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SARS-CoV-2 can infect wildlife, and SARS-CoV-2 variants of concern might expand into novel animal reservoirs, potentially by reverse zoonosis. White-tailed deer and mule deer of North America are the only deer species in which SARS-CoV-2 has been documented, raising the question of whether other reservoir species exist. We report cases of SARS-CoV-2 seropositivity in a fallow deer population located in Dublin, Ireland. Sampled deer were seronegative in 2020 when the Alpha variant was circulating in humans, 1 deer was seropositive for the Delta variant in 2021, and 12/21 (57%) sampled deer were seropositive for the Omicron variant in 2022, suggesting host tropism expansion as new variants emerged in humans. Omicron BA.1 was capable of infecting fallow deer lung type-2 pneumocytes and type-1-like pneumocytes or endothelial cells ex vivo. Ongoing surveillance to identify novel SARS-CoV-2 reservoirs is needed to prevent public health risks during human-animal interactions in periurban settings.
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COVID-19 , Ciervos , SARS-CoV-2 , Animales , SARS-CoV-2/inmunología , SARS-CoV-2/genética , COVID-19/epidemiología , COVID-19/veterinaria , Humanos , Ciervos/virología , Irlanda/epidemiología , Estudios Seroepidemiológicos , Población Urbana , Reservorios de Enfermedades/virología , Reservorios de Enfermedades/veterinaria , Animales Salvajes/virología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Femenino , MasculinoRESUMEN
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is associated with neurological sequelae including haemorrhage, thrombosis and ischaemic necrosis and encephalitis. However, the mechanism by which this occurs is unclear. Neurological disease associated with COVID-19 has been proposed to occur following direct infection of the central nervous system and/or indirectly by local or systemic immune activation. We evaluated the expression of angiotensin-converting enzyme-2 and transmembrane protease, serine 2 (TMPRSS2) in brain tissue from five healthy human donors and observed low-level expression of these proteins in cells morphologically consistent with astrocytes, neurons and choroidal ependymal cells within the frontal cortex and medulla oblongata. Primary human astrocytes, neurons, choroid plexus epithelial cells and pericytes supported productive SARS-CoV-2 infection with ancestral, Alpha, Delta and Omicron variants. Infected cells supported the full viral life cycle, releasing infectious virus particles. In contrast, primary brain microvascular endothelial cells and microglia were refractory to SARS-CoV-2 infection. These data support a model whereby SARS-CoV-2 can infect human brain cells, and the mechanism of viral entry warrants further investigation.
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Enzima Convertidora de Angiotensina 2 , Astrocitos , COVID-19 , Plexo Coroideo , Células Epiteliales , Neuronas , Pericitos , SARS-CoV-2 , Serina Endopeptidasas , Humanos , Pericitos/virología , SARS-CoV-2/fisiología , Astrocitos/virología , Plexo Coroideo/virología , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Neuronas/virología , COVID-19/virología , COVID-19/patología , Células Epiteliales/virología , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Células Cultivadas , Encéfalo/virología , Encéfalo/patología , Sistema Nervioso Central/virologíaRESUMEN
A novel proprietary formulation, ViruSAL, has previously been demonstrated to inhibit diverse enveloped viral infections in vitro and in vivo. We evaluated the ability of ViruSAL to inhibit SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) infectivity, using physiologically relevant models of the human bronchial epithelium, to model early infection of the upper respiratory tract. ViruSAL potently inhibited SARS-CoV-2 infection of human bronchial epithelial cells cultured as an air-liquid interface (ALI) model, in a concentration- and time-dependent manner. Viral infection was completely inhibited when ViruSAL was added to bronchial airway models prior to infection. Importantly, ViruSAL also inhibited viral infection when added to ALI models post-infection. No evidence of cellular toxicity was detected in ViruSAL-treated cells at concentrations that completely abrogated viral infectivity. Moreover, intranasal instillation of ViruSAL to a rat model did not result in any toxicity or pathological changes. Together these findings highlight the potential for ViruSAL as a novel and potent antiviral for use within clinical and prophylactic settings.
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Antivirales , COVID-19 , Humanos , Ratas , Animales , Antivirales/farmacología , SARS-CoV-2 , Células Epiteliales , BronquiosRESUMEN
The SARS-CoV-2 virus can utilize host cell proteases to facilitate cell entry, whereby the Spike (S) protein is cleaved at two specific sites to enable membrane fusion. Furin, transmembrane protease serine 2 (TMPRSS2), and cathepsin L (CatL) are the major proteases implicated, and are thus targets for anti-viral therapy. The human serpin (serine protease inhibitor) alpha-1 antitrypsin (A1AT) shows inhibitory activity for TMPRSS2, and has previously been found to suppress cell infection with SARS-CoV-2. Here, we have generated modified serpin inhibitors with increased specificity for these cellular proteases. Using SerpinB3 (SCCA-1), a cross-class inhibitor of CatL, as a scaffold, we have designed and produced reactive centre loop (RCL) variants to more specifically target both furin and TMPRSS2. Two further variants were generated by substituting the RCL P7-P1 with the spike protein S1/S2 cleavage site from either SARS-CoV-2 alpha or delta (P681R) sequences. Altered inhibitory specificity of purified recombinant proteins was verified in protease assays, with attenuated CatL inhibition and gain of furin or TMPRSS2 inhibition, as predicted, and modified serpins were shown to block S protein cleavage in vitro. Furthermore, the serpin variants were able to inhibit S-pseudoparticle entry into A549-ACE2-TMPRSS2 cells and suppress SARS-CoV-2 replication in Vero E6 cells expressing TMPRSS2. The construct designed to inhibit TMPRSS2 (B3-TMP) was most potent. It was more effective than A1AT for TMPRSS2 enzyme inhibition (with an eighteen-fold improvement in the second order inhibition rate constant) and for blocking SARS-CoV-2 viral replication. These findings advance the potential for serpin RCL mutagenesis to generate new inhibitors, and may lead to novel anti-viral biological molecules.
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Tratamiento Farmacológico de COVID-19 , Serpinas , Humanos , SARS-CoV-2 , Furina/genética , Furina/metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Serpinas/genética , Serpinas/farmacología , Catepsina L/metabolismo , Enzima Convertidora de Angiotensina 2 , Internalización del Virus , Antivirales/farmacología , Mutagénesis , Proteínas Recombinantes , Serina , Serina Endopeptidasas/genéticaRESUMEN
Understanding the dynamic properties of cellular proteins in live cells and in real time is essential to delineate their function. In this context, we introduce the Fluorescence Recovery After Photobleaching-Photoactivation unit (Andor) combined with the Nikon Eclipse Ti E Spinning Disk (Andor) confocal microscope as an advantageous and robust platform to exploit the properties of the Dendra2 photoconvertible fluorescent protein (Evrogen) and analyse protein subcellular trafficking in living cells. A major advantage of the spinning disk confocal is the rapid acquisition speed, enabling high temporal resolution of cellular processes. Furthermore, photoconversion and imaging are less invasive on the spinning disk confocal as the cell exposition to illumination power is reduced, thereby minimizing photobleaching and increasing cell viability. We have tested this commercially available platform using experimental settings adapted to track the migration of fast trafficking proteins such as UBC9, Fibrillarin and have successfully characterized their differential motion between subnuclear structures. We describe here step-by-step procedures, with emphasis on cellular imaging parameters, to successfully perform the dynamic imaging and photoconversion of Dendra2-fused proteins at high spatial and temporal resolutions necessary to characterize the trafficking pathways of proteins.
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Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Transporte de Proteínas/fisiología , Proteínas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/fisiología , Células HeLa , Humanos , Iluminación/métodos , FotoblanqueoRESUMEN
Background: The inflammatory changes that underlie the heterogeneous presentations of COVID-19 remain incompletely understood. In this study we aimed to identify inflammatory profiles that precede the development of severe COVID-19, that could serve as targets for optimised delivery of immunomodulatory therapies and provide insights for the development of new therapies. Methods: We included individuals sampled <10 days from COVID-19 symptom onset, recruited from both inpatient and outpatient settings. We measured 61 biomarkers in plasma, including markers of innate immune and T cell activation, coagulation, tissue repair and lung injury. We used principal component analysis and hierarchical clustering to derive biomarker clusters, and ordinal logistic regression to explore associations between cluster membership and maximal disease severity, adjusting for known risk factors for severe COVID-19. Results: In 312 individuals, median (IQR) 7 (4-9) days from symptom onset, we found four clusters. Cluster 1 was characterised by low overall inflammation, cluster 2 was characterised by higher levels of growth factors and markers of endothelial activation (EGF, VEGF, PDGF, TGFα, PAI-1 and p-selectin). Cluster 3 and 4 both had higher overall inflammation. Cluster 4 had the highest levels of most markers including markers of innate immune activation (IL6, procalcitonin, CRP, TNFα), and coagulation (D-dimer, TPO), in contrast cluster 3 had the highest levels of alveolar epithelial injury markers (RAGE, ST2), but relative downregulation of growth factors and endothelial activation markers, suggesting a dysfunctional inflammatory pattern. In unadjusted and adjusted analysis, compared to cluster 1, cluster 3 had the highest odds of progressing to more severe disease (unadjusted OR (95%CI) 9.02 (4.53-17.96), adjusted OR (95%CI) 6.02 (2.70-13.39)). Conclusion: Early inflammatory profiles predicted subsequent maximal disease severity independent of risk factors for severe COVID-19. A cluster with downregulation of growth factors and endothelial activation markers, and early evidence of alveolar epithelial injury, had the highest risk of severe COVID-19.
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Quantifying neutralising capacity of circulating SARS-COV-2 antibodies is critical in evaluating protective humoral immune responses generated post-infection/post-vaccination. Here we describe a novel medium-throughput flow cytometry-based micro-neutralisation test to evaluate Neutralising Antibody (NAb) responses against live SARS-CoV-2 Wild Type and Variants of Concern (VOC) in convalescent/vaccinated populations. Flow Cytometry-Based Micro-Neutralisation Test (Micro-NT) was performed in 96-well plates using clinical isolates WT-B, WT-B.1.177.18 and/or VOCs Beta and Omicron. Plasma samples (All Ireland Infectious Diseases (AIID) Cohort) were serially diluted (8 points, half-log) from 1:20 and pre-incubated with SARS-CoV-2 (1h, 37°C). Virus-plasma mixture were added onto Vero E6 or Vero E6/TMPRSS2 cells for 18h. Percentage infected cells was analysed by automated flow cytometry following trypsinisation, fixation and SARS-CoV-2 Nucleoprotein intracellular staining. Half-maximal Neutralisation Titres (NT50) were determined using non-linear regression. Our assay was compared to Plaque Reduction Neutralisation Test (PRNT) and validated against the First WHO International Standard for anti-SARS-CoV-2 immunoglobulin. Both Micro-NT and PRNT achieved comparable NT50 values. Further validation showed adequate correlation with PRNT using a panel of secondary standards of clinical convalescent and vaccinated plasma samples. We found the assay to be reproducible through measuring both repeatability and intermediate precision. Screening 190 convalescent samples and 11 COVID-19 naive controls (AIID cohort) we demonstrated that Micro-NT has broad dynamic range differentiating NT50s <1/20 to >1/5000. We could also characterise immune-escape VOC Beta and Omicron BA.5, achieving fold-reductions in neutralising capacity similar to those published. Our flow cytometry-based Micro-NT is a robust and reliable assay to quantify NAb titres, and has been selected as an endpoint in clinical trials.
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COVID-19 , Vacunas , Humanos , Citometría de Flujo , SARS-CoV-2 , Pruebas de Neutralización , Anticuerpos Neutralizantes , Anticuerpos AntiviralesRESUMEN
BACKGROUND AND AIMS: Patients with inflammatory bowel disease [IBD] have an attenuated response to initial COVID-19 vaccination. We sought to characterize the impact of IBD and its treatment on responses after the third vaccine against SARS-CoV-2. METHODS: This was a prospective multicentre observational study of patients with IBD [nâ =â 202] and healthy controls [HC, nâ =â 92]. Serological response to vaccination was assessed by quantification of anti-spike protein [SP] immunoglobulin [Ig]G levels [anti-SPIgG] and in vitro neutralization of binding to angiotensin-converting enzyme 2 [ACE2]. Peripheral blood B-cell phenotype populations were assessed by flow cytometry. SARS-CoV-2 antigen-specific B-cell responses were assessed in ex vivo culture. RESULTS: Median anti-SP IgG post-third vaccination in our IBD cohort was significantly lower than HCs [7862 vs 19 622 AU/mL, pâ <â 0.001] as was ACE2 binding inhibition [pâ <â 0.001]. IBD patients previously infected with COVID-19 [30%] had similar quantitative antibody response as HCs previously infected with COVID-19 [pâ =â 0.12]. Lowest anti-SP IgG titres and neutralization were seen in IBD patients on anti-tumour necrosis factor [anti-TNF] agents, without prior COVID-19 infection, but all IBD patients show an attenuated vaccine response compared to HCs. Patients with IBD have reduced memory B-cell populations and attenuated B-cell responses to SARS-CoV-2 antigens if not previously infected with COVID-19 [pâ =â 0.01]. Higher anti-TNF drug levels and zinc levels <65 ng/ml were associated with significantly lower serological responses. CONCLUSIONS: Patients with IBD have an attenuated response to three doses of SARS-CoV-2 vaccine. Physicians should consider patients with higher anti-TNF drug levels and/or zinc deficiency as potentially at higher risk of attenuated response to vaccination.
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SARS-CoV-2 neutralising antibodies provide protection against COVID-19. Evidence from early vaccine trials suggested binding antibody thresholds could serve as surrogate markers of neutralising capacity, but whether these thresholds predict sufficient neutralising capacity against variants of concern (VOCs), and whether this is impacted by vaccine or infection history remains unclear. Here we analyse individuals recovered from, vaccinated or with hybrid immunity against SARS-CoV-2. An NT50 ≥ 100 IU confers protection in vaccine trials, however, as VOC induce a reduction in NT50, we use NT50 ≥ 1000 IU as a cut off for WT NT50 that would retain neutralisation against VOC. In unvaccinated convalescent participants, a receptor binding domain (RBD) IgG of 456 BAU/mL predicts an NT50 against WT of 1000 IU with an accuracy of 80% (95%CI 73-86%). This threshold maintains accuracy in determining loss of protective immunity against VOC in two vaccinated cohorts. It predicts an NT50 < 100 IU against Beta with an accuracy of 80% (95%CI 67-89%) in 2 vaccine dose recipients. In booster vaccine recipients with a history of COVID-19 (hybrid immunity), accuracy is 87% (95%CI 77-94%) in determining an NT50 of <100 IU against BA.5. This analysis provides a discrete threshold that could be used in future clinical studies.
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COVID-19 , Vacunas , Humanos , SARS-CoV-2 , COVID-19/prevención & control , Anticuerpos Antivirales , Inmunoglobulina G , Anticuerpos NeutralizantesRESUMEN
Measurement of quantitative antibody responses are increasingly important in evaluating the immune response to infection and vaccination. In this study we describe the validation of a quantitative, multiplex serologic assay utilising an electrochemiluminescence platform, which measures IgG against the receptor binding domain (RBD), spike S1 and S2 subunits and nucleocapsid antigens of SARS-CoV-2. The assay displayed a sensitivity ranging from 73 to 91% and specificity from 90 to 96% in detecting previous infection with SARS-CoV-2 depending on antigenic target and time since infection, and this assay highly correlated with commercially available assays. The within-plate coefficient of variation ranged from 3.8-3.9% and the inter-plate coefficient of variation from 11 to 13% for each antigen.
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COVID-19 , Anticuerpos Antivirales , COVID-19/diagnóstico , Prueba de COVID-19 , Humanos , Inmunoglobulina G , SARS-CoV-2 , Sensibilidad y Especificidad , Glicoproteína de la Espiga del Coronavirus , VacunaciónRESUMEN
BACKGROUND: The multiplicity, heterogeneity, and dynamic nature of human immunodeficiency virus type-1 (HIV-1) latency mechanisms are reflected in the current lack of functional cure for HIV-1. Accordingly, all classes of latency-reversing agents (LRAs) have been reported to present variable ex vivo potencies. Here, we investigated the molecular mechanisms underlying the potency variability of one LRA: the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-AzadC). METHODS: We employed epigenetic interrogation methods (electrophoretic mobility shift assays, chromatin immunoprecipitation, Infinium array) in complementary HIV-1 infection models (latently-infected T-cell line models, primary CD4+ T-cell models and ex vivo cultures of PBMCs from HIV+ individuals). Extracellular staining of cell surface receptors and intracellular metabolic activity were measured in drug-treated cells. HIV-1 expression in reactivation studies was explored by combining the measures of capsid p24Gag protein, green fluorescence protein signal, intracellular and extracellular viral RNA and viral DNA. FINDINGS: We uncovered specific demethylation CpG signatures induced by 5-AzadC in the HIV-1 promoter. By analyzing the binding modalities to these CpG, we revealed the recruitment of the epigenetic integrator Ubiquitin-like with PHD and RING finger domain 1 (UHRF1) to the HIV-1 promoter. We showed that UHRF1 redundantly binds to the HIV-1 promoter with different binding modalities where DNA methylation was either non-essential, essential or enhancing UHRF1 binding. We further demonstrated the role of UHRF1 in the epigenetic repression of the latent viral promoter by a concerted control of DNA and histone methylations. INTERPRETATION: A better understanding of the molecular mechanisms of HIV-1 latency allows for the development of innovative antiviral strategies. As a proof-of-concept, we showed that pharmacological inhibition of UHRF1 in ex vivo HIV+ patient cell cultures resulted in potent viral reactivation from latency. Together, we identify UHRF1 as a novel actor in HIV-1 epigenetic silencing and highlight that it constitutes a new molecular target for HIV-1 cure strategies. FUNDING: Funding was provided by the Belgian National Fund for Scientific Research (F.R.S.-FNRS, Belgium), the « Fondation Roi Baudouin ¼, the NEAT (European AIDS Treatment Network) program, the Internationale Brachet Stiftung, ViiV Healthcare, the Télévie, the Walloon Region (« Fonds de Maturation ¼), « Les Amis des Instituts Pasteur à Bruxelles, asbl ¼, the University of Brussels (Action de Recherche Concertée ULB grant), the Marie Skodowska Curie COFUND action, the European Union's Horizon 2020 research and innovation program under grant agreement No 691119-EU4HIVCURE-H2020-MSCA-RISE-2015, the French Agency for Research on AIDS and Viral Hepatitis (ANRS), the Sidaction and the "Alsace contre le Cancer" Foundation. This work is supported by 1UM1AI164562-01, co-funded by National Heart, Lung and Blood Institute, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Neurological Disorders and Stroke, National Institute on Drug Abuse and the National Institute of Allergy and Infectious Diseases.
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Proteínas Potenciadoras de Unión a CCAAT , Represión Epigenética , Infecciones por VIH , VIH-1 , Ubiquitina-Proteína Ligasas , Latencia del Virus , Síndrome de Inmunodeficiencia Adquirida , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Metilación de ADN , Decitabina/metabolismo , Infecciones por VIH/genética , VIH-1/fisiología , Humanos , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Latencia del Virus/genéticaRESUMEN
BACKGROUND: The HIV-1 regulatory protein Rev, which is essential for viral replication, mediates the nuclear export of unspliced viral transcripts. Rev nuclear function requires active nucleocytoplasmic shuttling, and Rev nuclear import is mediated by the recognition of its Nuclear Localisation Signal (NLS) by multiple import factors, which include transportin and importin ß. However, it remains unclear which nuclear import pathway(s) predominate in vivo, and the cellular environment that modulates Rev nucleocytoplasmic shuttling remains to be characterised. RESULTS: In our study, we have identified the cellular protein HIC (Human I-mfa domain-Containing protein) as a novel interactor of HIV-1 Rev. We demonstrate that HIC selectively interferes with Rev NLS interaction with importin ß and impedes its nuclear import and function, but does not affect Rev nuclear import mediated by transportin. Hence, the molecular determinants mediating Rev-NLS recognition by importin ß and transportin appear to be distinct. Furthermore, we have employed HIC and M9 M, a peptide specifically designed to inhibit the transportin-mediated nuclear import pathway, to characterise Rev nuclear import pathways within different cellular environments. Remarkably, we could show that in 293T, HeLa, COS7, Jurkat, U937, THP-1 and CEM cells, Rev nuclear import is cell type specific and alternatively mediated by transportin or importin ß, in a mutually exclusive fashion. CONCLUSIONS: Rev cytoplasmic sequestration by HIC may represent a novel mechanism for the control of Rev function. These studies highlight that the multivalent nature of the Rev NLS for different import receptors enables Rev to adapt its nuclear trafficking strategy.
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Núcleo Celular/metabolismo , Regulación Viral de la Expresión Génica , VIH-1/metabolismo , Factores Reguladores Miogénicos/metabolismo , Señales de Localización Nuclear/metabolismo , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismo , Transporte Activo de Núcleo Celular , Animales , Células COS , Línea Celular , Chlorocebus aethiops , VIH-1/genética , Células HeLa , Humanos , Células Jurkat , Carioferinas/metabolismo , Factores Reguladores Miogénicos/genética , Señales de Localización Nuclear/genética , Unión Proteica , Células U937 , beta Carioferinas/genética , beta Carioferinas/metabolismo , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Adult T-cell leukemia (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1). Elevated expression of vascular endothelial growth factor (VEGF) in ATL patients is associated with leukemic cell invasion and infiltration in different organs. The regulatory protein Tax 1 encoded by HTLV-1 plays a pivotal role in T-cell transformation by deregulating the function and expression of several cellular factors. In the present study, we examined the effect of Tax 1 on VEGF expression at transcriptional and posttranscriptional levels in order to elucidate the regulatory mechanisms involved. Using functional assays, we demonstrate that Tax 1 downregulates the VEGF promoter through a cluster of Sp1 sites located close to the transcriptional start site. Using gel mobility shift assays, we show that Tax 1 reduced Sp1:DNA complex formation. We demonstrate that the level of secreted VEGF was significantly lower in Tax 1-transfected 293T cells compared to nontransfected cells, which is consistent with the observed downregulatory effect of Tax 1 at the transcription level. We showed that VEGF was secreted by HTLV-1-transformed and nontransformed cells, irrespective of Tax 1 expression. Overall our data indicate that, contrary to a previous report, Tax 1 downregulates VEGF expression and suggest there are Tax 1-independent mechanisms of VEGF activation in ATL.
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Moléculas de Adhesión Celular Neuronal/fisiología , Regulación hacia Abajo , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Leucemia-Linfoma de Células T del Adulto/virología , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Contactina 2 , ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Humanos , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
HIV-1 latency generates reservoirs that prevent viral eradication by the current therapies. To find strategies toward an HIV cure, detailed understandings of the molecular mechanisms underlying establishment and persistence of the reservoirs are needed. The cellular transcription factor KAP1 is known as a potent repressor of gene transcription. Here we report that KAP1 represses HIV-1 gene expression in myeloid cells including microglial cells, the major reservoir of the central nervous system. Mechanistically, KAP1 interacts and colocalizes with the viral transactivator Tat to promote its degradation via the proteasome pathway and repress HIV-1 gene expression. In myeloid models of latent HIV-1 infection, the depletion of KAP1 increased viral gene elongation and reactivated HIV-1 expression. Bound to the latent HIV-1 promoter, KAP1 associates and cooperates with CTIP2, a key epigenetic silencer of HIV-1 expression in microglial cells. In addition, Tat and CTIP2 compete for KAP1 binding suggesting a dynamic modulation of the KAP1 cellular partners upon HIV-1 infection. Altogether, our results suggest that KAP1 contributes to the establishment and the persistence of HIV-1 latency in myeloid cells.
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Regulación Viral de la Expresión Génica , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Células Mieloides/metabolismo , Transcripción Genética , Proteína 28 que Contiene Motivos Tripartito/metabolismo , Células HEK293 , Infecciones por VIH/genética , VIH-1/genética , Humanos , Células Mieloides/virología , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteína 28 que Contiene Motivos Tripartito/genética , 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
BACKGROUND: One facet of the complexity underlying the biology of HIV-1 resides not only in its limited number of viral proteins, but in the extensive repertoire of cellular proteins they interact with and their higher-order assembly. HIV-1 encodes the regulatory protein Tat (86-101aa), which is essential for HIV-1 replication and primarily orchestrates HIV-1 provirus transcriptional regulation. Previous studies have demonstrated that Tat function is highly dependent on specific interactions with a range of cellular proteins. However they can only partially account for the intricate molecular mechanisms underlying the dynamics of proviral gene expression. To obtain a comprehensive nuclear interaction map of Tat in T-cells, we have designed a proteomic strategy based on affinity chromatography coupled with mass spectrometry. RESULTS: Our approach resulted in the identification of a total of 183 candidates as Tat nuclear partners, 90% of which have not been previously characterised. Subsequently we applied in silico analysis, to validate and characterise our dataset which revealed that the Tat nuclear interactome exhibits unique signature(s). First, motif composition analysis highlighted that our dataset is enriched for domains mediating protein, RNA and DNA interactions, and helicase and ATPase activities. Secondly, functional classification and network reconstruction clearly depicted Tat as a polyvalent protein adaptor and positioned Tat at the nexus of a densely interconnected interaction network involved in a range of biological processes which included gene expression regulation, RNA biogenesis, chromatin structure, chromosome organisation, DNA replication and nuclear architecture. CONCLUSION: We have completed the in vitro Tat nuclear interactome and have highlighted its modular network properties and particularly those involved in the coordination of gene expression by Tat. Ultimately, the highly specialised set of molecular interactions identified will provide a framework to further advance our understanding of the mechanisms of HIV-1 proviral gene silencing and activation.
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Infecciones por VIH/metabolismo , VIH-1/fisiología , Interacciones Huésped-Patógeno , Proteínas Nucleares/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencias de Aminoácidos , Cromatografía de Afinidad , Regulación Viral de la Expresión Génica , Redes Reguladoras de Genes , Infecciones por VIH/virología , Humanos , Células Jurkat , Espectrometría de Masas , Proteínas Nucleares/química , Procesamiento Postranscripcional del ARN , ARN Viral/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
Despite efficient combination of the antiretroviral therapy (cART), which significantly decreased mortality and morbidity of HIV-1 infection, a definitive HIV cure has not been achieved. Hidden HIV-1 in cellular and anatomic reservoirs is the major hurdle toward a functional cure. Microglial cells, the Central Nervous system (CNS) resident macrophages, are one of the major cellular reservoirs of latent HIV-1. These cells are believed to be involved in the emergence of drugs resistance and reseeding peripheral tissues. Moreover, these long-life reservoirs are also involved in the development of HIV-1-associated neurocognitive diseases (HAND). Clearing these infected cells from the brain is therefore crucial to achieve a cure. However, many characteristics of microglial cells and the CNS hinder the eradication of these brain reservoirs. Better understandings of the specific molecular mechanisms of HIV-1 latency in microglial cells should help to design new molecules and new strategies preventing HAND and achieving HIV cure. Moreover, new strategies are needed to circumvent the limitations associated to anatomical sanctuaries with barriers such as the blood brain barrier (BBB) that reduce the access of drugs.
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Reservorios de Enfermedades , Infecciones por VIH/virología , VIH-1/fisiología , Microglía/virología , Encéfalo/virología , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Interacciones Huésped-Patógeno , Humanos , Microglía/efectos de los fármacos , Activación Viral , Latencia del VirusRESUMEN
One of the most explored therapeutic approaches aimed at eradicating HIV-1 reservoirs is the "shock and kill" strategy which is based on HIV-1 reactivation in latently-infected cells ("shock" phase) while maintaining antiretroviral therapy (ART) in order to prevent spreading of the infection by the neosynthesized virus. This kind of strategy allows for the "kill" phase, during which latently-infected cells die from viral cytopathic effects or from host cytolytic effector mechanisms following viral reactivation. Several latency reversing agents (LRAs) with distinct mechanistic classes have been characterized to reactivate HIV-1 viral gene expression. Some LRAs have been tested in terms of their potential to purge latent HIV-1 in vivo in clinical trials, showing that reversing HIV-1 latency is possible. However, LRAs alone have failed to reduce the size of the viral reservoirs. Together with the inability of the immune system to clear the LRA-activated reservoirs and the lack of specificity of these LRAs, the heterogeneity of the reservoirs largely contributes to the limited success of clinical trials using LRAs. Indeed, HIV-1 latency is established in numerous cell types that are characterized by distinct phenotypes and metabolic properties, and these are influenced by patient history. Hence, the silencing mechanisms of HIV-1 gene expression in these cellular and tissue reservoirs need to be better understood to rationally improve this cure strategy and hopefully reach clinical success.
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The human DEAD-box helicase DDX3 is a multi-functional protein involved in the regulation of gene expression and additional non-conventional roles as signalling adaptor molecule that are independent of its enzymatic RNA remodeling activity. It is a nucleo-cytoplasmic shuttling protein and it has previously been suggested that dysregulation of its subcellular localization could contribute to tumourigenesis. Indeed, both tumour suppressor and oncogenic functions have been attributed to DDX3. In this study, we investigated the regulation of DDX3's nucleocytoplasmic shuttling. We confirmed that an N-terminal conserved Nuclear Export Signal (NES) is required for export of human DDX3 from the nucleus, and identified three regions within DDX3 that can independently facilitate its nuclear import. We also aimed to identify conditions that alter DDX3's subcellular localisation. Viral infection, cytokine treatment and DNA damage only induced minor changes in DDX3's subcellular distribution as determined by High Content Analysis. However, DDX3's nuclear localization increased in early mitotic cells (during prophase) concomitant with an increase in DDX3 expression levels. Our results are likely to have implications for the proposed use of (nuclear) DDX3 as a prognostic biomarker in cancer.
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Núcleo Celular/metabolismo , ARN Helicasas DEAD-box/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Ciclo Celular , Secuencia Conservada , ARN Helicasas DEAD-box/química , Células HEK293 , Células HeLa , Humanos , Carioferinas/metabolismo , Mutación/genética , Señales de Exportación Nuclear , Señales de Localización Nuclear/metabolismo , Fosforilación , Fosfotreonina/metabolismo , Unión Proteica , Dominios Proteicos , Receptores Citoplasmáticos y Nucleares/metabolismo , Fracciones Subcelulares/metabolismo , Regulación hacia Arriba/genética , Proteína Exportina 1RESUMEN
Introduction of cART in 1996 has drastically increased the life expectancy of people living with HIV-1. However, this treatment has not allowed cure as cessation of cART is associated with a rapid viral rebound. The main barrier to the eradication of the virus is related to the persistence of latent HIV reservoirs. Evidence is now accumulating that purging the HIV-1 reservoir might lead to a cure or a remission. The most studied strategy is the so called "shock and kill" therapy. This strategy is based on reactivation of dormant viruses from the latently-infected reservoirs (the shock) followed by the eradication of the reservoirs (the kill). This review focuses mainly on the recent advances made in the "shock and kill" therapy. We believe that a cure or a remission will come from combinatorial approaches i.e. combination of drugs to reactivate the dormant virus from all the reservoirs including the one located in sanctuaries, and combination of strategies boosting the immune system. Alternative strategies based on cell and gene therapy or based in inducing deep latency, which are evoked in this review reinforce the idea that at least a remission is attainable.