RESUMEN
Individuals who are infected with HIV-1 accumulate damage to cells and tissues (e.g. neurons) that are not directly infected by the virus. These include changes known as HIV-associated neurodegenerative disorder (HAND), leading to the loss of neuronal functions, including synaptic long-term potentiation (LTP). Several mechanisms have been proposed for HAND, including direct effects of viral proteins such as the Tat protein. Searching for the mechanisms involved, we found here that HIV-1 Tat inhibits E2F transcription factor 3 (E2F3), CAMP-responsive element-binding protein (CREB), and brain-derived neurotropic factor (BDNF) by up-regulating the microRNA miR-34a. These changes rendered murine neurons dysfunctional by promoting neurite retraction, and we also demonstrate that E2F3 is a specific target of miR-34a. Interestingly, bioinformatics analysis revealed the presence of an E2F3-binding site within the CREB promoter, which we validated with ChIP and transient transfection assays. Of note, luciferase reporter assays revealed that E2F3 up-regulates CREB expression and that Tat interferes with this up-regulation. Further, we show that miR-34a inhibition or E2F3 overexpression neutralizes Tat's effects and restores normal distribution of the synaptic protein synaptophysin, confirming that Tat alters these factors, leading to neurite retraction inhibition. Our results suggest that E2F3 is a key player in neuronal functions and may represent a good target for preventing the development of HAND.
Asunto(s)
Factor de Transcripción E2F3/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/farmacología , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Línea Celular Tumoral , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Potenciación a Largo Plazo/efectos de los fármacos , Ratones , MicroARNs/genética , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Neuronas/metabolismo , Regiones Promotoras Genéticas/genética , Sinaptofisina/metabolismoRESUMEN
Involvement of the human immunodeficiency virus type 1 (HIV-1) trans-activator of transcription (Tat) protein in neuronal deregulation and in the development of HIV-1 associated neurocognitive disorders (HAND) has been amply explored; however the mechanisms involved remain unclear. In search for the mechanisms, we demonstrated that Tat deregulates neuronal functions through a pathway that involved p73 and p53 pathway. We showed that Tat uses microRNA-196a (miR-196a) to deregulate the p73 pathway. Further, we found that the Abelson murine leukemia (c-Abl) phosphorylates p73 on tyrosine residue 99 (Tyr-99) in Tat-treated cells. Interestingly, Tat lost its ability to promote accumulation and phosphorylation of p73 in the presence of miR-196a mimic. Interestingly, accumulation of p73 did not lead to neuronal cell death by apoptosis as obtained by cell viability assay. Western blot analysis using antibodies directed against serine residues 807 and 811 of retinoblastoma (Rb) protein was also used to validate our data regarding lack of cell death. Hyperphosphorylation of RB (S807/811) is an indication of cell neuronal viability. These results highlight the key role played by p73 and microRNA in Tat-treated neurons leading to their deregulation and it deciphers mechanistically one of the pathways used by Tat to cause neuronal dysfunction that contributes to the development of HAND.
Asunto(s)
Trastornos del Conocimiento/metabolismo , Infecciones por VIH/metabolismo , VIH-1/fisiología , MicroARNs/metabolismo , Animales , Apoptosis , Muerte Celular , Línea Celular Tumoral , Supervivencia Celular , Trastornos del Conocimiento/etiología , Trastornos del Conocimiento/virología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Infecciones por VIH/complicaciones , Infecciones por VIH/virología , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/metabolismo , Neuronas/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-abl/genética , Proteínas Proto-Oncogénicas c-abl/metabolismo , Proteína de Retinoblastoma/metabolismo , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
For over two decades, highly active antiretroviral therapy (HAART) was able to help prolong the life expectancy of people living with HIV-1 (PLWH) and eliminate the virus to an undetectable level. However, an increased prevalence of HIV- associated neurocognitive disorders (HAND) was observed. These symptoms range from neuronal dysfunction to cell death. Among the markers of neuronal deregulation, we cite the alteration of synaptic plasticity and neuronal communications. Clinically, these dysfunctions led to neurocognitive disorders such as learning alteration and loss of spatial memory, which promote premature brain aging even in HAART-treated patients. In support of these observations, we showed that the gp120 protein deregulates miR-499-5p and its downstream target, the calcineurin (CaN) protein. The gp120 protein also promotes the accumulation of calcium (Ca2+) and reactive oxygen species (ROS) inside the neurons leading to the activation of CaN and the inhibition of miR-499-5p. gp120 protein also caused mitochondrial fragmentation and changes in shape and size. The use of mimic miR-499 restored mitochondrial functions, appearance, and size. These results demonstrated the additional effect of the gp120 protein on neurons through the miR-499-5p/calcineurin pathway.
Asunto(s)
Infecciones por VIH , VIH-1 , MicroARNs , Humanos , VIH-1/metabolismo , Calcineurina/metabolismo , Calcineurina/farmacología , Encéfalo/metabolismo , Muerte Celular , MicroARNs/genética , MicroARNs/metabolismoRESUMEN
Over the last decade, small noncoding RNA molecules such as microRNAs (miRNAs) have emerged as critical regulators in the expression and function of eukaryotic genomes. It has been suggested that viral infections and neurological disease outcome may also be shaped by the influence of small RNAs. This has prompted us to suggest that HIV infection alters the endogenous miRNA expression patterns, thereby contributing to neuronal deregulation and AIDS dementia. Therefore, using primary cultures and neuronal cell lines, we examined the impact of a viral protein (HIV-1 Tat) on the expression of miRNAs due to its characteristic features such as release from the infected cells and taken up by noninfected cells. Using microRNA array assay, we demonstrated that Tat deregulates the levels of several miRNAs. Interestingly, miR-34a was among the most highly induced miRNAs in Tat-treated neurons. Tat also decreases the levels of miR-34a target genes such as CREB protein as shown by real time PCR. The effect of Tat was neutralized in the presence of anti-miR-34a. Using in situ hybridization assay, we found that the levels of miR-34a increase in Tat transgenic mice when compared with the parental mice. Therefore, we conclude that deregulation of neuronal functions by HIV-1 Tat protein is miRNA-dependent.
Asunto(s)
VIH-1 , MicroARNs/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/farmacología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/virología , Calcio/metabolismo , Línea Celular Tumoral , Perfilación de la Expresión Génica , Proteína p24 del Núcleo del VIH/metabolismo , VIH-1/fisiología , Humanos , Ratones , MicroARNs/genética , Neuronas/patología , Neuronas/virología , Reproducibilidad de los Resultados , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Studies have shown that HIV-infected patients develop neurocognitive disorders characterized by neuronal dysfunction. The lack of productive infection of neurons by HIV suggests that viral and cellular proteins, with neurotoxic activities, released from HIV-1-infected target cells can cause this neuronal deregulation. The viral protein R (Vpr), a protein encoded by HIV-1, has been shown to alter the expression of various important cytokines and inflammatory proteins in infected and uninfected cells; however the mechanisms involved remain unclear. Using a human neuronal cell line, we found that Vpr can be taken up by neurons causing: (i) deregulation of calcium homeostasis, (ii) endoplasmic reticulum-calcium release, (iii) activation of the oxidative stress pathway, (iv) mitochondrial dysfunction and v- synaptic retraction. In search for the cellular factors involved, we performed microRNAs and gene array assays using human neurons (primary cultures or cell line, SH-SY5Y) that we treated with recombinant Vpr proteins. Interestingly, Vpr deregulates the levels of several microRNAs (e.g. miR-34a) and their target genes (e.g. CREB), which could lead to neuronal dysfunctions. Therefore, we conclude that Vpr plays a major role in neuronal dysfunction through deregulating microRNAs and their target genes, a phenomenon that could lead to the development of neurocognitive disorders.
Asunto(s)
Calcio/metabolismo , Trastornos del Conocimiento/virología , Regulación Viral de la Expresión Génica , MicroARNs/metabolismo , Mitocondrias/metabolismo , Enfermedades del Sistema Nervioso/virología , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , Trastornos del Conocimiento/patología , Humanos , Inmunohistoquímica/métodos , Ratones , Ratones Transgénicos , Microscopía Fluorescente/métodos , Enfermedades del Sistema Nervioso/patología , Neuronas/metabolismo , Estrés OxidativoRESUMEN
BACKGROUND: p53 plays an important role in many areas of cellular physiology and biology, ranging from cellular development and differentiation to cell cycle arrest and apoptosis. Many of its functions are attributed to its role in assuring proper cellular division. However, since the establishment of its role in cell cycle arrest, damage repair, and apoptosis (thus also establishing its importance in cancer development), numerous reports have demonstrated additional functions of p53 in various cells. In particular, p53 appears to have important functions as it relates to neurodegeneration and synaptic plasticity. OBJECTIVE: In this review, we will address p53 functions as it relates to various neurodegenerative diseases, mainly its implications in the development of HIV-associated neurocognitive disorders. CONCLUSION: p53 plays a pivotal role in the development of neurodegenerative diseases through its interaction with cellular factors, viral factors, and/or small RNAs that have the ability to promote the development of these diseases. Hence, inhibition of p53 may present an ideal target to restore neuronal functions.
Asunto(s)
Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Proteína p53 Supresora de Tumor/fisiología , Complejo SIDA Demencia/genética , Complejo SIDA Demencia/metabolismo , Complejo SIDA Demencia/fisiopatología , Animales , Humanos , Enfermedades Neurodegenerativas/fisiopatologíaRESUMEN
HIV-associated neurocognitive disorders (HAND) remain an unsolved problem that persists despite using antiretroviral therapy. We have obtained data showing that HIV-gp120 protein contributes to neurodegeneration through metabolic reprogramming. This led to decreased ATP levels, lower mitochondrial DNA copy numbers, and loss of mitochondria cristae, all-important for mitochondrial biogenesis. gp120 protein also disrupted mitochondrial movement and synaptic plasticity. Searching for the mechanisms involved, we found that gp120 alters the cyclic AMP response element-binding protein (CREB) phosphorylation on serine residue 133 necessary for its function as a transcription factor. Since CREB regulates the promoters of PGC1α and BDNF genes, we found that CREB dephosphorylation causes PGC1α and BDNF loss of functions. The data was validated in vitro and in vivo. The negative effect of gp120 was alleviated in cells and animals in the presence of rolipram, an inhibitor of phosphodiesterase protein 4 (PDE4), restoring CREB phosphorylation. We concluded that HIV-gp120 protein contributes to HAND via inhibition of CREB protein function.
RESUMEN
St. John's Wort is commonly known for its antiviral, antidepressant, and cytotoxic properties, but traditionally St. John's Wort has also been used to treat inflammation. In this study, we sought to characterize the mechanisms used by St. John's Wort to treat inflammation by examining the effect of the recently isolated protein from St. John's Wort, p27SJ on the expression of MCP-1. By employing an adenovirus expression vector, we demonstrate that a low concentration of p27SJ upregulates the MCP-1 promoter through the transcription factor C/EBPbeta. In addition, we found that C/EBPbeta-homologous protein (CHOP) or siRNA-C/EBPbeta significantly reduced the ability of p27SJ to activate MCP-1 gene expression. Results from protein-protein interaction studies illustrate the existence of a physical interaction between p27SJ and C/EBPbeta in microglial cells. The use of chromatin immunoprecipitation assay (ChIP) led to the identification of a new cis-element that is responsive to C/EBPbeta within the MCP-1 promoter. Association of C/EBPbeta with MCP-1 DNA was not affected by the presence of p27SJ. The biological activity of MCP-1 produced by cultures of adenovirus-p27SJ transduced cells was increased relative to controls as measured by the transmigration of human Jurkat cells. Thus, we conclude that at high concentration, p27SJ is a potential agent that may be developed as a modulator of MCP-1 leading to the inhibition of the cytokine-mediated inflammatory responses.
Asunto(s)
Quimiocina CCL2/biosíntesis , Hypericum/química , Proteínas de Plantas/farmacología , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Línea Celular Tumoral , Quimiocina CCL2/genética , Regulación de la Expresión Génica , Humanos , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
Non-small cell lung cancer (NSCLC) represents about 85% of all lung cancer cases. Lung cancer is the most frequent non-AIDS-defining malignancies in HIV-infected patients. The mechanism of the increased risk for lung cancer in HIV-1 patients is poorly understood. HIV-1 Nef protein has been suggested to be one of the key players in HIV-related lung disease. In here, we showed the involvement of Nef protein in cell modifications such as fibroblasts (IMR-90) and normal (BEAS-2B) or cancerous (A549) epithelial cells. We demonstrated that Nef protein reprograms initial stages of lung cancer (e.g. changes in the metabolism, improved cell survival and invasion, increase the angiogenesis factor VEGF). Additionally, we showed that Nef is provoking a global decrease of mature miRNA and a decrease of DICER1 and AGO expression in lung cells. MiRNAs play a crucial role in cell signaling and homeostasis, functioning as oncogenes or tumor suppressors, and their dysregulation can contribute to the tumorigenic process. These results showed that HIV-1 Nef protein is directly involved in preventing cell death and contributes to tumor progression.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/virología , Proliferación Celular , Infecciones por VIH/virología , VIH-1/fisiología , Neoplasias Pulmonares/virología , MicroARNs/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Células A549 , Proteínas Argonautas/metabolismo , Autofagia , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Supervivencia Celular , ARN Helicasas DEAD-box/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Mitocondrias/virología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleasa III/metabolismo , Transfección , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Disruption of mitochondria axonal transport, essential for the maintenance of synaptic and neuronal integrity and function, has been identified in neurodegenerative diseases. Whether HIV-1 viral proteins affect mitochondria axonal transport is unknown, albeit HIV-associated neurocognitive disorders occur in around half of the patients living with HIV. Therefore, we sought to examine the effect of HIV-1 viral protein R (Vpr) on mitochondria axonal transport. Using mice primary neuronal cultures, we demonstrated that 4-day Vpr treatment reduced the ratio of moving mitochondria associated with (i) less energy (ATP) supply, (ii) reduction in Miro-1 and (iii) increase of α-synuclein which led to loss of microtubule stability as demonstrated by inconsecutive distribution of acetylated α-tubulin along the axons. Interestingly, the effect of Vpr on mitochondria axonal transport was partially restored in the presence of bongkrekic acid, a compound that negatively affected the Vpr-adenine nucleotide translocator (ANT) interaction and totally restored the ATP level in neurons. This indicated Vpr impaired mitochondria axonal transport partially related to its interaction with ANT. The above effect of Vpr was similar to the data obtained from hippocampal tissues isolated from 18-month-old aging mice compared to 5-month-old mice. In accord with previous clinical findings that HIV infection prematurely ages the brain and increases the susceptibility to HAND, we found that Vpr induced aging markers in neurons. Thus, we concluded that instead of causing cell death, low concentration of HIV-1 Vpr altered neuronal function related with inhibition of mitochondria axonal transport which might contribute to the accelerated neuronal aging.
Asunto(s)
Transporte Axonal/fisiología , Senescencia Celular/fisiología , VIH-1 , Mitocondrias/metabolismo , Neuronas/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Transporte Axonal/efectos de los fármacos , Ácido Bongcréquico/farmacología , Línea Celular Tumoral , Senescencia Celular/efectos de los fármacos , Células HEK293 , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Hipocampo/virología , Humanos , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Mitocondrias/virología , Neuronas/efectos de los fármacos , Neuronas/patología , Neuronas/virología , Fármacos Neuroprotectores/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección , alfa-Sinucleína/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Herpes simplex virus type 1 (HSV-1) establishes a lifelong latency in the neurons of its host. Sporadically, the latent virus reactivates and spreads back to the original site of infection and causes recrudescent diseases. The only gene actively transcribed during neuronal latency is the latency associated transcript (LAT) gene. Several transcripts have been detected in the important LAT promoter region. However, no polypeptides coded by these transcripts are known. In this communication, we reported the cloning, sequencing, and characterization of a transcript immediately upstream of LAT. We designated this gene UOL (Upstream of LAT). The UOL RNA is polyadenylated, expressed as a late gene in infected cells, transcribed in the same direction as LAT, and contains an open reading frame (ORF) capable of encoding a protein of 96 amino acids with a predicted molecular mass of 11 kDa. The UOL transcript contains 466 nucleotides in length. The 5' end of the UOL transcript starts at nucleotide 118,266 and the 3' end of the UOL transcript ends at nucleotide 118,731 based on the published 17syn+ genomic sequence. The UOL protein was detected in infected cell lysates by immunoprecipitation using an antibody raised against UOL ORF synthetic peptide. More importantly, sera from mice infected with wild-type HSV-1 but not sera from mice infected with a mutant with the UOL region deleted recognized the UOL ORF, expressed in Escherichia coli, on Western blots. These results suggest that a UOL protein is in HSV-1 infected tissue culture cells and in mice infected with HSV-1.
Asunto(s)
Herpesvirus Humano 1/genética , Regiones Promotoras Genéticas , Proteínas Virales/genética , Proteínas Virales/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/sangre , Secuencia de Bases , Western Blotting , Línea Celular , Chlorocebus aethiops , Clonación Molecular , Modelos Animales de Enfermedad , Escherichia coli , Herpes Simple/inmunología , Herpes Simple/virología , Ratones , MicroARNs , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta , Análisis de Secuencia de ADN , Regiones Terminadoras Genéticas , Sitio de Iniciación de la Transcripción , Proteínas Virales/químicaRESUMEN
Several reports have pointed to the negative involvement of p53 in transcriptional regulation of the human immunodeficiency virus type 1 long-terminal repeat (HIV-1 LTR). We recently demonstrated that through their physical interaction, cdk9 phosphorylates p53 on Ser-392, leading to p53 stability and accumulation. As a result, p53 stalled transcriptional elongation of the HIV-1 LTR and significantly reduced HIV-1 replication in primary microglia and astrocytes. Therefore, we sought to identify the mechanisms used by cdk9 to allow this p53 function. Using western blot analysis, we found that cdk9 promotes inhibition and phosphorylation of Mdm2 on Ser-395, thus preventing degradation of p53, a protein that is directly involved in promoting p53 ubiquitination. On the other hand, we showed that cdk9 phosphorylates Pirh2 on Ser-211 and Thr-217 residues through their physical interaction. Phosphorylation of Pirh2 renders it inactive and may contribute to p53-inhibition of transcriptional elongation of the HIV-1 LTR. Hence, we suggest that phosphorylation of Pirh2 may be a novel target for the inhibition of HIV-1 gene expression.
Asunto(s)
Quinasa 9 Dependiente de la Ciclina/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Línea Celular , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 9 Dependiente de la Ciclina/genética , Duplicado del Terminal Largo de VIH/genética , VIH-1/metabolismo , Humanos , Datos de Secuencia Molecular , Fosforilación , Mapeo de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transfección , Proteína p53 Supresora de Tumor/genética , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
The HIV-1 protein, Tat has been implicated in AIDS pathogenesis however, the amount of circulating Tat is believed to be very low and its quantification has been difficult. We performed the quantification of Tat released from infected cells and taken up by neurons using high performance capillary electrophoresis. This is the first report to successfully measure the amount of Tat in neurons and places Tat as a key player involved in HIV-associated neurocognitive disorders.
Asunto(s)
Electroforesis Capilar/métodos , Productos del Gen tat/análisis , VIH-1/química , Neuronas/virología , Complejo SIDA Demencia/etiología , Comunicación Celular , Línea Celular , Productos del Gen tat/sangre , Humanos , Neuronas/químicaRESUMEN
Several reports have pointed to the negative involvement of p53 in transcriptional regulation of the human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR). However, the mechanisms of this negative effect remain unclear. In here, we showed that over expression of p53 wild type prevented the phosphorylation of serine 2 in the carboxyl terminal domain (CTD) of RNA polymerase II. As a result of this inhibition, p53 stalled transcriptional elongation on the HIV-1 LTR leading to a significant reduction of HIV-1 replication in primary microglia and astrocytes. However, despite the delay/pause caused by p53, viral transcription and replication decreased and then salvaged. These studies suggest that the negative effect of p53 is alleviated by a third factor. In this regard, our Preliminary Data point to the involvement of the Pirh2 protein in p53 inhibition. Therefore, we suggest that p53 may be a novel therapeutic target for the inhibition of HIV-1 gene expression and replication and the treatment of AIDS.
Asunto(s)
Regulación Viral de la Expresión Génica , VIH-1/genética , Proteína p53 Supresora de Tumor/metabolismo , Células Cultivadas , Quinasa 9 Dependiente de la Ciclina/análisis , Quinasa 9 Dependiente de la Ciclina/metabolismo , Duplicado del Terminal Largo de VIH/genética , Humanos , Fosforilación , ARN Polimerasa II/metabolismo , Serina/metabolismo , Transcripción Genética , Proteína p53 Supresora de Tumor/análisis , Proteína p53 Supresora de Tumor/genética , Ubiquitina-Proteína Ligasas/metabolismo , Replicación ViralRESUMEN
The cytokine tumor necrosis factor alpha (TNFalpha) is a key factor in several inflammatory diseases and its levels increase in response to a variety of internal or external stimuli. The regulation of the TNFalpha promoter is mediated by several transcription factors including the nuclear factor kappa B protein (NF-kappaB). This study examines the role of NF-kappaB in the regulation of TNFalpha production by morphine in microglia. Using reverse transcriptase polymerase chain reaction, we demonstrated the presence of morphine receptors in these cells. We next demonstrated the ability of morphine to promote TNFalpha production and secretion by these cells using a cytokine array assay. Transient transfection experiments led to the identification of the region located between nucleotides -751 and -615 within the TNFalpha promoter as being responsive to morphine treatment. The DNA sequence of this region contains a motif indicative of a potential NF-kappaB binding site. The use of a small interfering RNA directed against p65, a subunit of NF-kappaB, demonstrated that TNFalpha induction by morphine is NF-kappaB-dependent. All of the effects of morphine were reversed by the morphine inhibitor, naloxone. These data provide important insights into the effects of morphine on microglia.
Asunto(s)
Analgésicos Opioides/farmacología , Morfina/farmacología , FN-kappa B/fisiología , Neuroglía/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Analgésicos Opioides/antagonistas & inhibidores , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Western Blotting , Células Cultivadas , Infecciones por VIH/virología , VIH-1 , Humanos , Microglía/efectos de los fármacos , Microglía/metabolismo , Morfina/antagonistas & inhibidores , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Neuroglía/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Células U937RESUMEN
The human immunodeficiency virus-1 (HIV-1) Vpr protein plays multiple roles in HIV-1 replication. In early infection, Vpr provides help in the nuclear localization of pre-integration complex. Subsequently, Vpr induces cell cycle arrest of infected cells at G2/M phase. Cell cycle arrest facilitates higher rate of viral gene transcription. Vpr is also capable of activating transcription of viral and heterologous genes. Vpr induces apoptosis in infected cells leading to loss of immune cells and onset of clinical AIDS. Interestingly, Vpr is also considered as a passenger protein in the virus particles as it is incorporated into the virus particles through interaction with Gag. The structure of full length Vpr has been resolved recently through NMR. In this review, we have analysed the functions of Vpr using the available data from structural perspective. Packing of the three helices of Vpr around a core formed by hydrophobic side chains and integrity of helical domains are critical for Vpr functions. The distinct functions of Vpr have been attributed to structural integrity of different domains. The unique distribution of acidic and basic residues in Vpr is an interesting feature. Two hydrophobic pockets on the structure of Vpr are proposed to be important targets for modulating Vpr functions. The inter-relationship between different functions of Vpr is discussed in the context of structure. Based on bioinformatics analysis, we propose new targets for modulating Vpr functions, which need to be validated experimentally.
Asunto(s)
VIH-1/química , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/química , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismo , Modelos Moleculares , Estructura Terciaria de Proteína , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The lack of productive infection of neurons by HIV-1 suggests that the neuronal damage seen in AIDS patients with cognitive disorders is caused indirectly via viral and cellular proteins with neurotoxic activity. Among HIV-1 proteins, Vpr has been shown to deregulate expression of various important cytokines and inflammatory proteins in infected and uninfected cells. However, the mechanisms underlying these changes remain unclear. Here, we demonstrate that neurons can take up Vpr that is released into the supernatant of HIV-infected microglia. We also found that administration of recombinant Vpr (rVpr) to human neurons resulted in a slow but sustained elevation of intracellular calcium [Ca(2+)]i. Interestingly, our data also show that [Ca(2+)]i elevation by Vpr leads to ROS production and impairs glutamate signaling in neuronal cells. Vpr disturbs calcium homeostasis through downregulation of endogenous PMCA. Finally, we found that the permeability of the plasma membrane increases in neurons treated with Vpr. Therefore, we conclude that soluble Vpr is a major viral factor that causes a disturbance in neuronal communication leading to neuronal dysfunction. The outcome of these studies will advance the understanding of HIV-1 pathogenesis and will help in the development of new therapeutic approaches.
Asunto(s)
Calcio/fisiología , VIH-1 , Neuronas/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/fisiología , Calcio/antagonistas & inhibidores , Comunicación Celular/fisiología , Línea Celular Tumoral , Células Cultivadas , Regulación hacia Abajo/fisiología , Humanos , Microglía/metabolismo , Microglía/virología , Neuronas/virología , Células U937 , Regulación hacia Arriba/fisiologíaRESUMEN
The detection of biomarkers of oxidative stress in brain tissue and cerebrospinal fluid of patients with human immunodeficiency virus, type 1 (HIV)-associated dementia indicates the involvement of stress pathways in the neuropathogenesis of AIDS. Although the biological importance of oxidative stress on events involved in AIDS neuropathogenesis and the HIV-1 proteins responsible for oxidative stress remain to be elucidated, our results point to the activation of hypoxia-inducible factor 1 (HIF-1) upon HIV-1 infection and its elevation in brain cells of AIDS patients with dementia. HIF-1 is a transcription factor that is responsive to oxygen. Under hypoxic conditions, HIF-1alpha becomes stable and translocates to the nucleus where it dimerizes with aryl hydrocarbon receptor nuclear translocator and modulates gene transcription. Activation of HIF-1 can also be mediated by the HIV-1 accessory protein Vpr. In addition, cellular components, including reactive oxygen species, contribute to the induction of HIF-1alpha. Our results show that Vpr induces reactive oxygen species by increasing H(2)O(2) production, which can contribute to HIF-1alpha accumulation. Interestingly, increased levels of HIF-1alpha stimulated HIV-1 gene transcription through HIF-1 association with HIV-1 long terminal repeat. These observations point to the existence of a positive feedback interplay between HIF-1alpha and Vpr and that, by inducing oxidative stress via activation of HIF-1, Vpr can induce HIV-1 gene expression and dysregulate multiple host cellular pathways.
Asunto(s)
Regulación de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismo , Encéfalo/metabolismo , Línea Celular , Dimerización , Células HeLa , Humanos , Peróxido de Hidrógeno/metabolismo , Microglía/metabolismo , Modelos Biológicos , Estrés Oxidativo , Regiones Promotoras Genéticas , Interferencia de ARN , Especies Reactivas de OxígenoRESUMEN
The polyomavirus JC (JCV) causes the demyelinating disease progressive multifocal leukoencephalopathy (PML). Infection by JCV is very common in childhood after which the virus enters a latent state, which is poorly understood. Under conditions of severe immunosuppression, especially AIDS, JCV may reactivate to cause PML. Expression of JC viral proteins is regulated by the JCV non-coding control region (NCCR), which contains an NF-kappaB binding site previously shown to activate transcription. We now report that C/EBPbeta inhibits basal and NF-kappaB-stimulated JCV transcription via the same site. Gel shift analysis showed C/EBPbeta bound to this region in vitro and ChIP assays confirmed this binding in vivo. Further, a ternary complex of NF-kappaB/p65, C/EBPbeta-LIP and JCV DNA could be detected in co-immunoprecipitation experiments. Mutagenesis analysis of the JCV NCCR indicated p65 and C/EBPbeta-LIP bound to adjacent but distinct sites and that both sites regulate basal and p65-stimulated transcription. Thus C/EBPbeta negatively regulates JCV, which together with NF-kappaB activation, may control the balance between JCV latency and activation leading to PML. This balance may be regulated by proinflammatory cytokines in the brain.
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Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Virus JC/fisiología , Transcripción Genética , Sitios de Unión , Línea Celular , Inmunoprecipitación de Cromatina , ADN Viral/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Humanos , FN-kappa B/metabolismo , Neuroglía/virología , Unión ProteicaRESUMEN
HIV-associated dementia (HAD) is the most common AIDS-associated neurological disorder and is characterized by the development of synaptodendritic injury to neurons. To advance HAD therapy, it is crucial to identify the mechanisms and factors involved. The viral protein HIV-1 Tat is among those factors and is released by HIV-1-infected cells and can be taken up by adjacent neuronal cells leading to neurotoxic effects. Multiple cellular host proteins have been identified as Tat cofactors in causing neuronal injury. Interestingly, most of these factors function through activation of the p53 pathway. We have now examined the ability of Tat to activate the p53 pathway leading to the induction of endogenous p53 and p73 in neuronal cells. We found that Tat induced p53 and p73 levels in SH-SY5Y cells and that this induction caused retraction of neurites. In the absence of either p53 or p73, Tat failed to induce dendritic retraction or to activate the proapoptotic proteins, such as Bax. Further, we found that p53-accumulation in Tat-treated cells depends on the presence of p73. Therefore, we conclude that Tat contributes to neuronal degeneration through activation of a pathway involving p53 and p73. This information will be valuable for the development of therapeutic agents that affect these pathways to protect CNS neurons and prevent HAD.