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

Lead Research Organisation: University of Warwick
Department Name: Biological Sciences

Abstract

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.

Technical Summary

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.
 
Description Cogent
Amount £37,500 (GBP)
Organisation Cogent 
Sector Private
Country Guernsey
Start  
 
Description Project grant
Amount £483,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2012 
End 03/2015
 
Description Strategic Impact Fund
Amount £37,000 (GBP)
Organisation University of Warwick 
Sector Academic/University
Country United Kingdom
Start  
 
Title Software 
Description Software developed by co-applicant (PS) as part of this proposal. 
Type Of Material Technology assay or reagent 
Year Produced 2007 
Provided To Others? Yes  
Impact In our laboratory - software has provided important insights into RNA structures in HCV and many other viruses. This software has enabled a number of new projects to be initiated - including studies on recombination (funded by the Wellcome Trust) and the role of RNA structures in subversion of the innate immune responses (funding obtained, but not yet initiated). In other laboratories - software has been downloaded over 300 times, but use and application has not been recorded. 
 
Description CJ 
Organisation University of Nottingham
Department School of Biomedical Sciences Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution Developed system to investigate translational control in hepatitis C virus and the influence of micro-RNA on this process.
Collaborator Contribution Intellectual and contribution of reagents
Impact Successful MRC grant application
Start Year 2010
 
Description EA 
Organisation University of Oxford
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Developed methods to study long-range interactions of RNA
Collaborator Contribution Intellectual
Impact Successful grant application using this approach. Multi-disciplinary - chemistry and biology/virology. Additional grant application with this collaborator in which I am the external collaborator.
Start Year 2009
 
Description JL 
Organisation University of Edinburgh
Department School of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Devised new way of detecting long-range RNA interactions
Collaborator Contribution Intellectual and - in due course - experimental.
Impact Successful MRC grant application. Multi-disciplinary - chemistry and biology/virology. Additional collaborations on other research council grant applications.
Start Year 2009
 
Title INHIBITOR FOR TREATMENT OF HCV 
Description Therapeutic target identified and inhibitors designed 
IP Reference WO2014155097 
Protection Patent application published
Year Protection Granted
Licensed Commercial In Confidence
Impact PhD. funding from Cogent Ltd.