FIG 3.

Effect of second-site mutations on replication of wild-type and NS4A mutants. (A) Vero E6 cells were electroporated with the reporter virus DVs-R2A containing the mutations specified on the bottom of each panel. Cell lysates were collected 24, 48, 72, and 96 h after transfection, and renilla luciferase activity was measured as surrogate for viral replication. Values were normalized to the 4-h time point, reflecting transfection efficiency. Data represent the mean and SD of three independent experiments. Significance has been calculated by Student’s t test using, for comparison, values from the same time point obtained with the primary mutant. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B) NS4A genetic interaction map as determined through reverse and forward genetic screens. Arrows connect primary mutations (squares) and the corresponding second-site compensatory mutations (circles).