Dissertation Submitted to the Combined Faculties for the Natural
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Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Diplom-Biochemiker Johannes Hermle born in: Offenbach a.M., Germany Oral-examination: July 26, 2017 siRNA SCREEN FOR IDENTIFICATION OF HUMAN KINASES INVOLVED IN ASSEMBLY AND RELEASE OF HIV-1 Referees: Prof. Dr. Hans-Georg Kräusslich Prof. Dr. Dirk Grimm ii Meiner Familie iii Summary Summary The replication of the human immunodeficiency virus type 1 (HIV-1) is as yet not fully understood. In particular the knowledge of interactions between viral and host cell proteins and the understanding of complete virus-host protein networks are still imprecise. An integral picture of the hijacked cellular machinery is essential for a better comprehension of the virus. And as a prerequisite, new tools are needed for this purpose. To create such a novel tool, a screening platform for host cell factors was established in this work. The screening assay serves as a powerful method to gain insights into virus-host-interactions. It was specifically tailored to addressing the stage of assembly and release of viral particles during the replication cycle of HIV-1. It was designed to be suitable for both RNAi and chemical compound screening. The first phase of this work comprised the setup and optimization of the assay. It was shown, that it was robust and reliable and delivered reproducible results. As a subsequent step, a siRNA library targeting 724 human kinases and accessory proteins was examined. After the evaluation of the complete siRNA library in a primary screen, all primary hits were validated in a second reconfirmation screen using different siRNAs. The purpose of this two-step approach was to identify and exclude false positives. In the end, 43 genes were reconfirmed to influence the assembly and release of HIV-1. Out of those, 39 were host dependency and 4 host restriction factors. Several of them had already been described in the literature to interact with HIV-1. However, various so far unknown host cell proteins were identified within this work. A subsequent combinatory pathway analysis including hits from other published screens identified several important signaling pathways to be important for HIV-1 assembly and release. The described single key proteins and their underlying protein networks provide a basis for the next steps toward understanding the virus and improving treatment in the future. iv Zusammenfassung Zusammenfassung Noch immer gibt es große Lücken im Verständnis der Replikationsmechanik des Humanen Immundefizienz-Virus Typ 1 (HIV-1). Im Besonderen das Wissen um Interaktionen von HIV-1 mit Wirtszellproteinen ist weiterhin unvollständig, sowie das Wissen über den Aufbau der Virus-Wirt Proteinnetzwerke. Ein umfassendes Bild der, durch das Virus zweckentfremdeten, Zellmaschinerie ist essentiell, um das Virus im Ganzen zu verstehen. Die vorliegende Arbeit beschreibt die Etablierung einer Hochdurchsatz- Screening Plattform als äußerst leistungsfähige Methode, um Einblicke in die Virus-Wirt Wechselbeziehungen zu generieren. Die Plattform ist spezifisch auf die Untersuchung der Partikelbildung und –freisetzung von HIV-1 zugeschnitten. Sie wurde entwickelt, um sowohl mit RNA- Interferenzbibliotheken, als auch mit Bibliotheken chemischer Moleküle verwendbar zu sein. Die erste Phase dieser Arbeit umfasste die Entwicklung und den Aufbau der Plattform unter Durchführung der notwendigen Qualitätstests. Die Ergebnisse zeigten, dass die entwickelte Plattform robust war und verlässliche und reproduzierbare Ergebnisse lieferte. Als erste Anwendung wurde eine Bibliothek von „short interfering RNAs“ (siRNAs) getestet, die 724 humane Kinasen und verwandte Proteine abdeckte. Zunächst wurde in einem primären Test die komplette Bibliothek untersucht. Um die gefundenen, potenziellen Wirtszellfaktoren zu bestätigen und um mögliche fälschlich- Positive auszusondern, wurden diese Treffer in einem zweiten Bestätigungssuchtest überprüft. Insgesamt wurden hierbei 43 Proteine bestätigt – davon 39 Abhängigkeits- und 4 Restriktionsfaktoren. Einige davon waren schon vorher in der Literatur in Bezug zu HIV-1 beschrieben, jedoch war auch ein Teil in diesem Kontext bisher unbekannt und stellt daher vielversprechende, neue Ziele für das Verständnis von der HIV-1-Replikation dar. Eine anschließend durchgeführte Kombinations-Netzwerk-Analyse unter Einbeziehung anderer Publikationen identifizierte wichtige Signalkaskaden. Die in dieser Arbeit gewonnen v Zusammenfassung Erkenntnisse bilden die Basis für zukünftige Untersuchungen, um die spezifischen Rollen dieser Proteine und Netzwerke für die Formation und Freisetzung von HIV-1 aufzudecken. vi Table of Contents Table of contents Summary ............................................................................................................... iv Zusammenfassung .................................................................................................. v Table of contents .................................................................................................. vii 1 Introduction .................................................................................................. 9 1.1 Human immunodeficiency virus type 1 ................................................... 9 1.2 Virus host interactions ............................................................................19 1.3 The ESCRT complex ............................................................................... 25 1.4 Kinases and HIV-1 .................................................................................. 28 1.5 Identification of host cell factors ........................................................... 30 2 Aim of the study .......................................................................................... 32 3 Materials & Methods ................................................................................... 33 3.1 Materials ................................................................................................. 33 3.2 Methods .................................................................................................. 38 4 Results ......................................................................................................... 43 4.1 Setup of the screening assay .................................................................. 45 4.2 Primary siRNA screen ............................................................................ 64 4.3 Reconfirmation screen ........................................................................... 73 4.4 Exemplary single hit characterization ................................................... 78 4.5 Bioinformatical analysis ......................................................................... 81 5 Discussion ................................................................................................... 87 5.1 Establishment of the screening assay .................................................... 87 5.2 Focus on the cellular conductors: Results from the kinase screen ....... 90 5.3 Individual hits in the context of current literature .............................. 102 5.4 The broader picture: Signaling pathways ............................................ 106 5.5 Conclusion ............................................................................................ 109 vii Table of Contents 6 List of figures .............................................................................................. 111 7 List of tables ............................................................................................... 112 8 List of abbreviations .................................................................................. 113 9 List of publications..................................................................................... 118 10 Acknowledgments ...................................................................................... 121 11 References .................................................................................................. 122 12 Appendix .....................................................................................................151 12.1 Appendix 1: Primary screen kinase library (Ambion) .......................... 151 12.2 Appendix 2: Reconfirmation screen library ......................................... 154 12.3 Appendix 3: KEGG protein pathway maps ........................................... 164 12.4 Appendix 4: NCBI HIV-1 interaction database .................................... 168 viii Introduction 1 Introduction Great advances have been made in the therapy of the human immunodeficiency virus type 1 (HIV-1), which is the causative agent of the acquired immunodeficiency syndrome (AIDS). However, it still poses an enormous burden for patients and health care systems worldwide. This is in part due to the fact that there are uncharted areas in its replication cycle - especially regarding its interactions with the host cells. 1.1 Human immunodeficiency virus type 1 1.1.1 Clinical relevance In 1983 HIV-1 was first described to be the cause of a newly emerging epidemic of an immunodeficiency syndrome called AIDS (1-3). According to the United Nations Joint Program on HIV/AIDS (UNAIDS) approximately 36.7 million people were estimated to be living with a HIV-1 infection at the end of 2015. Furthermore, the UNAIDS fact sheet 2016 records 1.1 million AIDS related deaths and approximately