FIG 7.

Phosphomimetic and dimerization-deficient IRF3 mutants. (A and B) Promoter reporter assays were performed under conditions of stimulation with a phosphomimetic, constitutively active IRF3(5D). (A) HEK293 cells were transfected with expression plasmids for NSs of RVFV, SFSV, PTV-A, or PTV-B or inactive control ΔMx, as well as firefly and Renilla luciferase reporters, under the control of the IFN-β and constitutively active simian virus 40 (SV40) promoters, respectively. IFN-β induction was stimulated by overexpression of IRF3(5D) and total plasmid adjusted to equal levels with empty vector. Firefly activities were normalized to those of Renilla, and the stimulation control was set to 100% (n = 3; means ± SD). (B) A dual-luciferase assay was performed in parallel with a PRDI-responsive firefly luciferase reporter (n = 3; means ± SD). (C and D) Interaction with IRF3 mutants. (C) 3×FLAG-tagged SFSV NSs or PTV-A NSs was coexpressed with IRF3(5D) in HEK293 cells. Cell lysates were then subjected to immunoprecipitation using an antibody against FLAG that was covalently coupled to magnetic beads beforehand. (D) GFP-IRF3(S385/386A), eGFP-IRF3, or eGFP, as well as 3×FLAG-tagged SFSV NSs, was obtained by transient transfection of HEK293 cells. Immunoprecipitation was performed via the use of GFP.