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Cutaneous T-cell lymphomas (CTCLs) are telomerase-positive tumors expressing hTERT, although neither gene rearrangement/amplification nor promoter hotspot mutations could explain the hTERT re-expression. As the hTERT promoter is rich in CpG, we investigated the contribution of epigenetic mechanisms in its re-expression. We analyzed hTERT promoter methylation status in CTCL cells compared with healthy cells. Gene-specific methylation analyses revealed a common methylation pattern exclusively in tumor cells. This methylation pattern encompassed a hypermethylated distal region from -650 to -150 bp and a hypomethylated proximal region from -150 to +150 bp. Interestingly, the hypermethylated region matches with the recently named TERT hypermethylated oncogenic region (THOR). THOR has been associated with telomerase reactivation in many cancers, but it has so far not been reported in cutaneous lymphomas. Additionally, we assessed the effect of THOR on two histone deacetylase inhibitors (HDACi), romidepsin and vorinostat, both approved for CTCL treatment and a DNA methyltransferase inhibitor (DNMTi) 5-azacytidine, unapproved for CTCL. Contrary to our expectations, the findings reported herein revealed that THOR methylation is relatively stable under these epigenetic drugs' pressure, whereas these drugs reduced the hTERT gene expression.

There are four human T-lymphotropic viruses (HTLV-1, 2, 3, 4) that have emerged from the transmission of simian viruses. HTLV-1 was the first retrovirus to be shown to be responsible for a human pathology. The expression of retroviral genes depends mostly on their 5'LTR, but it was revealed that HTLV have a promoter in their 3'LTR, capable of transcription from the antisense strand of their genome. These transcripts can be translated into proteins named HBZ, APH-2, APH-3 and APH-4. Antisense transcription in HTLV-1 and its encoded protein HBZ have been thoroughly studied and it has been suggested that HBZ plays an important role in viral replication and the development of ATL. Very few studies have been conducted on antisense transcription from the three other viruses, although it is likely that these genes are also implicated in viral replication.

International audience; Beyond their role in cellular RNA metabolism, DExD/H-box RNA heli-cases are hijacked by various RNA viruses in order to assist replication of the viral genome. Here, we identify the DExH-box RNA helicase 9 (DHX9) as a binding partner of chikungunya virus (CHIKV) nsP3 mainly interacting with the C-terminal hypervari-able domain. We show that during early CHIKV infection, DHX9 is recruited to the plasma membrane, where it associates with replication complexes. At a later stage of infection, DHX9 is, however, degraded through a proteasome-dependent mechanism. Using silencing experiments, we demonstrate that while DHX9 negatively controls viral RNA synthesis, it is also required for optimal mature nonstructural protein translation. Altogether, this study identifies DHX9 as a novel cofactor for CHIKV repli-cation in human cells that differently regulates the various steps of CHIKV life cycle and may therefore mediate a switch in RNA usage from translation to replication during the earliest steps of CHIKV replication. IMPORTANCE The reemergence of chikungunya virus (CHIKV), an alphavirus that is transmitted to humans by Aedes mosquitoes, is a serious global health threat. In the absence of effective antiviral drugs, CHIKV infection has a significant impact on human health, with chronic arthritis being one of the most serious complications. The molecular understanding of host-virus interactions is a prerequisite to the development of targeted therapeutics capable to interrupt viral replication and transmission. Here, we identify the host cell DHX9 DExH-Box helicase as an essential cofactor for early CHIKV genome translation. We demonstrate that CHIKV nsP3 protein acts as a key factor for DHX9 recruitment to replication complexes. Finally, we establish that DHX9 behaves as a switch that regulates the progression of the viral cycle from translation to genome replication. This study might therefore have a significant impact on the development of antiviral strategies. KEYWORDS chikungunya virus, DHX9, RNA helicase, nsP3, viral replication T he chikungunya virus (CHIKV), a mosquito-borne alphavirus transmitted by Aedes mosquitoes, represents an ongoing challenge to medicine and public health. The clinical manifestation of CHIKV infection is an acute syndrome (high fever, rash, myalgia, and intense arthralgia) that coincides with high viremia. In the absence of targeted therapeutics the infection evolves into a chronic incapacitating arthralgia in the distal joints in more than half of the cases, with patients requiring long-term administration of anti-inflammatory and immunosuppressive treatment (for a review, see reference 1). Because CHIKV recently caused major outbreaks worldwide with a disastrous socioeconomic impact and because antiviral molecules are still lacking, there is an urgent need to identify the mechanisms of infection that might be targeted therapeutically. Citation Matkovic R, Bernard E, Fontanel S, Eldin P, Chazal N, Hassan Hersi D, Merits A, Péloponèse J-M, Briant L. 2019. The host DHX9 DExH-box helicase is recruited to chikungunya virus replication complexes for optimal genomic RNA translation. J Virol 93:e01764-18.

International audience; 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.

International audience; More than thirty years have passed since human T-cell leukemia virus type 1 (HTLV-1) was described as the first retrovirus to be the causative agent of a human cancer, adult T-cell leukemia (ATL), but the precise mechanism behind HTLV-1 pathogenesis still remains elusive. For more than two decades, the transforming ability of HTLV-1 has been exclusively associated to the viral transactivator Tax. Thirteen year ago, we first reported that the minus strand of HTLV-1 encoded for a basic Zip factor factor (HBZ), and since then several teams have underscored the importance of this antisense viral protein for the maintenance of a chronic infection and the proliferation of infected cells. More recently, we as well as others have demonstrated that HBZ has the potential to transform cells both in vitro and in vivo. In this review, we focus on the latest progress in our understanding of HBZ functions in chronicity and cellular transformation. We will discuss the involvement of this paradigm shift of HTLV-1 research on new therapeutic approaches to treat HTLV-1-related human diseases.

International audience; Even though an estimated 10-20 million people worldwide are infected with the oncogenic retrovirus, human T-lymphotropic virus type 1 (HTLV-1), its epidemiology is poorly understood, and little effort has been made to reduce its prevalence. In response to this situation, the Global Virus Network launched a taskforce in 2014 to develop new methods of prevention and treatment of HTLV-1 infection and promote basic research. HTLV-1 is the etiological agent of two life-threatening diseases, adult T-cell leukemia and HTLV-associated myelopathy/tropical spastic paraparesis, for which no effective therapy is currently available. Although the modes of transmission of HTLV-1 resemble those of the more familiar HIV-1, routine diagnostic methods are generally unavailable to support the prevention of new infections. In the present article, the Taskforce proposes a series of actions to expand epidemiological studies; increase research on mechanisms of HTLV-1 persistence, replication and pathogenesis; discover effective treatments; and develop prophylactic and therapeutic vaccines.

Human T-cell leukemia virus type 1 (HTLV-1) Tax affects cellular genomic stability and senescence. As yet, the mechanism(s) for these events caused by Tax is incompletely understood. Here, we show that Tax expression in primary human cells induces reactive oxygen species (ROS), which elicits DNA damage and the expression of senescence marker. Treatment with a ROS scavenger or knockdown of Tax expression by small interfering RNA (siRNA) abrogated Tax-induced DNA damage and the expression of senescence marker. Our data suggest that ROS induction explains Tax-induced cellular DNA damage and cellular senescence.

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus etiologically causal of adult T-cell leukemia (ATL). The virus encodes a Tax oncoprotein that functions in transcriptional regulation, cell cycle control, and transformation. ATL is a highly virulent cancer that is resistant to chemotherapeutic treatments. To understand this disease better, it is important to comprehend how HTLV-1 promotes cellular growth and survival. Tax activation of NF-κB is important for the proliferation and transformation of virus-infected cells. We show here that prolyl isomerase Pin1 is over expressed in HTLV-1 cell lines; Pin1 binds Tax and regulates Tax-induced NF-κB activation.

Infection of T-cells by human T-cell leukemia virus type 1 (HTLV-1) causes a lymphoproliferative malignancy known as adult T-cell leukemia (ATL). ATL is characterized by abnormal lymphocytes, called flower cells, which have cleaved and convoluted nuclei. Tax, encoded by the HTLV-1 pX region, is a critical nonstructural protein that plays a central role in leukemogenesis; however, the mechanisms of HTLV-1 oncogenesis have not been clarified fully. In this review, we summarize current thinking on how Tax may affect ATL leukemogenesis.

Mitotic arrest–deficient protein 1 (MAD1) is a component of the mitotic spindle assembly checkpoint. We have created a knockout mouse model to examine the physiologic consequence of reduced MAD1 function. Mad1+/− mice were successfully generated, but repeated paired mating of Mad1+/− with Mad1+/− mice failed to produce a single Mad1−/− animal, suggesting that the latter genotype is embryonic lethal. In aging studies conducted for >18 months, Mad1+/− mice compared with control wild-type (wt) littermates showed a 2-fold higher incidence of constitutive tumors. Moreover, 42% of Mad1+/− (P < 0.03), but 0% of wt, mice developed neoplasia after treatment with vincristine, a microtubule depolymerization agent. Mad1+/− mouse embryonic fibroblasts (MEF) were found to be more prone than wt cells to become aneuploid; Mad1+/−, but not wt, MEFs produced fibrosarcomas when explanted into nude mice. Our results indicate an essential MAD1 function in mouse development and correlate Mad1 haploinsufficiency with increased constitutive tumors. [Cancer Res 2007;67(1):160–6]

International audience; AIDS vaccines now use a truncated version of 86 residues of the Tat protein related to the HIV-1 HXB2 strain predominant in Europe and North America. We compared antibodies raised in rabbits using a B subtype short Tat HXB2(86) and a full-length Tat HXB2(100). Serum against HXB2(86) recognizes only B and D subtypes while serum against HXB2(100) recognizes B, D, and C subtype variants. Conformational epitopes appear to be involved in the capacity of anti-Tat HXB2 sera to recognized non-homologous Tat variants. A linear B-epitope identified in sequence 71-81 in HXB2(86) disappears in HXB2(100), which has a new linear B-epitope identified at the C-terminus. Anti-HXB2(100) serum has a higher titer in neutralizing antibody against homologous and non-homologous variants compared to anti-HXB2(86) serum. We suggest that a Tat vaccine should contain a Tat variant with regular size, up to 99-101 residues now found in the field.

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 3β (GSK-3β), 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.

Brain-Derived Neurotrophic Factor (BDNF) is a neurotrophin that generally promotes neuronal proliferation, survival and plasticity. These effects occur through cellular secretion and subsequent binding of BDNF to its cognate receptor, TrkB, which promotes paracrine and autocrine signaling cascades. A BDNF/TrkB autocrine loop has been implicated in the survival of cells representing several human cancers and is associated with poor prognosis. The HTLV-1 protein, HBZ, is localized to the nucleus where it is capable of deregulating gene expression. In this study, we found that HBZ increases expression of BDNF. There are a variety of transcriptional variants of BDNF, and HBZ increased expression of several of these. Furthermore, multiple transcriptional variants of BDNF were upregulated in HTLV-1-infected cells compared to normal CD4 cells. Expression of the TrkB receptor was also higher in HTLV-1-infected cells than in normal CD4 cells. This trend similarly occurred when comparing ATL patient and healthy donor specimens, with patient cells exhibiting higher levels of BDNF and TrkB expression. TrkB appeared to localize to the cytoplasmic membrane of HTLV-1-infected cells, suggesting a BDNF/TrkB loop contributes to the survival of these cells. This hypothesis was confirmed by chemically blocking the binding of BDNF to TrkB or by inhibiting TrkB signaling, as both treatments led to a significant increase in apoptosis. Together these results suggest that HBZ activates a BDNF/TrkB autocrine loop that enhances survival of HTLV-1-infected cells. This mechanism may ultimately facilitate the survival of ATL cells.

Epigenetic drugs are known to regulate expression of tumor suppressor genes and activities of transcriptional factors involved in both cancer initiation and progression. Valproate (VPA) has been shown to enhance Tax expression and block HBZ-mRNA expression in HTLV-1 and HAM/TSP lymphocytes. HBZ is critical for immune escape and proliferation of ATL cells. We evaluated the impact of VPA on Tax, Gag and HBZ expression in ex vivo cultured ATL cells. Samples were obtained from 6 patients with acute ATL, 4 patients with HAM/TSP, and 3 asymptomatic carriers (AC). CD8+-cell-depleted PBMCs were cultured during a week with or without 5mM VPA. HTLV-1 mean proviral loads at day 0 of culture were 58 copies/100 cells, 7 copies/ 100 cells, and 4 copies/100 cells in ATL, HAM/TSP, and AC, respectively. At day 5, mean proviral loads decrease by 2 fold in VPA treated vs. untreated ATL samples. Mean±SD apoptosis at day 1 was 11%±6 in untreated versus 23%±11 in VPA-treated non-ATL samples, and 13%±9 in untreated versus 43%±25 in VPA-treated samples. Tax expression in CD4+ cells, measured by Facs and by qRT-PCR, peaked at day 1 of culture in AC and HAM/TSP samples but not in ATL samples. Tax and gag mRNAs were undetectable during culture of ATL untreated samples, whereas HBZ was expressed. VPA treatment of ATL samples significantly increased Tax and Gag mRNA mean levels, and blocked HBZ mRNA expression. The possibility to target HBZ expression using VPA at therapeutically useful concentrations opens an ew way for the treatment of ATL.

Human T-cell leukemia virus type-1 (HTLV-1) is the causative agent of a non-Hodgkin leukemia known as adult T cell leukemia (ATL). MicroRNAs (miRNAs) are a class of non-coding RNAs that regulate the expression of target genes at posttranscriptional level. Biogenesis of miRNAs is tightly regulated and starts with the production of pri-miRNAs by RNA polymerase II. The pri-miRNAs are then cleaved by Drosha in conjunction with DGCR8 in the nucleus, generating pre-miRNAs, which are then transferred to the cytoplasm where Dicer1 further processes the hairpin into the mature miRNAs. With exception of few types of neoplasm, a general downregulation of miRNAs expression has been observed in cancers cells, mainly due to chromosomal abnormalities, epigenetic changes or aberrant expression of the miRNA biogenesis factors. Involvement of miRNAs alteration in the HTLV-1 life cycle has recently come to light. However, the mechanisms that regulate the expression of miRNAs biogenesis factors in ATL cells are still largely unknown. In this study, we assessed Dicer1 expression levels in ATL patients and investigated its transcriptional regulation in HTLV infected cell lines. We reported a significant correlation between reduced miRNA biogenesis and expression of HTLV-1 basic leucine zipper factor (HBZ). We showed that Dicer1 expression was downregulated in ATL patients vs. asymptomatic patients by qRT-PCR and western blot analysis. We also demonstrated that Dicer1 was regulated by AP-1 factors, through luciferase and Chip assays. Our data suggest that Dicer1 expression may be used as a promising prognostic marker and therapeutic target for ATL.

International audience; AIDS in Africa is characterized by the equal distribution of mortality between the two genders because of highly virulent human immunodeficiency virus type 1 (HIV-1) strains. The viral protein Tat trans-activates viral gene expression and is essential for HIV-1 replication. We chemically synthesized six different Tat proteins, with sizes ranging from 86 to 101 residues, from HIV-1 isolates located in different parts of the world including highly virulent African strains. Protein purification, mass spectroscopy, and amino acid analysis showed that the synthesis was successful in each case but with different yields. We show that all have the ability to bind the HIV long terminal repeat (LTR) RNA trans-activation response element (TAR) region, involved in Tat-mediated trans-activation, but structural heterogeneities are revealed by circular dichroism. These Tat synthetic proteins cross membranes but differ in their ability to trans-activate an HIV LTR-reporter gene in stably transfected HeLa cells. Two Tat proteins from virulent African HIV-1 strains were much more active than those from Europe and the United States. The interferon-induced kinase (PKR), involved in cell antiviral defense, phosphorylates only Tat variants corresponding to less or nonvirulent HIV-1 isolates. Our results indicate that the high virulence of some African HIV-1 strains could be related to Tat activity.

International audience; The production of antisense transcripts from the 3 long terminal repeat (LTR) in human T-lymphotropic retroviruses has now been clearly demonstrated. After the identification of the antisense strand-encoded human T-lymphotropic virus type 1 (HTLV-1) bZIP (HBZ) factor, we reported that HBZ could interact with CRE-binding protein (CREB) transcription factors and consequently turn off the important activating potential of the viralTax protein on HTLV-1 5 LTR promoter activity. We have recently accumulated new results demonstrating that antisense transcripts also exist in HTLV-2,-3, and-4. Furthermore, our data have confirmed the existence of encoded proteins from these antisense transcripts (termed antisense proteins of HTLVs or APHs). APHs are also involved in the down-regulation of Tax-dependent viral transcription. In this review, we will focus on the different molecular mechanisms used by HBZ and APH-2 to control viral expression. While HBZ interacts with CREB through its basic zipper domain, APH-2 binds to this cellular factor through a five amino acid motif localized in its carboxyl terminus. Moreover, unlike APH-2, HBZ possesses an N-terminal activation domain that also contributes to the inhibition of the viral transcription by interacting with the KIX domain of p300/CBP. On the other hand, HBZ was found to induce T cell proliferation while APH-2 was unable to promote such proliferation. Interestingly, HTLV-2 has not been causally linked to human T cell leukemia, while HTLV-1 is responsible for the development of the adult T cell leukemia/lymphoma. We will further discuss the possible role played by antisense proteins in the establishment of pathologies induced by viral infection.

Background HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurological inflammatory disease associated with a predominant infiltration of CD4+ T lymphocytes, which are the main subset of HTLV-1-infected cells. It has been demonstrated that in cell line the viral Tax protein transcriptionnally regulate expression of osteopontin, an inflammatory cytokine associated with Th17-related pathologies. Objectives The aim of the study was to explore osteopontin expression in HTLV-1 asymptomatic carriers and in HAM/TSP patients and consequences on IL17 expression. Study design We quantified Tax, osteopontin, RORγ, IL17 and IL22 mRNA expressions in cells from 10 HAM/TSP patients, 6 asymptomatic HTLV-1 carriers (ASY) and 4 HTLV-1-negative healthy donors during ex vivo culture. Results We observed that the expression of osteopontin was higher in HAM/TSP patients and correlated with Tax expression levels. Positive regulation of RORγ, IL17 and IL22 were also observed during cell culture. Conclusions Our results propose a new mechanism which could contribute to HAM/TSP pathogenesis.

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.

A determinant of human T-lymphotropic virus-1 (HTLV-1)–associated myelopathy/tropical spastic paraparesis (HAM/TSP) development is the HTLV-1–infected cell burden. Viral proteins Tax and HBZ, encoded by the sense and antisense strands of the pX region, respectively, play key roles in HTLV-1 persistence. Tax drives CD4+-T cell clonal expansion and is the immunodominant viral antigen recognized by the immune response. Valproate (2-n-propylpentanoic acid, VPA), a histone deacetylase inhibitor, was thought to trigger Tax expression, thereby exposing the latent HTLV-1 reservoir to immune destruction. We evaluated the impact of VPA on Tax, Gag, and HBZ expressions in cultured lymphocytes from HTLV-1 asymptomatic carriers and HAM/TSP patients. Approximately one-fifth of provirus-positive CD4+ T cells spontaneously became Tax-positive, but this fraction rose to two-thirds of Tax-positive–infected cells when cultured with VPA. Valproate enhanced Gag-p19 release. Tax- and Gag-mRNA levels peaked spontaneously, before declining concomitantly to HBZ-mRNA increase. VPA enhanced and prolonged Tax-mRNA expression, whereas it blocked HBZ expression. Our findings suggest that, in addition to modulating Tax expression, another mechanism involving HBZ repression might determine the outcome of VPA treatment on HTLV-1–infected–cell proliferation and survival.

A determinant of HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) development is HTLV-1-infected cell burden. Viral proteins Tax and HBZ, encoded by the positive and negative strands of the pX region respectively, play a key role in HTLV-1 persistence. Tax drives CD4+ T-cell clonal expansion but is the immunodominant antigen. Valproate (VPA), an histone deacetylase inhibitor, has been proposed to trigger Tax expression and expose latent HTLV-1 reservoir to immune destruction. We evaluated the impact of VPA treatment on Tax, Gag and HBZ expression in cultured lymphocytes from asymptomatic HTLV-1 carriers and HAM/TSP patients. Around one-fifth of provirus-positive CD4+ T cells spontaneously became Tax-positive. The estimation rose up to two-thirds of Tax-positive infected cells when VPA was added. VPA enhanced Gag p19 protein release. Tax and Gag mRNA levels spontaneously peaked, before a decline concomitant to HBZ mRNA increase. VPA treatment enhanced and prolonged Tax mRNA expression, while it blocked HBZ expression. This is the first ex vivo study on the balance between Tax and HBZ expression (i.e. sense and antisense transcription). Our data suggest that besides modulation of the expression of Tax, another mechanism involving repression of HBZ may determine the outcome of VPA treatment on HTLV-1-infected cell proliferation and survival.

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.

Nuclear factor kappa B (NF-kappaB) is a key mediator of inflammation. Unchecked NF-kappaB signalling can engender autoimmune pathologies and cancers. Here, we show that Tax1-binding protein 1 (TAX1BP1) is a negative regulator of TNF-alpha- and IL-1beta-induced NF-kappaB activation and that binding to mono- and polyubiquitin by a ubiquitin-binding Zn finger domain in TAX1BP1 is needed for TRAF6 association and NF-kappaB inhibition. Mice genetically knocked out for TAX1BP1 are born normal, but develop age-dependent inflammatory cardiac valvulitis, die prematurely, and are hypersensitive to low doses of TNF-alpha and IL-1beta. TAX1BP1-/- cells are more highly activated for NF-kappaB than control cells when stimulated with TNF-alpha or IL-1beta. Mechanistically, TAX1BP1 acts in NF-kappaB signalling as an essential adaptor between A20 and its targets.

International audience; Replicated mammalian chromosomes condense to segregate during anaphase, and they de-condense at the conclusion of mitosis. Currently, it is not understood what the factors and events are that specify de-condensation. Here, we demonstrate that chromosome de-condensation needs the function of an inner nuclear membrane (INM) protein hsSUN1 and a membrane associated histone acetyltransferase (HAT), hALP. We propose that nascently reforming nuclear envelope employs hsSUN1 and hALP to acetylate histones for de-compacting DNA at the end of mitosis. The eukaryotic nucleus is separated from other organelles by an envelope containing two membrane layers continuous with the endoplasmic reticulum. Nuclear membrane proteins fall into three categories according to their localization. The first group is the trans-nuclear membrane proteins resident in the nuclear pore complex (NPC). 2 The second group contains the inner membrane proteins (INM), which include the lamin B receptor (LBR), emerin, and lamin-associated polypeptides (LAPs). The third group includes proteins underlying the nuclear membrane such as nuclear lamina (1). Functionally, the INM provides a physical barrier; the NPC serves for the transport of material between the nucleus and the cytoplasm (2); and the nuclear lamina erects a meshwork, which maintains nuclear structure and assists indirectly in DNA replication and RNA processing (3, 4). Most INM proteins are associated with the nuclear lam-ina. In a proteomic study of INM proteins, in addition to 13 known proteins, 67 uncharacterized open reading frames (ORFs) were identified (5)

Aneuploidy is frequent in cancers. Recently it was found that pituitary tumor transforming gene (PTTG; also called Pds1p or securin) is overexpressed in many different tumors. Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that primarily infects CD4 + T lymphocytes and causes adult T-cell leukemia. Here, we report that overexpression of human PTTG cooperated with the HTLV-I Tax oncoprotein in cellular transformation. Coexpression of Tax and PTTG enhanced chromosomal instability and neoplastic changes to levels greater than overexpression of either factor singularly. Cells that overexpressed both PTTG and Tax induced tumors more robustly in nude mice than cells that expressed either PTTG alone or Tax alone.

International audience; Human T-cell leukemia virus type 1 is an oncogenic retrovirus etiologically causal of adult T-cell leukemia. The virus encodes a Tax oncoprotein, which functions in transcriptional regulation, cell cycle control, and transformation. Because adult T-cell leukemia is a highly virulent cancer that is resistant to numerous chemotherapeutic treatments, to understand better this disease it is important to comprehend how human T-cell leukemia virus type 1 promotes cellular growth and survival. Most of the existing data point to Tax activation of NF-kappaB as important for cellular proliferation and transformation. We show here that Tax, in the absence of NF-kappaB signaling, can activate activator protein-1 to promote cellular proliferation and survival. Tax is shown to activate activator protein-1 through the phosphatidylinositol 3-kinase/Akt pathway.

International audience; Activation of the nuclear factor kappa B (NF-ϰB) transcription factor family by different stimuli, such as inflammatory cytokines, stress inducers, or pathogens, results in innate and adaptive immunity. While the main function of NF-ϰB is to promote the host's immune response, the NF-ϰB pathway is frequently dysregulated by invading viral pathogens. Human T cell leukemia virus type 1 (HTLV-1) is the causative agent of a fatal malignancy known as adult T cell leukemia (ATL) and an inflammatory disease named tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM). HTLV-1 encodes an oncoprotein, Tax, which plays a significant role in the initiation of cellular transformation and the elicitation of the host's inflammatory responses. Here, we review current thinking on how Tax may affect both diseases through activation of NF-ϰB signaling.

International audience; Human T cell leukemia virus type-1 (HTLV-1) is an oncogenic retrovirus etiologically causal of adult T cell leukemia. The virus encodes a Tax oncoprotein that functions in transcriptional regulation, cell cycle control, and transformation. Because adult T cell leukemia like many other human cancers is a disease of genomic instability with frequent gains and losses of chromosomes, to understand this disease it is important to comprehend how HTLV-1 engenders aneuploidy in host cells. In this regard, loss of cell cycle checkpoints permits tolerance of aneuploidy but does not explain how aneuploidy is created. We show here that HTLV-1 Tax causes abnormal centrosome fragmentation in the mitotic phase of the cell cycle. We report that Tax directly binds Ran and Ran-binding protein-1, locates to centrosomes/spindle poles, and causes supernumerary centrosomes.

Human T-cell leukemia virus type 1 (HTLV-1) encodes a 40-kDa Tax phosphoprotein. Tax is a transcriptional activator which modulates expression of the viral long terminal repeat and transcription of many cellular genes. Because Tax is a critical HTLV-1 factor which mediates viral transformation of T cells during the genesis of adult T-cell leukemia, it is important to understand the processes which can activate or inactivate Tax function. Here, we report that ubiquitination of Tax is a posttranscriptional mechanism which regulates Tax function. We show that ubiquitination does not target Tax for degradation by the proteasome. Rather, ubiquitin addition modifies Tax in a proteasome-independent manner from an active to a less-active transcriptional form.

International audience; Nuclear factor-kappaB essential modulator (NEMO), also called IKKgamma, has been proposed as a 'universal' adaptor of the I-kappaB kinase (IKK) complex for stimuli such as proinflammatory cytokines, microbes, and the HTLV-I Tax oncoprotein. Currently, it remains unclear whether the many signals that activate NF-kappaB through NEMO converge identically or differently. We have adopted two approaches to answer this question. First, we generated and targeted intracellularly three NEMO-specific monoclonal antibodies (mAbs). These mAbs produced two distinct intracellular NF-kappaB inhibition profiles segregating TNFalpha from Tax activation. Second, using NEMO knockout mouse fibroblasts and 10 NEMO mutants, we found that different regions function in trans either to complement or to inhibit dominantly TNFalpha, IL-1beta, or Tax activation of NF-kappaB. For instance, NEMO (1-245 amino acids) supported Tax-mediated NF-kappaB activation, but did not serve TNFalpha- or IL-1beta signaling. Altogether, our findings indicate that while NEMO 'universally' adapts numerous NF-kappaB activators, it may do so through separable domains. We provide the first evidence that selective targeting of NEMO can abrogate oncogenic Tax signaling without affecting signals used for normal cellular metabolism.

International audience; The human immunodeficiency virus type 1 (HIV-1) encodes a potent transactivator, Tat, which functions through binding to a short leader RNA, called transactivation responsive element (TAR). Recent studies suggest that Tat activates the HIV-1 long terminal repeat (LTR), mainly by adapting co-activator complexes, such as p300, PCAF and the positive transcription elongation factor P-TEFb, to the promoter. Here, we show that the proto-oncoprotein Hdm2 interacts with Tat and mediates its ubiquitination in vitro and in vivo. In addition, Hdm2 is a positive regulator of Tat-mediated transactivation, indicating that the transcriptional properties of Tat are stimulated by ubiquitination. Fusion of ubiquitin to Tat bypasses the requirement of Hdm2 for efficient transactivation, supporting the notion that ubiquitin has a non-proteolytic function in Tat-mediated transactivation.

The HIV-1 transactivator protein, Tat, is an atypical transcriptional activator that functions through binding, not to DNA, but to a short leader RNA, TAR. Although details of its functional mechanism are still unknown, emerging findings suggest that Tat serves primarily to adapt co-activator complexes such as p300, PCAF and P-TEFb to the HIV-1 long terminal repeat. Hence, an understanding of how Tat interacts with these cofactors is crucial. It has recently been shown that acetylation at a single lysine, residue 50, regulated the association of Tat with PCAF. Here, we report that in the absence of Tat acetylation, PCAF binds to amino acids 20–40 within Tat. Interestingly, acetylation of Tat at Lys28 abrogates Tat–PCAF interaction. Acetylation at Lys50 creates a new site for binding to PCAF and dictates the formation of a ternary complex of Tat–PCAF–P-TEFb. Thus, differential lysine acetylation of Tat coordinates the interactions with its co-activators, cyclin T1 and PCAF. Our results may help in understanding the ordered recruitment of Tat co-activators to the HIV-1 promoter.

International audience; Clinical studies show that in the absence of anti-retroviral therapy an immune response against the human immunodeficiency virus type 1 (HIV-1), transacting transcriptional activator (Tat) protein correlates with long term non-progression. The purpose of this study is to try to understand what can trigger an effective immune response against Tat. We used five Tat variants from HIV strains identified in different parts of the world and showed that mutations of as much as 38% exist without any change in activity. Rabbit sera were raised against Tat variants identified in rapid-progressor patients (Tat HXB2, a European variant and Tat Eli, an African variant) and a long term non-progressor patient (Tat Oyi, an inactive African variant). Enzyme-linked immunosorbent assay (ELISA) results showed that anti-Tat Oyi serum had the highest antibody titer and was the only one to have a broad antibody response against heterologous Tat variants. Surprisingly, Tat HXB2 was better recognized by anti-Tat Oyi serum compared with anti-Tat HXB2 serum. Western blots showed that non-homologous Tat variants were recognized by antibodies directed against conformational epitopes. This study suggests that the primary and tertiary structures of the Tat variant from the long term non-progressor patient are critical to the induction of a broad and effective antibody response against Tat.