FIG. 6.

Kinetics of 3Cpro inhibition of translation in vitro. (A) The graph indicates the accumulation of Luc (expressed in RLUs) in nuclease-treated RRL translation extracts pretreated for 5 min at 30°C with buffer (Cont), 5 ng of 3Cpro/μl, or 5 ng of 2Apro/μl before capped and polyadenylated Luc RNA was added and the incubation was shifted to 36°C (time point 0). At each time point, samples were transferred to Luc assay buffer and immediately assayed for Luc. (B) The graph indicates Luc accumulation in experiments in which HeLa extracts were similarly pretreated with 2Apro or 3Cpro and then translation cocktail and capped and polyadenylated Luc RNAs were added to start translation reactions (time point zero). (C) 2Apro (5 ng/μl) was used to pretreat HeLa lysate or was added at 0, 4, 8, or 12 min after the addition of Luc RNA and translation cocktail. The graph shows the accumulation of Luc RLUs plotted as percentages of the translation in protease-treated lysates compared to the results seen with mock-treated control lysates. The legend (depicted in panel D) is applicable to both panels C and D. (D) 3Cpro (5 ng/μl) was used to pretreat HeLa lysate or was added at 0, 4, 8, or 12 min after the addition of Luc RNA and translation cocktail. (E) HeLa translation extracts were supplemented with 2Apro (5 ng/μl) or 3Cpro (5 ng/μl) or both at the 11-min time point (arrow), and translation was allowed to continue. (F) Translation schematic.