Because activated PKR undergoes a structural alteration and autophosphorylation [30] , we tested whether NSs prevented PKR autophosphorylation. 293 cells were infected with rMP12-NSs-Flag, rMP12-rLuc-Flag or rMP12-PKRDE7, and mock-treated or immediately treated with ActD. Cell lysates were harvested at 16 h.p.i., and PKR was immunoprecipitated by anti-human PKR antibody, and the PKR bound to the protein A beads was subjected to an immunoprecipitation (IP)-kinase assay by using [c-32 P]ATP. We used 293 cells for this assay, because anti-human PKR antibody efficiently immunoprecipitated human PKR in 293 cells, but not non-human primate PKR in VeroE6 cells (data not shown). PKR is induced by IFN-a/b treatment [38] and the abundance of PKR could affect the results of the IP-kinase assay. We suspected that replication of MP-12 mutants lacking NSs may induce IFN-b production, leading to PKR induction [38] , whereas ActD treatment prevented the IFN-b production [39] . Indeed, IFN-b mRNA accumulation occurred at 8 h.p.i. in rMP-12-rLucinfected cells in the absence of ActD, but not in the presence of ActD ( Figure 6C ), a finding which suggested that ActD treatment inhibited the transcriptional induction of PKR which was induced by the type I IFN in infected 293 cells. We noted an efficient accumulation of rMP12-rLuc N mRNA at 8 h.p.i. in the presence of ActD ( Figure 6C ). Because rMP-12-rLuc replication in the presence of ActD did not induce an accumulation of phosphorylated eIF2a early in the course of the infection ( Figure 4A ), this efficient rMP12-rLuc replication probably occurred prior to 8 h.p.i. in the presence of ActD. As shown in Figure 6D , immunoprecipitated PKR from rMP12-rLuc-Flag-infected cells was phosphorylated both in the presence and absence of ActD, which led us to suggest that rMP12-rLuc-Flag replication activated PKR. In contrast, PKR phosphorylation did not occur in mockinfected cells or cells infected with rMP12-NSs-Flag or rMP12-PKRDE7 ( Figure 6D ). Most probably, a dominant-negative PKRDE7 suppressed PKR activation and prevented PKR phosphorylation [36] . To determine why we failed to detect the presence of PKR autophosporylation in rMP12-NSs-Flag-infected cells, we examined the amounts of the immunoprecipitated PKR in these samples ( Figure 6D, bottom) . Strikingly, substantial reductions in the abundance of cytoplasmic PKR occurred only in the cells supporting rMP12-NSs-Flag replication both in the presence and absence of ActD.