Figure 2.

Sandfly virus infection decreases the phosphorylation of STAT1 and 2. (A) SFNV, and SFSV inhibits STAT1 phosphorylation. A549 cells were infected with SFSV or SFNV at an MOI of 1. The cells were stimulated with IFNα (1000 U/ml) for 30 min 24 h post-infection. The lysates were analyzed by western blot using specific antibodies for phosphorylated or total STAT1. Tubulin was used as a loading control. Numbers below the bands represent average bands intensity from three independent experiments quantified using ImageJ and are expressed as a percent of mock-infected and stimulated control. (B) SFNV and SFSV affect STAT1 localization. Vero cells were infected with SFNV at the indicated MOIs (left). The next day, cells were treated with IFNα (1000 U/ml) for 30 min, then fixed and co-stained with anti-Naples (red) and anti-STAT1 (green) specific antibodies. DAPI was used for nuclei labeling (blue). A similar experiment was performed following SFSV infection. Staining was with anti-Sicily antibodies (right). Scale bar=50µM. (C) SFNV and SFSV infection inhibit STAT2 phosphorylation. Cells were infected and stimulated and analyzed as described above for STAT1. Analysis was performed using STAT2 and phospho-STAT2 specific antibodies. (D) Subcellular fractionation of Infected cells. A549 cells were infected with SFSV or SFNV at an MOI of 1. The cells were stimulated with IFNα (1000 U/ml) for 30 min 24 h post-infection. The cells were fractionated, and the lysates were analyzed by western blot using specific antibodies for phosphorylated STAT1. Tubulin was used as a cytoplasmic marker, and lamin was used as a nuclear marker.