The role of SAMHD1 in restriction and immune sensing of retroviruses and retroelements Die Rolle von SAMHD1 in der Restriktion und Immunerkennung von Retroviren und Retroelementen Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr. rer. nat. vorgelegt von Alexandra Herrmann aus Biberach an der Riß Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 31.07.2018 Vorsitzender des Promotionsorgans: Prof. Dr. Georg Kreimer Gutachter: Prof. Dr. Lars Nitschke Prof. Dr. Manfred Marschall Table of content Table of content I. Summary ......................................................................................................................... 1 I. Zusammenfassung ......................................................................................................... 3 II. Introduction ..................................................................................................................... 5 1. The human immunodeficiency virus .................................................................................... 5 2. Transposable elements ......................................................................................................... 7 3. Host restriction factors ........................................................................................................ 10 4. The restriction factor SAMHD1 ........................................................................................... 11 5. Involvement of SAMHD1 in autoimmunity and antiviral responses ............................... 14 6. The cGAS/STING pathway of cytosolic DNA sensing ...................................................... 15 III. Objectives ......................................................................................................................19 1. The role of SAMHD1 in retroviral infection ........................................................................ 19 2. The role of SAMHD1 in the inhibition of endogenous retroelements ............................. 19 IV. Material and Methods ....................................................................................................21 1. Materials ................................................................................................................................ 21 1.1. Chemicals............................................................................................................................... 21 1.2. Buffers, solutions, and media ................................................................................................. 21 1.2.1. Cell culture .............................................................................................................. 21 1.2.2. Bacterial culture ...................................................................................................... 21 1.2.3. Standard buffers and solutions ............................................................................... 21 1.2.4. Protein methods ...................................................................................................... 22 1.2.5. Virus preparation .................................................................................................... 22 1.2.6. LEAP assay ............................................................................................................ 22 1.3. Vectors ................................................................................................................................... 23 1.3.1. Empty vectors ......................................................................................................... 23 1.3.2. SAMHD1 expression vectors .................................................................................. 23 1.3.3. Expression vectors for retroelements ..................................................................... 25 1.3.4. Vectors for virus preparation ................................................................................... 25 1.3.5. Others ..................................................................................................................... 26 1.4. Oligonucleotides ..................................................................................................................... 26 1.4.1. Cloning of SAMHD1 mutants .................................................................................. 26 1.4.2. Cloning of LINE-1 expression vectors .................................................................... 27 1.4.3. LEAP assay ............................................................................................................ 28 1.4.4. cDNA synthesis and qRT-PCR ............................................................................... 28 1.4.5. Genotyping.............................................................................................................. 28 1.5. Antibodies............................................................................................................................... 29 1.5.1. Primary antibodies .................................................................................................. 29 Table of content 1.5.2. Secondary antibodies ............................................................................................. 30 1.5.3. Direct antibody ........................................................................................................ 30 1.6. Enzymes................................................................................................................................. 30 1.7. Biological materials ................................................................................................................ 31 1.7.1. Cell lines ................................................................................................................. 31 1.7.2. Bacterial strains ...................................................................................................... 31 1.7.3. Mouse strains ......................................................................................................... 32 2. Methods................................................................................................................................. 32 2.1. Cell culture ............................................................................................................................. 32 2.1.1. Bone marrow preparation and BMDC isolation ...................................................... 32 2.2. Nucleic acid methods and transfections ................................................................................ 33 2.2.1. Cloning and plasmid preparation ............................................................................ 33 2.2.2. RNA preparation ..................................................................................................... 33 2.2.3. cDNA synthesis and qRT-PCR ............................................................................... 33 2.2.4. Transfections .......................................................................................................... 34 2.3. Virus preparations .................................................................................................................. 35 2.3.1. HIV-GFP ................................................................................................................. 35 2.3.2. HIV-Luc ................................................................................................................... 35 2.3.3. shRNA-encoding lentiviral particles: ....................................................................... 35 2.4. Infection assays ..................................................................................................................... 36 2.4.1. Infection of THP-1 cells with HIV-GFP ................................................................... 36 2.4.2. Infection of THP-1 cells with HIV-Luc ..................................................................... 36 2.4.3. Infection of mBMDCs with HIV-GFP ....................................................................... 36 2.4.4. Infection of mBMDCs for RNA isolation .................................................................. 36 2.5. Transcriptome analysis of murine BMDCs ............................................................................. 37 2.6. Retrotransposition assays ...................................................................................................... 37 2.6.1. GFP-based retrotransposition assay ...................................................................... 37 2.6.2. Neomycin-based retrotransposition assays ............................................................ 37 2.7. LINE-1 promoter assay .......................................................................................................... 38 2.8. LEAP assay ............................................................................................................................ 38 2.9. Protein methods ..................................................................................................................... 39 2.9.1. Preparation of cell lysates ....................................................................................... 39 2.9.2. Co-immunoprecipitation .......................................................................................... 39 2.9.3. SDS-Polyacrylamide