INTRACELLULAR TRAFFICKING OF AAV2 CAPSID MUTANTS AND EFFECTS ON GENE EXPRESSION By FIKRET AYDEMIR A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2016 1 © 2016 Fikret Aydemir 2 To my wife, Dr. Tolunay Beker Aydemir 3 ACKNOWLEDGMENTS I would like to thank my dissertation adviser Nicholas Muzyczka and committee members Mavis Agbandje-McKenna, Sergei Zolutukhin and Arun Srivastava. They invested a lot of time, energy and funding for my doctoral training. This dissertation wouldn’t have happened without them. I would like to thank them for their patience and understanding. I have been very fortunate to have an opportunity to work in Dr. Muzyczka’s lab. I have been lucky to share my work space with very productive lab members. I am specifically thankful to Mrs. Weijun Chen, Dr. Hector Mendez-Gomez, Dr. Jasbir Singh and Maxim Salganik. University of Florida is home of Powell Gene Therapy Vector Core. I would like to thank Mr. Mark Potter and his colleagues for providing not only very valuable vectors but also troubleshooting experimental problems, as well. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 LIST OF ABBREVIATIONS ........................................................................................... 10 ABSTRACT ................................................................................................................... 13 CHAPTER 1 INTRODUCTION .................................................................................................... 15 AAV Genome Organization ..................................................................................... 15 Binding and Entry ................................................................................................... 18 pH Dependent Structural Changes ......................................................................... 19 Significance ............................................................................................................ 22 2 MATERIALS AND METHODS ................................................................................ 24 Cell Culture ............................................................................................................. 24 Site-directed Mutagenesis ...................................................................................... 24 Virus Production ..................................................................................................... 24 Infectivity Assay ...................................................................................................... 25 Sub-cellular Fractionation ....................................................................................... 25 Large Scale Sub-cellular Fractionation ................................................................... 26 FLAG-column Purification ....................................................................................... 27 Mass Spectrometry ................................................................................................. 27 Data Analysis .......................................................................................................... 28 QPCR ..................................................................................................................... 28 DNase Protection Assay ......................................................................................... 29 RT-PCR Assay ....................................................................................................... 29 Statistical Analysis .................................................................................................. 30 3 RESULTS ............................................................................................................... 31 Dead-zone Mutants and Transcription .................................................................... 31 Mutant Particle to Infectivity Ratios ......................................................................... 34 Cell Entry ................................................................................................................ 34 Nuclear Accumulation ............................................................................................. 35 Uncoating in the Nucleus ........................................................................................ 36 Second-strand Synthesis ........................................................................................ 36 Steady State Transcription at 24 hr ........................................................................ 37 5 Complementation of the Mutant Phenotype ............................................................ 38 Discussion .............................................................................................................. 39 Summary ................................................................................................................ 44 Figures .................................................................................................................... 45 4 CELLULAR PARTNERS OF AAV ASSEMBLY ...................................................... 59 Introduction ............................................................................................................. 59 Assembly models .................................................................................................... 61 Myers Model ..................................................................................................... 61 AAP Model ....................................................................................................... 62 Results .................................................................................................................... 64 DNA Replication and Chromosome Maintenance ............................................ 66 Histone Modification and Transcription Proteins .............................................. 68 rRNA Processing .............................................................................................. 71 Splicing ............................................................................................................. 72 Discussion .............................................................................................................. 76 Figures .................................................................................................................... 78 5 CONCLUSION AND FUTURE DIRECTIONS ......................................................... 83 Conclusion .............................................................................................................. 83 AAV2 Trafficking and Gene Expression ........................................................... 83 Cellular Partners of AAV Assembly .................................................................. 84 Future Directions .................................................................................................... 87 AAV2 Trafficking and Gene Expression ........................................................... 87 Cellular Partners of Assembly .......................................................................... 88 LIST OF REFERENCES ............................................................................................... 90 BIOGRAPHICAL SKETCH .......................................................................................... 104 6 LIST OF TABLES Table page 2-1 Primers for inserting Flag epiope (DYKDDDDK) into capsid gene. .................... 24 3-1 Conservation of residues at 2 fold interface between serotypes. ....................... 41 4-1 Number of proteins according to functional groups. ........................................... 66 4-2 DNA replication and chromosome maintenance proteins. .................................. 68 4-4 RNA processing proteins. ................................................................................... 72 4-5 Splicing proteins. ................................................................................................ 74 7 LIST OF FIGURES Figure page 3-1 Genomic map of AAV. ........................................................................................ 45 3-2 Conserved and variable regions on the AAV2 capsid surface.. .......................... 46 3-3 Representation of intracellular trafficking pathways for efficient AAV transduction.. ...................................................................................................... 47 3-4 AAV2 pH responsive domain. ............................................................................. 48 3-5 The position of the mutants in the 30Å diameter dead zone. .............................. 49 3-6 Particle to infectivity ratio of mutants carrying a single stranded GFP expression cassette. ........................................................................................... 50 3-7 Time course of wt and mutant cell entry.. ........................................................... 51 3-8 Distribution of virus between the nucleus and cytoplasm. ............................... 52 3-9 Efficiency of