Fig 3.

Establishment of a novel permissive cell line for robust propagation of HCVcc by expression of miR122 in Hep3B cells. (A) Huh7, Hep3B/Cont, and Hep3B/miR122 cells were infected with HCVcc at an MOI of 0.1 or 1, and the levels of expression of viral proteins were determined by immunoblotting using appropriate antibodies at 72 h postinfection. (B) Huh7, Hep3B/Cont, and Hep3B/miR122 cells were infected with HCVcc at an MOI of 1 and incubated with 1% methylcellulose in DMEM containing 5% FCS for the indicated time. Cells were fixed with 4% paraformaldehyde and subjected to indirect immunofluorescence assay using anti-NS5A antibody, followed by AF594-conjugated anti-rabbit IgG (red). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). (C) Huh7 and Hep3B cells were lysed and subjected to immunoblotting using appropriate antibodies. (D) Huh7, Hep3B/Cont, and Hep3B/miR122 cells were infected with HCVcc at an MOI of 1, the culture supernatants were collected at 72 h postinfection, and the viral titers of the supernatants were determined by focus-forming assay using Huh7.5.1 cells.