Functions of hepatitis C virus glycoprotein E2 variable regions

HCV chronically infects ~3% of the global human population, including 200,000 Australians, causing recurring, progressively worsening liver disease, and cirrhosis and hepatocellular carcinoma. Preventative vaccines are not available. However, the advent of direct acting antivirals for the treatment of HCV results in viral clearance in at least 90% of treated individuals. Hepatitis C virus encodes two structural glycoproteins; E1 and E2. Glycoproteins E1 and E2 associate covalently and non-covalently to form heterodimers that mediate HCV binding to cell surface receptors. The E2 ectodomain region spanning residues 384-661 constitutes the receptor binding domain (RBD; E2661) that includes three highly variable regions: hypervariable region 1 (HVR1), HVR2 and the intergenotypic variable region (igVR). The HVR1 is known to elicit type-specific neutralizing antibodies (NAb). The HVR2 is located downstream of HVR1 within a region flanked by two cysteine residues (Cys-459 and Cys-486). HVR2 is not a direct target of the antibody response and the reason for variation within its sequence is unknown. Similar to HVR2, the igVR is flanked by two cysteine residues (Cys-569 and Cys-581). The igVR is relatively conserved within subtypes but the sequence and length of this region varies widely between genotypes. Patients infected with HCV develop NAb during their infection. The natural targets for NAb are the envelope glycoproteins E1 and E2 especially within the E2 RBD. The majority of NAb are directed to the binding sites within E2 for the major cell surface receptor CD81. In this study, the role of HVRs in modulating the exposure of both neutralizing and non-neutralizing epitopes within E2 has been examined. Examination of sequence evolution within HCV infected patients revealed that in addition to HVR1, both HVR2 and igVR are under selective pressure. In the case of the igVR, this contrasts to earlier reports suggesting that the igVR does not vary within genotypes. Mutations in the igVR were shown to directly modulate the exposure of non-neutralizing antibody epitopes in the patient that cleared their infection. In addition, the CD81 binding site was more occluded in E2 RBDs isolated during the chronic phase of the disease and was partly attributable to mutation in HVR2. In another approach, intragenotypic HVR2 and igVR replacement attenuate genotype 2 (G.2) HCVcc virus, whereas intergenotypic HVR2 and igVR chimeras between G.2a and G.1a results in non-infectious HCVcc. Passaging attenuated virus restored replication due to acquisition of adaptive mutations in the E2 transmembrane domain. With the aid of neutralizing antibodies, we also show that neutralizing epitope I encompassing 411-428 is more accessible when replacing HVR1 of G.1a to that of G.1b, but becomes occluded by replacing igVR. In contrast, neutralizing epitope III covering residues 512-529 is occluded by HVR1 and becomes inaccessible by exchanging igVR of G.1a to that of G.1b. The results show that HCV variable regions play a role in modulating the exposure of NAb epitopes and access to the CD81 binding site on the E2 RBD.