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Sophie Bouchat, Jean-Stéphane Gatot,
Kabamba Kabeya,
Christelle Cardona,
Laurence Colin,
Georges Herbein,
Stéphane De Wit,
Nathan Clumeck,
Olivier Lambotte,
Christine Rouzioux,
Olivier Rohr,
Carine Van Lint
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ABSTRACT: Reactivation of HIV-1 expression in persistent reservoirs together with an efficient HAART has been proposed as an adjuvant therapy aimed at reaching a functional cure for HIV. Previously, H3K9 methylation was shown to play a major role in chromatin-mediated repression of the HIV-1 promoter. Here, we evaluated the therapeutic potential of histone methyltransferase inhibitors (HMTIs) in reactivating HIV-1 from latency.
We evaluated the reactivation potential of two specific HMTIs (chaetocin and BIX-01294, two specific inhibitors of Suv39H1 and G9a, respectively) in ex-vivo cultures of resting CD4 T cells isolated from HIV-1-infected HAART-treated individuals.
We measured HIV-1 recovery in ex-vivo cultures treated with an HMTI alone or in combination with other HIV-1 inducers (in absence of IL-2 and of allogenic stimulation) of CD8-depleted peripheral blood mononuclear cells (PBMCs) or of resting CD4 T cells isolated from 67 HIV-infected, HAART-treated patients with undetectable viral load.
We demonstrated, for the first time, that chaetocin induced HIV-1 recovery in 50% of CD8-depleted PBMCs cultures and in 86% of resting CD4 T-cell cultures isolated from HIV-1-infected, HAART-treated patients, whereas BIX-01294 reactivated HIV-1 expression in 80% of resting CD4 T-cell cultures isolated from similar patients. Moreover, we showed that combinatory treatments including one HMTI and either the histone deacetylase inhibitor suberoylanilide hydroxamic acid or the non-tumor-promoting NF-κB inducer prostratin had a higher reactivation potential than these compounds alone.
Our results constitute a proof-of-concept for the therapeutic potential of HMTIs in strategies aiming at reducing the pool of latent reservoirs in HIV-infected, HAART-treated patient.
AIDS (London, England) 05/2012; 26(12):1473-82. · 4.91 Impact Factor
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Sophie Reuse,
Miriam Calao,
Kabamba Kabeya,
Allan Guiguen, Jean-Stéphane Gatot,
Vincent Quivy,
Caroline Vanhulle,
Aurélia Lamine,
Dolores Vaira,
Dominique Demonte, [......],
Michel Moutschen,
Arsène Burny,
Christine Rouzioux,
Stéphane De Wit,
Georges Herbein,
Olivier Rohr,
Yves Collette,
Olivier Lambotte,
Nathan Clumeck,
Carine Van Lint
[show abstract]
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ABSTRACT: The persistence of transcriptionally silent but replication-competent HIV-1 reservoirs in Highly Active Anti-Retroviral Therapy (HAART)-treated infected individuals, represents a major hurdle to virus eradication. Activation of HIV-1 gene expression in these cells together with an efficient HAART has been proposed as an adjuvant therapy aimed at decreasing the pool of latent viral reservoirs. Using the latently-infected U1 monocytic cell line and latently-infected J-Lat T-cell clones, we here demonstrated a strong synergistic activation of HIV-1 production by clinically used histone deacetylase inhibitors (HDACIs) combined with prostratin, a non-tumor-promoting nuclear factor (NF)- kappaB inducer. In J-Lat cells, we showed that this synergism was due, at least partially, to the synergistic recruitment of unresponsive cells into the expressing cell population. A combination of prostratin+HDACI synergistically activated the 5' Long Terminal Repeat (5'LTR) from HIV-1 Major group subtypes representing the most prevalent viral genetic forms, as shown by transient transfection reporter assays. Mechanistically, HDACIs increased prostratin-induced DNA-binding activity of nuclear NF-kappaB and degradation of cytoplasmic NF-kappaB inhibitor, IkappaBalpha . Moreover, the combined treatment prostratin+HDACI caused a more pronounced nucleosomal remodeling in the U1 viral promoter region than the treatments with the compounds alone. This more pronounced remodeling correlated with a synergistic reactivation of HIV-1 transcription following the combined treatment prostratin+HDACI, as demonstrated by measuring recruitment of RNA polymerase II to the 5'LTR and both initiated and elongated transcripts. The physiological relevance of the prostratin+HDACI synergism was shown in CD8(+)-depleted peripheral blood mononuclear cells from HAART-treated patients with undetectable viral load. Moreover, this combined treatment reactivated viral replication in resting CD4(+) T cells isolated from similar patients. Our results suggest that combinations of different kinds of proviral activators may have important implications for reducing the size of latent HIV-1 reservoirs in HAART-treated patients.
PLoS ONE 02/2009; 4(6):e6093. · 4.09 Impact Factor
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Tetsuya Tanaka,
Amandine Legat,
Emmanuelle Adam,
Jonathan Steuve, Jean-Stéphane Gatot,
Michel Vandenbranden,
Liliana Ulianov,
Caroline Lonez,
Jean-Marie Ruysschaert,
Eric Muraille,
Marcel Tuynder,
Michel Goldman,
Alain Jacquet
[show abstract]
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ABSTRACT: DiC14-amidine cationic liposomes were recently shown to promote Th1 responses when mixed with allergen. To further define the mode of action of diC14-amidine as potential vaccine adjuvant, we characterized its effects on mouse and human myeloid dendritic cells (DC). First, we observed that, as compared with two other cationic liposomes, only diC14-amidine liposomes induced the production of IL-12p40 and TNF-alpha by mouse bone marrow-derived DC. DiC14-amidine liposomes also activated human DC, as shown by synthesis of IL-12p40 and TNF-alpha, accumulation of IL-6, IFN-beta and CXCL10 mRNA, and up-regulation of membrane expression of CD80 and CD86. DC stimulation by diC14-amidine liposomes was associated with activation of NF-kappaB, ERK1/2, JNK and p38 MAP kinases. Finally, we demonstrated in mouse and human cells that diC14-amidine liposomes use Toll-like receptor 4 to elicit both MyD88-dependent and Toll/IL-1R-containing adaptor inducing interferon IFN-beta (TRIF)-dependent responses.
European Journal of Immunology 06/2008; 38(5):1351-7. · 5.10 Impact Factor
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[show abstract]
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ABSTRACT: The IkappaB kinases (IKKs) IKK-alpha and IKK-beta, and the IKK-related kinases TBK1 and IKK-epsilon, have essential roles in innate immunity through signal-induced activation of NF-kappaB, IRF3 and IRF7, respectively. Although the signaling events within these pathways have been extensively studied, the mechanisms of IKK and IKK-related complex assembly and activation remain poorly defined. Recent data provide insight into the requirement for scaffold proteins in complex assembly; NF-kappaB essential modulator coordinates some IKK complexes, whereas TANK, NF-kappaB-activating kinase-associated protein 1 (NAP1) or similar to NAP1 TBK1 adaptor (SINTBAD) assemble TBK1 and IKK-epsilon complexes. The different scaffold proteins undergo similar post-translational modifications, including phosphorylation and non-degradative polyubiquitylation. Moreover, increasing evidence indicates that distinct scaffold proteins assemble IKK, and potentially TBK1 and IKK-epsilon subcomplexes, in a stimulus-specific manner, which might be a mechanism to achieve specificity.
Trends in Biochemical Sciences 05/2008; 33(4):171-80. · 10.85 Impact Factor
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Jean-Stéphane Gatot,
Romain Gioia,
Tieu-Lan Chau,
Félicia Patrascu,
Michael Warnier,
Pierre Close,
Jean-Paul Chapelle,
Eric Muraille,
Keith Brown,
Ulrich Siebenlist,
Jacques Piette,
Emmanuel Dejardin,
Alain Chariot
[show abstract]
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ABSTRACT: Type I interferon gene induction relies on IKK-related kinase TBK1 and IKKepsilon-mediated phosphorylations of IRF3/7 through the Toll-like receptor-dependent signaling pathways. The scaffold proteins that assemble these kinase complexes are poorly characterized. We show here that TANK/ITRAF is required for the TBK1- and IKKepsilon-mediated IRF3/7 phosphorylations through some Toll-like receptor-dependent pathways and is part of a TRAF3-containing complex. Moreover, TANK is dispensable for the early phase of double-stranded RNA-mediated IRF3 phosphorylation. Interestingly, TANK is heavily phosphorylated by TBK1-IKKepsilon upon lipopolysaccharide stimulation and is also subject to lipopolysaccharide- and TBK1-IKKepsilon-mediated Lys(63)-linked polyubiquitination, a mechanism that does not require TBK1-IKKepsilon kinase activity. Thus, we have identified TANK as a scaffold protein that assembles some but not all IRF3/7-phosphorylating TBK1-IKKepsilon complexes and demonstrated that these kinases possess two functions, namely the phosphorylation of both IRF3/7 and TANK as well as the recruitment of an E3 ligase for Lys(63)-linked polyubiquitination of their scaffold protein, TANK.
Journal of Biological Chemistry 11/2007; 282(43):31131-46. · 4.77 Impact Factor
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Jean-Stéphane Gatot,
Romain Gioia,
Tieu-Lan Chau,
Félicia Patrascu,
Michael Warnier,
Pierre Close,
Jean-Paul Chapelle,
Eric Muraille,
Keith Brown,
Ulrich Siebenlist,
Jacques Piette,
Emmanuel Dejardin,
Alain Chariot
[show abstract]
[hide abstract]
ABSTRACT: Type I interferon gene induction relies on IKK-related kinase TBK1 and IKKϵ-mediated phosphorylations of IRF3/7 through the
Toll-like receptor-dependent signaling pathways. The scaffold proteins that assemble these kinase complexes are poorly characterized.
We show here that TANK/ITRAF is required for the TBK1- and IKKϵ-mediated IRF3/7 phosphorylations through some Toll-like receptor-dependent
pathways and is part of a TRAF3-containing complex. Moreover, TANK is dispensable for the early phase of double-stranded RNA-mediated
IRF3 phosphorylation. Interestingly, TANK is heavily phosphorylated by TBK1-IKKϵ upon lipopolysaccharide stimulation and is
also subject to lipopolysaccharide- and TBK1-IKKϵ-mediated Lys63-linked polyubiquitination, a mechanism that does not require TBK1-IKKϵ kinase activity. Thus, we have identified TANK as
a scaffold protein that assembles some but not all IRF3/7-phosphorylating TBK1-IKKϵ complexes and demonstrated that these
kinases possess two functions, namely the phosphorylation of both IRF3/7 and TANK as well as the recruitment of an E3 ligase
for Lys63-linked polyubiquitination of their scaffold protein, TANK.
Journal of Biological Chemistry 10/2007; 282(43):31131-31146. · 4.77 Impact Factor
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[show abstract]
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ABSTRACT: Bovine leukemia virus (BLV) and human T-cell lymphotropic virus type 1 (HTLV-1) belong to the genus of deltaretroviruses. Their entry into the host cell is supposed to be mediated by interactions of the extracellular (SU) envelope glycoproteins with cellular receptors. To gain insight into the mechanisms governing this process, we investigated the ability of SU proteins to interact with specific ligands. In particular, by affinity chromatography, we have shown that BLV SU protein specifically interacted with zinc ions. To identify the protein domains involved in binding, 16 peptides distributed along the sequence were tested. Two of them appeared to be able to interact with zinc. To unravel the role of these SU regions in the biology of the virus, mutations were introduced into the env gene of a BLV molecular clone in order to modify residues potentially interacting with zinc. The fusogenic capacity of envelope mutated within the first zinc-binding region (104 to 123) was completely abolished. Furthermore, the integrity of this domain was also required for in vivo infectivity. In contrast, mutations within the second zinc-binding region (218 to 237) did not hamper the fusogenic capacity; indeed, the syncytia were even larger. In sheep, mutations in region 218 to 237 did not alter infectivity or viral spread. Finally, we demonstrated that the envelope of the related HTLV-1 was also able to bind zinc. Interestingly, zinc ions were found to be associated with the receptor-binding domain (RBD) of Friend murine leukemia virus (Fr-MLV) SU glycoprotein, further supporting their relevance in SU structure. Based on the sequence similarities shared with the Fr-MLV RBD, whose three-dimensional structure has been experimentally determined, we located the BLV zinc-binding peptide 104-123 on the opposite side of the potential receptor-binding surface. This observation supports the hypothesis that zinc ions could mediate interactions of the SU RBD either with the C-terminal part of SU, thereby contributing to the SU structural integrity, or with a partner(s) different from the receptor.
Journal of Virology 09/2002; 76(16):7956-67. · 5.40 Impact Factor
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[show abstract]
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ABSTRACT: The IκB kinases (IKKs) IKK-α and IKK-β, and the IKK-related kinases TBK1 and IKK-ɛ, have essential roles in innate immunity through signal-induced activation of NF-κB, IRF3 and IRF7, respectively. Although the signaling events within these pathways have been extensively studied, the mechanisms of IKK and IKK-related complex assembly and activation remain poorly defined. Recent data provide insight into the requirement for scaffold proteins in complex assembly; NF-κB essential modulator coordinates some IKK complexes, whereas TANK, NF-κB-activating kinase-associated protein 1 (NAP1) or similar to NAP1 TBK1 adaptor (SINTBAD) assemble TBK1 and IKK-ɛ complexes. The different scaffold proteins undergo similar post-translational modifications, including phosphorylation and non-degradative polyubiquitylation. Moreover, increasing evidence indicates that distinct scaffold proteins assemble IKK, and potentially TBK1 and IKK-ɛ subcomplexes, in a stimulus-specific manner, which might be a mechanism to achieve specificity.
Trends in Biochemical Sciences.
