For the radiolabelling of host and viral proteins in infected cells, VeroE6 cells were incubated at 14.5 h post-infection for 30 min at 37uC with medium made up with MEM lacking methionine, cystine, and L-glutamine (M2289, Sigma); 1% dialyzed FBS (Invitrogen); 20 mM L-glutamine; penicillin (100 U/ml) and streptomycin (100 mg/ml). Then, Trans[ 35 S]label metabolic reagent (MP biomedicals) was directly added into the medium (100 mCi/ml). After 1 h labelling, cells were washed with PBS once and lysed in sample buffer. Equal amounts of samples were subjected to SDS-PAGE in 10% polyacrylamide gel. The gel was dried and exposed to X-ray film (KODAK BioMax XAR) overnight at 280uC. For the radiolabelling of PKR, 293 cells were mock-treated or transfected with pcDNA3.1-Myc-PKRK296R, and labelled with Trans[ 35 S]label metabolic reagent between 14 and 16 h post-DNA transfection. In some samples, cell extracts were prepared with chase using a lysis buffer. In other samples, cells were transfected with in vitro-synthesized RNA transcripts encoding rLuc or MP-12 NSs. Then, cells were washed, and incubated for 8 h in the presence of 2 mM methionine/ cysteine. At 8 h post-RNA transfection, cell extracts prepared using lysis buffer, were employed for immunoprecipitation with anti-Myc antibody (Santa Cruz: sc-40), as described in an IPkinase assay. Immunoprecipitated samples were separated on a 7.5% poryacrylamide gel and visualized on an autoradiograph. dsRNA-binding assay 293 cells were mock-infected or infected with rMP12-rLuc-Flag, rMP12-NSs-Flag or rMP12-PKRDE7 at an moi of 3. Alternatively, 293 cells were transfected with in vitro-synthesized RNA transcripts encoding MP-12 NSs. The cytoplasmic lysate was harvested at 16 h.p.i. or 16 h post-transfection, and incubated with poly C beads or poly I:C beads on ice for 45 minutes. After washing beads with buffer-A for 4 times, the beads were mixed with 26 sample buffer, and bound proteins were analyzed with anti-Flag antibody (Sigma) on a Western blot.