Role of the herpesvirus telomeric repeats and the protein U94 in human herpesvirus 6 integration Dissertation zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.) eingereicht im Fachbereich Biologie, Chemie, Pharmazie der Freien Universität Berlin vorgelegt von Nina Claudia Wallaschek aus Würzburg, Deutschland Berlin 2015 Diese Promotionsarbeit wurde im Zeitraum von Juni 2011 bis November 2015 am Institut für Virologie der Freien Universität Berlin unter der Leitung von Professor Dr. Nikolaus Osterrieder angefertigt. 1. Gutachter: Prof. Dr. Nikolaus Osterrieder 2. Gutachter: Prof. Dr. Petra Knaus Disputation am 17.02.2016 ‘I’m still confused, but on a higher level.’ Enrico Fermi (1901-1954) Table of contents 1 Table of contents 1 Table of contents .......................................................................................... I 2 List of figures and tables............................................................................. V 3 Abbreviations ............................................................................................. VII 4 Prolog ........................................................................................................... 1 5 Introduction .................................................................................................. 3 5.1 Herpesvirales ....................................................................................................... 3 5.2 Human herpesvirus 6 ........................................................................................... 4 5.2.1 History and general facts .................................................................................. 4 5.2.2 Animal models .................................................................................................. 6 5.2.3 Genome structure ............................................................................................. 6 5.2.4 Immunobiology ................................................................................................. 7 5.2.5 Replication cycle ............................................................................................... 8 5.2.5.1 Nuclear domains 10 (ND10s) .................................................................... 9 5.2.5.2 Lytic phase ..............................................................................................10 5.2.5.3 Latent phase ............................................................................................12 5.3 HHV-6 integration ................................................................................................14 5.3.1 Telomere biology .............................................................................................15 5.3.2 Integration during latency vs. the germline .......................................................18 5.3.3 Viral integration factors ....................................................................................20 5.3.3.1 Telomeric repeat sequences ....................................................................21 5.3.3.2 U94 protein ..............................................................................................21 5.4 Project outline .....................................................................................................22 6 Materials and Methods .............................................................................. 24 6.1 Materials .............................................................................................................24 6.1.1 Chemicals, consumables and equipment .........................................................24 6.1.2 Enzymes and markers .....................................................................................29 6.1.3 Antibodies ........................................................................................................29 I Table of contents 6.1.4 Kits… ...............................................................................................................30 6.1.5 Antibiotics ........................................................................................................30 6.1.6 Bacteria, cells, viruses and plasmids ...............................................................31 6.1.7 Buffers and media ...........................................................................................32 6.1.8 Primers ............................................................................................................35 6.2 Methods ..............................................................................................................37 6.2.1 Molecular biology methods ..............................................................................37 6.2.1.1 The Red recombination system and en passant mutagenesis .................37 6.2.1.1.1 Generation of recombinant plasmids ................................................40 6.2.1.1.2 DNA mini- and midi-preparation........................................................41 6.2.1.1.3 Electrocompetent bacteria ................................................................42 6.2.1.1.4 Generation of HHV-6A mutants ........................................................42 6.2.1.1.5 Sequencing PCR ..............................................................................43 6.2.1.2 Southern blot ...........................................................................................43 6.2.2 Cell culture methods ........................................................................................44 6.2.2.1 Culture of eukaryotic cells ........................................................................44 6.2.2.2 Lentiviral transduction ..............................................................................44 6.2.2.3 Immunofluorescence ...............................................................................44 6.2.2.4 Western blot ............................................................................................45 6.2.2.5 Virus reconstitution ..................................................................................45 6.2.2.6 Cell-free virus production, titration and growth kinetics ............................46 6.2.2.7 In vitro integration assay ..........................................................................46 6.2.2.8 qPCR .......................................................................................................46 6.2.2.9 Metaphase preparation and fluorescence in situ hybridization (FISH) ......47 6.2.2.10 Preparation of clonal cell lines .................................................................48 6.2.2.11 Reactivation assay ..................................................................................48 6.2.2.12 Statistical analysis ...................................................................................48 7 Results ........................................................................................................ 49 7.1 Establishment of ND10 knockdown JJHan cells using lentiviral transduction ......49 II Table of contents 7.2 Evaluating the role of the HHV-6A telomeric repeats in replication and integration ...........................................................................................................50 7.2.1 Telomeric repeats are not essential for HHV-6 replication ...............................50 7.2.2 Development of an in vitro latency/integration model .......................................51 7.2.3 Integration of the double TMR mutant is severely impaired in U2OS cells .......53 7.2.4 The pTMRs are crucial for HHV-6 integration ..................................................54 7.2.5 Integration analysis of TMR mutant viruses in clonal U2OS cell lines ..............60 7.3 Evaluating the role of the HHV-6A protein U94 in replication and integration ......61 7.3.1 Disruption of U94 expression ...........................................................................61 7.3.1.1 Complete deletion of open reading frame leads to growth defect .............62 7.3.1.2 Introduction of stop codon mutations to abrogate U94 expression ...........63 7.3.2 Integration of HHV-6A is not altered in the absence of U94 .............................64 7.3.3 Role of U94 in the integration into T-cells ........................................................67 7.3.4 Establishment of clonal cell lines for the ∆U94 mutant .....................................69 7.3.5 Reactivation of HHV-6A in latently infected cells ..............................................70 8 Discussion .................................................................................................. 71 8.1 ND10 knockdown cells as tool for more efficient virus reconstitution ...................71 8.2 TMRs are required for HHV-6A integration ..........................................................71 8.3 Presence of TMRs in a number of herpesviruses ................................................74 8.4 U94 is dispensable for HHV-6A integration .........................................................76 8.5 Other viral and cellular factors potentially involved in integration .........................78 8.6 Why telomeres? ..................................................................................................79 8.7 ciHHV-6 disease associations .............................................................................81 8.8 Virus reactivation from its integrated state ...........................................................82 8.9 Therapeutic intervention ......................................................................................83