Induction of IRF-3 and IRF-7 phosphorylation following activation of the RIG-I pathway.
The induction of type I interferon (IFN) and the development of the innate antiviral response are mediated by the activation of interferon regulatory factor (IRF)-3 and IRF-7 under the control of the non-canonical kinases TBK-1 and IKKepsilon. The initial sensing of infection by RNA viruses is mediated by the cytoplasmic, retinoic acid inducible gene I (RIG-I), via a Toll-like receptor (TLR) independent signaling pathway. In the present study, we identify key residues involved in IRF-3 and IRF-7 phosphorylation using TAP-tag purification of TBK-1 and IKKepsilon proteins. Based on the identification of an extended sequence motif--SxSxxxS--common to both IRF-3 and IRF-7, an IRF-7 pSer477/479 phosphospecific antibody was generated. Virus infection, TBK-1/IKKepsilon expression or co-expression of different signaling adaptors such as RIG-I, MAVS and TRIF, all stimulated pSer477/479 phosphorylation. Furthermore, the newly identified adaptor of the RIG-I pathway (MAVS/IPS-1/VISA/Cardif) was able to induce IRF and NF-kappaB dependent promoter activity as efficiently as the constitutively active form of RIG-I (DeltaRIG-I). Co-expression of the NS3/4A protease activity of hepatitis C virus however blocked MAVS-mediated gene activation in a dose dependent manner. These studies link RIG-I sensing of viral RNA to downstream kinase signaling and phosphorylation of IRF-3 and IRF-7 via the MAVS/IPS/VISA/Cardif adaptor.
Paz
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Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research, 3755 Cote Ste. Catherine, Montreal, Quebec, Canada H3T 1E2.
Sun
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Nakhaei
P
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Romieu-Mourez
R
R
Goubau
D
D
Julkunen
I
I
Lin
R
R
Hiscott
J
J
D048868:Adaptor Proteins, Signal Transducing / Q000037:antagonists & inhibitors / Q000378:metabolism; D000918:Antibody Specificity; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases; D018178:Flaviviridae Infections / Q000276:immunology; D005786:Gene Expression Regulation; D016174:Hepacivirus / Q000276:immunology; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D007113:Immunity, Innate / Q000276:immunology; D007369:Interferon Inducers / Q000378:metabolism; D050838:Interferon Regulatory Factor-3 / Q000378:metabolism*; D050839:Interferon Regulatory Factor-7 / Q000378:metabolism*; D016898:Interferon-alpha / Q000235:genetics; D016899:Interferon-beta / Q000235:genetics; D024101:Mitochondrial Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D008954:Models, Biological; D052002:NF-kappa B p50 Subunit / Q000235:genetics; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000378:metabolism*; D011971:Receptors, Immunologic; D020218:Response Elements / Q000235:genetics; D015398:Signal Transduction; D013379:Substrate Specificity; D015533:Transcriptional Activation; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D000085242:Viral Proteases
D048868:Adaptor Proteins, Signal Transducing; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases; D051550:I-kappa B Kinase; D007369:Interferon Inducers; D050838:Interferon Regulatory Factor-3; D050839:Interferon Regulatory Factor-7; D016898:Interferon-alpha; D016899:Interferon-beta; D024101:Mitochondrial Proteins; D052002:NF-kappa B p50 Subunit; D017346:Protein Serine-Threonine Kinases; D020365:RNA Helicases; D011971:Receptors, Immunologic; D017361:Viral Nonstructural Proteins; C505371:MAVS protein, human; C489548:NFKB1 protein, human; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D000085242:Viral Proteases; C093542:NS4 protein, hepatitis C virus; C403191:TBK1 protein, human; C452820:RIGI protein, human