Hepatitis C virus (HCV) replication in vitro and the effect of HCV on human immunodeficiency virus (HIV-1) coreceptor usage and diversity in the coinfected patient

This dissertation explores HCV replication and the effect of HCV infection on HIV-1 in the HCV-HIV coinfected patient. To examine HCV replication, an in vitro binary replication system was used to explore the following hypotheses: (1) HCV structural proteins affect positiveand negative-strand RNA synthesis and (2) ethanol treatment of cells increases HCV replication. For the first hypothesis the effects of structural gene deletions on HCV replication were examined. Negative-strand RNA synthesis was significantly lower only when core was deleted. This evidence suggested two possible mechanisms for core in negative-strand synthesis: (1) core may shuttle the positive-strand RNA to the replication complex, or (2) core may inhibit translation to allow synthesis of negative-strand RNA from the positive-strand RNA. For the second hypothesis, two in vitro systems were utilized to assess replication in the presence of ethanol. Data from the binary replication system indicated that ethanol only affects negative-strand synthesis in both cell lines, however negative-strand RNA production was decreased in HepG2 cells and increased in Huh7 cells suggesting fundamental differences in gene expression between the two cell lines.;The second goal of this research was to examine the effect of HCV on HIV coreceptor usage and HIV diversity in the coinfected person. Analysis of coinfected hemophiliacs with low CD4 counts and a CD4-matched cohort of HIV singly infected patients indicated that the coinfected subjects had less immunosuppression by two measures: sequencing of gp120 V3 regions and heteroduplex tracking assay analysis of the gp120 regions V1-V2 and V4-V5. Coinfected patients developed X4 viral sequences less frequently than did the singly infected patients and demonstrated more diversity in both V1-V2 and V4-V5. Therefore, coinfection with both HCV and HIV does not lead to increased immunosuppression. In summary, we have demonstrated that HCV core is important for negative-strand RNA production. Additionally, we have shown that ethanol appears to affect production of negativestrand RNA. Finally, we have demonstrated that HIV-HCV coinfected patients with low CD4 have fewer X4 viral sequences and less HIV diversity than HIV singly infected persons with low CD4, suggesting that the immune systems of coinfected patients are not as compromised as those of singly infected patients and that low circulating CD4 counts may not reflect immunosuppression in the coinfected population.