Impact of Intra-Epitopic Resistance-Associated Substitutions on Peptide–HLA-A*02:01 Binding in Hepatitis C Virus

Abstract

In hepatitis C virus (HCV), non-structural proteins targeted by antiviral therapy also contain CD8⁺ T-cell epitopes, linking resistance-associated substitutions (RAS) to changes in antigen presentation. Once these substitutions occur within epitopes, they can change the peptide displayed by HLA-A*02:01 on infected hepatocytes. Nevertheless, it is not clear whether intra-epitopic RAS affects peptide-HLA-A02:01 binding affinity. In this study, intra-epitopic RAS in HLA-A*02:01-restricted NS3, NS5A, and NS5B epitopes were examined to determine their effects on peptide-HLA-A*02:01 binding of HCV subtypes 1b and 3a. A total of 2,636 NS3, 7,463 NS5A, and 3,107 NS5B sequences were retrieved from the euHCVdb database. HLA-A*02:01-restricted epitopes were predicted and aligned with known RAS positions. In NS5B subtype 3a, no RAS overlapped predicted epitopes; therefore, no intra-epitopic variants were analysed. Five representative intra-epitopic RAS were selected for detailed structural analysis using 200 ns molecular dynamics simulations and MM-GBSA binding free energy calculations. All peptide–HLA complexes remained structurally stable throughout the simulations, indicating that single substitutions do not disrupt complex formation. Binding free energy analysis (MM-GBSA) revealed substitution-specific bidirectional effects. V170I (NS3) and C316N (NS5B) reduced binding affinity by 17.5% and 12.9%, respectively, relative to wild-type. In contrast, L31I (NS5A) and V321I (NS5B) increased binding affinity by 67.6% and 70.8%, while L132I (NS3) showed no significant change. These results suggest that the intra-epitopic RAS, possess substitution-specific, bidirectional effects on the affinity of peptide-HLA-A*02:01 binding, and thus indicate a structural relationship between resistance-associated variation and antigen presentation. Substitutions that lead to reduced binding may be linked to immune escape. The results suggest that certain intra-epitopic RAS maintain or increase peptide-HLA-A*02:01 binding, and others decrease it, and reveal resistance mutations like V170I and C316N that can be further experimentally validated.