Key result
The addition of viral protein 3CDpro to HeLa cell-free in vitro translation-RNA replication reactions programmed with viral RNA resulted in a 100-fold increase in virus yield by enhancing virus maturation.
In a HeLa cell-free system, the addition of viral protein 3CDpro significantly enhances poliovirus RNA synthesis and virus maturation, dependent on an intact RNA binding site and VPg-linked RNA template.
Answers are grounded in this paper and cite their sources.
Identifies 3CDpro as maturation enhancer in cell-free poliovirus system; leaves open relevance to in vivo models or antivirals.
Poliovirus protein 3CDpro possesses both proteinase and RNA binding activities, which are located in the 3Cpro domain of the protein. The RNA polymerase (3Dpol) domain of 3CDpro modulates these activities of the protein. We have recently shown that the level of 3CDpro in HeLa cell-free in vitro translation-RNA replication reactions is suboptimal for efficient virus production. However, the addition of either 3CDpro mRNA or of purified 3CDpro protein to in vitro reactions, programmed with viral RNA, results in a 100-fold increase in virus yield. Mutational analyses of 3CDpro indicated that RNA binding by the 3Cpro domain and the integrity of interface I in the 3Dpol domain of the protein are both required for function. The aim of these studies was to determine the exact step or steps at which 3CDpro enhances virus yield and to determine the mechanism by which this occurs. Our results suggest that the addition of extra 3CDpro 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 3CDpro 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 3Cpro domain of 3CDpro. In addition, the integrity of interface I in the 3Dpol domain of 3CDpro 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 3CDpro. Our results indicate that the stimulation of RNA synthesis and virus maturation by 3CDpro in vitro is dependent on the presence of a VPg-linked RNA template.
No takes yet. Share an insight, caveat, or question.
Franco et al. (2005) studied Poliovirus replication. Viral protein 3CDpro vs. No extra 3CDpro was evaluated on Virus yield (100-fold increase). The addition of viral protein 3CDpro to HeLa cell-free in vitro translation-RNA replication reactions programmed with viral RNA resulted in a 100-fold increase in virus yield by enhancing virus maturation.
Synapse has enriched 5 closely related papers on similar clinical questions. Consider them for comparative context: