Effect of 3CDpro(3CproH40A) on the early stages of poliovirus assembly in vitro. Translation-RNA replication reactions were carried out in the presence of [35S]TransLabel, as described in Materials and Methods. When indicated purified 3CDpro(3CproH40A) protein (5.5 nM) or mRNA (1.4 μg/ml) was added to the reactions at t = 0 hr and the samples were incubated for 16 hr at 34°C. Following RNase treatment and dialysis the samples were loaded on a 5–20% sucrose gradient (Materials and Methods). The samples were centrifuged for 15 hr at 40,000 RPM in a SW41 rotor at 4°C for the separation of 5S protomers and 14S pentamers. The amount of radioactivity at the bottom of the tubes of the gradients was not determined. (A) Comparison of samples obtained in the absence or presence of 3CDpro(3CproH40A) and mutant 3CDpro protein 3Dpol(H40G, R455A/R456A) or mRNA 3Cpro(R84S/I86A). (B) The 14S peak from section (A) is shown enlarged; (C) Western blot analysis with anti VP2 antibodies of samples from the 5S and 14S peaks from the gradient shown on Fig. 4A. The same analysis of the 80S and 155S peaks from the gradient shown on Fig. 5.