The DNA constructs of the prototypic isolate H77 (41) for E2c (truncation of N and C termini and VR2 and removal of N448 and N576 glycosylation sites) and E2c3 (additional removal of VR3) were engineered on the basis of the soluble E2 ectodomain (E2ΔTM, amino acids 384 to 717) as previously described (28, 29). On the basis of sequence similarity to H77, E2c constructs of HCV1 (42), CON1 (43), UKN1b12.6 (44), J6 (45), UKN3A1.28c (44), S52 (46), ED43 (47), SA13 (48), and HK6a (34) isolates and the E2c3 constructs of H77, ED43, and HK6a isolates were engineered and synthesized by GeneArt (Invitrogen, USA; fig. S3A). Notably, the E1E2 envelope glycoprotein complex of the HK6a isolate was previously shown to be fully functional in vitro using the HCVpp and HCVcc systems and in vivo in chimpanzee and human liver chimeric mouse models (34, 49, 50). The E2 constructs were expressed and purified as previously described (29).

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