Figure 1

dsRNA species activate PKR. (A) Sindbis vector (SV) infection leads to activation of PKR. Lysates collected at 2, 4 and 6 h.p.i. from mock (-) or SV infected (+) MOSEC cells were analyzed by immunoblotting using a polyclonal antibody for PKR (upper arrow represents the phosphorylated form, lower arrow, non-phosphorylated form). (B) PKR is threonine phosphorylated following infection. Lysates were subjected to immunoprecipitation with PKR and blotted for phospho-threonine. SV infected MOSEC cells but not mock infected cells show phosphorylation indicating that this event is the result of infection. (C) MOSEC and Pan02 cells are efficiently transfected with siRNA. MOSEC and Pan02 cells were transfected with siGLO, a fluorescently conjugated control siRNA. Cells were collected and subjected to FACS analysis. (D) SV-GFP efficiently infects both MOSEC and Pan02 cells. 16 h.p.i. with SV-GFP, cells were harvested for FACS analysis monitoring GFP expression. (E) eIF2α phosphorylation is inhibited in cells with diminished PKR expression. Lysates were collected from MOSEC or Pan02 cells, transfected with siPKR or siGLO and infected with SV or mock infected, at 6 h.p.i. Immunoblotting for phospho-eIF2α indicates a lack of phosphorylation in siPKR-transfected cells. Immunoblots in panels A and E were stripped and reprobed with β actin for loading control. (F) Transfection of siPKR inhibits Sindbis-induced translational arrest. MOSEC cells transfected with either siPKR or siGLO were infected with SV or mock infected and subjected to ³⁵S labeling. Decreased translation was observed in the siGLO transfected/SV infected sample however not the similarly treated siPKR sample.