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R W Compans

Publications and source records attributed to R W Compans.

At least 199 records · Page 11Linked to original sources
PubMed1981

Glycosylation sites of influenza viral glycoproteins: characterization of tryptic glycopeptides from the A/USSR(H1N1) hemagglutinin glycoprotein.

Glycosylated tryptic peptides of the hemagglutinin (HA) glycoprotein of influenza A/USSR/90/77(H1N1) virus were separated by a combination of ion-exchange chromatography and gel filtration. Seven different glycosylated tryptic peptide classes were obtained from the HA1 polypeptide, and only one glycosylated peptide was obtained from the HA2 polypeptide. Several of the tryptic fragments of HA1 and the HA2 glycopeptides were sulfated. The nature of the carbohydrate chain in each of the glycosylated tryptic peptides was determined from observations of the incorporation of different sugar precursors and susceptibility to cleavage by the enzyme endoglycosidase H and by compositional analysis by gas chromatography. Such analyses showed that three types of carbohydrate chains were present in HA1 (type I [complex], type II [high mannose], and hybrid type), whereas HA2 contained only type I oligosaccharide chains. The amino acid composition of each of the glycosylated tryptic peptides was also determined.

Amino Acids↗
PubMed1981

Glycoproteins of murine leukemia viruses. III. Glycosylation of env precursor glycoproteins.

We have compared the glycopeptides obtained after extensive pronase digestion of the env precursors (PrENV proteins) of ecotropic, xenotropic, and dual-tropic murine leukemia viruses. Two glycopeptide size classes, having molecular weights of approximately 2,200 and 1,500, were shown to be associated with the PrENV proteins of all murine leukemia viruses studied. Glycopeptides associated with the env precursors were totally susceptible to endo-beta-N-acetyglucosaminidase H. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partial endo-beta-N-acetylglucosaminidase H digestion products of the env precursor of dual-tropic mink cell focus-forming virus (MCF 247) revealed the presence of seven bands, suggesting that six glycosylation sites were present on the precursor molecule. The MCF 247 PrENV protein had been previously shown to be accessible to lactoperoxidase-catalyzed radioiodination on the surface of infected cells. The cell surface PrENV molecules had the same electrophoretic mobility as pulse-labeled PrENV protein, and after endo-beta-N-acetylglucosaminidase H treatment a similar shift in electrophoretic mobility was observed for the cell surface PrENV protein and the pulse-labeled precursors, a finding which indicated that the PrENV protein located on the cell surface also possessed only mannose-rich oligosaccharides. These results indicated that the env precursor glycoproteins of dual-tropic viruses had the unusual property of migrating to the cell surface without undergoing the normal oligosaccharide processing and proteolytic cleavage events that had been observed for ecotropic and xenotropic murine leukemia virus glycoproteins.

AKR murine leukemia virus↗
PubMed1981

Delayed appearance of pseudotypes between vesicular stomatitis virus influenza virus during mixed infection of MDCK cells.

In intact Madin-Darby canine kidney (MDCK) cell monolayers, vesicular stomatitis virus (VSV) matures only at basolateral membranes beneath tight junctions, whereas influenza virus buds from apical cell surfaces. Early in the growth cycle, the viral glycoproteins are restricted to the membrane domain from which each virus buds. We report here that phenotypic mixing and formation of VSV pseudotypes occurred when influenza virus-infected MDCK cells were superinfected with VSV. Up to 75% of the infectious VSV particles from such experiments were neutralized by antiserum specific for influenza virus, and a smaller proportion (up to 3%) were resistant to neutralization with antiserum specific for VSV. The latter particles, which were neutralized by antiserum to influenza A/WSN virus, are designated as VSV(WSN) pseudotypes. During mixed infections, both wild-type viruses were detected 1 to 2 h before either phenotypically mixed VSV or VSV(WSN) pseudotypes. Coincident with the appearance of cytopathic effects in the monolayer, the yield of pseudotypes rose dramatically. In contrast, in doubly infected BHK-21 cells, which do not show polarity in virus maturation sites and are not connected by tight junctions, VSV(WSN) pseudotypes were detected as soon as VSV titers rose to the minimum levels which allowed detection of pseudotypes, and the proportion observed remained relatively constant at later times. Examination of thin sections of doubly infected MDCK monolayers revealed that polarity in maturation sites was preserved for both viruses until approximately 12 h after inoculation with influenza virus, when disruption of junctional complexes was evident. Even at later periods, the majority of each virus type was associated with its normal membrane domain, suggesting that the sorting mechanisms responsible for directing the glycoproteins of VSV and influenza virus to separate surface domains continue to operate in doubly infected MDCK cells. The time course of VSV(WSN) pseudotype formation and changes in virus maturation sites are compatible with progressive mixing of viral glycoproteins at either intracellular or plasma membranes of doubly infected cells.

Animals↗
PubMed1980

Glycopeptides of murine leukemia viruses. II. Comparison of xenotropic and dual-tropic viruses.

The glycosylation patterns of the gp70 glycoproteins of xenotropic and dualtropic murine leukemia virus (MuLV) were compared with those of ecotropic viruses. Ecotropic viruses contain a large glycopeptide size class designated G1 (molecular weight, approximately 5100), and such glycopeptides were not detected in xenotropic viruses grown in mink cells nor in dual-tropic viruses grown in mouse or mink lung cells. Both xenotropic and dual-tropic MuLV had glycopeptide size classes designated G2, G3, and G4 (molecular weights, approximately 2900, 2,200, and 1,500, respectively). G2 glycopeptides of xenotropic and dual-tropic MuLV were shown to be resistant to endo-beta-N-acetylglucosaminidase H, whereas G3 and G4 glycopeptides were susceptible. The relative abudance of glycopeptide G3 was increased in xenotropic and dual-tropic viruses as compared with ecotropic viruses, whereas the relative amount of G4 was decreased in xenotropic viruses. The similarity in the glycosylation patterns of a number of xenotropic and dual-tropic viruses suggests that glycosylation sites are highly conserved within the env gene products of each of these classes of viruses.

Acetylglucosaminidase↗
PubMed1980

Antibody-resistant spread of vesicular stomatitis virus infection in cell lines of epithelial origin.

In MDCK cells, vesicular stomatitis virus (VSV) buds exclusively from the basolateral plasma membranes beneath tight junctions, whereas influenza virus forms only at the free apical surface. Anti-VSV antiserum did not prevent the formation of plaques on MDCK cell monolayers infected with VSV, whereas plaque formation in BHK-21 cells was completely inhibited by such antiserum. Under similar conditions, homologous antiserum completely prevented plaque formation by influenza virus on MDCK cells. In several other epithelioid cell lines, VSV also formed plaques in the presence of specific antiserum. These results suggest that VSV receptors are present on basolateral membranes in the cells studied and that junctional complexes present between cells may exclude antibody from intercellular spaces and thus permit the lateral spread of virus infection in the presence of neutralizing antibody.

Animals↗
PubMed1979

Effect of cytochalasin B on the maturation of enveloped viruses.

The maturation of two enveloped viruses, influenza and vesicular stomatitis, occurs in cells treated with cytochalasin B. Virions produced in the presence of 50 microgram/ml cytochalasin B (CB) appear to be as infectious as those from control cells, indicating that polymerized actin is not required for the assembly of functional viral components. CB inhibits the release of influenza virus from treated cells, a phenomenon which appears to be a result of the synthesis of an aberrant neuraminidase (NA) glycoprotein; virions grown in CB-treated cells had a 90% reduction in specific enzymatic activity. We found that both influenza viral glycoproteins (NA and Hemagglutinin glycoprotein) had faster electrophoretic mobilities and were more heterogeneous in CB-treated cells as compared with controls. We also observed complete inhibition of incorporation of labeled glucosamine into viral glycoproteins in the presence of the drug. It was of interest that CB-induced inhibition of glycosylation appeared to cause loss of neuraminidase function, whereas hemagglutinating activity was not noticeably impaired. The presence of altered glycoproteins did not significantly diminish the infectivity of either influenza virus or vesicular stomatitis virus. Our results indicate that no step in the maturation of enveloped viruses is dependent upon an intact cytoskeletal network.

Cytochalasin B↗
PubMed1979

Biosynthesis of the oligosaccharides of influenza viral glycoproteins.

Glycosylation of influenza viral glycoproteins was investigated by pulse-labeling of infected BHK21-F cells with radioactive sugar precursors and by cell fractionation and analysis of Pronase-digested viral glycopeptides by gel filtration. The results with short pulses of [3H]mannose suggested that the initial event in glycosylation is the en bloc transfer of oligomannosyl cores to viral glycoproteins associated with rough membranes. The molecular weight of the glycopeptides which represent the cores was estimated to be approximately 1600-2200. Some mannose residues appear to be subsequently removed from oligosaccharide cores. [3H]mannose-labeled glycopeptides obtained either from cells pulsed for brief periods or from rough membranes, which contain predominantly oligosaccharide cores, were sensitive to digestion by endo-p-N-acetylglucosaminidase H (endo-H). On the other hand, glycopeptides larger than oligosaccharide cores, which appeared during chases or after migration of viral glycoproteins from rough to smooth membranes, were resistant to endo-H treatment. The branched sugars (glucosamine, galactose, and fucose), which are contained only in the complex (type I) oligosaccharide chains of virions, appear to be added in a stepwise manner to the trimmed oligosaccharide cores primarily on smooth membranes. Mannoserich glycopeptides of virions (type II) are similar in size to oligosaccharide cores detected in infected cells and are totally sensitive to endo-H, suggesting that type II glycopeptides may represent oligomannosyl cores which escape trimming as well as addition of branched sugars. Comparison of glycopeptides of infected and uninfected BHK21-F cells suggests that influenza viral glycoproteins contain oligosaccharide chains similar in size to those of host cells except for the absence of sialic acid in viral glycoproteins. Further, we observed that intracytoplasmic membranes from infected cells contain much less sialic acid than those from uninfected cells, indicating that viral neuraminidase present in the interior of infected cells possesses enzymatic activity.

Animals↗
PubMed1979

Polarity of influenza and vesicular stomatitis virus maturation in MDCK cells: lack of a requirement for glycosylation of viral glycoproteins.

We have investigated whether glycosylation of membrane glycoproteins is a determinant of the site of maturation of enveloped viruses in Madin-Darby canine kidney (MDCK) cells. In MDCK cell monolayers, vesicular stomatitis virus buds exclusively from the basal or lateral plasma membranes and contains a sialylated glycoprotein, whereas influenza virus buds exclusively from the apical plasma membrane and lacks neuraminic acid. In order to study the possible relationship between glycosylation of viral glycoproteins and the budding site, infected MDCK cells were treated with tunicamycin at a concentration that completely inhibits glycosylation of viral glycoproteins and the site of virus maturation was examined by electron microscopy. When tunicamycin-treated monolayers were compared to controls, the polarity in the maturation sites of both viruses was maintained. These results indicate that glycosylation of viral glycoproteins is not required for the determination of the cellular maturation site of these enveloped viruses.

Cell Line↗
PubMed1979

Carbohydrate components of influenza C virions.

The carbohydrate components of influenza C virions grown in chicken kidney (CK) cells were analyzed by gel filtration following exhaustive digestion with Pronase. The [(3)H]glucosamine-labeled glycopeptides were larger and more heterogeneous than those of influenza A/WSN virions; three major size classes (G(1), G(2), and G(3)) were resolved. Treatment with Vibrio cholerae neuraminidase caused a decrease in size of G(1) and G(2), along with release of about 16% of the (3)H label. The released sugar components were identified as N-acetylneuraminic acid by thin-layer chromatography. Peak G(3) was highly labeled with [(3)H]mannose, whereas G(1) and G(2) contained lower levels of mannose. The three major viral glycoproteins gp88, gp65, and gp30 were isolated from sodium dodecyl sulfate-polyacrylamide gels, and their glycopeptide components were analyzed after Pronase digestion. The three size classes of glycopeptides were obtained from any of the three glycoproteins; however, the relative amounts of the three components varied among the glycoproteins. Host cell-derived components, which appear to be mucopolysaccharides and glycoproteins, were found associated with influenza C virions grown in CK cells. These components contained glycopeptides that were mainly of sizes similar to peak G(2) from influenza C virions. Previous studies have shown that influenza A/WSN virus grown in several cell types contained only two size classes of glycopeptides. Two size classes comparable to peaks G(2) and G(3) from influenza C virions were also observed in influenza A/WSN grown in CK cells. Thus the large G(1) glycopeptides appear to be characteristic of influenza C virions.

Animals↗
PubMed1979

Structure of the membrane protein of influenza virus. I. Isolation and characterization of cyanogen bromide cleavage products.

After cleavage of the membrane (M) protein of influenza A/WSN virus by using cyanogen bromide (CNBr), six peptide peaks representing approximate molecular weights of 6,000, 4,000, 2,200, 1,600, 1,200, and 1,000 were resolved by gel filtration on BioGel P6. Analysis by thin-layer chromatography indicates that the first, second, fourth, and fifth peaks contain single-peptide components, whereas the third and sixth peaks contain more than one peptide. By using Whatman CM52 ion-exchange chromatography in 5 M urea, four peptides were resolved from the third BioGel P6 peak. The amino acid composition of each of the purified peptides has been determined, and partial sequences were obtained for several peptides. Based on finding a blocked amino terminal residue, the 6,000-dalton fragment appears to contain the amino terminus of the M protein, whereas the carboxy terminal peptide was identified as a 2,000-dalton peptide.

Amino Acid Sequence↗
PubMed1979

Glycopeptides of murine leukemia viruses. I. Comparison of two ecotropic viruses.

The glycopeptides obtained by pronase digestion of two ecotropic strains of murine leukemia virus (MuLV) were compared by gel filtration. Four different glycopeptide size classes, designated G(1), G(2), G(3), and G(4), with molecular weights of approximately 5,100, 2,900, 2,200, and 1,500, respectively, were shown to be associated with Rauscher MuLV virions grown in JLS-V9 cells. Various sugar precursors, including glucosamine, galactose, fucose, and mannose were incorporated into G(1) and G(2), suggesting that these are complex (type I) glycopeptides. The two smaller glycopeptide size classes, G(3) and G(4), were shown to be mannoserich (type II) glycopeptides. G(4) was more sensitive to digestion with endo-beta-N-acetylglucosaminidase H than G(3), suggesting that the core of G(3) may contain fewer mannose residues. Glycopeptides of the same size class as G(1) and G(2) were associated with both Rauscher MuLV and AKR-MuLV grown in III6A (mouse embryo) cells. Previous studies have shown that gp52, a proteolytic cleavage product of gp70, possessed primarily G(1) glycopeptides and that gp52 was more highly sulfated than gp70. We observed that G(1) is approximately twofold more highly sulfated than G(2), explaining the observed difference in sulfation of gp52. The unusually large size of G(1) suggested that infection with MuLV may alter the host cell glycosylation pattern. To test this possibility, glycopeptides from Sindbis virions grown in uninfected and Rauscher MuLV-infected JLS-V9 cells were compared, and no differences were observed. G(1) was not detected in Sindbis virions, indicating that acquisition of G(1) depends on properties of the virus-coded polypeptide backbone of the gp70 molecule.

AKR murine leukemia virus↗
PubMed1979

Inhibition of influenza C virus replication by actinomycin D, alpha-amanitin, and UV irradiation.

Actinomycin D and alpha-amanitin caused similar reductions in the yields of influenza A/WSN and influenza C/JBH/1/66 viruses in a chicken kidney cell culture system. Irradiation of host cells with UV light before virus infection also produced a similar reduction in yields of the two viruses. The results indicate a close similarity between the replication processes of influenza C and other orthomyxoviruses.

Amanitins↗