FIG 8.

Generation of mutant viruses using an entry route that is less dependent on endosomal pH and clathrin. (A) Huh-7 cells were pretreated for 2 h with monensin and then infected with JFH1-EtOH or JFH1-Mon for 2 h in the presence of monensin or EtOH (CTL). Cells were then incubated for 30 h in complete medium supplemented with monensin at the indicated concentrations. (B) Huh-7 cells were infected with JFH1 or FL-8 as described for panel A. (C) Huh-7 cells were pretreated for 2 h with chloroquine at the indicated concentrations and then infected with JFH1-EtOH or JFH1-Mon viral populations for 2 h in the presence of chloroquine. Cells were then incubated for 30 h in complete medium supplemented with chloroquine or H₂O (CTL). (D) Huh-7 cells were infected with JFH1 or FL-8 as described for panel C. (E) Dose-response curves for the JFH1 and FL-8 viruses and monensin or chloroquine. Infections and treatments were performed as described for the previous panels. (F) Huh-7 cells were pretreated with chlorpromazine (5 μg/ml) for 30 min and then infected with JFH1 or FL-8 for 2 h in the presence of chlorpromazine or H₂O (CTL). Cells were then incubated for 30 h in complete medium supplemented with chlorpromazine. Results are presented as means ± SD for three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.