Françoise Bex - Academia.edu (original) (raw)

Papers by Françoise Bex

Journal of Biological Chemistry

The human T cell leukemia/lymphotropic virus type 1 (HTLV-1) Tax protein strongly activates viral... more 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 t...

Viruses, 2011

The HTLV-1 Tax protein both activates viral replication and is involved in HTLV-1-mediated transf... more The HTLV-1 Tax protein both activates viral replication and is involved in HTLV-1-mediated transformation of T lymphocytes. The transforming properties of Tax include altering the expression of select cellular genes via activation of cellular pathways and perturbation of both cell cycle control mechanisms and apoptotic signals. The recent discovery that Tax undergoes a hierarchical sequence of posttranslational modifications that control its intracellular localization provides provocative insights into the mechanisms regulating Tax transcriptional and transforming activities.

Retrovirology, 2005

In this study, we have examined the role of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1)... more In this study, we have examined the role of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) in viral gene expression in T lymphocytes transformed by HTLV-1. We have previously observed that hnRNP A1 (A1) down-modulates the post transcriptional activity of Rex protein of HTLV-1. Here, we tested whether the ectopic expression of a dominant negative mutant (NLS-A1-HA) defective in shuttling activity or knockdown of the hnRNPA1 gene using RNA interference could inhibit Rex-mediated export of viral mRNAs in HTLV-1 producing C91PL T-cells. We show that the expression of NLS-A1-HA does not modify the export of Rex-dependent viral mRNAs. Conversely, inhibiting A1 expression in C91PL cells by RNA interference provoked an increase in the Rex-dependent export of unspliced and singly spliced mRNAs. Surprisingly, we also observed a significant increase in proviral transcription and an accumulation of unspliced mRNAs, suggesting that the splicing process was affected. Finally, A1 knockdown ...

Frontiers in Microbiology, 2013

Human T cell leukemia viruses (HTLVs) are complex human retroviruses of the Deltaretrovirus genus... more Human T cell leukemia viruses (HTLVs) are complex human retroviruses of the Deltaretrovirus genus. Four types have been identified thus far, with HTLV-1 and HTLV-2 much more prevalent than HTLV-3 or HTLV-4. HTLV-1 and HTLV-2 possess strictly related genomic structures, but differ significantly in pathogenicity, as HTLV-1 is the causative agent of adult T cell leukemia and of HTLV-associated myelopathy/tropical spastic paraparesis, whereas HTLV-2 is not associated with neoplasia. HTLVs code for a protein named Tax that is responsible for enhancing viral expression and drives cell transformation. Much effort has been invested to dissect the impact of Tax on signal transduction pathways and to identify functional differences between the HTLV Tax proteins that may explain the distinct oncogenic potential of HTLV-1 and HTLV-2. This review summarizes our current knowledge of Tax-1 and Tax-2 with emphasis on their structure, role in activation of the NF-κB (nuclear factor kappa-B) pathway, and interactions with host factors.

Virology, 2009

HTLV-1 is more pathogenic than HTLV-2B. The difference is generally attributed to the properties ... more 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.

Retrovirology, 2011

The Tax oncoprotein of HTLV-1 initiates T-cell transformation by dysregulating cell cycle progres... more 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-B 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-B activation cascade, including the regulatory subunit of the IKK complex, IKKgamma/NEMO, the RelA subunit of NF-B and TAX1-BP1, which are Tax interacting partners. Overexpression of TAB2 strongly stimulated Taxmediated activation of the NF-B pathway indicating that the concentration of these NF-B factors in Taxcontaining cytoplasmic punctate structures is a step important for activation of the NF-B 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-B pathway via initiation of events in the nucleus leading to the transport of IKKgamma from the nucleus to the cytoplasm will be discussed.

Retrovirology, 2011

IFN-alpha and high dose ascorbic acid (AA) have a modest clinical benefit in HAM/TSP (Nakagawa, 1... more 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.

Retrovirology, 2009

The trophoblastic cell represents the main functional unit of the placenta. It proliferates, migr... more 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.

Nature Immunology, 2003

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) is phosphorylated by Ins(1,4,5)P(3) 3-kinase, gener... more Inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) is phosphorylated by Ins(1,4,5)P(3) 3-kinase, generating inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)). The physiological function of Ins(1,3,4,5)P(4) is still unclear, but it has been reported to be a potential modulator of calcium mobilization. Disruption of the gene encoding the ubiquitously expressed Ins(1,4,5)P(3) 3-kinase isoform B (Itpkb) in mice caused a severe T cell deficiency due to major alterations in thymocyte responsiveness and selection. However, we were unable to detect substantial defects in Ins(1,4,5)P(3) amounts or calcium mobilization in Itpkb(-/-) thymocytes. These data indicate that Itpkb and Ins(1,3,4,5)P(4) define an essential signaling pathway for T cell precursor responsiveness and development.

Molecular and Cellular Biology, 2004

inhibitor that correlated temporally with the prolonged intranuclear binding and presence of NF-B... more inhibitor that correlated temporally with the prolonged intranuclear binding and presence of NF-B. This delay was due neither to a defect in IB␣ mRNA production nor to a nuclear retention of IB␣ but was rather due to a persistent proteasome-mediated degradation of IB␣. A prolongation of IB kinase activity could explain, at least partially, the delayed IB␣ cytoplasmic reappearance observed in presence of TNF plus TSA.

Methods, 1998

The human T-cell leukemia virus type I or HTLV-I is the causative agent of adult T-cell leukemia.... more The human T-cell leukemia virus type I or HTLV-I is the causative agent of adult T-cell leukemia. A protein encoded by HTLV-I, Tax, activates viral gene expression and is essential for transforming T-lymphocytes. Tax activates HTLV-I gene expression via interactions with the ATF/CREB proteins and the coactivators CBP/p300 which assemble as a multiprotein complex on regulatory elements known as 21-bp repeats in the HTLV-I LTR. Tax can also activate expression from cellular genes including the interleukin-2 (IL-2) and the IL-2 receptor genes via increases in nuclear levels of NF-kappaB. Tax modulation of gene expression via the ATF/CREB and NF-kappaB pathways is linked to its transforming properties. This review discusses the mechanisms by which Tax regulates viral and cellular gene expression.

Journal of Virology, 2004

Human T-cell leukemia virus type 1 (HTLV-1) is the retrovirus responsible for adult T-cell leukem... more 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 ubiquitinproteasome pathway and therefore reveal that Tax conjugation to ubiquitin mediates a nonproteolytic function.

Journal of Cell Science, 2007

In an attempt to elucidate the mechanism by which actin disruption induced IKK activation, we stu... more 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-B 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-B 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 detergentinsoluble pellet was specific to actin cytoskeleton. Confocal analysis also revealed a Nod2 colocalization with membrane-associated F-actin. Colocalization and coimmunoprecipitation 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-B signaling. The recruitment of Nod2 in Rac-induced dynamic cytoskeletal structures could be a strategy to both repress the Nod2-dependent NF-B signaling in unstimulated cells and rapidly mobilize Nod2 during bacterial infection.

Journal of Biological Chemistry, 2007

IKKalpha regulates many chromatin events in the nuclear phase of the NF-kappaB program, including... more 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.

PloS one, 2007

Although STAT5 promotes survival of hematopoietic progenitors, STAT5-/- mice develop mild neutrop... more Although STAT5 promotes survival of hematopoietic progenitors, STAT5-/- mice develop mild neutrophilia.

Gene, 2007

The human T-cell leukemia virus HTLV-1 encodes regulatory proteins, Tax, Rex and p30(II), which a... more The human T-cell leukemia virus HTLV-1 encodes regulatory proteins, Tax, Rex and p30(II), 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 p30(II). 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 p30(II) 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, p30(II) 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.

Cloning and Stem Cells, 2004

The genetic make-up of a cell resides entirely in its DNA. Now that the nucleotide sequence of se... more 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.

Biotechnology & Bioengineering, 2000

Viral expression systems offer the ability to generate high levels of a particular protein within... more Viral expression systems offer the ability to generate high levels of a particular protein within a relatively short period of time. In particular, alphavirus constructs based on Sindbis virus (SV) and Semliki Forest virus (SFV) are promising vehicles as they are cytoplasmic vectors with the potential for high expression levels. Two such alphavirus vectors were utilized during the current study to infect two commercially relevant cell lines, baby hamster kidney (BHK) and Chinese hamster ovary (CHO); the first was a fully competent SV derivative carrying the gene for chloramphenicol acetyltransferase (dsSV-CAT), while the second was a replication deficient SFV construct containing the human interleukin-12 (IL-12) p35 and p40 genes (SFV-IL-12). Since infection with these vectors induced apoptosis in both cell lines, the present effort was dedicated to determining the ability of anti-apoptosis genes to limit the cell death associated with these virus constructs. Infection with the dsSV-CAT vector resulted in the rapid death of BHK and CHO cells within 4 days, a phenomenon which was considerably delayed by stably overexpressing bcl-2 or bcl-x(L). In fact, cellular lifespans were doubled in both BHK-bcl2 and CHO-bclx(L) cells relative to the parental cell lines. Furthermore, the presence of these gene products provided increases of up to 2-fold in recombinant CAT production. Overexpression of bcl-2 and bcl-x(L) also altered the response of these cells upon infection with SFV-IL-12. While the parental cell lines were completely nonviable within 1 week, the BHK-bcl2, BHK-bclx(L), and CHO-bclx(L) cells each recovered from the infection, resuming exponential growth and regaining viabilities of over 90% by 9 days post-infection. Total IL-12 productivities were nearly doubled by Bcl-2 and Bcl-x(L) in the CHO cells, although this effect was apparently cell-line specific, as the native BHK cells were able to secrete more IL-12 than either of its transfected derivatives. Regardless, the presence of the anti-apoptosis genes allowed the production of IL-12 to be maintained, albeit at low levels, from each of the cell lines for the duration of the culture process. Therefore, overexpression of bcl-2 family members can have a significant impact on culture viabilities and recombinant protein production during alphavirus infections of mammalian cells.

Journal of Biological Chemistry

The human T cell leukemia/lymphotropic virus type 1 (HTLV-1) Tax protein strongly activates viral... more 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 t...

Viruses, 2011

The HTLV-1 Tax protein both activates viral replication and is involved in HTLV-1-mediated transf... more The HTLV-1 Tax protein both activates viral replication and is involved in HTLV-1-mediated transformation of T lymphocytes. The transforming properties of Tax include altering the expression of select cellular genes via activation of cellular pathways and perturbation of both cell cycle control mechanisms and apoptotic signals. The recent discovery that Tax undergoes a hierarchical sequence of posttranslational modifications that control its intracellular localization provides provocative insights into the mechanisms regulating Tax transcriptional and transforming activities.

Retrovirology, 2005

In this study, we have examined the role of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1)... more In this study, we have examined the role of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) in viral gene expression in T lymphocytes transformed by HTLV-1. We have previously observed that hnRNP A1 (A1) down-modulates the post transcriptional activity of Rex protein of HTLV-1. Here, we tested whether the ectopic expression of a dominant negative mutant (NLS-A1-HA) defective in shuttling activity or knockdown of the hnRNPA1 gene using RNA interference could inhibit Rex-mediated export of viral mRNAs in HTLV-1 producing C91PL T-cells. We show that the expression of NLS-A1-HA does not modify the export of Rex-dependent viral mRNAs. Conversely, inhibiting A1 expression in C91PL cells by RNA interference provoked an increase in the Rex-dependent export of unspliced and singly spliced mRNAs. Surprisingly, we also observed a significant increase in proviral transcription and an accumulation of unspliced mRNAs, suggesting that the splicing process was affected. Finally, A1 knockdown ...

Frontiers in Microbiology, 2013

Human T cell leukemia viruses (HTLVs) are complex human retroviruses of the Deltaretrovirus genus... more Human T cell leukemia viruses (HTLVs) are complex human retroviruses of the Deltaretrovirus genus. Four types have been identified thus far, with HTLV-1 and HTLV-2 much more prevalent than HTLV-3 or HTLV-4. HTLV-1 and HTLV-2 possess strictly related genomic structures, but differ significantly in pathogenicity, as HTLV-1 is the causative agent of adult T cell leukemia and of HTLV-associated myelopathy/tropical spastic paraparesis, whereas HTLV-2 is not associated with neoplasia. HTLVs code for a protein named Tax that is responsible for enhancing viral expression and drives cell transformation. Much effort has been invested to dissect the impact of Tax on signal transduction pathways and to identify functional differences between the HTLV Tax proteins that may explain the distinct oncogenic potential of HTLV-1 and HTLV-2. This review summarizes our current knowledge of Tax-1 and Tax-2 with emphasis on their structure, role in activation of the NF-κB (nuclear factor kappa-B) pathway, and interactions with host factors.

Virology, 2009

HTLV-1 is more pathogenic than HTLV-2B. The difference is generally attributed to the properties ... more 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.

Retrovirology, 2011

The Tax oncoprotein of HTLV-1 initiates T-cell transformation by dysregulating cell cycle progres... more 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-B 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-B activation cascade, including the regulatory subunit of the IKK complex, IKKgamma/NEMO, the RelA subunit of NF-B and TAX1-BP1, which are Tax interacting partners. Overexpression of TAB2 strongly stimulated Taxmediated activation of the NF-B pathway indicating that the concentration of these NF-B factors in Taxcontaining cytoplasmic punctate structures is a step important for activation of the NF-B 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-B pathway via initiation of events in the nucleus leading to the transport of IKKgamma from the nucleus to the cytoplasm will be discussed.

Retrovirology, 2011

IFN-alpha and high dose ascorbic acid (AA) have a modest clinical benefit in HAM/TSP (Nakagawa, 1... more 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.

Retrovirology, 2009

The trophoblastic cell represents the main functional unit of the placenta. It proliferates, migr... more 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.

Nature Immunology, 2003

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) is phosphorylated by Ins(1,4,5)P(3) 3-kinase, gener... more Inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) is phosphorylated by Ins(1,4,5)P(3) 3-kinase, generating inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)). The physiological function of Ins(1,3,4,5)P(4) is still unclear, but it has been reported to be a potential modulator of calcium mobilization. Disruption of the gene encoding the ubiquitously expressed Ins(1,4,5)P(3) 3-kinase isoform B (Itpkb) in mice caused a severe T cell deficiency due to major alterations in thymocyte responsiveness and selection. However, we were unable to detect substantial defects in Ins(1,4,5)P(3) amounts or calcium mobilization in Itpkb(-/-) thymocytes. These data indicate that Itpkb and Ins(1,3,4,5)P(4) define an essential signaling pathway for T cell precursor responsiveness and development.

Molecular and Cellular Biology, 2004

inhibitor that correlated temporally with the prolonged intranuclear binding and presence of NF-B... more inhibitor that correlated temporally with the prolonged intranuclear binding and presence of NF-B. This delay was due neither to a defect in IB␣ mRNA production nor to a nuclear retention of IB␣ but was rather due to a persistent proteasome-mediated degradation of IB␣. A prolongation of IB kinase activity could explain, at least partially, the delayed IB␣ cytoplasmic reappearance observed in presence of TNF plus TSA.

Methods, 1998

The human T-cell leukemia virus type I or HTLV-I is the causative agent of adult T-cell leukemia.... more The human T-cell leukemia virus type I or HTLV-I is the causative agent of adult T-cell leukemia. A protein encoded by HTLV-I, Tax, activates viral gene expression and is essential for transforming T-lymphocytes. Tax activates HTLV-I gene expression via interactions with the ATF/CREB proteins and the coactivators CBP/p300 which assemble as a multiprotein complex on regulatory elements known as 21-bp repeats in the HTLV-I LTR. Tax can also activate expression from cellular genes including the interleukin-2 (IL-2) and the IL-2 receptor genes via increases in nuclear levels of NF-kappaB. Tax modulation of gene expression via the ATF/CREB and NF-kappaB pathways is linked to its transforming properties. This review discusses the mechanisms by which Tax regulates viral and cellular gene expression.

Journal of Virology, 2004

Human T-cell leukemia virus type 1 (HTLV-1) is the retrovirus responsible for adult T-cell leukem... more 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 ubiquitinproteasome pathway and therefore reveal that Tax conjugation to ubiquitin mediates a nonproteolytic function.

Journal of Cell Science, 2007

In an attempt to elucidate the mechanism by which actin disruption induced IKK activation, we stu... more 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-B 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-B 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 detergentinsoluble pellet was specific to actin cytoskeleton. Confocal analysis also revealed a Nod2 colocalization with membrane-associated F-actin. Colocalization and coimmunoprecipitation 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-B signaling. The recruitment of Nod2 in Rac-induced dynamic cytoskeletal structures could be a strategy to both repress the Nod2-dependent NF-B signaling in unstimulated cells and rapidly mobilize Nod2 during bacterial infection.

Journal of Biological Chemistry, 2007

IKKalpha regulates many chromatin events in the nuclear phase of the NF-kappaB program, including... more 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.

PloS one, 2007

Although STAT5 promotes survival of hematopoietic progenitors, STAT5-/- mice develop mild neutrop... more Although STAT5 promotes survival of hematopoietic progenitors, STAT5-/- mice develop mild neutrophilia.

Gene, 2007

The human T-cell leukemia virus HTLV-1 encodes regulatory proteins, Tax, Rex and p30(II), which a... more The human T-cell leukemia virus HTLV-1 encodes regulatory proteins, Tax, Rex and p30(II), 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 p30(II). 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 p30(II) 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, p30(II) 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.

Cloning and Stem Cells, 2004

The genetic make-up of a cell resides entirely in its DNA. Now that the nucleotide sequence of se... more 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.

Biotechnology & Bioengineering, 2000

Viral expression systems offer the ability to generate high levels of a particular protein within... more Viral expression systems offer the ability to generate high levels of a particular protein within a relatively short period of time. In particular, alphavirus constructs based on Sindbis virus (SV) and Semliki Forest virus (SFV) are promising vehicles as they are cytoplasmic vectors with the potential for high expression levels. Two such alphavirus vectors were utilized during the current study to infect two commercially relevant cell lines, baby hamster kidney (BHK) and Chinese hamster ovary (CHO); the first was a fully competent SV derivative carrying the gene for chloramphenicol acetyltransferase (dsSV-CAT), while the second was a replication deficient SFV construct containing the human interleukin-12 (IL-12) p35 and p40 genes (SFV-IL-12). Since infection with these vectors induced apoptosis in both cell lines, the present effort was dedicated to determining the ability of anti-apoptosis genes to limit the cell death associated with these virus constructs. Infection with the dsSV-CAT vector resulted in the rapid death of BHK and CHO cells within 4 days, a phenomenon which was considerably delayed by stably overexpressing bcl-2 or bcl-x(L). In fact, cellular lifespans were doubled in both BHK-bcl2 and CHO-bclx(L) cells relative to the parental cell lines. Furthermore, the presence of these gene products provided increases of up to 2-fold in recombinant CAT production. Overexpression of bcl-2 and bcl-x(L) also altered the response of these cells upon infection with SFV-IL-12. While the parental cell lines were completely nonviable within 1 week, the BHK-bcl2, BHK-bclx(L), and CHO-bclx(L) cells each recovered from the infection, resuming exponential growth and regaining viabilities of over 90% by 9 days post-infection. Total IL-12 productivities were nearly doubled by Bcl-2 and Bcl-x(L) in the CHO cells, although this effect was apparently cell-line specific, as the native BHK cells were able to secrete more IL-12 than either of its transfected derivatives. Regardless, the presence of the anti-apoptosis genes allowed the production of IL-12 to be maintained, albeit at low levels, from each of the cell lines for the duration of the culture process. Therefore, overexpression of bcl-2 family members can have a significant impact on culture viabilities and recombinant protein production during alphavirus infections of mammalian cells.