scholarly journals Structural and Functional Analysis of Interferon Regulatory Factor 3: Localization of the Transactivation and Autoinhibitory Domains

1999 ◽  
Vol 19 (4) ◽  
pp. 2465-2474 ◽  
Author(s):  
Rongtuan Lin ◽  
Yael Mamane ◽  
John Hiscott
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Virus Infection ◽  
Nuclear Translocation ◽  
Sendai Virus ◽  
Interferon Regulatory Factor ◽  
Phosphorylation Sites ◽  
Transactivation Domain ◽  
Regulatory Factor ◽  
Interferon Regulatory Factor 3

ABSTRACT 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.

scholarly journals A study on the relationship between HCV NS3 and endogenous IRF-3

2018 ◽  
Vol 16 ◽  
pp. 205873921878445 ◽  
Author(s):  
Hong-Ping Liang ◽  
Hotta Hak ◽  
Jian-Min Ji
Keyword(s):  
Virus Infection ◽  
Hela Cells ◽  
Nuclear Translocation ◽  
Sendai Virus ◽  
Interferon Regulatory Factor ◽  
Immunofluorescence Assay ◽  
Regulatory Factor ◽  
Before And After ◽  
The Relationship ◽  
Published Paper

This study aims to investigate the relationship between hepatitis C virus (HCV) NS3/4A and endogenous interferon regulatory factor-3 (IRF-3). The localization of endogenous IRF-3 protein before and after virus infection was analyzed by immunofluorescence assay (IFA). IFA results revealed that the synergistic action of transfection and HCV virus infection could more effectively reduce the nuclear translocation of endogenous IRF-3 in HeLa cells, compared to the activation of Sendai virus infection alone. The highest nuclear translocation of endogenous IRF-3 in transfected HeLa cells occurred at 24 h after Sendai virus infection. Our study was consistent with a published paper, which revealed that HCV NS3/4A protease could suppress the activation of IRF-3 and was indispensable in the transcription of interferon (IFN)-α/β.

scholarly journals Analysis of Genes Induced by Sendai Virus Infection of Mutant Cell Lines Reveals Essential Roles of Interferon Regulatory Factor 3, NF-κB, and Interferon but Not Toll-Like Receptor 3

2005 ◽  
Vol 79 (7) ◽  
pp. 3920-3929 ◽  
Author(s):  
Christopher P. Elco ◽  
Jeanna M. Guenther ◽  
Bryan R. G. Williams ◽  
Ganes C. Sen
Keyword(s):  
Virus Infection ◽  
Cell Lines ◽  
Sendai Virus ◽  
Mutant Cell ◽  
Gene Induction ◽  
Interferon Regulatory Factor ◽  
Toll Like Receptor ◽  
Regulatory Factor ◽  
Interferon Regulatory Factor 3 ◽  
Infected Cells

ABSTRACT Sendai virus (SeV) infection causes the transcriptional induction of many cellular genes that are also induced by interferon (IFN) or double-stranded RNA (dsRNA). We took advantage of various mutant cell lines to investigate the putative roles of the components of the IFN and dsRNA signaling pathways in the induction of those genes by SeV. Profiling the patterns of gene expression in SeV-infected cells demonstrated that Toll-like receptor 3, although essential for gene induction by dsRNA, was dispensable for gene induction by SeV. In contrast, Jak1, which mediates IFN signaling, was required for the induction of a small subset of genes by SeV. NF-κB and interferon regulatory factor 3 (IRF-3), the two major transcription factors activated by virus infection, were essential for the induction of two sets of genes by SeV. As expected, some of the IRF-3-dependent genes, such as ISG56, were more strongly induced by SeV in IRF-3-overexpressing cells. Surprisingly, in those cells, a number of NF-κB-dependent genes, such as the A20 gene, were induced poorly. Using a series of cell lines expressing increasing levels of IRF-3, we demonstrated that the degree of induction of A20 mRNA, upon SeV infection, was inversely proportional to the cellular level of IRF-3, whereas that of ISG56 mRNA was directly proportional. Thus, IRF-3 can suppress the expression of NF-κB-dependent genes in SeV-infected cells.

scholarly journals The Ebola Virus VP35 Protein Inhibits Activation of Interferon Regulatory Factor 3

2003 ◽  
Vol 77 (14) ◽  
pp. 7945-7956 ◽  
Author(s):  
Christopher F. Basler ◽  
Andrea Mikulasova ◽  
Luis Martinez-Sobrido ◽  
Jason Paragas ◽  
Elke Mühlberger ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Virus Infection ◽  
Transcriptional Activation ◽  
Ebola Virus ◽  
Nuclear Translocation ◽  
Sendai Virus ◽  
Vero Cells ◽  
Nuclear Accumulation ◽  
Regulatory Factor ◽  
Cellular Transcription Factor

ABSTRACT The Ebola virus VP35 protein was previously found to act as an interferon (IFN) antagonist which could complement growth of influenza delNS1 virus, a mutant influenza virus lacking the influenza virus IFN antagonist protein, NS1. The Ebola virus VP35 could also prevent the virus- or double-stranded RNA-mediated transcriptional activation of both the beta IFN (IFN-β) promoter and the IFN-stimulated ISG54 promoter (C. Basler et al., Proc. Natl. Acad. Sci. USA 97:12289-12294, 2000). We now show that VP35 inhibits virus infection-induced transcriptional activation of IFN regulatory factor 3 (IRF-3)-responsive mammalian promoters and that VP35 does not block signaling from the IFN-α/β receptor. The ability of VP35 to inhibit this virus-induced transcription correlates with its ability to block activation of IRF-3, a cellular transcription factor of central importance in initiating the host cell IFN response. We demonstrate that VP35 blocks the Sendai virus-induced activation of two promoters which can be directly activated by IRF-3, namely, the ISG54 promoter and the ISG56 promoter. Further, expression of VP35 prevents the IRF-3-dependent activation of the IFN-α4 promoter in response to viral infection. The inhibition of IRF-3 appears to occur through an inhibition of IRF-3 phosphorylation. VP35 blocks virus-induced IRF-3 phosphorylation and subsequent IRF-3 dimerization and nuclear translocation. Consistent with these observations, Ebola virus infection of Vero cells activated neither transcription from the ISG54 promoter nor nuclear accumulation of IRF-3. These data suggest that in Ebola virus-infected cells, VP35 inhibits the induction of antiviral genes, including the IFN-β gene, by blocking IRF-3 activation.

scholarly journals Hsp90 Regulates Activation of Interferon Regulatory Factor 3 and TBK-1 Stabilization in Sendai Virus-infected Cells

2006 ◽  
Vol 17 (3) ◽  
pp. 1461-1471 ◽  
Author(s):  
Kai Yang ◽  
Hexin Shi ◽  
Rong Qi ◽  
Shaogang Sun ◽  
Yujie Tang ◽  
...  
Keyword(s):  
Sendai Virus ◽  
Specific Interaction ◽  
Hybrid Approach ◽  
Specific Inhibitor ◽  
Interferon Regulatory Factor ◽  
Dominant Negative ◽  
Regulatory Factor ◽  
Interferon Regulatory Factor 3 ◽  
Infected Cells ◽  
Irf3 Activation

Interferon regulatory factor 3 (IRF3) plays a crucial role in mediating cellular responses to virus intrusion. The protein kinase TBK1 is a key regulator inducing phosphorylation of IRF3. The regulatory mechanisms during IRF3 activation remain poorly characterized. In the present study, we have identified by yeast two-hybrid approach a specific interaction between IRF3 and chaperone heat-shock protein of 90 kDa (Hsp90). The C-terminal truncation mutant of Hsp90 is a strong dominant-negative inhibitor of IRF3 activation. Knockdown of endogenous Hsp90 by RNA interference attenuates IRF3 activation and its target gene expressions. Alternatively, Hsp90-specific inhibitor geldanamycin (GA) dramatically reduces expression of IRF3-regulated interferon-stimulated genes and abolishes the cytoplasm-to-nucleus translocation and DNA binding activity of IRF3 in Sendai virus-infected cells. Significantly, virus-induced IRF3 phosphorylation is blocked by GA, whereas GA does not affect the protein level of IRF3. In addition, TBK1 is found to be a client protein of Hsp90 in vivo. Treatment of 293 cells with GA interferes with the interaction of TBK1 and Hsp90, resulting in TBK1 destabilization and its subsequent proteasome-mediated degradation. Besides maintaining stability of TBK1, Hsp90 also forms a novel complex with TBK1 and IRF3, which brings TBK1 and IRF3 dynamically into proximity and facilitates signal transduction from TBK1 to IRF3. Our study uncovers an essential role of Hsp90 in the virus-induced activation of IRF3.

scholarly journals Positive Regulation of Interferon Regulatory Factor 3 Activation by Herc5 via ISG15 Modification

2010 ◽  
Vol 30 (10) ◽  
pp. 2424-2436 ◽  
Author(s):  
He-Xin Shi ◽  
Kai Yang ◽  
Xing Liu ◽  
Xin-Yi Liu ◽  
Bo Wei ◽  
...  
Keyword(s):  
Sendai Virus ◽  
Specific Binding ◽  
Ectopic Expression ◽  
Interferon Regulatory Factor ◽  
Type I Interferons ◽  
Type I ◽  
Regulatory Factor ◽  
Interferon Regulatory Factor 3 ◽  
Antiviral Responses ◽  
Irf3 Activation

ABSTRACT Virus infection induces host antiviral responses, including induction of type I interferons. Transcription factor interferon regulatory factor 3 (IRF3) plays a pivotal role and is tightly regulated in this process. Here, we identify HERC5 (HECT domain and RLD 5) as a specific binding protein of IRF3 by immunoprecipitation. Ectopic expression or knockdown of HERC5 could, respectively, enhance or impair IRF3-mediated gene expression. Mechanistically, HERC5 catalyzes the conjugation of ubiquitin-like protein ISG15 onto IRF3 (Lys193, -360, and -366), thus attenuating the interaction between Pin1 and IRF3, resulting in sustained IRF3 activation. In contrast to results for wild-type IRF3, the mutant IRF3(K193,360,366R) interacts tightly with Pin1, is highly polyubiquitinated, and becomes less stable upon Sendai virus (SeV) infection. Consistently, host antiviral responses are obviously boosted or crippled in the presence or absence of HERC5, respectively. Collectively, this study characterizes HERC5 as a positive regulator of innate antiviral responses. It sustains IRF3 activation via a novel posttranslational modification, ISGylation.

Interferon regulatory factor 3-dependent responses to lipopolysaccharide are selectively blunted in cord blood cells

Blood ◽  
2006 ◽  
Vol 109 (7) ◽  
pp. 2887-2893 ◽  
Author(s):  
Ezra Aksoy ◽  
Valentina Albarani ◽  
Muriel Nguyen ◽  
Jean-Francois Laes ◽  
Jean-Louis Ruelle ◽  
...  
Keyword(s):  
Cord Blood ◽  
Blood Cells ◽  
Nuclear Translocation ◽  
Interferon Regulatory Factor ◽  
Creb Binding Protein ◽  
Regulatory Factor ◽  
Interferon Regulatory Factor 3 ◽  
Altered Expression ◽  
Neonatal Blood ◽  
Inducible Genes

AbstractThe synthesis of interferon-β (IFNβ) and IFN-inducible factors elicited by lipopolysaccharide (LPS) depends on the transcriptional activity of interferon regulatory factor 3 (IRF-3) downstream of Toll-like receptor-4 (TLR4). To examine the ability of human newborns to mount TLR4-mediated IRF-3–dependent responses, we analyzed the pattern of genes expressed on the addition of LPS to cord blood or cord blood monocyte-derived dendritic cells (moDCs). Expression of IFNβ and IFN-inducible genes was selectively impaired in neonatal blood and moDCs as compared with their adult counterparts. This selective defect was confirmed by microarray experiments on moDCs. Altered expression of IFN-inducible genes was related to impaired IFNβ synthesis because IFNβ signaling was functional in neonatal moDCs. However, addition of exogenous IFNβ failed to restore LPS-induced IL-12p70 synthesis which was previously shown to be defective in neonatal moDCs. Although LPS-induced IRF-3 nuclear translocation was observed both in adult and neonatal moDCs, IRF-3 DNA-binding activity and association with the coactivator CREB-binding protein (CBP) were decreased in neonatal as compared with adult moDCs. We conclude that impaired IRF-3/CBP interaction in neonatal blood cells exposed to LPS is associated with impaired expression of IFNβ and IFN-inducible genes. Because IRF-3 activity is also required for IL-12p70 synthesis, our findings provide a molecular basis for the decreased ability of LPS-stimulated neonatal moDCs to elicit Th1-type responses.

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