Transfection of in vitro synthesized RNA was performed by using TransIT-mRNA Transfection Kit (Mirus) according to manufacturer's instruction. Figure 8 . Co-expression of truncated NSs in RVFV-infected cells. 293 cells were mock-infected or infected with rMP12-NSs-Flag at an moi of 3, and mock-transfected or immediately transfected with in vitro transcribed RNA encoding CAT (control) or NSs with indicated NSs mutants. Cells were collected at 16 hpi, and NSs-Flag/NSs (a-RVFV antibody), NSs-Flag (a-Flag antibody), PKR (anti-PKR antibody), CAT-myc (anti-myc antibody) and b-actin (anti-actin antibody) were detected by Western blot. (B) A549 cells were mock-infected or infected with rMP12-NSs-Flag at an moi of 3, and mock-transfected or immediately transfected with in vitro transcribed RNA encoding CAT or NSs with indicated NSs mutants. As a control to induce IFN-b and ISG56 mRNA synthesis, A549 cells were infected with rMP12-C13type (C13type) at an moi of 3. Total RNA was extracted at 7 hpi, and IFN-b mRNA, ISG56 mRNA and RVFV anti-viral-sense S-RNA/N mRNA were detected by Northern blot with specific RNA probe. Representative data from at least 3 independent experiments are shown. (C) A549 cells were infected with MP-12 at moi of 0.01, and mock-transfected or immediately transfected with in vitro transcribed RNA encoding CAT or NSs of NSD249-265 or NSs-E253-255A. Culture supernatants were harvested at 72 hpi, and virus titers were measured by plaque assay. P-values of unpaired Student's t-test are shown (*; p,0.05, ns; not significant). doi:10.1371/journal.pone.0045730.g008