The IP-kinase assay was performed as described previously [65] . Briefly, 293 cells were mock-infected or infected with recombinant MP-12 at an moi of 3. Cells were dissolved in lysis buffer containing 10 mM Tris-HCl pH 7.6, 50 mM KCl, 2 mM Magnesium acetate, 10 mM 2-mercaptoethanol, 1% Triton X-100, 1 mM EDTA, phosphatase inhibitor cocktail (Sigma) and proteasome inhibitor cocktail (Roche). After centrifugation at 10,0006g for 5 min, a cytoplasmic fraction was collected. Cytoplasmic lysates were subjected to immunoprecipitation with anti-PKR antibody (Santa Cruz Biotech, K-17). Protein A beads that bound to the immunoprecipitated PKR were washed twice with a buffer-A containing 20 mM Tris-HCl, pH 7.6; 50 mM KCl; 400 mM NaCl; 5 mM 2-mercaptoethanol; 1% Triton X-100; 1 mM EDTA; phosphatase inhibitor cocktail (Sigma); proteasome inhibitor cocktail (Roche); and 20% glycerol. The beads were further washed twice with buffer-B containing 20 mM Tris-HCl, pH 7.6; 100 mM KCl; 5 mM 2-mercaptoethanol; 1% Triton X-100; 0.1 mM EDTA; proteasome inhibitor cocktail (Roche); and 20% glycerol. Then, washed beads were resuspended in 26 kinase buffer containing 30 mM Hepes-KOH, pH 7.4; 2 mM dithiothreitol; 2 mM MgCl 2 ; proteasome inhibitor cocktail (Roche); and 10 mCi of [c-32 P] ATP (MP Biomedicals). The suspension (20 ml) was incubated at 30uC for 20 min, and then 26 SDS sample buffer was added to terminate the reaction. The samples were separated on 10% SDS-PAGE gel and visualized on an autoradiograph. A portion of the samples were also used for Western blot analysis by employing anti-PKR monoclonal antibody (BD biosciences) to show the abundance of immunoprecipitated PKR.