Characterisation of the oxidation state of hepatitis C virus (HCV) envelope glycoproteins E1 and E2 during glycoprotein biosynthesis and virus entry

2017-01-31T05:04:59Z (GMT) by Dean, Johanna Elisabeth
Hepatitis C virus (HCV) is a major cause of chronic liver disease, including cirrhosis and hepatocellular carcinoma (HCC) and is a leading reason for liver transplantation in industrialised countries. The most recent estimate of global prevalence is 2-3% representing 130-170 million HCV-positive people [1]. HCV is an enveloped, positive strand RNA virus which comprises the hepacivirus genus within the Flaviviridae family. The virus displays two glycoproteins on its surface, E1 and E2, which are required to mediate entry into hepatocytes [2,3,4]. Cellular infection is dependent on receptors CD81, SR-BI, claudin-1, 6 or 9, and occludin [5,6,7,8,9,10]. Following attachment HCV is thought to be internalised via clathrin mediated endocytosis where the low pH of the endosome is required for fusion of the viral and cellular membranes [11]. Productive infection occurs following delivery of the viral nucleocapsid to the cytoplasm where RNA translation, replication and virus assembly takes place. This project investigated the significance of the redox state of HCV envelope glycoproteins E1 and E2 during glycoprotein biosynthesis and virus entry. It was found that virion incorporated forms of E1 and E2 contain at least one free cysteine residue each prior to cellular attachment. Alkylation of reduced sulfhydryl groups prior to cellular attachment abolished virus infectivity. However, it was found that cell culture derived HCV (HCVcc) became insensitive to thiol-reactive agents after binding to cells indicating that the thiol groups were no longer available for modification following receptor engagement. These data suggest that free sulfhydryl groups within E1 and/or E2 are required to mediate virus entry and that receptor engagement may induce a switch in glycoprotein redox state from reduced to oxidised. A highly conserved C226VPC thiol isomerisation motif was identified in E1 critical for formation of a functional E1E2 heterodimer. Mutation of Val227 to Ile or Leu allowed normal E1 and E2 biosynthesis and virion incorporation. However,mutant viruses demonstrated significantly reduced entry compared to wild type. These findings indicate that the C226VPC motif is required to function during virus entry. Domain organisation within E1 was examined by expression of E1 ectodomain residues in bacteria. A stable domain consisting of N-terminal amino acids 191-259 was identified and produced as a soluble monomer. When expressed in mammalian cells, ectodomain residues 191-329 were efficiently expressed and secreted from cells suggesting that when glycosylated these residues may fold independently from other HCV protein sequences. The findings from this study indicate that HCV functionality is highly dependent on careful regulation of the redox state of the viral envelope glycoproteins, possibly mediated by the E1 C226VPC motif.