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Impact of Hypoxia on Hepatitis B Virus Replication

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Impact of Hypoxia on Hepatitis B Virus Replication i IMPACT OF HYPOXIA ON HEPATITIS B VIRUS REPLICATION NICHOLAS ROSS BAKER FRAMPTON A thesis submitted to the University of Birmingham for the degree of Doctor of Philosophy Institute of Immunology and Immunotherapy College of Medical and Dental Sciences University of Birmingham September 2017 ii University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ABSTRACT Hepatitis B virus (HBV) is one of the world’s unconquered diseases, with 370 million chronically infected globally. HBV replicates in hepatocytes within the liver that exist under a range of oxygen tensions from 11% in the peri-portal area to 3% in the peri- central lobules. HBV transgenic mice show a zonal pattern of viral antigen with expression in the peri-central areas supporting a hypothesis that low oxygen regulates HBV replication. We investigated this hypothesis using a recently developed in vitro model system that supports HBV replication. We demonstrated that low oxygen significantly increases covalently closed circular viral DNA (cccDNA), viral promoter activity and pre-genomic RNA (pgRNA) levels, consistent with low oxygen boosting viral transcription. Hypoxia inducible factors (HIFs) regulate cellular responses to low oxygen and we investigated a role for HIF-1α or HIF-2α on viral transcription. A combination of HIF inhibitors and silencing of HIF-1α and HIF-2α ablated the effect of low oxygen on cccDNA and pgRNA, suggesting a role in regulating HBV transcription. This study highlights a new role for hepatic oxygen levels to regulate multiple steps in the HBV life cycle and this may impact on future treatments for viral associated pathologies. iii DEDICATION & ACKNOWLEDGEMENTS Firstly, I’d like to thank Professor Jane McKeating for her supervision throughout my PhD and great help in preparing my thesis, her input was invaluable. I’d also like to thank everyone who has ever helped me in the lab. In particular, Peter Balfe, who taught me how to run and analyse PCRs; and Ke Hu, who helped me learn a great many techniques. I’d like to thank all my colleagues from throughout the PhD, both for the fun times and support in the lab. I’d also like to thank Dan Tennant and members of his lab who helped me during the early stages of my PhD. I’d like to thank Chunkyu Ko, Maarten van de Klundert and the Protzer Laboratory for a productive collaboration and help in developing some techniques. I’d also like to thank the Ratcliffe Laboratory for providing many of the reagents used in this study. Finally, I’d like to thank Jane and Peter for giving me the opportunity to study for this PhD and the European Commission for funding my research. Thank you to my friends and fellow PhD students for supporting me throughout the PhD and providing great memories. Thank you to my parents Helen and Jon for their great advice during my PhD and continued financial, educational and emotional support throughout my life. Lastly, I’d like to thank my wife Rosalind, my constant companion and light of my life. iv TABLE OF CONTENTS Abstract ............................................................................................................. iii Dedication & Acknowledgements ...................................................................... iv Table of Contents ............................................................................................... v List of Figures ................................................................................................... vii List of Tables ...................................................................................................... x List of Common Abbreviations ........................................................................... xi 1. Introduction ................................................................................................ 12 1.1 The Liver as the Sight for Hepatitis B Infection ............................................. 12 1.1.1 Liver Structure ....................................................................................... 12 1.1.2 Liver Differentiation ................................................................................ 16 1.2 Hepatitis B Virus ........................................................................................... 17 1.2.1 HBV Epidemiology and Vaccinology ...................................................... 17 1.2.2 HBV Morphology ................................................................................... 25 1.2.3 HBV Entry and Nuclear Import ............................................................... 30 1.2.4 cccDNA Formation................................................................................. 39 1.2.5 cccDNA Transcription and Translation ................................................... 43 1.2.6 HBV Protein Roles and Functions .......................................................... 55 1.3 Hypoxia Inducible Factors ............................................................................. 68 1.3.1 HIF1α .................................................................................................... 72 1.3.2 HIF2α .................................................................................................... 73 1.3.3 HIF3α .................................................................................................... 74 1.4 HBV and Hypoxia ......................................................................................... 75 1.5 Aims ............................................................................................................. 80 2. Materials and Methods............................................................................... 81 2.1 Tissue Culture .............................................................................................. 81 2.2 Transfections ................................................................................................ 81 2.3 HRE Luciferase Assay .................................................................................. 84 2.4 Generation of HBV ........................................................................................ 84 2.5 De novo Infections ........................................................................................ 85 2.6 HBV E and S antigen ELISAs ....................................................................... 87 2.7 Western Blotting ........................................................................................... 88 2.7.1 Lysate preparation and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) ................................................................. 88 2.7.2 Immuno-blotting and chemiluminescent detection of proteins ................ 88 2.8 Quantitative RT-PCR .................................................................................... 90 2.9 SYBR Green PCR ........................................................................................ 90 2.10 PCR Array ................................................................................................... 95 v 2.11 Immunofluorescent Staining of HBV S Antigen ............................................ 97 2.12 Chromatin Immunoprecipitation (ChIP) ........................................................ 97 2.13 Statistical Analysis ....................................................................................... 98 3. Examining the hepatoma cell response to low oxygen ............................ 100 3.1 Introduction ................................................................................................. 100 3.2 Huh-7.5 cell response to low oxygen .......................................................... 103 3.3 HepG2 cell response to low oxygen ............................................................ 105 3.4 The effect of HIF targeting drugs on hepatoma cell lines ............................ 108 3.5 HIF over expression in hepatoma cells ....................................................... 115 3.6 HIF target gene activation in hepatoma cells .............................................. 117 3.7 Hepatoma cell lines and differentiation ....................................................... 123 3.8 Cellular differentiation status and response to low oxygen .......................... 125 3.9 Cell density, differentiation and HIF responses ........................................... 127 3.10 Discussion .................................................................................................. 132 3.11 Summary .................................................................................................... 134 4. Studying HBV and HIF Stabilisation ........................................................ 135 4.1 Introduction ................................................................................................. 135 4.2 HBV models and HIF expression ................................................................ 139 4.3 HIF stabilisation of HIF is independent of HBx ............................................ 141 4.4
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