Electron Microscopic Study of the Morphogenesis of Vesicular Stomatitis Virus (original) (raw)
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Separate pathways of maturation of the major structural proteins of vesicular stomatitis virus
Journal of virology, 1977
Cell fractionation and protein electrophoresis were used to study the intracellular sites of synthesis and intermediate structures in the assembly of the virion proteins of vesicular stomatitis virus. Each of the three major virion proteins assembled into virions through a separable pathway. The nucleocapsid (N) protein was first a soluble protein and later incorporated into free, cytoplasmic nucleocapsids. A small amount of N protein was bound to membranes at later times, presumably representing either nucleocapsids in the process of budding or completed virions attached to the cell surface. The matrix (M) protein also appeared to be synthesized as a soluble protein, but was then directly incorporated into membranous structures with the same density as whole virus. Very little M protein was ever found in membranes banding at the density of plasma membranes. The M protein entered extracellular virus very quickly, as though it moved directly from a soluble state into budding virus. I...
Journal of virology, 1977
Maturation of viral proteins in cells infected with mutants of vesicular stomatitis virus was studied by surface iodination and cell fractionation. The movement of G, M, and N proteins to the virion bud appeared to be interdependent. Mutations thought to be in G protein prevented its migration to the cell surface, allowed neither M nor N protein to become membrane bound, and blocked formation of viral particles. Mutant G protein appeared not to leave the endoplasmic reticulum at the nonpermissive temperature, but this defect was partially reversible. In cells infected with mutants that caused N protein to be degraded rapidly or prevented its assembly into nucleocapsids, M protein did not bind to membranes and G protein matured to the cell surface, but never entered structures with the density of virions. Mutations causing M protein to be degraded prevented virion formation, and G protein behaved as in cells infected by mutants in N protein. These results are consistent with a model ...
Pathway of vesicular stomatitis virus entry leading to infection
Journal of Molecular Biology, 1982
The entry of vesicular stomatitis virus into Madin-Darby canine kidney (MDCK) cells was examined both biochemically and morphologically. At low multiplicity and 0°C viruses bound to the cell surface but were not internalized. Binding was very dependent on pH. More than ten times more virus bound at pH 65 than at higher pH values. At the optima1 pH, binding failed to reach equilibrium after more than two hours. The proportion of virus bound was irreproducible and low, relative to the binding of other enveloped viruses. Over 90% of the bound viruses were removed by proteases. When cells with pre-bound virus were warmed to 37% a proportion of the bound virus became protease-resistant with a half-time of about 30 minutes, After a brief lag period, degraded viral material was released into the medium. The protease-resistant virus was capable of infecting the cells and probably did so by an intracellular route, since ammonium chloride blocked the infection and slightly reduced the degradation of viral protein. When the entry process was observed by electron microscopy, viruses were seen bound to the cell surface at 0°C and, after warming at 37"C, within coated pits, coated vesicles and larger, smooth-surfaced vesicles. No fusion of the virus wit,h t,he plasma membrane was observed at pH 7.4. When pre-bound virus was incubated at a pH below 6 for 30 seconds at 37"(', about 40 to SO?, of the pre-bound virus became protease-resistant. On the basis of this result' and previously published experiments (White et al.. 1981). it was c.oncluded that vesicular stomatitis virus fuses to the MDCK cell plasma membrane at low pH. These experiments suggest that vesicular stomatitis virus enters MDCK cells by rndocytosis in coated pits and coated vesicles, and is transported to the lysosome where the low pH triggers a fusion reaction ultimately leading to the transfer of the penome into the cytoplasm. The entry pathway of vesicular stomatitis virus thus resembles that described earlier for both Semliki Forest virus and fowl plague virus.
Entry pathway of vesicular stomatitis virus into different host cells
1987
A biochemical and morphological investigation of the mechanism of entry of vesicular stomatitis virus (VSV) into host cells of mammalian (HeLa), avian (CER), piscine (EPC) and arthropod (Aedes albopictus) origin, is described. VSV was capable of infecting all cell lines tested by a endosome-and/or a lysosome-dependent step since ammonium chloride and amantadine blocked the early stages of infection. Complement-dependent immune lysis of infected host cells provided evidence that in none of the four different cell types examined did insertion of VSV antigens occur from the outside to any great extent on the cell surface. When the entry process was studied by electron microscopy, virus particles were seen to be bound to the cell surface at 0 °C. After warming at 37 °C for homeothermic cells or at 26 °C for poikilothermic cells, virus was detected within coated pits and coated vesicles and, later, in lysosomes. VSV entry was seen to take place by endocytosis in all four cell lines, which were derived from phylogenetically unrelated species.
The Propagation, Quantification, and Storage of Vesicular Stomatitis Virus
Current Protocols in Microbiology
This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/cpmc.110.
Journal of Virology, 1977
We report here an in vitro system designed to study the interactions of vesicular stomatitis virus (VSV) proteins with cellular membranes. We have synthesized the VSV nucleocapsid (N) protein, nonstructural (NS) protein, glycoprotein (G protein), and membrane (M) protein in a wheat germ, cell-free, protein-synthesizing system directed by VSV 12 to 18S RNA. When incubated at low salt concentrations with purified cytoplasmic membranes derived from Chinese hamster ovary cells, the VSV M and G proteins bind to membranes, whereas the VSV N and NS proteins do not. The VSV M protein binds to membranes in low or high divalent cation concentrations, whereas binding of significant amounts of G protein requires at least 5 mM magnesium acetate concentrations. Vesicular stomatitis virus (VSV) is a simple, lar membranes, whereas the VSV N and NS enveloped virus that contains two membrane proteins do not. proteins: the glycoprotein (G protein), which forms the spikes of the virion (4, 23), and the MATERIALS AND METHODS membrane (M) protein, which lines the inner Cells and viruses. Membranes were prepared surface of the viral membrane (3). There are from CHO cells. Stocks of VSV (pure B particles of three other known viral proteins, the VSV nuthe Indiana serotype) were grown in CHO cells and cleocapsid (N) protein, the nonstructural (NS) purified as described previously (20). protein, and the viral transcriptase (L) protein. Preparation of VSV 12-186 polyribosomal RNA. These three proteins are associated with the The procedure described by Palmiter (15) was used core ofthe virus particle (16, 23, 24). Each of the with several modifications for the preparation of five viral proteins is synthesized from a mono-VSV 12-18S polyribosomal RNA. CHO cells growing cistronic mRNA (9, 13, 14). at 37°C were infected with VSV at a multiplicity of 3 risngtic m arNAl(9 13, 14). PFU/cell as described previously (20), except that 5 During the early stages in the maturation Of~.tg of actinomycin D per ml was added at the beginthis virus, host cell membranes are modified ning of infection. [3H]uridine (70 Ci/mmol, 25 ,uCi/ with the VSV G and M proteins. Cell fractionaml; New England Nuclear Corp.) was added 2 h tion studies of VSV-infected cells have shown postinfection. Infected cells were harvested at 4.5 h that the VSV M and G proteins rapidly become postinfection, suspended in sucrose-TKM buffer associated with the membrane fraction of the (0.05 M Tris [pH 7.5], 0.025 M KCl, 0.005 M magnecells after their synthesis (5, 11, 12, 24). Nucleo-sium acetate, 0.25 M sucrose), and disrupted with 10 capsid structures containing the genome RNA strokes of a tight-fitting Dounce homogenizer. Nuas well as the viral N, NS, and L proteins are clei were removed by centrifugation (1,000 x g for 2 asswemlld as the vira plaNsm. andsLptroteisures min). The resulting cytoplasmic extract was centriassembled in the cytoplasm. These structures fuged at 20,000 x g for 20 min, and the pellet (mem
Journal of Virology, 1985
Dark-field scanning transmission electron microscopy was used to perform mass analyses of purified vesicular stomatitis virions, pronase-treated virions, and nucleocapsids, leading to a complete self-consistent account of the molecular composition of vesicular stomatitis virus. The masses obtained were 265.6 +/- 13.3 megadaltons (MDa) for the native virion, 197.5 +/- 8.4 MDa for the pronase-treated virion, and 69.4 +/- 4.9 MDa for the nucleocapsid. The reduction in mass effected by pronase treatment, which corresponds to excision of the external domains (spikes) of G protein, leads to an average of 1,205 molecules of G protein per virion. The nucleocapsid mass, after compensation for the RNA (3.7 MDa) and residual amounts of other proteins, yielded a complement of 1,258 copies of N protein. Calibration of the amounts of M, NS, and L proteins relative to N protein by biochemical quantitation yielded values of 1,826, 466, and 50 molecules, respectively, per virion. Assuming that the r...
Migration of vesicular stomatitis virus glycoprotein to the nucleus of infected cells
Proceedings of the National Academy of Sciences, 1996
A new means of direct visualization of the early events of viral infection by selective fluorescence labeling of viral proteins coupled with digital imaging microscopy is reported. The early phases of viral infection have great importance for understanding viral replication and pathogenesis. Vesicular stomatitis virus, the best-studied rhabdovirus, is composed of an RNA genome of negative sense, five viral proteins, and membrane lipids derived from the host cell. The glycoprotein of vesicular stomatitis virus was labeled with fluorescein isothiocyanate, and the labeled virus was incubated with baby hamster kidney cells. After initiation of infection, the fluorescence of the labeled glycoprotein was first seen inside the cells in endocytic vesicles. The fluorescence progressively migrated to the nucleus of infected cells. After 1 h of infection, the virus glycoprotein was concentrated in the nucleus and could be recovered intact in a preparation of purified nuclei. These results sugg...