FIG. 4.

Activity of PLP1 and nsp5 (3CLpro) in processing replicase nsp1 and nsp8. Cytoplasmic lysates were generated from radiolabeled DBT-9 cells that were either mock infected or infected with the WT, Alb/ts/nsp5/V148A (Alb/ts/nsp5), nsp5/V148A (V148A), nsp5/S133N/V148A (S133N), or nsp5/H134Y/V148A (H134Y) virus at 30°C with labeling from 26 to 28 h p.i. (A and C) or at 30°C for 24 h followed by a shift to 40°C from 24 to 28 h p.i. with labeling from 26 to 28 h p.i. (B and D). Labeled proteins were immunoprecipitated with antiserum specific for nsp8 (A and B) or nsp1 (C and D), as indicated below each gel. Proteins were resolved by SDS-PAGE in 4 to 12% polyacrylamide gradient gels and visualized using fluorography. The numbers on the left side of the gel represent molecular mass markers (in kDa), and arrows to the right of gels point to the indicated proteins. α, anti.