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Journal of Virology, October 2009, p. 10460-10471, Vol. 83, No. 20
0022-538X/09/$08.00+0     doi:10.1128/JVI.00819-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Turnip Mosaic Virus RNA Replication Complex Vesicles Are Mobile, Align with Microfilaments, and Are Each Derived from a Single Viral Genome ^,

Sophie Cotton,¹ Romain Grangeon,² Karine Thivierge,¹ Isabelle Mathieu,² Christine Ide,¹ Taiyun Wei,³ Aiming Wang,³ and Jean-François Laliberté^2*

Department of Plant Science, McGill University, 21,111 Lakeshore, Ste-Anne-de-Bellevue, Quebec H9X 3V9, Canada,¹ INRS-Institut Armand-Frappier, 531 Boulevard des Prairies, Laval, Quebec H7V 1B7, Canada,² Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, Ontario N5V 4T3, Canada³

Received 22 April 2009/ Accepted 1 July 2009

Nicotiana benthamiana plants were agroinoculated with an infectious cDNA clone of Turnip mosaic virus (TuMV) that was engineered to express a fluorescent protein (green fluorescent protein [GFP] or mCherry) fused to the viral 6K₂ protein known to induce vesicle formation. Cytoplasmic fluorescent discrete protein structures were observed in infected cells, corresponding to the vesicles containing the viral RNA replication complex. The vesicles were motile and aligned with microfilaments. Intracellular movement of the vesicles was inhibited when cells were infiltrated with latrunculin B, an inhibitor of microfilament polymerization. It was also observed that viral accumulation in the presence of this drug was reduced. These data indicate that microfilaments are used for vesicle movement and are necessary for virus production. Biogenesis of the vesicles was further investigated by infecting cells with two recombinant TuMV strains: one expressed 6K₂GFP and the other expressed 6K₂mCherry. Green- and red-only vesicles were observed within the same cell, suggesting that each vesicle originated from a single viral genome. There were also vesicles that exhibited sectors of green, red, or yellow fluorescence, an indication that fusion among individual vesicles is possible. Protoplasts derived from TuMV-infected N. benthamiana leaves were isolated. Using immunofluorescence staining and confocal microscopy, viral RNA synthesis sites were visualized as punctate structures distributed throughout the cytoplasm. The viral proteins VPg-Pro, RNA-dependent RNA polymerase, and cytoplasmic inclusion protein (helicase) and host translation factors were found to be associated with these structures. A single-genome origin and presence of protein synthetic machinery components suggest that translation of viral RNA is taking place within the vesicle.

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* Corresponding author. Mailing address: Institut National de la Recherche Scientifique, Institut Armand-Frappier, 531 Boulevard des Prairies, Laval, Québec H7V 1B7, Canada. Phone: (450) 687-5010. Fax: (450) 686-5501. E-mail: jean-francois.laliberte{at}iaf.inrs.ca

Published ahead of print on 5 August 2009.

Supplemental material for this article may be found at http://jvi.asm.org/.

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Journal of Virology, October 2009, p. 10460-10471, Vol. 83, No. 20
0022-538X/09/$08.00+0     doi:10.1128/JVI.00819-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.