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University of Florida Thesis Or Dissertation Formatting BIOPHYSICAL CHARACTERIZATION OF THE ASSEMBLY AND DISASSEMBLY OF THE ssDNA VIRUSES By ANTONETTE DENISE BENNETT 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 2009 1 © 2009 Antonette D Bennett 2 To my mother Iris Barrett-Browne, my brother Matthew Browne, my sister Shema Barrett, nephew Luke Walters and my son John-Claude Hutchinson 3 ACKNOWLEDGMENTS I am indebted to Professor Mavis McKenna for her mentorship, her patience and for allowing me the opportunity to work with her, as well as, in her lab for the last 5 years. Her commitment to excellence in all aspects of science has inspired and motivated me. I would also like to thank Dr Nicholas Muzyczka for his Co-chairing of my committee as well as allowing me to work in his lab for part of this study and his co-mentorship is greatly appreciated. I would also like to thank the members of my supervisory committee, Dr. Michael Bubb, Dr. J. Bert Flannegan, and Dr Jane Polston. They have been helpful and supportive in the work that I am presenting. I would like to thank the members of the McKenna lab and the Muuzyczka lab (past and present) for their assistance, friendship and their kindness. I would also like to show my appreciation of the Biochemistry staff and the IDP recruiting and clerical staff, without whom I could not have completed this work. I want to thank my parents Iris and Eric Browne, my brother Mathew Browne, my sister Shema Barrett, cousins, my aunts, my uncles, and my son John-Claude Hutchinson. I want to thank them for supporting and believing in me. I want to especially thank my mother for loving, motivating, inspiring and supporting me, and without her none of this would have been possible. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................................... 4 LIST OF TABLES................................................................................................................................ 8 LIST OF FIGURES .............................................................................................................................. 9 LIST OF ABBREVIATIONS ............................................................................................................ 12 ABSTRACT ........................................................................................................................................ 13 CHAPTER 1 FUNDAMENTALS OF VIRUS ASSEMBLY AND DISASSEMBLY ................................. 15 Virus Capsid Function ................................................................................................................ 15 Virus Assembly ........................................................................................................................... 15 Experimental Techniques Used to Study Virus Assembly ....................................................... 17 The ssDNA Virus Families......................................................................................................... 18 Geminiviridae .............................................................................................................................. 19 MSV-N Assembled Capsid Architecture ........................................................................... 20 The Structure of MSV-N CP Monomer and its Symmetry Related Interactions ............ 21 Geminivirus Assembly/Disassembly Studies ............................................................................ 22 Significance of Plant Virus Assembly/Disassembly Studies ................................................... 23 Parvoviridae ................................................................................................................................. 24 AAV2 Assembled Capsid Architecture ............................................................................. 25 The Structure of AAV2 CP Monomer and its Symmetry Related Interactions............... 26 Parvovirus assembly/disassembly Studies ................................................................................. 27 Significance of AAV2 Assembly Studies.................................................................................. 28 Overall Goals ............................................................................................................................... 29 2 MATERIALS AND METHODS ............................................................................................... 35 Introduction ................................................................................................................................. 35 Purification of MSV-N CP ......................................................................................................... 35 Biophysical Characterization of MSV-N CP Components ...................................................... 36 Negative Stain EM ............................................................................................................... 36 12 % SDS PAGE and Western Blot ................................................................................... 36 Genome extraction and agarose gel electrophoresis.......................................................... 37 Native Gel Analysis ............................................................................................................. 37 Analytical Gel Filtration Chromatography ........................................................................ 37 Scanning Transmission Electron Microscopy (STEM)..................................................... 38 Dynamic Light Scattering (DLS)........................................................................................ 39 Development of MSV-N Capsid Disassembly and Reassembly Assay .................................. 39 MSV-N CP Post Translation Modification Determination ...................................................... 39 5 Baculovirus MSV-N (Wildtype (wt) and Mutant (mt)) CP Production and Purification....... 40 Cloning of MSV-N Recombinant Donor Plasmid (pFastBac) ......................................... 41 Transformation of DH10 Bac E.Coli Cells ........................................................................ 41 Transfection of sf9 Cells ..................................................................................................... 43 Baculovirus MSV-N CP Expression .................................................................................. 43 Baculovirus MSV-N CP Purification ................................................................................. 44 Cell Culture and Virus Infection ......................................................................................... 44 Virus Purification................................................................................................................. 45 Biophysical Characterization of AAV2 VP1-3 Components ................................................... 45 SDS Gel Electrophoresis and Immunoblots....................................................................... 45 The Production and Characterization of AAV2 VP1-3 Mutants ............................................. 46 Selection of residues for mutagenesis ................................................................................ 46 Site Directed Mutagenesis of AAV2 VP3 ......................................................................... 47 Cell Culture .......................................................................................................................... 47 Transfection and Recombinant AAV2 Production ............................................................ 47 Biophysical Characterization of AAV2 VP1-3 Mutants .......................................................... 48 Gel Electrophoresis and Immunoblots ............................................................................... 48 Total Virus Capsid, Genome and Infectivity Quantification ............................................ 48 AAV2 Capsid Wildtype and Mutant Purification ............................................................. 49 3 MSV-N ASSEMBLY/DISASSEMBLY COMPONENTS ...................................................... 55 Introduction ................................................................................................................................. 55 Results .......................................................................................................................................... 57 MSV-N Wt Components Purification and Characterization .................................................... 57 MSV-N Wt Components Isolated ....................................................................................... 57 MSV-N Wt Components Genome Extraction ............................................................ 57 Native Polyacrylamide Gel .......................................................................................... 58 Size Exclusion Chromatography ................................................................................. 58 Dynamic Light Scattering (DLS) ................................................................................ 58 STEM Analysis ............................................................................................................ 59 Discussion for MSV-N CP Purification and Characterization ................................................. 60 MSV-N Assembly/Disassembly Assay
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