Complete suppression of viral gene expression is associated with the onset and progression of lymphoid malignancy: observations in Bovine Leukemia Virus-infected sheep

BACKGROUND: During malignant progression, tumor cells need to acquire novel characteristics that lead to uncontrolled growth and reduced immunogenicity. In the Bovine Leukemia Virus-induced ovine leukemia model, silencing of viral gene expression has been proposed as a mechanism leading to immune evasion. However, whether proviral expression in tumors is completely suppressed in vivo was not conclusively demonstrated. Therefore, we studied viral expression in two selected experimentally-infected sheep, the virus or the disease of which had features that made it possible to distinguish tumor cells from their nontransformed counterparts. RESULTS: In the first animal, we observed the emergence of a genetically modified provirus simultaneously with leukemia onset. We found a Tax-mutated (TaxK303) replication-deficient provirus in the malignant B-cell clone while functional provirus (TaxE303) had been consistently monitored over the 17-month aleukemic period. In the second case, both non-transformed and transformed BLV-infected cells were present at the same time, but at distinct sites. While there was potentially-active provirus in the non-leukemic blood B-cell population, as demonstrated by ex-vivo culture and injection into naïve sheep, virus expression was completely suppressed in the malignant B-cells isolated from the lymphoid tumors despite the absence of genetic alterations in the proviral genome. These observations suggest that silencing of viral genes, including the oncoprotein Tax, is associated with tumor onset. CONCLUSION: Our findings suggest that silencing is critical for tumor progression and identify two distinct mechanisms-genetic and epigenetic-involved in the complete suppression of virus and Tax expression. We demonstrate that, in contrast to systems that require sustained oncogene expression, the major viral transforming protein Tax can be turned-off without reversing the transformed phenotype. We propose that suppression of viral gene expression is a contributory factor in the impairment of immune surveillance and the uncontrolled proliferation of the BLV-infected tumor cell.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Etude du potentiel oncogène de la protéine Tax du virus de la leucémie bovine :implication dans le dérégulation de l'homéostasie des lymphocytes B primaires

Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Etude du potentiel oncogène de la protéine Tax du virus de la leucémie bovine :implication dans le dérégulation de l'homéostasie des lymphocytes B primaires

Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Suppression of Viral Gene Expression in Bovine Leukemia Virus-Associated B-Cell Malignancy: Interplay of Epigenetic Modifications Leading to Chromatin with a Repressive Histone Code▿

Ovine leukemia/lymphoma resulting from bovine leukemia virus infection of sheep offers a large animal model for studying mechanisms underlying leukemogenesis. Silencing of viral information including Tax, the major contributor to the oncogenic potential of the virus, is critical if not mandatory for tumor progression. In this study, we have identified epigenetic mechanisms that govern the complete suppression of viral expression, using a lymphoma-derived B-cell clone carrying a silent provirus. Silencing was not relieved by injection of the malignant B cells into sheep. However, exogenous expression of Tax or treatment with either the DNA methyltransferase inhibitor 5′azacytidine or the histone deacetylase (HDAC) inhibitor trichostatin A rescued viral expression, as demonstrated by in vivo infectivity trials. Comparing silent and reactivated provirus, we found mechanistic connections between chromatin conformation and tumor-associated transcriptional repression. Silencing is associated with DNA methylation and decreased accessibility of promoter sequences. HDAC1 and the transcriptional corepressor mSin3A are associated with the inactive but not the reactivated promoter. Silencing correlates with a repressed chromatin structure marked by histone H3 and H4 hypoacetylation, a loss of methylation at H3 lysine 4, and an increase of H3 lysine 9 methylation. These observations point to the critical role of epigenetic mechanisms in tumor-specific virus/oncogene silencing, a potential strategy to evade immune response and favor the propagation of the transformed cell

Cytotoxic responses to BLV tax oncoprotein do not prevent leukemogenesis in sheep.

Delta retrovirus-mediated leukemogenesis is dependent on the oncogenic potential of Tax. It is not clear, however, whether Tax-specific immune responses play a role in leukemia onset and progression. Using the BLV-associated leukemia model in sheep, we found that Tax-specific cytotoxic responses induced by DNA immunization or viral infection of naïve animals were not predictive of disease outcome and did not prevent tumor development. Furthermore, provirus and tax may be absent from blood for extended periods, emphasizing the relevance of surveying other compartments during chronic lymphoproliferative disorders. Our results support the conclusion that Tax-specific cytotoxic responses, even during the initial phase of infection, are not sufficient to prevent leukemogenesis.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Insights into gene expression changes impacting B-cell transformation: cross-species microarray analysis of bovine leukemia virus tax-responsive genes in ovine B cells.

Large-animal models for leukemia have the potential to aid in the understanding of networks that contribute to oncogenesis. Infection of cattle and sheep with bovine leukemia virus (BLV), a complex retrovirus related to human T-cell leukemia virus type 1 (HTLV-1), is associated with the development of B-cell leukemia. Whereas the natural disease in cattle is characterized by a low tumor incidence, experimental infection of sheep leads to overt leukemia in the majority of infected animals, providing a model for studying the pathogenesis associated with BLV and HTLV-1. Tax(BLV), the major oncoprotein, initiates a cascade of events leading toward malignancy, although the basis of transformation is not fully understood. We have taken a cross-species ovine-to-human microarray approach to identify Tax(BLV)-responsive transcriptional changes in two sets of cultured ovine B cells following retroviral vector-mediated delivery of Tax(BLV). Using cDNA-spotted microarrays comprising 10,336 human genes/expressed sequence tags, we identified a cohort of differentially expressed genes, including genes related to apoptosis, DNA transcription, and repair; proto-oncogenes; cell cycle regulators; transcription factors; small Rho GTPases/GTPase-binding proteins; and previously reported Tax(HTLV-1)-responsive genes. Interestingly, genes known to be associated with human neoplasia, especially B-cell malignancies, were extensively represented. Others were novel or unexpected. The results suggest that Tax(BLV) deregulates a broad network of interrelated pathways rather than a single B-lineage-specific regulatory process. Although cross-species approaches do not permit a comprehensive analysis of gene expression patterns, they can provide initial clues for the functional roles of genes that participate in B-cell transformation and pinpoint molecular targets not identified using other methods in animal models.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Disruption of B-cell homeostatic control mediated by the BLV-Tax oncoprotein: association with the upregulation of Bcl-2 and signaling through NF-kappaB.

Transactivating proteins associated with complex onco-retroviruses including human T-cell leukemia virus-1 (HTLV-1) and bovine leukemia virus (BLV) mediate transformation using poorly understood mechanisms. To gain insight into the processes that govern tumor onset and progression, we have examined the impact of BLV-Tax expression on ovine B-cells, the targets of BLV in experimentally infected sheep, using B-cell clones that are dependent on CD154 and gammac-common cytokines. Tax was capable of mediating progression of B-cells from cytokine dependence to cytokine independence, indicating that the transactivator can over-ride signaling pathways typically controlled by cytokine receptor activation in B-cells. When examined in the presence of both CD154 and interleukin-4, Tax had a clear supportive role on B-cell growth, with an impact on B-cell proliferation, cell cycle phase distribution, and survival. Apoptotic B-cell death mediated by growth factor withdrawal, physical insult, and NF-kappaB inhibition was dramatically reduced in the presence of Tax. Furthermore, the expression of Tax was associated with higher Bcl-2 protein levels, providing rationale for the rescue signals mediated by the transactivator. Finally, Tax expression in B-cells led to a dramatic increase of nuclear RelB/p50 and p50/p50 NF-kappaB dimers, indicating that cellular signaling through NF-kappaB is a major contributory mechanism in the disruption of B-cell homeostasis. Although Tax is involved in aspects of pathogenesis that are unique to complex retroviruses, the viral strategies associated with this transactivating oncoprotein may have wide-ranging effects that are relevant to other B-cell malignancies.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Complete suppression of viral gene expression is associated with the onset and progression of lymphoid malignancy: observations in Bovine Leukemia Virus-infected sheep-0

<p><b>Copyright information:</b></p><p>Taken from "Complete suppression of viral gene expression is associated with the onset and progression of lymphoid malignancy: observations in Bovine Leukemia Virus-infected sheep"</p><p>http://www.retrovirology.com/content/4/1/51</p><p>Retrovirology 2007;4():51-51.</p><p>Published online 23 Jul 2007</p><p>PMCID:PMC1948017.</p><p></p> parameters. WBC counts per mmare indicated. Provirus load and integration were examined by Southern blot hybridization of - and -digests respectively, showing increasing provirus load and the progression from polyclonal to monoclonal integration as leukemia develops. The nucleotide sequence of the 3' end of the proviral DNA is illustrated by a polyacrylamide gel autoradiography of dideoxynucleotide sequenced PCR-amplified DNA. Boxes highlight nucleotides at positions 8149, 8150 and 8151 of the BLV sequence [29]. Arrows indicate the nucleotide identified at position 8149: a G at pre-leukemic stages (yellow arrow); a G to A transition at the time of the first documented WBC increase (17-month post-infection, red arrow). The resulting amino acid at position 303 of the corresponding Tax proteins is shown below. The transactivation potential of the putative S2531 proviral Tax proteins were examined in a luciferase reporter assay following co-transfection of HeLa cells with the pSGTaxexpression vectors containing sequences cloned from S2531 PBMCs collected at different times post-infection and the reporter plasmid pLTR-Luc as detailed in B. "+" indicates a luciferase activity equivalent to that resulting from transfection with the wild-type pSGTax; "-" indicates the background level activity similar to that obtained when the empty expression vector pSG5 is co-transfected with pLTR-Luc. (B) Luciferase assay reflecting the transactivation potential of a selection of four S2531-derived sequences. Each pSGTaxconstruct containing the different S2531-derived sequences downstream of the CMV promoter was used in HeLa co-transfection with pLTR-Luc which expresses the firefly luciferase under the control of the BLV-LTR promoter. Luciferase activities were measured in cell lysates 42 h posttransfection and were normalized to protein concentrations as previously described [19]. Results are represented as histograms indicating basal luciferase activities (arbitrary units). pSGTaxand pSGTaxcontain sequences amplified from PBMCs isolated during the aleukemic stage, 6 and 14 months post-inoculation respectively; pSGTaxcontains sequences from leukemic PBMC isolated 18 months post-inoculation, and the pSGTaxconstruct resulted from the insertion of lymphoma-derived sequences collected 18 months post-infection. pSGc is the empty control vector. Values represent the means of the results of triplicate samples. The results from a representative experiment of four independent experiments are shown

Complete suppression of viral gene expression is associated with the onset and progression of lymphoid malignancy: observations in Bovine Leukemia Virus-infected sheep-2

<p><b>Copyright information:</b></p><p>Taken from "Complete suppression of viral gene expression is associated with the onset and progression of lymphoid malignancy: observations in Bovine Leukemia Virus-infected sheep"</p><p>http://www.retrovirology.com/content/4/1/51</p><p>Retrovirology 2007;4():51-51.</p><p>Published online 23 Jul 2007</p><p>PMCID:PMC1948017.</p><p></p> parameters. WBC counts per mmare indicated. Provirus load and integration were examined by Southern blot hybridization of - and -digests respectively, showing increasing provirus load and the progression from polyclonal to monoclonal integration as leukemia develops. The nucleotide sequence of the 3' end of the proviral DNA is illustrated by a polyacrylamide gel autoradiography of dideoxynucleotide sequenced PCR-amplified DNA. Boxes highlight nucleotides at positions 8149, 8150 and 8151 of the BLV sequence [29]. Arrows indicate the nucleotide identified at position 8149: a G at pre-leukemic stages (yellow arrow); a G to A transition at the time of the first documented WBC increase (17-month post-infection, red arrow). The resulting amino acid at position 303 of the corresponding Tax proteins is shown below. The transactivation potential of the putative S2531 proviral Tax proteins were examined in a luciferase reporter assay following co-transfection of HeLa cells with the pSGTaxexpression vectors containing sequences cloned from S2531 PBMCs collected at different times post-infection and the reporter plasmid pLTR-Luc as detailed in B. "+" indicates a luciferase activity equivalent to that resulting from transfection with the wild-type pSGTax; "-" indicates the background level activity similar to that obtained when the empty expression vector pSG5 is co-transfected with pLTR-Luc. (B) Luciferase assay reflecting the transactivation potential of a selection of four S2531-derived sequences. Each pSGTaxconstruct containing the different S2531-derived sequences downstream of the CMV promoter was used in HeLa co-transfection with pLTR-Luc which expresses the firefly luciferase under the control of the BLV-LTR promoter. Luciferase activities were measured in cell lysates 42 h posttransfection and were normalized to protein concentrations as previously described [19]. Results are represented as histograms indicating basal luciferase activities (arbitrary units). pSGTaxand pSGTaxcontain sequences amplified from PBMCs isolated during the aleukemic stage, 6 and 14 months post-inoculation respectively; pSGTaxcontains sequences from leukemic PBMC isolated 18 months post-inoculation, and the pSGTaxconstruct resulted from the insertion of lymphoma-derived sequences collected 18 months post-infection. pSGc is the empty control vector. Values represent the means of the results of triplicate samples. The results from a representative experiment of four independent experiments are shown