FIG 6.

B5 inhibits a late step of HCV entry. (A) B5 inhibits HCVpp entry. Huh-7 cells were infected with pseudoparticles containing the HCV envelope glycoproteins of JFH1 (HCVpp) or with pseudoparticles containing the envelope glycoprotein of the feline endogenous retrovirus RD114 (RD114pp). Cells were treated with B5 for 1 h before infection and for the whole duration of the experiment. At 48 h postinfection, cells were lysed to quantify the luciferase activity. A control experiment with anti-CD81 MAb JS-81 was performed in parallel. HCVpp infectivity varied between 10⁵ and 10⁶ relative light units (RLUs). Results are expressed as percentage of infection compared to the control infection in the presence of solvent. Error bars indicate standard errors of the means from at least two independent experiments. (B) Effect of B5 on HCV binding and internalization. Huh-7 cells were inoculated for 1 h at 4°C with purified HCVcc at an MOI of 10 in the presence of solvent (PBS), B5, or 500 μg/ml of heparin. Cells were washed thrice with ice-cold PBS, and total RNA was extracted. For internalization, the binding step was followed by a 30-min incubation at 37°C, cells were then chilled on ice, and noninternalized virions were removed by trypsinization for 1 h at 4°C. Bound HCV virions or internalized viral particles were detected by quantification of HCV genomic RNA by quantitative RT-PCR. Mean values ± SD (error bars) from three different experiments are presented. Turkey's multiple-comparison test was used for statistical analysis. To verify the efficacy of trypsin treatment, virus was bound to cells at 4°C and then removed by trypsinization (data not shown). No significant difference (ns) was observed between treated and untreated cells. (C) B5 does not induce alkalization of intracellular organelles. Huh-7 cells were treated for 90 min with B5 (10 μM) or monensin (1 μM) or were left untreated (control). After incubation with acridine orange, cells were observed by confocal microscopy.