FIG 2.

Effects of mutations on viral protein expression and infectivity. (A) Huh-7 cells were electroporated with viral RNA transcribed from JFH1-derived mutants, and they were lysed at 48 h postelectroporation. Viral proteins were separated by SDS-PAGE and detected by Western blotting with MAbs A4 (anti-E1), 3/11 (anti-E2), and anti-NS5A. Western blotting with an anti-beta-tubulin antibody was performed in parallel to verify that equal amounts of cell lysates had been loaded. (B) Infectivity of E1 mutants. Huh-7 cells were electroporated with viral RNA transcribed from JFH1-derived mutants. At 48, 72, and 96 h postelectroporation, infectivities of the supernatants and intracellular viruses were determined by titration. Error bars indicate standard errors of the means from at least three independent experiments. Results were compared to those for the wild type, and a P value of <0.05 was determined for intracellular mutants Q193A, V194A, I212A, T213A, W214A, Q215A, V220A, L221A, H222A, R231A, G233A, W239A, V240A, P244A, I262A, D263A and M264A. A P value of <0.05 was determined for extracellular mutants I212A, T213A, W214A, Q215A, H222A, W239A, and D263A. WT, wild type; FFU, focus-forming units.