Structural basis for RNA recognition and activation by human innate immune receptor RIG-1

Innate immunity provides the first line of host defense against pathogenic microbial and viral invasion. Activation of innate immune responses relies on the specific recognition of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs). Retinoic acid Inducible Gene- I (RIG-I) is a crucial PPR in the cytoplasm that induces antiviral and inflammatory immune responses against RNA viruses by selectively detecting PAMP RNAs. The RIG-I signaling pathway is highly regulated. Aberrant signaling can lead to apoptosis and altered cell differentiation, which have been implicated in the development of inflammation, autoimmune diseases including type 1 diabetes, and cancer. We have collaborated with Dr. Michael Gale at University of Washington School of Medicine to identify the poly-uridine motif of the Hepatitis C virus (HCV) genome 3′ non-translated region and its replication intermediate as a PAMP substrate of RIG-I (Saito et al., 2008). To this end, I have developed efficient expression and purification methods for human RIG-I, and characterized the protein using biochemical and biophysical methods. Highly purified RIG-I protein was then used to verify HCV PAMP RNA in vitro by gel shift assay and limited proteolysis. RIG-I consists of two N-terminal caspase recruitment domains (CARDs), a central DExD/H box RNA helicase/ATPase domain, and a C- terminal repressor domain (RD). To understand how the RIG-I helicase binds RNA and leads to activation, I have determined the crystal structure of the human RIG-I helicase-RD domain bound to dsRNA and ADP•BeF3 in collaboration with Dr. Smita Patel’s group at UMDNJ. The structure of ternary complex reveals a major contribution from the helicase domain to RNA binding and a synergy between the helicase and RD in recognition of blunt-ended dsRNA (Jiang et al., 2011). Furthermore, I have determined the crystal structures of RIG-I bound to panhandle-like short hairpin RNAs in the presence or absence of 5’-triphosphorylated modification, and chimeric RNA-DNA duplex at high resolution. These recent structures provide further insights into the molecular mechanics of RNA recognition and RIG-I activation upon viral infection.Ph. D.Includes bibliographical referencesIncludes vitaby Fuguo Jian