scholarly journals The glycoprotein isolated from vesicular stomatitis virus is mitogenic for mouse B lymphocytes.

1981 ◽  
Vol 153 (6) ◽  
pp. 1489-1502 ◽  
Author(s):  
G Goodman-Snitkoff ◽  
R J Mannino ◽  
J J McSharry
Keyword(s):  
G Protein ◽  
B Lymphocyte ◽  
Lipid Vesicles ◽  
Murine Splenocytes ◽  
Intact Virion ◽  
Glycoprotein G ◽  
Polyclonal Activator ◽  
Vesicular Stomatitis ◽  
Viral Components ◽  
Triton X

The glycoprotein (G protein) of VSV was purified from the intact virion by Triton X-100 extraction. The isolated G protein has been shown to be a T cell-independent, B lymphocyte mitogen and polyclonal activator. Neither G protein nor the intact virion are stimulatory for murine T lymphocytes. The greater the density of G protein in lipid vesicles or the degree of aggregation of isolated G protein, the more highly stimulatory it is for murine splenocytes. As G protein is spread out in artificial vesicles, it becomes less mitogenic. It is probable that other viral components are also stimulatory since the Triton-insoluble pellet and VSV from which the G protein has been enzymatically removed retain mitogenic activity. To out knowledge, this is the first time a purified viral component has been demonstrated to be lymphocyte mitogen.

Intracellular transport of the glycoprotein of VSV is inhibited by CCCP at a late stage of post-translational processing

1989 ◽  
Vol 92 (4) ◽  
pp. 633-642
Author(s):  
J.K. Burkhardt ◽  
Y. Argon
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Late Stage ◽  
Intracellular Transport ◽  
Post Translational Modifications ◽  
Glycoprotein G ◽  
Golgi Compartment ◽  
Vesicular Stomatitis

The appearance of newly synthesized glycoprotein (G) of vesicular stomatitis virus at the surface of infected BHK cells is inhibited reversibly by treatment with carbonylcyanide m-chlorophenylhydrazone (CCCP). Under the conditions used, CCCP treatment depleted the cellular ATP levels by 40–60%, consistent with inhibition of transport at energy-requiring stages. The G protein that accumulates in cells treated with CCCP is heterogeneous. Most of it is larger than the newly synthesized G protein, is acylated with palmitic acid, and is resistant to endoglycosidase H (Endo H). Most of the arrested G protein is also sensitive to digestion with neuraminidase, indicating that it has undergone at least partial sialylation. A minority of G protein accumulates under these conditions in a less-mature form, suggesting its inability to reach the mid-Golgi compartment. The oligosaccharides of this G protein are Endo-H-sensitive and seem to be partly trimmed. Whereas sialylated G protein was arrested intracellularly, fucose-labelled G protein was able to complete its transport to the cell surface, indicating that a late CCCP-sensitive step separates sialylation from fucosylation. These post-translational modifications indicate that G protein can be transported as far as the trans-Golgi in the presence of CCCP and is not merely arrested in the endoplasmic reticulum.

scholarly journals Measles Virus Assembly within Membrane Rafts

2000 ◽  
Vol 74 (21) ◽  
pp. 9911-9915 ◽  
Author(s):  
Séverine Vincent ◽  
Denis Gerlier ◽  
Serge N. Manié
Keyword(s):  
G Protein ◽  
Measles Virus ◽  
Virus Assembly ◽  
Membrane Rafts ◽  
Virus Envelope ◽  
N Protein ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Triton X ◽  
N Proteins

ABSTRACT During measles virus (MV) replication, approximately half of the internal M and N proteins, together with envelope H and F glycoproteins, are selectively enriched in microdomains rich in cholesterol and sphingolipids called membrane rafts. Rafts isolated from MV-infected cells after cold Triton X-100 solubilization and flotation in a sucrose gradient contain all MV components and are infectious. Furthermore, the H and F glycoproteins from released virus are also partly in membrane rafts (S. N. Manié et al., J. Virol. 74:305–311, 2000). When expressed alone, the M but not N protein shows a low partitioning (around 10%) into rafts; this distribution is unchanged when all of the internal proteins, M, N, P, and L, are coexpressed. After infection with MGV, a chimeric MV where both H and F proteins have been replaced by vesicular stomatitis virus G protein, both the M and N proteins were found enriched in membrane rafts, whereas the G protein was not. These data suggest that assembly of internal MV proteins into rafts requires the presence of the MV genome. The F but not H glycoprotein has the intrinsic ability to be localized in rafts. When coexpressed with F, the H glycoprotein is dragged into the rafts. This is not observed following coexpression of either the M or N protein. We propose a model for MV assembly into membrane rafts where the virus envelope and the ribonucleoparticle colocalize and associate.

scholarly journals Intercompartmental transport in the Golgi complex is a dissociative process: facile transfer of membrane protein between two Golgi populations.

1984 ◽  
Vol 99 (1) ◽  
pp. 260-271 ◽  
Author(s):  
J E Rothman ◽  
R L Miller ◽  
L J Urbani
Keyword(s):  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Golgi Complex ◽  
Golgi Stack ◽  
Compartment Boundary ◽  
Glycoprotein G ◽  
Transport Vesicles ◽  
Protein Molecules ◽  
Vesicular Stomatitis ◽  
To Receive

The transfer of the vesicular stomatitis virus-encoded glycoprotein (G protein) between Golgi populations in fused cells (Rothman, J. E., L. J. Urbani, and R. Brands. 1984. J. Cell Biol. 99:248-259) is exploited here to study and to help define the compartmental organization of the Golgi stack and to characterize the mechanism of intercompartmental transport. We find that G protein that has just received its peripheral N-acetylglucosamine in the Golgi complex of one cell is efficiently transferred to the Golgi complex of another cell to receive galactose (Gal). Remarkably, this transport occurs at the same rate between these two compartments whether they are present in the same or different Golgi populations. Therefore, a dissociative (presumably vesicular) transport step moves G protein from one part of the Golgi in which N-acetylglucosamine is added to another in which Gal is added. Minutes later, upon receiving Gal, the same G protein molecules are very poorly transferred to an exogenous Golgi population after cell fusion. Therefore, once this intercompartmental transfer has already taken place (before fusion), it cannot take place again (after fusion); i.e., transport across the compartment boundary in the Golgi complex that separates the sites of N-acetylglucosamine and Gal incorporation is a vectorial process. We conclude that transfers between Golgi cisternae occur by a stochastic process in which transport vesicles budding from cisternae dissociate, can diffuse away, and then attach to and fuse with the appropriate target cisterna residing in the same or in a different stack, based on a biochemical pairing after a random encounter. Under these circumstances, a transported protein would almost always randomize among stacks with each intercisternal transfer; it would not progress systematically through a single stack. Altogether, our studies define three sequential compartments in the Golgi stack.

scholarly journals Fatty acid acylation at the single cysteine residue in the cytoplasmic domain of the glycoprotein of vesicular-stomatitis virus

1988 ◽  
Vol 256 (3) ◽  
pp. 1021-1027 ◽  
Author(s):  
D Mack ◽  
J Kruppa
Keyword(s):  
Palmitic Acid ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Transmembrane Domain ◽  
Cytoplasmic Domain ◽  
Cysteine Residue ◽  
Reaction Products ◽  
Glycoprotein G ◽  
Thioester Bond ◽  
Vesicular Stomatitis

The cysteine residue in the cytoplasmic domain at position 489 of the sequence of the glycoprotein (G protein) isolated from vesicular-stomatitis virions is completely blocked for carboxymethylation. After release of covalently bound fatty acids by hydroxylamine at pH 6.8, this cysteine residue could be specifically labelled by iodo[14C]acetic acid. Reaction products were analysed after specific cleavage of labelled G protein at asparagine-glycine bonds by hydroxylamine at pH 9.3, which generated a C-terminal peptide of Mr 15,300 containing only the single cysteine residue. Bromelain digestion of [3H]palmitic acid-labelled membrane fractions of vesicular-stomatitis-virus-infected baby-hamster kidney cells removed almost completely the 3H radioactivity from the cytoplasmic domain of the G protein, whereas the ectodomain was completely protected by the microsomal membrane. This result indicates that the acylation site of the G protein is exposed on the cytoplasmic side of intracellular membranes. Taken together, both biochemical techniques strongly suggest that the single cysteine-489 residue, which is located six amino acid residues distal to the putative transmembrane domain, is the acylation site. The thioester bond between palmitic acid and the G protein is quite resistant to hydroxylamine treatment (0.32 M at pH 6.8 for 1 h at 37 degrees C) compared with the reactivity of the thioester linkage in palmitoyl-CoA, which is cleaved at relatively low concentrations of hydroxylamine (0.05 M).

scholarly journals PtK1 cells contain a nondiffusible, dominant factor that makes the Golgi apparatus resistant to brefeldin A.

1991 ◽  
Vol 113 (5) ◽  
pp. 1009-1023 ◽  
Author(s):  
N T Ktistakis ◽  
M G Roth ◽  
G S Bloom
Keyword(s):  
Golgi Apparatus ◽  
G Protein ◽  
Golgi Complex ◽  
Brefeldin A ◽  
Cell Types ◽  
Live Cells ◽  
Dominant Factor ◽  
Glycoprotein G ◽  
Numerous Cell ◽  
Vesicular Stomatitis

Brefeldin A (BFA) was shown in earlier studies of numerous cell types to inhibit secretion, induce enzymes of the Golgi stacks to redistribute into the ER, and to cause the Golgi cisternae to disappear. Here, we demonstrate that the PtK1 line of rat kangaroo kidney cells is resistant to BFA. The drug did not disrupt the morphology of the Golgi complex in PtK1 cells, as judged by immunofluorescence using antibodies to 58- (58K) and 110-kD (beta-COP) Golgi proteins, and by fluorescence microscopy of live cells labeled with C6-NBD-ceramide. In addition, BFA did not inhibit protein secretion, not alter the kinetics or extent of glycosylation of the vesicular stomatitis virus (VSV) glycoprotein (G-protein) in VSV-infected PtK1 cells. To explore the mechanism of resistance to BFA, PtK1 cells were fused with BFA-sensitive CV-1 cells that had been infected with a recombinant SV-40 strain containing the gene for VSV G-protein and, at various times following fusion, the cultures were exposed to BFA. Shortly after cell fusion, heterokaryons contained one Golgi complex associated with each nucleus. Golgi membranes derived from CV-1 cells were sensitive to BFA, whereas those of PtK1 origin were BFA resistant. A few hours after fusion, most heterokaryons contained a single, large Golgi apparatus that was resistant to BFA and contained CV-1 galactosyltransferase. In unfused cells that had been perforated using nitrocellulose filters, retention of beta-COP on the Golgi was optimal in the presence of cytosol, ATP, and GTP. In perforated cell models of the BFA-sensitive MA104 line, BFA caused beta-COP to be released from the Golgi complex in the presence of nucleotides, and either MA104 or PtK1 cytosol. In contrast, when perforated PtK1 cells were incubated with BFA, nucleotides, and cytosol from either cell type, beta-COP remained bound to the Golgi complex. We conclude that PtK1 cells contain a nondiffusible factor, which is located on or very close to the Golgi complex, and confers a dominant resistance to BFA. It is possible that this factor is homologous to the target of BFA in cells that are sensitive to the drug.

Infectivity-deficient vesicular stomatitis virus produced in the presence of interferon has a functional virion core

1989 ◽  
Vol 35 (2) ◽  
pp. 334-339
Author(s):  
Francis T. Jay ◽  
Magdy R. Dawood ◽  
Sandy K. S. Luk
Keyword(s):  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Peptide Mapping ◽  
Surface Glycoprotein ◽  
Structural Abnormality ◽  
Viral Protein Synthesis ◽  
Virion Production ◽  
Glycoprotein G ◽  
Vesicular Stomatitis ◽  
Virion Core

Interferon induces two antiviral actions against vesicular stomatitis virus by (i) inhibiting viral protein synthesis which leads to a reduction in virion production, and (ii) producing progeny which are deficient in infectivity (VSV1F). At low or physiological concentrations of interferon, while the virion production was decreased by less than 10-fold, the virion infectivity yield was suppressed more than 1000-fold. The VSVIF was found to be deficient (quantitatively) in envelop glycoprotein G and protein M. Tryptic peptide mapping indicated mat there was no detectable structural abnormality in the G, M, and N proteins of VSVIF. The virion cores, lacking only the envelop G protein, isolated from VSVIF and control VSV have essentially identical specific infectivity. This indicated that the virion proteins L, N, NS, and M, as well as viral RNA that make up the virion core, must be functionally normal, and the observed deficiency in G protein was likely to be the cause of the functional deficiency of the virion. Low concentrations of DEAE-dextran, which is known to partially overcome the virion's dependence on the G protein for adsorption to the cell during infection, were found to enhance the infectivity of VSVIF more than the control virion. These results together indicated that the loss of infectivity in the VSV1F was due to the deficiency of the surface glycoprotein G.Key words: interferon, vesicular stomatitis virus, defective virions.

scholarly journals Organization of the Vesicular Stomatitis Virus Glycoprotein into Membrane Microdomains Occurs Independently of Intracellular Viral Components

2003 ◽  
Vol 77 (7) ◽  
pp. 3985-3992 ◽  
Author(s):  
Erica L. Brown ◽  
Douglas S. Lyles
Keyword(s):  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Plasma Membranes ◽  
Cytoplasmic Domain ◽  
Immunogold Labeling ◽  
Membrane Microdomains ◽  
Virus Budding ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Viral Components

ABSTRACT The glycoprotein (G protein) of vesicular stomatitis virus (VSV) is primarily organized in plasma membranes of infected cells into membrane microdomains with diameters of 100 to 150 nm, with smaller amounts organized into microdomains of larger sizes. This organization has been observed in areas of the infected-cell plasma membrane that are outside of virus budding sites as well as in the envelopes of budding virions. These observations raise the question of whether the intracellular virion components play a role in organizing the G protein into membrane microdomains. Immunogold-labeling electron microscopy was used to analyze the distribution of the G protein in arbitrarily chosen areas of plasma membranes of transfected cells that expressed the G protein in the absence of other viral components. Similar to the results with virus-infected cells, the G protein was organized predominantly into membrane microdomains with diameters of approximately 100 to 150 nm. These results indicate that internal virion components are not required to concentrate the G protein into membrane microdomains with a density similar to that of virus envelopes. To determine if interactions between the G protein cytoplasmic domain and internal virion components were required to create a virus budding site, cells infected with recombinant VSVs encoding truncation mutations of the G protein cytoplasmic domain were analyzed by immunogold-labeling electron microscopy. Deletion of the cytoplasmic domain of the G protein did not alter its partitioning into the 100- to 150-nm microdomains, nor did it affect the incorporation of the G protein into virus envelopes. These data support a model for virus assembly in which the G protein has the inherent property of partitioning into membrane microdomains that then serve as the sites of assembly of internal virion components.

scholarly journals Identification and characterization of a mouse cell mutant defective in the intracellular transport of glycoproteins.

1987 ◽  
Vol 105 (2) ◽  
pp. 647-657 ◽  
Author(s):  
F Tufaro ◽  
M D Snider ◽  
S L McKnight
Keyword(s):  
G Protein ◽  
Intracellular Transport ◽  
Mutant Cell ◽  
Mouse Cell ◽  
Virus Maturation ◽  
Glycoprotein G ◽  
Oligosaccharide Processing ◽  
Vesicular Stomatitis ◽  
Glycoprotein Transport ◽  
Simplex Virus

We have isolated a mutant line of mouse L cells, termed gro29, in which the growth of herpes simplex virus (HSV) and vesicular stomatitis virus (VSV) is defective. The block occurs late in the infectious cycle of both viruses. We demonstrate that HSV and VSV enter gro29 cells normally, negotiate the early stages of infection, yet are impaired at a late stage of virus maturation. During VSV infection of the mutant cell line, intracellular transport of its glycoprotein (G protein) is slowed. Pulse-chase experiments showed that oligosaccharide processing is impeded, and immunofluorescence localization revealed an accumulation of G protein in a juxtanuclear region that contains the Golgi complex. We conclude that export of newly made glycoproteins is defective in gro29 cells, and speculate that this defect may reflect a lesion in the glycoprotein transport apparatus.

scholarly journals Transmembrane biogenesis of the vesicular stomatitis virus glycoprotein.

1979 ◽  
Vol 80 (2) ◽  
pp. 416-426 ◽  
Author(s):  
F N Katz ◽  
H F Lodish
Keyword(s):  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Amino Terminal ◽  
Glycoprotein G ◽  
Microsomal Membranes ◽  
Protease Treatment ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Polypeptide Backbone

Previous work has shown that the mRNA encoding the vesicular stomatitis virus (VSV) glycoprotein (G) is bound to the rough endoplasmic reticulum (RER) and that newly made G protein is localized to the RER. In this paper, we have investigated the topology and processing of the newly synthesized G protein in microsomal vesicles. G was labeled with [35S]methionine ([35S]met), either by pulse-labeling infected cells or by allowing membrane-bound polysomes containing nascent G polipeptides to complete G synthesis in vitro. In either case, digestion of microsomal vesicles with any of several proteases removes approximately 5% (30 amino acids) from each G molecule. These proteases will digest the entire G protein if detergents are present during digestion. Using the method of Dintzis (1961, Proc. Natl. Acad. Sci. U. S. A. 47:247--261) to order tryptic peptides (8), we show that peptides lost from G protein by protease treatment of closed vesicles are derived from the carboxyterminus of the molecule. The newly made VSV G in microsomal membranes is glycosylated. If carbohydrate is removed by glycosidases, the resultant peptide migrates more rapidly on polyacrylamide gels than the unglycosylated, G0, form synthesized in cell-free systems in the absence of membranes. We infer that some proteolytic cleavage of the polypeptide backbone is associated with membrane insertion of G. Further, our findings demonstrate that, soon after synthesis, G is found in a transmembrane, asymmetric orientation in microsomal membranes, with its carboxyterminus exposed to the extracisternal, or cytoplasmic, face of the vesicles, and with most or all of its amino-terminal peptides and its carbohydrate sequestered within the bilayer and lumen of the microsomes.

scholarly journals Intracellular transport of the transmembrane glycoprotein G of vesicular stomatitis virus through the Golgi apparatus as visualized by electron microscope radioautography.

1982 ◽  
Vol 94 (1) ◽  
pp. 36-41 ◽  
Author(s):  
J J Bergeron ◽  
G J Kotwal ◽  
G Levine ◽  
P Bilan ◽  
R Rachubinski ◽  
...  
Keyword(s):  
Electron Microscope ◽  
Golgi Apparatus ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Intracellular Transport ◽  
Transmembrane Glycoprotein ◽  
Glycoprotein G ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Endoglycosidase H

The intracellular migration of G protein in vesicular stomatitis virus-infected cells was visualized by light and electron microscope radioautography after a 2-min pulse with [3H]mannose followed by nonradioactive chase for various intervals. The radioactivity initially (at 5-10 min) appeared predominantly in the endoplasmic reticulum, and the [3H]mannose-labeled G protein produced was sensitive to endoglycosidase H. Silver grains were subsequently (at 30-40 min) observed over the Golgi apparatus, and the [3H]mannose-labeled G protein became resistant to endoglycosidase H digestion. Our data directly demonstrate the intracellular transport of a plasmalemma-destined transmembrane glycoprotein through the Golgi apparatus.

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