RESUMEN
HIV infects astrocytes in a restricted manner but leads to abundant expression of Nef, a major viral factor for HIV replication and disease progression. However, the roles of Nef in HIV gene expression and replication in astrocytes and viral transfer from astrocytes to CD4+ T cells remain largely unclear. In this study, we attempted to address these issues by transfecting human primary astrocytes with HIV molecular clones with intact Nef and without Nef (a nonsense Nef mutant) and comparing gene expression and replication in astrocytes and viral transfer from astrocytes to CD4+ T cells MT4. First, we found that lack of Nef expression led to increased extracellular virus production from astrocytes and intracellular viral protein and RNA expression in astrocytes. Using a HIV LTR-driven luciferase reporter gene assay, we showed that ectopic Nef expression alone inhibited the HIV LTR promoter activity in astrocytes. Consistent with the previously established function of Nef, we showed that the infectivity of HIV derived from astrocytes with Nef expression was significantly higher than that with no Nef expression. Next, we performed the co-culture assay to determine HIV transfer from astrocytes transfected to MT4. We showed that lack of Nef expression led to significant increase in HIV transfer from astrocytes to MT4 using two HIV clones. We also used Nef-null HIV complemented with Nef in trans in the co-culture assay and demonstrated that Nef expression led to significantly decreased HIV transfer from astrocytes to MT4. Taken together, these findings support a negative role of Nef in HIV replication and pathogenesis in astrocytes.
Asunto(s)
Infecciones por VIH , VIH-1 , Humanos , Linfocitos T , Astrocitos , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Replicación Viral/genética , VIH-1/genética , Linfocitos T CD4-Positivos , Infecciones por VIH/genética , Expresión GénicaRESUMEN
HIV-associated neurocognitive disorders (HAND) in the era of combination antiretroviral therapy are primarily manifested as impaired behaviours, glial activation/neuroinflammation and compromised neuronal integrity, for which there are no effective treatments currently available. In the current study, we used doxycycline-inducible astrocyte-specific HIV Tat transgenic mice (iTat), a surrogate HAND model, and determined effects of PNU-125096, a positive allosteric modulator of α7 nicotinic acetylcholine receptor (α7 nAChR) on Tat-induced behavioural impairments and neuropathologies. We showed that PNU-125096 treatment significantly improved locomotor, learning and memory deficits of iTat mice while inhibited glial activation and increased PSD-95 expression in the cortex and hippocampus of iTat mice. Using α7 nAChR knockout mice, we showed that α7 nAChR knockout eliminated the protective effects of PNU-125096 on iTat mice. In addition, we showed that inhibition of p38 phosphorylation by SB239063, a p38 MAPK-specific inhibitor exacerbated Tat neurotoxicity in iTat mice. Last, we used primary mouse cortical individual cultures and neuron-astrocytes co-cultures and in vivo staining of iTat mouse brain tissues and showed that glial activation was directly involved in the interplay among Tat neurotoxicity, α7 nAChR activation and the p38 MAPK signalling pathway. Taken together, these findings demonstrated for the first time that α7 nAChR activation led to protection against HAND and suggested that α7 nAChR modulator PNU-125096 holds significant promise for development of therapeutics for HAND.
Asunto(s)
Complejo SIDA Demencia/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/toxicidadRESUMEN
Astrocytes are susceptible to HIV infection and potential latent HIV reservoirs. Tat is one of three abundantly expressed HIV early genes in HIV-infected astrocytes and has been shown to be a major pathogenic factor for HIV/neuroAIDS. In this study, we sought to determine if and how Tat expression would affect HIV infection and latency in astrocytes. Using the glycoprotein from vesicular stomatitis virus-pseudotyped red-green HIV (RGH) reporter viruses, we showed that HIV infection was capable of establishing HIV latency in astrocytes. We also found that Tat expression decreased the generation of latent HIV-infected cells. Activation of latent HIV-infected astrocytes showed that treatment of GSK126, a selective inhibitor of methyltransferase enhancer of zeste homolog 2 (Ezh2) that is specifically responsible for tri-methylation of histone 3 lysine 27 (H3K27me3), led to activation of significantly more latent HIV-infected Tat-expressing astrocytes. Molecular analysis showed that H3K27me3, Ezh2, MeCP2, and Tat all exhibited a similar bimodal expression kinetics in the course of HIV infection and latency in astrocytes, although H3K27me3, Ezh2, and MeCP2 were expressed higher in Tat-expressing astrocytes and their expression were peaked immediately preceding Tat expression. Subsequent studies showed that Tat expression alone was sufficient to induce H3K27me3 expression, likely through its regulation of Ezh2 and MeCP2 expression. Taken together, these results showed for the first time that Tat expression induced H3K27me3 expression and contributed to HIV latency in astrocytes and suggest a new role and novel mechanism for Tat in HIV latency.
Asunto(s)
Astrocitos/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/genética , Histonas/genética , Interacciones Huésped-Patógeno/genética , Proteína 2 de Unión a Metil-CpG/genética , Latencia del Virus/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Astrocitos/efectos de los fármacos , Astrocitos/virología , Línea Celular Tumoral , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/virología , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , VIH-1/efectos de los fármacos , VIH-1/genética , VIH-1/crecimiento & desarrollo , VIH-1/metabolismo , Histonas/metabolismo , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Indoles/farmacología , Células Jurkat , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteína 2 de Unión a Metil-CpG/metabolismo , Metilación , Piridonas/farmacología , Transducción de Señal , Vesiculovirus/genética , Vesiculovirus/metabolismo , Latencia del Virus/efectos de los fármacos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Proteína Fluorescente RojaRESUMEN
In the original article the name of author Chris Sanborn was misspelled. It is correct here. Also, in the Acknowledgments section there was an error in the NIH/NINDS grant numbers.
RESUMEN
Tip110, an important regulator of several oncogenic proteins, was significantly downregulated in human metastatic melanoma cells exposed to a hypoxic condition. Therefore, in this study, we set to determine whether differential expression of Tip110 could be an important indicator for melanoma tumorigenesis and metastasis. We found that in melanoma, but not in other cancer types, Tip110 knockdown enhanced significant expression and secretion of IL-8 and melanoma cells invasions. This induction was further potentiated under hypoxia and by inflammatory cytokine and found independent of TNF-α autocrine signaling. We further showed that Tip110 knockdown-mediated IL-8 induction involved IL-8 mRNA stability. Furthermore, the transcriptomic profiling data and survival from 455 melanoma patients demonstrated that the correlation between Tip110 expression and the clinical outcomes in melanoma was stage-dependent. These findings uncover important roles of Tip110 in melanoma tumorigenesis and metastasis through regulation of IL-8 and hope to provide new clues for future therapeutic strategies.
Asunto(s)
Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Perfilación de la Expresión Génica/métodos , Interleucina-8/genética , Melanoma/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Interleucina-8/química , Interleucina-8/metabolismo , Masculino , Melanoma/genética , Melanoma/patología , Persona de Mediana Edad , Invasividad Neoplásica , Estadificación de Neoplasias , Pronóstico , Estabilidad del ARNRESUMEN
HIV-1 Tat is known to be neurotoxic and important for HIV/neuroAIDS pathogenesis. However, the overwhelming majority of the studies involved use of recombinant Tat protein. To understand the contributions of Tat protein to HIV/neuroAIDS and the underlying molecular mechanisms of HIV-1 Tat neurotoxicity in the context of a whole organism and independently of HIV-1 infection, a doxycycline-inducible astrocyte-specific HIV-1 Tat transgenic mouse (iTat) was created. Tat expression in the brains of iTat mice was determined to be in the range of 1-5 ng/ml and led to astrocytosis, loss of neuronal dendrites, and neuroinflammation. iTat mice have allowed us to define the direct effects of Tat on astrocytes and the molecular mechanisms of Tat-induced GFAP expression/astrocytosis, astrocyte-mediated Tat neurotoxicity, Tat-impaired neurogenesis, Tat-induced loss of neuronal integrity, and exosome-associated Tat release and uptake. In this review, we will provide an overview about the creation and characterization of this model and its utilities for our understanding of Tat neurotoxicity and the underlying molecular mechanisms.
Asunto(s)
Complejo SIDA Demencia/genética , Disfunción Cognitiva/genética , Modelos Animales de Enfermedad , VIH-1/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Complejo SIDA Demencia/metabolismo , Complejo SIDA Demencia/fisiopatología , Animales , Astrocitos/metabolismo , Astrocitos/patología , Encéfalo/metabolismo , Encéfalo/patología , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/fisiopatología , Doxiciclina/farmacología , Exosomas/química , Exosomas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , VIH-1/química , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
HIV-1 Tat-interacting protein of 110 kDa, Tip110, plays important roles in multiple biological processes. In this study, we aimed to characterize the function of Tip110 in embryonic development. Transgenic mice lacking expression of a functional Tip110 gene (Tip110-/- ) died post-implantation, and Tip110-/- embryos exhibited developmental arrest between 8.5 and 9.5 days post coitum. However, in vitro cultures of Tip110-/- embryos showed that Tip110 loss did not impair embryo growth from the zygote to the blastocyst. Extended in vitro cultures of Tip110-/- blastocysts showed that Tip110 loss impaired both blastocyst outgrowth and self-renewal and survival of blastocyst-derived embryonic stem cells. Microarray analysis of Tip110-/- embryonic stem cells revealed that Tip110 loss altered differentiation, pluripotency, and cycling of embryonic stem cells and was associated with downregulation of several major stem cell factors including Nanog, Oct4, and Sox2 through a complex network of signaling pathways. Taken together, these findings document for the first time the lethal effects of complete loss of Tip110 on mammalian embryonic development and suggest that Tip110 is an important regulator of not only embryonic development but also stem cell factors. Stem Cells 2017;35:1674-1686.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Células Madre Embrionarias de Ratones/metabolismo , Proteína Homeótica Nanog/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Proteínas de Unión al ARN/genética , Factores de Transcripción SOXB1/genética , Animales , Blastocisto/metabolismo , Blastocisto/patología , Diferenciación Celular , Células Cultivadas , Embrión de Mamíferos , Femenino , Eliminación de Gen , Perfilación de la Expresión Génica , Genes Letales , Ratones , Ratones Noqueados , Células Madre Embrionarias de Ratones/patología , Proteína Homeótica Nanog/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Embarazo , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción SOXB1/metabolismo , Transducción de SeñalRESUMEN
Alterations in adult neurogenesis have been noted in the brain of HIV-infected individuals and are likely linked to HIV-associated neurocognitive deficits, including those in learning and memory. But the underlying molecular mechanisms are not fully understood. In the study, we took advantage of doxycycline-inducible and astrocyte-specific HIV-1 Tat transgenic mice (iTat) and determined the relationship between Tat expression and neurogenesis. Tat expression in astrocytes was associated with fewer neuron progenitor cells (NPCs), fewer immature neurons, and fewer mature neurons in the dentate gyrus of the hippocampus of the mouse brain. In vitro NPC-derived neurosphere assays showed that Tat-containing conditioned media from astrocytes or recombinant Tat protein inhibited NPC proliferation and migration and altered NPC differentiation, while immunodepletion of Tat from Tat-containing conditioned media or heat inactivation of recombinant Tat abrogated those effects. Notch signaling downstream gene Hes1 promoter-driven luciferase reporter gene assay and Western blotting showed that recombinant Tat or Tat-containing conditioned media activated Hes1 transcription and protein expression, which were abrogated by Tat heat inactivation, immunodepletion, and cysteine mutation at position 30. Last, Notch signaling inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) significantly rescued Tat-impaired NPC differentiation in vitro and neurogenesis in vivo Together, these results show that Tat adversely affects NPCs and neurogenesis through Notch signaling and point to the potential of developing Notch signaling inhibitors as HIV/neuroAIDS therapeutics. SIGNIFICANCE STATEMENT: HIV infection of the CNS causes cognitive and memory deficits, which have become more prevalent in the era of combination antiretroviral therapy (cART). Neurogenesis is impaired in HIV-infected individuals. But the underlying molecular mechanisms remain largely unknown. In this study, we have discovered that HIV Tat impairs neurogenesis through the Notch signaling pathway. These findings are particularly important because Tat protein has recently been detected in the brain of HIV-infected individuals with HIV replication in the periphery being effectively controlled by cART. The current study not only further highlights the importance of HIV Tat protein in HIV/neuroAIDS, but also presents a new strategy to develop novel HIV/neuroAIDS therapeutics, particularly in the era of cART.
Asunto(s)
Células-Madre Neurales/metabolismo , Neurogénesis/fisiología , Neuronas/fisiología , Receptores Notch/metabolismo , Transducción de Señal/fisiología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Células Cultivadas , Femenino , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Células-Madre Neurales/citología , Neuronas/citología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Tat interaction with astrocytes has been shown to be important for Tat neurotoxicity and HIV/neuroAIDS. We have recently shown that Tat expression leads to increased glial fibrillary acidic protein (GFAP) expression and aggregation and activation of unfolded protein response/endoplasmic reticulum (ER) stress in astrocytes and causes neurotoxicity. However, the exact molecular mechanism of astrocyte-mediated Tat neurotoxicity is not defined. In this study, we showed that neurotoxic factors other than Tat protein itself were present in the supernatant of Tat-expressing astrocytes. Two-dimensional gel electrophoresis and mass spectrometry revealed significantly elevated lysosomal hydrolytic enzymes and plasma membrane-associated proteins in the supernatant of Tat-expressing astrocytes. We confirmed that Tat expression and infection of pseudotyped HIV.GFP led to increased lysosomal exocytosis from mouse astrocytes and human astrocytes. We found that Tat-induced lysosomal exocytosis was tightly coupled to astrocyte-mediated Tat neurotoxicity. In addition, we demonstrated that Tat-induced lysosomal exocytosis was astrocyte-specific and required GFAP expression and was mediated by ER stress. Taken together, these results show for the first time that Tat promotes lysosomal exocytosis in astrocytes and causes neurotoxicity through GFAP activation and ER stress induction in astrocytes and suggest a common cascade through which aberrant astrocytosis/GFAP up-regulation potentiates neurotoxicity and contributes to neurodegenerative diseases.
Asunto(s)
Astrocitos/metabolismo , Estrés del Retículo Endoplásmico , Exocitosis , VIH-1/metabolismo , Síndromes de Neurotoxicidad/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , Astrocitos/patología , Línea Celular Tumoral , Proteína Ácida Fibrilar de la Glía/biosíntesis , Proteína Ácida Fibrilar de la Glía/genética , VIH-1/genética , Humanos , Ratones , Ratones Transgénicos , Síndromes de Neurotoxicidad/genética , Síndromes de Neurotoxicidad/patología , Regulación hacia Arriba , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
HIV-1 Tat is a major culprit for HIV/neuroAIDS. One of the consistent hallmarks of HIV/neuroAIDS is reactive astrocytes or astrocytosis, characterized by increased cytoplasmic accumulation of the intermediate filament glial fibrillary acidic protein (GFAP). We have shown that that Tat induces GFAP expression in astrocytes and that GFAP activation is indispensable for astrocyte-mediated Tat neurotoxicity. However, the underlying molecular mechanisms are not known. In this study, we showed that Tat expression or GFAP expression led to formation of GFAP aggregates and induction of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress in astrocytes. In addition, we demonstrated that GFAP up-regulation and aggregation in astrocytes were necessary but also sufficient for UPR/ER stress induction in Tat-expressing astrocytes and for astrocyte-mediated Tat neurotoxicity. Importantly, we demonstrated that inhibition of Tat- or GFAP-induced UPR/ER stress by the chemical chaperone 4-phenylbutyrate significantly alleviated astrocyte-mediated Tat neurotoxicity in vitro and in the brain of Tat-expressing mice. Taken together, these results show that HIV-1 Tat expression leads to UPR/ER stress in astrocytes, which in turn contributes to astrocyte-mediated Tat neurotoxicity, and raise the possibility of developing HIV/neuroAIDS therapeutics targeted at UPR/ER stress.
Asunto(s)
Astrocitos/metabolismo , Estrés del Retículo Endoplásmico , Proteína Ácida Fibrilar de la Glía/metabolismo , VIH-1/metabolismo , Síndromes de Neurotoxicidad/metabolismo , Agregación Patológica de Proteínas/metabolismo , Respuesta de Proteína Desplegada , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , Astrocitos/patología , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular Tumoral , VIH-1/genética , Humanos , Ratones , Ratones Transgénicos , Síndromes de Neurotoxicidad/tratamiento farmacológico , Síndromes de Neurotoxicidad/genética , Síndromes de Neurotoxicidad/patología , Fenilbutiratos/farmacología , Agregación Patológica de Proteínas/tratamiento farmacológico , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/patología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Despite the success of antiretroviral therapies to control systemic HIV-1 infection, the prevalence of HIV-associated neurocognitive disorders (HANDs) has not decreased among aging patients with HIV. Autophagy pathway alterations, triggered by HIV-1 proteins including gp120, Tat, and Nef, might contribute to the neurodegenerative process in aging patients with HAND. Although no treatments are currently available to manage HAND, we have previously shown that sunitinib, an anticancer drug that blocks receptor tyrosine-kinase and cyclin kinase pathways, might be of interest. Studies in cancer models suggest that sunitinib might also modulate autophagy, which is dysregulated in our models of Tat-induced neurotoxicity. We evaluated the efficacy of sunitinib to promote autophagy in the CNS and ameliorate neurodegeneration using LC3-GFP-expressing neuronal cells challenged with low concentrations of Tat and using inducible Tat transgenic mice. In neuronal cultures challenged with low levels of Tat, sunitinib increased markers of autophagy such as LC3-II and reduced p62 accumulation in a dose-dependent manner. In vivo, sunitinib treatment restored LC3-II, p62, and endophilin B1 (EndoB1) levels in doxycycline-induced Tat transgenic mice. Moreover, in these animals, sunitinib reduced the hyperactivation of CDK5, tau hyperphosphorylation, and p35 cleavage to p25. Restoration of CDK5 and autophagy were associated with reduced neurodegeneration and behavioral alterations. Alterations in autophagy in the Tat tg mice were associated with reduced levels of a CDK5 substrate, EndoB1, and levels of total EndoB1 were normalized by sunitinib treatment. We conclude that sunitinib might ameliorate Tat-mediated autophagy alterations and may decrease neurodegeneration in aging patients with HAND.
Asunto(s)
Antineoplásicos/farmacología , Disfunción Cognitiva/tratamiento farmacológico , Infecciones por VIH/tratamiento farmacológico , Indoles/farmacología , Pirroles/farmacología , Transgenes , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Aciltransferasas/genética , Aciltransferasas/metabolismo , Animales , Autofagia/efectos de los fármacos , Autofagia/genética , Disfunción Cognitiva/complicaciones , Disfunción Cognitiva/genética , Disfunción Cognitiva/virología , Quinasa 5 Dependiente de la Ciclina/genética , Quinasa 5 Dependiente de la Ciclina/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación de la Expresión Génica , Infecciones por VIH/complicaciones , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/genética , VIH-1/metabolismo , Humanos , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/efectos de los fármacos , Neuronas/patología , Neuronas/virología , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismo , Transducción de Señal , Sunitinib , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
HIV-1 Nef plays important roles in HIV-1 replication and pathogenesis. It is translated from completely spliced HIV-1 RNA, and its expression is inherently regulated at the levels of viral DNA transcription and RNA splicing. Here we show that Sam68 cytoplasmic mutants potently suppress Nef expression. The suppression requires Sam68 domain aa 269-321 and is correlated with its ability to induce stress granules. In addition, the suppression is specific to Nef, and direct binding to nef mRNA 3'UTR confers the suppression specificity. Furthermore, nef mRNA is targeted to and enriched in these induced stress granules. Importantly, Nef suppression occurs in the context of HIV-1 infection of CD4+ T lymphocytes with little MHC I and CD4 downregulation. Taken together, these results demonstrate that stress granule induction and nef mRNA sequestration account for this translational suppression of Nef expression and offer a strategy for development of anti-HIV therapeutics to buttress our fight against HIV/AIDS.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Gránulos Citoplasmáticos/metabolismo , Proteínas de Unión al ADN/metabolismo , VIH-1/genética , Mutación/genética , Biosíntesis de Proteínas , Proteínas de Unión al ARN/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Regiones no Traducidas 3'/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Animales , Biomarcadores/metabolismo , Células COS , Chlorocebus aethiops , Proteínas de Unión al ADN/química , Regulación hacia Abajo , VIH-1/fisiología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Células Jurkat , Proteínas Mutantes/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Transporte de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Fracciones Subcelulares/metabolismo , Supresión Genética , Linfocitos T/citología , Linfocitos T/virología , Replicación ViralRESUMEN
PURPOSE OF REVIEW: Hematopoiesis is regulated by cellular factors including transcription factors, microRNAs, and epigenetic modifiers. Understanding how these factors regulate hematopoiesis is pivotal for manipulating them to achieve their desired potential. In this review, we will focus on HIV-1 Tat-interacting protein of 110âkDa (Tip110) and its regulation of hematopoiesis. RECENT FINDINGS: There are several pathways in hematopoiesis that involve Tip110 regulation. Tip110 is expressed in human cord blood CD34 cells; its expression decreases when CD34 cells begin to differentiate. Tip110 is also expressed in mouse marrow hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC). Tip110 expression increases the number, survival, and cell cycling of HPC. Tip110-mediated regulation of hematopoiesis has been linked to its reciprocal control of proto-oncogene expression. Small noncoding microRNAs (miRs) have been shown to play important roles in regulation of hematopoiesis. miR-124 specifically targets 3'-untranslated region of Tip110 and subsequently regulates Tip110 expression in HSC. SUMMARY: Our recent findings for manipulating expression levels of Tip110 in HSC and HPC could be useful for expanding HSC and HPC and for improving engraftment of cord blood HSC/HPC.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Regulación de la Expresión Génica , Hematopoyesis/fisiología , MicroARNs/genética , Proteínas de Unión al ARN/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , Diferenciación Celular/genética , Autorrenovación de las Células/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , MicroARNs/metabolismo , Proto-Oncogenes MasRESUMEN
Antiretroviral therapy has increased the life span of HIV+ individuals; however, HIV-associated neurocognitive disorder (HAND) occurrence is increasing in aging HIV patients. Previous studies suggest HIV infection alters autophagy function in the aging CNS and HIV-1 proteins affect autophagy in monocyte-derived cells. Despite these findings, the mechanisms leading to dysregulated autophagy in the CNS remain unclear. Here we sought to determine how HIV Tat dysregulates autophagy in neurons. Tat caused a dose-dependent decrease in autophagosome markers, microtubule-associated protein-1 light chain ß II (LC3II), and sequestosome 1(SQSTM1), in a membrane-enriched fraction, suggesting Tat increases autophagic degradation. Bafilomycin A1 increased autophagosome number, LC3II, and SQSTM1 accumulation; Tat cotreatment diminished this effect. Tat had no effect when 3-methyladenine or knockdown of beclin 1 blocked early stages of autophagy. Tat increased numbers of LC3 puncta and resulted in the formation of abnormal autophagosomes in vitro. Likewise, in vivo studies in GFAP-Tat tg mice showed increased autophagosome accumulation in neurons, altered LC3II levels, and neurodegeneration. These effects were reversed by rapamycin treatment. Tat colocalized with autophagosome and lysosomal markers and enhanced the colocalization of autophagosome with lysosome markers. Furthermore, co-IP studies showed that Tat interacts with lysosomal-associated membrane protein 2A (LAMP2A) in vitro and in vivo, and LAMP2A overexpression reduces Tat-induced neurotoxicity. Hence, Tat protein may induce autophagosome and lysosome fusion through interaction with LAMP2A leading to abnormal neuronal autophagy function and dysregulated degradation of critical intracellular components. Therapies targeting Tat-mediated autophagy alterations may decrease neurodegeneration in aging patients with HAND.
Asunto(s)
Autofagia , Lisosomas/metabolismo , Neuronas/metabolismo , Fagosomas/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Complejo SIDA Demencia/metabolismo , Animales , Línea Celular Tumoral , Células Cultivadas , VIH-1/genética , Proteína 2 de la Membrana Asociada a los Lisosomas/genética , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Ratones , Unión Proteica , Ratas , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/toxicidadRESUMEN
BACKGROUND: Synaptodendritic damage is a pathological hallmark of HIV-associated neurocognitive disorders, and HIV-1 Tat protein is known to cause such injury in the central nervous system. In this study, we aimed to determine the molecular mechanisms of Tat-induced neurite shortening, specifically the roles of miR-132, an important regulator of neurite morphogenesis in this process. METHODS: The relationship between Tat expression and miR-132 expression was first determined using reverse transcription quantitative PCR (qRT-PCR) in Tat-transfected astrocytes and neurons, astrocytes from Tat-transgenic mice, and HIV-infected astrocytes. qRT-PCR and Western blotting were performed to determine Tat effects on expression of miR-132 target genes methyl CpG-binding protein 2, Rho GTPase activator p250GAP, and brain-derived neurotrophic factor. Exosomes were isolated from Tat-expressing astrocytes, and exosomal microRNA (miRNA) uptake into neurons was studied using miRNA labeling and flow cytometry. The lactate dehydrogenase release was used to determine the cytotoxicity, while immunostaining was used to determine neurite lengths and synapse formation. Tat basic domain deletion mutant and miR-132 mimic and inhibitor were used to determine the specificity of the relationship between Tat and miR-132 and its effects on astrocytes and neurons and the underlying mechanisms of Tat-induced miR-132 expression. RESULTS: Tat significantly induced miR-132 expression, ensuing down-regulation of miR-132 target genes in astrocytes and neurons. miR-132 induction was associated with phosphorylation of cAMP response element-binding protein and required the basic domain of Tat. miRNA-132 induction had no effects on astrocyte activation or survival but was involved in the direct neurotoxicity of Tat. miR-132 was present in astrocyte-derived exosomes and was taken up by neurons, causing neurite shortening. CONCLUSIONS: Tat-induced miR-132 expression contributes to both direct and astrocyte-mediated Tat neurotoxicity and supports the important roles of miR-132 in controlling neurite outgrowth.
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Regulación Viral de la Expresión Génica/fisiología , MicroARNs/metabolismo , Neuritas/fisiología , Neuronas/citología , Neuronas/efectos de los fármacos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/farmacología , Animales , Antibacterianos/farmacología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitoma/patología , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Línea Celular Transformada , Citocinas/genética , Citocinas/metabolismo , Doxiciclina/farmacología , Células Epiteliales , Regulación Viral de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Proteína 2 de Unión a Metil-CpG/genética , Proteína 2 de Unión a Metil-CpG/metabolismo , Ratones , MicroARNs/genética , Neuritas/efectos de los fármacos , Neuroblastoma/patología , Neuronas/virología , ARN Nuclear Pequeño/metabolismoRESUMEN
HIV-1 Tat is an indispensible transactivator for HIV gene transcription and replication. It has been shown to exit cells as a free protein and enter neighboring cells or interact with surface receptors of neighboring cells to regulate gene expression and cell function. In this study, we report, for the first time, exosome-associated Tat release and uptake. Using a HIV-1 LTR-driven luciferase reporter-based cell assay and Western blotting or in combination with exosome inhibitor, OptiPrep gradient fractionation, and exosome depletion, we demonstrated significant presence of HIV-1 Tat in exosomes derived from Tat-expressing primary astrocytes, Tat-transfected U373.MG and 293T, and HIV-infected MT4. We further showed that exosome-associated Tat from Tat-expressing astrocytes was capable of causing neurite shortening and neuron death, further supporting that this new form of extracellular Tat is biologically active. Lastly, we constructed a Tat mutant deleted of its basic domain and determined the role of the basic domain in Tat trafficking into exosomes. Basic domain-deleted Tat exhibited no apparent effects on Tat trafficking into exosomes, while maintained its dominant-negative function in Tat-mediated LTR transactivation. Taken together, these results show a significant fraction of Tat is secreted and present in the form of exosomes and may contribute to the stability of extracellular Tat and broaden the spectrum of its target cells.
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Astrocitos/metabolismo , Exosomas/metabolismo , Duplicado del Terminal Largo de VIH , Neuroglía/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Astrocitos/patología , Línea Celular Transformada , Expresión Génica , Células HEK293 , VIH-1/química , Humanos , Linfocitos/metabolismo , Linfocitos/patología , Neuroglía/patología , Cultivo Primario de Células , Dominios Proteicos , Transporte de Proteínas , Activación Transcripcional , Transgenes , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/química , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Transcription plays an important role in both HIV-1 gene expression and replication and mandates complicated but coordinated interactions between the host and virus. Our previous studies have shown that an HIV-1 Tat-interacting protein of 110 kDa, Tip110, binds to and enhances Tat function in Tat-mediated HIV-1 gene transcription and replication (Liu, Y., Li, J., Kim, B. O., Pace, B. S., and He, J. J. (2002) HIV-1 Tat protein-mediated transactivation of the HIV-1 long terminal repeat promoter is potentiated by a novel nuclear Tat-interacting protein of 110 kDa, Tip110. J. Biol. Chem. 277, 23854-23863). However, the underlying molecular mechanisms by which this takes place were not understood. In this study, we demonstrated that Tip110 bound to unphosphorylated RNA polymerase II (RNAPII) in a direct and specific manner. In addition, we detected Tip110 at the HIV-1 long terminal repeat (LTR) promoter and found that Tip110 expression was associated with increased phosphorylation of serine 2 of the heptapeptide repeats within the RNAPII C-terminal domain and increased recruitment of positive transcription elongation factor b to the LTR promoter. Consistent with these findings, we showed that Tip110 interaction with Tat directly enhanced transcription elongation of the LTR promoter. Taken together, these findings have provided additional and mechanistic evidence to support Tip110 function in HIV-1 transcription.
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Antígenos de Neoplasias/metabolismo , Regulación Viral de la Expresión Génica , Duplicado del Terminal Largo de VIH , VIH-1/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Proteínas de Unión al ARN/metabolismo , Elongación de la Transcripción Genética , Antígenos de Neoplasias/genética , Línea Celular , VIH-1/genética , Humanos , Fosforilación/genética , Unión Proteica , ARN Polimerasa II/genética , Proteínas de Unión al ARN/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Astrocytes are the most abundant cells in the central nervous system and play important roles in human immunodeficiency virus (HIV)/neuro-acquired immunodeficiency syndrome. Detection of HIV proviral DNA, RNA, and early gene products but not late structural gene products in astrocytes in vivo and in vitro indicates that astrocytes are susceptible to HIV infection albeit in a restricted manner. We as well as others have shown that cell-free HIV is capable of entering CD4- astrocytes through human mannose receptor-mediated endocytosis. In this study, we took advantage of several newly developed fluorescence protein-based HIV reporter viruses and further characterized HIV interaction with astrocytes. First, we found that HIV was successfully transferred to astrocytes from HIV-infected CD4+ T cells in a cell-cell contact- and gp120-dependent manner. In addition, we demonstrated that, compared to endocytosis-mediated cell-free HIV entry and subsequent degradation of endocytosed virions, the cell-cell contact between astrocytes and HIV-infected CD4+ T cells led to robust HIV infection of astrocytes but retained the restricted nature of viral gene expression. Furthermore, we showed that HIV latency was established in astrocytes. Lastly, we demonstrated that infectious progeny HIV was readily recovered from HIV latent astrocytes in a cell-cell contact-mediated manner. Taken together, our studies point to the importance of the cell-cell contact-mediated HIV interaction with astrocytes and provide direct evidence to support the notion that astrocytes are HIV latent reservoirs in the central nervous system.
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Astrocitos/virología , Linfocitos T CD4-Positivos/virología , Regulación Viral de la Expresión Génica , Proteína gp120 de Envoltorio del VIH/genética , VIH-1/genética , Virión/genética , Astrocitos/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Comunicación Celular , Línea Celular Tumoral , Técnicas de Cocultivo , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/crecimiento & desarrollo , VIH-1/metabolismo , Interacciones Huésped-Patógeno , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Transducción de Señal , Virión/crecimiento & desarrollo , Virión/metabolismo , Latencia del Virus , Replicación Viral , Proteína Fluorescente RojaRESUMEN
Betulinic acid (1) has been modified to ionic derivatives (2-5) to improve its water solubility and biological activities. The binding properties of these derivatives with respect to human serum albumin (HSA) was examined and found to be similar to current anti-HIV drugs. These compounds did not inhibit HIV reverse transcriptase, however, 1, 2 and 5 inhibited herpes simplex type 2 (HSV-2) replication at concentrations similar to those reported for acyclovir (IC50 â¼ 0.1-10 µM) and with minimal cellular cytotoxicity. IC50 values for antiviral activity against HSV-2 186 were 1.6, 0.6, 0.9, 7.2, and 0.9 µM for compounds 1-5, respectively.