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.     

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 x 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 exogenous 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.     

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.     

Isolation and characterization of a new mouse mammary tumor virus from BALB/c mice

A novel mouse mammary tumor virus (MMTV) has been isolated from mice of a subline of the BALB/cCrl Med mouse strain designated BALB/cV. Whereas breeding females of the parent BALB/cCrl Med colony have a mammary tumor incidence of 1%, 47% of the breeding females of the BALB/cV subline develop mammary tumors before 10 months of age. Foster nursing experiments demonstrated this virus, termed MMTV (BALB/c), was transmitted only by milk. The novel MMTV isolate was shown to be immunologically related to, but distinct, from the MMTV variants of C3H, GR, and RIII mice by a series of competition radioimmunoassays for the MMTV 28,000-dalton major core protein (p28), and the 52,000 (gp52)- and 36,000-dalton (gp36) major envelope glycoproteins. Monoclonal antibodies directed against MMTV gp36 were also used to clearly distinguish MMTV(BALB/c) from MMTV(C3H), MMTV(RIII), MMTV(GR), MMTV(C3HfC57BL), and MMTV(A). MMTV-specific proviral DNA content of mammary adenocarcinomas arising in the BALB/cV subline was examined with restriction endonucleases and the Southern blot technique, and compared to the MMTV proviral DNA content of BALB/cAnDe mammary tumors. The virus arising from these latter tumors has been termed MMTV(O). Analysis of EcoRI digests of high-molecular-weight DNA from both types of mammary tumors demonstrated additional MMTV-related proviral sequences when compared to the DNA of normal BALB/c tissues. The patterns generated with the restriction endonuclease SacI distinguished the additional MMTV-specific proviral information in the mammary tumors of the BALB/cV mice from the proviral information in tissues containing the GR, C3H, or RIII MMTVs, as well as from the proviral information in the BALB/cAnDe mammary tumors. These immunological and molecular studies thus define MMTV(BALB/c) as a novel MMTV variant.     

Subversion of the innate immune system by a retrovirus

Retroviruses evolve rapidly to avoid the immune response of the infected host. We show here that the wild-type mouse mammary tumor virus MMTV(C3H) persisted indefinitely in C3H/HeN mice. However, it was rapidly lost in mice of the closely related C3H/HeJ strain and was replaced by a virus recombinant with an endogenous Mtv provirus. Maintenance of the wild-type virus was dependent on Toll-like receptor-4 (TLR4) signaling, which triggered production of the immunosuppressive cytokine interleukin-10. In the presence of mutant TLR4 in C3H/HeJ mice, wild-type virus was eliminated by the cytotoxic immune response, promoting selection of the immune escape recombinant MMTV variants. Thus, subversion of the innate immune system is yet another survival strategy used by retroviruses.     

Effect of lifetime administration of dimethylaminoethanol on longevity, aging changes, and cryptogenic neoplasms in C3H mice

The effects of lifetime treatment with dimethylaminoethanol on longevity and cryptogenic neoplasm formation were studied in females of two mouse sub-lines, the C3H/HeN which carries a germinal mammary tumor provirus and the C3H/HeJ(+) which also carries the exogenous mammary tumor virus. Administration in the drinking water of 10 mM dimethylaminoethanol to the C3H/HeN mice or 15 mM to the C3H/HeJ(+) mice did not result in significant differences between treated and untreated groups in average survival. No changes in age-related organ structure or morphology were observed with dimethylaminoethanol treatment, except for an apparent decrease in the amount of lipofuscin in the liver judged in histological sections. Among untreated C3H/HeJ(+) females, 89% developed neoplasms of the mammary gland, ovary, liver, lung and reticuloendothelial system, while the incidence was 88% in the treated mice. In C3H/HeN females, neoplasms of the mammary gland, ovary, liver, lung and lymphatic system occurred in 57% and in 60% of treated mice. Also, there was no statistically significant difference between control and treated animals in the age of onset or the type of specific neoplasms. Dimethylaminoethanol did not induce any neoplasms.     

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.     

Detection of virus antigens in Swiss albino mice infected by milk-borne mouse mammary tumour virus: the effect of age, sex and reproductive status. II. Radioimmunoassay of two virus components, gp47 and p28 in serum and organ extracts.

Extracts of various organs, mammary tumours and sera from milk-borne MMTV infected Swiss albino mice of different age, sex and physiological conditions were tested by radioimmunoassay for the presence of gp47, the main envelope polypeptide, and p28, the main core protein of the virus. Except in brain, ovaries and testes, both antigens were found in all organs of old animals and of females after the onset of their first pregnancy. Antigens were not present in organs of weanlings or in whole foetuses. Higher values were found in mammary glands, mammary tumours, epididymis and seminal vesicles. These organs also harboured a greater amount of gp47 than p28. The serum generally contained gp47 but rarely p28. This indicates that gp47 is not virion-bound in blood. Pregnancy, lactation and especially the presence of mammary tumours increased the concentration of gp47 in serum. The results do not allow localization of target organs of MMTV infection in the interval between ingestion of the virus by the suckling mouse and the first pregnancy. Moreover, results obtained with one group of mice devoid of exogenous virus show that, as endogenous MMTV genome expresses p28, it might account for part of the p28 detected exogenous MMTV-infected mice.     

Genetic analysis of the effects of Mtv-2 on the T cell repertoire in the WXG-2 mouse strain.

In this report, the T cell repertoire was studied in a natural genetic model system using a novel mouse strain (WXG-2) carrying a single pathogenic mouse mammary tumor virus (MMTV) provirus (Mtv-2) on an otherwise MMTV-free genetic background. The Mtv-2 provirus has complete biological activity, produces infectious milk-transmitted virus, and contributes to mammary carcinogenesis by an insertion mutation mechanism. In mice carrying the Mtv-2 provirus, T cells expressing V beta 14 were specifically deleted in mice with a functional MHC class II I-E gene but not in I-E- controls. The deletion of V beta 14+ T cells was more rapid in mice with the Mtv-2 provirus than in Mtv-2-free control mice infected with exogenous MMTV. In addition, the Mtv-2 deletion phenotype was age dependent. A slow depletion of V beta 14+ T cells was observed, and greater than 95% of the V beta 14+ T cells were eliminated by 6 months of age. These experiments indicate that (i) the Mtv-2 provirus encodes or regulates expression of a V beta 14-specific superantigen, (ii) interactions between Mtv-2 and other MMTV proviruses are not necessary for the V beta 14 deletion phenotype, (iii) the presence of a retroviral superantigen in all cells is not sufficient for T cell depletion during neonatal development in the thymus, and (iv) the Mtv-2 provirus and its associated exogenous provirus have the same V beta specificity.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Induction of Natural Killer Cells and Interferon during Mouse Hepatitis Virus Infection of Resistant and Susceptible Inbred Mouse Strains

Following infection by mouse hepatitis virus (JHM strain), an induction of natural killer (NK) cell activity was observed in C3H mice, which are considered to be sensitive to JHM virus infection. In contrast, mice of the resistant SJL strain did not show any increase of NK cell activity after JHM virus infection. However, infection of both SJL and C3H mice with mouse hepatitis virus type 3 (MHV3) resulted in an increase of NK level, comparable to that observed with the JHM virus infection in the C3H strain. No significant differences were observed in the NK cell activity of the peritoneal exudate or spleen cells of infected mice. Low levels of interferon were detected in serum or peritoneal exudate of C3H mice infected with JHM virus 18 or 24 hours before, but no detectable early interferon production was found. Also no interferon could be detected in the resistant SJL mice. After JHM virus infection, the number of peritoneal exudate cells (PEC) was increased significantly in C3H mice but not in SJL mice. Macrophages obtained from the C3H mice supported virus replication, whereas SJL macrophages did not. Our data suggest that NK cells do not play a role in the resistance of SJL mice against JHM virus infection but may participate in the defence mechanisms against this virus in C3H mice.     

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.     

Maternal transfer of infectious mouse mammary tumor retroviruses does not depend on clonal deletion of superantigen-reactive Vβ14+ T cells

Female C3H/HeJ mice maternally transmit through their milk an infectious mouse mammary tumor retrovirus (MMTV) which causes clonal deletion of T cell receptor (TcR)Vβ14⁺ T cells reactive to the retroviral superantigen (SAG). To test whether CD4⁺ or CD8⁺ T cells are crucial for intestinal infection and maternal transfer of exogenous retroviruses, newborn mice lacking CD4 or CD8 molecules after gene targetting were raised by surrogate C3H/HeJ mothers. In CD8^?/? mice, clonal deletion of TcRVβ14⁺ cells reactive to the SAG from this exogenous MMTV occured with delayed kinetics. Deletion of TcRVβ14⁺ cells was not observed in CD4^?/? mice up to 12 months after exposure to the retrovirus. In both CD4^?/? and CD8^?/? mice TcRVβ5⁺ and TcRVβ11⁺ T cells were deleted in the presence of genomically integrated endogenous MMTV (Mtv), indicating that the lack of SAG-induced clonal deletion was not due to a general defect in these mutant mouse strains. Although TcRVβ14⁺ T cells were not deleted in CD4^?/? mice, female CD4^?/? mice nursed on C3H/HeJ milk maternally transmitted the retrovirus to their offspring, albeit with delayed kinetics. These data demonstrate that CD4⁺ and CD8⁺ lymphocytes influence clonal deletion events and that the mechanisms responsible for clonal deletion of SAG-reactive TcRVβ14⁺ T cells may be different from mechanisms which allow the mammary tumor virus to enter the mammary gland and complete its infectious cycle.     

Mouse mammary tumor virus (MMTV) infection in SWISS and RIII mice

Summary Host-virus relationships were examined in mice from the two mouse mammary tumor virus (MMTV)-infected strains SWISS MB+ and RIII, which harbour the same MMTV variant, and from the derived sublines Swiss MB-and RIIIf, which were freed of milk-borne MMTV by foster-nursing. These two strains are not phylogenetically related, the SWISS strain bearing the endogenousMtv-3 locus in its DNA. In RIII and SWISS MB+ mice, the incidence of early mammary tumors, which was of 96% and 8%, respectively, was correlated to the level of MMTV expression in milk. In the SWISS MB-line, a non-coordinate expression of the provirus associated with theMtv-3 locus was observed in the mammary glands, the salivary glands and the spleen. This expression was not tumorigenic and was characterized by the presence of the p28gag antigen and the absence of the gp52env antigen, except, however, in mammary glands of elder mice where traces of gp52 were found. In the mammary glands of SWISS MB+ mice, the expression of theMtv-3 locus was masked by large amounts of antigens resulting from exogenous virus expression. RIIIf mice were MMTV-negative.Viral antigens coexisted with anti-MMTV antibodies in the serum of infected and tumor-bearing mice, but not in the form of immune complexes as verified by a method that allowed to detect specific antigen-containing-soluble immune complexes. An anti-MMTV serum reactivity was also detected in SWISS MB-and RIIIf mice. However, the serum response was higher in the two SWISS lines than in the two RIII lines. Except in tumor-bearing mice, the anti-MMTV response was not significantly modified by the presence of exogenous virus and thus resulted essentially from exposure to endogenous MMTV expression. In experimental infection studies, RIII mice were more susceptible to MMTV infection than SWISS mice. The correlation between resistance to MMTV infection and serum response to endogenous MMTV expression, suggests that the non-tumorigenic expression of an endogenous provirus can protect at least partially, against exogenous MMTV infection.     

Pseudotypes of vesicular stomatitis virus with envelope antigens provided by murine mammary tumor virus.

Infection of two mouse mammary carcinoma cell lines with vesicular stomatitis virus (VSV) resulted in the formation of at least two types of particles containing the VSV genome but expressing different envelope characteristics (VSV pseudotypes). One of these VSV pseudotypes was infectious for a cell line derived from normal mouse mammary epithelial cells and mouse embryo cells but noninfectious for 3T3 cells, mink lung cells, and Vero cells. If mouse mammary tumor cells were treated with dexamethason some days prior to infection with VSV, the titer of this pseudotype was significantly increased. In contrast, the second pseudotype was infectious for mink cells, but not for the other cell lines tested, and the titer of this second pseudotype was unaffected by the presence of dexamethasone. The first pseudotype was found to be almost completely neutralized by anti-murine mammary tumor virus (MuMTV) serum whereas the second pseudotype was only partially neutralized at a higher antiserum concentration. Neither pseudotype showed the neutralization, host range, or interference properties of either ecotropic or xenotropic murine C-type viruses. These results suggest that the first pseudotype is VSV(MuMTV). The other pseudotype is less well defined but conceivably may represent a xenotropic MuMTV. In the course of these studies, a filterable agent was observed in GR mammary carcinoma cultures that reactivated the infectivity of VSV neutralized by antiserum. This agent was transmissible to mink cells.     

Age related differences in immunocompetence and incidence of mammary adenocarcinoma in murine mammary tumor virus-infected C3H/Bi mice

In breeder C3H/Bi female mice, infected neonatally by murine mammary tumor virus (MTV), the incidence of spontaneous mammary tumors is greater than 95% between 5 and 9 months of age. In young (2–3 months) female the probability for developing a tumor in the next month is negligible, higher than 80% in mice of middle age (5–6 months) but lower than 4% in aged (10–12 months) females. The age-related changes of some immune functions of spleen cells from these tumor free female mice have been evaluated. While the proliferative capacity of cells to Phytohemagglutinin (PHA) increases, the T cell-dependent antibody response against sheep red blood cells (SRBC) and the antibody-dependent cellular cytotoxicity (ADCC) are significantly decreased in 5–6-month-old mice as compared to the young (2–3 months) female mice.The antibody response against SRBC and the mitogenic response to PHA decline markedly in 10–12-month-old mice but the ADCC increases in this group of mice. In addition, assays with monoclonal anti-Lyt-1 and anti-Lyt-2 antibodies indicate that percentage of Lyt 1⁻ 2⁺ cells (suppressor and cytotoxic T cells) is lower in 10–12-month-old female as compared to 5–6-month-old animals. These results show that the immune alterations observed in 10–12-month-old C3H/Bi mice are not closely associated with an increase in incidence of spontaneous tumors and suggest that a high non-T killer cell activity could protect some of these older C3H/Bi female mice against mammary tumor development.     

The Role of superantigens in virus infection

Murine mammary tumor viruses are retroviruses which encode superantigens capable of stimulating T cells via superantigen-reactive T-cell receptor Vβ chains. Murine mammary tumor viruses are transmitted to the suckling offspring through the milk. We have established that B cell-deficient pups which were foster-nursed by virus-secreting mice do not transfer infectious murine mammary tumor viruses to their offspring. No murine mammary tumor virus proviruses could be detected in the spleen and mammary tissue of these mice. We conclude that B cells are essential for the completion of the viral life cyclein vivo. This indicates that B cells are infected first and that viral amplification takes place only if infected B cells present the murine mammary tumor virus superantigen on their surface, which, in turn, results in activation of T cells expressing the appropriate T-cell receptor Vβ chains. These activated T cells secrete factors which stimulate B cells, enabling viral replication.     

Detection of virus antigens in Swiss albino mice infected by milk-borne mouse mammary tumour virus: the effect of age, sex and reproductive status. I. Localization by immunofluorescence of four antigens in mammary tissues and other organs.

The indirect immunofluorescence technique was used to investigate the expression of virus antigens in a Swiss albino mouse strain infected by mouse mammary tumour virus (MMTV). The antisera used were monospecific for: the glycopolypeptides gp160 and gp47, and the internal polypeptides p28 and p8. In mice infected with milk-borne MMTV, all four antigens were detected in the mammary gland during the first pregnancy and then throughout life, and in mammary tumours. Antigen gp160, located at the secretory border of all glandular cells, is shared by the plasma membrane of the mammary cell, whether virus-producing or not, and by the virus envelope, as shown by the use of absorbed antisera. The other three antigens are virus-specific. Anti-p47 serum stained the secretory border of cells whereas the fluorescence related to p28 and p8 consisted of a few large intracytoplasmic spots. The specific antibodies for p28 and p8 were only absorbed by disrupted virions, confirming that these polypeptides are internal antigens. With serial sections of glands from pregnant and old female mice, the alveoli stained with anti-p28 or anti-p8 serum were found to react also with anti-gp47 serum. However, during lactation, the presence of p28 and p8 could not be detected in the alveoli stained with anti-gp47 serum. Thus, infected alveoli express virus antigens differently. In the mammary glands of all mice studied, some alveoli did not display any staining when the latter three antisera were used. The specific antigenic pattern, maintained in differentiated mammary tumours, became a diffuse and unlocalized staining after transformation into the undifferentiated state. With the exception of the kidney, where gp47 was found in the glomeruli, all other organs of the mouse did not stain with any of the antisera. The presence of gp47 in the glomeruli, probably related to immune-complex deposits, increases with the age of the mouse and is most noticeable in tumour-bearing females. In mice free of milk-borne MMTV, examined during the first week of lactation, antigen gp47 could not be detected. Internal antigens p28 and p8 were detected in nearly all mammary cells as numerous small cytoplasmic dots. This suggests a limited expression of an endogenous virus genome; this expression would be modified when milk-borne virus is produced.     

Detection of MMTV gp52 in C3H and C3Hf strain mice

Immunofluorescence in paraffine sections demonstrated the difference in expression of the membrane glycoprotein of mouse mammary tumor virus (gp52) in two mouse sublines: C3H and C3Hf. In the C3Hf the glycoprotein becomes detectable in the mammary gland in the second part of pregnancy while in C3H line mature females before pregnancy. No viral glycoprotein could be found in the spleen, liver, kidneys and other organs in males and females of the two sublines or in C3H embryos.