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Journal of Virology, April 2008, p. 3864-3871, Vol. 82, No. 8
0022-538X/08/$08.00+0     doi:10.1128/JVI.02416-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Higher-Order RNA Structural Requirements and Small-Molecule Induction of Tombusvirus Subgenomic mRNA Transcription

Sheng Wang, Leyla Mortazavi, and K. Andrew White^*

Department of Biology, York University, Toronto, Ontario, Canada M3J 1P3

Received 8 November 2007/ Accepted 28 January 2008

Subgenomic (sg) mRNAs are small viral messages that are synthesized by polycistronic positive-strand RNA viruses to allow for the translation of certain viral proteins. Tombusviruses synthesize two such sg mRNAs via a premature termination mechanism. This transcriptional process involves the viral RNA-dependent RNA polymerase terminating minus-strand RNA synthesis prematurely at internal RNA signals during copying of the viral genome. The 3'-truncated minus-strand RNAs generated by the termination events then serve as templates for sg mRNA transcription. A higher-order RNA structure in the viral genome, located just upstream from the termination site, is a critical component of the RNA-based polymerase attenuation signal. Here, we have analyzed the role of this RNA structure in mediating efficient sg mRNA2 transcription. Our results include the following: (i) we define the minimum overall thermodynamic stability required for an operational higher-order RNA attenuation structure; (ii) we show that the distribution of stability within an attenuation structure affects its function; (iii) we establish that an RNA quadruplex structure can act as an effective attenuation structure; (iv) we prove that the higher-order RNA structure forms and functions in the plus strand; (v) we provide evidence that a specific attenuation structure-binding protein factor is not required for transcription; (vi) we demonstrate that sg mRNA transcription can be controlled artificially through small-molecule activation using RNA aptamer technology. These findings provide important new insights into the premature termination mechanism and present a novel approach to regulate the transcriptional process.

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* Corresponding author. Mailing address: 247 Farquharson Science Building, 4700 Keele St., Toronto, Ontario, Canada M3J 1P3. Phone: (416) 736-2100, ext. 40890. Fax: (416) 736-5698. E-mail: kawhite{at}yorku.ca

Published ahead of print on 6 February 2008.

Present address: School of Life Science, Ningxia University, Yinchuan, Ningxia, People's Republic of China 750021.

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Journal of Virology, April 2008, p. 3864-3871, Vol. 82, No. 8
0022-538X/08/$08.00+0     doi:10.1128/JVI.02416-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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