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 IKK epsilon. 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 TAO-tag purification of TBK-1 and IKK epsilon proteins. Based on the identification of an extended lsequence motif - SxSxxxS - common to both IRF-3 and IRF-7, an IRF-7 pSer477/479 phosphospecific antibody was generated. Virus infection, TBK-1/IKK epsilon 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-kappa B (dependent promoter activity as efficiently as the constitutively active form of RIG-I (Delta RIG-1). Co-expression of the NS3/4A protease activity of hepatitis) 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.