Evolutionary study of hepatitis C virus envelope genes during primary infection

Abstract

Hepatitis C virus (HCV) envelope genes encoding glycoproteins E1 and E2 exhibits a high degree of variability that gives rise to differing phenotypic traits; including alterations in receptor-binding affinity and immune recognition and escape. This study aims to elucidate the relationship of the evolutionary patterns for HCV envelope glycoproteins to viral persistence. HCV quasispecies were characterized in specimens collected every two to six months from a cohort of acutely HCV-infected subjects. We evaluated two individuals who spontaneously cleared viremia and three individuals with persistent viremia by cloning 33 1-kb amplicons that spanned E1 and the 5' half of E2; including hypervariable region 1 (HVR1). To detect representative variants for sequencing thirty-three cloned cDNAs representing each specimen were assessed by a method that combined analysis of a single-stranded conformational polymorphism (SSCP) method and heteroduplex analysis (HDA). For each patient, the rates of both synonymous and nonsynonymous substitutions for the E1, HVR1 and E2 regions outside HVR1 were evaluated. The amino acid sequences and predicted antigenic profiles were analyzed. The genetic diversity within HVR1 was consistently higher than that in the E1 and E2 regions outside HVR1 in individuals with persistent viremia, but did not change markedly over time in those with clearance of viremia. For individuals with persistent viremia, the rate of nonsynonymous substitutions within the HVR1 region predominated and gradually increased, compared to that in the E1 and E2 regions outside HVR1. By contrast, the rates of both nonsynonymous and synonymous substitutions for the E1 and E2 regions, including HVR1, were consistently lower in individuals with clearance of viremia. HVR1 had a higher antigenic variable and lower positive charge in subjects with persistent viremia. All cysteine residues and N-linked glycosylation sites, some of which were known to play a major role in protein folding and others play a role in HCV entry, were 100% conserved among the sequenced cloned cDNAs from the two outcome groups. HCV persistence may be associated with positive selection pressures on HVR1, rather than functional constraints in the envelope region.