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D F Summers

Publications and source records attributed to D F Summers.

At least 73 records · Page 4Linked to original sources
PubMed1979

Oligosaccharide chains of avian RNA tumor virus glycoproteins contain heterogeneous oligomannosyl cores.

Chicken embryo fibroblasts (C/E phenotype) infected with subgroups B and C of the Prague strain of Rous sarcoma virus were radiolabeled with either [6-(3)H]-glucosamine or [2-(3)H]mannose, and virus was purified from the growth medium. The large envelope glycoprotein, gp85, was the only major radiolabeled component of purified virus. Pronase-digested glycopeptides from purified virus were analyzed by a combination of (i) gel filtration with columns of Sephadex G15/G50 and Bio-Gel P4 and (ii) enzymatic digestion of the oligosaccharide chains with specific exoglycosidases and endo-beta-N-acetylglucosaminidases. The rather broad molecular weight distribution (approximately 2,000 to 4,000) for glycopeptides in these studies and previous studies in other laboratories was shown to represent actual heterogeneity in the carbohydrate moieties: (i) the glycopeptides contained both mannose-rich, neutral chains and complex, acidic chains with terminal sialic acid; and (ii) both classes of asparagine-linked carbohydrate structures exhibited heterogeneity in the size of the oligomannosyl core (a mixture of approximately 5 to 9 mannose units for the neutral structures, and 3 or 5 mannose units for the acidic structures). With the [2-(3)H]mannose-labeled glycopeptides from Rous sarcoma virus, Prague strain subgroup C, most of the oligosaccharide chains were high-molecular-weight, acidic structures, with similar numbers of 3-mannose and 5-mannose core structures.

Acetylglucosaminidase↗
PubMed1979

RNA synthesis of vesicular stomatitis virus-infected cells: in vivo regulation of replication.

Pulse-labeling of vesicular stomatitis virus-infected HeLa and BHK cells with [3H]uridine throughout the infectious cycle demonstrated two peaks of uridine incorporation into virus-specific RNA molecules. By separating total RNA synthesis into replication and transcription products, we showed that replication occurs over a shorter period of time in one peak synthesis. The biphasic nature of uridine incorporation is in part due to a general membrane phenomenon of reduced metabolite transport during vesicular stomatis virus infection and in part due to the apparent uncoupling of replication and transcription. A change in the ratio of newly synthesized plus and minus strands of the genome length (42S) RNA was found as the infection proceeded. Early in the infection, plus-stranded 42S RNA comprised 40% of the total genome length RNA synthesis, whereas late in infection, only 15 to 20% of the 42S RNA synthesized was complementary to the virion minus strand. Our data suggest that the rate of synthesis of plus-stranded 42S RNA was constant throughout the infection. The rate of virus release was determined by monitoring the uptake of [3H]uridine into released virus particles. Virus maturation and release are closely associated with the assembly of 42S RNA-containing nucleocapsids.

Animals↗
PubMed1979

Further characterization of the replicative complex of vesicular stomatitis virus.

Replicating vesicular stomatitis virus ribonucleoprotein (RNP) complexes were isolated in nonequilibrium Renografin density gradients. These nascent RNPs had the same buoyant density as virion nucleocapsids in both isopycnic Renografin and CsCl gradients. Both transcribing and replicating RNP complexes were shown to be stable in sucrose gradients, whereas only replicating RNP complexes were stable in Renografin gradients. Size analysis of the 5-min-pulse-labeled RNA species from the replicating RNPs using methylmercury gels revealed that the nascent strands were primarily less than full-length molecules. Longer times of radiolabeling demonstrated that the nascent RNA accumulated as 42S RNA, which was primarily of the same sense as the virion strand when it was radiolabeled at 5 h postinfection. The percentage of this radiolabeled RNA which was plus stranded was higher at 2.5 h postinfection, reflective of the shift in plus- to minus-stranded full-length 42S RNA synthesis which occurs in the cell. Addition of cycloheximide to the infected cells before the addition of the radiolabel prevented the formation of these RNP complexes. Both the change in the percentage of minus strands found in the RNP complexes at the different times postinfection and the sensitivity to cycloheximide indicate that the RNP complex which was isolated was indeed the replicative complex.

Cycloheximide↗
PubMed1979

Rhabdoviridae. Report of the Rhabdovirus Study Group, International Committee on Taxonomy of Viruses.

The family Rhabdoviridae comprises approximately 75 viruses infecting vertebrates, invertebrates and plants. The main characteristics of the member viruses are: (i) the viruses infecting vertebrates and invertebrates are bullet-shaped and the viruses infecting plants are usually bacilliform; (ii) the viruses have particle lengths varying from 130 to 380 nm and widths varying from 60 to 95 nm; (iii) the viruses possess unit-membrane envelopes from which protrude spikes 5 to 10 nm long; (iv) the viruses have precisely coiled helical nuecleocapsids with a diameter of approx. 50 nm; (v) most of the viruses which have been studied contain 5 proteins; the prototype, vesicular stomatitis virus, contains proteins designated L (large), G (glycoprotein), N (nucleoprotein), NS (nonstructural) and M (matrix); N or NS is phosphorylated in most members which have been studied; (vi) the viruses contain single-stranded RNA which is transcribed into several messenger RNA species with sizes corresponding to the structural proteins; (vii) the nucleocapsid contains the RNA-dependent RNA polymerase and is infectious; and (viii) many of the viruses produce morphologically distinct defective-interfering (T) particles.

RNA, Viral↗
PubMed1978

Specific changes in the oligosaccharide moieties of VSV grown in different lectin-resistnat CHO cells.

The carbohydrate moieties of the G glycoprotein of vesicular stomatitis virus (VSV) grown in three distinct lectin-resistant (LecR) Chinese hamster ovary (CHO) cell lines have been compared by fine structural analysis of radiolabeled glycopeptides. The mutant WgaRIII, selected for resistance to wheat germ agglutinin (WGA), produces VSV containing G glycoprotein specifically lacking in sialic acid. The mutant PhaRI, selected for resistance to phytohemagglutinin (PHA) and previously shown to lack a particular glycoprotein N-acetyl-glucosaminyl-transferase activity, produces VSV containing G glycoprotein specifically lacking terminal N-acetylglucosamine-galactose-sialic acid sequences and possessing an increased number of mannose residues in the "core" region of its carbohydrate moieties. The mutant PhaRIConARII, a "double" mutant selected from PhaRI cells for resistance to concanavalin A (ConA), produces VSV containing G glycoprotein with a further alteration in the mannose residues of the "core" oligosaccharide region. We discuss the relevance of these findings to the mechanisms of glycoprotein biosynthesis in mammalian cells and to the biochemical bases of lectin resistance in CHO cells.

Cell Line↗
PubMed1978

Oligosaccharide chains are trimmed during synthesis of the envelope glycoprotein of vesicular stomatitis virus.

The biosynthesis and maturation of the oligosaccharide moieties of the envelope glycoprotein of vesicular stomatitis virus were investigated in virus-infected HeLa and BHK21 cells after pulse labeling with [2-3H]mannose. Two major forms of the virus glycoprotein were detected by polyacrylamide gel electrophoresis, which appear to correspond to the viral glycoprotein with either "precursor" or "mature" oligosaccharide chains. The precursor chains in both HeLa and BHK21 cells infected with vesicular stomatitis virus obtained after a 30-min pulse were large oligomannose structures containing approximately 7--9 mannose residues as estimated by gel filtration analysis. The size of the oligomannose structures initially transferred to the protein may have been even larger. Mature, virus-size oligosaccharide chains, which could be detected after a 20- to 30-min delay, contained only three mannose residues and, in addition, contained branch structures terminating in sialic acid. A precursor--product relationship of these two forms of oligosaccharide chains was demonstrated by pulse--chase labeling of virus-infected HeLa cells. These studies indicated that the large oligomannosyl core structures initially added to the glycoprotein were being "trimmed" by the removal of mannose residues prior to (and/or during) the addition of the branch chains terminating in sialic acid.

Cells, Cultured↗
PubMed1977

Glycosylation of VSV glycoprotein is similar in cystic fibrosis, heterozygous carrier, and normal human fibroblasts.

The single envelope glycoprotein of vesicular stomatitis virus was used as a specific probe of glycosyltransferase activities in fibroblasts from two cystic fibrosis patients, an obligate heterozygous carrier and a normal individual. Gel filtration of pronase-digested glycopeptides from both purified virions and infected cell-associated VSV glycoprotein which had been labeled with[3H] glucosamine did not reveal any significant differences in the glycosylation patterns between the different cell cultures. All 4 cell lines were apparently able to synthesize the mannose- and glucosamine- containing core structure and branch chains terminating in sialic acid which are characteristic of asparagine-linked carbohydrate side chains in cellular glycoproteins. Analysis of tryptic glycopeptides by anion-exchange chromotography indicated that the same 2 major sites on the virus polypeptide were recognized and glycosylated in all 4 VSV-infected cell cultures. These studies suggest that the basic biochemical defect(s) in cystic fibrosis is not an absence or deficiency in enzymes responsible for the biosynthesis of complex carbohydrate side chains.

Cell Line↗
PubMed1976

Oligosaccharide moieties of the glycoprotein of vesicular stomatitis virus.

Vesicular stomatitis virus contains a single structural glycoprotein whose carbohydrate sequences are probably specified by the host cell. The glycopeptides derived by Pronase digestion of the glycoprotein of vesicular stomatitis virus grown in HeLa cells have an average molecular weight of 1,800. There are multiple oligosaccharide chains on the vesicular stomatitis virus glycoprotein with protein-carbohydrate linkages that are cleaved only by strong alkali under reducing conditions, suggesting that they contain asparagine and N-acetylglucosamine. The oligosaccharide moieties, in addition, appear to be heterogeneous in sequence on the basis of their mobilities during electrophoresis and their sensitivities to cleavage by an endoglycosidase. The carbohydrate-peptide linkage region of the major class of oligosaccharides of the vesicular stomatitis virus glycoprotein has the proposed sequence: (see article).

Acetylglucosamine↗
PubMed1976

Interactions of vesicular stomatitis virus with murine cell surface antigens.

The process of maturation of vesicular stomatitis virus (VSV) results in the loss of 70% of the H-2k antigenic activity from L-cell plasma membranes. This phenomenon is also demonstrated during VSV infection of cells of the H-2d haplotype. Using the method of inhibition of immune cytolysis, VSV-infected L5178Y tissue culture cells and VSV-infected METH A fibrosarcoma cells grown in vivo show a loss of H-2d activity of 73 and 76%, respectively. Using monospecific antisera, it is seen that VSV infection results in a significant loss of antigenic activity of the gene products of both the H-2D and H-2K regions in cells of the H-2d and H-2k haplotypes. In hybrid cells expressing H-2k as well as H-2b, VSV infection results in the decrease of both H-2 antigenic activities to the same extent. VSV purified from L cells shows considerable H-2k activity, but the reaction of this virus with anti-H-2k serum does not prevent a normal subsequent infection with this virus. VSV may associate with H-2 antigen in the culture medium, but the results of mixing VSV with uninfected H-2-containing homogenates suggest that this association occurs only when the host cell and the cell homogenate share the same H-2 haplotype. Velocity sedimentation of VSV, which would remove contaminating cellular membrane fragments, does not separate H-2 activity from VSV. H-2 activity is also stably associated with VSV throughout sequential sucrose gradient centrifugation steps. It is possible that H-2 antigen is a structural component of VSV grown in murine cells.

Antigens, Viral↗
PubMed1976

Glycosylation sites of vesicular stomatitis virus glycoprotein.

Detailed analysis on DEAE-Sephadex of the tryptic digestion products of the glycoprotein from vesicular stomatitis virus grown in HeLa suspension cultures revealed the presence of two major and several minor sugar-labeled species. The minor tryptic glycopeptides were converted to one of the two major glycopeptide species by treatment with neuraminidase. Thus, vesicular stomatitis virus glycoprotein contains only two oligosaccharide side chains that are heterogeneous in their sialic acid content.

Glycopeptides↗
PubMed1976

Association of vesicular stomatitis virus proteins with HeLa cell membranes and released virus.

The association of vesicular stomatitis virus proteins with intracellular and plasma membranes was examined by pulse and pulse-chase labeling of virus-infected HeLa cells with [35S]methionine and separation of cell homogenates into three major membrane fractions in discontinuous sucrose gradients. The glycoprotein G was primarily associated with rough endoplasmic reticulum-like membranes after short radioactive pulses (2 to 4 min) but accumulated in the plasma membrane-enriched fraction and the smooth internal membrane fraction with longer pulse or chase periods. The nucleocapsid protein N and the matrix protein M accumulated in the rough endoplasmic reticulum and plasma membrane-like fractions but not in the smooth internal membrane fraction. Only a fraction (35 to 40%) of the viral protein synthesized during a short pulse in the mid-cycle of infection was apparently utilized in released virus. The newly synthesized virus proteins first appeared in released virus in the order: M, N and L, and G.

Capsid↗
PubMed1976

Glycosylation of vesicular stomatitis virus glycoprotein in virus-infected HeLa cells.

Glycosylation of the envelope glycoprotein of vesicular stomatitis virus was examined using virus-infected HeLa cells that were pulse-labeled with radioactive sugar precursors. The intracellular sites of glycosylation and the stepwise elongation of the carbohydrate side chains of the G protein were monitored by membrane fractionation and gel filtration of Pronase-digested glycopeptides. The results with short pulses of sugar label (5 to 10 mtein linkage (glucosamine and mannose) are added to G which was associated with the rough endoplasmic reticulum-enriched membrane fraction, whereas the more distal sugars (galactose, sialic acid, fucose, and possibly more glucosamine) are added in the light-density internal membrane fraction. Accumulation of mature G was observed in the plasma membrane-enriched fraction. The gel filtration studies indicated that the initial glycosylation event may be the en bloc addition of a mannose and glucosamine oligomer, followed by the stepwise addition of the more distal sugars.

Chromatography↗
PubMed1974

Phosphorylation of vesicular stomatitis virus in vivo and in vitro.

The structural protein, NS, of purified vesicular stomatitis virus (VSV) is a phosphoprotein. In infected cells phosphorylated NS is found both free in the cytoplasm and as part of the viral ribonucleoprotein (RNP) complex containing both the 42S RNA and the structural proteins L, N, and NS, indicating that phosphorylation occurs as an early event in viral maturation. VSV contains an endogenous protein kinase activity, probably of host region, which catalyzes the in vitro phosphorylation of the viral proteins NS, M, and L, but not of N or G. The phosphorylated sites on NS appear to be different in the in vivo and in vitro reactions, and are differentially sensitive to alkaline phosphatase. After removal of the membrane components of purified VSV with a dextran-polyethylene glycol two-phase separation, the kinase activity remains tightly associated with the viral RNP. However, viral RNP isolated from infected cells shows only a small amount of kinase activity. The protein kinase enzyme appears to be a cellular contaminant of purified VSV because an activity from the uninfected cell extract can phosphorylate in vitro the dissociated viral proteins NS and M. The virion-associated activity may be derived either from the cytoplasm or the plasma membrane of the host cell since both of these cellular components contain protein kinase activity similar to that found in purified VSV.

Alkaline Phosphatase↗
PubMed1974

Newcastle disease virus infection of L cells.

Newcastle disease virus (NDV) California strain reportedly grows poorly in L cells but replicates very well in chicken embryo cells. NDV-infected L cell cultures show a characteristic virus growth curve with respect to uridine incorporation, but plaque assays of the virus produced 24 h postinfection (PI) show no infectious particles when assayed on L cell monolayers and only a very low titer on chick cell monolayers. Plasma membranes isolated and purified from infected L cells 8 h PI contain all of the major virion proteins. In addition, NDV-infected L cells show a 50% loss of H-2 antigenic activity, a phenomenon previously observed in cells productively infected with vesicular stomatitis virus. These results suggest that at least part of the normal process of NDV maturation occurs in NDV-infected L cells. Sodium dodecyl sulfate-polyacrylamide gel patterns of supernatant virus purified from cells radiolabeled with amino acids from 3 to 24 h PI in the presence of actinomycin D show that all the major NDV structural proteins are present. Electron micrographs of NDV-infected L cells show extensive virus maturation at cell membranes. It can be concluded that infection of L cells with NDV results in a normal production of virus-specific RNA, synthesis of all the major structural proteins, association of the viral envelope proteins with the L cell plasma membrane, and the loss of cell surface H-2 antigenic activity. However, most of the virus particles produced are noninfectious.

Animals↗