Pathogen host interactions: proteomics of Influenza NEP during infection reveals an antagonistic role in the formation of tight junctions Garrett Robert Poshusta A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2015 Reading Committee: John Aitchison, Chair Wilhelmus Hol Kenneth Stuart Program Authorized to Offer Degree: Biochemistry ©Copyright 2015 Garrett Robert Poshusta University of Washington Abstract Pathogen host interactions: proteomics of Influenza NEP during infection reveals an antagonistic role in the formation of tight junctions Garrett Robert Poshusta Chair of the Supervisory Committee: Affiliate Professor John Aitchison Department of Biochemistry Host-pathogen interaction networks are key to understanding the molecular mechanisms driving disease and can provide new targets for therapeutic intervention. We discovered new host factors interacting with the influenza nuclear export protein (NEP) using an engineered influenza virus expressing NEP with an N-terminal 3xFLAG tag. We collected immunopurification mass spectrometry data for 3xFLAG-NEP during an active infection and during plasmid expression in HEK293T cells. Network analysis of these complementary datasets revealed an enrichment of tight junction proteins in the NEP interactome. Expression of NEP in MDCK cells results in inhibition of tight junction formation as measured by transepithelial electrical resistance and inulin diffusion across the polarized cell monolayer. These findings reveal a new role for NEP as a tight junction antagonist. Table of Contents List of Figures .................................................................................................................................. iv List of Tables ................................................................................................................................... vi Chapter 1: Introduction .................................................................................................................. 1 1.1 Influenza Virus .................................................................................................................. 1 1.1.1 Overview ................................................................................................................... 1 1.1.2 Avian Influenza .......................................................................................................... 5 1.1.3 Influenza Nuclear Export Protein .............................................................................. 6 1.2 Systems Biology .............................................................................................................. 11 1.3 Methodologies for Studying Host Pathogen Interactions ............................................. 14 Chapter 2: Discovering new host interactors of NEP using IP-MS ................................................ 23 2.1 Summary ........................................................................................................................ 23 2.2 Introduction.................................................................................................................... 23 2.2.1 Control strategies for IP-MS experiments .............................................................. 23 2.2.2 Strategies and considerations for tagging NEP ....................................................... 28 2.3 Methods ......................................................................................................................... 34 2.3.1 Plasmid Constructs .................................................................................................. 34 2.3.2 Protein Expression .................................................................................................. 36 2.3.3 Protein Purification ................................................................................................. 36 2.3.4 Protein Analysis ....................................................................................................... 36 2.3.5 Cell Culture .............................................................................................................. 37 2.3.6 Viral Production ...................................................................................................... 37 2.3.7 Affinity purification for the SBP tag ........................................................................ 37 i 2.3.8 Immunopurification for the 3xFLAG-tag ................................................................. 38 2.3.9 Mass spectrometry ................................................................................................. 39 2.4 Results ............................................................................................................................ 41 2.4.1 Expression and purification of recombinant GST-NEP ........................................... 41 2.4.2 Genetic engineering of influenza for the expression of tagged NEP ...................... 43 2.5 Discussion ....................................................................................................................... 63 Chapter 3: Influenza NEP Interactome ......................................................................................... 71 3.1 Summary ........................................................................................................................ 71 3.2 Introduction.................................................................................................................... 71 3.3 Methods ......................................................................................................................... 74 3.3.1 Stable incorporation of labeled amino acids in cell culture (SILAC) ....................... 74 3.3.2 Viral I-DIRT Immunopurification ............................................................................. 74 3.3.3 Immunopurification for FLAG from plasmid expression ........................................ 75 3.3.4 Sample preparation for Mass Spectrometry by in solution digest ......................... 75 3.3.5 Proteomics Data Analysis ........................................................................................ 76 3.4 Results ............................................................................................................................ 77 3.4.1 Viral I-DIRT IP-MS .................................................................................................... 77 3.4.2 FLAG IP with plasmid expression of 3xFLAG-NEP ................................................... 89 3.4.3 Gene function classification with DAVID shows an enrichment of proteins involved in cellular processes important for the viral lifecycle ........................................................... 95 3.4.4 Network Analysis in Cytoscape ............................................................................... 97 3.5 Discussion ..................................................................................................................... 103 Chapter 4: NEP antagonizes tight junction function .................................................................. 108 4.1 Summary ...................................................................................................................... 108 ii 4.2 Introduction.................................................................................................................. 108 4.3 Methods ....................................................................................................................... 112 4.3.1 Lentiviral Production ............................................................................................. 112 4.3.2 Fluorescein isothiocyanate (FITC)-inulin Diffusion Assay ..................................... 112 4.3.3 Transepithelial electrical resistance (TEER) measurements ................................. 112 4.3.4 Immunofluorescence ............................................................................................ 112 4.4 Results .......................................................................................................................... 114 4.4.1 Transepithelial barrier function in MDCKII cells is impaired by expression of NEP but not GFP .......................................................................................................................... 114 4.4.2 Tight junction functioning in high resistance MDCKI cells is impaired by expression of NEP but not GFP .............................................................................................................. 117 4.4.3 Tight junction protein occludin is mislocalized when NEP is induced .................. 120 4.5 Discussion ..................................................................................................................... 123 Chapter 5: Perspectives .............................................................................................................. 128 5.1 Lessons from the study of host interactors of NEP ...................................................... 128 5.2 Potential roles for tight junctions in the influenza lifecycle ........................................ 130 5.3 Future directions .......................................................................................................... 135 Biliography .................................................................................................................................. 137 iii List of Figures Figure