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Genetics and Functions of Herpes Simplex Virus Type-1 Membrane

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Genetics and Functions of Herpes Simplex Virus Type-1 Membrane Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2014 Genetics and Functions of Herpes Simplex Virus Type-1 Membrane Proteins UL20/gK in Virion Envelopment and Entry Anu Susan Charles Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Veterinary Pathology and Pathobiology Commons Recommended Citation Charles, Anu Susan, "Genetics and Functions of Herpes Simplex Virus Type-1 Membrane Proteins UL20/gK in Virion Envelopment and Entry" (2014). LSU Doctoral Dissertations. 2619. https://digitalcommons.lsu.edu/gradschool_dissertations/2619 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]. GENETICS AND FUNCTIONS OF HERPES SIMPLEX VIRUS TYPE-1 MEMBRANE PROTEINS UL20/gK IN VIRION ENVELOPMENT AND ENTRY 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 Anu Susan Charles DVM, Kerala Agricultural University, 2005 M.S., University of Connecticut, 2009 December 2014 Dedicated to my Parents, Mrs. Susamma Charles and Dr. Charles Philip ii ACKNOWLEDGEMENTS First and foremost I would like to thank my Parents, Susamma Charles and Charles Philip and my sister Linu for their love, support and encouragements during my education. I am also grateful to my husband Pratheesh, sons Joel and Noah, and parents-in-law Jemini George and Thomas George for being understanding and supportive during my pursuits. I would sincerely extent my gratitude to Dr. Gus Kousoulas for giving me an opportunity to pursue Ph. D under his guidance and without his constant support and advice I would not be able to succeed in graduate studies along with family commitments. I specially thank Dr. Vladimir Chouljenko for all his help and technical assistance. I am grateful to all post docs Drs Nithya Jambunathan, Jason walker, Arun Iyer, Ramesh Subramanian and Paul Rider and to fellow graduate students Sona, Dimitry, Andrew, Beth, Ahmed, Misagh and Brent for their help and also making our laboratory a happy work place. I am also thankful to administrative staffs Ms. Pam Stratton and Mr. Anand Ramachandran and members of the GeneLab, Ms. Thaya Guedry and Tori for their sequencing and lab supplies ordering help. I would like to thank the faculty members that served on my committee, Drs Nobuko Wakamatsu, Maria Antonieta Guerrero Plata, Christopher N. Mores and Rendy Kartika for their advice and recommendations. I am also indebted to all teachers in Mount Carmel School, Bishop Choolaparambil Memorial College and College of Veterinary and Animal Sciences, Kerala who gave the best education out of the available resources. Last but not least, I thank God almighty for all his blessing and grace throughout my life. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS .............................................................................................. iii ABSTRACT ...................................................................................................................... vi CHAPTER I: INTRODUCTION ..................................................................................... 1 Statement of Problem and Hypothesis .................................................................. 1 Statement of Research Objectives ........................................................................ 3 CHAPTER II: REVIEW OF LITERATURE ................................................................... 5 History of Herpesviruses....................................................................................... 5 Taxonomy of Herpesviruses ................................................................................. 6 Structure of the Virus ............................................................................................ 7 HSV-1 genome and origins of DNA replication ................................................... 9 Clinical Manifestation ........................................................................................... 10 Epidemiology ........................................................................................................ 12 The molecular basis of Herpes Simplex Virus latency ......................................... 13 Immune Response against HSV-1 ........................................................................ 15 Life cycle of HSV-1 .............................................................................................. 17 Transport and Egress of HSV-1 ............................................................................ 20 gK/UL20 Role in Envelopment and Egress .......................................................... 24 Membrane Proteins of HSV-1 .............................................................................. 25 UL20 Protein/ Glycoprotein K (UL20/gk) ............................................... 25 Glycoprotein B .......................................................................................... 29 Glycoprotein C (gC) ................................................................................. 29 Glycoprotein D (gD) ................................................................................. 30 Glycoprotein G (gG) ................................................................................. 31 Glycoprotein E and I (gE/gI) .................................................................... 31 Glycoproteins H and L (gH/gL) ................................................................ 32 Glycoprotein J (gJ) .................................................................................... 33 Glycoprotein M (gM) ................................................................................ 34 Glycoprotein N (UL49.5) ......................................................................... 35 Tegument Proteins ................................................................................................ 35 References ............................................................................................................ 36 CHAPTER III: PHENYLALANINE RESIDUES AT THE CARBOXYL-TERMINUS OF THE HERPES SIMPLEX VIRUS TYPE-1 UL20 MEMBRANE PROTEIN REGULATE CYTOPLASMIC VIRION ENVELOPMENT AND INFECTIOUS VIRUS PRODUCTION Importance ............................................................................................................ 60 Introduction ........................................................................................................... 61 Materials and Methods .......................................................................................... 64 Cells .......................................................................................................... 64 iv Construction of HSV-1 mutant viruses ................................................... 64 SDS-PAGE and Western Immunoblot Assay ......................................... 65 Plaque Morphologies, Replication Kinetics and Electron Microscopy ..... 66 Quantification of cell-to-cell fusion ........................................................ 67 Quantitative PCR ...................................................................................... 67 Confocal Microscope ................................................................................ 68 Proximity Ligation Reaction (PLA) .................................................... 68 Results ................................................................................................................... 69 Construction and Molecular Analysis of recombinant viruses ............. 69 Replication and Phenotypic characteristics of the mutant viruses ........... 70 Quantification of cell-to-cell fusion......................................................... 71 Ultrastructural Characterization of wild-type and mutant viruses ......... 75 Characterization of the relative efficiency of cytoplasmic virion envelopment ........................................................................................... 76 Intracellular localization of the UL20 mutant proteins and UL20 interactions with the UL37 protein .......................................................... 76 Discussion ............................................................................................................. 80 References ............................................................................................................. 85 CHAPTER IV: ENTRY CHARACTERISTICS OF HSV-1 gK 31-68 MUTANT IN VERO CELLS AND NEURONS STUDIED USING UL37 PROTEIN/DYNEIN PLA ASSAY ............................................................................................................................ 89 Significance........................................................................................................... 89 Introduction ........................................................................................................... 89 Materials and Methods .......................................................................................... 95 Cell lines ..................................................................................................
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