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.

The 119 kDa and 124 kDa polyproteins of arabis mosaic nepovirus (isolate S) are encoded by two distinct RNA2 species.

Arabis mosaic virus (ArMV) is a nepovirus that is serologically distantly related to grapevine fanleaf virus (GFLV). Both ArMV and GFLV induce grapevine degeneration disease. Several ArMV isolates, unlike isolates of GFLV, produce upon in vitro translation of RNA2 a polyprotein (P2) that forms a double band in polyacrylamide-SDS gels. Cloning of full-length copies of RNA2 of an ArMV isolate from grapevine (ArMV-S) revealed that this isolate contained two RNA2s of different length, called RNA2-U and RNA2-L. The two species were not readily separated by electrophoresis of the virion RNA under denaturing gel electrophoresis conditions but could be distinguished by analysis of primer extension and in vitro translation products. The size difference of the two RNA2s is due mostly if not exclusively to differences in their coding regions. The 124 kDa RNA2-U-encoded polyprotein P2' and the 119 kDa RNA2-L-encoded polyprotein P2", which co-migrate, respectively, with the upper and lower polyprotein bands produced by RNA2 of ArMV-S, were more than 95% identical except in their N-terminal domains. In vitro maturation experiments and sequence comparisons indicate that the N-terminal products of P2' and P2" have a molecular mass of 31 kDa and 26 kDa. The genomic organization proposed is similar to that of GFLV RNA2.

Primary structure and location of the genome-linked protein (VPg) of grapevine fanleaf nepovirus.

The genome linked protein VPg covalently linked to the RNAs of grapevine fanleaf nepovirus has been sequenced. The VPg (Mr = 2931) composed of 24 residues is linked by its N-terminal Ser beta-OH group to the viral RNAs. The VPg mapped from residues 1218 to 1241 of the 253K polyprotein encoded by GFLV RNA1.