Since NSs expression did not decrease the abundance of PKR mRNA ( Figure 7C ), we next tested whether putative NSsmediated translational inhibition leads to a reduction in the abundance of PKR. To this end, 293 cells were mock-infected, infected with MP-12, or transfected with in vitro-synthesized RNA transcripts encoding NSs. Then cells were incubated with 100 mg/ ml of puromycin to completely shut off cellular translation or puromycin untreated, and cell extracts were harvested at 16 h.p.i. or post-transfection. As expected, puromycin treatment completely abolished the synthesis of N and NSs proteins in MP-12-infected cells ( Figure 7D, lane 4) and that of NSs in cells transfected with the RNA transcripts encoding NSs ( Figure 7D, lane 6) . We found that treatment of 293 cells with puromycin for 16 h decreased the abundance of PKR only slightly ( Figure 7D, lane 2) . Accordingly, it is highly unlikely that putative NSs-induced translational We next performed pulse-chase experiments to know whether NSs expression promoted PKR degradation. Because immunoprecipitation experiments using various anti-PKR antibodies failed to convincingly demonstrate a radiolabelled endogenous PKR signal from extracts of 293 cells (data not shown), we examined the effect of NSs expression on the stability of an expressed mutant PKR, PKRK296R, lacking kinase activity [40] and carrying a Nterminal myc tag; expression of wild-type PKR was not used due to its strong host translation suppression effects (data not shown). 293 cells were mock-transfected or transfected with pcDNA3.1-Myc-PKRK296R, a plasmid encoding PKRK296R under cytomegalovirus promoter and radiolabelled with [ 35 S] methionine/cycteine between 14 and 16 h post-DNA transfection. After pulse-radiolabelling cell extracts were prepared from some samples. In other samples, cells were transfected with in vitrosynthesized RNA transcripts encoding rLuc or NSs, and cell extracts were harvested at 8 h post-RNA transfection. Then the cell extracts were subjected to radioimmunoprecipitation analysis using anti-myc antibody, which immunoprecipitated the pulseradiolabelled, myc-tagged PKR ( Figure 7E , lane 2). After 8 h chase, the amount of the radiolabelled myc-tagged PKR was clearly reduced in the cells expressing NSs ( Figure 7E, lane 4) , but not those expressing rLuc ( Figure 7E, lane 3) . Instead, the amount of radiolabelled myc-tagged PKR was increased slightly in rLucexpressing cells, presumably due to radiolabelling of continuously synthesized myc-tagged PKR by residual [ 35 S] methionine/ cysteine.