Figure 1. CoFIM selection of a full-length HCV that is less dependent on CyPA.

(A) A diagram of CoFIM selection #1. Sh-A161 was introduced into infected cells and selected with puromycin. Stable cells were then passaged until all cells became infected. (B) The selected mutant virus (J-LA) efficiently infects CyPA and CyPB knockdown cells. Supernatant collected from (A) was used to infect cells harboring indicated shRNAs; infected cell lysates were collected 4 days after infection and analyzed by SDS-PAGE and western blotting. A cellular protein, Ku80, was detected as a loading control. (C) J-LA is more resistant to CsA treatment than WT JFH-1. Huh-7.5 sh-Luc cells were infected with JFH-1 or J-LA for 24 h before CsA was added to the culture medium. The cells were incubated for 4 more days before being lysed and analyzed for NS3 expression. (D) J-LA virus efficiently infects CyPA knockdown cells but not PI4K or Occludin knockdown cells. Equal numbers of Huh-7.5 cells harboring either control or the indicated shRNAs were seeded and infected with either JFH-1 or J-LA virus. Five days after infection, cells were fixed and subjected to anti-NS3 antibody staining. Data shown in (B), (C), and (D) represent two independent experiments.