Triple alanine mutants induce ultrastructural changes similar to membranous web structures in PI4KIIIα knockdown cells.
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Huh7-Lunet T7 cells (A) or Huh7-Lunet T7 cells with stable PI4KIIIα knockdown (B) were transfected with pTM constructs expressing wt or mutant NS3 to NS5B polyproteins or eGFP. Cells were fixed and prepared for EM analysis 24 h post transfection. Consecutive enlargements of the boxed areas are shown from left to right. Note the heterogeneous membranous web (MW, yellow arrows) in cells expressing the wt polyprotein and the clusters of smaller double-membrane vesicles (DMVs) in shPI4KIIIα cells (B) and in cells expressing mutant polyproteins. Scale bars are given in the lower right of each panel. N, nucleus; LD, lipid droplet; rER, rough endoplasmic reticulum; m, mitochondrium. The number in the upper left of right panels shows the average diameter of 70 double-membranous vesicles (DMV) +/− SD measured for each condition. (C) Average diameter of DMVs detected in cells that had been transfected with constructs and conditions specified on the left and shown in panel A and B. Error bars indicate the mean +/− SD of seventy vesicles. Significance of differences in DMV sizes was measured by a paired t-test and is indicated ***, p<0.001.