scholarly journals Binding of viral glycoprotein mRNA to endoplasmic reticulum membranes is disrupted by puromycin.

1977 ◽  
Vol 74 (2) ◽  
pp. 358-364 ◽  
Author(s):  
H F Lodish ◽  
S Froshauer
Keyword(s):  
Endoplasmic Reticulum ◽  
Ionic Strength ◽  
Vesicular Stomatitis Virus ◽  
Direct Interaction ◽  
Viral Glycoprotein ◽  
Glycoprotein G ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Low Ionic Strength ◽  
Er Membrane

Previous studies showed that the glycoprotein (G) of vesicular stomatitis virus is synthesized in association with the endoplasmic reticulum (ER) membrane and that all G mRNA co-fractionates with ER membrane. Here, we show that treatment of infected cells with puromycin results in dissociation of G mRNA, and presumably the associated ribosomes, from the ER membrane. Even it extracts from treated cells are kept at low ionic strength (0.01 M KCl), over 80% of G mRNA is found unattached to membranes. There is no evidence for direct interaction of GmRNA with membranes; rather, the linkage apparently is mediated by the nascent G polypeptide.

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 Antibody-induced modulation of proteins in vesicular stomatitis virus-infected fibroblasts.

1983 ◽  
Vol 3 (9) ◽  
pp. 1580-1588 ◽  
Author(s):  
E J O'Rourke ◽  
W H Guo ◽  
A S Huang
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Cytochalasin B ◽  
Viral Glycoprotein ◽  
Host Proteins ◽  
Antigenic Modulation ◽  
Persistent Infections ◽  
Glycoprotein G ◽  
Vesicular Stomatitis ◽  
Degree Of Degradation ◽  
Baby Hamster Kidney Cells

When vesicular stomatitis virus-infected baby hamster kidney cells were treated with rabbit anti-vesicular stomatitis virus serum, there was a loss of the viral glycoprotein G into acid-soluble products. This degradation occurred within minutes at 37 degrees C and required the presence of G protein at the cell surface. The degree of degradation depended on antiserum concentration. The antiserum, also, prevented maturation of extracellular virions and induced partial degradation of the intracellular viral proteins, without affecting host proteins. The degradation could not be prevented by the presence of lysosomotropic agents, protease inhibitors, colchicine, or cytochalasin B. Similar kinetics and specificity of degradation was obtained with cells infected with vesicular stomatitis virus mutants that were less cytopathic. These results characterize a model system for studying the parameters and consequences of antigenic modulation as well as for studying the fate of viral antigens during persistent infections.

Transport pathway, maturation, and targetting of the vesicular stomatitis virus glycoprotein in skeletal muscle fibers

1996 ◽  
Vol 109 (6) ◽  
pp. 1585-1596
Author(s):  
P. Rahkila ◽  
A. Alakangas ◽  
K. Vaananen ◽  
K. Metsikko
Keyword(s):  
Skeletal Muscle ◽  
Endoplasmic Reticulum ◽  
Sarcoplasmic Reticulum ◽  
Vesicular Stomatitis Virus ◽  
Muscle Fibers ◽  
Viral Glycoprotein ◽  
Transverse Tubules ◽  
Skeletal Muscle Fibers ◽  
Vesicular Stomatitis ◽  
Terminal Cisternae

We have infected isolated skeletal muscle fibers with the vesicular stomatitis virus or the mutant tsO45, whose glycoprotein is blocked in the endoplasmic reticulum at 39 degrees C. Immunofluorescence analysis for the viral glycoprotein indicated that the fibers were infected over their entire length at a virus dose of 10(9)/ml. When we infected the myofibers with the tsO45 mutant at 39 degrees C, the viral glycoprotein appeared to be localised to the terminal cisternae of the sarcoplasmic reticulum. Upon shifting the cultures to the permissive temperature, 32 degrees C, in the presence of dinitrophenol, which blocks vesicular transport, the viral glycoprotein proceeded to completely fill the sarcoplasmic reticulum. Thus, both the endoplasmic reticulum located at the terminal cisternae of the sarcoplasmic reticulum, and the entire endoplasmic and sarcoplasmic reticulum appeared to be continuous. Shifting the culture temperature from 39 degrees C to 20 degrees C, resulted in prominent perinuclear staining throughout the fibers, accompanied by the appearance of distinct bright dots between the nuclei. Electron microscopic immunoperoxidase labeling indicated that these bright structures represented the Golgi apparatus. When either the tsO45-infected or wild-type virus-infected fibers were incubated at 32 degrees C, the viral glycoprotein showed a staining pattern that consisted of double rows of punctate fluorescence. Immunogold labeling showed that the viral glycoprotein was present in both the transverse tubules as well as the endoplasmic/sarcoplasmic reticulum endomembranes. In addition, extensive viral budding was observed in the transverse tubules. Metabolic labeling experiments revealed that only half of the glycoprotein was processed in the Golgi, and this processed form had become incorporated into the budding viral particles. Thus, the processed viral glycoprotein was targeted to the transverse tubules. The other half of the glycoprotein remained endoglycosidase H-sensitive, suggesting its retention in the endoplasmic/sarcoplasmic reticulum endomembranes.

Antibody-induced modulation of proteins in vesicular stomatitis virus-infected fibroblasts

1983 ◽  
Vol 3 (9) ◽  
pp. 1580-1588
Author(s):  
E J O'Rourke ◽  
W H Guo ◽  
A S Huang
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Cytochalasin B ◽  
Viral Glycoprotein ◽  
Host Proteins ◽  
Antigenic Modulation ◽  
Persistent Infections ◽  
Glycoprotein G ◽  
Vesicular Stomatitis ◽  
Degree Of Degradation ◽  
Baby Hamster Kidney Cells

When vesicular stomatitis virus-infected baby hamster kidney cells were treated with rabbit anti-vesicular stomatitis virus serum, there was a loss of the viral glycoprotein G into acid-soluble products. This degradation occurred within minutes at 37 degrees C and required the presence of G protein at the cell surface. The degree of degradation depended on antiserum concentration. The antiserum, also, prevented maturation of extracellular virions and induced partial degradation of the intracellular viral proteins, without affecting host proteins. The degradation could not be prevented by the presence of lysosomotropic agents, protease inhibitors, colchicine, or cytochalasin B. Similar kinetics and specificity of degradation was obtained with cells infected with vesicular stomatitis virus mutants that were less cytopathic. These results characterize a model system for studying the parameters and consequences of antigenic modulation as well as for studying the fate of viral antigens during persistent infections.

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.

Effects of altered cytoplasmic domains on transport of the vesicular stomatitis virus glycoprotein are transferable to other proteins

1988 ◽  
Vol 8 (7) ◽  
pp. 2869-2874
Author(s):  
J L Guan ◽  
A Ruusala ◽  
H Cao ◽  
J K Rose
Keyword(s):  
Endoplasmic Reticulum ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Cytoplasmic Domain ◽  
Secretory Proteins ◽  
Vesicular Stomatitis Virus Glycoprotein ◽  
Virus Glycoprotein ◽  
Vesicular Stomatitis

Alterations of the cytoplasmic domain of the vesicular stomatitis virus glycoprotein (G protein) were shown previously to affect transport of the protein from the endoplasmic reticulum, and recent studies have shown that this occurs without detectable effects on G protein folding and trimerization (R. W. Doms et al., J. Cell Biol., in press). Deletions within this domain slowed exit of the mutant proteins from the endoplasmic reticulum, and replacement of this domain with a foreign 12-amino-acid sequence blocked all transport out of the endoplasmic reticulum. To extend these studies, we determined whether such effects of cytoplasmic domain changes were transferable to other proteins. Three different assays showed that the effects of the mutations on transport of two membrane-anchored secretory proteins were the same as those observed with vesicular stomatitis virus G protein. In addition, possible effects on oligomerization were examined for both transported and nontransported forms of membrane-anchored human chorionic gonadotropin-alpha. These membrane-anchored forms, like the nonanchored human chorionic gonadotropin-alpha, had sedimentation coefficients consistent with a monomeric structure. Taken together, our results provide strong evidence that these cytoplasmic mutations affect transport by affecting interactions at or near the cytoplasmic side of the membrane.

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