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1.
Nucleic Acids Res ; 50(6): 3190-3202, 2022 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-35234910

RESUMO

Bovine leukemia virus (BLV)-induced tumoral development is a multifactorial phenomenon that remains incompletely understood. Here, we highlight the critical role of the cellular CCCTC-binding factor (CTCF) both in the regulation of BLV transcriptional activities and in the deregulation of the three-dimensional (3D) chromatin architecture surrounding the BLV integration site. We demonstrated the in vivo recruitment of CTCF to three conserved CTCF binding motifs along the provirus. Next, we showed that CTCF localized to regions of transitions in the histone modifications profile along the BLV genome and that it is implicated in the repression of the 5'Long Terminal Repeat (LTR) promoter activity, thereby contributing to viral latency, while favoring the 3'LTR promoter activity. Finally, we demonstrated that BLV integration deregulated the host cellular 3D chromatin organization through the formation of viral/host chromatin loops. Altogether, our results highlight CTCF as a new critical effector of BLV transcriptional regulation and BLV-induced physiopathology.


Assuntos
Vírus da Leucemia Bovina , Latência Viral , Fator de Ligação a CCCTC/metabolismo , Cromatina , Vírus da Leucemia Bovina/genética , Vírus da Leucemia Bovina/metabolismo , Regiões Promotoras Genéticas , Sequências Repetidas Terminais/genética
2.
PLoS Pathog ; 11(7): e1005063, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26225566

RESUMO

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.


Assuntos
Briostatinas/farmacologia , Linfócitos T CD4-Positivos/virologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , HIV-1/efeitos dos fármacos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Diterpenos/metabolismo , HIV-1/fisiologia , Humanos , Fator B de Elongação Transcricional Positiva/metabolismo , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos
3.
EBioMedicine ; 79: 103985, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35429693

RESUMO

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). METHODS: 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 p24Gag protein, green fluorescence protein signal, intracellular and extracellular viral RNA and viral DNA. FINDINGS: 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. INTERPRETATION: 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. FUNDING: Funding was provided by the Belgian National Fund for Scientific Research (F.R.S.-FNRS, Belgium), the « Fondation Roi Baudouin ¼, the NEAT (European AIDS Treatment Network) program, the Internationale Brachet Stiftung, ViiV Healthcare, the Télévie, the Walloon Region (« Fonds de Maturation ¼), « Les Amis des Instituts Pasteur à Bruxelles, asbl ¼, 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.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Repressão Epigenética , Infecções por HIV , HIV-1 , Ubiquitina-Proteína Ligases , Latência Viral , Síndrome da Imunodeficiência Adquirida , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Metilação de DNA , Decitabina/metabolismo , Infecções por HIV/genética , HIV-1/fisiologia , Humanos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Latência Viral/genética
4.
J Biol Chem ; 285(25): 19434-49, 2010 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-20413592

RESUMO

Bovine leukemia virus (BLV) proviral latency represents a viral strategy to escape the host immune system and allow tumor development. Besides the previously demonstrated role of histone deacetylation in the epigenetic repression of BLV expression, we showed here that BLV promoter activity was induced by several DNA methylation inhibitors (such as 5-aza-2'-deoxycytidine) and that overexpressed DNMT1 and DNMT3A, but not DNMT3B, down-regulated BLV promoter activity. Importantly, cytosine hypermethylation in the 5'-long terminal repeat (LTR) U3 and R regions was associated with true latency in the lymphoma-derived B-cell line L267 but not with defective latency in YR2 cells. Moreover, the virus-encoded transactivator Tax(BLV) decreased DNA methyltransferase expression levels, which could explain the lower level of cytosine methylation observed in the L267(LTaxSN) 5'-LTR compared with the L267 5'-LTR. Interestingly, DNA methylation inhibitors and Tax(BLV) synergistically activated BLV promoter transcriptional activity in a cAMP-responsive element (CRE)-dependent manner. Mechanistically, methylation at the -154 or -129 CpG position (relative to the transcription start site) impaired in vitro binding of CRE-binding protein (CREB) transcription factors to their respective CRE sites. Methylation at -129 CpG alone was sufficient to decrease BLV promoter-driven reporter gene expression by 2-fold. We demonstrated in vivo the recruitment of CREB/CRE modulator (CREM) and to a lesser extent activating transcription factor-1 (ATF-1) to the hypomethylated CRE region of the YR2 5'-LTR, whereas we detected no CREB/CREM/ATF recruitment to the hypermethylated corresponding region in the L267 cells. Altogether, these findings suggest that site-specific DNA methylation of the BLV promoter represses viral transcription by directly inhibiting transcription factor binding, thereby contributing to true proviral latency.


Assuntos
Fator 1 Ativador da Transcrição/metabolismo , Linfócitos B/metabolismo , Linfócitos B/virologia , Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Citosina/metabolismo , Metilação de DNA , DNA/genética , Vírus da Leucemia Bovina/genética , Linfoma/metabolismo , Regiões Promotoras Genéticas , Cromatina/química , AMP Cíclico/metabolismo , Citosina/química , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Plasmídeos/metabolismo , Sulfitos/química
5.
Brain ; 132(Pt 6): 1523-35, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19336457

RESUMO

Pilocytic astrocytomas are WHO grade I gliomas that occur predominantly in childhood. They share features of both astroglial and oligodendroglial lineages. These tumours affect preferentially the cerebellum (benign clinical course) and the optic pathway, especially the hypothalamo-chiasmatic region (poor prognosis). Understanding the molecular basis responsible for the aggressive behaviour of hypothalamo-chiasmatic pilocytic astrocytomas is a prerequisite to setting up new molecular targeted therapies. We used the microarray technique to compare the transcriptional profiles of five hypothalamo-chiasmatic and six cerebellar pilocytic astrocytomas. Validation of the microarray results and comparison of the tumours with normal developing tissue was done by quantitative real-time PCR and immunohistochemistry. Results demonstrate that cerebellar and hypothalamo-chiasmatic pilocytic astrocytomas are two genetically distinct and topography-dependent entities. Numerous genes upregulated in hypothalamo-chiasmatic pilocytic astrocytomas also increased in the developing chiasm, suggesting that developmental genes mirror the cell of origin whereas migrative, adhesive and proliferative genes reflect infiltrative properties of these tumours. Of particular interest, NOTCH2, a gene expressed in radial glia and involved in gliomagenesis, was upregulated in hypothalamo-chiasmatic pilocytic astrocytomas. In order to find progenitor cells that could give rise to hypothalamo-chiasmatic pilocytic astrocytomas, we performed a morphological study of the hypothalamo-chiasmatic region and identified, in the floor of the third ventricle, a unique population of vimentin- and glial fibrillary acidic protein-positive cells highly suggestive of radial glia cells. Therefore, pilocytic astrocytomas of the hypothalamo-chiasmatic region should be considered as a distinct entity which probably originates from a unique population of cells with radial glia phenotype.


Assuntos
Astrocitoma/diagnóstico , Neoplasias do Nervo Óptico/diagnóstico , Adolescente , Adulto , Astrócitos/metabolismo , Astrocitoma/genética , Astrocitoma/patologia , Proliferação de Células , Neoplasias Cerebelares/diagnóstico , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Criança , Pré-Escolar , DNA de Neoplasias/genética , Diagnóstico Diferencial , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Hipotálamo/metabolismo , Lactente , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/patologia , Neuroglia/patologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Quiasma Óptico/citologia , Quiasma Óptico/embriologia , Quiasma Óptico/metabolismo , Neoplasias do Nervo Óptico/genética , Neoplasias do Nervo Óptico/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Regulação para Cima , Vimentina/metabolismo , Adulto Jovem
6.
J Acquir Immune Defic Syndr ; 80(5): 605-613, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30768485

RESUMO

OBJECTIVES: Few single latency-reversing agents (LRAs) have been tested in vivo, and only some of them have demonstrated an effect, albeit weak, on the decrease of latent reservoir. Therefore, other LRAs and combinations of LRAs need to be assessed. Here, we evaluated the potential of combined treatments of therapeutically promising LRAs, disulfiram and romidepsin. SETTING AND METHODS: We assessed the reactivation potential of individual disulfiram or simultaneous or sequential combined treatments with romidepsin in vitro in latently infected cell lines of T-lymphoid and myeloid origins and in ex vivo cultures of CD8-depleted peripheral blood mononuclear cells isolated from 18 HIV-1 combination antiretroviral therapy-treated individuals. RESULTS: We demonstrated heterogeneous reactivation effects of disulfiram in vitro in various cell lines of myeloid origin and no latency reversal neither in vitro in T-lymphoid cells nor ex vivo, even if doses corresponding to maximal plasmatic concentration or higher were tested. Disulfiram+romidepsin combined treatments produced distinct reactivation patterns in vitro. Ex vivo, the combined treatments showed a modest reactivation effect when used simultaneously as opposed to no viral reactivation for the corresponding sequential treatment. CONCLUSIONS: Exclusive reactivation effects of disulfiram in myeloid latency cell lines suggest that disulfiram could be a potential LRA for this neglected reservoir. Moreover, distinct reactivation profiles pinpoint heterogeneity of the latent reservoir and confirm that the mechanisms that contribute to HIV latency are diverse. Importantly, disulfiram+romidepsin treatments are not potent ex vivo and most likely do not represent an effective drug combination to achieve high levels of latency reversal in vivo.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Depsipeptídeos/uso terapêutico , Dissulfiram/uso terapêutico , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Fármacos Anti-HIV/administração & dosagem , Linhagem Celular , Depsipeptídeos/administração & dosagem , Dissulfiram/administração & dosagem , Quimioterapia Combinada , HIV-1/fisiologia , Humanos
7.
J Clin Invest ; 111(9): 1319-27, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12727923

RESUMO

Although the role of estradiol in maintaining bone mass is well established, the relative contributions of the estradiol receptors ERalpha and ERbeta and of the androgen receptor (AR) remain controversial. To determine the role of ERalpha-mediated, ERbeta-mediated, and non-ER-mediated mechanisms in maintaining bone mass, gonadectomy and estradiol treatment were studied in ER-knockout mice. Estradiol treatment of ovariectomized ERalphabeta(-/-) mice failed to prevent bone loss, precluding significant effects of estradiol on bone through non-ER-signaling pathways. In contrast, estradiol prevented ovariectomy-induced bone loss in ERbeta(-/-) mice, as in WT males and females, indicating that ERalpha is the major mediator of estradiol effects in bone. No response of bone to estradiol was detected in orchidectomized ERalpha(-/-) mice, suggesting estradiol cannot protect bone mass via the AR in vivo. In contrast to female ERalphabeta(-/-) and male ERalpha(-/-) mice, female ERalpha(-/-) mice were partially protected against ovariectomy-induced bone loss by estradiol, confirming that ERbeta mediates estradiol effects in bone, but only in females and with a lower efficacy than ERalpha. We conclude that ERalpha is the main effector of estradiol's protective function in bone in both male and female mice, and that, in its absence, AR is not sufficient to mediate this response.


Assuntos
Osso e Ossos/efeitos dos fármacos , Estradiol/análogos & derivados , Estradiol/farmacologia , Orquiectomia , Ovariectomia , Receptores Androgênicos/metabolismo , Receptores de Estrogênio/metabolismo , Antagonistas de Androgênios/farmacologia , Animais , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Estradiol/metabolismo , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Feminino , Masculino , Camundongos , Camundongos Knockout , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Estrogênio/genética , Testosterona/análogos & derivados , Testosterona/metabolismo
8.
Mol Cell Biol ; 23(17): 6200-9, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12917341

RESUMO

Previous studies have implicated acetylases and deacetylases in regulating the transcriptional activity of NF-kappa B. Here, we show that inhibitors of deacetylases such as trichostatin A (TSA) and sodium butyrate (NaBut) potentiated TNF-induced expression of several natural NF-kappa B-driven promoters. This transcriptional synergism observed between TNF and TSA (or NaBut) required intact kappa B sites in all promoters tested and was biologically relevant as demonstrated by RNase protection on two instances of endogenous NF-kappa B-regulated gene transcription. Importantly, TSA prolonged both TNF-induced DNA-binding activity and the presence of NF-kappa B in the nucleus. We showed that the p65 subunit of NF-kappa B was acetylated in vivo. However, this acetylation was weak, suggesting that other mechanisms could be implicated in the potentiated binding and transactivation activities of NF-kappa B after TNF plus TSA versus TNF treatment. Western blot and immunofluorescence confocal microscopy experiments revealed a delay in the cytoplasmic reappearance of the I kappa B alpha inhibitor that correlated temporally with the prolonged intranuclear binding and presence of NF-kappa B. This delay was due neither to a defect in I kappa B alpha mRNA production nor to a nuclear retention of I kappa B alpha but was rather due to a persistent proteasome-mediated degradation of I kappa B alpha. A prolongation of I kappa B kinase activity could explain, at least partially, the delayed I kappa B alpha cytoplasmic reappearance observed in presence of TNF plus TSA.


Assuntos
Citoplasma/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Proteínas I-kappa B/metabolismo , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Ácido Butírico/farmacologia , Cisteína Endopeptidases , Células HeLa , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Quinase I-kappa B , Proteínas I-kappa B/efeitos dos fármacos , Proteínas I-kappa B/genética , Leupeptinas/farmacologia , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Complexos Multienzimáticos/antagonistas & inibidores , Inibidor de NF-kappaB alfa , NF-kappa B/efeitos dos fármacos , NF-kappa B/genética , Complexo de Endopeptidases do Proteassoma , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico/efeitos dos fármacos , Fator de Transcrição RelA
9.
Nucleic Acids Res ; 33(13): 4285-310, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16061936

RESUMO

We have previously identified in the pol gene of human immunodeficiency virus type 1 (HIV-1) a new positive transcriptional regulatory element (nt 4481-4982) containing recognition sites for nuclear proteins (sites B, C, D and a GC-box) [C. Van Lint, J. Ghysdael, P. Paras, Jr, A. Burny and E. Verdin (1994) J. Virol. 68, 2632-2648]. In this study, we have further physically characterized each binding site and have shown that the transcription factors Oct-1, Oct-2, PU.1, Sp1 and Sp3 interact in vitro with the pol region. Chromatin immunoprecipitation assays using HIV-infected cell lines demonstrated in the context of chromatin that Sp1, Sp3, Oct-1 and PU.1 are recruited to the HS7 region in vivo. For each site, we have identified mutations abolishing factor binding to their cognate DNA sequences without altering the underlying amino acid sequence of the integrase. By transient transfection assays, we have demonstrated the involvement of the pol binding sites in the transcriptional enhancing activity of the intragenic region. Our functional results with multimerized wild-type and mutated pol binding sites separately (i.e. in the absence of the other sites) have demonstrated that the PU.1, Sp1, Sp3 and Oct-1 transcription factors regulate the transcriptional activity of a heterologous promoter through their respective HS7 binding sites. Finally, we have investigated the physiological role of the HS7 binding sites in HIV-1 replication and have shown that these sites are important for viral infectivity.


Assuntos
Regulação Viral da Expressão Gênica , Produtos do Gene pol/genética , HIV-1/genética , Elementos de Resposta , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Análise Mutacional de DNA , Proteínas de Ligação a DNA/metabolismo , Produtos do Gene tat/metabolismo , HIV-1/fisiologia , Humanos , Camundongos , Fator 1 de Transcrição de Octâmero , Mutação Puntual , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3 , Timidina Quinase/genética , Transativadores/metabolismo , Fatores de Transcrição/química , Ativação Transcricional , Replicação Viral , Dedos de Zinco , Produtos do Gene tat do Vírus da Imunodeficiência Humana
10.
AIDS ; 31(2): 181-189, 2017 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-27755105

RESUMO

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. DESIGN 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. RESULTS: 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. CONCLUSION: 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.


Assuntos
Infecções por HIV/virologia , HIV-1/crescimento & desenvolvimento , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Ativação Viral , Latência Viral/efeitos dos fármacos , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
11.
Sci Rep ; 7: 43221, 2017 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-28256531

RESUMO

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.


Assuntos
Repressão Epigenética , Interações Hospedeiro-Patógeno , Vírus Linfotrópico T Tipo 1 Humano/crescimento & desenvolvimento , Vírus Linfotrópico T Tipo 1 Humano/genética , Fator de Transcrição Sp1/metabolismo , Sequências Repetidas Terminais , Transcrição Gênica , Sítios de Ligação , Imunoprecipitação da Cromatina , Células HEK293 , Humanos , Células Jurkat , Ligação Proteica , Fator de Transcrição Sp3/metabolismo
12.
Sci Rep ; 6: 31125, 2016 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-27545598

RESUMO

Bovine leukemia virus latency is a viral strategy used to escape from the host immune system and contribute to tumor development. However, a highly expressed BLV micro-RNA cluster has been reported, suggesting that the BLV silencing is not complete. Here, we demonstrate the in vivo recruitment of RNA polymerase III to the BLV miRNA cluster both in BLV-latently infected cell lines and in ovine BLV-infected primary cells, through a canonical type 2 RNAPIII promoter. Moreover, by RPC6-knockdown, we showed a direct functional link between RNAPIII transcription and BLV miRNAs expression. Furthermore, both the tumor- and the quiescent-related isoforms of RPC7 subunits were recruited to the miRNA cluster. We showed that the BLV miRNA cluster was enriched in positive epigenetic marks. Interestingly, we demonstrated the in vivo recruitment of RNAPII at the 3'LTR/host genomic junction, associated with positive epigenetic marks. Functionally, we showed that the BLV LTR exhibited a strong antisense promoter activity and identified cis-acting elements of an RNAPII-dependent promoter. Finally, we provided evidence for an in vivo collision between RNAPIII and RNAPII convergent transcriptions. Our results provide new insights into alternative ways used by BLV to counteract silencing of the viral 5'LTR promoter.


Assuntos
Genoma Viral , Vírus da Leucemia Bovina/enzimologia , Vírus da Leucemia Bovina/genética , RNA Polimerase III/genética , RNA Polimerase II/genética , Regiões 3' não Traduzidas , Animais , Sítios de Ligação/genética , Bovinos , Linhagem Celular , Epigênese Genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Regiões Promotoras Genéticas , Subunidades Proteicas , RNA Polimerase II/química , RNA Polimerase II/metabolismo , RNA Polimerase III/química , RNA Polimerase III/metabolismo , RNA Interferente Pequeno/genética , Ovinos , Transcrição Gênica
13.
Sci Rep ; 6: 24090, 2016 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-27076174

RESUMO

Akt signaling plays a central role in many biological processes, which are key players in human immunodeficiency virus 1 (HIV-1) pathogenesis. We found that Akt interacts with HIV-1 Nef protein. In primary T cells treated with exogenous Nef or acutely infected with Nef-expressing HIV-1 in vitro, Akt became phosphorylated on serine(473) and threonine(308). In vitro, Akt activation mediated by Nef in T-cells was blocked by HIV protease inhibitors (PI), but not by reverse transcriptase inhibitors (RTI). Ex vivo, we found that the Akt pathway is hyperactivated in peripheral blood lymphocytes (PBLs) from cART naïve HIV-1-infected patients. PBLs isolated from PI-treated patients, but not from RTI-treated patients, exhibited decreased Akt activation, T-cell proliferation and IL-2 production. We found that PI but not RTI can block HIV-1 reactivation in latently infected J-Lat lymphoid cells stimulated with various stimuli. Using luciferase measurement, we further confirmed that Nef-mediated reactivation of HIV-1 from latency in 1G5 cells was blocked by PI parallel to decreased Akt activation. Our results indicate that PI-mediated blockade of Akt activation could impact the HIV-1 reservoir and support the need to further assess the therapeutic use of HIV-1 PI in order to curtail latently infected cells in HIV-1-infected patients.


Assuntos
HIV-1/fisiologia , Inibidores de Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Linfócitos T/virologia , Latência Viral , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Células Cultivadas , Infecções por HIV/patologia , Infecções por HIV/virologia , Humanos , Fosforilação , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Linfócitos T/fisiologia
14.
EMBO Mol Med ; 8(2): 117-38, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26681773

RESUMO

Reactivation of HIV gene expression in latently infected cells together with an efficient cART has been proposed as an adjuvant therapy aimed at eliminating/decreasing the reservoir size. Results from HIV clinical trials using deacetylase inhibitors (HDACIs) question the efficiency of these latency-reversing agents (LRAs) used alone and underline the need to evaluate other LRAs in combination with HDACIs. Here, we evaluated the therapeutic potential of a demethylating agent (5-AzadC) in combination with clinically tolerable HDACIs in reactivating HIV-1 from latency first in vitro and next ex vivo. We showed that a sequential treatment with 5-AzadC and HDACIs was more effective than the corresponding simultaneous treatment both in vitro and ex vivo. Interestingly, only two of the sequential LRA combinatory treatments tested induced HIV-1 particle recovery in a higher manner than the drugs alone ex vivo and at concentrations lower than the human tolerable plasmatic concentrations. Taken together, our data reveal the benefit of using combinations of 5-AzadC with an HDACI and, for the first time, the importance of treatment time schedule for LRA combinations in order to reactivate HIV.


Assuntos
Azacitidina/análogos & derivados , Inibidores Enzimáticos/farmacologia , HIV-1/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Ativação Viral/efeitos dos fármacos , Azacitidina/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Decitabina , Humanos , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
Cloning Stem Cells ; 6(2): 140-9, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15268788

RESUMO

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


Assuntos
Regulação da Expressão Gênica , Inativação Gênica , Heterocromatina/genética , Acetilação , Animais , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Montagem e Desmontagem da Cromatina , Heterocromatina/metabolismo , Histonas/metabolismo , Humanos , Regiões Promotoras Genéticas , Retroviridae/genética , Ativação Transcricional
16.
PLoS One ; 6(4): e19084, 2011 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-21526160

RESUMO

Our laboratory has previously identified an important intragenic region in the human immunodeficiency virus type 1 (HIV-1) genome, whose complete functional unit is composed of the 5103 fragment, the DNaseI-hypersensitive site HS7 and the 5105 fragment. These fragments (5103 and 5105) both exhibit a phorbol 12-myristate 13-acetate (PMA)-inducible enhancer activity on the herpes simplex virus thymidine kinase promoter. Here, we characterized the three previously identified AP-1 binding sites of fragment 5103 by showing the PMA-inducible in vitro binding and in vivo recruitment of c-Fos, JunB and JunD to this fragment located at the end of the pol gene. Functional analyses demonstrated that the intragenic AP-1 binding sites are fully responsible for the PMA-dependent enhancer activity of fragment 5103. Moreover, infection of T-lymphoid Jurkat and promonocytic U937 cells with wild-type and mutant viruses demonstrated that mutations of the intragenic AP-1 sites individually or in combination altered HIV-1 replication. Importantly, mutations of the three intragenic AP-1 sites led to a decreased in vivo recruitment of RNA polymerase II to the viral promoter, strongly supporting that the deleterious effect of these mutations on viral replication occurs, at least partly, at the transcriptional level. Single-round infections of monocyte-derived macrophages confirmed the importance of intragenic AP-1 sites for HIV-1 infectivity.


Assuntos
Genes pol/genética , HIV-1/genética , HIV-1/fisiologia , Sequências Reguladoras de Ácido Nucleico/genética , Fator de Transcrição AP-1/metabolismo , Replicação Viral/genética , Sequência de Bases , Sítios de Ligação , Elementos Facilitadores Genéticos/genética , Genes Dominantes/genética , Humanos , Células Jurkat , Macrófagos/efeitos dos fármacos , Macrófagos/virologia , Dados de Sequência Molecular , Monócitos/efeitos dos fármacos , Monócitos/virologia , Mutação Puntual/genética , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Polimerase II/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/virologia , Acetato de Tetradecanoilforbol/farmacologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana
17.
PLoS One ; 4(6): e6093, 2009 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-19564922

RESUMO

The persistence of transcriptionally silent but replication-competent HIV-1 reservoirs in Highly Active Anti-Retroviral Therapy (HAART)-treated infected individuals, represents a major hurdle to virus eradication. Activation of HIV-1 gene expression in these cells together with an efficient HAART has been proposed as an adjuvant therapy aimed at decreasing the pool of latent viral reservoirs. Using the latently-infected U1 monocytic cell line and latently-infected J-Lat T-cell clones, we here demonstrated a strong synergistic activation of HIV-1 production by clinically used histone deacetylase inhibitors (HDACIs) combined with prostratin, a non-tumor-promoting nuclear factor (NF)- kappaB inducer. In J-Lat cells, we showed that this synergism was due, at least partially, to the synergistic recruitment of unresponsive cells into the expressing cell population. A combination of prostratin+HDACI synergistically activated the 5' Long Terminal Repeat (5'LTR) from HIV-1 Major group subtypes representing the most prevalent viral genetic forms, as shown by transient transfection reporter assays. Mechanistically, HDACIs increased prostratin-induced DNA-binding activity of nuclear NF-kappaB and degradation of cytoplasmic NF-kappaB inhibitor, IkappaBalpha . Moreover, the combined treatment prostratin+HDACI caused a more pronounced nucleosomal remodeling in the U1 viral promoter region than the treatments with the compounds alone. This more pronounced remodeling correlated with a synergistic reactivation of HIV-1 transcription following the combined treatment prostratin+HDACI, as demonstrated by measuring recruitment of RNA polymerase II to the 5'LTR and both initiated and elongated transcripts. The physiological relevance of the prostratin+HDACI synergism was shown in CD8(+)-depleted peripheral blood mononuclear cells from HAART-treated patients with undetectable viral load. Moreover, this combined treatment reactivated viral replication in resting CD4(+) T cells isolated from similar patients. Our results suggest that combinations of different kinds of proviral activators may have important implications for reducing the size of latent HIV-1 reservoirs in HAART-treated patients.


Assuntos
Fármacos Anti-HIV/farmacologia , Inibidores Enzimáticos/farmacologia , Infecções por HIV/tratamento farmacológico , HIV-1/enzimologia , HIV-1/metabolismo , Ésteres de Forbol/farmacologia , Latência Viral/efeitos dos fármacos , Adulto , Idoso , Linfócitos T CD8-Positivos/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Sinergismo Farmacológico , Humanos , Proteínas I-kappa B/metabolismo , Pessoa de Meia-Idade , Monócitos/virologia , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , Nucleossomos/metabolismo
18.
J Virol ; 76(21): 11091-103, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12368351

RESUMO

The transcription factor NF-kappaB plays a central role in the human immunodeficiency virus type 1 (HIV-1) activation pathway. HIV-1 transcription is also regulated by protein acetylation, since treatment with deacetylase inhibitors such as trichostatin A (TSA) or sodium butyrate (NaBut) markedly induces HIV-1 transcriptional activity of the long terminal repeat (LTR) promoter. Here, we demonstrate that TSA (NaBut) synergized with both ectopically expressed p50/p65 and tumor necrosis factor alpha/SF2 (TNF)-induced NF-kappaB to activate the LTR. This was confirmed for LTRs from subtypes A through G of the HIV-1 major group, with a positive correlation between the number of kappaB sites present in the LTRs and the amplitude of the TNF-TSA synergism. Mechanistically, TSA (NaBut) delayed the cytoplasmic recovery of the inhibitory protein IkappaBalpha. This coincided with a prolonged intranuclear presence and DNA binding activity of NF-kappaB. The physiological relevance of the TNF-TSA (NaBut) synergism was shown on HIV-1 replication in both acutely and latently HIV-infected cell lines. Therefore, our results open new therapeutic strategies aimed at decreasing or eliminating the pool of latently HIV-infected reservoirs by forcing viral expression.


Assuntos
Regulação Viral da Expressão Gênica , Repetição Terminal Longa de HIV , HIV-1/genética , Inibidores de Histona Desacetilases , Proteínas I-kappa B , NF-kappa B/metabolismo , Ativação Transcricional , Acetilação , Butiratos/farmacologia , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Infecções por HIV/tratamento farmacológico , Humanos , Ácidos Hidroxâmicos/farmacologia , Mutagênese , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Subunidade p50 de NF-kappa B , Fator de Transcrição RelA , Fator de Necrose Tumoral alfa/farmacologia , Células U937 , Latência Viral , Replicação Viral/efeitos dos fármacos
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