Synthesis and processing of the three major envelope glycoproteins of Epstein-Barr virus.

In pulse-chase experiments, the three major Epstein-Barr virus envelope glycoproteins, gp350/300, gp250/200, and gp85, were shown to be synthesized from separate precursors of 190,000, 160,000, and 83,000 daltons, respectively. These three pulse-labeled species were chased into the mature forms of the glycoproteins between 1 and 3 h after transfer to nonradioactive medium. Digestion of precursor forms with endo-beta-N-acetylglucosaminidase H (endo H) yielded polypeptides of 160,000, 120,000, and 75,000 daltons. Comparison of these results with those from experiments with tunicamycin, which specifically blocks N-linked glycosylation, indicated that some other post-translational modification(s), probably O-linked glycosylation, contributes about 100,000 and 60,000 daltons of apparent molecular mass to gp350/300 and gp250/200, respectively. Experiments with endo H showed that mature gp350/300 and gp250/200 contain complex-type (endo H-resistant) N-linked glycosyl chains, whereas gp85 contains both high-mannose (endo H-sensitive)- and complex-type oligosaccharides. In contrast to the results obtained with the three envelope glycoproteins, no precursor forms of the two unglycosylated protein, p160 (the major Epstein-Barr virus capsid antigen) and p140 (an envelope protein), were detected. The partial proteolytic maps of gp350/300 and gp250/200 were quite similar, suggesting that polypeptide sequence homology could account for at least part of the observed serological cross-reactivity of the two proteins. Taken together, these results demonstrate that the polypeptide portions of gp350/300 and gp250/200 are closely related but not derived from a common precursor. Furthermore, the polypeptide portions comprise half or less of the apparent molecular weight of the mature glycoproteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Characterization of herpesvirus sylvilagus glycoproteins released into the culture medium of infected cells: antisera to gp13 and gp32 neutralize viral infectivity in vitro and identify antigens on plasma membranes of infected cells.

Polypeptides released into the culture medium of herpesvirus sylvilagus-infected cells were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracellular fluid from [35S]methionine- and [3H]glucosamine-labeled cell cultures. Virus-induced glycoproteins 31, 32, and 33 (molecular weights of 62,000, 59,000, and 54,000, respectively) were the most abundant species and appeared predominantly in the culture medium. This observation, together with the known cell-associated nature of herpesvirus sylvilagus, suggested that virus-induced glycoproteins 31, 32, and 33 were specifically released. Immunization of rabbits with virus-induced glycoproteins 13 (molecular weight of 130,000) and 32 resulted in the production of antibodies that neutralized viral infectivity in vitro. Both antiserum to gp13 and antiserum to gp32 immunoprecipitated gp13, gp26, gp33a, gp45, and virus-induced polypeptide 39 (molecular weights of 130,000, 77,000, 49,000, 27,000, and 36,000, respectively) from [35S]methionine-labeled cell extracts as well as virus-induced glycoproteins 31, 32, and 33 from the culture medium. In addition, membrane immunofluorescence assays indicate that an antigen(s) reactive with anti-gp13/32 serum was located on the plasma membrane of infected cells.     

Rous sarcoma virus p19 and gp35 can be chemically crosslinked to high molecular weight complexes. An insight into virus assembly

We have used the method of chemical crosslinking in order to determine the spatial interactions between components of Rous sarcoma virus. A high molecular weight complex formed by crosslinking has been isolated by ultracentrifugation on sucrose density gradients containing 0.1% (w/v) sodium dodecyl sulphate. This complex is composed of the two viral glycoproteins gp85 and gp35, the gag protein p19, and the viral RNA. Two types of bonding are important for the formation and stability of the complex: first, native disulphide bonds between gp85 and gp35 and between individual p19 molecules; and second, hetero-crosslinking between gp35 and p19 as well as homo-crosslinking between p19. Although viral RNA is quantitatively present in the complex, experiments with RNase treatment show that it is not essential for its formation or stability. A small amount of lipid is present in the complex and appears to be crosslinked to p19. In vitro-labelling of purified virus with the lipophilic photoactivatable reagent [125I] iodonaphthylazide resulted in the labelling of gp35 and p19/23. In vivo-labelling of virus with [3H]palmitate resulted in only gp35 becoming labelled. These results substantiate the membrane association of these proteins. The significance of the interactions in the high molecular weight complex for the stability of the virus and, by implication, the role which they may play in viral assembly are discussed.     

Proteolytic release of glycopeptides from glycoproteins transferred to nitrocellulose following gel electrophoresis

To determine whether glycopeptides could be released from glycoproteins bound to nitrocellulose, the glycoproteins of murine mammary tumor virus (MuMTV) were radiolabeled by the periodate oxidation/tritiated sodium borohydride reduction technique and separated by gel electrophoresis followed by diffusion transfer. Pronase digestion of nitrocellulose filter strips containing labeled glycoproteins (gp55 or gp34) revealed a rapid release of glycopeptides, i.e., approximately total release within 4 h. The released glycopeptides were similar in size, as determined by molecular sieving chromatography, to glycopeptides obtained by proteolytic digestion of MuMTV glycoproteins from dried gel strips (A. Zilberstein et al., 1980, Cell 21, 417-427) or in solution (M. J. Yagi et al., 1978, Virology 91, 291-304).     

Complex-type N-linked oligosaccharides of gp120 from human immunodeficiency virus type 1 contain sulfated N-acetylglucosamine.

The major envelope glycoproteins gp120 and gp41 of human immunodeficiency virus type 1, the causative agent for human AIDS, contain numerous N-linked oligosaccharides. We report here our discovery that N-acetylglucosamine residues within the complex-type N-linked oligosaccharides of both gp120 and its precursor, gp160, are sulfated. When human Molt-3 cells persistently infected with human T-cell leukemia virus IIIB were metabolically radiolabeled with 35SO4, gp160, gp120, and to some extent gp41 were radiolabeled. The 35SO4-labeled oligosaccharides were quantitatively released by N-glycanase treatment and were bound by immobilized Ricinus communis agglutinin I, a lectin that binds to terminal beta-galactosyl residues. The kinetics of release of sulfate upon acid hydrolysis from 35SO4-labeled gp120 indicate that sulfation occurs in a primary sulfate ester linkage. Methylation analysis of total glycopeptides from Molt-3 cells metabolically radiolabeled with [3H]glucosamine demonstrates that sulfation occurs at the C-6 position of N-acetylglucosamine. Fragmentation of the gp120-derived 35SO4-labeled glycopeptides by treatment with hydrazine and nitrous acid and subsequent reduction generated galactosyl-anhydromannitol-6-35SO4, which is the expected reaction product from GlcNAc-6-sulfate within a sulfated lactosamine moiety. Charge analysis of the [3H]galactose- and [3H]glucosamine-labeled glycopeptides from gp120 and gp160 indicates that approximately 14% of the complex-type N-linked oligosaccharides are sulfated.     

Subcellular localization of the env-related glycoproteins in Friend erythroleukemia cells.

A scheme was developed for the subcellular fractionation of murine erythroleukemia cells transformed by Friend leukemia virus. The subcellular localization of the env-related glycoproteins was determined by immune precipitation with antiserum against gp70, the envelope glycoprotein of the helper virus, followed by gel electrophoresis. In cells labeled for 2 h with [35S]methionine, the glycoprotein encoded by the defective spleen focus-forming virus, gp55SFFV, was found primarily in the nuclear fraction and in fractions containing dense cytoplasmic membranes such as endoplasmic reticulum. A similar distribution was noted for gp85env, the precursor to gp70. The concentration of viral glycoproteins in the nuclear fraction could not be accounted for by contamination with endoplasmic reticulum. In pulse-chase experiments, neither glycoprotein underwent major redistribution. However, labeled gp85env disappeared from intracellular membranes with a half-time of 30 min to 1 h, whereas labeled gp55SFFV was stable during a 2-h chase. In plasma membrane preparations with very low levels of contamination with endoplasmic reticulum, gp70 was the major viral env-related glycoprotein detected; a minor amount of gp55SFFV and no gp85env could be detected. The unexpected result of these experiments is the amount of viral glycoproteins found in the nuclear fraction. Presence of viral proteins in the nucleus could be relevant to the mechanism of viral leukemogenesis.     

Analysis of three late varicella-zoster virus proteins, a 125,000-molecular-weight protein and gp1 and gp3

Two monoclonal antibodies were prepared against varicella-zoster virus proteins. One of the monoclonal antibodies (10.2) reacted only with the nuclei of infected cells and immunoprecipitated one nonglycosylated late viral protein (125,000 molecular weight). The other monoclonal antibody (19.1) with neutralizing activity, reacted with membrane antigens of infected cells and with the varicella-zoster virus envelope and immunoprecipitated two late major viral glycoproteins (gp1 and gp3). Synthesis of the 125,000-molecular-weight protein, gp1, and gp3 began at 20 to 22 h postinfection, 2 h after the peak of viral DNA synthesis, and continued until 29 h postinfection, when the first progeny virus appeared in infected cells. Pulse-chase experiments showed that during pulse-labeling, only gp1 was detected, whereas during the chase period, gp1 as well as gp3 was detected in infected cells. Under nonreducing conditions, gp3 migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 130,000-molecular-weight protein as compared with the 62,000-molecular-weight species obtained when gels were resolved under reducing conditions. This finding indicates that gp3 is a dimer that is disulfide linked.     

Immunochemical characterization of gp115, a yeast glycoprotein modulated by the cell cycle

A cell cycle-modulated glycoprotein (gp115, 115 kDa, isoelectric point 4.8-5) of Saccharomyces cerevisiae has been purified by Concanavalin A-affinity chromatography, followed by preparative two-dimensional gel electrophoresis, from yeast membrane proteins solubilized in Triton X-100. Antisera have been generated against the electrophoretically purified protein. Their specificity has been established by immunoblot analysis and by comparison of the partial proteolytic map obtained for the immunoprecipitated 35S-labeled 115 kDa polypeptide with that of the in vivo [35S]methionine-labeled gp115 isolated from two-dimensional gels. In tunicamycin-treated cells the immunoblot analysis identifies an unglycosylated precursor (86-88 kDa) and in sec18 mutant cells at the restrictive temperature an intermediary precursor of about 100 kDa. Six to seven carbohydrate chains have been estimated to be present on the gp115 protein, accounting for an electrophoretic shift corresponding to about 27 to 29 kDa of its relative molecular mass. Affinity-purified antibodies against the unglycosylated precursor (86-88 kDa) of gp115 were prepared and used to localize gp115 by indirect immunofluorescence microscopy. The similarity between the pattern of fluorescence obtained with these antibodies and that obtained using anti-plasma membrane H+-ATPase antibodies suggests an association of gp115 with the plasma membrane.     

Molecular Characterisation and Structural Relationship of the Synapse-Enriched Glycoproteins gp65 and gp55

gp65 and gp55 are glycoprotein components of CNS synapses that are recognised by a single monoclonal antibody, SMgp65. This antibody has now been used to investigate the molecular properties of these two glycoproteins and the structural relationship between them. Both gp65 and gp55 occur in most brain regions as doublets of apparent molecular masses of 63 and 67 kDa, and 52 and 57 kDa, respectively. Striatal samples, however, are enriched in a novel gp65 isoform of 69 kDa. Removal of oligosaccharide residues from gp65 and gp55 with trifluoromethanesulphonic acid shows that gp65 and gp55 are composed of single polypeptide chains of 40 and 28 kDa, respectively. Removal of sialic acid residues with neuraminidase lowers the apparent molecular mass of both glycoproteins by 5-6 kDa. Triton X-114 phase partitioning and alkaline extraction of synaptic membranes indicate that both gp65 and gp55 are integral membrane glycoproteins. Treatment of synaptic membranes with phosphatidylinositol-specific phospholipase C does not solubilise either glycoprotein. One-dimensional peptide and epitope maps obtained by digestion of gp65 and gp55 with endoproteinase lys C or subtilisin are consistent with a close structural relationship between the two molecules. Tryptic digestion of samples enriched in gp65 and/or gp55 results in the formation of a novel immunoreactive 53-kDa species that is resistant to further trypsin degradation except in the presence of 0.1% (wt/vol) sodium dodecyl sulphate. Trypsin treatment of cultures of forebrain neurones in situ lowers the apparent molecular mass of gp65 to 53 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the N- and O-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni schistosomula

This report describes the structural analyses of the O- and N-linked oligosaccharides contained in glycoproteins synthesized by 48-hr-old Schistosoma mansoni schistosomula. Schistosomula were prepared by mechanical transformation of cercariae and were then incubated in media containing either [2-3H] mannose, [6-3H]glucosamine, or [6-3H]galactose to metabolically radiolabel the oligosaccharide moieties of newly synthesized glycoproteins. Analysis by SDS-polyacrylamide gel electrophoresis and fluorography demonstrated that many glycoproteins were metabolically radiolabeled with the radioactive mannose and glucosamine precursors, whereas few glycoproteins were labeled by the radioactive galactose precursor. Glycopeptide were prepared from the radiolabeled glycoproteins by digestion with pronase and fractionated by chromatography on columns of concanavalin A-Sepharose and pea lectin-agarose. The structures of the oligosaccharide chains in the glycopeptides were analyzed by a variety of techniques. The major O-linked sugars were not bound by concanavalin A-Sepharose and consisted of simple O-linked monosaccharides that were terminal O-linked N-acetylgalactosamine, the minor type, and terminal O-linked N-acetylglucosamine, the major type. The N-linked oligosaccharides were found to consist of high mannose- and complex-type chains. The high mannose-type N-linked chains, which were bound with high affinity by concanavalin A-Sepharose, ranged in size from Man6GlcNAc2 to Man9GlcNAc2. The complex-type chains contained mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. No sialic acid was present in any metabolically radiolabeled glycoproteins from schistosomula.     

Detection of glycoproteins in the Acanthamoeba plasma membrane

In the present study we have shown that glycoproteins are present in the plasma membrane of Acanthamoeba castellanii by utilizing different radioactive labeling techniques. Plasma membrane proteins in the amoeba were iodinated by 125I-lactoperoxidase labeling and the solubilized radiolabeled glycoproteins were separated by lectin-Sepharose affinity chromatography followed by polyacrylamide gel electrophoresis. The periodate/NaB3H4 and galactose oxidase/NaB3H4 labeling techniques were used for labeling of surface carbohydrates in the amoeba. Several surface-labeled glycoproteins were observed in addition to a diffusely labeled region with Mr of 55,000-75,000 seen on electrophoresis, which could represent glycolipids. The presence of glycoproteins in the plasma membrane of Acanthamoeba castellanii was confirmed by metabolic labeling with [35S]methionine followed by lectin-Sepharose affinity chromatography and polyacrylamide gel electrophoresis.     

Structural proteins of Herpesvirus saimiri.

Herpesvirus saimiri particles were purified from productively infected owl monkey kidney cell cultures, and the virion polypeptides were analyzed by polyacrylamide gel electrophoresis. A total of 21 predominant proteins were found in lysates of H. saimiri 11 particles by Coomassie blue staining or by [35S]methionine labeling and autoradiography; all proteins were between 160,000 and 12,000 daltons in size. They are most probably virion constituents, as most of them were precipitated by immune sera, and no dominant proteins of equivalent sizes were found in mock-infected cultures. Four glycoproteins (gp 155/160, gp 128, gp 84/90, gp 55) and three polypeptides that appeared not to be glycosylated (p71, p35, p28) were assigned to the envelope or matrix of virions, whereas at least four phosphoproteins (pp132, pp118, pp55, pp13) and ten polypeptides without apparent secondary modification (p155/160, p106, p96, p67, p53, p36, p32, p15, p14, p12) were found in the nucleocapsid fraction. Analysis of virion proteins from different H. saimiri strains did not reveal appreciable differences in the migration behavior of most polypeptides, including all glycoproteins; however, determination of a strain-specific size pattern was possible for three of four phosphoproteins. The overall similarity in protein architecture of H. saimiri strains obviously does not reflect the variability in biology, such as oncogenic properties. In comparison, DNA sequence divergences appear to remain a better taxonomic criterion for strain distinction.     

Envelope polypeptides of Friend leukemia virus: purification and structural analysis.

Roughly 10% of surface glycoproteins in the envelope of mature Friend murine leukemia virus are coupled to membrane polypeptides by disulfide bridges. The remaining 90% of these glycoproteins are associated noncovalently. However, they could also be linked to membrane polypeptides by the treatment of purified Friend murine leukemia virus with 2,2'dithiobis(m-nitropyridine). These amphiphilic heterodimer polypeptides, gp84/86, were recovered almost quantitatively in the form of aggregates, termed rosettes, when prepared by solubilization of the viral membrane with Triton X-100 and subsequent velocity sedimentation. gp69/71 and p12(E)/15(E) were purified from these protein micelles after reduction of the disulfide bonds by gel chromatography. Electron micrographs of rosettes, as well as of purified p12(E)/15(E), showed structures different from native viral knobs. Isolated gp84/86 could be reassociated and then displayed more similarity to these viral surface projections. As shown by peptide mapping, the primary structures of the glycoproteins gp69/71 are highly related as are those of the membrane polypeptides p12(E) and p15(E). Furthermore, it was shown by two-dimensional polyacrylamide gel electrophoresis and re-electrophoresis of purified gp84/86 that the larger component, gp86, was composed of gp71 associated with p15(E) and p12(E), whereas the smaller component, gp84, was formed by gp69 bound only to p12(E).     

Structural protein markers in the avian oncoviruses.

The proteins of purified avian oncoviruses were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and isoelectric focusing. Certain members of the avian leukosis-sarcoma viruses (ALSV) had group-specific antigens with altered electrophoretic properties. (i) The p27 protein of Rous-associated virus 0 (RAV-0) had a lower electrophoretic mobility in SDS gels and a lower isoelectric point than the p27 of other ALSV. (ii) The p19 proteins of RAV-1, RAV-2, and the Bryan high-titer strain of Rous sarcoma virus had higher mobilities in SDS gels than did the corresponding protein of other viruses. This altered electrophoretic mobility was correlated with specific differences in the tryptic peptides of radioiodinated p19s. (iii) The p15 protein of RAV-7 had a lower mobility in SDS gels than did the p15 of other ALSV. These markers were used in a study of the structural proteins of subgroup E RAV-60 produced after infection of chicken embryo cells by exogenous ALSV. Although exogenous group-specific protein markers could often be identified in the subgroup E isolates, one RAV-60 had a p27 that comigrated with the p27 of RAV-0. The p19s of two other RAV-60 isolates had electrophoretic properties that were different than those of p19s from either RAV-0 or the exogenous viruses. These results support the hypothesis that RAV-60 is generated by recombination between endogenous and exogenous oncoviruses and indicate that at least the p27 encoded by RAV-0 is closely related to a protein specified by endogenous viral information in chicken cells.     

Physical and chemical properties of the major envelope glycoprotein gp85 from avian myeloblastosis virus

The major envelope glycoprotein gp85 of avian myeloblastosis virus, observed by electron microscopy as nearly spherical knobs projecting from the virus surface, was purified to homogeneity by gel filtration in 6 M guanidinium chloride followed by ion-exchange chromatography. The purified glycoprotein has a molecular weight of 80 000 from sedimentation equilibrium analysis. Glycoprotein gp85 contains approx. 45% carbohydrate including 25% N-acetylglucosamine, while the remaining weight consists of a polypeptide chain of approx. 45 000 daltons. Based on the oligosaccharide chain molecular weight data of Lai and Duesberg (Lai, M.M.C. and Duesberg, P.H. (1972) Virology 50, 359-372), the carbohydrate is calculated to be distributed between seven to nine oligosaccharide side chains. No self-association of gp85 was observed up to 2.0 mg/ml in dilute salt solution. The hydrodynamic properties of gp85 in dilute salt solution indicate a highly elongated molecule with an axial ratio of 7. One structural model which reconciles the hydrodynamic properties of gp85 with the nearly spherical architecture observed by electron microscopy requires the organization of the polypeptide chain and approx. 50% of the carbohydrate into a globular form. The remaining covalently linked oligosaccharides would by necessity extend outwardly from the globular structure as randomly oriented chains.     

Extracellular cleavage of the glycoprotein precursor of Rous sarcoma virus.

The kinetics of cleavage of pr92gp, the precursor of the two glycoproteins of Rous sarcoma virus gp85 and gp35, were followed. Viral glycoproteins were detected by immunoprecipitation with anti-gp85 and anti-gp35 serum. It could be shown in pulse-chase experiments that little or no intracellular cleavage of the precursor took place during the time in which the majority of newly synthesized viral glycoprotein was exported from the cells. Soon after its synthesis, however, pr92gp underwent some modification that made it migrate slightly faster on sodium dodecyl sulfate-polyacrylamide gels. Under steady state conditions the precursor was shown to be the predominant form of intracellular viral glycoprotein. Virus which was harvested every 2 min from infected cells prelabeled for 90 min with [3H]mannose contained mostly uncleaved and only a little mature glycoprotein. By incubation of this freshly released virus in serum-free buffer, the majority of the glycoprotein precursor could be cleaved into mature gp85 and gp35. Virus harvested every 10 min contained only mature glycoproteins.     

The p185(neu)-containing glycoprotein complex of a microfilament-associated signal transduction particle. Purification, reconstitution, and molecular associations with p58(gag) and actin.

Microfilaments associate with the microvillar membrane of 13762 ascites mammary adenocarcinoma cells via a large transmembrane complex (TMC) comprising the major glycoproteins TMC-gp120, -110, -80, -65, and -55, the receptor kinase p185(neu), and the cytoplasmic proteins actin and p58(gag), linking the receptor with microfilaments in a signal transduction particle. Immunoblot screening with polyclonal antisera to TMC glycoproteins showed selective epithelial expression in normal rat tissues and epithelially derived tumor cells. The TMC glycoproteins were isolated by solubilization of microfilament core preparations in SDS, dilution, and separation on a concanavalin A-agarose affinity column. The large p185(neu)-containing complex was reconstituted from the column eluate after displacement of SDS with nonionic detergent, demonstrated by gel filtration and co-immunoprecipitation of the glycoproteins with anti-gp55 or anti-p185(neu). Exhaustive biotinylation of the glycoproteins gave a stoichiometry of gp120:gp110:gp80:gp65:gp55 of approximately 1:1:1:0.5:1. Overlay blots with biotinylated actin and in vitro translated, [(35)S]methionine-labeled p58(gag), respectively, showed specific interactions of actin with gp55 and gp120 and of p58(gag) with gp65 and gp55. These results provide evidence for a specific complex of microfilament-associated glycoproteins containing p185(neu) and p58(gag) and suggest a role for the complex in signal transduction scaffolding.