Student Theses and Dissertations

Date of Award

2007

Document Type

Thesis

RU Laboratory

Rice Laboratory

Keywords

hepatitis c virus, capsid gene

Abstract

In populations worldwide, the Hepatitis C Virus displays great diversity. The capsid gene, however, is remarkably conserved and this has led to predictions of RNA secondary structure and an overlapping, alternative reading frame (ARF). We investigated a role of the ARF in HCV infection, by introducing four stop codons into the ARF of a genotype 1a H77 molecular clone. These changes did not alter the capsid protein sequence, but were predicted to disrupt the RNA secondary structures, SLV and SLVI. An infection was launched after inoculation of the mutant HCV RNA into an HCV naïve chimpanzee. The acute infection was attenuated with low peak viremia, minimal ALT elevation, and early virus control by a diverse adaptive immune response. Sequencing circulating virus revealed progressive reversions at the third and then fourth stop codon mutations. In cell culture, replication of a genome with the four stop codons was severely impaired, but the revertant genomes showed a marked improvement in replicative fitness. Consistent with the chimpanzee infection, reversions at stop codons 3 and 4 were also selected by passage of mutant, infectious HCV in cell culture. Genetic evidence for RNA structures that were disrupted by the stop codons was provided by structure-restoring compensating mutations that relieved the defective HCV replication. Further mutagenesis identified bases and secondary structure features critical for function; mutations in the top stem of SLVI severely impaired HCV replication. RNA bearing these mutations was less efficiently translated in cell free extracts. The effect of mutations in SLVI was reduced when the HCV replicase proteins were translated using a heterologous IRES. This data suggests that these RNA structures are important for translation from the HCV IRES and that the defective translation exhibited by the SLVI mutants contributed to their impaired replication. Thus, RNA structures within the capsid coding region are responsible for modulating translation from the HCV IRES, and do not encode an ARF protein as had been postulated. While dispensable for the viability of subgenomic replicons, strong selective pressure for the integrity of these structures, both in vivo and in vitro, highlights their importance for the HCV lifecycle.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Permanent URL

http://hdl.handle.net/10209/125

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