Murine mammary tumor virus pol-related sequences in human DNA: characterization and sequence comparison with the complete murine mammary tumor virus pol gene.

Sequences in the human genome with homology to the murine mammary tumor virus (MMTV) pol gene were isolated from a human phage library. Ten clones with extensive pol homology were shown to define five separate loci. These loci share common sequences immediately adjacent to the pol-like segments and, in addition, contain a related repeat element which bounds this region. This organization is suggestive of a proviral structure. We estimate that the human genome contains 30 to 40 copies of these pol-related sequences. The pol region of one of the cloned segments (HM16) and the complete MMTV pol gene were sequenced and compared. The nucleotide homology between these pol sequences is 52% and is concentrated in the terminal regions. The MMTV pol gene contains a single long open reading frame encoding 899 amino acids and is demarcated from the partially overlapping putative gag gene by termination codons and a shift in translational reading frame. The pol sequence of HM16 is multiply terminated but does contain open reading frames which encode 370, 105, and 112 amino acid residues in separate reading frames. We deduced a composite pol protein sequence for HM16 by aligning it to the MMTV pol gene and then compared these sequences with other retroviral pol protein sequences. Conserved sequences occur in both the amino and carboxyl regions which lie within the polymerase and endonuclease domains of pol, respectively.     

Mouse mammary tumor virus DNA in infected rat cells: characterization of unintegrated forms.

Mouse mammary tumor virus (MMTV) DNA in chronically infected rat hepatoma cells is maintained in both the integrated and unintegrated state. Fractionation of DNA by the procedure of Hirt (1967) as well as by sedimentation through alkaline sucrose suggests that about two thirds of the viral DNA is associated with high molecular weight cell DNA. The remainder of the viral DNA is unintegrated and is present primarily as linear or open circular duplexes consisting of a genome-length strand complementary to the viral RNA ("minus" strand) and "plus" strands of subgenomic length. Approximately 20% of the unintegrated MMTV DNA is present as double-stranded, covently closed circles (form I) with a molecular weight of 6 X 10(6) daltons. Form I viral DNA is found primarily in the nucleus, whereas the open forms are both nuclear and cytoplasmic.     

Murine mammary tumor cells with a claudin-low genotype

In adults, bone is the preferential target site for metastases from primary cancers of prostate, breast, lungs and thyroid. The tendency of these cancers to metastasize to bone is determined by the anatomical distribution of the blood vessels, by the genetic profile of the cancer cells and by the biological characteristics of the bone microenvironment that favour the growth of metastatic cells of certain cancers. Metastases to bone may have either an osteolytic or an ostoblastic phenotype. The interaction in the bone microenvironment between biological factors secreted by metastatic cells, and by osteoblasts and osteoclasts, and the osteolytic and osteoblastic factors released from the organic matrix mediate a vicious cycle characterized by metastatic growth and by ongoing progressive bone destruction. This interaction determines the phenotype of the metastatic bone disease.     

Preferential infection of immature dendritic cells and B cells by mouse mammary tumor virus

Until now it was thought that the retrovirus mouse mammary tumor virus preferentially infects B cells, which thereafter proliferate and differentiate due to superantigen-mediated T cell help. We describe in this study that dendritic cells are infectable at levels comparable to B cells in the first days after virus injection. Moreover, IgM knockout mice have chronically deleted superantigen-reactive T cells after MMTV injection, indicating that superantigen presentation by dendritic cells is sufficient for T cell deletion. In both subsets initially only few cells were infected, but there was an exponential increase in numbers of infected B cells due to superantigen-mediated T cell help, explaining that at the peak of the response infection is almost exclusively found in B cells. The level of infection in vivo was below 1 in 1000 dendritic cells or B cells. Infection levels in freshly isolated dendritic cells from spleen, Langerhans cells from skin, or bone marrow-derived dendritic cells were compared in an in vitro infection assay. Immature dendritic cells such as Langerhans cells or bone marrow-derived dendritic cells were infected 10- to 30-fold more efficiently than mature splenic dendritic cells. Bone marrow-derived dendritic cells carrying an endogenous mouse mammary tumor virus superantigen were highly efficient at inducing a superantigen response in vivo. These results highlight the importance of professional APC and efficient T cell priming for the establishment of a persistent infection by mouse mammary tumor virus.     

Critical role of dendritic cells in mouse mammary tumor virus in vivo infection

Mouse mammary tumor virus (MMTV) is a milk-transmitted betaretrovirus that causes mammary tumors in mice. Although mammary epithelial cells are the ultimate targets of MMTV, the virus utilizes components of the host immune system to establish infection. Previous studies indicated that dendritic cells play a role in MMTV infection. Here we show that dendritic cells are the first cells to be infected by MMTV in vivo and that they are capable of producing infectious virus that can be transmitted to other cell types. Moreover, upon contact with the virus, dendritic cells became more mature and migrated in response to the chemokine macrophage inflammatory protein 3beta. Finally, we demonstrate that targeted ablation of dendritic cells in vivo dramatically attenuated MMTV infection. These data indicate that MMTV infection of dendritic cells is critical to initial propagation of the virus in vivo.     

Transgenic mouse mammary tumor virus superantigen expression prevents viral infection.

Endogenous mouse mammary tumor virus (MMTV) proviruses have recently been shown to cosegregate genetically with the minor lymphocyte-stimulating loci, also termed self-superantigens. The antigenic activity has been localized to the open reading frame (ORF) protein encoded in the long terminal repeat of MMTV. We show here that unlike their nontransgenic littermates, transgenic mice expressing high levels of an ORF protein derived from the C3H exogenous MMTV specifically delete their V beta 14+ T cells and do not become infected with this virus when it is present in their mother's milk. Thus, it appears that MMTV utilizes cells of the immune system in its infection pathway, and mice that retain endogenous MMTVs should be immune to infection by exogenous virus. These results offer possible new approaches to anti-viral therapy or immunization.     

Initial stages of mammary tumor virus infection are superantigen independent

Exogenous mouse mammary tumor virus (MMTV) is transmitted via the milk from infected mothers to newborn pups. Efficient MMTV transmission is dependent on proliferation of T cells with particular TCR beta-chains, which occurs upon recognition of virally encoded superantigen (SAg) bound to MHC class II molecules. It is assumed that infection of these dividing cells favors MMTV amplification. SAg is important for MMTV infection, as mice that lack SAg-cognate T cells due to expression of endogenous Mtv loci or mice that express inappropriate MHC haplotypes unable to present viral SAg efficiently were shown to be resistant to MMTV infection. However, this resistance was not absolute, as these mice developed late onset MMTV-induced mammary tumors. In this study, we show that the success of initial MMTV infection in neonates is independent of SAg function but depends on the developmentally regulated proliferation of target cells. However, SAg was absolutely required for virus spread following completion of this proliferative stage.     

Murine infection by vesicular stomatitis virus: initial characterization of the H-2d system.

BALB/c mice and congenic H-2Ld-deficient BALB/c-H-2dm2 (dm2) mice were experimentally infected intranasally with isolates of vesicular stomatitis virus (VSV). The survival of infected hosts, viral replication in lungs and brains, and histopathologic in the two mouse strains were compared. In both strains of mice, mortality occurred during the period 7 to 10 days postinfection. However, dm2 mice were relatively resistant to lethal infections. Viral replication occurred at low levels in the lungs of both strains and did not evoke significant pathologic changes. In contrast, viral replication in the brains was much greater; in the BALB/c strain, this was accompanied by more frequent and more severe pathologic changes. In general, mice surviving at day 10 had effectively cleared virus from central nervous system but not respiratory sites. Evidence is presented that viral replication occurs first in the nasal cavity and is transmitted both to the lungs and to the olfactory bulb where focal cytopathology occurs. Virus enters the ventricles, causing encephalitis; necrosis occurs around the ventricles and in the lumbosacral region of the spinal cord. Necrotic lesions were accompanied by mononuclear infiltration. Mice immunized with virus of the same serotype or with a vaccinia virus hybrid encoding the VSV glycoprotein were protected from lethal infection; in contrast, mice immunized with heterotypic virus were susceptible to challenge.     

Murine mammary tumor virus structural protein interactions: formation of oligomeric complexes with cleavable cross-linking agents

Murine mammary tumor virus protein interactions in the intact virion structure were studied with the use of the cleavable cross-linking reagents dithiobis(succinimidyl propionate) and methyl 4-mercaptobutyrimidate hydrochloride. Cross-linked oligomeric complexes of murine mammary tumor virus proteins were analyzed by two-dimensional gel electrophoresis. Among the complexes most consistently formed were a heterodimer of the two glycoproteins gp36 and gp52, the homodimer of gp36, and the homotrimer of gp52. A very prominent oligomer formed at higher concentrations of dithiobis(succinimidyl propionate) was a complex of about 230,000 molecular weight, made up of three molecules each of gp36 and gp52. A number of lines of evidence, including electron microscopic analysis, suggest that the 230,000-molecular-weight complex actually represents the murine mammary tumor virus spike structure. Of the murine mammary tumor virus core proteins, p14 forms homooligomers most readily. Upon cross-linking with methyl 4-mercaptobutyrimidate hydrochloride a small amount of what seems to be a heterodimer made up of the N-terminal gag protein p10 and the hydrophobic membrane glycoprotein gp36 can be observed.     

Infection of cultured rat hepatoma cells by mouse mammary tumor virus.

A continuous line of buffalo rat hepatoma (HTC) cells has been successfully infected with mouse mammary tumor virus (MMTV) produced by the GR mammary tumor cell line. Uniform infection required initial exposure of the HTC cells to greater than 10(5) MMTV particles per cell. The resultant chronically infected cell population was found to have stably acquired 20-30 copies of MMTV DNA. The infected cells contain viral RNA and express viral antigens; however, very few MMTV particles are released into the culture medium. In spite of the biochemical evidence for infection, we have not detected any alterations in the morphology or growth properties of the infected HTC cells. As is the case in mammary tumor cells, the intracellular concentration of viral RNA is strongly stimulated (50-150 fold) by the synthetic glucorcorticoid, dexamethasone. Thus it appears that the mechanisms by which glucorticoids regulate MMTV gene expression in mouse cells are maintained when this virus infects nonmurine cells.     

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.     

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.     

Murine mammary tumour cells in vitro. II. Recruitment of quiescent cells

The development of a pure quiescent (Q) tumour cell population can be induced in three mouse mammary tumour lines (66, 67 and 68H) by nutrient deprivation. When these Q cells were removed from nutrient-deprived cultures and replated in fresh medium at a lower cell concentration within 72 hr of entering quiescence virtually all of the Q cells could re-enter the proliferating (P) state. This recruitment was characterized by an increase in cell volume, an increase in total cellular RNA, and a resumption of cell division. The length of the Q to P transition varied among the three cell lines and the depth of the quiescent state depended on the amount of time the cells had been quiescent. Once re-entry into the P compartment was completed, cell-cycle times, as estimated by the culture doubling time, were the same as the cells that had not entered the Q state, however, after 72 hr in quiescence, not all of the 66 cells could reattach after trypsinization and of those that could reattach approximately equal to 50% were incapable of either increasing their RNA levels to that of proliferating G1 cells or entering S. Clonogenicity of the nutrient-deprived Q cells in these lines decreases exponentially from time the cells enter quiescence with approximate half-times of 32, 34, and 96 hr for the 66, 68H and 67 cells, respectively. Since clonogenicity was already declining at a time when all the Q cells could re-enter the P compartment, the ability of a Q cell to form a colony is not determined solely by its capacity to re-enter the proliferating compartment.     

Early increases in superantigen-specific Foxp3+ regulatory T cells during mouse mammary tumor virus infection

Mouse mammary tumor virus (MMTV) is a milk-borne betaretrovirus that has developed strategies to exploit and subvert the host immune system. Here, we show in a natural model of MMTV infection that the virus causes early and progressive increases in superantigen (SAg)-specific Foxp3⁺ regulatory T cells (T_reg) in Peyer's patches (PP). These increases were shown to be dependent on the presence of dendritic cells. CD4⁺ CD25⁺ T cells from the PP of infected mice preferentially suppress the proliferative response of T cells to SAg-expressing antigen-presenting cells ex vivo. We investigated the influence of the depletion of CD25⁺ cells at different stages of the infection. When CD25⁺ cells were depleted before MMTV infection, an increase in the number of PP SAg-cognate Foxp3⁻ T cells was found at day 6 of infection. Since the SAg response is associated with viral amplification, the possibility exists that T_reg cells attenuate the increase in viral load at the beginning of the infection. In contrast, depletion of CD25⁺ cells once the initial SAg response has developed caused a lower viral load, suggesting that at later stages T_reg cells may favor viral persistence. Thus, our results indicated that T_reg cells play an important and complex role during MMTV infection.     

The purification and characterization of a major glycoprotein of the murine mammary tumor virus.

Affinity chromatography of solubilized murine mammary tumor virus on concanavalin A-Sepharose was clearly affected by different mixtures of detergent present in the elution buffer: A complex consisting of a glycoprotein of 52,000 daltons (gp52), and a glycoprotein of 36,000 daltons (gp36), besides free gp52 were isolated. The gp36 could be purified by gel filtration of the complex in the presence of a high concentration of sodium deoxycholate. The elution of gp36 in the void volume of the Sephadex column and the results obtained with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed strong hydrophobic interactions within the molecule. The glycoprotein was immunochemically characterized by competitive radioimmunoassay and immunoelectrophoresis. No cross-reactivity of gp36 with gp52 or two nonglycosylated viral polypeptides was observed.