Deep sequencing reveals abundant noncanonical retroviral microRNAs in B-cell leukemia/lymphoma.

Viral tumor models have significantly contributed to our understanding of oncogenic mechanisms. How transforming delta-retroviruses induce malignancy, however, remains poorly understood, especially as viral mRNA/protein are tightly silenced in tumors. Here, using deep sequencing of broad windows of small RNA sizes in the bovine leukemia virus ovine model of leukemia/lymphoma, we provide in vivo evidence of the production of noncanonical RNA polymerase III (Pol III)-transcribed viral microRNAs in leukemic B cells in the complete absence of Pol II 5'-LTR-driven transcriptional activity. Processed from a cluster of five independent self-sufficient transcriptional units located in a proviral region dispensable for in vivo infectivity, bovine leukemia virus microRNAs represent ∼40% of all microRNAs in both experimental and natural malignancy. They are subject to strong purifying selection and associate with Argonautes, consistent with a critical function in silencing of important cellular and/or viral targets. Bovine leukemia virus microRNAs are strongly expressed in preleukemic and malignant cells in which structural and regulatory gene expression is repressed, suggesting a key role in tumor onset and progression. Understanding how Pol III-dependent microRNAs subvert cellular and viral pathways will contribute to deciphering the intricate perturbations that underlie malignant transformation.

Chromatin disruption in the promoter of bovine leukemia virus during transcriptional activation.

Bovine leukemia virus expression relies on its chromatin organization after integration into the host cell genome. Proviral latency, which results from transcriptional repression in vivo, represents a viral strategy to escape the host immune system and likely allows for tumor progression. Here, we discriminated two types of latency: an easily reactivable latent state of the YR2 provirus and a 'locked' latent state of the L267 provirus. The defective YR2 provirus was characterized by the presence of nuclease hypersensitive sites at the U3/R junction and in the R/U5 region of the 5'-long terminal repeat (5'-LTR), whereas the L267 provirus displayed a closed chromatin configuration at the U3/R junction. Reactivation of viral expression in YR2 cells by the phorbol 12-myristate 13-acetate (PMA) plus ionomycin combination was accompanied by a rapid but transient chromatin remodeling in the 5'-LTR, leading to an increased PU.1 and USF-1/USF-2 recruitment in vivo sustained by PMA/ionomycin-mediated USF phosphorylation. In contrast, viral expression was not reactivated by PMA/ionomycin in L267 cells, because the 5'-LTR U3/R region remained inaccessible to nucleases and hypermethylated at CpG dinucleotides. Remarkably, we elucidated the BLV 5'-LTR chromatin organization in PBMCs isolated from BLV-infected cows, thereby depicting the virus hiding in vivo in its natural host.

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.

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.

Lentiviral-mediated gene delivery in human monocyte-derived dendritic cells: optimized design and procedures for highly efficient transduction compatible with clinical constraints.

Gene delivery to dendritic cells (DCs) could represent a powerful method of inducing potent, long-lasting immunity. Although recent studies underline the intense interest in lentiviral vector-mediated monocyte-derived DC transduction, efficient gene transfer methods currently require high multiplicities of infection and are not compatible with clinical constraints. We have designed a strategy to optimize the efficiency and clinical relevance of this approach. Initially, using a third generation lentiviral vector expressing green fluorescent protein, we found that modifying the vector design, the DC precursor cell type, and the DC differentiation stage for transduction results in sustained transgene expression in 75-85% of immature DCs (transduction at a multiplicity of infection of 8). This high efficiency was reproducible among different donors irrespective of whether DCs were expanded from fresh or cryopreserved CD14(+) precursors. We then developed procedures that bypass the need for highly concentrated lentiviral preparations and the addition of polybrene to achieve efficient transduction. DCs transduced under these conditions retain their immature phenotype and immunostimulatory potential in both autologous and allogeneic settings. Furthermore, genetically modified DCs maintain their ability to respond to maturation signals and secrete bioactive IL-12, indicating that they are fully functional. Finally, the level of transgene expression is preserved in the therapeutically relevant mature DCs, demonstrating that there is neither promoter-silencing nor loss of transduced cells during maturation. The novel approach described should advance lentiviral-mediated monocyte-derived DC transduction towards a clinical reality.

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.

Epigenetics and leukemia: unraveling oncogenic processes in the BLV ovine model.

Bovine Leukemia Virus (BLV)-induced B-cell leukemia in sheep is a valuable large animal model for investigating oncogenic mechanisms, particularly those associated with human T-cell leukemia virus 1 (HTLV-1). Multiple factors including viral genes, genetic and epigenetic alterations, and the host immune system are likely to contribute and cooperate in the leukemogenesis of adult T-cell leukemia (ATL) in human and B-cell leukemia in sheep. While considerable effort has been made to explore the role of viral determinants in the transformation process, the participation of host-related mechanisms has been poorly addressed. We discuss recent evidence from sheep studies in the context of the growing knowledge that has accumulated in the field of epigenetics in human cancer. These results support the hypothesis that epigenetic events, which were initially identified as a causative mechanism of virus silencing, are also major players in host gene regulation. Future studies in sheep will increase the number of genes identified that are aberrantly regulated by epigenetic processes and identify potential biomarkers which may be used as therapeutic targets in leukemia.

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.

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.