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University of Florida Thesis Or Dissertation CHARACTERIZATION AND COMPARISON OF SQUAMATE DEPENDOPARVOVIRUS TYPE I TO MAMMALIAN ADENO-ASSOCIATED VIRUSES By VICTORIA E. FIELDING A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2016 © 2016 Victoria E. Fielding This work is dedicated to Trevor, my parents, Marshall, Rebekah, my grandparents, and all of the friends that have always believed in me. ACKNOWLEDGMENTS I would like to thank my parents, Charles and Angela, and my siblings, Marshall and Rebekah, for their love and support throughout this process. My heart-felt thanks is also extended to my grandparents, who have been my biggest fans and strongest supporters throughout my scientific career. I would also like to extend my sincere thanks to my mentor, Dr. Mavis Agbandje-McKenna, for pushing me to be the best scientist that I can be, for her endless patience, and for believing in me. I would like to thank Dr. Robert McKenna for his unique constructive insight into my project, and his encouragement. I would also like to thank my committee members, Dr. James B. Flanegan, Dr. Joanna Long, Dr. Arun Srivastava, and Dr. Gail Fanucci, for their time, invaluable ideas, and encouragement. I would also like to extend my thanks to our collaborators, Dr. Peter Tijssen, Qian Yu, Maude Boisvert, and Judit Pénzes (Institut National de la Recherché Scientifique, Québec) for providing us with the original sAAV constructs that were used throughout the course of this project, and to Dr. Mario Mietzsch (UF, Charité Medical School) and Dr. Regine Heilbronn (Charité Medical School) for supplying rAAV sample. I would also like to thank my undergraduate student Megan R. Lee for all her assistance on this project, and for reminding me of my love for teaching. I would like to extend my most sincere gratitude to Dr. Bridget Lins, Dr. Antonette Bennet, Paul Chipman, and all of the other members of the Agbandje- McKenna lab for their endless patience as they taught me the different techniques utilized in this laboratory. Finally, I would like to extend my heartfelt thanks to my partner and best friend, Trevor Makal, without whose love and support, none of this work would have been possible. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 LIST OF ABBREVIATIONS ............................................................................................. 9 ABSTRACT ................................................................................................................... 11 CHAPTER 1 BACKGROUND AND INTRODUCTION ................................................................. 13 Dependoparvovirus Taxonomy ............................................................................... 13 Adeno-associated Virus Genome Structure ............................................................ 14 Structural Features of the AAV Viral Protein ........................................................... 16 Lifecycle and Trafficking ......................................................................................... 17 Tropism and Receptor Determinants ...................................................................... 18 AAVs as Gene Therapy Vectors ............................................................................. 20 Seropositivity as a Limitation of Treatment ............................................................. 21 Approaches for Overcoming Pre-Existing Anti-AAV Immune Response ................ 22 Reptilian Dependoparvoviruses and Potential Therapeutic Applications ................ 23 Global Significance and Impact .............................................................................. 24 2 MATERIALS AND METHODS FOR BIOCHEMICAL CHARACTERIZATION ......... 32 Maintenance of Sf9 Insect Cell Lines ...................................................................... 32 Maintenance of Mammalian CHO Cell Line Variants .............................................. 32 Production of Virus-Like Particles ........................................................................... 33 Virus Purification by Sucrose Cushion and Gradient .............................................. 33 Virus Purification by Iodixanol Gradient .................................................................. 35 Ion Exchange Chromatography .............................................................................. 36 OneBac Virus Production Platform ......................................................................... 36 Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis .................................. 37 Negative Stain Electron Microscopy ....................................................................... 37 Native Dot Blot – Mouse Monoclonal Antibody Panel ............................................. 38 Fluorescent Labeling of Virus Capsid for FACS ...................................................... 39 Fluorescence Activated Cell Sorting ....................................................................... 40 Low pH Studies ....................................................................................................... 41 3 MATERIALS AND METHODS FOR CRYO ELECTRON MICROSCOPY AND 3D STRUCTURE DETERMINATION...................................................................... 44 5 Vitrification .............................................................................................................. 44 Cryo-Electron Microscopy Data Collection .............................................................. 44 Cryo-EM Data Processing and 3D Structure Determination ................................... 44 Model Building ........................................................................................................ 45 Secondary Structure Matching and Variable Region Assignment ........................... 46 Visualization of Structural Comparison ................................................................... 47 4 RESULTS AND DISCUSSION ............................................................................... 49 Structural Determination of Squamate Dependoparvovirus Type I ......................... 49 Characterization of Squamate Dependoparvovirus I .............................................. 52 sAAV is Capable of Escaping Recognition by Mouse Monoclonal Antibodies that Target the mammalian AAV Capsid ....................................................... 52 sAAV is Capable of Recognizing and Binding to Mammalian Cell Lines .......... 53 sAAV Conserves pH-Induced Proteolytic Cleavage Activity Observed in Mammalian AAVs .......................................................................................... 54 sAAV is Highly Thermostable and Conserves the pH Stability Profile Observed in Mammalian AAVs ..................................................................... 55 5 SUMMARY AND FUTURE DIRECTIONS .............................................................. 67 Overall Summary of Experimental Findings ............................................................ 67 Improvement of sAAV High Resolution Cryo-EM Structure .................................... 67 Challenging sAAV with Polyclonal Human Serum .................................................. 68 Reconfirming sAAV Cell Binding and Transduction of Specific Mammalian Cell Lines .................................................................................................................... 69 Determination of HSPG as a Possible sAAV Cell Surface Receptor ...................... 70 Identification of Autolytic Cleavage Products by Mass Spectrometry ..................... 70 Characterization of Other Reptilian Dependoparvoviruses ..................................... 71 LIST OF REFERENCES ............................................................................................... 72 BIOGRAPHICAL SKETCH ............................................................................................ 82 6 LIST OF TABLES Table page 1-1 Mammalian AAV VP3 sequence identity in comparison to Squamate Dependoparvovirus Type I (sAAV). .................................................................... 26 1-2 Known reptilian dependoparvoviruses. ............................................................... 27 2-1 Mouse monoclonal antibody panel details. ......................................................... 43 3-1 Equations used in 3D image reconstruction. ...................................................... 48 4-1 sAAV variable region assignment and corresponding mammalian AAV residues. ............................................................................................................. 57 7 LIST OF FIGURES Figure page 1-1 Taxonomy of the Family Parvoviridae. ............................................................... 28 1-2 Dependoparvovirus genome organization. ....................................................... 29 1-3 AAV Capsid Structure. ........................................................................................ 30 1-4 The viral lifecycle. ............................................................................................... 31 4-1 Purification of sAAV. .........................................................................................
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