1. Suzanne Vroman Katze Lab University of Washington Protein-Protein Interactions of P58 IPK

2. Aim 3 Use functional genomics approaches to examine the role of P58 in the presence and absence of virus infection – –Identification of P58 interactive partners   Protein Expression – –Mammalian, E. coli   Yeast two-hybrid screens

3. Hsp40 P52 rIPK Characterization of P58 pathway P58 IPK PKR ? ? PERK Inactive active eIF2α eIF2α – independent? Virus InfectionER stress Enhanced viral replication Return to ER homeostasis Regulation of apoptosis and other functions Post-transcriptional activation Transcriptional activation

4. pTriEx expression vector mammalian promoter E. coli promoter HSV/HIS -tags

5. Bacterial Protein Expression Transformation of mP58 and hP58 pTriEx vectors into BL21 E. coli cells

6. Bacterial Protein Expression Transformation of mP58 and hP58 pTriEx vectors into BL21 E. coli cells Inoculate LB-Amp-Cam Media for expression

7. Bacterial Protein Expression Transformation of mP58 and hP58 pTriEx vectors into BL21 E. coli cells Inoculate LB-Amp-Cam Media for expression Check OD throughout the day (approx. every hour)

8. Bacterial Protein Expression Transformation of mP58 and hP58 pTriEx vectors into BL21 E. coli cells Inoculate LB-Amp-Cam Media for expression Check OD throughout the day (approx. every hour) - At OD of.6 add IPTG inducer and grow for 3-5 hrs.

9. Bacterial Protein Expression Transformation of mP58 and hP58 pTriEx vectors into BL21 E. coli cells Inoculate LB-Amp-Cam Media for expression Check OD throughout the day (approx. every hour) - At OD of.6 add IPTG inducer and grow for 3-5 hrs. Freeze and harvest pellet

10. Transient Transfection: Mammalian Protein Expression Plasmid vector DNA hP58, mP58 pTriEx Transfection (lipofection) Reagent

11. Transient Transfection: Mammalian Protein Expression Plasmid vector DNA hP58, mP58 pTriEx Transfection (lipofection) Reagent DNA lipid liposome

12. Transient Transfection: Mammalian Protein Expression Plasmid vector DNA hP58, mP58 pTriEx Transfection (lipofection) Reagent DNA lipid liposome Endocytosis Cell 293/293T

13. Transient Transfection: Mammalian Protein Expression Plasmid vector DNA hP58, mP58 pTriEx Transfection (lipofection) Reagent DNA lipid liposome Endocytosis DNA released Cell 293/293T

14. Transient Transfection: Mammalian Protein Expression Plasmid vector DNA hP58, mP58 pTriEx Transfection (lipofection) Reagent DNA lipid liposome Endocytosis DNA released Cell 293/293T -24 hours -48 hours Lysis/cell harvest

15. Transient Transfection: Mammalian Protein Expression Plasmid vector DNA hP58, mP58 pTriEx Transfection (lipofection) Reagent DNA lipid liposome Endocytosis DNA released Cell 293/293T -24 hours -48 hours Lysis/cell harvest Anti P58 western blotting

16. Targeted proteomics to identify interacting partners Affinity chromatography and mass spectrometry Affinity chromatography and mass spectrometry –Discovery of new proteins that interact with the given “bait” protein –Complex protein environment native to the environment of the bait protein Immobilized protein (P58) “bait” Protein mixture “prey”

17. Dual-Reporter 2-Hybrid Assay HIS3 Gal4BD Gal4AD bait prey -“Bait” protein fused to a DNA binding domain of a transcription factor and placed in a yeast expression vector -A library of proteins (the “prey) are fused to an activation domain of another transcription factor, and placed in separate yeast expression vector -When the bait and prey are transformed in yeast, the binding domain binds to a DNA-binding site on a promoter sequence that drives a reporter protein (HIS3) -An interaction between bait and prey brings the activation domain in the vicinity of the DNA binding domain

18. Possible outcomes of protein interaction data ScenarioPossible interpretations Present in allNon-specific Present in mock Involve in P58 regulation Novel P58IPK regulatory pathway Present during influenza virus infection Involved in P58 activation? Novel antiviral pathway Present during ER stress Involved in PERK inhibition Novel UPR pathway? Present during ER stress and influenza infectionCommon P58 IPK regulator or effector Present in all Vector alone

19. Possible outcomes of protein interaction data ScenarioPossible interpretations Present in allNon-specific Present in mock Involve in P58 regulation Novel P58IPK regulatory pathway Present during influenza virus infection Involved in P58 activation? Novel antiviral pathway Present during ER stress Involved in PERK inhibition Novel UPR pathway? Present during ER stress and influenza infectionCommon P58 IPK regulator or effector Present in all Mock infection/treatment Present in mock

20. Possible outcomes of protein interaction data ScenarioPossible interpretations Present in allNon-specific Present in mock Involve in P58 regulation Novel P58IPK regulatory pathway Present during influenza virus infection Involved in P58 activation? Novel antiviral pathway Present during ER stress Involved in PERK inhibition Novel UPR pathway? Present during ER stress and influenza infectionCommon P58 IPK regulator or effector Present in all Influenza infection Present in mock Present in infection Present during ER stress and infection

21. Possible outcomes of protein interaction data ScenarioPossible interpretations Present in allNon-specific Present in mock Involve in P58 regulation Novel P58IPK regulatory pathway Present during influenza virus infection Involved in P58 activation? Novel antiviral pathway Present during ER stress Involved in PERK inhibition Novel UPR pathway? Present during ER stress and influenza infectionCommon P58 IPK regulator or effector Present in all ER stress Present in mock Present in infection Present during ER stress and infection Present in ER stress