Stimulation of poliovirus RNA synthesis and virus maturation in a HeLa cell-free in vitro translation-RNA replication system by viral protein 3CDpro.
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.
Franco
David
D
Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, NY 11790, USA. davidfranco72@yahoo.com
Pathak
Harsh B
HB
Cameron
Craig E
CE
Rombaut
Bart
B
Wimmer
Eckard
E
Paul
Aniko V
AV
D000086042:3C Viral Proteases; D002474:Cell-Free System; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D006367:HeLa Cells; D006801:Humans; D017955:Poliovirus / Q000378:metabolism*; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism*; D019065:Virus Assembly
D012367:RNA, Viral; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases