Analysis of functional domains of interferon regulatory factor 7 and its association with IRF-3.
IRF-7 plays an essential role in virus-activated transcription of IFNA genes. To analyze functional domains of IRF-7 we have constructed an amino-terminal deletion mutant of IRF-7 (237-514) which exerted a dominant negative (DN) effect on virus-induced expression of the endogenous Type I IFN genes. Focusing on the molecular mechanism underlying the dominant negative effect of IRF-7 DN, we found that virus-activated transcription of endogenous IFNA genes requires full-length IRF-7 and that Serine 483 and 484 play an essential role. While IRF-7 DN had no effect on virus-stimulated nuclear translocation of IRF-3 and IRF-7, the binding of IRF-7 DN to IRF-3 and IRF-7 was detected by GST pull-down assay as well as by immunoprecipitation in infected cells, indicating that IRF-7 DN targets both IRF-7 and IRF-3. The region by which IRF-7 interacts with IRF-3 was mapped between amino acid 418 and 473. Overexpression of IRF-7 DN in virus-infected 2FTGH cells resulted in an inhibition of IFN synthesis and in a significant reduction of binding of both IRF-3 and IRF-7 to the IFNA1 promoter. Interestingly, the IRF-7 DN-mediated suppression of IFNA gene expression can be negated by overexpression of IRF-3. Altogether these results suggest that the IRF-3/IRF-7 complexes are biologically active and are involved in virus-activated transcription of endogenous IFNA genes.
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Oncology Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA.
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Pitha
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D005786:Gene Expression Regulation; D006801:Humans; D050838:Interferon Regulatory Factor-3; D050839:Interferon Regulatory Factor-7; D016898:Interferon-alpha / Q000235:genetics; D016899:Interferon-beta / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
D004268:DNA-Binding Proteins; C494232:IRF3 protein, human; C494234:IRF7 protein, human; D050838:Interferon Regulatory Factor-3; D050839:Interferon Regulatory Factor-7; D016898:Interferon-alpha; C494233:Irf3 protein, mouse; C494235:Irf7 protein, mouse; D014157:Transcription Factors; D016899:Interferon-beta