Lights, Camera, Actin: Divergent roles of β- and γ- cytoplasmic actin in vaccinia virus infection NOORUL BISHARA MARZOOK A thesis submitted in fulfillment of requirements for the degree of Doctor of Philosophy FACULTY OF SCIENCE SCHOOL OF MOLECULAR BIOSCIENCE UNIVERSITY OF SYDNEY 2017 i TABLE OF CONTENTS Table of Contents ........................................................................................................... ii Acknowledgements ....................................................................................................... v Declaration ................................................................................................................... vii Abstract ....................................................................................................................... viii List of Figures ................................................................................................................ x List of Publications Arising From This Work.............................................................. xi Abbreviations Used ..................................................................................................... xii Chapter 1: Introduction ............................................................................................... 1 1.1 The Cytoskeleton ............................................................................................................ 2 1.1.1 The Eukaryotic Cytoskeleton ...................................................................................... 3 1.1.1.1 The Actin Cytoskeleton......................................................................................................... 5 1.1.1.2 Actin Dynamics ..................................................................................................................... 5 1.2 Host-Pathogen Interactions At The Cytoskeleton ......................................................... 9 1.2.1 Knocking On Actin’s Door – Cell Entry ...................................................................... 11 1.2.1.1 Virus-cell surfing ................................................................................................................. 11 1.2.1.2 Clathrin-mediated entry ...................................................................................................... 11 1.2.1.3 Macropinocytosis ................................................................................................................ 13 1.2.2 Viral Revolution – Seizing the Means of Cellular Transportation ............................... 15 1.2.2.1 Intracellular transport .......................................................................................................... 15 1.2.2.2 Intracellular replication........................................................................................................ 16 1.2.2.3 Post-replicative transport and assembly ............................................................................ 17 1.2.3 Pathogen Exit ........................................................................................................... 18 1.2.4 Pathogens Are Doing It For Themselves – Hijacking Actin-Based Motility ................ 19 1.3 Poxviruses ..................................................................................................................... 24 1.3.1 Vaccinia Virus and its Life Cycle ............................................................................... 27 1.3.1.1 Vaccinia virus and the actin cytoskeleton........................................................................... 30 1.4 Project Aims .................................................................................................................. 37 Chapter 2: Materials and Methods ........................................................................... 38 2.1 Building blocks .............................................................................................................. 39 2.1.1 Reagents .................................................................................................................. 39 2.1.2 Cell lines ................................................................................................................... 40 2.1.3 Viruses ..................................................................................................................... 41 2.1.4 Buffers and solutions ................................................................................................ 42 2.1.5 Primary antibodies used for immunoblots ................................................................. 43 2.1.6 Secondary antibodies used for immunoblots ............................................................ 43 2.1.7 Reagents for immunofluorescent staining ................................................................. 44 2.1.8 Primers ..................................................................................................................... 45 2.1.9 Vector constructs made and/or used ........................................................................ 46 2.2 Fantastic viruses and how we use them ...................................................................... 48 2.2.1 Viral infection ............................................................................................................ 48 2.2.2 Transfection .............................................................................................................. 48 2.2.3 Plaque assays .......................................................................................................... 48 2.2.3.1 Plaque picking for virus purification ........................................................................ 48 2.2.3.2 Plaque visualisation ............................................................................................... 48 2.2.3.3 Plaque size measurement ..................................................................................... 49 2.2.4 EEV release assays ................................................................................................. 49 2.2.5 Virus DNA extraction ................................................................................................ 49 2.3 Under the Microscope ................................................................................................... 51 2.3.1 Immunofluorescence assays .................................................................................... 51 2.3.2 Image acquisition ...................................................................................................... 51 2.3.2.1 Wide-field microscopy ............................................................................................ 51 2.3.2.2 Confocal microscopy ............................................................................................. 51 ii 2.3.2.3 Live-cell wide-field microscopy .............................................................................. 52 2.3.3 Image analysis ......................................................................................................... 52 2.3.3.1 Actin tail measurements ........................................................................................ 52 2.3.3.2 Virus particles at the cell surface ........................................................................... 52 2.3.3.3 Measuring virus speed ........................................................................................... 52 2.4 DNA ................................................................................................................................ 53 2.4.1 Polymerase chain reaction (PCR) and cloning .......................................................... 53 2.4.2 Plasmid vector construction ...................................................................................... 53 2.5 Proteins .......................................................................................................................... 55 2.5.1 Bacterial expression of proteins ................................................................................ 55 2.5.2 Protein purification using GST-pull-down assays ...................................................... 55 2.5.3 SDS-PAGE gel electrophoresis ................................................................................ 55 2.5.4 Immunoblot assays for proteins of interest ................................................................ 56 2.6 The Silent Treatment ..................................................................................................... 57 2.6.1 siRNA ....................................................................................................................... 57 2.6.2 siRNA protocol ......................................................................................................... 57 Chapter 3: Developing an optimised VACV gene-tagging method ....................... 58 3.1 Introduction ................................................................................................................... 59 3.1.1 Fluorescent Markers: The Highlights ........................................................................ 61 3.1.2 Fluorescent Labelling Goes Viral: Applications for Virology ...................................... 65 3.1.3 Creating Recombinant VACV ................................................................................... 66 3.1.4 Dominant Selection and Fluorescent Markers – With Their Powers Combined ......... 68 3.1.5 VACV Genes Of Interest ..........................................................................................