The Novel Interacting Partners of Viperin and Their Role in Establishing a Host Antiviral State Onruedee Khantisitthiporn, M.Sc. Discipline of Molecular and Cellular Biology School of Biological Sciences The University of Adelaide A dissertation submitted to The University of Adelaide in candidature for the degree of Doctor of Philosophy in the Faculty of Sciences October 2017 Table of content LIST OF FIGURES ........................................................................................................ XI LIST OF TABLES ......................................................................................................... XV ABSTRACT .................................................................................................................. XVI DECLARATION.......................................................................................................... XIX ACKNOWLEDGEMENTS .......................................................................................... XX PRESENTATIONS AND PUBLICATIONS ARISING FROM THIS PHD ......... XXI MATERIALS PROVIDERS .................................................................................... XXIII ABBREVIATIONS USED ......................................................................................... XXV CHAPTER 1 ...................................................................................................................... 1 INTRODUCTION............................................................................................................. 1 1.1 Hepatitis C Virus......................................................................................................... 1 1.1.1 Discovery and Epidemiology ................................................................................. 1 1.1.2 Transmission .......................................................................................................... 2 1.1.3 Pathogenesis ........................................................................................................... 2 1.1.4 Treatment ............................................................................................................... 3 1.1.5 Classification and genotypes .................................................................................. 7 1.1.6 HCV genome and proteins ..................................................................................... 7 1.1.7 HCV virion/particles ............................................................................................ 15 1.1.8 HCV life cycle ..................................................................................................... 15 1.1.9 HCV model systems ............................................................................................ 20 1.1.9.1 HCV replicon systems .................................................................................. 21 ii 1.1.9.2 Infectious cell culture model ......................................................................... 23 1.2. Innate immune response to viral infection ............................................................ 24 1.2.1 Overview .............................................................................................................. 24 1.2.2 Innate immune response to RNA virus infection ................................................. 25 1.2.2.1 Cellular recognition of RNA viruses ............................................................ 25 1.2.2.1.1 Retinoic acid-inducible gene I (RIG-I) .................................................. 27 1.2.2.1.2 Melanoma differentiation-associated antigen 5 (MDA5) ...................... 29 1.2.2.1.3 Laboratory of genetics and physiology 2 (LGP2) ................................. 30 1.2.2.2 RIG-I-like receptors (RLRs) downstream signalling cascade ...................... 30 1.2.2.3 The interferon response to viral infection ..................................................... 36 1.2.2.4 Interferon Transduction Pathway .................................................................. 37 1.2.2.5 Interferon-stimulated genes (ISGs) ............................................................... 38 1.2.3 Innate immune recognition of HCV .................................................................... 38 1.2.3.1 Cellular recognition and downstream signalling events upon HCV infection ................................................................................................................................... 38 1.2.3.2 ISGs targeting HCV ...................................................................................... 39 1.3. Viperin ...................................................................................................................... 42 1.3.1 Viperin protein structure and subcellular localisation ......................................... 43 1.3.2 Regulation of viperin expression ......................................................................... 45 1.3.3 Antiviral functions of viperin............................................................................... 46 1.3.3.1 In vitro antiviral function .............................................................................. 47 1.3.3.2 In vivo antiviral function ............................................................................... 49 1.3.4 Evade and co-opt of viperin by viruses................................................................ 50 1.3.5 Roles of viperin in immune signalling ................................................................. 51 1.4 Peroxisomes and the antiviral response .................................................................. 55 1.4.1 Peroxisomes and their functions .......................................................................... 55 1.4.2 Peroxisomes and RLR signalling ......................................................................... 57 1.4.3 Peroxisome evasion by viruses ............................................................................ 60 1.5. Hypothesis and Aims ............................................................................................... 61 iii CHAPTER 2 .................................................................................................................... 62 MATERIALS AND METHODS ................................................................................... 62 2.1 General Molecular Methods .................................................................................... 62 2.1.1 Synthetic oligonucleotides (primers) ................................................................... 62 2.1.2 Bacterial transformation....................................................................................... 62 2.1.3 Small scale plasmid DNA extraction (Mini-preparation) .................................... 63 2.1.4 Large scale plasmid DNA extraction (Maxi-preparation) ................................... 63 2.1.5 Restriction endonuclease digestion ...................................................................... 64 2.1.6 Agarose gel electrophoresis ................................................................................. 65 2.1.7 DNA ligation ........................................................................................................ 65 2.1.8 DNA purification from agarose gel ..................................................................... 65 2.1.9 DNA sequencing .................................................................................................. 66 2.1.10 Total RNA extraction ......................................................................................... 67 2.1.11 Nucleic acid quantification ................................................................................ 67 2.1.12 First-strand cDNA synthesis .............................................................................. 68 2.1.13 Polymerase Chain Reaction (PCR) .................................................................... 68 2.1.14 Real-Time Quantitative PCR (qPCR) ................................................................ 69 2.1.15 Extraction of cellular proteins ............................................................................ 69 2.1.16 SDS-PAGE and protein transfer ........................................................................ 70 2.1.17 Western blotting ................................................................................................. 70 2.1.18 Co-immunoprecipitation .................................................................................... 71 2.1.19 Dual Renilla luciferase assay ............................................................................. 71 2.1.20 Duolink® In situ Proximity Ligation Assay ....................................................... 72 2.2 Yeast Two-Hybrid system ........................................................................................ 73 2.2.1 Competent yeast cell preparation ......................................................................... 74 2.2.2 Transformation ..................................................................................................... 74 2.2.2.1 Small scale transformation ............................................................................ 74 2.2.2.2 Library scale co-transformation .................................................................... 75 2.2.3 Plasmid DNA extraction from yeast cells ............................................................ 75 iv 2.2.4 Construction and testing of the bait (viperin) protein expression plasmid