The molecular interactions of the Marek’s disease virus-encoded oncoprotein Meq James S. Green BSc (Hons) 2011 Imperial College London Department of Virology Faculty of Medicine Submitted to Imperial College London for the degree of Doctor of Philosophy 1 Abstract Meq is a viral c-Jun analog and member of the AP-1 transcription factor family that acts as the primary oncoprotein encoded by Marek’s disease virus. Previous studies have shown that Meq interacts with a variety of proteins as part of its pivotal function in the development of Marek’s disease virus-induced lymphoma. The primary focus of this study was to identify the global interactome of the Meq oncoprotein. This was initially carried out by review of the Meq sequence and the subsequent identification of BATF and BATF3 as potential analogous partners. Interactions with novel proteins were also predicted based on the nature of charged interactions that mediate a leucine zipper dimerisation event. Furthermore, the Meq protein was used as bait in a yeast two-hybrid screen to produce a large data set of both known and novel interacting proteins. Interactions with JunB, JunD and CtBP1 were confirmed along with a set of novel proteins including Par-4, ATF3, RACK1, N4BP1 and Pin1. The chicken Par-4 and ATF3 genes were cloned and expressed to confirm co-localisation with Meq in the cell nucleus while further biochemical investigations for each interaction were carried out by co-immunoprecipitation. The Par-4 and ATF3 proteins have been shown to play a variety of key roles in cellular mechanisms such as apoptosis, cell cycle regulation and transformation but we failed to see expression of these proteins in either normal or transformed lymphocytes. However, Par-4 and ATF3 are expressed in the feather follicle epithelium of infected birds providing the potential for a role during lytic infection in the skin. It is clear that more work is required in order to explore this hypothesis further but this thesis describes a number of novel interactions made by Meq and in doing so we have contributed to the greater understanding of Marek’s disease virology. 2 Table of Contents Abstract ...................................................................................................................................... 2 Table of Contents ....................................................................................................................... 3 Table of Figures ......................................................................................................................... 8 Table of Tables ........................................................................................................................ 10 Abbreviations ........................................................................................................................... 11 Acknowledgements .................................................................................................................. 15 Declaration ............................................................................................................................... 16 1 Introduction ...................................................................................................................... 17 1.1 Introduction to oncogenesis ...................................................................................... 17 1.2 Viral oncogenesis ...................................................................................................... 19 1.3 Introduction to Marek’s disease virus ....................................................................... 21 1.4 History of Marek’s disease virus ............................................................................... 22 1.5 Classification and genome structure ......................................................................... 22 1.6 Marek’s disease virus infection, life cycle and pathology ....................................... 26 1.6.1 Infection ................................................................................................................. 26 1.6.2 Early cytolytic phase ............................................................................................. 26 1.6.3 Latency .................................................................................................................. 27 1.6.4 Late cytolytic phase and productive infection ....................................................... 28 1.6.5 Cell transformation ................................................................................................ 28 1.7 Immunity to Marek’s disease .................................................................................... 30 1.8 Vaccination................................................................................................................ 30 1.9 Marek’s disease virus-encoded proteins of interest .................................................. 31 1.10 Marek’s Disease virus-encoded microRNAs ............................................................ 33 1.11 Marek’s disease virus-encoded EcoRI-Q fragment (Meq) gene ............................... 33 1.11.1 The Meq protein .................................................................................................... 36 1.11.2 Meq protein function ............................................................................................. 40 1.11.3 Meq as a transcription factor ................................................................................. 41 1.11.4 Meq during apoptosis ............................................................................................ 45 1.11.5 Meq interacts with CtBP1...................................................................................... 45 1.12 Prostate apoptosis response-4 ................................................................................... 46 1.13 Activating transcription factor 3 ............................................................................... 55 3 1.14 Yeast two-hybrid screen ............................................................................................ 57 1.15 Aim of this thesis....................................................................................................... 59 2 Methods ............................................................................................................................ 60 2.1 Solutions and buffers in appendix I........................................................................... 60 2.2 Plasmids in appendix II ............................................................................................. 60 2.3 Antibodies ................................................................................................................. 60 2.4 Oligonucleotides........................................................................................................ 61 2.5 Methods ..................................................................................................................... 62 2.5.1 Leucine zipper interaction prediction .................................................................... 62 2.5.2 Phylogenetic analysis ............................................................................................ 62 2.5.3 Plasmid DNA extraction – miniprep ..................................................................... 63 2.5.4 Plasmid DNA extraction – maxiprep..................................................................... 63 2.5.5 Glycerol stock production ..................................................................................... 64 2.5.6 Molecular cloning .................................................................................................. 64 2.5.7 Polymerase chain reaction ..................................................................................... 65 2.5.8 Reverse transcription polymerase chain reaction .................................................. 66 2.5.9 Transformation of chemically competent E. coli .................................................. 66 2.5.10 Transformation of electro-competent E. coli ......................................................... 67 2.5.11 Nucleic acid electrophoresis .................................................................................. 67 2.5.12 SDS protein electrophoresis .................................................................................. 67 2.5.13 Transfection of DF-1 cells ..................................................................................... 68 2.5.14 Transfection of HeLa cells .................................................................................... 68 2.5.15 Transfection of PC-3 cells ..................................................................................... 69 2.5.16 Transfection of CEF cells ...................................................................................... 69 2.5.17 Coomassie brilliant blue staining .......................................................................... 70 2.5.18 Silver staining ........................................................................................................ 70 2.5.19 Western blot ........................................................................................................... 70 2.5.20 Immunofluorescent staining of cells ...................................................................... 71 2.5.21 Immunofluorescent staining of tissues .................................................................. 71 2.5.22 GST protein