Investigating virus entry using cell-culture adapted hepatitis C virus

Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Present estimates predict that between 120-130 million people worldwide are infected with HCV with the majority of all infections progressing to chronicity, ultimately leading to fibrosis, cirrhosis and hepatocellular carcinoma. The virus, which belongs to the family Flaviviridae, has a single-stranded RNA genome of positive polarity that codes for a unique polyprotein of approximately 3000 amino acids. The structural proteins E1 and E2 constitute the viral envelope glycoproteins. These glycoproteins have multiple functions in the viral life cycle such as promoting viral entry and fusion, assembly of infectious virions and aid in viral persistence through immune escape. Numerous cell culture-adaptive mutations have been reported within the HCV glycoproteins. The value of such mutations in understanding the virus interaction(s) with cellular receptors and neutralizing antibodies was first recognised from studies characterizing the E2 cell culture adaptive mutation G451R. This single mutation altered the affinity of HCV to the cell surface receptors CD81 and SRB1 as well as increasing its sensitivity to neutralizing antibodies targeting the viral glycoproteins. A striking observation from previously reported E2 cell culture adaptive mutations is their frequent occurrence within a highly conserved region of E2, spanning residues 412-423. Indeed, the long-term passaging of JFH1 infected cells here in this study also created an adapted virus with a substitution at residue 415. The aim of this study was to determine the phenotypic changes to viral entry caused by mutations in this region. To do this, four JFH1 viruses containing the mutations N415D, T416A, N417S and I422L were constructed and characterized. These mutant viruses were found to have very similar phenotypes to the G451R virus, suggesting all E2 adaptive mutations are selected to alter a specific function in viral entry. Residues 412-423 of HCV E2 also constitute the epitope of the in-house generated broadly neutralizing antibody AP33. ELISA binding and virus infection inhibition assays using AP33 with the E2 mutant viruses provided important information regarding the E2 contact residues of this antibody. In a separate study, intergenotypic chimeric JFH1 viruses were generated and characterised. Viable intra- and intergenotypic JFH1 chimeric viruses have previously been generated by different research groups by replacing the core to NS2 genes of JFH1 with those from different genotypes. Many of these chimeric viruses required numerous cell culture adaptive mutations to permit efficient infectious virus production. In the present study, 5 intergenotypic viruses were constructed by replacing the JFH1 envelope genes with those from other HCV genotypes. Despite these chimeric genomes replicating efficiently, none were capable of producing infectious virus. These viral genomes also failed to acquire infectivity during pro-longed cell passaging, suggesting that replacing the JFH1 envelope glycoproteins with those from other genotypes may confer total incompatibility for virus assembly. In addition to this work, the infectivity of a previously generated genotype 4a/JFH1 chimera was improved by repeatedly passaging the virus infected cells. The chimeric virus contained the core to NS2 genes of a genotype 4a strain in place of the those from the original JFH1 sequence. A total of six-adaptive mutations were identified throughout the adapted genome that enhanced infectivity by more than 100-fold. Achieving higher titers with this chimera permitted studies on its viral entry properties as well as its sensitivity to neutralizing antibodies. The ability of the adapted virus 4a/JFH1 virus to spread during multiple rounds of infection was greatly reduced compared to WT/JFH1 due to its inefficient cell-to-cell spread. The 4a/JFH1 virions were also highly sensitive to neutralizing antibodies targeting both linear and conformational E2 epitopes, suggesting that the glycoproteins are more exposed on the surface of this virus. In its totality, this study has provided key insights into the viral entry and antibodymediated neutralization properties of cell-culture adapted and intergenotypic chimeric forms of the JFH1 virus.