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The Molecular Interaction of Feline Immunodeficiency Virus Vif with Feline APOBEC3 and Cullin 5

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The Molecular Interaction of Feline Immunodeficiency Virus Vif with Feline APOBEC3 and Cullin 5 The molecular interaction of feline immunodeficiency virus Vif with feline APOBEC3 and Cullin 5 INAUGURAL-DISSERTATION zur Erlangung des Grades eines Dr.biol.anim. beim Fachbereich Veterinärmedizin der Justus-Liebig-Universität Gießen Qinyong Gu Aus dem Institut für Virologie der Justus-Liebig-Universität Gießen Betreuer: Prof. Dr. Gergely Tekes und Aus der Klinik für Gastroenterologie, Hepatologie und Infektiologie der Heinrich-Heine-Universität Düsseldorf Betreuer: Prof. Dr. Carsten Münk The molecular interaction of feline immunodeficiency virus Vif with feline APOBEC3 and Cullin 5 INAUGURAL-DISSERTATION zur Erlangung des Grades eines Dr.biol.anim. beim Fachbereich Veterinärmedizin der Justus-Liebig-Universität Gießen Eingereicht von Qinyong Gu Aus China Giessen 2018 Mit Genehmigung des Fachbereichs Veterinärmedizin der Justus-Liebig-Universität Gießen Dekan: Prof. Dr. Martin Kramer Gutachter: Prof. Dr. Gergely Tekes Prof. Dr. Carsten Münk Tag der Disputation: 29-06-2018 Declaration I declare under oath that I have compiled my dissertation independently and without any undue assistance by third parties under consideration of the ‘Principles for the Safeguarding of Good Scientific Practice at Justus -Liebig- Universität Gießen’ Gießen/ Düsseldorf Date: 29-06-2018 Qinyong Gu I Publications This thesis is based on the following publications: 1. Gu Q, Zhang Z, Cano Ortiz L, Franco AC, Häussinger D, Münk C. 2016. Feline Immunodeficiency Virus Vif N-Terminal Residues Selectively Counteract Feline APOBEC3s. Journal of virology 90:10545-10557. 2. Gu Q, Zhang Z, Gertzen CGW, Häussinger D, Gohlke H, Münk C. 2017. Identification of a conserved interface of HIV-1 and FIV Vifs with Cullin 5. Journal of virology. 2017 Dec 20. pii: JVI.01697-17. doi: 10.1128/JVI.01697-17. This thesis is also partially based on the following publication: 1. Zhang Z, Gu Q, Jaguva Vasudevan AA, Hain A, Kloke BP, Hasheminasab S, Mulnaes D, Sato K, Cichutek K, Häussinger D, Bravo IG, Smits SH, Gohlke H, Münk C. 2016. Determinants of FIV and HIV Vif sensitivity of feline APOBEC3 restriction factors. Retrovirology 13:46. Statement Most of data in current thesis are produced by me. To retain the integrity of the written thesis, several figures produced by my project cooperators are included with their permission. The contribution of these data is clearly indicated. II Table of contents List of figures ......................................................................................................................................... VII List of tables ........................................................................................................................................... IX List of abbreviations .............................................................................................................................. XI 1. Introduction ........................................................................................................................................ 1 1.1 Retroviruses .............................................................................................................................. 1 1.1.1 Discovery of retroviruses ............................................................................................... 1 1.1.2 Classification of retroviruses ......................................................................................... 1 1.1.3 Retrovirus genome structure ........................................................................................ 2 1.1.4 Retroviral lifecycle ......................................................................................................... 4 1.2 Human immunodeficiency virus .............................................................................................. 5 1.3 Feline immunodeficiency virus................................................................................................. 8 1.3.1 FIV-caused disease ......................................................................................................... 8 1.3.2 FIV subgroups and cross-species transmission ............................................................. 9 1.3.3 FIV genome structure .................................................................................................. 10 1.3.4 FIV based lentivirus vectors ........................................................................................ 11 1.4 Feline restriction factors ......................................................................................................... 12 1.4.1 Tetherin: ....................................................................................................................... 14 1.4.2 Trim5α: ......................................................................................................................... 14 1.4.3 SAMHD1： ................................................................................................................... 15 1.4.4 MxB: ............................................................................................................................. 16 1.4.5 SERINC: ......................................................................................................................... 17 1.4.6 APOBEC3: ..................................................................................................................... 17 1.5 Cullin 5-E3 ubiquitin complex ................................................................................................. 19 1.6 Feline APOBEC3 and FIV Vif .................................................................................................... 21 1.7 Molecular interaction of APOBEC3 with Vif .......................................................................... 22 1.8 Objectives of current study .................................................................................................... 24 III 2. Materials and Methods .................................................................................................................... 25 2.1 Laboratory instruments .......................................................................................................... 25 2.2 Chemicals ................................................................................................................................ 25 2.3 Enzymes................................................................................................................................... 26 2.4 Kits ........................................................................................................................................... 26 2.5 Buffers and solutions .............................................................................................................. 26 2.5.1 Buffers for gel electrophoresis .................................................................................... 26 2.5.2 6 x DNA loading dye ..................................................................................................... 27 2.5.3 10 x SDS PAGE buffer (Table 2.5.3) ............................................................................. 27 2.5.4 20 x TBS ........................................................................................................................ 27 2.6 Bacterial strains (Competent cells) ........................................................................................ 27 2.7 Cells ......................................................................................................................................... 28 2.8 Vif and A3 plasmids. ............................................................................................................... 28 2.9 Fusion PCR ............................................................................................................................... 34 2.10 Transfection .......................................................................................................................... 36 2.11 Viruses and infection ............................................................................................................ 37 2.12 Immunoblot analysis ............................................................................................................ 39 2.13 Immunofluorescence ............................................................................................................ 40 2.14 GST-pull down ....................................................................................................................... 41 2.15 Immunoprecipitation ............................................................................................................ 41 2.16 Vif sequences from naturally infected cats ......................................................................... 43 2.17 Homology Modeling ............................................................................................................. 44 2.18 Nucleotide sequence accession numbers ............................................................................ 45 2.19 Statistical analysis ................................................................................................................. 45 3. Results ............................................................................................................................................... 46 3.1 Identification of FIV Vif domains responsible for feline A3 degradation ............................. 46 3.1.1 Identification of FIV Vif determinants specific for feline A3Z2 degradation.
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