Figure 3.

HAV‐RNA replication is independent of PI4KIIIα or β. (A) HAV‐ and HCV‐RNA replication upon treatment with PI4KIIIα inhibitor AL‐9. Huh7‐Lunet cells were electroporated with subgenomic replicon RNA of HAV or HCV (JFH, Con1, and H77) and treated with indicated concentrations of PI4KIIIα inhibitor AL‐9 4 hours p.t. Then, 48 hours upon electroporation, cells were lysed for measurement of luciferase activity. Data are derived from two independent experiments performed with triplicates and normalized to untreated controls. Graph depicts mean and standard deviation (SD) in logarithmic scale (n = 2). (B) HAV‐ and HCV‐RNA replication in PI4KIIIα knockdown cells. HAV or HCV subgenomic replicon RNAs were transfected into Huh7‐Lunet cells stably expressing either a nontargeting shRNA (shONT) or a PI4KIIIα‐specific shRNA (shPI4KIIIα). Then, 4, 24, 48, and 72 hours p.t., cells were lysed to determine luciferase activity. Graph depicts mean and SD of two independent experiments performed with triplicates. Data were normalized to the 4‐hour value and are presented in logarithmic scale (n = 2). (C) HAV‐ and HCV‐RNA replication upon treatment with PI4KIIIβ inhibitor PIK93. Cells were treated with PIK93 and analyzed as described in (A). (D) Western blot analysis of PI4KIIIβ in CRISPR/Cas9‐PI4KIIIβ cells. Parental cells, a pool of CRISPR/Cas9‐PI4KIIIβ cells (Pool3) and three single‐cell clones (A1, C1, and D2), were examined for PI4KIIIβ protein expression by using a mouse monoclonal anti‐PI4KIIIβ antibody. (E) HAV‐ and HCV‐RNA replication in CRISPR/Cas9‐PI4KIIIβ cells. Parental cells, Pool3, or Clone C1 cells were transfected with HAV or HCV subgenomic replicon RNA. Then, 4, 24, 48, and 72 hours upon electroporation, cells were harvested to measure luciferase activity. Graph depicts mean and SD of a representative experiment (n = 2). Data were normalized to the 4‐hour value and are presented in logarithmic scale. Abbreviations: kDa, kilodaltons; p.t., post‐transfection.