Using evolutionary tools to refine the new hypervariable region 3 within the envelope 2 protein of hepatitis C virus

The envelope 2 protein of hepatitis C virus (HCV) presents three hypervariable regions, named HVR1, HVR2 and HVR3, in which the presence of antigenic sites has been described. Genetic variability in these regions may reflect the generation of escape mutants as a consequence of the immune response. Therefore, these regions would tend to accumulate amino acid changes along the infection process, an effect that could be accelerated by antiviral treatments. In this study, we have analyzed the E1–E2 region of 23 HCV patients non-responders to antiviral treatment, 7 of which were infected with subtype 1a, 15 with subtype 1b, and 1 with a new HCV-1 subtype, before and after 6 and/or 12 months of peg-interferon + ribavirin treatment. We have sequenced about 100 clones from each sample, analyzing a total of 4906 sequences. A detailed analysis of the evolutionary forces acting along the genome region studied confirmed the existence of the three hypervariable regions, characterized by significant changes in amino acid composition between samples taken at different times from the same patient and a high number of sites evolving under positive selection. Moreover, for the recently described HVR3, our results suggest that its location could be restricted to residues 434–450, instead of the originally postulated 431–466.