Hepatitis C virus (HCV) is an important human pathogen that previously could be studied only in the chimpanzee model because the virus could not be grown in cultured cells. Growth of the virus in cultured hepatoma cells was recently achieved by Wakita et al. but even now growth is strain-specific and inefficient and infectious titers rarely reach even 100,000 viruses per ml of culture medium. Prior to discovery of this culture system, a retrovirus pseudotyped particle system was the only means to study neutralization of HCV and to perform mutational analysis of the two glycoproteins of HCV. Cell entry by enveloped viruses is mediated by viral glycoproteins, and generally involves a short hydrophobic peptide (fusion peptide) that inserts into the cellular membrane. An internal hydrophobic domain within E1 (aa262-290) of hepatitis C virus (HCV) may function as a fusion peptide. In FY2009, we generated retrovirus-based HCV-pseudotyped viruses (HCVpp;genotype 1a) containing Ala or Pro substitutions at conserved amino acid positions within this putative fusion peptide. Mutation of conserved residues significantly reduced efficiency of HCVpp entry into Huh-7 cells. The majority of amino acid substitutions appeared to disrupt necessary interactions between E1 and E2. For some mutants, reductions in HCVpp-associated E1 were associated with the incorporation of a high molecular weight, hyperglycosylated E2 that displayed decreased CD81-binding, and therefore might affect viral fusion. One mutant (S283P) consistently displayed two- to threefold higher infectivity than did wild-type. Three mutations that decreased HCVpp infectivity also reduced levels of HCVcc infectious virus production. However, the S283P mutation had a different effect in the two systems as it did not increase production of infectious HCVcc. This comprehensive mutational analysis of the putative HCV fusion peptide provides insight into the role of E1 in its interaction with E2 and in HCV entry. In FY2009 we performed mutagenesis studies, utilizing pseudoparticles, which demonstrated that the core protein, which was not a part of most pseudoparticles, increased the efficiency of pseudoparticle formation for certain strains. The hypervariable region 1 (HVR1) in the E2 glycoprotein of HCV is a target for neutralizing antibodies and is implicated in entry into cells. In FY2009, we showed that a shotgun approach could be used to clone E1E2 co-evolved genes into an infectious cDNA vector. The clones recovered reflected the quasispecies distribution of the original sample. Transfection of cells with the quasispecies transcripts followed by serial passage in cell culture was used to determine which set of glycoproteins in the quasispecies was most efficient at promoting virus spread in culture cells. In FY2009, we collaborated with Thomas Leto in the Laboratory of Host Defenses, NIAID, to study HCV pathogenesis. Expression of HCV core protein alone, viral RNA, or replicating virus induced NOX4 (a member of the NADPH oxidase family) mRNA expression resulting in an increase of reactive oxygen species. Therefore, HCV may induce liver disease by its role in production of reactive oxygen species.
