Functional and bioinformatic analysis of Hepatitis C virus cis-acting replication elements
Lead Research Organisation:
University of Warwick
Department Name: Biological Sciences
Abstract
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Technical Summary
Hepatitis C virus (HCV) is a leading cause of chronic liver disease, infecting more than 170 million people worldwide. Persistent HCV infection leads to irreversible cirrhosis, can cause hepatocellular carcinoma, and is the principal indication for liver transplantation in US and European adults. Some patients respond to antiviral drug therapy, though this is limited by cost, availability, unpleasant side-effects and poor responses. In the absence of a protective vaccine for HCV, there is an urgent need to develop new therapeutics. New targets must therefore be identified; this requires a better understanding of the fundamental mechanism of virus replication.
Small RNA viruses such as HCV probably all contain multi-functional RNA sequences ? with roles in the control of replication in addition to their protein-coding capacity. We have developed robust predictive techniques to identify such sequences and conducted preliminary experiments that support our predictions.
Our bioinformatic approaches exploit phylogenetic information derived from comparative sequence information. These methods predict up to 12 new stem-loop structures in the core- and RNA polymerase-encoding regions of HCV. We have mapped many of these structures using classical enzymatic analysis, and have conducted preliminary mutagenesis on three ? designated SL9011, SL9061, SL9118 ? to test our predictions. The mutations selected were designed to disrupt the predicted RNA structures and ? since HCV cannot properly replicate in cell culture ? were built into a sub-genomic replicon that confers antibiotic-resistance to cells in which it replicates. Disruption of SL9061 or SL9011 respectively prevented or partially inhibited replication, confirming the value of our bioinformatic predictions.
We propose to determine whether other RNA structures we predict have a role in virus replication. Using the replicon system we will investigate the structure and function of the seven most highly conserved structures. We will quantify replicon activity in different hepatoma cell types and analyse recovered replicons for adaptive mutations. We will determine the position-dependence of any RNA structures we show have a role in replication, and will investigate the interaction of cellular and viral proteins with the RNAs. We already have bioinformatic and experimental to suggest that SL9061 interacts with sequences elsewhere in the virus genome. This will be tested by further mutagenesis and the results used to iteratively improve our predictive computational methods.
The functional definition of HCV cre sequences will enable the identification of future targets for therapeutic intervention and the subsequent reduction of the disease burden on society caused by this virus.
Small RNA viruses such as HCV probably all contain multi-functional RNA sequences ? with roles in the control of replication in addition to their protein-coding capacity. We have developed robust predictive techniques to identify such sequences and conducted preliminary experiments that support our predictions.
Our bioinformatic approaches exploit phylogenetic information derived from comparative sequence information. These methods predict up to 12 new stem-loop structures in the core- and RNA polymerase-encoding regions of HCV. We have mapped many of these structures using classical enzymatic analysis, and have conducted preliminary mutagenesis on three ? designated SL9011, SL9061, SL9118 ? to test our predictions. The mutations selected were designed to disrupt the predicted RNA structures and ? since HCV cannot properly replicate in cell culture ? were built into a sub-genomic replicon that confers antibiotic-resistance to cells in which it replicates. Disruption of SL9061 or SL9011 respectively prevented or partially inhibited replication, confirming the value of our bioinformatic predictions.
We propose to determine whether other RNA structures we predict have a role in virus replication. Using the replicon system we will investigate the structure and function of the seven most highly conserved structures. We will quantify replicon activity in different hepatoma cell types and analyse recovered replicons for adaptive mutations. We will determine the position-dependence of any RNA structures we show have a role in replication, and will investigate the interaction of cellular and viral proteins with the RNAs. We already have bioinformatic and experimental to suggest that SL9061 interacts with sequences elsewhere in the virus genome. This will be tested by further mutagenesis and the results used to iteratively improve our predictive computational methods.
The functional definition of HCV cre sequences will enable the identification of future targets for therapeutic intervention and the subsequent reduction of the disease burden on society caused by this virus.
