The Pennsylvania State University. The Graduate School. College of Medicine

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1 The Pennsylvania State University The Graduate School College of Medicine EPSTEIN-BARR VIRUS INFECTION AND REPLICATION IN STRATIFIED EPITHELIAL CELLS A Dissertation in Microbiology and Immunology by Rachel Mary Temple 2014 Rachel M. Temple Submitted in Partial Fulfillment of the Requirements for the Degree of Doctoral of Philosophy December 2014

2 This dissertation of Rachel Mary Temple was reviewed and approved* by the following: Clare Sample Professor of Microbiology and Immunology Dissertation Advisor Chair of Committee Laura Carrel Associate Professor of Biochemistry and Molecular Biology Craig Meyers Distinguished Professor of Microbiology and Immunology Jeffery Sample Professor of Microbiology and Immunology Todd Schell Associate Professor of Microbiology and Immunology Aron Lukacher Professor of Microbiology and Immunology Department Chair of Microbiology and Immunology *Signatures are on file in the Graduate School ii

3 ABSTRACT Epstein-Barr virus (EBV) is a ubiquitous human herpes virus associated with epithelial and lymphoid tumors. EBV is transmitted between human hosts in saliva, and must cross the oral mucosal epithelium before infecting B lymphocytes where it establishes a life-long infection. While there are numerous model systems to study infection of B cells in vitro, our knowledge of infection of epithelial cells is limited due to the inability to readily infect epithelial cells in vitro or generate cell lines from EBV-infected epithelial tumors. Because epithelium exists in vivo as a stratified tissue, organotypic 'raft' cultures may serve as a better model for EBV in epithelial tissue than monolayer cultures. Using the raft culture system, we were able to study EBV infection and replication in stratified epithelium for the first time. Here, we demonstrate that EBV readily infects stratified epithelium, with infection limited to the non-replicating, terminally differentiating cells. Infection resulted in the spontaneous expression of lytic cycle proteins, amplification of the viral genome, and production of infectious viral particles. These de novo synthesized viral particles infected the adjacent epithelial cells, forming large foci of infection and generating the first multicycle growth curve for EBV. Blocking lytic replication inhibited dissemination of the virus. Although infected cells did express latency-associated proteins while concurrently expressing lytic proteins, a population of cells which exclusively expressed the latency-associated proteins could not be identified. Furthermore, infection did not induce cellular proliferation as it does in B cells, but resulted in cytopathic effects more commonly associated with productive viral replication. This is the first in vitro system in which infected cells bypass latency and iii

4 immediately undergo productive replication. These data suggest infection of epithelial cells is an integral part of the viral lifecycle, which does not typically result in the immortalization or enhanced growth of the infected epithelial cells, but instead results in the production of high quantities of infectious viral particles. Despite the high prevalence of EBV in the human population, we still do not know the primary site of infection or where the virus replicates for dissemination to a new host. However, our observation that EBV infection in raft cultures results in high levels of virus production suggests the oral epithelium would be an ideal site to amplify virus during primary infection and again during viral egress. To refine our understanding of the role of epithelium in the EBV lifecycle, we investigated how infection can be initiated in stratified epithelium and assessed the tropism of virus produced by raft cultures. Stratified epithelium could only be infected through the apical surface, and not from the basal surface. In fact, basal epithelial cells consistently appeared to be refractory to infection. Consistent with previous observations, we determined that the cellular source of the virus strongly influenced viral tropism. The virus produced by stratified epithelium was highly infectious for B cells, but not for epithelial tissue. Instead, infection was most readily initiated in the raft cultures by virus-producing B cells or slightly less efficiently with B-cell-derived cell-free virus. Infection with epithelial-derived virus could be enhanced if the virus was pre-incubated with an anti-gp350 antibody or transferred by virus-producing epithelial cells. These data have practical implications for vaccine development, suggesting the vaccine- iv

5 mediated immune response should target the oral epithelium rather than the peripheral B cells to successfully prevent infection. v

6 Table of Contents List of Figures... x List of Tables... xiii Abbreviations... xiv Acknowledgements... xvii Chapter 1: Introduction... 1 Chapter 2: Literature Review EBV EBV infection and associated diseases... 5 EBV lifecycle... 5 Acute infection Persistent infection and latency Latency 0/I Latency II Latency III Lytic replication Lytic gene expression Lytic genome replication Egress EBV-associated disease Epithelial associated disease Epithelial tumors OHL Infection of B cells Entry Viral protein expression in B cells Infection of epithelial cells Entry vi

7 Viral protein expression in epithelial cells Immune response to EBV infection Experimental systems to study EBV biology Primary B cells and B cell lines Epithelial cells growing in monolayer culture Primary epithelial raft cultures Chapter 3: Materials and Methods Cell lines Primary cells Primary epithelial raft cultures Virus production and infection Immunostaining EBER ISH Isolation and quantification of encapsidated EBV genomes Isolation and quantification of total EBV genomes Termini analysis TEM Isolation and infection of primary B cells Immortalization of B cells Evaluation of anti-viral compounds against EBV in raft cultures Multiplex human cytokine array Statistical analysis Chapter 4: EBV Infection of Stratified Epithelium Results in Spontaneous Virus Production in the Absence of a Detectable Latent Phase Introduction Results EBV infection of raft tissue results in discrete foci which expand over time 89 vii

8 EBV-infected epithelial cells concurrently express gene products from both the latent and lytic cycle EBV does not affect cellular proliferation or early differentiation but does induce cytopathology Infection of raft cultures results in high levels of virus production The anti-viral drug acyclovir inhibits productive replication and dissemination Discussion Chapter 5: EBV Transmission: Using Raft Cultures to Model the Oral Epithelium Introduction Results Replication of ΔTK virus is attenuated in stratified epithelium Raft cultures can be infected at the apical surface but not the basal lateral surface Raft-derived virus displays enhanced tropism for primary B cells CFV produced in raft tissue exhibits decreased tropism for stratified epithelium relative to B-cell derived virus Homogenate isolated from infected raft cultures mildly inhibits virus production Virus-producing epithelial cells can transmit infection between raft cultures Discussion Chapter 6: General Discussion and Conclusions Infection of stratified epithelium results in spontaneous productive replication Absence of a detectable latent phase in stratified epithelium viii

9 6.3 Dissemination throughout the stratified epithelium is dependent on lytic, not latent, replication Exclusion of infection from basal cells The dual-tropism of EBV Response of epithelial cells to infection Primary infection Virus egress and shedding Implications for vaccine design and prevention Bibliography Appendix A: The Effects of ART Compounds on EBV Replication in Stratified Epithelium Appendix B: Expression of p16, p21, and p53 in EBV-Infected Stratified Epithelium Appendix C: The Cytokine Expression Profile Induced by EBV- Infection in Stratified Epithelium ix

10 List of Figures Figure 2.1. Morphology of the EBV virion...7 Figure 2.2. Expression profiles of the various forms of EBV latency...15 Figure 2.3. Replication of the EBV genome...28 Figure 2.4. EBV attachment and entry...41 Figure 2.5. Diagram of a raft culture...63 Figure 3.1. Diagram demonstrating the production and infection of primary raft cultures...72 Figure 4.1. EBV infection results in discrete foci of lytically replicating cells which expand over time...92 Figure 4.2. EBV infection does not affect cellular proliferation or differentiation, but induces cytopathology...97 Figure 4.3. Infection results in viral genome amplification and the production of viral particles Figure 4.4. Viral replication and spread are inhibited by treatment with acyclovir Figure 5.1. Replication of ΔTK virus was attenuated in stratified epithelium Figure 5.2. EBV from virus-producing B cells initiated infection at the apical but not the basal surface of epithelial tissue Figure 5.3. Epithelial-derived virus displays enhanced tropism for primary B cells x

11 Figure 5.4. Epithelial-derived CFV exhibits decreased tropism for stratified epithelium relative to B-cell derived virus Figure 5.5. Homogenate from infected raft cultures mildly inhibits virus replication in raft cultures Figure 5.6. Virus-producing epithelial cells can transmit infection between raft cultures Figure 6.1. Diagram summarizing the general EBV expression profile in different cell types Figure 6.2. Models for EBV infection and spread in stratified epithelium Figure 6.3. Model for EBV infection of stratified epithelium Figure Appendix A.1. The properties and chemical structure of Kaletra and amprenavir Figure Appendix A.2. Kaletra reduces the level of EBV replication in raft cultures Figure Appendix A.3. The effects of Kaletra and amprenavir on the expression of cellular and viral proteins Figure Appendix A.4. Treatment with Kaletra reduces the level of LMP1 expression in raft cultures Figure Appendix B.1. Expression of p16, p21, and p53 in EBV-infected raft cultures Figure Appendix C.1. Schematic of the VeriPlex TM Human Cytokine 16-Plex ELISA Kit Figure Appendix C.2. Cytokine production by EBV-infected raft cultures xi

12 Figure Appendix C.3. Analysis of the cytokines present in CFV preparations and raft cultures infected with the CFV preparations xii

13 List of Tables Table 2.1. List of EBV proteins and transcripts...17 Table 2.2. EBV-associated diseases...30 Table 4.1. Percentage of Zta-positive cells expressing each of the indicated viral proteins...94 Table 4.2. Percentage of Zta-positive cells expressing each of the indicated cellular proteins...98 Table 4.3. Ability of raft-derived virus to immortalize primary B cells in vitro xiii

14 Abbreviations aa: Ab: ART: BAC: BART: Bim: BL: Blimp1: bp: BrdU: CD: CDK: CFV: CMV: DC: DDR: DMEM: DNA: ds: EA-D: EBER: EBNA: EBV: EGF: ELISA: FBS: GC: GFP: gp/g: amino acid antibody antiretroviral therapy bacterial artificial chromosome BamHI-A fragment rightward transcript bcl2-interacting mediator Burkitt lymphoma B lymphocyte-induced maturation protein-1 base pair 5-bromo-2'-deoxyuridine cluster of differentiation cyclin-dependent kinase cell-free virus cytomegalovirus dendritic cell DNA damage response Dulbecco's-modified Eagle medium deoxyribonucleic acid double stranded early antigen-diffuse Epstein-Barr virus-encoded RNA Epstein-Barr virus nuclear antigen Epstein-Barr virus epithelial growth factor enzyme-linked immunosorbent assay fetal bovine serum Gastric carcinoma green fluorescent protein glycoprotein xiv

15 H&E: hematoxylin and eosin HIV: human immunodeficiency virus HL: Hodgkin's lymphoma HLA: human leukocyte antigen HPV: human papillomavirus HSV: herpes simplex virus htert: human telomerase reverse transcriptase IF: immunofluorescence IFN: interferon Ig: immunoglobulin IHC: immunohistochemistry IL: interleukin IM: infectious mononucleosis ISH: in situ hybridization K: keratin KSHV: Kaposi's sarcoma-associated herpesvirus LC: Langerhans cell LCL: lymphoblastoid cell line LMP: latent membrane protein LOH: loss of heterozygosity LP: leader protein MFI: mean fluorescence intensity MOI: multiplicity of infection nm: nanometer NPC: Nasopharyngeal carcinoma OHL: oral hairy leukoplakia ORF: open reading frame ori: origin of replication PBS: phosphate buffered saline PCNA: proliferating cell nuclear antigen PGEC: primary gingival epithelial cells xv

16 PI: post infection PK: protein kinase PTEC: primary tonsillar epithelial cells PTLD: posttransplant lymphoproliferative disease qpcr: quantitative polymerase chain reaction qrt-pcr: quantitative reverse-transcription polymerase chain reaction rebv: recombinant EBV RFP: red fluorescent protein RIG-I: retinoic acid inducible gene I RNA: ribonucleic acid RR: ribonucleotide reductase SD: standard deviation SDS: sodium dodecyl sulphate Skp2: S-phase kinase-associated protein 2 SV40: simian virus 40 TEM: transmission electron microscopy TGF: transforming growth factor TK: thymidine kinase TNF: tumor necrosis factor TPA: 12-O-tetradecanoylphorbaol-13-acetate TR: terminal repeat UPR: unfolded protein response VLP: virus-like particle VCA: viral capsid antigen VZV: varicella zoster virus WT: wild type XBP-1: X-box binding protein 1 xvi

17 Acknowledgements When I started in this laboratory, I was originally on a project involving EBNA3A and latency. Over the course of the my first year in the laboratory, my focus shifted to the raft culture project, which was originally just a side project. I would like to thank Dr. Clare Sample for giving me the support and freedom to pursue this fun and exciting project even though it was completely different than all the other projects in the laboratory at the time. I have learned a lot about science and research in this laboratory. I would also like to thank each and every member of my committee for their guidance and council during this rather long journey. I have been lucky to have the help of many collogues and collaborators on this project, including Craig Meyers, Lynn Budgeon, Catherine Abendroth, Nate Sheaffer, Roland Myers, and many others. I would like to thank all the members of the department of Microbiology and Immunology for their assistance, whether great or small. Graduate school, and research in general, can be difficult at time, and I would be remise if I did not thank the may friends I have made along the way that have helped to see me through these tough times. As they say, laughter is the best medicine. Throughout my life, my family has been my biggest supporter, always encouraging me to keep trying my best. Thank you for all you have done for me. And finally, I have been so blessed to have had the most amazing daughter supporting me, being patient with me, and constantly reminding me of what matters most in life though out my studies. Thanks Annie. xvii

18 Chapter 1: Introduction The herpesvirus Epstein-Barr virus (EBV) has a very high prevalence in the human population. While EBV is the etiological agent of infectious mononucleosis (IM), the EBV-associated tumors which develop years after primary infection contribute most significantly to the disease burden associated with this virus. EBV is transmitted between hosts through saliva and eventually gains access to the B-cell compartment where it will establish a persistent latent infection. The oral mucosal epithelium is an ideal candidate site for primary infection and production of viral particles for transmission between hosts. In support of this model, immunosuppressed individuals frequently develop oral hairy leukoplakia (OHL), a benign lesion of the lingual epithelium characterized by productive EBV replication. Detection of EBV in the epithelium of healthy individuals has been more difficult. In a retrospective study EBV was detected in only 3 of 517 oral epithelium samples 65. Similar to OHL, EBV sustains lytic replication rather than latency in these samples. This pattern of viral gene expression differs from the EBV-associated epithelial malignancies where predominately the latency-associated proteins are expressed. Whether EBV can establish latency in epithelial cells in vivo, or if not, what cellular changes are required to allow latency in tumor cells is not known, although recent publications suggest that inactivation of the p53 and cyclin D pathways are required 181,235,313. Given that the two most common EBV-associated malignancies are both of 1

19 epithelial origin, understanding the dynamics of infection in this tissue is of upmost importance. While numerous models are available to study EBV latency in B cells and have contributed greatly to our understanding of the role of these cells in the viral lifecycle, models for EBV infection of epithelial cells in vitro are lacking, in part because epithelial cells are poorly infected in vitro. The majority of studies with epithelial cells have relied on monolayer culture, where the cells display basallike characteristics. Under these conditions, EBV generally establishes a nonpersistent latent infection in which the viral genome fails to replicate and the infected cells senesce. To date, no in vitro model recapitulates the productive replication observed in the stratified epithelium of the oral cavity, leaving many gaps in our understanding of infection of this tissue. In vivo epithelial cells grow as a stratified tissue with the suprabasal layers consisting of terminally differentiating cells. This process can be recapitulated in vitro in organotypic 'raft' cultures grown from primary oral mucosal epithelial cells. The goal of the research presented in this dissertation is to use the raft culture system to study EBV infection of epithelial tissue. We hypothesize EBV infects stratified epithelium and undergoes productive replication resulting in genome amplification and dissemination of the virus. The following specific aims will be used to test this hypothesis. AIM 1: Determine the type(s) and kinetics of EBV infection in stratified epithelium. 2

20 Rationale: Previous data from OHL lesions and a limited number of normal patient samples suggest EBV establishes a productive infection in stratified epithelium, yet no in vitro experiments have been able to corroborated if EBV will undergo spontaneous lytic replication in epithelial tissue. For this aim, we will determine whether EBV infects normal stratified epithelial tissue, and if so, which viral protein are expressed in infected cells. We will examine whether infection results in virus production, and quantify the kinetics of viral replication. We will also establish whether EBV can spread between epithelial cells, as well as the capacity of this virus to persist in stratified epithelium. Finally, we will investigate the effects of viral infection on the epithelial cell. Findings: We found that infection of stratified epithelium resulted in lytic replication with high levels of virus production. These viral particles were able to spread to neighboring epithelial cells, allowing the virus to disseminate throughout the superficial layers of the tissue. We could not identify a persistent latent infection in the tissue. Infected tissue displayed cytopathology typical of active viral replication with no semblance to those cellular changes observed in the EBV-associated malignancies. AIM 2: Define the modes EBV can use to initiate infection of stratified epithelium. Rationale: Using the raft culture system, we would like to model infection in the oral cavity as closely as possible. It is believed that virions within saliva are responsible for initial infection, though it has also been postulated that the virus 3

21 may enter a new host through EBV-infected epithelial cells. Therefore, we will determine which viral sources are capable of initiating infection in stratified epithelium and where this infection can be initiated within the tissue. We will also assess the tropism of virus produced by stratified epithelium in comparison to virus produced by B cells. Findings: We determined that EBV initiates infection in the superficial layers of stratified epithelium while being excluded from the basal cells. Infection was initiated most efficiently by virus-producing B cells or virus-producing epithelial cells, and less efficiently by cell-free virus (CFV). The virus produced by the epithelial tissue displayed a strong tropism for B cells, but a relatively weak tropism for epithelial cells. These data demonstrate that virus produced by the mucosal epithelium can transmit infection to a new host, but the resulting infection would likely be limited to a few discrete foci, as is observed in healthy individuals. Overall significance By addressing each of the questions listed above, we hope to increase our understanding of where primary infection occurs in a naive host (a requirement for adequate vaccine design), and where viral replication occurs for transmission. Furthermore, we hope to gain insight as to why, in a small fraction of individuals, a relatively benign viral infection progresses to form a malignant carcinoma. 4

22 Chapter 2: Literature Review 2.1 EBV EBV is a ubiquitous human herpesvirus which persistently infects over 90% of the human population. The distinct virion morphology observed by transmission electron microscopy (TEM) categorizes this virus in the Herpesviridae family. The cellular tropism of EBV further classifies it in the genus Lymphocryptovirus in the Gammaherpesvirinae subfamily. The virion is composed of linear double stranded (ds) DNA ~172,000 base pairs (bp) in length which encodes up to 84 open reading frames (ORF) and numerous non-coding RNAs 45. The DNA is encased in a T=16 icosahedral capsid shell comprised of 6 capsid proteins 76,105. This capsid shell is surrounded by tegument proteins and an envelope (Figure 2.1) EBV infection and associated diseases EBV lifecycle As with all members of the Herpesviridae family, EBV has a biphasic life cycle consisting of both lytic and latent phases. For the purposes of this dissertation, latency is defined as viral persistence in the absence of virion production. Viral persistence is accomplished by vertical transmission of the viral genome to daughter cells following viral genome replication in conjunction with cellular division as will be discussed in more detail in subsequent sections. Lytic replication is defined as the synthesis of viral proteins and genomes for the 5

23 Figure 2.1. Morphology of the EBV virion. Illustration showing the general morphology of an EBV virion. An electron micrograph is also shown with the appropriate labels for comparison purposes. The virion contains linear dsdna encased in a capsid shell. The capsid is surrounded by the tegument proteins and an envelope which contains numerous viral glycoproteins. The black line indicates 100 nanometers (nm). 6

24 Figure 2.1 7

25 production of new viral particles. Lytic replication can be further subdivided as productive (replication resulting in the release of viral particles) or abortive (lytic protein expression, occasionally with genome amplification, which does not result in virion production). An additional distinction between latent and lytic EBV infection is that for the virus to persist, the cell must survive latency whereas lytic replication culminates in cell death. EBV is transmitted between human hosts in saliva. From the oral cavity, EBV gains access to the B-cell compartment where it infects B cells, and persists in a latent state for the life of the host. Currently, we do not know the primary site of infection nor do we understand how EBV is transmitted through the oral mucosa to the B cell compartment. These questions will be a focus of this dissertation. Current models propose EBV initially infects the epithelial tissue in the oral cavity, and following replication in this tissue, gains access to the B cell compartment. While EBV persists in B cells in vivo in a restricted state of latency (latency 0/I), occasionally the virus reactivates in a subset of B cells, a process believed to be linked to B cell activation and plasma cell differentiation, and produces infectious viral particles 11,161,297. Another poorly defined step in virus transmission is how EBV gains access to the oral cavity for secretion in saliva. Within the natural host, the oral cavity and lymphatic system are physically separated by the epithelial tissue and basement membrane, with B cells rarely observed crossing the basement membrane and migrating into the epithelium 316. Multiple theories have been proposed to address how EBV may transverse this 8

26 barrier during both entry and egress. Microabrasions or related trauma which result in bleeding in the oral cavity would give B cells access to the oral epithelium while also providing the virus access to the circulatory system. Although EBV-infected B cells are rare (1-50 per 1 x 10 6 peripheral B cells 152 ) and the frequency of productively replicating B cells is significantly lower, contact with epithelial cells can induce productive replication in B cells 221, increasing the likelihood of infection should B cells infiltrate the oral cavity. Alternatively, the virus may transit the epithelial tissue by transcytosis (a process predominately observed in epithelial cells in which macromolecules are transported across the interior of a cell within a vesicle, e.g. transcytosis is used to transport immunoglobulin (Ig) A across mucosal epithelium for secretion into bodily fluids), which would not require infection of the epithelium 318. An additional model recently proposed by two independent groups suggests that the tissue resident dendritic cells (DC), namely Langerhans cells (LC), are latently infected with EBV and the virus reactivates as these cell terminally differentiate resulting in transmission of EBV to neighboring cells 316,330. In support of this model, EBV latently infects pre-lc in the peripheral blood while mature LC in the oral mucosa demonstrate both latent and lytic protein expression profiles 330. Chemical treatment of LC (12-O-tetradecanoylphorbaol-13-acetate (TPA) and sodium butyrate) isolated from the oral mucosa induces lytic replication 330. Furthermore, EBV-infected macrophages and LC can be identified at sites of active viral replication in the oral mucosa 316. When infected macrophages and LC are added to ex vivo tongue and buccal tissue explants, these cells migrate from the 9

27 lamina propria (the dermis) to the mucosal epithelium and initiate infection of the epithelial cells, while B cells do not 316. These mechanisms are not mutually exclusive, and in the human host, EBV likely uses multiple routes to transit through the oral epithelium. Regardless of the mechanism, EBV is very efficient at gaining access to the oral cavity with high levels of virus actively shed into the saliva (up to 3 x x genomes per day) in 30-90% of healthy individuals at any given time 94,123,175. This level of shedding is considered too high to be completely accounted for by viral replication in the B cells located in the tonsils alone, but instead requires replication in epithelial cells, likely at 1-3 foci 94. Mathematical modeling suggests that viral replication in only a small number of epithelial cells could evade detection by the adaptive immune response while continuously shedding relatively high levels of virus into the oral cavity 94,102. Moreover, based on the ratio of glycoproteins in the virion envelope, the virus secreted into saliva is thought to be produced predominately by epithelial cells 134. Acute infection The majority of individuals acquire EBV in the first decade of life, without infection causing any recognizable symptoms. When primary infection occurs post adolescence, more common in developed countries, infection can result in the development of IM. IM presents as a self-limiting lymphoproliferative disease accompanied by fever, sore throat, fatigue, splenomegaly, and adenopathy approximately 6 weeks after initial exposure to the virus 109. At the time of clinical 10

28 manifestations, between 0.1-1% of the peripheral B cells are infected with EBV 154, these cells displaying a latency III expression profile 305. The symptoms of IM are thought to result from the robust cluster of differentiation (CD) 8+ T cell response directed primarily towards EBV-infected B cells 108,238. Over time, the virus transitions to the more restricted latency 0/I expression profile, which is poorly detected by the immune system, resulting in disease regression but not viral clearance. Both virus and EBV-infected epithelial cells can be isolated from the saliva of IM patients 167,285, but multiple attempts to locate infected epithelial tissue within the tonsils of IM patients have been unsuccessful 4,147,228,230. This inability to detect EBV in the tonsillar epithelial cells of IM patients has lead some investigators to question the role of epithelial cells within the EBV lifecycle, though alternate oral epithelial tissues in IM patients have not been thoroughly investigated. Furthermore, IM presents well after primary infection, at which point infection within the epithelium might be resolved. Although the host mounts a strong innate and adaptive immune response following EBV infection, the virus is able to reactivate and release progeny virions throughout the life of the host, demonstrating that while viral infection and replication are controlled, they are not ablated. Historically it was thought that infection with EBV resulted in neutralizing immunity, but analysis of clinical isolates reveals individuals are repeatedly exposed to and infected with EBV throughout their lifetime 58,332. The dominate strain of EBV in an individual varies over time, with some individuals exhibiting infection with multiple strains simultaneously 58,

29 Viral infections are generally controlled by the use of prophylactic therapy, such as vaccination, or administration of anti-viral compounds post-infection. There are currently no effective vaccines or therapeutics for EBV infection. The majority of vaccination strategies target the viral glycoprotein gp350 (which is required for entry into B cells) alone or in combination with a subset of latencyassociated proteins 53,55,88,213,214,251,264,290. In the only phase 2 trial for an EBV vaccine, adults receiving soluble gp350 showed a 78% reduction in the development of IM compared to the placebo control group, but there was no difference in the actual rate of infection between these two groups 290. Therefore, while these vaccines can reduce the symptoms associated with acute infection, they do not prevent establishment of a persistent latent infection, and may not protect against the formation of EBV-associated tumors later in life. To induce neutralizing immunity, which is especially important for a virus which establishes a persistent latent infection, viral infection needs to be inhibited at the site of primary infection. Therefore, it is of upmost importance to identify this site in the host. Furthermore, as will be discussed in more detail later, while gp350 may be a good target to prevent infection of B cells, it may not be an adequate target to block infection of epithelial cells. Productive EBV replication can be inhibited by the guanine nucleoside analogues acyclovir and ganciclovir. These anti-viral compounds are administered in a pro-drug form, requiring phosphorylation by the virus encoded protein kinase (PK), for conversion to a biologically active nucleotide 5,77,199. This kinase is an early protein, only expressed during lytic replication. Furthermore, 12

30 acyclovir and ganciclovir triphosphate are better substrates for the viral DNA polymerase, BALF5, than the cellular DNA polymerase, displaying enhanced inhibition of viral genome synthesis over cellular DNA replication 44,77. Due to these two factors, these anti-viral drugs are highly efficient at selectively inhibiting lytic viral replication, but have little to no effect on latent infection. Additional nucleoside analogues, such as 5-bromo-2'-deoxyuridine (BrdU) which is phosphorylated by the viral thymidine kinase (TK), can inhibit the viral DNA polymerase 90. Given that most EBV-associated diseases are associated with latent EBV infection, administration of these anti-viral compounds can reduce viral burden, but do not result in the elimination of latently infected cells or disease regression. Persistent infection and latency Once EBV infects a cell, it can establish any of four different transcription programs: latency 0/I, latency II, latency III, or lytic replication with each transcription program characterized by a unique protein expression profile (Figure 2.2) 150,256,342. In all in vitro systems studied thus far, EBV initially establishes a latent infection, and as such, EBV latency and persistence are the most well studied aspects of this virus. A defining characteristic of EBV is its ability to immortalize primary B cells through expression of the latencyassociated proteins following infection in vitro. The repertoire of latencyassociated transcripts and their known function in B cells and epithelial cells are 13

31 Figure 2.2. Expression profiles of the various forms of EBV latency. The expression pattern of EBV-encoded proteins and non-coding RNAs (EBER and BART) during each of the indicated forms of latency. Following infection of a B cell, EBV will express all the latency-associated proteins and RNAs, termed latency III, leading to B cell proliferation and differentiation. In vivo, this protein expression profile is down-regulated until only the non-coding RNAs are constitutively expressed with transient expression of EBNA1 preceding cellular division, termed latency 0/I. The arrows indicate the order of down-regulation from latency III to latency 0/I. 14

32 Figure 2.2 EBER BART EBNA1 LMP1 LMP2A LMP2B EBNALP EBNA2 EBNA3A EBNA3B EBNA3C EBER BART EBNA1 LMP1 LMP2A LMP2B latency III latency II EBER BART EBNA1 EBER BART EBNA1(intermittent) latency I latency 0/I 15

33 described below and summarized in Table 2.1, with special emphasis placed on those proteins and transcripts examined in my research. Latency 0/I Within the host, EBV has evolved to evade immune surveillance and persist in the memory B cell pool by maintaining a restricted latency (termed latency 0/I) in which a limited number of transcripts are expressed. These include the EBV-encoded RNA (EBER) 1 and 2 transcripts, which are the most abundant transcript in latently infected cells and are expressed in all EBV-associated tumors examined thus far. While the EBERs can elicit cytokine production through retinoic acid inducible gene I (RIG I), activating the type I interferon (IFN) pathway and inducing interleukin (IL) -10 production in B cells, these RNAs also protect cells from IFN-α induced apoptosis 220,260,261. It is unclear if the EBERs are expressed in productively infected epithelial cells in vivo 78,234. EBV encodes a second set of noncoding RNAs, the BamHI-A fragment rightward transcripts (BARTs) that are expressed during all forms of latency and in all known EBVassociated pathologies. RNA expressed from the BART region of the genome is processed into micro RNAs, displaying anti-apoptotic properties which are predicted to contribute to the transformation of infected epithelial cells 20,86,191,192,248. The only protein expressed during latency I is EBV nuclear antigen 1 (EBNA1) which is required for efficient B cell immortalization 118,166. EBV persists in latently infected cells as an episome in the nuclear compartment. EBNA1 binds to the latent origin of replication, orip, on the EBV episome and 16

34 Table 2.1 List of EBV proteins and transcripts Protein or transcript name (alternate name) Brief description of protein function EBER BART EBNA1 EBNA2 EBNALP EBNA 3A, 3B, 3C LMP1 LMP2A, 2B Zta Rta EA-D BHRF1 BALF5 BGLF4 (PK) BXLF1 (TK) BCRF1 gp350 gp42 gh gl gb (gp110) BMRF2 VCA Latency-associated noncoding RNA expressed by all latently infected cells, possibly involved in immune evasion encodes micrornas required for replication and segregation of viral genomes during latency transcription factor cooperates with EBNA2 to activate promoters transcription factors oncoprotein, acts as constitutively active CD40 molecule blocks tyrosine kinase signaling in B cells, thought to prevent reactivation in latently infected cells Lytic transcription factor, responsible for activation of lytic replication transcription factor which triggers lytic replication in concert with Zta polymerase processivity factor for viral DNA polymerase, replaces PCNA anti-apoptotic protein, bcl-2 homologue virus encoded DNA polymerase protein kinase, activates acyclovir thymidine kinase, required for DNA synthesis on nonreplicating cells vil-10, anti-inflammatory binds to receptor, CD21, on B cells bind to co-receptor, HLA class II, on B cells part of fusion complex chaperon for gh type III viral fusion protein glycoprotein which possibly binds to receptor on epithelial cells viral capsid antigen 17

35 initiates replication of the viral genome using cellular replication machinery in coordination with replication of the cellular genome 283,348. After replication, EBNA1 binds to the EBV episome and tethers it to the host chromosomes to ensure proper segregation of the viral genomes during cellular division 186,187,239. In memory B cells not undergoing active cellular division, the noncoding RNAs are the only EBV transcripts expressed (termed latency 0) 110. Prior to cellular division, EBNA1 is transiently expressed to allow for maintenance of the genome. As will be discussed in more detail in later sections, data have suggested that infected epithelial cells may not express sufficient levels of EBNA1 for genome replication and maintenance, resulting in loss of the viral genome 272. EBNA1 can be expressed from four different promoters, with the promoter usage corresponding to the viral transcription program 353. Immediately following infection and during latency III EBNA1 is expressed, along with the other EBNA proteins, from the W then C promoters 342. During latency I and II, EBNA1 is expressed from Qp 262,266. The role of EBNA1 during lytic replication is not well defined, but EBNA1 can be expressed from a fourth lytic specific promoter, Fp 16,164,265,353. Recently, Sivachandran et al. demonstrated that while expression of EBNA1 suppresses lytic reactivation, once the lytic cycle is induced, EBNA1 can contribute to genome replication and gene expression by disrupting promyelocytic leukemia nuclear bodies 284. Latency II The protein expression profile during latency II is broadened to include the 18

36 latent membrane proteins LMP1, LMP2A and LMP2B. Although B cells with a latency II expression profile are not routinely isolated from healthy patients, this form of latency is commonly detected in EBV-associated tumors. LMP1 is expressed during latency II and III. Despite the fact that LMP1 can be expressed during lytic replication from a second ORF 62,115, the biological significance of LMP1 expression during lytic replication is under debate. One group reports expression of LMP1 enhances virion release 1 while other reports claims LMP1 expression is expendable or even incompatible with lytic reactivation 73,185. LMP1 is required for B cell transformation 148, mimicking a constitutively active CD40 molecule 101,130,131,218, which in turn activates the NF-ƙB, JNK/p38, phosphoinositide 3 (PI3)-kinase, and ERK-MPK pathways (as reviewed in 215 ). In B cells, activation of the NF-ƙB pathway induces cellular differentiation and upregulation of bcl-2, leading to resistance to apoptosis 104. In epithelial cell lines, on the other hand, expression of LMP1 alone is insufficient to induce upregulation of bcl-2 279,280. LMP1 is expressed in OHL, in a subset of nasopharyngeal carcinoma (NPC) tumors, and a minority of gastric carcinoma (GC) tumors 57,234,355 ; these epithelial pathologies will be discussed in greater detail in later sections. LMP2A is expressed in latency II and III and is often expressed in NPC and in approximately one half of GC 107,355. LMP2B, expressed during latency II and II, modulates LMP2A activity 259. Exogenous expression of LMP1, LMP2A, and LMP2B in epithelial cell lines can inhibit epithelial cell differentiation and increased cellular replication if the cells are induced to differentiate 40,59,

37 Latency III During latency III, also referred to as the growth program, EBV expresses the full repertoire of latency-associated genes. In addition to the transcripts expressed during latency I and II, this repertoire includes EBNA leader protein (LP), EBNA2, EBNA3A, EBNA3B, and EBNA3C. EBNA2 is a transcription factor which activates both viral and cellular genes required for B cell immortalization 21,33,34. Although often considered a strictly latent protein, EBNA2 is detected in OHL lesions. EBNALP cooperates with EBNA2 in promoter activation 100,236 and is also required for efficient B cell immortalization 190. The EBNA3 proteins are transcriptional regulators 193, which exhibit sequence homology, though only EBNA3A and 3C are required for B cell immortalization 306,307. While the essential role of each of the individual EBNA3 proteins is currently being investigated, EBNA3C, and to a lesser extent EBNA3A, inhibit p14 ARF, p16 INK4A, and bcl2- interacting mediator (Bim) allowing cell cycle progression 194,287. Lytic replication EBV is relatively unique among viruses in that in all in vitro systems examined thus far, EBV initially establishes a latent infection which, under specific circumstances, can persist. For lytic replication to occur, the latent genome must be reactivated; a process which appears to be directly linked to the differentiation state of the cell likely due to the differentiation dependent expression of transcription factors such as X-box binding protein 1 (XBP-1) and B lymphocyte-induced maturation protein-1 (Blimp1), which can activate the Zta 20

38 and Rta promoters 11,18,27,60,65,161,170,234,296,297,351. In any given population of latently infected cells, a small fraction of cells will spontaneously reactive, the rate varying between cell types, viral strains, and even between individual clones 311. This spontaneous reactivation is generally abortive 7. Productive viral replication can be induced in B cells by stimulating the B-cell receptor, treatment with transforming growth factor-beta (TGF-β), hypoxia, or chemical treatment such as TPA and sodium butyrate 47,56,122,133,241,299. When productive replication is induced, only a subpopulation of the cells that express Zta will go on to express the full repertoire of lytic proteins. Therefore, it is important to identify the expression of multiple lytic cycle proteins to ensure productive lytic replication. Currently, there are no in vitro systems for EBV which result in robust viral production. Few experiments have been performed using virus stocks derived from epithelial cells. The majority of these experiments have been conducted simply to confirm production of infectious viral particles in cells undergoing spontaneous reactivation 311 or in cells induced to differentiate in monolayer culture 27,170,317.The only noted exception is the AGS cell line, a gastric carcinoma cell line which is of a slightly differentiated phenotype. This cell line is semipermissive to EBV infection and productive replication. Following infection of AGS cells, approximately 30% of the infected cells will spontaneously express Zta, though only 4% of the cell population will go on to express the late viral proteins 272. These cells can be induced to undergo productive viral replication for the isolation of viral particles for experimental analysis using TPA and sodium butyrate 12. An additional system for the production of EBV is HEK 293 cells 21

39 carrying a recombinant EBV (rebv) bacterial artificial chromosome (BAC), which can be induced to produce viral titers similar to those isolated from induced B cells 46,273. In many reports, virus produced from HEK 293 cells is considered epithelial derived 275,311. Lytic gene expression The general scheme of lytic replication for EBV is similar to that observed for other herpesviruses. The lytic viral proteins are expressed in three successive phases; immediate-early, early, and late. The immediate-early proteins, Zta and Rta, are transcription factors which activate their own promoters and the promoters of the early genes 36,37,257. Zta also binds to the lytic origins of replication, orilyt, to recruit the virally-encoded DNA polymerase complex and additional cellular replication factors for genome replication 267. The expression of Zta alone is sufficient to induce lytic replication, though in B cells, the expression of both Zta and Rta are required for maximal expression of the early genes 99. Immediately following infection of B-cells and epithelial cells, Zta is transiently expressed, at least in part from mrnas that are transduced into the cell by the virion and virus-like particles (VLP) 136,145. This early expression of Zta initiates the pre-latent phase, which is thought to enhance expression of proteins associated with cell cycle progression and interfere with the expression of genes influencing growth arrest 136,145,195. Zta is unique among transcription factors in that it was the first transcription factor identified which preferentially binds methylated DNA in a promoter-dependent manner 10. This attribute is thought to 22

40 be vital for reactivation given that during latency the EBV genome is heavily methylated in many cell types. Conversely, Rta preferentially activate unmethylated promoters, with this activity most pronounced in a telomeraseimmortalized epithelial cell line in which the promoters of the lytic genes remained predominately unmethylated 340. This suggests that while EBV has evolved to reactivate and express lytic proteins from a heavily methylated genome, it has retained the capacity to express these genes from an unmethylated genome as well. The early proteins include those proteins involved in viral genome replication and cell survival. The viral DNA polymerase is encoded by the BALF5 gene. Early antigen-diffuse (EA-D), encoded by the BMRF1 gene, acts as the viral DNA polymerase processivity factor, replacing proliferating cell nuclear antigen (PCNA) as the DNA clamp, and is essential for lytic replication 314,315. EBV produces a TK protein, encoded by the BXLF1 gene, which converts deoxythymidine to deoxythymidine monophosphate 41,176. Deoxythymidine monophosphate is further phosphorylated by cellular enzymes to form deoxythymidine triphosphate, which is a substrate for DNA synthesis. The expression of the viral TK protein is believed to be important for continued DNA synthesis in terminally differentiated cells, which no longer express the cellular thymidine kinase required for DNA synthesis. Despite being highly conserved among herpesviruses, the EBV-encoded TK has not been shown to be essential for productive replication in any in vitro systems thus far 207. The BGLF4 gene encodes a PK, which can phosphorylate the guanine nucleoside analogue 23

41 antiviral compounds (acyclovir and ganciclovir) to produce the biologically active monophosphate forms as discussed earlier 199,289. The early protein BHRF1 is an anti-apoptotic bcl-2 homologue 246. BHRF1 is also expressed in B-cells upon initial infection, peaking at 24 hours post infection (PI) 3,104. BHRF1 expression has also been detected in NPC tumors 177. The proteins incorporated into the virion, i.e. the nucleocapsid, tegument, and envelope glycoproteins, comprise the late proteins. By definition, the late viral proteins require viral DNA synthesis for expression, but EBV can express some of the late proteins independent of genome replication 61. Glycoprotein B (gb) and gp350 are late proteins essential for infection of B cells, which will be described in more detail in the following sections. The fusion protein gb, expressed from the BALF4 ORF 198 is also required for proper virion maturation and egress 83,106. Interestingly, the level of gb incorporated into the virion can vary by viral strain, with the level of incorporation directly influencing infectivity and viral tropism 226. The viral capsid antigen (VCA) is a true late protein, only expressed after lytic replication of the viral genome. The BCRF1 gene of EBV encodes an IL-10 homologue with 70% amino acid (aa) sequence identity to cellular IL ,210. This vil-10 inhibits inflammatory cytokine production, including IFN-γ, by multiple cell types while promoting B cell proliferation and differentiation 42,80,112,113,258,322. Similar to some of the other lytic viral genes, BCRF1 is transiently expressed immediately following infection (6-9 hrs PI) 206,310, and this expression is essential for B cell immortalization 126,

42 Lytic genome replication Most DNA viruses rely on the cellular replication machinery for viral DNA synthesis and therefore preferentially infect actively replicating cells. Given that herpesviruses encode their own replication machinery, these viruses were postulated to replicate in resting or terminally differentiated cells. Indeed, when cells undergoing lytic replication were examined, many appeared to be arrested between the G 0 /G 1 phases of the cell cycle. Unfortunately, many of the inducing agents used to illicit lytic replication can have this effect on cells. Using a tetracycline-inducible Zta expression system, Kudoh et al. found that during reactivation in immortalized cell lines, EBV halts the cell cycle between G1 and S phase, expressing many S phase proteins while inhibiting host DNA replication EBV is thought to halt cell cycle progression through de-ubiquitination of PCNA 338. One hurdle for investigations into EBV lytic replication has been the propensity of EBV to establish a latent infection. Consequently, in many studies, the effects of lytic replication are deduced from an entire population of cells in which many of the cells had failed to achieve full lytic replication. Using live cell imaging of two tetracycline-inducible cell lines containing an EBV amplicon or rebv genome, Chiu et al. found that cells already undergoing mitosis at the time of induction continue through the cell cycle, only initiating viral DNA replication hrs after the completion of mitosis depending on the cell type 31. Furthermore, once a cell begins to productively replicate the viral genome, the cell no longer undergoes mitosis 31. These data suggest that lytic genome replication and cellular division are incompatible. Prior to viral genome 25

43 replication, the nuclear architecture begins to transform, with the cellular chromatin eventually migrating to the margins of the nuclei and the viral DNA and replication machinery forming replication factories, excluding the host encoded PCNA and histones H2B, H3.1, and H3.3 from the sites of active viral DNA synthesis 31. Lytic cycle induction activates the DNA damage response (DDR), and interestingly, this activation actually enhances viral genome replication and gene expression 95,159,171. Lytic replication of the EBV genome occurs at discrete sites in the nuclei of the host cell in two phases, starting with theta-form replication then transitioning to rolling circle replication (Figure 2.3) 31. Both phases rely on the viral DNA polymerase, BALF5, which is recruited to either of two origins of lytic replication, orilyt L and R, by the immediate-early protein Zta 98. During thetaform replication, the episome continues to be amplified as a circular genome for subsequently use as a template for further DNA synthesis or transcription. The genomes ultimately packaged into newly synthesized capsids are produced by rolling circle replication. The signal that causes the switch to from theta-form to rolling circle replication is unknown. During rolling circle replication, the genome is synthesized as a long concatemer which is cut into genome size units at the terminal repeats (TR) and packaged into capsids as a linear genome 96,97. Egress The capsid is assembled in the nucleus, and although the capsid is of icosahedral symmetry, one of the twelve pentameric vertices, termed the portal, 26

44 Figure 2.3. Replication of the EBV genome. The various forms of viral genome replication are indicated with the appropriate origins of replication and some of the necessary co-factors as discussed in the text. The size of the arrows indicate the relative frequency of each step observed in vitro. 1) Within the virion, the EBV genome exists as linear dsdna flanked on each end by multiple terminal repeats, each approximately 500 bp in length. 2) Upon entering the nucleus, the genome circularizes through homologous recombination of the terminal repeats. 3) In latently infected cells, the viral episome replicates in conjunction with cellular division using the cellular DNA polymerase and replication machinery, which is recruited to the latent origin of replication, orip, by EBNA1. 4) When the virus reactivates, the viral genome undergoes lytic replication using the virally encoded DNA polymerase, BALF5, which is recruited to either of the lytic origins of replication, orilyt, by Zta. Initially, the genome will replicate using theta-form replication, creating multiple episomes which can be used as templates for DNA synthesis or RNA production. 5) Eventually, the virus will transit to rolling circle replication in which the genome is synthesized as long a concatemer, which is cleaved into individual genome size units and packaged into the viral capsid. WHile the viral genome could theoretically undergo lytic replication immediately following infection, the virus is initially latent in all in vitro systems characterized thus far, requiring reactivation for productive replication to occur. 27

45 Figure 2.3 Latent replication (3) (1) TR (2) Circular genome in nucleus Reactivation (4) TR Linear genome in virion TR Lytic replication Theta-form replication Figure legend orip orilyt EBNA1 (5) Rolling circle replication Zta Host DNA polymerase BALF5 28

46 is unique 255. This portal is the site for genome packaging and later, during entry, the site for DNA expulsion. The capsid is too large (~100 nm) to exit the nucleus through a nuclear pore complex and instead must bud through the inner and outer nuclear membrane in a process of envelopment and de-envelopment 54,82. Quite commonly, the nuclear membrane in virus-producing cells becomes fragmented, with virions frequently detected in cells which no longer appear to be viable by TEM 54. In the cytoplasm, the naked capsid acquires the tegument proteins, viral RNAs, and eventually an envelope, though it is currently unknown if the plasma membrane or an internal membrane is the site of final envelopment 82. The mature enveloped virion is then released from the cell by budding or exocytosis through the plasma membrane. EBV-associated disease EBV is able to infect B cells and epithelial cells in vivo. Acute infection can result in IM, the symptoms relating to the strong immune response elicited towards the latently infected B cells. EBV is routinely identified in the neoplastic cells in epithelial carcinomas (GC and NPC) and B-cell lymphomas (Hodgkin's lymphoma, HL, and Burkitt lymphoma, BL) (Table 2.2). EBV is also associated with OHL, a benign lesion on the lateral tongue margins of immunocompromised individuals. Post-transplant lymphoproliferative disease (PTLD) is one of the most common malignancies following transplantation. PTLD frequently results from the uncontrolled growth of EBV-infected B cells displaying a latency III phenotype, a consequence of the severe immunosuppression in these patients. 29

47 Malignancy Table 2.2. EBV associated diseases Progenitor cell type EBV gene expression profile Annual frequency of EBV associated cases worldwide GC Epithelial Latency I or II 84, NPC Epithelial Latency I or II with low level spontaneous abortive lytic replication 78, HL B cell Latency II 28, BL B cell Latency I 6, PTLD B cell Latency III ND 30

48 PTLD is most common in patients with primary EBV infection; these patients often acquiring EBV from the graft itself. Epithelial associated disease Infection of epithelial cells can result in multiple pathologies including OHL, NPC, and GC; with GC and NPC being the two most common EBVassociated tumors worldwide 35. However, our understanding of EBV infection of the oral mucosa is still very limited due to the lack of an accurate model of EBV infection of differentiated epithelium. Furthermore, since there are no models for infection of stratified epithelium, the role EBV plays in these epithelial pathologies is still unresolved. Numerous groups have reported an association between EBV and cervical cancer 144,263, breast cancer 142,292, and gingivitis 323, though these associations are still controversial and as such will not be discussed further. Epithelial tumors NPC was the first epithelial tumor to be associated with EBV infection, with almost 100% of the nonkeratinizing subtype of NPC (the more common subtype) EBV-associated 153,231,343,357. These tumors are not common worldwide, but instead show a distinct geographic distribution with high incidence in southeast Asia, the Arctic, and Northern Africa 227,229. The reason for this geographic distribution is not known, but is thought to be related to the local diet, host genetic factors, and viral genetic variations. In the tumors, the neoplastic cells are poorly differentiated or undifferentiated. These tumors are further 31

49 characterized by a large T cell response and tumor infiltrate, resulting in the name lymphoepithelioma. All of the tumor cells exhibit a clonal EBV infection, as determined by termini analysis, while the infiltrating lymphocytes are not infected with EBV 244,249. Within the tumor cells, EBV expresses a latency I or latency II profile with a low level of spontaneous abortive lytic replication. More specifically, the tumor cells always express the EBER transcripts and EBNA1 while LMP1, LMP2A, LMP2B, Zta, BARF1, and BHRF1 are detected in only some tumors 17,32,89,107,177,216,233,249,271,350. Although NPC is associated with latent viral infection, both serum levels of Ig specific for EBV lytic cycle antigens and cell-free, DNaseresistant viral genomes increase in conjunction with the development of NPC 25,26,183. In fact, this correlation is so strong that the presence of high titers of antibodies (Abs) directed towards lytic cycle antigens and an increase in the number of cell-free viral genomes in the blood are used as a clinical screen for NPC in endemic areas 182. Therefore, while the tumor is latently infected with EBV, high levels of lytic viral replication does appear to be a predisposing factor for the development of NPC. More recently, EBV infection has been associated with GC, the fourth most common cancer and the second most common cause of cancer-related deaths worldwide 245,281. GC include any cancer occurring in the stomach. There are no specific symptoms associated with early disease and as such patients often present with advanced disease resulting in an overall poor prognosis for GC patients. Like many of the other EBV-associated cancers, GC shows a geographic distribution, with incidence rates highest in Asia and South America 32

50 245. GC also shows a gender bias, with rates about twice as high in males as in females 245. In addition to the lymphoepithelioma-like GC, a rare form of GC that histologically resembles nonkeratinizing NPC and is always EBV-associated, the poorly and moderately differentiated GC tumors can also be EBV-associated. Overall, approximately 10% of all GC cases are EBV-associated 282. Within these tumors, the infected cells express a more restricted latency profile than that observed in NPC, with all tumor cells expressing the EBER transcripts, EBNA1 initiated from Qp, and the BARTs 124,232,355. A subset of tumors also express LMP2A and BARF1 while very few tumors express LMP1 or Zta 232,271,355. Similar to NPC, the role of EBV in tumorigenesis is unknown. To understand the role EBV plays in the development of epithelial tumors, it is important to understand precisely when infection occurs. Given that all tumor cells carry clonal copies of the EBV genome in NPC and GC, infection must occur before the expansion of malignant cells. To further refine the timing of infection, Chan et al. used microdissection to examine normal epithelium, dysplastic lesions, and carcinoma tissue from the nasopharynx for the presence of EBV and the loss of heterozygosity (LOH) at specific genetic loci commonly inactivated or deleted in NPC 23,24. Genetic aberrations on chromosome 3p (containing the RASSF1A gene and fragile histidine triad protein tumor suppressors) and 9p (containing the cyclin-dependent kinase (CDK) inhibitors p14 ARF, p15 INK4B, and p16 INK4A tumor suppressors) are present in histologically normal tissue, low-grade lesions, high-grade lesions, and tumors while latent EBV infection can only be detected in the high-grade lesions and tumors 23,24,178-33

51 181. Similarly for GC, dysplastic and other precancerous lesions are EBV-negative but all the neoplastic cells within the tumor are EBV-infected 356. As such, infection appears to occur somewhat late in tumorigenesis likely during progression from a low-grade lesion to a high-grade lesion with pre-existing genetic mutations being a prerequisite for persistent latent infection 23,24,181,244. The clonality of EBV in these tumors and the presence of latent EBV in 100% of tumor cells suggest the tumors arise from a single infected cell, and that infection is a necessary for progression to a high-grade dysplastic lesion and tumor 23,24,181,244. As discussed in more detail in later sections, nasopharyngeal and gastric epithelial cells in monolayer culture undergo senescence following latent EBV-infection, due to p16 INK4A and p21 activation 235,313. This halt to the cell cycle can be overcome by overexpression of cyclin D or inactivation of the p53 or p16/cdk4 pathways, possibly explaining the requirement for genetic alterations to epithelial cells before persistent latent EBV-infection can occur 235,312,313. When EBV-associated epithelial tumors are cultured in vitro, the epithelial cells consistently lose the EBV genome, suggesting maintenance of the EBV genome only offers a growth advantage to these epithelial cells in the tumor microenvironment, which is not recapitulated in vitro 19,48,79. Together these data suggest that EBV cannot generally persist in a latent state in epithelial cells, but infection of an epithelial cell with pre-existing genetic aberrations that permit latent infection may result in progression to cancer. 34

52 OHL OHL is a benign lesion commonly located on the lateral tongue margins of immunocompromised individuals, but also occurring on the dorsal and ventral surfaces of the tongue, the buccal mucosa (cheeks), and the gingiva (gum). These lesions are frequent among human immunodeficiency virus (HIV) infected individuals, with a prevalence of 25-53% in this population 14. OHL can also occur among individuals with other forms of immunosuppression, such as transplant patients, and occasionally among immune competent individuals 14. Smoking is co-factor for the development of OHL 242,293. Unlike all other EBV-associated pathologies, OHL is the only disease directly associated with lytic viral replication 84. Although OHL is directly associated with productive infection, viral replication alone is not believed to be responsible for the development of OHL lesions. Instead, these lesions are thought to be a result of the convergence of productive viral replication with concurrent expression of a subset of latency-associated proteins, genetic alterations to the viral genome, and immune evasion. The lesions are often asymptomatic, but can present with mild pain, dysesthesia (alteration to the sense of touch), and alterations to taste. The appearance of the lesions can change daily with lesions often appearing and disappearing spontaneously. Due to the benign nature of OHL, treatment is not often indicated, but OHL lesions can be resolved by targeting EBV replication directly with antiviral compounds such as acyclovir or by treating the immunosuppression of an HIV patient by administration of antiretroviral therapy (ART). Upon examination, OHL presents with the following five histopathology 35

53 characteristics: (1) hyperkeratosis (an altered pattern of keratin expression) in the upper epithelial layers, (2) parakeratosis of the superficial epithelium (an abnormal persistence of the nuclei in the stratum corneum suggestive of incomplete differentiation), (3) acanthosis of the stratum spinosum (abnormal thickening of the stratum spinosum due to foci comprised of ballooning koilocytes), (4) minimal or no inflammation at the site of infection and surrounding tissue, and (5) histologically normal basal epithelial layer. A definitive diagnosis of OHL can only be made by the detection of EBV DNA, RNA, or proteins in the lesions. Interestingly, OHL lesions are also characterized by a marked decrease or complete absence of LC, the tissue resident DC, possibly explaining the immune evasion and lack of local inflammation 38,81,327. Within these lesions, EBV can be detected productively replicating in discrete foci in the suprabasal layers with no evidence of a latent infection and no detectable infection in the basal layer 84,234,351. Productive viral infection has been verified by the detection of lytic cycle proteins (Zta, EA-D, and VCA) and the detection of viral particles by TEM 84,234,351. Interestingly, a subset of latencyassociated proteins can also be detected in these lesions. In addition to EBNA1, weak expression of the latency-associated protein EBNA2 can be detected in the most superficial layers of some lesions, as well as weak expression of LMP1 in some samples 234. Genetic recombination of the EBNA2 gene resulting in a truncation of the carboxyl-terminal which alters protein function such that the truncated EBNA2 acts in a dominant negative manner is tightly associated with the development of OHL 326,328,329. Whether EBV expresses the EBER transcripts 36

54 in OHL lesions is currently unresolved 78,234. Based on the sequence of the LMP1 gene, which is highly variable among viral strains, OHL lesions were found to frequently contain multiple different strains of EBV 331. Despite the observed acanthosis, these lesions are not associated with alterations to cellular proliferation, as determined by expression of the proliferation marker ki Given the close association of OHL with immunosuppression, it is currently not known whether the presentation of EBV infection in the epithelium of these individuals is typical, an exaggerated form of what would be observed in a healthy individual, or unique to an immunosuppressed individual. Therefore, the characteristics of EBV infection observed in OHL lesions are generally not extrapolated to predict infection of epithelial tissue in an immune competent host. 2.3 Infection of B cells For an enveloped virus such as EBV to successfully infect a cell, multiple steps must be completed. The virus must first attach to the target cell, and the viral envelope fuse with a cellular membrane, releasing the viral nucleocapsid. Finally, the capsid must successfully traffic to the site of viral replication within the cell and uncoat, releasing the viral genome. EBV, like other members of the herpesvirus family, uses multiple glycoproteins for binding and entry. Different glycoproteins or distinct complexes of glycoproteins are used to mediate binding and entry into various cell types. Interactions of these glycoproteins during virion production or within the extracellular milieu influence the resulting tropism of the viral particle. While the focus of this research is the role of epithelial cells within 37

55 the virus lifecycle, relatively little is known about infection of this cell type. Therefore, our knowledge of infection of B cells serves as a foundation for studying infection of epithelial cells. Entry One of the most abundant glycoproteins in the virion envelope is the heavily glycosylated gp350/220, so named for the approximate mass of the two protein isoforms produced due to alternate splicing of the transcript 9,119,138. Gp350/220 mediates viral attachment to the B cell surface through high affinity interactions with the complement receptor 2, alternately named CD21 63,64,212,224,225,300,301. Antibodies to gp350 which block virus binding and soluble CD21 neutralize infection of B cells, though a Δgp350 mutant strain is still capable of immortalizing B cells, with severely reduced efficiency, suggesting additional glycoproteins may be able to initiate entry 132,211,301. These observations made gp350 an attractive target for vaccination strategies. Interactions between CD21 and gp350 bring the viral and cell membranes within approximately 50 nm of each other, though the inherent flexibility of CD21 and transfer of binding from gp350 to gp220 could theoretically bring these two membranes even closer 223,336. Additionally, these interactions between gp350/220 and CD21 induce capping of CD21 on the B cell surface, triggering endocytosis and additional downstream signaling events 223,300. Fusion between the viral envelope and the cell membrane occurs in an endosomal compartment using the core fusion machinery, gb and ghgl, although low-ph is not required 38

56 204,291. While many viruses encode a single peptide responsible for fusion, EBV requires the activity of at least three proteins for fusion. Crystallography data identify gb as a member of the class III viral fusion proteins, and membrane fusion assays confirm gb acts as a fusion protein 8,51,92,198. gh is also required for fusion and is believed to initiate hemi-fusion of the two membranes 93,207,294. gl is a chaperone for gh, necessary for proper folding and incorporation of gh into the virion 168,347. gp42, which noncovalently associates with ghgl, triggers the fusion process by interacting with the co-receptor, human leukocyte antigen (HLA) class II, on the cell membrane, sending a signal to gh to initiate fusion, as outlines in Figure ,169,203. Following this fusion event, the capsid and tegument proteins are released into the cytosol and the capsid is transported to the nucleus. The capsid, which is too large to enter the nucleus, docks at a nuclear pore complex to allow the viral genome to enter the nucleus (reviewed in 174 ). Currently, it is unknown whether this process of DNA insertion is active or passive. Within the nucleus, the linear genome undergoes homologous recombination using the TR located at both ends of the linear genome to form a circular genome. The number of TR on each end of the genome is variable as a result of cleaving the viral genome for packaging. The number of TR in the resulting episome will also vary depending on which TRs are used for homologous recombination. Therefore, each viral episome contains a unique number of TR, generally between 1-20 copies. Digesting the viral genomes with a restriction endonuclease, such as BamHI, and probing for the genomic fragments containing the TR, a process known as termini analysis, reveals the 39

57 Figure 2.4. EBV attachment and entry. Illustration demonstrating the viral and cellular factors responsible for virus binding and entry into B cells and epithelial cells. (1) Interactions between gp350 and CD21 attach the virus to the B cell and trigger endocytosis. (2) The close proximity of the virus and cell membranes allow HLA class II on the cell to interact with gp42 on the virus (3) triggering the ghglgb fusion complex to initiate fusion of the membranes. For entry into epithelial cells, (4) an as yet unknown receptor on the cell, possibly integrins, bind to an unidentified viral protein, possibly BMRF2 (5) triggering the ghglgb fusion complex to initiate fusion between the membranes. 40

58 Figure 2.4 (1) CD21 gp350 B cell (2) HLA class II gp42 ghgl EBV (3) gb (4) Integrin? BMRF2? Epithelial cell (5) ghgl EBV gb 41

59 clonality of the virus population and whether the viral genome is episomal, linear, or integrated into the host genome 120,249. Due to the many proteins which must be incorporated into a virion for successful infection, not all viral particles are fully infectious (this is actually quite common for viruses). The specific infectivity for B cells inoculated with virus produced from HEK 293 cells is approximately ~1% of the encapsidated genomes 31. Within 16 hrs of engagement of CD21 on the B cell surface, the viral genome traffics to the nucleus, circularizes, and begins to express RNA, though some reports claim this process may be completed in as little as 30 minutes 2,30. Viral protein expression in B cells Upon infection of B cells, EBV expresses a tightly regulated transcription program with infected cells initially expressing the nuclear antigens EBNALP and EBNA2 2,30. Approximately two days PI, the other four EBNA proteins (EBNA1, EBNA3A, EBNA3B, and EBNA3C) are expressed at detectable levels 2. The last latency-associated proteins to be detected are the latent membrane proteins LMP1 and LMP2A, along with the noncoding EBER transcripts between 2-4 days PI 2. The expression of EBNA1, EBNA2, EBNA3A, EBNA3C, EBNALP, and LMP1 are required for B cell immortalization, LMP1 being the prototypic viral oncogene, activating multiple cellular pathways including the up-regulation of cellular bcl-2. In addition to the latency-associated transcripts, at least four lytic proteins, BHRF1, vil-10, Zta, and EA-D are transiently expressed early after infection 3,126,136,145,206,272,310,337. The expression of BHRF1, a bcl-2 homologue, 42

60 peaks approximately 24 hours PI, protecting newly infected B cells from apoptosis until there are sufficient levels of LMP1, between day 2-4 PI, to upregulate expression of the cellular bcl-2 3,104. The vil-10 is expressed within hours of infection, followed hours later by the expression of cellular IL IL-10 acts as an autocrine growth factor, promoting B cell proliferation and survival. The mrna for Zta, as well as some additional lytic and latencyassociated transcripts, is carried into newly infected cells in the virion itself, and protein is translated from this viral mrna 136 3,104. The early expression of Zta transactivates additional viral and cellular promoters resulting in a pre-latent state, helping to drive proliferation of the infected cell 136,145. rebv encoding a green fluorescent protein (GFP) reporter under the control of the ubiquitously expressed simian virus 40 (SV40) and cytomegalovirus (CMV) promoters express optimal levels of GFP three to four days PI Infection of epithelial cells Unfortunately, even though EBV can routinely be detected as a latent infection in epithelial tumors and productively replicating in OHL lesions in immunocompromised individuals, this virus has only rarely been detected in the stratified epithelium of healthy patients 65. Epithelial cell lines and primary epithelial cells growing in monolayer culture are generally refractory to infection with cell-free EBV, further limiting our ability to study and understand infection of epithelial cells and causing some investigators to question the importance of epithelial cells in the viral lifecycle. Therefore, we know very little about infection 43

61 of stratified epithelium, including how infection is initiated, the type(s) of infection present (i.e. latent or lytic), the kinetics of infection, or how EBV disseminates within an infected individual and between hosts. These questions have been the focus of my research. Entry Investigations into EBV-infection of epithelial cells have been hampered by the reported difficulties infecting both primary epithelial cells and epithelial cell lines with EBV. As described above, CD21 acts as the EBV receptor on B cells, binding to gp350. Initially, it was assumed EBV would also use gp350 - CD21 interactions to enter epithelial cells. In fact, early experiments which confirmed EBV's ability to infect an epithelial cell were only successful if the target cell expressed exogenous CD21 170,277, and many studies continue to use these CD21 expressing epithelial cell lines 13,29,207,334. Following these reports, efforts were made to identify epithelial cells which could express CD21 in vivo 135,276,312. CD21 mrna has been isolated from tonsillar epithelial cells, but due to Ab crossreactivity, expression of CD21 protein has not been confirmed 135,349. The majority of oral epithelial cells generally do not express CD21, with the mrna for CD21 not detected in uvula, soft palate, lingual, or buccal mucosal epithelial cells and CD21 protein expression not detected in nasopharyngeal epithelial cells 135,312. Given the high concentration of B cells in the tonsils and the possibility that tonsil epithelial cells may express the EBV receptor, CD21, the tonsil epithelium became an ideal candidate for the initial site of EBV infection. EBV 44

62 has been detected productively replicating in the lingual tissue of a very limited number of healthy individuals (3 of 232 samples) 65 and in OHL lesions 84,234. Recently, one group isolated EBV-positive epithelial cells from gingival tissue demonstrating that these cells can be infected in vivo 323. Unfortunately, only a limited analysis was performed on these gingival cells, and the analysis used cells in suspension and not intact tissue. There is a published case report describing productive EBV infection of the nasal mucosa resulting in nasal obstruction which persisted for over a year 74. These data suggest EBV can infect multiple epithelial tissues within the oral and nasal cavity and is not limited to CD21-expressing cells. Therefore, for my research, we have examined infection of primary cells isolated from both tonsils and gingiva. Within the last 15 years, investigators have determined that co-culturing epithelial cells with virus-producing or virus-coated B cells results in a more efficient infection than infection with CFV 27,125,247,272,275,312. Infection by co-culture is not dependent on the target cell expressing CD21 27,125,312,317. Infection by coculture requires the epithelial cell and virus-producing or virus-coated B cell come into direct contact with each other, forming a conjugate which allows efficient transfer of the virus 27,274,275. More specifically, interactions between gp350 on the virion and CD21 on the B cell surface initiate capping of CD21 and activation of adhesion molecules on the B cell and then the epithelial cell resulting in the formation of a virological synapse 274,275. This interaction between CD21 and gp350 is also thought to expose an otherwise masked envelope component on the virus that is essential for infection of epithelial cells. The enhanced level of 45

63 infection routinely detected in the presence of B cells has led some investigators to hypothesize EBV must come into direct contact with a B cell before it is capable of infecting oral epithelium, suggesting epithelial tissue may not be the primary site of infection 274,275. Infection of epithelial cells with CFV is improved using a Δgp350 mutant virus, supporting the hypothesis that the presence of gp350 on the virion surface blocks infection of epithelial cells 274,275. The efficiency of infection with CFV can also be improved by the presence of antigp350 Abs, which are believed to cap gp350 in the virus envelope, though enhancement in the presence of anti-gp350 Abs has only been observed by one group 319, whereas a second group saw no enhancement 275. It has been proposed that the presence of gp350 on the virus surface is inhibitory to infection of epithelial cells, the large size of gp350 possibly sterically hindering interactions with additional receptors. Consequently, for efficient infection of epithelial cells, gp350 must be sequestered either by binding to CD21 on a B cell or by the presence of anti-gp350 Abs. This sequestering of gp350 is thought to reveal an otherwise hidden glycoprotein required for entry into epithelial cells. The presence of a second glycoprotein, gp42, on the viral envelope also appears to inhibit infection of epithelial cells, at least as CFV. Within the human host, stratified epithelial cells generally do not express HLA class II, and therefore, in addition to lacking the EBV receptor, CD21, these cells also lack the co-receptor, HLA class II, which interacts with gp42 to trigger fusion. In fact, the presence of gp42 in complex with ghgl is thought to inhibit fusion events initiated by ghgl in the absence of HLA class II interactions 334. Therefore, the 46

64 virus must incorporate two distinct fusion complexes in the virion, one composed of ghglgp42, which enables infection of B cells, and ghgl, which enables infection of epithelial cells 12,334. Stoichiometric analysis of virions has verified that a higher amount of ghgl is incorporated into virions than gp42, supporting the notion that two forms of the complex exist in the virions 334.The ratio of ghglgp42 complexes in the virion relative to ghgl complexes is dictated by the type of cell producing the virus. When virus is produced in cells expressing HLA class II (e.g. B cells), gp42 interacts with HLA class II within a cytoplasmic compartment leading to sequestration and eventual degradation of gp Consequently, less gp42 is available for incorporation into the final virion. Virus produced in B cells contains low levels of gp42, resulting in enhanced infectivity of epithelial cells. On the other hand, epithelial cells, which do not constitutively express HLA class II, produce viral particles with high levels of gp42 and enhanced tropism for B cells 12. The differential rate of incorporation of each of these complexes into the final virion is referred to as the dual-tropism model. Of note, while the presence of gp42 in the virion may inhibit infection with CFV, multiple studies have determined the presence or absence of gp42 in the virion has no impact on the efficiency of co-culture infection 274,275. Though we generally assume B cells express HLA class II but epithelial cells do not, this distinction is not absolute (as reviewed in 252 ). For example, once B cells differentiate into plasma cells (the cell type thought to produce infectious viral particles), they no longer express de novo synthesized HLA class II. Conversely, epithelial cells can be induced to express HLA class II as non-professional antigen-presenting cells during inflammation, 47

65 most notably in response to IFN-γ. Therefore, while the level of gp42 incorporated into the virion does appear to play a significant role in the tropism of CFV, it is intriguing to think this dual-tropism may not be as much cell type dependent as it is context dependent, with the local cytokine milieu influencing the tropism of the viral particles. While many of the details regarding EBV attachment and entry into epithelial cells are unresolved, some insight has been gained through the study of epithelial cell lines and primary epithelial cells in monolayer culture. At least four mechanisms have been proposed for attachment of cell-free EBV to epithelial cells. The first mechanism postulates virus coated by anti-gp350 IgA binds to the IgA receptor on epithelial cells 286, although if the epithelial cells are polarized, this interaction results in transcytosis of intact viral particles across the cells and not productive infection 70. A second mechanism proposes a direct interaction between the fusion complex, ghgl, on the viral surface with α 5 β 5, α 5 β 6, and α 5 β 8 integrins on the cell surface, though much of the early work identifying a role for ghgl in attachment was performed using the SVKCR2 cell line which constitutively expresses exogenous CD21 28,29,207,237. However, in CD21-negative epithelial cells viral entry initiated through ghgl interactions is inefficient 13. Furthermore, α 5 β 6 and α 5 β 8 integrins may not be expressed at biologically significant levels by target cells in vivo 28. Even though α 5 β 6 may not be used for viral entry in normal epithelium, epithelial cells upregulate expression of this integrin during carcinogenesis, suggesting a possible role for this integrin during EBV-associated tumorigenesis 15,29,278,304. Nevertheless, these 48

66 interactions between ghgl and the various integrins can directly trigger fusion, negating the requirement for gp42 HLA class II interactions. Therefore, while ghgl can be used for attachment, the primary role for the interactions between ghgl and integrins is thought to be the initiation of fusion in the absence of gp42 - HLA class II interactions 13. In polarized epithelial cell lines, the most biologically relevant system examined thus far, the viral protein BMRF2 interacts with β 1 or α 5 β 1 intergrins, and this interaction, which is limited to the basolateral surface of the cell, is required for efficient infection with CFV 317. By contrast, infection can be initiated at both the apical and basolateral surface of polarized epithelial cells by virus-producing cells 317. The virus produced by the polarized epithelial cells is subsequently able to spread across the lateral membranes of the cells 317. Importantly, infection with CFV is neutralized by the presence of anti-ebv Abs in human serum, but neither form of cell-to-cell spread (B cell to epithelial cell or lateral spread between adjacent epithelial cells) is inhibited by these Abs 317. Transfer of virus between cells may not only enhance infection of epithelial cells, but it may shield the virus from anti-viral Abs. The fourth mechanism for EBV attachment to epithelial cells was proposed by the only group to efficiently infect primary epithelial cells with CFV, which was achieved by growing primary sphenoidal sinus epithelial cells to a high passage number and under conditions which encouraged terminal differentiation in monolayer culture 60. Under these conditions, EBV preferentially infects terminally differentiating, non-basal-like, involucrin-positive cells (between % of cells following inoculation with 8 49

67 infectious units per cell), suggesting that expression of the EBV receptor may be limited to terminally differentiating cells 60. For cell-to-cell transfer between virus-coated B cells and epithelial cells growing in traditional monolayer culture, LFA-1 on the B cell interacts with ICAM- 1 on the epithelial cell, initiating conjugate formation 274. In polarized primary epithelial cells, infection with virus-coated B cells is limited to the basolateral surface despite the fact that ICAM-1 is restricted to the apical surface of the cell, suggesting ICAM-1 may not play a role during infection under physiological relevant conditions 274. Following attachment, fusion, at least with nonpolarized epithelial cell lines, occurs in a ph independent manner and is not dependent on endocytosis, possibly occurring at the plasma membrane 204. Successful fusion with an epithelial cell requires higher levels of gb than fusion with B cells 226. After fusion, the capsid traffics to the nucleus, possibly using an alternate pathway than that used in B cells 320. Regardless of the system used, integrins appear to play a role during infection of epithelial cells. While there are numerous candidates for a potential receptor on epithelial cells, verification of a biologically relevant receptor has not been achieved, due to the lack of an accurate model system. Given that infection with CFV and cell-associated virus may occur by different mechanisms, it is important to study each mode of entry separately, as demonstrated in chapter 5. Furthermore, the changes to the route of infection observed after polarization of the epithelial cells highlight the importance of using the most biologically relevant system available to study infection, with fully stratified epithelial tissue being the 50

68 most biologically relevant in vitro system for epithelial tissue. Finally, the local cytokine milieu may influence the ability of EBV to infect epithelial cells. Treatment of both primary and immortalized nasopharyngeal epithelial cells with TGF-β1 or tumor necrosis factor (TNF) -α enhances the ability of virus-producing cells to initiate infection 312,313, and alveolar epithelial cells become susceptible to EBV infection following IL-4 treatment through up-regulation of CD Viral protein expression in epithelial cells A majority of the early experiments designed to study EBV infection of epithelial cells were performed using epithelial cell lines and not primary epithelial cells. Because epithelial cell lines can differ quite dramatically in their epithelial cell characteristics, the use of various epithelial cell lines for these experiments has resulted in conflicting data, with some cell lines demonstrating latent infection while in others EBV can undergo at least partial lytic replication. Thus far, a comprehensive analysis of EBV protein expression has not been performed on primary epithelial cells infected in vitro. The most complete analysis to date tracks the expression profile of viral transcripts in primary B cells, primary epithelial cells (tonsillar), and two epithelial cell lines (AdAH and AGS) for 5 days following infection with virus-coated B cells (~25% infection rate) using quantitative reverse-transcription polymerase chain reaction (qrt-pcr) 272. Within 2-3 days PI epithelial cell lines express the EBER and BART transcripts, Qp-driven EBNA1 (at a level equivalent to that detected in the latency I Rael cells), LMP1, low levels of LMP2A, and Zta. By contrast, only 51

69 the EBERs, Qp-driven EBNA1, and LMP1 are expressed, each at relatively low level, by primary epithelial cells, with only transient expression of LMP2A at 3 days PI 272. Zta expression is detected 2 days PI in the primary cells and continues to increase over time. Both primary epithelial cells and epithelial cell lines show little transcription from the W and C promoters, implying little to no expression of EBNA2 or the EBNA3 proteins. Furthermore, the primary epithelial cells show very low or no expression of the BARTs. Using immunofluorescence (IF) staining and in situ hybridization (ISH) to examine individual cells, essentially all infected AdAH cells (identified by GFP expression) express the EBERs, while less than 30% express EBNA1 and less than 10% expressed LMP This low level of EBNA1 expression corresponds with a rapid loss of the viral genome from these cells, with over 80% of the infected cells losing the genome by 15 days PI. Zta is seen in approximately 15% of infected AdAH cells, although very few (<0.1%) express the early or late proteins, EA-D and gp350 respectively, suggesting abortive lytic replication 272. Similar expression occurs following infection of primary tonsillar keratinocytes, with detection of LMP1, inconsistent expression of EBNA1, and no expression of EBNA2 nor LMP2A 247. Involucrinpositive primary keratinocytes in monolayer culture (a slightly differentiated phenotype) express the EBERs, EBNA1, LMP2A, and BARF1 following infection, as detected by RT-PCR 60. Almost 100% of these infected cells express LMP1, while 90% express Zta, and approximately 45% express gp Therefore, unlike the other primary cultures, these slightly differentiated epithelial cells appear to support lytic replication, though attempts were not made to determine 52

70 whether infectious viral particles are produced. In addition to the differential expression of viral genes observed in B cell and epithelial cells, expression from a constitutively active GFP reporter gene is maximally expressed 3-4 days PI in B cells, but within 24 hours in epithelial cells 274. Latently infected primary epithelial cells do not persist in culture and cannot be passaged 60,67,247, demonstrating that EBV is not sufficient to immortalize epithelial cells (previously noted by Shannon-Lowe, et. al. 272 ). The inability of EBV to establish a persistent latent infection in epithelial cells may be due, at least in part, to the low level of EBNA1 expression which may be insufficient to maintain the viral genome. Following infection of B cells, the viral genome is amplified so that by 30 days PI the majority of cells contain episomes, this amplification occurring independent of lytic replication 272. By contrast, the viral genome does not amplify following infection of primary epithelial cells and some epithelial cell lines 272. The semi-permissive AGS epithelial cell line does show amplification of the viral episome following infection, but unlike B cells, this amplification is dependent on expression of the lytic protein Zta, though it does not result in the production of viral particles 272. In addition to the lack of amplification and maintenance of the viral episome, the presence of latent EBV may even be detrimental to epithelial cells. High levels of EBNA1 expression are toxic to epithelial cells 141. Furthermore, only one EBVpositive cell line has been generated from primary epithelial cells, and the parental gastric epithelial cells used to produce this line displayed dysregulation of the cell cycle pathway, as evidenced by over expression of p53, the ability of 53

71 the parental line to be maintained for 30 passages, and inactivation of the tumor suppressor gene RASSF1A upon propagation. It is likely these genetic alterations contributed to the growth of the gastric epithelial cells following EBV infection since gastric epithelial cells isolated from an additional 20 patients failed to produce EBV positive cell lines 235. EBV infection of human telomerase reverse transcriptase (htert)-immortalized nasopharyngeal epithelial cells results in up-regulation of p16 and p21 suggesting cell cycle arrest 313. Moreover, latently infected cells stop proliferating and senesce, leading to the rapid loss of EBV within the cultures 313. EBV-infected cell lines can only be produced by htert-immortalized nasopharyngeal epithelial cells following inactivation of the cyclin D pathway through overexpression of cyclin D1, deletion or shrna mediated knockdown of p16, or expression of a dominant active CDK4 R224 which is insensitive to p16 activity 312,313. Cyclin D1 overexpression also results in increased expression of EBNA1 and EBER transcripts, with a concurrent decrease in expression of the lytic cycle proteins, and resistance to serum induced terminal differentiation 313. Numerous reports have suggested just as EBV relies on a B cell differentiating into a plasma cells to switch from latent to lytic replication, EBV relies on the epithelial cell differentiation program for lytic replication, with lytic protein expression detected following differentiation by the addition of serum or TPA, though the expression of these lytic proteins does not always result in the production of infectious viral particles 11,27,60,65,161,170,234,297,351. A few groups have noticed spontaneous lytic replication following infection of epithelial cells using 54

72 highly lytic viral strains 311 or polarized epithelial cells 317,345. During polarization, the epithelial cells cease proliferating and form tight junctions with desmosomes, closely resemble cells from the stratum spinosum and not basal cells. The best characterized of these showed that following infection of a polarized epithelial cell line, foci of infected cells could be detected, with these cells expressing gp350 and producing viral particles 317. The infected cells did not show cytopathic effects, though this could be due to the fact that these experiments were conducted using immortalized epithelial cell lines generated from NPC tissue (Detroit-562) and not primary epithelial cells 317. At 21 days PI, after allowing 4 days of accumulation in the media, the polarized epithelial cells released 9.5 x 10 3 and 1.2 x 10 3 infectious particles per ml from the apical and basolateral surfaces, respectively. The only group to infect stratified epithelium in vitro used punch biopsies derived from tonsils 316. Following inoculation with virusproducing DC, the tissue showed foci of gp350 and VCA expressing cells in the suprabasal layers of the epithelium. Unfortunately, these are the only two viral proteins assessed by this group. In summary, based on experiments conducted in vitro predominately in monolayer culture, EBV infection of undifferentiated epithelial cells appears to result in a latency 0, I, or II expression profile with the EBERS always detected, but the level of EBNA1 often intermittent or low. Given that maintenance of the viral genome generally offers no growth advantage to epithelial cells, cells which express sufficient levels of EBNA1 for genome maintenance may not be selected for during sustained culture. It is currently unresolved if primary epithelial cells 55

73 express LMP1 or LMP2A following infection 60,65,125,247,272. Differentiated cells often initiate lytic replication, though in many systems, this does not result in the production of viral particles. In the three biopsy samples where EBV has been identified in normal epithelial tissue, infection is characterized by the expression of lytic cycle proteins including Zta, EA-D, VCA, and gp Infected cells localize to discrete foci restricted to the suprabasal layers of the lingual epithelium, with no infection evident in the basal layer. Latent infection cannot be detected by ISH for the viral DNA or the EBER transcripts. This expression pattern is reminiscent of OHL, which is also characterized by lytic EBV replication in the suprabasal layers, with no detectable latent infection in the basal epithelium 84,234,351. Conversely, as discussed previously, the EBV-associated epithelial tumors are characterized by latent EBV infection, sometimes with a low level of abortive lytic replication. The fact that latent infection has thus far only been identified in neoplastic cells and the difficulties encountered trying to propagate latently infected epithelial cells in vitro have led many investigators to question whether EBV is capable of persisting latently in epithelial cells in normal epithelium. Immune response to EBV infection A transcriptome analysis of acute EBV infection showed that while active infection induces a strong type I IFN response, some of the key IFN-induced genes are not up-regulated 50. EBV encodes multiple proteins that can alter the host response to infection or allow immune evasion. Of note for the research 56

74 presented here, EBV can induce expression of the immunosuppressive cellular IL-10 cytokine through RIG-I signaling from the EBERs in addition to encoding an IL-10 homologue, vil ,260. Zta can directly induce transcription of IL-10 in B cells 188, but not in an NPC line 165. The expression of cellular IL-10 can also be induced through activation of PI3-kinase by LMP1 in at least some B cell lymphomas 163,325. Importantly, this IL-10 acts as an autocrine growth factor for many EBV-associated lymphomas, and may serve to protect infected cells from immune surveillance 43,126,137,188. While essentially nothing is known about the immune response to infection of epithelial cells in the oral cavity, a mathematical model of oral infection based on the rate of viral shedding and the cytotoxic T cell response within a host suggests that productive infection may not elicit an adaptive immune response if the infection is limited to a small number (between 1-3) of infected foci 94, Experimental systems to study EBV biology Primary B cells and B cell lines Primary B cells and B cell lines are the most common model systems employed to study all aspects of the EBV lifecycle. Our broad understanding of the biology of EBV within the B-cell compartment has been facilitated by the ability of EBV to infect and immortalize primary B cells in vitro and of some EBVpositive tumor cells (notably BL) to give rise to cell lines that maintain a restricted latency similar to that seen in vivo. Infection of primary B cells in vitro results in immortalization of 1-10% of infected cells due to the continued expression of the 57

75 latency III proteins and noncoding RNAs 103,295. Following infection in vivo, B cells down-regulate expression of most latency-associated viral proteins resulting in a latency 0/I expression profile. While this down-regulation is not recapitulated following infection of primary B cells in vitro, it can occur following infection of some EBV-positive and EBV-negative B cell lines 116 ; the mechanisms contributing to this down-regulation currently under investigation. Additional studies takes advantage of the diverse set of EBV-positive B cell lines established from lymphomas which display different forms of latency. Many of these lines are amenable to various forms of experimental manipulation, allowing investigation into the functions of individual viral and cellular proteins. Primary B cells and some EBV-positive and EBV-negative B cell lines are readily infected by cell-free EBV, allowing detailed investigation into the mechanisms involved in attachment and entry as previously discussed. A limited number of the EBVpositive cell lines can be induced to produce infectious viral particles, with the Akata and B95.8 cell lines being the most amendable to virus production. Unfortunately, there are currently no lines in which induction results in the production of high titers of virus, which has severely limited the ability to perform detailed molecular and biochemical analysis of this virus. Epithelial cells growing in monolayer culture Much of the experimental data discussed in this review have come from epithelial cells growing in monolayer culture. While these data are informative, infection of epithelial cells in monolayer culture does not appear to recapitulate 58

76 infection in vivo, and for conclusions to be drawn, the data must be interpreted in the context of the differentiation state of the cells. Some of the more common cell lines employed to investigate infection of epithelial cells in monolayer culture are SVKCR2 cells (SV40 transformed epithelial cell line stably transfected with a CD21 expression vector), htert-immortalized nasopharyngeal epithelial cells, AGS cells (CD21-negative gastric carcinoma-derived cell line with a slightly differentiated phenotype), AdAH cells (nasopharyngeal epithelial cells line), and 293 cells (embryonic kidney-derived cell line which express low levels of CD ). Some groups also work with primary epithelial cells, the most common being tonsillar epithelial cells, though tongue, nasopharyngeal, foreskin, and sphenoidal sinus epithelium have also been used 60,247,272,316,317. SVKCR2, 293, and AGS cells can be infected with CFV, though the efficiency is quite low. For infection of other model systems, especially primary cells, epithelial cells are generally coculture with virus-producing B cells (often at a 5:1 or 10:1 B cell to epithelial cell ratio) or peripheral B cells coated with CFV (the B cells are generally coated with CFV at an MOI of genome equivalents then added to the epithelial cells at a ratio of 5 B cells per epithelial cell). To examine the effects differentiation might have on EBV, epithelial cells in monolayer culture can be induced to differentiate to some degree by the addition of serum, treatment with chemicals such as TPA (also used to induce lytic EBV replication in B cells), or polarization of the monolayer. In undifferentiated monolayer culture EBV infection most often results in nonpersistent latency (i.e. latent infection without persistence of the viral genome in 59

77 culture), but use of any of the indicated methods to differentiation of the cells induces lytic replication. Unfortunately, this lytic replication is often incomplete and at best results in low levels of virus production. While these data strongly suggest that lytic replication is dependent on the differentiation state of the cell, it is not clear why latently infected cells have never been identified in normal epithelium. Furthermore, these data do not address the inability to detect infection in the basal layers of stratified epithelium in both normal and OHL tissue samples. If EBV is able to establish a persistent latent infection in stratified epithelium, due to the inherent nature of epithelial tissue, infection must occur in the basal cells since keratinocytes above the basal layer are no longer mitotically active, and therefore persistence in these cells would require that EBV override the cessation to cellular division that occurs during terminal differentiation. Furthermore, suprabasal cells are pushed up and eventually off the tissue as the underlying cells continue to divide and stratify. The two model systems most often employed to produce viral particles from epithelial cells are 293 cells carrying a rebv on a BAC backbone and the AGS cells. 293 cells will maintain the viral genome under constant drug selection and can be induced to produce infectious viral particles by exogenous expression of Zta followed by treatment with TPA and sodium butyrate. To enhance the infectivity of the resulting virus, the 293 cells are often transfected with an expression vector for the fusion protein gb 226. AGS cells will also maintain the viral genome under constant drug selection and can be induced to produce infectious viral particles with TPA and sodium butyrate treatment, though the 60

78 resulting virus stock is of a lower titer than that from induced Akata cells 12,13. Primary epithelial raft cultures The stratified epithelium of the oral cavity is a multilayered tissue that comprises a basal layer, which is in contact with the basement membrane and contains the mitotically active stem cells, and suprabasal layers which contain epithelial cells in various stages of terminal differentiation. During terminal differentiation, epithelial cells follow a programmed pattern of gene expression characterized by the expression of a unique set of keratin proteins (intermediate filaments) at each stage of differentiation. Based on the stage of terminal differentiation, the suprabasal layers can be further divided into stratum spinosum, granulosum, and corneum. Primary epithelial cells and some epithelial cell lines retain the capacity to differentiate in vitro. When these cells are grown to confluence in monolayer culture, the cells will form tight junctions between the individual cells and can polarize. If these cells are seeded onto a collagen containing dermal equivalent and grown at an air-liquid interface, a portion of the cells will leave the basal layer and begin to terminally differentiate. These raft cultures recapitulate terminal differentiation, forming all the layers of stratified epithelium 155. The formation of differentiated tissue can be verified by hematoxylin and eosin (H&E) staining and immunostaining tissue for cellular proteins expressed in a differentiationdependent manner. The following differentiation markers will be used in these studies (see Figure. 2.5). Keratin 5 (K5) is exclusively expressed in basal 61

79 Figure 2.5. Diagram of a raft culture. (A) Illustration of a raft culture with the localization of pertinent proteins indicated. The basal layer is the bottom most layer, which is in contact with the collagen dermal equivalent. The majority of cell division, as indicated by the expression of ki67, occurs in this basal layer. K5 is expressed in the basal layers and stably maintained throughout all the layers of the epithelium. Involucrin is expressed as soon as cells lose contact with the dermal equivalent. Cleavage of caspase 3 can be detected in a few cells in all layers of the tissue. (B) Micrograph of an H&E stained section of a raft culture 10 days post airlifting showing the four layers of stratified epithelium. The cornified layer is sloughing off the tissue, as commonly occurs during tissue processing. 10x magnification. 62

80 Figure 2.5 A cleaved caspase 3 Cornified Granular involucrin Spinous K5 ki67 Basal Collagen B Cornified Granular Spinous Basal Collagen 63

81 keratinocytes and is incorporated into intermediate filaments which can be detected in all layers of the epithelial tissue, though detection in the cornified layer can be sporadic 222. Ki67 is a tightly regulated protein, with a half-life between minutes, expressed in the G 1, S, G 2, and M phase of the cell cycle, but not expressed in G Therefore, the expression of ki67 can be used to identify actively replicating cells. Within raft cultures, the majority of ki67 cells will be in the basal layer, but ki67 positive cells can routinely be detected within the lower 2-3 layers of tissue. As a primary raft ages, and the primary cells approach the Hayflick limit, fewer cells will retain the capacity to replicate, and the number of ki67 expressing cells will decline. When epithelial cells leave the basal layer and begin to differentiate, they start to express involucrin (a precursor protein involved in crosslinking the cell membranes), making involucrin an early marker for terminal differentiation 253. Involucrin can also be expressed in monolayer culture if the cells are allowed to grow in layers. The terminal differentiation process not only effects the expression of epithelial specific proteins, but also many cell cycle proteins. These include the cell cycle regulators p21 and p53, and the anti-apoptotic protein bcl-2, all of which are expressed in basal cells; expression ceasing in conjunction with terminal differentiation 111,196,205,309,344. In normal epithelium, expression of p63 in the basal cells inhibits activation of p21 by p53 while inducing expression of the proproliferative gene S-phase kinase-associated protein 2 (Skp2) 196. Even though p21 and p53 levels decrease with terminal differentiation, the block in cell-cycle progression is not alleviated. Within any culture generated from primary cells, a 64

82 portion of the cells will undergo apoptosis, and can be identified by activation of the caspase cascade. Caspase 3, an executioner caspase, is present as an inactive zymogen that is cleaved and activated by caspase 8 during intrinsic activation of the caspase cascade or by caspase 9 after extrinsic activation of the caspase cascade. Raft cultures have been used to study the lifecycle of multiple viruses, the most notable being human papillomavirus (HPV), which is dependent on the terminal differentiation of epithelial cells for viral replication 200. Though all herpesviruses are capable of infecting epithelial cells, some do so much more readily in vitro than others. The alphaherpesviruses herpes simplex virus (HSV)-1 and HSV-2 display a broad tropism in vitro and easily infect epithelial cells in monolayer and raft culture. Infection of raft cultures with HSV-1 and HSV-2 results in virus production and cytopathic effects, such as ballooning of infected cells (swelling of cytoplasm without vacuolization), formation of multinucleated giant cells, and reticular degeneration (rupture of epithelial cells) similar to those observed in vivo 5,117,298,324. Following infection of raft cultures, two patterns of viral spread have been observed for HSV-1, HSV-2, and varicella-zoster virus (VZV). The virus can infect the basal cells and spread to all layers of the epithelium due to cellular division, the virus thought to gain access to the basal cells through microabrasions or at the margins (edges) of the tissue 5,117,324. In addition some, but not all, groups have also noted infection of the superficial layers of the stratified epithelium in raft cultures produced from HaCat and primary cells accompanied by lateral spread of the virus 5,117. The raft culture 65

83 system has successfully been used to evaluate the role of individual viral proteins and anti-viral compounds on viral replication in stratified epithelium 5,324. Interestingly, at 10 days or later post airlifting, inoculation of raft cultures with VZV no longer results in virus production and inoculation with HSV-1 or HSV-2 results in a more limited area of infection, with a reduction in the level of virus replication noted for all three viruses as early as 6 days post airlifting 5. The gammaherpesvirus Kaposi s sarcoma-associated herpesvirus (KSHV), a close relative of EBV, has also been studied in raft culture. Although the precise route of transmission for KHSV is not known, salivary transmission is suspected 139. The examine the effects of terminal differentiation on viral replication, primary tonsillar epithelial cells were infected with rkshv.219 (a recombinant virus which constitutively expresses GFP from the cellular EF-1α promoter while red fluorescent protein (RFP) is expressed from the PAN early lytic promoter in cells undergoing lytic replication 321 ) 139. In monolayer culture, infected cells only express GFP, indicative of a latent infection 139. When infected cells are used to generate raft cultures, RFP can be detected at the apical surface of the epithelium while GFP is expressed in all layers of the stratified epithelium, suggesting lytic replication is occurring in conjunction with terminal differentiation. A subset of the cells expressing RFP also express the late lytic protein K8.1 resulting in production of viral particles ( infectious units per culture, 0.9 cm 2, after incubation with medium overnight) 139. During terminal differentiation, stratification, or more specifically a loss of substratum contact and changes in cell signaling resulting from loss of integrin engagement, and not 66

84 necessarily early differentiation, is required for KSHV reactivation 270. No experiments have been conducted to study KSHV infection or dissemination in raft cultures. Thus far, there are no published reports describing EBV infection and replication in raft cultures, though some raft cultures have been generated from epithelial cell lines that express individual EBV proteins, namely LMP1, LMP2A, LMP2B, and BHRF1 39,40,59,268. Expression of any of these proteins during stratification results in a thicker, less organized tissue with abnormal expression of adhesion molecules including a lack of tight desmosomal junctions 39,40,59,268. Expression of the LMP proteins results in abnormal differentiation, with stratified cells failing to expressing appropriate levels of involucrin 40,59,268. Ectopic expression of LMP2A can also result in hyperproliferation, the epithelial cells continuing to replicate throughout all layers of the epithelium, and pseudo invasion into the dermal equivalent 59,268. In each of these experiments, the viral proteins were not expressed in the context of viral infection. Furthermore, these viral proteins were expressed by cells in all layers of the tissue whereas in normal epithelium in vivo, the expression of these proteins has only been detected in the postmitotic, suprabasal cells which have already initiated terminal differentiation. Therefore, it is not know in expression of these proteins will have a similar effect during a natural infection. Using primary raft cultures we can study EBV infection and replication in stratified epithelium. Additionally, we can investigate how the virus behaves in these cells as well as what effect, if any, viral infection has on the cell. 67

85 Chapter 3: Materials and Methods 3.1 Cell lines The EBV-positive BL cell line Akata was maintained in RPMI (HyClone) with 10% fetal bovine serum (FBS). Akata cell carrying a recombinant Akata virus in which a neomycin resistance gene, under control of a TK promoter, and a modified GFP gene expressed under the control of the CMV promoter (UF5) inserted 1,395 bp from the initiation codon of the BXLF1 ORF 207 (ΔTK, gift of L. Hutt-Fletcher) were maintained in RPMI with 10% FBS and 500 μg/ml Geneticin. Lymphoblastoid cell lines (LCL) were generated by infecting primary B lymphocytes with rebv Akata BAC (a gift from K. Takada) produced from 293 cells, and were maintained in RPMI with 15% FBS. Mouse fibroblast 3T3 cells were maintained in Dulbecco's-modified eagle medium (DMEM, Hyclone) with 10% heat inactivated newborn calf serum and 25 μg/ml gentamicin sulfate (Lonza). 3.2 Primary cells Primary gingival and tonsil epithelial cells (PGEC and PTEC, respectively) were isolated from tissue obtained from patients undergoing dental surgery or surgical treatment for chronic tonsillitis, respectively and grown in K-154 media with human keratinocyte growth supplement (Cascade Biologics, Inc.) as previously described with minor modifications 128,269. Primary tissue was pooled and incubated in wash buffer (PBS containing 67 μg/ml gentamicin sulfate 68

86 (Lonza) and 1x nystatin (Sigma)) for minutes. Tissue was washed three times in this wash buffer. The connective tissue and dermis were removed and discarded. The epithelial tissue was minced and to disassociate the cells 0.05% trypsin-edta (Gibco) was used in a sterile glass universal containing a stir bar at 37 C for ~3 hours. The supernatant fraction was removed and replaced with fresh trypsin every hour. The supernatant fractions were neutralized with E- media 201 supplemented with 5% FBS and the cells were pelleted by centrifugation. Cells were resuspended in mls K-154 media and cultured until the cells reached 70% confluence or 2 weeks in culture, and frozen for later use. Gingival cells were thawed and passaged one time prior to seeding in raft culture. 3.3 Primary epithelial raft cultures Primary raft cultures were grown as previously described with minor modifications 128. In brief, mouse 3T3 fibroblasts were trypsinized and resuspended in 10X reconstitution buffer (62 mm NaOH, 260 mm NaHCO 3, 200 mm Hepes, ph 8.2), 10X DMEM (Life Technologies), 80% collagen (Dickinson), and 2.4 μl/ml 10M NaOH for a final concentration of 2.5 x 10 5 cells per ml. This mixture was allowed to solidify to form a collagen matrix in 2.5 ml aliquots in a 6 well plate at 37 C for 1-2 hours. Once the matrix had solidified, 2 mls of E-media supplemented with 5 ng/ml epidermal growth factor (EGF, BD Biosciences) was added to each well and the matrices were allowed to equilibrate. Primary gingival or tonsil epithelial cells were trypsinized and resuspended to a final concentration 69

87 of 1.5 x 10 6 cells per ml E-media supplemented with 5% FBS and 5 ng/ml EGF. One ml of this cell suspension was added to each collagen matrix and cells were allowed to adhere for ~4 hours at 37 C. At this point, the collagen matrices were lifted onto stainless steel grids at the air-liquid interface and fed E-media by diffusion through the dermal equivalent. Raft cultures were fed E-media every other day. 3.4 Virus production and infection To induce virus-producing B cells, Akata cells (5 x 10 5 cells/ml) were incubated with 100 µg/ml goat anti-human IgG F(ab) 2 (ICN/Cappel) for 24 hours. The induced Akata cells were harvested, washed once in phosphate buffered saline (PBS), then resuspended in PBS at a final concentration of 2.5 x 10 6 cells/200 µl. To inoculate the primary raft cultures, 200 µl of this cell suspension was added to the top of a 4 day old raft with or without prior wounding, which we achieved by scoring the tissue with a scalpel, as indicated (Figure 3.1). To embed the virus-producing cells in collagen matrix, Akata cells were induced as described above and after washing in PBS, cells were resuspended in PBS at a final concentration of 2.5 x 10 6 cells/100 µl. The cell suspension or PBS for mock infected tissue was then mixed 1:1 with the collagen mixture, which had been stored on ice to prevent solidification. 200 µl of this slurry was added to the top of a 4 day old raft or to the top of a solidified collagen matrix prior to seeding with epithelial cells. The samples were immediately incubated at 37ºC to encourage rapid solidification of the collagen encasing the B cells (less 70

88 Figure 3.1. Diagram demonstrating the production and infection of primary raft cultures. Illustration of the general protocol used to grow and infect primary raft cultures. (1) A collagen matrix containing J2 3T3 mouse fibroblast feeder cells was allowed to solidify in a 6-well plate, (2) the collagen matrix was seeded with 1.5 x 10 6 PTEC or PGEC, (3) these cells were allowed to adhere for 2-4 hours, (4) the collagen matrix with adherent epithelial cells was lifted onto a stainless steel support grid and E-media was added below the support grid, (5) 4 days after airlifting, the raft was cut multiple times with a scalpel to wound the tissue, (6) 2.5 x 10 6 virus-producing Akata cells were added to the top of the tissue in 200 μl PBS, (7) the raft cultures were harvested at multiple times PI for analysis. 71

89 Figure

90 than 10 minutes). After 1 hour incubation, the collagen matrices were seeded with epithelial cells. For production of B cell-derived CFV, Akata cells, 1 x 10 6 cells/ml, were induced as described above and incubated at 37ºC for 48 hours. The virus containing supernatant was harvested, clarified by centrifugation (2143 x g for 5 minutes), and filtered (0.45 μm pore size). Virus stocks were concentrated using either a centriprep centrifugal concentrator (Millipore) with a 10,000 molecular weight cut-off (MWCO) for small preps or by tangential flow using a Model EP-1 Econo Pump (Bio-Rad) with a MidiKros hollow fiber membrane module with a 500,000 MWCO for large preps. Virus stocks were titrated by quantifying the total number of genomes using quantitative PCR (qpcr) as described below. The concentrated virus stocks were diluted in PBS prior to inoculating raft cultures when applicable µl of virus was added to the top of a wounded raft four days post air-lifting. For pre-incubation with anti-ebv Abs, the non-neutralizing mouse monoclonal Abs L2 (Chemicon), which recognizes gb, or 2L10 (hybridoma), which recognizes gp350, were added to virus stocks (1 x 10 9 genomes per raft ) at identical dilutions with a final concentration of 2 μg/ml for L2. The Abs were incubated with the virus stocks at room temperature for ~1 hour, then used to inoculate the top of a wounded raft 4 days after airlifting. Epithelial-derived virus stocks were generated by harvesting the encapsidated genomes from raft cultures inoculated with 2.5 x 10 6 induced Akata cells, as described above, 6 or 8 days PI. The virus was harvested and quantified as described below. The virus stocks were diluted in PBS prior to inoculating raft 73

91 cultures where applicable µl of virus was added to the top of a wounded raft culture four days post air-lifting. The epithelial-derived virus stocks were preincubated with the L2 and 2L10 Abs as described above. To determine whether the virus-containing homogenate harvested from infected raft tissue contained anti-viral properties, raft cultures were infected with virus-producing B cells or B cell-derived virus stock, diluted in either PBS or homogenate isolated from raft cultures 6 or 8 days PI. Specifically, 2.5 x 10 6 induced Akata cells were resuspended in 200 µl PBS or 200 µl infected raft homogenate and used to inoculate a four day old wounded raft culture. B cellderived CFV, 5 x 10 8 genomes, was used to inoculate a 4 day old wounded raft culture with or without the prior addition of 200 µl of infected raft homogenate. The raft homogenates used in these experiments contained 3.78 x 10 9 ± 2.55 x 10 8 encapsidated genomes per 200 µl. To account for the level of virus replication resulting from the homogenate itself, 200 µl of homogenate and 200 µl homogenate diluted 1:10 in PBS were used to inoculate additional raft cultures. The level of infection and replication were assessed by isolating and quantifying the number of encapsidated genomes produced from inoculated raft cultures at 6 and 8 days PI. To isolate virus-producing epithelial cells, wounded raft cultures were infected with 2.5 x 10 6 induced Akata, or mock infected with PBS, 4 days after airlifting and harvested 6 days PI. PBS, 1.5 mls, was added to the tissue and pipetted up and down multiple times to encourage cells to disassociate from the raft. The tissue was discarded and the cells were pelleted by centrifugation (

92 x g for 5 minutes). The supernatant fraction was aspirated from the cells and the cell pellet was resuspended in 750 μl PBS. Cell clumps were removed from the cell suspension using a 40 μm nylon cell strainer (BD Falcon). Wounded raft cultures produced from a different patient sample were inoculated with 200 μl of this cell suspension 4 days after airlifting. 3.5 Immunostaining At the indicated time points PI, infected, mock infected, and control raft cultures were harvested, fixed in 10% buffered formalin phosphate (Fisher Scientific) 2-4 hours, and paraffin embedded. Five micrometer thick sections were cut and stained with hematoxylin and eosin. Additional sections were assessed for the expression of viral and cellular proteins by IF staining. Briefly, the tissue sections were deparaffinized in xylene 2 x 10 minutes. The xylene was removed by 3 x 3 minute washes in 100% ethanol, followed by a 10 minute rehydration in dh 2 0. Antigen retrieval was performed in a boiling water bath using citrate buffer (10 mm sodium citrate, 0.05% Tween, ph 6.0) for 25 minutes. After the slides had cooled, they were washed with 0.1% Triton-X 100 in PBS, then permeabilized and blocked with 1% Triton-X 100, 10% goat serum in PBS for 30 minutes. Sections were incubated with the indicated primary Ab overnight at 4 C. After incubation with the primary Ab, cell were washed, and incubated with the appropriate goat anti-mouse, rabbit, or chicken secondary Ab conjugated to Alexa 488 or Alexa 594 (Molecular Probes) diluted 1:200 for 2 hour at room temperature. Cells were then stained with 10 μg/ml Hoechst (Molecular 75

93 Probes/Invitrogen) for 10 minutes at room temperature, washed, and mounted with an aqueous mounting media 68. The tissue sections were immunostained for EBNA1 1:300 (R4 rabbit serum, a gift from L. Frappier), LMP1 1:10 (S12), EBNA2 1:10 (PE2), Zta 1:25 (BZ1, Santa Cruz), Zta 1:6000 (polyclonal rabbit serum, a gift from G. Miller), EA- D 1:1500 (48180, Capricorn), BHRF1 1:100 (5B11, Millipore), gp350 1:1 (2L10), gb 1:500 (L2, Chemicon), GFP 1:500 (ab13970 polyclonal chicken serum, Abcam), K5 1:2000 (ab24647 polyclonal rabbit serum, Abcam), involucrin 1:2 (SY5, Neomarkers), ki67 1:500 (ab15580 polyclonal rabbit serum, Abcam), cleaved caspase 3 1:1500 (ab13847 polyclonal rabbit serum, Abcam), p16 1:25 (D25, Santa Cruz), p21 1:25 (F-5, Santa Cruz), and p53 1:25 (D0-1, Santa Cruz). Positive control raft tissue was generated by injecting EBV-positive cells into raft tissue 7 day after airlifting using a 25 gauge needle. Control rafts were harvested ~1 hr after injecting 2.5 x 10 7 LCL (latency-associated proteins and EBERs) or 2.5 x 10 7 induced Akata (lytic EBV proteins). Mock infected raft tissue served as negative controls for all EBV proteins. To quantify the percentage of Zta positive cells expressing additional viral or cellular proteins, at least 100 Zta positive cells were evaluated per time point on raft tissue grown from three sets of patient samples. Tissue sections were visualized using a Nikon Eclipse 80i microscope. Images were captured using a Photometrics CoolSnap cf2 camera and Nikon Digital Sight SD-Fi1 camera using NIS-Elements 3.10 software. To quantify the size of the infected foci, all LMP1 positive foci in 10 fields of view per sample 76

94 were measured using NIS-Elements 3.10 software. Minimal editing was performed to adjust brightness and contrast with experimental and control images edited in an identical manner. Three days after infection with CFV, the number of EBV-positive primary B cells was assessed by immunostaining. EBV-infected cells were identified and quantified by EBNA2 expression. Cells were immunostained for EBNA2 1:2 (PE2, hybridoma). In brief, B cells were washed, fixed in 4% paraformaldehyde for 10 min at room temperature, washed, permeabilized with 0.1% Triton-X in PBS, blocked in 5% goat serum, then incubated with the indicated primary Ab for 1 hour at room temperature. After incubation with the primary Ab, cell were washed, and incubated with goat anti-mouse secondary Ab conjugated to Alexa 594 (Molecular Probes) diluted 1:1000 for 1 hour at room temperature. Cells were then stained with 2 μg/ml Hoechst for 10 minutes at room temperature prior to mounting with an aqueous mounting media 69. LCLs were used as a positive control for the expression of EBNA EBER ISH ISH for the EBER transcripts was performed on formalin fixed paraffin embedded tissue sections harvested 2 and 4 days PI or mock infected tissue, according to the manufacturer's instructions (Vector Laboratories). Tissue sections from rafts injected with LCLs served as a positive control for EBER ISH. Briefly, tissue was deparaffinized in xylene, hydrated in a graded series of ethanol and dh 2 O, digested with 5 ug/ml proteinase K at 37 C for 30 minutes, 77

95 dehydrated in a graded series of ethanol, and incubated with a fluorescienconjugated EBER or control probe for 2 hours at 37 C. After blocking for 10 minutes in TBS, 3% bovine serum albumin (BSA, Fisher Scientific), 0.1% Tritonx, 20% rabbit serum (Sigma), the tissue sections were incubated with a rabbit F(ab') anti-fitc/ap Ab diluted 1:200 in TBS, 3% BSA, 0.1% Triton-x for 30 minutes at room temperature. Tissue sections were washed, and alkaline phosphatase activity was demonstrated by incubating the sections with enzyme substrate diluted 1:50 and inhibitor diluted 1:1000 in 100 mm Tris, 50 mm MgCl 2, 100 mm NaCl ph 9.0 over night at room temperature. Sections were counterstained with hematoxylin (Vector Laboratories) and mounted with an aqueous mounting media. Tissue sections were visualized with a Nikon Eclipse 80i microscope. Images were captured using a Nikon Digital Sight SD-Fi1 camera. 3.7 Isolation and quantification of encapsidated EBV genomes Encapsidated viral genomes were isolated from the raft tissue for quantification by qpcr. Briefly, after adding 0.5 ml homogenization buffer (0.05M Na-phosphate, ph 8) to the tissue, the tissue was freeze-thawed 3 times in a dry ice ethanol slurry and 37 C water bath. The tissue was then homogenized 30 times using a disposable plastic pellet pestle. The supernatant was collected and the tissue and pestle were washed with an additional 0.25 mls homogenization buffer. Benzonase, 1.5 μl (Sigma-aldrich), and MgCl 2 (2 mm final concentration) were added to the pooled supernatant and incubated at 37 C for 1 hour at which 78

96 time NaCl was added to a final concentration of 1 M. The sample was centrifuged at 2500 x g at 4 C for 10 minutes. The supernatant was collected and considered the virus stock. To titer the virus stock, 10 μl virus stock was mixed with 166 μl Hirt buffer (400 mm NaCl; 10 mm Tris-HCl, ph 7.4; 10 mm EDTA), 2 μl of 20 mg/ml proteinase K (Sigma-aldrich), and 2 μl 10% sodium dodecyl sulfate (SDS) and incubated for 2 hours at 37 C with constant agitation. DNA was phenolchloroform (Sigma-aldrich) extracted, ethanol precipitated and resuspended in 20 μl dh 2 O. Each DNA extraction was performed in duplicate. The number of EBV genomes in the virus stock was quantified by qpcr amplification of the BALF5 gene as previously described with minor modifications 143. Briefly, a 25 μl amplification reaction containing 12.5 μl Taqman Universal PCR master mix (Applied Biosystems), 0.25 μl of 20 μm BALF5 forward (5'AGT-CCT-TCT-TGG- CTA-GTC-TGT-TGA-C 3') and reverse (5' CTT-TGG-CGC-GGA-TCC-TC 3') primers (Sigma-aldrich), 0.5 μl of 5μM Taqman dual-labeled fluorogenic probe (5' (FAM) CAT-CAA-GAA-GCT-GCT-GGC-GGC-CT (TAMARA) 3', Applied Biosystems), and 1 μl DNA was performed. The uracil-n-glycosylase was activated for 2 minutes at 50 C, followed by activation of the Amplitaq Gold at 95 C for 10 minutes. The reaction mixture was amplified for 40 cycles consisting of 15 second denaturation at 95 C and 60 second extension at 60 C, with the fluorescence signal generated detected at the end of each cycle by a DNA Engine Opticon system (Bio-Rad). A standard curve was generated using 10 fold serial dilutions of a known copy number ( ) of the rebv Akata BAC (gift of K. Takada)

97 The number of viral genomes present in the B cell-derived virus stocks was quantified in an identical manner. Virus containing supernatant was not benzonase treated due to the high level of salts present in the B cell supernatant which would inactivate the benzonase. 3.8 Isolation and quantification of total EBV genomes The total DNA from rafts cultures was isolated using the QIAamp DNA mini kit (Qiagen) according to manufacturer's instructions. The number of EBV genomes in 1 μl of the isolated DNA was quantified by qpcr analysis of the BALF5 gene as described above. 3.9 Termini analysis Termini analysis was used to examine the circular and linear forms of the EBV genomes as previously described 249. Briefly, total DNA from the raft cultures was isolated using the QIAamp DNA mini kit as described above. 10 ug of purified DNA was digested with BamHI, seperated by electrophoresis through a 0.8% agarose gel (Lonza) and transferred to a GeneScreen Plus nylon membrane (PerkinElmer). The probe, LMP1 cdna fragment specific for unique DNA adjacent to the left terminal repeats, was labeled with α- 32 P-dCTP using a Nick Translation System (Invitrogen), and hybridized in 0.5M Na 2 HPO 4, 7% SDS, 1 um EDTA at 65 C overnight. 80

98 3.10 TEM Wounded raft tissue was inoculated with virus-producing Akata cells or mock infected with PBS as previously described was harvested six days PI. Cells from the upper surface of the tissue were isolated by pipetting up and down in 1 ml PBS. The intact tissue was discarded and the cell suspension was pelletted at 2500 x g for 10 minutes at 4 C. The cell pellet was fixed at 4 C for 3 hours in a solution of 1% glutaraldehyde and 4% paraformaldehyde buffered with 0.1M sodium cacodylate, ph 7.3. Following fixation, cells were washed in 0.1M sodium cacodylate buffer and postfixed overnight in buffered 1% osmium/1.5% potassium ferrocyanide. After postfixation cells were rinsed with buffer, dehydrated in a graded series of ethanol, and embedded in EMbed 812 (Electron Microscopy Sciences). A diamond knife mounted in a porter-blum MT-2B ultramicrotome was used to cut nm thin sections. The sections were mounted on 200 mesh copper grids and stained with 2% aqueous uranyl acetate and lead citrate. The sections were examined in a JEOL JEM 1400 transmission electron microscope. An Orius SC1000 bottom mounted camera was used to capture the images Isolation and infection of primary B cells B cells were isolated from peripheral blood using a Ficoll gradient (lymphocyte separation media, MP Biomedicals, LLC) and magnetically sorted using anti-human CD19 microbeads (MACS, Miltenyi Biotec) according to the manufacturers' instructions with minor modifications. Briefly, the blood was 81

99 incubated with RPMI supplemented with 1% FBS during the Ficoll gradient. B cells were plated in a 96 well plate (1-3 x 10 4 cells per well) and infected with serial dilutions of B cell or epithelial-derived virus stock or mock infected with PBS. Virus stock was diluted in RPMI supplemented with 15% FBS and 50 μg/ml gentamicin for a final volume of μl/well. B cells were infected by centrifugation at 238 x g at 10 C for 1 hour. Following centrifugation, cells were incubated at 37ºC for 3 days. These experiments were conducted on primary B cells isolated from a single donor infected with one B cell-derived and four epithelial-derived virus preparations. The number of infected cells was quantified by the detection of EBNA2 3 days PI by IF staining Immortalization of B cells B cells were isolated from peripheral blood using a Ficoll gradient and magnetically sorted using anti-human CD19 microbeads as described above. B cells were seeded in a 96 well plate (~1 x 10 4 cells per well) and infected with serial dilutions of virus stock harvested from raft cultures infected with virusproducing Akata cells, or mock infected with PBS. Virus stock was diluted in media for a final volume of 200 μl/well. B cells were infected by centrifugation at 238 x g at 10 C for 1 hour. Cells were maintained in RPMI supplemented with 15% FBS and 50 u/ml gentamicin, with media changed once per week or more often as needed. After 6 weeks of culture, the number of wells with viable B cells was assessed by B cell proliferation and trypan blue staining. 82

100 3.13 Evaluation of anti-viral compounds against EBV in raft cultures Acycloguanosine (acyclovir, Sigma-aldrich) was prepared as a 50 mg/ml stock solution in 1 M HCl. Wounded raft cultures were prepared and infected with virus-producing Akata cells as described above. The virus-producing Akata cells were allowed to infect the raft culture for 24 hours before the anti-viral drug was introduced. One day PI, the media was replaced with E-media containing the indicated concentration of acyclovir or vehicle control (1 M HCl). The drug containing media was replaced every other day until the rafts were harvested and formalin fixed for histological examination or harvested for qpcr analysis as described above. To study the effects of ART compounds on viral replication in stratified epithelium, the protease inhibitors amprenavir (GlaxoSmithKline) and lopinavir/ritonavir (trade name Kaletra, Abbott Labortaries) were added to raft cultures following infection. Amprenavir was dissolved in 100% ethanol for a final stock concentration of 10 mg/ml and stored at 4 C. A 200 mg Kaletra tablet (purchased from the pharmacy at the Milton S. Hershey Medical Center, Pennsylvania State University) was ground with a mortal and pestle and suspended in 70% ethanol for a final stock concentration of 20 mg/ml. Wounded raft cultures were infected four days after airlifting with virus-producing Akata cells, or mock infected with PBS, as previous described. Immediately following infection, the rafts were fed E-media containing the indicated concentration of amprenavir or Kaletra. The drug containing media was replaced every other day. 83

101 At 6 days PI, raft cultures were harvested for histological examination or quantification of viral replication using qpcr as described above Multiplex human cytokine array The level of 16 cytokines in the various virus stocks and tissue homogenate were quantified using the VeriPlex TM Human Cytokine 16-plex enzyme-linked immunosorbent assay (ELISA) kit (Pestka Biomedical Laboratories, Inc). Using a standard curve with known values for each cytokine, this kit can quantify the level of IFN-α (good reactivity with 10 of the 15 subtypes), IFN-β, INF-γ, IFN-λ (good reactivity with 2 of the 3 subtypes), IL-1α, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-15, IL-17, IL-23, and TNF-α in a sample. The ELISA was performed according to manufacturer's instructions. Briefly, following reconstitution of the antigen standard, serial dilutions of the standard and test samples were made using the sample diluent. 50 μl assay diluent and 50 μl standard, test, or blank were added to each well and incubated for 2 hours at room temperature with shaking. After washing, 50 μl detection Ab was added to each well and incubated for 1 hour at room temperature with shaking. The samples were again washed, than incubated with 50 μl streptavidinhorse radish peroxidase for 15 minutes at room temperature with shaking. After thorough washing, 50 μl of substrate were added to each well and the plate was immediately imaged with the ChemiDoc MP imaging system (Bio-Rad) using the Image lab 4.1 software (Bio-Rad). Data analysis was performed using the Q- View TM software (Quansys Biosciences). 84

102 3.15 Statistical analysis Statistical analysis was performed by Junjia Zhu, Ph.D., of the Penn State Cancer Institute, Division of Biostatistics, at Penn State Milton S. Hershey Medical Center. A growth curve statistical model (based on linear mixed model) was built to compare the growth rate of WT, ΔTK, or acyclovir treated raft cultures based on the number of encapsidated genomes per raft culture. The number of encapsidated genomes per raft culture was log-transformed to ensure the validity of the statistical assumptions. The size of the infected foci for each sample group was also analyzed using the log-transformed area by a linear mixed model. The Dunnett test was used to compare multiple groups. All analyses were performed using statistical software SAS version 9.3 (SAS Institute, Cary, NC, USA). The level of statistical significance used was

103 Chapter 4: EBV Infection of Stratified Epithelium Results in Spontaneous Virus Production in the Absence of a Detectable Latent Phase 4.1 Introduction Although the association between EBV and epithelial malignancies has been known for more than three decades, our understanding of the EBV lifecycle within the epithelium is still only poorly understood. By contrast, our broad understanding of the biology of EBV within the B-cell compartment has been facilitated by the ability of EBV to infect and immortalize primary B cells in vitro and of some EBV-positive B cell tumors (notably BL) to give rise to cell lines that maintain a restricted latency similar to that seen in vivo. Following infection of B lymphocytes in vitro and in vivo, expression of the entire complement of EBV latency-associated nuclear proteins (EBNAs 1, 2, 3A, 3B, 3C, and LP), membrane proteins (LMPs 1, 2A, and 2B), and noncoding RNAs (EBERs and BARTs) promote cellular proliferation and survival (termed latency III). EBV transitions to a more restricted pattern of viral gene expression as it enters the germinal center (latency II, associated with expression of EBNA1, LMP1, LMP2A, LMP2B, EBERs and BARTs) with further down-regulation of the LMPs as the infected B cells enter the memory B-cell pool where the virus establishes a lifelong latent infection. Because EBNA1 functions to maintain the viral genome during cell division, it is required in all dividing cells including proliferating memory B cells (latency I) but not in resting memory B cells, in which the only 86

104 viral transcripts are the noncoding RNAs (latency 0) rendering the infected cells invisible to the immune system 110. These different forms of latency are also observed in the EBV-associated lymphomas such as PTLD (latency III), HL (latency II), and BL (latency I). Because EBV is transmitted in saliva, it must cross the oral epithelial mucosa to gain access to the B-cell compartment. Several mechanisms have been proposed that focus on microabrasions of the oral epithelium, transcytosis of the viral particle across the epithelium, infection of tissue resident DC, or infection of the oral mucosal epithelial cells 70,316,318,330. In the latter, productive viral replication could expand the incoming virus, with or without establishment of a latent infection, and the resulting virus could then access the B-cell pool or be released into the oral cavity to infect a new host. In biopsies of OHL (a benign hyperplastic lesion that occurs in immunocompromised individuals), productively replicating EBV DNA, lytic cycle proteins, and mature virions are detected in the suprabasal layers of the lingual epithelium providing convincing evidence that EBV can infect epithelial cells and undergo productive replication 84. Similar patterns have be found in a small number of normal tongue biopsies (3 of 217) in an extensive study 65. By contrast, no evidence of latently infected cells is observed in these samples. EBV is associated with 100% of undifferentiated NPC, 100% of lymphoepithelioma-like GC, and less frequently with the poorly and moderately differentiated GC where it exhibits a latency I or II pattern of gene expression 17,107,232,233,350. The presence of latent EBV in these epithelial 87

105 malignancies suggests the possibility that EBV might establish a latent infection in primary epithelial cells, but this has been difficult to substantiate. Unlike B cells, EBV-infected epithelial cell lines are not readily isolated from NPC or GC tumors, and primary epithelial cells are very poorly infected in vitro. In monolayer culture, EBV-infected primary keratinocytes often exhibit a latent protein expression profile, but the infected cells fail to proliferate 60,67,247,272. Although these problems have hindered progress in understanding the lifecycle of EBV in epithelial cells, a few EBV-negative epithelial cell lines can be infected with EBV, commonly resulting in latency, and have provided some advances in our knowledge. One notable example is EBV tropism, which is controlled by differences in the glycoprotein profile of the viral particles derived from either B or epithelial cells 12,204,334. The glycoprotein profile of virus isolated from the saliva of normal individuals is consistent with an epithelial origin, suggesting EBV replicates within the epithelial compartment prior to transmission between hosts 134. Individuals with a persist infection frequently shed high levels of virus into the oral cavity, with the rate of shedding thought to be too high to be accounted for by virus-producing B cells alone 94. In all in vitro systems, after infection EBV establishes latency which, under specific circumstances can persist. For productive viral replication to occur, the latent genome must be reactivated, a process which appears to be directly linked to the differentiation state of the cell 11,27,161,170,297,351. While these model systems have been valuable tools for the study of EBV, infection of undifferentiated epithelial cells (which is frequently latent) does not recapitulate natural infection 88

106 in the host (which appears to be exclusively lytic). Raft cultures may be a more appropriate model system because the proliferating cells in the basal layer stratify and differentiate, mimicking epithelial stratification in vivo. Here, we demonstrate that EBV infects keratinocytes in raft cultures, resulting in high levels of productive replication in the suprabasal layers similar to that observed in OHL. Although we were unable to detect latently infected cells, the de novo synthesized viral particles readily disseminated throughout the epithelium. By 6 days PI a large numbers of cells were productively infected, resulting in the release of very high titers of virus. 4.2 Results EBV infection of raft tissue results in discrete foci which expand over time To infect organotypic cultures generated with primary human gingival epithelial cells (PGEC), virus-producing Akata cells were applied to the scored surface of a raft culture 4 days after lifting to the air-liquid interface. Because the immediate-early protein Zta is transiently expressed immediately following infection in both B cells and epithelial cells 136,272,337, we first used Zta expression to identify EBV-infected cells at various times PI. No Zta-expressing cells were detected in mock-infected raft cultures (Figure 4.1A). At 2 days PI, we detected individual or small clusters of Zta-expressing epithelial cells (K5-positive) in the suprabasal layers (Figure 4.1A). By 4 days PI, the Zta-expressing cells were localized in large clusters, and by 6 days PI the majority of the suprabasal cells expressed Zta. Similar results were obtained with primary tonsillar epithelial cells 89

107 (PTEC) (Figure 4.1B). This method of infection resulted in a 100% infection rate of inoculated cultures. EBV-infected epithelial cells concurrently express gene products from both the latent and lytic cycle Because EBV-infected epithelial cells in monolayer culture and EBVassociated epithelial malignancies exhibit a latent infection, we analyzed the expression of the latency-associated proteins EBNA1, EBNA2, and LMP1 in the raft cultures. LMP1 was readily detected in the majority of Zta-expressing cells at 2, 4, and 6 days PI, while EBNA1 was not detected at significant levels until 4 days PI and then only in a small percentage of cells (Figure 4.1C-D and Table 4.1). EBNA2 was also expressed at 4 and 6 days PI in a subset of Ztaexpressing cells (Figure 4.1E). None of these latency-associated proteins were detected in cells of the basal layer, in the absence of lytic cycle proteins, or in uninfected rafts. To determine whether EBV had established a restricted latency (latency 0), we examined expression of the EBERs, which are abundantly expressed in the nuclei of infected cells in all forms of latency, at 2 and 4 days PI, prior to the peak of productive replication. At both of these times points, EBER staining was detected in cells forming discrete foci confined to the suprabasal epithelium, localizing to sites of Zta expression on serial sections, and not in the basal cells (Figure 4.1F). Although Zta is expressed immediately following infection preceding the establishment of latency, it is best known as a lytic cycle transcriptional activator 90

108 Figure 4.1. EBV infection results in discrete foci of lytically replicating cells which expand over time. Four days after airlifting, wounded raft cultures were inoculated with 2.5 x 10 6 virus-producing Akata cells or mock infected with PBS. EBV-infected cells were identified by Zta expression in (A) PGEC or (B) PTEC raft cultures at 2, 4, and 6 days PI. Expression of the latency-associated gene products (C) EBNA1, (D) LMP1, and (E) EBNA2 was examined at 6 days PI; (F) ISH was performed for EBER transcripts at 4 days PI. Lytic proteins (G) EA-D, (H) BHRF1, (I) gb, and (J) gp350 were detected at 6 days PI. Micrographs are representative of data from 3 sets of patient samples. Rafts spiked with induced Akata or LCL cells served as positive controls for the lytic and latency-associated proteins respectively (data not shown). DNA was stained with Hoechst (A-E and G-J) or hematoxylin (F). 10x magnification. 91

109 Figure

110 Figure 4.1 continued Latency-associated proteins and transcripts C EBNA1 Zta EBNA1 D Zta LMP1 E EBNA2 Zta EBNA2 F EBER Lytic proteins G Zta EA-D H Zta BHRF1 I Zta gb J Zta gp350 93

111 Table 4.1. Percentage of Zta-positive cells expressing each of the indicated viral proteins Lytic Latent EA-D BHRF1 gb gp350 EBNA1 LMP1 EBNA2 2 days PI 94.2 ± ± ± ± ± ± ± days PI 98.4 ± ± ± ± ± ± ± days PI 97.3 ± ± ± ± ± ± ± 2.6 N=3 ±SD 94

112 that initiates the cascade of protein expression culminating in virus production. Because Zta was expressed in the absence of convincing evidence of latency, we examined the expression of lytic proteins. At 2, 4, and 6, days PI, the majority of Zta-positive cells expressed the early proteins BHRF1 (the viral anti-apoptotic bcl-2 homologue) and EA-D (the EBV polymerase processivity factor), and to a lesser extent the late glycoproteins gp350 (the viral glycoprotein which binds to the viral receptor, CD21, on B cells) and gb (the fusion protein) demonstrating that the infected cells were undergoing lytic replication (Figure 4.1G-J and Table 4.1). EBV does not affect cellular proliferation or early differentiation but does induce cytopathology In raft cultures generated from epithelial cell lines, exogenous expression of LMP1, LMP2A, LMP2B, or BHRF1 alone or in combination promotes epithelial cell proliferation with a corresponding decrease in differentiation, functions likely to contribute to tumorigenesis 39,40,59,268. By contrast, EBV infection had no effect on the expression of the early differentiation markers K5 and involucrin (Figure 4.2A and Table 4.2) and could not be detected in cells expressing the proliferation marker, ki67 (Figure 4.2B). Furthermore, the infected raft cultures exhibited no evidence of hyperproliferation when compared to the mock infected tissue. Despite expression of the anti-apoptotic viral protein BHRF1, infected tissue exhibited increased cleavage of caspase 3 (a marker for apoptotic cells) relative to mock infected tissue (Figure 4.2C). This increase in caspase 3 95

113 Figure 4.2. EBV infection does not affect cellular proliferation or differentiation, but induces cytopathology. Sections of mock infected or EBVinfected PGEC raft cultures at 6 days PI were analyzed by IF staining for (A) the early epithelial differentiation marker involucrin, (B) the replication marker ki67, or (C) the apoptosis marker cleaved caspase 3 (casp3). EBV-infected cells were identified by the presence of Zta and Hoechst was used to stain DNA. (D) H&E staining to examine cellular morphology and tissue integrity. Koilocytes are indicated by white arrows. EBV-infected cells were identified by staining of adjacent sections and the region of Zta expression is indicated by the bracket. Micrographs are representative of data produced from 3 sets of patient samples. 10x magnification. 96

114 Figure 4.2 A Mock infected Zta involucrin Infected Zta involucrin B Zta ki67 Zta ki67 C Zta casp 3 Zta casp 3 D H&E H&E * 97

115 Table 4.2. Percentage of Zta-positive cells expressing each of the indicated cellular proteins Ki67 Involucrin Cleaved caspase 3 (mock infected)* 2 days PI 0.2 ± days PI 0.3 ± ± ± 3.2 (2.4 ± 0.3) ± ± 1.9 (0.8 ± 0.5) N=3 ±SD 6 days PI 0.4 ± ± ± 3.6 (1.9±1.1) *Percentage of cleaved caspase 3 positive cells in matching layers of mock infected tissue 98

116 cleavage was not restricted to the Zta-positive cells, but could also be detected in Zta-negative cells adjacent to infected foci. Table 4.2 summarizes the percentage of Zta-expressing cells that co-express each of the cellular markers. Additionally, the EBV-infected cells exhibited cytopathic effects commonly observed with productive viral replication, including koilocytes, reduced cytoplasmic glycogen, decreased staining likely due to hypokeratinization, and ground glass nuclei (Figure 4.2D). Infection of raft cultures results in high levels of virus production Since EBV expressed lytic proteins in the raft culture, we wanted to determine if expression of these proteins resulted in amplification of the viral genome. Using qpcr analysis of the BALF5 gene (the EBV encoded DNA polymerase) to quantify the number of viral genome, we found that by 8 days PI, the total number of viral genomes in the PGEC raft cultures had increased ~100 fold relative to Day 0 (D0, measured at 1 hour PI) (Figure 4.3A). Encapsidated genomes, identified by resistance to endonuclease treatment, increased exponentially following an initial lag phase before attaining a plateau by 8 days PI, resulting in a fold increase relative to D0. Similar results were obtained with cultures generated using PTEC. Because the EBV genome is maintained as an episome in latently infected cells while it is both episomal and linear during productive replication, these two phases of the viral lifecycle can be distinguished by the differential mobility of the DNA on a polyacrylamide gel following restriction endonuclease digestion 249. If latent infection precedes 99

117 productive replication, we would expect to see an increase in the intensity of the episomal form of the genome prior to an increase in the linear form of the genome. Instead, linear viral genomes, indicative of productive replication, were detected as early as 1 day PI and were the predominant form of the genome by 2 days PI (Figure 4.3B). By contrast, the detection of episomal genomes was relatively weak at early times PI. TEM of infected raft cultures showed de novo synthesized capsids of the appropriate size (~100 nm) in the nuclei of epithelial cells (Figure 4.3C). Both naked and enveloped capsids were visible in the cytoplasm, while extracellular capsids were predominately enveloped, typical of a productive herpesvirus infection. No viral particles were detected in the mock infected tissue. Because the hallmark property of EBV is its ability to immortalize primary B cells in vitro, we infected primary B lymphocytes with serial dilutions of virus derived from raft cultures at 1 hour and 8 days PI. While virus isolated from raft cultures 1 hour PI was unable to immortalize B cells at dilutions of 1:100 and 1:1,000, virus isolated from raft cultures 8 days PI efficiently immortalized primary B cells even at a dilution of 1:100,000 (Table 4.3), suggesting that the virus is not likely to be surviving B cell-derived virus. Data is reported for raft cultures produced from PGEC isolated from two sets of patient samples. The anti-viral drug acyclovir inhibits productive replication and dissemination The absence of a detectable latent infection and the short time to virus production suggested that the spread of the virus throughout the tissue could be 100

118 Figure 4.3. Infection results in viral genome amplification and the production of viral particles. Wounded raft cultures were infected with 2.5 x 10 6 virus-producing Akata cells or mock infected with PBS four days after airlifting. (A) Total ( PGEC) or encapsidated viral genomes ( PGEC, PTEC) were quantified using qpcr analysis. Numbers represent average values plus standard deviation (SD) from cultures generated from 3 or 4 patient samples. The EBV genome was not detected in mock infected tissue from any patient sample. (B) Termini analysis of the EBV genomes in infected raft cultures. Uninduced and induced Akata cells served as positive controls for episomal and linear forms of the genome, respectively, while mock-infected raft tissue was used as a negative control. A darker exposure showing the early time points is shown on the right. Data is representative of experiments conducted using 2 sets of patient samples. (C) TEM of cells isolated from PGEC 6 days PI. A higher magnification image, of the area outlines in red, is shown on the right. Naked capsids can be seen in the nuclei of infected cells (top) and enveloped virions can be seen aligning on the plasma membrane of infected epithelial cells (bottom). No viral particles were detected in cells isolated from mock infected tissue. 101

119 Akata (-) Akata (+) Mock Akata (-) Akata (+) Mock Number of EBV genomes / raft Figure 4.3 A Gingival total genomes Gingival encapsidated genomes Tonsil encapsidated genomes Days PI B Raft days PI Raft days PI 10 kb 8 kb 6 kb 5 kb 4 kb C 3 kb C 102

120 Table 4.3. Ability of raft-derived virus to immortalize primary B cells in vitro Raft culture used to produce virus stock Dilution of virus stock Tissue sample 1 Tissue sample 2 MOI Percentage of wells immortalized MOI Percentage of wells immortalized Mock infected hr PI hr PI days PI days PI days PI

121 due to production of new virions and infection of neighboring cells. To examine this possibility, we treated the raft cultures with the anti-viral drug acyclovir, which inhibits lytic, but not latent, viral replication. The virus-producing Akata cells were allowed to initiate infection in the raft cultures for 24 hours before acyclovir treatment. We observed a significant reduction in viral replication in raft cultures treated with acyclovir relative to those treated with vehicle control (Figure 4.4A; p<0.001) with an IC50 of 0.88 μg/ml (Figure 4.4B). Furthermore, the size of the infected foci in the presence of 5 or 50 μg/ml acyclovir was significantly reduced compared to vehicle control (p= and p<0.0001, respectively; Figure 4.4C), demonstrating that acyclovir was effectively preventing spread to adjacent cells and restricting infection to isolated Zta-positive cells. 4.3 Discussion Although we have known for almost 40 years that EBV is transmitted by saliva and is associated with epithelial malignancies, relatively little is known about the lifecycle of EBV in normal oral epithelium, largely due to the lack of in vitro models to study this stage of the lifecycle. Using raft cultures, we demonstrated that EBV can infect stratified oral epithelium in vitro resulting in the expression of proteins from all phases of the lytic cycle by 2 days PI followed by a rapid increase in the number of productively infected cells. The presentation of infection closely resembled OHL where substantial productive replication in the suprabasal layers culminates in virion production 65,84,234,351. Similar results are observed in the few normal tongue biopsies in which EBV has been detected

122 Figure 4.4. Viral replication and spread are inhibited by treatment with acyclovir. (A) Wounded PGEC raft cultures were infected 4 days after airlifting with 2.5 x 10 6 virus-producing Akata cells or mock infected with PBS and treated 1 day PI with either vehicle ( ) or 50μg/ml acyclovir ( ). Encapsidated EBV genomes were quantified by qpcr. Numbers shown are averages plus standard deviation from 3 experiments using cells from different donors. The number of genomes was log-transformed and used in a linear mixed model, which demonstrated statistical difference (p<0.001). The EBV genome was not detected in mock infected tissue from any patient sample. (B) Quantification of the dose response of EBV replication in raft cultures following treatment with of acyclovir. (C) Quantification of the size of the infected foci based on LMP1 expression at 6 days PI in rafts cultured in the presence of vehicle or various concentrations of acyclovir. The size of the infected foci was measured from at least 10 fields of view at 10x magnification. Bars represent average plus SD using tissue from 3 different sets of patient samples. Analysis of these values by the Dunnett test revealed that the size of the infected foci in the raft cultures treated with acyclovir was statistically different than that in the vehicle control (5ug/ml, p=0.0004; 50ug/ml, p<0.0001). 105

123 Size of infected foci (x1000 μm 2 ) Encapsidated genomes / raft Encapsidated genomes / raft Figure 4.4 A WT Akata-Vehicle WT Akata-50 ug/ml acyclovir 10 9 * B *p Days PI C Mock, 50 ug/ml Vehicle 0.1 ug/ml 0.5 ug/ml 5 ug/ml 50 ug/ml Concentration acyclovir p p= Vehicle 0.1 ug/ml 0.5 ug/ml 5 ug/ml 50 ug/ml Concentration of Acyclovir 106

124 We detected LMP1 in the productively replicating cells, while EBNA1 and EBNA2 expression was limited to a subset of these cells. Although these proteins are typically considered latency-associated, they can be expressed during lytic replication in B cells and are detected in OHL biopsies 234. Specific roles for EBNA1 and LMP1 during lytic replication have been proposed 1,284. Because the suprabasal cells were no longer replicating (ki67-negative), the large number of infected cells observed in the raft cultures was not due to the proliferation and expansion of infected cells. Rather, the inhibition to virus spread observed with acyclovir treatment, which specifically inhibits lytic genome replication, suggests that adjacent cells were being infected by de novo synthesized viral particles transmitted between cells. Ultimately, infection of the raft cultures resulted in the production of very high titer virus (up to 5 x encapsidated genomes per PGEC and 7 x encapsidated genomes per PTEC raft culture) that, unlike most in vitro cultures, occurred without the need for any external stimulus. This spontaneous virus production and dissemination allowed us to produce the first multicycle growth curve ever reported for EBV. The high level of replication we observed is consistent with the level of viral replication thought to be necessary to generate the viral titers detected in saliva 94. Interestingly, the virus produced by the raft cultures was more efficient at infecting or immortalizing primary B cells than B cell-derived virus harvested at the time of infection. These data support a model in which the glycoprotein composition of the epithelial-derived viral envelope is distinct from that of B cell-derived virus, causing epithelial-derived 107

125 virus to be more infectious for B cells 12,334. We investigated this further and will describe the results from those experiments in the next chapter. Although it is accepted that EBV infects cells in the oropharynx, the epithelial cells of the oral cavity that are susceptible to EBV infection are not known. The tonsils have been proposed as a primary target for infection due to their close association with the lymphatic system and identification of EBVinfected naïve B cells within the tonsils, but clinical data supporting EBV infection of tonsillar epithelium is lacking. Though the epithelial receptor for EBV is not known, the mrna for the EBV receptor on B cells, CD21, is expressed in tonsillar epithelial cells, but not uvula, soft palate, tongue, or buccal mucosa epithelial cells 135. On the other hand, OHL is observed on tongue, buccal, and even gingival surfaces, supporting the likelihood that EBV can infect epithelial cells through an alternate receptor. We observed no significant differences in the levels of infection or virus production between PGEC or PTEC raft cultures, suggesting that EBV does not preferentially target tonsil epithelium and as such models of EBV infection of the oral mucosa should not be limited to this anatomical site. Given that lytic replication is induced by terminal differentiation in B cells and epithelial cells in monolayer culture 11,27,60,161,170,297, the productive replication observed in the suprabasal layers was not unexpected. The gammaherpesvirus KSHV, like EBV, relies on the terminal differentiation of B cells and epithelial cells for induction of lytic replication 139,341. During plasma cell differentiation, the UPR is activated, inducing expression of the spliced form of the transcription factor 108

126 XBP-1. For KSHV, expression of XBP-1 alone is sufficient to activate the Rta promoter, a homologue of the EBV Rta protein, and induce lytic replication 160,184,308,341,352. Expression of XBP-1 can also weakly induce expression of the immediate-early EBV proteins Zta and Rta in B cells 11,297. The UPR is activated during terminal differentiation of epithelial cells 296. Therefore, EBV may use signals from the UPR, possibly including XBP-1, to induce lytic replication in the raft cultures. Unlike EBV, the alphaherpesvirus (HSV-1, HSV-2, and VZV) can productively replicate in basal cells of raft cultures 5,117,324. When KSHV infected cells are grown in raft culture, KSHV can be detected latently replicating in the basal cells 139. EBV is the first herpesvirus we are aware of which cannot be detected replicating in the basal cells of raft cultures. This is reminiscent of EBV infection in OHL and normal tongue samples, where viral infection appears to be excluded from basal cells 65,234,351. A possible explanation for the absence of EBV in the basal layer is that the epithelial specific viral receptor may only be expressed on terminally differentiating epithelial cells, as previously proposed 60. Infected epithelial cells expressed latency-associated viral proteins while simultaneously expressing lytic proteins. A population of cells which exclusively expressed latent proteins could not be identified in these experiments. Furthermore, as discussed above, we were unable to identify infected cells in the basal layer, where a persistent latent infection would have to occur. Because EBNA1 is required to replicate and partition the viral genome during cell division, EBNA1 expression is required for persistent latent infection in actively replicating cells. Importantly, EBNA1 was only detected in a subset of the infected epithelial 109

127 cells, and not in actively replicating cells. It should be noted it is possible a small population of cells were latently infected but expressed the latency-associated proteins below the level of detection for our assays or expressed no latencyassociated proteins at all, although the latter would not result in a persistent latent infection. The EBERs are the gold standard for detection of EBV, being highly expressed in all forms of latency including the epithelial tumors NPC and GC. The staining from the EBER ISH was restricted to the suprabasal layers. Due to the high level of EBV replication in these cells, we cannot ensure the EBER probe was binding to the EBER transcript and not the viral genome. None the less, the absence of a signal in the basal layer is strong evidence that viral infection is not occurring in these cells. We were also unable to detect amplification of the episomal form of the genome prior to lytic replication, further supporting the notion that EBV did not initially establish even a transient latent infection in the raft cultures. These experiments provide the first evidence of an in vitro system in which EBV undergoes lytic replication in the absence of latency. Therefore, replication of EBV in stratified epithelium may not require reactivation from latency, but instead immediately following infection the virus productively replicates, possibly due to activation of the UPR. Furthermore, these data suggest EBV does not routinely establish a persistent latent infection in stratified epithelium, with B cells acting as the sole latent reservoir. Raft cultures have been generated with epithelial cell lines expressing exogenous LMP1, BHRF1, LMP2A, and LMP2B 39,40,59,268. Cellular changes typically seen with EBV-associated carcinomas, such as hyperproliferation and 110

128 decreased differentiation were observed in these cultures 39,40,59,268. Infection of primary raft cultures resulted in cytopathic effects typical of productive viral replication including koilocytosis and signs of apoptosis (cleavage of caspase 3). We speculate the differences we observed in our raft culture system from that of previous reports are due to the location within the stratified epithelium in which the viral proteins are being expressed (non-replicating cells), and the fact that the viral proteins were expressed in the context of productive viral replication and not as individual proteins. Interestingly, expression of the viral anti-apoptotic bcl-2 homologue, BHRF1, and expression of the major EBV oncoprotein, LMP1, appeared to be insufficient to completely protect epithelial cells from apoptosis. When HPV infected cells are grown in raft cultures, the virus induces a low level of caspase 3, 7, and 9 cleavage which enhance viral replication without inducing apoptosis 209. We cannot exclude the possibility that a similar process is occurring in the EBV-infected raft cultures. Our findings are in stark contrast to the EBV gene expression profile found in EBV-associated epithelial tumors. EBV is latent in these tumors, supporting the possibility that EBV might establish latency in normal epithelium. But our data, as well as that from others suggest the changes that occur during tumorigenesis, including mutations in the p53 and cyclin D pathways, facilitate the establishment of latency 235,313,313. Indeed, development of NPC is characterized by early chromosomal aberrations followed by infection with EBV. These EBV-infected cells then undergo clonal expansion and form the highgrade dysplastic lesions and invasive carcinoma 181. These data are consistent 111

129 with a model where EBV does not routinely persist in epithelial cells, but if infection of an epithelial cell capable of maintaining latency occurs, this could provide an additional stimulus for progression of the infected cell to cancer. It would be of interest to explore what cellular changes are required for EBV to latently persist in stratified epithelium given that this is the form of infection most commonly associated with epithelial tumors. Furthermore, if EBV is generally unable to latently persist in epithelial cells in vivo, then latent infection may serve as a biomarker for pre-malignant epithelial cells. These data demonstrate the raft culture system can be used to further our understanding of EBV infection of stratified epithelium. Within this system infection resulted in a phenotype quite distinct from those reported for infection of epithelial cells growing in monolayer culture. Our data highlight the need to study viral infection in appropriate model systems when possible. Using the raft culture system we have gained further insight into how EBV can replicate in epithelial tissue for transmission between hosts. Raft cultures will be an invaluable tool to further evaluation many questions concerning productive EBV replication and transmission. 112

130 Chapter 5: EBV Transmission: Using Raft Cultures to Model the Oral Epithelium 5.1 Introduction EBV is a ubiquitous human herpes virus typically known for its ability to latently infect B cells where it persists for the life of the host. Although B cells are the most intensely studied and well-defined target cell for EBV, this virus is not typically considered a bloodborne pathogen. Instead, EBV transmission occurs through saliva, with infection beginning and ending in the oral mucosa. At any given time, EBV can be detected in the saliva of 30-90% of the population, often at relatively high titers, and virus is detected more frequently in the saliva of healthy volunteers than in the blood 94,123,175. Despite the frequency and efficiency of virus production and shedding in the human population, there are no readily available in vivo or in vitro systems to study EBV transmission or infection of the oral cavity. Therefore, we currently do not know the primary site of infection or the site where virus is produced for transfer between hosts. The stratified epithelium in the oral cavity is proposed to be the site for both of these events. In support of this hypothesis, a previous report suggests virus in the oral cavity is produced by epithelial cells based on the ratio of glycoproteins in the viral envelope 134. Furthermore, OHL, a benign lesion on the lateral tongue margins of immunocompromised individuals, is characterized by productive EBV infection and virus shedding 84,234,351. Understanding the EBV lifecycle in epithelial tissue is clinically important given the two most common EBV- 113

131 associated tumors, GC and NPC, are both of epithelial origin 66 and EBV infection of gingival epithelium may play a role in periodontal disease 323. Interactions between gp350 and the complement receptor CD21 binds EBV to the B cell surface, triggering endocytosis 63,64,212, ,300,301. As the virus and cell membranes come into close contact, gp42 interacts with the co-receptor HLA class II on the cell surface 168. Within the endosomal compartment, fusion is initiated by the core fusion machinery, gb and ghgl, which is present in a complex with gp42 203,204,291. By contrast, epithelial cells generally do not express the receptor, CD21, or the co-receptor, HLA class II 135,312. Consequently, both gp350 and gp42 appear to inhibit infection 274,275,319,334, resulting in an inability to infect CD21-negative epithelial cells in vitro, severely hindering exploration of EBV infection of epithelial cells 125,170,277,317. More recently, numerous investigators have shown that epithelial cells are infected most efficiently by coculture with virus-producing or virus-coated B cells rather than with CFV 27,125,247,292. During co-culture, gp350 on the virus binds to CD21 on the B cell resulting in the formation of a virological synapse between the virus-producing or virus-coated B cell and epithelial cell, allowing for efficient transfer of EBV between cells 274,275. Alternatively, epithelial cells can be infected with CFV in which gp350 has been deleted (Δgp350) or sequestered following pre-incubation with anti-gp350 Abs 275,319. During replication in B cells, gp42 interacts with HLA class II within a cytoplasmic compartment, leading to sequestration and eventual degradation of gp42, effectively reducing the accumulation of gp42 in virion 12,334. This gp42-low virus has reduced ability to infect B cells, and an increased 114

132 tropism for epithelial cells. Virus produced in epithelial cells, where HLA class II is not expressed, contains high levels of gp42 enhancing its ability to infect B cells 12,334. Thus gp42 acts as a major determinant of viral tropism. Additional unknown factors also influence the tropism of EBV given that different viral strains display distinct preferences for B cells or epithelial cells, the Akata strain being epitheliotropic 170,311,334. According to current models for EBV transmission, viral particles are produced by epithelial cells in the oral cavity, shed into the saliva, and transferred to a naive host. This virus initially infects the oral epithelium, resulting in amplification of the number of virions in the newly infected host, while providing the virus enhanced access to the B-cell compartment where EBV establishes a persistent latent infection. Throughout the life of the individual, EBV reactivates in a small number of B cells, gain access to the oral epithelium, where it replicates for secretion in saliva. An important assumption of this model is that the virus shed into the oral cavity is capable of infecting epithelial cells of a naive host. Whether EBV actually enters and replicates in epithelial cells during transmission is not clear. Given the high levels of gp42 in epithelial-derived virus, epithelialderived CFV may not efficiently infect a new host. In Chapter 4 we demonstrated that EBV infected the stratified epithelium in raft cultures, and this infection resulted in the spontaneous production of high titer virus. We have used this system to gain further insight into how EBV can be transmitted between hosts. We found that infection of raft cultures could be initiated through the apical surface of the tissue, but not through the basal lateral 115

133 surface. Cell-free and cell-associated virus produced by B cells were most efficient at infecting raft cultures. Virus produced in stratified epithelium exhibited a strong tropism for B cells, but poorly infected stratified epithelium. Epithelial-toepithelial transmission could be enhanced slightly by pre-incubating CFV with an anti-gp350 Ab or by using virus-producing epithelial cells. 5.2 Results Replication of ΔTK virus is attenuated in stratified epithelium During the course of our experiments described in Chapter 4, we had routinely been unable to detect EBV infection of the basal cells in the raft cultures. Thus far, to identify infected cells we have relied on the expression of numerous viral proteins and transcripts, including the immediate-early protein Zta which is expressed immediately following infection at least in part from viral RNAs incorporated into virions 136. To verify the absence of infection in the basal cells, we used a recombinant Akata strain in which the viral TK gene has been replaced with GFP 207 to identify infected cells within the raft cultures independent of viral protein expression. This virus has been shown to express detectable levels of GFP in latently infected epithelial cells by 48 hrs PI 319. GFP localized to the Zta-expressing foci in the suprabasal layers; the majority of GFPpositive cells concurrently expressing Zta (Figure 5.1A). Using this GFP reporter, we were still unable to detect infection of the basal cells. While the WT virus was able to form large foci of Zta-positive cells, we noticed the ΔTK virus 116

134 predominately formed small foci, especially evident at 4 and 6 days PI (Figure 5.1A). The size of the infected foci were measured based on expression of the viral membrane protein LMP1. A statistically significant reduction in the size of infected foci was observed at 4 and 6 days PI compared to WT virus (Figure 5.1B). Mock-infected tissue showed no evidence of infection. Because these results are similar to those observed following acyclovir treatment (Figure 4.4), we performed a multicycle growth curve, and determined that the ΔTK virus showed little evidence of virus production later than 2 days PI (Figure 5.1C). Theoretically, since the viral TK gene is only expressed during lytic replication, the attenuation observed with this strain should not influence our ability to detect latently infected cells, if present. Raft cultures can be infected at the apical surface but not the basal lateral surface For our initial studies, we wounded the raft cultures prior to adding virusproducing B cells to the tissue, giving these cells access to all layers of the stratified epithelium. Therefore, we cannot be certain where within the stratified epithelium EBV can initiate infection and whether wounding is necessary for infection. To address these questions, we first restricted access of the virusproducing B cells to the apical surface by not wounding the tissue prior to inoculation. Infected epithelial cells were indentified at 6 days PI by coexpression of Zta and the epithelial marker K5. The level of infection and subsequent replication in raft cultures were quantified 6 days PI by qpcr. The 117

135 Figure 5.1. Replication of ΔTK virus was attenuated in stratified epithelium. Wounded raft cultures were infected with 2.5 x 10 6 WT or ΔTK virus-producing B cells 4 days after airlifting and analyzed at various times PI. (A) Zta expression (WT) or Zta and GFP expression (ΔTK) detected by IF staining. (B) Quantification of the size of LMP1-expressing foci (μm 2 ) in WT or ΔTK infected raft cultures. The size of the infected foci was measured from at least 10 fields of view per sample. (C) Quantification of the number of encapsidated genomes produced by the ΔTK virus in stratified epithelium. Day 0 is 1 hr PI. Data represent average plus SD from tissue generated from 3 separate patient samples. 118

136 Figure

137 level of infection and the production of viral particles were similar for both the wounded and non-wounded tissue (Figure 5.2A-B). The mock infected tissue showed no evidence of infection or viral replication. During a persistent infection, EBV is stably maintained in memory B cells, and following reactivation these cells are thought to transmit infection to the oral cavity. Previous studies have noted that B cells accumulate in the dermis of ex vivo tonsil explants, but do not readily cross the basement membrane and migrate through the epithelium to initiate infection 316. Given that we have never been able to detect infection in basal cells, we wanted to examine whether infection of stratified epithelium could be initiated through the basal lateral surface of the tissue. To mimic virus-producing B cells localized in the dermis, we encased the virus-producing B cells in the collagen dermal equivalent prior to seeding the epithelial cells, restricting access of the virus-producing cells to the basal surface of the tissue. To ensure the collagen embedding process did not prevent infection, we added collagen-encased virus-producing B cells to the apical surface of raft cultures 4 days post airlifting. The collagen-encased B cells applied to the apical surface of the raft initiated infection in the culture, as demonstrated by expression of the viral protein Zta, but no infection was observed when EBV-producing B cells were restricted to the basal surface (Figure 5.2C). 120

138 Figure 5.2. EBV from virus-producing B cells initiated infection at the apical but not the basal surface of epithelial tissue. (A) Raft cultures were infected with 2.5 x 10 6 virus-producing B cells or mock infected with or without wounding prior to infection. Expression of the viral protein Zta and the epithelial marker K5 were detected by IF staining on raft cultures harvested 6 days PI. (B) Quantification of the number of encapsidated genomes produced by raft cultures infected as described in (A) 6 days PI. Bars represent the average from three different patient samples plus SD. (C) The virus-producing B cells were encased in collagen and applied to the apical surface of a 4 day old raft or to the basal surface of a raft prior to seeding. Infected cells were detected as in (A). Images are representative of data obtained from tissue generated from 4 (A) or 3 (C) separate patient samples. 121

139 Figure

140 Raft-derived virus displays enhanced tropism for primary B cells If EBV infects and productively replicates in oral epithelium prior to both entry and egress, we would anticipate that virus produced in epithelial cells should be able to infect both B cells and epithelial cells. According to the dualtropism model in which the level of gp42 regulates tropism, B cell-derived virus will display an enhanced tropism for epithelial cells while epithelial-derived virus will preferentially infect B cells. To explore how efficiently EBV can shuttle between the epithelial and B-cell compartment, we first compared the ability of virus produced by either raft cultures or B cells to infect primary lymphocytes. Peripheral B cells were isolated and inoculated with serial dilutions of raft-derived or B cell-derived CFV. EBV-infected B cells were identified 3 days PI by expression of EBNA2. At each MOI, the virus produced in raft cultures infected primary B cells more efficiently than virus produced by B cells (Figure 5.3). CFV produced in raft tissue exhibits decreased tropism for stratified epithelium relative to B-cell derived virus Although cell-free EBV readily infects B cells, primary epithelial cells and epithelial cell lines in monolayer culture are generally refractory to infection with CFV 27,125,247,272,275. To determine if CFV was capable of infecting stratified epithelium, raft cultures were inoculated with serial dilutions made from high titer stocks of B cell-derived CFV. Given the high number of epithelial-derived virions presumably shed into the oral cavity of an individual with an active infection, we also investigated the ability of epithelial-derived CFV to initiate infection of raft 123

141 Figure 5.3. Epithelial-derived virus displays enhanced tropism for primary B cells. Primary B cells were infected with serial dilutions of CFV derived from B cells (Akata) or raft cultures. The number of infected cells was quantified by counting the number of EBNA2-expressing cells 3 days PI. Bars represent the average plus SD of experiments conducted on 1 patient samples using 1-4 different virus preps. Experiments were conducted in triplicate with at least 100 cells were counted per sample. LCL generated from the rebv Akata BAC served as positive control for EBNA2 expression while mock infected B cells served as a negative control. 124

142 Percent infected Figure % 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Mock LCL B cell derived virus Epithelial derived virus MOI 125

143 cultures. To measure the efficiency of infection, the number of virions present in the raft culture 6 days PI was quantified. Successful infection was verified by expression of Zta as described above. The B cell-derived CFV was considerably more efficient at infecting the stratified epithelium than the epithelial-derived CFV (Figure 5.4A-D). While infection of the raft cultures with 1 x 10 8 genome equivalents of B cell-derived CFV resulted in amplification of the input virus in all samples (between 3-18 fold increase in the number of encapsidated genomes), inoculation of raft cultures with 1 x 10 8 epithelial-derived genome equivalents did not result in amplification of the viral genome. Furthermore, infected epithelial cells were not identified in any of these raft cultures (Figure 5.4C). Inoculation with very high titers of epithelial-derived CFV (1 x 10 9 and 1 x genome equivalents) resulted in detectable infection with the formation of Zta expressing foci (Figure 5.4D), though the level of viral replication was on average lower than that observed for a 100-fold lower inoculum of B cell-derived CFV. Pre-incubating CFV with anti-gp350 Abs, which can be detected in the saliva of EBV-infected individuals 346, can enhance infection of epithelial cell lines growing in monolayer culture 319. To determine whether similar enhancement would be observed for infection of raft cultures, CFV was pre-incubated with an Ab recognizing gp350. As a control, CFV was pre-incubated with an anti-gb Ab, ensuring observed enhancement was due to specific interactions with gp350 and not a general effect from Ab binding. Pre-incubation with the anti-gp350 Ab increased the efficiency of infection for epithelial-derived CFV in 3 of the 4 samples tested (Figure 5.4A and E). The level of enhancement varied, but was 126

144 Figure 5.4. Epithelial-derived CFV exhibits decreased tropism for stratified epithelium relative to B-cell derived virus. (A-E) Wounded raft cultures were inoculated with the indicated genome equivalents, or mock infected with PBS, in the absence or presence of an anti-gb or anti-gp350 mab 4 days after airlifting. (A) The number of encapsidated genomes produced by infected raft cultures was quantified 6 days PI by qpcr. (B-E) Expression of the viral protein Zta and the epithelial marker K5 (B-D), or Zta alone (E) in raft cultures 6 days PI inoculated with (B) 1 x 10 8 B-cell-derived CFV, (C) 1 x 10 8 epithelial-derived CFV, (D) 1 x 10 9 epithelial-derived CFV, (E) 1 x 10 9 epithelial-derived CFV pre-incubated with anti-gp350 mab. Micrographs are representative images from staining of infected tissue generated from 3 separate patient samples. 127

145 Figure

146 be as much as 18 fold greater than the anti-gb Ab control. We did note enhancement with the anti-gp350 Ab was most pronounced when the initial level of infection was lower, due to variations in our virus preparations. No significant change was observed following infection with B cell-derived CFV pre-incubated with anti-gp350 Ab. Homogenate isolated from infected raft cultures mildly inhibits virus production Despite the low level of virus isolated from the raft cultures inoculated with 1 x 10 9 and 1 x epithelial-derived genome equivalents, we were frequently able to detect multiple foci of infection in these tissues. We considered that productive replication could be delayed in cells infected with epithelial-derived virus or that the tissue homogenate used as the inoculum might contain anti-viral factors. To investigate the first possibility, we examined virus production at both 6 and 8 days PI. We observed approximately 9 fold increase in the titer between 6 and 8 days PI in raft cultures inoculated with epithelial-derived virus while all other cultures increased 3.5 fold between these two time points. Even though this result suggested that the raft cultures inoculated with epithelial-derived virus might have delayed replication kinetics, the titers at 8 days PI varied considerably, and therefore, we cannot be certain of the significance of this finding. To address the second possibility, we inoculated raft cultures with virusproducing B cells, which produce the most robust infection, in the presence or absence of homogenate from infected raft cultures. Raft cultures inoculated with 129

147 homogenate alone were used to account for the level of replication resulting from the homogenate itself. We detected a slight inhibition to virus production in the presence of homogenate at both 6 and 8 days PI (Figure 5.5). To ensure that the homogenate was not having an effect on the virus-producing B cells, we also added homogenate to raft cultures inoculated with B cell-derived CFV, and again, we detected a similar reduction to virus production in the presence of homogenate. Overall, there was a subtle but consistent reduction in the number of encapsidated genomes isolated from samples inoculated with homogenate from infected raft tissue (64% ± 5% reduction in virus production) regardless of the form of input virus (i.e. cell-free or cell-associated). At this time, we do not know whether this decrease is due to a reduced level of infection or reduced replication. Virus-producing epithelial cells can transmit infection between raft cultures The efficiency of epithelial cell infection is enhanced when EBV is B cellassociated rather than cell-free, possibly a result of gp350 interacting with its ligand, CD21, on the B cell exposing a glycoprotein required for infection of epithelial cells 274,275. Although EBV is largely cell-free in saliva, EBV-infected epithelial cells have also been observed 167,285. The ability of epithelial cells to transmit infection to another epithelial cell has never been examined, but the ability of EBV to spread between adjacent epithelial cells in raft cultures suggests this may occur. We noticed that infected epithelial cells expressing the highest levels of gb were frequently dissociating from the tissue, and we considered 130

148 Figure 5.5. Homogenate from infected raft cultures mildly inhibits virus replication in raft cultures. Wounded raft cultures were inoculated 200 µl of homogenate isolated from infected raft cultures (homogenate contained ~3.8 x 10 9 epithelial-derived CFV) or a 1:10 dilution of the homogenate and harvested 6 and 8 days PI. The number of encapsidated genomes produced by each culture was quantified by qpcr. Additional raft cultures were inoculated with 2.5 x 10 6 virus-producing B cells or 5 x 10 8 genome equivalents of B cell-derived CFV in the presence or absence of homogenate (200 µl) harvested from infected raft tissue. Bars represent the average plus SD for experiments conducted on raft tissue generated from 2 separate patient samples. 131

149 Figure

150 whether these cells were capable of transmitting infection. To examine this possibility, we isolated virus-producing epithelial cells from infected raft culture through gentle agitation and used these cells to inoculated an uninfected raft culture. Raft cultures were harvested at 1 hr PI (D0), to quantify the input virus, and 6 days PI to analyze infection. We found that the number of encapsidated genomes increased in the raft cultures between day 0 and 6 PI, suggesting the virus-producing epithelial cells were transmitting infection to another raft culture, albeit at a lower efficiency than virus-producing B cells (Figure 5.6A). Infection was confirmed by the expression of Zta in 5/5 inoculated cultures (Figure 5.6C). Control raft cultures were inoculated with epithelial cells isolated from mock infected tissue and did not show evidence of infection by IF staining or qpcr (Figure 5.6B). 5.3 Discussion The lack of an in vivo model to study EBV transmission has greatly hindered our understanding of how this virus is transmitted between hosts. Furthermore, once within a host, we do not fully understand how the virus moves between the oral epithelium and the B cell compartments, which are physically separated by the basement membrane. Current models for EBV transmission assume EBV infects the stratified epithelium during viral entry and egress to amplify the number of virions during transmission, though alternate models have been proposed. The data from the experiments presented here suggest that EBV can use multiple methods to infect stratified epithelium, including epithelial-to- 133

151 Figure 5.6. Virus-producing epithelial cells can transmit infection between raft cultures. Virus-producing epithelial cells were isolated from raft cultures 6 days PI and used to inoculate another wounded raft culture 4 days after airlifting. Cells isolated from a mock infected raft were used to inoculate the control raft tissue. (A) Quantification of the number of encapsidated genomes present in raft cultures 1 hr (D0) and 6 days PI, each line representing data obtained from cultures produced from one set of patient samples (B-C) Expression of the viral protein Zta and the epithelial marker K5 were used to verify infection of the raft cultures following inoculation with virus-producing epithelial cells isolated from (B) mock infected or (C) infected raft cultures. Micrograph representative of data obtained from infecting raft tissue generated from 5 separate patient samples. 134

152 Encapsidated genomes per raft culture Figure 5.6 A D0 Days PI D6 B Z K5 C Z K5 135

153 epithelial transmission, with variable efficiency, and it is likely that multiple routes are used with the human host. Using a ΔTK virus, we demonstrated the viral TK gene was necessary for efficient viral replication and spread in raft cultures, although it is dispensable in cell lines 207.These differences are likely due to the fact that cell lines express the cellular TK gene while in stratified epithelium, the cellular TK gene is expressed by proliferating cells located in the basal layer, but expression down-regulates as the cells terminally differentiate 156. These data further support the hypothesis that the basal epithelium is not a significant site of viral replication in stratified epithelium. Given both the Akata and B95.8 rebv BACs contain a selection marker or reporter gene in the TK locus, the usability of these mutant viral strains in raft culture may be limited. Infection could be initiated through the apical surface of stratified epithelium, with wounding not required for infection of raft cultures 4 days post airlifting, at which time the cultures have not developed a significant cornified layer. In vivo, however, there could be a well developed cornified layer through which the virus or virus-producing cells must transit, and wounding might facilitate infection. Restricting access of the virus-producing B cells to the basal surface of the tissue prevented infection. We consistently could not detect infection in the basal layer, even with a viral strain which encodes a GFP reporter, suggesting CFV or virus-producing cells may require direct contact with the suprabasal layers of the epithelium to initiate infection. Therefore, B cells localized in the dermis may not be able to transit both the basement membrane 136

154 and basal epithelial layer to initiate infection of the stratified epithelium unless there is a wounding event that allows B cells access the oral cavity (e.g. bleeding in the oral cavity). An alternative route for transfer of infection between the B-cell compartment and the epithelium are LC, which can localize to all layers of the epidermis but predominately localize to the stratum spinosum. Following stimulation, LC can release infectious viral particles 316,330. Virus from the HLA class II expressing LC would presumably be epitheliotropic 208. The alphaherpesviruses HSV and VZV infect and productively replicate in the basal cells of raft cultures 5,117,197,324. The gammaherpesvirus KSHV latently replicates in basal cells of raft cultures 139. EBV is the first herpesvirus we are aware of that appears to be excluded from the basal layer. At this time, the biological cause and consequence for this exclusion is not known. It is worth noting that EBVassociated epithelial tumors are predominately of an undifferentiated phenotype, resembling basal cells. These tumors are characterized by a latent, not lytic, infection which appears to occur only after the accumulation of multiple genetic aberrations in the cell 23,24,181. Replication in the epithelial tissue resulted in the production of high titers virus which exhibited an enhanced tropism for B cells, and possibly other HLA class II expressing cells. Therefore, virus produced in the oral epithelium should be highly efficient at progressing to the B cell compartment. Infection of raft cultures with B cell-derived CFV resulted in low, but consistent amplification of the input virus (generally less than 10 fold). Inoculation with epithelial-derived CFV did not result in an increase in the overall number of viral particles, and no 137

155 infection was detected in any cultures inoculated with less than 1 x 10 9 epithelialderived genome equivalents. Between 6 and 8 days PI, cultures inoculated with the diluted raft homogenate (~3.8 x 10 8 genome equivalents) actually decreased, suggest infection was not established in these cultures or the level of infection and viral replication were insufficient to exceed the rate of virus decay. The homogenate isolated from infected raft cultures contained mild anti-viral activity, suppressing viral output by ~0.6 fold. This slight suppression does not account for the 25 fold reduction observed when the inoculating virus was produced by epithelial cells rather than B cells. Therefore, in agreement with previous reports, the level of gp42 incorporated into the virion, dictated by the cell type producing the virus, appears to strongly influences the tropism of the virus 12,334. Cultures inoculation with epithelial-derived CFV never exhibited an increase in the number of viral particles relative to the inoculum. As such, even though stratified epithelium can be infected with CFV, if the inoculating virus is produced by epithelial cells, this mode of transmission is inefficient and may not result in amplification of the overall number of viral particles during primary infection, though it could lead to local areas of relatively high viral concentrations. We detected a slight enhancement to infection with epithelial-derived CFV following pre-incubation with an anti-gp350 Ab, though we did not observe enhancement with B cell-derived virus. Our findings may differ slightly from previously published reports due to (i) inherent differences between epithelial cell lines in monolayer culture and stratified epithelium generated from primary cells, (ii) the fact that we used a different anti-gp350 Ab than that used in the previous 138

156 report, (iii) we used a lower concentration of Ab, or (iv) the antibody enhanced infection, but the level of enhancement was too low to be detected by our assay given the relatively robust level of infection with the B cell-derived CFV. It is plausible we would detect enhancement of infection with B cell-derived CFV with this Ab if we used different concentrations of Ab or lower titers of virus. Coating of CFV with anti-gp350 Ab may be most beneficial when the inoculating titer is low or only poorly infectious. Virus-producing epithelial cells were able to transfer infection to an uninfected raft, even with an inoculating dose as low as 6 x 10 6 genome equivalents, adding yet another mode of viral transmission, and demonstrating for the first time that successful co-culture does not require CD21-expressing B cells. Regardless of the method of infection, the virus readily spread to adjacent cells once infection occurred. Therefore, the limiting factor for infection of stratified epithelium seems to be the initiation of infection. In agreement with previous reports, the presence of gp42 and gp350 on the surface of CFV hindered infection of stratified epithelium. The data from these experiments suggest that virus produced by the oral epithelium is most readily transferred between hosts by virus-producing epithelial cells or as Ab-coated CFV. The glycoprotein gp350 is capable of eliciting a strong humoral immune response following primary infection and gp350 Abs are present in saliva, so it is reasonable to assume that virus produced by an infected individual would be coated with anti-gp350 Abs. Abs isolated from the saliva of EBV-seropositive donors can enhanced rather than neutralize infection 139

157 of epithelial cells in monolayer culture 319. Given the relatively low level of amplification that resulted from any pathway that involved epithelial-to-epithelial transmission, an intriguing alternate hypothesis is that during transmission between hosts, EBV does not primarily infect the oral epithelial cells to amplify the incoming virus. Given that virus shed from an EBV-positive host would be coated with anti-gp350 Abs, many of which neutralize infection of B cells, this virus may not be capable of direct transmission to B cells. Indeed, previous reports found that the virus shed into the saliva was very inefficient at binding to B cells, likely a result of the virus being coated with neutralizing Abs 94,319. An initial round of replication in the epithelium of a naive host may instead be vital to produce virus highly infectious for B cells that is devoid of neutralizing Abs. Furthermore, this initial replication in the epithelium and the subsequent production of the viral IL-10 homologue, along with additional cytokines, may act to recruit immune cells, such as B cells and dendritic cells, to the site of infection, providing the virus easy access to these target cells. Amplification of the inoculating virus was observed following infection with virus-producing epithelial cells. Both these factors complicate the implementation of viral control mechanisms, given that the current vaccination strategies for EBV involve eliciting a strong humoral response, frequently to gp350, to neutralize infection with CFV 35,290. Virus may be capable of shuttling from the epithelial compartment to the B-cell compartment through transcytosis of the basal epithelial cells. Tugizov et al. showed that EBV can be transcytosed bidirectionally through polarized epithelial cells in monolayer culture without productive infection

158 This later method of transfer is of interest given our inability to detect active infection of the basal epithelial cells. It is plausible if EBV enters a basal cell, it trafficks through the transcytosis pathway instead of eliciting an active infection in order to gain access to the underlying B cells. Using the raft culture system to model the oral epithelium, we have refined our understanding of how EBV may be transmitted between individuals. Infection of epithelial cells during egress would result in robust virus amplification and high levels of viral shedding. This epithelial-derived virus is relatively inefficient at infecting stratified epithelium, especially as CFV. This would suggest that within a host, infection of the oral cavity occurs as rare, discrete foci of infected cells and not widespread infection of the tissue. Indeed, this is what is observed in a limited number of clinical samples and predicted to occur in most individuals based on mathematical modeling of the dynamics of viral shedding 65,94. This continuous low level of viral replication and shedding may help the virus avoid triggering a robust adaptive immune response. We have also verified that EBV can directly move between the B cell and epithelial compartment as cellassociated and CFV, displaying distinct tropisms depending on the source of the virus. Consequently, infection of the oral epithelium may not only serve to amplify the quantity of incoming virus during primary infection, but depending on the source of the inoculating virus, this infection may produce virions with altered tissue tropism, enhancing the ability of the virus to infect B cells. For EBV, it appears to be more beneficial to shed low levels of virus from a limited number of cells than to shed very high titer virus from a large population of cells. Therefore, 141

159 the virus may have evolved to temper its ability to infect the tissue that actively produces virus. EBV has evolved exquisite mechanisms to evade the immune system and infect two very distinct tissues in the host. Infection of these tissues results in two divergent yet highly orchestrated cascades of viral protein expression, resulting in viral latency or productive replication, which we are now only beginning to understand. 142

160 Chapter 6: General Discussion and Conclusions The overall goal of the research presented in this dissertation has been to define the role of epithelial cells within the natural EBV lifecycle using the raft culture system. The oral epithelium is thought to be the site of primary infection and virus production for transmission, yet experimental evidence supporting this hypothesis was lacking. Furthermore, because the epithelium of the digestive tract is the most common site for EBV-associated tumors, the implications for this research are numerous. After establishing the kinetics of EBV infection in stratified epithelium using the raft culture system, we refined our understanding of how infection can be initiated within epithelial tissue and the tropism of the resulting virus. Finally, we investigated the effects infection had on the epithelial cells. 6.1 Infection of stratified epithelium results in spontaneous productive replication EBV is unique among herpesviruses in that in all in vitro systems studied thus far, EBV initially establishes a latent infection. The viral genome can be reactivated in a small percentage of cells following treatment with various chemicals, cross-linking the B cell receptor, or exogenous expression of the immediate-early protein Zta 47,56,122,133,241,299. With each of these methods, only a subset of the cells will reactivate, and for many of the reactivated cells, induction of the lytic cycle is abortive. Spontaneous reactivation is infrequent, and is most commonly abortive. Therefore, the study of productive EBV replication has been 143

161 difficult. Previous reports suggested that productive replication could occur in stratified epithelium, and that induction of the lytic cycle would be dependent on terminal differentiation of the epithelial cells 65,234,351. In agreement with these predictions, we found that infection of stratified epithelium did result in lytic replication in the terminally differentiating cells (Figure 4.1 and 4.3). This replication occurred spontaneously, e.g. without the addition of chemicals or expression of exogenous proteins. While previous data suggested EBV may not establish a persistent latent infection in epithelial cells, the absence of even a transient latency was unexpected. Productive replication simultaneously occurring in the entire population of infected cells has never been observed in vitro for EBV. Not only did EBV productively replicate in the stratified epithelium, but the level of virus production from these raft cultures was 500 fold higher than that reported for any other system 31,273. The inability to produce high titer EBV stocks has severely limited analysis of the virion. The high titer virus produced from raft cultures should now allow for many of these analyses. One particular area of interest is the production of a high resolution structure of the EBV virion using cryoem. The current EBV structure, published in 2012, is only at a resolution of 20 Å due to the low number of viral particles available for the reconstruction (for this analysis, a virus stock containing 3.6 x 10 9 genome equivalents was used) 76, while in 2000, a high resolution structure of HSV (8.5 Å), for which high titer stocks can easily be produced, was published in the journal Science 354. Negative staining we performed on virus preparations isolated from infected raft cultures were promising, requiring some purification 144

162 and minimal concentration before use in cryoem. These high titer virus stocks could also be used to further explore the viral RNAs present in mature virions and VLP 136, as well as the composition of tegument proteins. Within the raft cultures, the majority of infected epithelial cells expressed the full repertoire of lytic cycle proteins, based on the detection of immediateearly, early, and late proteins in as many as 68% of infected cells (Table 4.1). In B cells numerous cellular proteins, including the B-cell specific transcription factors Oct-2 and Pax5, directly and indirectly inhibit lytic replication 250,254, while ZEB1 and ZEB2, the myocyte enhancer binding factor 2 family, and the p65 subunit of NF-κB inhibit lytic replication in multiple cell types 52,85,91. The high level of productive replication observed in the raft cultures suggests that these inhibitory proteins may not be present, at least not at significant levels, in stratified epithelium, allowing for a more robust induction of lytic replication once infection occurs. Furthermore, the cellular factors required for productive replication should be present, though many of these are currently unknown. The raft culture system is ideal to delineate the cellular factors required for induction of productive replication. During terminal differentiation, epithelial cells activate the UPR and in turn may express XBP-1 18,296. For KSHV, a close relative of EBV, productive replication is initiated by the transcription factor Rta, a homologue of the EBV Rta immediate-early protein 184. Expression of XBP-1 alone is sufficient to activate the Rta promoter of KSHV resulting in productive replication in B cells 160,308,341,352. An early report suggested XBP-1 alone could also activate the Zta and Rta promoters of EBV in B cells 297, but four 145

163 subsequent publications have failed to show a strong induction of these promoters by XBP-1 alone or a correlation between XBP-1 expression and lytic replication 11,18,160,302. Furthermore, in all reports XBP-1 alone is insufficient to activate productive replication in epithelial cell lines 11,160,297. Therefore, while the UPR may trigger productive replication in the stratified epithelium, possibly in part through the activity of XBP-1, the proteins responsible for this activation have not been identified. The transcription factor Blimp1 has recently been proposed to activate lytic replication in OHL 18. Interestingly, productive EBV replication itself can induce the UPR. Endoplasmic reticular stress resulting in the UPR induces the expression of LMP1 and IL-8 in an LMP1 dependent manner, both proteins detected in infected raft cultures (Figure 4.1D, Table 4.1, Figure Appendix C.2D, and Figure Appendix C.3D). Therefore, the interplay between the UPR and productive EBV replication does warrant further investigation. The raft culture system could be used to examine this, as well as other cellular and viral requirements for productive replication. Numerous methods could be used to examine the contribution of individual viral or cellular proteins or signaling pathways to viral replication in stratified epithelium. These methods include mutagenesis of the viral genome, RNAi targeting cellular or viral transcripts or exogenous protein expression delivered by retrovirus transduction or transfection of primary epithelial cells growing in monolayer culture prior to seeding in raft cultures, or by chemical inhibitors. We verified the raft system can be used to examine the effects of viral mutations (Figure 5.1, the ΔTK virus) and chemical inhibitors (Figure 4.4 and Appendix A, treatment with acyclovir and ART 146

164 compounds) on viral replication. We, as well as other groups, have demonstrated that primary epithelial cells can efficiently be transduced (~30% transduction of primary gingival cells) with retroviral vectors. Many of the mutant EBV strains, including both the Akata and B95.8 rebv BACs are lacking the TK gene, and as such would be attenuated in raft cultures. Unfortunately, not only are the rebv BAC strains attenuated in viral replication and spread, but the initiation of infection by the 293 cells carrying the rebv BAC strains is low. Our initial experiments in raft cultures used the WT Akata rebv BAC and when raft cultures were harvested at various times PI, we detected at most 3 foci of infected cells in a single tissue section, and quite frequently detected none making any analysis with this viral strain difficult. Following infection of B cells, EBV initially expresses a subset of the lytic and latency-associated genes (the mrna for many of these possibly being carried into the cell with the virion 136 ), but within a few days, the B cell progresses to latency. In epithelial cells, EBV may initially express the same set of RNAs, which appears to be sufficient to trigger expression of the second wave of lytic proteins instead of the latency-associated proteins. As such, infection of stratified epithelium results in a virus expression profile completely unique to that observed following infection of B cells. Figure 6.1 summarizes the various virus expression profiles detected in EBV-infected cells. 147

165 Figure 6.1. Diagram summarizing the general EBV expression profile in different cell types. The general expression profile of EBV following infection of (1) B cells in vivo, (2) epithelial cells growing in monolayer culture, (3) epithelial tumors, and (4) normal stratified epithelium is indicated with the arrows depicting the temporal regulation of expression where it is known. EBV infected cells are depicted in green with the viral genome depicted in purple. Black indicates the latency-associated transcripts and blue indicated the lytic proteins. For B cells and epithelial cells growing in monolayer culture, the protein expression profile following external stimulation or exogenous expression of the immediate protein Zta is also indicated with the size of the text for each set of proteins representing the relative frequency of expression following stimulation. 148

166 Figure

167 6.2 Absence of a detectable latent phase in stratified epithelium Even though latently infected epithelial cells have never been identified in normal epithelium, given the propensity of EBV to establish a latent infection in all in vitro systems examined thus far, we thought EBV might establish a persistent latent infection in a small subset of cells or at least transiently express the latency-associated genes prior to lytic replication in the raft cultures. Although we examined multiple raft cultures at multiple times PI and used numerous assays, we were never able to identify a population of latently infected cells or detect any evidence of a latent infection. Specifically, we could not identify a population of cells which exclusively expressed the latency-associated proteins or the EBER transcript, the gold standard in the field for identifying latently infected cells (Figure 4.1). Given that in all forms of latency identified thus far, EBV expresses the EBER transcripts, we are confident in claiming that we could not detect any recognized form a latent infection in the stratified epithelium. To examine the possibility that EBV may establish a unique form of latency in which no viral transcripts were expressed, we infected raft cultures with two different viral strain each constructed to express a GFP reporter in all cells containing the viral genome (Figure 5.1). Even with these viral strains, we could not identify latently infected cells. Finally, using termini analysis, we were unable to detect amplification of the episomal form of the EBV genome prior to lytic replication (Figure 4.3), further supporting the notion that EBV did not initially establish a latent infection in the raft cultures. Although the lack of even a transient latency was somewhat unexpected, these data do clarify the observations that latent 150

168 EBV infection has never been identified in normal epithelial cells in vivo. As stated before, these experiments provide the first evidence of an in vitro system in which EBV undergoes lytic replication without first establishing latency. Therefore, in normal epithelium, EBV may not require reactivation from a latent state as is frequently assumed, but instead upon initial infection the virus immediately undergoes lytic replication. Furthermore, if EBV does not routinely establish a persistent latent infection in stratified epithelium, B cells would be the sole latent viral reservoir during persistent infection. One important implication for these findings are if EBV does not routinely establish a latent infection in epithelial cells, yet latent infection occurs during tumorigenesis, detection of a latent infection may serve as a good biomarker for pre-malignant lesions, and could potentially be used as an early screen for cancer. This is of significance since researchers are actively trying to identify an early marker for GC development to improve the currently poor prognosis for these patients. It would also be of interest to identify the cellular changes necessary for EBV to latently persist in stratified epithelium given that this is the form of infection identified in epithelial tumors. When EBV-associated carcinomas are propagated ex vivo, the tumor cells quickly lose the viral genome, suggesting maintenance of the viral genome only offers these cells a growth advantage in the tumor microenvironment 19,48,79. Therefore, it may not be possible to recapitulate the requirements for EBV in these cells in vitro. An alternate approach to study the role of EBV within these tumor cells would be to mix EBVpositive tumor cells with normal primary epithelial cells and immediately grow 151

169 these cells in raft culture. This would ensure the presence of the viral genome for at least a short time to examine if the presence of EBV effects cellular proliferation, differentiation, interactions with neighboring epithelial cells, and invasion into the dermal equivalent. One hypothesis to explain the absence of latency in normal epithelium is that healthy epithelial cells, like B cells, require expression of the lytic cycle anti-apoptotic proteins (BHRF1 and/or BALF1) to survive initial EBV infection 3,149. If latency occurs in the epithelial tissue, without expression of BHRF1, the cell immediately undergoes apoptosis. Indeed, in the infected raft cultures, we identified uninfected (e.g. Zta-negative) epithelial cells adjacent to the infected cells which showed evidence of apoptosis. The alphaherspesviruses, like EBV, have a biphasic lifecycle with two primary cell targets, each characterized by a distinct form of infection. For each of these viruses, primary infection and high levels of lytic replication occurs predominately in epithelial cells while a second cell type, either neurons or B lymphocytes, respectively, are the site for persistent latent infection with rare episodes of reactivation functioning to re-inoculate the epithelial tissue. 6.3 Dissemination throughout the stratified epithelium is dependent on lytic, not latent, replication It is currently unknown whether EBV spreads between epithelial cells in the oral epithelium, or whether infected epithelial cells frequently occur as isolated cells. This lack of understanding has impeded investigations into and understanding of virus transmission and maintenance. In OHL, EBV-infected 152

170 cells are located in discrete foci comprised of productively replicating cells with no evidence of an underlying latent infection 234. These data demonstrate that multiple epithelial cells can be infected by EBV at a single time, but since OHL is a disease strongly associated with immunocompromised hosts, we do not know if this mimics infection of a healthy host. A large study examining biopsy samples from normal epithelial tissue only found a few areas of infection (1.4% of the samples) 65. In these samples, EBV again localized to discrete foci of productively replicating cells with no evidence of a latent infection. But because the EBV-infected samples were so rare (n=3), it is not known if these results are typical of EBV infection in healthy individuals or if they are an anomaly. Furthermore, even if infection occurs as foci within epithelial tissue, until now there have been no methods to examine how these foci formed. Mechanisms that could result in the formation of infected foci include latent infection and subsequent cellular division (vertical transmission; this is the mechanism most often employed by EBV to spread in vitro), productive replication with progeny virions infecting neighboring cells (horizontal transmission, this is how the majority of viruses spread), or multiple cells infected simultaneously from a single virus-producing B cell. Figure 6.2 illustrates how these various forms of infection and spread might appear in stratified epithelium. In the raft cultures, infection did appear as discrete foci which expanded over time. This viral dissemination was dependent on lytic replication since spread was attenuated with a ΔTK mutant (Figure 5.1) and completely blocked at high concentrations (50 μg/ml) of acyclovir (Figure 4.1), both of which specifically inhibit lytic, but not latent, 153

171 Figure 6.2. Models for EBV infection and spread in stratified epithelium. Illustrations modeling six possible mechanisms of spread within the raft cultures with a brief description of each. EBV infected cells are depicted as green. The black arrow indicates a virus-producing B cell. The last option, infection followed by spread of de novo synthesized viral particles to neighboring cells is the only phenotype we observed in the infected raft cultures. 154

172 Figure

173 genome replication. Termini analysis verified the absence of significant latent genome replication (episomal only) occurring prior to dissemination of EBV in the raft cultures (Figure 4.3). Furthermore, infection was limited to non-replicating cells (Figure 4.2 and Table 4.2) precluding the possibility of vertical transmission. Taken together, these data suggest that infection of stratified epithelium occurs as foci and the formation of these foci is completely dependent on productive replication and horizontal transmission. Based on the rate of EBV shedding in saliva, previous reports postulated that infection must occur in a few discrete locations in the oral epithelium and these areas, or plaques, would grow exponentially until viral replication was controlled 94,121,121. We were able to verify the feasibility of this model experimentally in the raft cultures. As the infected epithelial cell produced progeny virions, these viral particles went on to infect neighboring cells, allowing us to produce the first mutlicycle growth curve for EBV (Figure 4.3). The number of infected cells, rate of dissemination, and level of virus production correlate well with those predicted to occur in individuals actively shedding virus 94. The rate limiting step to EBV replication in epithelial tissue appears to be the initial infection event. Once this initial infection occurs, the virus rapidly and efficiently spreads to neighboring cells (Figure 4.1, Figure 5.1, Figure 5.2, Figure 5.4, and Figure 5.6). If we assume that in normal epithelial cells successful infection requires initiation of the lytic cycle (a reasonable assumption given the inability we and other groups have had detecting latently infected cells), we can hypothesize why certain modes of infection are more efficient and why once 156

174 infection occurs, it can spread so readily between epithelial cells. One possibility is that induction of lytic replication requires a relatively high number of viral particles and VLP in a single cell. This requirement could be due to the need of high levels of vrna to produce adequate quantities of the immediate-early proteins to activate the lytic promoters. Alternately, successful lytic replication may require the presence of multiple viral genomes in a single nucleus. Using TEM, we observed a small number of epithelial cells in the infected raft cultures which did not show evidence of viral replication (i.e. no capsids within the nucleus), but did contain multiple naked capsids surrounding the nucleus, possibly docked at nuclear pore complexes. The lack of evidence of active viral replication in these cells suggested the capsids we observed surrounding the nucleus were viral particles recently delivered to the cell that had trafficked to the nucleus for genome delivery. What was intriguing about this finding was the sheer number of capsids observed surrounding the nucleus of a single cell (~42 capsids in a single nm tissue section). This would suggest that during cellto-cell spread, numerous viral particles ( 100) are transferred between cells allowing newly infected cells to receive a high initial dose of viral genomes and vrnas, resulting in successful initiation of lytic replication. On the other hand, during infection with CFV, the number of viral particles entering a single cell would likely be significantly lower. Could this explain, at least in part, why cell-tocell transmission is always more efficient than infection with CFV? Another important finding was that within the superficial layers of the epithelium, all 157

175 epithelial cells appeared to be equally susceptible to infection, with infection not limited to a small subset of epithelial cells as others have proposed. Very little is known about EBV transmission between adjacent cells. It would be of interest to examine the requirements for cell-to-cell spread. Firstly, does cell-to-cell transmission between adjacent epithelial cells require mature virions with an intact fusion complex? This question could be examined using the ΔgH Akata virus constructed by Hutt-Fletcher 207. This virus also fails to incorporate gl and gp42 into the virion 207. These virus-producing Akata cells could be transfected with a gh expression vector prior to induction to ensure initiation of infection in the raft culture. Secondly, does the presence of neutralizing Ab have any effect on cell-to-cell transmission? Third, what viral and cellular proteins are required for cell-to-cell transmission, and are these requirements distinct from those required for infection with CFV? 6.4 Exclusion of infection from basal cells Throughout these experiments, we routinely noticed that viral infection could not be detected in the basal layer (Figure 4.1, Figure 4.2, Figure 5.1, Figure 5.2, Figure 5.4, Figure 5.5, Figure Appendix A.3) regardless of the method used to inoculate the tissue or the method used to identify infected cells (IF for Zta, EBNA1, LMP1, or GFP and ISH for EBERs). Furthermore, when exposure to the virus-producing B cells was restricted to the basal lateral surface of the raft culture, infection could not be initiated (Figure 5.2). It is possible the epithelial receptor for EBV is excluded from the basal lateral surface of the epithelial cells 158

176 in raft culture, but this is not likely since previous reports have shown the epithelial receptor is exclusively localized to the basal lateral surface of polarized epithelial cells 274,317. Moreover, the virus-producing B cells were already present when the epithelial cells were seeded on the collagen matrix, prior to polarization. The requirement for the viral TK gene for efficient viral replication and spread in the raft cultures further supports the hypothesis that the basal epithelium is not a significant site of viral replication in stratified epithelium (Figure 5.1). Given that HSV-1, HSV-2, and VZV can infect and productively replicate in basal cells in raft cultures 5,117,202,298,324, and if KSHV-infected cells are seeded into raft cultures, the virus can be detected latently replicating in the basal cells 139, EBV is the first herpesvirus which is specifically excluded from basal epithelial cells. The reason for this exclusion is not know, but likely these cells either fail to express a factor which is required for infection, such as the viral receptor, or these cells express a factor which protects them from infection. Along this line of reasoning, although infection of raft cultures did not alter the expression of p16, p21, or p53, we noticed that viral infection was generally excluded from the p16, p21, and p53 positive cells, suggesting that the natural expression of these or similar cellular proteins in stratified epithelium may be inhibitory to successful viral infection and replication. If this is indeed the case, EBV would be the first herpesvirus with such a unique cellular requirement. It would be of interest to determine if the block to infection in the basal cells occurs at the point of attachment, entry, uncoating, trafficking, gene expression, or survival. FISH targeting the viral genome, CFV in which the genome has been labeled with BrdU, and 159

177 fluorescently labeled viral particles could be used to explore some of these possibilities. 6.5 The dual-tropism of EBV The cellular tropism of EBV is directly linked to the level of gp42 incorporated into the virion, with the level of gp42 incorporated into the virion dictated by the cell type producing the virus 12,204,334. As such, it is predicted that virus produced by epithelial cells should be highly infectious for B cells, but less infectious for epithelial cells, while virus produced by B cells should display an enhanced tropism for epithelial cells, but a decreased tropism for B cells. Indeed, the virus produced from the raft cultures demonstrated enhanced infectivity for primary B cells compared to B cell-derived virus (8 fold increased), but a decreased infectivity for stratified epithelium relative to the B cell-derived virus ( 25 fold reduced, Figure 5.3 and 5.4). These distinct viral tropisms were evident even if the inoculum was very dilute and did not show variations in conjunction with the volume of raft homogenate or supernatant used in the inoculum (often less than 1 μl per reaction for the lower dilutions) suggesting these preferences were virus intrinsic. Furthermore, the presence of 200 μl of tissue homogenate isolated from infected raft cultures had minimal effect (0.6 fold reduction) on the infectivity of B cell-derived virus (Figure 5.5). Therefore, the differences we observed in viral tropism for the virus produced in raft cultures versus that isolated from B cells likely reflect differences in the incorporation of gp42 into the virion. The inhibitory effects of gp42 on infection of epithelial cells could be 160

178 directly tested using the Δgp42 Akata strain 333. Epithelial-derived virus for both the WT and Δgp42 Akata strains could be generated in raft cultures and then used to inoculate additional raft cultures to verify that the incorporation of gp42 into the viral particles is dictating the tropism of the resulting virus. Previous reports have shown the dual-tropism model only applies to infection with CFV and not to cell-to-cell transmission 274,275. Therefore, these experiments should be conducted with CFV. An additional benefit of this system would be the production of high titer virus which readily infects epithelial cells. This high titer virus stock could be used for additional assays such as virus overlay protein binding assay to identify the EBV receptor on epithelial cells, a technique recently employed to identify the cellular receptor for respiratory syncytial virus 303. The basis of the dual-tropism model is the interactions between gp42 and HLA class II and how cellular expression of HLA class II can subsequently alter the virus. While epithelial cells generally do not express HLA class II, these cells can be induced to express HLA class II if stimulated with IFN-γ or other proinflammatory mediators, acting as non-professional antigen presenting cells 252. Theoretically, this could influence incorporation of gp42 into the virion and the subsequent tropism of the virus. Essentially, the tropism of virus produced by epithelial cells could be dictated by the local cytokine milieu, producing B cell tropic virus immediately following infection, then later during infection when there is localized inflammation, the virus produced by the epithelial cells could switch affinity to become epitheliotropic. To test this hypothesis, exogenous IFN-γ, as well as other pro-inflammatory cytokines, could be added to infected raft cultures. 161

179 After establishing HLA class II expression by these cells following cytokine treatment (PCR, IF, or immunoblot depending on the most convenient assay to perform with a genetically diverse set of patient samples), virus could be isolated to assess the level of gp42 in purified virion (immunoblot) and the tropism of the virus (i.e. is the virus now epitheliotropic instead of B cell tropic). There are caveats to this experiment such as the presence of IFN-γ may inhibit viral replication (though this data itself would be of significant interest), and EBV encodes proteins and noncoding RNAs which can alter portions of the IFN signaling pathways 217 and therefore exogenous IFN-γ may have little or no effect on these cells (again, these data would still be of interest). 6.6 Response of epithelial cells to infection Very little is known about the epithelial cell response to EBV infection. This information is pertinent for understanding both the immune response to EBV infection in the oral cavity and the role of EBV in tumorigenesis. EBV infection of raft cultures did not alter the expression of the early differentiation markers K5 and involucrin, the proliferation marker ki67, or the cell cycle regulators p16, p21, and p53 (Figure 4.1A-B, Figure 4.2A-B, and Figure Appendix B), each of these proteins frequently deregulated during tumorigenesis. These data suggest that the cellular changes that occur in EBV-associated carcinomas are unique to these tumor cells and not typical of EBV infection in stratified epithelium. In monolayer culture, EBV infection induces up-regulation of the tumor suppressors p16 and p21 313, highlighting how stratification of the epithelial cells can influence 162

180 the cellular response to infection. Infection also did not result in activation of the IFN pathways (Figure Appendix C.2C and Figure Appendix C.3C), though the ELISA kit we used did not offer full coverage of all of the IFN-α or the IFN-λ isoforms. To verify the IFN pathways are inactive in the infected raft cultures, the phosphorylation status of STAT-1 (common to all of the IFN signaling pathways) could be assessed using a phosphospecific antibody. Alternatively, expression levels of an IFN-responsive gene could be used to determine if the IFN pathways are active. Infection did induce cytopathic effects, apoptosis, up-regulation of the pro-inflammatory cytokine IL-8, and expression of the anti-inflammatory cytokine IL-10 (Figure 4.2C-D, Figure Appendix C.2D, and Figure Appendix C.3D-E). A comprehensive analysis, such as RNA seq 335, should be performed on infected and uninfected raft cultures to comprehensively examine the cellular and viral RNA expression profiles. During these experiments we noticed that the infected tissue showed a loss of glycogen storage (the loss of vacuoles at the site of infected foci; Figure 4.2D) suggesting the possibility of an increased rates of glycolysis. Two factors could explain this observation. (i) One might imagine a scenario in which the infected cells have an increased metabolic rate relative to their uninfected counterparts, resulting in a higher rate of energy consumption. (ii) By TEM, we saw that cells containing a high number of viral particles frequently contained a lower number of intact mitochondria. The increase in the cleavage of caspase 3 in the infected cultures, which can be instigated by the release of cytochrome C from the mitochondria, further supports the notion that there might be alterations 163

181 to the mitochondria in infected tissue. Mitochondrial alterations have been reported following HSV-1 and HSV-2 infection 219, and expression of Zta has been shown to alter mitochondria and reduce mitochondrial DNA synthesis 162,339. It might be of interest to examine the infected tissue to determine if there is a difference in the number of intact mitochondria in the infected versus uninfected tissue. This could be accomplished by staining tissue sections for a mitochondria specific marker such as Hsp-60 or the MTCO2 mab. MitoTracker is a fluorescent dye which labels mitochondria in live cells, the accumulation of which is dependent on mitochondrial membrane potential, the dye being retained following fixation. Alternately, MitoTracker or a similar dye could be used to examine the membrane potential of the mitochondria in the cells which are disassociating from the raft cultures using flow cytometry. The raft culture system is a powerful tool to examine the innate immune response to EBV infection. These data would be biologically relevant given our raft cultures are produced from primary cells and as such should contain functioning innate immune sensors. It would be particularly interesting to examine whether the recently identified adaptor protein STING is activated in the EBV-infected raft cultures given that STING was shown to be activated in mice following infection with the murine gammaherpesvirus MHV68. STING acts as an innate immune sensor for cytosolic dsdna triggering activation of the type I IFN pathway following infection with microorganisms which contain a dsdna genome 127. Importantly, the interferon-inducible protein IFI16 was recently identified as a nuclear dsdna innate immune sensor, which is activated following infection by 164

182 CMV, HSV, and KSHV triggering the expression of anti-viral cytokines and blocking viral replication via activation of STING 72,140,151,172,173,240. The viral proteins pul83 and ICP0 have both evolved to inhibit activation of IFI16 and the inflammasome by CMV and HSV, respectively 140,172,240. The latent EBV genome can be detected by IFI16 6, though it is currently unknown if this activation inhibits viral replication and whether EBV has evolved mechanisms to evade this immune sensor. Given the raft culture system is the only model for infection of stratified epithelium, it would be interesting to examine the effects of various cytokines on virus replication and production. Furthermore, since EBV infection induces high levels of the immunosuppressive IL-10 (Figure Appendix C.2D), it would be interesting to examine how EBV infection might alter the tissues susceptibility to infection by other microorganisms. 6.7 Primary infection Due to the extreme difficulties infecting epithelial cells with CFV and the significant improvement observed when virus-producing or virus-coated B cells are used to initiate infection, some researchers have suggested EBV must come into contact with a B cells before it can infect the oral epithelium 274,275. This would make B cells, and not the oral epithelium, the primary site of infection. Our data showed that while infection of epithelial tissue is enhanced with virusproducing cells, CFV can initiate infection (Figure 5.4). Furthermore, while infection with CFV, especially epithelial-derived CFV, required high titer virus (1 x 10 9 genome equivalents to initiate infection), infection could be initiated using a 165

183 much lower inoculum by virus-producing epithelial cells (as low as 6 x 10 6 genome equivalents, Figure 5.6). Our data does not exclude the possibility that B cells could initially be infected following exposure to EBV, but it does demonstrate that initial infection of B cells is not a requirement for successful transmission. Instead, EBV can use multiple mechanisms to infect stratified epithelium with variable efficiency. To determine if the presence of gp350 inhibits infection of epithelial cells, a Δgp350 viral strain could be used 132. Although infection of epithelial tissue with virus produced by epithelium was not as robust as other methods of transfer, primary infection in the oral mucosa may serve a dual purpose. The virus shed in saliva was found to only weakly bind to B cells, possibly because the virus was coated with neutralizing Ab 94,319. Therefore, an initial round of replication in the epithelium of a naive host would result in the production of viral particles devoid of neutralizing Ab. Furthermore, the infected epithelial tissue produced IL-10 (Figure Appendix C.3D), a B cell growth factor. IL-10 is required for B cell survival immediately following EBV infection 137. As such, by infecting epithelial tissue, EBV not only produces virus highly infectious for B cells, but also produces cytokines which encourage survival of B cells following infection. Another concern has been to identify where within the stratified epithelium primary infection can occur, i.e. does the virus need access to the basal layer, thus requiring microabrasions, or can the virus infect at the apical surface? We determined that infection can be initiated at the apical surface of stratified epithelium, and likely many of the suprabasal layers (Figure 5.2), but was 166

184 excluded from the basal layer as previously discussed. Therefore, during primary infection, the virus or virus-producing cells only require contact with the superficial layers of the tissue and do not need to migrate through the epithelium. This may be especially important for transmission via virus-producing epithelial cells which might not express the appropriate adhesion molecules for tissue migration. When raft cultures were inoculated with virus-producing B cells, virus production peaked between days 4-6 PI (after this time point viral titers no longer increased exponentially, and instead at best doubled each day). Currently, we do not know why viral titers peaked at this time point. It is possible that the rate of viral spread no longer exceeded the rate of death of infected cells given that the majority of the raft tissue was already infected. One argument against this hypothesis is that raft cultures reached peak virus production at a similar time point regardless of the efficiency of initial infection. An alternate explanation for this phenomenon is that viral titers started to wane as the raft aged. Another group noticed that between 6-10 days after airlifting, raft cultures were more resistant to herpesvirus infection and showed lower levels of viral replication 5. The reason why older rafts are less efficient at viral replication is not understood, but it could be a lack of cell cycle proteins as fewer and fewer of the cells are actively replicating, or it could be the presence of inhibitory cytokines, such as IL- 1α and IFN-λ in the older raft cultures (Figure Appendix C.2B-C). Regardless of the reason, in the natural host loss of cellular proliferation and build up of inflammatory cytokines may not be as relevant. Therefore, while infection with 167

185 epithelial derived CFV did not result in overall amplification of the virus in the raft cultures by 8 days PI, in the body, given more time, more tissue, and the ability to spread to multiple sites in the oral cavity, it may eventually result in an overall amplification. 6.8 Virus egress and shedding During viral egress, EBV is thought to reactivate in a B cell which has been stimulated to differentiate into a plasma cell, with the resulting virus moving into the oral cavity either by transcytosis or infection of the epithelium. The virus isolated from saliva contains high levels of gp42, suggesting it is predominately produced by epithelial cells 134. The fact that infection could not be initiated by virus-producing B cells restricted to the basal lateral surface (Figure 5.2) suggests that direct infection and transcytosis of viral particles across the basement membrane and basal cells may not be a significant source of infection for the oral epithelium. Instead, virus-producing cells and CFV appear to require direct contact with the superficial layers of the stratified epithelium to initiate infection. This contact could readily occur if there is an open sore or bleeding in the mouth, which does occur with regular frequency. Additionally, the tissue resident DC and LC harbor latent EBV infection which can reactivate to infect nearby epithelial cells 316,330. It would be interesting to examine the ability of DC to initiate infection in raft cultures. Finally, at least a portion of the recurrent shedding from persistently infected individuals could be the result of re-exposure to EBV and super infection, given that after primary infection neutralizing 168

186 immunity is never achieved. In fact, since a pre-exposed individual should have high levels of anti-gp350 Ab in their saliva, re-infection may occur with an even higher efficiency than primary infection. Figure 6.3 summarizes our data on how EBV can initiate infection of stratified epithelium during entry and again during egress. 6.9 Implications for vaccine design and prevention Our data confirm that the oral mucosal epithelium is the likely site of primary infection and importantly, a site where high levels of virus replication can occur. During primary infection, EBV may most readily be transferred between individuals by virus-producing epithelial cells or as CFV coated with anti-gp350 Ab. Both of these factors complicate the implementation of viral control mechanisms, given that the current vaccination strategies for EBV involve eliciting a strong humoral response to neutralize infection with CFV, many vaccines specifically targeting gp350 (as reviewed in 35 ). First, the viral protein gp350 may not be a good target for vaccine development since anti-gp350 Abs would not prevent infection of the oral epithelium and in fact may even enhance infection of this tissue (Figure 5.4) 319. Vaccines which target gp350 have failed to prevent infection in phase II trials. It is important to keep in mind that vaccines are often designed to neutralize infection from a low dose challenge, and not after multiple rounds of virus replication and amplification as would occur if the epithelial tissue is infected. This is why it is essential to know the primary site of infection to design a vaccine which provides adequate coverage and protection. 169

187 Figure 6.3. Model for EBV infection of stratified epithelium. An illustration depicting modes EBV can use to infect epithelial cells grown in raft culture. Raft cultures can be infected with EBV when exposed to virus-producing B cells at the apical surface (whether or not the tissue was wounded), but not at the basal lateral surface. B cell-derived CFV can infect raft cultures while epithelial-derived CFV does so much less efficiently. The presence of anti-gp350 Ab can enhance infection of the epithelial-derived CFV. EBV can also be transmitted by virusproducing epithelial cells. The figure legend is given on the right. 170

188 Figure

189 GB, or another glycoprotein essential for membrane fusion, may serve as a better target to neutralize infection of all cell types. The vaccine should also elicit an Ab response which is targeted to and actively shed in the oral mucosa (IgA). Second, Abs are thought to have little to no effect on cell-to-cell virus transmission, and as such a humoral immune response may not block transmission by virus-producing epithelial cells. This could be tested experimentally by incubating virus-producing epithelial cells with neutralizing antigp350 Abs prior to inoculating raft cultures. It is possible the expression of viral glycoproteins on the membrane of infected cells will allow Ab to coat the cell, effectively blocking virus transmission. Control of EBV infection is especially important in the immunocompromised host, such as HIV-positive individuals and post transplant patients. These groups are at an increased risk of developing EBV-associated tumors (both carcinomas and lymphomas). Although EBV-associated tumors are characterized by latent infection, the development of these tumors often corresponds with high levels of lytic replication. As such, there is great interest in finding methods to reduce productive EBV replication, especially in the immunocompromised host, in hopes of impeding the development of EBVassociated tumors. The anti-viral compound acyclovir was very efficient at inhibiting viral replication and spread in the raft cultures (Figure 4.4). Therefore, treatment with the guanine nucleoside analogues should reduce EBV replication to negligible levels. Interestingly, the HIV protease inhibitors in Kaletra reduced EBV replication ~10 fold (Figure Appendix A.2). To define the mechanisms 172

190 involved in this reduction, it is vital to determine if it is lopinavir, ritonavir, or a combination of both which display this anti-viral activity. Given the reduction to viral replication observed in the raft cultures treated with Kaletra (Figure Appendix A.2), it would be of interest to determine if Kaletra is also able to inhibit viral replication in B cells, or is this inhibition specific for epithelial tissue. The only change we detected in the Kaletra treated raft cultures was a decreased level of LMP1 expression (Figure Appendix A.3 and Figure Appendix A.4). If activation of the UPR activates both productive replication and LMP1 expression in infected cells 302, the decrease in LMP1 expression suggests the presence of Kaletra might be blocking the UPR pathway. For those high risk patients (post-transplant or otherwise severely immunocompromised), prophylactic anti-viral therapy may be the most promising option for disease prevention. Although anti-viral treatment would not block EBV infection, it would limit viral replication, decreasing the likelihood of transfer to the B cell compartment, while lowering the viral load in these individuals should infection occur. Although EBV was identified decades ago, and the association between EBV and various tumors has been known for many years, the prevalence of this virus in the human population has continued, unabated by attempts to control or prevent infection. The raft culture system will be an invaluable tool for understanding the natural lifecycle of EBV, as well as developing methods to prevent infection and control viral replication. 173

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220 Appendix A: The Effects of ART Compounds on EBV Replication in Stratified Epithelium EBV is frequently associated with malignancies occurring in HIV patients, posing a significant health burden in this population 22. Finding methods to control EBV replication in these patients is of upmost importance. The progression of HIV infection to acquired immunodeficiency syndrome can be abated with ART treatment, but HIV patients on ART treatment, including the protease inhibitors Kaletra and amprenavir (Figure Appendix A.1), experience a significant increase in the occurrence of oral lesions, sometimes leading to treatment noncompliance. The cause of these lesions is currently not known, but is an active area of investigation. Recently, an HIV protease inhibitor was found to inhibit KSHV (a gammaherpesvirus) replication in vitro demonstrating these drugs can have off target effects 71. Previous data suggest treatment of primary raft cultures with amprenavir and Kaletra increases proliferation and alters the differentiation of the raft tissue to that of a wound healing environment 128,129. Given that we only detect productive EBV replication in non-proliferating and terminally differentiating epithelial cells, we hypothesized treatment of infected raft cultures with amprenavir and Kaletra would decrease EBV replication in raft cultures due to cellular hyperproliferation and deregulated differentiation. To address this hypothesis, we treated infected PGEC raft cultures with various concentrations of Kaletra and amprenavir based on the C max of these drugs and previously published reports starting at the time of infection, and measured the level of viral replication at 6 days PI 128,129. Treatment with Kaletra, but not 203

221 Figure Appendix A.1. The properties of Kaletra and amprenavir. The composition and basic properties of each of the ART compounds used in these experiments are listed for (A) Kaletra and (B) amprenavir. 204

222 Figure Appendix A.1 A Kaletra Protease inhibitor Fixed dose combination drug Lopinavir protease inhibitor Ritonavir protease inhibitor, in this formulation ritonavir is at a subtherapeutic dose, and is not included to directly treat HIV but because it inhibits host enzymes which metabolize other protease inhibitors B Amprenavir Protease inhibitor 205

223 amprenavir, inhibited EBV replication in the raft cultures (Figure Appendix A.2A). When we repeated this experiment in raft cultures derived from a second set of patient samples and with a broader range of concentration for Kaletra to include the C max, the presence of Kaletra again inhibited viral replication, in a dose dependent manner (Figure Appendix A.2B). Next, we wanted to examine possible causes for this inhibition using IF staining for viral proteins in tandem with cellular markers for proliferation and differentiation. The drug treated tissue appeared relatively normal by H&E staining with typical cytopathic effects following EBV infection; the only noted exception being the absence of cells dissociating from the infected foci in the raft cultures treated with 6 μg/ml Kaletra (Figure Appendix A.3A). K5 and involucrin, both markers for early stages of terminal differentiation, were not altered by treatment with either compound (Figure Appendix A.3B-C). Cellular replication in these raft cultures was also unaffected following drug treatment (Figure Appendix A.3D). The only viral protein which appeared to be altered following treatment with Kaletra was LMP1 (Figure Appendix a.3d). Though the size of the LMP1-expressing foci did not change following treatment (suggesting EBV is still able to spread within the epithelial tissue), there was a decrease in the level of LMP1 expression, as verified by quantifying the mean fluorescence intensity (MFI) of the LMP1- expressing foci (Appendix A.4). It was interesting to see that the HIV protease inhibitor Kaletra was able to inhibit EBV replication in stratified epithelium. At this time, we do not know the mechanism responsible for the decrease in viral replication in the raft tissue 206

224 Figure Appendix A.2. Kaletra reduces the level of EBV replication in raft cultures. Wounded raft cultures were inoculated with 2.5 x 10 6 WT virusproducing B cells or mock infected 4 days after airlifting and the number of encapsidated genomes produced by each raft cultures was quantified 6 days PI by qpcr. (A) Following infection the raft cultures were treated with the indicated concentration of Kaletra, amprenavir, or vehicle alone. Each bar represents the percentage of encapsidated genomes produced by each raft relative to the vehicle control. (B) Raft cultures were infected as in (A) and treated with the indicated concentration of Kaletra. The number of encapsidated genomes produced by each raft was quantified and reported as in (A). Each bar represents data from one experiment conducted on one patient set. The experiments in (B) were conducted on a different patient set than that used in part (A). 207

225 Percentage of encapsidated genomes per raft relative to vehicle control Percentage of encapsidated genomes per raft relative to vehicle control Figure Appendix A.2 A Infected B Mock, 9.8 μg/ml Vehicle 3 μg/ml 6 μg/ml 9.8 μg/ml Infected Concentration of Kaletra 208

226 Figure Appendix A.3. The effects of Kaletra and amprenavir on the expression of cellular and viral proteins. Four days after airlifting wounded raft cultures were infected with 2.5 x 10 6 WT virus-producing B cells or mock infected. Immediately following infection, the cultures were treated with the indicated concentrations of Kaletra, amprenavir, or vehicle. Raft cultures were harvested 6 days PI and stained as indicated with (A) H&E (B) Zta (green) and involucrin (red) IF, (C) K5 (green) and EA-D (red) IF, and (D) ki67 (green) and LMP1 (red) IF. These experiments have been conducted one time on one set of patient sample. Magnification 10x. 209

227 Figure Appendix A.3 A Mock Infected - 6 μg/ml Kaletra Infected - Vehicle Infected 3 μg/ml Kaletra Infected 6 μg/ml Kaletra Infected 2 μg/ml Amprenavir Infected 5 μg/ml Amprenavir 210

228 Figure Appendix A.3 continued B Zta Involucrin Mock infected Zta Involucrin Infected - Vehicle Infected 3 μg/ml Kaletra Zta Involucrin Infected 6 μg/ml Kaletra Zta Involucrin Infected 2 μg/ml Amprenavir Zta Involucrin Infected 5 μg/ml Amprenavir Zta Involucrin 211

229 Figure Appendix A.3 continued C K5 EA-D Mock infected K5 EA-D Infected - Vehicle Infected 3 μg/ml Kaletra K5 EA-D K5 EA-D Infected 6 μg/ml Kaletra Infected 2 μg/ml Amprenavir Infected 5 μg/ml Amprenavir K5 EA-D K5 EA-D 212

230 Figure Appendix A.3 continued D ki67 LMP1 Mock infected ki67 LMP1 Infected - Vehicle Infected 3 μg/ml Kaletra ki67 LMP1 Infected 6 μg/ml Kaletra ki67 LMP1 Infected 2 μg/ml Amprenavir ki67 LMP1 Infected 5 μg/ml Amprenavir ki67 LMP1 213

231 Figure Appendix A.4. Treatment with Kaletra reduces the level of LMP1 expression in raft cultures. Wounded raft cultures were infected with 2.5 x 10 6 WT virus-producing B cells or mock infected 4 days after airlifting, treated with the indicated concentration of Kaletra, amprenavir, or vehicle, and harvested 6 days PI. The expression of LMP1 was examined in tissue sections by IF staining. (A) Quantification of the size of LMP1-expressing foci (μm 2 ) in raft cultures. The size of the infected foci was measured from 10 fields of view per sample. (B) The MFI of the LMP1-expressing foci reported in (A). For the mock infected tissue, the MFI represents the background fluorescence following IF staining in matching layers of tissue. These experiments have been conducted one time on tissue from one set of patient sample. 214

232 MFI for LMP1 Average size of infected foci (μm 2 ) Figure Appendix A.4 A Mock Vehicle 3 µg/ml Kaletra 6 µg/ml Kaletra 2 µg/ml 5 µg/ml Amprenavir Amprenavir B 800 Infected Mock Vehicle 3 µg/ml Kaletra 6 µg/ml Kaletra 2 µg/ml 5 µg/ml Amprenavir Amprenavir Infected 215

233 treated with Kaletra. It is possible this compound has an effect on the tissue, other than hyperproliferation and alterations to early differentiation, that in turn limits viral replication. Alternately, this compound could directly affect viral replication. Kaletra was also shown to inhibit HPV replication in stratified epithelium (Israr and Meyers, manuscript in preparation). Importantly, a recent study demonstrated that following primary infection, EBV replication is suppressed more rapidly in HIV-positive infants treated with Kaletra than in HIVpositive infants receiving the non-nucleoside reverse transcriptase inhibitor nevirapine, suggesting the decrease we observed in EBV replication following Kaletra treatment in raft cultures may hold true in the clinical setting as well 288. Expression of the viral protein LMP1 appears to be down-regulated in the Kaletra treated rafts, though the cause and significance of this down-regulation is currently unknown. Additional experiments need to be performed to investigate how Kaletra is inhibiting EBV replication. These investigations could lead to novel drug targets for EBV and strategies to control EBV replication in HIV-positive individuals. 216

234 Appendix B: Expression of p16, p21, and p53 in EBV-Infected Stratified Epithelium A previous report showed that following EBV infection, htert immortalized epithelial cell lines in monolayer culture upregulate p16 and p21 expression resulting in cellular senescence 313. These pathways are also frequently mutated in NPC and GC 179,313. The cell cycle regulator p53 can also be activated by various pathways following viral infection. Little is known about the host response to EBV infection in stratified epithelium, therefore we wanted to investigate whether any of these cell cycle regulators were activated following infection of stratified epithelium. To this end, we examined infected raft cultures by IF staining for the expression of p16, p21, and p53 alone (Figure Appendix B.1A) and in combination with the immediate-early viral protein Zta (Figure Appendix B.1B). Raft cultures injected with HeLa cells, LCLs, and 293 cells served as positive controls for the detection of p16, p21, and p53 respectively. In addition, each of these proteins are expressed in basal cells during normal epithelial cell differentiation 196,205,309,344. After epithelial cells leave the basal layer, these proteins are down-regulated even though the cells do not resume proliferating. This fact suggests the possibility that these cell cycle regulator pathways are no longer active in terminally differentiated epithelial cells. As seen in Figure Appendix B.1A, we detected no changes in the expression of p16, p21, or p53 following viral infection. Instead, we noticed that viral infection, as detected by cytopathic effects, appeared to be excluded from the layers of tissue expressing these proteins. The diminished intensity of Hoechst staining evident in these 217

235 tissue sections was typical of infected tissue, a result of chromatin remodeling due to active viral replication. To examine whether the expression of these proteins was truly segregated from infection, infected raft cultures were costained for p21 and Zta (Figure Appendix B.1B). The majority of cells expressing p21 did not express Zta, but this exclusion was not absolute. A minority of cells at the margin of infection expressed both p21 and Zta. These data demonstrate that the cell cycle regulators p16, p21, and p53 are not activated following EBV infection. At this time we are not sure if the virus is able to circumvent activation of these pathways or, given that the infected cells have already ceased proliferating, these pathways are no longer active. Other groups have reported that productive EBV replication in immortalized cell lines activates the DDR through the ataxia telangiectasia-mutated signal transduction pathway resulting in p53 activation, though downstream p53 signaling is blocked, at least in part, by Zta 159. Therefore, it would be interesting to see if the DDR is activated in the infected raft cultures. 218

236 Figure Appendix B.1. Expression of p16, p21, and p53 in EBV infected raft cultures. Raft cultures were infected with 2.5 x 10 6 WT virus-producing B cells or mock infected 4 days after airlifting and harvested at the indicated times PI. (A) The expression of p16, p21, and p53 were assessed in raft cultures 6 days PI. Phase contrast images were taken of the infected tissue to identify the areas of infection based on the cytopathic effects evident in the tissue, as indicated by the red bracket. Raft cultures spiked with the following cells served as positive control: HeLa cells for p16, LCL for p21, and 293 cells for p53. The HeLa cells and the 293 cells are indicated by the white arrows. The LCLs did not express significant levels of p21 and therefore, are not indicated. In addition to the positive control cells, p16 can be detected as diffuse staining in the cytoplasm of basal cells, p21 can be detected in the nuclei of the J2 3T3 feeder cells in the collagen matrix and less intensely in the nuclei predominately of basal cells, while p53 can be detected in the nuclei of predominately the basal cells as expected. Magnification 10x. (B) The expression of p21 in infected raft cultures with the infected cells identified by the expression of Zta at 4 (left) and 6 (right) days PI. For enhanced visualization of these nuclear proteins, Hoechst staining has been removed from the merge image on the left while the merge image on the right is of a higher magnification. 219

237 Figure Appendix B.1 A p16 Positive control p16 Infected raft p21 p21 p53 p53 B Zta p21 10x Zta p21 20x 220

238 Appendix C: The Cytokine Expression Profile Induced by EBV- Infection in Stratified Epithelium Since EBV infection has not been studied in stratified epithelium, the cellular response to infection is largely unknown. During the course of our studies, we noticed a drastic difference in the ability of the epithelial-derived virus to infect epithelial tissue compared to the B cell-derived virus (Figure 5.4). This begged the question: does the epithelial-derived virus stock contain anti-viral factors, such as cytokines, which inhibit infection or viral replication? We addressed this question by adding tissue homogenate to raft cultures infected with virus-producing B cells and B cell-derived CFV and quantifying the resulting level of viral replication (Figure 5.5). The presence of the tissue homogenate only had a slight effect on virus production (~0.6 fold decrease). The differences we observed in infectivity were reversed when we infected primary B-cells (Figure 5.3), indicating this phenomenon is likely a result of the dual-tropism displayed by EBV. Furthermore, these distinct viral tropisms were evident at all dilutions used in each of these assays, and did not vary in relation to the total volume of virus containing supernatant or tissue homogenate used for the inoculum, again suggesting this property is virus intrinsic and not from the supernatant or homogenate. Nevertheless, it is still of significant interest to determine how primary epithelial tissue responds to EBV infection and if there is an innate immune response elicited by the epithelial cells following infection. Over the course of our experiments, we noticed the viral titers in the raft cultures infected with virus-producing B cells peaked between 4 and 6 days PI, generally only 221

239 doubling after day 6 PI regardless of the final viral titer or the extent of infection throughout the tissue. These data suggest the epithelial cells may be establishing an anti-viral state within the infected tissue, as would be expected following viral infection. Therefore, we performed preliminary experiments to determine the cytokine response elicited by EBV infection in raft cultures. Although we cannot perform an extensive study of the anti-viral response occurring in the infected tissue at this time, we did want to examine the level of IFNs present in the tissue homogenates. We particularly wanted to examine the type I IFNs (α and β) given their known ability to induce a strong anti-viral state, and the type III IFNs (λ), which are specifically produced by virally infected epithelial cells to induce an anti-viral state in the surrounding epithelial tissue (reviewed in 49 ). Using the VeriPlex TM Human Cytokine 16-Plex ELISA Kit, we were able to measure the level of 16 cytokines, namely IFN-α, IFN-β, INF-γ, IFN-λ, IL-1α, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-23, and TNF-α, in the infected raft cultures. A general schematic of how this multiplex ELISA assay is designed is outlined in Figure Appendix C.1. First, we wanted to establish if the cytokine profile of the raft tissue changed with viral infection. Therefore, we quantified the level of cytokines present in the homogenate isolated from PGEC and PTEC raft cultures at 0, 2, 4, and 6 days PI and from mock infected tissue at 8 days PI. The complete cytokine profile is shown in Figure Appendix C.2A, with the exclusion of IL-1α which is shown alone (Figure Appendix C.2B) due to the large scale necessary for this cytokine. The standard curves for both IL-23 and TNF-α were invalid and therefore the data obtained for these two cytokines are not reported. 222

240 Figure Appendix C.1. Schematic of the VeriPlex TM Human Cytokine 16-Plex ELISA Kit. A general schematic of the VeriPlex TM Human Cytokine 16-Plex ELISA Kit. Each well of a 96-well plate was coated with capture Ab arranged in the order shown on the right. The captured cytokines were detected using a second biotin labeled Ab followed by incubation with streptavidin-horse radish peroxidase. Horse radish peroxidase substrate was added to each well, resulting in chemiluminescence. The pixel intensity for each spot was captured and quantified by a ChemiDoc MP imaging system. Each well contains a reference spot used to normalize the values. The first two columns of the 96-well plate contained serial dilutions of a standard with known quantities of each cytokine. From these values, a standard curve was generated using the Q-View TM software (bottom image, a sample standard curve for human IL-10 is shown with the regression model used to generate the curve, 4PL, indicated). The values for each test sample were then assigned based on the standard curve for each cytokine. 223

241 Figure Appendix C.1 96-well plate Spot location within each well IFN-α IFN-β IFN-γ IFN-λ IFN-1α IL-4 IL-5 IL-6 IL-8 IL-10 IL-12 IL-13 IL-15 IL-17 IL-23 TNF-α Ref Spot 224