Aus dem Zentrum für Hygiene und medizinische Mikrobiologie der Philipps Universität Marburg Institut für Virologie Geschäftsführender Direktor: Prof. Dr. Stephan Becker Potent inhibition of highly pathogenic influenza virus infection using a peptidomimetic furin inhibitor alone or in combination with conventional antiviral agents Dissertation Zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) dem Fachbereich Biologie der Philipps-Universität Marburg vorgelegt von Yinghui Lu aus Shanghai, China Marburg an der Lahn 2014 Die Untersuchungen zur vorliegenden Arbeit wurden im Institut für Virologie, Direktor: Prof. Dr. Stephan Becker, Fachbereich Medizin der Philipps-Universität Marburg, unter der Anleitung von Prof. Dr. Wolfgang Garten durchgeführt. Vom Fachbereich Biologie der Philipps-Universität Marburg als Dissertation angenommen am: 18.08.2014 Erstgutachter: Prof. Dr. Wolfgang Garten Zweitgutachter: Prof. Dr. Wolfgang Buckel Weitere Mitglieder der Prüfungskommission: Prof. Dr. Erhard Bremer Prof. Dr. Susanne Önel Tag der mündlichen Prüfung: 01.10.2014 献给我亲爱的家人 For my parents and sister Für meine Eltern und Schwester Inhalt Summary .................................................................................................................... 1 Zusammenfassung ..................................................................................................... 1 1. Introduction ............................................................................................................. 3 1.1 Influenza ............................................................................................................ 3 1.1.1 Classification ............................................................................................... 3 1.1.2 Clinical features and pathogenesis of influenza .......................................... 3 1.1.2.1 Human influenza viruses ...................................................................... 3 1.1.2.2 Avian influenza viruses ........................................................................ 4 1.1.2.2.1 Avian influenza H7 viruses ............................................................ 4 1.1.2.2.2 Avian influenza H5 viruses ............................................................ 6 1.1.3 Epidemiology .............................................................................................. 7 1.1.3.1 Antigenic drift and antigenic shift ......................................................... 7 1.1.3.2 History of human influenza pandemics ................................................ 8 1.1.4 Virus morphology, genome and proteins ..................................................... 9 1.1.5 Virus life cycle ........................................................................................... 12 1.2 Hemagglutinin ................................................................................................. 14 1.2.1 The structure of the hemagglutinin ............................................................ 14 1.2.2 Receptor binding specificity of hemagglutinin ........................................... 15 1.2.3 Proteolytic activation of precursor hemagglutinin ...................................... 16 1.2.3.1 Monobasic cleavage site .................................................................... 17 1.2.3.2 Multibasic cleavage site ..................................................................... 18 1.3 Proprotein convertases ................................................................................... 18 1.3.1 Furin ...................................................................................................... 19 1.4 Prevention and treatment of influenza ............................................................. 21 1.4.1 Vaccination ............................................................................................... 21 1.4.2 Approved antiviral agents .......................................................................... 22 1.4.2.1 Adamantane derivatives .................................................................... 23 1.4.2.2 Neuraminidase inhibitors ................................................................... 24 1.4.3 Other antiviral agents ................................................................................ 25 1.4.3.1 Proprotein convertase inhibitors ......................................................... 26 1.4.3.1.1 Furin inhibitors ................................................................................ 26 1.4.4 Combination therapy ................................................................................. 28 1.5 Aim of the study ............................................................................................... 29 2. Materials ............................................................................................................... 31 2.1 Chemicals ....................................................................................................... 31 2.2 Consumed materials ....................................................................................... 32 2.3 Instruments...................................................................................................... 33 2.4 Kits .................................................................................................................. 33 2.5 Protein markers ............................................................................................... 33 2.6 Antibodies ....................................................................................................... 34 2.6.1 Primary antibodies .................................................................................... 34 2.6.2 Secondary antibodies ................................................................................ 34 2.7 Enzymes ......................................................................................................... 34 2.8 Primers ............................................................................................................ 34 2.9 Viruses ............................................................................................................ 35 2.10 Consumed materials for cell cultures ............................................................ 35 2.10.1 Cell culture medium ................................................................................ 35 2.10.2 Cell lines ................................................................................................. 36 2.11 Buffers ........................................................................................................... 36 2.12 Others ........................................................................................................... 38 3. Methods ................................................................................................................ 39 3.1 Molecular and biological methods ................................................................... 39 3.1.1 Antiviral compounds .................................................................................. 39 3.1.2 MTT viability assay .................................................................................... 39 3.1.3 Stability measurements of furin inhibitors with high performance liquid chromatography ................................................................................................. 39 3.1.4 Sodium dodecyl sulfate polyacrylamide gel electrophoresis ..................... 40 3.1.5 Western blots ............................................................................................ 41 3.1.6 Viral RNA extraction .................................................................................. 41 3.1.7 One-step reverse transcription polymerase chain reaction ....................... 42 3.1.8 Two-steps reverse transcription polymerase chain reaction ..................... 43 3.1.8.1 Reverse transcription ......................................................................... 43 3.1.8.2 Polymerase chain reaction ................................................................. 43 3.1.9 DNA electrophoresis ................................................................................. 44 3.1.10 DNA fragment extraction and purification ................................................ 44 3.1.11 DNA purification ...................................................................................... 44 3.1.12 DNA-sequencing ..................................................................................... 44 3.2 Cell culture methods ........................................................................................ 45 3.3 Virological methods ......................................................................................... 45 3.3.1 Virus propagation in eggs ......................................................................... 45 3.3.2 Virus propagation in cell cultures .............................................................. 46 3.3.3 Hemagglutination assay ............................................................................ 46 3.3.4 Plaque assay ............................................................................................ 46 3.3.5 Plaque reduction assay ............................................................................. 47 3.3.6 Immunostaining ......................................................................................... 47 3.3.7 Cleavage of HA0 protein in the presence of furin inhibitors ...................... 48