Conventional Protein Kinase C- (PKC-) and PKC- Negatively Regulate RIG-I Antiviral Signal Transduction

Retinoic acid-inducible gene I (RIG-I) is a key sensor for viral RNA in the cytosol, and it initiates a signaling cascade that leads to the establishment of an interferon (IFN)-mediated antiviral state. Because of its integral role in immune signaling, RIG-I activity must be precisely controlled. Recent studies have shown that RIG-I CARD-dependent signaling function is regulated by the dy-namic balance between phosphorylation and TRIM25-induced K63-linked ubiquitination. While ubiquitination of RIG-I is criti-cal for RIG-I’s ability to induce an antiviral IFN response, phosphorylation of RIG-I at S8 or T170 suppresses RIG-I signal-transducing activity under normal conditions. Here, we not only further define the roles of S8 and T170 phosphorylation for controlling RIG-I activity but also identify conventional protein kinase C- (PKC-) and PKC- as important negative regula-tors of the RIG-I signaling pathway. Mutational analysis indicated that while the phosphorylation of S8 or T170 potently inhibits RIG-I downstream signaling, the dephosphorylation of RIG-I at both residues is necessary for optimal TRIM25 binding and ubiquitination-mediated RIG-I activation. Furthermore, exogenous expression, gene silencing, and specific inhibitor treatment demonstrated that PKC-/ are the primary kinases responsible for RIG-I S8 and T170 phosphorylation. Coimmunoprecipita-tion showed that PKC-/ interact with RIG-I under normal conditions, leading to its phosphorylation, which suppresses TRIM25 binding, RIG-I CARD ubiquitination, and thereby RIG-I-mediated IFN induction. PKC-/ double-knockdown cells exhibited markedly decreased S8/T170 phosphorylation levels of RIG-I and resistance to infection by vesicular stomatitis virus. Thus, these findings demonstrate that PKC-/-induced RIG-I phosphorylation is a critical regulatory mechanism for control