Reduced PI4KIIIα interaction and RNA replication deficiency correlate with relative increase of NS5A hyperphosphorylation.
A: Huh7-Lunet T7 cells were transfected with plasmids encoding the NS3 to NS5B polyprotein of genotype 2a (JFH-1) containing a wt sequence or triple alanine mutants as indicated or with empty plasmid (mock). Newly synthesized proteins were radiolabeled and cell lysates subjected to immunoprecipitation using NS5A specific antibodies. Immunocomplexes were analyzed by SDS-PAGE and autoradiography. B: Quantitative analysis of the NS5A p58/p56 ratio. Bands corresponding to NS5A p58 and p56, respectively, as shown in panel A were individually quantified by phosphoimaging to obtain a p58/p56 ratio. Error bars indicate mean values +/− SD of two independent experiments analyzed in duplicates. Significance was compared to the wt polyprotein and calculated by a paired t-test. *, p<0.05; **, p<0.01; ***, p<0.001. A blow up of figure 6A focusing on the NS5A phosphorylation of PFIS mutants in comparison to the wildtype polyprotein is shown below.