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B M Sefton

Publications and source records attributed to B M Sefton.

At least 109 records · Page 6Linked to original sources
PubMed1980

Characterization of a small, nonstructural viral polypeptide present late during infection of BHK cells by Semliki Forest virus.

BHK cells, late in infection with Semliki Forest virus, were found to contain a small virus-specific polypeptide not found in the mature virion. This polypeptide had an apparent molecular weight of 6,000 and is referred to here as the 6K protein. No [2-3H]mannose was incorporated into 6K, and hence it does not appear to be a glycoprotein. This protein appears to be a primary translation product of the subgenomic 26S mRNA, which encodes the viral structural proteins. The genes encoding the viral structural proteins are arranged on the message in the order of 5'-C-E3-E2-E1-3'. We have found that the gene coding for 6K is located to the 3' side of the gene encoding E2. Subcellular fractionation of pulse-labeled cells infected with Semliki Forest virus demonstrated that 6K, like the viral glycoproteins p62 and E1, was present predominantly in the rough microsomal membrane fraction. 6K appears to be analogous, therefore, to the nonstructural 4.2K protein present in cells infected with Sindbis virus.

Animals↗
PubMed1979

Complete exchange of viral cholesterol.

The exchange of the cholesterol in the membranes of two enveloped viruses, Sindbis virus and vesicular stomatitis virus, with cholesterol present in lipid vesicles and in serum was measured. Biosynthetically labeled viral cholesterol underwent spontaneous and complete transfer to both lipid vesicles and to serum. The rate with which and the extent to which this process occurred were very similar for these two viruses. During incubation with lipid vesicles in excess, half of the viral cholesterol underwent transfer in approximately 4 h and more than 90% underwent transfer in 24h at 37 degrees C. Similar rates and extents of movement of viral cholesterol were observed when incubations were carried out with vesicles which contained cholesterol and phospholipid in the same molar ratio as in the virus or with egg lecithin vesicles which contained no cholesterol. When labeled cholesterol was present initially in the lipid vesicles, movement of cholesterol from the vesicles to the virus was observed. One implication of the fact that viral cholesterol undergoes extensive exchange with serum cholesterol is that cellular cholesterol is in equilibrium with that in the extracellular fluid.

Carbon Radioisotopes↗
PubMed1979

Two small virus-specific polypeptides are produced during infection with Sindbis virus.

We have identified and characterized two small virus-specific polypeptides which are produced during infection of cells with Sindbis virus, but which are not incorporated into the mature virion. The larger of these is a glycoprotein with an approximate molecular weight of 9,800 and is found predominantly in the medium of infected cells. Three independent lines of evidence demonstrate conclusively that this 9,800-dalton glycoprotein is produced during the proteolytic conversion of the precursor polypeptide, PE2, to the virion glycoprotein E2. This small glycoprotein is therefore analogous to the virion glycoprotein E3 of the very closely related alphavirus, Semliki Forest virus. The 9,800-dalton glycoprotein of Sindbis virus, unlike the E3 glycoprotein of Semliki Forest virus, is not, however, present in the viral particle. The other virus-specific polypeptide is 4,200 daltons in size, does not appear to be a glycoprotein, and is neither incorporated into the mature virus nor released into the culture medium. The gene for this small polypeptide is present in the viral 26S mRNA (the mRNA which encodes all the viral structural polypeptides) and appears to be located in the portion of the mRNA which encodes the two viral glycoproteins. The possibility that this 4,200-dalton polypeptide functions as a signal peptide during the synthesis of the viral membrane glycoproteins is discussed.

Cell Line↗
PubMed1979

Polymorphism of avian sarcoma virus src proteins.

The src gene products of seven different avian sarcoma viruses were compared. In vitro translation of virion RNA yielded products identified unambiguously as p60src in the case of two stocks of the Schmidt-Ruppin strain, three stocks of the Prague strain, the Bryan strain, and the Bratislava 77 strain of avian sarcoma virus. Differences in the electrophoretic mobility of these seven p60src proteins in sodium dodecyl sulfate-polyacrylamide gels, corresponding to variation in the apparent molecular weights ranging from 56,000 to 60,500, were observed. Antigenic variability was also found; only three of the seven viruses tested encoded a p60src, which was precipitated by antisera derived from rabbits bearing tumors induced by the Schmidt-Ruppin strain of Rous sarcoma virus. Examination of the methionine-containing tryptic peptides of the seven ;60src proteins by two-dimensional mapping revealed four common peptides but marked variability in the five to eight other peptides in each protein. Clear differences in the peptide maps of p60src were observed, both between different strains of virus and within strains. In the three cases examined, p60src synthesized in transformed cells was found to be essentially identical to that synthesized in vitro. We conclude that there is significant polymorphism in the p60src proteins of the avian sarcoma viruses.

Avian Sarcoma Viruses↗
PubMed1979

Product of in vitro translation of the Rous sarcoma virus src gene has protein kinase activity.

In vitro translation of Rous sarcoma virus virion RNA resulted in the synthesis of a protein kinase which, when immunoprecipitated with antitumor serum, phosphorylated the immunoglobulin heavy chain. Even though in vitro translation of virion RNA resulted in the synthesis of a number of polypeptides which were recognized by antitumor serum, control experiments demonstrated that an immunoprecipitable protein kinase activity was found only when an immunoprecipitable p60src, the polypeptide product of the src gene, was synthesized. A protein kinase with similar properties was therefore intimately associated with p60src which was synthesized in vitro in the reticulocyte lysate, just as it is with p60src which is obtained from transformed chick and mammalian cells. It is therefore highly unlikely that this association is artifactual. ts NY68 is a mutant of Rous sarcoma virus which is able to transform cells at 36 but not at 41 degrees C. In vitro translation of ts NY68 virion RNA at 30 degrees C resulted in efficient synthesis of immunoprecipitable p60src, but very inefficient synthesis of an immunoprecipitable protein kinase. The p60src obtained by in vitro translation of wild-type virion RNA was more than 20-fold more active as a protein kinase than was that obtained from ts NY68 RNA. The correlation in the case of ts NY68 of a deficiency in protein kinase activity with an inability to transform cells at high temperature suggests that the protein kinase activity associated with p60src is indeed critical to cellular transformation.

Avian Sarcoma Viruses↗
PubMed1978

The entry into host cells of Sindbis virus, vesicular stomatitis virus and Sendai virus.

We have compared the mechanisms of entry into host cells of three enveloped viruses: Sendai virus, vesicular stomatitis virus (VSV) and Sindbis virus. Virus entry by membrane fusion should antigenically modify the surface of a newly infected cell in such a way that it will be killed by anti-viral antibody and complement. On the other hand, virus entry by a mechanism involving uptake by the cell of the whole virion should not make cells sensitive to antibody and complement. As expected, cells newly infected with Sendai virus were readily and completely lysed by anti-Sendai antibody and complement. In marked contrast, however, cells newly infected with either Sindbis virus or VSV were killed by anti-viral antibody and complement only when infected at an extremely high multiplicity of infection, in excess of 1000 plaque-forming units per cell. We favor the following explanation for these results with Sindbis virus and VSV: a very large majority of the Sindbis and VSV virions entered the infected cells by some means other than membrane fusion, presumably engulfment of the whole particle. Efficient entry by way of membrane fusion may therefore not be a general characteristic of enveloped viruses.

Antigens, Surface↗
PubMed1978

Comparison of the expression of the src gene of Rous sarcoma virus in vitro and in vivo.

We have compared the polypeptide products of the src gene of several strains of Rous sarcoma virus produced by in vitro translation of heat-denatured 70S virion RNA in the nuclease-treated reticulocyte lysate with those present in chick cells transformed by these viruses. We have done this by immunoprecipitation, using sera from rabbits injected at birth with Schmidt-Ruppin Rous sarcoma virus. In vitro translation results in the synthesis of at least nine polypeptides which appear to be encoded by the src gene. These range in size from 17,000 to 60,000 daltons. The sera from tumor-bearing rabbits precipitated these polypeptides arising from the in vitro translation of RNA from Schmidt-Ruppin Rous sarcoma virus of both subgroup A and subgroup D and from one stock of Prague Rous sarcoma virus of subgroup C. In each case, all of this family of related polypeptides could be precipitated except the smallest, the 17,000-dalton polypeptide. No precipitation of analogous polypeptides resulting from the translation of RNA from other strains of Rous sarcoma virus was observed. Cells transformed by these three strains of Rous sarcoma virus contain easily detectable amounts of a polypeptide, p60src, essentially identical to the 60,000-dalton in vitro product. With one exception, they do not contain significant amounts of polypeptides analogous to the smaller in vitro products which can be precipitated by these sera. Cells transformed by one stock of Schmidt-Ruppin Rous sarcoma virus of subgroup A did contain a 39,000-dalton polypeptide, which was related, by peptide mapping, to the 60,000-dalton polypeptide and was similar in size to a precipitable in vitro product. The 60,000-dalton polypeptide present in transformed cells appeared to be phosphorylated 10 to 25 min after its synthesis, metabolically very stable, and not derived from a precursor polypeptide. All immunoprecipitates from transformed cells which contained p60src also contained an 80,000-dalton phosphoprotein. This polypeptide is unrelated to p60src, as determined by peptide mapping, and may well be a host cell polypeptide which is specifically associated with p60src.

Avian Sarcoma Viruses↗
PubMed1978

The initial glycosylation of the Sindbis virus membrane proteins.

The mechanism by which the membrane proteins of Sindbis virus are initially glycosylated during growth of the virus in chick cells was studied. The experiments suggest strongly that the 2 viral glycoproteins are glycosylated before release from the polysome and that this glycosylation involves transfer of a large, 1,800 dalton oligosaccharide to the polypeptide chains. The donor of the oligosaccharide is most probably a lipid.

Glycolipids↗
PubMed1977

Immediate glycosylation of Sindbis virus membrane proteins.

The mechanism by which the membrane proteins of Sindbis virus are initially glycosylated during growth of the virus in chick cells was studied. The experiments suggest strongly that the two viral glycoproteins are glycosylated before release from the polysome, and that this glycosylation involves transfer of a large 1800 dalton oligosaccharide to the polypeptide chains. The donor of the oligosaccharide is most probably a lipid.

Animals↗
PubMed1975

Virus-dependent glycosylation.

The oligosaccharides of the membrane glycoproteins of Sindbis virus, vesicular stomatitis virus, and Rous sarcoma virus were compared on the basis of apparent size and sugar composition. It appears that each virus acquires a different set of oligosaccharides during growth in a single type of cell.

Animals↗
PubMed1974

Glycoproteins of Sindbis virus: priliminary characterization of the oligosaccharides.

The carbohydrate content of Sindbis virus was determined by gas chromatographic analysis. The two viral glycoproteins were found to be approximately 8% carbohydrate by weight. Mannose is the sugar present in the largest amount. Smaller amounts of glucosamine, galactose, sialic acid, and fucose were also detected. Each of the two viral glycoproteins appears to contain two structurally unrelated oligosaccharides. Two of the three Sindbis-specific glycoproteins found in infected chick cells were shown to contain short, unfinished oligosaccharides.

Animals↗
PubMed1973

Enzymatic iodination of Sindbis virus proteins.

Sindbis virus was iodinated by using the enzyme lactoperoxidase, an iodination technique which labels only surface proteins. By this technique, the two viral glycoproteins are labeled, and the internal viral protein is not. The two glycoproteins are iodinated to strikingly different extents. This difference in susceptibility to iodination apparently is due to the position or conformation of the glycoproteins in the envelope spikes of the virion and not to differing contents of tyrosine, the amino acid substrate of lactoperoxidase. Both viral glycoproteins are iodinated by lactoperoxidase on the surface of Sindbis-infected chicken cells. Here, as in the virion, the glycoproteins are iodinated unequally, with the smaller glycoprotein again being preferentially iodinated. Another virus-specific protein found in large amounts in infected cells, and from which the preferentially iodinated virion glycoprotein is produced by a proteolytic cleavage, is not iodinated by lactoperoxidase. Thus it appears that the viral glycoproteins are present on the cell surface and that the precursor protein is not.

Animals↗
PubMed1973

Biosynthesis of the Sindbis virus carbohydrates.

The sequence in which sugars are added to the Sindbis virus glycoproteins was studied. Infected cells contain three glycosylated virus-specific proteins: the two virion glycoproteins and the immediate precursor to the smaller virion glycoprotein. Larger Sindbis-specific proteins are not glycosylated. The cell-associated forms of both of the virion glycoproteins contain glucosamine, mannose, galactose, and fucose. The glycosylated precursor contains only glucosamine, mannose, and some galactose. The conversion of precursor to virion protein involves both the addition of galactose and fucose and the loss of mannose. The apparent extent of glycosylation of each virus-specific protein is not influenced by the host cell.

Amino Acids↗
PubMed1971

Stimulation of glucose transport in cultures of density-inhibited chick embryo cells.

The rate of glucose transport in sparse, rapidly growing chick-embryo fibroblasts is much greater than that in density-inhibited cells. The addition of fresh chicken serum or trypsin to the medium of density-inhibited cells causes a large increase in the rate of glucose transport that is detectable 15 min after addition. The increase in glucose transport precedes the increase in DNA synthesis by 5-6 hr. Only small changes in rates of transport are seen with nucleosides or a nonmetabolizable amino acid. The increase in glucose transport requires protein synthesis but not RNA or DNA synthesis.

Aminoisobutyric Acids↗
PubMedDate not supplied

Direct evidence that oncogenic tyrosine kinases and cyclic AMP-dependent protein kinase have homologous ATP-binding sites.

p60src, the transforming protein of Rous sarcoma virus (RSV), is a protein kinase that has a strict specificity for tyrosine. The phosphorylation of cellular proteins by p60src (ref. 4) results in transformation. Recently, Barker and Dayhoff discovered that residues 259-485 of p60src have 22% sequence identity with residues 33-258 of the catalytic subunit of cyclic AMP-dependent protein kinase, an enzyme that has a specificity for serine. Because it was necessary to introduce eight gaps to align the two proteins, the question remained as to whether this apparent homology reflected a common evolutionary origin. We demonstrate here that the ATP analogue p-fluorosulphonylbenzoyl 5'-adenosine (FSBA) inactivates the tyrosine protein kinase activity of p60src by reacting with lysine 295. When aligned for maximum sequence identity, lysine 295 of p60src and the lysine in the catalytic subunit which also reacts specifically with FSBA are superimposed precisely. This functional homology is strong evidence that the protein kinases, irrespective of amino acid substrate specificity, comprise a single divergent gene family.

Adenosine↗