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“The Cellular Context of Rabies Virus Replication – Cell Culture Adaptation, Intra-Neuronal Virus Transport and Development of Host Gene Targeting Vectors”

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“The Cellular Context of Rabies Virus Replication – Cell Culture Adaptation, Intra-Neuronal Virus Transport and Development of Host Gene Targeting Vectors” “The Cellular Context of Rabies Virus Replication – Cell Culture Adaptation, Intra-Neuronal Virus Transport and Development of Host Gene Targeting Vectors” I n a u g u r a l d i s s e r t a t i o n zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald vorgelegt von Sabine Nemitz geboren am 14.02.1989 in Rathenow Greifswald, den 20.07.2018 1 Dekan: Prof. Dr. Werner Weitschies 1. Gutachter : PD Dr. Stefan Finke 2. Gutachter: Prof. Dr. Karl-Klaus Conzelmann Tag der Promotion: 05.12.2018 2 3 I. Table of Content I. Table of Content 4 II. List of Abbreviations 7 III. Publications 10 1. SUMMARY 11 2. INTRODUCTION 15 2.1. Classification 16 2.2. Transmission 18 2.3. RABV virion structure and genome organization 19 2.4. RABV proteins 21 2.5. Life cycle 26 2.6. Neuronal transport of RABV particles 28 2.7. Fixed and non-fixed RABV 30 2.8. RABV as vector and research tool 31 2.8.1. Gene regulation via post-transcriptional silencing: RNAi 32 2.8.2. Cytoplasmically replicating RNA viruses as vectors for shRNA synthesis 34 3. AIMS OF THE STUDY 35 4. MATERIAL 36 4.1. Technical equipment 36 4.2. Chemicals 36 4.3. Antibiotics 36 4.4. Standard buffers 37 4.5. Marker 38 4.6. Kits 38 4.7. Cell lines 38 4.8. Cell culture media 39 4.9. Bacteria 41 4.10. Viruses 41 4.11. Plasmids 42 4.12. Enzymes 44 4.13. Oligonucleotides 45 4.14. Serologic reagents 48 4.14.1. Primary antibodies 49 4.14.2. Secondary antibodies 49 4.14.3. Fluorescent dyes 49 4.15. Software 50 4.16. Sequencing technologies 50 5. METHODS 51 5.1. Eukaryotic cell culture 51 5.1.1. Cell cultivation 51 5.1.2. Transfection of mammalian cell lines 52 5.1.3. Complementation assay 52 5.2. Preparation of primary neuronal cells 53 5.2.1. Preparation and cultivation of hippocampal neurons from neonatal rats 53 4 5.2.2. Preparation and cultivation of dorsal root ganglions from embryonic rats 53 5.3. Working with nucleic acids 54 5.3.1. Nuclease treatment 54 5.3.2. RNA extraction with Trifast (Trizol) / Chloroform 55 5.3.3. Isolation of viral RNA for Deep sequence analysis 56 5.3.4. Determining RNA and DNA concentration 56 5.3.5. Reverse Transcription (RT)-PCR 57 5.3.6. Polymerase chain reaction (PCR) 57 5.3.7. Polymerase chain reaction for comparison of mRNA level 58 5.3.8. Restriction enzyme digestion 59 5.3.9. Ligation 60 5.3.10. Transformation of chemically competent bacteria 60 5.3.11. Preparation of plasmid DNA 60 5.3.12. Preparative and analytic agarose gel electrophoresis 61 5.3.13. Site-directed mutagenesis of full-length RABV c-DNA containing plasmids 61 5.3.14. Routine DNA sequencing (Sanger sequencing) 62 5.4. Working with proteins 63 5.4.1. Preparation of protein samples with protein lysis mix 63 5.4.2. Discontinuous sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 64 5.4.3. Semi-dry western blot 64 5.4.4. Indirect immunofluorescence 65 5.5. Working with viruses 66 5.5.1. Production of RABV virus stocks 66 5.5.2. Rescue of recombinant RABV (CaPO4 Method) 66 5.5.3. Virus titration by end-point-dilution 67 5.5.4. Replication kinetics 68 5.6. Cloning strategies 68 5.6.1. Truncation of full-length cDNA plasmids encoding shRNA against cellular targets 68 5.6.2. Cloning of G protein sequences from recombinant dog rabies virus (rRABV Dog) 69 5.6.3. Generation of recombinant RABV expressing ecto-domains of other Lyssavirus strains 70 5.7. Electron Microscopy 70 6. RESULTS 71 6.1. Axonal transport of RABV in dorsal root ganglions 71 6.1.1. Effect of various Lyssavirus G-sequences on the accumulation of RABV N particles in DRG axons 71 6.1.2. Accumulation of N and G in rRABV DogA and rRABV Fox infected DRG neurons 73 6.2. Molecular basis of RABV cell culture adaptation 77 6.2.1. Virus passages on different cell lines 77 6.2.2. Deep sequence analysis of cell culture passaged rRABV Dog and rRABV Fox 81 6.2.3. Release of new virus particles is impaired in non-fixed rRABV Dog 87 6.2.4. Cell line specific intracellular G accumulation and increased surface expression after virus passages. 90 6.3. RABV vectors for cytoplasmic shRNA expression 98 6.3.1. Generation of RABV expressing miR-shRNAs 98 6.3.2. Growth kinetics of rRABV expressing miR-shRNA 100 6.3.3. Downregulation of DYNLL1 in RABV-infected NA 42/13 cells 101 6.3.4. Infection of primary rat hippocampus neurons 107 5 7. DISCUSSION 110 7.1. Axonal transport of RABV 110 7.1.1. Accumulation of RABV N particles in DRG axons 111 7.1.2. Anterograde transport of non-fixed N and G virus particles in DRG axons 111 7.2. Cell culture passages of rRABV Dog and rRABV Fox lead to specific mutations in G and P 112 7.2.1. Increased virus titer after cell culture passage 113 7.2.2. Identification of adaptive mutations in the G protein 114 7.2.3. Accumulation of mutations in the P protein 116 7.2.4. Mutations in non-coding gene borders 117 7.2.5. Accumulation of non-fixed rRABV Dog-B at Endoplasmic reticulum 118 7.2.6. Cell culture adaptation and virulence 120 7.3. Generation of RABV expressing shRNA against cellular targets 121 7.3.1. Downregulation of DYNLL1 mRNA and protein level by RABV expressing miR-shRNAs 123 8. REFERENCES 127 APPENDIX 141 Supplementary data 141 Eigenständigkeitserklärung 153 Posters and Presentations on Conferences 154 Acknowledgement 155 6 II. List of Abbreviations aa amino acid AAV Adeno-associated virus AGO Agonaute protein ANP32B Acidic (Leucine-Rich) Nuclear Phosphoprotein 32 ATV Alsever’s Trypsin-Versene BHK baby hamster kidney CCLV Collection of Cell Lines in Veterinary Medicine cf. compare, latin: confer CLSM confocal laserscanning microscopy CNS central nervous system CR coding region C-terminus carboxy-terminus CVS challenge virus standard ddH2O deminaralized, destilled water DEPC Diethylpyrocarbonate DGCR8 DiGeorge syndrome critical region DMEM Dulbecco’s Modified Eagle Medium DMSO Dimethylsulfoxid DNA deoxyribonucleic acid dNTPs deoxyribonucleosid triphosphate DogA Dog Azerbaijan dpi days post infection DRG dorsal root ganglia neuron DYNLL1 dynein light chain 1 E. coli Escherichia coli e.g. for example, latin: exempli gratia eIF3H cellular eukaryotic translation initiation factor 3 subunit H ER endoplasmatic reticulum et al. and others; latin: et alii EXP5 Exportin 5 FCS fetal calf serum ffu focus forming units G glycoprotein G418 Geneticin Genta Gentamycin GFP green fluorescent protein HBSS Hanks balanced salt solution HEKT human embryonic kidney cells HRP horse raddish peroxidase i.e. that is; latin: id est ICTV imternational commitee for the taxonomy of viruses 7 IFN Interferon IgG immunoglobulin G iIF indirect immunofluorescence IRF interferon regulatory factor L large protein LB lysogeny broth LBV Lagos bat lyssavirus M matrixprotein MDCK Madin Darby canine kidney MEM Minimal Essential Medium miR micro RNA miR-shRNA micro RNA-adapted shRNA MNA murine neuroblastoma cells MOI multiplicity of infection MOKV Mokola bat lyssavirus mRNA messenger RNA N nucleoprotein NA 42/13 murine neuroblastoma cells NCBI National Center for Biotechnology Information NCR non- coding region NCS new born calf serum Nf-kappa-B Nuclear Factor Kappa B Subunit 1 nt nucleotide N-terminus amino-terminus o/n over night ORF open reading frame P passage P phosphoprotein p75NTR low affintiy nerve growth factor receptor PAGE polyacrylamid gelelectrophoresis PBS phosphate buffered saline PCR polymerase chain reaction PDZ structural domain in signal proteins PEI Polyethylenimine Pen/Strep Penicelline and Streptomycine PEP postexposure prophylaxis PFA paraform aldehyde pH potentia hydrogenii poly I:C polyinosinic:polycytidylic acid pre-miRNA precursor miRNA pri-miRNA primary miRNA q-PCR quantitative PCR RABV Rabies lyssavirus 8 RelAp43 NF-κB protein close to RelA/p65 RISC RNA-induced silencing complex RNA ribonucleic acid RNAi RNA interference RNP ribonucleoprotein RPMI medium for mammalian cell culture rRABV recombinant Rabies lyssavirus RT-PCR reverse transcription PCR SAD Street Alabama Dufferin SDS sodium dodecyl sulfate shRNA short hairpin RNA SINV Sindbis virus siRNA small interfering RNA interferon-induced signal transducer and activator of STAT transcription TCID50 50% tissue culture infective dose TEMED tetramethylethylendiamine TET tetracyclin inducible TIS transcription initiation signal TTS transcription termination signal VSV Indiana vesiculovirus WB western blot WHO world health organization WT wildtype α anti 9 III. Publications In Preparation: Rabies Virus Vectors for Specific Host Cell shRNA Knock-down in infected cells Nemitz S, Nolden T, Finke S Cell culture adaptation of recombinant Rabies virus field viruses from fox and dog Nemitz S, Nolden T, Christen M, Finke S Published: Oral vaccination of wildlife against rabies: Differences among host species in vaccine uptake efficiency. Vos A, Freuling CM, Hundt B, Kaiser C, Nemitz S, Neubert A, Nolden T, Teifke JP, Te Kamp V, Ulrich R, Finke S, Müller T. Vaccine. 2017 Jul 13;35(32):3938-3944. doi: 10.1016/j.vaccine.2017.06.022. Epub 2017 Jun 19. Reverse genetics in high throughput: rapid generation of complete negative strand RNA virus cDNA clones and recombinant viruses thereof. Nolden T, Pfaff F, Nemitz S, Freuling CM, Höper D, Müller T, Finke S. Sci Rep. 2016 Apr 5;6:23887. doi: 10.1038/srep23887. A Dynein Light Chain 1 Binding Motif in Rabies Virus Polymerase L Protein Plays a Role in Microtubule Reorganization and Viral Primary Transcription.
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