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.

Conditions necessary for inhibition of protein synthesis and production of cytopathic effect in Aedes albopictus cells infected with vesicular stomatitis virus

1982 ◽  
Vol 2 (1) ◽  
pp. 66-75
Author(s):  
S Gillies ◽  
V Stollar
Keyword(s):  
Protein Synthesis ◽  
Vesicular Stomatitis Virus ◽  
Aedes Albopictus ◽  
Rna Synthesis ◽  
Cytopathic Effect ◽  
Viral Gene ◽  
Inhibition Of Protein Synthesis ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Aedes Albopictus Cells

The relationship between the development of cytopathic effect (CPE) and the inhibition of host macromolecular synthesis was examined in a CPE-susceptible cloned line of Aedes albopictus cells after infection with vesicular stomatitis virus. To induce rapid and maximal CPE, two conditions were required: (i) presence of serum in the medium and (ii) incubation at 34 degrees C rather than at 28 degrees C. In the absence of serum, incubation of infected cultures at 34 degrees C resulted in a significant increase in viral protein and RNA synthesis compared with that observed at 28 degrees C. However, when serum was present in the medium, by 6 h after infection protein synthesis (both host and viral) was markedly inhibited when infected cells were maintained at 34 degrees C. RNA synthesis (host and viral) was also inhibited in vesicular stomatitis virus-infected cells maintained at 34 degrees C with serum, but somewhat more slowly than protein synthesis. Examination of polysome patterns indicated that when infected cultures were maintained under conditions which predispose to CPE, more than half of the ribosomes existed as monosomes, suggesting that protein synthesis was being inhibited at the level of initiation. In addition, the phosphorylation of one (or two) polysome-associated proteins was reduced when protein synthesis was inhibited. Our findings indicate a strong correlation between virus-induced CPE in the LT-C7 clone of A. albopictus cells and the inhibition of protein synthesis. Although the mechanism of the serum effect is not understood, incubation at 34 degrees C probably predisposes to CPE and inhibition of protein synthesis by increasing the amount of viral gene products made.

Protein synthesis in lysates of Aedes albopictus cells infected with vesicular stomatitis virus

1982 ◽  
Vol 2 (10) ◽  
pp. 1174-1186
Author(s):  
S Gillies ◽  
V Stollar
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Vesicular Stomatitis Virus ◽  
Aedes Albopictus ◽  
Synthetic Activity ◽  
Cell Biol ◽  
S 100 ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Aedes Albopictus Cells

Aedes albopictus cells (clone LT-C7) showed a marked cytopathic effect and inhibition of protein synthesis (both host and viral) after infection with vesicular stomatitis virus (VSV), but only if (i) cultures were incubated at 34 degrees C rather than 28 degrees C and (ii) serum was present in the medium (S. Gillies and V. Stollar, Mol. Cell. Biol. 2:66-75, 1982). To learn more about how protein synthesis is shut off in VSV-infected A. albopictus cells, we have compared cell-free protein synthesis in extracts prepared from VSV-infected cells and control cells. Extracts prepared 6 h after infection from VSV-infected cells maintained at 34 degrees C in the presence of serum reflected what was observed with intact cells in at least two respects: (i) they showed a markedly diminished capacity to carry out protein synthesis (whether directed by endogenous or exogenously added mRNA), and (ii) there was decreased phosphorylation in vitro by [gamma-32P]ATP of a specific ribosomal protein (Gillies and Stollar, Mol. Cell. Biol. 2:66-75, 1982). In addition, and consistent with a block at the level of initiation, the formation of 80S initiation complexes, as measured by binding of VSV 12 to 18S mRNA, was reduced in the inactive extracts. Addition of an S-100 fraction from uninfected cells to the inactive extract reversed each of the aforementioned changes; i.e., it restored protein synthetic activity, it stimulated the formation of 80S initiation complexes, and it increased phosphorylation of the specific ribosomal protein referred to above. The active component in the S-100 fraction was heat labile and non-dialyzable and, upon ammonium sulfate fractionation of the S-100 fraction, was found in the 40 to 70% saturation fraction. Our findings suggest that VSV infection of A. albopictus cells inhibits protein synthesis by inactivating a macromolecular component, probably a protein, in the S-100 fraction which may be involved in the initiation of protein synthesis. More specifically, we suggest that this component is involved in the joining of the ribosomal subunits to form 80S initiation complexes.

scholarly journals Selective retention of monoglucosylated high mannose oligosaccharides by a class of mutant vesicular stomatitis virus G proteins.

1989 ◽  
Vol 108 (3) ◽  
pp. 811-819 ◽  
Author(s):  
K Suh ◽  
J E Bergmann ◽  
C A Gabel
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Vesicular Stomatitis Virus ◽  
Plasmid Vector ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
High Mannose

Cells infected with a temperature-sensitive mutant of vesicular stomatitis virus, ts045, or transfected with the plasmid vector pdTM12 produce mutant forms of the G protein that remain within the ER. The mutant G proteins were isolated by immunoprecipitation from cells metabolically labeled with [2-3H]mannose to facilitate analysis of the protein-linked oligosaccharides. The 3H-labeled glycopeptides recovered from the immunoprecipitated G proteins contained high mannose-type oligosaccharides. Structural analysis, however, indicated that 60-78% of the 3H-mannose-labeled oligosaccharides contained a single glucose residue and no fewer than eight mannose residues. The 3H-labeled ts045 oligosaccharides were deglucosylated and processed to complex-type units after the infected cells were returned to the permissive temperature. When shifted to the permissive temperature in the presence of a proton ionophore, the G protein oligosaccharides were deglucosylated but remained as high mannose-type units. The glucosylated state was observed, therefore, when the G protein existed in an altered conformation. The ts045 G protein oligosaccharides were deglucosylated in vitro by glucosidase II at both the permissive and nonpermissive temperatures. G protein isolated from ts045-infected cells labeled with [6-3H]galactose in the presence of cycloheximide contained 3H-glucose-labeled monoglucosylated oligosaccharides, indicating that the high mannose oligosaccharides were glucosylated in a posttranslational process. These results suggest that aberrant G proteins are selectively modified by resident ER enzymes to retain monoglucosylated oligosaccharides.

scholarly journals Conditions necessary for inhibition of protein synthesis and production of cytopathic effect in Aedes albopictus cells infected with vesicular stomatitis virus.

1982 ◽  
Vol 2 (1) ◽  
pp. 66-75 ◽  
Author(s):  
S Gillies ◽  
V Stollar
Keyword(s):  
Protein Synthesis ◽  
Vesicular Stomatitis Virus ◽  
Aedes Albopictus ◽  
Rna Synthesis ◽  
Cytopathic Effect ◽  
Viral Gene ◽  
Inhibition Of Protein Synthesis ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Aedes Albopictus Cells

The relationship between the development of cytopathic effect (CPE) and the inhibition of host macromolecular synthesis was examined in a CPE-susceptible cloned line of Aedes albopictus cells after infection with vesicular stomatitis virus. To induce rapid and maximal CPE, two conditions were required: (i) presence of serum in the medium and (ii) incubation at 34 degrees C rather than at 28 degrees C. In the absence of serum, incubation of infected cultures at 34 degrees C resulted in a significant increase in viral protein and RNA synthesis compared with that observed at 28 degrees C. However, when serum was present in the medium, by 6 h after infection protein synthesis (both host and viral) was markedly inhibited when infected cells were maintained at 34 degrees C. RNA synthesis (host and viral) was also inhibited in vesicular stomatitis virus-infected cells maintained at 34 degrees C with serum, but somewhat more slowly than protein synthesis. Examination of polysome patterns indicated that when infected cultures were maintained under conditions which predispose to CPE, more than half of the ribosomes existed as monosomes, suggesting that protein synthesis was being inhibited at the level of initiation. In addition, the phosphorylation of one (or two) polysome-associated proteins was reduced when protein synthesis was inhibited. Our findings indicate a strong correlation between virus-induced CPE in the LT-C7 clone of A. albopictus cells and the inhibition of protein synthesis. Although the mechanism of the serum effect is not understood, incubation at 34 degrees C probably predisposes to CPE and inhibition of protein synthesis by increasing the amount of viral gene products made.

scholarly journals Exit of newly synthesized membrane proteins from the trans cisterna of the Golgi complex to the plasma membrane.

1985 ◽  
Vol 101 (3) ◽  
pp. 949-964 ◽  
Author(s):  
G Griffiths ◽  
S Pfeiffer ◽  
K Simons ◽  
K Matlin
Keyword(s):  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Protein Localization ◽  
Coated Pits ◽  
Extracellular Medium ◽  
Double Labeling ◽  
Intracellular Location ◽  
Golgi Stack ◽  
Infected Cells ◽  
Vesicular Stomatitis

The intracellular location at which the G protein of vesicular stomatitis virus accumulated when transport was blocked at 20 degrees C has been studied by biochemical, cytochemical, and immunocytochemical methods. Our results indicated that the viral G protein was blocked in that cisterna of the Golgi stack which stained for acid phosphatase. At 20 degrees C this trans cisterna became structurally altered by the accumulation of G protein. This alteration was characterized by extensive areas of membrane buds which were covered by a cytoplasmic coat. These coated structures were of two kinds--those that labeled with anti-clathrin antibodies and those that did not. The clathrin-coated pits consistently did not label with anti-G antibodies. Upon warming infected cells to 32 degrees C, G protein appeared on the surface within minutes. Concomitantly, the trans cisterna lost its characteristic structural organization. Double-labeling experiments were performed in which G protein localization was combined with staining for horseradish peroxidase, which had been taken up from the extracellular medium by endocytosis. The results suggest that the trans cisterna was distinct from the endosome compartment and that the latter was not an obligatory station in the route taken by G protein to the cell surface.

scholarly journals The M protein of vesicular stomatitis virus associates specifically with the basolateral membranes of polarized epithelial cells independently of the G protein.

1988 ◽  
Vol 107 (5) ◽  
pp. 1707-1715 ◽  
Author(s):  
J E Bergmann ◽  
P J Fusco
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
M Protein ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Polarized Epithelial Cells

Using monoclonal antibodies and indirect immunofluorescence microscopy, we investigated the distribution of the M protein in situ in vesicular stomatitis virus-(VSV) infected MDCK cells. M protein was observed free in the cytoplasm and associated with the plasma membrane. Using the ts045 mutant of VSV to uncouple the synthesis and transport of the VSV G protein we demonstrated that this distribution was not related to the presence of G protein on the cell surface. Sections of epon-embedded infected cells labeled with antibody to the M protein and processed for indirect horseradish peroxidase immunocytochemistry revealed that the M protein was associated specifically with the basolateral plasma membrane. The G and M proteins of VSV have therefore evolved features which bring them independently to the basolateral membrane of polarized epithelial cells and allow virus to bud specifically from that membrane.

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 Protein synthesis in lysates of Aedes albopictus cells infected with vesicular stomatitis virus.

1982 ◽  
Vol 2 (10) ◽  
pp. 1174-1186 ◽  
Author(s):  
S Gillies ◽  
V Stollar
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Vesicular Stomatitis Virus ◽  
Aedes Albopictus ◽  
Synthetic Activity ◽  
Cell Biol ◽  
S 100 ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Aedes Albopictus Cells

Aedes albopictus cells (clone LT-C7) showed a marked cytopathic effect and inhibition of protein synthesis (both host and viral) after infection with vesicular stomatitis virus (VSV), but only if (i) cultures were incubated at 34 degrees C rather than 28 degrees C and (ii) serum was present in the medium (S. Gillies and V. Stollar, Mol. Cell. Biol. 2:66-75, 1982). To learn more about how protein synthesis is shut off in VSV-infected A. albopictus cells, we have compared cell-free protein synthesis in extracts prepared from VSV-infected cells and control cells. Extracts prepared 6 h after infection from VSV-infected cells maintained at 34 degrees C in the presence of serum reflected what was observed with intact cells in at least two respects: (i) they showed a markedly diminished capacity to carry out protein synthesis (whether directed by endogenous or exogenously added mRNA), and (ii) there was decreased phosphorylation in vitro by [gamma-32P]ATP of a specific ribosomal protein (Gillies and Stollar, Mol. Cell. Biol. 2:66-75, 1982). In addition, and consistent with a block at the level of initiation, the formation of 80S initiation complexes, as measured by binding of VSV 12 to 18S mRNA, was reduced in the inactive extracts. Addition of an S-100 fraction from uninfected cells to the inactive extract reversed each of the aforementioned changes; i.e., it restored protein synthetic activity, it stimulated the formation of 80S initiation complexes, and it increased phosphorylation of the specific ribosomal protein referred to above. The active component in the S-100 fraction was heat labile and non-dialyzable and, upon ammonium sulfate fractionation of the S-100 fraction, was found in the 40 to 70% saturation fraction. Our findings suggest that VSV infection of A. albopictus cells inhibits protein synthesis by inactivating a macromolecular component, probably a protein, in the S-100 fraction which may be involved in the initiation of protein synthesis. More specifically, we suggest that this component is involved in the joining of the ribosomal subunits to form 80S initiation complexes.

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