Sulfated components of enveloped viruses.

The glycoproteins of several enveloped viruses, grown in a variety of cell types, are labeled with 35SO4(-2), whereas the nonglycosylated proteins are not. This was shown for the HN and F glycoproteins of SV5 and Sendai virus, the E1 and E2 glycoproteins of Sindbis virus, and for the major glycoprotein, gp69, as well as for a minor glycoprotein, gp52, of Rauscher leukemia virus. The minor glycoprotein of Rauscher leukemia virus is more highly sulfated, with a ratio of 35SO4- [3H]glucosamine about threefold greater than that of gp69. The G protein of vesicular stomatitis virus was labeled when virions were grown in the MDBK line of bovine kidney cells, although no significant incorporation of 35SO4(-2) into this protein was observed in virions grown in BHK21-F line of baby hamster kidney cells. In addition to the viral glycoproteins, sulfate was also incorporated into a heterogenous component with an electrophoretic mobility lower than that of any labeled with 35SO4(-2) and [3H]leucine, this component had a much greater 35S-3H ratio than any of the viral polypeptides and thus could not represent aggregated viral proteins. This material is believed to be a cell-derived mucopolysaccharide and can be removed from virions by treatment with hyaluronidase without affecting the amount of sulfate present on the glycoproteins.     

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 [³H]glucosamine-labeled glycopeptides were larger and more heterogeneous than those of influenza A/WSN virions; three major size classes (G₁, G₂, and G₃) were resolved. Treatment with Vibrio cholerae neuraminidase caused a decrease in size of G₁ and G₂, along with release of about 16% of the ³H label. The released sugar components were identified as N-acetylneuraminic acid by thin-layer chromatography. Peak G₃ was highly labeled with [³H]mannose, whereas G₁ and G₂ 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 G₂ 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 G₂ and G₃ from influenza C virions were also observed in influenza A/WSN grown in CK cells. Thus the large G₁ glycopeptides appear to be characteristic of influenza C virions.     

Origin of the minor glycoproteins of murine leukemia viruses.

Polyacrylamide gel electrophoretic analysis and immunoprecipitation were used to study glycoproteins from purified Rauscher murine leukemia virus (R-MuLV) and from AKR thymic lymphoblastoid cell membranes. In addition to gp70, a minor glycoprotein of approximately 52,000 daltons (gp52) was demonstrated in purified R-MuLV preparations, which was antigenically related to gp70. Analysis of R-MuLV glycopeptides obtained after exhaustive Pronase digestion showed that gp70 has at least two different glycopeptide size classes with molecular weights of 5,100 and 2,900, respectively. gp52, however, contained only a single glycopeptide size class of approximately 5,100 daltons, indicating that the two glycoproteins contain distinct carbohydrate components. Trypsin treatment of R-MuLV converted gp70 into a product with a molecular mass of approximately 52,000 daltons as well as a 45,000-dalton minor product, with little effect on virus infectivity. Similarly, trypsin treatment of 125I-labeled glycoproteins derived from AKR mouse lymphoblastoid cell membranes generated fragments antigenically related to gp70 and similar in size to those obtained by trypsin treatment of R-MuLV. In both cases, the appearance of cleavage products was accompanied by a decrease in gp70 during trypsin treatment. The occurrence of glycosylated components antigenically related to gp70 in AKR membrane glycoprotein preparations and in purified R-MuLV preparations which were similar to those generated by trypsin treatment supports the concept that these minor components arise from proteolytic cleavage of gp70.     

Characterization of Glycos-aminoglycans Associated with Rauscher Murine Leukemia Virions

Host-derived sulfated components that copurify and are physically associated with the envelope of Rauscher murine leukemia virions grown in JLS-V9 cells were characterized by digestion with chondroitinase ABC and chondroitinase AC II, as well as nitrous acid degradation. A dermatan-sulfate-chondroitin-sulfate copolymer and heparin or heparan sulfate were shown to be associated with the virions. Competitive binding studies indicated a specificity of the virions for association with heparan sulfate. The physiological importance of the association is discussed.     

Carbohydrate Structure of Sindbis Virus Glycoprotein E2 from Virus Grown in Hamster and Chicken Cells

Sindbis virus was used as a probe to examine glycosylation processes in two different species of cultured cells. Parallel studies were carried out analyzing the carbohydrate added to Sindbis glycoprotein E2 when the virus was grown in chicken embryo cells and BHK cells. The Pronase glycopeptides of Sindbis glycoprotein E2 were purified by a combination of ion-exchange and gel filtration chromatography. Four glycopeptides were resolved, ranging in molecular weight from 1,800 to 2,700. Structures are proposed for each of the four glycopeptides, based on data obtained by quantitative composition analyses, methylation analyses, and degradation of the glycopeptides using purified exo- and endoglycosidases. The largest three glycopeptides (S1, S2, and S3) have similar structures but differ in the extent of sialylation. All three contain N-acetylglucosamine, mannose, galactose, and fucose, in a structure similar to oligosaccharides found on other glycoproteins. Glycopeptide S1 has two residues of sialic acid, whereas glycopeptides S2 and S3 contain 1 and 0 residues of sialic acid, respectively. The smallest glycopeptide, S4, contains only N-acetyglucosamine and mannose, and is also similar to mannose-rich oligosaccharides found on other glycoproteins. Each of the complex glycopeptides (S1, S2, or S3) from virus grown in BHK cells is indistinguishable from the corresponding glycopeptides derived from virus grown in chicken cells. Glycopeptide S4 is also very similar in size, composition, and sugar linkages from virus derived from the two hosts. These results suggest that chicken cells and BHK cells have similar glycosylation mechanisms and glycosylate Sindbis glycoprotein E2 in nearly identical ways.     

Natural immunity in mice to the envelope glycoprotein of endogenous ecotropic type C viruses: neutralization of virus infectivity.

The ability of naturally immune mouse sera to neutralize ecotropic AKR murine leukemia virus (MuLV) was examined by using unfrozen virus preparations harvested for 1 h. In this assay several mouse sera significantly and consistently neutralized MuLV infectivity. The ability of these sera to neutralize was correlated with the presence of antibodies against MuLV detectable in a radioimmune precipitation assay using radioactively labeled intact virions. This neutralization was specific, in that either N- or B-tropic viruses, but not Friend MuLV, were neutralized. In addition, neutralization could be abrogated with purified AKR MuLV gp71 at concentrations that do not interfere with virus infectivity but could not be abrogated with Rauscher MuLV gp71. Neutralizing activity could be removed by absorption with intact AKR MuLV, but not by absorption with Friend MuLV, a BALB/c xenotropic virus, or with NZB xenotropic virus. All the neutralizing activity of (B6C3)F1 mouse sera was associated with the immunoglobulin G fraction.     

The errors in assembly of MuLV in interferon treated cells

Interferon treatment of JLSV-6 cells chronically infected with Rauscher MuLV leads to the formation of noninfectious particles (interferon virions) containing the structural proteins of env and gag genes as well as additional viral polypeptides. In the control virions the major glycoprotein detected is gp71, interferon virions contain in addition to gp71 and 85k dalton (gp85) glucosamine-containing, fucose-deficient glycoprotein which is recognized by antiserum to MuLV but not by the gp71 antiserum. The surface iodination of the intact virions indicates that both gp71 and gp85 are the major components of the external virions envelope. However, unlike the control virions in which gp71 associates with p15E (gp90), the gp71-p15E complex was not detected in interferon virions. The analysis of the iodinated proteins of the disrupted interferon virions revealed the presence of 85k and 65k dalton polypeptides preciptable with antiserum against MuLV, which are not present in the control virions. The difference in the polypeptide pattern of virions produced in the presence of interferon does not seem to be a consequence of the slowdown in the synthesis of viral proteins or their processing in the interferon-treated cells. Both the structural proteins of env and gag genes seem to be synthesized and processed at a comparable rate in the interferon-treated and -untreated cells. These results indicate an alteration of virus assembly in the presence of interferon.     

Identification of an FMR cell surface antigen associated with murine leukemia virus-infected cells.

FMR antigens are found on the surface of cells infected with Friend, Moloney, and Rauscher murine leukemia viruses (MuLV). These antigens are serologically distinct from the G cell surface antigens that are found on cells infected with endogenous MuLV (AKR and Gross virus). Cell surface antigens of both virus groups are immunogenic in mice, and immunization with appropriate virus-infected cells leads to the production of cytotoxic antisera. The cytotoxic activity of FMR antisera can be absorbed by disrupted preparations of Rauscher MuLV, but not by AKR MuLV. FMR antisera precipitate the viral envelope proteins gp70, pl5(E), and p12(E) from detergent-disrupted preparations of [3H]leucine-labeled MuLV. The reaction of these antisera with p15(E) and p12(E) proteins is directed against group-specific antigens and can be absorbed with AKR MuLV; in contrast, the reaction of these antisera with gp70 is directed against type-specific antigens and is absorbed only by viruses of the FMR group. In immune precipitation assays with detergent-disrupted 125I surface-labeled cells, FMR antisera react only with type-specific antigens of the viral envelpe protein. On the basis of these findings we conclude that the FMR cell surface antigen is a determinant on the MuLV env gene product.     

Identification of a mouse gene required for binding of Rauscher MuLV envelope gp70.

Mouse chromosome segregating somatic cell hybrids were established between a mouse thymic leukemai cell line (GRSL) and Chinese hamster E36 cells. The GRSL cells specifically bound purified Rauscher leukemia virus gp70 while the E36 cells exhibited no binding. The hybrids selectively bound Ruascher gp70 depending on the presence of a mouse cellular gene for the ecotropic murine luekemia gp70 receptor. A syntenic relationship was observed between the DIP-3 chromosome marker (on chromosome 5) and the gp70 receptor in primary clones and subclones of these hybrids; this was confirmed by chromosome analysis. The involvement of H-2 in the binding of Rauscher MuLV gp70 could be ruled out, because discordancies of the receptor presence and H-2 absence as well as of the receptor absence and H-2 presence type could be observed. Our results indicate that the Rec-1 (replication ecotropic MuLV) gene of Gazdar et al. (4) may well be the receptor gene for the ecotropic murine leukemia virus.     

Membrane association and defective transport of spleen focus-forming virus glycoproteins

The gp52 glycoprotein of the spleen focus-forming virus found in the Friend and Rauscher complexes of murine leukemia viruses (MuLV) has been previously identified as a recombinant molecule involving substitutions and deletions of the MuLV env gene. Unlike the MuLV structural glycoproteins, gp52 is defective in its transport to the cell surface. We have studied aspects of the intracellular transport and membrane association of gp52 to investigate the possible mechanisms underlying the defective transport process. It was found that a panel of monoclonal antibodies to different epitopes of p 15E, as well as an antiserum to a synthetic peptide corresponding to the carboxy terminus of MuLV envelope precursors, failed to react with gp52. Despite the possible absence of membrane-anchoring regions of MuLV envelope proteins known to reside on p 15E, gp52 was not found to be secreted into the culture fluids. Detergent extraction studies indicated that gp52 is associated with the membranes and not the contents of microsomal vesicles in speen focus-forming virus-infected cells. gp65, the processed form of gp52, could be labeled with [3H]palmitic acid, suggesting a membrane association. To determine whether a spontaneous denaturation occurs leading to aggregation and defective transport of gp52, we studied the surface expression of gp52 in cells grown at different temperatures, as well as the solubility of gp52 in low concentrations of Triton X-100. No evidence of aggregation or of a temperature-dependent difference in transport was obtained. gp52 appears to be a monotopic integral membrane protein, unlike MuLV envelope proteins which are bitopic integral membrane proteins; proteolytic digestion of intact microsomal vesicles did not reveal a detectable cytoplasmic tail under conditions where this could be demonstrated on MuLV envelope precursors. We suggest that a loss of putative signals involved in mediating intracellular transport is a likely cause for the defective transport of the spleen focus-forming virus glycoproteins.     

Biological, Chemical, and Immunological Studies of Rauscher Ecotropic and Mink Cell Focus-Forming Viruses from JLS-V9 Cells

Two murine leukemia viruses were isolated from JLS-V9 cells which had been infected with Rauscher plasma virus. One virus was XC positive and failed to grow on mink or cat cells and thus was an ecotropic virus. The other virus formed cytopathic foci on mink cells, was XC negative, and fell into the mink cell focus-forming (MCF) viral interference group and was thus an MCF virus. The glycoproteins of the two viruses could be distinguished immunologically, by peptide mapping, and by size in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The MCF virus produced gp69, and the ecotropic virus produced gp71, explaining the origin of the heterogeneous glycoprotein (gp69 and gp71) of Rauscher leukemia virus. Amino-terminal sequences of gp69 and gp71 were determined. The MCF sequence was distinct from the ecotropic sequence, but retained partial homology to it. The data show that the glycoproteins are encoded by related yet distinct genes. The protein structural data support the proposal that MCF virus gp70 molecules have nonecotropic sequences at the amino terminus, with ecotropic sequences occurring at the 3' end of the gene. The Rauscher MCF virus glycoprotein lacks a glycosylation site found at position 12 of the ecotropic sequence.     

Purification and serological characterization of the major envelope glycoprotein from AKR murine leukemia virus and its reactivity with autogenous immune sera from mice.

The major envelope glycoprotein (gp71) from AKR murine leukemia virus (MuLV) was purified and its serological reactivity with heterologous and autogenous immune mouse sera was examined. Homologous and interspecies competition radioimmunoassays using antisera to Rauscher-MulV gp69/71 or Friend-MuLV gp71 or antisera to feline leukemia virus to precipitate 125I-labeled gp71 from various MuLV showed that distinct differences exist between Rauscher- or Friend-MuLV and AKR-MuLV glycoproteins. Characteristically the AKR-MuLV gp71, in contrast to FLV or RLV gp71, does not compete fully in homologous or interspecies radioimmunoassays with iodinated Friend of Rauscher glycoproteins. Purified 125I-labeled AKR-MuLV gp71, in contrast to the Rauscher- or Friend-MuLV glycoproteins, reacts with normal (autogenous immune) mouse sera in direct radioimmune precipitation assays. Competition experiments further demonstrate that this is a predominant immunological reactivity of normal mouse sera which had previously been detected by radioimmune precipitation assay against intact virions.     

Comparison of Membrane Protein Glycopeptides of Sindbis Virus and Vesicular Stomatitis Virus

A comparison has been made of the membrane glycoproteins and glycopeptides from two enveloped viruses, Sindbis virus and vesicular stomatitis virus (VSV). Glycopeptides isolated from Sindbis virus and VSV grown in the same host appear to differ principally in the number of sialic acid residues per glycopeptide; when sialic acid is removed by mild acid treatment, the glycopeptides of the two viral proteins are indistinguishable by exclusion chromatography. Preliminary evidence argues that the carbohydrate moiety covalently bound to different virus-specified membrane proteins may be specified principally by the host.     

Structure of the high mannose oligosaccharides of a human IgM myeloma protein. I. The major oligosaccharides of the two high mannose glycopeptides.

The structures of the predominant high mannose oligosaccharides present in a human IgM myeloma protein (Patient Wa) have been determined. The IgM glycopeptides, produced by pronase digestion, were fractionated on DEAE-cellulonalysis shows that glycopeptide I contains Asn, Pro, Ala, Thr, and His and glycopeptide II contains Asn, Val, and Ser, which are the same amino acids found in the sequences around Asn 402 and Asn 563 respectively, to which high mannose oligosaccharides are attached in IgM (Patient Ou) (Putnman, F.W., Florent, G., Paul, C., Shinoda, T., and Shimizu, A. (1973) Science 182, 287-290). The high mannose glycopeptides in IgM (Wa) exhibit heterogeneity in the oligosaccharide portion. Structural analysis of the major oligosaccharides indicates that the simplest structure is: (see article of journal). The larger oligosaccharides present have additional mannose residues linked alpha 1 yields 2 to terminal mannose residues in the above structure. Glycopeptide I contains primarily Man5 and Man6 species, while glycopeptide II contains Man6 and Man8 species. The two Man6 oligosaccharides have different branching patterns.     

Quantitative determination of sulfated glycopeptide by two-dimensional electrophoresis on cellulose acetate membrane

Quantitative determination of the sulfated glycoproteins present in tissue and secretion fluid was performed. After digestion of the specimen with pronase in order to convert glycoproteins to glycopeptides, the sulfated glycopeptides were separated from a mixture of acidic glycans (glycosaminoglycans, sialoglycopeptides and sulfated glycopeptides) by two-dimensional electrophoresis on cellulose acetate membrane [(1986) J. Biochem. Biophys. Methods 12, 239-246]. After staining with alcian blue, the spot of sulfated glycopeptide on the cellulose acetate membrane was cut out, and then only the dye bound to the sulfated glycopeptide was extracted with a 5% cetylpyridinium chloride solution at 100 degrees C for 15 min. The extract was then measured by absorbance at 615 nm using an authentic sulfated glycopeptide as a standard. This method facilitated the determination of sulfated glycopeptides, which were separated from other acidic glycans, within the range 0-25 micrograms.     

Monosaccharide Sequence of Protein-Bound Glycans of Uukuniemi Virus

Uukuniemi virus, a member of the Bunyaviridae family, was grown in BHK-21 cells in the presence of [³H]mannose. The purified virions were disrupted with sodium dodecyl sulfate and digested with pronase. The [³H]mannose-labeled glycopeptides of the mixture of the two envelope glycoproteins G1 and G2 were characterized by degrading the glycans with specific exo-and endoglycosidases, by chemical methods, and by analyzing the products with lectin affinity and gel chromatography. The glycopeptides of Uukuniemi virus fell into three categories: complex, high-mannose type, and intermediate. The complex glycopeptides probably contained mainly two NeuNAc-Gal-GlcNAc branches attached to a core (Man)₃(GlcNAc)₂ peptide. The high-mannose-type glycans were estimated to contain at least five mannose units attached to two N-acetylglucosamine residues. Both glycan species appeared to be similar to the asparagine-linked oligosaccharides found in many soluble and membrane glycoproteins. The results suggested that the intermediate glycopeptides contained a mannosyl core. In about half of the molecules, one branch appeared to be terminated in mannose, and one appeared to be terminated in N-acetylglucosamine. Such glycans are a novel finding in viral membrane proteins. They may represent intermediate species in the biosynthetic pathway from high-mannose-type to complex glycans. Their accumulation could be connected with the site of maturation of the members of the Bunyaviridae family. Electron microscopic data suggest that the virions bud into smooth-surfaced cisternae in the Golgi region. The relative amounts of [³H]mannose in the complex, high-mannose-type, and intermediate glycans were 25, 62, and 13%, respectively, which corresponded to the approximate relative number of oligosaccharide chains of 2:2.8:1, respectively, in the roughly equimolar mixture of G1 and G2. Endoglycosidase H digestion of isolated [³⁵S]methionine-labeled G1 and G2 proteins suggested that most of the complex and intermediate chains were attached to G1 and that most of the high-mannose-type chains were attached to G2.     

A method for the identification of sulfated glycopeptide by two-dimensional electrophoresis on cellulose acetate membrane

Two-dimensional electrophoresis on cellulose acetate membrane permits the clean separation of sulfated glycopeptide in a mixture of acidic glycans (glycosaminoglycans and acidic glycopeptides). Two systems were used. In system 1, 0.1 M pyridine-0.47 M formic acid buffer (pH 3.0) was used in the first and 0.1 M barium acetate (pH 8.0) in the second dimension. In system 2, 0.1 M pyridine-0.47 M formic acid buffer (pH 3.0) was used in the first and 0.1 M HCl in the second dimension. All of the acidic glycans on electrophoretogram were stained with alcian blue in 70% ethanol. On the other hand, sulfated glycans alone were made visible with alcian blue in 0.1 M HCl. Alcian blue in 70% ethanol or 0.1 M HCl, when combined with periodic acid-Schiff's reagent identified sulfated glycopeptides on cellulose acetate membrane.     

Characterization of a progressive neurodegenerative disease induced by a temperature-sensitive Moloney murine leukemia virus infection.

A progressive neurodegenerative disease occurred following infection of mice with a temperature-sensitive (ts) isolate of Moloney (Mo) murine leukemia virus (MuLV), ts Mo BA-1 MuLV. This NB-tropic ecotropic MuLV, which was ts for a late function, induced a syndrome of tremor, weakness of the hind limbs, and spasticity following infection of several strains of laboratory neonatal mice, including NFS, C3H/He, CBA, SJL, and BALB/c. The latent period of 8 to 16 weeks was considerably longer than that observed for the acute paralytic diseases observed following neonatal infection with other ts Mo-MuLV, rat-passaged Friend MuLV, and some wild mouse ecotropic MuLVs. Spongiform pathology without inflammation and degeneration of neurons devoid of budding virions occurred in the cerebellar grey matter, brain stem, and upper spinal cord; but lower spinal cord anterior horn cells were less obviously affected than in other MuLV-associated neuroparalytic syndromes. ts Mo BA-1 MuLV differed from other ts Mo-MuLV mutants that are capable of inducing a neuroparalytic syndrome in that while infected nervous system tissue contained high levels of MuLV p30 and gp70, no evidence of precursor accumulation or abnormal processing of MuLV p30 or gp70 could be demonstrated. The localization of virus within the nervous system suggests that direct neuronal infection may not be the etiologic mechanism in this MuLV-induced neurodegenerative disease.     

Isolation,Characterization and Synthesis of Pimprinine,Pimprinethine and Pimprinaphine,Metabolites of Streptoverticillium olivoreticuli

The sulfated glycopeptides in ovomucin, chalazae and yolk membrane were isolated from the proteolytic digests by gel filtration on a Bio-Gel P-100 column and DEAE-Sephadex A-25 column chromatography. These sulfated glycopeptides contained N-acetylhexosamine (23.3-26.8%), hexose (23.6-24.4%), sialic acid (11.2-18.0%), sulfate (5-12.1%) and peptide (17.5-18.1%). The sulfate contents of glycopeptides in chalazae and yolk membrane were much higher than those in ovomucin, about two times in a molar ratio to hexosamine. The sedimentation patterns of each sulfated glycopeptide were single and the sedimentation constants were around 3 S, suggesting that these sulfated glycopeptides were macromolecular components. Thus, the presence of highly sulfated glycoproteins was confirmed in chalazae and yolk membrane, which were different from those in ovomucin.     

Sindbis envelope proteins as endogenous acceptors in reactions of guanosine diphosphate-[14C]Mannose with preparations of infected chicken embryo fibroblasts.

Preparations of Sindbis-infected chicken embryo fibroblasts incubated with GDP-[14C]mannose and UDP-N-acetylglucosamine catalyze the glycosylation of endogenous phospholipids and membrane-associated proteins. The proteins are identified as the viral envelope proteins by precipitation with anti-Sindbis antiserum, by comparison with authentic virion glycoproteins on sodium dodecyl sulfate-poly-acrylamide gel electrophoresis, and by comparison of the glycopeptides of the membrane-associated glycoproteins with the glycopeptides from Sindbis virions on gel filtration chromatography. Our results indicate that glycophospholipid participates in the mannosylation of the viral proteins since an inhibitor of oligosaccharide-lipid synthesis also inhibits the labeling of the glycoproteins.