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Journal of Virology, August 2005, p. 10608-10618, Vol. 79, No. 16
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.16.10608-10618.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Role of an Internal and Two 3'-Terminal RNA Elements in Assembly of Tombusvirus Replicase{dagger}

Zivile Panaviene,{ddagger} Tadas Panavas,{ddagger} and Peter D. Nagy*

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

Received 9 March 2005/ Accepted 8 May 2005

Plus-strand RNA virus replication requires the assembly of the viral replicase complexes on intracellular membranes in the host cells. The replicase of Cucumber necrosis virus (CNV), a tombusvirus, contains the viral p33 and p92 replication proteins and possible host factors. In addition, the assembly of CNV replicase is stimulated in the presence of plus-stranded viral RNA (Z. Panaviene et al., J. Virol. 78:8254-8263, 2004). To define cis-acting viral RNA sequences that stimulate replicase assembly, we performed a systematic deletion approach with a model tombusvirus replicon RNA in Saccharomyces cerevisiae, which also coexpressed p33 and p92 replication proteins. In vitro replicase assays performed with purified CNV replicase preparations from yeast revealed critical roles for three RNA elements in CNV replicase assembly: the internal p33 recognition element (p33RE), the replication silencer element (RSE), and the 3'-terminal minus-strand initiation promoter (gPR). Deletion or mutagenesis of these elements reduced the activity of the CNV replicase to a minimal level. In addition to the primary sequences of gPR, RSE, and p33RE, formation of two alternative structures among these elements may also play a role in replicase assembly. Altogether, the role of multiple RNA elements in tombusvirus replicase assembly could be an important factor to ensure fidelity of template selection during replication.

* Corresponding author. Mailing address: Department of Plant Pathology, University of Kentucky, 201F Plant Science Building, Lexington, KY 40546-0312. Phone: (859) 257-7445, ext. 80726. Fax: (859) 323-1961. E-mail: pdnagy2{at}uky.edu.

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/.

{ddagger} Z.P. and T.P. contributed equally to this work.

Journal of Virology, August 2005, p. 10608-10618, Vol. 79, No. 16
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.16.10608-10618.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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