scholarly journals Transfer of carbohydrates on to nascent glycoprotein of vesicular stomatitis virus

1979 ◽  
Vol 181 (2) ◽  
pp. 295-300 ◽  
Author(s):  
J Kruppa
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Viral Envelope ◽  
Pulse Labelling ◽  
Viral Envelope Protein ◽  
Viral Glycoprotein ◽  
Membrane Bound ◽  
Vesicular Stomatitis

I studied the glycosylation in vivo of a viral envelope protein, the glycoprotein of vesicular stomatitis virus (VSV), by pulse labelling of virus-infected HeLa cells with 3H-labelled monosaccharides (mannose, glucosamine). Radioactivity was incorporated into the fraction of membrane-bound polyribosomes, although metabolic conversion of [3H]-mannose into amino acids was negligible. Dissociation of bound polyribosomes revealed that the radioactively co-purified with the peptidyl-tRNA. The nascent peptides were released by alkaline hydrolysis, immunoprecipitated and analysed by polyacrylamide-gel electrophoresis. It is apparent from the size distribution of the [3H]mannose-labelled nascent chains that attachment of carbohydrate starts when approximately half of the amino acid sequence of the viral glycoprotein has been synthesized.

scholarly journals Studies on the mechanism for entry of vesicular stomatitis virus glycoprotein g mRNA into membrane-bound polyribosome complexes

1977 ◽  
Vol 74 (1) ◽  
pp. 43-57 ◽  
Author(s):  
MJ Grubman ◽  
JA Weinstein ◽  
DA Shafritz
Keyword(s):  
Protein Synthesis ◽  
Vesicular Stomatitis Virus ◽  
Cytosol Fraction ◽  
Initial Event ◽  
Glycoprotein G ◽  
Nonspecific Effects ◽  
Membrane Bound ◽  
Vesicular Stomatitis

Glycoprotein mRNA (G mRNA) of vesicular stomatitis virus is synthesized in the cytosol fraction of infected HeLa cells. Shortly after synthesis, this mRNA associates with 40S ribosomal subunits and subsequently forms 80S monosomes in the cytosol fraction. The bulk of labeled G mRNA is then found in polysomes associated with the membrane, without first appearing in the subunit or monomer pool of the membrane-bound fraction. Inhibition of the initiation of protein synthesis by pactamycin or muconomycin A blocks entry of newly synthesized G m RNA into membrane-bound polysomes. Under these circumstances, labeled G mRNA accumulates into the cytosol. Inhibition of the elongation of protein synthesis by cucloheximide, however, allows entry of 60 percent of newly synthesized G mRNA into membrane-bound polysomes. Furthermore, prelabeled G mRNA associated with membrane-bound polysomes is released from the membrane fraction in vivo by pactamycin or mucomycon A and in vitro by 1mM puromycin - 0.5 M KCI. This release is not due to nonspecific effects of the drugs. These results demonstrate that association of G mRNA with membrane-bound polysomes is dependent upon polysome formation and initiation of protein synthesis. Therefore, direct association of the 3' end of G mRNA with the membrane does not appear to be the initial event in the formation of membrane-bound polysomes.

Isolation and Analysis of Non-Histone Chromatin Proteins from Rat-Liver Nuclei by Three Different Methods

1974 ◽  
Vol 52 (12) ◽  
pp. 1143-1153 ◽  
Author(s):  
D. Suria ◽  
C. C. Liew
Keyword(s):  
Rat Liver ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Soluble Proteins ◽  
Polyacrylamide Gel ◽  
Pulse Labelling ◽  
Rat Liver Nuclei ◽  
Liver Nuclei ◽  
Chromatin Proteins

Non-histone chromatin proteins were isolated from rat-liver nuclei by three different methods, and defined as (I) phenol-soluble proteins, (II) SDS-soluble proteins and (III) proteins not adsorbed by cation-exchange chromatography. About 62–70% of chromatin proteins were recovered from the total nuclear proteins. The yield of non-histone chromatin proteins varied from 17 to 26% of chromatin proteins, depending on the method used. The amino-acid composition of these proteins showed that they are acidic in nature. Their phosphorus content was found to be 0.9, 1.1, and 1.4%, respectively, according to method I, II, or III. In-vivo pulse-labelling experiments indicated that chromatin proteins were highly labelled with 3H-acetate and 32P-phosphoric acid. In particular, the specific activities of 32P incorporation were higher in all non-histone chromatin proteins isolated as compared with histones. One-dimensional SDS–polyacrylamide gel electrophoresis showed that at least 26 similar fractions can be detected in the samples prepared by these three methods.The similarity of some of the proteins obtained from methods I and III was further confirmed by fractionation of the non-histone chromatin proteins in an isoelectro-focusing system followed by a second-dimensional SDS–polyacrylamide gel electrophoresis. It was found that more than 100 components could be identified. However, some minor variations of the non-histone chromatin proteins were detected by this system. The differences in proteins isolated by these methods are mainly quantitative rather than qualitative. The methods examined are not specific for the fractionation of a certain class of non-histone chromatin proteins.

scholarly journals A GP64-Null Baculovirus Pseudotyped with Vesicular Stomatitis Virus G Protein

2001 ◽  
Vol 75 (6) ◽  
pp. 2544-2556 ◽  
Author(s):  
J. T. Mangor ◽  
S. A. Monsma ◽  
M. C. Johnson ◽  
G. W. Blissard
Keyword(s):  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Viral Entry ◽  
Viral Envelope ◽  
Productive Infection ◽  
Pseudotyped Virus ◽  
Sf9 Cells ◽  
Viral Envelope Protein ◽  
Virion Protein ◽  
Vesicular Stomatitis

ABSTRACT The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) GP64 protein is an essential virion protein that is involved in both receptor binding and membrane fusion during viral entry. Genetic studies have shown that GP64-null viruses are unable to move from cell to cell and this results from a defect in the assembly and production of budded virions (BV). To further examine requirements for virion budding, we asked whether a GP64-null baculovirus, vAc64−, could be pseudotyped by introducing a heterologous viral envelope protein (vesicular stomatitis virus G protein [VSV-G]) into its membrane and whether the resulting virus was infectious. To address this question, we generated a stably transfected insect Sf9 cell line (Sf9VSV-G) that inducibly expresses the VSV-G protein upon infection with AcMNPV Sf9VSV-G and Sf9 cells were infected with vAc64−, and cells were monitored for infection and for movement of infection from cell to cell. vAc64− formed plaques on Sf9VSV-G cells but not on Sf9 cells, and plaques formed on Sf9VSV-G cells were observed only after prolonged intervals. Passage and amplification of vAc64− on Sf9VSV-G cells resulted in pseudotyped virus particles that contained the VSV-G protein. Cell-to-cell propagation of vAc64− in the G-expressing cells was delayed in comparison to wild-type (wt) AcMNPV, and growth curves showed that pseudotyped vAc64− was generated at titers of approximately 106 to 107 infectious units (IU)/ml, compared with titers of approximately 108 IU/ml for wt AcMNPV. Propagation and amplification of pseudotyped vAc64− virions in Sf9VSV-G cells suggests that the VSV-G protein may either possess the signals necessary for baculovirus BV assembly and budding at the cell surface or may otherwise facilitate production of infectious baculovirus virions. The functional complementation of GP64-null viruses by VSV-G protein was further demonstrated by identification of a vAc64−-derived virus that had acquired the G gene through recombination with Sf9VSV-G cellular DNA. GP64-null viruses expressing the VSV-G gene were capable of productive infection, replication, and propagation in Sf9 cells.

scholarly journals Vesicular stomatitis virus chimeras expressing the Oropouche virus glycoproteins elicit protective immune responses in mice

2021 ◽  
Author(s):  
Sarah Hulsey Stubbs ◽  
Marjorie Cornejo Pontelli ◽  
Nischay Mishra ◽  
Changhong Zhou ◽  
Juliano de Paula Souza ◽  
...  
Keyword(s):  
Antibody Response ◽  
Vesicular Stomatitis Virus ◽  
Amazon Basin ◽  
Variable Region ◽  
Febrile Illness ◽  
Neutralizing Antibody ◽  
Viral Envelope ◽  
Wild Type ◽  
Viral Glycoprotein ◽  
Vesicular Stomatitis

AbstractOropouche virus (OROV) infection of humans is associated with a debilitating febrile illness that can progress to meningitis or encephalitis. First isolated from a forest worker in Trinidad and Tobago in 1955, the arbovirus OROV has since been detected throughout the Amazon basin with an estimated 500,000 human infections. Like other members of the family Peribunyaviridae, the viral genome exists as 3 single-stranded negative-sense RNA segments. The medium sized segment encodes a viral glycoprotein complex (GPC) that is proteolytically processed into two viral envelope proteins Gn and Gc responsible for attachment and membrane fusion. There are no therapeutics or vaccines to combat OROV infection, and we have little understanding of protective immunity to infection. Here we generated a replication competent chimeric vesicular stomatitis virus (VSV), in which the endogenous glycoprotein was replaced by the GPC of OROV. Serum from mice immunized with VSV-OROV specifically neutralized wild type OROV, and using peptide arrays we mapped multiple epitopes within an N-terminal variable region of Gc recognized by the immune sera. VSV-OROV lacking this variable region of Gc was also immunogenic in mice producing neutralizing sera that recognize additional regions of Gc. Challenge of both sets of immunized mice with wild type OROV shows that the VSV-OROV chimeras reduce wild type viral infection and suggest that antibodies that recognize the variable N-terminus of Gc afford less protection than those that target more conserved regions of Gc.ImportanceOropouche virus (OROV), an orthobunyavirus found in Central and South America, is an emerging public health challenge that causes debilitating febrile illness. OROV is transmitted by arthropods, and increasing mobilization has the potential to significantly increase the spread of OROV globally. Despite this, no therapeutics or vaccines have been developed to combat infection. Using vesicular stomatitis (VSV) as a backbone, we developed a chimeric virus bearing the OROV glycoproteins (VSV-OROV) and tested its ability to elicit a neutralizing antibody response. Our results demonstrate that VSV-OROV produces a strong neutralizing antibody response that is at least partially targeted to the N-terminal region of Gc. Importantly, vaccination with VSV-OROV reduces viral loads in mice challenged with wildtype virus. This data provides the first evidence that targeting the OROV glycoproteins may be an effective vaccination strategy to combat OROV infection.

The biogenesis of the cyanellae of Cyanophora paradoxa . - II. Pulse-labelling of cyanellar polypeptides in the presence of transcriptional and translational inhibitors

1989 ◽  
Vol 238 (1290) ◽  
pp. 73-87 ◽  
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Specific Inhibitor ◽  
Protein Translation ◽  
Total Cell ◽  
Pulse Labelling ◽  
Cyanophora Paradoxa ◽  
Cytoplasmic Ribosomes ◽  
Specific Inhibitors

Cycloheximide and chloroamphenicol, specific inhibitors of protein translation in the cytoplasmic and cyanellar compartments, respectively, of Cyanophora paradoxa , have been employed in 30 min pulse-labelling experiments by using [NaH- 14 C]O 3 to label total cell proteins in vivo . Cyanellae purified from host cell lysates were separated into soluble and thylakoid fractions and analysed by one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) to determine the distribution of radio-activity in the cyanellar polypeptides. Analysis of the autoradiograms of electrophoretically resolved proteins of the cyanellae indicates that about 70% of the total number of cyanellar proteins visualized in the controls are synthesized on cytoplasmic ribosomes. The majority (81%) of the soluble cyanellar proteins appear to be cytoplasmically synthesized. In contrast, the majority (70%) of the thylakoid proteins are synthesized within the cyanellae. The observations also suggest that the polypeptides synthesized within the cyanellae include species that are the most abundant and rapidly turned over. A number of the polypeptides previously identified have now been characterized with regard to their sites of synthesis. In addition, we report on labelling experiments involving rifampicin, a specific inhibitor of cyanellar transcription, which indicate that different mRNAs within the cyanellae have markedly different stabilities.

In-vivo and in-vitro secretion of prolactin by lactating rat adenohypophyses as a function of intracellular age

1984 ◽  
Vol 101 (1) ◽  
pp. 27-32 ◽  
Author(s):  
F. Mena ◽  
G. Martínez-Escalera ◽  
C. Clapp ◽  
C. E. Grosvenor
Keyword(s):  
Pituitary Gland ◽  
Incubation Period ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Pituitary Glands ◽  
H 26

ABSTRACT Adenohypophysial prolactin of lactating rats was pulse-labelled by [3H]leucine injected i.v. at the time of removal of the pups. The [3H]prolactin concentration in the pituitary gland, analysed by polyacrylamide-gel electrophoresis, progressively fell as the time from labelling to removal of the pituitary gland increased from 8 to 24 h, which suggests that there was a loss of hormone as it aged within the gland. Suckling effectively provoked the depletion–transformation of total and [3H]prolactin (extracted at pH 7·2) when applied after 8 h but not when applied after either 16 or 24 h after removing the pups. In rats whose pups were removed for 8 h, suckling also depleted–transformed [3H]prolactin labelled 4 h, but not that labelled 1 h before suckling. The pituitary glands of other lactating rats were labelled with [3H]leucine injected i.v. at various times before removing the glands and incubating them in medium 199. The secretion into the medium of [3H]prolactin labelled either 4, 8, 16 or 24 h beforehand was maximal during the first 30 min then declined from 30 to 240 min of incubation. However, secretion of prolactin labelled 1 h and 10 min beforehand reached a maximum after 0·5–1 h and 2 h of incubation respectively, then remained constant during the remainder of the 4-h incubation period. The total 4-h secretion of [3H]prolactin was greatest (65% of preincubation concentration) from those glands labelled 4 h before in contrast to those labelled 10 min (15%) or 1 (38%), 8 (34%), 16 (18%) or 24 h (26%) before incubation. Taken together, these data suggest that prolactin synthesized 4 h earlier is more likely to be released in response to physiological stimuli than is more recently formed prolactin or prolactin which has remained in the pituitary gland for 16 h or longer. J. Endocr. (1984) 101, 27–32

scholarly journals Many Nonmammalian Cells Exhibit Postentry Blocks to Transduction by Gammaretroviruses Pseudotyped with Various Viral Envelopes, Including Vesicular Stomatitis Virus G Glycoprotein

2001 ◽  
Vol 75 (14) ◽  
pp. 6375-6383 ◽  
Author(s):  
Clarissa Dirks ◽  
A. Dusty Miller
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Reverse Transcription ◽  
Leukemia Virus ◽  
Culture Conditions ◽  
Viral Envelope ◽  
Division Rate ◽  
Retroviral Transduction ◽  
Vesicular Stomatitis ◽  
Viral Envelopes ◽  
Vector Transduction

ABSTRACT Previous studies have suggested that Moloney murine leukemia virus (MoMLV)-based vectors pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G) have extensive ability to transduce nonmammalian cells. However, we have identified multiple cell lines from fish (FHM), mosquitoes (Mos-55), moths (Sf9 and High-5), flies (S2), and frogs (XPK2) that are not efficiently transduced by MoMLV-based vectors pseudotyped with many different viral envelope proteins, including VSV-G, while the same vectors are functional in these cells following transfection. A comparison of MoMLV-based vector transduction in mammalian and nonmammalian cells shows that the nonmammalian cells exhibit blocks at either entry, reverse transcription, or integration. Additionally, VSV-G-pseudotyped MoMLV-based vector transduction is attenuated in the zebrafish cell line ZF4 at entry and/or reverse transcription, whereas other transduction processes are unaffected. We show that the variation of transduction by MoMLV-based vectors in mammalian and nonmammalian cells is not due to differences in culture conditions or cell division rate but is likely the result of divergence in cellular factors required for retroviral transduction.

scholarly journals In vivo biodistribution of a highly attenuated recombinant vesicular stomatitis virus expressing HIV-1 Gag following intramuscular, intranasal, or intravenous inoculation

Vaccine ◽  
2009 ◽  
Vol 27 (22) ◽  
pp. 2930-2939 ◽  
Author(s):  
J. Erik Johnson ◽  
John W. Coleman ◽  
Narender K. Kalyan ◽  
Priscilla Calderon ◽  
Kevin J. Wright ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
In Vivo Biodistribution ◽  
Vesicular Stomatitis ◽  
Intravenous Inoculation ◽  
Hiv 1
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