Grapevine fanleaf virus replication occurs on endoplasmic reticulum-derived membranes - PubMed (original) (raw)

Grapevine fanleaf virus replication occurs on endoplasmic reticulum-derived membranes

C Ritzenthaler et al. J Virol. 2002 Sep.

Abstract

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.

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Figures

FIG. 1.

FIG. 1.

Confocal immunomicroscopy on healthy or GFLV-infected T-BY2 protoplasts. The cell lines used were: wild type (A and B, N to U), transgenic cells expressing GFP targeted to the Golgi (C to F) or to the ER (G to M). Mock-inoculated and infected protoplasts were harvestedat 24 hpi (R to U) or 48 hpi (A to Q). To monitor the synthesis of viral RNA, cells were fed at 24 hpi with BrUTP in the presence of actinomycin D and incubated for 6 additional hours to pulse-label the viral RNA (R to U). Cells were fixed with glutaraldehyde, permeabilized, quenched with NaBH4, and treated with primary antibodies, followed by treatment with secondary antibodies coupled to fluorochromes (Ab1 and Ab2). Immunolabelings were performed with anti-VPg/A488 (A, B, N, P, and Q), anti-VPg/A568 (C, E, G, and I), anti-BrdU/A488 (R and T), and anti-dsRNA/A568 (L, M, O to Q, and S to U). Panels G and H show fields containing two adjacent cells; the left cell escaped infection and represents a healthy cell, whereas the right cell is infected and shows condensed ER (H; large full arrowheads). Panels J and K are orthogonal projections of two series of optical sections through a healthy cell with a typical cortical ER (J; single arrowheads) and an infected cell showing a depletion of the cortical ER (K; single arrowheads). Merged pictures were obtained by combining the green and red channels (E = C + D; I = G + H, right cells; M = K + L; P and Q = N + O; and T = R + S). The VPg-containing perinuclear aggregates and punctate dsRNA labeling are indicated in panel N by full arrowheads and in panel O by arrows. Colocalization between VPg and dsRNA labeling are designated by double arrowheads in panel P. Superposition of differential interference contrast in panels I, M, and Q allowed visualization of the cell content and, particularly, the nucleus. N, nucleus; Nu, nucleolus. Panels F, J, and U correspond to healthy cells. Bars, 5 μm.

FIG. 2.

FIG. 2.

Electron microscopy of endomembrane proliferation in GFLV-infected T-BY2 protoplasts. Mock-inoculated (A) and GFLV-infected (B and C) T-BY2 protoplasts were harvested at 48 hpi, fixed, dehydrated, and embedded in Epon. Ultrathin sections (90 nm) were stained with uranyl acetate and lead citrate. Note in panels B and C the abundance of small vesicles (arrows) near the nucleus in infected cells and in panel C the presence of virus-like particles (small arrows) trapped in filamentous material (star) located at the periphery of the vesicular aggregates. Arrowheads, Golgi; N, nucleus; P, plastids; M, mitochondria. Bars: 1 μm in panels A and B and 400 nm in panel C.

FIG. 3.

FIG. 3.

Sucrose gradient fractionation of a crude extract of GFLV-infected T-BY2 protoplasts harvested at 72 hpi. Clarified crude extracts prepared from 106 protoplasts were fractionated by a 4-h centrifugation at 29,000 rpm through an 11-ml linear 20 to 45% sucrose gradient containing 5 mM MgCl2. Sixteen fractions (670 μl each) were collected. (A) For Western blot analysis, 15-μl aliquots of each fraction were analyzed by SDS-12.5% PAGE, electrotransferred onto an Immobilon-P membrane, and then further immunoreacted with anti-VPg antibodies to reveal VPg-containing proteins. The molecular mass of protein standards are indicated in kilodaltons on the right side, and the apparent molecular masses of the VPg precursors are shown on the left side. (B and C) For dot blot assays, 5-μl aliquots of each fraction were applied as dots on two strips of Immobilon-P membrane (Millipore). The strips were treated either with JIM84 (B) or with anti-HDEL monoclonal antibodies (C). The membranes were then incubated with goat anti-rabbit IgG (A) or goat anti-mouse IgG (B and C) coupled to horseradish peroxidase and revealed by chemiluminescence.

FIG. 4.

FIG. 4.

Immunotrapping of vesicles obtained by sucrose gradient separation of crude extracts from GFLV-infected T-BY2 protoplasts harvested at 72 hpi (A and B) and healthy protoplasts (C). Formvar-coated electron microscopic grids were first coated with affinity immunopurified anti-VPg antibodies and then floated on aliquots of the gradient fractions combined three by three. The bound material was successively fixed with glutaraldehyde, tannic acid, and osmium tetroxide before observation under the electron microscope. Immunocapture of vesicles was observed only with bottom fractions 11 to 13 from infected samples. Vesicles were either isolated (A) or aggregated in rosette-like structures (B). Bar, 200 nm.

FIG. 5.

FIG. 5.

Effect of BFA and cerulenin on GFLV multiplication in infected T-BY2 protoplasts. Immediately after electroporation of the protoplasts with viral RNA, 10 and 30 μg of BFA/ml or 30 and 45 μM cerulenin (final concentrations) were added, and the protoplasts were further incubated for 48 h. Cell viability was assessed on aliquots of protoplasts by adding 0.1% FDA. The remaining protoplasts were fixed and immunolabeled with anti-VPg or anti-CP antibodies and A568 conjugate. The number of infected cells, as revealed by the anti-VPg or anti-CP signal, was counted versus the total number of protoplasts determined under UV illumination after staining the nucleus with Hoechst 33258 and corrected for cell viability. The results were expressed as the ratio between the percentage of infection and the percentage of viable cells. This ratio was normalized to 100% in the control samples in which only DMSO was added.

FIG. 6.

FIG. 6.

Confocal immunomicroscopy on healthy or GFLV-infected T-BY2 protoplasts. Cells were from transgenic lines expressing GFP in the ER (A, B, and C) or from wild type (D to M). Cells were harvested at 48 h p.i. and treated as in Fig. 1. Labelings were done with anti-CP/A568 (B, C, D, E, F, H, I, J, L, and M) or anti-MP/A488 (G, I, J, K, and M). Panels C, I, and M are merged pictures from panels A + B, G + H, and K + L, respectively. Differential interference contrast observations were superimposed in panels C, D, F, and M for visualization of the cell and its nucleus. Panel J is a 2.8-fold magnification of the area indicated in panel I. The arrowheads in panels B, C, E, and F indicate the perinuclear viral compartment. The arrows in panels E and F indicate the CP labeling in the nucleoplasm. The boxed region in panel I is shown enlarged in panel J. Bar, 5 μm except in panel J (1 μm).

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