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Biophysical Characterization of the Assembly and Disassembly of the Single Stranded DNA Viruses

Permanent Link: http://ufdc.ufl.edu/UFE0025005/00001

Material Information

Title: Biophysical Characterization of the Assembly and Disassembly of the Single Stranded DNA Viruses
Physical Description: 1 online resource (164 p.)
Language: english
Creator: Bennett, Antonette
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: assembly, disassembly, disease, gene, mutants, pathogen, plant, therapy, virus
Biochemistry and Molecular Biology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Viruses are the causative agents of many diseases that affect plants and animals and the mechanisms by which they assemble and disassemble represent critical steps in their replicative life cycle. The focus of this study was to elucidate the steps involved in the formation and disruption of the viral capsid of the Geminiviridae and Pavoviridae family of viruses. Maize Streak Virus (MSV-N) belongs to the Geminiviridae family of virus. The Geminiviridae have caused significant economic loses and hardship due to their infection of a number of horticultural crops worldwide. MSV-N is a severe pathogen of maize and is used in this study as a model for understanding assembly and disassembly of this unique virus family. The goal of the study was to decipher the steps involved in the formation and disruption of the MSV capsid. This was accomplished by isolating and characterizing the capsid components and intermediates from maize agro-inoculated with MSV-N. These components and intermediates include the characteristic geminate capsid, a T=1 icosahedral (single) capsid, pentamers, and dimers of pentamers. This study also included a pH assembly/diasassembly assay that has been generated for MSV and could be developed as a method to test peptide inhibitors of these processes. Adeno-Associated Virus serotype 2 (AAV2) belongs to the Parvoviridae family and it is an important therapeutic agent for clinical gene therapy. AAV2 has been extensively studied and it has been used in the treatment of several genetic diseases. However, there is still a large deficit in the amount of information available on the mechanism by which it assembles and disassembles. We have isolated and characterized subassemblies of recombinant AAV2. These include what appear to be pentamers, dimers of pentamers (DOPs) and pentamers of dimers (PODs). In the final aspect of this work, we used the crystal structure of AAV2, other AAV serotypes, other parvoviruses and site directed mutagenesis to identify residues and regions of the capsid that are important to the assembly and stability of the virus capsid.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Antonette Bennett.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Agbandje-McKenna, Mavis.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-02-28

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0025005:00001

Permanent Link: http://ufdc.ufl.edu/UFE0025005/00001

Material Information

Title: Biophysical Characterization of the Assembly and Disassembly of the Single Stranded DNA Viruses
Physical Description: 1 online resource (164 p.)
Language: english
Creator: Bennett, Antonette
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: assembly, disassembly, disease, gene, mutants, pathogen, plant, therapy, virus
Biochemistry and Molecular Biology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Viruses are the causative agents of many diseases that affect plants and animals and the mechanisms by which they assemble and disassemble represent critical steps in their replicative life cycle. The focus of this study was to elucidate the steps involved in the formation and disruption of the viral capsid of the Geminiviridae and Pavoviridae family of viruses. Maize Streak Virus (MSV-N) belongs to the Geminiviridae family of virus. The Geminiviridae have caused significant economic loses and hardship due to their infection of a number of horticultural crops worldwide. MSV-N is a severe pathogen of maize and is used in this study as a model for understanding assembly and disassembly of this unique virus family. The goal of the study was to decipher the steps involved in the formation and disruption of the MSV capsid. This was accomplished by isolating and characterizing the capsid components and intermediates from maize agro-inoculated with MSV-N. These components and intermediates include the characteristic geminate capsid, a T=1 icosahedral (single) capsid, pentamers, and dimers of pentamers. This study also included a pH assembly/diasassembly assay that has been generated for MSV and could be developed as a method to test peptide inhibitors of these processes. Adeno-Associated Virus serotype 2 (AAV2) belongs to the Parvoviridae family and it is an important therapeutic agent for clinical gene therapy. AAV2 has been extensively studied and it has been used in the treatment of several genetic diseases. However, there is still a large deficit in the amount of information available on the mechanism by which it assembles and disassembles. We have isolated and characterized subassemblies of recombinant AAV2. These include what appear to be pentamers, dimers of pentamers (DOPs) and pentamers of dimers (PODs). In the final aspect of this work, we used the crystal structure of AAV2, other AAV serotypes, other parvoviruses and site directed mutagenesis to identify residues and regions of the capsid that are important to the assembly and stability of the virus capsid.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Antonette Bennett.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Agbandje-McKenna, Mavis.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-02-28

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0025005:00001


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1 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 REQ UIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

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2 2009 Antonette D Bennett

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3 To my mother Iris Barrett -Browne, my brother Matthew Browne, my sister Shema Barrett, nephew Luke Walt ers an d my son John Claude Hutchinson

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4 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 excel lence 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 la b 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 JohnClaude 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.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................................... 4 LIST OF TABLES ................................................................................................................................ 8 LIST OF FIGURES .............................................................................................................................. 9 LIST OF ABBREVIATIONS ............................................................................................................ 12 ABSTRACT ........................................................................................................................................ 13 CH A P T ER 1 FUN DAMENTALS OF VIRUS ASSEMBLY AND DISASSEMBLY ................................. 15 Virus Capsid Function ................................................................................................................ 15 Virus Assembly ........................................................................................................................... 15 Experimental Techniques Used to Study Virus Assembly ....................................................... 17 T he 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 Nat ive Gel Analysis ............................................................................................................. 37 Analytical Gel Filtration Chromatography ........................................................................ 37 Scanning Transmission Electron Microscopy (STEM) ..................................................... 38 Dynamic Light Scattering (DLS) ........................................................................................ 39 Development of MSV N Ca psid Disassembly and Reassembly Assay .................................. 39 MSV N CP Post Translation Modification Determination ...................................................... 39

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6 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 Productio n and Characterization of AAV2 VP13 Mutants ............................................. 46 Selection of residues for mutagenesis ................................................................................ 46 Site Directed Mutagenesis of AAV2 VP3 ......................................................................... 47 Cell Culture .......................................................................................................................... 47 Transfection and Recombinant AAV2 Product ion ............................................................ 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 Characte rization .................................................... 57 MSV N Wt Components Isolated ....................................................................................... 57 MSV N Wt Components Genome Extraction ............................................................ 57 Native Polyacrylamide Gel .......................................................................................... 58 Size Exclusion Chromatograp hy ................................................................................. 58 Dynamic Light Scattering (DLS) ................................................................................ 58 STEM Analysis ............................................................................................................ 59 Discussion for MSVN CP Purification and Characterization ................................................. 60 MSV N Assembly/Disassembly Assay ..................................................................................... 60 MSV N pH Components Isolated ....................................................................................... 60 MSV N pH Components Genome Extraction ............................................................ 61 MSV N pH Components STEM Analysis .................................................................. 62 MSV N pH Assembly/D isassembly Assay in the Presence of Protease Inhibitor .......... 62 Discussion of MSVN pH Disassembly/Reassembly Assay .................................................... 63 Post translational Modification .................................................................................................. 64 Post Translational Modification Determination ........................................................ 64 Post Tr anslational Modification Prediction ................................................................ 65 Discussion .................................................................................................................................... 65

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7 4 WILDTYPE AND MUTANT MSVN CP BACULOVITUS CLONING AND EXPRESSION ............................................................................................................................. 91 Introduction ................................................................................................................................. 91 Results .......................................................................................................................................... 93 Cloning of MSV N CP (wt and mutant) into pFastBac .................................................... 93 pFast Bac (wt and mutant) Clone Selection ....................................................................... 93 BMSV -NCP, BMSV -NCP182 or BMSV -NCP201 recombinant identification ............. 94 Wild Type and Mutant BMSV N CP Expression .............................................................. 94 BMSV -NCP Purification (Wt and Mutant) ........................................................................ 95 Discussion .................................................................................................................................... 96 5 BIOPHYSICAL CHARACTERIZATION OF THE ASSEMBLY/DISASSEMBLY OF AAV2 ......................................................................................................................................... 104 Introduction ............................................................................................................................... 104 Results ........................................................................................................................................ 107 Selection of Residues for Mutagenesis ............................................................................ 107 Symmetry Related Location of VP3 Mutants .................................................................. 108 Immunoblots of Mutant Cell Lysate ................................................................................. 109 AAV2 Total Particle Titration .......................................................................................... 110 Packaging Efficiency ......................................................................................................... 110 Infectious Particle Titer ..................................................................................................... 110 Virus Purification and V isualization ................................................................................ 111 Baculovirus AAV2 VP Purification and Characterization.............................................. 111 Discussion .................................................................................................................................. 113 Assembly Mutants ............................................................................................................. 114 Internal Polar Mutants ....................................................................................................... 115 Packaging and Infectivity Mutants ................................................................................... 115 Structure to Function Correlation for AAV2 Capsid Based on Assembly Studies ....... 116 6 CONCLUSIONS AND FUTURE DIRECTIONS .................................................................. 142 MSV N Assembly/Disa ssembly Model ................................................................................... 142 AAV2 Assembly/Disassembly Model ..................................................................................... 144 Structure to function correlation for the ssDNA viruses ........................................................ 145 Future Directions ....................................................................................................................... 146 LIST OF REFERENCES ................................................................................................................. 150 BIOGRAPHICAL SKETCH ........................................................................................................... 164

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8 LIST OF TABLES Table page 1 1 List of the economically important viral diseases caused by geminiviruses ...................... 34 3 1 STEM statistical analysis of purified MSV N capsomers, single and geminate capsids. .................................................................................................................................... 86 3 2 STEM statistical analysis of MSV -N assembly/disassembly compone nts from the pH assay ........................................................................................................................................ 87 3 3 Comparison of the Molecular Mass (kDa) of the pH reassembled geminates and singless compared to wt. ........................................................................................................ 88 3 3 MSV N CP phosphorylation prediction table ...................................................................... 89 5 1 List and location of AAV2 mutants .................................................................................... 136 5 2 STEM statistical analysis of AAV2 assembly/disassembly components from baculovirus expression. ........................................................................................................ 137 5 3 Summary of AAV2 mutant phenotype. .............................................................................. 138 5 4 Summary of a ll AAV2 Assembly Mutants. ........................................................................ 139

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9 LIST OF FIGURES Figure page 1 1 MSV N Infection. ................................................................................................................... 31 1 2 MSV N and AAV2 virus capsid and CP/VP structures.. .................................................... 32 1 3 Capsid protein interfaces. ...................................................................................................... 33 2 1 Schematic of MSV -N CP components pu rification and characterization. ......................... 51 2 2 Diagram of the production of recombinant MSV N CP (wt and mutants). ....................... 52 2 3 Schematic AAV 2 VP components purification and characterization. ................................ 53 2 4 Schematic of the production, purification and characterization of AAV2 symmetry related mutants. ...................................................................................................................... 54 3 1 Schematic of the life cycle of MSV N within its plant host and insect vector. ................ 68 3 2 Characterization of purified MSV N components.. ............................................................. 69 3 3 Determination of the molecular mass and diameter of purified MSV N components. ..... 70 3 4 STEM micrograph of purified MSV -N components: C1 = fresh ca psomers, C2 = capsomers 1 month post purification, S = singles and G = geminate capsids. ................... 71 3 5 STEM histogram of MSV N a fresh capsomeric sample which appear to fit a Gaussian distribution. ............................................................................................................ 72 3 6 STEM histogram of an old capsomeric sample. ................................................................... 73 3 7 STEM histogram of purified MSV N single capsids. .......................................................... 74 3 8 STEM histogram of purified MSV N geminate. .................................................................. 75 3 9 MSV N pH assembly/disassembly assay.. ........................................................................... 76 3 10 MSV N pH Assay. ................................................................................................................. 77 3 11 STEM micrographs of MSV -N pH components. ................................................................. 78 3 12 STEM histogram of MSV N geminate capsi ds at pH 6.4. .................................................. 79 3 13 STEM histogram of MSV N pH assay reassembled singles (RS) at pH 4.8. .................... 80 3 14 Stem histogram of MSV pH assa y reassembled geminates (RG) at pH 4.8. ..................... 81

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10 3 15 MSV N pH assay in the presence of protease inhibitor. ...................................................... 82 3 16 STEM histogram of reassembled MSV N singles (pRS) at pH4.8 assayed in the presence of protease inhibitor. ............................................................................................... 83 3 17 STEM histogram of reassembled MSV N geminates (pRG) at pH 4.8.assayed in the presence of proteas e inhibitor. ............................................................................................... 84 3 18 Protease inhibited (PI) MSV -N pH assay assembly/disassembly. ...................................... 85 3 19 Kinase landscape of MSV N CP sequence kinase prediction with scores above 0.5 shown by uppercase letters and with size proportional to the scores. ................................ 90 4 1 Ribbon diagram of MSV -N CP. ............................................................................................ 97 4 2 pFast Bacmid map and the MSV NCP wt and mt gene inserted ........................................ 98 4 3 pFast Bac MSV N clones restriction digest analysis. .......................................................... 99 4 4 BMSV -NCP wt and mt clones restriction digest analysis. ................................................ 100 4 5 Western Blot of the time course experiment of the baculovirus expressed MSV NCP wt and mutant. ...................................................................................................................... 101 4 6 Ammonium sulphate precipitation fractions of baculovirus expressed MSV NCP wt and mt .................................................................................................................................... 102 4 7 Western blot analysis of ion exchange col umn chromatography of MSV NCP wt and mt ........................................................................................................................................... 103 5 1 Schematic of the life cycle of AAV2.. ................................................................................ 117 5 2 Ribbon diagram of AAV2 VP3 based on its crystal structure. ......................................... 118 5 3 Ribbon diagram of AAV2 VP3 based on its crytal structure, and the spheres show the position of each mutant. ................................................................................................. 119 5 4 Ribbon diagram of the AAV2 icosahedral 2 -fold axis with the orange spheres representing 2 -fold symmetry related interface mutants. .................................................. 120 5 5 Ribbon diagram of the AAV2 icosa hedral 3 -fold axis with the blue spheres representing 3 -fold symmetry related interface mutants. .................................................. 121 5 6 Ribbon diagram of the AAV2 icosahedral 5 -fold axis with the yellow spheres representing 5 -fold symmetry related interface mutants. .................................................. 122 5 7 Immunoblots of AAV2 mutant cell lysate. ......................................................................... 123

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11 5 8 Histogram of A20 value for e ach mutant versus A20 value of the recombinant wt AAV2.. .................................................................................................................................. 124 5 9 Histogram of the packaging ratio obtained for each A20 positive mutant. ...................... 125 5 10 Histogram of the log of the particle infectivty ratio obtained for each A20 positive mutant. .................................................................................................................................. 126 5 11 B1 dot blot of AAV2 mutants purified on a step iodixanol gradient. ............................... 127 5 12 Transmission Electron Micrographs of ion exchange column purified AAV2 mutants after iodixanol step gradients. .............................................................................................. 128 5 13 Visu alization of AAV2 capsids and intermediates. ........................................................... 129 5 14 STEM histogram of baculovirus expressed AAV2 intermediates. ................................... 130 5 15 STEM histogram of baculovirus expressed AAV2 capsid intermediates with intermediates attached. ......................................................................................................... 131 5 16 STEM histogram of baculovirus expressed AAV2 capsids. ............................................. 132 5 17 Immunoblot and SDS gel characterization of AAV2 capsids and intermediates.. .......... 133 5 19 Ribbon diagrams of different views of AAV2 internal polar cluster mutants (salmon colored) spheres. ................................................................................................................... 135 6 1 Proposed model for the assembly and disassembly of MSV N ........................................ 148 6 2 Proposed model for the assem bly of AAV2. ...................................................................... 149

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12 LIST OF ABBREVIATION S AAV Adenoassociated virus BMSV -NCP Baculovirus expressed maize streak virus CP Coat protein CPV Canine parvovirus dsDNA Double stranded DNA DLS Dynamic light scattering DSC Differential scanning calometry EDTA Ethylene diaminotetraacetic acid EM Electron microscopy MALDI Matrix assisted laser desorption/ionization MVM Minute virus of mice MSV Maize Streak Virus MSV N Maize Streak Virus (Nigerian Strain) MT Mutant ssDNA Single stranded DNA SDS Sodium dodecyl sulfate STEM Scanning transmission electron microscopy TMV Tobacco mosaic virus TYLCV Tomato yellow leaf curl virus VP Viral protein WT Wild type

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13 Abstract of Dissertation Presented to the Graduate School of the University of Florid a in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy BIOPHYSICAL CHARACTERIZATION OF THE ASSEMBLY AND DISASSEMBLY OF SSDNA VIRUSES By A ntonette D Bennett August 2009 Chair: Mavis -Agbanje McKenna Co-chair : Nicholas Muzyczka Major: Medical Sciences--Biochemistry and Molecular Biology V iruses are the causative agents of many diseases that affect plants and animals and the mechanisms by which they assemble and disassemble represent critical steps in their replicative life cycle. The focus of this study was to elucidate the steps involved in the formation and disruption of the viral capsid of the Geminiviridae and Pavoviridae family of viruses. Maize Streak Virus ( MSV -N ) belongs to the Geminiviridae family of virus. The Geminivir idae have caused significant economic loses and hardship due to their infection of a number of horticultural crops worldwide. MSV -N is a severe pathogen of maize and is used in this study as a model for understanding assembly and disassembly of this unique virus family. The goal of the study was to decipher the steps involved in the formation and disruption of the MSV capsid. This was accomplished by isolating and characterizing the capsid components and intermediates from maize agro inoculated with MSV -N These components and intermediates i nclude the characteristic geminate capsid a T=1 icosahedral (single) capsid, pentamers and dime rs of pentamers. This study also include d a pH assembly /diasassembly assay that has been generated for MSV and could be developed as a method to test peptide inhibitors of these processes.

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14 Adeno-Associated Virus serotype 2 (AAV2) belongs to the Parvoviridae family and it is an important therapeutic agent for clinical gene therapy. AAV2 has been extensively studied and it has b een used in the treatmen t of several genetic diseases. However, t here is still a large deficit in the amount of information available on the mechanism by which it assembles and disassembles. We have isolated and characterized subassembli es of recombinant A AV2. The se incl u de what appear to be pentamers, dimers of pentamers (DOPs) and pentamers of dimers (PODs) In the final as pect of this work, we use d the crystal structure of AAV2, other AAV serotypes, other parvoviruses and site directed mutagenesis to ide ntify residues and regions of the capsid that are important to the assembly and stability of the virus capsid.

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15 CHAPTER 1 FUNDAMENTALS OF VIRU S ASSEMBLY AND DISAS SEMBLY Virus Capsid Function Viruses are the causative pathogens of severe diseases and are ubiquitous in all organisms, infecting everything from bacteria to humans. To achieve such biodiversity they have evolved different strategies for host recognition, internalization, cellular trafficking, genome replication, capsid assembly and release of p rogeny to optimize their life cycle in their unique niche. This has resulted in viruses of different shapes and sizes, but invariably their viral coat proteins (CPs or VPs) form a protective shell (a viral capsid) around the infectious genomic nucleic acid which can be ssDNA, ssRNA, dsDNA or dsRNA. The packaged viral genome encodes all the required structural CPs (or VPs) and auxiliary non-structural proteins that are required, in combination with host proteins, for host infection. Some viruses, termed env eloped viruses, also incorporate their hosts lipids as either an internal and/or external envelope during assembly. For a number of viruses, CP recognition and encapsidation of the genomic nucleic acid is a prerequisite for infectious capsid formation, while for others the genome is packaged into preformed capsids via interactions with viral or host encoded proteins. In addition to genome encapsidation and protection during cellular entry and trafficking, the CP can also dictate many other viral functions, including host receptor/vector recognition, transmission and the genomic transduction efficiency during infection. The formation of the assembled mature viral capsid from CP (or VP) monomers is the focus of this study Virus Assembly Virus assembly, utili zing a limited number of VP or CP building blocks, is an excellent example of a directed macromolecular interaction occurring in nature. Two basic principles govern the assembly of spherical (icosahedral) viruses: (i) Genetic Economy the encapsidated

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16 gen ome encodes a single or few CPs that assemble a protective shell (the viral capsid); and (ii) specificity the CPs have to fold to recognize each other and form exact CP CP interfacial interactions during the assembly pathway. For spherical virus assembly, the CP organization in the capsid architecture takes on the form of an icosahedron (a platonic solid with point group symmetry 5.3.2). This symmetric CP shell (44) is a consequence of it consisting of identical (or almost identical) g ene products, consistent with the argument that there is insufficient volume inside a virus to accommodate a more complicated protein coding strategy. The exact 2 3 and 5 -fold symmetry of the icosahedron permits the (quasi) equivalence symmetry (31) re quired to construct structures with 60 or multiples (denoted by a T number) of 60 subunits. Significant effort has been dedicated toward understanding the driving forces behind icosahedral viral capsid assembly. Structural biology tools, such as X ray crys tallography and cryo -electron microscopy (cryo -EM) combined with 3D homology model building, enable the atomic visualization of interface interactions between protein protein subunits and protein nucleic acids in virus capsids (12, 71) Combined with biochemical and molecular biology analysis, these studies indicate a high degree of fidel ity in the steps that result in the assembly of mature infectious virus capsids. They also show that the fundamental principles governing successful viral capsid assembly, efficient polymerization of CP subunits utilizing specific interface interactions th at spontaneously terminate, often employ structural polymorphisms to facilitate t he r equired interactions ( e.g. (71, 72) ). However, there are still many unanswered questions regarding the numerous steps that ensure the polymerization of protein subunits, with precise accuracy, in the successful assembly of infectious virions. This study will limit itself to only include the simplest single stranded (ss) DNA families, which assemble a T=1 or twinned pseudo T = 1 icosahedral capsid.

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17 Experimental Techniques Used to Study Virus Assembly There are several methods available to study the forces involved in orchestrating the CP:CP or VP:VP interactions that occurs in virus a ssembly and disassembly. X ray c rystallography provide s the high resolution molecular information of purified viral CP and capsid in the crystalline state (71) Cryo -electr on m icroscopy provides low resolution structural information of virus capsids in the aqueous state (90) Negat ive stain electron microscopy provides information about the overall shape of the virus capsid and possibl y the shape of any intermediates (33, 176) Scanning transmission electron microscopy can be used to determine the molecular mass, as well as, visuali ze the virus capsid and any stable intermediates (109) Cryo electron tomography has been used to show viruses in different cellular organelles as well different stages of assembly (60) Analytical gel filtration chromatography, analytical ultra centrifugation and dynamic light scattering have been used to determine the molecular mass sedimentation coefficient, and the diameter of the viral capsid components, respectively (33, 176) The isolation of stable capsid intermediates has been shown to present numerous challenges based on the fact these intermediates are extremely transient (30) The use of CP mutants however, has been successfully used to generate capsid intermediates that were stable enough to be characterized (30, 32) The CP from many viruses can reversible assemble and disassemble in response to s olution conditions (174) for example, changes in pH (13, 14) CP concentration (65) ionic strength (146) temperature or the presence of a chaperone or scaffold (4, 100, 110, 124, 138) The determination of the specific chemical environment that facilitate assembly and disassembly of ssDNA virus es being studied in this study is a ddressed Similarly, certain small molecules have been shown to inhibit or misdirect assembly or disassembly. This is illustrated in the use of WIN

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18 compounds that stabil ize viral capsid of rhinovi rus (7, 10, 11, 99) which inhibit uncoati ng and infection (177) The detailed structures of viral capsids can illustrate how the CPs interact with each other and how they interact with their packaged genome (155) The determination of the CP interaction with each other and it s interaction with the viral genome is critical to the understanding of the assembly and disassembly processes. The polyoma VP1 protein for example cont barrel core and long flexible N and C terminal arms, the C terminus being extremely positive and may be important in binding the viral DNA (137) Both arms are dispensable for pentamer formation but indispensable for inter -pentameric interaction. In CCMV on the other hand 25 of the N termi nal residues of the CP contain a high proport ion of basic amino acids which have been modeled to interact with the viral RNA; deletion of these residues eliminates the ability of this virus to assemble RNA containing particles in vivo (158) To date significant progress has been made in the deciphering of the me chanism of assembly and disassembly of several ssRNA, dsRNA and dsDNA virus families (39, 118, 137) However, with the exception of the Microviridae very little is known about these mechanisms for the ssDNA virus families The ssDNA Virus Families There are six ssDNA virus families: Microviridae, Inoviridae, Nanovi ridae, Geminiviridae, Circoviridae and Parvoviridae (132) The first two families infect bacteria, the next two infect plants, and the last two infect mammals, with the Parvoviridae also infecting invertebrates. Despite this diverse host rang e and no genetic homology between them, the CPs/VPs for four of the six viruses ( Microviridae, Nanoviridae, Circoviridae and Parvoviridae ) assemble a T = 1 icosahedral capsid, the Geminiviridae assembles a unique twinned pseudo T = 1 icosahedral capsid, an d the Inoviridae are filamentous. This study discusses what is known

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19 about the structural architecture of the CPs/VPs and assembled capsids of two ssDNA virus families, the Geminiviridae and Parvoviridae T hese families provide two disparate examples of ca psid assembly that result in a T = 1 capsid or twinned pseudo T = 1 capsids: the Geminiviridae involving the assembly of two incomplete shells from a single CP to accommodate its genome; and the Parvoviridae which orchestrate interactions between t he commo n region of two to four overlapping VPs. Geminiviridae The Geminiviridae are a family of highly pathogenic viruses that cause significant crop losses due to their infection of horticultural plants such as maize, tomatoes, beans, squash, cassava and cotton (Table 1 1 (156)). They are normally transmitted from host to host via a whitefly or leafhopper vector. Efforts aimed at geminivirus control are mainly focused on identifying resistance/silencing genes that can be engineered into plant crops. The Geminiviridae package e ither one circular ssDNA genome (monopartite), of 2.5 2.8 kb, or two ssDNA molecules (bipa rtite, with component A and B) totaling 5.2 kb, into one or two different twined pseudo -icosahedral capsi ds, respectively. The capsid is ~ 200 x 400 in diameter. The Geminiviridae are divided into four genera: Mastrevirus, Curtovirus, Begomovirus and Topocuvirus based on the genome organization, host range and the insect vector used (126) Members of the Mastrevirus have a monopartite genome and are transmitt ed by specific species of leafhoppers ( Cicadellidae sp.). Members of this genus infect monocotyledonous plants; Bean yellow dwarf virus and Tobacco yellow dwarf virus infect dicotyledons. Maize streak virus (MSV -N ) is the prototype member of this genus. Th e Curtoviruses have monopartite genomes and are transmitted by leafhoppers to dicotyledonous Bemisia tabaci plants. Beet curly top virus is the prototype member of the genus. The Begomoviruses can have a single (e.g. Tomato yellow leaf curl virus (TYLCV) a nd Bean yellow vein mosaic virus or bipartite genome (e.g. Bean

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20 golden yellow mosaic virus ), and are transmitted by whiteflies and infect dicots. Bean golden mosaic virus is the prototype species. The genus Topocuvirus has only one member, Tomato pseudo-c urly top virus This virus has a monopartite genome and is transmitted by treehoppers. In this study, the Nigerian strain of MSV (MSV N) will serve as the viral model for the Geminiviridae (23) MSV N is one of the bes t characterized members of its family. The virus is transmitted specifically by the leafhopper Cicadulina mbila (Figure 1 1A ). The sy mptoms of MSV N infection include deformed cob and chlorotic stre aks on the maize leaves (Figure 1 1B ). The 2.7 kb genome e ncodes four gene products, CP, replication proteins Rep and RepA, and the movement protein, MP. The MSV N virion is assembled from 110 copies of a single CP (244 amino acids, 27 kDa). The CP is required for a variety of viral life cycle functions that incl ude; insect transmission, systemic infectio n and encapsidation of ssDNA (6, 21, 37, 94, 101103, 131) The expressed CP accumulates in the nucleus and facilitates nuclear and cell to -cell transport of the genomic ssDNA via an interaction with the MP. In addition, the Rep and RepA proteins have multifunctional roles that include genome replication (reviewed in (108) ). MSV -N Assembled Capsid Architecture The twinned quasi -isometric incomplete T = 1 icosahedral capsid (geminate) architecture for the geminiviruses was predicted from negative stain images and verified by the cryo EM image reconstruction of the MSV N capsid at 25 resolution (173) This geminate architecture is unique among all known virus families. The MSV N capsid dimensions are, 220 x 380 (Figure 1 2B). The 2.7 kb genome is packaged int o an internal capsid volume of 1.2 x 1063. The capsid consists of 22 pentameric capsomers and they are arranged with a 5.2 point group symmetry. Each incomplete icosahedr al head (55 CPs) is missing 5 CPs (a pentamer) compared to a regular T=1 capsid assembled with 60 CPs (singles) The structure is built from distinct pentamers that protrude radially outwards from the capsid surface (Figure 1 -2A) Three classes

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21 of pent americ buildin g blocks are observed. They are the two apical that are located at each end of the capsid; the 10 peripentonal that are adjacent to the apical pentamers; and the 10 equatorial that line the waist of the capsid at the interface between the two fused incomplete icosahedra (Figure 1 2A ). A gap of 25 containing fifteen regions of tubular density, which connects the two heads. A small protrusion is observed at the icosahedral 3 -fold axis and there is a small depression at the 2 -fold interfac es between non -equatorial pentamers. Recently, a cryo EM image reconstruction of single head capsids of MSV N with T = 1 icosahedral symmetry which package sub genomic ssDNA has been determined to 10.2 resolution (not shown; Reutzel et al. unpublished data). This structure shows a capsid constructed exclusively from pentameric building blocks that resemble the apical capsomers of the geminate The Structure of MSV N CP Monomer and its Symmetry Related Interactions A pseudoatomic model of the MSV N CP, residues 19 244, has been built into the cryo EM envelope of MSV N based on an amino acid sequence alignment with the CP of satellite tob acco necrosis virus (Figure 1 2B (173) ). The first 18 amino acids were not built because their location could not be unambiguously assigned. The MSV -N CP model contains an eight stra nded anti -barrel motif that forms the core contiguous capsid, the BIDG sheet is closest to the interior of the capsid. This core motif represents 60% of the CP amino acids. The MSV N CP model also contains two -helical regions, residues 19 34 a nd 138 (Figure 1 2B ). The N -ed in the EF loop (Figure 1 2B ). The 2 interaction at the c -termini of one CP (Figure 1 3A). In ea 2 he other CPs) to satisfy ten intops form the 3 -fold axis (Fig ure 1 3C). The main 5 -fold interface interaction includes the interaction

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22 The 5 -fold axis is formed by the GH loop, and it creates a channel that ru ns from the inside to the outside of the capsid (Fig ure 1 3E ). Geminivirus Assembly/Disassembly Studies The Geminivirus capsid is the most unique of all virus families. It has been illustrated that the assembled capsid is required for insect transmission and systemic host infection (94) However, while there is considerable data available about the infection (94) replication (24) transcription (24, 115) translocati on, and systemic infection (115) of the geminivirus within the host as well as its mode of circulation in the vector, further work is requir ed for the detailed dissection of the mechanism of assembly or disassembly. In geminiviruses, the CP can perform diverse roles. It is the only protein component of the virion and is required for vector transmission (9) and it determines insect specificity (26) .The C P is also important for the transport of the viral DNA in and out of the nucleus (89, 105, 153, 154) Deletion mutagenesis have been used to identify regi ons of the CP that are important for DNA binding and multimerization (153) The mechanism by which geminivirus es disassemble is not yet known except for the disassembly of African Cassava Mosaic Virus (ACMV) pH, heat tr eatment, EM and chemical cross -linking experiment s of ACMV was used to illustrate the expulsion of the viral genome while st ill attached to the viral capsid (70) In order to dec ipher regions of the geminivirus CP that are important for maintaining the capsid structure the mastrevirus CP sequence were aligned and residue K182 was identified as one of the most conserved residue. Mutations at this position, for example K182V have been shown experimentally to disrupt capsid assembly, proposed to be due to capsid instability (104) however, intermediates formed have not been characterized. In addition to this residu e the N terminal region also been shown to be important for MSV N assembly and DNA binding (101) Our study of the assembly and disassembly of MSV N will focus on isolating, purifying and

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23 characterizing assembly/disassembly components formed during a wild type infection. In addition, none of the antiviral therapies being explored is utilizing the disruption of geminivirus capsid assembly or the prevention of disassembly. The second part of the MSV N s tudies will focus on the environmental factors affecting these processes and the development of an assay that can be used to test geminivirus assembly or disassembly disrupters. The third part of this work will focus on the baculovirus expression of the MS V N mutant K182V, N terminal truncation and wt CP. Significance of Plant Virus Assembly/Disassembly Studies For several vector borne plant virus families, successful virus resistance has been reported by utilizing a process referred to as pathogen derive d resistance. Pathogen derived resistance is defined as the use of resistant gen es obtained from the viruses own genome. Two main examples, in which this method was successfully utilized by transforming the plant with viral CP gene, include tobacco resista nce to Tobacco mosaic virus (TMV) (120) and papaya resistance to Papaya ringspot virus (152) Resistance for TMV is caused by interference with viral uncoating. In the case of geminivirus es resistance has not been achieved using this approach because the mechanism of asse mbly and disassembly is not known and so it has been difficult to develop resistance strategies utilizing the CP (156) In the past, the pathogen derived resistance method utilized for g eminiviruses include the expression of the mutant or truncated viral proteins that in terferes with virus infection. The other method utilized to confer resistnce to geminiviruses is, the transcription of viral RNA sequences that silences the expression of virus genes. Recent methods include the use of Geminivirus inducible toxic proteins to kill the infected plant cell, or the expression of DNA binding proteins, peptide aptamers or GroEL homologues that either lessen th eir harmful effects or disrupt g eminivirus infection These methods have provided some success in several lab strains, the y have not provided durable resistance to g eminivirus infections

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24 ( reviewed in (144) ). The aim of our study is to decipher the mechanism of viral capsid assembly and disassembly and we b elieve that information on the se mechanism s will provide a platform for the design of improved control strategies Parvoviridae The Parvoviridae also exhibit diverse host range and tissue tropism, with an infection outcome spectrum of nonpathogenicity to lethality. The Parvoviridae package linear ( ) or (and +) sense ssDNA that is ~5 kb into capsids that are ~260 in diameter. The family is divided into two subfamilies: Parvovirinae which infect vertebrates, and the Densovirinae which infects insects (150) This study will focus specifically on the Parvovirinae, which infect a wide distribution of hosts including mice, cats, dogs, pigs, cows, monkeys and humans. This subfamily is subdivided into five genera: Parvovirus, Erythrovirus, Dependovirus, Amdovirus and Bocavirus The prototype member for the Parvovirus genera is Minute virus of mice (MVM) which infects mice, with other members infecting rats, cats, dogs and pigs. This genus contains both pathogenic and nonpathogenic strains. The genus Erythrovirus contains human parvovirus B19 as the prototype member and includes viruses that infect non -human primates such as rhesus monkeys and pigtailed macaques. The genus Dependovirus comprises the Adeno-associated viruses (AAVs), which require helper functions from viruses such as Adenovirus and Herpes virus for replic ation, hence the name of the genus. These viruses, for which there has been no disease association, have been isolated from a wide range of hosts, including mice, rat, cows, snakes, nonhuman primates and humans. The prototype member for this genus is AAV serotype 2 (AAV2). The Amdovirus genus currently contains one member, Aleutian mink disease parvovirus infecting minks. Members of the Bocavirus genus are bovine parvovirus which infect cows and human bocavirus recently discovered in children and associa ted with respiratory disease and gastroenteritis. For this study AAV2 will serve as the parvovirus model. This virus

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25 is also the best characterized member of the Dependovirus genus, mostly because of interest in its development and use for gene delivery a pplications. The AAV2 genome of 4.7 kb contains a replication (rep) and a capsid (cap) ORF. The rep ORF encodes four non-stru ctural overlapping proteins Rep78, Rep68, Rep52 and Rep 40 that are invol ved in viral DNA replication (116) The cap ORF encodes three overlapping structural proteins, VP1 (735 amino acids, 87 kDa), VP2 (598 amino acids, 72 kDa) and VP3 (533 amino acids, 62 kDa), that are alternatively spliced. These proteins have a common C -terminal region (the VP3 sequence) with VP2 and VP1 having N terminal extensions compared to VP3 of 65 and 202 amino aci ds, respectively. The AAV2 capsid is ~ 260 in diameter, is assembled from 60 copies (in total) of VP1:VP2:VP3 in a ratio of 1:1:8. The VP3 common region is known to be important for cell binding, antigenic recognition and genomic DNA packaging (20) VP1 and VP2 contain nuclear localization sequences and are suggested to play a role in transporting VP3 to the nucleus. VP1 has a unique N -terminal region (of 136 amino acids) which displays a phospholipase A2 activity required for endosomal escape during cellular trafficking (55) AAV2 Assembled Capsid Architecture The AAV2 capsid structure has been determined by cryoEM and X ray crystallography to 10 and 3.2 resolution, respectively (90, 168) The characteristic surface features of the cap sid include depressions at the icosahedral 2 -fold axes, three spike like protrusions around the icosahedral 3 -fold axes, and a cylindrical channel at the icosahedral 5 -fold axes surrounded by a moat (Figure 1 2C ). This surface topology forms a caspid that is, 220, 230 and 240 in diameter at the icosahedral 2 -, 3 and 5 -fold axes, respectively, with a diameter of 290 at the maximal extremity of the protrusions s urrounding the 3 -fold axes (Figure 1 -2D). The internal volume of the capsid that packages the 4.7 kb genome is ~3.5 x 106 3.

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26 The Structure of AAV2 CP Monomer and its Symmetry Related Interactions The crystal structure of the AAV2 capsid has provided information on the common VP3 amino acids 217 735 only, despite the fact that VP1, VP2 and VP3 were pre sent in the samples studied (168) The lack of ordering of the N terminal regions of VP1 VP3 is consistent for all parvovirus structures determined to date (reviewed in ref. (3)). The low copy numbers of VP1 and VP2 and possible multiple conformations of the N termini of all three capsid VPs, which is incompatible with the icosahedral symmetry imposed during the structure determination, are likely responsible for this apparent disorder. The main structural motif of the AAV2 common VP3 region is an eight -stranded anti barrel motif which forms the core contiguous capsi d with large inter -strand loops (Fig ure 1 2D). barrel i s oriented tangential to the capsid surface. The BIDG sheet also forms the interior surface of the AAV2 capsid. The inter strand an helical structure (Figure 1 2D ). A conserved loop region at the C -termini of the VP3 (residues 696 neighboring icosahedral 2 -fold related VP3 monomers to form the floor of the depression at t he 2 -fold axis (Figures 1 2D and 1 3B ). The 2 fold interface interac surface depre ssion at this axis (Figures 1 2C and 1 3B ). The 2 -fold is the thinnest region of the capsid shell, being only one polypeptide chain thick, as is also the case in the MSV -N c apsids. The interactions between the icosahedral symmetry 3 -fold related monomers are the most the most amino acid sequence variability among the parvoviruses and interdigitate to form the surface spike like protrusions around the icosahedral 3 -fold axes characteristic of members of the dependovirus gener a of the Parvoviridae (Figure 1 3D ). The spike like protrusions surround a depression at the icosahedral 3 -fold axi s (Fig ure 1 2C ). T he loop extensions from the narrow end

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27 -barrel wedge form the icosahedral 5 -fold VP3 symmetry related interactions with elements of the BIDG sheet and the N terminal residues in the adjacent monomer. In addition, -strands of adjacent VP3 monomers forming the floor of the conserved depression that surrounds t he icosahedral 5 -fold axis (Figure s 1 2D and 3F). As observed for MSV N AAV2 also has a channel that connects t he inside and outside of the capsid at the 5 ribbon which assembles a surface turret at this axis Parvovirus assembly/disassembly Studies The assembly/disassembly of several parvoviruses have been studied by: determining the fu nctions of the VPs in their life cy c le (27, 123) the characterization of any intermediates formed (107, 172) analyzing mutagenesis data, and by understanding the effects of other virally associated proteins (42, 66, 128) Genetic analysis of the expression of the major VPs of the parvoviruses proves that they are sufficient for capsid assembly, e.g. MVM (123) CPV (27, 136) ADV (38) B19 (27, 75) and AAV2 (57, 135, 165) The mutagenesis of several of the AAV2 VPs amino acids have shown that there are several conserved residues that are important for capsid assembly and disassembly (58, 145, 159, 165, 175) The first part of the AAV2 section of this study will focus on mutating residues involved in symmetry relate d in teractions of VP3 and combining the information with existing data to determine if there are regions of t he capsid that are imp ortant for capsid assembly. The examination of several parvovirus assembly systems have illustrated that there is an apparent connection between virus assembly and nuclear localization. For example, co expression of AAV2 VP3 with either VP1 or VP2 which both cont ain a NLS is important for the transport of VP3 to the nucleus, and VP2 is important for capsid assembly (66, 159, 160) N terminal VP2 deletion mutants that were unable to accumulate in the nucleus of COS cells were

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28 also not able to assemble into capsid; the N terminal addition of a NLS conferred assembly competence to VP3 (66) Similarly MVM VP2 mutants which affected nuclear transport also affected assembly (107) and a panel of CPV mu tants which are located within possible NLS also appeared to affect assembly (151) The identification of VP multimers or subassemblies represents potential steps in the assembly of the parvovirus capsid. Sedimentation analysis of soluble cytoplasmic pools of newly synthesized AAV2 VP1 3 showed capsid protein oligomerization and no capsid. This oligomer ization is consistent with VP monomers and pentamers (160, 161) which were presen t in the cytoplasm of the cell. The next obse rvation which represents a step in virus assembly is the formation or accumulation of empty AAV2 capsids in the nucleus. Trimeric subassemblies were identified for MVM and CPV (107, 133, 172) The final section of this study will focus on the purification and the biophysical characterization characterization of assembly/disassembly intermediates of baculovirus expressed AAV2. There are several proteins that are believed to be associated with the assembly and packaging of the parvovirus capsid. For example w hen CPV VPs were expressed alone in mammalian cells they assemble utilizing more VP1 compared to capsids formed in infected cells; this suggests an influenc e of other vi ral gene products in the assembly process (172) Nucleolin is a nonviral protein that has been identified to be associated with assembled AAV2 capsid (73, 128) The nonstructural proteins (NS1) e x pressed by the parvovirus genus and the Rep proteins from the dependoviruses have been shown to be important for the packaging of the viral genome (41 43, 47, 91, 125) Significance of AAV2 Assembly Studies AAV has several key features which makes it an ideal vector for gene therapy applications. AAV i s stable and exhibits no pathogenicity, it can be manufactured and purified in a high titer

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29 and it can als o be utilized in a form that does not express any viral gene product. Consequently, there are several clinical trials that are presently complete tha t utilized AAV vectors to treat diseases like hemophilia (76 78) cystic fibrosis (48 50, 5254) retinal diseases (1, 2, 5, 62) neurodegenerative diseases (Parkinson and Alzheimer) (79 84) and lysozomal storage diseases (45, 63, 134, 139, 140, 157, 170) These factors have generated significant interest in all aspects of the basic b iology of AAV To date there is a limited amount of data a vailable on the mechanism assembly of AAV viral particles. Furthermore, it has been shown that tissue specifi city, immune evasion and vector retargeting can be enhanced by using different serotypes and chimer ic vectors (36, 46, 59, 61, 97, 98, 122, 143, 149, 166) Our study of the assembly and disassembly of AAV2 will focu s on isolating, purifying and characterizing assembly/disassembly components formed during a baculovirus expression ; as well as, the mutational analysis of symmetry related interaction s of VP3. The information obtained from the proposed assembly and disass embly studies will expand our basic knowledge of the interactions/conditions controlling theses processes, a requirement for vector development. Understanding the basic mechanism of assembly of AAV2 will provide the ground work for understanding this mecha nism in other AAV vectors (different serotypes and chimeras) as well as determine residues or regions of the capsid that can or cannot tolerate change Overall Goals The overall goal of this project is to biophysically study the steps involved in the as sembly and disassembly of two families of ssDNA viruses, the Geminiviridae and Parvoviridae The study is divided into three specific aims. The first aim is focused on utilizing biophysical techniques to characterize assembly/disassembly intermediates. The second aim will expose the virus capsids to different host environmental conditions to di ssect their role in the

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30 assembly/disassembly process. The third aim is to utilize the available 3 dimensional structure s to generate assembly/disassembly mutants and to characterize the mutants generated.

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31 Figure 1 1. MSV N Infection. A) Symptoms caused by MSV N which includes deformed cob and chlorotic streaks on leaves B) Cicadellidae mbilla the leafhopper that transmits MSV N

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32 Figure 1 2. MSV N and AAV2 virus capsid and CP/VP structures. Surface representation of the virus capsid (A) MSV N with A=apical, P=peripentonal and E=equatorial pentamer locations and (C)AAV2. Ribbon diagram of (B ) MSV N CP and (D ) AAV2 VP3.

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33 Figure 1 3. Capsid protein in terfaces. Ribbon drawings of the icosahedral 2 3 and 5 -fold symmetry related interfaces for MSV N CP [ ( A), (C), and (E)] and AAV2 VP [ (B), (D), and (F)]

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34 Table 1 1. List of the economically important viral diseases caused by geminiviruses Disease na me Virus genus Virion structure Host crop Epidemic region Yield loss Maize streak disease Mastrevirus Monopartite Maize Sub Saharan Africa Average 20%, up to 100% Cassava mosaic disease Begomovirus Bipartite Cassava Africa, India Overall 15 24%, up to 90% Cotton leaf curl disease Begomovirus Monopartite (associated component) Cotton Pakistan Average 30%, up to 80% Bean golden mosaic disease/bean golden yellow mosaic disease Begomovirus Bipartite Bean Florida, Central and South Ameri ca 10% 100% Yellow mosaic disease Begomovirus Bipartite Grain legumes India 10% 90% Tomato leaf curl disease/tomato yellow leaf curl disease Begomovirus Monopartite Tomato Europe, Asia, Americas, Australia 20% 80%, up to 100%

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35 CHAPTER 2 MATE RIALS AND METHODS Introduction This chapter describes common experimental methods and reagents utilized throughout this thesis This study is divided into four main sections: two sections are based on the purification and biophysical characterization of MSV N and AAV2 CP/VP intermed iates; the other two sections are based on the generation and biophysical characterization of the CP/VP mutants that were predicted to be important for the assembly and disassembly of both viruses Purification of MSV -N CP Maize streak virus ( MSV N ) capsids and intermediates were purified from maize plant leaves agro inoculated with MSV N (25) The symptoma tic leaves were harvested t ~ 25 days post agro innoculation. Infected maize plant tissue were cut into small pieces (~3 cm in lengths), rapidly frozen and stored at 200 C (173) The infected plants were kept brittle with the use of liquid nitrogen, crushed with a mortar for approximately 30min or until the leaves were powder like in appearance. The crushed leaves were transferred to a blender along with 500ml of Buffer A (0.1 M NaAc pH 5.2 1mM Ascorbic acid and 1mM EDTA ), 5mg of cellulose, and 7.5mg of hemice llulase. The mixture was burst blended (1min every 10min ) for a total of 1hr using a blende r. Plant material was separated from the soluble virus containing fraction by adding 250ml of chloroform and the mixture was stirred at 4 for 30min .The mixtu re was centrifuged for 30min at 9961xg The clarified aqueous upper layer was transferred to anoth er flask with 10% ( PEG 8000) and 125mM NaCl added The mixture was allowed to stir overnight at 40C and the virus was pelleted at 10400xg for 90min The pell et was resuspended in 50 ml Buffer B (0.1 M NaAc pH 4.8). The mixture was further purified and concent rated on a 5 ml 15% sucrose c ushion and centrifuged at 210 000xg for 3hr. The pellet was resuspended in 1 ml Buffer B and loaded unto a

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36 5 40% step sucr ose gradient. The blue bands were dialyzed against Buffer B and the virus concentration determined at 260nm wavelength and extinc tion coefficient of 7.5. The supernatan t from the 15% sucrose cushion wa s diluted 2 times and loa ded unto a 5% sucrose cushion. The pellet formed was loaded onto a new sucrose gradient (1 ml 10% sucrose, 5 ml 15% sucrose, and 5 ml 20% sucrose). The virus sedimenting into tight blue bands which appears blue under white light were extracted with a needle an d syringe and dialyzed against Buffer B The virus bands were dialyzed into final Buffer (0.1M NaAc pH 4.8). The MSV -N purification scheme is illus trated in Figure 2 1A. Bioph ysical Characterization of MSV -N CP Components The MSV -N c ontaining sucrose gradient fractions were characterized by a series of biophysical techniques. These techni ques include negative stain EM, 12% sodium dodecyl sul phate (SDS) polyacrylamide gel e lectro phoresis (PAGE), western blot, analytical gel filtra tion chromatography, STEM and DLS Nega tive Stain EM 5 L of sample was loaded unto carbon coated copper grid s and negatively stained with 5 L 2% uranyl acetate. The g ri ds were air dried then examined in a JOEL 1200 EX transmission electron microscope. The instrument was set to collect images at 50,000x magnification and on film. 12 % SDS PAGE and Western Blot 10g of each sample was denatured and loaded unto two differ ent 12% SDS gel One gel was co o massie stained and the other gel was transferred to a ImmobilonP tr ansfer membrane (Millipore, cat#IPVH20200) for western blot analysis using polyclonal antibody rai sed against purified MSV N CP as the primary antibody and goat anti rabbit antibody (Biorad, cat # 170-

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37 6515) was used as the secondary antibody (1:10,000 dilution ). The MSV N a ntibody complex was detected with the EC L substrate (Amersham cat#RPN 2109) Genome extraction and a garose gel electrophoresis The genomic material of each sample was isolated by phenol chloroform extraction and ethanol precipitation. 10g of each sample was digested with 4l proteinase K (Roche, cat#1373196) 20L of 10x proteinase K Buffer (10mM Tris HCl pH 8.0, 10mM EDTA 10% SDS) and the solution diluted with sterile distilled water to a total volume of 200l The mixture was incubated for 1 hr in a water bath at 370 C, treated twice with an equal volume of phenol chloroform (Roche, cat#1373196) and the upper aqueous layer transferred to a clean eppendorf after each extraction. The aqueous fraction was then treated with chloroform (Fisher cat#C298500) and the aqueous layer transferred to a clean eppendorf. The DNA was precipitated overnight at 200C, after adding 10% NaAc pH 5.2, 1l glyc ogen, and 3x volume of 95% ethanol. The sample was then pelleted for 20 min at 13050xg The pellet was air dried and resuspended with 20L water The sample was loaded unto a 0. 8% agarose gel with ethidium bromide (Bio rad, cat#1610433) and the DNA bands detected with the program Gel Doc. Native Gel A nalysis Native gel a nalysis was performed by loading three known protein standards, bovine serum albumin (BSA), alcohol dehydrogenase mass s 66kDa, 150kDa and 20 0kDa respectively and 5g of MSV N fraction purified from the 10% suc rose gradient unto a native p hast gel (GE Healthcare cat#17 051701) ((4 -15%). The gel was then silver stained in order t o detect all the proteins Analytical Gel Filtration Chromatography Analytical gel filtration chromatography was performed with a superpose 6 analytical column (Amersham Pharmacia) calibrated with the same proteins used in the native gel as well

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38 as carbon ic anhydrase (30kDa) and apoferritin (400kDa) and Blue Dextran. The elution volumes of the standards were plotted against the log of their molecular mass ; this is defined as the calibration curve for the column. 250L of purified MSV N containing fractions were loaded unto the pre -calibrated and pre -equilibrated analytical superpose 6 gel filtration column using the AKTA FPLC system. The system pump was set at 0.5 ml /min and the elution volume fraction at 1 ml The elution volumes for MSV N fractions were det ermined and the molecular mass calculated from the calibration curve. Scanning Transmission Electron Microscopy (STEM) Scanning Transmission Electron Microscopy (STEM) was performed at Brookhaven National Laboratory (BNL). The technique has the advantage of accurately measuri ng molecular mass of relatively small amount s of heterogeneous, macromolecular assemblies. The technique utilizes 2L of unstained sample at a concentration between 50300 g/L. The sample are loaded onto 2.3m m carbon coated titanium grids and fre eze -dried in liquid nitrogen at ultra high vacuum. The grids are then transferred to the microscope which is operated at 40 keV and utilizes a 0.25nm focused probe for scan ning. An array of detectors collect s scattered electrons, with the intensity signal detected being proport ional to specific features of the specimen and can be use d t o calculate the mass of the sample in the path of the beam. The software packaged PCMASS 27 (developed at BNL and available at stem/bnl.gov) was used for v iewin g, manual mass measurement and statistical summaries The program calculates net signal (intensity minus background) for each pixel and multiplies it by a calibration cons tant. The calibration constant wa s determined from TMV for which the expected mass/length ratio is 133kDa/nm The absolute accuracy of the system ranges from 10% for a 100kDa molecule to 4 % for a 1MD molecule The MSV N samples masses were determined by PC MASS 27 and t he statistical analysis was performed using the program Graph Pad Prim (114)

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39 D ynamic Light Scattering (D LS ) The Precision Detectors Model PDDLS/Batch and Cool/Batch is the instrument that was used to collect and process D LS data for the differ ent MSV N components. The instrument was first equilibrated with 500 L of Buffer B. 500 L of MSV -N containing fractions at a concentration of 0.5 mg/ ml in a cuvette and they were placed in the instrument The fluctuations in scattered light intensity were monitored; their correlation function calculated and deconvoluted into a distribution of diffusion coefficients and a radii calculated for the scattering particles. Data acquisition, recording and analyses were performed by the pr ogra m Precision Deconvolution Development of MSV -N Capsid Disassembly and Reassembly Assay The final or stabilizing Buffer for MSV N g eminate capsids is 0.1M NaAc pH 4.8. To test the effect of pH on ca psid stability, 200L of sample in the final Buffer was d ialyzed into Buffer s with pH changes of 0. 2 pH units in the range 4.8 to 7.2, for a period of ~24 hr at 4C. 20l of the sample was removed at the end of each dialysis run for analysis by negative stain EM and gel electrophoresis. For each sequential chan ge in pH, all other chemical environmental factors, such as ionic strength, the presence or absence of divalent ions, temperature, and i nitial capsid concentration, were all kept c onstant. The same procedure was used for screening the effect of ionic stren gth, utilizing changes in NaCl concentration. Ethylene diaminotetraacetic acid ( EDTA ) is a divalent cation chelator, thus in addition to evaluating the effects of ions on the capsid structure; this chelator was added to remove divalent cations that might a lready be present in the viral capsids. MSV -N CP Post Translation Modification Determination All purified MSV N components with the exception of Gemini capsids, resolve as two CP bands when analyzed by SD S PAGE and western blot analysis To decipher this differential

PAGE 40

40 electrophoretic mobility of the CP the samples were checked to see if they were post translationally modified MSV N geminate ca psid at pH 7.2 was exposed to a sequential increase in conce ntration of DNase (Promega, cat # M10A) and calf intes tinal alkaline phosphatase (CIAP) (Invitrogen, cat #18009019). CIAP was used to digest any available phosphate in both the geminate capsid and disassembled geminate capsid at pH 7.2 and the DNase was used to degrade any unprotected DNA. The recommended st art ing concentration of CIAP is 10units for 10M of protein and for DNase it is 1 unit to 10 g of DNA. The samples were allowed to incubate at 37C for 1h r before loading unto a 12 % SDS gel To check if the CP is phosphorylated as well as if it binds DNA, the samples wer e treated with both CIAP and DNase The MSV -N sample was then treated with 20units DNase for 1 hr at 3 C followed by 80 units of CIAP and the mixture was allowed to incubate at 37C for 1 h r before it was loaded on a 12% SDS gel Baculoviru s MSV -N (Wildtype (wt) and Mutant (mt)) CP Production and Purification To clone MSV N CP (wt and mt) into the Spodoptera frugiperda (sf9) insect cells the genome sequence of MSV N CP wt ( MSV N CP) MSV N CP mutants K182V ( MSV -N CP182), and N terminal trunca tion (MSV N CP201) were digested from Eschericha Coli (E. coli) pET vector (101) and ligated into a pFAST baculovirus transfer vector The recombinant transfer vector is the n homologously recombined i n a specialized E.coli vector s (DH10 Bac) which contains the b acmid genome The expression protocol was used according to the manufacturers protocol (Invitrogen, cat#10359016) The recombinant baculo virus MSV N CP (BMCP), MSV N CP182 (BMCP182) and MSV N CP2 01 (BMCP201) were transfected into sf9 insect cells. The innoculum was then amplified and used for subsequent sf9 infection.

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41 Cloning of MSV -N Recombinant Donor P lasmid (pFastBac) The components of the transfer vector pFastBac are illustrated in figure 4 1 The vector is controlled by the Autographa Californica Multiple Nuclear Polyhedrosis Virus (AcMNPV) polyhedron promoter. The expression cassette is fla nked by the left and right arms of Tn7, it also contains a gentamicin resistance gene and a SV40 polyade nylation signal. The restriction enzymes EcoR I and XbaI were used to extract the region of the pFast Bac ORF th at will accommodate the MSV N CP, MSV -N CP182and MSV N CP201 genes. 5 g of pFas tBac was digested with EcoR I and XbaI in a 50L total volume. The MSV N CP and MSV N CP182 were released from the pET vector using the re striction enzymes BamH1 and XBa1 while the MSV N CP201 was digested with BamH1 and Nde 1 The newly generated protruding ends were blunted usin g Klenow fragment and incubated for 15 min at ro om temperatue. The blunt ended pFast Bac was further dephosphorylated with CIAP and purified by phenol chloroform extraction and ethanol precipitation. The pFastBac and the MSV N CP, MSV N CP182and MSV N CP201 genes were ligated with DNA ligase (NEB cat#M0202 S) 1L of ligation mixture was used to tra nsform electrocompetent JM109 cells The recombinant donor b acmid P MSV N CP, P MSV N CP182and P MSV N CP201 genes were screened on gentamicin agar plates and their proper ori entation determined by BamHI, FspI and NcoI restriction enzyme analysis Transformation of DH10 Bac E.Coli Cells DH10 Bac E. c oli c ells were purchased with the Bac to Bac Expression Systems (Invitrogen cat#10359016). The DH10Bac E coli contains a baculovirus shuttle vector (bacmid) and a helper pl asmid. The Bacmid is specialized as it contains a mini attTn7 target site adjacent a The helper plasmid encodes a transposase as well as tetracycline resistance The key feature of this DH10Bac Ecoli is that it facilitates the trans position of the P MSV N CP, P MSV N CP182 and P MSV N CP201 into the recombinant Ba cmid by homologous

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42 recombination between the pFastBac (Tn7) and the mini attTn7 Bacmid The main advantage of th is cell is the ability to generate blue and white colonies depending on whether the MSV N gene is inserted into the bacmid gene segment that encodes Lac Z This is the basis of the blue and white screen. Lac Z is the enzyme that interacts with galactosidase which is a component of bluo gal (Invitrogen cat#15519028). This reaction causes the substrate and consequently the colony to look blue If th e P MSV -N CP gene is inserted in the B acmid then the colony generate d will be white but if it is not properly inserted into th e B acmid then, Lac Z will be expres sed and the colony will be blue 5ng of recombinant P MSV N CP, P MSV -N CP182 or P MSV N CP 201was add ed to 100L of DH10 Bac cells in a pre-chilled 15 ml round-bottom polypropylene tube, the cells were gently mixed then incubat ed on ice for 30min The cell and DNA mixture were then heat shocked for 45sec at 45C then immediately transferred to ice and chill ed for 2min 900L of S.O.C medium (Invitrogen cat.# 15544034) was added to the newly transformed cells. The tube containing the transformants was shaken for 4hr at 225rpm. The transformation mix was then serially diluted and 100L of each dilution plated on Luria broth (LB) agar plates containing 50g/ ml kanamycin, 7g/ ml gentamicin, 100g/ ml bluo -gal, and 40 g/ ml IPTG (bluo gal plates). The plates were incubated for 48 hr at 37C and white colonies were pick ed and restreaked on fresh bluo -gal plates and incubated overnight at 37C. Once the phenotype was confirmed the clone was used to inoculate LB medium containing 50g/ ml kanamycin, 7g/ ml gentamicin with a shaking speed of 250 rpm for 24 hr at 37 C. 1ml of the cells were harvested in a 1.5 ml microce ntrifuge tube by cent rifugation. The supernatant were then removed by aspiration and the cells gently re suspended in 0.3 ml of solution I (15mM Tris pH 8.0; 10Mm EDTA, 100g/ ml RNase A), mixed gently with 0.3 ml of solution II (0.2M NaOH, 1%SDS), then incubated for

PAGE 43

43 5 min at room temperature. 0.3ml potass ium acetate (3M, pH 5.5) was slowly added and the contents of the tube was gently mixed and incubated for 10 min. After centrifugation at 14000xg for 10min, 1:1 ratio of the supernatant will be mixed with iso propanol and kept on ice for 10 min. The D NA was precipitated after centrifugation with 0.5 ml ethanol and the sample air dried. The bacmid was resuspended in 40l of Tris EDTA, pH 8.0 and analyzed by agarose gel electrophoresis The purified BMSV N CP, B MSV N CP182 or B MSV N CP 201 was used to tra nsfect sf9 cells to generate P1 stocks. Transfection of sf9 Cells 1g of purified bacmid DNA was added to100L unsupplemented Graces medium was added to a mixture of cellfectin (6L) and unsupplemented media (100L) The mixture was then incubated for 45 m in at rt it was then added to the 6 well tissue culture plate c ontaining 0.5 x 10 6 sf 9 cells and 0.8 ml of Graces m edium. The cells were allowed to incubate for 5 hr and then the DNA:Ce llfectin mix removed. 2 ml sf 9 medium was then added to the cells and it was allowed to incubate for 72 hr Th e 2 ml of media contained the recombinant bacmids and the sample represents the P1 viral stock. The schematic for the Baculovirus cloning and expression of B MSV -N CP, B MSV N CP a nd B MSV N CP is illustrated in Figure 2 2 Baculovirus MSV -N CP Expression The P1 viral stock was amplified to P2 then P3 using a moi of 0.1. The P3 viral s tock was then used to infect 300l of sf9 suspension ce lls at a moi of 5 for 72 hr in a 24 well dish. 300L of cells were collected 0hr, 24hr, 48hr, and 72hr The cells were harvested and resuspended in lysis Buffer (50 mM Tris, pH8.5, 20m M EDTA, 0.15 M NaCl, and 1%w/v Triton X 100), 50mM Tris pH 8.5) and recombinant B MSV N CP, B MSV N CP182 or B MSV N CP 201 proteins released from the cells by 3 freeze thaws. The cell lysate was then clarified by centrifugation at

PAGE 44

44 10,000 rpm for 15 min The expr essed proteins in the cell lysate were analyzed by SDS PAGE wi th coomassie blue staining and w estern blot analysis. Baculovirus MSV -N CP Purification T wo methods were utilized to purify MSV -N from the clarified cell lysate The first method attempted was the use of a 5 ml HiTrap SP column (GE Healthcare, cat#17 515801) This method was selected based on the reported pI o f MSV -N which is 10.8 and higher than what is reported for most known proteins and so would selectively bind the SP column at a pH below its pI The cell lysate was diluted 2 x with Buffer C (20mM Tris pH=8.5 and 15mM NaCl) loade d onto a SP column that was previously equilibrated with 25 ml of Buffer C and at a flow rate of 2.5 ml /min The column was washed with 50 ml of Buffer C then eluted with 50 ml Buffer D (20mM Tris pH=8.5 and 1M M NaCl) at 2.5 ml /min. All the samples were checked for the presence of MSV N by western blot analysis. The second method attempted was the use of ammonium sulphate precipitation. T he proteins were salted out of the cell lysate by sequentially adding 10% (NH4)2SO4, stir red at 40C for 30min, and centrifuged at 5,000xg for 30min. T he expr essed proteins in the cell lysate were analyzed by SDS -PAGE wi th coomassie blue staining and w estern blot analysis Purification of Baculovirus AAV2 VP1 -3 Cell Culture and Virus Infection The recombinant baculovirus vector containing AAV2 VP1 3 was donated by Sergei Zolotukin (Gene therapy Center, University of Florida ). An appropriate volume of the recombinant bAAV2 was used to infect sf9 cells ma intained at 2.0 x 106 cells/ml of suspens ion at 270 C. The cell s were harvested 72hr post infection by centrifugation at x1100g for 15 min and the pellet collected.

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45 Virus Purification The recombinant AAV2 was purified according to the published protocol (101) The pellet was resuspended in lysis Buffer (150 mM NaCl 50mM Tris, pH8.5) and freeze thawed 3x in a dry ice ethanol slurry and 370C water baths. 1l Benzonase ( Sigma, cat#E1014) was added to the cell lysate (50 U/ ml final concentration) and incubated for 30min at 370C. The crude cell lysate is clar ified by centrifugation at 4000xg for 20min and the clarified supernatant was then loaded onto a discontinuous 15 60% iodixanol step gradient prepared from Optiprep (Nycomed, cat#1114542) The iodixanol gradient was th en centrifuged at 350,000xg for 1hr at 180C. The gradient was fractionated and the fractions contain in g VP1 -VP3 d etermined by Western blots probed with B1 as primary antibody (178) The fractions from the higher percentage iodix anol gradient (40/60%, 40%, 40/25 and 25%) were further purif ied on an anion exchange 5 ml HiTrap Q column (GE Healthcare, cat#17 515901). The clarified cell lysate was diluted with 2 x Buffer E (20Mm Tris, 15mM NaCl, pH8.5) loaded onto the column at 2.5ml /m in, washed with 50 ml Buffer E, and the sample was eluted with 25 ml Buffer F (20Mm Tris, 15mM NaCl, pH8.5). T he fractions were collected and biophysically characterized The schematic for the purifica tion of Baculovirus AAV2 VP1 3 is illustrated in Figure 2 3 Biophysical Characterization of AAV2 VP1 -3 Components These techniques are the same as those used for the characterization of MSV N and so the main focus of this section will be on the use of the reagents relevant to AAV2. SDS Gel Electrophoresis and Immunoblots The primary antibody used for the detection of VP1 3 is B1 diluted 1:3,000and it recognizes an epitope in the C terminus of VP3 A20 and C37 were used to detect the conformational epitope present on assembled capsid. The secondary antibody use d to detect B1, C37 and A20 is the anti-mouse antibody diluted 1:5,000. T he AAV2 antibody complex was

PAGE 46

46 detected with the ECL substrate (Amersham, cat#RPN2109). The immunoblots were treated as b oth denatured and native dot blot s a s the names suggest the nati ve samples were unboiled and the denatured samples were boiled for 5 min at1000C. The Production and Characterization of AAV2 VP1 -3 Mutants The production and biophysical characterization of AAV2 assembly mutants is divided into 4 parts. The fi rst part is the visualization a nd structural comparison of the dimer, trimer and pentamer of AAV2 and MVM by using their crystal structure s The second part is the site directed muatagenesis of the VP. The third part is the transfection of the Human Embryonic Kidney 293 cell line (HEK 293 cells ) to express the mutant proteins. The fourth part is the biophysical characterization of the expressed mutant VP. Selection of residues for mutagenesis Symmetry operators were used to generate the dimers, trimers and pentamers of AAV2, MVM and MSV -N in the program O. The c o ordinates for the monomers of AAV 2 and MVM were accessed from the protein database (PDB, accession #s 1LP3, 1Z14, respectively). The coordinates for the dimer, trimer, and pentamer interface were th en written in the PDB format and saved. Input files for CNS were then generated and SEGID ID created for each interface. The data was then merged using the CNS subroutine called merge. A table of the interface residue distances was then generated in the CNS subroutine called contact, the maximum cut off distance used was 3.2 Pymol was used to generate the images of the monomers, dimer s, trime s and pentamers. A list of 19 mutants was generated based on residues that were st ructurally conserved between AAV2 and MVM. Another 4 residues were selected based on residues that were identified in previous mutagenesis studies that were believed to be important for AAV2 assembly (20, 57, 58, 165)

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47 Site Directed Mutagenesis of AAV2 VP3 The AAV2 plasmid pIM45 was used as a template for site directed mutagenesis. Site directed mutagenesis was performed according to the Stratagene site directed mutagenesis kit (Strategene, cat#200524) The primers were generated with silent mutation including a specific restriction enzyme site to facilitate the screening of the PCR clon es generated. The PCR product was then digested with DpnI to remove the parental DNA. The clones were selected based on ampicillin resistance and the restriction enzyme screen. Positive clones were sequenced and the mutated gene sub-cloned into the pIM45 backbone to eliminate any background mutations Cell Culture HEK 293 cells were maintained in Dubelccos modified Eagle s medium supplemented with penincillin and streptomycin at 100U/ ml and 10% FBS and they were maintained in 15cm3petri dish at 370C and 5% CO2. Transfection and Recombinant AAV2 Production The mutants for AAV2 were generated by site -directed mutagenesis in the non -infectious AAV plasmid pIM45 template (Figure 2 6) which contains all the AAV 2 DNA sequence e xcept the two terminal repeats required for DNA packaging(112) To produce rAAV with mutan t caps id protein, 293 cells were triple tr ansfected with mutant pIM45 (18g) pXX 6 (54g) which contains t he adenovirus helper genes (167) a nd pTRUF11 (18g) which contains the green fluorescent protein (gfp) driven by the CMV promoter and the AAV terminal repeats required for packaging (85) Transfection will utilize 293 ce lls at 75% confluency mixed at 3 :1:1 ratio of the three plasmids (pXX6: pIM 45: pTRUF 11) res pectively. The plasmids were Ce sium Chloride (CsCl) purified and used to transfect the cells by calcium phosphate precipitation, and incubated for 48hr at 370C. The cells were then harve sted by centrifugation at 1,140xg for 20min and the pellet resuspended in 10 m lysis Buffer (0.15 M NaCl 50mM Tris HCl pH 8.5) .T he virus was

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48 then released by three freeze/thaws and treatment with benzonase to remove nucleic acid. The lysate was clari fied by cent rifugation at 3,700xg for 20min and was further purifie d by a step iodixanol gradient and ion exchange chromatography. The purification is described in the Baculovirus expressed AAV2 purification section. Biop hysical Characterization of AA V2 VP1 -3 Mutants The characterizations of the AAV2 mutants generated were di vided into two main sections. The first section includes immunoblots, A20 Elisas, RT PCRs, and GFP assays of the cell lysate. The second section includes the purification by iodixa nol gradient and visualization by EM of mutant capsids and subassemblies. Gel Electrophoresis and Immunoblot s The crude cell lysate was analyzed similar to the purification of AAV2 baculovirus expressed VP The only additional detail was the use of the 1F antibody to determine the expression of the replication protein. Total Virus Capsid, Genome and Infectivity Quantification Total capsid t iter was determined by A20 enzyme linked immunosuorbent assay (ELISA) according to the manufacturers instruction (Amer ican Research Product, cat# PRATV). The clarified cell lysate were serially diluted and 100l added to the kit well. The readings that were within the detection limit of the kit were used to calculate the both empty and full particles. Total packaged genome or copy number was determined by real time PCR (RT PC R). 20 L of crude lysate was treated with DNAse1 for 1hr at 370C to degrade any DNA that is not encapsidated and each sample was digested with 4l proteinase K (Roche, cat#1373196), 20L of 10 x protei nase K Buffer (10mM Tris HCl pH 8.0, 10mM EDTA, 10% SDS) and the solution diluted with sterile distilled water to a total volume of 200l. The mixture was incubated for 1hr in a water bath at 370 C, treated twice with an equal volume of phenol chloroform ( Roche,

PAGE 49

49 cat#1373196), and the upper aqeous layer transferred to a clean eppendorf after each extraction. The aqueous fraction was then treated with chloroform (Fisher cat#C298500) and the aqueous layer transferred to a clean eppendorf. The DNA was precipit ated overnight at 200C, after adding 10% NaAc pH5.2, 1l glycogen, and 3xvolume of 95% ethanol. The sample was then pelleted for 20min at 13050xg. The pellet was air dried and resuspended with 20 l water. 1l of the viral DNA, 1l of primers (forward and reverse) to GFP, 12.5l of iQ SYBR Green Supermix which contains Taq DNA polymerase (Biorad, cat# 1708882 ) was combined to a total volume of 25L with sterile water. The sample was run on the Biorad my IQ system The infectvity or infectious units/ ml of mutants was determined by GFP Assay (165) The crude cell lysate w as serially diluted with DMEM containing Ad5 at a moi of 10, and a 100 L of each dilution was added to the 80% confluent HEK 293 cells, that were grown in a 96 well plate. The cells were then incubated for 24 hr at 370C and the number of green cells for each dilution counted using the fluorescent microscope AAV2 Capsid Wildtype and Mutant Purification The wt and mt cell lysates were loaded onto a discontinuous 15 60% iodixanol step gradient prepared from Optiprep (Nyco med, cat#1114542). The iodixanol gradient was then centrifuged at 350,000xg for 1hr at 180C. The gradient was fractionated and the fractions containing VP1 -VP3 determined by denatured dot blots pr obed with B1 as primary antibody (162) The fractions from the higher percentage iodixanol gradient (40/60%, 40%, 40/25 and 25%) were further purified on an anion exchange 5 ml HiTrap Q column (GE Healthcare, cat#17 515901). The clarified cell lysate was diluted with 2x Buffer E (20Mm Tris, 15mM NaCl, pH8.5) loaded onto the column at 2.5 ml /min, washed with 50 ml Buffer E, and the sample was eluted with 25 ml Buffer F (20Mm Tris, 15mM NaCl, pH8.5). The fractions were collected and

PAGE 50

50 biophysically characterized. The schematic for the production, purification, and characterization AAV2 mt and wt is illustrated in Figure 2 4

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51 Figure 2 1. Schematic of MSV N CP components purification and characterization

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52 pFastBac donor plasmid + Gene of interest Cloning Recombinant donor plasmid Donor Helper Bacmid Transformation Competent DH10 Bac E.coli cells Transposition Helper Bacmid Recombinant Bacmid DNA isolation Transfection of sf9 insect cells Infection of insect cells Recombinant gene expression and viral amplification Donor Figure 2 2. Diagram of the production of recombinant MSV N CP (wt and mutants)

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53 Figure 2 3. Schematic AAV2 VP components purific ation and characterization.

PAGE 54

54 Figure 2 4 Schematic of the production, purification and characterization of AAV2 symmetry related mutants.

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55 CHAPTER 3 MSV N ASSEMBLY/DISASSEMBLY COMPONENTS Introduction MSV N is a prototypic Mastrevirus and it causes the d evastation of maize crops in sub Saharan Africa. The architecture of MSV -N is a quasi isometric (geminate), pseudo T=1 icosahedral shell which is formed by 110 copies of the CP. The MSV N capsid encapsulates a circular ss DNA genome whic h is 2687 nucleotides in length (67, 93, 115) The MSV -N genome encodes four proteins and they are all required for viral infection. Rep and RepA are the early gene products and they are responsible for the viral DNA replication (94) The other two proteins are the CP (27kDa) and the MP (10.9kDa), which are expressed later in infection. The MP is important for the long distance transport of the virus and the CP is necessary for the accumulation and e ncapsulation of the viral ss DNA (22, 25, 94) To date MSV N is one of the most extensively studied Geminivirus. This study is focused on understan ding the mechanism by which the MSV N capsid assembles and disassembles during its replicative life cycle. The life cycle of MSV N is illustrated schematically in Figure 3 1. MSV N is transmitted to Cicadellidae mbila during an acquisition probe into the maize plant mesophyll during feeding. The ultimate goal of the Cicadellidae mbila is that its stylet reaches the phloem where it acqu ires a liquid meal The virus containing meal is then transported to the bucal cavity where powerful muscles in the pharynx transport the liquid meal to the esophagus It has been proposed based on viral vector specificity that MSV N is then internalized by cells of the vectors filter chamber by receptor mediated endocytosis. The aggregated virus is then enveloped in the end oplasmic reticulum It has also been proposed that MSV N is then transcytosed into cells of the ventriculus (foregut) with inclusions breaking off into the hemolymph MSV N is then transported across the insects gut lining into the hemocoel and eventually the salivary glands via

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56 specialize d secreatory cells MSV N is introduced into the cytoplasm of the phloem cell of an uninfected plant host from the infected vector during feeding (111) The geminate capsid has been shown to travel within the plant host in what is described as a phloem limited pathway (28) It is not known whether disassembly occurs in the maize cell cytoplasm or the nucleus. It is believed that t he viral genome once it is release d from the capsid is replicated via a dsDNA intermediate. The mechanism by which the virus genome is uncoated and delivered to the nucleus is not known. Replication has been shown to occur in the nucleus by a rolling circ le mechanism and utilizes a ds DNA replicative as well as host enzymes The dsDNA is bidirectionally transcribed to produce both viral and complimentary strand transcripts (113, 115) The complimentary strand transcripts are translated to form replica tion proteins Rep and RepA, and the viral strand transcripts from both the CP and the MP (101, 103) The translated viral proteins are transported to the nucleus where it is believed the unique geminate capsid is assembled. The mech anism by which the geminate capsid assembles is not known. This data begs the qu estion what are the steps involved the assembly of the geminivirus capsid? The first part of this chapter will report results from efforts to characrterize the components a ssembled from the MSV -N CP and it s genome. This includes the isolation and purific ation of pentameric intermediates as we ll as single and geminate capsid It has been previously reporte d that certain viruses assemble and disassemble with a change in their environmental pH, CP concentration and ionic strength (177) (147) Based on this information and what is known about the environmental conditions encountered by MSV N geminate s within its host and vector; purified MSV N geminates were exposed to varying concent rations of Ca2+, Na+ and pH. In this chapter we will show that intact geminate virus disassembles and reassembles reversibly in the presence of increasing and decreasing pH,

PAGE 57

57 respectively. The information obtained forms the basis for a model developed to explain the assembly and disassembly of MSV -N geminate capsid with changes in environmental pH. This assay may be used to test MSV -N assembly inhibitors or capsid stabilizers. Results MSV -N Wt Components Purification and Characterization MSV -N Wt C omponents I solated To identify MSV N CP and CP -DNA components isolat ed in agro inoculated m aize plants, infected leaves were purified according to the protocol described in the materials a nd methods section in Chapter 2 There were three gradient fractions that cont ained MSV N CP components in the sucrose gradient These were at the 8 10% interface, bet ween the 14 and 16%, and 25% to 3 0% sucrose gradient fractions and t hes e bands were enriched with MSV N intermediate s characterized as capsomers singles, and geminate s respectively. They were visualized by negative stain EM (Figure 3 2A). Western blot analysis and c oomassie stained SDS -PAGE confirmed that the three different size components isolated were composed purely of MSV N CP (Figure 3 2B). The capsomers, singles and geminates had CP species with different electrophoretic mobility Two MSV -N CP bands were observed at approximate ly 2 7 and 29kDa for the capsomer while only one main band at 29kDa was observe d for the singles and geminates. In some prepara tions (not sh own ) the single capsid samples also contains a small portion of the faster migrating CP. MSV -N Wt C omponents G enome E xtraction To determine if the geminate s, singles and capsomers are associated with DNA, they were protease treated and DNA extracted by ph enol chloroform. The DNA was precipitated using ethanol and the genomic material visualized on an agarose gel. The res ults show that the

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58 capsomers are not associated with any detectable genome, c ompared to the assembled singles and geminate which encapsula te sub genomic and genomic material respectively ( Figure 3 2C ). Native Polyacrylamide Gel To determine the approximate molecular mass of the purified capsomer a native polyacrylamide gel calibrated with three known protein standards whose molecular mass s are 66kDa, 150kDa and 200kDa was used. The protein bands obtained woul d indicate that the re are two capsomeric componen ts with different molecular masses The most abundant capsomer has an approximate molecular mass of 150kDa and the less abundant capso mer has a molecular mass greater than 200kDa (Figure 3 2D ). Size Exclusion Chromatography Size exclusion gel filtration chromatography was used to more accurately determine the molecular mass of the purified capsomer. A super ose 6 analytical column was cal ibrated with the same proteins used in the native gel as well as carbonic anhydrase (30kDa), apoferritin (400 kDa) and Blue Dextran (2000kDa). Using the calib ration equation in (Figure 3 3A), the eluti on volume of each MSV N intermediate was then used to calculate its approximate molecular mass The calculated molecular mass of the capsomer is 157.7kDa. As described above, there are two MS V N CP species observed i n a SDS PAGE for the capsomeric sample, one migrating at approximately 29kDa and the other at approximately 27kDa (Figure 3 2A). The calculated molecular mass of a pentamer (5CP) containing the 27kDa or 29kDa CP is approximately 135kDa or 145kDa. This suggests that the approximately 157.7 kDa specie observed by the size exclusion chromatography an alysis is likely to be a pentamer Dynamic Light Scattering (DLS) DLS was used to experimentally determine the diameter of the MSV N geminate single s and capsomers. The diameter of the capsomer was shown to be approximately 8 nm, the single s

PAGE 59

59 were app roxima tely 20nm and the geminate capsid approximately 20 40nm (Figure 3 3B ). This da ta is consistent with the sizes measured from the cryo -EM reconstructions of the single and geminate capsid structures and the models built into the reconstructed densities ( (173) Reutzel unpublished data ). STEM A nalysis To further visualize the structures and confirm the molecular mass of the different MSV N components they were sent to the Brookhaven National Laboratory for STEM analysis. The statistical results are pres ented as hi stograms (Figures 3 5 to 38) and numerical data (Table 3 1 ). The shape of each component was visualized on STEM micrographs (Figure 34). For the capsomeric components two different samples were sent for analysis: a fresh sample (C1) which wa s sent two to three days post purification and an old sample (C2) which was at least one month old (Figure 3 4) The C1 sample contains predominantly capsomers and the C2 samples contain predominantly interacting capsomers. The singles and geminates stru ctures are consistent with what was already observed by negative stain em The molecular mass distribution for the samples are presented as histograms (Figure 3 5 to 3 8) and in Table 31 The molecular mass of the C1 sample sample ranged from 54512kDa, w ith a mean at approximately 193kDa and a standard deviation of approximately 64kDa (Table 3 1). This molecular mass is consistent with the size of seven CP. In consideration of the standard deviation which is approximately equivalent to two MSV NCP and our previous molecular mass measurement by native PAGE and size exclusion chromatography, the mean could be interpreted as a pentamer plus a dimer In the C2 sample the the molecular mass ranged from 27898 kDa with a mean of 332 kDa and a standard deviation of approximately 165kDa (Table 31). This mean value is consistent with the size of 12 MSV CPs. Interestingly rather than a Gaussian distribution this sample clearly separated into three distinct peaks P1, P2 and P3 (Figure 3 6) at 162kDa, 297kDa and 513kDa,

PAGE 60

60 respectively. This would correspond to building blocks or subunits that are pentameric. The mean molecular mass of the single sample is approximately 2210kDa (Table 31 and Figure 3 7). The molecular mass of the geminate capsid is 4,198kDa (Table 31 a nd Figure 3 8). These two masses with calculations based on the predicted number of CPs that assemble these capsids and their packaged genome. The calculated standard error for each data set w as lower than 10% which is reported to be within the acceptable range (Table 3 1). Discussion for MSV -N CP Purification and Characterization Three stable components have been isolated from a wt infection. The data confirms what was already known about the molecular mass particle dimensions, and genomic content of gem inate single capsids (29) The capsomeric components appear to be assembled from from pentameric components with the three specie identified in the old sample characterized by STEM representing 5CPs, 10CPS, and approximately 20CPs This interpretation was also supported by native gel electrophoresis and gel filtration. The DLS data provided structural measurement also consistent with a pentame ric building block. These pentameric intermediates are not associated with any detectable genomic material. Interestingly, the capsomeric components contain two CP specie, one consistent with the calculated molecular mass at 27kDa and the other at approxim ately 29kDa (Figure 3 2B). The observation that the assembled geminate and single capsids contain predominantly the larger molecular mass CP, suggests the requirement for a CP modification for assembly (29) MSV -N Assembly/Di s assembly Assay MSV -N pH Components Isolated To determine the effect of different environmental conditions on the stability of the MSV N geminate capsid, it was exposed to increasing pH, and dif ferent concentrations of Ca2+, Na+ and EDTA. The viral capsid disassembled in inc reasing concentration of pH, Ca2+, Na+, but was

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61 unaffected by EDTA (data not shown) The data for change in pH which recapitulates conditions encounte red in the insect vector (pH4.5 5.8) and plant host cell (pH7.4) are discussed (Figure 31) The pH assay shows that the geminate virus appear s intact from pH 4.8 pH 6.0. The virus then starts to disassemble at pH 6.4 and completely falls apart by pH 7.2 (Figure 3 9 A ). I f the disassembled virus is returned sequentially to its original pH of 4.8, there appears to be the formation of reassembled geminate cap sids (RG) singles (RS) and capsomers (RC) (Figure 3 9B ). These three components appear to be similar t o those isolated in the wt infection. It should be noted that negative control geminates were transferred every 24 hr to a freshly made final Buffer (0.1M NaAc pH4.8) and at the end of the assembly /disassembly assay the sample that was kept in Buffer at 4 .8 was viewed by EM and the geminate capsids were still intact The MSV N CP species observed on a SDS -PAGE for the virus at pH6.4 and pH7.2 was consistent with the differential electrophoresis mobility observed wi th capsomers isolated from the wt infectio n (Figure 3 10A) A SDS PAGE of the purified reassembled geminate and single capsid contained predominantly the higher molecular mass CP specie (Figure 3 10A) MSV -N pH Components Genome Extraction The genome extracted from the sample at pH 6.4 and 7.2 was a mix ture of the complete genome (2.7 kb) and a smear (Figure 3 10B). Thus would imply that the increasing pH results in the release of the of the encapsulated genome aaand the also results in the sharing following deprotect ion. The RS and RG sample contai ned some full length genome and a smaller proportion of sheared DNA (Figure 3 10), while the RC sample was devoid of any DNA. Thus while the RC and RG recapitulates the the geminate and capsomers isolated in the wild type infection, the RS is able to assoc iate with full length genome. This observation may be due to the fact that DNase was not used to remove unencapsulated genomic material prior to purification.

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62 MSV -N pH Components STEM Analysis STEM micrographs of the different components observed at pH 6.4 and 7.2 and the different reassembled compon ents are shown in Figure 3 11. The predominant size of the disassembled MSV N CP specie at ph 6.4 had a me dian and modal molecular mass ~ 291 kDa and 270kDa respectively (Figure 3 12 and Table 3_2), consistent w ith that of 10 MSV N CP at 27 kDa each. The molecular mass of the geminate at pH 6 .4 is consistent with that of 10 CP at 29 kD a for each CP. The sample at pH 7.2 was too aggregated, even when diluted, to be adequately analyzed The distribution of molecul ar m asses for the RS and RG samples are illustrated in Figure 3 13 and Figure 3 14, respectively and detailed in Table3 2 The mean, median and modal molecular mass values for the RS were ~ 1643, ~ 1616, and ~ 1620, respectively. These values are consisten t with a capsid assembled from 60CPs of 27kDa without genomic material. This suggests that the DNA isolated form this sample reported above, is likely just a co -purification artifact. The mean, median and modal molecular mass values for the RG were ~ 3168, ~ 3185, and ~ 3240, respectively. These sizes are all smaller than that observed for the wt geminate capsids. Assuming that genomic interaction is required for geminate capsid assembly, as has been reported, the smaller molecular mass suggests that this c apsid assembly contains lessthan 110 copies of the CP, and is missing approximately 7 pentameric capsomers. MSV -N pH Assembly/Disassembly Assay in the Presence of Protease Inhibitor To determine if (i) the smaller size of the RS and RG and (ii) the two CP species observed for the RC and due to protein cleavage, the pH assembly/disassembly assay was repeated in the presence of protease inhibitors. Following disassembly with increasing pH, which mimicked the process in the absence of protease inhibitors, the reassembly conditions produced a much smaller number of RS (Figure 3 15A last panel) Western blot analysis showed the presence of a double CP banding pattern for the pH 6.4 and 7.2 samples while the purified reassembled sample

PAGE 63

63 (pRSG) which contains both s ingle and geminate capsids, showed only the larger molecular mass CP (Figure 3 15B). The STEM micrographs (Figure 315C), as well as the resulting histograms (Figure 3 16 and 3 17), also showed that t he reassembled sample contains fewer singles compared to the samples reassembled without protease inhibitor The mean molecular mass of the pRS is 2204 kDa which is very similar to the wild type singles (Table 3 3). T he molecular mass of the reassembled geminates with and without protease inhibitor and the wt g eminates is given in Table 3 3. The molecular mass of the reassembled geminates in the absence of protease inhibitor is the smallest of all three samples analyzed The reassembled geminates in the presence of protease inhibitors have a higher m olecular mas s than the reassembled geminates formed in the absence of protease and contain nine more CPs Thus, despite the increase in molecular mass, these geminate capsids are still significantly smaller than wt capsids These observations suggest that a host component may be required to play a role in the successful wt capsid assembly. Discussion of MSV -N pH Disassembly/Reassembly Assay MSV N geminates disassemble when there is an increase in environmental pH and reassemble when the pH is decreased. These observat ions are likely to be relevant to events that occur in a wt infection. The introduction of the geminate capsid into the maize leaf cytoplasm (pH7.4) likely facilitates the disassembly and the release of the viral genome for replication.The translated viral protein likely assembles into geminate capsids provided they are exposed to pH4.8 and they interact with ssDNA. Geminate capsid assembly in the presence of protease inhibitor is still missing several CPs, this would imply that either host factors may be r equired or the effective CP concentration in the assay is less than optimal to replicapitulate the in vivo assembly conditions.

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64 Post translational Modification The question of the differential electrophoretic mobility of the interm ediate MSV N CP speci es isolated components was first addressed by Casado 2004 (29) Mass Spec Analysis ( MS MALDI TO F) was used to identify the two species observ ed as MSV -N CPs. The C and N terminal 5 amino acids were blocked and could not be resolved. However based on peptide mass measurements it was predict ed that that the CP may be post translationally modified by either myristoylation, phosphorylation or glyco sylation were unresolved, but based on the mass measurements it was predicted that MSV N -A may be myristoylated, phosphorylated or glycosylated (29) Post Translational Modification Determination To address the question of the potential post translational modification of the MSV -N CP we hypothesize that if geminate capsid assembly requires CP post translation al modification then either the capsid contains an autoregulatory region/domain, or it packages an enzyme, or the sample preparation is contaminated with a modifying enzyme. This enzyme function would thus explain the conversion of the lower molecular mass CP species observed in the pH 6.4 and 7.2 samples to the larger migrating protein on pH decrease A second hypothesis is that if this modification is reversed then the higher molecular mass CP species will be chased into the lower one. The two modifications tested were phosphorylation and the interaction with DNA. The two enzymes used to treat the pH7.2 sample (generated in the presence of pI) were CIAP and DNase 1 which facilitates dephosphorylation and DNA removal, respectively.In the presence of increasing concentration of CIAP very little change was observed in the intensity of the two CP species migration at ~.27kDa AND 29kDa, respectively (Figure 13 18). However at the CP band that migrates between 45 and 66kDa which is interpreted as being a CP dimer, a shift to a lower

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65 molecular mass is observed, with the most dramatic shift observed at a CIAP concentration of 70U/l (Figure 1318) Post -Translational Modification Prediction T he program Net Phos K was used to predict whether the CP was phosphorylated and more specifically what res idues The program is based on the assumption that if there are enough homologous proteins both in terms of sequence and structure then it predicts a consensus phosphorylation site. The access site is http://www.cbs.dtu.dk/services/NetPhosK/ The results indicate that there were not enough homologous proteins to MSV N CP to predict a consensus phosphorylation sequence, but there was enough data about well known kinases to predict which residues may be phosphorylated. The re are several sub strate sites predicted for MSV N CP the highest score was Threonine 19 which had a score of 0 .94 out of a m aximum of 1(Table 3 3 and Figure 3 19). Several MSV N CP samples were selected an d submitted to MS to positi vely identify potential phosphorylation residues of MSV N CP The result s obtained for MS were inconclusive therefore using a phosphostain or specific antibody for phosphorylation is a part of the future directions. Discussion In this study we isolated and characterized sev eral intermediate s from maize plants agro ino culated with MSV N These include the characteristic geminate capsid, a complete T=1 icosahedra l (single) capsid, pentameric intermediates (capsomers) and possible dimers of pentamers (DOP) or pentamers of dimers (POD) and higher species These data provide the information about the biophysical characterization of possible intermediates involved in geminivirus assembly/disassembly that can be correlated to the pH conditions encountered by the v irus during its life cycle that includes trafficking through their insect vector and the host plant.

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66 This study also describes the development of an assay that can be used to test potential inhibitors of geminivirus assembly/disassembly The disassembly/a ssembly assay was discovered while trying to decipher the effects of different environmental conditions on the stability of the geminate capsid The f unction of the vector is to transport the virus from one host to the next. There is no report of viral amp lification in the vector This information is consistent with our experimental observation which shows the geminate capsid is intact between pH 4.8 6.0 Thus given that observation the internal passage of the vector utilized by the virus is between (pH 4.55.8 ) the capsid is likely to remain intact during transmission and assembly. If the geminate is exposed to a pH greater than or equal to 6.4 it disassemble to form a pentameric intermediate ( pentamer, DOP or a POD) that is still associated with the viral genome. If the pH is increased to a neutral pH (7.2) which is similar to the internal environment of the maize plant cell cytoplasm, the pentameric intermediate would continue to disassemble into a CP structure that is still associated with the viral genom e. Thi s data was confirmed by DNA gel electrophoresis analysis The generation of this pH assay ideally serve s three main functions; (i) it has illustrated a possible mechanism for the disassembly of the geminate capsid; (ii) it c an be utilized to test ass embly/disassembly inhibitors and (iii) it has provided some insights into the intermediates associated with the assembly/ disassembly of the geminate and single capsids In conclusion, though we have not totally deciphered the mechanism by which MSV N asse mbly and disassembly occurs we have illustrated some features of these mechanisms. T he first step in the assembly of the geminate capsid appears to be the formati on of a pentameric intermediate, followed by the addition of further pentamer The CP interacti ons during this assembly still needs to be tested by mutagenesis DNA extracted from the wt geminate and single

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67 capsids indicates that the geminates packages full length genomic material while the singles package subgenomic material confirming previous obs ervation (29) O ur data would suggest that there is some phosphorylation of the CP dimer however this needs to be further investigated. Th e development of an assembly system utilizing baculovi rus expressed CP s (wt and mutant ) could allow us to systematically decipher the possible components required to ass emble the unique geminate capsid.

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68 Figure 3 1. Schematic of the life cycle of MSV N within its plant host and insect vector. The virus once introduced within the cytoplasm of the maize plant the virus is exp osed to a pH of 7.4 The asse mbled virus when transmitted via the insect vector the new external enviro n ment of the virus ranges from pH 4.5 5.8

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69 Figure 3 2. Chara cterization of purified MSV -N components. A) Electron micrographs of purified MSV N capsomers (C), single capsids (S) and geminate capsids (G). B) Immunobl o t analysis of MSV N components with MSV N primary antibodies C) Ag arose gel of MSV N components. D) N ative gel of the MSV -N capsomer ic components The asterisks indicate the position of the capsomeric band s

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70 Figure 3 3 Det e rmination of the molecular mass and diameter of purified MSV N components. A) Cali bration c urve used to determine the molecular mass of MSV N capsomeric component separated and characterized on a superose 6 analytical column. The red circle indicates the pos ition of the MSV N capsomer and the blue diamonds are the calibration standards. (B) Dynamic Light Scattering histograms of MSV N capsome rs single capsids and geminate capsids

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71 Figure 3 4 STEM micrograph of purified MSV N components: C1 = fresh capsomers, C2 = capsomers 1 month post purification S = singles and G = geminate c apsids. There are two numbers beside eac h sampl e that has been selected the number on the left represents the component number and the number at the right represents the molecular mass in MDa.

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72 Figure 3 5. STEM h ist ogram of MSV N a fresh capsomeric s ample which appear to fit a Gaussian distribution

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73 F igure 3 6 STEM h is togram of an old capsomeric sample P1, P2 and P3 represent the three differ ent populations of capsomers and it does not appear to fit a Gaussian distribution

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74 A Figure 3 7 STEM h istogram of purified MSV N single capsids.

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75 Figure 3 8 STEM h istogram of purified MSV N geminate.

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76 Figure 3 9 MSV N pH a ssembly/disassembly assay A) Illustrates the disassembly of geminate capsid with increasing pH from 4.87.2 (blue a rrow) and reassembly of intermediates to form both geminate and single capsids in pH 7.2 4.8 (red arrow ). B) Purified reassembled MSV N components at pH 4.8.

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77 Figure 3 10. MSV N pH Assay A) Coomassie stained gel of disassembled/reassembled gemina te (G) capsid at pH 6.4, 7.2 and 4.8. B) DNA extraction of geminate treated at pH 6.4, pH 7.2, reassembled geminate capsids (RG), reassembled single capsid s (RS) and reassembled capsomers (RC).

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78 Figure 3 11. STEM micrographs of MSV N pH components. Th e samples are disassembled geminate s at pH 6.4, geminates at pH7.2 red arrows show two interacring capsomer reassembled singles (RS) and reassembled geminates and singles (RS and RG) at pH 4.8.

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79 Figure 3 12. STEM histogram of MSV N geminate capsid s at pH 6.4

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80 Figure 3 13. STEM histog ram of MSV N pH assay reassembled singles (RS) at pH 4.8.

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81 0 270 540 810 1080 1350 1620 1890 2160 2430 2700 2970 3240 3510 0 1 2 3 4 5 6 7 8 9 10 11Molecular Mass (kDa)Distribution of Molecular Mass Figure 3 14. Stem histogram of MSV pH assay reassembled geminates (RG) at pH 4.8.

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82 Figure 3 15. MSV N pH assay in the presence of protease inhibi tor. A) Electron micrographs of left to right wt geminate capsids; disassembled sample at pH 6.4, and pH7.2, and purified reassembled sample at pH4.8 B) Immunoblot analysis of MSV N sample as listed in (A). C) STEM micrograph of purified reassembled sa mple.

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83 Figure 3 1 6 STEM histogram of reassembled MSV N singles (pRS) at pH4.8 assayed in the presence of protease inhibitor

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84 Figure 3 1 7 STEM histogram of reassembled MSV N geminates (pRG) at pH 4.8.assayed in the presence of protease inhibitor

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85 Figure 3 1 8 Protease inhibited (PI) MSV N pH a ssay assembly/disassembly A) Immunoblot analysis of the MSV N components treated with DNase and CIAP separately and together. B) Immunoblot analysis of the MSV N subassembly formed at pH 7.2 treated with increasing concentrations of CIAP (U/L) and the final lane is treatment with D N ase I only.

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86 Table 3 1 STEM statistical analysis of purified MSV N capsomers, single and geminate capsids. Capsomer s (fresh) Capsomers ( old ) Singles Geminates Total n umber of measurements 436 170 59 35 Minimum 45.9 27.1 1776.9 3620 25% Percentile 153.3 187.1 2126.7 4081.3 Median 193. 4 306.7 2210.4 4198.6 75% Percentile 225.6 415.3 2273.1 4263.9 Maximum 512. 6 898.0 3077.8 4461.5 Mean 192. 7 332.1 2210.8 4132 .4 Std. Deviation 63.8 164.5 175.5 221.3 Std. Error 3. 1 12.6 22.85 37.4 Lower 95% CI of mean 186. 7 307.2 2165.0 4056.3 Upper 95% CI of mean 198. 7 356.9 2256.5 4208.4

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87 Table 3 2 STEM statistical analysis of MSV N assembly/disassembly components from the pH assay pH 6.4 RS RG pRS pRG Total number of measurements 514 25 25 13 37 Minimum 64.5 1144.2 2806.4 1447.4 3019.3 25% Percentile 258.3 1529.7 3033.9 1839.45 3280.7 Median 291.2 1616.3 3185.7 2192.9 3358.9 75% Percentile 400.5 1678.3 3288.7 2756.8 3532.5 Maximum 949.9 2608.3 3626.1 2917 4510.1 Mean 333.9 1643.1 3168.3 2204.4 3404.2 Std. Deviation 140.9 244.7 206.4 489.4 263.6 Std. Error 6.2 48.9 41.3 135.7 43.3 Lower 95% CI of mean 321.7 1542.1 3083.1 1908.6 3316.3 Upper 95% CI of mean 346.2 1744.1 3253.5 2500.1 3492.1

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88 Table 3 3 Comparison of the Molecular Mass (kDa) of the pH reassembled geminates and singles s compared to wt. Singles Geminates wt samples 2210 4132 Reassembled samples 1643 3168 Reassembled samples ( protease inhibitor) 2204 3404

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89 Table 3 3 MSV N CP phosphorylation prediction table Sequence site Kinase Score S 2 PKC 0.8 T 3 PKC 0.91 S 4 PKC 0.91 S 15 PKC 0.85* T 19 PKC 0.94 ** S 24 PKA 0.63 S 33 RSK 0.51 S 33 PKC 0.71 S 33 PKA 0.67 S 39 P KA 0.83* T 50 cdc2 0.51 T 51 PKC 0.58 T 66 DNAPK 0.52 S 79 PKA 0.64 T 81 PKC 0.7 T 83 PKC 0.8* Y 101 INSR 0.53 S 102 PKA 0.65 T 115 cdc2 0.54 T 116 p38MAPK 0.54 T 123 p38MAPK 0.5 T 123 cdk5 0.52 T 126 PKC 0.72 T 133 PKC 0.73 T 140 PKC 0.75 S 177 PKC 0.8* T 198 DNAPK 0.51 Y 217 INSR 0.53 T 227 PKC 0.55

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90 Figure 3 19. Kinase landscape of MSV -N CP sequence kinase prediction with scores above 0.5 shown by uppercase letters and with size proportional to the scores. Color codes and kinas e symbols are listed at the top of the figure.

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91 CHAPTER 4 W ILD T YPE AND MUTANT MSV N CP BACULOVITUS CLONING AND EXPRESSION Introduction The assembly and disassembly of the viral capsid represents key steps in the replicative life cycle of the virus. The CP CP and the CP DNA interactions are important to the formation or assembly of the viral capsid. The detailed structures of viral capsids can illustrate how the CPs interact with each other and how they interact with their packaged genome. The determination o f the CP interaction with each other and itsinteraction with the viral genome are critical to the understanding of the assembly and disassembly processes. The polyoma VP1 protein for barrel core and long flexible N and C terminal arms, the C terminus being extremely positive and may be importa nt in binding the viral DNA Both arms are dispensable for pentamer formation but indispensable for inter -pentameric interaction (137) CCMV on the other hand 25 of the N terminal resid ues of the coat protein contains a high proportion of basic amino acids which have been modeled to interact with the viral RNA, deletion of these residues eliminates the ability of this virus to assemble RNA containing particles in vivo (158) The previous chapter wa s focused on the purification of assembly/disassembly components of MSV N to decipher the possible steps involved in these processes. This chapter is focused on the c loning and expression of baculovirus MSV N CP wt and mt, the aim is to determine their str ucture and to correlate it, to their role in the assembly of the virus. MSV N is a pathogenic plant virus that encapsidate s the covalently closed 2.687 kb genome in a unique twinned pseudo T=1 icosahedral capsid. The capsid is composed of 110 copies of the CP. The CP of MSV N is translated from the viral transcript V1 (25) it is 244aa in leng th and 27kDa in molecular mass The MSV N CP is required for a variety of functions

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92 which includes insect transmission (94) syste mic infections of maize plants (93) binding of sin gle and double stranded DNA (101) and for the specific accumulation and encapsulation of single stra nded DNA in infected tissue (25) The CP accumulates in the nucleus and facilitates nuclear transport (103) and cell to cell transport of MSV -N DNA via an interaction with the movement protein (MP) (102) The N terminal residues 5 22 contain 9 basic amino acid domains that are important for binding both ss and dsDNA (101) These domains have also been predicted to contain the NLS important for the transport of the viral genome to the nucleus (101) There are certain CP residues along with N and C termini residues that have been reported to be essential for geminate capsid assembly. CD studies of MSV N and structure prediction (74) of the prototype members of the four geminivirus genera ( MSV N BCTV, TPCTV, BGMV ) have been used to validate the secondary structural element composition of our MSV N CP model. The CP str ucture was then used to guide a multi -sequence alignment generated by Clustal W (171) of 46 different viruses sampled from the four genera. The alignment results were mapped onto the MSV N CP model, and only five residues were identical for all 46 sequences compare with a requirement for forming the core CP structure. The alignment of geminivirus sequences also showed that residue K182 (104) conserved among the mastreviruses, is located at the base of a channel formed by pent americ CP interactions (Figure 4 1) Mutations at this position, for example, K182V, have been shown experimentally to disrupt capsid assembly ; however, intermediates formed have not been characterized. The biophysical c haracterization of capsid or intermediates resulting from this mutant protein would elucidate the contribution K182 to the stability and assembly of the MSV N viral capsid. In addition to this residue, the N terminal region has also been shown to be important for MSV N assembly, and DNA binding (25, 101)

PAGE 93

93 This chapter will focus on the use of a recombinant baculovirus expression system to express the wt MSV N CP as B MSV N CP, K182V mutant as B MSV N 182, and the N t erminal truncation as BMSV -N CP201. Results To generate the B MSV N CP B MSV N CP201 and B MSV N CP182, the Bac to Bac (Inv itrogen) expression system was used and the details are listed in Chapter 2 Cloning of MSV -N CP (wt and mutant) into pFastBac The pFast Bac donor vector was used to transpose the MSV -N CP wt and m t into th e baculovirus genome. The pFast Bac plasmid and the recombinant wt and mt MSV N CP are shown in Figure 4 2A -D. The MSV N CP gen e fragments are ligated into the digested pFastBac. pFastBac contains a gentamicin resistant gene which allows for screening of recombinant pFastBac and MSV N CP wt and mt colonies pFast Bac ( wt and mutant ) Clone Selection To determine which clones contai n the recombinant pFB MSV N CP, pFB MSV N CP182 and pFB MSV N CP 201, the clones were examined on a DNA gel before and after restriction digest analysis a nd the results are shown i n Figure 4 3A B All the clones are circularized except pFBMSV N CP2014 pFastBac c ontains a BamHI restriction site in the promoter and MSV NCP gene contains an Nco1 restriction site. Fsp1 is an additional site present in pFBMSV NCP182 and is used for screening bacterial colonies. Double digestion of the ligated pFastBac vector containin g MSV NCP wt and mt insert generated two DNA fragments. Clones 1 and 2 of pFBMSV NCP201, CP and 182, all contain the expected fragments (F igure 4 3B). The other clones 3 and 4 did not appear to contain the MSV NCP insert and CP201 appears to have two pFast Bac ligated to itself (Figure 4 3). The positive clones were then transformed into DH10Bac E. coli cells.

PAGE 94

94 BMSV -NCP, BMSV -NCP182 or BMSV -NCP201 recombinant identification To determine the MSV N CP that clone that was transposed into the the baculovirus vect or each white clone generated was analyzed by restriction digestion. Recombinant MSV NCP wt and mt clones were first selected as white colonies from a blue and white colony screen. The purified clones were then analyzed by restriction digest and undigested recombinant BMSV NCP, BMSV NCP201 and BMSV NCP182 were examined on an agarose gel (Figure 4 4A). Clone 1 of each BMSV NCP wt and mt were used for restriction digest analysis. The first digest was Nco1 and it linearized all the baculovirus recombinant clones, as illustrated in Figure 4 4B. The restriction enzyme, Nde1 was then used to digest, BMSV NCP201 and BMSV -NCP and Fsp1 was used to digest BMSV -NCP182. The data in lane 2 and 3 demonstrate the results of these digestions and show that each clone was linearized (Figure 4 4 B). The results in lane 4 illustrate the double digestion of BMSV NCP201 and BMSV NCP with Nde1 and Nco1; and BMSV -NCP182 with Nco1 and Fsp1. The data confirms that the recombinant clones contain the MSV NCP wt and mt genes. Wild Type an d Mutant BMSV N CP Expression To determine the time for maximum protein expression before CP degradation as well as the best MOI to use for virus amplification and titration several time course experiments were performe d. The time course experiment recorde d in Figure 4 5 A and B All the mutants appear to produce proteins that are positive to the MSV -N antibody but only MSV -N CP and the MSV N CP201N appear to show overexpression of the CP. The time course experiment was also able to show that maximum expre ssio n for the MSV N CP201 is at 48 hr after which the CP appears to be degrad ed The MSV N CP appear to be overexpressed by 72 hr but the study would need more time points in order to determine if this was the maximum expression that could be achieved before CP degradation.

PAGE 95

95 BMSV -N CP Purification (Wt and Mutant) Ammonium sulphate precipitation was used to purify the baculoviru s expressed MSV N CP wt and mt. The samples were precipitated by sequentially increasing the ammonium sulphate concentration 10% at a time up to 70%. The resuspended pellets were analyzed by coomassie stained SDS -PAGE and western blot analysis. BMSV NCP182 was observed in the c ell lysate (CL) and clarified su perna ta nt (S), no MSV NCP was observed in the pellet (P). Based on western blot analysis t he BMSV NCP182 pelleted at 30% ammonium sulphate. BMSV -NCP was observed in cell lysate at 3040% and 70% ammonium sulphate precipitation according to t he western blot (Figure 4 6 A). When compared to the corresponding coomassie stained gel, these fractio ns contained other protein bands apart from MSV NCP. BMSV NCP201 at 20% and 30% ammonium sulphate also contained MSV -NCP (Figure 4 6 B). Consistent with the results obtained for the BMSV NCP and 182, the coomassie stained gel for these fractions also appear ed to have other protein bands (Figure 4 6 C). Consequently, a mmonium sulphate precipitation may be used as a method to concentrate the recombinant BMSV NCP, but cannot be used to purify it and so other CP purification methods were explored. The calculated PI of MSV NCP has been predicted to be approximately 10. There are few proteins with this high pI, for example lysozyme, which is a rare occurrence. This concept forms the basis of using ion exchange chromatography a method of purification for MSV NCP as illustrated in Figure 4 7 The cell lysate (CL) contains over -expressed recombinant MSV NCP, most of which is in clarified supernatant The flow through (FT) and the eluant (E) were positive for MSV -NCP. It should be noted that the FT fraction contained CP species with differing molecular masses, some higher or lower than 30kDa, while the eluant contained CP species that were lower than 30kDa. Though this method of purification does offer some success, the purity of the samples eluted as well as the redu ction in degradation will have to be addressed.

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96 Discussion This chapter describes the successful cloning, expression and partial purification of the BMSV -N CP, B MSV N CP201 and B MSV N CP182. The mutants of MSV -NCP were generated based on the role of the diff erent regions of the CP on capsidassembly. It has been shown that the N terminus of the MSV -CP is important for binding ss and dsDNA as well as for virus assembly. Similarly CP mutants of 182 has been shown to bind the viral genome, replicate and transla te, causing local infection, without generating any observable capsids. The location of 182 is illustrated in Figure 4 1A and B, its position is a n intra -pentamer location and previous data implies that th is residue is important for capsid assembly. The ti me course experiment (Figure 4 5) shows that the majority of the expressed CP is retained in the cell lysate when compared to the secreted sample. The same experiment was repeated for different multiplicity of infection and time course repeated to determin e the all the appropriate conditions for maximum expression of the BMSV -NCP. The purification and characterization of the baculovirus expressed MSV -NCP wt and mt will provide information about the structure of the CP and their relevance to the assembly of the viral capsid. The ammonium sulphate purification method is insufficient on its own but may be combined with another method that requires pre -concentration. The success of ion exchange chromatography will be explored by first determining the purity of both the flow through and eluant. Depending on the purity of these fractions, this method may be further combined with other methods like gel filtration chromatography. The purification, characterization and crystallization of the MSV -N CP or any components formed, will provide valuable information about the assembly of the unique geminate virus capsid

PAGE 97

97 Figure 4 1 Ribbon diagram of MSV N CP. A) Monomer in cyan, orange ball indicate the location of K182V mutant, N is the N terminal residue and C is the C -terminal residue B) 5 -fold related monomers, orange ball represents K182V mutant position, T he pentagon represent s the 5 -fold axis, the ellipsoid represent the 2 -fold axis and the triangles represent the 3 -fold axis

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98 Figure 4 2 pFast Bacmid map and the MSV N CP wt a nd mt gene inserted A) pF MSV N CP wt B) pF MSV N CP182 and C) pF MSV N CP201.

PAGE 99

99 Figure 4 3 pFast Bac MSV N clone s restriction digest analysis. A) The circularized recombinant pFast Bac clones before restrict ion digest B) DNA fragments on a agaroge gel after restriction digest with appropria te enzymes.

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100 Figure 4 4 BMSV NCP wt and mt clones restriction digest analysis. A) The circularized recombinant clones before restriction digest. B) DNA fragments of clone 1, after restriction diges t, selected from panel of 3 mutants in top figure. Lane 1 for all three BM SV -NCP wt and mt are uncut.Lanes 2and 3 represents single restriction digest and lane 4 represents double digests. Nco1 was the enzyme used in all lane 2, Fsp1 was used for BMSV NCP1 82, Nde1 for BMSV NCP201, and Nde1 for BMSV -NCP

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101 Figure 4 5 Western Blot of the time course experiment of the baculovirus expressed MSV N CP wt and mutant. A) Coomassie stained gel of the s upernatant (on the left) and cell lysate (on the right) harve sted at 0, 24, 48 and 72 hr .post infection. B) Western blot analysis of the same fractions in A.

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102 Figure 4 6 Ammonium sulphate precipitation fractions of baculovirus expressed MSV N CP wt and mt A) B MSV N CP182, B) pF MSV N CP and C) pF MSV N CP201. CL is the crude c ell lysate, P is the pellet, S is the clarified supernatant and the numbers represent the percentage ammonium sulphate used The figures on the left are the coomassie stained samples and the figures on the right are western blots.

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103 Figure 4 7 Western blot analysis of ion exchange column chromato graphy of MSV N CP wt and mt A) B MSV -N CP182, B) pF MSV N CP and C) pF MSV N CP201. CL is the crude cell lysate, P is the pellet, and S is the clarified supernatant after centrifugation FT is the flow th rough after the clarified supernatant was loaded on unto the ion exchange column and E refers to the elu ant

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104 CHAPTER 5 BIOPHYSICAL CHARACTERIZATION OF THE ASSEMBLY/DISASSEMBLY OF AAV2 Introduction AAV2 is a non -pathogenic, non -enveloped p arvovirus. It i s a prototypic Dependovirus (64) as it requires the help of either Adenovir us or Herpes Simplex virus for a productive infection. The architecture of the AAV2 capsid is a T=1 icosahedral which is formed by 60 copies of VP1, VP2 and VP3 in a ratio of 1:1:8 respectively (130) The VPs share the same ORF, but different splice and translational start sites. The amino acid sequence of VP2 is encoded in VP1 and the sequence of VP3 is common to VP1 and VP2. The three dimensional structure of the AAV2 as described by X ray crystallography (168) and cryoem reconstruction (90) illustrates that each VPs forms an eight and each strand is connected by the interconnecting loops that are responsible for the capsid surface features. The AAV2 capsid is characterized by a shallow depression at the 2 -fold symmetry axis, fingerlike projections surrounding the 3 -fold symmetry axis and a pentameric pore surrounding the 5 -fold symmetry axis. To date, AAV2 is one of the most extensively studied AAV serotypes because of its use in the treatment of seve ral human genetic diseases (16, 51, 69, 142) A AV2 has several advantages as a gene therapy vector as compared to other viruses. It has a broad host range, infects both divi di ng and nond ividing cells, has low pathogeni city and the recombinant form of the virus can be produced and purified in large quantities (40, 86) There is a considerable amount of data available about almost all aspect s of the AAV2 replicative life cycle which is illustrated in Figure 1 1 It has been shown that AAV2 infection is initiated by binding to its primary receptor heparin sulphate proteoglycan (96, 119, 129, 148) This facilitates the binding of the v5 (95, 141) and or fibroblast growth factor1 (127) which are both secondary receptors The AAV2 capsid is then internalized

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105 by endocytosis in clathrin coated pits (15) The AAV2 capsid is acidified in the endosome, which is believed to facilitate the externalization of VP1. The externalized VP1 has two functions: phospholipase activity of the unique region of VP1 which all ows the virus to escape the endosome (55) and nuclear localization sequence s locate d within both VP1 and VP2 (145) which target the virus to the nucleus. The virus is transported to the nucleus by microfilaments and microtubules (141) The nucleus is the site of DNA replication and transcription. The viral transcripts are transported to the cyto plasm where they are translated, then the translated proteins are transported to the nucleus where the newly synthesized VP is believed to be assembled and the viral genom e packaged (68, 117, 160) In the case of a lytic infection the viral capsid is released or in the absence of helper virus the viral genome becomes integrated into the host genome (17, 34, 35, 87, 88, 92) There is limited information available on the mechanism involved in th e assembly of the ma ture particle. There is substantial information available about the roles of the VPs. It has been shown that mutagenesis of both t he N -terminus and C terminus of VP3 abolishes the formation of the viral capsid (135) has shown that VP1 is important for capsid infectivity (66), VP1 and VP2 are required for the nuclear transport of VP3 (66, 135) It has also been shown that VP2 alone can transport VP3 to the nucleus, as well as both VP2 and VP3 are required for the assembly of the viral capsid (135) and for the sequestering and accumulation of the virus progeny ssDNA, this is still not completely settled as both Samulski and Worring ton has made VP3 only capsids. Furthermore, time course immunofluorescence experiments illustrate the formation and accumulation of capsid intermediates (pentamers ) which are transported to the nucleus, specifically, the nucleolus which is believed to be the site of AAV2 capsid assembly (160, 161) This information now leads us to ask other questions, namely: what is the signal used by the

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106 three capsid protein to transport them to the nucleus and is this signal based on the three dimensional structure of the VPs and their interaction with each other, it also raises the question as to the possible order of capsid assembly. Is a trimer formed as in the case of MVM (133) and CPV (163) or is it a dime r or a pentamer? To address these questions a series of mutatio ns were made in the AAV2 capsid and all VP structure s ( capsid and intermediates ) expressed in a baculovirus system was characterized with the overall goal of deciphering the steps involved in the ass embly of the AAV2 capsids To date, at least 130 AAV2 capsid mutants have been made Wu, et al (165) made 67 AAV2 capsid mutants, 48 of which were charge cluster mutants and found 11 mutants that did not assemble. Lochrie, et al (106) made only surface mutants; none of which turned out to be assembly mutants. Blekker et al. (18) and Sonntag et al. (145) made 5 -fold pore mutants hoping to find packaging mutants; 3 of them were also assembly mutants AAV2 show s regions of high sequence conservation with other AAV serotypes as well as high structural conservation with other p arvovirus es which might suggest r esidues that are required for capsid integrity and stability (56, 121) Our first specific goal was to identify the residues that are essential for holding the capsid together. We hypothesize d that conserved residues at icosahedral symmetry related interface s are likely to participate in assembly enabling interactions and that mutation will disrupt assembly. Second, as stated above we also hoped that the mutational analysis might reveal the steps involved in capsid assembly. This would be accomplished by the isolation and purification of capsid intermediates that may be formed. Third, the biophysical characterization of baculovirus expressed intermediate would provide information about the steps in viral assembly.

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107 We constructed a total of 22 mutants. E xperimental data presented in this chapter indicates that the mutation of amino acid residues at the 2 -fold and 5 -fold symmetry rela ted interfaces either adversely affects or prevents the assembly of AAV2 capsids. Furthermore, in addition to 5 -fold interface mutations not assembling capsids, they also dont make any detectable capsid intermediates suggesting that the 5 -fold intermediat e is formed first during capsid assembly. This data is consistent with the purification of pentameric intermediates obtained from our baculovirus expressed AAV2 The mutations at the 3 fold interface had minimal or no effect on capsid assembly, suggesting redundant non-mutant residues can compensate. Results Selection of R esidues for M utagenesis The AAV2 residues selected for mutagenesis were based primarily on the available high resolution X ray crystal structure of AAV2 (90, 168) Residues were identified based on their interaction at the 2 fold, 3 -fold and 5 -fold symmetry related monomer interface s R esidues that are within a 3.4 (hydro gen bond distance ) from each other were generated by the program Contact. The contact list obtained for the 3 -fold monomer related interactions was almost 2.5 times the number of that obtained for the 5 -fold and the 2 -f old monomer related interactions. The list was then trimmed by comparison with similar lists obtained for other parvovirus whose st ructure s had been determined and residues /interactions that were conserved were selected. Finally some residues that had pre viously been implicated in assembly by alanine scanning mutagenesis (165) were included. Table 5 1 contains a list of the mutants as well as their location in the viral capsid monomer, and their symmetry related interactions. Figure 5 2 and Figure 5 3 illustrate the position s of the mutated amino acids within the VP monomer structure The mutants have been generated in the AAV2 plasmid pIM45 which expresses all three capsid proteins and all 4 replication proteins. All the mutations are located in VP3 since this protein is

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108 sufficient f or capsid assembly and is the only structure observed in all the 3 -D structures of AAV2 so far determined (90, 168, 169) A total of 22 residues were selected for site directed m utagenesis and their phenotypes were characterized Symmetry Related Location of VP3 M utants There are 7 mutants that are located within the 2 fold symmetry related monomer interface. Four are located within the wall of the 2 fold depression (R294 A Q297A R298A and R294/8A ) -A helix and 3 of them (K692A W694A and P696A ) are located on t he C terminal loop which forms the floor of the 2 -fold depression. K692 and W694 are exceptions within the group as these positions also have 3 -fold related interaction s (Fi g ure 5 4 A and Figure 5 4B ). The buried surface area of the 2 -fold interface is 3060 2 and represents the weakest of all the symmetry related interface interactions. Seven of the 22 mutants are involved in 3-fold related interactions. The 3 -fold interface is the strongest of all the symmetry related interactions. The buried surface area of this interface is 10352 2. The large area is related to the fact that the interactions at this interface are extensive and are generated predominantly by the -fold axis. The positions of these mutants are illustrated in Figure 5 5A and Figure 5 5 B. There are 8 mutant positions that are potentially involved in 5 -fold related interactions. Two of these are double mut ants V221/S224A and H255/K258A The 5 -fold pore is generated 338) for ming a turret like structure interacting with N terminus of the adjacent monomer (219229). The depressio n surrounding the 5 -fold pore contains location of all the other 5 -fold related mutants. The buried surface area of the 5 -fold interface is 4949 2 which is intermediate in size compared to that of the 2 -fold and 3 -fold monomer related interactions. The other distinguishing feature of the 5 -fold axis is the loop extending over each

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109 interface monomer from the highly conserved parvovirus HI loop. F661 is a noninterface mutant that sits on the corner of the HI loop (Fi g ure 5 6 A and Figure 5 6 B). Finally, two mutants, Y413 and F415 were chosen because they are highly conserved residues; they also do not sit on an interface Immunoblots of Mutant C ell L ysate The non infectious AAV2 plasmid pIM 45 (wt and mutants), GFP pl asmid pTRUF11 and and pXX6 which provides Adenovirus helper function were triple transfected in HEK 293 cells to make GFP packaged virus. Immunoblots were used to determine the effects of the mutations on VP1 3 expression and capsid assembly. A panel of 4 monoclonal antibodies (B1, 1F, A20 and C37B) were used to perform preliminary characterization of the mutants. B1 antibody recognizes a linear epitope in the carboxy terminal of all 3 capsid proteins; this antibody was used to determine whether the mutan ts were capable of producing the capsid proteins. 1F recognizes a linear epitope present in all 4 Rep proteins. Because Rep proteins form complexes with capsid intermediates it was important to demonstrate that Rep expression was not impa i red (161) The A20 and C37 antibodies (162) are neutralizing antibodies that recognize non linear structural epitopes on the AAV2 capsid surface. A20 is unique in that it recognizes only intact, fully assembled capsids, while C37 can recognize some assembl y intermediates. When the lysates from the mutants were probed with A20 and C37 it was apparent that all but one of the 2 -fold mutant s and three of the 5 -fold mutants were defective for capsid assembly. In contrast all of the 3 -fold mutants appeared to be capable of assembly of at least some capsids (Figure 5 7A) Western blot of all the mutants showed the expression of VP1 and VP3. Most of the mutants that were A20 positive, except Y441A, P622A, and H255A/K258A also showed the expression of VP2. All the ot her mutants

PAGE 110

110 either did not show VP2 expression or they appeared to have multiple bands at a molecular mass similar to that of VP2 and is illustrated in Figure 5 7B. AAV2 Total P article Titration To determine more precisely the severity of the capsid asse mbly defect we used A20 ELISA to determine the total amount of AAV2 capsid produced for each mutant. The total capsids produced for each mutant was compared to wt and the log of t he ratio was plotted in Fig. 5 8 When this was done it became apparent all o f the 2 -fold mutants were defective for assembly including K692A which assembled approximately 100 fold fewer than wt capsids. Similarly, all but one of the 5 -fold mutant s (F661) was found to be significantly defective for capsid assembly. Finally, althoug h none of the 3 -fold mutants were found to have defects that were statistically significant, all of them displayed a trend toward lower assembly (2 7 fold) compared to wt. Packaging Efficiency Twelve of the 22 mutants produced sufficient yields of intact c apsids to determine the relative ability to package DNA and to transduce cells. Real time PCR, with forward and reverse primers to GFP was used to determine the amount of genome packaged by each mutant. Packaging efficiency was determined by comparing the total number of capsids assembled (as determined by A20 E LISA ) with the total genome s packaged (as determined by RT PCR). As shown in Figure 5 9 only one mutant R389A (in the 3 fold group) was significantly defective for packaging compared to wt. R432 whic h assembles capsids, was not included in this analysis as we and others have shown previously that this mutant packages little or no detectable DNA (19) Infectious P article Titer To determine the effect of the mutations on particle infectivity, serial dilutions of mutant cell lysate was used to infect HEK 293 cells in the presence of Ad5. The number of green cells

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111 counted for each dilution, at 24 hr post infection, was used to determine the infectious units/ ml for each mutant. The number of viral genomes per infectious unit is referred to as the particle to infec tivity ratio. This ratio is equivalent to the number of genomes required to transduce one cell and provides a measure of the relative infectivity of the mutant compared to wt. Of the twelve mutants that were capable of assembling intact mature capsids fou r were sever e ly defective for infectivity (>7 logs) : K692 A Y 441, L510A and R404A (Fig ure 5 10). An additional 2 mutants P602A and H255/K258A were also partially defective (2 logs) for infectivity. Virus Purification and V isualization The mutant AAV2 VP capsid and subassembled capsid components were purified on a step iodixanol gradient followed by ion exchange column chromatography. The iodixanol fractions were screened using the monoclonal antibody B1, to determine which fractions are positive for the VPs. The capsid containing mutants were B1 positive from the 40/60%, 40/25% and 25% iodixanol fractions (Figure 5 11A, B, and C ). The non-capsid mutant s on the other hand were B1 posistive only from the 40/25% and 25% iodixanol fractions; this is shown in Fig ure 5 11A and Figure 5 11C The B1 iodixanol fractions were further purified by ion exchange chromatography, using a Q Sepharose column. The peak Q Sepharose column fractions were used to prepare EM grids and viral capsids, subassembly intermediates or mon o mers were visualized by TEM The electron micrographs clearly show AAV2 capsids for the mutants that were A20 positive; they are located predominantly in the 3 -fold symmetry related interface. There appears to be subassemblies for the 2 -fold non -capsi d mutant s, and is illustrated in Fig ure 5 12. Baculovirus AAV2 VP Purification and Characterization To determine all assembly components in a baculovirus expressed AAV2, the iodixanol fractions were further purified on a Q Sepharose column and the peak fra ctions were visualized

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112 b y EM illustrated in Figure 5 13A. Three main components were identified, a sub assembled intermediate labeled P, empty capsids with sub assembled component attached labeled I, and a closed capsid labeled C. The subassembled interme diate s were and intermediate capsids were purified from the 40/25% and 25% iodixanol fraction s and the closed capsids were purified from the 40/60% iodixanol fraction. To determine the molecular mass of the different bAAV2 components isolated, the samples were sent to BNL for STEM analysis. The shape of each component was visualized on STEM and EM micrographs as illustrated in Figure 5 13A and B. The sample labeled P had a mean, median and modal mass mea surement of approximately 600 kD a (Table 5 2 and Figur e 5 14) and the micrograph s illustrated what appears to be a mixture of single specie or two specie attached to each Figure 5 1 3B The molecular mass of the sample labeled P is consistent with the mass of ten VP3s. This data would imply that our subassembl ed intermediate is either two pentamers beside each other forming a dimer of pentamers (DOP) or five dimers int eracting to form a pentamer of dim er (POD). Also of interest were the empty capsids wi th what appeared to be subassembled intermediates or pentam eric intermediates attached. Both the molecular mass of the empty capsids calculated and the histogram showing the distribution of molecular mass in the sample is consistent with the pentameric intermediate attached to empty capsid (Table 5 2 and Figure 5 15) The molecular mass of the closed capsid according to STEM analysis is approximately 3,200 kDa, which is presented in Table 5 3, illustrated as one peak in Figure 5 16, and is consistent with the molecular mass of an empty capsid. To evaluate the assembly state of the different components, immunoblot analysis was conducted using antibodies against denatured and native baculovirus expressed AAV2 samples. The results are illustrated in Figure 5 17 A, which shows that the sample labeled P is B1 positive

PAGE 113

113 wh en denatured and the native sample is B1 negative The data prove that the pentameric intermediate contains the VP3 and the C terminus is not accessible in the native state. The data also show the pentameric intermediate s in the native state either does n ot contain VP1 or VP2, or they are not accessible to the A69 or A1 antibody. The pentameric sample is also A20 negative which would verify the subassemblies observed in the electron micrographs The intermediate and the closed sample are both A20 positive and they are not positive for A1 and A69 which would indicate that VP1 or VP2 is not externalized Figure 5 17B illustrates that the pentamer is compose d predominantly of VP3 while the capsid containing fraction is composed of VP1, VP2 and VP3. Discussion In this study we generated a panel of 22 mutants, 19 of which are icosahedral symmetry related interface mutants and 3 were selected based on the fact that their phenotype had previously been investigated and they appea red to have an assembly defect. Our e xperimental data clearly shows that the mutants generated were predominantly assembly mutants and, they are located in the 2 -fold and 5 -fold axis and symmetry related interface. It should be noted that the selection of these mutants were based largely on t he available crystal structure of AAV2, MVM, as well as other AAV serotypes, with the goal of identifying residues that were essential for assembling viral capsids and maintaining their integrity. Concurrently we isolated and characterized three different assembly components form a baculovirus expressed AAV2. These components include a pentameric intermediate, an intermediate capsid conformation and a closed capsid. The goal of this experiment was to increase our knowledge of the steps involved in the assem bly and disassembly of the virus.

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114 Assembly M utants The predominant symmetry related interactions that are considered assembly mutants are the 2 -fold and 5 -fold. All the mutants located at the 2 -fold axis are assembly mutants. Based on the crystal structur e, the forms the wall of the 2 -fold depression and the C -terminal loop interlocks with the adjacent monomer to form the floor. It has been shown that the 2 -fold axis is the thinnest region of the capsid (169) and we have shown that the mutations of several residues within this interface w ill either prevent the AAV2 capsid from assembling or it will destabilize the assembled capsid. This data is consistent with information obtai ned by Pei Wus mutants mut 24 and mut 19 (165) which are both double mutants with a non -capsid, noninfectious phenotypes Mut 24 is R294A/ D295A, and both residues are loc H229A/D231A where D231 interacts with K692A in a 2 -fold related interaction (165) The other assembly mutants are located either in the 5 -fold axis or at the 5 -fold symmetry related interface. The double mutants V221A/S224A are located in the wall of the 5 -fold pore as well as within the 5 -fold interface, M402A is located on the conserved sheet of a 5 -fold interface interaction and they are also assembly mutants All other 5 -fold related mutants, which include, H255/K258A ( mut 21), Y397A and R404A fo rm capsid in significantly less quantity than wt (Figure 5 8 ). Other 5 -fold symmetry related assembly mutants include V221W, V221C, V221Y (18, 19) H229/D231A (mut 19) which is also a 2 -fold related interaction, K321A/E322A (mut26) and N334W (165) The compilation of all AAV2 assembly mutants generated in this wor k (Table 5 3) as well as previously characterized assembly mutants clearly shows that both the 2 -fold and 5 -fold symmetry related interactions are critical for capsid assembly and stability (Table 5 4, and Figure 5 15).

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115 Internal Polar M utants There are ano ther set of mutants that are not lo cated on any symmetry related interface, but have been characterized as defective as well as partially defective for assembly. These mutants internal of the capsid (Figure 5 16) These residues are polar and a few are ringed which may be important for their and the overall icosahedral shape of t he viral capsid. Packaging and Infectivity M utants The mutants that make capsids belong to two 2 main groups, one is based on whether or not it packages genome. R432 and R389 are classified as packaging mutant, because they encapsulate less viral genome, than wt R432 has been extensively characterized as a packaging mutant. R389 is both a 3 -fold as well as a 5 -fold monomer related interface. The packaging and release of the viral genome through the AAV pentameric pore has been illustrated for AAV2 based a lanine mutants or the substitution of pore residues with larger residues and has been postulated to occur through its pentameric pore. This phenomenon has also been shown for other viruses; these include the autonomous parvoviruses as well as several pico rnaviruses (61) There are 4 mutants that are significantly defective for infectivity. It should be noted that the particle to infectivity ratio of R404A L510A Y441 A and K692A is reduced by approximately 8 logs compared to wild type which is recorded as 2 logs vector genome s per infectious unit. These mutants are located within three separate symmetry related interfaces, that is, R404A is a 5 -fold symmetry related mutant, L510 and Y441 are 3 -fold symmetry rela ted mutants and K692A is both 2 -fold and 3 -fold symmetry related mutant They appear to make capsid and package capsid just as well as wt but are defective in terms of their particle infectivity. In this cas e infectivity may be related to viral traffickin g, or genome release

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116 v 1) binding site, as well as, genome packaging (8). Resid ue G512P was also shown to be 5 -fold defective in terms of particle infectivity (106) NGR (511 513) has been predicted to be the site v 1). Structure to F unction Correlation for AAV2 C apsid B ased on Assembly Studies We have shown that the assembly of AAV2 to form its T=1 icosahedral is a n ordered process in which the first intermediate formed is a pentamer followed by a interaction at the 2 fold interface, we are not sure if the 3 -fold interface requires a scaffold or chaperone or if the interactions at the 3 -fold interface are so red undant that the effect of the disruption of one interaction does not affect how the capsid assembly. The symmetry related interactions of the T=1 icosahedral AAV2 are critical to the survival of the virus, for example, the 3 -fold axis has been shown to be important for host cell recognition, and receptor binding; the 5 -fold has been shown to be important for VP1 externalization and endosomal release, and viral genome packaging and based on our study both the 5-fold and 2 -fold symmetry related interface appe ars to be critica l to its assembly and stability These findings are important because the use of AAV2 in treating genetic diseases. The new trend in gene therapy is the generation of chimeric AAVs which will target specific tissues. Knowing the residues a s well as the symmetry related interface that are important for assembly and the stability of the virus will guide us in our decisions as to the regions of the capsid that cannot be changed.

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117 Figure 5 1. Schematic of the life cycle of AAV2. 1) AAV2 i nfection is initiated by bindi ng to its primary and secondary receptor. 2 ) Clathrin mediated endocytosis followed by endosomal acidification 3 ) Viral capsid disassembly, r elease of genome and transport to the nucleus 4) V iral replication and transcrip tion 5 ) E xport of viral mRNA from the nucleus 6 ) Translation of VPs and Rep s. 7) Assembly and transport of VPs o r empty capsids to the nucleus. 8) Resolution and packaging of viral genome 9) Egress from the nucleus. 10) Release from host cell duri ng a lytic infection. The purple hexagon represents the AAV2 capsid, blue half circles represent the primary receptor, black quadrilateral represents the secondary receptor, and the purple circles represent the VPs and reps.

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118 Figure 5 2 Ribbon diagra m of AAV2 VP3 based on its cry s tal structu re. The oblong, which marks the ico sahedral 2 -fold axis, the triangle mark s the icosahedral 3 -fold axis, and the pentagon represent s the icosahedral 5 -fold axis.

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119 Figure 5 3 Ribbon diagram of AAV2 VP3 based on its crytal structure and t he spheres show the position of each mutant. The orange spheres are the two fold symmetry related mutants and they are located in the immediate vicinity of the oblong, which marks the icsahedral 2 -fold axis. The blue spheres ar e the icosahedral 3 -fold mutants and the triangle marks the icosahedral 3 -fold axis. The yellow spheres represents the5fold symmetry related mutants and the pentagon represent the icosahedral 5 -fold axis.

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120 A B Figure 5 4 Ribbon diagram of the AAV2 ic osahedral 2 -fold axis with the orange sphere s representing 2 -fold symmetry related interface mutants. A) 2 -VP3 monomers viewed down the 2 -fold axis. B) Close up of 2 -fold axis showing location of mutated residues.

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121 A B Figure 5 5 Ribbon diagram of t he AAV2 icosahedral 3 -fold axis with the blue spheres representing 3 -fold symmetry related interface mutants. A) 3 -VP3 monomers viewed down the 3 -fold axis. B) Close up of 3 -fold axis showing location of mutated residues.

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122 A B Figure 5 6 Ribbon dia gram of the AAV2 icosahedral 5 -fold axis with the yellow spheres representing 5 -fold symmetry related interface mutants. A) 5 -VP3 monomers viewed down the 5 -fold axis. B) Close up of 5 -fold axis showing location of mutated residues.

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123 Figure 5 7 Immuno blots of AAV2 mutant cell lysate. A) Denatured D ot Blot utilizing B1 (row 1) for all viral capsid proteins (VP1 3) and 1F ( row 2) fo r all the replication protein (Rep 42, Rep 52, Rep 68 and Rep 78). The A20 and C37 lanes are n ative dot blots and are used to identify intact AAV2 capsids. B) Western blot utilizing B1 as the primary antibody used to detect all three viral capsid proteins (VP1 3)

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124 Figure 5 8 Histogram of A20 value for each mutant versus A20 value of the recombinant wt AAV2. The mutan ts wer e grouped into three categories; the 2 -fold, 3 -fold and 5 -fold symmetry related interactions. Group ANOVA statistics were determined for :2 -fold vs wt. F [6,19]=1294, P=0.0001, for 3 -fold vs wt: F [7,21]=1.363, P=0.2674 and for 5 -fold vs wt F[9,29] =11.16, p=0.0001. Tukeys post hoc results are indicated as *, **, *** = P < 0.05, 0.001, and 0.0001 vs wt.

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125 Figure 5 9 Histogram of the packaging ratio obtained for each A20 positive mutant. The packaging ratio is determined by dividing the A20 va lue obtained by the RT -PCR value for each mutant. Group ANOVA Statistics: F [12, 23] = 6.920, P=0.0001 with Tukeys post hoc results indicated as ***, P< 0.0001

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126 Figure 5 10. Histogram of the log of the particle infectivty ratio obtained for each A20 po sitive mutant. One Way Anova statistics: F[12, 22] = 98.40, P=0.0001. Tukeys post hoc results are indicated as *, *** = P < 0.05, and 0.0001

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127 Fig ure 5 11. B1 dot blot of AAV2 mutants purified on a step iodixanol gradient.

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128 Figure 5 12. Transmissi on Electron Micrographs of ion exchange column purified AAV2 mutants after iodixanol step gradients.

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129 A B Figure 5 13. Visualization of AAV2 capsids and intermediates. A) Electron micrographs of subassembli es refered to as (P) open conformation w ith pentamer beside capsid (I) to the left, and the red arr ow pointing to a flap, another view of the capsid with a region of the capsid appear to be partially opened (I) to the right, and I and P are isolated from 40/25 % iodixonal gradient and the closed conformation (C) which was isolated from a 40/60 % iodixonal gradient B) STEM micrographs of AAV2 components the red arrows indicating the presence of the pentamer apparently attached to the capsid

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130 Figure 5 14. STEM histogram of baculovirus expr es sed AAV2 intermediates.

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131 Figure 5 15. STEM histogram of baculovirus expressed AAV2 capsid intermediates with intermediates attached.

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132 Figure 5 16. STEM histogram of baculovirus expressed AAV2 capsids.

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133 A B.. Fig ure 5 17. Immunoblot and S DS gel characterization of AAV2 capsids and intermediates A) Dot Blot of AAV2 samples with different antib odies on the left B) Co o ma ssie stained SDS gel, l ane 1 contains intermediate components, lane 2 contains open conformation with the pentamer besid e the capsi d and lane 3 contains capsids with a clo sed conformation

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134 A B Figure 5 18. Ribbon diagrams of AAV2 assembly mutants. A) The yellow spheres representing 5 -fold symmetry related interface mutants and orange spheres representing 2 -fold symm etry related interface interactions. B) Close up of 5 2 3 -fold axis showing location of mutated residues .

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135 A B Figure 5 19. Ribbon diagrams of different views of AAV2 internal polar cluster mutants (salmon colored) spheres (A) outside looking in and (B) inside looking out.

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136 Table 5 1 List and location of AAV2 mutants Mutant Symmetry related interaction Position on secondary element Previously available, associated mutant R 294A 2 fold axis mut 24 (164) Q 297A 2 fold axis R 298A 2 fold axis R 294A/R298A 2 fold axis K 692A 2x3 fold axis C termi nal W694A 2x3 fold axis C terminal P696A 2 fold axis C terminal R389A 3x5 fold interface variable region III mut 29 (164) R432A 3 fold inter face GH loop mut 31 (164) Y441A 3 fold interface variable region IV L510A 3 fold interface variable region V P602A 3 fold axis variable region VIII P622A 3 fold interface GH loop D625A 3 fold interface GH loop V221A/S224A 5 fold axis A loop (20, 145) H255A/K258A 5 fold interface rand in variable region I Y397A 5x3 fold interface EF loop M402A 5 fold interface R404A 5 fold interface FG loop Y413A 5 fold non interface F415A 5 fold non interface GH loop mut 30 (165) F661A 5 fold non interface HI loop

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137 Table 5 2. STEM statistical analysis of AAV2 assembly/disassembly components from baculovirus expression. Pentamer Intermediate Capsid Closed Capsid Total number of values 411 61 35 Minimum Molecular Mass (kDa) 353 1080 2481 25% Percentile (kDa) 576 3074 3093 Median Molecular Mass ( kDa) 600 3145 3184 75% Percentile 629 3207 3294 Maximum Molecular Mass (kDa) 1664 3422 4105 Mean (kDa) 643 3079 3215 Standard Deviation (kDa) 180 382 219 Standard Error (kDa) 8 49 19 Lower 95% CI of mean 625 2981 3177 Upper 95% CI of mean 660 3177 3254

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138 Table 5 3 Summary of AAV2 mutant phenotype Mutant Capsid/ ml Particle titer (Genome/ ml ) IU/ ml mutant capsid/ ml wt capsid/ ml Packaging efficiency Log (particle/ ml infectivity/ ml ) wt 8.60E+11 2.38E+11 1.91E9 1 7.119 2.1 R 294A 4.65E+8 B D BD 7.13E 4 BD BD Q 297A 1.11E+8 BD BD 1.48E 4 BD BD R 298A 7.34E+7 BD BD 9.17E 5 BD BD R294 /R298A 5.15E+7 BD BD 6.12E 5 BD BD K 692A 2.72E10 2.37E10 10E+1 2.79E 2 0.1944 10.2 W694A 1.12E+8 BD BD 1.29E 4 BD BD P696A 3.91E+7 BD BD 5.13E 4 BD BD R389A 3.61E+11 1.19E+11 4.31E8 4.10E 1 29.7 2.5 R432A 3.47E+11 BD BD 3.56E 1 BD BD Y441A 3.64E+11 4.49E+10 10E+1 4.68E 1 10.47 9.7 L510A 6.86E+11 8.49E+10 10E+1 7.76E 1 4.636 9.9 P602A 2.51E+11 4.19E+10 1.02E7 3.16E 1 8.794 3.9 P622A 3.52E+11 2.10 E+11 4.90E+7 4.24E 1 3.73 4.0 D625A 5.67E+11 1.49E+11 1.09E+8 6.62E 1 6.571 3.4 V221 /S224A 8.35E+7 BD BD 1.01E 4 BD BD H255 /K258A 1.38E+10 1.79E+10 1.96E+6 1.58E 2 1.228 4.1 Y397A 9.10E+10 2.10E+10 4.58E+6 9.35E 2 1.627 4.0 M402A 1.51E+8 BD BD 1.61E 4 BD BD R404A 3.82E+11 6.02E+10 10E+1 4.32E 1 27.04 9.7 Y413A 9.96E+9 4.33E+9 2.46E+6 1.14E 2 2.156 3.2 F415A 3.34E8 BD BD 4.94E 4 BD BD F661A 1.09E12 3.25E11 1.74E8 1.14 3.812 3.3

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139 Table 5 4. Summary of all AAV2 Assembly Mutants. Mutant Oth er Name Pheno type Asse m bly Pack ag ing Part/ inf (log) Comment/ (Reference ) V221W pd X <3 fold 2 5 fold pore (19) V221C Pd X 3 fold 2 5 fold pore defective for extruding pla after heating (20) V221Y pd X 3 fold 3 5 fold pore (20) V221A,S224A X ? ? 4 5 log assembly defect, (AB) WHCDS 228 232 WACAS mut 19 ni X >5 No capsid, 5 fold axis, inside surface (165) M235L wt X (159) MGDRV 235 239 MGAAV mut 20 ni X >5 No capsid (165) NHLYK 254 258 NALYA mut 21 pd X Unstable capsid, N, L, K are on the surface (165) H255A/K258A pd X 2 log assembly defect, 5 fold axis (AB) NRFHC 285 289 NAFAC mut 23 ni X >5 No capsid, not on an axis of symmetry (165) FSPRD 291 295 FSPAA mut 24 ni X >5 No capsid, 2 fold axis (165) R294A ni X ? ? ~3 log assembly defect, 2 fold axis (AB) Q297A ni X ~ 4 log assembly defect, 5 fold axis (AB) R298A ni X ? ? ~ 4 log assembly defect, 2 fold axis (AB) R294A/R298A ni X ? ? ~ 4 log assembly defect, 2 fold (AB) RPKRL 307 311 APAAL mut 25 ni X > 5 No capsid, all residues on the inside surface (165) K309N/R310N ni X No capsid (58) VKEVT 320 324 VAAVT mut 26 hs X >5 5 fold pore, this ts mutant does not make capsids at 37C and capsid protein is degraded(165) (Hartledge thesis, unpublished)

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140 Table 5 4. Continued Mutant Other Name Pheno type Asse m bly Pack ag ing Part/ inf (log) Comment/ (Reference ) E322A ni X >5 5 fold pore (20) E3 22A/V323D/T324 N ni X >5 5 fold pore (20) T324Y pd <1 <2 5 fold pore (20) N334A pd 4 fold (20 ) N334W ni X 5 fold pore (19) N335A pd 2 5 fold p ore (20) L336A pd 2 5 fold pore, does not extrude VP1 even when heated (20) L336W pd X 5 fold 2 5 fold pore (19) S338A pd 9 fold 1 5 fold pore (19) L336W/D219A pd 3 fold 2 5 fold pore (19) S338A/V221W ni X 5 fold pore (19) TDSEY 344 348 TASAY mut 27 hs X >5 (165) Y397A pd X 1 2 ~ 1 log assembly defect, and 1 2 log infectivity defect, 5 fold axis (AB) M402A ni X ? 4 log reduced caps id, 5 fold axis (AB) R404A ni X 14 fold >8 Slight (~5x) reduced capsid (not statistically significant), reduced packaging, non infectious, 5 fold axis (AB) Y413A pd X <1 2 log assembly defect, 5 fold axis close to conserved F415 (AB) FEDVP 415 419 FAAVP mut30 pd 2 (165) F4 15A ni X >3 No capsid (AB)

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141 Table 5 4. Continued Mutant Other Name Pheno type Asse m bly Pack ag ing Part/ inf (log) Comment/ (Reference ) DIRD 469 472 AIAA mut 33 hs X >5 This virus is apparently ts for assembly but once assembled at 32, i ts normal for infection at all temperatures (165) (Van Fliet, unpublished) QDRDV 607 611 QAAAV mut 42 ni X >6 Not likely to be the C37 binding site (165) (162) EIE 681 683 AAA mut 46 ni X No capsid (165) K692A ni X 8 1 2 log deficit for assembly may reflect lower affinity for A20, severe defect for infectivity probably due to trafficking or uncoating, 2, 3, and 5 fold axes (AB) W694A ni X 4 log assembly defect, 2 fold axis (AB) P696A ni X 4 log assembly defect, 2 fold axis (AB)

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142 CHAPTER 6 CONCLUSIONS AND FUTURE DIRECTIONS The focus of this study was to understand the mechanis m by which two families of ssDNA viruses Geminiviridae and Parvoviridae, assemble and disassemb le MSV -N was selected as a model for the Geminivirida e and AAV2 was selected as a model for the Parvoviridae. Thes e viruses provided two different examples of capsid assembl y, in that, AAV2 infects animal cells, and it assembles to form a T = 1 capsid which is formed from a total 60 copies of the VPs (VP1 3). MSV N on the other hand is a plant virus which assembles to form a twin ned pseudo T = 1 capsid : the MSV N capsid is generated by the as sembly of two incompl ete T=1 capsid shell s and 110 copies of the same CP to accommodate its genome The conclusion obtained from the assembly disassembly of each virus, as well as the future directions will be summarized in this chapter. MSV -N Assembly/Disassembly Model The a ssembly/disassembly studies of MSV N were div i ded into two parts. The first part was the characterization of MSV N CP capsids a nd subassemblies present in infected maize leaves The second part is focused on the reversible disassemble and assemble of the g emininate capsid. A model of MSV N assembly / disassembly has been generated based on our experimental data (Figure 6 1) Three stable components have been isolated from a wild type infection; they include the geminate, singles and capsomers. The geminate a nd single capsids have been previously identified and characterized, and our data confirms what was already known about the genomic content, molecular mass and particle dimensions. The capsomeric components appear to be assembled from pentameric components and three species have been identified by STEM analysis, they are 5CPs, 10CPs and 20CPs ; this interpretation was supported by native gel

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143 electrophoresis and gel filtration. The dimension of this pentameric building block was confirmed by DLS. These pentam ers, unlike the singles and geminates, are not associated with any detectable genomic material. There is an interesting feature of our capsomer, namely the presence of 2CP species, one at 27kDa and the other at 29kDA. The larger CP species has been observe d to be the predominant species in the singles and geminate capsids suggesting that there is a requirement for a CP modification for assembly. This is summarized in the model of MSV assembly (Figure 6 1) and it is believed that both CP species exist in sol ution at the initiation of assembly. Stability studies of another geminivirus, African cassava mosaic virus (ACMV), have shown that it is stable between pH4.0 8 5, while aggretating at pH6.0 or lower. ACMV was reported to dis assemble by releasing the vi ral genome preferably from the apical and peritoneal region at pH8.0 and more. The virus also appears to extrude its DNA by the removal of a pentamer, similar to the parvovirus which appears to extrude their DNA through a pentameric pore. The dis assembly /assembly of MSV was examined in this study by varying the pH as well as ions of the sample buffer. In this study we report ed the dis a sse mbly of MSV N geminates with the increase of environmental pH, and re assembly with the pH is decreased. Similar to t he wt infection MSV geminates have been shown to dis assemble via a pentameric intermediate 5CPs and 10CPs units M ore importantly, the dis assembly/assembly assay may recapitulate the environmental conditions experienced by the virus while being trafficke d through its insect vector and plant host. There is no report of viral replication in the insect vector ; this information is consistent with our experimental observation which shows that the geminate capsid i s intact between pH4.8 and 6.0 This observatio n that the internal passage of the vector utilized by the virus is between pH4.5 5.8 implies that the capsid is likely to remain intact during transmission.

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144 The exposure of the MSV N geminate capsid to pH6.4 and greater results in its disassembly to form a pentameric intermediate (POD or DOP) that is still associated with the viral genome. The pentameric intermediate continues to disassemble with increasing pH (7.2), which is similar to the internal environment of the maize plant cell cytoplasm. The disasse mbled capsomer at pH7.2 is still associated with the viral genome and t his data is confirmed by DNA electrophoresis. Disassembly and the release of the viral genome is an important step in the viral life cycle as it allows the genome to be replicated. The replicated genome is transcribed and translated in the host cytoplasm; the pH of this environment (pH7.4) ensures that the CP is not assembled. The data shows that the CP likely assembles into geminate capsids provided they are exposed to pH4.8 and they in teract with ssDNA. The assembly of the geminate capsid in the presence of protease inhibitor is still missing several CPs, this would imply that either host factors may be required or the effective CP concentration in the assay is less than optimal to reca pitulate in vivo assembly conditions. AAV2 Assembly/Disassembly Model The assembly/disassembly studies of AAV2 were divided into two parts. The first part of this section was site directed mutagenesis of the VP to disrupt symmetry related interface inte ractions of the virus, to test to see if the interface is important for assembly. The second part of the AAV2 sect ion was focused on the chara cterization of all VP intermediate s and capsids from AAV2 baculovirus expression The concl usions for the assembly studies of AAV2 are illustrated in figure 6 2 The model illustrates that the first step in the assembly process is the formation of the VP pentamer. Based on the mutational analysis of the VP the mutants that were made in the 2 -fold interface fo rmed wh at appeared to be pentameric structures compared to the mutants made in the 5 -fold interface, which appears to form an aggregated mass once expressed. Our experimental

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145 data clearly shows that the mutants generated were predominantly assembly mutants and, t hey are located in the 2 -fold and 5 -fold axis and symmetry related interface Based on the crystal structure of AAV2 -fold depression and the C terminal loop interlocks with the adjacent monomer to form the floor. It ha s been shown that the 2 -fold axis is the thinnest region of the capsid and we have shown that the mutations of several residues within this interface will either prevent the AAV2 capsid from assembling or it will destabilize the assembled capsid. The other assembly mutants are located either in the 5 -fold axis or at the 5 -barrel wedge form the icosahedral 5 -fold VP3 symmetry related interactions with elements of the BIDG sheet and the N -strands of adjacent VP3 monomers forming the floor of the conserved depression that surrounds the icosahe dral 5 -fold axis. AAV2 also forms a channel that connects the inside and outside of the capsid at the 5-fold axis formed assembly mutants generated in this work (Table 5 3) as well as previously characterized assembly mutants clearly shows that both the 2 -fold and 5 -fold symmetry related interactions are critical for capsid assembly and stability. It is the data obtained from the wt AAV2 purific ation that points to the presence of either POD or a DOP. To date the other steps involved in the formation of the empty capsid is still not known. Structure to function correlation for the ssDNA viruses The bacteriophage Mi croviridae is the only family am ong the ssDNA viruses for which a model has been proposed to describe the mechan ism of assembly and disassembly The Microviridae phi X has been shown to utilize two different pentameric intermediates, one made from the F protein and the other from the G p rotein. 12 F protein pentamers interact to form the

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146 body of the T=1 capsid and 12 of the G proteins pentamers form the spikes which are important for binding the bacterial host glyco -protein, and for the formation of the 5 -fold pore which is important for the ejection of the viral genome. Assembly intermediates has been identified for the a utonomou s parvovirus CPV and MVM have been shown to utilize a trimeric intermediate to form their T=1 capsid. Both MSV N (member of the Ge miniviridae family of virus), a nd AAV2 (member of the P arvoviridae family of virus) appear to use a pentameric intermediate to form either a pseudo T=1 geminate capsid or a T=1 single Future Directions The assembly/disassembly studies of both AAV2 and MSV N has provided new informatio n about the steps involved in the assembly and disassembly of these two prototypic ssDNA viruses. Despite the availability of this new data there are still several unanswered questions that need to be addressed. The future plans for the MSV N and AAV2 disa ssembly assembly studies will be covered in this section. The first part of our aim was to determine the mechanism by which MSV N assembles based on the purification of the MSV N components The second aim was to decipher the environmental conditions criti cal to the function of the virus capsid. The pH assay developed has added further details to our model. The ultimate goal of our model is to use the information to assist in the development or the testing of small molecule inhibitors of viral assembly or d isassembly. The main future aim is to test our assembly model by purifying the baculovirus expressed MSV N CP, MSV N CP201, and MSV N K182Vand to crystallize the purified protein as well as any isolated assembly components The AAV 2 assembly study also has li mitations due to our inability to isolate and fully characterize pure intermediates generated by t he mutation of the VP. Our future plan s for AAV2 will be focused on generating VP intermediates which are tagged with a series of histidine s It

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147 has been show n that VP2 N -terminus that can tolerate insertions without affecting virus assembly. The characterization of a purified 2 -fold or 5 -fold mutant will c onfirm without a doubt the size of the intermediate in the assembly of the AAV2 capsid

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148 Figure 6 1. Proposed model for the assembly and disassembly of MSV -N

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149 Figure 6 2. Proposed model for the assembly of AAV2.

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164 BIOGRAPHICAL SKETCH Antonette Bennett was born in Kingston, Jam aica on July1, 1971. In August of 1989, Antonette started he r first year of her undergraduate career at the University of the West Indies (Mona Campus Jamaica) Her major was in Chemistry. Sh e graduated with a B.S. in July of 1993. She was then employed by the Ministry of National Security as a forensic officer in August of 1993. She worked as a Forensic for 5 years (19931998). She sta rted a second Bachelor of Science degree at Florida Atlant ic University in the year 2000 and completed it in 2001. In August of 2003 she joined the IDP program of University of Florida, College of Medicine to pursue her PhD. In May 2004, she joined the laboratory of Professor Mavis -Agbanje McKenna. Antonette spen t five years studying the mechanism by which the ssDNA vir uses Maize Streak Virus and Adeno Associated Virus serotype 2 assembles and disassembles under the guidance of Professor McKenna.