Generation of a tumorigenic milk-borne mouse mammary tumor virus by recombination between endogenous and exogenous viruses.
ABSTRACT: 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.
Project description: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.
Project description:Mls-1 is an endogenous superantigen that leads to in vivo deletion and in vitro stimulation of T cell receptor (TCR) V beta 6-, 7-, 8.1-, and 9-expressing cells. The MA/MyJ mouse deletes the identical set of TCR from its mature T cell repertoire; however, it does not contain Mtv-7, the murine mammary tumor provirus (MMTV), whose sag gene encodes Mls-1. Interestingly, the superantigen activity of this mouse strain segregates with a new mammary tumor provirus, Mtv-43, not seen in other inbred strains. The predicted amino acid sequence of the sag gene of Mtv-43 was compared with that of Mtv-7. Strikingly, the COOH terminus of the two molecules is very similar, while all other MMTV-encoded superantigens differ 100% in this segment.
Project description:We have previously reported new Mtv loci, Mtv-48 and -51, in the Japanese laboratory mouse strains CS and NC. Here we show by backcross analysis that both Mtv-48 and -51 cosegregate with very slow deletion of T cells bearing V beta 2. The nucleotide sequences of the open reading frames in the 3' long terminal repeats of Mtv-48 and -51 were very similar to those of Mtv-DDO, mouse mammary tumor virus C4 [MMTV(C4)], and MMTV(BALB/cV), which encode V beta 2-specific superantigens. Furthermore, backcross female mice carrying Mtv-48 but not Mtv-51 were found to be able to produce milk-borne MMTV(CS), which can vigorously stimulate V beta 2-expressing T cells after local injection in vivo in an I-E-dependent manner. On the other hand, mice carrying Mtv-51 but not Mtv-48 could not produce such an MMTV in milk. The nucleotide sequences of MMTV(CS) open reading frame were completely matched with those of Mtv-48. These results indicate that the provirus Mtv-48 but not Mtv-51 is capable of producing a milk-borne virus of which the superantigen stimulates V beta 2-expressing T cells.
Project description:Superantigens have the ability to stimulate subsets of T lymphocytes bearing particular T-cell receptor Vbeta chains. The best-known viral superantigen is Mls, a product of the murine mammary tumor virus (MMTV) sag gene. The MMTV superantigen is not displayed by the virus itself; however, after infection of B lymphocytes, the superantigen is expressed. The resulting immune stimulation is essential for viral transmission. We have analyzed the transcriptional elements which control Mls-1 expression. Here we present evidence that a region at the 3' end of Mtv-7 env, Penv2, controls B-cell-specific expression of sag. Penv2 has elements homologous with promoters of immunoglobulin H chain, the invariant chain, and major histocompatibility complex class II, suggesting a coordinate regulation of expression of these various B-cell-specific genes and indicating a possible eukaryotic origin of MMTV sag. We have determined that both an IgH heptamer element and a Y box are essential for Penv2 promoter activity and that tandem octamer motifs in the U3 region of the 3' MMTV long terminal repeat function as enhancers. We propose that Penv2 controls constitutive Mls expression in B lymphocytes.
Project description:Superantigens (SAgs) of mouse mammary tumor viruses (MMTVs) play a crucial role in T cell selection in the thymus in a T cell receptor (TCR) V?-specific manner and SAgs presented by B cells activate T cells in the periphery. The peripheral T cell repertoire is dynamically shaped by the steady induction of T cell tolerance against self antigens throughout the lifespan. We hypothesize that de novo somatic mutation of endogenous MMTV SAgs contributes to the modulation of the peripheral T cell repertoire.SAg coding sequences were cloned from the genomic DNAs and/or cDNAs of various tissues of female C57BL/6J mice. A total of 68 unique SAg sequences (54 translated sequences) were identified from the genomic DNAs of liver, lungs, and bone marrow, which are presumed to harbor only three endogenous MMTV loci (Mtv-8, Mtv-9, and Mtv-17). Similarly, 69 unique SAg sequences (58 translated sequences) were cloned from the cDNAs of 18 different tissues. Examination of putative TCR V? specificity suggested that some of the SAg isoforms identified in this study have V? specificities different from the reference SAgs of Mtv-8, Mtv-9, or Mtv-17.The pool of diverse SAg isoforms, generated by de novo somatic mutation, may play a role in the shaping of the peripheral T cell repertoire including the autoimmune T cell population.
Project description: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.
Project description:Horizontal gene transfer from retroviruses to mammals is well documented and extensive, but is rare between unrelated viruses with distinct genome types. Three herpesviruses encode a gene with similarity to a retroviral superantigen gene (sag) of the unrelated mouse mammary tumour virus (MMTV). We uncover ancient retroviral sags in over 20 mammals to reconstruct their shared history with herpesviral sags, revealing that the acquisition is a convergent evolutionary event. A retrovirus circulating in South American primates over 10 million years ago was the source of sag in two monkey herpesviruses, and a different retrovirus was the source of sag in a Peruvian rodent herpesvirus. We further show through a timescaled phylogenetic analysis that a cross-species transmission of monkey herpesviruses occurred after the acquisition of sag. These results reveal that a diverse range of ancient sag-containing retroviruses independently donated sag twice from two separate lineages that are distinct from MMTV.
Project description:Complex human-pathogenic retroviruses cause high morbidity and mortality worldwide, but resist antiviral drugs and vaccine development due to evasion of the immune response. A complex retrovirus, mouse mammary tumor virus (MMTV), requires replication in B and T lymphocytes for mammary gland transmission and is antagonized by the innate immune restriction factor murine Apobec3 (mA3). To determine whether the regulatory/accessory protein Rem affects innate responses to MMTV, a splice-donor mutant (MMTV-SD) lacking Rem expression was injected into BALB/c mice. Mammary tumors induced by MMTV-SD had a lower proviral load, lower incidence, and longer latency than mammary tumors induced by wild-type MMTV (MMTV-WT). MMTV-SD proviruses had many G-to-A mutations on the proviral plus strand, but also C-to-T transitions within WRC motifs. Similarly, a lymphomagenic MMTV variant lacking Rem expression showed decreased proviral loads and increased WRC motif mutations relative to those in wild-type-virus-induced tumors, consistent with activation-induced cytidine deaminase (AID) mutagenesis in lymphoid cells. These mutations are typical of the Apobec family member AID, a B-cell-specific mutagenic protein involved in antibody variable region hypermutation. In contrast, mutations in WRC motifs and proviral loads were similar in MMTV-WT and MMTV-SD proviruses from tumors in AID-insufficient mice. AID was not packaged in MMTV virions. Rem coexpression in transfection experiments led to AID proteasomal degradation. Our data suggest that rem specifies a human-pathogenic immunodeficiency virus type 1 (HIV-1) Vif-like protein that inhibits AID and antagonizes innate immunity during MMTV replication in lymphocytes.IMPORTANCE Complex retroviruses, such as human-pathogenic immunodeficiency virus type 1 (HIV-1), cause many human deaths. These retroviruses produce lifelong infections through viral proteins that interfere with host immunity. The complex retrovirus mouse mammary tumor virus (MMTV) allows for studies of host-pathogen interactions not possible in humans. A mutation preventing expression of the MMTV Rem protein in two different MMTV strains decreased proviral loads in tumors and increased viral genome mutations typical of an evolutionarily ancient enzyme, AID. Although the presence of AID generally improves antibody-based immunity, it may contribute to human cancer progression. We observed that coexpression of MMTV Rem and AID led to AID destruction. Our results suggest that Rem is the first known protein inhibitor of AID and that further experiments could lead to new disease treatments.
Project description:The inbred mouse strain RIII has long been known for shedding large amounts of mouse mammary tumor virus (MMTV) particles in milk and for the development of hormone-dependent early mammary tumors at a very high incidence (>90%). We have established one RIII subline (RIII/Sa) that shows a pattern of virus expression and tumor incidence similar to that in RIII mice. In the present study, we analyzed the milk and mammary tumors of RIII/Sa mice for virus characterization by reverse transcriptase PCR (RT-PCR) cloning and sequencing of the open reading frame (ORF) of the MMTV long terminal repeats (LTRs). Our results show that these mice express a mixture of at least three different MMTV strains, two of which, designated here as RIII/Sa MMTV-1 and RIII/Sa MMTV-2, are exogenous. The third virus, RIII/Sa MMTV-3, appears to carry the signature of an endogenous provirus, Mtv-17. Similar studies done with the milk and mammary glands of another subline, RIIIS/J, revealed that they do not express MMTV in their milk. The RIII/Sa and RIIIS/J mice also exhibited differences in their endogenous proviral contents. Twelve spontaneously developed mammary tumors of RIII/Sa mice were examined for possible Wnt-1 and/or int-2/Fgf3 mutations that are usually found to occur in most mouse mammary tumors as a consequence of MMTV proviral integration. This work led to the isolation of one MMTV-Wnt-1 junction fragment and one MMTV-int-2/Fgf3 junction fragment from 2 of the 12 tumors. Further analyses showed that both junction fragments contained the RIII/Sa MMTV-2-specific LTR ORF, indicating that this virus was involved in the development of both tumors. Whether RIII/Sa MMTV-1 and/or RIII/Sa MMTV-3 plays any role in mammary tumor development in RIII/Sa mice remains to be established. Overall, the present study demonstrates, to our surprise, that (i) RIII/Sa mice express, unlike other native mouse strains, three strains of MMTVs; and (ii) the virions are completely different from the virus expressed by another subline of RIII mice, the BR6 mice.
Project description:Protein tyrosine kinase 6 (PTK6, also called BRK) is an intracellular tyrosine kinase expressed in the majority of human breast tumors and breast cancer cell lines, but its expression has not been reported in normal mammary gland. To study functions of PTK6 in vivo, we generated and characterized several transgenic mouse lines with expression of human PTK6 under control of the mouse mammary tumor virus (MMTV) long terminal repeat. Ectopic active PTK6 was detected in luminal epithelial cells of mature transgenic mammary glands. Lines expressing the MMTV-PTK6 transgene exhibited more than a two-fold increase in mammary gland tumor formation compared with nontransgenic control animals. PTK6 activates signal transducer and activator of transcription 3 (STAT3), and active STAT3 was detected in PTK6-positive mammary gland epithelial cells. Endogenous mouse PTK6 was not detected in the normal mouse mammary gland, but it was induced in mouse mammary gland tumors of different origin, including spontaneous tumors that developed in control mice, and tumors that formed in PTK6, H-Ras, ERBB2 and PyMT transgenic models. MMTV-PTK6 and MMTV-ERBB2 transgenic mice were crossed to explore crosstalk between PTK6 and ERBB2 signaling in vivo. We found no significant increase in tumor incidence, size or metastasis in ERBB2/PTK6 double transgenic mice. Although we detected increased proliferation in ERBB2/PTK6 double transgenic tumors, an increase in apoptosis was also observed. MMTV-PTK6 clearly promotes mammary gland tumorigenesis in vivo, but its impact may be underrepresented in our transgenic models because of induction of endogenous PTK6 expression.