Investigation of the mechanism of Epstein- Barr Virus Latent Membrane Protein 1- mediated NF-κB activation. Dissertation zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.) eingereicht im Fachbereich Biologie, Chemie, Pharmazie der Freien Universität Berlin vorgelegt von Daniela Böhm aus Mittweida Februar 2010 Die Arbeit wurde von März 2006 bis September 2009 im Labor von Prof. Elliott Kieff und Prof. Ellen Cahir- McFarland im Department of Microbiology and Molecular Genetics, Harvard Medical School and Department of Medicine, Division of Infectious Disease, Brigham and Woman’s Hospital in Boston, MA, USA unter der Leitung von Prof. Ellen Cahir-McFarland angefertigt. 1. Gutachter: Prof. Ellen Cahir-McFarland 2. Gutachter: Prof. Volker Haucke Disputation am: 27.April 2010 2 Acknowledgments ACKNOWLEDGMENTS I would like to thank Prof. Ellen D. Cahir McFarland and Prof. Elliott Kieff for giving me the opportunity to complete my thesis in their laboratory. Furthermore, I sincerely thank Ellen D. Cahir McFarland for her great guidance and support throughout my work. In addition, I thank all the members of the Kieff/ Muenger/ Kaye and Wang laboratories at the 8 th floor of the Channing lab, especially Mike Calderwood, Ben Gewurz, Aline Habison, Chantal Beauchemin, Kevin Hall, Amy Holthaus, Olga Saturne, Vishal Soni, Thomas Sommermann, Nick Shinners, and Jae Song. 5 Table of contents 1. INTRODUCTION ................................................................................................................. 3 1.1 Epstein-Barr Virus.......................................................................................................................................3 1.1.1 Diseases associated with Epstein-Barr Virus........................................................................................ 3 1.1.2 Virus Structure and Genome ................................................................................................................ 5 1.1.3 EBV Infection of Cells ......................................................................................................................... 6 1.2 EBV Latent Membrane Protein-1 (LMP1) ................................................................................................9 1.3 Nuclear Factor-κB-Signaling.....................................................................................................................12 1.3.1 NF-κB-activating Pathways................................................................................................................ 12 1.3.2 NF-κB Essential Modulator (NEMO)................................................................................................. 16 1.4 Mechanism of EBV LMP1 mediated NF-κB activation..........................................................................21 1.5 Reason for my Work..................................................................................................................................23 2. MATERIAL AND METHODS .......................................................................................... 25 2.1 Material.......................................................................................................................................................25 2.1.1 Equipment........................................................................................................................................... 25 2.1.2 Kits and Consumables ........................................................................................................................ 26 2.1.3 Chemicals ........................................................................................................................................... 27 2.1.3.1 Inhibitors..................................................................................................................................... 30 2.1.4 Buffer and Solutions........................................................................................................................... 30 2.1.5 Antibiotics .......................................................................................................................................... 33 2.1.6 Bacteria Strains................................................................................................................................... 34 2.1.7 Tissue Culture Material and Solutions................................................................................................ 34 2.1.8 Cell Lines and Media.......................................................................................................................... 35 2.1.9 Antibodies........................................................................................................................................... 36 2.1.10 Beads ................................................................................................................................................ 38 2.1.11 Enzyms ............................................................................................................................................. 38 2.1.12 Peptides ……………………………………………………………………………………………..40 2.1.13 Nucleotids......................................................................................................................................... 40 2.1.14 Plasmids............................................................................................................................................ 40 2.1.15 Primer and Constructs ...................................................................................................................... 40 2.1.16 siRNA............................................................................................................................................... 44 2.1.17 Ladder............................................................................................................................................... 45 2.2 Methods.......................................................................................................................................................46 2.2.1 Cell Culture and Cell Transfections.................................................................................................... 46 2.2.1.1 Cell Culture................................................................................................................................. 46 2.2.1.2 Cell Counting.............................................................................................................................. 46 2.2.1.3 Freezing/ Thawing of Cell Lines................................................................................................. 46 2.2.1.4 Transient Transfection of Eucaryotic Cells ................................................................................ 46 2.2.1.4.1 Lipofection ........................................................................................................................................... 47 2.2.1.4.2 Electroporation..................................................................................................................................... 47 2.2.1.4.3 Transfection with siRNA...................................................................................................................... 48 2.2.1.5 Stable Transfection of Eucaryotic Cells ...................................................................................... 49 2.2.2 Microbiological Methods.................................................................................................................... 49 2.2.2.1 Transformation of Bacteria......................................................................................................... 49 2.2.2.2 Bacteria Culture ......................................................................................................................... 49 2.2.3 Molecularbiological Methods............................................................................................................. 50 2.2.3.1 DNA ............................................................................................................................................ 50 2.2.3.1.1 DNA Extraction.................................................................................................................................... 50 2.2.3.1.2 DNA Precipitation................................................................................................................................ 51 2.2.3.1.3 Polymerase Chain Reaction (PCR)....................................................................................................... 51 2.2.3.1.4 Enzymatic Modification of DNA ......................................................................................................... 51 2.2.3.1.5 Isolation of DNA from Agarose ........................................................................................................... 52 2.2.3.2 RNA............................................................................................................................................. 52 2.2.3.2.1 Isolation of RNA .................................................................................................................................. 52 2.2.3.2.2 Reverse Transcription........................................................................................................................... 52 2.2.3.3 Protein .......................................................................................................................................