Search

Transcription factor IRF-3 is post-translationally activated by Toll-like receptor (TLR) signaling and has critical roles in the regulation of innate immunity. Here we present the X-ray crystal structure of the C-terminal regulatory domain of IRF-3(175-427) (IRF-3 175C) at a resolution of 2.3 Å. IRF-3 175C is structurally similar to the Mad homology domain 2 of the Smad family. Structural and functional analyses reveal phosphorylation-induced IRF-3 dimerization, which generates an extensive acidic pocket responsible for binding with p300/CBP. Although TLR and Smad signaling are evolutionarily independent, our results suggest that IRF-3 originates from Smad and acquires its function downstream of TLR. [ABSTRACT FROM AUTHOR]

Infections of bacteria and viruses induce host defense reactions known as innate responses that include the production of cytokines and chemokines. The production of type I interferon (IFN) is known to be induced by viral double-stranded (ds) RNA or bacterial lipopolysaccharide (LPS). Although important functions for the transcription factors NF-κB and interferon regulatory factor-3 (IRF-3) are indicated, the molecular signals leading to the activation of IFN genes have yet to be elucidated. We provide several lines of evidence that LPS and dsRNA trigger distinct intracellular signals upstream. Notably, our investigation revealed a critical function for TIRAP/MAL, a signaling adapter for Toll-like receptor (TLR) 4, in LPS-induced but not dsRNA-induced activation of IRF-3. These results highlight cross-talk between TLR-mediated and virus/dsRNA-induced signals resulting in activation of the IFN system. [Copyright &y& Elsevier]

Backgrounds: Infection by virus or treatment with double-stranded RNA (dsRNA) results in the activation of transcription factors including IRF-3, IRF-7 and a pleiotropic regulator NF-κB by specific phosphorylation. These factors are important in triggering a cascade of antiviral responses. A protein kinase that is yet to be identified is responsible for the activation of these factors and plays a key role in the responses. Results: The signal cascade was analysed using sensitive assays for the activation of IRF-3 and NF-κB, and various inhibitors. We found that the activation of IRF-3 and NF-κB by dsRNA or virus involves a process that is sensitive to Geldanamycin. Although the induction of NF-κB by dsRNA/virus and TNF-α involves common downstream pathways including IKK activation, the upstream, Geldanamycin-sensitive process was unique to the dsRNA/virus-induced signal. By an in vitro assay using cell extract, we found an inducible protein kinase activity with physiological specificity of IRF-3 phosphorylation. Furthermore, the same extract specifically phosphorylated IRF-7 in a similar manner. Conclusions: Double-stranded RNA or virus triggers a specific signal cascade that results in the activation of the IRF-3/-7 kinase we detected, which corresponds to the long-sought signalling machinery that is responsible for triggering the early phase of innate response. The signal branches to a common NF-κB activation cascade, thus resulting in the activation of a set of critical transcription factors for the response. [ABSTRACT FROM AUTHOR]

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]

Interferon regulatory factor (IRF)-1 and IRF-2 have been implicated for the virus-induced expression of the interferon-α and β (type I IFN) genes. However, recent gene disruption studies in mice suggested the presence of other factor(s) interacting with overlapping promoter elements. In the present paper, we describe the characterization of a DNA binding factor which is strongly induced after virus infection and recognizes these promoter elements. After extensive purification, the factor was revealed to be identical to IFN-stimulated gene factor 3 (ISGF3), a transcription factor complex activated by IFN treatment. ISGF3 binds to the promoter element of IFN-β positive regulatory domain I (PRDI), with significantly higher affinity than IRF-1, 2, and mutational analysis of PRDI showed that the gene expression and binding of ISGF3, but not of IRF-1, 2, are highly correlated. Furthermore, our functional analysis involving a dominant negative inhibitor for ISGF3 activation and an anti-IFN neutralizing antibody clearly demonstrated the presence of a positive feedback pathway for type I IFN genes mediated by ISGF3. [ABSTRACT FROM AUTHOR]

Interferon stimulated gene factor 3 (ISGF3) is a trimeric transcription factor activated on treatment of cells with interferon-α and β (type I EFNs). Upon stimulation, the regulatory subunits, p84/91 and pll3, present in the cytoplasm are phosphorylated at specific tyrosine residues and assemble with the DNA binding subunit, ISGF3γ, into the active ISGF3 in the nucleus. Thus, ISGF3 plays a primary role in the transmission of a signal from the cell surface to the nucleus. In this report, we describe the cloning of a mouse cDNA encoding a polypeptide homologous to human ISGF3γ. Comparison of the deduced amino acid sequences revealed the middle region was significantly different between mouse and man. The mouse cDNA was shown to encode a functional ISGF3 subunit by means of an in vitro constitution assay. Furthermore, the locus of the ISGF3γ gene, designated as Isgf3g, was mapped to distal mouse chromosome 14 by linkage analysis using an intersubspecific backcross typing panel. [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]