Effect of 3CDpro(3CproH40A) on the late 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. As a control, poliovirus proteins labeled with [35S]TransLabel in vivo in HeLa cells, were used. Following RNase treatment and dialysis the samples were loaded on a 5–20% sucrose gradient (Materials and Methods). The samples were centrifuged for 80 min at 40,000 RPM in a SW41 rotor at 4°C for the separation of 80S empty capsids and 155S virus particles (provirions and virions). (A) Comparison of samples obtained in the absence or presence of 3CDpro(3CproH40A) and mutant 3CDpro protein 3Dpol(H40G, R455A/R456A) or mRNA 3Cpro(R84A/I86A). (B) The 155S peak from section (A) is shown enlarged. (C) Plaque assays of fractions 7–14 in the 155S peak.