JYVÄSKYLÄ STUDIES IN BIOLOGICAL AND ENVIRONMENTAL SCIENCE 154 Leona Gilbert Development of Biotehcnological Tools for Studying Infectious Pathways of Canine and Human Parvoviruses JYVÄSKYLÄN YLIOPISTO JYVÄSKYLÄ STUDIES IN BIOLOGICAL AND ENVIRONMENTAL SCIENCE 154 Leona Gilbert Development of Biotechnological Tools for Studying Infectious Pathways of Canine and Human Parvoviruses Esitetään Jyväskylän yliopiston matemaattis-luonnontieteellisen tiedekunnan suostumuksella julkisesti tarkastettavaksi yliopiston Ambiotica-rakennuksen salissa (YAA303) kesäkuun 18. päivänä 2005 kello 12. Academic dissertation to be publicly discussed, by permission of the Faculty of Mathematics and Science of the University of Jyväskylä, in the Building Ambiotica, Auditorium YAA303, on June 18th, 2005 at 12 o'clock noon. UNIVERSITY OF JYVÄSKYLÄ JYVÄSKYLÄ 2005 Development of Biotechnological Tools for Studying Infectious Pathways of Canine and Human Parvoviruses JYVÄSKYLÄ STUDIES IN BIOLOGICAL AND ENVIRONMENTAL SCIENCE 154 Leona Gilbert Development of Biotechnological Tools for Studying Infectious Pathways of Canine and Human Parvoviruses UNIVERSITY OF JYVÄSKYLÄ JYVÄSKYLÄ 2005 Editors Jukka Särkkä Department of Biological and Environmental Science, University of Jyväskylä Pekka Olsbo, Irene Ylönen Publishing Unit, University Library of Jyväskylä Cover picture: Molecular models of the fluorescent biotechnological tools for CPV and B19. Picture by Leona Gilbert. ISBN 951-39-2134-4 (nid.) ISSN 1456-9701 Copyright © 2005, by University of Jyväskylä Jyväskylä University Printing House, Jyväskylä 2005 ABSTRACT Gilbert, Leona Development of biotechnological tools for studying infectious pathways of canine and human parvoviruses. Jyväskylä, University of Jyväskylä, 2005, 104 p. (Jyväskylä Studies in Biological and Environmental Science, ISSN 1456-9701; 154) ISBN 951-39-2182-4 Parvoviruses are among the smallest vertebrate DNA viruses known to date. The production of parvovirus-like particles (parvo-VLPs) has been successfully exploited for parvovirus vaccine development. The baculovirus expression vector system (BEVS) has been the popular choice to correctly express and produce VLPs. These multimeric structures are morphologically and structurally identical to the original virus. The capsid proteins can also be produced individually or in combination to monitor structural protein functions in the parvovirus life cycle. Moreover, the ability of baculoviruses to transduce a wide range of mammalian cells has brought about the opportunity to use these viruses as gene vectors to express individual parvoviral structural proteins of interest. The present study was aimed at the development of biotechnological tools that could be used to gain insight into the trafficking events of parvoviral infections. The non-fluorescent and fluorescent fusion proteins, as well as recombinant baculoviruses encoding these constructs were successfully generated and EGFP was incorporated on the surface of the parvo- VLPs. These recombinant baculoviruses (rBVs) were shown to be practical when following parvoviral structural proteins in vivo. The chimeric parvo-VLPs were further scrutinized specifically with regard to their assembly capabilities, trafficking events and nuclear targeting. In addition, a baculovirus-mediated de novo vector system was created to shed additional light on the synthesis and trafficking of individual parvoviral structural proteins in mammalian cells. Together, these results showed that the surface of parvoviruses could be modified, display a large foreign moiety and assemble correctly. Similarly recombinant baculoviruses could be used as gene transfer vectors to monitor the individual parvoviral proteins in transduced mammalian target cells. The parvovirus display and transduction techniques show promise as potential tools for various scientific applications ranging from basic virological studies, to gene therapy applications, and developments in biomedicine. Key words: Capsids; display; enhanced green fluorescent protein; parvovirus; virus-like particles; VP1; VP2. L. Gilbert, University of Jyväskylä, Department of Biological and Environmental Science, P.O. Box 35, FI-40014 University of Jyväskylä, Finland Author’s address Leona Gilbert Department of Biological and Environmental Science University of Jyväskylä P.O. Box 35 FI-40014 University of Jyväskylä, Finland E-mail: [email protected] Supervisor Professor Christian Oker-Blom Department of Biological and Environmental Science University of Jyväskylä P.O. Box 35 FI-40014 University of Jyväskylä, Finland E-mail: [email protected] Reviewers Docent Sarah Butcher Institute of Biotechnology and Department of Biological and Environmental Science Viikinkaari 1 FI-00014 University of Helsinki, Finland E-mail: [email protected] Docent Maria Söderlund-Venermo Department of Virology and HUSLAB Haartman Institute P.O. Box 21 (Haartmaninkatu 3) FI-00014 University of Helsinki, Finland E-mail: [email protected] Opponent Professor Loy Volkman Department of Plant & Microbial Biology University of California 251 Koshland Hall Berkeley, CA 94720-3102 E-mail: [email protected] CONTENTS LIST OF ORIGINAL PUBLICATIONS.....................................................................9 RESPONSIBILITIES OF LEONA GILBERT IN THE ARTICLES OF THIS THESIS ..............................................................................................................10 ABBREVIATIONS .....................................................................................................11 1 INTRODUCTION ............................................................................................13 2 REVIEW OF THE LITERATURE ...................................................................14 2.1 Parvoviruses............................................................................................14 2.1.1 Parvovirus genome and proteins................................................16 2.1.2 Parvovirus virion structure..........................................................16 2.1.3 Parvovirus life cycle......................................................................20 2.2 Parvovirus applications.........................................................................24 2.2.1 Vaccines..........................................................................................26 2.2.2 Gene therapy applications ...........................................................27 2.3 Baculoviruses...........................................................................................31 2.3.1 Baculovirus life cycle ....................................................................31 2.3.2 Baculovirus applications..............................................................32 2.3.3 Baculovirus expression vector system .......................................33 2.3.4 Baculovirus-mediated mammalian transduction.....................35 2.3.5 Parvoviral proteins produced in BEVS ......................................36 3 AIMS OF THE STUDY ....................................................................................38 4 SUMMARY OF MATERIALS AND METHODS.........................................39 4.1 Genetic baculovirus constructs.............................................................39 4.2 Cell lines and viruses .............................................................................41 4.3 Purification of recombinant proteins and wild-type viruses ........... 43 4.4 Antibodies................................................................................................43 4.5 Western blotting......................................................................................45 4.6 Feeding and transduction of mammalian cells ..................................45 4.7 Immunofluorescence microscopy ........................................................46 4.8 Electron microscopy...............................................................................46 4.9 Fluorescence correlation spectroscopy................................................47 4.10 Immunoprecipitation of fluorescent VLPs..........................................47 4.11 Atomic force microscopy.......................................................................48 4.12 Molecular modeling ...............................................................................49 4.13 Phospholipase A2 activity......................................................................49 5 REVIEW OF THE RESULTS...........................................................................50 5.1 Assembly of parvo-VLPs.......................................................................50 5.1.1 Characterization of recombinant CPV proteins........................50 5.1.2 Characterization of B19 fluorescent VLPs .................................52 5.2 Behavior of recombinant parvovirus proteins in mammalian cells............................................................................................................54 5.2.1 Trafficking of CPV recombinant proteins..................................54 5.2.2 Trafficking of B19 recombinant proteins ...................................56 5.3 rBVs as tools for transducing mammalian cells.................................57 6 DISCUSSION ................................................................................................…59 6.1 Creation of parvoviral biotechnological tools ....................................59 6.2 Recombinant proteins used to investigate the parvovirus life cycle ..........................................................................................................62