A novel glycosylation phenotype expressed by Lec23, a Chinese hamster ovary mutant deficient in alpha-glucosidase I.

Lec23 Chinese hamster ovary (CHO) cells have been shown to possess a unique lectin resistance phenotype and genotype compared with previously isolated CHO glycosylation mutants (Stanley, P., Sallustio, S., Krag, S. S., and Dunn, B. (1990) Somatic Cell Mol. Genet. 16, 211-223). In this paper, a biochemical basis for the lec23 mutation is identified. The carbohydrates associated with the G glycoprotein of vesicular stomatitis virus (VSV) grown in Lec23 cells (Lec23/VSV) were found to possess predominantly oligomannosyl carbohydrates that bound strongly to concanavalin A-Sepharose, eluted 3 sugar eq beyond a Man9GlcNAc marker oligosaccharide on ion suppression high pressure liquid chromatography, and were susceptible to digestion with jack bean alpha-mannosidase. Monosaccharide analyses revealed that the oligomannosyl carbohydrates contained glucose, indicating a defect in alpha-glucosidase activity. This was confirmed by further structural characterization of the Lec23/VSV oligomannosyl carbohydrates using purified rat mammary gland alpha-glucosidase I, jack bean alpha-mannosidase, and 1H NMR spectroscopy at 500 MHz. [3H]Glucose-labeled Glc3Man9GlcNAc was prepared from CHO/VSV labeled with [3H]galactose in the presence of the processing inhibitors castanospermine and deoxymannojirimycin. Subsequently, [3H]Glc2Man9GlcNAc was prepared by purified alpha-glucosidase I digestion of [3H]Glc3Man9GlcNAc. When these oligosaccharides were used as alpha-glucosidase substrates it was revealed that Lec23 cells are specifically defective in alpha-glucosidase I, a deficiency not previously identified among mammalian cell glycosylation mutants.     

Monensin-resistant mouse Balb/3T3 cell mutant with aberrant penetration of vesicular stomatitis virus

A mutant (MO-5) resistant to monensin (an ionophoric antibiotic) derived from the mouse Balb/3T3 cell line, was a poor host for vesicular stomatitis virus (VSV) or semliki forest virus (SFV) multiplication. The yield of VSV particles in MO-5 is one 100-fold reduced as is VSV-dependent RNA synthesis. In contrast to a pH-remedial mutant, the abortive production of infectious VSV particles in MO-5 cells was not restored by low pH treatment. The pH values in the endosome and the lysosome of MO-5 cells were 5.2 and 5.4, respectively, values that were comparable to the pH value in Balb/3T3 cells. Assays with [3H]uridine-labeled VSV indicated similar binding of VSV in MO-5: percoll gradient centrifugation analysis of [35S]methionine-labeled VSV-infected Balb/3T3 showed accumulation of VSV in the lysosome fraction 20 min after VSV infection, whereas VSV can be found mainly in endosome/Golgi fraction of MO-5 cells after 40 to 60 min on the percoll gradients. Degradation of [35S]methionine-labeled VSV was observed at a significant rate in Balb/3T3 cells, but not in MO-5 cells. The monensin-resistant somatic cell may thus provide a genetic route to study the mechanism of endocytosis or transport of enveloped viruses.     

The relationship between glycosylation and glycoprotein metabolism of mouse neuroblastoma N18 cells.

Two inhibitors of glycosylation, glucosamine and tunicamycin, were utilized to examine the effect of glycosylation inhibition in mouse neuroblastoma N18 cells on the degradation of membrane glycoproteins synthesized before addition of the inhibitor. Treatment with 10 mM-glucosamine resulted in inhibition of glycosylation after 2h, as measured by [3H]fucose incorporation into acid-insoluble macromolecules, and in a decreased rate of glycoprotein degradation. However, these results were difficult to interpret since glucosamine also significantly inhibited protein synthesis, which in itself could cause the alteration in glycoprotein degradation [Hudson & Johnson (1977) Biochim. Biophys. Acta 497, 567-577]. N18 cells treated with 5 microgram of tunicamycin/ml, a more specific inhibitor of glycosylation, showed a small decrease in protein synthesis relative to its effect on glycosylation, which was inhibited by 85%. Tunicamycin-treated cells also showed a marked decrease in glycoprotein degradation in experiments with intact cells. The inhibition of glycoprotein degradation by tunicamycin was shown to be independent of alterations in cyclic AMP concentration. Polyacrylamide-gel electrophoresis of isolated membranes from N18 cells, double-labelled with [14C]fucose and [3H]fucose, revealed heterogeneous turnover rates for specific plasma-membrane glycoproteins. Comparisons of polyacrylamide gels of isolated plasma membranes from [3H]fucose-labelled control cells and [14C]fucose-labelled tunicamycin-treated cells revealed that both rapidly and slowly metabolized, although not all, membrane glycoproteins became resistant to degradation after glycosylation inhibition.     

Perturbation of N-linked oligosaccharide structure results in an altered incorporation of [3H]palmitate into specific proteins in Chinese hamster ovary cells

Increased [3H]palmitate incorporation into specific cellular proteins has been reported to occur in Chinese hamster ovary (Wellner, R. B., Ray, B., Ghosh, P. C., and Wu, H. C. (1984) J. Biol. Chem. 259, 12788-12793) and yeast (Wen, D., and Schlesinger, M. J. (1984) Mol. Cell. Biol. 4, 688-694) mutant cells. In this paper we report studies concerning the relationship between N-linked oligosaccharide structure and [3H]palmitate incorporation into proteins of Chinese hamster ovary (CHO) cells. We have compared the incorporation of [3H]palmitate into proteins of wild-type and four different mutant CHO cell lines defective in various steps of N-linked protein glycosylation. Sodium dodecyl sulfate-gel electrophoretic analysis showed that three of the mutants exhibited increased [3H]palmitate incorporation into several CHO cellular proteins (approximately 30,000-38,000 molecular weight) as compared to the wild-type cells. One of the affected mutants which accumulates the Man5Gn2Asn intermediate structure was examined in detail. In agreement with earlier reports, virtually all of the [3H] palmitate-labeled proteins of both wild-type and mutant cell lines are membrane-bound. Pretreatment of the mutant cell line with tunicamycin blocked the increased [3H]palmitate incorporation into the two specific proteins (both of approximately 30,000 molecular weight) observed in untreated cells; the decreased incorporation of [3H]palmitate into the 30,000 molecular weight species was accompanied by a concomitant increase in the incorporation of [3H]palmitate into two proteins of approximately 20,000 molecular weight. Pretreatment of wild-type cells with tunicamycin also caused increased [3H]palmitate incorporation into the 20,000 molecular weight species. Endoglycosidase H treatment of [3H]palmitate-labeled extracts from the mutant cell line resulted in the disappearance of the heavily labeled 30,000 molecular weight species and the appearance of intensely labeled 20,000 molecular weight species. Pretreatment of the mutant cell line with either castanospermine or deoxynojirimycin reduced the [3H]palmitate incorporation in to the 30,000 molecular weight species increased in untreated cells, but did not cause increased [3H]palmitate incorporation into the 20,000 molecular weight species. Our results indicate that perturbation of N-linked oligosaccharide structure results in altered incorporation of [3H]palmitate into specific proteins in CHO cells.     

Transport of glutamine by rat kidney brush-border membrane vesicles.

I studied the glycosylation in vivo of a viral envelope protein, the glycoprotein of vesicular stomatitis virus (VSV), by pulse labelling of virus-infected HeLa cells with 3H-labelled monosaccharides (mannose, glucosamine). Radioactivity was incorporated into the fraction of membrane-bound polyribosomes, although metabolic conversion of [3H]-mannose into amino acids was negligible. Dissociation of bound polyribosomes revealed that the radioactively co-purified with the peptidyl-tRNA. The nascent peptides were released by alkaline hydrolysis, immunoprecipitated and analysed by polyacrylamide-gel electrophoresis. It is apparent from the size distribution of the [3H]mannose-labelled nascent chains that attachment of carbohydrate starts when approximately half of the amino acid sequence of the viral glycoprotein has been synthesized.     

Sequential isolation of proteoglycan synthesis mutants by using herpes simplex virus as a selective agent: evidence for a proteoglycan-independent virus entry pathway.

A novel mouse L-cell mutant cell line defective in the biosynthesis of glycosaminoglycans was isolated by selection for cells resistant to herpes simplex virus (HSV) infection. These cells, termed sog9, were derived from mutant parental gro2C cells, which are themselves defective in heparan sulfate biosynthesis and 90% resistant to HSV type 1 (HSV-1) infection compared with control L cells (S. Gruenheid, L. Gatzke, H. Meadows, and F. Tufaro, J. Virol. 67:93-100, 1993). In this report, we show that sog9 cells exhibit a 3-order-of-magnitude reduction in susceptibility to HSV-1 compared with control L cells. In steady-state labeling experiments, sog9 cells accumulated almost no [35S]sulfate-labeled or [6-3H]glucosamine-labeled glycosaminoglycans, suggesting that the initiation of glycosaminoglycan assembly was specifically reduced in these cells. Despite these defects, sog9 cells were fully susceptible to vesicular stomatitis virus (VSV) and permissive for both VSV and HSV replication, assembly, and egress. HSV plaques formed in the sog9 monolayers in proportion to the amount of input virus, suggesting the block to infection was in the virus entry pathway. More importantly, HSV-1 infection of sog9 cells was not significantly reduced by soluble heparan sulfate, indicating that infection was glycosaminoglycan independent. Infection was inhibited by soluble gD-1, however, which suggests that glycoprotein gD plays a role in the infection of this cell line. The block to sog9 cell infection by HSV-1 could be eliminated by adding soluble dextran sulfate to the inoculum, which may act by stabilizing the virus at the sog9 cell surface. Thus, sog9 cells provide direct genetic evidence for a proteoglycan-independent entry pathway for HSV-1, and results with these cells suggest that HSV-1 is a useful reagent for the direct selection of novel animal cell mutants defective in the synthesis of cell surface proteoglycans.     

Isolation of a temperature-sensitive FM3A mutant deficient in asparagine-linked glycosylation by selecting for resistance to tritiated mannose suicide

Protein glycosylation mutants in the mouse mammary carcinoma cell line FM3A were selected for ability to withstand exposure to [2-3H]mannose at 39 degrees C. G258 , one of the mutant cells isolated, has been characterized. G258 cells were temperature-sensitive for cell growth. Moreover, G258 cells showed temperature sensitivity for [3H]mannose incorporation into the TCA-insoluble fraction. To study the biochemical basis of the defect in glycoprotein biosynthesis, the formation of lipid-linked saccharides was examined. The results showed that the formation of lipid-linked oligosaccharides was severely inhibited in G258 cells at 39 degrees C. At 33 degrees C, G258 cells synthesized Glc3Man9GlcNAc2-PP-Dol, the fully assembled lipid-linked oligosaccharides, but at 39 degrees C, G258 cells were able to synthesize merely the smaller lipid-linked oligosaccharides (approximately up to Man3GlcNAc2 -PP-Dol), but were unable to synthesize the larger lipid-linked oligosaccharides.     

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 pronasedigested 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.     

Changes of Sertoli cell glycoproteins induced by removal of the associated germ cells

Sertoli cell glycoproteins were studied in culture where these cells were in contact with germ cells (Sertoli cell enriched cultures, SCEC) and in pure Sertoli cell cultures. Sertoli cell only cultures (SCOC) were prepared by a short treatment of SCEC with hypotonic solution or by culturing seminiferous epithelium fragments from prenatally irradiated rats. After metabolic labeling with [3H]fucose. [14C]N-acetylglucosamine or [3H]leucine, SCEC and SCOC particulate fractions (105 000 g pellet) were analyzed by one-dimensional slab gel electrophoresis and fluorography. The comparison of the electrophoretic patterns obtained, demonstrated that a glycoprotein of MW 48 000, undetectable in SCEC, was present in SCOC after labelling with both sugar precursors. The MW 48 000 glycoprotein was also present in the electrophoretic profile of particulate fraction from [3H]fucos-labelled Sertoli cell cultures from prenatally irradiated rat. Such difference was not observed after labelling with [3H]leucine; in this experimental condition a MW 48 000 band was present in the electrophoretic profile of polypeptides from SCEC as well from SCOC. The synthesis of this glycoprotein represented a specific and stable cell response, since it occurred only a few hours after germ cell removal, and it was still detectable 3 days later. FSH stimulation did not influence the synthesis of the MW 48 000 glycoprotein, whereas it increased the synthesis of high MW glycoproteins. The hypothesis is discussed that the appearance of a new glycoprotein when Sertoli cells have lost their contact with germ cells could represent a product of glycosylation of preexisting molecules and their possible location in the Sertoli cell membrane. The results presented here provide additional evidence that Sertoli cell functions may be dependent on the association with the germ cell.     

Identification and characterization of a mouse cell mutant defective in the intracellular transport of glycoproteins

We have isolated a mutant line of mouse L cells, termed gro29, in which the growth of herpes simplex virus (HSV) and vesicular stomatitis virus (VSV) is defective. The block occurs late in the infectious cycle of both viruses. We demonstrate that HSV and VSV enter gro29 cells normally, negotiate the early stages of infection, yet are impaired at a late stage of virus maturation. During VSV infection of the mutant cell line, intracellular transport of its glycoprotein (G protein) is slowed. Pulse-chase experiments showed that oligosaccharide processing is impeded, and immunofluorescence localization revealed an accumulation of G protein in a juxtanuclear region that contains the Golgi complex. We conclude that export of newly made glycoproteins is defective in gro29 cells, and speculate that this defect may reflect a lesion in the glycoprotein transport apparatus.     

Binding partners for the myelin-associated glycoprotein of N2A neuroblastoma cells

The myelin-associated glycoprotein (MAG) has been proposed to be important for the integrity of myelinated axons. For a better understanding of the interactions involved in the binding of MAG to neuronal axons, we performed this study to identify the binding partners for MAG on neuronal cells. Experiments with glycosylation inhibitors revealed that sialylated N-glycans of glycoproteins represent the major binding sites for MAG on the neuroblastoma cell line N2A. From extracts of [3H]glucosamine-labelled N2A cells several glycoproteins with molecular weights between 20 and 230 kDa were affinity-precipitated using immobilised MAG. The interactions of these proteins with MAG were sialic acid-dependent and specific for MAG.     

Target antigens for H-2-restricted vesicular stomatitis virus-specific cytotoxic T cells.

Cytotoxic thymus-derived lymphocytes from mice infected with vesicular stomatitis virus (VSV) are H-2 restricted and virus specific for the Indiana and New Jersey strains of VSV. VSV-Indiana-immune T cells can lyse target cells infected with the temperature sensitive (ts) mutant ts 045 about 30 times better when target cell infection occurs at the permissive rather than the non-permissive temperature. Since this mutant fails to express the glycoprotein at the cell surface when grown at the nonpermissive temperature, our results support the view that the viral glycoprotein is involved in defining the major target antigen for VSV-specific T cells. However, the tl 17 mutant that expresses a mutant glycoprotein at the cell surface was lysed, suggesting that the expressed mutated glycoprotein can cross-react with Indiana wild-type glycoprotein. Targets infected at the nonpermissive temperature with VSV ts G31 (mutant in the matrix protein) are still susceptible to T cell-mediated lysis but at a lower level of sensitivity. These results are compatible with the interpretation that for VSV-specific T cell lysis of infected target cells, the viral glycoprotein is a major target antigen and must be expressed, and that the matrix protein plays a lesser role, probably by indirectly influencing glycoprotein configuration at the cell surface.     

A membrane glycoprotein that accumulates intracellularly: cellular processing of the large glycoprotein of LaCrosse virus

The intracellular transport and certain posttranslational modifications of the large glycoprotein (G1) of LaCrosse virus (LAC) in BHK cells have been studied. G1 from released LAC virus was characterized by complex oligosaccharides (endo H-resistant) and covalently attached fatty acid. Only a small fraction of total cellular G1 was present on the baby hamster kidney cell surface. Cell-surface G1 contained complex oligosaccharides, while total G1 in infected cells contained largely unprocessed (endo H-sensitive) oligosaccharides. In addition, cell G1 contained significantly less fatty acid than virion-associated G1. Pulse-chase experiments showed that the oligosaccharides of G1 were processed to the complex from much more slowly than the oligosaccharides of the vesicular stomatitis virus (VSV) glycoprotein (G). In addition, transit of LAC G1 to the cell surface and into extracellular virions was two to three fold slower than the transit of VSV G. Thus LAC G1 accumulates intracellularly and is only slowly processed by intracellular processing enzymes. Treatment with monensin caused accumulation in the cell of a form of G1 with partial sensitivity toward endo H, suggesting that monensin may act to inhibit the glycosylation process directly.     

Vesicular stomatitis virus envelope glycoprotein alterations induced by host cell transformation

Vesicular stomatitis virus (VSV) contains a single structural glycoprotein in which the sugar sequences are largely host specified. We have used VSV as a probe to study the changes in cell glycoprotein metabolism induced by virus transformation. Analysis of purified VSV grown in baby hamster kidney (BHK) or polyoma transformed BHK cells showed that the virus glycoproteins have identical apparent molecular weights. The glycopeptides derived from the glycoproteins by extensive pronase digestion have an identical molecular weight distribution.On the basis of labeling experiments with fucose, mannose, and glucosamine, the oligosaccharide moieties of the VSV glycoprotein were different in virus from the two cell lines. The VSV glycopeptides from transformed cells showed an increased resistance to cleavage by an endoglycosidase, indicating structural changes in the core region of the oligosaccharides. They also showed an increased ratio of sialic acid to N-acetylglucosamine.VSV grows in a wide variety of cell types, and the carbohydrate structures of its single glycoprotein are amenable to analysis with specific glycosidases. The virus thus provides an excellent tool with which to study alterations induced by cell transformation in the glycosylation of membrane proteins.     

The synthesis and secretion of gastric mucus glycoprotein by mucosal cells cultured in the presence of ethanol

The effect of ethanol on the synthesis and secretion of mucus glycoprotein in gastric mucosal cells was investigated. The mucosal cell suspensions were subjected to a short-term (4 h) culture in the presence of 0-1.5 M ethanol, with [3H]proline and [3H]palmitic acid as markers for glycoprotein synthesis and acylation. The synthesized labeled mucus glycoprotein was isolated from the incubation medium (extracellular glycoprotein) and from the mucosal cells (intracellular glycoprotein), and analyzed. Depending upon the ethanol concentration in the cell culture medium, two distinct effects on the synthesis and secretion of mucus glycoprotein were observed. The cells cultured in the presence of 0.02-0.1 M ethanol showed increased ability for the incorporation of [3H]proline and [3H]palmitic acid, and for the secretion of the newly assembled mucus glycoprotein. The synthesis of the glycoprotein increased 18-fold, acylation 5-fold, and secretion 10-fold. The synthesized glycoprotein, however, contained four to five times less of acyl-bound fatty acids. Ethanol at 0.1-1.5 M caused a marked reduction (62-64%) in the mucus glycoprotein synthesis, but the amount of glycoprotein released to the medium remained constant. This indicated that higher concentrations of ethanol caused the release of the preformed intracellular mucus glycoprotein reserves. The results demonstrate that gastric mucosal cells incubated in the presence of ethanol exhibit impaired synthesis and secretion of mucus glycoprotein, and that the severity of impairment depends upon the ethanol concentration.     

A fusion-defective mutant of the vesicular stomatitis virus glycoprotein.

We have recently described an assay in which a temperature-sensitive mutant of vesicular stomatitis virus (VSV; mutant tsO45), encoding a glycoprotein that is not transported to the cell surface, can be rescued by expression of wild-type VSV glycoproteins from cDNA (M. Whitt, L. Chong, and J. Rose, J. Virol. 63:3569-3578, 1989). Here we examined the ability of mutant G proteins to rescue tsO45. We found that one mutant protein (QN-1) having an additional N-linked oligosaccharide at amino acid 117 in the extracellular domain was incorporated into VSV virions but that the virions containing this glycoprotein were not infectious. Further analysis showed that virus particles containing the mutant protein would bind to cells and were endocytosed with kinetics identical to those of virions rescued with wild-type G protein. We also found that QN-1 lacked the normal membrane fusion activity characteristic of wild-type G protein. The absence of fusion activity appears to explain lack of particle infectivity. The proximity of the new glycosylation site to a sequence of 19 uncharged amino acids (residues 118 to 136) that is conserved in the glycoproteins of the two VSV serotypes suggests that this region may be involved in membrane fusion. The mutant glycoprotein also interferes strongly with rescue of virus by wild-type G protein. The strong interference may result from formation of heterotrimers that lack fusion activity.     

In vitro acylation of rat gastric mucus glycoprotein with [3H]palmitic acid

The incorporation of fatty acids into gastric mucus glycoproteins was studied by incubating rat gastric mucosal cell suspensions with [9,10-3H]palmitic acid and [3H]proline. The mucus glycoprotein polymer, secreted into the growth medium (extracellular) and that contained within the cells (intracellular), was purified from the other components of the secretion, thoroughly delipidated, and then analyzed for the radiolabeled tracers. Both pools of mucus glycoprotein, incubated in the presence of [3H]palmitic acid, contained radioactive label which could not be removed by gel filtration, CsCl density gradient centrifugation, sodium dodecyl sulfate-gel electrophoresis, or lipid extraction. Treatment of the purified mucus glycoprotein with 1 M hydroxylamine or 0.3 M methanolic KOH released the radioactivity, thus indicating that [3H]palmitic acid was covalently bound by ester linkage to the glycoprotein. The released radioactivity was associated mainly (87%) with palmitic acid. The incorporation ratio of [3H]proline to [3H]palmitic acid was 0.12:1.0 in the extracellular glycoprotein and 1.38:1.0 in the intracellular glycoprotein, which suggested that acylation of mucus glycoprotein occurs in the intracellular compartment after completion of its polypeptide core. The fact that incorporation of [3H]palmitic acid was greater in the glycoprotein subunits than in the glycoprotein polymer indicates that acylation takes place near the end of subunit processing but before their assembly into the high molecular weight mucus glycoprotein polymer.     

Cerulenin blocks fatty acid acylation of glycoproteins and inhibits vesicular stomatitis and Sindbis virus particle formation

Cerulenin, an antibiotic that inhibits de novo fatty acid and cholesterol biosynthesis, effectively inhibited the formation and release of virus particles from chicken embryo fibroblasts infected with Sindbis or vesicular stomatitis virus (VSV). When added for 1 h at 3 h postinfection, the antibiotic blocked VSV particle production by 80 to 90% and inhibited incorporation of [3H]palmitic acid into the VSV glycoprotein by an equivalent amount. The effect of this antibiotic on virus protein and RNA biosynthesis was significantly less than that on fatty acid acylation. Nonacylated virus glycoproteins accumulated inside and on the surface of cerulenin-treated cells. These data indicate that fatty acid acylation is not essential for intracellular transport of these membrane proteins, but it may have an important role in the interaction of glycoproteins with membranes during virus assembly and budding.     

Specific [3H]-N-methyl scopolamine binding without cholinergic function in cultured adult skin fibroblasts

Cultured adult skin fibroblasts were studied for binding and functional evidence of muscarinic receptors in order to assess their utility as a model of cholinergic function in affective illness. Saturable, specific, high affinity binding could be demonstrated in intact cells from some cell lines with [3H]-NMS, but not [3H]-QNB, presumably because of intracellular trapping of unbound [3H]-QNB. [3H]-NMS specific binding indicated a single site with a KD of approximately 210 pM. [3H]-NMS was displaced by cholinergic agonists and antagonists with relative affinities similar to muscarinic receptors in brain. Many cell lines, however, showed no specific binding. No functional response to carbachol could be demonstrated with respect to inhibition of isoproterenol-stimulated cyclic AMP formation, stimulation of cyclic GMP formation or stimulation of phosphoinositide hydrolysis in any cell line regardless of either high or no specific [3H]-NMS binding.     

Assembly of viral membranes: maturation of the vesicular stomatitis virus glycoprotein in the presence of tunicamycin.

The role of glycosylation in the maturation of the vesicular stomatitis virus (VSV) glycoprotein was studied by use of the antibiotic tunicamycin. Tunicamycin-treated VSV-infected cells synthesize an unglycosylated form of the VSV glycoprotein (R. Leavitt, S. Schlesinger, and S. Kornfeld, J. Virol. 21:375--385, 1977). We have found that tunicamycin has no effect on the attachment of the glycoprotein to intracellular membranes or on the transport of protein to the lumen of the endoplasmic reticulum. However, tunicamycin prevented the migration of the glycoprotein from the rough endoplasmic reticulum to smooth intracellular membranes.