NS5A phosphorylation is influenced by PI4KIIIα enzymatic activity.

A: Huh7-Lunet T7 cells expressing shRNA directed against PI4KIIIα (shPI4K) or a non-targeting control (shNT) were transfected with plasmids encoding the NS3 to NS5B polyprotein of HCV genotype 2a (JFH-1) or variants of genotype 1b (Con1 wt, ET, mutHIT). PI4KIIIα expression in shPI4K cells was reconstituted by expression of a knockdown-resistant escape variant of PI4KIIIα (sh+Esc) to exclude off-target effects. B: Quantitative analysis of the ratio of NS5A p58 and p56 obtained by phosphoimaging of experiments as shown in panel A. C: Naïve Huh7-Lunet T7 cells were transfected with plasmids encoding the NS3 to NS5B polyprotein of JFH-1 wt, Con1 wt or Con1 ET. Starting at 7 h post transfection, cells were incubated with indicated concentrations of AL-9. D: Quantitative analysis of the ratio of NS5A p58 and p56 obtained by phosphoimaging of experiments as shown in panel C. E: Huh7-Lunet T7 cells were cotransfected with plasmids encoding the NS3 to NS5B polyprotein of HCV genotype 2a (JFH-1) or variants of genotype 1b (Con1 wt, ET, mutHIT) and empty constructs (−) or plasmids encoding HA-tagged wt (wt) or inactive mutant (D1957A) PI4KIIIα. F: Quantitative analysis of the ratio of NS5A p58 and p56 obtained by phosphoimaging of experiments as shown in panel E. A, C, E: Newly synthesized proteins were radiolabeled and cell lysates subjected to immunoprecipitations using NS5A specific antibodies. Immunocomplexes were analyzed by 10% SDS-PAGE and autoradiography. B, D, F: Data represent mean values +/− SD from 2 independent experiments analyzed in duplicates. Significances were calculated by paired t-tests. *, p<0.05; **, p<0.01; ***, p<0.001.