We next tested the effect of co-expression of NSs-E253-255A/ D257-259A, NSsD257-259A or NSs-E253-255A in infected cells, as described above. 293 cells were mock-infected or infected with rMP12-NSs-Flag using a moi of 3, then cells were mocktransfected or transfected with in vitro synthesized RNA encoding CAT, NSD249-265, NSs-E253-255A/D257-259A, NSsD257-259A or NSs-E253-255A. Cells were collected at 16 hpi for Western blot analysis. As shown in Fig. 8A , co-expression of NSs-E253-255A/D257-259A or NSs-D257-259A or NSs-E253-255A did not inhibit PKR degradation by rMP12-NSs-Flag, while that of NSD249-265 very slightly increased the PKR abundance, which is consistent with the result in Fig. 5A .