Incoming RNA virus nucleocapsids containing a 5′-triphosphorylated genome activate RIG-I and antiviral signaling

Host defense to RNA viruses depends on rapid intracellular recognition of viral RNA by two cytoplasmic RNA helicases: RIG-I and MDA5. RNA transfection experiments indicate that RIG-I responds to naked double-stranded RNAs (dsRNAs) with a triphosphorylated 5′ (5′ppp) terminus. However, the identity of the RIG-I stimulating viral structures in an authentic infection context remains unresolved. We show that incoming viral nucleocapsids containing a 5′ppp dsRNA "panhandle" structure trigger antiviral signaling that commences with RIG-I, is mediated through the adaptor protein MAVS, and terminates with transcription factor IRF-3. Independent of mammalian cofactors or viral polymerase activity, RIG-I bound to viral nucleocapsids, underwent a conformational switch, and homo-oligomerized. Enzymatic probing and superresolution microscopy suggest that RIG-I interacts with the panhandle structure of the viral nucleocapsids. These results define cytoplasmic entry of nucleocapsids as the proximal RIG-I-sensitive step during infection and establish viral nucleocapsids with a 5′ppp dsRNA panhandle as a RIG-I activator.