Aus dem Institut für Virologie des Fachbereichs Veterinärmedizin der Freien Universität Berlin Generation of a Complete Single-Gene Knockout Bacterial Artificial Chromosome Library of Cowpox Virus and Identification of Genes involved in hemorrhagic lesions on infected chicken chorioallantoic membranes (CAMs) Inaugural-Dissertation zur Erlangung des akademischen Doktorgrades philosophiae doctor (Ph.D.) in “Biomedical Science” an der Freien Universität Berlin vorgelegt von Zhiyong Xu aus Jidong / Volksrepublik China Berlin 2014 Journal-Nr.: 3712 Gedruckt mit Genehmigung des Fachbereichs Veterinärmedizin der Freien Universität Berlin Dekan: Univ.-Prof. Dr. Jürgen Zentek Erster Gutachter: Univ.-Prof. Dr. Nikolaus Osterrieder Zweiter Gutachter: PD Dr. Martin G. Beer Dritter Gutachter: Prof. Dr. Robert Klopfleisch Deskriptoren (nach CAB-Thesaurus): Cowpox virus; bacterial artificial chromosomes; chorioallantoic membrane; deletions; lesions; genes, essential (MeSH) Tag der Promotion: 28.04.2014 Bibliografische Information der Deutschen Nationalbibliothek Die Deutsche Nationalbibliothek verzeichnet diese Publikation in der Deutschen Nationalbibliografie; detaillierte bibliografische Daten sind im Internet über <http://dnb.ddb.de> abrufbar. ISBN: 978-3-86387-482-7 Zugl.: Berlin, Freie Univ., Diss., 2014 Dissertation, Freie Universität Berlin D 188 Dieses Werk ist urheberrechtlich geschützt. Alle Rechte, auch die der Übersetzung, des Nachdruckes und der Vervielfältigung des Buches, oder Teilen daraus, vorbehalten. Kein Teil des Werkes darf ohne schriftliche Genehmigung des Verlages in irgendeiner Form reproduziert oder unter Verwendung elektronischer Systeme verarbeitet, vervielfältigt oder verbreitet werden. Die Wiedergabe von Gebrauchsnamen, Warenbezeichnungen, usw. in diesem Werk berechtigt auch ohne besondere Kennzeichnung nicht zu der Annahme, dass solche Namen im Sinne der Warenzeichen- und Markenschutz-Gesetzgebung als frei zu betrachten wären und daher von jedermann benutzt werden dürfen. This document is protected by copyright law. No part of this document may be reproduced in any form by any means without prior written authorization of the publisher. Alle Rechte vorbehalten | all rights reserved © Mensch und Buch Verlag 2014 Choriner Str. 85 - 10119 Berlin [email protected] – www.menschundbuch.de Table of Contents Table of Contents Table of Contents ......................................................................................................... I List of Figures ............................................................................................................ III List of Tables ............................................................................................................. IV List of Abbreviations .................................................................................................. X Chapter 1: Introduction .............................................................................................. 1 1.1. Taxonomy of Poxviruses ....................................................................................................... 1 1.2. Orthopoxviruses .................................................................................................................... 1 1.3. Cowpox virus ......................................................................................................................... 2 1.4. Lesions caused by OPVs on chicken CAMs ........................................................................ 2 1.5. Replication cycle, morphogenesis, and actin tail induction ............................................... 3 1.6. Genome structure and DNA replication of poxvirus ......................................................... 5 1.7. Regulation of Gene expression ............................................................................................. 5 1.8. Modulation of host immune response by OPVs ................................................................. 6 1.9. Outline of this thesis .............................................................................................................. 7 Chapter 2 Generation of a Complete Single-Gene Knockout Bacterial Artificial Chromosome Library of Cowpox Virus and Identification of Its Essential Genes ........................................................................................................................................ 8 2.1. Abstract .................................................................................................................................. 8 2.2. Introduction ........................................................................................................................... 8 2.3. Materials and Methods ....................................................................................................... 10 2.3.1. Cell lines and viruses .................................................................................................... 10 2.3.2. Reconstitution of infectious virus from BAC DNA ..................................................... 10 2.3.3. Generation of plasmid pACAA .................................................................................... 11 2.3.4. Generation of dual marker CPXV-BR BAC clone pBRFseR ...................................... 12 2.3.5. BAC mutagenesis ......................................................................................................... 12 2.3.6. Knockout BAC verification .......................................................................................... 13 2.3.7. Bioinformatics analysis ................................................................................................ 13 2.4. Results and Discussion ........................................................................................................ 14 2.4.1. Generation of the dual marker CPXV-BR BAC clone pBRFseR ................................ 14 2.4.2. Targeted knockout of all unique CPXV-BR ORFs ...................................................... 14 2.4.3. Virus reconstitution ...................................................................................................... 16 2.4.4. Different phenotypes upon virus reconstitution ........................................................... 21 2.4.5. Essential genes of CPXV ............................................................................................. 21 2.5. Acknowledgements ............................................................................................................. 25 2.6. References ............................................................................................................................ 25 2.7. Supplement Materials ......................................................................................................... 29 2.8. Contributions ....................................................................................................................... 41 Chapter 3. Identification of 10 cowpox virus proteins that are necessary for induction of hemorrhagic lesions (red pocks) on chorioallantoic membranes ..... 42 3.1. Abstract ................................................................................................................................ 42 3.2. Introduction ......................................................................................................................... 42 3.3. Materials and Methods ....................................................................................................... 43 I Table of Contents 3.3.1. Cell lines....................................................................................................................... 43 3.3.2. Viruses ......................................................................................................................... 43 3.3.3. BAC clones and BAC mutagenesis .............................................................................. 44 3.3.4. Preparation of plasmid and BAC DNA and verification of BAC mutagenesis ............ 44 3.3.5. Plasmid construction .................................................................................................... 44 3.3.6. Reconstitution of infectious virus from BAC DNA ..................................................... 45 3.3.7. CAM infections ............................................................................................................ 45 3.4. Results .................................................................................................................................. 45 3.4.1. Generation of CPXV with deletion of kelch-like genes ............................................... 45 3.4.2. Identification of mutant CPXV inducing non-hemorrhagic pocks on CAM ................ 49 3.4.3. The shape of pocks induced by mutant CPXV on CAM .............................................. 51 3.4.4. Histological examination of pocks from infected CAM. .............................................. 51 3.4.5. Generation of revertant viruses and CAM infection..................................................... 51 3.4.6. CPXV ORFs necessary for hemorrhagic pocks and bioinformatics analysis ............... 53 3.5. Discussion ............................................................................................................................ 54 3.6. Acknowledgements ............................................................................................................. 60 3.7. References ...........................................................................................................................