University of Florida Thesis Or Dissertation

CHARACTERIZATION AND COMPARISON OF SQUAMATE DEPENDOPARVOVIRUS TYPE I TO MAMMALIAN ADENO-ASSOCIATED VIRUSES

VICTORIA E. FIELDING

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2016

This work is dedicated to Trevor, my parents, Marshall, Rebekah, my grandparents, and all of the friends that have always believed in me.

ACKNOWLEDGMENTS

I would like to thank my parents, Charles and Angela, and my siblings, Marshall and Rebekah, for their love and support throughout this process. My heart-felt thanks is also extended to my grandparents, who have been my biggest fans and strongest supporters throughout my scientific career. I would also like to extend my sincere thanks to my mentor, Dr. Mavis Agbandje-McKenna, for pushing me to be the best scientist that I can be, for her endless patience, and for believing in me. I would like to thank Dr.

Robert McKenna for his unique constructive insight into my project, and his encouragement. I would also like to thank my committee members, Dr. James B.

Flanegan, Dr. Joanna Long, Dr. Arun Srivastava, and Dr. Gail Fanucci, for their time, invaluable ideas, and encouragement. I would also like to extend my thanks to our collaborators, Dr. Peter Tijssen, Qian Yu, Maude Boisvert, and Judit Pénzes (Institut

National de la Recherché Scientifique, Québec) for providing us with the original sAAV constructs that were used throughout the course of this project, and to Dr. Mario

Mietzsch (UF, Charité Medical School) and Dr. Regine Heilbronn (Charité Medical

School) for supplying rAAV sample. I would also like to thank my undergraduate student

Megan R. Lee for all her assistance on this project, and for reminding me of my love for teaching. I would like to extend my most sincere gratitude to Dr. Bridget Lins, Dr.

Antonette Bennet, Paul Chipman, and all of the other members of the Agbandje-

McKenna lab for their endless patience as they taught me the different techniques utilized in this laboratory. Finally, I would like to extend my heartfelt thanks to my partner and best friend, Trevor Makal, without whose love and support, none of this work would have been possible.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 7

LIST OF FIGURES ...... 8

LIST OF ABBREVIATIONS ...... 9

ABSTRACT ...... 11

CHAPTER

1 BACKGROUND AND INTRODUCTION ...... 13

Dependoparvovirus Taxonomy ...... 13 Adeno-associated Virus Genome Structure ...... 14 Structural Features of the AAV Viral Protein ...... 16 Lifecycle and Trafficking ...... 17 Tropism and Receptor Determinants ...... 18 AAVs as Gene Therapy Vectors ...... 20 Seropositivity as a Limitation of Treatment ...... 21 Approaches for Overcoming Pre-Existing Anti-AAV Immune Response ...... 22 Reptilian Dependoparvoviruses and Potential Therapeutic Applications ...... 23 Global Significance and Impact ...... 24

2 MATERIALS AND METHODS FOR BIOCHEMICAL CHARACTERIZATION ...... 32

Maintenance of Sf9 Insect Cell Lines ...... 32 Maintenance of Mammalian CHO Cell Line Variants ...... 32 Production of Virus-Like Particles ...... 33 Virus Purification by Sucrose Cushion and Gradient ...... 33 Virus Purification by Iodixanol Gradient ...... 35 Ion Exchange Chromatography ...... 36 OneBac Virus Production Platform ...... 36 Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis ...... 37 Negative Stain Electron Microscopy ...... 37 Native Dot Blot – Mouse Monoclonal Antibody Panel ...... 38 Fluorescent Labeling of Virus Capsid for FACS ...... 39 Fluorescence Activated Cell Sorting ...... 40 Low pH Studies ...... 41

3 MATERIALS AND METHODS FOR CRYO ELECTRON MICROSCOPY AND 3D STRUCTURE DETERMINATION...... 44

Vitrification ...... 44 Cryo-Electron Microscopy Data Collection ...... 44 Cryo-EM Data Processing and 3D Structure Determination ...... 44 Model Building ...... 45 Secondary Structure Matching and Variable Region Assignment ...... 46 Visualization of Structural Comparison ...... 47

4 RESULTS AND DISCUSSION ...... 49

Structural Determination of Squamate Dependoparvovirus Type I ...... 49 Characterization of Squamate Dependoparvovirus I ...... 52 sAAV is Capable of Escaping Recognition by Mouse Monoclonal Antibodies that Target the mammalian AAV Capsid ...... 52 sAAV is Capable of Recognizing and Binding to Mammalian Cell Lines ...... 53 sAAV Conserves pH-Induced Proteolytic Cleavage Activity Observed in Mammalian AAVs ...... 54 sAAV is Highly Thermostable and Conserves the pH Stability Profile Observed in Mammalian AAVs ...... 55

5 SUMMARY AND FUTURE DIRECTIONS ...... 67

Overall Summary of Experimental Findings ...... 67 Improvement of sAAV High Resolution Cryo-EM Structure ...... 67 Challenging sAAV with Polyclonal Human Serum ...... 68 Reconfirming sAAV Cell Binding and Transduction of Specific Mammalian Cell Lines ...... 69 Determination of HSPG as a Possible sAAV Cell Surface Receptor ...... 70 Identification of Autolytic Cleavage Products by Mass Spectrometry ...... 70 Characterization of Other Reptilian Dependoparvoviruses ...... 71

LIST OF REFERENCES ...... 72

BIOGRAPHICAL SKETCH ...... 82

LIST OF TABLES

Table page

1-1 Mammalian AAV VP3 sequence identity in comparison to Squamate Dependoparvovirus Type I (sAAV)...... 26

1-2 Known reptilian dependoparvoviruses...... 27

2-1 Mouse monoclonal antibody panel details...... 43

3-1 Equations used in 3D image reconstruction...... 48

4-1 sAAV variable region assignment and corresponding mammalian AAV residues...... 57

LIST OF FIGURES

Figure page

1-1 Taxonomy of the Family Parvoviridae...... 28

1-2 Dependoparvovirus genome organization...... 29

1-3 AAV Capsid Structure...... 30

1-4 The viral lifecycle...... 31

4-1 Purification of sAAV...... 58

4-2 Comparison of sAAV capsid surface to mammalian AAV serotypes 1, 2, 5, 8, and 9...... 59

4-3 sAAV differs from mammalian AAVs structurally in its variable regions...... 60

4-4 Sequence Alignment of sAAV with mammalian AAV serotypes 1, 2, 5, 8, and 9...... 62

4-5 sAAV is capable of escaping recognition by mouse monoclonal antibodies that target the mammalian AAV capsid...... 63

4-6 sAAV is capable of binding to mammalian cells. sAAV is capable of binding to Pro-5, Lec-2, and Lec-8 cell lines at varying levels of efficiency in comparison to AAV2...... 64

4-7 sAAV conserves the pH-induced autoproteolytic behavior observed in mammalian AAVs, and retains capsid integrity regardless of environmental pH...... 65

4-8 sAAV is highly thermostable and conserves the capsid stability trends observed in mammalian AAV serotypes 1, 2, 8, and 9...... 66

LIST OF ABBREVIATIONS

AAV Adeno-associated Virus

Ad Adenovirus

BDPV Bearded Dragon Parvovirus

BP Basepair

CHO Chinese Hamster Ovary

CLIC/GEEC Clathrin-Independent Carriers/GPI-Enriched Endocytic Compartments

CMV Cytomegalovirus

CNS Central Nervous System

CPV Canine Parvovirus

DSF Differential Scanning Fluorimetry

EM Electron Microscopy

FPLC Fast Protein Liquid Chromatography

FSC Fourier Shell Correlation

GFP Green Fluorescent Protein

GPI Glycophosphatidylinositol

HRP Horseradish Peroxidase

HSV-1 Herpes Simplex Virus

ITR Inverted Terminal Repeat kB Kilobases

Lec-2 CHO cell line variant expressing terminal N-linked galactose

Lec-8 CHO cell line variant expressing terminal N-acetyl glucosamine and

MVM Minute Virus of Mice

NAb Neutralizing Antibody

NHP Non-Human Primate

NLS Nuclear Localization Signal

PAM Point Accepted Mutation

PBS Phosphate Buffered Saline

PDB Protein Data Bank

PEG Polyethylene Glycol

PFT Polar Fourier Transform

PO2R Parallel Origin and Orientation Refinement

Pro-5 CHO cell line variant expressing terminal N-linked sialic acid rAAV Recombinant Adeno-Associated Virus

Rh Rhesus Macaque sAAV Serpentine Adeno-associated Virus; also known as Squamate Dependoparvovirus Type I

SF9 Spodoptera frugiperda Cell Line

SSM Secondary structure matching trs Terminal Resolution Site

VP Viral Protein

VP1u Viral Protein 1 Unique Region

VR Variable Region

WT Wild-type

Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

CHARACTERIZATION AND COMPARISON OF SQUAMATE DEPENDOPARVOVIRUS TYPE I TO MAMMALIAN ADENO-ASSOCIATED VIRUSES

Victoria E. Fielding

December 2016

Chair: Mavis Agbandje-McKenna Major: Biochemistry and Molecular Biology

Adeno-associated viruses (AAV) are small, single-stranded DNA viruses that are widely studied for their applications as therapeutic gene delivery vectors. They are non- pathogenic, able to transduce both dividing and non-dividing cells, and have relatively low immunogenicity in comparison to other existing gene delivery vectors. Currently, 13 serotypes and over 150 clonal variants of AAV have been isolated from mammalian hosts, including humans and non-human primates. The use of AAV vectors for treating human patients is limited by neutralizing antibodies in patient sera that target the mammalian AAV capsid. This seropositivity renders these patients ineligible for treatment. The field has shifted toward developing vectors that can escape the neutralizing immune response. Dependoparvoviruses like AAV have also been isolated from reptilian hosts, and the potential for environmental exposure to these reptilian viruses is significantly lower than the risk of exposure to a mammalian virus. Squamate

Dependoparvovirus Type I (Serpentine Adeno-associated Virus, or sAAV) presents a potential alternative to existing mammalian AAV vectors.

The structure of sAAV was determined to a resolution of 4.1 Ångstrom using cryo-electron microscopy, and was found to conserve the hallmark structural features of

the mammalian AAVs, such as the five-fold pore, three-fold protrusions, and the two- fold depression, but the three-fold protrusion appeared to be severely truncated in relation to AAV1, 2, 8, and 9. Given the unique features of the sequence and the surface of the capsid, sAAV was challenged with a panel of mouse monoclonal antibodies that target specific surface epitopes of mammalian AAV serotypes 1-9. sAAV was able to escape recognition by all of these antibodies. Fluorescence activated cell sorting experiments indicated that sAAV is capable of binding to mammalian cell lines with different terminal glycans, and differential scanning fluorimetry experiments found that sAAV is highly thermostable, and retains capsid integrity at temperatures up to

90.5°C. sAAV was also found to conserve the pH-related auto-proteolytic behavior previously described in mammalian AAVs. In summary, these data support the potential for sAAV as an alternative to existing mammalian gene therapy vectors.

CHAPTER 1 BACKGROUND AND INTRODUCTION

Dependoparvovirus Taxonomy

The family Parvoviridae is a family of small, non-enveloped single-stranded DNA viruses that have T=1 icosahedral symmetry and assembled particles are ~26 nm in diameter (1). Parvoviridae is further divided into two subfamilies, Parvovirinae and

Densovirinae (Figure 1-1) (2). Members of these subfamilies are classified based on the range of host species that they can infect, with Densovirinae being specific to arthropod

(insect and crustacean) hosts, and with Parvovirinae infecting a wide variety of vertebrate species (2). The subfamily Parvovirinae is divided into eight genera:

Amdoparvovirus, Aviparvovirus, Bocaparvovirus, Copiparvovirus, Dependoparvovirus,

Erythroparvovirus, Protoparvovirus, and Tetraparvovirus (2). All members of these genera are autonomously replicating, with the exception of genus Dependoparvovirus.

Members of the genus Dependoparvovirus are classified as replication deficient and require a helper virus such as Adenovirus (Ad), Herpes simplex (HSV-1),

Cytomegalovirus (CMV), or vaccinia in order to carry out productive infection (3–7).

Dependoparvoviruses have been isolated from a variety of species covering a range of taxonomic classes, including non-marsupial mammals, anseriform aves, and squamates(2). This project focuses on the comparison of Squamate Dependoparvovirus

Type I, also called Serpentine Adeno-associated Virus (sAAV), and mammalian Adeno- associated viruses of human and non-human primate origin. To date, Adeno-associated viruses are divided into two sub-species, Adeno-associated dependoparvovirus A

(serotypes AAV1-13) and Adeno-associated dependoparvovirus B (AAV5) (2, 8). These two groups contain serotypes and viral variants isolated from humans and non-human

primates(2). For the purposes of medical research, the mammalian serotypes isolated from humans and NHPs have been the focus of clinical trials and development as targeted therapeutics for genetic disorders.

Adeno-associated Virus Genome Structure

The AAV genome structure is made up of three open reading frames (ORF), rep, cap, and AAP (Figure 1-2). The 4.7 kB linear genome is flanked by two 145 base pair inverted terminal repeats (ITR) (9). The ITRs are cis-acting replication elements required for successful genome packaging, site-specific integration into the host genome, and viral replication. These nucleotide sequences have been found to act as primers, in order to promote second strand synthesis of the single-stranded DNA. Rep contains two promoters, p5 and p19, that encode for non-structural proteins (10). The

N-terminal region of the Rep ORF is involved in DNA binding activity, while the C- terminal region is required for multimerization and helicase activity (11). The C-terminal region is conserved in transcripts from both the p5 and p19 promoters. The p5 promoter is responsible for the transcription of the two high molecular weight Rep proteins, Rep78 and Rep 68 (12). Both Rep 78 and 68 are ATP-dependent and have site-specific endonuclease activity that cleaves at the terminal resolution site (trs) (13, 14). The p19 promoter transcripts, Rep52 and Rep 40, share the C-terminal sequence of the larger rep proteins, and conserve the helicase activity present in that region of the genome

(12). While these proteins play a role in viral DNA replication, they are not sufficient to carry out productive infection on their own. AAVs take advantage of the helper virus’ replication machinery in order to replicate its own genome. In the case of Adenovirus,

AAVs require Ad proteins E1, E2, E4, and VA in order to replicate efficiently (15). The

E1 protein acts as an anti-apoptotic factor by sequestering p53, a pro-apoptotic factor. It

is also directly involved in viral replication. E2 proteins provide replication and transcription machinery, while the E4 genes are involved in the promotion of viral DNA replication and downregulation of transcription of host cell proteins. The VA RNA also encoded by the Ad helper virus plays a regulatory role in translation. In cell culture, these proteins are provided in trans via co-transfected plasmids. This helps avoid the cytotoxic effects that result from coinfection with intact helper Adenovirus particles.

Similar proteins are required from other helper viruses like HSV-1 and CMV.

The second open reading frame, called cap, has a single p40 promoter that encodes for the structural proteins VP1 (~87 kDa), VP2 (~72 kDa), and VP3 (~62 kDa), which compose the assembled capsid (10, 16). A total of 60 individual monomers make up the assembled capsid, with all three viral proteins sharing the same C-terminal sequence that makes up the entirety of VP3 (17). The VPs are typically in a stoichiometric ratio of 1:1:10, with VP3 composing a majority of the capsid (18). VP2 has an additional ~60 amino acids on the N-terminal region that it shares with VP1.

Previous studies have shown that this region, called the VP1/2 common region, contains patches of basic residues that act as nuclear localization signals and aid the virus in targeting to the nucleus for uncoating and genome release (19). The largest viral protein, VP1, has a unique N-terminal sequence that it does not share with the other viral proteins. This region is termed the VP1-unique region (VP1u) that is required for infectivity, and contains a phospholipase A2 domain that cleaves the sn-2 acyl bonds of glycerophospholipids that compose the inner leaflet of a cell membrane lipid bilayer

(20). This enzyme activity has been widely studied for its role in the viral lifecycle and has been shown to result in the release of a lysolipid and a free fatty acid, and results in

positive membrane curvature that creates steric strain, eventually allowing the virus to escape during trafficking (21). All three of these viral proteins are generated from the same p40 promoter, but alternative splicing is required for production of the VP1 (17).

Additionally, VP2 is produced by an alternate ACG start codon just upstream of the

AUG start codon encoding the VP3 protein (17, 22). The p40 promoter also encodes a fourth protein, called Assembly Activating Protein, which is produced as result of an alternative open reading frame within cap and an alternative CUG start codon (17, 23).

Previous studies have shown that this protein is mandatory for viral assembly and targeting to the nucleus (19, 23).

Structural Features of the AAV Viral Protein

The 60 monomers of the AAV capsid create T=1 icosahedral symmetry. The capsid structure has 2-, 3, and 5-fold axes of symmetry (Figure 1-3). The interactions of these monomers produce characteristic structural features that are conserved in all members of the genus Dependoparvoviridae. The interactions between five viral protein monomers produce a pore at the five-fold axis, which is thought to play a role in the externalization of the VP1u and in genome release during trafficking. Three monomers overlap to produce protrusions at the three-fold axis, which have been associated with antibody binding in multiple serotypes, as well as receptor binding. The overall shape of these protrusions varies between serotypes, and these variations are thought to be responsible for differences in antibody binding profiles among the different AAVs. The interaction of two monomers produces a depression at the two-fold axis of symmetry, that connects with the depression surrounding the five-fold axis. Overall, these major capsid features are conserved within all known Dependoparvoviruses. The viral protein monomers that make up the assembled AAV capsid share a conserved core structure,

composed of an eight stranded anti-parallel beta sheet and an alpha helix that are connected by nine distinct regions of coil. The amino acid sequences at the apex of the coil are highly variable, and have been termed ‘variable regions’ (VR). These nine regions vary significantly between AAV serotypes and have been associated with multiple functions including transduction, receptor binding, and antibody recognition

(24).

Lifecycle and Trafficking

The AAV lifecycle follows the basic steps of viral entry: receptor recognition, attachment, internalization, trafficking, and genome replication (Figure 1-4). For AAVs, receptor recognition is mediated by a primary receptor and a secondary co-receptor.

The primary surface receptors are typically glycosaminoglycans and are required for initial attachment (25–28). Co-receptors, while only known for a few serotypes, are widely variable between serotypes and play a role in concentration of virus on the host cell surface as well as in dictating the ultimate fate of the virus by determining which biological pathway it will follow for trafficking upon internalization. Many studies have explored the variety of internalization mechanisms that are capable of internalizing AAV virus particles. One of the most well-studied mechanisms is clathrin-mediated internalization (29). This mechanism is dynamin-dependent and the virus:receptor complex is internalized into a clathrin-coated vesicle (29). Alternatively, AAV can also be internalized via caveolin-mediated endocytosis (30). This mechanism is clathrin- independent and relies on the oligomerization of caveolins in lipid rafts to form coated vesicles called caveolae (31). AAVs have also been shown to enter the host cell via transcytosis, macropinocytosis, and phagocytosis (32, 33). In addition, prior studies support evidence of a clathrin- and caveolin-independent mechanism that relies on the

use of Clathrin-Independent Carriers/GPI-Enriched Endocytic Compartments in order to traffic AAV particles (34). This method is known as the CLIC/GEEC pathway.

While there a variety of internalization mechanisms, the internalized virus particle ultimately follows the same pathway as it is trafficked through the host cell regardless of how it was internalized. For a generic receptor binding event, the virus:receptor complex is internalized into a vesicle that is then trafficked through the endo-lysosomal pathway.

During trafficking, the environmental conditions of the interior of the maturing endosome acidify, which is mediated by ion pumps and H+-ATP-ases that pump protons into the endosome resulting in a decrease in pH, from pH 7.4 in the extracellular space, to pH

6.0 in the early endosome, pH 5.5 in the late endosome, and pH 4.0 in the lysosome

(35–37). The acidification is thought to result in autolytic proteolysis and modification of the capsid surface, which are thought to play a role in improving capsid flexibility for the externalization of the VP1u, the VP1/2 common region, and genome release (38–40).

The externalization of the VP1u and the VP1/2 common region is required for PLA2- mediated escape from the endosome and translocation to the nucleus, respectively (41,

42). The VP1u phospholipase A2 activity is responsible for enzymatically modifying the inner leaflet of the membrane bilayer, and allowing the virus to escape. The VP1/2 common region contains basic residue motifs that target the virus capsid to the nucleus in order to aid in replication and future gene expression (42). While the purpose of the self-proteolytic modifications to the capsid remains to be determined, this acidification has been found to be critical for successful trafficking of the viral particle (29).

Tropism and Receptor Determinants

Tissue tropisms vary by serotype in mammalian AAVs, and this tropism is determined by which cell surface receptors that the virus can bind to. The selected

clinically relevant serotypes AAV1, 2, 5, 8, and 9 have a wide array of determinants that make them useful in a variety of different tissue types. In the case of AAV2, the most studied mammalian AAV serotypes, the primary cell surface receptor has been identified as heparin sulfate proteoglycan (HSPG) (25). Receptor binding determinants for AAV2 have previously been found to be localized to VR-V, VR-VIII, and VR-IX (24).

AAV1 and AAV5 have been shown to bind terminal N-linked sialic acid (26, 43). The receptor binding determinants for the sialic acid binding site on the surface of AAV1 have previously been shown to be contributed by VR-I, VR-IV, and VR-V (28). The sialic acid binding site on AAV5 has been mapped to residues associated with VR-VIII (44).

AAV9 has been shown to bind N-linked terminal galactose, and the binding site was found to be contributed by residues found near the external surface of the protrusions facing the 2- and 5-fold axes, and a protrusion between the 2- and 5-fold axes formed by VR-I (27, 45). AAV8 cell surface receptors are unknown at this time. In the case of co-receptors, many mammalian AAVs take advantage of growth factor receptors (ie. epidermal growth factor receptor, fibroblast growth factor receptor, hepatocyte growth factor receptor), integrins, or laminin receptors on the cell surface in order to improve the concentration of virus particles on the cell surface prior to internalization (46–50).

The select clinically relevant serotypes AAV1, 2, 5, 8, and 9 have previously been shown to have tropism for a wide range of tissues, including skeletal and cardiac muscle, the liver, lungs, the central nervous system (CNS), and the eye (51–57). At this time, the receptor determinants for reptilian Dependoparvoviruses are unclear, but necropsy results of the original animal specimens, including the snakes, bearded dragons, chameleons, and amphisbaenians, revealed the presence of virus in liver,

kidney, lung, heart, and the intestinal tissues (58–60). These animals were reported to have shown symptoms of clinical disease before they were sacrificed, but none of the pathology was able to be directly related to the presence of the dependoparvovirus itself.

AAVs as Gene Therapy Vectors

Gene therapy is defined as a method of therapeutic treatment that takes advantage of gene replacement in order to alleviate or cure a genetic disease. This can be done by replacing a ‘broken’ or otherwise defective copy of a gene with a healthy one, down-regulating a mutated gene, or introducing a healthy copy of a gene in order to alleviate the pathogenic effects of a defective gene copy without replacing that gene.

A variety of gene therapy techniques exist, which include the genetic engineering of human cells, bacteria, or viruses in order to deliver the desired gene into a person. In the past, viral vectors used for gene therapy included Adenoviruses, Herpesviruses,

Retroviruses, and Dependoparvoviruses, specifically AAVs, to name a few (61–63).

Dependoparvoviruses, Adeno-associated viruses in particular, are widely studied for their application as gene therapy vectors, and have been isolated in a wide range of host species. These viruses have been of great interest because they are considered to be non-pathogenic, meaning that there is very little current evidence to suggest any clinical disease outcome in infected hosts that can be directly attributed to the presence of the AAV (64). AAV vectors have been used in animal models and human clinical trials to effectively treat diseases such as lipoprotein lipase deficiency, Leiber’s congenital amaurosis, cystic fibrosis, haemophilia B, Duchenne muscular dystrophy, and Pompe disease (65–69). Several recent AAV clinical trials have focused on serotypes 1, 2, 5, 8, and 9 variants as the vector used for gene delivery (70).

Seropositivity as a Limitation of Treatment

AAVs are widely favored as vectors for gene therapy due to their ability to induce long-term transgene expression in dividing and non-dividing cells, and the relatively low immunogenicity of the AAV capsid in comparison to other viral vectors. However, this does not preclude the formation of a memory B-cell mediated immune response targeting the AAV capsid in infected hosts. Regardless of the non-pathogenic nature of the AAV capsid, immune responses still target surface epitopes and neutralizing antibodies are produced in the infected host. Neutralizing antibodies have been implicated in a dramatic loss of transgene expression in clinical trials, and have led to seropositive patients being deemed ineligible for treatment (71). This is attributed to the rapid neutralization of viral vectors before transgene expression can be established.

Given the high sequence similarity (Table 1-1) between mammalian AAV serotypes and the likelihood of environmental exposure to these viruses in a person’s lifetime, antibodies targeting one capsid are more likely to target multiple mammalian AAV variants.

Global seroprevalence studies have shown that approximately 70% of individuals are seropositive for neutralizing IgG antibodies (NAb) that target the AAV2 capsid, 67% seropositive for NAb targeting AAV1, and <40% seropositive for antibodies targeting

AAV5, AAV8, and AAV9 (72, 73). In addition, this study found that 100% of patients that were seropositive for NAb targeting AAV2 were seropositive for cross-reactive antibodies targeting AAV1, AAV5, AAV8, and AAV9 (72). Similar levels of cross- reactivity were observed for patients that were seropositive for AAV1 NAbs. This same group of patients had serum IgG levels ranging from 50-90% of coprevalent antibodies targeting AAV5, AAV8, and AAV9, and these patients would be considered ineligible for

treatment due to the potential for antibody cross-reactivity (72). Of the serotypes tested,

AAV2 and AAV5 were originally isolated in humans, while AAV8 and AAV9 were isolated from non-human primates, and AAV1 has been found to have both human and

NHP origin (3, 54, 74–76). As such, this panel demonstrated that human or non-human primate origin does not exclude a viral variant from being susceptible to antibody cross- reactivity and therefore drives the need for development of alternative vectors.

Approaches for Overcoming Pre-Existing Anti-AAV Immune Response

Patients that are deemed ineligible for treatment with AAV vectors are still in need of therapy in order to alleviate the symptoms of their particular condition. In order to treat these patients, there was a push to develop alternative approaches in an attempt to circumvent the neutralizing immune response that greatly limits the patient cohort. The current standard of treatment for seropositive patients is focused on two main approaches: reduction of patient NAb titers to avoid neutralization, or modification of the vector to avoid pre-existing NAbs.

In order to reduce the NAb blood titers in eligible patients, plasmapheresis has been used to physically filter antibodies from the blood sera before returning all of the other blood components to the patient (77). This results in temporary immunosuppression and allows for successful delivery of AAV vectors, but also increases patient risk of opportunistic infection as this technique removes all serum antibodies, and not just those that specifically target AAV capsids. Transient immunosuppression via administration of drugs like cyclosporine in conjunction with administration of non-depleting neutralizing antibodies that target T- or B-cells, have been shown to result in a reduction of antibody titers that is low enough to allow the

AAV vector to establish transgene expression (78). This approach faces similar

challenges as those encountered during plasmapheresis, in that depletion of immune cells can result in weakening the patient and causing opportunistic disease. As a result of these outcomes, a majority of the field’s focus has shifted toward capsid engineering in order to avoid the effects of NAbs without having to immunosuppress the patient.

Avoidance of NAbs can be accomplished via several methods. The first, and perhaps the most conceptually simple, requires the delivery of the AAV vector into an immune privileged site. This allows restriction of transgene expression to the target tissue, and expression can take place without the threat of an immune response in that particular tissue type. The effectiveness of this technique has already been confirmed in several studies, that have established long term transgene expression in the eye, brain, and some regions of the liver in animal models and humans (79–81). However, the primary limitation of this approach is that it restricts its applicability to tissues that are immune privileged, so all other tissues are unable to be targeted by this method.

Studies have also been conducted using empty rAAV capsids as decoys that would effectively soak up NAbs so that they would not be able to target the capsids that were actually packaging genome (82). Another method for circumventing pre-existing immune response requires the availability of a library of vectors that can avoid detection

(83). These variants may be naturally occurring in host species that are evolutionarily distant from the target host (ie. reptiles vs. mammals), or created by mutating specific residues or regions of the capsid to disrupt common epitopes for antibody binding. The discovery approach has led to the isolation of reptilian dependoparvoviruses.

Reptilian Dependoparvoviruses and Potential Therapeutic Applications

Reptilian parvoviruses are a distinct phylogenetic group that includes several species and viral variants (Table 1-2). Squamate Dependoparvovirus Type I (also called

Serpentine Adeno-associated Virus, or sAAV) was isolated from recently deceased ball python (Python regius) and boa constrictor (Boa constrictor) specimens in 2004 (58).

Similar variants were isolated from other species of colubrid snakes (Pantheropus guttatus), and another proposed variant, termed Squamate Dependoparvovirus Type II, has been described in Indonesian pit vipers (Parias hageni) (59, 84, 85). Other reptilian dependoparvoviruses have been isolated from bearded dragons (Pogona vitticeps), short-tailed pygmy chameleons (Rampholeon brevicaudatus), and amphisbaenians

(Trogonophis weigmanni) (59, 60). These species are phylogenetically distant from each other, and current research has shown that the chameleon and amphisbaenian isolates share genomic features with autonomous parvoviruses that are not present in sAAV or Bearded Dragon Parvovirus (59, 60). The purpose of these features and how they impact the viral lifecycle is unclear from the partial sequences that are currently available. Reptilian Dependoparvoviruses are proposed to be viable alternatives to mammalian AAV vectors because they have not been found to naturally infect humans.

In addition, it is hypothesized that the natural isolation between humans and reptiles will prevent environmental exposure, and thus reduce or potentially eliminate the complications created by the presence of pre-existing NAbs that typically target mammalian AAVs.

Global Significance and Impact

The overall purpose of this project is to characterize Squamate

Dependoparvovirus Type I (sAAV) as a potential therapeutic vector that can avoid detection by the pre-existing humoral immune response that limits the treatment applications of mammalian AAVs. AAV vectors isolated from humans and non-human primates are among the most well-characterized viral gene delivery vectors to date, and

are the subject of numerous clinical trials focused on treating a variety of human diseases. In addition, an AAV-based gene therapy vector is the only viral gene delivery vector currently approved for therapeutic use in the western world (86). However, the patient cohort eligible for treatment with AAV vectors is currently limited by pre-existing neutralizing antibodies circulating in patient serum that can elicit a memory B-cell- mediated immune response (73, 87). This immune response can result in the neutralization of the AAV capsid and result in clearance of the virus, thereby eliminating any therapeutic potential. Given the decreased likelihood of exposure to a non- mammalian virus during a human patient’s lifetime, the use of a reptilian AAV vector may expand the patient cohort that is eligible for treatment.

Table 1-1. Mammalian AAV VP3 sequence identity in comparison to Squamate Dependoparvovirus Type I (sAAV).

sAAV AAV1 AAV2 AAV5 AAV8 AAV9 sAAV ------

AAV1 59.3 - - - - -

AAV2 57.9 59.3 - - - -

AAV5 53.5 80.2 59.1 - - -

AAV8 58.8 80.2 82.6 58.8 - -

AAV9 57.9 79.2 81.2 57.8 83.7 - Multiple sequence alignment completed using the European Bioinformatics Institute (EMBL-EBI) Clustal- Omega algorithm (88).

Table 1-2. Known reptilian dependoparvoviruses.

Species of Origin Species Common Virus Tissue of Reference Name Origin Python regius Ball python Squamate Heart, liver, (58) Dependoparvovirus kidney Type I

Boa constrictor Boa constrictor Squamate Spleen, liver (58) Dependoparvovirus Type I

Pantherophis Corn Snake Squamate * (84) (Elapha) Dependoparvovirus guttatus Type I

Parias Indonesian Pit Squamate * (85) (Trimeresurus) Viper Dependoparvovirus hageni Type II

Pogona vitticeps Bearded Dragon Bearded Dragon Liver, small (59, 89) Parvovirus intestine

Rhampholeon Pygmy short- Pygmy Chameleon Lungs, liver, (59) brevicaudatus tailed Parvovirus kidney, chameleon intestine, gonads

Trogonophis Checker-board Non-serpentine Reptilian Kidney, liver, (60) wiegmanni worm Parvovirus lungs, lizard intestine

*The original literature describing the isolation of Squamate Dependoparvovirus Type I from Pantherophis gutattus and Squamate Dependoparvovirus Type II from Parias (Trimeresurus) hageni is currently only available in Hungarian without English translation, so tissue of origin information for this virus cannot be described in this text.

Figure 1-1. Taxonomy of the Family Parvoviridae. The family Parvoviridae contains two subfamilies, Densovirinae, which infect arthropod hosts, and Parvovirinae, which infect vertebrate hosts. Figure adapted from multiple sources (1, 2, 90).

Figure 1-2. Dependoparvovirus genome organization. The linear parvovirus genome is flanked by two ITRs, and the C-terminal end contains a Poly-A tail. The Rep ORF encodes for four non-structural proteins: Rep78/68 from the p5 promoter, and Rep52/40 from the p19 promoter. The Cap ORF is encoded by the p40 promoter, and it is responsible for the production three structural proteins: VP1, VP2, and VP3. The VP1 contains a unique N-terminal sequence called the VP1u, shown in green. The VP1/2 common region contains NLS motifs and is shown in dark blue. The p40 promoter also encodes for the Assembly Activating Protein, which is required for capsid assembly. Figure adapted from Drouin and Agbandje-McKenna, 2013 (91).

A B

C D

Figure 1-3. AAV Capsid Structure. A) The AAV1 viral protein (VP3) monomer. The two- fold axis is shown by an ellipse, the three-fold axis is indicated by a triangle, and the five-fold axis of symmetry is indicated by a pentagon. B) The assembled AAV1 capsid has a depression at the two-fold, protrusions at the three-fold, and a pore at the five-fold axis of symmetry. C) The nine variable regions of the AAV1 are color-coded: VR-I in purple, VR-II in blue, VR-III in yellow, VR-IV in red, VR-V in black, VR-VI in pink, VR-VII in cyan, VR-VIII in green, and VR-IX in brown. These regions are the apex of the loops that connect the core elements of secondary structure. D) The arrangement of the variable regions within the capsid. VR-II makes the five-fold pore, while VR-IX makes up the two-fold depression. The remaining VR’s make up the surface of the three-fold protrusion and the surface directly adjacent to the protrusion. Figures A and C were made in Pymol. Figures B and D were made in Chimera.

Figure 1-4. The viral lifecycle. The AAV lifecycle begins with the internalization of the virus:receptor complex, followed by trafficking through the endo-lysosomal pathway, VP1u-mediated escape, and localization to the nucleus for replication.

CHAPTER 2 MATERIALS AND METHODS FOR BIOCHEMICAL CHARACTERIZATION

Maintenance of Sf9 Insect Cell Lines

Spodoptera frugiperda insect cells (ATCC® CRL-1711™) in suspension culture were passaged and split when they reached approximately 2.0x106 cells per mL of media. Cells were maintained in Sf-900™ II Serum-Free Media (ThermoFisher

Scientific, cat. no. 10902088), supplemented with 1% Antibiotic-Antimycotic (100X,

ThermoFisher Scientific, cat. no. 15240062). Cells were incubated in a shaking incubator at 37°C until next passage.

Maintenance of Mammalian CHO Cell Line Variants

Pro5 (ATCC® CRL-1781™), Lec2 (ATCC® CRL-1736™), and Lec8 (ATCC® CRL-

1737™) cell lines were passaged and split when they reached 70-100% confluency.

Prior to splitting, old media was removed and plates were washed in sterile 1X PBS (5 mL/10 cm plate, or 10 mL/15 cm plate). Cells were then removed from the plate surface using 0.05% trypsin (ThermoFisher Scientific, cat. no. 25300054) into 10 or 15 cm cell culture treated plates. For a 10 cm plate, 1 mL of 0.05% trypsin (2 mL for a 15 cm plate) was added after washing and incubated in the hood for 2-3 minutes at room temperature before the reaction was terminated using Minimum Essential Medium

Alpha + Glutamax (GIBCO, cat. no. 32571-036) supplemented with 10% Fetal Bovine

Serum and 1% Antibiotic-Antimycotic to a total volume of 5 mL (or 10 mL for a 15 cm plate). Plates were incubated in a 37°C incubator under conditions of 5% CO2 until next passage.

Production of Virus-Like Particles

Virus-like Particles of sAAV and other mammalian AAVs were expressed as previously described, using recombinant baculovirus in SF9 insect cells (92, 93). A P2 titered baculovirus stock of sAAV was generously gifted to us for the purpose of this project by the Tijssen Lab (INRS-Institut Armand-Frappier). This P2 stock was used to infect SF9 insect cells at an MOI of 5 plaque-forming units per cell in order to drive production of AAV viral proteins. Mammalian serotypes produced from P3 stocks using similar methodology. After 72 hours, cell pellets were produced by centrifugation at

3,000 rpm in a JA-20 rotor (Beckman-Coulter) at 4°C for 15 minutes. Cell pellets were resuspended in 25-50 mL of sample buffer (25 mM Tris HCl, 100 mM NaCl, 2 mM

MgCl2, 0.2% Triton X-100, pH 8) for pellets purified via sucrose, or in 1X TD (1X PBS with 1 mM MgCl2 and 2.5 mM KCl) for pellets purified via iodixanol. The supernatant was retained and mixed with 10% w/v polyethylene glycol 8000 in order to produce a peg pellet. This would prevent the loss of virus that had been secreted into the supernatant. The supernatant was stirred overnight at 4°C to encourage precipitation.

The mixture was then centrifuged at 9,000 rpm in a JA-20 rotor for 2 hours at 4°C in order to separate the precipitated virus from the media. Supernatant was discarded, and the pellet was resuspended in 10 mL of sample buffer. All cell and PEG pellets were stored at -20°C until needed for purification.

Virus Purification by Sucrose Cushion and Gradient

PEG and cell pellets of virus-like particles, with the exception of AAV2, were subjected to three rounds of freeze-thaws using liquid nitrogen and a 37°C water bath.

During the third thaw, the pellets were treated with 1 μL of Benzonase (250 units per μL;

Sigma, cat. no. E1014) per 10 mL of sample and incubated for 30 minutes. Pellets were

then centrifuged for 10 minutes at 7k rpm at 4°C in a Beckman JA-20 rotor in order to remove cell debris. The supernatant was transferred to a clean 15 mL conical tube before loading into a sucrose cushion. The cushion was made by dividing the sample evenly between two ultracentrifuge tubes, and was mixed with 8 mL TNET (50 mM Tris

HCl, 100 mM NaCl, 1 mM EDTA, 0.2% Triton X-100, pH 8). The sample was then underlaid with 5 mL 20% sucrose w/v in TNET, and the remaining volume in the tube was topped off with TNET. The cushions were then centrifuged at 45,000 rpm at 4°C in a Ti70 rotor (Beckman) for 3 hours, under conditions of slow acceleration and maximum brake. The supernatant was then removed, and the pelleted virus was resuspended in

TNTM (25 mM Tris HCl, 100 mM NaCl, 2 mM MgCl2, 0.2% Triton X-100, pH 8). After manual resuspension, the samples were allowed to rest overnight at 4°C and centrifuged in a desktop microfuge at 10k rpm for 5 minutes in order to remove insoluble material before loading onto a gradient. Sucrose gradients were produced using dilutions of 40% sucrose w/v in TNTM. Gradients were loaded from 40% to 5% in order of most concentrated to least concentrated in a stepwise fashion. One milliliter of sample was loaded on top of the gradient, and balanced using TNTM. Gradients were then spun for three hours at 35k rpm in a Sw-41Ti rotor (Beckman) at 4°C under conditions of slow acceleration and slow deceleration. Bands were visualized under white light, and sample was recovered using needle puncture with an 18g needle and a

10 mL syringe. Samples were then dialyzed to remove remaining sucrose before undergoing quality control measures such as SDS-PAGE and negative stain EM. For sAAV in particular, the purified sample was dialyzed into sAAV Buffer (20 mM Tris, 350 mM NaCl, 2 mM MgCl2). All other viruses were dialyzed into 1X PBS.

Virus Purification by Iodixanol Gradient

AAV2 virus-like particles were purified using iodixanol gradients. Cell and PEG pellets of AAV2 were first subjected to the same freeze-thawing and Benzonase treatment process, and clarified by centrifugation as described in the sucrose purification described above. VLPs were then purified by a discontinuous iodixanol step- gradient, composed of 15%, 25%, 40%, and 60% iodixanol. Each solution was prepared

TM using 1X TD buffer (1X PBS with 1 mM MgCl2 and 2.5 mM KCl) and Optiprep solution

(Sigma-Aldrich, cat. no. D1556). The 15% solution was additionally supplemented with

5M NaCl, and the 25% and 60% fractions also contained phenol red as a pH indicator to delineate the different fractions of the gradient. Gradients were made in OptiSealTM tubes (Beckman Coulter, cat. # 361625). The discontinuous gradients were formed by underlaying the denser fraction, displacing the less dense solution. Gradients were composed of 7 mL 15% iodixanol, 5 mL 25% iodixanol, 4 mL 40% iodixanol, and 60% iodixanol on the bottom. Approximately 12 mL of clarified cell lysate was loaded on top of the gradient, and the remaining volume was filled with TD buffer. Gradients were centrifuged for one hour at 69,000 rpm in a 70Ti rotor (Beckman Coulter), and samples were fractionated using needle puncture. Fractions were collected for each band, and the interfaces between each band. Fractions were collected as follows: 2 mL 60%, 2 mL

40-60% interface, 2 mL 40% fraction, 5 mL of 25-40% interface, and the entire 25% fraction. The 15% fraction is typically discarded. Virus-like particles are most commonly observed in the 25% and 40% fractions, and the 25-40% interface. Samples were then run on a gel to identify which fractions contain sample before combining them and proceeding to the ion exchange chromatography step for further purification.

Ion Exchange Chromatography

Ion exchange chromatography was used to further purify samples purified by iodixanol gradient. The 25% fraction from iodixanol gradients were diluted 1:10, while the 25/40% interface and the 40% fraction were diluted 1:1 in binding buffer (20 mM Tris

Base, 15 mM NaCl, pH 8.5, filter sterile). 5 μL of Phenol Red was added as a visual marker for loading the sample onto columns. New Hi-Trap Q XL 5 mL sepharose anion exchange columns (GE Healthcare, cat. no. 17-5159-01) were washed with 10 mL sterile 20% ethanol, 10 mL sterile diH2O, and 10 mL binding buffer using a peristaltic pump at a flow rate of 1 mL/minute prior to use in order to remove all storage solution.

Columns were washed with binding buffer, charged with 25 mL elution buffer (20 mM

Tris Base, 500 mM NaCl, pH 8.5), and 50 mL binding buffer at a flow rate of 5 mL/minute in order to equilibrate the column using the AKTA FPLC system (GE

Healthcare). Charged columns were then loaded with sample before washing for five minutes with binding buffer at a flow rate of 5 mL/min. After washing, the column was eluted using a gradient ranging from 100% binding buffer to 100% elution buffer at a flow rate of 1 mL/minute over the course of 20 minutes. Fractions containing virus were identified by absorbance measurements, and confirmed using SDS-PAGE. Samples were further concentrated using Apollo conical concentrators (Orbital Biosciences), and visualized using negative stain EM to confirm sample purity and that the particles were intact.

OneBac Virus Production Platform

GFP-packaging constructs of recombinant AAV serotypes 1-9 were produced using the scalable One-Bac platform as previously described (94). These samples were

generously gifted to us for the purposes of this project by Dr. Mario Mietzsch (UF) and

Dr. Regine Heilbronn (Charité- Universitätsmedizin Berlin Campus Benjamin Franklin,

Institut für Virologie).

Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis

Concentration estimation and sample purity confirmation of virus-like particles was carried out using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-

PAGE). Samples were prepared by mixing 10 μL of sample with 2 μL of 6X SDS

Loading Buffer. Samples were then heat denatured by boiling at 100°C for 10 minutes.

Denatured samples were then briefly centrifuged in the desktop microfuge, before loading onto a 10% acrylamide protein gel. Samples were run alongside 8-10 μL of

Precision Plus All Blue Protein Molecular Weight Standard (Bio-Rad, cat. # 161-0373), and BSA standards ranging from 0.125 mg/mL to 1.0 mg/mL (Bio-Rad, cat. #500-0207).

Gels were electrophoresed at approximately 180 kilovolts for 45 minutes. Gels were then washed three times for ten minutes each in diH2O before staining with Gel Code

Blue Stain Reagent (CAT NO) for one hour at room temperature. After staining, gels were washed again three times for ten minutes each before imaging with the Bio-Rad

GelDocTM XR+ system.

Negative Stain Electron Microscopy

All negative stain electron microscopy (EM) grids using the same method.

CF400-400 carbon-coated holey copper mesh grids were glow discharged using the

PELCO easiGlow Glow Discharge Cleaning System (Ted Pella, Inc.) for one minute immediately before use. 5 μL of sample was applied to the carbon-coated surface of the grid, and allowed to adhere for two minutes. Grids were then washed in 15 μL droplets of filter sterile diH2O three times, at five seconds each to remove virus that did not

adhere to the surface of the grid. The excess water was wicked off of the surface of the grid using Whatman #1 filter paper (Sigma, cat # WHA1005090). The grids were then stained with two 10 μL droplets of filter sterile 2% uranyl acetate for 10 seconds each.

Remaining stain was wicked away using Whatman #1 filter paper, and grids were stored until viewing. Negative stain EM grids were imaged at the University of Florida

Interdisciplinary Center for Biotechnology core via a Gatan UltraScan 1000XP camera and FEI Tecnai G2 Spirit Transmission Electron Microscope (TEM) operating at 120 kV accelerating voltage.

Native Dot Blot – Mouse Monoclonal Antibody Panel

Mouse monoclonal antibodies targeting conformational epitopes on the surface of the mAAV capsid were used to challenge sAAV. Monoclonal antibodies including

ADK1a (AAV1), A20 (AAV2; ARP, cat. # 03-61055), ADK4 (AAV4), ADK5b (AAV5),

ADK6 (AAV6), ADK8 (AAV8), ADK9 (AAV9), and B1 (denatured capsids) were used for this panel. Mammalian AAV serotypes 1-9 and sAAV were diluted to approximately 50 ng/mL in 1X PBS. 50 μL of sample was then applied onto AmershamTM ProtranTM 0.45

μm NC Nitrocellulose Blotting Membrane (GE Healthcare Life Sciences, cat. no.

10600007) using vacuum suction and Minifold Dot-Blot apparatus (GE Healthcare, cat.

# 10447850). The membrane was then blocked in 10% non-fat dry milk (LabScientific,

M-0842) in filter sterile 0.05% Tween-PBS overnight at 4°C. The following day, blots were blocked one hour at room temperature. Blots were then washed twice for five minutes each in 0.05% Tween-PBS before moving into primary antibody. Primary antibodies were diluted 1:1000-1:5,000 in 5% milk in Tween-PBS. Primary antibodies were applied to the blot, and incubated on a rocker for one hour at room temperature.

Blots were then washed three times for 10 minutes each in 0.05% T-PBS before moving

into secondary antibody. Anti-mouse IgG HRP-conjugated secondary antibodies (GE

Healthcare, cat. # NA931) were diluted 1:10,000 in 1XPBS, and incubated on the blot for one hour at room temperature while rocking. Blots were washed again three times for ten minutes each before developing with Immobilon chemiluminescent substrate

(Millipore, cat. # WBKLS0500) and imaged for the detection of viral protein.

Fluorescent Labeling of Virus Capsid for FACS

sAAV and an AAV2 control were labeled with Alexa Fluor 488 Protein Labeling

Kit (ThermoFisher Scientific, cat. no. A10235). sAAV (100 μL, 2.5 mg/mL) was dialyzed from sAAV Buffer (described above) into 1X PBS buffer in order to remove Tris. This was done in order to prevent non-specific binding of the fluorescent label to lysine residues present in buffer components. After dialysis, sAAV and AAV2 were each diluted up to 500 μL in 1X PBS. Virus particles were labeled according to a modified version of the manufacturer’s specifications. Virus particles in 1X PBS were mixed with

40 μL of Borate Buffer (0.67 M, pH 8.5). The sample was then added to the DyLight

Reagent vial and incubated for 60 minutes at room temperature, protected from light.

After one hour, an additional 20 μL of Borate Buffer was added, and the virus was incubated an additional 30 minutes at room temperature. In a deviation from the manufacturer’s specification, the sample was not purified using the supplied column system. Instead, the sample was subjected to three rounds of dialysis into 4L of 1X PBS in order to remove unbound fluorescent dye in order to prevent loss of labeled capsids.

Successful labeling was confirmed using SDS-PAGE. The gel was run as described above, but was visualized using a UV light in order to observe the fluorescent probe on the gel before proceeding with Gel Code Blue staining.

Fluorescence Activated Cell Sorting

24 hours prior to conducting the experiment, Pro-5, Lec-2, and Lec-8 CHO variant cells were passaged into a 15 cm plate when they reached 70-100% confluency.

50 mL aliquots of filter sterile 1X PBS and unsupplemented MEM were chilled overnight at 4°C. Virus was diluted in a total volume of 300 μL per reaction to produce an MOI of

1x105.

On the day that the experiment was to be conducted, cells were viewed to confirm that cells were at 90-100% confluency, then removed from the culture dish using 0.5 M EDTA (2 mL per 15 cm plate) in a method similar to passaging. Cells were moved into a clean 50 mL conical tube, and the reaction was stopped with 4 mL of

MEM, for 6 mL total volume. This approach was used in the case of cells used in the sAAV experiments, given that trypsin cleaves off surface modifications and it is unknown if sAAV has any modifications that are required for cell binding. Cells were then centrifuged at 500 rpm in a desktop centrifuge for 5 minutes, and the supernatant was removed in order to remove all remaining EDTA. The pellet was resuspended into

6 mL of pre-chilled unsupplemented MEM. Following release from the cell culture plates, the cells were counted diluted 1:2 with Trypan Blue in order to identify live cells.

Cells were then diluted to 5x105 cells/mL, for each reaction. Cells were then chilled for

30 minutes at 4°C.

Following chilling, cells were aliquoted to 500 μL of cells at 5x105 cells/mL per each reaction, and mixed with 300 μL of fluorescently labeled VLPs at an MOI of 1x105

PFU/mL. The reactions were then incubated for one hour at 4°C on a shaker, shielded from light. Following incubation, the cells were washed to remove unbound virus-like particles. Cells were centrifuged for 10 minutes at 3000 rpm in a desktop microfuge and

the supernatant was removed. Pellets were resuspended in 300 μL of pre-chilled 1X

PBS, and centrifuged again at 3000 rpm for 10 minutes. The supernatant was removed, and the pellet was once again resuspended in pre-chilled 1X PBS. Cell binding was then determined by Fluorescence Activated Cell Sorting in the University of Florida

Interdisciplinary Center for Biotechnology using the FACS-Calibur instrument (BD

Biosciences). For this set of experiments, Alexa-488-labeled AAV2 was used as a control because it has previously been shown to bind to the surface of all three of the selected CHO variant cell lines (55). The experimental setup was conducted in technical triplicate for each cell line, and included an uninfected cell line control. The experiment was double-gated to select for live cells and bound cells. Raw data was processed using the FCS Express5 software suite (BD Biosciences). Statistical significance of the cell binding data was determined using standard deviation and standard error calculations, with n=3 for the technical triplicate.

Low pH Studies

sAAV underwent three rounds of dialysis into 4L citrate phosphate buffer at pH

7.4, 6.0, 5.5, and 4.0. Samples were then collected and run on an SDS-PAGE gel in order to confirm sample concentration and verify any banding patterns observed. In a

96-well PCR plate, 22.5 μL of sAAV at each pH was mixed with 2.5 μL of Sypro-Orange protein dye diluted 1% v/v in diH2O (Life Technologies, cat. # S-6651). The plate was then loaded into the Bio-Rad MyiQ2 Real-Time PCR machine. The samples were then subjected to a high temperature melting curve ranging from 30°C to 100°C, during which the temperature was raised 1°C per minute over the course of one hour. Melting temperature was determined from a plot of fluorescence emission against temperature.

Statistical significance of the trends observed was determined by calculating standard

deviation and standard error, for n = 7 trials for sAAV. All mammalian AAV data was determined previously (Bridget Lins, Dissertation), with error calculated in the same way.

Table 2-1. Mouse monoclonal antibody panel details. Antibody Recognizes Dilution in 0.05% T-PBS ADK1a Intact AAV1; cross-reactivity with AAV6 1:500

A20 Intact AAV2; cross-reactivity with AAV3b 1:1000

ADK4 Intact AAV4 1:500

ADK5b Intact AAV5 1:1000

ADK6 Intact AAV6 1:3000

ADK8 Intact AAV8; low level cross-reactivity 1:500 with AAV3b

ADK9 Intact AAV9 1:700

B1 C-terminus of denatured VP3 protein* 1:3000

* B1 does not recognize AAV4 due to a 3 amino acid difference in the sequence of AAV4 that disrupts the epitope.

CHAPTER 3 MATERIALS AND METHODS FOR CRYO ELECTRON MICROSCOPY AND 3D STRUCTURE DETERMINATION

Vitrification

Vitrification of grids for cryo electron microscopy was carried out using a Vitrobot

Mark 4 automatic plunge-freezing system. 3 μL of sample was applied to glow discharged C-flat holey carbon-coated grids (Protochips Inc., Raleigh, NC), and incubated at 4°C under 95% humidity for approximately 3.0 seconds. Grids were then blotted using filter paper (Whatman #1). Grids were then plunged into an ethane slush cooled with liquid nitrogen. Grids were stored under liquid nitrogen temperatures for data collection.

Cryo-Electron Microscopy Data Collection

For sAAV data collection, vitrified grids were processed for data collection at the

Florida State University Biological Science Imaging Resource Core. Samples were examined using the FEI Titan Krios instrument operating at an accelerating voltage of

300 kV. Micrographs were collected using a DE-20 camera system (Direct Electron) under dosage conditions of 61.72 e-/Å2 at a magnification of 29000X with a final step size of 1.26 Å/pixel.

Cryo-EM Data Processing and 3D Structure Determination

A total of 773 micrographs were used for sAAV 3D structure determination. The

RobEM subroutine of the Auto3DEM software suite was use to select individual particles for use in generation of the 3D reconstruction (95). The boxes used for particle selection were a box size of 291, which is approximately 3.5 times the radius of the particle in pixels. From this dataset, a total of 31,956 individual particles were manually selected and used in structure determination. The ctffind3 subroutine of the Auto3DEM

software suite was used to determine defocus estimates in order to estimate image quality. A separate Auto3DEM function was used to normalize and apodize a subset of particles to generate an ab initio random model under icosahedral symmetry constraints. This model was then used as the initial starting model for refinement, and was refined using the Auto3DEM Polar Fourier Transform (PFT) search mode refinement for the first 10 iterations, then Parallel Origin and Orientation Refinement

(PO2R) local refinement until map resolution failed to improve below 4.1 Å (95). The

PO2R local iterations of refinement resulted in the exclusion of particles based on the

Auto3DEM program’s internal criteria as well as score fractionation to select the highest quality particles. Inclusion of only the top 80% of particles resulted in approximately

17,000 particles being used to produce the final map. The final electron density map was refined to a resolution of ~4.1 Å at a Fourier Shell Correlation (FSC) value of 0.5, as opposed to the Auto3DEM gold standard value of 0.14. This FSC value was used to estimate the quality of the map, as well as provide provides a measure of signal-to- noise (Table 3-1)(96). The use of an FSC value of 0.5 is a more conservative estimate than the gold standard value of 0.14, and is typically a more accurate estimate of the current map resolution.

Model Building

A model of the sAAV VP3 was generated from the published amino acid sequence using Swiss Model, then converted into a standard orientation in order to generate a 60mer model in ViperDB (58, 97, 98). Using Chimera, sAAV 60mer coordinates were docked into the 4.1 Å resolution cryo-EM density map at a correlation coefficient of 0.79, with a voxel size of 1.22 (Table 3-1) (99). The correlation coefficient describes the goodness of fit, and is a quality estimate for how well the model fits into

the 3D map density. Voxels are a unit of measure for a 3D space, where pixels are used in 2D space. Voxels are a volumetric pixel that provide information about the sampling of the map. This may differ from the pixel estimate given during data collection, with

Angstrom per pixel measurements being higher or lower than the voxel size. Once the

60mer was fitted to the electron density map, a monomer was extracted in the fitted orientation and saved out as a new PDB file. The map was converted into a CCP4 format, and the new monomer was then fitted into the electron density map using the modeling program Coot and its real space refinement tool in order to fit the main chain of the viral protein into the density (100). and input into ViperDB Oligomer generator to produce a 60mer for fitting into the electron density map in the program Chimera (98,

99).

Secondary Structure Matching and Variable Region Assignment

Secondary structure matching was used to align sAAV with the PDB coordinates of pre-existing crystal structures of AAV1 (PDB ID 3NG9), AAV2 (PDB ID 1LP3), AAV5

(PDB ID 3NTT), AAV8 (PDB ID 2QA0), and AAV9 (PDB ID 3UX1) (101–105). These elements were used for alignment because they are the most highly conserved between

AAV serotypes. The mammalian AAVs were designated the reference structure while sAAV was compared to them. Variable regions were assigned using criteria established by Govindasamy et al., which defined the difference between the Cα backbone as two or more residues that deviated from the alignment by greater than 1Å at the apex of the loops connecting elements of secondary structure were considered to be variable (24).

These regions were compared for all five mammalian AAVs in order to establish an average range of variable residues for sAAV at the apex of the loops connecting the

strands of the beta sheet. If gaps were present in a series of residues that deviated by more than 1Å, those residues covered by the gap were included in the VR assignment.

Visualization of Structural Comparison

Structural comparison following secondary structure matching and variable region assignment was carried out in PyMol (The PyMOL Molecular Graphics System,

Version 1.2r3pre, Schrödinger, LLC.) in order to visualize structural differences between sAAV and the mammalian AAV serotypes 1, 2, 5, 8, and 9. PyMol was used to superimpose all of the viral protein PDB coordinate files onto each other, and analyze each individual variable region by structure and sequence. PyMol was also used to generate images.

Table 3-1. Equations used in 3D image reconstruction.

Equation

Map Fit 퐶표푟푟푒푙푎푡푖표푛 Correlation is defined in the Chimera Correlation < 푢 >< 푣 > software suite as “the inner = Coefficient |푢||푣| product of vectors u and v containing the fit map values and corresponding interpolated reference map values” (99)

Fourier Shell 퐹푆퐶(푟) The FSC variables are defined as: “F1 Correlation 훴퐹 (푟 ) ∙ 퐹 (푟 ) is the complex structure factor for = 1 푖 2 푖 2 2 volume 1 √(훴|퐹1(푟푖)| ∙ 훴|퐹2(푟푖)| ) F2 = conjugate of the structure factor for volume 2 ri = voxel element at radius r” (96)

CHAPTER 4 RESULTS AND DISCUSSION

Structural Determination of Squamate Dependoparvovirus Type I

sAAV was successfully produced and purified by sucrose gradient, dialyzed into sAAV buffer, and subjected to quality control measures by SDS-PAGE and negative stain EM (Figure 4-1). The three-dimensional capsid structure of Squamate

Dependoparvovirus Type I (sAAV) was determined to a resolution of 4.1 Å using cryo- electron microscopy and 3D image reconstruction. sAAV was found to conserve the hallmark structural features observed in the mammalian AAVs, such as protrusions at the three-fold axis, a pore at the five-fold, depression at the two-fold, and a wall at the interface between the two- and five-fold axes of symmetry (Figure 4-2). When compared to the pre-existing crystal structures for AAV1 (PDB ID 3NG9, 2.5 Å resolution), AAV2

(PDB ID 1LP3, 3.0 Å resolution), AAV5 (PDB ID 3NTT, 3.5 Å resolution), AAV8 (PDB ID

2QA0, 2.6 Å resolution), and AAV9 (PDB ID 3UX1, 2.8 Å resolution), the most observable difference, is that the three-fold protrusions on the surface of sAAV are more rounded, lobate, and less elevated than those observed in AAV1, 2, 8, and 9, whose three-fold protrusions are elongated (Figure 4-2). It appears to be most similar to AAV5 on a surface level, as AAV5 has truncated, rounded three-fold protrusions. The resolution of the map was high enough that density was observable for all main chain structures such as the strands of the βBIDG and CHEF sheets and the α helix, but not high enough to determine the orientation of side chains. Secondary structure matching with mammalian AAV VP3 monomers (described above) identified nine regions of variability, which was consistent with variable region assignments for the mammalian serotypes. While the amino acid composition of the loops differed between serotypes,

the arrangement of the variable regions within the capsid was conserved, in that the regions of high variability were localized to the apex of the surface loops. sAAV has some unique features in its variable regions that are not observed in the other serotypes. sAAV VR-I, VR-III, VR-IV, VR-VII, VR-VIII, and VR-IX possess significant variation, resulting in a Cα deviation ranging from 1.5-4 Å (Figure 4-3). The algorithm assigns ‘gaps’ to residues in the moving structure, in this case sAAV, for which there is no corresponding match in the reference mammalian AAV structure.

In the case of VR-I, the loop maintains the same shape as those from the other serotypes but is oriented differently. sAAV VR-I is tilted downward, and oriented more toward the midline of the VP monomer. VR-II did not differ significantly from the mammalian AAVs, and the loop maintained the same overall shape observed in the mammalian AAVs. VR-III also did not differ significantly in regards to Cα backbone alignment with the other AAVs, with the exception of sAAV residue G372. This is the only residue in this variable region that had a deviation greater than 1 Å. This particular residue differed by greater than 2 Å from each monomer compared. VR-IV differs significantly from the mammalian AAVs due to a seven amino acid deletion that truncates this loop. AAV5 has a similar deletion, but it is not as severe as that observed in sAAV. This loop is also tilted inward toward the interior of the three-fold protrusion in comparison to the mammalian AAVs, with the exception of AAV5. This, in conjunction with the deletion, are likely responsible for the truncated appearance of the sAAV three- fold protrusions. VR-V spans fewer amino acids in sAAV than it does in the mammalian

AAVs, with the exception of AAV5. There are several residues in this region that do not deviate from the mammalian AAVs 1, 2, 8, and 9 by more than 1 Å, and four residues

(I485-S488) that differ by greater than 1 Å. However, when compared to AAV5, all of the residues spanning VR-V differ by a range of 1-4 Å, with the same residues I485-S488 differing the most. AAV5 has gaps in the sequence in this variable region in comparison to sAAV, which likely accounts for the variation in VR-V. VR-VI is complex, because the number of residues that meet the criteria for being considered divergent varies between the mammalian serotypes that it is being compared to. For AAV1, residues F507-G519 differ by greater than 1 Å, while AAV2, AAV5, AAV8, and AAV9 differ by greater than 1

Å only at residues Y513-G515, with a gap at residue 514 for AAV2 and AAV5 or 513 for

AAV8. Overall, sAAV does not differ greatly from the VR-VI observed in the mammalian

AAVs, with the exception of AAV1. VR-VII corresponds with large regions of gap in the secondary structure matching output for sAAV residues 530-537, with intermittent residues measured as deviating from the Cα backbone from approximately 1-3.7 Å when compared to AAV1, AAV2, and AAV5. This region has fewer gaps with respect to

AAV8 and AAV9, and appears to deviate less from these two serotypes than AAV1, 2, and 5. VR-VIII differs visibly from the mammalian serotypes in that, much like VR-IV, the loop is folded inward toward the interior of the 3-fold protrusion. This loop is composed of sAAV residues Q570-T578, and the entire range is consistently above the 1 Å threshold for all the mammalian AAVs compared. VR-XI, similar to VR-IV, has sequence modifications that contribute to a change in surface features in comparison to the mammalian AAVs. Sequence alignment indicates that sAAV has a four amino acid

(P696-D699) insertion (Figure 4-3). These four additional residues, including a highly rigid proline residue, produce an additional protruding loop that is not present in any of the five mammalian AAV serotypes compared. However, this amino acid insertion does

not constitute the entire range of the variable region. The 1 Å deviation threshold criteria is met from T693-F703. A complete detailed alignment of sAAV with all five mammalian

AAVs and their corresponding mammalian AAV residues are shown in Table 4-1.

Characterization of Squamate Dependoparvovirus I sAAV is Capable of Escaping Recognition by Mouse Monoclonal Antibodies that Target the mammalian AAV Capsid

The antigenic profile of sAAV was determined using native dot blot (Figure 4-4).

The B1 positive control produced signal for all mammalian AAVs, with the exception of

AAV4. sAAV was also not recognized by the B1 antibody. This is because both AAV4 and sAAV have a three amino acid mutation in the eight amino acid linear epitope recognized by B1. A positive control for sAAV is currently in development. The antibodies used each produced signal or their targeted AAV serotype (ie. ADK1a recognizing AAV1 and AAV6) (Table 4-1). As expected, ADK1a recognized AAV1 and

AAV6, as previously described. A20 recognized AAV2 and AAV3b, as previously described (94) ADK4, ADK5b, and ADK6 all recognized their respective serotypes.

ADK8 demonstrated cross-reactivity with AAV8 and AAV3b, as previously described.

ADK8 is rather promiscuous, and has been shown to cross-react with several serotypes

(94). When challenged with a panel of mouse monoclonal antibodies, sAAV was able to escape recognition by all mouse monoclonal antibodies (Figure 4-4). This means that sAAV does not share any of these serotype-specific epitopes that are found on the surface of mammalian AAVs. It is likely that the variations in amino acid sequence and the overall structure of the variable regions of sAAV are responsible for its ability to escape recognition by antibodies that target the mammalian AAV capsid.

sAAV is Capable of Recognizing and Binding to Mammalian Cell Lines

Fluorescence Activated Cell Sorting data indicated that sAAV was capable of binding to all three CHO variant cell lines: Pro-5, Lec-2, and Lec-8 (Figure 4-6). All values reported are averages of binding events across all three trials in the technical triplicate. sAAV was able to bind to Pro-5 cells at comparable levels to AAV2, with an average of approximately 32% of cells being bound. However, sAAV bound Lec-2 with approximately 25% of the efficiency of AAV2. AAV2 bound approximately 29% of the

Lec-2 cells tested, while sAAV was only able to bind approximately 8% of Lec-2 cells.

With regard to Lec-8, AAV2 was found to bind approximately 34% of cells, while sAAV bound only 18%. Therefore, sAAV was able to bind Lec-8 cells with about half of the efficiency of AAV2.

These results answer a major question about the applicability of reptilian dependoparvoviruses, in that the ability of reptilian parvoviruses to bind to mammalian cells has not been previously established. This experiment marks the first time that a reptilian parvovirus has been shown to bind to mammalian cells, let alone multiple cell types. However, these data raise further questions in that all of the CHO cell line variants are also known to express HSPG on their cell surface, in addition to their respective terminal glycans. Therefore, while these data do provide information about sAAV’s relative binding efficiency in comparison to AAV2, they do not definitively resolve which terminal glycans sAAV is capable of binding to. It is possible that sAAV is capable of binding to terminal N-linked sialic acid (Pro-5), terminal N-galactose (Lec-2), and N-acetyl glucosamine (Lec-8) because the binding efficiency varies between cell types. AAV2 binds to HSPG on the surface of all three cell types and did not undergo such a drastic deviation in binding efficiency, while sAAV saw a wide variation in binding 53

between cell types. This indicates that sAAV is likely preferentially binding to the terminal glycans rather than heparin sulfate proteoglycan, but further experiments need to be undertaken in order to determine if this is true. Based on what is known about the receptor binding sites in the mammalian AAVs, it is likely that sAAV does not directly match the binding profiles observed in AAV1, 2, 5, and 9. The mutations in the variable regions indicate the likelihood of a unique receptor binding site on the sAAV capsid surface. However, given that the cell binding data shows that sAAV is capable of binding to 37% of Pro-5 cells, that sAAV is likely to bind sialic acid as its primary cell surface receptor. sAAV Conserves pH-Induced Proteolytic Cleavage Activity Observed in Mammalian AAVs

Previous studies have shown that mammalian AAVs are proteolytically modified as they are subjected to conditions of decreasing pH that mimic conditions of the endosomal pathway (39). In order to determine if this behavior was conserved in reptilian AAVs, sAAV was dialyzed into citrate phosphate buffer at pH 7.4, 6.0, 5.5, and

4.0, then run by SDS-PAGE and visualized by negative stain EM (Figure 4-7). The

SDS-PAGE gel depicted a pH-dependent increase in cleavage product, indicating that the capsid was being modified as pH became more acidic. The concentration of degradation products was greatest at pH 4.0, which is consistent with pre-existing data for AAV1, 2, 8, and 9 (38, Antonette Bennett, Unpublished Data). The degradation profile for AAV5 differs, with the onset of cleavage starting at pH 7.4. This is in contrast to AAV1, 2, 8, and 9, which follow a degradation pattern than demonstrates a substantial increase in cleavage products between pH 5.5 and 4.0. Between these two pH conditions, sAAV undergoes significant modification, as observed in the SDS gel.

There is a reduction in the concentration of whole VP3, and an increase in the other lower molecular weight bands below it, which is indicative of proteolytic cleavage.

When the pH-adjusted samples were viewed by negative stain EM, sAAV particles were found to remain intact, regardless of environmental pH. This is consistent with what has been observed for the mammalian AAVs. The concentration of the sAAV sample at pH 7.4 was slightly reduced in comparison to the other three pH samples, which is likely due to loss of virus to the membrane during dialysis. Regardless, the concentration was sufficient for use in negative stain EM and SDS-PAGE. Previously, questions have been posed regarding the distinction between true autolytic proteolysis and acid hydrolysis, regarding the pH cleavage profiles of mammalian AAVs (39). For the mammalian AAVs, it was established that this pH-dependent cleavage is confirmed even in the absence of environmental proteases (39). In summary, sAAV conserves the pH-dependent autolytic cleavage behavior observed in mammalian AAVs, and matches the cleavage trends observed in AAV1, 2, 8, and 9, with the most cleavage occurring between pH 5.5 and 4.0. sAAV is Highly Thermostable and Conserves the pH Stability Profile Observed in Mammalian AAVs

Differential Scanning Fluorimetry (DSF) experiments were used to determine how pH changes can modulate capsid thermostability, inconjunction with pre-existing

Differential Scanning Calorimetry (DSC) data from Bridget Lins. Previous studies have established a trend for mammalian AAV capsid stability, indicating that for serotypes

AAV1, 2, 8, and 9, the capsid demonstrates a trend of increasing stability from pH 7.4 to

6.0, peaks at 5.5, and sharply drops at pH 4.0 (Bridget Lins, Dissertation). This is countered by AAV5, which demonstrates a trend of being most stable at pH 7.4, then

steadily decreasing as pH becomes more acidic (Bridget Lins, Dissertation). Of these serotypes, AAV5 was found to be the most thermostable at pH 7.4, with a capsid melting temperature of approximately 89.8°C. All three technical triplicates were consistent, indicating that sAAV at pH 7.4 was stable up at temperatures up to 85.5°C,

89.5°C at pH 6.0, 90.0°C at pH 5.5, and 83.0°C at pH 4.0 (Figure 4-8). Standard error, in the case of sAAV, was calculated as 0.1 for both pH 7.4 and 6.0, 0.0 for pH 5.5, and

0.6 for pH 4.0.

Overall, these data indicate that sAAV conforms to the thermostability patterns observed in mammalian AAV serotypes 1, 2, 8, and 9. In addition, sAAV is the most thermostable out of all of the serotypes that have the same trend. AAV5 is the most stable at pH 7.4 in comparison to all other viruses studied, followed by sAAV, AAV1,

AAV9, AAV8, and AAV2 as the least stable. The viruses maintain this order, with the exception of AAV5 which rapidly becomes less thermostable. At pH 4.0, sAAV is most stable, followed by AAV1, AAV5, AAV8 and AAV9 at equal thermostability, and AAV2 as the least stable.

Table 4-1. sAAV variable region assignment and corresponding mammalian AAV residues.

sAAV AAV1 AAV2 AAV5 AAV8 AAV9

VR-I 250-259 265-270 261-269 252-261 263-272 263-272

VR-Il 316-319 329-330 326-328 318-325 329-322 316-319

VR-III 372-375 383 382 373-377 385 384

VR-IV 440-455 450-459 465-470 439-452 452-461 452-461

VR-V 482-487 500-502 490-501 474-493 502-505 502-505

VR-VI 514-515 528-529 527-528 511-519 530-531 530-531

VR-VII 535 545-550 549 532-544 548-552 548-552

VR-VIII 570-577 584-591 583-590 573-581 586-593 586-593

VR-IX 693-703 707-713 706-712 692-698 709-715 709-715 Amino acid assignments are indicated in VP1 numbering. Residues indicated are the amino acid ranges that were above the 1 Å threshold.

A B Figure 4-1. Purification of sAAV. A) sAAV was purified by sucrose gradient to a final volume of 10 mL, at a concentration of 0.125 mg/mL. BSA molecular weight standards were used to estimate concentration, which were confirmed by densitometry and absorbance readings. B) Negative stain EM images of purified sAAV reconfirm concentration and purity. sAAV particles were intact throughout the surface of the grid.

Figure 4-2. Comparison of sAAV capsid surface to mammalian AAV serotypes 1, 2, 5, 8, and 9. These images are radially depth cued, with blue corresponding to features that are closer to the center of the capsid interior, while red corresponds to features further from the center of the capsid. Capsid surface images were generated in Chimera.

Figure 4-3. sAAV differs from mammalian AAVs structurally in its variable regions. A) Superimposition of the VP3 monomer of sAAV, and AAV1, 2, 5, 8, and 9. B-J) Magnified images of each variable region. Figures were generated in Pymol.

SAAV ---MDFLDDFFADKYKETVNELGK--PVNPKPVKHISEAHSQPGSRRGFVVPGYRYLGPG 55 AAV1 MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKA----NQQKQDDGRGLVLPGYKYLGPF 56 AAV2 MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKP----AERHKDDSRGLVLPGYKYLGPF 56 AAV5 MSFVDHPPDWLEE-VGEGLREFLGLEAGPPKPKP----NQQHQDQARGLVLPGYNYLGPG 55 AAV8 MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKA----NQQKQDDGRGLVLPGYKYLGPF 56 AAV9 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKA----NQQHQDNARGLVLPGYKYLGPG 56 .. *:: : * :.: * * . : .. **:*:***.****

SAAV NSLDRGKPVNKADEAAKKHDQEYDQQLKAGDNPYIKYNHADEQFQKDLQGDTSLAGNAAN 115 AAV1 NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGR 116 AAV2 NGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGR 116 AAV5 NGLDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLGK 115 AAV8 NGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGR 116 AAV9 NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGR 116 *.**:*:*** ** .* :** *:.**.:*****::***** :**: * ***:.** ..

SAAV ALFQGKKTLLAPLGLVETPVGKTSEKHKLDEYYPKAKKAKQ-GLQIPA--PP------K 165 AAV1 AVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQ-EPDSSSGIGKTGQQPAKKRLNFGQ 175 AAV2 AVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPV-EPDSSSGTGKAGQQPARKRLNFGQ 175 AAV5 AVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPS------T 164 AAV8 AVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQ 176 AAV9 AVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQ-EPDSSAGIGKSGAQPAKKRLNFGQ 175 *:**.** :* *:**** . .: :: : * . .

SAAV GGEE---EATSSQSGGSPAGSDTSGTSVMATGGGGPMADDNQGAEGVGNSSGDWHCDTKW 222 AAV1 TGDSES-VPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTW 234 AAV2 TGDADS-VPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTW 234 AAV5 SSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTW 224 AAV8 TGDSES-VPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTW 235 AAV9 TGDTES-VPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQW 234 .: . * .*::*.*.*:.*:*:**:***.:**:****: *

SAAV MGDHVITKSTRTWVLPTYGNHLYGPINFDGTTGSGANAAYAGYKTPWGYFDFNRFHCHFS 282 AAV1 LGDRVITTSTRTWALPTYNNHLYKQISSAS-TGASNDNHYFGYSTPWGYFDFNRFHCHFS 293 AAV2 MGDRVITTSTRTWALPTYNNHLYKQISSQ--SGASNDNHYFGYSTPWGYFDFNRFHCHFS 292 AAV5 MGDRVVTKSTRTWVLPSYNNHQYREIKSGSVD-GSNANAYFGYSTPWGYFDFNRFHSHWS 283 AAV8 LGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYFDFNRFHCHFS 295 AAV9 LGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFS 294 :**:*:*.*****.**:*.** * *. . * **.************.*:*

SAAV PRDWQRLINNHTGIRPKGLKIKVFNVQVKEVTTQDSTKTIANNLTSTVQIFADENYDLPY 342 AAV1 PRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPY 353 AAV2 PRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPY 352 AAV5 PRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPY 343 AAV8 PRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPY 355 AAV9 PRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPY 354 ********** *:**: * .*:**:****** ::...*********:*:*:*.:*:*** Figure 4-4. Sequence Alignment of sAAV with mammalian AAV serotypes 1, 2, 5, 8, and 9. This table should be viewed in conjunction with Table 4-1. This alignment was generated with Clustal-W Multiple Sequence Alignment tool, and all assignments were created using the default algorithm values (88). Asterisks indicate fully conserved residues. A colon indicates residues with strongly similar properties, ranking with a Gonnet Point Accepted Mutation (PAM) 250 matrix score of greater than 0.5. A period indicates residues with weakly similar properties, ranking with a Gonnet PAM 250 matrix score of less than 0.5. PAM matrix scores are used by Clustal-W to calculate probabilities of an amino acid being replaced with an accepted point mutation during an evolutionary interval.

SAAV VLGSATQGTFPPFPNDVFMLPQYAYCTLQG-NSGKFVDRSAFYCLEYFPSQMLRTGNNFE 401 AAV1 VLGSAHQGCLPPFPADVFMIPQYGYLTLNNG--SQAVGRSSFYCLEYFPSQMLRTGNNFT 411 AAV2 VLGSAHQGCLPPFPADVFMVPQYGYLTLNNG--SQAVGRSSFYCLEYFPSQMLRTGNNFT 410 AAV5 VVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFE 403 AAV8 VLGSAHQGCLPPFPADVFMIPQYGYLTLNNG--SQAVGRSSFYCLEYFPSQMLRTGNNFQ 413 AAV9 VLGSAHEGCLPPFPADVFMIPQYGYLTLNDG--SQAVGRSSFYCLEYFPSQMLRTGNNFQ 412 *:*.. :* :* ** :** :***.* **: : . **:*:*******:********

SAAV FQFKFEEVPFHSGWAQSQSLDRLMNPLLDQYLIGDYG------TDASGNLIYHRAGPNDL 455 AAV1 FSYTFEEVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGM 471 AAV2 FSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDI 470 AAV5 FTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNK------NLAGRY 457 AAV8 FTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQTLGFSQGGPNTM 473 AAV9 FSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTIN-GSGQNQQTLKFSVAGPSNM 471 * : **:*****.:* **.* :* ***:****

SAAV NEFYKNWAPAPYECIQNINSSDNTKNANSINGSNSTNKWGLQGRQAWDAPGFVQASTYEG 515 AAV1 SVQPKNWLPGPCYRQQRVSKTK-TDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDD 530 AAV2 RDQSRNWLPGPCYRQQRVSKTS-ADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDD 529 AAV5 ANTYKNWFPGPMGRTQGWNLGS-GVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQ 516 AAV8 ANQAKNWLPGPCYRQQRVSTTT-GQNNNSNFAWTAGTKYHLNGRNSLANPGIAMATHKDD 532 AAV9 AVQGRNYIPGPSYRQQRVSTTV-TQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEG 530 :*: *.* * . * * . *:* . * :

SAAV AAAGQSLLNGVLTFDKSSATTSSP---AATAVNRTIEDEIQGTNNFGNARNNIVAINQQT 572 AAV1 ED-KFFPMSGVMIFGKESAGASN---TALDNVMITDEEEIKATNPVATERFGTVAVNFQS 586 AAV2 EE-KFFPQSGVLIFGKQGSEKTN---VDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQR 585 AAV5 GS-NTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQS 575 AAV8 EE-RFFPSNGILIFGKQNAARDN---ADYSDVMLTSEEEIKTTNPVATEEYGIVADNLQQ 588 AAV9 ED-RFFPLSGSLIFGKQGTGRDN---VDADKVMITNEEEIKTTNPVATESYGQVATNHQS 586 .. : *... : . : * *.* : .* .. . :: * *

SAAV KGTNPTTGSTSQFETMPGMVWSNRDIYLQGPIWAKIPNTDGHFHPSPRMGGFGLKHPPPM 632 AAV1 SSTDPATGDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQ 646 AAV2 GNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQ 645 AAV5 STTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPM 635 AAV8 QNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQ 648 AAV9 AQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQ 646 . :** ** :**:***********.*..:***** *****:*:***

SAAV ILIKNTPVPADPPTTFNPMPQTSFITEYSTGQVTVEMLWEVQKESSKRWNPEVQFTSNFG 692 AAV1 ILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYA 706 AAV2 ILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYN 705 AAV5 MLIKNTPVPGNI-TSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYN 694 AAV8 ILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYY 708 AAV9 ILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYY 706 :********.: : *. ****:******:**: **::**.*******:*:*.*:

SAAV TSDPAVDGIPFGINNLGTYVESRPIGTRYISKHL 726 AAV1 KSA----NVDFTVDNNGLYTEPRPIGTRYLTRPL 736 AAV2 KSV----NVDFTVDTNGVYSEPRPIGTRYLTRNL 735 AAV5 DPQ----FVDFAPDSTGEYRTTRPIGTRYLTRPL 724 AAV8 KST----SVDFAVNTEGVYSEPRPIGTRYLTRNL 738 AAV9 KSN----NVEFAVNTEGVYSEPRPIGTRYLTRNL 736 : * :. * * *******::: * Figure 4-4. Continued.

Figure 4-5. sAAV is capable of escaping recognition by mouse monoclonal antibodies that target the mammalian AAV capsid. B1 does not recognize AAV4 due to a 3 amino acid mutation in the epitope.

Figure 4-6. sAAV is capable of binding to mammalian cells. sAAV is capable of binding to Pro-5, Lec-2, and Lec-8 cell lines at varying levels of efficiency in comparison to AAV2. The values above the bars indicate the average percentage of live cells bound by the respective fluorescently labeled virus. All experiments were completed in technical triplicate, and error bars were assigned based on standard error (n=3).

B Figure 4-7. sAAV conserves the pH-induced autoproteolytic behavior observed in mammalian AAVs, and retains capsid integrity regardless of environmental pH. A) sAAV is proteolytically modified as pH becomes more acidic, with the most cleavage occurring between pH 5.5 and pH 4.0. B) sAAV capsids remain intact, regardless of pH.

Figure 4-8. sAAV is highly thermostable and conserves the capsid stability trends observed in mammalian AAV serotypes 1, 2, 8, and 9. sAAV is most stable at pH 5.5, at temperatures of up to 90.5°C. All experiments were completed in triplicate. Error was assigned based on standard error, where n=7 for sAAV. All other standard deviation and standard error calculations were completed by Bridget Lins (Dissertation). The values shown in the table below the graph are the peak Tm values, which were calculated as the peak of the melting curve, at the temperature that the majority of virus capsids had been degraded. Error bars are zero for AAV9, because the standard deviation within these trials was zero (Bridget Lins, Dissertation).

CHAPTER 5 SUMMARY AND FUTURE DIRECTIONS

Overall Summary of Experimental Findings

This study establishes the first structural characterization of a reptilian dependoparvovirus to date. Cryo-EM and 3D structure determination were used to generate the structure of sAAV to approximately 4.1 Å resolution. This electron density map was used to fit a model that was used to assign variable regions for comparison with the selected clinically relevant mammalian AAV serotypes 1, 2, 5, 8, and 9. This study also established that sAAV is capable of escaping recognition by mouse monoclonal that recognize epitopes on the surface of mammalian AAVs, and is capable of binding to mammalian cells. This is the first time that reptilian dependoparvoviruses have been shown to bind to mammalian cells. In addition, sAAV was determined to be highly thermostable in comparison to mammalian AAVs, in that it can withstand temperatures of up to 90.5°C, while still conserving the pH-related stability trends previously established for AAV1, 2, 8, and 9.

Improvement of sAAV High Resolution Cryo-EM Structure

In order to be able to carry out a more direct comparison with the crystal structures for AAV1, 2,5, 8, and 9, the sAAV dataset needs to be improved to a resolution that allows for the visualization of side chains. This may require further refinement of the existing dataset, or additional rounds of data collection using virus at a higher concentration. Additional cryo-EM data collection of a sample at a higher concentration would provide more particle orientations and therefore provide more information about side chain orientations within the electron density map. Improvement of the existing dataset can also be carried out by realigning the micrograph frames of

the dataset collected at FSU and correcting the Angstrom per pixel assignment during the alignment. The realigned micrographs will then be used to repeat the previously described refinement process in order to determine a new electron density map of sAAV at a higher resolution.

Challenging sAAV with Polyclonal Human Serum

It has been shown, as a result of this work, that sAAV is capable of escaping recognition by mouse monoclonal antibodies that target specific epitopes on the capsid surfaces of mammalian AAV serotypes 1-9. This demonstrates that sAAV does not share these serotype-specific epitopes with the selected mammalian AAVs. In order to prove the usefulness of sAAV as a potential therapeutic vector, seropositivity experiments must be conducted in order to determine whether or not pre-existing NAbs that target sAAV exist in the human population. It is proposed that given the typical environmental isolation between humans and reptiles, it is unlikely that environmental exposure will lead to the level of NAb production that is observed for mammalian AAVs of human and NHP origin. In order to determine the incidence of anti-sAAV NAbs in the human population, these panels will be completed using commercially available human donor serum samples that have previously been tested for anti-mammalian AAV NAbs

(106). sAAV will be challenged with this serum using native dot blot, and the same methods previously outlined for the monoclonal antibody panel. In addition to completing human serum panels, a positive control for sAAV will need to be developed.

Currently, experiments have been conducted to attempt to optimize a fluorescent Alexa-

488-labeled sAAV capsid in order to visualize the virus by dot blot or SDS-PAGE under

UV light. Alternatively, a capsid specific mouse monoclonal antibody is another option for a positive control against sAAV. This can be carried out using hybridoma cell lines 68

that are specific to antigens unique to the sAAV capsid in order to produce a polyclonal antibody supernatant. The polyclonal supernatant can then be used to detect sAAV, or it can be further purified in order to generate a monoclonal an anti-sAAV monoclonal antibody.

Reconfirming sAAV Cell Binding and Transduction of Human Cell Lines

The initial FACS cell binding data were collected in technical triplicate. These experiments will be completed with another biological replication with virus purified from another preparation of sAAV, in another technical triplicate to confirm that this data is consistent and statistically significant. Furthermore, FACS can also be used to test sAAV tissue tropism by expanding the cell line selections. For future studies, sAAV cell binding capability in relation to tissues will be tested using the cell lines HEK293 (human embryonic kidney), HEPG2 (human liver cancer), C2C12 (mouse muscle cells),

ARPE19 (human retina). These cell lines will be used because they have previously been shown to be specifically targeted by the natural tropism of the selected clinically relevant mammalian AAV serotypes AAV1, 2, 5, 8, and 9. HeLa human cervical cancer cell lines will be tested because they are known to be targeted by the oncolytic autonomous protoparvoviruses MVM and LuIII, which are structurally similar to AAV3b.

Transduction assays should be carried out using infectious AAV clones packaging GFP or luciferase reporter genes. These assays rely on the production of an infectious sAAV clone, that contains all three viral proteins. At this time, the only construct available is the VP3-only VLP construct, as the VP1-2-3 sAAV construct is still in development. In vitro assays will be conducted by infected cell lines with the reporter- packaging vectors, then observing the infected cells under confocal microscopy.

Alternatively, in vivo transduction assays can be completed using mice infected via intraperitoneal or tail vein injection, then viewed by bioluminescence imaging.

Determination of HSPG as a Possible sAAV Cell Surface Receptor

Results of the FACS cell binding experiments indicate that sAAV could potentially be binding to the terminal glycan expressed on the surface of all three CHO variant cell lines, but this is complicated by the fact that all three of these cell lines also express heparan sulfate proteoglycan. In order to clarify whether sAAV is binding to terminal sialic acid, N-linked galactose, or N-acetyl glucosamine, the Alexa-488 labeled sAAV that was used for FACS will be subjected to a heparin binding assay to determine which sAAV is actually binding to: terminal glycans, or HSPG. For viruses that possess the epitope for the B1 positive control or have a serotype-specific mouse monoclonal antibody, the results of the heparin binding assay can be visualized by native dot blot.

Alternatively, given that sAAV does not have a serotype-specific antibody, these results will have to be visualized by SDS-PAGE. However, the standard SDS-PAGE protocol commonly used in our laboratory requires staining with Gel Code Blue, which is not sensitive enough to visualize the low concentrations of viral protein in the heparin binding assay samples. This means that these gels will have to be visualized using the

Pierce Silver Stain kit (ThermoFisher, cat. no. 24612), as it is sensitive enough to detect protein at concentrations as low as 0.25 ng.

Identification of Autolytic Cleavage Products by Mass Spectrometry

Previous studies have shown that the proteolytic modifications to the capsid that occur under conditions of low pH are conserved in mammalian AAVs. Unpublished

LCMS data for AAV2 has indicated that the cleavage sites in the VP3 protein sequence are aspartic acid residues. For these seven known cleavage sites in total for the VP3

protein, two of these residues are structurally conserved in sAAV. In order to determine the other locations where the sAAV VP3 protein is being modified by acidic pH consitions during endo-lysosomal trafficking, the pH 4.0 sample will be run on SDS-

PAGE. The gels sent off for mass spectrometry analysis are fixed in a solution of 1:3:6 glacial acetic acid, ethanol, and water respectively. The fixed gels are then washed, stained in Gel Code Blue, and destained and washed again. Following washing, bands can be extracted from the acrylamide gel, and analyzed by LCMS. In the case of AAV2, samples were sent externally for analysis by Bruker. However, the University of Florida has recently expanded their mass spectrometry core to include instrumentation needed to be able to identify potential aspartic acid cleavage sites in sAAV. This will need to be repeated for the VP1-2-3 construct of sAAV in order to identify additional cleavage sites in the minor capsid proteins.

Characterization of Other Reptilian Dependoparvoviruses

In order to provide a more extensive array of options for patients that have already been treated with an sAAV-based vector but require readministration of the therapeutic vector, other reptilian dependoparvoviruses must be characterized and developed into useful vectors. This will allow for treatment readminstration without facing the same complications that mammalian AAVs face as a result of NAb development. Characterization of these vectors will follow the same experimental outline as proposed in this study, including production and purification, cryo-EM data collection and 3D structure determination, antibody and cell binding assays, and characterization of capsid autolysis and stability.

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BIOGRAPHICAL SKETCH

Victoria Fielding was born in Flowood, Mississippi in the fall of 1991, and raised in Cedar Park, Texas. She graduated from Leander High School with an International

Baccalaureate program diploma in Spring 2010, and was accepted to Texas A&M

University at College Station where she majored in wildlife and fisheries sciences, with a focus in vertebrate zoology. During her time at TAMU, she worked in several laboratories, ranging from bacterial pathogenesis, to entomology and bioinformatics, to museum curation internships, to external parasite studies in native Texas rodents, to mouse work focusing on viral-mediated induction of epilepsy and multiple sclerosis. This last experience led her to discovering her love of research and viruses in particular. In

Summer 2013, she presented a poster covering the epilepsy model to the Texas Brain and Spine Institute Symposium, and the TAMU Undergraduate Research Symposium.

Upon graduation from TAMU in May 2014, she received a Bachelor of Science degree and chose to continue her education by enrolling at the University of Florida in order to study biochemistry and molecular biology in Fall 2014. She joined the laboratory of Dr.

Mavis Agbandje-McKenna where she focused on the study of reptilian

Dependoparvoviruses in order to characterize their structural features and determine how they compare to mammalian adeno-associated viruses. Her research centered on a structural approach to characterization of the potential of Squamate

Dependoparvovirus Type I as a potential gene therapy vector. She presented this research at the XVIth International Parvovirus Workshop in the summer of 2016. She received her master’s degree in biochemistry and molecular biology in the winter of

2016.