RESUMEN
Genetic defects in the ability to deliver effective perforin have been reported in patients with hemophagocytic lymphohistiocytosis. We tested the hypothesis that a primary perforin deficiency might also be causal in severe SARS-CoV-2 infection. We recruited 54 volunteers confirmed as being SARS-CoV-2-infected by RT-PCR and admitted to intensive care units or non-intensive care units and age- and sex-matched healthy controls. Compared with healthy controls, the percentage of perforin-expressing CD3-CD56+ NK cells quantified by flow cytometry was low in COVID-19 patients (69.9 ± 17.7 versus 78.6 ± 14.6%, p = 0.026). There was no correlation between the proportions of perforin-positive NK cells and T8 lymphocytes. Moreover, the frequency of NK cells producing perforin was neither linked to disease severity nor predictive of death. Although IL-6 is known to downregulate perforin production in NK cells, we did not find any link between perforin expression and IL-6 plasma level. However, we unveiled a negative correlation between the degranulation marker CD107a and perforin expression in NK cells (r = -0.488, p = 10-4). PRF1 gene expression and the frequency of NK cells harboring perforin were normal in patients 1 y after acute SARS-CoV-2 infection. A primary perforin defect does not seem to be a driver of COVID-19 because NK perforin expression is 1) linked neither to T8 perforin expression nor to disease severity, 2) inversely correlated with NK degranulation, and 3) normalized at distance from acute infection. Thus, the cause of low frequency of perforin-positive NK cells appears, rather, to be consumption.
Asunto(s)
COVID-19 , Interleucina-6 , Humanos , Perforina/metabolismo , Interleucina-6/metabolismo , COVID-19/metabolismo , SARS-CoV-2/metabolismo , Células Asesinas Naturales/metabolismoRESUMEN
HIV-1 Tat is a key viral protein that stimulates several steps of viral gene expression. Tat is especially required for the transcription of viral genes. Nevertheless, it is still not clear if and how Tat is incorporated into HIV-1 virions. Cyclophilin A (CypA) is a prolyl isomerase that binds to HIV-1 capsid protein (CA) and is thereby encapsidated at the level of 200 to 250 copies of CypA/virion. Here, we found that a Tat-CypA-CA tripartite complex assembles in HIV-1-infected cells and allows Tat encapsidation into HIV virions (1 Tat/1 CypA). Biochemical and biophysical studies showed that high-affinity interactions drive the assembly of the Tat-CypA-CA complex that could be purified by size exclusion chromatography. We prepared different types of viruses devoid of transcriptionally active Tat. They showed a 5- to 10 fold decrease in HIV infectivity, and conversely, encapsidating Tat into ΔTat viruses greatly enhanced infectivity. The absence of encapsidated Tat decreased the efficiency of reverse transcription by ~50% and transcription by more than 90%. We thus identified a Tat-CypA-CA complex that enables Tat encapsidation and showed that encapsidated Tat is required to initiate robust viral transcription and thus viral production at the beginning of cell infection, before neosynthesized Tat becomes available. IMPORTANCE The viral transactivating protein Tat has been shown to stimulate several steps of HIV gene expression. It was found to facilitate reverse transcription. Moreover, Tat is strictly required for the transcription of viral genes. Although the presence of Tat within HIV virions would undoubtedly favor these steps and therefore enable the incoming virus to boost initial viral production, whether and how Tat is present within virions has been a matter a debate. We here described and characterized a tripartite complex between Tat, HIV capsid protein, and the cellular chaperone cyclophilin A that enables efficient and specific Tat encapsidation within HIV virions. We further showed that Tat encapsidation is required for the virus to efficiently initiate infection and viral production. This effect is mainly due to the transcriptional activity of Tat.
Asunto(s)
Proteínas de la Cápside , Ciclofilina A , Infecciones por VIH , VIH-1 , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Humanos , Proteínas de la Cápside/metabolismo , Ciclofilina A/metabolismo , Infecciones por VIH/virología , VIH-1/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/aislamiento & purificación , Complejos Multiproteicos/metabolismo , Resonancia por Plasmón de Superficie , Citosol/metabolismo , Línea CelularRESUMEN
SAMHD1 was previously characterized as a dNTPase that protects cells from viral infections. Mutations in SAMHD1 are implicated in cancer development and in a severe congenital inflammatory disease known as Aicardi-Goutières syndrome. The mechanism by which SAMHD1 protects against cancer and chronic inflammation is unknown. Here we show that SAMHD1 promotes degradation of nascent DNA at stalled replication forks in human cell lines by stimulating the exonuclease activity of MRE11. This function activates the ATR-CHK1 checkpoint and allows the forks to restart replication. In SAMHD1-depleted cells, single-stranded DNA fragments are released from stalled forks and accumulate in the cytosol, where they activate the cGAS-STING pathway to induce expression of pro-inflammatory type I interferons. SAMHD1 is thus an important player in the replication stress response, which prevents chronic inflammation by limiting the release of single-stranded DNA from stalled replication forks.
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Replicación del ADN , Interferón Tipo I/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Citosol/metabolismo , ADN de Cadena Simple/metabolismo , Células HEK293 , Células HeLa , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/prevención & control , Interferón Tipo I/inmunología , Proteína Homóloga de MRE11/metabolismo , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/metabolismo , RecQ Helicasas/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/deficienciaRESUMEN
BACKGROUND: Lymphopenia is predictive of survival in patients with coronavirus disease 2019 (COVID-19). OBJECTIVE: The aim of this study was to understand the cause of the lymphocyte count drop in severe forms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: Monocytic production of reactive oxygen species (ROSs) and T-cell apoptosis were measured by flow cytometry, DNA damage in PBMCs was measured by immunofluorescence, and angiotensin II (AngII) was measured by ELISA in patients infected with SARS-CoV-2 at admission to an intensive care unit (ICU) (n = 29) or not admitted to an ICU (n = 29) and in age- and sex-matched healthy controls. RESULTS: We showed that the monocytes of certain patients with COVID-19 spontaneously released ROSs able to induce DNA damage and apoptosis in neighboring cells. Of note, high ROS production was predictive of death in ICU patients. Accordingly, in most patients, we observed the presence of DNA damage in up to 50% of their PBMCs and T-cell apoptosis. Moreover, the intensity of this DNA damage was linked to lymphopenia. SARS-CoV-2 is known to induce the internalization of its receptor, angiotensin-converting enzyme 2, which is a protease capable of catabolizing AngII. Accordingly, in certain patients with COVID-19 we observed high plasma levels of AngII. When looking for the stimulus responsible for their monocytic ROS production, we revealed that AngII triggers ROS production by monocytes via angiotensin receptor I. ROSs released by AngII-activated monocytes induced DNA damage and apoptosis in neighboring lymphocytes. CONCLUSION: We conclude that T-cell apoptosis provoked via DNA damage due to the release of monocytic ROSs could play a major role in COVID-19 pathogenesis.
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Angiotensina II , COVID-19 , Linfopenia , Angiotensina II/sangre , Apoptosis , COVID-19/diagnóstico , COVID-19/patología , Daño del ADN , Humanos , Especies Reactivas de Oxígeno , SARS-CoV-2 , Linfocitos TRESUMEN
CXCR4 is a chemokine receptor that plays key roles with its specific ligand, CXCL12, in stem cell homing and immune trafficking. It is also used as a coreceptor by some HIV-1 strains (X4 strains), whereas other strains (R5 strains) use an alternative coreceptor, CCR5. X4 strains mainly emerge at late stages of the infection and are linked to disease progression. Two isoforms of this coreceptor have been described in humans: CXCR4-A and CXCR4-B, corresponding to an unspliced and a spliced mRNA, respectively. In this study, we show that CXCR4-B, but not CXCR4-A, mediates an efficient HIV-1 X4 entry and productive infection. Yet, the chemotactic activity of CXCL12 on both isoforms was similar. Furthermore, HIV-R5 infection favored CXCR4-B expression over that of CXCR4-A. In vitro infection with an R5 strain increased CXCR4-B/CXCR4-A mRNA ratio in PBMCs, and this ratio correlated with HIV RNA plasma level in R5-infected individuals. In addition, the presence of the CXCR4-B isoform favored R5 to X4 switch more efficiently than did CXCR4-A in vitro. Hence, the predominance of CXCR4-B over CXCR4-A expression in PBMCs was linked to the ability of circulating HIV-1 strains to use CXCR4, as determined by genotyping. These data suggest that R5 to X4 switch could be favored by R5 infection-induced overexpression of CXCR4-B. Finally, we achieved a specific small interfering RNA-mediated knockdown of CXCR4-B. This represents a proof of concept for a possible gene-therapeutic approach aimed at blocking the HIV coreceptor activity of CXCR4 without knocking down its chemotactic activity.
Asunto(s)
VIH-1/metabolismo , Receptores CXCR4/inmunología , Receptores del VIH/inmunología , Acoplamiento Viral , Línea Celular Tumoral , Quimiocina CXCL12/inmunología , Infecciones por VIH/inmunología , VIH-1/clasificación , VIH-1/genética , Células HeLa , Humanos , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Interferencia de ARN , ARN Interferente Pequeño , Receptores CCR5/inmunología , Receptores CXCR4/genética , Receptores del VIH/genética , Internalización del Virus , Replicación Viral/inmunologíaRESUMEN
Human immunodeficiency virus type 1 (HIV-1) transcription relies on its transactivating Tat protein. Although devoid of a signal sequence, Tat is released by infected cells and secreted Tat can affect uninfected cells, thereby contributing to HIV-1 pathogenesis. The mechanism and the efficiency of Tat export remained to be documented. Here, we show that, in HIV-1-infected primary CD4(+) T-cells that are the main targets of the virus, Tat accumulates at the plasma membrane because of its specific binding to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)). This interaction is driven by a specific motif of the Tat basic domain that recognizes a single PI(4,5)P(2) molecule and is stabilized by membrane insertion of Tat tryptophan side chain. This original recognition mechanism enables binding to membrane-embedded PI(4,5)P(2) only, but with an unusually high affinity that allows Tat to perturb the PI(4,5)P(2)-mediated recruitment of cellular proteins. Tat-PI(4,5)P(2) interaction is strictly required for Tat secretion, a process that is very efficient, as approximately 2/3 of Tat are exported by HIV-1-infected cells during their lifespan. The function of extracellular Tat in HIV-1 infection might thus be more significant than earlier thought.
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Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , VIH-1/patogenicidad , Fosfatidilinositol 4,5-Difosfato/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Sitios de Unión , Membrana Celular/metabolismo , Ensayo de Inmunoadsorción Enzimática , VIH-1/crecimiento & desarrollo , Humanos , Células Jurkat , Unión Proteica , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/análisisRESUMEN
OBJECTIVE: CCR5, a G protein-coupled receptor (GPCR), is used by most HIV strains as a coreceptor. In this study, we looked for other GPCR able to modify HIV-1 infection. DESIGN: We analyzed the effects of one GPCR coexpressed with CCR5, EBI2, on HIV-1 replicative cycle. METHODS: We identified GPCR expressed in primary CD4 + CCR5 + T cells by multi-RT-qPCR. We studied GPCR dimerization by FRET technology. Cell lines expressing EBI2 were established by transduction with HIV vectors. HIV-1 entry was quantified with virions harboring ß-lactamase fused to the viral protein vpr, early and late HIV-1 transcriptions by qPCR, NFkB nuclear activation by immunofluorescence and transfection, and viral production by measuring p24 concentration in culture supernatant by ELISA. RESULTS: We showed that EBI2 is naturally expressed in primary CD4 + CCR5 + T cells, and that CCR5 and EBI2 heterodimerize. We observed that this coexpression reduced viral entry by 50%. The amount of HIV reverse transcripts was similar in cells expressing or not EBI2. Finally, the presence of EBI2 induced the translocation of NFkB and activated HIV-1 genome expression. Globally, the result was a drastic HIV-1 R5, but not X4, overproduction in EBI2 -transduced cells. CONCLUSION: EBI2 expression in CD4 + CCR5 + cells boosts HIV-1 R5 productive infection. As the natural ligand for EBI2 is present in blood and lymphoid tissues, the constant EBI2 activation might increase HIV replication in CD4 + T cells. It might be of interest to test the effect of EBI2 antagonists on the residual viral production persisting in patients aviremic under treatment.
Asunto(s)
Linfocitos T CD4-Positivos , VIH-1 , Receptores CCR5 , Receptores Acoplados a Proteínas G , Replicación Viral , Humanos , Receptores CCR5/metabolismo , Linfocitos T CD4-Positivos/virología , Linfocitos T CD4-Positivos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Internalización del Virus , Células Cultivadas , Infecciones por VIH/virología , Infecciones por VIH/metabolismo , Multimerización de Proteína , Expresión GénicaRESUMEN
Oncogene-induced senescence (OIS) arrests cell proliferation in response to replication stress (RS) induced by oncogenes. OIS depends on the DNA damage response (DDR), but also on the cGAS-STING pathway, which detects cytosolic DNA and induces type I interferons (IFNs). Whether and how RS and IFN responses cooperate to promote OIS remains unknown. Here, we show that the induction of OIS by the H-RASV12 oncogene in immortalized human fibroblasts depends on the MRE11 nuclease. Indeed, treatment with the MRE11 inhibitor Mirin prevented RS, micronuclei formation and IFN response induced by RASV12. Overexpression of the cytosolic nuclease TREX1 also prevented OIS. Conversely, overexpression of a dominant negative mutant of TREX1 or treatment with IFN-ß was sufficient to induce RS and DNA damage, independent of RASV12 induction. These data suggest that the IFN response acts as a positive feedback loop to amplify DDR in OIS through a process regulated by MRE11 and TREX1.
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Senescencia Celular , Daño del ADN , Replicación del ADN , Exodesoxirribonucleasas , Proteína Homóloga de MRE11 , Fosfoproteínas , Transducción de Señal , Humanos , Exodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Proteína Homóloga de MRE11/metabolismo , Proteína Homóloga de MRE11/genética , Senescencia Celular/genética , Fibroblastos/metabolismo , Interferón beta/metabolismo , Interferón beta/genéticaRESUMEN
BACKGROUND: Morus alba has long been used in traditional Chinese medicine to treat inflammatory diseases; however, the scientific basis for such usage and the mechanism of action are not well understood. This study investigated the action of M. alba on leukocyte migration, one key step in inflammation. METHODS: Gas chromatography-mass spectrometry (GC-MS) and cluster analyses of supercritical CO2 extracts of three Morus species were performed for chemotaxonomy-aided plant authentication. Phytochemistry and CXCR4-mediated chemotaxis assays were used to characterize the chemical and biological properties of M. alba and its active compound, oxyresveratrol. fluorescence-activated cell sorting (FACS) and Western blot analyses were conducted to determine the mode of action of oxyresveratrol. RESULTS: Chemotaxonomy was used to help authenticate M. alba. Chemotaxis-based isolation identified oxyresveratrol as an active component in M. alba. Phytochemical and chemotaxis assays showed that the crude extract, ethyl acetate fraction and oxyresveratrol from M. alba suppressed cell migration of Jurkat T cells in response to SDF-1. Mechanistic study indicated that oxyresveratrol diminished CXCR4-mediated T-cell migration via inhibition of the MEK/ERK signaling cascade. CONCLUSIONS: A combination of GC-MS and cluster analysis techniques are applicable for authentication of the Morus species. Anti-inflammatory benefits of M. alba and its active compound, oxyresveratrol, may involve the inhibition of CXCR-4-mediated chemotaxis and MEK/ERK pathway in T and other immune cells.
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Antiinflamatorios/farmacología , Movimiento Celular/efectos de los fármacos , Leucocitos/citología , Leucocitos/inmunología , Morus/química , Extractos Vegetales/farmacología , Estilbenos/farmacología , Línea Celular , Quimiotaxis de Leucocito/efectos de los fármacos , Humanos , Leucocitos/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacosRESUMEN
It has been shown that living in risky environments, as well as having a risky occupation, can moderate risk-tolerance. Despite the involvement of dopamine in the expectation of reward described by neurobiologists, a GWAS study was not able to demonstrate a genetic contribution of genes involved in the dopaminergic pathway in risk attitudes and gene candidate studies gave contrasting results. We test the possibility that a genetic effect of the DRD4-7R allele in risk-taking behavior could be modulated by environmental factors. We show that the increase in risk-tolerance due to the 7R allele is independent of the environmental risk in two populations in Northern Senegal, one of which is exposed to a very high risk due to dangerous fishing.
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Dopamina , Receptores de Dopamina D4 , Alelos , Genotipo , Receptores de Dopamina D4/genética , Senegal , HumanosRESUMEN
Inflammation contributes to leukocyte migration, termed insulitis, and ß-cell loss in type 1 diabetes (T1D). Naturally occurring anthraquinones are claimed as anti-inflammatory compounds; however, their actions are not clear. This study aimed to investigate the effect and mechanism of catenarin on the inflammatory disease, T1D. Catenarin and/or its anthraquinone analogs dose-dependently suppressed C-X-C chemokine receptor type 4 (CXCR4)- and C-C chemokine receptor type 5 (CCR5)-implicated chemotaxis in leukocytes. Catenarin, the most potent anthraquinone tested in the study, prevented T1D in nonobese diabetic mice. Mechanistic study showed that catenarin did not act on the expression of CCR5 and CXCR4. On the contrary, catenarin inhibited CCR5- and CXCR4-mediated chemotaxis via the reduction of the phosphorylation of mitogen-activated protein kinases (p38 and JNK) and their upstream kinases (MKK6 and MKK7), and calcium mobilization. Overall, the data demonstrate the preventive effect and molecular mechanism of action of catenarin on T1D, suggesting its novel use as a prophylactic agent in T1D.
RESUMEN
BACKGROUND: Colorectal carcinoma (CRC) is the third most common cancer in the world and also the third leading cause of cancer-related mortality in Taiwan. CRC tumorigenesis is a multistep process, starting from mutations causing loss of function of tumor suppressor genes, canonically demonstrated in adenomatous polyposis coli pathogenesis. Although many genes or chromosomal alterations have been shown to be involved in this process, there are still unrecognized molecular events within CRC tumorigenesis. Elucidating these mechanisms may help improve the management and treatment. METHODS: In this study, we aimed to identify copy number alteration of the smallest chromosomal regions that is significantly associated with sporadic CRC tumorigenesis using high-resolution array-based Comparative Genomic Hybridization (aCGH) and quantitative Polymerase chain reaction (qPCR). In addition, microsatellite instability assay and sequencing-based mutation assay were performed to illustrate the initiation event of CRC tumorigenesis. RESULTS: A total of 571 CRC patients were recruited and 377 paired CRC tissues from sporadic CRC cases were used to define the smallest regions with chromosome copy number changes. In addition, 198 colorectal polyps from 160 patients were also used to study the role of 20q13.33 gain in CRC tumorigenesis. We found that gain in 20q13.33 is the main chromosomal abnormalities in this patient population and counts 50.9 and 62.8% in CRC and colon polyps, respectively. Furthermore, APC and KRAS gene mutations were profiled simultaneously and co-analyzed with microsatellite instability and 20q13.33 gain in CRC patients. Our study showed that the frequency of 20q13.33 copy number gain was highest among all reported CRC mutations. CONCLUSION: As APC or KRAS mutations are currently identified as the most important targets for CRC therapy, this study proposes that 20q13.33 copy number gain and the associated chromosomal genes function as promising biomarkers for both early stage detection and targeted therapy of sporadic CRCs in the future.
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Cromosomas Humanos Par 20 , Neoplasias Colorrectales/genética , Variaciones en el Número de Copia de ADN , Inestabilidad de Microsatélites , Biomarcadores/análisis , Carcinogénesis , Hibridación Genómica Comparativa , Femenino , Genómica , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia de ADN , TaiwánRESUMEN
R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops in the human genome, here, we map RNA:DNA hybrids, replication stress markers and DNA double-strand breaks (DSBs) in cells depleted for Topoisomerase I (Top1), an enzyme that relaxes DNA supercoiling and prevents R-loop formation. RNA:DNA hybrids are found at both promoters (TSS) and terminators (TTS) of highly expressed genes. In contrast, the phosphorylation of RPA by ATR is only detected at TTS, which are preferentially replicated in a head-on orientation relative to the direction of transcription. In Top1-depleted cells, DSBs also accumulate at TTS, leading to persistent checkpoint activation, spreading of γ-H2AX on chromatin and global replication fork slowdown. These data indicate that fork pausing at the TTS of highly expressed genes containing R-loops prevents head-on conflicts between replication and transcription and maintains genome integrity in a Top1-dependent manner.
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Replicación del ADN , ADN-Topoisomerasas de Tipo I/metabolismo , Estructuras R-Loop/genética , Regiones Terminadoras Genéticas/genética , Transcripción Genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Roturas del ADN de Doble Cadena , ADN-Topoisomerasas de Tipo I/genética , Técnicas de Silenciamiento del Gen , Inestabilidad Genómica , Células HEK293 , Células HeLa , Humanos , Fosforilación , Regiones Promotoras Genéticas , ARN Interferente Pequeño/metabolismoRESUMEN
The development of multidrug-resistant viruses compromises antiretroviral therapy efficacy and limits therapeutic options. Therefore, it is an ongoing task to identify new targets for antiretroviral therapy and to develop new drugs. Here, we show that an indole derivative (IDC16) that interferes with exonic splicing enhancer activity of the SR protein splicing factor SF2/ASF suppresses the production of key viral proteins, thereby compromising subsequent synthesis of full-length HIV-1 pre-mRNA and assembly of infectious particles. IDC16 inhibits replication of macrophage- and T cell-tropic laboratory strains, clinical isolates, and strains with high-level resistance to inhibitors of viral protease and reverse transcriptase. Importantly, drug treatment of primary blood cells did not alter splicing profiles of endogenous genes involved in cell cycle transition and apoptosis. Thus, human splicing factors represent novel and promising drug targets for the development of antiretroviral therapies, particularly for the inhibition of multidrug-resistant viruses.
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Antirretrovirales/farmacología , Farmacorresistencia Viral/genética , VIH/efectos de los fármacos , Indoles/farmacología , Isoquinolinas/farmacología , Precursores del ARN/efectos de los fármacos , Empalme del ARN/efectos de los fármacos , Carbazoles/farmacología , Evaluación Preclínica de Medicamentos , Ensayo de Inmunoadsorción Enzimática , VIH/genética , Humanos , Leucocitos Mononucleares/virología , Macrófagos/virología , ARN Viral/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Replicación Viral/efectos de los fármacosRESUMEN
Most HIV-1 Tat is unconventionally secreted by infected cells following Tat interaction with phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) at the plasma membrane. Extracellular Tat is endocytosed by uninfected cells before escaping from endosomes to reach the cytosol and bind PI(4,5)P2. It is not clear whether and how incoming Tat concentrates in uninfected cells. Here we show that, in uninfected cells, the S-acyl transferase DHHC-20 together with the prolylisomerases cyclophilin A (CypA) and FKBP12 palmitoylate Tat on Cys31 thereby increasing Tat affinity for PI(4,5)P2. In infected cells, CypA is bound by HIV-1 Gag, resulting in its encapsidation and CypA depletion from cells. Because of the lack of this essential cofactor, Tat is not palmitoylated in infected cells but strongly secreted. Hence, Tat palmitoylation specifically takes place in uninfected cells. Moreover, palmitoylation is required for Tat to accumulate at the plasma membrane and affect PI(4,5)P2-dependent membrane traffic such as phagocytosis and neurosecretion.
Asunto(s)
Membrana Celular/metabolismo , Ciclofilina A/metabolismo , VIH-1/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Aciltransferasas/metabolismo , Animales , Animales Recién Nacidos , Membrana Celular/virología , Ciclofilina A/genética , Células HEK293 , VIH-1/fisiología , Humanos , Células Jurkat , Lipoilación , Ratones , Ratones Endogámicos C57BL , Células PC12 , Fosfatidilinositol 4,5-Difosfato/metabolismo , Unión Proteica , Células RAW 264.7 , RatasRESUMEN
BACKGROUND: The presence of HIV-1 preintegration reservoir was assessed in an in vitro experimental model of latent HIV-1 infection, and in patients treated or not with highly active antiretroviral therapy (HAART). RESULTS: In resting CD4+ T lymphocytes latently infected in vitro with HIV-1, we demonstrated that the polyclonal activation induced a HIV-1 replication, which could be prevented by the use of an HIV-1 integrase inhibitor. We also showed that this reservoir was labile since the rescuable HIV-1-antigens production from unintegrated HIV-1 genomes declined over time. These data confirm that our experimental approach allows the characterization of a functional unintegrated HIV-1 reservoir. We then explored the preintegration reservoir in HIV-1-infected patients. This reservoir was detected in 11 of 12 untreated patients, in 4 of 10 sustained responders to HAART, and in one incomplete responder. This reservoir was also inducible, labile, and anti-HIV-1 integrase drug inhibited its induction. Finally, this reservoir was associated with the presence of spontaneous HIV-1 antigens producing CD4+ T cells in blood from 3 of 3 untreated patients and 2 of 2 sustained responders to HAART harboring a preintegration reservoir. CONCLUSION: This preintegration phase of HIV-1 latency could be a consequence of the ongoing viral replication in untreated patients and of a residual viral replication in treated patients.
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Fármacos Anti-VIH/uso terapéutico , Terapia Antirretroviral Altamente Activa , Infecciones por VIH/virología , VIH-1/fisiología , Latencia del Virus , Linfocitos T CD4-Positivos/virología , ADN Viral , Infecciones por VIH/tratamiento farmacológico , Humanos , Activación Viral , Integración Viral , Replicación ViralRESUMEN
OBJECTIVE: In this study, we looked for a new family of latency reversing agents. DESIGN: We searched for G-protein-coupled receptors (GPCR) coexpressed with the C-C chemokine receptor type 5 (CCR5) in primary CD4 T cells that activate infected cells and boost HIV production. METHODS: GPCR coexpression was unveiled by reverse transcriptase-PCR. We used fluorescence resonance energy transfer to analyze the dimerization with CCR5 of the expressed GPCR. Viral entry was measured by flow cytometry, reverse transcription by quantitative PCR, nuclear factor-kappa B translocation by immunofluorescence, long terminal repeat activation using a gene reporter assay and viral production by p24 quantification. RESULTS: Gαi-coupled sphingosine-1-phophate receptor 1 (S1P1) is highly coexpressed with CCR5 on primary CD4 T cells and dimerizes with it. The presence of S1P1 had major effects neither on viral entry nor on reverse transcription. Yet, S1P1 signaling induced NFκB activation, boosting the expression of the HIV LTR. Consequently, in culture medium containing sphingosine-1-phophate, the presence of S1P1 enhanced the replication of a CCR5-, but also of a CXCR4-using HIV-1 strain. The S1P1 ligand FTY720, a drug used in multiple sclerosis treatment, inhibited HIV-1 productive infection of monocyte-derived dendritic cells and of severe combined immunodeficiency mice engrafted with human peripheral blood mononuclear cells. Conversely, S1P1 agonists were able to force latently infected peripheral blood mononuclear cells and lymph node cells to produce virions in vitro. CONCLUSION: Altogether these data indicate that the presence of S1P1 facilitates HIV-1 replicative cycle by boosting viral genome transcription, S1P1 antagonists have anti-HIV effects and S1P1 agonists are HIV latency reversing agents.
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Linfocitos T CD4-Positivos/virología , VIH-1/fisiología , Receptores de Lisoesfingolípidos/agonistas , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Transducción de Señal , Latencia del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Animales , Células Cultivadas , Perfilación de la Expresión Génica , VIH-1/crecimiento & desarrollo , Humanos , Ratones SCID , Receptores CCR5/biosíntesis , Receptores de Lisoesfingolípidos/biosíntesisRESUMEN
OBJECTIVE AND DESIGN: The intensity of replication of CCR5-using HIV-1 strains is highly dependent on the number of CCR5 molecules on the surface of CD4-positive T cells. The molecular mechanisms responsible for this phenomenon remained so far unclear. As CCR5 co-receptors are coupled to G alpha i and G alpha q proteins, we tested the hypothesis that the activation triggered through these proteins secondary to the interaction between the viral envelope and CCR5 could account for the effect of the level of CCR5 expression on HIV-1 production. METHODS: We transduced the wild-type or a G-protein signalling-defective CCR5 gene into CD4/CCR5 HOS cells and peripheral blood mononuclear cells. The effect on cell activation in presence of a CCR5-binding chemokine and on HIV infection was monitored by measuring calcium mobilization and p24 antigen production, respectively. The role of G alpha i protein signalling was tested by adding pertussis toxin to the cell cultures or by transfecting small interfering (si) RNAs into the HOS cells. RESULTS: The over-expression of the wild-type form, but not of a G-protein signalling-defective form of CCR5, on the surface of CCR5 expressing peripheral blood mononuclear cells markedly increased their infectability. In addition, both pertussis toxin and G alpha i 1-specific siRNA drastically inhibited R5 infection. CONCLUSIONS: The signalling through G alpha i-protein induced upon R5 virion binding to CCR5 is responsible for the difference in HIV-1 infectability between CD4-positive T cells expressing low or high levels of cell surface CCR5 density. This observation sheds new light on the physiopathology of HIV infection, and opens new therapeutic opportunities targeting G alpha i signalling.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Infecciones por VIH/inmunología , VIH-1/fisiología , Receptores CCR5/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Infecciones por VIH/virología , Humanos , Toxina del Pertussis/farmacología , ARN Interferente Pequeño/genética , Receptores CCR5/genética , Transducción de Señal/inmunología , Transducción Genética , Células Tumorales Cultivadas , Virulencia/efectos de los fármacos , Replicación ViralRESUMEN
Cyclophilin A is involved in many cellular processes, including protein folding and intracellular transports. Because cyclophilin A has been shown to interact with HIV-1 gag proteins and to enhance the viral infectivity, nonimmunosuppressive cyclophilin A ligands may represent a new class of therapeutic agents against HIV. Here, we report a virtual screening using structure- and pharmacophore-based design to identify original nonpeptidic cyclophilin ligands. Following a lead identification of compounds 1 [1-(3-benzyloxypyridin-2-yl)-3-(3-chlorophenyl)urea] and 2 [1-(3-benzyloxypyridin-2-yl)-3-(3-trifluoromethylphenyl)urea] (IC(50) = 0.3 microM), a series of molecules were synthesized from a diarylurea scaffold and evaluated for their in vitro ability to inhibit the cis-trans isomerase activity of cyclophilin A. Molecular modifications provided several more potent compounds, in particular analogues 4d and 4i with IC(50) of 14 and 20 nM, respectively. Then, we evaluated the effect of analogues 1 and 2 on HIV virion infectivity in both immortalized and primary cells. Both 1 and 2 reduced virion infectivity in the replication-defective one-round infection assay, but only 1 impaired wild-type HIV infection in human peripheral blood mononuclear cells.
Asunto(s)
Fármacos Anti-VIH/síntesis química , Ciclofilina A/antagonistas & inhibidores , Compuestos de Fenilurea/síntesis química , Piridinas/síntesis química , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Sitios de Unión , Células Cultivadas , Diseño de Fármacos , VIH-1/efectos de los fármacos , Humanos , Ligandos , Modelos Moleculares , Estructura Molecular , Compuestos de Fenilurea/química , Compuestos de Fenilurea/farmacología , Piridinas/química , Piridinas/farmacología , Relación Estructura-Actividad , Virión/efectos de los fármacosRESUMEN
Humans have colonized and adapted to extremely diverse environments, and the genetic basis of some such adaptations, for example to high altitude, is understood. In some cases, local or regional variation in selection pressure could also cause behavioural adaptations. Numerous genes influence behaviour, such as alleles at the dopamine receptor locus D4 (DRD4), which are associated with attitude toward risk in experimental settings. We demonstrate genetic differentiation for this gene, but not for five unlinked microsatellite loci, between high- and low risk environments around Mount Merapi, an active volcano in Java, Indonesia. Using a behavioural experiment, we further show that people inhabiting the high risk environment are significantly more risk averse. We provide evidence of a genetic basis for this difference, showing that heterozygotes at the DRD4 locus are more risk averse than either homozygotes. In the high risk environment, allele frequencies are equilibrated, generating a high frequency of heterozygotes. Thus it appears that overdominance (i.e. selective advantage of heterozygotes) generates negative frequency dependent selection, favouring the rarer allele at this locus. Our results therefore provide evidence for adaptation to a marginal habitat through the selection of a neurocognitive trait with a genetic basis.