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Journal of Virology, September 2002, p. 8808-8819, Vol. 76, No. 17
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.17.8808-8819.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Grapevine Fanleaf Virus Replication Occurs on Endoplasmic Reticulum-Derived Membranes

C. Ritzenthaler, C. Laporte, F. Gaire,,{dagger} P. Dunoyer, C. Schmitt, S. Duval, A. Piéquet, A. M. Loudes, O. Rohfritsch, C. Stussi-Garaud,,{dagger}* and P. Pfeiffer

Institut de Biologie Moléculaire des Plantes, 67084 Strasbourg Cedex, France

Received 27 December 2001/ Accepted 3 June 2002

Infection by Grapevine fanleaf nepovirus (GFLV), a bipartite RNA virus of positive polarity belonging to the Comoviridae family, causes extensive cytopathic modifications of the host endomembrane system that eventually culminate in the formation of a perinuclear "viral compartment." We identified by immunoconfocal microscopy this compartment as the site of virus replication since it contained the RNA1-encoded proteins necessary for replication, newly synthesized viral RNA, and double-stranded replicative forms. In addition, by using transgenic T-BY2 protoplasts expressing green fluorescent protein in the endoplasmic reticulum (ER) or in the Golgi apparatus (GA), we could directly show that GFLV replication induced a depletion of the cortical ER, together with a condensation and redistribution of ER-derived membranes, to generate the viral compartment. Brefeldin A, a drug known to inhibit vesicle trafficking between the GA and the ER, was found to inhibit GFLV replication. Cerulenin, a drug inhibiting de novo synthesis of phospholipids, also inhibited GFLV replication. These observations imply that GFLV replication depends both on ER-derived membrane recruitment and on de novo lipid synthesis. In contrast to proteins involved in viral replication, the 2B movement protein and, to a lesser extent, the 2C coat protein were not confined to the viral compartment but were transported toward the cell periphery, a finding consistent with their role in cell-to-cell movement of virus particles.

* Corresponding author. Mailing address: Institut de Biologie Moléculaire des Plantes, 12 rue du Général Zimmer, 67084 Strasbourg Cedex, France. Phone: 33-3-88-41-72-00. Fax: 33-3-88-61-44-42. E-mail: christiane.garaud{at}ibmp-ulp.u-strasbg.fr.

{dagger} Present address: Ventana Medical System, 67400 Illkirch Graffenstaden, France.

Journal of Virology, September 2002, p. 8808-8819, Vol. 76, No. 17
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.17.8808-8819.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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