Processing of gPr92env, the precursor to the glycoproteins of Rous sarcoma virus: use of inhibitors of oligosaccharide trimming and glycoprotein transport

A number of aspects of the processing of gPr92env, the precursor to the viral glycoproteins gp85 and gp35 of Rous sarcoma virus (RSV), have been studied. First, the kinetics of gPr92env processing have been examined, revealing that the precursor is overproduced in the infected cell and only a small percentage (less than 5%) is converted into mature glycoprotein in virus particles. Second, the effects of inhibitors of intracellular transport (monensin) and oligosaccharide trimming (N-methyl-1-deoxynojirimycin (MdN) and bromoconduritol (BC) ) on the processing of gPr92env have been examined. It could be shown with all three inhibitors that proteolytic cleavage of gPr92env could occur although oligosaccharide trimming was inhibited. The aberrant cleavage products, gp75mon and gp30mon, produced in the presence of monensin, carry oligosaccharides where only 1-3 mannose residues have been removed in comparison to the precursor gPr92env (this latter carries predominantly Man9(GlcNAc)2). Virus particles containing the aberrant glycoproteins were released in virtually normal amounts and were infectious. In the presence of MdN and BC, viral glycoprotein precursors carrying three (MdN) or one (BC) glucose on the high-mannose oligosaccharide could be detected intracellularly. The aberrant precursors could be proteolytically cleaved to gp80MdN and gp75BC which are equivalent to gp85 but carry the smaller glucose-containing high-mannose oligosaccharides instead of the large, complex, sialidated oligosaccharides of mature glycoprotein. In the presence of MdN, the abnormal glycoproteins were incorporated into virions which were fully infectious.     

Gene order of mouse mammary tumor virus precusor polyproteins and their interaction leading to the formation of a virus.

Mouse mammary tumor virus (MMTV) proteins are synthesized as two major precursor polyproteins; gPr75^env containing gp52 and gp36, and Pr75^gag containing p27, pp20, p14, and p10. The gene order for gPr75^env has been previously shown to be H₂N-gp52-gp36-COOH (Schochetman, et al., 1977). gag polyproteins undergo intracellular cleavage in cat cells infected with MMTV and GR mammary tumor cells. Based on immunoprecipitation studies with antisera against intermediate MMTV cleavage products we now report the gene order for Pr75^gag is H₂N-p10-pp20-p27-p14-COOH. These results were further substantiated by analyzing the binding to ssDNA of the intermediate cleavage products which contain p14. To analyze the interaction of MMTV proteins with the cell membrane leading to budding of a virus particle, we used (i) lactoperoxidase-catalyzed iodination of MMTV cell surface proteins, (ii) galactose oxidase-catalyzed radiolabeling of carbohydrates on cell surface MMTV glycoproteins, (iii) serum cytotoxicity based on [⁵¹Cr] release with monospecific MMTV antisera, and (iv) membrane immunofluorescence with monospecific MMTV antisera. Analysis of ¹²⁵I-labeled MMTV cell surface antigens by immune precipitation with MMTV anti-gp52, gp36, p27, p14, and p10 sera followed by SDS-PAGE revealed only ¹²⁵I-gp52. In contrast, cell surface glycoprotein labeling revealed [³H]gp52 and [³H]gp36, indicating that, although the protein portion of gp36 was buried, some carbohydrate regions were exposed. EDTA treatment of cells to alter cell membranes prior to iodination resulted in the labeling of both Pr75^gag and gp52 but not gPr75^env. Furthermore, anti-p10 but not anti-p27 serum was cytotoxic against EDTA-treated cells. Similar results were obtained when the same antisera were tested by membrane immunofluorescence, ruling out the possibility that anti-p27 serum was not cytotoxic because it was unable to fix complement. These results show that Pr75^gag molecules, presumably as MMTV cores, interact with cell membrane sites containing gp52 and gp36 via the hydrophobic p10 portion of the molecule.     

Expression and disposition of the murine mammary tumor virus (MuMTV) envelope gene products by murine mammary tumor cells

Three murine mammary tumor virus (MuMTV)-producing epithelial cell lines derived from murine mammary tumors were examined in order to identify the MuMTV-specific cell surface antigens and their distribution on the cell surface, to study the kinetics of the MuMTV envelope precursor processing, virus assembly, and release, and to characterize the soluble MuMTV antigens that are shed into culture medium. Cell surface labeling experiments showed that only the mature MuMTV envelope glycoproteins gp52 and gp36 were exposed on the cell surface, and that gp52 was more abundant than gp36. In cells producing large quantities of MuMTV, expression of gp52 on the cell surface was shown by immunoelectron microscopy to be localized predominantly on the surface of budding virions and not on smooth areas of the cell surface where virus was not budding. The cell surface associated gp36 was found not to be incorporated into budding virions. A few cells in all three cell lines were found to produce only a few or no MuMTV particles and in these cells, unlike in the high virus-producing cells, considerable quantities of gp52 were expressed on the surface membrane. All three cell lines were found to shed large amounts of the MuMTV env precursor polyprotein as well as the mature non-virion-associated glycoprotein, gp52, into the culture medium. The envelope precursor protein (P75^env) that was shed into the culture medium was found to differ from the predominant form of the cellular env precursor Pr70^env in that (1) P75^env migrated with an apparent higher molecular weight than Pr70^env in SDS gels; (2) Pr70^env contained only the core oligosaccharide, whereas P75^env contained fucose in addition to the core sugars; (3) two-dimensional gel electrophoretic analysis showed that Pr70^env could be resolved into three to four components migrating in the basic region of the isoelectric focussing gel (pH 7–8), whereas P75^env was resolved into 9–13 components migrating in a more acidic region of the gel (pH 5–7). The molecular structure of the exfoliated gp52 was found to be similar to that of the gp52 that was incorporated into the virions although the virion-associated gp52 was not the source of the gp52 in the medium. Our quantitative pulse-chase studies suggest that of the two populations of MuMTV env precursors that are present in MuMTV-producing cells, only Pr70^env is processed intracellularly to give rise to the mature MuMTV envelope proteins gp52 and gp36.     

An analysis of type-C retrovirus polypeptides and their associations in the virion.

The polypeptide compositions of [³H]leucine-labeled Prague-Rous sarcoma virus-subgroup C (Pr-RSV-C) and Friend murine leukemia virus (FLV) were investigated using guanidine hydrochloride gel filtration and a high-resolution SDS-polyacrylamide-gel system. These techniques resolved seven major structural components in Pr-RSV-C (gp85, gp35, p27, p19, p12, p15, and p10), as reported previously for other avian leukosis-sarcoma viruses. However, in the case of FLV two previously unresolved proteins (p15E and p12E) were clearly demonstrated in addition to gp71, p30, p15C, p12, and p10. FLV p15E, p12E, and gp71 were removed when intact virions were digested with bromelain, whereas the remaining components were not affected. These and other studies support the notion that gp71, p15E, and p12E are situated on the surface of the virion. The linkages (covalent and noncovalent) between viral polypeptides were also examined. The results of these studies indicate that more than 90% of avian gp85 and gp35 are disulfide linked as a viral glycoprotein complex (VGP). The data also suggests that p19 exists as a network of disulfide-linked molecules, some of which may be further linked to VGP. In contrast to the avian system, only about 10–15% of FLV gp71 is disulfide linked to p15E in the VGP complex; the remaining gp71 is apparently loosely attached to the virus, perhaps by a noncovalent interaction with p12E. The implications of these associations to virus structure and assembly are discussed.     

Cell-free synthesis of Rauscher murine leukemia virus "gag" and "env" gene products from separate cellular mRNA species.

RNA from cells infected with Rauscher murine leukemia virus (R-MuLV) has been translated in an mRNA-dependent cell-free protein synthesizing system. It was found that a cellular RNA species of about 35 S in size codes for polypeptides of approximately 65,000 MW (Pr65^gag) and 200,000 MW (Pr200^gag) which are immunoprecipitable with antisera directed against the R-MuLV gag proteins p30, p15, p12, and p10. The methionine-containing-tryptic peptides of the 65,000 MW polypeptide translated from cellular 35 S RNA were identical to those of authentic Pr65^gag. Translation of RNA in the 25–35 S size class suggests that while Pr65^gag can be translated by RNA throughout this size range, Pr200^gag-pol translation is restricted to mRNA which sediments at 35 S. Antiserum directed against the R-MuLV envelope protein gp69/71 recognized a polypeptide of 68,000 MW, designated Pr68^env, which was coded for by RNA which sedimented at about 22 S in sucrose gradients and which had a minimum size of about 1.25 × 10⁶ daltons as estimated by agarose gel electrophoresis. Tryptic maps of Pr68^env showed it to contain all of the methionine-labeled tryptic peptides and most of the tyrosine-containing tryptic peptides characteristic of gPr90^env the authentic R-MuLV glycosylated envelope precursor.     

Isolation of separate precursor polypeptides for the mouse mammary tumor virus glycoproteins and nonglycoproteins.

We have employed monospecific antisera to the major glycoproteins (gp52 and gp36) and the major nonglycoprotein (p27) of the mouse mammary tumor virus (MMTV), and we now report the first isolation of an intracellular MMTV precursor polypeptide to p27. The precursor polypeptide to p27 (Pr75) binds to single-stranded DNA (ssDNA) and can be easily separated from the precursor to gp52 and gp36 (gPr75) by ssDNA-Sepharose column chromatography. [³⁵S]Methionine-labeled Pr75 contained tryptic peptides of p27 and p14 of MMTV. Protein p14 has previously been shown to be capable of binding to ssDNA. In contrast, [³⁵S]methionine-labeled gPr75 contained tryptic peptides of only gp52 and gp36, neither of which binds to ssDNA.     

Glucocorticoid-dependent maturation of viral proteins in mouse lymphoma cells: Isolation of defective and hormone-independent cell variants

Maturation of mouse mammary tumor virus proteins is dependent on glucocorticoid hormones in W7MG1, a stably infected mouse T-lymphosarcoma cell line derived from WEH17. We used an immunological procedure to select variant cell lines with altered levels of viral glycoproteins on the cell surface. One variant, W7M329a, expressed lower-than-normal levels of the major viral glycoprotein, gp52 ^env ,on the cell surface before and after exposure to hormone. Two other variants, W7M302b and W7M326.4, expressed elevated levels of gp52 ^env on the cell surface even in the absence of hormone. Analysis of the levels and/or rates of synthesis of viral RNA and glycoproteins before and after hormone treatment indicated that the variant phenotypes resulted from changes in posttranslational steps of viral gene expression. The hormone-independent maturation of MMTV proteins is a novel variant phenotype that has not previously been reported.     

Analysis of cells transformed by defective leukemia virus OK10: production of noninfectious particles and synthesis of Pr76gag and an additional 200,000-dalton protein

Nonproducer cells transformed by the defective leukemia virus (DL V), OK10, have been analyzed. Unlike nonproducer cells transformed by the other avian defective leukemia viruses examined so far, the OK10-transformed cells were found to release noninfectious particles. Analysis of these particles indicated that they contained the viral gag gene proteins but lacked env and pol gene products. In agreement with these results analysis of [³⁵S]methio-nine-labeled cell extracts of these nonproducer clones by immune precipitation showed that of the three viral structural protein precursors Pr769^gag, gPr95^env, and Pr180^gag-pol only Pr76^gag could be detected. In addition, a 200,000 molecular weight protein (OK10-200K) was identified in the cell extracts which by using specific antisera, was shown to be related to the gag and pol gene products but not to the product of the env gene. Tryptic peptide analysis of the OK10-200K protein confirmed the immunological data in that the OK10-200K protein was shown to contain all but one of the Pr1809^gag-pol methionine tryptic peptides plus unique peptides which were specific for OK10 and not related to the env gene product. One of these OK10-specific peptides was also shown to comigrate with one of the putative mac gene product tryptic peptides of the MC29-110K protein. These data indicate a novel gene order for a DLV.     

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 15I₅ × 7₂ fibroblasts and plasma cells. As analyzed under nonreducing conditions, the RAV-O-associated and 15I₅ × 7₂ 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.     

Effect of interferon on the exogenous Friend murine leukemia virus infection

Interferon treatment (600 U/ml) of NIH/3T3 cells induced greater than 90% reduction in the de novo production of Friend MuLV when measured 24 hr postinfection. Early events in viral replication such as the synthesis of proviral DNA and its subsequent integration into the cell genome were not inhibited by interferon treatment indicating that the suppression of virus production by interferon appears to occur after synthesis of proviral DNA. Analysis of viral RNA species present in controls and interferon-treated cells 24 hr after infection show that the same RNA species were present in the presence and absence of interferon. Synthesis of viral polypeptides was reduced but not blocked in interferon-treated cells when measured within 24 hr after infection while processing of gag precursor, Pr65^gag, and glycoprotein precursor, gPr85^env, to viral proteins was not altered. Phosphorylation of viral protein p12 but not that of the precursor, Pr65^gag, was inhibited in newly infected interferon-treated cells. In contrast to the first replicative cycle, interferon did not inhibit synthesis of viral proteins, and phosphorylation of p12 in those cells chronically infected with F-MuLV.     

Purification and characterization of a murine tumor cell surface glycoprotein of 75,000 daltons that is related to the major envelope glycoprotein of murine leukemia virus.

An antigen which competes with murine C-type viral glycoprotein in an interspecies radioimmunocompetition assay has been purified from the surface of EL-4 tumor cells. The EL-4 tumor cell is virus particle negative. The amount of p30 was extremely low and there was no detectable p12 or p15, confirming that the tumor cell was not expressing a murine oncornavirus. In homologous competition radioimmunoassays, the tumor cell was negative for Rauscher and AKR gp71, but in an interspecies assay employing ¹²⁵I-Rauscher gp71 and anti-feline leukemia virus serum, there was a reactive antigen. The gp71-like antigen was on the surface of the tumor cell since, by immunofluorescence and immunoelectronmicroscopy, the EL-4 cell could be labeled with antiserum against Rauscher virus gp71. The gp71 cross-reactive antigen was purified by lithium diiodosalycilate extraction, DEAF; chromatography, and lentil lectin affinity chromatography. It is a glycoprotein of about 75,000 daltons which could be precipitated by various broadly reactive antisera to murine gp71s and antisera to murine virus. Based on radioimmunocompetition assays, the purified gp75 was not related to AKR or Rauscher gp71, but did compete in an assay using BALB/2 xenotropic gp71 and anti-C57/L virus serum. It also competed in an assay using ¹²⁵I-Moloney virus gp71 and anti-Moloney virus serum, but not in a more type-specific assay assay ¹²⁵I-Moloney virus gp71 and anti-Moloney gp71 serum. Tryptic peptide maps of iodinated proteins were prepared and the cell surface antigen was compared with AKR gp71, Moloney gp71, Rauscher gp71, and xenotropic BALB/2 and NZB gp71s. The EL-4 gp75 was not identical with or very similar structurally to any of the viral gp71s. The differences in the structures of the various viral gp71s shown here and also in other laboratories are consistent with the idea that the env genes of murine viruses which code for gp71s are sites of frequent recombinational events. Recombination between different endogenous viral sequences or between viral and host allelic genomes could have resulted in many immunologically related proteins on viruses, cell surfaces and, in sera of mice, which are immunologically related, about 70,000-dalton molecular weight, but have divergent structure.     

Moloney cell surface antigen (MCSA) has properties of an env gene product that is serologically distinct from the env gene products of the moloney strain of murine leukemia virus

Moloney cell surface antigen (MCSA) is serologically detectable on Moloney murine leukemia virus (Mo-MuLV)-induced lymphomas. It is probably related to the tumor-associated transplantation antigen of these lymphomas. We characterized MCSA by using the typing antiserum in radioimmunoprecipitations followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Surface-iodinated Mo-MuLV-infected cells were used to identify the molecular nature of MCSA. It appeared that MCSA was expressed as an entity of 82,000 apparent molecular weight (p82^MCSA). Translation of size-fractionated mRNAs from infected cells in oocytes of Xenopus laevis showed that p82^MCSA was made on an mRNA of the same size (22 S) as that coding for the Mo-MuLV env proteins. Moreover, p82^MCSA was processed in oocytes to proteins exactly comigrating in the gel with gp70, p15E, and p12E. Competition experiments with unlabeled virus showed that MCSA and the MCSA-related env-like proteins made in oocytes were not related to any of the Mo-MuLV structural proteins. We concluded that MCSA has the properties of the product of the env gene of a murine C-type virus different from Mo-MuLV.     

Visna/maedi virus Env protein expressed by a vaccinia virus recombinant induces cell-to-cell fusion in cells of different origins in the apparent absence of Env cleavage: role of glycosylation and of proteoglycans

Summary.  The in vivo productive infection by the ovine Visna/maedi lentivirus (VISNA) is restricted to cells of the monocyte/macrophage lineage. The basis for this restriction is not understood. Although the VISNA envelope (Env) glycoprotein is the main target for virus neutralization, studies on the role of this protein in virus infection are limited. A vaccinia virus recombinant (VV-env-MV) containing the entire VISNA env sequence was generated and shown to produce in infected cells a protein of about 165 kDa (referred to as gp150). During VV-env-MV infection, expression of env caused extensive cell-to-cell fusion in cell lines of different origins. Pulse-chase and Western blot analyses revealed that gp150 is not cleaved in VV-env-MV infected cells. The glycoprotein gp150 formed oligomers held by disulfide bonding. Cell-to-cell fusion was prevented in the presence of the inhibitor of glycosilation, tunicamycin, but it was markedly enhanced by an inhibitor of proteoglycan synthesis, β-D-xyloside. These findings showed that the receptor for VISNA Env is widely distributed within cells, that fusion-from-within of cells can occur in the apparent absence of proteolytic cleavage of gp150, and that fusion require a glycosylated Env but not the addition of proteoglycan chains at the cell surface. This recombinant virus could have utility as a potential vaccine against VISNA. Received September 20, 2001; accepted June 17, 2002     

The nucleotide sequence of the env gene and post-env region of bovine leukemia virus

The env gene of a bovine leukemia virus (BLV) tumor-derived proviral DNA clone has been located by comparison of the translated DNA sequence with amino acid sequence data on purified gp60 and p30^env (A. M. Schultz, T. D. Copeland, and S. Oroszlan (1984)Virology135, 417–427). There is a continuous open reading frame from the N terminus of gp60 for 1446 nucleotides; gp60 is predicted to contain 268 amino acids and p30^env 214. The predicted p30^env shows structural features typical of type C viral transmembrane proteins. It is also clearly related to that of the human T-cell leukemia virus (HTLV), as predicted from the DNA sequence of Seiki et al. (M. Seiki, S. Hattori, Y. Hirayama, and M. Yoshida (1983)Proc. Natl. Acad. Sci. USA80, 3618–3622) The two proteins show 36% identities in their amino acid sequence, in an alignment requiring six gaps. More distant relatedness is also seen between BLV p30^env and both murine leukemia virus p15E and Rous sarcoma virus gp36. The gp60s of BLV and HTLV are more distantly related than their p30^envs, but their homology is nonetheless statistically significant. Between the presumptive terminator of the env gene and the beginning of the 3′-long terminal repeat is a region of 1817 base pairs of unknown function. Just as in the HTLV post-envelope sequence, there are at least two reading frames which are open for a significant fraction of this region. In neither the tumor-derived clone nor a clone from a virus-producing cell line, however, is there a continuous open reading frame throughout the region. Comparison of the BLV and HTLV sequences within the post-envelope region revealed a very limited but possibly significant similarity.     

Evidence for a Gp85-related glycoprotein in cells transformed by the Bryan High Titer strain of Rous Sarcoma Virus

Summary The Bryan High Titer strain of Rous Sarcoma Virus (BH-RSV) is a deletion mutant in the env-gene coding for the viral envelope glycoproteins gp35 and gp85. In this report experimental evidence is described that cells, transformed by BH-RSV, express a glycoprotein immunologically related to gp85.Animals bearing BH-RSV induced tumors produce antibodies reacting with gp85 of nondefective RSV. Lysates of a BH-RSV transformed quail cell line, R(^–): Q, inhibit the immunoprecipitation of gp85 by antibodies against the group-specific determinant of gp85. In R(^–): Q cell lysates and in the culture supernatant a glycoprotein of an apparent molecular weight 40,000 (gp40) is found that reacts with monospecific antisera against gp85 of nondefective RSV. In newly synthesized BH-RSV a gp40 associated with the virion is detectable but is easily lost during purification of the virus. Further, a 95k glycoprotein and a 95k phosphoprotein are specifically precipitated from R(^–): Q cells by an antiserum against gp85.From these results we conclude that the deletion of the env-gene is incomplete such that part of gp85, bearing group-specific antigenic determinants, is expressed in BH-RSV transformed cells.Analysis of BH-RSV particles freshly harvested from R(^–): Q cells reveals that they contain almost exclusively the gag-precursor pr76 and little or no processed gag-proteins. Therefore the R(^–): Q cell line seems to be suitable for the study of virus maturation occuring after the budding process.With 6 Figures     

Two-dimensional analysis of murine leukemia virus gag-gene polyproteins.

The processing of gag translational products in a Gross Murine Leukemia virus (MuLV)-induced leukemia (E λ G2) was studied with two-dimensional gel electrophoresis, combining separation based upon charge in the first dimension and separation based upon size in the second dimension. In most experiments, the gag species were compared to the env species; gag species were precipitated from labeled cells or virus with antisera to the virion gag proteins p30 or p10, whereas env species were precipitated from labeled cells or virus with anti-gp70 serum. Three viral proteins were detected on the surface of E λ G2 cells with [¹²⁵I] lactoperoxidase labelings: these included gp70 and two glycosylated gag gene species (gpP95^gag and gpP85^gag). Neuraminidase treatment of [¹²⁵I] lactoperoxidase-labeled cells did not affect the antigenicity of gp70, gpP95^gag, or gpP85^gag. However, the neuraminidase treatment caused gp70, gpP95^gag, and gpP85^gag to migrate as more basic species, indicating that all three glycoproteins contain terminal sialic acid. The cytoplasmic gag-gene products were studied with [³⁵S]methionine labelings of E λ G2 cells; seven relatively stable gag species were identified. In general, none of the gag intermediates were single proteins; rather, each of the species exhibited multiple, specific modifications that resulted in complex yet reproducible patterns in the two-dimensional gel system. The core polyproteins Pr75^gag and Pr65^gag were formed rapidly after pulse-labelings, with Pr65^gag being processed into Pr55^gag involving cleavage of p10. The smaller gag species (Pr45^gag and p30) also appeared to result from processing of Pr65^gag. In contrast, Pr75^gag was directly processed to form gpP95^gag. A protein of approximately 58,000 daltons, designated P58^gag, qualified as a gag species since it was specifically precipitated by anti-p30 serum. However, P58^gag did not appear to be a precursor of p30 since it was long-lived in the cytoplasm. Multiple forms of p30 were precipitated from the cytoplasm and from the virion, with unique forms of p30 present in both the cytoplasm and the virion. Comparisons of the gag species from several AKR leukemias indicated that similar, but not identical gag gene products were present in the various leukemias.     

Avian endogenous provirus (ev-3) env gene sequencing: Implication for pathogenic retrovirus origination

The avian endogenous env gene product blocks the surface receptor and, as a result, cells become immune to related exogenous retroviruses. On the other hand, the same sequence can be included in the pathogenic retrovirus genome, as shown by oligonucleotide mapping. However, since the complete env gene sequence was not known, the comparison of genomic nucleotide sequences was not possible. Therefore an avian endogenous provirus with an intact env gene was cloned from a chicken gene bank and the regions coding for the C terminus of the gp85 and gp37 proteins were sequenced. Comparison of this sequence with those of other retroviruses proved that one of the pathogenic viruses associated with osteopetrosis is a cross between avian endogenous virus and Rous sarcoma virus.