Targeting Assembly and Function of Hepatitis C Virus Replicase

Hepatitis C virus (HCV) affects over 170 million people worldwide, and current treatment options are inadequate. Development of more effective treatments requires identification of new structural and functional targets, and understanding of the molecular basis of drug-resistant mutants. Like other positive-strand RNA viruses, HCV genome replication is characterized by the formation of a membrane-associated replicase complex, which is composed of viral proteins and replicating RNA. Given its critical role in the HCV life cycle, inhibiting replicase activity is an attractive therapeutic strategy. However, optimal targeting of the HCV replicase is limited by a technology gap that precludes study of replicase assembly and function in the membrane-associated state. To target these steps, we hypothesize that model membrane sensing platforms can support in situ reconstitution of the multi-protein replicase. We will first determine if biologically relevant model membranes can host full-length NS5B polymerase—the core enzyme of the HCV replicase. Based on successful membrane association of NS5B, we additionally hypothesize that engineering approaches will enable detailed study of replicase assembly and function. We will determine template RNA requirements for activation of membrane-associated NS5B, and develop a quantitative method to measure polymerase activity. Further, we will investigate how additional HCV nonstructural proteins interact with membrane-associated NS5B, and modulate replicase function. To demonstrate the platform’s utility for accelerating HCV drug development, we will also determine the specific mechanism of action of promising drug candidates that target replicase assembly and function. Taken together, the proposed studies will lead to the establishment of a technology platform that uniquely supports functional studies of membrane-associated HCV replicase. Importantly, this technology will help deliver the best possible treatment regimens by enabling development of pharmaceuticals that precisely target specific steps in replicase assembly and function, and by determining the molecular basis of drug-resistant mutations which arise within individual replicase components.