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

Franco David, Pathak, Cameron, Rombaut Bart, Wimmer Eckard, Paul

Description

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.

Sujets

• Poliovirus
• Transcription (genetics)

Informations

• Publié par: biomed
• Publié le: 01 janvier 2005
• Nombre de lectures: 8
• Langue: English

Extrait

Virology Journal

BioMedCentral

Open Access Research Stimulation of poliovirus RNA synthesis and virus maturation in a HeLa cell-free in vitro translation-RNA replication system by viral pro protein 3CD 1 22 3 David Franco, Harsh B Pathak, Craig E Cameron, Bart Rombaut, 1 1 Eckard Wimmerand Aniko V Paul*

1 Address: Departmentof Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, N. Y. 11790, USA, 2 3 Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802, USA andDepartment of Microbiology and Hygiene, Vrije Universiteit Brussel, B1090 Brussels, Belgium Email: David Franco  [e-mail]; Harsh B Pathak  [e-mail]; Craig E Cameron  [e-mail]; Bart Rombaut  [e-mail]; Eckard Wimmer  ewimmer!@ms.cc.sunysb.edu; Aniko V Paul*  [e-mail] * Corresponding author

Published: 21 November 2005Received: 30 June 2005 Accepted: 21 November 2005 Virology Journal2005,2:86 doi:10.1186/1743-422X-2-86 This article is available from: http://www.virologyj.com/content/2/1/86 © 2005 Franco et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PoliovirusRNA replicationvirus maturationHeLa cellfree translationRNA replication system

Abstract pro Poliovirus protein 3CDpossesses both proteinase and RNA binding activities, which are located pro polpro in the 3Cdomain of the protein. The RNA polymerase (3D) domain of 3CDmodulates pro these activities of the protein. We have recently shown that the level of 3CDin HeLa cell-free in vitro translation-RNA replication reactions is suboptimal for efficient virus production. pro pro However, the addition of either 3CDmRNA or of purified 3CDprotein to in vitro reactions, programmed with viral RNA, results in a 100-fold increase in virus yield. Mutational analyses of pro propol 3CD indicatedthat RNA binding by the 3Cdomain and the integrity of interface I in the 3D domain of the protein are both required for function. The aim of these studies was to determine pro the exact step or steps at which 3CDenhances virus yield and to determine the mechanism by pro which this occurs. Our results suggest that the addition of extra 3CDto 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 pro determined that 3CDhas 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 pro propol site within the 3Cdomain of 3CD. In addition, the integrity of interface I in the 3Ddomain pro of 3CDis 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, pro are not enhanced by the addition of extra 3CD. Our results indicate that the stimulation of RNA pro synthesis and virus maturation by 3CDin vitro is dependent on the presence of a VPg-linked RNA template.

Page 1 of 19 (page number not for citation purposes)