Ectopic expression of CD81 in HCV-resistant Huh-7 cells restores HCV permissivity. A, Huh-7 cells and R1 cell population infected with JFH-1 HCVcc were processed for double-label immunofluorescence for capsid protein (green) and nuclei (blue, Hoechst). B, Cells were infected with virus pseudotyped with HCV envelope proteins from 1a (HCVpp 1a) or 2a (HCVpp 2a) or VSV G envelope protein (VSVpp). C, Huh-7 cells and R1 individual cellular clones were infected with HCVcc expressing Renilla luciferase. In parallel, Huh-7 cells and some of the clones were infected with HCVpp 1a, HCVpp 2a or VSVpp (D). Results are presented as relative percentages to HCVcc (C) and HCVpp (D) infectivity on Huh-7 cells. HCVpp infections (D) were also normalized to VSVpp infections on Huh-7 cells. E, Surface biotinylated cell lysates were immunoprecipitated with anti-CD81 (5A6), anti-SR-BI (NB400-104H3) or anti-CLDN-1 (JAY.8) mAbs. Proteins were revealed by Western blotting with HRP-conjugated streptavidin. F, Flow cytometry analysis of CD81 cell surface expression. Cells were stained using an anti-hCD81 (22.214.171.124, left panel) or an anti-mCD81 (MT81, right panel), and secondary antibodies conjugated with PE. Ctrl corresponds to Huh-7 cells stained only with secondary antibodies. Cell lines were infected with HCVcc (G) and in parallel with HCVpp (H) generated with envelope proteins from different genotypes or virus pseudotyped with feline endogenous virus RD114 glycoprotein (Rd114pp). Results are presented as relative percentages to HCVcc (G) and HCVpp (H) infectivity on Huh-7 cells. P < 0.05 as calculated by the Mann-Whitney's test; *, statistically not significant difference in HCVpp entry compared to entry into Huh-7 cells.