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Journal of Virology, April 2005, p. 4859-4869, Vol. 79, No. 8
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.8.4859-4869.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Specific Binding of Tombusvirus Replication Protein p33 to an Internal Replication Element in the Viral RNA Is Essential for Replication{dagger}

Judit Pogany,1 K. Andrew White,2 and Peter D. Nagy1*

Department of Plant Pathology, University of Kentucky, Lexington, Kentucky,1 Department of Biology, York University, Toronto, Ontario, Canada2

Received 8 September 2004/ Accepted 9 December 2004

The mechanism of template selection for genome replication in plus-strand RNA viruses is poorly understood. Using the prototypical tombusvirus, Tomato bushy stunt virus (TBSV), we show that recombinant p33 replicase protein binds specifically to an internal replication element (IRE) located within the p92 RNA-dependent RNA polymerase coding region of the viral genome. Specific binding of p33 to the IRE in vitro depends on the presence of a C · C mismatch within a conserved RNA helix. Interestingly, the absence of the p33:p33/p92 interaction domain in p33 prevented specific but allowed nonspecific RNA binding, suggesting that a multimeric form of this protein is involved in the IRE-specific interaction. Further support for the selectivity of p33 binding in vitro was provided by the inability of the replicase proteins of the closely related Turnip crinkle virus and distantly related Hepatitis C virus to specifically recognize the TBSV IRE. Importantly, there was also a strong correlation between p33:IRE complex formation in vitro and viral replication in vivo, where mutations in the IRE that disrupted selective p33 binding in vitro also abolished TBSV RNA replication both in plant and in Saccharomyces cerevisiae cells. Based on these findings and the other known properties of p33 and the IRE, it is proposed that the p33:IRE interaction provides a mechanism to selectively recruit viral RNAs into cognate viral replicase complexes. Since all genera in Tombusviridae encode comparable replicase proteins, these results may be relevant to other members of this large virus family.

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

{dagger} This study is publication no. 04-12-034 of the Kentucky Agricultural Experiment Station.

Journal of Virology, April 2005, p. 4859-4869, Vol. 79, No. 8
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.8.4859-4869.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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