Figure 3.

NSs inhibits IFN mediated transcription and affects STAT1 localization. (A) Intracellular localization of NSs-N and NSs-S proteins. FLAG-tagged NSs proteins were cotransfected into U2OS cells with pEGFP-N1. The cells were fixed and stained with anti-FLAG antibodies. (B) NSs mediated inhibition of IFN mediated transcription. ISG56 luciferase reporter cells were transfected with plasmids expressing sandfly NSs, Dengue NS4A, or an empty plasmid. A Renilla luciferase-expressing plasmid was used as a transfection control. After 24 h, the cells were treated with IFNα (1000 U\ml). The cells were harvested after an additional 12 h, and luciferase activity was determined. Shown is a representative experiment from three independent experiments showing similar results (**P<0.01, ***P<0.001, One way ANOVA, Dunnett’s multiple comparisons test). The lysates from the luciferase assay were subjected to western blot analysis using FLAG antibodies. Tubulin was used as a loading control. (C, D) NSs affects STAT1 localization. Vero cells were transfected with FLAG-tagged NSs expressing plasmids. The cells were stimulated with IFNα (1000 U/ml) for 30 min 24 h post-transfection, then fixed and stained with anti-FLAG (red) and anti-STAT1 (green) specific antibodies. DAPI was used for nuclei labeling (blue). (E) The percentage of transfected cells with nuclear STAT1. Results are representative of three independent experiments (n=50 cells for each condition ****P<0.0001, One way ANOVA, Dunnett’s multiple comparisons test). Scale bar = 10 µM.