Figure 3
Inhibition of 3CDpro(3CproH40A)-stimulated RNA synthesis by 3Cpro(C147G) in vitro. (A) Inhibition of 3CDpro(3CproH40A)-stimulated total viral RNA synthesis by 3Cpro(C147G). Translation-RNA replication reactions were incubated for the indicated time periods in the presence of [α-35S]CTP (Method II) either in the absence or presence of 3CDpro(CproH40A) (5.5 nM). The total amount of label incorporated into polymer was determined with a filter-binding assay, as described in Materials and Methods. Where indicated 3Cpro(C147G) was added to the reactions at t = 0 either alone or together with 3CDpro(3CproH40A). (B), (C) Inhibition of 3CDpro(3CproH40A)-stimulated minus (B) and plus strand (C) RNA synthesis by 3Cpro(C147G). Translation-RNA replication reactions were carried out in the presence of guanidine HCl 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 of the RNAs the samples were analyzed on a nondenaturing agarose gel (Materials and Methods). RF: double stranded replicative form RNA; ssRNA: single stranded RNA; CRC: [32P]-labeled RNA products from crude replication complexes (Materials and Methods).