Mapping Determinants of Hepatitis C Virus E1/E2 Transmembrane Interactions Using Intergenotypic Chimeras

Hepatitis C virus (HCV) infection remains a major global health burden, and no vaccine preventing chronic infection is available. The envelope glycoproteins, E1 and E2, form a complex essential for viral entry; however, the mechanisms governing E1/E2 assembly and stability remain incompletely defined. Here, we investigated the role of the E1/E2 transmembrane (TM) regions in HCV infectivity using chimeras of JFH1-based recombinants with isolate-specific Core-NS2 sequences in which the C-terminal TM domains of E1 (TME1), E2 (TME2), or both (TME1E2) from the H77 isolate (genotype 1a) replaced those of isolates representing genotypes 1–6. We further introduced the TM domains of S52 (genotype 3a) or J6 (genotype 2a) into H77 and included reciprocal swaps between J6 and S52. Most TM-swap chimeras displayed impaired infectivity; however, serial passaging led to partial recovery associated with adaptive mutations in E1/E2 mapping not only to the C-terminal TM regions but also to the E1 stem and the internal E1 TM region (iTME1). Extending the TME1 swap to include upstream α-helical segments improved infectivity in selected chimeras, whereas inclusion of iTME1 abolished infectivity. These findings support functional interactions between membrane-associated regions of E1/E2 and their ectodomains and highlight their relevance for E1/E2-based HCV vaccine design.