Mammary tumors from BALB/c mice with a reported high mammary tumor incidence have acquired new mammary tumor virus DNA sequences

Various laboratories have reported differences in the mammary tumor incidence caused by the endogenous mouse mammary tumor virus (MMTV) in BALB/c mice. In order to resolve these differences, we have compared the MMTV specific nucleic acids extracted from BALB/c normal organs and tumor tissue obtained from laboratories reporting either a high or low BALB/c mammary tumor incidence. Hybridization kinetics and restriction endonuclease analysis indicate that mammary tumor tissue from laboratories reporting a high mammary tumor incidence contains integrated MMTV-specific DNA that is not found in normal organs from these mice and is therefore not in the germ line. Furthermore we cannot detect these acquired MMTV DNA sequences in a BALB/c mammary tumor from a laboratory reporting a low mammary tumor incidence.     

Serological and biochemical characterization of the mouse mammary tumor virus with localization of p10.

The identification of a cyclic nucleotide-independent protein kinase in purified adenovirus types 2 and 5 is described. Enzyme activity was detected only in virus preparations which had been mildly disrupted by dialysis against low ionic strength buffers and not in intact virus preparations. Virus polypeptides Ma, V, VI, VII, and X were phosphorylated in vitro. The enzyme transferred the -y-phosphate of ATP to serine and threonine residues of virus proteins. Other proteins (histones, casein, and protamine sulphate) were phosphorylated by the virion protein kinase.     

Molecular basis of altered mouse mammary tumor virus expression in the D-2 hyperplastic alveolar nodule line of BALB/c mice

The preneoplastic D-2 hyperplastic outgrowth line, which was derived from a hormone-induced hyperplastic alveolar nodule (HAN) of a BALB/c mouse, was used for a detailed analysis of mouse mammary tumor virus (MMTV) expression. The D-2 HAN line has previously been shown to express viral RNA representative of the entire genome, although viral particles have been noted only rarely. The MMTV-specific mRNA, protein, and DNA content of the D-2 tissues was defined in an effort to better understand the molecular basis of the aberrant virus expression. Northern blotting techniques demonstrated the presence of properly processed 8.9 kb (genomic) and 3.6 kb (envelope) mRNA. Protein electroblotting procedures established the presence of properly processed viral core protein p28. In contrast, the envelope precursor polyprotein was not processed into detectable levels of gp52. Analysis of MMTV proviral content by Southern blot methodology revealed the presence of a newly acquired provirus which serves as a marker for the clonal nature of the D-2 line. The origin of the new provirus is unknown. Methylation studies established that the new proviral insert is hypomethylated and, therefore, is likely serving as the template for the MMTV expression observed in the D-2 HAN line. These characteristics of the D-2 line make it an excellent system in which to study the role, if any, of MMTV in the progression of D-2 preneoplastic tissues to the tumor phenotype.     

Effect of dexamethasone on expression of endogenous mouse mammary tumor virus sequences in BALB/c tumor cell lines.

BALB/c mammary tumor cell lines which contain only endogenous murine mammary tumor virus (MMTV) sequences respond to dexamethasone (DXS) treatment with minimal (approximately 2-fold) increases in MMTV RNA. This is in marked contrast to the 10? to 20-fold increases observed with cell lines harboring exogenous MMTV variants. Comparison of hybridization results obtained with two complementary DNA probes representative of either the entire MMTV RNA genome or its poly(A)-adjacent sequences suggests that the DXS response of BALB/c lines is also qualitatively different from that of exogenous MMTV-producer cell lines. Thermal stability studies suggested a 2–3% divergence between the RNA sequences of endogenous BALB/c and exogenous C3H viruses, with the 3′-end of the viral RNA appearing to be conserved relative to the rest of the genome.     

A comparative analysis of radiation- and virus-induced leukemias in BALB/c mice

Endogenous murine leukemia virus (MuLV) proviral copies were analyzed in thymomas induced in normal BALB/c (Fv-1b) and in Fv-1n congenic mice by X-irradiation. Both strains of mice developed leukemia with similar kinetics, indicating that N-tropism of endogenous MuLV was not a rate-limiting factor in development of disease. Southern blot analysis, using a probe specific for ecotropic virus and for ecotropic-specific sequences retained in pathogenic, env-recombinant viruses, showed that the majority of radiation leukemias lacked newly acquired, clonally integrated, proviruses. This was in contrast to virus-induced leukemias, which routinely exhibited several new proviral integration sites. When an internal proviral DNA restriction fragment was monitored, some radiation leukemias showed evidence of nonclonal infection, accounting for more frequent isolation of infectious virus from such leukemias. Differences in expression of T-cell surface antigens were found in X-ray-induced and virus-induced leukemias. All radiation leukemias were TL positive, whereas virus-induced leukemias were primarily negative for TL. Some differences were also found in Lyt-1 and Lyt-2 expression. The data as a whole suggest that, in the majority of cases, radiation leukemogenesis is not initiated by a viral route--that is, the sort of viral mechanism for which exogenous infection by known pathogenic MuLV is the paradigm.     

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.     

Type-specific determinants on proteins of an endogenous C3H mouse mammary tumor virus (MMTV) distinguish this virus from highly oncogenic exogenous MMTVs

The genetically transmitted endogenous MMTV isolated from C3H mice after removal of the milk-transmitted virus by foster nursing is designated C3Hf MMTV to distinguish it from the highly oncogenic milk-transmitted exogenous virus designated C3H MMTV. We have isolated a MMTV-expressing C3Hf mammary tumor cell line which has no exogenous proviral sequences detectable by analysis of DNA fragments generated by Pst I restriction endonuclease. This cell line produced sufficient C3Hf MMTV to allow purification of the major proteins and an antigenic comparison of this virus with highly oncogenic exogenous MMTVs from C3H, GR, and RIII strains of mice. The envelope glycoproteins, gp52 and gp36, purified from the C3Hf MMTV, were found to have both group- and type-specific reactivities. Only C3H MMTV gave incomplete competition in the gp36 assay and, therefore, could be distinguished from C3Hf, RIII, and GR MMTVs which gave complete competition. Unique antigenic determinants exist on the gp52 of C3Hf MMTV since it is the only virus to give complete competition in the gp52 radioimmunoassay. GR MMTV competed only 60%, whereas C3H and RIII MMTVs gave 80% competition. This was the first demonstration that RIII and C3Hf MMTVs were immunologically distinct. Only group-specific reactivity was found with the gag-coded MMTV p27; however, group and class antigenic determinants were found on the gag-coded MMTV p10.     

Gene order of murine mammary tumor virus gag proteins and env proteins

The gene orders of murine mammary tumor virus (MuMTV) gag proteins and env proteins have been determined by pactamycin mapping techniques. A MuMTV producing cell line, Mm5mt, was pulse-labeled with [³¹S]methionine in the presence or absence of 5 × 10^?7 M pactamycin, an inhibitor of initiation of protein synthesis. Both pactamycin and the labeled amino acid were removed after the pulsing period and cells were further incubated in normal growth medium. Virus was harvested from the medium after 24 hr, and the individual protein constituents of the virus were analyzed by SDS-gel electrophoresis. For each protein, the ratio of the radioactivity incorporation in the presence of pactamycin and in its absence was determined. The lower this ratio, the closer would be the protein to the amino terminal of the precursor polyprotein. The following gene orders were derived from these experiments: gag, NH, p10(p28,pp23)p14 COOH, and env, NH₂ gp52gp36 COOH.     

Common surface receptors on both mouse and rat cells distinguish different classes of mouse mammary tumor viruses

Cell surface receptors which bind mouse mammary tumor virus (MMTV) were detected on mouse and rat cells. Virus binding was quantitated by measuring ¹²⁵T-protein A binding to immune complexes composed of a C3H MMTV gp52 type-specific monoclonal antibody and receptor-bound MMTV. C3H MMTV binding to normal mouse mammary epithelial cells (NMuMG) was dose dependent and was ≥50% inhibited by GR MMTV, but not by endogenous C3Hf MMTV or Gross murine leukemia virus. These results were confirmed in [³H]leucine MMTV binding inhibition assays in which GR MMTV and C3H MMTV blocked C3H [³H]MMTV binding while C3Hf MMTV did not block. The affinities of C3H [³H]MMTV binding to receptors on NMuMG, NIH Swiss (SLP), and Fischer rat embryo (FRE) cells were identical by Scatchard analysis. C3Hf [³H]MMTV also bound these cells, but with an affinity approximately 10 times weaker than the C3H [³H]MMTV binding. Interference assays using a Kirsten sarcoma virus (C3H MMTV) pseudotype confirmed the importance of C3H MMTV specific binding to viral penetration and expression. Pretreatment of SLP or FRE cells with 100 μg of C3H MMTV or GR MMTV inhibited focus formation by ≥50% while C3Hf MMTV and RIII MMTV did not inhibit. Therefore, the functional C3H MMTV cell receptors on mouse and rat cells were related and were able to distinguish C3H MMTV and GR MMTV from C3Hf MMTV and RIII MMTV. These comparable receptors may represent evolutionarily conserved surface components of murine cells.     

Identification and characterization of a mouse mammary tumor virus protein uniquely expressed on the surface of BALB/cV mammary tumor cells

A unique subline of BALB/c mice, designated BALB/cV, exhibits an intermediate mammary tumor incidence (47%) and harbors a distinct milk-transmitted mouse mammary tumor virus (MMTV). The BALB/cV subline was used to study the molecular basis of potential virus-host interactions involving cell surface-expressed MMTV proteins. Cell surface iodination identified virus-specific proteins expressed on BALB/cV primary mammary tumor cells grown in culture. In contrast to (C3H)MMTV-producing cell lines which expressed MMTV gp52, BALB/cV tumor cells lacked gp52 and expressed instead a 68K, env-related protein. The 68Kenv protein was also detected on the surface of metabolically labeled BALB/cV tumor cells by an external immunoprecipitation technique. The expression of 68Kenv was restricted to mammary tissues of BALB/cV mice that also expressed other MMTV proteins. Biochemical analysis established that 68Kenv was not modified by N-linked glycosylation. 125I-labeled 68Kenv was rapidly released into the media of tumor cell cultures and was recovered both in the form of a soluble protein and in a 100,000 g pellet. The biologic function of this cell surface-expressed viral protein remains unknown.     

Endogenous mouse mammary tumor virus DNA is distributed among multiple mouse chromosomes.

We have examined the distribution of endogenous mouse mammary tumor virus-specific DNA in the genome of A/HeJ mice by using molecular hybridization and restriction endonucleases to analyze DNA from mouse-hamster hybrid clones that segregate mouse chromosomes. We have found that MMTV sequences are located on at least three separate chromosomal pairs, including chromosome number four.     

Mouse mammary tumor virus and murine leukemia virus cell surface antigens on virus producer and nonproducer mammary epithelial tumor cells.

Mouse mammary tumor virus (MMTV)- and murine leukemia virus (MuLV)- specific cell surface antigens (CSA) on virus producer and nonproducer mammary epithelial tumor cells were studied using the techniques of lactoperoxidase catalyzed iodination of cell surface proteins followed by radioimmune precipitation with monospecific antisera to the major MMTV proteins gp52, gp36, p27, and p10 and to the major MuLV proteins gp70 and p30. The incorporation of iodinated CSA into extracellular virus was determined by analyzing labeled proteins in purified virus. On cells producing only MMTV both gp52 and gp70 were present on the cell surface. Furthermore, gp52 was the only labeled protein in extracellular MMTV produced by these cells. On cells producing both MMTV and MuLV, both gp52 and gp70 were present on the cell surface, and were the only labeled proteins present in their respective extracellular viruses indicating that gp70 and gp52 are present on mutually exclusive cellular viral budding sites. In addition, MuLV anti-p30 serum precipitated two iodinated proteins with molecular weights of 85,000 and 95,000 daltons, analogous to the Gross cell surface antigen (GCSA). Labeled gp52 and gp70 represent true CSA as demonstrated by the fact that they were also present on the surface of cells producing no virus, but producing large amounts of MMTV glycoproteins and nonglyco-proteins. These results further demonstrate that the precursor to the MMTV glycoproteins (gPr75-MMTV env) is cleaved prior to the appearance of gp52 on the cell surface.     

The structure of the mouse mammary tumor virus: isolation and characterization of the core.

A procedure was developed for preparing cell fractions rich in chloroplasts, nuclei, and pea enation mosaic virus (PEMV)-induced cytopathological structures (vesicles). Those fractions from infected pea plants which contained nuclei or vesicles also contained actinomycin D-insensitive RNA polymerase activity and PEMV-specific hybridizable RNA. The fraction from infected plants containing predominantly chloroplasts had little of this polymerase activity or RNA, as was the case with all fractions from healthy plants. The significance of the polymerase activity in the nuclei and vesicles is discussed, as well as the potential role of the vesicles in the virus infection cycle.     

Production of mouse mammary tumor virus upon transfection of a recombinant proviral DNA into cultured cells

We have investigated the intracellular proteins synthesized in rat XC and feline kidney cells transfected with endogenous mouse mammary tumor virus (MMTV) proviral DNA. The endogenous provirus GR40, associated with the Mtv-8 locus, directs the synthesis of gag proteins indistinguishable from those found in MMTV-infected cells. The env precursor Pr73env and the mature gp52 proteins could not be detected in these cells. Instead an env-related protein of 68K is synthesized. In contrast to this endogenous provirus, a cloned exogenous proviral variant directs the synthesis of apparently normal env proteins upon transfection into the same cell lines. These results suggest that the env gene of the endogenous MMTV provirus GR40 is defective. The exogenous proviral variant is not expected to synthesize virus particles since it carries a rearrangement in the gag gene. In order to obtain an MMTV provirus capable of correctly expressing both gag and env functions, we have constructed a hybrid endogenous-exogenous provirus containing the 5' long terminal repeat (LTR)-gag of GR40 and the pol-env-3' LTR of the exogenous provirus. Upon transfection into feline kidney cells, this hybrid provirus directed the synthesis of apparently authentic gag and env proteins. Further, virus particles can be detected in the culture medium of the transfected cells by electron microscopy. Viral proteins obtained from viral particles banded in a sucrose gradient were detected by immunoprecipitation.     

Structural components of mouse mammary tumor virus. I. Polypeptides of the virion.

Mouse mammary tumor virus (mMTV) obtained from MJY-alpha cell cultures and analyzed by polyacrylamide gel electrophoresis possesses four major polypeptides and six to eight minor components. Three of the major proteins, with estimated molecular weights of 60,000, 52,000 and 37,000, are glycoproteins, as demonstrated by labeling with [³H]glucosamine. Labeling with [³⁵S]sulfate revealed significant amounts of sulfate associated with the 60,000 (gp60) and 52,000 (gp52) glycoproteins, whereas sulfate was minimally incorporated into the 37,000 (gp37) dalton glycoprotein. At least three glycopeptide species containing both [³H]glucosamine and [³⁵S]sulfate labels were obtained after extensive Pronase digestion of mMTV, indicating that [³⁵S]sulfate is covalently linked to the carbohydrate component of mMTV glycoproteins. Variations in the polypeptide pattern of mMTV were observed when virions were grown and labeled under different culture conditions. The amount of gp60 decreased substantially, with an apparent increase in a minor polypeptide at 33,000 daltons, if virions were harvested from stationary cultures or if culture medium was not changed daily during viral harvests. When virions containing gp60 were incubated with medium from stationary cultures or with stationary cell layers, the level of gp60 decreased with a corresponding increase in the amount of radioactivity at 33,000 daltons. Cleavage of gp60 and gp52 was demonstrated by treatment of purified mMTV virions with trypsin or alpha-chymotrypsin (1–150 μg/ml) for 1 min to 3 hr. Virions were morphologically unaltered after incubation with either protease; however, both gp60 and gp52 were absent from the polypeptide patterns. The data suggest that gp52 is cleaved to form 33,000, 22,000, and possibly 37,000 dalton glycoproteins which remain associated with virions. Other mMTV virion polypeptides were resistant to treatment with protease.     

Coexistence of the mouse mammary tumor virus (MMTV) major glycoprotein and natural antibodies to MMTV in sera of mammary tumor-bearing mice.

Sera of mammary tumor-bearing mice contain the major envelope glycoprotein (gp52) of mouse mammary tumor virus (MMTV) and autogenous antibodies to MMTV. Although antibodies to MMTV are readily demonstrable in sera of both tumor-free and tumorbearing animals, detection of the viral glycoprotein appears to be dependent on the presence of a palpable mammary tumor. MMTV gp52 was detected both as the free protein and in a high molecular weight complex as demonstrated by velocity sedimentation centrifugation. MMTV gp52 was also detected in both mammary tissue and lymph nodes of female C3H/HeN mice. The reproductive organs of the male C3H/HeN mice, such as the vas deferens and vesicular, coagulating, and prostate glands, contained gp52, while no MMTV gp52 could be demonstrated in the testes, epididymis, or preputial glands. This antigen was not found in any tissues of male or female BALB/c mice.     

Variants of type-C retroviruses from DBA/2 mice: Protein-structural and biological properties

Ecotropic murine leukemia viruses isolated from normal and carcinogen-treated DBA/2 mice can be classified into three main groups that differ in structure and biology. Two groups, called E_a and E_b, consist of N-tropic viruses related to the standard endogenous ecotropic virus of AKR mice. E_a viruses replicate with reduced efficiency in cell lines derived from C3H mice, while E_b viruses essentially replicate normally in these cells. As elsewhere reported, E_a viruses appear apathogenic in C3H mice, while E_b viruses cause a moderate incidence of late leukemias. The biological differences are associated with modulations of the fine structure of the gag gene-encoded proteins.A third group of viruses, called E_c, is clearly more diverged. They differ extensively from E_a and E_b viruses in the products of the gag and env gene, and are related to Rauscher leukemia virus. E_c viruses are NB-ecotropic; they replicate efficiently in all mouse cells tested, and induce leukemias in C3H mice with shorter latency periods than E_b viruses.Since published nucleic acid hybridization data indicate that DBA/2 mice only carry one ecotropic provirus, we assume that the DBA/2 viruses represent a developmental series of variants evolving during the life of the animals.     

Mammary tumors from GR mice contain more than one population of mouse mammary tumor virus-infected cells

Spontaneous mammary tumors in the GR mouse strain contain several acquired copies of mouse mammary tumor virus (MMTV) DNA that are not present in normal organ DNA and that are detectable by restriction endonuclease analysis and the Southern blotting procedure. Hormone-responsive and -independent cell populations were selected from spontaneous GR mammary tumors by grafting the tumors in castrated male GR mice in the presence and absence of female sex hormones. Analysis of the acquired MMTV DNA copies revealed differences between hormone-responsive and -independent cells derived from the same tumor; however, specific MMTV DNA fragments could not necessarily be correlated with the hormone responsiveness of the tumor. In some cases more than one proviral pattern could be detected for both hormone-responsive and hormone-independent cells. These results suggest that spontaneous GR mammary tumors are made up of more than one population of hormone-responsive and -independent cells which can be distinguished by their MMTV-specific proviral restriction fragments.