Impact of mutations within PI4KIIIα binding region on subcellular localization of NS5A and PI4P levels.

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 representative triple alanine mutants as indicated or with empty plasmid (mock). 24 h post transfection NS5A (red) or PI4P (green), respectively, were detected with specific antibodies and nuclear DNA was stained with DAPI (blue). Note the punctuate NS5A staining pattern in 3-5B wt transfected cells compared to formation of NS5A “clusters” in cells expressing mutant polyproteins. Asterisks point to non-transfected cells. B: Quantitative analysis of NS5A staining patterns for wt and mutant NS3-5B polyproteins. 350 NS5A positive cells for each construct were counted and distinguished between wt (black) and clustered (red) structures by qualitative judgement, based on the representative examples for wildtype (panel A, second row) and cluster (panel A, row 3–5). C: Quantitation of intracellular PI4P levels by measuring PI4P fluorescence intensity using ImageJ analysis (IntDen read-out). Bars indicate the mean of arbitrary units (AU) +/− SD of 35 NS5A positive cells analyzed per condition. The blue line points the threshold which was set to the mean of PI4P IntDen values of untransfected cells (mock). Significance of increased PI4P levels was measured by a paired t-test and is indicated *, p<0.05; **, p<0.01; ***, p<0.001.