Cellular and Viral Determinants for Hepatitis C Virus Replication

Abstract

The recent discovery of an HCV isolate which replicates in cell culture has opened up opportunities to study the full viral life cycle in vitro. This genotype 2a isolate (JFH-1) and its derivatives are the only ones known to replicate efficiently in cell culture, and recent work has indicated that viral determinants for efficient replication may lie in the non-structural protein coding region of the genome. In this thesis chimaeric JFH-1 virus containing full length NS3, NS3 helicase and NS3 protease sequences from genotype 1a and 1b were constructed. The replication efficiencies of chimaeric viruses were tested in cell culture, and were shown not to replicate, indicating that vital viral determinants for JFH-1 replication exist in NS3. The JFH-1 model also provides the opportunity to study the effect of the full viral life cycle on the host cell. Microarray analyses were performed to identify gene expression changes in Huh7 and Huh7.5 cells that had been infected with JFH-1 for 6, 12, 18, 24 and 48 hours. A large number of host genes were found to be regulated during JFH-1 infection, including those involved in lipid metabolism, oxidative stress, apoptosis and intracellular transport. The microarray data were validated by quantitative PCR analyses of separate infection experiments. A selection of the most highly regulated genes was assessed for their necessity to HCV replication by RNA interference studies. The knockdown by siRNA of genes ABLIM3, SPTLC3 and CYP1A1 resulted in significant impairment of HCV replication. The knockdown by siRNA of gene TXNIP (thioredoxin interacting protein) resulted in up to 90% reduction in HCV replication. This is a novel finding which may be of importance to the study of HCV as TXNIP plays roles in oxidative stress, lipid metabolism and glucose metabolism, all of which have potential to influence the HCV lifecycle. Magnetic resonance spectroscopy indicated a change in levels of choline metabolites in JFH-1 infected cells, which has implications for the aspects of the HCV lifecycle associated with lipid membranes and other lipid structures.