PI4KIIIα binding to NS5A triple alanine mutants of the PI4KIIIα binding region (PBR).
A: Schematic representation of NS5A and NS5A D1-LCS1. The sequence encompassing deletions ΔS3B and ΔS3C (186–212) is named PI4KIIIα binding region (PBR) and is highlighted in red. PBR was subjected to closer analysis by generation of triple alanine substitutions. Mutants were designated according to the respective wt sequence as exemplified. The PI4KIIIα functional interaction site (PFIS) is indicated by a dark red line. Small letters indicate amino acids specifically found in the JFH-1 isolate. For details refer to the legend of Fig. 1A. B: Huh7-Lunet T7 cells were cotransfected with plasmids encoding the NS3 to NS5B polyprotein of genotype 2a (JFH-1) containing triple alanine mutations in NS5A domain 1 as indicated and HA-tagged PI4KIIIα (HA-PI4K). Newly synthesized proteins were radiolabeled and cell lysates subjected to immunoprecipitation using NS5A (lower panel) or HA-specific antibodies (upper panel). Samples were analyzed by SDS-PAGE and autoradiography. C: Quantitative analysis of PI4KIIIα pull-down. Experiments as shown in panel B were quantified by phosphoimaging. Coprecipitation efficiency was normalized to the total amounts of HA-PI4K and calculated relative to PI4KIIIα pull-down by NS5A wt. Note that data were not normalized to input NS5A levels due to a 20–100fold molar excess of NS5A compared to PI4KIIIα (data not shown). Error bars indicate mean values +/− SD of two independent experiments analyzed in duplicates. Significance was calculated by a paired t-test. *, p<0.05; **, p<0.01; ***, p<0.001. D: Huh7-Lunet T7 cells were transfected with plasmids encoding the NS3 to NS5B polyprotein of genotype 2a (JFH-1) and/or HA-tagged PI4KIIIα (HA-PI4K) or mock transfected as indicated. Newly synthesized proteins were radiolabeled and cell lysates subjected to immunoprecipitation using HA- (lower panel) or NS5A-specific antibodies (upper panel). Samples were analyzed by SDS-PAGE and autoradiography.