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Stephen Goodbourn - One of the best experts on this subject based on the ideXlab platform.
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The Npro product of classical swine fever virus and bovine viral diarrhea virus uses a conserved mechanism to target Interferon Regulatory Factor-3.
The Journal of general virology, 2020Co-Authors: Julian Seago, Louise Hilton, Kartyk Moganeradj, Elizabeth Reid, Virginie Doceul, Janan Jeyatheesan, John Mccauley, Bryan Charleston, Stephen GoodbournAbstract:Classical swine fever virus (CSFV) is a member of the genus Pestivirus in the family Flaviviridae. The N(pro) product of CSFV targets the host's innate immune response and can prevent the production of type I Interferon (IFN). The mechanism by which CSFV orchestrates this inhibition was investigated and it is shown that, like the related pestivirus bovine viral diarrhea virus (BVDV), this involves the N(pro) protein targeting Interferon Regulatory Factor-3 (IRF-3) for degradation by proteasomes and thus preventing IRF-3 from activating transcription from the IFN-beta promoter. Like BVDV, the steady-state levels of IRF-3 mRNA are not reduced markedly by CSFV infection or N(pro) overexpression. Moreover, IFN-alpha stimulation of CSFV-infected cells induces the antiviral protein MxA, indicating that, as in BVDV-infected cells, the JAK/STAT pathway is not targeted for inhibition.
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Short Communication The N pro product of classical swine fever virus and bovine viral diarrhea virus uses a conserved mechanism to target Interferon Regulatory Factor-3
2020Co-Authors: Julian Seago, Louise Hilton, John W. Mccauley, Elizabeth Reid, Virginie Doceul, Janan Jeyatheesan, Bryan Charleston, Stephen GoodbournAbstract:Classical swine fever virus (CSFV) is a member of the genus Pestivirus in the family Flaviviridae. The N pro product of CSFV targets the host’s innate immune response and can prevent the production of type I Interferon (IFN). The mechanism by which CSFV orchestrates this inhibition was investigated and it is shown that, like the related pestivirus bovine viral diarrhea virus (BVDV), this involves the N pro protein targeting Interferon Regulatory Factor-3 (IRF-3) for degradation by proteasomes and thus preventing IRF-3 from activating transcription from the IFN-b promoter. Like BVDV, the steady-state levels of IRF-3 mRNA are not reduced markedly by CSFV infection or N pro overexpression. Moreover, IFN-a stimulation of CSFV-infected cells induces the antiviral
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The Npro product of classical swine fever virus and bovine viral diarrhea virus uses a conserved mechanism to target Interferon Regulatory Factor-3.
Journal of General Virology, 2007Co-Authors: Julian Seago, Louise Hilton, Kartyk Moganeradj, John W. Mccauley, Elizabeth Reid, Virginie Doceul, Janan Jeyatheesan, Bryan Charleston, Stephen GoodbournAbstract:Classical swine fever virus (CSFV) is a member of the genus Pestivirus in the family Flaviviridae. The Npro product of CSFV targets the host's innate immune response and can prevent the production of type I Interferon (IFN). The mechanism by which CSFV orchestrates this inhibition was investigated and it is shown that, like the related pestivirus bovine viral diarrhea virus (BVDV), this involves the Npro protein targeting Interferon Regulatory Factor-3 (IRF-3) for degradation by proteasomes and thus preventing IRF-3 from activating transcription from the IFN-β promoter. Like BVDV, the steady-state levels of IRF-3 mRNA are not reduced markedly by CSFV infection or Npro overexpression. Moreover, IFN-α stimulation of CSFV-infected cells induces the antiviral protein MxA, indicating that, as in BVDV-infected cells, the JAK/STAT pathway is not targeted for inhibition.
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The NPro Product of Bovine Viral Diarrhea Virus Inhibits DNA Binding by Interferon Regulatory Factor 3 and Targets It for Proteasomal Degradation
Journal of Virology, 2006Co-Authors: Louise Hilton, Kartyk Moganeradj, Gang Zhang, Yun-hsiang Chen, Richard E. Randall, John W. Mccauley, Stephen GoodbournAbstract:Bovine viral diarrhea virus (BVDV) is a pestivirus that can establish a persistent infection in the developing fetus and has the ability to disable the production of type I Interferon. In this report, we extend our previous observations that BVDV encodes a protein able to specifically block the activity of Interferon Regulatory factor 3 (IRF-3), a transcription factor essential for Interferon promoter activation, by demonstrating that this is a property of the N-terminal protease fragment (NPro) of the BVDV polyprotein. Although BVDV infections cause relocalization of cellular IRF-3 from the cytoplasm to the nucleus early in infection, NPro blocks IRF-3 from binding to DNA. NPro has the additional property of targeting IRF-3 for polyubiquitination and subsequent destruction by cellular multicatalytic proteasomes. The autoprotease activity of NPro is not required for the inhibition of type I Interferon induction or the targeting of IRF-3 for degradation.
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inhibition of beta Interferon transcription by noncytopathogenic bovine viral diarrhea virus is through an Interferon Regulatory factor 3 dependent mechanism
Journal of Virology, 2002Co-Authors: Susan J Baigent, Gang Zhang, Stephen Goodbourn, M D Fray, Helen Flicksmith, John W. MccauleyAbstract:The induction and inhibition of the Interferon (IFN) response and apoptosis by bovine viral diarrhea virus (BVDV) has been examined. Here we show that prior infection of cells by noncytopathogenic BVDV (ncp BVDV) fails to block transcriptional responses to alpha/beta IFN. In contrast, ncp BVDV-infected cells fail to produce IFN-α/β or MxA in response to double-stranded RNA (dsRNA) or infection with a heterologous virus (Semliki Forest virus [SFV]). ncp BVDV preinfection is unable to block cp BVDV- or SFV-induced apoptosis. The effects of ncp BVDV infection on the transcription factors controlling the IFN-β induction pathway have been analyzed. The transcription factor NF-κB was not activated following ncp BVDV infection, but ncp BVDV infection was not able to block the activation of NF-κB by either SFV or tumor necrosis factor alpha. Furthermore, ncp BVDV infection did not result in the activation of stress kinases (JNK1 and JNK2) or the phosphorylation of transcription factors ATF-2 and c-Jun; again, ncp BVDV infection was not able to block their activation by SFV. Interferon Regulatory factor 3 (IRF-3) was shown to be translocated to the nuclei of infected cells in response to ncp BVDV, although DNA-binding of IRF-3 was not seen in nuclear extracts. In contrast, an IRF-3-DNA complex was observed in nuclear extracts from cells infected with SFV, but the appearance of this complex was blocked when cells were previously exposed to ncp BVDV. We conclude that the inhibition of IFN induction by this pestivirus involves a block to IRF-3 function, and we speculate that this may be a key characteristic for the survival of pestiviruses in nature.
John W. Mccauley - One of the best experts on this subject based on the ideXlab platform.
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Short Communication The N pro product of classical swine fever virus and bovine viral diarrhea virus uses a conserved mechanism to target Interferon Regulatory Factor-3
2020Co-Authors: Julian Seago, Louise Hilton, John W. Mccauley, Elizabeth Reid, Virginie Doceul, Janan Jeyatheesan, Bryan Charleston, Stephen GoodbournAbstract:Classical swine fever virus (CSFV) is a member of the genus Pestivirus in the family Flaviviridae. The N pro product of CSFV targets the host’s innate immune response and can prevent the production of type I Interferon (IFN). The mechanism by which CSFV orchestrates this inhibition was investigated and it is shown that, like the related pestivirus bovine viral diarrhea virus (BVDV), this involves the N pro protein targeting Interferon Regulatory Factor-3 (IRF-3) for degradation by proteasomes and thus preventing IRF-3 from activating transcription from the IFN-b promoter. Like BVDV, the steady-state levels of IRF-3 mRNA are not reduced markedly by CSFV infection or N pro overexpression. Moreover, IFN-a stimulation of CSFV-infected cells induces the antiviral
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The Npro product of classical swine fever virus and bovine viral diarrhea virus uses a conserved mechanism to target Interferon Regulatory Factor-3.
Journal of General Virology, 2007Co-Authors: Julian Seago, Louise Hilton, Kartyk Moganeradj, John W. Mccauley, Elizabeth Reid, Virginie Doceul, Janan Jeyatheesan, Bryan Charleston, Stephen GoodbournAbstract:Classical swine fever virus (CSFV) is a member of the genus Pestivirus in the family Flaviviridae. The Npro product of CSFV targets the host's innate immune response and can prevent the production of type I Interferon (IFN). The mechanism by which CSFV orchestrates this inhibition was investigated and it is shown that, like the related pestivirus bovine viral diarrhea virus (BVDV), this involves the Npro protein targeting Interferon Regulatory Factor-3 (IRF-3) for degradation by proteasomes and thus preventing IRF-3 from activating transcription from the IFN-β promoter. Like BVDV, the steady-state levels of IRF-3 mRNA are not reduced markedly by CSFV infection or Npro overexpression. Moreover, IFN-α stimulation of CSFV-infected cells induces the antiviral protein MxA, indicating that, as in BVDV-infected cells, the JAK/STAT pathway is not targeted for inhibition.
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The NPro Product of Bovine Viral Diarrhea Virus Inhibits DNA Binding by Interferon Regulatory Factor 3 and Targets It for Proteasomal Degradation
Journal of Virology, 2006Co-Authors: Louise Hilton, Kartyk Moganeradj, Gang Zhang, Yun-hsiang Chen, Richard E. Randall, John W. Mccauley, Stephen GoodbournAbstract:Bovine viral diarrhea virus (BVDV) is a pestivirus that can establish a persistent infection in the developing fetus and has the ability to disable the production of type I Interferon. In this report, we extend our previous observations that BVDV encodes a protein able to specifically block the activity of Interferon Regulatory factor 3 (IRF-3), a transcription factor essential for Interferon promoter activation, by demonstrating that this is a property of the N-terminal protease fragment (NPro) of the BVDV polyprotein. Although BVDV infections cause relocalization of cellular IRF-3 from the cytoplasm to the nucleus early in infection, NPro blocks IRF-3 from binding to DNA. NPro has the additional property of targeting IRF-3 for polyubiquitination and subsequent destruction by cellular multicatalytic proteasomes. The autoprotease activity of NPro is not required for the inhibition of type I Interferon induction or the targeting of IRF-3 for degradation.
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inhibition of beta Interferon transcription by noncytopathogenic bovine viral diarrhea virus is through an Interferon Regulatory factor 3 dependent mechanism
Journal of Virology, 2002Co-Authors: Susan J Baigent, Gang Zhang, Stephen Goodbourn, M D Fray, Helen Flicksmith, John W. MccauleyAbstract:The induction and inhibition of the Interferon (IFN) response and apoptosis by bovine viral diarrhea virus (BVDV) has been examined. Here we show that prior infection of cells by noncytopathogenic BVDV (ncp BVDV) fails to block transcriptional responses to alpha/beta IFN. In contrast, ncp BVDV-infected cells fail to produce IFN-α/β or MxA in response to double-stranded RNA (dsRNA) or infection with a heterologous virus (Semliki Forest virus [SFV]). ncp BVDV preinfection is unable to block cp BVDV- or SFV-induced apoptosis. The effects of ncp BVDV infection on the transcription factors controlling the IFN-β induction pathway have been analyzed. The transcription factor NF-κB was not activated following ncp BVDV infection, but ncp BVDV infection was not able to block the activation of NF-κB by either SFV or tumor necrosis factor alpha. Furthermore, ncp BVDV infection did not result in the activation of stress kinases (JNK1 and JNK2) or the phosphorylation of transcription factors ATF-2 and c-Jun; again, ncp BVDV infection was not able to block their activation by SFV. Interferon Regulatory factor 3 (IRF-3) was shown to be translocated to the nuclei of infected cells in response to ncp BVDV, although DNA-binding of IRF-3 was not seen in nuclear extracts. In contrast, an IRF-3-DNA complex was observed in nuclear extracts from cells infected with SFV, but the appearance of this complex was blocked when cells were previously exposed to ncp BVDV. We conclude that the inhibition of IFN induction by this pestivirus involves a block to IRF-3 function, and we speculate that this may be a key characteristic for the survival of pestiviruses in nature.
Eicke Latz - One of the best experts on this subject based on the ideXlab platform.
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the rab11a gtpase controls toll like receptor 4 induced activation of Interferon Regulatory factor 3 on phagosomes
Immunity, 2010Co-Authors: Harald Husebye, Marie Hjelmseth Aune, Jorgen Stenvik, Eivind O Samstad, Frode Skjeldal, Oyvind Halaas, Nadra Nilsen, Harald Alfred Stenmark, Eicke LatzAbstract:Toll-like receptor 4 (TLR4) is indispensable for recognition of Gram-negative bacteria. We described a trafficking pathway for TLR4 from the endocytic recycling compartment (ERC) to E. coli phagosomes. We found a prominent colocalization between TLR4 and the small GTPase Rab11a in the ERC, and Rab11a was involved in the recruitment of TLR4 to phagosomes in a process requiring TLR4 signaling. Also, Toll-receptor-associated molecule (TRAM) and Interferon Regulatory Factor-3 (IRF3) localized to E. coli phagosomes and internalization of E. coli was required for a robust Interferon-β induction. Suppression of Rab11a reduced TLR4 in the ERC and on phagosomes leading to inhibition of the IRF3 signaling pathway induced by E. coli, whereas activation of the transcription factor NF-κB was unaffected. Moreover, Rab11a silencing reduced the amount of TRAM on phagosomes. Thus, Rab11a is an important regulator of TLR4 and TRAM transport to E. coli phagosomes thereby controlling IRF3 activation from this compartment.
Michael David - One of the best experts on this subject based on the ideXlab platform.
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phospholipase cγ 2 and intracellular calcium are required for lipopolysaccharide induced toll like receptor 4 tlr4 endocytosis and Interferon Regulatory factor 3 irf3 activation
Journal of Biological Chemistry, 2012Co-Authors: Chihyuan Chiang, Ville Veckman, Kirsten Limmer, Michael DavidAbstract:Toll-like receptor 4 (TLR4) is unique among the TLRs in its use of multiple adaptor proteins leading to activation of both the Interferon Regulatory factor 3 (IRF3) and nuclear factor κB (NF-κB) pathways. Previous work has demonstrated that TLR4 initiates NF-κB activation from the plasma membrane, but that subsequent TLR4 translocation to the endosomes is required for IRF3 activation. Here we have characterized several components of the signaling pathway that governs TLR4 translocation and subsequent IRF3 activation. We find that phospholipase C γ2 (PLCγ2) accounts for LPS-induced inositol 1,4,5-trisphosphate (IP3) production and subsequent calcium (Ca2+) release. Blockage of PLCγ2 function by inhibitors or knockdown of PLCγ2 expression by siRNAs in RAW 264.7 macrophages lead to reduced IRF3, but enhanced NF-κB activation. In addition, bone marrow-derived macrophages from PLCγ2-deficient mice showed impaired IRF3 phosphorylation and expression of IRF3-regulated genes after LPS stimulation. Using cell fractionation, we show that PLCγ2-IP3-Ca2+ signaling cascade is required for TLR4 endocytosis following LPS stimulation. In conclusion, our results describe a novel role of the PLCγ2-IP3-Ca2+ cascade in the LPS-induced innate immune response pathway where release of intracellular Ca2+ mediates TLR4 trafficking and subsequent activation of IRF3.
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Inhibition of lipopolysaccharide-induced Interferon Regulatory factor 3 activation and protection from septic shock by hydroxystilbenes.
Shock, 2004Co-Authors: Oanh Dang, Lorena Navarro, Michael DavidAbstract:Interferon Regulatory factor 3 (IRF3) mediates the transcriptional induction of Interferon-stimulated genes (ISGs) in response to viral and bacterial infections. Here we show that the hydroxystilbene piceatannol inhibits the LPS-mediated activation of IRF3 and subsequent ISG induction. Consequently, piceatannol blocks the LPS-induced up-regulation of critical mediators of the inflammatory response such as interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), intercellular adhesion molecule 1 (ICAM-1), and macrophage chemoattractant protein (MCP-1). Furthermore, the LPS-mediated induction of tissue factor (TF), a cell surface protein responsible for initiating the coagulation cascade, is also inhibited by piceatannol. The effectiveness of piceatannol in blocking both the inflammatory response and the coagulation pathway is evidenced by its ability to confer protection against LPS-induced septic shock in a murine model. Thus, IRF3 appears to be a promising target for pharmacologic intervention in the prevention or treatment of septic shock syndrome.
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p38-dependent activation of Interferon Regulatory factor 3 by lipopolysaccharide.
Journal of Biological Chemistry, 1999Co-Authors: Lorena Navarro, Michael DavidAbstract:Abstract Interferon Regulatory factor 3 (IRF3) is known to participate in the transcriptional induction of Interferon (IFN) α and IFNβ genes, as well as of a number of Interferon-stimulated genes (ISGs), as a result of viral infection. In the present study we demonstrate the activation of IRF3 followed by ISG induction after exposure of cells to the bacterial cell wall component lipopolysaccharide. Engagement of Toll-like receptors by lipopolysaccharide triggered the nuclear translocation of IRF3, followed by its DNA binding and the subsequent induction of several Interferon-regulated genes. Transcriptional activation of ISGs occurred in a protein synthesis independent manner, but was sensitive to inhibition of the stress-activated protein kinase, p38. The activation of IRF3 by viral particles or bacterial membrane components suggests that this signaling pathway might contribute to the evolutionary conserved innate immune response.
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Cytomegalovirus Activates Interferon Immediate-Early Response Gene Expression and an Interferon Regulatory Factor 3-Containing Interferon-Stimulated Response Element-Binding Complex
Molecular and Cellular Biology, 1998Co-Authors: Lorena Navarro, Brian K. Weaver, Nancy C. Reich, Kerri A. Mowen, Steven M. Rodems, Deborah H. Spector, Michael DavidAbstract:Interferon establishes an antiviral state in numerous cell types through the induction of a set of immediate-early response genes. Activation of these genes is mediated by phosphorylation of latent transcription factors of the STAT family. We found that infection of primary foreskin fibroblasts with human cytomegalovirus (HCMV) causes selective transcriptional activation of the alpha/beta-Interferon-responsive ISG54 gene. However, no activation or nuclear translocation of STAT proteins was detected. Activation of ISG54 occurs independent of protein synthesis but is prevented by protein tyrosine kinase inhibitors. Further analysis revealed that HCMV infection induced the DNA binding of a novel complex, tentatively called cytomegalovirus-induced Interferon-stimulated response element binding factor (CIF). CIF is composed, at least in part, of the recently identified Interferon Regulatory factor 3 (IRF3), but it does not contain the STAT1 and STAT2 proteins that participate in the formation of Interferon-stimulated gene factor 3. IRF3, which has previously been shown to possess no intrinsic transcriptional activation potential, interacts with the transcriptional coactivator CREB binding protein, but not with p300, to form CIF. Activating Interferon-stimulated genes without the need for prior synthesis of Interferons might provide the host cell with a potential shortcut in the activation of its antiviral defense.
John Hiscott - One of the best experts on this subject based on the ideXlab platform.
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CuRTAiling Interferon Regulatory Factor Signaling with the E3 Ligase RAUL
Immunity, 2010Co-Authors: John HiscottAbstract:Interferon Regulatory factor 3 (IRF3) and IRF7 modulate the transcription of type I Interferon. In this issue of Immunity, Yu and Hayward (2010) identify RAUL, the bona fide ubiquitin ligase that regulates turnover of IRF3 and IRF7.
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Regulation of arginase II by Interferon Regulatory factor 3 and the involvement of polyamines in the antiviral response.
FEBS Journal, 2005Co-Authors: Nathalie Grandvaux, Benjamin R. Tenoever, François Gaboriau, Jennifer Harris, John HiscottAbstract:The innate antiviral response requires the induction of genes and proteins with activities that limit virus replication. Among these, the well-characterized Interferon β (IFNB) gene is regulated through the cooperation of AP-1, NF-κB and Interferon Regulatory factor 3 (IRF-3) transcription factors. Using a constitutively active form of IRF-3, IRF-3 5D, we showed previously that IRF-3 also regulates an IFN-independent antiviral response through the direct induction of IFN-stimulated genes. In this study, we report that the arginase II gene (ArgII) as well as ArgII protein concentrations and enzymatic activity are induced in IRF-3 5D-expressing and Sendai virus-infected Jurkat cells in an IFN-independent manner. ArgII is a critical enzyme in the polyamine-biosynthetic pathway. Of the natural polyamines, spermine possesses antiviral activity and mediates apoptosis at physiological concentrations. Measurement of intracellular polyamine content revealed that expression of IRF-3 5D induces polyamine production, but that Sendai virus and vesicular stomatitis virus infections do not. These results show for the first time that the ArgII gene is an early IRF-3-regulated gene, which participates in the IFN-independent antiviral response through polyamine production and induction of apoptosis.
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Multiple signaling pathways leading to the activation of Interferon Regulatory factor 3
Biochemical Pharmacology, 2002Co-Authors: Marc J. Servant, Nathalie Grandvaux, John HiscottAbstract:Abstract Virus infection of susceptible cells activates multiple signaling pathways that orchestrate the activation of genes, such as cytokines, involved in the antiviral and innate immune response. Among the kinases induced are the mitogen-activated protein (MAP) kinases, Jun-amino terminal kinases (JNK) and p38, the IκB kinase (IKK) and DNA-PK. In addition, virus infection also activates an uncharacterized VAK responsible for the C-terminal phosphorylation and subsequent activation of Interferon Regulatory factor 3 (IRF-3). Virus-mediated activation of IRF-3 through VAK is dependent on viral entry and transcription, since replication deficient virus failed to induce IRF-3 activity. The pathways leading to VAK activation are not well characterized, but IRF-3 appears to represent a novel cellular detection pathway that recognizes viral nucleocapsid (N) structure. Recently, the range of inducers responsible for IRF-3 activation has increased. In addition to virus infection, recognition of bacterial infection mediated through lipopolysaccharide by Toll-like receptor 4 has also been reported. Furthermore, MAP kinase kinase kinase (MAP KKK)-related pathways and DNA-PK induce N-terminal phosphorylation of IRF-3. This review summarizes recent observations in the identification of novel signaling pathways leading to IRF-3 activation.
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Transcriptional profiling of Interferon Regulatory factor 3 target genes: direct involvement in the regulation of Interferon-stimulated genes.
Journal of Virology, 2002Co-Authors: Nathalie Grandvaux, Marc J. Servant, Benjamin R. Tenoever, Siddarth Balachandran, Glen N. Barber, John HiscottAbstract:Ubiquitously expressed Interferon Regulatory factor 3 (IRF-3) is directly activated after virus infection and functions as a key activator of the immediate-early alpha/beta Interferon (IFN) genes, as well as the RANTES chemokine gene. In the present study, a tetracycline-inducible expression system expressing a constitutively active form of IRF-3 (IRF-3 5D) was combined with DNA microarray analysis to identify target genes regulated by IRF-3. Changes in mRNA expression profiles of 8,556 genes were monitored after Tet-inducible expression of IRF-3 5D. Among the genes upregulated by IRF-3 were transcripts for several known IFN-stimulated genes (ISGs). Subsequent analysis revealed that IRF-3 directly induced the expression of ISG56 in an IFN-independent manner through the IFN-stimulated responsive elements (ISREs) of the ISG56 promoter. These results demonstrate that, in addition to its role in the formation of a functional immediate-early IFN-β enhanceosome, IRF-3 is able to discriminate among ISRE-containing genes involved in the establishment of the antiviral state as a direct response to virus infection.
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Structural and Functional Analysis of Interferon Regulatory Factor 3: Localization of the Transactivation and Autoinhibitory Domains
Molecular and Cellular Biology, 1999Co-Authors: Yael Mamane, John HiscottAbstract:The Interferon Regulatory factor 3 (IRF-3) gene encodes a 55-kDa protein which is expressed constitutively in all tissues. In unstimulated cells, IRF-3 is present in an inactive cytoplasmic form; following Sendai virus infection, IRF-3 is posttranslationally modified by protein phosphorylation at multiple serine and threonine residues located in the carboxy terminus. Virus-induced phosphorylation of IRF-3 leads to cytoplasmic to nuclear translocation of phosphorylated IRF-3, association with the transcriptional coactivator CBP/p300, and stimulation of DNA binding and transcriptional activities of virus-inducible genes. Using yeast and mammalian one-hybrid analysis, we now demonstrate that an extended, atypical transactivation domain is located in the C terminus of IRF-3 between amino acids (aa) 134 and 394. We also show that the C-terminal domain of IRF-3 located between aa 380 and 427 participates in the autoinhibition of IRF-3 activity via an intramolecular association with the N-terminal region between aa 98 and 240. After Sendai virus infection, an intermolecular association between IRF-3 proteins is detected, demonstrating a virus-dependent formation of IRF-3 homodimers; this interaction is also observed in the absence of virus infection with a constitutively activated form of IRF-3. Substitution of the C-terminal Ser-Thr phosphorylation sites with the phosphomimetic Asp in the region ISNSHPLSLTSDQ between amino acids 395 and 407 [IRF-3(5D)], but not the adjacent S385 and S386 residues, generates a constitutively activated DNA binding form of IRF-3. In contrast, substitution of S385 and S386 with either Ala or Asp inhibits both DNA binding and transactivation activities of the IRF-3(5D) protein. These studies thus define the transactivation domain of IRF-3, two domains that participate in the autoinhibition of IRF-3 activity, and the Regulatory phosphorylation sites controlling IRF-3 dimer formation, DNA binding activity, and association with the CBP/p300 coactivator.