Structural Basis Of RIG-I Auto-inhibition And RNA-induced Activation

Abstract

RIG-I (retinoic-acid-inducible-gene I) plays a critical role in regulating innate immune response against RNA viruses. RIG-I binds to 5′ triphosphate RNA of viral origin and activates downstream signaling pathways resulting in the induction of type I interferons. RIG-I contains two caspase-recruitment activation domains (CARD) in its N-terminus, a DExD/H helicase domain and a C-terminal repressor domain (RD). The function of RIG-I is regulated by auto-inhibition. While CARD domains activate downstream signaling, RD inhibits the activity of CARD domains in the absence of RNA. To investigate the RNA dependent structural transitions that activate RIG-I, we determined the structures of free and 5′ triphosphate RNA bound RIG-I using negative stain electron microscopy (EM) and single particle reconstruction. Preliminary biochemical studies show that free RIG-I, which is a monomer, undergoes dimerization upon binding to RNA. Analysis of the EM structure of RIG-I:RNA complex shows that dimerization is mediated by bivalent and parallel interactions between the two ends of adjacent RIG-I monomers. Structural modeling suggests that the dimer interfaces involve inter-molecular, homotypic, CARD-CARD and RD-RD interactions. In contrast to RNA bound RIG-I, which adopts an extended ‘open’ conformation, free RIG-I adopts a compact ‘closed’ conformation mediated by extensive intra-molecular interactions. Structural modeling indicates that the auto-inhibition of RIG-I is potentially mediated by a direct intra-molecular interaction between CARD and RD. Indeed, biochemical analysis demonstrates that isolated RD and CARD domain regions of RIG-I interact in the absence of RNA. We propose that the binding of 5′ triphosphate RNA to RIG-I disrupts the intra-molecular interactions that mediate auto-inhibition, and promote inter-molecular interactions that result in dimerization and subsequent activation. These results provide novel structural insights into the mechanism of RIG-I auto-inhibition and RNA-dependent activation.