UNIVERISTY OF CALIFORNIA, SAN DIEGO Characterization of the BST2/Tetherin cytoplasmic tail and understanding BST2-mediated NF-κB activation A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biomedical Sciences by Maryan Girgis Soliman Rizk Committee in charge: Professor John C. Guatelli, Chair Professor Michael I. David Professor Daniel Donoghue Professor Douglas Richman Professor Bruce Torbett 2017 Copyright © Maryan Girgis Soliman Rizk, 2017 All rights reserved. The Dissertation of Maryan Girgis Soliman Rizk is approved, and it is acceptable in quality and form for publication on microfilm and electronically: ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ Chair University of California, San Diego 2017 iii Table of Contents Signature Page ............................................................................................................. iii Table of Contents ..........................................................................................................iv List of Figure and Tables .............................................................................................. vii List of Abbreviations ...................................................................................................... x Acknowledgements ..................................................................................................... xiii Vita ...............................................................................................................................xv Abstract of the Dissertation ........................................................................................ xvii Chapter I. Introduction of BST2/Tetherin as a multifunctional transmembrane protein..1 Overview of BST2 Functions .............................................................................. 2 Historical perspective of BST2/Tetherin ............................................................. 2 BST2 physically traps virions of enveloped viruses ............................................ 3 BST2 activates NF-κB through its cytoplasmic tail ............................................. 4 The human immunodeficiency virus 1 and BST2 ............................................... 4 Ebola virus and BST2 ......................................................................................... 7 References ....................................................................................................... 12 Chapter II. Understanding the role of the cytoplasmic tail of BST2 in trafficking and activation of NF-κB ...................................................................................................... 20 Abstract ............................................................................................................ 21 Introduction ....................................................................................................... 22 Materials and Methods ..................................................................................... 25 Tissue culture, plasmids, and reagents.................................................. 25 Yeast 2-hybrid assays............................................................................ 27 Immunoprecipitation, SDS/PAGE, Western blotting .............................. 27 Dual-luciferase signaling assay ............................................................. 28 Statistical analysis ................................................................................. 28 Results ............................................................................................................. 29 The YxYxxV sequence of the cytoplasmic tail of BST2 facilitates direct binding of BST2 to the μ-subunit of the clathrin adaptor protein 1 ......... 29 iv Tyrosine 6 of the BST2CD drives the activation of NF-κB, but the CD alone is not sufficient for activity ............................................................ 30 BST2 is tyrosine-phosphorylated at Y6 and Y8 ..................................... 32 The over-expression of Syk kinase increases NF-κB activation by BST2 ................................................................................................. 33 BST2 CD does not directly bind to signaling co-factors upstream of NF-κB in the Y2H assay .................................................................................... 35 Discussion ........................................................................................................ 36 Acknowledgements .......................................................................................... 41 References ....................................................................................................... 51 Chapter III. Cooperation of the Ebola proteins VP40 and GP1,2 with BST2 activates NF- κB independently of virus-like particle trapping ........................................................... 54 Abstract and Importance .................................................................................. 55 Introduction ....................................................................................................... 56 Materials and Methods ..................................................................................... 59 Tissue culture and plasmids .................................................................. 59 Antibodies and reagents ........................................................................ 60 SDS-PAGE/Western blotting ................................................................. 61 Measuring NF-κB activity using a luciferase reporter system ................ 61 VLP collection and detection .................................................................. 62 MLN4924 treatment ............................................................................... 62 Flow cytometry ....................................................................................... 63 Statistical analysis ................................................................................. 63 Results ............................................................................................................. 64 Ebola GP1,2 does not inhibit BST2-mediated activation of NF-κB ........ 64 GP1,2 from the Zaire and Reston species cooperate similarly with VP40 and BST2 to activate NF-κB .................................................................. 66 BST2 and the Ebola proteins activate the canonical NF=kB pathway in a manner dependent on neddylation ........................................................ 67 v Determinants of BST2-mediated activation of NF-κB in the context of Ebola protein-expression ....................................................................... 68 Activation of NF-κB by BST2 in concert with the Ebola proteins is independent of VLP-entrapment ............................................................ 70 Statistical analysis confirms that BST2 cooperates with Ebola VP40 and GP1,2 to induce maximal NF-κB activity ................................................ 71 Discussion ........................................................................................................ 72 Acknowledgements .......................................................................................... 79 References ..................................................................................................... 101 Chapter VI. Conclusion and Future Directions .......................................................... 106 Summary ........................................................................................................ 107 The BST2 CD plays a major role in the trafficking and signaling functions of the protein ............................................................................................................ 107 The Ebola virus glycoprotein does not act as an antagonist of BST2-mediated activation of NF-κB, but rather cooperates with BST2 to increase NF-κB activity ................................................................................................. 108 The HIV-2 Envelope protein does not inhibit BST2-mediated activation of NF-κB ............................................................... 111 Conclusion ...................................................................................................... 112 Acknowledgements ........................................................................................ 113 References ..................................................................................................... 123 vi List of Figures and Tables FIGURES Chapter I Figure I.1 Structural features of BST2/Tetherin within a phospholipid bilayer .......... 9 Figure I.2 Schematic of the Ebola virus glycoprotein (GP1,2) ................................ 10 Chapter II Figure II.1 Yeast 2-hybrid assaying showing interaction of the BST2 cytoplasmic domain (BST2 CD) and μ-subunits ........................................................ 43 Figure II.2 Structural features of the interaction between BST2 and μ1 .................. 44 Figure II.3 NF-κB activity requires tyrosine 6 of the BST2 cytoplasmic domain, while the CD alone is not sufficient for signaling ............................................. 45 Figure