Browsing by Subject "Mouse mammary tumor virus"
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Item CDP/Cutl1 controls differentiation-specific MMTV and cellular gene expression in the mammary gland(2006) Maitra, Urmila; Dudley, JaquelinMouse mammary tumor virus (MMTV) is a betaretrovirus that causes breast cancer in mice. Completion of the MMTV life cycle is linked to the differentiation status of the mammary gland. Virus expression is low during early stages of mammary development, but high levels of MMTV transcription occur during lactation to ensure optimal viral transmission through maternal milk to the offspring. Negative regulation has been shown to play a significant role in mediating tissue-specific MMTV expression. The cellular factor, CCAAT displacement protein (CDP), is a transcriptional repressor of MMTV. Virgin mammary glands have high levels of full-length CDP that binds to negative regulatory elements (NRE) to repress MMTV transcription. During late pregnancy, CDP DNA-binding activity declines concomitant with an increase in viral transcripts. This study demonstrated that reduction of full-length CDP levels in two independent strains of CDP knockout mice increased endogenous MMTV and other mammary-specific cellular genes in undifferentiated mammary glands, suggesting a critical role for CDP in the MMTV life cycle. Interestingly, CDP was developmentally regulated during mammary differentiation. During late pregnancy, the loss of full-length CDP coincided with the appearance of a novel, C-terminally truncated isoform of 150 kDa (CDP150). CDP is proteolytically processed within the homeodomain by a cysteine protease to generate a dominantnegative CDP150 protein that interferes with the DNA-binding and repressor activities of CDP. Furthermore, the loss of DNA-binding activity correlates with increased expression of MMTV and other mammary-specific genes, including β-casein, whey acidic protein and α-lactalbumin, indicating that CDP150 is a developmentally controlled, naturally occurring dominant-negative protein. Studies with mouse embryo fibroblasts lacking endogenous nuclear CDP confirmed that CDP150 lacks transcriptional repressor or activator function for the MMTV LTR and β-casein. Therefore, MMTV has adopted a normal cellular process to control viral expression during mammary differentiation. This study reveals a novel posttranslational cleavage event that controls CDP activity and gene expression during normal development of the mammary gland.Item Characterization of the MMTV-encoded Rem protein(2010-05) Ali, Almas Fatima, 1986-; Dudley, Jaquelin; Huibregtse, Jon M.Mouse mammary tumor virus (MMTV) is a betaretrovirus that causes mammary tumors in mice. MMTV is the only known complex murine retrovirus and encodes Rem, an HIV-1 Rev-like protein. Rem is a 301-amino-acid (33 kDa) protein that is cotranslationally targeted to the ER, where the first 98 amino acids constitute the signal peptide (SP). The SP is cleaved and retrotranslocated to the cytoplasm prior to nuclear entry. In this thesis, the results show that the presence of a leucine at position 71 allows more efficient cleavage of SP and increases Rem activity. Further, in Rem-transfected cells, the majority of SP appears in the nuclear fraction, consistent with fluorescent microscopy data. The C-terminal fragment of Rem (RemCT) is glycosylated in the ER and, although glycosylation sites are present outside the SP, mutations of both these sites abolish SP activity in a reporter assay. Indirect evidence suggests that unglycosylated RemCT is degraded by the proteasome, whereas glycosylated RemCT is likely secreted out of the cell. A variant of MMTV (TBLV) that lacks functional Sag and RemCT has been prepared and will be studied in mice to elucidate the role of RemCT in vivo. Development of an antibody to RemCT will allow tracking of the protein in virus-producing cells. Although there are many other similarities between complex retroviruses like HIV-1 and MMTV, current evidence suggests that Rem lacks an HIV Tat-like transactivator function.Item Host resistance and viral transcription as determinants of MMTV tumorigenesis(2005) Bhadra, Sanchita; Dudley, JaquelinMouse mammary tumor virus (MMTV) is an oncogenic retrovirus that causes mammary tumors in susceptible mice within 6 to 9 months of infection. Type B leukemogenic virus (TBLV) is a variant of MMTV that induces thymic lymphomas in mice, instead of mammary tumors, with a shorter latency. Exogenous MMTV has a complicated life cycle involving milk-borne virus transmission and tissue type and differentiation-specific regulation of viral transcription. Most inbred mouse strains also harbor endogenous MMTV proviruses (Mtvs) in their germline that are inherited according to Mendelian rules and are usually not associated with mammary tumorigenesis. This thesis reports studies on the intricate relationship of MMTV with its host by demonstrating that the outcome of MMTV infection is influenced by both the composition of viral transcriptional elements and by host genetics. This is the first demonstration that the absence of negative regulation of viral transcription and presence of a T-cell specific enhancer in lymphoid cells converts a mammotropic MMTV to a lymphotropic virus. Interestingly, the endogenous Mtv loci in the BALB/cJ mouse genome are required for susceptibility to infection and tumorigenesis by both MMTV and TBLV. A BALB/cJ congenic mouse strain lacking endogenous Mtvs (BALB/Mtv-null) is highly resistant to both MMTV and TBLV. The resistance is not mediated by the production of MMTV-specific neutralizing antibodies in the BALB/Mtv-null strain. However, cell-mediated immune response directed against MMTV-infected cells might contribute towards the tumor resistance phenotype. The BALB/Mtv-null mice also are resistant to oral infection with the bacterial pathogen Vibrio cholerae compared to BALB/cJ mice, and this resistance to V. cholerae correlates with the absence of endogenous Mtvs. Thus, the BALB/Mtv-null mice demonstrate a novel mode of resistance to multiple pathogens that appears to be linked to expression of MMTV genes. These data support a role for endogenous Mtvs in determining susceptibility to multiple pathogens.Item Identification and characterization of the T-cell-specific enhancer of type B leukemogenic virus(2003) Mertz, Jennifer Andrea; Dudley, JaquelinMouse mammary tumor virus (MMTV) is a retrovirus that causes mammary adenocarcinomas and T-cell lymphomas. T-cell lymphomas induced by MMTV have additional proviral integrations that invariably have alterations in the long terminal repeats (LTRs). Type B leukemogenic virus (TBLV) is a thymotropic strain of MMTV that induces rapidly appearing T-cell tumors in mice. TBLV is highly related to MMTV except that TBLV LTRs have a deletion of negative regulatory elements and a multimerization of sequences flanking the deletion. In this study, the multimerized sequences from the TBLV LTR were identified as a novel T-cell-specific enhancer that can act on the TBLV promoter as well as thymidine kinase and c-myc promoters. Substitution mutagenesis of the enhancer identified a critical region that contains binding sites for RUNX1, NFkB and two unknown factors, NF-A and NF-B. RUNX1, NF-A and NF-B all positively regulate TBLV enhancer activity. However, the role of NF-kB in enhancer function is unclear since it does not appear to contribute to the activity of the TBLV LTR in T cells. Additionally, expression of NF-kB antagonizes the positive effect of RUNX1 expression on the TBLV LTR in non-T cells. NF-A appears to be the major contributor to enhancer function and is a lymphoidrestricted factor. The TBLV enhancer also contains functional glucocorticoid receptor (GR) binding sites; however GR binding is not necessary for enhancer activity in T cells. A c-Myb binding site overlaps the GR binding site and expression of c-Myb in non-T cells activates transcription from the LTR. Characterization of TBLV enhancers in proviruses integrated near c-myc revealed that the number of enhancer elements varied, but the most clonal tumors had proviruses containing four enhancer elements. Reporter gene assays showed that LTRs containing four enhancer elements were less effective at activating transcription from either the TBLV or c-myc promoters. In vivo passage of tumor cells in immunocompetent mice revealed a selection for proviral integrations near c-myc with four enhancer elements. Since c-myc overexpression often leads to apoptosis, these results suggested that selection for clonal growth occurred in tumor cells that had modest c-myc overexpression after TBLV insertion to prevent apoptosis.Item Mouse mammary tumor virus activates Cdc42 leading to filopodia formation and transformation of mammary epithelial cells(2005) Smith, Gail Perry; Poenie, Martin F.Mouse mammary tumor virus (MMTV) is a B type retrovirus characterized by assembly of viral “A” particles in the cytoplasm that move to the membrane where they acquire an envelope and exit the cell. Viral exit is associated with reorganization of cortical actin and accumulation of virus in filamentous projections at the cell surface. Here we show that MMTV egress and exit from the cell depends on actin polymerization mediated by the activation of Cdc42 and WASP. Cytochalasin B treatment blocked both accumulation of virus in filopodia and viral exit. Active virus production correlated with accumulation of activated Cdc42 and MMTV Gag on immobilized Pak columns. Both Cdc42 and WASP co-localized with virus particles in the cytoplasm and at the cell surface. Alternatively, dominant-negative Cdc42 or a mutant WASP construct without the Cdc42-binding domain failed to co-localize with virus and prevented MMTV-induced cytoskeletal reorganization as well as viral egress and exit. Infection with virus conferred on these cells the ability to grow in serum-free media, to grow on soft agar and to form foci, features indicative of transformation. Uninfected cells expressing constitutively active Cdc42 exhibited similar characteristics. Furthermore, dominant negative Cdc42 blocked transformation by MMTV. These findings suggest that activation of the Cdc42/WASP/ARP2/3 pathway is required for viral exit and that viral activation of Cdc42 is both required and sufficient to cause transformation.Item The nuclear matrix affects SATB1-mediated MMTV suppression(2005) Seo, Jin; Dudley, JaquelinItem Role of CDP in MMTV transcriptional regulation and tumorigenesis(2001-08) Zhu, Quan; Dudley, Jacquelin P.