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Mass Spectrometry
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2024
Soufi G El, Jorio L Di, Gerber Z, Cluzel N, Assche J Van, Delafoy D, Olaso R, Daviaud C, Loustau T, Schwartz C, Trebouet D, Hernalsteens O, Marechal V, Raffestin S, Rousset D, Lint C Van, Deleuze J F, Boni M, and O Rohr, Villain-Gambier M, Wallet C
In: Water Res, vol. 249, pp. 120959, 2024, ISSN: 1879-2448.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid38070350,
title = {Highly efficient and sensitive membrane-based concentration process allows quantification, surveillance, and sequencing of viruses in large volumes of wastewater},
author = {G El Soufi and L Di Jorio and Z Gerber and N Cluzel and J Van Assche and D Delafoy and R Olaso and C Daviaud and T Loustau and C Schwartz and D Trebouet and O Hernalsteens and V Marechal and S Raffestin and D Rousset and C Van Lint and J F Deleuze and M Boni and and O Rohr and M Villain-Gambier and C Wallet},
doi = {10.1016/j.watres.2023.120959},
issn = {1879-2448},
year = {2024},
date = {2024-02-01},
urldate = {2024-02-01},
journal = {Water Res},
volume = {249},
pages = {120959},
abstract = {Wastewater-based epidemiology is experiencing exponential development. Despite undeniable advantages compared to patient-centered approaches (cost, anonymity, survey of large populations without bias, detection of asymptomatic infected peoples…), major technical limitations persist. Among them is the low sensitivity of the current methods used for quantifying and sequencing viral genomes from wastewater. In situations of low viral circulation, during initial stages of viral emergences, or in areas experiencing heavy rains, the extremely low concentrations of viruses in wastewater may fall below the limit of detection of the current methods. The availability during crisis and the cost of the commercial kits, as well as the requirement of expensive materials such as high-speed centrifuge, can also present major blocks to the development of wastewater-based epidemiological survey, specifically in low-income countries. Thereby, highly sensitive, low cost and standardized methods are still needed, to increase the predictability of the viral emergences, to survey low-circulating viruses and to make the results from different labs comparable. Here, we outline and characterize new protocols for concentrating and quantifying SARS-CoV-2 from large volumes (500 mL-1 L) of untreated wastewater. In addition, we report that the methods are applicable for monitoring and sequencing. Our nucleic acid extraction technique (the routine C: 5 mL method) does not require sophisticated equipment such as automatons and is not reliant on commercial kits, making it readily available to a broader range of laboratories for routine epidemiological survey. Furthermore, we demonstrate the efficiency, the repeatability, and the high sensitivity of a new membrane-based concentration method (MBC: 500 mL method) for enveloped (SARS-CoV-2) and non-enveloped (F-specific RNA phages of genogroup II / FRNAPH GGII) viruses. We show that the MBC method allows the quantification and the monitoring of viruses in wastewater with a significantly improved sensitivity compared to the routine C method. In contexts of low viral circulation, we report quantifications of SARS-CoV-2 in wastewater at concentrations as low as 40 genome copies per liter. In highly diluted samples collected in wastewater treatment plants of French Guiana, we confirmed the accuracy of the MBC method compared to the estimations done with the routine C method. Finally, we demonstrate that both the routine C method processing 5 mL and the MBC method processing 500 mL of untreated wastewater are both compatible with SARS-CoV-2 sequencing. We show that the quality of the sequence is correlated with the concentration of the extracted viral genome. Of note, the quality of the sequences obtained with some MBC processed wastewater was improved by dilutions or enzyme substitutions suggesting the presence of specific enzyme inhibitors in some wastewater. To the best of our knowledge, our MBC method is one of the first efficient, sensitive, and repeatable method characterized for SARS-CoV-2 quantification and sequencing from large volumes of wastewater.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Wastewater-based epidemiology is experiencing exponential development. Despite undeniable advantages compared to patient-centered approaches (cost, anonymity, survey of large populations without bias, detection of asymptomatic infected peoples…), major technical limitations persist. Among them is the low sensitivity of the current methods used for quantifying and sequencing viral genomes from wastewater. In situations of low viral circulation, during initial stages of viral emergences, or in areas experiencing heavy rains, the extremely low concentrations of viruses in wastewater may fall below the limit of detection of the current methods. The availability during crisis and the cost of the commercial kits, as well as the requirement of expensive materials such as high-speed centrifuge, can also present major blocks to the development of wastewater-based epidemiological survey, specifically in low-income countries. Thereby, highly sensitive, low cost and standardized methods are still needed, to increase the predictability of the viral emergences, to survey low-circulating viruses and to make the results from different labs comparable. Here, we outline and characterize new protocols for concentrating and quantifying SARS-CoV-2 from large volumes (500 mL-1 L) of untreated wastewater. In addition, we report that the methods are applicable for monitoring and sequencing. Our nucleic acid extraction technique (the routine C: 5 mL method) does not require sophisticated equipment such as automatons and is not reliant on commercial kits, making it readily available to a broader range of laboratories for routine epidemiological survey. Furthermore, we demonstrate the efficiency, the repeatability, and the high sensitivity of a new membrane-based concentration method (MBC: 500 mL method) for enveloped (SARS-CoV-2) and non-enveloped (F-specific RNA phages of genogroup II / FRNAPH GGII) viruses. We show that the MBC method allows the quantification and the monitoring of viruses in wastewater with a significantly improved sensitivity compared to the routine C method. In contexts of low viral circulation, we report quantifications of SARS-CoV-2 in wastewater at concentrations as low as 40 genome copies per liter. In highly diluted samples collected in wastewater treatment plants of French Guiana, we confirmed the accuracy of the MBC method compared to the estimations done with the routine C method. Finally, we demonstrate that both the routine C method processing 5 mL and the MBC method processing 500 mL of untreated wastewater are both compatible with SARS-CoV-2 sequencing. We show that the quality of the sequence is correlated with the concentration of the extracted viral genome. Of note, the quality of the sequences obtained with some MBC processed wastewater was improved by dilutions or enzyme substitutions suggesting the presence of specific enzyme inhibitors in some wastewater. To the best of our knowledge, our MBC method is one of the first efficient, sensitive, and repeatable method characterized for SARS-CoV-2 quantification and sequencing from large volumes of wastewater.
2023
Saeb Sepideh, Wallet Clémentine, Rohr Olivier, Schwartz Christian, Loustau Thomas
Targeting and eradicating latent CNS reservoirs of HIV-1: Original strategies and new models Journal Article
In: Biochem Pharmacol, vol. 214, pp. 115679, 2023, ISSN: 1873-2968.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid37399950,
title = {Targeting and eradicating latent CNS reservoirs of HIV-1: Original strategies and new models},
author = {Sepideh Saeb and Clémentine Wallet and Olivier Rohr and Christian Schwartz and Thomas Loustau},
doi = {10.1016/j.bcp.2023.115679},
issn = {1873-2968},
year = {2023},
date = {2023-08-01},
urldate = {2023-08-01},
journal = {Biochem Pharmacol},
volume = {214},
pages = {115679},
abstract = {Nowadays, combination antiretroviral therapy (cART) is the standard treatment for all people with human immunodeficiency virus (HIV-1). Although cART is effective in treating productive infection, it does not eliminate latent reservoirs of the virus. This leads to lifelong treatment associated with the occurrence of side effects and the development of drug-resistant HIV-1. Suppression of viral latency is therefore the major hurdle to HIV-1 eradication. Multiple mechanisms exist to regulate viral gene expression and drive the transcriptional and post-transcriptional establishment of latency. Epigenetic processes are amongst the most studied mechanisms influencing both productive and latent infection states. The central nervous system (CNS) represents a key anatomical sanctuary for HIV and is the focal point of considerable research efforts. However, limited and difficult access to CNS compartments makes understanding the HIV-1 infection state in latent brain cells such as microglial cells, astrocytes, and perivascular macrophages challenging. This review examines the latest advances on epigenetic transformations involved in CNS viral latency and targeting of brain reservoirs. Evidence from clinical studies as well as in vivo and in vitro models of HIV-1 persistence in the CNS will be discussed, with a special focus on recent 3D in vitro models such as human brain organoids. Finally, the review will address therapeutic considerations for targeting latent CNS reservoirs.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Nowadays, combination antiretroviral therapy (cART) is the standard treatment for all people with human immunodeficiency virus (HIV-1). Although cART is effective in treating productive infection, it does not eliminate latent reservoirs of the virus. This leads to lifelong treatment associated with the occurrence of side effects and the development of drug-resistant HIV-1. Suppression of viral latency is therefore the major hurdle to HIV-1 eradication. Multiple mechanisms exist to regulate viral gene expression and drive the transcriptional and post-transcriptional establishment of latency. Epigenetic processes are amongst the most studied mechanisms influencing both productive and latent infection states. The central nervous system (CNS) represents a key anatomical sanctuary for HIV and is the focal point of considerable research efforts. However, limited and difficult access to CNS compartments makes understanding the HIV-1 infection state in latent brain cells such as microglial cells, astrocytes, and perivascular macrophages challenging. This review examines the latest advances on epigenetic transformations involved in CNS viral latency and targeting of brain reservoirs. Evidence from clinical studies as well as in vivo and in vitro models of HIV-1 persistence in the CNS will be discussed, with a special focus on recent 3D in vitro models such as human brain organoids. Finally, the review will address therapeutic considerations for targeting latent CNS reservoirs.
2022
Verdikt Roxane, Bendoumou Maryam, Bouchat Sophie, Nestola Lorena, Pasternak Alexander O, Darcis Gilles, Avettand-Fenoel Véronique, Vanhulle Caroline, Aït-Ammar Amina, Santangelo Marion, Plant Estelle, Douce Valentin Le, Delacourt Nadège, Cicilionytė Aurelija, Necsoi Coca, Corazza Francis, Passaes Caroline Pereira Bittencourt, Schwartz Christian, Bizet Martin, Fuks François, Sáez-Cirión Asier, Rouzioux Christine, Wit Stéphane De, Berkhout Ben, Gautier Virginie, Rohr Olivier, Lint Carine Van
Novel role of UHRF1 in the epigenetic repression of the latent HIV-1 Journal Article
In: EBioMedicine, vol. 79, pp. 103985, 2022, ISSN: 2352-3964.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid35429693,
title = {Novel role of UHRF1 in the epigenetic repression of the latent HIV-1},
author = {Roxane Verdikt and Maryam Bendoumou and Sophie Bouchat and Lorena Nestola and Alexander O Pasternak and Gilles Darcis and Véronique Avettand-Fenoel and Caroline Vanhulle and Amina Aït-Ammar and Marion Santangelo and Estelle Plant and Valentin Le Douce and Nadège Delacourt and Aurelija Cicilionytė and Coca Necsoi and Francis Corazza and Caroline Pereira Bittencourt Passaes and Christian Schwartz and Martin Bizet and François Fuks and Asier Sáez-Cirión and Christine Rouzioux and Stéphane De Wit and Ben Berkhout and Virginie Gautier and Olivier Rohr and Carine Van Lint},
doi = {10.1016/j.ebiom.2022.103985},
issn = {2352-3964},
year = {2022},
date = {2022-05-01},
urldate = {2022-05-01},
journal = {EBioMedicine},
volume = {79},
pages = {103985},
abstract = {BACKGROUND: The multiplicity, heterogeneity, and dynamic nature of human immunodeficiency virus type-1 (HIV-1) latency mechanisms are reflected in the current lack of functional cure for HIV-1. Accordingly, all classes of latency-reversing agents (LRAs) have been reported to present variable ex vivo potencies. Here, we investigated the molecular mechanisms underlying the potency variability of one LRA: the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-AzadC).nnMETHODS: We employed epigenetic interrogation methods (electrophoretic mobility shift assays, chromatin immunoprecipitation, Infinium array) in complementary HIV-1 infection models (latently-infected T-cell line models, primary CD4 T-cell models and ex vivo cultures of PBMCs from HIV individuals). Extracellular staining of cell surface receptors and intracellular metabolic activity were measured in drug-treated cells. HIV-1 expression in reactivation studies was explored by combining the measures of capsid p24 protein, green fluorescence protein signal, intracellular and extracellular viral RNA and viral DNA.nnFINDINGS: We uncovered specific demethylation CpG signatures induced by 5-AzadC in the HIV-1 promoter. By analyzing the binding modalities to these CpG, we revealed the recruitment of the epigenetic integrator Ubiquitin-like with PHD and RING finger domain 1 (UHRF1) to the HIV-1 promoter. We showed that UHRF1 redundantly binds to the HIV-1 promoter with different binding modalities where DNA methylation was either non-essential, essential or enhancing UHRF1 binding. We further demonstrated the role of UHRF1 in the epigenetic repression of the latent viral promoter by a concerted control of DNA and histone methylations.nnINTERPRETATION: A better understanding of the molecular mechanisms of HIV-1 latency allows for the development of innovative antiviral strategies. As a proof-of-concept, we showed that pharmacological inhibition of UHRF1 in ex vivo HIV patient cell cultures resulted in potent viral reactivation from latency. Together, we identify UHRF1 as a novel actor in HIV-1 epigenetic silencing and highlight that it constitutes a new molecular target for HIV-1 cure strategies.nnFUNDING: Funding was provided by the Belgian National Fund for Scientific Research (F.R.S.-FNRS, Belgium), the « Fondation Roi Baudouin », the NEAT (European AIDS Treatment Network) program, the Internationale Brachet Stiftung, ViiV Healthcare, the Télévie, the Walloon Region (« Fonds de Maturation »), « Les Amis des Instituts Pasteur à Bruxelles, asbl », the University of Brussels (Action de Recherche Concertée ULB grant), the Marie Skodowska Curie COFUND action, the European Union's Horizon 2020 research and innovation program under grant agreement No 691119-EU4HIVCURE-H2020-MSCA-RISE-2015, the French Agency for Research on AIDS and Viral Hepatitis (ANRS), the Sidaction and the "Alsace contre le Cancer" Foundation. This work is supported by 1UM1AI164562-01, co-funded by National Heart, Lung and Blood Institute, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Neurological Disorders and Stroke, National Institute on Drug Abuse and the National Institute of Allergy and Infectious Diseases.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
BACKGROUND: The multiplicity, heterogeneity, and dynamic nature of human immunodeficiency virus type-1 (HIV-1) latency mechanisms are reflected in the current lack of functional cure for HIV-1. Accordingly, all classes of latency-reversing agents (LRAs) have been reported to present variable ex vivo potencies. Here, we investigated the molecular mechanisms underlying the potency variability of one LRA: the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-AzadC).nnMETHODS: We employed epigenetic interrogation methods (electrophoretic mobility shift assays, chromatin immunoprecipitation, Infinium array) in complementary HIV-1 infection models (latently-infected T-cell line models, primary CD4 T-cell models and ex vivo cultures of PBMCs from HIV individuals). Extracellular staining of cell surface receptors and intracellular metabolic activity were measured in drug-treated cells. HIV-1 expression in reactivation studies was explored by combining the measures of capsid p24 protein, green fluorescence protein signal, intracellular and extracellular viral RNA and viral DNA.nnFINDINGS: We uncovered specific demethylation CpG signatures induced by 5-AzadC in the HIV-1 promoter. By analyzing the binding modalities to these CpG, we revealed the recruitment of the epigenetic integrator Ubiquitin-like with PHD and RING finger domain 1 (UHRF1) to the HIV-1 promoter. We showed that UHRF1 redundantly binds to the HIV-1 promoter with different binding modalities where DNA methylation was either non-essential, essential or enhancing UHRF1 binding. We further demonstrated the role of UHRF1 in the epigenetic repression of the latent viral promoter by a concerted control of DNA and histone methylations.nnINTERPRETATION: A better understanding of the molecular mechanisms of HIV-1 latency allows for the development of innovative antiviral strategies. As a proof-of-concept, we showed that pharmacological inhibition of UHRF1 in ex vivo HIV patient cell cultures resulted in potent viral reactivation from latency. Together, we identify UHRF1 as a novel actor in HIV-1 epigenetic silencing and highlight that it constitutes a new molecular target for HIV-1 cure strategies.nnFUNDING: Funding was provided by the Belgian National Fund for Scientific Research (F.R.S.-FNRS, Belgium), the « Fondation Roi Baudouin », the NEAT (European AIDS Treatment Network) program, the Internationale Brachet Stiftung, ViiV Healthcare, the Télévie, the Walloon Region (« Fonds de Maturation »), « Les Amis des Instituts Pasteur à Bruxelles, asbl », the University of Brussels (Action de Recherche Concertée ULB grant), the Marie Skodowska Curie COFUND action, the European Union's Horizon 2020 research and innovation program under grant agreement No 691119-EU4HIVCURE-H2020-MSCA-RISE-2015, the French Agency for Research on AIDS and Viral Hepatitis (ANRS), the Sidaction and the "Alsace contre le Cancer" Foundation. This work is supported by 1UM1AI164562-01, co-funded by National Heart, Lung and Blood Institute, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Neurological Disorders and Stroke, National Institute on Drug Abuse and the National Institute of Allergy and Infectious Diseases.
2021
Saeb Sepideh, Ravanshad Mehrdad, Pourkarim Mahmoud Reza, Daouad Fadoua, Baesi Kazem, Rohr Olivier, Wallet Clémentine, Schwartz Christian
Brain HIV-1 latently-infected reservoirs targeted by the suicide gene strategy Journal Article
In: Virol J, vol. 18, no. 1, pp. 107, 2021, ISSN: 1743-422X.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid34059075,
title = {Brain HIV-1 latently-infected reservoirs targeted by the suicide gene strategy},
author = {Sepideh Saeb and Mehrdad Ravanshad and Mahmoud Reza Pourkarim and Fadoua Daouad and Kazem Baesi and Olivier Rohr and Clémentine Wallet and Christian Schwartz},
doi = {10.1186/s12985-021-01584-2},
issn = {1743-422X},
year = {2021},
date = {2021-05-01},
urldate = {2021-05-01},
journal = {Virol J},
volume = {18},
number = {1},
pages = {107},
abstract = {Reducing the pool of HIV-1 reservoirs in patients is a must to achieve functional cure. The most prominent HIV-1 cell reservoirs are resting CD4 + T cells and brain derived microglial cells. Infected microglial cells are believed to be the source of peripheral tissues reseedings and the emergence of drug resistance. Clearing infected cells from the brain is therefore crucial. However, many characteristics of microglial cells and the central nervous system make extremely difficult their eradication from brain reservoirs. Current methods, such as the "shock and kill", the "block and lock" and gene editing strategies cannot override these difficulties. Therefore, new strategies have to be designed when considering the elimination of brain reservoirs. We set up an original gene suicide strategy using latently infected microglial cells as model cells. In this paper we provide proof of concept of this strategy.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Reducing the pool of HIV-1 reservoirs in patients is a must to achieve functional cure. The most prominent HIV-1 cell reservoirs are resting CD4 + T cells and brain derived microglial cells. Infected microglial cells are believed to be the source of peripheral tissues reseedings and the emergence of drug resistance. Clearing infected cells from the brain is therefore crucial. However, many characteristics of microglial cells and the central nervous system make extremely difficult their eradication from brain reservoirs. Current methods, such as the "shock and kill", the "block and lock" and gene editing strategies cannot override these difficulties. Therefore, new strategies have to be designed when considering the elimination of brain reservoirs. We set up an original gene suicide strategy using latently infected microglial cells as model cells. In this paper we provide proof of concept of this strategy.
Ait-Ammar Amina, Bellefroid Maxime, Daouad Fadoua, Martinelli Valérie, Assche Jeanne Van, Wallet Clémentine, Rodari Anthony, Rovere Marco De, Fahrenkrog Birthe, Schwartz Christian, Lint Carine Van, Gautier Virginie, Rohr Olivier
Inhibition of HIV-1 gene transcription by KAP1 in myeloid lineage Journal Article
In: Sci Rep, vol. 11, no. 1, pp. 2692, 2021, ISSN: 2045-2322.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid33514850,
title = {Inhibition of HIV-1 gene transcription by KAP1 in myeloid lineage},
author = {Amina Ait-Ammar and Maxime Bellefroid and Fadoua Daouad and Valérie Martinelli and Jeanne Van Assche and Clémentine Wallet and Anthony Rodari and Marco De Rovere and Birthe Fahrenkrog and Christian Schwartz and Carine Van Lint and Virginie Gautier and Olivier Rohr},
doi = {10.1038/s41598-021-82164-w},
issn = {2045-2322},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Sci Rep},
volume = {11},
number = {1},
pages = {2692},
abstract = {HIV-1 latency generates reservoirs that prevent viral eradication by the current therapies. To find strategies toward an HIV cure, detailed understandings of the molecular mechanisms underlying establishment and persistence of the reservoirs are needed. The cellular transcription factor KAP1 is known as a potent repressor of gene transcription. Here we report that KAP1 represses HIV-1 gene expression in myeloid cells including microglial cells, the major reservoir of the central nervous system. Mechanistically, KAP1 interacts and colocalizes with the viral transactivator Tat to promote its degradation via the proteasome pathway and repress HIV-1 gene expression. In myeloid models of latent HIV-1 infection, the depletion of KAP1 increased viral gene elongation and reactivated HIV-1 expression. Bound to the latent HIV-1 promoter, KAP1 associates and cooperates with CTIP2, a key epigenetic silencer of HIV-1 expression in microglial cells. In addition, Tat and CTIP2 compete for KAP1 binding suggesting a dynamic modulation of the KAP1 cellular partners upon HIV-1 infection. Altogether, our results suggest that KAP1 contributes to the establishment and the persistence of HIV-1 latency in myeloid cells.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
HIV-1 latency generates reservoirs that prevent viral eradication by the current therapies. To find strategies toward an HIV cure, detailed understandings of the molecular mechanisms underlying establishment and persistence of the reservoirs are needed. The cellular transcription factor KAP1 is known as a potent repressor of gene transcription. Here we report that KAP1 represses HIV-1 gene expression in myeloid cells including microglial cells, the major reservoir of the central nervous system. Mechanistically, KAP1 interacts and colocalizes with the viral transactivator Tat to promote its degradation via the proteasome pathway and repress HIV-1 gene expression. In myeloid models of latent HIV-1 infection, the depletion of KAP1 increased viral gene elongation and reactivated HIV-1 expression. Bound to the latent HIV-1 promoter, KAP1 associates and cooperates with CTIP2, a key epigenetic silencer of HIV-1 expression in microglial cells. In addition, Tat and CTIP2 compete for KAP1 binding suggesting a dynamic modulation of the KAP1 cellular partners upon HIV-1 infection. Altogether, our results suggest that KAP1 contributes to the establishment and the persistence of HIV-1 latency in myeloid cells.
2020
Wallet Clémentine, Rohr Olivier, Schwartz Christian
Evolution of a concept: From accessory protein to key virulence factor, the case of HIV-1 Vpr Journal Article
In: Biochem Pharmacol, vol. 180, pp. 114128, 2020, ISSN: 1873-2968.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid32619426,
title = {Evolution of a concept: From accessory protein to key virulence factor, the case of HIV-1 Vpr},
author = {Clémentine Wallet and Olivier Rohr and Christian Schwartz},
doi = {10.1016/j.bcp.2020.114128},
issn = {1873-2968},
year = {2020},
date = {2020-10-01},
urldate = {2020-10-01},
journal = {Biochem Pharmacol},
volume = {180},
pages = {114128},
abstract = {Back in 1989 some studies have shown that the viral protein Vpr was dispensable for HIV-1 replication in vitro. From then the concept of accessory or auxiliary protein for Vpr has emerged and it is still used to date. However, Vpr soon appeared to be very important for in vivo virus spread and pathogenesis. Vpr has been involved in many biological functions including regulation of reverse transcriptase activity, the nuclear import of the pre-integration complex (PIC), HIV-1 transcription, gene splicing, apoptosis and in cell cycle arrest. Thus, we might rather consider Vpr as a true virulence factor instead of just an accessory factor. At present, Vpr can be regarded as a potential and promising target in different strategies aiming to fight infected cells including latently infected cells.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Back in 1989 some studies have shown that the viral protein Vpr was dispensable for HIV-1 replication in vitro. From then the concept of accessory or auxiliary protein for Vpr has emerged and it is still used to date. However, Vpr soon appeared to be very important for in vivo virus spread and pathogenesis. Vpr has been involved in many biological functions including regulation of reverse transcriptase activity, the nuclear import of the pre-integration complex (PIC), HIV-1 transcription, gene splicing, apoptosis and in cell cycle arrest. Thus, we might rather consider Vpr as a true virulence factor instead of just an accessory factor. At present, Vpr can be regarded as a potential and promising target in different strategies aiming to fight infected cells including latently infected cells.
Shadrina Olga, Garanina Irina, Korolev Sergey, Zatsepin Timofei, Assche Jeanne Van, Daouad Fadoua, Wallet Clementine, Rohr Olivier, Gottikh Marina
Analysis of RNA binding properties of human Ku protein reveals its interactions with 7SK snRNA and protein components of 7SK snRNP complex Journal Article
In: Biochimie, vol. 171-172, pp. 110–123, 2020, ISSN: 1638-6183.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid32105815,
title = {Analysis of RNA binding properties of human Ku protein reveals its interactions with 7SK snRNA and protein components of 7SK snRNP complex},
author = {Olga Shadrina and Irina Garanina and Sergey Korolev and Timofei Zatsepin and Jeanne Van Assche and Fadoua Daouad and Clementine Wallet and Olivier Rohr and Marina Gottikh},
doi = {10.1016/j.biochi.2020.02.016},
issn = {1638-6183},
year = {2020},
date = {2020-01-01},
urldate = {2020-01-01},
journal = {Biochimie},
volume = {171-172},
pages = {110--123},
abstract = {Human Ku heterodimeric protein composed of Ku70 and Ku80 subunits plays an important role in the non-homologous end-joining DNA repair pathway as a sensor of double strand DNA breaks. Ku is also involved in numerous cellular processes, and in some of them it acts in an RNA-dependent manner. However, RNA binding properties of the human Ku have not been well studied. Here we have analyzed interactions of a recombinant Ku heterodimer with a set of RNAs of various structure as well as eCLIP (enhanced crosslinking and immunoprecipitation) data for human Ku70. As a result, we have proposed a consensus RNA structure preferable for the Ku binding that is a hairpin possessing a bulge just near GpG sequence-containing terminal loop. 7SK snRNA is a scaffold for a ribonucleoprotein complex (7SK snRNP), which is known to participate in transcription regulation. We have shown that the recombinant Ku specifically binds a G-rich loop of hairpin 1 within 7SK snRNA. Moreover, Ku protein has been co-precipitated from HEK 293T cells with endogenous 7SK snRNA and such proteins included in 7SK snRNP as HEXIM1, Cdk9 and CTIP2. Ku and Cdk9 binding is found to be RNA-independent, meanwhile HEXIM1 and Ku co-precipitation depended on the presence of intact 7SK snRNA. The latter result has been confirmed using recombinant HEXIM1 and Ku proteins. Colocalization of Ku and CTIP2 was additionally confirmed by confocal microscopy. These results allow us to propose human Ku as a new component of the 7SK snRNP complex.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Human Ku heterodimeric protein composed of Ku70 and Ku80 subunits plays an important role in the non-homologous end-joining DNA repair pathway as a sensor of double strand DNA breaks. Ku is also involved in numerous cellular processes, and in some of them it acts in an RNA-dependent manner. However, RNA binding properties of the human Ku have not been well studied. Here we have analyzed interactions of a recombinant Ku heterodimer with a set of RNAs of various structure as well as eCLIP (enhanced crosslinking and immunoprecipitation) data for human Ku70. As a result, we have proposed a consensus RNA structure preferable for the Ku binding that is a hairpin possessing a bulge just near GpG sequence-containing terminal loop. 7SK snRNA is a scaffold for a ribonucleoprotein complex (7SK snRNP), which is known to participate in transcription regulation. We have shown that the recombinant Ku specifically binds a G-rich loop of hairpin 1 within 7SK snRNA. Moreover, Ku protein has been co-precipitated from HEK 293T cells with endogenous 7SK snRNA and such proteins included in 7SK snRNP as HEXIM1, Cdk9 and CTIP2. Ku and Cdk9 binding is found to be RNA-independent, meanwhile HEXIM1 and Ku co-precipitation depended on the presence of intact 7SK snRNA. The latter result has been confirmed using recombinant HEXIM1 and Ku proteins. Colocalization of Ku and CTIP2 was additionally confirmed by confocal microscopy. These results allow us to propose human Ku as a new component of the 7SK snRNP complex.
2019
Ait-Ammar Amina, Kula Anna, Darcis Gilles, Verdikt Roxane, Wit Stephane De, Gautier Virginie, Mallon Patrick W G, Marcello Alessandro, Rohr Olivier, Lint Carine Van
Current Status of Latency Reversing Agents Facing the Heterogeneity of HIV-1 Cellular and Tissue Reservoirs Journal Article
In: Front Microbiol, vol. 10, pp. 3060, 2019, ISSN: 1664-302X.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid32038533,
title = {Current Status of Latency Reversing Agents Facing the Heterogeneity of HIV-1 Cellular and Tissue Reservoirs},
author = {Amina Ait-Ammar and Anna Kula and Gilles Darcis and Roxane Verdikt and Stephane De Wit and Virginie Gautier and Patrick W G Mallon and Alessandro Marcello and Olivier Rohr and Carine Van Lint},
doi = {10.3389/fmicb.2019.03060},
issn = {1664-302X},
year = {2019},
date = {2019-01-01},
urldate = {2019-01-01},
journal = {Front Microbiol},
volume = {10},
pages = {3060},
abstract = {One of the most explored therapeutic approaches aimed at eradicating HIV-1 reservoirs is the "shock and kill" strategy which is based on HIV-1 reactivation in latently-infected cells ("shock" phase) while maintaining antiretroviral therapy (ART) in order to prevent spreading of the infection by the neosynthesized virus. This kind of strategy allows for the "kill" phase, during which latently-infected cells die from viral cytopathic effects or from host cytolytic effector mechanisms following viral reactivation. Several latency reversing agents (LRAs) with distinct mechanistic classes have been characterized to reactivate HIV-1 viral gene expression. Some LRAs have been tested in terms of their potential to purge latent HIV-1 in clinical trials, showing that reversing HIV-1 latency is possible. However, LRAs alone have failed to reduce the size of the viral reservoirs. Together with the inability of the immune system to clear the LRA-activated reservoirs and the lack of specificity of these LRAs, the heterogeneity of the reservoirs largely contributes to the limited success of clinical trials using LRAs. Indeed, HIV-1 latency is established in numerous cell types that are characterized by distinct phenotypes and metabolic properties, and these are influenced by patient history. Hence, the silencing mechanisms of HIV-1 gene expression in these cellular and tissue reservoirs need to be better understood to rationally improve this cure strategy and hopefully reach clinical success.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
One of the most explored therapeutic approaches aimed at eradicating HIV-1 reservoirs is the "shock and kill" strategy which is based on HIV-1 reactivation in latently-infected cells ("shock" phase) while maintaining antiretroviral therapy (ART) in order to prevent spreading of the infection by the neosynthesized virus. This kind of strategy allows for the "kill" phase, during which latently-infected cells die from viral cytopathic effects or from host cytolytic effector mechanisms following viral reactivation. Several latency reversing agents (LRAs) with distinct mechanistic classes have been characterized to reactivate HIV-1 viral gene expression. Some LRAs have been tested in terms of their potential to purge latent HIV-1 in clinical trials, showing that reversing HIV-1 latency is possible. However, LRAs alone have failed to reduce the size of the viral reservoirs. Together with the inability of the immune system to clear the LRA-activated reservoirs and the lack of specificity of these LRAs, the heterogeneity of the reservoirs largely contributes to the limited success of clinical trials using LRAs. Indeed, HIV-1 latency is established in numerous cell types that are characterized by distinct phenotypes and metabolic properties, and these are influenced by patient history. Hence, the silencing mechanisms of HIV-1 gene expression in these cellular and tissue reservoirs need to be better understood to rationally improve this cure strategy and hopefully reach clinical success.
Wallet Clementine, Rovere Marco De, Assche Jeanne Van, Daouad Fadoua, Wit Stéphane De, Gautier Virginie, Mallon Patrick W G, Marcello Alessandro, Lint Carine Van, Rohr Olivier, Schwartz Christian
Microglial Cells: The Main HIV-1 Reservoir in the Brain Journal Article
In: Front Cell Infect Microbiol, vol. 9, pp. 362, 2019, ISSN: 2235-2988.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid31709195,
title = {Microglial Cells: The Main HIV-1 Reservoir in the Brain},
author = {Clementine Wallet and Marco De Rovere and Jeanne Van Assche and Fadoua Daouad and Stéphane De Wit and Virginie Gautier and Patrick W G Mallon and Alessandro Marcello and Carine Van Lint and Olivier Rohr and Christian Schwartz},
doi = {10.3389/fcimb.2019.00362},
issn = {2235-2988},
year = {2019},
date = {2019-01-01},
urldate = {2019-01-01},
journal = {Front Cell Infect Microbiol},
volume = {9},
pages = {362},
abstract = {Despite efficient combination of the antiretroviral therapy (cART), which significantly decreased mortality and morbidity of HIV-1 infection, a definitive HIV cure has not been achieved. Hidden HIV-1 in cellular and anatomic reservoirs is the major hurdle toward a functional cure. Microglial cells, the Central Nervous system (CNS) resident macrophages, are one of the major cellular reservoirs of latent HIV-1. These cells are believed to be involved in the emergence of drugs resistance and reseeding peripheral tissues. Moreover, these long-life reservoirs are also involved in the development of HIV-1-associated neurocognitive diseases (HAND). Clearing these infected cells from the brain is therefore crucial to achieve a cure. However, many characteristics of microglial cells and the CNS hinder the eradication of these brain reservoirs. Better understandings of the specific molecular mechanisms of HIV-1 latency in microglial cells should help to design new molecules and new strategies preventing HAND and achieving HIV cure. Moreover, new strategies are needed to circumvent the limitations associated to anatomical sanctuaries with barriers such as the blood brain barrier (BBB) that reduce the access of drugs.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Despite efficient combination of the antiretroviral therapy (cART), which significantly decreased mortality and morbidity of HIV-1 infection, a definitive HIV cure has not been achieved. Hidden HIV-1 in cellular and anatomic reservoirs is the major hurdle toward a functional cure. Microglial cells, the Central Nervous system (CNS) resident macrophages, are one of the major cellular reservoirs of latent HIV-1. These cells are believed to be involved in the emergence of drugs resistance and reseeding peripheral tissues. Moreover, these long-life reservoirs are also involved in the development of HIV-1-associated neurocognitive diseases (HAND). Clearing these infected cells from the brain is therefore crucial to achieve a cure. However, many characteristics of microglial cells and the CNS hinder the eradication of these brain reservoirs. Better understandings of the specific molecular mechanisms of HIV-1 latency in microglial cells should help to design new molecules and new strategies preventing HAND and achieving HIV cure. Moreover, new strategies are needed to circumvent the limitations associated to anatomical sanctuaries with barriers such as the blood brain barrier (BBB) that reduce the access of drugs.
2017
Schwartz Christian, Bouchat Sophie, Marban Céline, Gautier Virginie, Lint Carine Van, Rohr Olivier, Douce Valentin Le
On the way to find a cure: Purging latent HIV-1 reservoirs Journal Article
In: Biochem Pharmacol, vol. 146, pp. 10–22, 2017, ISSN: 1873-2968.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid28687465,
title = {On the way to find a cure: Purging latent HIV-1 reservoirs},
author = {Christian Schwartz and Sophie Bouchat and Céline Marban and Virginie Gautier and Carine Van Lint and Olivier Rohr and Valentin Le Douce},
doi = {10.1016/j.bcp.2017.07.001},
issn = {1873-2968},
year = {2017},
date = {2017-12-01},
urldate = {2017-12-01},
journal = {Biochem Pharmacol},
volume = {146},
pages = {10--22},
abstract = {Introduction of cART in 1996 has drastically increased the life expectancy of people living with HIV-1. However, this treatment has not allowed cure as cessation of cART is associated with a rapid viral rebound. The main barrier to the eradication of the virus is related to the persistence of latent HIV reservoirs. Evidence is now accumulating that purging the HIV-1 reservoir might lead to a cure or a remission. The most studied strategy is the so called "shock and kill" therapy. This strategy is based on reactivation of dormant viruses from the latently-infected reservoirs (the shock) followed by the eradication of the reservoirs (the kill). This review focuses mainly on the recent advances made in the "shock and kill" therapy. We believe that a cure or a remission will come from combinatorial approaches i.e. combination of drugs to reactivate the dormant virus from all the reservoirs including the one located in sanctuaries, and combination of strategies boosting the immune system. Alternative strategies based on cell and gene therapy or based in inducing deep latency, which are evoked in this review reinforce the idea that at least a remission is attainable.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Introduction of cART in 1996 has drastically increased the life expectancy of people living with HIV-1. However, this treatment has not allowed cure as cessation of cART is associated with a rapid viral rebound. The main barrier to the eradication of the virus is related to the persistence of latent HIV reservoirs. Evidence is now accumulating that purging the HIV-1 reservoir might lead to a cure or a remission. The most studied strategy is the so called "shock and kill" therapy. This strategy is based on reactivation of dormant viruses from the latently-infected reservoirs (the shock) followed by the eradication of the reservoirs (the kill). This review focuses mainly on the recent advances made in the "shock and kill" therapy. We believe that a cure or a remission will come from combinatorial approaches i.e. combination of drugs to reactivate the dormant virus from all the reservoirs including the one located in sanctuaries, and combination of strategies boosting the immune system. Alternative strategies based on cell and gene therapy or based in inducing deep latency, which are evoked in this review reinforce the idea that at least a remission is attainable.
Fauquenoy Sylvain, Robette Gwenaëlle, Kula Anna, Vanhulle Caroline, Bouchat Sophie, Delacourt Nadège, Rodari Anthony, Marban Céline, Schwartz Christian, Burny Arsène, Rohr Olivier, Driessche Benoit Van, Lint Carine Van
Repression of Human T-lymphotropic virus type 1 Long Terminal Repeat sense transcription by Sp1 recruitment to novel Sp1 binding sites Journal Article
In: Sci Rep, vol. 7, pp. 43221, 2017, ISSN: 2045-2322.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid28256531,
title = {Repression of Human T-lymphotropic virus type 1 Long Terminal Repeat sense transcription by Sp1 recruitment to novel Sp1 binding sites},
author = {Sylvain Fauquenoy and Gwenaëlle Robette and Anna Kula and Caroline Vanhulle and Sophie Bouchat and Nadège Delacourt and Anthony Rodari and Céline Marban and Christian Schwartz and Arsène Burny and Olivier Rohr and Benoit Van Driessche and Carine Van Lint},
doi = {10.1038/srep43221},
issn = {2045-2322},
year = {2017},
date = {2017-03-01},
urldate = {2017-03-01},
journal = {Sci Rep},
volume = {7},
pages = {43221},
abstract = {Human T-lymphotropic Virus type 1 (HTLV-1) infection is characterized by viral latency in the majority of infected cells and by the absence of viremia. These features are thought to be due to the repression of viral sense transcription in vivo. Here, our in silico analysis of the HTLV-1 Long Terminal Repeat (LTR) promoter nucleotide sequence revealed, in addition to the four Sp1 binding sites previously identified, the presence of two additional potential Sp1 sites within the R region. We demonstrated that the Sp1 and Sp3 transcription factors bound in vitro to these two sites and compared the binding affinity for Sp1 of all six different HTLV-1 Sp1 sites. By chromatin immunoprecipitation experiments, we showed Sp1 recruitment in vivo to the newly identified Sp1 sites. We demonstrated in the nucleosomal context of an episomal reporter vector that the Sp1 sites interfered with both the sense and antisense LTR promoter activities. Interestingly, the Sp1 sites exhibited together a repressor effect on the LTR sense transcriptional activity but had no effect on the LTR antisense activity. Thus, our results demonstrate the presence of two new functional Sp1 binding sites in the HTLV-1 LTR, which act as negative cis-regulatory elements of sense viral transcription.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Human T-lymphotropic Virus type 1 (HTLV-1) infection is characterized by viral latency in the majority of infected cells and by the absence of viremia. These features are thought to be due to the repression of viral sense transcription in vivo. Here, our in silico analysis of the HTLV-1 Long Terminal Repeat (LTR) promoter nucleotide sequence revealed, in addition to the four Sp1 binding sites previously identified, the presence of two additional potential Sp1 sites within the R region. We demonstrated that the Sp1 and Sp3 transcription factors bound in vitro to these two sites and compared the binding affinity for Sp1 of all six different HTLV-1 Sp1 sites. By chromatin immunoprecipitation experiments, we showed Sp1 recruitment in vivo to the newly identified Sp1 sites. We demonstrated in the nucleosomal context of an episomal reporter vector that the Sp1 sites interfered with both the sense and antisense LTR promoter activities. Interestingly, the Sp1 sites exhibited together a repressor effect on the LTR sense transcriptional activity but had no effect on the LTR antisense activity. Thus, our results demonstrate the presence of two new functional Sp1 binding sites in the HTLV-1 LTR, which act as negative cis-regulatory elements of sense viral transcription.
Darcis Gilles, Bouchat Sophie, Kula Anna, Driessche Benoit Van, Delacourt Nadège, Vanhulle Caroline, Avettand-Fenoel Véronique, Wit Stéphane De, Rohr Olivier, Rouzioux Christine, Lint Carine Van
Reactivation capacity by latency-reversing agents ex vivo correlates with the size of the HIV-1 reservoir Journal Article
In: AIDS, vol. 31, no. 2, pp. 181–189, 2017, ISSN: 1473-5571.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid27755105,
title = {Reactivation capacity by latency-reversing agents ex vivo correlates with the size of the HIV-1 reservoir},
author = {Gilles Darcis and Sophie Bouchat and Anna Kula and Benoit Van Driessche and Nadège Delacourt and Caroline Vanhulle and Véronique Avettand-Fenoel and Stéphane De Wit and Olivier Rohr and Christine Rouzioux and Carine Van Lint},
doi = {10.1097/QAD.0000000000001290},
issn = {1473-5571},
year = {2017},
date = {2017-01-01},
urldate = {2017-01-01},
journal = {AIDS},
volume = {31},
number = {2},
pages = {181--189},
abstract = {OBJECTIVE: HIV-1 reservoirs are the major hurdle to virus clearance in combination antiretroviral therapy (cART)-treated patients. An approach to eradicating HIV-1 involves reversing latency in cART-treated patients to make latent cells visible to the host immune system. Stimulation of patient cell cultures with latency-reversing agents (LRAs) ex vivo results in heterogeneous responses among HIV-infected patients. Determinants of this heterogeneity are unknown and consequently important to determine.nnDESIGN AND METHODS: Here, we grouped and retrospectively analyzed the data from our two recent HIV-1 reactivation studies to investigate the role of the HIV-1 reservoir size in the reactivation capacity by LRAs in ex vivo cultures of CD8-depleted peripheral blood mononuclear cells (PBMCs) isolated from 54 cART-treated patients and of resting CD4 T cells isolated from 30 cART-treated patients.nnRESULTS: Our results established a statistically relevant positive correlation between the HIV-1 reservoir size measured by total cell-associated HIV-1 DNA and the frequency of positive HIV-1 recovery measurements in response to various LRAs in ex vivo cultures of cells isolated from cART-treated HIV aviremic patients. HIV-1 reservoir size also correlated with the extracellular HIV-1 RNA median level measured in supernatants of cell cultures following LRA treatments. However, we identified HIV patients whose positive measurements frequency and median level of extracellular HIV-1 RNA deviated from linearity relative to their corresponding HIV reservoir size.nnCONCLUSION: We demonstrated that the reservoir size is one predictive marker of LRA effectiveness but this parameter alone is not sufficient. The identification of other predictive markers is necessary to predict the success of HIV anti-latency approaches.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
OBJECTIVE: HIV-1 reservoirs are the major hurdle to virus clearance in combination antiretroviral therapy (cART)-treated patients. An approach to eradicating HIV-1 involves reversing latency in cART-treated patients to make latent cells visible to the host immune system. Stimulation of patient cell cultures with latency-reversing agents (LRAs) ex vivo results in heterogeneous responses among HIV-infected patients. Determinants of this heterogeneity are unknown and consequently important to determine.nnDESIGN AND METHODS: Here, we grouped and retrospectively analyzed the data from our two recent HIV-1 reactivation studies to investigate the role of the HIV-1 reservoir size in the reactivation capacity by LRAs in ex vivo cultures of CD8-depleted peripheral blood mononuclear cells (PBMCs) isolated from 54 cART-treated patients and of resting CD4 T cells isolated from 30 cART-treated patients.nnRESULTS: Our results established a statistically relevant positive correlation between the HIV-1 reservoir size measured by total cell-associated HIV-1 DNA and the frequency of positive HIV-1 recovery measurements in response to various LRAs in ex vivo cultures of cells isolated from cART-treated HIV aviremic patients. HIV-1 reservoir size also correlated with the extracellular HIV-1 RNA median level measured in supernatants of cell cultures following LRA treatments. However, we identified HIV patients whose positive measurements frequency and median level of extracellular HIV-1 RNA deviated from linearity relative to their corresponding HIV reservoir size.nnCONCLUSION: We demonstrated that the reservoir size is one predictive marker of LRA effectiveness but this parameter alone is not sufficient. The identification of other predictive markers is necessary to predict the success of HIV anti-latency approaches.
2016
Douce Valentin Le, Forouzanfar Faezeh, Eilebrecht Sebastian, Driessche Benoit Van, Ait-Ammar Amina, Verdikt Roxane, Kurashige Yoshihito, Marban Céline, Gautier Virginie, Candolfi Ermanno, Benecke Arndt G, Lint Carine Van, Rohr Olivier, Schwartz Christian
HIC1 controls cellular- and HIV-1- gene transcription via interactions with CTIP2 and HMGA1 Journal Article
In: Sci Rep, vol. 6, pp. 34920, 2016, ISSN: 2045-2322.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid27725726,
title = {HIC1 controls cellular- and HIV-1- gene transcription via interactions with CTIP2 and HMGA1},
author = {Valentin Le Douce and Faezeh Forouzanfar and Sebastian Eilebrecht and Benoit Van Driessche and Amina Ait-Ammar and Roxane Verdikt and Yoshihito Kurashige and Céline Marban and Virginie Gautier and Ermanno Candolfi and Arndt G Benecke and Carine Van Lint and Olivier Rohr and Christian Schwartz},
doi = {10.1038/srep34920},
issn = {2045-2322},
year = {2016},
date = {2016-10-01},
urldate = {2016-10-01},
journal = {Sci Rep},
volume = {6},
pages = {34920},
abstract = {Among many cellular transcriptional regulators, Bcl11b/CTIP2 and HGMA1 have been described to control the establishment and the persistence of HIV-1 latency in microglial cells, the main viral reservoir in the brain. In this present work, we identify and characterize a transcription factor i.e. HIC1, which physically interacts with both Bcl11b/CTIP2 and HMGA1 to co-regulate specific subsets of cellular genes and the viral HIV-1 gene. Our results suggest that HIC1 represses Tat dependent HIV-1 transcription. Interestingly, this repression of Tat function is linked to HIC1 K314 acetylation status and to SIRT1 deacetylase activity. Finally, we show that HIC1 interacts and cooperates with HGMA1 to regulate Tat dependent HIV-1 transcription. Our results also suggest that HIC1 repression of Tat function happens in a TAR dependent manner and that this TAR element may serve as HIC1 reservoir at the viral promoter to facilitate HIC1/TAT interaction.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Among many cellular transcriptional regulators, Bcl11b/CTIP2 and HGMA1 have been described to control the establishment and the persistence of HIV-1 latency in microglial cells, the main viral reservoir in the brain. In this present work, we identify and characterize a transcription factor i.e. HIC1, which physically interacts with both Bcl11b/CTIP2 and HMGA1 to co-regulate specific subsets of cellular genes and the viral HIV-1 gene. Our results suggest that HIC1 represses Tat dependent HIV-1 transcription. Interestingly, this repression of Tat function is linked to HIC1 K314 acetylation status and to SIRT1 deacetylase activity. Finally, we show that HIC1 interacts and cooperates with HGMA1 to regulate Tat dependent HIV-1 transcription. Our results also suggest that HIC1 repression of Tat function happens in a TAR dependent manner and that this TAR element may serve as HIC1 reservoir at the viral promoter to facilitate HIC1/TAT interaction.
2015
Darcis Gilles, Kula Anna, Bouchat Sophie, Fujinaga Koh, Corazza Francis, Ait-Ammar Amina, Delacourt Nadège, Melard Adeline, Kabeya Kabamba, Vanhulle Caroline, Driessche Benoit Van, Gatot Jean-Stéphane, Cherrier Thomas, Pianowski Luiz F, Gama Lucio, Schwartz Christian, Vila Jorge, Burny Arsène, Clumeck Nathan, Moutschen Michel, Wit Stéphane De, Peterlin B Matija, Rouzioux Christine, Rohr Olivier, Lint Carine Van
In: PLoS Pathog, vol. 11, no. 7, pp. e1005063, 2015, ISSN: 1553-7374.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid26225566,
title = {An In-Depth Comparison of Latency-Reversing Agent Combinations in Various In Vitro and Ex Vivo HIV-1 Latency Models Identified Bryostatin-1+JQ1 and Ingenol-B+JQ1 to Potently Reactivate Viral Gene Expression},
author = {Gilles Darcis and Anna Kula and Sophie Bouchat and Koh Fujinaga and Francis Corazza and Amina Ait-Ammar and Nadège Delacourt and Adeline Melard and Kabamba Kabeya and Caroline Vanhulle and Benoit Van Driessche and Jean-Stéphane Gatot and Thomas Cherrier and Luiz F Pianowski and Lucio Gama and Christian Schwartz and Jorge Vila and Arsène Burny and Nathan Clumeck and Michel Moutschen and Stéphane De Wit and B Matija Peterlin and Christine Rouzioux and Olivier Rohr and Carine Van Lint},
doi = {10.1371/journal.ppat.1005063},
issn = {1553-7374},
year = {2015},
date = {2015-07-01},
urldate = {2015-07-01},
journal = {PLoS Pathog},
volume = {11},
number = {7},
pages = {e1005063},
abstract = {The persistence of latently infected cells in patients under combinatory antiretroviral therapy (cART) is a major hurdle to HIV-1 eradication. Strategies to purge these reservoirs are needed and activation of viral gene expression in latently infected cells is one promising strategy. Bromodomain and Extraterminal (BET) bromodomain inhibitors (BETi) are compounds able to reactivate latent proviruses in a positive transcription elongation factor b (P-TEFb)-dependent manner. In this study, we tested the reactivation potential of protein kinase C (PKC) agonists (prostratin, bryostatin-1 and ingenol-B), which are known to activate NF-κB signaling pathway as well as P-TEFb, used alone or in combination with P-TEFb-releasing agents (HMBA and BETi (JQ1, I-BET, I-BET151)). Using in vitro HIV-1 post-integration latency model cell lines of T-lymphoid and myeloid lineages, we demonstrated that PKC agonists and P-TEFb-releasing agents alone acted as potent latency-reversing agents (LRAs) and that their combinations led to synergistic activation of HIV-1 expression at the viral mRNA and protein levels. Mechanistically, combined treatments led to higher activations of P-TEFb and NF-κB than the corresponding individual drug treatments. Importantly, we observed in ex vivo cultures of CD8+-depleted PBMCs from 35 cART-treated HIV-1+ aviremic patients that the percentage of reactivated cultures following combinatory bryostatin-1+JQ1 treatment was identical to the percentage observed with anti-CD3+anti-CD28 antibodies positive control stimulation. Remarkably, in ex vivo cultures of resting CD4+ T cells isolated from 15 HIV-1+ cART-treated aviremic patients, the combinations bryostatin-1+JQ1 and ingenol-B+JQ1 released infectious viruses to levels similar to that obtained with the positive control stimulation. The potent effects of these two combination treatments were already detected 24 hours post-stimulation. These results constitute the first demonstration of LRA combinations exhibiting such a potent effect and represent a proof-of-concept for the co-administration of two different types of LRAs as a potential strategy to reduce the size of the latent HIV-1 reservoirs.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The persistence of latently infected cells in patients under combinatory antiretroviral therapy (cART) is a major hurdle to HIV-1 eradication. Strategies to purge these reservoirs are needed and activation of viral gene expression in latently infected cells is one promising strategy. Bromodomain and Extraterminal (BET) bromodomain inhibitors (BETi) are compounds able to reactivate latent proviruses in a positive transcription elongation factor b (P-TEFb)-dependent manner. In this study, we tested the reactivation potential of protein kinase C (PKC) agonists (prostratin, bryostatin-1 and ingenol-B), which are known to activate NF-κB signaling pathway as well as P-TEFb, used alone or in combination with P-TEFb-releasing agents (HMBA and BETi (JQ1, I-BET, I-BET151)). Using in vitro HIV-1 post-integration latency model cell lines of T-lymphoid and myeloid lineages, we demonstrated that PKC agonists and P-TEFb-releasing agents alone acted as potent latency-reversing agents (LRAs) and that their combinations led to synergistic activation of HIV-1 expression at the viral mRNA and protein levels. Mechanistically, combined treatments led to higher activations of P-TEFb and NF-κB than the corresponding individual drug treatments. Importantly, we observed in ex vivo cultures of CD8+-depleted PBMCs from 35 cART-treated HIV-1+ aviremic patients that the percentage of reactivated cultures following combinatory bryostatin-1+JQ1 treatment was identical to the percentage observed with anti-CD3+anti-CD28 antibodies positive control stimulation. Remarkably, in ex vivo cultures of resting CD4+ T cells isolated from 15 HIV-1+ cART-treated aviremic patients, the combinations bryostatin-1+JQ1 and ingenol-B+JQ1 released infectious viruses to levels similar to that obtained with the positive control stimulation. The potent effects of these two combination treatments were already detected 24 hours post-stimulation. These results constitute the first demonstration of LRA combinations exhibiting such a potent effect and represent a proof-of-concept for the co-administration of two different types of LRAs as a potential strategy to reduce the size of the latent HIV-1 reservoirs.
2014
Douce Valentin Le, Cherrier Thomas, Riclet Raphael, Rohr Olivier, Schwartz Christian
The many lives of CTIP2: from AIDS to cancer and cardiac hypertrophy Journal Article
In: J Cell Physiol, vol. 229, no. 5, pp. 533–537, 2014, ISSN: 1097-4652.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid24122342,
title = {The many lives of CTIP2: from AIDS to cancer and cardiac hypertrophy},
author = {Valentin Le Douce and Thomas Cherrier and Raphael Riclet and Olivier Rohr and Christian Schwartz},
doi = {10.1002/jcp.24490},
issn = {1097-4652},
year = {2014},
date = {2014-05-01},
urldate = {2014-05-01},
journal = {J Cell Physiol},
volume = {229},
number = {5},
pages = {533--537},
abstract = {CTIP2 is a key transcriptional regulator involved in numerous physiological functions. Initial works have shown the importance of CTIP2 in the establishment and persistence of HIV latency in microglial cells, the main latent/quiescent viral reservoir in the brain. Recent studies have highlighted the importance of CTIP2 in several other pathologies, such as cardiac hypertrophy and various types of human malignancies. Targeting CTIP2 may therefore constitute a new approach in the treatment of these pathologies.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
CTIP2 is a key transcriptional regulator involved in numerous physiological functions. Initial works have shown the importance of CTIP2 in the establishment and persistence of HIV latency in microglial cells, the main latent/quiescent viral reservoir in the brain. Recent studies have highlighted the importance of CTIP2 in several other pathologies, such as cardiac hypertrophy and various types of human malignancies. Targeting CTIP2 may therefore constitute a new approach in the treatment of these pathologies.
Eilebrecht Sebastian, Douce Valentin Le, Riclet Raphael, Targat Brice, Hallay Houda, Driessche Benot Van, Schwartz Christian, Robette Gwenaëlle, Lint Carine Van, Rohr Olivier, Benecke Arndt G
HMGA1 recruits CTIP2-repressed P-TEFb to the HIV-1 and cellular target promoters Journal Article
In: Nucleic Acids Res, vol. 42, no. 8, pp. 4962–4971, 2014, ISSN: 1362-4962.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid24623795,
title = {HMGA1 recruits CTIP2-repressed P-TEFb to the HIV-1 and cellular target promoters},
author = {Sebastian Eilebrecht and Valentin Le Douce and Raphael Riclet and Brice Targat and Houda Hallay and Benot Van Driessche and Christian Schwartz and Gwenaëlle Robette and Carine Van Lint and Olivier Rohr and Arndt G Benecke},
doi = {10.1093/nar/gku168},
issn = {1362-4962},
year = {2014},
date = {2014-04-01},
urldate = {2014-04-01},
journal = {Nucleic Acids Res},
volume = {42},
number = {8},
pages = {4962--4971},
abstract = {Active positive transcription elongation factor b (P-TEFb) is essential for cellular and human immunodeficiency virus type 1 (HIV-1) transcription elongation. CTIP2 represses P-TEFb activity in a complex containing 7SK RNA and HEXIM1. Recently, the inactive 7SK/P-TEFb small nuclear RNP (snRNP) has been detected at the HIV-1 core promoter as well as at the promoters of cellular genes, but a recruiting mechanism still remains unknown to date. Here we show global synergy between CTIP2 and the 7SK-binding chromatin master-regulator HMGA1 in terms of P-TEFb-dependent endogenous and HIV-1 gene expression regulation. While CTIP2 and HMGA1 concordingly repress the expression of cellular 7SK-dependent P-TEFb targets, the simultaneous knock-down of CTIP2 and HMGA1 also results in a boost in Tat-dependent and independent HIV-1 promoter activity. Chromatin immunoprecipitation experiments reveal a significant loss of CTIP2/7SK/P-TEFb snRNP recruitment to cellular gene promoters and the HIV-1 promoter on HMGA1 knock-down. Our findings not only provide insights into a recruiting mechanism for the inactive 7SK/P-TEFb snRNP, but may also contribute to a better understanding of viral latency.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Active positive transcription elongation factor b (P-TEFb) is essential for cellular and human immunodeficiency virus type 1 (HIV-1) transcription elongation. CTIP2 represses P-TEFb activity in a complex containing 7SK RNA and HEXIM1. Recently, the inactive 7SK/P-TEFb small nuclear RNP (snRNP) has been detected at the HIV-1 core promoter as well as at the promoters of cellular genes, but a recruiting mechanism still remains unknown to date. Here we show global synergy between CTIP2 and the 7SK-binding chromatin master-regulator HMGA1 in terms of P-TEFb-dependent endogenous and HIV-1 gene expression regulation. While CTIP2 and HMGA1 concordingly repress the expression of cellular 7SK-dependent P-TEFb targets, the simultaneous knock-down of CTIP2 and HMGA1 also results in a boost in Tat-dependent and independent HIV-1 promoter activity. Chromatin immunoprecipitation experiments reveal a significant loss of CTIP2/7SK/P-TEFb snRNP recruitment to cellular gene promoters and the HIV-1 promoter on HMGA1 knock-down. Our findings not only provide insights into a recruiting mechanism for the inactive 7SK/P-TEFb snRNP, but may also contribute to a better understanding of viral latency.
2013
Eilebrecht Sebastian, Schwartz Christian, Rohr Olivier
Non-coding RNAs: novel players in chromatin-regulation during viral latency Journal Article
In: Curr Opin Virol, vol. 3, no. 4, pp. 387–393, 2013, ISSN: 1879-6265.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid23660570,
title = {Non-coding RNAs: novel players in chromatin-regulation during viral latency},
author = {Sebastian Eilebrecht and Christian Schwartz and Olivier Rohr},
doi = {10.1016/j.coviro.2013.04.001},
issn = {1879-6265},
year = {2013},
date = {2013-08-01},
urldate = {2013-08-01},
journal = {Curr Opin Virol},
volume = {3},
number = {4},
pages = {387--393},
abstract = {Chromatin structure plays an essential role during gene expression regulation not only in the case of the host cellular genome, but also during the viral life cycle. Epigenetic chromatin marks thereby define, whether a gene promoter is accessible for the transcription machinery or whether a repressive heterochromatin state is established. The heterochromatin-mediated repression of lytic viral genes results in viral latency, enabling the virus to persist dormant without being recognized by the host immune system, but keeping the potential for reactivation. Arising new systems biology approaches are starting to uncover an unexpected multiplicity and variety of non-coding (nc)RNAs playing important roles during chromatin structure control, likely constituting a novel layer in epigenetic regulation. In this review we give an overview of chromatin-regulatory viral and host cellular ncRNAs and their links to viral latency.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Chromatin structure plays an essential role during gene expression regulation not only in the case of the host cellular genome, but also during the viral life cycle. Epigenetic chromatin marks thereby define, whether a gene promoter is accessible for the transcription machinery or whether a repressive heterochromatin state is established. The heterochromatin-mediated repression of lytic viral genes results in viral latency, enabling the virus to persist dormant without being recognized by the host immune system, but keeping the potential for reactivation. Arising new systems biology approaches are starting to uncover an unexpected multiplicity and variety of non-coding (nc)RNAs playing important roles during chromatin structure control, likely constituting a novel layer in epigenetic regulation. In this review we give an overview of chromatin-regulatory viral and host cellular ncRNAs and their links to viral latency.
Cherrier Thomas, Douce Valentin Le, Eilebrecht Sebastian, Riclet Raphael, Marban Céline, Dequiedt Franck, Goumon Yannick, Paillart Jean-Christophe, Mericskay Mathias, Parlakian Ara, Bausero Pedro, Abbas Wasim, Herbein Georges, Kurdistani Siavash K, Grana Xavier, Driessche Benoit Van, Schwartz Christian, Candolfi Ermanno, Benecke Arndt G, Lint Carine Van, Rohr Olivier
CTIP2 is a negative regulator of P-TEFb Journal Article
In: Proc Natl Acad Sci U S A, vol. 110, no. 31, pp. 12655–12660, 2013, ISSN: 1091-6490.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid23852730,
title = {CTIP2 is a negative regulator of P-TEFb},
author = {Thomas Cherrier and Valentin Le Douce and Sebastian Eilebrecht and Raphael Riclet and Céline Marban and Franck Dequiedt and Yannick Goumon and Jean-Christophe Paillart and Mathias Mericskay and Ara Parlakian and Pedro Bausero and Wasim Abbas and Georges Herbein and Siavash K Kurdistani and Xavier Grana and Benoit Van Driessche and Christian Schwartz and Ermanno Candolfi and Arndt G Benecke and Carine Van Lint and Olivier Rohr},
doi = {10.1073/pnas.1220136110},
issn = {1091-6490},
year = {2013},
date = {2013-07-01},
urldate = {2013-07-01},
journal = {Proc Natl Acad Sci U S A},
volume = {110},
number = {31},
pages = {12655--12660},
abstract = {The positive transcription elongation factor b (P-TEFb) is involved in physiological and pathological events including inflammation, cancer, AIDS, and cardiac hypertrophy. The balance between its active and inactive form is tightly controlled to ensure cellular integrity. We report that the transcriptional repressor CTIP2 is a major modulator of P-TEFb activity. CTIP2 copurifies and interacts with an inactive P-TEFb complex containing the 7SK snRNA and HEXIM1. CTIP2 associates directly with HEXIM1 and, via the loop 2 of the 7SK snRNA, with P-TEFb. In this nucleoprotein complex, CTIP2 significantly represses the Cdk9 kinase activity of P-TEFb. Accordingly, we show that CTIP2 inhibits large sets of P-TEFb- and 7SK snRNA-sensitive genes. In hearts of hypertrophic cardiomyopathic mice, CTIP2 controls P-TEFb-sensitive pathways involved in the establishment of this pathology. Overexpression of the β-myosin heavy chain protein contributes to the pathological cardiac wall thickening. The inactive P-TEFb complex associates with CTIP2 at the MYH7 gene promoter to repress its activity. Taken together, our results strongly suggest that CTIP2 controls P-TEFb function in physiological and pathological conditions.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The positive transcription elongation factor b (P-TEFb) is involved in physiological and pathological events including inflammation, cancer, AIDS, and cardiac hypertrophy. The balance between its active and inactive form is tightly controlled to ensure cellular integrity. We report that the transcriptional repressor CTIP2 is a major modulator of P-TEFb activity. CTIP2 copurifies and interacts with an inactive P-TEFb complex containing the 7SK snRNA and HEXIM1. CTIP2 associates directly with HEXIM1 and, via the loop 2 of the 7SK snRNA, with P-TEFb. In this nucleoprotein complex, CTIP2 significantly represses the Cdk9 kinase activity of P-TEFb. Accordingly, we show that CTIP2 inhibits large sets of P-TEFb- and 7SK snRNA-sensitive genes. In hearts of hypertrophic cardiomyopathic mice, CTIP2 controls P-TEFb-sensitive pathways involved in the establishment of this pathology. Overexpression of the β-myosin heavy chain protein contributes to the pathological cardiac wall thickening. The inactive P-TEFb complex associates with CTIP2 at the MYH7 gene promoter to repress its activity. Taken together, our results strongly suggest that CTIP2 controls P-TEFb function in physiological and pathological conditions.
Suh Andrew, Douce Valentin Le, Rohr Olivier, Schwartz Christian, Scott Ken
Pseudomonas DING proteins as human transcriptional regulators and HIV-1 antagonists Journal Article
In: Virol J, vol. 10, pp. 234, 2013, ISSN: 1743-422X.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid23855931,
title = {Pseudomonas DING proteins as human transcriptional regulators and HIV-1 antagonists},
author = {Andrew Suh and Valentin Le Douce and Olivier Rohr and Christian Schwartz and Ken Scott},
doi = {10.1186/1743-422X-10-234},
issn = {1743-422X},
year = {2013},
date = {2013-07-01},
urldate = {2013-07-01},
journal = {Virol J},
volume = {10},
pages = {234},
abstract = {BACKGROUND: Anti-HIV-1 therapy depends upon multiple agents that target different phases of the viral replication cycle. Recent reports indicate that plant and human DING proteins are unique in targeting viral gene transcription as the basis of their anti-HIV-1 therapy.nnMETHODS: Two cloned DING genes from Pseudomonas were transiently expressed in human cells, and effects on NFκB-mediated transcription, HIV-1 transcription, and HIV-1 production were measured.nnRESULTS: Both DING proteins elevated NFκB-mediated transcription. In microglial cells, one protein, from P. aeruginosa PA14, suppressed HIV-1 transcription; the other protein, from P. fluorescens SBW25, was inactive. The PA14DING protein also reduces HIV-1 production in microglial cells.nnCONCLUSIONS: Structural differences between the two DING proteins highlight regions of the PA14DING protein essential to the anti-HIV-1 activity, and may guide the design of therapeutic agents.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
BACKGROUND: Anti-HIV-1 therapy depends upon multiple agents that target different phases of the viral replication cycle. Recent reports indicate that plant and human DING proteins are unique in targeting viral gene transcription as the basis of their anti-HIV-1 therapy.nnMETHODS: Two cloned DING genes from Pseudomonas were transiently expressed in human cells, and effects on NFκB-mediated transcription, HIV-1 transcription, and HIV-1 production were measured.nnRESULTS: Both DING proteins elevated NFκB-mediated transcription. In microglial cells, one protein, from P. aeruginosa PA14, suppressed HIV-1 transcription; the other protein, from P. fluorescens SBW25, was inactive. The PA14DING protein also reduces HIV-1 production in microglial cells.nnCONCLUSIONS: Structural differences between the two DING proteins highlight regions of the PA14DING protein essential to the anti-HIV-1 activity, and may guide the design of therapeutic agents.
2012
Bouchat Sophie, Gatot Jean-Stéphane, Kabeya Kabamba, Cardona Christelle, Colin Laurence, Herbein Georges, Wit Stéphane De, Clumeck Nathan, Lambotte Olivier, Rouzioux Christine, Rohr Olivier, Lint Carine Van
Histone methyltransferase inhibitors induce HIV-1 recovery in resting CD4(+) T cells from HIV-1-infected HAART-treated patients Journal Article
In: AIDS, vol. 26, no. 12, pp. 1473–1482, 2012, ISSN: 1473-5571.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid22555163,
title = {Histone methyltransferase inhibitors induce HIV-1 recovery in resting CD4(+) T cells from HIV-1-infected HAART-treated patients},
author = {Sophie Bouchat and Jean-Stéphane Gatot and Kabamba Kabeya and Christelle Cardona and Laurence Colin and Georges Herbein and Stéphane De Wit and Nathan Clumeck and Olivier Lambotte and Christine Rouzioux and Olivier Rohr and Carine Van Lint},
doi = {10.1097/QAD.0b013e32835535f5},
issn = {1473-5571},
year = {2012},
date = {2012-07-01},
urldate = {2012-07-01},
journal = {AIDS},
volume = {26},
number = {12},
pages = {1473--1482},
abstract = {OBJECTIVE: 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.nnDESIGN: 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.nnMETHODS: 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.nnRESULTS: 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.nnCONCLUSION: 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.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
OBJECTIVE: 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.nnDESIGN: 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.nnMETHODS: 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.nnRESULTS: 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.nnCONCLUSION: 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.
Douce Valentin Le, Colin Laurence, Redel Laetitia, Cherrier Thomas, Herbein Georges, Aunis Dominique, Rohr Olivier, Lint Carine Van, Schwartz Christian
LSD1 cooperates with CTIP2 to promote HIV-1 transcriptional silencing Journal Article
In: Nucleic Acids Res, vol. 40, no. 5, pp. 1904–1915, 2012, ISSN: 1362-4962.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid22067449,
title = {LSD1 cooperates with CTIP2 to promote HIV-1 transcriptional silencing},
author = {Valentin Le Douce and Laurence Colin and Laetitia Redel and Thomas Cherrier and Georges Herbein and Dominique Aunis and Olivier Rohr and Carine Van Lint and Christian Schwartz},
doi = {10.1093/nar/gkr857},
issn = {1362-4962},
year = {2012},
date = {2012-03-01},
urldate = {2012-03-01},
journal = {Nucleic Acids Res},
volume = {40},
number = {5},
pages = {1904--1915},
abstract = {Microglial cells are the main HIV-1 targets in the central nervous system (CNS) and constitute an important reservoir of latently infected cells. Establishment and persistence of these reservoirs rely on the chromatin structure of the integrated proviruses. We have previously demonstrated that the cellular cofactor CTIP2 forces heterochromatin formation and HIV-1 gene silencing by recruiting HDAC and HMT activities at the integrated viral promoter. In the present work, we report that the histone demethylase LSD1 represses HIV-1 transcription and viral expression in a synergistic manner with CTIP2. We show that recruitment of LSD1 at the HIV-1 proximal promoter is associated with both H3K4me3 and H3K9me3 epigenetic marks. Finally, our data suggest that LSD1-induced H3K4 trimethylation is linked to hSET1 recruitment at the integrated provirus.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Microglial cells are the main HIV-1 targets in the central nervous system (CNS) and constitute an important reservoir of latently infected cells. Establishment and persistence of these reservoirs rely on the chromatin structure of the integrated proviruses. We have previously demonstrated that the cellular cofactor CTIP2 forces heterochromatin formation and HIV-1 gene silencing by recruiting HDAC and HMT activities at the integrated viral promoter. In the present work, we report that the histone demethylase LSD1 represses HIV-1 transcription and viral expression in a synergistic manner with CTIP2. We show that recruitment of LSD1 at the HIV-1 proximal promoter is associated with both H3K4me3 and H3K9me3 epigenetic marks. Finally, our data suggest that LSD1-induced H3K4 trimethylation is linked to hSET1 recruitment at the integrated provirus.
Djeghader Ahmed, Aragonès Gerard, Darbinian Nune, Elias Mikael, Gonzalez Daniel, García-Heredia Anabel, Beltrán-Debón Raúl, Kaminski Rafal, Gotthard Guillaume, Hiblot Julien, Rull Anna, Rohr Olivier, Schwartz Christian, Alonso-Villaverde Carlos, Joven Jorge, Camps Jordi, Chabriere Eric
The level of DING proteins is increased in HIV-infected patients: in vitro and in vivo studies Journal Article
In: PLoS One, vol. 7, no. 3, pp. e33062, 2012, ISSN: 1932-6203.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid22427948,
title = {The level of DING proteins is increased in HIV-infected patients: in vitro and in vivo studies},
author = {Ahmed Djeghader and Gerard Aragonès and Nune Darbinian and Mikael Elias and Daniel Gonzalez and Anabel García-Heredia and Raúl Beltrán-Debón and Rafal Kaminski and Guillaume Gotthard and Julien Hiblot and Anna Rull and Olivier Rohr and Christian Schwartz and Carlos Alonso-Villaverde and Jorge Joven and Jordi Camps and Eric Chabriere},
doi = {10.1371/journal.pone.0033062},
issn = {1932-6203},
year = {2012},
date = {2012-01-01},
urldate = {2012-01-01},
journal = {PLoS One},
volume = {7},
number = {3},
pages = {e33062},
abstract = {DING proteins constitute an interesting family, owing to their intriguing and important activities. However, after a decade of research, little is known about these proteins. In humans, at least five different DING proteins have been identified, which were implicated in important biological processes and diseases, including HIV. Indeed, recent data from different research groups have highlighted the anti-HIV activity of some DING representatives. These proteins share the ability to inhibit the transcriptional step of HIV-1, a key step of the viral cycle that is not yet targeted by the current therapies. Since such proteins have been isolated from humans, we undertook a comprehensive study that focuses on the relationship between these proteins and HIV-infection in an infectious context. Hence, we developed a home-made ELISA for the quantification of the concentration of DING proteins in human serum. Using this method, we were able to determine the concentration of DING proteins in healthy and HIV-infected patients. Interestingly, we observed a significant increase of the concentration of DING proteins in non treated and treated HIV-infected patients compared to controls. In addition, cell cultures infected with HIV also show an increased expression of DING proteins, ruling out the possible role of antiretroviral treatment in the increase of the expression of DING proteins. In conclusion, results from this study show that the organism reacts to HIV-infection by an overexpression of DING proteins.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
DING proteins constitute an interesting family, owing to their intriguing and important activities. However, after a decade of research, little is known about these proteins. In humans, at least five different DING proteins have been identified, which were implicated in important biological processes and diseases, including HIV. Indeed, recent data from different research groups have highlighted the anti-HIV activity of some DING representatives. These proteins share the ability to inhibit the transcriptional step of HIV-1, a key step of the viral cycle that is not yet targeted by the current therapies. Since such proteins have been isolated from humans, we undertook a comprehensive study that focuses on the relationship between these proteins and HIV-infection in an infectious context. Hence, we developed a home-made ELISA for the quantification of the concentration of DING proteins in human serum. Using this method, we were able to determine the concentration of DING proteins in healthy and HIV-infected patients. Interestingly, we observed a significant increase of the concentration of DING proteins in non treated and treated HIV-infected patients compared to controls. In addition, cell cultures infected with HIV also show an increased expression of DING proteins, ruling out the possible role of antiretroviral treatment in the increase of the expression of DING proteins. In conclusion, results from this study show that the organism reacts to HIV-infection by an overexpression of DING proteins.
2011
Cherrier Thomas, Elias Mikael, Jeudy Alicia, Gotthard Guillaume, Douce Valentin Le, Hallay Houda, Masson Patrick, Janossy Andrea, Candolfi Ermanno, Rohr Olivier, Chabrière Eric, Schwartz Christian
Human-Phosphate-Binding-Protein inhibits HIV-1 gene transcription and replication Journal Article
In: Virol J, vol. 8, pp. 352, 2011, ISSN: 1743-422X.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid21762475,
title = {Human-Phosphate-Binding-Protein inhibits HIV-1 gene transcription and replication},
author = {Thomas Cherrier and Mikael Elias and Alicia Jeudy and Guillaume Gotthard and Valentin Le Douce and Houda Hallay and Patrick Masson and Andrea Janossy and Ermanno Candolfi and Olivier Rohr and Eric Chabrière and Christian Schwartz},
doi = {10.1186/1743-422X-8-352},
issn = {1743-422X},
year = {2011},
date = {2011-07-01},
urldate = {2011-07-01},
journal = {Virol J},
volume = {8},
pages = {352},
abstract = {The Human Phosphate-Binding protein (HPBP) is a serendipitously discovered lipoprotein that binds phosphate with high affinity. HPBP belongs to the DING protein family, involved in various biological processes like cell cycle regulation. We report that HPBP inhibits HIV-1 gene transcription and replication in T cell line, primary peripherical blood lymphocytes and primary macrophages. We show that HPBP is efficient in naïve and HIV-1 AZT-resistant strains. Our results revealed HPBP as a new and potent anti HIV molecule that inhibits transcription of the virus, which has not yet been targeted by HAART and therefore opens new strategies in the treatment of HIV infection.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The Human Phosphate-Binding protein (HPBP) is a serendipitously discovered lipoprotein that binds phosphate with high affinity. HPBP belongs to the DING protein family, involved in various biological processes like cell cycle regulation. We report that HPBP inhibits HIV-1 gene transcription and replication in T cell line, primary peripherical blood lymphocytes and primary macrophages. We show that HPBP is efficient in naïve and HIV-1 AZT-resistant strains. Our results revealed HPBP as a new and potent anti HIV molecule that inhibits transcription of the virus, which has not yet been targeted by HAART and therefore opens new strategies in the treatment of HIV infection.
Marban Céline, Su Trent, Ferrari Roberto, Li Bing, Vatakis Dimitrios, Pellegrini Matteo, Zack Jerome A, Rohr Olivier, Kurdistani Siavash K
Genome-wide binding map of the HIV-1 Tat protein to the human genome Journal Article
In: PLoS One, vol. 6, no. 11, pp. e26894, 2011, ISSN: 1932-6203.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid22073215,
title = {Genome-wide binding map of the HIV-1 Tat protein to the human genome},
author = {Céline Marban and Trent Su and Roberto Ferrari and Bing Li and Dimitrios Vatakis and Matteo Pellegrini and Jerome A Zack and Olivier Rohr and Siavash K Kurdistani},
doi = {10.1371/journal.pone.0026894},
issn = {1932-6203},
year = {2011},
date = {2011-01-01},
urldate = {2011-01-01},
journal = {PLoS One},
volume = {6},
number = {11},
pages = {e26894},
abstract = {The HIV-1 Trans-Activator of Transcription (Tat) protein binds to multiple host cellular factors and greatly enhances the level of transcription of the HIV genome. While Tat's control of viral transcription is well-studied, much less is known about the interaction of Tat with the human genome. Here, we report the genome-wide binding map of Tat to the human genome in Jurkat T cells using chromatin immunoprecipitation combined with next-generation sequencing. Surprisingly, we found that ~53% of the Tat target regions are within DNA repeat elements, greater than half of which are Alu sequences. The remaining target regions are located in introns and distal intergenic regions; only ~7% of Tat-bound regions are near transcription start sites (TSS) at gene promoters. Interestingly, Tat binds to promoters of genes that, in Jurkat cells, are bound by the ETS1 transcription factor, the CBP histone acetyltransferase and/or are enriched for histone H3 lysine 4 tri-methylation (H3K4me3) and H3K27me3. Tat binding is associated with genes enriched with functions in T cell biology and immune response. Our data reveal that Tat's interaction with the host genome is more extensive than previously thought, with potentially important implications for the viral life cycle.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The HIV-1 Trans-Activator of Transcription (Tat) protein binds to multiple host cellular factors and greatly enhances the level of transcription of the HIV genome. While Tat's control of viral transcription is well-studied, much less is known about the interaction of Tat with the human genome. Here, we report the genome-wide binding map of Tat to the human genome in Jurkat T cells using chromatin immunoprecipitation combined with next-generation sequencing. Surprisingly, we found that ~53% of the Tat target regions are within DNA repeat elements, greater than half of which are Alu sequences. The remaining target regions are located in introns and distal intergenic regions; only ~7% of Tat-bound regions are near transcription start sites (TSS) at gene promoters. Interestingly, Tat binds to promoters of genes that, in Jurkat cells, are bound by the ETS1 transcription factor, the CBP histone acetyltransferase and/or are enriched for histone H3 lysine 4 tri-methylation (H3K4me3) and H3K27me3. Tat binding is associated with genes enriched with functions in T cell biology and immune response. Our data reveal that Tat's interaction with the host genome is more extensive than previously thought, with potentially important implications for the viral life cycle.
2010
Redel Laetitia, Douce Valentin Le, Cherrier Thomas, Marban Céline, Janossy Andrea, Aunis Dominique, Lint Carine Van, Rohr Olivier, Schwartz Christian
HIV-1 regulation of latency in the monocyte-macrophage lineage and in CD4+ T lymphocytes Journal Article
In: J Leukoc Biol, vol. 87, no. 4, pp. 575–588, 2010, ISSN: 1938-3673.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid19801499,
title = {HIV-1 regulation of latency in the monocyte-macrophage lineage and in CD4+ T lymphocytes},
author = {Laetitia Redel and Valentin Le Douce and Thomas Cherrier and Céline Marban and Andrea Janossy and Dominique Aunis and Carine Van Lint and Olivier Rohr and Christian Schwartz},
doi = {10.1189/jlb.0409264},
issn = {1938-3673},
year = {2010},
date = {2010-04-01},
urldate = {2010-04-01},
journal = {J Leukoc Biol},
volume = {87},
number = {4},
pages = {575--588},
abstract = {The introduction in 1996 of the HAART raised hopes for the eradication of HIV-1. Unfortunately, the discovery of latent HIV-1 reservoirs in CD4+ T cells and in the monocyte-macrophage lineage proved the optimism to be premature. The long-lived HIV-1 reservoirs constitute a major obstacle to the eradication of HIV-1. In this review, we focus on the establishment and maintenance of HIV-1 latency in the two major targets for HIV-1: the CD4+ T cells and the monocyte-macrophage lineage. Understanding the cell-type molecular mechanisms of establishment, maintenance, and reactivation of HIV-1 latency in these reservoirs is crucial for efficient therapeutic intervention. A complete viral eradication, the holy graal for clinicians, might be achieved by strategic interventions targeting latently and productively infected cells. We suggest that new approaches, such as the combination of different kinds of proviral activators, may help to reduce dramatically the size of latent HIV-1 reservoirs in patients on HAART.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The introduction in 1996 of the HAART raised hopes for the eradication of HIV-1. Unfortunately, the discovery of latent HIV-1 reservoirs in CD4+ T cells and in the monocyte-macrophage lineage proved the optimism to be premature. The long-lived HIV-1 reservoirs constitute a major obstacle to the eradication of HIV-1. In this review, we focus on the establishment and maintenance of HIV-1 latency in the two major targets for HIV-1: the CD4+ T cells and the monocyte-macrophage lineage. Understanding the cell-type molecular mechanisms of establishment, maintenance, and reactivation of HIV-1 latency in these reservoirs is crucial for efficient therapeutic intervention. A complete viral eradication, the holy graal for clinicians, might be achieved by strategic interventions targeting latently and productively infected cells. We suggest that new approaches, such as the combination of different kinds of proviral activators, may help to reduce dramatically the size of latent HIV-1 reservoirs in patients on HAART.
2007
Marban Céline, Suzanne Stella, Dequiedt Franck, de Walque Stéphane, Redel Laetitia, Lint Carine Van, Aunis Dominique, Rohr Olivier
Recruitment of chromatin-modifying enzymes by CTIP2 promotes HIV-1 transcriptional silencing Journal Article
In: EMBO J, vol. 26, no. 2, pp. 412–423, 2007, ISSN: 0261-4189.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid17245431,
title = {Recruitment of chromatin-modifying enzymes by CTIP2 promotes HIV-1 transcriptional silencing},
author = {Céline Marban and Stella Suzanne and Franck Dequiedt and Stéphane de Walque and Laetitia Redel and Carine Van Lint and Dominique Aunis and Olivier Rohr},
doi = {10.1038/sj.emboj.7601516},
issn = {0261-4189},
year = {2007},
date = {2007-01-01},
urldate = {2007-01-01},
journal = {EMBO J},
volume = {26},
number = {2},
pages = {412--423},
abstract = {Following entry and reverse transcription, the HIV-1 genome is integrated into the host genome. In contrast to productively infected cells, latently infected cells frequently harbor HIV-1 genomes integrated in heterochromatic structures, allowing persistence of transcriptionally silent proviruses. Microglial cells are the main HIV-1 target cells in the central nervous system and constitute an important reservoir for viral pathogenesis. In the present work, we show that, in microglial cells, the co-repressor COUP-TF interacting protein 2 (CTIP2) recruits a multienzymatic chromatin-modifying complex and establishes a heterochromatic environment at the HIV-1 promoter. We report that CTIP2 recruits histone deacetylase (HDAC)1 and HDAC2 to promote local histone H3 deacetylation at the HIV-1 promoter region. In addition, DNA-bound CTIP2 also associates with the histone methyltransferase SUV39H1, which increases local histone H3 lysine 9 methylation. This allows concomitant recruitment of HP1 proteins to the viral promoter and formation of local heterochromatin, leading to HIV-1 silencing. Altogether, our findings uncover new therapeutic opportunities for purging latent HIV-1 viruses from their cellular reservoirs.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Following entry and reverse transcription, the HIV-1 genome is integrated into the host genome. In contrast to productively infected cells, latently infected cells frequently harbor HIV-1 genomes integrated in heterochromatic structures, allowing persistence of transcriptionally silent proviruses. Microglial cells are the main HIV-1 target cells in the central nervous system and constitute an important reservoir for viral pathogenesis. In the present work, we show that, in microglial cells, the co-repressor COUP-TF interacting protein 2 (CTIP2) recruits a multienzymatic chromatin-modifying complex and establishes a heterochromatic environment at the HIV-1 promoter. We report that CTIP2 recruits histone deacetylase (HDAC)1 and HDAC2 to promote local histone H3 deacetylation at the HIV-1 promoter region. In addition, DNA-bound CTIP2 also associates with the histone methyltransferase SUV39H1, which increases local histone H3 lysine 9 methylation. This allows concomitant recruitment of HP1 proteins to the viral promoter and formation of local heterochromatin, leading to HIV-1 silencing. Altogether, our findings uncover new therapeutic opportunities for purging latent HIV-1 viruses from their cellular reservoirs.
2003
Rohr Olivier, Lecestre Dominique, Chasserot-Golaz Sylvette, Marban Céline, Avram Dorina, Aunis Dominique, Leid Mark, Schaeffer Evelyne
In: J Virol, vol. 77, no. 9, pp. 5415–5427, 2003, ISSN: 0022-538X.
Abstract | Links | BibTeX | Tags: ROHR, Unité ARN
@article{pmid12692243,
title = {Recruitment of Tat to heterochromatin protein HP1 via interaction with CTIP2 inhibits human immunodeficiency virus type 1 replication in microglial cells},
author = {Olivier Rohr and Dominique Lecestre and Sylvette Chasserot-Golaz and Céline Marban and Dorina Avram and Dominique Aunis and Mark Leid and Evelyne Schaeffer},
doi = {10.1128/jvi.77.9.5415-5427.2003},
issn = {0022-538X},
year = {2003},
date = {2003-05-01},
urldate = {2003-05-01},
journal = {J Virol},
volume = {77},
number = {9},
pages = {5415--5427},
abstract = {The Tat protein of human immunodeficiency virus type 1 (HIV-1) plays a key role as inducer of viral gene expression. We report that Tat function can be potently inhibited in human microglial cells by the recently described nuclear receptor cofactor chicken ovalbumin upstream promoter transcription factor-interacting protein 2 (CTIP2). Overexpression of CTIP2 leads to repression of HIV-1 replication, as a result of inhibition of Tat-mediated transactivation. In contrast, the related CTIP1 was unable to affect Tat function and viral replication. Using confocal microscopy to visualize Tat subcellular distribution in the presence of the CTIPs, we found that overexpression of CTIP2, and not of CTIP1, leads to disruption of Tat nuclear localization and recruitment of Tat within CTIP2-induced nuclear ball-like structures. In addition, our studies demonstrate that CTIP2 colocalizes and associates with the heterochromatin-associated protein HP1alpha. The CTIP2 protein harbors two Tat and HP1 interaction interfaces, the 145-434 and the 717-813 domains. CTIP2 and HP1alpha associate with Tat to form a three-protein complex in which the 145-434 CTIP2 domain interacts with the N-terminal region of Tat, while the 717-813 domain binds to HP1. The importance of this Tat binding interface and of Tat subnuclear relocation was confirmed by analysis of CTIP2 deletion mutants. Our findings suggest that inhibition of HIV-1 expression by CTIP2 correlates with recruitment of Tat within CTIP2-induced structures and relocalization within inactive regions of the chromatin via formation of the Tat-CTIP2-HP1alpha complex. These data highlight a new mechanism of Tat inactivation through subnuclear relocalization that may ultimately lead to inhibition of viral pathogenesis.},
keywords = {ROHR, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The Tat protein of human immunodeficiency virus type 1 (HIV-1) plays a key role as inducer of viral gene expression. We report that Tat function can be potently inhibited in human microglial cells by the recently described nuclear receptor cofactor chicken ovalbumin upstream promoter transcription factor-interacting protein 2 (CTIP2). Overexpression of CTIP2 leads to repression of HIV-1 replication, as a result of inhibition of Tat-mediated transactivation. In contrast, the related CTIP1 was unable to affect Tat function and viral replication. Using confocal microscopy to visualize Tat subcellular distribution in the presence of the CTIPs, we found that overexpression of CTIP2, and not of CTIP1, leads to disruption of Tat nuclear localization and recruitment of Tat within CTIP2-induced nuclear ball-like structures. In addition, our studies demonstrate that CTIP2 colocalizes and associates with the heterochromatin-associated protein HP1alpha. The CTIP2 protein harbors two Tat and HP1 interaction interfaces, the 145-434 and the 717-813 domains. CTIP2 and HP1alpha associate with Tat to form a three-protein complex in which the 145-434 CTIP2 domain interacts with the N-terminal region of Tat, while the 717-813 domain binds to HP1. The importance of this Tat binding interface and of Tat subnuclear relocation was confirmed by analysis of CTIP2 deletion mutants. Our findings suggest that inhibition of HIV-1 expression by CTIP2 correlates with recruitment of Tat within CTIP2-induced structures and relocalization within inactive regions of the chromatin via formation of the Tat-CTIP2-HP1alpha complex. These data highlight a new mechanism of Tat inactivation through subnuclear relocalization that may ultimately lead to inhibition of viral pathogenesis.