RESUMEN
Opioid substitution and syringes exchange programs have drastically reduced hepatitis C virus (HCV) spread in France but HCV sexual transmission in men having sex with men (MSM) has recently arisen as a significant public health concern. The fact that the virus is transmitting in a heterogeneous population, with different transmission routes, makes prevalence and incidence rates poorly informative. However, additional insights can be gained by analyzing virus phylogenies inferred from dated genetic sequence data. By combining a phylodynamics approach based on Approximate Bayesian Computation (ABC) and an original transmission model, we estimate key epidemiological parameters of an ongoing HCV epidemic among MSMs in Lyon (France). We show that this new epidemic is largely independent of the previously observed non-MSM HCV epidemics and that its doubling time is ten times lower (0.44 years versus 4.37 years). These results have practical implications for HCV control and illustrate the additional information provided by virus genomics in public health.
Asunto(s)
Hepacivirus/genética , Hepatitis C/epidemiología , Hepatitis C/transmisión , Epidemias , Femenino , Francia/epidemiología , Homosexualidad Masculina , Humanos , Masculino , Filogenia , Conducta SexualRESUMEN
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.
Asunto(s)
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.
Asunto(s)
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
BACKGROUND: Sexually transmitted acute hepatitis C virus (HCV) infections (AHIs) have been mainly described in human immunodeficiency virus (HIV)-infected men who have sex with men (MSM). Cases in HIV-negative MSM are scarce. We describe the epidemic of AHI in HIV-infected and HIV-negative MSM in Lyon, France. METHODS: All cases of AHI diagnosed in MSM in Lyon University Hospital from 2014 to 2017 were included. AHI incidence was determined in HIV-infected and in preexposure prophylaxis (PrEP)-using MSM. Transmission clusters were identified by construction of phylogenetic trees based on HCV NS5B (genotype 1a/4d) or NS5A (genotype 3a) Sanger sequencing. RESULTS: From 2014 to 2017, 108 AHIs (80 first infections, 28 reinfections) were reported in 96 MSM (HIV-infected, 72; HIV-negative, 24). AHI incidence rose from 1.1/100 person-years (95 confidence interval [CI], 0.7-1.7) in 2014 to 2.4/100 person-years (95 CI, 1.1-2.6) in 2017 in HIV-infected MSM (P = .05) and from 0.3/100 person-years (95 CI, 0.06-1.0) in 2016 to 3.4/100 person-years (95 CI, 2.0-5.5) in 2017 in PrEP users (P < .001). Eleven clusters were identified. All clusters included HIV-infected MSM; 6 also included HIV-negative MSM. All clusters started with ≥1 HIV-infected MSM. Risk factor distribution varied among clusters. CONCLUSIONS: AHI incidence increased in both HIV-infected and HIV-negative MSM. Cluster analysis suggests initial transmission from HIV-infected to HIV-negative MSM through chemsex and traumatic sexual practices, leading to mixed patterns of transmission regardless of HIV status and no overlap with the general population.
Asunto(s)
Coinfección/epidemiología , Infecciones por VIH/epidemiología , Hepatitis C/epidemiología , Hepatitis C/transmisión , Homosexualidad Masculina , Adulto , Recuento de Linfocito CD4 , Genotipo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Seropositividad para VIH , Hepacivirus/clasificación , Hepacivirus/genética , Hepatitis C/tratamiento farmacológico , Hepatitis C/virología , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Filogenia , Vigilancia en Salud Pública , Factores de Riesgo , Conducta Sexual , Carga ViralRESUMEN
Acute hepatitis E virus infection during pregnancy has a high fatality rate in developing countries. Little data are available on chronic infection in pregnant women. We report a case of chronic hepatitis E during treatment with infliximab and azathioprine, without adverse event during pregnancy and with spontaneous resolution after delivery.
Asunto(s)
Azatioprina/uso terapéutico , Virus de la Hepatitis E/genética , Hepatitis E/virología , Infliximab/uso terapéutico , Adulto , Colitis Ulcerosa/tratamiento farmacológico , Femenino , Genotipo , Humanos , Embarazo , Resultado del Embarazo , ARN Viral/sangre , Carga ViralRESUMEN
BACKGROUND: Hepatitis C virus (HCV) infection is a recognised cause of secondary immune thrombocytopenia (ITP). While its incidence has been largely described during chronic HCV infection, only one case of ITP secondary to acute HCV infection has been reported at this time. CASE PRESENTATION: We report herein the case of severe ITP secondary to an acute HCV genotype 1a reinfection in a human immunodeficiency virus (HIV)-negative man having sex with men who had been cured several years before of a previous acute genotype 4d HCV infection. After an unsuccessful standard therapy with two courses of intravenous immunoglobulin (at 1 g/kg daily for 2 days) associated with methylprednisolone 1 mg/kg daily, antiviral treatment with sofosbuvir-ledipasvir rapidly achieved virological response and normalised the platelet count. CONCLUSIONS: As a direct effect of HCV on megakaryocytes could be the predominant cause of ITP during acute infection, early antiviral treatment may be beneficial in this case.
Asunto(s)
Bencimidazoles/administración & dosificación , Intervención Médica Temprana , Fluorenos/administración & dosificación , Hepatitis C/tratamiento farmacológico , Púrpura Trombocitopénica Idiopática/tratamiento farmacológico , Uridina Monofosfato/análogos & derivados , Enfermedad Aguda , Antivirales/uso terapéutico , Hepacivirus/aislamiento & purificación , Hepatitis C/complicaciones , Humanos , Masculino , Persona de Mediana Edad , Púrpura Trombocitopénica Idiopática/virología , Índice de Severidad de la Enfermedad , Sofosbuvir , Factores de Tiempo , Uridina Monofosfato/administración & dosificaciónRESUMEN
Sanger population sequencing (SPS) is the reference technique to monitor HIV-1-infected patients' therapy. Ultra-deep sequencing (UDS), which allows quantitative detection of drug resistance mutations, may be an alternative method. The study aimed to compare reproducibility and predictions of UDS versus SPS in a routine setting. A control containing low-abundance variants was repeatedly tested and clinical plasma samples from 100 patients were prospectively assayed by SPS and UDS using the Roche 454 system. Complete analysis by UDS was available for 88% of samples with various viral loads and subtypes. Comparison of detection thresholds found that SPS sensitivity was variable. Variations found by UDS between 5% to >20% were detected by SPS in 25% to more than 80% of samples. At the 5% cut-off, disagreements were rare and in most cases UDS detected an additional protease secondary mutation, suggesting a possible resistance to a protease inhibitor according to the 2015 ANRS algorithm. Mutations found on reverse transcriptase by only UDS were often explained by previous therapy. UDS with a variant detection threshold at 5% might allow therapy management with minimal differences compared to population sequencing while providing additional information for further determination of pertinent cutoff values for specific resistance mutations.
Asunto(s)
Fármacos Anti-VIH/farmacología , Farmacorresistencia Viral/genética , VIH-1/efectos de los fármacos , VIH-1/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutación , Fármacos Anti-VIH/uso terapéutico , Femenino , Genotipo , Técnicas de Genotipaje , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Secuenciación de Nucleótidos de Alto Rendimiento/instrumentación , Humanos , Masculino , Reproducibilidad de los Resultados , Programas Informáticos , Carga Viral/efectos de los fármacosRESUMEN
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.
Asunto(s)
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
Since 2016, an increase in the number of hepatitis A cases affecting mainly men who have sex with men (MSM) has been reported in low endemic countries in Europe. We calculated the attack rate in Lyon, France, in populations considered at high-risk: HIV-infected MSM and HIV-negative MSM receiving HIV pre-exposure prophylaxis (PrEP). In these populations, high level of immunity did not prevent the outbreak, indicating that vaccination should be reinforced, particularly in younger individuals.
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Fármacos Anti-VIH/administración & dosificación , Transmisión de Enfermedad Infecciosa/prevención & control , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/prevención & control , Hepatitis A/epidemiología , Homosexualidad Masculina , Profilaxis Pre-Exposición , Adulto , Fármacos Anti-VIH/uso terapéutico , Brotes de Enfermedades , Francia/epidemiología , Infecciones por VIH/epidemiología , Infecciones por VIH/transmisión , Hepatitis A/diagnóstico , Humanos , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND & AIMS: Ribavirin monotherapy is the preferred treatment for chronic hepatitis E, although occasional treatment failure occurs. We present a patient with chronic hepatitis E experiencing ribavirin treatment failure with a completely resistant phenotype. We aimed to identify viral mutations associated with treatment failure and explore the underlying mechanisms. METHODS: Viral genomes were deep-sequenced at different time points and the role of identified mutations was assessed in vitro using mutant replicons, antiviral assays, cell culture of patient-derived virus and deep-sequencing. RESULTS: Ribavirin resistance was associated with Y1320H, K1383N and G1634R mutations in the viral polymerase, but also an insertion in the hypervariable region comprising a duplication and a polymerase-derived fragment. Analysis of these genome alterations in vitro revealed replication-increasing roles for Y1320H and G1634R mutations and the hypervariable region insertion. In contrast, the K1383N mutation in the polymerase F1-motif suppressed viral replication and increased the in vitro sensitivity to ribavirin, contrary to the clinical phenotype. Analysis of the replication of mutant full-length virus and in vitro culturing of patient-derived virus confirmed that sensitivity to ribavirin was retained. Finally, deep-sequencing of hepatitis E virus genomes revealed that ribavirin is mutagenic to viral replication in vitro and in vivo. CONCLUSIONS: Mutations Y1320H, G1634R and the hypervariable region insertion compensated for K1383N-associated replication defects. The specific role of the K1383N mutation remains enigmatic, but it appears to be of importance for the ribavirin resistant phenotype in this patient. LAY SUMMARY: Ribavirin is the most common treatment for chronic hepatitis E and is mostly effective, although some cases of ribavirin treatment failure have been described. Here, we report on a particular case of ribavirin resistance and investigate the underlying causes of treatment failure. Mutations in the viral polymerase, an essential enzyme for viral replication, appear to be responsible.
Asunto(s)
Virus de la Hepatitis E , Antivirales , Farmacorresistencia Viral , Humanos , Mutación , Ribavirina , Insuficiencia del Tratamiento , Replicación ViralRESUMEN
The pretherapeutic presence of protease inhibitor (PI) resistance-associated variants (RAVs) has not been shown to be predictive of triple-therapy outcomes in treatment-naive patients. However, they may influence the outcome in patients with less effective pegylated interferon (pegIFN)-ribavirin (RBV) backbones. Using hepatitis C virus (HCV) population sequence analysis, we retrospectively investigated the prevalence of baseline nonstructural 3 (NS3) RAVs in a multicenter cohort of poor IFN-RBV responders (i.e., prior null responders or patients with a viral load decrease of <1 log IU/ml during the pegIFN-RBV lead-in phase). The impact of the presence of these RAVs on the outcome of triple therapy was studied. Among 282 patients, the prevalances (95% confidence intervals) of baseline RAVs ranged from 5.7% (3.3% to 9.0%) to 22.0% (17.3% to 27.3%), depending to the algorithm used. Among mutations conferring a >3-fold shift in 50% inhibitory concentration (IC50) for telaprevir or boceprevir, T54S was the most frequently detected mutation (3.9%), followed by A156T, R155K (0.7%), V36M, and V55A (0.35%). Mutations were more frequently found in patients infected with genotype 1a (7.5 to 23.6%) than 1b (3.3 to 19.8%) (P = 0.03). No other sociodemographic or viroclinical characteristic was significantly associated with a higher prevalence of RAVs. No obvious effect of baseline RAVs on viral load was observed. In this cohort of poor responders to IFN-RBV, no link was found with a sustained virological response to triple therapy, regardless of the algorithm used for the detection of mutations. Based on a cross-study comparison, baseline RAVs are not more frequent in poor IFN-RBV responders than in treatment-naive patients and, even in these difficult-to-treat patients, this study demonstrates no impact on treatment outcome, arguing against resistance analysis prior to treatment.
Asunto(s)
Antivirales/farmacología , Farmacorresistencia Viral , Hepacivirus/efectos de los fármacos , Hepatitis C Crónica/virología , Inhibidores de Proteasas/farmacología , Adulto , Anciano , Antivirales/uso terapéutico , Femenino , Hepacivirus/genética , Hepacivirus/aislamiento & purificación , Hepatitis C Crónica/tratamiento farmacológico , Humanos , Concentración 50 Inhibidora , Interferón-alfa/uso terapéutico , Masculino , Persona de Mediana Edad , Mutación Missense , Oligopéptidos/farmacología , Oligopéptidos/uso terapéutico , Prolina/análogos & derivados , Prolina/farmacología , Prolina/uso terapéutico , Inhibidores de Proteasas/uso terapéutico , Estudios Retrospectivos , Ribavirina/uso terapéutico , Resultado del Tratamiento , Proteínas no Estructurales Virales/genéticaRESUMEN
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
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.
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Virus de la Hepatitis B/genética , Receptores Citoplasmáticos y Nucleares/fisiología , Sirtuina 1/fisiología , Factores de Transcripción/fisiología , Transcripción Genética/fisiología , Secuencia de Bases , Northern Blotting , Western Blotting , Línea Celular Tumoral , Cartilla de ADN , Humanos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , ARN Interferente PequeñoRESUMEN
Chronic hepatitis C (CHC) is a major burden for public health worldwide. Although newer direct-acting antivirals show good efficacy, their cost precludes their wide adoption in resource-limited regions. Thus, strategies are being developed to help identify patients with high susceptibility to response to classic PEG-interferon + ribavirin therapy. IL28B polymorphism rs12979860 C/T is an important predictor for an efficient response to interferon-based therapy. A genetic variant in adiponutrin (PNPLA3) gene, rs738409 C/G, is associated with steatosis, severity, and progression of liver fibrosis in CHC patients, and predicts treatment outcome in difficult-to-cure HCV-infected patients with advanced fibrosis. We developed a rapid and inexpensive assay based on duplex high-resolution melting (HRM) for the simultaneous genotyping of these two polymorphisms. The assay validation was performed on synthetic DNA templates and 132 clinical samples from CHC patients. When compared with allele-specific PCR and sequencing, our assay showed 100% (95% CI: 0.9724-1) accuracy, with 100% sensitivity and specificity. Our assay was robust against concentration and quality of DNA samples, melting curve normalization intervals, HRM analysis algorithm, and sequence variations near the targeted SNPs (single nucleotide polymorphism). This duplex assay should provide useful information for patient-oriented management and clinical decision-making in CHC.
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Técnicas de Genotipaje/métodos , Hepatitis C Crónica/genética , Interleucinas/genética , Lipasa/genética , Proteínas de la Membrana/genética , Desnaturalización de Ácido Nucleico , Polimorfismo de Nucleótido Simple , Adulto , Estudios de Casos y Controles , Femenino , Humanos , Interferones , Masculino , Persona de Mediana EdadRESUMEN
Liver disease is a major cause of morbidity and mortality worldwide. As in other fields of medicine, there is a stringent need for non-invasive markers to improve patient diagnostics, monitoring and prognostic ability in liver pathology. Cell-free circulating RNA molecules have been recently acknowledged as an important source of potential medical biomarkers. However, many aspects related to the biology of these molecules remain to be elucidated. In this review, we summarize current concepts related to the origin, transportation and possible functions of cell-free RNA. We outline current development of extracellular RNA-based biomarkers in the main forms of non-inherited liver disease: chronic viral hepatitis, hepatocellular carcinoma, non-alcoholic fatty liver, hepato-toxicity, and liver transplantation. Despite recent technological advances, the lack of standardization in the assessment of these markers makes their adoption into clinical practice difficult. We thus finally review the main factors influencing quantification of circulating RNA. These factors should be considered in the reporting and interpretation of current findings, as well as in the proper planning of future studies, to improve reliability and reproducibility of results.
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Hepatopatías/patología , ARN/sangre , Biomarcadores/sangre , Humanos , Hepatopatías/sangre , Hepatopatías/diagnóstico , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/patología , MicroARNs/sangre , Enfermedad del Hígado Graso no Alcohólico/sangre , Enfermedad del Hígado Graso no Alcohólico/patología , PronósticoRESUMEN
Viruses have developed sophisticated strategies to control metabolic activity of infected cells in order to supply replication machinery with energy and metabolites. Dengue virus (DENV), a mosquito-borne flavivirus responsible for dengue fever, is no exception. Previous reports have documented DENV interactions with metabolic pathways and shown in particular that glycolysis is increased in DENV-infected cells. However, underlying molecular mechanisms are still poorly characterized and dependence of DENV on this pathway has not been investigated in details yet. Here, we identified an interaction between the non-structural protein 3 (NS3) of DENV and glucokinase regulator protein (GCKR), a host protein that inhibits the liver-specific hexokinase GCK. NS3 expression was found to increase glucose consumption and lactate secretion in hepatic cell line expressing GCK. Interestingly, we observed that GCKR interaction with GCK decreases DENV replication, indicating the dependence of DENV to GCK activity and supporting the role of NS3 as an inhibitor of GCKR function. Accordingly, in the same cells, DENV replication both induces and depends on glycolysis. By targeting NAD(H) biosynthesis with the antimetabolite 6-Amino-Nicotinamide (6-AN), we decreased cellular glycolytic activity and inhibited DENV replication in hepatic cells. Infection of primary organotypic liver cultures (OLiC) from hamsters was also inhibited by 6-AN. Altogether, our results show that DENV has evolved strategies to control glycolysis in the liver, which could account for hepatic dysfunctions associated to infection. Besides, our findings suggest that lowering intracellular availability of NAD(H) could be a valuable therapeutic strategy to control glycolysis and inhibit DENV replication in the liver.
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Virus del Dengue , Dengue , Glucoquinasa , Glucólisis , NAD , Proteínas no Estructurales Virales , Replicación Viral , Glucólisis/efectos de los fármacos , Virus del Dengue/efectos de los fármacos , Glucoquinasa/metabolismo , Glucoquinasa/antagonistas & inhibidores , Humanos , Replicación Viral/efectos de los fármacos , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Animales , Dengue/tratamiento farmacológico , Dengue/virología , Dengue/metabolismo , NAD/metabolismo , NAD/biosíntesis , Línea Celular , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Glucosa/metabolismo , Hígado/virología , Hígado/metabolismo , Antivirales/farmacología , Proteasas Virales , Serina Endopeptidasas , Nucleósido-Trifosfatasa , ARN Helicasas DEAD-boxRESUMEN
UNLABELLED: Hepatitis C virus (HCV) particles associate viral and lipoprotein moieties to form hybrid lipoviral particles (LVPs). Cell culture-produced HCV (HCVcc) and ex vivo-characterized LVPs primarily differ by their apolipoprotein (apo) B content, which is low for HCVcc, but high for LVPs. Recombinant nucleocapsid-free subviral LVPs are assembled and secreted by apoB-producing cell lines. To determine whether such subviral particles circulate in HCV-infected individuals, LVPs complexed with immunoglobulin were precipitated with protein A from low-density plasma fractions of 36 hepatitis C patients, and their lipid content, apolipoprotein profile, and viral composition were determined. HCV RNA in LVPs was quantified and molar ratios of apoB and HCV genome copy number were calculated. LVPs lipidome from four patients was determined via electrospray ionization/tandem mass spectrometry. Protein A-purified LVPs contained at least the envelope glycoprotein E2 and E2-specific antibodies. LVPs were present in every patient and were characterized by high lipid content, presence of apolipoproteins characteristic of triglyceride-rich lipoproteins (TRLs), HCV RNA, and viral glycoprotein. Importantly, save for four patients, LVPs fractions contained large amounts of apoB, with on average more than 1 × 10(6) apoB molecules per HCV RNA genome. Because there is one apoB molecule per TRL, this ratio suggested that most LVPs are nucleocapsid-free, envelope glycoprotein-containing subviral particles. LVPs and TRLs had similar composition of triacylglycerol and phospholipid classes. CONCLUSION: LVPs are a mixed population of particles, comprising predominantly subviral particles that represent a distinct class of modified lipoproteins within the TRL family.
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Apolipoproteínas B/metabolismo , Hepacivirus/metabolismo , Hepatitis C Crónica/sangre , Lipoproteínas HDL/metabolismo , Proteínas de la Nucleocápside/metabolismo , Adulto , Anciano , Western Blotting , Estudios de Cohortes , Femenino , Hepatitis C Crónica/fisiopatología , Humanos , Lipoproteínas IDL/metabolismo , Lipoproteínas LDL/metabolismo , Masculino , Persona de Mediana Edad , Proteínas de la Nucleocápside/análisis , Pronóstico , ARN Viral/análisis , Análisis de Regresión , Sensibilidad y Especificidad , Proteínas del Envoltorio Viral/metabolismo , Carga ViralRESUMEN
Hepatitis viruses modify the cellular metabolism of hepatocytes by interacting with specific enzymes such as glucokinase. The metabolic changes induced by viruses can have a direct impact on the innate antiviral response. The complex interactions between viral components, innate immunity, and hepatocyte metabolism explain why chronic hepatitis infections lead to liver inflammation, progressing to cirrhosis, fibrosis, and hepatocellular carcinoma. Metabolic regulators could be used in innovative therapies to deprive viruses of key metabolites and induce an antiviral defense.
Title: Rôle du métabolisme cellulaire dans le contrôle des hépatites virales chroniques. Abstract: Les virus des hépatites modifient le métabolisme cellulaire des hépatocytes en interagissant avec des enzymes spécifiques, telles que la glucokinase. Les changements métaboliques induits par les virus peuvent avoir un impact direct sur la réponse antivirale innée. Les interactions complexes entre les composants viraux, l'immunité innée et le métabolisme des hépatocytes expliquent pourquoi les infections hépatiques chroniques conduisent à l'inflammation du foie, évoluant vers la cirrhose, la fibrose et le carcinome hépatocellulaire. Des régulateurs du métabolisme pourraient être utilisés dans des thérapies innovantes pour priver les virus de métabolites clés et induire une défense antivirale.
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Carcinoma Hepatocelular , Hepatitis Viral Humana , Neoplasias Hepáticas , Humanos , Hepatitis Crónica , Antivirales/uso terapéuticoRESUMEN
BACKGROUND AND AIMS: HDV, a satellite of HBV, is responsible for the most severe form of human viral hepatitis, for which curative therapy is still awaited. Both HBV and HDV use the hepatic transporter of bile acids (ie, Na+-taurocholate cotransporting polypeptide) to enter hepatocytes. We have previously shown that ligands of the farnesoid-X-receptor alpha (FXR), a master regulator of bile acids metabolism, inhibit HBV replication. Here we asked whether FXR ligands can also control HDV infection. APPROACH AND RESULTS: In vitro HDV monoinfections or HDV/HBV coinfections and superinfections were performed in differentiated HepaRG cells (dHepaRG) and primary human hepatocytes. Following treatment with FXR ligands, HDV RNAs and antigens were analyzed by RT-qPCR, northern blot, immunofluorescence, and western blot. Virus secretion was studied by RNA quantification in supernatants, and the infectivity of secreted HDV particles was measured by reinfection of naive HuH7.5-Na+-taurocholate cotransporting polypeptide cells. In HDV/HBV superinfection models, a 10-day treatment with FXR ligand GW4064 decreased intracellular HDV RNAs by 60% and 40% in dHepaRG cells and primary human hepatocytes, respectively. Both HDV genomic and antigenomic RNAs were affected by treatment, which also reduced the amount of intracellular delta antigen. This antiviral effect was also observed in HDV monoinfected dHepaRG cells, abolished by FXR loss of function, and reproduced with other FXR ligands. In HBV/HDV coinfected dHepaRG cells, HDV secretion was decreased by 60% and virion-specific infectivity by >95%. CONCLUSIONS: FXR ligands both inhibit directly (ie, independently of anti-HBV activity) and indirectly (ie, dependently of anti-HBV activity) the replication, secretion, and infectivity of HDV. The overall anti-HDV activity was superior to that obtained with interferon-α, highlighting the therapeutic potential of FXR ligands in HDV-infected patients.