FIG. 7.

EG PV exhibits delayed kinetics of P3 processing. (A) EG restores P3 processing. Processing was evaluated by Western blotting. HeLa cells were transfected with WT, GG, or EG subgenomic replicon RNA, held at 34°C, and harvested 20 h posttransfection. Extracts were prepared and processed for Western blotting as described in Materials and Methods. Antisera against VPg, 3C, and 3D were employed. The bands corresponding to the different precursor and processed proteins are indicated. (B) Kinetics of P3 processing for EG is delayed. Processing was evaluated by Western blotting. HeLa cells were transfected with WT or EG mutant subgenomic replicon RNA, held at 34°C, and harvested at 6, 8, and 20 h posttransfection. Extracts were prepared and processed for Western blotting as described in Materials and Methods. Antisera against VPg and 3D were employed. The bands corresponding to the different precursor and processed proteins are indicated. Purified 3CD and 3D (M1) or 3ABC and 3BC (M2) were loaded in lanes 8 and 17 and 9 and 18 as positive controls. Uninfected cells were used as negative controls (lanes 1 and 10). (C) Processing was evaluated by cell-free translation. HeLa cell-free translation extracts containing [³⁵S]methionine and [³⁵S]cysteine were programmed with WT or EG mutant RNA for 0.25, 1, and 2 h. Radiolabeled proteins were separated by 15% SDS-PAGE and detected by phosphorimaging. The bands corresponding to the different precursor and processed proteins are indicated.