FIG 8.

Phosphomimetic S386D directs IFN regulation by ANDV N protein. (A to D) HEK293T cells were cotransfected as described in the legend to Fig. 1 with plasmids expressing Flag-MDA5, ISRE/IFNβ firefly luciferase and Renilla luciferase reporters, the pcDNA3.1⁺ empty vector, and the indicated N proteins: wt ANDV N, ANDV N:S386H, ANDV N:S386A, ANDV N:S386D, MAPV N:H386D, or wt MAPV N. Luciferase activity was measured, Western blot analysis were performed, and the results were analyzed as described in the legend to Fig. 1. Assays were performed in triplicate with similar results in at least 3 separate experiments. Asterisks indicate statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001), as determined by one-way ANOVA with Tukey’s post hoc test. (E) HEK293T cells were cotransfected with plasmids expressing IRF3 and Flag-MDA5 as described in the legend to Fig. 2 and the plasmids expressing the indicated N protein: wt ANDV N, ANDV N:S386D, MAPV N:H386D, wt MAPV N, or the empty vector. Proteins were analyzed for Flag-MDA5, β-actin, total and phosphorylated IRF3, and N protein levels by Western blotting as described in the legend to Fig. 2, and the results are representative of those from ≥2 experiments.