ABSTRACT
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 3CDpro, 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 3CDpro in virus synthesis. We observed that purified 3CDpro 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 3CDpro is present early in the incubation period. The stimulation, however, is abolished by a mutation either in the RNA binding domain of 3CDpro, 3CproR84S/I86A, or by each of two groups of complementary mutations R455A/R456A and D339A/S341A/D349A at interface I in the 3Dpol domain of 3CDpro. Surprisingly, virus synthesis is strongly inhibited by the addition of both 3Cpro and 3CDpro 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, 3Dpol, and 3Cpro or with cellular protein PCBP2. These results suggest that 3CDpro has to be present in the reaction at the time the replication complexes are assembled and that both the 3Cpro and 3Dpol domains of the protein are required for its activity that stimulates virus production.
Automated in vitro evolution of a translation-coupled RNA replication system in a droplet flow reactor
