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Abstract: Interferon regulatory factor 3 (IRF-3) is a critical transcription factor that regulates innate immune responses against viral infection. Upon infection, IRF-3 is activated through phosphorylation of Ser/Thr residues in its C-terminal domain by the kinases, IKK-i and/or TBK-1. This phosphorylation triggers IRF-3 to interact with the co-activators to form a complex that activates target genes in the nucleus. However, the phosphorylation sites that determine the active/inactive status of IRF-3, estimated using biochemical methods such as mutagenesis and kinase assays, remain controversial. In the present study, phosphorylated IRF-3 189-427 (IRF-3 189C) was prepared by co-expression with IKK-i and was specifically fractionated into 3 major phosphorylation forms using anion-exchange chromatography. Identification of the phosphorylation sites was performed using systematic mass spectrometry approaches as follows: intact molecular mass analysis by nanoESI-MS, MS survey of phosphopeptides, and targeted MS/MS analysis of LC-MS/MS-based proteomics using a high-resolution Orbitrap mass spectrometer. Phosphorylated IRF-3 189C was clearly identified to exist as a mono-phosphoprotein (at Ser-402), and in two di-phosphoprotein forms (at Ser-386, -402 and Ser-396, -402). Thus, we demonstrated that Ser-386, -396 and -402 are directly phosphorylated by IKK-i in the co-expression system. These results will help provide new insights into the IRF-3 activation mechanism. [Copyright &y& Elsevier]

Abstract: IFN-stimulated gene 15 (ISG15), an ubiquitin-like protein, is rapidly induced by IFN-α/β, and ISG15 conjugation is associated with the antiviral immune response. Japanese encephalitis virus (JEV), a mosquito-borne neurotropic flavivirus, causes severe central nervous system diseases. We investigated the potential anti-JEV effect of ISG15 over-expression. ISG15 over-expression in human medulloblastoma cells significantly reduced the JEV-induced cytopathic effect and inhibited JEV replication by reducing the viral titers and genomes (p <0.05, Student''s t-test); it also increased activation of the interferon stimulatory response element (ISRE)-luciferase cis-acting reporter in JEV-infected cells (p <0.05, Chi-square test). Furthermore, Western blotting revealed that ISG15 over-expression increased phosphorylation of IRF-3 (Ser396), JAK2 (Tyr1007/1008) and STAT1 (Tyr701 and Ser727) in JEV-infected cells (P <0.05, Chi-square test). Confocal imaging indicated that nucleus translocation of transcription factor STAT1 occurred in ISG15-over-expressing cells but not in vector control cells post-JEV infection. ISG15 over-expression activated the expression of STAT1-dependent genes including IRF-3, IFN-β, IL-8, PKR and OAS before and post-JEV infection (p =0.063, Student''s t-test). The results enabled elucidation of the molecular mechanism of ISG15 over-expression against JEV, which will be useful for developing a novel treatment to combat JEV infection. [Copyright &y& Elsevier]

Abstract: Activation of interferon-β transcription is a highly ordered process beginning with the delivery of NF-κB to the IFN-β enhancer through a process involving stochastic interchromosomal interactions between the IFN-β enhancer and specialized Alu elements. NF-κB delivery is followed by the binding of ATF-2/c-Jun and IRF proteins in a highly cooperative fashion. The assembled “enhanceosome” then recruits PCAF/GCN5 which acetylates the histone tails of the adjacent nucleosomes. The transcriptional coactivator CBP, which binds in a complex with the RNA polymerase II holoenzyme is recruited by the enhanceosome replacing PCAF/GCN5. Next, SWI/SNF, which is part of the holoenzyme complex, induces a conformational change in a nucleosome positioned over the transcriptional start site allowing TFIID to bind, which promotes the sliding of this nucleosome to a new downstream position. At this point the full pre-initiation complex is assembled and transcription commences. This detailed picture of the IFN-β transcription program gathered through years of rigorous studies, now serves as a paradigm for understanding complex transcriptional switches in eukaryotic systems. [Copyright &y& Elsevier]

Fetal bovine lung (FBL) cells are used in the culture of viruses which infect cattle and ISG15 plays a role in innate immunity against viral infections. However, whether the expression of ISG15 gene can be induced in FBL cells is still unknown. In this work, the expression of ISG15 in cultured FBL cells was detected after stimulated with poly I:C or LPS. Real-time PCR analyses revealed that the transcript of ISG15 can be induced by poly I:C or LPS. The increased expression of free ISG15 was confirmed via Western blotting. Furthermore, immunofluorescence assays demonstrated that IRF-3 was translocated from the cytoplasm to the nucleus in the FBL cells treated with poly I:C. Chromatin immunoprecipitation assays showed that IRF-3 can bind to the promoter of the bISG15 gene. To demonstrate IRF-3 can promote the expression of bISG15, we establish a luciferase-reporter system of bovine ISG15 gene in 293 T cells. The luciferase assay showed that the over-expression of bovine IRF-3 could activate the promoter of bISG15 gene. Taken together, these results suggest that the expression of bISG15 can be induced in FBL cells stimulated with poly I:C or LPS, and IRF-3 may play a role in inducing the expression of ISG15 in FBL cells. [ABSTRACT FROM AUTHOR]

Abstract: The mechanism(s) by which DNA vaccines activate Ag-specific cellular immune responses is incompletely understood. Current findings indicate that IRF-3 plays an important role in this process. The IRF-3 dependent signaling pathway is triggered by the presence of intracytoplasmic DNA, and culminates in the production of type I IFNs. DNA vaccination of IRF-3 KO mice elicits a strong Ag-specific humoral response, yet CD4 and CD8 T cell responses (including the production of Th1, Th2 and Th17 cytokines) are severely impaired. Although expression of the immunogenic protein encoded by the DNA vaccine was similar in IRF-3 KO vs wild type mice, antigen presentation was severely impaired in the KO animals. This defect was remedied by the co-delivery of an IFNβ encoding plasmid. These findings suggest that the IRF-3/IFNβ pathways are key to the induction of cellular immunity following DNA vaccination. [Copyright &y& Elsevier]

Abstract: The cellular response to dsRNA or its synthetic analog polyinosinic–polycytidylic acid (poly I:C) results in IRF-3-, IRF-7- and NF-kB-mediated activation of type 1 IFNs and pro-inflammatory cytokines critical for innate antiviral immune responses. To investigate whether cigarette smoke compromises type 1 IFN signaling in humans, peripheral blood mononuclear cells (PBMCs) from non-smoking individuals were treated with smoke-conditioned media (SCM) and stimulated with poly I:C. We observed a marked attenuation of IRF-3 and NF-kB activation in PBMCs exposed to SCM compared to control PBMCs. Similarly, PBMCs from smokers or splenocytes from smoke-exposed mice also displayed marked reduction of poly I:C-induced antiviral responses compared with either non-smokers or sham-exposed mice. Cigarette smoke was found to block the production of type I IFNs following poly I:C treatment and inhibit subsequent STAT1 activation. Finally, we confirmed that inhibition of IFN-β, but not IFN-α, predominantly contributes to the cigarette smoke-mediated suppression of innate antiviral responses. These findings provide novel mechanistic insights to the susceptibility of cigarette smokers to viral infections. [Copyright &y& Elsevier]

Abstract: The innate cellular response to virus particle entry in non-immune cells requires the transcriptional activity of interferon regulatory factor 3 (IRF-3), but not production of type I interferon (IFN). Here, we characterize the IFN-independent innate cellular response to virus-derived stimuli in Vero cells, a monkey kidney epithelial cell line deficient for IFN production. We provide evidence that Vero cells are deficient in their ability to mount an IRF-3-dependent, IFN-independent antiviral response against either incoming virus particles or polyinosinic:polycytidylic acid (pIC), a dsRNA mimetic. We further demonstrate that abundance of IRF-3 protein is a determinant in the pIC-mediated antiviral signalling pathway. These observations further characterize the permissive nature of Vero cells to viral infection, and highlight the crucial involvement of IRF-3 in the innate antiviral response. [Copyright &y& Elsevier]

Abstract: Assembly of interferon-β enhanceosome from its individual protein components and of enhancer DNA has been studied in solution using a combination of fluorescence anisotropy, microcalorimetry, and CD titration. It was shown that the enhancer binds only one full-length phosphomimetic IRF-3 dimer at the PRDIII–PRDI sites, and this binding does not exhibit cooperativity with binding of the ATF-2/c-Jun bZIP (leucine zipper dimer with basic DNA recognition segments) heterodimer at the PRDIV site. The orientation of the bZIP pair is, therefore, not determined by the presence of the IRF-3 dimer, but is predetermined by the asymmetry of the PRDIV site. In contrast, bound IRF-3 dimer interacts strongly with the NF-κB (p50/p65) heterodimer bound at the neighboring PRDII site. The orientation of bound NF-κB is also predetermined by the asymmetry of the PRDII site and is the opposite of that found in the crystal structure. The HMG-I/Y protein, proposed as orchestrating enhanceosome assembly, interacts specifically with the PRDII site of the interferon-β enhancer by inserting its DNA-binding segments (AT hooks) into the minor groove, resulting in a significant increase in NF-κB binding affinity for the major groove of this site. [Copyright &y& Elsevier]

Epstein–Barr virus-encoded small RNA (EBER) is nonpolyadenylated, noncoding RNA, forms stem-loop structure by intermolecular base-pairing giving rise to double-stranded RNA (dsRNA)-like molecule and exists abundantly in EBV-infected cells. EBER induces IL-10 and thus supports the growth of Burkitt's lymphoma (BL) cells. In this study, the mechanism of IL-10 induction by EBER was analysed in the context of dsRNA signaling pathway. Knockdown of retinoic acid-inducible gene I (RIG-I) by small interfering RNA (siRNA), and expression of dominant-negative RIG-I downregulated IL-10 induction in EBER(+) EBV-infected and EBER plasmid-transfected BL cells. Transfection of EBER-expressing plasmid or in vitro synthesized EBER induced IL-10 in RIG-I-expressing cell clones, and activation of IL-10 promoter by EBER was blocked by dominant-negative RIG-I. Blocking of nuclear factor (NF)-κB by dominant-negative IκB-α plasmid did not block IL-10 expression, whereas knockdown of IRF-3 by siRNA resulted in downregulation of IL-10 in EBER(+) BL cells. NF-κB is reported to function downstream of RIG-I signaling pathway and is involved in the induction of proinflammatory cytokines. Our results indicate that EBER induces an anti-inflammatory cytokine IL-10 through RIG-I-mediated IRF-3 but not NF-κB signaling. These findings suggest a new mechanism of dsRNA signaling pathway that triggers the expression of IL-10, which acts as an autocrine growth factor in BL cells.Oncogene (2008) 27, 4150–4160; doi:10.1038/onc.2008.75; published online 24 March 2008 [ABSTRACT FROM AUTHOR]

Abstract: The type I interferon (IFN) response requires the coordinated activation of the latent transcription factors NF-κB, IRF-3 and ATF-2 which in turn activate transcription from the IFN-β promoter. Here we have examined the type I interferon response in rhinovirus type 14-infected A549 cells, with particular emphasis on the status of the transcription factor IRF-3. Our results indicate that although rhinovirus type 14 (RV14) infection induces the activation of NF-κB and ATF-2, only very low levels of IFN-β mRNA are detected. Analysis of ISG54 mRNA levels revealed very little induction of this IRF-3 responsive transcript and suggested that IRF-3 activation might be impaired. Examination of IRF-3 in RV14-infected cells demonstrated only low levels of phosphorylation, a lack of homodimer formation and an absence of nuclear accumulation indicating that this transcription factor is not activated. Inhibition of viral protein synthesis following infection resulted in an increase in IFN-β mRNA levels indicating that viral gene products prevent induction of this pathway. Collectively, these results indicate that RV14 infection inhibits the host type I interferon response by interfering with IRF-3 activation. [Copyright &y& Elsevier]

Abstract: Antiviral immunity in mammals involves several levels of surveillance and effector actions by host factors to detect viral pathogens, trigger α/β interferon production, and to mediate innate defenses within infected cells. Our studies have focused on understanding how these processes are regulated during infection by hepatitis C virus (HCV) and West Nile virus (WNV). Both viruses are members of the Flaviviridae and are human pathogens, but they each mediate a very different disease and course of infection. Our results demonstrate common and unique innate immune interactions of each virus that govern antiviral immunity and demonstrate the central role of α/β interferon immune defenses in controlling the outcome of infection. [Copyright &y& Elsevier]

Abstract: An effective type I interferon (IFN-α/β) response is critical for the control of many viral infections. Using an oncolytic strain of vesicular stomatitis virus, we have examined the cross-talk between virus-induced apoptosis and initiation of innate immune response. The intrinsic apoptotic cascade, specifically the Bax-Bcl-2-Caspase-9 cascade, was revealed as the primary pathway of VSV-induced apoptosis. Cell death was significantly reduced in BaxBak −/− murine embryonic fibroblasts (MEFs) and in human A549 epithelial cells treated with siRNA against Bax. Although inhibition of apoptosis resulted in enhanced virus replication in the BaxBak −/− MEFs as compared to wild-type cells, induction of the IFN antiviral response and expression of cytokine genes were attenuated in virus-infected cells. Moreover, Bax but not Bak pro-apoptotic protein was required for IRF-3 phosphorylation and activation, further substantiating a role for the intrinsic mitochondrial pathway in the innate immune response. Therefore, virus-induced apoptosis through a Bax-dependent mitochondrial pathway appears to enhance the full development of the IRF-3 mediated IFN antiviral response. [Copyright &y& Elsevier]

Abstract: The present study was undertaken to clarify the role of Sendai virus (SeV) V protein, which has been shown to downregulate IFN-β induction through inhibition of IRF-3 activation, in viral pathogenesis. Mice infected with rSeV mutants, deficient in V expression or expressing V lacking the C-terminus, had several-fold higher IFN activity levels in the lungs than those in wild-type virus-infected mice, and the mutant viruses were rapidly excluded from the lung from the early phase of infection before induction of acquired immunity. In addition, the unique early clearance of the mutants did not occur in IRF-3 knockout (KO) mice. However, high titers of IFN were detected even in the infected KO mice. Furthermore, early clearance of the mutant viruses was also observed in IFN signaling-deficient mice, IFN-α/β receptor KO mice and STAT1 KO mice. These results indicate that SeV V protein counteracts IRF-3-mediated innate antiviral immunity for efficient virus replication and pathogenesis in mice, but it is not IFN. [Copyright &y& Elsevier]

To characterize the mechanisms underlying apoptosis induced by viral infection, transcriptional activation of genes encoding members of the ‘BH3-only’ family of proteins was analysed during the course of virus infection. Among these genes, only NOXA is transcriptionally activated by vesicular stomatitis virus (VSV), sendai virus (SV), measles virus, herpes simplex virus, or dsRNA and required for efficient apoptosis of cells. Transcriptional activation of NOXA by VSV or SV is independent of p53, but requires the presence of interferon regulatory factor 1 (IRF-1), IRF-3 and cAMP-responsive element binding protein (CREB). Binding to and transactivation of the NOXA promoter by each of these transcription factors is governed by post-translational modification involving different pathways for each factor. Thus, SV infection activates IRF-3 and CREB by phosphorylation triggered by Toll like receptor 3 signalling, and a pathway involving calcium-independent phopholipase A2, respectively. In addition transactivation induced by IRF-1 during viral infection correlates with a 10 kDa increase in its molecular weight, suggesting a covalent linkage with a previously unknown regulatory polypeptide.Oncogene (2007) 26, 328–338. doi:10.1038/sj.onc.1209795; published online 10 July 2006 [ABSTRACT FROM AUTHOR]

Background/Aims: The development of an efficient in vitro infection system for HCV is important in order to develop new anti-HCV strategy. Only Huh7 hepatocyte cell lines were shown to be infected with JFH-1 fulminant HCV-2a strain and its chimeras. Here we aimed to establish a primary hepatocyte cell line that could be infected by HCV particles from patients’ sera. Methods: We transduced primary human hepatocytes with human telomerase reverse transcriptase together with human papilloma virus 18/E6E7 (HPV18/E6E7) genes or simian virus large T gene (SV40 T) to immortalize cells. We also established the HPV18/E6E7-immortalized hepatocytes in which interferon regulatory factor-7 was inactivated. Finally we analyzed HCV infectivity in these cells. Results: Even after prolonged culture HPV18/E6E7-immortalized hepatocytes exhibited hepatocyte functions and marker expression and were more prone to HCV infection than SV40 T-immortalized hepatocytes. The susceptibility of HPV18/E6E7-immortalized hepatocytes to HCV infection was further improved, in particular, by impairing signaling through interferon regulatory factor-7. Conclusions: HPV18/E6E7-immortalized hepatocytes are useful for the analysis of HCV infection, anti-HCV innate immune response, and screening of antiviral agents with a variety of HCV strains. [Copyright &y& Elsevier]

Abstract: Type I IFN (IFN-α/β) have important biological functions ranging from immune cell development and activation, to tumor cell killing and most importantly inhibition of virus replication. Following viral infection or activation of Toll-like receptors (TLRs) via distinct ligands, IFN-α/β are produced. Two members of the interferon regulatory factor (IRF) family – IRF-3 and IRF-7 – are the major modulators of IFN gene expression. Activation of IRF-3 and IRF-7 by TBK1/IKKɛ mediated phosphorylation promotes IFN gene expression and potentiates the production of IFN responsive genes important to the development of an effective antiviral immune response. IFN treatment can augment anti-tumor properties and they are potentially key players in cancer therapy. For example, adoptive transfer of IFN-γ-activated macrophages can mediate tumor cell killing via direct cell–cell contact, as well as release of soluble cytotoxic pro-inflammatory molecules. A recent study investigated whether IRF-3 and IRF-7 could mediate the acquisition of new anti-tumor effector functions in macrophages. Adenovirus mediated transduction of the active form of IRF-7 into primary macrophages resulted in the production of type I IFN, upregulation of target genes including TRAIL and increased tumoricidal activity of macrophages; in contrast, the active form of IRF-3 led to induction of cell death. These studies indicate that IRF-7 transduced macrophages may be an attractive candidate for in vivo adoptive therapy of cancer. [Copyright &y& Elsevier]

Abstract: Macrophages responding to viral infections may contribute to autoimmune demyelinating diseases (ADD). Macrophages from ADD-susceptible SJL/J mice responding to Theiler''s Virus (TMEV) infection, the TLR7 agonist loxoribine, or the TLR4 agonist-LPS expressed less IL-12 p35 but more IL-12/23 p40 and IFN-β than macrophages from ADD-resistant B10.S mice. While expression of IRF-1 and -7 was similar between B10.S and SJL/J TMEV-infected macrophages, SJL/J but not B10.S macrophages exhibited constitutively active IRF-3. In contrast to overexpressed IRF-1, IRF-5, and IRF-7, which stimulated p35 promoter reporter activity, overexpressed IRF-3 repressed p35 promoter activity in response to TMEV infection, loxoribine, IFN-γ/LPS, but not IFN-γ alone. IRF-3 lessened but did not eliminate IRF-1-stimulated p35 promoter activity. Repression by IRF-3 required bp −172 to −122 of the p35 promoter. The data suggest that pre-activated IRF-3 is a major factor in the differences in IL-12 production between B10.S and SJL/J macrophages responding to TMEV. [Copyright &y& Elsevier]

Background: Interferon (IFN)-γ-inducible protein of 10 kDa (IP-10/CXCL10) is a potent chemoattractant for activated T and NK cells, and elevated levels of IP-10 are identified in bronchoalveolar lavage fluids from patients with pulmonary disorders related to Th-1-type immunity, which is a prerequisite for elimination of viral pathogens. Bronchial epithelial cells play an important role in respiratory infections as the initiator of airway inflammation by releasing chemokines and expressing cell surface membrane molecules involved in leukocyte adhesion. Polyinosinic-polycytidylic acid (poly IC) is a synthetic double-stranded RNA (dsRNA) and induces antiviral reactions in cells. Objectives: We investigated the regulation of IP-10 in BEAS-2B bronchial epithelial cells in response to poly IC, and also addressed the possible role of retinoic-acid-inducible gene-I (RIG-I) and IFN-regulatory factor 3 (IRF-3), two genes involved in the signaling induced by viral infection. Methods: The expressions of IP-10 mRNA and protein were analyzed by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. The overexpression of RIG-I or IRF-3 was performed by transfection of BEAS-2B cells with each cDNA. Results: Poly IC enhanced the expression of IP-10 mRNA and protein in concentration- and time-dependent manners. Overexpression of RIG-I or IRF-3 potentiated the poly-IC-induced upregulation of IP-10. Conclusions: IP-10 may contribute to antiviral activity through the activation of Th-1-type immunity, and RIG-I and IRF-3 may be involved in this reaction. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Dendritic cells (DC), although a minor population in hematopoietic cells, produce type I interferons (IFN) and other cytokines and are essential for innate immunity. They are also potent antigen presenters and regulate adaptive immunity. Among DC subtypes plasmacytoid DC (pDC) produce the highest amounts of type I IFN. In addition, pro- and anti-inflammatory cytokines such as IL-12 and IL-10 are induced in DC in response to Toll like receptor (TLR) signaling and upon viral infection. Proteins in the IRF family control many aspects of DC activity. IRF-8 and IRF-4 are essential for DC development. They differentially control the development of four DC subsets. IRF-8−/− mice are largely devoid of pDC and CD8α+ DC, while IRF-4−/− mice lack CD4+DC. IRF-8−/−, IRF4−/−, double knock-out mice have only few CD8á -CD4−DC that lack MHC II. IRF proteins also control type I IFN induction in DC. IRF-7, activated upon TLR signaling is required for IFN induction not only in pDC, but also in conventional DC (cDC) and non-DC cell types. IRF-3, although contributes to IFN induction in fibroblasts, is dispensable in IFN induction in DC. Our recent evidence reveals that type I IFN induction in DC is critically dependent on IRF-8, which acts in the feedback phase of IFN gene induction in DC. Type I IFN induction in pDC is mediated by MyD88 dependent signaling pathway, and differs from pathways employed in other cells, which mostly rely on TLR3 and RIG-I family proteins. Other pro-inflammatory cytokines are produced in an IRF-5 dependent manner. However, IRF-5 is not required for IFN induction, suggesting the presence of separate mechanisms for induction of type I IFN and other pro-inflammatory cytokines. IFN and other cytokines produced by activated DC in turn advance DC maturation and change the phenotype and function of DC. These processes are also likely to be governed by IRF family proteins.Cell Research (2006) 16: 134–140. doi:10.1038/sj.cr.7310018; published online 13 February 2006 [ABSTRACT FROM AUTHOR]

Abstract: In this study, we found that infection with flaviviruses, such as Japanese encephalitis virus (JEV) and dengue virus serotype 2 (DEN-2), leads to interferon-β (IFN-β) gene expression in a virus-replication- and de novo protein-synthesis-dependent manner. NF-κB activation is essential for IFN-β induction in JEV- and DEN-2-infected cells. However, these two viruses seem to preferentially target different members of the interferon regulatory factor (IRF) family. The activation of constitutively expressed IRF-3, characterized by slower gel mobility, dimer formation, and nuclear translocation, is more evident in JEV-infected cells. Other members of the IRF family, such as IRF-1 and IRF-7 are also induced by DEN-2, but not by JEV infection. The upstream molecules responsible for IRF-3 and NF-κB activation were further studied. Evidently, a cellular RNA helicase, retinoic acid-inducible gene I (RIG-I), and a cellular kinase, phosphatidylinositol-3 kinase (PI3K), are required for flavivirus-induced IRF-3 and NF-κB activation, respectively. Therefore, we suggest that JEV and DEN-2 initiate the host innate immune response through a molecular mechanism involving RIG-I/IRF-3 and PI3K/NF-κB signaling pathways. [Copyright &y& Elsevier]

Viral infection or TLR3 engagement causes activation of the transcription factors IRF-3 and NF-κB, which collaborate to induce transcription of type I IFN genes. IKKɛ and TBK1 are two IKK-related kinases critically involved in virus- and TLR3-triggered activation of IRF-3. We identified a protein termed SIKE (for Suppressor of IKKɛ) that interacts with IKKɛ and TBK1. SIKE is associated with TBK1 under physiological condition and dissociated from TBK1 upon viral infection or TLR3 stimulation. Overexpression of SIKE disrupted the interactions of IKKɛ or TBK1 with TRIF, RIG-I and IRF-3, components in virus- and TLR3-triggered IRF-3 activation pathways, but did not disrupt the interactions of TRIF with TRAF6 and RIP, components in TLR3-triggered NF-κB activation pathway. Consistently, overexpression of SIKE inhibited virus- and TLR3-triggered interferon-stimulated response elements (ISRE) but not NF-κB activation. Knockdown of SIKE potentiated virus- and TLR3-triggered ISRE but not NF-κB activation. Moreover, overexpression of SIKE inhibited IKKɛ- and TBK1-mediated antiviral response. These findings suggest that SIKE is a physiological suppressor of IKKɛ and TBK1 and plays an inhibitory role in virus- and TLR3-triggered IRF-3 but not NF-κB activation pathways. [ABSTRACT FROM AUTHOR]

Abstract: Toll-like receptor (TLR) 3 and 4 mediate the expression of many genes, including NF-κB- and interferon-regulatory factor (IRF)-3/interferon (IFN)-inducible genes, in macrophages and dendritic cells (DCs) in response to their ligand stimuli, polyI:C and lipopolysaccharide (LPS). Toll-IL-1 receptor homology domain (TIR)-containing adapter molecule 1 (TICAM-1) facilitates expression of IFN-inducible genes via TLR3. Although MyD88 and Mal/TIRAP adapters function downstream of TLR4, they barely induce IFN-β. In addition, DC maturation as well as IFN-β induction are largely independent of MyD88 and Mal/TIRAP. TICAM-1 is the functional adapter for both TLR3 and TLR4 that induces type 1 IFN and MyD88-independent DC maturation. In LPS-mediated TLR4 activation, a complex of TICAM-1 and an additional TLR4-binding adapter serves as the adapter. We named this TLR4-TICAM-1-bridging adapter TICAM-2. Our results reveal the details of MyD88-independent pathways which separately recruit the distinct adapters downstream of TLR3 and TLR4 and variations of the TLR output are in part regulated by the two additional adapters in DCs. [Copyright &y& Elsevier]

Transcriptional activation of the interferon-ß(IFN-ß) gene requires assembly of an enhanceosome containing the transcription factors ATF-2/c-Jun, IRF-3/IRF-7, NF-?B and HMGI(Y). These factors cooperatively bind a composite DNA site and activate expression of the IFN-ßgene. The 3.0Åcrystal structure of the DNA-binding domains of ATF-2/c-Jun and two IRF-3 molecules in a complex with 31 base pairs (bp) of the PRDIV-PRDIII region of the IFN-ßenhancer shows that association of the four proteins with DNA creates a continuous surface for the recognition of 24 bp. The structure, together with in vitro binding studies and protein mutagenesis, shows that protein-protein interactions are not critical for cooperative binding. Instead, cooperativity arises mainly through nucleotide sequence-dependent structural changes in the DNA that allow formation of complementary DNA conformations. Because the binding sites overlap on the enhancer, the unit of recognition is the entire nucleotide sequence, not the individual subsites. [ABSTRACT FROM AUTHOR]

We here report a molecular basis for downregulation of interferon (IFN)-β production by V and C proteins of Sendai virus (SeV). The infection of HeLa cells with SeV poorly induced IFN-β even if the expression of C/C′ was disrupted. In contrast, when the expression of C/C′/Y1/Y2 or V/W was disrupted, SeV infection strongly induced IFN-β production and significantly activated the interferon regulatory factor (IRF)-3 pathway. The independent expression of C or V inhibited the double-stranded (ds) RNA- or Newcastle disease virus (NDV)-induced activation of IRF-3 and NF-κB, as well as the IFN-β promoter. This inhibitory effect was also observed when Y1, Y2, or a C-terminal half fragment (aa 85–204) of C was independently expressed. Phosphorylation and homodimer formation of IRF-3 were suppressed not only in cells infected with SeV capable of expressing both C/C′/Y1/Y2 (or Y1/Y2) and V/W, but also in HeLa cells constitutively expressing Y1. These results suggest that C, Y1, Y2, and V block signaling pathways leading to IRF-3 activation to downregulate IFN-β production. [Copyright &y& Elsevier]

The ubiquitously expressed interferon regulatory factor 3 (IRF-3) is directly activated following virus infection and functions as a key activator of the immediate-early Type 1 interferon (IFN) genes. Using DNA microarray analysis (8,556 genes) in Jurkat T cells inducibly expressing constitutively active IRF-3, several target genes directly regulated by IRF-3 were identified. Among the genes upregulated by IRF-3 were transcripts for a subset of known IFN-stimulated genes (ISGs), including ISG56, which functions as an inhibitor of translation initiation. Phosphorylation of C-terminal Ser/Thr residues-382 GGASSLENTVDLHISNSHPLSLTSDQY408-is required for IRF-3 activation. Using C-terminal point mutations and a novel phosphospecific antibody, Ser396 was characterized as the minimal phosphoacceptor site required in vivo for IRF-3 activation following Sendai virus (SeV) infection, expression of viral nucleocapsid, or double-stranded RNA (dsRNA) treatment. The identity of the virus-activated kinase (VAK) activity that targets and activates IRF-3 and IRF-7 has remained a critical missing link in the understanding of interferon signaling. We report that the IKK-related kinases-IKKϵ/TBK-1-are components of VAK that mediate IRF-3 and IRF-7 phosphorylation and thus functionally link the NF-κB and IRF pathways in the development of the antiviral response. [ABSTRACT FROM AUTHOR]

Type I interferons (IFN-α/β) affect many aspects of immune responses. Many pathogen-associated molecules, including bacterial lipopolysaccharide (LPS) and virus-associated double-stranded RNA, induce IFN gene expression through activation of distinct Toll-like receptors (TLRs). Although much has been studied about the activation of the transcription factor IRF-3 and induction of IFN-β gene by the LPS-mediated TLR4 signaling, definitive evidence is missing about the actual role of IRF-3 in LPS responses in vitro and in vivo. Using IRF-3 deficient mice, we show here that IRF-3 is indeed essential for the LPS-mediated IFN-β gene induction. Loss of IRF-3 also affects the expression of profile of other cytokine/chemokine genes. We also provide evidence that the LPS/TLR4 signaling activates IRF-7 to induce IFN-β, if IRF-7 is induced by IFNs prior to LPS simulation. Finally, the IRF-3-deficient mice show resistance to LPS-induced endotoxin shock. These results place IRF-3 as a molecule central to LPS/TLR4 signaling. [Copyright &y& Elsevier]

Virus infection results in the activation of a set of cellular genes involved in host antiviral defense. IRF-3 has been identified as a critical transcription factor in this process. The activation mechanism of IRF-3 is not fully elucidated, yet it involves a conformational change triggered by the virus-dependent phosphorylation of its C-terminus. This conformational change leads to nuclear accumulation, DNA binding and transcriptional transactivation. Here we show that two distinct sets of Ser/Thr residues of IRF-3, on phosphorylation, synergize functionally to achieve maximal activation. Remarkably, we find that activated IRF-3 lacks transcriptional activity, but activates transcription entirely through the recruitment of the p300/CBP coactivators. Moreover, we show that two separate domains of IRF-3 interact with several distinct regions of p300/CBP. Interference with any of these interactions leads to a complete loss of transcriptional activity, suggesting that a bivalent interaction is essential for coactivator recruitment by IRF-3. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

Cellular genes including the type I interferon genes are activated in response to viral infection. We previously reported that IRF-3 (interferon regulatory factor 3) is specifically phosphorylated on serine residues and directly transmits a virus-induced signal from the cytoplasm to the nucleus, and then participates in the primary phase of gene induction. In this study, we analyzed the molecular mechanism of IRF-3 activation further. The formation of a stable homomeric complex of IRF-3 between the specifically phosphorylated IRF-3 molecules occurred. While virus-induced IRF-7 did not bind to p300, the phosphorylated IRF-3 complex formed a stable multimeric complex with p300 (active holocomplex). Competition using a synthetic phosphopeptide corresponding to the activated IRF-3 demonstrated that p300 directly recognizes the structure in the vicinity of the phosphorylated residues of IRF-3. These results indicated that the phos-phorylation of serine residues at positions 385 and 386 is critical for the formation of the holocomplex, presumably through a conformational switch facilitating homodimer formation and the generation of the interaction interface with CBP/p300. [ABSTRACT FROM AUTHOR]

Vaccinia virus (VACV) is an enveloped DNA virus from the Orthopoxvirus family, various strains of which were used in the successful eradication campaign against smallpox. Both original and newer VACV-based replicating vaccines reveal a risk of serious complications in atopic individuals. VACV encodes various factors interfering with host immune responses at multiple levels. In atopic skin, the production of type I interferon is compromised, while VACV specifically inhibits the phosphorylation of the Interferon Regulatory Factor 3 (IRF-3) and expression of interferons. To overcome this block, we generated a recombinant VACV-expressing murine IRF-3 (WR-IRF3) and characterized its effects on virus growth, cytokine expression and apoptosis in tissue cultures and in spontaneously atopic Nc/Nga and control Balb/c mice. Further, we explored the induction of protective immune responses against a lethal dose of wild-type WR, the surrogate of smallpox. We demonstrate that the overexpression of IRF-3 by WR-IRF3 increases the expression of type I interferon, modulates the expression of several cytokines and induces superior protective immune responses against a lethal poxvirus challenge in both Nc/Nga and Balb/c mice. Additionally, the results may be informative for design of other virus-based vaccines or for therapy of different viral infections. [ABSTRACT FROM AUTHOR]

It has been hypothesized that certain viral infections directly activate a transcription factor(s) which is responsible for the activation of genes encoding type I interferons (IFNs) and interferon-stimulated genes (ISGs) via interferon regulatory factor (IRF) motifs present in their respective promoters. These events trigger the activation of defense machinery against viruses. Here we demonstrate that IRF-3 transmits a virus-induced signal from the cytoplasm to the nucleus. In unstimulated cells, IRF-3 is present in its inactive form, restricted to the cytoplasm due to a continuous nuclear export mediated by nuclear export signal, and it exhibits few DNA-binding properties. Virus infection but not IFN treatment induces phosphorylation of IRF-3 on specific serine residues, thereby allowing it to complex with the co-activator CBP/p300 with simultaneous nuclear translocation and its specific DNA binding. We also show that a dominant-negative mutant of IRF-3 could inhibit virus-induced activation of chromosomal type I IFN genes and ISGs. These findings suggest that IRF-3 plays an important role in the virus-inducible primary activation of type I IFN and IFN-responsive genes. [ABSTRACT FROM AUTHOR]

Type I interferons (IFNs) are produced by most cells in response to virus infection and stimulate a program of anti-viral gene expression in neighboring cells to suppress virus replication. Type III IFNs have similar properties, however their effects are limited to epithelial cells at mucosal surfaces due to restricted expression of the type III IFN receptor. Rotavirus (RV) replicates in intestinal epithelial cells that respond predominantly to type III IFNs, and it has been shown that type III rather than type I IFNs are important for controlling RV infections in vivo. The RV NSP1 protein antagonizes the host type I IFN response by targeting IRF-3, IRF-5, IRF-7, or β-TrCP for proteasome-mediated degradation in a strain-specific manner. Here we provide the first demonstration that NSP1 proteins from several human and animal RV strains antagonize type III as well as type I IFN induction. We also show that NSP1 is a potent inhibitor of IRF-1, a previously undescribed property of NSP1 which is conserved among human and animal RVs. Interestingly, all NSP1 proteins were substantially more effective inhibitors of IRF-1 than either IRF-3 or IRF-7 which has significance for evasion of basal anti-viral immunity and type III IFN induction in the intestinal epithelium. [ABSTRACT FROM AUTHOR]

• The first study showing PRRSV infection induces the activation of the Wnt/β-catenin signaling pathway via multiple structural proteins and Nsps. • β-catenin is an important antiviral factor in PRRSV infection. The Wnt/β-catenin signaling pathway is an evolutionarily highly conserved signaling pathway related to the replication of various viruses. However, the interaction between the Wnt/β-catenin pathway and porcine reproductive and respiratory syndrome virus (PRRSV) is unknown. In the present study, we showed that PRRSV-infected Marc-145 and PAM cells expressed high levels of c-myc and cyclinD1 mRNA and accumulation of β-catenin in the nucleus. PRRSV nonstructural proteins (Nsps) 1α, 1β, 3, 4, 7, 10, and 12, and proteins encoded by open reading frames (ORFs) 2b, 3, and 5 induced the activation of the Wnt pathway according to TOP/FOP luciferase reporter assay. But, Nsp5 inhibited the activation of the Wnt pathway. Pre-treatment with Wnt3a inhibited PRRSV replication in Marc-145 cells in a dose-dependent manner. Over-expression of β-catenin also inhibited PRRSV replication, while silencing of β-catenin by small hairpin RNA increased its replication in Marc-145 cells. Over-expression of β-catenin increased interferon regulatory factor (IRF)-3 expression and nuclear factor (NF)-κB phosphorylation, NF-κB and interferon-stimulated response element promoter activities, and interferon-β, DExD/H-box helicase 58 (DDX58), interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-8 mRNA expression. Conversely, silencing β-catenin decreased phosphorylated IRF-3 and NF-κB, NF-κB and IFIT1 promoter activities, and IFN-β, DDX58, IFIT1, IL-1β, TNF-α, and IL-8 mRNA levels in Marc-145 cells. Co-immunoprecipitation and immunofluorescence colocalization analyses confirmed that β-catenin interacted with NF-κB in Marc-145 cells. In conclusion, PRRSV infection activates the Wnt/β-catenin signaling pathway via Nsps 1α, 1β, 3, 4, 7, 10, and 12, and proteins encoded by ORFs 2b, 3, and 5. The Wnt/β-catenin pathway then inhibits PRRSV replication by enhancing the NF-κB-dependent innate immune response. These findings further our understanding of the role of the Wnt/β-catenin signaling pathway in regulating PRRSV replication and provide new insights into virus–host interactions. [ABSTRACT FROM AUTHOR]

Nipah and Hendra viruses are highly pathogenic, zoonotic henipaviruses that encode proteins that inhibit the host's innate immune response. The W protein is one of four products encoded from the P gene and binds a number of host proteins to regulate signalling pathways. The W protein is intrinsically disordered, a structural attribute that contributes to its diverse host protein interactions. Here, we review the role of W in innate immune suppression through inhibition of both pattern recognition receptor (PRR) pathways and interferon (IFN)-responsive signalling. PRR stimulation leading to activation of IRF-3 and IFN release is blocked by henipavirus W, and unphosphorylated STAT proteins are sequestered within the nucleus of host cells by W, thereby inhibiting the induction of IFN stimulated genes. We examine the critical role of nuclear transport in multiple functions of W and how specific binding of importin-alpha (Impα) isoforms, and the 14-3-3 group of regulatory proteins suggests further modulation of these processes. Overall, the disordered nature and multiple functions of W warrant further investigation to understand henipavirus pathogenesis and may reveal insights aiding the development of novel therapeutics. [ABSTRACT FROM AUTHOR]

Cytokines and chemokines are transcriptionally regulated by inflammatory transcription factors such as nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and interferon regulatory factor (IRF)-3. A daidzein derivative compound, 8-hydroxydaidzein (8-HD), isolated from soy products, has recently gained attention due to various pharmacological benefits, including anti-inflammatory activities. However, regulation of the inflammatory signaling mechanism for 8-HD is still poorly understood, particularly with respect to the IRF-3 signaling pathway. In this study, we explored the molecular mechanism of 8-HD in regulating inflammatory processes, with a focus on the IRF-3 signaling pathway using a lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid [Poly (I:C)] stimulated murine macrophage cell line (RAW264.7). The 8-HD downregulated the mRNA expression level of IRF-3-dependent genes by inhibiting phosphorylation of the IRF-3 transcription factor. The inhibitory mechanism of 8-HD in the IRF-3 signaling pathway was shown to inhibit the kinase activity of IKKε to phosphorylate IRF-3. This compound can also interfere with the TRIF-mediated complex formation composed of TRAF3, TANK, and IKKε leading to downregulation of AKT phosphorylation and reduction of IRF-3 activation, resulted in inhibition of IRF-3-dependent expression of genes including IFN-β, C-X-C motif chemokine 10 (CXCL10), and interferon-induced protein with tetratricopeptide repeats 1 (IFIT1). Therefore, these results strongly suggest that 8-HD can act as a promising compound with the regulatory function of IRF-3-mediated inflammatory responses. [ABSTRACT FROM AUTHOR]