Analysis of protein-RNA interactions in HCV non-structural proteins using reversible crosslinking and mass spectrometry

The structures of protein-RNA complexes are often difficult to obtain by X-ray crystallography or NMR spectroscopy. To rapidly map protein-RNA interactions, a method using reversible crosslinking, affinity purification, and mass spectrometry known as RCAP, was developed. The focus of my dissertation project is to apply and modify the RCAP method to analyze the interaction between RNA and three of the replication proteins of the human pathogen, the Hepatitis C Virus (HCV).;The HCV NS3 protein contains both a protease domain and a helicase domain, and is essential for the replication of HCV RNA, polyprotein processing, and evasion of innate immune signaling. Using the RCAP method, I showed that the protease domain binds RNA within its active site cleft. Furthermore, RNA binding in the active site was found to inhibit proteolysis. NS5A has no known enzymatic activity, but is required for HCV replication and impairs the host immune response. Using the RCAP method, both structured and intrinsically disordered regions of NS5A were shown to interact with RNA. Binding of the template RNA to NS5B, the HCV RNA-dependent RNA polymerase (RdRp), was previously analyzed (Kim et al., 2005). I manipulated the HCV RdRp to walk down the template in a systematic manner and produce an alkyn-labeled nascent RNA that could capture peptides that contacted the nascent RNA exit channel in the HCV polymerase ternary complex. The exit channel mapped to the fingers domain of the HCV RdRp underneath a structure known to regulate the transition from initiation to elongative RNA synthesis. Mutations within the putative nascent RNA binding region were found to be defective in RNA synthesis. The information gained from these studies contributed to the understanding of how RNA can regulate the HCV infection process.