Activation of cytosolic pathogen sensor Retinoic Acid-inducible Gene I

Retinoic-acid inducible gene I (RIG-I), also known as DDX58, is a critical cytosolic pathogen sensor that is activated upon binding to 5'-triphosphate (5'-ppp) and dsRNA containing ligands, such as those that are generated during infection with viruses or bacteria in cells. RIG-I is present in an inactive state in cells, where the CARD domains bind to the helicase domain. Mutagenesis experiments revealed critical residues arginine 109 and leucine 185 that keep RIG-I in an inactive state, as these mutations in CARD2 at the interface of CARD2 and Helicase rendered RIG-I hyperactive. RNA binding and subsequent ATP hydrolysis by RIG-I are believed to be critical for downstream signaling that leads to the induction of protective type-I interferon (IFN-I). However, the viral or bacterial RNA ligands of RIG-I produced during infection are largely unknown. Using RNA-protein pull down and deep sequencing, we identified that RIG-I preferentially associates with coding RNAs of Salmonella typhimurium. Studies with another ligand of RIG-I, Sendai virus defective-interfering RNA revealed that RIG-I oligomerizes in presence of this RNA. The oligomerization of RIG-I on Sendai virus RNA was dependent on the presence of terminal 5'-ppp and dsRNA motifs, the length of the dsRNA region, concentration of ATP, ATP hydrolysis and a functional ATPase domain. The level of ATPase-driven oligomerization correlated with the magnitude of IFN-I activation, identifying the previously unrecognized role of ATP hydrolysis in the activation of RIG-I-mediated IFN-I. Overall, this dissertation provided important insights into the activation process of RIG-I.