The present study explored a novel function of the RVFV NSs protein by testing the replication of RVFV lacking the NSs gene initially in type I IFN-incompetent VeroE6 cells [43, 44] in the presence of ActD or a-amanitin, which served as a surrogate of the host mRNA synthesis suppression function of the NSs. The NSs protein was essential for efficient virus replication in the presence of ActD or a-amanitin. We found that the replication of RVFV lacking the NSs gene in the presence of a transcription inhibitor induced an accumulation of phosphorylated eIF2a, The accumulation of phosphorylated eIF2a in the presence of transcriptional inhibitors, did not occur in VeroE6 cells that were infected with RVFV expressing a dominant-negative form of PKR (PKRDE7) and in MEF cells lacking functional PKR ( Figure 5 ). These findings suggested to us that PKR played a major role in increasing phosphorylated eIF2a; however, it is currently unclear whether other kinases, such as PERK or GCN2, have a possible role in eIF2a phosphorylation in rMP-12-rLuc-infected cells in the presence of transcriptional inhibitors. The endogenous PKRmediated eIF2a phosphorylation suppressed translation of the RVFV lacking the NSs gene, resulting in poor virus replication. Our data further suggested that the NSs promoted PKR degradation most probably through a proteasome-dependent pathway and prevented eIF2a phosphorylation, leading to efficient viral translation. (8 h) . Radiolabelled myc-tagged PKR was immunoprecipitated by anti-myc monoclonal antibody, analyzed by SDS-PAGE with 7.5% poryacrylamide gel, and visualized by autoradiography. (F) 293 cells were infected with rMP12-rLuc or MP-12 at an moi of 3, and, then, treated with 10 mM of MG132 (MG) or 50 mM of lactacystin (LA) or they were mock-treated (2) . Whole-cell lysates were collected at 8 h.p.i., and the abundance of PKR, NSs and b-actin were examined by Western blot analysis. (G) 293 cells were mock-infected (Mock) or infected with MP-12 (MP-12) at an moi of 3, and treated with MG132 (MG132) at 10 mM or they were untreated (no drug used). Whole-cell lysates were collected at 2, 4, 6, and 8 h.p.i. Anti-PKR antibody, anti-NSs antibody, and anti-b-actin antibody were used to detect PKR, NSs, and b-actin, respectively (F and G). Data are representative of two to three independent experiments (A-G). doi:10.1371/journal.ppat.1000287.g007