Biochemical, structural and functional analysis of a pseudoknot required for hepatitis C virus replication.

Hepatitis C virus infects a predicted 170 million people worldwide and causes chronic liver disease leading to cirrhosis and hepatocellular carcinoma. The virus is transmitted in contaminated blood and by needle-sharing. The virus is highly variable; prior infection does not appear to protect from re-infection and there is no realistic prospect of producing a vaccine. Extended drug treatment can clear the virus in a proportion of those infected, but treatment is expensive and has unpleasant side-effects. To make new and improved therapies we need to understand better how the virus replicates. Our previous studies have identified a small region of the genome that is critical for an early event in virus replication. It is highly conserved in all isolates of this variable virus. We have data to suggest that this element forms a molecular switch controlling two of the earliest events in the virus life cycle ? the translation of the virus to make proteins, and the replication of the virus genome. We propose to continue these studies to define exactly how this switch operates and, in doing so, expect to learn of ways in which we can prevent it from being ?tripped? and so halt the replication of the virus.

It is important to better understand the replication of hepatitis C virus (HCV) to identify potential new therapeutic targets to which anti-viral therapies can be directed. We have used bioinformatics to predict long-range interactions of structured RNA elements within the HCV genome, and confirmed these interactions by reverse genetic analysis using cell culture replication systems. Our studies suggest the structure forms a complex extended pseudoknot. In other viruses, such structures are implicated in control of translation and genome replication, and we have data that implies that this pseudoknot may have similar functions in HCV. We propose to conduct structural and biochemical studies to determine whether the pseudoknot forms one single structure, or can adopt two alternate configurations that implies it could operate as a molecular switch. We will investigate the interaction of proteins with this pseudoknot and determine their identity. We will rigorously investigate a role for the pseudoknot in virus translation using cell-based studies and will gain further insights into function by analysing replication in the HCV cell culture system. These studies will provide important information on fundamental aspects of the replication cycle of an important human pathogen.