Carbohydrates of human immunodeficiency virus. Structures of oligosaccharides linked to the envelope glycoprotein 120

Human T-cells (H9), persistently infected with the HTLV-III strain of human immunodeficiency virus, were metabolically labeled with D-[2-3H]mannose or D-[6-3H]glucosamine. The viral envelope glycoprotein, gp120, was isolated either from cell lysates or from cell-free culture supernatant. After proteolytic digestion, the radiolabeled oligosaccharides were sequentially liberated from glycopeptides by treatment with endo-beta-N-acetylhexosaminidase H and peptide:N-glycosidase F. Oligosaccharides released were separated from residual (glyco)peptides and fractionated according to size, charge, and fucose content. The individual oligosaccharide species obtained were characterized by digestion with exoglycosidases and by chromatographic comparison with standard oligosaccharides. Our results demonstrate that the intracellular gp120 carries predominantly oligomannosidic glycans comprising nine or eight mannose residues. The secreted glycoprotein is equally substituted by oligomannosidic species, containing seven to nine mannose residues, and by fucosylated, partially sialylated bi- and triantennary complex-type oligosaccharides.     

Carbohydrate structure of glycoprotein 52 encoded by the polycythemia-inducing strain of Friend spleen focus-forming virus

The primary envelope (env) gene product of the polycythemia-inducing variant of Friend spleen focus-forming virus (F-SFFVP), representing a glycoprotein with an apparent Mr of 52,000 (gp52), was isolated from F-SFFVP-infected normal rat kidney cells metabolically labeled with [2-3H]mannose in the presence or absence of glucose. Structures of the oligosaccharides present were determined by micromethylation analysis, acetolysis, and digestion with exoglycosidases. Gp52 radiolabeled in the presence of glucose contains solely oligomannosidic glycans comprising 6 to 9 mannose residues (Man6-9GlcNAc2), some of which carry additional glucose. The structures of the glycans found reflect the typical intermediates of oligosaccharide processing. The glycosylation of gp52 isolated from glucose-deprived cells (-Glc), however, is characterized by increased amounts of Man5-7 species comprising other structural isomers. Only gp52 (-Glc) glycans are, in part, further processed yielding incomplete complex-type oligosaccharides. Our results demonstrate that the limited post-translational processing of the primary F-SFFVP env gene product is neither due to aberrant trimming of its oligomannosidic glycans nor due to transfer of immature lipid-linked oligosaccharide-intermediates as observed in glucose-starved cells.     

Carbohydrate structure of Marburg virus glycoprotein.

Marburg virus was propagated in E6 cells, a cloned cell line of Vero cells, in the presence of [6-3H]glucosamine. Radiolabelled viral glycoprotein was digested with trypsin, and oligosaccharides were liberated by sequential treatment with endo-beta-N-acetylglucosaminidase H, peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and O-glycosidase, by beta-elimination, and by alkaline hydrolysis. After fractionation by HPLC and gel filtration, glycans were characterized chromatographically, by digestion with exoglycosidases and, in part, by methylation analysis and liquid secondary ion mass spectrometry. The oligosaccharide structures thus established include oligomannosidic and hybrid-type N-glycans, as well as neutral fucosylated bi-, tri- and tetraantennary species, most of which carry an additional bisecting N-acetylglucosamine. In addition, high amounts of neutral mucin-type O-glycans with type-1 and type-2 core structures were detected. None of the glycans present in this viral glycoprotein carried sialic acid residues.     

Carbohydrate structure of glycoprotein 65 encoded by the polycythemia-inducing strain of Friend spleen focus-forming virus

The secondary envelope-gene product, glycoprotein 65 (gp65), of the polycythemia-inducing variant of Friend spleen focus-forming virus (F-SFFVp) was isolated from F-SFFVp-infected normal rat kidney cells cultivated in the presence or absence (-Glc) of glucose. Oligosaccharide side chains present were sequentially liberated by treatment of tryptic glycopeptides with endo-beta-N-acetylglucosaminidase H and peptide N-glycosidase F and fractionated by high-performance liquid chromatography. The glycans were characterized by digestion with exoglycosidases, by chromatographic comparison with oligosaccharide standards and by methylation analysis. The results demonstrate that gp65 contains oligomannosidic, hybrid and N-acetyllactosaminic glycans. The oligomannosidic glycans represent the same partially glucosylated species with six to nine mannose residues present in F-SFFVp gp52, the biosynthetic precursor of gp65 [Strube, K.-H. Schott, H.-H. and Geyer, R. (1988) J. Biol. Chem. 263, 3762-3771]. Oligosaccharides of the hybrid type were found to comprise one sialylated lactosamine unit and three or four alpha-linked mannose residues. Analysis of the N-acetyllactosaminic glycans revealed that gp65 carries fucosylated, partially sialylated bi-antennary, tri-antennary and tetra-antennary oligosaccharides, in addition to incomplete species. The glycosylation of gp65(-Glc) is characterized by the presence of oligomannosidic glycans with five to nine mannose residues, similar hybrid-type species and by increased amounts of incomplete N-acetyllactosaminic oligosaccharides, a decrease in sialylation and the lack of tetra-antennary species.     

Glycosylation of the envelope glycoprotein from a polytropic murine retrovirus in two different host cells

A polytropic recombinant retrovirus containing the envelope gene of Friend mink cell focus-inducing virus plus the remainder of the genome of an amphoropic murine leukemia virus was propagated on mouse embryo fibroblasts and mink lung cells. Virus particles, metabolically labeled with [2-3H]mannose, were harvested from the culture supernatants and lysed with detergents. The viral envelope glycoprotein was isolated from the lysates by immunoaffinity chromatography and purified by preparative SDS/PAGE. Oligosaccharides were liberated by sequential treatment of tryptic glycopeptides with endo-beta-N-acetylglucosaminidase H and peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F and fractionated by high-performance liquid chromatography. Individual glycans were characterized chromatographically, by methylation analyses and in part, by enzymic microsequencing. The results demonstrated that viral glycoproteins, synthesized in mouse embryo fibroblasts, carried as major constituents partially fucosylated diantennary, 2,4- and 2,6-branched triantennary and tetraantennary complex type N-glycans with 0-4 sialic acid residues and only small amounts of high-mannose type species with 5-9 mannose residues. As a characteristic feature, part of the complex type glycans contained additional Gal(alpha 1-3) substituents. Glycoprotein obtained from virions propagated on mink lung cells, contained partially fucosylated diantennary and 2,4-branched triantennary oligosaccharides with 1-3 sialic acid residues, in addition to trace amounts of high-mannose type species with 8 or 9 mannose residues. Thus, the results reveal that predominantly, the complex type N-glycans of the retroviral envelope glycoprotein display cell-specific variations including differences in oligosaccharide branching, sialylation and substitution by additional Gal(alpha 1-3) residues.     

Processing of the glycoprotein of feline immunodeficiency virus: effect of inhibitors of glycosylation.

The processing and transport of the envelope glycoprotein complex of feline immunodeficiency virus (FIV) in the persistently infected Crandell feline kidney (CRFK) cell line were investigated. Pulse-chase analyses revealed that the glycoprotein is synthesized as a precursor with an Mr of 145,000 (gp145) and is quickly trimmed to a molecule with an Mr of 130,000 (gp130). Treatment of gp130 with endoglycosidase H (endo H) resulted in a protein with an Mr of 75,000, indicating that nearly half the weight of the gp130 precursor consists of endo H-sensitive glycans during biosynthesis. Chase periods of up to 8 h revealed intermediates during the further processing of this glycoprotein precursor. Initially, two minor protein species with apparent Mrs of 100,000 and 90,000 were detected along with gp130. At later chase times these two species appeared to migrate as a single dominant species with an Mr of 95,000 (gp95). Concomitant with the appearance of gp95 was another protein with an Mr of approximately 40,000 (gp40). Chase periods of up to 8 h revealed that approximately half of the precursor was processed into the gp95-gp40 complex within 4 h. gp95 was efficiently transported from the cell into the culture medium by 1 to 2 h after labeling, whereas gp40 was not observed to be released from infected CRFK cells. Analysis of the processing in the presence of monensin, castanospermine, and swainsonine also suggests the existence of these intermediates in the processing of this lentivirus glycoprotein. As with human immunodeficiency virus, virus produced in the presence of glucosidase inhibitors and reduced infectivity for T-lymphocyte cultures.     

A predominant group-specific neutralizing epitope of human immunodeficiency virus type 1 maps to residues 342 to 511 of the envelope glycoprotein gp120.

Recombinant native human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins gp160 and gp120 (residues 1 to 511) expressed in insect cells quantitatively adsorbed the group-specific neutralizing antibodies found in human sera. However, these antibodies were not adsorbed by envelope fragment 1 to 471 or 472 to 857 or by both fragments sequentially, even though together they add up to the full-length gp160 sequence. A hybrid envelope glycoprotein was constructed with residues 342 to 511 of the HIV-1 sequence and residues 1 to 399 of the simian immunodeficiency virus type 1 sequence to vary the HIV-1 sequence while preserving its conformation. This hybrid glycoprotein quantitatively adsorbed human neutralizing antibodies, while native simian immunodeficiency virus type 1 envelope glycoprotein did not. These results identify a new neutralizing epitope that depends on conformation and maps to residues 342 to 511 of gp120. It overlaps the extended CD4-binding site but is distinct from the V3 loop described previously (K. Javaherian et al., Proc. Natl. Acad. Sci. USA 86:6768-6772, 1989; J. R. Rusche et al., Proc. Natl. Acad. Sci. USA 85:3198-3202). Since it is conserved among diverse HIV-1 isolates, this new epitope may be a suitable target for future vaccine development.     

Role of N-linked glycans in a human immunodeficiency virus envelope glycoprotein: effects on protein function and the neutralizing antibody response

The envelope (Env) glycoprotein of human immunodeficiency virus (HIV) contains 24 N-glycosylation sites covering much of the protein surface. It has been proposed that one role of these carbohydrates is to form a shield that protects the virus from immune recognition. Strong evidence for such a role for glycosylation has been reported for simian immunodeficiency virus (SIV) mutants lacking glycans in the V1 region of Env (J. N. Reitter, R. E. Means, and R. C. Desrosiers, Nat. Med. 4:679-684, 1998). Here we used recombinant vesicular stomatitis viruses (VSVs) expressing HIV Env glycosylation mutants to determine if removal of carbohydrates in the V1 and V2 domains affected protein function and the generation of neutralizing antibodies in mice. Mutations that eliminated one to six of the sites for N-linked glycosylation in the V1 and V2 loops were introduced into a gene encoding the HIV type 1 primary isolate 89.6 envelope glycoprotein with its cytoplasmic domain replaced by that of the VSV G glycoprotein. The membrane fusion activities of the mutant proteins were studied in a syncytium induction assay. The transport and processing of the mutant proteins were studied with recombinant VSVs expressing mutant Env G proteins. We found that HIV Env V1 and V2 glycosylation mutants were no better than wild-type envelope at inducing antibodies neutralizing wild-type Env, although an Env mutant lacking glycans appeared somewhat more sensitive to neutralization by antibodies raised to mutant or wild-type Env. These results indicate significant differences between SIV and HIV with regard to the roles of glycans in the V1 and V2 domains.     

Isolation of glycopeptides containing individual glycosylation sites of Friend murine leukemia virus glycoprotein: studies of glycosylation by methylation analysis

Glycopeptides containing individual N-glycosylation sites of the glycoprotein from Friend murine leukemia virus were isolated by digestion of the viral glycoprotein with protease of S. aureus (V8) or with trypsin followed by fractionation of the resulting (glyco)peptides by gel filtration and reversed-phase, high-performance liquid chromatography at pH 6. Isolated glycopeptides were assigned to the known amino acid sequence of the protein by amino acid analysis and by determination of the NH2-termini. The carbohydrate moieties of each glycosylation site were analysed by methylation analysis. A high selectivity of the glycoprotein glycosylation was found with regard to the distribution of oligomannosidic, mixed, and N-acetyl-lactosaminic oligosaccharides.     

Analysis by lectin affinity chromatography of N-linked glycans of BHK cells and ricin-resistant mutants.

Normal baby hamster kidney (BHK) fibroblasts and ricin-resistant (RicR) mutants of BHK cells derived from them were labelled metabolically with [3H]mannose or [3H]fucose. Glycopeptides obtained by digestion of disrupted cells with Pronase were separated by affinity chromatography on concanavalin A-Sepharose. In the normal BHK cells major glycopeptide fractions were obtained consisting of tetra- and tri-antennary sialylated complex glycans, bi-antennary sialylated glycans, and neutral oligomannosidic chains. The majority of bi-antennary chains were shown to contain a fucosyl-(alpha 1-6)-N-acetylglucosaminyl sequence in the core region by their ability to bind to a lentil lectin affinity column. All of the mutant cell lines examined were found to accumulate oligomannosidic glycans in cellular glycoproteins: complex sialylated glycans were either absent or greatly reduced in amount. Analysis of fractions isolated from concanavalin A-Sepharose by Bio-Gel P-4 chromatography and glycosidase degradation indicated that the glycans accumulating in RicR14 cells have the general structure: (formula; see text) and derivatives having fewer alpha-mannosyl units. We have also analysed the glycopeptides released by trypsin treatment from the surface of the normal and mutant cells, as well as those obtained by proteolysis of fibronectin isolated from the medium. The glycopeptide profiles of the cell-surface-derived material and of fibronectin showed for the mutant cells a marked accumulation of oligomannosidic chains at the expense of complex oligosaccharide chains. Hence, the alterations in glycan structure detected in bulk cellular glycoproteins of RicR cells are expressed also in cell surface glycoproteins and in fibronectin, a secreted glycoprotein.     

Identification of the ZPC oligosaccharide ligand involved in sperm binding and the glycan structures of Xenopus laevis vitelline envelope glycoproteins

The Xenopus laevis egg vitelline envelope is composed of five glycoproteins (ZPA, ZPB, ZPC, ZPD, and ZPX). As shown previously, ZPC is the primary ligand for sperm binding to the egg envelope, and this binding involves the oligosaccharide moieties of the glycoprotein (Biol. Reprod., 62:766-774, 2000). To understand the molecular mechanism of sperm-egg envelope binding, we characterized the N-linked glycans of the vitelline envelope (VE) glycoproteins. The N-linked glycans of the VE were composed predominantly of a heterogeneous mixture of high-mannose (5-9) and neutral, complex oligosaccharides primarily derived from ZPC (the dominant glycoprotein). However, the ZPA N-linked glycans were composed of acidic-complex and high-mannose oligosaccharides, ZPX had only high-mannose oligosaccharides, and ZPB lacked N-linked oligosaccharides. The consensus sequence for N-linked glycosylation at the evolutionarily conserved residue N113 of the ZPC protein sequence was glycosylated solely with high-mannose oligosaccharides. This conserved glycosylation site may be of importance to the three-dimensional structure of the ZPC glycoproteins. One of the complex oligosaccharides of ZPC possessed terminal beta-N-acetyl-glucosamine residues. The same ZPC oligosaccharide species isolated from the activated egg envelopes lacked terminal beta-N-acetyl-glucosamine residues. We previously showed that the cortical granules contain beta-N-acetyl-glucosaminidase (J. Exp. Zool., 235:335-340, 1985). We propose that an alteration in the oligosaccharide structure of ZPC by glucosaminidase released from the cortical granule reaction is responsible for the loss of sperm binding ligand activity at fertilization.     

Diversity of oligosaccharide structures on the envelope glycoprotein gp 120 of human immunodeficiency virus 1 from the lymphoblastoid cell line H9. Presence of complex-type oligosaccharides with bisecting N-acetylglucosamine residues

The N-linked oligosaccharide structures on the envelope glycoprotein gp120 of human immunodeficiency virus 1 derived from chronically infected lymphoblastoid (H9) cells have been investigated by enzymatic microsequencing after release from protein by hydrazinolysis, labeling with NaB3H4, and chromatography on adsorbent columns of Phaseolus vulgaris erythrophytohemagglutinin and Ricinus communis agglutinin (Mr 120,000) and on Bio-Gel P-4. A substantially greater diversity of oligosaccharide structures was detected than among those released by hydrazinolysis from recombinant gp120 produced in Chinese hamster ovary cells and investigated by similar procedures (Mizuochi, T., Spellman, M.W., Larkin, M., Solomon, J., Basa, L.J., and Feizi, T. (1988) Biochem J. 254, 599-603) and among those released by endoglycosidases from virus-derived gp120 isolated from infected H9 cells after metabolic labeling with D-[2-3H]mannose or D-[6-3H]glucosamine (Geyer, H., Holschbach, L., Hunsmann, G., and Schneider, J. (1988) J. Biol. Chem. 263, 11760-11767). In this study, 16% of the oligosaccharides were identified as complex-type bi-, tri-, and tetraantennary sialo-oligosaccharides with bisecting N-acetylglucosamine residues. Such structures were lacking on recombinant gp120 and could not be detected on the metabolically labeled, virus-derived glycoprotein. As in the earlier investigations, complex-type chains lacking bisecting N-acetylglucosamine residues, hybrid-type chains, and a series of high mannose-type structures with 5-9 mannose residues were identified. In addition, an array of complex-type chains having one or more outer chains with beta-galactosyl residues were detected in this study, but with additional substitutions that require further investigation. The number of potential N-glycosylation sites on gp120 is on the order of 20, but the oligosaccharide structures are far more numerous. Thus, the salient conclusion from this and earlier investigations is that alternative structures occur on at least some of the glycosylation sites and that numerous glycosylation variants of this glycoprotein are produced even within a single cell line. Since the glycosylation is the product of host cell glycosyltransferases, an even greater number of glycosylation variants of gp120 are predicted to arise from the heterogeneous cell populations harboring the virus in in vivo infection.     

Oligosaccharide profiles of HIV-2 external envelope glycoprotein: dependence on host cells and virus isolates

The glycosylation pattern of the external envelope glycoproteinof human immunodeficiency virus type 2 (HIV-2) was studied independence on host cells and virus isolates. Strains HIV-2_ALT,HIV-2_ROD and HIV-2_D194, differing in their biological propertiesand in the amino acid sequences of their env genes, were propagatedin MOLT4, HUT78 and U937 cells, in human peripheral blood lymphocytesand monocytes/macrophages in the presence of [6-³] glucosamine.Radiolabelled viral glycoproteins were isolated from the cell-freesupernatants and digested with trypsin. Glycans were sequentiallyliberated by endo-ß-N-acetylglucosaminidase H andpeptide-N⁴-(N-acetyl-ß-glucosaminyl) asparagine amidaseF, and fractionated according to charge and size. Comparisonof the oligosaccharide profiles revealed that the envelope glycoproteinsof different virus isolates, propagated in the same host cells,yielded very similar glycan patterns, whereas cultivation ofan isolate in different host cells resulted in markedly divergentoligosaccharide maps. Variations concerned the proportion ofhigh-mannose-, hybrid- and complex-type substituents, as wellas the state of charge and structural parameters of the complex-typespecies. As a characteristic feature, complex-type glycans ofmacrophage-derived viral glycoprotein were almost exclusivelysubstituted by lactosamine repeats. Hence, glycosylation ofthe HIV-2 external envelope glycoprotein seems to be primarilygoverned by host cell-specific factors rather than by the aminoacid sequence of the corresponding polypeptide backbone. envelope glycoprotein glycosylation human immunodeficiency virus type 2     

cDNA cloning of a 30 kDa erythrocyte membrane protein associated with Rh (Rhesus)-blood-group-antigen expression.

Lactotransferrin was highly purified from lysates of human neutrophilic leucocytes by immuno-affinity chromatography. A comparative analysis of the molar carbohydrate compositions of human leucocyte lactotransferrin and human milk lactotransferrin reveals that the glycans of leucocyte lactotransferrin differ essentially by the absence of fucose residues. Structural analysis combining methylation-mass spectrometry and 400 MHz 1H-n.m.r. spectrometry of oligosaccharide alditols released from human leucocyte lactotransferrin shows the presence of two disialylated and non-fucosylated biantennary glycans of the N-acetyl-lactosaminic type. These results question a previously proposed mechanism for hyposideraemia in which the leucocyte lactotransferrin was involved and in which the fucose residues played a key role.     

Sulfation of the human immunodeficiency virus envelope glycoprotein.

Sulfation is a posttranslational modification of proteins which occurs on either the tyrosine residues or the carbohydrate moieties of some glycoproteins. In the case of secretory proteins, sulfation has been hypothesized to act as a signal for export from the cell. We have shown that the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein precursor (gp160) as well as the surface (gp120) and transmembrane (gp41) subunits can be specifically labelled with 35SO42-. Sulfated HIV-1 envelope glycoproteins were identified in H9 cells infected with the IIIB isolate of HIV-1 and in the cell lysates and culture media of cells infected with vaccinia virus recombinants expressing a full-length or truncated, secreted form of the HIV-1 gp160 gene. N-glycosidase F digestion of 35SO4(2-)-labelled envelope proteins removed virtually all radiolabel from gp160, gp120, and gp41, indicating that sulfate was linked to the carbohydrate chains of the glycoprotein. The 35SO42-label was at least partially resistant to endoglycosidase H digestion, indicating that some sulfate was linked to complex carbohydrates. Brefeldin A, a compound that inhibits the endoplasmic reticulum to Golgi transport of glycoproteins, was found to inhibit the sulfation of the envelope glycoproteins. Envelope glycoproteins synthesized in cells treated with chlorate failed to incorporate 35SO42-. However, HIV glycoproteins were still secreted from cells in the presence of chlorate, indicating that sulfation is not a requirement for secretion of envelope glycoproteins. Sulfation of HIV-2 and simian immunodeficiency virus envelope glycoproteins has also been demonstrated by using vaccinia virus-based expression systems. Sulfation is a major determinant of negative charge and could play a role in biological functions and antigenic properties of HIV glycoproteins.     

Positive and negative modulation of virus infectivity and envelope glycoprotein incorporation into virions by amino acid substitutions at the N terminus of the simian immunodeficiency virus matrix protein

The matrix (MA) protein of the simian immunodeficiency viruses (SIVs) is encoded by the amino-terminal region of the Gag precursor and is the component of the viral capsid that lines the inner surface of the virus envelope. Previously, we identified domains in the SIV MA that are involved in the transport of Gag to the plasma membrane and in particle assembly. In this study, we characterized the role in the SIV life cycle of highly conserved residues within the SIV MA region spanning the two N-terminal alpha-helices H1 and H2. Our analyses identified two classes of MA mutants: (i) viruses encoding amino acid substitutions within alpha-helices H1 or H2 that were defective in envelope (Env) glycoprotein incorporation and exhibited impaired infectivity and (ii) viruses harboring mutations in the beta-turn connecting helices H1 and H2 that were more infectious than the wild-type virus and displayed an enhanced ability to incorporate the Env glycoprotein. Remarkably, among the latter group of MA mutants, the R22L/G24L double amino acid substitution increased virus infectivity eightfold relative to the wild-type virus in single-cycle infectivity assays, an effect that correlated with a similar increase in Env incorporation. Furthermore, the R22L/G24L MA mutation partially or fully complemented single-point MA mutations that severely impair or block Env incorporation and virus infectivity. Our finding that the incorporation of the Env glycoprotein into virions can be upregulated by specific mutations within the SIV MA amino terminus strongly supports the notion that the SIV MA domain mediates Gag-Env association during particle formation.     

A Twin-Cysteine Motif in the V2 Region of gp120 Is Associated with SIV Envelope Trimer Stabilization

The V1 and V2 variable regions of the primate immunodeficiency viruses contribute to the trimer association domain of the gp120 exterior envelope glycoprotein. A pair of V2 cysteine residues at 183 and 191 (“twin cysteines”) is present in several simian immunodeficiency viruses, human immunodeficiency virus type 2 (HIV-2) and some SIV_cpz lineages, but not in HIV-1. To examine the role of this potentially disulfide-bonded twin-cysteine motif, the cysteine residues in the SIVmac239 envelope glycoproteins were individually and pairwise substituted by alanine residues. All of the twin-cysteine mutants exhibited decreases in gp120 association with the Env trimer, membrane-fusing activity, and ability to support virus entry. Thus, the twin-cysteine motif plays a role in Env trimer stabilization in SIV and may do so in HIV-2 and some SIV_cpz as well. This implies that HIV-1 lost the twin-cysteines, and may have relatively unstable Env trimers compared to SIV and HIV-2.     

Carbohydrate structure of recombinant human uterine tissue plasminogen activator expressed in mouse epithelial cells

Recombinant human uterine tissue plasminogen activator (tPA), in part metabolically labeled with [6-3H]glucosamine or [35S]sulfate, was isolated from mouse epithelial cells (C127). Oligosaccharides present were liberated by treatment of tryptic glycopeptides with endo-beta-N-acetylglucosaminidase H or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and fractionated by high-performance liquid chromatography. The glycans were characterized by digestion with exoglycosidases, methylation analysis and, in part, by acetolysis and 1H-NMR spectroscopy. Glycopeptides comprising individual glycosylation sites were identified by N-terminal amino acid sequencing. The results demonstrate that recombinant tPA from C127 cells carries at Asn117 oligomannosidic glycans with 5-8 mannose residues as well as small amounts of hybrid-type species. Asn184 is only partially glycosylated and substituted by fucosylated triantennary and small amounts of diantennary N-acetyllactosaminic glycans. Likewise, Asn448 carries predominantly fucosylated triantennary species, in addition to, small amounts of diantennary and tetraantennary oligosaccharides. As a characteristic feature, part of the triantennary glycans at Asn184 and Asn448 contain additional Gal(alpha 1-3) substituents and/or sulfate groups linked to position six of beta-galactosyl residues forming NeuAc(alpha 2-3)[HO3S-6]Gal(beta 1-4) units. Oligosaccharides attached to Asn448 are almost completely substituted by (alpha 2-3)- or (alpha 2-6)-linked sialic acid residues and carry the majority of sulfate groups present. Glycans at Asn184 were found to be less sialylated and sulfated.     

An epitope in the V1 domain of the simian immunodeficiency virus (SIV) gp120 protein is recognized by CD8+ cytotoxic T lymphocytes from an SIV-infected rhesus macaque.

Cytotoxic T-lymphocyte (CTL) responses against the external envelope glycoprotein (gp120) of the simian immunodeficiency virus (SIV) were studied in a rhesus macaque infected with SIVmac/239. CD8+ T cells enriched from concanavalin A-stimulated peripheral blood mononuclear cells lysed autologous target cells infected with recombinant vaccinia virus vectors expressing the SIVmac/239 or SIVsm/H4 envelope protein, which share approximately 80% identity in amino acid sequence. A CD8+ CTL line derived by limiting dilution culture of the concanavalin A-stimulated lymphocytes was also specific for the envelope proteins of both SIV isolates. Mapping studies revealed that this cell line recognized an epitope between amino acids 113 and 121 (CNKSETDRW) in the V1 domain of gp120. Amino acid substitutions are observed at positions 116 and 120 among viruses of the SIVsm/mac/human immunodeficiency virus type 2 group, and thus synthetic peptides representing these variants were tested for the ability to sensitize target cells for lysis by the CTL line. Autologous target cells sensitized with a synthetic peptide representing the SIVmac/239 sequence were efficiently killed. In contrast, recognition of target cells was reduced or abolished when peptides representing the amino acid substitutions at position 116 or 120 of other SIVmac, SIVsm, SIVmne, or SIVstm strains were tested. Further studies of CTL responses against this epitope could provide insights into mechanisms of variability within the gp120 V1 domain and its importance in evasion of immunity in infected or vaccinated monkeys.     

Truncation of the cytoplasmic domain of the simian immunodeficiency virus envelope glycoprotein increases env incorporation into particles and fusogenicity and infectivity.

Growth of macaque simian immunodeficiency virus (SIVmac) in certain cloned human T-cell lines, such as HUT.78, selects for isolates containing a premature stop codon within the cytoplasmic domain of the transmembrane envelope glycoprotein. In contrast, propagation of virus in macaques or in their cultured T cells favors replication of virus containing the full-length envelope glycoprotein. To elucidate the causes of this phenomenon, we used a human immunodeficiency virus pseudotyping system to assess the effects on infectivity of the cytoplasmic domains of envelope glycoproteins obtained from SIVmac1A11 and SIVmac239. These envelopes contain truncated and full-length cytoplasmic domains, respectively. By analyzing human immunodeficiency virus particles containing selectable genes pseudotyped with each glycoprotein or with chimeric derivatives, we found that truncation of the cytoplasmic domain resulted in a significant advantage in viral entry into HUT.78 T cells and CD4+ U87.MG glial cells. Truncation of the cytoplasmic domain significantly enhanced both envelope density on particles and envelope-mediated cell-to-cell fusion. It is likely that one or both of these effects contribute to the observed differences in infectivity and to the selection of virions with short cytoplasmic tails in human T cells.