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EBV)Episomes in Latently Ulfected B-Lymphocytes by Mediating Their

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EBV)Episomes in Latently Ulfected B-Lymphocytes by Mediating Their -----TU --:-* -- * - - -- - -- *.- -- RECONSTITUTION OF EPSTEIN-BARR NUCLEAR ANMGEN 1 (EBNA1)- MEDIATED PLASMID SEGREGATION IN BUDDING YEAST REQUIRES EUMAN EBP2 Priya Kapoor A thesis subrnitted in codomiity with the requirements for the de- of Master of Science Graduate Department of Molecular and Medical Genetics University of Toronto @ Copyright by Priya Kapoor, 2001 The author has gfaated a non- L'auteur a accordé une licence non exclusive licence dowing the exclusive permettant (i la National Li- of Canada to Biblioth&que nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microfonn, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format blectronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neitha the droit d'auteur qui protège cette thèse. thesis nor substantial extracts &om it Ni la thèse ni des extraits substantiels may be printed or othefwjse de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son Reconstitution of Epstein-Barr Nuclear Antigen 1 (EBNA1)-Mediateâ Plasrnid -Lu- -- A Segregation In Budding Yeast Rquires Human EBP2 by Priya Kapoor Masten of Science, Graduate Department of Molecular and Medical Geneticq University of Toronto, 2001 ABSTRACT Epstein-Barr nuclear antigm 1 (EBNAl ) govems the stable segregation of Epstein- Barr virus (EBV)episomes in latently Ulfected B-lymphocytes by mediating their attachment to the host metaphase chromosomes. The EBNAl residues that mediate chromosome attachrnent and DNA segregation activity bind to the human EBP2 (hEBP2) protein, which is a component of the cellular metaphase chromosomes. To investigate the importance of hEBP2 for EBNAl-mediateà DNA partitioning, 1 attempted to reconstitute the EBV-based segregation system in cerevisiue. 1 found that stable segregation of a yeast repücating plasmid containing the EBV segregation element, FR, required both EBNAl and hEBP2. An EBNAl mutant that cannot bind hEBP2 failed to support segregation of the FR plasmid, indicating that an EBNAI-hEBP2 interaction is necessary. My nsults pmvide dinct eviâence that hEBP2 is required for EBNAI- mediated segregation and dernonstrate that EBV-based segregation can occur in budding yeast . - A - First and fomnost, 1want to thank my supervisor, Dr. Lori Frappier. 1 thaak her for her encouragement, support and brilliant ideas. Moreover, 1thank her for being patient with me and for guidiag me through the tough times. 1also want to thank the wonderful members of the Frappier laboratory, both past and present, which includes Dr. Vicki Athanasopoulos, Tina Avolio, Dr. Derek Ceccarelli, Jennifer Cruickshank, Melissa Holowaty, Kathy Shire and Dr. Hong Wu. Words cannot express what each of them mean to me. 1 thank them al1 for their support and niendship. 1 also want to thank the members of my supervisory comrnittee, Dr. B. Andrews and Dr. B. Funnell, for their ideas and encouragement. 1 thank the Andrews laboratory for providing me with many of the yeast strains and plasmids and for helphil advice. From the bottom of my heart, 1 want to thank my parents and my brother, the people who mean the world to me. 1thank them for motivating me, loving me and understanding me. Last but ceriainly not the least, 1 thank my best fnend Rahul. Having him by my side has encouraged me to work harder. 1 thank him for pushing me to strive for the best and for always being by my side. TABLE OF CONTENTS L- ABSTRACT ii ACKNOWLEDGEMENTS iii TABLE OF CONTENTS IV LIST OF FIGURES vii LIST OF TABLES viii LIST OF ABBREVIATIONS ix 1, INTRODUCTION 1.1. Epstein-Barr Vh:Mode of Infection and Associated Diseases 1.2. Viral Protein Expression During EBV Latent Infection 1.3. EBV Latent Origin of Replication, oriP 1.4. Epstein-Barr Nuclear Antigen 1 (EBNAl ) I.4.a. Protein Domains and Structure of EBNA 1 I.4.b. EBNAl Functions 1.4.b.i. Transcriptional Activities of EBNAl I.4.b.ii. Replication Function of EBNAl 1.4.b.iii. Segregation Function of EBNAl L S. EBNA 1-hEBP2 (human EBNA 1-Binding Protein 2) Interaction 1.5.a. Roperties and Cellular Function of EBPZ 1.5.b. Significance of EBNAl -hEBP2 Interaction in EBV Segregation 1.6. Segregation Mechanism of Extrachromosomal DNA Molecules 1.6.a Papillomavirus 1.6.b. Kaposi Sarcoma-Associateci Herpesvirus 1.6,~.Double Minute Chromosomes I.6.d. Yeast 2-Micron Plasmids I.6.e. Yeast CEN Plasmids 1.7. Thesis Rationale II. MATERIALS AND METHODS II.1. Yeast Strains II.2. Construction of the Plasmid Loss Assay Constnicts II.3. Expression of EBNAl and hEBP2 in Buddhg Yeast U.4. Construction of the Yeast Two-Hybrid Plasmids 11.5. Construction of hEBP2-Integrated Yeast Strains 11.6. Plasmid Loss Assays n.7. Yeast Two-Hybnd Assays m.1. Expression of EBNAl and hEBP2 in Budding Yeast III.2. EBNAl Dots Not Mcdiate FR Plasmid Segregation in Yeast iII.3. Human EBP2 is RequKed for EBNAl-Mediateci Segregation in Yeast iII.4. EBNA 1-Mediated Segregation in Yeast Requues EBNA 1 Binding to hEBP2 IJI.5. EBNA1 Does Not Interact with the Yeast EBPZ Protein III.6. FR Plasmid Segregation in hEBP2-Integrated Yeast Sûains by EBNAl IV. DISCUSSION IV. 1. Cornparison of EBV-Bssed Segregation in Budding Yeast and Humans IV.2. hEBP2 Plays an Essential Role in EBNAl-Meâiated Segregation N.3.nie Function of hEBP2 in EBV Segregation Cannot be ProMded by yEBP2 N.4. A Role for hEBP2 in the Segregation of BPV and KSHV Episomes? V. FUTURE DIRECTIONS V. 1. Cellular Localization Studies of EBP2 and EBNAl in Budding Yeast V.2. Assessing hEBP2 Residues Involved in EBNAl- Mediated Segregation in Yeast V.2.a. Mapping hEBP2 Residues that Interact with EBNAl V.2.b. Role of the EBNAl-Binding Domain of hEBP2 in EBNA 1-Mediatecl Segregation V.2.c. Identifjing the Chromosome-Associathg Region of hEBP2 in Budding Yeast V.3. Identifjing hEBP2 Residues Required for Chromosome Association in Humans V.4. Using Yeast and Human EBP2 Hybnds to Assess the Inability of Yeast EBPZ to Support Segregation by EBNAl V.S. hEBP2-Mediated Segregation of a Plasmid Containhg GAIA Binding Sites in the Presence of the GALA DNA-Binding Domain V.6. Use of a Sectoring Assay to Messure Plasmid Stability in the EBV-Based Segregation System in Yeast VI. 1. Stability of Segregation Plasmids in the Presence and Absence Of EBNAl and hEBP2 - Results from Individual Experiments 85 VIT. REFERENCES 86 LIST OF FIGURES Figure 1 O*, the EBV latent origin of replication Figure 2 Schematic diagram of the functional regions of EBNAl Figure 3 Aiignrnent of EBP2 homologs Figure 4 Expression plasmids useci in the plasmid loss assay Figure 5 EBNAl and hEBP2 expression in budding yeast Figure 6 Segregation test plasmids Figure 7 EBNAl cannot support stable segregation of YRp7FR in budding yeast Figure 8 hEBP2 is required for EBNAl-mediated FR-plasmid segregation in budding yeast Figure 9 EBNAA325-376 expression in budding yeast Figure 10 Requirement of the hEBP2-binding region of EBNAl in FR plasrnid segregation in yeast Figure 11 Interaction of EBNAl with EBP2 Figure 12 Expression of hEBP2 in hEBP2-integrated yeast strains Figure 13 EBNAl-rneâiated segregation in hEBP2-ùltegrated yeast strains Figure 14 Schematic diagram of the hEBP2 protein LIST OF TABLES Table 1 Stability of Segregation Plasmids in the Resence 51 and Absence of EBNAI and hEBP2 LIST OF ABBREVIATIONS .- - ARS autonomously replicating sequence AT 3-aminotriazole NT adenindthymine ATP adenosine triphosphate bp base pairs BPV1 bovine papillomavirus type 1 BZLF 1 BamHI C teftward fiame 1 CAT chloramphenicol acetylûansferase CEN CIP calf intestinal alkaline phosphatase CTL cytotoxic T-lymphocyte m20 de-ionized water DMs doubleminute chromosomes DNA deoxyribonucleic acid ms deoxynucleotide triphosphate DS dyad symmetry DTT dithiothreitol EBER Epstein-Barr expmsed RNA EBNA1 Epstein-Barr nuclear antigen 1 EBP2 EBNAl-binding protein 2 EBV Epstein-Barr vinis ECL enbanced cherniluminescence A..-- 4 EDTA ethylenediaminetetraacetic acid FISH Fluorescence in-situ hybridization FR family of repeats GFP green fluorescent protein Gly-Ala gl ycine-alanine Gly -hg glycine-arginine hEBP2 human EBP2 HHV8 human herpesvirus 8 HMG-1 high-mobility group-1 Kb kilobase kD kilodaltons KSHV kaposi sarcoma-associated herpesvirus lac lactose LANA latency-associated nuclear antigen LB Luria Bertani broth LCLs lymphobiastoid ce11 lines LCV Iymphocrytovirus Leu leucine LiAc lithium acetate LMP latent membrane protein Mb megabase MCS multiple cloning sites MME minictuomosome maintenance element -- - - . -.-- - MO minimal o@n of replication NEB New England Biolabs NLS nuclear localization signal OD opticai density 0riP origin of plasmid replication PBS phosphate buffered saline PCR polymerase chah reactian PEG polyethylene glycol PMSF phenylmethylsulfonyl fluoride MG1 /2 recombination activating genes 1 and 2 RGG arginine glycine glycine RNPs ribonucleic proteins rRNA ribosomal ribonucleic acid RPA replication protein A SC synthetic complete SDS sodium dodecyl sulphate SDS-PAGE SDS polyacrylamide gel electrophoresis snRNPs small RNPs SV40 simian vinis 40 TBE tris, bric acid, ethylenediaminetetraacetic acid Trp tryptophan Ur a YACs yeast artificial chromosomes -----A %L --.-&A& - -- - yEBP2 yeast EBP2 YPD yeast extract peptone dextrose 2D two dimensional 1.1 Epstein-Barr Virus: Mode of Infection and Associateà Diseases A member of the lyrnphocryptovinis (LCV)genus of the gamma herpesvims, Epstein- Barr virus (EBV)affects humans worldwide. PNnary infection by EBV can take place during early childhood, when it is usually asymptomatic or during adolescence, when it cm cause infectious mononucleosis. Transmitted through the saliva, EBV infects the epithelial cells of the oropharynx, where it undergoes lytic infection, though latent infection has been observed in rare cases. The vhsalso infects the B-lymphocytes, where latent infection and long-term persistence occur.
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