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Impact of SR-BI and CD81 on Hepatitis C Virus Entry and Evasion Muhammad Nauman Zahid

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Impact of SR-BI and CD81 on Hepatitis C Virus Entry and Evasion Muhammad Nauman Zahid Impact of SR-BI and CD81 on Hepatitis C virus entry and evasion Muhammad Nauman Zahid To cite this version: Muhammad Nauman Zahid. Impact of SR-BI and CD81 on Hepatitis C virus entry and evasion. Biochemistry, Molecular Biology. Université de Strasbourg, 2012. English. NNT : 2012STRAJ014. tel-00818590 HAL Id: tel-00818590 https://tel.archives-ouvertes.fr/tel-00818590 Submitted on 28 Apr 2013 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. UNIVERSITÉ DE STRASBOURG ÉCOLE DOCTORALE _Sciences de la vie (ED414) _____________ [ Institute de virologie, Inserm U748 ] THÈSE présentée par : [ MUHAMMAD NAUMAN ZAHID ] soutenue le : 27 Avril 2012 pour obtenir le grade de : Docteur de l’université de Strasbourg Discipline/ Spécialité : Aspects Moléculaire et Cellulaire de la Biologie Impact of SR-BI and CD81 on Hepatitis C virus entry and evasion THÈSE dirigée par : [Baumert Thomas] Directeur de Recherche, Inserm U748, Université de Strasbourg RAPPORTEURS : [Boulanger Nathalie] MC, MD, PHD, Université de Strasbourg AUTRES MEMBRES DU JURY : [Hirsch Ivan] Directeur de Recherche, Inserm U1068, Marseille [Durantel David] Chargé de Recherche, Inserm U1052, Lyon Résumé Le virus de l’hépatite C (VHC) est l’une des causes majeures de cirrhose du foie et de carcinome hépatocellulaire. Il n’existe à ce jour pas de vaccin et les options thérapeutiques actuelles sont limitées par la résistance, la toxicité et le coût élevé du traitement. L’entrée du VHC dans les hépatocytes est un processus complexe impliquant les glycoprotéines de l’enveloppe virale E1 et E2, de même que de nombreux autres facteurs de l’hôte comme le récepteur scavenger de type BI (SR-BI), le CD81, la claudine 1, l’occludine et les récepteurs à activité tyrosine kinase tels que le récepteur du facteur de croissance épidermique (EGFR) et l’ephrine A2 (EphA2). Au courant de la première partie de ma thèse, nous nous sommes intéressés à caractériser plus en détail le rôle de SR-BI dans l’infection par le VHC. Le SR-BI humain est impliqué dans la capture sélective des esters de cholestérol HDL et le transport bidirectionnel du cholestérol libre à la membrane. Il a été démontré que SR-BI joue un rôle dans l’infection par le VHC lors de la liaison du virus à la cellule hôte et lors d’étapes suivant la liaison. Bien que les mécanismes impliquant SR-BI dans la liaison du virus à l’hépatocyte aient été partiellement caractérisés, le rôle de SR-BI dans les étapes suivant la liaison du VHC reste encore largement méconnu. Afin de mieux caractériser le rôle de l’interaction VHC/SR-BI dans l’infection par le VHC, notre laboratoire à généré une nouvelle classe d’anticorps monoclonaux anti-SR-BI inhibant l’infection virale. Nous avons pu démontrer que SR-BI humain jouait un rôle dans le processus d’entrée du virus à la fois lors de l’étape de liaison du virus à la cellule hôte mais aussi au cours d’étapes suivant cette liaison. Nos données indiquent que la fonction de SR-BI impliquée dans les processus suivant l’attachement du virus aux hépatocytes peut être dissociée de sa fonction de liaison du virus aux hépatocytes. Par ailleurs, nous avons démontré que cette fonction de SR-BI est également importante pour l’initiation et la dissémination du VHC. Ainsi il serait intéressant de cibler cette fonction de SR-BI dans le cadre d’une stratégie antivirale pour lutter contre l’infection par le VHC. Dans la seconde partie de ma thèse, nous avions pour but de caractériser les mécanismes moléculaires intervenant dans la réinfection du greffon lors de la transplantation hépatique (TH). En effet, la réinfection systématique du greffon hépatique est la limitation majeure de la TH. Il a été montré précédemment au sein de notre laboratoire que l’entrée virale et l’échappement aux anticorps neutralisants jouent un rôle déterminant dans la sélection des variants du VHC lors des phases précoces de TH. Cependant, les mécanismes moléculaires par lesquels le virus échappe à la réponse immunitaire de l’hôte ne sont toujours pas élucidés. Nous avons ainsi identifiés 3 mutations adaptatives dans la glycoprotéine d’enveloppe E2 responsables de l’entrée virale augmentée du variant hautement infectieux. Ces mutations influent sur la dépendance au récepteur CD81 du VHC résultant en une entrée virale accrue. Cette étude nous a permis d’identifier un nouveau mécanisme moléculaire de l’échappement viral dans lequel on observe une association entre l’utilisation des facteurs d’entrée par le VHC et l’échappement viral. L’identification de ces mécanismes va nous permettre une meilleure compréhension de la pathogénèse de l’infection par le VHC, et est un premier pas pour le développement d’une stratégie préventive antivirale ou vaccinale. De plus les anticorps anti-SR-BI développés au sein de notre laboratoire, compte tenu des mécanismes d'action novateur et du profil de toxicité potentiellement différent, représentent une nouvelle classe d'anticorps anti-SR-BI qui pourrait être utilisée comme antiviraux dans la prévention de l'infection par le VHC lors de la transplantation hépatique et / ou dans le traitement de l’infection chronique par le VHC. Abstract Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. Preventive modalities are absent and the current antiviral treatment is limited by resistance, toxicity and high costs. HCV entry into hepatocytes is a complex and multistep process involving the viral envelope glycoproteins E1 and E2, as well as several host factors such as SR-BI, CD81, CLDN1, OCLN, RTKs and NPC1L1. In the first part of my PhD, we aimed to further characterize the role of scavenger receptor class B type I (SR-BI) in HCV infection. Human SR-BI is a glycoprotein involved in the selective uptake of HDL cholesterol ester as well as the bidirectional free cholesterol transport at the cell membrane. SR-BI has been demonstrated to act during binding and post-binding steps of HCV entry. While the SR-BI determinants involved in HCV binding have been partially characterized, the post-binding function of SR-BI remains remained largely unknown. To further explore the role of HCV-SR-BI interaction during HCV infection, we generated a novel class of anti-SR-BI monoclonal antibodies inhibiting HCV infection. We demonstrated that human SR-BI plays a dual role in the HCV entry process during both binding and post-binding steps. Our data indicate that the HCV post- binding function of human SR-BI can be dissociated from its binding function. Moreover, we demonstrated that the post-binding function of SR-BI is most relevant for initiation of HCV infection and viral dissemination. Targeting the post-binding function of SR-BI thus represents an interesting antiviral strategy against HCV infection. In the second part of my PhD, we aimed to characterize the molecular mechanisms underlying HCV re-infection of the graft after liver transplantation (LT). A major limitation of LT is the universal re-infection of the liver graft with accelerated recurrence of liver disease. It had been previously shown in our laboratory that viral entry and escape from host neutralizing responses are important determinants allowing the virus to rapidly infect the liver during the early phase of transplantation. However, the molecular mechanisms by which the virus evades host immunity to persistently re-infect the liver graft are unknown. We identified three adaptive mutations in envelope glycoprotein E2 mediating enhanced entry and evasion of a highly infectious escape variant. These mutations markedly modulated CD81 receptor dependency resulting in enhanced viral entry. We identified a novel and clinically important mechanism of viral evasion, where co-evolution simultaneously occurs between cellular entry factor usage and escape from neutralization. The identification of these mechanisms advances our understanding of the pathogenesis of HCV infection and paves the way for the development of novel antiviral strategies and vaccines. Moreover, given the novel mechanism of action and the potential differential toxicity profile, our anti-SR-BI antibodies represent a novel class of antibodies that may be used as antivirals for prevention of HCV infection, such as during liver transplantation, and/or treatment of HCV infection. - 2 - Acknowledgements These three and half years in Strasbourg have been extremely rich for me. Now that this thesis arrives at its end, I have many people to thank for having made my stay and my work here so lively. It is a pleasure to convey my indebtedness to them in my humble acknowledgment. First and foremost I would like to thank God the most beneficent and merciful. I could never have done this without the faith I have in you, the Almighty. I would like to express my deep and sincere gratitude to my supervisor, Professor Dr. Thomas Baumert, for making it possible for me to work in such a prestigious scientific environment. Thomas has been very supportive, encouraging and kind. I owe a great deal of appreciation for his valuable advice, constructive criticism and extensive discussions around my work which will not only nourish my intellectual maturity but also be helpful in my future perspectives. I feel immense pleasure to gratefully acknowledge and to express deep sense of gratitude to my supervising guide and mentor, Dr.
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