Infectious Mouse Mammary Tumor Virus Research Articles

Mouse Mammary Tumor Virus Superantigen Expression Is Reduced by Glucocorticoid Treatment

Expression of mouse mammary tumor virus (MMTV)-encoded superantigens in B lymphocytes are required for viral transmission and pathogenesis. Due to problems with detection and quantification of the superantigen protein, most reports about the mechanism of superantigen expression from the viral sag gene rely on the quantitative analysis of putative sag mRNAs. The description of multiple promoters as a source of putative sag mRNA has complicated the situation even further. All conclusions about the level of superantigen protein expression based on these data remain circumstantial. To test the effect of the glucocorticoid hormone dexamethasone on the total superantigen expression from an infectious MMTV provirus we used a quantitative assay that is based on a superantigen–luciferase fusion protein. MMTV gene expression from the major promoter in the 5′ long terminal repeat (LTR) is strongly induced in the presence of glucocorticoid hormones. We now demonstrate that, in the presence of dexamethasone, sag gene expression is reduced despite increased transcription from the MMTV 5′ LTR and increased amounts of putative sag mRNA initiated at the LTR promoter. These data show that the expression of the MMTV sag gene does not correlate with the activity of the major LTR promoter and thus differs from all other MMTV genes.

Cloning of an Infectious Milk-Borne Mouse Mammary Tumor Virus (MMTV) DNA from a Mammary Tumor That Developed in an Endogenous MMTV-Free Wild Mouse

Molecular characterization of infectious mouse mammary tumor viruses (MMTVs) has been hampered due to the problem of cloning a full-length exogenous virus into a plasmid. The present report describes our strategy for obtaining a full-length clone of an exogenous MMTV from a mouse mammary tumor that arose spontaneously in a wild Chinese mouse free of endogenous MMTV and shows that the cloned virus (JYG-MMTV) is expressed in rat RBA cells. Four-week-old C58/J × CBA/CaJ female mice, free of both endogenous and exogenous MMTVs, were injected with virus-secreting RBA cells. The progeny of these mice were bred, and their offspring were tested for the presence of MMTV. These third-generation mice were found to actively produce MMTV that was shed in their milk and transmitted to their offspring. The virus was detected not only in the mammary glands of these young mice, but also in their spleens and bone marrow. These results suggest that our plasmid-cloned exogeneous JYG-MMTV is infectious. This virus can now be used effectively in manipulating the various genes of JYG-MMTV and other MMTV strains to understand their structure/function relationships.

Differential reactivity of TCR Vbeta10 alleles to a mouse mammary tumor virus superantigen.

Mouse mammary tumor virus (MMTV) expresses a superantigen (SAg) which plays a critical role in the viral life cycle. We have recently described the new infectious MMTV (SIM) encoding a Vbeta4-specific SAg in mice with a TCR-Vbeta(b) haplotype. We have now compared the SAg activity of this virus in BALB/c mice harboring the TCR-Vbeta(a), TCR-Vbeta(b) or TCR-Vbeta(c) haplotypes which differ by a central deletion in the TCR-Vbeta(a) and TCR-Vbeta(c) locus and by mutations in some of the remaining Vbeta elements. Injection of MMTV (SIM) led to a strong stimulation of Vbeta4+ CD4+ T cells in TCR-Vbeta(b) mice, but only to a weak stimulation of these cells in TCR-Vbeta(a) or TCR-Vbeta(c) mice. A large increase in the percentage of Vbeta10+ cells was observed among CD4+ T cells in mice with the Vbeta(a) or Vbeta(c), but not the Vbeta(b) TCR-Vbeta haplotype. Vbeta10+ cells dominated the response when Vbeta10(a/c) and Vbeta4 subsets were present together. This is the first report of a viral SAg interacting with murine Vbeta10+ cells. Six amino acid differences between Vbeta10(a/c) and Vbeta10(b) could account for the gain of reactivity of Vbeta10(a/c) to the MMTV(SIM) SAg. No mutations were found in the hypervariable region 4 (HV4) of the TCR. Mutations at positions 22 and 28 introduce into Vbeta10(a/c) the same amino acids which are found at these positions in the MMTV(SIM)-reactive Vbeta4. Tridimensional models indicated that these amino acids lie close to HV4 and are likely to be important for the interaction of the SAg with the TCR.

Mutational and functional analysis of the C-terminal region of the C3H mouse mammary tumor virus superantigen.

The mouse mammary tumor virus (MMTV) encodes within the U3 region of the long terminal repeat (LTR) a protein known as the superantigen (Sag). Sag is needed for the efficient transmission of milk-borne virus from the gut to target tissue in the mammary gland. MMTV-infected B cells in the gut express Sag as a type II transmembrane protein that is recognized by the variable region of particular beta chains (Vbeta) of the T-cell receptor (TCR) on the surface of T cells. Recognition of Sag by particular TCRs results in T-cell stimulation, release of cytokines, and amplification of MMTV infection in lymphoid cells that are needed for infection of adolescent mammary tissue. Because the C-terminal 30 to 40 amino acids of Sag are variable and correlate with recognition of particular TCR Vbeta chains, we prepared a series of C-terminal Sag mutations that were introduced into a cloned infectious MMTV provirus. Virus-producing XC rat cells were used for injection of susceptible BALB/c mice, and these mice were monitored for functional Sag activity by the deletion of C3H MMTV Sag-reactive (CD4+ Vbeta14+) T cells. Injected mice also were analyzed for mutant infection and tumor formation in mammary glands as well as milk-borne transmission of MMTV to offspring. Most mutations abrogated Sag function, although one mutation (HPA242) that changed the negative charge of the extreme C terminus to a positive charge created a weaker Sag that slowed the kinetics of Sag-mediated T-cell deletion. Despite the lack of Sag activity, many of the sag mutant viruses were capable of sporadic infections of the mammary glands of injected mice but not of offspring mice, indicating that functional Sag increases the probability of milk-borne MMTV infection. Furthermore, although most viruses encoding nonfunctional Sags were unable to cause mammary tumors, tumors were induced by such viruses carrying mutations in a negative regulatory element that overlaps the sag gene within the LTR, suggesting that loss of Sag function may be compensated, at least partially, by loss of transcriptional suppression in certain tissues. Together these results confirm the importance of Sag for efficient milk-borne transmission and indicate that the entire C-terminal region is needed for complete Sag function.

Both T and B cells shed infectious mouse mammary tumor virus.

Mouse mammary tumor virus (MMTV) infected both B and T tissue culture cells and primary B and T cells in vivo after milk-borne transmission of the virus. The infected tissue culture cells processed viral proteins, and both these and primary B and T cells shed virus when cultured in vitro. Moreover, the infected B and T tissue culture cells transmitted virus to uninfected mammary gland cells in vitro. The level of infection of these different cell types in vivo was dependent on the strain of mouse, with C3H/HeN mice showing greater B-cell infection and BALB/c mice greater T-cell infection after nursing on MMTV-infected C3H/HeN mothers. Although their B cells were less infected, BALB/c mice developed tumors more rapidly than C3H/HeN mice. These results indicate that both infected T and B cells are potential carriers of MMTV in vivo.

In vivo T cell response to viral superantigen. Selective migration rather than proliferation.

Superantigens induce T cell activation and proliferation in vitro, and some also induce cell activation in vivo. MMTV(SW) is an infectious mouse mammary tumor virus (MMTV) encoding a superantigen with the same Vbeta specificity as MIs-1a (Mtv-7), which induces a strong local response in vivo. injection of MMTV(SW) into mouse footpads leads to accumulation of superantigen-reactive T cells (Vbeta6+CD4+) and B cells in the draining lymph nodes (LN). We investigated the kinetics of this cell accumulation by measuring cell activation (blastogenesis, CD25 and CD69 expression), cell migration (using syngenic FITC-labeled CD4+ cells and L-selectin detection), and cell proliferation (using in vivo labeling with bromodeoxyuridine). Specific T cells selectively migrated to the draining LN. Accumulating Vbeta6+CD4+ T cells were large CD69+ cells, but remained CD25 negative and showed down-regulated L-selectin expression. Their DNA synthesis rate, studied by pulse labeling and continuous administration of bromodeoxyuridine, was increased, but remained too low to explain the draining LN hyperplasia. These data show that the local T cell response to MMTV(SW) mainly consists of selective migration followed by local activation of reactive T cells, and that cell proliferation is only a minor component of the response. By contrast, the optimal dose of staphylococcal enterotoxin B that, nevertheless, leads to a lower reactive T cell accumulation in the draining LN induces a very high proliferation rate.

Direct physical interaction involving CD40 ligand on T cells and CD40 on B cells is required to propagate MMTV

The propagation of mouse mammary tumor virus (MMTV) has been analyzed in mice defective for expnasion of CD40 ligand (CD40L). Mice with endogenous viral superantigen (SAG) delete T cells with cognate Vβ independent of CD40L expression. Nevertheless, CD40L − mice do not show deletion of cognate T cells after being exposed to infectious MMTV and have greatly diminished viral replication. The response of CD40L − T cells to SAG in vitro is also Impaired, but can be reconstituted by adding B cells activated by recombinant CD40L to express costimulatory molecules. Thus, direct CD40L-dependent B cell activation appears to be a critical step In the life cycle of MMTV. The initial step in SAG-dependent T cell activation, and hence the MMTV life cycle, may be mediated by non-B cells, because splenocytes from B cell-deficient SAG-transgenic mice are able to activate cognate T cells.

A new infectious mammary tumor virus in the milk of mice implanted with C4 hyperplastic alveolar nodules

We have previously characterized an infectious mouse mammary tumor virus [(MMTV(SW)] which induces a strong superantigen response in vivo. Here we describe the isolation and characterization of MMTV(C4) which was derived from milk of mice implanted with hyperplastic alveolar nodules. MMTV(C4) stimulates V beta 2 expressing T cells after local injection in vivo. Comparison with known open reading frame (orf) sequences revealed high homology to Mtv-6, an endogenous virus interacting with V beta 3-expressing T cells. The carboxyl-terminal amino acids were, however, altered. High homology including the carboxyl-terminal orf amino acids were found with MMTV(C3H-K). We show here that MMTV(C3H-K) has lost its superantigen function. Sequence comparisons permitted the characterization of few key amino acids which could be important for T cell receptor interaction and superantigen processing.

Expression of a MHC class II transgene determines both superantigenicity and susceptibility to mammary tumor virus infection.

Milk-borne mouse mammary tumor virus (MMTV) is a type B retrovirus that induces mammary carcinoma. Infectious MMTV, as well as genomically integrated mouse mammary proviruses, encode superantigens that are recognized by T cells that express appropriate T cell receptor V beta products. To determine the relationship between the superantigenic property of milk-borne MMTV and its in vivo infectivity, mice which were either positive or negative for expression of a transgene-encoded E alpha E beta class II major histocompatibility complex (MHC) product were exposed to milk borne C3H MMTV. Superantigen-mediated deletion of V beta 14-expressing T cells occurred only in E alpha transgene-positive mice, indicating that the deletion was E alpha E beta dependent. When mice were analyzed for viral infection by assaying viral p28 in the milk of recipient females, significant p28 levels were found only in E alpha E beta transgene-positive mice. Similarly, the presence of C3H MMTV LTR mRNA in mammary glands, as detected by PCR, paralleled p28 levels. These findings indicate that E alpha expression or the E alpha-dependent T cell response to viral superantigen is causally related to susceptibility to MMTV infection, and that lack of a permissive class II product can protect mice from virus infection.

Superantigen-reactive CD4+ T cells are required to stimulate B cells after infection with mouse mammary tumor virus.

Superantigens are defined by their ability to stimulate a large fraction of T cells via interaction with the T cell receptor (TCR) V beta domain. Endogenous superantigens, classically termed minor lymphocyte-stimulating (Mls) antigens, were recently identified as products of open reading frames (ORF) in integrated proviral copies of mouse mammary tumor virus (MMTV). We have described an infectious MMTV homologue of the classical endogenous superantigen Mls-1a (Mtv-7). The ORF molecules of both the endogenous Mtv-7 and the infectious MMTV(SW) interact with T cells expressing the TCR V beta 6, 7, 8.1, and 9 domains. Furthermore, the COOH termini of their ORF molecules, thought to confer TCR specificity, are very similar. Since successful transport of MMTV from the site of infection in the gut to the mammary gland depends on a functional immune system, we were interested in determining the early events after and requirements for MMTV infection. We show that MMTV(SW) infection induces a massive response of V beta 6+ CDC4+ T cells, which interact with the viral ORF. Concomitantly, we observed a B cell response and differentiation that depends on both the presence and stimulation of the superantigen-reactive T cells. Furthermore, we show that B cells are the main target of the initial MMTV infection as judged by the presence of the reverse-transcribed viral genome and ORF transcripts. Thus, we suggest that MMTV infection of B cells leads to ORF-mediated B-T cell interaction, which maintains and possibly amplifies viral infection.

Inhibition of mouse mammary tumor virus-induced T cell responses in vivo by antibodies to an open reading frame protein.

Minor lymphocyte stimulating (Mls) antigens specifically stimulate T cell responses that are restricted to particular T cell receptor (TCR) beta chain variable domains. The Mls phenotype is genetically controlled by an open reading frame (orf) located in the 3' long terminal repeat of mouse mammary tumor virus (MMTV); however, the mechanism of action of the orf gene product is unknown. Whereas predicted orf amino acid sequences show strong overall homology, the 20-30 COOH-terminal residues are strikingly polymorphic. This polymorphic region correlates with TCR V beta specificity. We have generated monoclonal antibodies to a synthetic peptide encompassing the 19 COOH-terminal amino acid residues of Mtv-7 orf, which encodes the Mls-1a determinant. We show here that these antibodies block Mls responses in vitro and can interfere specifically with thymic clonal deletion of Mls-1a reactive V beta 6+ T cells in neonatal mice. Furthermore, the antibodies can inhibit V beta 6+ T cell responses in vivo to an infectious MMTV that shares orf sequence homology and TCR specificity with Mtv-7. These results confirm the predicted extracellular localization of the orf COOH terminus and imply that the orf proteins of both endogenous and exogenous MMTV interact directly with TCR V beta.

Mammary tumorigenesis in feral mice: identification of a new int locus in mouse mammary tumor virus (Czech II)-induced mammary tumors.

A population of Mus musculus subsp. musculus (Czech II), recently isolated from the wild, lack endogenous mouse mammary tumor virus (MMTV) proviral genomes. Some of these mice carry an infectious MMTV [designated MMTV (Czech II)] that is transmitted in the milk and is associated with mammary tumor development. This virus is distinct from laboratory strains of MMTV present in inbred mice. An MMTV (Czech II) genome was found within a 0.5-kilobase region of the cellular genome in five of 16 Czech II mammary tumors. MMTV insertion at this site activates expression of a 2.4-kilobase species of RNA from a previously silent cellular gene. This region of the cellular genome was designated int-3 since it is unrelated to the int-1 and int-2 loci. The int-3 locus does not appear to correspond to other proto-oncogenes but is well conserved among mammalian species.

Mammary tumorigenesis in feral Mus cervicolor popaeus

A pedigreed breeding population of feral Mus cervicolor popaeus with a high incidence of mammary tumors, arising between 6 and 14 months of age, is described. These mice were chronically infected with a type B retrovirus which is distantly related to the mouse mammary tumor virus (MMTV) of inbred strains of Mus musculus. MMTV-induced mammary tumors in inbred mice frequently (80%) contained an insertion of the viral genome into the int-1 or int-2 loci of the tumor cellular genome. These two cellular genetic loci were also altered by viral insertion in 11 of 20 M. cervicolor popaeus mammary tumor cellular DNAs tested. Results of our study of mammary tumorigenesis in feral mice demonstrate that viral-induced rearrangement and activation of the int loci are not limited to the genetic background of inbred mice selected for highly infectious MMTV and a high incidence of mammary tumors.

Mouse mammary tumor virus proviral sequences congenital to C3H/Sm mice are differentially hypomethylated in chemically induced, virus-induced, and spontaneous mammary tumors.

C3H/Sm mice have lost the exogenous milk-borne mouse mammary tumor virus (MMTV) characteristic of the C3H strain and have a very low (1.5%) incidence of spontaneous mammary tumors, yet they are highly susceptible to mammary carcinogenesis by either chemical carcinogens or infection with the milk-borne virus. We have analyzed the MMTV proviral DNA content of normal tissues and of spontaneous, virus-induced, and chemically induced mammary tumors by restriction endonuclease digestion and Southern blot analysis. Although the results clearly showed additional MMTV sequences in the virus-induced tumor which are not present in normal liver DNA, none of the spontaneous or chemically induced tumors could be shown to contain either newly acquired exogenous or amplified endogenous MMTV sequences. Interestingly, mammary tumors arising in C3H/Sm mice treated simultaneously with infectious MMTV (C3H) and dimethylbenz[a]anthracene (DMBA) possessed new exogenous MMTV DNA even though no quantitative change in tumor production was observed when these mice were compared with C3H/Sm mice treated with DMBA alone (Smith et al., Int. J. Cancer 26:373-379, 1980). Our data indicate that the endogenous MMTV proviral units are extensively methylated in normal tissues, such as livers and normal nonlactating mammary glands. In the absence of MMTV (C3H), we found that in the rare, spontaneously occurring C3H/Sm mammary tumors, certain endogenous MMTV sequences were specifically hypomethylated. Hypomethylation of endogenous MMTV sequences was also noted in the chemically induced mammary tumors, even though radioimmune competition assays for MMTV gp52 and p28 are negative (Smith et al., Int. J. Cancer 27:81-86, 1981). Our results support the conclusion that amplification of endogenous MMTV sequences is not intrinsic to C3H/Sm mouse mammary tumors arising spontaneously or after induction by chemicals. On the other hand, integration of exogenous MMTV DNA into the genome was a constant feature of mammary tumors developing in MMTV (C3H)-infected C3H/Sm mice, even when DMBA was used as the carcinogen. Hypomethylation of some endogenous MMTV sequences is characteristic of C3H/Sm mammary tumors, whether spontaneous or induced by chemicals, which suggests that these sequences are located in actively transcribing regions of the tumor cell genome.