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

Virology Journal

Aniko V Paul¹  Eckard Wimmer¹  Bart Rombaut³  Craig E Cameron²  Harsh B Pathak²  David Franco¹ 

[1] Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, N. Y. 11790, USA;Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802, USA;Department of Microbiology and Hygiene, Vrije Universiteit Brussel, B-1090 Brussels, Belgium

关键词: HeLa cell-free translation-RNA replication system;    virus maturation;    RNA replication;    Poliovirus;   

Others  :  1158133
DOI  :  10.1186/1743-422X-2-86

 received in 2005-06-30, accepted in 2005-11-21,  发布年份 2005

【 摘 要 】

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.

【 授权许可】

   
2005 Franco et al; licensee BioMed Central Ltd.

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