Cowpea mosaic virus infection induces a massive proliferation of endoplasmic reticulum but not Golgi membranes and is dependent on de novo membrane synthesis

Abstract

Replication of cowpea mosaic virus (CPMV) is associated with small membranous vesicles that are induced upon infection. The effect of CPMV replication on the morphology and distribution of the endomembrane system in living plant cells was studied by expressing green fluorescent protein (GFP) targeted to the endoplasmic reticulum (ER) and the Golgi membranes. CPMV infection was found to induce an extensive proliferation of the ER, whereas the distribution and morphology of the Golgi stacks remained unaffected. Immunolocalization experiments using fluorescence confocal microscopy showed that the proliferated ER membranes were closely associated with the electron-dense structures that contain the replicative proteins encoded by RNA1. Replication of CPMV was strongly inhibited by cerulenin, an inhibitor of de novo lipid synthesis, at concentrations where the replication of the two unrelated viruses alfalfa mosaic virus and tobacco mosaic virus was largely unaffected. These results suggest that proliferating ER membranes produce the membranous vesicles formed during CPMV infection and that this process requires continuous lipid biosynthesis.

FIG. 1

Genetic organization and translational expression of the CPMV genome. Open reading frames in the RNA molecules (open bars) and VPg (black square) are indicated. Nucleotide positions of start and stop codons are indicated. Abbreviations: mp, movement protein; pro, proteinase; co-pro, cofactor for proteinase; hel?, putative helicase; pol, RdRp.

FIG. 2-3

Confocal fluorescence micrographs of healthy (A, B, E, G, and G′) and CPMV-infected (C, D, F, H, H′, and H") plant cells expressing GFP or YFP targeted to the ER (A to F) or the Golgi (G to H"). The confocal images were collected with focal depth of 1 μm, using standard FITC filter settings to detect GFP or YFP (pseudo-colored green) and standard rhodamine filter settings to detect autofluorescence of the chlorophyll (pseudo-colored red). (C, G, G′, H, and H′) Projections of serial optical sections; (A, B, D, E, F, and H") projections of single optical sections. (A to D) N. benthamiana mGFP5-ER epidermal cells. (A) Reticulate pattern of cortical ER network. (B) Fluorescent halo of mGFP5-ER in nuclear envelope. (C) Large body of proliferated ER adjacent to nucleus in CPMV-infected cell. (D) Small cortical body of proliferated ER early in infection. (E to H") Cowpea mesophyll protoplasts. (E) Disorganized ER tubules in cytoplasm surrounding chloroplasts and nucleus in uninfected cells. (F) Large body of proliferated ER in CPMV-infected cell. (G and G′) Golgi stacks in uninfected cell scattered through cytoplasm visualized by ERD2-smYFP fluorescence, shown in combination with autofluorescence of chloroplasts (G) or alone (G′). (H to H") Similar distribution in CPMV-infected cells using ERD2-smYFP shown in combination with autofluorescence of chloroplasts (H) or alone (H′). As illustrated in a single optical section, ERD2-smYFP faintly stains the ER, showing the nuclear envelope and the CPMV-induced large body of proliferated ER (H"). Bars = 5 μm. Immunofluorescence double labeling showing the intracellular distribution of mGFP5-ER targeted to the ER (A and B) and STtmd-eYFP targeted to the Golgi (C) and viral proteins in CPMV-infected cowpea protoplasts. Cells were fixed 48 h p.i. and processed for indirect immunofluorescence using rabbit antibodies against the viral proteins followed by anti-rabbit antibodies conjugated to Cy3. GFP or YFP retained its fluorescence throughout the procedure. Rows show GFP or YFP (left), viral protein (middle), and their superposition (right) of a representative cell. The antibodies used were raised against the replicative proteins 110K (A′) and VPg (C′) and the 48K movement protein (B′). The arrow indicates the tubular structure formed by the 48K movement protein. (C to C") Projections of serial optical sections; (A to B") projections of single optical sections. Bars = 5 μm.

FIG. 4

Electron microscopy of cytopathological structures in CPMV-infected N. benthamiana mesophyll cells carrying the mGFP5-ER transgene. (A) ER tubules (arrows) located near electron-dense structures (Eds) and small membranous vesicles (Ve). (B) Immunolabeling with anti-GFP shows labeling of the ER tubules (arrows) and not the vesicles. Ch, chloroplasts; Va, vacuole. Bars = 300 nm.

FIG. 5

Cerulenin inhibits CPMV but not TMV or AMV replication in cowpea protoplasts. Infected protoplasts were divided in four equal portions and incubated for 48 h in the presence of 0, 15, 30, or 50 μM cerulenin. Subsequently the protoplasts were processed for indirect immunofluorescence using antisera against CPMV 110K, TMV coat protein, and AMV coat protein, and the percentage of infected cells was determined. For each virus two independent experiments were performed; error bars indicate the standard deviation.