Select Paramyxoviral V Proteins Inhibit IRF3 Activation by Acting as Alternative Substrates for Inhibitor of κB Kinase ϵ (IKKe)/TBK1*

V accessory proteins from Paramyxoviruses are important in viral evasion of the innate immune response. Here, using a cell survival assay that identifies both inhibitors and activators of interferon regulatory factor 3 (IRF3)-mediated gene induction, we identified select paramyxoviral V proteins that inhibited double-stranded RNA-mediated signaling; these are encoded by mumps virus (MuV), human parainfluenza virus 2 (hPIV2), and parainfluenza virus 5 (PIV5), all members of the genus Rubulavirus. We showed that interaction between V and the IRF3/7 kinases, TRAF family member-associated NFκB activator (TANK)-binding kinase 1 (TBK1)/inhibitor of κB kinase ϵ (IKKe), was essential for this inhibition. Indeed, V proteins were phosphorylated directly by TBK1/IKKe, and this, intriguingly, resulted in lowering of the cellular level of V. Thus, it appears that V mimics IRF3 in both its phosphorylation by TBK1/IKKe and its subsequent degradation. Finally, a PIV5 mutant encoding a V protein that could not inhibit IKKe was much more susceptible to the antiviral effects of double-stranded RNA than the wild-type virus. Because many innate immune response signaling pathways, including those initiated by TLR3, TLR4, RIG-I, MDA5, and DNA-dependent activator of IRFs (DAI), use TBK1/IKKe as the terminal kinases to activate IRFs, rubulaviral V proteins have the potential to inhibit all of them.

Innate immunity is stimulated by viruses in part via RNA. dsRNA 2 specifically is present in several forms: viral genomes, single-stranded RNA virus replication intermediates, DNA virus symmetric transcription products, defective viral particles, and debris from lysed cells (1). Although extracellular dsRNA is sensed by Toll-like receptor 3 (TLR3), intracellular dsRNA is detected in part by the RNA helicases retinoic acidinducible gene 1 (RIG-I) and melanoma differentiation-associated gene 5 (Mda-5). These receptors signal through Toll-IL-1R (TIR) domain containing adaptor-inducing IFN␤ (TRIF) and IFN␤ promoter stimulator 1 (IPS1), respectively, to activate the kinases TANK-binding kinase 1 (TBK1) and inhibitor of B kinase ⑀ (IKKe). They, in turn, phosphorylate IFN regulatory factor 3 (IRF3), promoting its nuclear translocation and subsequent IFN-stimulated regulatory element-mediated transcription of IFN-stimulated genes (ISG), such as ISG56, as well as IFN and other cytokines. IFN then, through Janus kinase (JAK)/ STAT activation of IRF9, modulates microRNAs in addition to up-regulating itself and more ISGs to heighten the antiviral state and also to initiate the adaptive immune response by promoting dendritic cell maturation, memory T cell proliferation, and B cell differentiation (2)(3)(4).
To control this immune response, pathogens and hosts have developed methods of down-regulation and evasion at a variety of different points (5)(6)(7). At the level of sensing infection, NS1 from influenza virus binds to and sequesters dsRNA and also interacts with RIG-I (5,6,8). At the level of signal transduction, the NS3/4A protease from hepatitis C virus cleaves TRIF and IPS1 (9 -11), and vaccinia virus A46R, which contains a TIR domain, inhibits TRIF-dependent activation of IRF3 (12,13). At the level of IRF-mediated transcription, P proteins from Ebola (5,6) and borna disease (14) viruses and human papilloma virus E16 (15) interfere with IRF3 activation. For a more global effect, M protein from vesicular stomatitis virus inhibits host machinery to prevent gene transcription (5,6). Finally, at the level of IFN and cytokines binding to their receptors, human cytomegalovirus encodes a decoy for the chemokine RANTES (regulated on activation normal T cell expressed and secreted) to prevent further signaling (16).
The family Paramyxoviridae encompasses two subfamilies: Paramyxovirinae and Pneumovirinae. Viruses representing different genuses within the paramyxoviral subfamily are important pathogens for humans such as measles virus (MeV), a Morbillivirus, and mumps virus (MuV) and human parainfluenza virus 2 (hPIV2), both Rubulaviruses. For animals, examples include Hendra virus (HeV), a Henipavirus that infects flying * This work was supported, in whole or in part, by National Institutes of Health Grants CA62220 (to G. C. S.) and CA68782 (to G. C. S.) and Medical Scientist Training Grant GM07250 (to L. L. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 (17). Paramyxoviral V accessory proteins have been shown to be important in viral evasion of innate immunity. At the level of IFN signaling, V proteins act as ubiquitin-protein isopeptide ligases (E3) that target STATs for degradation or sequester them, preventing their nuclear translocation and subsequent transcriptional functions (18). At the level of sensing infection, V proteins bind to mda-5 and also to dsRNA itself to inhibit intracellular dsRNA signaling and protein kinase R activation, respectively (19 -21). These functions highlight the importance of immune signaling in the viral life cycle. Indeed, VDC PIV5, a mutant PIV5 virus lacking the unique C terminus of V, is no longer able to inhibit IRF3 activation and causes a greater cytopathic effect in a variety of different cells (22,23). Taken together, these lines of evidence indicate that V may inhibit dsRNA activation of IRF3 via TLR3.
Here we show that V proteins from hPIV2 (V H ), MuV (V M ), and PIV5 (V P ), but not HeV and MeV, inhibit TLR3 signaling. Analysis of the underlying mechanism revealed that the inhibitory V proteins interacted with the signaling kinases TBK1/ IKKe and served as their substrates, thus preventing IRF3 phosphorylation. Our results indicated that the above interaction led to modifications of both partners and their degradation. Therefore, the V proteins from Rubulaviruses and the IRF3activating kinases TBK1/IKKe are connected by a negative feedback loop.
Cell Survival Assay-TLR3 293 561-TK cells were transfected with pEF-V and treated with dsRNA and GCV. Cells were maintained under selection 4 days to assay survival and create pools expressing V proteins.
To assay V interaction with IKKe, TLR3 293 cells were transfected with relevant expression plasmids. Immunoprecipitations were performed as described previously (30) with agarose-conjugated HA F7 (Santa Cruz Biotechnology), FLAG M2 (Sigma), or protein A/G PLUS-agarose (Santa Cruz Biotechnology) with c-Myc 9E10 at 4°C. To assay for in vivo V phosphorylation, the same procedure was used to isolate V. The resulting samples were then treated with 100 units of calf intestine phosphatase (USB Corp.) 37°C 3 h where indicated.
IRF3 Activation Assays-To assess Ser-396 phosphorylation and IRF3 nuclear localization, HT1080 V M cells were treated with dsRNA for 3 h before collection of whole cell or nuclear lysates, respectively, described previously (27). To look at localization by immunofluorescence, the same cells were plated on coverslips and treated with dsRNA for 1 h and then LMB for 2 h. Cells were stained for IRF3 and 4Ј,6-diamidino-2-phenylindole (27). To determine IRF3 transcriptional activity, the same cells were treated with dsRNA for 1 h and then with LMB for 6 h. Whole cell extracts were prepared for immunoblotting.
In Vitro Kinase Assay-To purify FLAG-IRF3 and V M , the respective vectors were transfected for 24 h into TLR3 293 cells, and lysates were prepared as described (37). FLAG-tagged proteins were immunoprecipitated with FLAG M2 agarose (Sigma), eluted with FLAG peptide (Sigma), and concentrated with Microcon filter devices YM-3 (Millipore). Myc-TBK1 was purified similarly with c-Myc 9E10 and Trueblot immunoprecipitation beads (eBioscience). For in vitro kinase assays, purified FLAG-IRF3 or V M were incubated with GST⅐IKKe (Cell Signaling) or Myc-TBK1 in [␥-32 P]ATP (37). Quantitation for autoradiograph was performed by GE Healthcare PhosphorImager and for immunoblotting by Odyssey (Licor).
V M degradation-To follow V M degradation by IKKe, control empty or IKKe-expressing vectors were co-transfected with V-expressing vectors into TLR3 293 cells for 8 h, media were changed, and whole cell lysates were prepared 48 h later. To follow V M degradation by dsRNA, the same cells were simultaneously transfected with pEF-V and dsRNA-treated with either MG132 or control DMSO for 16 h.
TLR3 Antiviral Activity and PIV5 Plaque Assay-293 or TLR3 293 cells were treated with dsRNA for 16 h. The cells were then infected with WT or VDC PIV5 at a multiplicity of infection of 0.1. Supernatants were collected 6 days after infection. Virus yields were determined by plaque assay on Vero (24).

Some, but Not All, Paramyxoviral V Proteins Inhibit TLR3
Signaling-We used a cell survival assay to examine the potentials of V proteins from different paramyxoviral subfamilies to inhibit TLR3 signaling. This assay uses a TLR3-expressing 293 cell line (TLR3 293) in which a selection gene has been introduced; this gene is driven by the promoter of ISG56 (27) and encodes the herpesvirus TK protein (28). Any signaling that activates transcription factors containing IRF proteins, such as TLR3 signaling or type I IFN signaling, induces TK production and causes cell death in the presence of GCV. If an inhibitor of signaling blocks TK production in a cell, that cell survives and proliferates even in the presence of GCV. Using this assay, we determined that V proteins from the Rubulaviruses hPIV2 (V H ), MuV (V M ), and PIV5 (V P ) inhibited TLR3 signaling (Fig.  1A, top panel, lanes 4 -6). In contrast, V proteins from HeV and MeV were ineffective (Fig. 1A, top panel, lanes 2 and 3), although the different V proteins were expressed at similar levels in the transfected cells (Fig. 1B). Expression of the V proteins themselves, without any dsRNA treatment, did not affect cell survival at all (Fig. 1A, bottom panel). The above observations suggested that rubulaviral V proteins could block induction of cellular dsRNA-inducible genes as well, a conclusion that was confirmed by measuring the levels of ISG56 mRNA, using a quantitative reverse transcription-PCR assay in HT1080 cells permanently expressing different V proteins. The mRNA was induced strongly upon dsRNA treatment of cells (Fig. 1C, lane 5) and the induction was almost completely blocked by V H , V M , and V P (Fig. 1C, lanes 6 -8). Similar inhibitions of the induction of p56 protein were observed (Fig. 1D).
V Proteins Block IRF3 Activation-Once we established that the three V proteins blocked TLR3 signaling, we investigated the underlying mechanism. Exogenous expression of signaling proteins downstream of TLR3 is known to activate IRF3 and induce synthesis of p56, as shown in Fig. 2A. Expression of TRIF, IKKe, and TBK1 induced p56 (lane 1), and the induction was blocked by all three V proteins. In contrast, p56 induction from expression of a constitutively active IRF3 5D mutant was not blocked (Fig. 2A, lanes 2-4). These results indicated that the V proteins blocked a step in signaling downstream of TLR3, TRIF, and the IRF3 kinases and upstream of events following activation of IRF3. Inactive IRF3 shuttles in and out of the nucleus, whereas activated IRF3 translocates to the nucleus and induces transcription of target genes, such as ISG56 (38). IRF3 was not localized to the nucleus in cells expressing V M (Fig. 2B,  lane 4), V H , and V P (data not shown). By using LMB, a drug that blocks nuclear export of proteins (39), we examined whether V M blocked nuclear import of IRF3 or enhanced its export. As expected, in untreated cells, IRF3 was in the cytoplasm of both control and V-expressing cells (Fig. 2C, row 1). dsRNA treat-ment alone caused nuclear accumulation of IRF3 only in the absence of V protein (Fig. 2C, row 2). In both untreated and dsRNA-treated cells, LMB caused similar nuclear accumulation of IRF3 in the absence and the presence of V protein (Fig.  1-4) or dsRNA-treated (lanes 5-8) cells described in C. Immunoblotting for actin and FLAG-V served as controls. rows 3 and 4). These results suggested that either V M inhibited nuclear import of only activated IRF3 or V M enhanced export of activated IRF3 through exportin 1, which is blocked by LMB (39). To address these possibilities, we examined whether the IRF3 sequestered within the nucleus by LMB was transcriptionally active in the presence of dsRNA. Nuclear IRF3 in LMB-treated cells could not induce p56 in control cells unless they were dsRNA-treated as well (Fig. 2D, lanes 1 and 3). In dsRNA-treated V-expressing cells, although LMB treatment caused nuclear translocation of IRF3, p56 was not induced (Fig.  2D, lanes 2 and 4). These results indicated that V protein inhibited import of activated IRF3 to the nucleus and did not enhance its nuclear export. Furthermore, these results indicated that nuclear localization, although necessary, was insufficient for IRF3 to induce genes; additional activation was required by post-translational modifications of the protein. We then asked whether the observed block of IRF3 activation was due to a block in its phosphorylation, which is known to activate it. Indeed, phosphorylation of Ser-396, a hallmark of IRF3 activation, was inhibited in the presence of V M (Fig. 2E, lane 4). These results indicated that the major block caused by V M was at the level of IRF3 phosphorylation.

2C,
Interaction of V with IKKe Is Essential for Inhibiting TLR3 Signaling-To further analyze the mechanism of V-mediated blocking of TLR3 signaling, we examined possible interactions of the viral proteins with known components of the signaling pathways. V H , V M , and V P did not interact with TLR3, TRIF, IRF3, Src, phosphatidylinositol 3-kinase, and IKK ␣, ␤, and ␥ as revealed by co-immunoprecipitation assays, although the same assay demonstrated their known interactions with STAT2 (Fig.  3, A and B). In contrast, all three viral proteins interacted with both IKKe (Fig. 3C) and TBK1 (data not shown). IKKe co-im-  (Fig. 3C,  top panel). Note that the mobility of co-immunoprecipitated IKKe was slower than that of the protein in cell extracts. This was also the case for co-immunoprecipitated STAT2 (Fig. 3C, middle panel, lanes 3, 5,  and 7). Since the V proteins have E3 ubiquitin ligase activity (18), the observed mobility differences could be due to V-mediated polyubiquitination of the co-immunoprecipitated proteins. Indeed, exogenously introduced ubiquitin was covalently bound to IKKe purified by immunoprecipitation, and this ubiquitination was V M -dependent (Fig. 3D). The functional significance of the V-IKKe interaction was determined by testing mutant V M . Among six mutant proteins tested (Fig. 3E), only the V M mutant W174A/ W178A/W188A (V M-AAA ) failed to bind to IKKe (lane 3, left top panel). This mutant protein could not block TLR3 signaling as revealed by the cell survival assay (data not shown) and in HT1080 cells permanently expressing mutant V M-AAA (Fig. 3G, lane 8). In contrast, WT V M and mutant V C189A could inhibit signaling (Fig. 3G, lanes 4 and 6). Both bound to IKKe (Fig. 3E, lanes 7 and 8) and, as shown when electrophoresed longer, both shifted the mobility of co-immunoprecipitated IKKe, indicating its ubiquitination (Fig. 3F,  lanes 2 and 4, compared with lanes 1 and 3). These results strongly indicated that the V proteins blocked TLR3 signaling by blocking the action of the signaling kinases.
V Proteins Are Substrates for IKKe and TBK1-Because the V proteins bound to the kinases, in the next experiments, we tested whether they were phosphorylated as a consequence. Indeed, both V M (Fig. 4A) and V P (Fig. 4B) were phosphorylated by IKKe in vivo. As expected, the phosphorylated V proteins migrated more slowly (Fig. 4, A and B, lane 2), and upon phosphatase treatment, they co-migrated with the unphosphorylated proteins (Fig. 4, A and B, lane 3). Similar results were seen for TBK1 (data not shown). Expression of enzymatically inactive IKKe did not cause phosphorylation of the V proteins (Fig.  4, A and B, lane 4), suggesting that IKKe directly phosphorylated V. To test this suggestion, purified V M and IKKe were added to an in vitro protein kinase reaction in the presence of [␥-32 P]ATP, and radiolabeling of V M was measured. As a positive control, purified IRF3, a known substrate of IKKe, was used. Both IRF3 and V M were phosphorylated by IKKe (Fig. 4C, lanes  1 and 2). Control reactions showed that the addition of both IKKe and V M was needed to observe the radiolabeled protein (Fig. 4C, lanes 3 and 4). Quantification of the incorporated phosphates demonstrated that, on a molar basis, V M was a slightly better substrate than IRF3 (114 versus 100). Similar in vitro reactions demonstrated the ability of purified TBK1 to  4) were immunoblotted for IRF3, with histone as loading control. C, control and V M -expressing HT1080 cells were treated with dsRNA (rows 2 and 4) and LMB (rows 3 and 4). Subcellular location of IRF3 was determined by immunofluorescence. D, cell extracts from samples, as described for C, rows 3 and 4, were used for immunoblotting to detect p56, FLAG-V, and actin. E, IRF3 phosphorylation at serine 396 (P396) was detected by immunoblotting cell extracts with a phospho-IRF3specific antiserum, with total IRF3 as a control. Whole cell extracts were prepared from control cells ( lanes  1 and 3) or cells stably expressing V M (lanes 2 and 4) that were untreated (lanes 1 and 2) or treated with dsRNA (lanes 3 and 4).
As a consequence of phosphorylation, V appeared to be destined for faster degradation (Fig. 5A). Co-expression of V with kinase active (lane 2), but not kinase inactive (lane 3) IKKe, caused major diminution of the cellular level of V, as compared with control (lane 1). Furthermore, this was not caused just by overexpression of IKKe because dsRNA-mediated activation of the endogenous kinase caused a dramatic lowering of the level of V as well (Fig. 5B, lane 2), and this diminution could be inhibited by the proteasome inhibitor MG132 (Fig.  5B, lane 3). Finally, interaction between V and IKKe was required for degradation; V M-AAA , a mutant that did not co-immunoprecipitate with IKKe (Fig. 3E, lane 3), was not degraded (Fig. 5C).
V Protein Determines the Efficacy of Virus Replication in TLR3-activated Cells-To evaluate the biological significance of our observations, we chose to use the PIV5 mutant virus VDC, which encodes a C-terminally truncated V protein (22). VDC was not phosphorylated in vivo by IKKe as seen in WT PIV5 V protein (Fig. 6A). Furthermore, we determined that the truncated V protein could not block signaling induced by exogenous expression of IKKe (data not shown). Results showed that WT virus replicated better as compared with the mutant virus in both TLR3-expressing and non-expressing cells (Fig. 6B). Since the mda-5/RIG-I pathway is still intact in these cells, this difference may be due to the lack of ability of the truncated V protein to inhibit that signaling pathway (19). In cells not expressing TLR3, treatment with dsRNA did not have any effect on the replication of either virus. In contrast, in TLR3-expressing cells, dsRNA treatment strongly inhibited the replication of the mutant virus (5 logs), whereas the effect on WT virus was minor. These results demonstrated that inhibition of TLR3 signaling by V protein was relevant for effective virus replication.

DISCUSSION
The results presented above revealed several new features of the equilibrium established in an infected cell between the virus and the host (Fig. 7). Induction of viral stress-inducible genes, including IFNs, is blocked by rubulaviral V proteins by inhibiting IRF3 phosphorylation, which is required for its activation because the V proteins can themselves be phosphorylated by TBK1/IKKe. A similar observation has been made by Unterstab et al. (14) for borna disease virus P protein. It is not yet clear exactly how V proteins can FIGURE 3. Interaction of V with IKKe is essential for inhibition of TLR3 signaling. A, TRIF and V proteins were transiently expressed in TLR3 293 cells. V proteins were immunoprecipitated (IP), and co-immunoprecipitated proteins were immunoblotted to detect TRIF (indicated by arrow), HA-tagged V, stably expressed FLAG-tagged TLR3 and endogenous STAT2, phosphatidylinositol 3-kinase p85 (PI3K P85), c-Src (indicated by arrow), and IRF3 (indicated by arrow). Cell extracts (W) served as controls for expression levels. B, the same as A except that immunoblotting to detect endogenous IKK ␣, ␤, and ␥ was used in place of other TLR3 signaling mediators. C, IKKe and V proteins were transiently expressed in TLR3 293 cells. V proteins were immunoprecipitated, and co-immunoprecipitated proteins were immunoblotted to detect Myc-tagged IKKe, STAT2, and HA-tagged V; whole cell extracts served as controls for expression levels. inhibit IRF3 phosphorylation by TBK1/IKKe. Because they themselves are substrates of the same kinases, one possibility is that V simply competes out IRF3 as a substrate. In the in vitro kinase assays, quantitation of phosphorylation showed that IRF3 and V M were equally phosphorylated by IKKe, when present in equimolar amounts either singly or in combination (data not shown), indicating that the two proteins are equally competent substrates of the kinases. Hence, to compete out IRF3, V has to be present at a high molar excess, a possibility not unlikely because V proteins are produced in large quantities in Paramyxovirus-infected cells (17). Alternatively, V proteins can have stronger affinity for TBK1/IKKe, thus not allowing access to IRF3. The fact that V and IKKe were co-immunoprecipitated efficiently indicated that the two proteins could interact strongly, thus providing credence to the second scenario. Although our study was designed to determine whether different paramyxoviral V proteins could inhibit TLR3 signaling, our observations are equally relevant for other signaling pathways that converge on TBK1/IKKe. Thus, we can expect rubulaviral V proteins to block signaling initiated by the cytoplasmic receptors RIG-I, mda-5, and DNA-dependent activator of IRFs (DAI) or the membrane receptor TLR4, which are known to be triggered by RNA, DNA, or lipopolysaccharide (3). Indeed, we observed that RIG-I/mda-5 signaling to IRF3 triggered by transfected dsRNA was inhibited by V H , V M , and V P (data not shown).
An additional consequence of the action of V proteins as alternative substrates of IKKe could be inhibition of phosphorylation of STAT1. Because STAT1 phosphorylation by IKKe is essential for its ability to induce a subset of ISGs (40), in addition to causing STAT1 degradation (18), rubulaviral V proteins may block actions of IFN using this mechanism.
The interaction with V caused ubiquitination of IKKe as indicated by the observed slower mobility of co-immunoprecipitated IKKe and its V-dependent ubiquitination when co-transfected with tagged ubiquitin. A similar change in the mobility of IKKe was observed when C189A mutant of V M was used; this was unexpected because the mutant cannot bind DDB1 (data not shown), a component of the ubiquitin ligase complex (18). This indicates the existence of alternative pathways for the  lanes 3 and 5). HA immunoblotting was used to detect V. B, the same as A except that V P was used in place of V M . C, in vitro [␥-32 P]ATP kinase assays were conducted with GST⅐IKKe and V protein or IRF3. Radiolabeled proteins were visualized by autoradiography (top two panels) and immunoblotted (bottom two panels) to determine total amounts of IRF3 or V in the loaded samples.  ubiquitin ligase activity of V M . Here, polyubiquitination of IKKe leads to its proteasome-mediated degradation. Thus, V not only blocks IRF3 phosphorylation but destroys the activating kinase as well.
What was clear from our results was that the host could fight back by degrading V proteins. Just as phosphorylation of IRF3 leads to its degradation, phosphorylation of V protein was accompanied by its destruction (Fig. 5). This is consistent with the degradation of V M observed over the course of a viral infection (41). Because V was needed for the optimum replication of viruses, even in the absence of dsRNA signaling (Fig. 6), its destruction should be a potent antiviral mechanism in that context as well. Phosphorylation and degradation of V provide an opportunity for temporal regulation of IRF3 activation; when the level of V is high, IRF3 phosphorylation is blocked by competition. As more and more V is phosphorylated and degraded, the relative concentration of IRF3 will increase, leading to its phosphorylation, activation, and degradation. Thus, two negative feedback loops, one between V and IRF3 and the other between IKKe/TBK1 and V, regulate the equilibrium between the virus and the cell.
It is known that IKKe interacts with STAT1 and that STAT1 can interact with V (30,40). However, the interaction between IKKe and V reported here is not mediated by STAT1 because V M could block TLR3 signaling in U3B cells (42), which lack functional STAT1 (data not shown). The tryptophan residues of V that were required for IKKe binding are located in the C-terminal region of the protein (43). Because this region is not shared between viral V and P proteins, whose mRNAs are produced by alternative transcription of the same open reading frame, P proteins are not expected to bind to IKKe. This prediction is consistent with previous findings that V, but not P, blocks dsRNA-mediated IRF3 activation (22).
The experiments with LMB (Fig. 2, C and D) illuminated subtle, but important, aspects concerning IRF3 activation. According to the current paradigm of the activation process, IRF3 is phosphorylated at multiple serine residues, and this leads to its nuclear translocation, a process that is necessary for its action as a transcription factor (38). We found that forcing nuclear localization of IRF3 by LMB treatment of cells was not sufficient for its ability to induce genes. Therefore, there are consequences to IRF3 phosphorylation in addition to nuclear translocation that are necessary for activation.
It is interesting to note that among the V proteins from paramyxoviral subfamilies tested, all inhibit IFN signaling, but only members of the genus Rubulavirus were able to inhibit dsRNA-TLR3-mediated IRF3 activation (17). These observations seem to correlate with what seems to be a more important role for V in the life cycle of Rubulaviruses as compared with others. Rubulaviral V proteins, in contrast to P in other Paramyxoviruses, represent the default open reading frame transcribed at the P/V locus, resulting in higher levels of basal expression. Correspondingly, virions from Rubulaviruses contain a much higher level of V as compared with others and are conveniently present during the initial stages of infection and signaling (17). This importance of rubulaviral V proteins may be in part compensation for the lack of W or C accessory proteins, which Respiroviruses and Henipaviruses encode to counteract host defense (5,17,44).