Oligosaccharides of the Hazelhurst vesicular stomatitis virus glycoprotein are more extensively processed in Rous sarcoma virus-transformed baby hamster kidney cells

Because of the extensive oligosaccharide heterogeneity of the membrane glycoprotein (G) from the Hazelhurst strain of vesicular stomatitis virus, this virus has been used as a specific intracellular probe of altered protein glycosylation in Rous sarcoma virus-transformed versus normal baby hamster kidney cells. Over 70% of G protein from virus released from the transformed cells had acidic-type oligosaccharides at both glycosylation sites, compared to less than 50% from the corresponding normal host cells. The remaining G protein contained an acidic-type oligosaccharide at one site and an endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharide at the other. The major endoglycosidase-sensitive species were sialylated hybrid-type (NeuNAc-Gal-GlcNAc-Man5GlcNAc2-Asn) from the transformed and neutral-type (Man5-6GlcNAc2-Asn) from the normal host cells. The degree of branching of the acidic-type oligosaccharides was not increased in the transformed cells (approx. 80% biantennary for viral G protein from both cell types). At a reduced growth temperature (24 versus 37 degrees C), the G protein oligosaccharides were more extensively processed in both cell types (approximately 85-95% of G protein contained acidic-type structures at both sites), even though the level of viral protein synthesis and virus release was decreased. Essentially all of the minor, endoglycosidase-sensitive oligosaccharides on mature viral G protein were sialic acid-containing hybrid-type structures. At 24 degrees C the branching of the acidic-type oligosaccharides was increased in the virus released from the transformed cells versus normal cells.     

Both acidic-type and neutral-type asparaginyl-oligosaccharides of host-cell glycoproteins are altered in Rous-sarcoma-virus-transformed chick-embryo fibroblasts.

In comparisons of [3H]mannose-labelled glycopeptides from chick-embryo fibroblasts infected and transformed with non-defective Prague C Rous-sarcoma virus and from untransformed fibroblasts infected with a transformation-defective derivative of Prague C Rous-sarcoma virus, we have detected transformation-dependent alterations in both the acidic-type and the neutral-type asparagine-linked oligosaccharides of cellular glycoproteins. Pronase-digested glycopeptides were analysed by the combined techniques of gel filtration, exo- and endo-glycosidase digestion and concanavalin A-agarose affinity chromatography. The transformed cell glycoproteins contained more sialic acid and were enriched for more highly branched (versus biantennary) acidic-type structures compared with the untransformed cell glycoproteins, similarly to previously reported transformation-dependent alterations. In addition, the glycopeptides from the virus-transformed cells contained several neutral-type structures that were apparently absent from the untransformed cells: small neutral-type oligosaccharides (Man3GlcNAc2) that were sensitive to endo-beta-N-acetylglucosaminidase D but resistant to endo-beta-N-acetylglucosaminidase H, and oligosaccharides with the property of 'truncated' precursor oligosaccharides (endoglycosidase-resistant, alpha-mannosidase-sensitive). Endoglycosidase-released oligosaccharides with the properties of hybrid-type structures were derived from the glycoproteins of both transformed and untransformed cells.     

Unusual heterogeneity in the glycosylation of the G protein of the hazelhurst strain of vesicular stomatitis virus

The asparagine-linked oligosaccharides of the G protein of the Hazelhurst subtype of the New Jersey serotype of vesicular stomatitis virus (VSV) have been compared with the oligosaccharides from the G protein of the well-characterized Indiana serotype of VSV, with baby hamster kidney cells in monolayer culture as the host for both viruses. [3H]Glucosamine- and [3H]mannose-labeled glycopeptides from the G protein of purified virus were analyzed by the combined techniques of endo-beta-N-acetylglucosaminidase H (ENDO-H) digestion, concanavalin A and lentil lectin affinity chromatography, and Bio-Gel P-4 chromatography. Although almost all of the Indiana G protein oligosaccharides were acidic-type structures, as expected from previous studies; the Hazelhurst G protein contained a mixture of acidic-type, hybrid-type containing sialic acid, and neutral-type (predominantly Man5-6GlcNAc2-Asn) structures. The vast majority of acidic-type oligosaccharides from both the Hazelhurst and Indiana G proteins were diantennary structures, with less than half containing fucose linked to the innermost N-acetylglucosamine. Additional analysis of the Hazelhurst G protein by ENDO-H digestion and gel electrophoresis suggested that some of the mature G polypeptides contained acidic-type structures at both glycosylation sites, whereas the remainder contained an ENDO-H-resistant, acidic-type structure at one site and an ENDO-H-sensitive, hybrid- or neutral-type structure at the other site.     

Lectin affinity chromatography of glycopeptides and oligosaccharides from normal and lectin-resistant Chinese-hamster ovary cells.

The [3H]mannose-labelled glycopeptides from two lectin-resistant lines of Chinese-hamster ovary cells were fractionated by chromatography on lentil lectin-Sepharose and concanavalin A-agarose columns and subsequently analysed by gel filtration in comparison with the glycopeptides of the parental cell line. Essentially all of the [3H]mannose-labelled asparaginyl-oligosaccharides from the 'single-mutant' cells selected for resistance to phytohaemagglutinin and the 'double-mutant' cells selected for additional resistance to concanavalin A were not bound to lentil lectin, whereas approximately one-sixth of the parental-cell glycopeptides were bound and specifically eluted with alpha-methyl mannoside. These bound and eluted glycopeptides represented a specific subset of the complex acidic-type asparaginyl-oligosaccharides. The percentage of radiolabelled glycopeptides and oligosaccharides from each cell line that were specifically bound to concanavalin A was consistent with the relative sensitivities of the three cell lines to this lectin. The major radiolabelled species in the endoglycosidase digest of the 'double-mutant'-cell glycopeptides (Man4GlcNAc1-size neutral oligosaccharides) were not bound to concanavalin A, whereas essentially all of the other neutral-type oligosaccharides were bound. In addition, the larger neutral-type oligosaccharides (Man8--9GlcNAc1) were more strongly bound to concanavalin A than were either the smaller neutral-type or the di-antennary acidic-type structures.     

Hazelhurst-vesicular-stomatitis-virus G and Sindbis-virus E1 glycoproteins undergo similar host-cell-dependent variation in oligosaccharide processing.

We have examined and compared the host-cell-dependent glycosylation of the G glycoprotein of vesicular-stomatitis virus (Hazelhurst strain) and the E1 and E2 glycoproteins of Sindbis virus replicated by baby-hamster kidney, chicken-embryo fibroblast and mouse L929 monolayer cell cultures. The results of endo-beta-N-acetylglucosaminidase H digestion of viral proteins labelled with [3H]mannose or leucine and Pronase-digested glycopeptides labelled with [3H]mannose indicated that both the G protein and the E1 protein contained a similar mixture of endoglycosidase-resistant oligosaccharides of the complex acidic type and less extensively processed endoglycosidase-sensitive oligosaccharides of the neutral or hybrid type, with a relatively greater content of the endoglycosidase-sensitive oligosaccharides for virus replicated in the chicken as against hamster or mouse cells. A large fraction of the G protein and the majority of the E1 proteins from the mammalian host cells contained acidic-type oligosaccharides at both glycosylation sites, whereas most of the G and E1 glycoproteins from the avian host cells and essentially all of the E2 protein from all three host-cell types contained an acidic-type oligosaccharide at one site and neutral- or hybrid-type oligosaccharide at the other site. The relative increase in neutral- and hybrid-type oligosaccharides with five-mannose core structures observed for the G and E1 proteins of virus released from the avian host cells suggested that two specific steps in oligosaccharide processing (mediated by alpha-mannoside II and N-acetylglucosaminyltransferase I) were less efficient at one of the glycosylation sites of the vesicular-stomatitis-virus G protein and Sindbis-virus E1 protein in the avian as against mammalian host cells.     

Membrane glycopeptides from virus-transformed hamster fibroblasts and the normal counterpart.

Comparisons of membrane glycopeptides from baby hamster kidney fibroblasts (BHK21/C13) and a clone transformed by Rous sarcoma virus (C13/B4) were made by using cells metabolically labeled with radioactive D-glucose and L-fucose. Most of the glycopeptides were metabolically labeled with both the general and the specific glycoprotein precursors. The glycopeptides obtained from the cell surface by controlled trypsinization were representative of the surface membrane as shown by comparing them with those of purified membrane preparations. The trypsin-removable glycopeptides from both cell types were further processed and examined by successive chromatography on Sephadex G-50 and DEAE-cellulose. The chromatographic distribution patterns showed that each cell type had glycopeptides of similar characteristics, although the proportions of the glycopeptides differed dramatically between the two cell types. After transformation there was an increase in the larger, more highly charged glycopeptides. This was verified by the increased sialic acid content in these glycopeptides. Some of the glycopeptides were homogeneous after the size and charge separations, since a variety of procedures did not separate them further. The apparent homogeneity and reasonably few species obtained may be due to the methods of isolation, with the procedures selecting particular glycopeptides from the external portion of the membrane. These results corroborate the concept and show for the first time that virus transformation is accompanied by an increase in certain species of glycopeptides rather than de novo synthesis.     

Efficient transformation by Prague A Rous sarcoma virus plasmid DNA requires the presence of cis-acting regions within the gag gene.

A region in addition to and outside the long terminal repeats (LTRs) in the gag gene of the Prague A strain of Rous sarcoma virus was found to be essential in cis for efficient cell transformation by cloned viral DNA. Transformation in chicken embryo fibroblasts, which requires infectious virus production and reinfection, was facilitated in cis by sequences between nucleotides 630 and 1659. Efficient transformation of NIH 3T3 cells in which secondary spread of virus is not necessary (as it is in chicken embryo fibroblasts) required sequences between nucleotides 630 and 1149. A src cDNA clone which also lacks this region demonstrated low transformation efficiency, indicating that the role of the cis element cannot be attributed to interference with RNA splicing. The gag gene segment required in cis for transformation, between nucleotides 630 and 1149, could substitute for the simian virus 40 enhancer in either orientation, and cells transfected with Rous sarcoma virus LTR-driven plasmids containing the gag cis element had a two- to threefold increase in steady-state viral RNA levels compared with plasmids lacking this region. Thus, additional cis-acting regulatory elements located outside the viral LTRs may modulate viral gene expression and contribute to the efficiency of cell transformation.     

Differential effects of oncogenic transformation on N-linked oligosaccharide processing at individual glycosylation sites of viral glycoproteins

Hamster sarcoma virus (HSV) transformation of Nil-8 fibroblasts is associated with an increase in the average size of N-acetyllactosamine (complex) type N-linked glycans due to an increase in both the average number of branches/chain and in the fraction of N-linked glycans containing poly(GlcNAc(beta 1,3) Gal-(beta 1,4)) (polylactosaminylglycan) chains. Analysis of glycopeptides from the envelope glycoproteins of Sindbis virus and vesicular stomatitis virus (VSV) grown in Nil-8 and Nil/HSV cells indicated that the transformation-associated shift to larger N-linked oligosaccharides selectively affects some glycosylation sites far more than others. Glycosylation of the Sindbis virus glycoproteins and of Asn-179 of VSV G was similar in Nil-8 and Nil/HSV cells; oligosaccharide processing generally did not proceed beyond the biantennary complex stage. In contrast, Asn-336 of VSV G carried primarily biantennary complex glycans in Nil-8-grown virus (ratio, triantennary, and larger to biantennary complex glycans (tri+/bi) = 0.5) but more highly branched structures in Nil/HSV-grown virus (tri+/bi = 8.1). All of the triantennary or larger oligosaccharides from Asn-336 of Nil/HSV-grown VSV G bound to leukoagglutinating phytohemagglutinin-agarose, indicating the presence of a branch attached to the Man3GlcNAc2 core via a beta 1,6-linked GlcNAc residue and suggesting that increased UDP-GlcNAc:alpha-D-mannoside beta 1,6-N-acetylglucosaminyl transferase V (GlcNAc transferase V) activity accompanied transformation. At least 20% of these leukoagglutinating phytohemagglutinin-binding oligosaccharides were sensitive to an enzyme specific for polylactosaminylglycan chains, Escherichia freundii endo-beta-galactosidase.     

Altered synthesis and processing of oligosaccharides of vesicular stomatitis virus glycoprotein in different lectin-resistant Chinese hamster ovary cell lines.

To determine the particular intracellular steps in the glycosylation of the vesicular stomatitis virus (VSV) glycoprotein that were altered in several lectin-resistant CHO cell lines, VSV-infected parental and mutant cells were pulse-labeled for 30 and 120 min with [3H]mannose and [3H]glucosamine. Cell-associated viral glycopeptides were analyzed by gel filtration combined with specific glycosidase digestions and compared with the corresponding mature virion oligosaccharides. The intracellular glycosylation of the VSV glycoprotein in a mutant cell line resistant to phytohemagglutinin was identical to that in the normal cells except for a complete block in processing at a specific step in the final trimming of the oligomannosyl core from five to three mannoses. The results demonstrated that a double-mutant cell line selected from the phytohemagglutinin-resistant cells for resistance to concanavalin A had an additional defect in one of the earliest stages of glycosylation, resulting in smaller precursor oligosaccharides linked to protein.     

D-galactosyltransferase and its endogenous substrates in chick embryo fibroblasts transformed by Rous sarcoma virus

UDP-D-galactose: 2-acetamido-2-deoxy-beta-D-glucopyranosyl 4-beta-D-galactosyltransferase (GalTase) activity was purified, from primary chick embryo fibroblast (CEF) transformed by a temperature-sensitive, Rous sarcoma virus mutant (CEF-RSV), by chromatography on an affinity resin prepared with monoclonal antibodies to GalTase. Cellular glycopeptides from CEF, as well as CEF-RSV, maintained at permissive (37 degrees) [CEF-RSF (37 degrees)] and nonpermissive temperatures (41 degrees) [CEF-RSV (41 degrees)], were solubilized and galactosylated in vitro by incubation with purified GalTase substrates, composed of at least six discrete complex glycopeptides having bi- to tetra-antennary structures. The glycopeptides isolated from transformed cells, CEF-RSV (37 degrees), included the six types observed in nontransformed cells, but demonstrated alterations in their relative amounts, including an increase in the content of a glycopeptide containing 3 mannose and 4 glucosamine residues. Furthermore, two additional complex-type glycopeptides were isolated from CEF- but demonstrated alterations in their relative amounts, including an increase in the content of a glycopeptide containing 3 mannose and 4 glucosamine residues. Furthermore, two additional complex type glycopeptides were isolated from CEF-RSV (37 degrees). These malignant transformation-related glycopeptides were partially characterized and found to represent tri- and tetra-antennary complex glycopeptides. Endogenous galactosylation appeared to have occurred in a branched, nonspecific manner in these transformed cell-derived glycopeptides. These findings indicate that transformed cells may contain a greater preponderance of more highly branched, complex oligosaccharides which are randomly galactosylated at nonreducing termini by cellular GalTase.     

Expression of the Rous sarcoma virus src gene in avian macrophages fails to elicit transformed cell phenotype.

Infection of avian macrophages with Rous sarcoma virus does not induce any changes in the morphology, growth behavior, or expression of macrophage-specific proteins. The absence of cellular transformation does not result from a block in the synthesis of viral proteins, since infectious viruses are released from a majority of cells in the culture. In this report, we examine the synthesis, processing, and functional activity of pp60src in Rous sarcoma virus-infected macrophages to determine whether the absence of transformation is due to an alteration in the functional expression of pp60src. Although the absolute level of pp60src was reduced compared with fibroblasts, the protein exhibited the same phosphorylation pattern and subcellular distribution and was able to phosphorylate immunoglobulin in the immune complex-protein kinase assay. These results imply that the failure of Rous sarcoma virus to transform macrophage may be due to a restriction in the cellular response to a functional src protein, perhaps due to the absence of cellular products which are essential for mediating pp60src-induced transformation.     

Rous sarcoma virus-transformed baby hamster kidney cells express higher levels of asparagine-linked tri- and tetraantennary glycopeptides containing [GlcNAc-beta (1,6)Man-alpha (1,6)Man] and poly-N-acetyllactosamine sequences than baby hamster kidney cells

The alterations in complex-type N-linked oligosaccharides that can occur when an animal cell line is transformed by two dissimilar viruses were examined by comparing the N-linked oligosaccharides of baby hamster kidney (BHK) cells, metabolically radiolabeled with [2-3H]mannose, to the same class of oligosaccharides from BHK cells separately transformed by Rous sarcoma virus (RS-BHK), an RNA retrovirus, and polyoma virus (PY-BHK), a DNA papovavirus. Based on experiments that utilized serial lectin affinity chromatography, glycosidase digestions, and methylation analyses, both RS-BHK and PY-BHK cells demonstrated a significant increase in the relative amounts of tri- and tetraantennary complex-type N-linked oligosaccharides containing the branching sequence, [GlcNAc-beta(1,6)Man-alpha(1,6)Man], compared to the nontransformed BHK cells. In addition, almost all of the poly-N-acetyllactosamine sequence, [GlcNAc-beta(1,3)-Gal-beta(1,4)], was expressed on the tri- and tetraantennary N-linked oligosaccharides from BHK and RS-BHK cells that contain the sequence, [GlcNAc-beta(1,6)Man-beta(1,6)Man]. The increase in the relative amounts of this latter sequence in the transformed cells, therefore, most likely results in an increase in the amount of poly-N-acetyllactosamine sequence on the N-linked glycopeptides of these cells. The analysis of the degree of sialylation of the complex-type N-linked oligosaccharides from BHK and RS-BHK cells by ion exchange chromatography revealed no apparent differences, and in both of these cell types approximately 3% of the glycopeptide fraction radiolabeled with mannose was recovered in a highly negatively charged fraction that was identified by keratanase digestion to be keratan sulfate.     

Chromatographic Analyses of Isoaccepting tRNAs from Avian Tumor Viruses

Low-molecular-weight RNA from transforming viruses (Rous sarcoma virus-Rous-associated virus 1, Schmidt-Ruppin strain of Rous sarcoma virus, and sarcoma-B(77)), from nontransforming viruses (Rous-associated virus 1 and sarcoma-NTB(77)), and from chicken liver, chicken embryo fibroblast, and Rous sarcoma virus-Rous-associated virus 1-transformed chicken embryo fibroblast was isolated and purified. To determine if there are modified, qualitatively or quantitatively different isoaccepting species of tRNA in these avian sarcoma viruses as compared with the cell of virus origin, chicken embryo fibroblast or normal chicken liver, methionyl-, arginyl-, and lysyl-tRNA (with high amino acid acceptance activity), and aspartyl- and glutamyl-tRNA from viral-trans-formed cells (with low viral amino acid acceptance activity) were co-chromatographed on reversed phase-5 chromatography columns, and elution profiles were compared. Although in each case the elution profile between a particular viral and host cell tRNA differed quantitatively, there was no qualitative difference in the profiles of corresponding tRNAs from either transforming or nontransforming viruses examined. Minor quantitative differences in the elution profiles might be a reflection of the metabolic state of the cells, since all evidence points to acceptor activity being of host rather than viral origin. Since, with the exception of selective packaging of methionyl-tRNA (IV) species by both transforming and nontransforming viruses, no selectivity was found for isoacceptor species of other tRNAs, it seems that such preferential packaging of methionyl-tRNA (IV) species has no bearing on the event of viral transformation.     

Rous sarcoma virus glycoproteins contain hybrid-type oligosaccharides.

Examination of [3H]mannose-labeled glycopeptides from Prague C Rous sarcoma virus gp85 with gel filtration and sequential glycosidase digestions demonstrated the presence of hybrid-type asparaginyl-oligosaccharides. The major hybrid species had an oligomannosyl core (Man5GlcNAc2-ASN) characteristic of neutral structures, plus "branch" sugars (NeuNAc-Gal-GlcNAc-) characteristic of complex, acidic structures.     

Comparison between the viral transforming gene (src) of recovered avian sarcoma virus and its cellular homolog.

Recovered avian sarcoma viruses are recombinants between transformation-defective mutants of Rous sarcoma virus and the chicken cellular gene homologous to the src gene of Rous sarcoma virus. We have constructed and analyzed molecular clones of viral deoxyribonucleic acid from recovered avian sarcoma virus and its transformation-competent progenitor, the Schmidt-Ruppin A strain of Rous sarcoma virus. A 2.0-megadalton EcoRI fragment containing the entire src gene from each of these clones was subcloned and characterized. These fragments were also used as probes to isolate recombinant phage clones containing the cellular counterpart of the viral src gene, termed cellular src, from a lambda library of chicken deoxyribonucleic acid. The structure of cellular src was analyzed by restriction endonuclease mapping and electron microscopy. Restriction endonuclease mapping revealed extensive similarity between the src regions of Rous sarcoma virus and recovered avian sarcoma virus, but striking differences between the viral src's and cellular src. Electron microscopic analysis of heteroduplexes between recovered virus src and cellular src revealed a 1.8-kilobase region of homology. In the cellular gene, the homologous region was interrupted by seven nonhomologous regions which we interpret to be intervening sequences. We estimate the minimum length of cellular src to be about 7.2 kilobases. These findings have implications concerning the mechanism of formation of recovered virus src and possibly other cell-derived retrovirus transforming genes.     

Metabolism of cartilage proteins in cultured tissue sections.

The asparagine-linked oligosaccharides of the complex acidic-type from [3H]mannose-, [3H]glucosamine- or [3H]galactose-labelled membrane glycoproteins of BHK21 cells and Rous-sarcoma virus were analysed by gel filtration combined with extensive digestion with endo- and exo-glycosidases from bacterial and eukaryotic sources. The neutral products from the digestion with a mixture of exoglycosidases and endo-beta-N-acetylglucosaminidase D from Diplococcus pneumoniae included a series of [3H]mannose- and [3H]glucosamine-labelled neutral oligosaccharides that were all converted by digestion with eukaryotic beta-N-acetylglucosaminidases into free N-acetylglucosamine and a small oligomannosyl core containing two alpha-linked mannose residues and a third mannose residue beta-linked to N-acetylglucosamine. These studies suggested that the complex acidic-type oligosaccharides from cellular and viral membrane glycoproteins contained a common oligomannosyl core region (Man2 alpha leads to Man beta leads to GlcNAc2), with heterogeneity in the number and/or linkage of outer branch N-acetylglucosamine residues resulting in partial resistance to beta-N-acetylglucosaminidase from a bacterial source.     

Oligosaccharide Chains of Avian RNA Tumor Virus Glycoproteins Contain Heterogeneous Oligomannosyl Cores

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

Virus-Dependent Glycosylation

The oligosaccharides of the membrane glycoproteins of Sindbis virus, vesicular stomatitis virus, and Rous sarcoma virus were compared on the basis of apparent size and sugar composition. It appears that each virus acquires a different set of oligosaccharides during growth in a single type of cell.     

Resolution of the DNA strands of the specialized transducing bacteriophage lambda-h80C 1-857 dargF.

The oligosaccharides of the membrane glycoproteins of Sindbis virus, vesicular stomatitis virus, and Rous sarcoma virus were compared on the basis of apparent size and sugar composition. It appears that each virus acquires a different set of oligosaccharides during growth in a single type of cell.