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Journal of Virology, August 2004, p. 8552-8564, Vol. 78, No. 16
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.16.8552-8564.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Dissecting the Requirement for Subgenomic Promoter Sequences by RNA Recombination of Brome Mosaic Virus In Vivo: Evidence for Functional Separation of Transcription and Recombination

Rafal Wierzchoslawski,1,{dagger} Aleksandra Dzianott,1 and Jozef Bujarski1,2*

Plant Molecular Biology Center, Department of Biological Sciences, Northern Illinois University, De Kalb, Illinois 60115,1 Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland2

Received 17 December 2003/ Accepted 13 April 2004

Previously, we and others mapped an increased homologous recombination activity within the subgenomic promoter (sgp) region in brome mosaic virus (BMV) RNA3 (A. Bruyere et al., J. Virol. 74:4214-4219, 2000; R. Wierzchoslawski et al., J. Virol. 77:6769-6776, 2003). In order to correlate sgp-mediated recombination and transcription, in the present work we used BMV RNA3 constructs that carried altered sgp repeats. We observed that the removal or extension of the poly(U) tract reduced or increased recombination, respectively. Deletion of the sgp core hairpin or its replacement by a different stem-loop structure inhibited recombination activity. Nucleotide substitutions at the +1 or +2 transcription initiation position reduced recombination. The sgp core alone supported only basal recombination activity. The sites of crossovers mapped to the poly(U) region and to the core hairpin. The observed effects on recombination did not parallel those observed for transcription. To explain how both activities operate within the sgp sequence, we propose a dual mechanism whereby recombination is primed at the poly(U) tract by the predetached nascent plus strand, whereas transcription is initiated de novo at the sgp core.

* Corresponding author. Mailing address: Plant Molecular Biology Center, Department of Biological Sciences, Northern Illinois University, Montgomery Hall, De Kalb, IL 60115. Phone: (815) 753-0601. Fax: (815) 753-7855. E-mail: jbujarski{at}niu.edu.

{dagger} Permanent address: Plant Breeding and Acclimatization Institute, Bydgoszcz, Poland.

Journal of Virology, August 2004, p. 8552-8564, Vol. 78, No. 16
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.16.8552-8564.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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