Project Summary Hepatitis C Virus (HCV) is a RNA virus that currently infects three percent of the world's population. By chronically infecting hepatocytes, HCV leads to severe physiological changes of host cells. This often leads to liver cirrhosis, which is the leading cause for development of hepatocellular carcinoma (HCC) and need for liver transplantation. HCC is the second leading cause of cancer-related deaths worldwide, and current treatment of HCC is indirect and ineffective. Vaccines have not been successfully developed to prevent HCV infection, and direct-acting antiviral (DAA) therapy has only recently become successful, largely thanks to basic science research studying molecular aspects of HCV infection. HCV is a RNA-only virus, and recent advances in the study and analysis of RNA has lead to a revolution in understanding the complex role that diverse RNAs play in cells. This proposal seeks a molecular understanding of RNA-protein interactions between the virus and host cell during HCV infection using state-of- the-art approaches. In Aim 1, I will analyze the in vivo RNA targets of the HCV bi-functional protease/RNA helicase, NS3. Although NS3 is a target of current DAA therapy, no studies have analyzed what the RNA substrates for NS3 helicase activity are. It is presumed that NS3 targets viral RNA, but it is unknown whether the helicase impacts cellular gene expression. To resolve these unknowns, I will perform PAR-Clip on NS3. PAR-Clip permits unambiguous identification of regions of RNA that directly interact with proteins, and I will use a variety of mutants and/or drugs to probe the consequences of altering helicase or protease activity. In Aim 2, I will test whether the HCV RNA genome interacts with cellular proteins. The Pyle lab has uncovered conserved RNA structures that are required for normal HCV replication and infectivity. I will use RAP-MS to uncover whether host cell proteins interact with these structures. Follow-up experiments will confirm direct interactions and analyze the function of these interactions. These approaches will inform how RNA-protein interactions impact viral replication and cellular physiology. The ultimate goal of this work is to aid in the development of DAAs, uncover markers for HCC progression, and discover aspects of infection that can inform the study of other RNA viruses.