna disease virus," mouse hepatitis virus (mhv),-pseudorabies viru? and rabies virus " virus spread through the olfactory system is of particular importance since the olfactory receptor neuroepithelial cells are the only neurons with direct exposure to the environment. as such, the olfactory system is a likely route of entry for viruses and toxins into the cns and the distribution of lesions in some human diseases, such as alzheimer's disease, is consistent with spread of an environmental agent into the cns via the olfactory system. mhv belongs to the coronavirus family of enveloped, positive-stranded rna viruses. although some coronaviruses infect humans, causing respiratory inuo whom correspondence should be addressed. abbreviafions: hsv- , hernes simplex virus, type ; lc, locus coeruleus; mhv'-jhm, -mouse hepatitis virus, strain jhm; mob, main olfactorv bulb; pfu, plaque forming unit; p.i. post-inoculation; th, tyrosine hydroxylase; th + , tyrosine hydroxylase immunoreactive; th -, tyrosine hydroxylase immunonegative; vta, ventral tegmental area; wga-hrp, wheatgerm agglutinin-horseradish peroxidase. fections such as the common cold, coronavirus pathogenesis research has focused primarily on animal infections. rodent infections with neurotropic strains of mhv have been studied as models for acute and chronic virus-related neurological disease. , so strain jhm (mhv-jhm) is highly neurotropic and causes a fatal acute encephalitis following intranasal inoculation of suckling and adult c b / mice." mice passively immunized against the virus or infected with an attenuated strain do not develop an acute encephalitis, but instead often manifest a chronic demyelinating disease characterized by hindlimb paralysis. , , ' , , this virus-induced demyehnation serves as a model for multiple sclerosis. evidence from several studies indicates that mhv enters the cns via the olfactory nerve following intranasal inoculation and spreads to many of the primary and secondary connections of the main olfactory bulb (mob). '*o, - both chemical deafferentation and surgical removal of the mob prior to intranasal inoculation prevent cns entry and show that virus entry via the trigeminal nerve is insignificant. ,s the structures infected by mhv-jhm and the temporal sequence of their infection strongly suggests that this virus, like many other neurotropic viruses, spreads transneuronally into and throughout the cns. e. m. barnett pi u/ hsv-i is a human pathogen which usually causes recurrent, localized vesicular lesions. but is also an important cause of sporadic cases of severe encephalitis. hsv- causes encephalitis in mice and rats following intranasal inocuiation'y~ and, unlike mhv-jhm, can enter the rodent cns from peripheral inoculation sites such as the tongue, the eye ' and the tooth pulp (barnett e. m., jacobsen g. and perlman s., unpublished data). as is the case with mhv-jhm, the pattern of the resulting infection is suggestive of transneuronal movement of virus and, as such, hsv- has proved useful for tracing neural circuits. , , our initial studies with mhv-jhm suggested that after intranasal inoculation the virus spread along different pathways from the mob as compared to previous studies with hsv- . for example, several workers , reported hsv- infection of the locus coeruleus (lc) in rats and mice following mob infection, whereas we could not detect mhv-jhm in this structure. based on these preliminary results, we determined the extent to which these two neurotropic viruses infected different cns structures after infection of the mob. although studies have been performed describing the pattern of virus spread for wild-type and attenuated strains of several neurotropic viruses, , ,u this is the first to compare the transneuronal spread of two different viruses using exactly the same experimental design. the possible differential spread of viruses throughout the brain has important implications for the use of viruses such as hsv- as neuroanatomical tracers, as it has been generally assumed that such viruses spread nonspecifically throughout the cns along all the available pathways from an infected structure. such differential localization would also suggest that different viruses could cause different neurological diseases, depending on precisely which connections of the olfactory nerve were infected. twelve male suckling (lo-day-old) and male young adult (six-week-old) c b / mice purchased from sasco laboratories (omaha, ne) were used in these studies. for surgeries, mice were anesthetized by intraperitoneal injections of a sodium pentobarbital solution ( . mg/kg).@ all surgical procedures were approved by the institutional animal care and use committee at the university of iowa. was grown on balb/c cl- cells and hsv- on rk cells. the two viruses were titered and maintained frozen at - °c in tissue culture media. strain (kindly provided by dr moira brown), a highly neurovirulent strain of hsv-i," caused encephalitis after intranasal inoculation in suckling c bl/ mice and was used in all studies. consistent with other reports, adult c b / mice were resistant to even this strain of hsv- after intranasal inoculation, but preliminary experiments indicated that these mice could be infected with hsv- by intrabulbar inoculation. we compared the distributions of hsv-i and mhv-jhm in the brains of suckling c bli mice following intranasal inoculation and young adult c bl/ mice following intrabulbar inoculations. the use of both intranasal and intrabulbar inoculations served three purposes. first, intrabulbar inoculation eliminated trigeminal entry of hsv-i so that the pattern of infection would reflect viral spread from the mob. second the use of intranasal inoculations ensured that differences in the movement of the viruses into and throughout the internal circuitry of the mob would become apparent. third, inoculation of hsv- into the mob made possible the use of older c bl/ mice in which the infections spread more slowly, allowing for the examination of several time points during the course of the infection. ten-day-old mice received intranasal inoculations of approximately ' plaque forming units (pfu) of mhv-jhm or lo pfu of hsv-i strain in a volume of ~ . six-week-old mice underwent direct bulb inoculations with one of the two viruses as follows. following exposure of the skull over the olfactory bulbs, a small bone defect was produced over the left bulb using a dental drill. the dura was gently removed and nl of mhv-jhm ( ' pfu) or hsv-i (io pfu) was introduced into the center of the bulb by pressure injection using a glass micropipette (tip diameter nm) attached to a i ~ hamilton syringe. co-inoculation of nl of mhv-jhm and nl of wheatgerm agglutinin-horseradish peroxidase (wga--hrp; i%) was performed in four mice to confirm the site of inoculation. following inoculation the mice were observed over the next several days for signs of encephalitis such as irritability, ruffled fur, hunched backs and ataxia. mice were killed over a period of days post-inoculation (pi.), during which the appearance of the animals ranged from asymptomatic to nearly moribund. in situ hybridization was performed to determine the optimal times p.i. for neuroanatomical iocalization of viral nucleic acids, four to six mice at each age, for both of the viruses, were inoculated and killed at the optimal time(s) pi.: four days for all lo-day-old mice, three and four days for mhv-jhm-infected adult mice, and four and five days for hsv-l-infected adult mice. at earlier times p.i., virus had infected the mob and only a few mob connections. at later times, viral infection of the brain was so widespread as to make neuroanatomical localization of viral nucleic acid difficult. as a control for viral spread via the cerebrospinal fluid, direct inoculation into the lateral ventricle of nl of virus was performed on young adult c b / mice with each virus. stereotaxic coordinates for ventricular injections (ap - . ; lateral . ; horizontal mm) were taken from the stereotaxic mouse atlas of slotnick and leonard. the mhv-jhm-and hsv-l-infected mice were killed at four and five days p.i., respectively, and in siru hybridization was performed. an antisense ?+labeied rna probe for mhv-jhm was synthesized and in situ hybridization performed as described pretiously. briefly, mice were deeply anesthetized and exsanguinated by transcardiac perfusion with ml of phosphate-buffered saline and pm coronal brain sections were cut at pm intervals on a cryostat. sections were collected on silane-treated slides, fixed, treated with proteinase k and acetylated. approximately io c.p.m. of is-labeled antisense rna probe in hybridization solution was applied to each slide. after annealing overnight, slides were treated with rnase and washes of increasing stringency. slides were then placed on film for several days at °c followed by dipping in ntb- photographic emulsion (kodak, rochester, ny) for a two week exposure. after development and staining with cresyl violet, slides were examined by bright-field and dark-field light microscopy to localize viral nucleic acid in the brain. a plasmid construct contain-ing the coding sequence for the vp gene of hsv-i was obtained (kindly provided by dr ed wagner) and rna probe produced under the same protocol used for the production of mhv-jhm probe. ; situ hybridization for hsv- was performed as for mhv-jhm with two modifications: before hybridization the slides were heated at °c for min to denature viral dna and hvbridization took place overnight at °c rather than "c:since hsv- is a dna virus. the added denaturation steo enabled the rna probe to bind to both viral rna and genomic viral dna, producing a robust signal. the tetramethylbenzidine method of mesulam@ was used to visualize hrp in mob sections from mice co-inoculated with mhv-jhm and wga-hrp. six-week-old mice received intrabulbar inoculations with either virus, as described above. three mice each were killed at . days pi. for hsv-i and four days pi. for mhv-jhm. two additional mice were killed at three days p.i. for mhv-jhm. each mouse was anesthetized and killed by transcardiac perfusion fixation with ml of per& date-lysine-paraformaldehyde fixative. the brain was them removed and postfixed for h in the same fixative. cryostat sections ( pm) were cut through the caudal forebrain and brainstem at pm intervals and placed on silane-treated slides. the sections were incubated in phosphate-buffered saline for min, % normal goat serum in . % triton x-io&phosphate-buffered saline for hour, and rabbit anti-tyrosine hydroxylase (th; chemicon, temecula, ca) overnight in . % triton x-lo&phosphate-buffered saline at °c. the antigen-antibody complex was visualized using the vector abc method as specified by the manufacturer (vector labs, burlingame, ca) with , '-diaminobenzidine as the final substrate. rnasin ( u/ml; promega, madison, wi) and heparin ( mg/ml) were used to inhibit rnase activity during steps containing serum products. all buffers and solutions used during the procedure were treated with diethylpyrocarbonate and autobaved to eliminate rnase activit;. following immunohistochemistrv for th. in situ hvbridization was performed as above to detect viral nuclkic acid. brain sections were examined for cells containing both th immunoreactivity and silver grains. in this study, hsv- and mhv-jhm identical. the main difference, as expected, was hsv- infection of the spinal tract and sensory root of the trigeminal nucleus following intranasal, but not intrabulbar inoculation. although there is evidence for trigeminal innervation of the mob," hsv- did not infect trigeminal nuclei following inoculation into the mob. given the high similarity of the results across the two types of inoculations, only the results for intrabulbar inoculations in young adult mice will be presented. at the first time point examined (day mhv-jhm, day hsv- ) virus had already spread to the opposite hemisphere and most structures were infected bilaterally. although virus spread was more extensive on the side of bulbar inoculation, the pattern of labeling was highly similar on both sides by the second time point examined (day mhv-jhm, day hsv- ). consistent with our previous experience with mhv-jhm, some structures were only infected in a subset of cases at a particular time p.i. these structures eventually became infected in all cases at later times p.i., however, suggesting that this variability reflected differences between mice in the temporal course of infection rather than in the pattern of spread. (see table ) olfactory system. both viruses regularly infected cells in the periglomerular, mitral and internal granular layers of the mob. hsv- infected well-circumscribed regions of the internal granular layer, infecting large numbers of adjacent cells. in contrast, mhv-jhm infected more evenly distributed cells in this layer. infection of the mob did not differ significantly across the two types of inoculation for either virus. examination of mob sections from mice co-inoculated with wga-hrp showed that the injection site was restricted to the bulb, but commonly involved all layers. few hsv-l-or mhv-jhminfected cells were seen in the two white matter layers, the external and internal plexiform layers. the accessory olfactory bulb was commonly infected by both viruses. the anterior olfactory nucleus was infected by both viruses in all cases. mhv-jhm infected sparsely distributed cells in both the cellular and non-cellular layers of this nucleus (fig. a) . hsv- labeling was robust throughout the full extent of the cellular layer ( fig. b) . infection of the olfactory tubercle was seen in approximately one-half of the cases for either virus and was relatively light. the ventral tenia tecta was heavily infected by hsv- (fig. id) , but not at all by mhv-jhm (fig. c ). although both viruses regularly infected the dorsal endopiriform nucleus, the ventral endopiriform nucleus was infected by mhv-jhm only. labeling for mhv-ihm was commonly present over cells within the lateral olfactory tract by day (fig. ia) . cortex. hsv- infected a number of cortical areas, often producing extensive and specific laminar labeling. hsv- infection of the primary olfactory cortex was seen in every case. at day , virus was detected primarily in lamina b of the primary olfactory cortex, with scattered labeling in superficial lamina ( fig. a, c) . in more extensively infected brains, virus labeling was also seen in lamina a and throughout the full depth of lamina . medial prefrontal cortex was commonly infected by day , with virus labeling in laminae , and of anterior cingulate cortex and laminae and of medial and lateral orbital cortices. by day , the infection had spread to involve almost every lamina of these cortical areas. additional labeling was seen in laminae , and of medial agranular frontal cortex and , and of infralimbic cortex. entorhinal cortex was also commonly infected by hsv- (fig. c, d) . at day , infection of laminae , and was already detectable in each case. at day , the infection had sometimes spread to every lamina of entorhinal cortex. infection of perirhinal cortex was also detected in almost every case, beginning in lamina at day and spreading to involve laminae , , and at day . agranular insular cortex was commonly infected in laminae , and . other cortical regions were labeled in more extensively infected brains, including laminae and of parietal cortex and lamina of the granular insular, dysgranular insular and temporal co&es. mhv-jhm also infected a number of cortical areas, although the labeling in specific cortical laminae was less extensive than with hsv-i. primary olfactory cortex was infected by mhv-jhm in all cases. whereas hsv- infection was concentrated primarily in lamina b, mhv-jhm infection was most evident in lamina at both days and ( fig. c, d) . infected cells were seen throughout the depth of this lamina, although the labeling was less evenly dispersed than that seen with hsv- . virus was also detected in laminae and , with infection of sublaminae la and b being most common as with hsv-i, infection of the medial prefrontal cortex was common, particularly by day . labeling in the medial and lateral orbital cortices was first detected in lamina , extending into laminae , and at day . infection of the anterior cingulate cortex was also restricted to lamina in several cases, with eventual spread to all laminae at day . infralimbit cortex was infected in every case, virus first appearing in lamina ( fig. ic) , with spread to the other laminae, particularly lamina , in half of the cases. more caudally, mhv-jhm infected the entorhinal and perirhinal cortices. similar to the hsv- infection of entorhinal cortex, laminae , and were most commonly labeled, although laminae and were also infected in several mice. infection of perirhinal cortex was seen in all cases at day ? but was restricted to lamina only. by day , virus was detected in more superficial laminae in half of the cases. infection of agranular insular cortex by mhv-jhm was seen in all cases at day , most commonly in lamina , but also in laminae , and in more extensively infected cases. basal ,forebrain. mhv-jhm infected the vertical and horizontal nuclei of the diagonal band and the medial septal nucleus in all mice by day (fig. a, b) . the infection sometimes appeared to involve the entire extent of these nuclei, forming a continuous group of infected cells among these three structures. hsv- , however, regularly infected the horizontal nucleus of the diagonal band, but not the vertical nucleus. scattered hsv-l-infected cells were seen in the medial septal nucleus in half of the cases at day . by day , mhv-jhm had spread to other septal nuclei, including the lateral, triangular and septofimbrial nuclei. the lateral septal nucleus was also commonly infected by hsv- . although both viruses infected the ventral pallidum, mhv-jhm infected this structure both earlier in the infection and more intensely. labeled cells were evident from the rostra ventral pallidum to the most caudal aspect of this structure, where mhv-jhm mhv-jhm infected the anterior amygdaloid area also infected the ventral substantia innominata. more commonly (fig. ie) . with the exception of mhv-jhm-infected cells were seen in the white these structures, infection of the amygdala was more matter of the anterior commissure (fig. a ) in all prominent with hsv- . the posterior media and mice. overall, mhv-jhm infection of the basal posterior lateral nuclei were more commonly infected forebrain was more widespread than that with by hsv- (fig. c, d) , with labeling in both of these hsv- . structures in all day cases. hsv- labeling was amygdula. both viruses consistently infected the detected throughout the rest of the amygdala, includanterior and medial cortical amygdaloid nuclei, while ing the basolateral, basomedial and central nuclei. the nucleus of the lateral olfactory tract was also infected by hsv- in the majority of cases by day (fig. if) . primarily restricted to the midline nuclei, including the paratenial, paraventricular and reuniens nuclei (fig. a, b) . mhv-jhm labeling was detected less frequently in these structures. the ventroposterior parvocellular thalamus, however. was infected by mhv-jhm in all cases at day (fig. c, d) . this structure was never infected by hsv- , even at day . in the hypothalamus, hsv- infected scattered cells in the lateral hypothalamus at day . mhv-jhm infected this area in almost every case at both days and , although the labeling was commonly sparse. strong labeling was commonly detected in the nearby subthalamic nucleus and the posterior hypothalamic area (fig. c, d) . the stria medullaris also contained cells infected by mhv-jhm. hsv- was seen in every case at both four and five days. at day , ca pyramidal cells were infected in all four mice. by day , infection of the stratum oriens of ca and the pyramidal layer of ca occurred in half of the mice (fig. e. f) . labeling was also detected in the granule cell layer and less commonly in the polymorph layer of the dentate gyrus. in sharp contrast to this pattern of spread throughout the hippocampus, mhv-jhm sparsely infected only a small area of the ca region in one case at day . infection of the indusium griseum by mhv-jhm was seen in all cases by day and in almost half of the hsv-l-infected brains. mhv-jhm labeling in the indusium griseum continued caudally into the dorsal subicular portion of the hippocampal complex at the splenium of the corpus callosum. infection of the subiculum by hsv- also occurred by day , although the ventral subiculum was more commonly involved (fig. e. f) . midbrain. mhv-jhm was detected in the ventral tegmental area (vta) in all cases at day . the infection progressed to involve the entire structure at day , as well as the adjacent substantia nigra pars compacta. labeling was noted in a caudal and dorsal direction throughout the tegmentum, continuous with that in the vta. in contrast, hsv- infection of the vta was not commonly detected until day and was generally less intense. more caudally, hsv- infection of the caudal linear raphe nucleus was also seen at this time. both the median and dorsal raphe nuclei were infected by mhv-jhm and hsv- with and caudal pontine reticular nuclei and the nucleus equal frequency. gigantocellularis. the propensity of mhv-jhm to infect cholinergic structures, as seen in the basal forebrain, continued as the pedunculopontine tegmental nucleus was infected in almost every case (fig. sa, b) . this structure is continuous with the more caudal parabrachial complex, which was also commonly infected. viral rna was consistently located in the region of the medial parabrachial nucleus, which lies ventral to the superior cerebellar peduncle. hsv- did not infect these structures, but instead infected the nearby lc. hsv- infection was characterized by a dense infection of the full extent of the lc in every case. by day , labeling often continued caudally along the lateral borders of the fourth ventricle. pons and medulla. infection of structures caudal to the lc in hsv-l-infected mice was not common, even at day pi. the main exceptions were infected cells in the area of the al and a groups, which were present in more extensively infected brains. infection of brainstem raphe nuclei by hsv- occurred rarely, while the raphe magnus and raphe obscurus were infected by mhv-jhm in most cases. in addition, mhv-jhm regularly infected several parts of the brainstem reticular formation, including the oral inoculations into the lateral ventricle were performed to determine the possible contribution of cerebrospinal fluid spread to the observed distribution of virus. the pattern of infection seen following lateral ventricle inoculation with mhv-jhm at day was quite distinct from that seen following intranasal and intrabulbar inoculation. most of the structures infected were in close proximity to a ventricle, although not all juxtaventricular structures were labeled. among the regions heavily infected were the septum, the bulk of the hypothalamus and the hippocampus. detection of hsv- at day following a lateral ventricle inoculation revealed a similar pattern. in both cases, several structures normally infected following intrabulbar inoculation, such as the primary olfactory cortex, showed little or no labeling, the vta contains a dense collection of dopaminergic neurons which belong to the al group (fig. a) . although relatively few cells in the vta are infected by hsv- at . days p.i. (fig. b) . combined th immunohistochemistry and in situ hybridization for viral nucleic acids revealed that the vast majority of hsv- infected cells in the vta were also th immunoreactive (th+). mhv-jhm had already infected a larger number of cells in the vta at day (fig. c ) and the vast majority of infected cells were th+ (fig. a) . by day , the full extent of the vta was normally infected by mhv-jhm (fig. d) , although a smaller percentage of infected cells were also thf additionally, mhv-jhminfected th+ cells were detected in the substantia nigra pars compacta (a ) and throughout the a dopaminergic group on day . the percentage of mhv-jhm-infected cells which were also th + appeared to decrease significantly as the number of infected cells in these structures increased. double labeling for th and viral nucleic acid showed that hsv- infected the entire lc (a ), as dense silver grains were seen over th+ cells in this structure (fig. a, b) . in sections where a relatively small number of cells in the lc were infected, the infection was restricted almost entirely to neurons which were th+ (fig. b) . in sections where the infection of this structure was more widespread, the number of th-immunonegative (th -) cells infected by hsv-it increased dramatically. although mhv-jhm infected a rare th+ neuron in the dorsal lc, the vast majority of infected cells were clearly in the medial parabrachial nucleus (fig. c, d) . this is the first study to show by direct comparison using identical experimental protocols that two different neurotropic viruses move along different neural pathways following inoculation into a common site. our results indicate that mhv-jhm and hsv- spread along both common and unique pathways following intranasal and intrabulbar inoculations and that both viruses spread in a circuit-specific manner, since adjacent structures were infected by one virus or the other without contiguous (lateral) spread. the present findings are broadly consistent with earlier studies examining the spread of hsv-i and mhv after inoculation into the olfactory sys-tem ~ ~ '. although both viruses can spread via the cerebrospinal fluid, the pattern of infection following intraventricular inoculation suggests that virus spread via the cerebrospinal fluid following intranasal and intrabulbar inoculations was minimal at the times pi. examined in this study. many structures were infected on both the ipsilat-era and contralateral sides at early times pi., despite unilateral inoculation of virus. infection of the anterior olfactory nuclei, which project to both the ipsilateral and contralateral mobs,~~ was commonly bilateral for both viruses early in the infection. virus can also spread to the contralateral side across commissural connections between the olfactory cortices. contralateral primary olfactory cortex infection in hsv-l-infected brains was first seen in lamina b, from which commissural projections arise. mhv-jhm was detected in cells in the anterior commissure, through which these commissural projections cross. this is consistent with similar labeling in mice infected intranasally following unilateral bulbectomy. intranasal inoculation in that study quickly resulted in a bilateral infection also, despite the fact that mhv-jhm entry was restricted to the side with an intact mob. some of the labeling in heavily infected structures was likely related to infection of glia, and both mhv-jhm and hsv-i have been shown to infect glia during cns infections.s~' . . consistent with this, the percentage of th -cells in the lc infected with hsv- and in the a -a groups infected with mhv-jhm increased as the infections intensified. reactive gliosis, during which glia form a barrier around lysed neurons, may actually prevent the nonspecific spread of virions in the extracellular space, thereby contributing to the specificity of transneuronal movement of virus * epithelium to the mob and most likely from the olfactory neuroepithelial cells to mitral and periglomerular cells. most significantly, several primary mob connections were infected predominantly by one of the two viruses. hsv- produced an intense infection of both the ventral tenia tecta and the lc. neither of these were infected by mhv-jhm, despite the projections from both structures to the mob and piriform cortex.l , * similarly, the pyramidal cells of the ca region of the hippocampus were infected almost exclusively by hsv-i, although they project directly to the mob." these data show that despite the fact that both viruses had access to projections from the same structures, they were only infected by one of the two viruses. although both viruses infected primary olfactory cortex, the pattern of labeling differed considerably. laminae b and of this cortex provide the heaviest projection of all mob afferents. hsv- infection of this structure was consistent with retrograde movement along this pathway, as these laminae, particularly b, were infected early in the infection and laminae and a were not. in contrast, the early mhv-jhm infection was seen predominantly in lamina . these data show that hsv- and mhv-jhm preferentially infect different subsets of the neurons projecting from primary olfactory cortex to the mob. additional primary mob connections which were differentially infected include the posterolateral cortical amygdaloid nucleus and the nucleus of the lateral olfactory tract by hsv- , and the nucleus of the diagonal band, medial septal nucleus, ventral pallidum, anterior amygdaloid area and lateral hypothalamus by mi-iv-jhm (fig. ) . secondary and tertiary olfactory connections. the restriction of the spread of hsv- and mhv-jhm to specific structures and pathways continued as the secondary and tertiary connections of the mob were infected. the differential infection of these structures by the two viruses can in part be related to differences between the viruses in the infection of primary mob connections (fig. ). in the thalamus, hsv- infection of the thalamus involved midline nuclei such as the reuniens nucleus. this thalamic nucleus projects to a number of structures infected more heavily by hsv- , including the anterior olfactory nuclei, entorhinal cortex and the ca region of ammon's horn.*' the subthalamic nucleus, which projects to both the ventral pallidum and the lateral hypothalamus, * was infected exclusively by mhv-jhm. hsv- regularly infected several regions of the hippocampus, a structure infected only rarely by mhv-jhm. in addition to the previously mentioned infection of cal, ca pyramidal cells and granule cells of the dentate gytus were also infected by hsv- . the pattern of viral spread within the hippocampus was suggestive of retrograde transport. hsv- was detected in ca only in cases when the ca region, to which it projects,' was also infected. similarly, the granule cells of the dentate gyrus were only infected following ca infection, consistent with retrograde spread from the ca region, to which it projects. ' of the mhv-jhm-infected cells were th+ in this region. one possible explanation for this finding is that mhv-jhm infects both dopaminergic and nondopaminergic neurons in this structure, since onethird of vta neurons are non-dopaminergic and many projections arise from both dopaminergic and non-dopaminergic neurons ' another explanation is an increase in the infection of glial cells following virus spread from adjacent infected neurons. whereas hsv- infected the lc, mhv-jhm infected the pedunculopontine tegmental nucleus and the medial parabrachial nucleus. the latter two structures are continuous and together extend along the full rostralcaudal extent of the lc. the pedunculopontine tegmental nucleus projects to several structures infected exclusively or more prominently by mhv-jhm, including the vta, subthalamic nucleus and the lateral hypothaiamus. ', * the medial parabrachial nucleus projects to a number of mhv-jhm-infected structures, including insular cortex, the lateral hypothalamus and the ventroposterior parvocellular thalamus. ~so~ @' in the cortex, both viruses commonly infected laminae , and of entorhinal cortex, consistent with a projection from laminae and to the mob and projections from the deep layers of the entorhinal cortex to primary olfactory cortex." laminae , and of agranular insular cortex were also infected by both viruses. cells in lamina of this cortical area project to the lateral hypothalamic area, while those in all three laminae project to infralimbic cortex.' commonly, cortical infections were first restricted to specific lamina or laminae, with eventual involvement of additional laminae. in several cortical regions viral labeling was eventually seen in every lamina, suggesting intracortical spread of virus. combined assay for virus and th clearly showed that hsv- infected noradrenergic neurons in the lc, whereas mhv-jhm did not. this finding suggests that either mhv-jhm lacks the ability to infect noradrenergic fibers arising from the lc or, alternatively, that the virus does not cross the appropriate synapses to gain access to lc terminals. recent studies indicate that, in the rat, the noradrenergic input from the lc terminates in the internal and external plexiform layers and granule cell layer in the mob, but not in the glomerular layer. the granule cells of the mob form dendrodendritic contacts with mitral and tufted cells, which provide efferent fibers of the mob. a failure of mhv-jhm to enter the granule cells via the dendrodendritic contacts with the mitral and tufted cells would prevent access to the noradrenergic input from the lc. inability of mhv-jhm to infect lc neurons appears more likely since mhv-jhm infection of the internal granular layer of the mob was seen following intranasal inoculation. in addition, direct mob inoculations of mhv-jhm, which would be expected to expose lc terminals to virus, did not result in infection of this structure. finally, the lc projects to a number of other structures infected by mhv-jhm (e.g. anterior olfactory nuclei, primary olfactory cortex, the vta, entorhinal cortex), but was still not infected. there are a number of possible explanations for the differences described above between mhv-jhm and hsv- spread. the steps involved in the transneuronal spread of virus include, but are not restricted to, axonal transport of virus, viral exit from the presynaptic cell and uptake at the postsynaptic cell. differences between viruses at any of these steps would result in unique patterns of viral spread. both viruses were shown to infect cells in the vta, although anterograde axonal transport and which contains a dense collection of dopaminergic anterograde movement across the synapse are both neurons belonging to the a group. the probably involved in the spread of virus from the vta-substantia nigra projects to a number of strucolfactory epithehum to the brain, anterograde spread tures, including primary olfactory cortex, the nuclei once inside the brain is relatively inefficient, the of the diagonal band, and the medial prefrontal, direction of hsv- transneuronal movement is strain insular and entorhinal cortices. , , mhv-jhm independent, but our results suggest that hsv- strain fected this area more intensely and earlier in the utilizes retrograde axonal transport and transneucourse of the infection than hsv- , and also infected ronal movement once inside the brain. since both dopaminergic neurons in the a and a groups. both hsv- and mhv-jhm show preferential retrograde viruses primarily infected th + neurons early in their transport, this does not explain the observed differinfection of the vta. by day , only about one-half ences between them. e. m. baknett et al. viral exit from the cell may occur preferentially at a particular site (e.g. dendrites vs soma), exposing only neurons synapsing in that location to virus. hsv- , a dna virus which replicates in the nucleus, buds from the nuclear membrane. mhv, an rna virus: replicates in the cytoplasm, budding into transitional vesicles between the rough endoplasmic reticulum and golgi apparatus, which are then segregated into the constitutive exocytic pathway." while the relationship between virus budding and exit from specific sites in the cell has not been established for either mhv or hsv- , this could contribute to the pattern of virus spread. since both mhv-jhm and hsv- are believed to enter susceptible cells by a receptor-mediated process, the observed patterns of spread may result from the presence of a virus receptor on the postsynaptic neuron. the inability of mhv-jhm to infect the lc, as discussed above, may reflect the absence of the mhv receptor on these neurons. in addition, the cellular location of the receptor on neurons might determine whether anterograde transneuronal movement occurs. if the receptor is located only on the axon terminal: anterograde movement at an axodendritic or dendrodendritic synapse would be unlikely. the receptor for mhv has been identified as one or more members of the murine carcinoembryonic antigen family of glycoproteins.' ~ '~ although the cns receptor for hsv- has not been fully characterized, at least two hsv- receptors which bind different viral glycoproteins are expressed on some cells, ' possibly providing the basis by which strains which differ in surface glycoprotein expression can differ in the direction of transneuronal movement. as more is known regarding the distribution of the cellular receptors for these two viruses, it will be possible to correlate the pattern of viral spread with the distribution of the receptor. finally. viruses may differ in uptake between neurons of different neurotransmitter systems. in addition to its inability to infect the lc, mhv-jhm did not infect other noradrenergic cells groups in the brainstem, despite infection of a large number of brainstem structures. in contrast, both viruses infected the dopaminergic neurons of the vta. it is possible that the inability of mhv-jhm to infect noradrenergic neurons in part determined the pattern of spread observed. further studies are underway to determine the relative importance of this factor in viral spread. viruses that spread transneuronally have become increasingly popular as neural tracers. pseudorabies virus has been used primarily as a tracer for mapping out central connections of peripheral nerves and typically crosses two to three synapses.'@ both mhv and hsv- are useful as systems tracers after direct inoculation into the brain, since they can cross several synapses in a circuit-specific manner. a common assumption underlying the use of viruses for neural tracing is that they move non-specifically along all the available neural pathways, infecting all of the neurons which synapse on infected neurons. our results show quite clearly, hoaever, that mhv-jhm and hsv- infect only a subset of mob connections. the spread of these viruses along unique routes following a common inoculation site indicates that they may be most useful for identifying distinct transneuronal pathways from a structure. our results also have implication for the pathogenesis of neurological diseases, such as alzheimer's disease and parkinson's disease, which may result from virus destruction of neurons or may be triggered by a prior viral infection. , pathways exist from the mob to a number of cns structures affected in various neurological diseases, including, most notably, alzheimer's disease. , our results indicate that it is possible for a virus to enter the mob and infect only a subset of connected structures. in addition, our results also show that two different viruses could enter the cns via the olfactory epithelium (or possibly another entry site) and spread to infect different subsets of connected structures. the resulting clinical and pathological manifestations would be very different, even though the site of entry and spread via a transneuronal route would be the same. further studies using other strains of hsv- or mhv, or other neurotropic viruses, may provide additional evidence for an infectious component to some neurological diseases. both hsv- and mhv-jhm spread transneuronally, in a circuit-specific manner, after intranasal or intrabulbar inoculation. most significantly, while both viruses infected many of the same mob connections, a number of structures were infected exclusively by one of the two viruses. we conclude from these observations that each virus infects a distinct, but partially overlapping, set of neural pathways originating from the mob. some of this specificity appears to be determined by differential infection of neurons expressing specific neurotransmitters, although differences in access to synapses and in receptor distributions most likely also contribute to the observed patterns of infection. this 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of the carcinoembryonic antigen family of glycoproteins mouse hepatitis virus utilized two carcinoembryonic antigens as alternative receptors direction of transneuronal transport of herpes simplex virus in the primate motor system is strain-dependent thank drs m. brown and ed wagner for providing hsv-i, strain and a plasmid containing the vp- gene of hsv- : respectively. we thank gregory evans for helpful discussions and paul reimann key: cord- -f q j iu authors: nick, benjamin c.; pandya, mansi c.; lu, xiaotao; franke, megan e.; callahan, sean m.; hasik, emily f.; berthrong, sean t.; denison, mark r.; stobart, christopher c. title: identification of a critical horseshoe-shaped region in the nsp (mpro, clpro) protease interdomain loop (idl) of coronavirus mouse hepatitis virus (mhv) date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: f q j iu human coronaviruses are enveloped, positive-strand rna viruses which cause respiratory diseases ranging in severity from the seasonal common cold to sars and covid- . of the human coronaviruses discovered to date, emergent and severe human coronavirus strains (sars-cov, mers-cov, and sars-cov- ) have recently jumped to humans in the last years. the covid- pandemic spawned by the emergence of sars-cov- in late has highlighted the importance for development of effective therapeutics to target emerging coronaviruses. upon entry, the replicase genes of coronaviruses are translated and subsequently proteolytically processed by virus-encoded proteases. of these proteases, nonstructural protein (nsp , mpro, or clpro), mediates the majority of these cleavages and remains a key drug target for therapeutic inhibitors. efforts to develop nsp active-site inhibitors for human coronaviruses have thus far been unsuccessful, establishing the need for identification of other critical and conserved non-active-site regions of the protease. in this study, we describe the identification of an essential, conserved horseshoe-shaped region in the nsp interdomain loop (idl) of mouse hepatitis virus (mhv), a common coronavirus replication model. using site-directed mutagenesis and replication studies, we show that several residues comprising this horseshoe-shaped region either fail to tolerate mutagenesis or were associated with viral temperature-sensitivity. structural modeling and sequence analysis of these sites in other coronaviruses, including all human coronaviruses, suggests that the identified structure and sequence of this horseshoe regions is highly conserved and may represent a new, non-active-site regulatory region of the nsp ( clpro) protease to target with coronavirus inhibitors. importance in december , a novel coronavirus (sars-cov- ) emerged in humans and triggered a pandemic which has to date resulted in over million confirmed cases of covid- across more than countries and territories (june ). sars-cov- represents the third emergent coronavirus in the past years and the future emergence of new coronaviruses in humans remains certain. critically, there remains no vaccine nor established therapeutics to treat cases of covid- . the coronavirus nsp protease is a conserved and indispensable virus-encoded enzyme which remains a key target for therapeutic design. however, past attempts to target the active site of nsp with inhibitors have failed stressing the need to identify new conserved non-active-site targets for therapeutic development. this study describes the discovery of a novel conserved structural region of the nsp protease of coronavirus mouse hepatitis virus (mhv) which may provide a new target for coronavirus drug development. we used a combination of alanine-scanning mutagenesis and c-terminal additions and deletions to initially mutate the mhv nsp idl ( table ) . of the amino acids comprising the loop, a total of virus mutants were successfully recovered (p a, r a, a i, v i, v i, p a, q a, and y a), amino acid residues failed to permit virus recovery despite multiple attempts at rescue (y a, d a, q a, q a, and t a), and amino acid residues were not evaluated (l , v , and d ). among the unrecovered mutants, additional attempts to rescue using more conservative amino acid substitutions at residues d (d e) and q (q n) were also unsuccessful. a total of four different c-terminal modifications were also attempted, which included different c-terminal additions (a duplication of residues - and a duplication of residue ) and different c-terminal deletions (a deletion of residues - and a deletion of residue ). all four of these c- terminal modifications to the nsp idl failed to permit virus recovery. analyses of plaque formation, replication, and protease activity reveal a novel temperature-sensitive mutant in the mhv nsp idl. to evaluate the replication kinetics of each of the recovered mhv nsp idl mutants, we infected confluent dbt- cells with an moi of . of each of the idl mutants and titered aliquots over a h period ( fig. a) . all recovered mhv idl mutants exhibited indistinguishable replication kinetics compared to wt mhv. previously, we described a total of separate temperature-sensitive mutations (tsv a, tss a, and tsf l) in the mhv nsp protease whose phenotypes could be suppressed through long-distance second-site suppressor mutations ( , , ). to evaluate whether any of the recovered mhv nsp idl mutants may exhibit a temperature-sensitive phenotype, we performed an efficiency of plating (eop) analysis by comparing the titers of each idl virus by plaque assay determined at a physiologic ( °c) and elevated temperature ( °c) (fig. a) . average eop values were determined by the average ratios of titers at °c compared to °c, with those eop values less than - indicating a greater than -fold reduction in titers at the elevated temperature as being temperature-sensitive (ts). wt mhv exhibited an average eop of . x - . in contrast, previously described ts nsp mutant virus s a, exhibited an average eop of . x - , consistent with the eop previously reported ( ). two separate mhv nsp idl mutants exhibited average eop values less than - and were significantly lower than wt mhv (p< . ): p a and r a. mutant p a exhibited an average eop of . x - . in contrast, idl mutant r a resulted in a much lower average eop of . x - , which was not significantly different from the known ts mutant s a. no other idl mutants exhibited average eops significantly different from wt mhv. these data suggested that mutagenesis of two separate idl residues (p a and r a) have resulted in novel temperature-sensitive phenotypes. to determine whether the observed differences in phenotype for idl mutants p a and r a are due specifically to defects in nsp protease activity or some other long- distance effect, we performed a western blot to evaluate the ability for the p a and r a nsp proteases to process the maturation cleavage of a downstream replicase (pp ab) protein, nsp , during virus replication (fig. b) . lysates from wt-, p a-, and r a-infected dbt- cells were compared for nsp -mediated nsp processing at °c compared to °c. wt-mhv and p a exhibited approximately equivalent levels (ratios of . and . , respectively) of nsp protein detected at both temperatures. consistent with its temperature-sensitive eop, virus mutant r a exhibited reduced nsp protein detected at °c compared to °c (ratio of . ) and when normalized to wt, exhibited an approximate % reduction in mature nsp protein produced at the elevated temperature. these data demonstrate that mhv nsp idl mutation r a is associated with reduced nsp activity at °c, whereas no appreciable difference in processing at °c was detected for mutant p a. to assess the impact of elevated temperature on replication of the recovered mhv idl mutant viruses, we repeated the moi . replication assay in dbt- cells at °c (fig. b) . in contrast to replication at °c, the replication kinetics among the mhv idl strains were far more variable, with most strains exhibiting a delay in logarithmic growth compared to wt mhv. mutant p a, which had shown a temperature-sensitive eop of . x - , failed to exhibit replication kinetics that were significantly different for wild-type or the other mhv idl strains. in contrast, mutant strain r a showed significantly delayed replication kinetics to reach the maximal logarithmic growth rate (p< . ) compared to wt mhv consistent with its temperature-sensitive eop of . x - . collectively, these data indicate that mutant r a exhibits both significantly reduced capacity to form plaques and delayed replication kinetics at the elevated temperature of °c compared to wt mhv. reversion analysis of ts mhv nsp idl mutant r a reveals three compensatory second- site suppressor mutations. to identify potential interacting residues and novel regulatory networks within the mhv nsp protease associated with residue r , we performed reversion analysis at °c by expanding and sequencing formed plaques at the inhibitory temperature ( fig. a). a total of plaques were selected at expanded in t flasks for virus collection and sequencing. of these, of these plaques resulted in the original r a mutant virus while of these plaques yielded r a in addition to one of each of three different second-site putative suppressor mutations in nsp : p s, l v, and l i (fig. b) . additional sequencing was performed on these recovered viruses throughout the orf ab coding region and no other mutations were identified. the p s mutation arose within the mhv nsp idl, while residue l is located on the same loop housing the c catalytic residue of the active site. to evaluate whether the emergence of these second-site suppressor mutations aids in viral growth at °c, an eop analysis was performed using these viruses at °c and °c (fig. c) . bottom part of the binding pocket for residues p -p of the substrate (fig. a and b) . modeling using the crystal structure of sars-cov- , residues d and t formed a distinct pocket in and around the p residue of leu, residues t and q establish the back wall of the p binding pocket, and residues q , t , and q are responsible for forming the back (q and t ) and base (q ) of the p and p binding pockets. among the mhv idl mutants which failed to rescue were d a, q a, and q a. amino acid residues d and q are structurally conserved in all sequenced nsp proteases to date ( fig. b) . both d and q are located in a conserved horseshoe-shaped region in the n- terminus of the idl. the d side chain projects from the top of the horseshoe-shaped region towards domain and the protease active site and forms the inner wall pocket for the p binding site. in an alignment of the d residues of mhv, sars-cov, mers-cov, and sars-cov- , the positioning and orientation of the side chain are highly conserved with predicted polar contacts with two additional highly conserved residues r (which is immediately adjacent to the catalytic h ) and y (fig. c) . the q side chain is conserved in its positioning towards the center of the horseshoe-shaped region where it shares predicted polar contacts with several other idl residues including a and r (in mhv), r and r (in sars-cov- ), k (in mers-cov), and t (in sars-cov) (fig. d) (including the current sars-cov- pandemic) which collectively highlight both the importance for rapid development of effective therapeutics for the treatment of covid- , but also the need to be prepared for potential future coronavirus outbreaks. in the present study, we evaluated the structure and function of the nsp protease idl, a poorly studied and structurally conserved region of the protease. using site-directed mutagenesis, we demonstrated that some residues and regions of the protease were capable of accepting mutations without apparent defects in viral replication, however a number of residues mostly located within a horseshoe-shaped region in the n-terminus of the protease either failed to permit virus recovery or resulted in a viral temperature-sensitivity. of the amino acid residues comprising the loop, we were able to successfully recover viral mutants at different locations ( table ) . despite the overall structural conservation of the entirety of the loop, the majority of these mutations resulted in no apparent defects in viral replication compared to wt. a few of these residues (a , v , and v ) with no apparent viral defects are known to form the basis of part of the p -p substrate binding pockets of the protease ( , ). yet, compared to the rest of the idl, these residue positions showed among the least sequence conservation (figure b) , which may explain the plasticity with which these residues could tolerate mutagenesis as well as cleavage site variability among coronaviruses ( ). similarly, more c-terminal residues p , q , and y are also found in more variable sequence locations within the idl. collectively, these residue positions may simply represent flexible linker residues than serving additional structural supportive or enzymatic roles within the protease. residues p and r , while rescued when mutated to alanine amino acids, exhibited reduced capacity to form plaques at °c. p is found at a bend leading into the horseshoe shaped region of the idl and may be responsible for helping stabilize the n-terminal anchor of the loop within domain . replication analysis and western blots of the p a mutant virus failed to show significant differences from wt mhv, however the selection of a p s mutation in reversion analysis of r a may suggest that these two residues represent stabilizing and interacting nodes within the protease (figure b ). we previously described different temperature-sensitive mutations in mhv-a (s a, v a, and f l) which all shared overlapping compensatory second-site suppressor mutations ( , , ). all viruses selected for an h y mutation, while the temperature-sensitive v a mutation selected for an s n mutation. furthermore, second-site mutations were identified for f l which were located greater than Å away from the initial mutation. p a is located on an adjacent loop in domain to both s and h (less than Å) in distance (not shown). mhv viral mutant r a was found to exhibit delayed replication kinetics (figure ) , reduced capacity to form plaques (figure ) , and reduced nsp -mediated proteolytic processing at the elevated temperature of °c, consistent with a temperature-sensitive phenotype (figure ) . perhaps surprising, the r residue position was the most variable and least conserved structurally among all hcovs evaluated ( figure b) . structural analysis of the mhv, sars-cov, sars- cov- , and mers-cov revealed that the side chain of the % conserved q appears to form conserved polar interactions with the backbone amino and carboxyl termini of the residue position (figure d) . these data may suggest that q is stabilized within the horseshoe show a high level of amino acid conservation with two of these residues (d and q ) being % conserved across all known coronavirus nsp protease sequences to date (figure b) . all four of these residues are found within a conserved horseshoe-shaped region within the n- terminus of the nsp idl. we propose that this horseshoe-shaped region is a critical region of the protease for both structure and function based on the following observations: ( ) residues with the catalytic dyad h and c residues labeled. predicted polar contacts between q and other residues of the idl are shown. sars-cov has an additional and unique predicted polar interaction with t (shown in red coronaviruses: an overview of their replication and pathogenesis conservation of substrate specificities among coronavirus main inhibition of sars-cov cl protease by flavonoids prediction of novel inhibitors of the main protease (m-pro) of sars-cov- through consensus docking and drug reposition crystal structure of sars-cov- main protease provides a basis for design of improved α-ketoamide inhibitors evaluation of a non-prime site specificities of c and c-like proteases by zinc-coordinating and peptidomimetic insights for wide spectrum anti-coronavirus drug design structure of the main protease from a global infectious human coronavirus, hcov-hku human coronavirus oc cl protease and the potential of ml as a broad-spectrum lead compound: homology modelling and molecular dynamic studies modeling of the key: cord- -oqusfhei authors: ma, yanlin; tong, xiaohang; xu, xiaoling; li, xuemei; lou, zhiyong; rao, zihe title: structures of the n- and c-terminal domains of mhv-a nucleocapsid protein corroborate a conserved rna-protein binding mechanism in coronavirus date: - - journal: protein & cell doi: . /s - - -x sha: doc_id: cord_uid: oqusfhei coronaviruses are the causative agent of respiratory and enteric diseases in animals and humans. one example is sars, which caused a worldwide health threat in . in coronaviruses, the structural protein n (nucleocapsid protein) associates with the viral rna to form the filamentous nucleocapsid and plays a crucial role in genome replication and transcription. the structure of nterminal domain of mhv n protein also implicated its specific affinity with transcriptional regulatory sequence (trs) rna. here we report the crystal structures of the two proteolytically resistant n- (ntd) and c-terminal (ctd) domains of the n protein from murine hepatitis virus (mhv). the structure of ntd in two different crystal forms was solved to . Å. the higher resolution provides more detailed structural information than previous reports, showing that the ntd structure from mhv shares a similar overall and topology structure with that of sars-cov and ibv, but varies in its potential surface, which indicates a possible difference in rna-binding module. the structure of ctd was solved to . -Å resolution and revealed a tightly intertwined dimer. this is consistent with analytical ultracentrifugation experiments, suggesting a dimeric assembly of the n protein. the similarity between the structures of these two domains from sars-cov, ibv and mhv corroborates a conserved mechanism of nucleocapsid formation for coronaviruses. coronaviruses are large, enveloped, positive single-stranded rna viruses, which belong to coronaviridae family, nidovirales order. coronatviruses are the causative agent of many animal and human diseases (rota et al., ) . especially, in , sars-cov caused a worldwide health threat and accounted for over infection and death cases (drosten et al., ; fleischauer and cdc sars investigative team, ; ksiazek et al., ) . the coronavirus has an extraordinary large genome, ranging from~ to . kb. on the basis of antigenic cross-reactivity and sequence similarity, coronaviruses can be assigned to three groups, with hcov- e (group i), mouse hepatitis virus (mhv, group ii), and avian infectious bronchitis virus (ibv, group iii) being the representatives of each group. mhv, which causes liver or neuron infection in mice, is the best-studied coronavirus before the sars outbreak. mhv contains a . -kb positive-sense ssrna genome (lai and stohlman, ; sturman and holmes, ) . the genomic rna is encapsidated by the nucleocapsid (n) protein into a capsid core. the other four structural proteins, including spike (s), membrane (m), envelope (e) and hemagglutinin-esterase (he), surrounded the capsid core to form the crown-like viral particles (sturman and holmes, ) . upon infection into a cell, the virus produces two large polyproteins (pp a and pp ab). they are cleaved by papainlike proteinase (plp ) and the poliovirus c-like proteinase ( cl m pro ) into non-structural proteins, which function as the replication-transcription complexes (rtc) (sturman and holmes, ) . the mhv-a n protein is well-conserved among the various mhv strains. it interacts with genomic rna to form the helical nucleocapsid (macneughton and davies, ; robbins et al., ; baric et al., ; almazán et al., ; sawicki et al., ) , and associates with the membrane glycoprotein via its c-terminal to stabilize virion assembly (kuo and masters, ; hurst et al., ; bednar et al., ; verma et al., ) . it is also considered as an rna chaperone (mir and panganiban, ; zúñiga et al., ) . previous biochemical results indicated that the n protein binds specific rna sequences, e.g., the leader rna zhang et al., ; nelson et al., ) and the packaging signal (molenkamp and spaan, ) . the leader rna contains - nucleotides, which consist of two or three copies of penta-nucleotide sequence (ucuaa) that is critical for virus transcription. nelson et al. ( ) used a rna ligand binding assay to demonstrate that the n protein had a dissociation constant (k d ) of . nm when rna contains ucuaa sequence. they also located the smallest n protein fragment with a significant k d of nm as residues - . the specific interaction of mhv packaging signal and n proteins was observed in vitro, and similar packaging signal or (nucleo)capsid protein interactions have been observed in several other rna viruses, including alphaviruses and retroviruses (molenkamp and spaan, ) . it has been postulated that the packaging signal functions as a selective encapsidation initiation site by its specific interaction with the n protein (molenkamp and spaan, ) . recently, grossoehme et al. ( ) reported that the mhv-n (residues - ) selectively binds to trs (transcription regulatory sequence) rna with high affinity. moreover, van der meer et al. ( ) used immunofluorescence microscopy to prove the co-localization of the n protein with cl m pro , helicase protein and rna polymerase protein in early mhv-a infected cells. using the same assay, bost et al. ( ) reported that pp ab and n protein could be closely localized in vivo. furthermore, the reverse genetic results showed that the rescue of recombinant coronaviruses (tgev, ibv, mhv) from cells can be greatly enhanced when the cells express n protein (almazán et al., ; casais et al., ; coley et al., ) . the n protein of mhv-a is a highly basic phosphoprotein with the molecular weight of kda. it could be sub-divided into three conserved domains: domains i (residues m -a ) and ii (residues d -q ) are basic, and the c-terminal domain iii (residues e -v ) is acidic. a general rna binding region was initially located at residues h -r (masters, ; cologna et al., ; you et al., ) , while the conserved negatively charged amino acids in domain iii are believed to play an important role in n-m protein interactions during assembly . to gain insight into the precise mechanism of n protein, several crystallographic or nmr structural results were reported, including mhv n-terminal rna binding domain (residues - ) (grossoehme et al., ) , two proteaseresistant domains of the n protein from sars-cov (huang et al., ; luo et al., ; yu et al., ; chen et al., ; saikatendu et al., ; takeda et al., ) , and ibv (beaudette strain and gray strain) (fan et al., ; jayaram et al., ) . the two domains of ibv and sars-cov and the flexible linker between them provide a putative binding surface for viral rna. this is supported by reported structures, which also revealed the dimerization of the cterminal domain. thus, a hypothesis for nucleocapsid formation proposes that the n protein self-assembles via its c-terminal dimeric domain, and the viral rna entwines around the protein (jayaram et al., ) . in this work, we report the crystal structures of two proteolytically stable domains of mhv-a n protein. in overall ribbon posture, the high resolution structure of mhv-ntd determined using two forms of crystals with different packing modes is similar to previously reported sars-cov and ibv structures, with a remarkable difference in surface electrostatic distribution. the ctd displayed a tightly intertwined dimerization structure as expected, indicating a potential role in self-association of n protein. these results suggest a similar model, but with exceptions in certain details for rna binding style. mhv-ntd was crystallized into two different packing forms under various conditions. the rod-shaped ntd crystal diffracts to higher resolution ( . Å), comparing to the reported . -Å resolution (grossoehme et al., ). there are two ntd molecules in one asymmetric unit (asu), and they are related by twofold axis. the ntd molecule consists of five βsheets and a single short / helix in the stable core, surrounded by large loops on the periphery (fig. a) , which is consistent with the reported structure of mhv-a ntd (pdb number: hd ) (grossoehme et al., ) . it is notable that the loop corresponding to residues arg -gln was missed due to the lack of electron density, and another crystal structure (packing form of ntd ) provides a good supplement at this point. the crystal of ntd was obtained from another diamondshaped crystal and diffracts to . -Å resolution. its structure was determined by molecular replacement, using ntd monomer as a searching model. comparing to the structure of ntd , ntd has unambiguous density at arg -gln loop, especially at the side chain of lys , which was modeled as an ala in the reported mhv-a ntd structure (pdb code: hd ). the stabilization of this loop has a straightforward explanation based on the crystal packing ( fig. c) : the dotted loops, including residues arg -gln in ntd , are exposed to the solvent, but in ntd , the corresponding loops are fixed at their equilibrium position by the adjacent dimer via hydrogen bonds and hydrophobic interactions between side chains. moreover, the structures of mhv-ntd molecules in these two different crystal forms are identified to share high similarity with a root-mean-squaredeviation (rmsd) of . Å. in the . -Å-resolution structure of ctd, two molecules are related by a non-crystallographic twofold axis in one asymmetry unit (fig. b) . each monomeric subunit consists of two anti-parallel β-strands and five α-helices, among which one helix (α ) and two stands (β , β ) associate tightly with the adjacent monomer. the ctd dimer is a tightly intertwined, domain swapping homo-dimer that looks like a rectangular slab ( fig. a ). in the final refined structure, several residues of n terminus (pro -cys ), c terminus (asp -arg ), and the part between the two strands could not be observed due to the poor electron density. since several homologous structures of ntd and ctd have been reported, we performed a superposition of these structures ( fig. a and a). the rmsd for two mhv-a ntd structures (our structure and the reported hd ) is . Å, the ribbon diagram of ntd monomer. secondary structures (helix, strands and loops) are colored in a rainbow fashion, from blue (n terminus) to red (c terminus). a single helix is labeled as α , and β-strands are numbered from β to β . the disordered loop between strands β and β is sketched by a dotted line. (b) overviews of the homodimer, in which molecule a is in rainbow color. (c) packing mode in the two crystal forms. the comparison clearly explained why the flexible loop in ntd is not flexible in ntd . in ntd , the dotted loops corresponding to residues arg -gln of molecule a and molecule b are exposed to the solvent; while in ntd , colored molecules and form a dimer, in which the loop is fixed by adjacent molecules. (d) sedimentation analysis of ntd by analytical ultracentrifugation (auc). the two curves are the continuous sedimentation coefficient and molar mass distribution of the protein. the molar mass distribution shows a single peak with a molecular mass of . kda, which is consistent with the molecular mass of the monomer. while the ntd structures from different coronavirus showed difference, with a total rmsd for mhv-a (our structure) vs. sars-cov ntds of . Å and that vs. ibv of . Å. the cα backbones of large loops share less similarity than the helix and strands in the core region. the superposition of ctd structures gives the rmsd of mhv-a ctd vs. sars-cov ctd is . Å, and that vs. ibv is . Å. amino acid sequence alignment of n proteins from five representative strains of coronavirus also revealed their similarity (fig. ) . the highly conserved amino acid residues are located in the three strands (β , β and β ) of ntd, and the n-terminal loop in ctd (fig. ) . these fully conserved residues, in addition to many partially conserved residues, contribute to the majority of the secondary structures ( helices, α-helices and βsheets). some of them also play important roles in rna binding, which will be discussed in detail. the ntd exists as a dimer in the asu of crystal (fig. b) : each monomer looks like a bottle with a narrow neck and big belly. the ends of the "necks" in two subunits cross at an angle of approximately degrees, leaving a gap between the "belly" regions. the flexible loops (arg -gln ) in ntd correspond to the crossing necks, which are stabilized by the two bellies from adjacent asymmetric units. it is notable that the two necks seem tightly intertwined, but in fact, they are separated, with a minimum distance of . Å between two loops. since the ntd of mhv n protein exists in two oligomeric forms in the crystals, it is necessary to clarify its oligomerization state, which is monomer, dimer or an equilibrium between the two states. in the dimer structure of ntd , the calculated here the main peak corresponding . kda represents the ctd dimer, and the another peak is meaningless for its too large width and bad symmetry. interface area between two molecules is approximately Å , with a majority of nonpolar residues ( . %). these residues associate via hydrophobic interactions and dominate the dimerization. usually, the protein-protein complexes have a similar structural feature of - involved residues and a buried surface in the range of - Å (janin and chothia, ) . these suggest a weak interaction between the two molecules inside one homedimer, which is consistent with the sedimentation velocity experiment using analytical ultracentrifugation (auc). the auc result proved that the ntd exists as a monomer in solution with a mass of . kda (fig. d) . as suggested previously, the dimer of ctd is tightly intertwined and stable. within the dimer, two subunits are associated through hydrophobic interactions and several salt bridges. these interactions may play an important role in stabilizing the secondary structures of the protein. area calculations indicate that the buried interface area of each molecule is up to Å ( . %, comparing to the total surface area of ctd molecule), formed by a majority of nonpolar residues ( . % comparing to the complete ctd molecule). residues located on the β strand, including leu , ala , tyr , gly , phe and val , contribute to strong hydrophobic interactions for dimerization (fig. b) . the strong interaction between two subunits in the ctd dimer was also demonstrated by auc experiment. the molar mass distribution curve showed a main peak of ctd dimer (fig. c) . importantly, the auc experiment detected the existence of ctd dimers in solution but could not identify other higher-order oligomers. the potential rna binding surface of ntd and ctd unlike the similarity between ntd secondary structures from the three coronaviruses, there are remarkable difference in their rna binding surface. the electrostatic distribution on the surface of mhv-n ntd forms a significant positively charged region, which consists of lys , arg , arg , lys and lys (fig. b ). all these central residues, including the highly conserved arg and lys (fig. ) , form a large contiguous surface. another residue tyr is interpreted to be crucial as the mutant leads to abolish of ntd-trs binding affinity (grossoehme et al., ) , which could be caused by the contribution for the stability of secondary structure. the variation between the three electrostatic surface potentials may result in differences in their rna binding sites (fig. b) . the electrostatic surface of ctd also appears different. in mhv ctd, the dimer surface looks like a dumb-bell, with a positively charged region (including lys , arg , lys protein & cell and lys ) winding around the middle in a spiral (fig. b) . a second positive region consists of lys , lys and arg on the other diagonal. on the surface of sars-cov and ibv ctds, the positively charged regions all located at the middle of the dimer in spite of their different shapes and detailed sites. it is expected that this shared pattern might be important for viral nucleocapsid assembly. because n protein plays an essential role in the formation of viral genome via its self-association, the structural information of n protein from the ibv (group iii) and sars-cov (closely related to group ii) could help propose a possible model for coronavirus nucleocapsid formation. the model is based on two central events: first, both ntd and ctd have multiple putative rna binding sites. in the n protein of ibv, ntd provides a binding surface for viral rna through several crucial residues (lys , lys , lys , arg , lys , lys , arg and arg ) (fan et al., ) , while the ctd also provides a positively charged surface to rna binding (jayaram et al., ) . in the n protein of sars-cov, the residues (arg , arg , arg , arg , lys , arg , arg and arg ) of ntd contribute to rna binding (saikatendu et al., ) , residues thr -pro of ctd are the responsible interacting partner with rna (luo et al., ) , and the long disordered regions between ntd and ctd was also proved capable of binding rna (chang et al., ) . moreover, the ctd acts as a dimeric domain to mediate the clustering of n protein. crystallography and solution structures of ibv-ctd (jayaram et al., ) and sars-ctd (chen et al., ; takeda et al., ) also implicated that the ctd is dimeric characteristic. therefore, they purposed a model that the dimerization of ctd provides a scaffold, while both the ntd and ctd provide multiple rna binding sites. the structural alignments show that the overall folding of ntd and ctd domains of mhv n protein were consistent with that of ibv and sars-cov. previous rna binding assays (masters, ; cologna et al., ; grossoehme et al., ) and the structure surface analysis demonstrated that ntd and ctd both have large positively charged regions for rna binding. furthermore, the interface between two ctd molecules in the crystal and sedimentation velocity experiment confirmed a dimeric ctd architecture. considering the electrostatic distribution (fig. b) , positively charged residues (including lys , arg , lys and lys ) form a spiral line on the surface, which may provide a helical rna binding groove. all the information is consistent with the above models for ibv and sars-cov. the conserved model for coronavirus nucleocapsid formation is summarized as following: the n protein dimerizes via its c-terminal domain, providing a platform to recruit viral rna; the prominent ntd is responsible for recruiting specific or non-specific rnas; the linkers between ntd and ctd may act as a flexible arm to change the relative position of the two domains (fig. ). this conserved model can explain the fundamental mechanism how coronavirus n protein functions; however, there are still some differences among different coronavirus, e.g., the rna binding sites in ntd. although continuous positively charged regions exist in all of the three structures, they clearly show different shapes and locations. this region in ibv protein looks like a clamp to fix rna, and the positive regions in the sars-cov and mhv proteins seem to be a binding groove, but in opposite orientations. the surface structures of different proteins possibly determine the different manners of rna-ntd binding, including recognition sites, relative position, binding ratios and affinity. the gene encoding the mhv n protein (mhv-n) was amplified by polymerase chain reaction (pcr) from strain mhv-a (located at nucleotides , - , in the genome). following that, the gene of ntd (n - ) and ctd (n - ) of mhv-n, which are composed by nucleotides , - , and , - , , respectively, were sub-cloned for protein expression and crystallization. the ntd was amplified by pcr with the primers: ′-cgcggatccac-cacttgggctgaccaaac- ′ and ′-ccgctcgagttatcca-gagccttcaacat- ′. the pcr for ctd was performed with the primer pairs: ′-cgcggatccccagtgcagcagtgttttg-gaaag- ′ and ′-cgctcgagttaacgcccttttctttggggctt tg- ′. the pcr strategy introduced a bamhi site via the forward primer and an xhoi site (shown in bold) in the reverse such that the pcr products could be inserted into the pgex- p- vector (ge healthcare) using t ligase. the recombinant plasmids were subsequently transformed into escherichia coli strain bl (de ). for each plasmid, a well-isolated colony was transferred into ml lb medium containing . mg/ml ampicillin and incubated at °c overnight. the cell culture was further grown at °c in lb medium supplemented with ampicillin ( . mg/ml) until the cells reach od of . . protein expression was induced by the addition of . mm isopropyl-β-d-thiogalactopyranoside (iptg) for another h at °c. cells were harvested and lysed by mild sonication in × pbs (phosphate-buffered saline: mm nacl, . mm kcl, mm na hpo , and . mm kh po , ph . ). the supernatants containing the recombinant glutathione s-transferase (gst) fusion proteins, gst-ntd and gst-ctd, were applied to a glutathione sepharose b (ge healthcare) column, followed by on-bead cleavage with prescission protease (ge healthcare) to remove the gst tag. following cleavage, the protein was purified by two chromatography processes: ion exchange chromatography through a pre-packed column resource s (ge healthcare), and then gel exclusion chromatography through a superdex / column (ge healthcare). sds-page analysis showed the protein purity over %, with expected molar masses. the purified ntd and ctd were concentrated to mg/ml using a spin filter for crystallization. selenomethionine-labeled ntd and ctd were expressed in e.coil strain b , and purified by the same procedure as the native protein. as there is no methionine in the ntd, we introduced an i m mutation (numbering protein & cell figure . the corroborated conserved rna-protein binding mechanism in coronavirus. the ctds dimerize to providing a platform to recruit viral rna. the prominent ntd is also responsible for recruiting rna. the linkers between ntd and ctd may act as a flexible arm to change the relative position of the two domains. refers to full-length n protein) for se-met labeling. sedimentation velocity experiments were performed in a proteome-lab™xl- analytical ultracentrifuge (beckman coulter). fresh protein in its own comfortable buffer was centrifuged at , rpm for h in an an ti rotor at °c. protein absorbance was monitored by continuous scans at nm. the protein partial specific volume, buffer viscosity and buffer density were determined using a c(m) distribution model (schuck, ) . the protein samples for analytical ultracentrifugation were prepared at a concentration of od = . in the buffer containing . m hepes, ph . , mm nacl. crystals of the mhv-n ntd and ctd were both grown at °c using the hanging drop diffusion method. one microliter of protein at a concentration of mg/ml was mixed with µl well solution against µl well solution. two different crystal forms of the ntd (ntd is the i m mutant and ntd is wild type) were obtained. for the native and se-met derivation of ntd , the optimal rod-shaped crystals were obtained in . m tris-hcl, ph . and % (w/v) peg . the best diamondshaped crystals of ntd were obtained in the condition of . m ammonium sulfate, . m mes, ph . , and % (w/v) peg-mme within d. in the case of ctd and its se-met derivative, the crystals were obtained in the optimal condition containing . m sodium citrate (ph . ) using crystal seeds initially generated in . m sodium citrate (ph . ). prior to data collection, all these crystals were transferred to the reservoir solution (supplemented with m sodium formate) for - min dehydration before plunged into liquid nitrogen for storage. a . -Å resolution single wavelength desperation (sad) data set of the se-met labeling ntd was collected at k using an sbc × ccd detector on beamline bl -id at the advanced photon source (aps, argonne national laboratory) at the wavelength of res. in generously allowed regions ( %) ( %) ( %) where h i h i is the mean of the observations i ih of reflection h. b r work = Σ(||f p (obs)| − |f p (calc)||)/ Σ|f p (obs)|; r free = r factor for a selected subset ( %) of the reflections that was not included in prior refinement calculations. c numbers in parentheses are corresponding values for the highest resolution shell. . Å. data for ntd was collected to . Å resolution on beamline bl- a of the photon factory (japan) using an adsc q detector. data of the se-met labeling ctd was collected to . -Å resolution on bl- a of photon factory (japan) at the wavelength of . Å. the crystal of ntd belongs to the orthorhombic space group p with the cell parameter of a = . Å, b = . Å, c = . Å, α = β = γ = °, while ntd belongs to the space group of p with the cell parameters a = . Å, b = . Å, c = . Å, α = β = γ = °. the ctd belongs to the space group p with cell parameters of a = b = . Å, c = . Å, α = β = γ = °. diffraction processing, scaling and integration were performed by using the hkl software package (otwinowski and minor, ) . the structure of ntd was solved by the single-wavelength anomalous dispersion (sad) method from a se-met derivative. the initial phases were calculated by the program solve (terwilliger and berendzen, ) . density modification was performed using resolve (terwilliger, ). an initial model of ntd was automatically traced using the program arp/warp (perrakis et al., ) to approximately % of total residues and then further manually built and refined using the programs coot (emsley and cowtan, ) and refmac (bailey, ) at . -Å resolution to a final r work of . % and r free of . %. the residues from arg to gln missed due to lack of electron density. the structure of ntd were phased using molecular replacement (mr) in phaser (mccoy et al., ) , with the previously solved ntd structure as initial searching model and then was manually build using coot and refined using refmac (bailey, ) at . -Å resolution to a final r work of . % and r free of . %. the ctd structure of . -Å resolution was also determined using sad method. data was collected and phased following a similar procedure to ntd and finally refined to a final r work of . % and r free of . %. the stereochemistry of all the structures was validated by the program procheck (laskowski et al., ) . the statistics of data collection and structure refinement are summarized in table . the nucleoprotein is required for efficient coronavirus genome replication engineering the largest rna virus genome as an infectious bacterial artificial chromosome the ccp suite: programs for protein crystallography interactions between coronavirus nucleocapsid protein and viral rnas: implications for viral transcription importance of mhv-cov a nucleocapsid protein cooh-terminal negative charges four proteins processed from the replicase gene polyprotein of mouse hepatitis virus colocalize in the cell periphery and adjacent to sites of virion assembly reverse genetics system for the avian coronavirus infectious bronchitis virus multiple nucleic acid binding sites and intrinsic disorder of severe acute respiratory syndrome coronavirus nucleocapsid protein: implications for ribonucleocapsid protein packaging structure of the sars coronavirus nucleocapsid protein rna-binding dimerization domain suggests a mechanism for helical packaging of viral rna recombinant mouse hepatitis virus strain a from cloned, full-length cdna replicates to high titers in vitro and is fully pathogenic in vivo identification of nucleocapsid binding sites within coronavirus-defective genomes the pymol molecular graphics system identification of a novel coronavirus in patients with severe acute respiratory syndrome coot: model-building tools for molecular graphics the nucleocapsid protein of coronavirus infectious bronchitis virus: crystal structure of its n-terminal domain and multimerization properties outbreak of severe acute respiratory syndromeworldwide coronavirus n protein nterminal domain (ntd) specifically binds the transcriptional regulatory sequence (trs) and melts trs-ctrs rna duplexes structure of the n-terminal rna-binding domain of the sars cov nucleocapsid protein a major determinant for membrane protein interaction localizes to the carboxy-terminal domain of the mouse coronavirus nucleocapsid protein minireview:the structure of protein-protein recognition sites x-ray structures of the n-and c-terminal domains of a coronavirus nucleocapsid protein: implications for nucleocapsid formation a novel coronavirus associated with severe acute respiratory syndrome genetic evidence for a structural interaction between the carboxy termini of the membrane and nucleocapsid proteins of mouse hepatitis virus rna of mouse hepatitis virus main-chain bond lengths and bond angles in protein structures carboxyl terminus of severe acute respiratory syndrome coronavirus nucleocapsid protein: self-association analysis and nucleic acid binding characterization ribonucleoprotein-like structures from coronavirus particles localization of an rna-binding domain in the nucleocapsid protein of the coronavirus mouse hepatitis virus phaser crystallographic software characterization of the rna chaperone activity of hantavirus nucleocapsid protein identification of a specific interaction between the coronavirus mouse hepatitis virus a nucleocapsid protein and packaging signal high affinity interaction between nucleocapsid protein and leader/intergenic sequence of mouse hepatitis virus rna processing of x-ray diffraction data collected in the oscillation mode automated protein model building combined with iterative structure refinement rna-binding proteins of coronavirus mhv: detection of monomeric and multimeric n protein with an rna overlayprotein blot assay characterization of a novel coronavirus associated with severe acute respiratory syndrome ribonucleocapsid formation of severe acute respiratory syndrome coronavirus through molecular action of the n-terminal domain of n protein functional and genetic analysis of coronavirus replicase-transcriptase proteins size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling specific interaction between coronavirus leader rna and nucleocapsid protein the molecular biology of coronaviruses solution structure of the c-terminal dimerization domain of sars coronavirus nucleocapsid protein solved by the sail-nmr method maximum-likelihood density modification automated mad and mir structure solution localization of mouse hepatitis virus nonstructural proteins and rna synthesis indicates a role for late endosomes in viral replication identification of functionally important negatively charged residues in the carboxy end of mouse hepatitis coronavirus a nucleocapsid protein trafficking motifs in the sars-coronavirus nucleocapsid protein crystal structure of the severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein dimerization domain reveals evolutionary linkage between corona-and arteriviridae coronavirus leader rna regulates and initiates subgenomic mrna transcription both in trans and in cis coronavirus nucleocapsid protein is an rna chaperone abbreviations asu, asymmetric unit; auc, analytical ultracentrifugation; ctd, cterminal domain; hcov- e, human coronavirus; ibv, avian infectious bronchitis virus; mhv, murine hepatitis virus; n protein, nucleocapsid protein; ntd, n-terminal domain; sad, single-wavelength anomalous dispersion; sars-cov, severe acute respiratory syndrome coronavirus; sds-page, sodium dodecyl sulfate polyacrylamide gel electrophoresis; tgev, transmissible gastroenteritis virus; trs, transcriptional regulatory sequence key: cord- -kq wmavt authors: kasai, h.; morita, e.; hatakeyama, k.; sugiyama, k. title: characterization of haemagglutinin-esterase protein (he) of murine corona virus dvim by monoclonal antibodies date: - - journal: arch virol doi: . /s sha: doc_id: cord_uid: kq wmavt we analyzed the characteristics of seven monoclonal antibodies (mabs) raised against purified he (hemagglutinin-esterase) glycoprotein of the murine coronavirus dvim (diarrhea virus of infant mice). immunocrossreaction of these mabs with jhm and/or mhv-s suggest that antigenic epitopes of he of dvim are similar to those of jhm and/or mhv-s. four mabs ( b , aff , c , b ), designated as group a mabs, strongly inhibited both ha and ae activities. on the other hand, three mabs ( aff , aff , aff ), referred to as group b, had a comparatively weak ha inhibition activity. these results indicate that the antigenic epitopes of this glycoprotein can be classified into at least two groups and that the functional sites of ha and ae activities are similar but not identical. neutralizing activity was shown in group a mabs exclusively, suggesting that the ratio of ha and/or ae activities may play important roles in the cell fusion activity of dvim-infected cells. coronaviruses are enveloped viruses containing a single-stranded rna genome of positive polarity [ ] [ ] [ ] . murine coronavirus mhv-dvim is an enteropathogenic coronavirus causing severe diarrhea in newborn mice [ ] [ ] [ ] . as described in a previous report [ ] , by treatment of dvim with the non-ionic detergent np- we isolated the he protein, which carries the functional sites for both acetylesterase (ae) and the haemagglutination (ha) activities. the contribution of these activities to infection by mhv-dvim is not yet known, nor is their relation to the antigenic epitopes. to know the antigenic properties of he, several monoclonal antibodies (mabs) raised against the purified protein were established and used to characterize the he protein. in the present report, we provide evidence that the functional sites for ae and ha activities have separate locations on the he protein. furthermore, mabs that inhibit ae activity also suppress the ability of dvim to cause cell fusion from within. murine coronavirus mhv-dvim was grown in dbt cells using eagle's mem supplemented with % newborn calf serum (gibco) and % tryptose phosphate broth (difco). the virus was purified as described previously [ , ] . the ae activity was determined according to the methods described by vlasak et al. [ , ] . purified virions dissolved in l of pbs (l g/l) were reacted with l of mabs ( . g/l) (dilution rate − to − ) for h at room temperature. after addition of l of esterase substrate (l mm p-nitrophenilacetate: dissolved in % acetonitril), the solution was incubated for min at • c, and hydrolysis of the substrate was monitored at od nm with a spectrophotometer (hitachi - ). the inhibitory effect was expressed as the percentage against a blank that contained pbs instead of the virus. ha assays were performed in v-shaped microtiter plates as described previously [ ] using . % (vol/vol) erythrocytes of balb/c mice (in dulbecco's pbs, ph ) supplemented with . % bovine serum albumin (bsa: sigma). the titer was recorded as the reciprocal value of the highest dilution causing a detectable ha reaction. hai assays were also carried out in microtiter plates. aliquots of l of purified virions ( -hau) were reacted with l of serial two-fold dilutions of mabs in a v plate. after h at • c, l of . % (v/v) erythrocytes were added. after incubation for h at • c, the hi titer was recorded as the reciprocal value of the highest dilution causing a detectable inhibitory effect. the preparation method was the same as that described in a previous paper [ ] . neutralization tests by mabs were carried out by a semimicro reduction assay [ , ] . the virus ( . tcid /ml) was mixed with diluted antibodies, incubated for h at • c, and plated onto -well plates. after adsorption for h at room temperature, the plates were washed with pbs (ph . ), and the culture medium was added to the cells. the neutralization titer was expressed as the highest antibody dilution that prevented virus infection (tcid /ml). the ratio of fusion formation was calculated from the number of nuclei in which the cells formed a syncytium. since gp was identified as the he glycoprotein of dvim [ ] , we attempted to characterize the antigenic sites of this glycoprotein. we obtained seven clones a lgg / + + + a hai was determined as described in materials and methods b aei was determined at the dilution of : of mabs c neutralization activity was expressed as the reciprocal of the highest dilution of mab that showed fusogenic cpe at h post infection. cpe (syncytium) (+ + +) developed overall in the cell sheet fig. . reactivity of mabs with the he protein of dvim shown by western blot analysis. after solubilization in a sample buffer with -mercaptoethanol, dvim virions were subjected to electrophoresis on . % sds-polyacrylamide gels. following transfer to a nitrocellulose membrane, the proteins were incubated with an mab as described in materials and methods. as a negative control, pbs was reacted instead of the st antibody. the molecular weights are indicated on the left. the arrow on the right side points to the he protein of dvim of mabs ( c , b , a , b , a , a and a ) directed against the he glycoprotein. all of them were identified as belonging to the igg ( /) isotype (table ) and reacted with the polypeptide of the gp kda of dvim in western blot analysis, indicating that they recognize an epitope that exists on this glycoprotein (fig. ) . these mabs were also examined for the crossreactivity with other strains of mhv as well as with influenza c virus, which also contains an acetylesterase, by western blot analysis. as shown in fig. , six mabs recognized a major kda protein of mhv-jhm but no crossreactivity was observed with mhv- , mhv- and influenza c/miyagi/ virus (fig. ) . however, mab a also reacted with gp of mhv-s. these results indicate that the he glycoprotein of dvim has antigenic similarity with that of mhv-jhm and to some extent also with mhv-s. to determine the biological properties of these mabs, we examined their inhibitory activities against acetylesterase, hemagglutination, and fusion ( from within) activites, respectively. inhibition of acetylesterase (aei) was analyzed by determining the effect of the mabs on the ability of the viral enzyme to release p-nitrophenol from p-nitrophenylacetate. as shown in table , five mabs ( c , b , a , b and a ) showed high aei titers ( - %). two mabs ( a and a ) were found to have low levels ( - %) of aei titer. high hemagglutination inhibition (hai) titers (> ) were measured with three mabs ( b and c ). with two mabs ( b and a ), intermediate values ( - ) were obtained. the mabs ( a , a and a ) had low hai titers (< ∼ ) (table ) . these data suggest that these mabs were classifiable into at least two groups: group a ( c , b and b ) that has a high titer to both activities and an intermediate titer to the ha activity, and group b ( a , a and a ) that has low titers to both activities ( fig. and table ). to determine the correlations between ae and ha active sites, the inhibitory effects against ha and ae activities by dfp were also tested. inhibitory effects were shown only in the ae activity and not in the ha activity (data not shown). these results suggests that ha and ae active sites that exist on the he glycoprotein of dvim are separated etiologically. we examined the cytopathic effect of mabs with respect to the formation of syncytia in dvim-infected cells. fusion was evident at h postinfection (p.i.). the mabs a , a , and a were unable to prevent this cytopathic effects, and no syncytia formation was observed at h p.i. when the virus was treated with mabs b , a and c prior to infection (fig. ) . it should be noted, however, that these antibodies did not completely neutralize dvim. they only delayed control, v virus control, a b , b a , c c , d a , e a , f a . cytopathic effect (fusion from within) is shown by asteriks virus cell fusion activity, because syncytia were observed by h p.i. (fig. ) . these data indicate the existence of a neutralization factor on the he glycoprotein of dvim. the role of the he protein in virus infection is unclear [ ] [ ] [ ] . for some viruses, such as bovine coronavirus and human coronavirus oc , this protein appears to be essential, because no variants or mutants that lack this protein have been found [ , ] . the esterase activity of he has been suggested to play a role either in virus entry or in virus release [ ] [ ] [ ] . though mhv is serologically related, it differs from the other viruses of this serogroup in several aspects. while the bovine and human counterparts use -o-acetylated sialic acid on the plasma membrane for binding to cells, biliary glycoprotein serves as a receptor for mhv [ , , ] . in addition only some strains of mhv contain an he-protein, while others lack such a protein [ , ] . even strains like dvim that contain an he-protein and thus are able to release the o-acetyl groups from n-acetyl- -o-acetylneuraminid acid use biliary glycoprotein as a receptor [ ] . thus, the importance of the he-protein for mhv is not known. to obtain more information about the he protein, we raised antibodies to this protein and analyzed their effect on the biological activities of he. with respect to inhibition of hemagglutination activity and acetylesterase activity, there were antibodies that had a high titer to both activities or a high titer to one activity and an intermediate titer to the other activity. two antibodies had low titers to both activities. from these results, we conclude that there are several epitopes present on the he protein. the data also indicate that the functional sites of the hemagglutination and acetylesterase activities are similar but not identical. none of the antibodies completely neutralized dvim. however, some of the antibodies delayed the onset of syncytium formation and the cytopathic affect. all these antibodies had high aei activities and high or intermediate hai activities. the antibodies that had low titers to both activities did not delay the fusion of infected cells. therefore, a functional acetylesterase activity and/or hemagglutination activity appear to be necessary for optimal infection by dvim. further investigation is needed to determine the exact role of the he protein. the antibodies described here should be helpful in this respect. genetic resistance to mouse hepatitis virus correlates with absence of virus-binding activity on target tissues interaction of mouse hepatitis virus (mhv) spike glycoprotein with receptor glycoprotein mhvr is required for infection with an mhv strain that express the hemagglutininesterase glycoprotein coronaviridae and replication ter meulen v ( ) the structure and replication of coronaviruses the molecular biology of coronavirus hemagglutination and structural polypeptides of a new coronavirus associated with diarrhea in infant mice structural polypeptides of the murine coronavirus dvim haemagglutinin-esterase protein (he) of murine corona virus: dvim human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza c viruses the e protein of bovine coronavirus a receptor-destroying enzyme with acetylesterase activity we wish to h. ebina of hirosaki university, faculty of science for his helpful assistance of this work. key: cord- -qeltdch authors: graepel, kevin w.; lu, xiaotao; case, james brett; sexton, nicole r.; smith, everett clinton; denison, mark r. title: proofreading-deficient coronaviruses adapt for increased fitness over long-term passage without reversion of exoribonuclease-inactivating mutations date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: qeltdch the coronavirus (cov) rna genome is the largest among the single-stranded positive-sense rna viruses. covs encode a proofreading ′-to- ′ exoribonuclease within nonstructural protein (nsp -exon) that is responsible for cov high-fidelity replication. alanine substitution of exon catalytic residues [exon(-)] in severe acute respiratory syndrome-associated coronavirus (sars-cov) and murine hepatitis virus (mhv) disrupts exon activity, yielding viable mutant viruses with defective replication, up to -fold-decreased fidelity, and increased susceptibility to nucleoside analogues. to test the stability of the exon(-) genotype and phenotype, we passaged mhv-exon(-) times in cultured cells (p ), in parallel with wild-type mhv (wt-mhv). compared to mhv-exon(-) p , mhv-exon(-) p demonstrated enhanced replication and increased competitive fitness without reversion at the exon(-) active site. furthermore, mhv-exon(-) p was less susceptible than mhv-exon(-) p to multiple nucleoside analogues, suggesting that mhv-exon(-) was under selection for increased replication fidelity. we subsequently identified novel amino acid changes within the rna-dependent rna polymerase and nsp of mhv-exon(-) p that partially accounted for the reduced susceptibility to nucleoside analogues. our results suggest that increased replication fidelity is selected in exon(-) covs and that there may be a significant barrier to exon(-) reversion. these results also support the hypothesis that high-fidelity replication is linked to cov fitness and indicate that multiple replicase proteins could compensate for exon functions during replication. a paradigm of rna virus biology is error-prone genomic replication due to the lack of proofreading or postreplicative rna repair mechanisms ( ) ( ) ( ) . decreased replication fidelity may constrain rna genome size and complexity and risks the accumulation of deleterious mutations leading to population extinction ( ) ( ) ( ) ( ) . while genetic diversity allows viral populations to adapt rapidly under selective pressure, many mutations are neutral or detrimental to viral fitness ( ) ( ) ( ) ( ) ( ) . research performed with many rna viruses supports the hypothesis that the mutation rate of rna virus replicases has evolved to balance multiple characteristics of the viral population such as genetic diversity, genomic integrity, and virulence. high-or low-fidelity variants are described for many rna viruses infecting animals, including the coronaviruses (covs) murine hepatitis virus (mhv-a ) and severe acute respiratory syndrome-associated coronavirus (sars-cov) ( ) ( ) ( ) ( ) ( ) , as well as foot-and-mouth disease virus ( ) ( ) ( ) ( ) ( ) , poliovirus ( ) ( ) ( ) ( ) ( ) ( ) ( ) , chikungunya virus ( , ) , influenza virus ( ) , coxsackievirus b ( , ) , and human enterovirus ( ) ( ) ( ) . most altered-fidelity variants described to date harbor mutations within the viral rna-dependent rna polymerase (rdrp), are attenuated in vivo, and protect against reinfection, highlighting their potential utility as live attenuated vaccines ( , , , , ) . those studies underscored the importance of understanding the molecular mechanisms by which rna viruses regulate their replication fidelity. viruses in the coronavirinae subfamily have large single-stranded positive-sense rna genomes [(ϩ)ssrna] ( ) , ranging between and kb in length ( ) . covs encode a =-to- = exoribonuclease (exon) in the n-terminal half of nonstructural protein (nsp -exon) ( , ) . cov exon activity depends on conserved magnesiumcoordinating acidic amino acids in three motifs (de-e-d) that together constitute the active site ( fig. ) ( ) . the cov exon is grouped with the de-d-dh superfamily of exonucleases involved in proofreading during prokaryotic and eukaryotic dna replication ( ) ( ) ( ) ( ) ( ) . alanine substitution of cov motif i de residues (de-to-aa) reduces biochemical exon activity in sars-cov ( , ) and human coronavirus e ( ) . mhv-a and sars-cov lacking exon activity [exon(-)] have mutation frequencies -fold to -fold greater than are seen with wt viruses and are highly susceptible to the activity of nucleoside analogues ( ) ( ) ( ) ( ) ( ) ) . thus, all available data to date support the hypothesis that nsp -exon is the first known proofreading enzyme encoded by an rna virus. despite the critical role of exon in virus replication, fidelity, fitness, and virulence, reversion of the exon-inactiviting substitutions ( fig. ) has not been detected following passages in culture, acute passages of sars-cov-exon(-) in aged balb/c mice, and days of persistent sars-cov-exon(-) infection in immunodeficient rag Ϫ/Ϫ mice ( , , , , ) . in this study, we sought to determine whether long-term passage of mhv-a -exon(-) ( passages over year [p ])-here mhv-exon(-)-would result in virus extinction, exon(-) reversion, or compensation for the loss of proofreading. we demonstrate that mhv-exon(-) did not extinguish during passage and adapted for increased replication. mhv-exon(-) concurrently evolved reduced susceptibility to multiple nucleoside and base analogues, consistent with selection for increased replication fidelity. importantly, the exon-inactivating substitutions did not revert. the evolved mutations in mhv-exon(-) nsp and nsp , which encodes the rdrp, accounted for only part of the increased nucleoside analogue resistance of mhv-exon(-) p , implicating multiple replicase proteins in adaptation for viral fitness. the results of this study support the proposed link between cov fidelity and fitness, demonstrate the surprising stability of the exon-inactivating substitutions, and identify additional proteins outside nsp and nsp that may contribute to cov fidelity regulation. long-term passage of wt-mhv and mhv-exon(-). we serially passaged wt-mhv and mhv-exon(-) in delayed brain tumor (dbt) cells times (p ). virus from each passage was harvested once % to % of the monolayer was involved in syncytia, which occurred between and hours postinfection (hpi). passage conditions varied increased replication of mhv-exon(-) p was affected by the presence of potential defective viral genomes or by some other population-based phenomenon, both wt-mhv p and mhv-exon(-) p were plaque purified three times. the plaque-purified viruses replicated indistinguishably from the parent populations (fig. c ). together, these data demonstrate that wt-mhv and mhv-exon(-) populations had adapted for increased replication and that either individual genomes or those derived from a single virus plaque encoded the adaptive changes required by the total population. mhv-exon(-) accumulated -fold-more mutations than wt-mhv but did not revert exon-inactivating substitutions. to determine whether the increased replication of mhv-exon(-) p resulted from primary reversion of exon(-) motif i, we sequenced nsp from infected-cell total rna. mhv-exon(-) p retained the motif i de-to-aa substitutions, demonstrating that primary reversion of exon(-) motif i did not occur. to identify potentially adaptive consensus mutations, we performed full-genome di-deoxy sequencing of mhv-exon(-) p and wt-mhv p . within wt-mhv p , we identified mutations, of which were nonsynonymous (ns) (fig. a) . in contrast, mhv-exon(-) p had total mutations ( ns) (fig. b) . the full-genome sequences have been deposited in genbank, and the mutations for both viruses are listed in tables s and s in the supplemental material. we identified only one mutation shared by both viruses (nsp a t), though it was present in approximately % of the wt-mhv p population by di-deoxy sequencing. both viruses deleted most of the hemagglutinin esterase (he). in mhv-a , he mrna is not transcribed in vitro ( ) ( ) ( ) , and he protein expression is detrimental to mhv-a fitness in cell culture ( ) . wt-mhv p also deleted open reading frame a (orf a), which is dispensable for mhv replication in cell culture ( ) . the c-terminal region of ns within mhv-exon(-) p was truncated and fused to he with a Ϫ frameshift. ns is a phosphodiesterase (pde) that protects viral rna by degrading =-to- = oligoadenylate, the activating factor for cellular rnase l ( ) ( ) ( ) . the portion of ns deleted in mhv-exon(-) p lies outside the pde catalytic domain, in a region of unknown function. c-terminally truncated ns retains enzymatic activity ( ) , but whether these specific deletions and fusions disrupt pde activity remains to be tested. nevertheless, ns is dispensable for mhv replication in immortalized cells ( , ) . details about the deletion sites are provided in fig. s in the supplemental material. within proteins predicted to be part of the replicasetranscriptase complex (nsp - and nucleocapsid) ( ), wt-mhv p had only one ns change, located in the nsp -helicase ( fig. a and table s ). in contrast, mhv-exon(-) p had ns changes within this region ( fig. b and table s ). mhv-exon(-) p displays increased genomic rna accumulation and increased resistance to -fluorouracil. coronaviruses lacking exon consistently display defects in rna synthesis relative to wt strains ( , , ) . to determine whether the increased replication of mhv-exon(-) p was associated with restored genomic rna (grna) production, we measured grna accumulation over time using two-step realtime quantitative pcr ( , ) . mhv-exon(-) p accumulated levels of grna similar to those accumulated by wt-mhv p and wt-mhv p at early time points, while grna levels for mhv-exon(-) p were~ log lower (fig. a ). mhv-exon(-) p grna levels fell below those of wt-mhv and wt-mhv p after h and were similar to those of mhv-exon(-) p at hpi. normalizing to the grna abundance at h for each virus demonstrated that the rates of grna accumulation were similar for all four viruses (fig. b) . these data suggest that the increased replication of p viruses relative to wt-mhv is not fully accounted for by increased rna synthesis. in addition to rna synthesis defects, exon(-) covs have up to -fold-increased mutation frequencies and profoundly increased sensitivity to nucleoside and base analogues relative to wt covs ( , , , , ) . to determine whether the nucleoside analogue sensitivity of mhv-exon(-) was altered by long-term passage, we treated cells infected with parental and passaged viruses with the base analog, -fluorouracil ( -fu). -fu is converted intracellularly into a nucleoside analogue that incorporates into growing rna strands and causes a:g and u:c mutations. for simplicity, we will hereafter refer to -fu as a nucleoside analogue. incorporation of -fu is increased in the absence of exon activity ( ) . all viruses displayed a concentration-dependent decrease in viral titer but differed greatly in their levels of susceptibility to -fu (fig. c ). at m, wt-mhv p titers were reduced by~ log , while mhv-exon(-) p titers were undetectable (Ͼ log fold reduction). wt-mhv -fu sensitivity was not altered by passage. mhv-exon(-) p was less susceptible than mhv-exon(-) p to -fu treatment, with a decrease in titer of only~ . log at m. mhv-exon(-) p remained more sensitive to -fu than wt-mhv, suggesting that wt-like resistance requires an intact exon. these data demonstrate that mhv-exon(-) p evolved resistance to -fu through mutations outside exon(-) motif i. spike mutations in mhv-exon(-) p do not increase resistance to -fu. bacteriophage x acquired resistance to -fu by delaying cell lysis, thereby reducing the number of replication cycles in which -fu can be incorporated ( ) . mhv-exon(-) p had multiple mutations in the spike glycoprotein, including one in the spike furin cleavage site that reduced syncytium formation. to test whether the spike mutations manifested in resistance to -fu, we cloned the spike gene from mhv-exon(-) p into the isogenic mhv-exon(-) background. the recombinant virus demonstrated intermediate replication kinetics between mhv-exon(-) p and mhv-exon(-) p (fig. a ) and did not form syncytia. spike-p also increased the specific infectivity of viral particles (fig. b) . however, the mhv-exon(-) p spike did not affect the sensitivity of the recombinant virus to -fu (fig. c ). thus, any adaptive increase in -fu resistance must be located elsewhere in the genome. mhv-exon(-) passage resulted in unique mutations in nsp and nsp . to date, three proteins have been shown to alter cov sensitivity to -fu: nsp -rdrp, nsp -exon, and nsp (which stimulates exon activity) ( , , ) . neither wt-mhv nor mhv-exon(-) p contained an ns mutation in nsp , and wt-mhv p had no mutations within either nsp or nsp . in contrast, mhv-exon(-) p had ns mutations in nsp and ns mutations in nsp ( fig. and ) , none of which have been described previously in vitro or in viable viruses. within nsp , six mutations were in the predicted rdrp finger, palm, and thumb domains ( fig. a ) ( ) . four residues (h , f , s , and m ) can be visualized on a phyre -modeled structure of the mhv-nsp rdrp, while the remaining residues lie outside the modeled core rdrp (fig. a ) ( ) . one mutation, m t, lies in the cov-specific domain, which is conserved among nidoviruses. this domain has been implicated in membrane targeting in mhv-a ( ) and performs an essential nucleotidylation activity in the arterivirus equine arteritis virus ( ) . however, m t is not predicted to catalyze nucleotidylation. within nsp , ns mutations were identified in the exon domain, and ns mutations were in the c-terminal n -methyltransferase domain (fig. b ). we next modeled the structure of mhv nsp using phyre software ( ), resulting in highest-probability similarity to the sars-cov nsp -nsp complex (pdb c s) ( ) with high confidence (i.e., the calculated probability of true homology between the structures) of % for residues to of mhv-nsp . the model predicts that five mutations are located close to surface of the protein (fig. b ). all three modeled zinc finger domains contain one ns mutation (f y, y h, and l i). two mutations, d e and f y, are located near the interface between nsp and nsp , though neither site has previously been implicated in nsp -nsp interactions ( , , ) . one ns mutation resulted in a d e substitution in exon motif iii, a metal-coordinating active site residue. we previously reported that alanine substitution of d results in an exon(-) phenotype ( ) , but the viability or phenotype of a d e substitution was not tested in that study. these data suggest that a network of residues evolved to regulate nsp and nsp activity or stability in the exon(-) background. fixed mutations in nsp and nsp in mhv-exon(-) p directly correlate with increased resistance to multiple nucleoside analogues. to determine approximately when the mutations in nsp and nsp arose, we performed di-deoxy sequencing across these protein-coding regions roughly every passages (p , p , p , p , p , p , p , p , p , p , p , p , and ). by this method, we detected consensus ns mutations at p , p , and p for nsp and at p and p for nsp (fig. ). both nsp and nsp carried their full complement of p consensus mutations by p , except for a minority variant (d e) in nsp that was maintained at Ͻ % of the population between p and p . these passage levels correlated with increased replication of mhv-exon(-) (fig. b ) and with decreasing sensitivity to -fu (fig. c ). neither replication nor -fu sensitivity of mhv-exon(-) changed substantially between p and p . to determine whether mhv-exon(-) adaptation of proofreading-deficient coronaviruses ® evolved increased resistance to multiple nucleoside analogues, we treated virusinfected cells with three additional analogues that are substrates for viral rdrps: ribavirin (rbv), a guanine analogue that inhibits viral replication through multiple mechanisms, including mutagenesis and inhibition of purine biosynthesis ( ); -azacytidine (azc), an rna mutagen ( ) ; and =-c-methyladenosine (cmea), which is proposed to incorporate in viral rna and terminate nascent transcripts ( ) . as with -fu, we observed dose-dependent sensitivity to rbv, azc, and cmea in all mhv-exon(-) viruses that decreased with increasing passage number (fig. d to f). except for azc, mhv-exon(-) sensitivity did not change between p and p . together, these data demonstrate that mhv-exon(-) evolved increased resistance to multiple nucleoside analogues that correlated with the length of passage and the acquisition of mutations in nsp and nsp . importantly, this occurred in the absence of specific mutagenic selection and without reversion of exon motif i. this increased general selectivity toward all four classes of nucleotide strongly supports the idea of an overall increase in fidelity in mhv-exon(-) p . mutations in nsp partially account for increased resistance of mhv-exon(-) p to multiple nucleoside analogues. we hypothesized that mutations in mhv-exon(-) p nsp and nsp were most likely to impact replication and nucleoside analogue sensitivity based on their enzymatic activities and temporal association with phenotypic changes. to test this hypothesis, we engineered recombinant mhv-exon(-) to encode the p nsp and nsp sequences, alone and together. expression of nsp -p and nsp -p , alone or in combination, altered replication kinetics of mhv-exon(-) without affecting peak titers (fig. a) and increased grna levels above those of mhv-exon(-) p (fig. b ). nsp -p had a greater effect than nsp -p on the sensitivity of mhv-exon(-) to all analogues tested, and the combination of nsp and nsp -p did not increase resistance above that seen with nsp -p alone ( fig. c to e) . none of the recombinant viruses recapitulated the resistance phenotypes of the mhv-exon(-) p population. together, these data demonstrate that nsp -p mutations account only partially for the nucleoside analogue resistance of mhv-exon(-) p and that adaptations in nsp -p mask those in nsp -p . we also can conclude that the nsp -p d e active site mutation does not correct the defect caused by the motif i de-to-aa substitutions. resistance to nucleoside analogues correlates with mhv-exon(-) fitness. we hypothesized that mutations in nsp and nsp provided a fitness advantage to mhv-exon(-) p . we competed the recombinant viruses with a reference mhv-exon(-) virus (p stock) containing silent mutations in the nsp coding region. mutant and reference viruses were detected in the mixed infection by real-time quantitative pcr using dually labeled probes specific for each virus. mhv-exon(-) p showed a modest fitness advantage over the reference p mhv-exon(-) silent strain (fig. f, solid green) . mhv-exon(-) p profoundly outcompeted mhv-exon(-) silent, with Ͼ , -fold more mhv-exon(-) p genomes present at the end of passage (fig. f, dotted green line) . mhv-exon(-) nsp -p had greater relative fitness than mhv-exon(-) nsp -p , and mhv-exon(-) nsp / -p was intermediate between the single recombinants, implicating a complex evolutionary interaction between these two proteins. the measured fitness correlated with the patterns of nucleoside analogue . for panels c to e, the statistical significance of changes in the titer of swapped viruses relative to mhv-exon(-) p at the highest drug concentration tolerated was determined using the mann-whitney test (*, p Ͻ . ; **, p Ͻ . ; ***, p Ͻ . ; ns, not significant). for panel f, the statistical significance for the indicated comparisons was determined using the mann-whitney test. boxed points have the same p value. adaptation of proofreading-deficient coronaviruses ® resistance and rna synthesis associated with mutations in nsp and nsp , suggesting a link between the evolutions of these phenotypes. the result also confirms that nsp and nsp are important but not sufficient to account for the significantly increased fitness of mhv-exon(-) p relative to mhv-exon(-) p . in this report, we describe experimental adaptive evolution of wt-mhv and mhv-exon(-) during long-term passage in cell culture. wt-mhv evolved increased replication kinetics over passages, with few consensus mutations arising in the wt-mhv p genome. in contrast, mhv-exon(-) accumulated -fold-more mutations than wt-mhv, none of which occurred at the exon-inactivating substitutions. nevertheless, mhv-exon(-) p demonstrated increased replication kinetics and fitness compared to mhv-exon(-) p ( fig. and ) . our previous studies demonstrated that exon-mediated proofreading is required for cov fitness ( , , ) . thus, mhv-exon(-) was likely under selective pressure for restoration of high-fidelity replication or for tolerance of the increased mutational load. consistent with this hypothesis, mhv-exon(-) p exhibited increased resistance to multiple nucleoside analogues, a phenotype strongly associated with high-fidelity viruses ( , , , ) . our results raise several important questions. in the face of selection for increased fidelity, why did mhv-exon(-) not revert? can mhv replicase proteins mediate high-fidelity replication without exon proofreading? which mechanisms other than increased fidelity can compensate for the loss of proofreading? in the face of selective pressure for increased fidelity, why did mhv-exon(-) not revert? although our data suggest that mhv-exon(-) was under selective pressure for increased fidelity, we detected no primary reversion at the de-to-aa substitutions in mhv-exon(-) at any passage tested. these data are consistent with and significantly extend previous studies reporting genotypic stability of exon(-) motif i in mhv and sars-cov ( , , , , ) . complete reversion to de within exon(-) motif i would require four nucleotide changes. this likely represents a high genetic barrier to reversion, especially given that fitness can be increased by mutations outside nsp -exon (fig. f) ( ) . single and double nucleotide changes within motif i could restore an acidic charge to individual residues (e.g., motif i ea, ad, ed, etc.). however, the active site compositions of deddh exonucleases, such as the klenow fragment, are so stringent that even conservative mutations (d to e or e to d) reduce exon activity by Ͼ % ( ). thus, intermediate amino acid changes may not have a selective advantage compared to motif i aa, limiting the evolutionary pathways to reversion. however, nsp -p had detectable effects on rbv and azc resistance as well as on the competitive fitness of mhv-exon(-) (fig. f) , demonstrating a modest capacity for fitness adaptation in nsp outside the catalytic residues. whether these mutations resulted from genetic drift or positive selection remains unclear. nevertheless, our data show that mhv-exon(-) can adapt for increased fitness without fully restoring exoribonuclease activity. while some mutations in mhv-exon(-) p likely confer dbt cellspecific selective advantages, others may represent generalizable strategies for overcoming exon(-) defects in other cell types and in other coronaviruses. thus, understanding the mechanisms by which mhv-exon(-) p compensated for exon activity could allow recovery of exon(-) variants of other covs, such as transmissible gastroenteritis virus and human cov e, which to date have been nonviable as exon(-) recombinants ( , ) . can mhv replicase proteins mediate high-fidelity replication without exon proofreading? mhv-exon(-) p exhibits increased resistance to four nucleoside analogues after passage (fig. ) . although resistance to a single nucleoside analogue can evolve without increasing overall fidelity, resistance to multiple nucleoside analogues strongly suggests a broadly increased capacity to discriminate nucleotides ( , , , ) . increased-fidelity variants in rna viruses have most frequently been mapped to rdrps ( , , , ) . thus, if increased fidelity contributes to nucleoside analogue resistance in mhv-exon(-) p , the most likely protein involved would be nsp -p . three findings are consistent with the hypothesis that mutations within nsp -p increase rdrp fidelity. first, nonsynonymous mutations to nsp arose in the low-fidelity mhv-exon(-) strain but not in the presence of proofreading (wt-mhv). second, five of the mutations lie in or near structural motifs important for fidelity regulation in other rdrps. amino acid substitutions in the finger and palm domains have been repeatedly shown to affect viral rdrp fidelity ( , ) , and we have recently reported a finger mutation (nsp -v i) that likely increases the fidelity of the mhv rdrp ( ) . our modeled structure predicts that nsp -p contains three mutations in the palm domain and one in the finger domain, with the m k thumb domain mutation lying near the palm (fig. a) . third, exchange of nsp -p alone into the background of mhv-exon(-) reduced the susceptibility of mhv-exon(-) to three different nucleoside analogues (fig. ) . thus, all data support the hypothesis that nsp -p is a high-fidelity rdrp. we are actively developing biochemical, phenotypic, and deep sequencing assays to quantify the fidelity of nsp -p . importantly, nsp -p accounts only partially for the mhv-exon(-) p nucleoside analogue resistance phenotype (fig. ) , suggesting a possible limit to the compensation achievable by mutating the rdrp alone. further, the effects of mutations in nsp -p and nsp -p are not additive and may be antagonistic when they are isolated from the whole passaged virus (fig. ) , indicating that the relationships between nsp -and nsp -p mutations are likely evolutionarily linked with those in other mhv proteins. in fact, a substantial component of the evolved resistance to nucleoside analogues cannot be explained by the presence of nsp -p and nsp -p , alone or together. in support of this hypothesis, we identified several nonsynonymous mutations in other replicase proteins, such as nsp , nsp , nsp , and nsp . sars-cov nsp and nsp have functional interactions with nsp , acting as a primase/ processivity factor ( , ) and a helicase/ntpase, respectively ( ) . processivity factors in herpes simplex virus and mycobacterium tuberculosis regulate dna polymerase fidelity by balancing polymerase extension and exonuclease activity ( , ) , and helicases in chikungunya virus and foot-and-mouth disease virus can evolve to increase fidelity ( ) and alter the frequency of ribavirin-induced mutations ( ), respectively. sars-cov nsp has rna-binding activities and is proposed to participate in the multiprotein replicase complex ( , ) , and mhv nsp is a uridylate-specific endoribonuclease ( ) . both could plausibly be involved in modulating polymerase activity. additionally, it remains possible that evolution for increased fidelity could involve proteins outside the canonical replication complex (nsp to nsp ), including those in the structural and accessory cassette. thus, while the immediate studies will focus on testing whether replicase proteins nsp , nsp , nsp , and nsp regulate fidelity, it is exciting to consider the possibility that this virus-directed discovery approach will reveal novel interactions between multiple mhv proteins. which mechanisms other than increased fidelity might account for mhv-exon(-) p nucleoside analog resistance? genomic mutations in rna viruses are most commonly detrimental or lethal ( ) ( ) ( ) ( ) ( ) . one strategy to prevent extinction by mutational load is to increase replication fidelity, as discussed above. an alternative strategy is to increase mutational robustness. mutational robustness describes the capacity of a virus to buffer the fitness effects of mutations. in the setting of low-fidelity replication, as in mhv-exon(-), increased mutational robustness could have provided a selective advantage ( ) ( ) ( ) . selection for increased robustness could explain the~ synonymous changes in mhv-exon(-) p . synonymous changes can alter codons to reduce the probability of nonconservative amino acid changes ( ) . alternatively, the increased population size of mhv-exon(-) p could promote robustness by a "safetyin-numbers" effect, allowing efficient purging of low-fitness mutants while maintaining population fitness ( ) . large populations also increase the likelihood of coinfection, allowing complementation between viral genomes. although increased replication conferred by mutations in spike did not alter -fu resistance (fig. ) , results of a recent study performed with poliovirus suggest that mutagenized populations have elevated coinfection frequencies ( ) . thus, complementation may contribute to mhv-exon(-) p nucleoside analogue resistance. conflicting evidence exists regarding whether mutational robustness itself affects the sensitivity to rna mutagens ( , , ) ; nevertheless, the robustness of mhv-exon(-) p merits further investigation. conclusions. the proofreading activity of the nsp exoribonuclease is a critical determinant of cov replication, fidelity, and fitness. we showed that covs have the capacity to compensate for loss of exon activity through a network of mutations in nsp and nsp and elsewhere in the genome. thus, while nsp -exon appears to play a dominant role in cov replication fidelity, its activity is likely closely tied to a highly evolved network of proteins. the demonstrated coadaptation for replication, competitive fitness, and likely increased fidelity within mhv-exon(-) supports the hypothesis that these roles are linked functionally and evolutionarily. it will be interesting to test whether evolution in other cell types derived from different species or with different innate immune environments would result in similar adaptive strategies. genetic and biochemical testing of the rich mutational resource revealed in mhv-exon(-) p will likely inform the design of countermeasures for endemic and emerging covs by defining novel common targets for stable virus attenuation or direct inhibition. cell culture. dbt- (delayed brain tumor, murine astrocytoma clone ) cells were maintained as described previously ( ) . baby hamster kidney (bhk) cells stably expressing the mhv-a receptor ceacam (bhk-r; ) were maintained under conditions of selection with . mg/ml of g (mediatech) as described previously ( ). long-term passage of virus and stock generation. the infectious cdna clone for mhv-a and the recovery of mhv-exon(-) were described previously ( , ) . long-term passage was initiated by infecting subconfluent monolayers of dbt- cells in -cm flasks with either wild-type mhv-a or mhv-exon(-) at an moi of approximately . pfu/cell. one lineage of each virus was subjected to a total of passages (p ). supernatant was harvested at each passage and stored at Ϫ °c. total rna was harvested for most passages using ml of trizol reagent (ambion) per -cm flask and stored at Ϫ °c. virus stocks of select intermediate passages were generated by infecting a subconfluent -cm flask of dbt- cells at an moi of . pfu/cell. at approximately hpi, the flask was frozen at Ϫ °c and the supernatant was clarified by centrifugation at , ϫ g (sorvall rc- b plus; ha- a rotor) for min at °c. the virus titer of each stock was determined by plaque assay using dbt- cells as described previously ( , ) . for plaque assays of viruses containing the spike protein from mhv-exon(-) p , which does not form syncytia, plaques were visualized with neutral red (sigma catalog no. n ) (dilution at : in phosphate-buffered saline [pbs] containing calcium and magnesium). neutral red was added h after plating, and the reaction mixture was incubated for an additional to h before formaldehyde fixation. plaque purification was performed by infecting dbt cells with serial dilutions of virus and overlaying the cultures with agar. single plaques were isolated, resuspended in pbs containing calcium and magnesium, and inoculated onto fresh dbts. this process was completed times before experimental stocks were generated as described above. sequencing of virus stocks. following p , rna was purified from the harvested trizol samples according to the manufacturer's protocol and reverse transcribed (rt) using superscript iii (invitrogen) as described previously ( ) . full-genome di-deoxy sequencing was performed for both wt-mhv p and mhv-exon(-) p using overlapping amplicons approximately kb in length. all coding regions were sequenced fully, and, of , nucleotides, Ͼ % were sequenced for each virus [for wt-mhv p , to , ; for mhv-exon(-) p , to , ]. two microliters of rt product was used for each pcr ( ) . di-deoxy sequencing was performed by genhunter corporation (nashville, tn) and genewiz (south plainfield, nj). sequence analysis was performed using macvector version (macvector, inc., apex, nc) and the mhv-a infectious clone reference genome (genbank accession number ay ). the nucleotide sequences of the amplicon and sequencing primers are available upon request. sequencing of nsp and nsp from intermediate passages was performed using trizol-purified rna from infected monolayers and the primers listed below. primers m f ( =-ttttggcgagatggtagc- =) and m r ( =-ggtaagacagttttaggtgag- =) were used to generate a , nucleotide amplicon containing all of nsp . primers m f ( =-atgcttaccaactatgagc- =) and m r ( =-ccgatttgaatggcgta g- =) were used to generate a , nucleotide amplicon containing all of nsp . the pcr conditions for these reactions were the same as those used to generate the amplicons used for full-genome sequencing ( ) . replication and rna synthesis kinetics. viral replication kinetics in dbt- cells were determined at an moi of pfu/cell or an moi of . pfu/cell as described previously ( ) . supernatant ( l) was harvested at the indicated time points, and the virus titer was determined by plaque assay. the accumulation of genomic rna at an moi of pfu/cell was measured by two-step real-time quantitative rt-pcr. intracellular rna was harvested using trizol and reverse transcribed using superscript iii (invitrogen). the levels of cdna derived from intracellular positive-sense viral rna were measured using primers directed to nsp . values were normalized to levels of the endogenous control glyceraldehyde- -phosphate dehydrogenase (gapdh). no mutations within the primer binding sites emerged in either p population. the primers and amplification conditions were the same as reported previously ( ), except that the rt product was diluted : prior to use. samples were plated in technical duplicate to minimize well-to-well variation. data are presented as ϪΔct , where Δct denotes the threshold cycle (c t ) value for the target (nsp ) minus the c t value for the reference (gapdh). determination of specific infectivity. subconfluent monolayers of dbt- cells in -well plates were infected with the indicated virus at an moi of pfu/cell, and supernatant was harvested at hpi. the levels of genomic rna in supernatant were measured using one-step real-time quantitative rt-pcr (rt-qpcr) of trizol-extracted rna as described previously ( ) . briefly, genomic rna was detected with a = -carboxyfluorescein (fam)-labeled and = black hole quencher (bhq- )-labeled probe targeting nsp (biosearch technologies, petaluma, ca), and rna copy numbers were calculated by reference to an rna standard derived from the mhv a fragment. samples were plated in technical duplicate to minimize well-to-well variation. titers were determined by plaque assay in dbt- cells, and specific infectivity was calculated as pfu per supernatant genomic rna copy. nucleoside and base analogue sensitivity assays. -azacytidine (azc), -fluorouracil ( -fu), and ribavirin (rbv) were purchased from sigma (product numbers a , f , and r , respectively), and stock solutions were prepared in dimethyl sulfoxide (dmso). cmea ( =-c-methyladenosine) was received from gilead sciences (foster city, ca). sensitivity assays were performed as described previously ( ) , except that -well plates were used at an moi of . pfu/cell. supernatants were harvested at hpi, and titers were determined by plaque assay. phyre modeling of mhv-nsp . the mhv nsp structure was modeled with the phyre online program ( ) using nsp residues to , corresponding to residues , to , of the orf ab polyprotein. the model was analyzed using the pymol molecular graphics system, version . (schrödinger, llc). generation of nsp and nsp swaps. viruses containing nsp -p or nsp -p or both were generated using the mhv-a reverse genetics system ( ) . rna from the mhv-exon(-) p virus was reversed transcribed with superscript iii (invitrogen) and used to generate amplicons containing either nsp or nsp . each amplicon was flanked by bp that overlapped an amplicon generated from the backbone plasmid. amplicons were inserted into mhv-a fragments using an infusion hd cloning kit (takara bio usa, inc., mountain view, ca). nsp is split across mhv e and f, while nsp is contained within mhv f. reaction mixtures contained ng of vector, ng of insertion, and l of enzyme and were incubated for min at °c. errors were corrected by site-directed mutagenesis using pfu turbo polymerase (agilent, santa clara, ca). the nsp / -p swap was generated through restriction digestion of the individual swaps using bsmbi and stui followed by gel purification and assembly using t dna ligase (neb, ipswich, ma). viable viruses were constructed and rescued as described previously ( ) . competitive fitness assays. competitor viruses were competed with an mhv-exon(-) virus harboring silent mutations in the probe-binding region within nsp . subconfluent dbt- monolayers in -well plates were coinfected at a total moi of . pfu/cell with competitor and reference viruses at a : ratio and passaged times. for each passage, supernatants were harvested at h. rna was extracted from l of supernatant using l of trizol reagent and purelink rna minikit columns (thermo scientific, waltham, ma), and l of supernatant was used to infect fresh cells in a -well plate (total moi estimated at pfu/cell). the proportion of each virus was determined by real-time rt-qpcr from the infection supernatant using two taqman probes with different fluorescent dyes in separate reactions. competitor viruses were detected with the same probe used in specific infectivity analyses ( ) . reference viruses were detected by a probe targeting the same region but with silent mutations ( =-tccgaactactgcaaccccaagtg- =) and labeled with = quasar and = black hole quencher (bhq- ) (biosearch technologies, petaluma, ca). rna copy numbers were calculated by reference to an rna standard generated by in vitro transcription of the corresponding mhv a fragment, and relative rna abundances were calculated as ratios of competitor genomes to reference genomes. statistical analysis. graphpad prism (la jolla, ca) was used to perform statistical tests. only the comparisons shown (with statistical significance indicated as ЉnsЉ [nonsignificance] or asterisk[s]) within each figure or described in each legend were performed. in many cases, the data were normalized to the results obtained with untreated controls. this was performed using graphpad prism . the number of replicate samples is denoted within each figure legend. accession numbers. full-length genome sequences for wt-mhv p and mhv-exon(-) p have been deposited in genbank 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putative mouse hepatitis virus rna-dependent rna polymerase discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the rna polymerase-containing protein of all nidoviruses the phyre web portal for protein modeling, prediction and analysis coronavirus nsp , a critical co-factor for activation of multiple replicative enzymes murine hepatitis virus replicase protein nsp is a critical regulator of viral rna synthesis ribavirin's antiviral mechanism of action: lethal mutagenesis? -azacytidine and rna secondary structure increase the retrovirus mutation rate inhibition of hepatitis c virus rna replication by =-modified nucleoside analogs the the '- ' exonuclease of dna polymerase i of escherichia coli: contribution of each amino acid at the active site to the reaction mutagenesis of coronavirus nsp reveals its potential role in modulation of the innate immune response fidelity of nucleotide incorporation by the rna-dependent rna polymerase from poliovirus dna replication across taxa one severe acute respiratory syndrome coronavirus protein complex integrates processive rna polymerase and exonuclease activities a second, non-canonical rna-dependent rna polymerase in sars coronavirus mechanism of nucleic acid unwinding by sars-cov helicase the herpes simplex virus type dna polymerase processivity factor increases fidelity without altering pre-steady-state rate constants for polymerization or excision the ␤ clamp in the mycobacterium tuberculosis dna polymerase iii ␣␤ replicase promotes polymerization and reduces exonuclease activity viral polymerase-helicase complexes regulate replication fidelity to overcome intracellular nucleotide depletion involvement of a joker mutation in a polymerase-independent lethal mutagenesis escape mechanism the severe acute respiratory syndrome-coronavirus replicative protein nsp is a single-stranded rna-binding subunit unique in the rna virus world the severe acute respiratory syndrome coronavirus nsp protein is an endoribonuclease that prefers manganese as a cofactor selection for robustness in mutagenized rna viruses evolvability and robustness in populations of rna virus ⌽ evolution of mutational robustness in an rna virus codon usage determines the mutational robustness, evolutionary capacity, and virulence of an rna virus the role of mutational robustness in rna virus evolution plaques formed by mutagenized viral populations have elevated coinfection frequencies mutational robustness of an rna virus influences sensitivity to lethal mutagenesis does mutational robustness inhibit extinction by lethal mutagenesis in viral populations? systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a we thank members of the denison laboratory for valuable discussions. t -ai (n.r.s.), t -ai (e.c.s.), and f -ai (e.c.s.), all from the national institutes of health.the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health.we declare that we have no conflicts of interest. key: cord- - b sycgv authors: guo, qirui; zhao, yingchi; li, junhong; liu, jiangning; guo, xuefei; zhang, zeming; cao, lili; luo, yujie; bao, linlin; wang, xiao; wei, xuemei; deng, wei; chen, luoying; zhu, hua; gao, ran; qin, chuan; wang, xiangxi; you, fuping title: small molecules inhibit sars-cov- induced aberrant inflammation and viral replication in mice by targeting s a /a -tlr axis date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: b sycgv the sars-cov- pandemic poses an unprecedented public health crisis. accumulating evidences suggest that sars-cov- infection causes dysregulation of immune system. however, the unique signature of early immune responses remains elusive. we characterized the transcriptome of rhesus macaques and mice infected with sars-cov- . alarmin s a was robustly induced by sars-cov- in animal models as well as in covid- patients. paquinimod, a specific inhibitor of s a /a , could reduce inflammatory response and rescue the pneumonia with substantial reduction of viral titers in sasr-cov- infected animals. remarkably, paquinimod treatment resulted in % survival of mice in a lethal model of mouse coronavirus (mhv) infection. a novel group of neutrophils that contributed to the uncontrolled inflammation and onset of covid- were dramatically induced by coronavirus infections. paquinimod treatment could reduce these neutrophils and regain antiviral responses, unveiling key roles of s a /a and noncanonical neutrophils in the pathogenesis of covid- , highlighting new opportunities for therapeutic intervention. the ongoing corona virus disease caused by severe acute respiratory syndrome coronavirus- (sars-cov- ) has resulted in unprecedented public health crises, requiring a deep understanding of the pathogenesis and developments of effective covid- therapeutics (wu et al., b; zhu et al., ) . innate immunity is an important arm of the mammalian immune system, which serves as the first line of host defense against pathogens. most of the cells of the body harbor the protective machinery of the innate immunity and can recognize foreign invading viruses (akira et al., ) . the innate immune system recognizes microorganisms via the pattern-recognition receptors (prrs) and upon detection of invasion by pathogens, and prrs activate downstream signaling pathways leading to the expression of various cytokines and immune-related genes for clearing the pathogens including bacteria, viruses and others (akira et al., ) . with regards to sars-cov- infection, an overaggressive immune response has been noted which causes immunopathology zhang et al., ) . in addition, t cell exhaustion or dysfunction has also been observed (diao et al., ; zheng et al., a; zheng et al., b) . besides, some studies suggest that there may be a unique immune response evoked by coronaviruses (blanco-melo et al., ) . however, the nature of these responses elicited by the virus remains poorly understood. accumulating evidences suggest that the neutrophil count is significantly increased in covid- patients with severe symptoms (kuri-cervantes et al., ; liao et al., ; tan et al., ; wu et al., a) . it is believed that neutrophils migrate from the circulating blood to infected tissues in response to inflammatory stimuli, where they protect the host by phagocytosing, killing and digesting bacterial and fungal pathogens (nauseef and borregaard, ; nicolas-avila et al., ) . the role of such a response in host defense against viral infection has not been clearly characterized. a recent study observed a new subpopulation of neutrophils in covid- patients, which have been named developing neutrophils because they lack canonical neutrophil markers like cxcr and fcgr b (wilk et al., ) . however, it is still not clear how this type of neutrophil is activated. moreover, the precise function of these cells is also unknown. alarmins are endogenous, chemotactic and immune activating proteins/peptides which are released as a result of cell injury or death, degranulation, or in response to infection. they mediate the relay of intercellular defense signals by interacting with chemotactic and pattern-recognition receptors (prrs) to activate immune cells in host defense (oppenheim and yang, ; yang et al., ) . currently, the major categories of alarmins include defensins, high-mobility group (hmg) proteins, interleukins (ils), heat shock proteins (hsps), s proteins, uric acid, hepatoma derived growth factor (hdgf), eosinophil-derived neurotoxin (edn), and cathelin-related antimicrobial peptide (cramp) (giri et al., ; yang et al., ) . in response to microbial infection, alarmins are released to initiate and amplify innate/inflammatory immune responses, which involve the activation of resident leukocytes (e.g. macrophages, dendritic cells, mast cells, etc.), production of inflammatory mediators (cytokines, chemokines, and lipid metabolites), recruitment of neutrophils and monocytes/macrophages for the purpose of eliminating invading microorganisms and clearing injured tissues (bianchi, ; chen and nunez, ; nathan, ; oppenheim and yang, ; yang et al., ) . however, uncontrolled production of alarmins is harmful or even fatal to the host in some cases. hmgb protein acts as a late mediator of lethal systemic inflammation in sepsis (wang et al., ) . therefore, anti-hmgb therapeutics have shown to be beneficial in experimental models of sepsis. s a and s a make up approximately % of the cytoplasmic proteins present in neutrophils. both these proteins are members of the s group of proteins. s a and s a are also referred to as mrp and mrp , respectively. under physiological conditions, massive levels of s a and s a are stored in neutrophils and myeloid-derived dendritic cells, while low levels of s a and s a are expressed constitutively in monocytes (foell et al., ; wang et al., ) . s a and s a often form heterodimers (s a /a ) (ometto et al., ) . the major functions of s a /a reported so far include the regulation of leukocyte migration and trafficking, the remodeling of cytoskeleton, amplification of inflammation and exertion of anti-microbial activity (ometto et al., ; wang et al., ) . after being infected with bacteria, neutrophils, macrophages, and monocytes intensely induce the expression and secretion of s a /a to modulate inflammatory processes through the induction of inflammatory cytokines. s a / is an endogenous ligand of toll-like receptor (tlr ) and can trigger multiple inflammatory pathways mediated by tlr (vogl et al., ) . s a and s a also have antibacterial potential via their ability to bind zn + (foell et al., ; wang et al., ) . not much is known about the roles of s a /a in host defense responses against viruses. in the present study, we characterized the nature of the early innate immune responses evoked in rhesus macaques and mice during sars-cov- infection. s a was dramatically upregulated by sars-cov- and a mouse coronavirus (mouse hepatitis virus, mhv), but not by other viruses. a group of non-canonical neutrophils were also activated during sars-cov- infection. the abnormal immune responses were mediated by the s a /a -tlr pathway. s a /a specific inhibitor, paquinimod, significantly reduced the number of neutrophils activated by the coronavirus, inhibited viral replication and rescued lung damage a result of sars-cov- infection. these results highlight the potential of therapeutically targeting s a /a for suppressing the uncontrolled inflammation associated with severe cases of covid- and provide new information on an alarmin-mediated pathway for regulating neutrophil function. to characterize the early immune responses against coronavirus infection, we infected rhesus macaques with sars-cov- and analyzed the transcriptome of infected and non-infected animals ( figure a ). the whole genome wide rna-seq analysis of the lungs from infected rhesus macaques showed that a number of transcripts were induced or inhibited at day and day after sars-cov- infection (supplementary figure a) . the induction of primary antiviral factors, like type i ifns was completely blocked during sars-cov- infection (supplementary figure b) . gene ontology (go) analysis showed that a small group of genes involved in defense responses against viruses was induced ( figure b) . however, interestingly, a greater number of genes involved in regulating cellular responses to lipopolysaccharide (lps) were induced by sars-cov- at day post infection. an analysis of the kegg pathways also showed that sars-cov- induced genes were enriched with more from anti-bacterial pathways than those from anti-viral pathways (supplementary figure c ). in line with this, neutrophil chemotaxis associated genes were also upregulated as a result of sars-cov- infection. this upregulation was less significant at day , but more significant at day when compared to the levels of genes related to antiviral responses that were induced as a result of the viral infection ( figure b ). it is worthy to note that the number of neutrophils increases in covid- patients with severe symptoms (kuri-cervantes et al., ; liao et al., ; tan et al., ; wu et al., a) . our results of the preliminary characterization of the early innate response to a sars-cov- challenge indicate that neutrophils are significantly activated at the very beginning of sars-cov- infection. to confirm this, we examined the expression of neutrophil markers in the lungs from infected rhesus macaques. all the signature genes, from primary granules to tertiary granules, were significantly induced as a result of sars-cov- infection ( figure c ). the markers for monocytes and natural killer cells were slightly upregulated. t cells were unchanged, while interestingly, b cells were significantly down-regulated in the lungs of infected animals ( figure d and supplementary figure d ). taken together, the above data suggested that the sars-cov- infection provoked a non-canonical antiviral response, or an antibacterial response accompanied by increased neutrophils in the lung at the early stage. to explore how an infection by sars-cov- triggers the activation of antibacterial responses, we examined the differential expression of genes before and after coronavirus infection. the expression of alarmin protein s a was robustly upregulated with , -and , -fold induction at day and day post sars-cov- infection, respectively ( figure e ). s a acts as an alarmin through formation of heterodimers with s a , then these heterodimers function as danger associated molecular pattern (damp) molecules and activate innate immune responses via binding to pattern recognition receptors such as toll-like receptor (tlr ). in our studies, s a was also induced during the sars-cov- infection ( figure e ). the basal expression of s a was much higher, but its induction was not as significant as that of s a . in fact, among all the known alarmins, s a was the most significantly induced gene at both day and day post infection ( figure f ). qrt-pcr analysis showed that the level of s a / surged along with an increase in the viral load in the lung of infected animals ( figure g ). next, we examined the expression of alarmins in the blood samples taken from infected rhesus macaques. s a / as well as the neutrophil marker genes were also induced by the viral infection ( figure h ). based on these results, we investigated if s a /a were upregulated in covid- patients. remarkably, both s a and s a were upregulated in post-mortem lung samples from covid- -positive patients when compared with biopsied healthy lung tissue from uninfected individuals ( figure i ). the expression of neutrophil marker genes was also significantly increased in covid- -positive patients ( figure i ). concomitantly, the mrna level of s a was significantly higher in peripheral blood from covid- -positive patients when compared to healthy subjects (supplementary figure e) . a group of alarmins were induced in different types of blood cells of covid- -positive patients. for instance, s a was significantly induced in red blood cells, cd + monocytes, cd + monocytes, neutrophils and developing neutrophils ( figure j ). s a /a is the ligand of tlr , which is the primary prr that recognizes invading gram-negative bacterium and lps. we thus observed a significant induction of genes that were predominantly involved in responses to lps and anti-bacterial pathways. s a /a are also able to induce neutrophil chemotaxis and adhesion, which probably contributed to the increased infiltration of neutrophils. to further investigate the nature of immune responses triggered and the roles of s a in these responses, we challenged hace transgenic mice with sars-cov- and c bl/ mice with influenza a virus (iav), which also infects the respiratory tract. we performed whole genome wide rna-seq analysis to characterize the defense responses during viral infection ( figure d) . interestingly, sars-cov- was only able to induce ifnκ but not any other type i ifns (supplementary figure d ). both sars-cov- and iav were able to induce ifnγ expression. iav, but not sars-cov- , induced type iii ifns production (supplementary figure d) . qrt-pcr further confirmed that ifnβ and isg induction was impaired during sars-cov- infection ( figure c ). the induction of most isgs was attenuated during sars-cov- infection compared with iav infection (supplementary figure e) . however, sars-cov- but not iav induced s a and neutrophil marker genes ly g expression ( figure c ). consistently, other neutrophil marker genes were also induced by sars -cov- but not by iav ( figure d ). similar to the data from rhesus macaque experiments, compared to other alarmins, s a was robustly induced by sars-cov- but not by iav infection in mice ( figure e ). thus, sars-cov- infection specifically induces the transcription of alarmin s a . to further confirm this, we infected c bl/ mice with other rna-or dna-viruses including encephalomyocarditis virus (emcv), herpes simplex virus (hsv- ) and vesicular stomatitis virus (vsv) and measured the expression of s a in the lung of infected animals. none of these viruses were able to induce the expression of s a ( figure f ). we then investigated if other coronaviruses were able to induce the transcription of s a and activate neutrophils. we infected c bl/ mice with mouse hepatitis virus (mhv-a ) intranasally ( figure a) . surprisingly, we did not observe any obvious symptoms in infected mice. we then infected irf /irf double knockout mice and ifnar deficient mice with mhv. similar to the wild type mice, irf /irf double knockout mice were able to eliminate the virus rapidly and did not develop severe pneumonia. interestingly, ifnar deficient mice showed obvious damage in pulmonary interstitium and alveoli ( figure g ). all the mice died within days ( figure h ). we performed whole genome rna-seq analysis to determine if mhv induced similar immune responses as those induced by sars-cov- infection. the lungs of mhv infected mice were collected at day post infection and subjected to rna analysis. similarly, mhv induced genes were enriched in neutrophil chemotaxis and antibacterial pathways ( figure i ). s a was also significantly induced by mhv infection ( figure j ). when compared to iav, type i ifns induction was impaired, but neutrophil marker genes were significantly induced by mhv infection ( figure k and l). histological staining of samples taken from lungs showed more s a positive cells infiltration after mhv infection ( figure m ). since the expression of neutrophil marker genes was elevated in the lung after coronavirus infection, we examined if more neutrophils were infiltrated into lung during infection. surprisingly, the number of ly- g (high) and cd b (high) neutrophils decreased in the lung after mhv infection ( figure n ). interestingly, a group of cd b (high) ly- g (mid) cells showed up after mhv infection. further analysis revealed that these cells were cd + granulocytes. a recent single cell analysis study had reported that a group of developing neutrophils was detected in the peripheral blood of covid- patients with decreased expression of canonical neutrophil markers cxcr and fcgr b (wilk et al., ) . we purified these cells by using flow cytometry sorting. qrt-pcr analysis showed decreased expression of cxcr and fcgr ( figure o ). to explore if other types of viruses or stimuli are able to induce these cells, we challenged c bl/ mice with iav, emcv, vsv, hsv- and lps, hace transgenic mice with sars-cov- through nasal inoculation. interestingly, only sars-cov- and mhv induced this group of neutrophils ( figure p ). taken together, these results suggest that coronavirus infection induces s a and a group of noncanonical neutrophils specifically. s a /a functions as a tlr ligand that amplifies inflammation by triggering the activation of innate immune signaling, including neutrophil chemotaxis. we thus reasoned that the abnormal upregulation of s a /a probably resulted in activation of coronavirus specific neutrophils and dysregulation of immune responses. quinoline- -carboxamide paquinimod (also known as abr- ), is an inhibitor of s a which can prevent the binding of s a to tlr- (bjork et al., ; schelbergen et al., ) , suggesting that it can be used to block the function of s a /a and mitigate the antibacterial immune responses elicited during sars-cov- infection. to test this, we treated the sars-cov- infected mice with s a /a inhibitor paquinimod ( figure a ). expectedly, paquinimod was able to rescue the weight loss of infected mice and inhibited sars-cov- viral replication significantly ( figure b and c). consistently, the expressions of neutrophil markers (ly g, mmp , ltf), il- and s a were significantly reduced after paquinimod treatment ( figure d ). more strikingly, paquinimod completely rescued the death of mhv infected, but not iav infected ifnar deficient mice and significantly inhibited mhv replication ( figure e , f and supplementary figure a ). the expression of neutrophil marker genes in lung and blood from infected mice was also inhibited by paquinimod ( figure g , h and supplementary figure b ). the damages to pulmonary interstitium and alveoli were alleviated, and infiltration of s a + cells was also reduced by paquinimod ( figure i and j). to gain further insights into the modulation of immune responses after paquinimod treatment, we performed genome wide rna-seq analysis using the rna from mhv infected ifnar deficient mice. as expected, pathways related to neutrophil chemotaxis and antibacterial responses were primarily downregulated by paquinimod figure d) . the expression of these b cell related genes was rescued or induced by paquinimod during mhv infection, which was confirmed by qrt-pcr analysis ( figure m ). in line with this, sars-cov- but not iav infection, suppressed the expression of these b cell related genes ( figure n and supplementary figure d ), which were also rescued by paquinimod ( figure o ). we demonstrated that paquinimod inhibited the expression of neutrophil maker genes in lung and blood. to determine if paquinimod could reduce the number of these abnormal cells, we performed flowcytometry to analyze the cell population during coronavirus infection. coronavirus-specific neutrophils were significantly reduced in blood and lung after treatment with paquinimod ( figure a ). it is believed that s a /a can activate tlr or receptor for advanced glycation end products (rage) pathways. interestingly, tlr inhibitor (resatorvid) but not rage inhibitor (azeliragon) was able to reduce the number of these neutrophils suggesting that s a /a activated these neutrophils via tlr pathway ( figure a ). moreover, mhv infection also induced these neutrophils in liver and bone marrow, which could be suppressed by tlr inhibitor ( figure b ). consistently, resatorvid was able to inhibit viral replication in lung and blood of the infected mice ( figure c ). moreover, both paquinimod and resatorvid suppressed the activation of coronavirus related neutrophils in lung during sars-cov- infection ( figure d ). to confirm the role of tlr in activating s a related signaling, we examined s a induction upon lps treatment. lps induced s a expression through adaptor protein myd (supplementary figure a) . we then treated mouse macrophages with the serum from sars-cov- infected mice with or without paquinimod treatment. the serum from infected mice was able to induce the expression of s a and cxcl but not il- β or il- in a myd dependent manner ( figure e and supplementary figure b ). s a further activated tlr signaling to form a positive loop. we thus observed that paquinimod was able to inhibit the induction of s a ( figure d and g-h). cxcl is the primary chemokine that recruits neutrophils. consistent with the in vivo results ( figure a ), azeliragon could not inhibit induction of s a or cxcl by the serum ( figure e ). s a therefore exerted its pathogenic function through activating tlr signaling during coronavirus infections. a recent single cell sequencing data clarified the heterogeneity of neutrophils and identified subpopulations (xie et al., ) . we collected the cd + and ly- g + cells from bone marrow ( figure b ) and examined the expression of each marker gene during mhv infection with or without tlr inhibitor. compared to noninfected mice, mhv induced expression of all g maker genes and maker gene from g to g , but inhibited expression of other maker genes ( figure f ). interestingly, resatorvid was able to reverse the expression of marker genes back to the normal levels. the expression of canonical neutrophil markers cxcr , ly g and fcgr were decreased after mhv infection ( figure g ). we further analyzed the rna-seq data of lung samples taken from the mice infected with sars-cov- and iav. g b population, the matured neutrophil, was activated during iav infection ( figure h and supplementary figure c ). whereas sars-cov- , similar to the results from bone marrow experiments ( figure f ), activated g subpopulation significantly. in addition, sars-cov- also induced other premature neutrophil markers from g to g subpopulation in lung ( figure h and supplementary figure c ). interestingly, paquinimod was able to turn off the abnormal upregulation of these genes (supplementary figure d) . together, these results suggested that coronavirus infection expanded the population of premature neutrophils in s a / and tlr dependent manner, and caused dysregulation of the immune system. innate immunity, the first line of host defense, employs pattern-recognition receptors (prrs) for recognizing pathogen-associated molecular patterns (pamps) of invading pathogens and galvanizing the host defense machinery. the endogenous danger-associated molecular patterns (damps) are also able to trigger the activation of innate immune signaling. alarmins are a panel of proteins or peptides that can function as damps to activate various immune pathways (bianchi, ; yang et al., ) . the fine tuning of the transcription of alarmins is critical for maintaining immune homoeostasis. over or sustained expression of alarmins could result in uncontrolled inflammation (chan et al., ; cher et al., ; kang et al., ; patel, ) . such imbalanced immune responses and cytokine storm contribute to the development of severe acute respiratory distress syndrome (ards). here we demonstrated that sars-cov- and mhv, but not any other tested viruses, induced a robust and sustained transcription of the alarmin s a in animal models (figure d-g and figure ). in line with this, the substantial upregulation of s a was also observed in the lung and peripheral blood of infected macaques and covid- patients ( figure h and i) . although we did observe that the sars-cov- and the other types of viruses could induce s a expression slightly in blood at day post infection ( figure c) , however, the induction of s a by viruses other than sars-cov- was either unchanged or reduced at day post infection. s a expression increased dramatically by folds at day post infection ( figure c ). similar phenotypes were observed during mhv infection ( figure f ). thus, uncontrolled or sustained induction of s a could be a common feature of coronavirus infections. however, the mechanisms by which coronaviruses provoke the induction of s a is unknown. in addition to studying the innate immune responses elicited by sars-cov- infected hace transgenic mice and rhesus macaques, we also infected ifnar deficient mice with mouse coronavirus and characterized the resulting immune responses. the mhv infected mice exhibited a characteristic immune signature comprising of s a induction, initiation of antibacterial like responses and activation of coronavirus related neutrophils. these features of the immune response were similar to those observed in sars-cov- infected mice and rhesus macaques ( figure and figure a and b). untreated mhv infected mice developed more severe symptoms and died within days. mice administered with s a inhibitor, paquinimod, were rescued from lung damage and death against mhv infections. since these results were similar to those obtained during studies with sars-cov- , it seems like a shared mechanism that directs the pathogenesis of pneumonia during sars-cov- and mhv infections in animal models. thus, ifnar deficient mice and mhv through its ability to evoke similar immune responses as sars-cov- , could serve as useful models for investigating ards associated with sars-cov- infection. although we inoculated wild type, mavs knockout, sting knockout, elf knockout and irf /irf double knockout mice with mhv intranasally, none of these mice developed obvious ards related symptoms. irf and irf are key transcription factor of type i ifns. type i ifns activate interferon stimulated genes by binding ifnar. since the irf /irf double knockout phenotype was not susceptible to mhv infection, it seems like ifnar can exert its function in a type i ifns independent way. previous studies (hadjadj et al., ; zhou et al., ) and our above data showed that induction of type i ifns is completely blocked during sars-cov- and mhv infections. this further suggests that ifnar is involved in defense against coronavirus with an unknown mechanism. there is consensus that the neutrophil number is significantly increased in covid- patients with severe symptoms (kuri-cervantes et al., ; liao et al., ; tan et al., ; wu et al., a) . moreover, a recent study identified that a group of premature neutrophils in covid- patients that do not express canonical neutrophil markers like cxcr and fcgr b. bacterial coinfection is common in viral pneumonia, especially, in critically ill patients (bost et al., ) . neutrophils can be activated during bacterial infection and are critical for killing invading bacteria (deng et al., ; li et al., ) . the increased neutrophil number and developing neutrophils in patients could be attributed to coinfection of bacterium. however, results of our studies showed that the premature neutrophils were induced by coronavirus at the early stage of infection in animal models. we compared these cells with neutrophil subpopulations that were proposed in a recent study. coronavirus induced neutrophils in bone marrow and lung belong to the g population predominately ( figure e and supplementary figure b) . furthermore, the populations of neutrophils in lungs are more diverse than those in bone marrow. compared to iav induced neutrophils, coronavirus preferentially induces the expression of premature marker genes (supplementary figure b) . however, the exact mechanism by which coronavirus induces these abnormal neutrophils and the key transcription factors that direct development of these cells await to be further investigated. in summary, we have demonstrated that alarmin s a was specifically upregulated by sars-cov- infection. s a /a amplified inflammatory responses by activating tlr signaling pathway. a group of coronavirus-specific premature neutrophils were activated during infection. the inhibitors of s a /a -tlr axis were able to mitigate abnormal inflammation and inhibit viral replication. these results uncover the role of alarmins and neutrophils in the pathogenesis of sasr-cov- infection and provide new therapeutic targets for the treatment of covid- . cd , mmp , cd , and cd in the lung of sars-cov- -infected rhesus macaques at dpi, dpi, and dpi. n = . (e) rna-seq analysis of rna from lung of rhesus macaques infected intranasally with sars-cov- at dpi and dpi. differential expressed genes represented by s a and s a were analyzed (compared with mock). (f) analysis of all known alarmins showing that s a is the most significantly induced one (e). n = . (g) qrt-pcr analysis for viral load and the expression level of s a and s a in the lung of sars-cov- -infected rhesus macaque at dpi, dpi, and dpi. vehicle treated group and paquinimod treated group at dpi, dpi, dpi, dpi, and dpi. n = . (*p < . ; **p < . ; ***p < . ; ****p < . ). before being inoculated intranasally with reagents and viruses, hace mice were intraperitoneally anaesthetized by . % avertin with . ml/g body weight; wt c bl/ j mice, interferon-α receptor gene knockout mice (ifnar -/-), elf -/mice, ifnar -/mice and mavs -/mice were anaesthetized by isoflurane; rhesus macaques were anaesthetized with mg/kg ketamine hydrochloride. the health status and body weight of all mice were observed and recorded daily. mice were euthanized at , , , and dpi to collect different tissues and examine virus replication and histopathological changes. vsv (vesicular stomatitis virus, indiana strain) was a gift from j. rose (yale university), iav (influenza a virus, pr ) was a gift from feng qiang (fudan university), and hsv- (herpes simplex virus ) was from a. iwasaki (yale university). emcv (encephalomyocarditis virus, vr- b) was purchased from american type culture collection (atcc). mhv-a (mouse hepatitis virus a- ) has been described previously (yang et al., ) . the sars-cov- which has been used in this paper is named strain hb- . the complete genome for this sars-cov- had been put in to gisaid (betacov/wuhan/ivdc-hb- / |epi_isl_ ), and has been described previously . vsv, emcv, hsv- were propagated in vero cells followed by cycles of freezing and thawing, then the large debrises were spun down and the supernatants were used as a stock solution. the titer of the viruses was determined by plaque assay in vero cells. for mice infection assay, wt mice were inoculated intranasally with vsv ( pfu), emcv ( pfu), hsv- ( pfu) after anesthesia. pfu: plaque forming unit. mhv-a were propagated in cl- cells followed by cycles of freezing and thawing, the large debrises were spun down and the supernatants were used as a stock solution. the titer of the viruses was determined by plaque assay in cl- cells. for mice infection assay, ifnar -/mice were inoculated intranasally with mhv-a ( pfu) after anesthesia or ifnar -/mice were inoculated intraperitoneally with mhv-a ( pfu). iav was propagated in -day-old specific-pathogen-free embryonic chicken eggs. the allantoic fluid was collected and titrated to determine the % tissue culture infection dose (tcid ) in a cells and the median lethal dose (ld ) in mice following the reed-muench method. for mice infection assay, wt mice were inoculated intranasally with iav ( tcid ) after anesthesia. for the paquinimod rescue assay, wt mice, hace mice, and ifnar -/mice were inoculated intranasally with iav, sars-cov- , and mhv-a respectively after anesthesia. the mice were given intranasally . μg/day of paquinimod (targetmol; catalog no. t ) starting on dpi. the control group mice were given intranasally μl of pbs. stock solutions of mg/ml paquinimod were prepared with dmso in advance. the health status and body weight of all mice were observed and recorded daily. mice were euthanized at , , , and dpi to collect different tissues and examine virus replication and histopathological changes. for serum co-culture assay, μl peripheral blood was collected from each group of ifnar -/mice on day during paquinimod rescue assay. after blood coagulation at room temperature, samples were spun down and the supernatants were used as a stock serum. raw . cells were seeded on -well plates with cells/ml. after cell adherence, lps ( ng/ml) and mice serum which has been collected above ( μl/ml) were added. after hours co-culture, cells were harvested and lysed by trnzol reagent for rna extraction. whole rna of cells with specific treatment were purified using rneasy mini kit (qiagen no. ). the transcriptome library for sequencing was generated using vahtstm mrna-seq v library prep kit for illumina® (vazyme biotech co.,ltd, nanjing, china) following the manufacturer's recommendations. after clustering, the libraries were sequenced on illumina hiseq x ten platform using ( × bp) paired-end module. the raw images were transformed into raw reads by base calling using casava (http://www. illumina.com/ support/ documentation.ilmn). then, raw reads in a fastq format were first processed using in-house perl scripts. clean reads were obtained by removing reads with adapters, reads in which unknown bases were more than % and low quality reads (the percentage of low quality bases was over % in a read, we defined the low quality base to be the base whose sequencing quality was no more than ). at the same time, q , q , gc content of the clean data were calculated (vazyme biotech co.,ltd, nanjing, china). the original data of the rna-seq was uploaded to the geo datasets. total rna was isolated from the tissues by trnzol reagent (dp , beijing tiangen biotech, china). then, cdna was prepared using hiscript iii st strand cdna synthesis kit (r - , nanjing vazyme biotech, china). qrt-pcr was performed using the applied biosystems real-time pcr systems (thermo fisher scientific, usa) with sybr qpcr master mix (q - , nanjing vazyme biotech, china). the data of qrt-pcr were analyzed by the livak method ( −ΔΔct ). ribosomal protein l (rpl ) was used as a reference gene for mice, and gapdh for macaques. qrt-pcr primers are displayed in supplementary materials table s . the lungs were quickly placed in cold saline solution and rinsed after they were collected. then, lungs were fixed in % pfa, dehydrated and embedded in paraffin prior to sectioning at μm and sections were stained with hematoxylin and eosin (h&e). for immunohistochemical staining, the lung paraffin sections were dewaxed and rehydrated through xylene and an alcohol gradient. antigen retrieval was performed by heating the sections to °c for min in . m citrate buffer (ph . ) and repeated times. the operations were performed according to the instructions of the two-step detection kit (pv- , beijing zsgb biotechnology, china). the samples were treated by endogenous peroxidase blockers for min at room temperature followed by incubation with primary antibodies s a ( : , t, cell signaling technology) at °c for h, then after washed with pbs, sections were incubated with reaction enhancer for min at room temperature and secondary antibodies at °c for min, and finally sections were visualized by , -diaminobenzidine tetrahydrochloride (dab) and counterstained with haematoxylin. the lung tissues, peripheral blood and bone marrow were collected from the mice. the lungs were first ground with mesh copper sieve, and then transferred to dmem containing % fbs, . mg/ml collagenase d ( , roche, switzerland) and . mg/ml dnase i ( , stemcell technologies, canada) for a min digestion at ℃ to obtain single-cell suspensions. livers were harvested and homogenized into single-cell suspensions using mesh copper sieve. bone marrow were flushed out of the femurs using a -gauge needle in pbs containing mm edta and % fetal bovine serum (fbs) and dispersed into single cells through a pipette. single-cell samples were treated by red blood cell lysis buffer (r , beijing solarbio science & technology, china) for min at room temperature and passed through a -μm nylon mesh sieve before staining. peripheral blood was treated with red blood cell lysis buffer to remove red blood cells. after blocking non-specific fc-mediated interactions with cd /cd antibodies ( - - , ebioscience, usa), single-cell suspensions were stained with fluorophore-conjugated anti-mouse antibodies at ℃ for min. after washing the samples, flow cytometry acquisition was performed on a bd lsrfortessa. sorting were performed using a bd ariaiii (bd). all antibodies were purchased from ebioscience: cd -pe ( - - ), ly- g-apc ( - - ), cd b-fitc ( - - ). all analyses were repeated at least three times, and a representative experimental result was presented. two-tailed unpaired student's t test was used for statistical analysis to determine significant differences when a pair of conditions was compared. asterisks denote statistical significance (*p < . ; **p < . ; *** p < . ). the data are reported as the mean ± s.d. figure s (a)-(d) rna-seq analysis of rna from lungs of rhesus macaques infected intranasally with sars-cov- at dpi and dpi. heatmap depicting the differentially expressed genes (a). the expression of ifns was analyzed (b). kegg analysis was performed with the differentially expressed genes compared with mock (c) (fc > or < . , p value < . ). the expression of indicated marker genes was analyzed (d). n = . (e) analysis of s a expression in peripheral blood from health control and covid- patients. fold change to health control (log ). data from the peripheral blood of covid- patients and health control correspond to geo: gse . (*p < . ; **p < . ; ***p < . ; ****p < . ). continued figure s (a) qrt-pcr analysis for viral load in the lung of hace mice infected intranasally with sars-cov- and c bl/ mice infected intranasally with iav at dpi, dpi, dpi, dpi, and dpi. n = . (b)-(e) rna-seq analysis of rna from lung of c bl/ mice infected with iav and hace mice infected with sars-cov- at dpi, dpi, dpi, and dpi. go and kegg analysis was performed with the differentially expressed genes compared with mock (b). go and kegg analysis was performed with the differentially expressed genes between iav induced genes and sars-cov- induced genes (c) (fc > or < . , p value < . ). the expression of ifns (d) and isgs was analyzed (e). rna-seq analysis of rna from lung of ifnar -/mice infected intranasally with mhv between vehicle treated group and paquinimod treated group at dpi. analysis of the expression level of indicted genes (b). kegg analysis was performed with the differentially expressed genes (c) (fc > or < . , p value < . ). (d) qrt-pcr analysis for the expression level of cd in the lungs of hace mice infected intranasally with sars-cov- at dpi, dpi, dpi, dpi, and dpi. n = . (****p < . ). analysis of the change of several related genes of neutrophil groups from the lungs of c bl/ mice infected with iav and hace mice infected with sars-cov- at dpi, dpi, dpi, dpi, and dpi. (d) rna-seq analysis of the change of several related genes of neutrophil groups from the lungs of ifnar-/-mice infected intranasally with mhv between vehicle treated group and paquinimod treated group at dpi. (*p < . ; **p < . ). pathogen recognition and innate immunity damps, pamps and alarmins: all we need to know about danger identification of human s a as a novel target for treatment of autoimmune disease via binding to quinoline- -carboxamides imbalanced host response to sars-cov- drives development of covid- host-viral infection maps reveal signatures of severe covid- patients alarmins: awaiting a clinical response sterile inflammation: sensing and reacting to damage alarmins in frozen shoulder: a molecular association between inflammation and pain localized bacterial infection induces systemic activation of neutrophils through cxcr signaling in zebrafish reduction and functional exhaustion of t cells in patients with coronavirus disease (covid- ). front immunol phagocyte-specific calcium-binding s proteins as clinical laboratory markers of inflammation hepatoma derived growth factor (hdgf) dynamics in ovarian cancer cells impaired type i interferon activity and inflammatory responses in severe covid- patients clinical features of patients infected with novel coronavirus in wuhan hmgb in health and disease comprehensive mapping of immune perturbations associated with severe covid- a critical concentration of neutrophils is required for effective bacterial killing in suspension single-cell landscape of bronchoalveolar immune cells in patients with covid- points of control in inflammation neutrophils at work neutrophils in homeostasis calprotectin in rheumatic diseases alarmins: chemotactic activators of immune responses danger-associated molecular patterns (damps): the derivatives and triggers of inflammation prophylactic treatment with s a inhibitor paquinimod reduces pathology in experimental collagenase-induced osteoarthritis lymphopenia predicts disease severity of covid- : a descriptive and predictive study mrp and mrp are endogenous activators of toll-like receptor , promoting lethal, endotoxin-induced shock cholinergic agonists inhibit hmgb release and improve survival in experimental sepsis s a /a in inflammation. front immunol a single-cell atlas of the peripheral immune response in patients with severe covid- risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease a new coronavirus associated with human respiratory disease in china single-cell transcriptome profiling reveals neutrophil heterogeneity in homeostasis and infection alarmins and immunity coronavirus mhv-a infects the lung and causes severe pneumonia in c bl/ age-related rhesus macaque models of covid- clinical characteristics of cases of death from covid- elevated exhaustion levels and reduced functional diversity of t cells in peripheral blood may predict severe functional exhaustion of antiviral lymphocytes in covid- patients bacterial and fungal infections in covid- patients: a matter of concern a novel coronavirus from patients with pneumonia in china this work was supported by the national natural science foundation of china f.y., x.w., c.q. and q.g. conceived the study and analyzed the data. q.g., y.z. and j.l. performed most experiments and analyzed the data. f.y. and x.g. analyzed the rna-seq data. z.z., l.c. and y.l. helped with the mice experiments. y.l., x.w, x.wei, l. chen. provided support on literature search. f.y. wrote the paper. f.y., x.w. and c.q. revised the paper. the authors have no conflicts of interest to declare. key: cord- -zrtixzgu authors: delgado-chaves, fernando m.; gómez-vela, francisco; divina, federico; garcía-torres, miguel; rodriguez-baena, domingo s. title: computational analysis of the global effects of ly e in the immune response to coronavirus infection using gene networks date: - - journal: genes (basel) doi: . /genes sha: doc_id: cord_uid: zrtixzgu gene networks have arisen as a promising tool in the comprehensive modeling and analysis of complex diseases. particularly in viral infections, the understanding of the host-pathogen mechanisms, and the immune response to these, is considered a major goal for the rational design of appropriate therapies. for this reason, the use of gene networks may well encourage therapy-associated research in the context of the coronavirus pandemic, orchestrating experimental scrutiny and reducing costs. in this work, gene co-expression networks were reconstructed from rna-seq expression data with the aim of analyzing the time-resolved effects of gene ly e in the immune response against the coronavirus responsible for murine hepatitis (mhv). through the integration of differential expression analyses and reconstructed networks exploration, significant differences in the immune response to virus were observed in ly e [formula: see text] compared to wild type animals. results show that ly e ablation at hematopoietic stem cells (hscs) leads to a progressive impaired immune response in both liver and spleen. specifically, depletion of the normal leukocyte mediated immunity and chemokine signaling is observed in the liver of ly e [formula: see text] mice. on the other hand, the immune response in the spleen, which seemed to be mediated by an intense chromatin activity in the normal situation, is replaced by ecm remodeling in ly e [formula: see text] mice. these findings, which require further experimental characterization, could be extrapolated to other coronaviruses and motivate the efforts towards novel antiviral approaches. the recent sars-cov- pandemic has exerted an unprecedented pressure on the scientific community in the quest for novel antiviral approaches. a major concern regarding sars-cov- is the capability of the coronaviridae family to cross the species barrier and infect humans [ ] . this, along with the tendency of coronaviruses to mutate and recombine, represents a significant threat to global health, which ultimately has put interdisciplinary research on the warpath towards the development of a vaccine or antiviral treatments. given the similarities found amongst the members of the coronaviridae family [ , ] , analyzing the global immune response to coronaviruses may shed some light on the natural control of viral infection, and inspire prospective treatments. this may well be achieved from the perspective of systems biology, in which the interactions between the biological entities involved in a certain process are represented by means of a mathematical system [ ] . within this framework, gene networks (gn) have become an important tool in the modeling and analysis of biological processes from gene expression data [ ] . gns constitute an abstraction of a given biological reality by means of a graph composed by nodes and edges. in such a graph, nodes represent the biological elements involved (i.e., genes, proteins or rnas) and edges represent the relationships between the nodes. in addition, gns are also useful to identify genes of interest in biological processes, as well as to discover relationships among these. thus, they provide a comprehensive picture of the studied processes [ , ] . among the different types of gns, gene co-expression networks (gcns) are widely used in the literature due to their computational simplicity and good performance in order to study biological processes or diseases [ ] [ ] [ ] . gcns usually compute pairwise co-expression indices for all genes. then, the level of interaction between two genes is considered significant if its score is higher than a certain threshold, which is set ad hoc. traditionally, statistical-based co-expression indices have been used to calculate the dependencies between genes [ , ] . some of the most popular correlation coefficients are pearson, kendall or spearman [ ] [ ] [ ] . despite their popularity, statistical-based measures present some limitations [ ] . for instance, they are not capable of identifying non-linear interactions and the dependence on the data distribution in the case of parametric correlation coefficients. in order to overcome some of these limitations, new approaches, e.g., the use of information theory-based measures or ensemble approaches, are receiving much attention [ ] [ ] [ ] . gene co-expression networks (gcns) have already been applied to the study of dramatic impact diseases, such as cancer [ ] , diabetes [ ] or viral infections (e.g., hiv) in order to study the role of immune response to these illnesses [ , ] . genetic approaches are expected to be the best strategy to understand viral infection and the immune response to it, potentially identifying the mechanisms of infection and assisting the design of strategies to combat infection [ , ] . the current gene expression profiling platforms, in combination with high-throughput sequencing, can provide time-resolved transcriptomic data, which can be related to the infection process. the main objective of this approach is to generate knowledge on the immune functioning upon viral entry into the organism, which means mean a perturbation to the system. in the context of viral infection, a first defense line is the innate response mediated by interferons, a type of cytokines which eventually leads to the activation of several genes of antiviral function [ ] . globally, these genes are termed interferon-stimulated genes (isgs), and regulate processes like inflammation, chemotaxis or macrophage activation among others. furthermore, isgs are also involved in the subsequent acquired immune response, specific for the viral pathogen detected [ ] . gene ly e (lymphocyte antigen family member e), which has been related to t cell maturation and tumorogenesis, is amongst the isgs [ ] . this gene is transcriptionally active in a variety of tissues, including liver, spleen, lung, brain, uterus and ovary. its role in viral infection has been elusive due to contradictory findings [ ] . for example, in liu et al. [ ] , ly e was associated with the resistance to marek's disease virus (mdv) in chickens. moreover, differences in the immune response to mouse adenovirus type (mav- ) have been attributed to ly e variants [ ] . conversely, ly e has also been related to an enhancement of human immunodeficiency viruses (hiv- ) pathogenesis, by promoting hiv- entry through virus-cell fusion processes [ ] . also in the work by mar et al. [ ] , the loss of function of ly e due to gene knockout reduced the infectivity of influenza a virus (iav) and yellow fever virus (yfv). this enhancing effect of ly e on viral infection has also been observed in other enveloped rna viruses such as in west nile virus (wnv), dengue virus (den), zika virus (zikv), o'nyong nyong virus (onnv) and chikungunya virus (chikv) among others [ ] . nevertheless, the exact mechanisms through which ly e modulates viral infection virus-wise, and sometimes even cell type-dependently, require further characterization. in this work we present a time-resolved study of the immune response of mice to a coronavirus, the murine hepatitis virus (mhv), in order to analyze the implications of gene ly e. to do so, we have applied a gcn reconstruction method called engnet [ ] , which is able to perform an ensemble strategy to combine three different co-expression measures, and a topology optimization of the final network. engnet has outscored other methods in terms of network precision and reduced network size, and has been proven useful in the modeling of disease, as in the case of human post-traumatic stress disorder. the rest of the paper is organized as follows. in the next section, we propose a description of related works. in section , we first describe the dataset used in this paper, and then we introduce the engnet algorithm and the different methods used to infer and analyze the generated networks. the results obtained are detailed in section , while, in section , we propose a discussion of the results presented in the previous section. finally, in section , we draw the main conclusions of our work. as already mentioned, gene co-expression networks have been extensively applied in the literature for the understanding of the mechanisms underlying complex diseases like cancer, diabetes or alzheimer [ ] [ ] [ ] . globally, gcn serve as an in silico genetic model of these pathologies, highlighting the main genes involved in these at the same time [ ] . besides, the identification of modules in the inferred gcns, may lead to the discovery of novel biomarkers for the disease under study, following the 'guilt by association' principle. along these lines, gcns are also considered suitable for the study of infectious diseases, as those caused by viruses to the matter at hand [ ] . to do so, multiple studies have analyzed the effects of viral infection over the organism, focusing on immune response or tissue damage [ , ] . for instance, the analysis of gene expression using co-expression networks is shown in the work by pedragosa et al. [ ] , where the infection caused by lymphocytic choriomeningitis virus (lcmv) is studied over time in mice spleen using gcns. in ray et al. [ ] , gcns are reconstructed from different microarray expression data in order to study hiv- progression, revealing important changes across the different infection stages. similarly, in the work presented by mcdermott et al. [ ] , the over-and under-stimulation of the innate immune response to severe acute respiratory syndrome coronavirus (sars-cov) infection is studied. using several network-based approaches on multiple knockout mouse strains, authors found that ranking genes based on their network topology made accurate predictions of the pathogenic state, thus solving a classification problem. in [ ] , co-expression networks were generated by microarray analysis of pediatric influenza-infected samples. thanks to this study, genes involved in the innate immune system and defense to virus were revealed. finally, in the work by pan et al. [ ] , a co-expression network is constructed based on differentially-expressed micrornas and genes identified in liver tissues from patients with hepatitis b virus (hbv). this study provides new insights on how micrornas take part in the molecular mechanism underlying hbv-associated acute liver failure. the alarm posed by the covid- pandemic has fueled the development of effective prevention and treatment protocols for -ncov/sars-cov- outbreak [ ] . due to the novelty of sars-cov- , recent research takes similar viruses, such as sars-cov and middle east respiratory syndrome coronavirus (mers-cov), as a starting point. other coronaviruses, like mouse hepatitis virus (mhv), are also considered appropriate for comparative studies in animal models, as demonstrated in the work by de albuquerque et al. [ ] and ding et al. [ ] . mhv is a murine coronavirus (m-cov) that causes an epidemic illness with high mortality, and has been widely used for experimentation purposes. works like the ones by case et al. [ ] and gorman et al. [ ] , study the innate immune response against mhv arbitrated by interferons, and those interferon-stimulated genes with potential antiviral function. this is the case of gene ly e, which has been shown to play an important role in viral infection, as well as various orthologs of the same gene [ , ] . mechanistic approaches often involved the ablation of the gene under study, like in the work by mar et al. [ ] , where gene knockout was used to characterize the implications of ly e in influenza a infection. as it is the case of giotis et al. [ ] , these studies often involve global transcriptome analyses, via rna-seq or microarrays, together with computational efforts, which intend to screen the key elements of the immune system that are required for the appropriate response. this approach ultimately leads experimental research through predictive analyses, as in the case of co-expression gene networks [ ] . in the following subsections, the main methods and gcn reconstruction steps are addressed. first, in section . , the original dataset used in the present work is described, together with the experimental design. then, in section . , the data preprocessing steps are described. subsequently in section . , key genes controlling the infection progression are extracted through differential expression analyses. finally, the inference of gcns and their analysis are detailed in sections . and . , respectively. the original experimental design can be described as follows. the progression of the mhv infection at genetic level was evaluated in two genetic backgrounds: wild type (wt, ly efl/fl) and ly e knockout mutants (ko, ly e ∆hsc ). the ablation of gene ly e in all cell types is lethal, hence the ly e ∆hsc strain contains a disrupted version of gene ly e only in hematopoietic stem cells (hsc), which give rise to myeloid and lymphoid progenitors of all blood cells. wild type and ly e ∆hsc mice were injected intraperitoneally with pfu mhv-a . at and days post-injection (d p.i.), mice were euthanized and biological samples for rna-seq were extracted. the overall effects of mhv infection in both wt and ko strains was assessed in liver and spleen. in total samples were analyzed, half of these corresponding to liver and spleen, respectively. from the organ-specific samples, samples correspond to mock infection (negative control), to mhv-infected samples at d p.i. and to mhv-infected samples at d p.i. for each sample, two technical replicates were obtained. libraries of cdna generated from the samples were sequenced using illumina novaseq . further details on sample preparation can be found in the original article by pfaender et al. [ ] . for the sake of simplicity, mhv-infected samples at and d p.i. will be termed 'cases', whereas mock-infection samples will be termed 'controls'. the original dataset consists of files, one per sample replicate, obtained upon the mapping of the transcript reads to the reference genome. reads were recorded in three different ways, considering whether these mapped introns, exons or total genes. then, a count table was retrieved from these files by selecting only the total gene counts of each sample replicate file. pre-processing was performed using the edger [ ] r package. the original dataset by pfaender et al. [ ] was retrieved from geo (accession id: gse ) using the geoquery [ ] package. additional files on sample information and treatment were also used to assist the modeling process. by convention, a sequencing depth per gene below is considered neglectable [ , ] . genes meeting this criterion are known as low expression genes, and are often removed since they add noise and computational burden to the following analyses [ ] . in order to remove genes showing less than reads across all conditions, counts per million (cpm) normalization was performed, so possible differences between library sizes for both replicates would not affect the result. afterwards, principal components analyses (pca) were performed over the data in order to detect the main sources of variability across samples. pca were accompanied by unsupervised k-medoid clustering analyses, in order to identify different groups of samples. in addition, multidimensional scaling plots (mds) were applied to further separate samples according to their features. last, between-sample similarities were assessed through hierarchical clustering. the analyses of differential expression served a two-way purpose, (i) the exploration of the directionality in the gene expression changes upon viral infection, and (ii) the identification of key regulatory elements for the subsequent network reconstruction. in the present application, differentially-expressed genes (deg) were filtered from the original dataset and proceeded to the reconstruction process. this approximation enabled the modeling of the genetic relationships that are considered of relevance in the presented comparison [ ] [ ] [ ] . in the present work mice samples were compared organ-wise depending on whether these corresponded to control, d p.i. and d p.i. the identification of deg was performed using the limma [ ] r package, which provides non-parametric robust estimation of the gene expression variance. this package includes voom, a method that incorporates rna-seq count data into the limma workbench, originally designed for microarrays [ ] . in this case, a minimum log -fold-change (log fc) of was chosen, which corresponds to four fold changes in the gene expression level. p-value was adjusted by benjamini-hochberg [ ] and the selected adjusted p-value cutoff was . . in order to generate gene networks the engnet algorithm was used. this technique, presented in gómez-vela et al. [ ] , is able to compute gene co-expression networks with a competitive performance compared other approaches from the literature. engnet performs a two-step process to infer gene networks: (a) an ensemble strategy for a reliable co-expression networks generation, and (b) a greedy algorithm that optimizes both the size and the topological features of the network. these two features of engnet offer a reliable solution for generating gene networks. in fact, engnet relies on three statistical measures in order to obtain networks. in particular, the measures used are the spearman, kendall and normalized mutual information (nmi), which are widely used in the literature for inferring gene networks. engnet uses these measures simultaneously by applying an ensemble strategy based on major voting, i.e., a relationship will be considered correct if at least of the measures evaluate the relationship as correct. the evaluation is based on different independent thresholds. in this work, the different thresholds were set to the values originally used in [ ] : . , . and . for spearman, kendall and nmi, respectively. in addition, as mentioned above, engnet performs an optimization of the topological structure of the networks obtained. this reduction is based on two steps: (i) the pruning of the relations considered of least interest in the initial network, and (ii) the analysis of the hubs present in the network. for this second step of the final network reconstruction, we have selected the same threshold that was used in [ ] , i.e., . . through this optimization, the final network produced by engnet results easier to analyze computationally, due to its reduced size. networks were imported to r for the estimation of topology parameters and the addition of network features that are of interest for the latter network analysis and interpretation. these attributes were added to the reconstructed networks to enrich the modeling using the igraph [ ] r package. the networks were then imported into cytoscape [ ] through rcy [ ] for examination and analyses purposes. in this case, two kind of analyses were performed: (i) a topological analysis and (ii) an enrichment analysis. regarding the topological analysis, clustering evaluation was performed in order to identify densely connected nodes, which, according to the literature, are often involved in a same biological process [ ] . the chosen clustering method was community clustering (glay) [ ] , implemented via cytoscape's clustermaker app [ ] , which has yielded significant results in the identification of densely connected modules [ , ] . among the topology parameters, degree and edge betweenness were estimated. the degree of a node refers to the number of its linking nodes. on the other hand, the betweenness of an edge refers to the number of shortest paths which go through that edge. both parameters are considered as a measure of the implications of respectively nodes and edges in a certain network. particularly, nodes whose degree exceeds the average network node degree, the so called hubs, are considered key elements of the biological processes modeled by the network. in this particular case, the distribution of nodes' degree network was analyzed so those nodes whose degree exceeded a threshold were selected as hubs. this threshold is defined as q + . × iqr, where q is the third quartile and iqr the interquartile range of the degree distribution. this method has been widely used for the detection of upper outliers in non-parametric distributions [ , ] , as it is the case. however, the outlier definition does not apply to this distribution since those nodes whose degree are far above the median degree are considered hubs. on the other hand, gene ontology (go) enrichment analysis provides valuable insights on the biological reality modeled by the reconstructed networks. the gene ontology consortium [ ] is a data base that seeks for a unified nomenclature for biological entities. go has developed three different ontologies, which describe gene products in terms of the biological processes, cell components or molecular functions in which these are involved. ontologies are built out of go terms or annotations, which provide biological information of gene products. in this case, the clusterprofiler [ ] r package, allowed the identification of the statistically over-represented go terms in the gene sets of interest. additional enrichment analyses were performed using david [ ] . for both analyses, the complete genome of mus musculus was selected as background. finally, further details on the interplay of the genes under study was examined using the string database [ ] . the reconstruction of gene networks that adequately model viral infection involves multiple steps, which ultimately shape the final outcome. first, in section . , exploratory analyses and data preprocessing are detailed, which prompted the modeling rationale. then, in section . , differential expression is evaluated for the samples of interest. finally, networks reconstruction and analysis are addressed in section . . at the end, four networks were generated, both in an organand genotype-wise manner. a schematic representation of the gcn reconstruction approach is shown in figure . general scheme for the reconstruction method. the preprocessed data was subjected to exploratory and differential expression analyses, which imposed the reconstruction rationale. four groups of samples were used to generate four independent networks, respectively modeling the immune response in the liver, both in the wt and the ko situations; and in the spleen, also in the wt and the ko scenarios. in order to remove low expression genes, a sequencing depth of was found to correspond to an average cpm of . , which was selected as threshold. hence, genes whose expression was found over . cpm in at least two samples of the dataset were maintained, ensuring that only genes which are truly being expressed in the tissue will be studied. the dataset was log -normalized with priority to the following analyses, in accordance to the recommendations posed in law et al. [ ] . the results of both pca and k-medoid clustering are shown in figure a . clustering of the log -normalized samples revealed clear differences between liver and spleen samples. also, for each organ, three subgroups of analogous samples that cluster together are identified. these groups correspond to mock infection, mhv-infected mice at d p.i. and mhv-infected mice at d p.i. (dashed lines in figure a ). finally, subtle differences were observed in homologous samples of different genotypes ( figure a ). organ-specific pca revealed major differences between mhv-infected samples for ly e ∆hsc and wt genotypes, at both and d p.i. these differences were not observed in the mock infection (control situation). organ-wise pca are shown in figure b ,c. the distances between same-genotype samples illustrate the infection-prompted genetic perturbation from the uninfected status (control) to d p.i., where clear signs of hepatitis were observed according to the original physiopathology studies [ ] . on the other hand, the differences observed between both genotypes are indicative of the role of gene ly e in the appropriate response to viral infection. these differences are subtle in control samples, but in case samples, some composition biass is observed depending on whether these are ko or wt, especially in spleen samples. the comparative analysis of the top most variable genes confirmed the differences observed in the pca, as shown in figure a . among the four different features of the samples under study: organ, genotype, sample type (case or control) and days post injection; the dissimilarities in terms of genotype were the subtlest. in the light of these exploratory findings, the network reconstruction approach was performed as follows. networks were reconstructed organ-wise, as these exhibit notable differences in gene expression. additionally, a main objective of the present work is to evaluate the differences in the genetic response in the wt situation compared to the ly e ∆hsc ko background, upon the viral infection onset in the two mentioned tissues. for each organ, log -normalized samples were coerced to generate time-series-like data, i.e., for each genotype, samples will be considered as a set, namely control samples, case samples at d p.i. and case samples at d p.i. both technical replicates were included. this rational design seeks for a gene expression span representative of the infection progress. thereby, control samples may well be considered as a time zero for the viral infection, followed by the corresponding samples at and d p.i. the proposed rationale is supported by the exploratory findings, which position d p.i. samples between control and d p.i. samples. at the same time, the reconstruction of gene expression becomes robuster with increasing number of samples. in this particular case, measuring points are attained for the reconstruction of each one of the four intended networks, since two technical replicates were obtained per sample [ ] . the differential expression analyses were performed over the four groups of samples explained above, with the aim of examining the differences in the immune response between ly e ∆hsc and wt samples. limma -voom differential expression analyses were performed over the log -normalized counts, in order to evaluate the different genotypes whilst contrasting the three infection stages: control vs. cases at d p.i., control vs. cases at d p.i. and cases at vs. d p.i. the choice of a minimum absolute log fc ≥ , enabled considering only those genes that truly effect changes between wt and ly e ∆hsc samples, whilst maintaining a relatively computer-manageable number of deg for network reconstruction. the latter is essential for the yield of accurate network sparseness values, as this is a main feature of gene networks [ ] . for both genotypes and organs, the results of the differential expression analyses reveal that mhv injection triggers a progressive genetic program from the control situation to the mhv-infected scenario at d p.i., as shown in figure a . the absolute number of deg between control vs. cases at d p.i. was considerably larger than in the comparison between control vs. cases at d p.i. furthermore, in all cases, most of the deg in control vs. cases at d p.i. are also differentially-expressed in the control vs. cases at d p.i. comparison, as shown in figure . regarding genes fold change, an overall genetic up-regulation is observed upon infection. around % of deg are upregulated for all the comparisons performed for wt samples, as shown in figure b . nonetheless, a dramatic reduce in this genetic up-regulation is observed, by contrast, in knockout samples, even limiting upregulated genes to nearly % in the control vs. cases at d p.i. comparison of liver ly e ∆hsc samples. the largest differences are observed in the comparison of controls vs. cases at d p.i ( figures a and a ). these deg are of great interest for the understanding of the immune response of both wt and ko mice to viral infection. these genes were selected to filter the original dataset for latter network reconstruction. the commonalities between wt and ko control samples for both organs were also verified through differential expression analysis following the same criteria (log fc > , p value < . ). the number of deg between wt and ko liver control samples ( ) and between wt and ko spleen control samples ( ) were not considered significant, so samples were taken as analogous starting points for infection. as stated above, the samples were arranged both organ and genotype-wise in order to generate networks which would model the progress of the disease in each scenario. gcns were inferred from log -normalized expression datasets. a count of was added at log normalization so the problem with remaining zero values was avoided. each network was generated exclusively taking into consideration their corresponding deg at control vs. cases at d p.i., where larger differences were observed. four networks were then reconstructed from these previously-identified deg for liver wt samples ( genes), liver ko samples ( genes), spleen wt samples ( genes) and spleen ko samples ( genes). this approach results in the modeling of only those relationships that are related to the viral infection. each sample set was then fed to engnet for the reconstruction of the subsequent network. genes that remained unconnected due to weak relationships, which do not overcome the set threshold, were removed from the networks. furthermore, the goodness of engnet-generated models outperformed other well-known inference approaches, as detailed in appendix b. topological parameters were estimated and added as node attributes using igraph, together with log fc, prior to cytoscape import. specifically, networks were simplified by removing potential loops and multiple edges. the clustering topological scrutiny of the reconstructed networks revealed neat modules in all cases, as shown in figure a . the number of clusters identified in each network, as well as the number of genes harbored in the clusters is shown in table a . as already mentioned, according to gene networks theory, nodes contained within the same cluster are often involved in the same biological process [ , ] . in this context, the go-based enrichment analyses over the identified clusters may well provide an idea of the affected functions. only clusters containing more than genes were considered, since this is the minimum number of elements required by the enrichment tool clusterprofiler. the results of the enrichment analyses revealed that most go terms were not shared between wt and ko homologous samples, as shown in figure . in order to further explore the reconstructed networks, the intersection of ko and wt networks of a same organ was computed. this refers to the genes and relationships that are shared between both genotypes for a specific organ. additionally, the genes and relationships that were exclusively present at the wt and ko samples were also estimated, as shown in figure a . the enrichment analyses over the nodes, separated using this criterion, would reveal the biological processes that make the difference between in ly e ∆hsc mice compared to wt ones. the results of such analyses are shown in figure a . finally, the exploration of nodes' degree distribution would reveal those genes that can be considered hubs. those nodes comprised within the top genes with highest degree (degree > q + . × iq), also known as upper outliers in the nodes distribution, were considered hubs. a representation of nodes' degree distribution throughout the four reconstructed networks is shown in figure . these distributions are detailed in figure a . this method provided four cutoff values for the degree, , , and , respectively for liver wt and ko, spleen wt and ko networks. above these thresholds, nodes would be considered as hubs in each network. these hubs are shown in tables a -a . figure . enrichment analyses performed over the main clusters identified in wt and ko networks of (a) liver and (b) spleen networks. gene ratio is defined by the number of genes used as input for the ernichment analyses associated with a particular go term divided by the total number of input genes. . boxplots representative of the degree distributions for each one of the four reconstructed networks. identified hubs, according to the q + . × iqr criterion, are highlighted in red. the degree cutoffs, above which nodes would be considered as hubs, were , , and , respectively for liver wt, liver ko, spleen wt and spleen ko networks. note degree is represented in a log scale given that the reconstructed networks present a scale-free topology. in this work four gene networks were reconstructed to model the genetic response mhv infection in two tissues, liver and spleen, and in two different genetic backgrounds, wild type and ly e ∆hsc . samples were initially explored in order to design an inference rationale. not only did the designed approach reveal major differences between the genetic programs in each organ, but also, between different subgroups of samples, in a time-series-like manner. noticeably, disparities between wt and ly e ∆hsc samples were observed in both tissues, and differential expression analyses revealed relevant differences in terms of the immune response generated. hereby, our results predict the impact of ly e ko on hsc, which resulted in an impaired immune response compared to the wt situation. overall, results indicate that the reconstruction rationale, elucidated from exploratory findings, is suitable for the modeling of the viral progression. regarding the variance in gene expression in response to virus, pca and k-medoid clustering revealed strong differences between samples corresponding to liver spleen, respectively (figure a ). these differences set the starting point for the modeling approach, in which samples corresponding to each organ were analyzed independently. this modus operandi is strongly supported by the tropism that viruses exhibit for certain tissues, which ultimately results in a differential viral incidence and charge depending on the organ [ ] . in particular, the liver is the target organ of mhv, identified as the main disease site [ ] . on the other hand, the role of the spleen in innate and adaptive immunity against mhv has been widely addressed [ , ] . the organization of this organ allows blood filtration for the presentation of antigens to cognate lymphocytes by the antigen presenting cells (apcs), which mediate the immune response exerted by t and b cells [ ] . as stated before, pca revealed differences between the three sample groups on each organ: control and mhv-infected at and d p.i. interestingly, between-groups differences are specially clear for liver samples (figure b) , whereas spleen samples are displayed in a continuum-like way. this becomes more evident in organ-wise pca (figure ) , and was latter confirmed by the exploration of the top most variable genes and differential expression analyses ( figure a ). furthermore, clear differences between wt and ly e ∆hsc samples are observed in none of these analyses, although the examination of the differential expression and network reconstruction did exposed divergent immune responses for both genotypes. the differential expression analyses revealed the progressive genetic response to virus for both organs and genotypes (figures a and ) . in a wt genetic background, mhv infection causes an overall rise in the expression level of certain genes, as most deg in cases vs. control samples are upregulated. however, in a ly e ∆hsc genetic background, this upregulation is not as prominent as in a wt background, significantly reducing the number of upregulated genes (figure b) . besides, the number of deg in each comparison varies from wt to ly e ∆hsc samples. attending at the deg in the performed comparisons, for both the wt and ko genotypes, liver cases at d p.i. are more similar to liver cases at d p.i. than to liver controls, since the number of deg between the first two measuring points is significantly lower than the number of deg between control and case samples at d p.i. (figure a,b) . a different situation occurs in the spleen, where wt cases at d p.i. are closer to control samples (figure c ), whereas ko cases at d p.i. seem to be more related to cases at d p.i. (figure d ). this was already suggested by hierarchical clustering in the analysis of the top most variable genes, and could be indicative of a different progression of the infection impact on both organs, which could be modulated by gene ly e, at least for the spleen samples. moreover, the results of the deg analyses indicate that the sole knockout of gene ly e in hsc considerably affects the upregulating genetic program normally triggered by viral infection in wild type individuals (in both liver and spleen). interestingly, there are some genes in each organ and genotype that are differentially expressed in every comparison between the possible three sample types, controls, cases at d p.i. and cases at d p.i. these genes, which we termed highly deg, could be linked to the progression of the infection, as changes in their expression level occur with days post injection, according to the data. the rest of the deg, show an uprise or fall when comparing two sample types, which does not change significantly in the third sample type. alternatively, highly deg, shown in table a , exhibited three different expression patterns: (i) their expression level, initially low, rises from control to cases at d p.i. and then rises again in cases at d p.i. (ii) their expression level, initially high in control samples, falls at d p.i. and falls even more at d p.i cases. (iii) their expression level, initially low, rises from control to cases at d p.i. but then falls at cases at d p.i., when it is still higher than the initial expression level. these expression patterns, which are shown in figure a , might be used to keep track of the disease progression, differentiating early from late infection stages. in some cases, these genes exhibited inconsistent expression levels, specially at d p.i. cases, which indicates the need for further experimental designs targeting these genes. highly deg could be correlated with the progression of the disease, as in regulation types (i) and (ii) or by contrast, be required exclusively at initial stages, as in regulation type (iii). notably, genes gm and gm are predicted genes which, to date, have been poorly described. according to the string database [ ] , gm is associated with gene lst (leukocyte-specific transcript protein), which has a possible role in modulating immune responses. in fact, gm is homologous to human gene piro (progranulin-induced-receptor-like gene during osteoclastogenesis), related to bone homeostasis [ , ] . thus, we hypothesize that bone marrow-derived cell lines, including erythrocytes and leukocytes (immunity effectors), could also be regulated by gm . on the other hand, gm is not associated to any other gene according to string. protein gm is homologous to human protein dhrs (dehydrogenase/reductase sdr family member ) isoform precursor. nonetheless and to the best of our knowledge, these genes have not been previously related to ly e, and could play a role in the immune processes mediated by this gene. finally, highly deg were not found exclusively present in wt nor ko networks, instead, these were common nodes of these networks for each organ. this suggests that highly deg might be of core relevance upon mhv infection, with a role in those processes independent on ly e ∆hsc . besides, genes hykk, ifit and ifit b; identified as highly deg throughout liver ly e ∆hsc samples were also identified as hubs in the liver ko network. also gene saa , highly deg across spleen ly e ∆hsc samples was considered a hub in the spleen ko network. nevertheless, these highly deg require further experimental validation. the enrichment analyses of the identified clusters at each network revealed that most go terms are not shared between the two genotypes ( figure ), despite the considerable amount of shared genes between the two genotypes for a same organ. the network reconstructed from liver wt samples reflects a strong response to viral infection, involving leukocyte migration or cytokine and interferon signaling among others. these processes, much related to immune processes, are not observed in its ko counterpart. the liver wt network presented four clusters ( figure a a ). its cluster regulates processes related to leukocyte migration, showing the implication of receptor ligand activity and cytokine signaling, which possibly mediates the migration of the involved cells. cluster is related to interferon-gamma for the response to mhv, whereas cluster is probably involved in the inflammatory response mediated by pro-inflammatory cytokines. last, cluster is related to cell extravasation, or the leave of blood cells from blood vessels, with the participation of gene nipal . the positive regulation observed across all clusters suggests the activation of these processes. overall, hub genes in this network have been related to the immune response to viral infection, as the innate immune response to the virus is the mediated by interferons. meanwhile, the liver ko network showed three main clusters ( figure a b ). its cluster would also be involved in defense response to virus, but other processes observed in the liver wt network, like leukocyte migration or cytokine activity, are not observed in this cluster nor the others. cluster is then related to the catabolism of small molecules and cluster is involved in acids biosynthesis. these processes are certainly ambiguous and do not correspond the immune response observed in the wt situation, which suggests a decrease in the immune response to mhv as a result of ly e ablation in hsc. on the other hand, spleen wt samples revealed high nuclear activity potentially involving nucleosome remodeling complexes and changes in dna accessibility. histone modification is a type of epigenetic modulation which regulates gene expression. taking into account the central role of the spleen in the development of immune responses, the manifested relevance of chromatin organization could be accompanied by changes in the accessibility of certain dna regions with implications in the spleen-dependent immune response. this is supported by the reduced reaction capacity in the first days post-infection of ly e ∆hsc samples compared to wt, as indicated by the number of deg between control and cases at d p.i for these genotypes. the spleen wt network displayed three clusters ( figure a c ). cluster , whose genes were all upregulated in ly e ∆hsc samples at d p.i. compared to mock infection, is mostly involved in nucleosome organization and chromatin remodelling, together with cluster . cluster would also be related to dna packaging complexes, possibly in response to interferon, similarly to liver networks. instead, in spleen ko most genes take part in processes related to the extracellular matrix. in the spleen ko network, four clusters were identified ( figure a d ). cluster is related to the activation of an immune response, but also, alongside with clusters and , to the extracellular matrix, possibly in relation with collagen, highlighting its role in the response to mhv. cluster is implied in protease binding. the dramatic shut down in the ko network of the nuclear activity observed in the spleen wt network, leads to the hypothesis that the chromatin remodeling activity observed could be related to the activation of certain immunoenhancer genes, modulated by gene ly e. in any case, further experimental validation of these results would provide meaningful insights in the face of potential therapeutic approaches (see appendix a for more details). the exploration of nodes memebership, depending on whether these exclusively belonged to wt or ko networks or, by contrast, were present in both networks, helped to understand the impairment caused by ly e ∆hsc . in this sense, go enrichment analyses over these three defined categories of the nodes in the liver networks revealed that genes at their intersection are mainly related to cytokine production, leukocyte migration and inflammatory response regulation, in accordance to the phenotype described for mhv-infection [ ] . however, a differential response to virus is observed in wt mice compared to ly e-ablated. the nodes exclusively present at the wt liver network are related to processes like regulation of immune effector process, leukocyte mediated immunity or adaptive immune response. these processes, which are found at a relatively high gene ratio, are not represented by nodes exclusively present in the liver ko network. additionally, genes exclusively present at the wt network and the intersection network are upregulated in case samples with respect to controls ( figure a a) , which suggests the activation of the previously mentioned biological processes. on the other hand, genes exclusively-present at the liver ko networks, mostly down-regulated, were found to be associated with catabolism. as for the spleen networks, genotype-wise go enrichment results revealed that the previously-mentioned intense nuclear activity involving protein-dna complexes and nucleosome assembly is mostly due to wt-exclusive genes. actually, these biological processes could be pinpointing cell replication events. analogously to the liver case, genes that were found exclusively present in the wt network and the intersection network are mostly upregulated, whereas in the case of ko-exclusive genes the upregulation is not that extensive. interestingly, the latter are mostly related to extracellular matrix (ecm) organization, which suggest the relevance of ly e on these. other lymphocyte antigen- (ly- ) superfamily members have been related to ecm remodelling processes such as the urokinase receptor (upar), which participates in the proteolysis of ecm proteins [ ] . however and to the best of our knowledge, the implications of ly e in ecm have not been reported. the results presented are in the main consistent with those by pfaender et al. [ ] , who observed a loss of genes associated with the type i ifn response, inflammation, antigen presentation, and b cells in infected ly e ∆hsc mice. genes stat and ifit , selected in their work for their high variation in absence of ly e, were identified as hub genes in the networks reconstructed from liver wild type and knockout samples, respectively. it is to be noticed that our approach significantly differs to the one carried out in the original study. in this particular case, we consider that the reconstruction of gcn enables a more comprehensive analysis of the data, potentially finding the key genes involved in the immune response onset and their relationships with other genes. for instance, the transcriptomic differences between liver and spleen upon ly e ablation become more evident using gcn. altogether, the presented results show the relevance of gene ly e in the immune response against the infection caused by mhv. the disruption of ly e significantly reduced the immunogenic response, affecting signaling and cell effectors. these results, combining in vivo and in silico approaches, deepen in our understanding of the immune response to viruses at the gene level, which could ultimately assist the development of new therapeutics. for example, basing on these results, prospective studies on ly e agonist therapies could be inspired, with the purpose of enhancing the gene expression level via gene delivery. given the relevance of ly e in sars-cov- according to previous studies [ , ] , the overall effects of ly e ablation in hscs upon sars-cov- infection, putting special interest in lung tissue, might show similarities with the deficient immune response observed in the present work. in this work we have presented an application of co-expression gene networks to analyze the global effects of ly e ablation in the immune response to mhv coronavirus infection. to do so, the progression of the mhv infection on the genetic level was evaluated in two genetic backgrounds: wild type mice (wt, ly efl/fl) and ly e knockout mutants (ko, ly e ∆hsc ) mice. for these, viral progression was assessed in two different organs, liver and spleen. the proposed reconstruction rationale revealed significant differences between mhv-infected wt and ly e ∆hsc mice for both organs. in addition we observed that mhv infection triggers a progressive genetic response of upregulating nature in both liver and spleen. in addition, the results suggest that the ablation of gene ly e at hsc caused an impaired genetic response in both organs compared to wt mice. the impact of such ablation is more evident in the liver, consistently with the disease site. at the same time, the immune response in the spleen, which seemed to be mediated by an intense chromatin activity in the normal situation, is replaced by ecm remodeling in ly e ∆hsc mice. we infer that the presence of ly e limits the damage in the above mentioned target sites. we believe that the characterization of these processes could motivate the efforts towards novel antiviral approaches. finally, in the light of previous works, we hypothesize that ly e ablation might show analogous detrimental effects on immunity upon the infection caused by other viruses including sars-cov, mers and sars-cov- . in future works, we plan to investigate whether the over-expression of ly e in wt mice has an enhancement effect in immunity. in this direction, ly e gene mimicking (agonist) therapies could represent a promising approach in the development of new antivirals. the authors declare no conflict of interest. q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q organ q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q genotype q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q sample type q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q top most variable genes across liver samples row z−score top most variable genes across spleen samples table a . number of deg used as input to engnet for network reconstruction and their latter distribution in inferred networks. genes that were not assigned to a cluster (or were comprised in minoritary clusters) were not taken into consideration for enrichment analyses. input genes network genes cluster cluster cluster cluster figure a . enrichment analyses based on node exclusiveness of (a) liver and (b) spleen networks. wt refers to nodes exclusively present at those networks reconstructed from wt samples; ko refers to nodes exclusively present at networks reconstructed from ly e ∆hsc samples; both addresses shared nodes between wt and ko networks. gene ratio is defined by the number of genes used as input for the ernichment analyses associated with a particular go term divided by the total number of input genes. expression of highly deg across spleen ko samples (d) figure a . cpm-normalized expression values of highly deg identified across (a) liver wt samples, (b) liver ko samples, (c) spleen wt samples and (d) spleen ko samples. dashed lines separate samples from the three groups under study: controls, cases at d p.i. and cases at d p.i. note sample order within same group is exchangeable. the reconstruction method employed in this case study was validated against other thee well-known inference methods: aracne [ ] , wgcna [ ] and wto [ ] . the output of each reconstruction method, using default values (including engnet) was compared to a gold standard (gs), retrieved from the string database. four different gss were taken into consideration, since these were reconstructed from the deg that were identified in the comparison of control vs. case samples at d p.i., as shown in section . . these deg were mapped to the string database gene identifiers selecting mus musculus as model organism (taxid: ). a variable percentage of deg ( - %) could not be assigned to a string identifier, and were thus removed from the analysis. the interactions exclusively concerning the resulting deg in each case were retrieved from the string database. these interaction networks would serve as gss. the mentioned deg (without unmapped identifiers) would also serve as input for the four reconstruction methods to be compared. the aracne networks were inferred using the spearman correlation coefficient following the implementations in the minet [ ] r package. in this case, mutual information values were normalized and scaled in the range - . on the other hand, the wgcna networks were reconstructed following the original tutorial provided by the authors [ ] . the power was defined as . additionally, the wto networks were built using pearson correlation in accordance to the documentation. absolute values were taken as relationship weights. finally, engnet networks were inferred using the default parameters described in the original article by gómez-vela et al. [ ] . for the comparison, the receiver operating characteristic (roc)-curve was estimated using the proc [ ] r package. roc curves are shown in figure a . the area under the roc curve (auc) was also computed in each case for the quantitative comparison of the methods, as shown in figure a a . the auc compares the reconstruction quality of each method against random prediction. an auc ≈ corresponds to the perfect classifier whereas am auc ≈ . approximates to a random classifier. thus, the higher the auc, the better the predictions. on average, engnet provided the best auc results, whilst maintaining a good discovery rate. in addition, engnet provided relatively scarce networks compared to wgcna, as shown in figure a b . this is considered of relevance given that sparseness is a main feature of gene networks [ ] . hosts and sources of endemic human coronaviruses identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins an orally bioavailable 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networks for the identification of lung carcinoma biomarkers: an ensemble approach step-by-step construction of gene co-expression networks from high-throughput arabidopsis rna sequencing data limma powers differential expression analyses for rna-sequencing and microarray studies precision weights unlock linear model analysis tools for rna-seq read counts false discovery control with p-value weighting the igraph software package for complex network research cytoscape . : new features for data integration and network visualization network biology using cytoscape from within r gene co-opening network deciphers gene functional relationships community structure analysis of biological networks a multi-algorithm clustering plugin for cytoscape topological analysis and interactive visualization of biological networks and protein structures selectivity determinants of gpcr-g-protein binding boxplot-based outlier detection for the location-scale family outlier detection: how to threshold outlier scores? gene ontology consortium: going forward clusterprofiler: an r package for comparing biological themes among gene clusters systematic and integrative analysis of large gene lists using david bioinformatics resources the string database in : quality-controlled protein-protein association networks, made broadly accessible massive-scale gene co-expression network construction and robustness testing using random matrix theory uncovering biological network function via graphlet degree signatures. cancer inform viral pathogenesis structure-guided mutagenesis alters deubiquitinating activity and attenuates pathogenesis of a murine coronavirus crosstalk of liver immune cells and cell death mechanisms in different murine models of liver injury and its clinical relevance a disparate subset of double-negative t cells contributes to the outcome of murine fulminant viral hepatitis via effector molecule fibrinogen-like protein structure and function of the immune system in the spleen progranulin and a five transmembrane domain-containing receptor-like gene are the key components in receptor activator of nuclear factor κb (rank)-dependent formation of multinucleated osteoclasts rank is essential for osteoclast and lymph node development autologous intramuscular transplantation of engineered satellite cells induces exosome-mediated systemic expression of fukutin-related protein and rescues disease phenotype in a murine model of limb-girdle muscular dystrophy type i the intriguing role of soluble urokinase receptor in inflammatory diseases ly e restricts the entry of human coronaviruses, including the currently pandemic sars-cov- aracne: an algorithm for the reconstruction of gene regulatory networks in a mammalian cellular context wgcna: an r package for weighted correlation network analysis wto: an r package for computing weighted topological overlap and a consensus network with integrated visualization tool ar/bioconductor package for inferring large transcriptional networks using mutual information a general framework for weighted gene co-expression network analysis proc: an open-source package for r and s+ to analyze and compare roc curves this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -ppa gb authors: neuman, benjamin w.; adair, brian d.; yoshioka, craig; quispe, joel d.; milligan, ronald a.; yeager, mark; buchmeier, michael j. title: ultrastructure of sars-cov, fipv, and mhv revealed by electron cryomicroscopy date: journal: the nidoviruses doi: . / - - - - _ sha: doc_id: cord_uid: ppa gb nan * the scripps research institute, la jolla, california. the current understanding of coronavirus ultrastructure relies heavily on transmission electron microscopy of negatively stained images. such images typically show desiccated specimens and derive contrast from the accumulation of heavy metal negative stains, distorting the sample in the resulting image. electron cryomicroscopy (cryo-em) avoids some of the drawbacks of negative staining by imaging frozen specimens preserved in a fully hydrated state in vitreous ice. cryo-em images typically derive contrast solely from the density of the imaged sample and the surrounding ice matrix. a limited analysis of porcine transmissible gastroenteritis virus (tgev) imaged by cryo-em has been previously reported. in this report we present a more detailed description of the supramolecular design of three coronaviruses: sars coronavirus (sars-cov), feline infectious peritonitis virus (fipv), and murine hepatitis virus (mhv). coronaviruses are usually classified as non-icosahedral, pleomorphic, enveloped viruses. cryo-em has revealed that other pleomorphic viruses have a roughly spherical appearance, studded with projections that correspond to oligomers of the attachment and fusion protein. examples include influenza virus [ ] [ ] [ ] ; several retroviruses such as foamy virus, human immunodeficiency virus, - murine leukemia virus, and rous sarcoma virus , ; la crosse virus , ; sendai virus ; and pichinde, tacaribe, and lymphocytic choriomeningitis viruses. based on single-particle image analysis of arenaviruses imaged by cryo-em, we have proposed that pleomorphic arenavirus particles are constructed from overlapping paracrystalline lattices of proteins, and that these lattices span the viral membrane. we hypothesized that coronaviruses may contain a similar supramolecular arrangement of proteins comprising a membrane-proximal scaffold. here we used cryo-em to examine the ultrastructure of a selection of coronaviruses representing two of the three proposed phylogenetic groups. particles of sars-cov, fipv, and mhv were prepared from vero-e , ak-d, and dbt cells, respectively. mhv and sars-cov were also produced in cells cultured with tunicamycin, to form spike-depleted particles with low infectivity. for safety reasons, all particles were fixed with % (for sars-cov) or % (for fipv and mhv) formalin in ph . hepes-buffered physiological saline before imaging. all viruses were collected by sucrose gradient ultracentrifugation, and each remained highly infectious until fixed. each virus appeared approximately round in cryo-em images, with a fringe of spikes protruding from the viral membrane and a region of lower density near the virion center ( fig. a-b) . the average diameter of the membrane-enclosed part of each virus was similar, ranging from ~ Å for sars-cov to ~ Å for fipv (fig. c) . the diameters of mhv and sars-cov virions were distributed more tightly than diameters of fipv or spike-depleted, tunicamycin-grown mhv. the mean diameters of native and tunicamycin-grown mhv were similar. particles of sars-cov and mhv produced from tunicamycin-treated cells lacked the characteristic fringe of spikes, but were otherwise indistinguishable from particles grown under standard culture conditions (fig. ) . spike-depleted sars-cov particles appeared similar to spike-depleted mhv particles in negative stain, but were produced in lower yield, not suitable for effective cryo-em imaging. particles were imaged in several degrees of focus in order to emphasize different structural elements. fine features such as the phospholipids headgroup densities of the viral membrane and individual nucleocapsid protein densities are revealed more clearly in images recorded relatively near to focus (figs. a-b, a right) . images recorded farther from focus reveal spikes more clearly at the edge and center of each particle ( fig. a, left) . preparations of each virus contained a small amount of material that was consistent with the appearance of coronavirus ribonucleoprotein (rnp). a particularly interesting image of a sars-cov particle trapped in a partially uncoated state at the time of freezing (fig. a-b) shows the spiral rnp partially uncoiled from an approximately round rnp core. the rnp proximal to the extruded membrane segment remains roughly spherical, and appears to be connected to the inner face of the membrane at the ruptured fringe (fig. c ). each virus is covered with spikes that extend ~ Å from the peak density of the headgroups in the outer leaflet of the viral membrane. there appears to be a gap between adjacent end-projected spikes near the virion center ( fig. a right, for example) . the arrangement of spike densities near the center of some particles approximates a rhombus, which would not be inconsistent with a paracrystalline organization of spikes as observed in the virions of pleomorphic arenavirus particles, or a local hexagonal close-packing of structural proteins as observed in retroviral particles. coronavirus particles, as previously pleomorphic virions we have examined by cryo-em. the observed variability in shape and size of the coronavirus particle would typically be considered inconsistent with icosahedral organization. the observation that the helical rnp is retained in a rough sphere through apparent interaction with proteins resident in the viral membrane is consistent with the spherical arrangement of the viral nucleocapsid proposed for tgev. however, further image analysis and biochemical experimentation will be required to determine the supramolecular organization of the virion. some of the work described here was conducted at the national resource for automated molecular microscopy (nramm), which is supported by the national institutes of health though the national center for research resources' p program (rr ). this work was supported by nih grants ai , ai , and ns , and by nih/niaid contract hhsn c. the transmissible gastroenteritis coronavirus contains a spherical core shell consisting of m and n proteins electron microscopy of influenza virus. a comparison of negatively stained and ice-embedded particles fine structure of influenza a virus observed by electron cryo-microscopy morphological changes and fusogenic activity of influenza virus hemagglutinin specific interaction of a novel foamy virus env leader protein with the n-terminal gag domain structural organization of authentic, mature hiv- virions and cores cryo-electron microscopy reveals ordered domains in the immature hiv- particle projection structures of human further evidence of icosahedral symmetry in human and simian immunodeficiency virus organization of immature human immunodeficiency virus type supramolecular organization of immature and mature murine leukemia virus revealed by electron cryo-microscopy: implications for retroviral assembly mechanisms the organization of mature rous sarcoma virus as studied by cryoelectron microscopy characterization of rous sarcoma virus gag particles assembled in vitro electron microscopy of vitrified-hydrated la crosse virus structural variation of la crosse virions under different chemical and physical conditions cryoelectron microscopy of vitrified sendai virions complementarity in the supramolecular design of arenaviruses and retroviruses revealed by electron cryomicroscopy and image analysis ribonucleoprotein-like structures from coronavirus particles the membrane m protein carboxy terminus binds to transmissible gastroenteritis coronavirus core and contributes to core stability immunodeficiency virus type (hiv- ) observed with high resolution electron cryo-microscopy key: cord- -szezd vb authors: jacomy, hélène; talbot, pierre j title: vacuolating encephalitis in mice infected by human coronavirus oc date: - - journal: virology doi: . /s - ( ) - sha: doc_id: cord_uid: szezd vb involvement of viruses in human neurodegenerative diseases and the underlying pathologic mechanisms remain generally unclear. human respiratory coronaviruses (hcov) can infect neural cells, persist in human brain, and activate myelin-reactive t cells. as a means of understanding the human infection, we characterized in vivo the neurotropic and neuroinvasive properties of hcov-oc through the development of an experimental animal model. virus inoculation of -day postnatal c bl/ and balb/c mice led to a generalized infection of the whole cns, demonstrating hcov-oc neuroinvasiveness and neurovirulence. this acute infection targeted neurons, which underwent vacuolation and degeneration while infected regions presented strong microglial reactivity and inflammatory reactions. damage to the cns was not immunologically mediated and microglial reactivity was instead a consequence of direct virus-mediated neuronal injury. although this acute encephalitis appears generally similar to that induced by murine coronaviruses, an important difference rests in the prominent spongiform-like degeneration that could trigger neuropathology in surviving animals. although the etiology of most neuroautoimmune, neuroinflammatory, and/or neurodegenerative diseases remains unclear, virus infections could directly trigger neurodegeneration or initiate a cns-directed inflammatory process leading to central nervous system (cns) damage, or a combination of both. indeed, parkinson's disease, alzheimer's disease, amyotrophic lateral sclerosis (als), and multiple sclerosis (ms) could actually represent infectious diseases (calne et al., ; kristensson, ; kirk and zhou, ; allen et al., ; hayase and tobita, ; klein et al., ; boucher et al., ; jubelt and berger, ; giraud et al., ; sola et al., ) . moreover, psychiatric disorders were also investigated as a possible consequence of viral infections (waltrip et al., ; lewis, ) . the vertebrate cns was long thought to be inaccessible to cells of the immune system or to viruses. however, the presence of virus in the cns is more frequent than expected and viral detection in the cerebrospinal fluid of patients suggests the ability of viruses to cross the bloodbrain barrier (koskiniemi and vaheri, ; georgsson, ) . in fact, neuroinvasive viruses can damage the cns and produce neurological disease in sensitive hosts, due to the misdirected immune response of the host (virus-induced immunopathology) and/or viral replication in cells of the brain (virus-induced cytopathology). nevertheless, primary infections of the brain are not common and viruses are the leading cause of encephalitis, which results from either direct infection (acute encephalitis) or the immune response to an infection (postinfectious encephalitis or acute demyelinating encephalomyelitis). in acute encephalitis, viral replication occurs in the brain tissue itself, causing destructive lesions of the gray matter: this was reported after herpes simplex, rabies, or some arbovirus infections (rupprecht et al., ; shoji et al., ) . therefore, the knowledge of infectious agents involved in neurological diseases and mechanisms underlying the induction of neuropathology by these pathogens will be invaluable for preventing and developing novel clinical interventions. coronaviruses are enveloped positive-stranded rna viruses that infect multiple species of mammals, including man, causing diseases that range from encephalitis to enteritis. human coronaviruses (hcov) are recognized respiratory pathogens responsible for up to % of common colds (mcintosh, ; myint, ) and also involved in nocosomial infections (sizun et al., ) . they have occasionally been associated with other pathologies, such as pneumonia, meningitis, and enteritis (riski and hovi, ; resta et al., ) . moreover, hcov have the ability to replicate and persist in human neural cells (bonavia et al., ; arbour et al., a,b) and to have neuroinvasive properties (burks et al., ; murray et al., ; stewart et al., ; arbour et al., ) . this has stimulated research on their possible involvement in neurological disorders. of the two known hcov serotypes, designated oc and e, hcov-oc is antigenically related to murine coronaviruses (mhv) . given that, under certain conditions, mhv causes experimental cns inflammatory demyelination that pathologically resembles ms (bailey et al., ; lampert et al., ; weiner, ; wang et al., ) , the related human coronavirus represents a logical target for investigation. in the present study, we report the development of a mouse model to characterize in vivo hcov-oc -mediated neuropathogenesis. we describe the acute disease induced by hcov-oc infection, which resulted from neuronal infection and loss. this animal model constitutes a tool to study neuroinvasive and neurovirulence properties of a common cold virus and the mechanisms underlying the development of a diffuse vacuolating meningoencephalitis, an emerging medical problem (shoji et al., ; whitley and gnann, ) . balb/c and c bl/ mice were selected in view of their relative susceptibility to both respiratory and enteric strains of mhv (barthold and smith, ) . we tried different inoculation routes to establish the neurotropic and neuroinvasive properties of hcov-oc in mice. an intraperitoneal inoculation with a virus dose of tcid revealed that hcov-oc virus infection could be lethal until days postnatal (dpn) and the same doses were nonlethal at dpn. with an intraoral inoculation of - tcid of virus, we were unable to reveal the presence of virus or virus gene products in any tissue tested (brain, spinal cord, heart, lung, liver, and spleen), even by rt-pcr. mice were susceptible to intranasal (in) inhalation of the hcov-oc solution at - tcid . this infection was lethal in -week-old c bl/ mice. however, dpn mice infected this way did not show clinical signs of pathology, but of the animals were found positive for viral rna by rt-pcr analyses. viral rna was mainly found in the cns but some mice also showed virus rna in the spleen (data not shown). therefore, virus could spread from the periphery to the cns after in inhalation. having shown hcov-oc neuroinvasive properties, we chose for the remaining experimentation to use intracerebral inoculation (ic) so as to favor a cns infection. the ic route results in a more reproducible infection, a better control of viral doses introduced into the brain. the correlation between viral infectious dose and % mortality in the two strains of mice after inoculation is shown in table . mice became less susceptible with age and were resistant at dpn for c bl/ and at dpn for balb/c mice. we then determined the optimal experimental conditions to obtain a sublethal dose that still allowed virus replication and virus-induced cns pathology in -day-old mice. viral dose was administered under deep anesthesia and was determined to be l of a virus solution containing tcid for c bl/ and tcid for balb/c mice aged dpn. under these conditions, inoculated mice developed signs of acute disease characterized by loss of weight, apathy, ruffled fur, humped posture, and wasting (figs. a and b) . animals showed atrophy of skeletal muscles and occasionally exhibited paralysis of their forelimbs. during the second week postinfection, some of the animals recovered and clinical signs totally disappeared. for others, pathological signs increased and led to death. the infected animals became anorexic, inactive, and dehydrated, increasing percentages of mortality. we established survival curves for each mouse strain (fig. c) . eighty percent of the c bl/ mice died within the first days postinfection and only % of balb/c mice died during this period, even after receiving a higher viral dose. moreover, mice inoculated with supernatants from cell cultures infected with brain tissue from affected mice developed the same disease, demonstrating that the virus was responsible for pathology. viral rna could be detected in the brain as early as h postinfection, and after to days in the spinal cord. all c bl/ mice were positive for hcov-oc rna during the first days postinfection and during the first days for balb/c mice ( figs. a and b) . a screening of viral rep- note. this dose (expressed in tcid ) is in function of mouse age (days postnatal; dpn) at the time of inoculation. lication was performed by rt-pcr in a variety of tissues and results obtained indicated that infection was restricted to the cns during the first days postinfection. after that, in the most affected mice, viral rna was also found in heart, spleen, and lungs, and at lower levels in liver and muscles between the th and th days postinfection in c bl/ , suggesting a viremic spread or transneuronal transmission (fig. b) . the presence of hcov-oc rna was detectable in the brain until days postinfection for % of balb/c mice and until days postinfection for % of c bl/ mice. no viral rna could be found from tissues harvested after these times postinfection. it was also confirmed that the rt-pcr assay designed to specifically detect hcov-oc was indeed specific and could not have detected an enzootic mhv strain (fig. c) . infectious virus appeared around days postinfection and could be isolated from the cns of c bl/ mice during the first weeks postinfection ( fig. a) . the highest levels of infectious virions were found between and days postinfection (fig. c) . in balb/c mice, virus was detectable at day postinfection and reached the highest titer around days postinfection (figs. b and c). the highest infectious titers observed were tcid /g for brain and tcid /g for spinal cord extracts. no infectivity could be detected at and after days postinfection for c bl/ and days postinfection for balb/c mice. viral proteins were found in the brain and spinal cord of c bl/ mice between and days postinfection (fig. a) and were undetectable after days postinfection. we detected two forms of the n protein, as already noted in dpn hcov-oc -inoculated mice (jacomy and talbot, ) or after mhv- infection (talbot et al., ) . blood collected at different time points after infection revealed that serum contained antibodies specific for hcov-oc . humoral immunity started to appear at week postinfection and increased during the first month postinfection, and antiviral antibodies were still present at months postinfection, as shown by indirect immunofluorescence on infected hrt- cells (data not shown). no immunofluorescent cells were seen with serum obtain from control mice. histochemical labeling of viral distribution at different times after infection revealed that virus infection initiated by ic inoculation was quickly disseminated throughout the cns. cells positive for viral antigens were first observed at hcov-oc -infected mice gained weight normally during the first days after infection, after which they all lost weight during the acute phase of the disease. the more affected mice lost more weight more rapidly than less affected mice and died during this period. after days postinfection, mice which survived gained weight to reach the weight of control animals around days postinfection. (c) survival curves of mice after hcov-oc infection. balb/c mice received a higher dose than the c bl/ mice, , tcid versus tcid . however, c bl/ were less resistant, with versus % of death after infection. days postinfection in the gray matter of the brains of c bl/ mice. at this time, microglial activation was still undetectable as assessed by mac- immunostaining. at week postinfection, hcov-oc had spread to all cns regions, predominantly in the entire cerebral cortex, the striatum, the hippocampus, the hypothalamus areas, the colliculus superior, and the brain stem, including the spinal cord (figs. and ). the cerebellum was frequently spared, but purkinje cells were found positive for virus in some animals. astrogliosis revealed by gfap staining increased and activated microglial cells started to appear along the ventricles (figs. f, g, and h). activated microglial cells were not observed in the cns of noninfected control mice at any time during investigation, as monitored by the absence of staining for mac- , a marker not expressed in nonreactive microglia (walther et al., ) . in the spinal cord at days postinfection, an hcov-oc -specific mab labeled sensory and motor neurons, and microgliosis and astrogliosis were also detected in infected regions (fig. ) . the progression of the infection was accompanied by identical neuropathologic features in the two strains of mice: neurons exhibited severe signs of pathology, most of them showing necrosis and vacuolation. this started by the development of small and round empty vacuoles in the cytoplasm, which increased in size (figs. c, d, and e). these spongiform-like lesions were seen primarily within the neuronal cell bodies, the neuropil being generally unaffected (fig. c ). this feature was never observed in noninfected brain (fig. a) . ultrastructurally, numerous cells presented cytoplasm disorganization without lysis of the cellular membranes. degenerative changes included cytoplasm rarefaction, dilatation of the rough endoplasmic reticulum (rer), and disaggregation of polyribosomes leading to the appearance of free ribosomes. hematoxylin-eosin staining also revealed the presence of degenerated neurons with picknotic or small densely stained nuclei and eosinophilic cytoplasm (figs. c, e, h, and i). at an advanced stage of disease, loss of neurons was pronounced and was particularly evident in ca and ca hippocampal layers ( fig. d -i). histological examination of the brain or spinal cord revealed scattered infiltration of inflammatory cells, starting by mononuclear cell infiltrations (fig. b ) and perivascular cuffing. some macrophage-mediated elimination (neu- fig. . hcov-oc infectious virus and rna in the cns of dpn mice. (a) % of brains from c bl/ mice inoculated ic with tcid of hcov-oc were positive for viral rna between and days postinfection. only % of the surviving mice were found positive after days postinfection and rna was not found thereafter. infectious virus appeared later and disappeared before elimination of viral rna. between and days postinfection, % of brains contained infectious virus. (b) detection of hcov-oc rna in the brain of balb/c mice inoculated ic with tcid of hcov-oc revealed that % of these mice were positive until days postinfection. infectious virus was detectable in all mice only during the first days postinfection and gradually fewer mice were found positive. (c) histogram representing the amount of infectious virus detected in five brains from the two strains of mice at different intervals postinfection. the limit of the detection assay was . tcid . ronophagia) was also encountered. in the spinal cord, viral particles observed days postinfection at the electron microscopic level were mostly localized in the cell cytoplasm, closely associated with the golgi apparatus or in extracellular spaces (fig. ) . viral replication and transneuronal passage occurred in a stepwise fashion that utilized existing cellular processes. when hcov-oc replication and spread reached maximal levels, around days postinfection, astrogliosis and microgliosis progressively increased in all infected regions of the cns until the death of the animal (figs. g and h). therefore, a correlation between pathological signs of disease observed in mice and morphological injury of the brain was apparent. clearance of mhv from the cns appears to involve t cells (sussman et al., ) and age-acquired resistance to virus could be abolished in immunosuppressed animals (zimmer and dales, ) . therefore, we examined the effect of immunosuppression on hcov-oc -mediated neuropathogenesis. the immunosuppressive effects of cyclosporin a (csa) are clearly established (borel et al., ) . as csa causes a specific reversible inhibition of immunocompetent lymphocytes (preferentially t cells) and inhibits gene transcription for certain cytokines, in particular il- (kupiec-weglinski et al., ; elliott et al., ; shevach, ) , we investigated whether csa could modify the course of the acute hcov-oc infection on the development of cns lesions or on viral replication. it is known that csa injected into mice at mg/kg/day induces neurotoxicity (hypocellular and disorganized organs), whereas csa at . mg/kg/day induces no abnormalities and spread to all organs (boland et al., ) . therefore, csa doses were selected to avoid cytotoxic effects and mortality in mice and were in accordance with immunosuppression-inducing doses described in the literature (bolton et al., ; pasick et al., ) . control mice treated with csa at a daily dose of mg/kg did not show any apparent adverse effects: they gained weight normally and did not present ruffled fur or lethargy. for csa-treated and untreated mice, the kinetics of weight loss was similar after hcov-oc infection. nevertheless, immunosuppression by csa slightly precipitated the disease but increased mortality (fig. ) . this was more pronounced in mice treated with csa at mg/kg/day where % of mice died, whereas only % of oil-treated mice succumbed to hcov-oc infection. infection of mice by hcov-oc was dependent on a number of variables, including dose, route of inoculation, age of the host, and its genetic background. indeed, our results show striking susceptibility differences between two strains of mice: balb/c mice were more resistant than c bl/ . moreover, resistance increased with age in the two strains of mice. this suggests that susceptibility to human coronavirus neuropathogenesis may be linked to genetic factors. our study also confirms that human coronaviruses have neuroinvasive properties in mice, which was first shown in newborn mice (barthold et al., ) , and that such neuroinvasion is possible even after maturation of the immune system (king et al., ) , which is consistent with their detection in human brain (burks et al., ; murray et al., ; stewart et al., ; arbour et al., ) . even though our study does not confirm a specific route of entry into the cns, a transneuronal route already demonstrated for mhv (lavi et al., ; barthold et al., ; perlman et al., a) constitutes a likely possibility. twenty-one days postnatal mice infected by ic inoculation of hcov-oc developed signs of acute disease characterized by apathy, hunched posture, ruffled fur, and tremors, comparable to pathological signs described after mhv infection (kristensson et al., ) . following ic inocula- pathologic symptoms and mortality even with very high viral doses (lavi et al., ) . the clinical signs of pathology after hcov-oc ic inoculation coincided with the peak in virus yields observed at approximately to days postinfection for c bl/ mice. this indicates that virus replication in the cns apparently played a major role in the establishment of the pathology. infected mice showed extensive inflammatory responses characterized by mononuclear perivascular cuffing, neuronophagia, and a great number of reactive glial cells in the infected regions. to investigate whether infiltration of inflammatory cells contributed to neurodegeneration or if infectious virus was directly responsible for vacuolating lesions and neuronal death, we evaluated the effect of treating animals with cyclosporin a, a powerful immunosuppressant drug. with the dose used ( or mg/kg/day), csa is known to be distributed extensively throughout the body and not to cause neurotoxicity in mice (boland et al., ) . immunosuppression precipitated human coronavirus-induced disease and increased the percentage of acute death ( vs %). under csa treatment, neurons also presented vacuolation and degeneration. therefore, the pathology observed following hcov-oc infection was likely not immunologically mediated, unlike that induced by mhv-a and mhv-jhm (sussman et al., ; wang et al., ) , although experiments with immunodeficient mice of the same genetic background will be needed to definitely address this question with hcov-oc . moreover, macrophage/microglial reactivity was delayed when related to infection and appeared only when the virus was present in most parts of the brain. the inflammatory response and macrophage/microglial cell recruitment seem to be strongly correlated with virus clearance, as was also demonstrated after mhv infection (sussman et al., ) . some strains of mhv, including a and jhm, are neuroinvasive in rodents, eliciting either an acute encephalitis or a chronic paralytic disease (for review, see perlman, ) . unlike the slow neurodegenerative disease caused by mhv (bailey et al., ; lampert et al., ; weiner, ; lavi et al., ; wang et al., ) , hcov-oc resulted in a productive and cytotoxic infection of neuronal cells in the cns, which led to neurodegeneration. group of c bl/ mice treated with cyclosporin a at mg/kg/day (oc / mgcsa) and at mg/kg/day (oc / mgcsa) became more susceptible to hcov-oc infection, with and % of death after infection versus % in non-csa-treated animal. infected c bl/ mice treated with oil alone (oc /oil) presented similar survival curves as previously reported, with % of death after infection. noninfected hcov-oc mice treated with csa at mg/kg/day (control/ mgcsa) illustrated that csa was not toxic under these conditions. having previously demonstrated a persistent infection of hcov-oc in primary murine cns cell cultures and in the cns of mice inoculated at dpn (jacomy and talbot, ) , and given the observations that mhv antigens or rna were still detectable in the spinal cord several weeks after infection (woyciechowska et al., ; perlman et al., b) , we expected to detect a persistent infection in the cns of infected dpn mice. however, rt-pcr analysis revealed that viral rna could not be detected after the second week postinfection, suggesting a nonpersistent infection of hcov-oc virus in dpn mice. histological analysis of infected dpn mice showed virus spread throughout the brain and spinal cord, as we had previously described for dpn mice (jacomy and talbot, ) . neurons remained the major cellular targets for virus, which was probably disseminated from neurons to neurons by a cell-to-cell transport, as was described after mhv infection (lavi et al., ; . nevertheless, even though neurons were susceptible to mhv infection, oligodendrocytes and astrocytes represented the major infected cell types (perlman and ries, ; . this may explain why the pathology observed in mice was different after infection with these two antigenically related coronaviruses: mhv can cause demyelination (bailey et al., ; lampert et al., ; weiner, ; wang et al., ) with a spongy state observed in the white matter (lavi et al., ) , whereas hcov-oc encephalitis is accompanied by vacuolating degeneration of the gray matter. the latter lesions were not detected in a previous study (pearson and mims, ; barthold et al., ) , probably because mice were younger and died within to days postinfection. spongiform cellular changes were occasionally reported after mhv infection. for example, vacuolation was observed in the subthalamic-nigral region after ic inoculation of mhv-a (fishman et al., ) , and foci of vacuolation were observed in the hypothalamus, cerebellar peduncles, and pons regions after in inoculation of mhv-s ( barthold and smith, ) . nevertheless, these degenerative changes were not commonly observed after mhv infection and were restricted to small cell populations, even after infection with -to -fold higher virus doses into c bl/ mice. interestingly, the appearance of clear round vacuoles and neuronal death represents a hallmark of cns degeneration observed in prion diseases (prusiner, ) . nevertheless, mitochondrial disease (mckelvie et al., ) , leigh's disease (kimura et al., ; agapitos et al., ) , pick's disease (deruaz et al., ) , or alzheimer's disease (duffy et al., ; budka et al., ) also display spongiform cns lesions, which are independent of the prion protein. pathways and causal start points of transmissible spongiform encephalopathies or acute encephalitis remain unknown. occasionally, viral infections of the cns were described to induce spreading spongiosis, such as in human t cell leukemia virus associated myelopathy (htlv- ) or in hiv encephalitis (rhodes, ; goldwater and paton, ) . in rodents, mutants of vsv virus (rabinowitz et al., ) and moloney murine leukemia virus (mo-mulv) (gardner et al., ; czub et al., ) were shown to experimentally induce spongiosis. histologically, vacuolar degeneration induced by hcov-oc was mainly restricted to the neuronal cell bodies, whereas that caused by prion or retrovirus first affected the neuropil. moreover, the inflammatory response was very limited in prion encephalopathy, whereas hcov-oc induced extensive brain inflammation. these indicate different mechanisms underlying vacuolation and neuronal death. interestingly, noninflammatory neuropathologies have been considered evidence against a viral etiology. infections by opportunistic pathogens such as respiratory or enteric virus in immuosuppressed patients (hiv, transplantation, and cancer chemotherapy) may also cause cns pathology. it has been reported that immunocompromised patients have increased incidences of malignancies induced by viral infection (penn, ) . therefore, severe cases of encephalitis have devastating effects on the brain and spinal cord functions. given our previous observations of hcov-oc persistence in human brain (stewart et al., ; arbour et al., ) , we propose that respiratory pathogens with a neurotropic and neuroinvasive potential could be associated with neurodegenerative disease in susceptible individuals. this animal model of human coronavirus neuropathogenesis may prove helpful in the characterization of coronavirus-induced neurodegeneration in surviving infected animals and of transneuronal virus spread to the cns. moreover, susceptibilities of endothelial cells and leukocytes to viral infection need to be investigated as a possible alternate route of virus entry into the cns. finally, possible explanations for our observations of striking differences in susceptibility to hcov-oc infection of two strains of mice remain to be investigated. even though mice are not the natural host for hcov-oc infections, they may provide data that, together with studies using human neural cell cultures and post-mortem brain tissue, may contribute to our understanding of the underlying mechanisms and neuropathological consequences of coronavirus infections in humans. the oc strain of hcov was originally obtained from the american type culture collection (atcc, rockville, md), plaque-purified, and grown on the human rectal carcinoma cell line hrt- as previously described . hcov-oc virus stocks ( tcid / ml) were kept at Ϫ °c. to determine the susceptibility of mice to hcov-oc infection, two different strains of mhv-seronegative female mice (balb/c and c bl/ , from jackson laboratories, bar harbor, me, u.s.a.) were inoculated. inoculations were performed on mice at various days postnatal ( , , , , , dpn) using l of various dilutions of the initial virus stock and using different inoculation routes: intraperitoneal, intraoral, intranasal, and intracerebral, to investigate hcov-oc infection parameters, in particular its neuroinvasive property. the viral dose was administrated ic under deep anesthesia of ketamine-xylamine (ketamine at mg/kg and xylamine at mg/kg). in the present study, we chose to infect dpn mice with an ic inoculation of l containing tcid of hcov-oc for c bl/ and , tcid for balb/c mice. twenty mice of each strain were used to establish survival curves. every days postinfection, five animals from two groups of infected mice of each strain were sacrificed and processed for detection of viral rna, viral proteins, and infectious virus. moreover, two infected mice of each strain were perfused every days for histological analysis during the first month postinfection. for each experiment, age-matched control animals, which had received a virus-free solution containing culture medium from the hrt- cell line, were used. to confirm that hcov-oc was responsible for the observed pathology, we infected hrt- cell cultures with brain homogenates, prepared as described below, from infected mice. cell-free supernatants harvested days later were confirmed to contain infectious virus and l was reinoculated ic into other animals. cyclosporin a (sigma chemical co., st. louis, mo) was rehydrated in pure ethanol as specified by the manufacturer. it was then dissolved before use in olive oil to favor free diffusion of hydrophobic cyclosporin molecules through the plasma membrane into the cytoplasm (handschumacher et al., ) , and heated for h at °c. mice received a subcutaneous injection of or mg/kg/day. this drug was administered day prior to virus inoculation and daily thereafter for days. three groups of female c bl/ mice were infected with tcid of hcov-oc and control females were inoculated with hrt- medium. two groups of hcov-oc -infected mice received a single daily injection of csa, either or mg/kg/day. four control mice and the third group of hcov-oc -infected mice received injection of olive oil alone. the four remaining control mice were treated with csa at mg/kg/day, to verify the absence of csa toxicity. brain and spinal cords were dissected, homogenized in % (w/v) sterile pbs, centrifuged at °c, min at g; then supernatants were immediately frozen at Ϫ °c and stored until assayed. the extracts were processed for the presence and quantification of infectious virus by an indirect immunohistochemistry assay, as previously described (bonavia et al., ) . hcov-oc -susceptible hrt- cells were inoculated with serial logarithmic dilutions of each tissue sample in a -well linbro plate (icn biomedical canada ltd., costa mesa, ca). after days of incubation at °c in % (v/v) co , cells were washed in pbs and fixed with . % (v/v) hydrogen peroxide (h o ) in methanol for min. after washing with pbs, they were incubated for h at °c in / dilution of an ascites fluid from mouse mab - c. , directed against the nucleocapsid protein of hcov-oc (arbour et al., b) . afterwards, cells were washed in pbs and hrp goat antimouse immunoglobulins (dako, diagnostics canada inc., mississauga, on) were added and incubated for h at °c. antibody complexes were detected by incubation in . Јdiaminobenzidine tetrahydrochloride solution (dab, sigma), with . % (v/v) h o . mice were perfused by intraventricular injection of % (v/v) paraformaldehyde, under deep ketamine-xylazine anesthesia. brains and spinal cords were removed and tissue blocks were left in the fixative for h. coronal sections from brain and segments from cervical and lumbar spinal cord were sectioned at a thickness of m with a lancer vibratome. serial sections were collected in . m trisbuffered saline (tbs) and were then incubated overnight with primary antibodies, as previously described (jacomy and bosler, ) . for viral antigens, we used / dilutions of ascites fluids of the -e . hybridoma that secretes monoclonal antibodies specific for the nucleocapsid protein of the serologically related hemagglutinating encephalomyelitis virus of pigs (bonavia et al., ) . astrocytes were identified with a rabbit anti glial fibrillary acidic protein antibody (gfap, dako) diluted / , microglia/macrophages by an ascites fluid of the rat mac- antibody (atcc) diluted / . then, sections were rinsed and processed with vectastain abc kit (vector laboratories, burlingame, ca). labeling was revealed with . % (w/v) dab solution (sigma) and . % (v/v) h o , which yielded a dark brown product. some sections were counterstained with the classical cresyl violet stain. to further investigate histological changes occurring in mouse brains, half hemispheres from control and infected animals were paraffin-embedded and -m sections were stained with hematoxylin-eosin. this was performed by the pathology department, animal resources centre, mcgill university (montréal, québec, canada). samples for electron microscopy were postfixed for h with % (v/v) osmium tetraoxide in . m phosphate buffer at ph . , dehydrated in graded ethanol series, and eponembedded as previously described (jacomy and bosler, ) . one-micron sections were stained with toluidine blue and examined by light microscopy. subsequent ultrathin sections were collected on collodion-coated single-slot grids, stained with lead citrate, and examined with transmission electron microscope. blood from infected or control mice were collected at , , , or weeks and at , , and months postinfection. sera were collected and kept at Ϫ °c until use for the detection of antibodies against hcov-oc by indirect immunofluorescent labeling of infected hrt- cells. briefly, hrt- cells cultured on -well slides were infected by hcov-oc and fixed days later in cold methanol and then kept at Ϫ °c until needed. at the time of the assays, slides were incubated h at room temperature with serum from control and infected mice, diluted / , / , and / . after several washes in pbs, slides were incubated h at °c with alexa fluor f(abЈ) fragments of goat antimouse igg (hϩl), at a dilution of / , (molecular probes, inc., eugene, or) and observed under a fluorescence microscope. tissues were homogenized in sub buffer, containing m urea, . % (w/v) sds, and % (v/v) ␤-mercaptoethanol and then centrifuged for min at °c, in a microfuge at , g and supernatants were collected, as previously described (jacomy et al., ) . samples ( g total protein) were fractionated on a . % polyacrylamide gel (sds-page) and either visualized by coomassic blue staining or transferred to nitrocellulose for western blot analysis. nitrocellulose membranes were preincubated in % (w/v) skimmed milk powder in ts buffer ( . m tris, ph . , . m nacl) and then incubated overnight at °c with e . antiviral mab. after several washes with ts buffer containing . % (v/v) tween , membranes were incubated h with peroxidase-conjugated anti-mouse igg diluted / (dako). bands were visualized using a western blot chemoluminescent kit (super signal, pierce, rockford, md). tissues were dissected every days postinfection. total rna was extracted by homogenization in trizol (gibco-brl, burlington, ca). for rt-pcr, one pair of hcov-oc primers was designed to amplify a region containing nucleotides (primers o and o ) of the gene coding for the n protein (arbour et al., b) . the target sequences were specific to hcov-oc and did not amplify mhv. the suitability of rna for rt-pcr amplification was verified by an rt-pcr specific for a housekeeping gene encoding glyceraldehyde- -phosphate dehydrogenase (gapdh) using a pair of gapdh primers amplifying a region containing nucleotides (arbour et al., b) . one pair of mhv primers was also designed to amplify a conserved region of the mhv n protein gene. primers were Ј-cctctactgtaaaacct-gatatgg- Ј and Ј-ctaatttagatccaaagaaga-agc- Ј, corresponding to nucleotides - and - , respectively. approximately g of rna was reverse transcribed with expand moloney murine leukemia virus reverse transcriptase (gibco-brl) and the cdna products were incubated in pmol of each sense and antisense primers, . mm mgcl , ϫ pcr buffer ( mm tris-hcl, ph . ; mm kcl), and . mm of each deoxynucleotide triphosphate, heated at °c for min and °c (hcov-oc ) or °c (gapdh and mhv) for min. after the addition of expand high-fidelity pcr system dna polymerase (rtaq, u/l; amersham pharmacia biotech inc., baie d'urfé, qc), amplification cycles of min at °c, min at °c, and min at °c (hcov-oc ) or °c (gapdh and mhv) were performed. ten microliters of this reaction mix was loaded onto a . % (w/v) agarose gel containing l ethidium bromide. subacute necrotizing encephalopathy (leigh's disease): a clinicopathologic study of ten cases the significance of measles virus antigen and genome distribution in the cns in sspe for mechanisms of viral spread and demyelination acute and persistent infection of human neural cell lines by human coronavirus oc neuroinvasion by human respiratory coronaviruses and association with multiple sclerosis persistent infection of human oligodendrocytic and neuroglial cell lines by human coronavirus e a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin. ii mouse hepatitis virus s in weanling 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in c h mice in vivo and in vitro models of demyelinating diseases xxiv. the infectious process in cyclosporin a treated winstar lewis rats inoculated with jhm virus we thank annie boucher, inrs-institut armand-frappier, for the critical review of the manuscript and francine lambert for excellent technical assistance. we also thank dr. serge dea (who tragically passed away on january , ), inrs-institut armand-frappier, for the generous gift of the -e . antibody, and dr. yves robitaille, mcgill university, for constructive comments on neuropathology. we also thank the mcgill university animal resources centre for their help with some histology. this work was supported by grant mt- from the canadian institutes of health research (institute of infection and immunity). key: cord- -m va er authors: raaben, matthijs; groot koerkamp, marian ja; rottier, peter jm; de haan, cornelis am title: type i interferon receptor-independent and -dependent host transcriptional responses to mouse hepatitis coronavirus infection in vivo date: - - journal: bmc genomics doi: . / - - - sha: doc_id: cord_uid: m va er background: the role of type i ifns in protecting against coronavirus (cov) infections is not fully understood. while covs are poor inducers of type i ifns in tissue culture, several studies have demonstrated the importance of the type i ifn response in controlling mhv infection in animals. the protective effectors against mhv infection are, however, still unknown. results: in order to get more insight into the antiviral gene expression induced in the brains of mhv-infected mice, we performed whole-genome expression profiling. three different mouse strains, differing in their susceptibility to infection with mhv, were used. in balb/c mice, which display high viral loads but are able to control the infection, and genes were significantly differentially expressed (≥ . fold change) upon infection at and days post infection, respectively. functional association network analyses demonstrated a strong type i ifn response, with irf and irf as the central players. at days post infection, a type ii ifn response also becomes apparent. both the type i and ii ifn response, which were more pronounced in mice with a higher viral load, were not observed in svev mice, which are much less susceptible to infection with mhv. svev mice lacking the type i interferon receptor (ifnar-/-), however, were not able to control the infection. gene expression profiling of these mice identified type i ifn-independent responses to infection, with ifn-γ as the central player. as the balb/c and the ifnar-/- svev mice demonstrated very similar viral loads in their brains, we also compared their gene expression profiles upon infection with mhv in order to identify type i ifn-dependent transcriptional responses. many known ifn-inducible genes were detected, several of which have previously been shown to play an important protective role against virus infections. we speculate that the additional type i ifn-dependent genes that we discovered may also be important for protection against mhv infection. conclusion: transcriptional profiling of mice infected with mhv demonstrated the induction of a robust ifn response, which correlated with the viral load. profiling of ifnar-/- mice allowed us to identify type i ifn-independent and -dependent responses. overall, this study broadens our present knowledge of the type i and ii ifn-mediated effector responses during cov infection in vivo. cytokines are key regulators that dictate many aspects of innate and adaptive immunity. induction of type i interferons (ifns), a well-known subset of cytokines with antiviral activity, is triggered by a selection of cellular pattern recognition receptors, including tlrs (toll-like receptors), rig-i (retinoic acid-inducible gene i), and mda (melanoma differentiation-associated protein ). these receptors are activated in response to a range of pathogenspecific factors, which includes double-stranded rna produced during virus infection [ , ] . secreted type i ifns (i.e. ifn-α and ifn-β), subsequently induce an antiviral transcription program in the infected cell as well as in adjacent cells, thereby magnifying the "danger" signal and protecting against the infection. the role of type i ifns in controlling coronavirus (cov) infections is not well understood. a number of studies has shown that covs, like the mouse hepatitis virus (mhv) and the severe acute respiratory syndrome (sars)-cov, are poor inducers of type i ifns in cell culture, and even escape from detection by cytoplasmic pattern recognition receptors [ ] [ ] [ ] [ ] [ ] [ ] . consistently, virus-encoded ifn antagonistic functions have been described for both mhv and sars-cov [ , ] . in vivo, however, mhv infection appeared to induce the production of ifn-α in plasmacytoid dendritic cells (pdcs) by a tlr -dependent mechanism [ ] . moreover, mhv infections of primary neuronal cultures and of the central nervous system (cns) induced ifn-β gene expression, indicating that the production of type i ifns in vivo is not limited to pdcs [ , ] . furthermore, neuronal cultures infected with mhv exhibited increased expression of several type i ifninduced transcription factors [ ] . more recently, roth-cross and co-workers reported that macrophages and macrophage-like microglia cells produce ifn-β in the cns of mhv-infected mice in a mda -dependent manner [ ] . several studies have demonstrated the importance of the type i ifn response in controlling mhv infection in vivo. the exogenous delivery of type i ifns was shown to inhibit mhv infection of and spread to the mouse brain [ , ] . consistently, infection of mice lacking the functional type i ifn receptor (ifnar-/-) with mhv resulted in increased viral replication and extended tissue tropism [ , , ] . although many type i ifn-responsive genes have been identified [ ] , the protective effectors against mhv infection are yet unknown [ ] . in order to get more insight into the antiviral gene expression induced in the brains of mhv-infected mice, we performed whole-genome expression profiling. three different mouse strains (balb/c, svev and ifnar-/- svev mice), differing in their susceptibility to infec-tion with mhv, were used. previously, we have observed that svev mice are significantly more resistant to infection via the intranasal route than balb/c mice [ ] . the reason for the significant difference in susceptibility is not known, but may be related to different antiviral immune responses in these two mouse strains. furthermore, gene expression profiling of svev mice lacking the type i ifn receptor, which are not able to control the mhv infection [ ] , allowed us to identify type i ifn-independent transcriptional responses. we started by comparing the whole-genome expression profiles in the brains of the balb/c and the svev mice upon infection with mhv. to this end, mice were inoculated intranasally with tcid of mhv strain a or with pbs (control). groups of mice (n = ) were sacrificed at and days post inoculation after which the brains were harvested and total rna was isolated. the extent of virus replication was determined by quantitative reverse transcriptase (rt)-pcr targeting mhv-specific rna sequences as described earlier [ ] . previously, we demonstrated that the viral rna load correlates well with viral infectivity in tissue homogenates [ ] . while no viral rna could be detected yet at days post inoculation (data not shown), viral rna was observed in the brain of both mouse strains at day ( figure a ). as expected, the balb/ c mice displayed a much higher viral rna load than the svev mice. next, the rna extracts were processed for microarray analysis using the pbs-inoculated groups as the reference. in total, and genes were significantly differentially expressed (≥ . fold change) in balb/c mice at and days post infection, respectively. in contrast, in the svev mice, no significant induction of gene expression was observed. the results are depicted in figure b as a gene tree that was built based on the genes with a significantly altered expression level in balb/c mice at days post infection (i.e. expression-based cluster analysis). from these data we were able to identify host genes, the increased expression (≥ . fold) of which could already be detected at day (i.e. early genes; figure c ) or only at day (i.e. late genes; figure d ). the group of earlyinduced transcripts contained many ifn-inducible genes, including the well-known interferon regulatory factor (irf ), signal transducer and activator of transcription (stat ), and '- ' oligoadenylate synthetase (oas) genes (additional file a). within the cluster of "late" genes (additional file b) several chemokines (i.e. ccl , ccl , ccl , cxcl , and cxcl ) could be identified. next, in order to construct a functional association network, we applied the string . software [ ] to the list of proteins encoded by the "early" and "late" genes. we also included known interactors of our hits in this analysis, while proteins that did not demonstrate any known interactions were excluded for clarity. the results are shown in figure a and b. functional association network analysis of the proteins encoded by the "early" genes revealed two main modules. one module contained several proteins involved in antigen presentation, while the other module contained numerous proteins involved in the type i ifn response. the key player in this latter module appeared to be irf , which is the master regulator of type i ifn-dependent responses [ ] . functional association network analysis of the proteins encoded by the "late" genes revealed a large network of proteins involved in host-pathogen interactions. although the microarray analyses did not reveal the induction of ifn-γ gene expression itself, ifn-γ appeared at a central position in the network. in addition, the induction of a type i ifn response was also evident from this network as demonstrated by the presence of the transcription factors irf and irf , both of which demonstrated elevated mrna levels upon mhv infection. in conclusion, these results demonstrate that mhv infection induces a robust ifn response both at and days post infection, in which the transcription factors irf , irf , and irf appear to be the key players. at days post infection, a type ii ifn response also becomes apparent. to confirm and extend these observations, we next analyzed the induction of type i and ii ifn gene expression (i.e. ifn-α and ifn-β , and ifn-γ, respectively) by using quantitative rt-pcr. in agreement with the microarray expression profiles, significant induction of these type i and ii ifns could only be detected in the mhv-infected balb/c animals ( figure e ). the observation that the balb/c mice, unlike the svev mice, exhibited abundant expression of ifn-responsive genes upon mhv infection appears counter intuitive as the svev mice are much more resistant to the infection than the balb/c mice. apparently, the resistance of svev mice to mhv infection is not controlled by a more robust ifn response. the reason for the observed difference in susceptibility between the different mouse strains after intranasal inoculation is not known. mhv-a was recently shown to replicate efficiently in the liver of svev mice after intraperitoneal inoculation [ ] . interestingly, the resistance of svev mice after intranasal inoculation is not restricted to infection with mhv, as it was also observed for vesicular stomatitis virus [ ] . the microarray expression profiles described above suggested that the induction of an ifn response correlates with the viral load within the brain. to confirm this, we examined the data of the individual balb/c mice at days post infection in more detail. clearly, the animals with the highest viral loads (mouse and ; figure a ), also dis-played significantly higher levels of induction of type i and ii ifn expression ( figure b ). likewise, the amplitude of the gene expression profiles ( figure c and additional file ) of the individual mice also correlated with the viral loads in the brain. these observations are in agreement with results obtained by the profiling of sars-covinfected macaques [ ] . also in that study a positive correlation between virus load and the induction of gene expression was observed. a few genes (n = ), including isg , showed an inverse correlation with the viral load. we currently have no explanation for this observation as expression of isg is known to be induced by type i ifns [ , ] . interestingly, isg has been shown to exhibit antiviral activity against other viruses [ , ] . to study the role of type i ifn-independent and -dependent gene expression in the control of mhv infection in vivo in more detail, we next made use of the ifnar-/-mice [ ] . these mice are highly susceptible to mhv infection as compared to the parental svev mice [ , ] . indeed, when these mice were inoculated intranasally with tcid of mhv-a , viral rna levels in their brains became much higher than in animals from the parental strain at days post infection ( figure a ). interestingly, at this time point the viral rna levels in the ifnar-/-mice were comparable to those in the brains of the balb/c mice. however, efficient dissemination of the infection, resulting in high viral loads in the liver as determined by quantitative rt-pcr, was only observed in the ifnar-/-mice and not in the wild-type mice, which displayed viral rna levels just above background ( figure b ). thus, in agreement with previous studies, a type i ifn-dependent response is required to inhibit virus dissemination [ , ] . whole-genome expression profiling of brains of the ifnar-/-mice revealed the significantly induced expression of genes (≥ . fold) at days post infection. in contrast, at day , hardly any alterations in gene expression could be detected in these knock-out mice (additional file ). figure c shows an expression-based cluster analysis of these genes for the wild-type and ifnar-/mice. comparison of the complete expression profiles of these mice revealed that the transcriptional profile at day in the ifnar-/-mice has a larger similarity with the profile at day of the parental svev mice than with that of the knock-out mice at day post infection ( figure c ). this observation may suggest the presence of an early host response to infection with mhv in the parental mice, even though no significant induction (≥ . fold) of gene expression could be detected ( figure b) . such a response, may not be evident in transcriptional profiles of whole organs, but might only be apparent at the cellular level. we speculate that early decisive events are happening in initial target cell populations such as dcs and macro-early and late transcriptional responses to infection with mhv as the knock-out mice lack a functional type i ifn receptor, the upregulation of gene expression observed in these mice apparently occurs independently of type i ifn signalling. not much is known yet about type i ifn-independent responses to infection. the observation that the transcriptional upregulation of irf was independent of type i ifn signalling is consistent with the notion that ifn-γ can also induce expression of this gene [ , ]. the type i ifn receptor-independent expression profile within the brains of ifnar-/-mice after mhv infection likewise, we also observed increased transcription of ifitm and ifitm independent of type i ifn signalling, again corresponding with the literature [ , ] . interestingly, the expression of various genes encoding proteins involved in antigen presentation (i.e. h , b m, psmb , psmb , and ctss) was also increased in the absence of type i ifn signalling. psmb and psmb encode immunoproteasome subunits which facilitate antigen presentation to cd + t cells after virus infection, a process that is primarily regulated by ifn-γ [ ] . furthermore, also the expression of the major histocompatibility complex class ii (mhc ii) invariant chain, also called cd [ ], was increased upon infection of the knock-out mice. these data are in agreement with the observation that the induction of genes involved in antigen processing is independent of stat activation by . we also observed the transcriptional upregulation of the isoforms of metallothionein (mt , mt , and mt ), which encode proteins known to scavenge toxic metals [ ] . the induction of these genes, which was not apparent in either wild-type mice, could reflect an acute-phase reaction in the brain of mhv-infected ifnar-/-mice, which likely contributes to pathogenesis as has been shown for other viruses [ - ]. we constructed a functional association network by applying the string . software [ ] to the list of proteins encoded by the type i ifn-independent genes (additional file ). we also included known interactors of our hits in this analysis, while proteins that did not demonstrate any interactions were again excluded for clarity. the result is shown in figure . the analysis revealed ifn-γ as the central player in the type i ifn-independent antiviral network as this protein appeared to link a number of smaller modules. the induction of ifn-γ gene expression could be confirmed using quantitative rt-pcr (data not shown). the finding that ifn-γ-mediated transcriptional responses are not dramatically affected in the absence of type i ifn signalling is in agreement with reports referred to above and with a recent publication by ireland et al. [ ] , which shows that ifn-γ expression is significantly induced in the cns of mhv-infected ifnar-/-mice. while the production of ifn-γ by nk cells plays a major role in the protection against infection with mhv [ - ], the ifn-γ-mediated transcriptional responses that we observed were not protective against acute mhv infection in the ifnar-/-mice. several studies have shown that mhv [ , , ] as well as several other viruses [ - ] replicate to much higher levels (up to fold difference) in ifnar-/-mice than in their wild-type counterparts. in this study we show that a strong correlation exists between the amplitude of type i and ii ifn host responses with the viral load. the huge differences in virus replication between wild-type and ifnar-/-mice therefore do not permit a fair comparison between gene expression profiles of these mice, with the aim of identifying type i ifn-dependent responses. indeed, as no significant gene expression is observed in the wild-type svev mice, a comparison with the expression profile of the ifnar-/-mice only provides information about type i ifn-independent and not ifndependent responses. we now observe, in agreement with our previous study, that the brain of balb/c and ifnar-/ - svev mice contain very similar mhv loads at day and post infection [ ] . since the type i ifn-responsive pathway is very well conserved among many different species [ ], we considered it acceptable to compare the gene expression profiles of these mice with the aim of identifying type-i ifn-dependent responses, although comparing transcriptional profiles of wild-type and ifnar-/-mice from a different genetic background should obviously be done very cautiously. ideally, a comparison between wildtype balb/c and ifnar-/-balb/c mice would have been more accurate. while the induced expression of a number of genes was similar for the two mouse strains (i.e. type i ifn signalling-independent gene-expression), that of other genes was only observed in the balb/c mice (i.e. tentative type i ifn signalling-dependent gene expression). the expression of yet other genes appeared to be partially dependent of type i ifn signalling: increased expression of these genes was observed in the ifnar-/mice, but much more so in the balb/c mice. genes, the expression of which was upregulated (≥ . fold) in the balb/c mice but not significantly changed in the ifnar-/-mice upon infection with mhv, were tentatively designated as type i ifn-dependent. genes, the transcriptional upregulation of which was at least times higher in the balb/c mice than in the ifnar-/-mice, were also added to the list of tentative type i ifn-dependent genes. as expected, this set of genes (n = ) contained many known ifn-responsive genes like isg , ifit , ifit , isgf g, mx and ube l (additional file ). functional association network analyses showed irf and irf to be the key players in the network (additional file ). several of the tentative type i ifn-dependent genes (including mx and ube l) have previously been shown to play an important protective role against virus infections [ - ]. we speculate that other genes present in this list may also be important for full protection against mhv infection. transcriptional profiling of mice infected with mhv demonstrated the induction of a robust ifn response, which correlated with the viral load. profiling of ifnar-/-mice allowed us to identify type i ifn-independent anddependent responses. overall, this study broadens our present knowledge of the type i ifn-mediated effector responses during cov infection in vivo. [ ] [ ] [ ] week old balb/c were obtained from charles river laboratories, while type i ifn receptor knock-out mice (ifnar-/-) [ ] and the parental svev mice were obtained from b&k universal ltd. mice were inoculated intranasally with tcid of mhv strain a and sacrificed at the indicated time-points for organ dissection. control animals were treated with pbs. the study proto-col was approved by the animal ethics committee of the utrecht university, and all experiments were performed in accordance with accepted institutional and governmental policies. whole brains and livers were dissected from the mhvinfected and control mice. the tissues were added to lysing matrix d tubes (mp biomedical), containing ml of the type i ifn-independent gene expression network figure the type i ifn-independent gene expression network. the genes listed in additional file (n = ) were subjected to functional association network analysis by using the string . database as described in the legend of figure . the key player in the network, ifn-γ, is indicated in red. rnapro™ solution (q-biogene), and processed using a fastprep instrument (mp biomedical). the tissues were homogenized at , rpm for sec and immediately placed on ice. subsequently, the homogenates were centrifuged at , rpm for minutes at °c and supernatants were harvested and stored at - °c. total rna was isolated from the homogenates using the trizol reagent (invitrogen) according to the manufacturer's protocol. rna was further purified using the rneasy mini-kit with subsequent dnasei treatment on the column (qiagen). rna integrity was determined by spectrometry and by a microfluidics-based platform using a uv-mini device (shimadzu) and a bioanalyzer (agilent technologies), respectively. ], respectively), were measured by quantitative pcr using assay-on-demand reagents and equipment (pe applied biosystems), according to the manufacturer's instructions. the quantitative pcr reactions were performed in a total reaction volume of μl containing μl taqman ® universal pcr master mix ( ×), μl cdna, μl taqman ® gene expression assay mix ( ×), and μl water using an abi prism sequence detection system under the following conditions: °c for mins, followed by cycles of °c for secs and °c for min. for all assays, we performed "no-rt" (reaction using total rna as the substrate) and "no template" (reaction using water as the substrate) controls. in both cases, omitting cdna from the reaction resulted in a lack of pcr product generation. all assays were analyzed with abi prism software v . . f (pe applied biosystems). the comparative ctmethod was used to determine the fold change for each gene (primer efficiencies were similar for both the endogenous control primer set and genes of interest primer sets [data not shown]). note that the ct values of all samples were within the limits of the standard curves (data not shown). the housekeeping gene gapdh (nm_ . ) was used as a reference in all experiments, since expression of this gene was found constant among samples. the amounts of viral rna were determined by quantitative rt-pcr as described before [ ] . the microarray experiments were performed as described previously [ ] . briefly, mrna was amplified from μg of total rna by cdna synthesis with oligo(dt) doubleanchored primers, followed by in vitro transcription using a t rna polymerase kit (ambion). during transcription, -( -aminoallyl)-utp was incorporated into the single stranded crna. cy and cy nhs-esters (amersham biosciences) were coupled to μg crna. rna quality was monitored after each successive step using the equipment described above. corning ultragaps slides, printed with a mouse array-ready oligo set (operon; , spots), were hybridized with μg of each alternatively labeled crna target at °c for - h. two independent dyeswap hybridizations ( arrays) were performed for each experimental group. after hybridization the slides were washed extensively and scanned using the agilent g aa dna microarray scanner. after data extraction using imagene . software (biodiscovery), lowess normalization [ ] was performed on mean spot-intensities in order to correct for dye and printtip biases [ ] . the microarray data was analysed using anova (r version . . /maanova version . - ) http://www.r-project.org [ ] . briefly, in a fixed effect analysis, sample, array and dye effects were modelled. pvalues were determined by a permutation f -test, in which residuals were shuffled , times globally. genes with p < . after family wise error correction were considered significantly changed. cluster analysis (standard correlation) was performed with genespring gx . software (silicon genetics). when indicated, the confidence level was increased by applying a fold change cut-off. the resulting genelists were subjected to genespring . software for further analysis. arrayexpress accession numbers miame-compliant data in mage-ml format as well as complete descriptions of protocols have been submitted to the public microarray database arrayexpress http:// www.ebi.ac.uk/arrayexpress/ with the following accession numbers: microarray layout, p-umcu- ; gene expression data of mhv-infected mice, e-mexp- ; protocols for total rna isolation and mrna amplification, p-mexp- ; crna labeling, p-mexp- and p-mexp- ; hybridization and washing of slides, p-mexp- ; scanning of slides, p-mexp- ; data normalization, p-mexp- . innate immune recognition of viral infection type i interferons in host defense group coronaviruses prevent immediate early interferon induction by protection of viral rna from host cell recognition transcriptional profiling of acute cytopathic murine hepatitis virus infection in fibroblast-like cells mouse hepatitis coronavirus replication induces host translational shutoff and mrna decay, with concomitant formation of stress granules and processing bodies preferential infection of mature dendritic cells by mouse hepatitis virus strain jhm mouse hepatitis virus does not induce beta interferon synthesis and does not inhibit its induction by double-stranded rna inhibition of beta interferon induction by severe acute respiratory syndrome coronavirus suggests a two-step model for activation of interferon regulatory factor mouse hepatitis coronavirus a nucleocapsid protein is a type i interferon antagonist severe acute respiratory syndrome coronavirus nsp suppresses host gene expression, including that of type i interferon, in infected cells control of coronavirus infection through plasmacytoid dendritic-cell-derived type i interferon differential regulation of innate and adaptive immune responses in viral encephalitis inhibition of the alpha/beta interferon response by mouse hepatitis virus at multiple levels 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 protective effect of recombinant murine interferon beta against mouse hepatitis virus infection non-invasive imaging of mouse hepatitis coronavirus infection reveals determinants of viral replication and spread in vivo type i interferons are essential in controlling neurotropic coronavirus infection irrespective of functional cd t cells interferome: the database of interferon regulated genes interferon and cytokine responses to sarscoronavirus infection cyclooxygenase activity is important for efficient replication of mouse hepatitis virus at an early stage of infection this work was supported by grants from the m.w. beijerinck virology fund, royal netherlands academy of arts and sciences, and the netherlands organization for scientific research (nwo-vidi- . . ) to c.a.m. de haan. we thank connie bergmann for advice and monique oostra, marne hagemeijer, and mijke vogels for stimulating discussions. the authors declare that they have no competing interests. mr and mjagk conducted all the experiments. mr wrote the manuscript. pjmr and camdeh coordinated the research efforts and assisted with writing the manuscript. all authors read and approved the final manuscript. key: cord- -c hajb k authors: matthews, a. e.; weiss, s. r.; paterson, y. title: murine hepatitis virus—a model for virus-induced cns demyelination date: journal: j neurovirol doi: . / sha: doc_id: cord_uid: c hajb k most murine hepatitis virus (mhv) strains, as their name suggests, infect the liver. however, several murine strains are tropic for the central nervous system (cns) and cause encephalitis with subsequent cns demyelination. the cns demyelination shares pathological similarities with human cns demyelinating diseases such as multiple sclerosis (ms). these viruses are, therefore, used to study the role of the immune system in viral clearance from the cns, in cns demyelination, and in remyelination. nevertheless, it is still unclear exactly how mhv induces demyelination and to what extent the immune system plays a role in this pathology. here we review this field in the context of the immune response to mhv in the liver and the cns focusing on studies that have been published in the past years. in weanling mice compared to adult mice (weiner, ) . most studies are performed on -to -weekold mice. both mhv-a and jhm infect, to some extent, oligodendrocytes, astrocytes (lavi et al, ; stohlman et al, a) , neurons (parra et al, ; lavi et al, ) , hepatocytes (weiner, ; navas et al, ) , macrophages, including kuppfer cells (even et al, ; stohlman et al, a) , thymic epithelial cells (knobler and oldstone, ; godfraind et al, ) , and the endothelial cells which line blood vessels (joseph et al, ; lavi et al, ) . mhv-a has also been shown to infect b cells (coutelier et al, ) , and mhv-jhm has been detected in ependymal cells (stohlman et al, a) . except for glial cells and neurons, these cell populations closely match those demonstrated to express the known mhv receptor bgp a (biliary glycoprotein a), a member of the carcinoembryonic antigen (cea) family, which in turn is a member of the immunoglobulin superfamily (williams et al, ) . the bgp a receptor has been identi ed on hepatocytes, macrophages, epithelial cells (such as those in the intestine), endothelial cells, b cells, and a small proportion of thymic epithelial cells (williams et al, ) . a major determinant of tropism is the spike (s) protein (phillips et al, ; navas et al, ) , suggesting that variations in mhv receptor (such as posttranslational modi cations or expression of different isoforms) may affect recognition by different infectious mhv can be found in astrocytes, oligodendrocytes, and neurons after acute infection . late after infection, virus is detectable only as low levels of antigen or rna (lavi et al, b) . in animals with viral persistence due to the absence of ifn-°, antigen is detected mainly in oligodendrocytes (parra et al, ) . in mice infected in the presence of protective maternal antibody, antigen persists in astrocytes as well as other unidenti ed cells (perlman and ries, ) . these studies demonstrate that virus can persist in glial cells. after intracerebral (i.c.) or intranasal (i.n.) infection with mhv-a , virus enters the brain and causes encephalitis . intranasal infection with mhv-jhm or -a leads to viral invasion through the olfactory bulb and along olfactory tracts, as well as a slower approach along the trigeminal nerve to the mesencephalic nucleus perlman et al, ) . early spread of i.) appears to be along speci c neural pathways in the cns (perlman et al, ) and, therefore, through neural connections. later widespread infection can be blocked by passive transfer of antibody, suggesting spread through the blood, cerebrospinal uid, and/or interstitial uid (perlman et al, ) . liver infection can occur after any route of infection (i.n., i.c., i.g., or i.p.) with mhv-a . mhv-a liver titers peak dpi after i.c. infection (lavi et al, b) and hepatitis develops during the rst to weeks . mhv-jhm replicates transiently in the lungs, blood, and cervical lymph nodes after i.n. infection, and occasionally is found in the thymus and bone marrow (barthold and smith, ) . as would be expected in an infection that comes under immunological control, viral titers peak around d.p.i. in the brain (parra et al, ) , slightly later in the spinal cord (matthews et al, ) , and virus is cleared by - d.p.i. (sutherland et al, ) . the kinetics of viral antigen expression follow a pattern similar to that observed of viral titers during acute infection (wang et al, ) , but viral antigen is still detectable up to d.p.i. (lavi et al, a) . viral rna is detectable in the brain as late as - months postinfection, although the amount of rna decreases with time (lavi et al, a) . however, immunosuppression of cell-mediated immunity by drugs, radiation, or t-cell depletion at late time points after infection does not result in viral recrudescence from this rna pool (stohlman and weiner, ) . the blood-brain barrier is a major factor of the cns that limits immune function by preventing the easy entry of serum antibodies and infectious organisms in the absence of cns in ammation (cserr et al, ) . although it has been thought that the blood-brain barrier allows the entry of only activated t cells, a recent study demonstrated that naive t cells can enter the cns if there are few activated t cells in circulation (brabb et al, ) . it has been suggested that immunosuppressive cytokines such as transforming growth factor-beta, alpha-melanocyte stimulating hormone, and interleukin- (il- ) may limit or alter immune function within the cns (gordon et al, ; lipton and gonzales-scarano , ) . work with t cells responding to sindbis virus infection of the cns demonstrated that t cells lose their ability to proliferate and down-regulate il- production after entering the brain, supporting the hypothesis that the environment of the cns alters immune function (irani et al, ) . in addition, the brain fosters viral antigen or nucleic acid persistence, which is not observed in most peripheral organs and seems to be attributable at least in some instances to inhibited immunemediated clearance (kristensson and norrby, ) . the poor regenerative potential of neurons and the sensitivity of the cns to small lesions may have contributed to the development of a cns environment which downregulates the more destructive aspects of the immune response. the cells of the immune system successfully enter the cns after mhv infection. mononuclear cells of bone marrow origin peak d.p.i. they then decline, even in animals that do not control the infection (williamson et al, ) . in the absence of t cells in nude mice, infectious mhv-jhm is not cleared with normal kinetics (houtman and fleming, ) , demonstrating that t cells are important for clearance of mhv. cd c t cell response to mhv cd c t cells are critical for the early control of mhv infection in the cns and for prevention of mortality (gombold et al, ; stohlman et al, b; sutherland et al, ) . virus-speci c cd c t cells peak days after mhv infection in the brain (williamson et al, ) . the ability to detect mhv speci c cd c t cells in the draining cervical lymph nodes (cln) or the spleen varies parra et al, ) . this variability may be due to differences in the speci c injection sites. virus delivered to the parenchyma results in a delayed peripheral immune response compared to virus delivered to the ventricles and cerebrospinal uid (stevenson et al, ) . cd c t cells recognize peptides presented on cell surface mhc class i molecules. although neurons do not express mhc class i and therefore are not anticipated targets of cd c t cells, mhc class i is present on endothelial cells and microglia (horwitz et al, ) . in addition, class i expression can be induced in astrocytes and oligodendrocytes in culture if exposed to factors secreted by cells infected with mhv-a (suzumura et al, ) . after mhv-a infection of cd c t-cell-de cient mice, viral antigen clearance was delayed in microglia, lymphocytes, endothelial cells, and meningeal cells but not in neurons or glia . in another study, transfer of cd c t cells suppressed mhv-jhm antigen expression in astrocytes and microglia but not oligodendrocytes (stohlman et al, a) . cd c t cells can function through the lytic molecule perforin, through apoptosis induced by fas ligand (fasl), or by secreting cytokines such as ifngamma and tnf-alpha (kajino et al, ) . all of these mechanisms may be productively used in the immune response to mhv. in the cns of mice decient for perforin, viral clearance is delayed, thereby implicating a role for perforin in viral control . ef cient viral clearance in fas-de cient mice demonstrated that fasl-mediated lysis is not required for viral clearance. nonetheless, mice lacking both fasl-mediated apoptosis and perforin have delayed clearance compared to mice just lacking perforin, suggesting that fas-fasl interactions can contribute to viral control although they may not be very important (parra et al, ) . both cd c and cd c t cells persist in the cns for up to d.p.i., with cd c t cells being more prominent in the parenchyma both early and late after infection (stohlman et al, a) . the number of cd c t cells decreases with time and decreasing viral rna marten et al, ) . at d.p.i., about % of these cells are still virus-speci c, similar to the percentage of virus-speci c t cells observed during acute infection. many of these chronically present cells secrete ifn-gamma, but cytolytic activity is gradually lost . although activated , mhv-speci c, cd c t cells transferred to infected mice can control viral replication, they are not suf cient to clear infectious virus from oligodendrocytes (stohlman et al, b) . the presence of cd c t cells is necessary for transferred cd c t cell protection. indeed, these cells appear to be important for cd c t-cell lytic activity and survival in the cns, perhaps through the secretion of il- (stohlman et al, b) . virally activated cd c t cells protect against lethal viral infections after transfer, but as with transferred cd c t cells, they are not suf cient to clear the virus (stohlman et al, ) . depletion of either cd c or cd c t cells results in the inability of mice to control acute viral titers in the brain (williamson et al, ; sutherland et al, ) . thus, both cd c and cd c t cells are important for control of acute viral infection in the cns. unlike cd c t cells, which accumulate in the parenchyma, cd c t cells tend to accumulate perivascularly and in subarachnoid spaces (stohlman et al, a) , locations rich in mhc class ii positive macrophages and microglia (hickey and kimura, ; braun et al, ) . interestingly, cd c t cell numbers peak in the cns at d.p.i., a little later than cd c t cells (williamson et al, ) , and persist in the cns after viral clearance marten et al, ) . the effector function of many lymphocytes involves the production and secretion of cytokines. mrna from a wide number of in ammatory cytokines can be detected in the brain - days following i.c. infection with mhv-jhm. these include interferon (ifn) alpha and beta (wang et al, ) , interleukin (il)- alpha, il- beta, il- , il- , and tnf-alpha (parra et al, ) . immuno uorescence of mhv-jhm infected spinal cords demonstrated the presence of tnf-alpha protein as well as il- beta and il- in cells with the morphology of macrophages early after infection and in uninfected astrocytes late after infection . both th (ifn-gamma) and th (il- , il- , and il- ) cytokine mrnas increase after infection with various strains of mhv, peaking to days after mhv-jhm infection (stohlman et al, a; parra et al, ) . in several studies mhv infection resulted in a predominantly igg a antibody isotype pro le (coutelier et al, ; fleming et al, ) supporting the idea of a predominantly th response, although in another study igg a did not predominate over igg (parra et al, ) . these data, combined with the presence of both th and th cytokine mrnas, suggest that neither t-helper cell type necessarily predominates, although a th pro le is slightly more common. mhv-jhm infection of mice de cient for ifn-gamma revealed that ifn-gamma is not required for optimal antibody production or for ctl responses. ifn-gamma is, however, necessary for clearance of virus from the oligodendrocytes (parra et al, ) . clearance of an mhv strain that primarily infects neurons is delayed in the absence of ifn-gamma, suggesting that it may affect viral control in other cell types as well without being absolutely required for clearance (lane et al, ) . in mice decient for il- , mhv-jhm causes a transient increase in viral titers d.p.i. and greater mortality than in wild-type mice despite normal kinetics of viral clearance, which is thought to be due to an increase in in ammatory cytokines (lin et al, ) . the role of b cells in the control of mhv has been studied in rats and mice. in lewis rats, the transfer of b cells into irradiated recipients decreased mhv-jhm titers in the cns (schwender et al, ) . igm, the rst detectable antibody isotype, rst appears d.p.i. with mhv-jhm i.c. in mice (stohlman et al, ; williamson et al, ) . neutralizing antibodies are detected in the serum by d.p.i. with mhv-jhm given i.c., but were not suf cient to control viral titers within d.p.i. in the absence of cd c t cells . similarly, after mhv-a infection, serum antibody is detectable d.p.i. and reaches maximal levels - d.p.i. (lavi et al, b) . by electron microscopy, plasma cells can be observed in the cns as late as d.p.i. with mhv-jhm (stohlman and weiner, ) . antibody mostly of isotype igg a is detected after infection (lardans et al, ) , although sometimes igg a and igg eventually reach equivalent levels (parra et al, ) . transfer of virusspeci c antibodies increases survival, presumably by decreasing peak viral levels, delaying neuronal infection, and/or decreasing neuronal infection (fleming et al, ; yokomori et al, ) . the contribution of humoral immunity to viral clearance and persistent infection in the cns has been investigated using mice de cient in secreted antibodies or b cells bergmann et al, ; matthews et al, ) . mice homozygous for disruption of the ig mu gene (mumt mice) lack b cells and develop acute disease in the cns with similar kinetics and severity to wild-type c bl/ mice after mhv-jhm infection or mhv-a infection (matthews et al, ) . viral clearance during acute infection is similar in both groups. however, although virus is quickly cleared from the cns of wildtype mice, it reemerges and persists in the cns of mumt mice. b-cell-de cient mice have been shown to have impaired t cell immunity against a number of viral infections including mhv-jhm (bergmann et al, ) , possibly due to their absence as antigenpresenting cells in these mice. to clarify how b cells mediate viral clearance in the cns, we compared the role of b cells as antigen-presenting cells, using mice with b cells that are unable to secrete antibody, with that of secreted antibody, using mumt mice reconstituted with mhv-a immune immunoglobulin (matthews et al, ) . mumt mice that received a -speci c antibody had decreased virus in the cns, whereas mice with b cells decient in antibody secretion did not clear virus from the cns. these data suggest a major role for immune antibody in controlling virus replication in the cns. nonlymphocytic immune response to mhv natural killer (nk) cells (asialogm c cells) peak in the cns at days after i.c. inoculation with mhv-jhm. the d.p.i. cns mononuclear cells are actively cytotoxic on the classic nk target cell line yac- when assayed ex vivo (williamson et al, ) . in the absence of t cells, a transient decrease in viral titers to d.p.i. has been attributed to the nk cell response (williamson et al, ) . after immunosuppression by cyclophosphamide, in mice infected with a high dose of mhv-jhm, death occurs more quickly ( d.p.i.) than in untreated mice ( - d.p.i.) (weiner, ) . this observation suggests that the innate immune response that expands in response to infection, such as the nk cells, can be important in the early control of viral infection. macrophages are susceptible to infection by mhv (wijburg et al, ) , although only a subset is infected initially. it has been proposed that cd c t cells are necessary as a source of rantes to attract monocyte/macrophage in ltration of the cns . the role of macrophages in the immune response is dif cult to pinpoint. depletion of blood-borne macrophages had little effect on cns demyelination (xue et al, ) . on the other hand, depletion of macrophages before i.v. infection with a highly lethal isolate of mhv-a resulted in earlier mortality ( - d.p.i.) associated with increased viral titers in the spleen and liver (wijburg et al, ) . macrophage depletion before i.n. infection with mhv-jhm also resulted in rapid death (xue et al, ) . the early time point of mortality suggests that macrophages help control viral titers before the t and b cell populations are fully activated and effective. the immune response that controls mhv infection in the liver is similar in some ways to that important in the cns. pre-existing antibody can protect from lethal liver infections and decrease liver lesions, so antibody is capable of controlling liver viral titers (buchmeier et al, ) . however in contrast to the importance of antibody in controlling mhv-a replication in the cns, antibody is not required for viral clearance from the liver. in mumt mice, which lack mature b lymphocytes, and in igm-tg mice with b cells that do not secrete antibody, infectious virus was cleared from the livers with similar kinetics to wild-type mice after i.c. or i.h. infection (matthews et al, ) . cell-mediated immunity is required for the control of infection in the liver. depletion of cd c or cd c t cells has been shown to result in increased mhv-jhm titers in the liver and prolonged accumulation of infectious virus to d.p.i. when both t cell populations were depleted, viral titers were not controlled within d.p.i., although the mice did subsequently recover (kyuwa et al, ) . these data suggest that t cells play a role in controlling viral titers, even before d.p.i. beta -microglobulinde cient mice, which lack cd c t cells, have delayed clearance of virus from the liver . ifn-gamma-de cient mice experience greater mortality than do wild-type mice after i.p. infection with mhv-jhm or mhv-a (kyuwa et al, ; schijns et al, ) . virus persists up to d.p.i. in the liver and large lesions develop in these mice, although there is no chronic increase in liver enzyme activity indicative of loss of liver function. therefore, ifn-gamma is critical for viral control in the liver. in the absence of ifn-gamma, cd c t cell depletion further increased the severity of disease (kyuwa et al, ) . therefore, cd c t cell functions other than ifn-gamma production are also important for viral control. cns demyelination develops as active mhv-a or mhv-jhm infection resolves. these lesions are histologically very similar to those observed in ms patients. cns lesions are predominantly created by primary demyelination as evidenced by the intact, naked axons present in these lesions, although axonal damage is also detected . the peripheral nervous system is not affected (lampert et al, ) . lesions are scattered randomly throughout the spinal cord (lampert, ) . chronic lesions are associated with lipid-laden macrophages (presumably full of myelin), scattered lymphocytes, and naked axons. viral particles are not detected. astrocytic reactions and perivascular cuf ng are also sometimes found in lesions (weiner, ; stohlman and weiner, ) . chronic lesions can persist as late as d.p.i. (stohlman and weiner, ) , and scattered demyelinated axons can be detected even months after infection (herndon et al, ) . early demyelination after high-dose, lethal mhv-jhm infection is associated with polymorphonuclear cells, mononuclear cells, and extracellula r myelin debris. degeneratin g axons are sometimes detectable (lampert et al, ) . nevertheless, even in necrotic lesions formed during lethal infections, some axonal preservation is observed (weiner, ) . chronic disease in mhv-infected animals is characterized by a single major episode of demyelination associated with the development of ataxia, hind limb paresis, and paralysis (lavi et al, b) , following which animals usually recover. recovery is mediated by cns remyelination, sometimes accompanied by peripheral nervous system remyelination occurring in the cns (takahashi et al, ) . remyelination has been reported to begin anywhere from to d.p.i. (kristensson and norrby, ; takahashi et al, ) . as early as to weeks postinfection, there is a dramatic increase in mbp mrna at the edges of lesions suggestive of early remyelination before histological detection (kristensson and norrby, ) . at d.p.i., replicating oligodendrocytes were detectable near lesions (herndon et al, ) . it is thought that remyelination involves oligodendrocyte precursor o- a cell proliferation (godfraind et al, ) . how does cns demyelination occur? this is perhaps the major question posed with the mhv model of ms. neither acute encephalitis nor rna persistence is suf cient to cause demyelination, as demonstrated with two mutants of mhv-a (leparc-goffart et al, ; das sarma et al, ) . infectious virus does not have to be successfully cleared for demyelination to occur (houtman and fleming, ) . transfer of cd c t cells during acute infection results in no subsequent demyelination, although whether this treatment changes viral rna persistence is unclear (stohlman et al, a) . early mhv-jhm-induced demyelination is rare in immunode cient transgenic scid and rag knockout mice or irradiated mice, demonstrating a strong if not absolute requirement for lymphocytes (houtman and fleming, ; wu and perlman, ) . electron micrographs of demyelinating lesions demonstrate that macrophage processes slip between layers in the myelin sheath, suggesting that macrophages could be the direct mediators of demyelination (powell and lampert, ) . macrophages localize to demyelinating lesions, and their appearance correlates with the development of lesions. they do not appear in great numbers in the absence of lymphocytes, consistent with the observation that lymphocytes are important for demyelination (wu and perlman, ; lane et al, ) . despite the visual evidence that macrophages invade myelin sheaths, it is possible that macrophages could be localizing to areas of demyelination to clean up the damaged myelin caused by a nonmacrophage dependent mechanism. depletion of blood-borne macrophages does not affect the severity of demyelination, although similar techniques do affect demyelination in eae and theiler's virus-induced demyelination (huitinga et al, ; rossi et al, ; xue et al, ) . however, macrophages that are assumed to be derived from microglia or perivascular macrophages are present in the cns of these depleted mice and may still play a role beyond that of scavenging for disintegrating myelin (xue et al, ) . t cells could mediate demyelination through direct lysis of oligodendrocytes or through in ammation mediated by cd c t cell-dependent in ammation as demonstrated in experimental autoimmune encephalomyelitis . certainly t cells persist in the cns after virus is cleared and during active demyelination (marten et al, ) . there is a signi cant amount of data demonstrating that neither cd c nor cd c t cells are speci cally required for demyelination. demyelination occurs during acute infection of t cellde cient nude mice. beta- -microglobulin-de cient mice cannot express mhc class i and therefore do not develop mature cd c t cells. i-a b -de cient mice lack mhc class ii and therefore do not develop mature cd c t cells. both of these strains of t cell-de cient mice demonstrate early mhv-jhm induced demyelination (houtman and fleming, ) . infection with mhv-a of beta- microglobulin-de cient mice and mice ef ciently depleted of cd + t cells also results in demyelination, although less frequently than in wild-type mice (gombold et al, ; sutherland et al, ; lavi et al, ) . partial depletion of cd c and cd c t cells does not inhibit chronic demyelination either, although in this study t cells were not depleted until d.p.i. (sutherland et al, ) . a few studies suggested that the absence of cd c or cd c t cells resulted in decient demyelination. cd c or cd c t cell-de cient mice infected with mhv-jhm were less likely to develop acute demyelination, but the mice that did develop demyelination had similar severity to that observed in wild-type mice (houtman and fleming, ) . cd c t cell-de cient mice infected with mhv-jhm developed acute and chronic demyelination, but it was signi cantly less severe than that seen in cd de cient or wild-type mice . when splenocytes were transferred to immunodecient rag knockout mice, either cd c or cd c t cells were suf cient to generate signi cant levels of acute demyelination, although cd c t cells were less ef cient at inducing demyelination than cd c cells (wu et al, ) . these data suggest that each of these t-cell subpopulations has the capability to mediate cns demyelination after mhv infection and may induce slightly different levels or pathways of demyelination. on the other hand we have observed (matthews et al, ) moderately sized, typical demyelinating lesions as well as atypical small round necrotic lesions in mhv-a infected rag knockout mice, suggesting that, although t cells may be required for robust demyelination, other mechanisms must also be at work. knockout mice have been used to demonstrate that the mechanism by which demyelination takes place requires neither perforin nor fas-fasl interactions parra et al, ) . furthermore, removing tnf-alpha, il- , or ifn-gamma through depletion or the use of genetically de cient mice did not decrease demyelination (stohlman et al, b; lin et al, ; parra et al, ) . chemokines, however, may play a role in demyelination. gene expression for a number of chemokines has been found in the cns of mice undergoing chronic demyelination (lane et al, ) . of these the c-c chemokine rantes has been shown to possibly play a role in mhv-induced demyelination . systemic depletion of rantes with rantes-speci c antisera resulted in a signi cant reduction in macrophage in ltration and demyelination in c bl/ mice infected with mhv- . rantes is a pro-in ammatory chemokine produced by t cells, platelets, and endothelia that acts as a chemoattractant for a variety of lymphocytic and myeloid cell types including monocytes and granulocytes. lane and colleagues believe that the reduced demyelination observed in mhv-infected cd ¡=¡ mice may be due to the absence of cd c t cell-derived rantes . thus, rantes produced by cd c t cells may attract macrophages into the cns during viral infection. activated macrophages are a potent source of toxic nitric oxide intermediates produced by inducible nitric oxide synthases (inos). inhibition of one of these, nos- , has been shown to reduce mhv-induced demyelination (lane and buchmeier, ) , providing further evidence for a role for pro-in ammatory innate immune mechanisms in cns demyelination in this mouse model. among the cxc chemokines expressed in the cns during mhv-induced demyelinating disease is ifninducible protein (ip- ), suggesting a possible role for this chemokine in demyelination (lane et al, ) . however, ip- has also been shown to be crucial for control of mhv viral replicatio n and recovery from acute encephalitis in the c bl/ mouse . administration of anti-ip- antibody led to a signi cant reduction in t cells in ltrating the cns of mhv-infected mice, decreased levels of ifn-°in the cns and increased mortality of infected mice . thus, although chemokinemediated in ammatory responses in the cns may contribute to demyelination, as with all immune response elements examined, they also play a vital role in viral clearance. b cells and the antibodies they secrete could potentially also mediate demyelination. however, no correlation has been found in mhv-infected mice between antibody titers and severity of demyelination (koolen et al, ) . in some animal models of cns demyelination, antibodies against self-antigens in the brain induce or exacerbate demyelination (brehm et al, ; genain et al, ) but there is no evidence for the induction of antibodies that recognize host proteins after mhv infection of the cns. we have examined the role of b cells in demyelination using b cell-de cient mumt mice (matthews et al, ) . in infected b cell-de cient mice, robust demyelination not only occurred but was also statistically more severe than in wild-type animals and d.p.i. this increase in demyelination could be associated with the persistence of infectious virus in the absence of b cells. in mice lacking antibody fc receptors or complement pathway activity, thereby lacking the ability to utilize antibody effector functions, virus did not persist yet demyelination was similar to that observed in wild-type mice. antibody has also been suggested to facilitate remyelination. however, in mumt mice, remyelination was still detected. therefore, we nd no evidence that b cells are important for cns demyelination, nor are they required for remyelination. another hypothesis that has been proposed is that mhv invades the oligodendrocytes or cells that provide critical functions to the oligodendrocyte and damages them directly, thereby causing oligodendrocyte death or retraction of the myelin sheath. early work on mhv-jhm using electron microscopy and immuno ourescence demonstrated that the virus infects oligodendrocytes early and possibly late after infection, and thereby supported this hypothesis (lampert et al, ; powell and lampert, ; wu and perlman, ) . however, the degenerating oligodendrocytes detected during early paralysis rarely actually contained viral particles (powell and lampert, ) . viral particles were also detected in astrocytes d.p.i., and virus could potentially damage oligodendrocytes by disrupting the function of these cells . interestingly, mice immunosuppressed using cyclophosphamide with resultant loss of antibody production and decreased perivascular in ammation still displayed small demyelinating lesions (weiner, ) . likewise, lipid-laden macrophages and occasional small lesions were detected in rag knockout mice (wu and perlman, ) , and scid mice can develop demyelination, if rarely (houtman and fleming, ) . direct virally mediated demyelination is unlikely to be the sole cause of demyelination, however, because virus in the presence of t cells more reliably induces demyelinating lesions (wang et al, ) . it is clear that demyelination after mhv infection is a complex issue. it is possible that both direct viral destruction and immune mediated destruction contribute to the development of demyelinating lesions. past research has focused on dissecting the immune response to mhv in order to determine the role of single components in the demyelinating phase of the disease. however, in contrast to other mouse models of demyelination, there does not appear to be a clear role for any one particular lymphocytic or monocytic subset that mediates the demyelination. rather, it appears that a balance of immune components may be required for viral clearance and that various immune and nonimmune pathways may mediate the subsequent demyelinating events. it is this balance that determines the level of encephalitis in acute infection and sets up the conditions that lead to demyelination. one model that would account for the involvement of a variety of immune elements in demyelination would associate cns pathology with free radical production by in ammatory cells of the myeloid lineage. these may be attracted into the cns by chemokines secreted by viral speci c cd c t cells. in this model cytokines secreted by cd c t cells would assist in the state of activation of macrophages and astrocytes and therefore in uence the level of cns disease. such a model for cns pathology has been implicated for eae (koprowski et al, ; lin et al, ) and borna disease virus-induced cns pathology (hooper et al, ) . future studies are required to determine if this mechanism also prevails in mhv-induced demyelination. in this review we have described studies in which modulating the immune response has resulted in perturbation of the balance between replication/spread of the virus and clearance with consequential changes in acute disease and demyelination. knowledge of the contribution of different components of the disease would be useful in manipulating this balance so that robust viral clearance does not result in chronic cns demyelination. viremic dissemination of mouse hepatitis virus-jhm following intranasal inoculation of mice inverted immunodominance and impaired cytolytic function of cd c t cells during viral persistence in the central nervous system impaired t cell immunity in b cell-de cient mice following viral central nervous system infection in situ tolerance within the central nervous system as a mechanism for preventing autoimmunity cellular component s of the immune barrier in the spinal meninges and dorsal root ganglia of the normal rat: 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expression in the central nervous system: astrocytes fail to express major histocompatibility complex class i and ii molecules dissociation of demyelination and viral clearance in congenitally immunode cient mice infected with murine coronavirus jhm macrophages in t cell line-mediated, demyelinating, and chronic relapsing experimental autoimmune encephalomyelitis in lewis rats regulation of brainderived t cells during acute central nervous system in ammation organ speci c endothelial cell heterogeneity in uences differential replication and cytopathogenicity of mhv- and mhv- . implications in viral tropism fas-and perforinindependent mechanisms of cytotoxic t lymphocyte virus persistence and recurring demyelination produced by a temperature-sensitive mutant of mhv- infection and involution of mouse thymus by mhv- induction of demyelination by a temperature-sensitive mutant of the coronavirus mhv-a is associated with restriction of viral replication in the brain in vivo expression of 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cd c and cd c t cells are not major effectors of mouse hepatitis virus a -induced demyelinating disease coronavirus infection induces h- antigen expression on oligodendrocytes and astrocytes post in ammatory remyelination in the spinal cord of mice infected with mouse hepatitis virus, jhm strain sequential infection of glial cells by the murine hepatitis virus jhm strain (mhv- ) leads to a characteristic distribution of demyelination coronavirus mhv-a causes upregulation of interferon-beta rna in primary glial cell cultures pathogenesis of demyelination induced by a mouse hepatitis role of spleen macrophages in innate and acquired immune responses against mouse hepatitis virus strain a role of virus-speci c cd c cytotoxic t cells in recovery from mouse hepatitis virus infection receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins effective clearance of mouse hepatitis virus from the central nervous system requires both cd c and cd c t cells characterization of brain-in ltrating mononuclear cells during infection with mouse hepatitis virus strain jhm cd and cd t cells have redundant but not identical roles in virus-induced demyelination macrophage in ltration, but not apoptosis, is correlated with immune-mediated demyelination following murine infection with a neurotropic coronavirus depletion of blood-borne macrophages does not reduce demyelination in mice infected with a neurotropic coronavirus hemagglutinin-esterase-speci c monoclonal antibodies alter the neuropathogenicity of mouse hepatitis virus key: cord- -uypjqif authors: firpo, mason r.; mastrodomenico, vincent; hawkins, grant m.; prot, matthieu; levillayer, laura; gallagher, tom; simon-loriere, etienne; mounce, bryan c. title: targeting polyamines inhibits coronavirus infection by reducing cellular attachment and entry date: - - journal: acs infect dis doi: . /acsinfecdis. c sha: doc_id: cord_uid: uypjqif [image: see text] coronaviruses first garnered widespread attention in when the severe acute respiratory syndrome coronavirus (sars-cov) emerged from bats in china and rapidly spread in human populations. since then, middle east respiratory syndrome coronavirus (mers-cov) emerged and still actively infects humans. the recent sars-cov- outbreak and the resulting disease (coronavirus disease , covid ) have rapidly and catastrophically spread and highlighted significant limitations to our ability to control and treat infection. thus, a basic understanding of entry and replication mechanisms of coronaviruses is necessary to rationally evaluate potential antivirals. here, we show that polyamines, small metabolites synthesized in human cells, facilitate coronavirus replication and the depletion of polyamines with fda-approved molecules significantly reduces coronavirus replication. we find that diverse coronaviruses, including endemic and epidemic coronaviruses, exhibit reduced attachment and entry into polyamine-depleted cells. we further demonstrate that several molecules targeting the polyamine biosynthetic pathway are antiviral in vitro. in sum, our data suggest that polyamines are critical to coronavirus replication and represent a highly promising drug target in the current and any future coronavirus outbreaks. c oronaviruses are significant human pathogens with significant epidemic potential. recurring coronavirus outbreaks wreak havoc on human health and activity, as demonstrated by the original severe acute respiratory syndrome coronavirus (sars-cov) outbreak of , the middle east respiratory syndrome coronavirus (mers-cov) of to present day, and the newly emergent sars-cov- of to present. each of these viruses emerged from zoonotic sources and were rapidly transmitted through human populations, requiring urgent attention due to the number of infections, combined with mortality rates of up to % in the case of mers-cov. in addition to their epidemic potential, coronaviruses are common human pathogens, with several endemic coronaviruses causing recurrent infections with generally mild symptoms. , treatments for sars-cov- , as well as other coronaviruses, include remdesivir, a base analog that exhibited promising clinical efficacy. , a dearth of additional antivirals is available to treat or prevent infections. the omnipresence, unpredictable epidemic potential, and significant impact on human health make the investigation into potential antiviral compounds essential to battle current and future coronavirus outbreaks. polyamines are small aliphatic metabolites synthesized by mammalian cells to support cellular processes such as cell cycling, transcription, and translation. − the polyamines putrescine, spermidine, and spermine ( figure a ) are synthesized from an ornithine precursor through the enzyme odc . several specific and potent inhibitors of the polyamine biosynthetic pathway have been developed and are fda approved. the odc inhibitor difluoromethylornithine (dfmo) is approved for the treatment of trypanosomiasis. in fact, dfmo exhibits low toxicity and mild side effects. topical dfmo has also been developed and is sold as vaniqa to reduce hair growth. viruses rely on polyamines for varied stages in replication, including polymerase activity, cell binding, and viral protein translation. , polyamine depletion via dfmo effectively reduces the replication of diverse viruses in vitro and in vivo with minimal cytotoxicity. , prior work demonstrated that mers-cov was sensitive to the polyamine inhibitor dfmo in vitro at a dose of μm, suggesting that polyamines function in coronavirus replication. whether and how polyamines function in the replication of other coronaviruses remain to be investigated. the study of human epidemic coronaviruses is limited due to requisite biosafety protocols. thus, murine model systems have been developed to study coronavirus replication and pathogenesis with significantly reduced risk. murine hepatitis virus (mhv) is a common model system for coronavirus infection. mhv, a β-coronavirus, is related to the human epidemic coronaviruses, though it is safe to work with in the laboratory. the mhv model is well characterized, , and combined with its ease of use, it provides a significant opportunity to study coronavirus infection. using the mhv model system, we investigated a role for polyamines in coronavirus infection. we observe that polyamine depletion via dfmo limits virus replication and that polyamines support virus infection at the stage of attachment: polyamine depletion reduces virus binding to susceptible cells. we extend this phenotype to the human endemic coronavirus nl as well as the pandemic sars-cov- . additionally, we demonstrate that several molecules targeting the polyamine pathway, including fda-approved pharmaceuticals, exhibit significant antiviral activity. together, our data highlight a role for polyamines in coronavirus replication and suggest that targeting polyamines may be a viable preventative or treatment option for coronavirus infection. to determine if coronaviruses are susceptible to polyamine depletion, we treated vero-e cells with doses of dfmo ranging from μm to mm for days prior to infection with sars-cov- at a multiplicity of infection (moi) of . plaque forming units (pfu) per cell. released viral genomes were then measured at h post-infection (hpi) and converted to virus equivalents using a standard curve comparing known infectious titers and genomes. we observed that dfmo significantly reduced sars-cov- genomes, greater than fold at mm, with an ic ( % inhibitory concentration) value of μm ( figure b ). to confirm that dfmo was not cytotoxic, we measured the viability of vero-e cells treated with escalating doses of dfmo and calculated a cc ( % cytotoxic concentration) value of mm ( figure c ; summarized in table ). we further confirmed that dfmo treatment at these concentrations successfully depleted polyamines via thin layer chromatography (tlc; figure d ). to expand on this work, we turned to the mhv model system. as with our experiments with sars-cov- , bhk- figure e ). we confirmed that dfmo depleted polyamines in these cells by tlc ( figure g ), especially levels of putrescine and spermidine, as previously described. to test if dfmo remained antiviral over multiple rounds of infection, bhk-rs were treated with dfmo and infected at an moi of . . supernatant was collected and titered at the indicated time points ( figure h ). dfmo reduced viral titers compared to the no treatment group up to h, and viral titers in dfmotreated cells plateaued at a lower level than untreated controls, suggesting that mhv was unable to overcome polyamine depletion over several rounds of replication. we observed that dfmo had modest toxicity in bhk-r cells with a cc of . mm ( figure f ), giving a selectivity index (si) of . finally, we measured the relative number of genomes present in the supernatant from the infected cells. following treatment and infection, we purified, reverse-transcribed, and measured viral genomes by qpcr using specific primers and observed reduced genome levels with dfmo treatment ( figure i ). using the data from infectious titers ( figure e ) and viral genomes ( figure i ), we calculated the specific infectivity (genomes per pfu) and observed that dfmo treatment increased the ratio of genomes to titer ( figure j ), similar to previous findings with bunyaviruses and polyamine depletion. thus, polyamines enhance coronavirus infection and maintain a specific infectivity of mhv. to address the role of cell type, we tested the ability of dfmo to restrict virus replication in hela cells expressing the mhv receptor (hela-r). cells were pretreated with dfmo and infected at an moi of . for h. we observed a decline in virus replication, more pronounced than on bhk-r cells, with an ic value of approximately . μm ( figure k ). we confirmed that dfmo treatment of hela-r cells reduced polyamines ( figure m ) and verified that dfmo was not toxic to these cells ( figure l ). the cc of dfmo in hela cells was mm, and thus, the selectivity index was . polyamines facilitate coronavirus replication. dfmo restricts virus replication by reducing cellular polyamine pools. however, excess polyamines do not enhance virus replication, according to work based on chikungunya virus. recent work has suggested that polyamines may serve coronavirus replication. to determine if exogenously supplied polyamines serve an antiviral role, we treated bhk-r cells with increasing doses of the individual polyamines putrescine, spermidine, and spermine, from to μm. we measured cellular viability ( figure a ) and infected these cells to measure viral titers at hpi ( figure b ). we observed that the polyamines significantly reduced cellular viability at approximately μm, reflecting prior descriptions of high polyamine concentrations leading to apoptosis. , despite this reduction in viability, we observed modestly reduced viral titers ( figure b ). we measured polyamines in supplemented cells and observed that supplementation with any of the three polyamines resulted in only modest changes in cell-associated polyamines ( figure b , lower), as we previously described. the reduced signal with high concentrations of spermidine and spermine likely was an artifact of reduced cellular viability at these doses. to determine if dfmo-mediated virus restriction was a result of polyamine depletion, we treated bhk-r cells with dfmo and, at the time of infection, supplemented with the individual polyamines putrescine, spermidine, and spermine at μm, a level that did not affect cellular viability ( figure a ). we observed that dfmo reduced viral titers and that supplementation with any of the individual polyamines rescued virus replication to untreated levels ( figure c ). we confirmed the presence of these polyamines ( figure c , below) and observed that supplementation with any of the individual polyamines results in the accumulation of all three polyamines in the cells. to exclude the possibility that polyamines may directly inactivate viral particles, we directly incubated mhv with dfmo and each of the individual polyamines at μm for h and then titered the incubated virus. we observed that neither dfmo nor polyamines affected infectivity when incubated with virus, as titers were unchanged compared to untreated controls ( figure d ). in sum, our data suggest that polyamines support virus yield and that supplementation of polyamines supports but does not enhance this yield and can instead be cytotoxic. polyamine depletion is prophylactic. dfmo depletes polyamines by inhibiting odc , though time is required to deplete cellular polyamine pools and subsequently reduce viral titers. ideally, the treatment with antivirals commences after the initiation of infection, though identification of soon-to-beinfected individuals is impractical. thus, to determine if we could treat cells with dfmo at the time of infection, which would be more clinically relevant, we determined whether dfmo suppresses plaque development when present in overlay media, as previously described. we observed no changes in plaque size ( figure a ) or morphology ( figure b ) with dfmo supplementation to the media, suggesting that dfmo was not sufficiently antiviral when applied to cells after infection and highlighting that dfmo must be applied prophylactically to reduce coronavirus replication. we observed a small, though statistically insignificant change in hela-r cells ( figure c ). the use of dfmo as an anti-trypanosomal agent requires repeated dosing to reach a serum concentration high enough to inhibit trypanosome growth. , , to mimic this, we treated bhk-r cells with . mm dfmo in a single treatment, three μm treatments, or four μm treatments equally spaced over the days prior to infection. we observed that cells receiving multiple doses of the drug reduced cellular capacity to support virus production, similar to when the drug was delivered in a single dose ( figure d ), suggesting that repeated dosing is as effective as a single drug treatment in vitro. finally, we tested whether the antiviral effects of dfmo were reversed by removing the drug and replenishing cells with fresh media. we treated cells with mm for days and then removed and replaced the media with fresh media without drug for h before infection. when we measured viral titers, we observed the dfmo treatment reduced infectious virus production, and this effect was completely reversed by replenishing the cells with fresh media, suggesting that dfmo's antiviral effect can be reversed by removing the drug. polyamines facilitate coronavirus binding and entry. we previously demonstrated that enteroviruses, flaviviruses, and bunyaviruses rely on polyamines for viral attachment and entry. to test whether coronaviruses similarly rely on polyamines for attachment and entry, we performed a binding assay in cells treated with dfmo. we treated bhk-r cells with dfmo for days prior to infection with pfu. after min, unbound virus was washed away with pbs and cells were overlaid with agarose for a plaque assay, lacking dfmo in the overlay. thus, dfmo is present only during binding of mhv to target cells and not for any other portion of the viral life cycle. plaques were then allowed to form and were enumerated, representing bound and entered infectious virus. we observed that the attachment and plaque formation was significantly inhibited with dfmo treatment ( figure a , representative plaques for nt and mm in figure b ). we expanded these results by infecting dfmo-treated bhk-r cells with mhv for min, washing away unbound virus, and quantifying bound virus by qrt-pcr. we observed that, similar to our plaque formation assays, bound viral genomes were significantly reduced with dfmo treatment ( figure c ). we confirmed that the cells expressed the mhv receptor (cecam- ) by immunofluorescence ( figure d ), regardless of dfmo treatment, suggesting that dfmo treatment was not directly reducing receptor availability. we recapitulated these results with hela-r cells and observed a similar reduction in virus binding with dfmo treatment ( figure e ). to expand to human coronaviruses, we tested hcov-nl for binding to dfmo-treated vero-e cells. again, we figure e ). again, we noted no change in viral receptor expression (ace , figure g ). taking advantage of a pseudoparticle system as previously described and adapted to sars-cov- , we measured the attachment of sars-cov- to dfmo-treated cells. we verified that these pseudoparticles were competent to bind susceptible cells compared to "bald" particles, lacking viral spike ( figure h ), and we confirmed the presence of the spike protein on purified particles by western blot ( figure i ). we observed a reduction in cellular attachment of sars-cov- pseudoparticles in a dose-dependent manner ( figure h ). thus, human coronaviruses, including pandemic sars-cov- , are sensitive to polyamine depletion and rely on polyamines for efficient cellular attachment. additional molecules targeting the polyamine and hypusination pathway quell coronavirus infection. several pharmaceuticals target polyamine biosynthesis ( figure a ) and exhibit antiviral activity, , though coronaviruses have not been thoroughly tested. additionally, an offshoot of the polyamine pathway, the cellular hypusination pathway, in which spermidine is conjugated to eif a, plays roles in cellular translation, which is critical for filovirus replication. , to determine if additional compounds targeting the polyamine and hypusination pathways exhibited activity against coronaviruses, we treated cells with increasing doses of inhibitors of figure b ) with an ic of . μm. we observed minimal toxicity ( figure c ) with a cc value of μm resulting in a selectivity index of . because denspm activates sat to deplete polyamines, its action is much more rapid than dfmo. thus, we tested whether denspm exhibited antiviral activity after the initiation of infection. cells were treated with denspm at times before and after infection, and virus titers were measured at hpi. we observed that denspm was most effective when treatment was initiated before infection; however, significant antiviral activity was maintained when denspm was added as late as hpi ( figure d ). finally, we tested the hypusination inhibitors gc , def, and cpx. we observed in all cases that these molecules significantly reduced mhv replication in a dose-dependent manner ( figure e −g) and that cytotoxicity was minimal ( figure h ; summarized in table ). thus, molecules targeting an additional branch of the polyamine biosynthetic pathway exhibit anti-coronavirus activity. polyamines are crucial to virus infection; however, their role in coronavirus replication was previously unknown. here, we demonstrated that polyamines are essential to coronavirus infection and that attachment is at least one of the steps affected by polyamine depletion. we previously demonstrated that enteroviruses are similarly sensitive to polyamine depletion and rely on these polyamines for attachment. this phenotype is conserved among several viral families, suggesting that polyamines may mediate a common attachment factor. whether polyamines directly facilitate attachment by mediating virus−cell connections is unclear, and future work will be needed to delineate precisely how polyamines affect attachment. in addition to attachment, we observed that inhibitors of the hypusination pathway impact virus replication, suggesting that polyamines may facilitate infection through eif a hypusination, as observed for ebolavirus. given the role of hypusinated eif a in translation, inhibitors of this pathway may directly inhibit viral translation or may inhibit the translation of a cellular factor essential to virus replication. regardless, targeting polyamines may affect multiple steps of infection, and additional work will be needed to uncover additional mechanisms. polyamines play several important roles in the cell, and recent work has highlighted pathways that may modulate virus replication. spermidine, as a substrate for eif a hypusination, enhances cellular autophagy, and recent work has suggested that spermidine-enhanced autophagy limits sars-cov- replication. in contrast, our results suggest that spermidine does not inhibit or restrict virus replication at concentrations up to μm. we do observe that spermine supplementation to cells reduces virus replication; however, we also observe significant cytotoxicity with spermine treatment, likely the reason for reduced titers. additionally, we observed that dfmo treatment reduced polyamine content within cells, even at concentrations that did not affect viral titers. these data suggest that coronaviruses require a low concentration of polyamines for robust replication. in fact, bunyaviruses and alphaviruses replicate with supplementation of nm polyamines in polyamine depleted cells. , thus, these viruses may have evolved mechanisms to take advantage of polyamines even at low concentrations. this has implications for the molecular interactions between polyamines and coronaviruses, and perhaps coronaviruses evolved a strong affinity for polyamines. this also has implications for infection in an organism, as polyamines are transported between cells. in cancer therapies, polyamine transporter inhibitors can be used in conjunction with polyamine depletion agents to prevent both polyamine synthesis and internalization. while this has not been tested in the context of virus infection, the combination of inhibitors of synthesis and transport may more effectively reduce virus replication. precisely how polyamines play into cellular signaling and metabolic pathways to affect virus replication is not clear; however, the complex interplay between metabolism and virus replication affords several opportunities to target virus replication with small molecule inhibitors. with the current sars-cov- outbreak and the resulting coronavirus disease of (covid- ), novel antivirals are urgently needed. remdesivir has shown significant potential clinically, − and additional clinical testing will determine if this molecule can effectively treat infected individuals. our work with dfmo and polyamine depletion highlights another promising intervention. an important limitation of our study is the relatively high concentration of drug required to reduce coronavirus infection. given this caveat, the possibility of offtarget antiviral effects cannot be ignored. however, we show that dfmo's antiviral effects are reversed by the addition of exogenous polyamines, suggesting specificity. our prior results with chikungunya, zika, coxsackie, and rift valley fever viruses have demonstrated that low micromolar concentrations of inhibitor are sufficient to reduce virus replication. thus, coronaviruses may be less reliant on polyamines than other viral families, though this requires significant further investigation. importantly, we observe that polyamine depletion functions prophylactically to reduce virus replication and may not reduce viral titers after the initiation of infection. given the limits to our in vitro studies, we cannot say for certain whether polyamine depletion in a human could reduce virus replication or improve clinical outcome when treatment commences after exposure. however, polyamine depletion may be most effective as a prophylactic, preventing infection of vulnerable populations. with the significant spread of sars-cov- , protecting vulnerable populations, including the elderly and immunocompromised, is essential to reducing mortality. further, prophylactically treating frontline workers, including medical staff, could reduce the spread of the virus in a hospital setting. currently, dfmo is approved for human use as a topical and intravenous agent. nebulization and direct lung delivery, if effective at depleting polyamines in the lung, would be a benefit for reducing virus burden, but this remains untested. whether dfmo or other polyamine-targeting drugs are sufficient to do this is unclear and requires significantly more investigation, including in animal models. ■ methods cell culture. cells were maintained in dulbecco's modified eagle's medium (dmem; life technologies) with bovine serum and penicillin−streptomycin at °c and % co . vero cells were obtained through bei resources, and veroe cells were obtained through atcc (crl- ). vero and hela-r cells were supplemented with % new-born calf serum (nbcs; thermo-fischer). bhk-r cells were kindly provided by dr. susan baker and were supplemented with % fetal bovine serum (fbs; thermo-fischer). hek t cells were grown in % fbs. infection and enumeration of viral titers. mhv-a was derived from the first passage of virus in bhk-r cells from an infectious clone. sars-cov- , isolate betacov/france/ idf / c , was supplied by the national reference centre for respiratory viruses hosted at institut pasteur (paris, france). the human sample from which this strain was isolated was provided by dr. x. lescure and pr. y. yazdanpanah from the bichat hospital, paris, france. viral stocks were prepared by propagation in vero e cells in dmem supplemented with % fbs. all experiments involving live sars-cov- were performed in compliance with the institut pasteur guidelines for biosafety level work. for all infections, dfmo was maintained throughout the infection as designated. viral stocks were maintained at − °c. for infection, virus was diluted in serum-free dmem for a multiplicity of infection (moi) of . on bhk-r cells, unless otherwise indicated. viral inoculum was added to the cells, and supernatants were collected at specified time points. to quantify viral titers via plaque assay, dilutions of cell supernatant were prepared in serum-free dmem and used to inoculate confluent monolayers of bhk-r cells for to min at °c. cells were overlain with . % agarose in dmem containing % fbs. mhv-a samples were incubated for days at °c. cells were fixed with % formalin and revealed with crystal violet solution ( % crystal violet; sigma-aldrich). plaques were enumerated and used to back-calculate the number of plaque forming units (pfu) per milliliter of collected volume. drug treatments. difluoromethylornithine (dfmo; targetmol) was diluted to a mm solution in sterile pbs. for dfmo treatments, cells were trypsinized (zymo research) and reseeded with fresh medium supplemented with % fbs. cells were treated with mm dfmo unless otherwise indicated. cells were incubated with dfmo for h to allow for the depletion of polyamines. experiments involving polyamine rescues were performed using μm polyamines (sigma-aldrich) added to either the cell supernatant or viral inoculum as indicated. thin layer chromatography of polyamines. polyamines were separated by thin layer chromatography as acs infectious diseases pubs.acs.org/journal/aidcbc article previously described. for all samples, cells were treated as described prior to being trypsinized and centrifuged. pellets were washed with pbs and then resuspended in μl of % perchloric acid. samples were then incubated overnight at °c . supernatant or μmole of putrescine, spermidine, or spermine standards was combined with mg/ml dansyl chloride (sigma-aldrich) in acetone and saturated sodium bicarbonate. samples were incubated in the dark overnight at room temperature. excess dansyl chloride was cleared by incubating the reaction with proline (sigma-aldrich). dansylated polyamines were extracted with toluene (sigma-aldrich) and centrifuged. the sample was added in spots to the silica gel matrix tlc plates (sigma-aldrich) and exposed to ascending chromatography with : cyclohexane/ethyl acetate. the plate was dried and visualized via exposure to uv. plaque formation attachment assay. bhk-r cells were seeded in -well plates and grown to confluence in dmem with % fbs. the cells were treated for h with varying concentrations of dfmo. after the h dfmo treatment, the media were aspirated from the cells and replaced with μl of serum free media containing pfu mhv. the infected cells were incubated at room temperature or on ice for min. cells were washed × with pbs and then overlaid with . % agarose containing dmem with % fbs. the plates were incubated at °c for days for plaques to develop. the cells were fixed with % formalin, and the plaques were visualized with crystal violet staining. plaque size measurement. bhk-r or hela-r cells were seeded in -well plates and grown to confluence. approximately pfu of mhv-a was diluted in a μl inoculum of serum free dmem. the inoculum was incubated on the cells for approximately min at °c. after min, an overlay of ml of . % agarose containing dmem with % fbs (bhk-r) or nbcs (hela-r) was added to each well. the dishes were incubated at °c for days to allow plaque formation. the cells were fixed with % formalin, and the agarose plugs were removed. the fixed cells were stained with crystal violet. plaque size was determined using imagej software. immunofluorescence imaging. cells grown on coverslips were either treated with mm dfmo or untreated. cells were fixed with % formalin for min, washed with pbs, permeabilized, and blocked with . % triton x- and % bsa in pbs (blocking solution) for min at room temperature (rt). cells were sequentially incubated as follows: primary rabbit anti-hace (invitrogen, sn ) (vero e ) or rabbit anti-mceacam (bhk-r) antibodies ( : ) with blocking for h at room temperature. cells were subsequently washed then incubated with secondary goat antirabbit antibodies ( : with blocking, min, rt). mounting media with dapi was used to visualize nuclei. samples were imaged with a zeiss axio observer with lumencor spectra x led light system and a hamamatsu flash camera using appropriate filters using zen blue software with a × objective. western blot. samples were collected with bolt lds buffer and bolt reducing agent (invitrogen) and run on polyacrylamide gels. gels were transferred using the iblot gel transfer device (invitrogen). membranes were probed with primary antibodies for mouse anti-rhodopsin (emd millipore, mab ), anti-vsv-m ( h ), and anti-mouse igg (goat, hrp-labeled, nef ea). membranes were treated with supersignal west pico plus chemiluminescent substrate (thermofisher scientific) and visualized on a proteinsimple fluorchem e imager. rna purification and cdna synthesis. media were cleared from cells, and trizol reagent (zymo research) was added directly. lysate was then collected, and rna was purified through a zymo rna extraction kit. purified rna was subsequently used for cdna synthesis using high capacity cdna reverse transcription kits (thermo-fischer), according to the manufacturer's protocol, with − ng of rna and random hexamer primers. qpcr-based attachment assay. bhk-r, hela-r, and vero cells were seeded at × cells per well in -well plates in dmem with either % fbs or nbcs. the cells were treated for h with varying concentrations of dfmo. after h, the media were aspirated from the cells and replaced with μl of serum free media containing virus. the infected cells were incubated for min at room temperature or on ice. the cells were then washed × with pbs, and then, μl of trizol was added to the cells. the rna was extracted with the zymo rna extraction kit, converted to cdna, and quantified by real-time pcr with sybr green (dotscientific) using the one-step protocol quantstudio (thermofisher scientific). relative genomes were calculated using the Δc t method, normalized to the β-actin qrt-pcr control, and calculated as the fraction of the unwashed samples. primer sequences are as follows: nl , ′-tgt-caa-cga-ggt-ttt-gca-tta-aat- ′ (f) and ′-act-ggc-cta-cca-ttg-tgt-gta-aga- ′ (r); mhv, ′-atc-ctc-aag-aag-acc-act-tgg-gct-gac- ′ (f) and ′-gag-taa-tgg-gga-acc-aca-ctc−ccg- ′ (r); β-actin, ′-cac-tct-tcc-agc-ctt-cct-tc- ′ (f) and ′-gta-cag-gtc-ttt-gcg-gat-gt- ′ (r). primers were verified for linearity using fold serial diluted cdna and checked for specificity via melt curve analysis. sars-cov- genomes were detected using a taqman assay (ip set) as described on the who web site (https://www.who.int/docs/default-source/coronaviruse/realtime-rt-pcr-assays-for-the-detection-of-sars-cov- -institutpasteur-paris.pdf?sfvrsn= fcb _ ) synthesis of sars-cov- pseudovirus. t cells were seeded into a mm tissue culture dish h prior to transfection. cells were transfected with a sars-cov- spike using lipod and following the manufacture's protocol. after h, the media were removed and replaced with fresh, prewarmed % fbs media. the following day, cells were infected with a to dilution of fluc-vsv-junin virus and allowed to infect for h. after the h, the cells were washed × with serum free dmem, and fresh, prewarmed media were placed onto the cells. on the following day, cell supernatant was collected and spun down via differential centrifugation ( g for min at °c, g for min at °c) and stored at − °c. pseudovirus attachment assay. vero e cells were seeded at × in -well plates in dmem with % nbcs. cells were then treated with varying concentrations of dfmo for h. after h, the media were aspirated from the cells and replaced with μl of serum free media containing pseudovirus. the infected cells were incubated for min at room temperature. pseudovirus was then aspirated off and washed × with pbs (bound), and new, prewarmed % nbcs media were added to the cells and allowed to sit overnight (input and bound). the following day, the media were aspirated from the cells, and the cells were then washed with × pbs. subsequently, cells were then lysed with μl of × sars and mers: recent insights into emerging coronaviruses return of the coronavirus: -ncov middle east respiratory syndrome coronavirus (mers-cov) mosaic structure of human coronavirus nl , one thousand years of evolution burden of disease due to human coronavirus nl infections and periodicity of infection broad spectrum antiviral remdesivir inhibits human endemic and zoonotic deltacoronaviruses with a highly divergent rna dependent rna polymerase broad-spectrum antiviral gs- inhibits both epidemic and zoonotic coronaviruses polyamines and cancer: old molecules, new understanding synthetic polyamines stimulate in vitro transcription by t rna polymerase depletion of cellular polyamines, spermidine and spermine, causes a total arrest in translation and growth in mammalian cells eflornithine for the treatment of human african trypanosomiasis efficacy and toxicity of eflornithine for treatment of trypanosoma brucei gambiense sleeping sickness vaniqa -eflornithine . % cream interferon-induced spermidine-spermine acetyltransferase and polyamine depletion restrict zika and chikungunya viruses polyamine depletion abrogates enterovirus cellular attachment polyamines and hypusination are required for ebolavirus gene expression and replication diverse functions of polyamines in virus infection inhibition of polyamine biosynthesis is a broad-spectrum strategy against rna viruses the cellular and molecular pathogenesis of coronaviruses advances in virus research coronaviruses: propagation, quantification, storage, and construction of recombinant mouse hepatitis virus polyamine depletion inhibits bunyavirus infection via generation of noninfectious interfering virions analysis of sars-cov- -controlled autophagy reveals spermidine, mk- , and niclosamide as putative antiviral therapeutics polyamines: mysterious modulators of cellular functions the functional role of polyamines in eukaryotic cells virion-associated spermidine transmits with rift valley fever virus particles to maintain infectivity eflornithine hcl study group. randomized, double-blind clinical evaluation of the efficacy and safety of topical eflornithine hcl . % cream in the treatment of women with facial hair distinct roles for sialoside and protein receptors in coronavirus infection hypusine, a polyamine-derived amino acid critical for eukaryotic translation differential mechanisms for the involvement of polyamines and hypusinated eif a in ebola virus gene expression polyamines control eif a hypusination, tfeb translation, and autophagy to reverse b cell senescence virion-associated polyamines transmit with bunyaviruses to maintain infectivity and promote entry actt- study group members gs-us- − investigators thin-layer chromatographic method for assaying polyamines nih image to imagej: years of image analysis the authors declare no competing financial interest. we thank susan baker for generous assistance with the mhv system and for providing the virus and cells as well as ivana kuo for microscopy assistance. we also thank susan uprichard for critical discussion of the data. e.s.-l. acknowledges funding from the inception program (investissements d'avenir grant anr- -conv- ), from the institut pasteur corona task force, and from the laboratoire d'excellence "integrative biology of emerging infectious diseases" (grant no. anr- -labx- -ibeid). key: cord- -xpj c vx authors: piñón, josefina d.; teng, henry; weiss, susan r. title: further requirements for cleavage by the murine coronavirus c-like proteinase: identification of a cleavage site within orf b date: - - journal: virology doi: . /viro. . sha: doc_id: cord_uid: xpj c vx abstract the coronavirus mouse hepatitis virus strain a (mhv-a ) encodes a c-like proteinase ( clpro) that is proposed to be responsible for the majority of the processing events that take place within the replicase polyproteins pp a and pp ab. in this study we demonstrate that the q ↓s peptide bond, located between the polymerase and zn-finger regions of pp ab (the pol↓zn site), is processed by the clpro, albeit inefficiently. mutagenesis of the pol↓zn site, as well as the previously identified hd ↓ c site in the a region of pp a and pp ab, demonstrated that the amino acid residues at the p and p positions of the cleavage site, occupied by l and q, respectively, were important determinants of clpro substrate specificity. finally, a direct comparison of the clpro-mediated cleavages at the hd ↓ c and pol↓zn sites was made by determining the rate constants using synthetic peptides. the results show that while a larger polypeptide substrate carrying the hd ↓ c site was processed more efficiently than a polypeptide substrate carrying the pol↓zn site, cleavage of the synthetic peptide substrates containing these two cleavage sites occurred at similar efficiencies. this indicates that the overall conformation of a large polyprotein substrate is important in the accessibility of the cleavage site to the proteinase. the family coronaviridae is composed of a group of viruses that cause a variety of diseases in different animal hosts. the murine coronavirus, mouse hepatitis virus (mhv), causes a range of diseases in mouse, including enteritis, hepatitis, encephalitis, and a demyelinating disease (holmes and lai, ; houtman and fleming, ; lai, ) . coronaviruses, along with the arteriviruses, are classified under the newly established order nidovirales based on the similarities in their genome organization and replication strategy (cavanagh, ; de vries et al., ) . the name nidovirales originates from the latin word nidus, meaning "nest," and refers to the Ј nested set of subgenomic mrnas that is produced during viral infection (de vries et al., ) . as with all positive-strand rna viruses, entry of the viral genome into the cytoplasm is followed by the translation of the positive-strand rna genome, resulting in the expression of viral proteins. the coronavirus genome is organized into seven genes that are separated by stop codons and intergenic sequences (holmes and lai, ; lai, ) . thus, translation of the viral genome results only in the expression of gene proteins. the rest of the viral genome is expressed through subgenomic mrnas that are transcribed by the viral rna-dependent rna polymerase encoded in gene . replication of the viral genome also requires the replicase proteins encoded in gene . thus, for both viral replication and subgenomic mrna transcription to take place, the expression of gene products is essential. the replicase gene (gene ) ( fig. ) of coronaviruses, spanning - kb, is organized into two overlapping open reading frames, orf a and orf b (bonilla et al., ; lee et al., ) . the expression of the downstream orf b is mediated by a ribosomal frameshift event that is aided by the formation of a pseudoknot structure within the overlapping region (bredenbeek et al., ; brierley et al., ; . thus, two polypeptides, pp a and pp ab, are expressed from gene , with the translation of pp ab being only - % as efficient as that of pp a in in vitro studies (bredenbeek et al., ; brierley et al., ) . through a series of intricate cotranslational and posttranslational processing events, these polyproteins are converted into a functional complex that in turn is responsible for both genomic rna replication and subgenomic mrna transcription (de vries et al., ) . responsible for these processing events are at least two or three viral proteinases encoded within the orf a region of gene (fig. ) . two of these proteinase domains, by sequence analysis, share similarities with the cellular proteinase papain. a third proteinase, resembling the poliovirus c proteinase, has also been identified (gorbalenya et al., ; lee et al., ) . the coronavirus c-like proteinase ( clpro), flanked on either side by hydrophobic, possibly membrane-spanning regions (hd and hd ), is believed to be the prinicipal viral proteinase responsible for the processing events leading to the formation of the viral replicase complex, with as many as potential cleavage sites identified throughout pp ab (gorbalenya et al., ; lee et al., ) (see fig. ). the presence of the clpro is conserved in all coronavirus genomes studied to date (bonilla et al., ; boursnell et al., ; eleouet et al., ; lee et al., ) . the clpro of mhv-a has been identified as a -kda protein (p ) both in in vitro study and in mhv-a infected cells (piñó n et al., ) . reported a molecular weight of kda for the same polypeptide.) the catalytic residues of the mhv-a clpro, his , and cys have also been identified . treatment of infected cells with e- d, a known inhibitor of the clpro, results in the inhibition of viral rna replication in these cells (kim et al., ) , demonstrating the importance of the action of the clpro in the events leading to viral replication. van dinten et al. ( ) demonstrated the importance of clpro cleavages using an infectious clone of the related arterivirus eav; introduction of mutations into the candidate orf b clpro cleavage sites had drastic effects on rna synthesis and virus replication. these fndings indicate that this proteinase is a good potential target for antiviral therapy. the cleavage sites of the coronavirus clpro ( fig. ) conform to the consensus xq z (arrow indicates site of cleavage), with x being a hydrophobic residue, usually l, although the amino acids i, m, v, and f are also found in this position (de vries et al., ) . at the p Ј position, z is usually a small uncharged residue such as s, a, g, or c (de vries et al., ) , with s being the most common residue at this position. data recently obtained for the avian infectious bronchitis virus (ibv), the human coronavirus (hcv- e), and mhv-a demonstrated that many of these predicted cleavage sites are functional. for all three strains, many of the cleavage sites in pp a have been identified, including a noncanonical lq n site that had not previously been predicted liu and brown, ; liu et al., ; ng and liu, ; piñó n et al., ; tibbles et al., ; ziebuhr et al., ; ziebuhr and siddell, ) . in addition, several of the processing sites in orf b have also been identified in both ibv and hcv- e (grötzinger et al., ; heusipp et al., a,b; liu et al., liu et al., , . however, for mhv, there has been greater difficulty in demonstrating processing by the clpro at any of the predicted orf b cleavage sites. in this study we demonstrate processing by the clpro at the putative orf b the following domains are shown: papain-like proteinase (plp- and plp- ), x domain (x), poliovirus c-like proteinase ( clpro), hydrophobic domains (hd and hd ), growth factor-like domain (gfl), rna-dependent rna polymerase (pol), zinc-finger domain (zn), and helicase (hel). the predicted clpro cleavage sites are indicated by the numbers and the cleavage site sequences from the p to the p Ј position are listed in table form (arrow indicates site of cleavage) (bonilla et al., ; bredenbeek et al., ; gorbalenya et al., ; lee et al., ). cleavage site situated between the putative polymerase (pol) and zinc finger (zn) domains (the pol zn site) (fig. ) . furthermore, identification of this site allowed us to make comparisons between the efficiencies of processing at this orf b site and a previously identified site in orf a located at the junction between hydrophobic domain (hd ) and the clpro (the hd c site). we have previously demonstrated processing at an orf a site in mhv-a , located at the junction between hd and the clpro (hd c), by a recombinant mhv-a clpro expressed as a fusion protein with the maltose binding protein (mbp) (piñó n et al., ) . the plasmid pet -nx. c c a, encoding the carboxy-terminal amino acids of hd and the inactivated clpro, is in vitro transcribed and translated to yield a -kda substrate nx. c (fig. a, lane ) . as previously demonstrated (piñó n et al., ), upon addition of the recombinant mbp- clpro, this substrate is efficiently processed into the -kda proteinase ( c) and the -kda hd -derived product (nx) (lane ). other studies using similar methods have led to the identification of orf a cleavage sites downstream of the clpro, including a previously unidentified, noncanonical lq n site . however, the demonstration of processing at orf b sites has proven to be difficult. our work with papain-like proteinase (plp- ) showed that substrate length, and possibly substrate conformation, played an important role in the ability of a substrate to be cleaved efficiently by the proteinase (teng et al., ) . we therefore created several substrates of various lengths, encoding different putative cleavage sites in orf b, in order to investigate processing by the recombinant mbp- clpro. of these, only the substrate expressed from pet -pol.zn, encoding mhv-a orf b amino acids l -q , proved to be useful in our investigations. in vitro transcription-translation of the construct pet -pol.zn resulted in the expression of a -kda full-length substrate, pol.zn (predicted molecular weight kda) (fig. b , lane ). upon incubation of this substrate with the recombinant mbp- clpro enzyme, cleavage between q and s , would give rise to two products, an n-terminal product with a predicted molecular weight (bonilla et al., ; bredenbeek et al., ; lee et al., ) . the positions of the catalytic residues, his and cys , are shown. pet -ha-hd . c encodes mhv-a amino acids from s to g directly downstream of the influenza hemagglutinin (ha) tag under the control of the t promoter (Á). pet -nx. c encodes mhv-a amino acids from k to q . pmal- c.wt encodes mhv-a amino acid sequences from s to g fused to the mal e gene encoding the maltose-binding protein (mbp). the construct pmal- c is used for the overexpression of the clpro in escherichia coli. (c) enlarged map of pol and zn region of orf b and schematic representation of plasmids derived from this region. the position of the q s cleavage site at the junction between pol and zn is shown. pet -pol.zn encodes mhv-a orf b amino acids from l to q under the control of the t promoter. of kda (pol) and a c-terminal product with a predicted molecular weight of kda (zn). in fig. b , lane , the addition of the recombinant mbp- clpro to the pol.zn substrate resulted in the production of a -kda protein which presumably corresponds to the n-terminal processing product. mutagenesis of q to k (lane ) or r (lane ) abolished this processing, suggesting that the cleavage is occurring at the predicted site and that, in view of the substrate specificity demonstrated, the observed processing event is in fact due to the action of the recombinant mbp- clpro. we were unable to detect the c-terminal -kda product by sds-page analysis. one explanation could be that p cannot be resolved from p in our gel system. (there have been previous reports of viral proteins migrating with electrophoretic mobilities different from that expected. the mhv-a clpro (p ) itself migrates with an electrophoretic mobility faster than its predicted molecular weight of kda (piñó n et al., ; lu et al., ) ). in addition, the predicted cleavage product p has approximately half the methionine content of p , which may contribute to the difficulty in its detection. the identification of these two cleavage sites, one in orf a (hd c) and one in orf b (pol zn), allowed us to further define the amino acids required for efficient processing by the clpro. for these experiments, we chose to use the pet -ha.hd . c construct, rather than other plasmids encoding clpro, because the ha.hd . c polypeptide can be efficiently cleaved both in cis and in trans and does not require membranes for its cleavage (piñó n et al., ) . several sets of mutations, from the p to the p Ј position, were introduced into the construct pet -ha.hd . c by pcr mutagenesis using the mutagenesis primers outlined in table . the effect of these cleavage site mutations on the autocatalytic cis release of the -kda clpro was assayed by the expression of the radiolabeled, in vitro transcribed, and translated substrate from pet -nx. c was incubated with mbp- clpro (lane ) or an equal volume of column buffer/ % glycerol (lane ) and the processed products were separated on a % sds-page gel. the arrows on the right of the panel indicate the electrophoretic migration of the -kda clpro-and the -kda hd -derived cleavage products (nx). (b) trans processing at the pol zn site. the plasmid pet -pol.zn was in vitro transcribed and translated. radiolabeled substrate was incubated either with mbp- clpro or with an equal volume of column buffer/ % glycerol (denoted by plus or minus signs above the lanes, respectively). processed products were analyzed on a % sds gel. the electrophoretic migration of p is indicated by an arrow on the right of the panel. the molecular weight in kilodaltons of prestained protein markers is indicated on the left of each panel. mutated substrates using in vitro transcription-translation, followed by sds-page analyses of the protein products (fig. ) . we observed that the l and q residues, at the p and p positions, respectively, were most sensitive to mutations. any mutation at either one of these positions inhibited the autocatalytic cis processing by the clpro (fig. , lanes - ). in contrast, mutations at any of the other positions studied were tolerated and the expression of precursor proteins harboring mutations at these sites still resulted in the autocatalytic release of the -kda clpro. exceptions are the s c substitution at position p Ј (lane ) and the g p substitution at position p Ј (lane ). these mutations also abolish the cis processing by the clpro. the effect of the g p mutation, however, is expected since the introduction of a p at this site could result in a drastic change in the conformation at the cleavage site. to investigate whether the cleavage sequence requirements for trans cleavage at the hd c site by the recombinant mbp- clpro parallel that observed for cis cleavage, the same set of cleavage site mutations were introduced into the construct pet -ha.hd . c c a, which also carries an inactivating mutation in the catalytic cysteine residue of the proteinase. the release of the -kda clpro from this precursor can only be accomplished by incubation with the recombinant mbp- clpro. figure showed that the effect of these mutations on trans cleavage paralleled the effects on cis cleavage. those mutations centering around the p and p positions of the cleavage site (l and q , respectively) affected trans processing the most. for the pol zn cleavage site in orf b, similar mutagenesis studies were conducted in order to determine the cleavage specificity requirements at this site (fig. ) . mutations from the p to the p Ј position were introduced into the plasmid pet -pol.zn. the mutant substrates were expressed using an in vitro transcriptiontranslation system and then incubated with the recombinant mbp- clpro. the effect of the mutations on the production of p was assayed by sds-page and com-pared to the processing of the wildtype pol.zn substrate (fig. , lanes and ) . the results were similar to that observed with the orf a hd c site in that the l and q residues, at the p and p positions respectively, were most sensitive to mutations. the l i mutation (fig. , lanes and ) resulted in the inhibition of p processing, indicating the sensitivity of this site to a conservative change. however, the l m substitution (fig. , lanes and ) did not abolish processing of p . mutation of q to either k (lanes and ) or r (lanes and ) abolished processing at this site. substitution of s with an a (lanes and ) was tolerated, whereas mutating s to the bulkier n (lanes and ) resulted in the inhibition of p processing. the results of mutagenesis studies on both the hd c and pol zn cleavage sites demonstrate that the p and p positions at the cleavage site are the primary determinants of cleavage specificity by the clpro in both cis and trans processing. we investigated the efficiency of processing at the orf a site compared to the orf b site. the substrates, nx. c and pol.zn, were incubated with a fixed amount of recombinant proteinase for increasing periods of time. we observed that the orf a substrate, nx. c, is readily processed within h, with the substrate completely converted into product by h (fig. a) . processing of the orf b substrate, however, is not observed until after h of incubation with the recombinant proteinase. furthermore, cleavage of pol.zn occurs at a much lower level and does not reach completion even after h (fig. b ). this inefficiency of processing of the pol zn site compared to the hd c site may be explained in two ways. first, although the primary sequences of the two sites reveal no obvious reason why one is processed more efficiently than the other, it is possible that the subtle differences in the sequences of the two sites are enough to make the hd c site a more efficient substrate than the pol zn site. alternatively, the difference may not be inherent to the primary sequence of the cleavage sites, but rather to the conformation of the entire substrate as a whole. it is possible that the substrate conformation of nx. c allows the recombinant proteinase better access to the cleavage site. in the pol.zn substrate, the cleavage site may be more obscured, and in this manner the virus regulates both when and how much of its encoded proteins are produced. to determine whether the difference in processing efficiencies at the hd c and pol zn sites can be attributed to the primary sequences of the cleavage sites, we synthesized a -mer peptide, representing the p -p Ј residues of the hd c cleavage site, and a -mer peptide, representing the p -p Ј amino acids of the pol zn cleavage site. each peptide was then incubated with the recombinant mbp- clpro and the resulting cleavage products were separated from each other and from the substrate by reverse-phase chromatography. for the orf a peptide, reverse-phase chromatography of the reaction at zero time resulted in a single peak, representing the peptide substrate, in the elution profile (fig. a) . incubation with mbp- clpro followed by chromatography resulted in two additional peaks, representing the cleavage products, and a reduction in the fig. . hd c cleavage site mutagenesis: trans processing at the hd c site. the same cleavage site mutants used in the study of cis processing were introduced into pet -ha.hd . c c a, which also harbors a mutation at the catalytic cysteine residue of the proteinase. equivalent counts per minute of radiolabeled substrates expressed from these mutant plasmids by tnt were incubated with mbp- clpro or an equivalent volume of column buffer/ % glycerol (ϩ/Ϫ mbp- clpro). cleavage products were then separated on a % sds-gel. the electrophoretic migration of p is indicated by an arrow on the right. molecular weight markers are indicated on the left. substrate peak. microsequencing of the cleavage products confirmed that cleavage had occurred between q and s . for the orf b peptide similar results were observed in that the peptide substrate eluted as a single peak at zero time (fig. b) . incubation with mbp- clpro prior to separation resulted in two new peaks, representing the two cleavage products. a reduction in the substrate peak was also observed. again, the authenticity of cleavage of the synthetic peptide was confirmed by microsequencing of the cleavage products, which showed that cleavage had occurred between q and s . the level of cleavage of the orf a peptide was not any different from, and in some cases was less than, that of the orf b peptide. we observed that under identical reaction conditions no more than % of the orf a peptide was cleaved by the mbp- clpro, whereas with the orf b peptide the level of cleavage was between and %. in order to allow direct comparison between the cleavage efficiencies of the nx c and pol zn sites, we determined the k cat and k m values for the reactions with the synthetic peptides described above. results show that cleavage of the orf a peptide by the recombinant mbp- clpro yielded a k cat of . s Ϫ , and a k m of . Ϯ . mm. with the orf b peptide the k cat was slightly slower, with a value of . s Ϫ , and the k m was calculated at . Ϯ . mm. our results therefore show that cleavage (k cat ) of the orf a peptide occurred at only a slightly higher rate ( . -fold) than that of the orf b peptide. the lack of substantial difference between the k cat values is not surprising given that the sequences of the peptides are very similar. interestingly, when the catalytic efficiency (k cat /k m ) was taken into consideration, the orf b peptide (k cat /k m ϭ . ϫ Ϫ m Ϫ s Ϫ ) was a slightly better substrate than the orf a peptide (k cat /k m ϭ . ϫ Ϫ m Ϫ s Ϫ ). taken together, the peptide cleavage results presented here suggest that the cleavage efficiencies of the two peptides are similar. the action of viral-encoded proteinases is essential to viral replication (dougherty and semler, ) . this makes viral-encoded proteinases potentially good targets for antiviral drugs. in the murine coronavirus, two such proteinases are under continued investigation in order to better understand the manner in which these proteinases function. papain-like proteinase (plp- ) fig. . pol zn cleavage site mutagenesis. recombinant mbp- clpro was used in posttranslational trans cleavage assays with radiolabeled substrates generated from pet -pol.zn that encoded a wildtype cleavage sequence or harbored mutations around the pol zn site. substrate volumes containing equivalent counts per minute were incubated with mbp- clpro (denoted by plus sign above the lanes) or an equivalent volume of column buffer/ % glycerol (denoted by a minus sign above the lanes). the electrophoretic migration of p is indicated by the arrow to the right of the panel. molecular weight markers are indicated on the left. has been linked to the processing of several nonstructural proteins encoded in the Ј end of the viral genome (baker et al., ; bonilla et al., ; denison et al., ; hughes et al., ) . these cleavage products, p and p , are not yet linked with any known viral function. however, those viral proteins with presumed functions in viral replication and viral rna transcription, such as the rna-dependent rna polymerase (pol) and the zinc-finger/helicase (zn-hel) proteins, are believed to be processed by the c-like proteinase of the virus. the clpro is predicted to cleave at, at least, sites in pp ab. many of the cleavage sites located in the a region of pp ab have been shown to be functional cleavage sites and processing by the clpro at these sites has been demonstrated in the coronaviruses ibv (liu et al., ; liu and brown, ; ng and liu, ; tibbles et al., ) , hcv- e (ziebuhr et al., ; ziebuhr and siddell, ) , and mhv-a piñó n et al., ) . some of the mature viral products resulting from these cleavages have also been identified in infected cells (liu et al., ; lu et al., ; ng and liu, ; piñó n et al., ; ziebuhr and siddell, ; denison et al., ) . according to computer predictions, further processing at the q s , q c , q s , and q a sites located in the b region of mhv-a pp ab would result in mature viral products of , , , , and kda, respectively, corresponding to pol, zn-hel, and the three c-terminal-most cleavage products. in hcv- e, viral products of , , and kda, corresponding to the pol, zn-hel, and the second c-terminal-most proteins, have been identified in infected cells and the role of the clpro in the processing of these products has been authenticated in vitro (grötzinger et al., ; heusipp et al., a,b) . similarly, in the case of ibv, viral proteins of , , and kda have been identified in infected cells (corresponding to pol and the two c-terminal-most proteins) and cotransfection experiments have implicated the clpro in the processing of these viral products (liu et al., . for mhv-a , however, demonstration of processing at any mhv cleavage site in the b region of pp ab has lagged behind that of hcv and ibv. here we report the first demonstration of processing at the site between pol and zn in pp ab by the mhv-a clpro. the cleavage at the pol zn site is highly inefficient compared to the processing observed at the hd c site, as evidenced by the time course assays illustrated in fig. . phosphorimager analyses indicate that the pol.zn substrate is cleaved fivefold less efficiently than the nx. c substrate (data not shown). the differences in these efficiencies, however, could not be explained by the subtle differences in the primary sequences of the cleavage sites alone. in fact, when presented to the enzyme in the context of a -or -mer peptide substrate, there was no substantial difference in cleavage efficiency between these two peptides, an observation that would seem to contradict the results obtained with the larger polyprotein substrates. recently, ziebuhr and siddell ( ) investigated the efficiency of processing at several hcv- e clpro sites located at the c-terminus of pp a or the central region of pp ab. they observed that several viral products were produced less efficiently than others, with reduced cleavage activity at two orf a sites, between v-q s and l-q n , compared with cleavage at the sites flanking the clpro domain. the corresponding lq n site in mhv-a has also been shown to be less effi-ciently cleaved than the lq s sites flanking the clpro domain . in the case of l-q n in hcv- e, additional peptide cleavage data demonstrated that the properties of the cleavage sequence itself, rather than the overall conformation of the polypeptide and the accessibility of the cleavage site, contribute to the observed inefficiency of processing at this site (ziebuhr and siddell, ) . while our results do not contradict theirs, they do demonstrate that, in vivo, the conformation of a larger polypeptide substrate is likely to be as important a determinant of cleavage as is the primary structure and sequence of the cleavage site. our results clearly show that, at least with the case of the pol zn site of mhv-a , the observed inefficiency of cleavage was likely due to the overall conformation of the polypeptide, which may directly translate into the accessibility of the cleavage site, rather than the primary sequence of the site. taken together, both sets of results highlight important regulatory mechanisms employed by the virus to coordinate the temporal production and the accumulation of the various replicase proteins. thus, the very slow in vitro processing at the orf b cleavage site, compared with that at the orf a site, generally correlates with the levels of orf a and orf b polypeptides found in infected cells. however, there are inherent differences between in vitro cleavage reactions with either recombinant proteins or peptide substrates and in vivo processing; these include the lengths of the substrates, the concentration of the enzyme and substrates, as well as the subcellular localization of replication complexes in vivo (denison et al., ; ziebuhr and siddell, ) . these differences may all contribute to the extended lengths of time necessary for in vitro cleavages. the substrate specificity of the coronavirus clpro has been determined mainly through the identification of functional cleavage sites and a visual inspection of these cleavage site sequences. mutagenesis has been done primarily to verify the authenticity of the cleavage site and most mutagenesis studies have not extended beyond the q residue that is absolutely conserved at the p position in all clpro cleavage sites identified to date. we have extended our mutagenesis studies to cover the p to p Ј positions of the cleavage site sequence. our results demonstrated that the substrate sequence specificity of the clpro is primarily influenced by the amino acid residues present at the p and p positions of the cleavage site. substitution of the q residue at the p position with any other amino acid has resulted in substrates that could not be cleaved by the clpro, demonstrating the importance of this residue in the substrate. we could not detect any processing in substrates containing mutations at this position (figs. - ) . the p position of the cleavage site is most often occupied by an l; however, in some cleavage sites identified in hcv- e, this position is occupied by a v or an i (grötzinger fig. . chromatograms of cleavage products of the synthetic peptides corresponding to orf a amino acids and orf b amino acids. a and b show chromatograms of orf a peptide ( mm) and orf b peptide ( mm), respectively, at h and after h of incubation with mbp- clpro ( . m enzyme) at °c. in both panels the chromatograms for t ϭ h are offset to allow comparison between the two time points. the asterisks (*) indicate the cleavage products that were used in peptide sequencing in order to confirm the sites of cleavage. ziebuhr and siddell, ) , suggesting that this position is not as strictly conserved as the p position and may thus be able to tolerate some mutations. however, our results demonstrated otherwise, in that even a conservative change to i resulted in a reduction of cleavage , to below %, as measured by phosphorimager analysis (data not shown). this suggests that perhaps an i at the p position of the cleavage site can be functional only when compensated for elsewhere in the substrate sequence. the only change tolerated at this position was a change to m in the p position at the pol zn site (fig. ) . interestingly, the jhm strain of mhv encodes an m instead of an l at this position (lee et al., ) . thus such a mutation resulted in a wildtype jhm pol zn site and a cleavage efficiency equivalent to wildtype levels. this provides indirect evidence that the pol zn site in jhm is a functional clpro site. although the s found at the p Ј position of both the hd c and pol zn sites is not as sensitive to mutations, we observed that some mutations, such as s c (in the hd c site) (fig. ) and s n (in the pol zn site) (fig. ) , are not tolerated. additionally, both s a and s a substitutions, though tolerated, resulted in a reduction, but not complete inhibition of cleavage , suggesting that the residue at the p Ј position also plays a role in substrate recognition, although to a lesser extent than those at the p and p positions. aside from the amino acid residues at the p , p , and p Ј positions, the amino acid sequences surrounding the scissile q s(a,g) peptide bonds that are recognized by the clpro do not share any other significant primary structure similarity. furthermore, the presence of an lqa tripeptide not cleaved by the proteinase would again suggest the existence of a common conformational determinant shared by all clpro susbstrates that is necessary for clpro-mediated processing. the pol zn site is the first functional cleavage site identified in the orf b region of the mhv-a pp ab. further work remains to be done in order to identify other functional cleavage sites in orf b. additionally, the mature viral products resulting from these processing events remain to be identified in infected cells. a direct comparison of the cleavage efficiencies of each site will help to elucidate the complex posttranslational processing pattern of the mhv-a gene polyprotein, as well as provide insight into the regulatory mechanisms employed by the virus to maintain the production of its proteins under control. the parental plasmids used in this study are illustrated in fig. . the plasmids pet -nx. c, pet -ha.hd . c, and pmal- c.wt have all been described elsewhere (piñó n et al., ) . pet -nx. c encompasses mhv-a nucleotides - , encoding the last amino acids of hd and the entire -aminoacid region encompassing the clpro (from k to q ). pet -nx. c c a is the same as pet -nx. c but carries the inactivating c a mutation in the catalytic cysteine residues of the proteinase. the plasmid pet -ha.hd . c encodes mhv-a nucleotides - , encoding hd and the clpro from s to q , directly behind the influenza hemagglutinin (ha) epitope under the control of the t promoter in pet a. the plasmid pet -ha.hd . c c a contains the clpro inactivating mutation c a in the background of the parental pet -ha.hd . c plasmid. the plasmid pmal- c.wt encodes mhv-a nucleotides - , corresponding to the clpro region from s to q , behind the mal e gene in the pmalc vector (new england biolabs). this plasmid encodes the clpro domain fused to the coding sequence of the maltose binding protein and is used for overexpression of the recombinant mbp- clpro enzyme. a region of mhv-a gene , from nucleotides to , corresponding to orf b amino acids l to q , was pcr amplified from a plasmid encoding the entire orf b sequence using the primers f bp - ( Ј-ttcgaattccccgggggatcccttatggcatg-caatggacac- Ј) and r bp - ( Ј-cgaattc-ctctagaaagcttgctgaaacgtctcaggcacact- Ј). the resulting pcr fragment was digested with bamhi and hindiii (denoted by the underlined sequences in the primers) and cloned into the corresponding sites of pet a, resulting in the plasmid pet -pol.zn. the pet -ha.hd . c cleavage site mutants l i, l s, q k, q r, and s a were created by two rounds of pcr as described previously hughes et al., ) using the fmp and rmp primers listed in table and the primers fij ( Ј-tg-gcttgtcatgtatggtgc- Ј) and rsp - ( Ј-aacatatcctacagaacc- Ј). the resulting mutant fragments were digested with kpni and bamhi and cloned into the same sites in pet -ha.hd . c. all other cleavage site mutants in pet -ha.hd . c were created using the quikchange mutagenesis kit (stratagene) following the manufacturer's protocols. the mutagenic primers used are listed in table . following pcr amplification, the amplified plasmids were digested with the restriction enzyme dpni, which digests methylated and hemimethylated dna, thus destroying the parental plasmid and any hybrids containing one parental strand and one mutated strand. escherichia coli xl -blue supercompetent cells (stratagene) were then transformed with the mutated plasmids. the presence of the desired mutation was verified by sequencing. the fragments containing the hd . c cleavage site mutations were also subcloned into pet -ha.hd . c c a using the ndei and bamhi sites in order to create plasmids carrying both the inactivating c a mutation and mutations at the cleavage site. these plasmids were used to express substrates used in trans cleavage assays. all pet -pol.zn cleavage site mutants v s, l i, l m, q k, q r, s a, and s n were created using the quikchange mutagenesis kit as described above. the primers used in creating these mutants are also listed in table . cell-free expression of plasmid dnas was carried out using the tnt rabbit reticulocyte lysate-coupled transcription-translation system (promega) at °c for h, as previously described (piñó n et al., ) . the incorporation of [ s]methionine into acid precipitable counts was used as an indicator of protein synthesis. equivalent amounts of acid precipitable counts were directly analyzed by sds-polyacrylamide gel electrophoresis (sds-page) or used in posttranslational proteolytic assays as indicated. radioimmunoprecipitations were carried out as described previously denison et al., ) . expression of the recombinant clpro from pmal- c.wt (neb), which expresses clpro as an mbp- cl fusion protein, and the purification of the fusion protein was carried out according to the manufacturer's protocol and as described by herold et al. ( ) . briefly, e. coli tb cells transformed with pmal- c.wt were grown at °c in the presence of ampicillin ( g/ml) until the a nm reached . , at which point the cells were induced with isopropylthio-␤-d-galactoside at a final concentration of . mm for h at °. cells were harvested and then resuspended in ml of column buffer [ mm tris-cl (ph . )], mm nacl, mm edta, mm dtt] per gram of cells. the cell suspension was then lysed by sonication. cell debris was pelleted by centrifugation at g for min. the crude lysates were diluted : in column buffer and then loaded onto an amylose column (bed volume ml), preequilibrated with column buffer, at a flow rate of ml/min. the column was then washed with column volumes of column buffer and the mbp- clpro fusion protein was eluted with column buffer containing mm maltose. fractions of ml were collected and those containing the -kda recombinant mbp- clpro were identified by analyzing -l aliquots by sds-page. fractions containing the recombinant proteinase were pooled and the concentration of the fusion proteinase was determined using the bradford assay against known concentrations of bovine serum albumin. the recombinant proteinase was stored at Ϫ °c in column buffer supplemented with % glycerol. radiolabeled substrates containing the cleavage sequences of hd . c or pol.zn (fig. ) were generated using the tnt rabbit reticulocyte lysate system. lysate volumes containing equivalent counts per minute were incubated with approximately - g of recombinant proteinase or an equivalent volume of column buffer/ % glycerol at °c for - h or, where applicable, the specified lengths of time. the processed products were analyzed by sds-page followed by autoradiography. phosphorimager analysis was carried out as previously described teng et al., ) . recombinant mbp- clpro enzyme ( . mg/ml in elution buffer supplemented with % glycerol and mm dtt, . m enzyme) was incubated with synthetic peptide orf a ( . to . mm in % dmso), with the sequence h n-thr-thr-ser-phe-leu-gln ser-gly-ile-val-lys-met-val-ser-cooh, corresponding to orf a amino acids to (arrow indicates cleavage site) or orf b peptide ( . to . mm in % dmso), with the sequence h n-arg-ser-ala-val-leu-gln ser-val-gly-ala-cys-val-val-cys-ser-cooh, corresponding to orf b amino acids to (arrow indicates cleavage site) in a final reaction volume of l. the reactions were allowed to proceed at °c for - min, at which time the reactions were quenched by addition of trichloroacetic acid (tca) to % final concentration. the samples were then chilled on ice, and the denatured protein was precipitated by centrifugation. for zero time point samples, the enzyme was mixed with tca prior to the addition of peptide substrates and the reactions were carried out as described above. separation of cleavage product from substrate was carried out with the Ä ktapurifier system (amersham pharmacia biotech) equipped with a sephasil peptide c -m st . / reverse-phase column (amersham pharmacia biotech). elution was performed with a linear gradient of % eluent a ( . % aqueous tfa)/ % eluent b ( . % tfa in % acetonitrile/ % water) up to % eluent b (seybert et al., ) over min (flow rate . ml/min, detection at nm). with the orf a peptide the level of cleavage was no more than %, whereas with the orf b peptide the level of cleavage was between and %. the data obtained were fitted to the michaelis-menten equation and the k cat and k m values were obtained using kaleidagraph . (synergy software). identification of a domain required for autoproteolytic cleavage of murine coronavirus gene a polyprotein mouse hepatitis virus strain a rna polymerase gene orf a: heterogeneity among mhv strains characterization of the leader papain-like proteinase of mhv-a : identification of a new in vitro cleavage site completion of the sequence of the genome of the coronavirus avian infectious bronchitis 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 an efficient ribosomal frame-shifting signal in the polymerase encoding region of the coronavirus ibv nidovirales: a new order comprising coronaviridae and arteriviridae identification and characterization of a -kda protein processed from the gene polyprotein of the murine coronavirus mhv-a 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 intracellular processing of the n-terminal orf a proteins of the coronavirus mhv-a requires multiple proteolytic events identification of polypeptides encoded in open reading frame b of the putative polymerase gene of the murine coronavirus mouse hepatitis virus a the genome organization of the nidovirales: similarities and differences between arteri-, toro-, and coronaviruses expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes complete sequence ( kb) of the polyprotein-encoding gene of transmissible gastroenteritis virus coronavirus genome: prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis characterization of a -kda polypeptide encoded in gene of the human coronavirus hcv e nucleotide sequence of the human coronavirus e rna polymerase locus an "elaborated" pseudoknot is required for high frequency frameshifting during translation of hcv e polymerase mrna characterization of coronavirus rna polymerase gene products identification and subcellular localization of a kda polyprotein ab processing product in human coronavirus e-infected cells identification of an atpase activity associated with a -kilodalton polypeptide encoded in gene of the human coronavirus e coronaviridae: the viruses and their replication pathogenesis of mouse hepatitis virus-induced demyelination identification of the murine coronavirus p cleavage site coronavirus protein processing and rna synthesis is inhibited by the cysteine proteinase inhibitor e d coronavirus: organization, replication and expression of genome the complete sequence ( kilobases) of murine coronavirus gene encoding the putative proteases and rna polymerase a -kilodalton polypeptide encoded by open reading frame (orf) b of coronavirus infectious bronchitis virus is processed by orf a products characterization and mutational analysis of an orf a-encoding proteinase domain responsible for proteolytic processing of the infectious bronchitis virus a/ b polyprotein proteolytic mapping of the coronavirus infectious bronchitis virus b polyprotein: evidence for the presence of four cleavage sites of the c-like proteinase and identification of two novel cleavage products proteolytic processing of the coronavirus infectious bronchitis virus a polyprotein: identification of a -kilodalton polypeptide and determination of its cleavage sites identification and characterization of a serine-like proteinase of the murine coronavirus mhv-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 identification of a -kda polypeptide processed from the coronavirus infectious bronchitis virus a polyprotein by the c-like proteinase and determination of its cleavage sites efficient autoproteolytic processing of the mhv-a c-like proteinase from the flanking hydrophobic domains requires membranes expression and characterization of a recombinant murine coronavirus c-like proteinase expression, purification, and activity of recombinant mhv-a clpro expression of murine coronavirus recombinant papain-like proteinase: efficient cleavage is dependent on the lengths of both the substrate and the proteinase polypeptides characterization in vitro of an autocatalytic processing activity associated with the predicted c-like proteinase domain of the coronavirus avian infectious bronchitis virus proteolytic processing of the open reading frame b-encoded part of the arterivirus replicase is mediated by nsp serine protease and is essential for virus replication characterization of a human coronavirus (strain e) c-like proteinase assay processing of the human coronavirus e replicase polyproteins by the virus-encoded c-like proteinase: identification of proteolytic products and cleavage sites common to pp a and pp ab the authors thank ravi mayreddy for the construction of the orf bencoding plasmids. peptide sequencing was provided by the protein chemistry laboratory of the school of medicine (university of pennsylvania), supported by core grants of the diabetes and cancer centers (dk- and ca- ). this work was supported by nih grant ai- . key: cord- -us dybue authors: kanjanahaluethai, amornrat; chen, zhongbin; jukneliene, dalia; baker, susan c. title: membrane topology of murine coronavirus replicase nonstructural protein date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: us dybue mouse hepatitis virus (mhv) is a member of the family coronaviridae. these positive strand rna viruses encode a replicase polyprotein that is processed into nonstructural proteins (nsps). the nsps assemble with membranes to generate double membrane vesicles, which are the sites of viral rna synthesis. mhv nsp contains multiple domains including two papain-like protease domains, plp and plp , and a predicted transmembrane (tm) domain. in this study, we determined the membrane topology of nsp -tm and showed that tm-mediated tethering of plp is important for processing at cleavage site . biochemical analysis revealed that nsp is an integral membrane protein that is inserted into endoplasmic reticulum (er) membranes co-translationally and glycosylated at asparagine- . proteinase k digestion experiments indicate that the tm domain of nsp has membrane-spanning helices. we show that nsp -tm is sufficient to mediate er membrane association of a cytosolic protein. this study is the first detailed analysis of the topology and function of the coronavirus nsp tm domain. proteolytic processing of a replicase polyprotein and the generation of a membrane-associated replication complex are common themes in studies analyzing the replication of positive strand rna viruses. for the majority of coronaviruses, the proteolytic processing of the viral replicase polyprotein is mediated by three distinct viral proteinases to generate replicase products [reviewed in (ziebuhr, ) ]. both coronavirus and the related arterivirus replicase products have been shown to assemble with cellular membranes to generate double-membrane vesicles that are the sites of viral rna synthesis (goldsmith et al., ; gosert et al., ; pedersen et al., ; snijder et al., ) . the goal of our research is to characterize the coronavirus replicase proteolytic processing cascade and identify factors that mediate membrane-association of the replication complex. our model system is the replication of mhv, one of the prototype coronaviruses. mhv is a ∼ . -kilobase (kb) positivestrand rnavirus that replicates in the cytoplasm of infected cells. the ′-most kb of the mhv genomic rna contains two large open reading frames (orfs), termed orf a and orf b (lee et al., ) . during translation of the genomic rna, the two orfs are joined via a ribosomal frameshifting mechanism to produce a polyprotein of ∼ kilodaltons (kda) in size (brierley et al., ; lee et al., ) . this polyprotein is the viral rnadependent rna polymerase, termed the replicase. the mhv replicase is processed by three distinct proteases contained within orf a of the replicase polyprotein. two of the proteases are papain-like cysteine proteases, termed plp and plp . the third protease domain is distantly related to the picornavirus family of c-like proteases, and is termed clpro. these three proteases process the replicase polyprotein to produce intermediates and products that function in the replication of genomic rna and the synthesis of a nested set of subgenomic mrnas [reviewed in (brian and baric, ; sawicki and sawicki, ) ]. studies have shown that the replicase products alone are sufficient to mediate viral rna synthesis , and to generate the double-membrane structures that serve as the sites for viral rna synthesis (pedersen et al., ) . however, the role of proteolytic processing in regulating the assembly and function of the mhv replication complex is not yet understood. therefore, we wanted to identify regions of the replicase polyprotein that may direct or regulate proteolytic processing and/or membrane association. the focus of this study was to identify the regions of mhv nsp that direct membrane association and plp activity. previously, we showed the mhv plp can act in trans to process cleavage site (cs ) at the nsp /nsp junction (kanjanahaluethai and baker, ; kanjanahaluethai et al., ) . other coronaviruses, such as the human coronavirus e, have been shown to utilize plp (also termed pl pro) to process sites both upstream and downstream of the catalytic domain (ziebuhr et al., ) . the papain-like protease (plpro) encoded by the coronavirus that causes severe acute respiratory syndrome (sars-cov) processes three sites in the replicase polyprotein (harcourt et al., ) , and has recently been shown to have de-ubiquitinating activity (barretto et al., ; lindner et al., ) . the crystal structure of this enzyme has been resolved and is currently being targeted for anti-viral drug development (ratia et al., ) . a better understanding of the coronavirus papain-like proteases may facilitate anti-viral drug development for sars and also other recently identified human coronavirus infections caused by nl (van der hoek et al., ) and hku (woo et al., ) , which can cause pneumonia and respiratory tract infections in children and the elderly. the aims of this study were to determine the topology of mhv nsp and to identify the regions in nsp required for plp activity. we used a trans-cleavage assay to determine if an expressed plp domain was sufficient for the recognition and processing of a substrate containing cs . we found that constructs containing plp and the downstream putative tm domain were able to efficiently process the substrate. bioinformatic analysis of the nsp -tm domain indicated the presence of putative membrane-spanning helices and consensus sites for n-linked glycosylation. site-directed mutagenesis of the asparagine residues and analysis of endoglycosidase h (endo h) sensitivity revealed that asparagine- in nsp -tm is glycosylated. to investigate the topology of nsp -tm, we tested for sensitivity to digestion with proteinase k and found that at least two lumenal domains are protected, consistent with membranespanning regions in nsp -tm. we showed that nsp -tm alone is sufficient to confer membrane association to a normally cytosolic protein, enhanced green fluorescent protein (egfp), and that a predicted multi-spanning tm domain is conserved in nsp of all coronaviruses. thus, the nsp -tm domain is important for membrane association of the replicase and tethering the plp domain for viral polyprotein processing activity. the mhv nsp -tm domain is important for plp processing at cleavage site analysis of sars-cov plpro activity showed that the nsp hydrophobic domain downstream of plpro is essential for processing at cs (harcourt et al., ) . to determine if a similar domain is important for mhv plp activity, we generated a series of plp c-terminal deletion constructs and tested each construct for expression and processing activity. plp expression constructs were generated by polymerase chain reaction (pcr) amplification and cloning of the amplified region into pcdna . /v -his, as described in materials and methods. to determine if each clone was expressing a protein of the expected size, we analyzed the products after t -mediated expression and immunoprecipitation (fuerst et al., ; kanjanahaluethai and baker, ) . we detected the expected series of truncated plp proteins that ranged in size from ∼ kda to ∼ kda (fig. b, lanes - ) . to determine if these plp products were sufficient to mediate processing of cs , we tested each construct in the trans-cleavage assay by co-transfection with the substrate (fig. c, lanes - ) . we found that only two of the plp expression products, pplp - and pplp - , were able to efficiently process the substrate and produce the -kda cleavage product, nsp (fig. c, lanes and ). these two constructs encompass all or a major part of the predicted nsp -tm domain indicating that membrane tethering of plp is important for recognition and processing at cs . thus, both sars-cov plpro (harcourt et al., ) and mhv plp require the downstream tm domain for recognition and processing at the nsp /nsp junction. bioinformatic analysis of nsp -tm predicts a series of putative membrane-spanning sequences previously, we showed that mhv nsp is indeed an integral membrane protein, but the role of the tm in mediating this membrane association was not investigated (gosert et al., ) . initial bioinformatic analysis indicated two transmembrane helices in nsp (ziebuhr et al., ) . to extend these studies of membrane association of coronavirus replicase products, we analyzed the amino acid sequence of mhv-jhm nsp (from glycine- to glycine- ) for probability of transmembrane helices using the five different programs designed to search for putative membrane-spanning sequences: phobius, tmhmm, hmmtop, sosui and tmpred (fig. ) . interestingly, each program generated a unique prediction for the topology of nsp (fig. c ). the number of predicted membrane-spanning domains varied from three (phobius) to seven (tmpred). however, since both the n-and c-termini of nsp are cleaved in the cytosol, the number of membrane-spanning helices must be either two [as previously predicted (ziebuhr et al., ) ], four or six. to better understand the topology of the nsp -tm domain, we performed membrane-association, fractionation and proteinase k protection experiments. to determine if the nsp -tm is indeed required for membrane association, we performed in vitro transcription and translation of the plp expression constructs in the absence or presence of canine microsomal membrane (cmm) and assayed for membrane association. the newly translated proteins were metabolically radiolabeled with [ s]-translabel, subjected to centrifugation to separate the membrane-associated pelleted fraction from the soluble fraction. protein products of both fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page), and visualized by autoradiography (fig. ) . the percentage of total proteins detected in soluble and pelleted fractions was quantitated by phosphoimaging analysis. in the absence of cmm, the translated protein products were detected predominantly in the soluble fraction (fig. a) . in contrast, when cmms were added to the mixture, protein products that included all or part of nsp -tm (plp - , - and - ) were detected predominantly in the pelleted fraction, consistent with membrane association (fig. b ). to determine if the membrane association occurred co-translationally or post-translationally, we added the cmms after termination of translation, and assessed membrane association. previous studies with hepatitis c virus revealed post-translational insertion of the ns a replicase protein (brass et al., ) . we found that the translation product of plp - is inserted co-translationally, with no detectable post-translational insertion into membranes (fig. c ). these results indicate that mhv nsp membrane insertion likely occurs via the normal, signal recognition particle (srp)-dependent er translocation mechanism, and not via a tail-anchoring or post-translational mechanism. to distinguish between membrane association versus integral membrane insertion, membrane extraction experiments were performed. protein products expressed from pplp - , pplp - and pplp - constructs were translated in vitro in the presence of cmm and the pelleted fraction was subsequently subjected to differential extraction methods as indicated in fig. . as expected, treatment with nte buffer alone had no effect and the proteins pelleted with membranes, whereas treatment with detergent triton x- disrupted the membranes, and the proteins were detected in the soluble fraction (fig. a) . treatments with urea, nacl or sodium carbonate, ph . have been shown to disrupt the association between peripheral, membrane-associated proteins, but such treatments do not disrupt integral membrane proteins (bordier, ) . we tested for disruption of plp -tm proteins and found that majority of the proteins remained in the pelleted membrane fraction, consistent with integral membrane proteins (fig. b ). we noted that the protein product expressed from pplp - , which contains only one or two predicted membrane spanning helices, was dissociated by treatment with high ph, but that the protein products expressed from constructs that extended further into the tm were not disrupted from the membranes by these treatments, consistent with the characteristics of integral membrane proteins. bioinformatic analysis also revealed two consensus sequences for n-linked glycosylation [the consensus being nx(t/s), where x is any amino acid (helenius and aebi, ) ] in nsp -tm. if one or more of the predicted sequences was indeed a transmembrane helix, then the sites for n-linked glycosylation could be lumenal, and subject to modification. to determine if either of the putative sites was modified by glycosylation, we the amino acid numbering is according to ncbi accession number nc . conserved domains previously identified by comparative sequence analysis (ziebuhr et al., ) and the recently described adp-ribose- ʺ-phosphatase (adrp) domain (saikatendu et al., ) are indicated. two putative sites for n-linked glycosylation were predicted at asparagine residues and . (b) the diagram shows the results of an analysis of the nsp for predicted transmembrane helices using tmhmm program. the predicted tm domain designates the boundaries of the putative membrane-spanning regions a, b, c, d, and e. (c) sequence of the plp -tm (amino acid residues to ) were analyzed for the prediction of transmembrane domains using phobius, tmhmm, hmmtop, sosui and tmpred programs. subjected the proteins expressed from wild-type pplp -cen (kanjanahaluethai and baker, ) and two asparagine-toalanine substitution mutants to endo h treatment (fig. ) . expressed proteins were radiolabeled with [ s]-translabel, lysates were prepared, and subjected to immunoprecipitation with anti-v antibody as described in materials and methods. the plp -cen protein was either untreated, or treated with endo h for h, then analyzed for mobility by electrophoresis through a % polyacrylamide gel. we found that the untreated wild-type protein migrated at approximately kda (lane ). however, after digestion with endo h, the protein migrated at kda (lane ), consistent with the loss of one n-linked modification. the plp -cen-n a protein with an asparagine-to-alanine substitution migrated more quickly than the wild type or n a protein, indicating that n is the site modified by n-linked glycosylation (compare lanes - ). furthermore, proteins generated by endo h treatment of the pplp -cen-n a and pplp -cen-n a migrated more quickly than wild type plp -cen (lanes - ). these results indicate that nsp is glycosylated at asparagine- . overall, these experiments demonstrate that nsp -tm does have transmembrane and lumen sequences that can tether the plp domain to intracellular membranes. to further investigate the topology of nsp -tm, we tested for sensitivity to proteinase k digestion. cytosolic domains are sensitive to proteinase k, whereas transmembrane and lumenal domains are protected from digestion with proteinase k. analysis of plp - revealed two major fragments of kda and kda protected from proteinase k digestion (fig. , lane ) . we noted the predicted size of the tm domain from k -y is . kda, and the predicted size of the k -i (a and b helices) is . kda. analysis of plp - revealed two protected fragments of kda and kda, respectively (fig. , lane ) . the predicted size of the k -s fragment is . kda. by combining the results of the integral membrane assays (figs. and ) , the glycosylation assay (fig. ) , and the proteinase k sensitivity assay (fig. a ), we were able to generate a topology model for nsp -tm that is consistent with all the data (figs. b and c). our results indicate that nsp -tm has four membrane-spanning sequences and two lumenal domains (fig. b ) and that the kda fragment likely represents the proteinase k resistant fragment generated by the first two membrane-spanning sequences. our model is most similar to the predicted models generated by the tmhmm and sosui programs with the exception that the fourth predicted membrane-spanning sequence (which is the only domain not consistent in these two predictions) is not a membrane-spanning sequence, but instead remains lumenal, and the final membranespanning domain is in the reverse orientation. our results, and the results of others (hugle et al., ; miller et al., ) , demonstrate the importance of experimental validation of bioinformatics predictions of membrane-spanning sequences. for example, in the case of dengue virus type nonstructural protein b, miller and co-workers found that two computerpredicted transmembrane helices were in fact lumenal and one was glycosylated (miller et al., ) . in addition, we note the value of using multiple programs to better estimate the complexity of the bioinformatic prediction. the differences in the predicted models could then be tested experimentally. here, we provide an initial model of nsp -tm that should be refined by further experimentation. similar studies should also be performed to determine the topology of nsp and nsp , the other coronavirus replicase products with multiple predicted transmembrane helices. finally, to determine if the tm domain of mhv nsp was sufficient to confer membrane association to a cytosolic protein, we appended nsp -tm to egfp and determined the localization of the fusion protein using confocal microscopy (fig. ) . egfp normally is distributed throughout the cell (fig. a) . we found that appending the mhv nsp -tm sequence to egfp fig. . membrane extraction experiments of the plp expressed protein products treated with triton x- , urea, nacl or sodium carbonate solution ph . . in vitro transcription and translation reactions of pplp - , pplp - or pplp - were performed in the presence of cmm. subsequently, reaction mixtures were centrifuged to sediment cmm containing associated plp protein. the supernatant were removed and the pellets were resuspended in nte buffer or . % triton x- (a), m urea, m nacl or mm sodium carbonate solution ph . (b) and incubated for min at °c. subsequently, membrane sedimentation analyses were performed as described under materials and methods. soluble (s) and pellet (p) fractions were applied in equivalent amounts and subjected to % sds-page analysis. quantitation was performed by phosphoimaging and values expressed in % were given at the bottom and depicted as bars. was sufficient to tether it to membranes, as shown by the intense, perinuclear localized signal (fig. b ). to determine if the nsp -tm domain is retained in the er membranes or is transported through the medial golgi, we radiolabeled the efgp-nsp tm protein in transfected cells, immunoprecipitated the protein and subjected the immunoprecipitated products to endo h. we found that efgp-nsp tm is sensitive to treatment with endo h, indicating that the protein is retained in the er and does not pass through the medial golgi. thus, the nsp -tm domain is sufficient to confer membrane-localization and retention in the er. these studies are in agreement with our previous findings showing that the tm domain of sars-cov nsp (previously termed the hd) confers membrane association of egfp (harcourt et al., ) . in summary, using biochemical fractionation and proteinase k protection assays, we show that nsp -tm likely has four membrane-spanning domains, and that lumenal residue asparagine- is modified by glycosylation. furthermore, we found the region nsp -tm domain is required for efficient mhv plp process activity at cleavage site in the polyprotein. why does mhv plp require membrane association for proteolytic processing of the plp cleavage site? one possible explanation is that membrane-tethering brings plp into close proximity with a membrane-associated substrate. it is also possible that the nsp -tm membrane tether is important for anchoring the replicase complex to intracellular membranes. bioinformatic analysis of the nsp of other coronaviruses revealed that the membrane-spanning features of nsp -tm are conserved in all viruses (fig. , analysis using the tmhmm program is shown as an example), even though the amino acid identity is relatively low ( - % identity within nsp ). therefore, the tm domain is likely to be important for both plp activity and assembly of the replication complex. further studies will be required to determine the precise topology of the tm domain in other coronaviruses, and if the lumenal sequences in nsp -tm play any role in interacting with host factors during viral replication. hela cells expressing the mhv receptor, hela-mhvr cells (gallagher, ) were used for all transfection experiments. the cells were grown in dulbecco's modified eagle's medium supplemented with % fetal bovine serum (invitrogen, carlsbad, ca), . % penicillin/streptomycin, % glutamine, and mm sodium n- -hydroxyethylpiperazine-n′- ethanesulfonic acid, ph . . recombinant plasmid dna constructs expressing the plp coding region were generated using specific primers (listed in table ) to amplify the designated region from the parental plasmid pplp -cen (kanjanahaluethai and baker, ) . the region of interest was generated by pcr amplification using la-taq polymerase according to the manufacturer's instructions (clontech, palo alto, ca). the amplified region was then digested with restriction enzymes bamhi and xhoi and ligated into the corresponding sites in the pcdna . /v -his expression vector (stratagene, la jolla, ca) using t ligase (new england biolabs). the ligated dna product was transformed into xl- blue competent cells according to the manufacturer's instructions (stratagene), except that the bacteria were grown at °c. hela-mhvr cells were infected with a recombinant vaccinia virus expressing the bacteriophage t polymerase (vtf - ) at a multiplicity of infection of . then, infected cells were co-transfected with recombinant plasmid dnas encoding the mhv-jhm indicated protease domain and the substrate using lipofectamine according to manufacturer's instruction as previously described (fuerst et al., ; kanjanahaluethai and baker, ) . newly synthesized proteins were metabolically labeled with μci/ml [ s]-translabel (icn, costa mesa, ca) from . to . h post-infection (hpi). to harvest the cells, radioactive labeled cells were washed with phosphate buffered saline (pbs), and cell lysates were prepared by scraping the cells in lysis buffer a [ % sds, % dtt, % glycerol and . m tris, ph . (schiller et al., ) ]. the lysates were either used directly for immunoprecipitation assays or stored at − °c for future studies. radiolabeled cell lysate was diluted in . ml ripa buffer [ . % triton x- , . % sds, mm nacl, mm edta, and mm tris-hcl, ph . (schiller et al., ) ] and subjected to immunoprecipitation with anti-v monoclonal antibody (invitrogen) and protein-a sepharose beads (amersham biosciences, piscataway, nj). the immunoprecipitated products were eluted with × laemmli sample buffer, incubated at °c for min, and analyzed by electrophoresis on a . - . % gradient polyacrylamide gel containing . % sds. following electrophoresis, the gel was fixed in % methanol- % acetic acid, enhanced with amplify (amersham biosciences) for min, dried, and exposed to kodak x-ray film at − °c. site-directed mutagenesis of putative glycosylation sites in mhv-jhm nsp -tm domain plasmid dna pplp -cen which encompasses mhv-jhm gene amino acid residues - (kanjanahaluethai and baker, ) was subjected to site-directed mutagenesis at positions and for pplp -n a and positions and for pplp -n a using synthetic oligonucleotides with mismatches encoding specific nucleotides changes as shown in table . mutagenesis was performed according to the manufacturer's instructions (quickchange site-directed mutagenesis; stratagene), and as previously described (kanjanahaluethai and baker, ) . mutations were confirmed by dna sequence analysis. the tnt t -coupled reticulocyte lysate system (promega, madison, wi) was used according to the manufacturer's instructions. the recombinant plasmid dna encoding the designated plp region was linearized by digestion with pmei. in vitro transcription and translation was performed for min at °c in the presence of . μci of [ s]-translabel per ml in a volume of μl. where indicated, . μl of cmm (promega) was added prior to the incubation. for analysis of membrane association, the products of in vitro transcription and translation were centrifuged at , rpm for min. the supernatant was removed, the pellet that may contain aggregated or membrane-associated protein was suspended in × laemmli sample buffer, heated at °c for min, and both fractions were analyzed by sds-page and subjected to autoradiography. protection of translation products by microsomal membranes was examined by digestion with proteinase k (hugle et al., ) . following translation, reaction mixtures (after incubation with rnase a) were adjusted to . mg/ml of proteinase k (roche, indianapolis, in) and incubated for min on ice. proteinase k digestion was terminated by addition of phenylmethylsulfonyl fluoride to mg/ml and incubation was continued for min on ice. a portion of each reaction mixture (generally μl) was mixed with μl of × laemmli sample buffer, heated at °c for min, analyzed by % sds-page and subjected to autoradiography. for endo h treatment, lysates from vtf . -infected and pplp -tm transfected cells were prepared and subjected to immunoprecipitation as described above. protein-a sepharoseantibody-antigen complexes were washed once in ripa buffer and endo h treatment was performed as suggested by the manufacturer (roche). briefly, the complexes were resuspended in μl of mm sodium phosphate buffer, ph . , and incubated in the presence or absence of a final concentration of unit/μl of endo h for h at °c. following the incubation, μl of × laemmli sample buffer was added to each sample, mixed, and incubated for min at °c. the sepharose beads were pelleted by a brief, high-speed spin in a microfuge, and the supernatant loaded directly for analysis by % sds-page and subjected to autoradiography. for membrane extraction experiments, the pellets from μl in vitro transcription-translation reactions performed in the presence of cmm were resuspended in μl nte buffer, . % triton x- , m urea, m nacl, or mm sodium carbonate (ph . ) and incubated for min at °c (hugle et al., ) . subsequently, supernatant and pellet fractions were separated by centrifugation at , rpm for min, and analyzed by sds-page and autoradiography. quantitation was performed by using phosphoimaging analysis. the putative transmembrane domain region of mhv-jhm nsp (nt to ) was pcr amplified from the pplp -cen (kanjanahaluethai and baker, ) with primers b and b (table ) , cloned into the mammalian expression vector for egfp, pegfp-c (bd biosciences), and designated pegfp-nsp tm. the plasmid dna encoding egfp or egfp-nsp tm was transfected into hela-mhvr cells in well chamber culture slides with lipofectamine according to manufacturer's instructions, for h. expression of egfp and egfp-nsp tm fusion protein products was detected by confocal microscopy (zeiss lsm lazer-scanning confocal microscope). egfp-nsp tm was amplified by primers zcp and zcp (table ) using pegfp-nsp tm as template, and then cloned into bamhi and xbai sites of pcdna . /v -hisb (invitrogen) to generate the construct of pcdna . -egfp-nsp tm. the plasmid dna was expressed via the vaccinia virus-t expression system, proteins radiolabeled with stranslabel, cell lysates were subjected to immunoprecipitation with anti-v antibody, and products were either incubated with endo h or buffer alone, and analyzed by electrophoresis on % sds-page. the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity phase separation of integral membrane proteins in triton x- solution an amino-terminal amphipathic alpha-helix mediates membrane association of the hepatitis c virus nonstructural protein a coronavirus genome structure and replication an efficient ribosomal frame-shifting signal in the polymerase-encoding region of the coronavirus ibv eukaryotic transientexpression system based on recombinant vaccinia virus that synthesizes bacteriophage t rna polymerase murine coronavirus membrane fusion is blocked by modification of thiols buried within the spike protein rna replication of mouse hepatitis virus takes place at double-membrane vesicles identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity intracellular functions of n-linked glycans the hepatitis c virus nonstructural protein b is an integral endoplasmic reticulum membrane protein identification of mouse hepatitis virus papain-like proteinase activity identification of the murine coronavirus mp cleavage site recognized by papain-like proteinase the complete sequence ( kilobases) of murine coronavirus gene encoding the putative proteases and rna polymerase the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme subcellular localization and membrane topology of the dengue virus type non-structural protein b open reading frame a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme structural basis of severe acute respiratory syndrome coronavirus adp-ribose- ʺ-phosphate dephosphorylation by a conserved domain of nsp coronavirus transcription: a perspective processing of the coronavirus mhv-jhm polymerase polyprotein: identification of precursors and proteolytic products spanning kilodaltons of orf a ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex viral replicase gene products suffice for coronavirus discontinuous transcription identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku , from patients with pneumonia the coronavirus replicase the autocatalytic release of a putative rna virus transcription factor from its polyprotein precursor involves two paralogous papain-like proteases that cleave the same peptide bond we thank nicole kreuziger, kari severson and ami knop ullrich for excellent technical assistance, and dr. alexander gorbalenya, leiden university medical center for useful suggestions during the course of this work. this research was supported by public health service research grants ai and hhsn c. key: cord- -b y authors: vanleuven, james t.; ridenhour, benjamin j.; gonzalez, andres j.; miller, craig r.; miura, tanya a. title: lung epithelial cells have virus-specific and shared gene expression responses to infection by diverse respiratory viruses date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: b y the severity of respiratory viral infections is partially determined by the cellular response mounted by infected lung epithelial cells. disease prevention and treatment is dependent on our understanding of the shared and unique responses elicited by diverse viruses, yet few studies compare host responses to viruses from different families while controlling other experimental parameters. murine models are commonly used to study the pathogenesis of respiratory viral infections, and in vitro studies using murine cells provide mechanistic insight into the pathogenesis observed in vivo. we used microarray analysis to compare changes in gene expression of murine lung epithelial cells infected individually by three respiratory viruses causing mild (rhinovirus, rv b), moderate (coronavirus, mhv- ), and severe (influenza a virus, pr ) disease in mice. rv b infection caused numerous gene expression changes, but the differential effect peaked at hours post-infection. pr altered an intermediate number of genes whose expression continued to change through hours. mhv- had comparatively few effects on host gene expression. the viruses elicited highly overlapping responses in antiviral genes, though mhv- induced a lower type i interferon response than the other two viruses. signature genes were identified for each virus and included host defense genes for pr , tissue remodeling genes for rv b, and transcription factors for mhv- . our comparative approach identified universal and specific transcriptional signatures of virus infection that can be used to distinguish shared and virus-specific mechanisms of pathogenesis in the respiratory tract. viruses from several different families, including picornaviridae, orthomyxoviridae, paramyxoviridae, coronaviridae, and adenoviridae, infect and cause diseases in the respiratory tract. these diseases range from mild infections of the upper respiratory tract, to severe diseases when viruses infect the lungs. respiratory viruses commonly target epithelial cells of the a a a a a airways and lungs. these epithelial cells are responsible for detecting viral pathogens and initiating antiviral responses at the level of infected cells and the immune system, and therefore their response to infection has an important role in determining disease outcomes. knowledge of the shared and virus-specific responses of respiratory epithelial cells to infection by diverse viruses is fundamental to understanding viral pathogenesis and developing therapies to treat severe respiratory infections. murine models of respiratory viral infections have been widely used to identify the mechanisms that determine disease severity in the respiratory tract. while these models are invaluable for evaluating pathology and host responses to infection, parallel in vitro studies can be used to identify gene expression and signaling pathway changes that occur in infected cells to mediate pathogenesis. in this study, we compare the gene expression response of murine lung epithelial cells to infection by three respiratory viruses used in murine models: rhinovirus (serotype rv b) in the family picornaviridae, mouse hepatitis virus (mhv strain ) in the family coronaviridae, and influenza a virus (strain pr ) in the family orthomyxoviridae. viruses from different families and with different replication strategies were chosen to identify which responses to infection in respiratory epithelial cells are shared and which are virus-specific. in the following paragraphs, we describe some of the key features of these three viruses in murine models. minor group rhinoviruses, including rv b, use low-density lipoprotein receptor to enter either human or murine cells. because rv b can replicate in mouse cells, it has been used to study immune responses and mechanisms of asthma exacerbation in infected mice [ ] [ ] [ ] [ ] [ ] . intranasal inoculation of mice with a high dose of rv b results in viral replication and an early inflammatory response in the respiratory tract without clinical signs of disease [ , [ ] [ ] [ ] . rv b antigens have been detected in cells of the epithelia and lamina propria of the upper respiratory tract of infected mice [ , ] . mhv- is a strain of mouse hepatitis virus that preferentially replicates and causes disease in the respiratory tract of specific mouse strains and thus serves as a model for respiratory coronaviral infections [ , ] . mhv- causes severe disease in a/j-strain mice and milder pathology in other mouse strains [ , ] . mouse strain-dependent disease severity corresponds to inflammatory responses, fibrin deposition, and reduced type i interferon (ifn) production [ ] . further, type i ifn-mediated signaling is required for protection from severe disease during mhv- infection of resistant mice [ ] . mhv- has been detected in pulmonary macrophages of infected mice, but has not been reported to infect respiratory epithelial cells in vivo [ ] . although primary murine alveolar epithelial cells are susceptible to mhv- infection in vitro, their role during in vivo infection is not clear [ ] . mice have been used for decades to study the pathogenesis of influenza. one of the most commonly used strains of influenza a virus, pr , has been serially passaged in mice to produce a model for pulmonary infection. pr infection results in a range of disease severities that is mouse strain-dependent [ ] . although susceptible mice mount a type i ifn response to pr infection, lethal infection is associated with spread of virus to the alveoli and an excessive inflammatory response [ ] [ ] [ ] [ ] . pr replicates in bronchiolar and alveolar epithelial cells of the lower respiratory tract in vivo and in primary murine respiratory epithelial cells in vitro [ , , , ] . in this study, we used a murine lung epithelial cell line (la ) to compare the gene expression response to these three unrelated viruses. la cells were derived from neoplastic lung epithelia from strain a (a/he) mice and have some properties of alveolar type ii cells, although they do not maintain a highly differentiated type ii phenotype [ ] . strain a (a/j) mice are highly susceptible to respiratory viral infections, including mhv- and influenza a viruses [ , ] . other studies have demonstrated that la cells are susceptible to infection by pr and rv b [ , ] . in this study, we show that la cells are also susceptible to infection by mhv- (hereafter referred to as mhv). the gene expression response of la cells to infection by mhv, pr , and rv b (hereafter referred to as rv) differed in timing and magnitude of the changes. while we expected to see highly divergent transcription responses to these three viruses, they induced expression of a surprisingly large overlapping set of shared genes, including genes involved in antiviral responses. however, and more in line with our expectation, each virus also altered expression of unique genes, which highlight their different replication strategies and mechanisms of pathogenesis in murine hosts. virus stocks were generated by h of growth from a low dose inoculum in the cell lines described below. supernatant medium from infected cells was centrifuged to remove cell debris, aliquoted, flash frozen and stored at - ˚c. pr (a/puerto rico/ / (h n )), obtained from bei resources (nr- ), was grown and titrated by plaque assay in mdck cells from atcc (ccl- ) [ ] . mhv, obtained from atcc (vr- ), was grown and titrated by plaque assay in cl. cells [ ] (provided by dr. kathryn holmes, university of colorado denver school of medicine). rv, obtained from atcc (vr- ), was grown and titrated by tissue culture infectious dose % (tcid ) assay in hela (atcc: ccl- ) cells [ ] . la , a murine lung epithelial cell line from atcc (ccl- ), was cultured in ham's f k medium (mediatech) with % fbs (atlanta biologicals) and x antibiotic/antimycotic (gibco). our experimental approach was to inoculate la cells with the three viruses at times t = h and t = h and harvest rna for microarray analysis at t = h. controls were mock-inoculated at both time points. preliminary experiments were done to establish the growth kinetics of each virus and determine a multiplicity of infection (moi) that resulted in comparable numbers of cells positive for viral antigen at h post-infection (s fig) . based on this, la cells were inoculated with tcid /cell rv, pfu/cell pr , or pfu/cell mhv. triplicate wells of la cells in -well plates were inoculated with each virus diluted in serum-free medium or were mock-inoculated with serum-free medium for h at ˚c. viral inocula were removed and the cells were rinsed twice with serum-free medium. the cells were incubated in ham's f k medium with % fbs until the h time point at which time rna was isolated from cell cultures using an rnaeasy plus kit (qiagen) and transcript levels were measured by microarray (nimblegen mus musculus mm expression array). for the h samples, the media were removed and replaced with fresh media h after inoculation, which is the same time that h samples were inoculated. raw and processed data are available from ncbi gene expression omnibus under accession number gse . nimblescan v . software (niblegen, madison, wi) was used to extract raw intensity data for each probe on each array. intensity data were read into the r statistical computing environment and checked for quality [ ] . data were prepared for processing using the pdinfobuilder package and then normalized using the robust multichip average (rma) function in the oligo package [ ] . statistical tests for differences in expression between treatments were conducted on the normalized expression data using a linear mixed-effect model followed by linear contrasts corrected for multiple comparisons. more specifically, expression was modeled as a function of treatment, while probes for a particular gene were treated as random effects. the models used the nlme::lme function in r. the data contained seven treatments: three viruses tested at two time points ( h and h) each plus the mock treatment (rv , rv , mhv , mhv , pr , pr , and mock). the following nine post hoc, two-sided contrasts were then performed on the fitted models using the multcomp::glht function in r: each virus-time combination vs. mock (rv vs. mock, rv vs. mock, mhv vs. mock, mhv vs. mock, pr vs. mock, pr vs. mock) and each pairwise combination of viruses at the h time point (rv vs. mhv , rv vs. pr mhv vs. pr ). these tests had their p-values adjusted by the multcomp::summary.glht function according to their joint distribution. any factors detected to be significant at the family (gene) level were then subsequently corrected using the benjamini-hochberg algorithm [ ] with a false discovery rate set at %. genes associated with type i ifn responses were identified among the sets of genes with differential expression for each virus compared to mock at h and h using the interferome v. . database (http://interferome.its.monash.edu.au; [ ] ). this database was queried using the search criteria: type i ifn (all), in vitro, mus musculus, . fold change (up or down). interferome genes were manually sorted into functional categories: antiviral, ifn signaling, viral detection, immune response, mhc class i, inhibitory, apoptosis, ubiquitination, miscellaneous, and unknown. the significance of each virus having genes in the specific categories was tested using a chi-squared test. gene expression responses to rv b were compared between our data from mouse cells and published data using human cells [ ] using the mgi vertebrate homology database provided by the jackson laboratory [ ] as well as the annotate package in r. mhv, pr , and rv alter cellular gene expression by different magnitudes and with different timing our goal was to evaluate the gene expression response of murine respiratory epithelial cells to infection by three unrelated respiratory viruses studied in murine models. a preliminary study was undertaken to identify a murine cell line that was susceptible to infection by the three viruses. la cells were chosen because mhv, rv, and pr established productive infections in this line, as shown by increased viral titers released from infected cultures over a h infection (s fig). additionally, comparable numbers of viral antigen-positive cells were observed for the three viruses h after inoculation of la cells (s fig) . to compare how unrelated respiratory viruses (mhv, pr , and rv) alter gene expression of murine epithelial cells, we inoculated la cells with each of the three viruses and evaluated cellular gene expression by microarray analysis after and h of infection compared to mock-inoculated controls. fig shows the log -fold change in expression level of genes that were differentially expressed in virus-infected, compared to mock-inoculated, cells. by plotting the changes in gene expression at vs. hours, we observed differences in magnitude and timing of gene expression changes mediated by the three viruses. the genes with significantly different expression in mhv-infected cells had low fold change values (fig a) . at h, when gene expression changes were the highest, genes that were up-regulated by mhv infection had log -fold change values of less than five. in contrast, pr and rv induced expression of many genes by greater than five log -fold at h, and genes were spread consistently across the full range of values. by h, the genes most strongly up-regulated by pr and rv induced changes of - . log -fold and - . log -fold compared to mock, respectively. this same pattern was observed with the down-regulated genes (fig ) . in addition to the differences in fold change values, the three viruses also differed in the timing of gene expression changes. mhv altered expression of relatively few host genes, most of which were only significantly different from mock at h ( fig a) . while both pr and rv induced expression of large subsets of host genes, they did so with different timing. pr -induced changes in gene expression occurred at a constant rate: the expression level of most genes at h was approximately twice the expression value at h ( fig b) . in contrast, rv infection altered expression of a large number of genes by h and the expression levels were maintained at approximately the same levels at the h time point (fig c) . taken together, we observed differences in magnitude and timing of gene expression changes mediated by the three viruses. the limited response to mhv infection is in agreement with studies of other coronaviruses, such as mhv-a [ ] and sars-cov [ , ] . in addition to inducing minor transcriptional up-regulation of host genes, mhv-a shuts down host gene expression by enhancing mrna degradation [ ] . a related coronavirus, sars-cov, also induces degradation of host mrnas [ ] . the low numbers of host mrnas that were altered in response to mhv infection in our study could be due to one or both of these mechanisms. while rhinoviruses are known to down-regulate host gene expression by inhibiting transcription [ ], upon rv infection we saw robust, early increases in host mrna expression ( fig c) . this is in agreement with other transcriptome studies of major and minor serogroup rhinoviruses in human respiratory epithelial cells and experimental infections of humans [ , [ ] [ ] [ ] [ ] . the plateau in gene expression changes in rv-infected cells at h may be due to transcriptional inhibition later in infection, or the death of infected cells. pr infection induced a strong transcriptional response in la cells, which has also been seen with multiple strains of influenza a viruses in primary human and mouse airway or lung epithelial cells [ ] [ ] [ ] [ ] . interestingly, the differences in kinetics of host cell gene expression did not correspond with differences in the kinetics of viral replication in this cell line (s fig). despite inducing ) . these data also demonstrate that the limited response of cellular gene expression to mhv infection was not due to limited infection of la cells (s fig). host genes have shared and unique responses to rv, pr , and mhv infection we identified which genes were altered by each virus at h compared to mock and the degree of overlap among the differentially expressed genes. at h rv infection resulted in up-regulation of the largest number of genes, followed by pr then mhv (fig a) ; a similar pattern was seen with down-regulated genes (fig b) . while one might worry that some of the small number of significant genes that were altered by mhv could be false positives, the majority of these genes ( % of up-regulated and % of down-regulated genes) were also significantly altered by at least one other virus suggesting that most of these genes are true positives. for both up-and down-regulated gene sets, rv had the largest proportion of unique genes, while the majority of genes affected by pr and mhv were shared by at least one other virus. s table contains the list of genes whose expression was significantly up-regulated by all three viruses compared to mock-inoculated cells. these genes may reflect a global response of epithelial cells to viral infection. several of the genes with the highest fold change values are involved in antiviral defense at the level of infected cells (eg., mx , bst , oas , gbp ) or recruitment of immune cells (eg. , cxcl , cxcl , cxcl ) . these genes are up-regulated by type i ifns, suggesting that induction of a type i ifn response is shared by these viruses. in contrast to the shared up-regulated genes, genes that were significantly down-regulated by all three viruses have diverse functions (s table) . some examples of genes that were down-regulated by all three viruses included genes that encode transmembrane proteins (tmem , , , a, and c), extracellular matrix proteins (spon , ogn, aspn), and apoptotic signaling proteins (sdpr, bmf, bnip l). as a measure of validation, the expression levels of five genes (tnf, cxcl , bst , icam , and oas a) were also quantified by rt-qpcr at h post-infection (s fig). a strong correlation was observed between rt-qpcr and microarray measurements of gene expression (slope = . , r = . ). identification of signature genes that were uniquely altered by each virus comparing the number of genes altered by each virus provides insight into shared and unique cellular responses elicited by the viruses, but it does not provide information on the relative magnitudes of gene expression changes between viruses. to compare gene expression changes between viruses, we plotted the log -fold change of each gene at h for mhv vs. rv vs. pr (fig a) . we only included genes that were differentially expressed in at least one viral infection compared to mock. like fig , this d plot illustrates that pr and rv not only caused a larger number of genes to be up-regulated compared to mhv, but they also induced higher fold change values (fig a) . for each of the three viruses, we defined a signature gene as a gene that is both differentially regulated at h compared to the mock treatment and has an effect size significantly larger than the other two viruses (i.e. fold change on the x axis is significantly different from y-axis, z-axis, and mock). these genes are colored in fig a and appear along the diagonal in fig b. as expected, rv had the largest number of signature genes, followed by pr , then mhv ( fig b) . interestingly, the genes with the highest fold change values compared to mock were not signature genes, but were up-regulated by both pr and rv infection. a pairwise analysis was performed to identify the number of genes with altered expression compared with mock in two viruses compared with the third. this analysis, shown in fig b, reveals that rv and pr had the most similarities in both up-and down-regulated genes (fig b, purple blocks) . the pattern of up-regulated gene expression changes during mhv infection was more similar to pr ( genes) than rv ( genes). this may reflect the fact that pr and mhv cause severe pathogenesis in mice, whereas rv-infected mice do not exhibit clinical signs of disease. among the six genes co-upregulated by mhv and pr was tnf-α, a key proinflammatory cytokine (not shown). several host defense genes were identified as signature genes uniquely up-regulated by pr infection (s table) . these genes included cytokines and chemokines (cxcl , ccl , il b, ccl ), ifn response genes (ifitm , ifi l a, ifna , ifit , ifitm , ifna ) , and genes involved in processing mhc class i antigens (psmb , tap , h -q , h -k , psmd , psme , psme ) . the significant up-regulation of host defense genes in response to pr in the la cell line corresponds with the expression profile of murine type ii alveolar epithelial cells in response to pr infection in mice [ ] . pr infection in highly susceptible mouse strains results in dramatic up-regulation of inflammation-associated genes when compared to resistant mouse strains [ ] . many studies in murine models of influenza a virus infection have demonstrated a relationship between an excessive inflammatory response and disease severity [ ] [ ] [ ] ] . furthermore, infection of tlr -/-mice with influenza a virus results in a reduced inflammatory response and increased survival [ ] . our data support the role of alveolar epithelial cells in generating this excessive inflammatory response in vivo. several genes that were uniquely down-regulated by pr are involved in cellular metabolic pathways (cdo , aldh a , acad , hsd b ) or intracellular transport (myl b, ift , anxa ). although rv induced expression of several genes involved in host defense, these were largely shared by pr so were not identified as signature genes. the signature genes up-regulated by rv included kallikrein- and ten kallikrein- -related peptidases and additional proteins involved in tissue remodeling (s table) . although tissue remodeling is not likely to be relevant in murine models of rhinovirus infection alone, due to the limited damage, it may be an important factor in murine models of rhinovirus-induced allergic asthma [ , , ] . rhinovirus infections are a significant cause of asthma exacerbations, which correspond with inflammatory responses in the airways. kallikreins generate kinins and contribute to many disease processes, including inflammation. kinins are induced by rhinovirus infections and kallikrein- is up-regulated by rhinovirus infection in humans, especially those with asthma [ , ] . upregulation of these genes in mouse cells upon rv infection would provide a tractable animal model in which to study the roles of kallikreins in rhinovirus-induced asthma exacerbations. mucins, which contribute to mucus hypersecretion, are up-regulated by rhinovirus infection of airway epithelial cells in vitro and in mice [ , ] . muc was the only mucin gene up-regulated by rv in our study, and was unique to rv infection (s table) . mhv infection resulted in regulation of a small set of signature genes (fig b, s table) . signature genes that were uniquely up-regulated by mhv infection included multiple transcription factors from the double homeobox (duxf , dux, dux ) and zinc finger and scan domain (zscan d, zscan c, zscan -ps , and ) families. despite up-regulating expression of transcription factors, mhv infection had a minor impact on the host cell transcriptome. this may be due to enhanced degradation of mrnas as discussed above, which has been shown to occur during other coronaviral infections [ , ] . therefore, la cells may be up-regulating transcription in response to mhv infection through expression of various transcription factors while mhv causes degradation of these transcripts, which would reflect the small number of up-regulated transcripts in mhv infected samples. in contrast to mhv-a , mhv- infection did not cause down-regulation of a substantial number of host genes. differences could be due to virus strain, host cell type, and timing differences between the studies. in contrast to the robust up-regulation of genes involved in innate immunity and inflammatory responses by pr and rv, the limited response of infected epithelial cells to mhv infection may reflect the ability of mhv to replicate (s fig) without being detected by the host cell. coronaviruses, including mhv, delay induction of antiviral responses. multiple mechanisms have been proposed to account for this, including replicating within double membrane vesicles, ribose '-omethylation of viral mrna, and endonuclease activity within the rna polymerase complex [ ] [ ] [ ] [ ] . this would allow mhv to replicate to higher levels before triggering antiviral defenses, which might promote pathogenesis in the murine respiratory tract [ ] . alternatively, the reduced response to mhv by epithelial cells may reflect the different cellular tropism of mhv. in contrast to pr and rv, which are known to infect epithelial cells in the murine respiratory tract, mhv- has only been reported in alveolar macrophages [ , , ] . type i ifn-related genes had increased expression in la cells infected by pr , rv, and mhv as described above, several of the genes with up-regulated expression in response to all three viruses, as well as those that were unique to pr , are induced by type i ifns. to specifically evaluate how ifn response genes were altered by the three viruses, genes that were significantly up-regulated by each virus at the h time point were used to query the interferome v . database (see materials and methods). a venn diagram was generated to visualize the degree of overlap in ifn-related genes whose expression was induced by at least one of the three viruses (fig ) . pr induced expression of the greatest number of ifn-related genes, a majority of which were shared by at least one other virus. rv up-regulated slightly fewer ifninduced genes compared to pr and mhv infection resulted in up-regulation of the fewest ifn-induced genes. it was somewhat surprising that pr induced a higher type i ifn response than rv, given that rv induced expression of nearly twice as many genes than pr (fig ) . however, some of these genes contribute to inflammatory responses, which could explain the excessive inflammatory response to pr infection vs. the self-limited inflammation during rv infection in mice [ ] [ ] [ ] ] . there was strong overlap between the ifn-induced genes up-regulated by each virus. the timing of ifn-related gene expression followed the same trend as was seen for all significant genes in fig (data not shown) . most of the ifn-related genes up-regulated by mhv were only increased at h. pr induced expression of ifn-related genes at h and these genes were a subset of the genes up-regulated at h. in contrast, rv infection induced expression of more ifn-related genes at h ( genes) than at h ( genes). relative to up-regulation, few ifn-related genes were down-regulated at the h time point (mhv = , pr = , rv = ). type i ifns induce expression of genes with different functions during an antiviral response. to determine whether there were specific patterns in expression of ifn-induced genes that correspond with function, the ifn-induced genes that had significantly increased expression by any of the three viruses were separated into functional groups. heatmaps that demonstrate differences in fold change (color scale) and significant differences (outlined boxes) in expression compared to mock-inoculated controls were generated (figs and s ) . as shown in the venn diagram, this analysis also demonstrates that pr infection resulted in up-regulation of the most genes involved in type i ifn responses, followed by rv then mhv. the fold change values induced by pr infection were also generally higher than the other two viruses. however, there was not a significant association between virus identity and functional group. for most of the functional groups, mhv up-regulated expression of a smaller subset of the same genes as pr and rv, with the exception of the mhc class i pathway (fig ) . mhv significantly up-regulated expression of only one gene involved in the mhc class i pathway (blmh), which was not significantly up-regulated by the other two viruses. this observation suggests that cytotoxic t cell responses may differ in mhv infections compared to pr and rv. t cell responses have complex roles in mhv- infections, as they contribute to protection in resistant mouse strains but mediate pathology in susceptible strains [ ] . however, mice with the cd + t cell repertoire of a resistant strain in the background of a susceptible strain remain susceptible to severe mhv- infection [ ] . the failure of mhv- to activate processing and presentation of mhc class i antigens could explain the inability of a broadly reactive cd + t cell response to protect these mice. the interferome analysis focuses on ifn-induced gene expression, but not expression of the type i ifns that induce these responses. multiple type i ifns exist, including ifn-β and subtypes of ifn-α, all of which signal through the type i ifn α/β receptor [ ] . type i ifns can induce autocrine and paracrine signaling; thus the ifn-induced genes we detected could be from both infected and uninfected cells in the cultures. to determine if differential expression of type i ifns explains the differences in ifn-induced gene expression upon infection by pr , rv, and mhv, we analyzed the expression of type i ifn and receptor genes for each virus compared to mock (fig ) . probes for ifn-β and ten subtypes of ifn-α were present on the arrays. in agreement with expression of ifn-induced genes, pr induced expression of the largest set of type i ifns, followed by rv. both viruses induced expression of ifnb and ifna , which encode type i ifns known to be expressed early during antiviral responses [ , ] . five subtypes of ifna were up-regulated by both pr and rv, while three ifna subtypes were uniquely up-regulated by pr and only ifnab was uniquely up-regulated by rv. only pr induced expression of ifnar , which encodes the high affinity chain of the type i ifn α/β receptor [ ] . this may allow for enhanced positive-feedback signaling and account for the larger number of ifn-induced genes up-regulated by pr infection. differential signaling through mda- and rig-i pathways may contribute to the differences in type i ifn responses by rv and pr . rhinoviruses and influenza a viruses are known to induce type i ifn responses through recognition by mda- and rig-i, respectively [ , ] . furthermore, both viruses are recognized by tlr in infected epithelial cells [ , ] . however, tlr predominantly induces expression of pro-inflammatory genes, rather than type i ifn-dependent genes, during influenza a virus infection [ ] . zaritsky et al. have demonstrated that the type i ifn response to sendai virus differs when cells are infected by different doses [ ] . they further showed that these differences were mediated by differential signaling through the ifn α/β receptor, with robust signaling in uninfected cells. this supports our findings that pr induces expression of ifnar and additional type i ifn genes that are not up-regulated by rv (fig ) . none of the type i ifns or receptors had significantly altered expression upon mhv infection (fig ) , despite up-regulation of a modest number of ifn-stimulated genes (fig ) . this could be due to ifn-independent expression of these genes, or induction by a type i ifn that was not represented on the microarray. coronaviruses are notorious for being able to replicate within cells without triggering type i ifn responses, or delaying ifn induction until late in the replication cycle [ , [ ] [ ] [ ] . other studies have shown that the ifn response to mhv- is a critical determinant of susceptibility. severe disease in a/j mice compared to c bl/ mice correlates with lower type i ifns detected in the lungs of a/j mice upon mhv- infection [ , ] . similarly, the expression of various type i ifns in response to mhv- infection in vitro is cell line-dependent [ ] . because the cell line we used, la , was derived from the lungs of a/ he mice, we would expect it to have a similar response as a/j mice. thus the lack of type i ifns induced by mhv- in la cells in vitro corresponds with pathogenesis observed in a/j mice in vivo. the finding that la cells mount a stronger response to pr than rv or mhv infection may be due to differences in the viral recognition and signaling pathways used to detect these different viruses and amplification of the type i ifn response as discussed above. alternatively, it could be due to the timing of our analysis. rv-infected cells have started dying by the h time point (not shown), therefore expression of host genes may be declining by that time point. in contrast, coronaviruses are known to delay cellular responses to infection [ ] , so the h time point may be too early to evaluate the innate response to mhv infection. alternatively, the cells may detect mhv and up-regulate transcription of ifn response genes, but mrna degradation would mask this process. by quantifying mrna transcripts at two time points after viral infection, our study cannot distinguish between these possibilities. one limitation of our study is the analysis of three viruses that do not share a natural host. mhv is a natural pathogen of mice and pr is a highly mouse-adapted strain of influenza a virus. however, rv b is a human rhinovirus whose receptor is conserved between mice and humans. rv b is increasingly being used in mouse studies [ ] [ ] [ ] [ ] ] . despite the difference in host, we found similar changes to gene expression in murine cells as studies with rv b in human cells [ ] . of the , and , genes represented on our mouse microarray and the human microarray chip used by chen et al., respectively, , genes are shared. using the same -fold increase in expression cut-off and restricting our list only to homologous human genes studied by chen et al., we found that mouse genes were up-regulated by rv b infection. comparing this list of genes to the up-regulated human genes identified by chen et al., we found that genes (s table) were up-regulated by rv b infection in both human and mouse cells. a chi-squared test confirmed the significance of this shared number of up-regulated genes (χ = . , d.f. = , p< . ). interestingly, all of the shared genes we identified are involved in type i ifn responses. while far from identical, the similarity of the responses in the two cell types suggests conserved activation of type i ifn responses by these different hosts and supports the validity of a murine model for studying rhinovirus infections in humans. alveolar epithelial cells have a key role in alerting the immune system to infection by respiratory viruses and shaping immune responses [ , , ] . as viruses from several different families all target respiratory epithelial cells, it is important to understand the similarities and differences in how these cells respond to a diverse set of viruses. a significant number of genes were up-or down-regulated in response to infection by three distinct viruses from different families. genes that were associated with a shared response to the three viruses included those involved in defense against viruses and particularly genes induced by type i ifns. however, there were differences in the timing, numbers of genes altered, and expression levels of these genes. this may reflect differences in viral replication cycles and signaling pathways that are activated by infection, which may reflect differences in pathogenesis of these viruses in murine models. la cells were inoculated with virus using the same moi's as for the microarray study and rna was extracted at h post-infection using trizol (ambion). rna was converted to cdna using random hexamers and super-script vilo (invitrogen). five genes with differential expression by microarray analysis were validated by qpcr analysis using sybr green (powerup, applied biosystems) and the primer pairs listed in the figure on a stepone plus instrument (applied biosystems). ct values from triplicate qpcr reactions were averaged and normalized to β-actin before calculating the fold change values of virus-infected samples vs. mock. linear regression was used to compare fold change values between qpcr and microarray with removal of outliers ( à ) with cook's distance > . . (pdf) genes with significantly up-regulated expression compared to mock at h (see fig ) were used to query the interferome v. . database. interferon-regulated genes were divided into functional groups and heat maps were generated using log -fold change values for each virus at h compared to mockinoculated controls. heat maps of additional functional groups can be found in fig . gene names are indicated to the right of each row and statistically significant values are outlined in black. (pdf) s table. genes whose expression was significantly up-regulated by all three viruses compared to mock-inoculated cells. these genes were from the center of the venn diagram in fig a. (xlsx) s table. genes whose expression was significantly down-regulated by all three viruses compared to mock-inoculated cells. these genes were from the center of the venn diagram in fig b. (xlsx) s table. signature genes for pr . these genes were significantly different in pr infection compared to mock, rv, and mhv. (xlsx) s table. signature genes for rv. these genes were significantly different in rv infection compared to mock, pr , and mhv. (xlsx) s table. signature genes for mhv. these genes were significantly different in mhv infection compared to mock, rv, and pr . (xlsx) s table. genes up-regulated by rv in both murine and human cells. these genes were identified to be up-regulated in our study by rv b in murine cells and also were found by chen et al. ( ) to be up-regulated by rv b in human cells. (xlsx) mouse models of rhinovirus-induced disease and exacerbation of allergic airway inflammation rhinovirus infection in murine chronic allergic rhinosinusitis model cxcr is required for neutrophilic airway inflammation and hyperresponsiveness in a mouse model of human rhinovirus infection human rhinovirus b exposure induces phosphatidylinositol -kinase-dependent airway inflammation in mice mda and tlr initiate pro-inflammatory signaling pathways leading to rhinovirus-induced airways inflammation and hyperresponsiveness murine hepatitis virus strain produces a clinically relevant model of severe acute respiratory syndrome in a/j mice genetic determinants of mouse hepatitis virus strain pneumovirulence toll-like receptor deficiency increases disease and mortality after mouse hepatitis virus type infection of susceptible c h mice differentiated phenotypes of primary murine alveolar epithelial cells and their susceptibility to infection by respiratory viruses host genetic background 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hepatitis virus nonstructural proteins and rna synthesis indicates a role for late endosomes in viral replication mouse hepatitis virus replicase proteins associate with two distinct populations of intracellular membranes early endonuclease-mediated evasion of rna sensing ensures efficient coronavirus replication ribose '-o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda protective and pathologic roles of the immune response to mouse hepatitis virus type : implications for severe acute respiratory syndrome t cell epitope specificity and pathogenesis of mouse hepatitis virus- -induced disease in susceptible and resistant hosts characterization of the murine alpha interferon gene family regulation of inducible and tissue-specific gene expression. science differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor- . the embo journal the interferons and their receptors-distribution and regulation cutting edge: influenza a virus activates tlr -dependent inflammatory and rig-i-dependent antiviral responses in human lung epithelial cells role of double-stranded rna pattern recognition receptors in rhinovirus-induced airway epithelial cell responses virus multiplicity of infection affects type i interferon subtype induction profiles and interferon-stimulated genes murine coronavirus cell type dependent interaction with the type i interferon response group coronaviruses prevent immediate early interferon induction by protection of viral rna from host cell recognition mouse hepatitis virus does not induce beta interferon synthesis and does not inhibit its induction by double-stranded rna distinct signature type i interferon responses are determined by the infecting virus and the target cell dynamic innate immune responses of human bronchial epithelial cells to severe acute respiratory syndrome-associated coronavirus infection toll-like receptor -expressing macrophages are required and sufficient for rhinovirus-induced airway inflammation host-pathogen interactions during coronavirus infection of primary alveolar epithelial cells virus-infected alveolar epithelial cells direct neutrophil chemotaxis and inhibit their apoptosis the authors are grateful to dr. kathryn holmes, university of colorado at denver school of medicine, dr. elizabeth fortunato, university of idaho, and dr. julian leibowitz, texas a&m university for cells and antibodies that were used in this study. the following reagents were key: cord- -srb ac l authors: leparc-goffart, isabelle; hingley, susan t.; chua, ming ming; jiang, xinhe; lavi, ehud; weiss, susan r. title: altered pathogenesis of a mutant of the murine coronavirus mhv-a is associated with a q l amino acid substitution in the spike protein date: - - journal: virology doi: . /viro. . sha: doc_id: cord_uid: srb ac l abstract c , an attenuated, fusion delayed, very weakly hepatotropic mutant of mouse hepatitis virus strain a (mhv-a ) has been further characterized. we have previously shown that c has two amino acid substitutions relative to wild type virus in the spike protein, q l (within a region of s shown to bind to viral receptor in anin vitroassay) and h d (in the proteolytic cleavage recognition site). we have sequenced the rest of the -kb genome of c and compared it to wild type virus. only three additional amino acids substitutions were found, all encoded within the replicase gene. analysis of c in vivoin c bl/ mice has shown that despite the fact that this virus replicates in the brain to titers at least as high as wild type and causes acute encephalitis similar to wild type, this virus causes a minimal level of demyelination and only at very high levels of virus inoculation. thus acute encephalitis is not sufficient for the induction of demyelination by mhv-a . analysis of mutants isolated at earlier times from the same persistently infected glial cell culture as c , as well as mutants isolated from a second independent culture of persistently infected glial cells, suggests that both the weakly demyelinating and the weakly hepatotropic phenotypes of c are associated with the q l amino acid substitution. for the altered pathogenic properties of the mutant viruses, we compared the sequence of the spike (s) genes the murine coronavirus, mouse hepatitis virus strain of wild type and mutant viruses isolated from persistently a (mhv-a ), produces both hepatitis and neurologiinfected glial cells cultures. we focused on the s protein cal disease in susceptible mice. neurological disease because this protein mediates both binding to the viral includes both acute meningoencephalitis and chronic receptor (holmes and compton, ) and cell to cell demyelinating disease (lavi et al., b) . in vitro, mhvfusion. this, in combination with the fact that viruses a causes a lytic infection in mouse fibroblast cells, with mutations in the s gene have altered pathogenic but a persistent productive, nonlytic infection of primary properties (fleming et al., ; gallagher et al., ) cultured glial cells (lavi et al., ) . the mechanism of suggest that s is a major determinant of viral tropism. demyelination is not well understood but it is thought to the fusion-delayed phenotype was shown to result from involve elements of direct viral infection as well as an the substitution of an aspartic acid residue for a histidine immune mediated component (houtman and fleming, (h d) in the basic sequence that serves as a recogni- a). tion signal for proteolytic cleavage of the s protein (gom-we have been using a group of mhv-a mutants with bold et al., ) . this amino acid substitution prevents altered pathogenic properties to map the determinants of cleavage of the -kda precursor s protein into the two central nervous system pathogenesis and to understand approximately -kda subunits, s and s (luytjes et al., what features of infection of mice lead to demyelination. ; gombold et al., ) . aside from the cleavage we have previously characterized this group of attenurecognition site mutation (h d), all the mutant viruses ated, fusion-delayed mutants, which were isolated from examined contained one, and only one, additional amino persistently infected glial cells. these mutants had acid substitution in s, q l, located within the amino greatly reduced ability to replicate in the liver and cause terminal portion of the s subunit. analysis of fusion hepatitis (hingley et al., ) . competent revertant viruses, however, showed that atten-in order to begin to identify the mutations responsible uation and loss of hepatotropism were not linked to the fusion phenotype and did not map to the h d amino acid substitution. an association between the q l mu-hypothesis that this mutation may play a role in tissue fin and sectioned for staining with luxol fast blue to detect plaques indicative of demyelination. for each of five mice tropism. interestingly, q l lies within the amino terminal amino acids of s , a region of the spike protein at each dose of virus, demyelination was quantitated by examining one spinal cord section (four quadrants) from demonstrated to bind to the viral receptor in an in vitro assay (kubo et al., ; suzuki and taguchi ) , sug-each of five levels of spinal cord; thus there were quadrants examined for each dose of virus. the experi-gesting the possibility that this amino acid may influence the interaction of the spike protein with the viral receptor. ments were designed so that there would be five animals surviving acute infection even at high doses of virus rela-earlier studies (see table ) also demonstrated that viruses with identical s gene sequences had different tive to the ld ; however, in the case of the highest dose of c and all doses of b , there were less than five virulence and hepatotropism phenotypes (hingley et al., ) . thus, to determine which additional genes may surviving animals (figs. and ) . demyelination was also verified in the brain by staining with luxol fast blue. influence pathogenesis, we have sequenced the entire genome of the c mutant of mhv-a , one of the viruses isolated from persistently infected glial cells (gom-genome sequencing bold et al., ; hingley et al., ) . in addition to the two mutations found in the s gene, there were only five for sequencing of the viral genome, reverse transcriptase/polymerase chain reaction amplification (rt/ other mutations; of these, three resulted in amino acid substitutions, all within the viral replicase gene. pcr) was carried out, using as templates, cytoplasmic rna extracted from l cell monolayers infected with wild we describe here the further characterization of the c mutant. we have shown that despite the fact that c type mhv-a , c , c , c , c , b , or b . complementary dna was synthesized using random oligomers as replicates in the brain and causes encephalitis similar to that of wild type virus, at a dose of pfus ( wild primers. mhv-a -specific primers were designed to amplify fragments of about base pairs. double-type ld ), at which wild type virus induces extensive demyelination in all infected animals, c does not in-stranded pcr products were gel purified and analyzed by automated sequencing using the taq dye terminator duce detectable demyelination. analysis of other mutants isolated at earlier time points from persistently infected procedure according to the manufacturer's protocol (taq dyedeoxy terminator cycle sequencing kit, applied bio-glial cell cultures (hingley et al., ) as well as mutants isolated from an independent culture of persistently in-systems) and the same primers used for amplification. each fragment was sequenced in both directions. the fected glial cells, suggests that the q l mutation, and not the three replicase mutations, is associated with loss entire c genome was sequenced; fragments of each of the other viruses were sequenced as described under of the ability to demyelinate efficiently as well as the loss of the ability to induce hepatotropism. results. sequence comparison of the genomes of the c mutant and mhv-a mhv-a was obtained originally from dr. lawrence sturman (albany, ny). the mutants were plaque purified we have shown previously that the spike (s) protein from the supernatant of either the ''c'' culture of persisencoded by the c mutant has two amino acid substitutently infected glial cells at week (c ), weeks (c ), tions as compared with the wild type s protein. earlier weeks (c ), and weeks (c ) postinfection or from studies demonstrated that viruses with the identical s the independent ''b'' culture of persistently infected glial protein sequence had different virulence and hepatotropcells at weeks postinfection (b , b ) and characterism phenotypes (see table ). for example the mutant ized as previously described (gombold et al., ; hing-b appeared to be blocked in its spread from the central ley et al., , ) . nervous system to the liver, while the mutant c was inhibited in its ability to replicate in hepatocytes even demyelination when inoculated directly into the liver (hingley et al., ) . the b mutant, while nonhepatotropic like c , four-to -week-old mhv-free c bl/ mice (jackson laboratories, bar harbor, me) were inoculated intracere-retained its virulence (hingley et al., ) . thus, we concluded that these pathogenic properties must also brally with -fold serial dilutions of wild type or mutant mhv-a . at days postinfection, mice surviving acute involve genes outside of s. this, in combination with the idea that it is difficult to assign phenotypes to specific infection were perfused with phosphate-buffered saline, followed by % buffered formalin. brains and spinal sequences in s without knowing if there are any other mutations outside of s, led us to sequence the rest of cords were removed and tissue was embedded in paraf- with, the n protein (fischer et al., ) and the hemagglutinin esterase (he) protein, which is encoded, but not pathogenic phenotypes of mutants containing q l expressed in mhv-a (luytjes et al., (luytjes et al., ) . needed to kill half the weanling c bl/ mice after intracerebral inoculation. a minus sign indicates the ld is at least log greater than the deduced sequences of the nonstructural proteins wild type. these data were taken from hingley et al. ( ) . from the first amino acid in each orf.) of the two substitutions in orf a, p s is in the predicted papain-like proteinase- (plp- ) domain; the substitution in orf b, the c genome to identify other possible mutations that may play a role in these different pathogenicities. r s, is in the predicted helicase domain (lee et al., ; bredenbeek et al., ; bonilla et al., ) . there we have sequenced the entire -kb genome of c . sequencing was carried out by rt/pcr of viral rna, is only one silent mutation in the genome of c ; nucleotide of orf a is u rather than c, which is in the present in infected cells, as described under materials and methods. this generates a consensus sequence wild type genome. we have also compared the noncoding regions of c because it is derived from the rna genome and not cloned cdnas; we compared the c sequence with the and wild type mhv-a . we have found no differences between c and wild type in the or noncoding published sequence for wild type a (from our lab as well as from other labs) (budzilowicz et al., ; budzi- regions. with the exception of the conserved intergenic region preceding the m gene, there were also no differ-lowicz and weiss, ; zoltick et al., ; bonilla et al., ; weiss et al., ; bredenbeek et al., ; luytjes ences between c and wild type mhv-a intergenic sequences. there is one nucleotide change in the in-et , armstrong et al., armstrong et al., , . whenever we found a difference between c and the published tergenic region preceding the m gene. in c the sequence is aaucuaaac instead of aauccaaac present mhv-a wild type sequence, we made a new preparation of cdna from intracellular c rna, amplified the in wild type mhv-a (budzilowicz et al., ) . we examined the early mutants from the c culture for same double stranded dna fragment, and verified the c sequence. we then determined if our wild type se-the presence of the three replicase mutations as well as the intergenic nucleotide difference. these include the quence was the same as the published one. the c genome had surprisingly few mutations compared to the c , c , and c mutants isolated at various times after infection ( , , and weeks, respectively). neither the wild type genome after weeks of culture in glial cells. figure shows the comparison of the wild type and c replicase mutations (table ) nor the intergenic region mutation were present in any of the early mutants. this genomes. with the exception of the two previously discussed suggested that these mutations arose in the culture later than either of the s mutations. amino acid substitutions in s, h d and q l (fig. ) , there are no amino acid substitutions in the deduced in order to determine if these replicase substitutions were a general characteristic of the attenuated, tropism sequences of any of the structural proteins; this includes spike (s) (luytjes et al., ; hingley et al., ) , matrix mutants that arise in persistently infected glial cells, we examined the genomes of two mutants, b and b , (m) (armstrong et al., ) , nucleocapsid (n) (armstrong et al., ) , and small membrane (e) (budzilowicz and isolated from the independent (''b'') culture of persistently infected glial cells (gombold et al., ) . none of the weiss, ) proteins as well as the open reading frames (orfs) for the i protein, encoded within, but out of frame three replicase mutations were found in either of these fig. . sequence comparison of the c and wild type mhv-a genomes. the entire c genome was sequenced and the sequence compared to that of wild type mhv-a (as described in the text). (a) schematic diagram of the viral genome and the location of the five amino acid substitutions and the one nucleotide change in an intergenic region of c . the structural proteins are spike, he (hemagglutinin esterase), m (matrix), n (nucleocapsid), e (small membrane), and i (internal orf). the nonstructural replicase proteins are encoded in orf a and orf b. the domains of the replicase plp- (papain-like proteinase- ) and plp- (papain-like proteinase- ), c (poliovirus c like proteinase), pol (polymerase), and hel (helicase) were all predicted by lee et al. ( ) . the nonstructural, nonessential protein products of orfs a, , and a are not shown. the one silent mutation (nucleotide is u rather than c, present in the wild type genome) is not shown. (b) genes and predicted functional domains which contain these amino acid substitutions. the amino acids are numbered from the beginning of each open reading frame (orf) (bredenbeek et al., ; bonilla et al., ; luytjes et al., ) . the cleavage site of s was mapped by luytjes et al. ( ) and the mapping of the receptor binding domain is from kubo et al. ( ) . the intergenic regions are thought to be involved in transcriptional regulation (van der most and spaan, ). genomes. we then sequenced a larger region around lab (bonilla et al., ; budzilowicz et al., ; budzilowicz and weiss, ; weiss et al., ; , the mutation in the predicted plp- domain ( nucleotides) and the mutation in the predicted helicase domain , unpublished sequences of orf b). mutations encoding amino acid substitutions included two in orf a, ( nucleotides) to determine if there were other mutations within either of these predicted domains in b or two in orf b, one in s, one in n, and one each in orfs and a. additional silent mutations included three in b . b did have another amino acid substitution within the plp- domain, v m. orf a, two in orf b, and two in n. there were also two nucleotide changes and two insertions in the non-we also found several mutations, both silent and coding, in the comparison of wild type mhv-a that we coding region of the genome. have used to derive the mutants (obtained from dr. l. sturman) as compared to the published sequences demyelination of c (bredenbeek et al., ; bonilla et al., ; luytjes et al., ; luytjes et al., ; hingley et al., ; arm- we have shown previously that the c mutant of mhv-a has a fusion delayed phenotype in vitro (gom-strong et al., (gom-strong et al., , budzilowicz et al., ; budzilowicz and weiss, ; weiss et al., ; zoltick et bold et al., ) . furthermore, c replicates to titers equal to wild type virus in the cns and causes a similar al., ), some of which came from the sequencing of another mhv-a (obtained from dr. j. leibowitz) in our level of encephalitis. however, it is very weakly hepato- e fusion was defined as previously described gombold et al., ( ) . a negative sign signifies a delayed fusion phenotype in which fusion is negative when measured in l cells at h postinfection but eventually does occur by about h postinfection. tropic (hingley et al., ) . in an earlier study (gombold analysis of the mutants isolated earlier from persistently infected cultures (hingley et al., ) . we have used et al., ), we observed that c appeared to demyelinate strikingly less well than wild type virus. to assess the same mutants to ask whether demyelination is also associated with this mutation or perhaps with the repli-the difference in demyelination more quantitatively, we measured the amount of demyelination (as described case mutations that arise later during persistence. thus, the c , c , and c mutants were analyzed for the ability under materials and methods) induced by wild type and c viruses as a function of dose of virus inoculated. to induce demyelination. these results are shown in fig. and summarized in table . the c mutant was able figure shows these results. for wild type mhv-a , the amount of demyelination was approximately linear to induce demyelination with the same efficiency as wild type virus, while the c and c viruses exhibit a very with the log of the amount of virus inoculated. with a dose of pfu (approximately ld s) of wild type weakly demyelinating phenotype, similar to c . this suggests that the demyelination phenotype correlated virus, all the animals infected had demyelination ( / ) and demyelination was present in approximately % of with the appearance of the q l mutation and before the h d and the replicase amino acid substitutions in the spinal cord quadrants examined. we have analyzed demyelination by wild type mhv-a in four experiments. the evolution of the ''c'' culture. the range of demyelination was from to % of the quadrants for a dose of to pfu; thus the discussion amount of demyelination is quite reproducible. at the we have previously shown that the group of fusion same dose c caused no detectable demyelination. delayed, nonhepatotropic mhv-a mutants isolated even at -fold higher dose of inoculation, demyelinfrom two independently infected glial cells had the same ation was observed in / of c infected animals and two, and only two, amino acid substitutions relative to in only % of the quadrants (fig. ) . thus, despite the wild type in the gene encoding the spike glycoprotein s. fact that c replicates to high titer in the brain and the cleavage recognition site amino acid substitution causes encephalitis to a similar extent as wild type virus (h d) clearly correlates with the delayed fusion pheno- (hingley et al., ) , it is a poor inducer of demyelination. type, but not with loss of hepatotropism (hingley et al., similar results were obtained with the b and b mu- ; gombold et al., ) . the other amino acid substitants, isolated from an independent culture of persistution, q l, appeared to correlate with the hepatotroptently infected glial cells (fig. ) . ism phenotype (hingley et al., (hingley et al., , see table ). we concluded this because the q l mutation first ap-association of demyelination with q l peared in virus plaque purified from two independent persistently infected glial cell cultures at weeks postin-an association between the q l substitution in s and the loss of hepatotropism was demonstrated by the fection, at the same time that the altered hepatotropism phenotype became evident. the h d mutation did not esis of this virus. since there is not yet available an infectious cdna clone for mhv, we were not able to appear until weeks postinfection, along with the delayed fusion phenotype (hingley et al., ; table ). introduce individual mutations into the viral genome. therefore, we used an alternative method to map muta-it was not possible to map unambiguously the hepatotropic phenotype to q l when the sequence of the tions that alter pathogenic properties; we sequenced the entire -kb genome of c . rest of the genome was not known. furthermore, the observation that c , b , and b have somewhat dif-surprisingly, there were only five amino acid substitutions in the entire -kb genome of c after weeks ferent pathogenic properties while possessing the same sequence of s (table ; hingley et al., ) suggested of persistent culture in glial cells. this suggests that the fidelity of the mhv polymerase is higher than commonly that mutations outside of s must influence the pathogen- assumed (lai, ; adami et al., ) and/or that, dur-and for sequencing of genes , a, , and (bonilla et al., ; budzilowicz and weiss, ; weiss et al., ; ing persistence in glial cell cultures, there is a strong selective pressure against mutation within the genome. zoltick et al., ) . the only amino acid substitutions in the c genome consistent with this observation was our finding that, in the comparison of our mhv-a lab strain with the other than the two encoded in the s gene were three amino acid substitutions in the replicase gene. there are published sequences, there were just amino acid substitutions and silent mutations. we do not think these two amino acid substitutions encoded in orf a (p s and m k) and one in orf b, r s. the p s sequences differences between the wild type mhv-a parent of c and the published sequences are signifi-substitution is in the predicted plp- domain of orf a and the r s substitution is in the predicted helicase cant in terms of pathogenesis. the mhv-a used as our wild type in these experiments (obtained from dr. l. domain of orf b (bredenbeek et al., ; lee et al., ) (see fig. ). it is difficult to predict whether the sturman) exhibits the same pathogenic properties as the mhv-a (obtained from dr. j. leibowitz) that we used mutation in plp- is likely to be significant. the b mutant also has a mutation, albeit a different one from for our previous pathogenesis studies (lavi et al., b) c , v m in plp- . numerous attempts in our lab rather than the replicase mutations. the relationship of q l to virulence is, however, not as clear. the obser-to detect an activity for plp- had been unsuccessful, suggesting that the plp- domain does not encode an vation that the b mutant retains its virulence (hingley et al., , table ) demonstrates that q l is not active proteinase; if this were the case, then mutations in this region of the replicase gene would not be ex-sufficient for attenuation and furthermore that the loss of hepatotropism does not necessarily lead to attenuation. pected to have a phenotype. however, recently, we have found that a recombinant protein encoded in the pre- the hepatotropic, demyelinating c mutant also retains its virulence while the nonhepatotropic, weakly demyelin-dicted plp- domain does indeed have a proteinase activity (h. teng and s. r. weiss, unpublished results) . ating c and c mutants are somewhat attenuated although probably not as attenuated as c (data not thus, if plp- is a necessary activity for replication, these mutations may be significant. the c mutation in orf shown). thus there may be other mutations in these viruses, absent in c , that modify the virulence pheno- b is in the predicted helicase domain. the recent demonstration of an atpase activity for a recombinant protein type. the data discussed above and shown in table suggest there must be mutations outside of s that result encoded in the predicted helicase domain of orf b of the human coronavirus e (heusipp et al., ) in the differences in hepatotropism between b and c , and differences in virulence between b and the supports the prediction that this is indeed a helicase domain. it is likely that such a polypeptide might interact other mutants. it is possible that the three replicase mutations in c modify the pathogenic properties. we cannot with host cell proteins and thus influence tropism. the third mutation is in orf a, m kl, in a region of the be sure of the effects of these mutations until we can analyze recombinant viruses containing only the q l replicase that has not been assigned a function. the aaucuaaac sequence observed preceding the mutation; such work is in progress. the mechanism of mhv induced demyelination is to m gene of c (different from the wild type aauccaaac) is the sequence found in most of the intergenic regions this date still not well understood. there is mounting evidence that both immune mediated components as in the wild type genome (budzilowicz et al., ; bonilla et al., ; luytjes et al., ) . thus it is clear that this well as direct infection of virus are factors in the establishment of demyelination (houtman and fleming, sequence can function as an intergenic region. since the mechanism of generation of coronavirus mrna is still a). our observation that for wild type mhv-a the amount of demyelination was proportional to the log of not clearly understood, the role of coronavirus conserved intergenic regions is also not known. however, it is be-the virus inoculum (fig. ) argues that the amount of virus in the cns is important in the development of demyelin-lieved that intergenic regions play a role in the control of subgenomic mrna transcription, perhaps serving as ation. this is consistent with the hypothesis that a direct infection mechanism plays a significant role in demyelin-promoters for subgenomic mrna synthesis (van der most and spaan, ) . thus, it is possible that this ation (houtman and fleming, a) . alternatively, the larger virus inoculum could result in a larger number of nucleotide change could effect transcription of the c mrna encoding the m gene. we doubt that this is the persistently infected cells in the cns to be damaged by the immune response. case as the levels of m protein in cells infected with c is approximately the same as in wild type infected cells mhv acute infection includes encephalitis and virus replication at high titer in the cns. it has been shown (unpublished data). while there is no evidence suggesting that any of the mutations in the c genome, that while infectious virus cannot be recovered from the cns after the acute infection, viral rna persists in the either silent or encoding amino acid substitutions, can produce a change in replication or pathogenesis by ef-white matter of the cns probably throughout the lifetime of the mouse (lavi et al., a; adami et al., ) . the fecting the secondary structure of the genome rna, we cannot completely rule out this possibility. group of mutants that we are working with all cause acute encephalitis, but at least in the case of the three we show here that the appearance of the q l mutation within the ''c'' culture of persistently infected glial that we have examined carefully (c , b , b ), there are only minimal levels of demyelination and only at very cells, in addition to correlating with the loss of hepatotropism, also coincides with the development of the high levels of virus inoculated. thus encephalitis does not necessarily lead to demyelination. several other labs weakly demyelinating phenotype. further evidence that the q l mutation is important in the demyelination have reported mutants of the jhm strain of mhv, in which encephalitis and demyelination are dissociated. for ex-phenotype comes from our observations that the b and b mutants, isolated from another parallel glial cell ample the attenuated jhm . -v- mutant (fleming et al., (fleming et al., , and the mhv- mutant a . mutant (dalziel culture, are also weakly demyelinating (fig. ) and contain this mutation in the s gene (hingley et al., ) . et al., ; fazakerley et al., ) cause little encephalitis but do induce significant demyelinating disease. both thus it appears that the demyelination and hepatotropism phenotypes of c are both associated with q l, of these mutants were selected by resistance to mono-clonal antibodies directed against epitopes in the s gene; to a change in viral tropism, perhaps through receptor utilization. thus, the change in pathogenic phenotype is likely to we are currently using targeted recombination techmap to s. the mutants we have selected from persisnology developed by dr. paul masters (masters et al., tently infected glial cells (c , b , and b for example) fischer et al., ) to introduce q l alone into are, to our knowledge, the first examples of mhv mutants the genome of wild type mhv-a . this will allow us to in which encephalitis occurs without subsequent demyedetermine unambiguously the effects of this amino acid linating disease. substitution alone on pathogenesis and interaction of the the location of the q l amino acid substitution mutant protein with the mhv receptor. within the predicted receptor binding domain (kubo et al., ) of s suggests that this mutation may play a acknowledgments role in the development of demyelination and hepatitis by affecting the interaction of s and the viral receptor. this work was supported by public health service grants ns- the inability of c to interact with the receptor on one (s.r.w.) and ns- (s.r.w.) as well as grants rg- a / (s.r.w.) and rg- / (e.l.) from the national multiple sclerosis society. we or more liver cell types would be a reasonable explanathank drs. julian leibowitz and henry teng for reading and commenttion for the lack of hepatitis following infection with c . ing on the manuscript. the difference in the ability of c and wild type virus to induce demyelination could also be related to the tro-references pism for a certain cell type. while we have shown that similar localization in the limbic system (hingley et al., virology , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ), we are currently studying the spread of the two armstrong, j., smeekens, s., and rottier, p. ( ) . sequence of the nucleocapsid gene of the murine coronavirus mhv-a . nucleic viruses to various cell types and the persistence in the acids res. , - . cns after the acute disease and viral clearance occurs. armstrong, j., niemann, h., smeekens, s., rottier, p., and warren, g. the region defined as a receptor binding domain in- ( ) . sequence and topology of a model intracellular membrane cludes the amino terminal amino acids of s (kubo protein, e glycoprotein, from a coronavirus. nature (london) , - . et al., ) . amino acid q , and the region sur- hepatotropism and demyelination via another mecha-nucleic acids res. , res. , - nism, perhaps due to a step in replication after binding budzilowicz, c. j., wilczynski, s. p., and weiss, s. r. ( ) . three inand entry. tergenic regions of coronavirus mouse hepatitis virus strain a contain a common nucleotide sequence that is homologous to the another possible explanation for the different patho- pathogenicity of antigenic variants of murine of self reactive t cells after murine coronavirus infection interaction of immune and central nervous systems: tence of mhv-a rna in a slow virus demyelinating infection in contribution of thy- / cells to demyelination induced by coronavirus mice as detected by in situ hybridization experimental demyelination produced by the a strain of tion resistant variants of a neurotropic coronavirus are generated by mouse hepatitis virus fusion-defective tis virus (mhv)-a causes a persistent, productive infection in primutants of mouse hepatitis virus a contain a mutation in the spike mary glial cell cultures mouse hepatitis virus a -induced demyelination can occur sequence of the murine coronavirus gene encoding the putative in the absence of cd / t cells primary strucencoded in gene of the human coronavirus e sequence of mouse hepatitis virus a mrna : virology , - . indications for rna recombination between coronaviruses and influ analysis of the receptor binding site dissociation of demyelination transcription and rna recombination. in ''the coronaviradae'' (s. g. and viral clearance in congenitally immunodeficient mice infected siddell localization of neutralizmouse hepatitis virus (mhv), strain a contains two orfs and thus ing epitopes and the receptor binding site within the amino-terminal differs from ns of the jhm and s strains - . mouse hepatitis virus orf a is expressed in the cytosol of infected mouse fibroblasts key: cord- -c e rai authors: daniel, claude; lamarre, alain; talbot, pierre j. title: increased viral titers and enhanced reactivity of antibodies to the spike glycoprotein of murine coronavirus produced by infection at ph date: - - journal: journal of virological methods doi: . / - ( ) - sha: doc_id: cord_uid: c e rai abstract infection of cell monolayers by murine coronavirus a at ph rather than yielded a ten-fold increase in the infectious titer and a remarkable enhancement of the reactivities of monoclonal and polyclonal antibodies against the spike glycoprotein in immunoblotting, immuno-precipitation and enzyme-linked immunosorbent assays. these observations are very useful for detecting antibodies against the s glycoprotein of coronaviruses and enhancing infectious titers. murine hepatitis viruses (mhv) are members of the coronaviridue, a family of enveloped, positive-stranded rna viruses. these pathogens are associated with various respiratory, gastrointestinal and neurological infections in mammals and fowls (wege et al., ) . indeed, infection of mice by the neurotropic strains a and jhm provides an excellent animal model of virus-induced diseases of the central nervous system (ter meulen et al., ) . three structural proteins were identified on mhv-a virions (sturman and holmes, ) : the nucleoprotein (n, kda), the membrane glycoprotein (m, kda) and the spike glycoprotein (s, kda; sl/s , ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the s glycoprotein is essential for infectivity; as it mediates viral attachment to a cell receptor (williams et al., ) and virus-induced cell fusion (vennema et al., ) . recent studies have shown that fusion induced in cell monolayers infected with mhv-a was reduced in slightly acidic culture medium . the conformation of s was also shown to be affected by ph variations. this was demonstrated by aggregation of the c-terminal membrane-anchored s and the release of the n-terminal sl after treatment of virions at ph . and °c . moreover, this treatment modified the antigenicity of sl (weismiller et al., ) . in this study, we show that infection by mhv-a in culture medium at ph , instead of the usual ph , not only increased infectious viral titers, but also greatly enhanced the reactivity of the spike glycoprotein to antibodies. the murine hepatitis virus, strain a. , was obtained from the american type culture collection (rockville, md), plaque purified twice, and passaged on dbt cells, a murine cell line established from a delayed brain tumor in a cdfl mouse inoculated intracerebrally with strain schmidt-ruppin of rous sarcoma virus (hirano et al., ) , as described previously (daniel and talbot, ) . viral growth kinetics in media initially adjusted to ph or were compared. dbt cell monolayers of identical surface and cell passage were infected at a multiplicity of infection of . in medium at either phs. media from different culture flasks were harvested at h intervals after infection and frozen at - °c until assayed. cell monolayers were lysed by three cycles of freezing and thawing in an equivalent volume of medium at the appropriate ph. viral titers were determined by a plaque assay (daniel and talbot, ) . as shown in fig. , growth kinetics at either phs were similar, with optimal titers reached between and h post-infection. however, viral titers were reproducibly - times higher in ph samples throughout the course of the infection. examination of virions in cell supernatants and lysates under the electron microscope did not reveal significant morphological changes that would indicate a variation in peplomer density or conformation (data not shown). interestingly, cytopathic effects observed after infection, which consisted of cell-to-cell fusion into multinucleated syncytia, were very similar (extent and time course of syncytia formation) when cells were infected in medium at ph or . indeed, the dbt cells were almost completely fused at the time of optimal infectious viral titers. this is in contrast to the previously reported decreased virus-induced fusion index of -cl-l cells infected at ph . presumably, different cell types are differentially susceptible to the effect of ph on the development of virus-induced cytopathic effects. indeed, host-dependent differences in mhv-a -induced cell fusion have been reported (frana et al., ) . the antigenicity of the structural proteins of viruses produced by infection at ph or were evaluated by western immunoblotting, immunoprecipitation, and elisa. the monoclonal antibodies (mabs) used in these experiments were raised against mhv-jhm or mhv-a as described previously ( b : collins et al., ; -loa, - lg and l- f: daniel and talbot, ; e : wege et al., ) . the mabs b and el were kindly provided by drs. m.j. buchmeier (research institute of scripps clinic, la jolla, ca) and h. wege (university of wiirzburg, germany), respectively. mabs b , -loa, - lg and l- f are neutralizing in vitro and, with the exception of l- f, inhibit fusion in mhv-infected cell cultures. the epitope recognized by mab l- f has been mapped to sl, and those recognized by mabs b , e , - a and - lg to s (daniel et al., ; luytjes et al., ) . to prepare antigen for western immunoblotting, dbt cell monolayers were infected as described above, and medium harvested at h post-infection. concentrated viral proteins (daniel and talbot, ) were solubilized in a sample buffer containing sds and p-mercaptoethanol, separated by sds-page on a - % linear polyacrylamide gel (laemmli, ) , and electro-transferred to nitrocellulose paper for h at v. this was sufficient to transfer all proteins, as shown by the absence of coomassie blue staining of the gels after electrotransfer. immunoblotting was performed with a mouse hyperimmune serum specific for mhv-a (diluted l/ ), or with anti-s mabs ( b , culture supernatant diluted l/ ; e , ascites fluid diluted l/ ), according to talbot et al. ( a) , except that . % (v/v) tween- in phosphate-buffered saline (pbs) was used as blocking and washing buffer, and bound mabs were revealed by incubation with horseradish peroxidase-labeled, goat antibodies to mouse ig (kirkegaard & perry laboratory, gaithersburg, md; diluted l/ ), followed by incubation with hydrogen peroxide and -chloro- -naphthol (hawkes et al., ) . the reactivity of the anti-s mabs (shown here: b and e ) against the sl/s ( kda) subunits of virus produced by infection at ph was evident, whereas reaction against viral antigen produced by infection at ph was barely detectable (fig. ) . the mhv-a hyperimmune serum also reacted more intensely with antigen produced by infection at ph , although reactivity with the s glycoprotein was very faint. the prominent reactivity of this antiserum in western blot against the nucleoprotein ( kda) suggests a greater immunogenicity of this protein or its inherent property to bear more linear epitopes. to determine whether this enhanced reactivity could be observed with antibodies that bind to discontinuous, conformation-sensitive epitopes, immuno-precipitation was carried out as described previously (daniel and talbot, fig. . radio-immunoprecipitation of virus-infected cell lysates produced at ph or with mhv-specific antibodies. metabolically labeled virus-infected cell lysates were immunoprecipitated with mhv-specific antibodies ((y-as : hyperimmune serum; - a: anti-s mab; : control ascites fluid) and resolved by sds-page on a - % gel. a hyperimmune serum against mhv-a ( ~ of a l/ dilution), anti-s or control hybridoma ascites fluids ( ~ ). from this experiment (fig. , it appears that a large proportion of the anti-s antibodies in the mhv-a hyperimmune serum recognize conformational epitopes, since this antiserum reacted strongly with s in immunoprecipitation (fig. ) but not in western immunoblotting (fig. ) . reactions against the nucleoprotein (n) were similar between the two antigens, whereas the membrane protein (m) was hardly detectable. it is also obvious that the enhanced reactivity was observed with discontinuous epitopes, since mab - oa and mhva hyperimmune serum reacted more strongly with s and sl/s of virus produced at ph . finally, the reactivity of antisera or mabs against antigens produced by infection at ph or were compared in elisa. the antigen used in the elisa was prepared by infecting and mock-infecting l cell monolayers with mhv-a. in medium at ph or . confluent monolayers were lysed by freezing and thawing, sonicated x min in a bransonic sonicating water bath (branson cleaning equipment company, shelton, ct), and clarified by centrifugation at x g for min. membranes (viral and cellular) were pelleted by ultracentrifugation at x g for h, resuspended in pbs ( / .of the original volume) and total protein concentrations evaluated. elisa with hyperimmune serum against mhv-a , or anti-s mabs was performed according to previously published procedures (talbot et al., b) . as shown in fig. , the anti-s mabs reacted much more strongly with antigen produced by infection at ph , in contrast to the mhv-a hyperimmune serum, which reacted equally well with both antigens. in the representative experiments shown in fig. , the reactivity of mab - a was enhanced fold, and those of mabs l- f, e , and -llg, -fold, fold and s-fold, respectively. in elisa, mhv-jhm antigen produced by infection at ph also showed enhanced antigenicity, in comparison with ph -produced antigen, using jhm-specific mabs (data not shown). from these results, it is concluded that the optimal virus stability at ph observed previously (daniel and talbot, ) contributed to the higher titer observed for virus produced at this ph, since prolonged cell culture survival resulting from reduced cell fusion was not observed in dbt cells. furthermore, antibody reactivity to the s glycoprotein produced after infection in acidic medium was much enhanced. comparison of viral antigens produced at ph . or . by sds-page and coomassie blue staining did not reveal significative differences in the total amount of s glycoprotein (data not shown). however, further studies using the s protein purified in native conditions will be needed to demonstrate that a structural modification of the protein produced at ph . is responsible for the enhanced reactivity of antibodies. this study shows that the production of murine coronavirus antigen in slightly acidic medium would be very advantageous for the detection of anti-s reactivity in sera containing antibodies of low titers and/or low affinity. indeed, this procedure was successfully used to reveal cross-reactivity between anti-peptide antibodies and the s glycoprotein (daniel et al., ) . moreover, the increased infectious viral titers observed after infection at ph will have an important practical value for studies with these viruses. monoclonal antibodies to murine hepatitis virus- (strain jhm) define the viral glycoprotein responsible for attachment and cell-cell fusion physico-chemical properties of murine hepatitis virus, strain a protection from lethal coronavirus infection by affinity-purified spike glycoprotein of murine hepatitis virus, strain a identification of an immunodominant linear neutralization domain on the s portion of the murine coronavirus spike glycoprotein and evidence that it forms part of a complex tridimensional structure mapping of linear antigenic sites on the s glycoprotein of a neurotropic murine coronavirus with synthetic peptides: a combination of nine prediction algorithms fails to identify relevant epitopes and peptide immunogenicity is drastically influenced by the nature of the protein carrier proteolytic cleavage of the e glycoprotein of murine coronavirus: host-dependent differences in proteolytic cleavage and cell fusion a dot-immunobinding assay for monoclonal and other antibodies replication and plaque formation of mouse hepatitis virus (mhv- ) in mouse cell line dbt culture cleavage of structural proteins during the assembly of the head of bacteriophage t amino acid sequence of a conserved neutralizing epitope of murine coronaviruses the molecular biology of coronaviruses conformational change of the coronavirus peplomer glycoprotein at ph . and °c correlates with virus aggregation and virus-induced cell fusion western and dot immunoblotting analysis of viral antigens and antibodies: application to murine hepatitis virus topographical mapping of epitopes on the glycoproteins of murine hepatitis virus- (strain jhm): correlation with biological activities intracellular transport of recombinant coronavirus spike proteins: implications for virus assembly hybridoma antibodies to the murine coronavirus jhm: characterization of epitopes on the peplomer protein (e ) the biology and pathogenesis of coronaviruses monoclonal antibodies to the peplomer glycoprotein of coronavirus mouse hepatitis virus identify two subunits and detect a conformational change in the subunit released under mild alkaline conditions receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins we thank francine lambert for excellent technical assistance. we also thank m.j. buchmeier and h. wege for the generous gift of their respective mabs. this study was supported by grant ogp from the natural sciences and engineering research council of canada (nserc) and in part by grant mt- from the medical research council of canada (mrc) to p.j.t., who also acknowledges a scholarship from nserc.c.d. and a.l. are grateful to nserc and 'fond de la recherche en sante du quebec', and 'fond pour la formation des chercheurs et 'aide 'a la recherche', respectively, for studentship support. key: cord- -q rsfzgw authors: lavi, ehud; wang, qian; weiss, susan r.; gonatas, nicholas k. title: syncytia formation induced by coronavirus infection is associated with fragmentation and rearrangement of the golgi apparatus date: - - journal: virology doi: . /viro. . sha: doc_id: cord_uid: q rsfzgw abstract coronavirus mouse hepatitis virus (mhv) possesses a membrane glycoprotein (m) which is targeted to the golgi apparatus (ga). we used immunocytochemistry with an organelle-specific antiserum to investigate the morphologic changes of the ga during infection of l murine fibroblasts with mhv-a . twenty-four hours after infection the ga was fragmented and translocated in the center of syncytia, while the microtubular network was also rearranged displaying radiating elements toward the center of syncytia. two fusion-defective mutants, which contain an identical amino acid substitution in the cleavage signal sequence of the spike glycoprotein (s), induced fragmentation of the ga. however, the ga migrated only partially to the centers of syncytia during infection with these mutants. revertant viruses, in which the above mutation was corrected, had fusion properties and ga staining similar to wtmhv-a . experiments with brefeldin a (bfa), which induces redistribution of the ga into the rough endoplasmic reticulum (rer), revealed that an intact ga for a period of – hr postinfection, is required for coronavirus replication and syncytia formation. thus, during mhv infection, syncytia formation is associated with fragmentation of the ga, followed by a previously undescribed phenomenon of migration of the organelle into the centers of syncytia. the fragmentation of the ga, however, may occur without the formation of syncytia. therefore, two distinct mechanisms may be responsible for the fragmentation of the ga and its subsequent migration to the center of syncytia. undergo endocytosis into the trans-golgi-network (tgn), while shiga toxin is internalized into the ga and the rer polypeptides synthesized in the rough endoplasmic (sandvig et al., ; . pretreatment of cells reticulum (rer) and destined for plasma membranes, with bfa protects them against the lethal effects of cerlysosomes, and secretion are transported through the tain toxins, implying that the toxin's entry into tgn and golgi apparatus for posttranslational modifications and the ga is necessary for translocation into their cytosolic targeting (farquhar and palade, ; mellman and sitargets (yoshida et al., ) . in the human disease mons, ; rothman and orci, ) . the ga is a dyamyotrophic lateral sclerosis (als), the ga of spinal cord namic organelle which undergoes morphologic and funcmotor neurons is fragmented into numerous small eletional modifications under physiologic and pathologic ments which resemble the dispersion of the organelle conditions. in mitotic hela cells the ga disperses in early induced by agents depolymerizing microtubules (moureprophase and reaggregates in telophase (robbins and latos et al., , ; gonatas et al., ) . a similar gonatas, ) . during mitosis, the golgi apparatus fragfragmentation of the ga has been observed in motor ments into numerous small groups of vesicles which neurons of transgenic mice expressing a mutant cu,zn have been referred to as the mitotic form of the organelle superoxide dismutase (mourelatos et al., ) . (lucocq et al., ) . in interphase cells treated with migration and rearrangements of the ga have been drugs which depolymerize microtubules, the ga fragreported during syncytia formation and fusion of vero ments into small randomly distributed elements (robbins cells infected with sindbis virus (ho et al., ) . within and gonatas, ; turner and tartakoff, ) . in many - hr after infection, individual elements of the ga, cells the secretion blocker brefeldin a (bfa) reversibly which are associated initially with separate microtubuleredistributes membranes and enzymes of the ga back organizing centers in perinuclear areas of fused cells, into the rer, but does not inhibit endocytosis (doms et congregate in the center of syncytia and form an exal., ; lippincott-schwartz et al., ; johnston et al., tended network of nondisrupted intact golgi complexes ). ricin, cholera toxin, and wheat germ agglutinin (ho et al., ) . in contrast to sindbis infections, the golgi apparatus is fragmented in cells infected with herpes simplex virus (campadelli et al., ) . is characterized by retention of the viral envelope glyco- ). the primary structure of mg- revealed significant homology with a chicken fibroblast growth factor protein m (previously known as e ) within the ga (tooze et al., ; klumperman et al., ; krijnse-locker et receptor and a ligand to e-selectin (gonatas et al., ; steegmaier et al., ) . in chicken, mg- appears al., ) and budding of virions from internal membranes (holmes and behnke, ; tooze et al., ) . the close early in development and the gene coding the protein, named glg , has been assigned to chromosome interaction between coronavirus particles with the ga provides an unusual opportunity to study morphologic (stieber et al., ; mourelatos et al., ) . and functional properties of the ga and various aspects of virus-cell interactions. in this study, we used organ-immunohistochemistry elle-specific antibodies, immunohistochemistry, and transmission electron microscopy to examine the fate of cells, grown on poly-d-lysine-treated coverslips, were the ga during cell fusion and syncytia formation in mouse fixed with % paraformaldehyde for min at room tem-l- cells infected with mhv-a and fusion defective perature, washed three times in pbs, then incubated for mutants. fragmentation of the ga into small immuno- min in . % saponin/ % goat serum (gs) in pbs stained elements occurred prior to their migration into and washed three times in % gs in pbs. cells were the centers of syncytia. experiments with bfa revealed then incubated with primary antibody ( : dilution in that the period of - hr postinfection is critical for pbs of immunoaffinity-purified rabbit anti-mg- anticoronavirus replication and syncytia formation. the rebodies or with a supernatant from the anti mg- hysults of studies with fusion defective mutants suggest bridoma ( a ) overnight at room temperature. cultures that different mechanisms are responsible for the initial were then washed, incubated with a biotinylated goat fragmentation of the ga and its subsequent migration to anti-rabbit igg antibody, incubated with the avidin-biotin the centers of syncytia. complex (abc), and stained with diaminobenzidine tetrahydrochloride (dab) ( mg dab/ ml tris-saline con-materials and methods taining mm imidazole and . % h o ), according to standard methods (graham and karnovsky, ; gues-cell cultures don et al., ) . l- cells (murine fibroblasts), originally obtained from american type culture collection (atcc; rockville, md), electron microscopy were used to maintain viral growth cultures, preparation of viral stocks, and viral plaque assays. in some experi-cells grown on thermanox plastic (em sciences, fort ments cl- murine fibroblasts were used. the cultures washington, pa), were fixed overnight at Њ with . % were maintained in dmem/heat-inactivated % fetal boglutaraldehyde / % paraformaldehyde in . % cacodylvine serum/ % pen/strep/ mg/liter d-glucose/l-gluate buffer, ph . , / . % cacl . subsequently cultures tamine. were postfixed in % osmium tetroxide / . % potassium ferrocyanide, dehydrated in ethanol, and embedded in viruses and infections araldite. sections ( - nm thick) were stained with lead and uranyl salts and viewed in a transmission electron three times plaque purified mhv-a stock virus was microscope (jeol cx) at kv (karnovsky, ; used as previously described (lavi and weiss, ) . stieber et al., ) . fusion defective mutants and revertants were prepared as previously described (gombold et al., ; hingley et al., ) . infections of cultures were done by incuba-viral infectivity assay tion of virus with cells for hr at a multiplicity of infection (m.o.i.) of , followed by washing of cells with fresh me-viral titers were determined by duplicate plaque dium three times. assays of several -fold dilution of samples in l- cell grown in six-well plates (lavi et al., ) . mg- is a conserved sialoglycoprotein of the medial brefeldin a (bfa) treatment cisternae of the ga. the preparations of the anti-mg- monoclonal antibody ( a ), and the immunoaffinity bfa (sigma) stock solution ( mg/ml in ethanol) was diluted in pbs and applied to cultures at a concentration purified anti-mg- polyclonal antibodies, were described in previous publications (gonatas et al., ; of mg/ml for periods of hr or longer, as specifically indicated in the text (fig. ) . in control experiments cul- croul et al., croul et al., , . the preparation of the monoclonal antibody h , a rer marker of a -to -kda tures were incubated for the same time periods with the same dilution of ethanol used to dissolve bfa. protein, has been previously described (chen et al., and cyncytia formation when infected with mhv-a . following infection, cells were fixed and examined by immu-changes of the ga during mhv infection and nohistochemistry with identical methods used for l formation of syncytia cells. the changes in the ga observed in these cells were identical to those seen in l -infected cells. morphological changes of the ga during coronavirus infection of l cells ( , , , , and hr) were examined by immunocytochemistry using organelle-specific changes of the ga following infection of l cells with antibodies (croul et al., (croul et al., , gonatas et al., ) . fusion-defective mutants and a fusion-negative strain typical infection of l cells with mhv-a caused syncyof mhv tia formation of the entire monolayer within hr and complete cytolysis within hr. at hr postinfection, to investigate whether the fragmentation of the ga is dependent on cell fusion, we examined the organelle in cell borders were indistinct and the nuclei were aggregated; by hr postinfection, the syncytia acquired their l cells infected with two fusion defective mutants of mhv-a . the c and b mutants, used in these ex-typical morphology consisting of a ring of nuclei surrounding a cytoplasmic center devoid of nuclei. between periments, were isolated from primary cultures of glial cells infected with mhv-a . these mutants are fusion- and hr postinfection, cells underwent pyknosis, karyorhexis, and then died and detached from the culture delayed and defective but not fusion-negative and produce reduced number of syncytia formations. thus at dish. the immunostained ga of uninfected cells formed con-any given time point postinfection with the mutant viruses, only a small percentage of l cells form syncytia, tiguous coarsely granular focal or ring-like perinuclear profiles (figs. a and d). following infection of l cells as compared to cells infected with mhv-a (gombold et al., ) . these fusion-defective mutants have a histi-with mhv-a , the coarsely granular stain of the ga became fine and smaller individual elements of the ga dine to aspartic acid mutation (h d) within the cleavage signal of the spike (s) glycoprotein. cleavage of s were discernible. at hr postinfection, the majority of the syncytia consisted of clustered nuclei within a cyto-is necessary for efficient cell fusion during mhv-a infection (gombold et al., ) . infection with these mutant plasmic mass, lacking distinguishable cell borders; in those syncytia, the immunostained ga appeared as viruses does not affect the efficiency of viral replication or titers of infectious virus (gombold et al., ) . immu-strands of finely granular elements forming a honeycomb-like network with interspersed nuclei (figs. b and nostaining of the ga in l cultures infected with the fusion defective mutants b and c showed fragmen- e). however, before full syncytia formation, in cells surrounding the forming syncytia the distribution of the ga tation of the ga similar to that found in cells infected with mhv-a . however, the translocation and rear-was perinuclear, similar to that of controls but with finer granular ga elements (compare figs. e and d) . this rangement of the ga during infection with these two mutant viruses was distinctly different from the changes phenomenon probably represented an early stage of fragmentation and rearrangement of the ga. at hr of the organelle seen during infections with the wild-type mhv-a . specifically, cells infected with either b or postinfection, the process of fragmentation of the ga and its relocation in the centers of syncytia, as well as the c mutants displayed both central and peripheral aggregates of finely granular ga (figs. h and i). the rearrangement of the nuclei in a ring formation within the syncytia, was complete (figs. c and f) . the honey-granular stain of the ga at the periphery of syncytia, and specifically around the peripherally located nuclei, was comb morphology due to the interspersed nuclei was replaced by a typical central aggregate of finely granular observed only in cultures infected with these two mutant viruses. this pattern of ga distribution was not seen in or fragmented ga, surrounded by a rim of nuclei (compare figs. f with e). furthermore, the nuclei in the any of the stages of mhv-a viral replication (compare figs. i and f ). in order to rule out the possibility that syncytium were not surrounded by any residual ga. thus, in the fully developed syncytium, all elements of the frag-the fusion-defective variants showed only delay in translocation of the ga into the center of cyncytia, immunocy-mented ga had moved into the center, while the more peripherally arranged nuclei formed a ring devoid of adja-tochemical analysis was performed at and hr after viral infection. these experiments showed that the typical cent elements of the ga (fig. f) . staining of syncytia with rabbit anti-mhv polyclonal antibodies revealed an complete central translocation of the ga, similar to that seen in a -infected cultures, never occurred in cultures abundance of viral proteins in the center of syncytia (not shown). infected with the two fusion defective mutants, even prior to or at the stage of complete cell death. in order to determine whether the fragmentation and rearrangement of the ga was cell-type dependent, an-to further explore the link between fragmentation of the ga and fusion we then infected l cells with mhv-other cell line, cl- murine fibroblasts, was infected with mhv-a . this cell line supports both cell fusion , a fusion-negative strain of mhv. l cells are suscepti- the network of microtubules is rearranged during mhv-a infection pletely destroyed after hr with titers similar to mhv-a . however, there was no cell fusion or syncytia forma-since the ga is associated with microtubules of tion in these cultures. infection with mhv- produced interphase cells (robbins and gonatas, ; turner dispersion and fragmentation of the ga in individual and tartakoff, ) , we investigated whether microtucells, some of which appeared to be balooned after bules are affected during coronavirus infection. spehr (fig. g) . these observations are consistent with the cifically, we investigated whether the fragmentation of conclusion that the fragmentation of the ga during infecthe ga within the centers of the syncytia is associated tion with mhv is independent of cell fusion. however, with a similar change of the microtubules. the immuthe translocation of the ga in the center of the syncytia nocytochemical staining of the ga at , , , , and is probably linked to cell fusion since fusion-defective hr after mhv-a infection was compared with the mutants were also defective in their ability to induce immunostaining of microtubules with antibodies translocation of the ga to the center of the syncytia. against alpha and beta tubulin. while the ga appeared fragmented early during infection and syncytia forma-ultrastructural changes of the ga in mhv-infected tion, fragmentation and disintegration of microtubules cells occurred late ( hr), when cells die. the kinetics of to further investigate the morphologic aspects of the the microtubule changes after infection with mhv-a fragmented ga in syncytia of l cells infected with mhvwas depicted by immunofluorescence on l- cells a , an electron microscopic examination was perafter infection with mhv-a (m.o.i. Å pfu/cell) and formed in cells hr after infection and at a multiplicity after staining with anti-tubulin antibodies and fitcof infection (m.o.i.) of plaque forming unit per cell. areas conjugated secondary antibodies (fig. ) . in uninof syncytia with a typical peripheral rim of nuclei were fected cells and at hr postinfection the microtubules selected from semithin ( . - mm) sections. in uninwere distributed throughout the entire cytoplasm of fected cells the ga was seen in a perinuclear location the individual cells. at hr postinfection the syncytia and consisted of several groups of stacked cisternae were beginning to form and the microtubules were surrounded by numerous coated and uncoated vesicles still distributed within the entire cytoplasm. at hr (fig. b) . in contrast to this typical morphology of the ga, postinfection, when the ga was fragmented and transin infected cells the stacks of the cisternae were marklocated to the centers of syncytia, the microtubules edly diminished in size and were replaced by numerous were rearranged in a characteristic pattern (fig. ) . tubulovesicular structures, some of which containing vi-specifically, at the periphery of the syncytia, an inrus particles ( fig. a) . furthermore, in infected cells the tense stain for tubulins suggested that the nuclei were region containing remnants of ga cisternae and the surrounded by a rich network of microtubules. at the abundant tubulovesicular structures was rich in intermediate zone, between the periphery and the centransversing microtubules ( fig. a) . ter of syncytia, the microtubules formed a radiating network. at the center of the syncytia, the immunostain the distribution of the rer is not affected by mhvfor alpha and beta tubulin was less intense and amor-a infection phous (fig. ) . these changes suggest that during coronavirus infection the microtubules undergo rear-the distribution of the rer in mhv-a -infected and rangement and perhaps provide guidance for the control l cells was investigated by immunocytochemtranslocation of the fragmented elements of the ga istry with the organelle-specific monoclonal antibody into the center of the syncytia. h . there was no detectable difference between the infected and uninfected cells in the the effect of brefeldin a on coronavirus infection immunostaining of the rer. in both cases the fine granular staining of the rer was evenly distributed since coronavirus infection is associated with the processing of viral proteins through the ga, including within the entire cytoplasm including the centers of syncytia (not shown). within the organelle, we investigated the effect of bfa hr later. uninfected cells, however, had completely recovered from the bfa effect - hr later. these on the morphology and kinetics of coronavirus infection. as summarized in fig. , bfa was introduced at findings suggest that virus infection accentuates and prolongs the bfa effect (fig. ) . various time points during infection of l cell with mhv-a . in uninfected l cells incubated for hr when cells were treated continuously with bfa during infection there was dispersion of the ga, lack of syncytia with mg/ml of bfa, immunostaining with anti-mg- showed a diffuse cytoplasmic pattern consistent formation, and no detectable viral titers (fig. ) . if bfa treatment began at or hr after viral inoculation, the with the known redistribution of mg- and other golgi markers within the rer (doms et al., ; lip- effect of this treatment on infection was similar to the effect observed after continuous bfa treatment (i.e., re-pincott- schwartz et al., ; johnston et al., ) . following hr of bfa treatment at the beginning of duced syncytia formation and no viral titers). however, when the bfa treatment began hr or more after viral infection cells had diffuse staining of the ga when observed hr postinfection as did uninfected cells inoculation, there were relatively minimal effects on syncytia formation, and viral titers were - logs lower as following a similar treatment with bfa. however, at hr postinfection (and hr of treatment with bfa at the compared to viral titers without bfa treatment (which usually fluctuate about - . logs in various experi-beginning of infection) there was incomplete recovery from the bfa effect. in uninfected cells treated in a ments). when bfa was introduced min or an hour before similar fashion with bfa, the recovery was complete. therefore, when cells were infected with mhv-a inoculation and continued through the entire infection or , , or hr after inoculation, there was no significant prior to or at the same time of treatment with bfa, the effect of the drug on the ga was not abolished or effect on the ability of the virus to form syncytia hr by cisternae derived from the intermediate compartment between the er and the golgi stacks, thus acquiring two membranes in one step (sodeik et al., (sodeik et al., , . the second wrapping cisternae in vaccinia virus assembly is derived from the trans golgi network (schmelz et al., ) . in cmv, ultrastructural as well as biochemical studies suggested that short-term exposure of infected cultures to bfa during the late infectious cycle primarily prevented golgi-dependent processes, e.g., envelopment of naked cytoplasmic nucleocapsids in the trans-golgi network (tgn) and normal processing of glycoprotein b (eggers et al., ) . in uukuniemi virus, a member of the bunyaviridae, immunofluorescent staining indicated that g glycoprotein expressed alone localized to the ga. g expressed alone was associated with the rer (melin et al., ) . coronavirus mhv m glycoprotein (previously known as e ) is targeted to the ga and contains a retention signal for the ga (machamer et al., ; swift and machamer, ; armstrong and patel, ) . previous reports have shown that the ga undergoes rearrangement (ho et al., ) and fragmentation (campadelli et al., ) in viral infections. the data presented here detail the changes in the morphology of the ga in cells infected by a virus which induces the formation of syncytia. in coronavirus infection, the virus displays cultures were incubated with virus (") for hr and exposed to bfa (ٖ) for various periods. at the end of -hr incubation the cultured cells complex and close interactions with the ga. in coronaviwere stained for mg- by immuohistochemistry and the supernatants rus-induced syncytia formation there is a unique translowere titered for virus by plaque assay. titers labeled indicate levels cation and aggregation of the ga into the center of the below pfu/ml, which is the lowest level of detection in this assay. syncytia which is not accompanied by similar changes of the rer and cytoskeleton (fig. f) . the observed fragmentation of the ga is not related to the formation of postinfection and viral titers at that time were only - . syncytia as demonstrated by the experiments using a logs lower than without treatment. thus bfa did not fusion-negative strain of mhv and fusion-defective mublock the endocytosis and the initial processing of the tants in which fragmentation of the ga also occurs. fragvirus into cells. bfa reduced viral replication only by mentation and translocation of the ga may be important - logs when introduced at - or - hr after in the life cycle of coronavirus replication. further studies infection. the results of the exposure to bfa at the beginare necessary to determine whether there are viruses ning of infection for hr and for various periods at the that replicate within cells without causing alteration of end of infection (fig. ) indicated that the bfa effect on ga morphology. the ga during - hr postinoculation was associated central translocation and aggregation of the ga may with reduced viral replication. if bfa was introduced after be unique to coronaviruses as it was not associated with or before this stage ( - hr), there was no effect on other fusion and syncytia forming viruses such as sindbis viral replication, assembly, and maturation. these results virus infection (ho et al., ) or herpes simplex virus suggest that the interval of - hr postinfection is the infection (campadelli et al., ) . since the aggregation most significant period requiring an intact ga for viral of the fragmented ga in the centers of syncytia has not replication and syncytia formation. this conclusion is been previously reported, we investigated the ga in cells based on infection at an m.o.i. of pfu/cell which may infected with lacrosse bunyavirus, which is known to be an asynchronous infection. in a more synchronous induce the formation of syncytia (gonzalez-scarano et infection the period of bfa effect may terminate earlier al., ) . immunostaining of the ga in bhk cells infected than hr postinfection. with lacrosse virus for hr showed network formation and fragmentation of the ga, without the aggregation of discussion the ga into centralized zones and without the formation of amorphous centers of syncytia which is characteristic the ga plays an important role in the life cycle of many viruses such as vaccinia, cmv, bunyaviruses, and of mhv infection (unpublished observations). thus the translocation of the ga to the center of the syncytia may coronaviruses. vaccinia virus dna becomes enwrapped evolution of a coronavirus during persistent infection in vitro use of ferrocyanide-reduced osmium tetroxide cessed through the golgi apparatus after infection with herpes simin electron microscopy coronavirus m proteins accumulate in the - kd membrane polypeptide of the rough endoplasmic reticulum golgi complex beyond the site of virion budding an anti-organelle antibody in pathology. the chromatolytic reaction studied with a characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the rer to the golgi complex rothman experimental demyelination produced by the a strain of ture and toxic effect assembly of vaccinia virus: the second lar aspects of neurotropic viral infections assembly of vaccinia virus: incorporation of p treated with brefeldin a: evidence for membrane cycling from golgi and p into the membrane of the intracellular mature virus form of the golgi apparatus in hela cells assembly of vaccinia virus: role of the intermediate compartment between the endoplasmic reticulum and the golgi stacks proteolytic cleavage golgi complex ronnholm, r., and fusion activity virus contains a signal for localization to the golgi complex the golgi complex: in vitro veritas? fragmentation of the golgi apparatus of motor anti-organelle antibody neurons in amyotrophic lateral sclerosis revealed by organelle-spe-stieber mg- , a membrane protein of the golgi apparatus assignment of the glg gene for mg- , a fibroblast ligand for e-selectin, is found only in the golgi apparatus and apgrowth factor and e-selectin binding membrane sialoglycoprotein of pears early in chicken embryo development - . membrane-spanning domain of coronavirus e protein fusion formation by the uncleaved spike protein of in transgenic mice expressing mutant cu,zn superoxide dismutase murine coronavirus jhmv variant cl- acetyl-galactosamine to the e glycoprotein of mouse hepatitis virus-mourelatos histochemical and ultrastrucand membrane traffic in golgi complex organization the ultrastructure of a mammaof the golgi apparatus by brefeldin a inhibits the cytotoxicity of ricin, modeccin, and pseudomonas toxin monoclonal antibody against the golgi apparatus. am. j. pathol. , be related to a unique interaction between coronavirus - . proteins with the membranes of the ga. croul, s. e., mezitis, s. g. e., stieber, a., chen, y., gonatas, j. o., goud, since mutant viruses that are defective in their ability b., and gonatas, n. k. ( ) . immunohistochemical visualization of to cause efficient fusion are also inefficient in translocatthe golgi apparatus in several species, including human, and tissue ing the ga to the center of the syncytia, the translocation with antiserum against mg- , a sialoglycoprotein of rat golgi apparatus. j. histochem. cytochem. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] of the ga appears to be linked to the ability of the virus doms, r. w., russ, g., and yewdell, j. w. ( ) . brefeldin a redistribto cause fusion. although the fusion property has been utes resident and itinerant golgi proteins to the endoplasmic reticuassociated with the s gene and the site encoding cleav- lum. j. cell biol. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] age of the s protein (gombold et al., ) , other reports eggers, m., bogner, e., agricola, b., kern, h. f., and radsak, k. ( ). showed that cleavage of the s gene in coronaviruses is inhibition of human cytomegalovirus maturation by brefeldin a. j. gen. virol. , - not an absolute requirement for fusion (stauber et al., farquhar, m. g., and palade, g. e. ( ) . the golgi apparatus (com- ; taguchi et al., ) . however, fragmentation of plex)- useful in future studies of aspects of both viral replication gonzalez-scarano, f., pobjecky, n., and nathanson, n. ( ) . lacross and the biology of the ga.bunyavirus can mediate ph-dependent fusion from without. virology , - . key: cord- -qlqavi t authors: chiow, k.h.; phoon, m.c.; putti, thomas; tan, benny k.h.; chow, vincent t. title: evaluation of antiviral activities of houttuynia cordata thunb. extract, quercetin, quercetrin and cinanserin on murine coronavirus and dengue virus infection date: - - journal: asian pac j trop med doi: . /j.apjtm. . . sha: doc_id: cord_uid: qlqavi t objective: to evaluate the in vitro activities of the ethyl acetate (ea) fraction of houttuynia cordata (h. cordata) thunb. (saururaceae) and three of its constituent flavonoids (quercetin, quercitrin and rutin) against murine coronavirus and dengue virus (denv). methods: the antiviral activities of various concentrations of the ea fraction of h. cordata and flavonoids were assessed using virus neutralization tests against mouse hepatitis virus (mhv) and denv type (denv- ). cinanserin hydrochloride was also tested against mhv. the ea fraction of h. cordata was tested for acute oral toxicity in c bl/ mice. results: the ea fraction of h. cordata inhibited viral infectivity up to d. cinanserin hydrochloride was able to inhibit mhv for only d. the % inhibitory concentrations (ic( )) of the ea fraction of h. cordata added before the viral adsorption stage were . μg/ml for mhv and . μg/ml for denv- with absence of cytotoxicity. the mice fed with the ea fraction up to mg/kg did not induce any signs of acute toxicity, with normal histological features of major organs. certain flavonoids exhibited comparatively weaker antiviral activity, notably quercetin which could inhibit both mhv and denv- . this was followed by quercitrin which could inhibit denv- but not mhv, whereas rutin did not exert any inhibitory effect on either virus. when quercetin was combined with quercitrin, enhancement of anti-denv- activity and reduced cytotoxicity were observed. however, the synergistic efficacy of the flavonoid combination was still less than that of the ea fraction. conclusions: the compounds in h. cordata contribute to the superior antiviral efficacy of the ea fraction which lacked cytotoxicity in vitro and acute toxicity in vivo. h. cordata has much potential for the development of antiviral agents against coronavirus and dengue infections. severe acute distress syndrome (sars) is a highly contagious respiratory illness caused by sars coronavirus, which emerged in and rapidly spread throughout the world, with a mortality rate of %- %. although the disease disappeared in mid- , its re-emergence cannot be excluded since sars-like coronaviruses are zoonotic and exist in animal reservoirs (e.g., bats, raccoon dogs and palm civets), thus posing a potential risk for future epidemics [ ] [ ] [ ] . sars coronavirus is a large, enveloped, single-strand, positive-sense rna virus. the viral genome is about kbp, containing open reading frames that encode the polymerases required for viral rna synthesis, while the remaining open reading frames encode structural proteins (i.e., spike, envelope, membrane and nucleocapsid) and accessory proteins. first identified in , the emerging middle east respiratory syndrome (mers) coronavirus causes severe pneumonia with multiorgan involvement (including acute renal failure), and a mortality rate of % ( deaths out of cases documented by who as of june ). it is considered to be a zoonotic virus being transmitted from camels in the middle east. currently, no specific treatment exists against mers coronavirus. the murine coronavirus, mouse hepatitis virus (mhv), is a coronavirus that causes an epidemic murine illness with high mortality. generally, mhv is extremely infectious to colonies of mice and causes hepatitis upon infection. sars, mers and mhv belong to the group coronaviruses and are classified under the genus betacoronavirus. in view of the relatedness of these coronaviruses, mhv was selected in this study to act as a surrogate model for sars and mers coronaviruses which necessitate bsl- facilities, whereas mhv is considered a bsl- pathogen. belonging to the genus flavivirus, dengue virus (denv) represents the most important mosquito-borne viral disease with considerable resurgence in many parts of the world. there are four antigenically-related but distinct denv serotypes, such that infection with one serotype does not confer life-long immunity against the other serotypes. denv infection causes dengue fever, and occasionally the more serious conditions of dengue hemorrhagic fever and dengue shock syndrome [ ] . antibodydependent enhancement is thought to play a central role in dengue pathogenesis, with the risk of developing dengue hemorrhagic fever and/or dengue shock syndrome being greater in secondary infections with denv- compared to other serotypes [ ] . the different manifestations of dengue may also be attributed to denv variants with varying degrees of virulence [ ] , while viral load is also a contributing factor in the development of potentially fatal complications [ ] . in addition, being a highly prevalent serotype in tropical and subtropical regions worldwide, denv- was selected for this study. although several dengue candidate vaccines are undergoing clinical trials, there are currently no effective antiviral therapies which are urgently needed to control dengue. belonging to the saururaceae family, houttuynia cordata (h. cordata) thunb. is a perennial plant native to mountainous regions of eastern asia, with an indefinite spread as a creeping rhizome in moist locations. this herb possesses very promising antiviral properties especially against clinically important enveloped viruses such as herpes simplex virus- (hsv- ), influenza virus, and human immunodeficiency virus- in vitro. interestingly, the steam distillate of h. cordata can strikingly inactivate an enveloped virus but is incapable of inactivating a non-enveloped virus [ ] [ ] [ ] [ ] [ ] . to support these observations, we and others have demonstrated that the ethyl acetate (ea) fraction of h. cordata is effective in inhibiting the infectivity of enveloped viruses such as denv [ ] . meng et al. discovered common peaks in the hplc-dad ms fingerprint of fresh h. cordata [ ] . in our previous project, we verified some of these peaks in the ea extract as polyphenols or flavonoids (chlorogenic acid, hyperoside, quercetin and quercetrin) and have investigated their antiviral efficacy against denv [ ] . flavonoids are a class of natural products with high pharmacological potency, are ubiquitous in photosynthesizing cells and hence likely to be consumed daily [ ] . flavonoids are known to display antiviral activities, e.g., glabranine and -o-methyl-glabranine against dengue virus, procyanidin and pelargonidin against hsv, and catechins against influenza virus [ , ] . using virus-specific neutralization tests, this study tested the ea fraction of h. cordata for its antiviral efficacy against mhv (for the first time) and denv- (as further verification using a different batch of h. cordata specimen). three flavonoid components of h. cordata, i.e., quercetin, quercitrin and rutin, were also investigated for their antiviral activities against both viruses. they were also selected since they are common and naturally occurring flavonoids, whose molecular structures share high degrees of similarity. in the mhv experiments, we also compared the potency of cinanserin hydrochloride which has been proven to neutralize sars coronavirus in vitro. all the compounds were also tested for cytotoxicity in vitro, while the ea fraction was also tested for acute oral toxicity in mice. . . plant material, ethanolic extraction, aqueous-ea fractionation, and flavonoids of h. cordata the aerial parts (fresh leaves) of h. cordata were collected from a farm in johor, malaysia and authenticated in the department of pharmacology, national university of singapore. the voucher specimen of h. cordata was deposited in the singapore botanic gardens and assigned the identification number sing - . prior to extraction, the herb (dry weight of g) was washed with de-ionized water, homogenized to a fine powder, and soaked overnight in % (v/v) ethanol. the next day, the ethanolic extract was removed and stored. more solvent was added to the blended herb, which was left to soak till exhaustion. the extract was filtered, concentrated with a rotary evaporator (buchi rotavapor r- ) and freeze-dried to yield . g of crude extract in powder form. the crude extract was then dissolved in ea and de-ionized water. the water and ea phases were separated with a separating funnel. the ea phase was concentrated with a rotary evaporator, and the concentrated ea fraction was subsequently stored at − c overnight and freeze-dried. the flavonoids tested were quercetin dihydrate ( % hplc), quercitrin, and rutin hydrate ( %), all purchased from sigma-aldrich. ccl . , a normal mus musculus (mouse) liver epithelial cell line, was used for propagation of mhv and for the mhv neutralization test. compounds that were tested against mhv were the ea fraction of h. cordata, its flavonoid components, and cinanserin hydrochloride (tocris bioscience). each compound was -fold serially diluted with the corresponding diluent, and the dilutions were then added to wells of a -well microtiter plate. next, tcid of mhv was added to each diluted compound and incubated at c for h with % co . also tested was treatment with diluent only at various concentrations. virus-infected controls and uninfected cell controls were included in each batch of assays. confluent ccl . cells were cultured in dmem supplemented with % horse serum, and × cells were seeded into each well of another -well microtiter plate, and incubated at c with % co until the cells were % confluent. cell culture fluid from each well was discarded. each virus-compound mixture and controls were transferred to these wells in duplicate, followed by addition of dmem with % (v/v) horse serum. the plate was sealed and incubated at c with % co , and observed daily for d. the highest dilution of compound that inhibited cytopathic effects (cpe) was considered as the minimum inhibitory concentration (mic). the new guinea c strain of denv- was propagated in the c / aedes albopictus mosquito cell line and maintained in leibovitz l medium supplemented with % (v/v) fetal bovine serum at c under % co atmosphere. the anti-denv- activity of h. cordata and its flavonoids were evaluated by plaque reduction neutralization test or prnt [ , ] . bhk- (baby hamster kidney) fibroblasts were cultured to form cell monolayers in -well plates with rpmi- supplemented with % (v/v) fetal bovine serum at c under % co . the test compounds were dissolved in the relevant diluents, and -fold serial dilutions were prepared to obtain different concentrations. denv- new guinea c neutralizing monoclonal antibody h from chemicon served as the positive control [ ] . diluent only (at various concentrations), virus, and cell controls were also included by adding the corresponding diluent, virus suspension, and medium without the treatment compounds. each experiment was performed in duplicate. denv- ( plaque-forming units or pfu) were incubated for h with various concentrations of each compound together with controls before adding to the cells. the virus-sample mixtures were incubated with the cells at c under % co for another hour with rocking at -min intervals before the cells were overlaid with % carboxymethylcellulose at c under % co for d. the cells were then fixed with % formaldehyde and stained with % crystal violet, and the number of plaques was counted. the percentage plaque reduction of the compounds at every dilution was determined as follows: (mean number of plaques in virus control) − (average number of plaques in sample) × % divided by (mean number of plaques in virus control). the percentage plaque reduction was plotted against various concentrations of the test agents to determine the concentration that causes % plaque reduction (ic ). the mtt cell proliferation assay was performed to determine the cell viability following exposure to the test compounds. various concentrations of each test compound were added to wells containing cell monolayers and incubated at c under % co for h. after incubation, mtt reagent was added to each well, and further incubated for h or until purple precipitates were visible under an inverted microscope. then, ml of g/l sds in . mol/l hcl were added to each well and incubated overnight in the dark at room temperature. the optical density (od) at nm was then read, and the cell inhibition rate calculated from the formula: [ the inhibition rates were plotted against various sample concentrations to ascertain the concentration that causes % cytotoxicity (cc ). the ea fraction and flavonoids (either individually or in combination) were tested at the same concentrations as those for neutralization tests. the assay included wells containing medium only as well as untreated control cells. each experiment was repeated, and the mean and standard deviation were calculated. this approach was adapted from the oecd guideline for testing of chemicals, : acute oral toxicity -fixed dose procedure, and relied on the observation of clear signs of toxicity or even mortality. c /bl nulliparous and nonpregnant female mice about -weeks old were obtained from the laboratory animals center, national university of singapore. upon arrival, they were kept in cages for d prior to commencement of the study. on the actual dose-feeding day, the mice were fasted for about h. doses of , , and mg/kg were prepared for administration using water as vehicle to suspend the ethanolic extract of h. cordata at a constant volume of ml/ g of body weight. in total, the five groups of mice (n = each) were fed with a single dose of the extract (including the vehicle control) by oral gavage. a sighting study was then conducted frequently from the first min, with special attention to the first h. thereafter, the condition of each mouse was observed daily to detect abnormalities such as changes in physical appearance, behavioral signs and body weight. all mice were finally euthanized after d of observation, and major organs (brain, heart, lungs, liver and kidneys) were harvested for histopathological examination after staining with hematoxylin and eosin (h&e). the ea fraction of h. cordata was evaluated at concentrations of . mg/ml down to . mg/ml. table and figure show that the ea fraction of h. cordata exhibited anti-mhv activity at a mic of . mg/ml without any apparent cytotoxic effects on ccl . cells. tested at concentrations from . to . mg/ml, the anti-mhv effect of quercetin was significantly less, with mic of . mg/ml (figure ), cytotoxicity cc value of . mg/ml, and selectivity index of . . however, quercitrin and rutin did not exhibit antiviral activity on days postinfection (dpi) at concentrations of . mg/ml and below (table ). cinanserin hydrochloride was tested at concentrations ranging from . to . mg/ml. table indicates that cinanserin exerted anti-mhv activity at mic of . mg/ml on dpi and . mg/ml on dpi (figure ) , with minimal or no cytotoxicity. however, on dpi, no viral inhibition was observed. the ea fraction of h. cordata was tested starting with the highest concentration of . mg/ml followed by -fold dilutions down to . mg/ml. by means of prnt, a distinct trend of denv- inhibition was observed at various concentrations of the ea fraction without cytotoxicity to bhk cells, with the ic being . mg/ml. the efficacy of the ea fraction in inhibiting denv- infection was clearly evident at concentrations of . mg/ml and above, at which complete viral inhibition was achieved ( figure ). individual flavonoids were tested at concentrations of . mg/ml down to . mg/ml. table table . the ea fraction, quercetrin and rutin were dissolved in % dmso. quercetin was dissolved in aqueous alkali ( . mol/l naoh) as it was not very soluble in dmso. various concentrations of ea fraction and compounds were subjected to the corresponding viral neutralization tests as well as the mtt assay. as negative controls, dmso and aqueous alkali were also tested separately by denv- prnt, mhv neutralization test and the mtt assay which revealed absence of non-specific denv- and mhv inhibition as well as lack of cytotoxicity of these solvents (data not shown). throughout the -day period of observation, the control group as well as the four groups of mice fed with varying doses of h. cordata extract showed no mortality and no significant weight loss (data not shown). moreover, all mice appeared to be active and well-groomed before euthanasia. from the histopathological sections of all major organs stained with h&e, there was no significant difference between the control group and all the test groups, as exemplified in figure which depicts the control group compared with the group fed with the highest dose of mg/kg. the experimental strategy in this study was to investigate the prophylactic antiviral effects since the compounds were allowed to interact with the viruses for h before introduction into the cells. this approach was employed as we previously found no therapeutic effect when the compounds were added after prior infection of cells with denv [ ] . overall, this study demonstrated that ea fraction of h. cordata and its flavonoid component, quercetin, could inhibit both mhv and denv- in vitro. this was followed by quercitrin which could inhibit denv- but not mhv, whereas rutin did not exert any inhibitory effect on both viruses. we and others have provided evidence to show that different flavonoid components in the ea fraction of h. cordata exert varying degrees of antiviral activity against different viruses. our findings corroborate previous evidence that among the flavonoid components, quercetin is the most effective against denv- [ ] . wleklik et al. emphasized that there is a structural basis for the distinct differences in antiviral activities of flavonoids [ ] . hence, it is noteworthy that being the most bioactive, quercetin possesses the hydroxyl group at the r position compared to the other two flavonoids tested, i.e., rhamnose in quercitrin and rubinose in rutin [ ] . quercetin is an aglycone present at high concentration in onions. this compound has virucidal activity against enveloped viruses such as mengovirus, herpes simplex, parainfluenza type , pseudorabies, respiratory syncytial, and sindbis viruses [ ] [ ] [ ] [ ] . quercetin is able to inhibit h + -atpase of lysosomal membrane and thus prevent virus coat removal [ ] . moreover, quercetin exhibits significant inhibitory effects on the atpase of multidrug resistance-associated proteins, thus increasing the bioavailability of anticancer and antiviral drugs in vivo [ ] . hence, quercetin can be considered for its potential efficacy in antiviral drug therapies. quercitrin (quercetin- -l-rhamnoside) and rutin (quercetin- rutinoside) occur as glycosides. quercitrin appears to show the highest content in fingerprint analysis of h. cordata by hplc [ ] . among the flavonoids, quercetin also exerts the highest activity on hsv- , whilst rutin has no effect at all [ ] . this characteristic implies that the substitution or addition of free hydroxyl group at certain positions may culminate in a decreased or even completely abolished antiviral effect. such the ea fraction of h. cordata was tested at concentrations of . mg/ ml down to . mg/ml. the upper plot reveals a distinct trend of denv- inhibition, with the ic being . mg/ml, and complete viral inhibition being achieved at concentrations of . mg/ml and above. the lower plot shows that no cytotoxicity to bhk- cells was evident at various concentrations of the ea fraction tested. structural-activity relationships can lead to the design and development of more active and less toxic flavonoids with appropriate pharmacokinetic properties. hence, they may pave the way for novel compounds that can match or even exceed the efficacies of existing antiviral drugs [ , , ] . another interesting aspect was the enhancement of anti-denv- activity of quercetin when combined with quercitrin. besides augmenting the antiviral effect, this combination also reduced the cytotoxicity relative to that induced by quercetin alone. this characteristic reiterates that combinations of components with greater anti-dengue efficacy but lower toxicity are potentially promising, as discovered previously using combinations of quercetin, chlorogenic acid and/or hyperoside [ ] . indeed, several reports have already documented the synergistic antiviral effects of combinations of individual flavonoids against viral pathogens, such as herpesviruses and fowlpox virus [ , ] . although cinanserin was able to inhibit mhv at . mg/ ml on dpi and . mg/ml on dpi, its effect was relatively short-lived and it could not inhibit mhv from dpi onwards. in comparison, however, we observed that the ea fraction and quercetin yielded a longer-lasting antiviral effect of up to d. this finding may be explained by superimposition of the two c-like proteinases of mhv and sars cov which illustrates that certain amino acid residues residing within Å of the active site (c ) are different (not shown). thus, these amino acid disparities may influence recognition and binding of cinanserin hydrochloride to the active site and may also lead to its lower affinity for c-like proteinase of mhv than sars-coronavirus. competitive binding of cinanserin hydrochloride to the active site of c-like proteinase hinders the processing of the precursor polyprotein to generate functional replicase necessary for viral replication [ ] [ ] [ ] . importantly, the ea fraction of h. cordata was capable of inhibiting both denv- and mhv in vitro at relatively low concentrations with negligible cytotoxicity and very good selectivity indices. moreover, the ea fraction was also more potent than all the individual compounds tested (including their combination) and did not cause any cytotoxicity to mammalian kidney (bhk) and liver (ccl . ) cell lines in vitro, nor any acute toxicity or pathology of major murine organs in vivo. this feature reiterates the synergistic effects of the combination of multiple flavonoids and other constituents within h. cordata that confer optimal antiviral activities and minimal toxicity [ ] , in keeping with the majority of successful traditional chinese medicine. our study lends support to the accumulating literature that the ea fraction of h. cordata indeed represents a highly promising prophylactic agent against coronaviruses and dengue viruses [ ] [ ] [ ] . to complement this study, future in vivo studies are warranted to assess the efficacy of the ea fraction of h. cordata against coronaviruses and dengue viruses using the relevant mouse models [ ] . however, further in vivo experiments are necessary to further assess the long-term safety of h. cordata as an antiviral agent in biological systems, including acquiring crucial information on its adsorption, distribution, metabolism and elimination. to address the important issue of batch-to-batch variation, greater efforts should also focus on the quality control of extracts derived from this medicinal plant. finally, the mechanisms of action of the ea fraction and its constituent flavonoids against viruses at the molecular level need to be further elucidated [ ] . isolation and characterization of viruses related to the sars coronavirus from animals in southern china bats are natural reservoirs of sars-like coronaviruses severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats monitoring of dengue viruses in field-caught aedes aegypti and aedes albopictus mosquitoes by a type-specific polymerase chain reaction and cycle sequencing pathogenesis of dengue. challenge to molecular biology molecular evolution of dengue type virus in thailand dengue viremia titer, antibody response pattern, and virus serotype correlate with severity virucidal effects of the steam distillate from houttuynia cordata and its components on hsv- , influenza virus, and hiv the constituents and their bioactivities of houttuynia cordata houttuynia cordata blocks hsv infection through inhibition of nf-kb activation a current update on the phytopharmacological aspects of houttuynia cordata thunb houttuynia cordata targets the beginning stage of herpes simplex virus infection houttuynia cordata extracts and 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antiviral effect of flavonoids on human viruses antiviral activity of flavones and potentiation by ascorbate present status and prospects of flavonoids as anti-viral agents antiviral activity of quercetin -rhamnoside against porcine epidemic diarrhea virus modulatory effects of plant phenols on human multidrug-resistance proteins , and (abcc , and ) inhibitory effects of quercetin -rhamnoside on influenza a virus replication flavonoids as noncompetitive inhibitors of dengue virus ns b-ns protease: inhibition kinetics and docking studies combined antiviral effects of flavonoids and -ethyl- -deoxyuridine on the multiplication of herpesviruses inhibition of fowlpox virus by an aqueous acetone extract from galls of guiera senegalensis j. f. gmel (combretaceae) design and synthesis of cinanserin analogs as severe acute respiratory syndrome coronavirus cl protease inhibitors flavonoid-mediated inhibition of sars coronavirus c-like protease expressed in pichia pastoris atlas of coronavirus replicase structure chemical composition and hepatoprotective effects of polyphenol-rich extract from houttuynia cordata tea immunomodulatory and anti-sars activities of houttuynia cordata in vitro and in vivo effects of houttuynia cordata on infectious bronchitis virus the inhibitory actions of houttuynia cordata aqueous extract on dengue virus and dengueinfected cell development of animal models against emerging coronaviruses: from sars to mers coronavirus houttuynia cordata thunb: a review of phytochemistry and pharmacology and quality control we thank annie hsu and s.h. lau for laboratory assistance, t. narasaraju for helpful advice, and mary ng for providing the dengue virus strain. this study was supported by a research grant from the national university of singapore. the authors declare no conflict of interest with respect to the publication of this paper. key: cord- -jal tkra authors: cervin, marguerite; anderson, robert title: modulation of coronavirus‐mediated cell fusion by homeostatic control of cholesterol and fatty acid metabolism date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: jal tkra cellular susceptibility to fusion mediated by murine coronavirus (mouse hepatitis virus, mhv strain a ) was separated into lipid‐dependent and lipid‐independent mechanisms with the use of subclones and selected mutants of mouse l‐ fibroblasts. fusion‐resistant l‐ cell mutants had similar cholesterol and fatty acid composition as did their fusion‐susceptible parent subclone, and were presumably deficient in a genetically mutable non‐lipid, host cell factor (e.g., fusion protein receptor). on the other hand, cellular sensitivity to virus fusion, which is known to be influenced by cell cholesterol content [daya et al., ], was shown further to be modulated by homeostatic alterations in fatty acid metabolism. cholesterol supplementation of mouse l‐ fibroblasts or of peritoneal macrophages from mhv‐susceptible mice elevated susceptibility to viral fusion. increased fusion susceptibility occurred in cholesterol‐supplemented l‐ cells in the absence of any detectable alterations i n host cell fatty acid composition, thus demonstrating fusion enhancement by cholesterol alone. l‐ cells cloned by limiting dilution in normal (not cholesterol‐supplemented) medium were found to be heterogeneous i n cholesterol content. interestingly, high cholesterol‐containing subclones had increased levels of c‐ : , c‐ : , c‐ : , and c‐ : and markedly reduced levels of c‐ :l fatty acids when compared to low cholesterol‐containing subclones. high cholesterol‐containing subclones did not show enhanced susceptibility to viral fusion, suggesting that homeostatic alteration of fatty acid metabolism compensated for the increased cholesterol levels and countered the normally fusion‐enhancing effect of cholesterol alone. since these observations have potentially important consequences regarding the effects of dietary cholesterol on the severity of virus infection, we examined liver titres and pathology of normal and hypercholesterolemic mice infected with mhv. hypercholesterolemia had no significant effect on virus replication or on liver pathology in two mhv‐ sensitive strains (balb/c and aij) or in one mhv‐resistant (sjlij) of mice. lipid analyses of the livers from normal and hypercholesterolemic mice showed evidence of two homeostatic mechanisms (cholesterol esterification and alteration of fatty acid composition) which likely counteracted the normally exacerbating effect of cholesterol on mhv cytopathology. of subclones and selected mutants of mouse l- fibroblasts. fusion-resistant l- cell mutants had similar cholesterol and fatty acid composition as did their fusion-susceptible parent subclone, and were presumably deficient in a genetically mutable non-lipid, host cell factor (e.g., fusion protein receptor). on the other hand, cellular sensitivity to virus fusion, which is known to be influenced by cell cholesterol content [daya et al., , was shown further to be modulated by homeostatic alterations in fatty acid metabolism. cholesterol supplementation of mouse l- fibroblasts or of peritoneal macrophages from mhvsusceptible mice elevated susceptibility to viral fusion. increased fusion susceptibility occurred in cholesterol-supplemented l- cells in the absence of any detectable alterations i n host cell fatty acid composition, thus demonstrating fusion enhancement by cholesterol alone. l- cells cloned by limiting dilution in normal (not cholesterol-supplemented) medium were found to be heterogeneous i n cholesterol content. interestingly, high cholesterol-containing subclones had increased levels of c- : , c- : , c- : , and c- : and markedly reduced levels of c- :l fatty acids when compared to low cholesterol-containing subclones. high cholesterolcontaining subclones did not show enhanced susceptibility to viral fusion, suggesting that homeostatic alteration of fatty acid metabolism compensated for the increased cholesterol levels and countered the normally fusion-enhancing effect of cholesterol alone. since these observations have potentially important consequences regarding the effects of dietary cholesterol on the severity of virus infection, we examined liver titres and pathology of normal and hypercholesterolemic mice infected with mhv. hypercholesterolemia had no significant effect on virus replication or on liver pathology in t w o mhvsensitive strains (balbic and aij) or in one mhv- wiley-liss, inc. resistant (sjlij) of mice. lipid analyses of the livers from normal and hypercholesterolemic mice showed evidence of two homeostatic mechanisms (cholesterol esterification and alteration of fatty acid composition) which likely counteracted the normally exacerbating effect of cholesterol on mhv cytopathology. cholesterol plays an important role in determining the physical properties and functions of animal cell membranes. in addition to having a modulating effect on membrane fluidity and permeability [demel et al., , there is evidence that cholesterol may interact directly with certain membrane proteins [johnson et al., ; asano and asano, and possibly regulate their functional activity [mcmurchie, ; asano and asano, . cholesterol is a n important dietary lipid and has been shown to modulate resistance of mice to infections by some bacteria and viruses, including listeria monocytogenes [kos et al., ; kos et al., and coxsackie b virus [campbell et al., and also mhv- [pereira et al., . mhv- is strongly hepatotropic in balb/c mice but causes only a mild liver infection in a/j mice. pereira et al. [ , hypothesized that kupffer cells (kc) , the liver macrophages, are involved in resistance to mhv- in aij mice, and that such resistance may be overcome by cholesterol supplementation. this and previous studies [ruebner et al., ; ruebner and bramhall, on modulation of mhv infection by cholesterol or fats employed complex food mixtures, in which cholesterol was not the sole variable constituent. as a result it has not been possible to relate biological effects to cholesterol per se. we have shown previously [daya et al., that supplementation of cultured mouse l- fibroblasts with cholesterol results in a marked increase in cellular susceptibility to fusion mediated by mouse hepatitis virus (mhv). roos et al. [ have also presented evidence suggesting a role for other lipids, particularly saturatediunsaturated fatty acids, in modifying cellular responsiveness to mhv-induced fusion. since these results have potentially important consequences for the spread and severity of virus infections as a function of dietary cholesterol and other lipids, we undertook a study of cholesterol metabolism and mhv infection in vitro and in vivo. the results identify important homeostatic control mechanisms which counteract the fusion-enhancing effects of excess cholesterol. materials and methods cells, virus, and culture conditions mouse l- fibroblasts [rothfels et al., were cultured as monolayers in minimal essential medium (mem), supplemented with % fetal calf serum. mouse l- cell mutants selected for partial resistance against mhv infection were those described by daya et al. [ . cholesterol-supplemented medium was prepared as previously described [daya et al., . peritoneal macrophages, obtained by peritoneal lavage of starch-primed mice, were also cultured in normal or cholesterol-supplemented medium in the same manner. the a strain of mhv [manaker et al., was used throughout. cell membrane preparation cell monolayer cultures ( mm tissue culture plates) were washed with phosphate-buffered saline (pbs), scraped from the plastic plates and spun into pellets ( min at x g). cells were resuspended in reticulocyte standard buffer (rsb), allowed to swell on ice for min and then disrupted by manual glass homogenization. following removal of nucleii by brief centrifugation ( min a t x g); total membranes were recovered by centrifugation a t , x g for h. for the assessment of fusion susceptibility, contact fusion assays similar to those described previously [mizzen et al., were performed. experiments were performed three times on triplicate cultures. sparsely seeded mhv-infected l- cells (l - subclone) were overlaid with a ten-fold excess of uninfected test cells. following a two-hour incubation a t "c, syncytial formation was evaluated by light microscopy and expressed as a fusion index [mizzen et al., . mice, diet, and mhv infection three strains of mice were used for the in vivo studies. balbic mice were purchased from charles river, quebec, canada, while a/j and sjlij mice were obtained from jackson laboratories, bar harbour, maine, usa. the control diet consisted of % (wiw) corn oil mixed in with ground wayne rodent blox. the cholesterol-supplemented diet was adapted from the hypercholesterolemic diet used for inducing atheroscle- rosis in rabbits [frank and fogelman, and consisted of % (w/w) crystalline cholesterol in % (wiw) corn oil in ground wayne rodent blox. mice were fed fresh food and water daily. following days feeding on either the control or cholesterol diet, eight mice on each diet were either mock infected with pl pbs or infected with lo pfu/ml mhv-a in pl pbs. at days post-inoculation (pi), mice were sacrificed, the livers extracted and either frozen at - °c for titration and lipid analysis or preserved in % formalin. paraffin sections from formalin-fixed mouse livers were stained with hematoxylin/eosin (hie), and the number of lesions present per section was counted. cell lipid determinations were performed on triplicate cultures from three separate experiments. lipids from l- cells or from membrane fractions were extracted with chloroformimethanol ( : ). liver lipids were extracted by first homogenizing . g portions of liver in ml pbs and then mixing pl of homogenate with ml chloroformimethanol ( :l). after h stirring at room temperature, extracts were filtered through glass wool. aliquots ( p ) of the lipid extracts were methanolyzed ( % methanolic hc for h a t °c) and acetylated (pyridineiacetic anhydride ( :l) overnight at room temperature) in order to convert total fatty acids and cholesterol to their methyl and acetyl derivatives, respectively. fatty acid methyl esters and cholesterol acetate were then analyzed quantitatively by gas chromatography as previously described [daya et al., . to differentiate free cholesterol from cholesterol ester, aliquots of the lipid extracts were applied to pasteur pipette columns of biosil a (biorad) and neutral lipid fractions obtained by elution with chloroformimethanol ( : ). trimethylsilylation of the neutral lipids, before and after methanolic hc hydrolysis, yielded the tms derivatives of free and total cholesterol, respectively, which were then determined by gas chromatography. since membrane fluidity is determined primarily by cholesterol content and fatty acid chain lengthiunsaturation, we examined the possibility that cholesterol-enhanced fusion could be partly brought about by alterations in the host cell fatty acid composition. cultures of l- cells were maintained in normal or cholesterol-supplemented medium for or hours and subjected to analysis of cholesterol and fatty acid. as illustrated in a representative chromatogram ( fig. ) and quantitated in table i, duration of cholesterol supplementation from to hours did not further raise the cellular contents of either free or esterified cholesterol. analysis of the membrane fraction also showed increased cholesterol content in response to cholesterol supplementation (table i) . it is important to note that although the major cellular response to cholesterol supplementation was an increase in cholesterol ester level, there was also considerable increase in free cholesterol which was found predominantly in the membrane fraction. cholesterol ester was not found in the membrane fraction and was presumably present as a cytoplasmic storage form (data not shown). analysis of the cellular fatty acids revealed that essentially no alterations in fatty acid composition had taken place as a result of the considerable infiltration of cholesterol documented above (table i) . therefore, the enhancement of viral fusion observed in cholesterol-supplemented cells [(daya et al., ) and table i cannot be ascribed to fatty acid-dependent membrane changes. analysis of a number of l- cell subclones (all grown in the same medium without added cholesterol) showed a surprising variability in cholesterol contents (table ) . notably however, and in contrast to l- cells supplemented exogenously with cholesterol, the high cholesterolcontaining subclones (l - and l - ) showed marked alterations in their fatty acid composition (table ). in particular, the high cholesterol-containing subclones had elevated amounts of c- : , c- : , c- : , and c- : , along with drastically reduced levels of c- :l. we exploited this observation to examine the susceptibility of the various subclones to viral fusion in a contact fusion assay. despite the elevated cholesterol levels found in two of the subclones (l - and l - ) these subclones showed no increased susceptibility to mhv-induced fusion over subclones of low cholesterol content (l - , l - , and l - ). taken together, the results so far suggest that cholesterol-enhanced fusion can be counterbalanced by cellular alterations in fatty acid metabolism. in light of the observations above, we investigated the fatty acid and cholesterol compositions of l- cell mutants selected for their ability to survive acute mhv infection and which show a relatively fusion-resistant phenotype [daya et al., . it was found that all five mutants examined had a cholesterol content and fatty acid composition similar to the parental l- cell clone from which they were generated (table ) . thus, an alteration in fatty acid or cholesterol metabolism does not underly the genetic lesion which is responsible for diminished susceptibility to mhv-induced fusion in these mutants. nevertheless, each of the five l- mutant cells showed cholesterol-dependent fusion enhancement when supplemented with cholesterol (data not shown), although the degree of fusion enhancement was not as great as that observed with wild type l- cells. it would therefore seem that even cells of disparate susceptibility to mhv-induced fusion possess a window within which cell fusion is subject to cholesterol-dependent modulation. cholesterol enhances mhv-mediated fusion of infected macrophages. in a n effort to extend our in vitro results to the in vivo situation, we subjected peritoneal macrophages from three strains of mice (balblc, aij and sjl/j) to cholesterol supplementation. both control and cholesterol-supplemented macrophages were then challenged with mhv and scored for the development of syncytia. macrophages are important in mhv infection for several reasons. first, hepatotropic viruses may require replication in macrophages prior to invading liver parenchymal cells [mims, ; allison, ; sabesin and koft, . second, mouse strain susceptibility to mhv has been reported to correlate with virus replication in explanted macrophages ivirelizier and allison, . third, the ability of a virus to replicate in macrophages, or any cell involved in the immune response, may compromise their role in virus clearance. we chose three strains of mice, two of which are permissive for mhv-a (balbic and a/j) and one which is nonpermissive (sjlij) due to a single genetic locus [smith et al., . cholesterol supplementation of peritoneal macrophages taken from balb/c and aij mice resulted in enhanced fusion following infection with mhv. sjlij macrophages, on the other hand, whether supplemented with cholesterol or not showed no evidence of mhv-induced fusion. thus, while cholesterol enhances viral fusion in mhv-permissive macrophages, it is unable to overcome the block to replication of macrophages which are non-permissive to mhv. cholesterol enhances cellular susceptibility to mhvinduced fusion, we examined whether cholesterol supplementation in vivo has a n effect on the course or severity of mhv-induced disease. three strains of mice (balbic, a/j and sjl/j) were maintained for days on either normal or cholesterol-supplemented diet and examined for evidence of hypercholesterolemia and any changes in susceptibility or severity of mhv-induced hepatic disease. in contrast to the results of loria et al. [ and pereira et al. [ , we found no difference in the rate of growth (approx. g/week), or in the liver weights, of mice maintained in normal or cholesterol-supplemented diet. respective liver weights (as percentage body weight) for normal and cholesterol-supplemented animals were: . * . % and . ? . % for balb/c, . . % and . * . % for am, and . . % and . . % for sjlij. livers from cholesterol-supplemented mice had a generally paler colour than did those from the control animals. microscopically, the hepatocytes showed extensive fatty infiltrations (data not shown). all three mouse strains responded to the cholesterol-supplemented diet with elevated liver cholesterol contents. respective liver cholesterol contents (as mgig liver) for normal and cholesterol-supplemented mice were: . . mg and . t . mg for balbic, . ? . mg and . f . mg for aij, and . ? , mg and . ? . mg for sjlij animals. analysis of the liver lipids by thin layer chromatography (tlc) indicated that the cholesterol was present primarily in the form of cholesterol ester. free cholesterol concentration was similar in livers from both control and cholesterol-supplemented mice but the concentration of cholesterol ester was markedly increased in cholesterol-supplemented mice as shown in figure . other lipids, were present in approximately the same amounts. balblc, . x lo pfdg liver in aij and . x ' pfulg liver in sjl/j mice. similarly, the numbers and sizes of necrotic lesions found in livers from control and cholesterol-supplemented mice were unchanged (data not shown). the present results confirm and extend our previous findings on the effects of cholesterol on mhv infection and its associated cell fusion. in particular, the fusionenhancing effect of cholesterol is directly attributable to the sterol and not to the possibility of alterations in cellular fatty acid composition. it is clear however that cell subclones which inherently have high cholesterol levels, show distinct alterations in fatty acid metabolism, which act homeostatically to regulate membrane function and to counteract the fusion-enhancing effect of cholesterol. the present study also underscores the importance of determining membrane cholesterol levels in addition to fatty acid composition, the latter of which might otherwise be considered to be the major determinant of susceptibility to viral-induced fusion [roos et al., . given the demonstrated fusion-modulating roles of membrane cholesterol and fatty acid, it is important to realize that these lipid constituents do not account for all instances of variations in fusion susceptibility. for example, in our studies, fusion-resistant l- cell mutants have wild type cholesterol and fatty acid compositions and are presumably deficient in another cellular gene product (e.g., fusion protein receptor) which is required for fusion. in contrast to the dramatic enhancement of mhvinduced fusion by cholesterol in vitro, the effects of cholesterol supplementation on mhv infection in vivo were minimal. despite the induction of hypercholesterolemic conditions in the three strains of mice examined, there was little effect on either virus replication or the production of hepatic lesions. this may be due to the fact that cell fusion is not an outstanding feature of mhv-induced hepatitis. (note, however, that hepatocytes from mhv-permissive mice are susceptible to viral fusion [arnheiter et al., ) . moreover, as documented in the present study there is clear evidence for two important homeostatic mechanisms which would be expected to diminish the effects of excess cholesterol on liver cell membrane function (and virusmediated membrane cytopathology). thus, while liver cholesterol levels were considerably elevated in mice given the cholesterol diet, most of the cholesterol was found as the esterified form. the cholesterol would therefore be mainly sequestered in lipid storage droplets within the cytoplasm rather than accumulated in the cell membranes. in addition, the increased incorporation of cholesterol into the livers of cholesterolsupplemented mice was accompanied by alterations in fatty acid composition, specifically raising the unsaturated fatty acid content, thereby counteracting the normally rigidifying effect of cholesterol. this appar-cervin and anderson ently homeostatic response of fatty acid metabolism to cholesterol is qualitatively similar to that observed in the rat liver [garg and sabine, . the present study underscores the requirement for a precisely defined diet for examining cholesterol effects in vivo. despite the development of a purely cholesterol-supplemented diet by fillios et al. [ ] , many animal studies have employed complex food mixtures which, apart from their cholesterol contents, bear little resemblance to their respective control diets. in such cases it is difficult to ascribe biological effects to specific dietary components. prior to the present study, there have been three reports of dietary modulation of hepatitis caused by mhv. two of these reports noted little effect of a high fat diet (and presumably cholesterol-rich) on mhv-%induced hepatitis in swiss webster mice [ruebner et al., ; ruebner and bramhall, . on the other hand, pereira et al. [ found an increase in susceptibility to mhv- among normally resistant a/j mice maintained for - days on a high cholesterol diet consisting of sucrose, casein, lard, cholesterol, cholic acid and vitamin supplements. it is important to note that mice which are genetically resistant to mhv (e.g., mhv-a resistant sjlij mice) are not rendered susceptible by dietary cholesterol supplementation, despite dramatic alterations in their liver cholesterol levels. these results are in contrast to those obtained by pereira et al. [ who concluded that cholesterol feeding overcame the genetic block of infection of mhv- in normally resistant aij mice. the differences may in part be due to the strain of mhv used. thus, the mechanism of mhv- resistance in aij mice may differ from that of mhv-a resistance in sjlij mice. however, the complexity of the dietary mixture used by these authors makes a direct comparison with our results difficult and it is possible that some element in their diet other than cholesterol affected the course of mhv infection. while it would be of interest to test a variety of mouse strains with distinct strains of mhv as to their possible modulation of susceptibility/resistance by cholesterol, we can conclude from our study that in at least one system (mhv-a resistant sjlij mice) genetic resistance is not determined by cholesterol. admittedly, lipid metabolism in the liver is much more complex than that which occurs in cultured cells in a defined medium. in particular, the parameters of cholesterol and fatty acid metabolism in hepatocytes and other liver cell types as well as the subcellular distribution of these lipids await further analysis. nevertheless, the results from the present study indicate certain parallels in the response of both cultured l- fibroblasts and mouse liver to exogenous cholesterol. the interrelationship between fatty acid metabolism and the uptake, membrane incorporation and esterification of cholesterol are aspects of lipid metabolism which have important consequences for cell membrane physiology and which invite continuing investigation as to the role of lipid homeostasis in modulating membrane-related aspects of viral (and other microbial) pathogenesis. on the role of mononuclear phagocytes in immunity against viruses adult mouse hepatocytes in primary monolayer culture express genetic resistance to mouse hepatitis virus type binding of cholesterol and inhibitory peptide derivatives with the fusogenic hydrophobic sequence of f-glycoprotein of hvj (sendai virus): possible implication in the fusion reaction dietary hepatic cholesterol elevation: effects on coxsackie b infection and inflammation cholesterol enhances mouse hepatitis virus-mediated cell fusion mutation of host cell determinants which discriminate between lytic and persistent mouse hepatitis virus infection results in a fusion-resistant phenotype the properties of polyunsaturated lecithins in monolayers and liposomes and the interaction of those lecithins with cholesterol experimental production of gross atherosclerosis in the rat ultrastructure of the intima in whhl and cholesterol-fed rabbit aortas prepared by ultra-rapid freezing and freeze-etching homoeostatic control of membrane cholesterol and fatty acid metabolism in the rat liver binding of cholesterol by sulfhydryl-activated cytolysins inhibition of host resistance by nutritional hypercholesteremia impaired function of immune reactivity to listeria monocytogenes in diet-fed mice infection of hypercholesterolemic mice with coxsackie b a hepatitis virus complicating studies with mouse leukemia dietary lipids and the regulation of membrane fluidity and function aspects of the pathogenesis of virus diseases fusion resistance and decreased infectability as major host cell determinants of coronavirus persistence increased susceptibility of mice to mhv- infection induced by hypercholesterolemic diet: impairment of kupffer cell function control of virus-induced cell fusion by host cell lipid composition the origin of altered cell lines from mouse, monkey and man as indicated by chromosome and transplantation studies experimental virus hepatitis in choline-deficient mice with fatty livers the effect of changes in dietary protein on experimental viral hepatitis in mice control of mouse hepatitis virus replication in macrophages by a recessive gene on chromosome correlation of persistent mouse hepatitis (mhv- ) infection with its effects on mouse macrophage cultures council of canada. key: cord- -z bdiuvx authors: ulasli, mustafa; verheije, monique h.; de haan, cornelis a. m.; reggiori, fulvio title: qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus date: - - journal: cell microbiol doi: . /j. - . . .x sha: doc_id: cord_uid: z bdiuvx coronaviruses (cov) are enveloped positive‐strand rna viruses that induce different membrane rearrangements in infected cells in order to efficiently replicate and assemble. the origin, the protein composition and the function of these structures are not well established. to shed further light on these structures, we have performed a time‐course experiment in which the mouse hepatitis virus (mhv)‐induced membrane rearrangements were examined qualitatively and quantitatively by (immuno)‐electron microscopy. with our approach we were able to confirm the appearance of , previously reported, membranous structures during the course of a complete infection cycle. these structures include the well‐characterized double‐membrane vesicles (dmvs), convoluted membranes (cms) and virions but also the more enigmatic large virion‐containing vacuoles (lvcvs), tubular bodies (tbs) and cubic membrane structures (cmss). we have characterized the lvcvs, tbs and cmss, and found that the cov‐induced structures appear in a strict order. by combining these data with quantitative analyses on viral rna, protein synthesis and virion release, this study generates an integrated molecular and ultrastructural overview of cov infection. in particular, it provides insights in the role of each cov‐induced structure and reveals that lvcvs are ergic/golgi compartments that expand to accommodate an increasing production of viral particles. compartments that expand to accommodate an increasing production of viral particles. viruses require cellular membranes in one or more steps of their infection cycle for replication, assembly and/or release, and therefore they have developed sophisticated mechanisms to opportunistically rearrange host membranes for their needs. for example, a common feature among positive (+) strand rna viruses is the assembly of their replication-transcription complexes (rtcs) in association with cytoplasmic membranes (salonen et al., ; miller and krijnse-locker, ) . the potential benefit of anchoring the rtcs to lipid bilayers is still unclear, but it may facilitate and co-ordinate different steps of the viral life cycle, and/or delay induction of the host immune response (ahlquist, ; haller et al., ) . enveloped viruses are another example; they generate their new virions by budding through cellular membranes (garoff et al., ) . cov are enveloped (+) strand rna viruses (weiss and navas-martin, ; gorbalenya et al., ) . they are pathogens of veterinary importance, but the relevance of this family of viruses has increased considerably due to the recent emergence of new human viruses such as the cov nl and the severe acute respiratory syndrome-cov (sars-cov). these viruses cause severe respiratory tract diseases and patients often have evidence of other organ dysfunctions (godfraind and coutelier, ; peiris et al., ; saif, ) . after fusion of the viral and cellular membranes, cov disassemble thereby releasing their genomic rna, which allows the production of non-structural proteins (nsp's) (brian and baric, ; sawicki et al., ) . these nsp's collectively form the rtcs and induce the formation of cytoplasmic dmvs into which the rtcs are anchored (ziebuhr et al., ) . the mechanism underlying the biogenesis of cov-induced dmvs is completely unknown even if experimental evidences indicate an endoplasmic reticulum (er) origin (harcourt et al., ; kanjanahaluethai et al., ; oostra et al., ; knoops et al., ) . the cov genomes encode for a common set of four structural proteins: the envelope (e), the membrane (m), the nucleocapsid (n) and the spike (s) proteins . e, m and s proteins are integral membrane components and after insertion in the er limiting membrane, they are transported to the er-to-golgi intermediate compartment (ergic), where together with the n protein and the genomic rna, they assemble into virions by inducing the invagination and luminal pinching off of the limiting membrane of this organelle (tooze et al., ; ng et al., ; goldsmith et al., ; stertz et al., ) . the resulting luminal virions subsequently reach the extracellular environment following the conventional secretory pathway (tooze et al., ) . the first ultrastructural analyses of cov-infected cells by electron microscopy (em) were already preformed in the s (svoboda et al., ; david-ferreira and manaker, ; ruebner et al., ) . the presence of dmvs and virions was the obvious morphological changes described at that time. these initial observations have successively been corroborated by numerous studies. more recently, other membranous rearrangements have been described. an electron tomography analysis of sars-cov-infected cells has confirmed the presence of reticular inclusions in between dmvs (knoops et al., ) , which were already reported in one of the original works (david-ferreira and manaker, ) . the recent study has proposed that these reticular inclusions, re-named cms, are the precursors of dmvs (knoops et al., ) . in addition to cms, it has been shown that the cov triggers the formation of highly organized crystalloid conformations, tubular rearrangements and vacuoles enclosing viral particles that have been named cmss, tbs and lvcvs respectively (david-ferreira and manaker, ; ruebner et al., ; tooze et al., ; ng et al., ; goldsmith et al., ; almsherqi et al., ; knoops et al., ) . the function of cms, cmss, tbs and lvcvs in cov infection is largely unknown. a major difficulty in understanding the role of these different structures has been the absence of a quantitative and qualitative em analyses over time that could help ordering them during the infection cycle. we have now filled this gap by performing a time-course em and immunoelectron microscopy (iem) examination of mhv-infected cells. by combining these qualitative and quantitative data with the measurement of viral rna synthesis, viral protein production and progeny virus release, we have, for the first time, integrated ultrastructural analyses with molecular information. this approach has allowed us to establish that mhv induces the formation of six membranous rearrangements in the following order: dmvs, cms, virions, lvcvs, tbs and cmss. importantly, we were able to show that most membrane rearrangements (lvcvs, tbs, cmss and possibly cms) observed in addition to the key structures in the infection (dmvs and virions) actually appear to be the consequence of a massive synthesis of viral proteins. in particular, lvcvs are ergic/golgi compartments that expand to accommodate an increasing production of virions. all together, our study provides an overall comprehensive picture of the ultrastructural events taking place inside a cell in the course of a cov infection. to understand the relationship between the different membranous structures induced by mhv and their role during the infection, we infected the cells at high multiplicity of infection (moi) and we analysed, in a timecourse manner, their ultrastructure by em as well as various other infection parameters during a period of h as described under experimental procedures. in order to be able to correlate our em and iem analyses with the progression of a cov infection inside the host cells, we first measured important known parameters that reflect the cov life cycle: viral rna replication/transcription, viral protein synthesis and secretion of progeny virus. to study the rna replication/transcription rate during mhv infection, the amount of genomic rna (grna) and that of subgenomic rna encoding for the n protein (sgrna n) was determined at each time point by rt-pcr as described in experimental procedures. both grna and sgrna n were already detected at h post infection (p.i.; fig. a ). their amount gradually increased until the h time point, after which the levels remained constant, resulting from a decline in rna synthesis (sawicki et al., ) . at each time, the amount of sgrna was about -fold higher than that of grna. this analysis allowed us to ascertain that the observed changes in the levels of grna and sgrna synthesis are identical to those measured using similar or different assays in various cell lines infected with diverse cov (sethna and brian, ; versteeg et al., ; sawicki et al., ) . next, we analysed the synthesis of the structural proteins during the infection. to this end, we measured the production rate of the m protein at the different time points by short pulse radio-labelling experiments followed by sds-page analysis of the crude cell lysates. the synthesis of the m protein was already detectable at h p.i. and continued to increase until h p.i. with the main increase occurring between and h p.i. (fig. b) . essentially, identical results were obtained when we analysed the production of the structural proteins s and n in the same way (data not shown). this correlates perfectly with the synthesis kinetics of the sgrna (fig. a) and consequently indicates that these mrnas are immediately available for translation. after h of infection, we observed a drop in the production of the m protein (fig. b) . these results again correspond with those of others (rottier et al., ; hilton et al., ; tahara et al., ) . to monitor the assembly and release of mhv over time, the infectivity levels in the culture supernatants collected at the different p.i. times were determined. secretion of mhv virions was first detected at h p.i. and increased until h p.i. before slowing down (fig. c ). this observation matches with the analysis of the viral rna and structural protein production ( fig. a and b) because as expected, it shows that mhv assembly and release are processes that follow intracellular mhv replication. all together, these measurements demonstrate that mhv infection in hela-ceacam a cells progresses following the typical, established dynamics, thereby validating the use of this cell line. in addition, they also show that the h time window used in our time-course analysis comprises all the phases of a cov infection and therefore our examinations allow obtaining a complete overview of a cov life cycle. as a first ultrastructural analysis, we compared the morphology of cells at h p.i. with that of those at h p.i. by em in order to make a repertoire of all the membranous rearrangements that mhv induces. we identified six different structures. the most abundant of them were large vesicles with an average diameter of - nm, which are limited by a double-membrane and often were clustered together ( fig. a and b; and fig. s a, arrows) . these are the characteristic dmvs induced by cov (svoboda et al., ; david-ferreira and manaker, ; ruebner et al., ; pedersen et al., ; gosert et al., ; snijder et al., ; knoops et al., ) . surprisingly, most of the observed dmvs appeared to have an invagination, which, from time to time, was associated to what looked as a small vesicle with a diameter of - nm (fig. s b, arrow) . this structural peculiarity is cell type-specific because it was also observed in mhv-a -infected mouse embryonic fibroblasts (data not shown) but not in mouse lr cells (knoops et al., ) . in the centre of the dmv clusters, we frequently observed a small network of membranes with a diameter varying from to nm, which have recently been described in sars-cov-infected cells and called cms [ fig. a and b, arrowheads; (knoops et al., ) . the cms were often in close proximity of the er (< - nm distance), and sometimes appeared to be connected with this organelle (fig. s c , arrow). virions were the third structure that we identified. these dark circular structures with a diameter of - nm were found in the lumen of either a stack of adjacent cisternae, very likely the golgi, or what appeared to be secretory vesicles (fig. c ) as well as extracellularly ( fig. s d ) in complete agreement with their known assembly and secretion mechanisms. the virions were also observed inside large circular organelles with a diameter of approximately - nm (fig. d, arrow) . interestingly, we observed virion particles assembling by invagination at the limiting membrane of these compartments through a process identical to the one occurring at the ergic (fig. d, arrow) . because similar organelles have previously been described (ng et al., ; goldsmith et al., ; knoops et al., ) , we called them in the same way: the lvcvs. the fifth conformation that we detected was a condensed rearrangement of membranes with a diameter of approximately - nm but without an apparent ultrastructural organization, which seemed to be connected to the er (fig. e ). these structures have already been described a long time ago and called tbs ( david-ferreira and manaker, ) . the sixth classified structure was a rectangular, extended (up to nm in length) and highly organized membranous conformation always continuous to what appeared to be a swollen er cisterna (fig. f , arrow; the arrowhead indicates the swollen er). a morphologically similar subcellular arrangement has been observed in sars-cov infected cell sections and consequently, we also called these structures cms (almsherqi et al., ; almsherqi et al., ) . to see whether the changes observed at the ultrastructural level correlate with the other measured infection parameters ( fig. ) , we first morphologically determined the number of cell sections at each time point that demonstrate visible signs of infection. to this end, the number of cell sections demonstrating at least one of the six structures induced by the mhv was determined at each time point. at h p.i., % of the cell sections showed visible signs of infection and this percentage gradually increased during time until reaching % at h p.i. (fig. a) . importantly, the percentages of cell sections with visible signs of infection as determined with the em analysis were very similar to those as evaluated by if (data not shown) and correlated well with the rest of the measured parameters, demonstrating that this is a reliable alternative approach to follow the mhv infection. to understand the role of the six mhv-induced structures during an infection and to unravel their relationship, we the quantitatively analysed the em sections obtained at the different p.i. time points. two values were calculated: (i) the percentage of cell profiles containing a specific structure and (ii) the average number of a given structure per cell section. our analysis revealed that the dmvs are the first membrane rearrangement to be detected in the infected cells. dmvs were already observed at h p.i. in about % of the cell sections and the number of cell sections positive for these vesicles gradually increased over time (fig. b ). the average number of dmvs per cell section reached a maximum, e.g. dmvs/cell section, at h p.i. (fig. c ). interestingly, the localization and morphology of the dmvs changed during the infection. at early time points, from to h p.i., dmvs were small ( - nm diameters) with a regular circular shape and distributed throughout the cytoplasm. from h onwards, dmvs organized in clusters mostly found in the perinuclear region of the cell. the dmv invaginations became more pronounced at h p.i. after which the small vesicles located in their interior also became more prominent (fig. s b , arrow). two hours later, the shape of the dmvs started to change acquiring a less circular form and with protuberances emerging from their surface that entered invaginations of adjacent dmvs (fig. s e , arrows). the next structures to be detected during the mhv infection were the cms, which became apparent at h p.i. the cms were always found in close proximity to at least one dmv (< - nm distance). at the early infection time points, e.g. - h p.i., the cms had small sizes ( - nm diameters) and were present in only % of the cells (fig. d ). their number per cell and their size, however, increased during the progression of the infection reaching a plateau at h p.i. (fig. e ; cm/cell section, - nm in diameter). a dramatic change in the percentage of cms was observed at h p.i. when the number of cm positive cells had increased from % to %. overall, these data suggested that the cms are structures that are functionally connected with dmvs as suggested (knoops et al., ) . virions appeared at h p.i. and their intracellular number became constant ( virions/cell section) already at h p.i., probably upon reaching an equilibrium between synthesis and secretion ( fig. f and g). the virions were mostly observed in the golgi cisternae at h p.i. in agreement with the fact that mhv particles assemble at the ergic and are released into the extracellular space by passing through the secretory pathway (ng et al., ; stertz et al., ) . lvcvs became detectable at h p.i. (fig. h ) in coincidence with a more than twofold increase in the number of virions per cell profile (fig. i ). this observation suggested that the formation of lvcvs is probably induced by a higher production of virions in the cells. the tbs also became visible at h p.i. (fig. j ). the number of tb-positive cell sections increased during time as well as the size of these structures (fig. k) , with an average diameter of - nm at h p.i. to one of about nm at h p.i. initially, each infected cell profile contained only one tb but, after h p.i., we occasionally observed more than one tb per cell section. the cmss, in contrast, were even more rare (observed in only % of the cells) and only detectable after h p.i.; hence a significant statistical analysis could not be performed. we concluded that the tbs and the cmss are not required for the early steps of the mhv infection cycle but rather the result of an advanced infection. to further understand the role of the mhv-induced structures, we explored by iem the presence or absence of viral non-structural and structural proteins in the six identified structures. the antibodies used were recognizing either nsp /nsp , nsp , nsp , the n, the m or the e proteins. we immunolabelled cryo-sections obtained from cells fixed at and h p.i. to be able to detect compositional changes. however, the labelling profiles at these two infection time points were identical; the only major difference was the higher immunoreactivity of the h p.i. samples due to the higher amounts of viral proteins. as expected (gosert et al., ; snijder et al., ; stertz et al., ) , the nsp /nsp , nsp and nsp proteins were decorating the surface of the dmvs (fig. a-c) . importantly, we discovered that these nsp's were also present in the cms but not in the other mhvinduced structures (fig. a -c and fig. s a -h). the n protein was also distributed on both dmvs and cms (fig. d ) suggesting a possible direct relationship between these two structures. like the two other struc-tural proteins, i.e. m and e, the n protein was found in virions present in the golgi complex and lvcvs ( fig. e-h) . the m and e proteins were additionally observed in the limiting membrane of the golgi cisternae but not on other mhv induced membranous rearrangements (fig. s ) . interestingly, we discovered that the tbs contain the e protein but are negative for the other tested viral proteins (fig. i, fig. s a and c) . this result, plus the fact that the tbs appear at the late stage of the mhv infection ( fig. j and k) , indicates that they could be generated by self-assembly, possibly in the er, of high levels of e protein. this hypothesis is sustained by the observation that the individual expression of the e protein induces the formation of a complex of tubular and smooth membranes with morphology reminiscent to that of the tbs (raamsman et al., ) . none of the tested antibodies labelled the cmss. because these structures appear at the late stage of the infection (data not shown), we speculate that they are induced by aggregation of the s protein for which we do not have an antibody compatible with our iem procedure. to acquire information about the host organelle origins of the mhv-induced structures, cryosections were obtained from cells harvested at and h p.i., and labelled with antibodies recognizing the protein disulfide isomerize pdi (er), ergic (ergic), gm (cis-golgi), tgn- (trans-golgi network, tgn) and lamp (late endosomes and lysosomes). these antibodies localized to the expected compartments in non-infected cells (fig. s ) . none of the employed organelle protein markers labelled the dmvs or the cms (data not shown), even though the latter ones were often observed in proximity of the er (fig. s c) . as previously reported, forming and completed virions were observed in the ergic and golgi cisternae ( fig. a and data not shown respectively). importantly, ergic , gm and tgn were found on the lvcvs as well, albeit at very low levels ( fig. b-d) . notably, despite its close proximity and almost clear continuity with the er, tbs were not positive for pdi (fig. e ). in contrast, the cmss contained this er protein marker (fig. f ) in agreement with morphological connection with this organelle (fig. f ). the low labelling of lvcvs with antibodies against ergic , gm and tgn- indicated that these organelles are derived from the ergic and/or golgi complex. it has been shown that golgi cisternae can increase in size in order to accommodate large luminal cargo proteins such as collagen (bonfanti et al., ) . therefore, we hypothesized that lvcvs are golgi cisternae that have expanded to increase their capacity to contain a higher number of viral proteins and/or forming virions. to sustain this notion, we first examined if the golgi changes its organization and subcellular distribution during the course of an mhv infection. we took advantage of the hela-galnact -gfp stable cell line (storrie et al., ) , which expresses the fluorescent golgi protein marker galnactt -gfp, and inoculated these cells with mhv-srec before analysing them by if at , and h p.i. the presence of viral proteins and virions in the golgi was assessed using anti-m protein antibodies. in non-infected cells, the golgi appeared as a juxtanuclear concentration of ribbon-like structures as expected (fig. a , upper panel; storrie et al., ) . the mhv infection caused three major changes, which were already detectable at h p.i. but became more prominent at h p.i. (fig. a, middle and lower panels) . first, the golgi lost its compact organization; the organelle was scattered throughout the cytoplasm. second, the intensity of the galnact -gfp signal in the golgi decreased. third, galnact -gfp appeared to partially localize to the er. to unravel the effects of mhv infection on the golgi at the ultrastructural level, the same samples were also processed in parallel for iem and immunolabelled with anti-gfp antibodies. at h p.i the galnact -gfp was exclusively concentrated in the golgi complex, where it distributes into several cisternae (fig. b ). in contrast, at h p.i., the labelling was found onto two different types of structures. the first were fragmented golgi complexes (fig. c ). this morphological change during an mhv infection phenomenon has previously been reported (lavi et al., ) . the second types of labelled structures were lvcvs, in keeping with our notion that these compartments have an ergic/golgi origin (fig. d) . to substantiate that lvcvs derive from the golgi, we statistically evaluated whether there is a numeric relationship between these two compartments and whether the appearance of lvcvs correlates with a decrease of golgi complexes. to this end, we determined the number of lvcvs and golgi complexes per cell profile at the different p.i. time points by counting these two organelles in the em preparations of the mhv infection time-course experiment. as shown in fig. e , the lvcvs were first detected at h p.i. and their number subsequently increased concomitant with a reduction of golgi complexes. all together, our data strongly suggest that lvcvs are ergic/golgi cisternae that expand as a consequence of a large local production of virions. -caplen et al., ; david-ferreira and manaker, ; ruebner et al., ; salanueva et al., ; escorcia et al., ; gosert et al., ; snijder et al., ; stertz et al., ; knoops et al., ; banacha et al. ), cms, lvcvs and cmss have exclusively been described in sars-cov-infected cells (goldsmith et al., ; almsherqi et al., ; knoops et al., ) and (table ). this apparent discrepancy is probably due to various reasons, including the rarity of some of these structures, the examination of a single infection time point and the use of em procedures lacking high resolution. in our study, we have detected all these types of membrane rearrangements during the course of an mhv infection because we performed a time-course ultrastructural analysis using state-of-the-art em and iem procedures. combining our data with the previous ones, we can assert that sars-cov is inducing the same type of structures and therefore we hypothesize that these membrane rearrangements are also formed during infections with other cov. as a result, the data presented in this study generate a model that could be applicable to all cov, even if some minor differences could exist. a major difficulty in understanding and studying the role of dmvs, cms, virions, lvcvs, tbs and cmss in the cov life cycle has been the absence of information concerning their appearance and fate in the course of an infection. another obstacle has also been the very limited characterization of lvcvs, tbs and cmss but also in part of cms. our time-course qualitative and quantitative examination of the mhv infection fills these two gaps (table ) , and thus generates a more comprehensive chronological picture of the ultrastructural transformations occurring in the host cells that are induced by this virus. mhv replication (fig. a) double-membrane vesicles were the first structure (knoops et al., ) that we observed after inoculating the cells with mhv ( h p.i.; fig. b and c). these vesicles are known to play a crucial role in viral rna synthesis (gosert et al., ; prentice et al., ; ahlquist, ; haller et al., ; sawicki et al., ) . in agreement with this notion but also with previous studies (gosert et al., ; snijder et al., ; stertz et al., ; knoops et al., ) , we found that the tested components of the rtcs, i.e. nsp , nsp , nsp and nsp , localize to dmvs (fig. ) and that viral rna production becomes detectable simultaneous with the appearance of dmvs ( h p.i.; fig. a) . moreover, the rna levels correlate with the number of dmvs throughout the course of the mhv infection (fig. s ) . a particularly interesting observation was that the production of rna reaches a steady-state coincident with a diminution in the generation of dmvs (fig. a, fig. c and d, fig. s ). this observation could support a new concept in which viral rna synthesis is dictated by the number of dmvs (and thus rtcs) rather than by, or in addition to, the regulation of the rate of rna replication and transcription. alternatively, depletion of one or more host factors required for dmv biogenesis could lead to an identical outcome. obviously, future studies are necessary to address this issue. a remarkable new feature that we observed while studying dmvs generated in hela cells was the presence of an invagination, which often contained a small vesicle ( fig. a and b, fig. s b ). this characteristic has not previously been documented perhaps because it is cell type specific. yet, it may provide valuable information about the dmv biogenesis. the small vesicles could carry newly synthesized viral proteins and/or host components to dmvs. alternatively; these profiles may also represent the fusion events between dmvs described for the sars-cov (knoops et al., ) . unfortunately, the tokuyasu cryosectioning technique that we have used for our iem analyses does not allow the preservation of inner content of the dmvs and the small vesicles intimately associated with them (fig. d, e and i, fig. s b and h, fig. s b , c and e; gosert et al., ; snijder et al., ; stertz et al., ) . hence, we could not determine the presence of viral proteins and double-stranded rna (dsrna) in these small vesicles. it has recently been proposed that cms are the site of generation of dmvs (knoops et al., ) . our data showing that the cms have the same viral protein composition as the dmvs may support this hypothesis ( fig. a-e, table ). in addition, they reinforce the model (knoops et al., ) that cms are also involved in the overview of the mhv-induced structure composition established by iem. the presence (+) or absence (-) of nsp's, the various structural proteins and the analysed organelle protein markers in the dmvs, cms, virions, lvcvs, tbs and cmss is illustrated. the p.i. appearance time of these mhv-induced structures is also indicated. replication and transcription of the viral rna. as already observed (knoops et al., ) , however, the cms appear after dmv formation ( fig. b and d) . because of this observation, we favour the idea that cms are unlikely to be the precursor structure of dmvs. potentially, they could originate from the dmvs but we never observed continuity between these two structures. another possibility is that cms are structures generated either for nsp's storage or by accumulation of excess nsp's that cannot be incorporated into dmvs. the possibility that the dmv biogenesis could require host components makes it possible that depletion of these components causes an accumulation of the newly synthesized transmembrane nsp's in the er (harcourt et al., ; kanjanahaluethai et al., ; oostra et al., ; oostra et al., ) , which leads to a clustering of rtcs at this site that results in the formation of cms. in accordance with this hypothesis, cms are frequently connected with the er (fig. s c ; knoops et al., ) . in addition, their size increases over time especially after h p.i. when dmv formation slows down. it remains to be investigated whether cms are able to synthesize viral rna and to eventually pack dsrna into their interior, which seems not to be the case so far (knoops et al., ) . virion assembly and release (fig. b) as expected, the virions were positive for the structural proteins, m, e and n, but not for the nsp's ( fig. and fig. s , table ). their immunolabelling with the anti-e protein antiserum, however, was weak, consistent with the low number of e proteins per virion (vennema et al., ; de haan and rottier, ) . in agreement with the previous literature about their formation and release, virions were seen assembling in the ergic/golgi (fig. c) and complete viral particles were present in these organelles but also in secretory vesicles and the extracellular space (fig. g, fig. a, fig. c, fig. s d ). these observations were first recorded at h p.i. (fig. f and g). from h p.i., virions were also forming and contained in the lvcvs (fig. d, fig. h and i, fig. e, f and h, fig. b-d, fig. d, fig. s a and c, table ). lvcvs have already been described in sarsinfected cells (ng et al., ; goldsmith et al., ; knoops et al., ) and the observation that viral particles assemble at the limiting membrane of these compartments has led us to hypothesize and to show that they have a golgi origin (fig. b-d and fig. ). the enlargement of the ergic/golgi compartments that leads to the formation of the lvcvs is probably a consequence of the accumulation of massive amounts of viral proteins and/or luminal virions that require an expansion of this organelle to accommodate them. this phenomenon has already been reported in the case of large cargo molecules passing through the golgi (bonfanti et al., ) . it cannot be excluded a priori, however, that an overloading in viral proteins causes a fusion of the golgi cisternae, which results in the lvcv formation. the fact that the secretory pathway is flooded by viral components is also emphasized by the formation of virions in the er from h p.i. (fig. s ) . the labelling efficiency of lvcvs for ergic and golgi protein markers, however, was reduced as compared with that of the same organelles in non-infected cells ( fig. b and fig. s b-d) . these observations indicate that some of the standard golgi functions of this compartment are probably altered by the high content of viral components. this is exemplified by the observation that the golgi cisternae fragment and the golgi protein marker galnact -gfp partially localizes to the er during the course of an mhv infection ( fig. a and c) . in addition, the secretion of gaussia luciferase is reduced during mhv infection (verheije et al., ) . nevertheless, the lvcvs are functional structures because the extracellular release of mhv is still effective at the advanced stages of the mhv infection when numerous lvcvs are present in each cell (fig. c, fig. h and i). lvcvs are probably not an organelle exclusively induced by covs, as lvcv-like structures have also been observed in cells infected with the (+) rna rubellavirus (risco et al., ; novoa et al., ) , which also assembles its progeny virions in association with the golgi compartment. our data show that tbs and cmss are infrequent structures ( fig. j and data not shown), that they appear at the late stages of the cov infection ( fig. j and k, and data not shown) and that they probably contain a single viral protein (fig. i, table ). these observations indicate that they are the result of the massive production of viral proteins that cannot be incorporated into the virions (fig. b ). the tbs are only positive for the e protein but not for the nsp's, the n and the m protein ( fig. s d -e, fig. s a and c; table ). we cannot formally exclude that they also contain the s protein but it is unlikely because the individual expression of the e protein is sufficient to induce the formation of a complex of smooth tubular membranes with morphology reminiscent to that of the tbs (raamsman et al., ) . the e protein is a transmembrane component that is inserted in the er before being transported to the ergic/golgi (lim and liu, ; corse and machamer, ) that is also able to selfinteract (raamsman et al., ; von brunn et al., ) . importantly, even if not positive for the er resident chaperone pdi, the tbs have clear membrane continuity with the er (fig. e, fig. e, fig. s d and f) . consequently, a very likely scenario is that the tbs are generated by excess e protein that self-aggregates in the er. the cmss were negative for any of the tested viral proteins ( fig. s g and h, fig. s d and f, table ), and therefore we speculate that they are induced by the s protein, for which we do not have an antibody compatible with our iem procedure. the cmss are clearly connected with er and positive for pdi ( fig. f and fig. f ). interestingly, it has previously been shown that overexpression of transmembrane proteins that have the capacity to self-interact can lead to the formation of geometrical and crystalloid conformations in the er that have been called either 'cubic membranes' or 'organised smooth er', which resemble the cmss (snapp et al., ; almsherqi et al., ; . therefore, the simplest model that we propose is that cmss are formed by the self-assembly of excess s protein in the er. in conclusion, our study has ordered and characterized the different membranous rearrangements induced during a cov infection. this information paves the way for future investigations about the biogenesis and the function of these structures, which is crucial to understanding the cov life cycle and eventually for the development of more effective therapies against these pathogens. hela-ceacam a cells (verheije et al., ) , the hela-galnact -gfp stable cell line (storrie et al., ) and lr cells, used to propagate and titrate mhv-a and mhv-srec, were maintained in dulbecco's modified eagle medium (dmem; cambrex bioscience, walkersville, md, usa) containing % fetal calf serum (bodinco, alkmaar, the netherlands), iu of penicillin·per millilitre and mg ml - of streptomycin (both from life technologies, rochester, ny, usa). hela cells expressing the mouse ceacam a receptor (hela-ceacam a cells), which allows infecting these human cells with mhv-a (verheije et al., ) , were used for the time-course mhv infection. we decided to employ hela cells because many antibodies against human proteins are available and those cells have previously been successfully used for the study of cov replication (gosert et al., ) . the hela-ceacam a cells were inoculated with mhv-a at an moi of tissue culture infection dose (tcid ) as determined on lr cells, in phosphate-buffered saline (pbs) containing mg ml - diethylaminoethyl-dextran (pbs-deae). after min, hela-ceacam a cells were washed and maintained in complete dmem containing mm of the fusion inhibitor mhr peptide (genscript, piscataway, nj; bosch et al., ) . the mhr peptide prevents fusion of adjacent cells upon interaction of expressed s protein and mceacam a receptor as well as additional infection by the residual inoculum in the medium or, at later times, secreted by the infected cells (bosch et al., ) . as a result, the mhv infection is synchronized. subsequently, aliquots of infected cells and culture supernatants were collected for analysis at , , , , , , , , and h p.i. the hela-galnact -gfp stable cell line was infected with mhv-srec, a recombinant mhv strain with an extended host range (de haan et al., ) . these cells were then fixed at , and h p.i. before being processed for if and iem. the total rna was isolated from the infected cells using the trizol reagent (invitrogen, san diego, ca, usa). rna was further purified using the rneasy mini-kit (qiagen, hilden, germany) according to the manufacturer instructions with subsequent dnasei treatment on the column. rna integrity was determined by spectrometry using a uv-mini device (shimadzu, kyoto, japan). taqman single-tube reverse transcription-pcr (rt-pcr) assay (pe biosystems, foster city, ca) was performed as described (de haan et al., ) and used to analyse both the orf b region (de haan et al., ) and the n protein-encoding region (raaben et al., ) of the viral genome. the reactions were performed in triplicate according to the manufacturer's instructions using an abi prism sequence detector (foster city, ca, usa). thirty minutes before the indicated time points, cells were starved for min in cysteine-and methionine-free modified eagle's medium containing % fetal calf serum and mm hepes, ph . . cells were subsequently radiolabelled in the same medium containing mci of s in vitro cell-labelling mixture (amersham pharmacia biotech, freiburg, germany) for min and lysed in the tesv lysis buffer ( mm tris-hcl, ph . , mm edta, mm nacl, mm pmsf, % triton x- ). the lysates were analysed by sds-page. due to the preferential and sustained synthesis of viral proteins during the course of an infection, the radioactive bands corresponding to the m protein ( - kda) and the structural proteins s and n were the most prominent on the autoradiographs. the m protein was quantified using a phosphorimager system (molecular dynamics, uppsala, sweden). the amount of virions present in the culture supernatants was determined by end-point dilutions on lr cells and then calculating the tcid values. cells were fixed with karnovsky ( % para-formaldehyde, . % glutaraldehyde, mm cacl , mm mgcl in . m sodium cacodylate ph . ), first at room temperature for min and then overnight at °c. cell pellets were then post fixed with % oso / . then % kcnfe in . m cacodylate buffer for h on ice and dehydrated stepwise with increasing concentrations of ethanol. the dehydrated pellets were rinsed with propylene oxide at room temperature and then embedded in epon (degtyarev et al., ) . after resin polymerization, - nm sections were cut using an ultra-microtome (leica) and contrasted with uranyl acetate and lead citrate before being viewed in a jeol or a jeol electron microscope (jeol, tokyo, japan). for the quantitative analyses, cell profiles were randomly selected at each p.i. time point and three different quantifications were performed. first, the number of cells containing at least one of the six mhv-induced structures was counted to determine morphologically the proportion of infected cells. second, we calculated the percentage of cell sections positive for each mhvinduced structure. third, we determined the average number of each structure per cell section and calculated the standard deviation. the standard deviation values were subsequently used to perform the t-test, which revealed the significance of the data about dmvs (p = . ), cms (p = . ), virions (p = . ), lvcvs (p = . ) and golgi (p = - ) but not for the tb (p = . ). the calculation of the standard deviation and the t-test could not be performed for the cmss because of the rarity of these structures. cells were directly fixed by adding double-strength fixative ( % para-formaldehyde/ . % glutaraldehyde or % paraformaldehyde, both in . m phosphate buffer, ph . ) to the culture for min at room temperature. this fixative was replaced by fresh standard strength fixative ( % paraformaldehyde/ . % glutaraldehyde or % para-formaldehyde, both in . m phosphate buffer, ph . ) and incubated overnight at °c. fixation was subsequently continued for h at room temperature before washing the cells times with . m phosphate buffer, ph . and scraping them from the plate in the same buffer containing % gelatin. cells were successively centrifuged and embedded in % gelatin (slot and geuze, ) . the obtained pellets were cut into small cubes, which were cryo-protected in . m sucrose and subsequently frozen in liquid nitrogen. finally, after trimming to a suitable block shape, - nm ultrathin sections were cut at - °c on dry diamond knives (diatome ag, biel, switzerland) using either an uc or an uct ultra-microtome (leica). cryo-sections were labelled with rabbit antisera recognizing nsp and nsp [a kind gift of s. baker (schiller et al., ) ], nsp [a kind gift from s. baker (schiller et al., ) ], nsp [a kind gift of m. denison (lu et al., ) ], m protein (krijnse-locker et al., ) , e protein (de haan et al., ) , gm [a kind gift of e.s. sztul (styers et al., ) ], gfp (abcam, cambridge, uk) or mouse monoclonal antibodies against the nucleocapsid n (a kind gift from stuart siddell), pdi (stressgen, ann arbor, mi, usa), ergic (alexis biochemicals, lausen, switzerland) or sheep anti-tgn- (serotec, oxford, uk) antiserum. the specificity of the antibodies against viral proteins was tested on mockinfected cells (data not shown). antibodies were visualized with or nm gold particles conjugated to protein a (slot and geuze, ) . rabbit antibodies were directly detected with protein a-gold particles whereas mouse and sheep antibodies where bridged with polyclonal anti-mouse (dako, carpinteria, ca, usa) and anti-sheep (nordic, tady, sweden) igg antibodies. labelled cryosections were finally contrasted with an uranyl acetate-uranyl acetate methyl cellulose mixture. all specimens were imaged as described for the conventional em. e and f. the e protein is not distributed on the dmvs, cms and cmss. arrows and arrowheads indicate tbs and cmss, respectively, whereas asterisks mark the dmvs. cm, convoluted membranes; er, endoplasmic reticulum; g, golgi; l, lysosome; lvcv, large virion-containing vacuole; m, mitochondria. white bar, nm; black bar, nm. fig. s . specific immunolabelling of intracellular organelles. mock-infected cells were fixed and processed with antibodies against (a) pdi (er), (b) ergic (ergic), (c) gm (cis-golgi cisternae), (d) tgn (trans-golgi) and (e) lamp (late endosomes and lysosomes). er, endoplasmic reticulum; l, lysosome; m, mitochondria; n, nucleus; pm, plasma membrane. white bar, nm; black bar, nm. fig. s . correlation between the viral rna synthesis and dmv biogenesis. the number of dmvs/ cells at each time point of the mhv infection was calculated by multiplying the percentage of dmv-containing cells with the average number of dmvs per cell. results are plotted on a log graph together with the grna and sgrna n amounts expressed using arbitrary units. fig. s . virion assembly can occur in the er. immunolabelling of hela-ceacam a infected cells with anti-pdi antibodies at h p.i. assembly of virions (arrows) can be observed in the er. er, endoplasmic reticulum; m, mitochondria. bar, nm. please note: wiley-blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. any queries (other than missing material) should be directed to the corresponding author for the article. parallels among positive-strand rna viruses, reverse-transcribing viruses and double-stranded rna viruses direct template matching reveals a host subcellular membrane gyroid cubic structure that is associated with sars virus cubic membranes: a legend beyond the flatland* of cell membrane organization 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associated with the severe acute respiratory syndrome coronavirus replication complex the intracellular sites of early replication and budding of sarscoronavirus recycling of golgi-resident glycosyltransferases through the er reveals a novel pathway and provides an explanation for nocodazoleinduced golgi scattering depletion of beta-cop reveals a role for cop-i in compartmentalization of secretory compartments and in biosynthetic transport of caveolin- an electron microscopic study of viral hepatitis in mice coronavirus translational regulation: leader affects mrna efficiency replication of coronavirus mhv-a in sac-cells: determination of the first site of budding of progeny virions sorting of progeny coronavirus from condensed secretory proteins at the exit from the trans-golgi network of att cells nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes mouse hepatitis coronavirus rna replication depends on gbf -mediated arf activation redirecting coronavirus to a nonnative receptor through a virusencoded targeting adapter transcriptional profiling of acute cytopathic murine hepatitis virus infection in fibroblast-like cells coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus virusencoded proteinases and proteolytic processing in the nidovirales the authors thank s. baker, m. denison, t. nilsson, s. siddell and e.s. sztul for reagents, matthijs raaben and eddie te lintelo for technical assistance, peter rottier and judith klumperman for the critical reading of the manuscript, and marc van peski and rené scriwanek for assistance with the preparation of the figures. f.r. and c.a.m.h. are supported by the utrecht university (high potential grant). additional supporting information may be found in the online version of this article: b. dmvs with invaginations that appear to contain a small vesicle. c. a cm (arrow) that appears to be connected with the er. d. virions (arrows) released in the extracellular space. e. dmvs with protuberances emerging from their surface entering the invagination of an adjacent dmv (arrows). er, endoplasmic reticulum; m, mitochondria; n, nucleus; pm, plasma membrane. white bar, nm; black bar, nm. fig. s . mhv-induced structures negative for non-structural proteins. hela-ceacam a cells infected with mhv-a were fixed at h p.i. before being processed for iem and immunolabelled as described in experimental procedures. a-c. nsp /nsp , nsp and nsp do not localize to the lvcvs (arrows). d-f. nsp /nsp , nsp and nsp are not present in the tbs (arrow). g and h. the cmcs (arrows) are not positive for nsp and nsp . asterisks mark the dmvs. m, mitochondria; n, nucleus; pm, plasma membrane. bar, nm. fig. s . mhv-induced structures negative for structural proteins. hela-ceacam a cells inoculated with mhv-a were fixed at h p.i. before being processed for iem and immunolabelled as described in fig. . a. the n protein does not localize to the tbs. b. the m protein is not present on both dmvs and cms. c and d. the tbs and the cmss are not positive for the m protein. key: cord- -y x x h authors: cai, yingyun; liu, yin; yu, dongdong; zhang, xuming title: down-regulation of transcription of the proapoptotic gene bnip in cultured astrocytes by murine coronavirus infection date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: y x x h murine coronavirus mouse hepatitis virus (mhv) causes encephalitis and demyelination in the central nervous system of susceptible rodents. astrocytes are the major target for mhv persistence. however, the mechanisms by which astrocytes survive mhv infection and permit viral persistence are not known. here we performed dna microarray analysis on differential gene expression in astrocyte dbt cells by mhv infection and found that the mrna of the proapoptotic gene bnip was significantly decreased following mhv infection. this finding was further confirmed by quantitative reverse transcription–polymerase chain reaction, western blot analysis, and bnip -promoter-luciferase reporter system. interestingly, infection with live and ultraviolet light-inactivated viruses equally repressed bnip expression, indicating that the down-regulation of bnip expression does not require virus replication and is mediated during cell entry. furthermore, treatment of cells with chloroquine, which blocks the acidification of endosomes, significantly inhibited the repression of the bnip promoter activity induced by the acidic ph-dependent mhv mutant oblv , which enters cells via endocytosis, indicating that the down-regulation of bnip expression is mediated by fusion between viral envelope and cell membranes during entry. deletion analysis showed that the sequence between nucleotides and of the -base-pair bnip promoter is necessary and sufficient for driving the bnip expression and that it contains signals that are responsible for mhv-induced down-regulation of bnip expression in dbt cells. these results may provide insights into the mechanisms by which mhv evades host antiviral defense and promotes cell survival, thereby allowing its persistence in the host astrocytes. viruses can cause different types or patterns of infection in susceptible hosts; host cells in turn respond to viral infection in many different ways. some virus-infected cells undergo rapid destruction, whereas others become immortalized. in addition to immune response, host cells often devise countermeasure mechanisms against virus invasion. one of the common antiviral defense strategies operated by host cells is programmed cell death or apoptosis, for apoptosis would eliminate virus-infected cells, thereby blocking the production of progeny virus and limiting the spread of virus to neighboring uninfected cells. however, apoptosis can also be a viral strategy to invade hosts. many viruses can induce apoptosis in host cells during late stages of the virus life cycle to speed up their release from infected cells and spread to neighboring cells. on the other hand, many viruses have also devised strategies to counter host antiviral defense by inducing an antiapoptotic state in host cells. for examples, human immunodeficiency virus tat protein (zauli et al., ) and epstein-barr virus-ebna and -lmp- proteins (silins and sculley, ; henderson et al., ) induce the expression of antiapoptotic protein bcl- , which is the master checkpoint protein of apoptosis through the mitochondrial pathway (hockenbery et al., ) . members of the bcl- family share up to four conserved regions of homology known as bcl- homology (bh) domains, which mediate interactions among themselves (kelekar and thompson, ) . a delicate balance of interaction between proapoptotic and antiapoptotic members of the bcl- family has been shown to modulate the sensitivity of cells to mitochondrial integrity in response to apoptotic signals . many viral proteins are bcl- ho-mologs such as adenovirus e b -kda protein (ad-e b k) (chiou et al., ) . bnip (bcl- and nineteen-kilodakton-protein interacting protein ) is the proapoptotic member of the bcl- family. it was first identified as an ad-e b k and bcl- interacting protein (boyd et al., ) . bnip contains only the bh domain (yasuda et al., ) . it functions as a proapoptotic protein that both induces apoptosis and increases the sensitivity of cells to apoptosis induced by drugs and granzymes b upon transient expression . moreover, bnip can form heterodimers with antiapoptotic bcl- family members such as bcl- and bcl-xl and promotes apoptosis by sequestering the antiapoptotic members (imazu et al., ) . recent evidence from studies in t cells suggests that the bh -dependent apoptotic pathways can also be turned on by surface receptor via interaction with cd (lamy et al., ) . it has been reported that the expression of bnip gene is induced by hypoxia (bruick, ; kubasiak et al., ) and a zinc-finger protein, plagl (mizutani et al., ) , leading to cellular apoptosis. murine coronavirus mouse hepatitis virus (mhv) is an enveloped, positive-strand rna virus. it contains four or five structural proteins, depending on virus strains. the spike protein (s) is the major peplomer protruding from the virion envelope. it is responsible for virion attachment to host cells via interaction with receptors, induction of fusion between viral envelope and cell membranes during infection, and elicitation of neutralizing antibodies and cellular immune responses (lai, ) . thus, the s protein is essential for virus infectivity. the hemagglutinin/esterase (he) has sequence homology with the hef protein of influenza c virus (luytjes et al., ) . it contains an esterase activity but lacks the receptor-binding activity . it is present on the virion surface only in some mhv strains (such as jhm) but not others (such as a ) la monica et al., ) , and it is thus not essential for virus infectivity. the small envelope (e) protein is acetylated (yu et al., ) and the membrane (m) protein is glycosylated (niemann et al., ) . both proteins are hydrophobic and largely embedded in the virion envelope. they are essential for virion assembly and morphogenesis (vennema et al., ; bos et al., ) . the nucleocapsid (n) protein is a phosphoprotein that is tightly associated with the viral rna genome to form the nucleocapsid inside the envelope shell (stohlman and lai, ) . it has been shown that dephosphorylation of the n protein following viral entry into host cells is a prerequisite step for virus replication (kalicharran and dales, ; kalicharran et al., ) . during the initiation of virus infection, the s protein binds to a specific receptor on the surface of susceptible cells, which is a murine biliary glycoprotein belonging to the carcinoembryonic antigen (cea) in the immunoglobulin superfamily (williams et al., ) . upon viral binding, fusion between viral envelope and plasma membrane is then triggered, allowing the nucleocapsid to release into the cytoplasm. interaction between the s protein and the receptor can also mediate endocytosis (nash and buchmeier, ) . in this case, fusion occurs between viral envelope and endosomal membrane at an acidic environment. once in the cytoplasm, the n protein is presumably dephosphorylated and dissociates from the genomic rna, thereby completing the uncoating process. the viral rna-dependent rna polymerase is then translated from the incoming viral genome and viral replication proceeds subsequently. mhv infects rodents, causing hepatitis, nephritis, enteritis, and central nervous system (cns) diseases. infection of rodent cns results in encephalitis and demyelination, the latter of which resembles the multiple sclerosis seen in humans (lampert et al., ; herndon et al., ) . therefore, mhv has been extensively used as an animal model for studying the pathogenesis of multiple sclerosis and other cns degenerative diseases. in certain mouse strains, demyelination develops during the first week of infection (woyciechowska et al., ) and reaches to a peak at approximately days postinfection (p.i.), at which time infectious virus can no longer be isolated but viral rnas are still detectable (fleming et al., ; adami et al., ) . the predominant cell type in the cns for harboring persistent viruses is the astrocyte (perlman and ries, ) . indeed, persistent infection can be readily established in animal cns (adami et al., ; rowe et al., ) and cultured astrocytes (maeda et al., ; chen and baric, ) . in contrast to the infection in vivo, infectious virus can be recovered from infected, cultured astrocytes after continuous passage of cells for more than days (chen and baric, ) . because astrocyte is one of the antigen-presenting cells in the cns (fierz et al., ) , it can produce diverse proinflammatory molecules such as cytokines, chemokines, and nitric oxide upon stimulation (van wagoner and benveniste, ; huang et al., ; chen and swanson, ) . it has been shown that coronaviruses induce the expression of transforming necrosis factor-(tnf) ␣, interleukin-(il) , matrix metalloproteinases, and inducible nitric oxide synthase in primary and established astrocytes (joseph et al., ; zhou et al., ; grzybicki et al., ; kyuwa et al., ) . thus, it is conceivable that infection of astrocytes with mhv can modulate the development of the cns pathology and alter the course of the disease. in the past, most studies have been focused on how mhv has evolved during persistent infection in the cns and in cultured cells (adami et al., ; rowe et al., ) . information on coevolvement of the host has been limited to the alteration of receptors by mhv infection (chen and baric, ; sawicki et al., ) . to understand how mhv infection alters the gene expression of astrocytes, we employed the dna microarray technology to analyze the mrna expression profiles in mhv-infected murine astrocytoma cell line dbt and compared them with those in mock-infected dbt cells. we found that the expression of a substantial number of genes was either increased or decreased upon mhv infection. of these, the expression of the proapoptotic gene bnip was significantly decreased. a series of subsequent experiments further confirmed this conclusion. interestingly, the downregulation of bnip expression does not require viral replication and is mediated during cell entry. our results may provide insight into the mechanisms by which mhv evades host antiviral defense and promotes cell survival, thereby allowing its persistence in the host astrocyte. to determine altered gene expression of dbt cells by virus infection, dbt cells were infected with mhv strain jhm at an m.o.i. of or mock-infected. at h p.i., intracellular rnas were isolated with the rnaeasy kit (qiagen inc.). the mrna expression profiles were determined using the affymetrix genechip-the murine genome u av array, which contains oligonucleotides representing approximately known genes and est of the mouse genome. a Ն -fold difference in mrna amount between virus-infected and mock-infected cells was considered statistically significant. based on this criteria, we identified known genes that were significantly up-regulated by mhv infection and known genes that were significantly down-regulated (data not shown). among those, we found that the mrna of the proapoptotic gene bnip was decreased approximately . -fold in mhv-infected dbt cells as compared to that in mock-infected dbt cell. to verify this microarray data, we employed three different approaches. the first approach was to use reverse transcription-polymerase chain reaction (rt-pcr) to directly measure the amounts of the bnip mrna expression during the course of virus infection. dbt cells were infected with mhv strain jhm at an m.o.i. of . at , , and h p.i., intracellular rnas were isolated and cdnas were synthesized by rt with a hexomer random primer. cdnas were then amplified by pcr using a primer pair specific to bnip (fig. a) . we found that cdnas representing the bnip gene decreased at and h p.i. in virus-infected cells as compared to those in mock-infected cells after normalizing with ␤-actin gene (data not shown). to further provide quantitative assessment on the extent of bnip mrna reduction by mhv infection, we employed a competitive rt-pcr. as depicted in fig. a , we generated a competitor dna fragment that contains an internal deletion of about nt but which contains the same two primer binding sites on the target dna. as the result, the amount of the target dna fragment being amplified by pcr would be decreased with an increased amount of competitor dna being added in the pcr reac-tion. the pcr products were analyzed by agarose gel electrophoresis and the intensity of the dna bands following ethidium bromide staining was determined by densitometry. rnas were isolated and were subjected to rt with a random primer. cdnas were amplified by pcr using the primer pair specific to bnip as shown in a in the presence of a known amount of the competitor dna. the competitor dna was amplified from a deletion construct and was purified from agarose gel. from lanes to and to , a -fold serially diluted competitor dna (indicated as copy number) was added to the pcr. the rt-pcr products were analyzed by electrophoresis in % agarose gel and visualized by staining with ethedium bromide. images were taken with the digital camera (uvp). the intensity of each band was determined with the software (labworks ). arrows indicate the target pcr products (bnip ) and the competitor pcr product (comp), respectively. an equal amount of the target pcr product to the competitor pcr product is considered the amount of the target template in the original rna sample, which is expressed as percentage of virus-infected samples to mockinfected samples as shown in c. a representative gel is shown in fig. b . the amounts of the target dnas and competitor dnas were then plotted, and the value, at which an equal amount of target and competitor dnas was obtained, was considered to be the amount of the target gene. to insure that the pcr products quantitatively reflected on the target gene, we optimized the pcr reaction by reducing the amount of input cdna and the number of pcr cycles so that the amount of the dna in each band was not saturated by ethidium bromide staining. we used this technique to determine the amount of bnip mrnas at different time points p.i. as shown in fig. c , there was virtually no difference in the amount of bnip cdna between virus-infected and mock-infected cells at h p.i., but an approximately % reduction was detected in virus-infected cells at h p.i., as compared with that of mock-infected cells. at h p.i., the reduction in bnip gene reached to approximately % in virus-infected cells. these results were reproducible in at least three independent experiments. these results demonstrate that mhv infection down-regulated bnip gene expression at the transcriptional level, thus confirming the results from the dna microarray analysis. the second approach was to determine the expression of the bnip protein. we employed western blot in combination with immunoprecipitation to determine the bnip protein expression at various time points p.i. as expected, a slight decrease in bnip protein was detected in virusinfected cells at and h p.i., as compared to those in mock-infected cells (fig. ) . the reduction of bnip protein in virus-infected cells was more pronounced at h p.i., when normalized with the ␤-actin protein. although the degree of reduction of the bnip protein in mhv-infected cells varied among experiments, the pattern of reduction was consistent in several independent experiments. these results indicate that, consistent with the pattern of mrna expression, bnip protein expression was inhibited by mhv infection. the third approach was to use a reporter system to determine the promoter activity of the bnip gene in re-sponse to mhv infection. the Ј upstream sequence (promoter) of the bnip gene was placed in front of the firefly luciferase gene in the pgl-bnip construct ( fig. a ) (bruick, ) . transient transfection of the pgl-bnip dna into dbt cells that were either infected with mhv at dbt cells were infected with mhv jhm at an m.o.i. of or mockinfected. at h postinfection (p.i.), cells were transfected with ng of pgl -bnip plasmid or the promoterless plasmid pgl . at h p.i., cell lysates were harvested and luciferase activities measured as described under materials and methods. the relative luciferase activity in pgl -bnip -transfected cells is expressed as fold increase over the luciferase activity in cells transfected with the promoteless pgl . data indicate the means of three separate infection/transfection results. error bars denote standand deviations. the data are representative of six independent experiments. (c) same as in b, except that the virus was purified through sucrose-gradient ultracentrifugation. (d) same as in b, except that the transfection was carried out h prior to virus infection and the luciferase activity was determined at and h p.i. o.i. of or mock-infected. at , , and h p.i., the cells were harvested and lysed. equal amounts of cell lysates were immunoprecipitated with an antibody specific to bnip and protein g-sepharose beads. the precipitates were separated by polyacrylamide gel ( %) electrophoresis. proteins were then transfered to nitrocellulose membranes and were detected with the same bnip antibody and enhanced chemiluminescence by western blot analysis. an aliquot of an equal amount of cell lysates for each sample was used for analyzing the ␤-actin protein with the ␤-actin-specific antibody in western blot as an internal control. the data are representative of three experiments. m, lysates from mock-infected cells; i, lysates from virus-infected cells. an m.o.i. of or mock-infected as a control resulted in the expression of luciferase activity. consistent with the results from dna microarray, rt-pcr and protein detection described above, the luciferase activity was significantly decreased following mhv infection (fig. b ). this result was reproducible in more than independent transfectioninfection experiments. it was also confirmed by using purified virus and uv-irradiated virus (see below). these data thus unequivocally establish that mhv infection downregulated bnip gene expression in dbt cells. because the luciferase reporter system is a very sensitive assay that has been well established and widely used for determining promoter activity and because our results also demonstrated its sensitivity and utility, we used this reporter system as a standard method for all subsequent experiments. because our original dna microarray study used rna samples isolated from dbt cells that were cultured or infected with mhv in the presence of . % newborn calf serum, it is possible that the presence of a low level of serum and other unidentified factors secreted by infected-dbt cells in the inoculum might have contributed to the observed down-regulation of bnip expression. to rule out this possibility, we used the virus purified through sucrose gradient ultracentrifugation. as shown in fig. c , infection of dbt cells with purified virus resulted in a six-fold decrease of luciferase activity as compared to that from mock-infected cells. this result is comparable to that obtained with unpurified virus (fig. b) , indicating that the down-regulation of bnip gene expression specifically resulted from mhv infection and not from factors present in the viral inoculum. it is also possible that the observed reduction in luciferase activity resulted from inhibition of transfection efficiency by virus infection rather than from direct virus infection because mhv infection could potentially cause alteration of cell membrane permeability. to exclude this possibility, we carried out dna transfection for h to allow complete delivery of the plasmid dna into cells prior to virus infection. we found that a significant reduction of luciferase activity was detected in mhv-infected cells as compared to mock-infected cells (fig. d) , although the fold reduction in luciferase activity was less prominent when compared with the results from prior infection (fig. c ). these data argue against an indirect effect by dna transfection efficiency. it is known that productive mhv replication results in inhibition of host cell translation hilton et al., ) . to establish that the reduction of luciferase activity expressed from the pgl -luc reporter was not due to mhv-replication-induced inhibition of cell translation, dbt cells were infected with uv-irradiated mhv at an m.o.i. of for h and then transfected with the pgl -luc reporter dna. mock-infected cells were used as controls. as shown in fig. , luciferase activities were reduced at similar levels in cells infected with live virus and uvinactivated virus as compared to those in mock-infected cells. this result strongly supports the conclusion that the bulk of the reduction of luciferase activity in mhv-infected cells indeed resulted from mhv infection and not from indirect, general inhibition of cellular translation. to establish further the specificity of the down-regulation of bnip gene expression, we used vesicular stomatitis virus (vsv) in a parallel experiment, because vsv is also an enveloped rna virus whose infection has a broad host cell range and is mediated by the interaction between the envelope g protein and cell membranes (riedel et al., ) . as expected, the luciferase activity was completely inhibited by infection with live vsv because it is known that vsv replication shuts down host cell transcription (fig. ) . in contrast, little reduction of luciferase activity was detected in dbt cells that were infected with uv-inactivated vsv. nevertheless, the difference in reduction of luciferase activity between uv-inactivated mhv and vsv was significant (fig. ) . taken together, these results demonstrate that the down-regulation of bnip gene expression resulted specifically from mhv infection. because uv-irradiated viruses are no longer capable of replicating themselves, although they are still able to bind cell receptors and to be up-taken by the cells, uv-irradiation of viruses can be used to distinguish the involvement of various steps either before or after virus replication. the data presented in fig. also indicate that the down-regulation of bnip gene expression does not require virus replication. this result was reproducible in both neurotropic mhv strains jhm and a and in at least six independent experiments with triplicates for each experiment (representative data shown in fig. , further data not shown). these results thus establish that mhv infection induces the downregulation of bnip gene expression during early stages of the virus life cycle and requires no virus replication. as the above results pinpointed the involvement of virus entry in regulation of bnip gene expression, we were interested in further dissecting the individual steps of the infectious process. we designed experiments to first block downstream processes so that the role of upstream events can be assessed. however, most mhv strains, including jhm and a , enter cells both via fusion with plasma and endosomal membranes (nash and buchmeier, ) . this property poses a technical dilemma for not being able to block fusion by the common lysosomotropic agents. gallagher et al. ( ) isolated several mutants from obl cells persistently infected with mhv jhm (mhv- ). one of these mutant isolates, oblv , strictly depends on acidic ph for entry and infectivity. we therefore used this ph-dependent mutant for this purpose. dbt cells were transfected with the pbnip -luc reporter plasmid dna for h, treated with , , and m of chloroquine for h prior to virus infection, and then infected with oblv or the wild-type jhm at an m.o.i. of in the presence of chloroquine. at h p.i., cell lysates were isolated and luciferase activity determined. mock-infected and untreated cells were used as controls. as shown in fig. , treatment of cells with an increasing concentration of chloroquine resulted in an increase of luciferase activities by approximately three fold in oblv infected cells (fig. a) , concomitant with a decrease of virus titer (fig. b ). in contrast, neither the luciferase activities nor the virus titers were significantly affected by chloroquine treatment in jhm-infected dbt cells. these results suggest that fusion between viral envelope and endosomal membranes during oblv infection provides at least in part the signal(s) that triggers the down-regulation of bnip gene expression. by extrapolating the data from the mutant virus, we can envision that fusion between viral envelope and plasma/endosomal membranes during wild-type mhv infection is involved in the down-regulation of bnip gene expression. the above results suggest that the promoter region of the bnip gene contains both the cis-acting elements that are essential for bnip expression and the negative elements that repress bnip expression in response to mhv infection. to identify and dissect such cis-acting elements, we made a series of reporter constructs that contain various deletions in the bnip promoter. in an initial experiment, we determined the minimal promoter sequences required for bnip gene expression. dbt cells were transfected with various deletion reporter constructs. transfection with the promoterless vector was used as a negative (basal level) control. at h posttransfection, cells were lysed and assayed for luciferase activities. as summarized in fig. , when deletions were made from the Ј-end to nt (construct pr- ) and from nt to (construct pr- ) of the promoter, the luciferase activities retained at . and . %, respectively, as compared to the full-length promoter, indicating that the upstream half ( nt) of the promoter is not essential for bnip gene expression. in contrast, deletions of nt to (in constructs pr- and pr- ) essentially abolished the luciferase activity, indicating that the sequence from nt to of the promoter is the cis-acting element required for driving bnip gene expression. further deletion of the downstream sequence (from nt to in pr- ) resulted in approximately % reduc- tion of luciferase activity, suggesting that this sequence can influence the transcription efficiency but is not essential for bnip gene expression. partial or complete deletions of the -nt sequence (from nt to ) in constructs pr- , pr- , pr- , pr- , pr- , and pr- essentially abolished the luciferase activity. we thus conclude that the -nt sequence (nt - ) of the promoter contains the cisacting elements essential for bnip gene expression. we next determined the cis-acting elements responsive to mhv infection. cells were transfected with the fulllength and various deletion reporter constructs for h and were then infected with uv-inactivated mhv at an m.o.i. of or mock-infected as controls. as shown in fig. , the luciferase activities were decreased approximately four-to sixfold in mhv-infected cells for various reporter constructs (full-length, pr- , pr- , pr- , pr- , and pr- ), as compared with those in mock-infected control cells. the reduction of luciferase activities must have resulted from the infection process rather than from general inhibition by mhv replication, because the virus was inactivated by uv irradiation. these results suggest that the negative elements responsive to mhv infection reside within the same region of the promoter that is required for bnip expression. we were unable to narrow down further the minimal responsive elements because further deletions within this region in the reporter constructs drastically abrogate the reporter gene expression even in the absence of virus infection (constructs pr- , pr- , pr- , pr- , pr- , and pr- ). in this study, we employed dna microarray, rt-pcr, western blot, and a promoter-reporter gene system to explore the potential alteration of cellular gene expression by mhv infection. using such multitude approaches, we discovered that the expression of the cellular proapoptotic gene bnip was significantly down-regulated in cultured dbt cells by mhv infection. this finding supports the hypothesis that mhv infection in dbt cells may induce an anti- the luciferase activity was expressed as fold increase over that expressed from the promoterless pgl . the data were the means and standard deviations (sd) of at least three independent experiments. the luciferase activities for the deletion constructs were also expressed as percentage to that derived from mock-infected and full-length plasmid-transfected cells, which was set as %. (b) details of the cis-acting elements in the bnip promoter region between nt and . the sequence was analysed with the computer software signal scan (prestridge, ) . the putative consensus sequences for the binding of transcription factors are shown schematically. the numbers in brackets indicate the approximate nt positions in the promoter. apoptotic state through down-regulation of expression of proapoptotic genes. it has been shown that mhv infection induces apoptosis in macrophages and fibroblast cells but not in dbt cells (belyavsky, et al., ; an et al., ; chen and makino, ) . however, overexpression of the single viral e protein in dbt cells by a recombinant vaccinia virus results in apoptosis, which can be blocked by overexpression of an antiapoptotic protein bcl- , indicating the involvement of the mitochondrial pathway in e proteininduced apoptosis in dbt cells . how mhv infection in dbt cells overcomes the apoptotic effect induced by the e protein expression remained unanswered in that study. it is possible that mhv infection of dbt cells induces an antiapoptotic state during an early stage of the virus life cycle through down-regulation of proapoptotic genes such as bnip (this study) or up-regulation of antiapoptotic genes (cai and zhang, unpublished results). such an antiapoptotic response would prevent cells from succumbing to apoptosis induced by the viral apoptotic e protein. consistent with this hypothesis is the observation that when dbt cells were treated with apoptotic inducers, fewer cells underwent apoptosis when infected with mhv than mock-infected cells (cai and zhang, unpublished results). although a direct correlation between down-regulation of bnip and inhibition of e protein-induced apoptosis in dbt cells has not been determined, the general proapoptotic effect exerted by bnip mizutani et al., ; kubasiak et al., ; lamy et al., ) makes the hypothesis conceivable. moreover, the involvement of the mitochondrial/bcl- pathway in e protein-induced apoptosis correlates well with the functional role of bnip as a member of the proapoptotic, mitochondrial/bcl- family. thus, it is conceivable that down-regulation of bnip expression can shift the balance to an antiapoptotic state at the mitochondrial checkpoint. using uv-inactivated virus for infection and chloroquine for blocking the acidification of the endosomes, we demonstrated that the down-regulation of bnip gene expression by oblv infection was mediated during cell entry and required no virus replication (figs. and ) . however, treatment of dbt cells with chloroquine did not completely block the down-regulation of bnip expression by oblv infection (fig. ) . one possible interpretation is that a part of the signals that trigger the down-regulation of bnip expression has already been induced during virus attachment and endocytosis but prior to fusion between viral envelope and endosomal membranes (in the case of oblv virus), so that inhibition of fusion by chloroquine cannot completely derepress the bnip promoter activity. alternatively, the partial restoration of bnip promoter activity may be due to incomplete blockage of fusion by chloroquine. this is possible and even likely because the virus infectivity was reduced by approximately only log by chloroquine treatment (at m). although gallagher et al. ( ) reported a reduction of log in virus titer when dbt cells were treated with chloroquine at m, they used approximately log less virus for infection. in our infection-transfection experiments, we had to use a higher m.o.i. to insure that each transfected cell is infected with a virus. the higher virus infectivity used in this study likely contributes to a lesser effective inhibition by chloroquine. therefore, although it cannot exclude the possibility that virus attachment to cell receptors and the subsequent endocytosis are also involved in signaling bnip down-regulation, our results clearly establish that virus-cell fusion during entry triggers the signaling cascade. little is known about the cis-acting elements that regulate bnip gene expression. our systematic deletion analyses identified the -nt sequence (from nt to ) of the bnip promoter, which is necessary and sufficient for the expression of bnip gene. interestingly, this region also contains the two previously identified hypoxia response elements (hre- and hre- ) (bruick, ) , the tata box, four transcription factor sp -binding sites, the cac or ccacc consensus sequence, the ccaat box, and the nuclear factor-(nf) -binding site (fig. b) . it is not known whether and how these transcription factors are involved in the regulation of bnip gene expression. it has been reported that the transcription factor hypoxia-inducing factor- binds to the hres on the bnip promoter and induces the bnip expression (bruick, ; kubasiak et al., ) , whereas the zinc-finger protein plagl induces the expression of bnip via the same hres, but independent of the hypoxia-inducing factor- (mizutani et al., ) . our finding that further deletions within this -nt region inactivated the promoter suggests that bnip transcription in dbt cells likely requires multiple transcription factors acting in concert with these cis-elements. the mechanism by which mhv infection of dbt cells downregulates bnip gene expression is currently unknown. we have attempted to identify the negative elements that are responsive to mhv infection. our results showed that the negative, mhv-responsive elements largely overlap with the cis-acting elements essential for bnip transcription (fig. ) . one hypothesis is that mhv infection triggers the induction of one or more transcription factors that act on the -nt sequence of the bnip promoter to compete with the positive transcription regulators, thereby down-regulating the transcription of bnip on infection. in our dna microarray analysis, we found that the expression of a class of early response transcription factors such as c-fos ( -fold increase) and early growth response gene- (egr- ) ( -fold increase) was significantly induced by mhv infection (data not shown). preliminary studies employing transient cotransfection of dbt cells with plasmids expressing c-fos or egr- gene and the bnip promoter reporter plasmid demonstrated that the bnip promoter was down-regulated by egr- in a dose-dependent manner but not by c-fos, suggesting that the down-regulation of bnip gene expression by mhv infection might be mediated through the induction of egr- (data not shown). it has been shown that egr- and sp share the consensus binding sequence and that egr- can act as an antagonist of sp by binding to the sp consensus sequence and represses transcription (huang et al., ) . our analysis of the -nt bnip promoter sequence (from nt to nt ) identified four sp -binding sites: two upstream at nt and and two downstream at nt and , respectively. coincidently, mhv infection of dbt cells inhibited the luciferase expression in all reporter plasmids that contain the two or four sp -binding sites (fig. ) . however, direct evidence for the role of egr- in mhv-dependent transcriptional regulation of bnip gene remains to be established. a recent study showed that overexpression of egr- resulted in down-regulation of bnip transcription in prostate cancer cells (virolle et al., ) , thus providing supporting evidence for the role of egr- in regulating bnip gene. the mouse hepatitis virus (mhv) strain jhm ) was used throughout this study. for some experiments, mhv strain a (leibowitz et al., ) and the mutant oblv (gallagher et al., ) were also used. oblv (kindly provided by michael buchmeier, the scripps research institute, la jolla, california) was originally isolated from the obl cells that were persistently infected with mhv jhm (mhv- ). this mutant enters cells via receptor-mediated endocytosis and thus its entry depends on acidic ph. the mouse astrocytoma cell line (dbt) (hirano et al., ) was used for virus propagation, plaque assay, and all other experiments involving cell culture throughout this study. dbt cells were cultured in x minimum essential medium (mem) containing % trypton phosphate broth (tpb) and . % newborn calf serum (ncs) (gibco brl, gaithersburg, md). all mhv strains were propagated in dbt cells in mem either containing % ncs or serum-free. for propagation of oblv mutant virus, no nahco was added to the medium. a recombinant vesicular stomatitis virus (vsv) expressing the green fluorescence protein (gfp) originally provided by michael whitt (university of tennessee at memphis) was obtained from marie chow (uams). vsv was propagated and titered in hela cells. antibodies specific to mouse bnip protein and ␤-actin were purchased from santa cruz biochem and sigma, respectively. dbt cells were infected with mhv at a multiplicity of infection (m.o.i.) of or mock-infected. at various time points postinfection (p.i.), intracellular total rnas were isolated and purified with the rneasy kit according to the manufacturer's instruction (qiagen, inc.). rnas were then treated with rnase-free dnase i (promega). the concen-trations of the rna samples were determined by spectrophotometry (hitatchi, model no. ) . for detection of the proapoptotic gene bnip mrna, a semiquantitative, competitive rt-pcr was employed. briefly, the rnas were reverse-transcribed into cdnas with maloney murine leukemia virus (mmlv) reverse transcriptase (promega) by using a random hexomer oligonucleotide primer (invitrogen, inc.) in a standard rt reaction as described previously (zhang et al., ) . cdnas were then used as templates for pcr amplification with two pairs of primers specific for bnip gene and ␤-actin gene as an internal control, respectively. the sense primer for bnip of taq dna polymerase, l of the rt products, for cycles with each cycle consisting of denaturation at °c for s, annealing at °c for min and extension at °c for min with a final extension of min. pcr was performed in a thermocycler dna engine (model ptc- , mj research). for competitive pcr, a plasmid dna containing a -nt deletion between the primer sequences ( Јbnip and Јbnip ) was constructed (see below) and used as a competitor. for each rna sample, at least pcr reactions were performed with an incremental amount of the competitor dna. pcr products were analyzed by agarose gel electrophoresis, and the gels were stained with ethidium bromide and photographed with a uvp gel document center. the intensity of the dna bands was determined by densitometry, analyzed with manufacture-installed software (uvp), and plotted. the point at which the amount of target pcr products equals to a known amount of competitor dna being added in the pcr reaction is the amount of target cdna of the virus-infected cells, which is expressed as percentage relative to the amounts of target cdna in mock-infected cells (which were set as %). mhv strains jhm and a and vsv were inactivated by exposing the viruses to uv light for min on ice in a uv crosslinker (fisher scientific). inactivation was confirmed by titration of samples before and after uv-light exposure and by the absence of cytopathic effect after inoculation of dbt cell monolayers with uv-irradiated virus or by the absence of green fluorescence cells (for vsv). dbt cells were grown to monolayer in -mm petri dishes and were infected with mhv jhm at an m.o.i. of or mock-infected. at , , and h p.i., cells were harvested and lysed in l of radioimmunoprecipitation assay (ripa) buffer ( mm tris-hcl, ph . , mm nacl, . % sodium deoxycholate, % np- ) containing protease inhibitor cocktail tablets (roche, mannheim, germany). the cell lysates were passed through a -gauge needle several times to sheer the dna and were clarified from cell debris by centrifugation. the protein concentration was measured by using bio-rad protein assay kit (bio-rad, ca). equal amounts of cell lysates (cell number equivalent) were immunoprecipitated with an anti-bnip peptide antibody ( g/ml) (santa cruz, ca) for h at °c in a rocking platform (bellco inc.). the antigen-antibody complexes were then precipitated with protein g-sepharose beads ( l of the % slurry) (roche) for h at °c in a rocking platform. protein g beads were pelleted down by centrifugation and washed three times with the ripa buffer. twenty microliter of x protein sample loading buffer [ mm tris-hcl, ph . , mm dithiothreitol (dtt), % sodium dodecyl sulfate (sds), . % bromphenol blue, % glycerol] were added to each sample. samples were boiled for min, chilled on ice, and pelleted by centrifugation before loading onto gels. proteins were separated by sdspolyacrylamide gel ( %) electrophoresis (page). the proteins were then transferred to nitrocellulose membrane (msi, westborough, ma) for h at v in a transfer buffer ( mm tris, mm glycine, % methanol, . % sds). after being blocked with % skin milk in tris-buffered saline (tbs) for h at room temperature, the membrane was washed three times in tbs containing . % tween- and immunoblotted with the bnip antibody ( g/ml) for h at room temperature, followed by a secondary ab coupled to horseradish peroxidase ( : dilution) (sigma) for h at room temperature. the presence of the bnip protein was detected by enhanced chemiluminescence (ecl) using peracid as a substrate (amersham) followed by autoradiography. the luciferase reporter plasmid that contains nt Ј upstream sequence (the promoter) of bnip gene in front of the luciferase orf was kindly provided by richard bruick (university of texas, southwestern medical center in dallas). for construction of a competitor dna for quantitative pcr, the full-length orf of bnip gene was cloned. rnas were isolated from dbt cells and cdnas were synthesized by rt using the random hexomer primer described above. the bnip gene was amplified by pcr using the sense primer Јbnip ecori ( Ј-agt gaa ttc acc atg tcg cag agc- Ј, corresponding to the first nt of the bnip orf, genbank accession number af ), which contains an ecori site at the Ј-end (underlined), and the antisense primer Јbnip xhoi ( Ј-ctc gag gaa ggt gct agt gga- , complementary to the last nt of the bnip orf), which contains an xhoi site (underlined). the pcr products were directly cloned into pcr . ta cloning vector (invitrogen), resulting in pta-bnip . the sequence of the clone was confirmed by automatic dna sequencing (ibi, prizm- ) in the departmental core facility. an internal deletion was made in three steps. in the first pcr, a -nt fragment of the bnip gene was amplified from the full-length clone pta-bnip using the primer pair Јbnip -rt and Јbnip -cl ( Ј-ttt ctc gcc aaa gct gtg gc- Ј, complementary to the sequence at nt - ). in the second pcr, a -nt fragment of the bnip gene was synthesized using the primer pair Јbnip -cl ( Ј-gc cac agc ttt ggc gag aaa cca gaa aat att ccc ccc aag- Ј, corresponding to sequence at nt - with a -nt sequence overhang at the Ј-end that corresponds to nt - ) and Јbnip -rt. the pcr products from these two sets of reactions were separated by agarose gel electrophoresis; the corresponding dna fragments were excised from the gels and purified with a gel extraction kit (qiagen, inc.). the two pcr fragments were then mixed and used as templates for a third pcr. the third pcr was carried out with the primer pair Јbnip -rt and Јbnip -rt. the products from the third pcr were directly cloned into the pcr . -ta cloning vector (invitrogen), resulting in generation of pta-bnip d . this clone contains the region of bnip gene from nt to nt with a deletion between nt and nt . this clone was then used as a competitor for the competitive pcr described above. for construction of Ј-deletion mutants of the bnip promoter, plasmid pgl -bnip , which contains the fulllength promoter, was doubly digested with hindiii and bstxi, pf mi, or stui, respectively. the digested plasmids were purified with the gel extraction kit (qiagen) following agarose gel electrophoresis, blunt-ended with t dna polymerase, and self religated with t dna ligase, resulting in generation of pgl -bnip -pr , pgl -bnip -pr , and pgl -bnip -pr , respectively (fig. ) . deletion mutants pgl -bnip -pr , pgl -bnip -pr , pgl -bnip -pr , pgl -bnip -pr , pgl -bnip -pr , and pgl -bnip -pr were made similarly by deleting the internal fragments through double digestion with stui and ncoi, pf mi and ncoi, bstxi and ncoi, pf mi and stui, bstxi and pfl mi, or apai alone (two sites), respectively. for construction of pgl -bnip -pr and pgl -bnip -pr , the -nt pf mi-stui fragment and the -nt apai-apai fragment were blunt-ended and cloned into the smai site of the pgl -basic vector. the orientation of these fragments in the plasmids were confirmed by dna sequencing. for dna transfection, the lipofectamine reagent was used according to the manufacturer's instruction (gibco-life technologies, inc.). briefly, dbt cells were seeded in a -well culture plates at a density of ϫ cells per well. when the cells reached to approximately % confluency, Ϸ ng of plasmid dnas and l of the lipofectamine reagent were diluted separately in l of serum-free opti medium without antibiotics, mixed, and incubated at room temperature for min before adding to each well of the -well plate. dbt cells were washed with pbs and l of opti medium were added to each well of the cell culture. the dna-lipofect amine mixture ( l) was added to each well and the cell culture plates were incubated at °c for various time periods as indicated. for luciferase assay, cells were harvested along with culture medium and lysed by freezing and thawing. one hundred microliters of the cell lysate was assayed for luciferase activity using the luciferase assay system (promega) in a microtiter plate luminometer (dynex technologies, inc.). evolution of mouse hepatitis virus (mhv) during chronic infection: quasispecies nature of the persisting mhv rna induction of apoptosis in murine coronavirus-infected cultured cells and demonstration of e protein as an apoptosis inducer coronavirus mhv- -induced apoptosis in macrophages the production of recombinant infectious di-particles of a murine coronavirus in the absence of helper virus adenovirus e b kda and bcl- proteins interact with a common set of cellular proteins expression of the gene encoding the proapoptotic nip protein is induced by hypoxia murine coronavirus-induced apoptosis in cl- cells involves a mitochondria-mediated pathway and its downstream caspase- activation and bid cleavage the e b k/bcl- -binding protein nip is a dimeric mitochondrial protein that activates apoptosis the e b k/bcl- -binding protein nip is a dimeric mitochondrial protein that activates apoptosis astrocytes and brain injury molecular anatomy of mouse hepatitis virus persistence: coevolution of increased host cell resistance and virus virulence function of a Ј-end genomic rna mutation that evolves during persistent mouse hepatitis virus infection in vitro functional complementation of the adenovirus e b -kilodalton protein with bcl- in the inhibition of apoptosis in infected cells astrocytes as antigen-presenting cells. i. induction of ia antigen expression on astrocytes by t cells via immune interferon and its effect on antigen presentation persistence of viral rna in the central nervous system of mice inoculated with mhv- alteration of the ph dependence of coronavirus-induced cell fusion: effect of mutations in the spike glycoprotein nitric oxide synthase type ii expression by different cell types in mhv-jhm encephalitis suggests distinct roles for nitric oxide in acute versus persistent virus infection induction of bcl- expression by epstein-barr virus latent membrane protein protects infected b cells from programmed cell death regeneration of oligodendroglia during recovery from demyelinating disease translational control in murine hepatitis virus infection replication and plaque formation of mouse hepatitis virus (mhv- ) in mouse cell line dbt culture bcl- is an inner mitochondrial membrane protein that blocks programmed cell death chemokines and chemokine receptors in inflammation of the nervous system: manifold roles and exquisite regulation reciprocal modulation between sp and egr- bcl- /e b kda-interacting protein -like protein (bnip l) interacts with bcl- /bcl-xl and induces apoptosis by altering mitochondrial membrane permeability interleukin- induction in vitro in mouse brain endothelial cells and astrocytes by exposure to mouse hepatitis virus (mhv- , jhm) dephosphorylation of the nucleocapsid protein of inoculum jhmv may be essential for initiating replication regulation of the initiation of coronavirus jhm infection in primary oligodendrocytes and l- fibroblasts bcl- -family proteins: the role of the bh domain in apoptosis hypoxia and acidosis activate cardiac myocyte death through the bcl- family protein bnip modulation of cellular macromolecular synthesis by coronavirus: implication for pathogenesis coronavirus mrna synthesis: identification of novel transcription initiation signals which are differentially regulated by different leader sequences coronaviruses: organization, replication and expression of genome mechanism of demyelination in jhm virus encephalomyelitis: electron microscopic studies cd and the bh -only protein bnip in t-cell apoptosis the virus-specific intracellular rna species of two murine coronaviruses: mhv-a and mhv-jhm sequence of mouse hepatitis virus a mrna : indications for rna recombination between coronaviruses and influenza c virus characterization of dbt cell clones derived from cells persistently infected with the jhm strain of mouse hepatitis virus evolution of the Ј-end of genomic rna of murine coronaviruses during passages in vitro a zinc-finger protein, plagl , induces the expression of a proapoptotic protein nip , leading to cellular apoptosis entry of mouse hepatitis virus into cells by endosomal and nonendosomal pathways the carbohydrates of mouse hepatitis virus (mhv) a : structures of the o-glycosidically linked oligosaccharides of glycoprotein e the astrocyte is a target cell in mice persistently infected with mouse hepatitis virus computer software for eukaryotic promoter analysis cell surface expression of fusogenic vesicular stomatitis virus g protein from cloned cdna quasispecies development by high frequency rna recombination during mhv persistence persistent infection of cultured cells with mouse hepatitis virus (mhv) results from the epigenetic expression of the mhv receptor identification of a new transcriptional initiation site and the corresponding functional gene b in the murine coronavirus rna genome bcl- family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel vdac burkitt's lymphoma cells are resistant to programmed cell death in the presence of the epstein-barr virus latent antigen ebna- phosphoproteins of murine hepatitis viruses coronavirus translational regulation: leader affects mrna efficiency interleukin- expression and regulation in astrocytes egr promotes growth and survival of prostate cancer cells. identification of novel egr target genes nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins acute and subacute demyelination induced by mouse hepatitis virus strain a in c h mice adenovirus e b- k/bcl- interacting protein bnip contains a bh domain and a mitochondrial targeting sequence biosynthesis, structure, and biological activities of envelope protein gp of murine coronavirus heterogeneity of gene expression of the hemagglutinin-esterase (he) protein of murine coronaviruses mouse hepatitis virus gene b protein is a new virion envelope protein the human immunodeficiency virus type- tat protein upregulates bcl- gene expression in jurkat t-cell lines and primary peripheral blood mononuclear cells comparison of the nucleotide and deduced amino acid sequences of the s genes specified by virulent and avirulent strains of bovine coronaviruses regulation of matrix metalloproteinase (mmp) and tissue inhibitor of matrix metalloproteinase (timp) genes during jhmv infection of the central nervous system this work was supported by a public health research grant (ai ) from the national institutes of health and in part by the graduate student research fund from the university of arkansas for medical sciences. we thank richard bruick (university of texas southwestern medical center, dallas) and mike buchmeier (the scripps research institute, la jolla, ca) for kindly providing the bnip promoter-reporter construct and mhv mutant oblv , respectively. we also thank marie chow for valuable suggestions during the course of this study. key: cord- - b yji n authors: yamaguchi, kenjiro; goto, naoaki; kyuwa, shigeru; hayami, masanori; toyoda, yutaka title: production of mice from a lethal coronavirus infection in the central nervous system by adoptive transfer of virus-specific t cell clones date: - - journal: journal of neuroimmunology doi: . / - ( ) -f sha: doc_id: cord_uid: b yji n abstract the protective effect of a mouse hepatitis virus type- (mhv- )-specific cd + cytotoxic t cell clone and a cd + helper t cell clone was examined by the adoptive transfer into brains of mice lethally infected with mhv- . mice survived acute encephalitis if more than × cells of either type of the virus-specific t cell clones had been transfered into h- -matched recipients by day post-infection. although the adoptive transfer of both types of the t cell clones suppressed viral growth and viral antigen-positive cells in the brains, a significant inhibition of virus replication by the cytotoxic t cell clone was detected prior to that induced by the helper t cell clone. histologically, cell destruction was prominent in the brains of mice which received the cytotoxic t cell clone. these results demonstrate that both the cd + cytotoxic t cell and the cd + helper t cell can protect mice from a lethal mhv- infection in the central nervous system. mouse hepatitis virus type- (mhv- ) infection in mice is one of the best examples of virus-in-duced central nervous system (cns) disease in an animal model (wege et al., ; kyuwa and stohlman, ) . intracerebral (i.c.) infection with mhv- induces an acute fatal encephalomyelitis in almost all the laboratory strains of naive mice (stohlman and frelinger, ; knobler et al., ) . survivors show evidence of chronic demyelination and occasionally suffer severe hind leg paralysis. various factors including virus strain, route of infection, genetic background and immune status of the host are known to modify the infection. recently, some reports have focused on immunological therapy of mhv- infection in the cns and demonstrated that in vivo treatment with antiviral monoclonal antibodies protected mice from a lethal mhv- infection (buchmeier et al., ; fleming et al., ) . in addition, adoptive transfer of cd + helper t cell (th) clones which induce a delayed-type hypersensitivity (dth) response also protects mice (stohlman et al., ) . however, viral replication in the brains of these mice is scarcely affected by these therapeutic treatments. since t cells, particularly cytotoxic t lymphocytes (ctl), are thought to play an important role in viral infections (blanden and gardner, ; yap et al., ; byrne and oldstone, ; oldstone et al., ) , we established mhv- -specific cd + ctl clones and cd + th clones from infected balb/c mice to better understand the role of these cells in mhv- -induced diseases (kyuwa et al., ; yamaguchi et al., ) . in this study we transferred mhv- -specific t cell clones into infected mice and examined whether these t cell clones could protect mice from acute fatal encephalitis. both types of t cell clones protected h- -matched mice, accompanied by a significant inhibition of virus replication and clearance of viral antigen-positive cells in the cns; however, the ctl clone was more active in terms of viral clearance. thus, not only cd + th but also cd + ctl can protect mice from a lethal mhv- infection in the cns. balb/c mice were obtained from colonies in this institute. b .d , b .a and b .br mice were purchased from shizuoka laboratory animal center, hamamatsu, japan. female, -to -weekold mice were used throughout the experiments. breeding colonies were routinely screened serologically for the absence of mhv. mhv- was propagated and assayed for plaque-forming units (pfu) on dbt cells as described (hirano et al., ) . virus was stored at - ° c until use. cd + ctl clones plld, p c , p a , p a and . and cd + dth-inducer th clone p b were established from the spleens of mhv- -infected mice (kyuwa et al., ; yamaguchi et al., ) . briefly, balb/c mice were inoculated intraperitoneally with pfu of mhv- . one week later the spleens were removed, and splenocytes were co-cultured with x balb/c spleen cells, which had been gamma irradiated ( rad) and infected with mhv- (multiplicity of infection . ), in ml of rpmi medium supplemented with % heat-inactivated fetal calf serum (fcs), x -s m mercaptoethanol and /xg/ml of gentamicin in cm flasks at °c. one to weeks later limiting dilution was performed in the presence of % rat concanavalin a (cona)-conditioned medium and irradiated, mhv- -infected balb/c spleen cells in -well flat-bottom plates. after expansion of growing cells, cells were recloned by the same procedure. t cell clones were maintained in iscove's modified dulbecco's medium supplemented with % fcs, % rat cona-conditioned medium, x - m mercaptoethanol, mm hepes and gg/ml of gentamicin and stimulated with mhv- -infected balb/c spleen cells weekly. the sendai virusspecific t cell line sv- was kindly provided by dr. yamamoto, institute of public health, tokyo (yamamoto et al., ) . mice were inoculated i.c. in the right cerebral hemisphere with pfu of mhv- in /~ of eagle's minimum essential medium (mem). the i.c. lds of mhv- in young adult balb/c mice is approximately pfu. five or days after stimulation with mhv- -infected spleen cells, t cell clones were centrifuged on ficoll-conray gradients to exclude dead cells. one million t cell clones were injected i.c. into the right cerebral hemisphere of recipients in a volume of /~ on day post-infection (p.i.) except for the time course study. control mice having received either t cell clone or rpmi medium without infection did not show any clinical symptoms. mice were sacrificed on days , and p.i. the brains, spleens and livers were removed aseptically and infectious virus titer was determined by plaque assay on dbt cells as described above. significance was determined using the student t-test. groups of balb/c mice were sacrificed and the brains were removed on days and p.i. the brains were fixed in % formalin in phosphatebuffered saline (pbs) and then embedded in paraffin. sections ( #m thick) were stained with hematoxylin and eosin (he). viral antigen was detected by immunoperoxidase staining. deparaffinized sections were rinsed in pbs and treated with % normal swine serum in pbs for rain at room temperature. the sections were then incubated for h at °c with rabbit anti-mhv- serum, purified by sepharose-protein a column chromatography. after washing for min in pbs the sections were exposed to peroxidase-conjugated anti-rabbit igg swine serum (dakopatts, denmark) diluted / in pbs for min. negative controls included sections of mhv- -infected brains incubated with normal rabbit serum and sections of uninfected brains incubated with the anti-mhv- serum. the peroxidase reaction was performed according to the methods of kaplow ( ) . sections were counterstained with mayer's hematoxylin. the number of mhv- antigen-positive cells was determined as follows. two coronal sections of brains approximately and mm from the tip of the brain were prepared from each mouse. the total number of viral antigen-positive cells per two sections from each mouse was counted microscopically. neurons and glial cells were classified according to the morphology. we have established virus-specific cd ÷ ctl clones and cd ÷ th clones from mhv- -infected a balb/c mice which had been infected i.c. with pfu of mhv- received × t cell clones on day p.i., and were observed for more than weeks. balb/c mice (kyuwa et al., ; yamaguchi et al., ) . adoptive transfers were used to examine the effect of these t cell clones on an acute fatal mhv- infection. groups of balb/c mice were infected i.c. with pfu of mhv- . the next day × cells of each t cell clone were transferred into the brains. subsequently, mice were observed for more than weeks. as controls, the sendai virus-specific cd + t cell line sv- , also established from balb/c mice, was transferred into the brains of mhv- -infected mice. table shows that the mhv- -specific ctl clones plld, pic , p a , p a and . and the th clone p b protected recipients from a lethal mhv- infection and that the recipients survived for more than weeks. survivors showed no clinical symptoms of chronic disease. in contrast, both the mice which received the sendai virus-specific t cell line sv- and the infected untreated mice had symptoms of hyperirritability, rough coat and loss of body weight within days p.i. and died between and days p.i. the protective effect of the ctl clone p d and the th clone p b was studied further. although the sera of each group of mice were examined on days , , and p.i., if possible, neutralizing antibodies were detected neither in mhv- -infected mice without t cell clones nor in mice having received the t cell clone p d or p b (less than : ). when transferred day before, on the same day or day after virus one million cells of each t cell clone were adoptively transferred into lethally infected balb/c mice day before, on the same day, or , or days after infection. b one mouse died on day p.i. c one mouse died on day p.i. inoculation, both the t clones protected mice from a lethal mhv- infection. however, adoptive transfer carried out on day or p.i. was not effective ( table ). the protective effect of both t cell clones was dose dependent and required more than x cells to protect a mouse (table ) . the t cell clones plld and p b were derived from balb/c (h- d). since the proliferation re- sponse of the t cell clones plld and p b is h- -restricted (kyuwa et al., ) , h- restriction of the protective effect of these t cell clones was examined using h- -congeneic strains derived from c bl/ mice, b .d (h- d), b .a (h- a) and b .br (h- k). table shows that adoptive transfer of ctl clone plld prevented a lethal infection in b .d mice; however, neither b .a nor b .br mice were protected. similarly, adoptive transfer of the th clone p b protected b .d mice but failed in b .a and b .br mice. thus, the t cell clones plld and p b were able to protect mice from a lethal mhv- infection only in the context of the appropriate genetic environment, presumably involving recognition of viral antigen in the context of the products of h- genes. to examine the effect of ctl and th clones on virus replication, titers of mhv- in the brains were examined. the brains of mhv- -infected mice, which had received the mhv- -specific ctl clone plld, the th clone p b or the sendai virus-specific t cell line sv- , were assayed for virus on days , and p.i. a % drop in virus titers in the cns was observed as early as day after adoptive transfer in mice which had received the ctl clone p d (table ). however, complete virus clearance was not observed by days p.i. in contrast, only a slight reduction in virus titer was observed day following adoptive transfer of the th clone p b. significant decreases in virus titer were detected by days after adoptive transfer in mice having received the th clone p b. the adoptive transfer of the sendai virusspecific t cell line sv- did not restrict mhv- replication. on days and p.i. histological changes in the brains were compared among mice having received the ctl clone plld or the th clone p b and infected untreated mice. basically, there was little difference between the mice having received the ctl clone and the infected untreated mice; the degree of cellular infiltration was at a minimum in the brains of mice in these groups. however, cell destruction looked prominent in the brains having received the ctl clone plld on day p.i. (fig. ) . by contrast, typical changes following transfer of the th clone p b were moderate infiltration of mononuclear cells and meningitis (fig. ) . the numbers of mhv- antigen-positive neurons and glial cells were determined in mice which had received the mhv- -specific ctl clone plld or the th clone p b and compared to infected untreated mice (fig. ) . the adoptive transfer of the ctl clone plld drastically reduced the num- balb/c mice which had been infected i.c. with pfu of mhv- received × t cell clones. on day p.i. brains were removed and viral antigen was detected as described in materials and methods. b viral antigen-positive cells on two coronal sections of brain from each mouse were counted microscopically. bers of viral antigen-positive cells in the brains ( % reduction in neurons and % reduction in glial cells). similarly, a % reduction in antigenpositive cells was also observed following the adoptive transfer of the th clone p b (table ). these data indicated that both t cell types can effectively limit the number of antigen-positive cells in the cns of infected mice. the immune response is an important factor which determines the fate of mhv- infection in the cns of mice. the adoptive transfer of antiviral monoclonal antibodies specific for three structural proteins (s, m and n) can protect mice from a lethal infection (buchmeier et al., ; fleming et al., ; s.a. stohlman, personal communication) . in addition, the adoptive transfer of cd ÷ dth-inducer t cell clones also resuits in protection of mice from the infection (stohlman et al., ) . interestingly, protection mediated by adoptive transfer into c bl/ mice was not associated with a decrease in viral replication within the cns but rather with neuronal protection. in the present study we demonstrate that the adoptive transfer of virus-specific cd ÷ ctl clones also protects mice from a lethal mhv- infection. in this case, however, protection is associated not only with decreased neuronal infection but also with a significant inhibition of viral growth. thus, protection of mice from a lethal mhv- infection is always accompanied by inhibition of neuronal infection, irrespective of the therapeutic treatment. these findings support the hypothesis that neuronal infection is a major fatal factor in acute fatal encephalomyelitis induced by mhv- (knobler et al., . the adoptive transfer of either ctl or th clones at day p.i. or later failed to protect the mice. this may suggest that approximately lds virus given i.c. had produced an overwhelming infection which would not be restrained by the action of the t cell clones. recently, we have made an interesting finding in this regard. expression of class i molecules on j . cells decreased after high multiplicity of mhv- infection in vitro (s. kyuwa, unpublished data) . for example, this down-regulation of class i molecules, which is indispensable for recognition by cd + ctl, may provide an escape mechanism for mhv- from ctl-mediated protection as described below. cd ÷ th cell-mediated protection was first reported by stohlman et al. ( ) . in this study we have confirmed that the adoptive transfer of virus-specific cd + th cells protects mice from a mhv- lethal infection. while viral replication in the cns was not inhibited in their study, viral replication was slightly suppressed following transfer of the cd ÷ th clone p b. the absence of detectable antibody in the recipients suggests that this was probably not due to provisional help for the secretion of neutralizing antibody. although both cd ÷ th clones ( b and p b) induce a virus-specific dth response, no other functions have been identified, which may be important for virus clearance. for example, it is not known whether these cd + th clones provide help for virus-specific ctl, secreting a variety of lymphokines in vivo. in addition, differences in virus and mouse strain may account, at least potentially, for this discrepancy. although ctl clone plld and th clone p b required almost the same number of cells and transfer timing for the protection, there was a slight difference in time when a significant drop in virus titer began. virus titers in mice which received the ctl clone plld decreased significantly day after transfer. in contrast, a significant drop in virus titer in recipients of the th clone p b was detected on and after days following the transfer and the inhibition was less than that induced by the ctl clone plld. these findings suggest that cd ÷ t cells are indeed responsible for the reduction in virus titer and fit with the hypothesis that cd ÷ t cells may function as helper t cells for the generation of virusspecific cd ÷ ctl ; however, we cannot rule out the possibility that the difference is due to the antigen specificity of t cell clones rather than the type of t cell clones. in addition, this hypothesis is supported by recent findings that the in vivo treatment with not only anti-cd but also anti-cd monoclonal antibodies inhibits virus clearance from the cns (williamson and . the mechanism(s) of protection conferred by virus-specific ctl is a most interesting point. generally, cd ÷ t cells recognize a small fragment of endogenously synthesized protein bound to major histocompatibility complex (mhc) class i molecules on target cells prior to lysis of the targets (sweetser et al., ; townsend and bodmer, ; whitton and oldstone, ) . therefore, two components are necessary for target cell lysis. first, potential targets must be infected with mhv- . in this regard mhv- can infect almost all of the major cell types in the cns: neurons, oligodendrocytes and astrocytes. second, the targets must express mhc class i molecules. although there is little expression of class i molecules on cells in the cns, recent reports indicate that some cytokines including interferon- (ifn-"t), ifn-a/fl and tumor necrosis factor increase expression of mhc class i molecules on cells in the cns (wong et al., (wong et al., , lavi et al., ) . in addition, elevation of mhc class i antigen expression on glial cells following murine coronavirus infection has been described (suzumura et al., ) . taken together, these results suggest that glial cells represent the most probable targets of mhv- -specific ctl. in fact, the adoptive transfer of the cd ÷ ctl clone plld induced cell destruction and decreased the number of viral antigen-positive cells. to our knowledge, this finding is notable because there is little direct evidence that ctl can kill target cells in vivo as they do in vitro (mullbacher and ada, ) . complete clearance of infectious virus from the brains was not observed by days p.i. this phenomenon is similar to incomplete clearance of lymphocytic choriomeningitis virus from the cns following adoptive transfer of cd + t cells (oldstone et al., ) . these findings may be due to the limited expression of mhc antigens in the cns (vitteta and capra, ; williams et al., ) in addition to lysis of virus-infected cells, cd + ctl can secrete a variety of lymphokines including ifn-t after target recognition. in fact, we have detected ifn activity in the culture supernatant of ctl clone plld after stimulation with mhv- -infected j . cells, which we used as the targets in the ctl assays (unpublished data). although direct lysis rather than secretion of ifn-t was suggested as the antiviral mechanism of ctl (lukacher et al., ; mullbacher and ada, ) , it might be still worthy of consideration, especially in the cns, an immunologically unique site. furthermore, ifn-~, secreted by ctl would also facilitate in target recognition by ctl as a result of the up-regulation of mhc class i molecules on cells in the cns. a recent study has demonstrated that cd + t cells play an important role not only in acute virus clearance, but also in the development of mhv- -induced demyelination (j.o. fleming, personal communication) . although adoptive transfer of virus-specific monoclonal antibodies or cd + t cells protects mice from a lethal infection, these therapeutic treatments cannot protect mice from demyelination. their data suggest that mhv- -induced demyelination is immune-mediated and that cd + t cells are responsible for the development of demyelination. although we did not examine the effect of our cd + ctl clones on virus-induced demyelination in this study, future experiments may reveal the interaction between mhv- , cd + t cells and cells in the cns, providing important information on the mechanism of mhv- -induced demyelination. the cell-mediated immune response to ectromelia virus infection. i. kinetics and characteristics of the primary effector t cell response in vivo biology of cytotoxic t lymphocytes specific for lymphocytic choriomeningitis virus: clearance of virus in vivo murine hepatitis virus (strain jhm)-induced neurologic disease is modulated in vivo by monoclonal antibody monoclonal antibodies to the matrix (el) glycoprotein of mouse hepatitis virus protect mice from encephalitis utility of mouse cell line dbt for propagation and assay of mouse hepatitis virus substitute for benzidine in myeloperoxidase stains selective localization of wild type and mutant mouse hepatitis virus ohm strain) antigens in cns tissue by fluorescence, light and electron microscopy mouse hepatitis virus type (jhm strain)-induced fatal central nervous system disease. i. genetic control and the murine neuron as the susceptible site of disease selected mutants of mouse hepatitis virus type (jhm strain) induce different cns disease pathogenesis of a murine coronavirus, strain jhm infection in the central nervous system of mice characterization of mouse hepatitis virus-reactive t cell clones tumor necrosis factor induces expression of mhc class i antigens on mouse astrocytes in vivo effector function of influenza virus-specific cytotoxic t lymphocyte clones is highly specific how do cytotoxic t lymphocytes work in vivo? microb cytoimmunotherapy for persistent virus infection reveals a unique clearance pattern from the central nervous system resistance to fatal central nervous system disease by mouse hepatitis virus, strain jhm. i. genetic analysis in vivo effect of coronavirus-specific t cell clones: dth inducer cells prevent a lethal infection but do not inhibit virus replication t-cell-mediated clearance of jhm virus from the central nervous system coronavirus infection induces h- antigen expression on oligodendrocytes and astrocytes recognition of pre-processed endogenous antigen by class i but not class ii mhc-restricted t cells antigen recognition by class i-restricted t lymphocytes the protein products of the murine th chromosome: genetics and structure the biology and pathogenesis of coronavirus distribution and quantitation of hla-a, b, c and dr (ia) antigens on human kidney and other tissues effective clearance of mouse hepatitis virus from the central nervous system requires both cd + and cd + t cells class i mhc can present an endogenous peptide to cytotoxic t lymphocytes inducible expression of h- and ia antigens on brain ceils interferon-t induces the expression of h- and ia antigens on brain cells establishment of cytotoxic t-cell clones specific for cells infected with mouse hepatitis virus establishment of tuberculous antigen-specific t cell line and its effect on hepatic granuloma formation in bcg-infected nude mice transfer of specific cytotoxic lymphocytes protects mice inoculated with influenza virus we thank dr. s. yamamoto for his kind gift of the sendai virus-specific t cell line, dr. i. sugawara for technical advice and drs. k.yamanouchi and s.a. stohlman for critical reading of the manuscript. this work was supported in part by a grant-in-aid for scientific research from the ministry of education, science and culture, japan. 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- -mgrf odm authors: wang, xiaojing; yan, weiming; lu, yulei; chen, tao; sun, ying; qin, xiaomin; zhang, jiangguo; han, meifang; guo, wei; wang, hongwu; wu, di; xi, dong; luo, xiaoping; ning, qin title: cd –cd -t cells contribute to the persistence of viral hepatitis by striking a delicate balance in immune modulation date: - - journal: cellular immunology doi: . /j.cellimm. . . sha: doc_id: cord_uid: mgrf odm abstract viral hepatitis remains the most common cause of liver disease and a major public health problem. here, we focus on the role of cd cd double negative t (dn t) cells involved in the mechanisms of viral persistence in hepatitis. c h/hej mice infected with murine hepatitis virus strain (mhv- ) were used to display chronic viral hepatitis. dn t cells dramatically increased in mhv- infected mice. adoptive transfer of dn t cells from mhv- infected mice led to a significant increase in mice survival. the dn t cells with production of ifn-γ and il- are able to kill virus-specific cd + t cells via the fas/fasl dependent pathway. the delicate balance of multiple effects of dn t cells may lead to viral persistence in mhv- induced hepatitis. in short, our study identified dn t cells contributing to viral persistence in mhv- induced hepatitis in c h/hej mice, which provides a rationale for modulating dn t cells for the management of viral hepatitis. viral hepatitis remains a major public health problem in the world with considerable morbidity and mortality. it is estimated that there are over million carriers of hepatitis b virus (hbv) and million carriers of hepatitis c virus (hcv) worldwide [ , ] . persistent infection may progress to chronic liver disease, cirrhosis and primary liver cancer [ ] . the viral proteins expressed in hepatocytes may influence the severity and progression of liver disease. however, extensive studies suggest that the mechanisms of liver injury in viral hepatitis are due to the host immune responses, but not to the direct cytopathic effects of viruses. the direct killing of infected cells by virusspecific cd + cytotoxic t lymphocytes (ctls) is considered as the central mechanism resulting in both liver damage and virus control. in addition, cd + t helper response is also found to have a strong association with viral clearance in the acute phase of hepatitis [ ] . viral persistence during hepatitis infection may be the direct result of a weak antiviral immune response to the viral antigens, with corresponding inability to eradicate virus within the infected cells. accumulating evidence has indicated that regulatory t (treg) cells play an important role in the suppression of virus specific immune responses [ ] [ ] [ ] . many subsets of treg cells have been studied including cd + cd + tregs [ ] [ ] [ ] [ ] [ ] [ ] , cd + dx + t cells [ ] , agspecific cd + or cd + t cells that secrete the immuno-regulatory cytokines il- (tr cells) or tgf-b (th cells), tcrcd + t cells [ , ] , and tcrab + cd + cd À cd À double-negative (dn) t cells [ ] [ ] [ ] . among treg cells, cd + cd + tregs are the most extensively studied. a transcription factor foxp is considered as the optimal marker of classic natural treg [ ] . in patients with hbv or hcv infection, cd + cd + foxp + tregs have increased levels and impair the immune responses directed against hepatitis viruses, leading to persistent infections and chronic liver injury [ ] [ ] [ ] . lately, shalev et al. reported that adoptive transfer of tregs from fgl +/+ mice into fgl À/À mice resulted in increased mortality to mhv- infection, demonstrating the critical role for cd + cd + tregs in the pathogenesis of mhv- induced fulminant hepatitis [ ] . dn t cells are a novel subset of treg cells which is first identified by zhang and colleagues. in mice, dn treg cells could kill activated cd + t cells with the same tcr specificity, and infusion of in vitro-activated dn treg cells led to significant prolongation of donor-specific skin and heart graft survival [ , [ ] [ ] [ ] . recently, increasing attention has been focused on these novel treg cells. crispín et al. demonstrated that dn t cells produce il- /ifn-c and contribute to the pathogenesis of kidney damage in patients with sle [ ] . another study showed that dn t splenic cells from young nod mice could provide long-lasting protection against type diabetes [ ] . in siv-induced cd + t cell depletion in sooty mangabeys which do not present immune dysfunction and clinical aids, dn t cells partially compensates for cd + t cell function in these animals [ ] . in cutaneous leishmaniasis infection, tcrab + dnt cells present an increased bias in their capacity to induce inflammatory immune responses, and tcrcd + dnt cells show a regulatory profile [ ] . in the present study, we investigated the characteristics and contribution of splenic dn t cells in a mhv- induced chronic viral hepatitis murine model. and our study provides a rationale for modulating dn t cells for the management of viral hepatitis. all animal studies were carried out according to the guidelines of the chinese council on animal care, and were approved by the tongji hospital of tongji medical school committees on animal experimentation (no. ). mhv- was obtained from the american type culture collection (atcc), plaque-purified on a monolayer of dbt cells, and titered on l cells using a standard plaque assay. c h/hej mice were purchased from shanghai laboratory animal center of chinese academy of science (shanghai, roc). mhv- ( pfu/ ll) was individually injected into the peritoneal cavity of c h/hej mice as previously described [ ] . peripheral blood and livers were obtained on different time points after mhv- infection. c h/hej mice receiving ll of . % nacl were used as controls. the survival, serum biochemistry parameters including alt, ast, tp, alb, and liver histology were observed. virus titers were determined in the liver tissue of mhv- infected c h/hej mice at various time points by standard plaque assay as described before [ ] . blood, spleen, and liver were collected from mhv- infected c h/hej mice on indicated days post infections. at each time point, triplicate samples were examined. c h/hej mice treated with ll nacl were used as control. three-color cytofluorometric analysis was performed on the facsaria flow cytometer (bd biosciences, san jose, ca, usa). in all experiments, approximately cells/ l (from the single cell suspensions of processed pbmc, spleen and liver) were stained by fitc-anti-cd , pe-anti-cd and percp-anti-cd (ebioscience, san diego, ca, usa) monoclonal antibodies. corresponding isotype antibodies were used as controls. five days after mhv- infection, three groups ( mice in each group) of mice were treated respectively with dn t cells, splenocytes or dn t-depleted splenocytes from mhv- infected mice via the tail vein. another c h/hej mice treated with pbs were used as controls. liver tissue samples of recipient mice from each group ( - mice in each group) were harvested and stained with h&e days post the adoptive transfer. the knodell hepatitis activity index (hai) was used to evaluate the severity of the necroinflammation. the knodell hai score consists of four separate scores, including periportal necrosis with or without bridging necrosis ( - ), intralobular degeneration and focal necrosis ( - ), portal inflammation ( - ) and fibrosis ( - ) [ ] . virus titers in the liver tissues of recipient mice from each group were tested by standard plaque assay at indicated time points. expression of dn t cell surface markers were determined by flow cytometry analysis. cell suspensions of processed spleens from c h/hej mice on days , , , , and post mhv- infection were stained with pecy . -anti-cd , fitc-anti-cd , fitc-anti-cd , apc-anti-cd , apc-anti-tcrb, pe-anti-tcrcd, pe-anti-cd /pe-anti-cd , pe-anti-cd , pe-anti-cd l, peanti-cd monoclonal antibodies or isotype control ab (ebioscience, san diego, ca, usa). cells were harvested from the spleen of mhv- infected c h/ hej mice. flow cytometric detection of intracellular cytokines was performed as previously described. the cells were stimulated with lmol/l ionomycin and ng/ml pma in the presence of monensin ( lg/ml) for h at °c in % co . cells were washed and fixed in ml % ice-cold paraformaldehyde for min. before the initial staining, cells/tube were washed with saponin/pbs buffer to permeabilize the plasma and intracellular membranes. pe-anti-il- , pe-anti-il- , pe-anti-ifn-c, pe-anti-il- , pe-anti-tnfa, pe-anti-perforin, pe-anti-granzyme b or isotype-matched, irrelevant control ab (ebioscience, san diego, ca,usa) was respectively added to the suspension and incubated for min. after two further washes in saponin/pbs buffer, cells were stained with pecy . -anti-cd , fitc-anti-cd , fitc-anti-cd (ebioscience, san diego, ca, usa) for min. then the cells were analyzed by flow cytometry. the spleens from c h/hej mice were made into suspensions and incubated with magnetic bead sorting buffer. non-t cells, i.e. b cells, nk cells, dendritic cells, and macrophages were magnetically labeled by a cocktail of biotin-conjugated antibodies and anti-biotin microbeads (miltenyi biotec, cologne, germany). isolation of highly pure t cells was achieved by depletion of magnetically labeled cells. using the cd + t cell isolation kit (miltenyi biotec, cologne, germany), cd + t cells were then isolated from t cells obtained previously by depletion of non-cd + t cells (negative selection). cd + t cells were isolated from the non-cd + t cells using the cd + t cell isolation kit (miltenyi biotec, cologne, germany). the unlabeled non-cd + , non-cd + t cells were dn t cells. cytotoxicity of dn t cells was measured using the cytotox non-radioactive cytotoxicity assay kit (promega, madison, wi, usa) following the manufacturer's instruction. this cytotoxicity kit measures the lactate dehydrogenase (ldh) activity released into the culture medium by lysed cells. mhv- specific cd + t cells, normal cd + t cells, mhv- infected hepatocytes, and normal hepatocytes were used as target cells, dn t cells were used as effector t cells. the experiment was repeated after fasl blockade with anti-mfasl antibodies (r&d minneapolis, mn, usa) with a : ratio of dnt cells to cd + t cells. mice infected with the smith strain of murine cytomegalovirus (mcmv) were taken as an unrelated control. cd + t cells were iso-lated from the spleens of c h/hej mice post mcmv infection. the cytotoxic effect of dn t cells (from the spleens of mhv- infected mice) on cd + t cells (from the spleens of mcmv infected mice) was then examined. both dn t cells and cd + t cells were isolated from the spleen of mhv- infected c h/hej mice. transwell (corning, lowell, ma, usa) experiments were conducted in wells in . ml of complete tissue culture medium. the semipermeable membrane separating the upper and lower chambers allows diffusion of soluble materials but not cells. to measure the effect of non-contact cytotoxicity of dn t cells on cd + t cells, cd + t cells ( ) were cultured in the lower chamber and dn t cells (  or .  ) in the upper chamber. cd + t cells were cultured in the lower chamber alone as control. after an incubation period of h, cd + t cells were stained with fitc-anti-cd , washed, and then stained with apc-anti-annexin v (bender med system, vienna, austria) and pi (jingmei biotech, shanghai,china). at least , cd + t events were collected for annexin v/pi analysis. in this approach, the percent of annexin v À /pi À events (viable cell population) were used to correct for spontaneous apoptosis with the following formula: % cytotoxicity ¼ ½% control À viable cells À ½% of coincubated À viable cells ½% control À viable cells the result reflects the rate of cells undergoing apoptosis/death at the time of sampling. all data are presented as mean ± sem. comparisons of data were performed by using the student's two-tailed t-test or oneway analysis of variance (anova), followed by the student-newman keuls test. statistical package for the social science (spss) was used for data analysis. in all cases, significance was determined at p < . . in order to investigate the role of dn t cells in antiviral responses, we developed a chronic hepatitis model by infecting c h mice with mhv- as formerly reported. for the first time we extensively characterized the disease chronicity, liver function, and pathology of viral hepatitis in this model. the infected c h/hej mice began to have anorexia and acratia - days post infection. between day and day post mhv- infection, approximately . % mice died, and the survivors became persistently infected afterwards (fig. a) . the virus in the liver tissues of infected mice could be detected during the whole course of observation. the virus replication peaked on day , and then decreased and maintained at a low level. (fig. b) various degrees of hydropic degeneration, hepatocellular necrosis and inflammatory infiltration were observed in the liver of c h/hej mice after days of infection. the most severe liver injury was observed between day and day , with dramatically impaired liver function, including increased alt and ast as well as decreased total protein and albumin (table. ). after that, the hepatocellular injury in the survived mice was alleviated, accompanied with less infiltration and improved liver function. but all the inflammatory changes could still be observed in the livers of survivors during the period of observation (fig. c) . the proportion of dn t cell in the blood, liver, and spleen rose significantly after mhv- infection in c h/hej mice ( fig. a, c) , peaked between day and and then decreased gradually. the absolute count of dn t cells in either liver or spleen presented a similar change as the proportion did (fig. b ). . . adoptive transfer of dn t cells from mhv- infected c h/hej mice increased the survival rate and improved liver histology of recipient mice infected by the same virus strain but had little impact on the virus titer of liver tissue fig. a showed that no significant difference in virus titer of liver tissue was observed among dn t cells group, splenocytes group, dnt-depleted splenocytes group and pbs control group. after adoptive transfer of either dn t cells or splenocytes from mhv- infected c h/hej mice, the survival rate of the recipient mice raised notably to . % and . %, respectively, while only . % of mice receiving pbs or % of mice receiving dn t-depleted splenocytes survived (fig. b) . as shown in fig. c , mice receiving dn t cells or splenocytes had alleviated hydropic degeneration, inflammatory cells infiltration, as well as bridge necrosis in their livers compared with mice receiving pbs or dn t-depleted splenocytes. compared with splenocytes group, the hepatocytes swelling in the recipient mice in dn t cells group were not as much severe. comparison of knodell score (hai) among the four groups on day after the adoptive transfer was shown in fig. d . significant difference was found when dn t cells group vs. dnt-depleted splenocytes group ( . ± versus . ± . , ⁄ p < . ), and dn t cells group vs. pbs control group ( . ± versus . ± . , ⁄ p < . ). to further characterize the dn t cells, we detected the cell surface markers of dn t cells from spleens of c h/hej mice post mhv- infection (fig. a) . the majority of dn t cells expressed tcrab + while only a small part expressed tcrcd + . adhesion molecule cd was highly expressed on dn t cells, while activation markers cd , cd or cd were rarely detected. moreover, these dn t cells did not recognize aÀgalcer (which nkt cells do in an ag-specific fashion), demonstrating that dn t cells are different from nkt cells (data not shown). intracellular cytokines of dn t cells were detected on day , , , , , and after mhv- infection. the infected dn t cells either producing il- or ifn-c have increased since day after mhv- infection, peaked at day and maintained a stable level hence. only marginal levels of il- , il- , tnf-a, perforin and granzyme b were detected, and there was no significant difference between infected and noninfected mice in the expression of these intracellular cytokines at any time points (fig. b, c, d) . the cytotoxic effects of dn t cells from the spleen of c h/hej mice , , , , days after mhv- infection were examined. dnt cells had significant cytotoxic effects on mhv- infected cd + t cells, but no apparent effect on infected hepatocytes or non-infected cd + t cells and hepatocytes (fig. a) . to further investigate the cytotoxic specificity of dn t cells, a murine cytomegalovirus (mcmv) was used as an unrelated control. after peritoneal mcmv infection, the livers of c h/hej mice were evidently infected by mcmv, and virus replication was observed in the liver (as revealed by a standard plaque assay; data not shown). dn t cells from the spleens of mhv- infected mice showed no obvious cytotoxic effect on cd + t cells from the spleens of mcmv-infected mice, strongly suggesting that dn t cells only specifically kill cd + t cells with same virus specificity, and that this effect is not due to bystander cytotoxicity (fig. b) . a transwell experiment was performed to determine whether the cytotoxic effect is contact-dependent or is mediated by cytokines. results showed that dn t cells cytotoxicity was remarkably weaker when dn t cells and cd + t cells were in different chambers as opposed to being in the same chamber (fig. c) . to identify the precise mechanism involved in dn t cells cytotoxicity, the expression of fasl, perforin, and granzyme b in dn t cells was studied. perforin and granzyme b were rarely detected (fig. b) , while ( . ± . ) % of dn t cells express fasl. moreover, dnt cells cytotoxicity dramatically decreased when fasl was blocked (effector:target = : ) (fig. d ). lacking of adequate animal model has long being a major obstacle in studying viral hepatitis. only chimpanzee and a few other primates are susceptible to hepatitis viruses, but the endangered status and financial considerations limit their widespread use. researchers have also used surrogate animal models, such as ground squirrel, duck, tamarins and woodchuck. in addition to the wild-type animal models, inbred strains of animals are valuable to investigate the pathogenesis of viral infection. there are transgenic mouse models as well as immunodeficient mice or tolerized mice or rats, transplanted with human hepatocytes or hepatoma cells. animal models mentioned above enable us to better understand the pathogenesis of viral hepatitis, but all have limitations [ , ] . using mhv- , which produces a strain-dependent viral hepatitis in inbred strains of mice, has brought insights into the pathogenesis of viral hepatitis from our group and others [ , [ ] [ ] [ ] [ ] [ ] . mhv- belongs to the coronavirus family and has a singlestranded positive-sense rna genome. as reported, susceptible inbred mouse strains such as balb/cj or c bl/ develop fulminant hepatitis and die within - days following peritoneal inoculation of the virus. in contrast, a/j mice are resistant and develop no clinical signs of hepatitis, and clear the virus within days of infection [ ] . c h/hej mouse, a semisusceptible mouse strain develops acute hepatitis which progresses to mild chronic hepatitis [ ] . in our study, the mice have presented acute inflammation from to days after infection, and a proportion of the infected mice died. however, after this phase, the liver injury in the survived mice was alleviated, accompanied with the recovery of the liver function. only mild inflammation has been observed in the livers of the survivors, with persistent low-level virus replication, suggesting the persistence of viral infection. since the disease course, histopathology, and serum biochemical changes observed in the mouse model are similar to those observed in patients with chronic viral hepatitis, this mouse model could be useful to understand the pathogenesis of chronic viral hepatitis in humans. host immune response exercises a great influence on the pathogenesis and outcome of viral hepatitis. some viruses can be eliminated by the host immune system in the acute phase of infection, but certain viruses, like hbv and hcv, can evade the host immune responses, resulting in viral persistence [ ] . in order to better understand the mechanisms leading to virus persistence in the model of mhv- infected c h/hej mice, we studied the variations of different subgroups of t cells in c h/hej mice after mhv- infection. the results showed that the frequency of dn t cells rose significantly in blood, liver, and spleen after mhv- infection. we also observed an increase of cd + cd + tregs (data not shown) in the liver, but the increase of dn t cells was more pronounced than the increase of cd + cd + tregs, indicating that dn t cells might play a critical role in controlling the pathogenesis of the disease. based on our observation, the liver injury of infected mice began around - days after infection. we assumed that the adoptive transfer of dn t cells at this time point might interfere the development of liver injury. therefore, we chose to transfer dn t cells to recipient mice at days post infection. the results demonstrated that dn t cells could lead to both the dramatic increase in the survival rate of recipient mice and amelioration in liver histology. however, differences in the virus titers of liver tissue had barely been observed between dn t group and control groups. we presume that dn t cells may play a role in inhibiting the host immune responses which cause liver injury. nevertheless, the virus replication and clearance appear to reach a dynamic balance in the process. the following cytotoxic assays revealed that after mhv- infection, dn t cells showed significant cytotoxic effects on virusspecific cd + t cells. since the virus-specific cd + t cells play a key role in causing liver damage, this result made a sensible explanation to the survival increase and improvement of liver histology in recipient mice in adoptive transfer experiment. transwell experiments indicated that cell-cell contact is necessary to the cytotoxicity mediated by dn t cells. moreover, the fas/fasl pathway instead of perforin/granzyme pathway plays a vital part in dn t cell killing. thus we presume that, in mhv- infected c h mice, dn t cells rapidly proliferated following the development of liver inflammation, and contributed to the control of liver injury by killing the virus-specific cd + t cells. the phenomenon observed here is in agreement with what has been described for tcrab + cd + cd + dn t cells, which are able to prevent the rejection of skin and heart allografts by specifically inhibiting cd + t cells through fas-fasl interaction [ ] . however, zhang et al. reported that b + cd + dn t cells can control b and t cell responses in perforin/granzyme-dependent mechanisms [ ] . voelkl also reported that human dn tregs do not eliminate effector t cells by fas/fasl-mediated apoptosis, but to suppress by an active cell contact-dependent mechanism [ ] . thus, it seems that the functions of dn tregs change in accordance with the different phenotypes in specific settings. we found that the splenic dn t cells of the infected mice bear a distinctive array of phenotypes (tcrab + cd -cd -cd -cd -cd -cd + ) which were completely different from previously described t cells [ , ] . meanwhile, these dn t cells showed a relatively increased production of ifn-c and il- , but low il- , il- , tnf-a. it is reported that, in other disease models, dn t cells with different phenotypes present different cytokine profiles. in autoimmune diseases like sle, tcrab + tcrva À dn t cells are the major producers of il- , one of the key inflammatory cytokine involved in sle. and the results suggested that these dn t cells contribute to the pathogenesis of kidney damage in patients with sle [ ] . in cd -low mangabeys, dn t cells have increased expression of ifn-c(th ), il- (th ) and il- (th ), suggesting these cells may be capable of performing multiple functions, including cd helper-like function and regulatory function. and the latter could potentially contribute to controlling immune activation during nonpathogenic siv infection [ ] . in our study, we assume that the dn t cells may, to some degree, exert the role of th cells, which promote viral elimination. nevertheless, the limited quantity of these cytokines constrained their power for viral clearance and allowed the low-level viral persistence. we postulate that it was the delicate balance of multiple effects of dn t cells contributing to the viral persistence in this model. in conclusion, we report herein for the first time that splenic dn t cells with production of ifn-c/il- have profound immunomodulatory effects by killing viral-specific cd + t cells via fas/fasl pathway in mhv- induced viral hepatitis, suggesting the contribution of these dn t cells to viral persistence. our findings may provide a rationale for modulating dn t cells for the management of viral hepatitis. the authors have no financial conflict of interest. hepatitis, virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures the global spread of hepatitis c virus a and b: a phylodynamic and phylogeographic analysis hepatitis virus. the major etiology of hepatocellular carcinoma mechanisms of viral hepatitis induced liver injury regulatory t cells in autoimmmunity⁄ suppressor t cells -they're back and critical for regulation of autoimmunity! the role of cd + t-cell subsets in determining transplantation rejection or tolerance cytotoxic t lymphocyte-associated antigen plays an essential role in the function of cd (+) cd (+) regulatory cells that control intestinal inflammation stimulation of cd 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tcr-alphabeta+cd À cd À double-negative t cells potent and selective stimulation of memory-phenotype cd + t-cells in vivo by il- modifications of cd + t-cell function during in vivo memory or tolerance induction the authors thank prof. li zhang, for her inspiring advices and critical reading for the manuscript. the authors also thank dr. guanxin shen and gary levy for their comments in the process of this project, and dr. feng fang for providing mcmv. key: cord- -c auspti authors: weiss, susan r. title: coronaviruses sd and sk share extensive nucleotide homology with murine coronavirus mhv-a , more than that shared between human and murine coronaviruses date: - - journal: virology doi: . /s - ( ) - sha: doc_id: cord_uid: c auspti a cdna probe representing the genome of mouse hepatitis virus (mhv) strain a (mhv-a ) was used to measure nucleotide sequence homologies among murine and human coronaviruses and the sd and sk coronaviruses isolated by burks et al. since sd and sk were isolated by inoculation of multiple sclerosis (ms) central nervous system (cns) tissue into mice or cultured mouse cells, it is important to determine their relationships to other murine and human coronavirus isolates. our results indicate that sd and sk share almost complete nucleotide homology (approximately %) with mhv-a and generate subgenomic rnas of the same sizes as mhv-a . the human coronavirus (hcv) strains tested show less homology with mhv-a . the immunologically unrelated hcv- e shows no nucleotide homology with mhv-a . the immunologically crossreactive hcv-oc shows nucleotide homology with mhv-a by blot hybridization but not when hybridized in solution and assayed by s nuclease digestion. coronaviruses have been associated with acute and chronic neurological diseases in many species of animals (mcintosh, ) . infection of rodents with the murine coronavirus, mouse hepatitis virus mhv strain jhm, has been used as a model system to study virus-induced demyelination (weiner, ; nagashima et al., ; stohlman and weiner, ) . after initial panencephalitis caused by mhv-jhm, this virus produces a persistent infection with primary demyelination with some evidence for remyelination (weiner, ) . thus persistent mhv-jhm infection of rodents has been cited as a model to study the human demyelinating disease multiple sclerosis (ms). human coronaviruses (hcv) are ubiquitious in nature with a large portion of the human population possessing neutralizing antibodies (mcintosh, ) . these viruses were isolated usually as respiratory, and occasionally as enteric viruses. to whom reprint requests should be addressed. they are estimated to be responsible for % of common colds (mcintosh, ) . there are no reports thus far of involvement of human coronaviruses with persistent neurological disease. some strains of hcv such as c , are antigenically related to murine coronaviruses such as mhv strain jhm (mcintosh, ; gerdes et al., a, b) and may be grown in the brains of suckling mice (mcintosh et al., ) . others such as hcv- e are unrelated antigenically to mhv or hcv- c (mcintosh, ; pederson et al., ) . because ( ) murine coronaviruses are associated with chronic demyelinating disease in rodents (weiner ; nagashima et al., ) , ( ) antibody against hcv is very common in the human population (mcintosh, ) , and ( ) there is evidence suggesting that ms may be caused by a virus, various workers have undertaken comparisons of human and murine coronaviruses and have started to search for coronaviruses in central nervous system (cns) tissue from ms patients. there is one report of particles with coronavirus-like morphology seen by electron microscopy in brain tissue from an ms patient (tanaka et al., ) . more recently burks and co-workers have isolated two coronaviruses, designated sd and sk, by intracerebral inoculation of unfrozen ms cns autopsy tissue either into weanling mice or into cl- mouse cells in culture (burks et al., ) . gerdes et al. ( a, b) and we (this manuscript) have examined the relationship between these viruses and other known murine and human coronaviruses. gerdes et al. ( a, b) showed that sd and sk are antigenically related to mhv-a and to hcv-oc but not hcv- e. they were inconclusive about which strains their isolates were more related to. we have further compared murine and human coronaviruses and sd and sk by using molecular hybridization of virus-specific rna with cdna probes. our results show extensive nucleotide homology between sd and sk and mhv-a , more than that between the human viruses and mhv-a . viruses and cells. mhv-a was grown in cl- cells as previously described (weiss and leibowitz, ) . sd and sk viruses (burks et al., ) were obtained from dr. j. gerdes and were also grown in cl- cells. hcv- e was obtained from the american type culture collection (atcc) and grown in human embryonic lung (l- ) cells also obtained from the atcc. these viruses were plaque purifled two times and grown in dulbecco's medium with % fetal calf serum (robb and bond, ) . hcv-oc was obtained as a % suckling mouse brain suspension from dr. j. hierholzer at the center for disease control (cdc), atlanta. it was inoculated intracerebrally into c bl/ suckling mice, harvested days later, and a % brain suspension was made in phosphate-buffered saline (pbs) containing . % bovine serum albumen. the mothers of the suckling mice were obtained from jackson labs as mhv-free animals. all were negative for antibodies against mhv-a (as de-termined by an enzyme-linked immunosorbent assay) and hcv-oc (as shown by a lack of c hemagglutination inhibiting activity in the sera of these animals (hierholzer et al., ) ) and thus were considered uninfected by these coronaviruses. virus in brain homogenates was assayed by hemagglutination of chicken red blood cells at room temperature (hierholzer et al., ) . virus was further verified as hcv-oc since hemagglutination was inhibited by an anti- c reference antisera obtained from cdc. hcv-oc was also grown in human rectal tumor (hrt) cells (laporte et al., ) obtained from dr. david brian. infected mouse brain homogenates were adsorbed onto monolayers of hrt cells for hr at room temperature. the cells were extensively washed, medium added, and the cells incubated at ~ virus in the supernatant was titered at various times postinfection by hemagglutination (hierholzer et al., ) . mock-infected cells were adsorbed with a brain homogenate from uninfected suckling mice. cdna probes, cdnas were synthesized using purified genome rna as template, oligomers of calf thymus dna as primers, and reverse transcriptase (taylor et al., ) . cdnas were labeled with [ p]dctp to specific activity of approximately l s cpm/#g. when used for liquid hybridization, cdna was synthesized in the presence of actinomycin d and was > % single stranded. such cdnas were validated to be highly virus specific and to represent the majority of the genome rna as previously described in detail leibowitz, , ) . rna extraction. mhv-a , sd, sk, and hcv- e virus infections were carried out with a multiplicity of infection of between . and plaque-forming units per cell. rna was extracted hr after infection with a , sd, and sk viruses, when massive syncytia were present. e-infected cells were labeled with [ h]uridine in the presence of ttg/ml actinomycin d from to hr postinfection when rna was extracted. rna was extracted from oc -infected hrt cells at days post-infection. rna was extracted from the cytoplasm of infected cells by sds-proteinase k treatment followed by phenol extraction as previously described (weiss and leibowitz, ) . rna was extracted from suckling mouse brain homogenates by sds-proteinase k treatment followed by phenol extraction (weiss, varmus and bishop, ) . rna analysis. ( ) gel electrophoresis. rna was electrophoresed in % agarose gels, in the presence of methyl mercury hydroxide (bailey and davidson, ) or formaldehyde (lehrach et al., ) as denaturant. gels were either fluorographed with sodium salicylate (chamberlain, ) or blotted onto nitrocellulose (thomas, ) . ( ) blots. dot blots. rna was adsorbed onto nitrocellulose filters in various amounts (as designated in figure legends), dried, and the filters were baked and hybridized with cdna (thomas, ) . northern blots: rna was electrophoresed in gels, blotted onto nitrocellulose filters, and hybridized with cdna (alwine et al., ; thomas, ) . ( ) hybridization in solution was carried out at ~ . mnac , and assayed by $ nuclease digestion as previously described (leong et al, ) . in crt curves, increasing amounts of rna were hybridized with a fixed amount of cdna to achieve increasing crt values where crt = concentration of rna • time of hybridization. hcv-oc has been difficult to grow and assay in cell culture and this has impaired the study of viral nucleic acids. this virus is usually grown in the brains of suckling mice and titered either by infection of suckling mice or by hemagglutination (hierholzer et al., ) . schmidt et al. ( ) have reported growing and plaquing hcv-oc on human rhabdomyosarcoma (rd) cells. although we had difficulty with growing the virus in rd cells, we have had some success with growth in human rectal tumor (hrt) cells (laporte et al., ) . we have used hemagglutination to detect and quantitate hcv-oc in both infected suckling mouse brain homogenates and hrt cell supernatants. as shown in table brain homogenates from infected mice contained , hau/ml of c and homogenates from control mock-infected animals had none. this activity could be specifically inhibited by anti-oc reference antisera but not by a antisera or preimmune sera (data not shown). also shown in table after infection of hrt cells with oc -infected mouse brain homogenates ( hemagglutinating units/ cells), hemagglutinating activity could be measured in the medium. as expected pretreatment of brain homogenates with antisera directed against oc prevented hemagglutinating units/ml of oc . one hau is defined as the amount of virus present in . ml of the highest dilution of brain homogenate or supernatant capable of agglutinating . ml of . % chicken erythrocytes in the standard assay described by hierholzer et al. ( ) . b mock-infected mice or cells are mock-infected with a % homogenate or uninfected suckling mouse brains. c oc -infected suckling mouse brain homogenate was incubated with anti-oc reference antiserum for hr at room temperature before infecting cells. b), the reciprocal e x p e r i m e n t using hcv- e cdna was also c a r r i e d out. as i l l u s t r a t e d in fig. , c d n a r e p r e s e n t i n g the hcv- e g e n o m e h y b r i d i z e d only to its homologous r n a a n d not to hcv-oc r n a or to mhv-a rna. the blot e x p e r i m e n t s i l l u s t r a t e d in the above sections show the h o m o l o g y between the nucleic acids of c o r o n a v i r u s s t r a i n s mhv-a and sd, sk, a n d hcv- c a n d the lack of homology w i t h hcv- e. these techniques, however, do not q u a n t i t a t e the p e r c e n t a g e of the g e n o m e sequences t h a t are homologous. to be more q u a n t i t a rna from infected or mockinfected cells or mouse brain homogenates was spotted onto nitrocellulose filters. in the case of intracellular rnas, . , . , and . ttg amounts were used. in the case of purified genome rna, . , . , and . #g were used. filters were hybridized to cpm ( cpm/#g) of a [sup]cdna and autoradiographed (alwine et al, ; thomas, ) . similar hybridizations were carried out with rnas from cells infected with the other viruses and the plateau values for the percentage cdna hybridized are summarized in table . sd and sk are almost completely homologous to mhv-a , more so t h a n another mhv strain, jhm. hcv- e showed no homology with a cdna as predicted from the blot experiments. hcv-oc showed no homology with the cdna by this assay. this is probably due to stringency of hybridization and s nuclease assay for hybridization (see discussion). to f u r t h e r compare the rna of the murine and h u m a n strains the intracellular subgenomic rnas were examined by gel electrophoresis. as illustrated in fig. , cells infected with mhv-a contain six m a j o r subgenomic rnas as well as genome-sized rna (band ) (cheley et al., a; leibowitz et al, ; spaan et al., ) . these range in molecular weight from . • daltons for rna to . • daltons for r n a . intraeellular rnas extracted from eells infected with sd or sk, were eleetrophoresed in parallel with mhv-a rna, blotted onto nitrocellulose and the virusspecific species detected by hybridization with mhv-a edna. seven rna bands were observed, all comigrating with the m a j o r mhv-a rnas. rnas , , , and, in the ease of sk, rna , are less abundant than with mhv-a and the extra band between rnas and is more prominent. the extra bands between rnas and are also more p r o m i n e n t in the sd rna. the intraeellular rnas of hcv- e were also compared to those of mhv. (in this experiment mhv- rna was used instead of mhv-a . the genome of mhv- is % homologous to mhv-a and mhv- generates the same size intraeellular rnas as mhv-a (cheley et al., b, weiss and ). since hcv- e rna does not cross-hybridize fig. , dot blot hybridizations of hcv- e edna with coronavirus rnas. rna from infected or mockinfected cells or brain homogenates or from purified virions was spotted onto a nitrocellulose filter. in the case of intracellular rna, . , . , and . ~g were used and in the case of purified genome rna . , . , and . ug were used. the filter was hybridized with cpm ( s cpm/#g) of e [ p]cdna and autoradiographed (alwine et al, ; thomas, ) . with a edna, e intracellular rna was labeled with [ h]uridine in the presence of actinomycin d (to inhibit host dna-dependent r n a synthesis). as shown in fig. , e also generates six subgenomic rnas, but they are of different sizes from mhv- and hence from sd and sk. (genome rna was difficult to observe in this experiment probably due to some rna degradation). m a m m a l i a n coronaviruses have been divided into two antigenic groups (pederson et al, ) . one includes mhv, hcv-oc , a rna from virions, infected cells, or brain homogenates was hybridized with cpm ( s cpm/ t~g) of a [azp]cdna to completion, the plateau portion of a crt curve. hybridization was assayed by $ nuclease digestion. these values have been normalized to % hybridization of a cdna with its homologous a rna. the actual values of hybridization of a cdna with a rna ranged from to %. gastroenteritis virus of swine, and canine coronavirus. gerdes et al. ( a, b) have shown that the sd and sk viruses fall into the first group. they showed that all a intracellular proteins are immunoprecipitable with antisera directed against sd, sk, or hcv-oc . from these experiments, however, it was impossible to determine whether sd and sk were more related to human ( c ) or murine (a ) viruses. this is important in determining the origin of sd and sk and the possible link to ms. we have used nucleic acid hybridization with cdna to further explore the relationship among these viruses. our cdnas are representative of most if not all of the genome rna sequences leibowitz, , ) and thus are appropriate reagents for quantitating sequence homologies. the relationship between sd and sk and mhv-a as determined by molecular hybridization experiments basically agrees with immunological studies. the sd and sk viruses show extensive homology in nucleotide sequence (approximately %) to the a strain of mhv even when assayed by the stringent $ nu-clease assay. this is more homology than that shared between mhv-a and another mhv strain, jhm leibowitz, , ) . furthermore, the pattern of intracellular rnas generated by sk and sd is very similar to that of mhv-a . this is not surprising, considering that gerdes et al., ( a, b) showed that cells infected by sd or sk have patterns of viral proteins similar to a -infected cells (gerdes et al., a, b) . an extra polypeptide of , daltons molecular weight was observed in , or sk (lane c) were electrophoresed in a % agarose gel containing formaldehyde as a denaturant (lehrach et al, ) . after electrophoresis the gel was stained with ethidium bromide to locate ribosomal rnas, blotted onto nitrocellulose (thomas, ) , hybridized with l s cpm of a [ p]cdna, and autoradiographed (alwine et al., ) . arrows designate the position of s and s ribosomal rnas. viral rnas are numbered according to (bailey and davidson, ) . after electrophoresis the gel was stained with ethidium bromide to locate ribosomal rnas and fluorographed (chamberlain et al, ) . the positions of s and s ribosomal rnas are designated by arrows. there is no extra major rna band seen in the sd and sk samples. the molar ratios of some of the rnas are different in sd-and sk-infected cells, specifically, rnas and are less prominent and the extra rna band between and is quite prominent. the extra bands between rnas and are also more prominent in the sd sample. however, there is similar variation among the mhv strains (cheley et al., b; leibowitz et al., ; weiss and leibowitz, ) and we do not understand its significance. perhaps this is due to a small amofint of degradation in the extraction of these very large rnas. comparison of the human coronaviruses oc and e with mhv-a reveals less homology than between mhv-a and sk and sd. rna extracted either from brain homogenates of oc -infected suckling mice or from hrt cells infected with oc shows homology with a cdna when assayed by blot hybridization. this homology is not detected when liquid hybridization followed by the more stringent s nuclease assay is used. there is precedence for this in at least two other systems. homology between murine and human papovaviruses (howley et al., ) and murine and human rotaviruses (schroeder et al, ) is detected only under relaxed hybridization and assay conditions. these viruses are antigenically related; this immunological cross-reactivity is detected without difficulty as is the immunological cross-reactivity between mhv-a and hcv-oc . there is no homology detected between hcv- e and mhv-a or hcv-oc in cross-hybridizations using dot blots and cdna representing either virus. it is unlikely that there is homology betwen hcv- e and sd and sk since the latter viruses are so closely related to mhv-a . this is not surprising considering these viruses fall into different antigenic groups (pederson et al., ) . however, there are reports of immunological cross-reactivity between the nucleocapsid proteins of these viruses (gerdes et al., b) . these sequences if related, are probably too diverged to be detected by our assay. perhaps under less stringent blotting conditions, homology could be detected. these results show that sd and sk are more closely related to mhv-a than are the human viruses c and e. because sd and sk are almost completely homologous ( %) to mhv-a it is unlikely that they share much homology with either hcv-oc or hcv- e. it is possible that the % of sd and sk sequences that are not homologous to mhv-a are homologous to either hcu or hcv- e. however, if that were the case, it would still suggest that these isolates are more related to murine than human coronaviruses. this, in addition to the fact that sd and sk grow only in murine and not human cells (gerdes et al., b) , suggests that these viruses are not of human, but murine origin. method for detection of specific rnas in agarose gels by transfer to diazobenzyloxymethyl paper and hybridization with dna probes methylmercury as a reversible denaturing agent for agarose-gel electrophoresis two coronaviruses isolated from central nervous system tissue of two multiple sclerosis patients fluorographic detection of radioactivity in polyacrylamide gels with the water soluble fluor, sodium salicylate intracellular murine hepatitis virus-specific rnas contain common sequences rna and polypeptide homology among murine coronaviruses antigenic relationships of coronaviruses detectable by plaque neutralization, competitive enzyme linked immunosorbent assay, and immunoprecipitation coronavirus isolates sk and sd from multiple sclerosis patients are serologically related to murine coronaviruses a and jhm and human coronavirus oc but not to human coronavirus e standardized viral hemagglutination and hemagglutination-inhibition tests ti. description and statistical evaluation a rapid method for detecting and mapping homology between heterologous dnas some characteristics of hemagglutination of certain strains of "ibv-like" virus mouse hepatitis virus a : mrna structure and genetic localization of the sequence diversion from hepatropic strain mhv- comparative analysis of rna genomes of mouse hepatitis virus une lign e cellulaire particuli~rement sensible a la r p-lication du coronavirus ent ritique: les cellules hrt rna molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination the virus-specific intracellular rna species of two murine coronaviruses: mhv-a and mhv-jhm virus-specific ribenucleic acid in cells producing rous sarcoma virus: detection and characterization growth in suckling mouse brain of "ibvlike" viruses from patients with upper respiratory tract disease: prec coronaviruses: a comparative review coronavirus induced subacute demyelinating encephalomyelitis in rats: a morphological analysis antigenic relationship of the feline infectious peritonitis virus to coronaviruses of other species pathogenic murine coronaviruses. l characterization of biological behavior in vitro and virus specific intracellular rna of strongly neurotropic jhmv and weakly neurotropic a v viruses plaque assay and improved yield of human coronaviruses in a human rhabdomyosarcoma cell line sequence relationship between the genome segments of human and animal rotavirus strains isolation and identification of virus-specific mrnas in cells infected with mouse hepatitis virus (mhv-a ) chronic central nervous system demyelination in mice after jhm virus infection intracisternal virus-like particles in the brain of a multiple sclerosis patient efficient transcription of rna into dna by avian sarcoma virus polymerase hybridization of denatured rna and small dna fragments transferred to nitrocellulose. prec pathogenesis of demyelination induced by a mouse hepatitis virus (jhm virus) comparison of the rnas of murine and human coronaviruses characterization of murine coronavirus rna by hybridization with virus-specific cdna probes the size and genetic composition of virus-specific rnas in the cytoplasm of cells producing avian sarcoma-leukosis viruses this research was supported by nih grant a and grant rg from the multiple sclerosis society. i thank maureen highkin and david petcu for help with some of the experiments and roseann femia for typing the manuscript. key: cord- -vfutmwxq authors: sturman, lawrence s.; holmes, kathryn v. title: the molecular biology of coronaviruses date: - - journal: advances in virus research doi: . /s - ( ) - sha: doc_id: cord_uid: vfutmwxq publisher summary coronaviruses have recently emerged as an important group of animal and human pathogens that share a distinctive replicative cycle. some of the unique characteristics in the replication of coronaviruses include generation of a ' coterminal-nested set of five or six subgenomic mrnas, each of which appears to direct the synthesis of one protein. two virus-specific rna polymerase activities have been identified. many of the distinctive features of coronavirus infection and coronavirus-induced diseases may result from the properties of the two coronavirus glycoproteins. the intracellular budding site, which may be important in the establishment and maintenance of persistent infections, appears to be due to the restricted intracytoplasmic migration of the e glycoprotein, which acts as a matrix-like transmembrane glycoprotein. e also exhibits distinctive behavior by self-aggregating on heating at °c in sodium dodecyl sulfate (sds) and by its interaction with rna in the viral nucleocapsid. the e of mouse hepatitis virus (mhv) is an o-linked glycoprotein, unlike most other viral glycoproteins. thus, the coronavirus system may be a useful model for the study of synthesis, glycosylation, and transport of o-linked cellular glycoproteins. this contribution is dedicated to the memory of our colleague and friend, frederik bang, who uncovered some of the most intriguing questions about the biology of coronaviruses. beginning with the discovery that mouse hepatitis virus (mhv) could grow in and destroy macrophages in culture (bang and warwick, ) and that macrophage susceptibility in uitro reflected a genetic determinant for susceptibility in the mouse, bang and warwick, ( ; kantoch et al., ) constructed a simple model to explain genetic resistance and susceptibility to this virus. for more than two decades, bang ( bang ( , and his colleagues explored genetic and environmental aspects of host resistance and susceptibility to mhv, and the effects of mhv on macrophages. at the wurzburg symposium on the biochemistry and biology of coronaviruses in , dr. bang ( ) recounted the changes in his thinking about mechanisms of host resistance, as many of the conclusions drawn from earlier experiments were reinterpreted in the light of subsequent findings. as always, he spoke with humility and humor, and displayed his distinctive interest in host-parasite interrelations. unfortunately, that was his last opportunity to address his colleagues in this rapidly expanding field. we shall miss him. coronaviruses have been known, although not by that name, for almost five decades. this previously unrecognized group emerged during the s in the aftermath of the discovery of several new human respiratory pathogens. avian infectious bronchitis virus (ibv), mouse hepatitis virus, and some newly described human respiratory viruses were noted t o have a similar appearance (almeida and tyrrell, ; mcintosh et al., ; becker et al., ) . in contrast to myxoviruses, with which they had been previously compared (berry et al., ; mallucci, ) , these viruses displayed a characteristic fringe of large, distinctive, petal-shaped peplomers or spikes which resembled a crown, like the corona spinarum in religious art; hence the name coronaviruses ( fig. a ; tyrrell et al., ) . in addition to their morphological similarities, some of the human coronaviruses (hcv) were noted to be antigenically related to mhv (tyrrell et al., ; mcintosh et al., ; bradburne, ) . between and , research on coronaviruses emphasized morphologic and immunologic relationships and comparative biology. several new viruses were added t o the coronavirus group: porcine transmissible gastroenteritis virus (tgev), porcine hemagglutinating encephalomyelitis virus (hew, rat coronavirus (rcv), sialodacryoadenitis virus of rats (sdav), turkey bluecomb disease virus (tcv), and neonatal bovine diarrhea coronavirus (bcv). a comprehensive review by mcintosh ( ) provides an excellent overview of early coronavirus research. in , the international committee on the taxonomy of viruses approved the creation of a new family, coronaviridae, with one genus, coronavirus . additional species were added later including canine coronavirus (ccv), feline infectious peritonitis virus (fipv), and human enteric coronaviruses (hecv) (tyrrell et al., ) , rabbit coronaviruses (small et al., ; lapierre et al., ) , and several others (reviewed by wege et al., ) . table i shows the coronaviruses with their natural hosts and the major diseases which they induce. clearly the coronaviruses are important pathogens of man and domestic animals. timely reviews of several aspects of the biology and pathogenesis of coronaviruses have been published by virelizier ( , macnaughton and davies ( ) , . during the past several years, substantial progress has been made in understanding the structure and replication of coronaviruses. the symposium on the biochemistry and biology of coronaviruses held in wurzburg in october, dramatized the emergence of exciting frontiers in coronavirus research . siddell et al. ( ) have written an excellent review of the structure and replica- the purpose of this article is to describe the development of our present understanding of the molecular biology of coronaviruses. we have taken an historical approach to the subject, using figures from some of the key papers to illustrate the development of our present concepts. we are grateful to our colleagues for making these figures and their unpublished data available to us. we hope that this contribution will convey the sense of excitement and cooperation which has characterized this period in coronavirus research. negative stains of coronaviruses from eggs, clinical specimens or media over tissue, or organ cultures infected with coronaviruses revealed the characteristic viral structure shown in fig. a (berry et al., ; tyrrell and almeida, ; mcintosh et al., a; almeida and tyrrell, ; apostolov et al., ; oshiro et al., ; johnson-lussenberg, - ) . the virions were spherical, enveloped particles ranging from nm to nm in diameter. they showed some pleiomorphism, and frequently had a shallow central hollow containing some negative stain. indeed, on the basis of negatively stained images of ibv, bingham and almeida ( ) suggested that the morphology of coronavirus virions might resemble a punchedin sphere. whether this represents the true morphology of the virions or is a deformation resulting from drying and negative staining remains to be determined. coronavirus morphology has been reviewed by mcintosh ( ) , oshiro ( , pensaert and callebaut ( ) , and robb and bond ( a) . most coronaviruses appear to have only one morphologic type of surface projection or peplorner. the peplomers of coronaviruses are large and roughly club shaped. for mhv, each peplomer is about nm long by nm wide a t the tip . there are approximately peplomers per virion. it is not known how many glycoprotein molecules form each peplomer. the peplomers of different coronaviruses have somewhat different appearances in negatively stained preparations (caul and egglestone, ; davies and macnaughton, ) . for bcv, bridger et al. ( ) have suggested that there may be two morphologically distinct types of peplomers. analysis of the structural proteins of bcv (section iic) also suggested that there might be an additional species of glycoprotein in the virion (king and brian, ) . possibly this additional glycoprotein forms the second type of peplomer. two types of peplomers have been observed rarely on hev and mhv virions (greig et al., ; sugiyama and amano, ) . it is not clear to what extent the morphological differences between peplomers of coronaviruses reflect differences in amino acid sequence, glycosylation, proteolytic cleavage, or reduction of disulfide bonds (see sections ii,c and iv,a). many preparations of cornaviruses contain some virus particles which partially or completely lack peplomers. storage of virions may lead to detachment of peplomers, and under some conditions virions lacking peplomers can be formed in infected cells (holmes et ( ) . the e l glycoprotein forms aggregates of irregular sizes x . . (d) the peplomeric glycoprotein e of mhv purified by the same technique forms rosettes or single peplomers. arrows indicate rosettes. x , . (c and d from k. v. holmes.) a,b; see section ,d). isolated noninfectious, empty ibv particles. such particles have not yet been identified for other coronaviruses. the virions of coronaviruses are rather fragile and tend t o disrupt upon storage and/or during the negative-staining procedure (apos-tolov et al., ) . this can be prevented by fixation of virions with glutaraldehyde prior to negative staining. in spontaneously disrupted preparations of coronaviruses, fragments of viral envelopes are frequently seen. however, the internal component of coronaviruses has been more difficult to visualize in negatively stained preparations. thin sections of infected cells or virions demonstrated a flexible, cylindrical nucleocapsid which was probably helical (see section ,f). johnson-lussenberg ( - ) showed that threadlike nucleocapsids, - nm in diameter, were released from disrupted hcv- e virions. these were very pleiomorphic and may represent strands which had uncoiled from helical nucleocapsids. the most tightly coiled helical nucleocapsids were obtained from virions which were spontaneously disrupted by storage at °c overnight. found hollow, helical nucleocapsids, - nm in diameter and up to . pm long, released from purified preparations of hcv- e and mhv- virions. the unit length of coronavirus nucleocapsids has not yet been determined, although nucleocapsids up to pm long have been observed . caul et al. ( ) purified helical nucleocapsids from hcv- e (fig. b) . the nucleocapsid was about - nm in diameter. coronavirus nucleocapsids appeared to be more flexible and easier to uncoil than paramyxovirus nucleocapsids. recent studies of helical nucleocapsids of negative-stranded rna viruses (heggeness et al., (heggeness et al., , showed that these properties depended upon the ionic strength and cation composition of the buffers. it appears likely that the appropriate conditions for further characterization of the structure of coronavirus nucleocapsids will be identified in the near future. in addition t o permitting analysis of the viral nucleocapsid, disruption of coronaviruses with nonionic detergents such as np- or triton x- has permitted the isolation of the envelope glycoproteins of coronaviruses (section ,g). the morphology of the two envelope glycoproteins of mhv isolated from detergent-disrupted virions on sucrose density gradients is shown in fig. c and d . early studies of coronaviruses were hampered by limited virus growth and difficulties with virus purification. coronaviruses exhibited restricted host ranges in cell culture, low virus yields were usually obtained, and the viruses were highly labile. these difficulties have now been overcome for many coronaviruses . suitable permissive cell types and virus strains have been identified and conditions have been described which result in greater virus stability. many human coronaviruses were first identified by their growth and cytopathogenicity in human embryonic tracheal and nasal organ cultures (tyrrell and bynoe, ; almeida and tyrrell, ; mcintosh et al., a) . some of these viruses have not yet been grown in continuous cell lines, and great difficulties are still encountered with primary isolation of hcvs. although permissive cell hosts have been described for hcv- e (hamre and procknow, ; hamre et al., ) as well as for tissue culture-"adapted" hcv-oc (bradburne and tyrrell, ; kapikian et al., ; bruckova et al., ; gerna et al., ; , virus yields of <lo pfu per milliliter are usually obtained. however, . w. obtained high yields (up to lo pfu per milliliter) of tissue cultureadapted strains of hcv-oc and - in a human rhabdomyosarcoma cell line. good growth of some human enteric coronaviruses has also been reported in a human rectal carcinoma cell line (laporte and bobulesco, ) . early studies of mhv revealed that productive infection could be obtained in nctc and l mouse cell lines (manaker et al., ; tanaka et al., ; hartley and rowe, ; mosley, ) . however, both of these cell lines contained endogenous retroviruses (david-ferreira and manaker, ; dales and howatson, , thus limiting their usefulness for propagation and biochemical studies of mhv. in , sturman and takemoto reported that the growth of mhv was enhanced in al/n and balb/c t mouse cells which were transformed by papovaviruses or retroviruses. a "spontaneously" transformed balb/c t cell line, designated c , was also shown to be a highly permissive host for mhv, yielding >lo pfu per milliliter. two additional transformed cell lines have also been found to produce high yields of mhv: dbt, a schmidt-ruppin rous sarcoma virus-induced mouse tumor cell line (hirano et al., (hirano et al., , (hirano et al., , , and sac (- , a maloney sarcoma virus-transformed mouse cell line which is defective in retrovirus production . in contrast to hcv and mhv, no continuous cell line is available which produces large amounts of ibv. limited growth of a few strains of ibv has been obtained in vero and bhk- cells (cunningham et al., ; coria and ritchie, ; otsuki et al., ) . however, high yields of some strains of ibv have been obtained in primary chick embryo kidney cells (otsuki et al., ; stern and kennedy, a) . another development which facilitated progress in this field was the recognition that coronaviruses are most stable between ph . and . alexander and collins, ; sturman, ) (fig. ) . at ph . , the half-life of mhv infectivity at °c in the presence of % fetal bovine serum was hours, whereas at ph . , a % loss in virus infectivity occurred in less than hour. the rapid loss stability of viral infectivity is shown as the ratio of viral titer at hoursititer at time, x . at °c the virus is quite stable from ph to . however, at °c the virus exhibits marked thermolahility at phs c . and > . . (reproduced from sturman, , with permission.) of infectivity at ph . was associated with aggregation of the peplomeric glycoprotein (sturman, ) . several unique features of the coronavirus glycoproteins, as well as the lack of suitable permissive cell types for some coronaviruses and residual host-cell contamination of other coronaviruses, delayed recognition that coronaviruses all possess a similar pattern of structural proteins. recently, the general organization of coronavirus structural proteins has become apparent. figure illustrates a model of the structure of mhv-a . we will use this model and the nomenclature developed during our studies of the structural proteins of mhv for the following discussion of coronavirus structural proteins and their organization. the envelope of the intact virions contains two envelope glycoproteins, e l and e , in a lipid bilayer. the helical nucleocapsid is composed of a single long strand of message sense, genomic rna with the nucleocapsid protein, n. glycosaminoglycan is associated with the viral envelope. (b) treatment of the virions with pronase or bromelain removed the bulk of e , a k glycosylated portion of e , and the glycosaminoglycan, but leaves the nucleocapsid intact. (c) virions released from cells treated with tunicamycin lack e but contain normal amounts of glycosylated e l and nucleocapsid. in , garwes and pocock characterized the structural polypeptides of tgev, a porcine coronavirus. although analysis of coronavirus polypeptides had been attempted earlier, those studies were less definitive since they utilized virus produced in animals or eggs which may have been contaminated with some host-cell components (hierholzer et al., ; bingham, ) . working with radiolabeled tgev produced in cell culture, garwes and pocock identified four major polypeptide peaks on sodium dodecyl sulfate (sds)-polyacryl-amide gels. treatment of virions with bromelain removed the peplomers and the largest glycoprotein ( k , which is analogous to the one which we have called e in fig. . a single, nonglycosylated, arginine-rich, k species similar to n, and two smaller ( and k) glycoproteins, analogous to e l in fig. , were also identified and partially characterized (garwes et al., ) . using double-labeled virus grown in tissue culture, the structural polypeptides of the a strain of mhv were described next (sturman, ; sturman and holmes, ) . this virus was shown to contain a nonglycosylated basic polypeptide, n ( k) and five glycoprotein peaks which were separated into two families based on the ratios of incorporation of different radiolabeled precursors. the e glycopro- and %-amino acids ( - ) were solubilized in sds and analyzed by page. two classes of glycoproteins, e l and e , are identified by the ratios of labels, and a nonglycosylated nucleocapsid protein, n, is seen at k. designation and molecular weights of the viral structural proteins are indicated above the arrows. ( b ) virions labeled with c glucosamine ( - ) and sh fucose (o---o) showed two classes of glycoproteins. the e l glycoproteins were labeled with glucosamine but not with fucose, whereas the e glycoproteins were labeled with both. (adapted from sturman and holmes, , with permission.) teins (gp and gp ) were labeled with both fucose and glucosamine, whereas the e l glycoproteins (gp , gp , and gp ) were labeled with glucosamine but not fucose (fig. b) . a surprising finding was the demonstration that e l (gp ) aggregated when heated to °c in the presence of sds and mercaptoethanol, generating several forms of higher apparent molecular weights (sturman, ; fig. b) . when virions were solublized in sds at "c, only a single broad peak at k was observed (fig. a) . the k form of e could be converted quantitatively to k by treatment of intact virions with trypsin, which did not remove the peplomers ( fig. ; sturman and holmes, ) . when the and k e glycoproteins were extracted from polyacrylamide gels and further digested with trypsin, virtually identical tryptic peptide patterns resulted. these results suggested either that proteolytic were prepared for page by incubating at °c for minutes instead of boiling. the e l glycoprotein did not aggregate into dimers and trimers as shown in fig. , but migrated as a broad peak of k. designations and molecular weights of the viral structural proteins are indicated above the arrows. (b) when virions similarly labeled were incubated with the proteolytic enzyme bromelain, the e glycoprotein and the k glycosylated portion of e l were removed, leaving an k portion protected within the viral envelope with the nucleocapsid protein n (vp k). (adapted from sturman and holmes, , with permission.) virions with pgiml trypsin, the k form of e was quantiatively converted into the k formb), while the other two structural proteins were unchanged. (adapted from sturman and holmes, , with permission.) cleavage of the k form of e yielded two different k forms which comigrated, or that the k e might be a covalently linked dimer of a single k species. thus, although the mhv virion was composed of only three major structural proteins, multiple forms of e l and e were generated by aggregation and proteolysis. similar observations have been made with some other coronaviruses (see below). the structures and functions of these two glycoproteins will be considered in detail in section iv. the study of mhv-a also provided information about the relative ratios of the structural proteins and their orientation in the virion. on the basis of incorporation of radioisotopic labels, we estimated that the proteins occur in virions in a ratio of n : el:l e . as with tgev, treatment of virions with bromelain or pronase resulted in the loss of e (fig. b ) and removal of the peplomers or spikes on the virion (sturman and holmes, ) . pronase also removed a k glycosylated portion of e l , which suggested that a terminal glycosylated region of e l was exposed on the outer surface of the viral envelope, while a larger ( k) nonglycosylated region was protected within the envelope. since pronase treatment of intact virions did not affect n ( k , n was thought to be an internal protein. the structural relationships of these three viral polypeptides are summarized in the model in fig. . wege et al. ( ) , investigating the structural proteins of the jhm strain of mhv, detected two species of e l on sds-polyacrylamide gel electrophoresis (sds-page) which appeared to correspond to nonglycosylated and glycosylated forms of e l . in addition, they showed that the e ( k) of mhv-jhm could be resolved into two distinct bands. in contrast to e from mhv-a , the e of jhm became aggregated with e l when heated to °c and remained near the origin of the resolving gel (wege et al., ; siddell et al., b) . comparison of a variety of strains of mhv revealed that some of the homologous polypeptides from different strains were distinguishable by page (stohlman and lai, ; anderson et al., ; bond et al., ; cheley et al., b) . this may be useful in genetic and complementation studies. stohlman and lai ( ) demonstrated that the n polypeptide of mhv was phosphorylated on serine residues (fig. ) . subsequently, '*i siddell et al. ( a) identified a cyclic amp-independent protein kinase activity which copurified with the virion (fig. ) . the functional significance of phosphorylation for transcription, translation, or maturation of the viral nucleocapsid is not yet known. the nucleotide sequence of the rna encoding the nucleocapsid protein of mhv-a was recently determined by armstrong et al. ( ) . this sequence contained a single long open reading frame encoding a protein of molecular weight , which was enriched in basic residues. there was also a second short, open reading frame in this sequence predicting a polypeptide of amino acids. however, no such product has yet been identified. the pattern of three major structural proteins and their organization in the virion as shown for mhv-a in fig. is generally applicable to most other species of coronaviruses (reviewed in detail by siddell et al., ) . although the proteins of different coronaviruses have different molecular weights, similar polypeptide patterns have been obtained with the porcine coronaviruses, transmissible gastroenteritis virus, and hemagglutination encephalomyelitis virus pocock and garwes, ; callebaut and pensaert, ; k. moreau and d. a. brian, personal communication) , and with canine coronavirus i (garwes and reynolds, ; carmichael and binn, ; see the review by garwes, ) . the structural polypeptides of several of the mammalian coronaviruses, including those from rats, cats, and rabbits, have not yet been investigated. macnaughton ( ) and schmidt and kenny ( ) reexamined the polypeptide composition of the human coronaviruses and oc and obtained results similar to mhv. schmidt and kenny demonstrated that the e l of oc aggregated upon heating in sds under reducing conditions, whereas e l from did not. several coronaviruses may have an additional envelope glycoprotein. in some bovine and porcine coronaviruses, three or four large glycoprotein peaks associated with the virus peplomers have been identified by sds-page (king and brian, ; callebaut and pensaert, , and more than one morphologically distinguishable spike has been detected by electron microscopy (bridger et al., ) . it has been suggested that these viruses possess several different types of peplomers. however, the relationship between the components detected on sds gels and the morphologic subunits of these viruses has not yet been elucidated. for some time it appeared that the structural polypeptides of an avian coronavirus, ibv, were more complex than those of mammalian fig. . demonstration of virion-associated protein kinase activity. virion-associated protein kinase activity is demonstrated in detergent-disrupted mhv virions by the incorporation of p from orthophosphate into protein. the page pattern on the right indicates that the only viral structural protein phosphorylated during this reaction is n. lanes indicate times after initiation of the reaction; molecular weight standards are shown on the right. (reproduced from siddell et al., a, with permission.) coronaviruses (bingham, ; collins et al., ; alexander and collins, ) . however, macnaughton and madge ( a) demonstrated that harsh conditions of sample treatment generated spurious additional bands on sds gels. these studies were extended by collins and alexander ( a,b) and lancer and howard ( ) . by , it was apparent from the work of several investigators (cavanagh, ; wadey and westaway, ; lomniczi and morser, ; macnaughton, ; that there were three major classes of ibv structural polypeptides. contamination by host polypeptides seems to have caused many of the problems associated with studies using virus grown in embryonated egge (wadey and westaway, ; cavanagh, ) . lomniczi and morser ( ) showed that the n polypeptide of ibv was phosphorylated like that of mhv (stohlman and lai, ) . they found that actin was bound to the surface of purified ibv virions. in contrast, actin has not been detected in purified mammalian coronaviruses. the polypeptide composition of another avian coronavirus, turkey bluecomb disease virus, has not yet been reported. differences in the carbohydrate compositions of e l and e for mhv were first indicated by results obtained from metabolic labeling of virions with radioisotopic precursors. e was labeled with both fucose and glucosamine, whereas e l was labeled with glucosamine but not with fucose (sturman and holmes, ) . the carbohydrate compositions of e l and e were analyzed by niemann and klenk ( a,b) . their results are shown in table . as coronaviruses were often grouped with myxoviruses in early classification schemes, it is interesting to note that both e l and e contained sialic acid, unlike the myxovirus glycoproteins. e also contained substantial amounts of mannose and galactose plus fucose, glucose, and n-acetylglucosamine. these sugars are all found in high mannose and complex oligosaccharides which are derived from a mannose-trisaccharide core nglycosidically linked to asparagine residues in the protein. the oligosaccharide side chains of e l were strikingly different from those of e , in that they lacked fucose and contained a high proportion of nacetylgalactosamine, which was absent from e . recent evidence indicates that in mhv-infected cells labeled with glucosamine, the glucosamine is converted to n-acetylgalactosamine prior to incorporation into the e l glycoprotein (h. niemann, personal communication). the carbohydrate composition of the e l glycoprotein suggested that e l oligosaccharides might possess -glycosidic linkages to serine or threonine residues in the protein. the same conclusion was reached by others (holmes et al., a,b; siddell et al., c; rottier et al., b) based on the resistance of the glycosylation of e l to tunicamycin, an inhibitor specific for n-glycosylation. such -linked glycoproteins had not been found previously in viruses. the glycopeptides of e l and e were readily distinguishable by electrophoresis on borate-polyacrylamide gels at high ph (sturman, ; holmes et al., a) and by column chromatography on biogel p (niemann and klenk, a) . niemann and klenk ( b) demonstrated conclusively that the oligosaccharide moieties on e l were attachd by glycosidic linkages since they could be released by p-elimination with sodium borohydride. recently, h. niemann et al. (personal communication) have used high-performance liquid chromatography (hplc) to characterize two species of oligosaccharide chains released from e by p-elimination. these are shown in fig. . it is highly probable that the virus makes use of host-cell enzymes for -linked glycosylation as other viruses do for n-linked glycosylation. thus, the e l glycoprotein of mhv is of considerable interest as a model for studying the synthesis and glycosylation of -linked cellular glycoproteins (see sections ii , d and iv, b) . there appears to be considerable variation in the glycosylation of the e l glycoproteins of different coronaviruses. the e l of some mhv strains could not be labeled with glucosamine (anderson et al., ) and the e l of tgev was labeled with both fucose and glucosamine ; also see review by games, ) . recent neunac la neunacz~ cal --- galnac-oh evidence from endoglycosidase h sensitivity and tunicamycin inhibition studies indicated that the e l glycoprotein of ibv is n-linked, rather than o-linked . the biological signficance of n-versus o-linkage of the oligosaccharides is not known. other constituents of e l and e have been identified. e l and e could both be labeled with s sulfate (garwes et al., ; l. s. sturman, unpublished data). by analogy with glycoproteins of orthomyxoparamyxo-, and rhabdoviruses (nakamura and compans, ; prehm et al., ; hsu and kingsbury, ) , these sulfate moieties may be ester linked to glucosamine or galactosamine residues of oligosaccharide side chains and may contribute to the charge heterogeneity of the viral glycoproteins. protein sulfation on tyrosine resides may also occur (huttner, ) , but this has not yet been reported for viral proteins. the significance of sulfate groups for antigenicity or other biological functions of viral glycoproteins is not known. m. f. g. schmidt and his co-workers ( ; schlesinger, , ; schmidt, a,b) demonstrated that palmitic acid was covalently attached to certain glycoproteins from a variety of enveloped viruses. niemann and klenk a) showed that palmitic acid was present on the e glycoprotein of mhv, but not on e l or n. schmidt reported similar results with bcv ( ) . recently, we separated two e ( k) species from trypsin-treated mhv-a by sds-hydroxyapatite chromatography (l. s. sturman and k. v. holmes, unpublished data) . only one of these k species contained palmitic acid. this palmitic acid may be useful as a marker to identify one k subunit of the e molecule. minor protein species associated with some coronaviruses have also been identified, such as gp in mhv-jhm (wege et al., ; siddell et al., b) . the reiationship of such minor components to the other structural proteins of the virion is not yet known. these may represent virus-specific polypeptides which are found in infected cells (see section ,d). as with the structural proteins, the earliest data on the coronavirus genome were misleading. initial observations suggested that coronaviruses contained segmented or multimeric genomes (tannock, ; . there were also indications of possible rna polymerase activity associated with the virion . these findings were in accord with the belief then current that coronaviruses were probably similar to myxoviruses. then watkins et al. ( ) demonstrated that high-molecular-weight rna could be extracted from ibv virions. the large, single-stranded, linear rna molecules obtained from ibv were shown to be polyadenylated and infectious (schochetman et al., ; lomniczi, ; lomniczi and kennedy, ; macnaughton and madge, ) . lomniczi and kennedy ( ) characterized the ibv genome by electrophoresis on methyl mercury gels and by t oligonucleotide fingerprinting. this approach also proved to be very useful in the comparative analysis of coronavirus genomes and subgenomic virus-specific mrnas (see section iid). extracted high-molecular-weight ( - s) rna from tgev and hev. however, they observed that the rna was dissociable above °c into and s species. tannock and hierholzer ( ) also reported releasing s rna from hcv-oc rna and fragmentation of the s rna after heating at °c. lai and stohlman ( ) found a variable amount of s rna in mhv preparations, in addition to s rna. garwes et al. ( , pocock and garwes ( , tannock and hierholzer ( , and lai and stohlman ( ) demonstrated that the size and heterogeneity of the virion rna which was isolated was affected by the time of virus harvest, the ph of the culture medium, the method of virus purification and duration of virus storage, and the method of rna extraction. greater fragmentation of coronavirus rna was observed if virions were harvested at hours postinfection versus hours, if cultures were kept at ph . or . and not at ph . , if virus was purified on potassium tartrate equilibrium gradients instead of sucrose gradients, if virions were stored for hours at °c before rna extraction, and if the rna was extracted in the presence of phenol. fragmentation was readily detected after heating the rna at °c or higher, and by centrifugation through dimethyl sulfoxide gradients. this behavior of coronavirus rna, as well as other aspects of the structure and physiocochemical properties of coronaviruses, is discussed in the excellent review by garwes ( ) . it now appears that coronavirus genomes are . - . x lo daltons in size, which corresponds to about , - , nucleotides. rna genomes in this size range have been detected in virions of ibv (schochetman et al., ; macnaughton and madge, ; macnaughton, ; stern and kennedy, a) , mhv (yogo et al., ; lai and stohlman, ; wege et al., b; macnaughton, ; spaan et al., ; weiss and leibowitz, , hcv (tannock and hierholzer, ; macnaughton and madge, ; macnaughton, ) , tgev and hev brian et al., , and bcv (brian et al., ) . the genomic rna is infectious (lomniczi, ; schochetman et al., ; wege et al., ; brian et al., , capped (lai and stohlman, ; lai et al., a) , and polyadenylated (schochetman et al., ; lomniczi, ; yogo et al., ; madge, b, ; wege et al., ; lai and stohlman, ; guy and brian, ; . for further details, see the reviews by garwes ( ) and siddell et al. ( ) . in , stern and kennedy ( b) mapped the location of the t oligonucleotides of the genome in a partial ' to ' order. large fragments of rna were produced by partial alkali fragmentation of the genome. these fragments were fractionated into different size classes by sedimentation on sucrose density gradients, and polyadenylated rnas were selected for t oligonucleotide analysis. the same approach was applied to mhv by lai et al. ( ) . these studies disclosed that there was no significant reiteration of oligonucleotides within the genome. analysis of the genetic complexity of the virion rna indicated that it was all of the same sense. since genomic rna was infectious, the virion rna must be of positive or message sense. t oligonucleotide analysis of coronavirus genomes has been employed for comparisons of virus isolates and strains, for characterization of mutants, and for epidemiological investigations. the t oligonucleotide fingerprints of the two isolates of the beaudette strain of ibv which were studied by lomniczi and kennedy ( ) and stern and kennedy ( a) were found to be quite different. subsequently, clewley et al. ( ) distinguished oligonucleotide fingerprint patterns from isolates of ibv, revealing differences between ibv serotypes, and also between different preparations of a single serotype. this suggested that considerable variation in the genome may be characteristic of ibv, and possibly of other coronaviruses as well. it is characteristic of rna viruses that the frequency of spontaneous mutations is high (holland et al., ) , and coronaviruses appear t o be no exception to this rule. analyses of murine coronaviruses have also indicated that there is considerable diversity between different strains. wege et al. ( a) and found that the oligonucleotide fingerprint pattern of the -jhm strain differed significantly from those of mhv- , - , - , -s, and -a , and that mhv- and -a were closest in oligonucleotide patterns. have attempted to correlate strain-specific oligonucleotide differences with hepatotropism and neurotropism of mhv- and -a . stohlman et al. ( b) have characterized oligonucleotide patterns of plaque morphology variants of j h m that differ in neuropathogenicity. the genetic relatedness of coronaviruses has also been analyzed by nucleic acid hybridization. using a cdna probe representative of the entire genome of mhv-a , weiss and leibowitz ( ) found that mhv- and -a were more closely related to one another than either was to -jhm. hcv- e appeared to be quite unrelated to mhv by this technique. cheley et al. ( b) , using a cdna probe prepared against the mrna of mhv-a which coded for the nucleocapsid protein, obtained evidence of % homology by analysis of hybridization kinetics of viral rna from cells infected with mhv- , - , -s, and -jhm. the lipid composition of coronavirus virions has not been analyzed in detail. it appears likely that the lipids of the viral envelope will reflect the lipid composition of the intracellular membranes from which virus budding occurs (see section ,f). indeed, pike and garwes ( ) observed that tgev contained less cholesterol and fatty acid than was found in the plasma membrane of the cells in which the virus was grown. when the virus was grown in different cell types, the viral lipids reflected the overall lipid composition of the cells in which it was grown. these studies need t o be extended using various cell-membrane fractions. the role of the lipid composition of cellular membranes in coronavirus maturation remains to be elucidated. it would be interesting to know whether e l and the viral nucleocapsid preferentially associate with membranes of a certain lipid composition. boundary lipids associated with the hydrophobic portions of e l and e have not been identified, although palmitic acid has been shown to be covalently attached to e (niemann and klenk, a,b; schmidt, a ,b) (see section ,c). the derivation of coronavirus envelopes from intracellular membranes may render the viral envelope less susceptible to solubilization by bile salts and other detergents than enveloped viruses which bud from the plasma membrane. greater stability of the viral envelope to solubilization by bile salts would be consistent with the survival and replication of these viruses in the enteric tract. comprehensive studies on the relative susceptibility to solubilization of coronavirus envelopes and other viral envelopes have not yet been carried out. several components of normal host cells or tissues have been found to copurify with coronaviruses. the difficulty of obtaining ibv free from host-cell contamination and the association of actin with ibv have already been mentioned (section ii,b and c). in early studies of coronavirus antigens, host antigens were often detected in association with the purified virions. some components of fetal bovine serum adsorb to and copurify with coronaviruses (kraaijeveld et al., ) . it is not yet clear whether the protein kinase associated with coronavirus particles is a host contaminant or a product of the viral genome. another class of host molecules which copurified with several different coronaviruses was glycosaminoglycans (gag) (garwes et al., ; sturman, ) . these are polyanionic, linear polysaccharides such as hyaluronic acid, chondroitin sulfate, keratin sulfate, heparan sulfate, and heparin, which are secreted by cells and which may remain associated with their external surfaces (roden, ; oldberg et al., ; prinz et al., ) . they tend to aggregate spontaneously with like molecules to form large complexes. multiple chains of gag molecules which are linked to core proteins are called proteoglycans. garwes et al. ( ) showed that some sulfated gag was associated with tgev. glycosaminoglycan associated with virions of mhv-a was partially characterized by enzymatic and chemical degradation (sturman, ) . the gag associated with mhv-a resembled the heparan sulfate species produced by uninfected transformed mouse cells, which contained a reduced level of - sulfated glucosamine residues (sturman, ; keller et al., ; winterbourne and mora, ) . the virion-associated gag illustrated in fig. is known to be on the external surface of the viral envelope since it can be removed by protease treatment of intact virions. the number of gag molecules associated with each virion is not yet known. cellular gags have been found in association with many types of enveloped viruses pinter and compans, ; lindenmann, ; kemp et al., ) , but the biological significance of this association is not known. in cells, heparan sulfate may play an important role in cell-cell and cell-substrate adhesion, growth control, and masking of cell surface receptors (vannucchi and chiarugi, ; rollins and culp, ; oldberg et al., ; kraemer and smith, ) . one could speculate that virion-associated gags or proteoglycans could alter viral functions by modifying viral structure, antigenicity, or susceptibility to proteolytic enzymes. coronavirus particles may spontaneously disrupt to yield membrane fragments and threadlike or helical nucleocapsids (see section ,a). viral subunits have also been generated for structural and immunologic analysis by solubilization of the envelope with nonionic detergents such as np- or triton x- in low salt concentrations. after detergent disruption of the viral envelope, solubilized glycoproteins could be separated from the nucleocapsid by sedimentation in sucrose density gradients. garwes et al. ( ) were the first to report isolation of the surface projections and nucleocapsid of a coronavirus, tgev, by this technique. immunization with np- -solubilized, gradient-purified tgev suface projections (e ) induced neutralizing antibodies (see section iv,a). johnson-lussenberg ( - ) isolated a nucleoprotein from by a similar method. similar studies were carried out with hev and mhv-jhm (pocock and garwes, ; wege et al., ) . in these studies, the nucleocapsid sedimented at a density of . - . gm/ml and was found to consist of rna in association with both n and e l structural proteins. electron microscopy revealed spherical "cores" to nm in diameter, sometimes with a strand approximately nm in diameter inside. helical nucleocapsids (fig. b) were not observed in these studies. in similar studies on np- -disrupted mhv-a (sturman et al., , we found that the temperature of solubilization determined whether e l would be isolated separately or in association with the nucleocapsid. solubilization a t °c yielded separate peaks of e l , e , and nucleocapsid (p = . gm/ml; fig. loa) , whereas incubation of the viral extract at °c for minutes resulted in quantitative binding of e l t o the nucleocapsid forming an e -n-rna complex (p = . gm/ml; fig. b ). in negatively stained preparations, purified e l formed irregular aggregates of varied size, whereas purified e was in the form of single peplomers or rosette-like aggregates of about peplomers ( fig. c and d). purified e l and e were used to raise monospecific antisera for the analysis of the functions of the glycoproteins (holmes et al., b; section iv) . the reasons for the difficulty in isolating helical nucleocapsids from coronaviruses are not yet clear. e. . caul (personal communication) has suggested that the helical nucleocapsid in the virion may be in the sturman et al., , with permission.) form of a labile supercoiled structure which may be identical to the to -nm spherical forms described above. alternatively, the to nm particles could represent incompletely solubilized nucleocapsids within a membrane-like structure containing e l . in some intracellular inclusions of nucleocapsids, such a structure has been visualized by transmission electron microscopy (dubois-dalcq et al., ; section ,f) . holmes and behnke ( ) observed that mhv-a virions changed from spherical to flattened, disk-shaped particles during migration from the rough endoplasmic reticulum to the golgi (section ,f). such a change may be correlated with supercoiling of the nucleocapsid strands. several investigators have studied the susceptiblity of isolated coronavirus nucleocapsids to digestion with proteases or rnase. using an electron microscopic assay, davies et al. ( ) showed that the nucleocapsid of ibv was destroyed by trypsin or pronase and was partially susceptible to degradation by pancreatic ribonuclease. during the past few years, a comprehensive understanding of coronavirus replication has begun to emerge. our current concept of coronavirus replication is shown in fig. . although coronaviruses have structural similarities to the large, enveloped, negative-stranded orthomyxo-and paramyxoviruses, the coronaviruses demonstrate several unique features in their replicative cycle. a nested set of five or six subgenomic mrnas are elaborated, each of which codes for a single protein. there is some evidence which suggests that rna fusion may play a role in coronavirus replication. in some cases an o-linked envelope glycoprotein is formed which exhibits restricted intracellular transport. the cellular secretory apparatus may be used for release of virions. while much clearly remains to be learned about the replication of coronaviruses, it is already apparent that they utilize many novel ways of solving the problems of virus replication. relatively little is known about the earliest stages in coronavirus replication. although the marked host and tissue tropisms of coronaviruses have frequently been ascribed to possible host-cell receptor specificities, studies on early virus-cell interactions have been limited. in studies analogous to those done on myxoviruses, early studies of the interaction of coronaviruses with membranes used hemagglutination as a model for binding to the cell surface receptor. several coronaviruses, including hev (greig et al., , ibv (corbo and cun- the entire replicative cycle occurs in the cytoplasm. the genomic rna acts as mrna to direct the synthesis of viral rna-dependent rna polymerase. this enzyme copies the genomic rna to form full-length, negative-stranded templates. from these templates, using the viral rna polymerase, a series of subgenomic mrnas are synthesized. these mrnas form a nested set with common ' ends. all are capped and polyadenylated. each of the mrnas apparently codes for a single gene product. the functions of the nonstructural gene products ns k and ns k are not known. rna codes for the synthesis of n protein on free ribosomes. the n protein associates with newly formed genomic rna to form the viral nucleocapsid. rna and - are translated on membranebound ribosomes t o yield e and e l , respectively. the peplomeric glycoprotein e is cotranslationally glycosylated at asparagine residues and the core oligosaccharides are trimmed as the glycoprotein migrates through the golgi apparatus. the membrane glycoprotein, e , migrates to the golgi apparatus where oligosaccharides are added to the serine residues posttranslationally. virions are formed by budding in the rough endoplasmic reticulum and golgi apparatus, but not at the plasma membrane. e migrates readily to the plasma membrane, but intracellular transport of e l is limited to the golgi apparatus. virions are released from intact cells by fusion of post-golgi vesicles with the plasma membrane, possibly using the cellular secretory apparatus. numerous virions adsorb to the plasma membrane of infected cells. coronavirus-induced cytopathic effects include cell fusion and rounding of infected cells. ningham, ; bingham et al., , bcv (sharpee et al., ) , rabbit enteric coronavirus (lapierre et al., , hcv-oc / (kaye and dowdle, , and mhv (sugiyama and amano, ; walker and clantor, , can cause hemagglutination (see section iv,a). mengeling et al. ( ) showed that hev bound to avian erythrocytes by the tips of the viral peplomers. binding of ibv to erythrocytes was studied by bingham et al. ( , who found that the binding was inhibited by protease and neuraminidase treatment, and enhanced by phospholipase c treatment of erythrocytes. shif and bang ( ) suggested that mhv- bound equally well to macrophages from mice genetically susceptible or resistant to mhv- . they demonstrated equivalent amounts of infectious virus remaining in the supernatant medium over macrophage cultures after virus adsorption. binding of mhv-a to l cells in spinner culture was studied by richter ( , who found that the cell surface receptor activity was removed by protease treatment of the cells. this suggested that the receptor was a protein. attachment occurred at c and more rapidly at °c. saturation of virus receptor sites was achieved with only about virus particles per cell. additional studies showed that at °c mhv-a bound to splenic lymphocytes from susceptible and resistant mice, but not to thymocytes. the interaction of mhv- with l cells and cells from susceptible and resistant strains of mice was studied by krystyniak and dupuy ( ) . at "c, radioactive virions bound to macrophages, spleen cells, t lymphocytes, and thymocytes. the virus bound equally well to macrophages from genetically susceptible and resistant mice. this supports the conclusion of shif and bang ( ) that genetic susceptibility and resistance are not determined at the level of virus receptors. the binding of coronaviruses to cells appeared to be via the peplomeric glycoprotein since antibody to the peplomeric glycoprotein e inhibited virus infectivity (garwes et al., - , and isolated e competed with intact virions for the same cell surface receptor (k. v. holmes, unpublished observation; see section iv,b). scanning electron microscopic analysis of the binding of hcv- e to cultures of mrc human diploid cells showed that the virions bound randomly to the cell surface at °c (patterson and macnaughton, ) . warming the cultures to °c resulted in the loss of virions from the cell periphery, apparently by an energy-dependent capping mechanism. the penetration and uncoating of enveloped rna viruses has been studied extensively in recent years using biochemical and electron microscopic techniques. semliki forest virus, influenza virus, and vesicular stomatitis virus (vsv) virions bind to cell surface receptors and are internalized via coated pits (helenius et al., a; marsh and hel-enius, ; matlin et al., matlin et al., , . the vesicles containing virions, which may be similar to the receptosomes of willingham and pastan ( ) , then appear to fuse with endosomes. at the low ph within the endosomes, the viral envelopes fuse with the endosomal membranes releasing nucleocapsids into the cytoplasmic sap. proteolytic cleavage of the peplomeric ha glycoprotein is required for fusion of influenza virus envelopes (huang et al., ; white et al., ) . although detailed studies of coronavirus penetration and uncoating have not yet been performed, there are some indications that a similar pathway of virus uptake may occur. electron microscopic studies of the uptake of mhv and ibv suggested that viral entry was by means of viropexis or endocytosis (tanaka et al., ; david-ferreira and manaker, ; sabesin, ; patterson and bingham, ) . however, other investigators suggested that coronaviruses entered the cell by fusion with the cell membrane (doughri et al., ) . the possibility that mhv- may enter the cell by fusion with the plasma membrane was supported by the observation of krystyniak and dupuy ( ) that mhv- could infect cells treated with cytochalasin b to prevent phagocytosis. adsorbed virions rapidly became associated with lysosomes (david-ferreira and manaker, ; sebesin, ) . virus uptake via coated pits was also observed (chasey and alexander, ; arnheiter et al., ) . chloroquine, a lysosomotropic drug that elevates the ph in lysosomes and prevents penetration of semliki forest virus and influenza virus through the endosomal membrane (helenius et al., b, , was found to inhibit the replication of mhv by affecting a stage subsequent to virus adsorption (mallucci, ) . in , in the first report on virus-specific rnas in coronavirusinfected cells, robb and bond ( b) identified multiple size classes of virus-specific rnas in mhv-jhm-and -a -infected cells by fractionation of mrnas on sucrose density gradients. soon thereafter, siddell et al. t ) demonstrated that different size classes of poly(a)containing intracellular mhv-jhm rnas fractionated on sucroseformamide gradients directed the synthesis of different structural proteins in cell-free translation systems. the n protein was translated from the smallest ( s) fraction, whereas e l was translated from a larger ( s) class of rna. at almost the same time, stern and kennedy ( a,b) showed, in a classic study of ibv rna, that coronavirus infection resulted in the production of a nested set of subgenomic messenger rnas with common ' ends. in ibv-infected cells, six virus-specific rna species were in this diagram each oligonucleotide in the rnase t fingerprint is labeled according to the smallest subgenomic mrna species in which it appears. the unlabeled nucleotides were not included in subgenomic rna e and hence, presumably, represent sequences between the ' end of the genome and rna e. (adapted from stern and kennedy, a,b, with permission.) identified by electrophoresis on agarose-glyoxal gels. all of these rnas were polyadenylated and therefore likely to be of message sense. together, the five subgenomic species greatly exceeded the total size of the genomic rna, suggesting that the subgenomic rnas shared some sequences. comparison of t oligonucleotide digests of these intracellular rnas, as shown in figs. and , revealed that they formed a "nested' set of sequences. the oligonucleotides of the smallest mrna were contained within the next larger mrna, and so on. these data also showed that the genomic rna was of the same sense as the mrna since they shared the same oligonucleotides. the possible origin of several unique oligonucleotides found in rnas a and c will be discussed later. by ordering the t oligonucleotides of the genomic rna through analyses of poly(a)-containing fragments produced by limited alkaline hydrolysis (see section ii,d), stern and kennedy b) showed that all of the subgenomic mrnas shared common sequences extending from the ' terminus of the genome. a similar structure for the intracellular rnas of mhv was also demonstrated. spaan et al. ( ) isolated six subgenomic, virus-specific rnas from polysomes of mhv-a infected cells, identified six intracellular rna species in cells infected with mhv-jhm. weiss and leibowitz ( ) and cheley et al. ( a) showed, by hybridization with a cdna probe against the ' end of the smallest message, that all of the subgenomic rnas of mhv-a contained sequences common to the ' end. t oligonucleotide fingerprints of subgonomic rnas of mhv were analyed by leibowitz et al. ( , lai et al. ( , and spaan et al. ( ) . the studies showed that, like avian coronaviruses, for the murine coronavirus mhv-a , the oligonucleotides of each of the subgenomic rnas were included within the next larger species, starting from the ' end of the genome. a tentative map of the mhv-a genome based on these data is shown in fig. . recently, a similar pattern of overlapping mrnas has been demonstrated by heilman et al. ( , and personal communication) for the early and late regions of the dna-containing bovine papilloma virus. using a cdna probe for the ' end of mhv rna, cheley et al. ( b) found differences in the elctrophoretic mobility of several subgenomic rnas of mhv-s in comparison with homologous rnas of mhv- , - , -a , and -jhm. found that the intracellular rnas appeared to be present in different ratios in cells infected with different strains of mhv (table ). the ratios of mrnas to each other did not change significantly during the course of viral infection (stern and kennedy, a; wege et al., b; . messenger rna coding assignments for the structural proteins of mhv have been established by eell-free translation experiments by siddell et al. , ~ , leibowitz and weiss ( , and leibowitz et al. ( b) , and from translation experiments in xenopus laeuis oocytes by rottier et al. ( a) (summarized in fig. , and see section ,d). the intracellular rnas of mhv have been designated through , beginning with genomic rna , and proceeding to the smallest mrna, . each virus-specific primary translation product has been shown to be associated primarily with one mrna (fig. ). fig. . tentative map of the genomic rna of mhv. based on the in uztro translation of isolated mrnas for several mhv strains by siddell et al. ( siddell et al. ( , c , leibowitz et al. ( b , and rottier et al. ( a , and the oligonucleotide mapping of lai et al. ( ) , we have drawn a tentative map of the mhv genome. although additional details will become available, it seems clear that the genes which have been identified to date are arranged in this order. comparison of each rna with the size of its translation products suggested that only the gene proximal to the ' end of each mrna was translated. as shown in fig. from the study by rottier et al. ( a , the product of rna was protein n (see section iii,c,l), that of rnag was e l , and of rna , e . the coding assignments for several nonstructural proteins were deduced by siddell et al. ( , ~ , leibowitz and weiss ( ,and leibowitz et al. ( ) . rnab directed the synthesis of a - k nonstructural (ns) protein; rna or - , a k ns protein; and rna , several related ns proteins which were > k. these data have been incorporated into the tentative map for the genomic rna of mhv-a shown in fig. . thus, the current dogma of coronavirus replication is that each mrna directs the synthesis of a single protein using only the gene at the ' end of the rna. however, some caution must be exercised in accepting this as proven. in all of the in uitro translation studies, rnag has been shown to direct the synthesis of e l , plus a fairly large amount of n (siddell, (siddell, , c rottier et al., a; leibowitz and weiss, ; fig. ). this has been ascribed to contamination of rnag with rna and a higher efficiency of translation of rna . it is possible that rnag alone might direct the synthesis of several gene products, both e l and n. in semliki forest virus, an enveloped, positive-stranded rna containing alphavirus, a large polycistronic mrna is translated to yield both glycosylated envelope glycoprotein and nonglycosylated capsid protein (schlesinger and kaariainen, ) . the translation of rna and - of mhv has been difficult to study because these rna species are present in such small amounts. so far only the k ns protein has been identified as a product for these two mrnas. in vitro translation studies using microsomal membrane fractions have not yet been done with coronavirus mrnas. such studies would permit the analysis of glycosylation, acylation, and processing of the glycoproteins. initial studies of intracellular virus-specific proteins anderson et al., ) identified the three major structural proteins, e l , e , and n, and several nonstructural proteins. no high-molecular-weight polyproteins were detected in infected cell . concurrent studies of the structural proteins of the virion and analyses of the mrnas and their translation products set limits on the probable number of virus-specific polypeptides, and established that the structural proteins were not derived from a large polyprotein precursor. the intracellular proteins of mhv and ibv were identified in many laboratories at almost the same time. much of this information was presented at the coronavirus symposium and was summarized in the review by siddell et al. ( ) . the intracellular proteins identified included n, e l , and e , and three nonstructural species: - k and k nonglycosylated polypeptides, and a k glycoprotein. the - k species has not been detected in virions. however, minor proteins of approximately k and k have been found in some coronaviruses (wege et al., ; siddell et al., a; rottier et al., b , though not in others (e.g., sturman, ; macnaughton, ; rottier et al., b; lomniczi and morser, ; niemann and klenk, a; bond et al., ; see siddell et al., , for further references). the significance of these findings is not known. the k species has not yet been associated with a virus-specific mrna. much of our understanding about the synthesis and processing of coronavirus proteins in uiuo has been obtained from pulse-labeling and pulse-chase experiments with mhv. several different virus-cell systems have been studied ranging from highly lytic ones involving l, dbt, and sac(-) cells, in which virus infection induced syncytia formation and destroyed the cells within - hours (anderson et al., ; cheley et al., b; siddell et al., ; spaan et al., ) , to a more moderate infection in c cells, in which the infected cells could survive for more than hours without significant cytopathic effects (cpe) (robb and bond, b; sturman et al., ; holmes et al., b) . not surprisingly, differences were detected in the rates of shutdown of cellular protein synthesis in these different systems. other differences between lytic and moderate coronavirus-cell interactions were also noted. synthesis of mhv proteins in lytic infections appeared to be coordinated throughout the replication cycle rottier et al., b) , whereas in cells showing minimal cpe, the synthesis of n was detected much earlier than that of e l and e (j. behnke, personal communication). pulse-labeling experiments at different times after infection and at different temperatures revealed that the synthesis of e l and e in these cells was coordinated, but different from the synthesis of n. the transit time for the synthesis of the viral glycoproteins, their incorporation into virions, and release of the virions from the infected cells also differed in moderate and lytic infections. in moderate infec- fig. . synthesis and processing of mhv structural polypeptides (cont, control; inf, infected). at hours after infection with mhv-a , cells were pulse labeled for minutes with [ h]-leucine and then incubated with excess cold leucine. there were no polyproteins observed. e is synthesized as the high-molecular-weight form, and no k e was observed in these cells. the n protein was synthesized in large amounts and appeared to be processed into two faster migrating species. the e l was synthesized as a nonglycosylated species which was posttranslationally glycosylated. both the glycoproteins were chased out of the cell into mature virions within - hours after labeling. only a small fraction of the n protein was chased into mature virions. (adapted from holmes et al., b, with permission.) tion, e l and e were quantitatively chased out of cells and into virions within about hours (holmes et al., b; fig. ). however, in lytic infection of sac(-) cells, e l chased into virions within - . hours after labeling, while much of the e remained cell associated for up to hours . another difference between lytic and moderate infections related to the appearance of e l and e on the surface of infected cells. both e l and e antigens of mhvjhm were detected on the surface of lytically infected l cells prior to the release of progeny virions (collins et al., ) . in contrast, e was the predominant viral glycoprotein on the surface of c cells during the early stages of a moderate infection with mhv-a (doller and holmes, ; section iii,d, ). to date, cleavage of the e glycoprotein has been detected only in lytically infected cells. indeed, cleavage of e may be responsible for the extensive cell fusion seen in lytically infected cultures (l. s. sturman and k. v. holmes, unpublished observation) . these host-dependent differences may reflect differences in host-dependent processes required for maturation of coronaviruses. the features of the synthesis, processing, and transport of coronavirus structural proteins which are consistent in all cell systems are described in the following paragraphs. although the time of appearance of labeled n paralleled the time of appearance of the labeled glycoproteins in virions, no significant decrease in the amount of n in the infected cell was detected during a to -minute chase period (anderson et al., ; bond et al., ; holmes et al., b; rottier et al., b; siddell et al., b) . this suggested that there was a large intracellular pool of n, most of which did not chase into virions, but was incorporated into nucleocapsids or replicative intermediates which never left the cell. recently, it was shown that n was synthesized on free ribosomes (niemann et al., ) . intracellular n was phosphorylated at serine residues, as was n in the virion (siddell et al., a,b) . during a -hour chase, an intracellular form of n with slightly greater electrophoretic mobility was detected within infected l cells (anderson et al., ) . this process was believed to be due to proteolytic processing of n (cheley and anderson, ) . the proportion of the smaller species increased following immunoprecipitation (siddell et al., ; rottier et al., a) , which led to the suggestion that these additional species represented molecules of n partially degraded by serum or cellular proteases. in pulse-labeling studies, these forms of n have been detected in cells without immunoprecipitation late in the infectious cycle of mhv-a (j. n. behnke and k. v. holmes, unpublished observations) . analysis of peptide maps of these different forms of n will be required to determine their relationships. e l appeared to be synthesized as a nonglycosylated k apoprotein which was posttranslationally glycosylated (holmes et al., b; niemann and klenk, a; rottier et al., b; siddell et al., ~) . the addition of sugars to this o-linked glycoprotein began to minutes after completion of the apoprotein, and continued for - hours (holmes et al., b; rottier et al., b) with production of two or three discrete glycosylated species. however, not all of the e l which entered the virion was glycosylated (rottier et al., b) . the oligosaccharide side chains of the e l glycoprotein are shown in fig. . niemann et al. (submitted for publication) have estimated that there are three oligosaccharide chains per e l molecule. glycosylation of e l is resistant to tunicamycin (holmes et al., a,b; niemann and klenk, a; rottier et al., b; siddell et al., ~) . recent cell fractionation studies (niemann et al., ; m. frana, unpublished observation) showed that e l was translated on membrane-bound polysomes. monensin inhibited glycosylation of e l (niemann et al., ) . the intracellular transport of the e l glycoprotein is markedly different from that of other viral glycoproteins. immunofluorescent staining with monospecific anti-el antibody showed that early in the infectious cycle e l was restricted to the perinuclear area of infected cells and accumulated in the golgi apparatus (doller et al., ; fig. ). in contrast, e rapidly dispersed throughout the cell membranes and appeared on the plasma membrane. the mechanisms and signals for intracellular transport of e l are not yet understood. possibly e l is transported by cellular mechanisms which transport cellular glycoproteins destined for the golgi apparatus. since e l is a transmembrane protein and has been shown to interact with viral nucleocapsid in uitro, it appears likely that the localization of e l on the rough endoplasmic reticulum and golgi membranes determines the site of virus budding. e , the peplomeric glycoprotein, was recently shown to be synthesized on membrane-bound ribosomes (niemann et al., ) . pulselabeling studies showed that a large, - k glycoprotein was the first form detected in infected cells (holmes et al., a; rottier et al., b; siddell et al., ~) . as noted below, there is considerable controversy over the relationship between the and k forms of e . this controversy may relate to differences in processing of e in different cell types. a - k apoprotein was detected in mhv-a infected sac(-) and c cells treated with tunicamycin rottier et al., b; niemann and klenk, a) . this is similar to the in uitro translation product of rna in l-cell or reticulocyte lysates in which glycosylation does not occur (rottier et al., a; siddell et al., l , c) , and probably represents the protein moiety of the e glycoprotein. translation of mrna inxenopus laeuis oocytes permitted glycosylation and yielded a k e glycoprotein (rottier et al., a; fig. ). pulse-labeling experiments with mhv-a and -jhm in sac(-) cells showed that the first form of e detected fig. . localization of e l in the golgi apparatus. (a) monospecific anti-el antiserum was used to stain e l antigens within the cytoplasm of infected cells. the e l accumulated in a sharply demarcated region near the nucleus and did not migrate to the plasma membrane as readily as did e . (b) the same cells were labeled by the thiamine pyrophosphatase histochemical reaction which labels the terminal saccules of the golgi apparatus. this demonstrates that the e l accumulates in the golgi apparatus. x . (from e. w. doller.) in these cells was a k species (siddell et al., b,c; rottier et al., b) . similar experiments using mhv- and -a in l and c cells, respectively, demonstrated a k, rather than a k, product (cheley and anderson, ; holmes et al., b) . the difference between the and k moieties has not been identified, but could relate to additional glycosylation, trimming, or sulfation of e . the appearance of the k species of e is different in different cell types. although in moderate infection of mhv-a in c cells, little or no k e was detected at - hours after infection (holmes et al., b; fig. ) , in sac ( -) cells infected with mhv-jhm or -a , a substantial amount of k e was detected after -to -minute chase (siddell et al., c; rottier et al., b) . siddell et a . ( ~) have suggested that the k precursor to e may be cleaved to a k protein which dimerizes to form the k e . based on our studies with mhv-a , we believe that two different k forms of e result from proteolytic cleavage of the k e which occurs as a late step in the intracellular transport or processing of e and the maturation of the virions. recently, stern and sefton ( ) showed by tryptic peptide mapping that the two large virion glycoproteins of ibv, gp , and gp , were produced by cleavage of a k glycoprotein precursor (gp ). although the cellular location at which the e glycoprotein undergoes acylation has not been identified, acylation of the vsv glycoprotein has been shown to occur near the golgi complex (schmidt, ) . the intracellular transport of e appears to be similar to that of nlinked glycoproteins of other viruses such as orthomyxo-, paramyxo-, rhabdo-, and alphaviruses. immunofluorescent staining of e with monospecific antiserum stained the cytoplasm diffusely, e appeared on the plasma membrane at a time when intracellular transport of e l was directed to the golgi apparatus (holmes et al., b; doller et al., ) . treatment of mhv-infected cells with tunicamycin resulted in a marked inhibition in the synthesis of e (holmes et al., b) , although late in infection some nonglycosylated e could be detected (niemann and klenk, a) . virions isolated from tunicamycintreated cells contained no e (sturman, ) and no peplomers (holmes et al., a,b) . this suggested that e was not required for virus budding or for the release of virions from infected cells. brian ( , ) and b. w. j. mahy et al. ( ) first detected rna-dependent rna polymerase activity in cells infected with tgev and mhv. this activity was insensitive to antinomycin d and was associated with cytoplasmic membrane fractions like that of alphaviruses. brayton et al. ( ) and lai et czl. ( b) have characterized the viral rna polymerase activity in mhv-a -infected cells. by pretreatment of cells with actinomycin d for hour before infection and synchronization of infection by adsorbing virus at - "c, brayton et al. ( ) detected virus-specific rna synthesis in mhv-a -infected cells as early as hour after infection. two peaks of virusspecific rna synthesis were demonstrated, one early ( hours) and the other late ( hours) after infection (fig. ). corresponding to these, two different virus-specific rna polymerase activities were detected, early and late in infection. these two polymerases were distinguished by different responses to potassium and different ph optima. the rna product of the early polymerase was of negative-strand polarity, complementary to the genomic rna, whereas the products of the late polymerase activity were predominantly of positive polarity (p. r. brayton, personal communication) . only a single virus-specific, negative-stranded rna species was detected. this was the size of the complete viral genome. a double-stranded rna form was isolated which contained rna of genomic size. when this double-stranded rna was isolated without rnase treatment and heated, subgenomic mrnas were released. this suggested that the virus-specific mrnas were incorporation of radiolabel into virus-specific rna in cells infected with mhv-a occurs in two peaks which show different ionic requirements. the early peak is believed to represent synthesis of full-length, negative-stranded rna templates, and the later peak is believed to represent synthesis of mrnas and genomic rna. (reproduced from brayton et al., , with permission.) transcribed from a negative-strand rna template of genomic size . the mechanism which regulates the frequency of transcription of each mrna species from the negative-stranded rna template remains t o be elucidated. the relative rates of synthesis of the mrnas appear to be constant throughout infection (stern and kennedy, a; wege et al., b; spaan et al., ; . the smaller mrnas (numbers and , which represent the ' end of the genome, are far more abundant than the sum of all the others (jacobs et az., ; table ). in different virus strains, the relative abundance of the different viral mrna species varies considerably. have shown ( table ) that the relative molar amount of rna synthesized in mhv-jhm-infected cells is about one tenth of that found in cells infected with mhv-a . a central question in the molecular biology of coronaviruses is how the subgenomic mrnas and genomic rna are transcribed from the negative-strand template. ultraviolet transcription mapping was done to identify the size of the template(s) for the synthesis of the mrnas. jacobs et al., ( ; table iv ) and stern and sefton ( a) found that b k x t was calculated from the relationship in (n,no) = k x t x t, where n t represents the incorporation of ["iuridine into rna after t seconds of uv irradiation, n o is the rna synthesis in the unirradiated culture, t is the target size, and k is a constant. the calculation was made from the data points by using linear regression analysis. the value ofk was calculated as . x - s - by substituting a value of . x for the target size of r n a l (experiment l), or as . x by inserting a value of . x for rna (experiment ). c by using the two values for k described in footnote b, the target sizes for the other rnas were calculated. d the molecular weights of the denatured, virus-specific rnas were determined by agarose gel electrophoresis. the uv target sizes of the templates for the mrnas of mhv-a and ibv were the same as the sizes of the respective mrnas. since the uv mapping studies were done at . and . hours postinfection, when synthesis of the negatively stranded template may have been completed, it appears likely that only the synthesis of the mrna species was being inactivated by uv. comparison of the oligonucleotide fingerprints of subgenomic mrnas and virion rna of mhv suggested that some rna splicing mechanism or other modification of the rna may take place during mrna synthesis. stern and kennedy ( a,b) found several t oligonucleotides in mrna species which were not present in larger mrnas. , , and spaan et al. ( ) also identified several t oligonucleotides in subgenomic mrnas which were not present in the viral genome of mhv, or were not found in the same region in the genome. preliminary evidence indicated that some of these oligonucleotides from several subgenomic rnas had similar sequences . it has been suggested that these may represent junction sequences from the splicing of two unlinked stretches of rna . since coronaviruses can replicate in enucleated cells brayton et al., ) , it appears unlikely that this splicing is done by cellular mechanisms located in the cell nucleus. lai et al. ( a) found that the nucleotides adjacent t o the cap structures of each of the subgenomic mrnas contained the same sequence, '-cap-n-uaag. it is not known how many additional nucleotides are shared at the ' end of these mrnas. it is not clear how the cap with its adjacent nucleotide sequences is added to each mrna. one possibility would involve rna splicing. a leader sequence which originates by splicing from large precursor molecules would appear to be contraindicated by the results of uv transcriptional mapping. however, a leader sequence with or without an attached cap may be derived from a small rna of viral or cellular origin. since viral rna synthesis was not inhibited by antinomycin d, this leader rna may be virus specific or perhaps derived from a stable, small, cellular rna. the sequence -uaag which was found adjacent to the cap structure at the ' end of viral subgenomic mrnas and genomic rna (lai et al., a) is also present in some small host coded cytoplasmic rnas, including s ribosomal rna (delihas and andersen, ) and s rna (busch et al., ) , and u and u small nuclear rnas (busch et al., ) . furthermore, the same order is contained in the consensus sequence a t the ' exon-intron boundary of many splice junctions (lerner et al., ) . the complementary sequence which is present in u snrna is thought to assist in the proper orientation of exons for splicing of rna. several models have been postulated for the utilization of a leader sequence in coronavirus transcription (lai et al., a; spaan et al., ) . . such a leader could act as a primer for the initiation of rna transcription along the full-length, negative-strand template. . alternatively, a leader sequence could be fused onto the ' end of newly synthesized viral transcripts. this sequence could come from viral or cellular rna. . an rna polymerase jumping mechanism could explain the common nucleotide sequences at the ' end of each viral mrna and genomic rna. thus, a short segment of the ' region would be transcribed from the ' end of the negative-strand template, and then the polymerase would translocate through intervening sequences on the negative-strand template and resume transcription at the beginning of a particular cistron. the uv transcription data do not contradict any of these models, since the leader sequence may be too small a target to have been detected. some of the early studies on coronavirus replication emphasized ultrastructural changes in infected cells (svoboda et al., ; tanaka et al., ; david-ferreira and manaker, ; hamre et al., ; becker et al., ) . all of these studies showed that the entire replicative cycle of coronaviruses occurred in the cytoplasm. indeed, mhv has been shown to replicate in enucleated cells wilhelmsen et al., ) , although replication of ibv in enucleated bhk- cells was significantly reduced (evans and simpson, ) . ultrastructural studies on the binding and penetration of coronaviruses have been described (section ii ,b.i. the early morphological events associated with coronavirus infection were rather nonspecific ones, such as increase in cytoplasmic membranes or in the size of polysomes (david-ferreira and manaker, ; sebesin, ) . toward the end of the viral latent period, about - hours postinfection, spherical virions approximately - nm in diameter were observed in the lumens of the rough endoplasmic reticulum, golgi apparatus, and smooth-walled vesicles (david-ferreira and manaker, ; massalski et al., ; ducatelle et al., ; dubois-dalcq et al., ; fig. a-c) . in most instances these virions had electron-lucent centers with electron-dense granular or tubular nucleocapsids associated with the inner surface of the viral envelope. tubular nucleocapsids were also observed under the membranes of the rough endoplasmic reticulum or golgi apparatus (oshiro, ; holmes et al., a; massalski et al., massalski et al., , dubois-dalcq et al., ; fig. b and c) . chasey and alexander ( ) showed that the envelopes of budding virions were covered with peplomers. during the early stages of viral infection, virions appeared singly or in small clusters, whereas later in infection, scores of virions were found within large vesicles and occasionally within the lumen of the nuclear membrane (fig. ) . budding of virions from the plasma membrane was almost never visualized. an exception was a single particle visulaized several days after virus infection of a neural cell culture (dubois-dalcq et al., ) . although sugiyama and amano ( ) reported that the virions were budding from the plasma membrane, the scanning electron microscopic (semi images were not of sufficient resolution to demonstrate budding. in some studies, although numerous intraluminal virions were observed, no budding virions were detected. detection of budding images apparently depended on the viral strain, the host cell type, and the time after virus inoculation (watanabe, ) . budding virions were most likely to be detected late in the infectious cycle. this suggests that these images represent arrested buds, and that during the early stages of infection coronavirus budding may be a very rapid process. several investigators have suggested that coronaviruses were released by lysis of the infected cells (hamre et al., ; oshiro et al., ; takeuchi et al., ; chasey and alexander, ) . release by fusion of virus-filled, smooth-walled vesicles with the plasma membrane has also been observed (doughri et al., ) . when the kinetics of release of infectious virus was correlated with cell lysis by microcinematography, however, it was apparent that the infectious virions were released from intact cells (k. v. holmes, unpublished observations) . indeed, it appears that release of virus depends upon the good condition of the host cells. the generally accepted mechanism of coronavirus release from infected cells is via fusion of virus-filled vesicles with the plasma membrane (doughri et al., ) . thus, the coronaviruses may be released from cells by utilizing a cellular transport mechanism developed for secretion or exocytosis of the contents of secretory vesicles. i n uiuo, the target cells for replication of many coronaviruses are epithelial cells with tight junctions in the respiratory or gastrointestinal tract. in polarized cells such as these, influenza virus and vsv have been shown to bud specifically from the apical or basilar plasma membranes (rodriguez-boulan and sabatini, ) . there is as yet little evidence to show whether the fusion with the plasma membrane of vesicles filled with coronaviruses exhibits specificity for basal or apical membranes. secretory cells do demonstrate strong polarity in the direction of secretion of cellular secretory products such as enzymes from pancreatic acinar cells. it is possible that some polarity of coronavirus release may be identified. to date, in the only study which addresses this point, doughri and storz ( ) observed that porcine coronavirus could be seen on both apical and basal cell surfaces of intestinal epithelial cells. fig. . coronavirus virions in the rough endoplasmic reticulum and nuclear envelope. late in the infectious cycle, numerous spherical virions accumulate within the rough endoplasmic reticulum and the nuclear envelope (n) (arrows). ~ , . (from j. n. behnke.) late in the infectious cycle, it was common to find very large numbers of virions adsorbed to the surface of infected cells (oshiro et al., ; oshiro, ; doughri and storz, ) . scanning electron microscopy showed these most effectively (sugiyama and amano, it is not clear what function, if any, may be served by this adsorption to infected cells. most of the virions appeared to remain on the surface without being internalized, although some were found in lysosomes (sabesin, ) . characteristic features of coronavirus infection were vacuolization of cells and virus-induced cell fusion (oshiro, ; mcintosh, ) . the time of appearance of these depended on the virus and the host cell type. it is noteworthy that large intracytoplasmic inclusions of nu- holmes.) cleocapsids were not observed in most early studies of coronavirusinfected cells. caul and egglestone ( ) did observe such inclusions in cells infected with hecv, and others have seen them also (watanabe, ) . massalski et al. ( ) suggested that nucleocapsid inclusions accumulate after the cessation of virus budding. dubois-dalcq et al. ( ) showed that several types of inclusions that could be produced by the intracytoplasmic nucleocapsids of different strains of mhv in differentiated cultures of central nervous system (cns) cells ( fig. d and e) . they also found an increase in "myelin figures" in the cytoplasm of mhv-infected cells. additional features of coronavirus cpe included several types of intracytoplasmic inclusions whose origins and functions remain unclear. david-ferreira and manaker ( ) found "reticular inclusions" in mhv-infected cells. these consisted of masses of interconnected tubules of smooth membranes in continuity with the rough endoplasmic reticulum (fig. a) . they have only occasionally been observed with other coronaviruses. a second type of inclusion was observed near the reticular inclusions. this consisted of vacuoles about nm in diameter containing flexible coiled filaments about nm in diameter. these filamentous structures were surrounded by a double membrane ( fig. b ; david-ferreira and manaker, ; takeuchi et al., ) . third, "tubular inclusions" consisting of interconnected tubules to nm in diameter were observed near the reticular inclusions (david-ferriera and manaker, ; watanabe, ) . inclusions consisting of interconnected virions within the lumen of smoothwalled vesicles have also been observed (oshiro et al., ) . within the lumen of the rough endoplasmic reticulum and smooth-walled vesicles, long, rigid, cylindrical structures about nm in diameter have occasionally been detected (fig. c; dubois-dalcq et al., ) . these may represent an excess of the e l glycoprotein since they occur frequently in tunicamycin-treated infected cells, where synthesis of e is greatly reduced (holmes et al., a) . it is evident from the preceding descriptions that coronaviruses produce a wide variety of effects in different cell types. further studies are needed to determine the composition of these different virus-associated structures. in other virus systems, conditional lethal mutants have been important in elucidating many steps in virus replication. with coronaviruses, this effort is still at an early stage. although many variants have been isolated from natural infections. there are few well-charac-terized, chemically induced mutants of coronaviruses. recently, several groups have obtained and partially characterized temperature-sensitive (ts) mutants of mhv. almost all of the mhv mutants have been selected for failure to induce syncytium formation. unfortunately, most of these mutants grow rather poorly, even under permissive conditions. there are few, if any, chemically induced mutants of other coronaviruses. clearly, other phenotypes and additional mutants are needed. the first collections of chemically induced mutants of mhv-jhm were made by haspel et al. ( ) with -azacytidine or -fluorouracil, and by robb et al. ( ) using n-methyl-n'-nitrosoguanidine and fluorouracil. temperature-sensitive mutants were selected for failure to induce fusion of susceptible cells at the nonpermissive temperature ( . " or °c). the majority of the mutants identified by robb et al. were found to be rna negative. only three were rna positive and exhibited synthesis of viral proteins. some of these mutants produced altered neuropathogenesis in mice. leibowitz et al. ( a) performed complementation analysis of ts mutants of mhv-jhm and identified seven complementation groups. six of these affected virus-specific rna synthesis. the gene product affected by each mutation has not yet been identified. several of the mutants described by haspel and his co-workers, including the ts mutant, are of particular interest because they induce demyelination with a much higher frequency than the wild type (haspel et al., ; knobler et al., c, , and because they may form aberrant inclusions in cns cells in uitro (dubois-dalcq et al., ) . wege et al. ( c wege et al. ( , ) also isolated temperature-sensitive mutants of mhv-jhm with -fluorouracil and found that these mutants caused higher rates of subacute and chronic neurological diseases than did wild type virus in suckling and weanling rats. twenty chemically induced (with -fluorouracil), temperature-sensitive mutants of mhv-a have been partially characterized by koolen et al. ( koolen et al. ( , . most of these mutants, selected for their inability to induce syncytium formation at "c, were rna negative also. several of the mutants exhibited altered neuropathogenic properties. a variety of coronavirus mutants have been isolated from persistently infected cultures of mhv, and are described in section ii ,h. clearly, the potential contributions of these and other coronavirus mutants to the study of coronavirus replication are enormous. in combination with cloning of the viral mrnas and genomic rna, which is being done in several laboratories, mutants will be invaluable for the analysis of coronavirus genetics, pathogenesis, and replication. coronaviruses are capable of inducing persistent infection in animals (robb and bond, a; wege et al., ) and in tissue cultures. many persistent infections were summarized at the wurzburg symposium on coronaviruses siddell et al., ) . the salient feature of these infections was that in most cases the majority of cells remained antigen negative, yet all of the cells were resistant to superinfection with wild-type virus. derived cold-sensitive mutants of jhm from persistently infected neuroblastoma cells. these were rescued from latently infected cells by polyethylene glycol-induced fusion to permissive cells. hirano et al. ( ) obtained small plaque mutants of jhm, and holmes and behnke ( ) isolated small plaque and temperature-sensitive mutants of a from persistently infected cells. these mutants have not yet been fully characterized. in order to understand the balance between virus and host which permits this persistent infection, it is necessary to identify the host functions which are utilized by the viruses during the replicative cycle and to characterize in detail the controls exerted upon coronavirus transcription and translation. there are important host controls over coronavirus replication at several levels. immune response genes may play an important role in resistance to coronavirus-induced disease . however, genetic factors are also important at the single-cell level. these are the focus of our discussion. bang and warwick ( ) demonstrated that while the pri strain of mhv (mhv- ) caused fatal hepatitis in mice of the pri strain, this virus did not kill c h mice. this susceptibility of pri mice to death induced by mhv- was found t o be inherited as a dominant gene (bang and warwick, ; kantoch et al., ) . by backcrossing, created a strain of mice congenic t o c h mice, but bearing the dominant susceptibility to mhv- (c h-ss strain). extensive studies were done comparing the effects of mhv- on c h and c h-ss mice taylor et al., ; bang, ) . by various manipulations, bang and his associates were able to modulate the effects of mhv on the susceptible and resistant mice. treatment of resistant animals with cortisone (gallily et al., ) or a proteindeficient diet (bang, ) rendered them susceptible so that they died from a small dose of mhv- , and treatment of susceptible animals with concanavalin a rendered them resistant so that they could survive a normally fatal dose of mhv- (weiser and bang, ) . the susceptibility or resistance of different mouse strains to mhv- , as measured by survival of animals, was directly correlated with the response of peritoneal macrophages from each strain in culture to this virus (bang and warwick, ; bang, ) . however, the cell culture conditions greatly affected these results. when fetal bovine serum was substituted for horse serum in the medium, the difference in virus yields between macrophages from resistant and susceptible mice was significantly reduced (lavelle and bang, ; bang, ) . the mechanism for this cellular restriction of mhv- synthesis in cells from resistant animals under defined conditions has not yet been characterized. shif and bang ( ) demonstrated that the restriction was at a stage subsequent to virus adsorption, and suggested that degradation of virions within the resistant cell might be responsible. later, cody showed that the virus grew equally well in resistant and susceptible cells, but was one-twentieth as infective for resistant cells (cody, ; bang, ) . in his last publications (bang and cody, ; bang, ) , bang described recent experiments which suggested that macrophage resistance to mhv- was also dependent upon associated lymphocyte action. he also suggested that cell-bound interferon might play a role in protecting the genetically resistant cell. the pioneering studies of bang and his co-workers were extended by other investigators using different strains of mhv. these studies demonstrate that mhv- , mhv- , and mhv-jhm exhibit different patterns of host susceptibility and resistance. although replication of mhv- was restricted in c h mice, replication of mhv- was only partially reduced (leprevost et al., ; virelizier and allison, ; yamada et al., ; taguchi et al., ) . furthermore, a/j mice, which were resistant to mhvs, were susceptible to mhv-jhm (knobler et al., b) . resistance to mhv- and mhv-jhm correlated with failure of the virus to replicate and with delayed appearance of cpe after low-multiplicity infection of cultures of peritoneal macrophages (virelizier and allison, ; krzystyniak and dupuy, ; knobler et al., a,b; stohlman and frelinger, ; stohlman et al., a , neuronal cells (knobler et al., a , and hepatocytes (arnheiter and haller, ; arnheiter et al., ) . resistance was partially overcome by infection at higher multiplicities, resulting in cell destruction, although virus yields remained low (virelizier and allison, ; arnheiter et al., ; knobler et al., b) . arnheiter et al. ( ) showed by fluorescent antibody staining that in the first cycle of virus replication, resistant cultures of hepatocytes had fewer cells expressing viral antigen. however, after infection at high multiplicty (multiplicity of infection of loo), all cells contained viral antigens, including e and e l , but virus production was delayed and virus yields remained low. the characterization of this cellular restriction to mhv replication remains incomplete. much of our present knowledge about the structure and functions of coronavirus glycoproteins comes from studies of mhv. a model for the structure of the e glycoprotein of mhv-a is shown in fig. . some of the important features of this model include ( ) anchoring of one end of the protein in the viral envelope; ( ) covalent attachment of palmitic acid; ( ) a single trypsin-sensitive site accessible in the native glycoprotein on the virion; ( ) the presence of sh groups and disulfide bonds; ( ) noncovalent association between the subunits ( a and b) of e ; and ( ) oligosaccharide side chains on both subunits. e is probably anchored to the viral envelope through a short hydrophobic region, as pronase or bromelain quantitatively removed both k and k species from the intact virions, while the peplomers were removed (sturman and holmes, ) . palmitic acid is covalently attached, probably at or near the hydrophobic domain which anchors the peplomer in the viral envelope, since this has been demonstrated for the influenza virus hemagglutinin (schmidt, a) and the g protein of vsv (petri and wagner, . although detergent-sol-fig. . model of the peplomeric glycoprotein e . this is a provisional model of e which shows some of the important structural features of the peplomeric glycoprotein such as the acylation, glycosylation, presence of both sulfhydryl groups and disulfide bonds, and the existence of a trypsin-sensitive cleavage site in the center of the molecule which results in the formation of two species, tentatively called e a and e b, which comigrate a t k. ubilized e could be digested by trypsin into many peptide fragments, there appears to be only a single trypsin-sensitive cleavage site accessible in the native k molecule on the mhv-a virion (sturman and holmes, . disulfide linkages mask proteolytic cleavage in the hemagglutinin of hev. upon reduction, the k hemagglutinin (e ) of strain vw disappeared and a new polypeptide species (gp ) appeared, indicating that the k e was composed of two k subunits which were linked through disulfide bonds (callebaut and pensaert, ) . the hemagglutinin of hev strain fs was also sensitive to treatment with sulfhydryl reagents; exposure to dithiothreitol resulted in the loss of hemagglutinating activity and release of a from the virion (pocock, ) . several investigators have reported that coronavirus infectivity was most stable below ph alexander and collins, ; sturman, ) . the ph-dependent thermolability of mhv infectivity shown in fig. appeared to be the result of a conformational change in e which led to aggregation of the peplomeric glycoprotein (sturman, ) . intrachain disulfide and sulfhydryl groups appeared to be important in determining the conformation of e , as ph and temperature-dependent aggregation of e on virions or of isolated, np- -solubilized e were enhanced by reducing agents and sulfhdryl blocking reagents (sturman, ) . there is no evidence for interchain disulfide bridges between the a and b subunits of mhv-a e ; they remained associated noncovalently on the virion after trypsin cleavage of the peplomer (sturman, ; sturman and holmes, ) . during ph inactivation, however, some k e was released, and incubation with reducing agents caused release of more k e . this was probably associated with a change in the conformation of this molecule following reduction of intramolecular disulfide bonds. the same appears to be true for the liberation of gp from hev strain fs (pocock, ) . present evidence indicates that e possesses six or more biological activities (table v): . binding of virions to receptors on the plasma membrane of susceptible cells (adsorption and hemagglutination) appears to be mediated by e . purified, radiolabeled e bound to susceptible cells but not to cells lacking in virus receptors, such as erythrocytes (k. v. holmes, unpublished data) . binding of e was inhibited by preincubation of cells with excess mhv. virions from which the peplomers had been removed, or particles without peplomers, exhibited markedly reduced capacities for cell attachment and infection (holmes et al., a) . in hcv-oc , monospecific antibody to e inhibited hemagglutination (schmidt and kenney, ) . coronaviruses which hemagglutinate include some strains of ibv, hev, hcv-oc / , mhv- , a murine enteric coronavirus, rabbit enteric coronavirus, and bcv (see section ,b). initially, trypsin treatment of virions appeared to be necessary for activation of ibv hemagglutinin (corbo and cunningham, ) . subsequently, however, the ibv hemagglutinin was shown to be inactivated by trypsin . trypsin also destroyed the hemagglutinating activity of oc viruses (kaye and dowdle, ) . there appeared to be significant strain differences in ibv hemagglutination , and the response of ibv t o trypsin was also strain dependent. . e is responsible for the induction of neutralizing antibody. garwes et al. ( , ( ) ( ) were the first to show that antibody against purified surface projections (of tgev) possessed virus-neutralizing activity in vivo. in accordance with this, found that most of the antibody induced during infection of human volunteers with hcv- e was directed against the surface projections of the virus. hasony and macnaughton ( ) also showed that immunization of mice with e protected them against infection with mhv- , whereas immunization with e l or n failed to provide protection against virus challenge. holmes et al. ( b) demonstrated that monospecific antibody against mhv-a e neutralized infectivity in cell culture, and similarly kenny ( , ) showed that monospecific antibody to e of hcv-oc neutralized the fig. . virus in uitro. monoclonal antibodies against mhv-jhm e were shown by collins et al. ( ) to neutralize mhv-jhm virus infectivity in the absence of complement, whereas monoclonal antibody to e l exhibited neutralizing activity only in the presence of complement. . e on the surface of infected cells renders them susceptible to cytotoxic effects of spleen cells. the cell-mediated cytotoxicity of spleen cells from uninfected mice to mhv-a -infected cells was also inhibited by antibody to e . the ph-dependent thermolability of coronavirus virions is due to aggregation of e . this conformational change in e , which occurs above ph . a t "c, is sensitive to sulfhydryl reagents (sturman, ) . . e is responsible for cell fusion. cell fusion was frequently a prominent feature of coronavirus infection in uiuo and in uitro. the extent of cell fusion depended upon the virus strain, host cell, and the conditions of infection. the role of e in the induction of cell fusion is indicated by the observation that coronavirus-induced fusion was inhibited by monospecific and monoclonal antibodies to e (holmes, b; collins et al., ; fig. a and b) , and by the finding that treatment of infected cells with tunicamycin inhibited both the synthesis of e and cell fusion (holmes et al., a) . trypsin in the overlay medium enhanced plaque formation of an enteropathogenic bovine coronavirus and several strains of ibv (storz et al., a,b; otsuki and tsubokura, ) . in the presence of trypsin, infection with coronaviruses was associated with cell fusion (storz et al., a,b; toth, ) . similar findings were obtained with a mutant of mhv-s which did not ordinarily induce cell fusion (yoshikura and tejima, ) . trypsin treatment of infected cells also enabled mhv-s to form fusion plaques on otherwise resistant cells, and allowed mhv- to form fusion-type plaques. this effect of trypsin on the ability of coronaviruses to induce cell fusion is similar to that however, if monospecific antibody against the e glycoprotein is added to the culture from hours after infection, the virus-induced cell fusion is prevented (b ). monospecific antiserum against the e glycoprotein does not prevent coronavirus-induced cell fusion; this suggests that e is the fusion factor of mhv(c). ~ . (from k. v. holmes.) ( d and e ) fusion of uninfected l cells by direct action of concentrated mhv-a on the plasma membrane does not occur within hours (d). however, if the virions had been previously treated with trypsin to cleave the e k to e k, then rapid fusion of uninfected cells was observed (e). this and other observations suggest that coronaviruses have a protease-activated cell fusion factor like those found in other enveloped rna viruses. (from l. s. sturman.) observed with the f protein of paramyxoviruses and the ha protein of myxoviruses . sturman and holmes ( ) showed that trypsin treatment of mhv-a resulted in cleavage of k e to k. recently, direct evidence has been obtained for the role of proteolytic cleavage of e in cell fusion. regardless of the multiplicity of infection, efforts to obtain rapid cell fusion with a coronavirus had been unsuccessful until we employed virus which had been treated with trypsin which caused cleavage of e wok) to e ( a + b). cell fusion occurred rapidly after addition of this virus and in the absence of viral protein synthesis (fig. c and d) . . proetolytic cleavage of e may be required for viral infectivity. however, this has not yet been proven. many investigators have studied the effects of trypsin on coronavirus infectivity with mixed results. for example, trypsin treatment at low concentrations ( p/mu enhanced mhv-a infectivity two-to threefold, whereas at high concentrations ( mg/ml), infectivity was reduced by the same relative degree (sturman and holmes, ) . since a large proportion of mhvassociated e was already in the k form, it has not been possible to assess the role of proteolytic cleavage of e as was done with the f, glycoprotein of sendai virus and the ha, glycoprotein of influenza virus . when a source of coronavirus with uncleaved e is identified, the effect of specific cleavage of e on virus infectivity can be demonstrated conclusively. e l is in many ways a unique viral glycoprotein. it performs functions associated with matrix proteins of other viruses, yet it is glycosylated and protrudes from the viral envelope. e l comprises approximately % of the protein of the virion and contains % of the methionine label. at first, e l was difficult to study because of its unusual tendency to aggregate upon heating at °c in sds (figs. and ; sturman, ) . the generation of multimers of e l produced a variety of polypeptide patterns on sds-page depending upon the conditions of sample treatment. aggregation of e l in sds has been described for other strains of mhv (wege et al., ) and for other coronaviruses as well (callebaut and pensaert, ; schmidt and kenny, ) . multiple forms of e l were also distinguished within the - k apparentmolecular-weight range (wege et al., ; siddell et al., b; rottier et al., b; holmes, et al., b) . these may reflect differences in the number or heterogeneity of the oligosaccharide chains on e l . fig. . model of the membrane glycoprotein el. this is a provisional model of the e l glycoprotein which is associated with the coronavirus envelope, showing some of the characteristic features of the molecule. these include the amino-terminal end (n) of the protein which protrudes from the envelope and bears the -linked carbohydrate side chains, and the extensive domains of the e l within and beneath the membrane where they can interact with other e l molecules, with the nucleocapsid, or with e . many of the characteristics of coronaviruses, such as their intracellular budding site, may be determined by the properties of the e l glycoprotein. a model for the e l glycoprotein of mhv-a is shown in fig. . e l appears to possess three domains: . a small k hydrophilic region, containing all of the carbohydrate on the molecule, extends outside the viral envelope and can be removed by pronase of bromelain (sturman and holmes, ) . have shown that for the e l of ibv, this external domain represents the amino-terminal portion of the molecule. we do not know anything about the functions of this glycosylated portion of e l which may have important cooperative effects with e . . a hydrophobic domain resides within the lipid bilayer. disulfide bonds are illustrated in this region because aggregation of e l in sds at °c was markedly enhanced in the presence of reducing agents, which indicated that reduction of disulfide bonds exposed a highly hydrophobic domain (sturman and holmes, ) . . the third domain of e l resides on the inner surface of the envelope and may be associated with the nucleocapsid. as described earlier, after solubilization of the viral envelope with np- at "c, the nucleocapsid could be separated from both e l and e , but at "c, e l reassociated with the nucleocapsid see fig. b ). e l has been shown to bind to rna in the nucleocapsid. however, this interaction was not specific for mhv rna. nucleocapsid structures of other coronaviruses also interacted with e l (garwes et al., ; pocock and garwes, ) . e l appears to be the only glycoprotein required for coronavirus budding, as shown by the fact that in mhv-infected, tunicamycin-treated cells, in which e was made in markedly reduced amounts and not incorporated into virions, mhv virions were formed and released normally. therefore, we infer that e l is responsible for the formation of the viral envelope. e l of ibv contained n-linked, rather than linked, oligosaccharides . glycosylation of the e l of ibv was inhibited by tunicamycin, but virions which lacked e and contained nonglycosylated e l were produced. thus, glycosylation of e l was not essential for formation of ibv virions. the intracellular localization of e on intracytoplasmic membranes ( fig. ) may determine the characteristic budding sites of coronaviruses, which are limited to the endoplasmic reticulum and the golgi apparatus (holmes et al., b; doller et al., ) . e l may also contain sites for interaction with the viral peplomers. some monoclonal antibodies to e l have been shown to exhibit neutralizing activity, but only in the presence of complement (collins et al., ) . the possibility has not been excluded that e l may also have a role in other functions which are presently thought to involve e , such as the interaction of virions with cell receptors, production of a cell-mediated immune response, and induction of cell fusion. v. antigenic relationships among coronaviruses antigenic relationships among ibv, hcv, and mhv strains were first studied in detail, by immunofluorescence, hemagglutination inhibition, and neutralization, by mcintosh et al. ( ) and bradburne ( ) . these investigators established that mhv and some human coronaviruses were antigenically related, while ibv did not cross-react with hcv or mhv. mcintoshs data indicated that there were at least two subgroups of human coronaviruses. hcv-oc , and the very similar oc , showed a close antigenic relationship to mhv, but none of these cross-reacted with hcv- e. bradburne also found an antigenic relationship between oc and mhv; however, he detected some cross-reactivity between hcv- e and both oc and mhv. more recently, schmidt and kenny ( , using rocket immunoelectrophoresis, found no evidence of cross-reactivity between any of the structural proteins of hcv- e and oc , whereas gerdes et al. ( a,b) and hasony and macnaughton ( ) detected some antigenic cross-reactivity between the n proteins of hcv- e and mhv by immunoprecipitation and enzyme-linked immunoassay. using immunofluorescence, pedersen et al. ( ) separated eight mammalian coronaviruses into two antigenically distinct groups. one group consisted of mhv, hev, hcv-oc , and bcv. the second group included tgev, hcv- e, fipv, and ccv. additional anti-genic cross-reactions between other coronaviruses have been detected: the rat coronaviruses, rcv and sdav, were antigenically related to mhv (parker et al., ; bhatt et al., ) , and the rabbit coronavirus which produces pleural effusion disease (rbcv) cross-reacted with both hcv- e and hcv-oc (small et al., ) . another mammalian coronavirus, the procine enteropathogenic coronavirus designated cv , did not cross-react with any other coronavirus tested, including tgev, hev, fipv, ccv, bcv, and ibv . these antigenic relationships are summarized in table vi . recent studies of antigenic relationships among coronaviruses have focused on antigens of individual structural proteins and have employed monospecific and monoclonal antibodies. gerdes, burks, and their co-workers showed that two coronaviruses (sd and sk) isolated from fresh autopsy brain tissue from two patients with multiple sclerosis (burks et al., ) were serologically related to mhv-a and hcv-oc (gerdes et al., a,b) . antisera prepared against each of the four viruses, sd, sk, a , and oc , precipitated all three structural proteins ( e l , e and n) of the other three viruses, demonstrating that the structural proteins of these viruses were antigenically related. gerna et al. ( ) found a high degree of cross-reactivity between e s of oc and bcv. reynolds et al. ( ) showed cross-reactivity between e s of tgev and ccv, and horzinek et al. ) have demonstrated cross-reactivity between analogous e l , e , and n proteins of tgev, fipv, and ccv using radioimmune precipitation, electroblotting, and enzyme-linked immunosorbent assay. at the present time, coronaviruses can be classified into two major subgroups: avian coronaviruses and mammalian coronaviruses. mammalian coronaviruses can be further subdivided into a t least two subtypes. within each subtype, individual virus species can be readily distinguished. there appears to be a great degree of antigenic diversity within some coronavirus species which have been studied extensively, including ibv and mhv. this suggests that there may be considerable antigenic drift in these coronaviruses (see section ,g). coronaviruses have recently emerged as an important group of animal and human pathogens which share a distinctive replicative cycle. some of the unique characteristics in the replication of coronaviruses illustrated in fig. include generation of a ' coterminal-nested set of five or six subgenomic mrnas, each of which appears to direct the synthesis of one protein. two virus-specific rna polymerase activities have been identified. early rna polymerase snythesizes a negative strand of genome size. a double-stranded form has been identified in the infected cell. the subgenomic mrnas are synthesized from a fulllength, negative-stranded template by a second (late) rna polymerase. rna fusion or some other type of rna modification appears to be involved in mrna synthesis. many of the distinctive features of coronavirus infection and coronavirus-induced diseases may result from the properties of the two coronavirus glycoproteins. the intracellular budding site, which may be important in the establishment and maintenance of persistent infections, appears to be due to the restricted intracytoplasmic migration of the e l glycoprotein, which acts as a matrix-like transmembrane glycoprotein. e also exhibits distinctive behavior by self-aggregating on heating at °c in sds and by its interaction with rna in the viral nucleocapsid. the e l of mhv is an o-linked glycoprotein, unlike most other viral glycoproteins. thus, the coronavirus system may be a useful model for the study of synthesis, glycosylation, and transport of o-linked cellular glycoproteins. e is a large, multifunctional, peplomeric glycoprotein which exhibits unusual ph-and temperature-dependent conformational changes. as for the myxo-and paramyxoviruses, it appears that specific proteolytic cleavage of e is necessary for coronavirus-induced cell fusion, and may also be an important determinant of coronavirus pathogenicity. future research must address the difficult problems of determining the functional relationships and their roles in infection and disease. thus, in recent years a large collection of facts about the replication of coronaviruses has been compiled. the interaction of these facts 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selected for high tumorigenicity or transformed by simian virus synthesize heparan sulfate with reduced degree of sulfation t-lymphocyte dependent difference in susceptibility between ddd and c h mice to mouse hepatitis virus, mhv- polyadenylate in the virion rna of mouse hepatitis virus role of proteins in mhv-induced cell fusion we wish to thank the many investigators who generously shared their knowledge, illustrations, and unpublished data with us for this article. excellent secretarial assistance was provided by kathleen cavanagh, mary tribley, and elinore dunphy. this work was supported by grants numbered a and gm from the national institutes of health, and r from the uniformed services university of the health sciences. the opinions expressed are the private views of the authors and should not be construed as official or necessarily reflecting the views of the uniformed services university school of medicine or the department of defense. key: cord- - dfc xjt authors: taguchi, fumihiro; marino, shinji; fujiwara, kosaku title: antigenic differentiation of mouse hepatitis viruses by neutralization test date: - - journal: microbiol immunol doi: . /j. - . .tb .x sha: doc_id: cord_uid: dfc xjt nan mouse hepatitis viruses (mhv) are members of the coronavirus group, which contain a single-stranded polyadenylated rna genome ( , , ) . the discovery of cell lines suitable for mhv replication and the development of assay systems have made it possible to investigate the mode of replication and other important characteristics of mhv ( , , ) . as a result, some information has become available on the molecular aspects of mhv multiplication in cultured cells ( , , , ) . mhv is also known to cause hepatitis and demyelinating disease in mice. variation in pathogenicity and organ tropism among many strains has been shown to occur ( , ) . in addition, studies of recent isolates of mhv from athymic nude mice dying with wasting disease ( ) or from suckling mice with severe diarrhea ( , , ) revealed some new aspects of the pathogenicity of mhv in mice ( , ). however, despite extensive studies of mhv multiplication and many pathological examinations, little is known about the serological differences among mhv strains ( , ) , though cross-reactivity of some strains of mhv with other coronaviruses has been demonstrated ( ) . in this communication, we report serological differences among six established and six freshly isolated strains of mhv, obtained by the neutralization test (nt) which is the only currently available method for the differentiation of closely related mhv strains. the mhv strains mhv-l ( ), mhv- ( ), mhv- ( ), jhm ( ), mhv-s ( ) , and mhv-a ( ) were kindly supplied by dr. ].e. parker, microbiological associates, inc., bethesda, md. mhv-a, mhv-u, mhv-nu ( ), mhv-k ( ), and mhv-nu were isolated from the livers of nude mice with hepatitis, and mhv-d was isolated from the intestines of suckling mice with severe diarrhea ( ) . these viruses were propagated in dbt cells ( ) which were grown in eagle's minimal essential medium (emem, nissui, tokyo) supplemented with % tryptose phosphate broth (tpb, difco, detroit, mich.) and % calf serum. the virus stock used in the nt was prepared as described previously ( ) . antisera to mhv- , jhm, and mhv-s were produced in rabbits by injection of purified viruses emulsified with freund's complete adjuvant, as previously reported ( ) . for nt, a modification of the method of dulbecco ( ) was employed, which was more convenient than the original method and more sensitive than the serum dilution method ( ) for the the differentiation of mhv strains. a preliminary test with the serum dilution method indicated that jhm was distinct from mhv-s. therefore, nts were performed with antisera to jhm and mhv-s and, as antigens, jhm, mhv-s, and mhv- which is clearly different from the other two viruses in terms of cytopathic effect ( , ) . antiserum was diluted in -fold steps with emem containing % tpb (emem-tpb) and . ml of the dilution was mixed with an equal volume of emem-tpb containing x pfu . ml of virus. the mixture was incubated at c for min in a humid c incubator and titrated for remaining infectious virus. the results, shown in fig. i , indicate that anti-jhm serum neutralized jhm very efficiently and mhv- moderately, but did not neutralize mhv-s (fig. ia) , and that the anti-mhv-s serum gave a reverse pattern of neutralization (fig. ib) . these results are compatible with those obtained by the serum dilution method (data not shown). figure also shows that it is sufficient for the differentiation of mhv strains to perform the nt with one antiserum dilution; the antiserum neutralized almost completely the infectivity ( x pfu( . ml) of the homologous virus. as a next step, the mixture of virus and antiserum was tested at various times during the incubation period to determine the equilibrium of neutralization. an aliquot of . ml of -fold-diluted anti-jhm or -fold-diluted anti-mhv-s serum was mixed with . ml of jhm or mhv-s containing x pfu( . ml in emem-tpb and the mixture was incubated at c for to min. figures c and d show that neutralization reached an equilibrium to min after incubation in all cases under these conditions. on the basis of these results, nt was performed with strains as follows. anti-mhv- , anti-jhm, and anti-mhv-s sera were diluted with emem-tpb to the point at which the homologous virus at a titer of x pfu( . ml was neutralized. to each antiserum, an equal volume of the desired mhv strain titering x pfu( . ml was added, and the mixture was incubated at c for min in a humid co incubator and assayed for remaining infectious virus. as shown in fig ( , , ) . high-virulence strains mhv- , mhv- , and mhv-a were neutralized by anti-mhv- serum, but the high-virulence of these strains did not coincide with serological relatedness, because many other low-virulence strains were also neutralized by anti-mhv- serum. this indicates that there is no correlation between virulence and serological type in mhv strains. demyelinating disease can be produced in mice by infection with jhm ( ) and mhv-s ( , ) , but these viruses are serologically different. this also suggests that there is no correlation between the disease picture and serological type of mhv. mhv-d, which is different from all the other strains tested, produces in suckling mice primary enteritis due to infection of intestinal epithelial cells by the orally administered virus ( ) . such enteritis was not produced by the other strains tested in the present study, though enteritis as a result of severe hepatitis has been reported for mhv-s ( ) and mhv- ( ) . in preliminary experiments with anti-mhv-d mouse serum, mhv-s and mhv- were neutralized to the same extent as mhv-d, while mhv- andjhm were not neutralized (data not shown). on the other hand, anti-mhv-s rabbit serum reacted with mhv-s but not with mhv-d. similar data were obtained with mhv-sjcdc isolated from infant mice with diarrhea ( ). anti-mhv-sjcdc serum neutralized mhv-s as well as mhv-sjcdc, but antibodies to mhv-s neutralized mhv-sjcdc less efficiently. it was shown in the same study, by complement fixation, that antiserum to mhv-sjcdc unilaterally reacted with another agent of infant diarrhea, lethal intestinal virus of infant mice (livim) ( ) . these observations suggest the possibility that mhv-d, mhv-sjcdc and livim comprise a group distinct from other mhv strains with regard to serological cross-reactivity and pathogenicity for suckling mice. recently, wege et al determined the antigenic relationships among several mhv strains by nt ( ); their results generally agree with those obtained by hierholzer et al ( ) . they showed that individual strains of mhv were neutralized most effectively by the antiserum to the homologous virus, though cross-reactivity was observed among some strains. the essential difference between our experimental system and those of the other authors ( , ) are the animal species used for the production of antisera and the procedures used for immunization, i.e., we injected purified virus with complete freund's adjuvant into the footpad of rabbits ( ) , while the other authors injected infectious virus into mice by the peritoneal route ( ) . these differences might be responsible for the inconsistency in the results. we thank dr. n. hirano of the department of veterinary microbiology, faculty of agriculture, iwate university, for supplying the mhv-a and mhv-nu strains. a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin. i. isolation and biological properties of the virus a virus related to that causing hepatitis in mice (mhv) a study of the basic aspects of neutralization of two animal viruses, western equine encephalitis virus and poliomyelitis virus a hepatitis virus of mice new strain of mouse hepatitis virus as the cause of lethal enteritis in infant mice replication and plaque formation of mouse hepatitis virus (mhv- ) in mouse cell line dbt culture utility of mouse cell line dbt for propagation of mouse hepatitis virus comparison of mouse hepatitis virus strains for pathogenicity in weanling mice infected by various routes isolation of lowvirulent mouse hepatitis virus from nude mice with wasting syndrome and hepatitis isolation of mouse hepatitis virus from infant mice with fatal diarrhea hepatosplenic myelosis in naturally occurring mouse hepatitis virus infection in the nude mouse an apparently new lethal virus disease of infant mice mouse hepatitis virus a : mrna structure and genetic localization of the sequence divergence from hepatotropic strain mhv- a hepatitis virus complicating studies with mouse leukemia acute hepatitis associated with mouse leukemia. . pathological features and transmission of the disease experimental viral hepatitis pathogenic murine coronaviruses. . characterization of biological behavior in vitro and virus-specific intracellular rna of strongly neurotropic jhmv and weakly neurotropic a viruses mouse hepatitis virus infection as a highly contagious, prevalent, enteric infection of mice isolation and identification of virus-specific mrnas in cells infected with mouse hepatitis virus (mhv-a ) enhanced growth of a murine coronavirus in transformed mouse cells pathogenesis of mouse hepatitis virus infection. the role of nasal epithelial cells as a primary target of low-virulence virus difference in response to mouse hepatitis virus among susceptible mouse strains genetic variation of neurotropic and non-neurotropic murine coronaviruses key: cord- - bf eiix authors: nan title: coronavirus induction of class i major histocompatibility complex expression in murine astrocytes is virus strain specific date: - - journal: j exp med doi: nan sha: doc_id: cord_uid: bf eiix neurotropic strains of mouse hepatitis viruses (mhv) such as mhv-a (a ) and mhv- (jhmv) cause acute and chronic encephalomyelitis and demyelination in susceptible strains of mice and rats. they are widely used as models of human demyelinating diseases such as multiple sclerosis (ms), in which immune mechanisms are thought to participate in the development of lesions in the central nervous system (cns). the effects of mhv infection on target cell functions in the cns are not well understood, but a has been shown to induce the expression of mhc class i molecules in glial cells after in vivo and in vitro infection. changes in class i expression in infected cells may contribute to the immunopathogenesis of mhv infection in the cns. in this communication, a large panel of mhv strains was tested for their ability to stimulate class i expression in primary astrocytes in vitro. the data show that the more hepatotropic strains, such as mhv-a , mhv- , mhv- , mhv- , mhv-d, mhv-k, and mhv-nuu, were potent inducers of class i expression in astrocytes during acute infection, measured by radioimmunoassay. the kb molecule was preferentially expressed over db. by contrast, jhmv and several viral strains derived from it did not stimulate the expression of class i molecules. assays of virus infectivity indicated that the class i-inducing activity did not correlate with the ability of the individual viral strain to replicate in astrocytes. however, exposure of the viruses or the supernatants from infected astrocytes to ultraviolet light abolished the class i-inducing activity, indicating that infectious virus is required for class i expression. these data also suggest that class i expression was induced directly by virus infection, and not by the secretion of a soluble substance into the medium by infected astrocytes. finally, analyses of a /jhmv recombinant viral strains suggest that class i-inducing activity resides in one of the a structural genes. m ouse hepatitis viruses (mhv) are members of the coronaviridae, which cause gastrointestinal, neurological, and respiratory diseases in a wide variety of mammalian species ( ) ( ) ( ) ( ) ( ) ( ) ( ) . although some mhv strains, such as a and mhv- , can cause both gastrointestinal and neurological disease in mice and rats, many tend to induce pathology that is restricted to either system, prompting their classification as hepatotropic or neurotropic. there has been considerable interest in the study of the more neurotropic strains of mhv, such as jhmv and a , because of their ability to produce demyelinating lesions that resemble the demyelinating plaques observed in the human neurological disease, multiple sclerosis (ms). in addition, mhv and human coronavirus iso- lates are capable of inducing demyelination in primates ( ) , and coronavirus rna and antigen have been detected in demyelinating lesions in the brains of ms patients ( , ) . as coronaviridae, all mhv strains are enveloped viruses that contain a single-stranded, positive-sense ilna genome of -kb that is expressed as or mrnas encoding both structural and nonstructural proteins ( ) . the structural proteins have been well characterized, and include the nucleocapsid (n) protein, which interacts with the viral rna, and two envelope glycoproteins, the spike (s) and membrane (m) proteins. the s protein forms the virion surface spikes and is responsible for binding the cellular mhv receptor, inducing cell-to-cell fusion and providing a target for neutralizing antibodies. it is the least conserved of the structural proteins among the mhv strains ( ) ( ) ( ) ( ) ( ) . m interacts with the n protein-r.na comply, and may be involved in virus assembly. it is the most conserved structural protein among the mhv. a third envelope glycoprotein, hemagglutinin-esterase (he), has been identified in mhv-s and several jhmv isolates, and may participate in the pathogenesis of jhmv infection in the central nervous system (cns) ( , ) . nonstructural proteins include an rna-dependent rna polymerase, and three additional proteins that do not appear to be required for viral replication, but whose functions and biological activities have not been identified. after intracerebral (i.c.) inoculation in mice and rats, the parent strains of both jhmv and a cause an acute encephalomyelitis and demyelination from which a varying number of animals survive to exhibit chronic demyelination ( ) ( ) ( ) ( ) ( ) ( ) ( ) ) . the severity of the disease varies with the individual virus strain and with the age, genetic background, and immune status of the infected host. the neuropathological features of acute infection can be somewhat distinguished from those of chronic disease by a prevalence of gray matter involvement in which virus replicates in neurons, oligodendrocytes, and astrocytes to cause extensive cns damage ( ) ( ) ( ) ( ) . in chronic disease, lesions are more prevalent in the white matter and consist of primary demyelination with axonal sparing ( , ) . infectious virus is rarely isolated during chronic jhmv disease, though viral rna can be detected by reverse transcriptase (rt)-pcr in the brains of infected mice as late as yr post infection (p.i., fleming, j., personal communication). viral antigen and rna tend to be restricted to astrocytes in chronic infection ( , ( ) ( ) ( ) ( ) , though there is evidence that neurons are also affected ( ) . this pattern of white matter involvement is also characteristic of infection with attenuated or mutant strains of a ( , ) or jhmv ( , ( ) ( ) ( ) , which cause demyelination in the relative absence of encephalitis. in vitro, jhmv and a cause a lytic infection in primary oligodendrocyte cultures that is rapidly selflimiting, but both virus strains can establish a productive and relatively nonlytic, acute, and long-term chronic infection in mixed glial cell cultures and in cultures enriched for astrocytes ( ) ( ) ( ) . these data support early reports that acute demyelination is the result of virus-mediated oligodendrocyte death ( , ) , but more importantly, they also suggest that the astrocyte plays an important role in acute encephalitis and chronic demyelination. currently, there is very little known about the ability of mhv strains to influence specific activities of the host cells that they infect. early reports indicate that a infection enhances the expression of mhc class i molecules in primary cultures of murine glial cells ( ) and that the enhancement also occurs in the brain after i.c. infection in c b / mice ( ). since class i molecules play a key role in the interaction between infected target cells and cd + cytotoxic t cells (ctl; , ) , any change in their expression after virus infection has potential implications for the outcome of the infection. cells in the cns generally express little, if any, constitutive class i antigen in vivo, but astrocytes have been consistently reported to express both class i and class ii antigens in vitro after the addition of ifn- ' and/or tnf-ot to the culture medium ( ) ( ) ( ) ( ) ( ) ( ) . several reports indicate that astrocytes are capable of acting as antigen-presenting cells for in vitro antigen-or a ospecific cd § and cd § t cell responses ( , ) , which suggests that they also have potential to participate in immune responses occurring within the confines of the cns. since clearance of infectious jhmv from the cns requires class i-restricted cd + t cells ( , ( ) ( ) ( ) , it is of particular interest to characterize the effect of mhv infection on class i expression in one of its principal cellular targets. in this communication, we have examined the ability of a panel of mhv strains to induce class i expression in cultures enriched at least % for astrocytes. the data indicate that class i expression occurred in response to all the mhv strains tested except jhmv and virus strains derived from jhmv. additional studies show that class i expression requires the presence of infectious virus. finally, the testing of a /jhmv recombinant viral strains suggests that the class i-inducing activity resides in the ' end of the a genome, possibly in one of the genes encoding the structural proteins. primary astrocyte cultures. astrocytes were isolated from mixed glial cell cultures prepared from the brains of newborn c b / mice (bantin and kingman, fremont, ca) at postnatal day - according to mccarthy and devellis ( ) . briefly, single cell suspensions were prepared from cerebri dissected free of brain stems and cerebelli, plated at - brains per t- flask and allowed to grow to confluence at - d in vitro. culture medium consisted of dmem/ham's f ( : ; jrh biosciences, lenexa, ks) supplemented with % fcs (gemini bioproducts, inc., calabasas, ca), mm hepes, . mm t-glutamine, and penicillin/streptomycin ( u/ml- ~g/ml). at confluence the cultures were mechanically shaken to dislodge microglia and oligodendroglia, resulting in preparations enriched % or greater for cells staining for glial fibrillary acidic protein (gfap). immunoperoxidase or immunofluorescent staining (described below) revealed that the cell preparations contained ,- - % of cells of microglia/macrophage lineage, expressing f / , t- , the mouse equivalent of human common leukocyte antigen (cd ), and/or mac- surface markers. coronavirus strains and infection. table presents a summary of the mhv strains used and the principal types of diseases they cause in mice. the derivation, propagation, and sources of mhv-a- , jhm-dl, jhm-ds, mhv- , mhv- , mhv- , mhv-d, mhv-k, and mhv-nuu have been described ( ) . the neutralization-resistant mhv- strains . -v- and . / . -v- ( , ) were the kind gift of dr. john fleming (university of wisconsin, madison, wi). the development and properties of jhm-x and the jhm/a recombinant mhv strains have been reviewed ( ) . all virus strains were propagated using the murine astrocytoma dbt as previously described ( ) . virus titers were determined for each virus preparation using dbt as indicator cells. infectious center assays. the number of infected astrocytes was determined on day p.i. to provide a measure of the relative efficiency of infection by several mhv strains. briefly, astrocytes were trypsinized into single cell suspensions and, after washing, were plated on dbt cell monolayers at . , , , , and , cells/ mm petri dish. cells were allowed to attach for h at ~ before the addition of agarose at . % in ikpmi supplemented with % heat-inactivated fcs, mm hepes, and penicillin/streptomycin. plaques were counted after h incubation at ~ virus inactivation by exposure to ultraviolet light. inactivation of virus was accomplished by exposure to uv light under a uvp transilluminator (uvp inc.,; san gabriel, ca) at mw/cm z for the possible presence of oligodendrocytes was identified using polyclonal rabbit antigalactocerebroside (gal c, a gift from m. smith, stanford university, stanford, ca). finally, viral antigen was identified using j. . , a mab specific for the n protein of jhmv that crossreacts with all of the mhv strains used in this study ( ) . rta. the expression of class i molecules was measured in astrocytes cultured in flat-bottomed -well plates at a density of cells/well and infected with various virus strains at a multiplicity of infection (m.o.i.) of - . mock infected cells served as controls. on day or p.i., cells were washed twice using a wash buffer of . % bsa in . m pbs before the addition of /a of the appropriate mabs in triplicate. after a rain incubation at room temperature, cells were washed three times, followed by the addition of , cpm/well of si-labeled protein a ( /~ci//zg; icn biomedicals, costa mesa, ca). at the end of a second min incubation, cells were extensively washed in pbs to remove unbound radiolabeled protein a and detached with . % trypsin- . % edta (jrh biosciences). data are presented as cpm bound radioactivity + sd, or percent increase in class i expression in infected cells compared with that in uninfected controls, corrected for background binding in the absence of antibody. a result was considered positive when the percent increase in expression was % or greater. nonspecific staining was identified by the inclusion of kd-specific mab sf - . . . in some experiments, supernatants from infected cells were substituted for virus and ria performed to detect class i d later. immunoperoxidase and immunofluorescent staining. cell phenotypes and the number of infected ceus were determined by immunoperoxidase staining in astrocytes cultured in tissue culture chamber slides. avidin-biotin immunoperoxidase staining kits (vectastain; vector laboratories inc., burlingame, ca) were used according to the manufacturer's instructions as previously described ( ) . cells were infected with virus d after plating and fixed on day or p.i. in acetone/methanol ( : ). the number of positively stained cells in each well was determined in three fields/well at a magnification of x and reported as the percentage of the number of total cells in the same fields. background staining was determined in cells stained in the absence of primary antibody. class i expression was also evaluated by facs | using an indirect immunoftuorescent staining procedure. briefly, single cell suspensions of astrocytes were adjusted to to cells/tube before the addition of class -specific primary antibodies. fitc-labeled goat anti-mouse igg f(ab) was used as secondary antibody (jackson immunoresearch laboratories, west grove, pa). fluorescence data were collected on x to viable cells, indicated by forward light scatter intensity, using a facstar cell sorter (becton dickinson & co.). again, background fluorescence was determined in cells stained in the absence of primary antibody. detection oflnterferons. the possible presence oflfn in the supernatants of infected astrocytes was determined by bioassay in a vesicular stomatitis virus (vsv) neutralization assay using vital dye uptake as a spectrophotometrically measured endpoint in l cells ( , ) . supernatants were collected from infected and control uninfected astrocytes on day and p.i., uv irradiated, and added in triplicate to l cells plated at s cells/well in -well plates. after a -h incubation at ~ and % co , supernatants were aspirated and cells infected with vsv (indiana strain) at approximately tissue culture infectious doses (tcids )/well. control wells included uninfected l cells (cell control) and vsv-infected l cells cultured in the absence of interferon (virus control). at - h p.i., when cpe was maximum in the control wells, cells were stained for h at ~ with . % neutral red. they were then lysed to release retained dye using . m hc in % ethanol, and ods measured at nm in an automated microelisa reader (dynatech laboratories, inc., chantilly, va). ifn concentrations were deduced by comparison of sample ods with those of standard concentrations of recombinant mouse ifn- ' (genzyme corp., cambridge, ma) and murine ifn-c~// (lee biomolecular, san diego, ca). immunoneutralization was also used to identify the possible presence of ifn in uv-irradiated supernatants of a qnfected astrocytes. for this purpose, rat monoclonal anti-mouse ifn- ' antibody (hybridoma r - a ; atcc hb ) or rabbit polyclonal anti-mouse ifn-a/ (lee biomolecular) was added to the astrocytes before the addition of a , and class i expression measured by ria on day p.i. anti-ifn- ' ( - /zg/ml) completely inhibited the protective activity of u/ml of recombinant mouse ifn- ', whereas - u/ml anti-ifn-a/r effective in neutralizing u/ml purified mouse ifn-c~//~. detection of tne tnf activity was measured in a cytotoxicity assay using actinomycin d-treated l fibroblasts as targets, again with vital dye uptake as a spectrophotometric endpoint. serial -fold dilutions of uv-irradiated supernatants, or -fold dilutions of murine recombinant tnf-ot (genzyme corp.), as a standard were added to l monolayers in -well plates in the presence of actinomycin d ( #g/ml; sigma chemical co., st. louis, mo). after an -h incubation at ~ % co , neutral red was added as described for the ifn assay, and od read at nm. to investigate the effects of coronavirus infection on mhc class i expression in primary astrocytes~ cultures were routinely used at - d in vitro, or - d after mechanical shaking to remove oligodendrocytes and microglia. fig. shows that a induced abundant class i expression in astrocytes, measured by ria using the kb-specific mab af . . . . class i expression was routinely measured on days and p.i. after infection at an m.o.i, of , though it was detectable within - h p.i. k b was preferentially expressed over d b after a infection. this was not due to a lack of inducibility of the d b molecule, since both k b and d b were expressed spontaneously with time in culture, and also in response to u/ml of ifn- /(data not shown). under these conditions, k b was expressed earlier than d b, and usually at significantly higher levels. in infected astrocytes, the class i-inducing activity of a was consistently - % more potent than the single dose of u/ml of ifn- ' (fig. ) . finally, facs | analyses revealed that k b was expressed on '~ % of the astrocytes in the infected cultures. by contrast, the large plaque morphology variant of jhmv designated jhm-dl did not stimulate class i at all, or at best, stimulated minimal expression at - % over that observed in uninfected ceus ( fig. and ) . these data indicate that the ability to induce class i expression is not a general property of mhv strains. to determine whether the lack of ability to stimulate class i expression in astrocytes is specific for jhmv, a panel of virus strains derived from wild-type jhmv or from jhm-dl were tested. the results indicate that none of the jhmv strains isolated in our laboratories stimulated class i ( table ). in addition, jhm-x, which was derived from jhmv passage in japan and has been shown to have extensive deletions in the s gene ( ), did not affect dass i expression. by contrast, all of the viral strains in a panel of more hepatotropic isolates of mhv, including mhv-nuu, mhv-k, mhv-d, mhv- , mhv- , and mhv- , were effective stimulators of class i expression. the magnitude of stimulation, expressed as percent change relative to uninfected cells, varied with the individual viral strain - fold over that in uninfected cells. a and mhv- were the most potent inducers of the class i k b molecule, and both inconsistently induced a low-level expression of d b. it should be mentioned that the kd-specific mab sf - . . occasionally yielded a low level of nonspecific binding; however, it was significantly lower than the specific binding observed with the kb-specific antibody. determine whether a specific a gene or genes are responsible for the class i-inducing activity, a panel of recombinant viruses between a and jhmv that retain various portions of a sequences were tested. the results, represented in fig. and summarized in fig. , indicate that all strains that retain a sequences at the ' end of the recombinant genome were potent class i inducers. thus, ca and ca , which retain • - % jhmv sequences at the ' end, were completely devoid of class i-inducing activity. this end of the mhv genome contains the genes encoding the structural coronavirus induction of class i mhc expression is virus strain specific proteins s, m, and n in addition to two nonstructural proteins, which suggests that the class i-inducing activity resides in one of these proteins. often, though not exclusively, the most potent inducers of class i were strains that retain the highest percentage of a sequences at the ' end, while those retaining the least a character, or exhibiting more than one crossover site for recombination, were significantly less potent. in addition, it was observed in three out of four experiments that rl was • % less potent than el , as illustrated in fig. b. el retains slightly more a sequences in the s gene than rl . this observation suggests that class i-inducing activity may involve s. however, it was not possible to attribute the class i-stimulating activity to an individual gene or gene product, since none of the recombinants had crossover sites that sufficiently isolated individual a from jhmv genes. however, it was clear that ifjhmv sequences were retained at the ' end, class -inducing activity was abolished, and that ' retention of a sequences did not salvage it. the ability_ of mhv to induce class i is not dependent on replication efficiency. in our laboratory, a grows to higher titer in vitro than the jhmv strains in dbt cells, often ex-ceeding jhmv growth - -fold. in addition, recombinant viruses containing a leader predominate over those containing jhmv leader, suggesting that a leader provides a growth advantage ( ) . thus, it was important to determine whether the lack of class i inducing activity by jhmv was due to poor replication efficiency in primary astrocytes. for this purpose, the number of astrocytes staining for viral antigen was determined by immunoperoxidase staining, and the yield of infectious viral particles/cell was measured in infectious center assays. the data, presented in table , indicate that on day p.i., % of the jhm-dl-infected astrocytes were positive for viral antigen, compared with % of cells infected with a . in addition, cells infected with the recombinant virus ca , which retains ' jhmv and ' a sequences and does not stimulate class i expression, showed % antigen positive staining. cells infected with ca produced the highest level of infectious virus at . plaques/cell, in spite of its inability to stimulate class i expression (table ) . by contrast, el , which was an effective class i inducer, replicated at relatively low efficiency at . plaques/cell. thus, class -inducing activity was not a function of the ability of the virus to infect or replicate in pri- infected glial cell cultures is not a direct consequence of infection, but is instead due to the release of a soluble factor into the medium that requires continual virus production ( , , ) . the presence of a non-ifn-like soluble factor was demonstrated in supernatants from a -infected glial cells that had been exposed to uv light to inactivate the virus. similarly, our data show that uv-inactivated a and a like recombinant coronavirus strains are not able to induce class i activity in purified astrocytes (table ). virus inactivation in the uv-treated virus preparations was confirmed by plaque assay using dbt cells (data not shown). however, class i-inducing activity was not detected in the supernatants collected on days and p.i. from a -infected astrocytes and exposed to uv light to inactivate the virus (fig. ) , while supernatants that were not exposed to uv light and thus, contained infectious virus, were able to induce class i. induction was not inhibited by the addition of antibodies specific for ifn-'y or ifn-ot/~ to the astrocytes before a infection (table ). finally, there was no evidence of the presence of tnf-ot in uv-treated supernatants of a -infected astrocytes (data not shown). these data suggest that class i induction is a direct consequence of a infection itself, and does not occur indirectly as a result of the release of a soluble class i inducer into the astrocyte medium. data are also included for uninfected astrocytes exposed to ifn-y ( u/ml) for h before assay on day p.i. a and b are two experiments that are representative of four experiments yielding similar results. in this communication, we report that the ability of a to stimulate mhc class i expression during acute infection in primary murine astrocytes, previously observed by suzumura et al. ( , , ) , is not an inherent property of mhv strains. it is interesting to note that the strains that did not stimulate class i are derivatives of the highly neurotropic mhv- , or jhmv strain, whereas most of the mhv strains that did are the more hepatotropic strains. however, class i-inducing in jhmv was also not due to poor replication efficiency in astrocytes, since jhmv infected the same percentage of cells and produced similar levels of infectious virus as the class i-inducing mhv strains (table ) . thus, some other mhv characteristic must be responsible for class i-inducing activity, or the lack thereof. since the expression of class i genes is regulated by interferons ( ) , it seemed possible that class i induction might correlate with the ability of mhv strains to stimulate ifn production. this possibility gains credibility from reports that jhmv is a notoriously poor inducer of interferon after in vitro ( ) and in vivo ( ) infection. however, suzumura et al. ( ) did not detect ifns in uv-treated supernatants from a -infected mixed glial cells in spite of the presence of class i-inducing activity, and our findings indicate that a -induced class i expression was not inhibited by antibodies to ifn-a/ or - ' (table ). unlike suzumura et al. ( ), we did not find that class i-inducing activity was retained after uv-inactivation of a or the a - ike recombinant mhv strains (table ) , and it was not detected in supernarants from a -infected astrocytes that were uv-treated to inactivate infectious virus (table and fig. ). in addition, we have recently found that class i expression is no longer observed when a replication is controlled by the culture of persistently infected astrocytes in the presence of mhvspecific polyclonal antiserum, in spite of the persistence of viral protein and rna ( ) . overall, our data suggest that class i induction by mhv in astrocytes requires infectious virus and does not involve the secretion of a soluble factor. in this respect, it is interesting to note that the class i-inducing activity originally reported by suzumura et al. ( , ) was also dependent on continuous virus production in glial cell cultures prepared from the brains of infected mice ( ) . however, it is possible that our lack of detection of class i-inducing activity in the supernatants could reflect culture, or other technical conditions. although it was not possible to attribute class i-inducing activity to a single gene or gene product using the a /jhmv recombinant virus strains, the data clearly indicate that class i-inducing activity maps to the ' end of the a genome, which contains the genes encoding the primary structural proteins s, m, and n, and two poorly understood nonstructural proteins. however, there are some indications that s is the most likely candidate. for example, recombinant strains which retain more a sequences in the s gene tended to be more potent than those retaining less a , or morejhmv character. in addition, the s gene shows the greatest antigenic divergence of the structural proteins between a and jhmv ( ) ( ) ( ) ( ) ( ) and so might be more likely to exhibit differences in activity. this possibility is supported by data indicating that a andjhmv differ considerably in their ability to cause receptor-independent fusion and syncytium formation ( ) . in addition, preliminary data suggest that a and jhmv may show selective binding to the cellular receptors for mhv ( ) , which are members of the carcinoembryonic antigen (cea) family of proteins ( ) . the mechanism by which a , mhv- , and the a like recombinant mhv strains induce class i expression in astrocytes is currently unknown. massa et al. ( , ) have shown that mhc class i promoter activity is upregulated in astrocytes treated with ifn-y and that the upregulation is associated with an increase in the binding activities of the mhc class i regulatory element (mhc-cre) and the ifn consensus sequence (ics). whether or not these regulatory elements are engaged by a infection remains to be determined. perhaps one of the more interesting findings in these studies is the prevalence of k b expression over that of d b after mhv infection, and to a lesser extent, ifn- " treatment. differential modulation of h- k and h- d molecules has been reported to follow in vitro jhmv infection in mouse brain endothelial cells, and the data indicate that the nature of the regulation was dependent on the mouse strain and did not necessarily correlate with susceptibility to jhmv-induced cns disease ( ) . thus, k was decreased and d increased in endothelial cells from susceptible balb/c mice, but both were upregulated in susceptible b .s and (balb/c x sjl)f and resistant sjl endothelial cells. differential expression of k and d molecules has not always been observed after virus infection of neural cells; both k and d molecules appear to be equally enhanced by west nile virus infection in astrocytes from cba/h mice ( ) , and the murine neuroblastoma c does not show differential k and d expression when persistently infected with measles virus ( ) . however, the possibility that differential modulation of class i molecules may be linked to cns disease, or may be a marker of susceptibility to virus-induced cns disease, is suggested by studies using theiler's murine encephalomyelitis virus (tmev; , ). after i.c. tmev inoculation, resistant b mice showed minimal class i expression in the cns that did not differ between k and d, whereas susceptible b .q and b .rbq mice showed a greater increase in d expression compared with that of k ( ) . the inability of jhmv to stimulate class i expression in c bl/ astrocytes in the current studies was not reported for astrocytes from balb/c, cxj- , sjl, and b .s mice by joseph et al. ( ) . in these experiments, jhmv infection at an m.o.i, of . was followed by a two-to threefold increase in the expression of h- k molecules, measured by facs | analyses, but astrocytes from c bl/ mice were not tested. the disparity in our findings may again reflect differences in the regulation of class i expression that are mouse strain specific. however, it is also possible that the disparity may be due to the cellular composition of the astrocyte cultures used for infection. in their report, joseph et al. ( ) , did not indicate whether or not they purified the astrocytes from mixed glial cell cultures. in our hands, mixed glial cells that have not been subjected to mechanical shaking for purification of astrocytes show up to % contamination by microglia, or cells staining for macrophage/monocyte surface antigens (data not shown). microglial cells readily express class i molecules both in vivo and in vitro ( ) ( ) ( ) , and so might be expected to express class i after jhmv infection. the cultures used in our experiments routinely show % or greater purity, containing - % microglial cells, and "~ % express class i after a infection. thus, class i expression in these cultures occurs primarily in astrocytes, with little, if any contribution by microglia. finally, it should also be mentioned that class i expression in astrocytes, like that in endothelial cells, does not appear to be linked to susceptibility to jhmv-induced cns disease, since both balb/c and c bl/ develop encephalitis and demyelination in spite of their differences in class i inducibility after jhmv infection. in addition, astrocytes from sjl mice express class i after jhmv infection in spite of being resistant to cns disease. the different effect of a and jhmv on class i expression in astrocytes suggests that class i may play different roles in their pathogenesis. as discussed by maudsley et al. ( ) , the classical picture of mhc expression after virus infection is that of an increase that is most likely mediated by the release of ifns by infiltrating immune cells, and is thought to facilitate the ability of t cells to recognize the infected cells and control or eliminate the infection. unfortunately, clearance of virus from infected tissue by ctls is often accompanied by significant cellular destruction, which is not well tolerated in tissues with limited regenerative capacity, especially the cns. thus, the ability of a virus to stimulate class i expression in host cells may facilitate a rapid ctl response that in turn accelerates tissue destruction originally begun by virus-mediated cell lysis ( ) . this may be the case for a infection, since the a strains used in these experiments cause a severe encephalitis after i.c. inoculation that begins on day p.i., and results in death of the majority of infected mice by day - p.i. (our unpublished data). however, it is not known if the infiltration of t cells into the cns after i.c. a infection is associated with the onset of encephalitis. by contrast, jhmv causes a similar clinical dis-ease, but the onset of encephalitis is delayed in comparison with that of a , beginning on day - p.i. ( ) . death occurs on day - at a similar incidence. the onset of encephalitis coincides with the appearance of immune cells in the cns, which, after jhm-ds infection, peaks on days - p.i. ( ) . it is possible that class i expression is upregulated at this time by the secretion of interferon by the infiltrating immune cells, facilitating jhmv-specific, ctl-mediated tissue destruction. thus, the difference in the ability of jhmv and a to stimulate class i expression in astrocytes may contribute to the speed of disease progression in the cns. it is interesting to note that fazakerley et al. ( ) reported that the jhmv variant, v a . , is neuroattenuated relative to parental jhmv by its slower rate of spread in the cns of balb/c mice. variant v a . differs from parental jhmv by the deletion of amino acids in one of the subunits of the s protein ( , ) , suggesting that s plays an important role in the rate of virus spread in the cns. in this respect, it would be of interest to test the ability of neuroattenuated a strains to stimulate class i expression in astrocytes. in conclusion, we present data indicating that significant differences exist among mhv strains in their ability to stimulate class i expression in murine astrocytes, and that these differences may contribute to their ability to cause cns disease. since class i expression has been reported on astrocytes in the brains of ms patients ( ) , the data have implications for understanding the role of class i in human cns disease. we wish to thank michael lai for the recombinant virus strains and steve stohlman, michael lai, and minnie mcmiuan for helpful critiques of the data and manuscript. we are also grateful for help with the manuscript provided by sonia garcia. a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin pathogenesis of demyelination induced by a mouse hepatitis virus ohm virus) pathogenic murine coronaviruses. iii. biological and biochemical characterization of temperature-sensitive mutants of jhmv hydrocephalus in suckling rats infected intracerebrally with mouse hepatitis virus mhv-a in vivo and in vitro models of demyelinating diseases chronic central nervous system demyelination in mice after jhm virus infection relapsing subacute demyelinating encephalomyelitis in rats during the course of eoronavirus jhm infection coronavirus infects and causes demyelination in primate central nervous system coronavirus isolates sk and sd from multiple sclerosis patients are serologically related to routine coronaviruses a and jhm gilmore et al. and 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localization of wild type and mutant mouse hepatitis virus (jhm strain) antigens in cns tissue by fluorescence, light and electron microscopy infection of the basal ganglia by a murine coronavirus sequential infection of glial cells by the murine hepatitis virus jhm strain (mhv- ) leads to a characteristic distribution of demyelination temperature-sensitive mutants of mouse hepatitis virus produce a high incidence of demyelination cell tropism and expression of mouse hepatitis viruses (mhv) in mouse spinal cord cultures the astrocyte is a target cell in mice persistently infected with mouse hepatitis virus the v a . envelope glycoprotein deletion mutant of mouse hepatitis virus type- is neuroattenuated by its reduced rate of spread in the central nervous system restricted repli- cation of mouse hepatitis virus a in primary mouse brain astrocytes correlates with reduced pathogenicity induction of demyelination by a temperature-sensitive mutant of the coronavirus mhv-a is associated with 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as perpetuators but not initiators of cd § t cell responses in the central nervous system t-cell-mediated clearance of mouse hepatitis virus strain jhm from the central nervous system effective clearance of mouse hepatitis virus from the central nervous system requires both cd + and cd § t cells protection of mice from a lethal coronavirus infection in the central nervous system by adoptive transfer of virus-specific t cell clones preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue rna recombination in animal and plant viruses dye uptake methods for assessing viral cytopathogenicity and their application to interferon assays automated bioassay of interferons in micro-test plates high-frequency kna recombination of murine coronaviruses induction of glial cell mhc antigen expression in neurotropic coronavirus infections: characterization of the h- -inducing soluble factor elaborated by infected brain cells induction of mhc class i antigens on 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antigen expression on mouse brain endothelial cells by mhv- infection flavivirus infection up-regulates the expression of class i and class ii major histocompatibility antigens on and enhances t cell recognition of astrocytes in vitro persistent measles virus infection enhances histocompatibility complex class i expression and immunogenicity of murine neuroblastoma cells differential expression of h- k and h- d in the central nervous system of mice infected with theiler's virus coexpression of class i major histocompatibility antigen and viral rna in central nervous system of mice infected with theiler's virus: a model for multiple sclerosis regulation of mhc class i and ii antigens on cerebral endothelial cells and astrocytes following mhv- infection perivascular microglia cells of the cns are bone-marrow derived and present antigen in vivo mhc antigen expression on bulk-isolated macrophage-microglia from newborn mouse brain: induction of ia antigen expression by y-interferon isolation and direct characterization of resident microglial cells from the normal and inflamed central nervous system modulation of mhc antigen expression by viruses and oncogenes viral persistence characterization of brain-infiltrating mononuclear cells during infection with mouse hepatitis virus strain jhm neutralization-resistant variants of a neurotropic coronavirus are generated by deletions within the amino-terminal half of the spike glycoprotein multiple sclerosis: relevance of class i and class ii mhc-expressing cells to lesion development key: cord- -epylkxdx authors: das sarma, jayasri title: a mechanism of virus-induced demyelination date: - - journal: interdiscip perspect infect dis doi: . / / sha: doc_id: cord_uid: epylkxdx myelin forms an insulating sheath surrounding axons in the central and peripheral nervous systems and is essential for rapid propagation of neuronal action potentials. demyelination is an acquired disorder in which normally formed myelin degenerates, exposing axons to the extracellular environment. the result is dysfunction of normal neuron-to-neuron communication and in many cases, varying degrees of axonal degeneration. numerous central nervous system demyelinating disorders exist, including multiple sclerosis. although demyelination is the major manifestation of most of the demyelinating diseases, recent studies have clearly documented concomitant axonal loss to varying degrees resulting in long-term disability. axonal injury may occur secondary to myelin damage (outside-in model) or myelin damage may occur secondary to axonal injury (inside-out model). viral induced demyelination models, has provided unique imminent into the cellular mechanisms of myelin destruction. they illustrate mechanisms of viral persistence, including latent infections, virus reactivation and viral-induced tissue damage. these studies have also provided excellent paradigms to study the interactions between the immune system and the central nervous system (cns). in this review we will discuss potential cellular and molecular mechanism of central nervous system axonal loss and demyelination in a viral induced mouse model of multiple sclerosis. demyelination is the process by which axons lose their normal insulating myelin. several central nervous system demyelinating disorders have been described in humans including multiple sclerosis, neuromyelitis optica (devic's disease), acute disseminated encephalomyelitis, and osmotic demyelination (central pontine myelinolysis, extrapontine myelinolysis). multiple sclerosis (ms) is a chronic, progressive, or relapsing and remitting demyelinating disorder that affects the central nervous system (cns) specifically and ranks as a major cause of nervous system disability in young adults aged to [ ] [ ] [ ] . it has long been hypothesized that oligodendrocytes (olgs) and/or the myelin sheath are the target of immune system-mediated destruction in ms. recent studies have demonstrated that axonal damage [ , ] also occurs and is likely to be a major component of long-term disability observed in ms. the etiology of ms is not very clearly known but the process of demyelination is believed to involve a tcell-mediated phenomenon that may be triggered by one or more viral infections. clinical studies show that infectious agents encountered during adolescence prime the diseases, which appear clinically later in the adult after a variable period of quiescence [ , ] . despite numerous attempts, a particular responsible virus has not been identified. our research has focused on understanding the mechanisms of demyelination. we use the mouse hepatitis virus (mhv) model of murine demyelination in order to dissect the inflammatory and molecular mechanisms of viral-induced demyelination. the most important evidence that ms might be infectious is based on the fact that the brain and cerebrospinal fluid (csf) of more than % ms patients contain increased amounts of igg, manifest as oligoclonal bands (ogbs) [ ] . there are other but not many cns disorders in which increased amounts of igg and ogbs are found. all those diseases are inflammatory, and most are infectious. furthermore, when the specificity of the increased igg and ogbs in interdisciplinary perspectives on infectious diseases those diseases was studied, the igg in every condition was shown to be antibody directed against the agent that caused disease. for example, the oligoclonal igg found in subacute sclerosing panencephalitis (sspe) brain and csf is directed against measles virus [ ] not herpes simplex virus or mumps virus; in cryptococcal meningitis, the igg is directed against cryptococcus and not another fungus such as candida [ ] . these findings provide a rationale for the notion that the oligoclonal igg in ms brain and csf is antibody directed against the agent that causes disease. moreover, a number of studies have documented viruses as triggers for ms. the fact that viruses are associated with multifocal leukoencephalopathy (pml), sspe, and postinfectious encephalitis explains the continued interest in viruses as triggers for ms [ , ] . studies in herpes virus have been quite extensive, especially in human herpes virus (hhv- ) [ ] [ ] [ ] and epstein-barr virus (ebv) [ , ] . herpes viruses are of particular interest because of their neurotropic, ubiquitous nature and their tendency to produce latent, recurrent infections. however, there are no studies to date that clearly demonstrate the underlying pathophysiology, correlating the trigger of viral infection with induction of the demyelination process. one key to a better insight into the molecular and cellular mechanisms of ms lies in the development of a reliable animal model. towards this goal several experimental animal models have been developed to study the mechanisms of virus-induced demyelination. mouse model studies using theiler's murine encephalomyelitis virus (tmev) [ , ] infection and neurotropic strains of mouse hepatitis virus (mhv) infection have given useful information on putative ms mechanisms [ ] [ ] [ ] [ ] . in virus-induced demyelination, infection is a necessary prerequisite for demyelination, and the cause/effect relationship makes this model an attractive platform for exploring the etiology and pathogenesis of demyelinating diseases. chronic viral-induced dem-yelination is associated with viral persistence [ , ] and concomitant enhancement of major histocompatibility complex class i antigens [ ] [ ] [ ] [ ] [ ] . these features parallel many of the pathologic findings seen in ms, in contrast to monophasic viral or postviral human demyelinating diseases such as acute disseminated encephalomyelitis (adem) [ ] and pml [ ] . infection of susceptible strains of mice with some strains of tmev or mhv causes biphasic disease of the cns, consisting of early acute disease and late chronic demyelinating disease that appears to days post infection (p.i) [ ] . as early as days p.i, infected mice develop late chronic demyelinating disease with extensive demyelinating lesions of the white matter and cell infiltrates in the spinal cord, consisting primarily of cd + t cells and cd + t cells, some monocots/macrophages, and a few b cells and plasma cells. in general, viral infection causes damage to the nervous system by two mechanisms: direct infection of neural cells and immune-mediated tissue injury (immunopathology). virtually all types of immune response have been proposed to play important roles in the pathogenesis of tmev-induced viral clearance and demyelination. it has been proposed that demyelination is caused by an immunemediated mechanism, in which cd + th cells mediate a delayed type of hypersensitivity response with epitope spreading [ ] . tmev-induced cd + t cells have been suggested to function as autoreactive cytotoxic cells or regulatory cells [ ] . antibody against tmev cross-reacts with galactocerebroside, and passive transfer of anti-tmev antibody can augment demyelination in experimental allergic encephalomyelitis (eae) [ ] . intracerebral inoculation with a tmev-infected macrophage cell line induces acute focal demyelination [ ] , and depletion of macrophages ameliorates tmev-induced demyelination [ ] . evidence from highly neurovirulent jhm strains of mhv suggests that mhv-induced demyelination is also primarily immune mediated [ , ] . demyelination can be completely eliminated in jhm-infected, rag − / − mice that lack functional t and b cells, and this process can be reversed upon transfer of splenocytes from immunocompetent mice [ ] . it has also been shown by depletion and transfer studies in the jhm model that either cd + or cd + t cells can induce demyelination. however, mhv-a -induced demyelination has been shown to develop in the absence of b and t cells [ ] . furthermore, depletion of cd + or cd + t cells after the acute stage of infection does not reduce demyelination [ ] . thus, different related strains of mhv may induce demyelination via unique mechanisms, and it is likely that in the absence of an intact immune response, some strains of mhv infection in the cns are responsible for the onset of demyelination, possibly through the direct destruction of olgs. mhv-induced demyelination can serve as a model for oligodendroglial tropic, nonimmune-mediated mechanisms of demyelination that may have important relevance for our understanding of ms. mhv is a member of the coronavirus family [ ] [ ] [ ] . the virus infects many vertebrate hosts and induces a variety of diseases ranging in severity. the outcome and degree of mhv-induced disease are dependent on several factors, including the age and strain of the mouse, the strain of mhv, and the route of virus inoculation. even very closely related strains of mhv differ in pathogenic properties. some strains of mhv are purely hepatotropic (e.g., mhv- ) [ ] ; some are primarily neurotropic (e.g., jhm, mhv- , an isolate of jhm) [ , ] ; while others (e.g., mhv-a and mhv ) [ , ] mhv virions are - nm in diameter, somewhat pleomorphic (figure (a) ), containing an internal helical rna genome-nucleocapsid phosphoprotein (n) complex, surrounded by an envelope containing spike glycoprotein (s), transmembrane glycoprotein (m), envelope protein (e), and hemagglutinin-esterase protein (he) (figures (b) and (c)) [ ] . structural proteins expressed at the half of the mhv genome are believed to be responsible for mhv pathogenesis. s is a highly glycosylated protein. the outward radial projection of s gives the virus its family name corona (like a crown). he forms smaller spike on virions, but its function in the virus life cycle is yet not clearly defined. both m and e play a role in the assembly of virions [ ] . the virions also have the i protein of unknown function [ ] . the mhv genome is a singlestranded, and non-segmented, polyadenylated rna, of positive polarity [ ] . it is one of the largest known viral rna genomes with a length of - kb [ , , ] . the rna genome contains genes, termed to , encoding structural as well as nonstructural genes (figure (d) ). there is a leader rna sequence at the end of the genome that regulates the transcription of mhv-rnas. the next two-thirds of the end of the genome are covered by gene , which encodes a set of polyproteins with polymerase and replicase functions. gene encodes two proteins including a kd product of orf a. gene also transcribes a kd hemagglutinin-esterase protein in some mhv strains. genes , , and code for the spike protein (s), transmembrane glycoprotein (m), and nucleocapsid protein (n), respectively. gene encodes a - kd product while gene encodes a kd product of a still unidentified nonstructural protein and a membrane-associated e protein. it also contains a large open reading frame embedded entirely in the half of its n gene. this internal gene (i) encodes a mostly hydrophobic kd structural protein [ ] . during lytic infection, mhv enters the host cell via attachment of the s protein to specific receptors on the host pericellular surface. carcinoembryonic antigen is one receptor implicated in infection of cultured mouse cells [ ] . the receptor interaction triggers fusion of the viral and plasma membranes, allowing entry of the nucleocapsid into the cytoplasm, where all virus-specific rna and proteins are synthesized. the nascent mhv virions acquire their membrane envelope by implanting themselves into the lumen of the intermediate compartment between the endoplasmic reticulum and golgi complex of the host. mature virions are thought to move through the vesicles via the secretory pathways and exit the cell when the vesicles fuse with the plasma membrane [ ] . viral recombination has been extremely useful for systematically exploring the possible biological consequences of viral genomic differences. the recombination technique has been extensively exploited to study reovirus disease in mice, where the segmented nature of the viral rna has enabled resorting of viruses with rna segments from different strains with different biological properties [ ] . in sindbis virus and picornaviruses, a full-length infectious clone of the viral genome has been exceptionally useful in the study of viral properties [ , ] . in mhv, the replicase carries out "discontinuous transcription" in the fusion of body and leader sequences in subgenomic rnas and also during recombination events which occur at high frequency during mhv replication. high-frequency rna-rna genomic recombination events are archetypal for coronaviruses [ ] . these serve as mechanisms of virus evolution and modulation of viral pathogenesis, a largely unexplored area of study which offers deep insight into the genomic control of biological properties. but for a long time the large genome size was a technical obstacle to achieving such a goal with coronaviruses. this problem was circumvented by molecularly cloning defective interfering-like rna, leveraging the high rna recombination frequencies during mixed infection. this targeted recombination technique in mhv was developed by dr. paul s. masters and colleagues and used extensively to introduce alterations into the end of the mhv genome [ ] [ ] [ ] . it was able not only to exchange specifically the structural and non structural genes among the mhv strains but also to introduce single amino acid substitutions. in the last few years there have been reports of full-length infectious clones for many coronaviruses including mhv and a human coronavirus causing severe acute respiratory syndrome (sars) [ ] [ ] [ ] [ ] [ ] . upon intracranial (i.c) infection of neurotropic mhvs acute meningoencephalitis (with or without hepatitis) is the major pathologic process (figure ). viral titer reaches its peak at days and post infection (p.i) [ ] . infectious virus is cleared within the first - days; however, at this time mice begin to develop demyelination, either clinical or interdisciplinary perspectives on infectious diseases accompanied by chronic hind limb paralysis [ , ] . both mhv-jhm and mhv-a cause inflammatory demyelination in the brain and spinal cord (figure (a)) whereas mhv only causes vasculitis [ , ] . it was formerly believed that in primary mhv-induced demyelination neuronal axons remain relatively preserved. recently, it has been observed that adoptive transfer of spleen cells from immune mhv b mice into mhv-infected rag − / − mice (that are defective in recombinase activating gene expression, and thus lack mature b or t cells) resulted in demyelination with increased axonal damage [ ] . they also showed that axonal damage is, in large part, immune mediated in mhv-infected mice and occurs concomitantly with demyelination. concurrent axonal loss and demyelination have recently also been observed with s protein recombinant dm strain-infected mouse spinal cord as shown in figure ((a) demyelination; (b) axonal loss) [ ] . clearance and demyelination. mhv clearance requires both cd + and cd + t cells [ ] [ ] [ ] . cd + t cells are necessary for proper cd + t cell activation, survival, and retention in the infected cns [ ] . clearance of infectious virus is mediated by both cytolyticand cytokine-mediated mechanisms. exact mechanisms of mhv-induced demyelination are unclear although both macrophages and t cells modulate pathologic changes [ , ] . severe combined immunodeficient (scid) mice, rag- knockout mice, and uv-irradiated mice infected with mhv-jhm have few lesions - days p.i despite viral replication [ , , [ ] [ ] [ ] . these data suggest that lymphocytes are required for demyelination. however, demyelination mediated by γδt cells still occurs in nude mice that lack cd + and cd + t cells [ ] . interestingly, demyelination occurs to a similar extent in wild type, b cell-deficient, and rag- knockout mice and in mice lacking antibody receptors or active complement pathways, when infected with mhv-a [ ] . viral rna persistence is essentially the failure of the immune system to clear viral rna from infected organs, mainly the cns. viral rna persistence has been demonstrated in infections with all of the neurotropic demyelinating strains of mhv including jhm and mhv-a [ , , ] . viral rna persistence appears to be an important factor and perhaps even a prerequisite for mhv-induced demyelination during chronic immune-mediated demyelination. mhv strains that do not persist, such as mhv- and penn - (laboratory recombinant strains of mhv) [ , ] , also do not demyelinate. however, the persistencepositive, demyelination-negative phenotypes of penn - and penn - (s protein recombinant strain of mhv) [ ] indicate that viral persistence per se is insufficient to induce demyelination. penn - and penn - (s protein recombinant strain) [ ] may persist in neuronal cells of gray matter while demyelinating strains, such as mhv a , persist in white matter, suggesting that cell-specific persistence is necessary for demyelination. during chronic infection, mhv rna persistence in the white matter has previously been demonstrated [ ] . however, it is not known which glial cells are mainly responsible for the induction of demyelination. a temperature-sensitive demyelinating mutant of jhm is known to infect mainly nonneuronal cells and specifically to have a strong affinity for astrocytes as well as to cause white matter lesions in the mouse [ , ] . on the other hand, neurotropic nondemyelinating mhv has an in vitro affinity for neurons, ependymal cells and meningothelial cells but not for astrocytes or oligodendrocytes. mhv can induce an initial ependymitis, meningitis, and encephalitis in the absence of white matter lesions [ ] . these observations reinforce the importance of glial cell infection in the onset of demyelination. astrocytes and microglia play an important defensive role in mhv pathogenesis by secreting cytokines [ [ ] [ ] [ ] . cytokines are believed to aid in host defense against mhv by contributing to elimination of the virus from the cns. it has been reported that specific cytokines and chemokines (such as interferon α, β, and γ), tumor necrosis factor (tnf-α), il- , il- , il- α, and il- β are all induced during the acute stage of mhv infection in the cns [ ] . tnf-α, il- , and il- β are reportedly secreted by astrocytes of persistently infected spinal cords [ ] . the role of individual cns cells in the induction of demyelination remains to be further elaborated. natural and genetically constructed recombinant mhv strains (generated by targeted rna recombination) with differential pathological properties were used to understand the mechanisms of demyelination and concomitant axonal loss. these encompass both demyelinating (dm) and nondemyelinating (ndm) strains of mhvs, on which we have performed comparative studies correlating the phenotypes, genomic sequences, and their pathogencity. as a first step mice were inoculated mice with plaque-purified demyelinating strain mhv-a [ ] and nondemyelinating strain mhv- [ ] . mhv-a , a wild type parental strain of mhv, infects a variety of cell types, including neurons, astrocytes, oligodendrocytes, microglia, and ependymal cells [ , , , ] in the central nervous system (cns) and causes acute meningitis, encephalitis, hepatitis, and chronic demyelination. in contrast, mhv- , a closely related fusion negative [ ] strain to mhv-a , has limited ability to invade brain and spinal cord, causing meningitis without encephalitis or demyelination [ , ] but causing severe hepatitis ( figure ), making it an appropriate experimental negative control for our understanding of mechanisms of mhv-a -induced cns inflammatory disease processes. some cd -stained infiltrating t cells were also found (data not shown) although nonspecific background staining of neurons with available anti-cd antibodies made staining difficult to quantify. together, data suggests that mhvinduced cns inflammation consists of mixed inflammatory cells, predominantly macrophages/microglia [ , ] . area of plaques were larger, and mhv- -infected mice did not exhibit any demyelination. to avoid a high mortality rate of mhv- due to hepatitis, . ld doses ( pfus) were used. however, to ensure that the inability of mhv- to cause encephalitis or demyelination is not dose dependent, mice were also inoculated with mhv-a at pfus. mhv-a produced larger demyelinating lesions given at , pfus than at pfus, but with both doses, % of mice were affected. results confirm earlier findings that mhv-a induces demyelination whereas mhv- is nondemyelinating at day [ ] . demyelination was not previously assessed at earlier time points. demyelination begins as early as day post inoculation, indicating that mhv-a -induced myelin damage begins at the time of acute inflammation, similar to what is observed in ms and eae lesions [ ] . (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) interdisciplinary perspectives on infectious diseases mhv-a viral antigen, d mhv- h and e, d mhv- viral antigen, d the degree of optic nerve inflammation was scored on a (no inflammation) to (severe inflammation) point scale described previously in [ ] [ ] [ ] , with any amount of inflammation (score - ) considered positive for on. on was detected as early as days post inoculation with mhv-a , with peak incidence at day (figure (a)) and resolution by day . at the peak of on (day ), almost % ( of ) of optic nerves examined from mhv-a -infected mice had on (figure (b)), with an average relative inflammation score of . ± . whereas, only one of nerves from mhv- -infected mice had even mild inflammation (score . ). incidence of on in eae was % ( of ) with a . ± . average inflammation score, similar to prior studies [ , ] and comparable to the incidence induced by mhv-a infection. to examine whether mhv-a -induced on also leads to axonal loss, optic nerves were stained by bielschowsky silver impregnation, and the area of axonal staining was quantified as described previously [ ] . days post inoculation, mhv-a -infected mice had significantly decreased axonal staining compared to control nerves or nerves from mhv- infected mice (figure (c) ). comparative studies demonstrate that mhv-a , but not mhv- , induces demyelination in the cns during acute encephalitis, as early as days post inoculation, in addition to chronic demyelination previously observed at day [ ] . inflammation triggered by mhv infection consists of mixed inflammatory cells, predominantly macrophages/microglia, which differs somewhat from immune-mediated models of ms where infiltrating t cells are significant contributors to pathology [ , ] . importantly, mhv-a also induces on with similar severity and incidence as seen in the autoimmune antigen-triggered model eae. experimental on is an important model being used to characterize neuronal damage and develop novel therapies for ms [ , [ ] [ ] [ ] , but studies have shown that different eae models, induced by distinct antigens, lead to different mechanisms of rgc loss [ , ] . the mhv-a -induced on model will provide a critical adjunct to study the pathophysiology of on secondary to viral-mediated inflammation as this is one mechanism that can cause on and ms in human patients. sequence comparison between mhv- (gene bank accession number: af ) and mhv-a (gene bank accession number ) revealed %- % sequence homology of the replicase genes, %- % sequence homology of genes a, , b, , and , and a marked difference in the sequence of genes b, , and a among two strains. among the structural proteins the s protein interacts with the host receptor and mediates viral-cell membrane fusion during viral entry [ ] . the ∼ amino acid spike protein in mhv-a is usually posttranslationally cleaved into two domains which associate with each other to form a functional dimer. in the s protein from mhv- strain, however, this cleavage does not occur, the reasons for which are not clearly evident. a look at the pairwise alignment between the s proteins from mhv-a and mhv- reveals that around % residues are common in both the chains and an additional % residue is similar. the % residues with no identity or similarity, include a -residue insertion in mhv- . this insertion is by far the most significant difference in the sequence. the role of s protein as agent of organ tropism and pathogenesis was hypothesized from comparative studies of different naturally occurring mhv strains [ , ] . nucleotide sequencing revealed that alterations in virus virulence were most closely associated with differences in the s gene. these findings were reinforced using targeted rna recombination to exchange specific gene/genes of interest between different strains of mhv [ ] [ ] [ ] [ ] [ ] [ ] . several targeted rna recombination studies have directly demonstrated that the s gene is a major determinant of virulence of mhv in mouse brain and liver. to determine whether the s gene of mhv also contains determinants of demyelination and whether demyelination is linked to encephalitis and chronic stage viral persistence, targeted rna recombination was used to create new mhv strains, penn - and penn - , by replacing the s gene of the encephalitic and ds (mhv-a ) with the s gene of a closely related, nonencephalitic nds (mhv- ) [ ] . molecularly cloned vector pmh [ , ] , which contains the entire ' end of the genome from mhv-a , was used for construction of the recombinant viruses. comparative pathological studies between recombinant ds (wtr ) and the nds penn - and penn - demonstrated that penn - and penn - exhibit similar inflammatory infiltration (encephalitis) (figures (a) and (b) ) in the cns during the acute stage. while wtr can induce demyelination at day p.i. (figure (c)), penn - and penn - are unable to induce demyelination (figure (d) ). penn - and penn - also exhibited chronic stage persistence in the spinal cord [ ] . for convenience and clarity, in subsequent sections these recombinant strains were labeled in different names though they are the same but presented in different names, penn - and penn - are referred to as rsmhv (viruses expressing the mhv- spike in the mhv-a background) and wtr as rsa (viruses expressing the mhv-a spike in the mhv-a background). as alteration of the mhv s protein influences pathogenesis, it is important to understand how alteration of the s protein alters the virus-host interaction. previously this type of study was carried out using specific antibodies that detect viral antigen in tissues. targeted recombination was used to select mhv isolates with stable and efficient expression of the gene encoding egfp to facilitate the in vivo detection of virus in the mouse cns as well as to trace the viral entry and spread in tissue culture [ ] . the egfp gene was inserted into the mhv genome in place of the nonessential gene , as interruption of the orf did not decrease neurovirulence in jhm [ ] . the viruses replicated with similar kinetics as wild-type virus both in tissue culture and in the mouse cns. they caused similar encephalitis and demyelination in animals as the wild-type virus or their recombinant strains; however, they were somewhat attenuated in virulence. isogenic egfpexpressing viruses differ only in the s gene and express either the s gene of the highly neurovirulent. previously used names for rsjhm egfp and rsa egfp are s r egfp and s a r egfp , respectively. jhm spike-mediated neurovirulence was associated with extensive viral spread in the brain [ ] . the difference in virulence and patterns of spread of viral antigen between the two isogenic recombinant strains reflected the differences between parental viruses expressing each of these s genes [ , ] . these egfp-expressing viruses are powerful tools that can be used to follow viral spread over time without terminating infection by the fixation necessary for immunofluorescence. in order to further compare the differential pathogenesis and the cns cell tropism between dm and ndm strain of mhv, an egfpexpressing nds of mhv rsmhv egfp , which contains the mhv- (nds) s gene in the mhv-a background, was constructed [ ] . a cartoon representation of construction of egfp-tagged strains of mhv has been shown in figure . detailed molecular studies show the egfp gene is inserted and expressed at high levels that can be readily detected by microscopy [ ] . frozen sagittal brain sections were obtained from rsa egfp -and rsmhv egfp -infected mice, postfixed, and observed by fluorescent microscopy. one of such representative egfp expressing sagittal brain sections has been shown in figure . egfp fluorescence was observed in similar regions of the brain in mice infected with both the viruses; however, the number of infected cells differed between strains. despite the similarity in regional localization, the distribution of rsmhv egfp (ndm) strain is more localized in the brain parenchyma compared to rsa egfp (dm) strain . to confirm that egfp positive cells were also positive for viral antigen, frozen sections were labeled with antinucleocapsid antiserum as primary antibody and texas red goat antimouse igg as secondary antibody. egfp fluorescent was colocalized with viral antigen in both rsa egfp -rsmhv egfp and-infected brain cortical sections (data not shown). there is a high degree of colocalization of egfp and viral antigen in the majority of sections analyzed. complete colocalization demonstrated that egfp fluorescence can be used to detect viral antigen without performing any immunostaining. pathology was also assessed in five to seven cross-sections of spinal cord from cervical, thoracic, and lumbar regions at day (peak inflammation) and day (peak demyelination) p.i. demyelinating plaques were detected by lfb stains for myelin at day p.i. in dm-infected mice (figure (a) ) and were quantified on a - scale [ ] in four quadrants from two spinal cord levels for each mouse [ ] . day figure : detection of viral antigen spread by egfp fluorescence. mice ware sacrificed at day p.i. brain was removed and processed for frozen sections. a portion of sagittal brain sections was cut from the middle of the brain of infected mice. frozen sections were processed for detection of viral antigen distribution by egfp fluorescence. bright egfp fluorescence was spread throughout the brain section as showed by montage assembled from serial images (a). higher magnification image (b) from the same brain section shows the cellular distribution of egfp fluorescence. (adapted from the wprk of [ ] ). showed a similar pattern of demyelination as on day , but the number and area of plaques ware larger [ ] . ndminfected mice did not exhibit any demyelination either at day ( figure (b) ) or at day p.i. (data not shown) [ ] . though comparative histopathological studies demonstrated that rsa egfp could induce demyelination (figure (a) ) whereas rsmhv egfp cannot induce demyelination (figure (b) ), both rsa egfp and rsmhv egfp viruses produced meningoencephalitis (figures (c)- (f)). brain pathology consisted of encephalitis, characterized by parenchymal lymphocytic infiltrates and microglial nodules with focal neuronophagia (figures (c) and (d) ). brain sections from rsa egfp showed comparatively more parenchymal inflammation compared to rsmhv egfp . associated lymphocytic meningitis was also present in both the strains (figures (e) and (f)). demyelination following rsa egfp infection. though mhv-induced demyelination is characteristically described as sparing axons within areas of demyelination, axonal loss with demyelination in the optic nerve of mhv-a -(parental dm) infected mice has been recently observed [ ] . this led to hypothesize that dm mhv may induce axonal loss in the spinal cord. to examine this, serial sections of spinal cords from rsa egfp -(dm) and rsmhv egfp -(ndm)-infected mice were stained parallaly by lfb and bielchowsky silver impregnation. dm-infected spinal cord sections at both day and day p.i. showed a significant decrease of axonal staining in demyelinating plaque ( figures (a) and (b) ). no demyelination and no axonal loss ware evident in the ndm-infected mouse spinal cords either at day (figures (e) and (f)) or at day p.i. to further evaluate the loss and/or preservation of myelin in dm-and ndm-induced inflammatory plaques, semithin spinal cord sections cut at -micron intervals from five infected mice at day p.i. were stained with toluidine blue. control mock-infected mouse spinal cord was used to evaluate for background fixation and/or postfixation artifacts. dm-infected spinal cords showed significant myelin loss within plaques (figure (c) ). moreover, high magnification images showed infiltrating macrophages filled with myelin debris (figure (d) ). in contrast, in ndm strain rsmhv infected spinal cord sections, myelin is almost completely preserved, with only rare examples of early axonal degeneration, characterized by loss of the central axon and collapse of the myelin sheath (figures (g) and (h) ). the presence of this focal axonal degeneration was not detectable in prior studies of paraffin-embedded tissues by silver impregnation. to further characterize axonal pathology at the ultrastructural level, representative foci of demyelination and axonal injury were selected from toluidine blue-stained sections, and Å ultrathin sections from poly-bed-embedded blocks were processed for tem. high-resolution tem images show a combination of axonal degeneration and demyelination. there are extensive axonal loss and many residual empty vacuoles corresponding to totally degenerated fibers. in addition, there are numerous hypomyelinated fibers and naked axons without any myelin sheath, indicative of a demyelinating process (figures (b)- (d) ). these naked axons are fully intact with surrounding axolemmas and axoplasm with preserved microtubules and intermediate filaments (figure (e) ). in contrast, ndm-infected mouse spinal cords demonstrate no appreciable axonal loss and no features of demyelination. specifically, there are no hypomyelinated fibers, no naked axons, no macrophages, and no evidence of macrophage-mediated myelin stripping. instead, there are only rare examples of early axonal degeneration characterized by loss of the central axon and collapse of the myelin sheath (figure (f)) as previously seen in toluidine blue-stained sections. normal myelinated region is denoted in figure (a). spinal cord section. one mechanism of demyelination is revealed as macrophage-mediated myelin stripping. this is demonstrated in figure in which a macrophage is observed surrounding a myelinated axon. the myelin is unraveling, yet the axon is completely intact. at the inner border of the myelin sheath, multiple vacuoles are present as the entire myelin sheath is lifting off the axon. the macrophage cell membrane is in intimate contact with the outer portion of the myelin sheath as the macrophage strips away and engulfs the myelin sheath. multiple vacuoles with myelin fragments are seen within the cytoplasm of the macrophage. (figures (e) and (f)). at day p.i., lca + cells were still present in abundance in demyelinating plaques produced following dm infection (figures (g) and (i)), but in ndm-infected mice, no demyelinating plaques were present and very few lca + cells were retained in the gray matter (figures (h) and (j) ). immunohistochemical data demonstrate that in dm-and ndm-strain-infected spinal cord there is an increase of lca + /cd b + cells; however, the distribution of these inflammatory cells is significantly different between the two strains. to determine whether differential localization of viral antigen is responsible for the observed distribution of inflammatory cells, crosssections from spinal cord of dm-and ndm-infected mice were examined. since the dm and ndm viruses used in these studies express egfp, viral distribution was assessed directly by fluorescence microscopy. on day p.i., the dm fluorescence was mainly restricted to white matter (figures (a) and (b) ) with limited involvement of gray matter whereas ndm fluorescence was restricted to gray matter (figures (c) and (d) ). high-magnification fluorescence images in brain from day p.i. demonstrate numerous neurons and their axons following dm infection (figure (e)) whereas far fewer fluorescent neurons and axons are observed following ndm infection (figure (f) ). the ability of dm and ndm to replicate and spread in vitro was compared using primary hippocampal neuronal cultures and described in very recent studies . immunostaining of map b [ ] and gfap [ ] demonstrated that cultures consisted primarily of neurons. at hours intervals, from to hours p.i., cultures were monitored for viral antigen spread by fluorescence microscopy. at hours p.i. several infected foci were observed in dm-infected cultures ( figure (g) ) whereas, in ndm-infected cultures, very few discrete cells were positive for egfp (data not shown). at hours p.i., the average number of egfp positive cells was significantly increased following infection with dm ( figure (h) ). in contrast, ndm-infected cultures had viral infection restricted only to the initially infected neurons, with little or no spread to adjacent cells, consistent with in vivo studies (figure (i)) [ ] . to confirm the differential distribution of viral antigen in gray and white matter in very recent studies [ ] cross-sections from several regions of all spinal cords were stained with viral antinucleocapsid antiserum. at day post-infection viral antigens were mainly observed in the gray matter of the spinal cord in rsa egfp -and rsmhv egfp -infected mice some viral antigen was also detected in the white matter. at day , most of the viral antigen in rsa egfp was found in white matter whereas rsmhv egfp viral antigen remained mainly restricted to gray matter with occasional distribution to white matter. in rsmhv egfp -infected mice, viral antigen was occasionally observed in the white matter, and in some spinal cord sections of rsmhv egfp viral antigen was detected at the graywhite matter junction as shown in very recent studies [ ] . tropism. the severity of tissue destruction in mhv infection is mediated by direct viral infection and immunemediated destruction. the relative contributions of these two components differ depending on a number of virus and host factors including viral tropism, rate of viral spread, and specificity of the immune response. during acute infection, neurotropic and demyelinating strains (dm) of mhv infect a number of cell types in the cns such as neurons, astrocytes, oligodendrocytes, and microglia [ , , , , ] . recently by using the s gene recombinant virus (s r, containing s gene from a highly neurovirulent mhv- on the background of mhv-a ) it has been demonstrated that s gene-mediated neurovirulence of mhv is associated with extensive viral spread in the brain in both neurons and astrocytes [ ] . thus far it was not known whether ndm of mhv infects the same cell types as ds. the phenomenon of demyelination may be due to primary infection of a single cell type, or alternatively, infection of diverse cns cell types leading to the final destruction of olgs/myelin sheath and the axon. to compare the cellular tropism of dm and ndm mhv double-label immunofluorescence on sagittal brain sections was performed [ ] . egfp fluorescence was used as a viral antigen marker, map b as a neuron-specific marker, and gfap as an astrocyte-specific marker. sections were systematically scanned in a blinded fashion. representative pictures of dual color fluorescence of viral antigen and map b are shown in figure . visual microscopic observations show that rsjhm egfp infects and spreads more in neurons in comparison to the other two strains. in rsjhm egfp -infected mice only a very small number of viral antigen positive cells were also positive for the astrocytic marker gfap, whereas in rsa egfp -infected mice, egfp positive cells partially overlapped with gfap staining (figure ). in addition, in rsa egfp -infected mice egfp positive cells were not only astrocytes but also other cell types. in contrast, in rsmhv egfp -infected mice egfp completely colocalized with gfap ( figure ) . localization studies showed that mhv strains differ in their neurovirulence and ability to demyelinate and also differ in their ability to infect particular cns cell types. rsjhm egfp infects neurons with greater efficiency, and infection may spread more rapidly than in rsa egfp , while rsmhv egfp infects neurons with limited efficiency. this is consistent with previous findings that the degree of neurovirulence is not only dependent on neuronal tropism but also on the number of infected neurons and spread of the infection through neurites [ ] . evidence from closely related strains of neurotropic viruses including hiv, tmev, and reovirus supports the hypothesis that cns cell tropism and spread in cns cells play a major role in the distribution and type of cns lesions [ , ] . studies with lcmv and hiv also suggest that virus strains that exhibit rapid spread are associated with increased immune-mediated pathology [ , ] . recently, it has been demonstrated that mhv spread to the spinal cord white matter occurs very rapidly, and protection from demyelination can be achieved by inhibiting this viral spread during the acute phase of infection by recruiting high numbers of mhvspecific cd + t cells [ ] . nondemyelinating strain is less able to infect neurons and spread through neurons; on the other hand, demyelinating strains are highly neuron tropic and spread rapidly through neurites. in contrast, it has been observed that a temperature-sensitive demyelinating mutant of jhm infects mainly nonneuronal cells, having (adapted from das [ ] ). a strong tropism specifically for astrocytes and causes white matter lesions. similarly, it has been demonstrated that a monoclonal antibody selected jhm variant, a s protein mutant, infects the glial cells predominantly in the cns, causing subacute demyelination. interestingly, the neurotropic and nondemyelinating mhv strain, which has an in vitro tropism for neurons, ependymal cells, and meningeal cells but not for astrocytes and oligodendrocytes, can induce initial ependymitis, meningitis, and encephalitis in the absence of white matter lesions. our in vivo data demonstrate that the highly neurovirulent strain rsjhm egfp has very little tropism for astrocytes. rsa egfp and rsmhv egfp infect astrocytes with similar efficiency. as demyelinating and nondemyelinating strains produce similar infections in astrocytes, it appears that astrocytic infection during the acute stage alone may not determine the onset of demyelination. our observations suggest that astrocyte tropism may be a relevant factor in persistent infection but direct astrocytic infection alone may not be sufficient to induce demyelination. this study highlights the important role of neural cell tropism of viral antigen in mhv-induced demyelination in the cns. the development of chronic demyelination involves replication and spread of viral antigen in the spinal cord during the acute stage of infection and the persistence of viral rna in white matter. this is substantiated by current observations on viral spread of ndm mhv in the spinal cord, where viral antigen during the acute phase of infection is mainly restricted to gray matter. in contrast, viral antigen from demyelinating strains spreads from gray to white matter during the acute stage of infection [ , ] based on experimental evidences it can also be argued that the spread of viral antigen from neuron to neuron and from neuron to glial cells plays a critical role in the induction of chronic stage demyelination. both dm and ndm strains infect neurons; however, these strains differ in their capacity to translocate to white matter as determined by fluorescence of egfp tagged viral particles. the current experiments indicate that axonal transport of viral particles is an important mechanism mediating not only the extent of axonal damage but also the subsequent induction of demyelination in the spinal cord. dm strain shows white matter involvement early (by day p.i.) and at later time points that gray matter involvement is essentially absent in the spinal cord, with only minimal involvement in the brain. this observation further supports earlier findings that dm and ndm strains differ in their viral antigen distribution during acute infection in the spinal cord [ ] . evaluation of axonal loss and demyelination in the spinal cord, where there is clear separation of gray matter and large tracts of white matter, demonstrated that dm mhv infection begins in the neuronal cell body and propagates centripetally to the axon [ ] which subsequently induces axonal degeneration and demyelination. ndm strains were unable to propagate from gray to white matter and as a result and were unable to induce demyelination. the migration and activation of numerous cd b+ macrophages/microglia to the white matter following viral spread of dm mhv [ ] suggests that recruitment of these cells mediates demyelination. during dm strain chronic infection at day p.i., macrophages/microglia were still present within areas of demyelination whereas macrophages/microglia and viral antigen were cleared from the gray matter following ndm strain infection. one plausible explanation is that mhv spreads intraaxonally in an anterograde manner within gray matter and when it reaches the white matter, viral particles are able to infect oligodendrocytes via direct cell-cell contact. a less likely possibility is that infection proceeds indirectly from neurons to oligodendrocytes by intermediary cells such astrocytes, microglia, or endothelial cells. regardless of the mechanism of viral spread, ultimately, macrophages and microglia are recruited to the areas of infection. in this way, mhv infection triggers axonal loss and macrophagemediated demyelination. however, we have also observed in ndm strain infection, that axonal degeneration and myelin disruption can occur in the absence of macrophages. therefore, mhv infection can both directly and indirectly cause axonal loss and demyelination. it may be that the failure of ndm strain to trigger macrophage-mediated damage is entirely a function of transport failure to the white matter. although there are no experimental data to demonstrate this, it can be suggested, based on our current neural cell tropism studies, that neuroinvasion by mhv may require entry into the nerve endings, transport to the cell body, replication in the cell body, axonal transport to the synapse, and transneuronal viral spread. similar mechanisms of axonal transport of virus particles have been observed for the alpha herpesviruses, herpes simplex virus and pseudorabies virus [ , ] . furthermore, theiler's murine encephalitis virus (tmev), a nonenveloped virus that, like mhv, causes chronic demyelination in mice, is able to traffic from the axon into the surrounding myelin in the absence of cell lysis [ ] . thus, by a mechanism involving transneuronal spread, mhv may gain access to synaptically linked neuronal circuits and glial cells. it is not clear, however, which type (s) of glial cells must be infected in order to promote the development of mhv-induced chronic demyelinating disease. available studies focus mainly on neurons and astrocytes, as they represent the majority of infected cells during the acute stage of mhv infection, but oligodendrocytes cannot be disregarded. it has been previously demonstrated that mhv can infect oligodendrocytes [ ] , but the direct cytolytic effect of mhv on oligodendrocytes is unclear [ , ] . direct viral infection of oligodendrocytes may contribute to mhv-induced demyelination, as observed in another human demyelinating disease, progressive multifocal leukoencephalopathy (pml), which is caused by jc virus infection of oligodendrocytes [ , ] . the current studies further demonstrate that the molecular mechanisms underlying axonal damage are mediated by the s glycoprotein. the recombinant dm and ndm viruses used are isogenic on a background of the dm mhv-a strain and differ only by the virus-host attachment s glycoprotein. in vivo, the s protein host receptor is carcinoembryonic antigen (cea), which functions as an intercellular adhesion molecule [ , , ] . however, there is evidence that other viral entry host factors may also be permissive for viral infection [ ] . evidently, the difference in the s proteins between the dm and ndm strains does not impair host-receptor interactions or viral entry since both strains effectively cause encephalitis following transcranial inoculation. the s protein does, however, appear to play a critical role in axonal transport, with a lack of viral antigen spread and subsequent inflammation extending into spinal cord white matter following infection with the ndm strain in contrast to extensive white matter involvement secondary to dm strain infection being observed. as mentioned earlier, mhv s is synthesized as a kda glycosylated precursor that is posttranslationally cleaved into two kda subunits, s and s [ ] , with a receptor-binding domain in the s subunit [ ] that is responsible for the initial attachment of mhv to cell surface receptors. this binding event triggers a conformational change in s that allows s to initiate fusion of the virus and host membranes [ ] [ ] [ ] . a candidate fusion peptide domain has been identified within s [ ] ; however, the actual fusion peptide for the mhv s has not been definitively identified. mhv-a and mhv- s proteins have % amino acid sequence identity and % similarity, with the s domain relatively more conserved and s more variable [ ] . they also differ in their cleavage signal site whereby mhv-a s is cleaved posttranslationally into s and s subunits, but mhv- s protein is not cleaved and unable to cause fusion in vivo and in vitro. thus, variable regions of the s domain or differences in the cleavage signal site between dm and ndm strains are candidate domains that may potentially explain the differential axonal transport and demyelination observed in our studies and need to be evaluated further in future studies. genes other than the s sometimes referred to as "background genes" also influence pathogenesis. in addition to structural proteins and replicase, all mhvs encode small unique proteins with unknown functions (orf a, , and a) [ ] . the proteins encoded in these three orfs appear to be nonessential for replication. it is instructive to note that, apart from the molecular role of the s gene in mhv pathogenesis, several additional genes have also been implicated in the disease etiology. asides from their structural roles, n and m proteins are believed to function in host interactions. in porcine coronavirus, tgev, mutations in the m protein ectodomain that impair n-glycosylation decrease the interferogenic activity and antibodies directed to the tgev m ectodomain block ifn-γ induction [ ] . for mhv, the glycosylation state of m protein may effect the ability to induce ifnγ and also to replicate in the liver [ ] . in measles infection, a defective m transmembrane protein has been related to the difference in the ability of the virus to persist in the cns and cause subacute sclerosing panencephalitis (sspe) [ ] . the n protein has been implicated in mhv-induced hepatitis [ , ] . in addition, the replicase and other non structural proteins could affect tropism and pathogenesis by influencing the rate of viral replication perhaps by interactions with cell type specific factors or with elements of the immune response [ ] . current available studies demonstrate that the mechanisms of white matter injury in this model of cns demyelinating disease are due to a combination of both axonal injury/loss and myelin damage. in dm infection, there is concomitant axonal loss and demyelination, and at least some demyelination is due to macrophage-mediated myelin stripping. one can hypothesize that axonal degeneration follows in this immune-mediated pathogenesis. partial axonal loss also appears to occur secondary to direct viral-induced cytopathic effects, a non-immune-mediated mechanism. following ndm infection, although relatively rare, myelinated fibers in the spinal cord white matter exhibit early axonal degeneration in the absence of any associated inflammation. therefore, mhv infection exhibits diverse mechanisms of axonal loss and demyelination that are both immune and nonimmune mediated. while the underlying mechanisms leading to the development of ms in patients are not known, numerous studies have provided evidence to suggest that a viral infection may trigger this autoimmune demyelinating disease. the critical role of the s glycoprotein in viral transport and subsequent axonal and myelin damage demonstrated in the current studies suggests that targeted disruption of the s glycoproteinhost interaction has the potential to prevent the onset or progression of demyelination. future experiments need to be geared toward identifying how the s proteins interact with the axonal transport system. clues may be provided by other human viral neurologic infections, including those caused by such diverse viruses as herpes simplex and rabies, as they take advantage of the axonal transport system in order to maximize spread and neurovirulence. experimental mouse model provides an excellent means by which the particular host-virus interactions responsible for successful axonal transport can be dissected and clarified. national multiple sclerosis society-about research multiple sclerosis (first of two parts) multiple sclerosis. 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virus type induces transcription of the novel fgl prothrombinase gene optimization of targeted rna recombination and mapping of a novel nucleocapsid gene mutation in the coronavirus mouse hepatitis 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 viral-induced experimental animal model to understand the mechanism of demyelination research was supported by national multiple sclerosis society, m. e. groff surgical medical research and education charitable trust, and lindback foundation career enhancement award. the success of this work has largely depended on contribution and devotion of many researchers whose names are listed in the references. dr. lawrence. c kenyon is acknowledged for the pathological data analysis and mr. ryan marek for image processing. 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 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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- -j ld q authors: wu, z. g.; yan, w. m.; guo, w.; chen, t.; zou, y.; wang, h. w.; wang, x. j.; yang, x. j.; lu, y. l.; luo, x. p.; ning, q. title: telbivudine preserves t‐helper cytokine production and downregulates programmed death ligand in a mouse model of viral hepatitis date: - - journal: j viral hepat doi: . /j. - . . .x sha: doc_id: cord_uid: j ld q summary. telbivudine is an orally bioavailable l‐nucleoside with potent and specific anti‐hepatitis b virus activity. the higher rate of hepatitis b e antigen (hbeag) seroconversion during telbivudine treatment than other potent anti‐hbv agents suggests a potential immunomodulatory effect. we sought to determine the effects of telbivudine on the immune system, particularly on cytokine production and t‐cell response, using an animal model with mouse hepatitis virus strain (mhv‐ )‐induced hepatitis. the effects of telbivudine on virus replication and cytokine production were investigated in vitro using mhv‐ ‐infected macrophages, and the effects on t‐cell response were investigated in vivo in an mhv‐ ‐induced viral hepatitis model. telbivudine had no effect on mhv‐ replication in macrophages. however, the production of tumour necrosis factor‐α and interleukin‐ was increased significantly in mhv‐ ‐induced macrophages treated with telbivudine. in vivo survival was enhanced in telbivudine‐treated mice, with marked normalization in clinical conditions and histological lesions. serum levels of interferon‐γ were elevated significantly after telbivudine treatment in mhv‐ ‐infected c h mice. in contrast, serum interleukin‐ levels were decreased significantly. furthermore, telbivudine treatment enhanced the ability of t cells to undergo proliferation and secrete cytokines but did not affect cytotoxicity of infected hepatocytes. of note, we found that telbivudine treatment suppressed programmed death ligand expression on t cells. the results demonstrate the immunomodulatory properties of telbivudine, independent of its antiviral activity, in a mouse model of mhv‐ ‐induced hepatitis. hepatitis b virus (hbv) infection is a significant cause of liver-related morbidity and mortality, particularly in regions of high endemicity [ ] . the natural course and clinical outcome of hbv infection is mediated by a complex interaction between the virus and the hostÕs immune response. successful control of hbv replication is associated with a strong, multi-specific cd + and cd + t-cell response in conjunction with a humoral immune response [ ] [ ] [ ] [ ] . based on current understanding of host-virus interactions and the natural course of hbv infection, management strategies have focussed on enhancement of the hostÕs hbv-specific tcell response and direct suppression of hbv replication to attain sustained viral control and remission of liver disease [ , ] . of the currently available anti-hbv therapies, only interferon (ifn)-a and pegylated ifn-a have demonstrated an immunomodulatory effect on hbv [ ] . the immunomodulatory effect has been reported with the nucleoside ribavirin; the mechanisms of action are considered to be th cell differentiation and upregulation of activity of doublestranded (ds) rna-activated protein kinase [ ] [ ] [ ] . very little is known about the influence of newer oral nucleosides on the t-cell-specific immune response. telbivudine, an l-nucleoside analogue of thymidine, is a potent and specific inhibitor of hbv dna polymerase, and it is approved for the treatment of patients with chronic hepatitis b (chb) [ ] [ ] [ ] [ ] [ ] . in the -year analysis of the globe study, the rate of hepatitis b e antigen (hbeag) seroconversion during telbivudine administration was higher than during lamivudine administration in patients with serum alanine aminotransferase (alt) levels greater than two times the upper limit of normal (uln) [ ] . the relatively high hbeag seroconversion rate attained with telbivudine differs from the rates obtained with other potent anti-hbv agents and suggests that telbivudine potentially has immunomodulatory activities. the aims of the present study were to characterize the effects of telbivudine on cytokine profile and t-cell response in vitro and in vivo using a previously characterized mouse model of viral hepatitis induced by the coronavirus mouse hepatitis virus strain (mhv- ) [ , ] . the effect of telbivudine on the programmed cell death pathway, a possible mechanism by which cd + t-cell function was influenced, was also investigated. telbivudine (pure substance) was obtained from novartis pharma (basel, switzerland) and lamivudine was purchased from moravek biochemical (brea, ca, usa). substance concentrations were determined by high-performance liquid chromatography. mouse hepatitis virus strain was originally obtained from the american type culture collection (rockville, md, usa) and it was plaque purified on monolayers of dbt cells and grown to titres of · plaque-forming units (p.f.u.)/ml. virus was harvested by centrifugation at g for h at °c and was assayed on monolayers of l cells in a standard plaque assay. female balb/cj and c h mice at - weeks of age were obtained from the hubei provincial institute of science and technology (wuhan, china) and the vital river company (beijing, china), respectively. all animal studies were conducted in accordance with the chinese council on animal care and they were approved by tongji hospital (tongji medical college, huazhong university of science and technology, wuhan, china). macrophage isolation peritoneal macrophages were harvested from balb/cj mice days after an intraperitoneal injection of . ml of % thioglycollate, as outlined previously [ ] . macrophages were ‡ % pure, as determined by morphology. viability exceeded % by trypan blue exclusion. macrophages were incubated in the presence of indicated concentrations of telbivudine ( , or lg/ml) with mhv- . macrophages were harvested h after infection and viral titre was determined in a standard plaque assay. cytokine level measurement by pcr and enzyme-linked immunosorbent assay to determine the effect of telbivudine on tumour necrosis factor (tnf)-a and interleukin (il)- cytokine production, one million macrophages from balb/cj mice were stimulated with mhv- (multiplicity of infection, . ) and telbivudine was added at indicated concentrations. relative quantitative real-time polymerase chain reaction (pcr) was performed to measure the mrna levels on a roche light-cycler Ò (roche diagnostics, nutley, nj, usa) following the manufacturersÕ instructions. cytokine protein levels were determined in cell supernatants following appropriate dilution using enzyme-linked immunosorbent assay (elisa) (r&d systems, abingdon, uk). the cytokine levels in lipopolysaccharide (lps)-stimulated macrophages with or without telbivudine added from the same strain of mice were also investigated. in vivo study animal model fifteen c h mice each infected with p.f.u. mhv- received telbivudine mg/kg in ll phosphate-buffered saline (pbs) via intraperitoneal injection for days, starting h before infection and daily thereafter. an additional mhv- -infected mice in each group received lamivudine mg/kg per day or pbs as a control. clinical conditions and survival were observed. animals were killed on day after infection, and spleens, livers and blood were collected for experiments. the effect of treatment with telbivudine on mhv- -induced liver disease was determined by monitoring mice twice daily for changes in clinical signs, including abnormalities of fur texture, increased respiration, and the presence of tremor. conditions were graded on a scale of - , with indicating the marked presence of an abnormality and indicating normalcy. a composite score for all parameters was calculated by two independent examiners who were blinded to the treatment regimens to minimize bias. at the time the mice were killed, livers were removed, weighed and fixed in % buffered formalin for a minimum of h. tissues were processed and paraffin embedded by routine methods, and they were stained with haematoxylin and eosin. spleens from each group of mice were harvested days after mhv- infection. spleens were dissected and single-cell suspensions were made by passage through a nylon cell strainer. after density gradient centrifugation and lysis of red cells in ammonium chloride solution, cells were prepared for study. cd + t cells were purified from single-cell splenocyte suspension using an isolation kit (miltenyi biotec, bergisch gladbach, germany). mouse hepatitis virus strain -infected c h mice were anaesthetized and their livers were perfused in situ with a ca +free hankÕs balanced salt solution containing mm ethylenediaminetetraacetic acid for min at °c, followed by a . % collagenase solution (type iv; sigma-aldrich, st louis, mo, usa) for min. after digestion, the livers were removed and placed into a sterile petri dish. single-cell suspensions were prepared. the viability of hepatocytes both before and after treatment was ‡ %, as assessed by trypan blue staining. on the day the mice were killed, sera were collected and assayed for il- and ifn-c using the appropriate elisa (r&d systems). mice were sacrificed at the indicated time points. purified cells were cultured at · cells/ml in the presence of ng/ml phorbol myristate acetate plus lg/ml ionomycin for h in -well plates. cells were then stained with monoclonal antibodies specific for surface markers, permeabilized, and fixed using cytofix/cytopermä (bd, franklin lakes, nj, usa) according to the manufacturerÕs instructions. different cell subsets were identified using antibodies to il- , il- , ifn-c, tnf-a, perforin, granzyme b (ebioscience, san diego, ca, usa) and their isotypes. the antibodies used for staining were anti-cd (pe-cy . ), anti-cd (apc), anti-cd [fluorescein isothiocyanate (fitc)], anti-programmed death (pd- ), anti-programmed death ligand (pdl- ), anti-fas ligand (fasl), anti-perforin, anti-granzyme b, anti-tnf-a-r / , anti-tnf-a, anti-ifn-c (pe), and isotype control (pe) (ebioscience). all analyses were performed on a facsariaä cell sorter (bd), and subsequent analysis was performed with facsdiva software (bd). freshly isolated hepatocytes from mhv- -infected c h mice were used as target cells and cd + t cells as effector cells. cytolytic activity was determined with the cytotox Ò non-radioactive cytotoxicity assay (promega, madison, wi, usa). for each effector:target cell ratio (ranging from . : to . : ), specific target cell lysis was calculated as follows: [(experimental release ) spontaneous release)/(maximum release ) spontaneous release)] · . each effector:target cell ratio was measured in quadruplicate. statistical analysis was conducted with a studentÕs t-test, and a p-value of £ . was considered statistically significant. results were reported as the mean ± standard deviation for three or more separate experiments, each performed in triplicate. first, we investigated whether telbivudine has no cytotoxic effect on macrophages. the addition of up to lg/ml telbivudine to peritoneal macrophages freshly isolated from balb/cj mice had no toxic effects, as demonstrated by trypan blue exclusion ( % viable cells). to examine the efficacy of telbivudine on viral replication directly, isolated macrophages were treated with telbivudine at indicated concentrations. the starting concentration ( lg/ml) was chosen based on maximal concentration and area under the concentration-time curve from zero to infinity, and it was as close to twice the dose as that for patient administration [ , ] . telbivudine had no effect on mhv- replication from the starting dose of lg/ml to doses as high as and lg/ml (fig. ) . experiments were performed to test the ability of telbivudine to modulate the immune response of mhv- -infected macrophages. macrophages produced significantly higher levels of tnf-a and il- in response to mhv- infection in comparison with basal values. telbivudine significantly enhanced the production of tnf-a and il- in mhv- -infected macrophages compared with controls and this effect occurred in a dose-dependent manner (figs b,d) . a similar pattern of increase in tnf-a and il- mrna production was also observed in response to telbivudine (figs a,c) . however, telbivudine had no effect on tnf-a and il- levels or mrna production in lps-stimulated macrophages (figs a,b) . telbivudine improves survival and clinical outcome in vivo telbivudine increased the survival rate in mhv- -infected c h mice from . % to . % compared with the pbstreated controls (fig. a) . a composite clinical score was obtained by combining the clinical scores for all measured symptoms. telbivudine-treated mice maintained improved composite clinical scores in all conditional categories to the end of the study (fig. b) . in addition to improving overall survival and clinical scores, telbivudine reduced the histological signs associated with mhv- infection (fig. c) . the livers recovered from the pbs-treated mice showed remarkable necrosis by days after infection compared with the livers obtained from the uninfected control mice (the arrows in fig. c indicate necrosis). in contrast, although not completely free of disease, the livers recovered from the telbivudine-treated mice showed a marked reduction in histological evidence of hepatic necrosis (fig. c) . telbivudine treatment resulted in an additional . -fold increase in ifn-c production in sera compared with mhv- infected controls (table ). in contrast, a . -fold inhibition of il- production was observed in the telbivudine-treated group compared with the controls. telbivudine increases the total number of t cells, the percentage of ifn-c-and tnf-a-producing cd + t cells, and the percentage of ifn-c-producing cd + t cells phenotypic analysis of spleen cells from telbivudine-treated mice revealed an increase in the total number of t cells compared with pbs-and lamivudine-treated controls (fig. a) . the frequency of ifn-c-producing cd + and cd + t cells in the telbivudine-treated group was significantly increased compared with the pbs-and lamivudine-treated groups (figs b,c,f) . the frequency of tnf-a-producing cd + t cells in the telbivudine-treated group was increased significantly compared with the pbs-and lamivudine- treated groups (figs d,g) . the frequency of the tnf-a-producing cd + t cells was similar among all of the treatment groups (figs e,g) . no difference was observed in the frequency of il- -and il- -producing cd + and cd + t cells (data not shown). telbivudine has no effect on cytolytic function but downregulates pd-l expression on t cells to determine whether telbivudine treatment enhanced killer t-cell cytotoxicity, we performed cytotoxicity assays using spleen-isolated cd + t cells as effector cells. an increase in cytotoxicity (fig. a) was observed in all infected groups, but no differences were observed among groups with or without telbivudine treatment, suggesting that telbivudine has no effect on the cytolytic function of t cells. pd- and fasl expression were increased in infected groups compared with the uninfected group, but no differences were observed among infected groups (fig. b) . there were no significant differences for tumour necrosis factor receptor (tnfr)- , tnfr- , perforin or granzyme b expression on the cd + t cells among infected groups and the uninfected group (fig. c) . to further understand this phenomenon, we investigated the expression of pd-l and found that telbivudine treatment significantly downregulated the expression of pd-l on cd + and cd + t cells compared with pbs and lamivudine treatment (figs d,e) . this is the first study to demonstrate, both in vitro and in vivo, that telbivudine has immunomodulatory effects, independent of its potent antiviral activity. our data show that telbivudine has no inhibitory effects on the replication of mhv- in macrophages. this finding is consistent with the fact that telbivudine is highly selective for hbv and lacks activity against other viruses [ ] . this study showed that telbivudine, at concentrations that are achieved in vivo, enhanced the production of tnf-a and il- in balb/cj macrophages in vitro when challenged with mhv- . telbivudine was unable to cause a similar effect in lps-induced inflammatory cytokines. this may be because the mecha-nism of induction of tnf-a and il- by lps is different from that by virus [ ] . previous studies of the immune response during antiviral treatment with nucleoside or nucleotide analogues in patients with chb have shown that potent suppression of hbv replication is associated with enhanced t-cell reactivity and cytokine production [ ] [ ] [ ] . however, these studies in patients with chb do not allow dissection of whether the effect on adaptive immunity is due to direct immunomodulatory properties of the antiviral agent or a secondary to reduced viraemia and antigen levels. the advantage of our mouse model is that it allows investigation of potential immunomodulatory effects independent of antiviral activity. the use of mhv- , which produces a strain-dependent pattern of viral hepatitis in inbred strains of mice, has brought insights into the pathogenesis of viral hepatitis [ ] [ ] [ ] [ ] [ ] . a previous study using an mhv- virus-induced hepatitis model from our group showed that ribavirin preserves th cytokine production but inhibits th cytokine response; the study explored the remarkable increased sustained virological response post interferon and ribavirin combination therapy for hepatitis c patients [ ] . the results from the in vitro experiments on the mhv- infected macrophages support a further investigation of the immunomodulatory effect of telbivudine in vivo. because mhv- belongs to the coronavirus family of positive-stranded rna viruses, it is a suitable model with which to study the pure immunomodulatory effect of telbivudine without the influence of its viral inhibitory effect, which in turn may indirectly affect the immune system. in this study, telbivudine treatment significantly improved survival in mhv- infected c h mice and it improved clinical conditions and histological outcomes. the treatment of mhv- -infected c h mice with telbivudine resulted in the inhibition of il- production, and similar doses of telbivudine enhanced the production of ifn-c, and furthermore increased total t cell numbers, ifn-c-producing cd + and cd + t cells and tnfa-producing cd + t cells, indicating a predominantly th response post telbivudine treatment. one mechanism of the immune response involves naive cd + t cells, which, through the production of cytokines, control the development of immune-effector mechanisms concentrations of ifn-c and il- were measured in sera of c h mice days after infection. data are presented as mean ± sd from three separate experiments with three mice in each group. *p < . compared with pbs-treated mice. [ ] . a th response has been associated with host resistance whereas a th response has been associated with susceptibility in murine models of mhv- . studies have suggested that resistance to mhv- is associated with a predominant th response, the production of ifn, and cytotoxic t cells [ , ] . cytotoxic lymphocytes kill target cells by two major pathways: one is dependent on the exocytosis of granule effector molecules, including perforin and granzymes a and b; the other is dependent on the engagement of target cell tnfr- family death receptors (e.g. fas, tnfr- , tnfr- ) by effector cell-expressed fasl, membrane-bound or secreted tnf, or tnf-related apoptosisinducing ligand [ ] [ ] [ ] . our results demonstrate that fasl plays an important role in cytotoxic t lymphocyte (ctl) cytotoxicity to hepatocytes in mhv- -infected c h mice. however, telbivudine treatment did not promote ctl cytotoxicity. previous studies have established that tnf-a and ifn-c are key cytokines for non-cytolytic control of hbv replication [ ] [ ] [ ] . the release of these cytokines from t cells leads to inactivation of hbv without killing the infected hepatocytes. our results suggest that telbivudine is altering the balance of th /th cytokines which is independent of the direct antiviral effect against mhv- . programmed death- is a receptor that is expressed on a subset of activated t and b cells and pd-l (also known as b -h ) is expressed on both haematopoietic and parenchymal cells [ , ] . in chronic viral infection, cd t cells express high levels of pd- , but its ligand pd-l is also highly upregulated [ ] . in vivo blockade of the interaction of pd- with its ligand pd-l has been reported to enhance t-cell responses [ ] . reduction of pd- during the treatment of chb was shown to correlate with reduction of hbv dna levels [ ] . given the role of pd- /pd-l is an important inhibitory pathway in t-cell function, the effect of telbivudine on the expression of pd- and pd-l was investigated to further define the effect of telbivudine on t-cell response. although telbivudine does not alter the expression of pd- , of note is that it significantly downregulated pd-l expression on t cells in mhv- -infected c h mice. this result provides initial evidence for a role of telbivudine in this inhibitory pathway in regulating t-cell function that may favour the development of immune clearance of virus infection. however, the effects of telbivudine on pd- and pd-l expression on other cell types should be clarified in further studies. also, the underlying molecular mechanism requires further investigation. one of the proposed mechanisms is upregulation of the activity of cellular kinases to facilitate efficient phosphorylation. on the other hand, many nucleoside analogue-like substances [e.g. cpg-containing dna, as found in dna viruses and bacteria; viral dsrna; the viral mimic polyinosinic:polycytidylic acid (polyi:c)] can exert immunomodulatory activities via tolllike receptors. as telbivudine is a thymidine nucleoside analogue, it may exert immunomodulatory activities via tolllike receptors. in summary, the present study demonstrates that the beneficial effects of telbivudine in mhv- -induced hepatitis may be mediated by its effect on the immune response rather than the inhibition of viral replication in this animal model. the immunomodulatory effect of telbivudine may be explained partly by a shift in the balance between th -and th -like immune responses. this in turn may explain the benefit of telbivudine on the amelioration of mhv- infection and increased survival as well as improvements in clinical scores and liver histology. these data identify an immunomodulatory mechanism of telbivudine treatment in the mhv- -induced viral hepatitis model and provide an insight into a potential additional mode of action for the management of viral hepatitis infection. cellular immune response to hepatitis b virus-encoded antigens in acute and chronic hepatitis b virus 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infection molecular and functional analysis of the human prothrombinase gene (hfgl ) and its role in viral hepatitis the fgl /fibroleukin prothrombinase contributes to immunologically mediated thrombosis in experimental and human viral hepatitis novel mfgl antisense plasmid inhibits murine fgl expression and ameliorates murine hepatitis virus type -induced fulminant hepatitis in balb/cj mice th and th cells: different patterns of lymphokine secretion lead to different functional properties the immune response to mouse hepatitis virus: genetic variation in antibody response and disease resistance of naive mice to murine hepatitis virus strain (mhv- ) requires development of th , but not th response, whereas pre-existing antibody protects against primary infection fas and perforin pathways as major mechanisms of t cell-mediated cytotoxicity cytotoxic t cells deficient in both functional fas ligand and perforin show residual cytolytic activity yet lose their capacity to induce lethal acute graft-versus host disease lymphocyte-mediated cytotoxicity hepatitis b virus transgenic mice: insights into the virus and the disease noncytolytic control of viral infections by the innate and adaptive immune response non-cytolytic inhibition of hepatitis b virus replication in human hepatocytes expression of the pd- antigen on the surface of stimulated mouse t and b lymphocytes pd- : an inhibitory immunoreceptor involved in peripheral tolerance restoring function in exhausted cd t cells during chronic viral infection we thank jinshang hu for secretarial work and members of our institution for discussion and technical help. key: cord- - bbmo m authors: yao, qianqian; masters, paul s.; ye, rong title: negatively charged residues in the endodomain are critical for specific assembly of spike protein into murine coronavirus date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: bbmo m coronavirus spike (s) protein assembles into virions via its carboxy-terminus, which is composed of a transmembrane domain and an endodomain. here, the carboxy-terminal charge-rich motif in the endodomain was verified to be critical for the specificity of s assembly into mouse hepatitis virus (mhv). recombinant mhvs exhibited a range of abilities to accommodate the homologous s endodomains from the betacoronaviruses bovine coronavirus and human sars-associated coronavirus, the alphacoronavirus porcine transmissible gastroenteritis virus (tgev), and the gammacoronavirus avian infectious bronchitis virus respectively. interestingly, in tgev endodomain chimeras the reverting mutations resulted in stronger s incorporation into virions, and a net gain of negatively charged residues in the charge-rich motif accounted for the improvement. additionally, mhv s assembly could also be rescued by the acidic carboxy-terminal domain of the nucleocapsid protein. these results indicate an important role for negatively charged endodomain residues in the incorporation of mhv s protein into assembled virions. coronaviruses are a family of enveloped, positive-sense singlestranded rna viruses (spaan et al., ) . since the first disease caused by a coronavirus, feline infectious peritonitis, was described one century ago, these viruses have come to be recognized as important pathogens that cause a variety of diseases in respiratory, digestive, and nervous systems of avian and mammalian hosts (siddell, ) . before the identification of a new human coronavirus, severe acute respiratory syndrome-associated coronavirus (sars-cov) in , there were only two known human coronaviruses, both associated with the common cold (peiris et al., ; lai, et al., ) . in the past decade numerous new coronaviruses have been isolated, including three additional human coronaviruses and numerous previously unknown bat coronaviruses. a revised and updated taxonomy has divided the family into four genera-the alpha-, beta-, gamma-, and deltacoronaviruses (adams and carstens, ) . the coronavirus spike (s) protein, a glycosylated class i viral fusion protein, is critical for viral infectivity, species and tissue tropism, and pathogenesis (gallagher and buchmeier, ) . in many coronaviruses, including the prototype betacoronavirus mouse hepatitis virus (mhv), most s molecules incorporated into virions are cleaved by a cellular furin-like enzyme into two equal-sized subunits, s and s (de haan et al., ) . the receptor binding domain is located in the n-terminal subunit, s , while components involved in membrane fusion, such as the fusion peptide and heptad repeats, are located in the ectodomain portion of the c-terminal subunit, s (cavanagh, ; holmes et al., ; masters, ) . the carboxy terminus of s is composed of a hydrophobic transmembrane (tm) domain and a hydrophilic endodomain (endo), and virus-like particle studies originally mapped to these two domains the ability of s protein to be recruited by the membrane (m) protein for virion assembly (de haan et al., godeke et al., ) . endo is further divided into two regions of roughly equal size: a membraneproximal cysteine-rich motif and a carboxy-terminal charge-rich motif (bos et al., ; chang et al., ; godeke et al., ) . the cysteine-rich segment of endo is the target for multiple modifications by s-palmitoylation. a minimum number of cysteine residues is required for viral viability (yang et al., ) , and the cysteine-rich motif appears to be principally required for cell-cell fusion (bos et al., ; chang et al., ; ye et al., ; petit et al., ; shulla and gallagher, ; mcbride and machamer, a) . the charge-rich motif, on the other hand, has been shown to be the major determinant for s protein incorporation into assembling virions (ye et al., ; bosch et al., ) . however, some evidence also suggests an effect of the cysteine-rich motif on assembly (thorp et al., ) . targeted rna recombination is a reverse genetics system for coronaviruses that has been efficiently used to study the interactions of coronavirus structural proteins (masters and rottier, ; masters et al., ) , as well as for the expression of foreign genes engineered to replace the nonessential genes and of mhv (das sarma et al., ; hurst et al., ; yang et al., yang et al., , ye et al., ) . we previously combined both of these properties to develop a method to dissect the tm and endo domains of s protein and to distinguish between the effects of mutations on the assembly of s into virions versus other functions of s (ye et al., ) . in this strategy, one set of recombinants was created in which mutations were introduced directly into the s protein tm or endo and their effects on virus viability and growth properties were evaluated (fig. a) . a second set of recombinants was also generated in which wild-type s protein remained unaltered, and the same mutations were moved into the tm and endo domains of a heterologous membrane protein, designated hook (hk), which was expressed in place of the nonessential genes a/he. hk is small ( -kda), epitope-tagged synthetic protein composed of a signal peptide, ectodomain, tm, and endodomain from various cellular sources (chesnut et al., ; ye et al., ) . the effects of the mutations on the incorporation of hk protein into purified virions could then be directly assayed, independent of whether they had an impact on s protein (fig. b ). in the current study, we analyzed the effects of the endo charge-rich motif on virion incorporation of mhv s protein through substitutions of the homologous regions from the alphacoronavirus porcine transmissible gastroenteritis virus (tgev), the betacoronaviruses bovine coronavirus (bcov) and sars-cov, or the gammacoronavirus avian infectious bronchitis virus (ibv). the results showed that the ability of recombinant mhvs to accommodate tm and endo domains from other virus species depended on their phylogenetic relatedness to mhv. thus, the bcov and sars-cov substitutions were completely allowed, while the tgev substitution retained only partial functionality, and the ibv substitution was lethal. further analysis of tgev substitutions and revertants obtained there from revealed the importance of negatively charged endo residues for the incorporation of mhv s protein into assembled virions. finally, we were able to replace the mhv endo charge-rich motif with the acidic carboxy-terminal domain of the nucleocapsid (n) protein. there are currently some twenty to thirty species of coronaviruses, which are now classified into four genera (formerly groups), designated the alpha-, beta-, gamma-, and deltacoronaviruses (adams and carstens, ; spaan et al., ) . although the s portions of coronavirus s proteins show some degree of conservation, the tm and endo domains are highly divergent, with the exception of a conserved cluster of seven hydrophobic residues (wpwyvwl) at the start of tm. to evaluate the functionality of different c-terminal sequence motifs in the mhv s protein, we constructed two sets of mutants in which the ectodomain of either s protein or hk protein was fused to the tm and endo domains from tgev (an alphacoronavirus), bcov (a betacoronavirus), sars-cov (a betacoronavirus), or ibv (a gammacoronavirus) ( fig. a) . substitution of the c-terminal sequence from bcov s did not significantly hinder the assembly of mhv s into virions. as shown in fig. b , the chimeric s recombinant formed slightly smaller plaques than wild-type mhv. correspondingly, the bcovsubstituted hk protein was incorporated into the virions at a lower level than the mhv version of hk protein ( b). more significant assembly was observed with the c-terminal replacement from sars-cov s. the chimeric s recombinant formed plaques similar in size and morphology to the mhv wild type, and much more sars-cov-substituted hk protein became incorporated into purified virions. by contrast, substitution of the c-terminal sequence from ibv s protein was lethal to recombinant mhv; consistent with this finding, ibv-substituted hk protein was not incorporated into purified virions. intermediate between these extremes, an mhv s mutant containing the c-terminus of the tgev s protein was markedly debilitated but viable. this recombinant had a much lower titer and formed smaller, heterogeneous plaques compared to those of wild-type mhv (larger plaques in fig. b were later found to be revertants). however, there was no detectable incorporation of the tgev-substituted hk protein into virions. in general, the toleration of mhv s protein for replacement of its tm and endo domains by the homologous regions of other s proteins was highest within the same genus (betacoronaviruses) and marginal for the somewhat more closely related alphavirus genus. a substitution originating from the more distantly related gammacoronavirus genus was unallowed. we previously demonstrated, through the construction of deletion and point mutations, that the charge-rich motif of endo has a dominant role in the incorporation of s protein into virions (ye et al., ) . we thus hypothesized that this motif might be the critical element allowing or precluding the substitution of c-termini from other coronavirus s proteins in place of that of mhv. to test this notion, we constructed two groups of recombinant mhvs with chimeric c-terminal sequences (fig. a ), in each case as separate s protein and hk protein mutants. in mut-ssm and mut-ttm, the tm and the cysteine-rich motif were from sars-cov s or tgev s, respectively, while the charge-rich motif was from mhv s. in mut-mms and mut-mmt, the tm and the cysteine-rich motif were from mhv s, and the charge-rich motif was from either sars-cov s or tgev s, respectively. for negative controls, we used the truncation mutant mut- (mutant Δ in ye et al., ) , which essentially lacks the entire mhv charge-rich motif. mut- is minimally viable as an s protein mutant and its hk protein counterpart fails to be incorporated into virions. as we anticipated, recombinants bearing the charge-rich motif of mhv s (mut-ssm and mut-ttm) or the charge-rich motif of sars-cov (mut-mms) produced wild type-like plaques (fig. b) and their corresponding hk proteins were incorporated into virions (fig. c) , irrespective of the source of their tm and cysteine-rich motifs. conversely, the s protein recombinant containing the tgev charge-rich motif (mut-mmt) produced tiny and irregular plaques (fig. b ) that were similar to the mutant with the entire c-terminus of tgev s (fig. b) , despite that fact that both the tm and cysteine-rich motif of mut-mmt were derived from mhv s. moreover, the mut-mmt hk protein exhibited no incorporation into virions (fig. c) . these results confirmed that the charge-rich motif in endodomain plays a more important role in the specific assembly of mhv s into virions than does the tm domain or the cysteine-rich motif. reverting mutations in tgev chimeras improved s assembly by eliminating positively charged residues in the endodomain mhv s protein mutants containing the entire carboxy terminus or just the charge-rich motif of tgev s (mut-tgev and mut-mmt) produced irregular plaques (figs. b, b, a). most of these plaques were very small and morphologically similar to those produced by the c-terminal truncation mutant, mut- (ye et al., ) . larger plaques arose following multiple passages of the tgev chimeric mutants, while plaques produced by mut- maintained a stable small morphology after passage under the same conditions. a number of larger plaques were randomly picked and subjected to multiple rounds of plaque purification, during which their large-plaque morphology remained stable (fig. a) . rt-pcr and sequencing analyses of the relevant segment of the s gene and downstream genes showed that each of the isolated revertants had acquired a deletion in endo (fig. b) . moreover, the observed deletions fell into two classes. in one class (revertants tgev-r and mmt-r ), the charge-rich motif of tgev s was totally deleted, and a new carboxyterminal sequence of seven residues, tenlnnl, was created through juxtaposition of a normally untranslated open reading frame beginning nucleotides downstream of the s gene. in the second class (revertants tgev-r and mmt-r ), the tgev s protein carboxy terminus was retained, but a portion of the charge-rich motif containing two adjacent positively-charged arginine residues was deleted. no other mutations were observed in these revertants in the s tm domain or in the structural proteins e, m, or n (data not shown). significantly, these reverting mutations displayed a tendency to delete positively-charged residues, either two arginines (rr) from the charge-rich motif of tgev s or two lysines (kk) from the cysteine-rich motif of mhv s (fig. b ). this suggested that positively-charged residues in those positions were harmful to the assembly of mhv s. additionally, the heptapeptide tenlnnl, created by two of the reverting mutations, introduced one negatively-charged glutamic acid (e) plus four polar residues (t and n) that might be beneficial to the assembly of s. to directly test the role of the heptapeptide tenlnnl in mhv s protein incorporation, three further sets of mutants were generated (fig. a ). mut- was designed to reconstruct the revertant mmt-r . mut- was a mimic of revertant mmt-r , but did not contain the heptapeptide. finally, mut- maintained just three residues (ten) of the heptapeptide, to check the importance of the negatively-charged e residue. the chimeric s recombinant of mut- formed homogenous plaques (fig. b ) whose morphology and size relative to the wild type was very similar to that of revertant mtt-r (fig. a ). in accord with this, the mut- hk protein was incorporated into virions almost as well as wild-type hk protein (fig. c ). this confirmed that the identified deletion in revertant mtt-r was indeed responsible for its observed gain of s protein function. the chimeric s recombinant of mut- produced plaques that were smaller than those of mut- but were still larger and clearer than those of the mut- s-mutant control (fig. b) . however, both the mut- and mut- hk proteins displayed weak incorporation into virions (fig. c) . thus, the tripeptide ten is not sufficient to fully recapitulate the advantage conferred by the heptapeptide tenlnnl. curiously, the weak incorporation into virions of mut- hk protein was still stronger than the consistently undetectable incorporation of mut- hk protein, despite the fact that the mut- endodomain was residues longer than that of mut- . we speculate that, in the absence of multiple negatively charged residues elsewhere in endo, the deletion of two adjacent positively-charged lysines (kk) in mut- removed a charge and spatial hindrance, thereby allowing some limited enhancement of s (or hk) protein incorporation into virions. the coronavirus m protein is the central organizer of virion assembly and budding through interactions that it carries out with both the s and the n proteins (masters, ) . m-s interactions encompass the carboxy-terminal endodomain of m protein but do not involve the extreme carboxy terminus of m protein (de haan et al., ; mcbride and machamer, b) . conversely, m-n interactions are predominantly or solely mediated by the extreme carboxy terminus of m protein and the carboxy-terminal domain of n protein (kuo and masters, ; hurst et al., ; verma et al., ; hurst et al., ) . to determine if it was possible to replace the m-interacting region of the s protein with the m-interacting region of the n protein, we created s protein and hk protein mutants in which the charge-rich motif was replaced with mhv n protein domain (mut-mn) (fig. a) . we also made recombinants substituting the related domain of the bcov n protein (mut-bn) or else the short endodomain of the mhv hemagglutinin-esterase (he) protein (mut-he). he is a membrane envelope structural protein that is expressed in a subset of betacoronaviruses (lissenberg et al., ) ; the m-interacting region of he has not been mapped. the chimeric s recombinant of mut-mn formed plaques that were smaller than those of the wild type but slightly larger than those of mut-bn or mut-he (fig. b) . the mut-mn hk protein displayed weaker incorporation into virions than wild-type or mut- hk protein, but incorporation of mut-mn hk protein was substantially stronger than that of mut-bn hk protein (fig. c ). from these results we conclude that the mhv n protein domain could partially function to replace the s protein endodomain charge-rich motif. however, it is unlikely that this transplanted n domain appropriated the normal m-n interaction, since the bcov domain in mut-bn was unable to play the same role. in the mhv m-n interaction, domain of bcov n protein is entirely functionally interchangeable with its mhv counterpart (hurst et al., ) . additionally, our results show that the he protein endodomain could not replace the s protein charge-rich motif. the coronavirus s protein is the largest described class i viral fusion protein, comprising as many as amino acid residues. its ectodomain, which contains the elements of receptor recognition and membrane fusion, accounts for almost the entirely of the protein. a small tm domain and endo region, at most some amino acid residues, governs membrane anchoring and incorporation of s into virions. at the boundary between these two regions, either proximal to, or within the outer leaflet of the membrane, there is a cluster of aromatic hydrophobic residues (wpwyvwl) that is highly conserved among all coronaviruses (bos et al., ; sainz et al., ) . in contrast to this conservation, many of the other parts of s protein show considerable divergence, both across genus boundaries and even within a given genus. the functional division between the s protein ectodomain and the tm plus endo was first demonstrated in virus-like particle studies . it was later shown that exchanging the s ectodomain at the wpwyvwl motif could switch the species specificity of coronaviruses, provided that the native transmembrane domain and endodomain were retained. this served as the basis to establish a strong host-range-based selection for targeted rna recombination as a reverse genetics system (kuo et al., ; haijema et al., ) . we have used this system to dissect the requirements for s incorporation into virions through construction of mutations in the mhv s protein tm plus endo region. since such mutational alteration of s may possibly affect s maturation, trafficking, or membrane fusion, we also constructed the same mutations in an epitope-tagged artificial membrane protein, hk, which was derived from heterologous components (originally in phook , chesnut et al., ) . this strategy previously allowed us to map the assembly competence of s to endo (ye et al., ) , a conclusion also reached by others (bosch et al., ) . in the present work, we further probed s protein incorporation by engineering interspecies chimeras in which the tm and endo segments of mhv s were replaced with those of closely or distantly related coronaviruses. we observed that these domains taken from the betacoronaviruses bcov and sars-cov could readily substitute for their mhv counterparts (fig. ) . the functionality of the bcov components in mhv is not surprising, given the generally high sequence homology between many proteins of these two viruses, and the interchangeability of other domains between them hurst et al., ) . the ability of the sars-cov s protein tm and endo to operate in mhv further underscores the phylogenetic relatedness of these two viruses. although initial characterizations of sars-cov placed it in a unique grouping, it has since become firmly established that this virus falls in the betacoronaviruses and that mhv and bcov are among its closer relatives (snijder et al., ; weiss and navas-martin, ) . s protein tm and endo domains derived from other genera of the coronavirus family were much less effective in mhv s. the most far-reaching substitution, that of ibv, was lethal. the tgev substitution, however, was marginally functional. construction of additional chimeras within the tm plus endo region more finely mapped the major determinant of virion incorporation to the charge-rich motif of endo (fig. ) , consistent with our previous results (ye et al., ) . examination of allowed and unallowed substitutions suggested that the number and location of negatively-charged residues in the charge-rich motif was critical for s protein incorporation into virions. additional work provided strong support for this surmise. first, we were able to obtain revertants of tgev chimeras that restored some of the functionality of the chimeric endo segments. analysis of these mutants showed that the quality they had in common was the gain of negatively-charged residues and the loss of positively-charged residues in the charge-rich motif (figs. and ) . second, replacement of the charge-rich motif of s protein with the acidic carboxy-terminal domain of the mhv n protein at least partially restored endo function (fig. ) . the evidence suggests that this came about through mimicking the essential attributes of the charge-rich motif and not through the interaction that n domain normally makes with the extreme carboxy terminus of m protein. the bcov n domain was unable to replace the endo charge-rich motif of mhv s, even though bcov n domain is completely active as a replacement for the mhv n domain (hurst et al., ) . the region of the mhv m endodomain that interacts with s protein has been previously localized to be upstream of the extreme carboxy terminus of m (de haan et al., ) . our data suggests that this s-interacting surface must have one or more key positively-charged residues that contact the negatively-charged charge-rich motif of the mhv s endo. it also appears that endodomain interactions with m protein are not the sole means by which viral membrane proteins can partition into assembling virions. the failure of the mut-he mutant to become incorporated into virions suggests that there exist other types of interactions that lead to the effective inclusion of he protein in the viral envelope. additionally, it is possible that the constraints on s protein endodomain composition reflect its more complex range of interactions with membranes. thus, a charge-rich region of a certain length may be uniquely required by s protein for virion assembly, in order to counterbalance the properties of other elements required for the fusion function of s, such as the wpwyvwl motif and the cysteine-rich motif. the m protein endodomain has thus far been resistant to structural determination. thus, there is a large gap in our understanding of the complexity of its interactions with multiple proteins, including other molecules of m. the identification and characterization of all m protein interactions within the coronavirus virion remains an important future challenge. mouse clone ( cl ) cells were used for propagation of wild-type mhv-a and recombinant mhvs. mouse fibroblast l cells were used for plaque assays and plaque purification of wildtype and recombinant mhvs. felis catus whole fetus cells (fcwf) were used to grow the interspecies chimeric virus fmhv.v in which the ectodomain of the s gene of mhv is replaced with that of fipv (goebel, et al., ; kuo, et al., ) . the three cell lines were cultured in dmem (gibco, invitrogen) supplemented with % fbs and . % sodium bicarbonate in a humidified % co atmosphere at c. for the generation of recombinants in which mutations were introduced directly into the s protein tm and endo region, donor rna transcription vectors were derived from plasmid pmh (kuo et al., ) . dna fragments with deletion or replacement mutations were created as described previously (yang et al., ; ye et al., ) using primer-pair pcr and splicing overlap extension pcr. mutated fragments were then transferred to pmh by replacement of the unique mlui-sbfi fragment running from the s ectodomain to the intergenic region downstream of s. for the generation of recombinants in which mutations were introduced into the hk protein tm and endo region, donor rna transcription vectors were derived from the previously described vector phkp (ye et al., ) . the pmh -derived plasmids were used as the templates for the amplification of pcr fragments of the mutated tm and endo regions, which were then used to replace the unique sali-asci fragment of phkp that contains the hk protein tm and endo region. all constructs were identified by restriction digest analysis, and cloning sites and junctions in plasmids were confirmed by sequencing. mhv mutants were produced by targeted rna recombination (masters and rottier, ) . briefly, fcwf cells were infected with fmhv and digested with trypsin at h post-infection (hpi) and washed twice with mg ++ -and ca ++ -free pbs. for preparation of donor rna transcription, the constructed pmh or phkm plasmids were digested with paci and transcribed into mrna using mmessage mmachine t ultra kit (ambion). approximately μl of each recombinant transcript was used for transfection of ∼ infected fcwf cells. rna transfection was performed using a gene pulser xceii electroporation system (bio-rad), with one pulse at settings of μf and . kv. the co-transfected fcwf cells were loaded onto l monolayers grown in -well plates. cytopathic effects were monitored and the supernatants were collected at , , and hpi. recombinant mhvs were purified by two rounds of plaque purification on l cells, and all mutations were verified by rt-pcr and sequencing. l cells were grown in -mm dishes to - % confluence and infected with ml of media containing viruses at dilutions ranging from − to − . seven ml of . % agar (amresco) in dmem with % fbs was overlaid onto cells at hpi. plaques were picked between and hpi. for plaque staining, ml of agar with . % neutral red (sigma-aldrich) was overlaid onto cells. six to eight hours later, the stained plaques were counted or photographed using a white light transilluminator (upland). mhv recombinants expressing wild-type and mutant hk proteins were purified as described previously (ye et al., ) . briefly, viral supernatants were precipitated by polyethylene glycol, and resuspended virus was isolated through two cycles of gradient centrifugation with to % potassium tartrate contrasting to % glycerol in tme buffer ( mm tris-maleate, ph . and mm edta). gradients were centrifuged at ,  g in a beckman sw rotor at c for - h. after centrifugation, viral bands were collected from the gradients and diluted with tme with mm nacl. virions were pelleted through a glycerol cushion by centrifugation for h at ,  g and resuspended in pbs. infected cl cells were washed twice with ice-cold pbs and collected in ipp buffer ( mm tris-hcl, ph . , mm nacl, . % nonidet p- ) containing completer protease inhibitor cocktail (roche). cell lysates were incubated on ice for min and clarified by centrifuging at ,  g for min at c. protein samples from either lysates or purified virions were mixed with an equal volume of  sample buffer and heated at c for min. one set of cell lysates and two identical sets of purified virions samples were separated by sds-polyacrylamide gel electrophoresis. after electrophoresis, one gel with a set of purified virions was stained with coomassie blue to illustrate n protein. another gel with a set of purified virions and the gel with cell lysates were transferred onto pvdf membranes using a criterion blotter (bio-rad). membranes were blocked for h in % non-fat milk and incubated with . μg/ml mouse mab to ha tag (roche) overnight at c, followed by incubation with horseradish peroxidase-conjugated secondary antibody (ge, amersham). the signal was developed using ecl system (ge, amersham). ratification vote on taxonomic proposals to the international committee on taxonomy of viruses mutational analysis of the murine coronavirus spike protein: effect 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coronaviridae unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group lineage order nidovirales palmitoylations on murine coronavirus spike proteins are essential for virion assembly and infectivity importance of the penultimate positive charge in mouse hepatitis coronavirus a membrane protein coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. microbiol replication of murine coronavirus requires multiple cysteines in the endodomain of spike protein partial deletion in the spike endodomain of mouse hepatitis virus decreases the cytopathic effect but maintains foreign protein expression in infected cells genetic analysis of determinants for spike glycoprotein assembly into murine coronavirus virions: distinct roles for charge-rich and cysteine-rich regions of the endodomain this study was supported in part by grants from the national institutes of health (national institute of allergy and infectious diseases), usa (r ai -p.s.m.) and the national natural science foundation, china ( -r.y.). key: cord- -q inrxcm authors: lai, michael m. c. title: sars virus: the beginning of the unraveling of a new coronavirus date: - - journal: j biomed sci doi: . /bf sha: doc_id: cord_uid: q inrxcm severe acute respiratory syndrome (sars) virus caused a severe outbreak in several regions of the world in . the virus is a novel coronavirus, which may have an origin in wild animals such as civet cats in southern china. its genome structure, gene expression pattern and protein profiles are similar to those of other coronaviruses. however, distinct patterns of several open reading frames in the sars virus genome may contribute to its severe virulence. the potential mutability of the coronavirus genome may pose problems in the control of future sars outbreaks. the mechanism of sars pathogenesis may involve both direct viral cytocidal effects on the target cells and immune-mediated mechanisms. the life cycle of the sars virus is largely unknown; however, based on the analogy with other coronaviruses, several potential targets for antiviral development are identified. vaccines offer an important preventive measure for possible future recurrences of sars, but the prospect for their development is still unknown because of the uncertainty regarding the role of immune responses in sars virus pathogenesis. the comparative studies of other coronaviruses offer insights into the understanding of sars virus. severe acute respiratory syndrome (sars) virus caused a severe outbreak in several regions of the world in . the virus is a novel coronavirus, which may have an origin in wild animals such as civet cats in southern china. its genome structure, gene expression pattern and protein profiles are similar to those of other coronaviruses. however, distinct patterns of several open reading frames in the sars virus genome may contribute to its severe virulence. the potential mutability of the coronavirus genome may pose problems in the control of future sars outbreaks. the mechanism of sars pathogenesis may involve both direct viral cytocidal effects on the target cells and immune-mediated mechanisms. the life cycle of the sars virus is largely unknown; however, based on the analogy with other coronaviruses, several potential targets for antiviral development are identified. vaccines offer an important preventive measure for possible future recurrences of sars, but the prospect for their development is still unknown because of the uncertainty regarding the role of immune responses in sars virus pathogenesis. the comparative studies of other coronaviruses offer insights into the understanding of sars virus. the identification of a novel coronavirus as the etiological agent of severe acute respiratory syndrome (sars) has given an impulse to research on the coronavirus family, which has long been relegated to the status of a scientifically interesting but medically unimportant virus family. it is not that coronavirus has never been important; in fact, coronavirus infections of farm animals, such as pigs or chickens, are so common and devastating that vaccinations against these viruses are routinely performed. in humans, coronavirus infections are responsible for almost / of common colds in wintertime [ , ] , and the virus has been repeatedly implicated, but never confirmed as one of the causative agents of multiple sclerosis [ , ] . nevertheless, the lack of a firm association of coronaviruses with any serious human illnesses had dampened the public's interest in this virus family until the sudden emergence of the sars coronavirus [ , , ] , which caused the first new infectious disease of this millennium. the numerous past studies of the coronavirus family then quickly provided a blueprint for understanding the sars virus. however, the contagiousness and high mortality rate of the sars virus are unparalleled to the other known human and animal coronaviruses; thus, sars coronavirus is unique in multiple ways. the understanding of this virus, including its origin, molecular properties and pathogenesis, will be crucial for the future management of this infectious disease. since the studies on the sars virus are very limited so far, this review aimed at investigating the possible properties of the virus based primarily on comparative studies of other coronaviruses. coronaviruses are divided into three antigenically distinct groups, which are also consistent with the genetic relatedness of these viruses [ ] . group i consists of human coronavirus e (hcov- e), porcine transmissible gastroentefitis virus (tgev), porcine respiratory coronavirus (prcv), feline infectious peritonitis virus (fipv) and enteritis virus (fecov), canine coronavirus (ccov) and others. group ii consists of human coronavirus oc (hcov-oc ), mouse hepatitis virus (mhv), bovine coronavirus (bcov) and others. group iii consists of avian species, including chicken infectious bronchitis virus (ib¥) and turkey coronavirus (tcov). the newly discovered sars virus cross-reacts with antibodies of group i coronaviruses [ ] . however, its genetic sequence indicates that it belongs to none of these three groups. it is of about equal distance from both groups ii and iii in the coronavirus family phylogenetic tree in nucleic acid or protein sequence [ , , ] . a similar conclusion is reached irrespective of which viral rna region is used for comparison. thus, sars virus represents a new group of coronavirus. although this virus most likely originated from a wild animal (see next section), the sars virus has been well adapted in humans, as evidenced by the high person-to-person transmissibitity of the virus. thus, it is most appropriately named human coronavirus sars strain. however, its official taxonomic status remains to be decided. since the first reported occurrence of sars in southern china, speculation on the origin of sars virus has largely focused on the animal species in that region. because the genetic sequence of sars virus is distinct and distant from that of any known coronaviruses in domestic animals, the sars virus most likely originated from a wild animal. the search for coronaviruses in wild animals eventually turned up a coronavirus in civet cats in a market in guangdong province. the civet cat coronavirus is very closely related to the sars virus, with more than % sequence homology [ , a, ] . recent reports indicate that sars virus is distinct from the civet cat virus by limited deletions and mutations. the extent of the coronavirus infection among wild animals is not yet clear [ a] . the animal handlers appear to be at risk for the infection as a higher proportion of them have antibodies against sars virus [ a]. however, none of these data have so far been confirmed. although plausible, the civet cat virus as the origin of sars virus remains to be established. it is possible that civet cats were infected with the virus from another animal species. in either case, the critical questions are whether there is extensive horizontal transmission between animals, and whether the jump of the virus from animals to human was a rare and accidental event or portends frequent occurrences in the future. the answers to these questions will determine whether these animals are viable reservoirs for future sars outbreaks. thus, epidemiological studies of coronavirus infections in wild animals are critical tbr control of future sars outbreaks. although coronaviruses, in general, infect only the animal species of their natural origin, they are known to jump to other species relatively easily. the human coronavirus oc is very closely related in genetic sequence to bcov [ , ] ; thus, there is no doubt that the virus jumped from one species to the other. bcov has also been reported to infect humans occasionally, causing diarrhea [ , ] . when mhv was passaged in tissue culture cells, the virus eventually adapted to growing in human cells [ , ] . although the mechanism of such an adaptation is not known, it is likely that it was the result of mutations in certain viral genes. thus, there is considerable latitude in species specificity of coronaviruses. so far, sars virus has been shown to infect humans and macaque monkeys [ ] . whether it can infect other animal species in natural infections and experimental inoculations may determine the extent of natural reservoirs for sars virus and the feasibility of establishing animal models for it. complete sars virus genome sequences have been determined from more than twenty different isolates so far. these sequences showed extremely high conservation, indicating that they were all derived from a common source and did not diverge significantly during the first [ , ] . sequence comparison with those of orfs of the other known coronaviruses reveals a similar pattern of gene organization, namely, gene la-lb (replicase and protease genes), spike (s), envelope (e), membrane (m) and nucleocapsid (n) in the order of ' to ' ends [ ] . notably, the sars virus genome does not encode a hemagglutinin-esterase (he) protein, which is present in most of the group ii coronaviruses. interspersed between these well-characterized genes are a series of orfs of unknown functions. at a quick glance, the sars virus rna has rfs between the s and e genes and an additional - rfs between the m and n genes. this gene organization most closely resembles that of group iii coronaviruses. in the sars virus genome, the organization of gene la-lb, which accounts for more than two-thirds of the viral rna, is very similar to that of the murine coronavirus mhv, except that it contains only one papain-like protease (plpro- ) ( fig. ) . thus, the plpro- of sars virus is likely responsible tbr the cleavage of all the nterminal proteins of gene l a, which is normally carried out by plpro- in murine comnavirus [ ] . it is noted that group iii coronaviruses have only one plpro. however, the sars virus genome has remnants of the plpro-i sequence; furthermore, the sars virus has the equivalent of the leader peptide (p ), which is missing in the group iii viruses. the remaining sequences of gene la-lb are highly conserved among all of the coronaviruses [ , ] . therefore, orfs la and lb are likely translated into a polyprotein by a ribosomal frameshifting mechanism [ ] . all of the potential gene products in gene la-lb are relatively conserved between sars and other coronaviruses. the c-like protease ( clpro) is likely responsible for the cleavage of all the remaining proteins in gene a- b [ ] . based on the predicted cleavage site specificity, the sars virus gene la-lb is likely processed into thirteen final protein products. the functions of these gene products are mostly unknown; nevertheless, they are likely involved in viral rna replication. in mhv, all the rnanegative temperature-sensitive mutants are mapped in the genetic regions covering almost the entire gene [ ] . [ ]. it should be noted that some of the protein-processing intermediates, in addition to the final products, may also function in viral replication. overall, from the perspective of gene organization, the "-half of the sars virus genome appears to be more closely related to group ii viruses, whereas the "-half more closely resembles group iii virnses. therefore, sars virus may have been derived from recombination between a group ii and a group iii virus. however, the published sequence analysis indicated that the entire sars virus rna resembled that of group ii viruses; no evidence of recombination was noted [ , ] . since the genetic sequence of sars virus is approximately equidistant between group ii and iii viruses, the possible presence of recombination might not have been evident from sequence analysis alone. regardless, recombination is probably not the triggering event for the jumping of the virus from animals to humans, since the recombination most likely occurred between the ancestral coronaviruses. coronavirus genes (except for gene ) are typically expressed from subgenomic mrnas, which share a common leader sequence at the '-end, but initiate at different places from the consensus intergenic sequences or transcription regulatory sequences (trs) in the genome and extend toward the '-end of the genome [ ]. the transcription initiation sequences are typically rich in u, c, and a residues and highly conserved in front of each gene. each mrna (except for the smallest) is physically polycistronic, but can be used to translate only the '-most orf. in rare situations, several orfs are translated from a single mrna, e.g. mrna of ibv and mrna of mhv, by internal initiation or other mechanisms, such as an internal ribosomal entry site, which are not completely understood. these unconventional translation mechanisms are not efficient; as a result, these gene products are usually not very abundant. these orfs typically encode viral nonstructural proteins, except for the e protein in some coronaviruses. significantly, the orfs between the structural protein genes are extremely heterogeneous among different coronaviruses in terms of the number of potential gene products and the method of their expression. they are not essential for viral replication in cell culture; however, recent studies suggest that deletions of these 'nonessential' orfs could result in the reduction of viral virulence [ , ] . conceivably, some of these orfs in sars virus could be responsible for the high virulence of this virus. a report shows that five subgenomic mrnas were detected in vero cells infected with the sars virus [ ] . thus, some of the orfs are expected to be expressed by internal initiation if they are translated at all. however, it is possible that additional subgenomic mrnas are transcribed; the amounts of the various subgenomic mrnas vary tremendously among different coronaviruses, depending on the strength of the transcription-start signals [ ] . the less abundant subgenomic mrnas frequently were missed in northern blot analysis. indeed, the analysis of the sars virus genome detected many more consensus intergenic sequences (ucuaaac and related sequences), which can potentially be used for transcription initiation [ , ] . a recent study detected eight subgenomic mrna species in sars virus-infected cells [ ] . in general, coronaviruses have four envelope proteins: s, m, e and he. the spike protein forms the characteristic spikes that are the namesake of the virus. the s protein is often cleaved into s and $ domains by intra-and extracellular proteases; the cleavage often enhances the viral infectivity [ ] . however, the s protein of some viruses, such as feline, is not cleaved, and yet the virus is fully infectious [ ] . sequence analysis of the s protein of sars virus suggests that it will not be cleaved. the spikes bind to the receptor on the target cells; the receptor-bind-ing domain is typically localized in the n-terminus of the s domain [ , ] . the spike consists of oligomeric structures, which are formed by the heptad repeats in the $ domain ( fig. ) . typically, there are two heptad repeats in the coronavirus $ proteins. $ also contains a fusion peptide sequence, which is responsible for the fusion activity associated with coronaviruses. significantly, the optimum ph for the spike-induced fusion differs from virus to virus; while most mhv strains cause fusion at the neutral ph [ ] , some variant viruses induce fusion at ph . [ ] . since the ph requirement of membrane fusion dictates the mechanism of virus entry (either by fusion at the plasma membrane or by endocytosis), different coronaviruses may use different entry mechanisms. sars virus also causes syncytia formation in vivo, but not in the cultured vero cells [ ] . as the s protein of sars virus possesses most of the features of the s proteins of other coronaviruses, it will be interesting to know the conditions, such as protease sensitivity and ph dependence of membrane fusion, that enhance the sars virus infectivity. it should be noted that both s and $ contain the neutralization epitopes of mhv [ ] and that there is a hypervariable region in s , which is frequently mutated or deleted in the coronaviruses. such mutations very often change the biological or pathogenic properties of the virus. the m and e proteins are the minimum protein units for virus assembly [ , ] . both are integral membrane proteins. m protein resides not only on the viral envelope, but in the viral internal core as well [ ] . in the cells, m protein is anchored in the golgi complex, thus dictating the site of virus assembly to the er-golgi complex [ ] . the expression of m and e proteins together is sufficient to trigger the formation of virus-like particles (vlp). when s protein is coexpressed with m and e proteins, the s protein is incorporated into vlp with presumably authentic conformation. such a vlp can infect cells [ ] . thus, these vi_ps will be an excellent candidate as a potential vaccine. intriguingly, it has recently been shown that e protein may not be absolutely required for viral infectivity [ ] . sars virus has comparable structural proteins. he protein is seen only in some group ii coronaviruses; it is not required for viral infectivity even in those viruses (e.g. mhv, hcov-oc ) that contain the he protein. however, he protein may be involved in the infection of bcov, as a monoclonal antibody against he protein can inhibit bcov infection [ ] . the he protein binds to certain sialic acid residues and possesses an acetyl-esterase activity. it is conceivable that he protein provides the initial binding contact between the virus and the target cells; however, subsequent tight and specific binding may be mediated by the s protein. he protein bears sequence homology with the hemagglutinin protein of influenza c virus, thus prompting suggestions that recombination has taken place between an ancestral coronavirus and an influenza virus [ ] . sars virus does not encode the he protein. the final structural protein is n protein, which likely interacts with viral rna and makes up the viral core and nucleocapsid. it is interesting to note that the coronavirus particles appear to include both an icosahedral core and an internal helical nucteocapsid [ ] . n protein is present in both structures. sars virus has typical n protein structural motifs, including several rna-binding domains. coronaviruses have an envelope that reflects the lipid composition of the cellular membranes. however, remarkably, coronavirus is one of the very few enveloped viruses that are able to cause enteric infections. thus, the viral envelope certainly can resist the harsh environment of the gastrointestinal tract, namely, the acidic environment of the stomach and the bile and lytic enzymes of the small intestines. the structural and chemical basis for such unusual resistance is not yet clear. correspondingly, sars patients very often show gi symptoms, and the virus is usually detected in the stool [ ] . therefore, the envelope of sars virus likely consists of unusual structures. indeed, it has been shown that sars virus can survive in diarrheal stool for as long as days and on a dry surface for h (world health organization bulletin). these unusual envelope properties have implications for the control of the sars outbreaks. as an rna virus, sars virus can be expected to undergo mutation at a very high frequency. with an estimated error frequency of x . for rna-dependent rna polymerases in general, the sars virus genome can be expected to accumulate an average of three mutations per round of rna replication. thus, sars virus, like all other rna viruses, probably consists of a collection of rna quasispecies [ ] . such a high frequency of mutation implies that the tkna viral genome is inherently limited in size. coronaviruses are obvious aberrations. yet, despite having an extraordinarily long rna genome, sars virus and other coronaviruses appear to be relatively genetically stable. to date, the predominant rna species from different sars patients appear to be quite homogeneous. the various sars virus isolates from different geographical regions differ by no more than ten amino acids in the entire genome. it is not clear whether these differences confer any differences in the biological properties of the virus. nevertheless, they allow epidemiological tracing of the virus transmission routes. it appears that two different lineages of the sars virus can be independently traced [ ] . whether the virus will undergo a higher frequency of mutation in the future once seasonal changes occur or after specific drug treatments are introduced is an issue of concern. however, there has been no investigation of the nature of rna quasispecies of sars virus derived from different patients so far. such rna quasispecies may represent potential sources of viral divergence. coronaviruses have another powerful genetic means for evolution: rna recombination, which occurs at a very high frequency [ ] . in theory, recombination may not only introduce genome alterations but, paradoxically, balance the deleterious effects of mutations by removing the undesirable defects [ ] . this ability may explain why coronavirus can maintain such a long rna genome. the ability of coronaviruses to recombine may stem from the nonprocessive nature of coronavirus rna polymerase, which mediates discontinuous rna synthesis during mrna transcription [ ] . in natural infections, both mutation and recombination have been demonstrated to contribute to the evolution of the coronaviruses. avian coronavirus ibv continues to undergo antigenic changes in the field through both mutation and recombination involving the s gene. recombination could occur between the field isolates and the vaccine strains of ibv [ , ] . this evolution causes problems in vaccination strategies, as multiple vaccines directed against different viral serotypes must be used simultaneously. another example is the emergence of porcine respiratory coronavirus (prcov) from the enteric tgev in europe as well as in the us in the s [ ] . again, this shift in viral tissue tropism and virulence was due to deletions and mutations in the s gene. this evolution turned out to be beneficial to the livestock industry because the resultant prcov is considerably less virulent than the parental tgev and, in fact, was used as an attenuated virus vaccine for tgev. a feline comnavirus has also been demonstrated to have undergone recombination with a canine coronavirus, resulting in changes in the biological properties of the virus [ ] . the ability of the coronavirus to recombine has been utilized as a genetic tool for manipulating the coronavirus genome. until recently it had been impossible to apply reverse genetic methods for studying coronaviruses because of the large size of its genome. taking advantage of the ability of coronavirus to recombine with not only other coronaviruses but also viral rna fragments, masters and his colleagues have developed techniques to introduce mutations into mhv defective interfering rna and allow it to recombine with a temperature-sensitive mutant (the mutation being in the n protein-coding region) of mhv [ ] . by selecting wild-type viruses capable of growing at the high temperature and using other selection markers, it is now possible to engineer mutations into certain viral rna regions or exchange certain viral genes between different coronaviruses [ ] . this technique has been applied only to mhv so far. however, a flood of reports in the last years has shown that full-length infectious cdna clones can be obtained for several coronaviruses, including tgev, mhv, hcov- e, pedv and ibv [ , , , ] . these cdna clones have added to the arsenal of coronavirus genetic tools. it is anticipated that the full-length sars virus cdna will be available soon. both this cdna and the targeted recombination approach may provide the reverse genetic tools for studying sars virus. the temptation to manipulate the sars virus genome will be great. however, i must add a word of caution before such attempts are made. the scientific community must be vigilant in guarding against the misuse of these genetic tools to alter the sars virus genome or create chimeric coronaviruses involving sars virus genes. i propose a moratorium on these types of experiments until the various concerned international communities agree upon specific guidelines. the potential for unleashing a more virulent new sars virus is simply too onerous to be ignored. coronaviruses, in general, cause disease by both cytocidal and immune-mediated mechanisms. most coronavirus infections in cell culture result in lysis or apoptosis of the infected cells [ ] . furthermore, the virus causes cell fusion, resulting in the formation of cell syncytia. these cytopathic effects (cpe) are usually the results of viral replication. for example, the coronavirus mobilizes cellular vesicles to form the viral replication complex [ ] , which may disrupt the golgi complex [ ] . sars virus also causes cpe in vero cells; furthermore, it causes syncytia formation in the lung tissue, suggesting that it causes cytocidal effects. another eerie similarity between sars virus and other coronaviruses is their ability to induce fibrosis; e.g. certain strains of mhv cause fibrosis in the liver [ ] , and sars virus causes fibrosis in the lung [ ] . the n protein of mhv has been shown to stimulate the promoter activity of the prothrombinase gene [ ] , which correlates with the fibrin deposit in the liver associated with mhv infection. a similar mechanism may be involved in the fibrosis of the lung induced by sars virus. immune responses also contribute to the pathogenesis of several coronaviruses. most notably, cytotoxic t cells and cytokines play a very significant role in the neuropathology caused by mhv [ ] . on the other hand, humoral antibodies are important for the disease associated with fipv, in which certain antibodies against the viral s protein induce severe peritonitis symptoms [ , ] . in the case of sars, there is an occurrence of cytokine storms and accumulation of inflammatory cells, particularly macrophages in the lung, during the peak of the disease [ ] , suggesting that both innate and adaptive immune responses are important for the disease. thus, the clinical management of sars should consider not only antiviral but anti-inflammatory strategies as well. several viral genes are likely involved in viral pathogenesis. most importantly, the spike protein gene affects viral pathogenesis not only by determining the host target cell specificity but by other mechanisms as well. as exemplified above, the emergence of the weakly virulent prcv from the virulent enteric tgev was the result of mutations in the s gene [ ] . also, it has been shown that a single mutation in the s gene of mhv can significantly alter the tissue tropism and virulence of the virus [ ] . the so-called 'non-essential orfs' may also play a role in viral pathogenesis. these genes are not necessary for viral replication in tissue culture cells, as experimental or natural deletions of any one of these genes did not affect the ability of the virus to replicate. however, some of these deletion mutants show much lower virulence than the wild-type virus in infections in animals [ ] . tt is significant that sars virus shows significant divergence of these orfs from other coronaviruses. a preliminary report shows that the civet cat coronavirus has a nucleotide deletion, which caused two non-essential orfs to fuse into one, creating a new orf in the sars virus [ a]. it will be very interesting to find out if this new orf contributes to the virulence of the sars virus. other viral gene products may also alter cellular responses to the viral infection. for example, m protein of tgev can induce interferon [ ] , and e protein of mhv can induce apoptosis [ ] . they may modulate the course of viral infection. another important issue in the pathogenesis of sars virus is whether the virus can cause chronic persistent infection. this issue concerns the possible presence of chronic carriers, who may serve as the source of continuing sars outbreaks. although human coronaviruses typically cause short, self-limiting illnesses, most of the animal coronaviruses are known to cause persistent infection. the best studied coronavirus in this regard is feline coronavirus fecov; infected animals can continue to shed virus tbr as long as months after infection, even though these animals do not show symptoms [ ] . tgev also shows a similar tendency, in which the virus can be detected in the respiratory tract of the infected animals several months after infection [ , ] . mhv can persist in the central nervous system many months after intracraniat inoculation [ ] . thus, the ability to persist is a common feature of most coronaviruses under some conditions. sars virus may also have this property. so far, viral rna reportedly has been detected in patients' stool more than days after infection (world health organization bulletin). coronavirus primarily infects the epithelial cells of the gi and respiratory tracts, but the virus can replicate in many cell types, particularly macrophages. sars virus also infects a wide spectrum of cell types; it has been detected in the blood, lung, liver and kidney, and in various bodily secretions, including stool and urine [ ] . thus, sars virus may induce systemic infection. the primary determinant of target cell specificity of the virus is the nature of its cellular receptors. type i coronaviruses use aminopeptidase n (cd ) of various animal species as receptors in a species-specific manner [ , ] , while a type ii coronavirus (mhv) uses the carcinoembryonic antigen as recenor [ ] . the receptors for type iii viruses and sars virus are not yet known. it should be noted that the virus easily adapts to cell lines of different animal species, probably by using other related molecules as a receptor [ ] . thus, the specificity of the spike-receptor interaction may not be very rigid. interruption of the binding between the spike protein and the receptor will be a potential means of inhibiting viral infection. the next step of virus infection is virus entry. the mode of virus entry for coronavirus may vary between different viruses. although most viruses appear to enter cells by fusion at the plasma membranes, some viruses may enter cells by acidic ph-dependent endocytosis. regardless, the fusion activity is likely important for virus entry. several functional domains, including the heptad repeat, which is important for oligomerization of the spike protein, the putative fusion peptide, and the receptor-binding domain of the s protein, will be good targets for antivirals. macromolecular synthesis, including transcription, translation and protein processing, offers many potential molecular targets as well. most notably, proteases, rnadependent rna polymerases (rdrp) and helicases are good targets. unfortunately, in vitro rdrp assays are still neither efficient nor specific, precluding their use as a molecular tool for drug screening. in vitro protease assays for both clpro and plpro, in contrast, are well established [ , ] . the protease activity is essential for viral protein processing and rna replication; inhibition of the protease function shuts off viral rna synthesis instantaneously [ ] . thus, the proteases are potentially powerful targets for antivirals. furthermore, the x-ray crystallographic structure for the clpro is available [ ] , as is a computation model for the sars virus clpro [ ] . these in addition, the processes of virus assembly, which likely occurs in the er-golgi intermediate compartment [ ] , and virus exit, which occurs through the cellular secretory pathway, are also potential antiviral targets. it has been known that the e and m proteins are the minimum components for virus assembly, forming vlp in the absence of other viral proteins [ , ] . the cellular machinery involved in the formation of virus particles is still not clear; it is a potential antiviral target as well. the prospect for sars vaccines is uncertain. several potential approaches are feasible. the killed whole virus vaccine is attractive because of the availability of a robust cell culture system that yields a large quantity of virus particles. attenuated virus vaccines are still unattainable because the parameters for virus attenuation are not yet well defined. ultimately, the attenuation of viral virulence needs to be verified preclinically in animal models. recombinant subunit vaccines consisting of the spike protein alone or in combination with other viral proteins can be prepared by using the recombinant proteins themselves or delivered by virus vectors or as dna plasmids. these approaches are technically feasible. however, the timetable for the development of any of these vaccines is lengthy, and the potential success of vaccine development may be hindered by the lack of accessibility to animal models and a suitable population base for clinical trials. at the present time, the macaque monkey is an acceptable animal model. however, with the sars epidemic virtually over worldwide, it will be difficult to test the efficacy of these vaccines. eventually, 'ring' vaccination (vaccination of the at-risk populations around the exposed people) may have to be attempted. nevertheless, vaccine development is clearly a top priority, as vaccines are the main weapons in the arsenal against future outbreaks, whether they are natural or man-made (i.e. bioterrorism) in origin. a survey of the currently available animal coronavirus vaccines offers a glimpse of the potential efficacy of sars vaccines. live attenuated vaccines have been used effectively against ibv. sometimes, a killed virus vaccine is used as a booster to the primary attenuated virus vaccine [ ] . the drawbacks of these vaccines are that ibv exists in multiple antigenic types and that the virus undergoes t?equent antigenic changes as a result of mutations and recombination. therefore, multiple viruses of different serotypes must be used in combination vaccines to protect against most ibv serotypes. for tgev, live attenuated virus vaccines have been used to vaccinate the adult pigs. the vulnerable piglets receive the protective antibodies through milk. improvement in lactogenic immunity is therefore the aim of tgev vaccine development. killed canine coronavirus has proven to be efficacious as a vaccine experimentally [ ] , although it is not routinely used because of the lack of necessity. on the other hand, feline coronavirus vaccines have been proven to be not only inefficacious; worse yet, the?, have been shown to lead to even more severe viral infections in the vaccinated cats [ ] . enhancement of viral infections by vaccines is probably due to the phenomenon of antibody-dependent enhancement (ade), in which antibodies against certain epitopes of the spike protein complex with the virus particles and enhance their infectivity [ . it is difficult to predict how the candidate sars virus vaccines will behave. if immune response plays an important role in the pathogenesis of sars, then an effective sars vaccine may turn out to be as elusive as the fipv vaccines. similar difficulties have plagued the developments of other viral vaccines, such as dengue virus, hiv, hepatitis c virus. sars is the first newly emerged infectious disease in this century. it is not likely to be the last one. even though the sars outbreak is now under control after a devastating run of more than months that affected and threatened many countries worldwide, the virus will continue to pose a threat in the near future. because of possible human and animal reservoirs for the virus, sars may return in the coming months or years. in the short term, the isolation and quarantine of patients and exposed individuals remain the only effective means of containing an outbreak. valuable experience in disease control was gained from this first outbreak; thus, the future outbreaks of sars and other emerging and re-emerging respiratory infectious diseases will likely be limited to sporadic occurrences. hopefully, intensive studies of the sars virus in the near future will provide sufficient understanding of its molecular 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coronavirus mouse hepatitis virus identify two subunits and detect a conformationai change in the subunit released under mild alkaline conditions antibody-mediated enhancement of disease in feline infectious peritonitis: comparisons with dengue hemorrhagic fever receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins human aminopeptidase n is a receptor for human coronavirus e systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a biological and genetic characterization of a hemagglutinating coronavirus isolated from a diarrhoeic child processing ofthe human coronavirus e replicase polyproteins by the virus-encoded c-like proteinase: identification of proteolytic products and cleavage sites common to ppla and pplab the beginning of the unraveling of a new coronavirus key: cord- -vas b dt authors: wege, h.; koga, m.; stephenson, j. r.; siddell, s.; ter meulen, v. title: neurovirulence and persistency of mouse hepatitis viruses in rats supported by the deutsche forschungsgemeinschaft and hertie-stiftung date: - - journal: animal virus genetics doi: . /b - - - - . - sha: doc_id: cord_uid: vas b dt abstract the murine coronavirus jhm induces in weanling rats different types of central nervous diseases ranging from an acute panencephalitis to a late demyelinating encephalomyelitis. the occurrence and rate of these different disease types is associated with the virus variant used for inoculation. except for mhv , neurovirulence was not observed in four other murine coronavirus strains. the relationship of these coronaviruses to jhm was investigated by cross neutralization and oligonucleotide maps of their genomic rna. murine coronaviruses induce in mice a variety of diseases, ranging from acute hepatitis, enteritis and encephalomyelitis to inapparent infections. of particular interest are the central nervous system (cns) diseases associated with this virus group, especially the mouse hepatitis virus strain jhm reveals a distinct neurovirulence for mice and rats ( , , , ) . in rats different disease processes accompanied by demyelination are observed ( , , ) , providing the opportunity to investigate the virus host relationships which lead to myelin destruction. in the present communication the neurovirulence of four murine coronavirus strains (mhv , mhv , mhv and mhv a ) for rats is compared to the jhm virus. neurovirulence of mhv-jhm. infection of suckling rats with jhm-virus regardless of virus history resulted in an acute encephalomyelitis characterized by necrotic lesions in all parts of the cns followed by hepatitis and rapid death within eight days after infection. intracerebral inoculation of weanling rats however, led to three different courses of a central nervous disease ( , , ) as summarized in table . the acute panencephalitis (ape) is characterized by a rapid onset within days post infection (p.i.), severe necrosis and acute inflammations in most parts of the brain. the subacute demyelinating encephalomyelitis (sde) does not occur before days p.i. and shows a more protracted clinical course. neuropathologically, inflammatory changes are present consisting mainly of mononuclear lymphocytes, plasmacells and macrophages without lesions of necrosis. in contrast to the ape neuronal involvement is minimal or absent. moreover, a primary demyelination in restricted areas of the brain and spinal cord is noticed which can easily be detected by histological staining procedures. the late demyelinating encephalomyelitis (lde) develops after an incubation period of - months. the main histological lesions of the cns consist of demyelinating areas present in the spinal cord and cerebrum. demyelination is accompanied by remyelination of the cns and pns type. inflammatory infiltrations can be detected in the neighborhood of demyelinating plaques. the attempts to isolate infectious virus from diseased brain material were successful in all animals tested, regardless of the different cns disease process. it is noteworthy, that even in the animals with lde infectious virus was present in brain, indicating jhm virus persistency during the incubation period. the occurrence and rate of the different types of cns disease induced by jhm virus is associated with the properties of the virus preparation used as inoculum as summarized in table . uncloned wild type jhm virus induced in weanling rats mainly ape and sde but only occasionally lde. this finding suggests that uncloned jhm consists of a heterologous virus population with different biological properties. this is supported by the observation that cloned tissue culture adapted jhm virus always caused in weanling rats an ape, whereas certain temperature sensitive mutants derived from that jhm virus clone after mutagenization with fluoruracil caused sde or lde. no acute disease process could be induced with these ts-mutants. reisolated mutants from diseased animals with lde did not always maintain their temperature sensitivity but were different from revertants by the type of neurovirulence. moreover, some characteristics in tissue culture were similar to the ts-mutants originally inoculated. the reisolate ts - is an example for this phenomenon. this variant was reisolated from a diseased animal months after infection with ts . the reisolate is much less temperature sensitive than the original mutant. it had an efficiency of plating of , x ~ compared to , x ~ for ts . in animals, this reisolate caused predominantely a high rate of sde in contrast to its parental virus, which induced only few cases of lde. neurovirulence of other mhv-strains. the virulence of cloned mhv-strains (mhv , mhv , mhv and mhv a ) was compared to jhm virus after intracerebral inoculation of defined virus doses into weanling rats (table ) . with the highest virus dose used, only mhv induced one case of an acute encephalomyelitis. no clinical disease was observed in the other animals injected with mhv , mhv and mhv a . pathological investigations carried out days after virus inoculation revealed only mild inflammations in liver tissue without detectable neuropathological changes. no infectious virus was recovered from cns tissue - days p.i. all the inoculated animals developed a humoral immune response against the different murine coronaviruses except for mhv . this virus strain grows in tissue culture lines of murine origin but appears not to be infectious for rats. cross neutralization among mhv-strains. antigenic comparisons among the different cloned murine coronavirus strains were carried out by cross neutralization. the results are summarized in table and indicate that each virus strain is antigenically distinct in the structural protein(s) which induced neutralizing antibodies. however, some cross neutralization among the murine coronaviruses can be noted. mhv jhm and mhv demonstrated a significant bilateral cross reactivity. mhv a and mhv cross reacted only in one direction, since anti mhv serum hardly neutralized mhv a virus. relative oligonucleotide homology between strains of murine coronaviruses. the genomic rna of the cloned mhv strains was labeled by growth in presence of ^ pj s p] lorus# «p^ genome from the purified viruses was extracted and further purified as described previously ( , , ) . after rnase digestion oligonucleotides were analysed in a two dimensional gel electrophoresis ( ) . the position of the large oligonucleotides within the fingerprint was compared. the result of this study (manuscript in preparation) is summarized in table and indicates distinct differences between jhm virus and the other murine coronavirus strains. mhv , mhv and mhv a are more closely related to each other than with mhv jhm, underlining the unique properties of this virus. comments from the five coronaviruses tested, only jhm revealed significant neurovirulence for weanling rats. the other murine coronaviruses induce beside liver inflammations no cns lesion in general even after intracerebral inoculation. jhm virus infection resulted in three different cns processes depending on the property of the inoculated virus preparation. this association provides a basis to investigate the mechanisms by which the different pattern of diseases are induced. it is conceivable that biochemical analysis of different mutants might unravel a specific marker which contributes to neurovirulence. the observation that jhm virus persists in weanling rats for months before lde is recognized suggests that specific events, either host or virus derived, prevent a rapid spread of virus infection. the temperature sensitivity of clones provides only a marker for a genetic lesion, because this lesion might be independent from the regions influencing the type of neurotropism which develops after inoculation in rats. this is suggested by the reisolation of mutants with reduced temperature sensitivity which nevertheless retain a specific neurovirulence. at present the selective neurotropism of different jhm mutants cannot be directly correlated to biological or biochemical properties of this virus. however, the preliminary data obtained from the comparison of oligonucleotide patterns indicates that jhm contains unique rna sequences which are not found among the other murine coronaviruses. further studies will define the genetic basis which contributes to neurovirulence, virus persistency and virus host interactions which lead to a demyelinating cns disease. proc.natl. acad.sci. usa we thank hanna wege and margarete sturm for excellent technical assistance and helga schneider for typing the manuscript. key: cord- -fhie m u authors: mazaleuskaya, liudmila; veltrop, rogier; ikpeze, nneka; martin-garcia, julio; navas-martin, sonia title: protective role of toll-like receptor -induced type i interferon in murine coronavirus infection of macrophages date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: fhie m u toll-like receptors (tlrs) sense viral infections and induce production of type i interferons (ifns), other cytokines, and chemokines. viral recognition by tlrs and other pattern recognition receptors (prrs) has been proven to be cell-type specific. triggering of tlrs with selected ligands can be beneficial against some viral infections. macrophages are antigen-presenting cells that express tlrs and have a key role in the innate and adaptive immunity against viruses. coronaviruses (covs) are single-stranded, positive-sense rna viruses that cause acute and chronic infections and can productively infect macrophages. investigation of the interplay between covs and prrs is in its infancy. we assessed the effect of triggering tlr , tlr , tlr , and tlr with selected ligands on the susceptibility of the j a. macrophage cell line to infection with murine coronavirus (mouse hepatitis virus, [mhv]). stimulation of tlr , tlr , or tlr did not affect mhv production. in contrast, pre-stimulation of tlr with polyinosinic-polycytidylic acid (poly i:c) hindered mhv infection through induction of ifn-β in macrophages. we demonstrate that activation of tlr with the synthetic ligand poly i:c mediates antiviral immunity that diminishes (mhv-a ) or suppresses (mhv-jhm, mhv- ) virus production in macrophages. coronaviruses (covs), a genus in the coronaviridae family, order nidovirales, are emerging rna pathogens of many animal species, including humans [ ] . currently there are no approved treatments or completely successful vaccines against cov infections. mice infected with different strains of mouse hepatitis virus (mhv), the prototype of betacoronaviruses, provide animal models for human diseases. the neurotropic strains, mhv-jhm and mhv-a , are commonly used to study viral encephalitis and virus-induced chronic demyelination, respectively [ ] . mhv-a also triggers mild to moderate hepatitis. the highly hepatovirulent strain mhv- provides a model of fulminant viral hepatitis [ ] . the first few days after infection with mhv are characterized by a strong innate immune response with infiltration of macrophages, neutrophils, and natural killer cells to the site of infection. it is widely documented that the host immune response plays a dual role in cov infection. on the one hand, it limits virus spread and replication and initiates adaptive immunity; on the other hand, it triggers overproduction of cytokines and chemokines, thus contributing to the severity of the disease [ ] [ ] [ ] [ ] [ ] . macrophages are productively infected by murine covs [ ] [ ] [ ] and represent the largest group of innate immune cells that infiltrate the central nervous system (cns) after infection with neurotropic mhv strains [ ] and the lungs of patients infected with severe acute respiratory syndrome (sars)-cov [ ] . the adaptive immune response that occurs during cov infection is well characterized [ , ] , but our understanding of the interaction of covs with the innate immune system of the host is still emerging [ , ] . type i interferon (ifn) (ifn-α and ifn-β) is crucial for the control of mhv infection in vivo [ ] [ ] [ ] . in most cell lines, murine covs are poor inducers of type i ifn and are barely sensitive to pretreatment with ifn [ ] . in primary cells, however, mhvs trigger ifn-α in plasmacytoid dendritic cells (pdcs) [ ] and ifn-β in macrophages [ , ] and are sensitive to pre-treatment with ifn-β in macrophages [ ] . therefore, interaction between murine covs and the type i ifn response depends on the cell type. the importance of type i ifn in cov infection is highlighted by a number of countermeasures and evasion mechanisms that covs in general and mhvs in particular developed to suppress signaling or prevent induction of the ifn response [ ] [ ] [ ] . induction of type i ifn can occur in all nucleated cells on tlrs activation [ ] . tlrs comprise a family of pattern recognition receptors (prr) that sense conserved molecular motifs of pathogens and trigger innate immunity and prime the adaptive immune response [ ] . triggering of tlrs induces complex signaling cascades initiated by the toll/interleukin- receptor (tir) domain in the cytoplasmic tail of the tlr. tir domain-containing adaptor molecules, myd , which is utilized by all tlrs except for tlr , as well as tirap, trif, and tram (for tlr ), are recruited to the receptor and activate a complex containing iraks and trafs which signal through nf-kb leading to the expression of a variety of genes encoding pro-inflammatory cytokines, chemokines and/or type i interferons (ifns) that orchestrate anti-bacterial and anti-viral responses [ ] . in the context of rna virus infection, tlr , tlr , tlr , tlr , and tlr can potentially be activated. cell surface tlr and tlr may recognize viral structural components, whereas endosomal tlr and tlr / may sense viral double-stranded and single-stranded rna, respectively [ ] . all of the above-mentioned tlrs were shown to induce type i ifn through activation of transcription factors and interferon regulatory factors (irfs); the magnitude of response, however, depends on the stimulus and the cell system. tlr , tlr and tlr are known to be potent inducers of the ifn response depending on the cell type [ ] . in contrast, tlr has been considered until recently a poor inducer of ifn response, despite triggering of tlr with bacteria-derived ligands induces strong pro-inflammatory cytokine response. in this regard, emerging evidence suggests that tlr and tlr activation induces pro-inflammatory cytokine and type i ifn responses from distinct sub-cellular sites: the plasma membrane and the endolysosomal compartments, respectively [ , ] . interestingly, only a particular monocyte subset has been reported to induce type i ifn through tlr in response to viral ligands [ ] . once secreted, ifn-α/β act through the jak-stat signaling pathway that triggers an "antiviral state" and help to eliminate viral infection [ , ] . the ability of tlrs to trigger antiviral immunity makes them a promising target for antiviral therapeutics. stimulation with tlr agonists has been shown to provide protection from some viral infections, such as hepatitis b virus (through tlr , tlr , tlr , tlr , or tlr ) [ ] , herpes simplex virus encephalitis (through tlr ) [ ] , lethal influenza virus (through tlr or tlr ) [ ] , hiv strains bal and jago (through tlr ) [ ] , and hepatitis c virus (through tlr ) [ ] . this study was undertaken to assess the effect of ligand-mediated, tlr activation of macrophages on their susceptibility to infection with murine cov. we profiled tlr , tlr , tlr , and tlr agonists (heat-killed listeria monocytogenes (hklm), poly i:c, lipopolysaccharide (lps), and imiquimod, respectively) and observed differential effects of these ligands on mhv production in macrophages. of all the ligands tested, only the triggering of tlr with poly i:c induced a strong antiviral response. mechanistically, the antiviral effect of poly i:c was promoted in a type i ifn-dependent manner. ligand-mediated activation of tlrs has been reported to affect the infectivity of various viruses [ , [ ] [ ] [ ] [ ] [ ] [ ] . the potential immunomodulatory and antiviral effects of triggering tlrs against cov infections in macrophages have not yet been investigated. macrophages are antigen-presenting cells that express tlrs and play a pivotal role in cov pathogenesis. the goal of this study was to investigate the effect of activation of tlr , tlr , tlr , or tlr with selected ligands on macrophage susceptibility to infection with murine coronavirus. we chose these tlrs on the basis of their potential role in the recognition of mhv by macrophages. tlr has been shown to recognize mhv- in peritoneal macrophages [ ] ; tlr has been implicated in protection and pathogenesis in mhv- -induced respiratory infection [ ] . despite the fact that tlr is a sensor of dsrna and could sense cov intermediate replicative forms in infected cells, its role in the recognition of covs or in their pathogenesis has not yet been established. tlr senses mhv-a in pdcs [ ] . first, we developed an in vitro model suitable for this study. the mouse macrophage cell line j a. was profiled for tlr - gene expression by quantitative real-time polymerase chain reaction (pcr) using predeveloped taqman gene expression assays (appliedbiosystems life technologies corp, carlsbad, ca) ( figure a ). expression levels of target genes were normalized to the housekeeping gene β-actin (Δct). gene expression values were calculated based on the ΔΔct method, with data for all samples analyzed against the mean value of four replicates. tlr showed a -fold greater expression than that of tlr and tlr (student's t test, p < . ), and the latter two had a more than -fold greater expression than tlr , , , , and (student's t test, p < . ). tlr and tlr transcripts were expressed to the highest and lowest levels, respectively (student's t test, p < . ). the expression of tlr , tlr , tlr , and tlr proteins was analyzed using flow cytometry (facs). as shown in figure b , facs data demonstrated robust expression of cell-surface tlr and tlr and intracellular tlr and tlr in naïve j a. cells. next we determined whether tlr , tlr , tlr , and tlr are functional in j a. cells. activation of the cells with a tlr ligand (hklm, cells/ml), a tlr agonist (poly i:c, μg/ml), a tlr ligand (lps, μg/ml), or a tlr agonist (imiquimod (r ), μg/ml) for , , and h resulted in the robust production of il- and tnf-α ( figure a ). this result indicates that, in j a. macrophages, tlr - and tlr are fully functional and signal with cytokine secretion after stimulation. we assessed type i ifn mrna induction in tlr-activated j a. macrophages. expression of ifn-α and ifn-β genes was up regulated by poly i:c, lps, and r ligands in j a. cells with different kinetics ( figure b ). lps-induced ifn-β and ifn-α mrnas peaked at h and h post-stimulation, respectively. r -induced ifn-β and ifn-α mrnas peaked at h post-stimulation. the induction of type i ifn gene expression after lps and r was not sustained (in contrast to ifn-β gene expression after poly i:c stimulation). ifn-α and ifn-β levels were determined by elisa in cell-free supernatants collected after h of prestimulation with hklm ( cells/ml), lps ( μg/ml), r ( μg/ml), and poly i:c ( . μg/ml). interestingly, activation of tlr but not of tlr , tlr or tlr triggered robust production of ifn-β following pre-stimulation for h in macrophages ( figure c ). similar to ifn-β, ifn-α was secreted only in tlr -activated cells, albeit to a much lesser degree ( figure c ). these contrasting results suggest that type i ifn response may be regulated differentially on tlr stimulation at the post transcriptional level in j a. cells. their lack of ifn secretion (as measured by elisa) in response to stimulation with the bacterial ligands hklm and lps is somewhat unpredicted and deserves further investigations. in addition, although it is well established that type i ifn response against rna viruses is mainly mediated by pdcs via a tlr -dependent pathway, the role of tlr in macrophage activation remains poorly understood. in this regard, the absence of ifn-β secretion after r stimulation of tlr in j a. cells may suggest differences in the regulation of the tlr pathway and/or its effectors between macrophages and pdcs. we are currently investigating the role of macrophage tlr in the antiviral response against rna viruses. furthermore, mhvs have been shown to productively infect j a. cells [ ] . by combining these results, we established a valid in vitro model in which to investigate the effect of triggering tlrs with selected ligands on mhv infectivity in macrophages. effect of triggering tlr , tlr , tlr , and tlr on virus production in mhv-infected j a. macrophages. j a. cells were prestimulated with tlr ligands for h, infected with mhv-a , mhv-jhm or mhv- ( moi) by adsorption for h in the absence of the ligands, and activated for up to h p.i. with the appropriate tlr agonist. tlr ligands were used as follows: hklm (tlr ) at cells/ml; lps (tlr ) at μg/ml; imiquimod (r ) (tlr ) at μg/ml. poly i:c (tlr ) was tested at a range of concentrations ( . , . , and . μg/ml). because poly i:c triggered a comparable effect on mhv production at all concentrations (data not shown), viral titers at . μg/ml were included in the plot. cells incubated with the basal medium before and during infection served as a negative control for the effect of tlr triggering on virus production. mhv titers were assessed in cell-free supernatants using a plaque assay on l fibroblasts. the data shown are the mean viral titers of three independent experiments, each done in duplicate ± standard deviation (*p value relative to virus alone, p < . student's t test). to test whether treatment with the tlr ligands hklm, poly i:c, lps, and r affected the replication of mhv, j a. cells were prestimulated with the appropriate ligand at the above-mentioned concentrations for h; and cells were infected with mhv-a , mhv-jhm, or mhv- at a multiplicity of infection (moi) of by adsorption for h in the absence of the ligands and stimulated again for up to h postinfection (p.i.) with the appropriate tlr agonist. therefore, there were two challenges with tlr ligands: one before and one after virus adsorption. activation of macrophages with tlr , tlr and tlr did not noticeably affect mhv production in j a. macrophages ( figure ). conversely, the triggering of tlr with poly i:c significantly inhibited mhv-a , mhv-jhm, and mhv- production relative to virus alone (student's t test, p = . ; figure ). complete suppression was observed only in poly i:c-treated mhv-jhm-and mhv- -, infected macrophages, although all mhv strains showed a dramatic -log reduction in virus production. the lack of complete suppression in mhv-a infected cells could be explained by the ability of mhv-a to grow to higher titers ( - log) than mhv-jhm and mhv- in macrophages. additionally, these results may also suggest that mhv-a counteracts the tlr pathway in j a. macrophages. indeed, our data shows that tlr -mediated, ifn-β secretion is significantly reduced in mhv-a -infected macrophages ( figure ). interestingly, poly i:c triggered comparable antiviral effect regardless of its concentration ( . , . , and . μg/ml; data not shown). it will be of interest to determine the minimal antiviral concentration of poly i:c in future experiments. the optimal concentration range for poly i:c was selected based on the highest rate of cytokine production (il- elisa) and minimal cytotoxicity (ldh cytotoxicity assay) in j a. macrophages activated with poly i:c at various doses (data not shown). collectively, these data demonstrate that, depending on the receptor, ligand-mediated tlr stimulation exerts differential effects on mhv production. triggering tlr with poly i:c, but not activation of tlr , tlr , or tlr with their respective ligands, impairs mhv replication in macrophages with a comparable magnitude of suppression of viral titers for mhv-a , mhv-hjm, and mhv- strains. given that all four tlr ligands induced strong il- and tnf-α proinflammatory responses (figure a ), we concluded that the inability of tlr , tlr and tlr agonists to protect macrophages from mhv infection is not due to the lack of signaling through these receptors, rather it stems from the absence of ifn-α and ifn-β production after h of stimulation with their ligands ( figure c ). in contrast, the antiviral effect mediated by activation of tlr with poly i:c is associated with a sustained transcriptional upregulation and secretion of ifn-α and ifn-β. we investigated the optimal conditions for poly i:c antiviral effects in j a. macrophages infected with a recombinant mhv-a expressing the gfp protein (ra -gfp) ( moi) and treated as follows: ( ) prestimulated with poly i:c, with no drug present during infection (poly i:c +/−); ( ) treated with poly i:c only after virus adsorption (poly i:c −/+); ( ) treated with poly i:c before and after virus adsorption (poly i:c +/+). the tlr ligand was used at concentrations of . to . μg/ml for h of prestimulation and/or h p.i. a profound suppression of gfp expression in cells stimulated with . μg/ml poly i:c was observed with all of the above-mentioned treatments relative to infected macrophages in the absence of the drug ( figure a ). thus, a single challenge with the tlr ligand before or after virus adsorption was sufficient to trigger a robust antiviral effect comparable to cells challenged with poly i:c twice. to determine the level of mhv production, released virus was quantified by plaque assay in cell-free supernatants from macrophages stimulated with . and . μg/ml poly i:c as above and in the absence of the drug ( figure b ). regardless of the concentration of poly i:c, the triggering of tlr with poly i:c resulted in a -log reduction in ra -gfp titers in prestimulated and coactivated macrophages (poly i:c +/+) relative to infected cells in the absence of the drug ( figure b , p < . ). cells challenged with poly i:c once before (poly i:c +/−) or h after mhv adsorption (poly i:c −/+) also exhibited a significant suppression (p < . ) of virus production comparable to that of prestimulated and coactivated macrophages (poly i:c +/+). interestingly, the triggering of tlr before adsorption with mhv (poly i:c +/−) resulted in significantly lower virus production relative to coactivated macrophages (poly i:c −/+) (p = . and p = . for . and . μg/ml poly i:c, respectively), suggesting that a single challenge with poly i:c prior to infection dramatically reduces macrophage susceptibility to mhv infection. taken together, these results indicate that . μg/ml of poly i:c is sufficient to trigger a profound tlr -mediated antiviral effect and that prestimulation alone is enough to protect macrophages from infection with mhv. to investigate the mechanism of the poly i:c-triggered antiviral effect in mhv-infected macrophages, we wanted to determine if the tlr ligand induced soluble factors that mediated protective immunity against cov infection. we pretreated j a. cells for h with conditioned medium (cm) from macrophages prestimulated with tlr - and tlr ligands for h (figure a ). next, cells were infected with ra -gfp ( moi) for h adsorption in the absence of tlr ligands and incubated for additional h in basal medium. ra -gfp virus titers determined by plaque assay are shown in figure . as expected, cm from mock macrophages (unstimulated, uninfected) did not affect virus production. treatment with cm from macrophages stimulated with hklm, r , and lps did not affect virus production ( figure ). remarkably, there was a -log reduction in ra -gfp titers in cells pretreated with poly i:c cm ( figure , p < . ) that correlated with the inhibition of gfp expression in these cells (data not shown). i. cells pretreated with the cm from mock cells or with fresh basal medium served as negative controls for the tlr-triggered effect. ra -gfp titers were assessed in cell-free supernatants using a plaque assay on l fibroblasts. error bars represent the standard error of the mean of two replicates (p value relative to cells pretreated with cm from mock, p < . , student's t test). overall, these data suggest that prestimulation with poly i:c but not hklm, lps, or r triggers the production of soluble factors that further protect macrophages from infection with mhv on subsequent exposure. in addition to soluble factors, residual poly i:c in the cm may have also contributed to the antiviral effect in cells pretreated with tlr -stimulated supernatants. our data in figure c demonstrated that after h of stimulation with hklm, lps, r , and poly i:c, ifn-β and ifn-α were secreted only in tlr -activated j a. cells. these results together with the antiviral effect of poly i:c (figure ) suggested that the protective role of tlr against coronavirus infection in j a. macrophages may be mediated by type i ifn. to investigate the role of type i ifn in the poly i:c-mediated inhibition of murine cov production in macrophages, we focused on mhv-a and mhv-jhm strains. we hypothesized that the differential effect of tlr ligands on mhv production is due to their variable ability to induce type i ifn crucial for triggering an "antiviral state" and protecting cells from virus infection [ , ] . to test this hypothesis, we assessed type i ifn production in tlr-stimulated and/or mhv-infected j a. a second challenge with poly i:c for h resulted in a robust secretion of ifn-α and ifn-β ( figure ). in contrast, a single challenge with poly i:c for h induced lower levels of ifn-β and levels of ifn-α that were close to the limit of detection of the elisa assay ( figure c ). such a pattern of induction of type i ifn in cells treated with dsrna (like poly i:c) is consistent with the activation of two types of type i ifn genes, immediate-early and delayed-type genes (reviewed in ref. [ ] ). immediate-early genes, mostly ifn-β and some ifn-α (only in murine cells), are induced by a protein-synthesis-independent pathway. secreted ifn signals in both an autocrine and paracrine fashion through the type i ifn receptor and triggers delayed-type ifns (including other ifn-α subtypes). expression of the delayed-type ifn depends on de novo protein synthesis and results in amplification of the ifn response. similarly, poly i:c-activated j a. macrophages exhibit high levels of ifn-β and a modest secretion of ifn-α on induction of the immediate-early gene. later, these cells secrete comparably high levels of both ifn-α and -β as a result of delayed-type ifn gene expression. figure ). the effect of infection with mhv on poly i:c-triggered ifn-β induction was previously assessed in ci- murine fibroblasts [ ] . in that study, neither mhv-a nor mhv-jhm inhibited ifn-β induction after poly i:c was transfected into fibroblasts (a way to activate rig-i and mda cytoplasmic helicases but not endosomal tlr ). thus, the ability of mhv to counteract poly i:c-induced ifn-β is cell type-specific and depends on the mode of delivery of poly i:c into the cell. targeting rig-i and mda helicases by poly i:c transfection with a lipid carrier as reported by [ ] , did not result in mhv-mediated inhibition of poly i:c-induced ifn-β secretion. in contrast, our data suggest that mhv-a might counteract the ifn-β response when macrophages are stimulated with soluble poly i:c to trigger the tlr pathway. further experiments are needed to define how mhv-a might counteract the tlr pathway in macrophages. interestingly, mhv-a has been reported to develop various measures to counteract the type i ifn response [ ] [ ] [ ] . besides poly i:c, the tlr ligand r induced a modest level of secretion of ifn-β with h stimulation; tlr and tlr agonists did not promote type i ifn secretion in j a. macrophages as measured by elisa ( figure ). overall, these findings are in agreement with our hypothesis that poly i:c triggers an antiviral effect via ifn-α/β, whereas the lack of a strong type i ifn production during tlr , tlr , or tlr signaling is responsible for uncontrolled mhv production in j a. macrophages. figure . il- and tnf-α production in cell-free supernatants from j a. macrophages stimulated with tlr ligands, mhv-infected, and/or coactivated during mhv infection. j a. cells were prestimulated for h with hklm ( cells/ml), lps ( μg/ml), r ( μg/ml), and poly i:c ( . μg/ml); media was removed and: ) a second challenge of the corresponding tlr ligand (same concentrations) was added to the cells for h; ) cells were prestimulated for h with the tlr ligands as above; media was removed and cells infected with mhv-a or mhv-jhm at . moi for h of adsorption in the absence of the ligands; after virus adsorption cells were stimulated with a second challenge of the tlr ligands using the same concentrations as during prestimulation and samples were taken at h; ) cells were not tlr activated and only infected with mhv-a or mhv-jhm at . moi for h of adsorption in the absence of the ligands; fresh media was added to the cells for h. non-stimulated, non-infected j a. cells were used as mock control. cell-free supernatants were taken at h from tlr-activated alone, infected alone, and tlr-activated and infected and assessed for il- and tnf-α using elisa. error bars represent the standard error of the mean of two independent experiments, each done in duplicate. to further rule out the potential antiviral effect of il- and tnf-α in infected and co-stimulated cells, we measured proinflammatory cytokines in the same samples as above. stimulation for h resulted in a very high induction of both cytokines after lps (a tlr ligand that based on our data does not induce antiviral effect against infection with mhv in j a. macrophages) and poly i:c (albeit to a lesser extent than with lps) (figure ) . overall, these data argue against the potential antiviral effects of il- and tnf-α in tlr -activated macrophages. tnf-α levels were reduced in mhv-a and mhv-jhm infected macrophages relative to mock cells (figure ) . although it was not the focus of the present study, the inhibitory effect of mhv on the basal tnf-α levels may be mediated through the action of the anti-inflammatory cytokine il- . il- is a known negative regulator of tnf-α production and function in macrophages (reviewed in ref. [ ] ). mhv-a was reported to induce il- in infected primary bone marrow-derived macrophages [ ] , therefore, one could speculate that covs suppress basal macrophage tnf-α secretion through triggering of il- . future studies will be designed to test this hypothesis. although tnf-α was reported to induce a strong antiviral response against various influenza strains in lung epithelial cells [ ] , our data demonstrated that tlr-induced tnf-α does not affect mhv production in macrophages. collectively, these results indicate that il- and tnf-α are not responsible for and do not contribute to a poly i:c-triggered antiviral effect in mhv-infected macrophages. considering that soluble factors mediate the poly i:c-triggered antiviral effect ( figure ) and that poly i:c potently induces type i ifn before and during virus infection ( figures c and ) , we further confirmed the role of ifn-β in tlr -triggered mhv suppression in macrophages ( figure a-c) . we focused on ifn-β because unlike ifn-α, ifn-β was profoundly induced in prestimulated macrophages at h poststimulation ( figure c ), and cm from macrophages treated with poly i:c for h exhibited inhibition of mhv-a production sufficient to reduce viral infection ( figure ). titration of anti-ifn-β antibody (ab) was done to establish the optimal ab concentration for neutralization of poly i:c-stimulated ifn-β. j a. macrophages were stimulated with . μg/ml poly i:c in the presence or absence of anti-ifn-β ab at various concentrations. we chose . μg/ml poly i:c because prestimulation with such a low concentration of the tlr ligand was sufficient to promote a strong antiviral effect (figure ). poly i:c-triggered ifn-β was significantly reduced by u/ml to u/ml of the ifn-β neutralizing ab, and it was suppressed in the presence of a higher concentration ( u/ml) ( figure a ). after stimulation, cells were infected with ra -gfp ( moi) by adsorption for h and incubated in fresh medium for up to h p.i. cm from mock or ifn-β ab-treated macrophages did not affect mhv-a virus production in these cells ( figure b,c) . as expected on the basis of our previous results, pretreatment with poly i:c-conditioned medium resulted in a drastic reduction in ra -gfp expression and in a -log reduction in mhv production in infected macrophages ( figure b,c) . importantly, mhv-a infection was significantly restored (p = . ) in j a. cells incubated with the supernatant from macrophages activated with poly i:c in the presence of the ifn-β neutralizing polyclonal ab (poly i:c + ifn-β ab cm) ( figure b,c) . this result indicates that ifn-β is a crucial mediator in the antiviral response against mhvs elicited by triggering tlr with poly i:c in macrophages. murine covs are sensitive to pretreatment of macrophages with recombinant ifn-β [ ] . in the present study prestimulation of j a. macrophages with poly i:c, a potent type i ifn inducer, resulted in a strong ifn-β response that triggered antiviral immunity and protected macrophages from mhv infection before and after virus adsorption. in support of our data, a poly i:c analog ampligen tm (poly i:poly c u) was successfully tested in sars-cov animal models [ , ] . balb/c mice were treated with ampligen tm intraperitoneally (i.p.) h before sars-cov infection and then the virus titers in the lungs were assessed days after virus exposure. sars-cov titers in the lungs were below the limit of detection suggesting that poly i:poly c u completely protected mice from viral infection [ ] . in a different study, ampligen tm was given intraperitoneally to balb/c mice h before they were infected with the mouse-adapted sars-cov strain v . treated mice exhibited complete survival, suppressed virus titers in the lungs, significantly reduced lung scores and weight loss [ ] . these studies did not investigate the mechanism of the ampligen tm -triggered antiviral effect in sars-cov-infected mice; type i ifn, however, was proposed as a mediator of antiviral immunity. ampligen tm is indeed a potent type i ifn inducer that acts through tlr [ ] and triggers protection from hiv [ , ] , coxsackie virus [ ] , punta toro virus [ ] , venezuelan equine encephalitis virus [ ] , and influenza virus [ ] infections. overall, our data demonstrates that tlr triggered type i ifn inhibits murine cov infection of macrophages. j a. murine macrophages (attc, tib- ) were cultured in dulbecco's modification of eagle's medium (dmem) (cellgro, mediatech, manassas, va, usa) supplemented with % v/v heat-inactivated fetal bovine serum (hifbs) (hyclone, thermo scientific, rockford, il, usa), u/ml penicillin (cellgro), μg/ml streptomycin (cellgro), . g/l sodium bicarbonate (biowhittaker, lonza, walkersville, md, usa) and mm glutamine (cellgro). cells were incubated in a humidified atmosphere at c with % co . the dualtropic mhv-a , and neurotropic mhv-jhm strains were previously characterized [ , ] . the hepatotropic mhv- strain was provided by dr. julian leibowitz (texas a&m university). recombinant mhv-a virus expresses the gfp in place of the orf [ ] . cells were infected at moi by adsorption for h in the basal medium. then, excess virus inoculum was removed and cells were incubated for up to h p.i. plaque assays were performed on l murine fibroblasts [ ] . % confluent monolayers of l cells were infected with -fold dilutions of samples in dmem supplemented with % v/v hifbs. after virus adsorption, cells were overlaid with a : mixture of . % agarose and % fbs in dmem and incubated for h at c with % co . to visualize viral plaques, infected cells were overlayed with equal parts of . % agarose, % fbs in dmem and / neutral red (harleco, emd chemicals inc., gibbstown, nj, usa) and incubated for h at c with % co . all tlr agonists were purchased from invivogen (san diego, ca, usa). the following tlr ligands were used: hklm for tlr at cells/ml; poly i:c for tlr at . to μg/ml; ultrapure lps from e. coli for tlr at μg/ml; and imiquimod (r ), an imidazoquinoline amine analogue of guanosine, for tlr at μg/ml. cells were prestimulated with the appropriate tlr ligand for h, infected as necessary and coactivated during infection with the corresponding tlr agonist for up to h p.i. mouse il- and tnf-α were quantified by elisa (ebioscience, san diego, ca, usa) according to the manufacturer's instructions. limits of detection: . - pg/ml for il- and . - pg/ml for tnf-α. verikine mouse ifn-α and verikine mouse ifn-β elisa kits were purchased from pbl interferon source (thermoscientifc, rockford, il, usa). limits of detection: . - pg/ml for ifn-β, and . - pg/ml for ifn-α. to titrate anti-ifn-β ab, j a. macrophages were activated with . μg/ml poly i:c in the presence or absence of anti-ifn-β ab at , , or u/ml for h. rabbit polyclonal anti-ifnβ ab were purchased from calbiochem (emd chemicals inc.). poly i:c-triggered ifn-β with or without neutralizing ab was assessed with a verikine ifn-β elisa kit. cell-free cm from poly i:cactivated and/or u/ml anti-ifn-β ab-incubated macrophages was used to pretreat j a. cells for h. then macrophages were infected with moi mhv-a by adsorption for h and incubated in fresh medium for up to h p.i. total rna was isolated using trizol ® reagent (invitrogen, carlsbad, ca, usa). total rna ( ng) of was transcribed into cdna with the superscript ii reverse transcriptase kit (invitrogen), using a total reaction mix volume of μl; . μl cdna was combined with . μl of taqman universal master mix (applied biosystems, life technologies, carlsbad, ca, usa), μl diethyl polycarbonate (depc)-treated water, and . μl murine tlr to tlr taqman gene expression assays (applied biosystems). dna was amplified using the applied biosystems real-time pcr machine, and cycle threshold values (c t ) were recorded. basal tlr - mrna levels were expressed as ΔΔc t values relative to s rrna [ΔΔc t = Δc t(tlr) − Δc t( s rrna) ]. tlr mrna expression levels were expressed as fold changes relative to mock values, using the variable −ΔΔct . ifn- α and ifn-β gene expression was quantified as above using specific pre-developed taqman gene expression assays (applied biosystems). expression of cell surface tlr and tlr , and endosomal tlr and tlr was determined using a facscalibur flow cytometer (bd biosciences, san jose, ca) and data were processed using flowjo software (treestar, ashland, or). cells were stained and analyzed by using a fixation & permeabilization kit (ebioscience) following the recommended protocols. specific antibodies against murine tlr , , , and , fluorescence-labeled secondary antibodies, isotype controls, and anti-fcrr (for blocking) were purchased from invivogen, imgenex (san diego, ca), and ebioscience (san diego, ca). gfp expression was analyzed in live or % paraformaldehyde fixed cells. cells were incubated with nm dapi ( ', '-diamidino- -phenylindole dilactate; invitrogen, molecular probes, eugene, or, usa) for nuclei stain for min at rt. images were obtained with an olympus x motorized inverted fluorescence microscope (center valley, pa, usa) using digital microscopy software (slidebook™ . software, intelligent imaging innovations, denver, co). an unpaired two-tail student's t test was used to determine statistical significance. all data were analyzed with graphpad prism (graphpad software, inc., ca, usa) our goal was to investigate the effects of triggering tlr , tlr , tlr and tlr with selected ligands on j a. macrophages susceptibility to mhv infection. our data demonstrates that triggering of tlrs with hklm (tlr agonist), lps (tlr agonist), and r (tlr agonist) does not affect mhv-a , mhv-jhm, and mhv- production. the absence of tlr -, tlr -, and tlr -mediated antiviral effects is not explained by their lack of expression or activation in j a. macrophages; rather, it is based on the weak induction of type i ifn. in contrast, stimulation of macrophages with poly i:c added to the cells to trigger endosomal tlr , induces a strong type i ifn-dependent antiviral response. neutralization of ifn-β successfully restored the poly i:c-inhibited production of mhv-a in macrophages. taken together, activation of tlr by poly i:c may be a successful antiviral approach against covs in vivo; its therapeutic potential as a curative drug remains to be established in future investigations. coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. microbiol pathogenesis of murine coronavirus in the central nervous system coronaviruses post-sars: update on replication and pathogenesis coronavirus infection of the central nervous system: host-virus stand-off pathogenesis of acute and chronic central nervous system infection with variants of mouse hepatitis virus, strain jhm the pathogenesis of murine coronavirus infection of the central nervous system murine coronavirus mouse hepatitis virus is recognized by mda and induces type i interferon in brain macrophages/microglia macrophage interleukin- and tumour necrosis factor-alpha are 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toll-like receptor signaling pathway for protection against influenza virus infection a critical function of toll-like receptor- in the induction of anti-human immunodeficiency virus activities in macrophages activation of anti-hepatitis c virus responses via toll-like receptor tlr / triggering exerts opposing effects in acute versus latent hiv infection tlr signaling is either protective or pathogenic for the development of theiler's virus-induced demyelinating disease depending on the time of viral infection activation of toll-like receptor impairs the dengue virus serotype replication through induction of ifn-beta in cultured hepatoma cells decreased dengue replication and an increased anti-viral humoral response with the use of combined toll-like receptor and / agonists in macaques toll-like receptor deficiency increases disease and mortality after mouse hepatitis virus type infection of susceptible c h mice murine coronavirus replication-induced p mitogen-activated protein kinase activation promotes interleukin- production and virus replication in cultured cells master regulators of signalling by toll-like receptors and cytosolic pattern-recognition receptors mouse hepatitis virus does not induce beta interferon synthesis and does not inhibit its induction by double-stranded rna plp , a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production accessory protein a is a major antagonist of the antiviral action of interferon against murine coronavirus plp of mouse hepatitis virus a (mhv-a ) targets tbk to negatively regulate cellular type i interferon signaling pathway regulation of interferon and toll-like receptor signaling during macrophage activation by opposing feedforward and feedback inhibition mechanisms tumor necrosis factor alpha exerts powerful anti-influenza virus effects in lung epithelial cells evaluation of immunomodulators, interferons and known in vitro sars-cov inhibitors for inhibition of sars-cov replication in balb/c mice a new mouse-adapted strain of sars-cov as a lethal model for evaluating antiviral agents in vitro and in vivo tlr is essential for the induction of protective immunity against punta toro virus infection by the double-stranded rna (dsrna), poly(i:c u), but not poly(i:c): differential recognition of synthetic dsrna molecules mismatched dsrna (ampligen) induces protection against genomic variants of the human immunodeficiency virus type (hiv- ) in a multiplicity of target cells mismatched double-stranded rna (polyi-polyc( )u) is synergistic with multiple anti-hiv drugs and is active against drug-sensitive and drug-resistant hiv- in vitro the interferon inducer ampligen [poly(i)-poly(c u)] markedly protects mice against coxsackie b virus-induced myocarditis hen mouse model for the evaluation of antiviral agents for the treatment of venezuelan equine encephalitis virus infection polyi:polyc u adjuvant-combined intranasal vaccine protects mice against highly pathogenic h n influenza virus variants replicase genes of murine coronavirus strains a and jhm are interchangeable: differences in pathogenesis map to the ' one-third of the genome enhanced green fluorescent protein expression may be used to monitor murine coronavirus spread in vitro and in the mouse central nervous system murine coronavirus evolution in vivo: functional compensation of a detrimental amino acid substitution in the receptor binding domain of the spike glycoprotein this article is an open access article distributed under the terms and conditions of the creative commons attribution license the authors would like to thank drs. paul s masters, julian leibowitz, and susan r weiss for reagents. this work was supported in part by public health service grants ns , ai and dk to s.n.m, and ns to j.m.g., from the national institute of neurological disorders and stroke (ns), the national institute of allergy and infectious disease (ai), and the national institute of diabetes and digestive and kidney diseases; and by drexel university college of medicine's internal funds to snm. the founders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. pamela fried (drexel university college of medicine academic publishing services) is acknowledged for manuscript editing. the authors declare no conflict of interest. key: cord- -bam pa authors: lee, han-jung; shieh, chien-kou; gorbalenya, alexander e.; koonin, eugene v.; la monica, nicola; tuler, jeremy; bagdzhadzhyan, anush; lai, michael m.c. title: the complete sequence ( kilobases) of murine coronavirus gene encoding the putative proteases and rna polymerase date: - - journal: virology doi: . / - ( ) -i sha: doc_id: cord_uid: bam pa abstract the ′-most gene, gene , of the genome of murine coronavirus, mouse hepatitis virus (mhv), is presumed to encode the viral rna-dependent rna polymerase. we have determined the complete sequence of this gene of the jhm strain by cdna cloning and sequencing. the total length of this gene is , nucleotides long, which includes two overlapping, large open reading frames. the first open reading frame, orf a, is amino acids long. the second open reading frame, orf b, overlaps orf a for nucleotides, and is amino acids long. the overlapping region may fold into a pseudoknot rna structure, similar to the corresponding region of the rna of avian coronavirus, infectious bronchitis virus (ibv). the in vitro transcription and translation studies of this region indicated that these two orfs were most likely translated into one polyprotein by a ribosomal frameshifting mechanism. thus, the predicted molecular weight of the gene product is more than , da. the sequence of orf b is very similar to the corresponding orf of ibv. in contrast, the orf a of these two viruses differ in size and have a high degree of divergence. the amino acid sequence analysis suggested that orf a contains several functional domains, including two hydrophobic, membrane-anchoring domains, and three cysteine-rich domains. it also contains a picornaviral c-like protease domain and two papain-like protease domains. the presence of these protease domains suggests that the polyprotein is most likely processed into multiple protein products. in contrast, the orf b contains polymerase, helicase, and zinc-finger motifs. these sequence studies suggested that the mhv gene product is involved in rna synthesis, and that this product is processed autoproteolytically after translation. this study completes the sequence of the mhv genome, which is kb long, and constitutes the largest viral rna known. mouse hepatitis virus (mhv), a murine coronavirus, contains a single-stranded, positive-sense rna genome (lai and stohlman, ; wege eta/., ) . the genomic organization is well understood lai, ) . it contains genes, each of which is expressed from the '-end of a polycrstronic mrna species. these mrnas have a '-coterminal, nestedset structure (lai et al., ) . starting from the '-end of the genome, the genes are named , a, b, , and so on until gene (cavanagh eta/., ) . genes b, , , and encode the four known viral structural proteins, i.e., he (hemagglutinin-esterase), s (spike), m (membrane), and n (nucleocapsid) proteins, respectively. the remaining genes presumably encode nonstructural proteins, most of which are yet to be identified in the virus-infected cells. the nucleotide sequences of genes to have been determined for two strains, a and jhm, of mhv (armstrong eta/., (armstrong eta/., , skinner and siddell, , sequence data from this article have been deposlted with the embugenbank under accession no. m . ' to whom correspondence should be addressed. ; schmidt et a/., ; luytjes et a/., luytjes et a/., , shieh et al., ) . altogether these seven genes account for roughly . kb. the remaining gene, gene , which is the '-most gene, has been estimated to be longer than the size of all of the other genes combined (pachuk eta/., ; baker et al., ) . only the '-terminal . kb in jhm strain and the '-terminal . kb of this gene in a strain have so far been sequenced baker et al., ; pachuk et al,, ; bredenbeek et al., ) . the corresponding gene of an avian coronavirus, infectious bronchitis virus (ibv), has been completely sequenced and shown to be kb long . this ibv gene consists of two open reading frames (orfs), which can be translated into a polyprotein via a ribosomal frameshifting mechanism (brierley et a/., (brierley et a/., , . again, the gene products have yet to be detected in the virus-infected cells. the size of mhv gene has not been determined. from the approximate sizes of the cdna clones, it has been estimated to be roughly - kb (pachuk et al,, ; baker et al,, ) . comparison of the published partial sequences of gene showed that ibv and mhv share sequence similarity in the '-terminus of the gene ( , ) , and yet their '-ends are diverged (soe eta/., ; baker et al., ) . thus, the evolutionary relationship of these two viruses in gene is not clear. several pieces of evidence suggest that gene may encode proteins which are directly involved in viral rna synthesis: first, since mhv does not contain rna polymerase (brayton et al., ) , this enzyme has to be synthesized from the incoming virion genomic rna. this translation is only possible if the gene is located at the '-end of the genome. second, rna recombination studies using temperature-sensitive (ts) mutants indicated that the ts lesions affecting rna synthesis are localized within the gene region (keck et al., ) . this conclusion has been confirmed by rna recombination mapping studies (baric et a/., ) . third, the '-half of the gene sequences of ibv and mhv-a contains the sequence motifs for rna polymerase and helicase, which are the activities expected to be involved in rna synthesis gorbalenya et a/., ; bredenbeek et a/., ) . however, these postulated functions have not been directly demonstrated. at least one enzymatic activity, i.e., an autoprotease , has been associated with the gene product. the presence of the protease activity suggests that the gene product is likely to be processed into multiple proteins. the properties of the rna polymerase of coronavirus are of considerable interest since the coronavirus rna synthesis utilizes an unusual mechanism of discontinuous transcription, probably involving a free leader rna species (lai, ) . the understanding of the rna polymerase should shed further light on the mechanism of rna synthesis. to this end, we have obtained the complete sequence of gene i of the jhm strain of mhv. this gene is nearly , nucleotides long and contains two overlapping orfs, similar to the corresponding ibv gene. sequence analysis shows that the mhv gene may have undergone extensive divergence from the ibv gene, particularly at its '-half. several functional domains were identified, which may be important for the processing and the enzymatic activities of its gene product. virus and cells. the plaque-cloned jhm strain of mhv (makino et a/., ) was used throughout this study. the virus was propagated on dbt cells (hirano et a/., ) at m.o.i. of . virus was harvested and purified from the medium, and viral rna was prepared as previously described (makino et a/., ) . cdna cloning. the cdna c\ones encompassing , i i i i , i i i / , i i i i i orf la/lb orf lb , i i i i , i i i i , i i i i i ' ' fig. . hydropathy profiles of the predicted amino acid sequences of orf a and orf b. values above the lrne are hydrophobic and values below the line are hydrophilic. the hydropathicrty was calculated using a moving window of amino acids, with a value plotted every residues (kyte and doolittle, ) . gene were obtained by using specific synthetic oligo-gonucleotides were derived from rna sequence analynucleotides as primers and purified virion genomic sis of the rnase tl-resistant oligonucleotides which rna as template. initially, the sequences of these olihad been mapped to either gene or (shieh et al, , , , nucleotide number , fig. . dragram of the codon preference in the region between orf a and orf b. the codon usage patterns for the three reading frames of the predicted amino acid sequences at the junction between the orf a and orf b are shown. the two stop codons at (tag) and (taa) are marked. the codon usage table was generated for genes , , and , which encode the viral structural proteins, of mhv-ihm (schmidt er a/., ; skinner and siddell, ) and used for comparison with orfs a and b. the parameters used are a window length of and a maximum scale of . (gribskov et al., ) . l bv-m ' gauaagaauuauuuaaacggguacgggguagcagug----aggcucggcugauaccccuugcuagugg ' ii i lllll lllllli iiii i ii ii - iii illll mhv-jhm ' gacacgaauuuuuuaaacggguucgggguacaaguguaaaugcccgucuuguacccugugccagugg ' mhv-a ' llllllll iiiiiiiiiiiiiiiltriiiiiiiiiiiiiiiiiiiiiiiiiiii gacacgaacuuuuuaaacggauucgggguacaaguguaaaugcccgucuuguacccugugccagugg comparison of the rna sequences and the proposed secondary structure of the mhv-jhm, mhv-a and ibv rnas at the junction between orf a and orf b. (a) alignment of nucleotide sequences. the first nucleotides are numbered according to boursnell era/. ( ) for ibv, and bredenbeek ef al. ( ) for mhva , and termrnation codons are underlrned. (b) tertiary rna structure at the region of ribosomal frameshifting. the potential signal for ribosomal frameshifting is boxed, and the stop codon is underlined. arrows indicate the differences in the rna sequence of mhv-jhm in comparison with that of ibv (boldfaced) and mhv-a (outlined). soe et al., ) . cdna synthesis was were trimmed with t dna polymerase and ligated to performed by the general method of gubler and hoff-ptz u (united states biochemical corp.) either by man ( ) . the double-stranded cdna molecules blunt-end ligation or ecorl linker ligation. the recombi- , and ) . generating a . .kb rna. translation was performed in a rabbit reticulocyte lysate system using [ s]methionine. translation products were analyzed directly (lanes l- ) or after immunoprecipitation using the orf a-specific antiserum (lanes - ) or rabbrt preimmune serum (lanes - ). m indicates molecular weight markers in kilodaltons; lanes . , and , translation of ptz(orfaug). nant dnas were transformed into escherichia co/i strain mvl competent cells (dagert and ehrlich, ) . homopolymer dc tailing to the '-end of the cdnas using terminal transferase were also used to anneal to pstl-linearized pbr with oligo(dg) tails and transformed into e. co/i strain mc . specific cdna clones were identified using '-end-labeled oligonucleotides as probes and confirmed by subsequent hybridization to viral mrna . once the sequences of the cdna clones were obtained, oligonucleotides complementary to the '-ends of these clones were synthesized to serve as primers for addi-tional cdna cloning to obtain overlapping cdna clones. dna sequencing. sequencing was performed as previously described . both chemical modification (maxam and gilbert, ) and dideoxynucleotide chain termination @anger et a/., ) methods were used directly on plasmid dna (chen and seeburg, ) . construction of recombinant plasmids for the frameshifting analysis. subcloning and mutagenesis of cdna clone t-l was accomplished using synthetic oligonucleotides and polymerase chain reaction (pcr). briefly, oligomer # ( '-gatcgaattcctttacat-ggtgaaggggtg- ') which extends from nucleotide , to , of gene and contains mismatches at both nucleotides , and , , and oligomer # ( '-catatgacacaggatcctttatgcc- ') which is complementary to nucleotides , to , and includes the bamhl site at nucleotide , , were used for dna amplification by pcr according to the standard procedures (saiki et al., ) . the resulting pcr dna product encompasses sequences from nucleotide , to , with a specific mutation (t to a) at nucleotide , and another (t to g) at nucleotide , , resulting in the introduction of an atg codon. the dnawas then digested with ii i iill iii iiii lllll ir iii iiiii iii qts~gvcvvcnspti the overall alignment was generated by combining segments aligned by programs optal (gorbalenya et al., a) and multalin (corpet, ) . it consists of four distinct pieces separated by regions that could not be aligned with certainty. for the latter regions, only the total numbers of amino acid residues are indicated. the amino acid numbers of the first and the last residues of each aligned segment are indicated in parentheses. two dots, identical residues; single dots, similar residues. conserved cys residues are highlighted by boldface. asterisks, putative catalytic residues of proteases; arrows, putative cleavage sites for bc-like proteases. box, the putative cleavage site for clpr in ibv substituted by a kr dipeptide in mhv-jhm. the ibv sequence was from boursnell et al. ( ) . mhva: orf la of mhv. ibvfl: orf la of ibv. previously described . the resulting in vitro transcription and translation. recombinant rna was translated in the mrna-dependent rabbit replasmids ptz(orfaug) and ptz (frsh) shin and morrison ( ) and analyzed by electrophoresis on . to % polyacrylamide gel. computer analysis of nucleotide and amino acid sequences. sequence data were analyzed on a vax using the gcg sequence analysis software package developed by genetics computer group of university of wisconsin. detailed comparative analyses of coronavirus protein sequences were done by programs multalin (corpet, ) optal (gorbalenya et a/., a), dothelix (leontovich et al., ) , and site (koonin et a/., ) . the programs dothelix and site are parts of the genbee program package for biopolymer sequence analysis. to clone the gene i region, which represents more than two thirds of the mhv genome, a synthetic oligonucleotide (oligo ; '-ctgaatftgggg-gttggg- ') was initially used as a primer for cdna synthesis . the sequence of this oligonucleotide was based on the sequence analysis of the rnase tl-resistant oligonucleotide no. , which had previously been mapped to gene (makino et al., ) . the resulting cdna clones contained inserts ranging from . to kb in size. these cdna clones detected only the genomic rna on northern blots of intracellular rnafrom mhv-infected cells (data not shown). based on the nested-set structure of mhv . a schematic presentation of the relationship between the orf a of mhv-jhm and ibv. the two orf a are shown to scale. the designation of regions, for which specific functional predictions could be made, and of regions of similarity between the two viruses are shown in the bottom of the figure. high similarity, statistical significance over sd (standard deviation), when aligned by the program optal (gorbalenya eta/., a,b) ; moderate similarity, significance of to sd. the alignments in the regions, with predicted functions, were significant at the level of at least sd. regrons of similarity between the two viruses are joined. vertical arrows, putative cleavage sites for clpr '). horizontal arrows, putative papain-like proteases (two copies in mhv-jhm, and one copy in ibv). mrnas (lai et a/., ) , this result indicated that these cdna clones represent part of gene . the 'ends of these dnas were sequenced, and synthetic oligonucleotides complementary to these sequences were generated to prime further cdna synthesis for walking toward the '-end of gene . in this way, overlapping dna clones which encompass about kb at the '-end of gene were obtained (fig. ) . cdna clones representing the '-terminal . kb of gene were derived as described . the cdna clones spanning the gap between the two cdna groups were obtained by using specific primers representing both the sequences downstream and upstream of the gap as primers for first-strand and second-strand cdna synthesis, respectively. the overlap of these cdna clones was determined by southern blotting and confirmed by dna sequencing. the complete cloning of jhm gene indicated that the size of gene is approximately kb in length (fig. l) , longer than that of ibv , and agrees with the previous estimate for the gene of the a strain of mhv (pachuk et al., ) . analysis of the nucleotide sequence and the predicted amino acid sequence. the complete mhv-jhm gene sequence was obtained from the cdna clones as indicated in fig. . this sequence has been deposited with genbank (accession no. m ), and will not be duplicated in this publication. the complete sequence of gene contains , nucleotides preceding the ucuauac, which is the transcriptional initiation site for gene . analysis of the sequence revealed two large, overlapping open reading frames (orfs), orf a and orf b (fig. a) . orf a is amino acids long and has a predicted molecular weight of , , which includes the coding region for p protein at its n-terminus . the hydropathy plot (kyte and doolittle, ) shows that orf a has several long stretches of hydrophobic regions at the carboxy-terminal region, which indicate potential membrane-spanning domains (fig. ) . orf b, which overlaps orf a for nucleotides but is located at a different reading frame, is amino acids long with a predicted molecular weight of , . the orf b sequence is very similar to that of mhv-a in both nucleotide and predicted amino acid sequences (bredenbeek et al., ) . only minor substitutions were noted between the two strains (data not shown). the orf b starts with cug instead of aug. the first potential initiator codon aug is located nucleotides downstream of the first amino acid the numbers of amino acid residues to the known or postulated termini of the respective viral clpro and between the aligned segments are indicated. for mhv clpro, the postulated n-terminus is at amino acid residue ( fig. gorbalenya et a/. ( b) , except bwyv (veidt et a/., ) and sbmv (wu et al., ) . riore. in the aa position columns, the amino acid positions of the respective q residues are indicated. the arrows show the predicted cleavage sites, abbreviations: mpl , mp , putative membrane proteins flanking the clpr at the n-and c-sides, respectively. pol: polymerase motif. hel: helicase motif. gfl: growth factor-like domain. the data on ibv was obtained from gorbalenya ef al. ( b) . the sequence analysis was performed using the computer program as described under materials and methods. codon in orf b. nevertheless, the codon preference plot suggests that the nucleotides upstream of the first aug are most likely translated together with the downstream sequences using the same reading frame (fig. ) . in light of the corresponding sequences of ibv and mhv-a bredenbeek et a/., ) , this result suggests that this region could be translated via a ribosomal frameshifting mechanism (brierley et al,, ) . comparison of tertiary structure of rna in the frameshift regions. it has been proposed that the nucleotide sequences in the overlapping regions between orf a and orf b in ibv and mhv-a rnas are able to fold into a pseudoknot tertiary structure, which is essential for efficient frameshifting and, thus, expression of the downstream orf b (brierley et a/., ; bredenbeek et al., ) . comparison of the primary sequence revealed that the corresponding region of mhv-jhm contains a "slippery" sequence, uuuaaac, similar to that of ibv (fig. a) . the possible folding of rna in this region into a pseudoknot tertiary structure is similar among ibv, mhv-a , and mhv-jhm (fig. b) . it is interesting to note that the nucleotide changes between mhv-jhm and ibv in either the stem or loop regions are compensated by mutations at the complementary positions (fig. ) . this suggests the significance of the putative tertiary structure in ribosomal frameshifting. only two nucleotides differ between mhv-jhm and mhv-a in this region; they are located at the regions immediately upstream and downstream of the uuuaaac sequence. ribosomal frameshifting in vitro. to confirm that the orf a and b of mhv-jhm could be translated into one polypeptide by ribosomal frameshifting, we cloned the region spanning from nucleotide , to , of gene into an expression vector under the control of the t promoter for in vitro translation studies. because of the lack of a translational initiation codon, an atg codon was introduced by pcr-mediated mutagenesis at nucleotide , -l , . if the translation of this transcript terminates at the uaa stop codon in orf a, a -kda protein will be produced. however, if the - translational frameshift occurs, a -kda protein will be synthesized. as shown in fig. , the in vitro translation of this rna yielded both proteins (lane ). the -kda protein was heterogeneous; the smaller proteins may represent aberrant translational initiation or specific processing of the translation products. the addition of protease inhibitors in the rabbit reticulocyte lysates did not alter this translation pattern (data not shown). the antiserum prepared against the amino acid sequence just upstream of the frameshift (unpublished) precipitated both proteins (lane ). surprisingly, the major products precipitated by this anti- serum migrated faster than the respective primary translation products, suggesting that protein processing had occurred. none of the proteins was immunoprecipitated by the preimmune serum. as controls, the transcripts containing either the '-or the '-halves [ptz(orf""g)] of the orf did not yield the -kd protein. as predicted, only the products of the '-half were precipitated by this antibody (fig. b, lane ) . these results are in agreement with the results obtained with ibv (brierly et a/., ) and mhv-a (bredenbeek et a/., ) . analysis of sequence homology among mhv-jhm, mhv-a , and ibv. the comparison of nucleotide and predicted amino acid sequences between mhv-jhm and ibv revealed considerable similarity between the two. the dot matrix comparison of the amino acid sequences shows that orf b is very similar between mhv and ibv (fig. ) . overall, there are . % similarity at nucleotide level and . % at amino acid level. similar to the orf b of ibv, the mhv orf contains the polymerase and helicase motifs at the corresponding positions (gorbalenya et al., b ) (data not shown). the putative zinc-binding domain is also largely conserved between the two viruses. on the other hand, two of the residues implicated in metal binding for ibv (gorbalenya et al., ) are replaced in mhv, suggesting that the specific structures of the putative "fingers" may differ (fig. ) . the orf b of mhv-jhm and mhv-a are also very similar ( . % at nucleotide level, and . % at amino acid level) (data not shown). in contrast, the orf a is more diverged (fig. ) . the mhv orf a is longer than the corresponding ibv orf by amino acids. the c-terminal half of the orf a is relatively conserved between mhv-jhm and ibv, while the n-terminal half is very diverged (fig. ) . the alignment of amino acids in orf la of mhv-jhm and ibv showed that there are four possible stretches of moderate homology which are separated by highly diverged sequences (fig. ) . analysis of the functional domains of orf la. although orf la is highly diverged between mhv-jhm and ibv, common functional domains could be identified in this orf of both viruses by detailed amino acid sequence analysis (see materials and methods) (fig. ). two hydrophobic, potentially membrane-anchoring regions are present in the c-terminal half. there are three cysteine-rich domains, one of which contains a segment distantly resembling growth factors and their receptors (gorbalenya et al., b) . in both coronaviruses, homologous domains of about residues each have been identified to be related to the putative clike proteases ( clpro) of picorna-, coma-, nepo-, poty-, sobemo-and luteoviruses (gorbalenya et al., b) . the sequences of the putative coronavirus c-like proteases possess certain unusual features distinct from that of other viral clike proteases (fig. , and see discussion). the search for sequences resembling the cleavage sites for the c-like proteases revealed six conserved putative target sites for the mhv and ibv c-like proteases (table ) (see discussion). these potential cleavage sites are localized in the orf b and the c-terminal half of the orf la. interestingly, the n-terminal one of these cleavage sites marks the n-end of the putative clike protease itself. finally, there is a region of moderate conservation between mhv and ibv, which contains short segments resembling those around the catalytic cys and his residues of papain-like proteases (fig. ) . this region is duplicated in the mhv genome, but not in ibv, at an upstream site in the orf la. this upstream papain-like cysteine protease has been identified as the one responsible for the cleavage of p from the n-terminus of the gene protein . a domain of considerable conservation between mhv and ibv (x domain in fig. ) has been found next to the putative coronavirus papain-like proteases. interestingly, a homologous conservative domain also flanks the putative thiol proteases of alpha-and rubiviruses (a. e. gorbalenya, unpublished observations) . the complete sequence of gene of mhv presented in this paper shows that this gene is probably the largest known viral gene among rna viruses. evidence was presented suggesting that the two orfs in this gene may be translated into a large polyprotein. this interpretation is consistent with the lack of the transcriptional initiation signal (ucuaaac) in the entire gene sequence except at the extreme '-end. although the putative "slippery" sequence (uuuaaac) between the orf a and b (brierly et a/., ) is similar to the transcriptional initiation signal, no major subgenomic mrnas have been detected within this gene. thus, this gene most likely encodes a single polyprotein of at least kda. the total size of the rna genome of mhv is approximately kb, which is considerably larger than any of the other known viral rna. the evolution of the coronavirus rna genome into such a large rna may have reflected the unusual mechanism of coronavirus rna synthesis. the complexity of the discontinuous mode of coronavirus rna synthesis (lai, ) suggests that the coronavirus rna polymerase needs a variety of different enzymatic activities. the amino acid sequence of gene of mhv shows considerable similarity to that of ibv. the orf b is particularly conserved. its degree of conservation between mhv and ibv is higher than that for any of the other genes in the coronavirus genomes. the orf b contains the polymerase, helicase, and metal-binding motifs (gorbalenya el a/., b) , suggesting that this region may be directly involved in rna synthesis. these structural features are conserved between these viruses. the proposed pseudoknot structure which is important for the ribosomal frameshifting for cotranslation of orf a and orf b (brierley et a/., ) is also highly conserved. this fact has previously been recognized in the partial sequence of gene of mhv-a (bredenbeek eta/., ) . the sequence differences between mhv-a and mhv-jhm within this junction region are located at the nucleotides which do not affect the putative pseudoknot structure. in contrast, orf la is much more diverged. it is nearly kb longer than the orf la of ibv, and contains several stretches of sequence which are not present in the ibv genome. these nonhomologous stretches of sequence are interspersed between the conserved regions. furthermore, a papain-like protease domain, which is present once in the ibv genome, is duplicated in the '-half of the orf la of mhv. the n-terminal sequence including ~ , which is cleaved by the papain-like protease of mhv , is also highly diverged between mhv and ibv. thus, it appears that the '-end of orf a has undergone considerable sequence rearrangement and possibly recombination, while the remaining sequences in gene are almost colinear between mhv and ibv. in contrast to the orf b which contains sequence motifs related to the synthesis of rna, the orf la contains several domains suggestive of other functions. first of all, there are two long stretches of hydrophobic domains, which are conserved between ibv and mhv. the presence of these domains suggests that the gene products may be anchored to the membrane. this possibility is consistent with the finding that mhv rna synthesis occurs on the membrane fractions in the infected cells (brayton et al., ) . second, there are three cysteine-rich regions, which are also homologous between mhv and ibv. the function of the cys-rich domains is still not clear. however, it has been noted with ibv that the c-terminal cys-rich domain is related to that of the growth factors and their receptors (gorbalenya et al., b) . third, there is a c-like protease domain ( clpro) in the '-half of orf a, which is also conserved in ibv. the putative catalytic his and cys residues previously predicted in ibv have also been observed in mhv (fig. ) . however, the putative coronavirus proteases remain unique in that they do not contain a conserved asp(glu) residue that could serve as the third catalytic residue as suggested for the other c-like proteases (gorbalenya et al., b) . furthermore, the unusual substitution of tyr for gly in the putative substrate-binding region, described previously in ibv, is also observed in the putative mhv clp" (fig. ) . the potential cleavage sites for this c-like protease have been identified to be mainly in orf b and the c-terminus of orf a (gorbalenya et al., b) . these sites (qs) are either conserved or converted to qa in mhv (table ). the potential cleavage at q/s and q/a sites by picornavirus clp" has been demonstrated previously (parks and palmenberg, ) . two qg dipeptides proposed to be cleaved in ibv were substituted in mhv by qc in one case, and by kr dipeptide in another (table ) . substitution of a c (unlike several other residues) for g in a cleavage site for encephalomyocarditis virus protease did not abolish processing in an in vitro system (parks et a/., ) . dibasic dipeptides are cleaved in the polyproteins of flaviviruses (strauss and strauss, ) . thus, these postulated cleavage sites are potentially cleavable by mhv clpro despite the divergence. these cleavages could separate different functional domains of the gene polyprotein into distinct protein products. whether these sites are indeed cleaved in mhv-infected cells remains to be studied. fourthly, the n-terminal portion, which is the most diverged region, contains a papain-like protease domain as pointed out previously for ibv (gorbalenya et al., b) . the papain protease domain is duplicated in the mhv orf a (fig. ) and is homologous with the known proteases (fig. ) . this protease is probably involved in the cleavage of the n-terminus of the gene polyprotein (baker eta/., ) , which has been demonstrated in mhv-infected cells (denison and perlman, ) . site-specific mutagenesis studies demonstrated that this protease has cys and his at its active site (unpublished observation). the possible presence of the protease domains suggests that the gene polyprotein is processed into many proteins. it has been shown that there are at least five to six complementation groups involving mhv rna synthesis, five of which have been mapped within gene (leibowitz eta/., ; baric eta/., ) . these proteins conceivably participate in various aspects of mhv rna synthesis. none of the proteins have been detected so far. sequence of the nucleocapsid gene from murine coronavirus mhv-a sequence and topology of a model intracellular membrane protein, el glycoprotein, from a coronavirus murine coronavirus gene polyprotein contains an autoproteolytic activity identification of a domain required for autoproteolytic cleavage of murine coronavirus gene a polyprotein. . viroi establishing a genetic recombination map for murine coronavirus strain a complementation groups completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus characterization of two rna polymerase activities induced by mouse hepatitis virus the primary structure and expresslon of the second open reading frame of the polymerase gene of the coronavirus mhv-a ; a highly conserved polymerase is expressed by an efficient rlbosomal frameshifting mechanism an efficient ribosomal frame-shifting signal in the polymerase-encoding region of the coronavirus ibv characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot recommendations of the coronavirus study group for the nomenclature of the structural proteins, mrnas and genes of coronavirus supercoil sequencing: a fast and simple method for sequencing plasmid dna multiple sequence alignment with hierarchical clustering prolonged incubation in calcium chloride improves the competence of escherychia co/i cells identification of putative polymerase gene product in cells infected with murine coronavirus a a comprehensive set of sequence analysis programs for the vax purification and amino acid sequence of chicken liver cathepsin l an ntp-binding motif is the most conserved sequence in a highly diverged monophyletic group of proteins involved in positive strand rna viral replication coronavirus genome: prediction of putative functional domains in the nonstructural polyprotein by comparative amino acid sequence analysis the codon preference plot: graphic analysis of protein coding sequences and prediction of gene expression a simple and very efficient method for generating cdna libraries replication and plaque formation of mouse hepatitis virus (mhv- ) in mouse cell line dbt culture multiple recombination sites at the '-end of murine coronavirus rna a method for localization of motifs in amino acid sequences. biopolim. kletka a simple method for displaying the pathic character of a protein replication of coronavirus rna coronavirus: organization, replication and expression of genome mouse hepatitis virus a : messenger rna structure and genetic localization of the sequence divergence from the hepatotropic strain mhv the rnaof mouse hepatitis virus genetic analysis of murine hepatitis virus strain jhm. . viral a method for generation of complete local similarity maps between two amino acid sequences. dothelix program of the genbee package primary structure of the glycoprotein e of coronavirus mhv-a and identification of the trypsin cleavage site sequence of mouse hepatitisvirus a mrna : indications for rna recombination between coronavirus and influenza c virus analysis of genomic and intracellular viral rnas of small plaque mutants of mouse hepatitis virus sequencing end-labeled dna with base-specific chemical cleavages. ln evolutionary origin of a calcium-dependent protease by fusion of genes for a thiol protease and a calcium-binding protein cloning and characterization of a mouse cysteine proteinase molecular cloning of the gene encoding the putative polymerase of mouse hepatitis coronavirus strain a proteolytic cleavage of encephalomyocarditis viral capsid region substrates by precursors to the c enzyme site-specific mutations at a picornavirus vpb/vpl cleavage site disrupt in vitro processing and assembly of capsid precursors primer-directed enzymatic amplification of dna with a thermostable dna polymerase dna sequencing with chain-terminating inhibitors nucleotide sequence of the gene encoding the surface projection glycoprotein of coronavirus mhv-jhm identification of a new transcriptional initiation site and the corresponding functional gene b in the murine coronavirus rna genome the '.end sequence of the murine coronavirus genome: implications for multiple fusion sites in leaderpnmed transcription production and properties of chimeric antibody molecules coronavirus mhv-jhm mrna has a sequence arrangement which potentially allows translation of a second, downstream open reading frame. . gem viral coronavirus jhm: nucleotide sequence of the mrna that encodes nucleocapsid protein coding sequence of coronavirus mhv-jhm mrna sequence and translation of the murine coronavirus y-end genomic rna reveals the n-terminal structure of the putative rna polymerase. . viral coronaviruses: structure and genome expression. . gem viral replication of the rnas of alphaviruses and flaviviruses nucleotide sequence of beet western yellows rna genomic rna of the murine coronavirus jhm. sequence and organization of southern bean mosaic virus rna we thank dr. susan baker for advice throughout the course of the study. we also thank lisa banner and daphne shimoda for editorial assistance. a.e.g. and e.v.k. are most grateful to professor v. i, ago for constant support, to dr. l. i. brodsky for supply of the gen-bee program package, and to dr. key: cord- - e x e authors: talbot, pierre j. title: hemagglutination by murine hepatitis viruses date: - - journal: intervirology doi: . / sha: doc_id: cord_uid: e x e erythrocytes from twelve mammalian and avian sources in ten different buffers at three incubation temperatures could not be hemagglutinated with murine hepatitis virus (mhv) strains , a , or s grown on dbt cells. viral antigen preparation in the absence of fetal calf serum, partial virus purification, or various concentrations of red blood cells still failed to yield detectable hemagglutinating activity. thus, the newly described mhv-dvim remains the only hemagglutinating strain of murine coronavirus. hemagglutination represents a useful model for virus adsorption to cells, a very convenient assay for the presence of virus in biological samples, and may be relevant to viral pathogenesis. several members of the coronaviridae family of enveloped rna vi ruses were shown to possess such a hemag glutinating activity: namely bovine coronavi rus, hemagglutinating encephalomyelitis vi rus of pigs, human coronavirus strain oc , rabbit enteric coronavirus, and avian in-fectious bronchitis virus [ ] . recently, a hem agglutinin was demonstrated on transmissi ble gastroenteritis virus [ ] . however, hemag glutination by murine coronavirus had not been reported until sugiyama and amano [ ] identified a new enteric strain of murine hep atitis virus (mhv): the diarrhea virus of in fant mice (dvim). like bovine coronavirus [ ] and strain oc of human coronavirus [ ] , the hemagglutinating activity of mhv-dvim appeared to correlate with the pres ence of a structural glycoprotein of kd, comprised of subunits of - kd linked by disulfide bridges [ ] , this presumed hem agglutinin appears distinct from the peplomer glycoprotein (named e , s, or p) of - kd [ , ] and its proteolytically cleaved monomers of - kd [ , ] . on * * buffers as described in the text and incubation at , , or °. the same result was obtained at any of these temperatures. b prepared as described in the text and used at . or . %(v/v). the same result was obtained at any of these concentrations. c grown in serum-free medium and concentrated -fold with polyethylene glycol. d not done. c grown with or without serum and used as clarified medium. ' semipurified, as described in the text. the other hand, the hemagglutinating activity of infectious bronchitis virus [ , ] and trans missible gastroenteritis virus [ ] was local ized on the unique peplomer glycoprotein. thus, the preliminary report of hemaggluti nation by another strain of mhv (mhv- ; grown in murine liver and semipurified on sucrose gradient) [ ] , which lacks structural proteins of and - kd [ the origin and culture of mhv strains , a , and s and of dbt cells were described previously [ ] . for use as antigen for hemag glutination assays, mhv- was grown in the absence of fetal calf serum (fcs) and har vested from clarified medium by precipita tion with % (w/v) polyethylene glycol in . m nacl. after centrifugation at , g for min, the pellet was resuspended and dialyzed against tmen buffer: mm tris-hci (ph . ), . m nacl, m m edta. mhv-a , which replicates in vitro more efficiently than any other strain of m h v, was grown with or without % (v/v) fcs and used mouse cdi c b / balb/c day-old chick rooster -- directly in the form of medium freed of cell debris by centrifugation at f , g for min. finally, mhv-s was grown in me dium containing % (v/v) fcs, concentrated as described above, and partially purified on a discontinuous and % (w/v) nyco-denz® (nyegaard, oslo, norway) gradient centrifuged for h at , rpm in an sw rotor (beckman instruments, fullerton, cal if., usa). the virus band was dialyzed against tmen buffer. all viral antigens had infectious titers of . - . logm pfu/ml. red blood cells from various mammalian and avian sources were collected and washed in alsever buffer [ ] and resuspended in the appropriate buffers. ten different buffers were tested: (v) phos phate-buffered saline, ph . [ ] ; and (vi). phosphate-buffered saline with % kaolintreated fcs [ ] , and ( - ) babs, ph . , . , . , or . , respectively [ ] , the hemag glutination assay was performed as de scribed previously [ ] , except that various buffers were used, red blood cells were made up to . or . % (v/v) in the appropriate buffer, and microtiter plates were incubated at , , or °. control hemagglutinating antigens were either rabbit enteric coronavirus (titer / with rabbit red blood cells ) or pneumonia virus of mice (titer / with cdi mouse erythrocytes). both antigens were kindly provided by dr. jean-paul descoteaux (institut armand-frappier). as shown in table , all tested combina tions of buffer, source or concentration of erythrocytes, antigen, or incubation temper atures yielded negative hemagglutination ti ters. one antigen (mhv-a ) was prepared in the presence or absence of fcs to monitor for the possible presence of hemagglutina tion inhibitors in serum [ ] , however, simi lar results were obtained with either antigen (table ). concentrated or partially purified mhv- and mhv-s antigens, respectively, were used to both increase viral infectivity titers and prevent reported inconsistent hem agglutination by crude antigen [ ] . even though not all possible combinations of anti gen, erythrocyte, and buffer were tested, the results presented here show that hemaggluti nation by these three strains of mhv grown on dbt cells is at least not an obvious biolog ical activity, unlike mhv-dvim [ ] . it is possible that the previously reported hemag-glutinating activity of mhv- [ ] was de pendent on the different host used for virus growth or the virus purification method. fi nally, it can be concluded that the -kd structural protein observed under reducing conditions in polyacrylamide gels of purified mhv-s [ , ; pjt, unpubl. data] apparently is not a viral hemagglutinin. the molecular biology ofcoronaviruses hemagglu tination with transmissible gastroenteritis virus hemagglutination and structural polypeptides of a new coronavirus as sociated with diarrhea in infant mice bovine coronavirus hemagglutinin protein structural proteins of hu man respiratory coronavirus oc structural polypeptides of the murine coronavirus dvim coronaviruses ibv: virus retaining spike glycopolypeptide s but not si is unable to induce virusneutralizing or haemagglutination-inhibiting an tibody or induce chicken tracheal protection coronavirus ibv: remov al of spike glycopolypeptide si by urea abolishes infectivity and haemagglutination but not attach ment to cells haemagglutination by mouse hepatitis virus type . ann virol (inst pas teur comparison of polypeptides of two strains of murine hepatitis virus rna and polypeptide homology among murine coronaviruses comparative analysis of rna genomes of mouse hepatitis viruses analysis and localization of mouse hepatitis virus (m h v- )polypeptides physico-chemical properties of murine hepatitis virus, strain a effect of several salts on rubella virus hemaggluti nation techniques for haemagglu tination inhibition with arthropod-borne viruses plaque formation and isola tion of pure lines with poliomyelitis virus hemag glutination activity with bovine herpesvirus type i the expert technical assistance of francine lam bert, manuscript preparation by lucie summerside, and red blood cell preparations by michel boivin and claude duhamel are gratefully acknowledged. this work was supported by a grant (u ) and a scholar ship from the natural sciences and engineering re search council of canada. key: cord- -yaf cmx authors: foley, janet e.; leutenegger, christian title: a review of coronavirus infection in the central nervous system of cats and mice date: - - journal: j vet intern med doi: . /j. - . .tb .x sha: doc_id: cord_uid: yaf cmx feline infectious peritonitis (fip) is a common cause of death in cats. management of this disease has been hampered by difficulties identifying the infection and determining the immunological status of affected cats and by high variability in the clinical, pathological, and immunological characteristics of affected cats. neurological fip, which is much more homogeneous than systemic effusive or noneffusive fip, appears to be a good model for establishing the basic features of fip immunopathogenesis. very little information is available about the immunopathogenesis of neurologic fip, and it is reasonable to use research from the well‐characterized mouse hepatitis virus (mhv) immune‐mediated encephalitis system, as a template for fip investigation, and to contrast findings from the mhv model with those of fip. it is expected that the immunopathogenic mechanisms will have important similarities. such comparative research may lead to better understanding of fip immunopathogenesis and rational prospects for management of this frustrating disease. f eline infectious peritonitis (fip) is a fatal, immune-mediated disease produced as a result of infection of macrophages by mutant feline coronavirus strains (fipvs). the severity of fip is determined by virus strain and by host-specific, partially heritable immune responses. the causative agent, fip virus (fipv), is a macrophage-tropic mutant of the ubiquitous feline enteric coronavirus (fecv). [ ] [ ] [ ] these feline coronaviruses are closely related to transmissible gastroenteritis virus (tgev) of pigs and more distantly related to mhv (a coronavirus of rodents), human respiratory coronavirus, and others. as with most rna viruses, a high rate of point or other small-scale mutations and larger scale recombination events occur. type ii fecvs are viruses that arise as a result of recombinations between type i fecv and canine coronavirus (ccv). type ii fecvs acquired a canine s gene and express a canine s gene product. mutations are common in the b open reading frame (orf) of both type i and type ii fecvs and may be associated with reduced virulence. , the b orf arose in the fecv/ccv lineage and encodes a nonstructural secretory glycoprotein of undetermined function, which is not necessary for viral replication. deletions, point mutations, and frame-shift mutations leading to early truncation of the c orf also are common. , because fip-defining mutations may occur in the s, c, and b genes, polymerase chain reaction (pcr) with these genes as a target cannot discriminate between benign fecv and fatal fipv. [ ] [ ] [ ] murine hepatitis virus shares most genes with the fecvs, including m (membrane glycoprotein), e (small membrane), n (nucleocapsid), and s (spike glycoprotein), which is post-translationally modified to s and s . the mhv genome, however, also codes for an he protein and does not contain a b orf. mutations in the e and s proteins lead to attenuation of mhv. a third coronavirus, hcv- e, has been implicated in neurological disease in people. fip occurs most frequently in cats younger than years of age from multiple-cat homes (shelters and breeding catteries). , one-quarter to one-third of cats with noneffusive fip have either primary neurological fip or neurological abnormalities as a part of their overall disease presentation (foley and pedersen, unpublished data). the immunological and pathological characteristics of neurological fip, however, are much more stereotypic than those of systemic fip. thus, neurological fip may be useful for studying basic mechanisms of fip pathogenesis. the extent to which genetic differences among viral strains confer relative neurotropism is unknown, but some cats with severe neurological disease have mild or undetectable systemic disease. both neurological and generalized fip may present first as a nonspecific illness, with clinical signs including weight loss, weakness, fever, and lethargy. abdominal abnormalities are detected commonly on physical examination of cats with neurological fip, including mesenteric lymphadenopathy and irregular splenic and renal surfaces. , common historical findings in cats with neurological fip include dementia, pica, seizures, inappropriate elimination, incontinence (fecal and urinary), and compulsive licking. , neurological examination may identify ataxia, hyperesthesia, reduced consciousness, hyperreflexia, crossed-extensor reflexes, reduced conscious proprioception, caudal paresis, cerebellar-vestibular signs, or cranial nerve deficits. , , [ ] [ ] [ ] ophthalmic lesions also are common in neurological fip, including anterior uveitis, keratic precipitates, flocculent debris in the anterior chamber, retinitis, and anisocoria. murine hepatitis virus, like fipv, can produce either systemic infection or disease primarily affecting the liver. well-defined neurotropic genetic variants of mhv also occur, including a well-studied variant designated mhv-jhm, which is responsible for progressive encephalomyelitis and death in infected mice and rats. intranasal or intracranial inoculation of mhv-jhm in balb/c or c bl/ mice leads to rapid, fatal encephalitis. the syndrome in survivors (either because the virus is attenuated or the host is resistant, vaccinated, or a pup from a vaccinated dams) consists of chronic demyelination and hindlimb paralysis and has been proposed as a model for multiple sclerosis. determinants of neurovirulence are found in the mhv s gene. , a second variant of mhv, mhv-oblv , leads to neuronal infection specifically in the anterior olfactory bulb after intranasal challenge in mice. neurological disease the definitive lesion of fip is a pyogranuloma that results from immune-mediated phenomena secondary to coronaviral infection of macrophages. the most common sites are serosal, pleural, meningeal, ependymal, or uveal membranes. in the earliest stages of abdominal fip, diffuse alterations with activated mesothelial cells and a few coronavirus-infected macrophages or an exudative precipitate may be detected on serosal surfaces. larger pyogranulomas become grossly visible, ranging from small lesions, often on the renal capsular surface, to severe, generalized miliary granulomatous lesions that distort renal surfaces, disseminate throughout the omentum and gastrointestinal serosa, and invade splenic and hepatic parenchyma. gross lesions of fip in the cns may be subtle, with ependymitis, thickening and opacification of meninges, and ventricular dilatation, usually in the fourth ventricle and least often in the lateral ventricles. lesions occur most commonly on the ventral surface of the brain, often accompanied by secondary obstructive hydrocephalus. histopathologically, lesions in the brains of cats with fip consist of meningitis, ependymitis (ranging from mild ependymal infiltration to complete effacement of the ependymal lining by a heavy infiltrate of histiocytes and lymphocytes), periventriculitis, and choroiditis of varying severity, often superficial and oriented around the ventricles, with dense infiltrates of lymphocytes, plasma cells, neutrophils, and macrophages. , , [ ] [ ] [ ] [ ] [ ] meningitis may be more severe on the ventrocaudal surfaces of the brain, especially at the base of the cerebellum and the brainstem, including the medulla oblongata. in the meninges, the inflammatory cells may have a predominantly perivascular distribution, forming cuffs around arteries (periarteritis) and infiltrating the wall of veins and venules (phlebitis), with exudation of a cell-and protein-rich edema fluid, and periventricular reactive astrocytosis. the inflammation may extend into the superficial neuropil, as well as into cranial nerve roots. if lesions are deep, they usually are perivascular with scattered glial nodules. hydrocephalus is seen in association with leptomeningitis, meningeal fibrosis, accumulation of cellular debris, and obstruction of the cerebrospinal fluid flow. pathologic lesions after mhv infection in the brains of rats and mice are variable, depending on viral dose and route of administration and rodent genotype, age, and immunological characteristics. in severe acute jhm encephalitis, necrotizing lesions are found in the gray and white matter, with axonal changes, including disintegration of neurofilaments. within the necrotic areas, perivascular neutrophilic infiltrates occur especially in the subependyma, choroid, and meninges. in acute mhv-a infection in cd ϩ t-cell deficient mice, periventricular encephalitis occurs with lymphocytic infiltration into the choroid plexus, ependyma, and subependymal brain tissue. lesions in mhv-oblv consist of local neuronal infection, some mitral cell destruction, and t-cell inflammation with astrocytosis. some evidence suggests a pathological component of vasculitis, with mouse endothelial cell lines susceptible to mhv-jhm exhibiting cytopathology within several days of infection. the subacute to chronic immune-modulated pathological changes that occur with manipulation of the mhv-jhm system are particularly relevant to the murine model of fip. depending on mouse strain and immunological status, mhv-jhm produces meningeal inflammation associated with t-cells and macrophages and demyelination but relatively little disease in axons. demyelination occurs within and adjacent to areas of inflammation and astrocytic proliferation. , neutrophils and monocytes have been observed infiltrating through endothelial cell junctions and participating in the phagocytosis of myelin debris. perivascular cuffing by macrophages is observed in chronic encephalitis, as in fip. the route of entry of fipv into the cns is unknown. the fipv virus probably travels hematogenously in macrophages, and study reported a cat with positive immunohistochemical staining for fipv in monocytes in blood vessels of the choroid plexus. once in the cns, there is little evidence that fipv enters any cells other than macrophages. foley et al reported positive immunohistochemical staining (by means of a mouse monoclonal antibody against the fipv n protein) for fipv, primarily in macrophages in fip granulomas but also in some lymphocytes. virus-infected cells were numerous in some areas of intense ependymitis and choroiditis and free within the ventricular lumen, with very few positive cells in the meningeal infiltrates. no staining was observed in vascular basement membranes or in cells of the neuropil. macrophages in necrotic regions and in the center of lesions often are not infected with coronavirus. neurotropic mhv strains have several routes of entry into the cns. some studies show that mhv-jhm travels up the olfactory nerve and enters the cns. this is not surprising, because coronaviruses generally are epitheliotropic, and neurons share embryological origin with epithelial cells. cns infection with jhm also may occur after peripheral infection and viremia. , receptors for neurotropic human coronavirus hcv- e have been detected not only in human lung cells but also in human neuron, astrocyte, and oligodendrocyte cell cultures. hcv- e also infects macrophages and endothelial cells, however, suggesting hematogenous introduction into the cns, , similar to fip. once inside the cns, the major targets for mhv are glial cells and neurons. especially in neuroattenuated strains, mhv-jhm infects primarily oligodendrocytes and has been reported in astrocytes. mhv- infects mainly neurons. subsequent events in chronic disease pathogenesis include recruitment of inflammatory cells and the interactions of immune cells with the virus-infected cells. macrophages, activated t-cells, and some b-cells may cross into the intact cns through the blood-brain barrier, with the potential for major cytokine upregulation. , the immunopathogenesis of neurologic coronavirus infections after establishment of fipv in the cns, mechanisms of disease are primarily immune-mediated, involving humoral and cell-mediated immunity (cmi). coronavirus-infected macrophages can trigger massive complement activation and deposition of c on affected surfaces, disseminated intravascular coagulopathy, vessel necrosis, and effusion. , [ ] [ ] [ ] however, immunopathogenic events in neurological fip have not been well described. both in systemic and neurological fip, antibodies (especially to the spike protein) contribute to the opsonization of viral antigen and have the capacity to mediate or enhance disease. [ ] [ ] [ ] [ ] [ ] antibodies to fecv and fipv are identical. anti-fipv igg and igm-producing b-cells are present in fip lesions, at the interface of healthy tissue and granulomas, and in the serum and csf of cats with neurological fip. , apparently, some antibody production occurs locally in the cns, in response to viral antigen in the brain. in one study, serum coronavirus titers of cats with neurological fip were positive, with a median titer of : , whereas csf titers were positive in cats, with a median titer of : . two cats had high protein concentrations and increased cells, predominantly lymphocytes and neutrophils. the titer in csf was not statistically correlated with serum titer, and the ratio of serum : csf titer was not correlated with the serum : csf protein ratio. if passive leakage of protein from serum into the csf were invoked to explain the presence of antibodies, it would be expected that the total protein : anti-fipv igg ratios would be similar in both serum and csf. in contrast, csf igg titers tended to be proportionally much higher than serum titers. this finding suggests that anti-fipv igg may have been produced in the tissues of the brain in response to a locally replicating virus. although almost nothing is known about the mechanism, cell-mediated immunity has been hypothesized to be protective against fip. [ ] [ ] [ ] [ ] [ ] cd ϩ t-cells commonly are observed in fip granulomas. during acute experimental multisystemic fip, apoptosis and t-cell depletion were observed in the spleen and mesenteric lymph nodes. this effect was induced by heat-treated effusion fluid (presumably containing some cytokines) but not tissue culture fluid. likewise, little is known about the induction of cytokines during the course of fip. preliminary findings suggest that development of fip appears to be associated with a switch to predominantly th immunity, with increases in il- concentrations. goitsuka et al described increases in il- and il- , but gunn-moore et al reported reductions in th and th cytokines including il- , il- , il- , and il- , which they attributed to general immunosuppression. mildly increased amounts of il- ␣ mrna are detected inconsistently in association with lesions in cells of many organs in cats with fip. roles of il- in this setting may include vascular endothelial activation, regulation of macrophages, il- and il- , and chemotaxis. il- and il- can increase b-cell growth and differentiation and could exacerbate humoral contributions to the severity of fip. the inflammatory cytokines tnf-␣, il- ␣, and il- could circumvent the blood-brain barrier during systemic fip, or they could cross the blood-brain barrier after reorganization of the endothelial actin cytoskeleton. , no information is available regarding cytokine production in neurological fip, and how cytokines in the cns compare to those in abdominal tissues of cats with generalized or effusive fip is unknown. mhv neurological disease can range from severe, acute, necrotizing, rapidly fatal encephalitis to chronic immunemediated demyelination with little encephalitis, depending on mouse strain, age, and immunocompetence. the appreciation of the principal role of the immune system in producing demyelination emerged from a series of experiments performed over several decades. profoundly immunosuppressed mice develop high concentrations of virus in the cns, but demyelination and clinical signs are minimal. immunocompetent mice have variable or even low concentrations of virus but develop marked disease. immune reconstitution in immunocompromised mice results in the development of severe demyelinating disease. if mice are pretreated with passive infusions of antibodies or t-cells or if they receive neuroattenuated mhv strains, they develop chronic, but not fatal, disease after mhv-jhm infection. , immunocompetent c bl/ mice clear mhv-jhm virus from the brain but develop severe immune-mediated demyelination and paralysis. in contrast, severe combined immunodeficient (scid) mice have persistent viral loads but no neurologic impairment or detectable lesions. gamma-irradiated immunocompromised mice similarly were resistant to chronic demyelination, but reconstitution of the immune system with adoptive transfer of splenocytes restored the immune-mediated lesions. both cd ϩ and cd ϩ t-cells apparently are required to clear mhv from the cns. cd ϩ t-cells are the predominant infiltrating leukocyte in lewis rats with mhv-jhm and paralytic disease, whereas infiltration in clinically normal mhv-jhm-positive brown norway rats consists of cd ϩ cells. ctls may kill some virally infected cells and protect mice from fatal disease, but they do not completely eliminate virus. when cd ϩ-depleted mice are reconstituted, virus load is reduced, and infection is not detected in most infected cell types, except for oligodendroglial cells. mice with nucleocapsid or spike proteinspecific cd ϩ t-cells develop chronic demyelinating disease. , in the mhv-oblv model, depletion of cd ϩ cells is associated with delayed clearance of oblv (but infected mice did recover). ctls probably exacerbate lesions by contributing to tissue damage. mhv is relatively labile genetically (a common characteristic of rna viruses), and mutant strains with recognition sites that can evade ctl-mediated viral killing apparently increase and become the predominant viral strains in response to ctl-mediated natural selection. this feature results in disease progression in immunocompetent, but not immunosuppressed, hosts infected with these mutants. several studies have suggested that apoptosis may be important in clearing virus from the cns. specific ctl recognition promotes apoptosis of mhv-infected cells. in experimental allergic encephalomyelitis in rats, apoptosis appears to help control inflammation. the role of cd ϩ cells in mhv infection is also complex. nucleocapsid or spike protein-specific cd ϩ t-cells have been shown to protect mice from coronaviral encephalomyelitis in the absence of cd ϩ t-cells. if mice with mhv-oblv had cd ϩ but not cd ϩ cells, they developed persistent infection. if they were cd ϩ deficient, but cd ϩ cells were normal, they had delayed clearance of the virus, suggesting a primary role for cd ϩ cells in clearing this virus. sussman et al and williamson and stohlman documented the requirement for cd ϩ cells for clearance of jhm from mice. however, ␥-irradiated mice that were reconstituted with cd ϩ cells responded to mhv-jhm challenge with earlier and more severe onset of neurological disease. cd ϩ knockout mice had less inflammation (with fewer macrophages and microglial cells) and less demyelination than did cd ϩ competent mice. the presence of mhv-jhm in astrocytes triggers a cytokine cascade that contributes to demyelination. sun et al documented production of tnf-␣, il- ␤, and il- by astrocytes in the spinal cords of mice that were chronically infected with mhv-jhm, localized to areas of virus infection and demyelination. tnf-␣ and il- are also produced in the brains of acutely infected mice, but the major cell producing these cytokines is the macrophage. these cytokines may help recruit t-cells and monocytes and may increase vascular permeability. il- ␤ promotes leukocyte adhesion to endothelial cells, and tnf-␣ is toxic to oligodendrocytes. the role of il- is unclear. effects attributed to il- include recruitment and activation of t-cells and macrophages, expansion of ctls, modulation of plasma cell differentiation, increased vascular permeability, downregulation of acute phase proteins, and contributions to immune-mediated destruction in the cns. [ ] [ ] [ ] [ ] in a study of cytokine profiles in lethally compared to sublethally affected mice with jhm, both th -and th type cytokines, including ifn-␥, il- , and il- , were induced in all mice. in the mice that died, tnf-␣ was induced more rapidly, and il- ␣ was increased. in mice with nonlethal infections, il- and il- ␤ were increased, and il- was expressed early. minor differences were observed in the patterns of inflammation in il- -deficient compared to syngeneic mice, but the outcome of infection (eg, mortality and virus load) was not affected. these results, combined with those of sun et al, suggest that il- ␤ may allow mice to survive the early stages of the disease but may not allow for clearance of chronic infection. interferon-␥ also appears to be important for clearance of mhv infection. ifn-␥-deficient mice developed persistent jhm infection with increased clinical signs and mortality compared to mice competent to produce ifn-␥. antiviral antibody and ctl responses were normal in ifn-␥-negative mice, despite the fact that ifn-␥ modulates antibody production. viral antigen occurred in oligodendrocytes and in association with cd ϩ t-cells, suggesting that ifn-␥ is necessary for control of viral replication in oligodendrocytes. treatment of mice with anti-ifn-␥ antibody increased the mortality rate of mice, whereas immunotherapy with ifn-␥ reduced mortality and virus load. mhv-oblv infection in immunocompetent mice induced transient mrna upregulation for cytokines il- ␣, il- ␤, il- , tnf-␣, and ifn-␥. nude mice differed in their cytokine profiles in that they lacked mrna for ifn-␥, whereas concentrations of the other cytokines remained persistently high. the authors suggested that the increased cytokines in the nude mice were produced by cns glial cells and possibly cd ϩ and cd ϩ subsets. ifn-␥ treatment of human neuronal cell cultures markedly increased the susceptibility of cells to infection with hcv-oc (although cells were susceptible to e without ifn-␥ treatment). in this study, the hypothesized role of ifn-␥ was induction of mhc class i expression. ifn-␥ may increase superoxide dismutase and protect against oxygen radicals, kill virus directly, and increase the cytotoxicity of other effector cells. the chemokine crg- is expressed primarily by astrocytes during mhv infection and co-localizes with areas of viral rna and demyelination. the function of crg- is not known, but this chemokine is also found in simian immunodeficiency viral (siv) encephalitis and lymphocytic choriomeningitis. with very little information available about the immunopathogenesis of neurologic fip, research performed in mice can be used as a model for fip and to contrast findings from the mhv model with those in fip. the immunopathogenic mechanisms are expected to have important similarities, but the demyelination that occurs in mhv is a more extensive pathological sequela of coronaviral infection (but not more fatal for the patient) than the focal granulomas that occur in fip. it can be hypothesized that in fip, upregulation of cytokines il- ␤, il- , ifn-␥, and tnf-␣ may be important in mediation of destructive inflammation, but cytokines il- and ifn-␥ may be important in controlling viral infection, despite adverse inflammatory effects. cytokine and chemokine alterations in the brains of mice and cats with coronavirus infections may provide valuable clues to the immunopathogenesis of these diseases, as well as possible diagnostic markers and targets for immunological treatment of disease. it is tempting to consider cytokine modulation in the treatment of fip, but this application is premature without information regarding the cytokine profiles of affected cats. in addition to characterizing the concentrations of cytokines in the cns of cats with fip, it will be important to describe the timing of upregulated cytokine transcription. in mice with hmv-jhm, treatment with recombinant ifn-␥ resulted in reduced virus load in the liver but not in the brain. if specific pro-inflammatory cytokines are found to be consistently high in cats with neurological fip, use of cytokine antagonists may eventually be of benefit in treatment. successful management of fip may consist of better methods of prevention as well as management of disease once it occurs. a commercial fip vaccine is available that consists of a mucosally delivered, temperature-sensitive mutant form of fipv. the vaccine virus is supposed to undergo replication only in outer oronasal cavities at low temperatures, thus triggering protective antibodies but not fip. a few controversial studies have documented reduction in fip as a result of vaccination. , many vaccinated cats, however, fail to seroconvert with either igg or iga (foley and pedersen, unpublished data) , and independent studies failed to identify vaccine efficacy. , other vaccines, including dna vaccines, have not progressed beyond experimental stages either because of lack of seroconversion, lack of protection, or both. in contrast with fip vaccines, vaccines against mhv have been shown to protect mice from challenge with virulent virus. a vaccine with purified spike protein and synthetic s led to neutralizing antibodies in vivo. , recombinant subunits against s in tobacco mosaic virus were given subcutaneously or intranasally and were protective against mhv-jhm. the current standard of care for immunomodulation of fip is immunosuppressive doses of prednisone, to sustain an acceptable quality of life for as long as possible. the effects of steroids include lymphocytolysis, inhibition of arachidonic acid metabolism, reduction in cytokine rna transcription, and nitric oxide synthesis inhibition. other potentially useful antiinflammatory drugs include antileukocyte antibodies and antioxidants. these treatments are only palliative. a better understanding of the fundamental pathogenesis of fip will be necessary to offer better treatment and, ultimately, cure of this disease. 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coronavirus into primate cns following peripheral infection involvement of aminopeptidase n (cd ) in infection of human neural cells by human coronavirus e replication of human respiratory coronavirus strain e in human macrophages characterization of a variant virus selected in rat brains after infection by coronavirus mouse hepatitis virus jhm spread of a neurotropic coronavirus to spinal cord white matter via neurons and astrocytes afferent and efferent arms of the humoral immune response to csf-administered albumins in a rat model with normal blood-brain barrier permeability t-lymphocyte entry into the central nervous system pathogenesis of feline infectious peritonitis: pathologic changes and immunofluorescence jacobse-geels he, daha mr, horzinek mc. isolation and characterization of feline c and evidence for the immune complex pathogenesis of feline infectious peritonitis pathogenesis of feline infectious peritonitis: nature and development of viremia early death after feline 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alpha-and beta-chemokine expression in the central nervous system during mouse hepatitis virus-induced demyelinating disease chemokine expression in simian immunodeficiency virus-induced aids encephalitis chemokine gene expression in the brains of mice with lymphocytic choriomeningitis vaccination against naturally occurring fip in a single large cat shelter overview of the development of a modified live temperature-sensitive fip virus vaccine independent evaluation of a modified live feline infectious peritonitis virus vaccine under experimental conditions (university of liverpool experience) independent evaluation of a modified live fipv vaccine under experimental conditions (cornell experience) protection from lethal coronavirus infection by affinity-purified spike glycoprotein of murine hepatitis virus, strain a vaccination against lethal coronavirus-induced encephalitis with a synthetic decapeptide homologous to a domain in the predicted peplomer stalk immunogenic peptide comprising a mouse hepatitis virus a b-cell epitope and an influenza virus t-cell epitope protects against lethal infection protective immunity against murine hepatitis virus (mhv) induced by intranasal or subcutaneous administration of hybrids of tobacco mosaic virus that carries an mhv epitope adjunctive therapy for bacterial meningitis: rationale for use, current status, and prospects for the future key: cord- -fz vrnb authors: templeton, steven p.; perlman, stanley title: pathogenesis of acute and chronic central nervous system infection with variants of mouse hepatitis virus, strain jhm date: - - journal: immunol res doi: . /s - - -y sha: doc_id: cord_uid: fz vrnb infection of mice with variants of mouse hepatitis virus, strain jhm (mhv-jhm), provide models of acute and chronic viral infection of the central nervous system (cns). through targeted recombination and reverse genetic manipulation, studies of infection with mhv-jhm variants have identified phenotypic differences and examined the effects of these differences on viral pathogenesis and anti-viral host immune responses. studies employing recombinant viruses with a modified spike (s) glycoprotein of mhv-jhm have identified the s gene as a major determinant of neurovirulence. however, the association of s gene variation and neurovirulence with host ability to generate anti-viral cd t cell responses is not completely clear. partially protective anti-viral immune responses may result in persistent infection and chronic demyelinating disease characterized by myelin removal from axons of the cns and associated with dense macrophage/microglial infiltration. demyelinating disease during mhv-jhm infection is immune-mediated, as mice that lack t lymphocytes fail to develop disease despite succumbing to encephalitis with high levels of infectious virus in the cns. however, the presence of t lymphocytes or anti-viral antibody can induce disease in infected immunodeficient mice. the mechanisms by which these immune effectors induce demyelination share an ability to activate and recruit macrophages and microglia, thus increasing the putative role of these cells in myelin destruction. in the absence of infection or injury, the resting state of the central nervous system (cns) is highly resistant to peripheral immune cell infiltration and activation. access to cns tissues by peripheral blood leukocytes is prohibited by tight intercellular junctions and low expression of adhesion molecules by endothelial cells of the blood brain barrier (bbb). within the cns, immune activation is further diminished through downregulation of mhc molecules by resident cns cells, and through constitutive expression of immunoregulatory molecules such as tgf-b. furthermore, secreted factors which regulate normal brain function and development exhibit immunosuppressive activity. when cns infection or injury occurs, these barriers to immune infiltration and activation are altered to allow access to circulating leukocytes and protective antibody. during cns immune responses, however, a balance between immune activation and suppression is maintained. this conserves integrity and function of non-regenerative cns tissue, while permitting immune responses against invading pathogens. tilting the balance toward immune suppression could result in persistent or chronic infection, whereas excessive immune activation could result in autoimmunity or bystander destruction and loss of function of vital cns tissue [ ] [ ] [ ] . one pathogen that infects the cns of mice and thus challenges the local balance between immune activation and suppression is mouse hepatitis virus, strain jhm (mhv-jhm). contrary to its name, mhv-jhm is a neurotropic, not a hepatotropic, single stranded rna virus of the family coronaviridae. human coronaviruses related to mhv-jhm cause a variety of infection and disease ranging from the common cold to severe acute respiratory syndrome (sars) [ , ] . naïve susceptible mice infected with virulent variants of mhv-jhm develop lethal acute encephalitis. however, other variants of mhv-jhm provide tools for studying both acute and chronic infection of the cns [ ] . studies of cns infection with these variants have identified distinct mutations in virus structure, and have compared the effect of these structural differences on viral pathogenesis and host immune responses to infection. differences between these variants are often reflected by changes in cell tropism, neurovirulence, and in the quality of host anti-viral immune responses in the infected cns. in order to more closely compare virus structure and function with associated pathogenesis, additional mhv-jhm variants are generated through targeted recombination, with methods similar to those originally used to create mhv strain a recombinant viruses [ ] [ ] [ ] [ ] . initially, mutations or introduced coding sequences are generated by pcr from mhv-jhm, or from host genes encoded on plasmids, respectively. the site used for expression of non-mhv sequences is within gene , which is not essential for virus replication or infection [ , ] . rna is generated from the final linearized plasmid, containing the mhv-jhm s and gene sequences, by in vitro transcription. donor rna is then transfected into cells infected with a homologous recipient virus. desired recombinants are selected by host cell specificity, which is altered by donor/recipient recombination. in this manner, recombinant mhv have been generated for functional studies of mhv-jhm structural proteins, or for the production of immune effectors, and for functional studies of non-structural proteins of the human coronavirus sars-cov [ ] [ ] [ ] . while acute infection is characterized by rapid virus spread and cns inflammation, chronic disease during mhv-jhm infection is characterized by incomplete clearance of infectious virus and concomitant development of demyelinating disease [ ] . mhv-jhm infected mice with demyelinating disease serve as a relevant animal model of the human autoimmune disease multiple sclerosis (ms) [ , ] . as in ms, demyelination induced during mhv-jhm infection is characterized by macrophage infiltration into the white matter of the cns, with subsequent destruction of the protective myelin sheath surrounding the axons of cns neuronal cells. demyelination may be induced in mice through persistent infection with attenuated mhv-jhm strains, or by partial protection from virulent mhv-jhm by mhv-specific antibody [ , , ] . demyelinating disease induced during mhv-jhm infection is partly immune mediated, as mice lacking the ability to generate t cell responses fail to develop demyelination, despite high viral loads and widespread inflammation in the cns of infected mice [ , ] . to examine this relationship between virus infection and demyelination, immunodeficient mice infected with mhv-jhm receive adoptively transferred enriched populations of t cells or other immune effectors [ , ] . these models of mhv-jhm infection provide excellent tools for investigating both acute and chronic viral infection of the cns. however, the factors that mediate the initiation of host anti-viral immune responses, virus-induced pathology, and the regulation of these events by the specialized local environment of the cns, are only partly understood. susceptible mice infected intracerebrally with the mhv-jhm variant mhv.sd (also termed mhv- ) develop fatal acute encephalitis with a % mortality after inoculation with plaque forming unit (pfu) [ ] . in highly virulent variants of mhv-jhm, intranasal inoculation also results in uniformly fatal encephalitis in susceptible mice. the resulting pathology is generally attributed to sequence variation in the spike (s) glycoprotein. the contributions of non-s versus s genes to enhanced mhv.sd neurovirulence were determined by infection with recombinant viruses comprised of either the non-s genes of the less virulent mhv strain a with the s gene of neurovirulent mhv.sd, or the non-s genes of mhv-jhm with the s gene of mhv-a [ , [ ] [ ] [ ] . these studies demonstrate that recombinant viruses expressing the mhv.sd s protein are more neurovirulent than mhv-a s glycoprotein expressing viruses. in addition to differences in mortality between mhv.sd and mhv-a infected mice, host immune responses also differ. mice infected with mhv.sd exhibit a prolonged innate response characterized by ifn-b production in the cns beyond days post infection with decreased il- p and ifn-c transcription [ ] . in mhv-a infected mice, ifn-b transcripts decrease after days post infection, with increasing ifn-c mrna coincident with increasing t cell infiltration. proinflammatory cytokines and chemokines such as il- , il- , mip- a, mip- b, and mip- are upregulated in the cns of mhv.sd mice in comparison to relatively low cns expression during mhv-a infection. adaptive immunity is suppressed during mhv.sd infection. antigen specificity of adaptive immune responses of both cd and cd t cells are determined by recognition of defined immunodominant and subdominant epitopes within mhv strains [ , ] . the immunodominant cd t cell epitope recognized in mhv-jhm infected c bl/ mice is presented on the h- d b class i molecule and is located in the mhv-jhm spike glycoprotein from (amino acids - (s )). an h- k b cd t cell epitope is located in the s protein of both mhv-jhm and mhv-a spanning amino acids - (s ). mhv-a infected mice mount a robust cd t cell response to the shared s epitope, while mhv.sd infected mice mount a weak response to both s and s epitopes [ , ] . total t cell infiltration (antigen specific and non-specific) is also reduced in the cns of mhv.sd infected mice. consistent with increased inflammation in virulent mhv.sd infection, mononuclear cells expressing cd b and fccri/iii are significantly increased in the cns of mhv.sd infected mice. these results demonstrate that mhv.sd infection of the cns results in a greater inflammatory response and a weakened cd t cell response when compared to mhv-a . to evaluate the contribution of the spike (s) glycoprotein to the pathogenesis of mhv.sd infection, targeted recombination and reverse genetic techniques were used by phillips et al. to generate mhv.sd and mhv-a viruses that express the s glycoprotein of the other strain [ ] . rempel et al. further characterized the immune responses to these viruses, using recombinant mhv-a viruses with the s glycoprotein of either mhv-a (wtr ) or mhv.sd (s r ) [ ] . infection of mice with s r results in increased virulence compared to the mhv-a wild type recombinant wtr . however, this increased virulence is not accompanied by a suppressed cd t cell response or prolonged ifn-b production, which were observed in the highly virulent mhv.sd infection. increased virulence in s r infection is associated with increased cns macrophage infiltration and increased mip- a and mip- b transcription, while infection with either s r or wtr results in a robust cd t cell response and increased cns ifn-c transcription. these results were repeated and confirmed by iacono et al., who further explored the role of background (non-s) genes by generating an mhv-jhm virus that expressed the mhv-a s glycoprotein. mice infected with this virus (sa /rjhm) display an attenuated neurovirulence compared to wild type recombinant mhv-a (ra ). however, these mice mount a diminished antigen specific cd t cell response to infection similar to the suppressed response to rjhm infection. from these results, the authors concluded that although the s glycoprotein determines the neurovirulence of mhv strains, the background genes determine the extent of the cd t cell response. this conclusion conflicts with results obtained from infection of mice with a slightly attenuated, yet closely related mhv-jhm variant, mhv.ia [ ] . similar to the neurovirulent mhv.sd, % of mice infected intracerebrally with pfu of mhv.ia succumb to fatal encephalitis. however, in contrast to the weak response to mhv.sd infection, mhv.ia infected mice mount a robust mhv specific cd t cell response [ ] . furthermore, attenuation in neurovirulence in mice infected with mhv.ia is attributed solely to a single amino acid change in the spike glycoprotein [ ] . as with studies comparing mhv.sd and mhv-a virulence, examining the association of the mhv s glycoprotein with neurovirulence in mhv.ia and mhv.sd infection was performed through generation of recombinant rjhm.ia virus via targeted recombination. with this strategy, a background rjhm.ia virus expressing the spike glycoprotein of mhv.sd was generated (rjhm.sd). comparison of rjhm.ia and rjhm.sd infection revealed that the mhv.sd s expressed by rjhm.ia increases neurovirulence with accompanied increases in viral titers and lateral spread throughout the cns of infected mice. furthermore, the spike glycoprotein of rjhm.sd was shown to mediate increased receptor independent spread in infection of tissue culture cells lacking the carcinoembryonic antigen cell adhesion molecule (ceacam- , the cellular receptor for mhv-jhm entry). sequencing of these viruses indicated that the structures of the spike proteins of rjhm.ia and rjhm.sd differ by four amino acids. targeted mutation of the s protein of rjhm.ia resulting in a single change in the amino acid from a serine to a glycine generated a virus (rjia.s g) that exhibited increased neurovirulence in infected mice when compared to rjhm.ia infection. although these studies have identified structural determinants of virulence, the importance of the spike protein in the generation of contrasting immune responses to mhv.sd and mhv.ia infection is not fully understood. prolonged production of ifn-b in the cns of mhv.sd infected mice may provide one clue [ , ] . stimulation of ifn-b production in the cns through toll like receptor (tlr ) suppresses eae [ ] . in certain conditions, ifn-b induces the upregulation of numerous effectors that ultimately limit t cell responses. in cultured cns cells, ifn-b production limits the capacity of antigen presenting cells to activate t cells [ ] . in addition, mice deficient in ifn-b have increased antigen-specific cd t cell responses to peptide or dna vaccination, with decreased il- producing t regulatory cells (t reg ) [ ] . both t reg cells and il- have been implicated in regulating immune responses to viral infection as well as autoimmunity [ ] [ ] [ ] . by controlling the magnitude of anti-viral immune responses, the activity of t reg cells and il- effectively limit immune-mediated pathology while providing a potential opening to chronic viral infection. determining the source of ifn-b in the cns of mhv.sd infected mice is important for understanding the mechanism of mhv.sd mediated immune suppression. neurons are a major source of type i ifn in mice infected with theiler's murine encephalitis virus (tmev) [ ] . furthermore, neurons inhibit t cell responses and ameliorate disease in the cns of mice with experimental autoimmune encephalomyelitis (eae), via conversion of encephalitogenic t cells into regulatory t cells [ ] . determining the source of type i ifn and important associated factors in mhv.sd mediated immune suppression are the focus of current investigation. in addition to providing a model for examining structural components and determinants of virulence, targeted recombination of mhv-jhm has provided insight into the role of immunodominant epitopes in both cd and cd t cell responses to infection. persistent infection with viruses like hiv- or hepatitis b or c viruses may select viral mutants that evade the host cd t cell response [ ] . these mutations are commonly selected in the immunodominant cd epitopes recognized by a large portion of virus-specific cytotoxic t cells, thus directly diminishing their ability to clear viral infection. these cd t cell epitope, or ctl escape, mutations also occur during mhv-jhm infection, allowing for viral persistence and chronic demyelinating disease [ ] . mhv-jhm ctl escape mutations are selected in the immunodominant s , but not the subdominant s epitope. through targeted recombination, a second high avidity cd t cell immunodominant epitope from lymphocytic choriomeningitis virus (lcmv gp ) [ ] was added to recombinant jhm [ ] . in the presence of both s and gp high avidity epitopes, demyelinating disease associated with ctl escape is prevented. in contrast to the persistent infection associated with elimination of the immunodominant mhv-jhm cd t cell epitope, mice infected with recombinant mhv-jhm with a single mutation in the immunodominant cd t cell epitope m - (rj.m y q ) exhibit milder disease with no mortality [ ] . virus is ultimately cleared in rj.m y q -infected mice and antigen specific cd t cell responses are equivalent to those detected in mice infected with wild type recombinant jhm. the absence of disease in rj.m y q -infected mice is not attributed to decreases in either tnf-a or to increased th cytokine production in the cns. these results are striking, particularly because numerous studies have reported decreased viral clearance and increased mortality in the absence of a cd t cell response [ , [ ] [ ] [ ] . future studies will be aimed at further characterizing the roles of these specific epitopes in mhv-jhm pathogenesis, the role of other epitopes such as the subdominant k b s epitope in ctl escape selection, and the mechanism by which the loss of the immunodominant cd epitope attenuates disease in infected animals. in addition to studies of acute mhv-jhm infection using the mhv.sd and mhv.ia variants, infection of mice with an attenuated variant of mhv.sd, mhv-j . -v , provides a model for studying acute and chronic infection of mice with subsequent development of demyelinating disease [ , ] . j . v- differs from mhv.sd in structure by a single amino acid in the mhv s glycoprotein. early responses to j . v- infection are similar to those in mhv-jhm-infected mice. cns inflammation allows breakdown of the bbb, permitting peripheral blood neutrophil and monocyte infiltration. concomitant with a t cell response, infectious virus is mostly cleared from the cns by weeks post-infection [ ] , but may remain detectable by low levels of viral rna well after viral clearance [ ] . however, macrophage recruitment into areas of white matter within the cns continues in the absence of infectious virus, and mice develop plaques of demyelination in associated areas of macrophage infiltration. thus, infection of mice with mhv-j . v- provides a model for the study of persistent viral infection and chronic demyelination. ms is a debilitating human disease with a worldwide distribution, and is characterized by immune-mediated destruction of the myelin sheaths surrounding neuronal axons and, in some cases, degeneration of the axons themselves [ , ] . ms patients can exhibit disease with several different, yet potentially overlapping, clinical and pathological profiles. due to this diversity, the etiology of ms is likely diverse as well. although not completely understood, both genetic and environmental factors play a role in disease development and progression. ms patients in remission often experience relapses after common viral infections, indicating an environmental component may be necessary to trigger disease in susceptible individuals. due to the multiple factors that promote and affect ms in humans, numerous animals models of demyelinating disease have been developed to examine particular aspects of its pathogenesis [ ] . the well-established model experimental eae examines immune responses to myelin antigens in rodents. viral demyelination induced during infections with semliki forest virus (sfv), theiler's murine encephalomyelitis virus (tmev), or mhv-jhm provide models of pathogen associated demyelinating disease [ , ] . a hallmark of ms and associated animal models of the disease is the presence of infiltrating macrophages and resident microglia in demyelinating plaques located in the white matter of the cns. both cell types are able to phagocytose myelin, and therefore are potential contributors to autoimmune tissue destruction. specific contributions of macrophages and microglia to demyelinating disease within each animal model are less clear. in models of demyelinating disease in which cns inflammation results in a breakdown of the bbb, other peripheral blood leukocytes are also present in the cns. myelin-specific t cells are present in eae lesions, while during mhv-jhm infection, t cells are predominantly specific for viral antigens. b cells play a role in demyelinating disease, as the presence of myelin specific antibody exacerbates disease in eae. professional antigen presenting cells such as dcs are also present in the cns in mice in both the eae and tmev models, and are able to prime naïve t cells in situ [ ] . demyelination during mhv-jhm infection has been studied in several contexts. one particular model relies on infection of mice partially protected by nursing on mhv-jhm immune dams (suckling mouse model) [ ] . in this scenario, maternal mhv-specific antibody facilitates partial viral clearance. thus, these mice survive the acute infection, but subsequently develop demyelinating disease accompanied by clinical signs of hind limb paresis. in this model, development of chronic disease is associated with mutations in the s cd t cell epitope [ ] . these ctl escape mutations allow for viral persistence and chronic demyelination. emergence of ctl escape in mhv-jhm infected animals is strain dependent but mhc independent, as c bl/ mice allow ctl escape while balb/c or balb/b mice do not [ ] . subsequent studies indicate that this difference is due to increased endogenous anti-viral antibody production, specifically due to an increase in the amount of antibody secreting, or plasma cells, in the cns of balb mice. despite the clear link between prevention of ctl escape and anti-viral antibody production in the cns of mhv-jhm infected mice, important questions remain. one important facet of ctl escape is the selection of mutations in the immunodominant d b s , but not the subdominant k b s epitope. an explanation for this selection is likely found in the differences between functional avidity of the two epitopes, with the s epitope exhibiting higher functional avidity than the subdominant s epitope [ ] . current studies are aimed at examining these differences by alteration of epitope avidity in recombinant mhv-jhm. a second model of demyelinating disease during mhv-jhm infection involves infection of adult mice with the attenuated mhv-j. . v- variant [ ] . although generally resistant to intranasal infection, c bl/ mice infected intracerebrally with j . v- develop mild acute encephalitis, followed by viral clearance mediated by an anti-viral t cell response. demyelination with clinical signs of hindlimb weakness occurs during the process of virus clearance. as in other animal models of demyelinating disease, plaques of demyelination in j . v- infection are characterized by dense macrophage/microglial infiltration [ ] . due to their similar function and surface marker expression, specific contributions of macrophages and microglia to demyelination during mhv-jhm infection are not well understood. chemical depletion of blood borne macrophages prior to infection does not affect disease, suggesting that microglia and/or perivascular macrophages are sufficient for demyelination to occur [ ] . induction of demyelinating disease in j . v- infected mice is usually t cell or antibody mediated, as mice lacking the recombinase activating gene (rag ) or severe combined immunodeficient (scid) mice do not clear virus and ultimately succumb to encephalitis with little or no demyelination [ , , ] . infection of rag -/-or scid mice, which completely lack t or b lymphocytes, provides a basis to determine the role of adaptive immune components in the development of demyelinating disease (table ). j . v- infected rag -/-mice that receive adoptively transferred splenocytes from immunocompetent mhv-jhm immunized mice regain the demyelinating phenotype [ ] . depletion of both cd and cd t cells from the donor splenocyte population abrogates demyelination, while depletion of a single population does not. however, demyelination mediated by each t cell population is characterized by a distinct disease profile. infected rag -/-mice that receive cd t cell-enriched splenocytes develop severe acute table mediators of demyelination in mhv-j . v- infected immunodeficient mice mice mechanism reference cd t cells rag -/-? [ ] , [ ] cd t cells rag -/-cd ifn-c production [ ] , [ ] cd t cells nude, tcrb-/-ifn-c, nkg d [ ] , [ ] anti-mhv ab rag -/-fccri/iii, c activation [ ] vccl (j . .ccl ) rag -/-mac recruitment [ ] encephalitis with moderate amounts of demyelination. mice that receive cd t cellenriched splenocytes exhibit less encephalitis and a prolonged disease course that is characterized by high levels of demyelination. furthermore, cd , but not cd , mediated demyelination and associated macrophage infiltration into the spinal cord white matter is significantly reduced when donor mice lack the ability to produce ifn-c [ , ] . perforin, an essential component of ctl cytolytic activity, is not required. how ifn-c produced by cd t cells contributes to demyelination in mhv-jhm-infected mice is unclear. potent activation of macrophages/microglia by ifn-c likely plays a role in cell recruitment and demyelination. for example, ifn-c treatment of macrophages results in increased production of nitric oxide and increased phagocytosis. since ifn-c is required for clearance of j . v- from oligodendroglia [ ] , it is possible that ifn-c produced by cd t cells as part of the anti-viral response results in increased activation and recruitment of myelindestroying macrophages/microglia. ifn-c also plays a role in autoimmune destruction of myelin, by enhancing cd t cell-mediated eae [ ] . interestingly, ifn-c produced in the cns in mice with eae induces upregulation of ccl (mcp- ) [ ] , a macrophage recruiting chemokine that also promotes infiltration into the cns during mhv-jhm infection [ ] . furthermore, in a cd t-cell mediated model of spontaneous demyelination utilizing transgenic mice that constitutively express the costimulatory ligand cd on microglial cells within the cns, ifn-c receptor deficient mice exhibited no disease [ ] . these studies suggest that ifn-c responsiveness by macrophages/microglia may be critical for cd t cell-mediated demyelination in mhv-j . v- infected mice. although transfer of cd or cd t cells induces demyelination in rag -/-mice, j . infected athymic nude mice, also lacking cd or cd t cells, develop demyelination in the absence of adoptive cell transfer [ ] . however, nude mice lack only conventional ab t cells, but still retain functional cd t cells. depletion of cd t cells from infected nude mice significantly reduces demyelination [ ] . demyelination in infected mice lacking the tcrb gene, which also lack conventional ab t cells, provides further proof that cd t cells can mediate demyelination [ ] . furthermore, antibody depletion of ifn-c in j . infected tcrb-/-mice significantly reduces demyelination, showing that ifn-c is critical for demyelination induced by cd t cells, as it is in cd + t cell-mediated myelin destruction. in addition to the requirement for ifn-c, recognition of nkg d is also important for cd t cell mediated demyelination [ ] . antibody blockade of nkg d, a classic activating receptor of nk cells also expressed on a subset of mouse cd t cells, abrogates demyelination in tcrb deficient mice. therefore, cd t cells are capable of mediating demyelination during viral infection through the action of two critical effector molecules. in contrast, the role of cd t cells in eae is still controversial. depletion of cd t cells in eae results in either milder or more severe disease, depending on whether cells are depleted early or late in the inflammatory process [ ] [ ] [ ] [ ] [ ] . in addition to cell-mediated demyelination, anti-mhv antibody is also capable of induction of disease. mhv-j . v- infected rag -/-mice treated with anti-mhv antibodies develop demyelinating disease with associated white matter infiltration of macrophages/microglia [ ] . although the precise role of humoral immunity in ms is not completely understood, b cells and antibody production are believed to be involved in myelin destruction [ ] [ ] [ ] . oligoclonally expanded b cells and high levels of immunoglobulin are detected in the cerebrospinal fluid of ms patients [ ] , some of which are directed against myelin components, or against common viruses such as epstein-barr [ ] or varicella-zoster [ ] . antibody mediated demyelination during mhv-j . v- infection requires the activating fcc receptors i and iii, since anti-mhv antibody treated infected rag -/-mice deficient in these receptors fail to develop disease [ ] . furthermore, the complement pathway may play a role in antibody-mediated demyelination, as depletion of complement by treatment of mice with cobra venom factor (cvf) results in a significant decrease in demyelination. fccr and complement may function together to activate macrophages with subsequent demyelination. adoptively transferred antibody is detectable in the cns of infected rag -/-mice, supporting the idea of direct interaction with activating fcc receptors on macrophages. in the eae model of demyelination, mice lacking the macrophage activating fccri/iii exhibit attenuated disease, while mice lacking the inhibitory fccrii display increased disease [ ] . furthermore, c derived complement products play a critical role in the pathogenesis of eae [ ] . in addition to liver production of serum components of complement, many resident cns cells are capable of producing complement proteins. through the classical pathway, these proteins may be activated on the surface of antibody bound mhv infected cells or in mhv/antibody immune complexes, thus enhancing the recruitment and effector functions of cns macrophages/microglia. of note, rag -/-mice also deficient in c , the central component of the complement pathway, develop antibody-mediated demyelination (templeton and perlman, unpublished) . contrasting results between c deficiency and cvf depletion of complement are not surprising, since cvf exhibits considerable toxicity [ ] . furthermore, recent evidence indicates that c a may be produced independently of c , providing a possibility that complement products downstream of c may play a role in demyelination in mhv-jhm infected c -/-rag -/-mice [ ] . current studies are focused on examination of the role of complement in cns disease and in induction of immune responses to mhv-jhm infection in both immunocompetent and immunodeficient mice. inflammatory chemokines such as the macrophage chemoattractant protein ccl also play a role in demyelinating disease. expression of ccl during eae follows t cell entry into the cns [ ] , and may be induced by t cell-associated increases in ifn-c [ ] . ccl and the ccl receptor ccr are both important for recruitment of immune cells and in increased pathogenesis in eae [ ] . ccl /ccr also promote monocyte recruitment and viral clearance during mhv-jhm infection [ , ] . introduction of the mouse ccl gene into a recombinant j . (rj . .ccl ) virus results in an infectious virus capable of producing secreted ccl in infected cells in vitro [ ] . rag -/-mice infected with rj . .ccl develop demyelinating disease, whereas rag -/-mice infected with a control virus that lacks a functional ccl protein (rj . .dccl ) do not. therefore, t cells or anti-mhv antibody are not required for demyelination in mhv-jhm-infected mice. rather, it is likely that factors induced by adaptive immune responses result in the recruitment and/or activation of macrophages/microglia into white matter areas of the cns. therefore, these cells are the final effectors in the demyelinating process and any intervention that induces their migration and activation will likely result in demyelination. further studies involve introduction of other chemoattractants into recombinant mhv-jhm, to evaluate their role in demyelinating disease, cell recruitment, generation of immune responses, and clearance of infectious virus in mhv-jhm-infected mice. 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vasculopathy are directed against vzv fc receptors are critical for autoimmune inflammatory damage to the central nervous system in experimental autoimmune encephalomyelitis complement and demyelinating disease: no mac needed? attenuation of experimental autoimmune demyelination in complement-deficient mice generation of c a in the absence of c : a new complement activation pathway central nervous system chemokine mrna accumulation follows initial leukocyte entry at the onset of acute murine experimental autoimmune encephalomyelitis targeting monocyte recruitment in cns autoimmune disease lack of ccr results in increased mortality and impaired leukocyte activation and trafficking following infection of the central nervous system with a neurotropic coronavirus 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 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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- -klv jdm authors: smith, abigail l.; barthold, s. w.; de souza, m. s.; bottomly, kim title: the role of gamma interferon in infection of susceptible mice with murine coronavirus, mhv-jhm date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: klv jdm infection of balb/c mice with mouse hepatitis virus, strain jhm (mhv-jhm), at any of several intervals relative to ovalbumin (ova) administration resulted in elevated ova-specific igg a titers. since gamma interferon (ifn) has been implicated as an up-regulator of igg a production, attempts were made to determine whether levels of this cytokine were modified in sera of infected mice. serum ifn-γ was not detected, but treatment of mhv-jhm-infected mice with monoclonal anti-ifn-γ antibody resulted in high mortality with decreased survival times, enhanced virus titers in liver and spleen, and more severe virus-associated pathology, compared to mock-treated, infected mice. immunotherapy with recombinant ifn-γ ameliorated disease as reflected by mortality rates and virus titers in target organs. mice inoculated with soluble protein antigens preferentially produce igg and igg specific to that antigen [ , ] . in contrast, infection of mice with any of several viruses enhances the igg a response both to the infecting virus and to immunizing soluble protein antigens [ ] [ ] [ ] . gamma interferon (ifn), a cytokine preferentially produced by the th subset of cd + t cells [ ] , stimulates production of igg a by b lymphocytes activated in vitro and in vivo and inhibits igg secretion [ , , ] . thus, it has been postulated that the immune response triggered by viruses mainly activates the th subset of helper t cells [ -] . mouse hepatitis virus (mhv) is a singular name for a group ofcoronaviruses, family coronaviridae, infecting laboratory mice worldwide at high prevalence [ , , ] . mice infected with mhv can exhibit aberrant immune responses that interfere with their usefulness in biomedical research [ , , , [ ] [ ] [ ] . coutelier and co-workers [ ] showed altered protein-specific isotype responses of mice infected with the a strain of mhv one day prior to antigen administration. the initial goal of the currently reported experiments was to determine whether mhv-jhm infection yielded similar results and whether timing of virus exposure was critical to observing the effect, as has been reported for other experimental systems altered by mhv infection [ , ] . mhv-jhm infection of the balb/c mouse was exploited as representative of a combination of moderately virulent, pantropic virus in a commonly used permissive genotype. based on the observation of elevated ovalbumin (ova)-specific igg a levels in sera of mice infected with mhv-jhm at any of several intervals relative to antigen exposure, attempts were made to detect ifn- in sera of infected mice and to reverse the igg a elevation by administration of ifn-y-specific antibody. the effect of this antibody and of immunotherapy with exogenous recombinant ifn- on virus-associated mortality and pathology and on virus replication in target organs was examined. female balb/cbyj mice (the jackson laboratory, bar harbor, me) were five to seven weeks old at the time of virus exposure. young adult balb/c nu/nu (athymic) and nu/+ (euthymic) mice were obtained from life sciences, inc. (st. petersburg, fl). cr:orl sencar dams with litters were obtained from the animal genetics and production branch, nci (bethesda, md). randomly selected mice were free of antibody to common murine viruses on arrival, all mice were housed in micro-isolator cages (lab products, maywood, nj) and were given food and water ad libitum. manipulations and husbandry were performed in a class ii biological safety cabinet, and infected mice were housed in a facility separate from that used for control (uninfected) mice. an open-cage sentinel mouse seromonitoring program was in place during the course of the reported studies. seroconversion to none of common murine viruses or mycoplasma pulmonis was detected, except among mhv-inoculated, immunocompetent mice which seroconverted to the infecting virus. mhv-jhm (american type culture collection, rockville, md) was used in the form of an infected infant mouse brain homogenate. mice immunized with ova or given antibody to ifn- were exposed orally to infant mouse iclds of virus. mice treated with recombinant ifn- were exposed intranasally to the same dose of virus. intranasal exposure of balb/c mice generally results in nearly % mortality, whereas mortality rates after oral inoculation are less than % (unpubl. obs.). groups of mice were infected per os with mhv-jhm on days - , - , - , , + , or + relative to intraperitoneal immunization with ova ( ~tg/mouse) in freund's complete adjuvant (fca). additional groups of uninfected mice received ova plus fca or fca only. mice were killed with co gas and exsanguinated on days or post-ova or fca administration. data from three replicate experiments were pooled. the numbers of mice per group are shown in table . mice were monitored for seroconversion as evidence of infection by indirect immunofluorescent staining using individual sera diluted : and a bivalent antigen consisting of mhv-jhm and mhv-s, as previously described [ ] . results are expressed as geometric mean titers [ ] for sera with significant a values at a dilution of : or greater. selected sera from mice tested for igg a were eliminated from the analysis due to unacceptably high background readings. ten percent tissue homogenates were clarified by centrifugation, and the supernates were screened for infectivity by intracerebral inoculation of litters of two day old sencar mice. virus was quantified by inoculating serial log dilutions of tissue homogenates. mortality during a one week period was recorded, and virus titers were calculated by the method of reed and muench [ ] . monoclonal antibody to mouse gamma ifn, designated xmg . [ ] , was precipitated with saturated ammonium sulfate and extensively dialyzed against pbs, ph . . the final product contained x neutralizing units per ml of antibody when tested with units of recombinant gamma ifn in the wehi b cell lymphoma bioassay [ ] . mice were given x l neutralizing units daily by the intraperitoneal route, beginning one day prior to virus exposure through one day prior to necropsy. recombinant mouse ifn- (lot no. ; specific activity ~> x units/mg; purity > % according to manufacturer) was obtained from amgen biologicals (thousand oaks, ca). mice were given x units intraperitoneally and × units intranasally once daily on days (- ) through (+ ) relative to virus exposure. liver and spleen were fixed in % neutral buffered formalin, paraffin embedded, sectioned at ~tm and stained with hematoxylin and eosin. chi square analysis was used to test for differences in proportions, and student's unpaired t test was used to analyze differences in mean virus titers. sera from mice infected with mhv-jhm one day prior to ova administration contained substantially elevated levels of antigen-specific igg a on day (table ) with geometric mean titers that were times control (ova only) values at that interval. there was a suggestion of a delayed igg a response among mice infected with mhv-jhm three or five days after ova administration, since sera from one of mice tested on day contained ova-specific igg a, whereas four of eleven ova-immunized, uninfected mice had seroconverted at that interval (% = . ; p < . ). sera from higher proportions of mice in groups that received mhv-jhm on days - ( %), - ( %) or ( %) relative to ova contained ova-specific igg a on day than the proportion that were ova-immunized, but uninfected ( %); however, these differences were not statistically significant. mice infected four days prior to ova administration had ova-specific igg titers that were sevenfold lower than those of ova-immunized, uninfected mice (table ) , and the proportion of igg -positive sera ( %) was reduced compared to uninfected control sera ( %). results are shown as number positive at a dilution of : or greater/number tested, and geometric mean enzyme-linked immunosorbent assay titer ± sd for positive sera sera collected from mice at days after ova immunization yielded a more clear-cut pattern. igg a titers were elevated in sera from mice that received virus any time from seven days prior to ova through one day after ova ( table ) . as had been seen among mice bled seven days after administration of ova, the most marked increase in ova-specific igg a ( -fold increase over controls) occurred among mice infected one day prior to immunization. sera from all infected mice except those exposed to virus seven days prior to immunization contained reduced levels of ova-specific igg (table ) . however, the greatest reduction (mice infected three days after ova immunization) was three-fold. mice infected seven days prior to immunization yielded igg titers equivalent to uninfected controls. isotype distribution of ovalbumin-specific antibody was also analyzed by determining igg :igg a ratios for sera from mice bled on day (fig. ) . although the two tests may have inherently different sensitivities, this parameter would be expected to remain relatively constant, since the two assays were always run in parallel, and the same reagents were used throughout the study. this analysis revealed that igg levels in sera from uninfected, ova-immunized mice were -fold higher than igg a levels. in contrast, the ratio was less than one for sera from mice infected one day prior to immunization and measured on day . the elevated igg a ova-specific response of most groups of mhv-infected mice and the preferential production of this isotype by mice infected one day prior to antigen administration suggested that these mice were producing ifny. however, using the wehi b cell lymphoma bioassay [ ] , attempts to detect the cytokine in sera of infected mice failed, as had earlier attempts to ] . it was reasoned that, ififn-y was being produced below the level of assay detection and was responsible for the induction of an elevated igg a response, treatment of ova-immunized, mhv-infected mice with anti-ifn- should restore, at least partially, the igg ova-specific response. in two experiments, % of mice inoculated orally with mhv-jhm and treated with xmg . died with survival times substantially shorter than those of mock-treated, infected mice ( . - - . days vs . ± . days). this suggested that ifn-y was produced during mhv infection and was influencing the course of disease. in an attempt to delineate the role of ifn-y in the pathogenesis of mhv-jhm infection, groups of athymic or euthymic balb/c mice were treated with monoclonal anti-ifn-y (xmg . ) or were mock-treated with dialysis buffer (pbs) and exposed orally to virus. athymic mice were included because they produce ifn-y by virtue of fully competent natural killer cells [- ]. four mice per genotype and treatment group were necropsied daily on post-infection days one through five. liver and spleen sections from all mice were examined histologically, and virus in the same organs of mice necropsied on days four and five post-infection was quantified. on day , mock-treated athymic and euthymic mice and xmg . -treated euthymic mice had rare scattered foci of necrotizing hepatitis, with necrosis of hepatocytes and infiltration of neutrophilic leukocytes. xmg . -treated athymic mice had hepatitis that was similar in quality to that of their mock-treated counterparts, but necrotic foci were more numerous. on day , differences between treatment groups were more striking. xmg . treated athymic mice had numerous large, coalescing foci of hepatocellular necrosis, with only modest leukocytic response (fig. a) . in contrast, mocktreated athymic mice had fewer, smaller foci of hepatitis with less necrosis and infiltration with more leukocytes (fig. b) . xmg . -treated euthymic mice had more foci of hepatitis than either their mock-treated counterparts or mock treated athymic mice. lesions among the latter three groups were qualitatively similar, with infiltration of both neutrophilic and mononuclear leukocytes. microscopic differences between treatment groups were not seen in spleens of mice necropsied on day , but were evident on day . xmg . -treated athymic mice, and to a lesser extent xmg . -treated euthymic mice, had severe necrosis of the periarteriolar regions of the white pulp. necrosis was present in the same regions of spleens from mock-treated mice, but was less severe. virus titers in livers and spleens of xmg . -treated mice necropsied on days and post-inoculation, whether athymic or euthymic, were higher than those in the corresponding tissues of mock-treated mice ( table ) . for athymic mice, these differences were statistically significant for both tissues on both days. among euthymic mice, statistically significant differences in virus titers occurred in the spleen, but not the liver. since treatment of mhv-jhm-infected balb/cbyj mice with antibody to ifn-y resulted in decreased survival time and increased virus titers in two target (table ) . virus titers in liver, spleen and brain of ifn- treated mice at five days post-inoculation were lower than those in the corresponding organs of mock-treated mice (table ) , but only the differences in liver titers were statistically significant. there have been a few reports detailing the responses of mhv-infected mice to nonreplicating antigens or other viruses. mice infected intraperitoneally with mhv- two or four days prior to sheep red blood cell immunization yielded depressed igm and igg plaque-forming cell responses [ ] , whereas mice in-fected at the time of or one day after red cell administration yielded elevated plaque-forming cell responses. this could be correlated with the timing of ifna/ responses relative to antigen administration. more recently, intranasal administration of mhv-a one day prior to tetanus toxoid immunization resulted in elevated antigen-specific igg with a four-fold decrease in the percent of igg that was igg and a like increase in the proportion that was igg a [ ] . our results with mhv-jhm and a different immunizing antigen confirm and extend that finding by showing elevated igg a ova-specific responses over a wide range of infection intervals. timing is crucial, however, to the extent that igg a titers exceeded igg titers only among mice infected one day prior to antigen administration. mhv infection reportedly increases resistance to a variety of secondary viral infections. pre-infection of mice with mhv increased resistance to lethal sendai virus infection in a normally susceptible genotype and interfered with replication of pneumonia virus of mice in respiratory tract tissues [ ] . subclinical mhv infection delayed increased plasma lactic dehydrogenase (ldh) levels resulting from ldh-elevating virus [ ] . natural, inapparent mhv infection also increased resistance to encephalomyocarditis virus and reduced the protective effect of exogenous ifn-a or - [ ] . since mhv infection induces ifn-(z/ production [ , , ] , it was postulated that increased resistance to these secondary infections was due to endogenous ifn-a/ levels and that the effects of exogenous ifn were masked by the endogenous production [ ] . there are now a limited number of reports showing that ifn- ~ modulates the pathogenesis of virus infections. treatment of mice with a different rat antimouse ifn-~f monoclonal antibody or with a sheep-derived antibody against recombinant ifn- resulted in reduced clearance or enhanced replication of lymphocytic choriomeningitis virus (lcmv) in mice [ , ] . neutralization of ifn- in lcmv-infected athymic c bl/ resulted in enhanced virus replication [ as seen in the studies reported here. more recently, a rat antimouse ifn- monoclonal antibody prevented il -induced recovery of athymic mice infected with recombinant vaccinia virus encoding murine il [ ] , suggesting that il -induced ifn- production was responsible for rapid clearance of this construct. virus replication and associated mortality were unaffected among anti-ifn- -treated athymic mice infected with a control virus construct encoding hsv tk and a/pr/ / ha [ . however, administration of recombinant ifn- to mice infected with the control construct significantly increased their survival time, although all treated mice eventually died of disseminated infection [ ] . resistance of intraperitoneally inoculated a/j mice to mhv- also correlates with the ability of this genotype to produce ifn- after infection [ ] . the accumulated data strongly suggest that ifn- , whether induced during the course of infection or administered therapeutically, limits viral replication in vivo. in the case of vaccinia virus encoding il , ifn- apparently mediates recovery of athymic mice. our data with mice exposed by presumed natural routes to mhv-jhm show that endogenous ifn- modulates the pathogenesis of infection in both athymic and euthymic mice. in earlier studies with lcmv [ ] and the current experiments with mhv, ifn- was either present in very low concentration or undetected in sera of infected mice, despite the fact that anti-ifn-y treatment resulted in elevated virus titers. these observations may stem from insensitivity of the assays used to detect ifn- . the vesicular stomatitis virus cytopathic effect reduction assay was used in the lcmv studies, and the wehi cell bioassay was used in the current experiments. the wehi cell bioassay is as sensitive as the ia induction assay for detection of ifn- [ ] , but sensitivity comparisons with other methods have not been reported. use of the sandwich enzyme-linked immunosorbent assay, as reported by karupiah and co-workers [ ] , would likely reveal the presence of ifn- in sera or tissue homogenates from infected mice. based on both histologic evaluation and virus quantification, the effect of administration of anti-ifn- antibody was more marked in athymic than in euthymic mice. this may be due to higher endogenous concentrations of ifn- in euthymic mice by virtue of functional t cells as well as natural killer cells. neither natural killer cells nor endogenous levels of ifn- are sufficient to afford protection, however, since athymic mice do die after natural or experimental infection with mhv [- , ] . mhv-jhm also kills virtually all balb/ c mice exposed by the intranasal route. the lethal effect of infection could be partially overcome by administration of recombinant ifn- , with a concomitant and significant decrease in virus concentration in the liver. others have postulated that virus infections preferentially activate the ifny producing t h subset of cd + t cells [ ] , based on the fact that most infections result in enhanced igg a production. using ldh-elevating virus or the fl strain of mouse adenovirus, attempts were made to inhibit virusassociated igg a production by injection of several different ifn-y-specific monoclonal antibodies [ ] . these efforts failed, although it was not clear whether treatment simply did not affect isotype distribution or whether the mice succumbed to infection. in the current study, neutralization of ifn- in vivo resulted in high mortality with reduced survival times, thereby inhibiting our efforts to show a direct link between virus-induced ifn- production and enhanced igg a responses. among mouse strains susceptible to mhv after exposure by natural routes, lesions and mortality occur at or near the infectious dose level, and the severity of lesions is independent of virus dose [ , ] . in the case of mhv-jhm inoculation of the balb/c mouse by presumed natural routes, the median infectious and lethal doses are essentially identical (unpubl. data). therefore, subsequent studies designed to reveal whether neutralization of ifn- reverses preferential igg a production will require the use of mouse genotypes more resistant to the lethal effects of mhv-jhm infection [ ] or of a less virulent mhv strain. our studies with mhv-jhm in the balb/c mouse have shown, however, that endogenous ifn-y modulates coronavirusassociated mortality, virus replication and tissue damage in a susceptible host. mouse hepatitis virus nasoencephalopathy is dependent upon virus strain and host genotype response of genetically susceptible and resistant mice to intranasal inoculation with mouse hepatitis virus jhm the polyclonal and antigen-specific ige and igg subclass response of mice injected with ovalbumin in alum or complete freund's adjuvant alteration of viral respiratory infections of mice by prior infection with mouse hepatitis virus two types of mouse helper t cell clone. iii. further differences in lymphokine synthesis between th and th clones revealed by rna hybridization, functionally monospecific bioassays, and monoclonal antibodies in vivo polyclonal b-lymphocyte activation elicited by murine viruses virally induced modulation of murine igg antibody subclasses igg a restriction of murine antibodies elicited by viral infections effect of inapparent murine hepatitis virus infections on macrophages and host resistance infection of balb/cbyj mice with the jhm strain of mouse hepatitis virus alters in vitro splenic t cell proliferation and cytokine production delayed increase in plasma lactic dehydrogenase activity in mouse hepatitis virus-infected mice subsequently infected with lactic dehydrogenase virus ifn-gamma regulates the isotypes of ig secreted during in vivo humoral immune responses production of b cell stimulatory factor- and interferon gamma in the central nervous system during viral meningitis and encephalitis persistent mouse hepatitis virus infection in nude mice responses of mice susceptible or resistant to lethal infection with mouse hepatitis virus, strain jhm, after exposure by a natural route interferon gamma is involved in the recovery of athymic nude mice from recombinant vaccinia virus/interleukin infection gamma interferon in murine coronavirus infection cytotoxic t lymphocyte control of acute lymphocytic choriomeningitis virus infection: interferon gamma, but not turnout necrosis factor alpha, displays antiviral activity in vivo diagnosis of murine infections in relation to test methods employed enhanced virus replication and inhibition of lymphocytic choriomeningitis virus disease in anti-gamma interferon-treated mice new a (ed) viral and mycoplasmat infections of laboratory rodents. effects on biomedical research a major role of macrophage activation by interferongamma during mouse hepatitis virus type infection. i. genetically dependent resistance prevalence of natural virus infections in laboratory mice and rats used in canada a simple method of estimating fifty percent endpoints inhibition of b lymphocyte activation by interferon gamma activationofnaturalkillercells and induction of interferon after injection of mouse hepatitis virus type in mice an immunofluorescence test for detection of serum antibody to rodent coronaviruses altered splenic t cell function of balb/ cbyj mice infected with mouse hepatitis virus or sendai virus antigenic characterization of tettnang virus: complications caused by passage of the virus in mice from a colony enzootically infected with mouse hepatitis virus in vitro splenic tcell responses of diverse mouse genotypes after oronasal exposure to mouse hepatitis virus, strain jhm interferon-gamma and b cell stimulatory factor- reciprocally regulate ig isotype production ifn-gamma stimulates igg a secretion by murine b cells stimulated with bacterial lipopolysaccharide persistent infection with mouse hepatitis virus of low virulence in nude mice the role of circulating interferon in the modifications of immune responsiveness by mouse hepatitis virus (mhv- ) statistical methods in serum surveys this research was supported by nih grants rr and rr . the authors thank deborah f. winograd, deborah s. beck, and jane dunn for excellent technical assistance. received january , key: cord- -pl tmky authors: brian, d. a.; baric, r. s. title: coronavirus genome structure and replication date: journal: coronavirus replication and reverse genetics doi: . / - - - _ sha: doc_id: cord_uid: pl tmky in addition to the sars coronavirus (treated separately elsewhere in this volume), the complete genome sequences of six species in the coronavirus genus of the coronavirus family [avian infectious bronchitis virus-beaudette strain (ibv-beaudette), bovine coronavirus-ent strain (bcov-ent), human coronavirus- e strain (hcov- e), murine hepatitis virus-a strain (mhv-a ), porcine transmissible gastroenteritis-purdue strain (tgev-purdue ), and porcine epidemic diarrhea virus-cv strain (pedv-cv )] have now been reported. their lengths range from , nt for hcov- e to , nt for the murine hepatitis virus-a , establishing the coronavirus genome as the largest known among rna viruses. the basic organization of the coronavirus genome is shared with other members of the nidovirus order (the torovirus genus, also in the family coronaviridae, and members of the family arteriviridae) in that the nonstructural proteins involved in proteolytic processing, genome replication, and subgenomic mrna synthesis (transcription) (an estimated – end products for coronaviruses) are encoded within the ′-proximal two-thirds of the genome on gene and the (mostly) structural proteins are encoded within the ′-proximal one-third of the genome ( – genes for coronaviruses). genes for the major structural proteins in all coronaviruses occur in the ′ to ′ order as s, e, m, and n. the precise strategy used by coronaviruses for genome replication is not yet known, but many features have been established. this chapter focuses on some of the known features and presents some current questions regarding genome replication strategy, the cis-acting elements necessary for genome replication [as inferred from defective interfering (di) rna molecules], the minimum sequence requirements for autonomous replication of an rna replicon, and the importance of gene order in genome replication. introduction despite its unique property as the largest of the known plus-strand rna genomes, the coronavirus genome shares with those of other plus-strand rna viruses (excepting retroviruses) the properties of ( ) infectiousness [and not using a packaged rna-dependent rna polymerase (rdrp)] (brian et al. ; schochetman et al. ) and ( ) replication in the cytoplasm in close association with cellular membranes dennis and brian ; gosert et al. ; sethna and brian ; shi et al. ; van der meer et al. ). many of the basic features of coronavirus genome structure and replication have been described in recent reviews enjuanes et al. a enjuanes et al. , b lai and cavanagh ; lai and holmes ; luytjes ; van der most and spaan ) . with the advent of reverse genetics enabling site-directed mutagenesis of any part of the genome (almazan et al. ; casais et al. ; masters ; yount et al. yount et al. , , many of the mechanistic features of coronavirus genome replication that could previously be learned only from direct manipulation of defective interfering (di) rna can now be examined in the context of the whole virus genome. in this chapter, we review the current knowledge of coronavirus genome structure and organization and the cis-acting elements in coronavirus replication and raise selected questions that we believe are important for approaching a better understanding of coronavirus genome replication. in addition to the sars coronavirus (treated separately elsewhere in this volume), the genomes of six species of coronaviruses have now been fully sequenced and reported in genbank (as of november ): ibv-beaudette (nc , boursnell et al. ) , bcov-ent (nc , chouljenko et al. ) , mhv-a (nc , leparc-goffart et al. ) , hcov- e (nc , , tgev-purdue (nc , almazan et al. ; eleouet et al. ; penzes et al. ), and pedv-cv (nc , kocherhans et al. . these, representing all three coronavirus serogroups (siddell ) , are schematically depicted in fig. . additional strains of bcov [bcov-lun (af , chouljenko et al. ) ], bcov-mebus (u , nixon and brian, unpublished data) and bcov-quebec (af , yoo and pei ) , and mhv [mhv- (af , sarma et al. ) ] have also been reported. the genome sizes range from , nt for hcov- e to , nt for mhv-a , establishing them as the largest known among rna viruses (enjuanes et al. a; lai and cavanagh ) . the following similarities in genome structure among the six can be noted: . the utrs ranging in length from to nt contain a similarly positioned short, aug-initiated open reading frame (orf) relative to the end [ table ; a situation that, by current terminology, is problematic because the "untranslated region" now becomes in part potentially translatable and thus should preferably be called a "leader" (morris and geballe ) . the term "leader," however, has an established meaning in the nidovirus lexicon ; see subsequent chapters, this volume) of a -terminal, genome-encoded sequence of - nt appearing on the terminus of each subgenomic mrna species]. for purposes of this review, " utr" will refer to the sequence upstream of orf (gene ) despite the internally positioned short orf. the short aug-initiated orfs (except for hcov- e) begin in a suboptimal kozak context for translation (table ) (kozak ) and potentially encode peptides of - amino acids. . the utrs range from to nt [although some strains of ibv have utrs of greater length because of internal sequence duplications (williams et al. ) ], all possess an octameric sequence of ggaagagc beginning at base to upstream from the poly(a) tail, and all possess a -terminal poly(a) tail (table ) . the optimal kozak context for translation initiation is gccaugg (kozak the second amino acid in the bcov-mebus strain is l. . all have an extremely large gene (separated into orfs a and b and extending over approximately two-thirds of the genome) encoding nonstructural proteins involved in proteolytic processing of the gene polyprotein products, virus genome replication, and sgmrna synthesis (transcription). in each, gene is translated as orfs a and ab, with ab resulting from a pseudoknot-induced ribosomal frame shifting event at a slippery sequence of uuuaaac at the orf a/ b junction ( fig. ) (brown and brierley ) . . all encode the structural spike (s) glycoprotein, small envelope (e) protein, membrane (m) glycoprotein, and nucleocapsid (n) protein, in that order, ! within the -proximal one-third of the genome. a variable number of other orfs appearing to be virus-or group-specific, many apparently encoding nonstructural proteins, are also found here. these (and their potential products) include orf a ( , orf a ( . -kda protein), orf b ( . -kda protein), orf ( . -kda protein), and an orf internal to gene ( -kda i protein) in bcov; and orf a ( . -kda protein), orf b ( . -kda protein), orf a ( . -kda protein), and orf b ( . -kda protein) in ibv ( fig. ; brown and brierly , and references listed in the genbank information noted above). some of these, such as orfs a and b in tgev (mcgoldrick ; wesley et al. ) and orfs a fig. . pseudoknotted structures and slippery sequences responsible for highly efficient ( %- %) - ribosomal frameshifting at the orf a and b junction in gene of the six coronaviruses shown in fig. . the slippery sequence uuuaaac, identified in bold, is the same in all sequenced genomes. the ibv pseudoknot-induced frameshifting was the first nonretroviral example of ribosomal frameshifting in higher eukaryotes (brierley et al. (brierley et al. , . the pseudoknots in mhv (bredenbeek et al. ) and bcov (yoo and pei ) are nearly identical and are similar to the structure in ibv. in hcov- e an elaborated pseudoknot with three stems was shown by mutation analysis to be the functional frameshifting structure (harold and siddell ) . in tgev (eleouet et al. ) and in pedv (kocherhans et al. ) an elaborated pseudoknot was also predicted based on similarities to hcov- e t (schwarz et al. ), b (he) (luytjes et al. ), (weiss et al. yokomori and lai ) , a (yokomori and lai ), and i (fischer et al. ) in mhv, have been shown to be nonessential for replication in cell culture, and their function in virus replication remains undetermined (de haan et al. ) . presumably all coronavirus genomes are capped with a methylated nucleotide, but so far this has been demonstrated only in mhv (lai et al. ) . cis-acting rna elements in coronavirus genome replication as with all nonretroviral plus-strand rna viruses, a necessary early step in genome replication is translation of the genome for production of the rdrp and other proteins required for viral genome replication. the presence of a terminal methylated cap on mhv genomic and subgenomic mrnas (lai et al. ) would suggest that coronaviruses use a cap-mediated ribosomal entry mechanism for translation. mutation analyses of the utr of bcov indicate that a scanning mechanism is used for entry of ribosomes onto orf (senanayake and brian ) . curiously in light of these results, a methylated cap on di rna transcripts is not required for initiation of replication of bcov di rna, which contains a genomic utr. this molecule has a cis-acting dependence on translation for replication . it remains to be determined whether capping is required for translation and replication of the intact viral genome. it remains to be determined what enzyme functions to cap the viral rnas (ziebuhr et al. ) . in mhv it has been demonstrated that the viral nucleocapsid protein n binds tightly (k d = nm) to the ucuaaac intergenic region (also named transcription-regulating sequence, trs) of the genomic leader and consequently may influence translation rate (nelson et al. ; tahara et al. ) . is this property of n common to all coronaviruses? if so, what role does it play in the regulation of genome replication? does the intra- utr short orf play a role in translation (or in subsequent replication) of the genome? with reverse genetics, disruption of an analogous orf in equine arterivirus had no apparent effect on virus replication in cell culture (molenkamp et al. ) , but the orfs may not have homologous function in the two virus groups. certainly, short upstream orfs can have profound enhancing or suppressing effects on the translation of a downstream orf (morris and geballe ) , and their universal existence in coronavirus utrs, albeit with little or no conservation in size or amino acid sequence (table ) , would suggest that they function in the regulation of replication or gene expression. one possibility is that the intra- utr short orf or some other utr element, such as the binding site for n described above, is responsible for the repression of translation from the orf start codon in virus-infected cells (senanayake and brian ) . some observed phenomena in coronavirus genome and di rna replication hint that the utr might be bypassed altogether in order to meet the translation requirements for genome replication. one set of observations relates to a possible role for n in genome replication compton et al. ; kim k and makino ; laude and masters ; nelson et al. ; stohlman et al. ), a role that would set coronaviruses apart from arteriviruses in this regard because only gene products have been shown to be sufficient for arterivirus genome replication (molenkamp et al. ) . n protein, for example, binds leader sequence with high affinity (nelson et al. ) , is present in a subpopulation of coronavirus rna replication complexes (sethna and brian ; sims et al. ) , and is essential for infectivity of recombinant ibv full-length transcripts . if n is required, then some mechanism for the translation of n from the polycistronic genome, such as an internal entry of ribosomes onto genomic rna or formation of an early subgenomic mrna transcript, would be needed, at least when infection is initiated by the genome alone (as in transfection experiments). some evidence for internal ribosomal entry has been demonstrated for ibv mrna (liu and inglis ), mhv mrna (thiel and siddell ; jengrach et al. ) , and tgev mrna (oconnor and brian ), making it prudent to consider an internal entry at these or other sites on the genome for protein synthesis. another set of observations relates to a requirement for translation in cis of the di rna molecule to be replicated. although some di rnas with a single orf do not appear to require translation in cis for replication (liao and lai ) , others do de groot et al. ; van der most et al. ) . might a cis-acting requirement for di rna translation reflect a similar cis-translation-dependent mechanism for genome replication as described for picornaviruses (egger et al. ; gamarnik and andino ; novak and kirkegaard ) and flaviviruses (khromykh et al. ) ? if so, then perhaps an internal ribosomal entry for translation onto the proximal region of the genome might be needed for coronavirus genome replication. the pseudoknot and slippery sequence involved in the ribosomal frameshifting at the orf a/ b junction ribosomal frameshifting in coronaviruses was the first described nonretroviral example of ribosomal frameshifting in higher eukaryotes (brierly ) , and the earliest described higher-order rna structure recognized as a cis-acting element in coronavirus genome replication was the pseudoknot located immediately downstream of the uuuaaac slippery sequence in the ibv genome (brierly et al. (brierly et al. , brown and brierly ) (fig. ) . the pseudoknot in ibv was described as a hairpin-type and was shown by mutation analyses to be responsible for the highly efficient ( %- %) frameshifting. subsequently, a pseudoknot with similar properties was found in gene of mhv (bredenbeek et al. ) and bcov (yoo and pei ) . interestingly, the pseudoknot found in gene of hcov- e was found to be quite different in structure, possessing an extremely large loop and a stem (fig. ) . this structure was termed an "elaborated" pseudoknot and was shown to function as such in in vitro measurements of frameshifting . the predicted pseudoknots in tgev and pedv gene appear to be quite similar to that in hcov- e (eleouet et al. ; kocherhans et al. ). the pseudoknot-associated slippery sequence is uuuaaac in all sequenced coronaviruses described to date. once made, or possibly concurrent with synthesis, viral proteins and (possibly) associated cellular proteins function to form the membraneassociated rna replication complexes. membrane association is a hallmark of replication complexes of plus-strand rna viruses, but the origin of the membrane and the anatomy of the replication complexes appear to differ among virus families. a preliminary understanding of the coronavirus replication complex has come primarily from studies with mhv and partly from studies with tgev. the following features have been observed: . the membrane in the mhv replication complex has shown markers for the endoplasmic reticulum and golgi (shi et al. ; gosert at al. ) and, alternatively, the late endosomes sims et al. ) . . the replication complex is intimately associated with double membrane structures, and the anchored proteins are the hydrophobic sequencecontaining intermediate cleavage products p and p , and p and p , of orf a (gossert et al. ) . . there appear to be two populations of membrane-associated replication complexes separable by isopycnic sedimentation (sethna and brian ; sims et al. ) . in mhv the less dense fraction ( . - . g/ml) was found to contain p and p a- , products of orf a, whereas the denser fraction ( . - . g/ml) contained p and helicase from orf b, and n (sims et al. ) . in tgev two buoyant density populations ( . - . g/ml and . - . g/ml) were also found, and both had associated with them genomeand subgenome-length plus-and minus-strand rnas (sethna and brian ) . some s, m, and n proteins were associated with the denser population. the tgev membrane replication complexes, furthermore, appeared to have an unusual impermeability to micrococcal nuclease. it remains to be determined precisely what proteins, viral and cellular, function together to make up the coronavirus replication complexes and how they might be associated with the membranes and with one another. how might they differ between the processes of minus-and plusstrand synthesis? between replication and transcription? which proteins bind the rna, both genomic and subgenomic, both plus and minus strands, within the complex? what is the stoichiometry of the components in the various complexes? what is the relationship between the rna replication complex and the site of virus assembly at the golgi and intermediate golgi membranes? how is the genome selected and transported from the replication complex to the site of virus assembly? does the evidence of resistance of coronaviral rnas to ribonuclease suggest existence of a compartmentalized replication complex and have implications for resistance to rna silencing (ahlquist ) and long-term persistent coronaviral infections (adami et al. ; baric et al. ; okumura et al. ; stohlman et al. )? . and -proximal rna cis-acting elements for di rna (and presumably genome) replication since the first description of their cloning and replication in helper virus-infected cells, coronavirus di rnas have been used in attempts to define the minimal cis-acting sequence requirements for their replication makino et al. makino et al. , a makino et al. , b van der most et al. ) . through deletion analyses the regions harboring minimal cis-acting sequences have been mapped for di rnas from tgev, mhv, bcov, and ibv (noted as filled regions in the di rna maps in fig. ). for most of the di rnas it can be seen that these sequences reside at the termini of the viral genomes for distances of - , at the end and - , at the end. further reduction in the sizes of these regions may result from further deletion analyses. requirements for internal genome sequence elements appear to be di rna specific but may reflect requirements of the intact genome (see below). what is the nature of the terminal cis-acting rna elements? is a specific sequence alone sufficient, or are higher-order structures required? so far, these questions have focused primarily on the small ( . - . kb) di rnas of the group coronaviruses mhv and bcov. with regard to the utr of mhv-a and bcov-mebus, common replication signals exist between the two viruses. this was demonstrated by experiments in which the entire utr of the mhv genome was replaced with the equivalent region of the bcov genome without loss of virus viability (hsue and masters ) and in a bcov di rna chimera in which the bcov utr was replaced with the mhv utr with no detectable loss of replicating ability (ku, williams, and brian, unpublished data). more recently, bcov di rna has been shown to replicate in the presence of mhv as helper virus (wu et al. ) . to date, three higher-order cis-acting elements mapping within the utr have been characterized in mhv and bcov (fig ) : izeta et al. ; deletion analyses were done on derivatives of tgev di rna c ( . kb) (mendez et al. ) ; m contains minimal sequence elements for replication and inefficient packaging; m and m contain t small nonoverlapping regions of orfs a and b that contribute to packaging; (b) luytjes et al. ; van der most et al. van der most et al. , ; deletion analyses were done on derivatives of mhv-a dia rna ( . kb); (c) lin and lai ; makino et al. ; deletion analyses were done on dissf; (d) fosmire et al. ; ; deletion analyses were done on disse; (e) masters et al. ; di b is synthetic and was designed after the bcov-mebus di rna; (f) chang et al. ; deletion analyses were done on reporter-containing di drep ; (g) dalton et al. ; deletion analyses were done on derivatives of . -kb ibv di rna cd- (penzes et al. ) ; unknown regions within the utrs suffice for packaging of di rna, but packaging is inefficient . a -nt bulged stem-loop beginning immediately downstream of the n stop codon consists in mhv of four stems (b, c, d, and f) and a -nt terminal loop (hsue and masters ; hsue et al. ) . stems c, d, and f have been shown to be required for replication of both the di rna and virus genome. . a -nt hairpin-type pseudoknot beginning nt downstream of the bulged stem-loop (williams et al. ) . both stems of the pseudoknot have been shown to be required for replication. the pseudoknot sequence overlaps the downstream arm of stem f in the bulged stem-loop , a hairpin-type pseudoknot (williams et al. ), a helix formed at the base of a long stem-loop and adjacent to the phylogenetically conserved octameric sequence (liu et al. ). the poly(a) tail is required for replication (lin and lai ; , spagnolo and hogue ) , and the -terminal nt are the minimal sequence requirements for minus-strand rna synthesis in mhv (lin et al. ). the higher-order structures are a stem-loop iii and stemloop iv within the utr (raman et al. ) and stem v within the partial orf a sequence (brown et al. ) . b experimental evidence for replication (accumulation) of reporter-containing di rna but not mrna containing the same reporter after transfection into helper virus-infected cells ). the only difference between the two molecules is a sequence of nt mapping between nt and in the bcov di rna such that the two structures cannot exist simultaneously. this led hsue et al. ( ) to suggest a possible interaction between the two elements, with the alternative conformations acting as a possible "switching" mechanism. this switch has now been confirmed experimentally (goebel et al. ) .the pseudoknot appears phylogenetically conserved to some degree in all coronaviruses. . a -nt bulged stem-loop mapping from nt to from the terminus in mhv contains two stems that demonstrated importance as cisacting replication structures (referred to as stems a and b in fig. ) (liu et al. ) . stem b, which shows greater importance in di rna replication, is phylogenetically conserved in structure between mhv and bcov. stem b is immediately adjacent downstream to the phylogenetically conserved utr octamer ggaagagc (liu et al. ) . unidentified cellular proteins of , , , and kda molecular mass bind to nt - which is the upstream half of the internal loop in stem b (liu et al. ; yu and liebowitz ) . proteins identified to date that bind within the region (or the minus-strand counterpart of this region) include the poly(a) binding protein (spagnolo and hogue ) , mitochondrial aconitase, which binds within the -nt -terminal region in mhv (nanda and leibowitz ) , and the polypyrimidine tract-binding protein, which binds to minus-strand sequence complementary to nt - (strongly) and - (weakly) in mhv . what roles the utr higher-order structures play in rna replication are not known. because the -terminal nt were shown to be a minimal sequence requirement for minus-strand synthesis in mhv (lin et al. ) , the higher-order structures mapping upstream of the -nt sequence possibly play no role in minus-strand synthesis. do they play a role in initiating or regulating plus-strand synthesis? precedents in picornaviruses (barton et al. ; herold and andino ) , alphaviruses (frolov et al. ) ,and flaviruses (you et al. ) would suggest they might. certainly the poly(a) tail through the poly(a)-binding protein is a candidate for such a process, perhaps through genome circularization (spagnolo and hogue ) . with regard to the utr it is known that the -terminal sequence is required for di rna replication ) and at least two stem-loops (stem-loops iii and iv in fig. ) function as higher-order cis-acting signaling elements (raman et al. ; raman and brian, unpublished data) . a higher-order cis-acting structure mapping within the first nt of orf (stem-loop v in fig. ) has also been found (brown, nixon, senanayake, and brian, unpublished data) . proteins shown to bind within the utr include the viral n protein, which binds in and around the leader-adjacent intergenic sequence motif ucuaaac (nelson et al. ) , the polypyrimidine tract binding protein, which also binds near the leader-adjacent ucuaaac sequence motif (li et al. ) , and hnrnp a , which binds the minus-strand complement of the leader-adjacent ucuaaac sequence motif (li et al. ) . none of these has been reported to bind regions covered by stemloops iii, iv, or v depicted in fig. . might there be a process of leader priming of genome replication (zhang and lai ) , as suggested by the phenomenon of high-frequency leader switching on di rnas during di rna replication makino and lai ; stirrups et al. ) ? the question of what cis-acting sequences act in coronavirus rna replication has relevance not only for genome replication but also for poorly understood features of sgmrna behavior. it has been suggested that coronavirus sgmrnas amplify by a replication mechanism hofmann et al. ; sethna et al. ). this hypothesis made use of the argument that the termini on the sgmrnas and genome, identical at the end for the length of the leader ( - nt, depending on the virus species) and at the end for greater than the length of the utr (i.e., greater than nt), are larger than the known promoters for a viral rdrp [replication promoters in influenza and sindbis viruses are less than nt in length (levis et al. ; li and palese ) ] and are therefore large enough to harbor promoters for replication. the hypothesis was also consistent with the observations that ( ) the molar ratios of minus-strand to plus-strand rna are equivalent for sgmrna and genome (i.e., : ), ( ) the rate of sgmrna accumulation is inversely proportional to the length of the molecule, ( ) the rate of sgmrna minus strand disappearance parallels that of antigenome, and ( ) sgmrna minus strands possess -terminal sequences complementary to the leader (sethna et al. ). furthermore, ( ) double-stranded subgenomic mrna-length rfs and ris hofmann et al. ; sawicki and sawicki ; sethna et al. ) were shown to be active in subgenomic mrna synthesis (baric and yont ; sawicki , ; sawicki et al. ; schaad and baric ) . if the -terminal nt are the only requirement for minus-strand rna synthesis (lin et al. ) , the possibility is left open that the subgenomic mrnas function as a templates for minusstrand synthesis. at no time, however, has it been directly demonstrated that sgmrna transcripts, with or without a reporter, are replicated in the presence of a helper virus after transfection into helper virus-infected cells (fig. b) makino et al. ) . therefore, what features enable the replication of the di rnas but not sgmrnas on transfection into helper virus-infected cells? the answer could lie in the function of the -proximal stem-loops iii, iv, and v residing within the -nt region found in bcov di rna but not found in sgmrnas (fig. a) . do these higher-order structures bind viral or cellular proteins? might they be signals working through long-distance rna-rna or rna-protein interactions? . internal cis-acting signals for di rna (and possibly also for genome) replication most di rnas described for coronaviruses are comprised of more than just the terminal genomic sequences. that is, they are mosaics of internal and terminal genome sequences. replication of mhv-jhm di rnas has been found to be dependent on a -nt sequence mapping within orf a lin and lai ) . this sequence has been shown to form a secondary structure in the positive strand, and both the higher-order structure and its sequence are important for function as a replication signal (repass and makino ) . does this structure represent a cis-acting replication signal required for replication of the intact genome? perpetuation of coronavirus infection via cell-to-cell spread requires that the genome be packaged into virions via one or more cis-acting packaging signals. inasmuch as several small di rnas containing only terminal sequences are packaged, some form of signal sufficient for incorporation into virions must reside in the termini. this idea is consistent with the observed packaging of subgenomic mrnas in tgev (sethna et al. ) , bcov (hofmann et al. ) , and ibv (zhao et al. ) . however, these packaging signals may not be the ones used by the virus genome for packaging. a candidate -nt genome packaging signal has been identified in mosaic di rnas of mhv (fosmire et al. ; makino et al. ; van der most et al. ) that maps to a region within orf b, shows correlation of function with maintenance of secondary struc-ture (fosmire et al. ) , and confers packaging on reporter rna molecules (bos et al. ; woo et al. ) . a homologous structure in bcov orf b also leads to packaging of nonviral rnas (cologna and hogue ) . do these represent the bona fide packaging signals for the viral genome? is there perhaps more than one packaging signal, as suggested by the ability of more than a single region of orf b to contribute to packaging efficiency in large tgev di rnas (izeta et al. ) ? perhaps not since a recent study shows only a single packaging signal encoded within the -terminal nts of the tgeu genome is sufficient (escors et al. ) . in addition to the n protein (laude and masters ) , might the packaging signals interact with other components of the virion? perhaps so since in mhv the envelop (e) protein (narayanan and makino ) and m protein (narayanan et al. ) have been shown to play roles in packaging. although gene products are the only ones required for arterivirus genome replication and sgmrna synthesis (molenkamp et al. ) , the story might be different for coronaviruses. gene of hcov- e in the presence of the genomic and utrs was shown to be sufficient for sgmrna synthesis when the intergenic sequence for mrna (n mrna) and an mrna body (gene for the green fluorescence protein) were present just downstream of gene . the authors, however, were unable to conclude that these sequences alone were sufficient for rna replication or to rule out a role for n as an enhancer for transcription. these results, therefore, leave open the possibility that another gene function is important for replication. autonomous replicons of tgev containing only genes , , part of , and all of and have been described (curtis et al. ) . reverse genetics with these and other coronaviruses now make feasible the analysis of the minimal sequences required for genome replication and should lead to a definitive resolution of the question of the role of n protein in rna replication. the gene order for coronaviruses, as for many positive-and negativestranded rna virus families, is highly conserved. in coronaviruses the essential genes pol, s, e, m, and n are invariably found in that order, to , although they are sometimes interspersed with genes showing no essential function for virus growth in cell culture (discussed above). what is the significance of this gene order? if it is altered, what might the consequences be on virus growth? might pathogenesis be altered such that the variants could be used as vaccines or vectors for other uses? the presence of nonessential genes a and b in tgev for cell culture growth has enabled development of tgev as a heterologous expression vector (see the chapter by enjuanes et al., this volume) and as a virus to study the effects of gene rearrangements. in initial studies on the effect of gene rearrangement, the n gene has been duplicated (producing the genotype snemn) and repositioned (producing the genotype snem) by making use of gene positions a and b (k. curtis and r. baric, unpublished data) . the n gene was chosen for repositioning because it encodes the most abundantly expressed sgmrna and is translated into the most abundant of the viral proteins. on the basis of general gene expression patterns relative to the end of the genome in coronaviruses it was anticipated that expression of e and m would increase relative to n in the rearranged snem construct. when tested by transfection, the tgev mutants snemn and snem were found to be viable but to replicate at about -fold and , -fold less than wild-type virus levels, respectively. these results indicated that a specified gene order per se is not essential for coronavirus replication in cell culture, but that order contributes in some way to a more robust virus yield. when tgev snem was serially passaged times, the mutant gene order snem was maintained, but, surprisingly, virus growth was restored to near wild-type levels. restoration of tgev snem fitness as defined by virus yield was associated with changes within the n-(partial) d b-e junction region. these included removal of most of the residual (partial) dorf b sequence, deletion of the wt e intergenic sequence element, and activation of a new, highly transcriptionally active e intergenic sequence element just downstream of the newly inserted n gene (fig. b) . these results indicate that high-frequency rna recombination does not function to restore a specific coronavirus gene order, at least over the short term, because the new n gene position in snem was stable for many passages. rather, the fig. a , b. effects of moving the n gene within the tgev genome from its normal position to an upstream site. the n gene including its immediate transcription stimulating element (tse)-containing upstream sequence of nt was placed just downstream of the a tse sequence in a tgev genome from which the entire a and a portion of the b gene had been deleted (a). transcripts of the recombinant tgev genome, designated snem, were transfected into cells, and progeny viruses were studied (b). immediately after transfection (passage ) the titer of progeny was low (< pfu/ml) and the genome sequence was identical to the original construct. the progeny (snem- and snem- ) grew more efficiently (~ . pfu/ml) after passages and reached wild-type levels (~ . ) after passages. in all progeny the upstream a tse sequence was used for leader fusion of the n transcript. for expression of the e gene, however, the story was different. at passage (snem- ), transcripts of the e gene used the wt tse as well as two additional sites, designated a and b within the orf b residual sequence, for leader fusion. in the snem- and snem- viruses the wt e tse was deleted and transcripts of the e gene used the two new tses formed within the residual gene b sequence (a= / clones, b= / clones) in snem- . in snem- only the a site was used for e gene expression. thus the reordered tgev genome was stable with regard to the new (upstream) position of n coronavirus genome can rapidly develop compensatory changes to restore virus replication rate (fitness) while maintaining a new gene order. mechanisms of fitness restoration appeared to include recombination events and point mutations (baric et al., unpublished data) . it is likely that gene order mutants will provide novel insights into the regulation of coronanvirus transcription and replication, identify protein-protein interactions that function cooperatively to maintain robust virus fitness and growth, and assist in the identification of core sequence elements that function in sgmrna synthesis. it is anticipated that reverse genetics, which now enables an alteration of any part of the coronavirus genome, will facilitate examination of the cis-and trans-acting elements in rna replication and transcription within the context of the intact genome. these elements have until now been studied primarily in di rnas. in light of precedents established with many much smaller plus-strand rna viruses of animals and plants, it would not be surprising to find novel long-distance rna-rna and protein-rna interactions involving genome sequences not present in di rnas. long-distance interactions are hinted at in comparative studies of di rnas (which replicate) and sgmrnas (which do not replicate). what genes are important in regulation of replication and transcription, and how important is gene order in these processes? these questions can now be rigorously approached with reverse genetics. it is also anticipated that a greater understanding of the assembly, stoichiometry, and function of the rna synthesizing complexes will be gained through similar rigorous analyses. it is anticipated that one practical outcome of reverse genetics will be the development of safe coronavirus-based replicon vectors, not necessarily only those that become packaged, for vaccine and other biomedical uses. still in waiting is the development of an in vitro virus replication system such as that used for poliovirus (molla for over passages, but in snem- and snem- additional mutations were selected upstream of the atse and in the m gene that greatly enhanced virus fitness and n gene expression. in snem the sequences of the tses are aactaaact for a, and acaaaac for e, taactaaact for n, aactaaag for a, and aacacaaaac for b t et al. ), in which complete virus replication can be accomplished in cell lysates. this approach would enable still more detailed analyses of the requirements for genome replication beginning with the infectious genome transcript. all in all, it is likely that the next decade will bring significant breakthroughs regarding our understanding of the mechanisms involved in coronavirus genome replication and transcription, the function of the replication complexes, and the development and application of coronavirus recombinant vectors for the treatment of animal and human 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characterization of the rna components of a putative molecular switch in the untranstated region of the murine coronavirus rna replication of mouse hepatitis virus takes place at double-membrane vesicles poliovirus rna replication requires genome circularization through a protein-protein bridge nucleotide sequence of the human coronavirus e rna polymerase locus an "elaborated" pseudoknot is required for high frequency frameshifting during translation of hcv e polymerase mrna the end of coronavirus minus-strand rnas contains a short poly(u) tract bovine coronavirus mrna replication continues throughout persistent infection in cell culture characterization of an essential rna secondary structure in the untranslated region of murine coronavirus genome a bulged stem-loop structure in the untranslated region of the coronavirus mouse hepatitis virus genome is essential for replication polypyrimidine tract-binding protein binds to the complementary strand of the mouse hepatitis virus untranslated region, thereby altering rna conformation l ( ) replication and packaging of transmissible gastroenteritis coronavirusderived synthetic minigenomes characterization of an internal ribosome entry site within mrna of murine hepatitis virus trans-complementation analysis of the flavivirus kunjin ns gene reveals an essential role for translation of its n-terminal half in rna replication two murine coronavirus genes suffice for viral rna synthesis characterization of a murine coronavirus defective interfering rna internal cis-acting replication signal analysis of cis-acting sequences essential for coronavirus defective interfering rna replication generation and selection of coronavirus defective interfering rna with large open reading frames by rna recombination and possible editing completion of the porcine epidemic diarrhoea coronavirus (pedv) genome sequence structural features in eukaryotic mrnas that modulate the initiation of translation the molecular biology of coronaviruses coronaviridae: the viruses and their replication further characterization of mrnas of mouse hepatitis virus: presence of common -end nucleotides the coronavirus nucleocapsid protein altered pathogenesis of a mutant of the murine coronavirus mhv-a is associated with a q l amino acid substitution in the spike protein deletion mapping of sindbis virus di rnas derived from cdnas defines the sequences essential for replication and packaging polypyrimidine tract-binding protein binds to the leader rna of mouse hepatitis virus and serves as a regulator of viral transcription heterogeneous nuclear ribonucleoprotein a binds to the transcription-regulatory region of mouse hepatitis virus rna mutational analysis of the promoter required for influenza virus virion rna synthesis a cis-acting viral protein is not required for the replication of a coronavirus defective interfering rna deletion mapping of a mouse hepatitis virus defective interfering rna reveals the requirement of an internal and discontiguous sequence for replication identification of the cis-acting signal for minusstrand rna synthesis of a murine coronavirus: implications for the role of minus-strand rna in rna replication and transcription internal entry of ribosomes on a tricistronic mrna encoded by infectious bronchitis virus secondary structural elements within the untranslated region of mouse hepatitis virus strain jhm genomic rna a specific host cellular protein binding element near the end of mouse hepatitis genomic rna coronavirus gene expression replication of synthetic defective interfering rnas derived from coronavirus mouse hepatitis virus-a structure of the intracellular defective viral rnas of defective interfering particles of mouse hepatitis virus a system for study of coronavirus mrna synthesis: a regulated, expressed subgenomic defective interfering rna results from intergenic site insertion high-frequency leader sequence switching during coronavirus defective interfering rna replication defective interfering particles of murine coronavirus: mechanism of synthesis of defective viral rnas primary structure and translation of a defective interfering rna of murine coronavirus analysis of efficiently packaged defective interfering rnas of murine coronavirus: localization of a possible rna-packaging signal reverse genetics of the largest rna viruses optimization of targeted rna recombination and mapping of a novel nucleocapsid gene mutation in the coronavirus mouse hepatitis virus molecular characterization of transmissible gastroenteritis coronavirus defective interfering genomes: packaging and heterogeneity the arterivirus replicase is the only viral protein required for genome replication and subgenomic mrna transcription cell-free, de novo synthesis of poliovirus upstream open reading frames as regulators of mrna translation mitochondrial aconitase binds to the untranslated region of the mouse hepatitis virus genome nucleocapsid-independent specific viral rna packaging via viral envelope protein and viral rna signal cooperation of an rna packaging signal and a viral envelope protein in coronavirus rna packaging high affinity interaction between nucleocapsid protein and leader/intergenic sequence of mouse hepatitis virus rna improved method for detecting poliovirus negative strands used to demonstrate specificity of positive-strand encapsidation and the ratio of positive to negative strands in infected cells maintenance of pluripotency in mouse embryonic stem cells persistently infected with murine coronavirus complete genome sequence of transmissible gastroenteritis coronavirus pur -mad clone and evolution of the purdue virus cluster characterization of a replicating and packaged defective rna of avian coronavirus infectious bronchitis virus replication and packaging of coronavirus infectious bronchitis virus defective rnas lacking a long open reading frame importance of the positive-strand rna secondary structure of a murine coronavirus defective interfering rna internal replication signal in positive-strand rna synthesis direct submission to gen-bank coronavirus transcription: subgenomic mouse hepatitis virus replicative intermediates function in mrna synthesis coronaviruses use discontinuous extension for synthesis of subgenome-length negative strands a new model for coronavirus transcription the rna structures engaged in replication and transcription of the a strain of mouse hepatitis virus genetics of mouse hepatitis virus transcription: evidence that subgenomic negative strands are functional templates presence of infectious polyadenylated rna in the coronavirus avian bronchitis virus translation from the utr of mrna is repressed, but that from the utr of mrna is stimulated in coronavirus-infected cells coronavirus subgenomic and genomic minus-strand rnas copartition in membrane-protected replication complexes minus-strand copies of replicating coronavirus mrnas contain antileaders coronavirus subgenomic minus-strand rna and the potential for mrna replicons colocalization and membrane association of murine hepatitis virus gene products and de novo-synthesized viral rna in infected cells the coronaviridae the molecular biology of arteriviruses host protein interactions with the end of bovine coronavirus rna and the requirement of the poly(a) tail for coronavirus defective genome replication leader switching occurs during the rescue of defective rnas by heterologous strains of the coronavirus infectious bronchitis virus specific interactions between coronavirus leader rna and nucleocapsid protein selected animal models of viral persistence: mouse hepatitis virus translation effector properties of mouse hepatitis virus nucleocapsid protein infectious rna transcribed in vitro from a cdna copy of the human coronavirus genome cloned in vaccinia virus viral replicase gene products suffice for coronavirus discontinuous transcription internal ribosomal entry in the coding region of murine hepatitis virus mrna localization of mouse hepatitis virus nonstructural proteins and rna synthesis indicates a role for late endosomes in viral replication a domain at the end of the polymerase gene is essential for encapsidation of coronavirus defective interfering rnas translation but not the encoded sequence is essential for the efficient propagation of the defective interfering rnas of the coronavirus mouse hepatitis virus characterization of an equine arteritis virus replicase mutant defective in subgenomic mrna synthesis analysis of a hypervariable region in the non-coding end of the infectious bronchitis virus genome a phylogenetically conserved hairpin-type untranslated region pseudoknot functions in coronavirus rna replication murine coronavirus packaging signal confers packaging to nonviral rna common rna replication signals exist among group coronaviruses: evidence for in vivo recombination between animal and human coronavirus molecules full-length genomic sequence of bovine coronavirus ( kb) in vitro rna synthesis from exogenous dengue viral rna templates requires long range interactions between -and -terminal regions that influence rna structure strategy for systematic assembly of large rna and dna genomes: the transmissible gastroenteritis virus model systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a specific binding of host cellular proteins to multiple sites within the end of mouse hepatitis virus genomic rna a -proximal rna sequence of murine coronavirus as a potential initiation site for genomic-length mrna transcription presence of subgenomic mrnas in virions of coronavirus ibv virus-encoded proteinases and proteolytic processing in the nidovirales acknowledgements we thank cary brown, kimberley nixon, sharmila raman, gwyn williams, and hung-yi wu in the brian laboratory and kristopher curtis and boyd yount in the baric laboratory for invaluable discussions and experimentation. work in d. brians laboratory is supported by grant ai- from the national institutes of health and work in r. barics laboratory by grants ai- and gm- from the national institutes of health. key: cord- -g tgo cn authors: deming, damon j.; graham, rachel l.; denison, mark r.; baric, ralph s. title: mhv-a orf a replicase protein nsp -nsp processing in replication date: journal: the nidoviruses doi: . / - - - - _ sha: doc_id: cord_uid: g tgo cn nan polyprotein is processed by the main protease (m pro ) into mature products including nsp -nsp , which are associated with replication complexes and presumably involved with rna synthesis. [ ] [ ] [ ] [ ] the exact function of these proteins, in either their pre-or postcleaved forms, is unknown. however, disruption of m pro cleavage during any stage of the infection cycle blocks replication, suggesting that constitutive proteolytic processing of the nonstructural polyprotein is a requirement for efficient transcription. in this report, we describe preliminary data defining the requirement for the proteolytic processing of the nsp -nsp proteins in mhv-a replication. through use of an efficient mhv-a reverse genetics system, we ablated each of the m pro cleavage sites associated with the nsp -nsp cassette, and evaluated whether the mutated genome was capable of supporting a viable virus, and if so, characterized the m pro processing of the mutated protein, transcription function, and in vitro growth fitness. the m pro targets amino acid sequences q-s/n/a, cleaving after the essential glu at position (p ). to evaluate the importance of cleavage on virus replication, the cleavage sites flanking nsp , nsp , nsp , and nsp were individually disrupted by mutating the p glu to an ala (table ) . however, there are two potential m pro cleavage sites at the nsp / interface, an lqa (present at positions p -p ) and lqs (p -p '). although the lqs has been shown to be cleaved during m pro processing, it is possible that the upstream lqa site is also functional in the presence or absence of the lqs site. to address this possibility, both sites were mutated, either individually (nsp * .a and graham, mark r. denison, vanderbilt university medical center, nashville, tennessee. the highly conserved region at the carboxy-terminus of the mhv replicase orf a ralph s. baric* damon j. deming, ralph s. baric, university of north carolina, chapel hill, north carolina. rachel l. * table . amino-acid sequences for the nsp -nsp mpro cleavage sites for mhv-a (top) and mutant viruses (bottom). a there are two potential m pro cleavage sites, an lqa and lqs. b the upstream lqa site is mutated. c the downstream lqs site is mutated. d both sites are mutated. nsp * .b) or in combination (nsp * .a+b). the viability of the seven mhv-a mutants were tested by transferring the mutations (or combination of mutations) to the mhv-a infectious clone, driving full-length transcripts, and electroporating the rna into cells. viability was confirmed by the formation of cpe and detection of leadercontaining transcripts by rt-pcr. three constructs produced replicating viruses. of the viable mutants, the mhv-nsp * .a and mhv-nsp * .b cleavage mutants replicated to wild-type titers while mhv-nsp * replication was reduced by about two logs. the genetic stability of the attenuated mhv-nsp * mutant virus was analyzed after serial passages on dbt cells. plaque purified passage virus, mhv-nsp * p , displayed improved fitness of in vitro growth, as demonstrated by the near wild-type titers. surprisingly, the recovered virus did not revert to wild-type sequence at the nsp /nsp m pro cleavage site, indicating that an as of yet unidentified mutation(s) has compensated for the virus's inability to properly process the nsp -nsp precursor protein. immunoprecipitation (i.p.) using either anti-nsp (for the nsp * mutants) or anti-nsp antibody (for the nsp * mutants) and western blot analysis was used to verify whether or not mutation of the p glu prevented cleavage of the viable viruses (data not shown). bands corresponding to nsp ( -kda protein) were absent in lysates of mhv-nsp * and mhv-nsp * p . however, a band of approximately kda, which is the predicted size for uncleaved nsp -nsp , was present in the mutants, but absent in the wild-type control cells. analysis of the i.p.-western blot suggested that mhv-a was able to use either of the two potential m pro cleavage sites located at the nsp /nsp site, likely accounting for the wild-type growth kinetics. bands of approximately kda were precipitated with anti-nsp antibody for the wild-type control, mhv-nsp * .a, and mhv-nsp * .b. notably, the mhv-nsp * .b band was slightly larger than that of the control or the first nsp * mutant, consistent with the prediction that the lqa site was cleaved and yielded an nsp three amino acids larger than that of the lqs cleaved protein. the cleavage site mutants were found to be similar to mhv-a in their transcriptional activity and cellular localization (results summarized in figure ). northern blots hybridized with an rna probe complementing the ' end of the n-gene showed no notable alteration in either the pattern or relative amounts of subgenomic to genomic rna in mutant and control viruses. the distribution of the mutant proteins within cells was compared with their wild-type counterparts. the nsp -nsp proteins are known to colocalize with sites of viral replication while being excluded from regions of virion assembly. , in order to determine if ablation of the m pro processing interferes with the ability of the protein to traffic into the replication complex, an immunofluorescence (ifa) study was completed. cells infected with either mhv-a , mock, or mutant virus were dual-stained for either nsp or nsp (depending on the mutant, as above) and nucleocapsid (n), which co-localizes with sites of active viral replication, or membrane (m), which is targeted to regions of virus assembly. regardless of the virus, the nsp and nsp colocalized with n and was separate from m (data not shown). we were unable to find a difference between the localization of the nsp or nsp proteins between the mutants or wild-type. cleavage site ablation resulted in lethal, debilitated, or near wild-type viability, and mutation of most of the m pro cleavage sites was lethal (summarized in figure ). lethality could be due to disruption of nsp - proteolytic processing causing a failure of precursor, intermediate, or mature protein function within the replication complex. however, not all of the m pro cleavage site mutants were nonviable. based on the genetic analysis, mhv-a has two functional nsp -nsp m pro cleavage sites, lqa and lqs, and disruption of either of these potential sites fails to affect replication competence, m pro cleavage pattern, or cellular localization in vitro. possible in vivo effects need to be studied. in contrast, simultaneous mutation of both sites was lethal. the only other m pro cleavage site harboring a viable mutation was that shared by the nsp and nsp proteins. in this case, the mutant virus was highly attenuated in its replication efficiency and was unable to proteolytically process the fused nsp -nsp protein. serial passage of this virus restored wild-type replication but did so without reverting the mutated cleavage site or the ability to process the nsp -nsp protein. the data demonstrate that with the exception of cleavage between the nsp and nsp proteins, m pro processing of the nsp -nsp cassette is essential in coronavirus rna transcription and replication. four proteins processed from the replicase gene polyprotein of mouse hepatitis virus colocalize in the cell periphery and adjacent to sites of virion assembly 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 localization of mouse hepatitis virus nonstructural proteins and rna synthesis indicates a role for late endosomes in viral replication coronavirus protein processing and rna synthesis is inhibited by the cysteine proteinase inhibitor e d systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a the complete sequence ( kilobases) of murine coronavirus gene encoding the putative proteases and rna polymerase key: cord- -kyavg vu authors: masters, p. s. title: localization of an rna-binding domain in the nucleocapsid protein of the coronavirus mouse hepatitis virus date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: kyavg vu the interaction between the nucleocapsid (n) protein of mouse hepatitis virus (mhv) and rna was studied in an effort to define portions of the n molecule that participate in binding to rna. n mrnas transcribed from sp and t vectors were translated in a rabbit reticulocyte lysate. analysis of synthesized n protein in a nondenaturing gel system showed that it bound in vitro to an endogenous rna in the reticulocyte lysate but not to its own mrna. a set of deletion mutants was constructed in order to localize the rna-binding activity of the n protein. it was found that removal of as much as amino-terminal or carboxy-terminal amino acids from the molecule had little or no effect on rna binding. moreover, deletion mutants lacking both termini still retained rna-binding ability. by contrast, internal deletions or truncations extending beyond these two limits effectively abolished rna binding by n protein. thus, the rna-binding region of n has been mapped to the second (central) of the three structural domains of the molecule. coronaviruses constitute a family of viruses whose single-stranded, positivesense rna genomes have the largest coding capacities among the rna viruses [ , ] . paradoxically, coronaviruses have relatively few structural proteins. their large, roughly spherical virions are enclosed by a membrane envelope containing two (or, in some cases, three) types of membrane-bound glycoproteins. internal to this, multiple monomers of a nucleocapsid protein (n) encapsidate the viral genome, which, in the case of the prototype mouse hepatitis virus (mhv), is some , nucleotides in length [ , ] . the nucleocapsids of coronaviruses are helically symmetric [ ] , a feature seen in only one other group of positive-stranded rna viruses, those of the proposed family toro-viridae [ ] . consequently, these structures pose interesting problems with respect to genome translation as well as in understanding the processes of encapsidation and assembly. elucidation of coronavirus nucleocapsid structure and function will require description of the multiple interactions in which the n protein must participate: (i) the binding of n protein along the entire length of the rna genome; (ii) contacts between adjacent n monomers on the r n a and between n monomers that neighbor each other per helical turn; and (iii) association between n protein and the membrane glycoprotein (m), which may stabilize virion assembly [ ] . for at least some of these, it may be possible to map regions of the n molecule that are involved in a particular interaction. the work presented here focusses on the first type of interaction: the ability of n to bind to rna. coronavirus n proteins are markedly basic, and they form complexes with rna that tend to dissociate in high concentrations of salt and provide only limited protection against the action of ribonucleases [ , ] . in these respects, they resemble the np proteins of the orthomyxoviruses [ , ] . by contrast, the n -r n a complexes of the rhabdoviruses and the paramyxoviruses, which also form helically symmetric nucteocapsids, are stable in high salt and are largely resistant to ribonuclease [ , ] . few details are known about the association between n protein and rna in the coronaviral nucleocapsid. we have previously compared the amino acid sequences of the n proteins of five strains of mhv and, on the basis of this analysis, proposed a three domain structural model for the m h v n protein [ ] . in this paper it is shown that the central of these three domains is responsible for the rna-binding ability of the mhv n molecule. transcription vectors linking the n gene of mhv to the rna polymerase promoter of either bacteriophage t or bacteriophage sp were constructed by standard recombinant dna techniques [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . a cdna clone, pal , containing a nearly full-length copy of the n mrna of mhv-a [ ] (embl/genbank accession no. m ) was restricted with snabi and saci, made blunt-ended, and inserted in either orientation into the vector pgem zf ( -) (promega), which had been opened and blunt-ended at the kpni and sphi sites of the polylinker. the resulting constructs, pa and pa , contained the t or sp promoter, respectively, linked to nt of the nt ' untranslated region (utr) of rna of mhv-a i- , ] , followed by the entire n protein coding region and nt of the nt ' utr ( fig. ) . hindiii-linearized pa encoded run-off transcripts that were bounded by polylinker sequences of nt and nt at their ' and ' ends; saci-linearized pa encoded run-off transcripts that were bounded by polylinker sequences of nt and nt at their ' and ' ends ( fig. ). full-length n mrnas transcribed from these two plasmids had identical translation efficiencies in vitro. truncated n mrnas were transcribed from pa that had been linearized with bsmi, scai or spei and from pa that had been linearized with acci or ecori (fig. ). an internal deletion mutant of the n gene was constructed by restriction of pa with [ ] . the n mrna initiation codon is underlined nhei and spei ( fig. ) , followed by religation of these compatible sites in the vector fragment. the resulting plasmid, pa , lacked nt - of the n gene and encoded the mutant protein n/nhe-spe. a second internal deletion mutant was generated by cleavage of pa with apai and nhei (fig. kpni site had been inserted immediately preceding the start codon. details of the construction of pa will be described elsewhere. the kpni(blunted)-hindlii fragment of pa was exchanged for the stui-hindlii fragment of the mutant vsv n sp transcription vector pn [ ] . this yielded a plasmid, pa , containing nt - and - of the coding region of the vsv n gene followed by the entire coding region of the mhv n gene; the encoded protein was designated n/fusl. a deletion mutant of pa was created by exchange of the apai(blunted)-hindlii fragment of pa for the stui-hindlii fragment of pn . the resulting plasmid, pa , contained nt - and - of the vsv n gene followed by nt - of the mhv n gene; the encoded protein was designated n/fus . during the construction of pa , there serendipitously arose a similar plasmid, pa , containing the same extent of mhv n sequence preceded only by nt - of the vsv n gene; the protein encoded by this construct was designated n/fus . all plasmid constructs were checked by restriction analysis, and all newly formed junctions were directly verified by dideoxy sequencing [ ] . dna plasmids to be used as transcription templates were purified by two cycles of equilibrium centrifugation in csci gradients in the presence of propidium iodide [ ] . capped, run-off sp and t mrnas [ ] were typically synthesized in gl reactions containing mm tris hydrochloride (pht. ), mm mgc , mm spermidine, mm nac , mm dithiothreitoi, i u rnasin (promega), gm each atp and ctp, gm each gtp and utp, iim mvgpppg (new england biolabs), gci [ , - h]utp ( ci/ mmol; amersham), gg linearized dna template, and either u sp rna polymerase or u t rna polymerase (new england biolabs). reactions were carried out for rain at either °c (sp ) or °c (t ). products were extracted twice with phenol-chloroform, twice with chloroform and were twice precipitated with ethanol. the incorporation of labeled utp was determined by binding of product rna to deae filter paper (whatman de ); the size and homogeneity of synthesized rna were verified by electrophoresis in % polyacrylamide gels containing m urea, followed by fluorography. for the synthesis of high specific activity, p-labeled n mrna, all reaction conditions were identical, except that the labeling nucleotide was ~tci [a- p]utp ( ci/mmol; amersham). translation of synthetic mrnas was carried out in a micrococcal nuclease-treated rabbit reticulocyte lysate (amersham). in the standard reaction, . to . ~tg of mrna in to ~tl of h was used to program gl of reticulocyte lysate containing u of rnasin and to gci of [ s]methionine (> ci/mmol; new england nuclear). incubations were carried out for rain at °c. in many experiments, where noted, gl of either h or rnase a was then added, and samples were incubated an additional rain at °c. bovine pancreatic rnase a (ca. u/mg; sigma type xii-a) was rendered dnase-free by heat treatment [ ] . for translation of authentic mhv mrna, total rna was purified [ ] from mhv-a -infected (as well as mock-infected) mouse clone cells at h postinfection, and gg was translated as above. in vitro-synthesized proteins were analyzed both by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) [ ] and by nondenaturing page employing the standard laemmli discontinuous system in which sds was omitted from all gels and buffers, and samples were not heated prior to electrophoresis [ ] . reticulocyte lysate samples ( ) were run on sds-page gels ( % acrylamide- . % methylene bisacrylamide) and nondenaturing page gels ( . % acrylamide- . % methylene bisacrylamide) that were cm long by . mm thick. nondenaturing page was performed at v at ambient temperature (ca. h); under these conditions there was no detectable heating of the gel for the duration of the run. gels were fixed in % methanol- % acetic acid and were impregnated with m sodium salicylate prior to drying and fluorography at - °c. [ ss]methionine-labeled bands were quantitated either by (/) excision and solubilization in ml of % h for h at oc prior to counting in ml of aquasol (new england nuclear) or (ii) direct counting with a betascope blot analyzer (betagen). in order to develop in vitro assays for mhv n protein function, the n gene was inserted into bacteriophage t and sp transcription vectors. synthetic mrna containing the entire coding region of the authentic n mrna, as well as most of the ' and ' utrs, was used to program a protein synthesizing system from rabbit reticulocyte lysate. the translation product from this message, labeled with [ s]methionine, was analyzed by sds-page and had a mobility indistinguishable from that of n protein translated in vitro from total rna purified from mhv-a -infected cells (fig. a, lanes b and c) . in addition, n protein translated from synthetic mrna was immunoprecipitable by either anti-n or anti-mhv polyclonat antibodies (peng and masters, unpubl. data). in vitro-translated n protein had a slightly higher apparent molecular weight than n protein from [ s]methionine-labeled virus of mhv ( fig. a, lane a). this was possibly due to differential extents of n protein phosphorylation [ ] in vitro and in vivo, or it may indicate that the mature n protein found in mhv virions had undergone amino-terminal modification, as has been observed for the n protein of the coronavirus avian infectious bronchitis virus [ ] . this apparent difference does not bear on the present study. to examine the properties of native n protein, the in vitro translation product was analyzed by nondenaturing page. in this gel electrophoresis system, the major fraction (ca. %) of translated n protein migrated into the running gel, forming a discrete band with a mobility of . relative to bromphenol blue (fig. b, lane f). the mhv-a n protein contains a considerable excess of basic amino acid residues ( lysines and arginines as opposed to glutamates and aspartates) and has a calculated pi of . [ ] . initially it was surprising that such a molecule, expected to have a substantial net positive charge, was migrating toward the positive pole during electrophoresis. this result suggested that the n molecule was tightly bound to some polyanionic molecule and migrated as part of a complex having an overall negative charge. that this, in fact, had occurred was shown by incubation of translated n protein with rnase a prior to nondenaturing gel electrophoresis. rnase treatment abolished the ability of translated n protein to migrate into the nondenaturing gel ( fig. b, lane g) . thus, synthesized n protein had bound to some rna species present in the translation reaction, and as a consequence of this complex formation it was carried into the nondenaturing gel during electrophoresis. n protein translated from total rna purified from mhv-a -infected cells behaved in the same rnase-sensitive manner (fig. more immediately digested. the n -r n a complex was more sensitive to rnase a than to rnase t , possibly due to the different nucleotide specificities of these enzymes (rnase a cuts ' to pyrimidines; rnase t cuts ' to g residues). alternatively, the differential sensitivity may have reflected the relative accessibility of each enzyme to phosphodiester bonds within the n -r n a complex. the n proteins of coronaviruses provide their encapsidated genomes only a minimal degree of protection against the action of ribonucleases [ , ] . hence, in this respect, the n -r n a interaction in the nondenaturing gel assay resembled that found in the m h v nucleocapsid. the rnase sensitivity of the ability of n protein to migrate into nondenaturing gels was not due to possible contaminating protease activity in the rnase preparations, since the same rnase-treated samples of translated n p.s. masters protein showed no degradation when analyzed by sds-page (fig. , lanes an, lower) . moreover, inclusion of the placental rnase a inhibitor, rnasin, allowed at least partial inhibition of the effect of an intermediate concentration of rnase a (fig. , lanes p and q) . incubation with rnase-free dnase had no effect on the mobility of n protein in nondenaturing page (fig. , lane r) . it has been reported previously that the mhv n protein exhibits sequencespecific binding to nucleotides to of the mhv leader rna [ ] . since the n mrnas synthesized from the transcription vectors shown in fig. contained this portion of the leader sequence, it seemed possible that the n-rna complex observed in nondenaturing page was that of translated n protein binding to its own mrna. to test this notion, parallel translation reactions were carried out either with unlabeled n mrna or with n mrna labeled to high specific activity ( . x cpm/gg) with [a- p]utp. n protein translated from unlabeled mrna was labeled with [ s]methionine, while n protein translated from labeled mrna was left unlabeled (fig. ) . it was expected that if translated n protein were binding to its own mrna, then a p-labeled rna band migrating to the same position as that of [ s]methionine-labeled n protein should have been observed in nondenaturing page. as shown in fig. (lanes b', c', f' and g') this clearly did not occur. this was not due to failure to translate the p-labeled mrna, since unlabeled and labeled n mrnas had comparable translation efficiencies (fig. , lanes b, e, b' , and e'). during the course of the min translation reactions, the p-labeled mrna underwent limited nucleolyfic degradation, presumably due to low levels of rnase activity in the reticulocyte lysate. most of the p-labeled material produced by this limited breakdown was retained by sds-page, but virtually all of the same species migrated near the dye front in nondenaturing page. some minor p-labeled bands were seen in the nondenaturing gel at positions other than that expected for n protein-bound material. however, these could not have been due to complex formation with n protein, since the same bands were observed when n protein synthesis was abolished by inclusion of the protein synthesis inhibitor cycloheximide in translation reactions (fig. , lane i') . thus, the n-rna complex observed in the nondenaturing gel assay must have been formed by the binding of n to some endogenous rna species in the reticulocyte lysate. that the observed complex was not due to translated n protein binding to the leader portion of its own mrna was also supported by the observation that full-length n protein translated from a construct in which the mhv leader sequence was entirely deleted was also able to migrate into nondenaturing gels (data not shown). the same was observed for vsv-n : mhv-n fusion constructs which had the ' end of a heterologous mrna substituted for the mhv leader (see below). the complex reported to occur between n protein and mhv leader rna [ ] may not have formed under (a'-j') the conditions of the translation assay or may not have been detectable in the electrophoresis system used here. the identity of the r n a b o u n d by n protein in the reticulocyte lysate has not yet been determined. n protein formed a discrete band rather than a heterogeneous smear in nondenaturing p a g e , suggesting that it was binding p.s. masters to a particular rna species or at least to a set of species of fairly uniform size. moreover, a band of identical mobility was formed by n protein that had been translated in a wheat germ extract (data not shown), indicating that the unknown rna species was not unique to reticulocytes. this non-sequence-specific mode of rna binding was not unexpected. the n protein of mhv has been shown previously to bind in vitro to rna of nonviral origin by a labeled rna overlay protein blot assay [ , ] , and, indeed such non-sequence-specific binding must be the principal type of protein-rna contact along the extensive length of the coronavirus nucleocapsid. the ability of translated n protein to migrate into a nondenaturing gel was next used as an assay to map the rna-binding function of the n protein molecule. a set of n protein deletion mutants with successively larger carboxyterminal truncations (designated n/ace, n/bsm, n/sea, n/eco and n/spe) was generated by translation of run-off transcripts synthesized from transcription vectors that had been linearized with the restriction enzymes acci, bsmi, scai, ecori, or spei (fig. ) . these variants contained, respectively, deletions of , , , , and amino acids. in addition, the internal deletion contained in the n gene of a temperature-sensitive and thermolabile mhv-a mutant, albany- , was transferred to a transcription vector. the mutant n protein translated from this construct (n/alb ) contained an in-phase deletion of amino acids through but was everywhere else identical to wild type n ( [ ] ; koetzner et al., manuscript in prep.). these six mutants, as well as full-length n protein, were analyzed by sds-page and by nondenaturing page (fig. ) . on sds-page, all migrated as discrete bands of the expected relative mobilities and were largely unaffected by treatment with rnase a. the n/ace mutant showed some degree of heterogeneity in both the presence and absence of rnase a (fig. , lanes e and f). since this protein product terminated in the basic amino acid-rich sequence kpqrkgr, it possibly was rendered sensitive to degradation by trypsin-like proteolytic activities within the reticulocyte lysate. when analyzed for the ability to bind to rna, only two of the n mutants, n/acc and n/alb , were able to enter a nondenaturing get to a significant quantitative extent by comparison with full-length n protein (fig. , lanes a' to p') . this migration was completely inhibited by prior incubation with rnase a. the remainder of the carboxyterminal truncated n proteins were only minimally detectable or were undetectable by nondenaturing page. an estimate of the relative rna-binding ability of each n protein construct was made by determining the ratio of cpm of translated protein that had banded in a nondenaturing gel compared to the cpm of an identical sample that had banded in sds-page (table ) . by this analysis, the n/ace construct retained the major part of the rna-binding ability of full-length n protein, demon- strating that at least the carboxy-terminal amino acids of n were dispensable with respect to this function. however, carboxy-terminal truncation further into the n molecule, in the n/bsm, n/sca, n/eco and n/spe mutants, generally abolished the ability of n to bind to rna. the relative rna-binding ability of n/alb was intermediate between these latter and that of n/acc. thus, the left end of the albany- deletion may define the right-most boundary of the rna-binding domain of the mhv n protein, since the n/bsm mutant, which truncated two further residues, lost rna-binding ability. effect of amino-terminal or centrally located deletions on the ability of n protein to bind to r n a a set of deletion mutants collectively spanning the remainder of the n molecule was next constructed. due to the paucity of convenient restriction sites near p. s. masters defined as the average ratio of cpm of translated protein able to enter a nondenaturing gel compared to cpm of an identical sample binding in an sds gel. the number of independent determinations from which the average was obtained is indicated in parentheses b for the carboxy-terminal and internal deletions (upper set), this is calculated relative to full-length mhv n protein. for the vsv-n:mhv-n fusion constructs (lower set), this is calculated relative to the fulmength construct n/fusl the ' end of the mhv n gene, a fusion construct incorporating a portion of the ' end of a mutant vsv n gene was created in order to provide a start codon for a construct in which the ' end of the m h v n gene was then removed. consequently, both n fusion genes encoded proteins initiating with the vsvderived sequence: m a p t v k r i i n d s i i q p k l p a n e d p d r s a e d d k w l p i -yilg, corresponding to amino acids - and - of that protein [ ] . the full-length fusion protein, designated n/fusl, contained the entire m h v n protein following this sequence. the amino-terminal deletion mutant, n/ fus , lacking the first amino acids of the mhv n protein, contained the vsv sequence fused to the remainder of mhv n. two internal deletions mutants, encoding n/apa-nhe and n/nhe-spe, were generated by removal of the indicated restriction fragments from one of the original (nonfusion) n vectors (fig. ) . the resulting protein products transcribed and translated from these lacked regions of and amino acids, respectively. these mutants were analyzed by the two gel electrophoresis systems and on sds-page were observed to be homogeneous and of the anticipated relative mobilities (fig. , lanes a to k) . nondenaturing page revealed that the n/fus and n/fus proteins retained the ability to bind to rna, whereas the deletions in n/apa-nhe and n/nhe-spe abolished rna binding (fig. , lanes a' to k') . quantitation of the extent of rna binding showed that the n/fusl mutant was % as binding-competent as unaltered, full-length n protein (table ) . although the vsv n protein is, itself, an rna-binding protein, the mutant vsv n protein used in the construction of n/fus and n/fus is unable to bind indeed, for n/fus the vsv n-derived portion of the molecule appeared to somewhat impede the entry of mhv n into the nondenaturing gel. remarkably, the n/fus protein was a better rna-binding molecule than its parent, n/fusl, in spite of the loss of a large portion of the mhv n amino terminus (table ) . clearly, this deleted portion of the n molecule can play little or no role in rna binding. by contrast, the deletions in the n/apa-nhe and n/nhe-spe mutants reduced rna binding to residual levels similar to those observed for the four shortest carboxy-terminal truncation mutants ( table ). since removal of a substantial portion of either the amino or carboxy terminus of the n molecule allowed it to retain the ability to bind to rna, it became of interest to examine the effect of removal of both of these regions. this was accomplished by generating the doubly-truncated mutant n/fus /acc. included for comparison in these experiments was n/fus , an n mutant containing the same amino-terminal deletion as n/fus but having only amino acids derived from the vsv n protein (maptvkri). in addition, the corresponding accigenerated truncation of n/fus was examined. analysis of these n protein mutants and their full-length counterparts demonstrated that all were able to enter nondenaturing gels to a significant degree (fig. ) . quantitation of the gel assay showed that n/fus was at least as competent as full-length n protein and nearly twice as competent as n/fus in rna binding (table ) , thereby extending the prior result obtained with the n/fus mutant. the n mutants containing both amino-and carboxy-terminal deletions, n/fus /acc and n/fus /acc, were at least % as able as n/fusl (or at least % as able as full-length n) to bind to rna (table ) . therefore, the rna-binding domain of the mhv n protein could be localized to a portion of the molecule encompassing amino acids to . the purpose of this study was to learn what regions of the mhv n molecule participate in the most salient function of n, its binding to rna. the extent of the deletions and the rna-binding competence of the various mhv n protein mutants constructed for this study are summarized schematically in fig. . the results indicate that the rna-binding ability of the n molecule can be localized to an internal region comprising some amino acids. deletion of either amino acids from the amino terminus or amino acids from the carboxy terminus of n produced only a limited effect on the rna-binding function of this protein. moreover, mutants lacking both termini still retained fig. . effect of combined amino-terminal and carboxy-terminal deletions on the ability of n protein to bind to rna. in vitro-synthesized mrnas encoding full-length n protein, a vsv-n : mhv-n fusion protein (n/fusl), amino-terminal deletion mutants (n/fus and n/fus ), carboxy-terminal deletion mutants (n/acc and n/fusl/acc) or combined aminoterminal and carboxy-terminal deletion mutants (n/fus /acc and n/fus /acc) were translated and subsequently incubated for min at °c with either h or with gg/ml rnase a. control reactions contained no exogenous mrna (a, b, a' and b'). [ s]methionine-labeled samples were analyzed by sds-page (a-r) and by nondenaturing (nd)-page (a'-r') rna-binding ability. by contrast, deletions occurring within the internal region or entering further into it via the carboxy terminus of the molecule effectively abolished r n a binding. the regions of n defined by this analysis coincide strikingly with those of a three domain model for the molecule that we have proposed previously on the basis of a comparison of the sequences of the n proteins of five different strains of mhv [ ] . the present work suggests that these three domains of n are not only structurally distinct but are functionally separable as well. the rna-binding characteristic of the n molecule resides within domain ii, whereas domains i and iii are expendable with respect to this activity. since the deletions entering domain ii are mutations to loss of function, it [ ] or recognizable similarity to recently described arginine-rich [ ] or methionine-rich [ ] rna-binding domains. thus, the rules governing this particular mode of protein-rna interaction remain to be elucidated. in fig. , the rna-binding ability of n/alb has been scored as equivocal, based on our recent finding that the ability of this mutant protein to enter a nondenaturing gel is temperature-sensitive with respect to that of wild type n (koetzner and masters, unpubl, results) . in addition, revertants of the albany- mutant have been selected, and all of these have n proteins which no longer exhibit the rna-binding temperature-sensitivity of n/alb . for the one revertant that we have analyzed in detail, the single point mutation that causes this restoration of rna-binding ability resides in the distal third of domain ii. this finding further underscores the role of domain ii in rna binding, and supports the notion that the nondenaturing gel assay measures a biologically relevant property of the n molecule. although the n protein may recognize a specific nucleation sequence or secondary structure on the mhv genome, it must also possess a more general non-sequence-specific rna-binding capability, since it encapsidates the entire length of the , nucleotide genome, which does not appear to contain any iterated pattern of potential rna recognition sequences. this general mode of rna binding may be a common characteristic of the n (np) proteins of rna viruses having helical nucleocapsids. bacterially expressed np protein of influenza virus was shown to bind well to both viral and nonviral rna substrates when assayed in vitro [ ] . the n protein of vsv bound tightly to cellular rnas when synthesized in the absence of other viral proteins in an in vivo expression system [ ] , and it bound to endogenous reticulocyte rnas or added rna species when expressed by in vitro translation [ ] . the measles virus np protein, expressed in vivo by a vaccinia virus recombinant, has recently been shown to assemble into structures very similar to authentic measles nucleocapsids, as determined by both electron microscopy and equilibrium sedimentation on csc gradients [ ] . for mhv, it has been demonstrated previously that the n protein of this virus can bind to a variety of viral and nonviral rna species following separation of n by sds-page and blotting onto nitrocellulose [ , ] . thus, it is not unexpected that the mhv n protein should bind in vitro to nonviral rna species in the nondenaturing gel assay reported here. it should be noted that an alternative interpretation of the nondenaturing gel assay is that the mhv n protein was not directly bound to rna but, rather, to a protein constituent of an rnase-sensitive ribonucleoprotein in the reticulocyte lysate. this seems less likely in light of the previously demonstrated rna-binding activity of n [ , ] , but it cannot be ruled out until an mhv n rna-binding assay using purified components is established. if such an n protein-ribonucteoprotein complex is shown to occur, then this may indicate a previously unappreciated interaction between n protein and host cell components. the work presented here does not address the problem of sequence-specific rna recognition by n protein. only full-length genomic rna of mhv is packaged into virions. recently, analysis of defective interfering particles of mhv has identified the rna packaging signal as falling within a ( nucleotide locus near the ' end of the huge viral polymerase gene [ , ] , which is unique to genomic r n a and is not found in subgenomic r n a species. others have reported sequence-specific binding of n protein to nucleotides to of the m h v leader [ ] , which is c o m m o n to all m h v genomic and subgenomic rnas. this latter sequence was present on many of the synthesized m r n a s used in our study, but selective binding to this r n a region was not observed (fig. ) . it is possible that the concentrations of n protein and n o n -m h v r n a in the reticulocyte lysate in our assay were too high to allow demonstration of a selective recognition of m h v r n a . we are currently seeking to establish the assay conditions and the correct r n a substrate to examine sequence-specific r n a binding by the m h v n protein. nucleocapsids of large rna viruses as functionally active units in transcription coronavirus ibv: partial amino terminal sequencing of spike polypeptide $ identifies the sequence arg-arg-phe-arg-arg at the cleavage site of the spike precursor propolypeptide of ibv strains beaudette and m heterogeneous nuclear ribonucleoprotein particles and the pathway of mrna formation assembly of influenza ribonucleoprotein in vitro using recombinant nucleoprotein mechanism of interferon action. interferon-mediated inhibition of simian virus- early rna accumulation zinc fingers": a novel protein motif for nucleic acid recognition cleavage of structural proteins during the assembly of the head of bacteriophage t characterization of leader rna sequences on the virion and mrnas of mouse hepatitis virus, a cytoplasmic rna virus sequence-specific recognition of rna hairpins by bacteriophage antiterminators requires a conserved arginine-rich motif the complete sequence ( kilobases) of murine coronavirus gene i encoding the putative proteases and rna polymerase plus and minus strand leader rnas in negative strand virus-infected cells ribonucleoprotein-like structures from coronavirus particles rna-binding domain of mhv n protein analysis of efficiently packaged defective interfering rnas of murine coronavirus: localization of a possible packaging signal molecular cloning: a laboratory manual resolution of multiple complexes of phosphoprotein ns with nucleocapsid protein n of vesicular stomatitis virus complex formation with vesicular stomatitis virus phosphoprotein ns prevents binding of nucleocapsid protein n to nonspecific rna structure and function studies of the nucleocapsid protein of mouse hepatitis virus efficient in vitro synthesis of biological active rna and rna hybridization probes from plasmids containing a bacteriophage sp promoter molecular cloning of the gene encoding the putative polymerase of mouse hepatitis coronavirus, strain a sequence comparison of the n genes of five strains of the coronavirus mouse hepatitis virus suggests a three domain structure for the nucleocapsid protein a common rna recognition motif identified within a defined u rna binding domain of the k u snrnp protein rna-binding proteins of coronavirus mhv: detection of monomeric and multimeric n protein with an rna overlay-protein blot assay the -kd protein of signal recognition particle contains a methionine-rich rna binding domain dna sequencing with chain terminating inhibitors identification and primary structure of the gene encoding the berne virus nucleocapsid protein coronaviruses: structure and genome expression assembly of nucleocapsidlike struct~es in animal cells infected with a vaccinia virus recombinant encoding the measles virus nucleoprorein expression of a recombinant dna gene coding for the vesicular stomatitis virus nucleocapsid protein deans ill ( ) specific interaction between coronavirus leader rna and nucleocapsid protein synthesis and subcellular localization of the murine coronavirus nucleocapsid protein the molecular biology of coronaviruses i thank lawrence sturman for numerous valuable discussions and cynthia ricard, who first suggested the rnase experiment. i am grateful to monica parker for expert technical assistance and to arlene ramsingh for critically reading the manuscript.this work was supported in part by public health service grant gm from the national institutes of health. key: cord- -hduv ur authors: nan title: sorting of progeny coronavirus from condensed secretory proteins at the exit from the trans-golgi network of att cells date: - - journal: j cell biol doi: nan sha: doc_id: cord_uid: hduv ur murine hepatitis virus (strain a ), (mhv-a ) is a coronavirus that buds into pre-golgi compartments and then exploits the exocytic pathway of the host cell to reach the exterior. the fibroblastic cells in which replication of this virus is usually studied have only a constitutive exocytic pathway that the virus uses. mhv-a also infects, albeit inefficiently, att cells, murine pituitary tumor cells with a regulated as well as a constitutive exocytic pathway. here we examine att cells at early times after the infection, when the golgi apparatus retains its morphological and biochemical integrity. we observe that progeny coronavirus and secretory protein destined for the secretory granules of the regulated exocytic pathway traverse the same golgi stacks and accumulate in the trans-golgi network. their pathways diverge at this site, the condensed secretory proteins including the acth going to the secretory granules and the coronavirus to post-golgi transport vesicles devoid of acth. on very rare occasions there is missorting such that aggregates of condensed secretory proteins and viruses occur together in post-golgi vesicles. we conclude that the constitutive and regulated exocytic pathways, identified respectively by the progeny virions and the secretory protein acth, diverge at the exit from the trans-golgi network. i n cells of the anterior pituitary tumor cell line art there are two exocytic pathways, one is constitutive and the other is regulated, such that secretion is enhanced by seeretagogues (gumbiner and kelly, ) . the major envelope glycoprotein of an endogenous murine leukemia virus (gumbiner and kelly, ) and the secreted glycoprotein laminin (burgess et al., ) are examples of proteins that are exported via the constitutive pathway. on the other hand, part of the proopiomelanocortin made in these calls follows the regulated pathway in which it is cleaved proteolyrically to yield acth and is stored in secretory granules (gumbiner and kelly, ; moore and kelly, ) . foreign peptide hormones, specified by cloned genes from various endocrine and exocrine cells transfected into att cells, are sorted into the secretory granules of the regulated pathway (reviewed in kelly, ) . condensed secretory proteins first accumulate in the trans-most golgi compartment of att cells. this extreme trans-compartment, which lies beyond the tppase-positive cisternae of the golgi stack and has been termed the trans-golgi network or trans-golgi reticulum (griffiths and simons, ) , is the site of formation of the secretory granules of the regulated pathway . the corona~arus mouse hepatitis virus strain a (mhv-a ) i infects and replicates efficiently in some mouse . abbre~ations used in this paper: ecm, extracellular matrix; mhv-a , mouse hepatitis virus a ; pi, protease inhibitor cocktail ( ig/ml leupeptin fibroblastic cell lines in which the maximum rate of release of progeny virions occurs well before cell lysis. the virus, which buds into pre-golgi compartments, moves through the golgi cisternae and exploits vesicles of the constitutive exocytic pathway of these fibroblastic hosts, which lack a regulated pathway, to reach the cell surface; the post-golgi vesicles filled with progeny virions that can be seen near the cell surface apparently fuse with the plasma membrane to release the virus into the medium (for review see dubois-dalcq et al., ) . mhv-a can, however, also infect att cells inefficiently (tooze and tooze, ) . in att cells, as in fibroblastic cells, the virus buds into pre-golgi compartments. the progeny virions then traverse the golgi stacks and are transported thence to the cell surface. assuming that mhv-a utilizes the constitutive pathway in att cells, as it does in fibroblasts, we can use the viral particles as a morphological marker for that pathway and use condensing secretory proteins, including acth, as a morphological marker for the regulated pathway. we can then ask at what level do the two pathways diverge? which is the last compartment to contain both virions and condensing secretory in distilled hzo, .tg/ml antipain in distilled h , p.g/ml pepstatin in h , mm bertzamidine in dimethylsulfoxide, gtg/ml phenylmethylsulfonyl fluoride in anhydrous ethanol, and u/ml trasylol); ripa, immunoprecipitation buffer ( % [wt/vol] tx- , . % lwt/vol] sds, . m nac , % [wt/vol] sodium dodeeylate, ram tris, ph . ); vsv, vesicular stomatitis virus. proteins and are the two markers segregated into different populations of transport vesicles at the exit from this compartment? we report here our attempts to answer these questions by electron microscopy and immunocytochemistry. our observations indicate that the constitutive and regulated pathways in att cells do indeed diverge at the exit from the trans-golgi network. art d v cells were propagated at ~ in a % coz incubator as previously described (tooze and tooze, ) in dme supplemented with % horse serum and . g/liter of glucose. when propagated on normal tissue culture plastic surfaces or glass att cells form dense clumps many ceils deep. when plated on plastic or glass surfaces coated with extracellular matrix (ecm; international bio-technologies ltd., jerusalem, israel), the att cells adhere and flatten within h, and grow as a monolayer. this greatly facilitates optical microscopy. in addition we have found that art cells growing on ecm am about five times more susceptible to infection with the coronavirus mhv-a than are parallel cultures growing on plastic surfaces. only between and % of the cells on plastic can be infected, whereas - % of the cells on ecm are infected. this effect is specific for mhv-a . vesicular stomatitis virus (vsv) infects essentially all the art cells in cultures growing on plastic or on ecm. we assume that when art cells grow flattened on ecm, either the numbers or the accessibility of the plasma membrane receptors exploited by the virus increases. but whatever the mechanism the increased susceptibility of the cells to infection facilitated several experiments reported here. coronavirus mhv-a , propagated on sac-cells as previously described (tooze et al, ) , was used to infect art cells at a multiplicity of infection of - pfu/cell. the virus was applied in rrd of culture medium per -ram dish for - h at ~ in a % co incubator. a stock of vesicular stomatitis virus (indiana serotype) was grown and titered on baby hamster kidney cells (bhk- ) as described in fuller et al. ( ) . infection of art cells on plastic or ecm was performed by rinsing the cell monolayer with eagle's medium supplemented with . % bsa, mm hepes, mm glutamine, tig/ml penicillin, and gg/ml streptomycin, and then applying the virus at a multiplicity of infection of pill/ cell in a volume of . ml per -mm dish. the monolayers were returned to the ~ % co incubator for h, rinsed with the supplemented eagle's medium to remove unbound virus, and then with growth medium. the cells were returned to the incubator in growth medium for the remainder of the incubation period. cells growing on glass coverslips coated with ecm were infected with mhv-a and subsequently superinfected with vsv as detailed in results. the cells were then fixed in % paraformaldehyde in pbs containing i mm mgc and i mm cach and labeled for indirect immunofluorescence microscopy using a procedure based on that of ash et al. ( ) . briefly the fixed cells were first incubated with a rabbit antiserum against vsv-g protein, generously provided by dr. b. burke (european molecular biological laboratory, heidelberg), and then with rhodamine-conjugated sheep anti-rabbit antibody. after this labeling the cells were permeabilized with . % triton x- and then incubated with an affinity-purified mouse monoelonai antibody against the e glycoprotein of mhv-a followed by fluoresceln-conjugated sheep anti-mouse antiserum. all incubations with antibodies were at room temperature for rain. the double-labeled cells were examined in a zeiss photomicroscope hi with a planapo x oil immersion objective and appropriate filters. cells grown on plastic petri dishes were fixed for h with modified karnowsky's fixative containing % glutaraldehyde. fixed cultures were postfixed in % osmium tetroxide in . m cacndylate buffer ph . for i h and then dehydrated, removed from the dishes with propylene oxide, and embedded in epon. thin sections were viewed in phuips electron microscopes after contrasting with uranyl acetate and lead citrate. we followed the procedure given by reggio et al. ( ) . the fixative used was % paraformaldehyde and . % glutaraldehydr in . m sodium phosphate, ph . . we used an affinity-purified antibody against aci~ kindly provided by dr. r. kelly (university of california, san francisco) and an aflinity-purified antiserum against the carboxy-terminal domain of the e glycoprotein mhv-a ('iboze and stanley, ) . for some of the immunoperoxidase labeling experiments the cells were grown on plastic dishes coated with ecm. after immunoperoxidase labeling the cells were dehydrated, removed from the dishes with propylene oxide, embedded in epon, and thin sectioned for electron microscopy. at -h postinfection five -mm dishes of att cells infected with mhv-a were washed three times with methionine-free modified eagle's medium (mem-met) containing . gm/liter sodium bicarbonate, and labeled with . i~ci/gl of [ s]methionlne in mem-met containing % dialyzed fcs, mm hepes, mm glutamine, gg/ml penicillin, and ~tg/ml streptomycin for rain. the incorporation of [~s]methionine was stopped by adding ml of growth medium per dish supplemented with lo times the normal amount of methionine ( . mg/ml) for the chase, or by placing the cells on an aluminium slab in ice and washing them three times with calcium and magnesium-free pbs at ~ for the zero time point. each -ram dish of cells was solubilized in ripa buffer ( % (wt/vol) tx- , . % (wt/vol) sds, . m nac , % (wt/vol) sodium dodecylate, mm tris, ph . ) containing protease inhibitors (pi) ( ig/ml leupeptin in distilled h , ~g/ml antipain in distilled h , ~g/ml pepstatin in h , mm benzamidine in dimethylsulfoxide, gg/ml phenylmethylsulfouyl fluoride pmsf in anhydrous ethanol, and u/ml trasylol) at ~ the insoluble debris was pelleted in an eppendorf centrifuge (brinkmmm instruments, inc., westhury, ny) for min at top speed at ~ the supernatant was preprecipitated with an equal volume of washed pansorbin (calbiochem, frankfurt, federal republic of germany) in ripa/pi ( % wt/vol) (see below) for rain at room temperature. the pansorbin was pelleted for rain at room temperature at , rpm in an eppendorf centrifuge, and the clarified supernatant was removed. to each sample gg of an affinity-purified antibody against the el glycoprotein of mhv-a (tooze and stanley, ) was added, and the mixture was rotated at ~ overnight. to the immune complexes, gl of washed pansorbin in ripa]pi ( % wt/vol) was added and the mixture was incubated at ~ for rain and pelleted. the pellet was then washed three times with ripa/pi buffer; once with mm tris-hcl, ph . , . % sds, mm edta plus pi; once with mm tris-hcl, ph . , . % sds, mm edta plus pi; and once with . m naci, mm tris-hcl, ph . , mm edta. the final wash was with mm tris-hc , ph . , and mm edta. all washes were for min at , rpm at room temperature. the samples were divided in two during the final wash. one-half was then resuspended in gl of sample buffer containing . m tris-hci ph ~ , mm edta, m sucrose, % (wt/vol), % (wt/vol) sds, bromopbenol blue, and mm dithiothreitol by shaking at ~ for min, followed by freezing at - ~ for min, followed by an additional -min incubation at ~ with shaking. the insoluble material was pelleted in an eppendorf centrifuge (bdnkmann instruments, inc.) for rain at top speed at room temperature, and prepared for sds-page as previously described (green et ai., ) . the other half was washed twice and resuspended in gl of mm sodium acetate, ph . and mm cac at ~ then gl of mu/gl neuraminidase from clostridium perfringens (sigma chemical co., munich, frg) in mm sodium acetate, ph . , mm caci was added, and the volume was adjusted to i~l with the same buffer. after a -h incubation rotating end over end at ~ the pansorbin was pelleted, solubilized in sample buffer to release the digested e glycoprotein, and run on a % sds-polyacrylamide gel. the pulse chase to evaluate the extent of n-glycosylatiou of vesicular stomatitis g protein in cells infected with the corouavirus mhv-a was performed essentially as described by fuller et al. ( ) . infected cells were pulsed with [~ss]methionine ( i~ci in . ml of mem lacking methionine and supplemented with . % bsa per -ram dish) for min and then chased with ml of the same medium containing . mg/ml methionine for various times. the cells were harvested by cooling on ice, rinsing twice with ice-cold pbs, and then lysed with . ml of % triton x-i in pbs cor~nining mm phenylmethylsulfouyl fluoride. the cells were scraped from the dish with a plastic pipette tip and the lysate spun at g for rain at ~ to remove nuclei. the supernatant was then subjected to two rounds of phase partitioning bordier, ) to enrich for the g protein. equal numbers of counts (corresponding to approximately one-twentieth of the total lysate) were then prepared for sds-page on a - % polyacrylamide gel. replication of mhv-a in att cells ultimately results in disruption of the golgi apparatus and death of the cells. it was necessary, therefore, to establish that at early times postinfection the golgi apparatus maintains its structure and function and is not yet disrupted by the cytopathic effects of viral replication. until at least h after infection of att cells with mhv-a the golgi apparatus maintains its stacked cistemal structure. progeny virus particles, budded into pre-golgi compartments, can be seen in the dilated rims of the golgi cisternae and accumulating in dilations of the trans-golgi network (figs. and ). from the trans-golgi network virions are transported, packed into spherical vesicles, to the cell surface. at these early times postinfection the morphology of the golgi stack and trans-golgi network is normal and, except for the presence of virions, identical to that in uninfected cells (see also figs. and ) . it is noteworthy that in att cells and also fibroblastic host cells the coronavirions invariably occur in the dilated rims of golgi cistemae and not in the comparatively flat stacked central regions of the cisternae. as figs. , , and show, virions in the trans-golgi network are often kidney shaped and have a fairly uniform and high electron density. by contrast the virions in the golgi cisternae and pre-golgi compartments are spherical structures with ribonucleoprotein core material immediately below the viral envelope and an "empty" center. this morphological maturation of the virus, which also occurs in other host-cell types (for review see dubois-dalcq et al., ) , is particularly pronounced in att cells, and at early times postinfection is strictly correlated with delivery of the viruses to the trans-golgi network. virions with the mature morphology are, therefore, a convenient and reliable morphological marker of transand post-golgi compartments. to establish that at between - h postinfection the golgi apparatus is still biochemically competent, as well as mor-phologicauy intact, we assayed its ability to o-glycosylate the e glycoprotein of mhv-a . the only known posttranslational modification of the transmembrane e glycoprotein is the addition of o-linked glycans during transit of the golgi apparatus (niemann and klenk, ) . in fibroblasts these are added posttranslationally to serine and threonine residues at the amino terminus of the protein (niemann et al., ) , which is exposed on the lumenal surface of intracel- att cells. infected cells were pulsed for rain with [ s]methionine and then chased for the times indicated, up to min. the e glycoprotein of mhv-a was then immunoprecipitated from detergent-solubilized cells and analyzed by sds-page. during the -min pulse, some of the el becomes partially glyeosylated (first lane). by min of chase virtually all of the primary translation product has chased into the two higher molecular weight forms. as shown for the -min sample, digestion with neumminidase converts the highest molecular weight form to an intermediate form, indicating that the former is the sialylated or fully glycosylated form of the protein. at -h postinfection att cells were pulsed for min with [ s]methionine and then chased for up to min. fig. shows the result. after the -min labeling the e glycoprotein is resolved on polyacrylamide gels into two bands. after a -min chase a third higher molecular weight band is resolved. these data indicate that, as in fibroblastic saccells (tooze, s. a., j. tooze, and g. warren, manuscript in preparation), the o-glycosylation of e occurs in at least two steps and that some e molecules are partially glycosylated by the end of a -min pulse. by counting the radioactivity in the three bands during the chase we found that within min of its synthesis, - % of the e is found in the highest molecular weight form and presumably is fully glycosylated. about the same percentage has been partially glycosylated. to establish that the highest molecular weight species had terminal sialic residues on the sugar chains, and therefore was fully glycosylated, an aliquot of e was taken after the -min chase. one-half was digested with neuraminidase while the other was similarly incubated but without neuraminidase. this digestion converted the highest molecular weight form to an intermediate form (fig. ) . these results show that at - -h postinfection the golgi apparatus is biochemically, as well as morphologically, intact and adds o-linked sugar chains terminating in sialic acid residues to the viral e glycoprotein. to provide further evidence that the constitutive pathway functions normally at early times after infection with mhv-a we performed the following double-infection experiment. art cells growing on ecm-coated glass coverslips were infected with mhv-a for h at "/~ the cells were then washed and incubated at c for . h until . -h postinfection. the cultures were then superinfected with vsv for i h and incubated for a further . h at ~ they were then fixed, h after infection with mhv-a and . h after infection with vsv, and processed for immunofluorescence microscopy using a double-labeling procedure with a rabbit antiserum against vsv-g protein and an affinity-purified mouse monoclonal antibody against e of mhv-a . all of the cells were infected by the vsv and expressed g protein at their cell surfaces, including the - % of cells that had been infected by mhv-a and therefore contained the coronaviral e glycoprotein accumulated in the golgi region (fig. ) . this establishes that between . -and -h postinfection with the coronavirus the constitutive exocytic pathway of att cells continues to function and can transport vsv-g protein to the plasma membrane. the same is true for cells of the murine fibroblastic line sac-(data not shown). we used the well characterized n-glycosylation of vsv g protein to test further the integrity of its transport to the plasma membrane surface in mhv-infected cells (fig. ) . knipe et al. ( ) originally showed that the increase in apparent molecular weight of the g protein marked the acquisition of terminal sialic acid, the last identified step in the golgi processing. there were no detectable differences in the kinetics of this apparent molecular weight shift in the presence or the absence of mhv infection implying that neither the rate nor extent of sialylation are affected (fig. ) . hence the processing of a plasma membrane glycoprotein seems to be unaffected by up to h of mhv-a infection. using an antibody specific for the carboxy-terminal domain of the glycoprotein e of mhv-a (tooze and stanley, ) we labeled mhv-a -infected att cells using the immunoperoxidase procedure. as fig. shows, membranes of the stacked golgi cisternae were heavily labeled on their the journal of cell biology, volume , cytoplasmic face; by contrast membranes of the trans-golgi network, identified by the presence of mature virions as well as extensive surface coats, were unlabeled. post-golgi vesicles transporting progeny virions to the cell surface and secretory granules were also unlabeled (not shown). this establishes that, at - -h postinfection, the e glycoprotein in att cells enters the exocytic pathway and reaches the membranes of the stacked golgi cisternae where it accumulates and is apparently retained, perhaps being responsible for the eventual vesicularization of the golgi cisternae. e is not, thereafter, transported as an integral protein of cellular membranes to the trans-golgi network and beyond. this pattern of selective e transport is also seen in infected sacfibroblastic cells (tooze and stanley, ) and provides further evidence that the golgi apparatus retains its functional integrity at early times postinfection. budded virions are not labeled presumably because the carboxy-terminal epitopes of e recognized by the antibody are masked by association with nucleocapsid protein and genomic rna on the inner face of the virion's envelope (tooze and stanley, ) . condensation of secretory proteins and the formation of secretory granules of the regulated exocytic pathway occurs exclusively in the trans-golgi network in uninfected att cells . progeny coronavirions that have budded in pre-golgi compartments traverse the golgi stacks and are thence transported to the plasma membrane. we therefore examined thin sections of infected att cells under the electron microscope to determine which compartments contained both condensing secretory proteins and coronavirus. in many infected cells with progeny virions in the golgi stack there was no evidence of condensing secretory proteins in the trans-golgi network (fig. ) ; since mhv-a infections are asynchronous, in some cells viral protein synthesis may have competed with cellular protein synthesis. in other cells virions and condensing secretory proteins were in different dilations of the complex trans-golgi network (fig. ) . in a third class, however, clumps of condensing secretory proteins and progeny virions occurred in the same regions of the trans-golgi network (fig. , a and b ). note that although they are in proximity in the same compartment, the virions do not occur embedded in or surrounded by condensing secretory proteins. this separation is maintained as the secretory proteins condense further into secretory granule cores. fig. b, inset shows two morphologically mature cores and two virions in the same trans-golgi compartment. as we have recently shown in art cells, detachment of secretory granules from the trans-golgi network occurs at a late stage in tooze et infection. aft cells were infected with mhv-a for . h and then with vsv for a further . h. after infection with vsg the ceils were pulse-chased with [ ss]methionine (see materials and methods) and the g protein of vsv and the e protein of mhv-a were then enriched by tx-u phase partition as described in materials and methods. the increase in apparent molecular weight of g protein, which indicates its sialylation, was first detected between and min and occurs with the same kinetics in the presence or absence of mhv-a infection. the mhv-a e protein is seen in the infected cells (+ lanes) in a longer exposure of the bottom portion of the gel. the e is the middle band of the three bands within the bracket. this establishes that the e protein of mhv-a was being synthesized at the same time as the vsv g protein. the maturation of the cores, after they have become condensed and have assumed a spherical or ovoid shape with a well-defined surface. the two cores shown in fig. b , inset are at a stage at which they detach from the trans-golgi network. note also that there are no virions in the secretory granule close to the golgi stack shown in fig. . we have never seen virions embedded in secretory granule cores either in the golgi region or at the cell periphery, where the mature granules accumulate. fig. shows condensing secretory protein in the trans-golgi network of uninfected att cells. comparison of fig. , a and b with fig. shows that the only detectable change at these early times after infection with mhv-a is the presence of virions in the golgi complex. clathrin is a marker for the trans-side of the golgi apparatus (for review see griffiths and simons, ) and, as we have previously shown by inununocytochemistry , in uninfected att ceils the trans-golgi network and immature secretory granules in the golgi region often have clathrin coats on parts of their surface (fig. ) , and are the site of budding or fusion of clathrin-coated vesicles. coats that resemble clathrin in morphology, but were not positively identified as such by immunocytochemistry, were also often present in infected cells on the cytoplasmic face of parts of the trans-golgi network containing virions and on peri-golgi vesicles with virions (figs. a and ). the observations presented above indicate: (a) that virions and secretory proteins traverse the same golgi stacks and accumulate in the trans-golgi network; we have shown previously by immunocytochemistry that acth is present in all cisternae of the golgi stacks of att cells (tooze and tooze, note that only morphologically mature virions are found on the trans-side of the golgi complex. many dilations of the trans-golgi network contain either condensing secretory proteins (arrowheads) or condensed virions (/arge arrows). when both virions and condensing secretory proteins share the same dilation, they are well separated (lower lej? and inset). we have previously shown that the coat on the cytoplasmic surface of the trans-golgi network (between the small arrows) is clathrin . note the coated vesicle budding from or fusing with the trans-golgi network (curved arrow). (b) in this example the trans-golgi network contains condensing secretory protein (arrows) and one dilation also contains virions (open arrow). the virions have the mature morphology and are well separated from the condensing secretory protein. note that the latter is more uniformly compact than in a, and is beginning to assume the form of a secretory granule core. (insert) part of the trans-golgi network in another infected cell in which two mature virions (arrow) and two aggregates of secretory proteins that have condensed into ovoid granule core structures (arrowheads) are in the same compartment but still quite separate in the sense that there are no virions within the cores. bars, . lun. figures - . (fig. ) condensing secretory proteins in the trans-golgi network of an uninfected att cell. the condensing secretory protein (open arrows) is within two dilations of the trans-golgi network. note the clathrin coat on parts of the membrane (between the arrows) and also coated vesicles. interestingly, on the cis-side of the golgi stack a sheet of rough endoplasmic reticulum is differentiated in one region into annulate lamellae (al) and in another into a transitional element (te). apparently two morphological differentiations of the rough endoplasmic reticulum can occur in very close proximity in these tumor cells. bar, . inn. (fig. ) condensed virions in the trans-golgi network. note the prominent coat on the cytoplasmic face of one of the compartments enclosing a mature virion (arrow). the coat has the characteristic morphology of clathrin. bar, . ima. (fig. ) part of an infected cell exposed to i~m chloroquine from . -to -h postinfection. note the large accumulations of condensed virions in the trans-golgi network (arrows) and immature uncondensed virions in the golgi cisternae (arrowheads). post-golgi transport vesicles packed with mature virions can be seen below the plasma membrane (open arrows). bar, . ~tm. figure . post-golgi transport vesicles and secretory granules close to the lower surface of infected att cells, growing on extracellular matrix, after immunoperoxidase labeling with anti-acth antibody. in a note that the secretory granules are heavily labeled for acth (arrows). a completely unlabeled transport vesicle packed with virions (arrowhead) is close to the lower cell surface, which lies on the layer of extra~llular matrix (ecm). b shows, near the lower surface of a cell growing on ecm, one of the extremely rare classes of post-golgi transport vesicles which contain both virions (arrows) and a clump of condensed secretory protein that labels heavily with anti-acth antibody (arrowhead). some of the immunoperoxidase reaction product has diffused to coat the virions and the membrane of the vesicle. bars, . lain. ). (b) that within the trans-golgi network secretory proteins condense into granule cores that exclude virions. (c) beyond the trans-golgi network virions are packed in vesicles lacking condensed secretory proteins. we attempted to confirm this sorting ofvirions from secretory proteins entering granules of the regulated exocytic pathway. infected cultures were labeled using an affinity-purified antiserum against acrh by the immunoperoxidase method, and the post-golgi exocytic compartments were examined. this labeling reveals acth in all compartments of the exocytic pathway from the rough endoplasmic reticulum onwards . the post-golgi compartments that occurred below the plasma membrane at the cell periphery and lacked coats on their cytoplasmic surface fell into three categories: (a) labeled secretory granules that did not contain coronavirions and were identical to secretory granules in uninfected cells (fig. a ) ; (b) post-golgi vesicles packed with many virions but either not labeled for acth (fig. a) or with traces of reaction product on the vesicles inner surface (in this class of vesicles there was no evidence of clumps of condensed secretory proteins); (c) extremely rare vesicles containing both virions and clumps of condensed secretory proteins (fig. u b) that resemble in their labeling for acth the core material of secretory granules in both infected and uninfected cells (compare fig. a and b) . this third class comprised considerably < % of the total post-golgi vesicles containing mature virions. the fact that we have never observed post-golgi vesicles containing virions and aggregates of condensed secretory proteins in thin sections of conventionally fixed and embedded cells is further evidence of their rarity. their detection is dependent upon labeling the cells for acth by the immunoperoxidase procedure. the extreme trans-golgi cisterna is the most acidic compartment of the golgi apparatus (anderson and pathak, ) , but its ph is believed for a variety of reasons not to be below (anderson and pathak, ; griffiths and simons, ) . in att cells, as we have recently shown (unpublished results), the trans-golgi network is much less heavily labeled with -( , -dinitroanilino- "amino-n-methyldipropylamine (anderson et al., ) than are the secretory granules whose ph we estimate to be between . and . . to test whether the lower ph of this trans-most compartment of the golgi apparatus is necessary for the morphological maturation of coronavirus particles, we exposed infected cultures to ttm chloroquine from . - -h postinfection and then fixed and processed them for electron microscopy. as fig. shows, although exposure to chloroquine causes dilation of the trans-golgi network, the virions that accumulated in this compartment in the presence of this weak base were as compact and electron dense as those in untreated cells. we obtained the same result when infected cultures were exposed from . - -h postinfection to mm ni-i c (data not shown). it is also noteworthy that ~tm chloroquine did not inhibit the formation of the post-golgi vesicles packed with mature virus particles that occur close to the plasma membrane (fig. ) . however, in both infected and uninfected cells in the same cultures exposed for . h to either ttm chloroquine or d mm ni-i c there was little if any condensing secretory protein in the trans-golgi network of the many sections we examined (data not shown). exposure of the cells to these lysomotrophic agents apparently, therefore, had a differential effect inhibiting condensation of secretory proteins but not inhibiting the morphological maturation of the coronavirions. viral infection is a convenient way in which to introduce foreign genes into cells to study the synthesis, intracellular transport, and sorting of foreign proteins. the approach has one chief disadvantage, namely the cytopathic effects that accompany viral replication. this problem can, however, be largely avoided by studying the cells early after infection before the onset of the cytopathic changes. in the case of att ceils infected by mhv-a we believe that at - -h postinfection the exocytic pathways, and in particular the golgi apparatus, still function normally for the following reasons: (a) the golgi stack has a normal morphology. (b) the golgi enzymes responsible for o-linked glycosylation are functional. (c) vsv-g protein is n-glycosylated in the same way in both uninfected and mhv-a -infected att cells. furthermore the g protein is transported to the plasma membrane in both uninfected and mhv-a -infected cells. therefore the constitutive exocytic pathway is fully functional after infection with the coronavirus. (d) in infected cells, as in uninfected cells , the first compartment in which condensed secretory proteins occur is the trans-golgi network, indicating that secretory proteins of the regulated exocytic pathway are being synthesized, transported, and condensed normally. (e) the e envelope glycoprotein of the virus is not transported beyond the golgi stack, except as an integral component of the envelopes of progeny virions (fig. ) . by contrast the second viral envelope glycoprotein e is transported to the cell surface, as we have previously shown (tooze and tooze, ) . therefore the golgi apparatus in mi-iv-a -infected ceils at - -h postinfection can sort one coronaviral glycoprotein from the other. by these five criteria the golgi apparatus maintains its structural and functional integrity until at least -h postinfection with mhv-a . in this context it is interesting to note that in cells infected with uukuniemi virus (a bunyavirus) the constitutive exocytic pathway also continues to transport semliki forest virus glycoprotein to the plasma membrane even after the golgi stack has vacuolized as a result of the uukuniemi virus infection (gahmberg et al., ) . in this case the biochemical integrity of the golgi complex survives longer than its morphological integrity. unlike many other viruses, mhv-a does not, early in infection, efficiently inhibit cellular protein synthesis. there is nevertheless, during coronavirus infections, a progressive competitive inhibition of host-protein synthesis (tooze, s. a., unpublished data) . since the early stages of mhv-a infections are by no means synchronous, in any one culture at - -h post infection the extent of virus budding varies considerably from infected cell to cell. however, in most in-fected cells the number of virions increases rapidly after h of infection. concomittantly the number of infected cells with condensing secretory proteins in their trans-golgi network rapidly declines and acth can no longer be detected by the immunoperoxidase method in cisternae of the endoplasmic reticulum (data not shown). our observations depended, therefore, on examining the cells during the relatively short time after the budding of progeny virions had begun and before cellular protein synthesis was inhibited. in the pulse-chase experiment reported here, two glycosylated forms of the mhv-a e glycoprotein were resolved (fig. ) . however, the simplicity of the gel electrophoretic pattern of glycosylated e molecules from att cells contrasts with the ladder of variously glycosylated molecules obtained by gel electrophoresis of e isolated from infected murine fibroblasts (niemann et al., ; tooze, s. a., j. tooze, and g. warren, manuscript in preparation) . in murine fibroblasts the o-glycan chain added to e has the composition n-acetylgalactosamine-galactose-sialic acid and in ~o % of these chains an additional sialic acid residue is added to the n-acetylgalactosamine residue; moreover an average three of the four amino-terminal acceptor amino acids (nh -ser-ser-thr-thr) are glycosylated (niemann et al., ) . our data indicate that in att cells the o-glycosylation of e is considerably simpler than in fibroblastic cells. different patterns of o-glycosylation of the same protein in different cell types is, however, not unprecedented. cummings et al. ( ) have shown that the low density lipoprotein receptor is differently o-glycosylated in different cells. we have now shown the same to be true of the e glycoprotein of mhv-a . in att cells, at - -h postinfection with mhv-a , both progeny virions and condensing secretory proteins accumulate in the trans-golgi network. within that compartment condensed secretory proteins, perhaps by a phase separation process, form the secretory granule cores that exclude virions. on the other hand, the post-golgi vesicles transporting mature virions were either not labeled or only very weakly labeled by the immunoperoxidase reaction using antibody that recognizes both acth and its precursor, proopiomelanocortin. whether or not there exists in infected cells a further class of vesicles that constitutively transport uncleaved proopiomelanocortin but not virions is an open question. furthermore chloroquine, at lxm, does not block the formation of the vesicles that transport virions to the cell surface. these properties are consistent with the interpretation that post-golgi vacuoles transporting progeny mhv-a are part of the constitutive exocytic pathway. using the presence of virions as a morphological marker of the constitutive pathway in infected att cells, we can conclude that the trans-golgi network is the compartment from which the constitutive and regulated pathways diverge in these cells. the fact that extremely rare post-golgi vesicles located at the cell periphery and packed with virions also contain clumps of condensed secretory protein indicates that although sorting of condensed secretory proteins into the cores of secretory granules of the regulated pathway is efficient, it is not absolutely rigorous. a number of groups have invested a great deal of effort in attempting to identify the site of divergence of intracellular transport pathways to the cell surface. this compartment will be the major site for the sorting of newly synthesized plasma membrane and secretory proteins. the evidence accumulated so far has narrowed down but not identified the site of sorting either for components destined for the constitutive and regulated pathways of secretion or for membrane proteins destined for different plasma membrane domains (reviewed in griffiths and simons, ) . biochemical and morphological studies (rindler et al., ) indicate that proteins destined for separate cell surfaces colocalize throughout the golgi stack and that they separate before reaching the plasma membrane (kelly, ; marlin and simons, ; misek et al., ; pfeiffer et al., ) . griffiths and simons ( ) proposed that the sorting of these components occurs in the trans-golgi network since components to be sorted are in contact during the last identifable steps of golgi processing . a critical test of this hypothesis has been difficult since it requires simultaneous observation of components destined for the two pathways. the experiment must also be done with sufficient time resolution to rule out the possibility of transient formation of a compartment beyond the trans-golgi network which fissions to produce sorted transport vesicles. the system we use here fulfills these requirements because it allows us to observe directly the separation of the constitutive and regulated exocytic pathways. as far as we are aware, these observations are the first direct visualization of the sorting at the exit of the trans-golgi network of components taking different routes to the cell surface. they rely on having readily distinguishable morphological markers for each pathway, and they establish for this system that there is no sorting compartment beyond the trans-golgi network. in both att cells and in fibroblastic host cells (our unpublished observations) the post-golgi vesicles that apparently transport progeny coronavirus to the cell surface are large structures, holding several virions each ~ , a in diameter. these vesicles occur close to the plasma membrane and lack clathrin coats on their cytoplasmic surface. in uninfected cells there are no post-golgi vesicles of this size; no doubt the vesicles responsible for the constitutive transport of endogenous proteins are much smaller. with the exception of hepatocytes, which exocytose v. low density lipoprotein particles, mammalian cells normally never transport large particles along the entire exocytic pathway but only membrane proteins and soluble proteins. however, the very fact that cells of both epithelial (att ) and fibroblastic (sac-, cll cells) origin can accomodate to the transport of coronavirions from pre-golgi compartments to the cell surface proves that the transport vesicles at all stages of the exocytic pathway have the capacity to enlarge to carry large particulate macromolecular assemblies such as virions, even though normally they are never called upon to do so. it would be of interest to determine by immunocytochemical means whether or not the constitutive pathway vesicles that transport progeny virions from the trans-golgi network to the cell surface in either sac-or att cells also contain constitutively secreted cellular proteins such as laminin. such experiments are planned. the striking and irreversible morphological maturation of coronavirions, which takes place during their transit of the golgi apparatus in several types of host cells (see dubois-dalcq et al., ) , occurs in art cells at early times after infection exclusively in the trans-golgi network. this morphological maturation is not, however, inhibited by ~tm chloroquine, or by the presence of mm ni-hc . it cannot, therefore, be dependent on the lower ph in the transmost cisterna of the golgi apparatus compared with the earlier compartments (anderson and pathak, ) . by contrast, condensation of secretory proteins and their packaging into secretory granules in these cells seems to be ph dependent, since moore et al. ( ) established that ~tm chloroquine diverts acth from the regulated to the constitutive pathway. consistent with these results we observed little, if any, condensing secretory proteins in the trans-golgi network of either uninfected or infected art cells after their exposure to ~tm chloroquine or mm ni-hci for . h (data not shown). in conclusion, the trans-golgi network of att cells appears to be the site of sorting of material destined for the constitutive and regulated exocytic pathways, which diverge at the exit from this compartment. visualization of acidic organelles in intact cells by electron microscopy vesicles and cisternae in the trans golgi apparatus of human fibroblasts are acidic compartments antibody-induced linkages of plasma membrane proteins to intracellular actomyosin-containing filaments in cultured fibroblasts phase separation of integral membrane proteins in triton x- solution the exocrine protein trypsinogen is targeted into the secretory granules of an endocrine cell line: studies by gene transfer biosynthesis of n-and o-linked oligosaccharides of the low density lipoprotein receptor assembly of the coronvidrae an enzymatic assay reveals that proteins destined for the apical or basolateral domains of an epithelial cell share the same late golgi compartments vesicular stomatitis virus infects and matures only through the basolateral surface of the polarized epithelial cell line efficient transport of semliki forest virus glycoproteins through a golgi complex morphologically altered by uukuniemi virus glycoproteins passage of viral membrane proteins through the golgi complex the trans golgi network; sorting at the exit site of the golgi complex two distinct intracellular pathways transport secretory and membrane glycoproteins to the surface of pituitary tumor cells pathways of protein secretion in eukaryotes localization of two intracellular forms of the vesicular stomatitis viral glycoprotein sorting of a plasma membrane protein occurs before it reaches the cell surface in cultured epithelial cells biogenesis of epithelial cell polarity: intracellular sorting and vectorial exocytosis of an apical plasma membrane glycoprotein chloroquine diverts acth from a regulated to a constitutive pathway in art cells secretory protein targeting in a pituitary cell line: differential transport of foreign secretory proteins to distinct secretory pathways the carbohydrates of the o-glycosidically linked oligosaccharides of glycoprotein el coronavirus glycoprotein el, a new type of viral glycoprotein intracellular sorting and basolateral appearance of the g protein of vesicular stomatitis virus in mdck cells use of immunocytocbemical techniques in studying the biogenesis of cell surfaces in polarised epithelia viral glycoprotein destined for apical or basolateral plasma membrane domains traverse the same golgi apparatus during their intracellufar transport in doubly infected mdck cells identification of two epitopes in the carboxy terminal amino acids of the el glycoprotein of murine hepatitis virus a by using hybrid proteins infection of art murine pituitary tumour cells by mouse hepatitis virus strain a : virus budding is restricted to the golgi region clathrin-coated vesicular transport of secretory proteins during the formation of acth-containing secretory granules in art cells replication of coronavirus mhv-a in sac-cells: determination of the first site of budding of progeny virus we thank our colleagues stella hurtiey, jean gruenberg, and gareth griffiths (european molecular biological laboratory, heidelberg) for their constructive comments on the manuscript, and ines benner who patiently and skillfully typed this manuscript.received for publication march , and in revised form may . key: cord- -oq zrnuv authors: taguchi, f.; matsuyama, s.; saeki, k. title: difference in bgp-independent fusion activity among mouse hepatitis viruses date: - - journal: arch virol doi: . /s sha: doc_id: cord_uid: oq zrnuv mouse hepatitis virus (mhv) utilizes a mouse biliary glycoprotein (bgp) as a receptor. co-cultivation of mhv-nonpermissive hamster bhk cells devoid of mouse bgp with mouse dbt cells infected with mhv-a or jhmv induces syncytia formation on bhk cells (bgp-independent fusion). this study shows the difference in bgp-independent fusion activity among various mhv strains. under a phase contrast microscopy, jhmv (cl- , sp- ) induced the bgp-independent syncytia on bhk cells similar to those observed on dbt cells, while such syncytia were not seen with the infection of other mhv strains (mhv- , mhv- , mhv-a , mhv-s, srr , srr and srr ). tiny syncytia detectable only by immunofluorescence were produced with the latter mhv strains except for srr which failed to produce syncytia. mhvs except for srr grew in bhk cells after bgp-independent infection. the bgp-independent fusion by jhmv was inhibited either by anti-s or anti-s antibodies. these results showed that the jhmv spike protein had a remarkably high bgp-independent fusion activity. (s n ) in which a conformational structure plays an important role [ , ] , while the s is not involved in this activity [ ] . several different regions and amino acid residues in the s have been reported to be important for fusion activity [ , , , ] , although it is unclear which of those are actually involved in the fusion event. biliary glycoprotein (bgp) , a member of carcinoembryonic antigen (cea) gene family, serves as a highly functional receptor for mhv [ ] . several different bgp isoforms are generated by an alternative splicing [ ] and expressed in the liver, brain and other organs. the bgp has two allelic forms. bgp a and bgp b , expressed in mhv-susceptible balb/c and resistant sjl mice, respectively [ , ] . both of these proteins retain the mhv receptor function, although the bgp a has much higher virus-binding activity than does the bgp b [ ] . such difference could result in the difference of mhv susceptibility in the whole animal level [ ] . two other species of protein, bgp [ ] and brain specific cea [ ] , both of which are cea members, have been reported to work as mhv receptor in mice. mhv- (jhmv) and mhv-a are known to infect cells lacking mhvspecific receptors, i.e., bgp-independent infection [ , ] . these viruses fail to directly infect bgp-negative bhk (bhk) cells, but infect them after cocultivation with the bgp-positive dbt cells infected with these viruses [ , ] . the s protein is involved in the bgp-independent infection and fusion, since the antibodies against the s protein prevented the infection [ ] and the expression of the s protein alone induced fusion on cells devoid of bgp [ , ] . in this paper, we compared the bgp-independent fusion activity of various mhv strains. mhv strains mhv- , mhv- , mhv-s, mhv-a [ , ] , jhmv cl- [ ] , sp- [ ] as well as different soluble receptor-resistant (srr) variants derived from cl- [ ] were used in this study. of these the amino acid sequences of a [ ] and jhmv cl- [ ] s proteins have been reported. sp- s has a -amino acid deletion in the hypervariable region of the cl- s [ ] . srr and srr have amino acid mutations at positions (leu→phe) and (cys→his) in the s , respectively. srr has a mutation at position of (leu→his) in the s . to see the bgp-independent fusion activity, dbt cells cultured in well-plate (corning) were infected with those viruses at an m.o.i. and incubated at • c for h. after times of washing with dulbecco's modified minimal essential medium (dmem, nissui, tokyo), infected cells were cultured in ml of dmem supplemented with % fetal calf serum (fcs, gibco) for to h. the cells were then trypsinized and cells were overlaid onto bhk cell monolayer cultured in well-plate ( × cells). the syncytia of bhk cells were examined by a phase contrast microscopy during h after overlay of mhv-infected dbt cells. viral antigen was also examined by immunofluorescence using anti-s mabs [ ] . only jhmv cl- and sp- induced the bgp-independent syncytia on bhk cells detectable by a phase contrast microscopy. the syncytia could be observed from about h after overlay. the syncytia formed by cl- were significantly larger than those by sp- ( fig. a and b ). this size difference observed on bhk cells between cl- and sp- was in good agreement with the size difference in anti-s mabs and anti-mouse igg labelled with fitc for immunofluorescence plaque produced on dbt cells [ ] . interestingly, no syncytia formation was demonstrated on bhk cells with other strains or srr mutants under the phase contrast microscopy, though all of them produced syncytia on bgp-positive dbt cells [ ] . mhv-a induced tiny syncytia on bhk cells comprising roughly to cells detectable only by immunofluorescence (fig. c) . other mhv strains, mhv- , mhv- and mhv-s produced tinier syncytia consisting of less than cells ( fig. d and e) . srr mutants have also failed to induce syncytia comparable to those produced by wild type cl- . srr caused syncytia including about to cells (fig. g ). srr showed very tiny syncytia consisting of less than cells. no obvious syncytia was observed on bhk cells overlaid with srr -infected dbt cells. single cells were mhv antigen-positive (fig. f ). these single cells presumably represented srr -infected dbt cells overlaid on bhk cell monolayer. we have examined whether these viruses multiply in bhk cells. dbt cells infected with jhmv-cl- , mhv-a and srr mutants were either overlaid onto bhk cell monolayer as described above or allowed to seed alone in the -well plate and virus growth in these cells was examined. as shown in fig. , all viruses except srr grew significantly higher in bhk cells co-cultured with mhv-infected dbt cells than in dbt cells alone, indicating that all viruses examined but srr multiplied in bhk cells. though cl- produced significantly larger syncytia on bhk cells in a bgp-independent manner as shown in fig. , it replicated to almost the same extent as other viruses. no significant difference in the growth on bhk cells between cl- and other mhv strains can not be accounted for at present. the cl- s protein could have a very strong bgp-independent fusion activity, but its replication in bhk cells could be less efficient than the other mhv strains. no replication of srr in bhk may be due to its failure to spread from infected dbt cells to bhk cells. next we have examined whether the anti-s and anti-s antibodies inhibit the bgp-independent fusion. we used neutralizing mabs , and recognizing the s n as well as mabs and recognizing other regions in the s [ ] . anti-s antibodies with neutralizing activity were produced using rabbits. rabbits were immunized with a synthetic peptide consisting of amino acid sequence nespllgcigstcaed which encompassed an immunodominant epitope in the s recognized by a neutralizing mab b . [ ] . the rabbit sera were then affinity-purified using the synthetic peptide. bhk cell monolayer overlaid with cl- -infected dbt cells that produced about syncytia was cultured in -fold serial dilutions of the anti-s and anti-s antibodies. cells were stained with giemsa solution at to h after infection and the syncytium number was counted under the light microscopy. the inhibition of syncytium formation was calculated in comparison with the syncytium number formed in the absence of neutralizing antibodies. standard neutralization test using cl- was also performed with these antibodies on dbt cells as described previously [ ] . as shown in fig. , all of these antibodies showed fusion inhibition (fi) activity, though the fi titers were variable; anti-s mabs showed to higher fi and viral neutralization activities compared with anti-s antibodies. the antibodies with higher neutralization titer exhibited the higher fi titer. this could imply that fig. . growth of mhv in bhk cells after bgp-independent infection. ten thousands of dbt cells infected with jhmv cl- , mhv-a , srr , srr or srr were overlaied on × bhk cells or allowed to seed alone in -well plate. at intervals after overlay, virus titers in the cultures were measured by plaque assay the attachment of the s protein to an unidentified receptor on bhk cells, which is conceibably prevented by the anti-si mabs ( , and ) recognizing the receptor-binding domain on the s, is an inevitable step for the bgp-independent fusion events or that the s is more profoundly involved in the bgp-independent fusion activity than the s . these results appear to suggest that both s and s are involved in the bgp-independent fusion activity. on dbt cells mhv-a and mhv- produce large plaques similar to those produced by cl- , while other mhvs used in this study produce smaller plaques similar in size to those produced by sp- . the plaque is composed of a single, large syncytium. this indicates that all these mhvs have a potential to induce fusion on bgp-positive cells. present study showed that jhmv (cl- and sp- ) could induce syncytia on bhk cells detectable by microscopy, whereas the other mhvs and srr mutants failed. these findings have indicated that jhmv, particularly cl- with a large s protein [ ] , has a strong bgp-independent fusion activity. the high fusogenicity of jhmv was also reported by gallagher [ ] who described that exogenously added jhmv viral particles had a strikingly high fusion activity on bgp-positive cells as compared with mhv-a . the srr mutants showed extremely reduced bgp-independent fusogenicity in spite of their high fusogenicity on bgp-positive dbt cells [ ] . this suggests that mutated amino acids in the srr mutants could play a critical role for the bgp-independent fusion. srr with a mutation at a position in the s has a low receptor-binding activity [ ] which also may affect on the fusion activity in bgp-negative cells. srr and srr have mutations in the amino acids at positions and in the s , respectively [ ]. gallagher et al. reported that both of these residues were involved in the fusion on bgp-positive cells [ , ] . their mutant virus which had mutations at a position of as well as two other positions showed an acid-ph dependent fusion activity on bgp-positive cells [ ] . this virus had a reduced fusion activity on bgp-negative cells [ ] . gallagher also reported the importance of cystein residue at a position of jhmv s protein for syncytia formation on bgp-positive cells using anti-cystein reagent [ ] . both amino acids at positions and are conceivably important for overall fusion activity, whether it is bgp-dependent or bgp-independent. nash and buchmeier reported that the mab to bgp prevented mhv- (jhmv) infection on bgp-positive cells, yet it failed to prevent syncytia formation in infectious center assay on mouse dbt cells [ ] . this implies that the bgp is not necessary for syncytia formation. in the present study, we showed all the viruses except for jhmv failed to produce microscopically-detectable syncytia on bhk cells, while they produced syncytia on dbt. these results could suggest that the bgp plays an important role in the syncytia formation by mhv strains but jhmv. expression of the s proteins of these mhvs in bgp-positive and bgp-negative cells will clarify the importance of bgp in syncytia formation. present study suggested that both s and s were critical for the bgp-independent fusion. the s could be important for the binding to the molecule on bhk cells which is an alternative receptor for mhv. the s protein-receptor interaction is an inevitable step prior to fusion events. such receptor is conceivably not so efficient as the bgp and therefore work as a receptor only when large amounts of s protein expressed on mhv-infected dbt cells are in contact with it [ ] . it is interesting to note that the recombinant mhv between jhmv and mhv-a arisen in a mixed culture of dbt and bhk cells as well as mutants derived from persistent infections are able to infect bhk and other non-murine cells devoid of murine bgp [ , , ] . some of those mutants have recently been reported to utilize human cea and bgp as functional receptors [ ] . the receptor protein on bhk cells for these mutants may also interact with the jhmv s protein expressed on dbt cells. presistent infection promotes cross-species transmissibility of mouse hepatitis virus episodic evolution mediates interspecies transfer of a murine coronavirus mutational analysis of the murine coronavirus spike protein: effect on cell-to cell fusion a pregnancy-specific glycoprotein is expressed in the brain and serves as a receptor for mouse hepatitis virus cloning of the mouse hepatitis virus (mhv) receptor: expression in human and hamster cell lines confers susceptibility to mhv several members of the mouse carcinoembryonic antigen-related glycoprotein family are functional receptors for the coronavirus mouse hepatitis virus-a murine coronavirus membrane fusion is blocked by modification of thiols buried within the spike protein cell receptor-independent infection by a neurotropic murine coronavirus alteration of the ph dependence of coronavirus-in duced cell fusion: effect of mutations in the spike glycoprotein utility of mouse cell line dbt for propagation and assay of mouse hepatitis virus neutralization and fusion inhibition activities of monoclonal antibodies specific for the s subunit of the spike protein of neurovirulent murine coronavirus jhmv cl- variant localization of neutralizing epitopes and the receptor-binding site within the amino-terminal amino acids of the murine coronavirus spike protein roles in cell-cell fusion of two conserved hydrophobic regions in the murine coronavirus spike protein amino acid sequence of a conserved neutralizing epitope of murine coronaviruses primary structure of the glycoprotein e of coronavirus mhv-a and identification of the trypsin cleavage site spike glycoprotein-mediated fusion in biliary glycoprotein-independent cell-associated spread of mouse hepatitis virus infection bgp , a new member of the carcinoembryonic antigen-related gene family, encodes an alternative receptor for mouse hepatitis viruses mouse susceptibility to mouse hepatitis virus infection is linked to viral receptor genotype difference in virus-binding activity of two distinct receptor proteins for mouse hepatitis virus identification of spike protein residues of murine coronavirus responsible for receptor-binding activity by use of soluble receptor-resistant mutants the murine coronavirus mouse hepatitis virus strain a from persistently infected murine cell exhibits an extended host range nucleotide sequence of the gene encoding the surface projection glycoprotein of coronavirus mhv-jhm coronaviruses: structure and genome expression proteolytic cleavage of the e glycoprotein of murine coronavirus: activation of cell-fusing activity of virions by trypsin and separation of two different k cleavage fragments analysis of the receptor binding site of murine coronavirus spike glycoprotein the s subunit of the murine coronavirus spike protein is not involved in receptor binding comparison of six different murine coronavirus jhm variants by monoclonal antibodies against the e glycoprotein molecular cloning and expression of a spike protein of neurovirulent murine coronavirus jhmv variant cl- characterization of a variant virus selected in rat brain after infection by coronavirus mouse hepatitis virus jhm the receptor for mouse hepatitis virus in the resistant mouse strain sjl is functional: implications for the requirement of a second factor for viral infection technical assistance by nozomu ishida and reading manuscript by noralyn dudt are greatly appreciated. this work was financially supported by grants from science and technology agency and the ministry of education, science and culture of japan. authors' address: dr. f. taguchi, national institute of neuroscience, ncnp, - - ogawahigashi, kodaira, tokyo , japan.received february , key: cord- -vtrfqozl authors: makino, shinji; shieh, chien-kou; soe, lisa h.; baker, susan c.; lai, michael m.c. title: primary structure and translation of a defective interfering rna of murine coronavirus date: - - journal: virology doi: . / - ( ) - sha: doc_id: cord_uid: vtrfqozl abstract an intracellular defective-interfering (di) rna, disse, of mouse hepatitis virus (mhv) obtained after serial high multiplicity passage of the virus was cloned and sequenced. disse rna is composed of three noncontiguous genomic regions, representing the first nucleotides of the fend, an internal nucleotides of the polymerase gene, and nucleotides from the ′ end of the parental mhv genome. the disse sequence contains one large continuous open reading frame. two protein products from this open reading frame were identified both by in vitro translation and in di-infected cells. sequence comparison of disse and the corresponding parts of the parental virus genome revealed that disse had three base substitutions within the leader sequence and also a deletion of nine nucleotides located at the junction of the leader and the remaining genomic sequence. the ′ end of disse rna was heterogeneous with respect to the number of ucuaa repeats within the leader sequence. the parental mhv genomic rna appears to have extensive and stable secondary structures at the regions where di rna rearrangements occurred. these data suggest that mhv di rna may have been generated as a result of the discontinuous and nonprocessive manner of mhv rna synthesis. mouse hepatitis virus (mhv), a member of the coronaviridae, contains a single-stranded, positive-sense rna of approximately x lo da (lai and stohlman, ; wege eta/., ) . in infected cells, the genomic rna of mhv is first translated into an rna-dependent rna polymerase (brayton et al., mahy et a/., ) which is responsible for the synthesis of a genomic-sized negative-stranded rna (lai et a/., b) . the negative-stranded rna then serves as the template for the synthesis of six subgenomic and a genomic-sized mrna lai et a/., ) . these mrnas are arranged in the form of a ' coterminal "nested" set, i.e., the sequence of each mrna is contained entirely within the next larger mrna (lai et a/., ; leibowitz et al., ) . in addition, each mrna has a common leader sequence, which is derived from the ' end of the genome (lai et a/., a (lai et a/., , spaan et al., ) . several pieces of evidence demonstrated that mhv utilizes a novel mechanism of leader rna-primed transcription, in which a free leader rna species derived from the ' end of genomic rna is utilized as a primer for the transcription of subgenomic mrnas (baric eta/., (baric eta/., , makino et a/., b) . another unusual feature of coronavirus rna synthesis is that the virus undergoes rna-rna recombination at a very high frequency (makino eta/., a) . the ' to whom requests for reprints should be addressed. unusually high frequency, approaching % under some circumstances (makino et al., a) , of coronavirus rna recombination suggests that discontinuous rna transcripts might be generated during coronavirus rna synthesis. these incomplete rna intermediates may rejoin the original or different rna template to continue rna synthesis, resulting in rna recombination in the latter case. the detection of such rna intermediates in mhv-infected cells (baric et a/., (baric et a/., , suggests that coronavirus genomic rna synthesis involves a discontinuous and nonprocessive mechanism, which may account for the high frequency of recombination via a copy choice mechanism. defective-interfering (di) particles are naturally occuring deletion mutants that have been described for many virus groups. characteristically, di particles (a) lack part of the viral genome, (b) contain normal viral structural proteins, (c) replicate only with the aid of a helper standard virus, and (d) interfere with replication of homologous standard virus. deletion of genomic sequence can occur in various regions of the genome; however, all of the di rnas apparently retain signals for rna replication since they can be replicated in the presence of helper virus. the generation of di rna can be viewed as the result of abnormal rna replication or illigitimate rna recombination. therefore, the structure of di rna is of particular interest in elucidation of the mechanism of viral rna replication and recombination. we have previously reported the generation of di particles during high multiplicity passages of the jhm - $ . copyright q by academic press. inc. all rights of reproduction in any form reserved. in a % agarose gel without denaturation. numbers , , , , and represent the major mhv-jhm-specific mrna species. strain of mhv (mhv-jhm) (makino eta/., a) . in diinfected cells, the synthesis of most of the standard viral mrnas is inhibited. instead, three distinct virusspecific rna species could be detected (makino eta/., ) (fig. ). the first species, dlssa, is equivalent to dl virion rna in length and is eventually incorporated into virus particles. this rna differs from the standard virus genome in that it contains multiple deletions distributed throughout the genome, except for the ' and ' ends of the genomic rna (makino et a/., ) which encode rna polymerase (gene a) and nucleocapsid (n) protein, respectively. surprisingly, dlssa rna can replicate by itself in the absence of helper virus infection, suggesting that dlssa codes for functional rna polymerases (makino et a/., ) . thus, dlssa is not a defective rna in a strict sense. the second major rna species found in di-infected cells is indistinguishable from the mrna made by the standard virus. the synthesis of this mrna and its product n protein is not inhibited in di-infected cells. the third rna species is a novel single-stranded polyadenylated di rna species of varying size. oligonucleotide fingerprinting studies suggest that it represents se-quences derived from various noncontiguous parts of the genome. the size of this rna varies with the di passage level (makino et a/., ) . one of these rnas, dlsse, which is the smallest di rna detected, has been analyzed in greater detail (makino et al., ) . in contrast to dlssa, dlsse rna synthesis requires helper virus coinfection (makino et al,, ) . only a trace amount of it is incorporated into virus partcles to serve as a template for rna replication (makino et a/., ) . thus, it may lack packaging signals. on the other hand, since it is efficiently replicated in diinfected cells, dlsse rna must contain the sequences essential for viral rna replication. in the present study, we analyzed the primary structure of dlsse rna. the results revealed that dlsse consists of three noncontiguous regions of mhv-jhm genomic rna, including 'end leader rna and the 'end of genomic rna. one large open reading frame (orf) was demonstrated and the product of this orf was identified both in infected cells and by in vitro translation. possible mechanisms of di rna generation are discussed. viruses and cell culture mhv-jhm was used as a nondefective standard virus. serially passaged mhv-jhm stock at passage level was used as the source of di particles (makino et al., ) . all viruses were propagated in dbt cells as described previously (makino et al., a ). mhv-specific intracellular rna was extracted by procedures described previously (makino et a/., b) . poly(a)-containing rna was obtained by oligo(dt)-cellulose column chromatography (makino et a/., ) . agarose gel electrophoresis p-labeled virus-specific rna was analyzed by electrophoresis on % agarose gels without denaturing as described previously (makino et al., ) . poly(a)-containing rna was purified by preparative gel electrophoresis in o/o urea-agarose gels as previously described (makino et a/., a) . the rna was eluted from gel slices by the methods of langridge et al., ( ) . cdna cloning of dlsse cdna cloning followed the general method of gubler and hoffman ( ) . five hundred nanograms of oli-go(dt), -, was mixed with pg of gel-purified dlsse rna in ~ of distilled water. the rna and oligo(dt) mixture was heated at "for min and chilled quickly. the rna-dna hybrid was then incubated in ~ of first-strand cdna synthesis buffer containing units of rnasin (promega biotec), mll/l tris-hci (ph . at ') mll/l kci, ml\/l mgc , mm dlt, . mmeach of datp, dctp, dgtp, and ltp, and units of avian myeloblastosisvirus reverse transcriptase (life science) at " for hr. the cdna synthesis was stopped by adding . ~ of mm edta. nucleic acids were extracted with phenol-chloroform-isoamyl alcohol and precipitated with ethanol. second-strand synthesis was carried out in a reaction volume of ~ containing ml\/l tris-hci (ph . ), mfl/l mgc , m/l/l kci, pg/ml of bsa, mm (nh&s , . mlll / -nad, plm dntps, units of dna polymerase i, units of escherichia co/i dna ligase, . units of rnase h, and the product from the first strand reaction. the mixture was incubated at " for hr, and then at " for hr. the reaction was stopped by adding . ~ of mm edta, and products were extracted with phenol-chloroform-isoamyl alcohol, and precipitated with ethanol. doublestranded dna was dc-tailed in a ~-pi reaction mixture containing units of terminal transferase, mm potassium cacodylate, . rnn/l coci,, mm tris-hci (ph . ) mm dlt, pglml bsa, and pi\/i dctp at " for min. the dc-tailed double-stranded dna was annealed to ng of dg-tailed pstl-cut pbr plasmid in ~ of a buffer containing mm tris-hci (ph . ), mlli naci, and . mm edta. the dna mixture was heated at " for min and then cooled slowly overnight for annealing. the annealed molecules were used to transform e. co/i mc as described (dagert and ehrlich, ) . identification of large cdna clones containing dlsse sequence p-labeled mhv-jhm gene a cdna clones c and f (shieh eta/., ) and 'end p-labeled leader-specific -mer derived from leader sequence of mhv were used for colony hybridization to isolate dlsse-specific cdna clones. colonies yielding a strong signal were further analyzed by southern hybridization (maniatis et a/., ) . the gel-purified rnas were incubated in ~ of distilled water containing mn/r methyl mercury. after min incubation at room temperature, rna was incu-bated in ~ of first-strand cdna synthesis buffer with mm / -mercaptoethanol and ' end-labeled oligodeoxyribonucleotides at " for hr. reaction products were extracted with phenol-chloroform-isoamyl alcohol, precipitated with ethanol, and analyzed by electrophoresis on % polyacrylamide gels containing . m urea and were eluted from the gels according to the published procedures (maxam and gilbert, ) . sequencing was carried out by sanger's dideoxyribonucleotide chain termination method (sanger et a/., ) and maxam-gilbert chemical modification procedure (maxam and gilbert, ) as described previously . sequence analysis and predicted rna secondary structures were obtained with the lntelligenetics sequencing program. an mrna-dependent rabbit reticulocyte lysate (new england nuclear) was used as previously described . antisera a monoclonal antibody, j. . , directed against the mhv-jhm n protein has been described (fleming et a/., ) . the anti-p antibody was generated in rabbits against a synthetic peptide representing a portion of the mhv-jhm p protein (soe et a/., ) and will be described in detail elsewhere (s. c. baker et a/., manuscript in preparation). labeling of intracellular proteins, immunoprecipitation, and sds-polyacrylamide gel electrophoresis dbt cells were infected with either wild type mhv-jhm or mhv-jhm containing di particles at pfu per cell. at . hr postinfection, cells were labeled in methionine-free medium containing pci of l-[ s]methionine/ml (icn translabel) for min. cell extracts were prepared by treatment with lysolecithin (l-a-lysophosphatidylcholine, palmitoyl; sigma) at pg/ml for min at ". the treated cells were scraped in ~ hnd buffer ( . m hepes, ph . , . m nh&i, . m dtt), disrupted by pipetting with a pastuer pipet, and then centrifuged at g for min to remove nuclei and cell debris. the resulting supernatant was used for immunoprecipitation. lmmunoprecipitation was performed by the methods of kessler ( ) . the cell-free extracts were incubated with ~ of antisera for hr at ". the antigen-antibody complexes were collected by binding to pansorbin (calbiochem, la jolla, ca) and washed three times with washing buffer ( mll/ltris-hci, ph . , mlll naci, mm edta, and . % np- ) and eluted by boiling for min in electrophoresis sample buffer ( . n/rpmercaptoethanol, o/o sds, . / tris-hci, ph . , and % glycerol). the bacteria were removed by centrifugation and proteins were analyzed by electrophoresis on to % sds-polyacrylamide gels (laemmli, ) . cdna cloning and sequencing of dlsse rna to understand the primary structure of dlsse rna, dlsse-specific cdna clones were generated according to the general method of gubler and hoffman ( ) using oligo(dt) as a primer and gel-purified dlsse rna. since previous oligonucleotide fingerprinting analysis suggested that dlsse rna contains the leader sequence and the ' end region of genomic sequence (makino et al., ) cdna clones were screened by colony hybridization using ' end-labeled, leader-specific -mer, and two cdna clones f and c , which correspond to the ' end of genomic rna of mhv-jhm . several large cdna clones were isolated and their structure was further analyzed. a diagram representing the structure of the dlsse genome and that of mhv-jhm genomic rna and the strategy used for sequencing the cdna clones are shown in fig. . the dlsse sequence obtained is shown in fig. . sequence analysis of dlsse cdna clones revealed that dlsse rna consists of three different regions of mhv-jhm genomic rna. the first region represents nucleotides from the ' end of the genomic rna. the second region, nucleotides in length, is a region within the polymerase gene that corresponds to the region at . to kb from the ' end of genomic rna (shieh, unpublished observation) , and the third region contains a sequence of nucleotides derived from the extreme 'end of the genomic rna. the entire sequence of dlsse rna is identical to that of the corresponding regions of mhv genomic rna shieh et a/., unpublished data) , with some exceptions in the leader sequence region (see below). the cdna clones obtained does not appear to have a complete sequence at its extreme ' end. to understand the complete ' end sequence of dlsse, we performed primer-extension studies on dlsse rna using a specific primer ( '~aatgtcagcactatgaca- ') complementary to nucleotides - from the ' end of the genome of mhv-jhm . the ei'end-labeled primer was hybridized to gel-purified dlsse rna and extended with reverse transcriptase. primer extension products were then analyzed by electrophoresis on % polyacrylamide gels containing m urea. as shown in fig. a , two cdna products of and nucleotides were obtained, indicating heterogeneity at the 'end sequence of dlsse. these primerextended products were sequenced by the maxam-gilbert method. the sequences of both cdna products were identical except that the faster migrating cdna products contained three ucuaa repeats at the 'end of the leader sequence, while the slower migrating species contained four ucuaa repeats (fig. b ). in addition, the 'end sequences of dlsse and mhv-jhm genomic rna showed several differences. within the leader sequence, bases were substituted in dlsse rna (fig. b , asterisks) and nine nucleotides (uuuau-aaac) were deleted in dlsse at the junction between the leader rna and the remaining genomic sequences. the significance of the heterogeneity in the number of ucuaa repeats and of the nine-nucleotide deletion will be discussed below. another significant feature of dlsse rna is the presence of a single large orf (fig. ) . this orf is expected to share amino acid sequence identity with three different regions of the standard mhv-jhm. the first amino acids correspond to the n terminus of the mhv polymerase. this region represents the part of the n-terminus of the polymerase protein which is cleaved into a p protein (denison and perlman, ; soe et al., ) . the following amino acids were derived from the region of the polymerase at . to kb from the 'end of the genome. the 'end region of the orf of dlsse rna is the same as the orf utilized for the n protein . thus, the predicted product of this orf should contain the n-terminus of p and the c-terminus of the n protein. the predicted molecularweight mass of this orf product is , . to examine whether the orf of dlsse rna is utilized for translation, we first performed in vitro translation in a rabbit reticulocyte lysate of dlsse rna purified from the di-infected cells. two proteins with an apparent molecular mass of approximately , ( k) and , ( k) were detected (fig. a ). both proteins were immunoprecipitated with anti-n protein monoclonal antibody and anti-p antibody (fig. a, lanes and ) . therefore, these two proteins were likely the translation products of dlsse rna. a minor band of ap- proximately kda had the same electrophoretic mobility as the n protein of mhv-jhm, and was precipitated with anti-n monoclonal antibody, but not with anti-p antibody (fig a, lanes and ) . thus, this protein is most likely the n protein translated from the contaminated mrna in the dlsse rna preparation. the synthesis of dlsse-specific protein in di-infected cells was then examined. dbt cells were mock-infected (fig. b, lanes and ) , infected with mhv-jhm (fig. b, lanes and ) or infected with mhv-jhm containing di particles (fig. b, lanes and ) . both k and k proteins were specifically immunoprecipitated with anti-n monoclonal antibody and anti-p antibody from di-infected cells. the amount of these two proteins was low as compared to the n protein. nevertheless, they were reproducibly detected in di-infected cells. thus, the dlsse rna is a functional mrna. the relationship between the two protein species detected is not clear. the discrepancy between the predicted and observed molecular weights of the translation products of dlsse could be due to post-translational modification of the protein or aberrant migration of the protein. a small amount of p was immunoprecipitated with anti-p antibody in mhv-jhm-infected cells (fig. b, lane ) . however, this protein was hardly detectable in di-infected cells (fig. , lane ) . the absence of detectable amount of p in di-infected cells may be due to the inhibition of mhv-jhm genomic rna synthesis by di particles (makino et al,, ) . possible secondary structure at the di rna rearrangment sites sequence analysis revealed that dlsse rna consisted of three noncontiguous regions of mhv-jhm genomic rna. we have previously proposed that coronavirus rna synthesis proceeds by a discontinuous, nonprocessive mechanism, being interrupted at sites with hairpin loops (baric et a/., ) . this transcriptional interruption could account for the generation of soe et al., ) was zp-labeled at the ' end, hybridized to the gel-purified dlsse rna, and extended with reverse transcriptase. the products were electrophoresed on % polyacrylamide gels containing m urea. , origin of the gel. two primer-extended products are shown as a and b. (b) the dna sequences of these primerextended products were determined by the maxam-gilbert method. the '.end sequence of mhv-jhm genomic sequence was obtained from previous studies soe ef a/., ) . the letters a and b represent the canonical seven-nucleotide sequence ucuaaac and imperfectly repeated sequence of uauaaac, respectively. a bold solid line represents the nine-nucleotide sequence which is deleted in dlsse but present in mhv-jhm. dlsse (a) and dlsse (b) correspond to the sequences of primer-extended products, a and b, in fig. a , respectively. three base substitutions are indicated by asterisks. di rnas. we therefore examined whether any significant secondary structure existed at rearrangement sites on mhv-jhm genomic rna. the nucleotide sequences surrounding deleted regions of mhv-jhm genomic rna were analyzed by an rna secondary structure program of zuker and stiegler ( ) . the predicted secondary structures of these rearrangement regions are shown in fig. . all four genomic deletion sites have extensive and stable secondary structures. the free energies of these structures range from - . to - . kcal/mol. furthermore, as previously described for the standard mhv-jhm, the sequence surrounding the junction of leader rna and the remaining '-end genomic sequence also contains a stable secondary structure (soe eta/., ) . this junction region includes the nine-nucleotide deletion detected in dlsse rna (fig. b) . thus, an extensive and stable secondary structure exists at each parental mhv-jhm genomic region where deletion occurred. the present study demonstrated that the smallest di-specific rna, dlsse, is composed of three discontiguous parts of the viral genome, including the ' end and 'end of genomic rna. this structure is similar to many di rnas of other viruses, which typically retain both ends of the standard nondefective viral rnas. our previous study has demonstrated that dlsse is replicated from its negative template in the presence of helper virus (makino et al., ) . therefore, the dlsse sequence likely contains essential recognition signals for mhv rna replication. the structure of dlsse rna supports the likelihood that the recognition signals for the synthesis of negative-strand rna and positivestrand rna are localized at the ' end and ' end of genomic rna, respectively. one of the unique features of coronavirus di rna is that subgenomic di rna was poorly incorporated into virus particles (makino et a/., ) . one of the possible explanations is that the di subgenomic rnas lack a packaging signal. since all mhv-specific subgenomic mrnas contain the leader sequence, yet only genomic-sized rna is efficiently packaged into virus par-ticles, the packaging signal is probably located in gene a but not in leader sequence. the present study indicates that dlsse rna has a nine-nucleotide (uuuau-aaac) deletion at the junction between the leader rna and the remaining genomic rna sequence. however, this deletion is not likely to account for the failure of efficient di rna packaging into virus particles since dlssa and the genomic rna of a mutant mhv-jhm, both of which are packaged into virus particles, also have similar nine-nucleotide deletions (s. makino, unpublished data). thus, the packaging signals may be localized downstream of the ' end nucleotides. recently we found that another intracellular di-specific rna, dlssf, could be packaged more efficiently than dlsse (s. makino, unpublished data). the dlssf rna is approximately i . kb larger than dlsse and appears to contain more gene a sequences than dlsse, as determined from tl -oligonucleotide fingerprinting (makino et al., ) . sequence analysis of dlssf may reveal the possible reason for the poor incorporation of dlsse rna into virus particles. the data presented in this paper demonstrate extensive and stable secondary structures in the standard viral rna at sites where di rna underwent deletions. this observation is consistent with a model of di rna generation, in which rna transcription is interrupted at sites of hairpin loops on the template, and the rna intermediates then fall off and rebind at new sites on the template to generate an rna with extensive deletions. we have previously suggested that coronavirus rna synthesis may utilize a discontinuous, nonprocessive mechanism, in which rna transcription pauses at sites of secondary structures (baric et a/., ) . the incomplete rna intermediates dissociate from templates and then rejoin the temple for subsequent rna transcription. this mechanism is supported by the findings that mhv can undergo rna recombination at an extremely high frequency (makino et a/., a) , and that free incomplete rna transcription products of various sizes were detectable in the cytoplasm of mhv-infected cells (baric et al., (baric et al., , . furthermore, the sizes of these rna products correspond to the lengths between the ' end and the sites of hairpin loops (baric et al,, ) , in agreement with the notion that transcription pauses at these hairpin loops. thus, the potential hairpin loops present in the genomic rna at the di rna rearrangement sites could have interrupted rna transcription. the incomplete rna tran-script may join the rna template at the downstream rearrangement sites and create deleted rna as a result. however, there is no consensus sequence at the sites of rna deletion and reinitiation. it is not known how the reinitiation of rna synthesis occurred. the deletion of the nine nucleotides (uuuauaaac) at the ' end where the leader rna joins the genomic rna may have been caused by the same discontinuous and nonprocessive transcription mechanism. it is interesting to note that the ucuaaac, which is the consensus sequence for the leader rna binding is imperfectly repeated (uauaaac) at nine nucleotides downstream . it is these nine nucleotides which were deleted in dlsse rna. similar nine-nucleotide deletions have also been noted in the genomic rna of dlssa, and that of an mhv-jhm mutant virus (s. makino, unpublished data) . this rna structure suggests that rna synthesis may pause at the first repeat, and then reinitiate at the second repeat because of the binding of the incomplete rna transcript to the second repeat. finally, the heterogeneity in the number of ucuaa repeats in di rnas also supports the discontinuous nature of coronavirus rna replication. similar heterogeneity has been noted in the genomic rna of several different mhv strains (s. makino and m. m. c. lai, manuscript in preparation) . thus, di rna may be a product of discontinuous, nonprocessive rna replication of coronaviruses. there was a significant difference between the apparent molecular mass of the dlsse-specific protein products, k and k, and the predicted molecular mass of the potential product of the large orf of dlsse rna. this difference could be due to unusual configurations affecting electrophoretic migration, or due to the presence of phosphorylation, since the n protein is phosphorylated (stohlman and lai, ) and protein translated in vitro could be phosphorylated (chattopadhyay and banerjee, ) . a similar difference between the predicted and actual molecular mass of the n protein has previously been noted . the relationship between the two protein species is not clear. the n protein has also been shown to consist of multiple species (robbins et al., ) . it is not clear whether these proteins play any functional roles in di-infected cells. typically, di rnas do not synthesize any protein; however, in the sindbis virus system, translation products have been detected from a di rna (migliaccio et al., ) . although mhv genomic rna and dlsse rna are the major rna species among mhv-specific mrna species in virus-infected cells (makino et a/., (makino et a/., , (fig. l) , the gene products of these two mrnas, rna polymerase and both the k and k proteins, were present in small quantities in virus-infected ceils (fig. b) . we have previously demonstrated that the presence of stable secondary structure at the ' end noncoding regions of the polymerase gene reduced the amount of polymerse protein synthesized in vitro . also, as discussed previously, the presence of the small orf encoding eight amino acids (fig. ) may reduce the number of ribosomes reaching the downstream optimal translation site . since dlsse rna has a ' end structure similar to that of genomic rna, the dlsse rna may provide a tool to better our understanding of the mechanism of translational control of mhv rnas. furthermore, the fusion protein synthesized by dlsse rna may be useful for understanding the functional and structural domains of the mhv polymerase and n protein. analysis of intracellular small rnas of mouse hepatitis virus: evidence for discontinuous transcription characterization of replicative intermediate rna of mouse hepatitis virus: presence of leader rna sequences on nascent chains. . viral characterization of leader-related small rnas in coronavirus-infected cells: further evidence for leader-primed mechanism of transcription characterization of two rna polymerase activities induced by mouse hepatitis virus. . i/irol further characterization of mouse hepatitis virus rna-dependent rna polymerases phosphorylation within a specific domain of the phosphoprotein of vesicular stomatitis virus regulates transcription in vitro prolonged incubation in calcium chloride improves the competence of escherichia co/i cells translation and processing of mouse hepatitis virus virion rna in a cell-free system antigenic relationships of murine coronaviruses: analysis using monoclonal antibodies to jhm (mhv- ) virus a simple and very efficient method for generating cdna libraries use of protein a-bearing staphylococci for the immunoprecipitation and isolation of antigens from cells cleavage of structural proteins during the assembly of the head of bacteriophage t characterization of leader rna sequences on the virion and mrnas of mouse hepatitis virus, a cytoplasmic rna virus mouse hepatitis virus a : mrna structure and genetic localization of the sequence divergence from hepatotropic strain mhv- presence of leader sequences in the mrna of mouse hepatitis virus further characterization of mrnas of mouse hepatitis virus: presence of common '-end nucleotides. f. viral replication of mouse hepatitis virus: negative-stranded rna and replicative form rna are of genome length rna of mouse hepatitis virus extraction of nucleic acids from agarose gels the virus-specific intracellular rna species of two murine coronaviruses: mhv-a and mhv-jhm rna-dependent rna polymerase activity in murine coronavirusinfected cells structure of the intracellular defective viral rnas of defective interfering particles of mouse hepatitis virus high-frequency rna recombination of murine coronaviruses defective-interfering particles of murine coronavirus: mechanism of synthesis of defective viral rnas leader sequences of murine coronavirus mrnas can be freely reassorted: evidence for the role of free leader rna in transcription defective interfering particles of mouse hepatitis virus analysis of genomic and intracellular viral rnas of small plaque mutants of mouse hepatitis virus molecular cloning: a laboratory manual sequencing end-labelled dnawith base-specific chemical cleavages mrna activity of a sindbis virus defective-interfering rna rna-binding proteins of coronavirus mhv: detection of monomeric and multimeric n protein with an rna overlay-protein blot assay dna sequencing with chain-terminating inhibitors the '-end sequence of the murine coronavirus genome: implications for multiple fusion sites in leaderprimed transcription coronovirus jhm: nucleotide sequence of the mrna that encodes nucleocapsid protein sequence and translation of the murine coronavirus '-end genomic rna reveals the n-terminal structure of the putative rna polymerase coronavirus mrna synthesis involves fusion of non-contiguous sequence phosphoproteins of murine hepatitis viruses. f. viral genomic rna of the murine coronavirus jhm optimal computer folding of large rna sequences using thermodynamics and auxiliary information we thank ming-fu chang for his valuable suggestions for dna sequencing and david vannierfor excellent technical assistance. we also thank carol flores for typing the manuscript. this work was sup- key: cord- - qcoxl authors: nicklas, werner; bleich, andré; mähler, michael title: viral infections of laboratory mice date: - - journal: the laboratory mouse doi: . /b - - - - . - sha: doc_id: cord_uid: qcoxl viral infections of laboratory mice have considerable impact on research results, and prevention of such infections is therefore of crucial importance. this chapter covers infections of mice with the following viruses: herpesviruses, mousepox virus, murine adenoviruses, polyomaviruses, parvoviruses, lactate dehydrogenase-elevating virus, lymphocytic choriomeningitis virus, mammalian orthoreovirus serotype , murine hepatitis virus, murine norovirus, murine pneumonia virus, murine rotavirus, sendai virus, and theiler’s murine encephalomyelitis virus. for each virus, there is a description of the agent, epizootiology, clinical symptoms, pathology, methods of diagnosis and control, and its impact on research. in interpreting the microbiological status of laboratory animals, it must be understood that infection and disease are not synonymous. infection refers to the invasion and multiplication of microorganisms in body tissues and may occur with or without apparent disease. disease refers to interruption or deviation from normal structure and function of any tissue, organ or system. many of the infections with which we are concerned may not cause discernable disease in many strains of mice. however, they may cause inapparent or subclinical changes that can interfere with research. such interference often remains undetected, and therefore modified results may be obtained and published. the types of interference of an agent with experimental results may be diverse. there is no doubt that research complications due to overt infectious disease are significant and that animals with clinical signs of disease should not be used for scientific experiments. but clinically inapparent infections may also have severe effects on animal experiments. there are numerous examples of influences of microorganisms on host physiology and hence of the interference of inapparent infections with the results of animal experiments. many microorganisms have the potential to induce activation or suppression of the immune system, or both at the same time but on different parts of the the laboratory mouse Ó elsevier ltd. all rights reserved. isbn - - - - doi: . /b - - - - . - immune system, regardless of the level of pathogenicity. all infections, apparent or inapparent, are likely to increase interindividual variability and hence result in increased numbers of animals necessary to obtain reliable results. microorganisms, in particular viruses, present in an animal may contaminate biological materials such as sera, cells or tumours [ , ] . this may interfere with in vitro experiments conducted with such materials and may also lead to contamination of animals [ ] . mouse antibody production (map) testing or polymerase chain reaction (pcr) testing of biologics to be inoculated into mice is an important component of a disease prevention programme. finally, latent infections may be activated by environmental factors, by experimental procedures, or by the combination and interaction between various microorganisms. for all these reasons, prevention of infection, not merely prevention of clinical disease, is essential. unfortunately, research complications due to infectious agents are usually considered artefacts and published only exceptionally. information on influences of microorganisms on experiments is scattered in diverse scientific journals, and many articles are difficult to find. to address this problem, several meetings have been held on viral complications on research. the knowledge available is summarized in conference proceedings [ , ] and has later repeatedly been reviewed [ ] [ ] [ ] . viral infections of mice have been studied in detail, and comprehensive information on their pathogenic potential, their impact on research, and the influence of host factors such as age, genotype, and immune status on the response to infection is available. the nomenclature and taxonomy of viruses is described based on recent nomenclature rules by the international union of microbiological societies [ ] and the universal virus database of the international committee on the taxonomy of viruses (http://www. ictvdb.org). retroviruses are not covered in this chapter because they are not included in routine health surveillance programmes and cannot be eradicated with the methods presently available. this is because most of them are incorporated in the mouse genome as proviruses and thus are transmitted via the germline. the ability to accurately determine whether or not laboratory animals or animal populations have been infected with a virus depends on the specificity and sensitivity of the detection methods used. most viral infections in immunocompetent mice are acute or short term, and lesions are often subtle or subclinical. the absence of clinical disease and pathological changes has therefore only limited diagnostic value. however, clinical signs, altered behaviour or lesions may be the first indicator of an infection and often provide clues for further investigations. serology is the primary means of testing mouse colonies for exposure to viruses, largely because serological tests are sensitive and specific, are relatively inexpensive and allow screening for a multitude of agents with one serum sample. they are also employed to monitor biological materials for viral contamination using the map test. serological tests detect specific antibodies, usually immunoglobulin g (igg), produced by the host against the virus and do not actually test for the presence of the virus. an animal may have been infected, mounted an effective antibody response and cleared the virus, but remains seropositive for weeks or months or for ever, even though it is no longer infected or shedding the agent. active infection can only be detected by using direct detection methods such as virus isolation, electron microscopy or pcr. meanwhile, pcr assays have been established for the detection of almost every agent of interest. they are highly sensitive and, depending on the demands, they can be designed to broadly detect all members of a genus or only one species. however, good timing and selection of the appropriate specimen is critical for establishing the diagnosis. in practice, combinations of diagnostic tests are often necessary, including the use of sentinel animals or immunosuppression to get clear aetiological results or to avoid consequences from false-positive results. reports on the prevalence of viral infections in laboratory mice throughout the world have been published frequently. in general, the microbiological quality of laboratory mice has constantly improved during the last decades, and several agents (e.g. herpesviruses and polyomaviruses) have been essentially eliminated from contemporary colonies due to advances in diagnostic methodologies and modern husbandry and rederivation practices [ ] [ ] [ ] [ ] [ ] [ ] . they may, however, reappear, since most have been retained or are still being used experimentally. furthermore, the general trend towards better microbiological quality is challenged by the increasing reliance of biomedical research on genetically modified and immunodeficient mice, whose responses to infection and disease can be unpredictable. increasing numbers of scientists are creating genetically modified mice, with minimal or no awareness of infectious disease issues. as a consequence, these animals are more frequently infected than 'standard' strains of mice coming from commercial breeders, and available information on their health status is often insufficient. frequently they are exchanged between laboratories, which amplifies the risk of introducing infections from a range of animal facilities. breeding cessation strategies that have been reported to eliminate viruses from immunocompetent mouse colonies may prove to be costly and ineffective in genetically modified colonies of uncertain or incompetent immune status. it must also be expected that new agents will be detected, although only occasionally. infections therefore remain a threat to biomedical research, and users of laboratory mice must be cognizant of infectious agents and the complications they can cause. two members of the family herpesviridae can cause natural infections in mice (mus musculus). mouse cytomegalovirus (mcmv- ) or murid herpesvirus (muhv- ) belongs to the subfamily betaherpesvirinae, genus muromegalovirus. murid herpesvirus (muhv- ) or mouse thymic virus (mtv) has not yet been assigned to a genus within the family herpesviridae. both are enveloped, double-stranded dna viruses that are highly host-specific and relatively unstable to environmental conditions such as heat and acidic ph. both agents are antigenically distinct and do not cross-react in serological tests, but their epidemiology is similar [ ] . mcmv- is very uncommon in european and american colonies of laboratory mice and is found at a very low rate [ ] or reported as not found [ , ] . seropositivity has, however, been reported from asian countries [ , ] . testing for mtv is not frequently reported, and no sample tested positive in recent studies [ ] . the data available suggest that the prevalence of both viruses in contemporary colonies and thus their importance for laboratory mice is negligible. however, both mcmv- and mtv are frequently found in wild mice, which may be coinfected with both viruses [ , [ ] [ ] [ ] . mouse cytomegalovirus (mcmv- ) or murid herpesvirus (muhv- ) natural infection with mcmv- causes subclinical salivary gland infection in mice. like other cytomegaloviruses, mcmv- is strictly hostspecific. it persists in the salivary glands (particularly in the submaxillary glands) and also in other organs [ ] [ ] [ ] . the virus can be cultured in mouse fibroblast lines like t cells, but primary mouse embryo fibroblasts are more sensitive to infection and produce higher virus titres. however, passage in cell culture results in its attenuation. to maintain virulence, the virus is best propagated by salivary gland passages of sublethal virus doses in weanling mice of a susceptible strain (e.g. balb/c) [ ] . most information concerning the pathogenesis of mcmv- infection is based on experimental infection studies. these results are very difficult to summarize because the outcome of experimental infection in laboratory mice depends on various factors such as mouse strain and age, virus strain and passage history [ ] , virus dose and route of inoculation [ ] . in general, newborn mice are most susceptible to clinical disease and to lethal infection and develop higher levels of resistance with increasing age. infection of neonates leads to abnormal brain development [ , ] . virus replication is observed in newborn mice in many tissues and appears in the salivary glands towards the end of the first week of infection when virus concentrations in liver and spleen have already declined. resistance develops rapidly after weaning between days and of age. experimental infection of adult mice results in mortality only in susceptible strains and only if high doses are administered. not even intravenous or intraperitoneal injections of adult mice usually produce signs of illness in resistant strains [ ] . mice of the h- b (e.g. c bl/ ) and h- d (e.g. balb/c) haplotype are more sensitive to experimental infection than are mice of the h- k haplotype (e.g. c h), which are approximately -fold more resistant to mortality than those of the b or d haplotype [ ] . subclinical or latent infections can be activated by immunosuppression (e.g. with cyclophosphamide or cortisone) or critical illness such as sepsis [ ] . reactivation of mcmv- also occurs after implantation of latently infected salivary glands into prkdc scid mice [ ] . immunodeficient mice lacking functional t cells or natural killer (nk) cells, such as foxn nu and lyst bg mice are more susceptible than are immunocompetent animals. experimental infection in prkdc scid mice causes severe disease or is lethal, with necrosis in spleen, liver and other organs, and multinucleate syncythia with inclusion bodies in the liver [ ] . similarly to aids patients infected with human cytomegalovirus, athymic foxn nu mice experimentally infected with mcmv- also develop adrenal necrosis [ ] . the virus also replicates in the lungs, leading to pneumonitis, whereas replication and disease are not seen in heterozygous (foxn nu/þ ) littermates [ ] . the pathogenesis of mcmv- infection in immunocompetent and in immunocompromised mice, as well as the role of the immune system, have been reviewed by krmpotic et al. [ ] . the most prominent histological finding of cytomegaloviruses is enlarged cells (cytomegaly) of salivary gland epithelium with eosinophilic nuclear and cytoplasmic inclusion bodies. the inclusion bodies contain viral material and are found also in other organs such as liver, spleen, ovary and pancreas [ ] . depending on inoculation route, dose, strain, and age of mice, experimental infections may result in inflammation or cytomegaly with inclusion bodies in a variety of tissues, pneumonitis, myocarditis, meningoencephalitis or splenic necrosis in susceptible strains [ , , ] . virus is transmitted by the oronasal route, by direct contact and is excreted in saliva, tears and urine for several months. the virus is ubiquitous in wild house mice worldwide. they serve as a natural reservoir for infection and can even be infected with different virus strains [ ] . the virus is most frequently transmitted horizontally through mouse-to-mouse contact but does not easily spread between cages. sexual transmission and transmission with tissues or organs is also possible. the virus does not cross the placenta in immunocompetent mice, although infection of pregnant females results in fetal death or resorption and wasting of borne pups. however, fetal infection is possible by direct injection of mcmv- into the placenta [ ] and also occurs by transplacental transmission in mice with severe immunodeficiency [ ] . vertical transmission is also possible by milk during lactation [ ] . it is generally assumed that mcmv- has a very low prevalence in contemporary colonies of laboratory mice. the risk of introduction into facilities housing laboratory mice is very low if wild mice are strictly excluded. monitoring is necessary if populations of laboratory mice may have been contaminated by contact with wild mice. as for other viruses, different serological tests, including multiplex fluorescent immunoassay (mfia) [ ] , are used for health surveillance of rodent colonies. as the virus persists, direct demonstration of mcmv- in infected mice is possible by pcr [ ] [ ] [ ] or by virus isolation using mouse embryo fibroblasts ( t cells). although mcmv- does not play a significant role as a natural pathogen of laboratory mice, it is frequently used as a model for human cytomegalovirus infection [ ] . these aspects have been discussed in detail by shellam et al. [ ] . mcmv- has also been used as a vaccine vector aiming at a disseminating mouse control agent by inducing immunocontraception in mice [ ] . the virus is known to influence immune reactions in infected mice [ , ] and may therefore have an impact on immunological research [ , ] . mtv was detected during studies in which samples from mice were passaged in newborn mice. unlike other herpesviruses, mtv is difficult to culture in vitro and is usually propagated by intraperitoneal infection of newborn mice. the thymus is removed - days later, and thymus suspensions serve as virus material for further studies. the prevalence of mtv is believed to be low in laboratory mice, and for this reason, and also due to the difficulties in virus production for serological assays, it is not included in many standard diagnostic or surveillance testing protocols. limited data are available indicating that it is common in wild mice [ , ] . further, mtv obviously represents a significant source of contamination of mcmv- (and vice versa) if virus is prepared from salivary glands, since both viruses cause chronic or persistent salivary gland infections and can coinfect the same host. all mouse strains are susceptible to infection, but natural or experimental infection of adult mice is subclinical. gross lesions appear only in the thymus and only if experimental infection occurs at an age of less than about days. infection results in nuclear inclusions in thymocytes and their almost complete destruction within weeks. virus is present in the thymus but may also be found in the blood and in salivary glands of surviving animals. salivary glands are the only site yielding positive virus isolations if animals are infected as adults. the virus persists here and is shed via saliva for months. mtv also establishes a persistent infection in athymic foxn nu mice, but virus shedding is reduced compared to euthymic mice, with virus recovery possible only in a lower percentage of mice [ ] . pathological changes caused by mtv occur in the thymus, and reduced thymus mass due to necrosis in suckling mice is the most characteristic gross lesion [ ] . lymphoid necrosis may also occur in lymph nodes and spleen [ ] , with necrosis and recovery similar to that in the thymus. in mice infected during the first days after birth, necrosis of thymus becomes evident within - days, and the animals' size and weight are markedly reduced at day - . intranuclear inclusions may be present in thymocytes between days - after infection. the thymus and the affected peripheral tissues regenerate within weeks after infection. regardless of the age of mice at infection, a persistent infection is established in the salivary glands, and infected animals shed virus for life. several alterations of immune responses are associated with neonatal mtv infection. there is transient immunosuppression, attributable to lytic infection of t lymphocytes, but activity (e.g. response of spleen cells to t-cell mitogens) returns to normal as the histological repair progresses [ ] . selective depletion of cd þ t cells by mtv results in autoimmune disease [ , ] . information about additional influences on the immune system is given in textbooks [ , ] . in experimentally infected newborn mice, oral and intraperitoneal infections similarly result in thymus necrosis, seroconversion and virus shedding, suggesting that the oral-nasal route is likely to be involved in natural transmission [ ] . the virus spreads to cagemates after long periods of contact. it is transmitted between mice kept in close contact, and transmissibility from cage to cage seems to be low. mtv is not transmitted to fetuses by the transplacental route, and intravenous infection of pregnant mice does not lead to congenital damage, impairment in size or development, or abortion [ ] . mtv and mcmv- do not cross-react serologically [ ] . serological monitoring of mouse populations for antibodies to mtv is possible by indirect immunofluorescent assay (ifa) testing, which is commercially available; enzyme-linked immunosorbent assays (elisa) tests have also been established [ , ] . elisa and complement fixation yield similar results [ ] . serological tests based on recombinant proteins and direct detection of virus by pcr are currently not possible because the genome of the virus has not yet been sequenced. it must be noted that the immune response depends on the age at infection. antibody responses are not detectable in mice infected as newborns, whereas adult mice develop high titres that are detectable by serological testing. if neonatal infection is suspected, homogenates of salivary glands or other materials can be inoculated into pathogen-free newborn mice followed by gross and histological examination of thymus, lymph nodes and spleens for lymphoid necrosis [ ] . alternatives to the in vivo infectivity assay for detecting mtv in infected tissues include a competition elisa [ ] and map testing, although this is slightly less sensitive than infectivity assays [ ] . there is very little experience of eradication methods for mtv because of its low prevalence in contemporary mouse colonies. methods that eliminate other herpesviruses will likely eliminate mtv. procurement of animals of known negative mtv status is an appropriate strategy to prevent infection. strict separation of laboratory mice from wild rodents is essential to avoid introduction of the virus into laboratory animal facilities. murid herpesvirus (muhv- ) or rat cytomegalovirus infects rats and is also a member of the genus muromegalovirus. murid herpesvirus (muhv- ) is a member of the genus rhadinovirus in the subfamily gammaherpesvirinae and is also known as mouse herpesvirus strain (mhv- ). other murid herpesviruses are not yet assigned to a subfamily within the family herpesviridae. among these is murid herpesvirus (mouse thymic herpesvirus), but also murid herpesvirus (field mouse herpesvirus) which infects voles (microtus pennsylvanicus), murid herpesvirus (sand rat nuclear inclusion agent), and murid herpesvirus [ ] . furthermore, a gammaherpesvirus of house mice (mus musculus) has been described recently which is clearly distinct from mhv- [ ] . experimental infection of laboratory mice with mhv- is a frequently used model system for the study of human gammaherpesvirus pathogenesis, e.g. of kaposi's sarcoma-associated herpesvirus or epstein-barr virus (ebv) [ , ] which are members of the same subfamily. they are also important models to study viral latency and immune mechanisms controlling latency [ ] [ ] [ ] . mus musculus is not the natural host for this virus; it was first isolated in slovakia from bank voles (myodes glareolus). additional closely related strains (mhv- , mhv- ) exist from the same host species, and similar strains (mhv- , mhv- ) were isolated from wood mice (apodemus flavicollis and apodemus sylvaticus). apodemus sp. seem to be the major hosts for mhv- in great britain [ ] . different virus strains exhibit different genetic and biological properties and also differ in their pathogenicity, e.g. for prkdc scid mice [ ] . infections in laboratory mice take the same course as in their natural hosts [ ] . there are, however, some differences as, e.g. higher virus levels are reached in the lungs of balb/c mice, and wood mice develop higher titres of neutralizing antibodies [ ] . house mice develop an acute infection in the lungs after intranasal infection. a latent infection develops within weeks and the virus persists lifelong in epithelial cells in the lungs and also spreads to the spleen and other organs (e.g. bone marrow, peritoneal cells) where it persists in different cells of the immune system. it behaves like a natural pathogen in inbred strains of mice and persists without causing disease. the mousepox (ectromelia) virus (ectv) is a member of the genus orthopoxvirus belonging to the family poxviridae. it is antigenically and morphologically very similar to vaccinia virus and other orthopoxviruses. poxviruses are the largest and most complex of all viruses, with a diameter of nm and a length of - nm. mousepox (ectromelia) virus contains one molecule of double-stranded dna with a total genome length of nearly nucleotides [ ] . it is the causative agent of mousepox, a generalized disease in mice. experimental transmission to young rats (up to days of age) is possible [ ] . unlike various other orthopoxviruses, ectromelia virus does not infect humans [ ] . the virus is resistant to desiccation, dry heat and many disinfectants. it is not consistently inactivated in serum heated for min at c [ , ] and remains active for months when maintained at c in fetal bovine serum [ ] . effective disinfectants include vapour-phase formaldehyde, sodium hypochlorite and iodophores [ , ] . historically, ectv has been an extremely important natural pathogen of laboratory mice. the virus was widespread in mouse colonies worldwide and can still be found in several countries. between and almost individual ectromelia outbreaks were reported in the usa. the last major epizootic in the usa occurred in - and has been described in great detail [ ] . severe outbreaks were also described in various european countries [ ] [ ] [ ] . a more recent outbreak in the usa, which resulted in the eradication of almost mice in one institution, was described by dick et al. [ ] . another recent and well-documented case of mousepox was published by lipman et al. [ ] . few additional but unpublished cases of ectromelia have been observed since then; the latest report of an outbreak was published in [ ] . in general, positive serological reactions are occasionally reported from routine health surveillance studies [ ] but the virus is extremely rare in european and american colonies of laboratory mice [ ] [ ] [ ] . natural infections manifest differently, depending on many factors. mousepox may occur as a rapidly spreading outbreak with acute disease and deaths, or may be inconspicuous with slow spreading and mild clinical signs and may therefore be very difficult to diagnose [ ] . the mortality rate can be very low in populations in which the virus has been present for long periods. the infection usually takes one of three clinical courses: acute asymptomatic infection, acute lethal infection (systemic form) or subacute to chronic infection (cutaneous form) [ , [ ] [ ] [ ] . the systemic or visceral form is characterized clinically by facial oedema, conjunctivitis, multisystemic necrosis and usually high mortality. this form is less contagious than the cutaneous form because the animals die before there is virus shedding. the cutaneous form is characterized by typical dermal lesions and variable mortality. the outcome of infection depends on many factors including strain and dose of virus; route of viral entry; strain, age, and sex of mouse; husbandry methods and duration of infection in the colony. while all mouse strains seem to be susceptible to infection with ectv, clinical signs and mortality are strain-dependent [ ] [ ] [ ] . acute lethal (systemic) infection occurs in highly susceptible inbred strains such as dba/ , dba/ , balb/c, a and c h/hej. immunodeficient mice may also be very susceptible [ ] . outbreaks among susceptible mice can be explosive, with variable morbidity and high mortality (> %). clinical disease may not be evident in resistant strains such as c bl/ and akr, and the virus can be endemic in a population for long periods before being recognized. furthermore, females seem to be more resistant to disease than males, at least in certain strains of mice [ , ] . killer cells are necessary to control mousepox infections [ ] . mice that are resistant to mousepox may lose their resistance with increasing age, most likely due to the decreased number and activity of nk cells [ ] . the mechanisms determining resistance versus susceptibility are not fully understood but appear to reflect the action of multiple genes. the genetic loci considered to be important include h d b (termed rmp , resistance to mousepox), on chromosome [ ] ; the c genes (rmp , on chromosome ); rmp , localized to a region on chromosome encoding the nk cell receptor nkr-p alloantigens [ ] ; the nitric oxide synthase locus on chromosome [ ] ; and the signal transducer and activator of transcription locus on chromosome [ ] . mousepox infections are controlled for several days during the initial course of infection by the complement system until the adaptive immune system can react. loss of the complement system results in lethal infection [ ] . clearance of the virus by the immune system is dependent upon the effector functions of cd þ t cells while nk cells, cd þ t cells and macrophages are necessary for the generation of an optimal response [ , ] . t-and b-cell interactions and antibodies play a central role during recovery from a secondary infection [ ] . mousepox (ectromelia) virus usually enters the host through the skin with local replication and extension to regional lymph nodes [ , , ] . it escapes into the blood (primary viraemia) and infects splenic and hepatic macrophages, resulting in necrosis of these organs and a massive secondary viraemia. this sequence takes approximately week. many animals die at the end of this stage without premonitory signs of illness; others develop varying clinical signs including ruffled fur, hunched posture, swelling of the face or extremities, conjunctivitis and skin lesions (papules, erosions or encrustations mainly on ears, feet and tail; figure . . ). necrotic amputation of limbs and tails can sometimes be seen in mice that survive the acute phase, hence the common gross lesions of acute mousepox include enlarged lymph nodes, peyer's patches, spleen and liver; multifocal to semiconfluent white foci of necrosis in the spleen and liver; and haemorrhage into the small intestinal lumen [ , , , ] . in animals that survive, necrosis and scarring of the spleen can produce a mosaic pattern of white and red-brown areas that is a striking gross finding. the most consistent histological lesions of acute mousepox are necroses of the spleen (figure . . ), lymph nodes, peyer's patches, thymus and liver [ , , , , ] . occasionally, necrosis may also be observed in other organs such as ovaries, uterus, vagina, intestine and lungs. the primary skin lesion, which occurs about a week after exposure at the site of inoculation (frequently on the head), is a localized swelling that enlarges from inflammatory oedema. necrosis of dermal epithelium provokes a surface scab and heals as a deep, hairless scar. secondary skin lesions (rash) develop - days later as the result of viraemia ( natural transmission of ectv mainly occurs by direct contact and fomites [ , , , ] . the primary route of infection is through skin abrasions. faecal-oral and aerosol routes may also be involved [ ] . in addition, the common practice of cannibalism by mice may contribute to the oral route of infection [ ] . intrauterine transmission is possible at least under experimental conditions [ ] . virus particles are shed from infected mice (mainly via scabs and/or faeces) for about - weeks, even though the virus can persist for months in the spleen of an occasional mouse [ , ] . cage-to-cage transmission of ectv and transmission between rooms or units is usually low and largely depends on husbandry practices (e.g. mixing mice from different cages). importantly, the virus may not be transmitted effectively to sentinel mice exposed to dirty bedding [ ] . various tests have been applied for the diagnosis of ectromelia. previous epidemics were difficult to deal with because of limited published data and information on the biology of the virus and the lack of specific and sensitive assays [ ] . in the s, diagnosis relied on clinical signs, histopathology and animal passages of tissues from moribund and dead animals. culture of the virus on the chorioallantoic membrane of embryonated eggs was also used. serology is currently the primary means of routine health surveillance for testing mouse colonies for exposure to ectv. the methods of choice are mfia, elisa and ifa; they are more sensitive and specific than the previously used haemagglutination inhibition (hi) assay [ , , ] . serological tests based on virus particles detect antibodies to orthopoxviruses and do not distinguish between ectv and vaccinia virus or other orthopoxviruses, respectively. vaccinia virus is commonly used as an antigen for serological testing to avoid the risk of infection for mice. thus, false-positive serological reactions may be found after experimental administration of replication-competent vaccinia virus. it has been shown that even cage contact sentinels may develop antibodies, and vaccinia virus leading to seroconversion may even be transmitted by dirty bedding [ ] . confirmation of positive serological results is important before action is taken because vaccinia virus is increasingly prevalent in animal facilities as a research tool (e.g. for vaccination or gene therapy). as observed in different outbreaks, serological testing is of little value in the initial stages of the disease. for example, in the outbreak described by dick et al. [ ] depopulation was nearly completed before serological confirmation was possible. for this reason, negative serological results should be confirmed by direct detection methods (pcr, immunohistochemistry, virus isolation) or by histopathology, especially when clinical signs suggestive of mousepox are observed. pcr assays to detect different genes of poxviruses in infected tissues have been used [ , , ] . other pcr tests which were developed to detect smallpox virus have also been shown to detect ectromelia virus and can be used as well [ , ] . the key to prevention and control of mousepox is early detection of infected mice and contaminated biological materials. all institutions that must introduce mice from other than commercial barrier facilities should have a health surveillance programme and test incoming mice. perhaps even more important than living animals are samples from mice (tumours, sera, tissues). the virus replicates in lymphoma and hybridoma cell lines [ ] , and such cells or material derived from them may therefore be a vehicle for inadvertent transfer between laboratories. the last three published outbreaks of ectromelia were introduced into the facilities by mouse serum [ , , ] . lipman et al. [ ] found that the contaminated serum originated from a pooled lot of l that had been imported from china, but in both other cases, serum was obtained from animals in the usa. because mouse serum is commonly sold to the end user in small aliquots (a few millilitres), it has to be expected that aliquots of the contaminated lot may still be stored in freezers. these published cases of ectromelia outbreaks provide excellent examples of why testing should be performed on all biological materials to be inoculated into mice. in the case of ectromelia virus it was shown that pcr is more sensitive, and map testing failed to detect contamination [ ] . eradication of mousepox has usually been accomplished by elimination of the affected colonies, disinfection of rooms and equipment, and disposal of all infected tissues and sera. while culling of entire mouse colonies is the safest method for eradication of mousepox, it is not a satisfactory method because of the uniqueness of numerous lines of genetically modified animals housed in many facilities. several studies indicate that mousepox is not highly contagious [ , , ] and that it may be self-limiting when adequate husbandry methods are applied. therefore, strict quarantine procedures along with cessation of breeding (to permit resolution of infection) and frequent monitoring, with removal of clinically sick and seropositive animals, are a potential alternative. the period from the last births until the first matings after cessation of breeding should be at least weeks [ ] . sequential testing of immunocompetent contact sentinels for seroconversion should be employed with this option. in the past, immunization with live vaccinia virus was used to suppress clinical expression of mousepox. vaccination may substantially reduce the mortality rate, but it does not prevent virus transmission or eradicate the agent from a population [ , ] . after vaccination, typical pocks develop at the vaccination site, and infectious vaccinia virus is detectable in spleen, liver, lungs and thymus [ ] . vaccination also causes seroconversion so that serological tests are not applicable for health surveillance in vaccinated populations. it is therefore more prudent to control mousepox by quarantine and serological surveillance than by relying on vaccination. mortality and clinical disease are the major factors by which ectv interferes with research. severe disruption of research can also occur when drastic measures are taken to control the infection. the loss of time, animals and financial resources can be substantial. experimental mousepox infections are frequently used as a model to study various aspects of smallpox infections of humans [ ] [ ] [ ] . mousepox shares many aspects of virus biology and pathology, and models the course of human smallpox. experimental mousepox infections are used to study vaccination procedures [ , ] or anti-poxvirus therapies [ ] . murine adenoviruses (madv) are non-enveloped, double-stranded dna viruses of the family adenoviridae. two distinct strains have been isolated from mice. the fl strain (madv- ) was first isolated in the usa as a contaminant of a friend leukaemia [ ] and has been classified as a member of the genus mastadenovirus. the k strain (madv- ) was isolated in japan from the faeces of a healthy mouse [ ] and has not yet been assigned to a genus. both strains are considered to represent different species [ ] [ ] [ ] . they are host species specific and are not infectious for infant rats [ ] . madv- can be cultured in vitro in mouse fibroblasts (e.g. t or l cells), madv- is usually cultured in vitro in a mouse rectum carcinoma cell line (cmt- ). in laboratory mice, seropositivity to adenoviruses was reported to be very low [ , , , , , ] or negative [ , ] . antibodies to madv are also found in wild mice [ , ] and in rats [ , ] . neither virus is known to cause clinical disease in naturally infected, immunocompetent mice. however, madv- can cause a fatal systemic disease in suckling mice after experimental inoculation [ , , ] . disease is characterized by scruffiness, lethargy, stunted growth and often death within days. experimental infection of adult mice with madv- is most often subclinical and persistent but can cause fatal haemorrhagic encephalomyelitis with neurological symptoms, including tremors, seizures, ataxia and paralysis in susceptible c bl/ and dba/ j mice [ ] . balb/c mice are relatively resistant to this condition. athymic foxn nu mice experimentally infected with madv- develop a lethal wasting disease [ ] . similarly, prkdc scid mice succumb to experimental infection with madv- [ ] . gross lesions in response to natural madv infections are not detectable. occasional lesions observed after experimental infection with madv- include small surface haemorrhages in the brain and spinal cord of c bl/ and dba/ j mice [ ] , duodenal haemorrhage in foxn nu mice [ ] and pale yellow livers in prkdc scid mice [ ] . histologically, experimental madv- infection of suckling mice is characterized by multifocal necrosis and large basophilic intranuclear inclusion bodies in liver, adrenal gland, heart, kidney, salivary glands, spleen, brain, pancreas and brown fat [ , , , ] . in experimentally induced haemorrhagic encephalomyelitis, multifocal petechial haemorrhages occur throughout the brain and spinal cord, predominantly in the white matter, and are attributed to infection and damage to the vascular epithelium of the central nervous system (cns) [ ] . histopathological manifestations in madv- -infected prkdc scid mice are marked by microvesicular fatty degeneration of hepatocytes [ ] . in contrast to madv- , the tissue tropism of madv- is limited to the intestinal epithelium. naturally or experimentally infected mice develop intranuclear inclusions in enterocytes, especially in the ileum and caecum [ , , ] . transmission of madv primarily occurs by ingestion. madv- is excreted in the urine and may be shed for up to years [ ] . madv- infects the intestinal tract and is shed in faeces for only a few weeks in immunocompetent mice [ ] ; immunodeficient mice may shed the virus for longer periods [ ] . murine adenovirus infections are routinely diagnosed by serological tests. however, there is a one-sided cross-reactivity of madv- with madv- [ ] . serum from mice experimentally infected with madv- yielded positive reactions in serological tests with both viruses, while serum from mice infected with madv- reacted only with the homologous antigen [ ] . smith et al. [ ] reported that sera might react with madv- or madv- or both antigens. occasional reports of mice with lesions suggestive of adenovirus infections and negative serology (with madv- ) indicate that the infection may not be detected if only one virus is used as an antigen [ ] . it is therefore usual to test sera for antibodies to both madv- and madv- . the commonly used methods are ifa, elisa and mfia. the low prevalence in colonies of laboratory mice indicates that madv can easily be eliminated (e.g. by hysterectomy derivation or embryo transfer) and that barrier maintenance has been very effective in preventing infection. the low pathogenicity and the low prevalence in contemporary mouse populations are the main reasons why adenoviruses are considered to be of little importance, which is also indicated by the fact that recent publications about murine adenoviruses are very rare. however, the viruses might easily be spread by the exchange of genetically modified mice and therefore re-emerge. only a few influences on research attributable to madv have been published. for example, it has been shown that madv- significantly aggravates the clinical course of scrapie disease in mice [ ] . natural infections with madv could also interfere with studies using adenovirus as a gene vector. a novel murine adenovirus classified as a mastadenovirus has recently been isolated from a striped field mouse (apodemus agrarius) [ ] . it was cultured in vero e cells and named madv type (madv- ). it revealed the highest similarity to madv- but it represents a separate serotype. however, there is some cross-reactivity between madv- and both other mouse viruses [ ] . in addition to serological and antigenic differences it also shows a unique organotropism and infects predominantly the heart tissue of c bl/ n mice after experimental infection. experimentally infected mice show no clinical signs. the virus is not easily transmitted from experimentally infected mice to contact sentinels [ ] . polyomaviridae are enveloped, double-stranded dna viruses. two different agents of this family exclusively infect mice (mus musculus), and both belong to the genus polyomavirus. murine pneumotropic virus (mptv) was formerly known as 'newborn mouse pneumonitis virus' or 'k virus' (named after l. kilham who first described the virus). the second is murine polyomavirus (mpyv). both are related, but antigenically distinct, from each other [ ] , and also viruslike particles from the major capsid protein (vp ) do not cross-react [ ] . they are enzootic in many populations of wild mice but are very uncommon in laboratory mice. even older reports indicate that both have been eradicated from the vast majority of contemporary mouse colonies, and their importance is negligible [ ] . seropositivity to these viruses was not reported in a recent survey conducted in the usa [ ] , and other publications also indicate that these viruses do not presently play a significant role in laboratory mice [ , , ] . because of their low prevalence, neither virus is included in the list of agents for which testing is recommended on a regular basis by felasa [ ] . although polyomavirus genes, especially those of sv , are widely used in gene constructs for insertional mutagenesis, very few reports have been published on spontaneous or experimental disease due to mpyv or mptv in the last - years. the reader is therefore referred to previous review articles for details [ , ] . natural infections with mptv are subclinical. the prevalence of infection is usually low in an infected population. the virus may persist in infected animals for months and perhaps for life depending on the age at infection and is reactivated under conditions of immunosuppression. virus replicates primarily in endothelial cells, but renal tubular epithelial cells are the major site of viral persistence [ , ] . clinical signs are observed only after infection of infant mice less than - days of age. infected pups suddenly develop respiratory symptoms after an incubation period of approximately week, and many die within a few hours of onset of symptoms with an interstitial pneumonia caused by productive infection of and damage to pulmonary endothelium (figure . . ). endothelial cells in other organs are also involved in virus replication [ , ] (figure . . ). in older suckling mice, mptv produces a more protracted infection, and the virus or viral antigen can be detected for as long as months. in adult animals, the virus produces a transient asymptomatic infection. even in immunodeficient foxn nu mice, experimental infection of adults is clinically asymptomatic, although virus is detectable for a period of several months [ ] . in vitro cultivation of mptv is difficult. no susceptible permanent cell line is known to support growth. it can be cultured in primary mouse embryonic cells, but viral titres are not sufficient for use in serological assays [ ] . for this reason, the hi test using homogenates of livers and lungs of infected newborn mice is still frequently used, but ifa and elisa tests are also available [ ] . furthermore, a pcr test for demonstration of mptv in biological samples has also been published [ ] . mpyv was first detected as a contaminant of murine leukaemia virus (mulv) when sarcomas developed in mice after experimental inoculation of contaminated samples. it has later been shown to be a frequent contaminant of transplantable tumours [ ] . natural infection of mice is subclinical, and gross lesions including tumours are usually not found. tumour formation occurs when mice are experimentally infected at a young age or when inoculated with high virus doses. development of tumours may be preceded by multifocal necrosis and mortality during the viraemic stage [ ] . parotid, salivary gland and mammary tumours are common, and sarcomas or carcinomas of kidney, subcutis, adrenal glands, bone, cartilage, teeth, blood vessels and thyroid also occur. virus strains vary with regard to the tumour types or lesions that they induce, and mouse strains vary in their susceptibility to different tumour types. those of c bl and c br/cd lineage are considered to be the most resistant strains; athymic foxn nu mice are considered to be most susceptible; c h mice are particularly susceptible to adrenal tumours and a mice tend to develop bone tumours. immunosuppression or inoculation into immunodeficient strains (e.g. foxn nu ) also supports the growth of tumours. on the other hand, experimental infection of adult immunocompetent mice does not result in tumour formation because the immune response suppresses tumour growth, and newborn immunocompetent mice develop runting only if inoculated with high virus doses [ ] . after experimental intranasal infection, mpyv initially infects the respiratory tract followed by a systemic phase in which liver, spleen, kidney and colon become infected [ ] . the virus is shed in faeces and in all body fluids, and transmission occurs rapidly by direct contact between animals, but also between cages in a room. further, intrauterine transmission has been documented after experimental infection [ ] . mpyv persists in all organs in prkdc scid mice while viral dna is detectable in immunocompetent mice after experimental infection for only a limited period of about weeks [ ] . however, virus may persist and can be reactivated by prolonged immunosuppression [ ] or during pregnancy, at least in young mice [ ] . it has been shown that interferon-gamma is an important factor of the host defence against tumour formation and mpyv infection [ ] . biological materials of mouse origin are likely to be the most common source of contamination of laboratory mice, emphasizing the importance of map or pcr screening of biological materials to be inoculated into mice. the most frequently used tests for health surveillance of mouse colonies are elisa, mfia and ifa; in addition, the hi test is still used. latent infections can be detected by intracerebral inoculation of neonate mice or by map testing, but direct demonstration of virus in biological samples is also possible by pcr testing [ ] . while mpyv infections are of low importance for laboratory animal medicine, the virus is used in models of persistent virus infection [ , ] . virus-like particles from both murine polyomaviruses have been used as a vector for gene therapy or vaccines [ , ] . parvoviruses are non-enveloped small viruses (approximately nm in diameter) with a singlestranded dna genome of approximately nucleotides. murine parvoviruses are members of the family parvoviridae, genus parvovirus. they are remarkably resistant to environmental conditions like heat, desiccation, acidic and basic ph-values. up to date, two distinct species that infect laboratory mice are officially listed: the minute virus of mice (mvm), previously named mice minute virus (mmv), and the mouse parvovirus (mpv). non-structural proteins (ns- and ns- ) are highly conserved among both viruses whereas the capsid proteins (vp- , vp- , vp- ) are more divergent and determine the serogroup [ ] . both viruses require mitotically active cells for replication. severe clinical signs are therefore not found in mature animals because of the lack of a sufficient number of susceptible cells in tissues. general aspects of rodent parvovirus infections and their potential effects on research results have been reviewed [ , , [ ] [ ] [ ] [ ] [ ] . already in the mid- s mouse colonies were identified that gave positive reactions for mvm by ifa but not by hi tests. it was subsequently shown that these colonies were infected with a novel parvovirus, initially referred to as 'mouse orphan parvovirus'. the first isolate of mpv was detected as a contaminant of cultivated t-cell clones interfering with in vitro immune responses [ ] and was named 'mouse parvovirus'. it does not replicate well in currently available cell cultures, and sufficient quantities of virus for serological tests are difficult to generate. hitherto, only very few isolates of mpv have been cultured and subsequently characterized on a molecular basis [ , ] . on the basis of epidemiological analyses, further parvoviruses were recently identified in mice, sequenced, and tentatively named serially mpv- and mpv- [ ] , mpv- (genbank fj ) and mpv- (genbank fj ). in addition, several variants are published for mpv- [ , , ] . at present, mpv is among the most common viruses found in colonies of laboratory mice. the prevalence of sera positive for parvoviruses ranged from % to nearly % in western europe and north america, with the majority of sera being positive for mpv in studies differentiating between the two parvovirus species [ , , , ] . these prevalence data are based on testing at commercial laboratories and do not reflect that, despite highly specific and sensitive test methods, enzootic parvovirus infections are difficult to detect due to virus-associated characteristics [ , ] . a recent survey conducted in the usa showed that during a - month period mouse parvoviruses were detected at almost all facilities that responded to a questionnaire, with mpv being more often diagnosed than mvm [ ] . clinical disease and gross or histological lesions have not been reported for mice naturally or experimentally infected with mpv. infections are subclinical even in newborn and immunocompromised animals [ , ] . in contrast to many other viruses infecting mice, viral replication and excretion is not terminated by the onset of host immunity. tissue necrosis has not been observed at any stage of infection in infected infant or adult mice [ , ] . humoral immunity to mpv does not protect against mvm infections, and vice versa [ ] . serological surveys have indicated that mpv naturally infects only mice, with the exception that mpv- shows genetic similarity to hamster parvovirus, suggesting that a cross-species transmission has occurred, where the mouse probably served as the natural host [ , ] . differences in mouse strain susceptibility to clinical mpv infection do not exist. however, seroconversion seems to be strain-dependent. after experimental infection with mpv- b, seroconversion occurred in all c h/hen mice, fewer balb/c, dba/ and icr mice, and seroconversion could not be detected in c bl/ mice [ ] . upon mpv- f inoculation, antibody response was absent in balb/carc mice [ ] . diagnosis of mpv infection by pcr testing of small intestine and mesenteric lymph nodes also depended on the mouse strain. mpv dna was detected in all mouse strains evaluated except dba/ even though seroconversion was detected in these mice. after oral infection, the intestine is the primary site of viral entry and replication. the virus spreads to the mesenteric lymph nodes and other lymphoid tissues, where it persists for more than months [ ] , and seems to be excreted via the intestinal and the urinary tract. after experimental inoculation of weanling mice, mpv is transmitted to cagemates by direct contact for - weeks [ ] , and transmission by dirty bedding is also possible. these results implicate a role for urinary, faecal, and perhaps respiratory excretion of virus. another study showed that naturally infected mice might not transmit the virus under similar experimental conditions [ ] . serology is a useful tool to identify mpv infections in immunocompetent hosts, but reaching a diagnosis based on serological assays may be difficult and requires a good knowledge of the available techniques. neither the virion elisa nor hi is a practical screening test for mpv because they require large quantities of purified mpv, which is difficult to obtain. diagnosis of mpv infections has long been made on the basis of an mvm hi-negative result coupled with an mvm ifa-positive result. ifa provides the opportunity to detect both serogroup-specific vp proteins as well as ns proteins that are conserved among mouse parvoviruses. a generic rodent parvovirus elisa using a recombinant ns- protein as antigen has been developed [ ] , but mpv ifa and mpv hi assays are more sensitive techniques than the ns- elisa and the mvm ifa [ ] . in contrast, elisa tests that use recombinant vp- provide sensitive and serogroup-specific assays for the diagnosis of mpv infections in mice [ , ] , although considerable cross-reactivity with heterologous capsid antigens exists [ ] . nevertheless, when using the elisa technique, one needs to consider that mpv- may not consistently be detected by mpv- vp- elisa [ , ] , especially when antibody titres are low (own observations). therefore, elisas using mpv- vp- and mpv- vp- antigens are also used for diagnostics. as parvovirus diagnostics using recombinant assays should be based on a combination of antigens, bead-based mulitplex assays are a convenient extension of traditional elisa, allowing the use of multiple antigens simultaneously. in immunodeficient mice that do not generate a humoral immune response, pcr assays can be used to detect mpv [ , ] and other parvoviruses. mpv has been shown to persist for at least weeks in the mesenteric lymph nodes [ ] . this tissue is considered the best suited for pcr analysis, but spleen and small intestine can also be used with good success [ ] . for antemortem detection, shedding of parvoviruses can also be detected by pcr of faecal samples [ ] . the virus persists sufficiently long in mesenteric lymph nodes so that pcr assays may also be used as a primary screening tool for laboratories that do not have access to specific mpv antigen-based serological assays. the pcr is further a good confirmatory method for serological assays and has also been described for the detection of parvoviruses in cell lines and tumours [ ] . in addition, the map test has been reported as a sensitive tool to detect mpv [ ] . given the high environmental stability of the virus and the potential fomite transmission, together with the long virus persistence in infected animals, spontaneous disappearance from a mouse population (e.g. by cessation of breeding) is unlikely. eradication of infection is possible by elimination of infected animals and subsequent replacement with uninfected mice, and the agent can be eliminated from breeding populations by embryo transfer or by hysterectomy. it should be noted that recent studies suggest a risk of virus transmission by embryo transfer, though successful sanitation of immunodeficient mice was achieved despite antibody response in recipients and progeny after embryo transfer [ , ] . although there are few published reports of confounding effects of mpv on research, it is lymphocytotropic and may perturb immune responses in vitro and in vivo. infections with mpv have been shown to influence rejection of skin and tumour grafts [ ] . mvm is the type species of the genus parvovirus. the virus was intermediately named mice minute virus (mmv). it was originally isolated by crawford [ ] from a stock of mouse adenovirus, and this prototype isolate was later designated mvmp. its allotropic variant was detected as a contaminant of a transplantable mouse lymphoma [ ] and designated mvmi because it exhibits immunosuppressive properties in vitro. both variants have distinct cell tropisms in vivo and in vitro. mvmp infects fibroblast cell lines and does not cause clinical disease [ , ] . both strains are apathogenic for adult mice, but the immunosuppressive variant is more pathogenic for neonatal mice than is mvmp. a third strain, the cutter strain mvmc, was isolated from bhk- cells [ ] . in contrast to these three strains detected as cell culture contaminants, an isolate was obtained from naturally infected mice with a b-cell maturational defect maintained at the university of missouri and therefore denominated mvmm [ ] . serological surveys show that the mouse is the primary natural host [ , , ] , but the virus is also infective for rats, hamsters [ , ] , and mastomys [ ] during fetal development or after parenteral inoculation. natural infections are usually asymptomatic in adults and infants, and the most common sign of infection is seroconversion. kilham and margolis [ ] observed mild growth retardation a few days after experimental infection of neonatal mice with mvmp. studies of transplacental infection yielded no pathological findings in mice [ ] . the immunosuppressive variant, but not the prototype strain, is able to produce a runting syndrome after experimental infection of newborn mice [ ] . depending on the host genotype, experimental infections of fetal and neonatal mice with mvmi produce various clinical presentations and lesions. infection in c bl/ mice is asymptomatic, but the virus causes lethal infections with intestinal haemorrhage in dba/ mice. infection of strains such as balb/c, cba, c h/he and sjl is also lethal and mice have renal papillary haemorrhage [ ] . the mvmi also infects haematopoietic stem cells and mediates an acute myelosuppression [ , ] . because of its dependence on mitotically active tissues, the fetus is at particular risk for damage by parvoviruses. mvm and other parvoviruses may have severe teratogenic effects and cause fetal and neonatal abnormalities by destroying rapidly dividing cell populations, often resulting in fetal death. adult prkdc scid mice develop an acute leukopenia month after experimental infection with mvmi and die within months. the virus persists lifelong in the bone marrow of these mice [ ] . during a natural concurrent outbreak of mvmm and mpv, a runting syndrome with lymphohistiocytic renal inflammation and inclusion bodies in cells resembling splenic haematopoietic progenitor cells was reported in b-cell (ighm)deficient mice [ ] . mmv is shed in faeces and urine. in faecal samples, mvm was detected for up to - weeks by pcr [ , ] , although shorter periods ( - days) have been observed [ ] . notably, shedding re-occurred after immunosuppression by irradiation [ ] . contaminated food and bedding are important factors in viral transmission because the virus is very resistant to environmental conditions. direct contact is also important and the virus does not easily spread between cages. routine health surveillance is usually conducted by serological methods. unlike mpv, mvm can easily be cultured in cell lines so that antigen production for hi and elisa (using whole purified virions) is easy. hi is a highly specific diagnostic test whereas ifa always exhibits some degree of cross-reactivity with mpv and other closely related parvoviruses. elisa is probably the most frequently used test, but depending on the purity of the antigen preparation, cross-reactions with mpv may occur due to contamination with non-structural proteins that are common to both viruses. this problem can be avoided by the use of recombinant vp- antigen [ ] . by using serological methods, one needs to consider that the mouse strain has a considerable effect on seroconversion so that an antibody response might not be detectable despite infection; while c bl/ j mice showed good antibody response, seroconversion was observed only in some balb/c, akr/n, dba/ j, fvb/n and c h/hen, but not in nmri and icr mice upon contact exposure to mvmi-inoculated mice [ ] . viral detection is also possible by pcr in biological materials, organs (intestine, mesenteric lymph node, kidney, spleen) and faeces from infected animals [ , , , , ] . although mvm was not thought to cause persistent infection in immunocompetent mice, recent data show that it can be detected in spleens for up to weeks after exposure in some mouse strains [ ] . therefore, pcr may be considered as a confirmatory method for serology. the virus can be eliminated from infected breeding populations by caesarean derivation or by embryo transfer. however, certain precautions such as careful washing and accompanying testing need to be minded, as mvm has been detected in reproductive organs and gametes and this virus firmly attaches to the zona pellucida or might even cross it [ , ] . in experimental colonies, elimination of infected animals and subsequent replacement with uninfected mice is practical if careful environmental sanitation is conducted by appropriate disinfection procedures. it is important that reintroduction is avoided by exclusion of wild mice and by strict separation from other infected populations and potentially contaminated materials in the same facility. admission of biological materials must be restricted to samples that have been tested and found to be free from viral contamination. both allotropic variants of mvm have been used as models for molecular virology, and their small size and simple structure have facilitated examination of their molecular biology and expedited understanding of cell tropism, viral genetics and structure. the significance for laboratory mouse populations was considered low or uncertain because natural infections are inapparent. however, various effects on mouse-based research have been published [ , , , , ] . because of their predilection for replicating in mitotically active cells, they are frequently associated with tumour cells and have a marked oncosuppressive effect [ ] . special attention is also necessary for immunological research and other studies involving rapidly dividing cells (embryology, teratology). in addition, mvm is a common contaminant of transplantable tumours, murine leukaemias and other cell lines [ , , ] . lactate dehydrogenase-elevating virus (ldv) is a single-stranded rna virus of the genus arterivirus belonging to the family arteriviridae. the genome organization and replication of ldv and other arteriviruses, their cell biology and other molecular aspects have been reviewed by snijder and meulenberg [ ] . ldv has repeatedly been detected in wild mice (mus musculus), which are considered to be a virus reservoir [ , ] . after infection of mice, virus titres of - particles per ml serum are found within - h after infection. the virus titre drops to particles per ml within - weeks and remains constant at this level for life. it persists in infected mice for the whole lifetime although it stimulates various immune mechanisms [ ] [ ] [ ] [ ] . the virus can be stored in undiluted mouse plasma at À c without loss of infectivity, but it is not stable at room temperature and is very sensitive to environmental conditions. only mice and primary mouse cells are susceptible to infection with ldv. it replicates in a subpopulation of macrophages in almost all tissues and persists in lymph nodes, spleen, liver, and testes tissues [ ] . as suitable cell systems have not been available for virus production, routine serology has not been easily possible so that testing for ldv was not included in serological health monitoring programs. the prevalence of ldv in contemporary colonies of laboratory rodents is likely to be very low but detailed information about its prevalence comparable to most other agents is not available. ldv was first detected during a study of methods that could be used in the early diagnosis of tumours [ ] . it produces a persistent infection with continuous virus production and a lifelong viraemia despite ldv-specific immune reactions of the host [ ]. ldv has been found in numerous biological materials that are serially passaged in mice such as transplantable tumours including human tumours or matrigel prepared from such materials [ , , , ] , monoclonal antibodies or ascitic fluids [ ] , or infectious agents (e.g. haemoprotozoans, k virus, clostridium piliforme). these materials are contaminated after serial passage in an infected and viraemic mouse. contamination with ldv leads to the infection of each sequential host and to transmission of the virus by the next passage and remains associated with the specimen. it is therefore the most frequently detected contaminant in biological materials [ , ] . infection with ldv is usually asymptomatic, and there are no gross lesions in immunocompetent as well as in immunodeficient mice. the only exception is poliomyelitis with flaccid paralysis of hindlimbs developing in c and akr mice when they are immunosuppressed either naturally with ageing or experimentally. it has been shown that only mice harbouring cells in the cns that express a specific endogenous mulv are susceptible to poliomyelitis [ ] . the characteristic feature of ldv infection is the increased activity of lactate dehydrogenase (ldh) and other plasma enzymes [ , ] , which is due to the continuous destruction of permissive macrophages that are responsible for the clearance of ldh from the circulation. as a consequence, the activity of plasma ldh begins to rise by only h after infection and peaks - days after infection at - -fold normal levels, or can even be up to -fold in sjl/j mice. the enzyme activity declines during the next weeks but remains elevated throughout life. antigen-antibody complexes produced during infection circulate in the blood and are deposited in the glomeruli [ ] . in contrast to other persistent virus infections (e.g. lymphocytic choriomeningitis virus), these complexes do not lead to immune complex disease and produce only a very mild glomerulopathy. the only gross finding associated with ldv infection is mild splenomegaly. microscopically, necrosis of lymphoid tissues is visible during the first days of infection. in mouse strains that are susceptible to poliomyelitis, ldv induces lesions in the grey matter of the spinal cord and the brainstem. ldv is not easily transmitted between mice, even in animals housed in the same cage. fighting and cannibalism increase transmission between cagemates, most likely via blood and saliva. infected females transmit the virus to their fetuses if they have been infected few days prior to birth and before igg anti-ldv antibodies are produced, but developmental and immunological factors (e.g. gestational age, timing of maternal infection with ldv, placental barrier) are important in the regulation of transplacental ldv infection [ , ] . maternal immunity protects fetuses from intrauterine infection. immunodeficient prkdc scid mice also transmit virus to their offspring during chronic infection [ ] . an important means of transmission is provided by experimental procedures such as mouse-to-mouse passage of contaminated biological materials or the use of the same needle for sequential inoculation of multiple mice. in principle, serological methods such as ifa may be used for detecting ldv infection [ ] but they are not of practical importance. circulating virus-antibody complexes interfere with serological tests, and sufficient quantities of virus for serological tests are difficult to generate because ldv replicates only in specific subpopulations of primary cultures of murine macrophages and monocytes for one cell cycle [ ] . however, it is meanwhile possible to use recombinant viral proteins of ldv as antigens [ ] in elisa and mfia tests so that routine testing by serology is possible. in the past, diagnosis of ldv infection has primarily been based on increased ldh activity in serum or plasma of mice. ldv activity in serum or plasma can be measured directly, or samples (e.g. plasma, cell or organ homogenates) are inoculated into pathogen-free mice and the increase in ldh activity within - days is measured. an - -fold increase is indicative of ldv infection. detection of infectivity of a plasma sample by the induction of increased ldh activity in the recipient animal is the most reliable means of identifying an infected animal. however, it is important to use clear non-haemolysed samples because haemolysis will (falsely) elevate activities of multiple serum or plasma enzymes, including ldh. this assay was usually included in a 'map test', but antibody detection similar to other viruses was not involved for reasons mentioned earlier. persistent infection makes ldv an ideal candidate for pcr detection in plasma or in organ homogenates [ , ] . however, reports exist that pcr may produce false-negative results and should be used cautiously [ ] . just as important as detecting ldv in animals is its detection in biological materials. this may be done by assay for increased ldh activity after inoculation of suspect material into pathogenfree mice [ , ] or by pcr [ , , [ ] [ ] [ ] . ldv spreads slowly in a population because direct contact is necessary. therefore, ldv-negative breeding populations can easily be established by selecting animals with normal plasma ldh activity. embryo transfer and hysterectomy derivation are also efficient. the presence of ldv in experimental populations may be indicative of contaminated biological materials. in such cases, it is essential that the virus is also eliminated from these samples. this is easily achieved by maintenance of cells by in vitro culture instead of by animal-to-animal passages [ ] . due to the extreme host specificity of the virus, contaminated tumour samples can also be sanitized by passages in nude rats [ ] or other animal species. another method to remove ldv from contaminated cells, which is based on cell sorting, has recently been described [ ] . ldv is a potential confounder of any research using biological materials that are passaged in mice. once present in an animal, the virus persists lifelong. the most obvious signs are increased levels of plasma ldh and several other enzymes. ldv may also exhibit numerous effects on the immune system (thymus involution, depression of cellular immunity, enhanced or diminished humoral responses, nk cell activation, development of autoimmunity, and suppression of development of diabetes in nod mice); [ , , , [ ] [ ] [ ] [ ] [ ] and enhance or suppress tumour growth [ , , ] . interaction with other viruses has also been described [ ] . lymphocytic choriomeningitis virus (lcmv) is an enveloped, segmented single-stranded rna virus of the genus arenavirus family, arenaviridae. it can easily be propagated in several commonly used cell lines like bhk- cells. however, cells are not lysed and a cytopathic effect (cpe) is not visible. the virus name refers to the condition that results from experimental intracerebral inoculation of the virus into adult mice and is not considered to be a feature of natural infections. mice (mus musculus) serve as the natural virus reservoir [ ] , but syrian hamsters are also important hosts [ ] . additional species such as rabbits, guinea-pigs, squirrels, monkeys and humans are susceptible to natural or experimental infection [ ] . natural infection of callitrichid primates (marmosets and tamarins) leads to a progressive hepatic disease that is known as 'callitrichid hepatitis' [ , ] . antibodies to lcmv have been found in wild mice in europe [ , ] , africa [ ] , asia [ ] , australia [ ] and america [ ] . thus, it is the only arenavirus with worldwide distribution. infection with lcmv is rarely found in laboratory mice [ ] . seropositivity to lcmv in laboratory mice was reported to be low during the last decade [ , , , ] or negative [ ] [ ] [ ] . in addition to laboratory mice and other vertebrate hosts, the virus has frequently been found in transplantable tumours and tissue culture cell lines from mice and hamsters [ , ] . despite the low prevalence in laboratory mice, seropositivity to this zoonotic agent should raise serious concern for human health. lcmv is frequently transmitted to humans from wild mice and is also endemic to a varying degree in the human population [ ] [ ] [ ] [ ] [ ] due to contact with wild mice. it has also been transmitted to humans by infected laboratory mice [ ] and by pet and laboratory syrian hamsters [ ] [ ] [ ] [ ] . in addition, contaminated biological materials are important sources of infections for humans, and several outbreaks of lcm among laboratory personnel have been traced to transplantable tumours [ , ] . transmission of lcmv to humans also occurred repeatedly by organ transplantation and was most likely transmitted to organ donors by close contact with infected pets [ , ] . lcmv can cause mild-to-serious or fatal disease in humans [ , , ] . congenital infection in humans may result in hydrocephalus, or fetal or neonatal death [ ] . in mice, clinical signs of lcmv infection vary with strain and age of mouse, strain and dose of virus, and route of inoculation [ , , ] . two forms of natural lcmv infection are generally recognized: a persistent tolerant and an (acute) non-tolerant form. the persistent form results from infection of mice that are immunotolerant. this is the case if mice are infected in utero or during the first days after birth. this form is characterized by lifelong viraemia and viral shedding. mice may show growth retardation, especially during the first - weeks, but they appear otherwise normal. infectious virus is bound to specific antibodies and complement, and these complexes accumulate in the renal glomeruli, the choroid plexus, and sometimes also in synovial membranes and blood vessel walls. at - months of age, immune complex nephritis develops with ruffled fur, hunched posture, ascites and occasional deaths. this immunopathologic phenomenon is called 'late onset disease' or 'chronic immune complex disease'. the incidence of this type of disease varies between mouse strains. gross lesions include enlarged spleen and lymph nodes due to lymphoid hyperplasia. kidneys affected with glomerulonephritis may be enlarged with a granular surface texture or may be shrunken in later stages of the disease process. microscopically, there is generalized lymphoid hyperplasia and immune complex deposition in glomeruli and vessel walls, resulting in glomerulonephritis and plasmacytic, lymphocytic perivascular cuffs in all visceral organs [ ] . the non-tolerant acute form occurs when infection is acquired after the development of immunocompetence (in mice older than week). these animals become viraemic but do not shed virus and may die within a few days or weeks. natural infections of adults are usually asymptomatic. surviving mice are seropositive and in most cases clear the virus to below detection levels of conventional methods. however, virus may persist at low levels in tissues (particularly spleen, lung and kidney) of mice for at least weeks after infection as determined by sensitive assays such as nested reverse transcriptase (rt)-pcr or immunohistochemistry [ ] . such non-lethal infection leads to protection against otherwise lethal intracerebral challenge. protection from lethal challenge is also achieved by maternally derived anti-lcmv antibodies through nursing or by the administration of anti-ldv monoclonal igg a antibodies [ ] . in experimentally infected mice, the route of inoculation (subcutaneous, intraperitoneal, intravenous, intracerebral) also influences the type and degree of disease [ ] . intracerebral inoculation of adult immunocompetent mice typically results in tremors, convulsions and death due to meningoencephalitis and hepatitis. neurological signs usually appear on day after inoculation, and animals die within - days after the onset of symptoms, or recover within several days. the classic histological picture is of dense perivascular accumulations of lymphocytes and plasma cells in meninges and choroid plexus. while infection following subcutaneous inoculation usually remains inapparent, reaction of mice to intraperitoneal or intravenous inoculation depends on the virus strain and on the mouse strain. infection by these routes primarily causes multifocal hepatic necrosis and necrosis of lymphoid cells. athymic foxn nu mice and other immunodeficient mice do not develop disease but become persistently viraemic and shed virus. as a general rule, all pathological alterations following lcmv infection are immune-mediated; and mice can be protected from lcmvinduced disease by immunosuppression [ ] . lcmv disease is a prototype for virus-induced t-lymphocyte-mediated immune injury and for immune complex disease. for detailed information on the pathogenesis, clinical and pathological features of lcmv infection, the reader is referred to review articles [ , , ] . in nature, carrier mice with persistent infection serve as the principal source of virus. intrauterine transmission is very efficient, and with few exceptions all pups born from carrier mice are infected. furthermore, persistently infected mice and hamsters can shed large numbers of infectious virions primarily in urine, but also in saliva and milk. the virus can replicate in the gastric mucosa after intragastric infection [ , ] . gastric inoculation elicits antibody responses of comparable magnitudes as intravenous inoculation and leads to active infection with lcmv, indicating that oral infection is possible, e.g. by ingestion of contaminated food or by cannibalism. a self-limiting infection frequently results from infection of adult mice. the virus does not spread rapidly after introduction in populations of adult mice, and the infectious chain usually ends. however, if the virus infects a pregnant dam or a newborn mouse, a lifelong infection results, and soon a whole breeding colony of mice may become infected if the mice live in close proximity (which is the case under laboratory conditions). the virus is not easily transmitted to dirty-bedding sentinels, and it is important that colony animals or animals having had direct contact with a population are tested to exclude lcmv infection [ ] . lcmv is most commonly diagnosed by serological methods such as mfia, ifa and elisa [ ] . all strains show a broad cross-reactivity and are serologically uniform. however, subclinical persistent infections may be difficult to detect because they may be associated with minimal or undetectable levels of circulating antibody. it is important that bleeding of mice is done carefully because of a potential risk due to viraemic animals. historically, direct viral detection was performed by inoculating body fluids or tissue homogenates into the brain of lcmv-free mice or by subcutaneous injection into mice and subsequent serological testing (map test). more recently, pcr assays have been developed for the direct detection of viral rna in clinical samples or animals [ ] [ ] [ ] . both map test and pcr can also be used to detect contamination of biological materials [ , ] . specifically for exclusion of contamination by lcmv, it was requested by different authorities that virus is inoculated intracerebrally at a lethal dose - weeks after administration of the material to be tested. in case of contamination by lcmv and subsequent seroconversion, animals survive the challenge infection. vertical transmission of lcmv by transuterine infection is efficient so that this virus cannot reliably be eliminated by caesarean rederivation [ ] . caesarean derivation may be effective if dams acquired infection after the development of immunocompetence (non-tolerant acute infection) and subsequently eliminated the virus, but such a strategy is difficult to justify in light of lcmv's zoonotic potential. in breeding colonies of great value, virus elimination might be possible soon after introduction into the colony by selecting non-viraemic breeders. this procedure is expensive and time consuming and requires special safety precautions. fortunately, infections of laboratory mice with lcmv are very uncommon. however, once lcmv has been detected in animals, or in biological materials, immediate destruction of all contaminated animals and materials is advisable to avoid risk of human infection. foxn nu and prkdc scid mice may pose a special risk because infections are silent and chronic [ ] . cages and equipment should be autoclaved, and animal rooms should be fumigated with disinfectants such as formaldehyde, vaporized paraformaldehyde, hydrogen peroxide or other effective disinfectants. prevention of introduction into an animal facility requires that wild mice cannot get access to the facility. similarly important is screening of biological materials originating from mice and hamsters because these can be contaminated by lcmv. finally, it has been shown that the virus can also be introduced into a population by mice with an undetected infection [ ] . appropriate precautions are necessary for experiments involving lcmv, or lcmv-infected animals or materials. biological safety level (bsl) will be considered to be sufficient in most cases. bsl practices may be considered when working with infected animals owing to the increased risk of virus transmission by bite wounds, scratching or aerosol formation from the bedding. animal biosafety level (absl) practices and facilities are generally recommended for work with infected hamsters. appropriate precautions have been defined for different bsls or absls by cdc [ ] . lcmv is frequently utilized as a model organism to study virus-host interactions, immunological tolerance, virus-induced immune complex disease, and a number of immunological mechanisms in vivo and in vitro [ ] [ ] [ ] . accidental transmission may have a severe impact on various kinds of experiments [ , , , ] and also affect infection with other agents [ ] . mammalian orthoreoviruses (mrv) are nonenveloped, segmented double-stranded rna viruses of the family reoviridae, genus orthoreovirus. they have a wide host range and are ubiquitous throughout the world. the designation reo stands for respiratory enteric orphan and reflects the original isolation of these viruses from human respiratory and intestinal tract without apparent disease. the term 'orphan' virus refers to a virus in search of a disease. mammalian orthoreovirus can be grouped into three serotypes, numbered - . mammalian orthoreovirus- (synonyms: hepatoencephalomyelitis virus; echo virus) infection remains prevalent in contemporary mouse colonies and has been reported in wild mice [ , , ] . a study in france reported antibodies to mrv- in % of mouse colonies examined [ ] . in more recent studies in north america and western europe, such antibodies were detected in . - . % of mice monitored [ , , ] . schoondermark-van de ven et al. [ ] found antibodies to mrv- in . % of mouse samplings from western european institutions; and in a survey conducted by carty [ ] , about % of responding institutions in the usa reported mrv- infection in their mouse colonies. in addition, contamination of mouse origin tumours and cell lines by mrv- has been reported many times [ , , ] . experimentally, mrv- infection of infant mice has been used to model human hepatobiliary disease, pancreatitis, diabetes mellitus and lymphoma [ , ] . the literature on mrv- infections in mice is dominated by studies on experimentally infected animals. the virus can cause severe pantropic infection in infant mice [ ] [ ] [ ] . after parenteral inoculation, virus can be recovered from the liver, brain, heart, pancreas, spleen, lymph nodes and blood vessels. following oral inoculation, reoviruses gain entry by infecting specialized epithelial cells (m cells) that overlie peyer's patches. the virus then becomes accessible to leukocytes and spreads to other organs by way of the lymphatic system and the bloodstream. neural spread to the cns has also been well documented [ , ] . the mechanisms of viral pathogenesis and their interactions with the host cell as well as the host's immune response are reviewed in detail by tyler et al. [ ] , schiff et al. [ ] and ward et al. [ ] . natural infection by mrv- in a mouse colony is usually subclinical, although diarrhoea or steatorrhoea and oily hair effect in suckling mice may be noted [ , , [ ] [ ] [ ] . the latter term has been used to describe the matted, unkempt appearance of the hair coat that results from steatorrhoea due to pancreatitis, maldigestion and biliary atresia. in addition, runting (attributed to immune-mediated destruction of cells in the pituitary gland that produce growth hormone), transient alopecia, jaundice (due to excessive bilirubin in the blood, which is attributed to the liver pathology, especially biliary atresia) and neurological signs such as incoordination, tremors or paralysis may develop. when present in natural infections, clinical signs and lesions are similar to but milder than in experimental neonatal infections. early descriptions of naturally occurring disease may have been complicated by concurrent infections such as mhv (murine hepatitis virus) or murine rotavirus a (murv-a)/epizootic diarrhoea of infant mice (edim) virus that contributed to the severity of the lesions especially in liver, pancreas, cns and intestine. the outcome of mrv- infection depends on age and immunological status of mouse, dose of virus and route of inoculation. adult immunocompetent mice typically show no clinical signs and have no discernible lesions even in experimental infections. mucosal and maternally conferred immunity are considered to be important in protection from or resolution of disease [ , ] . experimental infection of adult prkdc scid mice is lethal [ ] . depending on the route of inoculation, experimental infection of adult foxn nu mice is subclinical or results in liver disease [ , ] . histological findings reported to occur after experimental mrv- infection of neonatal mice include inflammation and necrosis in liver, pancreas, heart, adrenal, brain, and spinal cord; lymphoid depletion in thymus, spleen, and lymph nodes; and hepatic fibrosis with biliary atresia [ , [ ] [ ] [ ] ] . transmission of reoviruses probably involves the aerosol as well as the faecal-oral route [ , ] . fomites may play an important role as passive vectors because reoviruses resist environmental conditions moderately well. serological screening with mfia, elisa or ifa is in widespread use for detection of antibodies to mrv- in diagnostic and health surveillance programmes. both elisa and ifa detect cross-reacting antibodies to heterologous mrv serotypes that can infect mice [ ] , although a recent report indicates that some ifa-positive mrv infections in mice may not be detected by commonly used elisas [ ] . the hi test does not detect such cross-reacting antibodies but is prone to give false-positive results due to nonspecific inhibitors of haemagglutination [ , ] . rt-pcr methods for the detection of mrv- rna [ , ] or mrv rna [ , ] are also available. reports on contamination of mouse origin tumours and cell lines by mrv- and its interference with transplantable tumour studies [ , ] emphasize the importance of screening of biological materials to be inoculated into mice by map test or pcr. natural seroconversion to mrv- without clinical disease is also observed in laboratory rats, hamsters and guinea-pigs [ , ] . caesarean derivation and barrier maintenance have proven effective in the control and prevention of mrv- infection [ , ] . the virus may interfere with research involving transplantable tumours and cell lines of mouse origin. it has the potential to alter intestinal studies and multiple immune response functions in mice. in enzootically infected colonies, protection of neonates by maternal antibody could complicate or prevent experimental infections with reoviruses. it could further complicate experiments that require evaluation of liver, pancreas, cns, heart, lymphoid organs and other tissues affected by the virus. the term murine hepatitis virus (mhv; commonly referred to as 'mouse hepatitis virus') designates a large group of antigenically and genetically related, single-stranded rna viruses belonging to the family coronaviridae, genus coronavirus. they are surrounded by an envelope with a corona of surface projections (spikes). mhv is antigenically related to rat coronaviruses and other coronaviruses of pigs, cattle and humans. numerous different strains or isolates of mhv have been described. they can be distinguished by neutralization tests that detect strainspecific spike (s) antigens, by use of monoclonal antibodies, or by sequencing [ ] . the beststudied strains are the prototype strains mhv- , mhv- , mhv- , jhm (mhv- ), a , and s, of which mhv- is regarded as the most virulent. like other coronaviruses mhv mutates rapidly, and strains readily form recombinants, so that new (sub)strains are constantly evolving. strains vary in their virulence, organotropism and cell tropism [ ] . based on their primary organotropism, mhv strains can be grouped into two biotypes: respiratory (or polytropic) and enterotropic. however, intermediate forms (enterotropic strains with tropism to other organs) also exist. murine hepatitis virus is relatively resistant to repeated freezing and thawing, heating ( c for min) and acid ph but is sensitive to drying and disinfectants, especially those with detergent activity [ ] . given the environmental conditions present in mouse rooms, mhv might remain infective for several days, at low humidity ( % relative humidity) or low temperatures ( c) even for weeks on surfaces [ ] . mus musculus is the natural host of mhv. it can be found in wild and laboratory mice throughout the world and is one of the most common viral pathogens in contemporary mouse colonies. while polytropic strains have historically been considered more common, this situation is thought to have reversed. monitoring results for research institutions across north america and europe indicate that the prevalence of mhv has decreased in the past, though it seems to have remained quite stable since the s [ , ] . recently . % of north american laboratory mouse serum samples tested positive [ ] . in europe, prevalence rates ranged from . % to % [ , , ] . a retrospective study in france covering the period from to reported antibodies to mhv in % of mouse colonies examined [ ] , and a survey performed in revealed that almost half of north american research institutions detected mhv in their mouse populations [ ] . suckling rats inoculated experimentally with mhv had transient virus replication in the nasal mucosa and seroconversion but no clinical disease [ ] . similarly, deer mice seroconverted but showed no clinical disease after experimental infection [ ] . mhv is also a common contaminant of transplantable tumours [ , ] and cell lines [ , ] . the pathogenesis and outcome of mhv infections depend on interactions between numerous factors related to the virus (e.g. virulence and organotropism) and the host (e.g. age, genotype, immune status, and microbiological status) [ , , , , , ] . mhv strains appear to possess a primary tropism for the upper respiratory or enteric mucosa. those strains with respiratory tropism initiate infection in the nasal mucosa and then may disseminate via blood and lymphatics to a variety of other organs because of their polytropic nature. respiratory (polytropic) strains include mhv- , mhv- , mhv- , a , s and jhm. infection of mice with virulent polytropic mhv strains, infection of mice less than weeks of age, infection of genetically susceptible strains of mice or infection of immunocompromised mice favour virus dissemination. virus then secondarily replicates in vascular endothelium and parenchymal tissues, causing disease of the brain, liver, lymphoid organs, bone marrow and other sites. infection of the brain by viraemic dissemination occurs primarily in immunocompromised or neonatal mice. additionally, infection of adult mouse brain can occur by extension of virus along olfactory neural pathways, even in the absence of dissemination to other organs. in contrast, enterotropic mhv strains (e.g. livim, mhv-d, mhv-y) tend to selectively infect intestinal mucosal epithelium, with no or minimal dissemination to other organs such as mesenteric lymph nodes or liver. all ages and strains are susceptible to active infection, but disease is largely age related. infection of neonatal mice results in severe necrotizing enterocolitis with high mortality within h. mortality and lesion severity diminish rapidly with advancing age at infection. adult mice develop minimal lesions although replication of equal or higher titres of virus occurs compared with neonates. the age-dependent decrease in severity of enterotropic mhv disease is probably related to the higher mucosal epithelium turnover in older mice, allowing more rapid replacement of damaged mucosa. another factor that is of considerable importance to the outcome of mhv infections is host genotype. for example, balb/c mice are highly susceptible to enterotropic mhv disease while sjl mice, at the other end of the spectrum, are highly resistant [ ] . unlike in polytropic mhv infection where resistance is correlated with reduced virus replication in target cells [ ] , enterotropic mhv grows to comparable titres in sjl and balb/c mice at all ages [ ] . therefore, the resistance of the sjl mouse to disease caused by enterotropic mhv seems to be mediated through an entirely different mechanism than resistance to polytropic mhv. furthermore, mouse genotypes that are susceptible to disease caused by one mhv strain may be resistant to disease caused by another strain [ ] . it is therefore not possible to strictly categorize mouse strains as susceptible or resistant. the genetic factors determining susceptibility versus resistance in mhv infections are as yet poorly understood. both polytropic and enterotropic mhv infections are self-limiting in immunocompetent mice. immune-mediated clearance of virus usually begins about a week after infection, and most mice eliminate the virus within - weeks [ , , ] . humoral and cellular immunity appear to participate in host defences to infection, and functional t cells are an absolute requirement [ ] [ ] [ ] [ ] . therefore, immunodeficient mice such as foxn nu and prkdc scid mice cannot clear the virus [ , ] . similarly, some genetically modified strains of mice may have deficits in antiviral responses or other alterations that allow the development of persistent mhv infection [ ] . recovered immune mice are resistant to reinfection with the same mhv strain but remain susceptible to repeated infections with different strains of mhv [ ] [ ] [ ] . similarly, maternal immunity protects suckling mice against homologous mhv strains but not necessarily against other strains [ , ] . however, maternal immunity, even to homologous strains, depends on the presence of maternally acquired antibody in the lumen of the intestine [ ] . therefore, the susceptibility of young mice to infection significantly increases at weaning. most mhv infections are subclinical and follow one of two epidemiological patterns in immunocompetent mice [ , ] . enzootic (subclinical) infection, commonly seen in breeding colonies, occurs when a population has been in contact with the virus for a longer period (e.g. several weeks). adults are immune (due to prior infection), sucklings are passively protected, and infection is perpetuated in weanlings. epizootic (clinical) infection occurs when the virus is introduced into a naive population (housed in open cages). the infection rapidly spreads through the entire colony. clinical signs depend upon the virus and mouse strains and are most evident in infant mice. typically, they include diarrhoea, poor growth, lassitude, and death. in infections due to virulent enterotropic strains, mortality can reach % in infant mice. some strains may also cause neurological signs such as flaccid paralysis of hindlimbs, convulsions and circling. adult infections are again usually asymptomatic. as the infection becomes established in the colony, the epizootic pattern is replaced by the enzootic pattern. in immunodeficient (e.g. foxn nu and prkdc scid ) mice, infection with virulent polytropic mhv strains is often rapidly fatal while less virulent strains cause chronic wasting disease [ ] . in contrast, adult immunodeficient mice can tolerate chronic infection by enterotropic mhv, with slow emaciation and diarrhoea, or minimal clinical disease [ , ] . subclinical mhv infections can be activated by a variety of experimental procedures (e.g. thymectomy, whole body irradiation, treatment with chemotherapeutic agents, halothane anaesthesia) or by coinfections with other pathogens (e.g. eperythrozoon coccoides, k virus; reviewed in [ , ] ). in most natural infections, gross lesions are not present or are transient and not observed. gross findings in neonates with clinical signs include dehydration, emaciation, and in contrast to edim, an empty stomach [ , , ] . the intestine is distended and filled with watery to mucoid yellowish, sometimes gaseous contents. haemorrhage or rupture of the intestine can occur. depending on the virus strain, necrotic foci on the liver [ , , ] and thymus involution [ , ] may also be seen in susceptible mice. liver involvement may be accompanied by jaundice and haemorrhagic peritoneal exudate. splenomegaly may occur as a result of compensatory haematopoiesis [ ] . histopathological changes in susceptible mice infected with polytropic mhv strains include acute necrosis with syncytia in liver, spleen, lymph nodes, gut-associated lymphoid tissue, and bone marrow [ , , , ] (figure . . ) . recently, pulmonary inflammation has been observed in susceptible mouse strains (c h/hej and a/j) after intranasal inoculation with polytropic mhv- [ , ] . neonatally infected mice can have vascular-oriented necrotizing (meningo)encephalitis with demyelination in the brainstem and periependymal areas. lesions in peritoneum, bone marrow, thymus and other tissues can be variably present. mice can develop nasoencephalitis due to extension of infection from the nasal mucosa along olfactory pathways to the brain, with meningoencephalitis and demyelination, the latter of which is thought to be largely t-cell mediated [ ] . this pattern of infection regularly occurs after intranasal inoculation of many mhv strains but is a relatively rare event after natural exposure. syncytium arising from endothelium, parenchyma or leukocytes is a hallmark of infection in many tissues including intestine, lung, liver, lymph nodes, spleen, thymus, brain and bone marrow. lesions are transient and seldom fully developed in adult immunocompetent mice, but they are manifest in immunocompromised mice. highly unusual presentations can occur in mice with specific gene defects. for example, granulomatous peritonitis and pleuritis were found in interferongamma-deficient mice infected with mhv [ ] . histopathological changes caused by enterotropic strains of mhv are mainly confined to the intestinal tract and associated lymphoid tissues [ , , , ] . the most common sites are terminal ileum, caecum and proximal colon. the severity of disease is primarily age-dependent, with neonatal mice being most severely affected. these mice show segmentally distributed areas of villus attenuation, enterocytic syncytia (balloon cells) and mucosal necrosis accompanied by leukocytic infiltration. intracytoplasmic inclusions are present in enterocytes. erosions, ulceration, and haemorrhage may be seen in more severe cases. lesions can be fully developed within - h, but are usually more severe at - days after infection. surviving mice may develop compensatory mucosal hyperplasia. mesenteric lymph nodes usually contain lymphocytic syncytia, and mesenteric vessels may contain endothelial syncytia. pathological changes in older mice are generally much more subtle and may only consist of transient syncytia. an occasional exception seems to occur in immunodeficient animals such as foxn nu mice, which can develop chronic hyperplastic typhlocolitis of varying severity [ ] , but other agents such as helicobacter spp. may have been involved. in general, enterotropic mhv strains do not disseminate, but hepatitis and encephalitis can occur with some virus strains in certain mouse genotypes. in t-cell deficient mice, multisystemic lethal infection was observed after experimental infection with the enterotropic strain mhv-y [ ] . mhv is highly contagious. it is shed in faeces and nasopharyngeal secretions and appears to be transmitted via direct contact, aerosol and fomites [ , ] . vertical (in utero) transmission has been demonstrated in experimental infections [ ] but does not seem to be of practical importance under natural conditions. mhv was transmitted by ovarian transplantation after reproductive organs became infected [ ] . however, risk of mhv transmission by sperm or oocytes (ivf) or by embryo transfer seems to be low, though thorough washing of gametes and embryos is required [ , [ ] [ ] [ ] . diagnosis during the acute stage of infection can be made by histological demonstration of characteristic lesions with syncytia in target tissues, but clinical signs and lesions can be highly variable and may not be prominent. suckling, genetically susceptible or immunocompromised mice are the best candidates for evaluation. active infection can be confirmed by immunohistochemistry [ ] or by virus isolation. virus recovery from infected tissues is difficult but can be accomplished using primary macrophage cultures or a number of established cell lines such as nctc or dbt [ ] . these cells, however, may not be successful substrates for some enterotropic mhv strains. virus in suspect tissue can also be confirmed by bioassays such as map testing or infant or foxn nu mouse inoculation [ , ] . amplification by passage in these mice increases the likelihood of detection of lesions and antigen, or virus recovery. other direct diagnostic methods that have been successfully utilized to detect mhv in faeces or tissue of infected mice include monoclonal antibody solution hybridization assay [ ] and a number of rt-pcr assays [ ] [ ] [ ] [ ] . because of the transient nature of mhv infection in immunocompetent mice, serology is the most appropriate diagnostic tool for routine monitoring. multiplex fluorescent immunoassay, elisa and ifa are well established and sensitive, and all known mhv strains cross-react in these tests [ , , ] . the magnitude of antibody response depends on mhv strain and mouse genotype [ , ] . dba/ mice are poor antibody responders whereas c bl/ mice produce a high antibody titre and are therefore good sentinels. antibody titres remain high over a period of at least months [ , ] . infected mice may not develop detectable antibodies for up to days after initial exposure [ ] . in such cases, a direct diagnostic method, as discussed above, may be useful. another drawback of serology is that mice weaned from immune dams can have maternal antibodies until they are weeks of age [ ] . this may impact serological monitoring because the possibility must be considered that low positive results are due to maternally derived passive immunity. because the virus can be transmitted by transplantable tumours and other biological materials from mice, including hybridomas [ ] and embryonic stem cells [ , ] these materials should also be routinely screened for mhv contamination. mouse inoculation bioassay, map test and rt-pcr can be used for this purpose. therefore, surveillance programmes should combine careful evaluation of clinically ill animals, testing of biological materials and routine health monitoring. soiled-bedding sentinel mice, which are frequently used for routine monitoring, are likely well suited for detecting enterotropic strains of mhv, but might not indicate the presence of less contagious respiratory strains of mhv [ ] equally well. the mouse strain used as sentinel should be considered as a critical factor. furthermore, duration of mhv shedding and stability of the virus, which seems to be lower in static microisolator cages than in ivc cages, might interfere with detection. the amount of bedding transferred seems not to be as critical as for, e.g. parvoviruses, at least for enterotropic strains [ ] . use of contact and exhaust air sentinels and testing of exhaust filters by pcr was also shown to be effective at detecting mhv [ ] . the best means of mhv control is to prevent its entry into a facility. this can be accomplished by purchasing mice from virus-free sources and maintenance under effective barrier conditions monitored by a well-designed quality assurance programme. control of wild mouse populations, proper husbandry and sanitation, and strict monitoring of biological materials that may harbour virus are also important measures to prevent infection. if infection occurs, the most effective elimination strategy is to cull the affected colony and obtain clean replacement stock. however, this is not always a feasible option when working with valuable mice (e.g. genetically modified lines, breeding stocks). caesarean derivation or embryo transfer can be used to produce virus-free offspring, and foster-nursing has also been reported to be effective [ ] . quarantine of an affected colony with no breeding and no introduction of new animals for approximately months has been effective in immunocompetent mice [ ] . the infection is likely to be terminated because mhv requires a constant supply of susceptible animals. this method works best when working with small numbers of mice. large populations favour the development of new mhv strains that may result in repeated infections with slightly different strains [ ] . it may be practical to select a few future breeders from the infected population and quarantine them for approximately weeks [ ] . this can be achieved in isolators, or in individually ventilated cages if proper handling is guaranteed. after this interval, breeding can resume. the -week interval should permit recovery from active infection, and the additional -week gestation period effectively extends the total quarantine to weeks. it is advisable to select seropositive breeders because the possibility of active infection is lower in such animals. the breeding cessation strategy may not be successful if immunodeficient mice are used because they are susceptible to chronic infection and viral excretion [ ] . genetically engineered mice of unclear, unknown or deficient immune status pose a special challenge because they may develop unusual manifestations of infection or may be unable to clear virus. rederivation is likely to be the most cost-effective strategy in such situations. along with the measures described, proper sanitation and disinfection of caging and animal quarters, as well as stringent personal sanitation, are essential to eliminate infection. careful testing with sentinel mice should be applied to evaluate the effectiveness of rederivation. if transplantable tumours are contaminated with mhv, virus elimination can be achieved by passage of tumours in athymic foxn rnu rats [ ] . mhv is one of the most important viral pathogens of laboratory mice and has been intensively studied from a number of research perspectives (e.g. as a model organism for studying coronavirus molecular biology or the pathogenesis of viral-induced demyelinating disease). numerous reports document the effects of natural and experimental infections with mhv on host physiology and research, especially in the fields of immunology and tumour biology (reviewed in [ - , , , ] ). noroviruses are non-enveloped, single-stranded rna viruses with high environmental resistance and belong to the family caliciviridae, genus norovirus. they were first identified after an outbreak of acute gastroenteritis at a school in norwalk (ohio, usa) in and cause about % of non-bacterial epidemic gastroenteritis in humans. noroviruses found in animals include bovine, porcine and murine noroviruses. noroviruses are not known to cross species. murine norovirus (mnv) is endemic in many research mouse colonies and currently the most commonly detected viral agent in laboratory mice [ , , ] . in the hitherto largest survey [ ] , about % of mouse serum samples examined had antibodies against mnv. the first norovirus to infect mice was described in [ ] . experimental inoculation studies with this murine norovirus (mnv- ) show that duration of infection and disease manifestation vary depending on the mouse strain [ ] [ ] [ ] . in immunocompetent strains, mnv infection is variable in length (e.g. ! - days in s mice, ! weeks in hsd:icr mice) and does not induce clinical signs. infection is associated with mild histopathological alterations in the small intestine (increase in inflammatory cells) and spleen (red pulp hypertrophy and white pulp activation) of s mice. in certain immunodeficient strains, however, infection can cause lethal systemic disease (encephalitis, vasculitis, meningitis, hepatitis and pneumonia in interferon-alpha-beta-gamma-receptor-deficient and stat tm mice) or persist without symptoms (! days in rag À/À and rag À/À mice). these findings indicate that components of the innate immune system are critical for resistance to mnv- induced disease. consistent with this hypothesis, it was demonstrated that mnv- replicates in macrophages and dendritic cells [ ] . meanwhile, many additional strains of mnv with diverse biological properties were isolated [ , ] . an analysis of mnv isolates revealed distinct mnv strains that comprise a single genogroup and serotype [ ] . experimental inoculation studies show that several mnv strains are able to persist in various tissues (small intestine, caecum, mesenteric lymph node, spleen) of immunocompetent (c bl/ j, hsd:icr, jcl:icr) and immunodeficient (cb -prkdc scid ) mice with viral shedding in faeces for the duration of at least - days [ ] [ ] [ ] . murine norovirus is transmitted via the faecaloral route and is efficiently transferred to sentinel mice by soiled bedding [ , ] . mnv infection can be detected directly by rt-pcr on faecal pellets or tissue specimens (see above) and indirectly by serology (mfia, elisa, ifa) [ , , ] . detection is facilitated by high stability of mnv rna in faeces (at least weeks at room temperature) [ ] and by broad serological cross-reactivity among different strains of mnv [ , ] . embryo transfer [ ] and hysterectomy [ ] are most likely effective means of eliminating mnv from mouse colonies. since -to -day-old pups are resistant to infection, elimination of mnv may also be achieved by transferring neonates from infected dams to uninfected foster dams ('cross-fostering') [ ] . this transfer should ideally be performed within h after birth. mnv is used as a surrogate to evaluate resistance of human noroviruses to disinfectants. the impact of mnv on animal experiments remains to be evaluated. recent studies show that mnv is immunmodulatory and may alter disease phenotypes in mouse models of inflammatory bowel disease [ ] [ ] [ ] and other experimental mouse models [ , ] . murine pneumonia virus, commonly referred to as 'pneumonia virus of mice' (pvm), is an enveloped, single-stranded rna virus of the family paramyxoviridae, genus pneumovirus. it is closely related to human respiratory syncytial virus (hrsv). the virus name is officially abbreviated as 'mpv' according to the international union of microbiological societies [ ] ; however, the former designation 'pvm' will be used in this chapter to avoid confusion with the official abbreviation of mouse parvovirus (mpv). pvm infection remains prevalent in contemporary colonies of mice and rats throughout the world. a serological survey in france demonstrated antibodies to pvm in % of mouse colonies examined [ ] . in more recent studies in north america and western europe, the prevalence of pvm-specific antibodies in mice ranged between % and . % [ , , ] . schoondermark-van de ven et al. [ ] found antibodies to pvm in . % of mouse samplings from western european institutions. antibodies to pvm have also been detected in hamsters, gerbils, cotton rats, guinea-pigs and rabbits [ , , ] . experimentally, pvm infection of mice is used as a model for hrsv infection and has therefore been extensively studied (reviewed by rosenberg and domachowske [ ] ). in immunocompetent mice, natural infection with pvm is transient and usually not associated with clinical disease or pathological findings [ , , ] . however, natural disease and persistent infection may occur in immunodeficient mice [ ] [ ] [ ] . in particular, athymic foxn nu mice seem to be susceptible to pvm infection, which can result in dyspnoea, cyanosis, emaciation and death due to pneumonia [ , ] . similar clinical signs have been reported for experimentally infected immunocompetent mice [ ] . necropsy findings in naturally infected foxn nu mice include cachexia and diffuse pulmonary oedema or lobar consolidation [ ] . pulmonary consolidation (dark red or grey in color) has also been found after experimental infection of immunocompetent mice [ ] . histologically, natural infection of foxn nu mice with pvm presents as interstitial pneumonia [ , ] . experimental intranasal inoculation of immunocompetent mice can result in rhinitis, erosive bronchiolitis and interstitial pneumonia with prominent early pulmonary eosinophilia and neutrophilia [ , ] . hydrocephalus may result from intracerebral inoculation of neonatal mice [ ] . susceptibility to infection is influenced by age and strain of mouse, dose of virus, and a variety of local and systemic stressors [ , , ] . in terms of the extent of the alveolar inflammatory response, /sv and dba/ mice are susceptible to pvm infection, while balb/c and c bl/ mice are relatively resistant [ ] . in terms of the control of viral replication, mice of strains /sv, dba/ , balb/c and c bl/ are susceptible to pvm infection, while sjl mice are relatively resistant. pvm is labile in the environment and rapidly inactivated at room temperature [ , ] . the virus is tropic for the respiratory epithelium [ , ] , and transmission is exclusively horizontal via the respiratory tract, mainly by direct contact and aerosol [ , ] . therefore, transmissibility in mouse colonies is low, and infections tend to be focal enzootics. serology (mfia, elisa, ifa or hi) is the primary means of testing mouse colonies for exposure to pvm. immunohistochemistry has been applied to detect viral antigen in lung sections [ , ] ; however, proper sampling (see chapter . , 'health management and monitoring') is critical for establishing the diagnosis due to the focal nature of the infection. an rt-pcr assay to detect viral rna in respiratory tract tissues has also been reported [ ] . however, the use of direct methods requires good timing because the virus is present for only up to about days in immunocompetent mice [ ] . embryo transfer or caesarean derivation followed by barrier maintenance can be used to rear mice that are free of pvm. because active infection is present in the individual immunocompetent mouse for only a short period, strict isolation of a few (preferably seropositive) mice with the temporary cessation of breeding might also be successful in eliminating the virus [ , ] . pvm could interfere with studies involving the respiratory tract or immunological measurements in mice. in addition, pvm can have devastating effects on research using immunodeficient mice because they are particularly prone to develop fatal disease [ , ] or become more susceptible to the deleterious effects of other agents such as p. murina [ ] . murv-a/edim (commonly referred to as 'mouse rotavirus' or 'epizootic diarrhoea of infant mice virus') is a non-enveloped, segmented double-stranded rna virus of the family reoviridae, genus rotavirus. it is antigenically classified as a group a rotavirus, similar to rotaviruses of many other species that cause neonatal and infantile gastroenteritis [ ] . murv-a/edim infection remains prevalent in contemporary mouse colonies and appears to occur worldwide. large commercial laboratories found . % to % of mouse sera from north american and european facilities to be positive for antibodies against murv-a/edim [ , , , ] , and up to % of mouse colonies in the usa were identified as affected in a survey performed in [ ] . experimentally, murv-a/edim infection in mice is used as a model for human rotavirus infection, especially in investigations on the mechanisms of rotavirus immunity and in the development of vaccination strategies [ ] . clinical symptoms following murv-a/edim infection range from inapparent or mild to severe, sometimes fatal, diarrhoea. 'epizootic diarrhoea of infant mice' describes the clinical syndrome associated with natural or experimental infection by murv-a/edim during the first weeks of life [ , , , , ] . diarrhoea usually begins around h after infection and persists for about week. affected suckling mice have soft, yellow faeces that wet and stain the perianal region (figure . . ) . in severe instances, the mice may be stunted, have dry scaly skin, or are virtually covered with faecal material. morbidity is very high but mortality is usually low. gross lesions in affected mice are confined to the intestinal tract. the caecum and colon may be distended with gas and watery to paste-like contents that are frequently bright yellow. the stomach of diarrhoeic mice is almost always filled with milk, and this feature has been reported to be a reliable means to differentiate diarrhoea caused by rotavirus from the diarrhoea caused by mhv infection. histopathological changes may be subtle even in animals with significant diarrhoea (figure . . ). they are most prominent at the apices of villi, where rotaviruses infect and replicate within epithelial cells; the large intestinal surface mucosa may also be affected. though inflammation is minimal, the lamina propria may be oedematous, lymphatics may be dilated and mild leukocytic infiltration in the large intestinal mucosa and submucosa has been observed in a recent outbreak of disease [ , ] . hydropic change of villous epithelial cells is the hallmark finding of acute disease. the villi become shortened, and the cells that initially replace the damaged cells are less differentiated, typically cuboidal instead of columnar, and lack a full complement of enzymes for digestion and absorption, resulting in diarrhoea due to maldigestion and malabsorption. undigested milk in the small intestine promotes bacterial growth and exerts an osmotic effect, exacerbating damage to the villi. intestinal fluid and electrolyte secretion is further enhanced by activation of the enteric nervous system [ ] and through the effects of a viral enterotoxin called nsp (for non-structural protein ) [ ] . it is hypothesized that nsp is released from virus-infected cells and then triggers a signal transduction pathway that alters epithelial cell permeability and chloride secretion. susceptibility to edim depends on the age of the host and peaks between and days of age [ , , , , ] . mice older than about weeks can still be infected with murv-a/edim, but small numbers of enterocytes become infected, there is little replication of virus and diarrhoea does not occur. the exact reason for this agerelated resistance to disease is unknown. pups suckling from immune dams are protected against edim during their period of disease susceptibility [ ] . in general, the infection is self-limiting and resolves within days. successful viral control and clearance is promoted by an intact immune response [ ] [ ] [ ] [ ] , and some immunodeficient mice (e.g. prkdc scid and rag tm fwa mice) may shed virus for extended periods or become persistently infected [ , ] . protection against murv-a/edim reinfection is primarily mediated by antibodies [ , ] . murine rotavirus-a/edim is highly contagious and transmitted by the faecal-oral route [ , , ] . dissemination of the virus occurs through direct contact or contaminated fomites and aerosols and is facilitated by the general property of rotaviruses that they remain infectious outside the body, show resistance to inactivation (e.g. low ph, non-ionic detergents, hydrophobic organic liquids, proteolytic enzymes), and are shed in high quantities (> particles/g faeces) [ ] . murv-a/edim is stable at À c but otherwise tends to be susceptible to extreme environmental conditions, detergents and disinfectants containing phenols, chlorine or ethanol [ ] . mfia, elisa and ifa are in widespread use for detection of serum antibodies to murv-a/ edim in diagnostic and health surveillance programmes; other assay systems such as those using latex agglutination are also used [ ] . as murv-a/edim shares the vp protein determined group a antigen, for example, with human, simian or bovine rotavirus strains, commercially available elisa assays utilizing polyclonal or monoclonal antibodies have been used to detect rotavirus antigen in mice; however, great care must be taken in interpreting the results because some feeds have been reported to cause false-positive reactions with certain elisa kits [ ] . electron microscopy of faeces of diarrhoeic pups should reveal typical wheel-shaped rotavirus particles, - nm in diameter. rt-pcr also can be used to detect rotavirus rna in faecal samples [ ] . good timing is critical for establishing the diagnosis from faeces because virus is shed for only a few days in immunocompetent mice. embryo transfer or caesarean derivation followed by barrier maintenance is recommended for rederivation of breeding stocks [ ] . in immunocompetent mice in which infection is effectively cleared, a breeding suspension strategy for - weeks combined with excellent sanitation, filter tops and conscientious serological testing of offspring and sentinel mice has also been reported to be effective, and prolongation of breeding cessation up to weeks resolved infection even in immunocompromised mice [ ] . murv-a/edim has the potential to interfere with any research using suckling mice. it may have a significant impact on studies where the intestinal tract of neonatal or infant mice is the target organ. the infection also poses a problem for infectious disease and immune response studies, particularly those involving enteropathogens in infant mice [ ] . a disease-induced stress-related thymic necrosis may occur and alter immunology experiments [ ] . in addition, runting could be interpreted erroneously as the effect of genetic manipulation or other experimental manipulation. sendai virus (sev) is an enveloped, singlestranded rna virus of the family paramyxoviridae, genus respirovirus. it is antigenically related to human parainfluenza virus . the virus was named after sendai, japan, where it was first isolated from mice. historically, infections were relatively common in mouse and rat colonies worldwide. in addition, there is evidence that hamsters, guinea-pigs and rabbits are susceptible to infection with sev [ , , , ] ; however, some apparently seropositive guinea-pigs may in fact be seropositive to other parainfluenza viruses instead of sev. a study in france reported antibodies to sev in % of mouse colonies examined [ ] . a low rate of seropositive mice ( . %) was found in a survey in north america [ ] . schoondermark-van de ven et al. [ ] also found antibodies to sev in . % of mouse samplings from western european institutions. in more recent surveys in north america and western europe, sev infection was not detected [ ] [ ] [ ] , indicating that sev, like most viruses, has meanwhile been eliminated from the majority of mouse colonies. sev can contaminate biological materials [ ] . sev is pneumotropic and can cause significant respiratory disease in mice. the pneumotropism is partially a consequence of the action of respiratory serine proteases such as tryptase clara, which activate viral infectivity by specific cleavage of the viral fusion glycoprotein [ ] . in addition, the apical budding behaviour of sev may hinder the spread of virus into subepithelial tissues and subsequently to distant organs via the blood. two epidemiologic patterns of sev infection have been recognized, an enzootic (subclinical) and epizootic (clinically apparent) type [ , , ] . enzootic infections commonly occur in breeding or open colonies, where the constant supply of susceptible animals perpetuates the infection. in breeding colonies, mice are infected shortly after weaning as maternal antibody levels wane. normally, the infection is subclinical, with virus persisting for approximately weeks, accompanied by seroconversion that persists for a year or longer. epizootic infections occur upon first introduction of the virus to a colony and either die out (self-cure) after - months or become enzootic depending on colony conditions. the epizootic form is generally acute, and morbidity is very high, resulting in nearly all susceptible animals becoming infected within a short time. clinical signs vary and include rough hair coat, hunched posture, chattering, respiratory distress, prolonged gestation, death of neonates and sucklings and runting in young mice. breeding colonies may return to normal productivity within months and thereafter maintain the enzootic pattern of infection. factors such as strain susceptibility, age, husbandry, transport and copathogens are important in precipitating overt disease. dba and strains of mice are very susceptible to sev pneumonia, whereas sjl/j and c bl/ /j and several outbred stocks are relatively resistant. resistance to sev infection is under multigenic control with epistatic involvement [ ] . there is no evidence for persistent infection in immunocompetent mice, but persistent or prolonged infection may occur in immunodeficient mice and can result in wasting and death due to progressive pneumonia [ , ] . clearance of a primary sev infection is mediated by cd þ and cd þ t-cell mechanisms [ , ] . heavier than normal, consolidated, plumcolored or grey lungs are a characteristic gross finding in severe sev pneumonia [ , , , ] . lymphadenopathy and splenomegaly reflect the vigorous immune response to infection. histologically, three phases of disease can be recognized in susceptible immunocompetent mice: acute, reparative and resolution phases [ , ] . lesions of the acute phase, which lasts - days, are primarily attributed to the cellmediated immune response that destroys infected respiratory epithelial cells and include necrotizing rhinitis, tracheitis, bronch(iol)itis and alveolitis. epithelial syncytiae and cytoplasmic inclusion bodies in infected cells may be seen early in this phase. alveoli contain sloughed necrotic epithelium, fibrin, neutrophils and mononuclear cells. atelectasis, bronchiectasis and emphysema may occur as a result of damage and obstruction of airways. the reparative phase, which may overlap the acute phase but continues through about the third week after infection, is indicated by regeneration of airway lining epithelium. adenomatous hyperplasia and squamous metaplasia (with multilayered flat epithelial cells instead of normal columnar cells) in the terminal bronchioles and alveoli are considered to be a hallmark of sev pneumonia. mixed inflammatory cell infiltrates in this phase tend to be primarily interstitial, rather than alveolar, as they are in the acute phase. the resolution phase may be complete by the fourth week after infection and lesions may be difficult to subsequently identify. residual, persistent lesions that may occur include organizing alveolitis and bronchiolitis fibrosa obliterans. alveoli and bronchioles are replaced by collagen and fibroblasts, foamy macrophages and lymphoid infiltrates, often with foci of emphysema, cholesterol crystals and other debris, which represent attempts to organize and wall off residual necrotic debris and fibrin. lesions are more severe and variable when additional pathogens such as mycoplasma pulmonis are present [ ] . otitis media has also been reported in natural infections with sev although some of these studies have been complicated by the presence of other pathogens [ ] . sev has been detected in the inner ear after experimental intracerebral inoculation of neonatal mice [ ] . sev is extremely contagious. infectious virus is shed during the first weeks of infection and appears to be transmitted by direct contact, contaminated fomites and respiratory aerosol [ , ] . serology (mfia, elisa, ifa, or hi) is the approach of choice for routine monitoring because serum antibodies to sev are detectable soon after infection and persist at high levels for many months, although active infection lasts only - weeks in immunocompetent mice. the short period of active infection limits the utility of direct methods such as immunohistochemistry [ ] and rt-pcr [ , ] . although sev is considered to be highly contagious, studies have shown that dirty bedding sentinel systems do not reliably detect the infection and that outbred stocks may not seroconvert consistently [ , ] . map testing and rt-pcr can be used to detect sev in contaminated biological materials. sev infection in mouse colonies has proved to be one of the most difficult virus infections to control because the virus is highly infectious and easily disseminated. depopulation of infected colonies is probably the most appropriate means of eliminating the virus in most situations. embryo transfer, or caesarean derivation, followed by barrier maintenance, can also be used to eliminate the virus [ , ] . a less effective alternative is to place the infected animals under strict quarantine, remove all young and pregnant mice, suspend all breeding and prevent addition of other susceptible animals for approximately months until the infection is extinguished, and then breeding and other normal activities are resumed. vaccines against the virus have been developed [ , , ] , but these probably do not represent a practical means to achieve or maintain the seronegative status of colonies that is in demand today. sev has the potential to interfere with a wide variety of research involving mice. reported effects include interference with early embryonic development and fetal growth; alterations of macrophage, nk-cell, and t-and b-cell function; altered responses to transplantable tumours and respiratory carcinogens; altered isograft rejection; and delayed wound healing (reviewed in [ ] [ ] [ ] ). pulmonary changes during sev infection can compromise interpretation of experimentally induced lesions and may lead to opportunistic infections by other agents. they could also affect the response to anaesthetics. in addition, natural sev infection would interfere with studies using sev as a gene vector. theiler's murine encephalomyelitis virus (tmev), or murine poliovirus, is a member of the genus cardiovirus in the family picornaviridae. members of this genus are non-enveloped viruses with single-stranded rna. the virus is rapidly destroyed at temperatures above c. it is considered to be a primary pathogen of the cns of mice and can cause clinical disease resembling that due to poliomyelitis virus infections in humans. antibodies to tmev have been identified in mouse colonies and feral populations worldwide, and mus musculus is considered to be the natural host of tmev [ ] . the best-known and most frequently mentioned tmev strain is gdvii, which is virulent for mice. infant or young hamsters and laboratory rats are also susceptible to intracerebral infection. the original isolate is designated to (theiler's original) and represents a group of tmev strains with low virulence for mice. many additional virus strains have been isolated and studied, and they all fall in the broad grouping of to and gdvii. a similar virus strain has also been isolated from rats, but in contrast to mouse isolates, this virus is not pathogenic for rats and mice after intracerebral inoculation [ ] . recently, another rat isolate has been characterized and shown to be most closely related to, but quite distinct from, other tmev viruses [ ] . antibodies to tmev (strain gdvii) have been detected in guinea-pigs and are considered to indicate infection with another closely related cardiovirus [ ] . seropositivity to tmev was reported in approximately % of french mouse colonies in a retrospective study [ ] . in more recent studies, the prevalence of tmev infections was found to be lower. schoondermark-van de ven et al. [ ] detected antibodies to tmev in . % of mouse samplings from western european institutions. in a survey conducted by carty [ ] , about % of responding institutions in the usa reported tmev infection in their mouse colonies. further surveys in north america and western europe revealed antibodies in . - . % of mice monitored [ , , ] . tmev is primarily an enteric pathogen, and virus strains are enterotropic. in natural infections, virus can be detected in intestinal mucosa and faecal matter, and in some cases it is also found in the mesenteric lymph nodes. however, histological lesions in the intestine are not discerned. virus may be shed via intestinal contents for up to weeks, sometimes intermittently [ ] , and transmission under natural conditions is via the faecal-oral route, by direct contact between mice, as well as by indirect contact (e.g. dirty bedding). the host immune response limits virus spread, but it does not immediately terminate virus replication in the intestines. virus is cleared from extraneural tissues, but persists in the cns for at least a year. clinical disease due to natural tmev infection is rare, with a rate of only in - infected immunocompetent animals [ ] . in immunodeficient mice, especially in weanlings, clinical signs may be more common and mortality may be higher [ ] . this group of viruses usually causes asymptomatic infections of the intestinal tract. they may spread to the cns as a rare event where they cause different neurological disease manifestations. the most typical clinical sign of tmev infection is flaccid paralysis of hindlimbs. the animals appear otherwise healthy, and there is no mortality. experimental infection in mice provides models of poliomyelitis-like infection and virusinduced demyelinating disease including multiple sclerosis [ ] . after experimental infection, tmev causes a biphasic disease in susceptible strains of mice. the acute phase is characterized by early infection of neurons in the grey matter. encephalomyelitis may develop during this phase and may be fatal, but most animals survive and enter the second phase of the disease at - months after the acute phase. this phase is characterized by viral persistence in the spinal cord white matter, mainly in macrophages, and leads to white matter demyelination. persistence and demyelination occur only in genetically susceptible mouse strains, while resistant strains clear the infection after early grey matter encephalomyelitis through a cytotoxic t lymphocyte response. the severity and nature of disease depend on virus strain, route of inoculation, host genotype and age [ , , ] . in general, virus isolates with low virulence produce persistent cns infection in mice whereas virulent strains are unable to cause persistent infection. intracerebral inoculation results in the most severe infections, but the intranasal route is also effective. experimental intracerebral infections with virulent fa and gdvii strains of tmev are more likely to cause acute encephalomyelitis and death in weanling mice - days after inoculation ('early disease'). death may be preceded by neurological manifestations of encephalitis such as hyperexcitability, convulsions, tremors, circling, rolling and weakness. animals may develop typical flaccid paralysis of hindlimbs, and locomotion is possible only by use of the forelimbs. interestingly, the tail is not paralyzed. experimental infections with low-virulence virus strains (e.g. to, da, ww) are more likely to cause persistent infection with development of mild encephalomyelitis followed by a chronic demyelinating disease after a few months ('late disease'). these virus strains infect neurons in the grey matter of the brain and spinal cord during the acute phase of viral growth, followed by virus persistence in macrophages and glial cells in the spinal cord white matter. sjl, swr and dba/ strains are most susceptible to this chronic demyelinating disease. cba and c h/he are less susceptible strains, and strains a, c bl/ , c bl/ and dba/ are relatively resistant [ ] . differences in humoral immune responses play a role in resistance to tmev infection [ ] , but genetic factors are also important. several genetic loci implicated in susceptibility to virus persistence, demyelination, or clinical disease have been identified, including the h- d region of the major histocompatibility complex [ ] . furthermore, the age at infection influences the severity of clinical disease. in infant mice, intracerebral infection with low-virulence virus strains (e.g. to) is often lethal. young mice develop paralysis after an incubation period of - weeks while adult mice often show no clinical signs of infection. the only gross lesions are secondary to the posterior paralysis and may include urine scald or dermatitis due to incontinence of urine and trauma to paralyzed limbs, or wasting or atrophy of the hindlimbs in long-term survivors. tmev infects neurons and glial cells, and histological changes in the cns include nonsuppurative meningitis, perivasculitis and poliomyelitis with neuronolysis, neuronophagia and microgliosis in the brainstem and ventral horns of the spinal cord [ ] . demyelination in immunocompetent mice is considered to be immunemediated. susceptible strains develop a specific delayed-type hypersensitivity response which is the basis for inflammation and demyelination. this reaction is mediated by t cells that release cytokines leading to recruitment of monocytes and macrophages as a consequence of infection of macrophages and other cns-resident cells [ ] [ ] [ ] . protection from chronic demyelinating disease is possible by vaccination with live virus given previously by subcutaneous or intraperitoneal inoculation [ , ] . early immunosuppression at the time of infection, e.g. by treatment with cyclophosphamide or antithymocyte serum, inhibits or diminishes demyelination. immunosuppression in mice chronically infected with tmev leads to remyelination of oligodendrocytes [ ] . further details related to the pathogenesis of tmev infections and the role of immune mechanisms have been reviewed by yamada et al. [ ] , kim et al. [ ] and lipton et al. [ ] . experimental infection of foxn nu mice results in acute encephalitis and demyelination. demyelination associated with minimal inflammation and neurological signs, including the typical hindlimb paresis, develop weeks after inoculation, and most animals die within weeks. in foxn nu mice, demyelination is caused by a direct lytic effect of the virus on oligodendrocytes [ ] . demyelination and lethality are reduced after administration of neutralizing antibodies [ ] . histopathological changes in prkdc scid mice are very similar to those in foxn nu mice [ ] . young mice born in infected populations usually acquire infection shortly after weaning and are almost all infected by days of age. intrauterine transmission to fetuses is possible during the early gestation period, but a placental barrier develops during gestation and later prevents intrauterine infection [ ] . all tmev isolates are closely related antigenically and form a single serogroup, as determined by complement fixation and hi [ ] . hemelt et al. 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and demyelinating disease caused by theiler's virus infection with theiler's murine encephalomyelitis virus directly induces proinflammatory cytokines in primary astrocytes via nf-kappab activation: potential role for the initiation of demyelinating disease innate immune response induced by theiler's murine encephalomyelitis virus infection cardioviruses: encephalomyocarditis virus and theiler's murine encephalomyelitis virus effect of immunization with theiler's virus on the course of demyelinating disease protection of sjl/j mice from demyelinating disease mediated by theiler's murine encephalomyelitis virus immunosuppression promotes cns remyelination in chronic virus-induced demyelinating disease pathogenesis of theiler's murine encephalomyelitis virus mechanism of theiler's virus-induced demyelination in nude mice survival of athymic (nu/nu) mice after theiler's murine encephalomyelitis virus infection by passive administration of neutralizing monoclonal antibody vacuolar neuronal degeneration in the ventral horns of scid mice in naturally occurring theiler's encephalomyelitis evolution of the placental barrier to fetal infection by murine enteroviruses enhanced detection of theiler's virus rna copy equivalents in the mouse central nervous system by real-time rt-pcr spontaneous demyelinating myelopathy in aging laboratory mice 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 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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- -it ahzj authors: lane, t. e.; hardison, j. l.; walsh, k. b. title: functional diversity of chemokines and chemokine receptors in response to viral infection of the central nervous system date: journal: chemokines and viral infection doi: . / - - - - _ sha: doc_id: cord_uid: it ahzj encounters with neurotropic viruses result in varied outcomes ranging from encephalitis, paralytic poliomyelitis or other serious consequences to relatively benign infection. one of the principal factors that control the outcome of infection is the localized tissue response and subsequent immune response directed against the invading toxic agent. it is the role of the immune system to contain and control the spread of virus infection in the central nervous system (cns), and paradoxically, this response may also be pathologic. chemokines are potent proinflammatory molecules whose expression within virally infected tissues is often associated with protection and/or pathology which correlates with migration and accumulation of immune cells. indeed, studies with a neurotropic murine coronavirus, mouse hepatitis virus (mhv), have provided important insight into the functional roles of chemokines and chemokine receptors in participating in various aspects of host defense as well as disease development within the cns. this chapter will highlight recent discoveries that have provided insight into the diverse biologic roles of chemokines and their receptors in coordinating immune responses following viral infection of the cns. coronaviruses are classified on the basis of several fundamental characteristics, including nucleic acid type, a lipid envelope, and their distinctive morphology [ , , ] . all members have characteristic petal-shaped proteins extending from the virion surface. coronaviruses infect numerous vertebrate hosts including humans, chickens, pigs, and mice, causing a wide variety of disorders involving a number of different organ systems; however, there are specific tropisms for the cns, lungs, gastrointestinal tract, and liver [ , , ] . receptor use among the varied coronaviruses is restricted to several well-defined proteins. human coronavirus infections result in acute enteritis as well as % of common colds indistinguishable from those caused by other viruses [ , , ] . more recently, a human coronavirus has been indicated to be the etiologic agent for severe acute respiratory syndrome (sars). sars is a potentially lethal disease and is recognized as a health threat internationally [ ] . the first murine coronavirus strain (mouse hepatitis virus, mhv), was isolated in [ ] . mhv is a pathogen of wild mice, and natural infection is due to horizontal transmission, resulting in acute hepatitis with death in young animals and a variable course of persistent gastrointestinal tract infection in adults [ ] . mhv is not an endemic mouse virus, but infects mouse colonies sporadically. it is very closely related to some human coronaviruses both at the genomic and protein levels. for example, human sera often contain antibody reactive to mhv. therefore, characterizing the immune response to murine coronaviruses may provide important insight to mechanisms of control and elimination which may have important implications with regards to understanding the immune response to human coronaviruses such as the sars coronavirus. coronavirus genomes are single-stranded positive-polarity rna molecules, larger than the size of any other known stable rna, ranging from kb for the avian infectious bronchitis virus, to kb for murine coronaviruses [ ] . genomic rna is infectious, contains a cap structure at the -end and poly(a) at the -end. the genome is organized into seven or eight genes, each containing one or more open reading frames (orf) separated by intergenic sequences that contain the signals for the initiation of transcription of the subgenomic viral messenger (m)rna species. upon entry, the viral rna encodes an rna polymerase that transcribes the genome into a negative-stranded rna [ ] . the latter serves as templates for positivesensed genomic rna and subgenomic mrnas. important viral structural proteins include the envelope glycoproteins (s) that bind to receptors on cell membranes [ , , ] . analysis of monoclonal antibody neutralization escape variants demonstrated that the viral s protein controls cellular tropism in vivo and the role of the s protein in tropism has recently been confirmed using stable recombinant viruses in which all genes except the s protein gene were held constant [ , ] . the protective immune response to mhv infection is characterized predominantly by cell-mediated immunity during acute infection. a number of unique aspects of cns viral infection have been described by analysis of the interactions between mhv and the immune response. antibody, although protective if administered prior to infection, is not present in the serum of infected mice until after the vast majority of virus has been cleared from the cns [ , ] . following infection, neutrophils, macrophages, and nk cells are rapidly recruited into the cns, followed by t cells and b cells [ ] . inflammation is accompanied by a progressive loss of blood-brain barrier (bbb) integrity that is apparent as early as days post-infection. the initial influx of innate effectors is important in facilitating t cell infiltration, as well as regulating viral replication [ ] . however, the ability to survive mhv infection appears to be predominantly due to an effective t cell-mediated response [ ] . recent data have confirmed that cell-mediated immunity is critical during acute infection [ , , , , ] ; however, the ability to prevent viral recrudescence is associated with the continued presence of plasma cells in the cns secreting neutralizing antibody [ , ] . the major effectors of anti-viral immunity are virus-specific cd + t cells. cytotoxic t lymphocyte (ctl) induction following mhv infection of the cns has been shown to require cd + t cell help [ ] . although the pre-cise mechanism or mechanisms by which cd + t cells assist cd + t cells have yet to be completely determined, recent studies have demonstrated that cd + t cells are important in preventing apoptosis of ctl entering the cns parenchyma [ ] . in addition, the quality of the ctl response is cd + t cell-dependent [ ] . an important concept derived from analysis of mhv infection is that although cd + t cells are the most prominent effectors for viral clearance during the acute infection, the mechanisms which control virus replication differ with the type of cns cell infected. cytolysis is important for the control of viral replication in microglia/macrophages and astrocytes while interferon (ifn)-γ is the critical effector responsible for control of virus replication in oligodendroglia [ ] . the demonstration that cd + ctl suppresses viral replication by two separate effector mechanisms, which function within the cns in a cell type-specific manner, is an important new concept. viral persistence in white matter tracts results in a chronic demyelinating disease in which foci of demyelination are associated with areas of viral rna/antigen [ ] . clinically, mice develop loss of tail tone and a partial to complete hind-limb paralysis. as a result of the clinical and histologic similarities between mhv-induced demyelination and the human demyelinating disease multiple sclerosis (ms), the mhv system is considered a relevant model for studying the underlying immunopathologic mechanisms contributing to immune-mediated demyelinating diseases [ ] . a variety of different mechanisms have been postulated to contribute to mhv-induced demyelination. several studies suggest that mhv-induced demyelination involves immunopathologic responses against viral antigens expressed in infected tissues [ , , , ] . although virus-specific antibody is considered important in suppressing viral recrudescence [ , ] , it may also have a role in promoting demyelination [ ] . mhv infection of immunosuppressed or immunodeficient mice results in high titers of virus within the cns and death but not robust demyelination [ , ] . adoptive transfer of mhv-immune splenocytes results in demyelination to the infected recipients, suggesting a role for immune cells in amplifying demyelination [ , ] . additional evidence for t cells in contributing to demyelination is provided by wu et al. [ ] who demonstrated that both cd + and cd + t cells are important in mediating myelin destruction. in support of this are studies derived from our laboratory demonstrating that adoptive transfer of mhv-specific cd + or cd + t cells to mhv-infected rag −/− mice results in demyelination [ , ] . however, demyelination was more severe in recipients of cd + t cell compared to cd + t cell recipients, and this supports a more important role for cd + t cells in amplifying demyelination in this model. indeed, we have demonstrated that mhv-infected cd −/− mice displayed a significant reduction in the severity of demyelination compared to cd −/− and immunocompetent wildtype mice, suggesting an important role for cd + t cells in amplifying the severity of white matter destruction [ ] . while t cells are generally considered important in driving demyelination in mice persistently infected with mhv, the mechanisms by which these cells participate in disease may vary and depend upon various factors including the ability to secrete interferon (ifn)-γ [ , ] . while conventional cd and cd αβ t cells are generally viewed as the primary t cell type important in disease, γδ t cells have also been shown to participate in demyelination in mhv-infected athymic mice [ ] . in addition, we and others have found that macrophages/microglia are also important in contributing to demyelination [ , , , , ] . the collective evidence points to a role for inflammatory t cells in contributing to macrophage/microglial infiltration and activation which ultimately results in myelin destruction. current evidence suggests that demyelination in mhv-infected mice is not the result of epitope spreading and induction of an immune response against neuroantigens as has recently been reported to occur during theiler's virus-induced demyelination [ ] . however, adoptive transfer of t cells from mhv-infected rats to naïve recipient's results in demyelination [ ] . whether a similar response occurs in mhv-infected mice and what the contributions are to demyelination is not clear at this time. chemokines represent a family of low molecular weight ( - kda) proinflammatory cytokines that are divided into four subfamilies based on structural and functional criteria [ , , ] . the two major subfamilies are the cxc and cc chemokines. the cxc subfamily is structurally characterized by two conserved cysteine residues that are separated by an amino acid, while the cc subfamily is structurally characterized by conserved cysteine residues adjacent to one another. lymphotactin, the sole member of the c family, is chemotactic for t cells [ ] . the cx c chemokine, fractalkine, is unique in that it is expressed on the surface of cells as well as being secreted into the surrounding environment [ ] . chemokines have been shown to selectively attract distinct leukocyte populations during periods of inflammation in various disease models. the cxc chemokines function primarily in attracting neutrophils, yet have a limited effect on t lymphocytes and monocytes [ , , ] . however, there are exceptions to this rule in that cxc chemokines that lack the glutamic acid-leucine-arginine (elr) motif on the amino terminus are chemotactic for t cells. for example, the non-elr chemokine cxcl is a potent chemoattractant for activated t cells and nk cells and functions by binding to cxcr expressed on the surface of these cells [ , , , ] . however, cxcl does not exert a chemotactic effect on neutrophils [ ] . the cc chemokines are thought to attract t cells, monocytes, and macrophages, but not neutrophils [ , , ] . the cc chemokine ligand (ccl ) is able to attract both t cells and macrophages by binding to one of several cc chemokine receptors including ccr and ccr [ , , ] . furthermore, there is increasing evidence that chemokines, such as ccl , influence other immune system activities including t h /t h development and t cell proliferation [ , ] . chemokines function by binding to seven-transmembranespanning g protein-coupled receptors. the chemokine receptors are divided into those that preferentially bind cxc and cc chemokines. in addition, cc and cxc chemokine receptors are capable of binding more than one cc or cxc chemokine, respectively. a variety of cell types including lymphocytes and macrophages, as well as resident cells of the cns such as neurons, astrocytes, and microglia, express chemokine receptors [ , ] . instillation of mhv into the cns of susceptible mice results in a wellorchestrated expression of chemokine genes, and the expression pattern correlates with the level of inflammation and disease [ ] . early (~ - days) following infection, transcripts for cxcl and ccl are detected within the cns, suggesting an important role in initiation of immune responses (see following section; table ). by day post-infection (p.i.), virus has spread throughout the brain parenchyma, and a robust inflammatory response, characterized primarily by cd + and cd + t cells and macrophages, is established within the brain. chemokines expressed at this time include cxcl , cxcl , ccl , ccl , ccl , ccl , and ccl (mip- ) ( table ) . analysis of chemokine receptor expression by both rnase protection assay (rpa), immunostaining, and flow cytometry reveals that ccr , ccr , ccr , and cxcr are the prominent receptors expressed within the cns at various stages of disease ( table ) . chemokine transcripts are detected almost exclusively in areas in which virus is present, indicating a localized response to infection and subsequent spread of the virus throughout the parenchyma. in situ hybridization indicates that astrocytes are the primary cellular source for many chemokines during the acute stage of disease [ ] . infection of primary cultures of mouse astrocytes with mhv and evaluating chemokine gene expression by rpa provide additional support for astrocytes as an important cellular source of chemokines in this model [ ] . moreover, viral replication appears to be a necessary prerequisite for inducing chemokine expression, as infection of astrocytes with inactivated virus results in a muted chemokine expression profile. additional analysis revealed that both infected and noninfected astrocytes are capable of secreting chemokines following instillation of virus into the brain, indicating that viral infection is not required for chemokine gene synthesis by target cells. these data indicate that a factor or factors (possibly type i interferons) derived from infected cells are capable of functioning in both an autocrine and paracrine manner and regulate chemokine gene expression in response to viral infection. other cell types that may also secrete chemokines following mhv infection include resident microglia/inflammatory macrophages as well as neurons [ , ] . by day p.i., mhv-infected mice that have survived the acute stage of disease develop an immune-mediated demyelinating disease. mice have cleared infectious virus (as determined by plaque assay) by days, yet viral rna and protein can be detected within white matter tracts for months after infection. as the level of cns infiltration subsides following reduction of viral burden there is a corollary reduction in the expression of chemokine transcripts. analysis of chemokine message expression within the brains and spinal cords of mhv-infected mice during the demyelinating phase of disease (days and onward) indicates that cxcl and ccl are the two prominent chemokines expressed [ ] . in situ hybridization for chemokine transcripts indicated expression was limited primarily to areas of viral persistence within white matter tracts undergoing active demyelination [ ] . similar to what was found during acute disease, astrocytes were determined to be the cellular source of cxcl at this stage of disease whereas inflammatory cells, presumably cd + t lymphocytes, expressed ccl . more recent data now indicate that mhv-infected astrocytes treated with ifn-γ can also express ccl mrna transcripts and protein (t.e. lane, unpublished observations). chemokine receptors expressed during chronic demyelination include cxcr and ccr , which are capable of binding cxcl and ccl , respectively. indeed, we have recently determined that the majority (~ %) of infiltrating virus-specific cd + and cd + t cells express cxcr (t.e. lane, unpublished observations). the presence of dendritic cells (dcs) within the cns has been debated for quite some time. however, a series of recent studies clearly indicates that during induction of an autoimmune demyelinating disease, there exists the presence of cell types within the brain that clearly have characteristics of dcs [ , ] . in addition, emerging evidence points to a previously unappreciated role for chemokines in activating and inducing the migration of differing populations of dcs in response to microbial infection of the cns [ , ] . these cells may be important in initiation and/or maintenance of disease by participating in the activation of t cells. given the potential importance of this population of cells with regards to linking innate and adaptive immune responses following viral infection of the cns, we investigated whether dc-like cells were present within the cns in response to mhv infection. in brief, our findings clearly indicate that a dc-like population of cells is detectable within the cns as early as day p.i. with mhv [ ] . the activation/maturation of these cells as well as the ability to accumulate within the draining cervical lymph node (cln) appeared to be dictated by localized expression of ccl [ ] . moreover, the ability of cultured dcs to secrete cytokines associated with the development of a t h response such as interleukin (il)- was profoundly altered in the absence of ccl [ ] . the importance of ccl signaling and the evolution of an effective t cell response was further confirmed by the demonstration that in the absence of ccl signaling, robust anti-viral effector responses, e.g., cytokine production and ctl activity, were dramatically compromised following mhv infection of ccl −/− mice [ , ] . collectively, these studies highlight a previously unappreciated role for the importance of chemokine signaling and dc maturation/activation following mhv infection of the cns. moreover, these studies demonstrate that generation of effective t cell responses relies upon ccl signaling to successfully combat mhv infection. ccl is a chemoattractant for both t cells and macrophages and has been implicated in host defense following infection with a wide variety of microbial pathogens. mice deficient in ccl production exhibit increased susceptibility to disease following infection with paramyxovirus [ ] , influenza virus [ ] , and coxsackievirus, as well as other microbial pathogens [ , ] . in all cases, alterations in an effective host response correlated with a paucity in leukocyte accumulation at sites of infection. although originally thought to participate in defense by attracting effector cells to infected tissue, recent reports also suggest that ccl expression is important in coordinating a t h response [ ] . numerous studies now indicate that dcs are capable of expressing various chemokines including ccl [ , , , ] . moreover, dc precursors express the ccl receptors ccr and ccr and are capable of responding to ccl in vivo and in vitro resulting in both mobilization and maturation [ , ] . indeed, flesch and colleagues have demonstrated an important role for ccl in dc-dependent priming of ctl to viral antigens [ ] . using ccl −/− mice, we have demonstrated a role for ccl in regulating trafficking as well as antiviral effector functions following mhv infection of the cns [ ] . specifically, our experiments revealed an important role for ccl signaling in tailoring t cell responses that allowed for egress out of draining cervical lymph nodes and trafficking into the cns. although generation of antigen-specific cd + t cells was not impaired following mhv infection of ccl −/− mice, a significant percentage of cd + t cells retained expression of lymph-node homing receptors cd l (l-selectin) and the cc chemokine receptor (ccr ) and did not display a dramatic increase in mrna transcripts for either cxcr or ccr , two receptors which are important in allowing mhv-specific t cells access to the cns [ ] . moreover, adoptive transfer of ccl −/− cd + t cells into mhv-infected rag −/− mice (which express ccl following mhv infection) resulted in homing back to secondary lymphoid organs, suggesting that lack of ccl imprinted on these cells carries an inability to remodulate surface tissue homing receptors. analysis of antiviral effector functions also revealed that ccl −/− cd + t cells displayed overall muted cytolytic activity as well as expression of ifn-γ when compared to ccl +/+ cd + t cells [ ] . collectively, these studies highlight that, in addition to chemotactic function, chemokines influence specific lymphocyte responses and ultimately effector functions that are required for optimal host defense against microbial pathogens. cxcl and cxcl attract activated t lymphocytes following binding to cxcr . analysis of cxcl and cxcl mrna expression within the cns of mhv-infected mice revealed that cxcl was clearly detectable by day p.i. and was prominently expressed at days , , and p.i. [ ] . in contrast, cxcl transcripts were only detected at days and p.i. [ ] . these data suggested that both cxcl and cxcl might be important in host defense by attracting antiviral t lymphocytes into the cns. in support of this is the observation that administration of neutralizing antibodies specific for either cxcl or cxcl to mhv-infected mice during the acute stage of disease results in a dramatic increase in mortality [ , ] . additionally, this treatment also resulted in a significant decrease in numbers of cd + and cd + t lymphocyte infiltrating into the cns which correlated with decreased expression of ifn-γ and increased levels of virus [ , ] . mhv infection of cxcl −/− mice supported and extended our previous work on antibodymediated neutralization of cxcl in that mhv-infected cxcl −/− mice display reduced t cell infiltration into the cns accompanied by reduced ifn-γ secretion and increased viral burden [ ] . therefore, the collective evidence points to pivotal roles for both cxcl and cxcl as important sentinel molecules in promoting a protective response following mhv infection of the cns by attracting t cells into the cns that participate in elimination of virus. ccl is a t cell and macrophage chemoattractant that has been shown to influence leukocyte migration during periods of inflammation. upon mhv infection of the cns of mice, ccl transcripts and protein are readily detected within the brain [ ] . initial studies in which cd −/− or cd −/− mice were infected with mhv indicated an overall reduction in ccl mrna transcripts within the brains of cd −/− mice, suggesting that cd + t cells were either a primary cellular source for ccl and/or influenced the expression of ccl by resident and inflammatory cells [ ] . we now know that both inflammatory cd + t cells as well as astrocytes are capable of expressing ccl following instillation of mhv into the cns [ , ] . furthermore, treatment with neutralizing anti-ccl antisera results in diminished t cell and macrophage accumulation within the cns, suggesting that in this model ccl is capable of regulating trafficking of these two populations of cells [ ] . ccr is a member of the cc chemokine receptor family that is expressed on various hematopoietic cells including lymphocytes and macrophages [ ] . chemokines that are capable of binding to ccr include ccl , ccl , and ccl [ , , ] . recent studies have clearly indicated that ccr expression correlates with leukocyte trafficking to sites of inflammation as well as regulating the immune response following microbial infection. for example, mice deficient in ccr (ccr −/− ) exhibit altered t cell activity and impaired macrophage function [ , ] . furthermore, macrophage trafficking in response to antigen is impaired in ccr −/− mice, indicating that ccr is required for migration of this population of cells [ ] . given that both t cells and macrophages express ccr following mhv infection of the cns and these cells clearly influence outcome in response to infection, we have defined the contributions of ccr to both host defense and disease in response to mhv infection. using an adoptive transfer model in which virus-expanded t cells are transferred into mhv-infected rag −/− mice, we have been able to examine how ccr expression influences trafficking of t cells into the cns. transfer of ccr +/+ -derived cd + t cells to mhv-infected rag −/− mice resulted in cd + t cell entry into the cns and a reduction in viral titers within the brain [ ] . these mice also displayed robust demyelination correlating with macrophage accumulation within the cns. conversely, cd + t cells from ccr −/− mice displayed an impaired ability to traffic into the cns of mhv-infected rag −/− recipients, which correlated with increased viral titers, diminished macrophage accumulation, and limited demyelination. analysis of chemokine receptor mrna expression by m - -expanded ccr −/− -derived cd + t cells revealed reduced expression of ccr , ccr , and cxcr , indicating that ccr signaling is important in increased expression of these receptors which aid in trafficking of cd + t cells into the cns. collectively these results demonstrate that ccr signaling is important to migration of cd + t cells to the cns following mhv infection. with regards to the role of ccr in cd + t cell trafficking, comparable numbers of virus-specific cd + t cells derived from immunized ccr +/+ or ccr −/− mice were present within the cns of mhv-infected rag −/− mice following adoptive transfer, indicating that ccr is not required for trafficking of these cells into the cns [ ] . rag −/− recipients of ccr −/−derived cd + t cells exhibited a modest yet significant (p≤ . ) reduction in viral burden within the brain that correlated with increased cytolytic activity and ifn-γ expression. histologic analysis of rag −/− recipients of either ccr +/+ or ccr −/− -derived cd + t cells revealed only focal areas of demyelination with no significant differences in white matter destruction. these data indicate that ccr signaling on virus-specific cd + t cells modulates antiviral activities but is not essential for entry into the cns. finally, mhv infection of ccr −/− mice resulted in a dramatic reduction in macrophage (defined as cd high f / + dual-positive cells) accumulation within the brains, and this correlated with a significant reduction in the severity of demyelination compared to ccr +/+ mice. collectively, these data suggest that ligand binding, e.g., ccl and/or ccl , and signaling via ccr results in macrophage migration and infiltration into the cns. however, we have previously demonstrated that ccl is expressed only at low levels during acute disease and is not detectable during chronic demyelination, whereas robust expression of ccl is detected during both phases of disease, and this suggests that ccl is the primary ccr signaling chemokine in this model. this is supported by earlier studies that showed an important role for ccl in attracting macrophages into the cns following mhv infection [ ] . therefore, the data presented in this study suggest that one mechanism by which ccl contributes to demyelination is via attracting macrophages into the cns through ccr -mediated signaling pathways. additional evidence supporting this is provided by the observation that even in the presence of increased ccl expression at day p.i., demyelination is reduced in ccr −/− mice. ccl is capable of regulating the pathobiology of various inflammatory diseases including ms and atherosclerosis [ , , , , , ] . in addition to its potent chemoattractant effect on monocytes and macrophages, ccl also influences t h polarization in response to certain antigenic challenge [ , , , ] . the influence of ccl on t cell polarization may be due to the fact that ccl is constitutively expressed within secondary lymphoid tissue and would be capable of affecting cellular responses following exposure to antigen [ ] . thus, available evidence indicates that expression of ccl is capable of influencing both innate as well as adaptive immune responses by regulating monocyte and t cell responses, respectively. analysis of chemokine receptor expression following mhv infection reveals that ccr is expressed by endogenous cells of the cns as well as by inflammatory t cells and macrophages, indicating a role for these receptors in regulating both the immune response and disease development [ , ] . indeed, mhv-infection of ccr −/− mice resulted in a dramatic increase in mortality and enhanced viral recovery from the brain that correlated with reduced t cell and macrophage entry into the cns compared to viral infection of ccr +/+ mice [ ] . mhv infection of ccl −/− mice does not result in a similar disease phenotype as observed in ccr −/− mice. this was somewhat surprising as ccr is currently the only known functional receptor for ccl . specifically, ccl −/− mice were able to clear virus from the brain in a similar time frame as wildtype mice, and this correlated with the ability to generate antigen-specific t cells [ ] . the deficiency in ccr −/− mice to clear virus from the brain is not the result of an inherent inability to generate an effective adaptive immune response to virus, as ccr −/− mice had a similar frequency of antigenpresenting cells (apc) and virus-specific t cells present within draining cln compared to either ccl −/− or wildtype mice. our findings from mhv infection of ccl −/− mice indicated that while ccl does influence leukocyte migration into the cns in response to viral infection, ccr is clearly more influential in directing t cell trafficking into the cns. in support of the role for ccl in promoting leukocyte migration into the cns of mhv-infected mice are recent studies by perlman and colleagues demonstrating that localized ccl expression within the cns promotes macrophage infiltration [ ] . these data highlight the possibility that ligand(s) other than ccl are important in signaling through the ccr receptor. alternatively, it is possible that ccr signaling by either endothelial cells and/or astrocytes regulates the permeability of the bbb, as recently suggested by stamatovic and colleagues [ ] . expression of chemokines has been associated with demyelinating plaque lesions present in ms patients [ , , , ] . elevated levels of chemokines, notably cxcl , were found in the cerebral spinal fluid (csf) of ms patients during periods of clinical attack [ , ] . indeed, the concentration of cxcl within the csf of ms patients correlated with numbers of inflammatory cells and the severity of clinical disease [ , , ] . moreover, when cxcl levels decreased, there was a corresponding decrease in inflammation and disease severity [ ] . astrocyte expression of cxcl has been reported in active plaque lesions present in ms patients, and the majority of t cells infiltrating into the cns of ms patients express the cxcl receptor, cxcr . collectively, these studies highlight a potentially important role for cxcl in the pathogenesis of demyelinating diseases such as ms by attracting cxcr -expressing t cells into the cns and support targeting chemokines and their receptors for therapeutic intervention in the treatment of ms [ , , , ] . studies from animal models of ms support this notion by demonstrating that blocking of cxcl often results in diminished disease severity accompanied by a marked reduction in neuroinflammation. for example, several recent reports indicate that treatment with anti-cxcl neutralizing antibodies resulted in delayed disease onset and diminished neuroinflammation in mice with the autoimmune demyelinating disease experimental autoimmune encephalomyelitis (eae) [ ] . these studies support the idea that localized expression of cxcl within the cns amplifies disease severity by attracting cxcr -expressing t cells into the cns. once present, these cells enhance neuroinflammation by secreting additional chemokines as well as cytokines that can activate resident glia cells. importantly, these studies also implicate cxcl as a potential therapeutic target and suggest that alternative cxcr ligands, e.g., cxcl and cxcl , do not exert a prominent effect on t cell infiltration into the cns. however, the role of cxcl in contributing to neurologic disease in eae has been questioned by results indicating that cxcl may actually exert a protective effect in mice with eae [ , ] . antibody-mediated neutralization following induction of eae in rats resulted in increased disease severity, and this was associated with smaller draining lymph nodes and increased numbers of cd + t cells infiltrating into the cns [ ] . in addition, cxcl −/− mice exhibited increased clinical disease severity following immunization with myelin peptides, and this correlated with diminished lymph node sizes although t cell infiltration into the cns was not dramatically altered when compared to wildtype mice [ ] . in these particular eae models in which mice are immunized peripherally with antigen, cxcl expression within secondary lymphoid tissue is considered important in dictating disease outcome by serving to retain lymphocytes and tailoring t cell responses. moreover, these findings highlight the different roles of cxcl in regulating cellular immune responses in different models of neuroinflammation and emphasize the need for a better understanding of how signaling by this chemokine regulates inflammation and disease. as indicated, we have determined that mhv infection of the cns results in an orchestrated expression of chemokine and chemokine receptor genes that are regulated, in large part, by the viral burden. similar to ms patients, cxcl is expressed primarily by astrocytes in areas undergoing demyelination, suggesting an important role in the pathogenesis of demyelination by attracting cxcr -expressing t cells into the cns [ , ] . indeed, our laboratory was the first to demonstrate that treatment of mice with established demyelination and paralysis with anti-cxcl neutralizing antibody resulted in a significant reduction in cd + -but not cd + -t cells present within the cns, and this correlated with improved motor skills and a reduction in the severity of demyelination [ ] . moreover, the dramatic regain of movement in anti-cxcl -treated mice corresponded with more than % of previously demyelinated axons undergoing remyelination, indicating that removal of cxcl promoted an environment capable of remyelination. in addition to reduced numbers of cd + t cells within the cns, there was a paucity of macrophage infiltration into the cns of anti-cxcl treated mice that correlated with a dramatic reduction in the levels of the macrophage-chemoattractant ccl . these data were consistent with previous studies indicating that cd + t cells were the major source for ccl in mhv-infected mice undergoing demyelination [ , ] . the influence of cxcl in contributing to t cell responses was also examined. t cells isolated from secondary lymphoid tissue of mice treated with anti-cxcl displayed muted expression of ifn-γ in response to viral antigen when compared to t cells isolated from control mice, suggesting that cxcl also serves to influence t cell effector functions during chronic disease (t.e. lane, unpublished observations). we have previously determined that ccl mrna transcripts and protein are present within the cns of mhv-infected mice during chronic demyelination, indicating a potentially important role for this chemokine in promoting inflammation [ , ] . in order to assess the functional role of ccl in participating in viral-induced immune-mediated demyelination, mhv-infected mice were treated via intraperitoneal (i.p.) injection with anti-ccl monoclonal antibody (mab) following onset of clinical disease and demyelination. such treatment resulted in a significant (p≤ . ) reduction in the severity of clinical disease compared to mice treated with an isotype (igg )-matched antibody [ ] . upon removal of anti-ccl treatment, clinical disease returned to mice such that there was no difference between the two experimental groups of mice. immunophenotyping the cellular infiltrate of mice treated with anti-ccl revealed reduced t cell and macrophage infiltration into the cns that is consistent with our earlier studies that ccl attracts these cells into the cns of mice with chronic demyelination. further, analysis of the severity of demyelination in experimental groups of mice indicated that anti-ccl treatment resulted in a significant (p< . ) reduction in the severity of demyelination compared to control-treated mice. a picture is slowly evolving from our experiments designed to test the functional contributions of cxcl and ccl to chronic demyelination within mhv-infected mice. antibody targeting of the t cell chemoattractant cxcl in mhv-infected mice selectively affects cd + t cell accumulation within the cns accompanied by improved motor skills and a reduction in the severity of demyelination [ ] . in contrast, ccl is capable of attracting both cd + and cd + t cells into the cns. it is also important to emphasize that our data on ccl and cxcl inhibition with regards to t cell and macrophage trafficking are corollary and it is possible that alternative scenarios exist. for example, studies by bergmann and colleagues suggest that during persistent mhv infection there is limited to no trafficking of t cells from the periphery into the cns. rather, upon entry during acute encephalomyelitis a certain percentage of cd + and cd + t cells is retained and participate in disease [ , ] . in this instance, cxcl expression would not be functioning as a t cell chemoattractant but rather to influence specific biologic functions of t cells as well as potentiating the retention of t cells within the cns. in support of this, it is possible that cxcl serves to enhance cd + t cell proliferation, as several recent studies indicate that cxcl is important in contributing to t cell proliferation [ , , ] . it is unlikely that cxcl contributes to t cell survival, as cxcl −/− mice do not display any abnormalities with regards to t cell half-life nor do we see any increase in numbers of apoptotic t cells following anti-cxcl treatment. in addition, narumi et al. [ ] speculate that cxcl actually serves to retain cxcr + t cells within tissues and this influences disease severity. therefore, the selective reduction in cd + t cells within the cns of mhv-infected mice may not be the result of impaired trafficking. rather, either cd + t cells are not undergoing a steady-state turnover or are actually migrating out of the cns in the absence of signals specifying their retention. in addition, recent studies indicate an important role for cxcl in imparting effector functions to t cells. for example, salomon and colleagues demonstrated that anti-cxcl treatment improved joint swelling in a rodent model of arthritis and this correlated in part with an altered t h /t h balance, suggesting that cxcl expression promotes and maintains a t h state in t cells in this model [ ] . similarly, we have shown that mhv-infection of cxcl −/− mice results in diminished ifn-γ expression by virus-specific t cells, supporting the idea that cxcl expression serves to maintain a t h -like state in t cells [ ] (t.e. lane, unpublished observations). ccl signaling also modulates cytokine production by t cells following antigenic challenge. in support of this is our demonstration that inhibition of ccl signaling results in enhanced ifn-γ expression by virus-specific t cells, supporting the idea that ccl expression serves to regulate a t h -like state in t cells [ ] . moreover, ablation of ccl signaling also modifies the cytolytic activity of mhv-specific cd + t cells [ ] . this chapter highlights mechanisms by which chemokines participate in both host defense and disease progression in response to mhv infection of the cns. an overview of the potential functional role for select chemokines in linking innate and adaptive immune responses in response to viral infection of the cns is provided in fig. . in brief, following mhv infection there is robust expression of chemokines by infected astrocytes including ccl that contribute to the maturation/activation of local dcs, which ultimately enables migration to draining cervical lymph nodes. activated dcs present antigen to t cells as well as secrete chemokines such as ccl and cxcl that enhance polarization to a t h response. in turn, mhv-specific t cells express chemokine receptors including cxcr and ccr that enable them to traffic into the cns as a result of localized expression of ligands cxcl and cxcl (ligands for cxcr ) as well as ccl (ligand for ccr ). in addition, our contention is that expression of ccr by endothelial cells of the bbb is also important in increasing the permeability of this structure. with regards to chronic disease, mhv persistence within the cns results in chronic expression of cxcl and ccl which together contribute to the maintenance of a chronic inflammatory disease by attracting both t cells and macrophages (fig. ) . local secretion of cxcl and ccl may also contribute to demyelination by enhancing specific t cell effector functions including ( ) secretion of ifn-γ that activates local inflammatory macrophage and resident microglia, as well as directly damaging oligodendrocytes and ( ) increasing ctl activity by cd + t cells. in addition, immature dc-like cells may also be susceptible to infection and secrete ccl (b) that functions in a paracrine and autocrine manner to bind to ccr expressed on immature dc-like cells. as a result of ccl signaling and mhv infection, the dc-like cells undergo maturation and activation (c) resulting in a remodulation of the plasma membrane characterized by decreased expression of ccr accompanied by increased expression of ccr as well as major histocompatibility complex (mhc) class i and ii. ccr -expressing, activated dcs home to the draining cervical lymph node (d). upon entry, activated dcs express a variety of soluble factors including ccl and cxcl (e) that activate and enhance polarization of virus-specific t cells to a t h phenotype (f). activated t cells exit the lymph node via the efferent lymph (g), 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viral persistence by neutralizing antibody molecular cloning and functional characterization of a novel human cc chemokine receptor (ccr ) for rantes, mip- beta, and mip- alpha targeting the function of ifn-gamma-inducible protein suppresses ongoing adjuvant arthritis defects in the generation of ifn-gamma are overcome to control infection with leishmania donovani in cc chemokine receptor (ccr) -, macrophage inflammatory protein- alpha-, or ccr -deficient mice expression of specific chemokines and chemokine receptors in the central nervous system of multiple sclerosis patients multiple sclerosis: a study of cxcl and cxcr co-localization in the inflamed central nervous system monocyte chemoattractant protein- regulation of blood-brain barrier permeability ctl effector function within the central nervous system requires cd + t cells the art of survival during viral persistence chemokines, inflammation and the immune system cc chemokine ligand (ccl ) regulates cd (+)-t-cell effector function and migration following viral infection the cc chemokine ligand regulates cd c+cd b+ cd alpha-dendritic cell maturation and activation following viral infection of the central nervous system: implications for a role in t cell activation cxc chemokine ligand controls viral infection in the central nervous system: evidence for a role in innate immune response through recruitment and activation of natural killer cells distinct roles for ip- /cxcl in three animal models, theiler's virus infection, eae, and mhv infection, for multiple sclerosis: implication of differing roles for ip- effect of c-c chemokine receptor (ccr ) knockout on type- (schistosomal antigen-elicited) pulmonary granuloma formation: analysis of cellular recruitment and cytokine responses adoptive transfer of eae-like lesions from rats with coronavirus-induced demyelinating encephalomyelitis chemokine monokine induced by ifngamma/cxc chemokine ligand stimulates t lymphocyte proliferation and effector cytokine production cxcr and its ligands participate in the host response to bordetella bronchiseptica infection of the mouse respiratory tract but are not required for clearance of bacteria from the lung effective clearance of mouse hepatitis virus from the central nervous system requires both cd + and cd + t cells characterization of brain-infiltrating mononuclear cells during infection with mouse hepatitis virus strain jhm cd and cd t cells have redundant but not identical roles in virus-induced demyelination antibody-mediated blockade of the cxcr chemokine receptor results in diminished recruitment of t helper cells into sites of inflammation identification of a cd + t cell epitope within the m protein of a neurotropic coronavirus mobilization of dendritic cell precursors into the circulation by administration of mip- alpha in mice impaired macrophage function and enhanced t cell-dependent immune response in mice lacking ccr , the mouse homologue of the major hiv- coreceptor the authors wish to thank craig walsh for helpful discussion. key: cord- -b xvycyk authors: nan title: envelope glycoprotein interactions in coronavirus assembly date: - - journal: j cell biol doi: nan sha: doc_id: cord_uid: b xvycyk coronaviruses are assembled by budding into smooth membranes of the intermediate er-to-golgi compartment. we have studied the association of the viral membrane glycoproteins m and s in the formation of the virion envelope. using coimmunoprecipitation analysis we demonstrated that the m and s proteins of mouse hepatitis virus (mhv) interact specifically forming heteromultimeric complexes in infected cells. these could be detected only when the detergents used for their solubilization from cells or virions were carefully chosen: a combination of nonionic (np- ) and ionic (deoxycholic acid) detergents proved to be optimal. pulse-chase experiments revealed that newly made m and s proteins engaged in complex formation with different kinetics. whereas the m protein appeared in complexes immediately after its synthesis, newly synthesized s protein did so only after a lag phase of > min. newly made m was incorporated into virus particles faster than s, which suggests that it associates with preexisting s molecules. using the vaccinia virus t -driven coexpression of m and s we also demonstrate formation of m/s complexes in the absence of other coronaviral proteins. pulse-chase labelings and coimmunoprecipitation analyses revealed that m and s associate in pre-golgi membranes because the unglycosylated form of m appeared in m/s complexes rapidly. since no association of m and s was detected when protein export from the er was blocked by brefeldin a, stable complexes most likely arise in the er-to-golgi intermediate compartment. sucrose velocity gradient analysis showed the m/s complexes to be heterogeneous and of higher order, suggesting that they are maintained by homo- and heterotypic interactions. m/s complexes colocalized with alpha-mannosidase ii, a resident golgi protein. they acquired golgi-specific oligosaccharide modifications but were not detected at the cell surface. thus, the s protein, which on itself was transported to the plasma membrane, was retained in the golgi complex by its association with the m protein. because coronaviruses bud at pre-golgi membranes, this result implies that the envelope glycoprotein complexes do not determine the site of budding. yet, the self-association of the mhv envelope glycoproteins into higher order complexes is indicative of its role in the sorting of the viral membrane proteins and in driving the formation of the viral lipoprotein coat in virus assembly. m and s associate in pre-golgi membranes because the unglycosylated form of m appeared in m/s complexes rapidly. since no association of m and s was detected when protein export from the er was blocked by brefeldin a, stable complexes most likely arise in the er-to-golgi intermediate compartment. sucrose velocity gradient analysis showed the m/s complexes to be heterogeneous and of higher order, suggesting that they are maintained by homo-and heterotypic interactions. m/s complexes colocalized with a-mannosidase ii, a resident golgi protein. they acquired golgi-specific oligosaccharide modifications but were not detected at the cell surface. thus, the s protein, which on itself was transported to the plasma membrane, was retained in the golgi complex by its association with the m protein. because coronaviruses bud at pre-golgi membranes, this result implies that the envelope glycoprotein complexes do not determine the site of budding. yet, the self-association of the mhv envelope glycoproteins into higher order complexes is indicative of its role in the sorting of the viral membrane proteins and in driving the formation of the viral lipoprotein coat in virus assembly. udding through cellular membranes is the final step in the assembly of enveloped viruses. it results in the envelopment of the viral nucleocapsid (nc) by a membrane modified by the viral envelope proteins. complex protein-protein interactions between the envelope proteins control the timing, location, and specificity of budding. the envelopment of the nc is believed to be driven by its interactions with the envelope proteins (simons and garoff, ; dubois-dalcq et al., ; simons (stephens and compans, ) , while others bud into intracellular compartments (pettersson, ; griffiths and rottier, ) . the location of virus budding is thought to be dictated by the envelope proteins because they usually accumulate at the site of budding (dubois-dalcq et al., ; stephens and compans, ; pettersson, ; hobman, ) . when the semliki forest virus spike proteins were arrested in the golgi complex by monensin, budding took place there rather than at the plasma membrane (griffiths et al., ) . however, the accumulation of the vesicular stomatitis virus (vsv) g protein in the trans-golgi network by incubation at °c prevented assembly (griffiths et al., ) . thus, although a local accumulation of envelope proteins may be instrumental in budding, additional yet unidentified factors may be involved in localizing this process. we studied the assembly of the mouse hepatitis coronavirus (mhv), strain a- . mhv is a large, enveloped, positive-strand rna virus with a simple protein composition (spaan et al., ; holmes, ) . the viral genome is packaged by the nucleocapsid (n) protein into an nc with helical symmetry, and this in turn is enveloped by a lipid bilayer containing the membrane (m) protein ( - kd) and the spike (s) protein ( kd). a small, nonglycosylated membrane protein (e) of ~ kd was recently identified as a minor third constituent of the viral envelope (yu et al., ) . coronaviruses are assembled at smooth membranes of the intermediate compartment (ic) (tooze et al., ; klumperman et al., ; krijnse-locker et al., ) . because coronaviruses lack a matrix protein, envelopment probably involves direct interactions between the nc and one or more of the envelope proteins. the m protein is a likely candidate because of its abundancy, and because it was found to associate with the nc in vitro (sturman et al., ) . moreover, the s protein is probably dispensable for budding since spikeless virions were produced when infected cells were treated with tunicamycin (holmes et al., ; rottier et al., ) . the role of the e protein is presently unknown. we have shown previously that neither of the envelope glycoproteins accumulates at the site of budding when expressed independently: the m protein alone localizes to the golgi complex (rottier and rose, ; krijnse locker et al., a; klumperman et al., ) , whereas the s protein is transported to the plasma membrane (vennema, h., and p. j. m. rottier, unpublished data) . the different fates of the independently expressed glycoproteins suggested to us that m and s, which are synthesized from separate mrnas in infected cells, are involved in intermolecular interactions during virus assembly. the present study was therefore aimed at detecting and characterizing such interactions in mhv-infected cells and in cells expressing m and s from their cloned genes. using proper solubilization conditions, m/s complexes were identified by immunoprecipitation and sedimentation analysis. we investigated whether the association of m and s is the factor determining the site of budding, as it might preclude their transport beyond the budding compartment. in addition, we asked whether lateral interactions between m and s lead to the formation of large envelope glycoprotein assemblies indicative of their role in driving the formation of the viral envelope. sac(-) cells were maintained in dulbecco's minimal essential medium containing % fcs, penicillin, and streptomycin (dmem- % fcs). bhk- cells were maintained in dmem- % fcs. ost - cells (elroy-stein and moss, ) , a kind gift of b. moss (national institutes of health, bethesda, md) were maintained in dmem- % fcs supplemented with ~g/ml g- (geneticin; gibco laboratories, grand island, ny). mhv-a was propagated in sac(-) cells as described previously (spaan et al., ) . the recombinant vaccinia virus vtf - expressing the bacteriophage t rna polymerase (fuerst et al., ) was obtained from b. moss. the production of the rabbit polyclonal antiserum to mhv-a , the rabbit antipeptide serum to the m protein, and the rabbit polyclonal antiserum to vesicular stomatitis virus have been described previously (krijnse locker et al., b; rottier et al., , vennema et al., b . the mabs j . and j . against s and m, respectively, were kindly provided by j. fleming (university of southern california, los angeles, ca). the polyclonal rabbit serum against a-mannosidase ii (man ii) (moremen et al., ) was a kind gift of k. moremen (university of georgia, athens, ga). mhv infection. subconfluent monolayers of sac(-) or ost - cells in or -mm dishes were washed with pbs containing ~g of deae-dextran per ml and % fcs (pbs-deae-i% fcs) and inoculated with mhv-a for min at a multiplicity of infection of - in pbs-deae- % fcs at °c. for expression of cloned genes, subconfluent monolayers of ost - cells in -mm dishes were washed with dmem and inoculated with vtf - at a multiplicity of infection of ~ in dmem for rain at °c. ceils were then washed with dmem and transfected with plasmid dna. the following vectors were used: ptum-m (opstelten et al., ) , ptum-s (the mhv s gene cloned as a bamhi fragment [vennema et al., b] into ptug ), and ptuv-g, which contain a cdna copy of the mhv-m, the mhv-s, and the vsv-g protein, respectively, under the control of the t promotor. routinely, ixl dmem containing - txg plasmid dna was mixed with ixl lipofectin reagent (gibco brl, life technologies, inc., gaithersburg, md) and added to the cells. after a -rain incubation at room temperature, ~ dmem was added and the cells were incubated further at °c. coexpression of the mhv m and s proteins in double-transfected ost- cells was monitored by immunofluorescence. approximately % of the cells expressed detectable levels of the viral glycoproteins at h after infection; > % of these cells expressed both m and s. labeling. the incubation temperature of vtft- -infected bhk and ost - cells was shifted to °c at h after inoculation. the incubation temperature of mhv-infected ost - cells was shifted to °c min before labeling. at . or . h after inoculation, the ceils were starved for min in mem without methionine (gibco laboratories). when indicated, brefeldin a (bfa) (boehringer mannheim biochemicals, indianapolis, in) was added to a concentration of ~g/ml. cells were pulse labeled with - p.ci s-in vitro labeling mix (amersham corp., arlington heights, il) for the times indicated, then washed once with dmem- % fcs supplemented with mm hepes, mm l-methionine, and mm l-cysteine (chase medium) and chased for various times in chase medium. the cells were lysed on ice in mm tris (ph . ), . mm edta, . % np- , . % na-deoxycholate (detergent solution) containing mm pmsf. the lysates were spun for min at , g at °c to remove nuclei and cell debris. in the experiment of fig. a cells were also lysed using % np- or % triton x- in mm tris (ph . ), . mm edta or % triton x- in mnt ( mm mes, mm tris [ph . ], mm naci, . mm edta, mm egta). samples of cell lysates were mixed with % (wt/wt) sucrose in detergent solution to a final concentration of % (wt/wt) before loading on gradients consisting of - % (wt/wt) sucrose in detergent solution. the gradients were centrifuged for min at , rpm in an sw . rotor (beckman instruments, inc., fullerton, ca) at °c and fractions of ~ p. were collected from the bottom of the tubes. aliquots of each fraction were subjected to immunoprecipitation with specific antibodies and the precipitates analyzed by sds-page. the sedimentation of the marker molecules catalase ( . s ,~) and thyroglobulin ( . s ,~) was performed in a parallel gradient. aliquots of the fractions were analyzed in an sds-page, and the proteins visualized by staining with coomassie brilliant blue. viral proteins were immunoprecipitated with the polyclonal mhv-a antiserum ( }~l), the mab j . ct m ( }xl), or with the mab j . ct s ( i~l). antibodies were added to aliquots of cell lysates diluted with detergent solution to a final vol of p~l. after overnight incubation at °c, immune complexes were collected using - }xl of a % (wt/vol) suspension of formaldehyde-fixed and heat-inactivated staphylococcus aureus cells (gibco brl, life technologies, inc.). after a -min incubation at °c the cells were washed three times with detergent solution and finally suspended in }~l . mm tris-hcl (ph . ), mm dtf, % sds, % glycerol (sample buffer). the samples were heated for min at °c before loading on or % sds-pag. endoglycosidase h (boehringer mannheim biochemicals) treatments were carried out as described by machamer er al. ( ) . quantification of the radioactivity in the protein bands in the dried gels was carried out using a phosphoimager and imagequant (version . ; molecular dynamics, inc., sunnyvale, ca) according to the manufacturer's instructions. transfected cells were labeled with s-in vitro labeling mix from - . h after infection and chased for h in chase medium. the culture media cleared by centrifugation for min at , rpm, °c were diluted with a / volume of a times concentrated stock detergent solution and subjected to immunoprecipitation using mabs against m and s. plates were put on ice and the cells washed with pbs/ % fcs and incubated for h in p, pbs/ % fcs containing the mab j . etm ( p, ), and/or mab j . as ( }~l), and/or the polyclonal anti-vsv serum ( p~l). thereafter, cells were extensively washed with pbs/ % fcs and lysed with detergent solution containing mm pmsf. the lysates were spun for min at , g at °c, and }~l of a % (wt/vol) suspension of formalin-fixed s. aureus cells was added to the supernatants to collect the immune complexes. after a -min incubation at °c, the cells were pelleted by centrifugation, washed three times with detergent solution, and finally suspended in sample buffer. the primary supernatants were subjected to a second round of immunoprecipitation using the same antibodies. ost - or bhk- cells grown on -mm, gelatin-coated coverslips, were infected and transfected as described above. h after infection cells were fixed with % paraformaldehyde for - min and washed three times with pbs containing mm glycine (pbs-glycine). cells were permeabilized with pbs/ % triton x- for rain followed by three washes with pbs-glycine. they were then treated for min with a mixture of two an-tisera diluted in pbs-glycine- % fcs: mab j . ~s ( : ); m peptide antiserum ( : ); polyclonal man ii rabbit antiserum ( : ). antibodies were washed away, and the cells were stained for min with affinitypurified rhodamine-conjugated goat anti-rabbit ig and fluorescein-conjugated goat anti-mouse ig (protos immunoresearch, san fransisco, ca) that were diluted : and : in pbs-glycine- % fcs, respectively. all incubations were done at room temperature. finally, the coverslips were washed extensively and mounted in fluorosave tm (calbiochem corp., la jolla, ca). fluorescence was viewed with a microscope (bhs-f; olympus corp., precision instruments division, success, ny). complex formation between the m and s protein of coronaviruses has never been demonstrated. we reasoned that this might have been due to a disruption of the interactions during analytical procedures since maintenance of the integrity of the complexes might require specific conditions. by analyzing the effects of different solubilization conditions we were able to demonstrate the existence of m/s complexes and found that the choice of the detergents is crucial for the preservation of the interaction between m and s. this is illustrated by the experiment shown in fig. irrespective of the detergent used, the anti-mhv serum precipitated the viral structural proteins n, m, and the s precursor (gp ). the mabets precipitated the s protein, as expected, but in one case also the m protein, when a combination of the nonionic detergent np- and the ionic detergent deoxycholic acid was used. the amount of coprecipitated m was similar to that obtained with the anti-mhv serum; virtually no n protein nor any cellular proteins was observed in the precipitate. we conclude that in infected cells a large fraction of m is physically complexed with s, and that the stability of these complexes is dependent on the detergents used for solubilization. to determine whether m and s associate in the absence of other coronaviral proteins, we used vaccinia virus ttdriven coexpression of their respective genes in ostt- cells (elroy-stein and moss, ) . the results with the anti-mhv serum show that m and s were efficiently labeled in cells coexpressing the proteins (fig. b) . when using the s-specific antibodies m was also precipitated, in addition to s, indicating that the proteins do associate independent of other coronaviral proteins. to exclude the possibility that their association had occurred after cell lysis, we performed the immunoprecipitations with a mixture of lysates from cultures in which m and s had been expressed separately. in this case m was not coprecipitated. we never observed specific coprecipitation of other labeled proteins: some bands appearing after longer exposures were also detected in analyses of cells expressing m or s alone. note that the rabbit anti-mhv serum precipitates some non-mhv proteins from expressing cells, not from mhv-infected cells. the identity of these proteins is unclear, but they are likely to be derived from vaccinia virus. the almost quantitative coprecipitation of m from mhv-infected cells indicates that all forms of the protein were associated with s. the incubation temperature ( °c) did not affect complex formation since the same pattern was observed at °c ( fig. a) . in contrast, predominantly glycosylated forms of m were associated with s in transfected cells. to determine the kinetics of protein association in infected cells we carried out a pulse-chase experiment. mhv-infected cells were labeled for min and chased for various time periods as indicated in fig. . the material precipitated with the anti-mhv serum represents the total pool of labeled viral structural proteins present in infected cells after the various chase periods. their amount decreased during the chase period due to assembly into virions and subsequent release from the cells. the changes in the mobilities of the glycoproteins show that they were processed; the unglycosylated form of m was converted into various slower-migrating species as a result of posttranslational o-glycosylation. the addition of the first sugar, n-acetyl-galactosamine, takes place in the ic (tooze et al., ; krijnse-locker et al., ) while the addition of galactose and sialic acid occur after the protein has reached the golgi complex (krijnse locker et al., a) . the s protein is synthesized as a core glycosylated precursor s/gp which is slowly converted into s/gpl (not clearly resolved in this gel) by maturation of its oligosaccharides. a fraction of this species is cleaved into subunits s and $ , both with a molecular mass of ~ kd (s/gp ). m/s complexes were analyzed by immunoprecipitation with the monospecific antibodies against m and s. using the mabe~s, coprecipitation of m was again observed after the pulse. the amount of coprecipitated m protein rapidly increased and reached its maximum after -- min of chase. apparently, newly synthesized m associated with s very quickly as evidenced also by the presence of the unglycosylated form of m in the m/s complexes. these observations indicate that the proteins associate in a pre-golgi compartment. the m protein present in m/s compl.exes was converted from its unglycosylated form into the various glycosylated species, which shows that its processing occurs after its association with s. when the m a b a m was used instead, coprecipitation of labeled s was not observed immediately, but only after - min of chase. later, the amount of coprecipitated s, including its cleaved form s/gp , gradually increased. these observations indicate that s associated with m before its processing to the mature forms s/gpl and s/gp . m and s thus engage in complex formation at different rates: m rapidly associates with s, while s does so only after a considerable lag time. this implies that newly synthesized m molecules associate with s molecules already present. if these interpretations were correct, the m and s proteins would be incorporated into virus particles with different kinetics. we therefore performed a pulse-chase labeling of infected cells and monitored the appearance of labeled proteins in extracellular virus. because mhv is assembled intracellularly, such an analysis provides an indirect measurement of the kinetics of incorporation of newly synthesized proteins into virus particles. virus was purified by pelleting and analyzed by sds-page (fig. a) . the results of the quantifications of the radioactivities in the bands representing m and s (s/gp + s/gp ) are shown in fig. b. virions containing labeled s protein appeared in the medium after a -min lag period. thereafter, roughly equal amounts of labeled s were found to be released during each chase interval for at least min. in contrast, labeled m protein started to appear in the culture medium already during the - min chase period, increased rapidly during the -- min chase period, and declined thereafter. this implies that newly synthesized m was incorporated into virions faster than s. in addition, the s molecules were assembled into virions in a more protracted fashion than the m molecules. to investigate whether the association of m and s is the factor determining the site of virus budding we analyzed the intracellular localization of m/s complexes by indirect double immunofluorescence. for this purpose, we coexpressed m and s in bhk- cells because these cells are appropriate for immunolocalization. both proteins localized to a distinct perinuclear region (fig. , a and b) . in contrast, when expressed by itself, s had a faint reticular appearance (fig. d) while it was also observed at the surface of nonpermeabilized cells (not shown). the intracellular localization of the m protein appeared not to be affected by s because its distribution was similar to that in cells which expressed m only (fig. c) . the suggestion that m and s coaccumulated in the golgi complex was confirmed by visualizing this compartment using a serum against man ii, a resident golgi protein (moremen et al., ) . the localization of man ii clearly overlapped with that of the s protein (fig. , e and f). the conclusion that m had retained s in the golgi region is consistent with the observation that m/s complexes predominantly contained mature forms of m (fig. b) . to analyze the transport of m/s complexes biochemically we labeled double-transfected cells for min and chased them for different periods. equal fractions of the cell lysates were subjected to immunoprecipitation with the anti-mhv serum (fig. a) , the mabols (fig. a) , or the mabetm (fig. b) . the precipitates obtained using the mab~m were split to perform an endoglycosidase h sensitivity assay. the time courses with which newly synthesized m and s engage in heterocomplexes in the absence of other viral components appeared to be similar to those observed in mhv-infected cells (fig. ) . after the pulse, a small frac-tion of m was coprecipitated by the mabots (fig. a) ; its amount increased during the chase and reached a maximum between and min of chase. in contrast, coprecipitation of labeled s appeared only after min of chase and reached its highest level ~ min later (fig. b) . the m protein already associated with s while still in its unglycosylated form (fig. a) indicating that the complexes are formed in a pre-golgi compartment. consistently, the s precursor gpl , coprecipitated by the m-specific antibodies ( fig. b) , appeared to be completely endoglycosidase h-sensitive, which means that it had not yet passed the medial-golgi. the finding that m/s complexes eventually consisted of golgi-modified forms both of m and of s confirms their transport to the golgi complex. surprisingly, the pulse-chase assay did not reveal any transient accumulation of the unglycosylated precursor form m nor of m . apparently, the accumulation of m/s complexes in the ic, which supposedly occurs in mhv-infected cells, does not occur when the proteins are coexpressed. instead, m/s complexes are transported efficiently beyond the budding site to the golgi complex in the absence of other coronaviral proteins. although the golgi-modified forms of m and s were all found in m/s complexes, a fraction of m and s remained immature and incompetent to associate even after longer chase periods. as far as the s protein is concerned, this is similar to what we found in mhv-infected cells (fig. ) . in that case, however, the m protein associated with s very rapidly and almost quantitatively. this suggests that a fraction of m is not properly processed in this vaccinia virusbased expression system, a phenomenon also found with other viral proteins (marquardt and helenius, ; vennema, h., g.-j. godeke, and p. j. m. rottier, unpublished data). however, the less efficient transport of m seems not to be directly caused by the vaccinia virus infection, since the m protein expressed by a recombinant vaccinia virus was transported almost quantitatively to the golgi complex (krijnse locker et al., a) . the data obtained so far suggest that the coexpressed m and s proteins associate during their transport to the golgi complex while they form complexes already in the er of mhv-infected cells (fig. b) . to establish whether m and s associate in the er or beyond this compartment we analyzed the formation of m/s complexes in the presence -schwartz et al., -schwartz et al., , klausner et al., ) . in the experiment of fig. , bfa was added to mhv-infected cells and to cells coexpressing m and s at min before the labeling, and it was kept present during further incubations. the activity of the drug was evident from its effect on the maturation of s. in the presence of bfa its conversion into s/gp and its subsequent cleavage were completely inhibited, consistent with the block of transport to a late golgi compartment. the maturation of the m protein was not affected by bfa. this indicates that the enzymes catalyzing the formation of m and m relocated into the er upon the addition of bfa. this result is somewhat different from findings in another cell type (krijnse locker et al., a) where n o m species was synthesized when bfa was present. in the coexpression system bfa prevented the formation of stable m/s complexes as judged by the absence of m in the immunoprecipitate of s. this indicates that transport of the coexpressed m and s proteins from the er is required for their complexation. in contrast, in mhvinfected cells the association of m and s was not affected by bfa since equal amounts of m were coprecipitated from bfa-treated and -untreated samples. thus, the failure of m and s to interact in coexpressing cells was not artificially induced by bfa. the effects of bfa appeared to be reversible; after its washout a significant fraction of m was coprecipitated. in addition, the appearance of s/gp and s/gp , though weak, indicates that transport from the er to the golgi complex was also restored to some extent. the immunofluorescence data indicated that s is retained intracellularly by its interaction with m. apparently, the signal that mediates the retention of m in the golgi complex (rottier and rose, ; krijnse locker et al., a , , is also functional in complexes of m with s. in contrast to m, the s protein is normally transported to the cell surface (vennema, h., and p. j. m. rottier, unpublished data; see below) . to test whether the efficacy of golgi retention of the m protein is affected by its association with s we performed cell surface immunoprecipitations. cells expressing s or both m and s proteins were labeled for min and chased for h to allow the proteins to reach their final destination. the culture media were screened for the presence of viral proteins and the mabs as and am, which recognize the ectodomain of m or s, were used for surface immunoprecipitations. the intracellular pool of viral proteins was collected in a second round of immunoprecipitation with the same antibodies. the m and s proteins were virtually absent from the medium and the plasma membrane of double-transfected cells, although most of them had reached the golgi complex as evidenced by their maturation state inside the cells (fig. ) . in contrast, we specifically detected s/gp and s/gp at the surface of cells expressing s alone; the precursor s/gp was only detected intracellularly confirming the reliability of the assay. some s protein also appeared at the plasma membrane when expressed with m; a tiny fraction apparently escapes from interacting with m and is transported to the cell surface. accordingly, cells coexpressing m and s still fused, albeit more slowly and less extensively than when s was expressed alone (not shown). relatively more s/gp accumulated in the presence of m, whereas most of it was cleaved in its absence. moreover, s/gp was detected predominantly inside the cells coexpressing m and s while it appeared more prominently at the cell surface when expressed on its own. in the figure . intracellular transport of m/s complexes, vtft- -infected ost - cells were cotransfected with ptum-m and ptum-s dna. cells were pulse labeled at h after infection for min and immediately lysed or chased for the times indicated. equal fractions of the cell lysates were used for immunoprecipitation using polyclonal anti-mhv serum (a), the mabas (a), or the mabam (b). precipitates obtained using mabam were split; one-half was mock treated and the other half was treated with endoglycosidase h (b). viral proteins were analyzed in two sds- % pags. latter situation a significant amount of s/gp was found in the culture medium presumably representing the s subunit dissociated from the membrane-anchored $ subunit. the results indicate that s, when caught in m/s complexes, was arrested in the golgi complex. despite its interaction with s, the m protein does not leak to the plasma membrane. to check whether the accumulation of m in the golgi complex does prevent protein transport through this compartment nonspecifically we expressed m together with the vsv-g protein. the result shows that the g protein appeared quantitatively at the plasma membrane. we conclude that golgi retention of the s protein is mediated by its specific interaction with the m protein and that trans- figure . effects of bfa on the formation of m/s complexes. vtf - -infected ost - cells were cotransfected with ptum-m and ptum-s dna (m/s). parallel cultures were infected with mhv. at h after infection cells were pulse labeled for min followed by a -min chase. when indicated bfa was added to the cells at . h after infection and it was kept present during labeling and chase (+). in one case, bfa was washed out and cells were further chased for another min in the absence of bfa (+/-). equal portions of the lysates were used for immunoprecipitation using polyclonal anti-mhv serum or mabas. viral proteins were analyzed in an sds- % page. port of other membrane proteins to the cell surface is unimpeded by the presence of m. to investigate the complexity of the m/s complexes we have analyzed their sedimentation in sucrose velocity gradients. cells coexpressing m and s were radiolabeled for min and lysed either immediately or after and min of chase. the cleared lysates were analyzed in - % sucrose gradients. after fractionation, aliquots of gradient fractions were subjected to immunoprecipitation with the mabas or with the mabam. newly synthesized s protein was almost exclusively detected at the top of the gradient, while some m protein was coprecipitated from samples of the lower part of the gradient (fig. a) . the virtual absence of labeled s in the latter samples indicates that newly synthesized m was associated with preexisting, unlabeled s. during the chase the m protein in the complexes matured and gradually more labeled s protein sedimented into the gradient (fig. , b and c). the m/s complexes sedimented heterogeneously and were found predominantly between fraction and the bottom of the gradient. in addition, some m coprecipitated with s from the two top fractions; this material might represent partially disrupted complexes. using the m-specific antibodies we found that the complexes formed after a min chase (fig. d) contained s precursor gpl , as well as s/gpl . in contrast, when the s protein was expressed alone, its mature forms s/gpl and s/gp remained at the top of the gradient indicating that the protein does not nonspecifically associate into large aggregates (fig. e) . we have used the sedimentation behavior of catalase and thyroglobulin in parallel gradients as markers to get an estimate of the size of m/s complexes. these proteins peaked in fractions and , respectively (data not shown). this indicates that most complexes migrated as structures with sedimentation values higher than s. both unglycosylated and glycosylated forms of m were found in heterocomplexes formed during the pulse. the figure . intracellular accumulation of m/s complexes, vqtt- -infected ost - cells were (co)transfected with ptum-s, ptum-m, and/or ptuv-g dna. cells were pulse labeled for min and chased for min. the media (m) were cleared and subjected to immunoprecipitation (ripa) using the mabets and mabotm. the cell-surface (s) immunoprecipitations were carried out as follows: antibodies against the expressed proteins were added to the cells to allow binding at the plasma membrane. after a -h incubation on ice, the cells were washed extensively and then lysed. the immune complexes were precipitated with s. aureus cells after which the remaining intracellular (i) pool of viral proteins was collected in a second round of immunoprecipitation with the same antibodies. viral proteins were analyzed in an sds- % page (a). fig. b represents a longer exposure of a part of fig. a. presence of the unglycosylated form again indicates (see fig. ) that the protein is taken up in large heterocomplexes before its arrival in the golgi complex. the m and s proteins of mhv have different destinations in cells when expressed independently, yet they coassemble into virions during infection. here we describe the molecular basis for this peculiarity by showing that the two proteins exhibit an intrinsic affinity for each other. the association of m and s occurs in an early compartment since newly synthesized, unmodified m molecules are immediately taken up into complexes with s, indicating that they are formed before or during budding. in contrast, newly made s associates slowly and less efficiently, and m and s are therefore assembled into virions at different rates. when coexpressed in cells, m and s form large heteromultimeric complexes that are transported beyond the site of budding and accumulate in the golgi complex. under conditions of analysis, the stability of the envelope glycoprotein complexes is critically dependent on the detergents used for their solubilization. m-s interactions are preserved in a combination of np- and sodium deoxycholate. several observations indicated that m/s complexes do not form nonspecifically after solubilization. first, other proteins were not significantly coprecipitated with m and s. second, newly synthesized s started to associate with m only after a lag time of about min. third, m/s complexes were not formed upon mixing lysates containing m and s that had been separately expressed. moreover, flotation analysis ruled out the possibility that m and s were coisolated as part of detergent-insoluble membranes (data not shown). collectively, these data provide evidence for the existence and specificity of m-s interactions. newly synthesized m and s molecules enter into heterocomplexes with different kinetics, probably due to their different maturation rates. folding of s occurs slowly and involves the formation of intramolecular disulfide bonds (opstelten et al., ) , the addition and processing of n-linked sugars, and the assembly into homo-oligomers (vennema et al., a) . in contrast, the m protein acquires its final conformation in the er rapidly, without being glycosylated, without the formation of disulfide bonds (opstelten et al., ) , and even without the need for atp . thus folding of s is probably rate limiting in its association with m. newly made m interacts with preexisting folded s, as evidenced by the presence of unlabeled s in complexes after short labelings. only completely oxidized s molecules associate with m, and s is unable to interact when its folding has been inhibited by in vivo reduction with dtt (opstelten et al., ; our unpublished results) . a similar situation has been observed for bunyaviruses: heterodimerization of uukuniemi virus glycoproteins occurs between newly made g and presynthesized g due to the slow maturation of the latter (persson and pettersson, ) . mhv buds in the rer late in infection (tooze et al., ; . several observations indicate that m and s associate in this compartment. first, m/s complexes are rapidly formed, much of m was associated with s after a min pulse. second, the presence of unglycosylated m in the complexes indicates its association before addition of the first sugar (n-acetyl-galactosamine). third, treatment of infected cells with bfa, a drug that prevents exit of proteins from the er, did not inhibit the formation of m/s complexes. although these data suggest that the interaction between m and s precedes budding, we cannot exclude that association occurs synchronously with assembly. the association of coexpressed m and s also occurs in an early compartment since newly synthesized, unmodified m molecules appear in m/s complexes rapidly. however, the failure to detect the interactions in the presence of bfa suggests that stable complexes are formed only after the proteins have left the er, i.e., in the ic. this suggests that other factors are involved in the early formation and/or stabilization of m/s complexes in infected cells. one such factor might be the nc. its binding to the envelope glycoprotein complexes might promote and stabilize the inter-actions between m and s and create nucleation sites for conglomeration of the complexes. alternatively, the difference might be just the result of a concentration effect since multimerization reactions are dependent on the con- centrations of the reacting molecules (braakman et al., ) . thus, under our coexpression conditions, stable association of m and s might not have occurred in the e r because the proteins did not accumulate to sufficient lev-els. membrane glycoproteins are indeed concentrated during their export from the er (copeland et al., ; balch et al., ) , and this might induce the formation of stable m/s complexes. in the course of an mhv infection, the synthesis of the envelope proteins reaches much higher levels than during expression. thus, the association of m and s in infected cells might take place in the ic initially and in the er at later stages of infection, coinciding with the temporal pattern of virus budding. the m/s complexes do not determine the site of budding, because their transit through the ic was neither blocked nor delayed. the envelope proteins of other viruses generally accumulate at the site of assembly (pettersson, ; griffiths and rottier, ; hobman, ) while this is clearly not the case for mhv. because the m and s protein are not retained in the ic individually, we had anticipated that their association might endow the complexes with new retention information. their accumulation in the golgi complex, however, indicates that (an)other viral factor(s) determines the site of budding. a likely candidate is the nc that might bind and recruit the m/s complexes early in the exocytic pathway. another candidate is the small envelope protein (e) which was recently identified as a minor virion component (yu et al., ) . due to its association with m, the s protein accumulated in the golgi complex, the intrinsic residence of the m protein. because the cell-surface expression of the vsv-g protein was not affected by m, we conclude that m specifically retains the s protein. similarly, the g protein of punta toro virus is transported to the cell surface when expressed by itself, but accumulates in the golgi complex after heterodimerization with g that contains the signal for golgi retention (chen et al., ; matsuoka et al., ) . cosorting by association is a well-known principle of protein targeting and retention in eukaryotic cells. recently, nilsson et al. ( nilsson et al. ( , proposed kin recognition as a mechanism for sorting and retention of golgi enzymes. accordingly, sorting of the enzymes is based on specific interactions between kin-oligomers, while retention is achieved by their involvement in complexes too bulky to enter transport vescicles. the self-association of m and s has similar features: the proteins interact specifically, segregate from other proteins, and are subsequently targeted to and retained in the golgi complex. however, the association of m and s into large complexes in pre-golgi membranes apparently does not inhibit their further transport to the golgi complex. the interaction between m and s probably functions to incorporate the s protein into virions. s is dispensable for particle assembly since spikeless virions are released from infected cells treated with tunicamycin (holmes et al., ; rottier et al., ) . moreover, recent data from our laboratory show that the assembly of coronavirus-like particles also occurs independently of the s protein (vennema, h., g.-j. godeke, and p. j. m. rottier, unpublished data) which implies that envelope formation does not depend on m-s interactions. we therefore assume that the higher-order m/s complexes are maintained primarily, if not only, by m-m interactions. this is supported by our recent findings that m associates into large complexes when expressed by itself (krijnse locker et al., ) . extensive envelope protein interactions have important functions in coronavirus assembly. first, they are responsible for host protein exclusion. second, local membrane domains harboring these large envelope protein assemblies serve as the sites for budding. third, the clustering of envelope proteins may alter the fluidity of the lipid bilayer, for instance to facilitate the induction of curvature (dubois-dalcq et al., ; simons and fuller, ) . this factor could promote budding. finally, the envelope protein clusters might serve as a template for the condensation of the nc. the nc of coronaviruses are long, loosely coiled strands in the cytoplasm of infected cells that condensate at the membranes of the budding compartment; other viruses having a helical nc, e.g., vsv, encode a matrix protein that controls the coiling of the nc (newcomb et al., ) . the emerging picture of coronavirus budding shows extensive local rafts (patches) of laterally interacting m molecules in pre-golgi membranes. only few copies of the e protein and variable numbers of s molecules are encountered in these structures. ncs bind to the cytoplasmically exposed domains of the m proteins, surround themselves with the modified membrane, and pinch off into the lumen. the rafts are continuously being replenished by the arrival and incorporation of newly synthesized viral envelope proteins. further work should show whether this model is correct and establish the interactions that compound it. vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum folding of influenza hemagglutinin in the endoplasmic reticulum the role of envelope proteins in hepatitis b virus assembly golgi complex localization of the punta toro virus g protein requires its association with the g protein folding, trimerization, and transport are sequential events in the biogenesis of influenza virus hemagglutinin assembly of enveloped rna viruses membrane protein lateral interactions control semliki forest virus budding cytoplasmic expression system based on constitutive synthesis of bacteriophage t rna polymerase in mammalian cells eukaryotic transient-expression system based on recombinant vaccinia virus that synthesize bacteriophage t rna polymerase cell biology of viruses that assemble along the biosynthetic pathway dissection of the golgi complex. monensin inhibits the transport of viral membrane proteins from medial-to trans-golgi cisternae in baby hamster kidney cells infected with semliki forest virus exit of newly synthesized membrane 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complex oligomerization of a trans-golgi/trans-golgi network retained protein occurs in the golgi complex and may be part of its retention rapid redistribution of golgi proteins into the er in cells treated with brefeldin a: evidence for membrane cycling from golgi to er microtubule-dependent retrograde transport of proteins into the er in the presence of brefeldin a suggests an er recycling pathway nucleocapsid-glycoprotein interactions required for assembly of alphaviruses the e glycoprotein of an avian coronavirus is targeted to the cis golgi complex misfolding and aggregation of newly synthesized proteins in the endoplasmic reticulum a signal for golgi retention in the bunyavirus g glycoprotein novel purification of the catalytic domain of golgi ct-mannosidase ii in vitro reassembly of vesicular stomafitis virus skeletons kin recognition. a model for the retention of golgi enzymes kin recognition between medial golgi enzymes in hela cells disulfide bonds in folding and transport of mouse hepatitis coronavirus glycoproteins formation and intracellular transport of a heterodimeric viral spike protein complex protein localization and virus assembly at intracellular membranes coronavirus e glycoprotein expressed from cloned cdna localizes in the golgi region viral protein synthesis in mouse hepatitis virus strain a -infected cells: effects of tunicamycin signal recognition particledependent insertion of coronavirus el, an intracellular membrane glycoprotein the budding of enveloped viruses: a paradigm for membrane sorting? the budding mechanisms of enveloped animal viruses isolation and identification of virus-specific mrnas in cells infected with mouse hepatitis virus (mhv-a ) coronaviruses: structure and genome expression assembly of animal viruses at cellular membranes isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid spike protein-nucleocapsid interactions drive the budding of alphaviruses replication of coronavirus mhv-a in sac-cells: determination of the first site of budding of progeny virions site of addition of n-acetyl-galactosamine to the e glycoprotein of mouse hepatitis virus-a biosynthesis and function of the coronavirus spike protein intracellular transport of recombinant coronavirus spike proteins: implications for virus assembly mouse hepatitis virus gene b protein is a new virion envelope protein we thank h. vennema and g.-j. godeke for providing the plasmid dnas used in this work.received for publication february and in revised form june . key: cord- - v cncid authors: raaben, matthijs; prins, henk‐jan; martens, anton c.; rottier, peter j. m.; de haan, cornelis a. m. title: non‐invasive imaging of mouse hepatitis coronavirus infection reveals determinants of viral replication and spread in vivo date: - - journal: cell microbiol doi: . /j. - . . .x sha: doc_id: cord_uid: v cncid bioluminescence imaging (bli) is a powerful new method to study virus dissemination in the live animal. here we used this method to monitor the spatial and temporal progression of mouse hepatitis coronavirus (mhv) infection in mice using luciferase‐expressing viruses. upon intranasal inoculation, virus replication could initially be observed in the nasal cavity and the cervical lymph nodes, after which the infection spread to the brain and frequently to the eyes. the kinetics of virus spread to and clearance from the brain appeared to depend on the inoculation dose. after intraperitoneal inoculation, virus replication was predominantly observed in the liver and occasionally in the intestines, but interestingly also in the tail and paws. bli thus elucidated new anatomic locations of virus replication. furthermore, mhv dissemination was shown to be critically depended on the viral spike protein, but also on the mouse strain used. widespread dissemination was observed in mice lacking a functional type i interferon response. the importance of the type i interferon system in limiting viral spread was also demonstrated by the administration of type i interferons to mice. our results provide new insights in coronavirus pathogenesis and demonstrate the potential of bli to study coronavirus–host interactions in vivo. current insights into infection processes of pathogens in their hosts and into the dynamics of their spread are largely based on studies using conventional methodologies. these methods have, however, many limitations. they require the experimental animals to be sacrificed in order to identify the sites of infection and to quantify the replication of the pathogens. they hence require large numbers of animals and are costly. they do not allow the real-time monitoring of spatial and temporal progression of infection in the same animal. important variations in host-pathogen interactions might therefore be overlooked, while dissemination of a pathogen to unexpected anatomical locations might be missed, simply because the infected tissue is not harvested and analysed (hutchens and luker, ; luker and luker, a) . as of recently many of these limitations of the conventional techniques can be overcome by powerful new methods that involve non-invasive imaging of pathogen replication and spread in infected live animals. as a member of the coronavirus (cov) family, the mouse hepatitis virus (mhv) provides a practical model system for studying cov-induced pathogenesis in mice. depending on the inoculation route, the virus strain and the genetic background of the host, mhv infection can result in a variety of pathological disorders (compton et al., ) . the most commonly used laboratory strains primarily infect the liver and the brain, thereby providing animal models for studying encephalitis and hepatitis as well as for immune-mediated demyelinating disease that sometimes develop during a later stage of infection (perlman, ) . inoculation of susceptible mice, either intracranially or intranasally, with neurotropic mhv strains can result in a number of different outcomes, ranging from acute encephalomyelitis to chronic demyelinating disease (houtman and fleming, ) . besides a mild encephalitis, mhv strain a also causes enteric disease and moderate hepatitis (lavi et al., ) . the differences in pathogenesis between various mhv strains have been linked mainly to the spike protein which mediates viruscell attachment and subsequent membrane fusion (phillips et al., ) . however, other viral genes have been shown to also significantly contribute to pathogenesis (de haan et al., ; sperry et al., ; iacono et al., ) . the role of the immune system in response to mhv infection has been extensively studied. both humoral and cellular immune reactions are essential to guard against mhv infections marten et al., ; morales et al., ) . clearance of virus during acute infection is predominantly regulated by a typical expression pattern of proinflammatory chemokines that attract cd + and cd + t lymphocytes to sites of infection (glass et al., ; lane et al., ; stiles et al., ) . also interferon (ifn)-and perforin-mediated mechanisms are involved in the clearance of mhv from different cell types (weiss and navas-martin, ) . the kinetics and the extent of the host immune response, which aims to limit the production of infectious virus and viral spread without inducing extensive deleterious effects, is important in determining survival of the host. infectious virus is usually cleared from mhv-infected mice within weeks. most animals, however, do not appear to obtain complete sterile immunity, since viral rna is often found to persist within the central nervous system (cns). recent biotechnological advances now allow the realtime imaging of pathogen replication and spread in living animals by making use of bioluminescence imaging (bli). to this end, light emitted by luciferase reporter proteins is detected by a cooled charge-coupled device (ccd) camera (contag et al., ; wu et al., ) . important advantages of bli are an intrinsically low background combined with a very high sensitivity for monitoring light emission in vivo (contag and bachmann, ; hutchens and luker, ) . furthermore, the substrate for firefly luciferase (fl), d-luciferin, is able to cross cellular membranes as well as the intact blood-brain barrier, thereby allowing the imaging of any anatomic location. in addition, d-luciferin is non-toxic, allowing the monitoring of individual mice over time through consecutive imaging. thus, fewer animals are typically needed to acquire statistically meaningful data at multiple time points as compared with conventional approaches (hutchens and luker, ) . initially, bli was used to identify sites of bacterial replication in intact animals (contag et al., ) . nowadays, this technique is widely used to study the formation and spread of cancer metastases and to visualize the effect of chemotherapy (shah et al., ) . bli has also been used to study the dissemination of herpes simplex virus, sindbis virus, vaccinia virus, and infectious hematopoietic necrosis virus (rodriguez et al., ; luker et al., ; cook and griffin, ; harmache et al., ) . all previous studies describing mhv infections and pathogenesis in mouse models have been relying on the sacrifice of infected mice in order to determine virus distribution and titres in various organs over time. although these conventional approaches have defined important factors that dictate replication and virulence of mhv in vivo as described above, non-invasive whole-body imaging of mhv infection in living mice is likely to offer new insights into virus replication, dissemination and pathogenesis. here, we have taken advantage of bli to study the replication and spread of fl-expressing mhv (de haan et al., ; a) in mice in real-time. we were also able to demonstrate differences in virus replication and spread, resulting either from differences in virus doses, mutations in the spike gene, differences in host susceptibility or from the administration of antiviral compounds. in addition, we identified new anatomic sites of virus replication. our results provide new insights in cov pathogenesis and demonstrate the potential of bli to study cov-host interactions in vivo. in our previous studies we extensively characterized various recombinant mhvs expressing luciferase reporter genes. the insertion of an fl expression cassette at different positions in the viral rna genome was shown not to appreciably affect virus multiplication in vitro, while the fl expression levels were demonstrated to be a reliable measure for virus replication (de haan et al., ; verheije et al., ) . to verify the feasibility of detecting mhv infection in vivo with bli, we compared two recombinant mhvs containing the luciferase gene at different positions in the viral genome with a recombinant wildtype mhv-a with respect to virus replication. therefore, we prepared new stocks of mhv-eflm (containing the fl gene as an additional expression cassette between the e and m gene), mhv- afls (containing the fl expression cassette at the position of the haemagglutinin esterase pseudo-gene) and wild-type mhv-a (fig. a ). in agreement with the previous studies we observed that the growth characteristics of the luciferaseexpressing viruses in tissue culture were highly similar to that of the parental mhv-a (fig. b) , with the fl reporter gene being expressed at high levels for both mhv-eflm and mhv- afls. for the characterization of the fl-expressing viruses in vivo we used balb/c mice, which have been shown to be susceptible to mhv infection (ohtsuka and taguchi, ) . as the inoculation route largely determines the dissemination of virus infection in an animal, we made use of both intranasal and intraperitoneal injection. the -to -week-old mice were inoculated with tissue culture % infectious dose (tcid ) of virus or with phosphate-buffered saline (pbs) (control) and sacrificed at days post inoculation, at which day virus titres peak (macnamara et al., ) . as mhv-a mainly targets the liver and brain (lavi et al., ; haring and perlman, ) , these organs were taken for analyses. tissue homogenates were prepared and subsequently tested for virus titres, rna levels and fl activity. in intranasally inoculated animals, we detected high virus titres in the brains of all infected mice, with no disparity between the wild-type and fl-expressing viruses ( fig. a) . infectious virus could not be recovered from the liver homogenates. also the viral rna levels in the brains of the animals, as determined by quantitative reverse transcriptase polymerase chain reaction (rt-pcr), were indistinguishable between groups. however, viral rna in the liver could only be detected in wild-type mhv-infected mice (fig. b ). all mice infected with the fl-expressing recombinants showed high fl activity in the brain, as well as some fl activity in the liver (fig. c ). although not statistically significant, the fl expression levels of mhv- afls were somewhat lower than those of mhv-eflm, which is consistent with the relative expression levels observed in vitro (de haan et al., ) . after intraperitoneal inoculation, infectious virus could only be recovered from the livers of wild-type mhv-a -infected animals ( fig. a) . although viral rna was detected in the livers of all groups, the levels were approximately a -fold lower for the fl-expressing mhv recombinants (fig. b) . furthermore, only in some livers of mhv-eflm-and mhv- afls-infected mice could fl activity above background level be detected. overall, these data show that the introduction of the fl expression cassette into the genome of mhv-a affected virus multiplication in the liver but, importantly, not in the brain of -to -week-old balb/c mice. high levels of fl activity were measured in the brains of mhv-eflm-and mhv- afls-infected mice at days post infection, indicating that these viruses are attractive candidates to be used for bli after intranasal inoculation. in vivo imaging of mhv infection thus, we applied whole-body bli to study the replication and spread of the two fl-expressing mhvs. to this end, balb/c mice were inoculated intranasally with tcid of virus after which virus replication was followed over time in individual mice. in general, very similar patterns of virus dissemination were observed for both viruses. at days post inoculation, a strong signal was observed, apparently emanating from the nasal cavity, when the mice were imaged at their dorsal side (fig. a ). when imaged from the ventral side, replication additionally could be observed in two distinct spots, probably representing the cervical lymph nodes of the animal. at this time point, no signal coming from the brain could yet be detected. three days later, dissemination of virus replication to the brain was apparent, while replication in the cervical lymph nodes was no longer observed. at days post inoculation, the signal coming from the brain appeared more dispersed, while its intensity was clearly declining. at days post inoculation, signal from the brain was the -to -week-old female balb/c mice were inoculated either intranasally or intraperitoneally with pbs or with tcid of wild-type mhv-a , mhv-eflm or mhv- afls. at days post inoculation, all mice were sacrificed and brains and livers were collected. homogenates were prepared as described in the experimental procedures. a. viral infectivity in the homogenates was determined by performing a plaque assay on lr cells. the viral titres are expressed as plaque-forming units per gram (pfu g - ) tissue. b. the amounts of viral genomic rna relative to total rna, isolated from the liver and brain homogenates, were determined by quantitative taqman rt-pcr. c. luciferase activities (in rlu) in each of the homogenates were determined by using a luminometer as described above. standard deviations (n = ) are indicated in all graphs. scarcely detectable in most mice, while it could no longer be detected at day (data not shown). with bli, it is important to note that the measurable photon flux decreases with increasing depth of the target tissue (luker and luker, b) . thus, the signal coming from the brain is significantly attenuated when compared with the signal from the olfactory epithelium, because photons emitted from brain cells must penetrate considerably more tissue to be detected (fig. b) . as a consequence, signal intensities should only be compared when photons emanate from the same tissue. photon quantification of the head regions demonstrated that mice infected with mhv-eflm showed significantly higher signal intensities than mice infected with mhv- afls at days post inoculation (fig. b ). in conclusion, with bli the temporal and spatial spread of mhv replication could be readily visualized in living mice. while mice infected with different luciferase-expressing viruses displayed very similar patterns of virus dissemination, bli allowed the quantitative detection of relatively small differences in fl-expression levels between mhv-eflm and mhv- afls, which is consistent with our earlier observations (de haan et al., ) . we next analysed whether we could visualize virus inoculation dose-dependent effects. to this end, balb/c mice were infected with two different doses of mhv-eflm, a high dose of ¥ and a low dose of ¥ tcid , and progression of infection was subsequently monitored by bli at different time points post inoculation (fig. a ). as expected, photon quantification of the head regions at days post inoculation demonstrated a clear correlation between the virus inoculation dose and the total signal produced from the olfactory epithelium ( fig. b ). in addition, spread of virus replication from the nasal cavity into the cns was more rapidly observed for the mice inoculated with the higher dose. this effect was quantified by dividing the head region into two sections (i.e. anterior versus posterior) after which the relative amount of photons emitted from these sections was determined (fig. c ). with a high virus inoculation dose, approximately % of the total amount of photons was coming from the posterior part at day , while with the low virus dose this degree of dissemination was delayed until around day . consistently, while mice that received the high dose showed virus replication in the cervical lymph nodes after days, this was apparent in the mice inoculated with the low dose only after days (table s a) . interestingly, mice infected with the higher dose appeared to clear the virus infection faster than animals infected with the low dose. mhv replication was easily detectable at day in mice inoculated with the low dose, whereas the luciferase signal of the high dose-inoculated group was almost undetectable at this time point. in some mice infected with a high virus dose, we could additionally visualize infection of the liver and lungs after days, whereas at later time points ( days), strong signals from the eyes were occasionally detectable. for a summary of the results see table s a . in conclusion, virus dissemination was shown to differ in mice infected with a low or a high dose. in mice that received the low dose virus replication was initially restricted to the nasal cavity, while much faster dissemination of virus infection was observed after inoculation with a high dose. strikingly, and counter intuitively, this rapid dissemination appeared to come at a cost, as these mice were able to clear the infection faster than the mice that received the low dose. we next analysed the essential contribution of the s protein to virus dissemination by using bli. the s protein of mhv is a major determinant of pathogenesis and tropism (phillips et al., ) . it mediates virus-cell attachment and fusion via binding of the mhv receptor ceacam a both in vitro and in vivo hemmila et al., ) . schickli et al. ( ) have described a limited set of mutations in the s protein of an mhv-a variant that had been acquired after extensive passaging in cell culture. we have recently shown that fig. . the inoculation dose affects virus dissemination. balb/c mice were inoculated intranasally with ¥ or ¥ tcid of mhv-eflm. at the indicated times post inoculation (days p.i.), mice were processed for bli. a. dorsal images of a representative mouse are portrayed. the emitted photons were measured at the indicated time points and are displayed as a heat map in an overlay representation. scaling [in arbitrary units (au)] is similar in all depicted images. the vertical line in all panels represents the arbitrary border between the anterior (nose) and posterior (brain) regions of the head, which were selected for photon quantification. b. the total amounts of emitted photons from the complete head regions at the different time points were quantified and are expressed as integrated intensities on a log scale with standard deviations (n = ). c. the relative amounts of emitted photons from the anterior and posterior regions of the head (i.e. brain versus nasal epithelium) at the different time points were quantified and are expressed as percentage of the total signal, with standard deviations for both virus doses (n = ). d. interesting phenotypes of mice at early (i.e. days; ventral images portrayed) or late (i.e. days; dorsal images portrayed) times post inoculation with ¥ tcid of mhv-eflm are shown: (a) liver, (b) paw, (c) lung, (d) eye and (e) cervical lymph nodes. note that the scaling [in arbitrary units (au)] differs between the ventral and dorsal images. these mutations enable the virus to use heparan sulfate as an additional attachment/entry factor. as a result, viruses carrying these mutations appeared to have acquired an extended host range as they were capable of entering cells also in a ceacam a-independent manner (schickli et al., ; de haan et al., b) . the physiological consequences of this adaptation in vivo, however, remain to be elucidated. here, we examined the infection of mice by an mhv with such an extended host range. therefore, we made use of a previously described recombinant virus (de haan et al., b) , which contains the mutant spike gene (srec) and the fl reporter gene. thus, this recombinant virus mhv- aflsrec only differs from its control virus mhv- afls in its spike protein. both viruses replicated to comparable titres in murine lr cells as demonstrated by the one-step growth curve shown in fig. b and displayed similar fl expression kinetics ( fig. c) . next, balb/c mice were inoculated via the intranasal route with these viruses. clearly, mhv- aflsrec replicated to a lower extent than the parental virus mhv- afls at all time points measured ( fig. a and b) . although the luciferase signal exhibited by the virus with the extended host range increased until days post infection, the infection did not progress towards the cns as was observed for the control virus. apparently, the acquisition of a heparan sulfate-dependent tropism significantly attenuated the ability of the virus to replicate and spread in vivo. replication of viruses in vivo is not only dependent on the genetic make-up of the virus, but also that of its host. it has been observed that the susceptibility of inbred mouse strains to different virus infections can vary significantly (parker et al., ; jubelt et al., ; thach et al., ) . in order to comparatively evaluate the mhv infection process in mice, we infected three commonly used mouse strains with mhv-eflm. to this end, balb/c, c bl/ and svev mice were inoculated in parallel each with tcid via the intranasal route after which virus replication was monitored over time. clearly, at days post inoculation balb/c mice displayed the highest signal in the brain, followed by c bl/ mice ( fig. a and b) . interestingly, svev mice were significantly less susceptible to mhv-eflm infection. in contrast to balb/c and c bl/ mice, virus replication was no longer detectable from days post inoculation in these animals (data not shown). to confirm these observations, we inoculated mice also with mhv-a wild-type virus and determined the viral rna load in the brain by quantitative rt-pcr at days post inoculation. in agreement with the bli results, balb/c mice showed the highest viral rna levels, while the brains from c bl/ and svev mice contained considerably less viral rna (fig. b ). next we studied virus replication and spread of the fl-expressing virus after intraperitoneal inoculation of balb/c and c bl/ mice. to this end -week-old mice were inoculated intraperitoneally with tcid of mhv-eflm (fig. c ). at days post inoculation we could visualize virus replication of the liver, with no apparent fig. . replication of mhv-eflm in mice of different genetic background. the -to -week-old female balb/c, c bl/ and svev mice were inoculated intranasally with tcid of mhv-eflm. a. bli was performed at days post inoculation as described in the experimental procedures. here, the biospace photon imager was used as detector for reporter gene expression. the emitted photon counts were measured and are displayed as a heat map in an overlay image. a representative dorsal image is shown for the different mouse strains. b. the emitted photons from the head regions of each individual mouse were quantified and are expressed as the total number of counts measured within the min imaging period. data are corrected for the background values (signal from uninfected mice). c. mice infected with wild-type mhv-a were sacrificed at day post infection, after which the individual brains were collected. total rna was isolated and taqman rt-pcr was performed targeting mhv genomic rna sequences. the relative viral rna (vrna) levels and standard deviations are depicted (n = ). d. the -week-old balb/c and c bl/ mice were inoculated intraperitoneally with tcid of mhv-eflm. bli was performed at days post inoculation. also here, the biospace photon imager was used. the emitted photons were measured and are displayed as a heat map in an overlay image. two representative ventral images are shown for the two different mouse strains. the anatomic locations displaying virus replication are indicated and include: (a) liver and (b) intestine. the arrows point to sites of virus replication in the tail and paws of the mice. difference between the two mouse strains. in some mice, infection of the intestine was also observed. interestingly, in all mice we could observe infection of the tail and paws, hitherto unidentified sites of infection. the signal rapidly decreased in time, with infection no longer being visible at - days post inoculation (data not shown). these results show that although replication of fl gene-containing viruses is decreased in -to -week-old mice (fig. ) , which hardly enabled the detection of mhv replication by bli (data not shown), the infection could easily be monitored in younger mice, which are known to be more susceptible to infection. in addition, the technology allowed the identification of new anatomic sites of mhv replication that had been missed with the conventional techniques (table s b) . next, bli was used to investigate the importance of the antiviral type i ifn system in controlling acute mhv infection. to this end, the effect of ifn alpha receptor knock-out (ifnar-/-) was studied using svev mice. wild-type and knock-out mice were inoculated via the intranasal route with tcid of mhv-eflm and the infection process was monitored as before. already at days post infection a significant difference between the ifnar-/-and wild-type mice was apparent when the animals were examined from the ventral side (fig. a) . a much higher signal was emitted from the nasal cavity in the ifnar-/-mice compared with the wild-type mice while, in addition, infection of the cervical lymph nodes was only detected in the knock-out mice. at later time points, infection was rapidly cleared from the wild-type mice, whereas dissemination to other organs, including liver and intestine, was manifest in the ifnar-/-mice. virus dissemination was accompanied by severe clinical signs, which included a significant decrease in body weight (fig. b) . mice had to be euthanized at days post infection. interestingly, virus replication in the tail and paws could again be detected in the ifnar-/-mice, similar to what we had observed after inoculation of balb/c and c bl/ mice via the intraperitoneal route ( fig. c) . ex vivo imaging of the abdominal region of the ifnar-/-mice clearly identified the liver as the major site of mhv-eflm replication (fig. c) , the organ showing a focal pattern of luciferase expression. this observation was confirmed by the histological analysis of liver sections, as focal lesions were only observed in ifnar-/mice, but not in the control mice (fig. d) . overall, these results indicate that mice lacking a functional type i ifn response exhibit disseminated infection after intranasal inoculation with mhv-a , which could be readily visualized using bli. in view of the increasing availability of all kinds of mutant mice, bli is an attractive approach to investigate the role of a particular host protein or pathway in virus replication and dissemination in vivo. as apparently uncontrolled virus dissemination was observed in mice lacking a functional type i ifn response, we next evaluated the anti-coronaviral effect of exogenous administration of type i ifn. thus, a cocktail of ifna/b was applied to balb/c mice intranasally. it has been established before that ifn can bypass the bloodbrain barrier after intranasal delivery (ross et al., ) . following subsequent intranasal inoculation with ¥ tcid of mhv-eflm, mice were processed for bli at the indicated time points as described above (fig. a) . at day , replication of mhv was not significantly affected by the application of ifn. however, while virus replication spread to the brains of the mock-treated animals, this was not observed in the ifn-treated mice. rather, the luciferase signal was lost by day . these observations were confirmed by the photon quantification of the head regions of the individual mice (fig. b) . thus, exogenous delivery of type i ifn did not prevent the initial replication in the nose, but prevented dissemination of virus infection to the cns. the results also indicate that bli is a promising technique for the non-invasive screening of antiviral compounds, the major advantage being that this system allows the detection of virus replication and spread after application of an antiviral compound over time within the same animal. b. the average body weight of both groups of mice is shown as the percentage relative to the initial weight at the beginning of the experiment. standard deviations are indicated (n = ). c. in vivo versus ex vivo imaging of an ifnar-/-mouse infected with mhv-eflm at days post inoculation. after the standard bli procedure, this mouse was sacrificed and immediately processed for imaging of the internal organs. note that the scaling [in arbitrary units (au)] in both images is different in order to visualize the focal infection pattern in the liver after ex vivo imaging. d. the liver of the ifnar-/-mouse in c was subsequently processed for histology as described in the experimental procedures. as a control, a liver section of a mhv-eflm-infected svev wild-type mouse is shown. typical focal lesions resulting from infection with mhv are indicated by the black arrows. mouse models are essential for defining factors that regulate replication and virulence of viruses. however, the conventional approaches for studying viral infections in mouse models are frequently limited by the need to sacrifice large numbers of animals to quantify viral titres, and establish the complete pattern of virus dissemination. the in vivo imaging of mhv infection application of non-invasive imaging methods for the monitoring of virus infections in living animals can significantly facilitate studies on determinants of viral spread and pathogenesis (hutchens and luker, ) . we used bli for the real-time monitoring of mhv infection by using recombinant mhv viruses that express fl reporter proteins. we confirm and extend previous observations by demonstrating that mhv dissemination is critically dependent on the virus inoculation route and dose, the viral spike protein, the genetic background of the host, and the type i ifn system. in addition, the technology provided insights into the kinetics and dynamics of the infection process under these different conditions. furthermore, bli revealed an animal-to-animal variation in viral spread and elucidated new anatomic locations of virus replication. despite its many attractive features for studying mhv replication and spread, the bli technology has also some inherent limitations. first of all, photon transmission is affected by hair and organ pigmentation. thus, photon flux from the surface of an animal will be superior to that from an internal organ, because light is attenuated about -fold for every centimetre of tissue through which it has to pass (contag and bachmann, ) . as a consequence it is impossible to quantitatively compare bioluminescent signals derived from different anatomic locations. this phenomenon also explains why the photon flux from the head region at days post infection is much higher than at later time points as the signal initially originates from the nasal cavity but thereafter increasingly derives from the cns, where it is much more shielded by surrounding tissue. to partly overcome this limitation, we routinely imaged the mice from both the ventral and the dorsal side. second, the introduction of a foreign gene into the viral genome may affect virus replication in vivo. interestingly, no significant differences could be observed between the replication of wild-type and fl-expressing viruses in the brain of intranasally infected mice, indicating that introduction of the foreign gene did not affect replication in vivo per se. however, after intraperitoneal application the replication of reporter gene expressing viruses in the liver was clearly much lower. the cause of this apparent discrepancy is currently unknown. importantly, however, fl expression was found at all the known sites of mhv replication (robbins et al., ; holmes and lai, ; komurasaki et al., ; mcintosh, ; perlman et al., ; macnamara et al., ) . the mhv spike protein is an important determinant of pathogenesis (phillips et al., ) . using bli, we monitored virus replication and spread of a recombinant virus that carried mutations in the spike gene shown to result in an extended host range in vitro due to the virus' ability to enter cells in a ceacam a-independent but heparan sulfate-dependent manner (schickli et al., ; de haan et al., b; . although it has been suggested that host range variants might arise in vivo during persistent infection of tissues that express low levels of the native mhv receptor (schickli et al., ; , this particular host range extension was obtained after serial passaging in vitro. clearly, the mutant virus replicated to a much lower extent in vivo and was not able to spread to the brain. many viruses adapt to heparan sulfate as an entry receptor upon propagation of the virus in cell culture (olmsted et al., ; mandl et al., ; lee et al., ) . often, but not always (liu and thorp, ; de haan et al., ) , this adaptation results in a reduction of virulence in living animals, consistent with our results. inbred mouse strains are known to differ in their susceptibility to many virus infections (parker et al., ; jubelt et al., ; compton et al., ; thach et al., ) . in our experiments we monitored the replication of mhv in three different mouse strains. c bl/ and svev mice appeared to be less susceptible to infection compared with balb/c mice after intranasal inoculation. similar results were obtained when mice were infected with wild-type mhv-a and viral rna load monitored using quantitative rt-pcr. strikingly, mhv replicated to approximately the same extent in balb/c and c bl/ mice after intraperitoneal inoculation. previously, mouse susceptibility to mhv infection was found to be linked to the viral receptor genotype (ohtsuka and taguchi, ) . however, quantitative rt-pcr analysis of the ceacam a expression levels in brain and liver revealed only relatively small differences in the mhv receptor levels between the different mice strains, which did not correlate with the observed differences in viral replication (data not shown). apparently, other yet unknown host factors contribute to or limit the dissemination of mhv. the resistance of svev mice to infection is not restricted to mhv, because also vesicular stomatitis virus replicated much more efficiently in balb/c than in svev mice after intranasal inoculation (durbin et al., ) . the difference in mhv replication between balb/c and c bl/ mice might be associated with the mouse strain variation in immune responses which have been described for other virus infections (scalzo et al., ; brenner et al., ; thach et al., ; leipner et al., ; weinberg et al., ) . in general, c bl/ mice have a propensity to elicit a predominant t helper cell response, while balb/c mice have a tendency to elicit a predominant t helper cell response. our results confirm and extend previous observations that dissemination of mhv infection is determined both by the route and by the dose of inoculation. interestingly, clear differences in the spatio-temporal dissemination of the infection could be observed after intranasal inoculation with different virus doses. as expected, the virus inoculation dose correlated with the luminescent signal at days post infection. however, inoculation with a low virus dose resulted in delayed spreading of the infection to the cns and subsequent delayed clearance. this result suggests that a certain level of replication in the nasal epithelium is required for subsequent dissemination. strikingly, at days post infection higher levels of virus replication could be observed after inoculation with the low dose compared with the high dose. thus after inoculation with the high dose, virus was cleared faster from the brain. apparently, clearance of the virus is triggered by the extent of virus replication in the brain. probably high levels of virus replication more effectively induce an antiviral innate immune response. these results are corroborated by the observation that the expression levels of type i ifn and other cytokines positively correlate with the viral load in the brain of mhvinfected mice (m. raaben, unpublished results). type i ifn is a fundamental component of the innate immune response, which upon induction elicits an important antiviral signalling cascade that controls and orchestrates the outcome of numerous virus infections (zuniga et al., ) . here, we analysed the full extent of mhv dissemination in mice lacking a functional type i ifn response. these mice appeared to be highly susceptible to mhv and showed severe spread of the infection throughout the body when analysed with bli after intranasal inoculation. in contrast, the parental svev animals only showed some low levels of viral replication in the nasal cavity. these results confirm and extend recent studies in which mhv-a -infected ifnar-/-mice were also shown to be highly susceptible to infection, both after intraperitoneal (cervantes-barragan et al., ) and after intracranial inoculation (roth-cross et al., ) . we additionally demonstrated the importance of the type i ifn system in controlling mhv dissemination by administration of type i ifns. intranasal delivery of recombinant type in vivo imaging of mhv infection i ifns inhibited the spread of the infection to the brain, consistent with a previous study using the mhv strain jhm (minagawa et al., ) . it was interesting to observe that, while the spread to the brain was affected, the initial replication in the nasal epithelium was not decreased by the intranasally administered ifns. the experiments demonstrate the power of the bli technology for studying the effects of antiviral agents on virus replication and dissemination in animal models as was also illustrated earlier (luker et al., ; . our observations show that virus dissemination in an organism is a multifactorial process in which the genetic make-up of pathogen and host, the inoculation dose and route, and the status of the immune system play important roles. in addition, through the application of bli it has now become evident that, besides the typical, reproducible dissemination characteristics of mhv in mice, clear animal-to-animal variation also occurs. thus, while the infection invariably targets organs like brain, cervical lymph nodes and liver, depending on the inoculation route/dose, other anatomical sites of replication are less frequently detected (see table s for an overview). occasionally, mhv replication was measured in the lungs, in the intestine or in the eyes. quite frequently infection was also detected in the tail and paws, body parts not described earlier as sites of mhv replication. these results suggest that mhv can spread to and replicate in bone marrow in vivo, consistent with earlier in vitro studies demonstrating infection of bone marrow-derived macrophages and dendritic cells (zhou and perlman, ; roth-cross et al., ) . strikingly, mhv replication in tail and paws was never observed throughout the entire length of these extremities, but occurred only in distinct sites that differed between animals. in this respect, the dissemination of mhv is reminiscent of cancer metastases, which often also occur at distinct anatomical locations (molloy and van't veer, ) . similar to the importance of micro-environmental host factors in determining the non-random pattern of tumour localization, replication of mhv may also be enabled by local conditions in a specific micro-environment. lr mouse fibroblast cells (kuo et al., ) were maintained in dulbecco's modified eagle's medium (cambrex bio science) containing % (v/v) fetal calf serum (bodinco b.v), u ml - penicillin, and mg ml - streptomycin, supplemented with geneticin g ( mg ml - ). mhv strain a and the derivates expressing the fl reporter gene (mhv-eflm, mhv- afls and mhv- aflsrec) have been described previously (de haan et al., ; b) . all viruses were grown in lr cells. virus stocks were concentrated by pelleting through sucrose-cushion centrifugation, and subsequently resuspended in pbs. balb/c and c bl/ mice at different ages were obtained from charles river, while type i ifn receptor knock-out mice (ifnar-/-) (muller et al., ) and the parental svev mice were obtained from b&k universal ltd. mice were inoculated either intraperitoneally or intranasally with various doses of the different viruses. infected mice were either processed for bli, or sacrificed at the indicated time points for organ dissection. when indicated, mice were (pre-)treated with u of a cocktail of recombinant mouse ifn alpha/beta (ifn-a/b; sigma-aldrich). the cytokines were applied intranasally on each of days - , - , , and relative to the inoculation with mhv-eflm. control animals were treated with pbs. whole brains and livers were dissected from the mhv-infected and control mice. the tissues were added to lysing matrix d tubes (mp biomedical), containing ml of pbs, and processed using a fastprep instrument (mp biomedical). the tissues were homogenized at r.p.m. for s and immediately placed on ice. subsequently, the homogenates were centrifuged at r.p.m. for min at °c and supernatants were harvested, aliquoted and stored at - °c. the aliquots were processed for the different assays as described below. when indicated, whole livers from mhv-infected mice were harvested, fixed in phosphate-buffered formalin, embedded in paraffin, sectioned and stained with haematoxylin. total rna was isolated from brain and liver homogenates using the trizol reagent (invitrogen) according to the manufacturer's protocol. rna was further purified using the rneasy mini-kit with subsequent dnasei treatment on the column (qiagen). the relative amounts of viral genomic rna were determined by quantitative taqman rt-pcr as described before (de haan et al., ) . titration of virus stocks was performed by determination of the tcid in lr cells (verheije et al., ) . the viral titres in tissue homogenates were determined by plaque assays. briefly, lr cells in well microplates were inoculated with fourfold serial dilutions of the homogenates. at h post inoculation, medium was replaced with an agar overlay. at approximately h post inoculation, the lr cell monolayers were fixed and stained with crystal violet, after which the total amount of plaques was counted. the viral titres are expressed as plaque-forming units per gram (pfu g - ) tissue. monolayers of mhv-infected lr cells were lysed with ¥ passive lysis buffer (promega). luciferase expression was measured according to the manufacturer's instructions, and relative light units (rlu) were determined in a turner designs td- / luminometer. in case of the tissue homogenates, ml was mixed with ml ¥ passive lysis buffer and incubated for min at room temperature after which the luciferase expression was measured as described above. when indicated, mhv replication in mice was assessed by in vivo bli with a highly sensitive, ccd camera (versarray b, roper scientific inc.) mounted in a light-tight imaging chamber (roper scientific inc.). imaging and quantification of signals was controlled by the acquisition software metavue (universal imaging corporation). prior to imaging, mice were anaesthetized by intraperitoneal injection of kxa: ketamine hydrochloride ( mg kg - ; vétoquinol bv) plus xylazine ( mg kg - ; eurovet animal health bv) and atropine ( . mg kg - ; pharmachemie bv). the substrate d-luciferin sodium salt (synchem laborgemeinschaft ohg) dissolved in pbs was injected intraperitoneal at a dose of~ mg kg - . mice were positioned in a specially designed box and placed onto the stage inside the light-tight camera box. four mice were imaged simultaneously exactly min after the injection of d-luciferin. the integrated light intensity of a stack of sequential min exposures ( from the dorsal side and from the ventral side) was used to calculate the amount of emitted light from the animals. a low-intensity visible light image was made and used to create overlay images. images were further analysed with metamorph imaging software (universal imaging corporation). in some bli experiments, the detection of emitted photons was performed by the equally sensitive photon imager from biospace laboratory. bli images obtained with the biospace ccd camera were analysed by photovision software (biospace laboratory). table s a . anatomical sites of mhv-eflm infection in balb/c mice inoculated with different doses (i.e. ¥ and ¥ tcid ). the numbers of mice from a group of four which showed infection at the indicated anatomical sites are indicated at the different time points post infection (days p.i.). table s b . anatomical sites of infection in mhv-eflm infected mice of different genetic background (i.e. balb/c, c bl/ , svev and ifnar-/-). note that mice were inoculated intranasally (i.n.) or intraperitoneally (i.p.) with tcid of mhv-eflm and subsequently imaged at different time points post inoculation. the numbers of mice from a group of eight or four animals which showed infection at the indicated anatomical sites at any time point post inoculation are indicated. please note: wiley-blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. any queries (other than missing material) should be directed to the corresponding author for the article. in vivo imaging of mhv 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interferon during viral infections: multiple triggers for a multifunctional mediator additional supporting information may be found in the online version of this article this work was supported by grants from the m.w. beijerinck virology fund, royal netherlands academy of arts and sciences, and the netherlands organization for scientific research (nwo-vidi- . . ) to c.a.m. de haan. we thank monique oostra, marne hagemeijer and mijke vogels for stimulating discussions.