Construction and characterization of chimeric hepatitis C virus E2 glycoproteins: analysis of regions critical for glycoprotein aggregation and CD81 binding

We compared the ability of two closely related truncated E2 glycoproteins ( E2(660)) derived from hepatitis C virus (HCV) genotype 1 a strains Glasgow (Gla) and H77c to bind a panel of conformation-dependent monoclonal antibod ies (MAbs) and CD81. In contrast to H77c, Gla E2(660) formed disulfide-link ed high molecular mass aggregates and failed to react with conformation-dep endent MAbs and CD81. To delineate amino acid (aa) regions associated with protein aggregation and CD81 binding, several Gla-H77c E2(660) chimeric gly coproteins were constructed. Chimeras C1, C2 and C6, carrying aa 525-660 of Gla E2(660), produced disulfide-linked aggregates and failed to bind CD81 and conformation-dependent MAbs, suggesting that amino acids within this re gion are responsible for protein misfolding. The presence of Gla hypervaria ble region 1 (aa 384-406) on H77 E2(660), chimera C4, had no effect on prot ein folding or CD81 binding. Chimeras C3 and C5, carrying aa 384-524 or 407 -524 of Gla E2(660), respectively, were recognized by conformation-dependen t MAbs and yet failed to bind CD81, suggesting that amino acids in region 4 07-524 are important in modulating CD81 interaction without affecting antig en folding. Comparison of Gla and H77c E2(660), aa sequences with those of genotype la and divergent genotypes identified a number of variant amino ac ids, including two putative N-linked glycosylation sites at positions 476 a nd 532. However, introduction of G476N-G478S and/or D532N in Gla E2(660) ha d no effect on antigenicity or aggregation.