Stimulation of poliovirus synthesis in a HeLa cell-free in vitro translation-RNA replication system by viral protein 3CDpro

The plus-strand RNA genome of poliovirus serves three distinct functions in the life cycle of the virus. The RNA is translated and then replicated, and finally the progeny RNAs are encapsidated. These processes can be faithfully reproduced in a HeLa cell-free in vitro translation-RNA replication system that produces viable poliovirus. We have previously observed a stimulation of virus synthesis when an mRNA, encoding protein 3CD(pro), is added to the translation-RNA replication reactions of poliovirus RNA. Our aim in these experiments was to further define the factors that affect the stimulatory activity of 3CD(pro) in virus synthesis. We observed that purified 3CD(pro) protein also enhances virus synthesis by about 100-fold but has no effect on the translation of the polyprotein. Optimal stimulation is observed only when 3CD(pro) is present early in the incubation period. The stimulation, however, is abolished by a mutation either in the RNA binding domain of 3CD(pro), 3C(pro)R84S/I86A, or by each of two groups of complementary mutations R455A/R456A and D339A/S341A/D349A at interface 1 in the 3D(pol) domain of 3CD(pro). Surprisingly, virus synthesis is strongly inhibited by the addition of both 3C(pro) and 3CD(pro) at the beginning of incubation. We also examined the effect of other viral or cellular proteins on virus synthesis in the in vitro system. No enhancement of virus synthesis occurred with viral proteins 3BC, 3ABC, 3BCD, 3D(pol), and 3Cpro or with cellular protein PCBP2. These results suggest that 3CD(pro) has to be present in the reaction at the time the replication complexes are assembled and that both the 3C(pro) and 3D(pol) domains of the protein are required for its activity that stimulates virus production.