Nuclear dengue virus NS5 antagonizes expression of PAF1-dependent immune response genes.

Dengue virus (DENV) disruption of the innate immune response is critical to establish infection. DENV non-structural protein 5 (NS5) plays a central role in this disruption, such as antagonism of STAT2. We recently found that DENV serotype 2 (DENV2) NS5 interacts with Polymerase associated factor 1 complex (PAF1C). The primary members of PAF1C are PAF1, LEO1, CTR9, and CDC73. This nuclear complex is an emerging player in the immune response. It promotes the expression of many genes, including genes related to the antiviral, antimicrobial and inflammatory responses, through close association with the chromatin of these genes. Our previous work demonstrated that NS5 antagonizes PAF1C recruitment to immune response genes. However, it remains unknown if NS5 antagonism of PAF1C is complementary to its antagonism of STAT2. Here, we show that knockout of PAF1 enhances DENV2 infectious virion production. By comparing gene expression profiles in PAF1 and STAT2 knockout cells, we find that PAF1 is necessary to express immune response genes that are STAT2-independent. Finally, we mapped the viral determinants for the NS5-PAF1C protein interaction. We found that NS5 nuclear localization and the C-terminal region of the methyltransferase domain are required for its interaction with PAF1C. Mutation of these regions rescued the expression of PAF1-dependent immune response genes that are antagonized by NS5. In sum, our results support a role for PAF1C in restricting DENV2 replication that NS5 antagonizes through its protein interaction with PAF1C.

Nonstructural Protein 11 of Porcine Reproductive and Respiratory Syndrome Virus Induces STAT2 Degradation To Inhibit Interferon Signaling.

Interferons (IFNs) play a crucial role in host antiviral response by activating the JAK/STAT (Janus kinase/signal transducer and activator of transcription) signaling pathway to induce the expression of myriad genes. STAT2 is a key player in the IFN-activated JAK/STAT signaling. Porcine reproductive and respiratory syndrome virus (PRRSV) is an important viral pathogen, causing huge losses to the swine industry. PRRSV infection elicits a meager protective immune response in pigs. The objective of this study was to investigate the effect of PRRSV on STAT2 signaling. Here, we demonstrated that PRRSV downregulated STAT2 to inhibit IFN-activated signaling. PRRSV strains of both PRRSV-1 and PRRSV-2 species reduced the STAT2 protein level, whereas the STAT2 transcript level had minimal change. PRRSV reduced the STAT2 level in a dose-dependent manner and shortened STAT2 half-life significantly from approximately 30 to 5 h. PRRSV-induced STAT2 degradation could be restored by treatment with the proteasome inhibitor MG132 and lactacystin. In addition, PRRSV nonstructural protein 11 (nsp11) was identified to interact with and reduce STAT2. The N-terminal domain (NTD) of nsp11 was responsible for STAT2 degradation and interacted with STAT2 NTD and the coiled-coil domain. Mutagenesis analysis showed that the amino acid residue K59 of nsp11 was indispensable for inducing STAT2 reduction. Mutant PRRSV with the K59A mutation generated by reverse genetics almost lost the ability to reduce STAT2. Together, these results demonstrate that PRRSV nsp11 antagonizes IFN signaling via mediating STAT2 degradation and provide further insights into the PRRSV interference of the innate immunity.IMPORTANCE PRRSV infection elicits a meager protective immune response in pigs. One of the possible reasons is that PRRSV antagonizes interferon induction and its downstream signaling. Interferons are key components in the innate immunity and play crucial roles against viral infection and in the activation of adaptive immune response via JAK/STAT signaling. STAT2 is indispensable in the JAK/STAT signaling since it is also involved in activation of antiviral activity in the absence of STAT1. Here, we discovered that PRRSV nsp11 downregulates STAT2. Interestingly, the N-terminal domain of nsp11 is responsible for inducing STAT2 degradation and directly interacts with STAT2 N-terminal domain. We also identified a crucial amino acid residue K59 in nsp11 since a mutation of it led to loss of the ability to downregulate STAT2. A mutant PRRSV with mutation of K59 had minimal effect on STAT2 reduction. Our data provide further insights into PRRSV interference with interferon signaling.

Heartland virus antagonizes type I and III interferon antiviral signaling by inhibiting phosphorylation and nuclear translocation of STAT2 and STAT1.

Heartland virus (HRTV) is a pathogenic phlebovirus recently identified in the United States and related to severe fever with thrombocytopenia syndrome virus (SFTSV) emerging in Asia. We previously reported that SFTSV disrupts host antiviral responses directed by interferons (IFNs) and their downstream regulators, signal transducer and activator of transcription (STAT) proteins. However, whether HRTV infection antagonizes the IFN-STAT signaling axis remains unclear. Here, we show that, similar to SFTSV, HRTV also inhibits IFN-α- and IFN-λ-mediated antiviral responses. As expected, the nonstructural protein (NSs) of HRTV (HNSs) robustly antagonized both type I and III IFN signaling. Protein interaction analyses revealed that a common component downstream of type I and III IFN signaling, STAT2, is the target of HNSs. Of note, the DNA-binding and linker domains of STAT2 were required for an efficient HNSs-STAT2 interaction. Unlike the NSs of SFTSV (SNSs), which blocks both STAT2 and STAT1 nuclear accumulation, HNSs specifically blocked IFN-triggered nuclear translocation only of STAT2. However, upon HRTV infection, IFN-induced nuclear translocation of both STAT2 and STAT1 was suppressed, suggesting that STAT1 is an additional HRTV target for IFN antagonism. Consistently, despite HNSs inhibiting phosphorylation only of STAT2 and not STAT1, HRTV infection diminished both STAT2 and STAT1 phosphorylation. These results suggest that HRTV antagonizes IFN antiviral signaling by dampening both STAT2 and STAT1 activities. We propose that HNSs-specific targeting of STAT2 likely plays an important role but is not all of the "tactics" of HRTV in its immune evasion.

Type I interferon-enhanced IL-10 expression in human CD4 T cells is regulated by STAT3, STAT2, and BATF transcription factors.

Type I IFN can exert pro- and anti-inflammatory activities in the immune system. Here, we have investigated the mechanism by which IFN-α enhances early expression of the anti-inflammatory cytokine IL-10 in human CD45RA+CD4+ T cells. With the use of transcriptomic and biochemical approaches, we found distinct and combined contributions of the IFN and the TCR signaling pathways to the induction of STAT1/2/3 and the basic leucine zipper activating transcription factor-like (BATF) family members. Moreover, IFN-induced STAT3 phosphorylation was prolonged by the TCR response, whereas IFN-induced STAT2 phosphorylation was of long duration. With the use of RNA interference (RNAi), we identified STAT3 as the major actor and STAT2 as a contributor of the IFN action on IL-10 Upon TCR/IFN costimulation, STAT3 directly bound at the IL-10 conserved noncoding sequence (CNS)- 9, an enhancer element known to recruit BATF in CD4 T cells. The cosilencing of the 3 BATFs resulted in an overall reduction of IL-10 expression, but the promoting activity of IFN-α was retained. These results support the notion that the IFN action is indexed on BATF function and provide evidence for a cooperation between BATFs and STAT3, the latter being activated via early IFN and delayed TCR effects.

The non-pathogenic Henipavirus Cedar paramyxovirus phosphoprotein has a compromised ability to target STAT1 and STAT2.

Immune evasion by the lethal henipaviruses, Hendra (HeV) and Nipah virus, is mediated by its interferon (IFN) antagonist P gene products, phosphoprotein (P), and the related V and W proteins, which can target the signal transducer and activator of transcription 1 (STAT1) and STAT2 proteins to inhibit IFN/STAT signaling. However, it is not clear if the recently identified non-pathogenic Henipavirus, Cedar paramyxovirus (CedPV), is also able to antagonize the STAT proteins. We performed comparative studies between the HeV P gene products (P/V/W) and CedPV-P (CedPV does not encode V or W) and demonstrate that differences exist in their ability to engage the STAT proteins using immunoprecipitation and quantitative confocal microscopic analysis. In contrast to HeV-P gene encoded proteins, the ability of CedPV-P to interact with and relocalize STAT1 or STAT2 is compromised, correlating with a reduced capacity to inhibit the mRNA synthesis of IFN-inducible gene MxA. Furthermore, infection studies with HeV and CedPV demonstrate that HeV is more potent than CedPV in inhibiting the IFN-α-mediated nuclear accumulation of STAT1. These results strongly suggest that the ability of CedPV to counteract the IFN/STAT response is compromised compared to HeV.

Rotavirus inhibits IFN-induced STAT nuclear translocation by a mechanism that acts after STAT binding to importin-α.

The importance of innate immunity to rotaviruses is exemplified by the range of strategies evolved by rotaviruses to interfere with the IFN response. We showed previously that rotaviruses block gene expression induced by type I and II IFNs, through a mechanism allowing activation of signal transducer and activator of transcription (STAT) 1 and STAT2 but preventing their nuclear accumulation. This normally occurs through activated STAT1/2 dimerization, enabling an interaction with importin α5 that mediates transport into the nucleus. In rotavirus-infected cells, STAT1/2 inhibition may limit the antiviral actions of IFN produced early in infection. Here we further analysed the block to STAT1/2 nuclear accumulation, showing that activated STAT1 accumulates in the cytoplasm in rotavirus-infected cells. STAT1/2 nuclear accumulation was inhibited by rotavirus even in the presence of the nuclear export inhibitor Leptomycin B, demonstrating that enhanced nuclear export is not involved in STAT1/2 cytoplasmic retention. The ability to inhibit STAT nuclear translocation was completely conserved amongst the group A rotaviruses tested, including a divergent avian strain. Analysis of mutant rotaviruses indicated that residues after amino acid 47 of NSP1 are dispensable for STAT inhibition. Furthermore, expression of any of the 12 Rhesus monkey rotavirus proteins did not inhibit IFN-stimulated STAT1 nuclear translocation. Finally, co-immunoprecipitation experiments from transfected epithelial cells showed that STAT1/2 binds importin α5 normally following rotavirus infection. These findings demonstrate that rotavirus probably employs a novel strategy to inhibit IFN-induced STAT signalling, which acts after STAT activation and binding to the nuclear import machinery.

3Cpro of foot-and-mouth disease virus antagonizes the interferon signaling pathway by blocking STAT1/STAT2 nuclear translocation.

UNLABELLED: Foot-and-mouth disease virus (FMDV) causes a highly contagious, debilitating disease in cloven-hoofed animals with devastating economic consequences. To survive in the host, FMDV has evolved to antagonize the host type I interferon (IFN) response. Previous studies have reported that the leader proteinase (L(pro)) and 3C(pro) of FMDV are involved in the inhibition of type I IFN production. However, whether the proteins of FMDV can inhibit type I IFN signaling is less well understood. In this study, we first found that 3C(pro) of FMDV functioned to interfere with the JAK-STAT signaling pathway. Expression of 3C(pro) significantly reduced the transcript levels of IFN-stimulated genes (ISGs) and IFN-stimulated response element (ISRE) promoter activity. The protein level, tyrosine phosphorylation of STAT1 and STAT2, and their heterodimerization were not affected. However, the nuclear translocation of STAT1/STAT2 was blocked by the 3C(pro) protein. Further mechanistic studies demonstrated that 3C(pro) induced proteasome- and caspase-independent protein degradation of karyopherin α1 (KPNA1), the nuclear localization signal receptor for tyrosine-phosphorylated STAT1, but not karyopherin α2, α3, or α4. Finally, we showed that the protease activity of 3C(pro) contributed to the degradation of KPNA1 and thus blocked STAT1/STAT2 nuclear translocation. Taken together, results of our experiments describe for the first time a novel mechanism by which FMDV evolves to inhibit IFN signaling and counteract host innate antiviral responses. IMPORTANCE: We show that 3C(pro) of FMDV antagonizes the JAK-STAT signaling pathway by blocking STAT1/STAT2 nuclear translocation. Furthermore, 3C(pro) induces KPNA1 degradation, which is independent of proteasome and caspase pathways. The protease activity of 3C(pro) contributes to the degradation of KPNA1 and governs the ability of 3C(pro) to inhibit the JAK-STAT signaling pathway. This study uncovers a novel mechanism evolved by FMDV to antagonize host innate immune responses.

Luteolin sensitizes the antiproliferative effect of interferon α/ß by activation of Janus kinase/signal transducer and activator of transcription pathway signaling through protein kinase A-mediated inhibition of protein tyrosine phosphatase SHP-2 in cancer cells.

New negative regulators of interferon (IFN) signaling, preferably with tissue specificity, are needed to develop therapeutic means to enhance the efficacy of type I IFNs (IFN-α/ß) and reduce their side effects. We conducted cell-based screening for IFN signaling enhancer and discovered that luteolin, a natural flavonoid, sensitized the antiproliferative effect of IFN-α in hepatoma HepG2 cells and cervical carcinoma HeLa cells. Luteolin promoted IFN-ß-induced Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway activation by enhancing the phosphorylation of Jak1, Tyk2, and STAT1/2, thereby promoting STAT1 accumulation in the nucleus and endogenous IFN-α-regulated gene expression. Of interest, inhibition of phosphodiesterase (PDE) abolished the effect of IFN-ß and luteolin on STAT1 phosphorylation. Luteolin also increased the cAMP-degrading activity of PDE bound with type I interferon receptor 2 (IFNAR2) and decreased the intracellular cAMP level, indicating that luteolin may act on the JAK/STAT pathway via PDE. Protein kinase A (PKA) was found to negatively regulate IFN-ß-induced JAK/STAT signaling, and its inhibitory effect was counteracted by luteolin. Pull-down and immunoprecipitation assays revealed that type II PKA interacted with IFNAR2 via the receptor for activated C-kinase 1 (RACK-1), and such interaction was inhibited by luteolin. Src homology domain 2 containing tyrosine phosphatase-2 (SHP-2) was further found to mediate the inhibitory effect of PKA on the JAK/STAT pathway. These data suggest that PKA/PDE-mediated cAMP signaling, integrated by RACK-1 to IFNAR2, may negatively regulate IFN signaling through SHP-2. Inhibition of this signaling may provide a new way to sensitize the efficacy of IFN-α/ß.

In silico simulations of STAT1 and STAT3 inhibitors predict SH2 domain cross-binding specificity.

Signal transducers and activators of transcription (STATs) comprise a family of transcription factors that are structurally related and which participate in signaling pathways activated by cytokines, growth factors and pathogens. Activation of STAT proteins is mediated by the highly conserved Src homology 2 (SH2) domain, which interacts with phosphotyrosine motifs for specific contacts between STATs and receptors and for STAT dimerization. By generating new models for human (h)STAT1, hSTAT2 and hSTAT3 we applied comparative in silico docking to determine SH2-binding specificity of the STAT3 inhibitor stattic, and of fludarabine (STAT1 inhibitor). Thus, we provide evidence that by primarily targeting the highly conserved phosphotyrosine (pY+0) SH2 binding pocket stattic is not a specific hSTAT3 inhibitor, but is equally effective towards hSTAT1 and hSTAT2. This was confirmed in Human Micro-vascular Endothelial Cells (HMECs) in vitro, in which stattic inhibited interferon-α-induced phosphorylation of all three STATs. Likewise, fludarabine inhibits both hSTAT1 and hSTAT3 phosphorylation, but not hSTAT2, by competing with the highly conserved pY+0 and pY-X binding sites, which are less well-preserved in hSTAT2. Moreover we observed that in HMECs in vitro fludarabine inhibits cytokine and lipopolysaccharide-induced phosphorylation of hSTAT1 and hSTAT3 but does not affect hSTAT2. Finally, multiple sequence alignment of STAT-SH2 domain sequences confirmed high conservation between hSTAT1 and hSTAT3, but not hSTAT2, with respect to stattic and fludarabine binding sites. Together our data offer a molecular basis that explains STAT cross-binding specificity of stattic and fludarabine, thereby questioning the present selection strategies of SH2 domain-based competitive small inhibitors.

Hepatitis B virus polymerase impairs interferon-α-induced STA T activation through inhibition of importin-α5 and protein kinase C-δ.

UNLABELLED: Treatment with exogenous interferon (IFN)-α is not effective in the majority of patients with chronic hepatitis B virus (HBV) infection. Recent evidence suggests that HBV has evolved strategies to block the nuclear translocation of signal transducer and activator of transcription (STAT) 1 to limit IFN-α-induced cellular antiviral responses. However, it remains unclear whether STAT1 translocation is impaired in chronic hepatitis B patients and what mechanisms are involved. Here we report that the expression of HBV polymerase (Pol) in human hepatic cell lines inhibited induction of IFN-stimulated genes and resulted in a weakened antiviral activity of IFN-α. Ectopic expression of Pol suppressed IFN-α-induced STAT1 serine 727 phosphorylation and STAT1/2 nuclear accumulation, whereas STAT1 tyrosine 701 phosphorylation, and STAT1-STAT2 heterodimer formation were not affected. Further studies demonstrated that Pol interacted with the catalytic domain of protein kinase C-δ (PKC-δ) and perturbed PKC-δ phosphorylation and its association with STAT1, which resulted in the suppression of STAT1 Ser727 phosphorylation. Moreover, Pol was found to interfere with nuclear transportation of STAT1/2 by competitively binding to the region of importin-α5 required for STAT1/2 recruitment. Truncation analysis suggested that the terminal protein and RNase H domains of Pol were able to bind to PKC-δ and importin-α5, respectively, and were responsible for the inhibition of IFN-α signaling. More importantly, the inhibition of STAT1 and PKC-δ phosphorylation were confirmed in a hydrodynamic-based HBV mouse model, and the blockage of IFN-α-induced STAT1/2 nuclear translocation was observed in HBV-infected cells from liver biopsies of chronic HBV patients. CONCLUSIONS: These results demonstrate a role for Pol in HBV-mediated antagonization of IFN-α signaling and provide a possible molecular mechanism by which HBV resists the IFN therapy and maintains its persistence.

HSV-2 inhibits type-I interferon signaling via multiple complementary and compensatory STAT2-associated mechanisms.

Type-I interferon (IFN)-mediated responses are a crucial first line of defense against viral infections and are critical for generating both innate and adaptive immunity. Therefore, viruses have necessarily evolved mechanisms to impede the IFN response. HSV-2 was found to completely abolish type-1 IFN-mediated signaling via multiple STAT2-associated mechanisms. Although the extent and kinetics of this inactivation were indistinguishable between the various cell-lines examined, there were distinct differences in the mechanisms HSV-2 employed to subvert IFN-signaling among the cell-lines. These mechanistic differences could be segregated into two categories dependent on the phase of the HSV replicative cycle that was responsible for this inhibition: (1) early phase-inhibited cells which exhibited abrogation of IFN-signaling prior to viral DNA replication; (2) late phase-inhibited cells where early phase inhibition mechanisms were not functional, but viral functions expressed following DNA replication compensated for their ineffectiveness. In early phase-inhibited cells, HSV-2 infection targeted STAT2 protein for proteosomal degradation and prevented de novo expression of STAT2 by degrading its mRNA. In contrast, HSV-2 infected late phase-inhibited cells exhibited no apparent changes in STAT2 transcript or protein levels. However, in these cells STAT2 was not activated by phosphorylation and failed to translocate to the cell nucleus, thereby preventing transactivation of antiviral genes. In primary human fibroblasts, HSV-2 failed to fully degrade STAT2 and therefore, both early and late phase mechanisms functioned cooperatively to subvert IFN-mediated antiviral gene expression. Taken together, these results indicate the importance that HSV-2 has assigned to STAT2, investing significant genomic currency throughout its replicative lifecycle for continuous targeted destruction and inhibition of this protein.

Negative regulation of the type I interferon signaling pathway by synthetic Toll-like receptor 7 ligands.

Ten Toll-like receptor (TLR) family members have been reported in humans. Here, the endoplasmatic receptors TLR9, TLR8, TLR7, and TLR3 respond to nucleic acids and derivatives or to small molecules (TLR7 and 8). Another cytoplasmic RNA receptor, retinoic acid inducible gene I (RIG-I), is stimulated by 5' triphosphate double-stranded RNA. We discovered that TLR7 small-molecule agonists inhibit nucleic acid-mediated TLR3, TLR7, TLR9, or RIG-I-dependent interferon-α (IFN-α) immune response. Other cytokines and chemokines stimulated by nucleic acid agonists remained unaffected. The observed blockage of TLR3, TLR7, TLR9, and RIG-I-mediated IFN-α response appears to be driven by a competitive mechanism at the type I IFN pathway. Besides type I IFN, IFN response genes such as IFIT-1, Mx1, OAS1, or IRF7 were affected, which indicates that the key element driving the inhibition is located in the type I IFN pathway. Indeed, the heterotrimeric complex formation of phosphor-signal transducer and activator of transcription factor 1 (STAT1), phosphor-STAT2, and IRF9 (called ISGF3, IFN-stimulated gene factor 3) is inhibited through the TLR7 small-molecule agonists by phosphor-STAT2 blockage. These findings provide novel insights into the use of synthetic TLR7 or TLR7/8 small molecules as ligands for immune activation and suppression.

The C proteins of human parainfluenza virus type 1 block IFN signaling by binding and retaining Stat1 in perinuclear aggregates at the late endosome.

Interferons (IFNs) play a crucial role in the antiviral immune response. Whereas the C proteins of wild-type human parainfluenza virus type 1 (WT HPIV1) inhibit both IFN-ß induction and signaling, a HPIV1 mutant encoding a single amino acid substitution (F170S) in the C proteins is unable to block either host response. Here, signaling downstream of the type 1 IFN receptor was examined in Vero cells to define at what stage WT HPIV1 can block, and F170S HPIV1 fails to block, IFN signaling. WT HPIV1 inhibited phosphorylation of both Stat1 and Stat2, and this inhibition was only slightly reduced for F170S HPIV1. Degradation of Stat1 or Stat2 was not observed. The HPIV1 C proteins were found to accumulate in the perinuclear space, often forming large granules, and co-localized with Stat1 and the cation-independent mannose 6-phosphate receptor (M6PR) that is a marker for late endosomes. Upon stimulation with IFN-ß, both the WT and F170S C proteins remained in the perinuclear space, but only the WT C proteins prevented Stat1 translocation to the nucleus. In addition, WT HPIV1 C proteins, but not F170S C proteins, co-immunoprecipitated both phosphorylated and unphosphorylated Stat1. Our findings suggest that the WT HPIV1 C proteins form a stable complex with Stat1 in perinuclear granules that co-localize with M6PR, and that this direct interaction between the WT HPIV1 C proteins and Stat1 is the basis for the ability of HPIV1 to inhibit IFN signaling. The F170S mutation in HPIV1 C did not prevent perinuclear co-localization with Stat1, but apparently weakened this interaction such that, upon IFN stimulation, Stat1 was translocated to the nucleus to induce an antiviral response.

IL28B inhibits hepatitis C virus replication through the JAK-STAT pathway.

BACKGROUND & AIMS: The combination of pegylated interferon (IFN) α and ribavirin (RBV) is the standard therapy for patients with chronic HCV infection. However, it produces a sustained virologic response (SVR) in only half of the treated individuals and is associated with significant side effects. Recently, several single-nucleotide polymorphisms (SNPs) near the IL28B locus, also known as IFNλ3, were identified to be strong predictors of SVR in patients receiving PEG-IFN and RBV. We sought to determine whether IL28B was capable of inhibiting HCV replication and to determine the pathway by which IL28B exhibits anti-HCV activity. METHODS: Using the full-length HCV replicon OR6 and the infectious HCV clones JFH1 and Jc1, we assessed the anti-HCV effect of IL28B on HCV and characterized the key steps of the JAK-STAT pathway by real time PCR, luciferase assay, and Western blot. Finally, we evaluated the anti-HCV effect of IL28B in the presence of JAK-STAT pathway inhibitors such as blocking antibodies, a pharmacological inhibitor, and siRNAs. RESULTS: We found that IL28B inhibits HCV replication in a dose- and time-dependent manner. Like IFNα, IL28B induces the phosphorylation of STAT1 and STAT2, ISRE-driven transcription, and expression of known ISGs. The anti-HCV effects of IL28A, IL28B, and IL29 were abrogated by an IL10R2 blocking antibody, a pharmacological inhibitor of JAK1/TYK2, and by siRNA against IL28R1, STAT1, STAT2, and IRF9. CONCLUSIONS: Our data demonstrate that IL28A, IL28B, and IL29 signal through the JAK-STAT pathway to inhibit HCV. These data suggest possible applications of new approaches in HCV treatment.

Two domains of the V protein of virulent canine distemper virus selectively inhibit STAT1 and STAT2 nuclear import.

Canine distemper virus (CDV) causes in dogs a severe systemic infection, with a high frequency of demyelinating encephalitis. Among the six genes transcribed by CDV, the P gene encodes the polymerase cofactor protein (P) as well as two additional nonstructural proteins, C and V; of these V was shown to act as a virulence factor. We investigated the molecular mechanisms by which the P gene products of the neurovirulent CDV A75/17 strain disrupt type I interferon (IFN-alpha/beta)-induced signaling that results in the establishment of the antiviral state. Using recombinant knockout A75/17 viruses, the V protein was identified as the main antagonist of IFN-alpha/beta-mediated signaling. Importantly, immunofluorescence analysis illustrated that the inhibition of IFN-alpha/beta-mediated signaling correlated with impaired STAT1/STAT2 nuclear import, whereas the phosphorylation state of these proteins was not affected. Coimmunoprecipitation assays identified the N-terminal region of V (VNT) responsible for STAT1 targeting, which correlated with its ability to inhibit the activity of the IFN-alpha/beta-mediated antiviral state. Conversely, while the C-terminal domain of V (VCT) could not function autonomously, when fused to VNT it optimally interacted with STAT2 and subsequently efficiently suppressed the IFN-alpha/beta-mediated signaling pathway. The latter result was further supported by a single mutation at position 110 within the VNT domain of CDV V protein, resulting in a mutant that lost STAT1 binding while retaining a partial STAT2 association. Taken together, our results identified the CDV VNT and VCT as two essential modules that complement each other to interfere with the antiviral state induced by IFN-alpha/beta-mediated signaling. Hence, our experiments reveal a novel mechanism of IFN-alpha/beta evasion among the morbilliviruses.

Dengue virus NS5 inhibits interferon-alpha signaling by blocking signal transducer and activator of transcription 2 phosphorylation.

Type I interferons (interferon [IFN]-alpha/beta) are key mediators of innate antiviral responses. Inhibition of IFN-mediated signal transduction by dengue viruses (DENVs), mosquito-borne flaviviruses of immense global health importance, probably plays a crucial role in determining the outcome of the virus-host interaction. Understanding the molecular basis of IFN antagonism by DENV would therefore provide critical insight into disease pathogenesis and new opportunities for development of antiviral therapies and rationally attenuated vaccines. Here we examine the effects of expression of DENV nonstructural proteins on cellular IFN responses. We show that expression of nonstructural protein 5 (NS5) alone inhibits IFN-alpha, but not IFN-gamma, signaling. Expression of the polymerase domain of NS5 is sufficient to inhibit IFN-alpha signaling. NS5 binds signal transducer and activator of transcription 2 (STAT2) and inhibits its phosphorylation. NS5 alone did not, however, induce degradation of STAT2, which occurs when all nonstructural proteins are expressed together. We conclude that DENV NS5 is a potent and specific type I IFN antagonist.