Viral Induction of the Zinc Finger Antiviral Protein Is IRF3-dependent but NF-κB-independent

The zinc finger antiviral protein (ZAP) is an interferon-stimulated gene that restricts the replication of retroviruses, alphaviruses, and filoviruses. Relatively little is known, however, regarding the detailed mechanism of ZAP induction during viral infections. We show that, although being inducible by either interferon or virus, expression of ZAP is more efficiently activated by virus than are several other classical interferon-stimulated genes and that viral induction of ZAP occurs under the direct control of interferon regulatory factor 3 (IRF3) independent of interferon paracrine/autocrine signaling. ZAP was up-regulated in cells unresponsive to type I and III interferons upon engagement of TLR3, retinoic inducible gene I/melanoma differentiation-associated gene 5 pathways, or ectopic expression of a constitutively active IRF3 mutant. Conversely, induction of ZAP by virus or dsRNA was severely impaired in cells expressing a dominant-negative mutant IRF3 and completely abrogated in cells lacking IRF3. In contrast to IRF3, ZAP induction was independent of NF-κB activity. Mutational analysis of the human ZAP promoter revealed that multiple interferon-stimulated response elements far distal to the transcription start site serve redundantly to control IRF3-dependent induction of ZAP transcription. Chromatin immunoprecipitation assays demonstrated that IRF3 selectively binds the distal interferon-stimulated response elements in human ZAP promoter following viral infection. Collectively, these data suggest that ZAP is a direct target gene of IRF3 action in cellular antiviral responses. The zinc finger antiviral protein (ZAP) is an interferon-stimulated gene that restricts the replication of retroviruses, alphaviruses, and filoviruses. Relatively little is known, however, regarding the detailed mechanism of ZAP induction during viral infections. We show that, although being inducible by either interferon or virus, expression of ZAP is more efficiently activated by virus than are several other classical interferon-stimulated genes and that viral induction of ZAP occurs under the direct control of interferon regulatory factor 3 (IRF3) independent of interferon paracrine/autocrine signaling. ZAP was up-regulated in cells unresponsive to type I and III interferons upon engagement of TLR3, retinoic inducible gene I/melanoma differentiation-associated gene 5 pathways, or ectopic expression of a constitutively active IRF3 mutant. Conversely, induction of ZAP by virus or dsRNA was severely impaired in cells expressing a dominant-negative mutant IRF3 and completely abrogated in cells lacking IRF3. In contrast to IRF3, ZAP induction was independent of NF-κB activity. Mutational analysis of the human ZAP promoter revealed that multiple interferon-stimulated response elements far distal to the transcription start site serve redundantly to control IRF3-dependent induction of ZAP transcription. Chromatin immunoprecipitation assays demonstrated that IRF3 selectively binds the distal interferon-stimulated response elements in human ZAP promoter following viral infection. Collectively, these data suggest that ZAP is a direct target gene of IRF3 action in cellular antiviral responses. IntroductionMammalian cells respond to viral infections by the rapid induction of a family of pleiotropic cytokines known as interferons (IFNs). 3The abbreviations used are: IFNinterferonZAPzinc finger antiviral proteinIRFinterferon regulatory factorIRF-EIRF elementNF-κBnuclear factor κBSTATsignal transducers and activators of transcriptionISGinterferon stimulated geneISREinterferon-stimulated response elementTLR3Toll-like receptor 3RIG-Iretinoic inducible gene IMDA5melanoma differentiation-associated gene 5MAVSmitochondrial antiviral signaling protein, also known as IPS-1/Cardif/VISATRIFToll-IL1 receptor homology domain containing adaptor inducing interferon-β, also known as TICAM1OASL2′,5′-oligoadenylate synthetase like geneChIPchromatin immunoprecipitationHCVhepatitis C virusSeVSendai virusdsdouble-strandedQ-PCRquantitative PCRDNdominant-negativehhumanIKKinhibitor of κB kinaseHAUhemagglutinin units. Once secreted, IFNs act in an autocrine and/or paracrine manner through the Jak-STAT pathway to stimulate the expression of hundreds of IFN-stimulated genes (ISGs), thereby establishing a cellular antiviral state. IFNs also exert critical immune regulatory functions, shaping subsequent adaptive immunity that helps clear the infection and establishes long term protective immunity (1.Borden E.C. Sen G.C. Uze G. Silverman R.H. Ransohoff R.M. Foster G.R. Stark G.R. Nat. Rev. Drug. Discov. 2007; 6: 975-990Crossref PubMed Scopus (860) Google Scholar, 2.Sen G.C. Annu. Rev. Microbiol. 2001; 55: 255-281Crossref PubMed Scopus (777) Google Scholar). The IFN family consists of three types, i.e. types I (mainly IFNβ and IFNα), II (IFNγ), and III (IFNλs). Although IFNγ production is restricted to cell types of lymphocyte origin, type I and III IFNs can be induced in most cell types (1.Borden E.C. Sen G.C. Uze G. Silverman R.H. Ransohoff R.M. Foster G.R. Stark G.R. Nat. Rev. Drug. Discov. 2007; 6: 975-990Crossref PubMed Scopus (860) Google Scholar). The importance of type I IFNs in the control of viral infection has been vividly demonstrated in IFNα/β receptor knock-out mice (3.Müller U. Steinhoff U. Reis L.F. Hemmi S. Pavlovic J. Zinkernagel R.M. Aguet M. Science. 1994; 264: 1918-1921Crossref PubMed Scopus (1974) Google Scholar).Upstream, IFNβ and IFNλ genes are transcriptionally activated by a latent cellular transcription factor, IFN regulatory factor 3 (IRF3) (4.Hiscott J. J. Biol. Chem. 2007; 282: 15325-15329Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 5.Osterlund P.I. Pietilä T.E. Veckman V. Kotenko S.V. Julkunen I. J. Immunol. 2007; 179: 3434-3442Crossref PubMed Scopus (246) Google Scholar, 6.Onoguchi K. Yoneyama M. Takemura A. Akira S. Taniguchi T. Namiki H. Fujita T. J. Biol. Chem. 2007; 282: 7576-7581Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). Predominantly residing in the cytoplasm in quiescent cells, IRF3 undergoes specific carboxyl-terminal serine phosphorylation mediated by the noncanonical IKK kinases, TBK1 or IKKϵ (7.Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. Latz E. Golenbock D.T. Coyle A.J. Liao S.M. Maniatis T. Nat. Immunol. 2003; 4: 491-496Crossref PubMed Scopus (2037) Google Scholar, 8.Sharma S. tenOever B.R. Grandvaux N. Zhou G.P. Lin R. Hiscott J. Science. 2003; 300: 1148-1151Crossref PubMed Scopus (1342) Google Scholar), following viral engagement of the toll-like receptor 3 (TLR3) and/or retinoic acid inducible gene I (RIG-I)/melanoma differentiation-associated gene 5 (MDA5) pathways. Phosphorylated IRF3 then dimerizes and translocates into the nucleus, where it associates with p300/cAMP-responsive element-binding protein-binding protein and binds to the IRF element (IRF-E) present in IFNβ and IFNλ gene promoters, activating their transcription (1.Borden E.C. Sen G.C. Uze G. Silverman R.H. Ransohoff R.M. Foster G.R. Stark G.R. Nat. Rev. Drug. Discov. 2007; 6: 975-990Crossref PubMed Scopus (860) Google Scholar, 4.Hiscott J. J. Biol. Chem. 2007; 282: 15325-15329Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). Because IRF-Es and the IFN-stimulated responsive elements (ISREs) present in some ISG promoters overlap in sequence, IRF3 is known to directly regulate a subset of ISGs, such as ISG56 and ISG54 (9.Grandvaux N. Servant M.J. tenOever B. Sen G.C. Balachandran S. Barber G.N. Lin R. Hiscott J. J. Virol. 2002; 76: 5532-5539Crossref PubMed Scopus (411) Google Scholar, 10.Peters K.L. Smith H.L. Stark G.R. Sen G.C. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 6322-6327Crossref PubMed Scopus (123) Google Scholar), independent of the IFN autocrine/paracrine loop. Given the pivotal role of IRF3 in antiviral innate immunity, IRF3 target genes may play important roles in the early host responses to viral infections. For this reason, identification of direct IRF3 target genes is important.During our investigation of hepatitis C virus (HCV)-mediated disruption of global antiviral gene expression, we observed that viral induction of hundreds of cellular genes (using Sendai virus (SeV) as an inducer) was blocked by HCV in Huh7 cells stably replicating genome length HCV RNAs. 4K. Li and S. M. Lemon, unpublished data. Because many of the virus-inducible genes were well known ISGs, it was unclear whether the disruption of their induction was a direct result of inhibition of IRF3 activation by HCV or secondary to the blockade of induction of type I/III IFNs, which signal through the JAK-STAT pathway to regulate the expression of these genes. In our efforts to determine the subset of genes whose transcription is directly controlled by IRF3, we identified the zinc finger antiviral protein (ZAP) as a novel IRF3 target gene. Previously known as an ISG, ZAP possesses antiviral activity against viruses of multiple families, including Retroviridae, Togaviridae, and Filoviridae (11.Gao G. Guo X. Goff S.P. Science. 2002; 297: 1703-1706Crossref PubMed Scopus (343) Google Scholar, 12.Müller S. Möller P. Bick M.J. Wurr S. Becker S. Günther S. Kümmerer B.M. J. Virol. 2007; 81: 2391-2400Crossref PubMed Scopus (151) Google Scholar, 13.Bick M.J. Carroll J.W. Gao G. Goff S.P. Rice C.M. MacDonald M.R. J. Virol. 2003; 77: 11555-11562Crossref PubMed Scopus (260) Google Scholar, 14.Zhang Y. Burke C.W. Ryman K.D. Klimstra W.B. J. Virol. 2007; 81: 11246-11255Crossref PubMed Scopus (188) Google Scholar, 15.MacDonald M.R. Machlin E.S. Albin O.R. Levy D.E. J. Virol. 2007; 81: 13509-13518Crossref PubMed Scopus (66) Google Scholar). The experimental studies reported here reveal that ZAP expression is directly regulated by IRF3 following virus infection or stimulation of cells with dsRNA or dsDNA and is not dependent on NF-κB. Although IFN can also stimulate ZAP expression, our data indicate that ZAP is a primary IRF3 response gene in the innate antiviral signaling pathway.DISCUSSIONZAP is an antiviral host factor that prevents cytoplasmic accumulation of viral RNAs by promoting their decay by the exosome (47.Guo X. Ma J. Sun J. Gao G. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 151-156Crossref PubMed Scopus (202) Google Scholar) and thereby restricts the replication of many RNA viruses, especially those within the families Retroviridae, Togaviridae, and Filoviridae (11.Gao G. Guo X. Goff S.P. Science. 2002; 297: 1703-1706Crossref PubMed Scopus (343) Google Scholar, 12.Müller S. Möller P. Bick M.J. Wurr S. Becker S. Günther S. Kümmerer B.M. J. Virol. 2007; 81: 2391-2400Crossref PubMed Scopus (151) Google Scholar, 13.Bick M.J. Carroll J.W. Gao G. Goff S.P. Rice C.M. MacDonald M.R. J. Virol. 2003; 77: 11555-11562Crossref PubMed Scopus (260) Google Scholar, 14.Zhang Y. Burke C.W. Ryman K.D. Klimstra W.B. J. Virol. 2007; 81: 11246-11255Crossref PubMed Scopus (188) Google Scholar, 15.MacDonald M.R. Machlin E.S. Albin O.R. Levy D.E. J. Virol. 2007; 81: 13509-13518Crossref PubMed Scopus (66) Google Scholar). Although ZAP is an ISG induced early after viral infection (38.Marcello T. Grakoui A. Barba-Spaeth G. Machlin E.S. Kotenko S.V. MacDonald M.R. Rice C.M. Gastroenterology. 2006; 131: 1887-1898Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar, 48.Ryman K.D. Meier K.C. Nangle E.M. Ragsdale S.L. Korneeva N.L. Rhoads R.E. MacDonald M.R. Klimstra W.B. J. Virol. 2005; 79: 1487-1499Crossref PubMed Scopus (73) Google Scholar), how ZAP is transcriptionally regulated has not been well studied. The present study identifies ZAP as a gene that is directly regulated by IRF3. IRF3 activation, which occurs via several distinct signaling pathways when cells sense virus infection, is both necessary and sufficient for up-regulation of ZAP transcription in the context of virus infection (FIGURE 1, FIGURE 2, FIGURE 3, FIGURE 4). Conversely, disruption of IRF3 function by DN-IRF3 significantly attenuated up-regulation of ZAP by virus or dsRNA (Fig. 1). In addition, IRF3 deficiency completely abrogated viral induction of ZAP (Fig. 2). The IRF3-dependent viral induction of ZAP is independent of IFN paracrine/autocrine action, because it occurs in cells either capable of (Fig. 2) or deficient in IFN signaling (FIGURE 1, FIGURE 2, FIGURE 3, FIGURE 4). Furthermore, disruption of NF-κB activation, which is known to inhibit IFN-β induction, does not impair activation of ZAP promoter by virus (Fig. 8). The latter data provide additional support for the concept that viral induction of ZAP does not rely on IFNs released by infected cells early after infection. Taken together, these data place ZAP in a subset of ISGs that are induced as a result of direct IRF3 action or IFN-induced Jak-STAT signaling, such as ISG56 and ISG15.OAS1 and MxA represent classical ISGs that require autocrine/paracrine action of IFN for induction during viral infections (53.Nakaya T. Sato M. Hata N. Asagiri M. Suemori H. Noguchi S. Tanaka N. Taniguchi T. Biochem. Biophys. Res. Commun. 2001; 283: 1150-1156Crossref PubMed Scopus (149) Google Scholar, 54.Ronni T. Matikainen S. Lehtonen A. Palvimo J. Dellis J. Van Eylen F. Goetschy J.F. Horisberger M. Content J. Julkunen I. J. Interferon Cytokine Res. 1998; 18: 773-781Crossref PubMed Scopus (76) Google Scholar). As a result they are more efficiently induced by IFN than by virus (Figs. 2B and 3). In contrast, ZAP induction by virus is at least as efficient, if not better, as induction by high IFN concentrations (Figs. 2B and 3). This is consistent with direct regulation of ZAP expression by IRF3 without a requirement for IFN signaling. Importantly, we have demonstrated this to be the case not only in cancer cell lines of various tissue origins (HeLa and A549) but also in non-neoplastic hepatocytes (PH5CH8) and in normal lung fibroblasts (MRC-5). An exception was the Huh7 hepatoma cell line, in which IFN seemed to be a more efficient inducer than virus for ZAP expression. This is not surprising, because Huh7 cells have an impaired RIG-I pathway and only mount a relatively weak IRF3 activation response following SeV infection (17.Li K. Chen Z. Kato N. Gale Jr., M. Lemon S.M. J. Biol. Chem. 2005; 280: 16739-16747Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 68.Foy E. Li K. Wang C. Sumpter Jr., R. Ikeda M. Lemon S.M. Gale Jr., M. Science. 2003; 300: 1145-1148Crossref PubMed Scopus (700) Google Scholar).Mutational analyses of the human ZAP promoter led us to identify five ISRE/IRF-Es far upstream of the transcription start site (−2.5 to −2.2 kb) that serve in a redundant role to regulate IRF3-dependent viral induction of ZAP (Fig. 5). In agreement with this, IRF3 was found to bind strongly and specifically to these distal ISREs following virus infection as determined by ChIP assays (Fig. 7B). This is in contrast to the transcriptional control of most other well known IRF3 direct target genes, such as IFN-β and ISG56, in which the ISRE/IRF-Es are proximal to the transcription start site (4.Hiscott J. J. Biol. Chem. 2007; 282: 15325-15329Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 9.Grandvaux N. Servant M.J. tenOever B. Sen G.C. Balachandran S. Barber G.N. Lin R. Hiscott J. J. Virol. 2002; 76: 5532-5539Crossref PubMed Scopus (411) Google Scholar). Whether this unique arrangement in transcriptional regulation of ZAP reflects an evolutionary advantage remains to be determined.Our study also identifies a proximal STAT site (STAT V, −111 to −91) that is critical for both IFN- and virus-induced hZAP promoter activity (Fig. 6). STAT1/STAT2 preferentially bound to this site following IFN stimulation in ChIP assays (Fig. 7). The distal ISREs are apparently dispensable for IFN stimulation of hZAP promoter (Figs. 5, C and E, and 6), because deletion of these ISREs did not affect the promoter activation. However, our data do not rule out the possibility that other distal STAT sites as well as other STAT proteins may also contribute to IFN regulation of hZAP promoter.In summary, ZAP is a primary response ISG in cellular antiviral responses. Its transcription can be directly controlled by IRF3 or as a result of IFN autocrine/paracrine signaling in viral infections. We propose the following model of ZAP induction. Early after infection, IRF3 is activated and translocated into the nucleus, whereupon it binds to the distal ISRE/IRF-Es in hZAP promoter to up-regulate ZAP transcription. This step is independent of IFN signaling. In the later phase, IFN-β is secreted by infected cells and activates, in turn, Jak-STAT signaling in infected cells and/or uninfected neighboring cells, resulting in the activation of STATs that translocate into the nucleus. These STATs interact with the proximal STAT V site and possibly other STAT sites in the hZAP promoter and sustain ZAP induction. It remains to be determined whether IRF7, induced by autocrine/paracrine IFN, may also contribute to the late phase of ZAP induction in parenchymal cells via the distal ISREs. IntroductionMammalian cells respond to viral infections by the rapid induction of a family of pleiotropic cytokines known as interferons (IFNs). 3The abbreviations used are: IFNinterferonZAPzinc finger antiviral proteinIRFinterferon regulatory factorIRF-EIRF elementNF-κBnuclear factor κBSTATsignal transducers and activators of transcriptionISGinterferon stimulated geneISREinterferon-stimulated response elementTLR3Toll-like receptor 3RIG-Iretinoic inducible gene IMDA5melanoma differentiation-associated gene 5MAVSmitochondrial antiviral signaling protein, also known as IPS-1/Cardif/VISATRIFToll-IL1 receptor homology domain containing adaptor inducing interferon-β, also known as TICAM1OASL2′,5′-oligoadenylate synthetase like geneChIPchromatin immunoprecipitationHCVhepatitis C virusSeVSendai virusdsdouble-strandedQ-PCRquantitative PCRDNdominant-negativehhumanIKKinhibitor of κB kinaseHAUhemagglutinin units. Once secreted, IFNs act in an autocrine and/or paracrine manner through the Jak-STAT pathway to stimulate the expression of hundreds of IFN-stimulated genes (ISGs), thereby establishing a cellular antiviral state. IFNs also exert critical immune regulatory functions, shaping subsequent adaptive immunity that helps clear the infection and establishes long term protective immunity (1.Borden E.C. Sen G.C. Uze G. Silverman R.H. Ransohoff R.M. Foster G.R. Stark G.R. Nat. Rev. Drug. Discov. 2007; 6: 975-990Crossref PubMed Scopus (860) Google Scholar, 2.Sen G.C. Annu. Rev. Microbiol. 2001; 55: 255-281Crossref PubMed Scopus (777) Google Scholar). The IFN family consists of three types, i.e. types I (mainly IFNβ and IFNα), II (IFNγ), and III (IFNλs). Although IFNγ production is restricted to cell types of lymphocyte origin, type I and III IFNs can be induced in most cell types (1.Borden E.C. Sen G.C. Uze G. Silverman R.H. Ransohoff R.M. Foster G.R. Stark G.R. Nat. Rev. Drug. Discov. 2007; 6: 975-990Crossref PubMed Scopus (860) Google Scholar). The importance of type I IFNs in the control of viral infection has been vividly demonstrated in IFNα/β receptor knock-out mice (3.Müller U. Steinhoff U. Reis L.F. Hemmi S. Pavlovic J. Zinkernagel R.M. Aguet M. Science. 1994; 264: 1918-1921Crossref PubMed Scopus (1974) Google Scholar).Upstream, IFNβ and IFNλ genes are transcriptionally activated by a latent cellular transcription factor, IFN regulatory factor 3 (IRF3) (4.Hiscott J. J. Biol. Chem. 2007; 282: 15325-15329Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 5.Osterlund P.I. Pietilä T.E. Veckman V. Kotenko S.V. Julkunen I. J. Immunol. 2007; 179: 3434-3442Crossref PubMed Scopus (246) Google Scholar, 6.Onoguchi K. Yoneyama M. Takemura A. Akira S. Taniguchi T. Namiki H. Fujita T. J. Biol. Chem. 2007; 282: 7576-7581Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). Predominantly residing in the cytoplasm in quiescent cells, IRF3 undergoes specific carboxyl-terminal serine phosphorylation mediated by the noncanonical IKK kinases, TBK1 or IKKϵ (7.Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. Latz E. Golenbock D.T. Coyle A.J. Liao S.M. Maniatis T. Nat. Immunol. 2003; 4: 491-496Crossref PubMed Scopus (2037) Google Scholar, 8.Sharma S. tenOever B.R. Grandvaux N. Zhou G.P. Lin R. Hiscott J. Science. 2003; 300: 1148-1151Crossref PubMed Scopus (1342) Google Scholar), following viral engagement of the toll-like receptor 3 (TLR3) and/or retinoic acid inducible gene I (RIG-I)/melanoma differentiation-associated gene 5 (MDA5) pathways. Phosphorylated IRF3 then dimerizes and translocates into the nucleus, where it associates with p300/cAMP-responsive element-binding protein-binding protein and binds to the IRF element (IRF-E) present in IFNβ and IFNλ gene promoters, activating their transcription (1.Borden E.C. Sen G.C. Uze G. Silverman R.H. Ransohoff R.M. Foster G.R. Stark G.R. Nat. Rev. Drug. Discov. 2007; 6: 975-990Crossref PubMed Scopus (860) Google Scholar, 4.Hiscott J. J. Biol. Chem. 2007; 282: 15325-15329Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). Because IRF-Es and the IFN-stimulated responsive elements (ISREs) present in some ISG promoters overlap in sequence, IRF3 is known to directly regulate a subset of ISGs, such as ISG56 and ISG54 (9.Grandvaux N. Servant M.J. tenOever B. Sen G.C. Balachandran S. Barber G.N. Lin R. Hiscott J. J. Virol. 2002; 76: 5532-5539Crossref PubMed Scopus (411) Google Scholar, 10.Peters K.L. Smith H.L. Stark G.R. Sen G.C. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 6322-6327Crossref PubMed Scopus (123) Google Scholar), independent of the IFN autocrine/paracrine loop. Given the pivotal role of IRF3 in antiviral innate immunity, IRF3 target genes may play important roles in the early host responses to viral infections. For this reason, identification of direct IRF3 target genes is important.During our investigation of hepatitis C virus (HCV)-mediated disruption of global antiviral gene expression, we observed that viral induction of hundreds of cellular genes (using Sendai virus (SeV) as an inducer) was blocked by HCV in Huh7 cells stably replicating genome length HCV RNAs. 4K. Li and S. M. Lemon, unpublished data. Because many of the virus-inducible genes were well known ISGs, it was unclear whether the disruption of their induction was a direct result of inhibition of IRF3 activation by HCV or secondary to the blockade of induction of type I/III IFNs, which signal through the JAK-STAT pathway to regulate the expression of these genes. In our efforts to determine the subset of genes whose transcription is directly controlled by IRF3, we identified the zinc finger antiviral protein (ZAP) as a novel IRF3 target gene. Previously known as an ISG, ZAP possesses antiviral activity against viruses of multiple families, including Retroviridae, Togaviridae, and Filoviridae (11.Gao G. Guo X. Goff S.P. Science. 2002; 297: 1703-1706Crossref PubMed Scopus (343) Google Scholar, 12.Müller S. Möller P. Bick M.J. Wurr S. Becker S. Günther S. Kümmerer B.M. J. Virol. 2007; 81: 2391-2400Crossref PubMed Scopus (151) Google Scholar, 13.Bick M.J. Carroll J.W. Gao G. Goff S.P. Rice C.M. MacDonald M.R. J. Virol. 2003; 77: 11555-11562Crossref PubMed Scopus (260) Google Scholar, 14.Zhang Y. Burke C.W. Ryman K.D. Klimstra W.B. J. Virol. 2007; 81: 11246-11255Crossref PubMed Scopus (188) Google Scholar, 15.MacDonald M.R. Machlin E.S. Albin O.R. Levy D.E. J. Virol. 2007; 81: 13509-13518Crossref PubMed Scopus (66) Google Scholar). The experimental studies reported here reveal that ZAP expression is directly regulated by IRF3 following virus infection or stimulation of cells with dsRNA or dsDNA and is not dependent on NF-κB. Although IFN can also stimulate ZAP expression, our data indicate that ZAP is a primary IRF3 response gene in the innate antiviral signaling pathway.