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The Role of Viral Glycoproteins and Tegument Proteins in Herpes

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The Role of Viral Glycoproteins and Tegument Proteins in Herpes Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2014 The Role of Viral Glycoproteins and Tegument Proteins in Herpes Simplex Virus Type 1 Cytoplasmic Virion Envelopment Dmitry Vladimirovich Chouljenko Louisiana State University and Agricultural and Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Veterinary Pathology and Pathobiology Commons Recommended Citation Chouljenko, Dmitry Vladimirovich, "The Role of Viral Glycoproteins and Tegument Proteins in Herpes Simplex Virus Type 1 Cytoplasmic Virion Envelopment" (2014). LSU Doctoral Dissertations. 4076. https://digitalcommons.lsu.edu/gradschool_dissertations/4076 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. THE ROLE OF VIRAL GLYCOPROTEINS AND TEGUMENT PROTEINS IN HERPES SIMPLEX VIRUS TYPE 1 CYTOPLASMIC VIRION ENVELOPMENT A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Interdepartmental Program in Veterinary Medical Sciences through the Department of Pathobiological Sciences by Dmitry V. Chouljenko B.Sc., Louisiana State University, 2006 August 2014 ACKNOWLEDGMENTS First and foremost, I would like to thank my parents for their unwavering support and for helping to cultivate in me from an early age a curiosity about the natural world that would directly lead to my interest in science. I would like to express my gratitude to all of the current and former members of the Kousoulas laboratory who provided valuable advice and insights during my tenure here, as well as the members of GeneLab for their assistance in DNA sequencing. I would like to thank the faculty members that served on my graduate committee, Dr. Shafiqul I. Chowdhury, Dr. Masami Yoshimura, Dr. Bret D. Elderd and Dr. Inder Sehgal for their guidance. Most of all, I would like to thank my mentor, Dr. Konstantin G. Kousoulas, for the tremendous amount of support, patience and guidance he has provided during my journey to graduation. ii TABLE OF CONTENTS ACKNOWLEDGMENTS ...................................................................................................................... ii LIST OF TABLES ................................................................................................................................ vi LIST OF FIGURES ............................................................................................................................. vii ABSTRACT ........................................................................................................................................ ix CHAPTER I: INTRODUCTION ............................................................................................................ 1 Statement of Problem and Hypothesis ............................................................................... 1 Statement of Research Objectives ...................................................................................... 2 CHAPTER II: LITERATURE REVIEW ................................................................................................... 6 History of Herpes ................................................................................................................ 6 Taxonomy of Herpesviruses .............................................................................................. 10 Herpesviral Disease ........................................................................................................... 17 Herpes simplex viruses (HHV-1 and HHV-2) ......................................................... 18 Varicella-zoster virus (HHV-3) ............................................................................... 23 Cytomegalovirus (HHV-5)...................................................................................... 23 Kaposi’s sarcoma-associated herpesvirus (HHV-8) ............................................... 24 Human herpesvirus 7 (HHV-7) .............................................................................. 24 Human herpesvirus 6 (HHV-6A and HHV-6B) ....................................................... 25 Epstein-Barr virus (HHV-4) .................................................................................... 26 Marek’s disease virus (GaHV-2) ............................................................................ 26 Prevention and Treatment of Herpes Simplex Virus Infection ......................................... 27 Structure of the Herpes Virion .......................................................................................... 31 HSV-1 genome ...................................................................................................... 31 HSV-1 capsid ......................................................................................................... 35 HSV-1 tegument .................................................................................................... 39 UL36 protein ............................................................................................. 44 UL37 protein ............................................................................................. 48 HSV-1 envelope ..................................................................................................... 55 Glycoprotein B .......................................................................................... 57 Glycoprotein K........................................................................................... 58 UL20 protein ............................................................................................. 60 Life Cycle of Herpes Simplex Virus .................................................................................... 62 Virus entry ............................................................................................................. 62 Capsid transport to the nucleus ............................................................................ 65 Viral gene expression and DNA replication .......................................................... 67 Capsid assembly .................................................................................................... 68 Primary envelopment ........................................................................................... 70 iii Secondary envelopment ....................................................................................... 72 Egress to extracellular spaces ............................................................................... 77 HSV-1 egress in neurons: “married” vs. “separate” models ................................ 79 References ........................................................................................................................ 85 CHAPTER III: FUNCTIONAL HIERARCHY OF HERPES SIMPLEX VIRUS 1 VIRAL GLYCOPROTEINS IN CYTOPLASMIC VIRION ENVELOPMENT AND EGRESS ............................................................. 118 Introduction .................................................................................................................... 118 Materials and Methods ................................................................................................... 121 Cells and antibodies ............................................................................................ 121 Construction of HSV-1 mutant viruses ............................................................... 121 Confirmation of the targeted mutations, recovery of infectious virus and marker-rescue experiments ................................................................................ 123 Plaque morphology of mutant viruses and relative plaque area measurements .................................................................................................... 123 One-step viral growth kinetics ............................................................................ 124 SDS-PAGE, Western immunoblotting, and indirect immunofluorescence assay .................................................................................................................... 124 Electron microscopy............................................................................................ 124 Preparation of cytoplasmic and extracellular virions ......................................... 125 Q-PCR .................................................................................................................. 126 Results ............................................................................................................................. 126 Construction and molecular analysis of recombinant viruses ............................ 126 Recovery and plaque morphologies of infectious viruses produced by HSV-1 BAC DNAs ............................................................................................................ 127 Replication characteristics of HSV-1 mutants ..................................................... 129 Protein expression profiles of viral mutants....................................................... 131 Ultrastructural characterization of wild-type and mutant viruses ..................... 132 Quantification of relative
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