Structural insights into recognition of adenovirus by immunologic and serum factors

TABLE OF CONTENTS

List of Tables

List ofFigures

List of Abbreviations

Acknowledgements

Abstract

Chapter 1 Introduction and background
1.1 Adenoviruses, from pathogens to therapeutics
1.1.1 Adenoviruses as pathogens
1.1.2 Adenovirus structure and cell entry
1.1.3 Adenoviruses as therapeutic agents
1.2 Adenovirus-host interactions
1.2.1 Host immune response to adenovirus
1.2.2 Recognition of adenovirus by human alpha defensin 5
1.2.3 Recognition of adenovirus by serum factors
1.2.4 Recognition of adenovirus capsid-incorporated HIV antigen
1.3 A hybrid structural approach to analyzing adenovirus recognition
1.3.1 Preparation of samples for cryo-EM
1.3.2. Virus imaging and three-dimensional reconstruction
1.3.3 Modeling of cryo-EM maps

Chapter 2 Recognition of adenovirus by human alpha defensin 5 involves intrinsic disorder
2.1 Abstract
2.2 Intro
2.3 Results
Cryo-EM structures of HD5 complexed with neutralization-sensitive and - resistant HAdVs
Modeling of HD5 monomers at the HAdV vertex
The vertex of the defensin-sensitive HAdV accommodates HD5 dimers
Intrinsic disorder at the HD5 binding site
Stabilization of defensin-sensitive HAdV vertex region by HD5
2.4 Discussion
2.5 Materialandmethods
Cryo-EM and image processing
Atomic model building
Molecular dynamics flexible fitting
2.6 Acknowledgements
2.7 Authorcontributions

Chapter 3 Virus-misplaced humoral factor activates innate immunity
3.1 Abstract
3.2 Report
Cryo-EM and MDFF analysis of the protein-protein interface between FX and HAdV5
Validation of the FX-HAdV5 interaction model
FX decorated virus activates an innate immune response
Conclusion
3.3 Materialandmethods
Animal studies
Viruses
Electron microscopy, image processing and modeling
Proteome profiler antibody arrays
Antibody for confocal microscopy
Surface plasmon resonance analyses
Microarray sample processing
Microarray data analysis
Ingenuity pathway analysis
Promoter analysis of differentially expressed genes
Gene ontology category analysis
Statistical analysis
3.4 Acknowledgements

Chapter 4 FVII dimerization on adenovirus capsid may influence infectivity
4.1 Abstract
4.2 Introduction
4.3 Materials and methods
Cells and viruses
Cryo-electron microscopy, image processing, and modeling
AdV infection in vitro
AdV attachment assay
SPR analyses
Statistical analysis
Protein structure assessmentnumber
4.4 Results
Cryo-EM structural analysis adenovirus-FVII interaction
Mutation of the HVR7 TET amino acid motif reduces the affinity of FVII binding to the virus
FVII is inefficient at supporting virus attachment and cell transduction, despite efficient binding to hexon
FVII binds HAdV5 hexon in an altered orientation compared to that of FX
FVII dimerizes via SP domain interactions when bound to HAdV5 hexon
FVII domain dimerization obscure putative receptor interacting residues within the dimer interface
4.5 Discussion
4.6 Acknowledgements

Chapter 5 Visualization of adenovirus capsid-incorporated HIV antigen
5.1 Abstract
5.2 Intro
5.3 Material and methods
Cryo-EM and image processing
Cryo-EM guided molecular dynamic simulations to model the MPER insertions
5.4 Results
Cryo-EM structure of AdV vector with capsid incorporated MPER peptide
Alpha helices are observed within the AdV capsid and for one MPER insertion
MPER conformation is constrained by the AdV capsid at one insertion site
Strong helical interactions between MPER insertions at the 3-mer sites
Transient interactions between MPER insertions at the 2-mer sites
Conformational flexibility ofMPER next to penton base
5.5 Discussion
5.6 Acknowledgements
5.7 Author contributions

Chapter 6 Summary of discoveries and future directions
6.1 Insights on immune recognition of adenoviruses
6.1.1 Human alpha defensin opposes capsid disassembly
6.1.2 A role for FX in adenovirus innate immunity
6.1.3 Capsid-incorporated HIV antigen adopts multiple conformations
6.1.4 Conclusion
References

LIST OF TABLES

2.1 Intermodular nonbonded energies for HD5 monomers with adenovirus vertex proteins

2.2 Intermodular nonbonded energies for HD5 dimers with adenovirus vertex proteins

2.3 Intermodular nonbonded energies for three HD5 dimers with each of the subunits of fiber and penton base at one defensin-sensitive adenovirus vertex

53.1 Distances at the FX-hexon interface before and after molecular dynamics flexible fitting runs with different starting FX orientations

53.2 Thirty-four genes co-activated in the spleens ofWT and Illrl-/- mice after challenge with HAdV5

53.3 Differential expression of34 gene set in the spleens of WT and Illrl-/- mice after challenge with HAdV5 and TEA mutant viruses

4.1 Binding ofFVII to adenovirus vectors and summary of SPR fitting parameters for l:l kinetic models

4.2 Intermolecular nonbonded energies between FVII molecules and hexons at the icosahedral 2-fold axis ofHAdV5 at the end of three l00-ps MDFF simulations

S5.1 Optimization of the helical interface at a 3-mer site with molecular dynamics flexible fitting

S5.2 Distances between hexon insertion sites at 2-mer regions

LIST OF FIGURES

1.1 Schematic representation of the structural organization of AdV based on cryo-EM and X-ray crystallography

2.1 Cryo-EM structures ofHD5 bound to neutralization-sensitive (Ad5.F35) and -resistant (Ad5.PB/GYAR) chimeric HAdVs

2.2 Modeling and cryo-EM guided molecular dynamics simulations of the interaction between HD5 monomers and vertex proteins of the defensin-sensitive (Ad5.F35) and defensin-resistant (Ad5.PB/GYAR) HAdV chimeras

2.3 Movement of the RGD-containing loop and HD5 during the molecular dynamics simulations of the defensin-sensitive (Ad5.F35) and defensin-resistant (Ad5.PG/GYAR) HAdV chimeras

2.4 Modeling and cryo-EM guided molecular dynamics simulations of the interaction between HD5 dimers and complete vertex regions of the defensin-sensitive (Ad5.F35) and defensin-resistant (Ad5.PB/GYAR) HAdV chimeras

2.5 Structural malleability of the binding pocket within the defensin-sensitive HAdV chimera (Ad5.F35)

S2.1 Subnanometer resolution of cryo-EM structures ofHD5 bound to neutralization- sensitive (Ad5.F35) and-resistant (Ad5.PB/GYAR) chimeric HAdVs

52.2 Prediction of intrinsically disordered regions with the HAdV5 and HAdV19c penton base proteins by the PrDOS Webserver

52.3 Space filling representation of the vertex region of Ad5.F35 with three bound HD dimers

3.1 Cryo-EM structure of the FX-HAdV5 complex and simulation of the FX-hexon interface using MDFF

3.2 A single amino acid substitution (T425A) abrogates FX binding to HAdV5

3.3 FX binding-ablated virus triggers blunted transcriptional response ofNFKBl- dependent genes in vivo

3.4 HAdV5 binding to FX induces NFKBl-dependent inflammatory cytokines and chemokines downstream ofTLR4-TRIF/MyD88-TRAF6 signaling axis in vivo

53.1 Robustness ofFX-hexon interface after molecular dynamics flexible fitting runs with different starting FX orientations

53.2 Comparison ofFX bound to hexon in the subnanometer resolution cryo-EM structure to stimulated FX/hexon density

53.3 Infectivity of and response to wild type and hexon-mutated viruses in cells cultured in vitro and mouse hepatocytes in vivo

53.4 Co-localization of virus particles (red) with splenic marginal zone macrophages (green) observed 1 h after virus injection for indicated viruses analyzed by confocal microscopy

53.5 Confocal microscopy analysis of virus particle localization with CD169+ and MARCO+ marginal zone macrophages in the spleen of mice 30 minutes after challenge with indicated viruses

53.6 Confocal microscopy analysis of virus particle localization with F4/80+ macrophages in the liver of mice 30 minutes after challenge with indicated viruses

53.7 Z-score map of transcription factor binding site distribution in the -1000br promoter regions of 34 genes co-activated in the spleens ofWT and Illrl-/- mice upon challenge with HAdV

53.8 Ingenuity Pathway analysis of networks of transcriptional targets for NFKB1, CREB1, and SRF transcription factors that respond to HAdV5 infection in the spleen of WT mice 30 minutes after intravenous virus injection

53.9 Ingenuity Pathway analysis of networks of transcriptional targets for NFKB1, CREB1, and SRF transcription factors that respond to HAdV5 in the spleen ofIL-1RI- deficient mice 30 minutes after intravenous virus injection

53.10 The mRNA expression for IL-1ß in spleens ofWT and indicated gene-deficient mice 30 minutes after virus injection analyzed by the RNAse protection assay

4.1 Cryo-EM structure of the HAdV5 complex and simulation of the FVII-hexon interface by molecular dynamics flexible fitting

4.2 Integrity, infectivity, and kinetic response data and dissociation constants for FVII

binding to wild-type AdV and AdV vectors with mutated hexons

4.3 Transduction of CHO-K1 cells with AdV-WT vector and attachment of AdV-WT and Ad5S vectors to CHO-K1 cells in the presence ofFVII and X

4.4 Cryo-EM and MDFF simulations indicate that FVII and FX adopt distinct binding orientations relative to hexon

4.5 Modeling of two molecules ofFVII at the icosahedral 2-fold axis

4.6 Localization of positively charged amino acids on the surface of serine protease domains of coagulation factors FX and FVII

4.7 Proximal FVII molecules interact and bury potential heparan sulfate proteoglycan binding residues that are exposed on FX

5.1 Cryo-EM structure of the Ad-HVR2-GP41-L15 vector at subnanometer resolution

5.2 Cryo-EM density showing a-helices for two hexons and two MPER insertions

5.3 MPER insertion within a narrow cavity between hexons at the icosahedral 2-fold axis

5.4 MPER forms a stable helical bundle at 3-mer sites

5.5 MPER interactions at 2-mer sites are weak and transient

5.6 Alternate model conformations for MPER next to penton base

5.7 Proposed vector modifications for optimizing MPER presentation at the AdV hexon HVR2 site

55.1 Resolution assessment of the Ad-HVR2-GP41-L15 cryo-EM structure

55.2 Secondary structure prediction for the inserted MPER and linker sequence

S5.3 Comparison of density at the icosahedral 3-fold axis with simulated hexon/MPER density

LIST OF ABBREVIATIONS

illustration not visible in this excerpt

ACKNOWLEDGEMENTS

A dissertation is not a work of individual effort. Though my name alone is listed as an author, there are so many others who have contributed to this work either directly or indirectly and supported me in ways that made this possible. Here, I attempt to acknowledge those who have helped me cross the finish line. Unfortunately, it is impossible to list everyone or to thank those listed enough.

I would like to begin by thanking the people who influenced me to enter the race. Jimmy Mills started my science addiction. Jimmy is truly one of the best teachers I have ever met. His excitement and passion for science was contagious and because of this I decided that I had to try it myself. Jimmy, thank you for teaching me that science is a privilege and responsibility. Pam Twigg gave me the opportunity to work in her lab as an undergraduate. I still think fondly of my time spent in her lab. Thanks Pam for such a positive undergraduate experience. Also thanks to the graduate students I worked with in Pam’s lab: Randall, Amy, and Ronny. You guys made the lab a fun place to work.

I would be extremely remiss to forget to mention Hassane Mchaourab. Hassane played a special role in this story because he was my connection to my PhD advisor Phoebe Stewart. I first met Hassane when he visited my undergraduate university to give a lecture on the application of EPR spectroscopy to protein dynamics. We continued to keep in touch by email and he encouraged me to apply to graduate school at Vanderbilt University. During my senior year as an undergraduate, I visited Hassane’s lab and it was at this time that I was introduced to my future supervisor Phoebe. Thanks Hassane, for helping out with the transition to graduate student and for introducing me to a Phoebe Stewart.

I can still remember the first scientific conversation that I had with Phoebe Stewart. It was wonderful. She talked about cryo-electron microscopy and showed me a stunning image of adenovirus. I enjoyed our conversation so much that I jumped at the first opportunity to work with her, which was the summer before I started graduate school at Vanderbilt. Phoebe has been an excellent PhD advisor. She has put a tremendous amount of time and effort into developing me as an independent scientist. Phoebe I can’t thank you enough for being a great advisor and friend.

This work would not have been possible without the help and support of our collaborators: Dmitry Shayakhmetov, David Curiel, Glen Nemerow, and Jason Smith. Thank you all for sharing your materials and skills with us. Also, my research was supported by a training grant. I was appointed to a T32 institutional training grant in the pharmacological sciences, entitled the Molecular Therapeutics Training Program.

I sincerely appreciate the intellectual support and guidance from the members of my dissertation committee: Jason Mears, Andreas Engel, Sichun Yang, and Phoebe Stewart. Thank you all for being patient, supportive, available and inquisitive throughout this journey. Additionally, I thank Amy Ruschak for her willingness to serve on my committee during the final phase of graduate school. Besides my committee I have also benefited from conversations with Derek Taylor and Vera-Moiseenkova-Bell. Vera and Derek have always had an open door for my impromptu visits.

The members of the Stewart lab, both past and present, have contributed immensely to my personal and professional time during graduate school. I would like to thank, in no particular order, Steffen, Jian, Susan, Tara, Seth, Dewight, Neetu, Mariena, and Rob. Thank you all for your help and friendship.

Heather Holdaway provided support for the electron microscopes and aided with sample preparation during my time at the Cleveland Center for Membrane and Structural Biology. Kris Palczewski and John Mieyal went above and beyond in helping me transition as a graduate student from Vanderbilt to Case Western. There was never a shortage of assistance from the administrative staff which included, Cami Thompson, Diane Dowd, Ivona Golczak, Vida Tripodo, and Jenny Yang. Thank you all for your help and support.

Of course, I owe all the thanks in the world to my family! I thank my parents, Ron and Julie, and my two sisters, Jordan and Jantzen. Thank you for providing me with an overwhelming amount of love and support throughout graduate school and life in general. Looking back, I cannot count the many times that I have heard “we believe in you”s. I guess you were right. I finally did it! I thank my grandparents, Dave and Carolyn King, who have enthusiastically followed me every step of the way during graduate school. I thank my wife, who is also my best friend. After staring at this computer screen for much longer than is advised, I have concluded that there is no way of truly expressing my love and gratitude to Victoria. I am without words. Thank you for all that you do. Finally, I thank God for putting me in the race and giving me the strength to run it to completion.

Upon entering graduate school I quickly realized that it would take nothing short of a miracle for me to obtain a PhD.

Structural Insights into Recognition of Adenovirus by Immunologic and Serum

Factors

ABSTRACT
by

JUSTIN WAYNE FLATT

Adenoviruses (AdVs) are common pathogens that are a major cause of acute infections of the respiratory and intestinal tracts, as well as the eye. Despite having a distinguished and extensive experimental history, there remain many unanswered questions about how AdVs are recognized and eliminated during infection. In order to advance therapy for infectious and inherited diseases, these challenging questions must be addressed. Here we have examined recognition of AdV by immunologic and serum factors using high-resolution cryo-electron microscopy (cryo-EM) and computational modeling. These factors include human alpha defensin 5 (HD5), human blood coagulation factor X (FX), and factor VII (FVII). We also analyzed the structure of an AdV-based vaccine that is designed to provide protective immunity against human immunodeficiency virus (HIV). Structural analysis and modeling studies on HD5 recognition of AdV implicated a key role for intrinsic disorder in mediating a stabilizing interaction that blocks viral infection. Cryo-EM and functional examination of serum factor binding to AdV showed that FX, a noninflammatory humoral factor of the coagulation cascade, binds to the surface of AdV and becomes a pathogen-associated molecular pattern that, upon viral entry into liver cells, triggers activation of innate immunity via the TLR/NF-kB pathway. In contrast, FVII does not support AdV entry into liver cells because it binds in an altered orientation compared to that of FX and dimerizes, which buries potential liver receptor binding residues within the dimer interface. Characterization of the AdV-based HIV vaccine demonstrated how the adenoviral capsid influences epitope structure, flexibility, and accessibility, all of which affect the host immune response.

CHAPTER 1: Introduction and background

1.1 Adenoviruses, from pathogens to therapeutic agents

Adenoviruses (AdVs) were first identified as pathogens over 50 years ago using expiants of adenoids and tonsils grown in cell culture (Rowe et al, 1953). Since this discovery, research efforts have accelerated knowledge on AdV interactions with host cells and even paved the way for developing strategies to adapt AdVs for therapeutic interventions in humans. This first section of the introduction will cover AdVs both as pathogens and as therapeutic agents. It is designed to be a general introduction for a non­virologist.

1.1.1 Adenoviruses as pathogens

It all began in the winter of 1953, when Wallace Rowe, a post-doctoral fellow at the National Institutes of Health in Robert Huebner’s laboratory, discovered a filterable cytopathogenic agent that causes spontaneous degeneration in cultures of human adenoids (Rowe et al, 1953). The cytopathic changes, which caused rounding and grape like clustering of the affected cells, were shown to be caused by a new virus. At the time of this discovery, there was a major outbreak of acute respiratory disease (ARD) in military recruits at the Fort Leonard Wood military base in the Missouri Ozarks. This outbreak, which was thought to be caused by influenza virus (IFV), was under investigation by Maurice Hilleman and colleague Jacqueline Werner from the Army Medical Service Graduate School in Washington D.C. Hilleman and Werner failed to isolate IFV, but they succeeded in recovering a new agent from throat swabs of patients suffering from ARD (Hilleman & Werner, 1954). Shortly after, it was established that the agent isolated by Hilleman and Werner was related to the virus recovered by Rowe and co-workers (Huebner et al, 1954) and identical to the virus responsible for large outbreaks of ARD in recruits of the armed forces during World War II (Ginsberg et al, 1955). Several of the early groundbreaking studies on these new respiratory agents were carried out by Harold Ginsberg at Western Reserve University (now Case Western Reserve University) in Cleveland, OH (Babiss, 2003). In 1956 this group of viruses, affecting primarily the respiratory tract, was named adenoviruses (Enders et al, 1956).

Human AdVs, whose members include >55 types, are classified into 7 species (A­G) on the basis of common biologic, morphologic, and genetic features. These viruses cause a variety of illnesses including acute respiratory, intestinal, and ocular infections. Transmission typically occurs from person-to-person via respiratory droplets, but the virus can also spread by the conjunctival and fecal-oral routes. Epidemiological data indicates that the majority of AdV infections occur in the first 5 years of life (Hong et al, 2001), with a peak incidence in the first 2 years (Pacini et al, 1987). Most AdV infections are mild and self-limiting. However, in immunosuppressed individuals, disseminated disease occurs frequently and is associated with a high fatality rate (Pham et al, 2003). Also, AdVs have emerged as important pathogens within the transplant population, where for example, infection occurs in up to 40% of pediatric stem cell transplant patients and in 5-10% of solid organ transplant recipients (de Mezerville et al, 2006; Humar et al, 2005; Kampmann et al, 2005; Walls et al, 2005). Although human AdVs have been investigated for several decades, there are currently no anti-viral drugs to treat infection.

1.1.2 Adenovirus structure and cell entry

AdVs have a non-enveloped, icosahedrally shaped capsid that protects a double- stranded DNA genome. The viral capsid is comprised of three major proteins (hexon, penton base, and fiber) and four minor proteins (Ilia, VI, VIII, and IX) that surround -36 kilobase (kb) pairs of DNA. The AdV genome encodes more than 40 different proteins; however, only 13 of these proteins have been identified as constituents of the virus particle (Russell, 2009). The virion has a molecular weight of -150 MDa and is among the largest non-enveloped viruses. Early structural studies focused on individual capsid proteins. The AdV capsid protein hexon was the first animal virus protein to be crystallized (Franklin et al, 1971). This hexon X-ray diffraction study set the stage for more complex structural analyses of the entire AdV virion by X-ray crystallography and cryo-EM. Two decades after hexon was crystallized, a structure of the intact virus was produced by cryo-EM at35Â, which provided detailed information on the hexon packing arrangement within the capsid as well as a first structure of the vertex proteins, including the penton base and its associated fiber (Stewart et al, 1991). Combining the X-ray crystal structure of hexon with the image reconstruction of the intact AdV particle yielded a three-dimensional difference map containing information on the minor proteins that stabilize the capsid (Stewart et al, 1993). Cryo-EM has continued to provide insights into AdV capsid architecture, including inter-capsomer interactions and assignments for the locations of the AdV minor proteins (Fabry et al, 2005; Liu et al, 2010; Reddy et al, 2010; Saban et al, 2006). The vertex region of the AdV capsid has been examined using both X-ray crystallography and cryo-EM. A partial crystal structure of the AdV homo- trimeric fiber protein revealed a novel triple beta-spiral fibrous fold for the shaft as well as atomic information on the receptor-binding head domain (van Raaij et al, 1999). X-ray crystallographic studies of the AdV penton base protein showed that each subunit contains a basaljellyroll domain similar to hexon and also yielded a structural description of how a segment of fiber binds to form the penton complex (Zubieta et al, 2005). A cryo-EM structure of AdV in complex with a monoclonal antibody revealed the presence of an RGD sequence in each of the five loops located at the top of the penton base protein that serve as a binding site for av integrins, which mediate virus internalization (Stewart et al, 1997). Steady improvements in cryo-EM and genetically modified virus samples (for example AdV type 5 modified to contain short AdV type 35 fibers referred to as Ad35F) have significantly enhanced resolution and in turn, understanding of AdV capsid assembly (Saban et al, 2005). High resolution structural comparisons of an immature adenovirus particle (AdV type 2 temperature-sensitive mutant tsl) with mature AdV showed that virus maturation does not involve large scale conformational changes in the capsid, but rather differences in the inner capsid region below that penton base (Perez- Berna et al, 2009; Silvestry et al, 2009).

Amazingly, in recent years, atomic resolution structures of the AdV virion have been determined using both X-ray crystallography and cryo-EM (Liu et al, 2010; Reddy et al, 2010). In terms of organization, the AdV capsid is built almost entirely of hexons except at the vertices (Fig 1.1). Each of the 12 vertices contains a penton complex, which is composed of a penton base protein non-covalently associated with a fiber protein. Capsid diameter is -920 Â not including the fibers. AdV fibers are highly elongated and vary in size (roughly 120 to 315 Â) depending on virus type. The four minor proteins IlIa, VI, VIII, and IX form interactions on the inner and outer surface of AdV that are critical for capsid stabilization (Vellinga et al, 2005). Exactly how these minor proteins serve as cement to hold together the AdV virion is not yet fully understood and is currently an active area of research. As structure determination methods improve, it is becoming more feasible to visualize the location, folds, and interactions of the minor capsid proteins. Inside the capsid the viral genome is condensed along with proteins V, VII, mu, Iva2, protease and terminal protein. There is a lack of structural information on this core region of the AdV virion containing viral DNA and associated proteins because the core lacks icosahedral symmetry, making it extremely challenging for analysis by cryo-EM and X-ray crystallography. Also, information is limited regarding adenovirus interactions with host proteins. Atomic resolution crystal structures of AdV fiber domains with fragments of CD46, CAR, and sialyl lactose have provided detailed knowledge on the molecular interactions necessary for attachment to host cells (Bewley et al, 1999; Burmeister et al, 2004; Persson et al, 2007). Cryo-EM analysis of AdV penton base-cell integrin interactions has shed light on how the virus is internalized into clathrin coated vehicles (Stewart et al, 1997; Lindert et al, 2009). Additionally, a cryo-EM structure of AdV complexed with anti-hexon antibody has been used to understand a postentry neutralization mechanism in which the hexon capsid is cross-linked by antibodies, thus blocking infection (Varghese et al, 2004). The research covered in this thesis in chapters 2-5 seeks to understand how immunologic and soluble serum factors impact AdV infection.

AdVs enter a variety of postmitotic eukaryotic cells using a stepwise entry program that ensures that their genome reaches the nucleus for replication (Greber et al, 1993; Puntener et al, 2011). For nuclear import to be effective, upstream steps for AdV entry into cells entails traversing several physical barriers and a stepwise uncoating program of the virus, where cellular factors support or restrict the entry program (Mercer & Greber, 2013; Suomalainen & Greber, 2013). AdVs do not directly cross the plasma membrane at the cell surface but are instead taken up into vesicles. Most AdV types utilize the coxsackie-adenovirus-receptor (CAR) for attachment to cells and integrins are required for internalization (Roelvink et al, 1998; Stewart & Nemerow, 2007). The AdV penton complex contains all necessary components to negotiate viral attachment and internalization into host cells. Attachment occurs via the fiber protein and the penton base protein interacts with cellular integrins through an Arginine-Glycine-Asparagine (RGD) motif located in a hypervariable surface loop to trigger internalization (Wickham et al, 1993). Some AdV types use alternative receptors for attachment including complement receptor CD46, sialic acid, and heparin sulfate proteoglycans (Arnberg et al, 2002; Gaggar et al, 2003; Tuve et al, 2008; Zhang & Bergelson, 2005). Internalization results in uptake of AdV into clathrin-coated vesicles, which are then transported to the endosomal network. Although this strategy facilitates entry into cells, it does not provide direct access to their intracellular space for transport of DNA into the nucleus. Thus, AdVs entering the endocytic network must quickly subvert their vacuoles to avoid transport to the degradative lysosomal compartments within host cells. To escape endosomes and deliver DNA into the host nucleus, AdVs undergo stepwise disassembly, which involves removal of the protein capsid from the viral genome. This process begins at the plasma membrane, where receptor binding causes loss of protruding fibers. It continues in the early endosomes, where mild acidification causes release of a few vertex regions leading to exposure of the membrane-lytic protein VI and enhanced viral escape from endosomes. The partially disrupted AdV particles then travel along microtubules until they reach the nucleus. The nucleus is protected by the nuclear envelope and AdVs can only gain access through nuclear pore complexes (NPCs) (Greber et al, 1997). The size restriction of NPCs precludes the AdV capsid from directly invading the nucleus. Upon docking at the NPC, final disassembly of AdV is mediated by the microtubule motor protein kinesin-1, NPC filament protein CAN/Nup214, and nuclear histone H1 allowing the viral genome to enter the nucleus for replication (Meier & Greber, 2004; Strunze et al, 2011; Trotman et al, 2001).

1.1.3 Adenoviruses as therapeutic agents

Over fifty years of intense research has resulted in AdVs being arguably one of the best-characterized of human DNA viruses. Detailed knowledge of the AdV replication live cycle has motivated numerous attempts to engineer AdVs as vectors for gene therapy, vaccination, and oncolysis (Russell, 2000). These viruses have several characteristics that make them well suited for therapeutic interventions in humans. First, they have low pathogenicity in immunocompetent individuals, with symptoms that are mild and often associated with the common cold. Second, they are popular for their ability to accommodate relatively large transgenes. AdV-based gene delivery vectors can tolerate foreign gene insertions of up to 7.5 kb pairs of DNA. This is significantly larger than the size of the average human gene, which is about ~1.4 kb pairs of DNA (Lander et al, 2001). Third, AdVs are able to rapidly infect a broad range of human cells and tend to yield high levels of gene transfer. Fourth, the viral genome doesn’t undergo rearrangement at a high rate, and inserted genes are stable over successive rounds of replication. Finally, AdV vectors are easy to manipulate using standard recombinant DNA techniques.

Indeed, AdV vectors have been extremely popular in efforts to advance therapy for infectious and inherited diseases, yet there has been limited clinical success. Major obstacles that hinder their use as therapeutic agents include: the innate immune response to AdV challenge, pre-existing immunity to AdV vectors, and poor efficiency at targeting gene transfer to specific cell types. Activation of innate immunity to AdV is associated with a reduction in gene transfer efficiency (Worgall et al, 1997) and an acute inflammatory response that can cause serious damage to healthy tissues and may even lead to death (Raper et al, 2003). The limitation posed by pre-existing immunity is anti- AdV antibodies, which can prevent AdV vectors from transducing target cells (Zaiss et al, 2009). In terms of targeting specific cell types, a common goal is delivery via the bloodstream; however, upon injection, the majority of virus particles accumulate in the liver. Furthermore, the primary receptor for AdV is virtually ubiquitous and causes non­specific uptake into multiple organs. Even with these present challenges, AdVs are among the most commonly used vectors for therapeutic interventions in humans, second only to retroviruses. Research is currently underway, to design safe vectors that can efficiently transduce target tissues.

1.2 Adenovirus-host interactions

AdVs deliver their genome into the nucleus of human cells for replication. They accomplish this task by co-opting host cell machinery whilst avoiding host immunity. In turn, just as these viruses commandeer cellular factors to replicate, cells have evolved elaborate mechanisms to recognize and eliminate AdVs during infection. This evolutionary struggle between virus and host is poorly understood. There is much to be learned from studying AdV-host interactions and this has been the focus of our research. This section will introduce our studies.

1.2.1 Host immune response to adenovirus

A major barrier to AdV replication is the host immune response. Immunity to viral infections occurs by multiple specific and non-specific mechanisms that vary with respect to activation, duration, and magnitude (Lowenstein & Castro, 2003; Muruve, 2004). Ultimately the reaction of the immune system is modulated by how AdV interacts with host cells and spreads during infection. Viral antigens will be present in different parts of the body depending on both the route of spread and phase of infection. Local infections, for example at mucosal surfaces, can elicit local cell-mediated and humoral (IgA) immune responses, but not necessarily systemic immunity (Lemiale et al, 2003). One type of local response involves defensins, which are naturally occurring immune peptides that block AdV infection (Nguyen et al, 2010; Smith & Nemerow, 2008; Smith et al, 2010a). Chapter 2 of this thesis reveals new details on the defensin anti-viral mechanism against AdV. Virus and/or virus-infected cells can also stimulate В lymphocytes to produce antibodies that are specific for viral antigens, primarily against the AdV capsid proteins penton base, hexon, and fiber (Bradley et al, 2012). IgG, IgM, and IgA all exert anti-viral activity against AdV (Ariyawansa & Tobin, 1976). Antibodies can neutralize AdV by: 1) preventing virus-host cell interactions, 2) blocking post-entry steps of AdV infection, or 3) recognizing antigens on infected cells, which can lead to antibody-dependent cytotoxic cells or complement-mediated lysis (Cichon et al, 2001; Varghese et al, 2004). IgG antibodies are responsible for most anti-viral activity in serum, whereas IgA activity is prevalent when AdVs infect mucosal surfaces (Parkin & Cohen, 2001). This type of immunity is commonly referred to as humoral. Cell mediated immunity involves leukocytes and their production of cytokines in response to virus and virus-infected cells. Cytotoxic lymphocytes, natural killer cells and antiviral macrophages can recognize and kill AdV-infected cells (Thaci et al, 2011; Zhu et al, 2007). Helper T cells can recognize virus-infected cells and produce a number of important cytokines. Cytokines produced by monocytes, T cells, and natural killer cells play important roles in developing anti-AdV immune responses (Chen & Lee, 2013). Cell-mediated immunity is principally thought to be regulated by toll-like receptors. Toll-like receptors mediate the anti-viral immune response by recognizing virus infections, activating signaling pathways and inducing the production of cytokines and chemokines (Xagorari & Chlichlia, 2008). Chapter 3 focuses on a new mechanism by which toll-like receptor 4 triggers an immune response to AdV infection. The early, non-specific responses (for example natural killer cell activity and interferon) serve to limit virus infection during the acute phase. The later specific immune responses (humoral and cell-mediated) function to eliminate viral infections at the end of the acute phase, and to maintain specific resistance to infection.

1.2.2 Recognition of adenovirus by human alpha defensin 5

AdVs gain access to their host cells using a multi-step process that involves clathrin-mediated endocytosis and acid activated penetration of endosomes (see section 1.1.2). Successful entry for these non-enveloped viruses depends on the orchestrated disassembly program of their outer capsids. Human alpha defensin 5 (HD5) has been shown to stabilize the AdV capsid and prevent disassembly of the virus during cell entry, thereby blocking infection (Nguyen et al, 2010; Smith & Nemerow, 2008; Smith et al, 2010a). HD5 is a protein of the innate immune system that is produced in Paneth cells of the small intestine. This protein contains 32 amino acids and shares a common structural fold of the alpha-defensin family characterized by an anti-parallel ß-sheet structure stabilized by three intramolecular disulfide bonds. Also it is mainly positively charged, amphipathic, and possesses the ability to multimerize both in solution and upon ligand binding. These properties dictate HD5 anti-viral activity against non-enveloped viruses (Gounder et al, 2012). Although it is has been clearly demonstrated that HD5 stabilizes the AdV virion and prevents release of viral proteins including the membrane lytic protein VI required for endosomal escape, the structural and functional organization that drives this process is poorly defined. Unraveling the mechanism underlying this AdV- host interaction is important for several reasons. First, this may be a common mechanism for restricting infection of non-enveloped viruses. For instance, HD5 has been shown to block human papillomavirus (HPV) from escaping endocytic vesicles (Parker et al, 2006). Thus, there is clear potential for eventual development of broad-spectrum anti­viral agents that target virus disassembly during cell entry. Second, insights gleaned from the mechanism may be useful for optimizing AdV vectors for gene delivery. For example, a capsid modified AdV vector may increase the efficacy of therapeutic gene delivery by avoiding interaction with HD5. Finally, studies of the AdV-HD5 interaction provide insight into AdV stepwise disassembly during cell entry.

What do we currently know about HD5 neutralization of AdV? From an initial investigation of how HD5 antagonizes AdV infection, it was discovered that HD5 is a potent inhibitor and is capable of complete inhibition with an IC5o between 3 and 4 pM (Smith & Nemerow, 2008). Evidence indicated that this potent anti-viral activity involves HD5 binding to AdV outside the cell and preventing release of the internalized AdV- HD5 complex from the endosome. This conclusion was based on failure of AdV to mediate the translocation of ribotoxin a-sarcin from the endosome into the cytoplasm in the presence of inhibitory concentrations of HD5. Consistent with this finding, HD5 was shown to inhibit release of the viral protein VI, which is required for AdV-mediated endosome penetration. Moreover, at late times post-infection, AdV particles colocalized with lysosomes instead of the nucleus. This further demonstrated that HD5 causes AdV to remain trapped in the endosomal/lysosomal pathway rather than trafficking to the nucleus. To gain deeper insight into the defensin-mediated mechanism of neutralization, the specificity of the AdV-HD5 interaction was analyzed. Infectivity studies revealed that sensitivity of AdV to HD5 inhibition is species specific. Sensitivity to HD5 was found for many AdV types from species A, B, C, and E, while types from species D and F were resistant to neutralization (Smith et al, 2010a). It was observed that neutralization is dependent upon the tertiary structure of a correctly folded HD5 molecule. HD5 derivatives in which six cysteines were replaced with L-a-aminobutyric acid, to prevent formation of the three intramolecular disulfide bonds that stabilize a correctly folded HD5 molecule, failed to inhibit AdV infection. A cryo-EM structure of a sensitive AdV vector in complex with HD5, combined with sequence analysis of capsid proteins from sensitive and resistant AdV types, led to a hypothesis that the critical HD5 neutralization site is located in a region spanning the penton base and fiber proteins (Smith et al, 2010a). A critical binding site at the interface of the non-covalently coupled penton base and fiber was supported by two key observations. First, there was an accumulation of extra density at the top of penton base and around the fiber shaft in the cryo-EM structure of the AdV vector complexed with HD5 that was not present in a cryo-EM structure of the AdV vector alone. Second, this hypothesis was supported by infectivity assays, where HD5 activity was tested against virus chimeras comprised of capsid proteins from sensitive and resistant AdV types. These experiments confirmed the presence of multiple binding determinants in the penton complex that are critical for neutralization and suggested how HD5 may bridge adjacent capsid proteins fiber and penton base to prevent disassembly during cell entry. The goal of our research has been to gain insight into the structural basis for AdV susceptibility to HD5 anti-viral activity and to better define the critical neutralization site. This research is covered in detail in chapter 2 of the thesis.

1.2.3 Recognition of adenovirus by serum factors

As research in AdV biology progresses, a new model for cell infection is emerging, which appreciates that AdV biodistribution, spread, viral persistence, and replication is less reliant on direct receptor binding and more influenced by other complex interactions between virus and host. A first glimpse into this reality came from directly injecting AdV vectors into the bloodstream of mice. The biodistribution of these vectors revealed no correlation with CAR expression (Akiyama et al, 2003; Alemany & Curiel, 2001). Instead, it was later discovered that virus particles accumulate in the liver and that virus entry into hepatocytes is mediated by interaction between the AdV capsid protein hexon and vitamin К-dependent blood coagulation factors (Kalyuzhniy et al, 2008; Parker et al, 2006; Shayakhmetov et al, 2005; Waddington et al, 2008). Thus, a novel fiber-independent AdV entry mechanism was identified. In vitro studies of this mechanism have shown that several homologous blood coagulation factors, including factors VII, IX, and X (FVII, FIX, and FX) can support virus infection of susceptible cells (Parker et al, 2006). However, in vivo, specific inactivation of FX alone is sufficient to completely abrogate hepatocyte transduction in mice after intravenous administration. A detailed description of this AdV-FX interaction is of fundamental importance both for understanding basic AdV biology in vivo and for refinement and optimization of AdV vectors for gene therapy. From a gene therapy perspective, FX-mediated sequestration of AdV vectors into liver hepatocytes and Kupffer cells induces an acute inflammatory response that may be solely responsible for the morbidity and mortality associated with infection (Ni et al, 2005; Raper et al, 2003), and thus, represents the greatest constraint on safety. Therefore, interference with this process could potentially improve the safety of AdV vectors. Furthermore, the highly efficient interaction between AdV-FX and liver cells is a significant hindrance if gene delivery to extrahepatic cells and tissues is required. Precise knowledge on the AdV-FX interaction may inform strategies to bypass this effect. Moreover, studies on AdV-FX interaction might impact development of effective drugs for treating disseminated AdV infections, which are frequently associated with liver failure and a high virus burden in the blood.

Although extensive in vitro, ex vivo, and in vivo analyses has defined a critical role for coagulation FX in facilitating AdV invasion of liver hepatocytes, the exact mechanism of FX binding to AdV has remained elusive. The earliest attempts to understand AdV liver tropism following intravenous administration focused on modifying penton base and fiber proteins. Mutations to ablate CAR and integrin binding worked as expected in vitro, but did not alter tropism in vivo (Akiyama et al, 2003; Alemany & Curiel, 2001). Shortly after, the focus shifted to investigating AdV interactions with coagulation factors after a seminal study reported that they play a major role in targeting intravenously injected AdV vectors to hepatic cells (Shayakhmetov et al, 2005). Both in vitro and ex vivo data from mice showed that blood factor mediated uptake of virus into hepatocytes is CAR-independent and instead occurs through cell surface heparan sulfate proteoglycans and low-density lipoprotein receptor-related protein. Later studies demonstrated that FX mediates in vivo delivery of AdV into hepatocytes and revealed an unanticipated function for hexon in defining virus infectivity (Kalyuzhniy et al, 2008; Vigant et al, 2008; Waddington et al, 2008). Moderate resolution (< 20 Â) cryo- EM structures of an AdV vector bound to FX localized attachment to the viral capsid protein hexon (Kalyuzhniy et al, 2008; Waddington et al, 2008). Furthermore, hexon- mutated virus bearing a large insertion within the top of hexon showed markedly reduced FX binding in vitro and failed to deliver a transgene to hepatocytes in vivo. In our study, we used high-resolution cryo-EM and guided molecular dynamics simulations to characterize the interaction interface between FX and human AdV type 5 hexon. We also provided a link between this interaction and innate immunity. This work is covered in chapter 3. In a follow-up study we analyzed the interaction between FVII and human AdV type 5 hexon. Coagulation FVII, like FX, binds hexon with very high affinity, yet has a poor capacity for supporting virus entry into hepatocytes. Our results indicated a structural basis for differential infectivity. An explanation is given in chapter 4.

1.2.4 Recognition of an adenovirus capsid-incorporated HIV antigen

AdVs are promising vectors for therapeutic interventions in humans. To date, they have been explored as vaccines against cancer and infectious diseases (Tatsis & Erti, 2004). They are appealing as vaccine carriers based on their ability to induce high antibody titers and robust cytotoxic T-lymphocyte responses. Additionally, it is thought that the high immunogenicity inherent in these vectors may have an adjuvant effect. These characteristics have underpinned their utility as carriers to deliver antigens from other infectious agents for vaccination. Major efforts are underway for their use to protect against HIV. HIV destroys CD4+ T cells eventually overwhelming the capacity of the immune system to regenerate or fight off other infections. A vaccine to HIV remains elusive. The requirements for eliciting protective neutralizing antibody and cellular responses remain a significant challenge. However, human clinical trials of candidate HIV vaccines show modest efficacy, suggesting that it should be possible to generate vaccine-elicited protection against HIV infection (Kwong et al, 2011). For example, AdV type 5 vectors containing HIV gene inserts are notable for their ability to induce strong HIV-specific cellular immune responses and have advanced to clinical trials. One major obstacle to the development of an effective AdV-based HIV vaccine is pre-existing immunity. Neutralizing antibodies, even present at moderate titers, can drastically restrict AdV vector uptake by cells, including antigen presenting cells (Fitzgerald et al, 2003). As a result, heterologous antigens that are packaged as transgenes within the viral genome are not efficiently expressed, thereby limiting the induction of an antigen-specific immune response. While neutralizing antibodies can inhibit AdV vectors extracellularly, AdV-specific CD8+ T cells destroy vector expressing cells (Mittal et al, 1993; Yang et al, 1996). Studies have shown these effects in animals (Fitzgerald et al, 2003; Xiang et al, 2002) and in humans (Knowles et al, 1995). The mechanisms underlying the dampening effect of pre-existing immunity are still being investigated. Meanwhile, alternative strategies have been developed for using AdV vectors as vaccines against HIV.

One alternative to bypass acquired immunity has focused on AdV types to which the human population is less exposed. In particular, promising results have been obtained from vaccine vectors based on AdVs that infect chimpanzees (Dicks et al, 2012; O’Hara et al, 2012; Xiang et al, 2006). Another approach involves direct incorporation of antigens into the viral capsid. The viral capsid protein hexon has been utilized for antigen display due to its natural role in generation of an anti-AdV immune response and abundance within the AdV virion (Crompton et al, 1994; Matthews et al, 2010; Matthews et al, 2008). Recently an AdV vector presenting an HIV antigen on the surface of the hexon capsid was shown to elicit an anti-HIV cellular response (Matthews et al, 2010). The HIV-1 antigen is 24 amino acids of the HIV membrane proximal ectodomain region (MPER). It is derived from the HIV glycoprotein gp41. Viral protein gp41 is part of a complex, often referred to as a “spike”, which decorates the envelope of HIV and is responsible for attaching to and fusing with host cell membranes. The conserved 24 amino acid MPER sequence is a target of two neutralizing human monoclonal antibodies, 2F5 and 4E10, and is therefore an important lead for vaccine design (Zwick et al, 2005). Here, it has been genetically incorporated within the AdV hexon hypervariable region 2 (HVR2). HVR2 is a highly immunogenic site located at the top of hexon. A critical aspect of understanding how this immunologically promising AdV vector elicits protection against HIV is knowledge of how MPER is displayed at the surface of the viral capsid. Therefore, we undertook a cryo-EM structural study to visualize heterologous antigen display. We found that the AdV capsid influences epitope structure, flexibility and accessibility, all of which affect the host immune response. The results of this work are shared in chapter 5.

1.3. A hybrid structural approach to analyzing adenovirus recognition

Studies on the structural biology of AdV and its interactions with host proteins have benefited tremendously from the combination of cryo-EM structures of full complexes with available atomic structures of components from X-ray crystallography. This approach was first utilized to study the structure of AdV in the 1990s (Stewart et al, 1993). We have used this method to extend our knowledge on how AdV is recognized during infection. A new hybrid modeling tool, molecular dynamics flexible fitting (MDFF), allowed us to merge detail from atomic-scale structures with the overall architecture of AdV-host complexes captured in cryo-EM density maps. This section contains information on our approach.

1.3.1 Preparation of samples for cryo-EM

For observation by cryo-EM, biological samples must be cryogenically frozen prior to being examined in a transmission electron microscope. The technique of vitrifying samples for cryo-EM studies was first introduced over 30 years ago. In particular, Dubochet and colleagues demonstrated that a thin layer of an unfixed and unstained specimen, such as a virus, could be vitrified for electron microscopy imaging (Adrian et al, 1984; Dubochet et al, 1981). Their new freezing method did not damage the specimen but rather cryo-EM images were produced that revealed an impressive level of structural detail. Since this breakthrough discovery, advances in sample preparation have made it possible to examine a wide range of biological specimens from intact tissue sections and plunge-frozen cells to bacteria, viruses, and proteins. In terms of sample preparation, cryo-EM has a distinct advantage over X-ray crystallography in that crystals are not required for structure determination. The size of AdV (-150 MDa) and complexity of AdV-host interactions makes crystallization extremely difficult, if not impossible. Another advantage is that cryo-EM requires relatively little material. A single grid covered by a 2-6 pL droplet of concentrated sample (1 x 1013 virus particles per ml) should easily contain enough particles for an entire data set. The droplet is frozen in a cryogen such that the virus or virus mixture is preserved in a near native or physiologically relevant state. Plunging is done either manually or automatically. Automatic plunging devices should increase the reproducibility of the grids. Once the sample is frozen, it must be maintained below the devitrification temperature, which is approximately -137°C (Dubochet et al, 1982), to avoid conversion of the amorphous vitreous ice within the specimen into cubic or hexagonal ice. Crystalline ice formation would cause mechanical stresses on the sample, and those stresses might distort the structures.

Preparing AdV for cryo-EM imaging is a multi-step process. AdV is a biosafety level 2 virus and thus requires the use of adequate containment equipment and practices. Purified virus must be dialyzed out of cesium chloride and into a low salt solution suitable for a high-resolution cryo-EM study. We used Thermo Scientific Slide-A-Lyzer mini unit dialysis devices (10 kDa cutoff) to facilitate removal of cesium chloride. The low salt solution used for buffer exchange contained 150 mM sodium chloride, 25 mM Tris and was at a pH of 8 (2 L total - 1 fresh L per hour). Exchange was done at 4°C for two hours under gentle stirring conditions. Afterwards, virus was incubated with protein to form a complex. For the AdV-defensin project, virus (160 pg/mL) was combined with HD5 (5 pM). For the study involving serum factors, 100 pL of AdV at 1 x 1013 virus particles per mL was mixed with either 1 pL of FVII or FX (2mg/mL). We did not mix any protein with the AdV-based HIV vaccine. It is worth noting that some of the AdVs used in these studies were modified to contain short fibers, making them easier to study by cryo-EM. Before plunging the sample, cryo-EM grids were prepared. Quantifoil holey carbon grids (Cu 400 mesh, R2/4, SPI Supplies, US) were freshly glow discharged under 25 mA for 30 seconds on both sides. Sample was then applied to the grids. A total of 6 pL of sample was applied to each grid. Initially, 3 pL of sample was added and immediately blotted by touching the back of the grid with filter paper. We used Whatman filter paper cut into rectangular strips for blotting. After applying an additional 3 pL, the grid was allowed to sit for 20 seconds and was then blotted. The blot time ranged from 10-20 seconds depending on room temperature and humidity. The sample was then vitrified by plunging into liquid ethane. Frozen grids could then be imaged or were stored in a liquid nitrogen dewar for later use.

1.3.2 Virus imaging and three-dimensional reconstruction

After plunging, the next critical step is grid transfer into the microscope. The main objective here is to place the cryo-EM sample in the microscope without warming it above the devitrification temperature and without collecting too much condensation from water droplets in the air. We used an FEI Polara cryo-workstation for grid transfer. The workstation comes equipped with a loading area, vacuum system and an airlock for docking to an FEI Polara transfer device, making it easy and safe to transfer grids into the microscope. The transfer device can preserve up to 5 cryo-grids under liquid nitrogen for transfer into the electron microscope. The design is such that once the device is mounted onto the microscope, one grid is manually inserted while the others are kept at liquid nitrogen temperature. The electron microscope that we used for imagining is an FEI Polara field emission gun (FEG) transmission electron microscope with a 300 kV electron beam and a highly stable specimen cryo-stage. The microscope was operated at liquid nitrogen temperature for data acquisition to avoid build-up of excessive contamination on the cold specimen. Also, a low dose of electrons (~20 e"/Â2) was used during imaging due to the fact that cryo-EM samples are extremely radiation sensitive. Images were collected at a magnification of 397,878X on a Gatan Ultrascan 4000 CCD camera. This magnification corresponds to a pixel size of 0.4 Â on the molecular scale. The defocus values of particle images ranged from -1 to -4 pm. Datasets for the projects varied in size, with the largest being 5,025 for the AdV-based vaccine. For the AdV-HD5 study, 3,515 and 3,620 particle images were collected of HD5 in the presence of a sensitive and a resistant AdV respectively. The dataset size for AdV with serum factor VII was 1,503 and for AdV with factor X was 1,101.

Each dataset was handled as described here. Individual particles were extracted from cryo-EM images with in house scripts that call IMAGIC subroutines (van Heel et al, 1996). Particle images were computationally binned for the initial stages of refinement. Averaging of adjacent pixels during the initial phases of image processing helped to improve the image contrast. This, in turn, aided in determining the initial, relatively crude, orientation parameters for each particle in the stack. The program CTFFIND3 (Mindell & Grigorieff, 2003) was used to determine initial estimates for the microscope defocus and astigmatism parameters. The image reconstruction software FREALIGN was used for determining three-dimensional cryo-EM structures. A6Â cryo-EM structure of AdV (Saban et al, 2006) served as a starting model for refinement. Essential steps in refinement included: 1) determining the center in x, and y for each particle in the image stack, and 2) assigning the three Euler angles that describe the projection view, or three­dimensional orientation, for each particle in the image stack. After these five parameters were estimated for each particle image, the particles could be averaged to generate a preliminary three-dimensional reconstruction. We performed many iterative rounds of refinement to improve the orientational parameters, and hence the resolution of our cryo- EM structures. In the later rounds of refinement we used particle images with smaller binning factors to make use of the higher resolution information contained in the finer pixel sizes. We also refined defocus on a per particle basis at this stage. A key aspect of our refinement was the viral capsid, which has 60-fold symmetry. Icosahedral symmetry averaging effectively increased the number of asymmetric units that were averaged to produce the final cryo-EM structure. In doing so, any features of the virus that followed icosahedral symmetry were strengthened, whereas any features, such as the protruding fibers, that did not follow this symmetry were weakened or averaged away. This procedure allowed us to determine subnanometer resolution cryo-EM structures for the projects discussed in chapters 2-5. Final resolutions were based upon the Fourier shell correlation 0.5 threshold criterion, which was first used to define the resolution of icosahedral viruses in the late 1990s (Bottcher et al, 1997; Conway et al, 1997).

1.3.3 Modeling of cryo-EM maps

State-of-the-art cryo-EM routinely allows direct visualization of whole viruses in their near native state at subnanometer resolutions. This impressive capability is largely due to rapid developments in microscope design, imaging hardware, automation, and image processing, and has yielded astonishing results, which for instance, allowed a huge leap from a stunning first image of AdV in vitrified ice in 1984 (Adrian et al, 1984) to a 3.6 Â cryo-EM structure in 2010 (Liu et al, 2010). Thus, we used this approach to study AdV-host interactions at subnanometer resolutions. Detail at this resolution level distinguished viral capsid proteins involved in host interactions and informed protein­protein interfaces for virus complexes. For the AdV vaccine vector, subnanometer resolution revealed how an HIV antigen is displayed at the surface of the capsid. Interpretation of cryo-EM density involved isolating individual subunits, identifying secondary structures, and accurately fitting atomic models. We used several tools for completing these steps. Cryo-EM density was visualized and segmented for study using the UCSF Chimera software package (Pettersen et al, 2004). This program was also used to initially dock atomic resolution structures or computational atomic models into segmented density. In the absence of crystal structures, computational model building was done using a combination of Chimera, I-TASSER (Zhang, 2008), and Rosetta (Rohl et al, 2004). After components were docked into density we used cryo-EM guided Molecular Dynamics Flexible Fitting (MDFF) (Chan et al, 2012; Trabuco et al, 2008) to further refine the docked models into cryo-EM density. Setup and analysis of MDFF simulations was carried out using a program called Visual Molecular Dynamics (VMD) (Humphrey et al, 1996). VMD has several useful plugins for analyzing simulations that do tasks such as measuring the root mean square deviation over time, calculating agreement between atomic model and input cryo-EM density map, and evaluating energies of the system over the course of simulation. This general protocol was used in all of our structural studies.

We used MDFF to generate atomic models based on our density maps and, for this reason, time is spent here to briefly highlight how this approach works. With MDFF, a molecular dynamics simulation is performed using a starting atomic model, often built from crystal structures. The goal is to refine these crystal structures or computationally constructed atomic models of components into cryo-EM density to better understand the interactions between the docked units for insight into how function is orchestrated. Additionally, this method reveals differences between conformations of structures in crystal form versus conformations captured by cryo-EM. Simulations are carried out such that external forces proportional to the gradient of the density map are applied to all atoms, driving the models to occupy regions of high density. Each of the steps described here were done using VMD. More information on each of these steps as well as how to setup and run a MDFF simulation can be read about in detail at the developer’s website located at http://www.ks.uiuc.edu/Research/vmd/plugins/mdff/. First, atomic models are checked for errors in stereochemistry and peptide bond configuration. Then, structural restraints are applied to preserve secondary structural elements so that over-fitting does not occur during the simulation. Afterwards, an MDFF simulation can be setup. The simulations we performed applied a g-scale factor of 0.3 for guiding secondary structural elements into density. The g-scale dictates how strongly the atomic models are pulled into density. A low g-scale of 0.3 was chosen to help prevent over-fitting. All MDFF simulations were carried out using NAMD 2.8 (Phillips et al, 2005) and the CHARMM27 force field. Simulations were setup to run for 100 picoseconds under implicit solvent conditions. They were performed on the Case Western Reserve University High­Performance Computing Cluster. Typically, these simulations would take ~6 hours to complete, but this time varies with the number of atoms in the system.

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^[1] (date) DECEMBER 13.2013

Frequently asked questions about "Structural Insights into Recognition of Adenovirus by Immunologic and Serum Factors"

What is the main focus of this document?

This document is a language preview of a dissertation titled "Structural Insights into Recognition of Adenovirus by Immunologic and Serum Factors." It includes the table of contents, list of tables and figures, acknowledgements, an abstract, chapter summaries, and key themes.

What topics are covered in the table of contents?

The table of contents covers topics such as: adenoviruses as pathogens and therapeutic agents, adenovirus-host interactions, recognition of adenovirus by human alpha defensin 5, the role of humoral factors in innate immunity, factor VII dimerization and its influence on infectivity, visualization of adenovirus capsid-incorporated HIV antigen, and a summary of discoveries with future directions.

What are some key themes explored in the chapters?

Key themes include: the interaction of human alpha defensin 5 with adenovirus, the role of factor X in activating innate immunity, the influence of factor VII dimerization on adenovirus infectivity, and the structure and function of an adenovirus capsid-incorporated HIV antigen.

What techniques are used in the research described?

The research utilizes cryo-electron microscopy (cryo-EM), computational modeling, molecular dynamics flexible fitting (MDFF), and other biochemical and immunological assays to analyze the structural and functional aspects of adenovirus recognition and interaction with host factors.

What is the significance of studying adenovirus-host interactions?

Understanding adenovirus-host interactions is crucial for developing effective therapies against adenovirus infections, optimizing adenovirus vectors for gene therapy, and designing vaccines against other infectious diseases such as HIV.

What is the importance of minor proteins in AdV capsids?

The minor proteins IlIa, VI, VIII, and IX form interactions on the inner and outer surface of AdV that are critical for capsid stabilization. Exactly how these minor proteins serve as cement to hold together the AdV virion is not yet fully understood and is currently an active area of research.

What roles do coagulation factors play in AdV infections?

Coagulation factors such as FX and FVII bind to the AdV capsid protein hexon and influence liver tropism and immune responses. FX promotes virus entry into liver cells and triggers innate immunity, while FVII, despite binding to hexon, is inefficient at supporting virus attachment and cell transduction, possibly due to its altered binding orientation and dimerization.

What is the significance of HD5?

HD5, a protein of the innate immune system, stabilizes the AdV capsid and prevents disassembly of the virus during cell entry, blocking infection.

What is the role of the MPER insertion?

An HIV membrane proximal ectodomain region (MPER) is genetically inserted within the AdV hexon hypervariable region 2 (HVR2) to create a vaccine and elicit an anti-HIV cellular response.

What is MDFF and how is it used?

Molecular Dynamics Flexible Fitting (MDFF) is a computational method that combines atomic-scale structures with cryo-EM density maps, allowing researchers to merge detail from atomic structures with the overall architecture of AdV-host complexes. It's used to refine models into cryo-EM density and understand interactions.