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.