A novel mechanism of resistance to mouse mammary tumor virus infection

Exogenous mouse mammary tumor virus (MMTV) is carried from the gut of suckling pups to the mammary glands by lymphocytes and induces mammary gland tumors. MMTV-induced tumor incidence in inbred mice of different strains ranges from 0 to as high as 100%. For example, mice of the C3H/HeN strain are highly susceptible, whereas mice of the I/LnJ strain are highly resistant. Of the different factors that together determine the susceptibility of mice to development of MMTV-induced mammary tumors, genetic elements play a major role, although very few genes that determine a susceptibility-resistance phenotype have been identified so far. Our data indicate that MMTV fails to infect mammary glands in I/LnJ mice foster nursed on viremic C3H/HeN females, even though the I/LnJ mammary tissue is not refractory to MMTV infection. Lymphocytes from fostered I/LnJ mice contained integrated MMTV proviruses and shed virus but failed to establish infection in the mammary glands of susceptible syngeneic (I x C3H.JK)F(1) females. Based on the susceptible-resistant phenotype distribution in N(2) females, both MMTV mammary gland infection and mammary gland tumor development in I/LnJ mice are controlled by a single locus.     

Expression pattern of mouse mammary tumor virus in transgenic mice carrying exogenous proviruses of different origins.

To study the tissue specificity of mouse mammary tumor virus (MMTV) gene expression, we developed two series of transgenic mice, containing the MMTV proviral DNA of mammary (GR) and kidney (C3H-K) origin. The expression pattern in the MMTV(GR) transgenic mice is very similar to that observed in infected animals, e.g., a strong preference for viral expression in the lactating mammary glands and lower levels of expression in salivary glands, lymphoid tissues, and male reproductive organs. One line of transgenic mice carrying the C3H-K provirus has a similar expression pattern, indicating that MMTV(C3H-K), despite a striking alteration in the U3 region of its long terminal repeat, can be expressed in the same tissues as the wild-type MMTV.     

Integration of the DNA of mouse mammary tumor virus in virus-infected normal and neoplastic tissue of the mouse.

We have used restriction endonucleases which cleave the DNA of mouse mammary tumor virus (MMTV) at one site (Eco RI) and several sites (Pst I, Sac I and Bam HI) to study infection and mammary tumorigenesis in mice. Proviruses acquired during infection of BALB/c mice foster-nursed by virus-producing C3H females can be distinguished from the MMTV proviruses endogenous to uninfected BALB/c mice by the nature of the fragments generated with Pst I and Bam HI. Using this assay, we show that lactating mammary glands as well as mammary tumors from BALB/cfC3H mice have acquired MMTV DNA, and that a minimum of approximately 10% of normal glandular cells can be infected. The new proviruses appear to be linked to cellular DNA of mammary tumors and infected lactating mammary glands within a limited region (0.2 x 10(6) daltons) of the viral DNA; the location of this region, based upon mapping studies with unintegrated MMTV DNA, suggests that the orientation of these proviruses is colinear with linear DNA synthesized in infected cells and thus approximately colinear with the viral RNA. Comparisons of many mammary tumors and studies of lactating mammary glands with a high proportion of independently infected cells indicate that a large number of sites in the cellular genome can accommodate a new provirus; the acquired proviruses are rarely, if ever, found in tandem with each other or with endogenous proviruses. We cannot, however, distinguish between random integration and integration into a large number of preferred sites in the host genome. Since Eco RI and Bam HI cleavage of DNA from each mammary tumor generates a unique set of viral-specific fragments, we propose that the tumors are composed principally of cells derived from a subset of the many infected cells in a mammary gland; this proposal is supported by our finding that Eco RI digestion of DNA from several transplants of a primary tumor yields the pattern characteristic of the primary tumor.     

Polyomavirus replication in mice: influences of VP1 type and route of inoculation.

Patterns of polyomavirus replication and spread have been studied following inoculation of virus into newborn mice. Levels of virus replication in different tissues were followed in situ by using whole mouse section blots and immunoperoxidase staining for the major capsid protein VP1, as well as by tissue extraction and direct quantitation of viral DNA and infectious virus. Patterns of replication and spread were compared between the "high tumor" strain (inducing a high incidence of tumors) PTA and and the "low tumor" strain (inducing a low incidence of tumors) RA, following different routes of inoculation. The ability to induce a high tumor profile correlated with the ability to establish disseminated productive infection, with the kidney as a major site of amplification. Furthermore, results with PTA-RA recombinant viruses and site-directed mutants showed that the VP1 specificity of PTA, demonstrated earlier to be a critical determinant for induction of a high tumor profile (R. Freund, A. Calderone, C. J. Dawe, and T. L. Benjamin, J. Virol. 65:335-341, 1991), is also critical for amplification in the kidney and for establishment of disseminated infections.     

The primary site of replication alters the eventual site of persistent infection by polyomavirus in mice.

Using DNA blot analysis, we monitored the course of polyomavirus infection in mice receiving an intranasal inoculation and compared this with the course of infection in mice receiving an intraperitoneal inoculation. Intranasal infection was characterized by an initial primary replication phase in the respiratory tract, followed by a systemic infection of the visceral organs. At 12 days postinfection, there was partial clearing of viral DNA in all organs; by 22 days postinfection, viral DNA persisted only in the lungs and kidneys, and the level of DNA slowly decreased during the next 3 months. Lungs have been a previously unrecognized site for polyomavirus persistent infection. In contrast to intranasal infection, intraperitoneal infection of mice was characterized by only three phases: an initial systemic phase in which viral DNA was found in the same respiratory and visceral organs as during intranasal infection, clearing of the virus from the organs, and ultimately, a persistent infection in the kidneys but not in the lungs. Thus, different organs became persistently infected when mice were inoculated via these different routes.     

Direct detection of exogenous mouse mammary tumor virus sequences in lymphoid cells of BALB/cfC3H female mice.

The presence of exogenous mouse mammary tumor virus (MMTV) (C3H) DNA sequences in lymphoid tissue (spleen, bone marrow, and thymus) and nonlymphoid tissue (liver and kidney) of BALB/cfC3H female mice was directly assessed by DNA hybridization methods. Lymphoid tissues were found positive for integrated MMTV(C3H) sequences in females as young as 4 weeks. In most samples, the level of splenic MMTV(C3H) infection was low (2 to 5%). Infection remained throughout the life of the animal. The percentage of spleen samples found positive for exogenous viral infection was significantly higher in females bearing mammary tumors, whether virgin or multiparous. Liver and kidney DNAs were negative for exogenous MMTV sequences, suggesting tissue type selectivity in MMTV infection.     

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.     

Survival of athymic (nu/nu) mice after Theiler''s murine encephalomyelitis virus infection by passive administration of neutralizing monoclonal antibody.

Little or no antiviral immune response is mounted in athymic nude mice infected with the Daniels strain of Theiler's murine encephalomyelitis virus. In these athymic mice, increasing levels of infectious virus could be detected in the central nervous system. Seventy-five percent (9 of 12) of the nude mice were moribund or dead by 4 weeks postinfection. In contrast, treatment of Theiler's virus-infected nude mice with a neutralizing monoclonal antibody (H7-2) against the viral protein VP-1 resulted in a dramatic reduction of infectious virus within the central nervous system. All antibody-treated nude animals survived beyond 4 weeks postinfection. Monoclonal antibody titers could be maintained by passive transfer in treated nude mice at levels comparable to those of polyclonal antibody titers found in heterozygous infected nu/+ littermates. Areas of demyelination were detected in the untreated animals as early as 7 days after infection with little or no remyelination present. In approximately one-half of the antibody-treated nude animals, no demyelinating lesions were found. However, the rest of these treated mice were found to have areas of both demyelination and remyelination. Thus, anti-Theiler's murine encephalomyelitis virus antibody against VP-1 can play a dramatic role in the survival of mice, clearance of virus, limiting viral spread, and altering the pattern of disease in the absence of a functional T-cell response.     

Systemic polyomavirus genome increase and dissemination of capsid-defective genomes in mammary gland tumor-bearing mice

BALB/c mice that developed tumors 7 to 8 months following neonatal infection by polyomavirus (PYV) wild-type strain A2 were characterized with respect to the abundance and integrity of the viral genome in the tumors and in 12 nontumorous organs. These patterns were compared to those found in tumor-free mice infected in parallel. Six mice were analyzed in detail including four sibling females with mammary gland tumors. In four of five mammary gland tumors, the viral genome had undergone a unique deletion and/or rearrangement. Three tumor-resident genomes with an apparently intact large T coding region were present in abundant levels in an unintegrated state. Two of these had undergone deletions and rearrangements involving the capsid genes and therefore lacked the capacity to produce live virus. In the comparative organ survey, the tumors harboring replication-competent genomes contained by far the highest levels of genomes of any tissue. However, the levels of PYV genomes in other organs were elevated by up to 1 to 2 orders of magnitude compared to those detected in the same organs of tumor-free mice. The genomes found in the nontumorous organs had the same rearrangements as the genomes residing in the tumors. The original wild-type genome was detected at low levels in a few organs, particularly in the kidneys. The data indicate that a systemic increase in the level of viral genomes occurred in conjunction with the induction of tumors by PYV. The results suggest two novel hypotheses: (i) that genomes may spread from the tumors to the usual PYV target tissues and (ii) that this dissemination may take place in the absence of capsids, providing an important path for a virus to escape from the immune response. This situation may offer a useful model for the spread of HPV accompanying HPV-induced oncogenesis.     

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.     

Exogenous mouse mammary tumor virus proviral DNA isolated from a kidney adenocarcinoma cell line contains alterations in the U3 region of the long terminal repeat.

Mouse mammary tumor virus (MMTV) is a B-type retrovirus which induces predominantly mammary carcinomas after a relatively long latency period. To date, very little is known about the reasons for the strict tissue specificity of MMTV. The BALB/cf/Cd strain of mice, which was infected with milk-borne MMTV (C3H), shows a high incidence of kidney adenocarcinomas, and our data suggest that MMTV might be involved in the formation of these tumors. Newly integrated exogenous MMTV proviruses were found in the genome of transplanted tumor cells as well as in the DNA of a cell line derived from one tumor, but not in normal cells of BALB/cf/Cd mice. The MMTV DNA in these tumor cells was transcribed and viral RNA synthesis was strongly stimulated by glucocorticoid hormones. Viral structural polypeptides, comparable in size and antigenicity to MMTV polypeptides of infected mammary tumor cells were synthesized and processed normally in the cell line and were organized correctly into intracytoplasmic particles. Heteroduplex analysis of the molecularly cloned MMTV proviral DNAs of kidney and mammary tumor origin revealed a high degree of homology in the gag, pol, and env genes. A striking difference, however, was observed in the U3 region of the two LTRs that might relate to the different tissue specificity of the two viruses.     

Expression of major capsid protein VP-1 in the absence of viral particles in thymomas induced by murine polyomavirus

Thymomas induced by polyomavirus strain PTA in mice are known to express the major capsid protein VP-1. Since the expression of a late structural protein such as VP-1 is considered a sign of virus replication, the present work attempted to clarify the implication of the presence of this protein in tumor cells. Electron microscopy of tumors showed a striking absence of viral particles in the vast majority of the cells. However, immunoelectron microscopy of the same samples demonstrated intranuclear VP-1 in most cells despite the absence of viral particles. Very little infectious virus was recovered from tumors. A change in the electrophoretic mobility of VP-1 from thymomas was detected compared with VP-1 from productively infected cells. The data presented in this work prove that the expression of VP-1 in polyomavirus-induced tumors is not synonymous with the presence of infectious virus, suggesting a possible defect in viral encapsidation.     

Oncogenesis of mammary glands, skin, and bones by polyomavirus correlates with viral persistence and prolonged genome replication potential.

A correlation between polyomavirus-induced oncogenesis and viral persistence on the one hand and/or prolonged genome replication potential on the other was established with respect to their respective organ distributions. Prolonged replication potential is defined as the capacity of a genome to replicate in a given organ from the time of infection up to the onset of oncogenesis. This conclusion was derived following intraperitoneal infection of BALB/c mice with wild-type strain A2. Viral genomes were used as parameters of persistence and replication and were detected by Southern blotting and PCR analysis. The major tumor target organs (mammary gland, skin, and bone), which have not been previously analyzed for persistence, were compared with other, non-tumor-prone organs (kidney, liver, lung, spleen, and salivary gland). A progressive loss of viral genomes was observed in all tissues as a function of time postinfection; however, genomes were shown to persist through 20 weeks postinfection in the mammary glands, skin, and bones to an extent similar to that in the previously described kidneys (D. J. McCance, J. Virol. 39:958-962, 1981; W. P. Rowe, J. W. Hartley, J. D. Estes, and R. J. Huebner, Natl. Cancer Inst. Monogr. 4:189-209, 1960). Thus, tumors arise among organs that sustain a persistent infection, but not all such organs develop tumors (e.g., the kidney). The capacity of organs to support de novo replication at various ages, including the age reached when the first tumors are detected, was also determined using a 3-day infection period for ages between 0 and 7 weeks. For all organs tested, a higher level of genomes was observed in organs of mice infected as neonates than in those infected after the age of 3 weeks. However, marked organ-specific differences were seen in the degree and timing of loss of replication. In particular, viral genome replication, although reduced, was maintained in the mammary glands, skin, and bones of adult animals, in contrast to the kidneys. We conclude that organ-specific oncogenesis correlates with two organ-specific parameters: persistence of viral genomes and prolonged viral genome replication potential. This may reflect a requirement for continued viral genome replication and/or gene expression for tumorigenesis. In turn, these parameters may be linked to the tissue-specific continued capacity for cellular division.     

Amplification and novel locations of endogenous mouse mammary tumor virus genomes in mouse T-cell lymphomas.

Endogenous mouse mammary tumor virus genomes are amplified and located in novel cell DNA sequences in many mouse T-cell lymphomas. Transplanted tumors recovered from the same mouse strain and shown to be of independent origin by chromosomal analysis, by the presence of JH immunoglobulin gene rearrangements, or by the integration patterns of exogenous Moloney MuLV genomes frequently showed similar patterns of novel mouse mammary tumor virus-containing cell DNA fragments. This process of amplification and relocation can occur within a limited number of cell generations and in C57BL/6 mice does not lead to the synthesis of mature virus-encoded proteins. In some instances, amplified mouse mammary tumor virus genomes contained novel restriction cleavage sites in the gag-pol region. The restricted time course of occurrence, lack of synthesis of mature virion proteins, and apparent site specificity indicate that this process of retrovirus amplification differs significantly from virus replication after exogenous infection.     

Genetic analysis of the enhancer requirements for polyomavirus DNA replication in mice.

In this report, we describe the first systematic analysis of the genetic requirements for polyomavirus (Py) enhancer-activated viral DNA replication during the acute phase of infection in mice. Four mutants were made which substituted XhoI sites for conserved enhancer consensus sequences (adenovirus type 5 E1A, c-fos, simian virus 40, and a glucocorticoidlike consensus sequence). Viral DNA replication in infected mouse organs was measured by DNA blot analysis. Only the loss of the glucocorticoidlike consensus sequence element significantly reduced Py DNA replication in the kidneys, the primary target organ for viral replication. The loss of the c-fos, adenovirus type 5 E1A, or simian virus 40 consensus sequences, however, expanded organ-specific viral DNA replication, relative to wild-type Py, by allowing high-level replication in the pancreas or heart or both. Analysis of Py variants selected for replication in undifferentiated embryonal carcinoma cell lines (PyF441, PyF111) showed that there was little change in levels of viral DNA replication in kidneys and other organs as compared with those in the wild-type virus. If the entire B enhancer is deleted, only low overall levels of viral replication are observed. Wild-type levels of replication in the kidneys can be reconstituted by addition of a single domain from within the A enhancer (nucleotides 5094 to 5132) to the B enhancer deletion virus, suggesting that a single domain from the A enhancer can functionally substitute for the entire B enhancer. This also indicates that the determinants for kidney-specific replication are not found in the B enhancer.