RESUMEN
The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr enhances viral replication in both macrophages and, to a lesser extent, cycling T cells. Virion-packaged Vpr is released in target cells shortly after entry, suggesting it is required in the early phase of infection. Previously, we described REAF (RNA-associated early-stage antiviral factor; RPRD2), a constitutively expressed protein that potently restricts HIV replication at or during reverse transcription. Here, we show that a virus without an intact vpr gene is more highly restricted by REAF and, using delivery by virus-like particles (VLPs), that Vpr alone is sufficient for REAF degradation in primary macrophages. REAF is more highly expressed in macrophages than in cycling T cells, and we detected, by coimmunoprecipitation assay, an interaction between Vpr protein and endogenous REAF. Vpr acts quickly during the early phase of replication and induces the degradation of REAF within 30 min of viral entry. Using Vpr F34I and Q65R viral mutants, we show that nuclear localization and interaction with cullin 4A-DBB1 (DCAF1) E3 ubiquitin ligase are required for REAF degradation by Vpr. In response to infection, cells upregulate REAF levels. This response is curtailed in the presence of Vpr. These findings support the hypothesis that Vpr induces the degradation of a factor, REAF, that impedes HIV infection in macrophages.IMPORTANCE For at least 30 years, it has been known that HIV-1 Vpr, a protein carried in the virion, is important for efficient infection of primary macrophages. Vpr is also a determinant of the pathogenic effects of HIV-1 in vivo A number of cellular proteins that interact with Vpr have been identified. So far, it has not been possible to associate these proteins with altered viral replication in macrophages or to explain why Vpr is carried in the virus particle. Here, we show that Vpr mitigates the antiviral effects of REAF, a protein highly expressed in primary macrophages and one that inhibits virus replication during reverse transcription. REAF is degraded by Vpr within 30 min of virus entry in a manner dependent on the nuclear localization of Vpr and its interaction with the cell's protein degradation machinery.
Asunto(s)
Antivirales/metabolismo , VIH-1/metabolismo , Replicación Viral/fisiología , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/fisiología , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Productos del Gen vpr/metabolismo , Productos del Gen vpr/fisiología , Células HEK293 , Infecciones por VIH/virología , VIH-1/fisiología , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Macrófagos/metabolismo , Cultivo Primario de Células , Ubiquitina-Proteína Ligasas/metabolismo , Virión/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
BACKGROUND: The discovery of novel anti-viral restriction factors illuminates unknown aspects of innate sensing and immunity. We identified RNA-associated Early-stage Anti-viral Factor (REAF) using a whole genome siRNA screen for restriction factors to human immunodeficiency virus (HIV) that act in the early phase of viral replication. RESULTS: We observed more than 50 fold rescue of HIV-1 infection, using a focus forming unit (FFU) assay, following knockdown of REAF by specific siRNA. Quantitative PCR was used to show that REAF knockdown results in an increase of early and late reverse transcripts which impacts the level of integration. REAF thus appears to act at an early stage of the viral life cycle during reverse transcription. Conversely when REAF is over-expressed in target cells less infected cells are detectable and fewer reverse transcripts are produced. Human REAF can also inhibit HIV-2 and simian immunodeficiency virus (SIV) infection. REAF associates with viral nucleic acids and may act to prevent reverse transcription. CONCLUSIONS: This report firmly places REAF alongside APOBECs and SAMHD1 as a potent inhibitor of HIV replication acting early in the replication cycle, just after cell entry. We propose that REAF is part of an anti-viral surveillance system destroying incoming retroviruses. This novel mechanism could apply to invasion of cells by any intracellular pathogen.
Asunto(s)
VIH-1/inmunología , VIH-2/inmunología , Inmunidad Innata , ARN Viral/metabolismo , Virus de la Inmunodeficiencia de los Simios/inmunología , VIH-1/fisiología , VIH-2/fisiología , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , Integración ViralRESUMEN
Activation of the major histocompatibility complex (MHC) by interferon-gamma (IFN-γ) is a fundamental step in the adaptive immune response to pathogens. Here, we show that reorganization of chromatin loop domains in the MHC is evident within the first 30 min of IFN-γ treatment of fibroblasts, and that further dynamic alterations occur up to 6 h. These very rapid changes occur at genomic sites which are occupied by CTCF and are close to IFN-γ-inducible MHC genes. Early responses to IFN-γ are thus initiated independently of CIITA, the master regulator of MHC class II genes and prepare the MHC for subsequent induction of transcription.
Asunto(s)
Interferón gamma/farmacología , Complejo Mayor de Histocompatibilidad , Proteínas Represoras/metabolismo , Sitios de Unión , Factor de Unión a CCCTC , Células Cultivadas , Cromatina/química , Cromatina/efectos de los fármacos , Humanos , Regiones de Fijación a la Matriz/efectos de los fármacos , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: BORIS (CTCFL), a paralogue of the multifunctional and ubiquitously expressed transcription factor CTCF, is best known for its role in transcriptional regulation. In the nucleus, BORIS is particularly enriched in the nucleolus, a crucial compartment for ribosomal RNA and RNA metabolism. However, little is known about cytoplasmic BORIS, which represents the major pool of BORIS protein. RESULTS: We show, firstly, that BORIS has a putative nuclear export signal in the C-terminal domain. Furthermore, BORIS associates with mRNA in both neural stem cells and young neurons. The majority of the BORIS-associated transcripts are different in the two cell types. Finally, by using polysome profiling we show that BORIS is associated with actively translating ribosomes. CONCLUSION: We have demonstrated the RNA binding properties of cellular BORIS and its association with actively translating ribosomes. We suggest that BORIS is involved in gene expression at both the transcriptional and post-transcriptional levels.
Asunto(s)
Nucléolo Celular/genética , Citoplasma/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Polirribosomas/genética , ARN Mensajero/genética , ARN Ribosómico/genética , Secuencia de Aminoácidos , Línea Celular Tumoral , Nucléolo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Datos de Secuencia Molecular , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neuronas/citología , Neuronas/metabolismo , Polirribosomas/metabolismo , Unión Proteica , Biosíntesis de Proteínas , Señales de Clasificación de Proteína , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , ARN Ribosómico/metabolismo , Transducción de Señal , Transcripción GenéticaRESUMEN
We report genetic aberrations that activate the ERK/MAP kinase pathway in 100% of posterior fossa pilocytic astrocytomas, with a high frequency of gene fusions between KIAA1549 and BRAF among these tumours. These fusions were identified from analysis of focal copy number gains at 7q34, detected using Affymetrix 250K and 6.0 SNP arrays. PCR and sequencing confirmed the presence of five KIAA1549-BRAF fusion variants, along with a single fusion between SRGAP3 and RAF1. The resulting fusion genes lack the auto-inhibitory domains of BRAF and RAF1, which are replaced in-frame by the beginning of KIAA1549 and SRGAP3, respectively, conferring constitutive kinase activity. An activating mutation of KRAS was identified in the single pilocytic astrocytoma without a BRAF or RAF1 fusion. Further fusions and activating mutations in BRAF were identified in 28% of grade II astrocytomas, highlighting the importance of the ERK/MAP kinase pathway in the development of paediatric low-grade gliomas.
Asunto(s)
Astrocitoma/enzimología , Neoplasias Encefálicas/enzimología , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Adolescente , Adulto , Astrocitoma/genética , Neoplasias Encefálicas/genética , Niño , Preescolar , Análisis Mutacional de ADN , ADN Complementario/análisis , Activación Enzimática , Proteínas Activadoras de GTPasa/genética , Humanos , Lactante , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas de Fusión Oncogénica/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-raf/genética , Adulto JovenRESUMEN
The worldwide prevalence of diabetes has reached 8.5% among adults, and this is characterised by elevated glucose concentrations and failing insulin secretion. Furthermore, most people with type 2 diabetes are either obese or overweight, with the associated dyslipidaemia contributing to the development of insulin resistance and increased cardiovascular risk. Here we incubated INS-1 pancreatic ß-cells for 72â¯h in RPMI-1640 media, or media supplemented with 28â¯mM glucose, 200⯵M palmitic acid, and 200⯵M oleic acid as a cellular model of diabetic glucolipotoxicity. Illumina HiSeq gene expression analysis showed the trace amine-associated receptor (TAAR) family to be among the most highly downregulated by glucolipotoxicity. Importantly, MetaCore integrated knowledge database, from Clarivate Analytics, indicated potential TAAR impact on insulin secretion through adenylyl cyclase signalling pathways. We therefore investigated the effect of TAAR ligands on cAMP signalling and insulin secretion, and found that only the branch of the TAAR family tree that is activated by isopentylamine, 2-phenylethylamine, p-tyramine, and agmatine significantly increased intracellular cAMP and resulted in increased insulin secretion from INS-1 cells and primary mouse islets under normal conditions. Crucially however, this enhancement was not evident when the receptor family was downregulated by glucolipotoxic conditions. This data indicates that a subset of TAARs are regulators of insulin secretion in pancreatic ß-cells, and that their downregulation contributes to glucolipotoxic inhibition of insulin secretion. As such they may be potential targets for treatment of type 2 diabetes.
Asunto(s)
Glucosa/farmacología , Secreción de Insulina/efectos de los fármacos , Células Secretoras de Insulina/efectos de los fármacos , Ácido Oléico/farmacología , Ácido Palmítico/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Línea Celular Tumoral , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Perfilación de la Expresión Génica/métodos , Humanos , Resistencia a la Insulina/genética , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Ligandos , Masculino , Ratones , Ratas , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Type 2 diabetes is a chronic metabolic disorder, where failure to maintain normal glucose homoeostasis is associated with, and exacerbated by, obesity and the concomitant-elevated free fatty acid concentrations typically found in these patients. Hyperglycaemia and hyperlipidaemia together contribute to a decline in insulin-producing ß-cell mass through activation of the transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription (STAT)-1. There are however a large number of molecules potentially able to modulate NF-κB and STAT1 activity, and the mechanism(s) by which glucolipotoxicity initially induces NF-κB and STAT1 activation is currently poorly defined. Using high-density microarray analysis of the ß-cell transcritptome, we have identified those genes and proteins most sensitive to glucose and fatty acid environment. Our data show that of those potentially able to activate STAT1 or NF-κB pathways, tumour necrosis factor receptor (TNFR)-5 is the most highly upregulated by glucolipotoxicity. Importantly, our data also show that the physiological ligand for TNFR5, CD40L, elicits NF-κB activity in ß-cells, whereas selective knockdown of TNFR5 ameliorates glucolipotoxic induction of STAT1 expression and NF-κB activity. This data indicate for the first time that TNFR5 signalling has a major role in triggering glucolipotoxic islet cell death.
Asunto(s)
Antígenos CD40/metabolismo , Glucosa/toxicidad , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Lípidos/toxicidad , FN-kappa B/metabolismo , Factor de Transcripción STAT1/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Ratones Endogámicos C57BL , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
BORIS (CTCFL) is the paralog of CTCF (CCCTC-binding factor; NM_006565), a ubiquitously expressed DNA-binding protein with diverse roles in gene expression and chromatin organisation. BORIS and CTCF have virtually identical zinc finger domains, yet display major differences in their respective C- and N-terminal regions. Unlike CTCF, BORIS expression has been reported only in the testis and certain malignancies, leading to its classification as a "cancer-testis" antigen. However, the expression pattern of BORIS is both a significant and unresolved question in the field of DNA binding proteins. Here, we identify BORIS in the cytoplasm and nucleus of a wide range of normal and cancer cells. We compare the localization of CTCF and BORIS in the nucleus and demonstrate enrichment of BORIS within the nucleolus, inside the nucleolin core structure and adjacent to fibrillarin in the dense fibrillar component. In contrast, CTCF is not enriched in the nucleolus. Live imaging of cells transiently transfected with GFP tagged BORIS confirmed the nucleolar accumulation of BORIS. While BORIS transcript levels are low compared to CTCF, its protein levels are readily detectable. These findings show that BORIS expression is more widespread than previously believed, and suggest a role for BORIS in nucleolar function.