Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2018 Antiviral function of Mx proteins: role of cellular factors and subcellular localization Steiner, Fiona Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-153552 Dissertation Published Version Originally published at: Steiner, Fiona. Antiviral function of Mx proteins: role of cellular factors and subcellular localization. 2018, University of Zurich, Faculty of Science. Antiviral Function of Mx Proteins - Role of Cellular Factors and Subcellular Localization Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Fiona Steiner von Freienbach (SZ) Promotionskommission Prof. Dr. Alexandra Trkola (Vorsitz) PD Dr. Jovan Pavlovic (Leitung der Dissertation) Prof. Dr. Cornel Fraefel Prof. Dr. Peter Stäheli Prof. Dr. Silke Stertz Zürich, 2018 1 Table of Contents Summary ............................................................................................................................. iv Zusammenfassung .............................................................................................................. v 1 Background .................................................................................................................. 1 1.1 Influenza A virus ...................................................................................................... 1 1.1.1 IAV virion structure ........................................................................................... 2 1.1.2 IAV replication cycle ......................................................................................... 4 1.1.3 A closer look on the nucleoprotein NP of IAV ................................................... 5 1.2 Innate immune system ............................................................................................ 8 1.3 Mx proteins ........................................................................................................... 11 1.3.1 Structure and function .................................................................................... 11 1.3.1 Mx proteins and IAV ....................................................................................... 13 1.4 Cellular RNA helicase UAP56 (or DDX39B) .......................................................... 14 1.5 Aims of this work ................................................................................................... 15 2 Nuclear MxB variant blocks influenza A virus replication....................................... 16 2.1 Abstract ................................................................................................................. 17 2.2 Introduction ........................................................................................................... 18 2.3 Experimental procedures....................................................................................... 20 2.3.1 Cell lines ........................................................................................................ 20 2.3.2 Plasmids ........................................................................................................ 20 2.3.3 Viruses ........................................................................................................... 20 2.3.4 Immunofluorescence assay ............................................................................ 20 2.3.5 Minimal replicon reconstitution assay ............................................................. 21 2.3.6 Cell-Titer-Glo assay........................................................................................ 21 2.3.7 Co-immunoprecipitation (co-IP) ...................................................................... 21 2.3.8 qRT-PCR ....................................................................................................... 22 2.4 Results .................................................................................................................. 23 2.5 Discussion ............................................................................................................. 24 3 The trimeric MxA-NP-UAP56 complex: Identifying molecular determinants to unravel their interplay ................................................................................................ 35 3.1 Abstract ................................................................................................................. 36 3.2 Introduction ........................................................................................................... 37 3.3 Experimental procedures....................................................................................... 38 i 3.3.1 Cell lines ........................................................................................................ 38 3.3.2 Plasmids ........................................................................................................ 39 3.3.3 siRNA transfection ......................................................................................... 39 3.3.4 Tripartite split-GFP complementation assay ................................................... 40 3.3.5 Non-denaturing PAGE analysis ...................................................................... 40 3.3.6 Minimal replicon reconstitution assay ............................................................. 40 3.3.7 Co-immunoprecipitation (co-IP) ...................................................................... 41 3.3.8 Subcellular fractionation ................................................................................. 41 3.4 Results .................................................................................................................. 42 3.4.1 Assessment of the general binding capacity and localization of MxA, viral NP and UAP56 ..................................................................................................... 42 3.4.2 Investigating the capability of MxA to compete for NP binding against UAP56 .. and the effect of reduced UAP56 levels on MxA-NP binding .......................... 43 3.4.3 Molecular determinants of MxA in NP binding and anti-IAV activity ................ 44 3.4.4 Enzymatic activity of UAP56 is not crucial for the binding to MxA and NP ...... 45 3.4.5 NP F412A/R416A substitutions in NP abrogate binding to MxA and UAP56 .. 46 3.5 Discussion ............................................................................................................. 47 4 An attempt at mapping the interaction domains in MxA and UAP56 to each other and to viral NP ............................................................................................................ 62 4.1 Introduction ........................................................................................................... 62 4.2 Experimental procedures....................................................................................... 63 4.2.1 Cell lines ........................................................................................................ 63 4.2.2 Plasmids ........................................................................................................ 63 4.2.3 Immunofluorescence assay ............................................................................ 64 4.2.4 Tripartite split-GFP complementation assay ................................................... 64 4.2.5 Co-immunoprecipitation (co-IP) ...................................................................... 64 4.3 Results .................................................................................................................. 65 4.3.1 Generating MxA deletion mutants to reveal domains necessary for binding to UAP56 and NP ............................................................................................... 65 4.3.2 Investigating the potential involvement of MxA loop L4 in its capability to bind to UAP56 and NP ............................................................................................... 65 4.3.3 Attempts at finding binding sites in UAP56 to MxA and NP ............................ 66 4.4 Discussion ............................................................................................................. 67 5 General discussion .................................................................................................... 80 5.1 The antiviral mechanism of MxA: an initial model .................................................. 81 5.2 Adjustments to the model ...................................................................................... 82 ii 5.3 Primary transcription: inhibition or not? .................................................................. 83 5.4 Mx proteins and their ability to bind to IAV NP ....................................................... 84 5.5 Is there a common antiviral mechanism for all Mx proteins? .................................. 85 5.6 Outlook .................................................................................................................. 86 Acknowledgements ........................................................................................................... 88 References ......................................................................................................................... 90 iii Summary Viruses and their hosts co-evolve