Retroviral Envelope Glycoprotein Research Articles

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.

Mutational analysis of the cleavage sequence of the human immunodeficiency virus type 1 envelope glycoprotein precursor gp160.

The envelope glycoproteins of the human immunodeficiency virus (HIV) type 1 are synthesized as a precursor molecule, gp160, which is cleaved to generate the two mature envelope glycoproteins, gp120 and gp41. The cleavage reaction, which is mediated by a host protease, occurs at a sequence highly conserved in retroviral envelope glycoprotein precursors. We have investigated the sequence requirements for this cleavage reaction by introducing four single-amino-acid changes into the glutamic acid-lysine-arginine sequence immediately amino terminal to the site of cleavage. We have also examined the effects of these mutations on the syncytium formation induced by HIV envelope glycoproteins. Our results indicate that a glutamic acid to glycine change at gp120 amino acid 516, a lysine to isoleucine change at amino acid 517, and an arginine to lysine change at amino acid 518 affect neither gp160 cleavage nor syncytium formation. The results obtained with the arginine to lysine change at amino acid 518 differ significantly from the results obtained with the same mutation at the envelope precursor cleavage site of a murine leukemia virus (E. O. Freed, and R. Risser, J. Virol. 61:2852-2856, 1987). An arginine to threonine mutation at gp120 amino acid 518, the terminal residue of gp120, abolishes both gp160 cleavage and syncytium formation. These findings demonstrate that despite its highly conserved nature, the basic pair of amino acids at the site of gp160 cleavage is not absolutely required for proper envelope glycoprotein processing. This report also supports the idea that cleavage of gp160 is required for activation of the HIV envelope fusion function.

Integration and expression of murine retrovirus-related sequences in schistosomes.

Antibodies against the retrovirus envelope glycoprotein (gp70) of mouse xenotropic retrovirus, BALB virus 2 (Bv2) reacted with the adult worms of Schistosoma japonicum and S. mansoni. This reaction was completely inhibited after adsorption of the antibodies with virions of retrovirus. The reactive schistosome antigen was located in the subtegumental layer of the adult male fluke and in the vitelline gland of the adult female of S. japonicum and S. mansoni. Proteins extracted from both parasites were examined by immunoblot analysis. Anti-Bv2 gp70 antiserum reacted with those proteins from both schistosomes and the band patterns were different among sexes and species. Southern hybridization of the DNA extracted from adults of S. japonicum and S. mansoni demonstrated the presence of sequences homologous to the env gene of mouse ecotropic and xenotropic retroviruses. DNA sequences homologous to the gag and pol regions of the ecotropic murine leukaemia virus were also detected in the DNAs of schistosomes.

Formation of infectious hybrid virions with gibbon ape leukemia virus and human T-cell leukemia virus retroviral envelope glycoproteins and the gag and pol proteins of Moloney murine leukemia virus.

The gibbon ape leukemia virus, SEATO strain, and human T-cell leukemia virus type I envelope glycoproteins can be functionally assembled with a Moloney murine leukemia virus core into infectious particles. The envelope-host cell receptor interaction is the major determinant of the host cell specificity for these hybrid virions.

Influence of env and long terminal repeat sequences on the tissue tropism of avian leukosis viruses.

Adsorption and penetration of retroviruses into eucaryotic cells is mediated by retroviral envelope glycoproteins interacting with host receptors. Recombinant avian leukosis viruses (ALVs) differing only in envelope determinants that interact with host receptors for subgroup A or E ALVs have been found to have unexpectedly distinctive patterns of tissue-specific replication. Recombinants of both subgroups were highly expressed in bursal lymphocytes as well as in cultured chicken embryo fibroblasts. In contrast, the subgroup A but not subgroup E host range allowed high levels of expression in skeletal muscle, while subgroup E but not subgroup A envelope glycoproteins permitted efficient replication in the thymus. A subgroup B virus (RAV-2), like the subgroup E viruses, demonstrated a distinct bursal and thymic tropism, further supporting the theory that genes encoding receptors for subgroup B and E viruses are allelic. The source of long terminal repeats (LTRs) or adjacent sequences also influenced tissue-specific replication, with the LTRs from endogenous virus RAV-0 supporting efficient replication in the bursa and thymus but not in skeletal muscle. These results indicate that ALV env and LTR regions are responsible for unexpectedly distinctive tissue tropisms.

Expression of endogenous retroviral envelope glycoprotein as a determinant of immunity to rous sarcoma

To analyze the effect of the expression of endogenous retroviral envelope glycoprotein on tumor immunity, patterns of sarcoma growth were compared in inbred FP line chickens infected with either of two strains of avian sarcoma virus, Pr-B (subgroup B) or cl.85 (subgroup G). These viruses were chosen for analysis because the envelope glycoprotein of Pr-B, but not of cl.85, is antigenically cross-reactive with the endogeneous retroviral envelope glycoprotein expressed in the FP line. Inoculation of 1-day-old hatchmates with either virus yielded a significant percentage of chickens with distal sarcomas localized to visceral organs. By contrast, a marked difference in the percentage of chickens bearing distal sarcomas was noted when sarcoma tissue excised from virus-inoculated donors was implanted in 1-day-old recipients; a high proportion of the recipients of Pr-B-induced sarcoma tissue (Pr-B-sarcoma recipients), but only a low proportion of the cl.85-sarcoma recipients, exhibited distal sarcomas. At 3 weeks posthatch, a significantly higher percentage of donor-derived cells was detected in the primary tumors of the cl.85- versus the Pr-B-sarcoma recipients. A model of immune control, premised on the tolerogenicity of endogenous viral glycoprotein, is proposed to rationalize these results.

Neutralization of the AIDS Retrovirus by Antibodies to a Recombinant Envelope Glycoprotein

Mammalian cell lines have been engineered to produce a secreted form of the AIDS retrovirus envelope glycoprotein. The recombinant protein has been isolated from growth-conditioned culture media and used to immunize animals. Antibodies directed against the recombinant molecule were found to react with the envelope glycoprotein produced in virus-infected cells. Furthermore, these antibodies were able to directly inactivate the AIDS retrovirus in a neutralization assay in vitro. The expression system reported here should provide sufficient quantities of the AIDS retrovirus envelope protein for biological and vaccination studies.

Characterization of the endogenous retroviral envelope glycoproteins found in the sera of ev3 and ev6 chickens.

Chicken sera may contain components that immunologically crossreact with avian retroviral glycoprotein. Sera from chickens carrying the endogenous viral loci ev3 and ev6 contain glycoproteins with molecular weights of approx. 85 000, which on the basis of tryptic peptide analysis are related to exogenous viral glycoprotein. Such endogenous viral glycoproteins are present in a free form, lacking disulphide-bonded gp35.

Antigens and circulating immune complexes related to the primate retroviral glycoprotein SiSVgp70: Prevalence and distribution in human sera

We have shown previously that antigens and also circulating immune complexes related to the primate retroviral envelope glycoprotein SiSVgp70 correlate with early mortality and survival of 56 patients with acute leukemias and chronic myelogenous leukemia in blast crisis. The prevalence and general distribution of these antigens and immune complexes in human sera was therefore of obvious interest. We now report an analysis of a total of 200 sera from 166 patients. Of these 113 sera were obtained from 84 patients with acute or chronic leukemias and 87 from 82 non-leukemic subjects, including laboratory workers and patients with non-leukemic neoplasias. Antigens and immune complexes were determined by enzyme-linked immunosorbent assays (ELISA). The anti-SiSVgp70 antiserum used predominantly recognized the protein moieties of the glycoproteins. The distribution of SiSVgp70-related antigens and immune complexes was similar among leukemic and non-leukemic sera. The prevalence of SiSVgp70-related antigens was 53% and of SiSVgp70-related immune complexes 49% in all sera. SiSVgp70-related antigens were detected in a somewhat higher proportion of non-leukemic (69%) than leukemic sera (40%), whereas SiSVgp70-related immune complexes and cross-reactive antibodies were more evenly distributed in leukemic and non-leukemic sera (in 46 and 51%) of leukemic and 54 and 51% of non-leukemic sera). Presence of antigens correlated with presence of SiSVgp70-related immune complexes in 71% of all sera, but in 13% of all sera antigens were detectable only by determining SiSVgp70-related immune complexes. Total circulating immune complexes did not correlate with SiSVgp70-related immune complexes. The origin and pathophysiological role of the antigens are discussed.

Recognition of a leukemia-related antigen by an antiidiotypic antiserum to an anti-gp70 monoclonal antibody.

The possibility that receptors for retroviral gp70 share structural elements with the antigen-binding sites of anti-retroviral gp70 antibodies was investigated. A monoclonal antibody (1416) was produced that reacted with the gp70 of a cloned recombinant leukemogenic retrovirus, termed P1. An antiidiotypic antiserum raised to 1416 was tested for its ability to bind to the thymic leukemia induced by P1 (P1 Thy). A membrane structure was identified on the surface of P1 Thy that reacted with the antibody against the idiotypic determinant of 1416. A similar structure was identified on the surface of several different, independently derived murine leukemias of T cell, B cell, and erythroid lineage. The expression of the idiotype-like determinant on these leukemia cells was independent of the serological relatedness of their expressed retroviral envelope glycoproteins to P1 gp70. The determinant recognized by the antiidiotype was not detected on normal lymphoid cells. The recognition by the anti-(anti-gp70) idiotype of determinants on unrelated murine leukemias suggests that receptors for different leukemogenic viruses may share common structures.

Antigens and circulating immune complexes related to the primate retroviral glycoprotein SiSVgp70. Indicators of early mortality in human acute leukemias and chronic myelogenous leukemias in blast crisis.

Human sera contain antigens and also circulating immune complexes that are related to the primate retroviral envelope glycoprotein gp70 of simian sarcoma/simian sarcoma associated virus (SiSV) and of gibbon ape leukemia virus (GaLV). SiSVgp70 related antigens (AG) and immune complexes (IC) are detected both in leukemic and in nonleukemic sera. In a further analysis of these data, the prognostic significance of SiSVgp70 related AG and IC in leukemic patients was examined. The data show that the presence of SiSVgp70 related AG and IC indicates an unfavorable clinical course and a shorter survival time in acute leukemias (AL) and in chronic myelogenous leukemia in blast crisis (CML-BC). Survival data of 56 of 64 patients tested were analyzed (38 patients with AL and 18 patients with CML-BC). Patients with AL whose sera were positive for SiSVgp70 related AG and IC had a median survival time of 9.5 months after diagnosis versus 16 months for patients negative for such AG and IC. This difference in survival time was more pronounced for patients with acute nonlymphocytic leukemia (ANLL) (6.5 versus 19 months). The difference in survival between SiSVgp70 related AG- and IC-negative and positive groups as tested by life table analysis (log-rank test) is significant (P less than 0.05). Patients with AL of the AG- and/or IC-positive group had fewer complete remissions. Patients who had no remissions belong to the AG- and/or IC-positive group (P = 0.06). Patients with CML-BC whose sera were positive for SiSVgp70 related AG and/or IC had a median survival time of 2 months after diagnosis versus 7 months for patients with sera negative for such AG and IC. As tested by log-rank test, survival curves between the two groups are significantly different (P less than 0.05). These findings suggest that SiSVgp70 related AG and IC may play an important role in the course of acute leukemia and can provide useful prognostic information.

Increased endogenous retroviral gene expression is a consequence of lymphocyte activation.

Plasma cells of line 151(5) chickens have been shown to express elevated levels of endogenous retroviral envelope glycoprotein (VEG), measured relative to levels expressed by both immature B cells and resting peripheral B lymphocytes. In this study we analyzed the relationship between peripheral blood lymphocyte (PBL) maturation and the level of VEG expression. A culture system was developed that would support maturation of pokeweed mitogen-activated peripheral B lymphocytes. As analyzed by cytofluorometry, both Ig+ and Ig- lymphoblasts present in the pokeweed mitogen-stimulated cultures expressed detectable levels of VEG in contrast to bursacytes and PBL. Similarly, Ig- blasts, which were present in concanavalin A-stimulated cultures of PBL and presumed to represent activated T cells, were also positive for the expression of VEG. Immature T cells, i.e., thymocytes, although negative by immunofluorescence analysis, expressed VEG at levels that were detectable by radioimmunochemical techniques. These results indicate that T cells as well as B cells constitutively express VEG, and that mitogenic activation of the resting lymphocyte induces an increase in VEG expression.

Differential reactivity of serum antibody from tumor-bearing 15I 5 × 7 2 chickens for cross-reactive species of endogenous retroviral envelope glycoprotein

Experiments were undertaken to determine if the sera of avian sarcoma virus-infected 15I 5 × 7 2 chickens exhibit antibody reactivity for species of endogenous retroviral envelope glycoprotein expressed in 15I 6 × 7 2 fibroblasts. Two viruses were used for infection of the 15I 5 × 7 2 chickens, Pr-A and cl. 85; the envelope glycoprotein of Pr-A, but not of cl. 85, is antigenically cross-reactive with 15I 5 × 7 2 endogenous retroviral envelope glycoprotein. Both the Pr-A and cl. 85-infected 15I 5 × 7 2 chickens exhibited serum antibody reactivity for the envelope glycoprotein of the endogenous retrovirus RAV-0. In contrast, neither group of infected chickens exhibited detectable serum antibody reactivity for a distinct species of endogenous retroviral envelope glycoprotein, one which though antigenically cross-reactive with the envelope glycoprotein of RAV-0 is expressed to much higher levels in 15I 5 × 7 2 fibroblasts. Possible mechanisms to account for the observed pattern of antibody reactivity are discussed.

B cell expression of endogenous retroviral envelope antigen: biochemical and immunofluorescence characterization.

Plasma cells of line 15I5 chickens have been shown to express antigen that is cross-reactive with endogenous retroviral envelope glycoprotein. In the present study, we isolated this antigen and showed that it is structurally homologous to envelope glycoprotein. As assayed by immunofluorescence, most, if not all, of the plasma cell-associated envelope glycoprotein is present on the cell surface although it is not bound to surface Ig or Ia antigen. Although envelope antigen was not detectable by immunofluorescence on the surface of 15I5 bursal cells or peripheral B lymphocytes, immunochemical assays established that retroviral envelope glycoprotein is present on the surface of the bursal cells but at much lower levels than on the surface of the plasma cells. Both bursal and plasma cells synthesize envelope glycoprotein. These findings indicate that an increased level of expression of endogenous retroviral envelope glycoprotein is a concomitant of the differentiation of B lymphocytes to plasma cells in 15I5 chickens.

Avian endogenous retroviral envelope glycoprotein is assembled in two structural complexes of gp85 and gp37 subunits

Experiments were undertaken to determine the subunit structure of endogenous retroviral envelope glycoprotein present on Rous-associated virus-O (RAV-O) or expressed by 15I 5 × 7 2 fibroblasts and plasma cells. As analyzed under nonreducing conditions, the RAV-O-associated and 15I 5 × 7 2 cell-associated envelope glycoprotein comprised two components, designated VGP1 and VGP2, with approximate molecular weights of 200,000 and 100,000, respectively. By contrast, the envelope glycoprotein of exogenous strains of virus comprised predominantly, if not exclusively, VGP2. Our interpretation of the structural data is that VGP1 and VGP2 represent, respectively, a disulfide-linked tetramer and dimer of equimolar gp85 and gp37 subunits.

Human antibodies recognizing the envelope glycoprotein of the baboon endogenous virus BaEV are of heterophil origin.

Human sera were previously shown to possess antibodies capable of recognizing purified retrovirus envelope glycoproteins. In an extension of earlier studies we investigated sera from various groups of patients for an immune reaction against purified glycoprotein of the baboon endogenous virus BaEV. Reproducible demonstrations of oncovirus-like particles in human teratocarcinomas focused our main interest on sera from patients with testicular tumors. The specificity of the positive immune reaction of sera from these patients against BaEV gp 70 was analyzed in a competition RIAs with haptens and different cell lysates and experiments employing deglycosylated BaEV envelope antigen. From these experiments we conclude that the sera from teratocarcinoma patients contain naturally occurring, heterophil antibodies that react with the carbohydrate moieties of retrovirus envelope antigens.

Low-calorie diet selectively reduces expression of retroviral envelope glycoprotein gp70 in sera of NZB x NZW F1 hybrid mice.

The effect of dietary restriction on the expression of retroviral envelope glycoprotein, gp70, and the formation of gp70-anti gp70 immune complexes was investigated in lupus-prone NZB x NZW F1 hybrid mice. Restricting total calorie intake from the usual 20 to only 10 calories per day after weaning markedly reduced serum levels of both free and antibody-complexed gp70, prevented renal disease, and increased the life spans of these mice. The reduction in serum gp70 was evident after only 2 wk of feeding these animals the low-calorie diet, and the concentration remained virtually unchanged throughout the course of 10 mon experimentation. However, serum concentrations of the major structural protein, p30, of endogenous retroviruses were not altered by restricting calories. Amounts of the serum glycoprotein, haptoglobin, decreased parallel to those of gp70 but amounts of albumin did not. These results suggest that the expression of gp70 in serum is controlled independently of the production of complete viral particles, and regulated by a mechanism similar to that for other serum glycoproteins, such as haptoglobin.

Retroviral gp70 immune complexes in NZB x NZW F2 mice with murine lupus nephritis.

NZB x NZW (NZB x W) F1 hybrid mice spontaneously develop a disease most prominently characterized by immune complex glomerulonephritis (GN), which seems to be associated with both antibodies to DNA and to the serum retroviral envelope glycoprotein, gp70. To evaluate the contribution of each of these autoimmune responses to the pathogenesis of the GN, we studied NZB x W F2 mice in which the two responses appeared to segregate relatively independently. Use of this model permitted analysis of possible correlations between each response and the G.N. The presence of circulating anti-gp 70-complexed gp70 correlated significantly with the development of fatal GN and one could predict the course of renal disease by computing the rising serum levels of gp70 complexed with antibodies. In contrast, the presence of free antibodies to either double-stranded or single-stranded DNA was not significantly associated with the development of fatal GN. This association of anti-gp70 antibody production with these animals' early death from GN strongly suggests that the gene(s) governing production of antibodies to serum retroviral gp70 may be one of the major genes responsible for spontaneous renal disease segregated in NZB x W F2 generations.

Expression of endogenous retroviral envelope antigen in Ig(+) B cells of immunized 15I5 X 71 chickens.

In a previous study (1), we observed that antigen cross-reactive with structural protein of the endogenous retrovirus RAV-O is expressed in splenic Ig(+) B lymphocytes of immunized 15I5 X 71 chickens. The present experiments were undertaken to determine the relationship of this antigen to viral gag- and env-coded antigenic specificities. As analyzed by immunofluorescence with several antisera, group-specific determinants of the gp85 envelope glycoprotein as well as viral-related type E-specific determinants were detectable in islands of Ig(+) splenocytes; antigenic determinants of viral gag protein were not detected in the splenic islands. These observations indicated that antigen cross-reactive with endogenous retroviral envelope glycoprotein is expressed in Ig(+) B cells.

Heterophil human antibodies recognize oncovirus envelope antigens: Epidemiological parameters and immunological specificity of the reaction

Human sera were previously shown to possess antibodies capable of recognizing purified retrovirus envelope glycoproteins in standard radioimmunoprecipitation assays (RIAs). In a logical extension of the earlier studies, epidemiological characteristics and the exact specificity of the human antiviral antibodies was analyzed. The antiviral antibodies are not detectable in cord sera but show an age-dependent peak in early childhood and subsequently persist lifelong. Based on epidemiological and additional data obtained by serurn absorptions to cell surfaces, competition RIAs with animal sera and experiments employing deglycosylated virus envelope antigens, we conclude that the majority (if not all) of normal human sera contain naturally occurring, heterophil antibodies that react with the carbohydrate moieties of retrovirus envelope antigens. Similar conclusions have independently been reached by M. Barbacid, D. Bolognesi, and S. A. Aaronson (1980, Proc. Nat. Acad. Sci. USA 77, 1617–1621) and by H. W. Snyder and E. Fleissner (1980, Proc. Nat. Acad. Sci. USA 77, 1622–1626).