FIG 4.

N protein HVD chimeras lack the ability to regulate IFN signaling. (A) HVD residues (252 to 296) between ANDV and MAPV N proteins were reciprocally swapped to generate ANDV N:Δhvd and MAPV N:Δhvd mutant proteins. (B and C) HEK293T cells were cotransfected as described in the legend to Fig. 1 with plasmids expressing ISRE/IFNβ promoter firefly luciferase reporters, Renilla luciferase, Flag-MDA5, and, as indicated, plasmids expressing wt ANDV N protein, ANDV N:Δhvd, MAPV N:Δhvd, or wt MAPV N protein. Cells were lysed at 24 h posttransfection, and firefly luciferase activity was normalized to internal control Renilla luciferase activity, evaluated as described in the legend to Fig. 1. Comparable protein expression levels are shown in the Western blots. Assays were performed in triplicate with similar results in at least 3 separate experiments. Asterisks indicate statistical significance (*, P < 0.05), as determined by one-way ANOVA with Tukey’s post hoc test. (D) HEK293T cells were transfected as described in the legend to Fig. 2 with plasmids expressing IRF3, Flag-MDA5, and wt ANDV N protein, ANDV N:Δhvd, MAPV N:Δhvd, or wt MAPV N protein. Phospho-IRF3 (pIRF3 S-396), Flag-MDA5, N protein, and β-actin expression levels were analyzed by Western blotting, and the results are representative of those from ≥2 experiments.