Fig 4.

PKR downregulation by TOSV NSs. (A) TOSV NSs mediates PKR degradation. 293 cells were mock transfected or transfected with in vitro-synthesized RNA for GFP (control) or TOSV NSs and harvested at 20 h posttransfection. Whole-cell lysates were analyzed by Western blotting with anti-PKR, anti-GFP, anti-TOSV, or anti-GAPDH antibodies (from top to bottom), respectively. The asterisk denotes a nonspecific band. (B) Activation of PKR is not required for downregulation. 293 cells were mock infected or infected with TOSV at an MOI of 3 and transfected with in vitro-synthesized RNA encoding myc-PKRK296R, a kinase-inactive form of PKR, or myc-CAT immediately after virus adsorption. Whole-cell lysates were collected at 16 hpi and analyzed by Western blotting with anti-myc, anti-TOSV NSs, anti-TOSV, or anti-GAPDH antibodies (from top to bottom), respectively.