FIG 9.

Generation of monensin-resistant viruses. (A) Huh-7 cells were pretreated for 2 h with monensin and then infected with mutant viruses 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 electroporated with JFH1, FL-8, or I399T or Y297H mutant RNAs. Supernatants were collected 24, 48, 72, and 96 h after electroporation, and virus titers as well as viral RNA amounts were determined. Specific infections were calculated by dividing the virus titer by the corresponding number of viral RNA copies quantified in the same volume of supernatant. Results are presented as means ± SD for four independent experiments. (C) (Top) Amino acid sequences of HVR1 of the JFH1 and I399T viruses. (Bottom) Analysis of infected cell lysates by 8% SDS-PAGE followed by 3/11 Western blotting. The dashed line allowed us to evaluate the shift in migration profile, which was due to the presence of an additional N-glycosylation site in E2 HVR1 of the FL-8 and I399T viruses. The sizes (in kilodaltons) of protein molecular mass markers are indicated on the left.