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1.2 Epstein Barr Virus A University of Sussex DPhil thesis Available online via Sussex Research Online: http://sro.sussex.ac.uk/ This thesis is protected by copyright which belongs to the author. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Please visit Sussex Research Online for more information and further details Mutational analysis and protein interactions of Epstein-Barr virus protein Zta, with reference to ZtaRh, a homologue from Cercopithecine herpesvirus-15 by Questa Hope Karlsson PhD Biochemistry University of Sussex Submitted December 2011 - 2 - I hereby declare that this thesis has not been and will not be, submitted in whole or in part to another University for the award of any other degree. Signature: - 3 - Acknowledgments I would like to take this opportunity to thank the many people who have helped me during my PhD studies. The members of the lab have always been fantastic in helping me, both through their friendship and technical expertise. Thank you to Michelle, Helen, Andrea and Sandra, from the West lab and to Sharada, Kay, Winston and Bryony from the Sinclair lab. In particular, the other PhD students, Celine, Sarah and Kirsty, have been wonderful in their help and support. My supervisor, Alison, has probably never expected me to still be her student after so long but she has always been so supportive and positive, especially when I have faced increasing demands in my life. I would like to thank my two wonderful sons and daughter, Oliver, Noah and Phoebe, who all made an appearance during my studies, for keeping me smiling and providing an inspiration for finishing. My husband and inspiration, Sam, has been instrumental in motivating me and helping at home and with our children. Without my wonderful mother coming from New Zealand to stay with us to look after the children, this journey would have been so much more difficult, if not impossible. Also: thank you to my friends, in particular, Dawn and Ellie, for helping with childcare and always being available when I needed a break. And thank you to both my sisters; to Melda for making me feel closer to home; and to Finda for her proof-reading help. I would like to remember two very special people who passed away during my PhD: Lizzie was a wonderful, enthusiastic friend and colleague and is still greatly missed. My father, David, was probably one of the few retired people without a scientific background who enjoyed reading books on genetics and biology and I cherish the support and encouragement he gave me. - 4 - Table of Contents Abstract ................................................................................................................... 11 Chapter 1 Introduction ............................................................................................... 12 1.1 Introduction to Herpes viruses ........................................................................... 12 1.1.1 Gamma herpes virus .................................................................................... 14 1.2 Epstein Barr Virus .............................................................................................. 14 1.2.1 Identification of EBV .................................................................................. 14 1.2.2 EBV Background ........................................................................................ 14 1.2.3 EBV Structure ............................................................................................. 15 1.2.4 EBV Infection ............................................................................................. 16 1.2.5 Latency ........................................................................................................ 19 1.2.6 Establishment of Latency in B cells ............................................................ 19 1.2.7 EBNAs ........................................................................................................ 20 1.2.8 LMPs ........................................................................................................... 23 1.2.9 EBERs ......................................................................................................... 24 1.2.10 BARTs ........................................................................................................ 24 1.2.11 miRNAs ...................................................................................................... 25 1.2.12 Latency Types ............................................................................................. 25 1.3 EBV Associated diseases ................................................................................... 27 1.3.1 Infectious Mononucleosis ........................................................................... 29 1.3.2 X-linked lymphoproliferative disease ......................................................... 29 1.3.3 Post Transplant Lymphoproliferative disease ............................................. 30 1.3.4 Nasopharyngeal carcinoma ......................................................................... 30 1.3.5 Hodgkin’s Lymphoma ................................................................................ 31 1.3.6 Burkitt’s Lymphoma ................................................................................... 31 1.4 Lytic Cycle ......................................................................................................... 32 1.4.1 Zta Structure ................................................................................................ 33 1.4.2 Regulation of the BZLF1 promoter (Zp) .................................................... 35 1.4.3 Regulation of the BRLF1 promoter (Rp) .................................................... 39 1.4.4 Zta as a transcription factor (viral and cellular) .......................................... 39 1.4.5 Interaction with Cellular proteins................................................................ 40 1.4.6 Zta and EBV replication.............................................................................. 41 1.4.7 The Cell Cycle............................................................................................. 42 1.4.8 EBV, Zta and the Cell Cycle ....................................................................... 44 1.4.9 Zta and immune response............................................................................ 45 1.4.10 Lytic cycle Therapies .................................................................................. 46 1.5 Lymphocryptoviruses ......................................................................................... 46 1.6 Aims ................................................................................................................... 47 Chapter 2 Materials and Methods .............................................................................. 48 2.1 Materials ............................................................................................................. 48 2.1.1 Plasmids ...................................................................................................... 48 2.1.2 Oligonucleotides ......................................................................................... 49 2.1.3 Buffers ......................................................................................................... 53 2.1.4 Antibodies ................................................................................................... 55 2.1.5 Enzymes ...................................................................................................... 55 2.1.6 Kits .............................................................................................................. 56 2.1.7 Radioactivity ............................................................................................... 56 2.2 Methods .............................................................................................................. 57 2.2.1 Cell Culture ................................................................................................. 57 2.2.1.1 Cell Lines ............................................................................................. 57 - 5 - 2.2.1.2 Storage and recovery of cell lines ........................................................ 57 2.2.1.3 Determination of cell count ................................................................. 57 2.2.1.4 Calcium Phosphate Transfection (HeLa cells) .................................... 58 2.2.1.5 Effectene Transfection (HEK293-ZKO and HEK cells) ..................... 58 2.2.1.6 FACS ................................................................................................... 58 2.2.2 Protein Gels ................................................................................................. 59 2.2.2.1 SDS-PAGE .......................................................................................... 59 2.2.2.2 Acrylamide gels ................................................................................... 59 2.2.2.3 Western Blotting .................................................................................. 59 2.2.3 In vitro transcription and translation ..........................................................
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