Figure 1.

Envelope substitutions shown to reduce AR4A binding at the protein level resulted in highly attenuated viruses. A, Huh7.5 cells were transfected with in vitro–transcribed RNA of the indicated H77/JFH1 recombinants. Supernatants were collected at the indicated time points, and HCV infectivity titers were determined. The lower level of quantification was 100 focus-forming units (FFU)/mL. An independent duplicate transfection yielded similar results. B, First passages of the indicated viruses were subjected to a dilution series of antibody AR4A, from 250 µg/mL to 0.0032 µg/mL. The virus/antibody mixes, along with virus only, were added to Huh7.5 cells for 4 hours prior to wash and addition of fresh medium. Following 48 hours of infection, the cells were immunostained, and the number of FFU per well was counted as described in Methods. Error bars represent standard error of the mean of 4 replicates normalized to 8 replicates of virus only. The data were analyzed using 3-parameter curve fitting to obtain a sigmoidal dose-response curve. Independent neutralization experiments yielded similar results. ^aThe indicated virus had the additional E1 substitution M345T.