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Journal of Virology, April 2005, p. 4848-4858, Vol. 79, No. 8
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.8.4848-4858.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The p92 Polymerase Coding Region Contains an Internal RNA Element Required at an Early Step in Tombusvirus Genome Replication

Sandra Monkewich,1 Han-Xin Lin,1 Marc R. Fabian,1 Wei Xu,1 Hong Na,1 Debashish Ray,1 Olena A. Chernysheva,1 Peter D. Nagy,2 and K. Andrew White1*

Department of Biology, York University, Toronto, Ontario, Canada,1 Department of Plant Pathology, University of Kentucky, Lexington, Kentucky2

Received 9 September 2004/ Accepted 3 December 2004

The replication of positive-strand RNA viral genomes involves various cis-acting RNA sequences. Generally, regulatory RNA sequences are present at or near genomic termini; however, internal replication elements (IREs) also exist. Here we report the structural and functional characterization of an IRE present in the readthrough portion of the p92 polymerase gene of Tomato bushy stunt virus. Analysis of this element in the context of a noncoding defective interfering RNA revealed a functional core structure composed of two noncontiguous segments of sequence that interact with each other to form an extended helical conformation. IRE activity required maintenance of several base-paired sections as well as two distinct structural features: (i) a short, highly conserved segment that can potentially form two different and mutually exclusive structures and (ii) an internal loop that contains a critical CC mismatch. The IRE was also shown to play an essential role within the context of the viral genome. In vivo analysis with novel RNA-based temperature-sensitive genomic mutants and translationally active subgenomic viral replicons revealed the following about the IRE: (i) it is active in the positive strand, (ii) it is dispensable late in the viral RNA replication process, and (iii) it is functionally inhibited by active translation over its sequence. Together, these results suggest that IRE activity is required in the cytosol at an early step in the viral replication process, such as template recruitment and/or replicase complex assembly.

* Corresponding author. Mailing address: Department of Biology, York University, 4700 Keele St., Toronto, Ontario, Canada M3J 1P3. Phone: (416) 736-2100, ext. 40890 or 70352. Fax: (416) 736-5698. E-mail: kawhite{at}yorku.ca.

Journal of Virology, April 2005, p. 4848-4858, Vol. 79, No. 8
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.8.4848-4858.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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