SENSORS FOR REPLICATING VIRUSES AND INNATE IMMUNITY

Recent studies show the involvement of cytoplasmic RNA helicase family, RIG-I, MDA5 and LGP2 in\nantiviral innate immune responses. RIG-I and MDA5 are primarily responsible for the detection of viral infection\nand triggering activation cascade for type I interferon genes in many cell types. RIG-I consists of N-terminal\nCAspase Recruitment Domain (CARD) and a domain with signatures of DExD/H box helicase (helicase domain).\nFunctional analyses revealed that the helicase domain detects viral RNA and CARD triggers the activation of\ndownstream signaling cascade, including activation of transcription factors, NF-κB, IRF-3 and IRF-7. RIG-I binds\nto double stranded (ds)RNA, however it does not simply function as a binding receptor for dsRNA, since RIG-I\nwith disrupted ATP binding site is incapable of signaling. A model is proposed that in the absence of dsRNA,\nRIG-I forms “closed” conformation and upon binding to dsRNA, it conforms into “open” structure exposing CARD.\nWe produced recombinant RIG-I protein using Baculo virus system and purified it to homogeneity. Biochemical\nproperties, including dsRNA binding activity, ATPase activity and helicase activity, of recombinant RIG-I were\ninvestigated. The results suggested that RIG-I requires certain structure of ligand RNA that is specifi c to viral (or\nnon-self) origin. Furthermore, we found evidence that RIG-I conforms a certain structure upon binding to dsRNA in\nthe presence of ATP. These results were consistent with the above model for activation of RIG-I. Furthermore, we\nobserved that RIG-I forms oligomers in virus-infected cells and artifi cial oligomerization of RIG-I CARD mimics virusinduced\nsignaling, resulting in the activation of interferon and other cytokine genes. These results highlight how viral\nreplication in cytoplasm is detected by RIG-I helicase and switch on signal cascades for initial antiviral responses.