Stimulation of poliovirus RNA synthesis and virus maturation in a HeLa cell-free in vitro translation-RNA replication system by viral protein 3CD(pro)

Poliovirus protein 3CD(pro )possesses both proteinase and RNA binding activities, which are located in the 3C(pro )domain of the protein. The RNA polymerase (3D(pol)) domain of 3CD(pro )modulates these activities of the protein. We have recently shown that the level of 3CD(pro )in HeLa cell-free in vitro translation-RNA replication reactions is suboptimal for efficient virus production. However, the addition of either 3CD(pro )mRNA or of purified 3CD(pro )protein to in vitro reactions, programmed with viral RNA, results in a 100-fold increase in virus yield. Mutational analyses of 3CD(pro )indicated that RNA binding by the 3C(pro )domain and the integrity of interface I in the 3D(pol )domain of the protein are both required for function. The aim of these studies was to determine the exact step or steps at which 3CD(pro )enhances virus yield and to determine the mechanism by which this occurs. Our results suggest that the addition of extra 3CD(pro )to in vitro translation RNA-replication reactions results in a mild enhancement of both minus and plus strand RNA synthesis. By examining the viral particles formed in the in vitro reactions on sucrose gradients we determined that 3CD(pro )has only a slight stimulating effect on the synthesis of capsid precursors but it strikingly enhances the maturation of virus particles. Both the stimulation of RNA synthesis and the maturation of the virus particles are dependent on the presence of an intact RNA binding site within the 3C(pro )domain of 3CD(pro). In addition, the integrity of interface I in the 3D(pol )domain of 3CD(pro )is required for efficient production of mature virus. Surprisingly, plus strand RNA synthesis and virus production in in vitro reactions, programmed with full-length transcript RNA, are not enhanced by the addition of extra 3CD(pro). Our results indicate that the stimulation of RNA synthesis and virus maturation by 3CD(pro )in vitro is dependent on the presence of a VPg-linked RNA template.