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Journal of Virology, June 2008, p. 5967-5980, Vol. 82, No. 12
0022-538X/08/$08.00+0     doi:10.1128/JVI.02737-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Authentic Replication and Recombination of Tomato Bushy Stunt Virus RNA in a Cell-Free Extract from Yeast{triangledown}

Judit Pogany and Peter D. Nagy*

Department of Plant Pathology, University of Kentucky, Lexington, Kentucky

Received 23 December 2007/ Accepted 6 April 2008

To study the replication of Tomato bushy stunt virus (TBSV), a small tombusvirus of plants, we have developed a cell-free system based on a Saccharomyces cerevisiae extract. The cell-free system was capable of performing a complete replication cycle on added plus-stranded TBSV replicon RNA (repRNA) that led to the production of ~30-fold-more plus-stranded progeny RNAs than the minus-stranded replication intermediate. The cell-free system also replicated the full-length TBSV genomic RNA, which resulted in production of subgenomic RNAs as well. The cell-free system showed high template specificity, since a mutated repRNA, minus-stranded repRNA, or a heterologous viral RNA could not be used as templates by the tombusvirus replicase. Similar to the in vivo situation, replication of the TBSV replicon RNA took place in a membraneous fraction, in which the viral replicase-RNA complex was RNase and protease resistant but sensitive to detergents. In addition to faithfully replicating the TBSV replicon RNA, the cell-free system was also capable of generating TBSV RNA recombinants with high efficiency. Altogether, tombusvirus replicase in the cell-free system showed features remarkably similar to those of the in vivo replicase, including carrying out a complete cycle of replication, high template specificity, and the ability to recombine efficiently.

* Corresponding author. Mailing address: Department of Plant Pathology, University of Kentucky, S-305, 201F Plant Science Building, Lexington, KY 40546. Phone: (859) 257-7445. Fax: (859) 323-1961. E-mail: pdnagy2{at}uky.edu

{triangledown} Published ahead of print on 16 April 2008.

Journal of Virology, June 2008, p. 5967-5980, Vol. 82, No. 12
0022-538X/08/$08.00+0     doi:10.1128/JVI.02737-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Wang, R. Y.-L., Stork, J., Nagy, P. D. (2009). A Key Role for Heat Shock Protein 70 in the Localization and Insertion of Tombusvirus Replication Proteins to Intracellular Membranes. J. Virol. 83: 3276-3287 [Abstract] [Full Text]  
  • Pogany, J., Stork, J., Li, Z., Nagy, P. D. (2008). In vitro assembly of the Tomato bushy stunt virus replicase requires the host Heat shock protein 70. Proc. Natl. Acad. Sci. USA 105: 19956-19961 [Abstract] [Full Text]  


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