Up-regulation of the ATP-binding cassette transporter A1 inhibits hepatitis C virus infection.

International audienceHepatitis C virus (HCV) establishes infection using host lipid metabolism pathways that are thus considered potential targets for indirect anti-HCV strategies. HCV enters the cell via clathrin-dependent endocytosis, interacting with several receptors, and virus-cell fusion, which depends on acidic pH and the integrity of cholesterol-rich domains of the hepatocyte membrane. The ATP-binding Cassette Transporter A1 (ABCA1) mediates cholesterol efflux from hepatocytes to extracellular Apolipoprotein A1 and moves cholesterol within cell membranes. Furthermore, it generates high-density lipoprotein (HDL) particles. HDL protects against arteriosclerosis and cardiovascular disease. We show that the up-regulation of ABCA1 gene expression and its cholesterol efflux function in Huh7.5 hepatoma cells, using the liver X receptor (LXR) agonist GW3965, impairs HCV infection and decreases levels of virus produced. ABCA1-stimulation inhibited HCV cell entry, acting on virus-host cell fusion, but had no impact on virus attachment, replication, or assembly/secretion. It did not affect infectivity or properties of virus particles produced. Silencing of the ABCA1 gene and reduction of the specific cholesterol efflux function counteracted the inhibitory effect of the GW3965 on HCV infection, providing evidence for a key role of ABCA1 in this process. Impaired virus-cell entry correlated with the reorganisation of cholesterol-rich membrane microdomains (lipid rafts). The inhibitory effect could be reversed by an exogenous cholesterol supply, indicating that restriction of HCV infection was induced by changes of cholesterol content/distribution in membrane regions essential for virus-cell fusion. Stimulation of ABCA1 expression by GW3965 inhibited HCV infection of both human primary hepatocytes and isolated human liver slices. This study reveals that pharmacological stimulation of the ABCA1-dependent cholesterol efflux pathway disrupts membrane cholesterol homeostasis, leading to the inhibition of virus-cell fusion and thus HCV cell entry. Therefore besides other beneficial roles, ABCA1 might represent a potential target for HCV therapy

Caracterisation a l'aide d'anticorps humains autoimmuns de deux antigenes apparentes au facteur TFIIIA et d'un antigene nucleaire lie au cycle cellulaire

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Hepatitis C virus: Morphogenesis, infection and therapy

Hepatitis C virus (HCV) is a major cause of liver diseases including liver cirrhosis and hepatocellular carcinoma. Approximately 3% of the world population is infected with HCV. Thus, HCV infection is considered a public healthy challenge. It is worth mentioning, that the HCV prevalence is dependent on the countries with infection rates around 20% in high endemic countries. The review summarizes recent data on HCV molecular biology, the physiopathology of infection (immune-mediated liver damage, liver fibrosis and lipid metabolism), virus diagnostic and treatment. In addition, currently available in vitro, ex vivo and animal models to study the virus life cycle, virus pathogenesis and therapy are described. Understanding of both host and viral factors may in the future lead to creation of new approaches in generation of an efficient therapeutic vaccine

The remarkable history of the hepatitis C virus

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The remarkable history of the hepatitis C virus

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The TZM-bl Reporter Cell Line Expresses Kynureninase That Can Neutralize 2F5-like Antibodies in the HIV-1 Neutralization Assay

International audienceInduction of broadly neutralizing antibodies targeting ectodomain of the transmembrane (TM) glycoprotein gp41 HIV-1 provides a basis for the development of a universal anti-viral vaccine. The HeLa cell-derived TZM-bl reporter cell line is widely used for the estimation of lentiviruses neutralization by immune sera. The cell line is highly permissive to infection by most strains of HIV, SIV, and SHIV. Here we demonstrated that TZM-bl cells express a 48 kDa non-glycosylated protein (p48) recognized by broadly neutralizing monoclonal antibody (mAb) 2F5 targeting the ELDKWA (aa 669–674) epitope of gp41TM of HIV-1. A significant amount of p48 was found in the cell supernatant. The protein was identified as human kynureninase (KYNU), which has the ELDKWA epitope. The protein is further called “p48 KYNU”. The HIV-1 neutralization by mAb 2F5 and 4E10 in the presence of p48KYNU was tested on Jurkat and TZM-bl cells. It was demonstrated that p48KYNU reduces neutralization by 2F5-like antibodies, but it has almost no effect on mAb 4E10. Therefore, p48KYNU can attenuate HIV-1 neutralization by 2F5-like antibodies and hence create false-negative results. Thus, previously tested immune sera that recognized the ELDKWA-epitope and demonstrated a “weak neutralization” of HIV-1 in TZM-bl assay should be reevaluated

Modèles d'infection de l'hépatite C

L'hépatite C (VHC) est l'une des causes principales de maladies du foie dans le monde, qui représentent un risque élevé d'évoluer vers la cirrhose et le carcinome hépatocellulaire. Actuellement, le traitement standard de l infection par le VHC est l'interféron pégylé-(peg-IFN) et la ribavirine. Bien que le taux de la réponse virale soutenue (RVS) au traitement se soit améliorée au cours de ces années, cette thérapie n'est pas efficace chez tous les patients. En outre, plusieurs effets secondaires toxiques, de complications et le coût élevé limitent la compliance du patient et l'efficacité du traitement. Il n'existe pas de modèle simple d'infection par le VHC et il est nécessaire de développer des modèles in vitro et in vivo utiles pour étudier la physiopathologie de l'infection par le VHC, y compris les événements précoces de l'infection aiguë (l'entrée du virus, des mécanismes immunologiques et génétiques prédictifs) ainsi que l'évaluation de la puissance des médicaments antiviraux contre le VHC. Nous rapportons ici, nos efforts visant à développer des modèles appropriés de l'infection par le VHC. Dans un premier temps, nous avons établi un modèle de petit animal pour étudier l'infection par le VHC. Tupaia est un petit animal, apparenté aux primates et peu couteux. Dans notre travail, nous avons étudié la susceptibilité du tupaia à l'infection par VHC. Douze tupaias adultes ont été inoculés avec le VHC provenant de sérum de patient et d'ARN du VHC (génotype 1a). Trois jeunes tupaias ont été artificiellement nourris pendant un mois et ensuite inoculés par le VHC provenant de sérum du patient. L'ARN du VHC, les anticorps anti-VHC et l évolution des quasi-espèces du VHC ont été déterminées chez l'animal avant et après l'inoculation. L'infection transitoire et intermittente s'est produite chez deux des 3 jeunes tupaias et l infection chronique par le VHC s est produite chez quatre tupaias sur 12 tupaias adultes. Le tupaia devrait représenter un modèle utile pour l'étude de l infection chronique par le VHC. Dans une deuxième étape, un système de culture in vitro d'hépatocytes primaires de Tupaia a été établi, dans lequel l'infection par le VHC ne pouvait être bloquée ni par le CD81 soluble ni par des anticorps dirigés contre le CD81. Pour comprendre ces résultats, nous avons cloné, séquencé la grande boucle extracellulaire (LEL) du CD81 chez le Tupaia et analysé l'interaction de la protéine d enveloppe E2 du VHC avec la LEL du CD81 chez le Tupaia par un test enzyme-linked immunosorbent assay (EIA). Nous avons constaté que chez le Tupaia, la séquence d'acides aminés du LEL de CD81 qui se lie au VHC présentait en 6 résidus d'acides aminés différents par rapport à la séquence humaine et la capacité de LEL de CD81 à se lier à la proteine d enveloppe E2 du VHC a également diminuée. La structure différente de CD81 chez l homme et chez le tupaia pourrait expliquer l'altération de l'interaction entre CD81 et la proteine E2 du VHC. Ce résultat démontre un rôle important de LEL du CD81 pour l'entrée du VHC. Dans une troisième étape, nous avons développé un modèle ex vivo de culture de tranches de foie humain et leur infection par le VHC. Le développement de lignées cellulaires provenant d hepatocarcinome, permissives à la réplication du VHC, a fourni d'importants nouveaux outils virologiques pour étudier les mécanismes de l'infection par le VHC, mais ce modèle expérimental reste relativement éloigné des conditions physiologiques et pathologiques. Nous rapportons ici le développement d'un nouveau modèle ex vivo utilisant la culture de tranches de foie humain adulte, démontrant, pour la première fois, la capacité d isolats primaires ainsi que JFH -1, H77/C3, Con1/C3 (HCVcc), de répliquer et de produire de novo des particules virales infectieuses ayant un titre viral élevéHepatitis C virus (HCV) is one of the major causes of liver disease all over the world which has a high risk to progress to cirrhosis and hepatocellular carcinoma. Currently, the licensed standard treatment of HCV infection is Pegylated-interferon (peg-IFN) and ribavirin. Although the sustained viral response (SVR) rate of treatment has improved during these years, this therapy is not effective in all patients. In addition, several toxic side effects, complication and high cost limit the patient compliance and the efficacy of the treatment. There is no easy model of HCV infection and it is necessary to develop useful in vitro and in vivo models to study the pathobiology of HCV infection, including early events of acute infection (viral entry, immunological mechanisms, and genetic predictors) as well as the evaluation of the potency of the HCV antiviral drugs. We report here in our efforts in developing suitable models of HCV infection. In a first step, we preliminary established a small animal model to study HCV infection. Tupaia is a small, closed related to primate and cost-effective animal. In our work, we investigated the susceptibly of tupaia to HCV infection. Twelve adult tupaias were inoculated with native HCV from patient serum and full-length HCV RNA (Genotype 1a). Three young tupaias were artificially breeded for a month and then inoculated by native HCV from patient serum. HCV RNA, anti-HCV and HCV quasi species evolution were determined in the animal before and after inoculation. Transient and intermittent infection occurred in two among 3 young tupaias and HCV chronic infection occurred in four among 12 adult tupaias. Tupaia should represent a useful model for study HCV chronic infection. In a second step, an in vitro culture system of primary tupaia hepatocytes has been established in which HCV infection could be blocked neither by the soluble CD81 nor by antibodies against CD81. To understand these results, we cloned, sequenced the large extracellular loop (LEL) of tupaia CD81 and analyzed the interaction of HCV E2 with the tupaia CD81 LEL by enzyme-linked immunosorbent assay (EIA). We found that in the tupaia the amino acids sequence of HCV CD81 LEL presented in 6 different amino acid residues compared with human CD81 LEL sequence and the CD81 LEL ability to bind to HCV E2 was also decreased. The different structure of CD81 between human and tupaia could explain the alteration of the interaction between HCV E2 and CD81. This result demonstrated an important role of CD81 LEL for HCV entry. In a third step, we developed an ex vivo model of human liver slices culture and their infection with HCV. The development of human cultured HCV-replication-permissive hepatocarcinoma cell lines has provided important new virological tools to study the mechanisms of HCV infection; however this experimental model remains distantly related to physiological and pathological conditions. Here, we report the development of a new ex vivo model using human adult liver slices culture, demonstrating, for the first time, the ability of primary isolates to undergo de novo viral replication with the production of high titer infectious virus, as well as JFH-1, H77/C3, Con1/C3 (HCVcc). This experimental model was validated by demonstrating the HCV neutralization or HCV inhibition, in a dose-dependent manner, either by CD81 or E2 specific antibodies or convalescent serum from a recovered HCV patient, or by anti-viral drugs. This new ex vivo model represents a powerful tool for studying the viral life cycle, dynamics of virus spread in the liver and also for evaluating the efficacy of the new antiviral drugs. In the last step, we evaluated the efficacy of the new antiviral drugs with our ex vivo model of human adult liver slices. HCV NS3/4A protease is essential for viral replication and has been one of the most important target for developing specific antiviral drugPARIS5-Bibliotheque electronique (751069902) / SudocSudocFranceF

Retinoic Acid: A New Old Friend of IL-17A in the Immune Pathogeny of Liver Fibrosis

International audienceDespite all the medical advances mortality due to cirrhosis and hepatocellular carcinoma, the end stages of fibrosis, continuously increases. Recent data suggest that liver fibrosis is guided by type 3 inflammation with IL-17A at the top of the line. The storage of vitamin A and its active metabolites, as well as genetics, can influence the development and progression of liver fibrosis and inflammation. Retinoic acid (active metabolite of vitamin A) is able to regulate the differentiation of IL-17A + /IL-22-producing cells as well as the expression of profibrotic markers. IL-17A and its pro-fibrotic role in the liver is the most studied, while the interaction and communication between IL-17A, IL-22, and vitamin Aactive metabolites has not been investigated. We aim to update what is known about IL-17A, IL-22, and retinoic acid in the pathobiology of liver diseases

Reply to “Precision-cut human liver slice cultures as an immunological platform”

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