Functions of the hepatitis C virus NS5A protein in viral replication. (360G-Wellcome-078358_Z_05_Z)

Hepatitis C virus (HCV) is one of the most common causes of chronic liver disease. Progress in understanding viral replication has been hampered by the lack of a robust in vitro culture system. We propose to exploit recent developments in our laboratory to analyse the function of the HCV NS5A protein in viral RNA replication. NS5A is a phosphoprotein that is a key component of the viral RNA replication complex, but its precise role remained to be elucidated. Our studies will proceed in two interrelated areas: Firstly, we will use baculovirus vectors capable of delivering replication competent HCV genomes and sub-genomic replicons into hepatocytes, coupled with a mass spectrometry based approach, to unambiguously define the sites of phosphorylation. This will allow us to identify the kinases involved and use this information to assess the role of phosphorylation in RNA replication. Secondly, we have recently introduced a biotin acceptor domain into NS5A in the context of the subgenomic replicon - the resulting constructs are replication competent. We will exploit this advance to purify RNA replication complexes using avidin-affinity chromatography and use proteomic approaches to identify the cellular proteins associated with these complexes.