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Einfluss Gezielter Mutationen Auf Die Biologische Aktivität Des Oberflächen-Glykoproteins Eines Afrikanischen Henipavi

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Einfluss Gezielter Mutationen Auf Die Biologische Aktivität Des Oberflächen-Glykoproteins Eines Afrikanischen Henipavi Aus dem Institut für Virologie des Fachbereichs Medizin der Philipps-Universität Marburg Direktor: Prof. Dr. Stephan Becker _____________________________________________________________________ Einfluss gezielter Mutationen auf die biologische Aktivität des Oberflächen-Glykoproteins eines afrikanischen Henipavirus Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) dem Fachbereich Biologie der Philipps-Universität Marburg vorgelegt von Laura Behner aus Kassel Marburg an der Lahn, 2017 Die Untersuchungen zur vorliegenden Arbeit wurden von September 2013 bis September 2017 im Institut für Virologie, Direktor: Prof. Dr. Stephan Becker, Fachbereich Medizin der Philipps-Universität Marburg, unter der Leitung von Prof. Dr. Andrea Maisner durchgeführt. Vom Fachbereich Biologie der Philipps-Universität Marburg (Hochschulkennziffer: 1180) als Dissertation angenommen am: Erstgutachter: Prof. Dr. Andrea Maisner Zweitgutachter: Prof. Dr. Hans-Ulrich Mösch Weitere Mitglieder der Prüfungskommission: Prof. Dr. Uwe Maier Prof. Dr. Susanne Önel Tag der mündlichen Prüfung: 19.12.2017 Inhaltsverzeichnis Inhaltsverzeichnis Inhaltsverzeichnis ................................................................................................................. I Zusammenfassung .............................................................................................................. 1 Summary .............................................................................................................................. 3 I Einleitung...................................................................................................................... 4 1.1 Henipaviren .................................................................................................... 4 1.1.1 Virusaufbau von Henipaviren ................................................................... 4 1.1.2 Genomaufbau von Henipaviren ............................................................... 5 1.2 Flughunde als Henipavirus-Reservoir ............................................................. 6 1.3 Hendravirus-Infektionen .................................................................................. 8 1.3.1 Hendravirus-Ausbrüche in Australien ....................................................... 8 1.3.2 Hendravirus-Impfstoff für Pferde .............................................................. 9 1.4 Nipahvirus-Infektionen .................................................................................... 9 1.4.1 Erster Nipahvirus-Ausbruch 1998/99 in Malaysia ..................................... 9 1.4.2 Aktuelle Nipahvirus-Ausbrüche .............................................................. 10 1.5 Henipavirus-Impfstoffe und Therapien für den Menschen ............................. 11 1.6 Das afrikanische Kumasivirus (KV) ............................................................... 12 1.6.1 Die Entdeckung neuer Henipaviren ....................................................... 12 1.6.2 Isolierung des Kumasivirus .................................................................... 13 1.6.3 Genomanalyse des Kumasivirus............................................................ 16 1.7 Die Henipavirus Oberflächen-Glykoproteine G und F ................................... 18 1.7.1 Das Fusionsprotein F ............................................................................. 18 1.7.2 Das Glykoprotein G ............................................................................... 20 1.7.3 Die Rolle der henipaviralen Glykoproteine im Replikationszyklus .......... 25 ............................................................................................................................... 26 1.8 Die Henipavirus-Rezeptoren Ephrin-B2 und -B3 ........................................... 26 1.9 Aufgabenstellung und Zielsetzung ................................................................ 28 I Inhaltsverzeichnis II Ergebnisse .................................................................................................................. 30 2.1 Untersuchung der Fusionshelferaktivität des KV-G Proteins ......................... 30 2.2 Untersuchung der Oberflächenexpression des KV-G Proteins ...................... 31 2.3 Intrazelluläre Lokalisation des KV-G Proteins ............................................... 35 2.4 Charakterisierung der N-Glykosylierungsstellen im KV-G Protein ................. 37 2.4.1 Nachweis von Mannose-reichen und komplexen Zuckerketten im KV-G 37 2.4.2 Charakterisierung von KV-G Glykosylierungsmutanten ......................... 40 2.4.3 Untersuchung der Fusionshelferaktivität der Glykosylierungsmutanten . 43 2.5 Charakterisierung der Cysteine in der Stieldomäne des KV-G Proteins ........ 45 2.5.1 Untersuchungen zur KV-G Oligomerisierung ......................................... 46 2.5.2 Charakterisierung der KV-G Stieldomänen-Mutanten ............................ 48 2.5.3 Fusionshelferaktivität der Cystein-Stieldomänen-Mutanten .................... 50 ............................................................................................................................... 53 ............................................................................................................................... 54 2.6 Untersuchung eines nicht konservierten Endozytose-Motivs in der zytoplasmatischen Domäne des KV-G Proteins ...................................................... 54 2.6.1 Qualitativer Nachweis der Internalisierung durch einen antibody uptake Assay 55 2.6.2 Hemmung der Clathrin-vermittelten Endozytose durch Chlorpromazin- Behandlung der Zellen ........................................................................................ 57 2.6.3 Untersuchung der Ko-Endozytose von NiV-F und KV-G Proteinen ........ 58 2.6.4 Quantifizierung der Endozytoserate von KV-G durch „MESNA-Reduktion“ 59 2.6.5 Einfluss der Y38A Mutation auf die Oberflächenexpression von KV-G .. 61 2.6.6 Fusionshelferaktivität der Endozytose-Mutante KV-GY38A ...................... 62 2.7 Untersuchung der Doppelmutante KV-GY38A+C314S ......................................... 63 2.7.1 Untersuchung der Kopfdomänen-Mutante KV-GC314S ............................. 63 2.7.2 Charakterisierung der KV-GY38A+C314S Doppelmutante ............................ 64 III Diskussion .................................................................................................................. 66 3.1 Heterotypische Fusionshelferaktivität des KV-G Proteins ............................. 66 II Inhaltsverzeichnis 3.2 Ineffizienter Oberflächentransport des KV-G Proteins ................................... 67 3.3 N-Glykane sind essentiell für den Transport und die Fusionshelferfunktion des KV-G Proteins ......................................................................................................... 68 3.4 KV-G bildet verstärkt hocholigomere Formen aus ......................................... 69 3.5 Veränderungen im Oligomerisierungsmuster führen zum Verlust der Fusionshelferfunktion .............................................................................................. 71 3.6 Ein nicht konserviertes Endozytose-Motiv Y38FGL verringert die Oberflächenexpression des KV-G Proteins ............................................................. 72 3.7 Die Kombination aus den Mutationen C314S und Y38A steigert die Fusionshelferfunktion des KV-G Proteins signifikant ............................................... 73 3.8 Charakterisierung der Funktion des KV-G Proteins im viralen Kontext .......... 74 3.9 Identifikation neuer Henipaviren.................................................................... 75 3.10 Einschätzung des zoonotischen Potentials von KV ................................... 77 IV Material ....................................................................................................................... 79 4.1 Materialien, Chemikalien und Verbrauchsmaterialien ................................... 79 4.2 Enzyme ........................................................................................................ 81 4.2.1 Restriktionsendonukleasen .................................................................... 81 4.3 Antikörper ..................................................................................................... 82 4.3.1 Primärantikörper .................................................................................... 82 4.3.2 Sekundärantikörper ............................................................................... 82 4.4 Kits ............................................................................................................... 82 4.5 Plasmide ....................................................................................................... 83 4.6 Primer ........................................................................................................... 84 4.6.1 Sequenzierungsprimer ........................................................................... 84 4.6.2 Klonierungsprimer ................................................................................. 84 4.6.3 Q5 Site-Directed-Mutageneseprimer ..................................................... 85 4.7 Zellen............................................................................................................ 86 4.7.1 Eukaryotische Zellen ............................................................................. 86 4.7.2 Prokaryotische Zellen ...........................................................................
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