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Journal of Virology, February 2001, p. 1941-1948, Vol. 75, No. 4
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.4.1941-1948.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Peanut Clump Virus RNA-1-Encoded P15 Regulates Viral RNA Accumulation but Is Not Abundant at Viral RNA Replication Sites

Patrice Dunoyer,1 Etienne Herzog,2 Odile Hemmer,1 Christophe Ritzenthaler,1 and Christiane Fritsch1,*

Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique, 67084 Strasbourg Cedex, France,1 and Friedrich Miescher Institute, CH-4002 Basel, Switzerland2

Received 8 September 2000/Accepted 27 November 2000

RNA-1 of peanut clump pecluvirus (PCV) encodes N-terminally overlapping proteins which contain helicase-like (P131) and polymerase-like (P191) domains and is able to replicate in the absence of RNA-2 in protoplasts of tobacco BY-2 cells. RNA-1 also encodes P15, which is expressed via a subgenomic RNA. To investigate the role of P15, we analyzed RNA accumulation in tobacco BY-2 protoplasts inoculated with RNA-1 containing mutations in P15. For all the mutants, the amount of progeny RNA-1 produced was significantly lower than that obtained for wild-type RNA-1. If RNA-2 was included in the inoculum, the accumulation of both progeny RNAs was diminished, but near-normal yields of both could be recovered if the inoculum was supplemented with a small, chimeric viral replicon expressing P15, demonstrating that P15 has an effect on viral RNA accumulation. To further analyze the role of P15, transcripts were produced expressing P15 fused to enhanced green fluorescent protein (EGFP). Following inoculation to protoplasts, epifluorescence microscopy revealed that P15 accumulated as spots around the nucleus and in the cytoplasm. Intracellular sites of viral RNA synthesis were visualized by laser scanning confocal microscopy of infected protoplasts labeled with 5-bromouridine 5'-triphosphate (BrUTP). BrUTP labeling also occured in spots distributed within the cytoplasm and around the nucleus. However, the BrUTP-labeled RNA and EGFP/P15 very rarely colocalized, suggesting that P15 does not act primarily at sites of viral replication but intervenes indirectly to control viral accumulation levels.

* Corresponding author. Mailing address: Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique, 12 rue du Général Zimmer, 67084 Strasbourg Cedex, France. Phone: 33(0)388417200. Fax: 33(0)388614442. E-mail: christiane.fritsch{at}ibmp-ulp.u-strasbg.fr.

Journal of Virology, February 2001, p. 1941-1948, Vol. 75, No. 4
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.4.1941-1948.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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