Rotaviruses, members of the Reoviridae family, are the most common causative agents of severe gastroenteritis in infants and young children, worldwide. Rotavirus infection is a major health and economic burden to many countries, and the avail- ability of a prophylactic vaccine that provides protection against severe disease remains a high global priority (23). Two oral vaccine candidates have recently been licensed for hu- mans, Rotarix and Rotateq (25), both of which are based on live attenuated viruses. Rotaviruses are nonenveloped viruses with segmented double-stranded RNA genomes that encode six structural proteins (VP1 to 6) and six nonstructural proteins (NSP1 to 6). Although the functions of several of the NSPs remain obscure, a function was recently ascribed to rotavirus NSP1. Using a yeast two-hybrid screen, it was shown that NSP1 interacts with interferonregulatoryfactor3 (IRF-3) and that regions in the C-terminal domain of NSP1 were important for mediating this interaction (26). IRF-3 is required for the early production of type I interferons (IFN) in most cell types; thus, this finding suggested that NSP1 plays a role in suppressing the host innate immune response. More recent studies using rota- virus mutants with C-terminal truncations of NSP1 demon- strated that viruses encoding wild-type (wt) NSP1, but not viruses encoding C-terminally truncated NSP1, caused rapid and profound degradation of IRF-3 in MA104 cells (7). Inter-
ABSTRACT Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent for Kaposi’s sarcoma (KS), which is one of the most common HIV-associated neoplasms. The endothelium is the thin layer of squamous cells where vascular blood endothelial cells (BECs) line the interior surface of blood vessels and lymphatic endothelial cells (LECs) are in direct contact with lymphatic vessels. The KS lesions contain a prominent compart- ment of neoplastic spindle morphology cells that are closely related to LECs. Further- more, while KSHV can infect both LECs and BECs in vitro, its infection activates genetic programming related to lymphatic endothelial cell fate, suggesting that lymphangio- genic pathways are involved in KSHV infection and malignancy. Here, we report for the ﬁrst time that viral interferonregulatoryfactor3 (vIRF3) is readily detected in over 40% of KS lesions and that vIRF3 functions as a proangiogenic factor, inducing hypersprout- ing formation and abnormal growth in a LEC-speciﬁc manner. Mass spectrometry analy- sis revealed that vIRF3 interacted with histone deacetylase 5 (HDAC5), which is a signal- responsive regulator for vascular homeostasis. This interaction blocked the phosphorylation-dependent cytosolic translocation of HDAC5 and ultimately altered global gene expression in LECs but not in BECs. Consequently, vIRF3 robustly induced spindle morphology and hypersprouting formation of LECs but not BECs. Finally, KSHV infection led to the hypersprouting formation of LECs, whereas infection with a ΔvIRF3 mutant did not do so. Collectively, our data indicate that vIRF3 alters global gene ex- pression and induces a hypersprouting formation in an HDAC5-binding-dependent and LEC-speciﬁc manner, ultimately contributing to KSHV-associated pathogenesis.
Interferon production and apoptosis in virus-infected cells are necessary to prevent progeny virus produc- tion and to eliminate infected cells. Paramyxovirus infection induces apoptosis through interferonregulatoryfactor3 (IRF-3), but the exact mechanism of how IRF-3 functions is unknown. We show that IRF-3 is involved in the transcriptional induction of TRAIL, a key player in the apoptosis pathway. IRF-3 upregulates TRAIL transcription following viral infection and binds an interferon-stimulated response element in the TRAIL promoter. The mRNA for TRAIL and its receptor, DR5, are induced following viral infection. These studies identify TRAIL as a novel IRF-3 transcriptional target.
ylation at site I may also be important for IRF3 activation (41). We thus generated IRF3-5E/E, in which all five Ser/Thr residues in site II were mutated to Glu (S394E, S396E, S400E, T402E, and S403E) with an additional S384E substitution at site I. Because S385 in human IRF3 was reported to have an autoinhibitory function rather than an activating function (38), the corresponding S383 in porcine IRF3 was not changed to Glu. IRF3-5E/E was found to be both a monomer and a dimer in solution, but the construct was unstable and degraded rather rapidly. To stabilize the IRF3 dimer, we coexpressed a CBP peptide along with IRF3-5E/E. Although CBP is a large ⬃ 250-kDa protein, its interaction with IRF3 is me- diated by the small interferon response binding domain (IBiD; 46 residues) (43). The binding of IBiD to the phosphomimetic mu- tants of IRF3 has been shown to induce the dimerization of IRF3 in solution (38). Thus, a 48-amino-acid domain of CBP (referred to here as CBP 48 ) was used for coexpression. CBP binds to both
Human noroviruses (HuNoV) are the major cause of epidemic, nonbacterial gastroenteritis in the world. The short course of HuNoV-induced symptoms has implicated innate immunity in control of norovirus (NoV) infection. Studies using murine no- rovirus (MNV) confirm the importance of innate immune responses during NoV infection. Type I alpha and beta interferons (IFN- ␣ / ␤ ) limit HuNoV replicon function, restrict MNV replication in cultured cells, and control MNV replication in vivo. Therefore, the cell types and transcription factors involved in antiviral immune responses and IFN-␣/␤-mediated control of NoV infection are important to define. We used mice with floxed alleles of the IFNAR1 chain of the IFN-␣/␤ receptor to identify cells expressing lysozyme M or CD11c as cells that respond to IFN- ␣ / ␤ to restrict MNV replication in vivo. Furthermore, we show that the transcription factors IRF-3 and IRF-7 work in concert to initiate unique and overlapping antiviral responses to restrict MNV replication in vivo. IRF-3 and IRF-7 restrict MNV replication in both cultured macrophages and dendritic cells, are required for induction of IFN-␣/␤ in macrophages but not dendritic cells, and are dispensable for the antiviral effects of IFN- ␣ / ␤ that block MNV replication. These studies suggest that expression of the IFN- ␣ / ␤ receptor on macrophages/neutrophils and dendritic cells, as well as of IRF-3 and IRF-7, is critical for innate immune responses to NoV infection.
Cell culture, transfection, and luciferase assays. rtTA-Neo and rtTA–IRF-3 5D Jurkat cells (16) were grown in RPMI 1640 medium containing 10% heat- inactivated calf serum, glutamine, antibiotics, 2.5 g of puromycin/ml, and 400 g of G418 (Gibco)/ml. Cells were induced with doxycycline (DOX) at 1 g/ml for the indicated time in the presence of neutralizing antibodies against IFN-␣/␤ (Sigma). HEC1B cells (American Type Culture Collection) were grown in min- imal essential medium supplemented with 10% heat-inactivated fetal bovine serum, nonessential amino acids, sodium pyruvate, glutamine, and antibiotics and then transfected with Fugene reagent according to the manufacturer’s in- structions (Roche). For luciferase assays, subconfluent cells in 24-well plates were transfected with 10 ng of pRLTK reporter (Renilla luciferase, internal control), 100 ng of pGL3 reporter and the indicated amounts of expression plasmids. Cells were assayed for reporter gene activities after 24 h. For immu- noblot analysis, cells in 60-mm plates were transfected with a total of 10 g of DNA constructs and collected after 36 to 48 h. Where indicated, cells were treated with Sendai virus (40 hemagglutinating units [HAU]/ml) for 2 h in serum-free medium and further cultured for the indicated time in complete medium.
The accessory HIV protein Vpu inhibits a number of cellular pathways that trigger host innate restriction mechanisms. HIV Vpu-mediated degradation of tetherin allows efficient particle release and hampers the activation of the NF- B pathway thereby limiting the expression of proinflammatory genes. In addition, Vpu reduces cell surface expression of several cellular molecules such as newly synthesized CD4. However, the role of HIV Vpu in regulating the type 1 interferon response to viral infection by degradation of the interferonregulatoryfactor3 (IRF3) has been subject of conflicting reports. We therefore systematically in- vestigated the expression of IRF3 in primary CD4 ⴙ T cells and macrophages infected with HIV at different time points. In addi- tion, we also tested the ability of Vpu to interfere with innate immune signaling pathways such as the NF- B and the IRF3 path- ways. We report here that HIV Vpu failed to degrade IRF3 in infected primary cells. Moreover, we observed that HIV NL4.3 Vpu had no effect on IRF3-dependent gene expression in reporter assays. On the other hand, HIV NL4.3 Vpu downmodulated NF- B-dependent transcription. Mutation of two serines (positions 52 and 56) involved in the binding of NL4.3 Vpu to the ␤ TrCP ubiquitin ligase abolishes its ability to inhibit NF- B activity. Taken together, these results suggest that HIV Vpu regulates anti- viral innate response in primary human cells by acting specifically on the NF-B pathway.
Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus termed SARS-CoV. We and others have previously shown that the replication of SARS-CoV can be suppressed by exogenously added interferon (IFN), a cytokine which is normally synthesized by cells as a reaction to virus infection. Here, we demonstrate that SARS-CoV escapes IFN-mediated growth inhibition by preventing the induction of IFN- ␤ . In SARS-CoV-infected cells, no endogenous IFN- ␤ transcripts and no IFN- ␤ promoter activity were detected. Nevertheless, the transcription factorinterferonregulatoryfactor3 (IRF-3), which is essential for IFN- ␤ promoter activity, was transported from the cytoplasm to the nucleus early after infection with SARS-CoV. However, at a later time point in infection, IRF-3 was again localized in the cytoplasm. By contrast, IRF-3 remained in the nucleus of cells infected with the IFN-inducing control virus Bunyamwera delNSs. Other signs of IRF-3 activation such as hyperphosphorylation, homodimer formation, and recruitment of the coactivator CREB-binding protein (CBP) were found late after infection with the control virus but not with SARS-CoV. Our data suggest that nuclear transport of IRF-3 is an immediate-early reaction to virus infection and may precede its hyperphosphorylation, homodimer formation, and binding to CBP. In order to escape activation of the IFN system, SARS-CoV appears to block a step after the early nuclear transport of IRF-3.
two-step model of IRF3 activation. First, Sarkar et al. observed that following poly(I:C)-mediated phosphorylation, dimeriza- tion, and nuclear translocation of IRF3, a second posttransla- tional modification on IRF3 via phosphoinositide-3 (PI3) ki- nase was required for full activation of IRF3 and subsequent ISG induction (25). In addition, we previously reported that antiviral response induction mediated by virus particle entry requires a novel phosphoinositide-3 kinase family member to activate IRF3 following its dimerization and nuclear translo- cation (15). In vitro biochemical analyses using purified IRF3 and TBK1 revealed that TBK1 was solely responsible for the phosphorylation of serine and threonine residues in site 2 (amino acids 396 to 405) and subsequent release of autoinhi- bition on IRF3 and interaction with CBP in the absence of dimerization, suggesting that a second kinase may be respon- sible for dimerization and full activation of IRF3 (18). Fur- thermore, Clement et al. described a mechanism in which phosphorylation of Ser396 on IRF3 enhances the antiviral re- sponse by exposing Ser339 for subsequent phosphorylation by an unidentified proline-directed kinase, resulting in hyper- phosphorylation, dimerization and association with CBP (5). Taken together, these data strongly suggest the existence of other protein kinases that function to further activate the IRF3-mediated antiviral response after initial phosphorylation by the virus-activated kinases, TBK1/IKKε. Regardless of the likelihood of these alternative pathways, however, current techniques are insufficient to monitor and characterize the subsequent modification profile of IRF3 and infer biological activity.
The type I interferon (IFN) response requires the coordinated activation of the latent transcription factors NF- B, interferonregulatoryfactor3 (IRF-3), and ATF-2, which in turn activate transcription from the IFN- ␤ promoter. Synthesis and subsequent secretion of IFN- ␤ activate the Jak/STAT signaling pathway, resulting in the transcriptional induction of the full spectrum of antiviral gene products. We utilized high-density microarrays to examine the transcriptional response to rhinovirus type 14 (RV14) infection in HeLa cells, with particular emphasis on the type I interferon response and production of IFN- ␤ . We found that, although RV14 infection results in altered levels of a wide variety of host mRNAs, induction of IFN- ␤ mRNA or activation of the Jak/STAT pathway is not seen. Prior work has shown, and our results have confirmed, that NF- B and ATF-2 are activated following infection. Since many viruses are known to target IRF-3 to inhibit the induction of IFN- ␤ mRNA, we analyzed the status of IRF-3 in infected cells. IRF-3 was translocated to the nucleus and phosphorylated in RV14-infected cells. Despite this apparent activation, very little homodimerization of IRF-3 was evident following infection. Similar results in A549 lung alveolar epi- thelial cells demonstrated the biological relevance of these findings to RV14 pathogenesis. In addition, prior infection of cells with RV14 prevented the induction of IFN- ␤ mRNA following treatment with double-stranded RNA, indicating that RV14 encodes an activity that specifically inhibits this innate host defense pathway. Collectively, these results indicate that RV14 infection inhibits the host type I interferon response by inter- fering with IRF-3 activation.
Apoptosis is a pathological hallmark of encephalitis and myocarditis caused by reovirus in newborn mice. In cell culture models, the antiviral transcription factorinterferonregulatoryfactor3 (IRF-3) enhances reovirus-induced apoptosis following activation via retinoic acid inducible gene I and interferon promoter- stimulating factor 1. To determine the role of IRF-3 in reovirus disease, we infected newborn IRF-3 ⴙ / ⴙ and IRF-3 ⴚ / ⴚ mice perorally with mildly virulent strain type 1 Lang (T1L) and fully virulent strain type 3 SA ⴙ (T3SA ⴙ ) and monitored infected animals for survival. Both wild-type and IRF-3 ⴚ / ⴚ mice succumbed with equivalent frequencies to infection with T3SA ⴙ . However, the absence of IRF-3 was associated with signifi- cantly decreased survival rates following infection with T1L. The two virus strains achieved similar peak titers in IRF-3 ⴙ / ⴙ and IRF-3 ⴚ / ⴚ mice in the intestine, brain, heart, liver, and spleen. However, by day 12 postin- oculation, titers in all organs examined were 10- to 100-fold higher in IRF-3 ⴚ / ⴚ mice than those in wild-type mice. Increased titers were associated with marked pathological changes in all organs examined, especially in the heart, where absence of IRF-3 resulted in severe myocarditis. Cellular and humoral immune responses were equivalent in wild-type and IRF-3 ⴚ / ⴚ animals, suggesting that IRF-3 functions independently of the adaptive immune response to enhance reovirus clearance. Thus, IRF-3 serves to facilitate virus clearance and prevent tissue injury in response to reovirus infection.
FIG. 2. Effect on delNS1 virus-induced nuclear accumulation of hIRF-3 in HEC-1b cells by transient transfection of influenza virus NS1 proteins. (A) Coverslips coated with HEC-1b cells were transfected with a plasmid expressing the wild-type influenza virus NS1 protein [pCAGGS-NS1(SAM)] or an NS1 RNA binding mutant [pCAGGS-NS1-R38AK41A(SAM)] (mut) or with an empty vector [pCAGGS]. Both NS1 open reading frames contain a splice accep- tor mutation (SAM) to ensure that spliced NEP mRNA is not produced. (The NEP is a second protein normally produced by alternative splicing from the influenza A virus NS gene.) Except with PR8-infected cells and with the no-DNA control, the amount of DNA in all transfected cells was standardized to 0.5 g per coverslip using pCAGGS. Coverslips were transfected with either 0, 0.02, 0.1, or 0.5 g of pCAGGS-NS1(SAM) or with 0.5 g of pCAGGS-NS1- R38AK41A(SAM) and infected with delNS1 virus at an MOI of 1 for 8 h. Fixed cells were stained with antibodies directed against the NS1 protein and endog- enous hIRF-3. Cells were scored according to NS1 expression and hIRF-3 dis- tribution. Bars represent the mean cell number from between 5 and 13 random fields per experimental condition. Asterisks indicate that there was no NS1 expression. (B) HEC-1b cells were transfected with 0.1 g of pCAGGS- NS1(SAM) and infected with delNS1 virus as indicated for panel A. Cells were stained with a rabbit polyclonal antibody against NS1 and a mouse monoclonal antibody against hIRF-3. Anti-rabbit IgG (fluorescein isothiocyanate) and anti- mouse IgG (Texas Red) were used as secondary antibodies. In the top panel, arrows indicate two cells expressing the NS1 protein (green). The surrounding cells do not express detectable levels of NS1. In the bottom panel, arrows indicate the same two cells, demonstrating a lack of nuclear accumulation of hIRF-3. Surrounding cells which did not express the NS1 protein show a bright, nuclear accumulation of endogenous IRF-3. (C
(DBD), which is characterized by the presence of five trypto- phan repeats. The C-terminal part of these proteins is distinct and may contain the IRF-association domain (IAD) that facil- itates the formation of IRF homo- or heterodimers and inter- action with other transcription factors. IRFs can function as transcriptional activators or repressors. Three of these IRFs (IRF-3, -5, and -7) play a critical role in the innate antiviral response (20–22). Interferonregulatoryfactor3 (IRF-3) acts as a direct transducer of virus-mediated signaling, and together with IRF-7 controls transcriptional activation of alpha/beta IFN (IFN- ␣ / ␤ ) genes (23–26). All characterized IRFs contain nuclear localization signals that allow their translocation to the nucleus. However, during homeostasis certain IRFs reside in the cytoplasm of the cell and translocate to the nucleus in re- sponse to viral infection, where they participate in the tran- scription of type I IFN genes, inflammatory cytokines and chemokines, and interferon-stimulated genes (ISGs). The function of IRFs is not limited to the innate immune response, as they also play a role in the modulation of cell growth, differ- entiation, and apoptosis. Thus, deregulation of these functions may lead to tumorigenesis (20, 27–30). Indeed, KSHV-en- coded viral homologues of IRFs (designated vIRFs) (Fig. 1A and B) have been identified as effective inhibitors of interferon signaling and modulators of cellular oncogenic pathways (Fig. 2 and Fig. 3). These functions of vIRFs may contribute to KSHV-associated pathogenesis.
ABSTRACT The impact of mosquito-borne ﬂavivirus infections worldwide is signiﬁ- cant, and many critical aspects of these viruses’ biology, including virus-host interac- tions, host cell requirements for replication, and how virus-host interactions impact pathology, remain to be fully understood. The recent reemergence and spread of ﬂaviviruses, including dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV), highlight the importance of performing basic research on this important group of pathogens. MicroRNAs (miRNAs) are small, noncoding RNAs that modulate gene expression posttranscriptionally and have been demonstrated to regulate a broad range of cellular processes. Our research is focused on identifying pro- and antiﬂaviviral miRNAs as a means of characterizing cellular pathways that support or limit viral replication. We have screened a library of known human miRNA mimics for their effect on the replication of three ﬂaviviruses, DENV, WNV, and Japanese en- cephalitis virus (JEV), using a high-content immunoﬂuorescence screen. Several fami- lies of miRNAs were identiﬁed as inhibiting multiple ﬂaviviruses, including the miRNA miR-34, miR-15, and miR-517 families. Members of the miR-34 family, which have been extensively characterized for their ability to repress Wnt/ ␤ -catenin signal- ing, demonstrated strong antiﬂaviviral effects, and this inhibitory activity extended to other viruses, including ZIKV, alphaviruses, and herpesviruses. Previous research suggested a possible link between the Wnt and type I interferon (IFN) signaling pathways. Therefore, we investigated the role of type I IFN induction in the antiviral effects of the miR-34 family and conﬁrmed that these miRNAs potentiate interferonregulatoryfactor3 (IRF3) phosphorylation and translocation to the nucleus, the in- duction of IFN-responsive genes, and the release of type I IFN from transfected cells. We further demonstrate that the intersection between the Wnt and IFN signaling pathways occurs at the point of glycogen synthase kinase 3 ␤ (GSK3 ␤ )–TANK-binding kinase 1 (TBK1) binding, inducing TBK1 to phosphorylate IRF3 and initiate down- stream IFN signaling. In this way, we have identiﬁed a novel cellular signaling net- work with a critical role in regulating the replication of multiple virus families. These ﬁndings highlight the opportunities for using miRNAs as tools to discover and char- acterize unique cellular factors involved in supporting or limiting virus replication, opening up new avenues for antiviral research.
BGLF4 kinase is a virion-associated kinase and is expressed at the early stage of the lytic cycle (36, 69). Several viral and cellular substrates including BMRF1, BZLF1, EBNA2, EBNA-LP, and translation factor EF-1 ␦ were found to be phosphorylated by BGLF4 at Cdk1 target sites (2, 38, 39, 72, 74). BGLF4 colocalizes with the viral DNA polymerase pro- cessivity factor BMRF1 at the viral DNA replication compart- ment in virus-replicating cells and phosphorylates BMRF1 at multiple sites in vitro and in vivo (69, 72). BGLF4 kinase also phosphorylates lamin A/C to promote the reorganization of the nuclear lamina to facilitate virion maturation, and the knockdown of BGLF4 resulted in an accumulation of viral nucleocapsids in the nucleus, suggesting that BGLF4 may reg- ulate the process of nuclear egress in virus-replicating cells (23, 44). Our study also indicates that BGLF4 recruits the nucleo- tide excision repair protein XPC to the viral replication com- partment, enhancing viral DNA replication (48). The expres- sion of BGLF4 alone induces premature chromosome condensation and phosphorylates the cellular replication ori- gin binding complex MCM4-MCM6-MCM7, leading to an in- hibition of its helicase activities (41, 43). Both events suggest the ability of BGLF4 to inhibit cellular DNA replication and thus save resources for efficient viral DNA replication.
A second class of cytosolic DNA sensors is represented by the absent in melanoma 2 protein (AIM2). Several laboratories iden- tified Aim2 as a DNA sensor that anchors formation of an AIM2- dependent inflammasome complex following DNA transfection (3, 9, 14, 36) (reviewed in reference 21). Aim2 binds DNA through the HIN-200 domain. It contains a C-terminal pyrin domain through which it binds ASC and demonstrates a cytosolic pres- ence, unlike other HIN-200 family members. Binding to ASC re- sults in caspase-1 activation, leading to conversion of pro-IL-1 ␤ to IL-1 ␤ . IL-1 ␤ activation contributes to autocrine-paracrine activa- tion through the IL-1 receptor. IL-1 receptor ligation triggers an MyD88-dependent TRAF6 cascade leading to activation of NF-B and cJUN transcription factors, stimulating proinflammatory genes (IL-6, tumor necrosis factor [TNF], IFN, and transforming growth factor ␤ [TGF␤]). Potent stimulation of inflammasome pathways leads to cell death through pyroptosis (reviewed in ref- erence 23). A recent report identified IFI16 as a nuclear DNA sensor for Kaposi’s sarcoma-associated herpesvirus (KSHV), leading to formation of an ASC-dependent inflammasome com- plex (20).
Abnormal prion protein (PrP Sc ) generated from the cellular isoform of PrP (PrP C ) is assumed to be the main or sole component of the pathogen, called prion, of transmissible spongiform encephalopathies (TSE). Because PrP is a host-encoded protein, ac- quired immune responses are not induced in TSE. Meanwhile, activation of the innate immune system has been suggested to partially block the progression of TSE; however, the mechanism is not well understood. To further elucidate the role of the in- nate immune system in prion infection, we investigated the function of interferonregulatoryfactor3 (IRF3), a key transcription factor of the MyD88-independent type I interferon (IFN) production pathway. We found that IRF3-deficient mice exhibited sig- nificantly earlier onset with three murine TSE strains, namely, 22L, FK-1, and murine bovine spongiform encephalopathy (mBSE), following intraperitoneal transmission, than with wild-type controls. Moreover, overexpression of IRF3 attenuated prion infection in the cell culture system, while PrP Sc was increased in prion-infected cells treated with small interfering RNAs (siRNAs) against IRF3, suggesting that IRF3 negatively regulates PrP Sc formation. Our findings provide new insight into the role
I nnate immunity against many viruses is initiated by the binding of viral RNA species to the repressor and helicase domains of RIG-I, a cytosolic protein that also contains two amino-terminal caspase activation recruitment domains (CARDs) (23). The RIG-I CARDs then associate with the CARDs of the mitochondrion- associated adaptor MAVS protein, thereby triggering the signaling pathway that leads to the activation of the interferonregulatoryfactor3 (IRF3) and NF-B transcription factors and the activation of interferon (IFN) transcription (6, 11, 16, 21). LGP2 has repres- sor and helicase domains similar to those of RIG-I but lacks CARDs and, hence, cannot trigger the signaling pathway that leads to the activation of IFN transcription (13, 22). The role of LGP2 in virus infection is controversial: it has been reported to either pos- itively or negatively affect the RIG-I-mediated activation of IFN transcription and the production of IFN in virus-infected cells (14, 15, 20).
The V protein of Sendai virus (SeV) suppresses innate immunity, resulting in enhancement of viral growth in mouse lungs and viral pathogenicity. The innate immunity restricted by the V protein is induced through activation of interferonregulatory fac- tor 3 (IRF3). The V protein has been shown to interact with melanoma differentiation-associated gene 5 (MDA5) and to inhibit beta interferon production. In the present study, we infected MDA5-knockout mice with V-deficient SeV and found that MDA5 was largely unrelated to the innate immunity that the V protein suppresses in vivo. We therefore investigated the target of the SeV V protein. We previously reported interaction of the V protein with IRF3. Here we extended the observation and showed that the V protein appeared to inhibit translocation of IRF3 into the nucleus. We also found that the V protein inhibited IRF3 activation when induced by a constitutive active form of IRF3. The V proteins of measles virus and Newcastle disease virus inhib- ited IRF3 transcriptional activation, as did the V protein of SeV, while the V proteins of mumps virus and Nipah virus did not, and inhibition by these proteins correlated with interaction of each V protein with IRF3. These results indicate that IRF3 is im- portant as an alternative target of paramyxovirus V proteins.
Viral infection activates interferonregulatoryfactor3 (IRF3), a cofactor for the induction of interferon- stimulated genes (ISGs). The role of IRF3 in the activation of ISGs by human cytomegalovirus (HCMV) is controversial despite the fact that HCMV has consistently been shown to induce ISGs during infection of fibroblasts. To address the function of IRF3 in HCMV-mediated ISG induction, we monitored ISG expression and global gene expression in HCMV-infected cells in which IRF3 function had been depleted by small interfering RNA or blocked by dominant negative IRF3. A specific reduction of ISG induction was observed, whereas other transcripts were unaffected. We therefore conclude that IRF3 specifically regulates ISG induc- tion during the initial phase of HCMV infection.