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HTLV-1 is more pathogenic than HTLV-2 despite having a similar genome and closely related transactivating oncoproteins. Both Tax-1 protein from HTLV-1 and Tax-2 from HTLV-2 activate the NF-κB pathway. The mechanisms involved in Tax-1 deregulation of this signalling pathway have been thoroughly investigated, but little is known about regulation by Tax-2. We have compared the interaction of Tax-1 and Tax-2 with two key NF-κB signalling factors: TAK1-binding protein 2 (TAB2), an adaptor involved in the activation of TAK1 kinase, and RelA, the active subunit of the canonical RelA/p50 NF-κB transcription factor. Tax-2 formed stable complexes with both RelA and TAB2. These two NF-κB factors colocalized with Tax proteins in dotted cytoplasmic structures targeted by calreticulin, a multi-process calcium-buffering chaperone. Co-expression of RelA and/or TAB2 markedly increased Tax-mediated NF-κB activation. These findings provide new insights into the role of RelA, TAB2 and Tax in the deregulation of the NF-κB pathway.

The human T-cell leukemia virus type 1 (HTLV-1) Tax transactivator is known to induce or repress various cellular genes, several of them encoding transcription factors. As Tax is known to deregulate various basic bHLH factors, we looked more specifically at its effect on TAL1 (T-cell acute lymphoblastic leukemia 1), also known as SCL (stem cell leukemia). Indeed, TAL1 is deregulated in a high percentage of T-cell acute lymphoblastic leukemia cells, and its oncogenic properties are well-established. Here we show that Tax induces transcription of this proto-oncogene by stimulating the activity of the TAL1 gene promoter 1b, through both the CREB and NF-κB pathways. It was also observed that TAL1 upregulates HTLV-1 promoter activity, in either the presence or the absence of Tax. The viral promoter is inhibited in trans by expression of the E2A protein E47, and TAL1 is able to abrogate this inhibition. These data show the existence of a positive feedback loop between Tax and TAL1 expression and support the notion that this proto-oncogene participates in generation of adult T-cell leukemia/lymphoma by increasing the amount of the Tax oncoprotein but also possibly by its own transforming activities.

IKKalpha regulates many chromatin events in the nuclear phase of the NF-kappaB program, including phosphorylation of histone H3 and removal of co-repressors from NF-kappaB-dependent promoters. However, all of the nuclear functions of IKKalpha are not understood. In this study, using mouse embryonic fibroblasts IKKalpha knock-out and reexpressing IKKalpha after retroviral transduction, we demonstrate that IKKalpha contributes to NF-kappaB/p65 DNA binding activity on an exogenous kappaB element and on some, but not all, endogenous NF-kappaB-target promoters. Indeed, p65 chromatin immunoprecipitation assays revealed that IKKalpha is crucial for p65 binding on kappaB sites of icam-1 and mcp-1 promoters but not on ikappabalpha promoter. The mutation of IKKalpha putative nuclear localization sequence, which prevents its nuclear translocation, or of crucial serines in the IKKalpha activation loop completely inhibits p65 binding on icam-1 and mcp-1 promoters and rather enhances p65 binding on the ikappabalpha promoter. Further molecular studies demonstrated that the removal of chromatin-bound HDAC3, a histone deacetylase inhibiting p65 DNA binding, is differentially regulated by IKKalpha in a promoter-specific manner. Indeed, whereas the absence of IKKalpha induces HDAC3 recruitment and repression on the icam-1 promoter, it has an opposite effect on the ikappabalpha promoter, where a better p65 binding occurs. We conclude that nuclear IKKalpha is required for p65 DNA binding in a gene-specific manner.

The human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia and a virus-associated neurodegenerative disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (31, 36, 47). These two diseases have been linked to the expression of the HTLV-1 regulatory protein Tax (10, 13, 29, 41). Tax is a potent transcriptional activator of viral genes as well as specific cellular genes and has a clear oncogenic potential since Tax is able to transform T lymphocytes and fibroblasts and induces tumors in transgenic mice (19). A substantial part of the oncogenic properties of Tax is associated with its ability to activate the expression of cellular genes that control T-cell proliferation and differentiation by inducing constitutive activation of the NF-κB pathway (2, 38). In nonstimulated cells, inactive NF-κB complexes composed of p50 and RelA heterodimers are retained in the cytoplasm by NF-κB inhibitors of the IκB family (15, 23, 40). Tax activation of the NF-κB pathway involves its interaction with the regulatory subunit of the IκB kinase (IKK) complex, NEMO/IKKγ, leading to the activation of the two catalytic subunits, IKKα and IKKβ (8, 14). Activation of the IκB kinase complex by Tax determines the phosphorylation of the IκB proteins, leading to their ubiquitination and degradation by the proteasome and to the migration of the NF-κB complexes to the nucleus (18, 45, 46). It also determines the phosphorylation of the RelA subunit of NF-κB, a prerequisite for activation of RelA and p50 heterodimers in the nucleus (30). However, Tax also colocalizes in discrete nuclear bodies (NB) with the two subunits of NF-κB, p50 and RelA, in addition to RNA polymerase II, and the assembly of these nuclear structures correlates with Tax transcriptional activity (4-6, 37). Thus, Tax-mediated activation of gene expression may require the presence of Tax both in the cytoplasm and in the nucleus (3). Ubiquitin (Ub) and the small ubiquitin-like modifier (SUMO) are polypeptides which are attached to the lysine residues of a number of proteins via isopeptidic bonds, leading to the subsequent formation of poly-ubiquitinated or poly-sumoylated branched molecules. Ubiquitination and sumoylation often compete for the same lysine residues and have opposite effects in regulating the function of a variety of transcription factors by altering their intracellular targeting, their interaction with specific partners, and/or their stability (9, 22, 24, 27). Although poly-ubiquitinated proteins are often targeted to the proteasome, SUMO modification results in the targeting to subnuclear domains called nuclear bodies, as was first observed for the promyelocytic leukemia protein (PML) (27, 48). Activation of the NF-κB pathway following genotoxic stress requires the sequential sumoylation and nuclear targeting of IKKγ, followed by its ubiquitination, permitting IKK activation in the cytoplasm (16). Tax is ubiquitinated on carboxy-terminal lysine residues, and this modification is critical for Tax interaction with the proteasome (7, 34). In this work, we demonstrate that Tax is sumoylated and ubiquitinated on overlapping lysine residues. These two modifications determine the partitioning of Tax in the nuclear and cytoplasmic compartments. Colocalized Tax and ubiquitin molecules were detected in the cytoplasm only, in association with IKK complexes, and this modification plays a critical role in the translocation of the RelA subunit of NF-κB to the nucleus. However, the sumoylation of Tax and the subsequent formation of the Tax nuclear bodies that contained both RelA and IKKγ must also occur for Tax-mediated activation of gene expression via the NF-κB pathway. These results demonstrate that both the sumoylation and the ubiquitination of Tax are critical for Tax-mediated transcriptional activity.

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] 3-kinase catalyses the phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate. cDNAs encoding three human isoenzymes of InsP3 3-kinase (A, B and C) have been reported previously [Choi, Kim, Lee, Moon, Sim, Kim, Chung and Rhee (1990) Science 248, 64–66; Dewaste, Pouillon, Moreau, Shears, Takazawa and Erneux (2000) Biochem. J. 352, 343–351; Dewaste, Roymans, Moreau and Erneux (2002) Biochem. Biophys. Res. Commun. 291, 400–405; Takazawa, Perret, Dumont and Erneux (1991) Biochem. Biophys. Res. Commun. 174, 529–535]. The localization of InsP3 3-kinase isoenzymes fused at their N-terminus to the green fluorescent protein has been studied by confocal microscopy. The A isoform appeared to associate with the cytoskeleton, whereas the C isoform was totally cytoplasmic. The B isoform had a more complex localization: it appeared in the plasma membrane, cytoskeleton and in the endoplasmic reticulum. The three human isoenzymes of InsP3 3-kinase can thus be distinguished by their N-terminal sequence, sensitivity to Ca2+/calmodulin and localization on transfection in COS-7 cells. We have compared the cytosolic Ca2+ responses induced by ATP in COS-7 cells transfected with the three isoenzymes. Cells expressing high levels of any of the three isoforms no longer respond to ATP, whereas cells expressing low levels of each enzyme showed a reduced response consisting of one to three Ca2+ spikes in response to 100 μM ATP. These effects were seen only in wild-type InsP3 3-kinase-transfected cells. 3-Kinase-dead mutant cells behaved as vector-transfected cells. The results highlight the potential role of the three isoforms of InsP3 3-kinase as direct InsP3 metabolizing enzymes and direct regulators of Ca2+ responses to extracellular signals.

The human T cell leukemia/lymphotropic virus type 1 (HTLV-1) Tax protein strongly activates viral and cellular gene transcription. It mainly functions by interacting with cellular transcription factors and the KIX domain of the p300/CBP coactivators. Tax can also repress the transcription of cellular genes through the basic helix-loop-helix (bHLH) protein family. To investigate the molecular mechanisms of this Tax-mediated inhibition, we analyzed its effect on the transcriptional activity of the myogenic MyoD protein, which was used as a paradigm of bHLH factors. In this study, we show that overexpression of the p300 coactivator in transient transfection assays was sufficient to rescue MyoD repression by Tax. Furthermore, an N-terminal domain of p300 (amino acids 379-654) containing the region of KIX serving as the Tax binding site was found, when overexpressed, to potentiate Tax-mediated transactivation of HTLV-1 proviral as well as MyoD-dependent transcription, and to antagonize the inhibition by Tax of the transcriptional activity of MyoD. These results revealing the presence of an N-terminal MyoD binding site were confirmed by in vitro protein-protein interaction assays that demonstrate that MyoD binds to the KIX domain of p300 and that Tax competes with MyoD binding in a nonreciprocal manner. These observations provide evidence that Tax binding to the KIX domain of p300 prevents bHLH proteins from contacting this N-terminal domain of the coactivator, thus resulting in their transcriptional repression. As bHLH proteins are implicated in many developmental fate decisions, especially during thymopoiesis, Tax-mediated inhibition of their transcriptional activity may contribute to the induction of HTLV-1-linked leukemogenesis.

The Tax transactivator protein of human T-cell leukemia virus type 1 (HTLV-1) plays a central role in the activation of viral gene expression. In addition, Tax is capable of activating the expression of specific cellular genes and is involved in the transformation of T-lymphocytes resulting in the development of adult T-cell leukemia. Tax is a phosphoprotein that colocalizes in nuclear bodies with RNA polymerase II, splicing complexes, and specific transcription factors including members of the ATF/CREB and NF-κB families. In this study, we identified adjacent serine residues at positions 300 and 301 in the carboxy terminus of Tax as the major sites for phosphorylation. Phosphorylation of at least one of these serine residues is required for Tax localization in nuclear bodies and for Tax-mediated activation of gene expression via both the ATF/CREB and NF-κB pathways. Introduction of amino acid substitutions which are phosphoserine mimetics at positions 300 and 301 restored the ability of a phosphorylation-defective Tax mutant to form nuclear bodies and to activate gene expression. These studies define sites for regulatory phosphorylation events in Tax which are critical for its ability to activate gene transcription.

Transformation by the Tax oncoprotein of the human T cell leukemia virus type 1 (HTLV-1) is governed by actions on cellular regulatory signals, including modulation of specific cellular gene expression via activation of signaling pathways, acceleration of cell cycle progression via stimulation of cyclin-dependent kinase activity leading to retinoblastoma protein (pRb) hyperphosphorylation and perturbation of survival signals. These actions control early steps in T cell transformation and development of Adult T cell leukemia (ATL), an aggressive malignancy of HTLV-1 infected T lymphocytes. Post-translational modifications of Tax by phosphorylation, ubiquitination, sumoylation and acetylation have been implicated in Tax-mediated activation of the NF-κB pathway, a key function associated with Tax transforming potential., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

Immunotherapy by adoptive transfer of lymphocytes was attempted in identical twins, one who was virus-free and the other who was infected with human immunodeficiency virus-1 (HIV-1), at the stage of acquired immunodeficiency syndrome. The noninfected twin was vaccinated by priming with a recombinant vaccinia virus expressing the envelope glycoprotein of one of his brother's viruses and boosting with the same purified gp160 adsorbed on alum. Vaccination elicited major histocompatibility complex class I-restricted CD8+ cytolytic T lymphocytes specific for HIV-1, but no antibody response. The diseased brother, a 38-year-old homosexual who had developed repeated opportunistic infections since 1990 and had a CD4+ count reduced to practically zero, was treated by infusions of lymphocytes collected from the vaccinated brother by lymphopheresis. After a first transfer of the whole lymphocyte population, no changes were observed in the clinical status and biologic or virologic parameters. A second transfer was then applied with activation of the cells with purified envelope glycoprotein before infusion. The outcome of the treatment was an increase in total lymphocytes, in CD4+ and activated CD8+ DR+ cell counts, and in proliferative responses to HIV antigens. A marked but transient 3-log increase in cellular and plasmatic virus loads was also observed after the second adoptive transfer. These observations will be considered with attention to improve the future adoptive transfer protocols, especially in patients with severe CD4+ depletion.

The Tax oncoprotein of HTLV-1 initiates T-cell transformation by dysregulating cell cycle progression and inhibiting DNA damage responses. The subsequent genomic instability might result in constitutive activation of the NF-κB pathway observed in HTLV-1-transformed T lymphocytes. Our previous results indicated that differential modifications of Tax by ubiquitination or sumoylation controlled its retention either in the cytoplasm or in the nucleus, respectively. Here we show that Tax is targeted to pre-existing punctate cytoplasmic structures which contain the TNF-receptor associated protein 2 (TAB2). Colocalization of Tax with TAB2 in these cytoplasmic structures induced the recruitment of additional components involved in the NF-κB activation cascade, including the regulatory subunit of the IKK complex, IKKgamma/NEMO, the RelA subunit of NF-κB and TAX1-BP1, which are Tax interacting partners. Overexpression of TAB2 strongly stimulated Tax-mediated activation of the NF-κB pathway indicating that the concentration of these NF-κB factors in Tax-containing cytoplasmic punctate structures is a step important for activation of the NF-κB pathway by Tax. Interestingly, calreticulin, a multifunctional calcium buffering chaperone previously involved in the nucleocytoplasmic transport of nuclear receptors such as glucocorticoid and thyroid receptors, was also recruited by Tax in these cytoplasmic structures. The fact that overexpressed IKKgamma concentrated in nuclear foci that included calreticulin and that co-expression of Tax and IKKgamma led to the redistribution of both IKKgamma and calreticulin from the nucleus to the cytoplasm suggested that calreticulin might be involved in the nuclear export of these factors and their recruitment in TAB2-containing cytoplasmic punctate structures. The possibility that Tax activates the NF-κB pathway via initiation of events in the nucleus leading to the transport of IKKgamma from the nucleus to the cytoplasm will be discussed.

The oncogenic protein BCL-3 activates or represses gene transcription through binding with the NF-κB proteins p50 and p52 and is degraded through a phospho- and GSK3-dependent pathway. However, the mechanisms underlying its degradation remain poorly understood. Yeast two-hybrid analysis led to the identification of the proteasome subunit PSMB1 as a BCL-3-associated protein. The binding of BCL-3 to PSMB1 is required for its degradation through the proteasome. Indeed, PSMB1-depleted cells are defective in degrading polyubiquitinated BCL-3. The N-terminal part of BCL-3 includes lysines 13 and 26 required for the Lys48-linked polyubiquitination of BCL-3. Moreover, the E3 ligase FBW7, known to polyubiquitinate a variety of substrates phosphorylated by GSK3, is dispensable for BCL-3 degradation. Thus, our data defined a unique motif of BCL-3 that is needed for its recruitment to the proteasome and identified PSMB1 as a key protein required for the proteasome-mediated degradation of a nuclear and oncogenic IκB protein. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc., SCOPUS: ar.j, info:eu-repo/semantics/published

Sixteen children over the age of 5 years (Group 1) have been identified out of 537 children infected by human immunodeficiency virus and born to HIV-infected mothers, in Kigali, Rwanda. They were followed up for 2 years and compared with 16 younger AIDS patients (Group 2) and with 16 age- and gender-matched HIV-1 seronegative children (Group 3). Fourteen Group 1 subjects had anti-HIV-1 IgM which persisted during the entire study period, in 11 cases directed to HIV-1 envelope proteins. In vitro, immortalization of B lymphocytes by the Epstein-Barr virus confirmed a high production of IgM to envelope proteins. All these patients had anti-p 17 IgG which was not observed in 7 patients from Group 2. All 16 children mounted significant titers of neutralizing antibodies to HTLV-IIIB, and, in 8 patients tested, against two other HIV-1 strains, RII and MN. HIV-1-specific major histocompatibility complex (MHC)-restricted cytotoxic T cells were demonstrated in 3 of 5 of the subgroup who were tested. Prolonged survival over 5 years in children with maternally acquired HIV-1 infection is associated with a high titer of neutralizing antibodies, a persistent production of IGM to HIV-1 envelope proteins and of IgG to p 17.

The oncogenic potential of the HTLV-1 Tax protein involves activation of the NF-kappaB pathway, which depends on Tax phosphorylation, ubiquitination and sumoylation. We demonstrate that the nuclei of Tax-expressing cells, including HTLV-1 transformed T-lymphocytes, contain a pool of Tax molecules acetylated on lysine residue at amino acid position 346 by the transcriptional coactivator p300. Phosphorylation of Tax on serine residues 300/301 was a prerequisite for Tax localization in the nucleus and correlated with its subsequent acetylation by p300, whereas sumoylation, resulting in the formation of Tax nuclear bodies in which p300 was recruited, favored Tax acetylation. Overexpression of p300 markedly increased Tax acetylation and the ability of a wild type HTLV-1 provirus, but not of a mutant provirus carrying an acetylation deficient Tax gene, to activate gene expression from an integrated NF-kappaB-controlled promoter. Thus, Tax acetylation favors NF-kappaB activation and might play an important role in HTLV-1-induced cell transformation.

Actin disruption by CytochalasinD (CytD) and LatrunculinB (LatB) induced NF-kappaB activation in myelomonocytic and intestinal epithelial cells. In an attempt to elucidate the mechanism by which actin disruption induced IKK activation, we studied the human Nod2 protein, which was able to induce NF-kappaB activation and whose expression was restricted to myelomonocytic and intestinal epithelial cells. Nod2 is thought to play key roles in pathogen defence through sensing bacteria and generating an inflammatory immune response. We showed that actin disruption by CytD significantly and specifically increased Nod2-mediated NF-kappaB signaling. Nod2 was fully partitioned in the Triton-X-100-insoluble fraction but translocated into the soluble fraction after CytD treatment, demonstrating that the presence of Nod2 in the detergent-insoluble pellet was specific to actin cytoskeleton. Confocal analysis also revealed a Nod2 colocalization with membrane-associated F-actin. Colocalization and co-immunoprecipitation assays with endogenous Rac1 have shown that Nod2 associated with activated Rac1 in membrane ruffles through both its N-terminal caspase recruitment domains (CARD) and C-terminal leucine-rich repeats (LRRs). Membrane ruffle disruption by a Rac1 dominant negative form primed Nod2-dependent NF-kappaB signaling. The recruitment of Nod2 in Rac-induced dynamic cytoskeletal structures could be a strategy to both repress the Nod2-dependent NF-kappaB signaling in unstimulated cells and rapidly mobilize Nod2 during bacterial infection.

Human papillomavirus type 16 is commonly implicated in cervical cancers. The viral genome encodes potential targets like the oncoprotein E7, expressed in transformed cells but thought to represent a poorly immunogenic antigen. We describe in this work a DNA-based vaccination protocol aimed at inducing an efficient anti-E7 immune response in vivo. Plasmids allowing the expression of the E7 protein in distinct cellular compartments were generated and assayed in an in vivo model of tumor growth. Our data demonstrate that mice vaccinated with a plasmid encoding for an E7 protein fused to a domain of the MHC class II-associated invariant chain (IiE7) were protected against tumor challenge. Mice immunized against an ubiquitinated form of E7 (Ub(Ala)E7) failed to control tumor growth. Protection induced by IiE7 was correlated with the development of CD8+ CTL and required the presence of CD4+ cells. In vitro studies confirmed that the IiE7 fusion protein was expressed at high levels in the endosomal compartment of transfected cells, while the natural and the ubiquitin-modified form of E7 were mainly nuclear. The present study suggests that an efficient anti-tumor response can be induced in vivo by DNA constructs encoding for E7 protein forms localizing at the endosomal compartment.

The human T-cell leukemia virus HTLV-1 encodes regulatory proteins, Tax, Rex and p30II, which are involved in the control of viral gene expression at the transcriptional and post-transcriptional levels. Tax localizes in unique nuclear bodies that contain components of the transcription and splicing complexes. In this work, we studied the relative intracellular localizations of Tax, Rex and p30II. Run-on transcription assays and immunocytochemistry at light and electron microscopy levels indicated that the Tax nuclear bodies included both de novo transcribed RNA and the RNA polymerase II form that is phosphorylated on its carboxy-terminal domain whereas contacts with chromatin were observed at the periphery of these nuclear bodies. Rex first accumulated in nucleolar foci and then spread across the whole nucleus to display a diffuse and punctuate nucleoplasmic distribution. This distribution of Rex was observed in HTLV-1 transformed lymphocytes and in COS cells expressing the HTLV-1 provirus. Rex colocalized with the cellular export factor CRM-1 in the nucleolar foci as well as in the nucleoplasmic foci that did not overlap with Tax nuclear bodies but were found at the boundaries of the Tax bodies. In addition, we demonstrate that p30II interacts with Rex and colocalizes with the Rex/CRM-1 complexes in the nucleoli leading to their clearance from the nucleoplasm. Our results suggest that transcripts originating from Tax-induced activation of gene expression at the boundaries of the Tax bodies are transported out of the nucleus by nucleoplasmic Rex/CRM-1 complexes that are first assembled in nucleolar foci. In addition, p30II might exert its negative effect on viral RNA transport by preventing the release of the Rex/CRM-1 complexes from sequestration in nucleolar foci. These data support the idea that the transcriptional and post-transcriptional regulation of HTLV-1 gene expression depends on the concentration of select regulatory complexes at specific area of the nucleus.

Human T-cell leukemia virus type 1 (HTLV-1) is the retrovirus responsible for adult T-cell leukemia and HTLV-1-associated myelopathy. Adult T-cell leukemia development is mainly due to the ability of the viral oncoprotein Tax to promote T-cell proliferation, whereas the appearance of HTLV-1-associated myelopathy involves the antigenic properties of Tax. Understanding the events regulating the intracellular level of Tax is therefore an important issue. How Tax is degraded has not been determined, but it is known that Tax binds to proteasomes, the major sites for degradation of intracellular proteins, generally tagged through polyubiquitin conjugation. In this study, we investigated the relationship between Tax, ubiquitin, and proteasomes. We report that mono- and polyubiquitinated Tax proteins can be recovered from both transfected 293T cells and T lymphocytes. We also show that lysine residues located in the carboxy-terminal domain of Tax are the principal targets of this process. Remarkably, we further demonstrate that mutation of lysine residues in the C-terminal part of Tax, which massively reduces Tax ubiquitination, impairs proteasome binding, and conversely, that a Tax mutant that binds poorly to this particle (M22) is faintly ubiquitinated, suggesting that Tax ubiquitination is required for association with cellular proteasomes. Finally, we document that comparable amounts of ubiquitinated species were found whether proteasome activities were inhibited or not, providing evidence that they are not directly addressed to proteasomes for degradation. These findings indicate that although it is ubiquitinated and binds to proteasomes, Tax is not massively degraded via the ubiquitin-proteasome pathway and therefore reveal that Tax conjugation to ubiquitin mediates a nonproteolytic function.

The genetic make-up of a cell resides entirely in its DNA. Now that the nucleotide sequence of several genomes has been determined, the major challenging problem is to understand how cell differentiation, proliferation or death are controlled. Major steps include analysis of the determinants of the cell cycle, the unravelling of RNAs and proteins involved in the control of gene expression and the dissection of the protein-destruction machinery. The successive steps to be considered are transcription of RNA on the DNA template, mRNA stabilization or degradation, and mRNA translation and protein localization in the right cell compartment. Gene expression or gene silencing is the result of many DNA-RNA-protein interactions and chromatin is among the key regulators of gene expression. Open chromatin (euchromatin) allows expression of the DNA message. This chromatin structure is generally characterized by the presence on the gene promoters of transcription complexes associated with histone acetyltransferases (HATs). On the contrary, closed chromatin (heterochromatin) is poorly acetylated and more condensed. It contains histone deacetylases (HDACs), potentially associated with DNA methyltransferases (DNMTs). DNMT activity leads to methylation and silencing of the DNA. Thus, a major problem in the field of gene regulation resides in understanding chromatin structure at each promoter, a formidable task for the years to come.

IFN-alpha and high dose ascorbic acid (AA) have a modest clinical benefit in HAM/TSP (Nakagawa, 1996). We investigated the effect of ex vivo and in vitro AA and IFN-alpha treatment on HAM/TSP PBMC and HTLV-1-infected cell lines, respectively. We treated cells for 48-72h ex vivo and in vitro with low-high (10-100µg/ml) dose of AA and 1000U/ml of IFN-alpha and quantified lymphoproliferation by [3H]thymidine incorporation, tetraploid DNA content and PCNA expression (flow cytometry). Viral expression was measured at the RNA (tax, LTR) and protein (Tax, p19) level by RT-PCR, Western blot and ELISA, respectively. Apoptosis was quantified by subdiploid DNA content (flow cytometry). Th1/Th2/Th17 cytokines were quantified by cytometric bead array. AA induced a dramatic 95% decrease (control 3689±755 cpm vs. AA 121±52 cpm, p=0.001) in spontaneous, virus-driven lymphoproliferation and a decrease in tax and LTR transcription in HAM/TSP PBMC. In addition, AA decreased the exacerbated ex vivo IFN-gamma production in HAM/TSP PBMC. In HTLV-1 infected cell lines (MT-2 and MT-4), AA induced a dose-dependent increase in DNA fragmentation (p=0.02, p=0.005), paralleled by a decrease in PCNA (p=0.003), as well as p19 (p

A mutant of Saccharomyces cerevisiae, unable to grow at the expense of non fermentable carbon sources at 37°C, has been selected; at 25°C the mutant strain behaves like the parental wild strain. Evaluations of respiration rates during aerobic growth at restrictive temperature on one hand, enzymatic and/or spectral evaluations of the individual components of the respiratory chain on the other hand show that the respiratory deficiency is specifically correlated with a reduced level of cytochrome oxidase. The decrease of enzyme activity is the direct consequence of a lowering of hemoprotein (a, a3) concentration. Temperature-activity relationship of cytochrome oxidase elaborated at the permissive temperature by the mutant strain is modified as far as the particulate enzyme is concerned, but no difference is observed after partial solubilization of the enzyme by non ionic surfactant. Genetic analysis shows that the mutant phenotype results from a nuclear gene mutation. © 1977 Masson, Paris., SCOPUS: ar.j, info:eu-repo/semantics/published

HTLV-1 is more pathogenic than HTLV-2B. The difference is generally attributed to the properties of their individual transactivating Tax proteins. By using internal Flag-6His tagged Tax-1 and Tax-2B, which display transcriptional activities comparable to the untagged proteins and can be recognized by a single anti-Flag antibody, we demonstrate that Tax-2B is modified by ubiquitination and sumoylation. In addition, Tax2B is distributed in punctuate nuclear structures that include the RelA subunit of NF-κB, as has been previously demonstrated for Tax-1.

The elements which ensure the maintenance of the F plasmid are located in its f5 EcoRI restriction fragment. This f5 fragment constitutes a mini-F plasmid showing the same stability and copy number control as the entire F plasmid. The proteins expressed in minicells by wild-type or mutated f5 fragments were analysed by pH gradient two-dimensional electrophoresis. We identified seven f5-encoded polypeptides and located their genes on the F map. Among them, H1, an acidic polypeptide of mol. wt. 10.5 K, had not been detected before. It is in fact the most abundant f5-encoded polypeptide identified so far. In addition, we showed that both 10.5-K and 12-K protein bands detected by SDS-polyacrylamide gel electrophoresis are, respectively, composed of two polypeptides, H1 and H2, G1 and G2, of different isoelectric points. Polypeptides H2 and G2, respectively, share common coding sequences with polypeptides H1 and G1. Their possible biological significance is discussed. The sequences coding for polypeptides H1/H2 and G1/G2 are clustered in a 800-bp long region located between the two mini-F origin sites and are proposed to be organized as an operon. The results reported in the accompanying paper point out the importance of polypeptides G1/G2 and H1/H2 in the relationship between the F plasmid and its host.

SCOPUS: ar.j, info:eu-repo/semantics/published

The trophoblastic cell represents the main functional unit of the placenta. It proliferates, migrates, and invades the maternal tissue in a way that is similar to malignant tumors. Nevertheless, these processes are tightly controlled by stringent spatial and temporal confines. Therefore, the trophoblastic cell, as 'a well-behaved tumor', represents an ideal model system to investigate several oncogenic processes. Several studies reported that HPV viruses could infect trophoblasts during pregnancies. Surprisingly, HPV can replicate in vitro in trophoblasts. Higher HPV infection frequency has been reported to be associated with some spontaneous abortion and gestational trophoblastic diseases.

The functional analysis of regulatory proteins Tax-1 and Tax-2 can provide useful information to further understand the difference in pathogenicity between HTLV-1 and HTLV-2. Tax-1 molecules phosphorylated at serine residues 300 and 301, ubiquitinated or sumoylated at lysine residues 280 and 284 and acetylated at lysine 346 have been identified. These modifications control Tax-1 intracellular localization, the formation of Tax-1 nuclear bodies and sequential steps in Tax-1-mediated activation of the NF-κB pathway, a critical event thought to be involved in HTLV-1 transforming capacity [1]. By comparing internally tagged Tax-1 and Tax-2B, we demonstrated that Tax-2B activates gene expression via the NF-κB pathway, is present in nuclear bodies and in the cytoplasm and is modified by ubiquitination and sumoylation similarly to its homologue Tax-1 [2]. In the present study, we constructed a series of Tax-2B mutants with substitutions of specific lysine residues by arginines. The post-translational modifications, subcellular localization and capacity to activate gene expression of these Tax-2B mutants will be compared to those obtained for the corresponding mutants of Tax-1.

Background: Retroviruses HTLV-1 and HTLV-2 have homologous genomic structures but differ significantly in pathogenicity. HTLV-1 is associated with Adult T cell Leukemia (ATL), whereas infection by HTLV-2 has no association with neoplasia. Transformation of T lymphocytes by HTLV-1 is linked to the capacity of its oncoprotein Tax-1 to alter cell survival and cell cycle control mechanisms. Among these functions, Tax-1-mediated activation of cellular gene expression via the NF-κB pathway depends on Tax-1 post-translational modifications by ubiquitination and sumoylation. The Tax-2 protein of HTLV-2B (Tax-2B) is also modified by ubiquitination and sumoylation and activates the NF-κB pathway to a level similar to that of Tax-1. The present study aims to understand whether ubiquitination and sumoylation modifications are involved in Tax-2B-mediated activation of the NF-κB pathway.Results: The comparison of Tax-1 and Tax-2B lysine to arginine substitution mutants revealed conserved patterns and levels of ubiquitination with notable difference in the lysine usage for sumoylation. Neither Tax-1 nor Tax-2B ubiquitination and sumoylation deficient mutants could activate the NF-κB pathway and fusion of ubiquitin or SUMO-1 to the C-terminus of the ubiquitination and sumoylation deficient Tax-2B mutant strikingly restored transcriptional activity. In addition, ubiquitinated forms of Tax-2B colocalized with RelA and IKKγ in prominent cytoplasmic structures associated with the Golgi apparatus, whereas colocalization of Tax-2B with the RelA subunit of NF-κB and the transcriptional coactivator p300 in punctate nuclear structures was dependent on Tax-2B sumoylation, as previously observed for Tax-1.Conclusions: Both Tax-1 and Tax-2 activate the NF-κB pathway via similar mechanisms involving ubiquitination and sumoylation. Therefore, the different transforming potential of HTLV-1 and HTLV-2 is unlikely to be related to different modes of activation of the canonical NF-κB pathway. © 2012 Turci et al. licensee BioMed Central Ltd., SCOPUS: ar.j, info:eu-repo/semantics/published