Using Live Cell Selective 2' Hydroxyl Acylation Analyzed by Primer Extension (SHAPE) to Investigate the Dynamic Structure of Hepatitis C Virus (HCV) RNA during the Viral Life Cycle

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Hepatitis C virus (HCV) infection is a global health problem with 200 million people infected worldwide. HCV-infected individuals typically develop chronic hepatitis, cirrhosis and liver cancer. To date, there is no vaccine available, and current treatment protocols are expensive, associated with severe side effects, and only 50-70% of patients respond to therapy. Thus, there is a pressing need for novel antiviral strategies. However, before novel treatment strategies can be rationally designed, a better understanding of the biology of the virus is required. As a (+)-sense RNA virus, the HCV genome itself must serve as a template for translation, replication, and packaging. The viral RNA must therefore be a dynamic structure that is able to readily accommodate unwinding, elongation and exposure of different regions of the RNA to viral and cellular proteins for viral protein production, RNA replication, and packaging. Although much has been uncovered about the 2° structures of the HCV genome required for translation and replication, these RNA regulatory motifs have been identified by focusing on 5’ and 3’ noncoding regions plus a few internal sequences. Approximately 85% of the genome and the negative-strand intermediate remain uncharacterized. One of the hallmarks of RNA is the presence of a 2’-hydroxyl group. Probing of 2’-hydroxl groups using Selective 2’ Hydroxyl Acylation analyzed by Primer Extension (SHAPE) has become the gold standard in interrogating RNA structure. Nucleotides constrained by base-pairing or other interactions exhibit poor SHAPE reactivity, while flexible or single-stranded nucleotides are more likely to sample numerous conformations that make the 2’-hydroxyl amenable to nucleophilic attack. The resultant 2’-O-adducts are then detected as stops to primer extension reactions using end-labeled primers. SHAPE has improved the accuracy of RNA structure models and increased our understanding of RNA regulatory motifs. Recently, SHAPE reagents have been developed that are amenable to interrogating RNA structure in live cells. The main hypothesis of this proposal is that the HCV genomic RNA forms multiple dynamic structures that are critical to the HCV life cycle. To test this hypothesis, we will perform SHAPE on the HCV genome in vitro, in virio and in live cells to identify the RNA structures that mediate the various stages of the viral life cycle. The following specific aims will be explored: 1) Characterization of the HCV genome in vitro, in virio and ex virio using SHAPE. While previous reports suggest that packaging signals may be present in the 5’ NCR, to date no viral packaging signals have been identified. To this end, we will carry out SHAPE analysis of viral RNA (in vitro) and in virions (in virio), as well as in deproteinized virions (ex virio). We anticipate that this analysis will help define packaging signals and reveal the complex structure of the HCV genome inside virions. 2) Characterization of the RNA regulatory motifs in the HCV life cycle using live cell SHAPE. In this aim, we will use a panel of HCV mutants that are stalled in one or more stages of the HCV life cycle to investigate RNA regulatory elements important for viral translation, replication and packaging. Identified regulatory RNA motifs will be subject to mutagenesis and the effects on translation, replication or particle production will be assessed. The proposed research is highly significant as gaining insight into RNA structures important for the viral life cycle will improve our understanding of HCV biology. The identification of novel RNA regulatory motifs will pave the way for the design of RNA structure-based inhibitors, refine models for HCV replication, and define new RNA regulatory elements in the HCV genome.