Glucocorticoid-dependent maturation of mouse mammary tumor virus glycoproteins in mouse lymphoma cells: isolation of variants with constitutive viral protein maturation and normal glucocorticoid receptor function

The posttranslational maturation and cell surface localization of mouse mammary tumor virus (MMTV) envelope glycoproteins is regulated by glucocorticoid hormone in mouse T-lymphoma cell line W7MG1. Only when the cells are cultured with glucocorticoid is the MMTV envelope precursor, Pr74, converted efficiently to the two mature proteolytic products, gp52 and gp33. By immunological selection we have isolated protein-processing variants that express the mature viral proteins constitutively on the cell surface. The rate of synthesis of Pr74 is indistinguishable in variant and wild-type cells, but the variants efficiently convert Pr74 to gp52 and gp33 even when grown without the hormone. The variant phenotype persists when the variant cells are fused with uninfected wild-type cells to form somatic cell hybrids, indicating that the variant phenotype resulted from expression of a new or altered function that is not expressed in wild-type cells grown without glucocorticoid. Although the specific gene whose structure or regulation is altered in the variant has not yet been determined, some possibilities have been eliminated. First, the number and function of the glucocorticoid receptors in the variant cells was normal, suggesting that alterations in this protein were not responsible for the variant phenotype. Second, comparison by two-dimensional gel electrophoresis of gp52 produced in variant and wild-type cells revealed no differences in size or charge, indicating no gross differences in the processing of the viral proteins in the variant and wild-type cells.     

Characterization of oligosaccharide chains on mouse mammary tumor virus envelope proteins and the implications for the mechanism of their glucocorticoid regulated processing

Glucocorticoid hormone is required for complete posttranslational processing of the glycosylated mouse mammary tumor virus envelope precursor, Pr74env in the murine T-lymphosarcoma cell line, W7MG1. Metabolic labeling studies with [35S]methionine, [3H]galactose, and [3H]mannose, combined with enzymatic digestion analyses with a variety of endoglycosidases, demonstrated that both proteolytic processing and N-linked oligosaccharide maturation depended, either directly or indirectly, on glucocorticoid action. Pr74 is found in both control and hormone-treated cells. In both cases Pr74 molecules carry high mannose and/or hybrid, but not complex, oligosaccharide chains with very little or no sialic acid. When cells are grown with glucocorticoid, Pr74 is converted to gp52 and gp33 with greatly increased efficiency, and these mature glycoproteins carry complex oligosaccharides containing sialic acid. No O-linked carbohydrate was detected on any of these species. According to this evidence, the glucocorticoid-regulated step in this pathway must occur at or before the final mannose trimming step in the Golgi that is required for formation of complex carbohydrate chains.     

The order of processing events in mouse mammary tumor virus envelope protein maturation: implications for the location of the glucocorticoid-regulated step.

Treatment of the W7MG1 mouse T lymphoma cell line with glucocorticoid stimulates directly or indirectly two observable steps in the processing of mouse mammary tumor virus (MMTV) envelope glycoprotein precursor Pr74: cleavage of Pr74 to yield the mature glycoprotein products gp52 and gp33, and processing of the N-linked oligosaccharides to endoglycosidase H (endo H)-resistant forms found on the mature products but not on the precursor. Therefore, the primary hormone-regulated event in this pathway must occur at or before the point where MMTV envelope proteins become endo H resistant. Pulse-chase analyses identified a novel endo H-resistant 80-kDa species (designated gp80) as a processing intermediate. Therefore, in contrast to conclusions drawn for the envelope proteins of several other retroviruses, proteolytic cleavage of MMTV envelope proteins occurs after acquisition of endo H resistance. Also, proteolytic cleavage cannot be the primary hormone-regulated step. Second, inhibition of mannosidase II by the drug swainsonine did not prevent Pr74 from being proteolytically processed, thus demonstrating that conversion of oligosaccharide chains from endo H-sensitive to -resistant forms was not a prerequisite for proteolytic cleavage. Therefore, the requisite hormone-regulated event in MMTV glycoprotein processing must precede both acquisition of endo H resistance and proteolytic cleavage. This places the regulated event in the endoplasmic reticulum or early Golgi.     

A novel intermediate in processing of murine leukemia virus envelope glycoproteins. Proteolytic cleavage in the late Golgi region.

The intracellular processing of the murine leukemia virus envelope glycoprotein precursor Pr85 to the mature products gp70 and p15e was analyzed in the mouse T-lymphoma cell line W7MG1. Kinetic (pulse-chase) analysis of synthesis and processing, coupled with endoglycosidase (endo H) and neuraminidase digestions revealed the existence of a novel high molecular weight processing intermediate, gp95, containing endo H-resistant terminally glycosylated oligosaccharide chains. In contrast to previously published conclusions, our data indicate that proteolytic cleavage of the envelope precursor occurs after the acquisition of endo H-resistant chains and terminal glycosylation and thus after the mannosidase II step. In the same W7MG1 cell line, the type and order of murine leukemia virus envelope protein processing events was identical to that for the mouse mammary tumor virus envelope protein. Interestingly, complete mouse mammary tumor virus envelope protein processing requires the addition of glucocorticoid hormone, whereas murine leukemia virus envelope protein processing occurs constitutively in these W7MG1 cells. We propose that all retroviral envelope proteins share a common processing pathway in which proteolytic processing is a late event that follows acquisition of endo H resistance and terminal glycosylation.     

Expression of a defective mouse mammary tumor virus envelope glycoprotein precursor which binds stably to GRP78 within the lumen of the endoplasmic reticulum is associated with decreased glucocorticoid-induced apoptosis in mouse lymphoma cells

Viral proteins inhibit apoptosis in host cells by a variety of mechanisms. This report proposes an additional mechanism, based on the interaction of a mutant mouse mammary tumor virus (MMTV) envelope glycoprotein precursor, Pr74, with the stress protein GRP78 (BiP) within the lumen of the endoplasmic reticulum (ER) (J. Biol. Chem. 268 7482-7488, 1993). We show that WEHI7.2 (W7.2) mouse lymphoma cells, which do not express Pr74, are more sensitive to cell death induction by the glucocorticoid hormone dexamethasone (dex), than W7MG1 cells, which were derived by infecting W7.2 cells with MMTV and therefore express Pr74 under control of the glucocorticoid-inducible MMTV promoter. Moreover, W7-ENV/N cells, derived by stably transfecting W7.2 cells with a constitutively expressed cDNA encoding mutant Pr74, were less sensitive to dex-induced cell death than control transfectant W7-ENV/- cells. Among multiple W7-ENV/N subclones, susceptibility to dex-induced cell death was inversely related to the level of Pr74 synthesis. The interaction of Pr74 with GRP78 induces an increase in GRP78 synthesis. Thus, the repression of cell death associated with Pr74 expression may be secondary to elevated synthesis of GRP78, a stress protein previously implicated in protection against cell death.     

Impaired Maturation of Mouse Mammary Tumor Virus Precursor Polypeptides in Lymphoid Leukemia Cells, Producing Intracytoplasmic A Particles and No Extracellular B-Type Virions

Processing of polypeptides of the mouse mammary tumor virus, a type B retrovirus, was investigated in a transplanted thymic lymphoma cell line of the GR strain (GRSL). This cell line was maintained in vivo in ascites form and in vitro as a suspension culture. GRSL cells produce clusters of intracytoplasmic A particles and are virtually deficient in the production of mature extracellular B-type particles. As control, a mammary tumor cell line of the same mouse strain capable of complete virion synthesis was used. The kinetics of viral polypeptide synthesis were studied by pulse labeling with various isotopes (including (35)S and (32)P), followed by immunoprecipitation of cell lysates with monospecific antisera to the major mouse mammary tumor virus gag and env proteins, p27 and gp52, respectively. Both the primary gag and env precursor polypeptides were synthesized in the GRSL cells, but their conversion into viral proteins was impaired. The major gag precursor, Pr73(gag), was stable over a period of 8 h, and mature viral core polypeptides could not be detected. Also, the highly phosphorylated intermediates in the proteolytic processing of Pr73(gag) in virus-producing cells were absent in GRSL cells. By immunoprecipitation, Pr73(gag) was detected in a GRSL particle fraction with the density of intracytoplasmic A particles. The precursor for envelope proteins, Pr73(env), was turned over without the generation of mature viral envelope components gp52 and gp36. The in vivo-transplanted ascites GRSL cells, however, were shown to express gp52 on the cell surface together with a 73,000-dalton polypeptide, as indicated by cell surface iodination and immunoprecipitation.     

Isolation of glucocorticoid-unresponsive rat hepatoma cells by fluorescence-activated cell sorting

We have used a mouse mammary tumor virus (MMTV)-infected rat hepatoma cell line as a model system for studying glucocorticoid action. These cells induce tyrosine aminotransferase and MMTV in response to the synthetic glucocorticoid, dexamethasone. The major viral antigen, a glycoprotein of 52,000 daltons (gp52), appears on the surface of infected cells in amounts which reflect the cytoplasmic content of viral RNA. Using an anti-gp52 antiserum and a fluorescence-activated cell sorter (FACS), we have selected variants which display low levels of pg52 in the presence of the hormone. Multiple cycles of enrichment for cells that fluoresce weakly in the presence of hormone have generated a population which fails to produce a detectable increase in cell surface gp52 in response to dexamethasone. This population of nonresponders and a number of independent clones derived from this population were analyzed for their ability to induce gp52 and TAT and for these presence of glucocorticoid receptors. All nonresponder clones exhibited little or no induction of either glucocorticoid-inducible marker. Two of the clones contained reduced levels of glucocrticoid receptor, while the remainder of the clones showed no detectable specific hormone binding. These results provide genetic evidence that a single class of glucocorticoid receptors is involved in the induction of both MMTV and TAT in HTC cells.     

Glucocorticoids stimulate the release of a viral tumor marker (MMTV gp52) while inhibiting tumor cell growth

The influence of glucocorticoid treatments on the release of mouse mammary tumor virus (MMTV) envelope antigen (gp52) has been studied in C3H mammary tumor cell cultures and compared to treatment-mediated effects on tumor cell growth. Simultaneous assessment of extracellular viral antigen levels and tumor cell growth has indicated that both are coordinately affected by glucocorticoid treatment. While gp52 release is stimulated by treatment, this effect is accompanied by an inhibition of tumor cell growth. These stimulatory and inhibitory effects are mediated by dexamethasone (DEX) in a dose-dependent fashion, and both effects are more pronounced with the synthetic glucocorticoids DEX or triamcinolone acetonide (TA). Quantitation of media gp52 levels by RIA revealed the following hierarchy of glucocorticoid enhancement: TA greater than DEX greater than prednisolone greater than hydrocortisone greater than triamcinolone. A similar order of activity was observed in terms of inhibition of cell growth. The ability of TA to enhance gp52 release was 2.4-2.7 times greater than DEX, a previously proven stimulator of MMTV expression. Cell density of B9 mammary tumor cells was reduced 73% following 72 h of 10(-8) MTA treatment while C3H Mm5mt/cl mammary tumor cells were reduced by 53%. Hormone-mediated changes in in vitro gp52 release suggest that hormones might also influence plasma levels of MMTV gp52 as a systemic marker for the presence and status of murine mammary tumors. Coordinate stimulatory and inhibitory effects suggest that glucocorticoids may play a complex role in murine mammary tumorigenesis and subsequent mammary disease.     

Temperature-sensitive transport of glycoproteins to the surface of a variant mouse lymphoma cell line.

The expression of mouse mammary tumor virus (MMTV) glycoproteins on the surface of stably infected mouse lymphoma cell line W7MG1 is dramatically increased by glucocorticoid hormones. A variant cell line, W7M.TS1, was selected from W7MG1 for its lack of expression of MMTV glycoproteins on the cell surface in response to treatment with glucocorticoid. Hormonal stimulation of MMTV RNA levels and hormone-induced cytolysis occurred normally in the variant cells. Furthermore, the rates of production of the precursor and mature forms of MMTV glycoproteins in the presence of glucocorticoid were similar in variant and wild-type cells. However, the accumulation of MMTV glycoproteins on the cell surface after hormone treatment was delayed by about 8 h in the variant relative to wild-type cells. The steady-state level of a constitutively expressed cellular protein, T200, on the variant cell surface was comparable to that on wild-type cells. However, in pulse-chase experiments, the appearance of newly synthesized T200 on the cell surface was delayed in the variant compared with wild-type cells. Another glucocorticoid hormone response, removal of H-2 class I antigens from the cell surface, was also delayed in the variant relative to wild-type cells, suggesting that turnover or internalization of cell surface glycoproteins may also be affected in the variant. The defects in the variant cell line were observed at 37 degrees C, but not at 31 degrees C; the variant cells grew normally at both temperatures. This variant phenotype defines a new genetic entity that is important for transport of glycoproteins between internal microsomal compartments and the cell surface.     

Mammary tumor virus induction by glucocorticoids. Characterization of specific transcriptional regulation.

Dexamethasone (1,4-pregnadiene-9-fluor-16alpha-methyl-11beta,17alpha,21-triol-3,20-dione), a potent synthetic glucocorticoid, stimulates mouse mammary tumor virus expression 10- to 20-fold in tissue culture cells. This hormone effect was observed at concentrations as low as 1 times 10-10 M and was maximal at 10-7 to 10-8 M. The time course of induction indicated that detectable increases in extracellular viral DNA polymerase were first noted 18 to 24 hours following the addition of dexamethasone, and cells produced the highest polymerase levels at the time monolayers approached confluence. Steroid responsiveness was associated with specific increases in type B murine mammary tumor virus structural polypeptide (gp52(sl) expression and murine mammary tumor virus RNA that quantitatively paralleled the increase in extracellular virus production as measured by electron microscopy and supernatant RNA-dependent DNA polymerase activity. Another virally transformed murine cell line, KA 31, did not contain detectable levels of murine mammary tumor virus gp52(sl) or RNA before or after dexamethasone stimulation; thus induction was noted only in murine cells with pre-existing murine mammary tumor virus expression. No increase in basal levels of type C murine leukemia viral proteins or RNA was detected in dexamethasone-treated mammary cell lines which were producing increased levels of murine mammary tumor virus. Therefore, increases in murine mammary tumor virus gene products are specific for murine mammary tumor virus DNA sequences under these conditions.     

Detection and characterization of mouse mammary tumor virus cell surface antigens: estimation of antigen abundance by protein A assay.

Antisera against the following mouse mammary tumor virus (MMTV) structural proteins were used to detect MMTV cell surface antigens: (i) the 27,000-dalton nucleoid protein, p27; (ii) the 36,000-dalton envelope glycoprotein, gp36; and (iii) the 52,000-dalton exterior envelope glycoprotein, gp52. We report here the development of an adherent-cell isotopic staphylococcal protein A (SPA) test (ISPAT) for MMTV structural proteins which allows for the detection of an MMTV membrane-associated antigen as well as an estimate of its relative abundance on the cell surface. This test demonstrated that the gp52 was the predominant MMTV cell surface antigen detected on both C3H and GR mouse mammary tumor cells. In a comparative study with anti-gp52 and anti-gp36 sera, SPA-specific binding with anti-gp36 serum was found to be only 5 to 6% of that obtained for the external virion glycoprotein, gp52. Both direct and indirect ISPAT indicated the presence of a low but detectable number of gp36 determinants on GR-MMTV cells; however, these gp36 determinants, unlike gp52 determinants, appeared to be exposed by the fixation procedure used. Only 0.9 to 1.1% of the gp52-specific binding was detected when anti-gp36 serum was allowed to react with viable cells. The binding of [125I]SPA achieved with anti-p27 serum was even less than that detected with gp36-directed reagents, indicating that p27 is not a cell surface antigen. The use of fluoresceinated SPA further demonstrated that p27 and gp36 reactivity was only associated with a small number of cells in each of the mammary cultures tested. When N-[4-(5-nitro-2-furyl)-2-thiazoly]-formamide-induced C3H bladder tumor cells were subjected to a gp52-directed ISPAT, the failure to detect gp52-specific binding demonstrated the specificity of this assay for MMTV gp52-expressing cells. In addition to detecting and characterizing MMTV cell surface antigens, the newly developed adherent cell assay could measure changes in the abundance of cell surface gp52. When dexamethasone-treated and untreated GR cells were compared, measurements of gp52-specific SPA binding indicated that dexamethasone stimulation leads to a 12.2-fold increase in the amount of cell surface gp52 detected.     

A unique glycoprotein containing GR-mouse mammary tumor virus peptides and additional peptides unrelated to viral structural proteins

A glycoprotein of molecular weight 130,000 (gP130) has been precipitated from the cytoplasm of GR-strain mouse mammary tumor (GR-MMT) cells by a rabbit antiserum (anti-MMTV) to GR-strain mouse mammary tumor virus (GR-MMTV). This protein was not precipitated by antisera specific for detergent-disrupted C3H-strain MMTV (C3H-MMTV); C3H-MMTV glycoproteins; C3H-MMTV nonglycosylated proteins; GR-MMTV p25 or p12; RIII strain (milk) MMTV proteins; or Rauscher murine leukemia virus (R-MuLV) proteins; nor was it precipitated by normal rabbit serum. Two-dimensional thin layer analysis of 35S-methionine-containing tryptic peptides revealed that five of nine gp33 peptides and one of seven gp55 peptides are shared by gP130 and gPr76env. The envelope protein precursor, gPr76env, contains all of the gp33 peptides and six of seven gp55 peptides. One peptide in gPr76env, possibly a gp55-gp33 junction peptide, is also apparently present in gP130. Six of ten p25 peptides and four more gag-related peptides are shared by PR78gag and gP130. Protein gP130 also contains several tryptic peptides not found in gPr76env or in the core protein precursors Pr78gag, Pr110gag or Pr180gag-pol. Radioimmunoprecipitation experiments showed that gP130 could be precipitated from extracts of GR-MMTV cells with anti-MMTV serum even after antibodies to the known MMTV structural proteins had been removed from the serum by absorption. Both gP130 and a second protein, p30, were found in immunoprecipitates of detergent-disrupted isotopically labeled GR-MMTV treated with the absorbed anti-MMTV serum. These results suggest that antibodies to gP130 in the anti-MMTV serum are capable of recognizing those protein sequences unique to gP130; that is, those protein sequences which are not related to viral structural proteins. In light of these data and data published previously, gP130 is apparently a polyprotein containing juxtaposed components translated from the 5' and 3' end of the MMTV genome and protein components not previously identified as virus-specific.     

Effect of 5-azacytidine on expression of DNA sequences homologous to ENV gene of murine mammary tumor virus in normal human lymphocytes

Expression of DNA sequences, related to MMTV env gene, in peripheral blood lymphocytes, which was strictly specific for human mammary carcinoma, has been previously reported. These sequences (homologous to env gene site coding for MMTV gp52 envelope antigen) expressed in T cells can play the key role in virus infection transmission and propagation. In order to elucidate the possible routes of env MMTV-homologous sequences expression, we tried to induced it in donot T lymphocytes by various methods: hormone and virus treatment (related genome "saving" at the expense of the added virus envelope), T cell culturing with conA, interferon-2, and 5-azacytidine. RT-PCR with primers specific for the gp52-coding area of MMTV env gene showed expression of env-homologous sequences in donor T cells cultured in medium with 5-azacytidine. Indirect immunofluorescence with monospecific serum to MMTV gp52 detected gp52 analogous genes only in cultures with 5-azacytidine but not other agents. We therefore suggested that MMTV env-homologous sequences in donors are situated in the methylated promoter zone. Expression of these sequences in T cells, specific for human mammary carcinoma, can be due to demethylation of the promoter and induction of env-homologous sequences to the level of translation of gp52 analogous antigens or by initial location of some of the expressed sequences in the demethylated zone of the genome.     

Identification of a cellular receptor for mouse mammary tumor virus and mapping of its gene to chromosome 16

Pseudotypes of vesicular stomatitis virus (VSV) containing envelope glycoproteins provided by C3H mammary tumor virus (MTV) instead of the normal VSV G-proteins were prepared and used to assay the presence of an MTV receptor on cells. The assay was specific as demonstrated by competition studies with excess MTV particles and neutralization of the pseudotypes with anti-MTV serum or monoclonal antibodies directed against MTV gp52. The MTV receptor was abundantly present on mouse cells but hardly detectable on nonmurine cells, including the Chinese hamster cell line E36. Somatic cell hybrids between E36 cells and GRS/A spontaneous leukemia cells (GRSL cells) and between E36 and GRS/A primary mammary tumor cells were made. The hybrids retained all Chinese hamster chromosomes but segregated mouse chromosomes. From the analysis of the isoenzymes and chromosomes of the hybrid cell lines we conclude that the gene for the receptor (MTVR-1) is located on mouse chromosome 16.     

Immunoelectron microscopic localization of mammary tumor virus (MTV) antigens in normal and cancerous mammary glands of mice

Peroxidase-labeled Fab' fragments of rabbit antisera against gp52 (major envelope protein) and A-particles of mammary tumor virus (MTV) were prepared and used for investigation by immunoelectron microscopy of the replication cycle of MTV-specific envelope and core antigens in normal and malignant mammary gland cells of female mice. The specificity of the antisera was proven by absorption tests and lack of reactivity to MTV-free mammary tissues. Periodate-lysine-paraformaldehyde (PLP) fixation sufficiently preserved the antigenicity of gp52, while Zamboni's fixative was useful to preserve the core antigen. Saponin pretreatment was necessary to reveal the intracellular antigen of A particles but had no influence on gp52. In addition to its presence at the envelope of D particles, gp52 was clearly associated with the biomembrane system, including the nuclear membrane, endoplasmic reticulum, Golgi apparatus and plasma membrane independent of the presence of virus particles. In mammary tumors, a significant level of gp52 antigen was often expressed on the entire cell surface membrane. In contrast, it was localized only to the apical plasma membrane in normal mammary gland cells. A particle antigens were confined to the intracytoplasmic A particles, usually visible as clusters, and to the inner part of B particles. These ultrastructural findings support the available biochemical data on the morphogenesis of MTV particles.     

Terminal amino acid sequences and proteolytic cleavage sites of mouse mammary tumor virus env gene products

The mature envelope glycoproteins of mouse mammary tumor virus (gp52 and gp36) were isolated by reversed-phase high-pressure liquid chromatography. The N-terminal amino acid sequence of gp36 was determined for 28 residues. The C-terminal amino acid sequences of gp52 and gp36 were determined by carboxypeptidase digestion. The N-terminal amino acid sequence of gp52 has been reported previously (L. O. Arthur et al., J. Virol. 41:414-422, 1982). These data were aligned with the predicted amino acid sequence of the env gene product obtained by translation of the DNA sequence (S. M. S. Redmond and C. Dickson, Eur. Mol. Biol. Org. J. 2:125-131, 1983). The amino acid sequences of the mature viral proteins were in agreement with the predicted amino acid sequence of the env gene product over the regions of alignment. This alignment showed the sites of proteolytic cleavages of the env gene product leading to the mature viral envelope glycoproteins. The N-terminal amino acid sequence of gp52 starts at residue 99 of the predicted structure indicating proteolytic cleavage of a signal peptide. A dipeptide (Lys-Arg) is excised between the C-terminus of gp52 and the N-terminus of gp36. The C-terminal amino acid sequence of gp36 is identical to the sequence predicted by the codons immediately preceding the termination codon for the env gene product. The data show that there is no proteolytic processing at the C-terminal of the murine mammary tumor virus env gene product and that the env gene coding region extends into the long terminal repeat.     

Regulation of the mouse mammary tumor virus receptor by phosphorylation and internalization in mammary epithelial cells

The mouse mammary tumor virus enters mammary epithelial cells via a plasma membrane protein that binds to a viral envelope glycoprotein, gp52. In intact cells, this gp52 receptor can be phosphorylated by activators of protein kinase A and protein kinase C (PKC), but this modification does not occur in response to epidermal growth factor, whose receptor is a tyrosine kinase, or to gp52. Phosphorylation of the gp52 receptor rapidly leads to internalization and gradual loss of binding activity. Both the phosphorylation and the intrnalization induced by PKC are abolished by prior downregulation of this kinase. Although the physiological function of the gp52 receptor is unknown, its binding to gp52 can stimulate several biological activities, including amino acid accumulation. Receptor processingimpairs this gp52-induced amino acid uptake, as well as viral infection, by depleting the binding protein at the cell surface. In contrast, PKC augments insulin-induced amino acid transport, and PKC downregulation abolishes the action of insulin, suggesting that insulin and gp52 utlize partially separate pathways leading to amino acid transport. These data further suggest that PKC may be involved in this insulin-stimulated activity. © 1994 Wiley-Liss, Inc.     

Structure of the Mouse Mammary Tumor Virus: Characterization of Bald Particles

The polypeptide, antigenic, and morphological structure of the mouse mammary tumor virus was studied following protease digestion of intact virions. Intact, untreated virions (rho = 1.17 g/ml) had characteristic envelope spikes, five major polypeptides, and were precipitated by antisera against gp52. Two of the major polypeptides, with molecular weights of 52,000 (gp52) and 36,000 (gp36), had carbohydrate moieties. Protease treatment resulted in spikeless, "bald" particles (rho = 1.14 g/ml), which had altered surface antigenicity and which contained neither gp52 nor gp36. These data indicated that gp52 and gp36 were on the viral envelope. Bald particles retained a 28,000 dalton polypeptide (p28) which was proposed as the major internal polypeptide.