We subsequently examined the requirement of the ActD treatment for the PKR downregulation. 293 cells were transfected with in vitro-synthesized RNA transcripts encoding NSs or rLuc in the absence of ActD. Analysis of cell extracts harvested at 8 h posttransfection showed the PKR downregulation in the NSsexpressing cells ( Figure 7B, right panel) , demonstrating that the NSs-mediated PKR downregulation occurred in the absence of ActD.