Exogenous Mouse Mammary Tumor Virus Research Articles

Viral superantigens

Superantigens (SAgs) are microbial proteins produced by various microorganisms that elicit excessive and strong stimulation of T cells via an unconventional mechanism. They cause polyclonal activation of T cells in a non-specific manner, by binding to a particular variable-beta (Vβ) chain of T-cell receptor (TCR) and MHC class II molecule, in unprocessed form and outside of peptide-binding cleft, forming a bridge between the antigen presenting cell and the T cell. SAgs are classified into three groups, namely 1) exogenous (soluble proteins and exotoxins secreted by microorganisms), 2) endogenous (transmembrane proteins encoded by viruses which are integrated into the genome) and 3) B-cell SAgs (proteins which stimulate predominantly B cells). The best characterized and mostly studied SAgs are staphylococcal and streptococcal exotoxins, however it is well-known that many other microorganisms also possess SAg activities. Despite the presence of several viruses that cause severe infections in humans, the number of viruses that have proteins identified with SAg property in their pathogenesis, is relatively low. To date, the defined viruses that encoded SAgs are as follows; mouse mammary tumor virus (MMTV) (Marrack, et al. 1991), rabies virus (Lafon, et al. 1992), Epstein-Barr virus (EBV) (Sutkowski, et al. 1996), human endogenous retrovirus (HERV) (Conrad, et al. 1997), human immunodeficiency virus (HIV) (Posnett, et al. 1995; Torres, et al. 1996; Townsley-Fuchs, et al. 1997) and Ebola virus (Leroy, et al. 2011). SAgs were first described in the MMTV, a polymorphic B-type retrovirus that is either contained in the genome as an endogenous provirus (germline transmission) or exogenous infectious virus that transmits vertically via breast milk. Both MMTV forms encode SAgs. The SAg-mediated massive T cell activation is required for the spread of exogenous MMTV from intestines to mammary glands, facilitating the transmission of infectious virus. On the other hand, expression of endogenous SAgs leads to thymic deletion of responding T cells (bearing Vβ6-9+ TCR) due to self-tolerance induction during the fetal life, and protects the host against future exogenous MMTV infections. The SAg of rabies virus is the N protein found in nucleocapsid structure and stimulates Vβ8+TCR-bearing T cells. The SAg-induced polyclonal activation of T cells leads to turn-off the specific immune response, to enhance the immunopathogenesis and facilitates viral transmission from the initial site of infection (the muscle tissue) to the nerve endings. In case of EBV-associated SAg that activates Vβ13+TCR-bearing T cells, it was detected that the SAg activity was not encoded by EBV itself, but instead was due to the transactivation of HERV-K18 by EBV latent membrane proteins, whose env gene encodes the SAg (Sutkowski, et al. 2001). It has been denoted that EBV-induced SAg expression plays a role in the long-term persistence and latency of virus in memory B cells, in the development of autoimmune diseases and in the oncogenesis mechanisms. The proteins which are identified as SAgs of HIV are Nef and gp120. It is believed that, the massive activation of CD4+ T cells (selectively with Vβ-12+, Vβ-5.3+ and Vβ-18+ TCRs) in early stages of infection and clonal deletion, anergy and apoptosis of bystander T cells in the late stages may be due to SAg property of Nef protein, as well as the other mechanisms. However there are some studies indicating that Nef does not act as a SAg (Lapatschek, et al. 2001). HIV gp120 glycoprotein is a B-cell SAg that binds to VH3-expressing B cell receptors and causes polyclonal B cell activation. In addition, binding of gp120 to IgE on the surface of basophiles and mast cells causes activation of those cells, secretion of high level proinflammatory mediators leading to allergic reactions and tissue damage. In a recent study, the depletion (anergy or deletion) of T cell populations bearing Vβ12+, Vβ13+ and Vβ17+ TCR have been shown, in patients infected with Zaire Ebola virus, whatever the clinical outcome (death or recovery), these results also suggest the presence of SAg activity. In this review article, following a brief description of the general characteristics of SAgs, virus-encoded SAgs and their roles in the diseases have been discussed.

Mouse mammary tumor virus derived from wild mice does not target Notch-4 protooncogene for the development of mammary tumors in inbred mice

The colony of wild mice, named Jyg, has been shown to express an exogenous mouse mammary tumor virus (Jyg-MMTV). This virus induces mammary tumors in its natural host at a high incidence (≈ 80%) resulting from insertion mutations in Notch-4 (43%), Wnt-1 (26%), and Fgf-3 (13%). Since the activation of Notch-4 is not common in mammary tumors of standard laboratory strains of mice infected with various MMTV strains, we examined the consequences of Jyg-MMTV infection in BALB/c and C57BL/6 mice. The results show that Jyg-MMTV induces mammary tumors in both mouse strains, but the incidence of mammary tumors in BALB/c mice is greater than in C57BL/6 mice. Surprisingly, however, none of the 75 mammary tumors, analyzed both by Southern and Northern hybridizations, showed insertion mutations in or expression of Notch-4. In contrast, both Wnt-1 and Fgf-3 were found to be involved in these tumors. Our findings may suggest, among other possibilities, the existence of a structural difference(s) between laboratory and wild mice at the Notch-4 locus that regulates the integration of Jyg-MMTV proviral DNA.

BALB/ Mtv -Null Mice Responding to Strong Mouse Mammary Tumor Virus Superantigens Restrict Mammary Tumorigenesis

The absence of endogenous mouse mammary tumor viruses (MMTVs) in the congenic mouse strain, BALB/Mtv-null, restricts the early steps of exogenous C3H MMTV infection, preventing the superantigen (Sag) response and mammary tumorigenesis. Here we demonstrate that BALB/Mtv-null mice also resist tumor induction by FM MMTV, which encodes a stronger Sag compared to C3H MMTV. In contrast to infections with C3H MMTV, Mtv-null mice show FM-MMTV Sag-specific responses comparable to those observed in susceptible BALB/c mice. Neither virus shows significant replication in the spleen or mammary gland. Thus, Mtv-null mice restrict MMTV replication and mammary tumorigenesis even after a robust Sag response.

Sequences within thegagGene of Mouse Mammary Tumor Virus Needed for Mammary Gland Cell Transformation

Previously, we identified a group of replication-competent exogenous mouse mammary tumor viruses that failed to induce mammary tumors in susceptible mice. Sequence comparison of tumorigenic and tumor-attenuated virus variants has linked the ability of virus to cause high-frequency mammary tumors to the gag gene. To determine the specific sequences within the gag gene that contribute to tumor induction, we constructed five distinct chimeric viruses that have various amino acid coding sequences of gag derived from a tumor-attenuated virus replaced by those of highly tumorigenic virus and tested these viruses for tumorigenic capacities in virus-susceptible C3H/HeN mice. Comparing the tumorigenic potentials of these viruses has allowed us to map the region responsible for tumorigenesis to a 253-amino-acid region within the CA and NC regions of the Gag protein. Unlike C3H/HeN mice, BALB/cJ mice develop tumors when infected with all viral variants, irrespective of the gag gene sequences. Using genetic crosses between BALB/cJ and C3H/HeN mice, we were able to determine that the mechanism that confers susceptibility to Gag-independent mammary tumors in BALB/cJ mice is inherited as a dominant trait and is controlled by a single gene, called mammary tumor susceptibility (mts), that maps to chromosome 14.

Endogenous MMTV Proviruses Induce Susceptibility to Both Viral and Bacterial Pathogens

Most inbred mice carry germline proviruses of the retrovirus, mouse mammary tumor virus (MMTV) (called Mtvs), which have multiple replication defects. A BALB/c congenic mouse strain lacking all endogenous Mtvs (Mtv-null) was resistant to MMTV oral and intraperitoneal infection and tumorigenesis compared to wild-type BALB/c mice. Infection of Mtv-null mice with an MMTV-related retrovirus, type B leukemogenic virus, also resulted in severely reduced viral loads and failure to induce T-cell lymphomas, indicating that resistance is not dependent on expression of a superantigen (Sag) encoded by exogenous MMTV. Resistance to MMTV in Mtv-null animals was not due to neutralizing antibodies. Further, Mtv-null mice were resistant to rapid mortality induced by intragastric inoculation of the Gram-negative bacterium, Vibrio cholerae, but susceptibility to Salmonella typhimurium was not significantly different from BALB/c mice. Susceptibility to both MMTV and V. cholerae was reconstituted by the presence of any one of three endogenous Mtvs located on different chromosomes and was associated with increased pathogen load. One of these endogenous proviruses is known to encode only Sag. Therefore, Mtv-encoded Sag appears to provide a unique genetic susceptibility to specific viruses and bacteria. Since human endogenous retroviruses also encode Sags, these studies have broad implications for pathogen-induced responses in mice and humans.

Ecotropic murine leukemia viruses and exogenous mouse mammary tumor viruses are not essential for pristane-induced lupus.

Ecotropic murine leukemia viruses and exogenous mouse mammary tumor viruses are not essential for pristane-induced lupus.

Sequence and genetic analyses of the 3' terminus and integration sites of the RIII/Sa mouse mammary tumor (MMTV) exogenous provirus.

The RIII/Sa mouse mammary tumor virus (MMTV) is one of several exogenous MMTV viruses transmitted to suckling mice through the milk. We report herein the nucleotide sequence of the exogenous RIII/Sa provirus from the central Eco RI site through to the end of the U5 region encoded by the 3' LTR. We also provide a detailed sequence analysis often different 3' terminal exogenous MMTV proviral integration sites within mammary tumor DNA obtained by the technique of genome walking. Using a combination of Southern blotting with 3' end probes and PCR utilizing a unique RIII/Sa specific 3' primer, we confirm that the RIII/Sa provirus integrates multiple times in mouse mammary tumors but that little or no integration occurs in various normal tissues. Southern blotting analyses with 3' end probes also indicate that RIII/Sa mice contain two additional endogenous MMTV loci, mtv-6 and mtv- 17, not previously reported. The combined genetic analyses reported herein distinguish between exogenous proviral integrants and endogenous germline MMTV.

Genetics of mouse mammary tumor virus-induced mammary tumors: linkage of tumor induction to the gag gene.

Retroviruses are believed to induce tumors by acting as insertional mutagens that activate expression of cellular protooncogenes. Indeed, almost 90% of mouse mammary tumor virus (MMTV)-induced mammary tumors in C3H/He mice show upregulation of Int protooncogenes. We have analyzed three different MMTV variants [MMTV(C3H), MMTV(HeJ), and a genetically engineered MMTV hybrid provirus (HP)] for tumorigenicity in mice from two distinct genetic backgrounds. All three viruses were tumor causing in BALB/cJ mice. However, only MMTV(C3H), but not MMTV(HeJ) or HP, induced mammary tumors in C3H/He mice. All of the viruses were infectious on either background and up-regulated expression of Int genes in tumors they induced. Like HP, MMTV(HeJ) was found to be a genetic recombinant between endogenous Mtv1 provirus and exogenous MMTV(C3H). Sequence comparison of MMTV variants linked the tumorigenicity of MMTV(C3H) to the gag region of the retrovirus.

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.

Expression of Mouse Mammary Tumor Virus Envelope Protein Does Not Prevent Superinfection In Vivo or In Vitro

Inbred mice expressing endogenous mouse mammary tumor virus envelope proteins can be infected with exogenous virus, and the mammary tumors that develop in these mice usually have many proviruses integrated in their genomes, indicating that this virus is not subject to receptor interference. We show here that transgenic mice expressing an exogenous mouse mammary tumor virus (C3H) envelope protein can still be infected with this virus. Moreover, cultured mammary gland cells expressing the mouse mammary tumor virus (C3H) envelope protein can be superinfected with pseudotyped viruses bearing that same protein. Thus cellular expression of the mouse mammary tumor virus envelope protein does not block superinfection in vivo or in vitro.

Strain-Specific Expression of Spliced MMTV RNAs Containing thesuperantigenGene

The transmission of milk-borne or exogenous mouse mammary tumor virus (MMTV) requires infection of B cells in the gut-associated lymphoid tissue and expression of the superantigen (Sag) protein at the B-cell surface. Presentation of Sag at the B-cell surface is required for the transmission of MMTV to T cells and subsequent infection of the target mammary gland tissue. Because several different promoters have been reported for MMTVsagmRNA expression, we investigated whether the detection of splicedsagRNAs was dependent upon the cell type infected or the particular MMTV strain examined. In this study, we detected expression of splicedsagRNA from the standard promoter and from an internal U3 promoter in B-cell lines expressing endogenousMtv-6by RT-PCR, although expression from the standard promoter appeared to be at least 10-fold higher than that observed from the internal U3 promoter.SagRNA originating from exogenous C3H MMTV was not observed from either of the U3 promoters in any cell type examined. However, spliced mRNAs containing the exogenous C3H MMTV, endogenousMtv-8,or endogenousMtv-17 saggenes could be detected from a previously described promoter in theenvelopecoding region regardless of the cell type infected. Becausesag-specific RNAs can be initiated independently of the LTR promoters, there may be selection for independent control of MMTVsagand structural gene expression.

T cells bearing Vbeta8 are preferentially infected with exogenous mouse mammary tumor virus.

We previously reported that a mouse mammary tumor virus (MMTV(II-TES14)), encoding a superantigen specific for TCR Vbeta14, can infect lymph node (LN) cells of mice in an I-E-independent manner. Here we examined the kinetics of cell types infected with exogenous MMTV in the draining LN after s.c. injection of II-TES milk containing MMTV(II-TES14). The infectivity was assessed in LN cells sorted into each cell subset by a semiquantitative analysis of MMTV provirus using PCR with a primer specific for MMTV(II-TES14). Only B cells in the LN were infected by the MMTV on day 6 after injection, but CD8+ T cells and, to a lesser extent, CD4+ T cells were also found to be detectably infected on day 14 after the injection of II-TES milk. Among the T cells we examined, Vbeta8 T cells were most preferentially infected with MMTV, but no Vbeta14 T cells specific for MMTV(II-TES14) superantigen were infected on day 14 after infection. The transfer of Vbeta8 T cells sorted from mice injected with II-TES milk 14 days previously resulted in the deletion of CD4+ Vbeta14 T cells and in the MMTV infection of normal B6 mice. No MMTV infection of T cells occurred in IgM knockout mice, which lack a mature B cell compartment. These results suggest that MMTV surviving in B cells is transferred to Vbeta8 T cells, which may play an important role in MMTV longevity.

Generation of a tumorigenic milk-borne mouse mammary tumor virus by recombination between endogenous and exogenous viruses.

Two novel exogenous mouse mammary tumor viruses (MMTV), BALB2 and BALB14, that encode superantigens (Sags) with Vbeta2+ and Vbeta14+ specificities, respectively, were found in the BALB/cT mouse strain. BALB/cT females were crossed with AKR/J males to generate F1 females. Foster nursing of BALB/cT mice on (BALB/cT x AKR/J)F1 mothers resulted in the generation of a new mouse strain, BALB/cLA, that had acquired a new exogenous MMTV (hereafter called LA) with a Vbeta6+/Vbeta8.1+-T-cell-specific Sag. Sequence analysis of the long terminal repeats of the BALB2, BALB14, and LA viruses indicated that LA virus resulted from recombination between BALB14 and the endogenous Mtv-7 provirus. Mtv-7 is expressed only in lymphoid tissues but not the mammary glands of Mtv-7-containing mouse strains such as AKR. In contrast, LA virus was highly expressed in the mammary gland, although it had the sag-specific region from Mtv-7. The LA virus, as well as different recombinant viruses expressed in the mammary glands of (BALB/cT x AKR/J)F1 mice, acquired a specific DNA sequence from BALB14 virus that is required for the mammary-gland-specific expression of MMTV. Since the Sag encoded by LA virus strongly stimulated cognate T cells in vivo, selection for recombinant virus with the Mtv-7 sag most likely occurred because the increased T-cell proliferation resulted in greater lymphoid and mammary gland cell infection. As a result of the higher virus titer, 80% of BALB/cLA females developed mammary gland tumors, although the incidence was only 40% in BALB/cT mice.

Mouse mammary tumor virus production by thymic epithelial cells in vivo.

Exogenous mouse mammary tumor viruses (MMTV) replicate in the mammary glands of infected females, and so infect the suckling pups. We have previously shown that the virus is rapidly disseminated to all the lymphoid organs, including the thymus. The present electron microscope immunohistochemical study describes the viral production site in the thymus. Viral buds and viral proteins were restricted to the thymus medullary epithelial cells. MMTV-encoded proteins were identified on the free viral particles and on the budding ones, the ribosomes, the membrane of the endoplasmic reticulum, and on the membrane of the medullary type II epithelial cell vacuolar network. The thymus medullary epithelial cells can thus integrate the virus and allow viral replication. The results support earlier results indicating that in some experimental conditions, epithelial cells may be involved in MMTV-induced negative selection by showing that thymic epithelial cells do express MMTV-encoded proteins.

Deletion of CD4+T Cells by Mouse Mammary Tumor Virus (FM) Superantigen with Broad Specificity of T Cell Receptor β-Chain Variable Region

We previously identified a superantigen from the exogenous mouse mammary tumor virus carried by FM mice [MMTV (FM)], which can preferentially activate Vβ8.2+CD4+T cells by subcutaneous injection. In the present study we investigated the effect of neonatal infection with the virus on the T cell receptor (TCR) β-chain variable region (Vβ) repertoire, T cell immune response, and development of experimental allergic encephalomyelitis (EAE). The infection, surprisingly, resulted in deletion of a large portion of CD4+T cells including Vβ2+, 6+, 8.1+, 8.3+, and 14+CD4+T cells in addition to Vβ8.2+CD4+T cells. Nevertheless, the infection marginally affected T cell immune response to various antigens such as ovalbumin (OVA) and alloantigen except the abrogated response to anti-Vβ8.2 antibody-mediated receptor cross-linking. Moreover, the infection exerted a protective effect on the development of EAE in (PL/J × SJL)F1 mice. Thus, MMTV (FM) superantigen has the ability to delete a large portion of CD4+T cells with broad TCR Vβ specificity, including Vβ8.2+CD4+T cells, and may have potential as a therapeutic agent against autoimmune diseases.

CD4 Expression is Important but not Essential for Infection with Exogenous Mouse Mammary Tumor Virus

We studied local events in the popliteal lymph nodes of CD4-deficient mice following foot pad injection with an MMTV strain which carries the gene for a Vβ14-specific superantigen. Injection of the Vβ14-specific MMTV induced vigorous expansion of Vβ14 + CD4 + T cells and B cells in their lymph nodes of CD4+/- heterozygous control mice. On the other hand, CD4-/- mice injected with the MMTV showed a proliferation of Vβ14 + T cells among the population of TCRáβ + CD4 -CD8 -T cells, although to a lesser extent. This phenomenon was not accompanied by vigorous B cell expansion. A PCR assay revelated that the MMTV definitely infected the lymph nodes cells of the CD4-/- mouse. However, the infectivity of the MMTV in CD4-/- mice was approximately 20 times lower than that in CD4+/- mice. These findings indicate that, in MMTV infection of CD4-deficient mice, the superantigen-reactive T cells among the population of TCRαβ +CD4 -CD8 -T cells substitute for the superantigen-reactive CD4 + T cells of normal mice, and that the absence of CD4 molecules decreased the infectivity of MMTV because of insufficient expansion of the superantigen-reactive T cells.

A V beta 4-specific superantigen encoded by a new exogenous mouse mammary tumor virus.

The superantigen (SAg) expressed by mouse mammary tumor virus (MMTV) has been shown to play an essential role in the course of the viral life cycle. In the present study, we describe a V beta 4-specific SAg encoded by a new exogenous MMTV carried by the SIM mouse strain. This is the first report of a viral or bacterial SAg reacting with mouse V beta 4+ T cells. Injection of MMTV(SIM) into adult BALB/c mice leads to a rapid and strong stimulation of V beta 4+ CD4+ T cells, followed by a slow deletion of these cells. Neonatal exposure to the virus also leads to a progressive deletion of V beta 4+ T cells. In contrast to other strong MMTV SAg, this new SAg requires the presence of major histocompatibility complex class II I-E molecules to be presented efficiently to T cells. Sequence analysis revealed a new predicted amino acid sequence in the C-terminal polymorphic region of this SAg. Furthermore, sequence comparisons to the most closely related SAg with different V beta specificities hint at the specific residues involved in the interaction with the T cell receptor.

A novel endogenous mouse mammary tumor virus locus in Asian wild mice and its evolutionary divergency.

The divergency of endogenous mouse mammary tumor virus (MMTV) in European mouse strains indicates acquisition of exogenous MMTV into their genomes during evolution. In the present study, we selected two strains of Asian wild mice, Cas-Hmi (Mus musculus castaneus) and Sub-Kjr (M. m. subspecies) to investigate a possible divergency of endogenous MMTVs among Asian mouse strains. Southern blot analysis and polymerase chain reaction (PCR) demonstrated that Cas-Hmi and Sub-Kjr have the full structure of integrated endogenous MMTVs, suggesting that these mice were diverged during evolution after MMTV endemics.

Deletion of Peripheral Vβ14+T Cells byMtv-2-Encoded Viral Superantigen Preceded by Blastogenesis and DNA Synthesis but Not by Specific Expansion

Exogenous and endogenous mouse mammary tumor viruses encode superantigen (SAG) in the 3′ long terminal repeat. We investigated the immune response of lymph node cells to the viral SAG encoded by endogenousMtv-2(vSAG-2) by iv injecting splenocytes fromMtv-2+mice intoMtv-2−congenic counterparts. vSAG-2 stimulation induced blastogenesis and DNA synthesis but not subsequent specific expansion of Vβ14+CD4+or CD8+T lymphocytes. Instead, immediate deletion of these T cells progressed after vSAG-2 stimulation, and it was more prominent in both rapidity and degree in CD4+than CD8+population and in I-E+than I-E−mice. vSAG-7 stimulation caused specific expansion of Vβ6+T cells prior to their deletion as reported. vSAG-7 but not vSAG-2 induced IL-2R expression in the specific T cells. Moreover, the percentage of 5-bromo-2′-deoxyuridine-incorporated cells was about twofold higher in Vβ6+T cells stimulated with vSAG-7 than in Vβ14+T cells stimulated with vSAG-2. The results suggest that vSAG-2 may be a weak mitogen against specific T cells and that T cells weakly activated by SAG may die without preceding expansion. In addition, proliferation of lymphocytes, especially B cells, and enhancement of the expression of IL-2, IL-4, and IFN-γ messenger RNA were observed. Thus, Vβ14+T cells stimulated with vSAG-2 were activated to produce cytokines and depleted quickly.

ウイルス性スーパー抗原とその生物学的意義

Mouse mammary tumor virus (MMTV), a replication-competent B-type murine retrovirus, causes mammary adenocarcinomas in some strains of laboratory mice and it can be transmitted exogenously via milk or endogenously through the germ line. It has been established that the immune system is directly involved in the infection pathway of MMTV, because thymectomy in neonates can decrease or delay mammary tumor incidence, and both T and B cells become infected with the virus. It has been shown that exogenous and endogenous MMTV have an open reading frame (ORF) encoding superantigen in the 3' long terminal repeat, which binds to MHC class II molecules and leads to stimulation and subsequent deletion of T cells bearing particular Vβ gene products.MMTVORF product is a type II transmembrane molecules where the COOH-terminal is extracellular and the COOH-terminal is responsible for the Vβ specificity of the superantigens. As T cell recognition of superantigens is predominantly mediated by the TCR Vβ domain, superantigen can stimulate much higher proportions of T cells than conventional peptide antigens. This characteristic of superantigens plays an important role in infection with exogenous MMTV.