Polymorphism and structural maturation of bunyamwera virus in Golgi and post-Golgi compartments - PubMed (original) (raw)
Polymorphism and structural maturation of bunyamwera virus in Golgi and post-Golgi compartments
Iñigo J Salanueva et al. J Virol. 2003 Jan.
Abstract
The Golgi apparatus is the assembly site for a number of complex enveloped viruses. Using high-preservation methods for electron microscopy, we have detected two previously unknown maturation steps in the morphogenesis of Bunyamwera virus in BHK-21 cells. The first maturation takes place inside the Golgi stack, where annular immature particles transform into dense, compact structures. Megalomicin, a drug that disrupts the trans side of the Golgi complex, reversibly blocks transformation, showing that a functional trans-Golgi is needed for maturation. The second structural change seems to take place during the egress of viral particles from cells, when a coat of round-shaped spikes becomes evident. A fourth viral assembly was detected in infected cells: rigid tubular structures assemble in the Golgi region early in infection and frequently connect with mitochondria. In Vero cells, the virus induces an early and spectacular fragmentation of intracellular membranes while productive infection progresses. Assembly occurs in fragmented Golgi stacks and generates tubular structures, as well as the three spherical viral forms. These results, together with our previous studies with nonrelated viruses, show that the Golgi complex contains key factors for the structural transformation of a number of enveloped viruses that assemble intracellularly.
Figures
FIG. 1.
Progression of infection by Bunyamwera virus in BHK-21 cells. (A and B) Immunofluorescence detection of G1 protein at 5 h p.i. (A) and 10 h p.i. (B). (C) EM of the Golgi area in a BHK-21 cell at 10 h p.i. (conventional processing). At least four potential types of virus-related structures are distinguished: budding arcs (white arrowheads), annular spherical particles (black arrowheads), dense spherical particles (arrows), and elongated tube-like structures (asterisk). (D) Typical view of perinuclear Golgi membranes with tubular structures: arrows point to longitudinal sections of tubes, while the double white arrowhead points to cross-sectioned tubes. The single white arrowhead points to a budding arc, while a viral particle is marked with v. (E) Some tubes are seen inside Golgi sacculi (arrow). (F) The three types of round viral assemblies detected in Golgi stacks are shown together: the double white arrowhead points to the cross-section of a tube, the black arrowhead points to an IAV, and the arrow points to an IDV. (G to L) Freeze-substituted infected cells showing the structure and dimensions of the different viral assemblies after high-preservation processing methods. (G) Three annular, less dense particles (IDVs) are seen on the right, while a dense virus (IDV) is seen on the left. (H) EDVs contain a dense surface layer of spikes (arrow) absent in IDVs. The white arrowhead in panel H points to a surface section of an extracellular particle, where the arrangement of surface spikes can be seen. (I) tube; (J) IAV; (K) IDV; (L) EDV. Bars: 200 nm (C and D); 100 nm (E to L).
FIG.2.
Cytochemical characterization and quantification of the different virus-related assemblies in BHK-21 cells (A to H). An anti-Bunyamwera virus antiserum (A to D) and a complex of RNase and colloidal gold (E to H) were used. A cryosection is shown in panel D, while sections of freeze-substituted samples are shown in panels A to C and E to J. The specificity of RNase-gold labeling was assessed with positive (I) and negative (J) controls. (K) Distribution of the different viral assemblies in BHK-21 cells at early times p.i. For quantification, 25 infected cells were studied at 6, 7, 8, 10, 24, and 48 h p.i. On the other hand, 50 cells were studied at 5 h p.i. The total number of structures is indicated on the y axis for each time p.i. A total of 1,194 viral structures were included in the quantification. Bars representing the number of viral tubes are marked with asterisks. (L) These tubes were frequently observed as structures with both ends closed (arrows), interrupted membranes (black arrowheads), and different arrangements within the Golgi stack (arrows and double white arrowhead). (M) Tube (arrow) associated to the Golgi stack (G), as visualized by freeze-fracture. (N) Golgi-associated tubes and mitochondria were frequently connected (arrow). G, Golgi complex; mi: mitochondria. Bars, 100 nm.
FIG.3.
MGM treatment of BHK-21 cells. (A and B) Immunofluorescence localization of the trans side of the Golgi complex (with the lectin WGA) in noninfected untreated (A), and MGM-treated (B) BHK-21 cells. (C) Reversal of the drug's effects. (D to F) WGA labeling shows similar MGM effects on Bunyamwera virus-infected BHK-21 cells. (D) Cells at 10 h p.i. (E) Cell at 10 h p.i. but with the last 6 hours in the presence of MGM; (F) Cell subjected to the same treatment as in panel E but incubated for 2 h after removing the drug. (G) Ultrastructure of BHK-21 cells after MGM treatment. Asterisks mark the swollen _trans_-Golgi subcompartments, some of them still attached to the Golgi sacculi. (H and I) Localization of the cis side of the Golgi complex (with an anti-giantin antiserum) in noninfected untreated (H) and MGM-treated (I) cells. (J and K) Localization of Bunyamwera virus G1 protein during MGM treatment in infected cells (J) and after reversal of the drug's effects (K). (L) Accumulation of tubes (double white arrowheads) and annular viruses (black arrowheads) in Golgi membranes of MGM-treated cells. (M) Accumulation of dense intracellular viruses (arrows) in Golgi membranes after reversal of the effects of MGM in the absence of cycloheximide. (N) After reversal of the effects of MGM in the presence of cycloheximide, the number of intracellular viruses decreases and the edvs (v) are more abundant. (O) Quantification of the different viral assemblies in untreated infected cells and in infected cells subjected to the different treatments: “untreated” cells are BHK-21 cultures at 10 h p.i. without MGM treatment; “MGM” cells at 4 h p.i. were treated with the drug until 10 h p.i.; “Rev-cy” corresponds to cells treated with the drug as in “MGM” and incubated for a further 2 h after removing the drug from the cultures (total, 12 h p.i.); finally, “Rev+cy” corresponds to cells also subjected to 2 h of reversion of the effects of MGM but in the presence of 100 μg of cycloheximide per ml. A total of 1,254 viral structures were included in the quantification. N, nucleus. Bars, 0.5 μm (G and N); 100 nm (L and M).
FIG. 4.
Second maturation step for Bunyamwera virus in BHK-21 cells. (A to D) Labeling with anti-Bunyamwera virus antiserum on cryosections of infected cells. (A) Viral particles inside the Golgi complex (G) are marked with arrows (moderate labeling), while viruses in the post-Golgi area are marked with arrowheads (weak to moderate labeling). (B) Viral particles inside secretory vesicles (sv) reaching the plasma membrane exhibiting weak labeling. They are near an area where viral particles are already exiting the cell and are strongly labeled (arrows). (C and D) Extracellular viral particles attached to the cell surface, exhibiting strong labeling on their periphery. (E and F) Extracellular viral particles visualized after negative staining with uranyl acetate (E) and sodium phosphotungstate (F). The surface spikes are clearly distinguished in the latter. Bars, 100 nm.
FIG. 5.
Progression of Bunyamwera virus infection in Vero cells. (A and B) Immunofluorescence localization of Bunyamwera virus G1 protein at 5 h p.i. (A) and 12 h p.i. (B). (C to E) Immunofluorescence detection of Golgi with an anti-giantin antiserum in noninfected cells (C) and in infected cells at 3 h p.i. (D) and 6 h p.i. (E). (F to H) Detection of ERGIC with an anti-ERGIC-53 monoclonal antibody in noninfected cells (F) and in cells infected at 4 h p.i. (G) and 6 h p.i. (H). (I and J) Detection of the RER with an anti-protein disulfide isomerase (PDI) antiserum, both in noninfected cells (I) and in Bunyamwera virus-infected cells at 4 h p.i. (J). (K and L) Double-immunofluorescence localization of Bunyamwera virus G1 (K) and Golgi membranes (L) at 6 h p.i. (M and N) The organization of the microtubular network is shown in noninfected (M) and infected (N) cells at 5 h p.i., using an anti-tubulin antibody.
FIG. 6.
Ultrastructural analysis of Bunyamwera virus infection in Vero cells. (A) Control, noninfected Vero cell with the characteristic organization of Golgi (G), RER, and mitochondria (mi). (B and C) Ultrastructure of infected Vero cells at 8 h p.i. A fragmented Golgi stack is shown in panel B, while general fragmentation of endomembranes is marked with arrows in panel C. (D to H) Bunyamwera virus-relatedassemblies in Vero cells: tube in Golgi membranes showing connections with the Golgi stack (arrow) and areas open to the cytoplasm (arrowheads) (D); cross-sectioned tube attached to a mitochondrion (arrow) (E); iav in Golgi membranes (F); idv (G); and edv (H). Samples were processed by conventional processing in panels A to E, while freeze-substituted cells were used in panels F to H. (I) Quantification of the different viral assemblies found in Vero cells at different times p.i. Bars representing the amount of viral tubes are marked with asterisks. A total of 25 infected cells were studied at each time p.i., and 1,285 viral structures were included in the quantification. (J) Growth curves and plaque morphologies of Bunyamwera virus in BHK-21 and Vero cells. Bars, 0.5 μm (A); 1 μm (C); and 100 nm (B and D to H).
FIG. 7.
Model for Bunyamwera virus assembly pathway showing the formation of the viral factory and the main maturation steps. Golgi stacks and mitochondria form a large complex, with viral tubes connecting them. Budding profiles, viral tubes, immature annular viruses, and mature dense viruses have been introduced in different locations within the Golgi stack, according to our experimental data.
References
- Anderson, G. W., and J. F. Smith. 1987. Immunoelectron microscopy of Rift Valley Fever viral morphogenesis in rat primary hepatocytes. Virology 161:91-100. - PubMed
- Bonay, P., S. Munro, M. Fresno, and B. Alarcón. 1996. Intra-Golgi transport inhibition by megalomicin. J. Biol. Chem. 271:3719-3726. - PubMed
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