Fig. 5
PPP2R5D was required for HCV infection in Huh7.5 cells demonstrated by gene knockdown, knockout, and stable complementation. A shRNAs targeting PPP2R5D (shPPP2R5D-1 and -2) suppressed PPP2R5D mRNA expression in Huh7.5 cells. The mRNA level of PPP2R5D in the shRNA-treated Huh7.5 cells was determined by real-time RT-PCR using GAPDH as internal control. Data are the mean of three determinations with the standard error of the mean (SEM). B HCV infectivity titers (FFU/ml, log10) were decreased in shPPP2R5D-knockdown Huh7.5 cells, in comparison with non-treated WT and control shRNAs (shScramble-1 and -2)-treated Huh7.5 cells. Culture supernatant was collected on day 7 post infection, and HCV infectivity titers were determined by FFU assay. Data are the mean of three determinations plus SEM. C PPP2R5D disruption in puromycin-selected monoclonal Huh7.5 cell line. Monoclonal PPP2R5D-KO cell line was sequenced, and one nucleotide insertion was found in PPP2R5D gene. D HCV infection was dramatically decreased in PPP2R5D-KO cells, compared to WT Huh7.5 cells at day 7. E Transient expression of PPP2R5D restored HCV infection in PPP2R5D-KO cells. The wild-type (WT) and PPP2R5D-KO Huh7.5 cells were transfected with HA-tagged PPP2R5D for 24 h (“#1” and “#2” represent two transfections), HCV infection was performed with MOI of 0.01, and immunostaining of HCV Core was performed at 48 h post infection. F Western blot analysis of PPP2R5D expression and HCV Core in transfected WT and PPP2R5D-KO Huh7.5 cells. The cells were collected from the experiment shown in panel E. “#1” and “#2” indicate the cells from duplicate transfections; “m.” indicates mock transfection; “b.” indicates the cells non-transfected (blank control). G Stable complementation of PPP2R5D in PPP2R5D-KO cells (PPP2R5D-Compl) rescued HCV infection. The WT, PPP2R5D-Compl, and irrelative knockout control cells (IRR-KO) Huh7.5 cells were infected with HCV (MOI = 0.01) in duplicate (#1 and #2), and the culture supernatant was collected at day 5 and determined for the infectivity titers by FFU assay. The means of three determinations plus FFU are shown