A common mouse mammary tumor virus integration site in chemically induced precancerous mammary hyperplasias

Mammary carcinomas can be induced by chemical and hormonal as well as viral carcinogens. Irrespective of the class of inducer, these tumors develop in discrete stages, of which alveolar hyperplasia is one of the earliest identifiable. Since carcinogenesis by the mammary tumor virus is now thought to involve proviral activation of adjacent cell genes at specific loci, we sought to determine if a similar mechanism also played a role in chemical and hormonal carcinogenesis and if its role was stage specific. Three high-tumor-incidence BALB/c hyperplastic alveolar nodule outgrowths of two different etiologies were found to have exogenous mouse mammary tumor virus proviruses integrated at the same site in the genome. This common site of integration is not within the bounds of the int-1 and int-2 loci into which proviruses detected at these loci are clustered in MMTV-induced mammary tumors. All three HANs are commonly impaired in end-point differentiation. We propose that mouse mammary tumor virus integration at this site is responsible for a specific abnormality in differentiation associated with the preneoplastic phenotype.     

A rare common integration site of proviruses of the mouse mammary tumor virus in P-type mammary tumors of mouse strain GR

The mouse mammary tumor virus (MMTV) can induce mammary tumors in mice by proviral activation of the cellular oncogenes int-1 or int-2. Activation of these genes, however, is observed in only a few hormone- and pregnancy-dependent mammary tumors of the mouse strain GR. To study the possible involvement of other oncogenes we cloned three MMTV proviral-host fragments (MT 40, 42, and 53) from different mammary tumors of GR with a single acquired MMTV provirus. From a genomic library of normal mouse DNA we isolated phages with insert DNAs that covered 20-30 kb of the uninterrupted regions. Suitable probes devoid of repetitive DNA sequences were isolated in order to screen other mammary tumors for MMTV proviral integrations in these regions. Only two mammary tumors, MT 40 and 42, showed integration of extra MMTV proviruses within the same region. The integrations occurred only 60 bp apart. The other mammary tumors, however, did not contain MMTV proviral integrations in this region, nor in the MT 53 region. Using mouse-hamster somatic cell hybrid DNA, the MT 40/42 integration region was assigned to mouse chromosome 7, and the second region, MT 53, to chromosome 16. The two regions bear no homology to known cellular oncogenes. We did not observe any mRNA being expressed from these cloned segments either in tumors or in normal mammary glands. These findings indicate that plaque(P)-type mammary tumors in mouse strain GR do not originate from MMTV provirus insertions in a particularly favored integration region, but that there may be a variety of integration sites in these tumors.     

Surface structure of mouse mammary tumor virus

The structure of the envelope of mouse mammary tumor virus (MuMTV) was studied by using negative stain, thin sections and freeze-drying-shadowing. The surface of the viral membrane was found to contain a reticular structure composed mainly of hexagons and occasional pentagons. The spacing between the nearest corners was about 74 Å. The viral projections were attached to this reticulum and each projection was surrounded by five or six immediate neighbors. The average interprojection distance was 73.7 ± 10.3 Å. The projections have two distinguishable components, knobs at the distal ends and thin stalks that connect the knobs to the viral member-reticulum. The knobs measure 54.4 ± 10.3 Å in diameter and the average length of the projections was 94.9 ± 8.4 Å. The projection knobs were found to be composed of at least three subunits 15–25 Å in diameter. To explain the pattern of distribution of the surface projections in relation to the reticular structure, we propose that MuMTV contains two types of projections.Freeze-drying and shadowing revealed a highly regular hexagonal array of pits on the viral surface. Pits in fivefold symmetry were also observed. They were spaced 218 ± 43 Å apart and were about 80–100 Å in diameter.Considering the symmetry of the surface projections, reticular structure and pits, we conclude that the shape of the envelope of mouse mammary tumor virus is quasi-icosahedral or like a geodesic dome.     

Gene order of the mouse mammary tumor virus glycoproteins

Immunochemical and tryptic peptide mapping techniques were used to show that the mouse mammary tumor virus (MMTV) envelope glycoproteins gp52 and gp36 are distinct components derived from a common glycosylated precursor polypeptide of 75,000 daltons (gPr75). Because both gp52 and gp36 are derived from a common precursor polypeptide and therefore have a common initiation site, we have been able to determine their gene order within the viral genome. The gene order was deduced from three different types of experiments. The first approach measured the differential inhibition by NaCl hypertonic shock on initiation of gp52 and gp36 synthesis. The second approach measured the kinetics of appearance of various MMTV proteins following the synchronized reinitiation of polypeptide synthesis resulting from NaCl hypertonic shock. The third approach analyzed polypeptides released from polyribosomes after a series of variable-length short pulses with [3H]amino acids. Our results indicate that the gene order for gPr75 is H2N-gp52-gp36-COOH and 5'-gp52-gp36-3' within the MMTV genome.     

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.     

Identification of a protein that recognizes a distal negative regulatory element within the mouse mammary tumor virus long terminal repeat.

The mouse mammary tumor virus (MMTV) long terminal repeat contains a distal negative regulatory element (dNRE) that selectively represses activity of the proviral promoter in the absence of steroid hormone receptor-mediated activation. A protein, termed MMTV NRE-binding protein 1 (MNBP-1), that recognizes long terminal repeat sequences between -433 and -418 was identified by gel electrophoresis mobility shift assays and methylation interference footprinting in nuclear extracts of HeLa and Ltk(-) cells. Mutations within the defined binding site affect dNRE-mediated promoter repression in vivo. MNBP-1 has an apparent molecular mass of approximately 100 kDa as determined by gel filtration chromatography.     

The polypeptide composition of mouse mammary tumor virus

The polypeptide composition of mouse mammary tumor virus isolated from several strains of mice, has been examined by polyacrylamide gel electrophoresis. Gel Patterns, as revealed by staining and ¹⁴C amino acid labelling, showed 10 polypeptide species reproducibly associated with the virus. Three of these contain carbohydrate moieties as determined by incorporation of labelled glucosamine.     

Antisense downregulation of a mouse mammary tumor virus activated protooncogene in mouse mammary tumor cells reverses the malignant phenotype

Activation of the protooncogene Wnt-1 by insertion of the mouse mammary tumor virus (MMTV) is known to cause mammary tumors in mice. Wnt-1 expression in mammary glands has been postulated to confer direct local growth stimulation of mammary epithelial cells leading to their acquisition of a preneoplastic state. Wnt-1 expression also induces morphological alterations in cultured normal mammary cells. However, it has not been determined whether or not transformed mammary cells require continuous Wnt-1 expression for their ability to form tumors in vivo. To address this question, we constructed antisense and sense Wnt-1 expression vectors containing a synthetic promoter composed of five high-affinity glucocorticoid response elements (GRE5). This promoter is at least 50-fold more inducible by dexamethasone than the promoter contained in the long terminal repeats of MMTV. The vectors were introduced into a mouse mammary tumor cell line (R/Sa-MT) that expresses high levels of endogenous Wnt-1 mRNA and forms rapidly growing tumors when transplanted into syngeneic hosts. Of the 12 stably transfected cell lines established (9 with antisense and 3 with sense constructs), 2 antisense cell lines (R/Sa-MT/antisense) and 1 sense cell line (R/Sa-MT/sense) were examined for inducibility by dexamethasone of antisense and sense Wnt-1 RNAs, changes in endogenous Wnt-1 RNA expression, and changes in cell morphology. The growth patterns of the cells in vitro and in vivo were also examined. Our results show that (1) the levels of the expression of endogenous Wnt-1 mRNA and protein were reduced significantly (>80%) in those cells (R/Sa-MT/antisense) that expressed antisense Wnt-1 RNA at high levels following exposure to dexamethasone, compared to the R/Sa-MT/sense and R/Sa-MT control cells and (2) transplantation of the R/Sa-MT/antisense cells produced smaller tumors ( approximately 0.2 cm in 16 weeks) compared to the tumors ( approximately 2.0 cm in 8 weeks) that were produced by the R/Sa-MT/sense and R/Sa-MT cells. We therefore suggest that Wnt-1 expression is required not only for the transformation of normal mammary cells into tumor cells, but also for the maintenance of their tumorigenicity.     

Identification of human homologues of the mouse mammary tumor virus receptor

Summary.  Human breast tumors contain DNA sequences with high homology to the Mouse Mammary Tumor Virus (MMTV) env gene. env encodes the membrane glycoprotein that binds to the MMTV receptor (Mtvr) in mouse tissues and is required for infection. If humans become infected with MMTV, identification of a human Mtvr might shed light on the mechanism of infection. Here, we identified two human genes, Mtvr1 and Mtvr2, encoding proteins highly related to the mouse receptor. Mtvr-related sequences were also detected in other mammalian species. Thus, zoonotic transmission of MMTV is a real possibility given the existence of highly homologous MMTV receptors. Received July 23, 2001 Accepted October 20, 2001     

Superantigens and retroviral infection: insights from mouse mammary tumor virus

Superantigens induce a vigorous immune response by stimulating T cells that express particular T-cell receptor Vβ chains. Mouse mammary tumor virus is a milk-transmitted retrovirus that encodes such a superantigen. Paradoxically, as discussed by Werner Held and colleagues, the strong superantigen-induced immune response permits the survival of the virus via T-cell dependent clonal expansion of infected B cells.     

Interplays between mouse mammary tumor virus and the cellular and humoral immune response

Summary: Mouse mammary tumor virus has developed strategies to exploit the immune response. It requires vigorous immune stimulation to achieve efficient infection. The infected antigen-presenting cells present a viral superantigen on the cell surface which stimulates strong CD4-mediated T-cell help but CDS T-cell responses are undetectable. Despite the high frequency of superantigen-reactive T cells, the superantigen-induced immune response is comparable to classical antigen responses in terms of T-cell priming, T-cell—B-cell collaboration as well as follicular and extra-follicular B-cell differentiation. Induction of systemic anergy is observed, similar to classical antigen responses wbere antigen is administered systemically but does not influence the role of the superantigen-reactive T cells in the maintenance of the chronic germinal center reaction. So far we have been unable to detect a cytotoxic T-cell response to mouse mammary tumor virus peptide antigens or to the superantigen. This might yet represent another step in the viral infection strategy.     

Maturation of mouse mammary tumor virus envelope protein is blocked by a specific point mutation.

Expression of a mouse mammary tumor virus (MMTV) envelope gene, ENV2, in COS-7 cells resulted in the synthesis of a precursor protein Pr74 that underwent complete proteolytic processing to the mature envelope species gp52 and gp33. In contrast, expression of a second MMTV envelope gene, ENV1, in COS-7 cells resulted in the synthesis of a precursor protein Pr74 that failed to undergo proteolytic processing to the mature products. Expression and sequencing of various chimeric ENV1/ENV2 genes determined that a single glycine-to-glutamic acid mutation at position 54 was responsible for the nonprocessable phenotype of ENV1-encoded Pr74. The significance of this point mutation in relation to the requirements for precursor protein folding and maturation are discussed.     

Effects of cytoskeletal disrupting agents on mouse mammary tumor virus replication

Alterations in mouse mammary tumor virus (MMTV) production and composition were induced by exposure of mammary tumor cells to cytodisruptive agents. Treatment with 2.1 microM cytochalasin D (CD) for 24 h reduced MMTV yield by 80% and electron microscopic examination of these cells did not reveal budding virions. Immune precipitation and quantitative immunofluorescence studies demonstrated that CD had no significant effect on MMTV polypeptide synthesis or surface expression suggesting that CD inhibited late steps in MMTV maturation. Decreases in MMTV production were also observed as a result of 24 h exposure of the cells to 2.1 microM cytochalasin B (CB). However, an initial 70% increase in the levels of extracellular virions within the first 18 h of treatment preceded diminution of virus production. In addition, CB was unable to abrogate maturation and release of MMTV particles as revealed by electron microscopic evaluation of thin sections of treated cells. Colcemid at 0.28 microM had no effect on virus production during the first 24 h of exposure although MMTV yield was reduced by 60-70% after 36 h of treatment. Polypeptide profiles of MMTV purified from cell cultures treated with any of the three cytodisruptive agents were altered and included 5-7 polypeptides not typically present in MMTV from untreated cells. These cytodisruptive agents did not significantly affect viability and protein metabolism of MJY-alpha cells; the data suggest that alterations in MMTV replication were due to disruption of the cellular cytoskeleton.     

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.