The helicase-like transcription factor redirects the autophagic flux and restricts human T cell leukemia virus type 1 infection.

Retroviruses and their host have coevolved in a delicate balance between viral replication and survival of the infected cell. In this equilibrium, restriction factors expressed by infected cells control different steps of retroviral replication such as entry, uncoating, nuclear import, expression, or budding. Here, we describe a mechanism of restriction against human T cell leukemia virus type 1 (HTLV-1) by the helicase-like transcription factor (HLTF). We show that RNA and protein levels of HLTF are reduced in primary T cells of HTLV-1-infected subjects, suggesting a clinical relevance. We further demonstrate that the viral oncogene Tax represses HLTF transcription via the Enhancer of zeste homolog 2 methyltransferase of the Polycomb repressive complex 2. The Tax protein also directly interacts with HLTF and induces its proteasomal degradation. RNA interference and gene transduction in HTLV-1-infected T cells derived from patients indicate that HLTF is a restriction factor. Restoring the normal levels of HLTF expression induces the dispersal of the Golgi apparatus and overproduction of secretory granules. By synergizing with Tax-mediated NF-κB activation, physiologically relevant levels of HLTF intensify the autophagic flux. Increased vesicular trafficking leads to an enlargement of the lysosomes and the production of large vacuoles containing viral particles. HLTF induction in HTLV-1-infected cells significantly increases the percentage of defective virions. In conclusion, HLTF-mediated activation of the autophagic flux blunts the infectious replication cycle of HTLV-1, revealing an original mode of viral restriction.

Specific DMPK-promoter targeting by CRISPRi reverses myotonic dystrophy type 1-associated defects in patient muscle cells.

Myotonic dystrophy type 1 (DM1) is a neuromuscular disease that originates from an expansion of CTG microsatellites in the 3' untranslated region of the DMPK gene, thus leading to the expression of transcripts containing expanded CUG repeats (CUGexp). The pathophysiology is explained by a toxic RNA gain of function where CUGexp RNAs form nuclear aggregates that sequester and alter the function of MBNL splicing factors, triggering splicing misregulation linked to the DM1 symptoms. There is currently no cure for DM1, and most therapeutic strategies aim at eliminating CUGexp-DMPK transcripts. Here, we investigate a DMPK-promoter silencing strategy using CRISPR interference as a new alternative approach. Different sgRNAs targeting the DMPK promoter are evaluated in DM1 patient muscle cells. The most effective guides allowed us to reduce the level of DMPK transcripts and CUGexp-RNA aggregates up to 80%. The CUGexp-DMPK repression corrects the overall transcriptome, including spliceopathy, and reverses a physiological parameter in DM1 muscle cells. Its action is specific and restricted to the DMPK gene, as confirmed by genome-wide expression analysis. Altogether, our findings highlight DMPK-promoter silencing by CRISPRi as a promising therapeutic approach for DM1.

BLV: lessons on vaccine development.

Vaccination against retroviruses is a challenge because of their ability to stably integrate into the host genome, undergo long-term latency in a proportion of infected cells and thereby escape immune response. Since clearance of the virus is almost impossible once infection is established, the primary goal is to achieve sterilizing immunity. Besides efficacy, safety is the major issue since vaccination has been associated with increased infection or reversion to pathogenicity. In this review, we discuss the different issues that we faced during the development of an efficient vaccine against bovine leukemia virus (BLV). We summarize the historical failures of inactivated vaccines, the efficacy and safety of a live-attenuated vaccine and the economical constraints of further industrial development.

Inhibition of EZH2 methyltransferase decreases immunoediting of mesothelioma cells by autologous macrophages through a PD-1-dependent mechanism.

The roles of macrophages in orchestrating innate immunity through phagocytosis and T lymphocyte activation have been extensively investigated. Much less understood is the unexpected role of macrophages in direct tumor regression. Tumoricidal macrophages can indeed manifest cancer immunoediting activity in the absence of adaptive immunity. We investigated direct macrophage cytotoxicity in malignant pleural mesothelioma, a lethal cancer that develops from mesothelial cells of the pleural cavity after occupational asbestos exposure. In particular, we analyzed the cytotoxic activity of mouse RAW264.7 macrophages upon cell-cell contact with autologous AB1/AB12 mesothelioma cells. We show that macrophages killed mesothelioma cells by oxeiptosis via a mechanism involving enhancer of zeste homolog 2 (EZH2), a histone H3 lysine 27-specific (H3K27-specific) methyltransferase of the polycomb repressive complex 2 (PRC2). A selective inhibitor of EZH2 indeed impaired RAW264.7-directed cytotoxicity and concomitantly stimulated the PD-1 immune checkpoint. In the immunocompetent BALB/c model, RAW264.7 macrophages pretreated with the EZH2 inhibitor failed to control tumor growth of AB1 and AB12 mesothelioma cells. Blockade of PD-1 engagement restored macrophage-dependent antitumor activity. We conclude that macrophages can be directly cytotoxic for mesothelioma cells independent of phagocytosis. Inhibition of the PRC2 EZH2 methyltransferase reduces this activity because of PD-1 overexpression. Combination of PD-1 blockade and EZH2 inhibition restores macrophage cytotoxicity.

How Does HTLV-1 Undergo Oncogene-Dependent Replication Despite a Strong Immune Response?

In 1987, Mitsuaki Yoshida proposed the following model (Yoshida and Seiki, 1987): "... T-cells activated through the endogenous p40x would express viral antigens including the envelope glycoproteins which are exposed on the cell surface. These glycoproteins are targets of host immune surveillance, as is evidenced by the cytotoxic effects of anti-envelope antibodies or patient sera. Eventually all cells expressing the viral antigens, that is, all cells driven by the p40x would be rejected by the host. Only those cells that did not express the viral antigens would survive. Later, these antigen-negative infected cells would begin again to express viral antigens, including p40x, thus entering into the second cycle of cell propagation. These cycles would be repeated in so-called healthy virus carriers for 20 or 30 years or longer...." Three decades later, accumulated experimental facts particularly on intermittent viral transcription and regulation by the host immune response appear to prove that Yoshida was right. This Hypothesis and Theory summarizes the evidences that support this paradigm.

Hijacking of the AP-1 Signaling Pathway during Development of ATL.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of a fatal malignancy known as adult T-cell leukemia (ATL). One way to address the pathology of the disease lies on conducting research with a molecular approach. In addition to the analysis of ATL-relevant signaling pathways, understanding the regulation of important and relevant transcription factors allows researchers to reach this fundamental objective. HTLV-1 encodes for two oncoproteins, Tax and HTLV-1 basic leucine-zipper factor, which play significant roles in the cellular transformation and the activation of the host's immune responses. Activating protein-1 (AP-1) transcription factor has been linked to cancer and neoplastic transformation ever since the first representative members of the Jun and Fos gene family were cloned and shown to be cellular homologs of viral oncogenes. AP-1 is a dimeric transcription factor composed of proteins belonging to the Jun (c-Jun, JunB, and JunD), Fos (c-Fos, FosB, Fra1, and Fra2), and activating transcription factor protein families. Activation of AP-1 transcription factor family by different stimuli, such as inflammatory cytokines, stress inducers, or pathogens, results in innate and adaptive immunity. AP-1 is also involved in various cellular events including differentiation, proliferation, survival, and apoptosis. Deregulated expression of AP-1 transcription factors is implicated in various lymphomas such as classical Hodgkin lymphomas, anaplastic large cell lymphomas, diffuse large B-cell lymphomas, and adult T-cell leukemia. Here, we review the current thinking behind deregulation of the AP-1 pathway and its contribution to HTLV-induced cellular transformation.

Impaired expression of DICER and some microRNAs in HBZ expressing cells from acute adult T-cell leukemia patients.

Global dysregulation of microRNAs (miRNAs), a class of non-coding RNAs that regulate genes expression, is a common feature of human tumors. Profiling of cellular miRNAs on Adult T cell Leukemia (ATL) cells by Yamagishi et al. showed a strong decrease in expression for 96.7% of cellular miRNAs in ATL cells. However, the mechanisms that regulate the expression of miRNAs in ATL cells are still largely unknown. In this study, we compared the expression of 12 miRs previously described for being overexpress by Tax and the expression of several key components of the miRNAs biogenesis pathways in different HBZ expressing cell lines as well as in primary CD4 (+) cells from acute ATL patients. We showed that the expression of miRNAs and Dicer1 were downregulated in cells lines expressing HBZ as well as in fresh CD4 (+) cells from acute ATL patients. Using qRT-PCR, western blotting analysis and Chromatin Immunoprecipitation, we showed that dicer transcription was regulated by c-Jun and JunD, two AP-1 transcription factors. We also demonstrated that HBZ affects the expression of Dicer by removing JunD from the proximal promoter. Furthermore, we showed that at therapeutic concentration of 1mM, Valproate (VPA) an HDAC inhibitors often used in cancer treatment, rescue Dicer expression and miRNAs maturation. These results might offer a rationale for clinical studies of new combined therapy in an effort to improve the outcome of patients with acute ATL.

Recent Advances in BLV Research.

Different animal models have been proposed to investigate the mechanisms of Human T-lymphotropic Virus (HTLV)-induced pathogenesis: rats, transgenic and NOD-SCID/γcnull (NOG) mice, rabbits, squirrel monkeys, baboons and macaques. These systems indeed provide useful information but have intrinsic limitations such as lack of disease relevance, species specificity or inadequate immune response. Another strategy based on a comparative virology approach is to characterize a related pathogen and to speculate on possible shared mechanisms. In this perspective, bovine leukemia virus (BLV), another member of the deltaretrovirus genus, is evolutionary related to HTLV-1. BLV induces lymphoproliferative disorders in ruminants providing useful information on the mechanisms of viral persistence, genetic determinants of pathogenesis and potential novel therapies.

Modes of Human T Cell Leukemia Virus Type 1 Transmission, Replication and Persistence.

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes cancer (Adult T cell Leukemia, ATL) and a spectrum of inflammatory diseases (mainly HTLV-associated myelopathy-tropical spastic paraparesis, HAM/TSP). Since virions are particularly unstable, HTLV-1 transmission primarily occurs by transfer of a cell carrying an integrated provirus. After transcription, the viral genomic RNA undergoes reverse transcription and integration into the chromosomal DNA of a cell from the newly infected host. The virus then replicates by either one of two modes: (i) an infectious cycle by virus budding and infection of new targets and (ii) mitotic division of cells harboring an integrated provirus. HTLV-1 replication initiates a series of mechanisms in the host including antiviral immunity and checkpoint control of cell proliferation. HTLV-1 has elaborated strategies to counteract these defense mechanisms allowing continuous persistence in humans.

HBZ stimulates brain-derived neurotrophic factor/TrkB autocrine/paracrine signaling to promote survival of human T-cell leukemia virus type 1-Infected T cells.

UNLABELLED: Brain-derived neurotrophic factor (BDNF) is a neurotrophin that promotes neuronal proliferation, survival, and plasticity. These effects occur through autocrine and paracrine signaling events initiated by interactions between secreted BDNF and its high-affinity receptor, TrkB. A BDNF/TrkB autocrine/paracrine signaling loop has additionally been implicated in augmenting the survival of cells representing several human cancers and is associated with poor patient prognosis. Adult T-cell leukemia (ATL) is a fatal malignancy caused by infection with the complex retrovirus human T-cell leukemia virus type 1 (HTLV-1). In this study, we found that the HTLV-1-encoded protein HBZ activates expression of BDNF, and consistent with this effect, BDNF expression is elevated in HTLV-1-infected T-cell lines compared to uninfected T cells. Expression of TrkB is also higher in HTLV-1-infected T-cell lines than in uninfected T cells. Furthermore, levels of both BDNF and TrkB mRNAs are elevated in peripheral blood mononuclear cells (PBMCs) from ATL patients, and ATL patient sera contain higher concentrations of BDNF than sera from noninfected individuals. Finally, chemical inhibition of TrkB signaling increases apoptosis in HTLV-1-infected T cells and reduces phosphorylation of glycogen synthase kinase 3ß (GSK-3ß), a downstream target in the signaling pathway. These results suggest that HBZ contributes to an active BDNF/TrkB autocrine/paracrine signaling loop in HTLV-1-infected T cells that enhances the survival of these cells. IMPORTANCE: Infection with human T-cell leukemia virus type 1 (HTLV-1) can cause a rare form of leukemia designated adult T-cell leukemia (ATL). Because ATL patients are unresponsive to chemotherapy, this malignancy is fatal. As a retrovirus, HTLV-1 integrates its genome into a host cell chromosome in order to utilize host factors for replication and expression of viral proteins. However, in infected cells from ATL patients, the viral genome is frequently modified to block expression of all but a single viral protein. This protein, known as HBZ, is therefore believed to modulate cellular pathways necessary for the leukemic state and the chemotherapeutic resistance of the cell. Here we provide evidence to support this hypothesis. We found that HBZ promotes a BDNF/TrkB autocrine/paracrine signaling pathway that is known to enhance the survival and chemotherapeutic resistance of other types of cancer cells. It is possible that inhibition of this pathway may improve treatments for ATL.

Human T-cell leukemia virus type 1 (HTLV-1) bZIP factor requires cellular transcription factor JunD to upregulate HTLV-1 antisense transcription from the 3' long terminal repeat.

Infection with the human T-cell leukemia virus type 1 (HTLV-1) results in a variety of diseases including adult T-cell leukemia (ATL), a fatal malignancy characterized by the uncontrolled proliferation of virally infected CD4(+) T cells. The HTLV-1 basic leucine zipper factor (HBZ) is believed to contribute to development and maintenance of ATL. Unlike the other HTLV-1 genes, the hbz gene is encoded on the complementary strand of the provirus and therefore is not under direct control of the promoter within the 5' long terminal repeat (LTR) of the provirus. This promoter can undergo inactivating genetic or epigenetic changes during the course of ATL that eliminates expression of all viral genes except that of hbz. In contrast, repressive modifications are not known to occur on the hbz promoter located in the 3' LTR, and hbz expression has been consistently detected in all ATL patient samples. Although Sp1 regulates basal transcription from the HBZ promoter, other factors that activate transcription remain undefined. In this study, we used a proviral reporter construct deleted of the 5' LTR to show that HBZ upregulates its own expression through cooperation with JunD. Activation of antisense transcription was apparent in serum-deprived cells in which the level of JunD was elevated, and elimination of JunD expression by gene knockout or shRNA-mediated knockdown abrogated this effect. Activation through HBZ and JunD additionally required Sp1 binding at the hbz promoter. These data favor a model in which JunD is recruited to the promoter through Sp1, where it heterodimerizes with HBZ thereby enhancing its activity. Separately, hbz gene expression led to an increase in JunD abundance, and this effect correlated with emergence of features of transformed cells in immortalized fibroblasts. Overall, our results suggest that JunD represents a novel therapeutic target for the treatment of ATL.