We subsequently tested the possibility that the NSs downregulated PKR expression or sequestered PKR into the nuclear compartment, leading to the suppression of eIF2a phosphorylation. 293 cells were mock-infected or infected with rMP12-NSs-Flag, rMP12-rLuc-Flag or rMP12-PKRDE7, treated with ActD, and cytoplasmic and nuclear fractions of cell extracts were prepared at 16 h.p.i. Western blot analyses showed the presence of RVFV NSs both in the cytoplasmic and nuclear fractions, while rLuc and PKRDE7 signals were observed only in the cytoplasmic fraction. A substantial reduction in PKR abundance occurred in both the cytoplasmic and nuclear fractions of cells infected with rMP-12-NSs-Flag, but not in mock-infected cells and in those infected with rMP12-rLuc-Flag or rMP12-PKRDE7 ( Figure 7A ), demonstrating that NSs induced the PKR downregulation in the infected cells. The PKR downregulation also occurred in MP-12infected VeroE6 cells and in MRC-5 cells that were infected with the wt ZH501 strain of RVFV (data not shown).