FIG 8.

Impact of phosphoablative mutations of potential phosphoacceptor sites on T242 phosphorylation. Huh7-Lunet T7 cells were transfected with pTM vectors expressing NS3-5B of isolate JFH-1, either WT or harboring the indicated phosphoablative alanine mutations. Mutant “N-termA” comprises all five serine and threonine mutations from T242 to T249. Mutant “C-termA” comprises both substitutions at positions T268 and S272. (A) Cells were lysed 24 h after transfection and analyzed by Western blotting using anti-NS5A (monoclonal antibody 9E10, upper)-, anti-NS5A-pT242-, anti-NS5A-pS235-, anti-NS3, and anti-β-actin (ACTB)-specific antibodies. (B) Ratio of p58 and pT242 to total NS5A. Intensities of the p58 bands, the pT242 bands, and the sum of p56 and p58 (total NS5A) as shown in panel A were quantified and used to calculate the indicated ratios. An additional staining of NS3 was performed subsequently on the same membranes previously used for detection of NS5A or pT242. These NS3 signals were used to obtain relative NS5A or pT242 levels for each membrane, which built the basis for pT242/NS5A levels. Bars represent mean values and SD from one representative experiment with three technical replicates. A second biological replicate was performed with a similar outcome (n = 2). n.d., not detectable. ***, P < 0.01 (homoscedastic, two-tailed t test).