Fig 6. The substitutions L665S and S680T in E2 are both required to confer complete AR5A resistance to J6/JFH1_ΔHVR1 without affecting virus fitness.

Huh7.5 cells were transfected with in-vitro transcribed RNA of the recombinant viruses with (A) single or (B) combined substitutions I345V, L665S, and S680T. Supernatants were collected at the indicated time-points and HCV infectivity titers were determined. 1st passages of the viruses with (C) single or (D) combined substitutions I345V, L665S and S680T were subjected to a ten-fold dilution series of AR5A starting at 50 μg/ml. The virus/antibody mixes along with virus only were added to Huh7.5 cells and after 48 hour infection the cells were immunostained and the number of FFUs per well were counted as described in Materials and Methods. Neutralization values are the mean of four replicates and normalized to eight replicates of virus only. Three-parameter curve-fitting was used to obtain sigmoidal dose-response curves. Error bars represent the standard error of the mean.