CAPSID STRUCTURE AND CELL SURFACE INTERACTION OF HUMAN BOCAVIRUS 2

By

MENGXIAO LUO

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

2017

© 2017 Mengxiao Luo

To my parents and mentors

ACKNOWLEDGMENTS

First of all, I would like to thank my parents, Huihong Xiao and Zhandong Luo, who provide all their love and encouragement for me at any time. They always understand and support my decision especially for studying abroad. I am grateful that I am able to pursue my academic dreams and have a meaningful life in UF with their wishes. Also, I want to say thank you to my boyfriend Yuxuan Wang, who always bring me happiness and strength. I am so honored to have studied under my great mentor,

Dr. Mavis-Agbandje McKenna, who gave me the opportunity to study in McKenna lab and help me to become more competent compared to two years ago. She is thoughtful and always gives me some useful advice when I encounter difficulties. Besides, without the help from my committee members and amazing teachers, Dr. Robert McKenna and

Dr. Linda B. Bloom, the master degree would have not been possible and I would not make progress in my study. Particularly, I really want to thank Dr. Mario Mietzsch, a patient and excellent teacher who guides me to finish my project step by step. Also, I woule like to thank Dr. Shweta Kailasan and undergraduate student Maria Ilyas for providing protocols of generation as well as purification of VLPs, Dr. Antonette Bennett for teaching me baculovirus expression, purification of VLPs and heparin binding assay,

Paul Chipman for negative stain EM and cryo-EM, and Joshua A. Hull, the lab manager for generating VLPs sample. I would like to say special thank you to the graduate students, Nikea Pittman for providing help for labeling VLPs and culturing CHO cell lines, Justin Kurian for providing AAV2 samples and J. Kennon Smith for giving me some advice of experiments, Victoria E. Fielding for helping me to solve problems from classes. I really enjoy and will miss all the happy moments of sharing food, chatting about life and funny stories. I am grateful to have some amazing friends Wenying,

4 Peiyao, and Ziyan who go shopping, traveling with me and give me a lot of support during these two years. Last, but not the lease, I would like to say thank you to my grandparents, Youhuan, Xiaolian and Sue, my aunt Huiji, my cousin Zinxin and Xuhong, my closest friends Junxiang, Zhixin, Jiamin and Shaoqun for their constant love, support and memory of joy, laughter and happiness.

5 TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 8

LIST OF FIGURES ...... 9

LIST OF ABBREVIATIONS ...... 10

ABSTRACT ...... 13

CHAPTER

1 INTRODUCTION TO BOCAPARVOVIRUSES ...... 15

Overview of Family ...... 15 Classification ...... 15 Genome Structure and Capsid Composition ...... 16 Pathogenicity ...... 17 Features of Genus Bocaparvovirus ...... 18 Viral Emergency and Epidemiology ...... 18 Bocaparvovirus Structure of Genome and Capsid ...... 19 Bocaparvovirus Infectious Pathway ...... 21 Knowledge of Human Bocavirus ...... 21 Significance ...... 23

2 STRUCTURAL INSIGHTS INTO HUMAN BOCAVIRUS 2 ...... 28

Introduction ...... 28 Experimental Methods ...... 29 Production of Baculovirus Stocks ...... 29 Generation of HBoV2 Baculovirus-Infected Cell Lysate ...... 29 Purification of HBoV2 VLPs ...... 30 SDS-PAGE ...... 31 Negative-Stain EM ...... 31 Cryo-EM and Data Collection ...... 32 Structure Determination of HBoV2 Capsid ...... 32 VP3 Model Building and Structure Refinement ...... 33 Structure Alignment of HBoVs ...... 34 Result and Discussion ...... 34 Purification of HBoV2 VLPs ...... 34 The Capsid Structure of HBoV2 ...... 35 Characterization and Comparison of HBoV VP3 Structures ...... 37 Summary ...... 40

6 3 IDENTIFICATION OF VIRAL-CELL RECEPTOR INTERACTION ...... 52

Introduction ...... 52 Methods and Materials...... 53 Labeling Capsids with Dylight Dyes ...... 53 Cell Lines ...... 53 Cell Binding Assay ...... 53 Heparin Binding Assay ...... 54 Result and Discussion ...... 55 CHO Cell Binding Assay...... 55 Heparin Binding Assay ...... 56 Summary ...... 56

4 CONCLUSION AND FUTURE DIRECTIONS ...... 62

Structural Features of Bocaparvoviruses ...... 62 Identify the Receptors and Tissue Tropism of HBoVs ...... 63

LIST OF REFERENCES ...... 65

BIOGRAPHICAL SKETCH ...... 75

7 LIST OF TABLES

Table page

2-1 Summary of cryo-EM data collection, image-processing parameters and refinement statistics...... 42

2-2 Range of Cα distances for the aligned Human bocaviruses in the VRs ...... 43

8 LIST OF FIGURES

Figure page

1-1 Classification of the family Parvoviridae...... 25

1-2 Genome and capsid structure of Bocaparvovirus...... 26

1-3 A simplified view of the parvovirus life cycle...... 27

2-1 Purification of HBoV2 VLPs...... 41

2-2 The capsid structure of HBoV2...... 44

2-3 Cross-sectional view of the HBoV2 structure...... 45

2-4 βG of HBoV2 density map and atomic model...... 46

2-5 The N termini of HBoV2...... 47

2-6 The HBoV VP3 structures...... 48

2-7 Structural alignment of the HBoVs...... 49

2-8 The HBoV VP3 VRs...... 50

2-9 Surface representation of HBoV2 capsid...... 51

3-1 SDS-PAGE analysis of labeling HBoV2 VLPs...... 58

3-2 The result of cell binding assay of HBoV2...... 59

3-3 Average values of triplicate cell binding assay...... 60

3-4 Heparin binding assay for HBoV1 and HBoV2...... 61

9 LIST OF ABBREVIATIONS

AAP Assembly activating protein

AAV2 Adeno-associated parvovirus 2

AMDV Aleutian mink disease

ARTI Acute respiratory tract infection

A488 Alexa Fluor 488

BAAV Bovine AAV

BC Basic amino acid cluster

BDPV Barbarie duck parvovirus

BPV Bovine parvovirus

Bu Buffalo lung fibroblasts

BuPV Human bufavirus

B19 Human parvovirus B19-V9

CaBoV Canine Bocavirus

C.C Correlation coefficient

ChPV Chicken parvovirus

CHO Chinese hamster ovarian cells

CnMV Canine Minute Virus

CPV Canine parvovirus

Cryo-EM Cryo-electron microscopy

CslBoV California Sea Lion Bocavirus

CTF Contrast transfer function

EBTr Host bovine tracheal cells

FACs Fluorescence-activating cell sorting

FBoV Feline Bocavirus

10 FBS Fetal bovine serum

FPV Feline parvovirus

FSC Fourier shell correlation

GAG Glycosaminoglycan

GBoV Gorilla Bocavirus

Gb4 Globoside

GlcNAc N-acetylglucosamine

GPA Glycophorin A

GPV Goose parvovirus

HBoV Human bocavirus

HSPG Heparan sulfate proteoglycan

H1-PV H-1Parvovirus

ICTV International Committee on Taxonomy of

ITR Inverted terminal repeat

LBoV Rabbit bocavirus

MBoV Mink bocavirus

MDPV Muscovy duck parvovirus

MmBoV Bat bocavirus

MOI Multiplicity of infection

MVMi Minute virus of mice (immunosuppressive)

MVMp Minute virus of mice (prototype)

NP Phosphorylated non-structural protein

NS1 Non-structural protein 1

ORF Open reading frame

PARV4 Human parvovirus 4

11 pLA2 Phospholipase A2

PPV Porcine parvovirus

PWMS Post-weaning multi-systemic wasting syndrome

RBoV Rat bocavirus

RHR Rolling-hairpin replication

R.M.S.D Root mean square deviation

Sf9 Spodoptera frugiperda ssDNA Single stranded DNA

SSM Secondary structure matching

Tm Melting temperature

VLP Virus-like particle

VP Viral protein

VP1u VP1 unique N-terminal region

VR Variable region

12 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

CAPSID STRUCTURE AND CELL SURFACE INTERACTION OF HUMAN BOCAVIRUS 2

By

Mengxiao Luo

August 2017

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

Human Bocavirus is a non-enveloped, single stranded DNA virus that belongs to genus Bocaparvovirus, family Parvoviridae. HBoV, the second pathogenic human parvovirus identified and the first human bocaparvovirus, consists of four strains:

HBoV1, which causes respiratory disease and HBoV2-4 that cause gastrointestinal disease. Due to the lack of an animal model and a versatile cell culture system, information of bocaparvovirus replication is limited and there is no specific treatment or vaccine for severe infection. There is therefore, a need to understand the replication mechanisms of these viruses at the molecular and structural level. Towards this, in this study the structure of the HBoV2 capsid was determined to 2.9 Å by cryo-EM, and the topology of HBoV2 capsid compared to those available for HBoV1, HBoV3, and HBoV4 as well as members of the Parvovirinae subfamily. The capsid displays features common to some members of Parvovirinae subfamily, including a depression at the two- fold symmetry axis, protrusions around the three-fold symmetry axis, and a channel at five-fold symmetry axis. Significantly, this study provides evidence that the five-fold channel extending into the interior capsid to form a ‘basket’ structure and the additional

α-helix (αB) present in viral protein 3 (VP3) is unique for Bocaparvovirus. These

13 features may have genus-conserved functions. The VP3 structure contains an eight- stranded anti-parallel-β barrel core (BIDG-CHEF) and α-helix (αA) with loops between the β-strands, which are observed in other parvoviruses. By comparing the VP3 structures of all the HBoVs, significant differences were identified in some variable regions (VRs) that are involve in tissue tropism, pathogenicity, and antigenicity in other parvoviruses. Among them, VR-III differed the most between HBoV1 and the other

HBoV strains and is thus considered as a possible determinant of tissue tropism for

HBoVs. Since the recognition and attachment between viruses and host cells is the first step in the virus life cycle, this study also aimed to identify the cell binding receptor for

HBoV2 by cell and heparin binding assays. The data showed that glycans with terminal sialic acid, galactose, GlcNAc moieties or heparin do not facilitate HBoV2 and HBoV1 cellular attachment. Overall, the results provide insight for future studies of HBoV tissue tropism, pathogenicity, antigenicity, and the development of antiviral strategies.

14 CHAPTER 1 INTRODUCTION TO BOCAPARVOVIRUSES

Overview of Parvoviridae Family

Classification

Members of the Parvoviridae family are non-enveloped icosahedral viruses packaging single stranded DNA (ssDNA) genomes. They are further divided into two subfamilies according to their ability to infect vertebrates or invertebrates: Parvovirinae and (1) (Figure 1-1). Based on genome characteristics and pairwise sequence alignments of their non-structural (NS) gene products, eight genera are included in Parvovirinae that has a wide host range of mammalian and bird species with type members: Amdoparvovirus (Aleutian mink disease virus, AMDV), Aveparvovirus

(chicken parvovirus, ChPV), Bocaparvovirus (bovine parvovirus, BPV), Copiparvovirus

(bovine parvovirus 2, BPV2), Dependoparvovirus (adeno-associated parvovirus 2,

AAV2), Erythroparvovirus (human parvovirus B19-V9, B19), Protoparvovirus (minute virus of mice (prototype strain), MVMp) and Tetraparvovirus (human parvovirus 4,

PARV4). Members of Dependoparvovirus except goose parvovirus (GPV), Barbarie duck parvovirus (BDPV), and Muscovy duck parvovirus (MDPV) require a helper virus

(e.g. adenovirus, herpesvirus, papillomavirus or vaccinia virus) co-infection in host cells for successful infection and replication. Otherwise, the virus genome integrates into the host’s chromosomes (2). In 2014, a novel genus Marinoparvovirus was proposed following the isolation of Sesavirus (3), and it is pending the International Committee on

Taxonomy of Viruses (ICTV) approval. For the subfamily Densovirinae that only infect insects and arthropods, viruses are classified into five genera: Ambidensovirus,

Brevidensovirus, Hepandensovirus, Iteradensovirus and Penstyldensovirus.

15 Genome Structure and Capsid Composition

The hallmark of the parvovirus genome is that the linear ss-DNA is bracketed by inverted terminal repeats (ITRs) at the 5’ and 3’ ends (4). The length of parvovirus genome ranges from 4–6 kb, while the termini is about 120–420 bases in size, folding back on themselves to form the hairpin structures. This specific structure contains the sequences that create viral origins of DNA replication, and therefore plays an important role in ‘rolling-hairpin replication’ (RHR) strategy. The hairpin can present in Y-/T-shape in diverse size (117 to 418 nucleotides) as well as sequence, depending on viral genera, and the variability is even observed between species within the genus Dependovirus, whose hairpins may be minimally small (125 bases in AAV2) or the largest currently on record (418 bases in MDPV) (4).

The parvovirus coding sequence contains two major open reading frames

(ORFs), a non-structural (ns or rep) ORF and a viral protein (vp or cap) ORF. ns/rep

ORF that locates in the left half of the genome encodes a NS1 (autonomous viruses) or

Rep (dependoparvovirus viruses) protein acting as DNA helicases play a role in several processing steps during DNA replication to regulate gene expression. vp/cap ORF encodes a single structure protein in several forms (VP1-4) by alternative mRNA splicing of the transcript and alternative translation initiation codon usage (2). For genus

Dependoparvovirus, a AAP ORF within the cap ORF encodes a capsid assembly activating protein (AAP), which is essential for capsid assembly. All the VPs share a common C terminus and assemble the capsid which is involved in cell entry, endosomal trafficking to the nucleus, cell egress pathogenicity, and antigenicity. Previous structural studies show that the parvovirus capsid is arranged with T=1 icosahedral symmetry with

16 60 copies of VPs in different ratio (1:10 for VP1 and VP2 and 1:1:10 for VP1:VP2:VP3), containing thirty 2-fold, twenty 3-fold, and twelve 5-fold vertices (5). Besides, a phospholipase A2 (pLA2) domain is identified in the VP1 N-termini of almost all parvoviruses, except for Brevidensovirus members and Aleutian mink disease virus

(AMDV) (6). It externalizes from inner to outer capsid after heat or pH treatments and its lipolytic enzyme activity is required during entry between late endosome/lysosome and nuclear entry in life cycle (7).

Pathogenicity

The clinical manifestations of autonomous parvoviruses infection vary depending on age of host, in vitro and in vivo tropism, et al (8). Among Dependoparvovirus, AAVs which need adenovirus or herpes simplex virus for successful replication are not associated with any known disease, and are being developed as vectors for gene therapy (9, 10), while MDPV and GPV that can autonomously replicate can cause

Derzsy’s disease that may be lethal to their hosts (11). In genus Protoparvovirus, there are both pathogenic and non-pathogenic variants of PPV (porcine parvovirus) and

MVM, but are only pathogenic for hosts at certain ages and also contain non-pathogenic strains (2). Besides, the difference exists between in vivo and in vitro base on the fact that MVMp strain is non-pathogenic and cause an asymptomatic infection in vivo while the immunosuppressive MVMi strain can cause a lethal infection in vitro (12, 13). For human beings, the pathogens include HBoV (Bocaparvovirus), B19 (Erythroparvovirus),

BuPV1-3, Cutavirus, Tusavirus (Protoparvovirus) and PARV4 (Tetraparvovirus) (14-18)

Some of parvoviruses have specific host and tissue tropism while others can infect more than one host. For example, from genus protoparvovirus, both CPV (canine

17 parvovirus) and FPV (feline parvovirus/panleukopenia virus) can replicate in feline cells, but FPV only infects the thymic cells of dogs in vivo (16, 19). In genus Bocaparvovirus, there are twelve different host species and viruses are restricted to their own hosts.

Interestingly, all the infections cause similar disease of the gastrointestinal and respiratory tracts (20, 21). During the process of infection, it has been reported that some kinds of glycan particularly terminal sialic acid are common as receptors for virus- cell binding, despite of the diversity of host/tissue tropism (22). For dependoparvovirus, the AAVs that can transduce multiple organs in the human body, AAV1, AAV4 and AAV5 use terminal N -acetyl neuraminic acid (sialic acid) as their primary receptor; AAV2,

AAV3b and AAV13 bind to heparan sulfate proteoglycan (HSPG); AAV6 can bind to both sialic acid and HSPG while AAV9 preferably binds to galactose (22). A kind of ceramide- based glycolipid (ganglioside) and β-1–4-linked N-acetyl glucosamine (chitotriose) are utilized by bovine AAV (BAAV) for transduction and cellular transcytosis, respectively

(23, 24). For autonomous parvoviruses, the only available information of

Bocaparvovirus is BPV which binds to the glycan with terminal sialic acid. For genus

Erythroparvovirus, B19 has wide tissue tropism and its receptors are several tissue- specific glycosphingolipids as well as globoside (Gb4) (25). Among Protoparvovirus,

MVM, PPV and H1-PV (H-1Parvovirus) use sialic acid as receptor while CPV and FPV prefer bind to N-Glycolylneuraminic acid.

Features of Genus Bocaparvovirus

Viral Emergence and Epidemiology

The genus Bocaparvovirus was originally named according to the first two members, Bovine Parvovirus (BPV) reported in dead cattle in 1961 (26, 27) and Canine

18 Minute Virus (CnMV) isolated in the rectal samples of German shepherd dogs in 1967

(28). In 2005, Human Bocavirus (HBoV1) was discovered in 17 respiratory samples from children (< 2 years of age) suffering from acute respiratory tract infections (ARTI) in Sweden (29). The other three subtypes are named HBoV2, HBoV3, and HBoV4, isolated from pediatric patients with acute gastroenteritis (30-32). In recent years several new bocaviruses were identified: from 2009-2010, six Porcine Bocavirus (PBoV) were identified worldwide in healthy piglets as well as those suffering from post-weaning multi-systemic wasting syndrome (PWMS) which consists of progressive weight loss, tachypnea, dyspnea, and jaundice in piglets (33, 34); California Sea Lion Bocavirus

(CslBoV) was isolated in stool samples of California sea lions that suffered enteric viral infections in 2011 (35). In 2012, three novel members were discovered: Gorilla

Bocavirus (GBoV) was isolated from captive North American Western Gorillas ailing from gastroenteritis (36); Feline Bocavirus (FBoV) and Canine Bocavirus (CaBoV) were discovered in Thai stray cats and dogs, respectively (37). From 2015-2016, the genus expanded with the addiction of Rabbit bocavirus (LBoV) (38), Rat bocavirus (RBoV)

(39), Bat bocavirus (MmBoV) (40) and Mink bocavirus (MBoV) (41). Although bocaviruses have a wide range of host, they cause similar disease of the gastrointestinal and respiratory tracts in their respective host species.

Bocaparvovirus Structure of Genome and Capsid

Bocaparvoviruses are unique among parvoviruses since they contain a third ORF between ORF1 and ORF2 in their genomes (7, 42) (Figure 1-2A). ORF1 codes for a non-structural protein 1 (NS1) that controls genomic DNA replication. NS1 is cytotoxic to host cells and plays a role in apoptosis (43). The ORF in the center of the viral genome,

19 called ORF3, codes for the NP protein that is a highly phosphorylated non-structural protein (42). In some bocaviruses, NP protein is important for DNA replication, the expression of capsid proteins or regulates the interferon signaling pathway (44, 45).

ORF2, which is located at the 5’-end of the genome encodes the VP1 (~74 kDa), VP2

(~64 kDa) and VP3 (~60 kDa) proteins that assemble the icosahedral viral capsid

(Figure 1-2B). The different VPs share a common C terminus since they are expressed from different start codons within the same transcript (46-48). As the minor component of the virus capsids, VP1 contains a unique N-terminal region (VP1u) that has a phospholipase A2 (pLA2) domain conserved in all parvoviruses. The enzymatic pLA2 activity is required for endosomal escape and nuclear entry during the viral replication cycle (7, 49-50). Moreover, in the N-terminal region there are four basic amino acid clusters (BC1-4) whose sequences are highly conserved among parvoviruses. Some previous experiments show that those elements are related to nuclear transport during the viral infection but further studies are needed to testify if it is common in all bocaviruses (51, 52). According to the structural studies of BPV, HBoV1, -3 and -4, the structure of VP3 is conserved despite of the low identity of amino acid between the

HBoVs and BPV (53, 54). The core VP structure consists of a α-helix (αA) and an eight- stranded anti-parallel motif (βB to βI, named from N- to the C-terminus) organized in two

β-sheets, BIDG and CHEF with an additional βA that is anti-parallel with βB. Besides, a unique αB for bocaviruses is observed. Between β-strands, loops (with variable regions,

VRs, at their apex) with different lengths and conformation are inserted and named after the strands between them. For example, DE loop is inserted between βD and βE and HI loop is inserted between βH and βI. The loops characterize features at and around the

20 icosahedral two-, three- and five-fold axes of symmetry and consequently contribute to the capsid surface topology (53).

Bocaparvovirus Infectious Pathway

The first step of virus infection is attachment and recognition by a specific receptor on the cell surface. Since the capsid surface structures are diverse among different viruses, the recognition is restricted which dictates tissue tropism and pathogenicity (2, 22). Proteoglycans, glycolipids, glycoproteins or proteins are the common primary/secondary receptors. For bocaparvoviruses, the only available information is for BPV, which uses N- and O-linked sialic acid moieties on buffalo lung fibroblasts (Bu) or EBTr (Host bovine tracheal cells) as receptors (55, 56). However, the previous lab member, Dr. Shweta Kailasan discovered that BPV could also recognize glycans with terminal N-acetylglucosamine on CHO (Chinese hamster ovarian) cells

(unpublished data). After attached to the receptor, bocaparvoviruses are internalized and form clathrin-coated vesicles that traffic through the endocytic pathway to the nucleus (57). In this process, pH decreases from the early endosome to lysosome which is essential for infection and virus escape from the late endosome, facilitated by

VP1u pLA2 activity that can destroy the membrane (7). After the viruses enter the nucleus, they follow several steps including capsid disassembly, genome release, genome transcription, protein translation, capsid assembly, and genome packaging to generate new progenies (Figure 1-3).

Knowledge of Human Bocavirus

As the second pathogenic human parvovirus discovered and the first human bocaparvovirus, HBoV that is detected in respiratory and gastrointestinal infections is

21 found worldwide including all over Europe, North and South American, Asia and

Australia (58-63). There are four subtypes of HBoV: HBoV1 is mainly related to respiratory diseases, while HBoV2, HBoV3, and HBoV4 are associated with gastrointestinal infections. Since some previous studies report that the recombination between HBoV1 and HBoV2 generates HBoV3, and between HBoV2 and HBoV3 generates HBoV4, HBoVs may have a high frequency of recombination among each other (64). At first HBoVs were considered as a possible harmless passenger, as it was co-detected with more additional pathogens than any other respiratory virus (15).

Recently, some diagnoses suggested that HBoV1 was the third most prevalent virus detected in samples from young English and Thai children suffering from lower respiratory tract infections, after adenovirus and respiratory syncytial virus (65, 66).

Besides, epidemiological studies reported the presence of epidemiological HBoV1 and

HBoV3 DNA in infected patients, suggesting the ability of the viruses to cause persistent infections. The common patient symptoms of infection include diarrhea, wheezing, nausea, vomiting, cough, dyspnea, pneumonia, pharyngitis and bronchitis, similar to those caused by animal bocaparvoviruses. Stool as well as nasopharyngeal aspirates are two prevalent sources where viruses are isolated, and this fact predicts the oral- fecal route for transmission but needs further studies to confirm (67-69). In addition, viruses also appear in serum, blood, tonsils, urine, and saliva from healthy as well as diseased individuals. As for the structural study, the capsid structures of HBoV1, -3 and

-4 in high resolution are available and display features in common with other parvoviruses in 2-, 3-, and 5-fold symmetry axis (54). And three strain-specific antigenic epitopes on the HBoV1 capsid and a cross-reactive epitope on the HBoV1, HBoV2, and

22 HBoV4 have also been identified (70). However, more efforts should be made to have a better understanding for pathogenesis and antigenicity of HBoVs.

Significance

The first paper recording the parvoviruses was in 1959 (71) and they have been studied for decades. To date there are some achievements especially for AAVs, which are being developed as vectors for gene delivery and applied in more than 70 approved clinical trials worldwide for a variety of diseases (Huntington’s disease, Alzheimer’s disease, Hemophilia B, heart failure, heart failure, et al.) (72, 102). However, it is necessary to expand the knowledge of parvoviruses since some of them can cause economically important diseases of animals or threaten human’s health. For Human

Bocavirus, due to the lack of an animal model and a versatile cell culture system, the information of virus replication is limited and therefore it is difficult to develop specific treatment or vaccine for severe respiratory and gastrointestinal infections. Therefore, there is a desire to understand the disease-causing mechanisms of these viruses.

As the parvovirus capsids that have been studied play a significant role in cell entry, endosomal trafficking to the nucleus, capsid assembly, genome packaging, and antibody recognition, the goal of this study is to determine the capsid structure of

HBoV2, identify properties as well as important regions of HBoV capsids, and try to identify the viral cell binding receptor. In chapter 2, the capsid structure of HBoV2 is determined by using cryo-electron microscopy (cryo-EM) and image reconstruction and surface properties are also analyzed. Besides, the model of HBoV2 VP3 protein is generated in order to understand how the residues decorate the capsid surface and identify some important ones that may have function related to infection. By aligning all

23 the HBoV VP structures, several variable regions are defined and some of them are predicted as the possible determinant for tissue tropism, since HBoV1 can infect respiratory tracts while HBoV2-4 can infect gastrointestinal tracts. Since recognition and attachment to the host cell surface is the first step of the virus life cycle, chapter 3 will focus on efforts to identify the receptor for HBoV1 and HBoV2. Capsids labeled by a fluorophore are used for cell and heparin binding assays and a preference to bind specific glycans is analyzed.

Overall, this study aimed to obtain a cryo-EM reconstructed to high resolution and identify the specific receptor for cell binding. Together with the HBoV1, -3 and -4,

3D information is now available for capsid structures of all strains of human bocavirus, which give insights into systematic study for pathogenesis and antigenicity. Significantly, the results will give some new information to the previous guesses that for bocaparvoviruses, the five-fold channel extends into the interior capsid to form a

‘basket’ structure and there is a unique α-helix (αB) in VP. Besides, structural alignment guides to identify important regions that may determine HBoVs tissue tropism. That information helps to understand the molecular infection mechanism. In all, this new knowledge will help to control HBoV infection and spread of disease in human beings.

24

Parvovirinae

Protoparvovirus

Amdoparvovirus

Aveparvovirus

Bocaparvovirus

Dependoparvoviru

Erythroparvovirus

Copiparvovirus

Tetraparvovirus

Densovirinae Ambidensovirus

Parvoviridae Iteradensovirus

Hepandensovirus

The family family The Penstyldensovirus

Brevidensovirus

Figure 1-1. Classification of the family Parvoviridae. The family is divided into two subfamilies, Parvovirinae and Densovirinae. The eight genera under Parvovirinae with some of their members discussed in the text are listed. The figure is from Cotmore et al. (2013) Arch Virol.

25 A

B

Figure 1-2. Genome and capsid structure of Bocaparvovirus. (A) Organization of Bocaparvovirus genome. ORF1 codes for a non-structural protein (NS1) that control genomic replication. ORF in the center of the viral genome also called ORF3, codes for the NP protein that is a highly phosphorylated non-structural protein. ORF2, which is located at the 5’-end of the genome encodes the VP1 (~74 kDa), VP2 (~64 kDa) and VP3 (~60 kDa) proteins that form the viral capsid. (B) The capsid structure of BPV. The reconstructed maps are colored according to radial distance from the particle center (blue to red), as indicated by the scale bar at the right. The figure is from Kailasan S, Halder S, Gurda B, et al (2015).

26

Figure 1-3. A simplified view of the parvovirus life cycle. The virus binds to a cell surface receptor and is internalized into clathrin-coated vesicles. The virus then traffics from the early to late endosome/lysosome. A pLA2 enzyme motif found within the VP1 N terminus facilitates viral escape into the nucleus, After the virus enters the nucleus, it follows several steps including capsid disassembly, genome release, genome transcription, protein translation, capsid assembly, and genome packaging to generate new progenies.

27 CHAPTER 2 STRUCTURAL INSIGHTS INTO HUMAN BOCAVIRUS 2

Introduction

It has been previously reported that the parvovirus capsid plays significant roles in the virus lifecycle, including host/tissue cell recognition, entry, endosomal trafficking, genome release, capsid assembly. and antibody recognition (2, 7). Especially for some parvovirus such as AAV2, AAV9 and B19, the function roles of regions in or around icosahedral axes of symmetry on the capsid surface have been confirmed (53, 74).

However, the structural knowledge of Bocaparvovirus is incomplete since the high- resolution capsid structures are only available for BPV (3.2 Å) (53), HBoV1 (2.9 Å),

HBoV3 (2.8 Å), and HBoV4 (3.0 Å) (54). More information is needed to determine conserved bocaparvovirus-specific features and the important sites of the VP that may be related to specific functions. For HBoVs, as mentioned in chapter one, HBoV1 is more related to respiratory diseases while the others are more related to gastrointestinal diseases. The comparison of variable regions (VRs) on capsid structures will provide insight into determinants for tissue tropism and antigenicity. The goal of this chapter is to provide structural information of HBoV2 capsid and characterize unique features of

HBoVs.

In this chapter, virus-like particles (VLPs) assembled by HBoV2 VP3 proteins were used for structure determination since the previous study showed that overexpressed HBoV1 VP3 proteins can self-assemble into capsid-like structures (53,

75). The capsid structure was determined by cryo-EM and three-dimensional (3D) image reconstruction. The overall VP topology was studied and compared to that of the other HBoV members. VP3 3D model was built for structure alignment, which was

28 necessary to identify the conserved regions, VRs and important residues in capsid.

Overall, this study gives insight for future study of determinants of tissue tropism and antigenicity.

Experimental Methods

Production of Baculovirus Stocks

The baculovirus-expression system in Spodoptera frugiperda (Sf9) (ATCC) cells was adopted to express the bocaparvovirus VP3 gene of HBoV2. The overall process involves three stages: (1) incorporation of the VP2 gene into a suitable shuttle vector to introduce our gene of interest into a bacmid, which contains the entire baculovirus genome, (2) purification and transfection of the bacmid DNA into Sf9 cells, and (3) collection of baculovirus particles, which will express the protein of interest (i.e. VP3) upon infection of Sf9 cells. To obtain the baculovirus stock in high titers, the baculovirus needed to be amplified. A standard plaque assay was applied and after 96 h incubation, plaques were large enough to pick by plucking the agarose overlay using a glass pipet followed by resuspension into 500 µL of Sf9 II media (Life Technologies). Those stocks were denoted as P0 and stored at 4°C. Later the P0 stocks were amplified (multiplicity of infection (MOI) of 0.1) to obtain P1 stocks. After the P1 stocks were tittered by plaque assay, they were amplified to P2 stocks which was used for VLP production (53).

Generation of HBoV2 Baculovirus-Infected Cell Lysate

250 ml of Sf9 cells (1.7~2.0x106 cells/mL) were infected by P2 baculovirus stocks

(8x107 pfu/mL) of HBoV2 with a MOI of 5 and incubated at 28°C for 3 days. The infected cells were harvested by centrifugation at 3500 rpm (JA-20) for 20 min at 4°C. The cell pellet was resuspended in 15 mL of TNET buffer (50 mM Tris-HCl, pH 8.0, 100 mM

29 NaCl, 1 mM EDTA, 0.2% Triton X-100). The supernatant was collected and mixed with polyethylene glycol, PEG 8000 (1 g/10 mL) to precipitate the VLPs. The PEG- supernatant mixture was stirred at 4°C overnight and centrifuged on the following day at

9,000 rpm (JA-20) for 90 min at 4°C. The pellet was resuspended in TNET and labeled as PEG pellet. Both cell and PEG pellet were stored in -20°C until the purification.

Purification of HBoV2 VLPs

The cell pellet underwent three rounds of freeze/thaw cycles (using dry ice bath/37°C water bath) in order to break the cells and release the VLPs. After the second cycle, the completely thawed pellet was treated with Benzonase (250 U/µL, Millipore,

Novagen) (1 µL/10 mL pellets). The sample was incubated in 37°C water bath for 30 min followed by the third freeze. In addition, the PEG pellet was thawed and then combined with the cell pellet. After that, the pellets were centrifuged (JA-20, 9000 rpm,

15 mins, 4°C) to remove any large cell debris and VLPs in the supernatant were pelleted through sucrose cushion. 5 mL of 20% (wt/vol) sucrose (in TNET) were layered underneath the supernatant and ultracentrifuged at 45,000 rpm (45K) for 3 hour at 4°C

(Beckman 70Ti Rotor). Subsequently, the pellet was resuspended in 500 µL~2 mL

TNTM buffer (25 mM Tris-HCl, pH 8.0, 100 mM NaCl, 2 mM MgCl2, 0.2% Triton X-100) and stored overnight at 4°C. Large, insoluble aggregates were removed by low speed spin (8000 rpm) and the sample was further purified by sucrose gradient. In this step,

1.35 mL of each % sucrose (from 40%, 35%, 30%, 25%, 20%, 15%, 10% to 5%, wt/vol) were layered in ultra-clear tubes, followed by loading the sample to the top and spinning at 35000 rpm (35K) for 3 h at 4°C (Beckman SW41Ti Rotor). 20-25%, 25-30% and 30-

35% fractions of sucrose gradient were collected and analyzed the presence of VLPs.

30 Then the collections were dialyzed into 1×PBS (phosphate buffer saline, pH 7.4) at 4°C

3 times (3 h each). The samples were concentrated to between 0.5 to 2 mg/mL using

Amicon concentrators (EMD Millipore) and the concentration was determined by DU

730 UV/Vis Spectrophotometer (Beckman Coulter®) at wavelength of 280 nm. SDS-

PAGE and negative-stain electron microscopy (EM) were applied to analyze the purity as well as integrity of the VLPs.

SDS-PAGE

The gel system was composed of 12% polyacrylamide gel (4 mL H20, 3.3 mL

30% Acrylamide/Bis solution, 2.5 mL 1.5 M Tris (pH 8.8), 0.1 mL 10% SDS, .1 mL 10%

APS, 7 µL TEMED), which was layered underneath the stacking gel (3.4 mL H20, 0.83 mL 30% Acrylamide/Bis solution, 0.63 mL 1 M Tris (pH 6.8), 0.05 mL 10% SDS, 0.05 mL 10% APS, 5 µL TEMED). Before loading on the gel, the samples were mixed with

1× Protein Loading Dye (Morganville Scientific®) with 9% 2-mercaptoethanol (Bio-

Rad®) and boiled for 10 min at 100°C. Then the denatured samples migrated through the gel system by using a current of 75 V in the stacking gel and 120 V in the 12% polyacrylamide gel to separate the individual proteins by size. When it was done, the gel was washed 3 times with distilled water (diH20) and stained with Blue Protein Safe stain

(Life Technologies) for 1 h, followed by destaining with diH20 for 30 min. prior to imaging using a GelDock EX system (Bio-Rad).

Negative-Stain EM

Carbon-coated copper EM films (Ted Pella) were treated by the Glow Discharge

Cleaning System (PELCO easiGlow™) to be made hydrophilic. 5 µL samples were loaded on the grids and incubated for 1~5 min. The residual liquid was blotted away by

31 filter paper. Then the grids were washed by three 10 µL drops of diH20, followed by staining with 5 µL Nano-W® (Nanoprobes) or 1% solution of uranyl acetate (UA) for 10 second and stain solution was blotted away. The grids were imaged on a Tecnai G²

Spirit TEM (FEI) microscope operated at an accelerating voltage of 120 kV.

Cryo-EM and Data Collection

HBoV2 VLPs (~1 mg/mL) were applied to a thin carbon coated over holey

QUANTIFOIL® grids to overcome aggregation observed when using holey carbon grids.

Then the samples were vitrified using a Vitrobot™ Mark IV (FEI Co.) and screened on a

16-megapixel CCD camera (Gatan, Inc.) in a Tecnai (FEI Co.) G2 F20-TWIN

Transmission Electron Microscope (200 kV, ~ e-/Å2) prior to data collection. Micrographs of virus particles were collected by using a Titan Krios electron microscope (FEI Co.)

(300 kV) with a DE20 (Direct Electron) direct electron detector. Images were recorded as ‘movies’ consisting of multiple frames with a defocus range of ~0.78 to 4.39 µm using a pixel size of 0.95 Å/pixel, summarized in Table 3-1. Subsequently, multiple frames of micrographs were aligned to enhance the signal-to-noise ratio by using

DE_process_frames software (Direct Electron) with corresponding dark and bright reference images without radiation dose damage compensation (76).

Structure Determination of HBoV2 Capsid

Several subroutines within the AUTO3DEM software package (77) were applied to generate the particle database used for the structure determination: RobEM was used to extract the individual virus particles in each cryo-micrographs followed by pre- processing for image background linearization and normalization to bring the entire data set to a common greyscale); ctffind4 (78) was used to estimate the defocus values for

32 the micrographs that enable correction of the microscope-related contrast transfer functions (CTFs). Then 100 particles were used to build a random model map at low- resolution (28 Å) while imposing icosahedral symmetry (79), which can determine the orientations and origins of each particle. This was followed by multiple refinement steps for all the images including particle recentering (particles are accurately centered in their boxes), solvent flattening (the density of solvent region is modified to a low, constant value), and individual particle CTF refinement (the movement of Z-direction that might change the CTF of movie frame is modified). The reconstructed density maps were generated based on 100%, 90%, 80%, 70% data set respectively (some bad particles are eliminated) and the one at highest resolution was chosen (90% data set, 85,155 particles). The FSC used to measure the resolution, represents the correlation between two 3D maps, each calculated from an independent half of the data. The density map in high resolution on the basis of Fourier shell correlation (FSC) threshold criteria of 0.143 and 0.5 was obtained. Inverse temperature factor of 1/50 Å2, 1/100 Å2 and 1/150 Å2 were applied to sharpen the final density map, respectively, and the maps with different temperature factor were compared in UCSF-Chimera (80). The 1/100 Å2 one showed the best high-resolution features and was used for subsequent model building.

VP3 Model Building and Structure Refinement.

A model of HBoV2 VP3 monomer was generated based on the protein sequence

(NCBI accession number: AFW98869.1) in SWISS-MODEL using the crystal structure of

BPV (Research Collaboratory for Structural Bioinformatics [RCSB] PDB code 4QC8) as the template (81). That monomer was utilized to build an icosahedral 60-mer capsid model in VIPERdb2 (82) The 60-mer capsid model was adjusted into the HBoV2

33 cryoreconstructed density map by using the ‘fit in map’ subroutine in UCSF-Chimera and changing the pixel size of the reconstructed map. Using the e2proc3d.py subroutine in EMAN2, the reconstructed map with new pixel size was converted into a format that could present in the Coot program (83, 84). The reference VP3 model was fitted into the density map by adjusting the position of residues through interactive model building and the real-space-refine options available in Coot (83). After that, the model processed the real space refinement for two macrocycles in PHENIX (85). The refined model was analyzed in Coot with the density map and side chains were modified if necessary, followed by the rigid-body and B-factor refinement in PHENIX.

Structure Alignment of HBoVs

The VP3 structure of HBoV1-4 were superimposed with each other based on backbones by using the secondary structure matching (SSM) program in Coot (86) The program calculated root mean square deviations (RMSDs) for the superposed structures and the distances between the aligned Cα positions. Regions with two or more consecutive residues with an RMSD of 2.0 Å between the superposed structures were defined as variable regions (VRs) (74), and that definition was previously applied for identification of VRs in autonomously replicating viruses. The labeling of VRs on the capsid surface, cartoon representations of the VP3 structures and side chain density images were generated by PyMOL (87).

Result and Discussion

Purification of HBoV2 VLPs

Self-assembled HBoV2 VLPs were generated from infected insect cells and purified by sucrose cushion and gradient. Their purity and composition were analyzed

34 by SDS-PAGE gel, which presented a single ~60 kDa sized band corresponding to

HBoV2 VP3 (Figure 2-1). Empty, circular capsids filled with stain in negative-stain EM image confirmed the homogeneity and integrity of VLPs (Figure 2-1), which were qualified for the following structure study.

The Capsid Structure of HBoV2

The 3D reconstructed structure of HBoV2 was generated by using 85,155 particles, with resolution of 2.9 Å estimated by using a Fourier shell correlation (FSC) plot on the basis of threshold criteria of 0.143 (Table 2-1). The topology of HBoV2 capsid (Figure 2-2) presents some common features of Parvovirinae subfamily structures: there is a narrow-depressed region at each 2-fold symmetry; a trimeric protrusion that surrounds each three-fold symmetry axis (except for some viruses such as MVM, CPV, FPV and PPV) (2); a cylindrical channel at each five-fold axis surrounded a wide, canyon-like region and a ‘wall’ between the two- and five-fold depressions. The most obvious difference among HBoVs is the conformations of three- fold protrusion, which are more prominent in HBoV3-4. Besides, the pointed HBoV2 protrusions is so different from the others while HBoV4 has the rounder ones. The cross-sectional view of HBoV2 cryoreconstruction (Figure 2-3) indicates the 5-fold channel extends into the interior of capsid and form a ‘basket’ structure beneath the 5- fold axis. To date, this feature is only observed in BPV (53) as well as other HBoVs (54), and HBoV4 has longer and larger extensions than HBoV1-3 (Figure 2-3).

The VP3 model was fitted into the high-resolution map to position of amino acids side chains. The refined model built from N-terminal residues 33 to the last C-terminal residues 542 showed a high correlation coefficient (CC) (0.812) with density map. The

35 statistics of refinement by using PHENIX program and evaluation of model fit into map are summarize in Table 2-1. Residues are assigned well in the core secondary region

(βB-βI) and most loops inserted between strands. However, disordered density appears in some glutamic acid and aspartic acid residues, especially in their terminal acidic groups, which indicates the flexibility of conformation in those regions (Figure 2-4).

Those disorders result from the high sensitivity of the negatively charged side chain to radiation damage and were previously reported for other high-resolution cryo-EM maps

(88). The first 32 residues of VP3 protein are not presented in the refined model because of the lack of density (map rendered at lower density thresholds (0.7 to 0.8 sigma) can present the main chains of residue 27-32). Similar phenomenon is also reported for most other parvoviruses, except B19 (2, 89), with the hypothesis that there is inherent disorder in this region. This is confirmed by the fact that the N-terminal residues 33 in the model locates after a glycine-rich region (Figure 2-5B), which is predicted to be disordered for HBoV1, -3 and -4 (54).

The N-terminus of the VP3 model is a part of the interior extension in 5-fold channel (Figure 2-5A), which is consistent with a previous hypothesis that the N- terminal residues of all VP proteins of bocaparvoviruses are situated under the 5-fold axis of the capsid (70, 74). In the future study, it is necessary to examine if VP1 and

VP2 also display that feature when the capsid assemble by all VP. The function of the 5- fold channel of bocaparvoviruses is needed to be confirmed, while in other parvoviruses, VP1 unique region externalize through the 5-fold channel during the life cycle in order to enable its PLA2 function (7, 49-50). Therefore, one direction of the structural study of bocaparvoviruses can focus on the analysis of the change of the 5-

36 fold channel, using the capsid protein treated with different pH to mimic the condition in different step of life cycle.

In general, VP3 structure of HBoV2 display the conserved features of other parvoviruses (53), including of a α-helix (αA), an eight-stranded anti-parallel motif organized in two β-sheets, BIDG and CHEF with an additional βA that is anti-parallel with βB and loops (containing variable regions, VRs) with different length and conformation inserted between β-strands (Figure 2-6A). Those loops named after the strands between which they are inserted (DE loop, between D and E), characterize features at and around the icosahedral two-, three- and five-fold axes of symmetry and consequently contribute to the capsid surface topology. Another α-helix (αB) existing in the large GH surface loop, is a unique feature for all HBoVs (54) and BPV (53), suggesting that it may have genus level function.

Characterization and Comparison of HBoV VP3 Structures

All HBoV VP3 models are superimposed with each other based on their backbones in COOT. The identity of sequence ranges from 78~91% with HBoV3 and

HBoV4 being the most conserved. The structural alignment resulted in Cα RMSDs of

0.88 Å (HBoV1 and HBoV3), 0.87 Å (HBoV1 and HBoV2), 0.97 Å (HBoV1 and HBoV4),

0.66 Å (HBoV2 and HBoV3), 0.84 Å (HBoV2 and HBoV4) and 0.86 Å (HBoV3 and

HBoV4). Those values suggest that the main chain of HBoV1 and HBoV4 are the most structurally different while of HBoV2 and HBoV3 are most structurally similar. Both the sequence alignment (Figure 2-7) and the superimposed VP3 models (Figure 2-6C) show that the secondary elements in the core region (β-barrel motif and αA) are highly conserved, while the surface loops are variable in different extent that characterize the

37 features of HBoV capsids (Table 2-2). There are ten VRs defined in genus level for

Bocaparvovirus (labeled in Figure 2-6C), but only seven of them are identified as VRs for HBoVs: VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VII and VR-VIIIB, while VR-VI, VR-VIII and VR-IX are structurally conserved. The most obvious differences appear in VR-I, VR-

II, VR-III, VR-IV, VR-V, and VR-VIIIB (Figure 2-6C, 2-8 and 2-9). Among HBoVs, HBoV1 is more divergent at VR-III, VR-V, and VR-VIIIB and HBoV4 is more divergent at VR-I,

VR-II and VR-IV. When compare HBoV2 and HBoV3, the most structurally similar pair, most divergent regions were observed in VR-III, VR-V and VR-VI (Figure 2-6B).

Differences locating at the 2-fold, 2/5-fold wall (between the depressions at the 2- and

5-fold axes), the 3-fold protrusions, the 5-fold channel, and the floor of the 5-fold depression contribute to the various topology of virus capsid surface.

The analysis of VRs based on the amino acid sequence alignment (Figure 2-7) and superimposed models (Figure 2-8). The two-fold axis is decorated by VR-IX whose function is related to receptor binding for MVMp (20) and transduction efficiency for AAV

(90). VR-IX is so observed between the HBoVs although there is variability in residues ranging from 507-511. This may suggest that in some cases various amino acid does not cause a great difference of structural conformation. The 2/5-fold wall consists of VR-

I, VR-III, VR-VII as well as a part of VR-IX, and previous studies indicate that this region is important in AMDV tissue tropism, pathogenicity (91, 92) as well as antibody reactivity for AAV2 (93). In VR-I, the sequence shows more diversity in HBoV1 and HBoV4, which results in a more divergent structural conformation of HBoV1 and HBoV4 compared to

HBoV2 and HBoV3. VR-III of HBoV1 that contains α-helix B has four amino acid insertions compared to the others. This difference may contribute to a higher wall on

38 HBoV1 capsid surface and determine the tissue tropism for HBoVs, since the tract- infection is different between HBoV1 and other strains. Besides, it has been reported that residues in this VR are part of an HBoV1-specific antigenic region (70). In VR-VII, the amino acid sequences are conserved. The three-fold protrusion surrounding the three-fold axis consists of VR-IV and VR-V. VR-IV is a determinant of antibody recognition for other parvoviruses such as adeno-associated virus (AAV), human parvovirus B19, and Aleutian mink disease parvovirus (92, 94-98). HBoV4 has two insertions in amino acid sequence in this VR. These additional residues cause a longer loop that generates a more “spikier” appearance of the protrusions. In VR-V, which is part of an HBoV1-specific antibody footprint (70) and has been shown to be important for transduction efficiency and antigenicity in other parvoviruses, the amino acid sequences are not so conserved that structures are diverse especially for HBoV1.

Besides, VR-VI, VR-VIII and partial VR-I as well as VR-VII form the side of the 3-fold protrusions. Both amino acid sequences and structures of HBoVs are conserved in VR-

VI and VR-VIII. VR-II in the DE loop locates in five-fold symmetry of the capsid axis involve in the VP1u externalization and endosomal escape (99, 100), genomic DNA packaging and uncoating for the parvoviruses. The structural flexibility of this region may be required to despite that amino acid sequences are so conserved in this VR. The canyon floor around the five-fold channel is modified by VR-VIIIB that forms a part of the footprint of an antibody generated against HBoV1 that cross-reacts with HBoV2 and

HBoV4 (70), but this finding still needs to be confirmed for its role. In this region, the fact that the sequence is different yet there is cross-reactivity means that this VR-VIIIB region does not play a role in the antigenic reactivity for this cross-reactivity antibody.

39 Summary

This study determined the HBoV2 capsid structure at high resolution and expand the knowledge of Bocaparvovirus structure. The topology of HBoV2 capsid displays common features of Parvovirinae subfamily structures at the two-, three-, five-fold symmetry. Also, the VP3 structure contains an eight-stranded anti-parallel-β barrel core

(BIDG-CHEF) and α-helix (αA) with loops between the β-strands, which are observed in other parvoviruses. The unique second α-helix (αB) proximal to VR-III for

Bocaparvovirus suggests a genus level function, but this possibility needs confirmation.

For HBoVs, capsid structures of all strains are now available due to this study, which builds a good foundation for systematic study of the tissue tropism, pathogenicity and infectious pathway for the bocaparvoviruses. Since HBoV1 is more likely to infect respiratory tracts while HBoV2-4 are likely to infect gastrointestinal tracts, it is possible that they have different tissue tropism. As mentioned before, virus capsid plays an important role in recognition and attachment with the host cell. Therefore, the diversity of the capsid surface topology may result in the interaction with different kinds of cells.

The comparison among capsid structures helps to identify the important regions or residues for determining tissue tropism. Significant differences present in some variable regions, e.g. VR-III, is suggested as the possible determinant of tissue tropism. Other regions that characterize the appearance of capsid are also identified, but their function for Bocaparvovirus need to be determined. In all, these results can guide future studies of virus infection, antigen development, and control of diseases.

40 A B HBoV2

250

150 100 75

50

37

25

20 15

Figure 2-1. Purification of HBoV2 VLPs. (A) SDS-PAGE analysis shows presence of VP3 (expected size ~60 kDa) for the purified samples for HBoV2. Molecular mass markers are indicated. (B) Micrograph of negatively stained HBoV2 VLPs imaged at a magnification of 42,000X.

41 Table 2-1. Summary of cryo-EM data collection, image-processing parameters, and refinement statistics.

Parameter HBov2 No. of micrographs 2,921 Defocus range (µm) 0.78-4.39 Electron dose (e-/Å2) 64 No. of frames/micrograph 36 Pixel size (Å/pixel) 0.95 Starting no. of particles 94,590 No. of particles used for final map 85,155 B factor used for final map (Å2) -100 Resolution of final map (Å) 2.9

PHENIX model refinement statistics Residue range 33-537 Map CC 0.812 RMSD (Å) Bonds 0.01 Angles 0.97 All-atom clash score 15.30

Ramachandran plot (%) Favored 86.9 Allowed 11.9 Outliers 1.2 Rotamer outliers 0.23

42 Table 2-2. Range of Cα distances for the aligned Human bocaviruses in the VRs

Comparison HBoV1 vs HBoV1 vs HBoV1 vs HBoV2 vs HBoV2 vs HBoV3 vs HBoV2 HBoV3 HBoV4 HBoV3 HBoV4 HBoV4

VR-I 0.9-3.8 0.6-5.2 0.1-4.4 0.3-3.8 0.6-7.0 0.6-6.1

VR-II 0.9-3.0 0.1-2.2 0.4-3.5 1.0-2.3 0.8-3.9 0.3-2.1 VR-III 0.7-6.8 0.2-4.9 0.5-4.3 0.4-3.8 0.6-3.9 0.1-1.2 VR-IV 0.9-3.2 0.2-1.8 0.3-5.6 0.5-2.3 0.7-3.9 0.2-3.9

distances(Å) VR-V 0.5-6.0 0.5-6.6 0.5-5.5 0.2-2.5 0.1-2.1 0.4-2.8

α VR-VI 1.1-1.7 0.2-1.1 0.4-1.2 1.1-1.8 1.2-1.8 0.7-1.2 VR-VII 0.7-2.1 0.7-2.9 0.8-1.8 0.9-2.1 0.8-1.9 0.5-2.0

VR-VIII 0.9-1.6 0.4-1.3 0.3-1.9 0.6-1.4 0.7-2.5 0.4-2.3 Range of C Range

VR-VIIIB 0.8-3.6 0.3-4.7 0.5-3.9 0.9-2.4 1.1-2.4 0.3-1.6 VR-IX 0.7-1.9 0.4-2.5 0.6-1.9 0.3-1.4 0.3-1.2 0.4-1.7

43

HBoV2

Figure 2-2. The capsid structure of HBoV2. The reconstructed maps are colored according to radial distance from the particle center (blue to red), as indicated by the scale bar at the right. The image was generated with UCSF-Chimera (85). The images of HBoV1, HBoV3 and HBoV4 are from Mietzsch M, Kailasan S, Garrison J, et al (2017).

44

HBoV1 HBoV2

HBoV3 HBoV4

Figure 2-3. Cross-sectional view of the HBoV2 structure. Arrowheads point to the locations of the 5-fold (5f) symmetry axes and channel. Under each 5-fold channel, density forming a basket can be observed. The image was generated with UCSF-Chimera (85). The images of HBoV1, HBoV3 and HBoV4 are from Mietzsch M, Kailasan S, Garrison J, et al (2017).

45

Phe 237 Phe 239 Phe 241 Thr 235

Glu 236 Thr 238 Asp 240

Figure 2-4. βG of HBoV2 density map and atomic model. The resolution of the map enabled the interpretation of side chains. The truncated density of the glutamic acid side chain (Glu236) is due to high sensitivity to radiation damage. The amino acid residues are shown as a stick representation inside a gray mesh density map and colored according to atom type as follows: C, green; O, red; N, blue; S, yellow. This image was generated with PyMOL (87).

46 A

Gly 33

B

Figure 2-5. The N termini of HBoV2. (A) Closeup of a cross-sectional view of the basket under the 5-fold channel in HBoV2. The ribbon diagram of the docked HBoV2 VP3 structure is shown within its semitransparent density. The first residue glycine 33 is labeled. (B) Sequence alignment of the VP3 N terminus of HBoVs. Orange-shaded residues are identical amino acids among the HBoVs, while yellow-shaded residues are conserved in more than 50% of HBoVs and green- shaded ones are similar amino acids

47 A B VR-III αB VR-IV VR-V

VR-I VR-VIII VR-VIIIB VR-VI VR-VII

βH βC βE βF βG VR-II βD VR-IX C-Term βI βB αA βA N-Term

C VR-III

VR-IV VR-V VR-I VR-VIII

VR-VIIIB/HI loop VR-VI VR-VII

VR-IX C-Term VR-II/DE loop

N-Term

Figure 2-6. The HBoV VP3 structures. (A) Diagram of the HBoV2 VP3 structure. The conserved β-barrel core motif (B-I), the αA and αB helices (orange) are indicated. The loops inserted between these secondary structure elements also contain β-strand regions, as indicated. (B) Structural superposition of HBoV2 (pink) and HBoV3 (green), which are most structurally conserved. (C) Structural superposition of HBoV1 (blue), HBoV2 (pink), HBoV3 (green), and HBoV4 (red) VP3 shown with the positions of VR-I to VR-IX indicated. The positions of the icosahedral 2-, 3-, and 5-fold symmetry axes are also indicated as a filled oval, triangle and a pentagon, respectively. This image was generated with PyMOL (87).

48

Figure 2-7. Structural alignment of HBoVs. Secondary structures elements identified for HBoV1, β-strands and α-helices are indicated by blue arrows and red cylinders, respectively. The previously defined variable regions (VR) are also indicated. In the alignment, the different background colors reflect the extent of conservation: the orange color indicates complete conservation in all HBoVs, while yellow-shaded residues are conserved in more than 50% of HBoVs and green-shaded ones are similar amino acids.

49 VR-I VR-II

VR-III VR-IV

VR-V VR-VI

VR-VII VR-VIII

VR-VIIIB VR-IX

Figure 2-8. The HBoV VP3 VRs. Shown are closeup views of the VRs when HBoV1 (blue), HBoV2 (pink) HBoV3 (green) and HBoV4 (red). Next to each VR, a sequence alignment of the loops of the different bocaviruses is shown. Orange highlighting indicates conservation among the HBoVs, while yellow-shaded residues are conserved in more than 50% of HBoVs and green-shaded ones are similar amino acids. The VR structure images were generated with PyMOl (87).

50

HBoV1 HBoV2

.

HBoV3 HBoV4

Figure 2-9. Surface representation of HBoV2 capsid. 3D capsid surface representation is generated with PyMOL (87) viewed down the 2-fold axis. The VRs are colored as indicated in the legend at the bottom. The images of HBoV1, HBoV3 and HBoV4 are from Mietzsch M, Kailasan S, Garrison J, et al (2017).

51 CHAPTER 3 IDENTIFICATION OF VIRAL-CELL RECEPTOR INTERACTION

Introduction

The information for viral-cell binding interaction that enables the virus to enter the cell is unknown for Bocaparvoviruses, with the exception of BPV, what previous studies indicated used glycophorin A (GPA) with terminal α-2,3- O-linked sialic acid present on the O-linked oligosaccharides on erythrocytes as the hemagglutination receptor (101). If the host cells such as buffalo lung fibroblasts (Bu) and bovine host bovine tracheal cells

(ETBr) are treated by sialidase, which cleaves terminal α-2,3- O-linked and α-2,3-N- linked sialic acids, the infection is disrupted (102, 103). Furthermore, Dr. Shweta

Kailasan discovered that BPV could also recognize glycans with terminal N- acetylglucosamine on CHO (not published). These finding lead to a hypothesis that

HBoVs may also use those glycan as receptor since those viruses are in the same genus.

In this chapter efforts to identify the receptor for HBoV1 and HBoV2 under the guide of previous study are described. Cell binding assays using CHO cell lines were used in order to test if they can bind to terminal sialic acids, galactose or N- acetylglucosamine on glycans, respectively. In addition, a heparin binding assay was also utilized to provide more choices for the identification of a possible receptor. As

HBoV1 and HBoV2 infect respiratory and gastrointestinal tracts, respectively, and may have diverse tissue tropism, those assays can also suggest if they have different preference to bind specific glycans. Overall, this study will help to understand the interaction between HBoV viruses and cells, and reveal cellular and molecular mechanisms of replication.

52 Methods and Materials

Labeling Capsids with DyLight Dyes

Before labeling, the integrity of purified HBoV1, HBoV2 and AAV5 (provide by Dr.

Mario Mietzsch) VLPs were confirmed by negative-stain EM and the concentration were tested by DU 730 UV/Vis Spectrophotometer (Beckman Coulter®) at wavelengths of

280 nm. 300~500 µL VLPs (0.5~0.8 mg/mL) were enough for labeling by The Alex Fluor

488 labeling kit (Molecular Probes, Life Technologies). According to the manufacturer’s instructions, 40 µL borate buffer was added to the sample and then mixed with DyLight

Reagent by vortex. The mixture was incubated in room temperature for one hour protected from light. After one hour, the mixture was added with 20 µL borate buffer and incubated for another half hour. Subsequently, 10 µL mixture was analyzed by SDS-

PAGE and successfully labeled protein was green fluorescent in UV transilluminator.

Labeled VLPs were aliquoted at 100 µL /Eppendorf and stored at -80°C .

Cell Lines

Three CHO cell lines were used for the binding assays: Pro5 and Lec2 cell lines were generously provided by Dr.Jude Samulski (UNC Chapel Hill), and Lec8 cell line by

Dr. Aravind Asokan (UNC Chapel Hill). The culture medium was α-MEM (Minimum

Essential Medium) + GlutaMax (GIBCO) composed of 10% FBS (fetal bovine serum) and 1% Antibiotic-Antimycotic (mixture of penicillin, streptomycin, and amphotericin B).

The cell lines were cultured as monolayers in a 5% CO2 37°C incubator.

Cell Binding Assay

When different CHO cell lines grew to 70~100% confluency in 15 cm tissue culture dish, each dish was added with 1 mL 0.5 M EDTA and incubated for 1 min at

53 room temperature. Then dishes were washed with pipette, and cells were collected in

50 mL falcon tubes, followed by centrifugation at 500 rpm (Eppendorf 5810) for 5 min to remove the supernatant. Cell pellets were washed by 10 mL 1×PBS (Dulbecco, Sigma) and centrifuged again to remove the supernatant, followed by resuspension by 6 mL ice-cold MEM (no FBS). Subsequently, cells were diluted to 5×105 cells/mL and pre- chilled for 30 min at 4°C . Then cells were aliquoted to 500 µL /Eppendorf and incubated with A488-labeled VLPs at a MOI of 106 for 2~4 hour at 4°C . After the incubation, the mixtures were centrifuged at 3000 rpm (Eppendorf 5418) for 10 min and the supernatant was discarded. Unbound VLPs were removed by washing the cells with

300 µL ice-cold 1×PBS by vortex, followed by another centrifugation. Pellets were resuspended in 300 µL 1×PBS by vortex and analyzed by fluorescence-activating cell sorting (FACs) using standard excitation and emission wavelengths defined for A488.

Heparin Binding Assay

Microspin columns (732–6204; Biorad) were washed by 200 µL TNTM buffer followed by 1 ml 1×TD buffer (1×PBS, 1 mM MgCl2, 2.5 mM KCl). Then 50 µL of heparin-conjugated agarose type I resin (H-6508; Sigma) were loaded in columns. The affinity columns were equilibrated with 1 mL of 1×TD buffer, and charged by washing with 500 µL of 1×TD/1 M NaCl buffer followed by three sequential washes with 1 mL of

1×TD buffer. Subsequently, 10 µg HBoV1, HBoV2 and AAV2 (positive control) VLPs were diluted in 60 µL 1×TD buffer respectively and the columns were loaded with 30 µL of samples followed by sequential collection of flow through, five column washes with

1×TD buffer (30 µL each), and five elution fractions with 1×TD/1 M NaCl buffer (30 µL

54 each). The input, flow through, washes, and elution fractions were analyzed by SDS-

PAGE.

Result and Discussion

CHO Cell Binding Assay

In cell binding assay, three CHO cell lines were used to test the extent of preferential glycan binding by HBoV1 and HBoV2 with AAV5 used as a positive control: the parental Pro5 CHO cell line displays terminal sialic acid while the mutant Lec2 cell line displays terminal galactose and Lec8 cell line displays N-acetylglucasaminyl, resulting from defects in UDP-galactose translocation, and N-acetylglucasaminyl transferase translocation pathways, respectively (104). Although there is no stable cell line or animal model available and whether HBoVs are infectious to CHO cell lines is unknown, the cell binding assay based on those cell lines was designed to identify the cell surface receptor for HBoV1 and HBoV2. AAV5 which requires sialic acid for infection and used as positive control. SDS-PAGE analysis suggested that purified

HBoV1, HBoV2 and AAV5 VLPs were successfully labeled (Figure 3-1). The assay was repeated thrice and absorption at a wavelength of 494 nm, which corresponds to the absorption wavelength of A488, was measured to confirm presence of tagged-VLPs. An important tool for evaluating FACs data is the dot plot (Figure 3-2), where cell detected by the instrument is showed as a point on an x-y graph. In each graph, x axis represents the related intensity of green fluorescence, y axis represents the side scatter that determines the granularity and complexity of each cell, and the green circled area in the right distinguishes the cells binding with labeled viruses. In the negative control group, there was no labeled viruses added into the cell lines and therefore only some

55 background noise appears in the green circled arear (Figure 3-2). As predicted, the positive control AAV5 has significant binding with Pro5 cell line (Figure 3-2A) and significantly decreased binding to sialic acid-lacking mutants Lec2 and Lec8 cell lines

(Figure 3-3). However, HBoV2 shows similarly weak binding to all three cell lines

(Figure 3-2, Figure 3-3). It seems that HBoV2 does not use glycans with terminal sialic acid (Pro5), galactose (Lec2) or GlcNAc moieties (Lec8) as receptors for cell binding.

Heparin Binding Assay

This assay attempts to test if virus can bind to heparin. Accoding to the result of

SDS-PAGE, the positive control AAV2 VLPs that require heparin for infection bound to the heparin-conjugated agarose column as there is no proteins in the flow through and washes but they appear in the elution fractions (Figure 3-4A). For HBoV1 and HBoV2

VLPs, proteins in expected size exist in the flow through and washes instead of elution fractions, which suggests that both of them do not bind to heparin (Figure 3-4B, -4C).

The result of HBoV2 VLPs is consistent with the obervation of cell binding assay, as

CHO cell lines also contain heparin sulfate proteoglycan but HBoV2 VLPs dose not have significant binding to those cells.

Summary

The virus capsid is involved in several roles during infection and the first and important step is the recognition by host cells. As viruses have to locate to the correct tissues/cells of the organism being infected to guarantee successful infection, they rely on the recognition based on the binding between specific receptor and some recognition sites on the capsid surface. These sites are conserved and a few changes in sequence may lead to interaction with different glycans. Due to previous reports that BPV uses

56 glycans with terminal sialic acid as receptor and has weak binding to GlcNAc moieties, studies were conducted with HBoV2 (and HBoV1) to determine if these viruses have similar glycan recognition. However, none of these three glycans serve as the receptor for HBoV2. Also, HBoV2 is not able to bind to heparin which is the receptor of AAV2.

Although the specific receptor for HBoV1 and HBoV2 are not identified in this study, it suggests that the receptors vary even among the viruses in the same genus, despite conserved features of the capsid structure at the genus-level.

57 A B

250 150

100

75

50

37

25

Figure 3-1. SDS-PAGE analysis of labeling HBoV2 VLPs. (A) Gel stained with Blue Protein Safe stain analysis shows presence of VP3 (expected size ~60 kDa). (B) Gel before staining shows that successfully labeled protein was fluorescent in UV Transilluminator. The band at the bottom is the unbound Dye.

58 A B C

Figure 3-2. The result of cell binding assay of HBoV2. (A) Pro5 cells were incubated without virus (control), with HBoV2 and with AAV5 VLPs, respectively. (B) Lec2 cells were incubated without virus (control), with HBoV2 and with AAV5 VLPs, respectively. (C) Lec8 cells were incubated without virus (control), with HBoV2 and with AAV5 VLPs, respectively. Dots in the left represent cells that do not bind to labeled VLPs, while in the green circled arear represent cells that bind to labeled VLPs. The value in each graph is the percentage of cells that bind to labeled VLPs.

59 100

90

80

70

60

50

40

% Cells with A488 with Cells % 30

20

10

0 Pro5 Lec2 Lec8 Control AAV5 HBoV2

Figure 3-3. Average values of triplicate cell binding assay. Analysis of binding of A488- tagged AAV5 (red bars) and HBoV2 (green bars) VLPs to parental and mutant CHO cell lines, Pro5, Lec2, and Lec8 by FACs. Control: blue bars. Error bars represent standard error.

60 A

B

C

Figure 3-4. Heparin binding assay for HBoV1 and HBoV2. (A) SDS-PAGE analysis for positive control AAV2. (B) SDS-PAGE analysis for HBoV1. (C) SDS-PAGE analysis for HBoV2. W: wash; E: elution.

61 CHAPTER 4 CONCLUSION AND FUTRURE DIRECTIONS

Structural Features of Bocaparvoviruses

In this project, the capsid structure of HBoV2 was determined to at 2.9 Å by cryo- electron microscopy and image reconstruction. The results support previous observations that the five-fold channel extending into the interior capsid to form a

‘basket’ structure and that there is a unique α-helix (αB) in VP of the bocaparvoviruses.

Comparative structural analysis for all HBoVs provided insight into regions of their VP, including VR-III, which may determine their differential tissue tropism. As mentioned in chapter 2, for other parvoviruses, VP1u is reportedly externalized through the 5-fold channel to enable their pLA2 function (105, 106). pLA2 has lipolytic enzyme activity and is essential for the escape of viral particles from the late endosome or lysosome. This is one of the important steps during infection and facilitated by the endosomal acidification

(2). For Bocaparvovirus, the hypothesis is that viruses also apply a similar strategy but this needs further confirmation. It is observed that the N-terminal residues of HBoV VP3 locate under the 5-fold channel, which suggests that the VP1u may also display this feature. Therefore, future studies can focus on two directions that help to determine the process of infection and the function of 5-fold channel: (1) To obtain the capsid structure assembled from all three VPs, rather than VP3 alone and analyze if the location of N- terminus is the same; (2) To determine the effects of different treatments (pH or heat) on the density of 5-fold basket. This future study will explain the thermodynamic properties of capsid and how the structure changes to expose the pLA2 domain (107). Dr. Shweta

Kailasan, our previous lab member did a thermal denaturation assay called DSF to measure the capsid stability of the all HBoVs and discovered that for any of the HBoVs,

62 the capsid is most stable at pH 5.5 (Tm ranges from 70-68°C) and least stable at pH 2.6

(Tm ranges from 56-42°C). In addition, ligand-interactions or salt conditions may also contribute to the stability of the capsid (108). This information provides some guides for testing the structural variability under different conditions.

Identify the Receptors and Tissue Tropism of HBoVs

Cell and heparin binding assays suggest that HBoV2 does not bind to glycan with terminal sialic acid, galactose, N-acetylglucasaminyl or heparin, which are common receptors utilized by other parvoviruses for infection, and HBoV1 does not have interactions with heparin, either. Future studies will include cell binding assays for

HBoV1, HBoV3, and HBoV4. To improve the positive control in cell binding assay, AAV2 which can interact with galactose and N-acetylglucasaminyl will also be used as a positive control. Since all HBoVs did not bind to the sialylated glycans on the CFG array

(Dr. Shweta Kailasan, not published), another possible approach is to screen the VLPs on GAG (Glycosaminoglycans) arrays (109). The future studies will illustrate if different

HBoV strains have different preferences for receptors that may govern the tissue tropism. Once the receptors are identified, the follow up studies could be conducted to determine the capsid-receptor complex structures, identify capsid residues important for receptor attachment, and confirm the role of the identified residues by mutation. As

HBoV1 causes respiratory diseases while HBoV2-4 causes gastrointestinal ailments, it is necessary to identify cell lines that confirm the tissue tropism among HBoVs proposed based on epidemiological studies. Some binding assays using respiratory and gastrointestinal cell lines, such as human lung/gut epithelial cells can be applied in these future studies. Subsequently, VR-III that is proposed as a determinant of the

63 tissue tropism can be analyzed to confirm its function. Based on the knowledge of virus capsid structure and cell surface receptor, information can be obtained to be applied for the development of anti-virals to interfere with viral infection and control the spread of disease.

64

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BIOGRAPHICAL SKETCH

Mengxiao Luo, the only child of Zhandong Luo and Huihong Xiao, was born in

Guangzhou, China. She completed the high school study at Guangdong GuangYa High

School in 2011. After that, she discovered her interest in biology and entered South

China Agriculture University, where she graduated with a B.S. in biotechnology in 2015.

She enjoyed spending her spare time on student association activities and learning guitar. When she was a sophomore, she was honored to study in Dr. Linghua Zhang’s

Lab, where she learned a lot of experiment skills and knowledge of sirtuins, the NAD- dependent deacetylases. With the help of a graduate student Miaopeng Ma, she wrote a thesis based on experience in expression and purification of the porcine sirtuin,

SIRT3. In the meanwhile, she decided to pursue higher studies in the United States after graduation with her parents’ encouragement and support. Fortunately, Dr. Mavis

Agbandje-McKenna gave her the opportunity to realize her dream. In August 2015, she joined McKenna’s lab as a master student and focused on the structural study of CnMV

(Canine minute virus) and PBoV capsid (Porcine Bocavirus). However, she re-directed her project at characterizing the structure of Human bocavirus 2 (HBoV2) and its cellular interactions in fall 2016 due to difficulties in the expression and purification of the CnMV and PBoV capsid. With the help of Dr. Mario Mietzsch and the other lab members, she was able to finish her project and graduate in summer 2017. During those two years in

Gainesville, she lived a happy and meaningful life and enjoyed shopping, charting, traveling with her friends at weekend.

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