Effect of 3CDpro(3CproH40A) on viral RNA synthesis in the translation-RNA replication system. (A) Comparison of the stimulating activities of purified 3CDpro(3CproH40A) with mutant 3CDpro(3CproR84S/I86A) or 3CDpro(3CproH40G; 3DpolR455A/R456A) on total viral RNA synthesis. Translation-RNA replication reactions were carried out in the presence of [α-35S]CTP. Where indicated purified 3CDpro proteins (5.5 nM) or mRNA (1.4 μg/ml) was added at t = 0 hr. Samples were taken at the indicated time points (Method I) and the total amount of label incorporated into polymer was determined with a filter-binding assay, as described in Materials and Methods. (B), (C) Comparison of the stimulating activities of purified 3CDpro(3CproH40A) with that of mutants 3CDpro(3CproH40G, 3DpolR455A/R456A) and 3CDpro(3CproR84S/I86A), respectively, on plus strand RNA synthesis. Translation-RNA replication reactions were carried out for 4 hr and the replication complexes were isolated by centrifugation (Materials and Methods). The pellets were resuspended in translation reactions lacking viral RNA in the presence of [α-32P]CTP and the samples were incubated for 1 hr at 34°C. Following extraction and purification the RNA products were applied to a nondenaturing agarose gel (Materials and Methods). A [32P]UMP-labeled PV transcript RNA was used as a size marker for full length PV RNA.