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.

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.

Orobol: An Inhibitor of Vesicular Stomatitis Virus that Blocks the Synthesis of Viral Nucleic Acids and the Glycosylation of G Protein

1994 ◽  
Vol 5 (2) ◽  
pp. 99-104 ◽  
Author(s):  
M. J. Almela ◽  
A. Irurzun ◽  
L. Carrasco
Keyword(s):  
Protein Synthesis ◽  
Nucleic Acids ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Viral Protein Synthesis ◽  
Viral Translation ◽  
Naturally Occurring ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Virus Influenza

The naturally occurring isoflavonoid orobol exhibits antiviral effects against some animal viruses. Addition of the compound after virus entry inhibits the appearance of late viral protein synthesis in Vesicular Stomatitis Virus, influenza, or vaccinia virus-infected cells, but has no effect on poliovirus protein synthesis. Concentrations of the compound above 10–50 Mg ml−1 are sufficient to decrease the synthesis of VSV proteins when added early during infection, but have no effect on viral translation if added later, indicating that orobol does not block VSV translation directly. The synthesis of VSV nucleic acids is one of the targets of this flavonoid. The synthesis of both minus and plus-stranded viral RNA are inhibited by orobol when added during the first 2 h of infection. In addition, this compound interferes potently with the glycosylation of VSV G protein, indicating that orobol has several targets of antiviral action. The possibility that orobol interferes with the function of the cellular vesicular system is discussed.

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 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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