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Journal of Virology, January 2003, p. 1195-1203, Vol. 77, No. 2
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.2.1195-1203.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Two Distinct Mechanisms Ensure Transcriptional Polarity in Double-Stranded RNA Bacteriophages

Hongyan Yang, Eugene V. Makeyev, Sarah J. Butcher, Ausra Gaidelyte, and Dennis H. Bamford*

Department of Biosciences and Institute of Biotechnology, FIN-00014 University of Helsinki, Finland

Received 13 May 2002/ Accepted 8 October 2002

In most double-stranded RNA (dsRNA) viruses, RNA transcription occurs inside a polymerase (Pol) complex particle, which contains an RNA-dependent RNA Pol subunit as a minor component. Only plus- but not minus-sense copies of genomic segments are produced during this reaction. In the case of {phi}6, a dsRNA bacteriophage from the Cystoviridae family, isolated Pol synthesizes predominantly plus strands using virus-specific dsRNAs in vitro, thus suggesting that Pol template preferences determine the transcriptional polarity. Here, we dissect transcription reactions catalyzed by Pol complexes and Pol subunits of two other cystoviruses, {phi}8 and {phi}13. While both Pol complexes synthesize exclusively plus strands over a wide range of conditions, isolated Pol subunits can be stimulated by Mn2+ to produce minus-sense copies on {phi}13 dsRNA templates. Importantly, all three Pol subunits become more prone to the native-like plus-strand synthesis when the dsRNA templates (including {phi}13 dsRNA) are activated by denaturation before the reaction. Based on these and earlier observations, we propose a model of transcriptional polarity in Cystoviridae controlled on two independent levels: Pol affinity to plus-strand initiation sites and accessibility of these sites to the Pol in a single-stranded form.

* Corresponding author. Mailing address: Department of Biosciences and Institute of Biotechnology, P.O. Box 56, Viikinkaari 5, FIN-00014 University of Helsinki, Finland. Phone: (358) 9 191 59100. Fax: (358) 9 191 59098. E-mail: dennis.bamford{at}helsinki.fi.

Journal of Virology, January 2003, p. 1195-1203, Vol. 77, No. 2
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.2.1195-1203.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.





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