Position L665 in HCV E2 is highly sensitive to amino-acid substitutions. (A) The leucine at position 665 in H77/JFH1 was substituted for the indicated amino acids, and Huh7.5 cells were transfected with in vitro transcribed RNA of recombinants harboring these substitutions. Supernatants were collected at the indicated time-points and HCV infectivity titers were determined as described in Materials and Methods. (B) 293T cells were transiently transfected with vectors expressing L665 mutants of H77 E1E2 in cis. E1/E2 protein complexes were gently released using 1% nDDM detergent with protease inhibitors and benzonase. The samples were subjected to AR3A (conformational E2-specific antibody) immunoprecipitation and SDS-PAGE and western blot transfer was performed on the enriched fractions. E2 was detected using antibody H52 and E1 was detected using antibody A4.