Fig 6.

Cured Hep3B/miR122 cells facilitate efficient propagation of HCVcc through enhanced expression of miR122. (A) (Left) Hep3B/miR122 parental cells and cured cells of clone 5 were cotransfected with pIFNβ-Luc and pRL-TK and then infected with the VSV NCP mutant at an MOI of 0.01 or transfected with 1 μg of poly(I·C) at 24 h posttransfection, and luciferase activities were determined at 48 h posttreatment; (right) the cells were cotransfected with pISRE-Luc and pRL-TK and then infected with VSV at an MOI of 0.01 or treated with IFN-α (100 IU/ml) at 24 h posttransfection, and luciferase activities were determined at 48 h posttreatment. (B) (Upper) Hep3B/miR122 parental cells and the cured cells were infected with VSV at an MOI of 0.01, fixed with 4% phosphonoformic acid at 18 h postinfection, and subjected to indirect immunofluorescence assay using rabbit anti-IRF3 antibody, followed by AF488-conjugated anti-rabbit IgG (red); (lower) the cells were treated with IFN-α (100 IU/ml), fixed with 4% paraformaldehyde at 1 h postinfection, and subjected to indirect immunofluorescence assay using rabbit anti-STAT2 antibody, followed by AF488-conjugated anti-rabbit IgG (red). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). (C) Total RNA was extracted from parental Huh7 and Hep3B/miR122 cells and their cured cells, and the relative expression of miR122 was determined by qRT-PCR by using U6 snRNA as an internal control.