RESUMEN
Most nuclear factor-kappaB (NF-kappaB) inducers converge to activate the IkappaB kinase (IKK) complex, leading to NF-kappaB nuclear accumulation. However, depending on the inducer and the cell line, the subset of NF-kappaB-induced genes is different, underlining a complex regulation network. Recent findings have begun to delineate that histone and non-histone protein acetylation is involved, directly and indirectly, in controlling the duration, strength and specificity of the NF-kappaB-activating signaling pathway at multiple levels. Acetylation and deacetylation events, in combination with other post-translational protein modifications, generate an 'NF-kappaB-signaling code' and regulate NF-kappaB-dependent gene transcription in an inducer- and promoter-dependent manner. Indeed, the intricate involvement of histone acetyltransferases and histone deacetylases modulates both the NF-kappaB-signaling pathway and the transcriptional transactivation of NF-kappaB-dependent genes.
Asunto(s)
Histona Acetiltransferasas/metabolismo , Histona Desacetilasas/metabolismo , FN-kappa B/metabolismo , Transducción de Señal/fisiología , Animales , Humanos , Modelos BiológicosRESUMEN
Human Immunodeficiency Virus type 1 (HIV-1) infection can now be treated effectively in many patients in the developed world, using combinations of antiretroviral therapeutics, called Highly Active Anti-Retroviral Therapy (HAART). However, despite prolonged treatment with HAART, the persistence of latently HIV-1-infected cellular reservoirs harboring transcriptionally silent but replication-competent proviruses represents the major hurdle to virus eradication. These latently infected cells are a permanent source for virus reactivation and lead to a rebound of the viral load after interruption of HAART. Therefore, a greater understanding of the molecular mechanisms regulating proviral latency and reactivation should lead to rational strategies aimed at purging these cellular reservoirs of HIV-1. This review summarizes our current knowledge and understanding of the elements involved in HIV-1 transcriptional reactivation: (1) the site of integration; (2) the transcription factor NF-kappaB, which is induced by proinflammatory cytokines (such as TNFalpha) and binds to two kappaB sites in the HIV-1 promoter region; (3) the specific remodeling of a single nucleosome (called nuc-1 and located immediately downstream of the HIV-1 transcription start site under latency conditions) upon activation of the HIV-1 promoter; (4) post-translational acetylation of histones and of non-histone proteins (following treatment with deacetylases inhibitors, which induce viral transcription and nuc-1 remodeling); and (5) the viral trans-activator Tat, which promotes transcription by mediating the recruitment to the HIV-1 promoter of histone-modifying enzymes and ATP-dependent chromatin remodeling complexes required for nucleosome disruption and transcriptional processivity. Finally, this review highlights experimental therapies aimed at administrating HIV-1 gene expression activators (such as HDAC inhibitors) combined with an effective HAART in order to reactivate and decrease/eliminate the pool of latently HIV-1-infected cellular reservoirs
Asunto(s)
Cromatina/metabolismo , VIH-1/metabolismo , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , Transcripción Genética , Acetilación/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Fármacos Anti-VIH/farmacología , Ensamble y Desensamble de Cromatina , Desoxirribonucleasa I/metabolismo , Inhibidores Enzimáticos/farmacología , Productos del Gen tat/metabolismo , Duplicado del Terminal Largo de VIH/efectos de los fármacos , VIH-1/efectos de los fármacos , VIH-1/genética , Histona Acetiltransferasas/metabolismo , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/metabolismo , Humanos , Nucleosomas/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Integración Viral , Latencia del Virus , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
HIV-1 latency represents a major hurdle to the complete eradication of the virus from patients under highly active anti-retroviral therapy (HAART) regimens. One solution to this problem would be to eliminate the latently infected cellular reservoirs by forcing gene expression in presence of HAART to prevent spreading of the infection by the newly synthesized viruses. Many studies have reported that a combination of a histone deacetylase inhibitor (HDACi) (i.e. TSA, NaBut, Valproic acid, ...) with a pro-inflammatory cytokine (i.e. TNFalpha, IL-1, ...) reactivates in a synergistic manner HIV-1 transcription in latently infected cells. The aim of the present study was to determine whether HIV-1 protease inhibitors (PIs) used in HAART (such as Saquinavir, Indinavir, Nelfinavir, Lopinavir, Ritonavir and Amprenavir) could interfere with the potential purge of the cellular reservoirs induced by a combined treatment involving TSA and TNFalpha. We showed, in two HIV-1 latently infected cell lines (ACH-2 and U1) that all PIs efficiently inhibited release of mature viral particles but did neither affect cell apoptosis nor NF-kappaB induction and HIV-1 transcription activation following combined treatment with TNFalpha+TSA. This study is encouraging in the fight against HIV-1 and shows that PIs should be compatible with an inductive adjuvent therapy for latent reservoir reduction/elimination in association with efficient HAART regimens.
Asunto(s)
Apoptosis/efectos de los fármacos , Inhibidores de la Proteasa del VIH/farmacología , Ácidos Hidroxámicos/farmacología , Provirus/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Terapia Antirretroviral Altamente Activa , Apoptosis/fisiología , Interacciones Farmacológicas , VIH-1/efectos de los fármacos , VIH-1/genética , Humanos , FN-kappa B/metabolismo , Provirus/genéticaRESUMEN
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.
Asunto(s)
Citoplasma/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Proteínas I-kappa B/metabolismo , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Ácido Butírico/farmacología , Cisteína Endopeptidasas , Células HeLa , Histona Desacetilasas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Quinasa I-kappa B , Proteínas I-kappa B/efectos de los fármacos , Proteínas I-kappa B/genética , Leupeptinas/farmacología , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Complejos Multienzimáticos/antagonistas & inhibidores , Inhibidor NF-kappaB alfa , FN-kappa B/efectos de los fármacos , FN-kappa B/genética , Complejo de la Endopetidasa Proteasomal , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Factor de Transcripción ReIARESUMEN
Persuasive evidence has accumulated that reversible acetylation of proteins is key post-translational modification regulating transcription in eukaryotes. Deacetylase inhibitors (such as trichostatin A) modulate the expression of approximately 2% of all cellular genes. We and others have demonstrated a marked transcriptional activation of the human immunodeficiency virus type 1 (HIV-1) promoter in response to deacetylase inhibitors. Deacetylation events seem to be an important mechanism of HIV-1 transcriptional repression during latency, whereas acetylation events play critical functional roles in HIV-1 reactivation from latency. These deacetylation/acetylation events are implicated in chromatin remodeling of the viral promoter region, as well as in modulating the functional properties of cellular and viral transcription factors binding to this promoter region. Thereby, the HIV-1 promoter constitutes a unique regulatory model system to study the complex relationship between acetylation processes and transcriptional activity.
Asunto(s)
VIH-1/genética , Transcripción Genética/fisiología , Acetilación , Acetiltransferasas/metabolismo , Animales , Cromatina/fisiología , Regulación Viral de la Expresión Génica , Productos del Gen tat/fisiología , Humanos , Modelos Biológicos , Nucleosomas/fisiología , Regiones Promotoras Genéticas , Transducción de Señal , Transactivadores , Factores de Transcripción/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
Evidence has accumulated that deacetylation and acetylation events are implicated in the regulation of NF-kappaB transcriptional activity. Several groups have reported potentiation of NF-kappaB-mediated gene induction [by specific inducers (such as TNFalpha)], following deacetylase inhibition by trichostatin A or sodium butyrate. This potentiation reflects a complex acetylation-dependent regulation of NF-kappaB-dependent transactivation. This acetylation-dependent regulation occurs at multiple levels. First, acetylation of histones regulates the NF-kappaB-dependent gene accessibility. Second, unidentified acetylation events modulate temporally the IKK activity and subsequently the duration of NF-kappaB presence and DNA-binding in the nucleus. Third, direct acetylation of the NF-kappaB subunits p65 and p50 regulates different NF-kappaB functions, including transcriptional activation, DNA-binding affinity and IkappaBalpha assembly. Finally, acetyltransferases and deacetylases interact directly with several proteins involved in the NF-kappaB signaling pathway, including NF-kappaB itself, IkappaBalpha, IKKalpha and IKKgamma. These interactions probably allow acetylation of NF-kappaB itself, of other transcription factors and of histones associated with NF-kappaB-regulated genes. The present review discusses these recent data obtained on the role of protein acetylation in the regulation of the NF-kappaB cascade.
Asunto(s)
FN-kappa B/metabolismo , Activación Transcripcional , Factor de Necrosis Tumoral alfa/farmacología , Acetilación , Animales , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/fisiología , Cromatina/metabolismo , Inhibidores Enzimáticos/farmacología , Histona Desacetilasas/metabolismo , Humanos , Proteínas I-kappa B/metabolismo , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/fisiología , Inhibidor NF-kappaB alfa , FN-kappa B/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/fisiología , Sinaptotagmina I , SinaptotagminasRESUMEN
The discovery of powerful antiviral compounds in the 90's raised the hope that the human immunodeficiency virus type 1 (HIV-1) might be eradicated. However, if these drugs succeed in decreasing and controlling viral replication, complete eradication of the virus is nowadays impossible. The persistence of virus even after long periods of highly active antiretroviral therapy (HAART) mainly results from the presence of cellular reservoirs that contain transcriptionally competent latent viruses capable of producing infectious particles after cellular activation. These latently infected cells are a permanent source for virus reactivation and lead to a rebound of the viral load after interruption of HAART. Activation of HIV gene expression in these cells combined with an effective HAART has been proposed as an adjuvant therapy that could lead to the elimination of the latently infected cells and then to the eradication of the infection. In this context, we have previously demonstrated that deacetylase inhibitors (HDACi) synergize with TNF-induced NF-kappaB to activate the HIV-1 promoter. The physiological relevance of the TNF/HDACi synergism was shown on HIV-1 replication in both acutely and latently HIV-infected cell lines. Based on these results, we propose the administration of deacetylase inhibitor(s) together with continuous HAART as a new potential therapeutic perspective to decrease the pool of latent HIV reservoirs by forcing viral expression.
Asunto(s)
Fármacos Anti-VIH/farmacología , Inhibidores Enzimáticos/farmacología , VIH-1/efectos de los fármacos , Inhibidores de Histona Desacetilasas , Replicación Viral/efectos de los fármacos , Fármacos Anti-VIH/uso terapéutico , Terapia Antirretroviral Altamente Activa , Inhibidores Enzimáticos/uso terapéutico , Regulación Viral de la Expresión Génica , Infecciones por VIH/tratamiento farmacológico , VIH-1/fisiología , HumanosRESUMEN
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.
Asunto(s)
Regulación de la Expresión Génica , Silenciador del Gen , Heterocromatina/genética , Acetilación , Animales , Núcleo Celular/genética , Núcleo Celular/ultraestructura , Ensamble y Desensamble de Cromatina , Heterocromatina/metabolismo , Histonas/metabolismo , Humanos , Regiones Promotoras Genéticas , Retroviridae/genética , Activación TranscripcionalRESUMEN
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.
Asunto(s)
Fármacos Anti-VIH/farmacología , Inhibidores Enzimáticos/farmacología , Infecciones por VIH/tratamiento farmacológico , VIH-1/enzimología , VIH-1/metabolismo , Ésteres del Forbol/farmacología , Latencia del Virus/efectos de los fármacos , Adulto , Anciano , Linfocitos T CD8-positivos/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Sinergismo Farmacológico , Humanos , Proteínas I-kappa B/metabolismo , Persona de Mediana Edad , Monocitos/virología , Inhibidor NF-kappaB alfa , FN-kappa B/antagonistas & inhibidores , Nucleosomas/metabolismoRESUMEN
NF-kappaB is a crucial transcription factor tightly regulated by protein interactions and post-translational modifications, like phosphorylation and acetylation. A previous study has shown that trichostatin A (TSA), a histone deacetylase inhibitor, potentiates tumor necrosis factor (TNF) alpha-elicited NF-kappaB activation and delays IkappaBalpha cytoplasmic reappearance. Here, we demonstrated that TSA also prolongs NF-kappaB activation when induced by the insulino-mimetic pervanadate (PV), a tyrosine phosphatase inhibitor that initiates an atypical NF-kappaB signaling. This extension is similarly correlated with delayed IkappaBalpha cytoplasmic reappearance. However, whereas TSA causes a prolonged IKK activity when added to TNFalpha, it does not when added to PV. Instead, quantitative reverse transcriptase-PCR revealed a decrease of ikappabalpha mRNA level after TSA addition to PV stimulation. This synthesis deficit of the inhibitor could explain the sustained NF-kappaB residence in the nucleus. In vivo analysis by chromatin immunoprecipitation assays uncovered that, for PV induction but not for TNFalpha, the presence of TSA provokes several impairments on the ikappabalpha promoter: (i) diminution of RNA Pol II recruitment; (ii) reduced acetylation and phosphorylation of histone H3-Lys(14) and -Ser(10), respectively; (iii) decreased presence of phosphorylated p65-Ser(536); and (iv) reduction of IKKalpha binding. The recruitment of these proteins on the icam-1 promoter, another NF-kappaB-regulated gene, is not equally affected, suggesting a promoter specificity of PV with TSA stimulation. Taken together, these data suggest that TSA acts differently depending on the NF-kappaB pathway and the targeted promoter in question. This indicates that one overall histone deacetylase role is to inhibit NF-kappaB activation by molecular mechanisms specific of the stimulus and the promoter.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Ácidos Hidroxámicos/farmacología , Proteínas I-kappa B/biosíntesis , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Vanadatos/farmacología , Acetilación/efectos de los fármacos , Inhibidores Enzimáticos/agonistas , Células HeLa , Histonas/metabolismo , Humanos , Ácidos Hidroxámicos/agonistas , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Inhibidor NF-kappaB alfa , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas/fisiología , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , ARN Polimerasa II/metabolismo , ARN Mensajero/biosíntesis , Transducción de Señal/efectos de los fármacos , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/agonistas , Vanadatos/agonistasRESUMEN
Fas ligand (FasL/CD95L/TNFSF6), a member of the tumor necrosis factor family, initiates apoptosis in lymphoid and nonlymphoid tissues by binding to its receptor Fas (CD95/TNFRSF6). Although the transcriptional control of TNFSF6 gene expression is subjected to intense study, the role of its chromatin organization and accessibility to the transcriptional machinery is not known. Here, we determined the chromatin organization of TNFSF6 gene 5' regulatory regions. Using the indirect end-labeling technique, a unique region named HSS1 and encompassing nucleotides -189 to +185 according to the transcriptional start site, was identified throughout a 20-kilobase nucleosomal DNA domain surrounding the promoter. The HSS1 region displayed hypersensitivity to in vivo DNase I digestion in TNFSF6-expressing cells only, including upon T cell activation. Hypersensitivity to micrococcal nuclease digestion and to specific restriction enzyme digestion suggested the precise positioning of two nucleosomes across the transcription start site and minimal promoter region, likely interfering with TNFSF6 active transcription in T lymphocytes. Indeed, HSS1 hypersensitivity to nuclease digestion strictly correlated with TNFSF6 transcription, including in primary and leukemia T cells. HSS1 chromatin remodeling preceded detectable TNFSF6 mRNA accumulation and was blocked by cycloheximide that also prevented TNFSF6 transcription. However, DNA methylation levels of the TNFSF6 HSS1 region did not correlate with transcriptional activation. Induction of global protein acetylation by treatment with histone deacetylase inhibitors was not accompanied by HSS1 chromatin remodeling and/or TNFSF6 transcription. We conclude that chromatin remodeling is a primary event in the activation of TNFSF6 expression in primary and leukemia T cells and that mechanisms independent of protein deacetylation and of DNA methylation of the TNFSF6 promoter region are involved in the repression of TNFSF6 gene expression.
Asunto(s)
Cromatina/metabolismo , Metilación de ADN , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Transcripción Genética , Factores de Necrosis Tumoral/genética , Factores de Necrosis Tumoral/metabolismo , Islas de CpG , Cicloheximida/farmacología , Inhibidores Enzimáticos/farmacología , Proteína Ligando Fas , Inhibidores de Histona Desacetilasas , Humanos , Células Jurkat , Células Asesinas Naturales/metabolismo , Nucleasa Microcócica/metabolismo , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , Linfocitos T/metabolismoRESUMEN
The human immunodeficiency virus (HIV) Vpr protein plays a critical role in AIDS pathogenesis, especially by allowing viral replication within nondividing cells such as mononuclear phagocytes. Most of the data obtained so far have been in experiments with endogenous Vpr protein; therefore the effects of extracellular Vpr protein remain largely unknown. We used synthetic Vpr protein to activate nuclear transcription factors activator protein-1 (AP-1) and NF-kappaB in the promonocytic cell line U937 and in primary macrophages. Synthetic HIV-1 Vpr protein activated AP-1, c-Jun N-terminal kinase, and MKK7 in both U937 cells and primary macrophages. Synthetic Vpr activated NF-kappaB in primary macrophages and to a lesser extent in U937 cells. Because synthetic Vpr activated AP-1 and NF-kappaB, which bind to the HIV-1 long terminal repeat, we investigated the effect of synthetic Vpr on HIV-1 replication. We observed that synthetic Vpr stimulated HIV-1 long terminal repeat in U937 cells and enhanced viral replication in chronically infected U1 promonocytic cells. Similarly, synthetic Vpr stimulated HIV-1 replication in acutely infected primary macrophages. Activation of transcription factors and enhancement of viral replication in U937 cells and primary macrophages were mediated by both the N-terminal and the C-terminal moieties of synthetic Vpr. Therefore, our results suggest that extracellular Vpr could fuel the progression of AIDS via stimulation of HIV-1 provirus present in such cellular reservoirs as mononuclear phagocytes in HIV-infected patients.
Asunto(s)
Productos del Gen vpr/metabolismo , VIH-1/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Macrófagos/citología , Monocitos/citología , FN-kappa B/metabolismo , Factor de Transcripción AP-1/metabolismo , Transcripción Genética , Síndrome de Inmunodeficiencia Adquirida/patología , Síndrome de Inmunodeficiencia Adquirida/virología , Western Blotting , Núcleo Celular/metabolismo , Progresión de la Enfermedad , Activación Enzimática , Genes Reporteros , Proteína p24 del Núcleo del VIH/metabolismo , Humanos , Leucocitos Mononucleares/virología , MAP Quinasa Quinasa 7/metabolismo , Macrófagos/enzimología , Monocitos/enzimología , Fagocitos/patología , Fagocitos/virología , Fagocitosis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Células U937 , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
The binding subunit of pertussis toxin (PTX-B) has been shown recently to inhibit the entry and postentry events in HIV-1 replication in primary T lymphocytes and monocyte-derived macrophages. While the effect of PTX-B on HIV-1 entry was shown to involve CCR5 desensitization, the mechanism of postentry inhibition remained unclear. In T lymphocytes, PTX-B affected transcription or stability of Tat-stimulated HIV-1 mRNAs. In this study, we sought to identify the mechanism of postentry inhibition of HIV-1 replication by PTX-B in U-937 promonocytic cells. We demonstrate that in these cells PTX-B inhibits expression of luciferase reporter gene controlled by the HIV-1 LTR promoter. This effect is Tat-independent and is not restricted to the HIV-1 LTR promoter. Instead, PTX-B activity is mediated through suppression of the cellular transcription factor, NF-kappaB. PTX-B inhibits phosphorylation and nuclear translocation of the p65 subunit of NF-kappaB. This effect is independent of the cytoplasmic NF-kappaB inhibitor, IkappaBalpha, as PTX-B stimulates phosphorylation and subsequent degradation of this protein. The suppressive activity of PTX-B on NF-kappaB p65 phosphorylation and nuclear translocation is delayed, suggesting that PTX-B signaling might initiate synthesis and cytoplasmic accumulation of a p65 phosphorylation inhibitor.
Asunto(s)
Regulación Viral de la Expresión Génica/efectos de los fármacos , Duplicado del Terminal Largo de VIH/efectos de los fármacos , VIH-1/genética , FN-kappa B/metabolismo , Toxina del Pertussis/farmacología , Transcripción Genética/efectos de los fármacos , Sitios de Unión , Núcleo Celular/metabolismo , Genes Reporteros , Humanos , Proteínas I-kappa B/metabolismo , Luciferasas/genética , Inhibidor NF-kappaB alfa , Fosforilación , Factor de Transcripción ReIA , Células U937RESUMEN
The human immunodeficiency virus (HIV) Nef protein plays a critical role in AIDS pathogenesis by enhancing replication and survival of the virus within infected cells and by facilitating its spread in vivo. Most of the data obtained so far have been in experiments with endogenous Nef protein, so far overlooking the effects of exogenous soluble Nef protein. We used recombinant exogenous Nef proteins to activate nuclear transcription factors NF-kappaB and AP-1 in the promonocytic cell line U937. Exogenous SIV and HIV-1 Nef proteins activated NF-kappaB and AP-1 in a dose- and time-dependent manner. Activation of NF-kappaB by exogenous Nef was concomitant to the degradation of the inhibitor of NF-kappaB, IkappaBalpha. In agreement with increased AP-1 activation, a time- and dose-dependent increase in JNK activation was observed following treatment of U937 cells with exogenous Nef. Since exogenous Nef activates the transcription factors NF-kappaB and AP-1, which bind to the HIV-1 long terminal repeat (LTR), we investigated the effect of exogenous Nef on HIV-1 replication. We observed that exogenous Nef stimulated HIV-1 LTR via NF-kappaB activation in U937 cells and enhanced viral replication in the chronically infected promonocytic cells U1. Therefore, our results suggest that exogenous Nef could fuel the progression of the disease via stimulation of HIV-1 provirus present in such cellular reservoirs as mononuclear phagocytes in HIV-infected patients.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/virología , Productos del Gen nef/fisiología , VIH/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Monocitos/enzimología , FN-kappa B/metabolismo , Factor de Transcripción AP-1/metabolismo , Western Blotting , Línea Celular , Relación Dosis-Respuesta a Droga , Activación Enzimática , Genes Reporteros , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Monocitos/virología , Transducción de Señal , Virus de la Inmunodeficiencia de los Simios/metabolismo , Factores de Tiempo , Células U937 , Replicación Viral/genética , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
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.