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Journal of Virology, May 2005, p. 6358-6367, Vol. 79, No. 10
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.10.6358-6367.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Stimulation of Poliovirus Synthesis in a HeLa Cell-Free In Vitro Translation-RNA Replication System by Viral Protein 3CDpro

David Franco,1 Harsh B. Pathak,2 Craig E. Cameron,2 Bart Rombaut,3 Eckard Wimmer,1 and Aniko V. Paul1*

Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, New York 11790,1 Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802,2 Department of Microbiology and Hygiene, Vrije Universiteit Brussel, B-1090 Brussels, Belgium3

Received 20 August 2004/ Accepted 30 December 2004

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.

* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, NY 11790. Phone: (631) 632-9777. Fax: (631) 632-8891. E-mail: apaul{at}notes.cc.sunysb.edu.

Journal of Virology, May 2005, p. 6358-6367, Vol. 79, No. 10
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.10.6358-6367.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Oh, H. S., Pathak, H. B., Goodfellow, I. G., Arnold, J. J., Cameron, C. E. (2009). Insight into Poliovirus Genome Replication and Encapsidation Obtained from Studies of 3B-3C Cleavage Site Mutants. J. Virol. 83: 9370-9387 [Abstract] [Full Text]  
  • Shen, M., Reitman, Z. J., Zhao, Y., Moustafa, I., Wang, Q., Arnold, J. J., Pathak, H. B., Cameron, C. E. (2008). Picornavirus Genome Replication: IDENTIFICATION OF THE SURFACE OF THE POLIOVIRUS (PV) 3C DIMER THAT INTERACTS WITH PV 3Dpol DURING VPg URIDYLYLATION AND CONSTRUCTION OF A STRUCTURAL MODEL FOR THE PV 3C2-3Dpol COMPLEX. J. Biol. Chem. 283: 875-888 [Abstract] [Full Text]  
  • Belov, G. A., Habbersett, C., Franco, D., Ehrenfeld, E. (2007). Activation of Cellular Arf GTPases by Poliovirus Protein 3CD Correlates with Virus Replication. J. Virol. 81: 9259-9267 [Abstract] [Full Text]  


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