Four different kinases, including PKR, PKR-like ER kinase (PERK), heme-regulated inhibitor and the general control, nondepressible-2 (GCN2), are known to phosphorylate eIF2a [35] . To determine the role of PKR in the accumulation of phosphorylated eIF2a in rMP12-rLuc-infected cells treated with ActD or aamanitin, we generated a recombinant MP-12, rMP12-PKRDE7, which expresses a dominant-negative form of PKR, PKRDE7 [36] carrying the N-terminal Flag epitope tag, in place of the NSs ( Figure 5A ). If the replication of MP-12 lacking the NSs gene in cells subjected to host transcriptional suppression activates PKR, which in turn phosphorylates eIF2a, then the virally-encoded PKRDE7 in rMP12-PKRDE7-infected cells would interfere with the PKR function, resulting in the inhibition of PKR-mediated eIF2a phosphorylation and thereby leading to efficient viral translation and virus production. VeroE6 cells were mock-infected or independently infected with rMP12-rLuc, rMP12-PKRDE7 and MP-12 at an moi of 3. After the removal of the inocula, cells were treated with ActD or a-amanitin, and cell extracts were harvested at 16 h.p.i. As expected, rMP12-rLuc replication in the presence of ActD or a-amanitin induced eIF2a phosphorylation, resulted in reduced virus replication ( Figure 5B -5D). In contrast, efficient N protein accumulation and efficient virus replication, with no significant accumulation of phosphorylated eIF2a occurred in both MP-12-infected cells and rMP12-PKRDE7infected cells in the presence of ActD-or a-amanitin ( Figure 5B -5D). This finding strongly suggested that PKR is important for eIF2a phosphorylation in cells infected with the RVFV lacking the NSs in the presence of transcription inhibitors.