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Identification of a Human Cytomegalovirus-Encoded Jak1 Antagonist Using a Novel Screening Strategy

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Identification of a Human Cytomegalovirus-Encoded Jak1 Antagonist Using a Novel Screening Strategy Identification of a Human Cytomegalovirus-encoded Jak1 antagonist using a novel screening strategy Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf vorgelegt von Sebastian Howe aus Pinneberg Düsseldorf, Dezember 2016 Aus dem Institut für Virologie der Heinrich-Heine-Universität Düsseldorf Gedruckt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Referent: Herr Prof. Dr. Hartmut Hengel Koreferent: Herr Prof. Dr. Johannes Hegemann Tag der mündlichen Prüfung: 19. Oktober 2017 Table of contents Table of contents ...................................................................................................................................... i Abbreviations ........................................................................................................................................... v Figure Summary ...................................................................................................................................... ix Abstract ................................................................................................................................................... 1 Zusammenfassung ................................................................................................................................... 2 1. Introduction ................................................................................................................................. 3 1.1 Viruses ....................................................................................................................................... 3 1.1.1 Herpesviruses ......................................................................................................................... 3 1.1.1.1 Cytomegaloviruses (CMV) ................................................................................................... 4 1.1.1.2 The Human Cytomegalovirus (HCMV) ................................................................................. 5 1.2 Interferons (IFN) ........................................................................................................................ 6 1.2.1 Interferon induction ............................................................................................................... 6 1.2.2 Jak-STAT signaling pathway .................................................................................................... 6 1.3 HCMV-mediated immune evasion ............................................................................................ 9 1.3.1. HCMV-encoded IFN signaling antagonists assigned to a defined HCMV gene ..................... 9 1.3.2 HCMV-encoded IFN signaling antagonists unidentified ....................................................... 12 1.3.3 HCMV-encoded ISG antagonists assigned to a defined HCMV gene ................................... 12 1.3.4 HCMV-encoded ISG antagonists unidentified ...................................................................... 14 1.4 MCMV-mediated immune evasion ......................................................................................... 15 1.4.1 MCMV-encoded IFN antagonists assigned to a defined MCMV gene ................................. 15 1.4.2 MCMV-encoded IFN antagonists unidentified ..................................................................... 16 1.5 Cellular protein degradation ................................................................................................... 17 1.5.1 Proteasomal degradation ..................................................................................................... 18 1.5.2 Endosomal degradation ....................................................................................................... 18 1.5.3 Autophagy ............................................................................................................................ 19 1.6. Herpes simplex thymidine kinase (HSV-TK) ............................................................................ 20 1.7 Aim of the thesis ...................................................................................................................... 20 2. Results ....................................................................................................................................... 23 2.1 HCMV downregulates protein amounts of Jak1 and STAT2.................................................... 23 2.2 Generation of stable cell lines expressing thymidine kinase (TK) fusion constructs .............. 23 2.3 TK-fusion proteins seem to be properly folded and able to reconstitute IFN signaling events ....................................................................................................................................................... 25 i 2.4 Expression of TK-fusion proteins results in susceptibility towards ganciclovir (GCV) treatment ....................................................................................................................................................... 26 2.5 Using a previously described collection of cloned HCMV open-reading-frames, no HCMV- encoded STAT2 antagonist was identified .................................................................................... 27 2.6 Global screening with lentiviral cDNAs leads to survival of cell clones expressing potential HCMV Jak1 antagonists ................................................................................................................. 29 2.7 UL74A does not suffice to reduce protein amounts of Jak1 ................................................... 31 2.8 UL42 reduces protein amounts of TK-Jak1 and Jak1 dose-dependently ................................ 32 2.9 The antagonistic capacity of pUL42 does not affect untransfected bystander cells .............. 33 2.10 HCMV pUL42 is subjected to complex N-glycosylation in the Golgi apparatus .................... 34 2.11 gpUL42 is a surface protein ................................................................................................... 35 2.12 gpUL42 reduces Jak1 amounts post-transcriptionally .......................................................... 36 2.13 Translation of gpUL42 is essential for the negative effect on Jak1 ....................................... 37 2.14 The conserved tyrosine Y45 is important for Jak1 protein reduction ................................... 39 2.15 No indication for gpUL42-dependent Jak1 protein reduction via autophagy ....................... 41 2.16 The proteasome controls Jak1 amounts in uninfected cells ................................................. 42 2.17 Overexpression of gpUL42 affects several co-transfected proteins ..................................... 43 2.18 HCMV mutants lacking the ability to express gpUL42 exhibit reduced and protracted Jak1 reduction ....................................................................................................................................... 44 2.19 gpUL42mut-HCMVAD169varL has comparable growth characteristics as wild-type HCMV in absence of IFN ............................................................................................................................... 45 2.20 gpUL42mut-HCMVAD169varL and wt-HCMVAD169varL are susceptible towards IFN-J pre- treatment ...................................................................................................................................... 46 2.21 gpUL42mut-HCMVAD169varL is more susceptible towards IFN-J treatment in the first 3 days than wt-HCMVAD169varL .................................................................................................................... 49 3. Discussion .................................................................................................................................. 50 3.1 HCMV gpUL42 was identified as a Jak1 antagonist using a novel screening system .............. 50 3.2 Importance of Jak1 and IFN signaling in anti-viral activity ...................................................... 51 3.3 Redundancy in HCMV-mediated Jak1 protein reduction ........................................................ 53 3.4 Cellular cDNAs apparently leading to a selection advantage in TK-Jak1 cells ......................... 55 3.5 Cellular degradation mechanism exploited by gpUL42 ........................................................... 57 3.6 Possible co-factors involved in gpUL42-mediated Jak1 protein reduction ............................. 57 3.7 Expanded ‘antagonistic’ capacity of HCMV gpUL42 in co-transfection experiments ............. 59 3.8 Comparative analysis of MCMV-expressed gpUL42 homologues ........................................... 60 3.9 HCMV mutants lacking IFN antagonists as live attenuated vaccine viruses? ......................... 60 3.10 General applicability of the herein established screening system ........................................ 61 ii 4. Materials and Methods ............................................................................................................. 64 4.1 Materials .................................................................................................................................. 64 4.1.1 Equipment ............................................................................................................................ 64 4.1.2 Chemicals .............................................................................................................................
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