ALY is a common coactivator of RUNX1 and c-Myb on the type B leukemogenic virus enhancer

Type B leukemogenic virus (TBLV), a mouse mammary tumor virus (MMTV) variant, often induces T-cell leukemias and lymphomas by c-myc activation following viral DNA integration. Transfection assays using a c-myc reporter plasmid indicated that the TBLV long terminal repeat (LTR) enhancer is necessary for T-cell-specific increases in basal reporter activity. The sequence requirements for this effect were studied using mutations of the 62-bp enhancer region in an MMTV LTR reporter vector. Deletion of a nuclear factor A-binding site dramatically reduced reporter activity in Jurkat T cells. However, a 41-bp enhancer missing the RUNX1 site still retained minimal enhancer function. DNA affinity purification using a TBLV enhancer oligomer containing the RUNX1 binding site followed by mass spectrometry resulted in the identification of ALY. Subsequent experiments focused on the reconstitution of enhancer activity in epithelial cells. ALY overexpression synergized with RUNX1B on TBLV enhancer activity, and synergism required the RUNX1B-binding site. A predicted c-Myb binding site in the enhancer was confirmed after c-myb overexpression elevated TBLV LTR reporter activity, and overexpression of c-Myb and RUNX1B together showed additive effects on reporter gene levels. ALY also synergized with c-Myb, and coimmunoprecipitation experiments demonstrated an interaction between ALY and c-Myb. These experiments suggest a central role for ALY in T-cell enhancer function and oncogene activation.     

Mouse mammary tumor virus with rearranged long terminal repeats causes murine lymphomas.

Mouse mammary tumor virus (MMTV) is a slowly transforming retrovirus associated primarily with the induction of mammary tumors. It is widely accepted that T-cell lymphomas of various mouse strains are associated with extra proviruses of MMTV. These extra proviruses showed site-specific rearrangements in the U3 region of long terminal repeats (LTRs), consisting of about 400 nucleotide deletions and occasional substitution resulting in unique tandem repeats. However, the question of whether these mutant MMTVs cause lymphomas has not been experimentally resolved. Here we present distinct evidence that they do. We constructed chimeric MMTVs by replacing the LTR of the recently constructed pathogenic MMTV provirus clone with rearranged LTRs of MMTV proviruses obtained from two DBA/2 mouse lymphoma cell lines, MLA and DL-8, and inoculated them into BALB/c mice. These mice developed lymphomas, but no mammary tumors, 4 to 11 months postinoculation, whereas the original pathogenic MMTV clone alone induced mammary tumors. These results showed that the tissue specificity of MMTV tumorigenesis is determined by the LTR structures.     

Rearrangements in the long terminal repeat of extra mouse mammary tumor proviruses in T-cell leukemias of mouse strain GR result in a novel enhancer-like structure.

Male GR mice develop T-cell leukemia at low frequency late in life. These leukemia cells invariably contain large amounts of mouse mammary tumor virus (MMTV) RNA and MMTV proteins and have extra MMTV proviruses integrated in their DNA. We show here that the extra MMTV proviruses are all derived from the endogenous MMTV provirus associated with the Mtv-2 locus and that the T-cell leukemias are clonal with respect to the acquired MMTV proviruses. The extra MMTV proviruses in six transplantable T-cell leukemia lines studied had rearranged, shortened long terminal repeats (LTRs); each T-cell leukemia, however, had a different LTR rearrangement within its extra MMTV provirus. The alteration within the extra LTRs of T-cell leukemia line 42 involved deletion of 453 nucleotides and generation of a tandem repeat region consisting of regions flanking the deletion. This alteration generated a sequence similar to the adenovirus enhancer core sequence. The viral RNAs in the T-cell leukemias contained corresponding alterations in their U3 regions. These results demonstrate that expression of MMTV in T-cell leukemias of GR mice may be the consequence of the generation of a novel enhancer, which could also stimulate expression of any adjacent cellular oncogene.     

Rorgamma (Rorc) is a common integration site in type B leukemogenic virus-induced T-cell lymphomas

The retrovirus type B leukemogenic virus (TBLV) causes T-cell lymphomas in mice. We have identified the Rorgamma locus as an integration site in 19% of TBLV-induced tumors. Overexpression of one or more Rorgamma isoforms in >77% of the tumors tested may complement apoptotic effects of c-myc overexpression.     

Alterations in the U3 region of the long terminal repeat of an infectious thymotropic type B retrovirus.

We isolated and characterized a type B thymotropic retrovirus (DMBA-LV) which is highly related to mouse mammary tumor virus (MMTV) isolates and which induces T-cell thymomas with a high incidence and a very short latent period. Regions of nonhomology between the DMBA-LV genome and the MMTV genome were identified by heteroduplex mapping and nucleotide sequence studies. In the electron microscope heteroduplex mapping studies the EcoRI-generated 5' and 3' fragments of the DMBA-LV genome were compared with the corresponding fragments of the MMTV (C3H and GR) genome isolated from mammary tumors. The results indicated that DMBA-LV contained a region of nonhomologous nucleotide sequences in the 3' half of the U3 region of the long terminal repeat (LTR). Nucleotide sequence studies confirmed these results and showed that in this region 440 nucleotides of the MMTV (C3H) sequences were deleted and substituted with a segment of 122 nucleotides. This substituted segment in the form of a tandem repeat structure contained nucleotide sequences derived exclusively from sequences which flanked the substitution loop. The distal glucocorticoid regulatory element was unaltered, and two additional copies of the distal glucocorticoid regulatory element-binding site were present in the substituted region. The restriction endonuclease map of the reconstructed molecular clone of DMBA-LV was identical to that corresponding to unintegrated linear DMBA-LV DNA present in DMBA-LV-induced tumor cell lines. Since the nucleotide sequences of the LTRs present in four different DMBA-LV proviral copies isolated from a single thymoma were identical, we concluded that they were derived from the same parental virus and that this type B retrovirus containing an alteration in the U3 region of its LTR could induce thymic lymphomas. Thus, DMBA-LV represents the first example of a productively replicating type B retrovirus that contains an LTR modified in the U3 region and that has target cell and disease specificity for T cells.     

Mouse mammary tumor virus carrying a bacterial supF gene has wild-type pathogenicity and enables rapid isolation of proviral integration sites

Mouse mammary tumor virus (MMTV) has frequently been used as an insertional mutagen to identify provirally activated mammary proto-oncogenes. To expedite and facilitate the process of cloning MMTV insertion sites, we have introduced a bacterial supF suppressor tRNA gene into the long terminal repeat (LTR) of MMTV, thus allowing selection of clones containing it in lambda vectors bearing amber mutations. The presence of supF in the LTR should circumvent the screening process for proviral insertion sites, since only those lambda clones with supF-containing proviral-cellular junction fragments should be able to form plaques on a lawn of wild-type Escherichia coli (i.e., lacking supF). The resulting virus (MMTVsupF) induced mammary tumors at the expected rate in infected mice, deleted the appropriate T-cell population by virtue of its superantigen gene, and stably retained the supF gene after passage via the milk to female offspring. To test the selective function of the system, size-selected DNA containing two proviral-cellular junction fragments from an MMTV supF-induced mammary tumor was ligated into lambdagtWES.lambdaB, packaged, and plated on a supF-deficient bacterial host for selection of supF-containing clones. All plaques tested contained the desired cloned fragments, thus demonstrating the utility of this modified provirus for the rapid cloning of MMTV insertion sites.     

Prevalence of mouse mammary tumor virus (MMTV) in wild house mice (Mus musculis) in southeastern Australia

We determined the prevalence of mouse mammary tumor virus (MMTV) in introduced, free-roaming, wild house mice (Mus musculus domesticus) [corrected] and compared envelope (env) and long terminal repeat (LTR) nucleotide sequences of viruses from wild mice and other sources. Mice were trapped on two occasions, in October (spring) and the following May (autumn) of 2003-2004 in the Mallee region of northwestern Victoria, Australia. Animals were assigned to three cohorts (subadult, young, and old adults) based on their body length. The DNA from salivary glands (62 of 62 mice) and mammary glands (19 of 32 female mice) was screened for the MMTV envelope (env) gene, and the long terminal repeat (LTR) region including the superantigen (SAg) sequence was amplified from a subset. Positive polymerase chain reaction (PCR) results for the MMTV env PCR were detected from salivary gland tissues from 60 of 62 (97%) mice and from mammary gland tissues from 19 of 19 (100%) female mice. All but two mice were positive for MMTV env across both sexes and the three cohorts. Similarity of the SAg carboxy-terminal nucleotide sequence between free-roaming wild house mice varied from 64% to 99%, although most of this variation was due to DNA sequences from two mice (M4 and M5). Phylogenetic analysis of the LTR region did not result in distinct grouping of sequences derived from mice when comparisons were made among sequences from mice in the US, Europe, and Australia, and MMTV-like virus (MMTV-LV) env sequences derived from human hosts. We report a high prevalence of the MMTV env sequence during a sampling period when peak mouse density was low. This indicates that MMTV is an enzootic virus in a population of wild, free-ranging mice in northwestern Victoria, in Australia. Phylogenetic analysis, based upon env and LTR sequence data, indicated minor variation among all isolates. This represents the first report on the prevalence of MMTV in mouse populations in Australia.     

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.     

Major histocompatibility complex class II-regulated immunity to murine leukemia virus protects against early T- but not late B-cell lymphomas

We studied the relative importance of class I and class II major histocompatibility complex (MHC) immunoregulation in the control of T- and B-cell lymphomas induced by murine leukemia virus. Previously, we have described a mink cell focus-inducing (MCF) murine leukemia virus, MCF 1233, which induces not only lymphoblastic T-cell lymphomas but also follicle center cell or lymphoblastic B-cell lymphomas. We now report that the outcome of neonatal infection with MCF 1233 in H-2-congenic C57BL/10 and C57BL/6 mice is decisively influenced by the H-2 I-A locus. A total of 64% of H-2 I-Ak, d mice [B10.BR, B10.D2, B10.A(2R), B10.A(4R), and B10.MBR] developed T-cell lymphomas after MCF 1233 infection (mean latency, 37 weeks). In contrast, H-2 I-Ab [B10, B10.A(5R), B6], H-2 I-Ab/k [(B10.A x B10)F1 and (B10 x B10.A)F1], and H-2 I-Abm12 (bm12) mice were resistant against T-cell lymphomagenesis, but 65% of these H-2 I-Ab, b/k, bm12 animals developed B-cell lymphomas (mean latency, 71 weeks). Animals of T-cell lymphoma-susceptible strains that escaped from T-cell lymphomagenesis developed B-cell lymphomas with similar frequency as animals of T-cell lymphoma-resistant strains, but with a shorter latency. H-2 class II-determined regulation of antiviral immunity was reflected in the presence of high titers of antiviral envelope antibodies in T-cell lymphoma-resistant B-cell lymphoma-susceptible H-2 I-Ab, b/k, bm12 mice, whereas in T-cell lymphoma-susceptible H-2 I-Ak,d mice no antiviral antibodies were found. At week 4 after neonatal MCF 1233 infection, a high percentage of thymocytes were virally infected in both T-cell lymphoma-susceptible and -resistant mice. However, T-cell lymphoma-resistant animals cleared the thymic infection between weeks 4 and 10 of age, coinciding with a sharp rise in serum levels of antiviral antibodies. We conclude that the pleiotropic effects of MCF 1233 infection in H-2-congenic mice result from MHC class II I-A-determined T-cell response differences.