Grapevine fanleaf virus replication occurs on endoplasmic reticulum-derived membranes.
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.
Ritzenthaler
C
C
Institut de Biologie Moléculaire des Plantes, 67084 Strasbourg Cedex, France.
Laporte
C
C
Gaire
F
F
Dunoyer
P
P
Schmitt
C
C
Duval
S
S
Piéquet
A
A
Loudes
A M
AM
Rohfritsch
O
O
Stussi-Garaud
C
C
Pfeiffer
P
P
D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000648:ultrastructure* / Q000821:virology; D049452:Green Fluorescent Proteins; D007425:Intracellular Membranes / Q000648:ultrastructure* / Q000821:virology*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D017860:Nepovirus / Q000472:pathogenicity* / Q000502:physiology*; D030821:Plants, Genetically Modified; D011523:Protoplasts / Q000648:ultrastructure / Q000821:virology; D019685:Rosales / Q000821:virology*; D014026:Nicotiana; D014779:Virus Replication
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins