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.     

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.     

The effect of glucocorticoid on the subcellular localization, oligomerization, and processing of mouse mammary tumor virus envelope protein precursor Pr74.

Mouse lymphoma cell line W7MG1 is stably infected with mouse mammary tumor virus and produces the viral envelope glycoprotein precursor Pr74, but the mature envelope proteins gp52 and gp33, which are derived from Pr74 by posttranslational processing, are produced only when the cells are cultured with a glucocorticoid agonist. The current study demonstrated that even when W7MG1 cells are grown with hormone, the conversion of Pr74 to gp52 and gp33 is an inefficient process in this cell line. At least 2 h of exposure to glucocorticoid were required to induce the appearance of gp52 and gp33; furthermore, Pr74 labeled in the absence of hormone was not converted to gp52 and gp33 upon subsequent addition of hormone. RNA synthesis inhibitors blocked the hormonal induction of gp52 and gp33, indicating that the hormone acts by promoting the expression of a new gene(s) required for the production of gp52 and gp33, rather than by inhibiting the expression of a gene(s) that prevents processing of Pr74. Subcellular fractionation studies demonstrated that Pr74 produced in either the presence or absence of hormone was associated primarily with the ER, whereas gp52 and gp33 were found in the Golgi and plasma membrane fractions. The Pr74 molecules from W7MG1 cells grown either with or without glucocorticoid coimmunoprecipitated with BiP/GRP78 and sedimented as aggregates of heterogeneous size. In contrast, Pr74 from virus-producing GR3A mouse mammary tumor cells, which process Pr74 more efficiently, sedimented as apparent monomers, dimers, and trimers.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Processing and amino acid sequence analysis of the mouse mammary tumor virus env gene product.

The envelope proteins of mouse mammary tumor virus (MMTV) are synthesized from a subgenomic 24S mRNA as a 75,000-dalton glycosylated precursor polyprotein which is eventually processed to the mature glycoproteins gp52 and gp36. In vivo synthesis of this env precursor in the presence of the core glycosylation inhibitor tunicamycin yielded a precursor of approximately 61,000 daltons (P61env). However, a 67,000-dalton protein (P67env) was obtained from cell-free translation with the MMTV 24S mRNA as the template. To determine whether the portion of the protein cleaved from P67env to give P61env was removed from the NH2-terminal end of P67env and as such would represent a leader sequence, the NH2-terminal amino acid sequence of the terminal peptide gp52 was determined. Glutamic acid, and not methionine, was found to be the amino-terminal residue of gp52, indicating that the cleaved portion was derived from the NH2-terminal end of P67env. The NH2-terminal amino acid sequences of gp52's from endogenous and exogenous C3H MMTVs were determined though 46 residues and found to be identical. However, amino acid composition and type-specific gp52 radioimmunoassays from MMTVs grown in heterologous cells indicated primary structure differences between gp52's of the two viruses. The nucleic acid sequence of cloned MMTV DNA fragments (J. Majors and H. E. Varmus, personal communication) in conjunction with the NH2-terminal sequence of gp52 allowed localization of the env gene in the MMTV genome. Nucleotides coding for the NH2 terminus of gp52 begin approximately 0.8 kilobase to the 3' side of the single EcoRI cleavage site. Localization of the env gene at that point agrees with the proposed gene order -gag-pol-env- and also allows sufficient coding potential for the glycoprotein precursor without extending into the long terminal repeat.     

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.     

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.     

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.     

Cloned mouse mammary tumor virus DNA exhibits glucocorticoid-dependent expression in simian virus 40-transformed mink cells.

Mink lung epithelial cells were transfected with two cloned mouse mammary tumor virus (MMTV) DNAs, a 9-kilobase clone derived from an unintegrated exogenous viral genome and a 14-kilobase clone containing an integrated endogenous provirus along with cellular flanking sequences. Mink lung cells were chosen because they do not contain endogenous MMTV sequences. On the basis of our observation that simian virus 40 DNA efficiently transforms these cells, we isolated cell clones containing MMTV DNA by using transformation with simian virus 40 DNA as a selective marker in cotransfection experiments. Levels of the 9-kilobase MMTV mRNA representing the entire viral genome and of the spliced 4.4-kilobase mRNA which codes for the viral envelope proteins were glucocorticoid dependent in transformed cells. Expression of low levels of Pr77gag, the precursor of the group-specific viral core proteins, and of gPr73env, the precursor of the viral envelope proteins, was also hormone dependent. We conclude that these cloned MMTV DNAs contain all the information necessary for the synthesis of normal viral RNAs and proteins. These findings also provide further evidence that the DNA sequences involved in the hormone responsiveness of MMTV expression are contained within the viral genome.     

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.     

Establishment of a C3Hf Mammary Tumor Cell Line Expressing Endogenous Mouse Mammary Tumor Virus: Antigenic and Genetic Relationships of This Virus with Highly Oncogenic Mouse Mammary Tumor Viruses

A single-cell clone of C3Hf mammary tumor cells (clone 14) was developed into a continuous cell line expressing high levels of endogenous mouse mammary tumor virus (MMTV) with less than 0.1% murine leukemia virus expression. Comparison of the C3Hf MMTV protein profile on sodium dodecyl sulfatepolyacrylamide gel electrophoresis with that of C3H MMTV revealed that the protein content of the two viruses was quite similar. However, oligonucleotide fingerprints obtained of MMTV 70S RNA revealed that approximately 20% of the large oligonucleotides examined were unique to each virus. The oligonucleotide fingerprint indicated that although the viruses were similar, they differed in their genetic content. The differences in the two viruses extended to immunological differences in the major envelope glycoprotein, gp52. C3Hf MMTV competed only partially in a homologous radioimmunoassay for gp52 of C3H MMTV, whereas C3H MMTV gave complete competition, indicating that gp52 of C3H MMTV contained type-specific determinants not present on gp52 of C3Hf MMTV. Comparison of C3Hf MMTV with highly oncogenic C3H, GR, and RIII MMTVs in a homologous C3H MMTV gp52 assay gave two patterns of reactivity: complete competition by GR and C3H MMTV and incomplete competition by C3Hf and RIII MMTV. Absorption of anti-C3H MMTV serum by either C3Hf MMTV or RIII MMTV removed all antibodies against both viruses but not against GR and C3H MMTVs. These results indicate that C3H and GR MMTVs are more closely related to each other than to RIII and C3Hf MMTVs.     

Polyprotein Precursors to Mouse Mammary Tumor Virus Proteins

Mouse mammary tumor virus (MMTV) derived from the culture medium of GR cells contained seven proteins, identified as gp55, gp33, p25, pp20, p16, p12, and p10. The major viral phosphoprotein was the 20,000-molecular-weight protein, pp20. Immunoprecipitation of cytoplasmic extracts from pulse-labeled GR cells identified three MMTV gag-specific proteins, termed Pr78(gag), Pr110(gag), and Pr180(gag+). These intracellular polyproteins were precipitable from cytoplasmic extracts by antisera to virions p25 and p12 but not by antisera to gp55. The major intracellular gag-specific precursor polyprotein, Pr78(gag), contained antigenic determinants and tryptic peptides characteristic of p25, p12, p10, and presumably pp20. This precursor is presumably derived from nascent chain cleavage or rapid posttranslational cleavage of the larger intracellular precursor-like protein, designated Pr110(gag). Pr110(gag) contained all but one of the leucine-containing tryptic peptides of Pr78(gag), plus several additional peptides. In addition to Pr78(gag) and Pr110(gag), monospecific antisera to virion p12 and p25 were also capable of precipitating from pulse-labeled cells a small amount of a 180,000-molecular-weight precursor-like protein, designated Pr180(gag+). This large polyprotein contained nearly all of the leucine-containing tryptic peptides of Pr78(gag) and Pr110(gag) plus several additional peptides. By analogy to type C viral systems, Pr180(gag+) is presumed to represent a gag-pol common precursor which is the major pathway for synthesis of MMTV polymerase. Immunoprecipitation of cytoplasmic extracts from pulse-labeled cells with antisera to gp55 identified two env-specific proteins, designated gPr76(env) and gP79(env). The major env precursor, gPr76(env), could be labeled with radioactive glucosamine and was shown to contain antigenic determinants and tryptic peptides characteristic of gp55 and gp33. A minor glycoprotein, gP79(env), contained both fucose and glucosamine and was precipitable from cytoplasmic extracts with monospecific serum to gp55. It is suggested that gP79(env) represents fucosylated gPr76(env) which is transiently synthesized and cleaved rapidly into gp55 and gp33.     

Type-specific antigenic determinants on the major external glycoprotein of high- and low-oncogenic murine mammary tumor viruses.

We recently showed that the 52,000-dalton external glycoprotein (gp52) of the highly oncogenic mouse mammary tumor viruses (MMTVs) of RIII, GR, and C3H mice contains both type- and group-specific antigenic determinants. This was demonstrated by using a competition radioimmunoassay with 125I-externally labeled virions and antisera to the gp52 of MMTV from RIII mice (Proc. Natl. Acad. Sci. U.S.A. 74:3564-3568, 1977). We report here that we were able to distinguish between the gp52's of the high-oncogenic MMTV of C3H mice [MMTV(C3H)] and the low-oncogenic MMTV of that same mouse strain [MMTV(C3Hf)]. This was accomplished by use of a competition radioimmunoassay with 125I-externally labeled virions of MMTV(C3H) and antisera prepared against MMTV(C3H). A comparison of the intact virion and purified gp52 radioimmunoassays showed that MMTV type-specific differences were enhanced with the intact virion radioimmunoassay. These differences were further magnified with appropriately absorbed antisera. These findings thus allow an immunological distinction between the surface glycoproteins of a low-oncogenic endogenous and a high-oncogenic exogenous MMTV of the same mouse strain.     

Viral protein expression in producer and nonproducer clones of friend erythroleukemia cell lines.

Erythroleukemia cell lines HFL/d and HFL/b, derived from tumors induced in vivo in BALB/c (H-2d) and congenic BALB.B (H-2b) mice, respectively, by a polycythemia-inducing strain of Friend virus, produced both spleen focus-forming virus (SFFV) and its native NB-tropic helper virus (Friend murine leukemia virus [FMuLV]) during early-passage generations in culture. Eventually each line ceased production of both infectious viruses but retained its tumorigenic potential in syngeneic hosts. Virus-producer and -nonproducer clones of these cell lines were examined for expression of proteins encoded by the SFFV or FMuLV genomes. Lysates of labeled cells were treated with various antiviral sera, and the precipitates were examined by gel electrophoresis. Expression of the FMuLV env gene-encoded precursor protein, gPr84env, was observed in all producer and most nonproducer clones, but the FMuLV gag and pol gene products, Pr65gag and Pr200gag-pol, were uniformly undetectable in nonproducer clones. All HFL/d and HFL/b clones expressed appreciable amounts of the SFFV-encoded envelope protein, gp52, including one exceptional clone which had ceased to express any FMuLV-encoded proteins. The molecular weight of this SFFV-encoded envelope protein was consistently smaller in all HFL/b clones than in HFL/d clones, regardless of their producer or nonproducer status. The virus-nonproducer phenotype thus appears to be due to shutdown of expression of the 5' portion of the FMuLV genome in two independent cell lines.     

An antigen related to the mouse mammary cancer virus env gene product, detected in human lymphocytes, is associated with human breast cancer]

Expression of DNA sequences homologous to sequences of env gene of mouse mammary tumor virus (MMTV) in the lymphocytes of patients with breast cancer and in subjects at a high risk of breast cancer has been reported. Antigen analogous to envelope protein gp52, product of MMTV env gene, is detected in T lymphocytes of virtually all patients with breast cancer and extremely rarely in T cells of controls, where its expression is confined to B cells. For explaining such unexpected results, we studied the molecular basis of this antigen synthesis. Specific PCR products were obtained using primers to gp52-coding region of MMTV env gene. One of them (957 nucleotides) was used as a probe for hybridization of DNA and RNA from lymphocytes of patients with breast cancer and controls. This sequence was hybridized with 90% frequency with genome DNA of breast cancer patients and with 85% frequency with genome RNA of such patients, which is almost 4-fold more than in the controls (patients with gynecological tumors or donors). These results correlate with the frequency of detection of the studied antigen in patients with breast cancer and control group patients.     

Immunochemical Characterization of Two Major Polypeptides from Murine Mammary Tumor Virus

A major murine mammary tumor viral (MMTV) antigen, sl, originally described by Nowinski et al. (1967, 1968, 1971), has been purified from RIII mouse milk MMTV by sequential ion-exchange and gel chromatography. The purified protein with sl antigenic reactivity contains carbohydrate, and has an apparent minimal molecular weight of 52,000. It can be designated as gp52 (sl). Another major MMTV viral protein with a molecular weight of 27,000 has also been isolated, and antisera have been prepared against it. Both MMTV gp52 (sl) and p27 viral polypeptides have been iodinated with (125)I and used in immunoprecipitation and competition assays. The two MMTV proteins differ absolutely from each other and from major mouse type C viral polypeptides in molecular weight, immunological reactivity, and amino acid composition. Purified gp52 (sl) in radioimmunoprecipitation inhibition assays reacted in two distinct patterns. One pattern showed partial displacement of antibody which could be converted to the second, a complete displacement, by heating the antigen, presumably by exposing additional reactive determinants. Biologically, the patterns of major MMTV polypeptide expression in milk correlated with spontaneous mammary tumor incidence in different strains of mice, indicating that the sl antigen is group specific for MMTV or that several mouse strains contain the same virus type.