key: cord-0001513-ojh7dw9v
authors: Bhattacharyya, Sankar
title: Can’t RIDD off viruses
date: 2014-06-18
journal: Front Microbiol
DOI: 10.3389/fmicb.2014.00292
sha: ef58c6e2790539f30df14acc260ae2af4b5f3d1f
doc_id: 1513
cord_uid: ojh7dw9v

The mammalian genome has evolved to encode a battery of mechanisms, to mitigate a progression in the life cycle of an invasive viral pathogen. Although apparently disadvantaged by their dependence on the host biosynthetic processes, an immensely faster rate of evolution provides viruses with an edge in this conflict. In this review, I have discussed the potential anti-virus activity of inositol-requiring enzyme 1 (IRE1), a well characterized effector of the cellular homeostatic response to an overloading of the endoplasmic reticulum (ER) protein-folding capacity. IRE1, an ER-membrane-resident ribonuclease (RNase), upon activation catalyses regulated cleavage of select protein-coding and non-coding host RNAs, using an RNase domain which is homologous to that of the known anti-viral effector RNaseL. The latter operates as part of the Oligoadenylate synthetase OAS/RNaseL system of anti-viral defense mechanism. Protein-coding RNA substrates are differentially treated by the IRE1 RNase to either augment, through cytoplasmic splicing of an intron in the Xbp1 transcript, or suppress gene expression. This referred suppression of gene expression is mediated through degradative cleavage of a select cohort of cellular RNA transcripts, initiating the regulated IRE1-dependent decay (RIDD) pathway. The review first discusses the anti-viral mechanism of the OAS/RNaseL system and evasion tactics employed by different viruses. This is followed by a review of the RIDD pathway and its potential effect on the stability of viral RNAs. I conclude with a comparison of the enzymatic activity of the two RNases followed by deliberations on the physiological consequences of their activation.

Establishment of infection by a virus, even in permissive host cells, is beset with a plethora of challenges from innate-antiviral and cell-death pathways. Therefore, the host response to a virus infection might prove to be inhibitory for the viral life cycle in a direct or an indirect manner. The direct mechanism involves expression of multiple anti-viral genes that have evolved to recognize, react, and thereby rid the infected host of the viral nucleic acid (Zhou et al., 1997; Thompson et al., 2011) . On the other hand the pathways, e.g., those that culminate in initiating an apoptotic death for the host cell, indirectly serve to limit the spread of virus (Roulston et al., 1999) . A major difference between these two mechanisms is that while the former response is transmissible to neighboring uninfected cells through interferon (IFN) signaling, the latter is observed mostly in cis. Recent reports, however, have demonstrated transmission of an apoptotic signal between cells that are in contact through gap junctions, although such a signaling from an virus infected host cell to an uninfected one is not known yet (Cusato et al., 2003; Udawatte and Ripps, 2005; Kameritsch et al., 2013) . Successful viral pathogens, through a process of active selection, have evolved to replicate and simultaneously evade or block either of these host responses. The viral nucleic acids which could be the genome (positive-sense singlestranded RNA virus) or RNA derived from transcription of the genome [negative-stranded single-sense RNA or double-stranded RNA (dsRNA) or DNA virus], offer critical targets for both detection and eradication. The viral nucleic acid targeting armaments in the host arsenal include those that recognize the associated molecular patterns like toll-like receptors (TLRs), DDX58 (or RIG-1), IFIH1 (or MDA5), IFIT proteins [IFN-stimulated genes (ISG)56 and ISF54], etc. (Aoshi et al., 2011; Bowzard et al., 2011; Jensen and Thomsen, 2012) . This is followed by IFN signaling and expression or activation of factors that target the inducer for degradation or modification like OAS/ribonuclease L (RNaseL) system, APOBEC3, MCPIP1, the ZC3HAV1/exosome system and RNAi pathways (Gao et al., 2002; Sheehy et al., 2002; Guo et al., 2007; Daffis et al., 2010; Sidahmed and Wilkie, 2010; Schmidt et al., 2012; Cho et al., 2013a; Lin et al., 2013) . In this review we focus on two proteins containing homologous RNase domains, RNaseL with a known direct antiviral function and Inositolrequiring enzyme 1 (IRE1 or ERN1) which has an RNaseL-like RNase domain with a known role in homeostatic response to unfolded proteins in the endoplasmic reticulum (ER) and a potential to function as an antiviral (Figure 1 ; Tirasophon et al., 2000) .

In mammalian cells the tell-tale signs of RNA virus infection, like the presence of cytosolic RNA having 5 -ppp or extensive (>30 bp) dsRNA segments are detected by dedicated pathogen associated molecular pattern receptors (PAMPs) or pattern recognition receptors (PRRs) in the host cell, like RIG-1, MDA5, and the IFIT family of proteins (Aoshi et al., 2011; Bowzard et al., 2011; Vabret and Blander, 2013) . The transduction of a signal of this recognition results in the expression of IFN genes the products www.frontiersin.org FIGURE 1 | Schematic representation of the ribonuclease activity of IRE1 and RNaseL showing cross-talk between the paths catalysed by the enzymes. The figure shows activation of RNase activity following dimerization triggered by either accumulation of unfolded proteins in the ER-lumen or synthesis of 2-5A by the enzyme OAS, respectively, for IRE1 and RNaseL. The cleavage of Xbp1u by IRE1 releases an intron thus generating Xbp1s. The IRE1 targets in RIDD pathway or all RNaseL substrates are shown to undergo degradative cleavage. The cleavage products generated through degradation of the respective substrate is shown to potentially interact with RIG-I thereby leading to Interferon secretion and trans-activation of Oas genes through Interferon signaling. Abbreviations: RIG-I = retinoic acid inducible gene-I, Ifnb = interferon beta gene loci, IFN = interferons, ISG = interferon-sensitive genes, 2-5A = 2 -5 oligoadenylates.

of which upon secretion outside the cell bind to cognate receptors, initiating further downstream signaling (Figure 1 ; Randall and Goodbourn, 2008) . The genes that are regulated as a result of IFN signaling are termed as IFN-stimulated or IFN-regulated genes (ISGs or IRGs; Sen and Sarkar, 2007; Schoggins and Rice, 2011) . Oligoadenylate synthetase or OAS genes are canonical ISGs that convert ATP into 2 -5 linked oligoadenylates (2-5A) by an unique enzymatic mechanism (Figure 1 ; Hartmann et al., 2003) . Further, they are RNA-binding proteins that function like PRRs, in a way that the 2-5A synthesizing activity needs to be induced through an interaction with dsRNA (Minks et al., 1979; Hartmann et al., 2003) . In a host cell infected by an RNA virus, such dsRNA is present in the form of replication-intermediates (RI), which are synthesized by the virus-encoded RNA-dependent RNA polymerases (RdRp) and subsequently used by the same enzyme to synthesize more genomic RNA, through asymmetric transcription (Weber et al., 2006) . However, the replications complexes (RCs) harboring these RI molecules are found secluded inside host-membrane derived vesicles, at least in positive-strand RNA viruses, a group which contains many human pathogens (Uchil and Satchidanandam, 2003; Denison, 2008) . Reports from different groups suggest OAS proteins to be distributed both in the cytoplasm as well as in membrane-associated fractions, perhaps indicating an evolution of the host anti-viral methodologies towards detection of the membrane-associated viral dsRNAs (Marie et al., 1990; Lin et al., 2009) . DNA viruses on the other hand, produce dsRNA by annealing of RNA derived from transcription of both strands in the same viral genomic loci, which are probably detected by the cytoplasmic pool of OAS proteins (Jacobs and Langland, 1996; Weber et al., 2006) . Post-activation the OAS enzymes synthesize 2-5A molecules in a non-processive reaction producing oligomers which, although potentially ranging in size from dimeric to multimeric, are functionally active only in a trimeric or tetrameric form (Dong et al., 1994; Sarkar et al., 1999; Silverman, 2007) . These small ligands, which bear phosphate groups (1-3) at the 5 end and hydroxyl groups at the 2 and 3 positions, serve as co-factor which can specifically interact with and thereby allosterically activate, existing RNaseL molecules (Knight et al., 1980; Zhou et al., 1997 Zhou et al., , 2005 Sarkar et al., 1999) . As part of a physiological control system these 2-5A oligomers are quite unstable in that they are highly susceptible to degradation by cellular 5 -phosphatases and PDE12 (2 -phosphodiesterase; Silverman et al., 1981; Johnston and Hearl, 1987; Kubota et al., 2004; Schmidt et al., 2012) . Viral strategies to evade or overcome this host defense mechanism ranges from preventing IFN signaling which would hinder the induction of OAS expression or thwarting activation of expressed OAS proteins by either shielding the viral dsRNA from interacting with it or modulating the host pathway to synthesize inactive 2-5A derivatives (Cayley et al., 1984; Hersh et al., 1984; Rice et al., 1985; Maitra et al., 1994; Beattie et al., 1995; Rivas et al., 1998; Child et al., 2004; Min and Krug, 2006; Sanchez and Mohr, 2007; Sorgeloos et al., 2013) . Shielding of viral RNA from interacting with OAS is possible through enclosure of dsRNA replication intermediates in membrane enclosed compartments as observed in many flaviviruses (Ahlquist, 2006; Miller and Krijnse-Locker, 2008; Miorin et al., 2013) .

RNaseL is a 741 amino acid protein containing three predominantly structured region, an N-terminal ankyrin repeat domain (ARD), a middle catalytically inactive pseudo-kinase (PK) and a C-terminal RNase domain (Figure 2A ; Hassel et al., 1993; Zhou et al., 1993) . The activity of the RNase domain is negatively regulated by the ARD, which is relieved upon binding of 2-5A molecules to ankyrin repeats 2 and 4 followed by a conformational alteration (Figure 1 ; Hassel et al., 1993; Tanaka et al., 2004; Nakanishi et al., 2005) . In support of this contention, deletion of the ARD has been demonstrated to produce constitutively active RNaseL, although with dramatically lower RNase activity (Dong and Silverman, 1997) . However, recent reports suggest that while 2-5A links the ankyrin repeats from adjacent molecules leading to formation of dimer and higher order structures, at sufficiently high in vitro concentrations, RNaseL could oligomerize even in the absence of 2-5A . Nonetheless, in vivo the RNaseL nuclease activity still seems to be under the sole regulation of 2-5A (Al-Saif and Khabar, 2012) . In order to exploit this dependence, multiple viruses like mouse hepatitis virus (MHV) and rotavirus group A (RVA) have evolved to encode phosphodiesterases capable of hydrolysing the 2 -5 linkages in 2-5A and thereby attenuate the RNaseL cleavage activity (Zhao et al., 2012; Zhang et al., 2013) . In addition to 5 -phosphatases and 2 -phosphodiesterases to reduce ClustalW alignment of primary sequence from a segment of the PK domain indicating amino acid residues which are important for interacting with nucleotide cofactors. The conserved lysine residues, critical for this interaction (K599 for IRE1 and K392 in RNaseL) are underlined. (C) Alignment of the KEN domains in RNaseL and IRE1. The amino acids highlighted and numbered in IRE1 are critical for the IRE1 RNase activity (Tirasophon et al., 2000) .

the endogenous 2-5A levels, mammalian genomes encode posttranscriptional and post-translation inhibitors of RNaseL activity in the form of microRNA-29 and the protein ABCE1 (RNaseL inhibitor or RLI), respectively (Bisbal et al., 1995; Lee et al., 2013) . Direct inhibition of RNaseL function is also observed upon infection by Picornaviruses through, either inducing the expression of ABCE1 or exercising a unique inhibitory property of a segment of the viral RNA (Martinand et al., 1998 (Martinand et al., , 1999 Townsend et al., 2008; Sorgeloos et al., 2013) .

Once activated by 2-5A, RNaseL can degrade single-stranded RNA irrespective of its origin (virus or host) although there seems to exist a bias towards cleavage of viral RNA (Wreschner et al., 1981a; Silverman et al., 1983; Li et al., 1998) . RNA sequences that are predominantly cleaved by RNaseL are U-rich with the cleavage points being typically at the 3 end of UA or UG or UU di-nucleotides, leaving a 5 -OH and a 3 -monophosphate in the cleavage product (Floyd-Smith et al., 1981; Wreschner et al., 1981b) . A recent report shows a more general consensus of 5 -UNN-3 with the cleavage point between the second and the third nucleotide (Han et al., 2014) . Cellular targets of RNaseL include both ribosomal RNA (rRNA) and mRNAs, the latter predominantly representing genes involved in protein biosynthesis (Wreschner et al., 1981a; Al-Ahmadi et al., 2009; Andersen et al., 2009) . Additionally, RNaseL activity can also degrade specific ISG mRNA transcripts and thereby attenuate the effect of IFN signaling (Li et al., 2000) . Probably an evolution towards insulating gene expression from RNaseL activity is observed in the coding region of mammalian genes where the UU/UA dinucleotide frequency is rarer (Bisbal et al., 2000; Khabar et al., 2003; Al-Saif and Khabar, 2012) . Perhaps not surprisingly, with a much faster rate of evolution, similar observations have been made with respect to evasion of RNaseL mediated degradation by viral RNAs too (Han and Barton, 2002; Washenberger et al., 2007) . Moreover, nucleoside modifications in host mRNAs, rarely observed in viral RNAs, have also been shown to confer protection from RNaseL (Anderson et al., 2011) . In addition to directly targeting viral RNA, the reduction in functional ribosomes and ribosomal protein mRNA affects viral protein synthesis and replication in an indirect manner. Probably, as a reflection of these effects on cellular RNAs, RNaseL is implicated as one of the factors determining the anti-proliferative effect of IFN activity . The anti-viral activity of RNaseL extends beyond direct cleavage of viral RNA, through stimulation of RIG-I by the cleavage product (Malathi et al., , 2007 (Malathi et al., , 2010 . A global effect of RNaseL is observed in the form of autophagy induced through c-jun N-terminal kinase (JNK) signaling and apoptosis, probably as a consequence of rRNA cleavage (Li et al., 2004; Chakrabarti et al., 2012; Siddiqui and Malathi, 2012) . RNaseL has also been demonstrated to play a role in apoptotic cell death initiated by pharmacological agents extending the physiological role of this pathway beyond the boundary of being only an anti-viral mechanism (Castelli et al., 1997 (Castelli et al., , 1998 .

The ER serves as a conduit for maturation of cellular proteins which are either secreted or destined to be associated with a membrane for its function. An exclusive microenvironment (high Calcium ion and unique ratio of reduced to oxidized glutathione) along with a battery of ER-lumen resident enzymes (foldases, chaperones, and lectins) catalyse/mediate the necessary folding, disulfide-bond formation, and glycosylation reactions (Schroder and Kaufman, 2005) . A perturbation of the folding capacity, due to either physiological disturbances or virus infection, can lead to an accumulation of unfolded proteins in the ER lumen, which signals an unfolded protein response (UPR). UPR encompasses a networked transcriptional and translational gene-expression program, initiated by three ER-membrane resident sensors namely IRE1 or ERN1, PKR-like ER Kinase (PERK or EIF2AK3) and activating transcription factor 6 (ATF6; Hetz, 2012) . IRE1 is a type I single-pass trans-membrane protein in which, similar to what is observed with RNaseL, the N-terminal resident in the ER lumen serves as sensor and the cytosolic C-terminal as the effector (Figure 1 ; Chen and Brandizzi, 2013) . The IRE1 coding gene is present in genomes ranging from yeast to mammals and in the latter is ubiquitously expressed in all tissues (Tirasophon et al., 1998) . Signal transduction by stimulated IRE1 initiates multiple gene regulatory pathways with either pro-survival or pro-apoptotic consequences (Kaufman, 1999) . During homeostasis or unstressed conditions the sensor molecules are monomeric, a state maintained co-operatively by the " absence" of unfolded proteins and the "presence" of HSPA5 (GRP78 or Bip, an ERresident chaperone) molecules bound to a membrane-proximal disordered segment of the protein in the ER-lumen-resident Nterminus (Credle et al., 2005) . Accumulated unfolded proteins in the lumen triggers coupling of this domain from adjacent sensor molecules through a combination of (a) titration of the bound HSPA5 chaperone molecules and (b) direct tethering by malfolded protein molecules (Shamu and Walter, 1996; Credle et al., 2005; Aragon et al., 2009; Korennykh et al., 2009) . Abutting of the luminal domains juxtapose the cytosolic C-terminal segments, leading to an aggregation of the IRE1 molecules into distinct ER-membrane foci (Kimata et al., 2007; Li et al., 2010) . The C-terminal segment has a serine/threonine kinase domain and a RNase domain homologous to that of RNaseL (Figure 1 ; Tirasophon et al., 1998 Tirasophon et al., , 2000 . A trans-autophosphorylation by the kinase domain allosterically activates the RNase domain (Tirasophon et al., 2000; Lee et al., 2008; Korennykh et al., 2009) . In fact, exogenous over-expression of IRE1 in mammalian cells lead to activation suggesting that, under homeostatic conditions, the non-juxtaposition of cytosolic domains maintains an inactive IRE1 (Tirasophon et al., 1998) . Once activated, IRE1 performs cleavage of a variety of RNA substrates mediated by its RNase domain, in addition to phosphorylating and thereby activating JNK (Cox and Walter, 1996; Urano et al., 2000) . Depending on the RNA substrate, the cleavage catalyzed by IRE1 RNase produces differential consequence. Although scission of the Xbp1 mRNA transcript at two internal positions is followed by splicing of the internal segment through ligation of the terminal cleavage products, that in all other known IRE1 target RNA is followed by degradation (Figure 1 ; Sidrauski and Walter, 1997; Calfon et al., 2002) . The latter mode of negative regulation of gene expression is termed as the regulated IRE1-dependent decay (RIDD) pathway (Hollien and Weissman, 2006; Oikawa et al., 2007; Iqbal et al., 2008; Lipson et al., 2008) . Gene transcripts regulated by RIDD pathway includes that from IRE1 (i.e., selftranscripts), probably in a negative feedback loop mechanism (Tirasophon et al., 2000) . In addition to protein coding RNA, RIDD pathway down-regulates the level of a host of microRNA precursors (pre-miRNAs) and can potentially cleave in the anticodon loop of tRNA Phe (Korennykh et al., 2011; Upton et al., 2012) .

The IRE1 RNase domain cleaves the Xbp1u (u for unspliced) mRNA transcript at two precise internal positions within the open reading frame (ORF) generating three segments, the terminal two of which are ligated by a tRNA ligase in yeast and by an unknown ligase in mammalian cells, to produce the Xbp1s (s for spliced) mRNA transcript (Figure 1 ; Yoshida et al., 2001) . The Xbp1s thus generated has a longer ORF, which is created by a frame-shift in the coding sequence downstream of the splice site (Cox and Walter, 1996; Calfon et al., 2002) . A similar dual endonucleolytic cleavage is also observed to initiate the XRN1 and Ski2-3-8 dependent degradation of transcripts in the RIDD degradation pathway (Hollien and Weissman, 2006) . The RIDD target transcript genes are predominantly those that encode membrane-associated or secretory proteins and which are not necessary for ER proteinfolding reactions (Hollien and Weissman, 2006) . The cleavage of Xbp1 and the RIDD-target transcripts constitute homeostatic or pro-survival response by IRE1 since XBP1S trans-activates genes encoding multiple chaperones (to fold unfolded proteins) and the ERAD pathway genes (to degrade terminally misfolded proteins) whereas RIDD reduces flux of polypeptides entering the ER lumen (Lee et al., 2003; Hollien and Weissman, 2006) . On the other hand, cleavage of pre-miRNA transcripts which are processed in the cell to generate CASPASE-2 mRNA (Casp2) controlling miRNAs, constitutes the pro-apoptotic function of IRE1 (Upton et al., 2012) . Another pro-apoptotic signal from IRE1 emanates from signaling through phosphorylation of JNK1 (Urano et al., 2000) . Although in the initial phase RIDD activity does not cleave mRNAs encoding essential ER proteins, at later stages of chronic UPR such transcripts are rendered susceptible to degradation promoting apoptosis induction (Han et al., 2009; Bhattacharyya et al., 2014) .

Infection of mammalian cells by a multitude of viruses induce an UPR which is sometimes characterized by suppression of signaling by one or more of the three sensor(s; Su et al., 2002; Tardif et al., 2002; He, 2006; Yu et al., 2006 Yu et al., , 2013 Medigeshi et al., 2007; Zhang et al., 2010; Merquiol et al., 2011) . Among these at least two viruses from diverse families, HCMV (a DNA virus) and hepatitis C virus (a hepacivirus), interfere with IRE1 signaling by different mechanism (Tardif et al., 2004; Stahl et al., 2013 ). An observed inhibition of any cellular function by a virus infection could suggest a potential anti-virus function for it, which the virus has evolved to evade through blocking some critical step(s). In both the cases mentioned above, stability of the viral proteins seems to be affected by ERAD-mediated degradation, although other potential anti-viral effect of IRE1 activation are not clear yet (Isler et al., 2005; Saeed et al., 2011) . Interestingly, host mRNA fragments produced following IRE1 activation during bacterial infection, has been shown to activate RIG-I signaling (Figure 1 ; Cho et al., 2013b) . Theoretically, other functions of IRE1 can also have anti-viral effect necessitating its inhibition for uninhibited viral replication. It is, however, still not clear whether IRE1 is able to cleave any viral RNA (or mRNA) in a manner similar to that of other RIDD targets (Figure 1) . The possibilities of such a direct anti-viral function are encouraged by the fact that all these viruses encode at least one protein which, as part of its maturation process, requires glycosylation and disulfide-bond formation. Such a necessity would entail translation of the mRNA encoding such a protein, which in case of positive-sense single-stranded RNA viruses would mean the genome, in association with the ER-membrane (Figure 1 ; Lerner et al., 2003) . Additionally for many RNA viruses, replication complexes are housed in ER-derived vesicular structures (Denison, 2008; den Boon et al., 2010) . Considering the proximity of IRE1 and these virus-derived RNAs it is tempting to speculate that probably at some point of time in the viral life cycle one or more virus-associated RNA would be susceptible to cleavage by IRE1. However, studies with at least two viruses have shown that instead of increasing viral titre, inhibiting the RNase activity of activated IRE1 has an opposite effect (Hassan et al., 2012; Bhattacharyya et al., 2014) . This implies potential benefits of IRE1 activation through one or more of the following, (a) expression of chaperones or other pro-viral molecules downstream of XBP1Supregulation or JNK-activation, (b) cleavage of potential anti-viral gene mRNA transcripts by RIDD activity. However, the mode of protection for the viral RNA from RIDD activity is still not clear. It is possible that the viral proteins create a subdomain within the ER membrane, which through some mechanism excludes IRE1 from diffusing near the genomic RNA, thereby protecting the replication complexes (Denison, 2008) . It is therefore probably not surprising that single-stranded plus-sense RNA viruses encode a polyprotein, which produces replication complexes in cis, promoting formation of such subdomains (Egger et al., 2000) . The fact that IRE1 forms bulky oligomers of higher order probably aggravates such an exclusion of the activated sensor molecules from vicinity of the viral replication complexes. The UPR signaling eventually attenuate during chronic ER-stress and since that is what a virus-induced UPR mimics, probably the viral RNA needs protection only during the initial phase of UPR activation (Lin et al., 2007) . Since the choice of RIDD target seems to be grossly driven towards mRNAs that encode ER-transitory but are not ER-essential proteins, it is also possible that one or more viral protein have evolved to mimic a host protein the transcript of which is RIDD-resistant (Hollien and Weissman, 2006) . Most of the RIDD target mRNA are observed to be ER-membrane associated, the proximity to IRE1 facilitating association and cleavage (Figure 1 ; Hollien and Weissman, 2006) . Although ER-association for an mRNA is possible without the mediation of ribosomes, Gaddam and co-workers reported that continued association with polysomes for a membrane-bound mRNA can confer protection from IRE1 cleavage (Cui et al., 2012; Gaddam et al., 2013) . This would suggest important implications for the observed refractory nature of Japanese encephalitis virus (JEV) and influenza virus RNA to RIDD cleavage (Hassan et al., 2012; Bhattacharyya et al., 2014) . In contrast to Influenza virus, flaviviruses (which include JEV) do not suppress host protein synthesis implying the absence of a global inhibition on translation as would be expected during UPR (Clyde et al., 2006; Edgil et al., 2006) . Therefore, a continued translation of viral RNA in spite of UPR activation can in principle confer protection from the pattern of RNA cleavage observed in the RIDD pathway.

IRE1 and RNaseL, in addition to biochemical similarities in protein kinase domain and structural similarities in their RNase domain, share the functional consequences of their activation in initiating cellular apoptosis through JNK signaling (Table 1 and Figure 2 ; Liu and Lin, 2005; Dhanasekaran and Reddy, 2008) . Though initial discoveries were made in the context of homeostatic and anti-viral role for the former and latter, differences between the pathways are narrowed by further advances in research. In the same vein, while inhibition of IRE1 signaling in virus infected cells indicates a potential anti-viral role, www.frontiersin.org (Tirasophon et al., 1998; Dong and Silverman, 1999; Papa et al., 2003; Lin et al., 2007) Nature of RNase substrates Both 28S rRNA and mRNAs IRE1β can cleave both 28S rRNA and mRNA while IRE1α substrates include only mRNAs (Iwawaki et al., 2001) Dissimilarities

Cleavage substrates Beside 28S rRNA, predominantly cleaves mRNAs encoding ribosomal proteins (Andersen et al., 2009) Xbp1u and other mRNAs in addition to microRNA precursors which are targeted as part of the RIDD pathway Selection of cleavage site Cleaved between 2nd and 3rd nucleotide positions of UN/N sites (Han et al., 2014) RNA sequence with the consensus of 5 -CUGCAG-3 in association with a stem-loop (SL) structure essential for recognition of Xbp1u and other mRNAs (Oikawa et al., 2010) association of RNaseL mutations with generation of prostate cancer extends the ambit of influence of this anti-viral effector to more non-infectious physiological disorders (Silverman, 2003) . Biochemically, the similarity in their RNase domains does not extend to the choice of either substrates or cleavage point, which are downstream of UU or UA in RNaseL and downstream of G (predominantly) for IRE1 ( Figure 2C ; Yoshida et al., 2001; Hollien and Weissman, 2006; Upton et al., 2012) . Further, while RNaseL cleaves pre-dominantly in single-stranded region, IRE1 seems to cleave equally well in single-and double-stranded region (Upton et al., 2012) . However, a recent report suggested a consensus cleavage site with the sequence UN/N, in RNaseL targets and in those mRNAs that are cleaved by IRE1 as part of the RIDD pathway (Han et al., 2014) . Access to potential cleavage substrate for RNaseL is conjectured to be facilitated through its association with polyribosomes, while no such association is known for IRE1 (Salehzada et al., 1991) . Possibilities exist that IRE1 would have preferential distribution in the rough ER which, upon activation, would give it ready access to mRNAs for initiating the RIDD pathway.

In the context of a virus infection, the pathway leading from both these proteins have the potential to lead to cell death. Notwithstanding the fact that this might be an efficient way of virus clearance, it also portends pathological outcomes for the infected organism. Future research would probably lead to design of drugs targeting these proteins based on the structural homology of their effector domains, regulating the pathological denouement of their activation without compromising their anti-viral or potential anti-viral functions.

Parallels among positive-strand RNA viruses, reversetranscribing viruses and double-stranded RNA viruses

Downmodulation of the RNA-binding protein, HuR, and cellular growth

UU/UA dinucleotide frequency reduction in coding regions results in increased mRNA stability and protein expression

Ribosomal protein mRNAs are primary targets of regulation in RNase-Linduced senescence

Nucleoside modifications in RNA limit activation of 2 -5 -oligoadenylate synthetase and increase resistance to cleavage by RNase L

Innate and adaptive immune responses to viral infection and vaccination

Messenger RNA targeting to endoplasmic reticulum stress signalling sites

Reversal of the interferon-sensitive phenotype of a vaccinia virus lacking E3L by expression of the reovirus S4 gene

Regulated IRE1-dependent decay pathway is activated during Japanese encephalitis virus-induced unfolded protein response and benefits viral replication

Cloning and characterization of a RNAse L inhibitor. A new component of the interferon-regulated 2-5A pathway

The 2 -5 oligoadenylate/RNase L/RNase L inhibitor pathway regulates both MyoD mRNA stability and muscle cell differentiation

PAMPer and tRIGer: ligand-induced activation of RIG-I

IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA

The role of 2 -5 oligoadenylate-activated ribonuclease L in apoptosis

A study of the interferon antiviral mechanism: apoptosis activation by the 2-5A system

Activation of the ppp(A2 p)nA system in interferon-treated, herpes simplex virus-infected cells and evidence for novel inhibitors of the ppp(A2 p)nA-dependent RNase

RNase L triggers autophagy in response to viral infections

IRE1: ER stress sensor and cell fate executor

Evasion of cellular antiviral responses by human cytomegalovirus TRS1 and IRS1

A role for Ifit2 in restricting West Nile virus infection in the brain

The unfolded protein response element IRE1alpha senses bacterial proteins invading the ER to activate RIG-I and innate immune signaling

Recent advances in deciphering viral and host determinants of dengue virus replication and pathogenesis

A novel mechanism for regulating activity of a transcription factor that controls the unfolded protein response

On the mechanism of sensing unfolded protein in the endoplasmic reticulum

p180 promotes the ribosomeindependent localization of a subset of mRNA to the endoplasmic reticulum

Gap junctions mediate bystander cell death in developing retina

2 -O methylation of the viral mRNA cap evades host restriction by IFIT family members

Seeking membranes: positive-strand RNA virus replication complexes

JNK signaling in apoptosis

A bipartite model of 2-5A-dependent RNase L

Alternative function of a protein kinase homology domain in 2 , 5 -oligoadenylate dependent RNase L

Intrinsic molecular activities of the interferon-induced 2-5A-dependent RNase

Dengue virus utilizes a novel strategy for translation initiation when cap-dependent translation is inhibited

Formation of the poliovirus replication complex requires coupled viral translation, vesicle production, and viral RNA synthesis

Interferon action: RNA cleavage pattern of a (2 -5 )oligoadenylate-dependent endonuclease

Comparison of mRNA localization and regulation during endoplasmic reticulum stress in Drosophila cells

Inhibition of retroviral RNA production by ZAP, a CCCH-type zinc finger protein

The zinc-finger antiviral protein recruits the RNA processing exosome to degrade the target mRNA

IRE1alpha kinase activation modes control alternate endoribonuclease outputs to determine divergent cell fates

Activation and evasion of the antiviral 2 -5 oligoadenylate synthetase/ribonuclease L pathway by hepatitis C virus mRNA

Structure of human RNase L reveals the basis for regulated RNA decay in the IFN response

Innate immune messenger 2-5A tethers human RNase L into active high-order complexes

Crystal structure of the 2 -specific and double-stranded RNA-activated interferon-induced antiviral protein 2 -5 -oligoadenylate synthetase

Influenza A viral replication is blocked by inhibition of the inositol-requiring enzyme 1 (IRE1) stress pathway

A dominant negative mutant of 2-5A-dependent RNase suppresses antiproliferative and antiviral effects of interferon

Viruses, endoplasmic reticulum stress, and interferon responses

Simian virus 40-infected, interferon-treated cells contain 2 ,5 -oligoadenylates which do not activate cleavage of RNA

The unfolded protein response: controlling cell fate decisions under ER stress and beyond

Decay of endoplasmic reticulum-localized mRNAs during the unfolded protein response

IRE1beta inhibits chylomicron production by selectively degrading MTP mRNA

Human cytomegalovirus infection activates and regulates the unfolded protein response

Translational control by the ER transmembrane kinase/ribonuclease IRE1 under ER stress

When two strands are better than one: the mediators and modulators of the cellular responses to double-stranded RNA

Sensing of RNA viruses: a review of innate immune receptors involved in recognizing RNA virus invasion

Purification and characterization of a 2 -phosphodiesterase from bovine spleen

Gap junctional communication promotes apoptosis in a connexin-type-dependent manner

Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls

RNase L mediates transient control of the interferon response through modulation of the double-stranded RNA-dependent protein kinase PKR

Two regulatory steps of ER-stress sensor Ire1 involving its cluster formation and interaction with unfolded proteins

Radioimmune, radiobinding and HPLC analysis of 2-5A and related oligonucleotides from intact cells

The unfolded protein response signals through high-order assembly of Ire1

Structural and functional basis for RNA cleavage by Ire1

Identification of 2 -phosphodiesterase, which plays a role in the 2-5A system regulated by interferon

XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response

Structure of the dual enzyme Ire1 reveals the basis for catalysis and regulation in nonconventional RNA splicing

Regulation of human RNase-L by the miR-29 family reveals a novel oncogenic role in chronic myelogenous leukemia

Partitioning and translation of mRNAs encoding soluble proteins on membrane-bound ribosomes

An apoptotic signaling pathway in the interferon antiviral response mediated by RNase L and c-Jun NH2-terminal kinase

Mammalian endoplasmic reticulum stress sensor IRE1 signals by dynamic clustering

RNase L mediates the antiviral effect of interferon through a selective reduction in viral RNA during encephalomyocarditis virus infection

RNase-L-dependent destabilization of interferon-induced mRNAs. A role for the 2-5A system in attenuation of the interferon response

IRE1 signaling affects cell fate during the unfolded protein response

MCPIP1 ribonuclease exhibits broad-spectrum antiviral effects through viral RNA binding and degradation

Distinct antiviral roles for human 2 ,5 -oligoadenylate synthetase family members against dengue virus infection

The role of IRE1alpha in the degradation of insulin mRNA in pancreatic beta-cells

Role of JNK activation in apoptosis: a double-edged sword

HIV-1 TAR RNA has an intrinsic ability to activate interferoninducible enzymes

Small self-RNA generated by RNase L amplifies antiviral innate immunity

A transcriptional signaling pathway in the IFN system mediated by 2 -5 -oligoadenylate activation of RNase L

RNase L releases a small RNA from HCV RNA that refolds into a potent PAMP

Differential expression and distinct structure of 69-and 100-kDa forms of 2-5A synthetase in human cells treated with interferon

RNase L inhibitor is induced during human immunodeficiency virus type 1 infection and down regulates the 2-5A/RNase L pathway in human T cells

RNase L inhibitor (RLI) antisense constructions block partially the down regulation of the 2-5A/RNase L pathway in encephalomyocarditis-virus-(EMCV)-infected cells

West Nile virus infection activates the unfolded protein response, leading to CHOP induction and apoptosis

HCV causes chronic endoplasmic reticulum stress leading to adaptation and interference with the unfolded protein response

Modification of intracellular membrane structures for virus replication

The primary function of RNA binding by the influenza A virus NS1 protein in infected cells: Inhibiting the 2 -5 oligo (A) synthetase/RNase L pathway

Structural requirements of double-stranded RNA for the activation of 2 ,5 -oligo(A) polymerase and protein kinase of interferon-treated HeLa cells

Three-dimensional architecture of tick-borne encephalitis virus replication sites and trafficking of the replicated RNA

2-5A induces a conformational change in the ankyrin-repeat domain of RNase L

Identification of a consensus element recognized and cleaved by IRE1 alpha

Site-specific cleavage of CD59 mRNA by endoplasmic reticulum-localized ribonuclease, IRE1

Bypassing a kinase activity with an ATP-competitive drug

Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures

Novel 2 ,5 -oligoadenylates synthesized in interferon-treated, vaccinia virus-infected cells

Vaccinia virus E3L protein is an inhibitor of the interferon (i.f.n.)-induced 2-5A synthetase enzyme

Viruses and apoptosis

Role of the endoplasmic reticulum-associated degradation (ERAD) pathway in degradation of hepatitis C virus envelope proteins and production of virus particles

Polyclonal antibodies against RNase L. Subcellular localization of this enzyme in mouse cells

Inhibition of cellular 2 -5 oligoadenylate synthetase by the herpes simplex virus type 1 Us11 protein

Enzymatic characteristics of recombinant medium isozyme of 2 -5 oligoadenylate synthetase

Sensing of viral nucleic acids by RIG-I: from translocation to translation

Interferon-stimulated genes and their antiviral effector functions

ER stress and the unfolded protein response

The interferon-stimulated genes: targets of direct signaling by interferons, double-stranded RNA, and viruses

Oligomerization and phosphorylation of the Ire1p kinase during intracellular signaling from the endoplasmic reticulum to the nucleus

Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein

Endogenous antiviral mechanisms of RNA interference: a comparative biology perspective

RNase L induces autophagy via c-Jun Nterminal kinase and double-stranded RNA-dependent protein kinase signaling pathways

The transmembrane kinase Ire1p is a site-specific endonuclease that initiates mRNA splicing in the unfolded protein response

Implications for RNase L in prostate cancer biology

Viral encounters with 2 ,5 -oligoadenylate synthetase and RNase L during the interferon antiviral response

rRNA cleavage as an index of ppp(A2 p)nA activity in interferon-treated encephalomyocarditis virus-infected cells

Synthesis, characterization and properties of ppp(A2 p)nApCp and related high-specificactivity 32P-labelled derivatives of ppp(A2 p)nA

Evasion of antiviral innate immunity by Theiler's virus L* protein through direct inhibition of RNase L

Cytomegalovirus downregulates IRE1 to repress the unfolded protein response

Japanese encephalitis virus infection initiates endoplasmic reticulum stress and an unfolded protein response

Structural basis for recognition of 2 ,5 -linked oligoadenylates by human ribonuclease L

Hepatitis C virus suppresses the IRE1-XBP1 pathway of the unfolded protein response

Hepatitis C virus subgenomic replicons induce endoplasmic reticulum stress activating an intracellular signaling pathway

Pattern recognition receptors and the innate immune response to viral infection

The endoribonuclease activity of mammalian IRE1 autoregulates its mRNA and is required for the unfolded protein response

A stress response pathway from the endoplasmic reticulum to the nucleus requires a novel bifunctional protein kinase/endoribonuclease (Ire1p) in mammalian cells

A viral RNA competitively inhibits the antiviral endoribonuclease domain of RNase L

Architecture of the flaviviral replication complex. Protease, nuclease, and detergents reveal encasement within double-layered membrane compartments

The spread of apoptosis through gap-junctional channels in BHK cells transfected with Cx32

IRE1alpha cleaves select microRNAs during ER stress to derepress translation of proapoptotic Caspase-2

Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1

Sensing microbial RNA in the cytosol

Hepatitis C virus RNA: dinucleotide frequencies and cleavage by RNase L

Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses

Ribosomal RNA cleavage, nuclease activation and 2-5A(ppp(A2 p)nA) in interferon-treated cells

Interferon action-sequence specificity of the ppp(A2 p)nA-dependent ribonuclease

XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor

Tick-borne encephalitis virus triggers inositol-requiring enzyme 1 (IRE1) and transcription factor 6 (ATF6) pathways of unfolded protein response

Flavivirus infection activates the XBP1 pathway of the unfolded protein response to cope with endoplasmic reticulum stress

Coxsackievirus B3 infection activates the unfolded protein response and induces apoptosis through downregulation of p58IPK and activation of CHOP and SREBP1

Homologous 2 ,5 -phosphodiesterases from disparate RNA viruses antagonize antiviral innate immunity

Antagonism of the interferon-induced OAS-RNase L pathway by murine coronavirus ns2 protein is required for virus replication and liver pathology

Expression cloning of 2-5A-dependent RNAase: a uniquely regulated mediator of interferon action

Mapping of the human RNASEL promoter and expression in cancer and normal cells

Interferon action and apoptosis are defective in mice devoid of 2 ,5 -oligoadenylatedependent RNase L

The research carried out was supported by intramural funding from THSTI. I would like to thank Dr. Manjula Kalia for careful reading of the manuscript.

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.