RESUMEN
We present a drug design strategy based on structural knowledge of protein-protein interfaces selected through virus-host coevolution and translated into highly potential small molecules. This approach is grounded on Vinland, the most comprehensive atlas of virus-human protein-protein interactions with annotation of interacting domains. From this inspiration, we identified small viral protein domains responsible for interaction with human proteins. These peptides form a library of new chemical entities used to screen for replication modulators of several pathogens. As a proof of concept, a peptide from a KSHV protein, identified as an inhibitor of influenza virus replication, was translated into a small molecule series with low nanomolar antiviral activity. By targeting the NEET proteins, these molecules turn out to be of therapeutic interest in a nonalcoholic steatohepatitis mouse model with kidney lesions. This study provides a biomimetic framework to design original chemistries targeting cellular proteins, with indications going far beyond infectious diseases.
Asunto(s)
Gripe Humana , Virus , Animales , Ratones , Humanos , Proteoma , Péptidos/farmacología , Descubrimiento de DrogasRESUMEN
Hexokinases (HKs) control the first step of glucose catabolism. A switch of expression from liver HK (glucokinase, GCK) to the tumor isoenzyme HK2 is observed in hepatocellular carcinoma progression. Our prior work revealed that HK isoenzyme switch in hepatocytes not only regulates hepatic metabolic functions but also modulates innate immunity and sensitivity to Natural Killer (NK) cell cytotoxicity. This study investigates the impact of HK2 expression and its mitochondrial binding on the resistance of human liver cancer cells to NK-cell-induced cytolysis. We have shown that HK2 expression induces resistance to NK cell cytotoxicity in a process requiring mitochondrial binding of HK2. Neither HK2 nor GCK expression affects target cells' ability to activate NK cells. In contrast, mitochondrial binding of HK2 reduces effector caspase 3/7 activity both at baseline and upon NK-cell activation. Furthermore, HK2 tethering to mitochondria enhances their resistance to cytochrome c release triggered by tBID. These findings indicate that HK2 mitochondrial binding in liver cancer cells is an intrinsic resistance factor to cytolysis and an escape mechanism from immune surveillance.
RESUMEN
The hypoxia response pathway enables adaptation to oxygen deprivation. It is mediated by hypoxia-inducible factors (HIF), which promote metabolic reprogramming, erythropoiesis, angiogenesis and tissue remodeling. This led to the successful development of HIF-inducing drugs for treating anemia and some of these molecules are now in clinic. However, elevated levels of HIFs are frequently associated with tumor growth, poor prognosis, and drug resistance in various cancers, including hepatocellular carcinoma (HCC). Consequently, there are concerns regarding the recommendation of HIF-inducing drugs in certain clinical situations. Here, we analyzed the effects of two HIF-inducing drugs, Molidustat and Roxadustat, in the well-characterized HCC cell line Huh7. These drugs increased HIF-1α and HIF-2α protein levels which both participate in inducing hypoxia response genes such as BNIP3, SERPINE1, LDHA or EPO. Combined transcriptomics, proteomics and metabolomics showed that Molidustat increased the expression of glycolytic enzymes, while the mitochondrial network was fragmented and cellular respiration decreased. This metabolic remodeling was associated with a reduced proliferation and a lower demand for pyrimidine supply, but an increased ability of cells to convert pyruvate to lactate. This was accompanied by a higher resistance to the inhibition of mitochondrial respiration by antimycin A, a phenotype confirmed in Roxadustat-treated Huh7 cells and Molidustat-treated hepatoblastoma cells (Huh6 and HepG2). Overall, this study shows that HIF-inducing drugs increase the metabolic resilience of liver cancer cells to metabolic stressors, arguing for careful monitoring of patients treated with HIF-inducing drugs, especially when they are at risk of liver cancer.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Carcinoma Hepatocelular , Proliferación Celular , Subunidad alfa del Factor 1 Inducible por Hipoxia , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Proliferación Celular/efectos de los fármacos , Línea Celular Tumoral , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Isoquinolinas/farmacología , Glicina/análogos & derivados , Glicina/farmacología , Estrés Fisiológico/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacosRESUMEN
Vaccination is one of the most widely used strategies to protect horses against pathogens. However, available equine vaccines often have limitations, as they do not always provide effective, long-term protection and booster injections are often required. In addition, research efforts are needed to develop effective vaccines against emerging equine pathogens. In this review, we provide an inventory of approved adjuvants for equine vaccines worldwide, and discuss their composition and mode of action when available. A wide range of adjuvants are used in marketed vaccines for horses, the main families being aluminium salts, emulsions, polymers, saponins and ISCOMs. We also present veterinary adjuvants that are already used for vaccination in other species and are currently evaluated in horses to improve equine vaccination and to meet the expected level of protection against pathogens in the equine industry. Finally, we discuss new adjuvants such as liposomes, polylactic acid polymers, inulin, poly-ε-caprolactone nanoparticles and co-polymers that are in development. Our objective is to help professionals in the horse industry understand the composition of marketed equine vaccines in a context of mistrust towards vaccines. Besides, this review provides researchers with a list of adjuvants, either approved or at least evaluated in horses, that could be used either alone or in combination to develop new vaccines.
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Adyuvantes Inmunológicos , Nanopartículas , Caballos , Animales , Adyuvantes Inmunológicos/farmacología , Vacunación/veterinaria , Nanopartículas/uso terapéutico , PolímerosRESUMEN
Hepatitis C virus (HCV) relies on cellular lipid metabolism for its replication, and actively modulates lipogenesis and lipid trafficking in infected hepatocytes. This translates into an intracellular accumulation of triglycerides leading to liver steatosis, cirrhosis and hepatocellular carcinoma, which are hallmarks of HCV pathogenesis. While the interaction of HCV with hepatocyte metabolic pathways is patent, how viral proteins are able to redirect central carbon metabolism towards lipogenesis is unclear. Here, we report that the HCV protein NS5A activates the glucokinase (GCK) isoenzyme of hexokinases through its D2 domain (NS5A-D2). GCK is the first rate-limiting enzyme of glycolysis in normal hepatocytes whose expression is replaced by the hexokinase 2 (HK2) isoenzyme in hepatocellular carcinoma cell lines. We took advantage of a unique cellular model specifically engineered to re-express GCK instead of HK2 in the Huh7 cell line to evaluate the consequences of NS5A-D2 expression on central carbon and lipid metabolism. NS5A-D2 increased glucose consumption but decreased glycogen storage. This was accompanied by an altered mitochondrial respiration, an accumulation of intracellular triglycerides and an increased production of very-low density lipoproteins. Altogether, our results show that NS5A-D2 can reprogram central carbon metabolism towards a more energetic and glycolytic phenotype compatible with HCV needs for replication.
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Glucoquinasa/metabolismo , Hepacivirus/fisiología , Hepatitis C/metabolismo , Hepatitis C/virología , Hepatocitos/metabolismo , Hepatocitos/virología , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas no Estructurales Virales/metabolismo , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Glucógeno/metabolismo , Glucólisis , Interacciones Huésped-Patógeno , Humanos , Metabolismo de los Lípidos , Lipogénesis , Mitocondrias/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , ARN Polimerasa Dependiente del ARN/química , Proteínas no Estructurales Virales/químicaRESUMEN
La Reunion island in the South West Indian Ocean is now endemic for dengue following the introduction of dengue virus serotype 2 (DENV-2) cosmopolitan-I genotype in 2017. DENV-2 infection causes a wide spectrum of clinical manifestations ranging from flu-like disease to severe dengue. The nonstructural glycoprotein 1 (NS1) has been identified as playing a key role in dengue disease severity. The intracellular NS1 exists as a homodimer, whereas a fraction is driven towards the plasma membrane or released as a soluble hexameric protein. Here, we characterized the NS1 glycoproteins from clinical isolates DES-14 and RUN-18 that were collected during the DENV-2 epidemics in Tanzania in 2014 and La Reunion island in 2018, respectively. In relation to hepatotropism of the DENV, expression of recombinant DES-14 NS1 and RUN-18 NS1 glycoproteins was compared in human hepatoma Huh7 cells. We observed that RUN-18 NS1 was poorly stable in Huh7 cells compared to DES-14 NS1. The instability of RUN-18 NS1 leading to a low level of NS1 secretion mostly relates to lysine residues on positions 272 and 324. Our data raise the issue of the consequences of a defect in NS1 stability in human hepatocytes in relation to the major role of NS1 in the pathogenesis of the DENV-2 infection.
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Virus del Dengue/metabolismo , Dengue/epidemiología , Dengue/metabolismo , Epidemias , Genotipo , Lisina/química , Proteínas no Estructurales Virales/química , Sustitución de Aminoácidos , Antígenos Virales/química , Antígenos Virales/genética , Línea Celular Tumoral , Dengue/virología , Células HEK293 , Hepatocitos/metabolismo , Hepatocitos/virología , Humanos , Multimerización de Proteína , Estabilidad Proteica , Proteínas Recombinantes/química , Reunión/epidemiología , Serogrupo , Tanzanía/epidemiología , Transfección , Proteínas no Estructurales Virales/genéticaRESUMEN
In less than 20 years, three deadly coronaviruses, SARS-CoV, MERS-CoV and SARS-CoV-2, have emerged in human population causing hundreds to hundreds of thousands of deaths. Other coronaviruses are causing epizootic representing a significant threat for both domestic and wild animals. Members of this viral family have the longest genome of all RNA viruses, and express up to 29 proteins establishing complex interactions with the host proteome. Deciphering these interactions is essential to identify cellular pathways hijacked by these viruses to replicate and escape innate immunity. Virus-host interactions also provide key information to select targets for antiviral drug development. Here, we have manually curated the literature to assemble a unique dataset of 1311 coronavirus-host protein-protein interactions. Functional enrichment and network-based analyses showed coronavirus connections to RNA processing and translation, DNA damage and pathogen sensing, interferon production, and metabolic pathways. In particular, this global analysis pinpointed overlooked interactions with translation modulators (GIGYF2-EIF4E2), components of the nuclear pore, proteins involved in mitochondria homeostasis (PHB, PHB2, STOML2), and methylation pathways (MAT2A/B). Finally, interactome data provided a rational for the antiviral activity of some drugs inhibiting coronaviruses replication. Altogether, this work describing the current landscape of coronavirus-host interactions provides valuable hints for understanding the pathophysiology of coronavirus infections and developing effective antiviral therapies.
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Infecciones por Coronavirus/metabolismo , Coronavirus/metabolismo , Interacciones Huésped-Patógeno/fisiología , Mapas de Interacción de Proteínas , Proteínas Virales/metabolismo , Animales , Betacoronavirus/fisiología , COVID-19 , Coronavirus/química , Infecciones por Coronavirus/virología , Bases de Datos de Proteínas , Humanos , Proteínas Mitocondriales/metabolismo , Pandemias , Neumonía Viral/metabolismo , Neumonía Viral/virología , Prohibitinas , SARS-CoV-2 , Factores de Transcripción/metabolismo , Replicación Viral/genéticaRESUMEN
Hepatitis B virus (HBV) infection and bile acid (BA) metabolism are interdependent: infection modifies the expression of the BA nuclear receptor farnesoid X receptor (FXR)-α, and modulation of FXRα activity by ligands alters HBV replication. Mechanisms of HBV control by FXRα remain to be unveiled. FXRα silencing in HBV-infected HepaRG cells decreased the viral covalently closed circular (ccc)DNA pool size and transcriptional activity. Treatment with the FXRα agonist GW4064 inhibited FXRα proviral effect on cccDNA similarly for wild-type and hepatitis B viral X protein (HBx)-deficient virus, whereas agonist-induced inhibition of pregenomic and precore RNA transcription and viral DNA secretion was HBx dependent. These data indicated that FXRα acts as a proviral factor by 2 different mechanisms, which are abolished by FXRα stimulation. Finally, infection of C3H/HeN mice by a recombinant adeno-associated virus-2/8-HBV vector induced a sustained HBV replication in young mice in contrast with the transient decline in adult mice. Four-week GW4064 treatment of infected C3H/HeN mice decreased secretion of HBV DNA and HB surface antigen in adult mice only. These results suggest that the physiologic balance of FXRα expression and activation by bile acid is a key host metabolic pathway in the regulation of HBV infection and that FXRα can be envisioned as a target for HBV treatment.-Mouzannar, K., Fusil, F., Lacombe, B., Ollivier, A., Ménard, C., Lotteau, V., Cosset, F.-L., Ramière, C., André, P. Farnesoid X receptor α is a proviral host factor for hepatitis B virus that is inhibited by ligands in vitro and in vivo.
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Regulación de la Expresión Génica , Virus de la Hepatitis B/patogenicidad , Hepatitis B/virología , Provirus/patogenicidad , Receptores Citoplasmáticos y Nucleares/metabolismo , Replicación Viral , Animales , ADN Viral/genética , Femenino , Células Hep G2 , Hepatitis B/metabolismo , Hepatitis B/patología , Virus de la Hepatitis B/genética , Humanos , Técnicas In Vitro , Ligandos , Ratones , Ratones Endogámicos C3H , Provirus/genéticaRESUMEN
Cell metabolism now appears as an essential regulator of immune cells activation. In particular, TLR stimulation triggers metabolic reprogramming of dendritic cells (DCs) with an increased glycolytic flux, whereas inhibition of glycolysis alters their functional activation. The molecular mechanisms involved in the control of glycolysis upon TLR stimulation are poorly understood for human DCs. TLR4 activation of human monocyte-derived DCs (MoDCs) stimulated glycolysis with an increased glucose consumption and lactate production. Global hexokinase (HK) activity, controlling the initial rate-limiting step of glycolysis, was also increased. TLR4-induced glycolytic burst correlated with a differential modulation of HK isoenzymes. LPS strongly enhanced the expression of HK2, whereas HK3 was reduced, HK1 remained unchanged, and HK4 was not expressed. Expression of the other rate-limiting glycolytic enzymes was not significantly increased. Exploring the signaling pathways involved in LPS-induced glycolysis with various specific inhibitors, we observed that only the inhibitors of p38-MAPK (SB203580) and of HIF-1α DNA binding (echinomycin) reduced both the glycolytic activity and production of cytokines triggered by TLR4 stimulation. In addition, LPS-induced HK2 expression required p38-MAPK-dependent HIF-1α accumulation and transcriptional activity. TLR1/2 and TLR2/6 stimulation increased glucose consumption by MoDCs through alternate mechanisms that are independent of p38-MAPK activation. TBK1 contributed to glycolysis regulation when DCs were stimulated via TLR2/6. Therefore, our results indicate that TLR4-dependent upregulation of glycolysis in human MoDCs involves a p38-MAPK-dependent HIF-1α accumulation, leading to an increased HK activity supported by enhanced HK2 expression.
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Células Dendríticas/inmunología , Regulación Enzimológica de la Expresión Génica/inmunología , Hexoquinasa/inmunología , Subunidad alfa del Factor 1 Inducible por Hipoxia/inmunología , Monocitos/inmunología , Receptor Toll-Like 4/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/inmunología , Células Cultivadas , Células Dendríticas/patología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Lipopolisacáridos/toxicidad , Monocitos/patología , Estabilidad Proteica , Receptor Toll-Like 4/agonistasRESUMEN
PB1-F2 is a viral protein encoded by influenza A viruses (IAVs). PB1-F2 is implicated in virulence by triggering immune cell apoptosis and enhancing inflammation. To obtain an insight into the molecular mechanisms of PB1-F2-mediated virulence, we used the yeast two-hybrid approach to find new PB1-F2 cellular interactors. This allowed us to identify calcium-binding and coiled-coil domain 2 (CALCOCO2, also known as NDP52) as a binding partner of PB1-F2. Binding of PB1-F2 to CALCOCO2 was confirmed by pull-down. Surface plasmon resonance binding experiments enabled us to estimate the dissociation constant (Kd) of the two partners to be around 20 nM. Using bioinformatics tools, we designed a CALCOCO2 interaction map based on previous knowledge and showed a strong connection between this protein and the type I interferon production pathways and the I-κB kinase/NF-κB signalling pathway. NF-κB reporter assays in which CALCOCO2, MAVS and PB1-F2 were co-expressed showed a cooperation of these three proteins to increase the inflammatory response. By contrast, PB1-F2 inhibits the TBK1-dependent activation of an ISRE reporter plasmid. We also demonstrated that the signal transducer TRAF6 is implicated in the enhancement of NF-κB activity mediated by PB1-F2/CALCOCO2 binding. Altogether, this report provides evidence of an interaction link between PB1-F2 and human proteins, and allows a better understanding of the involvement of PB1-F2 in the pathologic process mediated by IAV.
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Interacciones Huésped-Patógeno , Inmunidad Innata , Virus de la Influenza A/inmunología , Virus de la Influenza A/patogenicidad , Proteínas Nucleares/metabolismo , Proteínas Virales/metabolismo , Factores de Virulencia/metabolismo , Biología Computacional , Humanos , Cinética , Unión Proteica , Mapeo de Interacción de Proteínas , Resonancia por Plasmón de Superficie , Técnicas del Sistema de Dos HíbridosRESUMEN
The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is a sequence-specific DNA-binding protein that plays an essential role in viral episome replication and segregation, by recruiting the cellular complex of DNA replication onto the origin (oriP) and by tethering the viral DNA onto the mitotic chromosomes. Whereas the mechanisms of viral DNA replication are well documented, those involved in tethering EBNA1 to the cellular chromatin are far from being understood. Here, we have identified regulator of chromosome condensation 1 (RCC1) as a novel cellular partner for EBNA1. RCC1 is the major nuclear guanine nucleotide exchange factor for the small GTPase Ran enzyme. RCC1, associated with chromatin, is involved in the formation of RanGTP gradients critical for nucleo-cytoplasmic transport, mitotic spindle formation and nuclear envelope reassembly following mitosis. Using several approaches, we have demonstrated a direct interaction between these two proteins and found that the EBNA1 domains responsible for EBNA1 tethering to the mitotic chromosomes are also involved in the interaction with RCC1. The use of an EBNA1 peptide array confirmed the interaction of RCC1 with these regions and also the importance of the N-terminal region of RCC1 in this interaction. Finally, using confocal microscopy and Förster resonance energy transfer analysis to follow the dynamics of interaction between the two proteins throughout the cell cycle, we have demonstrated that EBNA1 and RCC1 closely associate on the chromosomes during metaphase, suggesting an essential role for the interaction during this phase, perhaps in tethering EBNA1 to mitotic chromosomes.
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Proteínas de Ciclo Celular/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Mitosis , Proteínas Nucleares/metabolismo , Dominios y Motivos de Interacción de Proteínas , Secuencias de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Cromosomas Humanos/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/química , Antígenos Nucleares del Virus de Epstein-Barr/genética , Transferencia Resonante de Energía de Fluorescencia , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/genética , Células HEK293 , Células HeLa , Humanos , Metafase , Microscopía Confocal , Proteínas Nucleares/química , Proteínas Nucleares/genética , Análisis por Matrices de Proteínas , Mapeo de Interacción de Proteínas , Huso Acromático/metabolismoRESUMEN
Hepatitis B virus (HBV) and bile salt metabolism seem tightly connected. HBV enters hepatocytes by binding to sodium taurocholate cotransporting polypeptide (NTCP), the genome of which contains 2 active farnesoid X receptor (FXR) α response elements that participate in HBV transcriptional activity. We investigated in differentiated HepaRG cells and in primary human hepatocytes (PHHs) effects of FXR activation on HBV replication and of infection on the FXR pathway. In HepaRG cells, FXR agonists (6-ethyl chenodeoxycholic acid and GW4064), but no antagonist, and an FXR-unrelated bile salt inhibited viral mRNA, DNA, and protein production (IC50, 0.1-0.5 µM) and reduced covalently closed circular DNA pool size. These effects were independent of the NTCP inhibitor cyclosporine-A, which suggests inhibition occurred at a postentry step. Similar results were obtained in PHHs with GW4064. Infection of these cells increased expression of FXR and modified expression of FXR-regulated genes SHP, APOA1, NTCP, CYP7A1, and CYP8B1 with a more pronounced effect in PHHs than in HepaRG cells. FXR agonists reversed all but one of the HBV-induced FXR gene profile modifications. HBV replication and FXR regulation seem to be interdependent, and altered bile salt metabolism homeostasis might contribute to the persistence of HBV infection.-Radreau, P., Porcherot, M., Ramière, C., Mouzannar, K., Lotteau, V., André, P. Reciprocal regulation of farnesoid X receptor α activity and hepatitis B virus replication in differentiated HepaRG cells and primary human hepatocytes.
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Diferenciación Celular/fisiología , Virus de la Hepatitis B/fisiología , Hepatocitos/fisiología , Receptores Citoplasmáticos y Nucleares/metabolismo , Replicación Viral/fisiología , Línea Celular , ADN Viral , Regulación de la Expresión Génica/fisiología , Humanos , ARN Viral , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/genética , Replicación Viral/efectos de los fármacosRESUMEN
UNLABELLED: Ovine pulmonary adenocarcinoma is a naturally occurring lung cancer in sheep induced by the Jaagsiekte sheep retrovirus (JSRV). Its envelope glycoprotein (Env) carries oncogenic properties, and its expression is sufficient to induce in vitro cell transformation and in vivo lung adenocarcinoma. The identification of cellular partners of the JSRV envelope remains crucial for deciphering mechanisms leading to cell transformation. We initially identified RALBP1 (RalA binding protein 1; also known as RLIP76 or RIP), a cellular protein implicated in the ras pathway, as a partner of JSRV Env by yeast two-hybrid screening and confirmed formation of RALBP1/Env complexes in mammalian cells. Expression of the RALBP1 protein was repressed in tumoral lungs and in tumor-derived alveolar type II cells. Through its inhibition using specific small interfering RNA (siRNA), we showed that RALBP1 was involved in envelope-induced cell transformation and in modulation of the mTOR (mammalian target of rapamycin)/p70S6K pathway by the retroviral envelope. IMPORTANCE: JSRV-induced lung adenocarcinoma is of importance for the sheep industry. While the envelope has been reported as the oncogenic determinant of the virus, the cellular proteins directly interacting with Env are still not known. Our report on the formation of RALBP/Env complexes and the role of this interaction in cell transformation opens up a new hypothesis for the dysregulation observed upon virus infection in sheep.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Transformación Celular Viral/fisiología , Proteínas Activadoras de GTPasa/metabolismo , Productos del Gen env/metabolismo , Retrovirus Ovino Jaagsiekte/fisiología , Adenomatosis Pulmonar Ovina/fisiopatología , Enfermedades de las Ovejas/fisiopatología , Enfermedades de las Ovejas/virología , Animales , Western Blotting , Cartilla de ADN/genética , Regulación Neoplásica de la Expresión Génica/fisiología , Células HEK293 , Humanos , Inmunoprecipitación , Complejos Multiproteicos/metabolismo , Sistemas de Lectura Abierta/genética , ARN Interferente Pequeño/genética , Ovinos , Estadísticas no Paramétricas , Técnicas del Sistema de Dos HíbridosRESUMEN
Statin treatment of hypercholesterolemia can lead to chronic myotoxicity which is, in most cases, alleviated by drug withdrawal. Cellular and molecular mechanisms of this adverse effect have been elusive, in particular because of the lack of in vitro models suitable for long-term exposures. We have taken advantage of the properties of human pluripotent stem cell-derived mesodermal precursors, that can be maintained unaltered in vitro for a long period of time, to develop a model of repeated exposures to simvastatin during more than 2 weeks. This approach unveiled major differences, both in functional and molecular terms, in response to single versus repeated-dose exposures to simvastatin. The main functional effect of the in vitro simvastatin-induced long-term toxicity was a loss of proliferative capacity in the absence of concomitant cell death, revealing that cytostatic effect could be a major contributor to statin-induced myotoxicity. Comparative analysis of molecular modifications induced by simvastatin short-term versus prolonged exposures demonstrated powerful adaptive cell responses, as illustrated by the dramatic decrease in the number of differentially expressed genes, distinct biological pathway enrichments, and distinct patterns of nutrient transporters expressed at the cell surface. This study underlines the potential of derivatives of human pluripotent stem cells for developing new approaches in toxicology, in particular for chronic toxicity testing.
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Hipercolesterolemia/tratamiento farmacológico , Mesodermo/efectos de los fármacos , Células Madre Pluripotentes/efectos de los fármacos , Simvastatina/efectos adversos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Hipercolesterolemia/complicaciones , Hipercolesterolemia/patología , Mesodermo/citología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Células Madre Pluripotentes/citología , Simvastatina/administración & dosificación , Transcriptoma/efectos de los fármacosRESUMEN
More than 170 million people worldwide are infected with the hepatitis C virus (HCV), for which future therapies are expected to rely upon a combination of oral antivirals. For a rapidly evolving virus like HCV, host-targeting antivirals are an attractive option. To decipher the role of novel HCV-host interactions, we used a proteomics approach combining immunoprecipitation of viral-host protein complexes coupled to mass spectrometry identification and functional genomics RNA interference screening of HCV partners. Here, we report the proteomics analyses of protein complexes associated with Core, NS2, NS3/4A, NS4B, NS5A, and NS5B proteins. We identified a stringent set of 98 human proteins interacting specifically with one of the viral proteins. The overlap with previous virus-host interaction studies demonstrates 24.5% shared HCV interactors overall (24/98), illustrating the reliability of the approach. The identified human proteins show enriched Gene Ontology terms associated with the endoplasmic reticulum, transport proteins with a major contribution of NS3/4A interactors, and transmembrane proteins for Core interactors. The interaction network emphasizes a high degree distribution, a high betweenness distribution, and high interconnectivity of targeted human proteins, in agreement with previous virus-host interactome studies. The set of HCV interactors also shows extensive enrichment for known targets of other viruses. The combined proteomic and gene silencing study revealed strong enrichment in modulators of HCV RNA replication, with the identification of 11 novel cofactors among our set of specific HCV partners. Finally, we report a novel immune evasion mechanism of NS3/4A protein based on its ability to affect nucleocytoplasmic transport of type I interferon-mediated signal transducer and activator of transcription 1 nuclear translocation. The study revealed highly stringent association between HCV interactors and their functional contribution to the viral replication cycle and pathogenesis.
Asunto(s)
Hepacivirus/genética , Interacciones Huésped-Patógeno/genética , Proteómica , Proteínas Virales/biosíntesis , Genómica , Humanos , Espectrometría de Masas , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/metabolismo , Interferencia de ARNRESUMEN
The Epstein-Barr virus (EBV) nuclear antigen 3 family of protein is critical for the EBV-induced primary B-cell growth transformation process. Using a yeast two-hybrid screen we identified 22 novel cellular partners of the EBNA3s. Most importantly, among the newly identified partners, five are known to play direct and important roles in transcriptional regulation. Of these, the Myc-interacting zinc finger protein-1 (MIZ-1) is a transcription factor initially characterized as a binding partner of MYC. MIZ-1 activates the transcription of a number of target genes including the cell cycle inhibitor CDKN2B. Focusing on the EBNA3A/MIZ-1 interaction we demonstrate that binding occurs in EBV-infected cells expressing both proteins at endogenous physiological levels and that in the presence of EBNA3A, a significant fraction of MIZ-1 translocates from the cytoplasm to the nucleus. Moreover, we show that a trimeric complex composed of a MIZ-1 recognition DNA element, MIZ-1 and EBNA3A can be formed, and that interaction of MIZ-1 with nucleophosmin (NPM), one of its coactivator, is prevented by EBNA3A. Finally, we show that, in the presence of EBNA3A, expression of the MIZ-1 target gene, CDKN2B, is downregulated and repressive H3K27 marks are established on its promoter region suggesting that EBNA3A directly counteracts the growth inhibitory action of MIZ-1.
Asunto(s)
Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Regulación de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética , Oxidorreductasas de Alcohol/metabolismo , Núcleo Celular/metabolismo , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/biosíntesis , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Antígenos Nucleares del Virus de Epstein-Barr/química , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/química , Proteínas Nucleares/metabolismo , Nucleofosmina , Regiones Promotoras Genéticas , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Represoras/químicaRESUMEN
BACKGROUND & AIMS: Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a multifunctional protein playing a crucial role in diverse steps of the viral replication cycle and perturbing multiple host cell pathways. We showed previously that removal of a region in domain 2 (D2) of NS5A (mutant NS5A(D2Δ)) is dispensable for viral replication in hepatoma cell lines. By using a mouse model and immune-competent cell systems, we studied the role of D2 in controlling the innate immune response. METHODS: In vivo replication competence of NS5A(D2Δ) was studied in transgenic mice with human liver xenografts. Results were validated using primary human hepatocytes (PHHs) and mechanistic analyses were conducted in engineered Huh7 hepatoma cells with reconstituted innate signaling pathways. RESULTS: Although the deletion in NS5A removed most of the interferon (IFN) sensitivity determining-region, mutant NS5A(D2Δ) was as sensitive as the wild type to IFN-α and IFN-λ in vitro, but severely attenuated in vivo. This attenuation could be recapitulated in PHHs and was linked to higher activation of the IFN response, concomitant with reduced viral replication and virus production. Importantly, immune-reconstituted Huh7-derived cell lines revealed a sequential activation of the IFN-response via RIG-I (retinoic acid-inducible gene I) and MDA5 (Myeloma differentiation associated factor 5), respectively, that was significantly higher in the case of the mutant lacking most of NS5A D2. CONCLUSIONS: Our study reveals an important role of NS5A D2 for suppression of the IFN response that is activated by HCV via RIG-I and MDA5 in a sequential manner.
Asunto(s)
ADN Viral/genética , Hepacivirus/genética , Hepatitis C/tratamiento farmacológico , Interferón-alfa/uso terapéutico , Mutación/genética , Proteínas no Estructurales Virales/genética , Animales , Antivirales/uso terapéutico , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Femenino , Genotipo , Hepacivirus/efectos de los fármacos , Hepatitis C/patología , Hepatitis C/virología , Hepatocitos , Humanos , Masculino , Ratones , Ratones Transgénicos , Proteínas no Estructurales Virales/metabolismoRESUMEN
UNLABELLED: The study of cellular central carbon metabolism modulations induced by viruses is an emerging field. Human cytomegalovirus (HCMV), herpes simplex virus (HSV), Kaposi's sarcoma-associated herpesvirus (KSHV), and hepatitis C virus (HCV) have been shown recently to reprogram cell metabolism to support their replication. During HCV infection the global glucidolipidic metabolism of hepatocytes is highly impacted. It was suggested that HCV might modify glucose uptake and glycolysis to increase fatty acids synthesis, but underlying mechanisms have not been completely elucidated. We thus investigated how HCV may modulate glycolysis. We observed that in infected Huh7.5 cells and in subgenomic replicon-positive Huh9.13 cells, glucose consumption as well as lactate secretion was increased. Using protein complementation assays and coimmunoprecipitation, we identified a direct interaction between the HCV NS5A protein and cellular hexokinase 2 (HK2), the first rate-limiting enzyme of glycolysis. NS5A expression was sufficient to enhance glucose consumption and lactate secretion in Huh7.5 cells. Moreover, determination of HK activity in cell homogenates revealed that addition of exogenous NS5A protein, either the full-length protein or its D2 or D3, but not D1, domain, was sufficient to increase enzyme activity. Finally, determination of recombinant HK2 catalytic parameters (V(max) and K(m)) in the presence of NS5A identified this viral protein as an activator of the enzyme. In summary, this study describes a direct interaction between HCV NS5A protein and cellular HK2 which is accompanied by an increase in HK2 activity that might contribute to an increased glycolysis rate during HCV infection. IMPORTANCE: Substantial evidence indicates that viruses reprogram the central carbon metabolism of the cell to support their replication. Nevertheless, precise underlying mechanisms are poorly described. Metabolic pathways are structured as connected enzymatic cascades providing elemental biomolecular blocks necessary for cell life and viral replication. In this study, we observed an increase in glucose consumption and lactate secretion in HCV-infected cells, revealing higher glycolytic activity. We also identified an interaction between the HCV NS5A nonstructural protein and cellular hexokinase 2, the first rate-limiting enzyme of glycolysis. This interaction results in an enhancement of catalytic parameters of the enzyme, which might explain, at least in part, the aerobic glycolysis shift observed in HCV-infected cells.
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Hepacivirus/enzimología , Hepatitis C/enzimología , Hexoquinasa/metabolismo , Regulación hacia Arriba , Proteínas no Estructurales Virales/metabolismo , Línea Celular , Glucosa/metabolismo , Glucólisis , Hepacivirus/genética , Hepatitis C/genética , Hepatitis C/metabolismo , Hepatitis C/virología , Hexoquinasa/genética , Humanos , Unión Proteica , Proteínas no Estructurales Virales/genéticaRESUMEN
Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.
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Adenosina Desaminasa/metabolismo , Interacciones Huésped-Patógeno , Virus de la Influenza A/fisiología , Proteínas no Estructurales Virales/metabolismo , Factores de Virulencia/metabolismo , Replicación Viral , Adenosina Desaminasa/química , Adenosina Desaminasa/genética , Transporte Biológico , Línea Celular , Virus del Dengue/enzimología , Humanos , Subtipo H1N1 del Virus de la Influenza A/fisiología , Gripe Humana/metabolismo , Gripe Humana/patología , Gripe Humana/virología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes/metabolismo , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Mucosa Respiratoria/virología , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Especificidad de la Especie , Técnicas del Sistema de Dos Híbridos , Proteínas no Estructurales Virales/genética , Factores de Virulencia/genéticaRESUMEN
Hepatitis B virus (HBV) genome transcription is highly dependent on liver-enriched, metabolic nuclear receptors (NRs). Among others, NR farnesoid X receptor α (FXRα) enhances HBV core promoter activity and pregenomic RNA synthesis. Interestingly, two food-withdrawal-induced FXRα modulators, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and deacetylase SIRT1, have been found to be associated with HBV genomes ex vivo. Whereas PGC-1α induction was shown to increase HBV replication, the effect of SIRT1 on HBV transcription remains unknown. Here, we showed that, in hepatocarcinoma-derived Huh-7 cells, combined activation of FXRα by GW4064 and SIRT1 by activator 3 increased HBV core promoter-controlled luciferase expression by 25-fold, compared with a 10-fold increase with GW4064 alone. Using cell lines differentially expressing FXRα in overexpression and silencing experiments, we demonstrated that SIRT1 activated the core promoter in an FXRα- and PGC-1α-dependent manner. Maximal activation (>150-fold) was observed in FXRα- and PGC-1α-overexpressing Huh-7 cells treated with FXRα and SIRT1 activators. Similarly, in cells transfected with full-length HBV genomes, maximal induction (3.5-fold) of core promoter-controlled synthesis of 3.5-kb RNA was observed in the same conditions of transfection and treatments. Thus, we identified a subnetwork of metabolic factors regulating HBV replication, strengthening the hypothesis that transcription of HBV and metabolic genes is similarly controlled.