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. 2015 Jun;89(12):6184-95.
doi: 10.1128/JVI.00401-15. Epub 2015 Apr 1.

Morphogenesis of Endoplasmic Reticulum Membrane-Invaginated Vesicles during Beet Black Scorch Virus Infection: Role of Auxiliary Replication Protein and New Implications of Three-Dimensional Architecture

Xiuling Cao  1 , Xuejiao Jin  1 , Xiaofeng Zhang  1 , Ying Li  2 , Chunyan Wang  3 , Xianbing Wang  1 , Jian Hong  3 , Xiaofeng Wang  4 , Dawei Li  1 , Yongliang Zhang  5
Affiliations

Morphogenesis of Endoplasmic Reticulum Membrane-Invaginated Vesicles during Beet Black Scorch Virus Infection: Role of Auxiliary Replication Protein and New Implications of Three-Dimensional Architecture

Xiuling Cao et al. J Virol. 2015 Jun.

Abstract

All well-characterized positive-strand RNA viruses[(+)RNA viruses] induce the formation of host membrane-bound viral replication complexes (VRCs), yet the underlying mechanism and machinery for VRC formation remain elusive. We report here the biogenesis and topology of the Beet black scorch virus (BBSV) replication complex. Distinct cytopathological changes typical of endoplasmic reticulum (ER) aggregation and vesiculation were observed in BBSV-infected Nicotiana benthamiana cells. Immunogold labeling of the auxiliary replication protein p23 and double-stranded RNA (dsRNA) revealed that the ER-derived membranous spherules provide the site for BBSV replication. Further studies indicated that p23 plays a crucial role in mediating the ER rearrangement. Three-dimensional electron tomographic analysis revealed the formation of multiple ER-originated vesicle packets. Each vesicle packet enclosed a few to hundreds of independent spherules that were invaginations of the ER membranes into the lumen. Strikingly, these vesicle packets were connected to each other via tubules, a rearrangement event that is rare among other virus-induced membrane reorganizations. Fibrillar contents within the spherules were also reconstructed by electron tomography, which showed diverse structures. Our results provide the first, to our knowledge, three-dimensional ultrastructural analysis of membrane-bound VRCs of a plant (+)RNA virus and should help to achieve a better mechanistic understanding of the organization and microenvironment of plant (+)RNA virus replication complexes.

Importance: Assembly of virus replication complexes for all known positive-strand RNA viruses depends on the extensive remodeling of host intracellular membranes. Beet black scorch virus, a necrovirus in the family Tombusviridae, invaginates the endoplasmic reticulum (ER) membranes to form spherules in infected cells. Double-stranded RNAs, the viral replication intermediate, and the viral auxiliary replication protein p23 are all localized within such viral spherules, indicating that these are the sites for generating progeny viral RNAs. Furthermore, the BBSV p23 protein could to some extent reorganize the ER when transiently expressed in N. benthamiana. Electron tomographic analysis resolves the three-dimensional (3D) architecture of such spherules, which are connected to the cytoplasm via a neck-like structure. Strikingly, different numbers of spherules are enclosed in ER-originated vesicle packets that are connected to each other via tubule-like structures. Our results have significant implications for further understanding the mechanisms underlying the replication of positive-strand RNA viruses.

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Figures

FIG 1
FIG 1
BBSV infection induced severe morphological changes in the endoplasmic reticulum. BBSV virions were inoculated onto N. benthamiana line 16C, in which the ER is decorated with GFP (37). The treatment is indicated on the left of each row. Both leaf epidermis (A) and mesophyll (B) cells were analyzed by CLSM. N. benthamiana 16C leaves were inoculated with BBSV virions (BBSV/Ino). At 3 dpi, the leaf epidermis was peeled from these leaves, and the remainder was treated with enzymes to liberate protoplasts from the mesophyll. Systemically infected leaves (BBSV/Sys) were harvested at about 8 dpi and similarly processed for CLSM analysis. Mock-inoculated leaves (Mock) from line 16C were processed following the same procedures. The arrowheads indicate the ER aggregates. Bars = 10 μm.
FIG 2
FIG 2
BBSV remodels the ER membranes. TEM was carried out to analyze the cytopathological changes during BBSV infection of N. benthamiana. (A) BBSV infection led to ER aggregation and large amounts of viral particles intermingled within the convoluted ER membrane. The arrow indicates the ER. (B to F) BBSV-induced vesiculation of the ER and VPs were often present in the dilated ER cisternae. (B) VPs were observed in the aggregates of branched ER cisternae (⭑), and a virus crystal (Vi) was in proximity to the VPs. Electron-dense globules (DG) were occasionally visible in the cytoplasm. The arrow indicates the ER. (C) Different numbers of spherules were enclosed in the VPs, and sometimes spherules appeared in the lumen of the perinuclear membrane (arrowheads). (D) Some spherules seem to be suspended in the cytoplasm (arrowheads). The enlarged image (top right corner) shows the region pointed to by the arrowhead. (E) Higher magnification of an area in panel D (dashed rectangle) shows the presence of fine fibrils in the VPs. (F) Membrane-associated spherules (further enlargement of the dashed rectangle in panel E) showing potential connections between the spherules and the outer ER membrane (arrowheads). Chl, chloroplast; Vi, virus particles or virus crystals; Mit, mitochondria; CW, cell wall; Nu, nucleus; Va, vacuole. Bars, 200 nm.
FIG 3
FIG 3
BBSV replication occurs in ER membrane-associated spherules. (A and B) Immunogold labeling of the auxiliary replication protein p23 in BBSV-infected N. benthamiana cells. (A) p23 is localized to the ER. (B) p23 is localized to the aggregated ER or in close proximity to the spherules. The arrow indicates the ER. The arrowheads point to spherule-associated 10-nm gold particles. (C and D) Examples of immunogold labeling of dsRNA in membrane-associated spherules. The arrowheads indicate areas of spherules labeled with 10-nm gold particles. Note that the distance between the gold particles and the target epitopes may span up to 20 nm, as reported previously (44). CW, cell wall. Scale bar, 200 nm.
FIG 4
FIG 4
Subcellular localization of p23-p82 complexes. (A) The BiFC assay indicates that p23 interacts with p82 and that the complexes colocalize with the aggregated ER. (B) No fluorescence is observed in leaves agroinfiltrated with control plasmid combinations. Bars = 10 μm. (C) Western blot analyses of protein expression in control agroinfiltrated leaves. The asterisks indicate the target bands.
FIG 5
FIG 5
p23, an ER-localized membrane protein, induces the formation of ER aggregates in N. benthamiana cells. (A) Schematic diagram of BBSV genome organization and fluorescent fusion proteins of p23 used for subcellular localization. (B) CLSM analysis of the subcellular localization of p23 protein in agroinfiltrated leaves of N. benthamiana at 3 dpi. The arrowheads point to the fluorescent halo of perinuclear ER-mCherry. The region in the dashed rectangle in the merged image of p23-GFP is enlarged in the top left corner. Bars = 10 μm. (C) Extraction of p23 from the membrane fraction. The P30 precipitate of BBSV-infected leaf homogenate was treated with either an extraction solution (2% Triton X-100) or elution solutions (4 M urea, 0.1 M Na2CO3 [pH 10.5], or 1 M KCl), followed by Western blotting of the membrane (P) and the solubilized (S) fractions with anti-p23 (α-p23) antibody. (D) Membrane flotation assays revealed that p23 cofractionates with the ER marker BiP. The P30 precipitates derived from leaves agroinfiltrated with p23-GFP or infected by BBSV were subjected to differential centrifugation fractionation. As a control, free GFP in the S3 fraction was also subjected to a membrane flotation assay. The primary antibodies used for immunoblot analyses are indicated on the right.
FIG 6
FIG 6
p23 is at least partially responsible for ER rearrangement during BBSV infection. Agroinfiltrated leaf tissues were embedded, ultrasectioned, and observed under TEM. (A and B) Transient expression of p23 alone in N. benthamiana leaf tissues induces convolution and aggregation of the ER. (C) Electron-dense structures were also frequently observed in leaf tissues agroinfiltrated with p23. (D) Higher magnification of the area enclosed by the dashed line in panel C. The electron-dense areas are connected to the ER (arrowheads). (E) There was no obvious abnormality in the ER structure in leaves agroinfiltrated with empty vector. The expression of p23 in the agroinfiltrated leaves was confirmed by Western blotting (see Fig. S3C in the supplemental material, 35S-p23). Chl, chloroplast; Mit, mitochondria; CW, cell wall. Scale bars = 200 nm.
FIG 7
FIG 7
Three-dimensional model of BBSV-induced, ER-derived spherules and vesicle packets by electron tomography. (A and B) A series of slices through a tomographic reconstruction show BBSV-induced ER membrane rearrangements (see Movie S1 in the supplemental material). The arrowheads indicate two spherules that appear to be free vesicles in one slice but connect to the outer membrane in the other slice (see Movie S1). I, II, and III represent different units of VPs. (C) Vesicle packets were also connected with each other through a tubule-like structure (arrowhead). (D) 3D models of the BBSV-modified ER and 3D surface reconstruction of the tomogram corresponding to the intact spherules in panels A and B. Gold, outer ER membrane; gray, BBSV-induced spherules; green, fibrillar materials inside the spherules. (E) Enlargement of the connections between the outer ER membrane and the spherules. (F) 90° counterclockwise rotation of panel E highlighting the pore-like openings (red arrowheads) (see Movie S4). (G to G3) 3D models of the fibrillar contents within the spherules. Panel G shows the electron density map from which the 3D surface-rendered models in panels G1 to G3 were derived. The arrowhead in panel G marks the channel connecting the interior of a representative spherule to the cytoplasm. Scale bars: 100 nm (A to D), 20 nm (E and F), and 10 nm (G to G3).
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