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Investigation and Comparison of the Human Interactions of Flaviviral NS5 Protein

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Investigation and Comparison of the Human Interactions of Flaviviral NS5 Protein This electronic thesis or dissertation has been downloaded from Explore Bristol Research, http://research-information.bristol.ac.uk Author: Hales, Jack Title: Investigation and comparison of the human interactions of flaviviral NS5 protein General rights Access to the thesis is subject to the Creative Commons Attribution - NonCommercial-No Derivatives 4.0 International Public License. A copy of this may be found at https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode This license sets out your rights and the restrictions that apply to your access to the thesis so it is important you read this before proceeding. Take down policy Some pages of this thesis may have been removed for copyright restrictions prior to having it been deposited in Explore Bristol Research. However, if you have discovered material within the thesis that you consider to be unlawful e.g. breaches of copyright (either yours or that of a third party) or any other law, including but not limited to those relating to patent, trademark, confidentiality, data protection, obscenity, defamation, libel, then please contact [email protected] and include the following information in your message: •Your contact details •Bibliographic details for the item, including a URL •An outline nature of the complaint Your claim will be investigated and, where appropriate, the item in question will be removed from public view as soon as possible. Investigation and comparison of the human interactions of flaviviral NS5 protein JACK HALES SCHOOL: CELLULAR AND MOLECULAR MEDICINE SUPERVISOR: DR ANDREW DAVIDSON START DATE: 22/09/2016 This project is my own work except where indicated. All text, figures, tables, data, or results which are not my own work, are indicated and the sources acknowledged. In addition, I confirm that the hardcopy and the e-submission are identical. Signed:_________________ Dated:_________________ In memory of: Richard John Hales (1945 – 2017) Who part funded this master’s degree And instilled the values of hard work and doing what you love Acknowledgements I would like to acknowledge first the input of Dr Andrew Davidson who supervised this project. Having been my supervisor through both my undergraduate project and master’s project, there is no doubt that his patient guidance and support throughout the years have been instrumental in developing my skills and helping me to progress onto a PhD project and into future research. For that, I am extremely grateful. I would also like to acknowledge Dr Lisa Stevens and Mr Joshua Lee for their input at the beginning of this project (as stated within) and also their advice and support during my time in the lab. Finally, I would like to acknowledge the constant support from family throughout the duration of this project and beyond. Table of contents 1: Introduction 1.1: Flaviviruses 15 1.2: The global impact of dengue virus 15 1.3: The global impact of Zika virus 17 1.4: Presentation of disease 1.4.1: Dengue 19 1.4.2: Zika 21 1.5: Flavivirus physiology, lifecycle, and pathogenesis 1.5.1: Flavivirus genome organisation and physiology 22 1.5.2: Transmission of dengue virus and Zika virus 24 1.5.3: Dengue lifecycle 26 1.5.4: Pathogenesis of Zika 29 1.5.5: Antibody-dependent enhancement 31 1.6: Flavivirus proteins 1.6.1: Capsid 34 1.6.2: Pre-membrane/membrane 34 1.6.3: Envelope 35 1.6.4: NS1 36 1.6.5: NS2A 37 1.6.6: NS2B 38 1.6.7: NS3 38 1.6.8: NS4A 39 1.6.9: NS4B 40 1.6.10: NS5 41 1.6.11: Similarities between ZIKV and DENV NS5 42 1.7: Flavivirus treatment and control methods 1.7.1: Drugs 43 1.7.2: Vaccines 44 1.7.3: Vector control 44 1.7.4: Host factor targets 45 1.8: Methods for investigating host-virus protein-protein interactions 46 1.9: Project aims 48 1 2: Methods and Materials 2.1: Cell culturing 2.1.1: Cell culture conditions 49 2.1.2: Cell lines 49 2.2: DNA/RNA techniques 2.2.1: DNA gel electrophoresis 50 2.2.2: DNA concentration determination 50 2.2.3: Primer design and synthesis 50 2.2.4: Primer sequences 50 2.2.5: DNA sequencing 51 2.2.6: RT-PCR 51 2.2.7: Transfection of HEK-293-Flp cells (transient) 52 2.2.8: Transfection of HEK-293-Flp cells (stable) 52 2.2.9: Bacterial transformation 53 2.2.10: Growth/selection of bacteria 53 2.2.11: Restriction enzyme (RE) digest 53 2.2.12: Ligation 53 2.3: Protein techniques 2.3.1: Cell lysis 53 2.3.2: BCA assay 54 2.3.3: Immunofluorescence assay (IFA) 54 2.3.4: Coomassie blue staining 54 2.3.5: HRP-conjugated western blot 55 2.3.6: Fluorescent western blot 55 2.3.7: Wet transfer 55 2.3.8: FLAG-bead pulldown 57 2.3.9: Biotin labelling 57 2.3.10: Streptavidin pulldown 57 2.3.11: Protein G-bead pulldown 58 2.3.12: Antibody and binding partner information 58 2.4: Proteomics and bioinformatics techniques 2.4.1: TMT-labelling and LC-MS/MS 59 2.4.2: Bioinformatic analysis 60 2 3: Results 3.1: Experimental background to the study 61 3.2: Introduction to the 293 Flp-in™ system 62 3.3: Production and testing of a ZNS5-FLAG cell line 63 3.4: Production and testing of a ZNS5-BirA cell line 68 3.5: Pulldowns: 3.5.1: NS5-FLAG pulldown 74 3.5.2: Streptavidin pulldown 75 3.6: FLAG TMT-MS/MS and bioinformatic programme comparison 3.6.1: TMT-MS/MS 76 3.6.2: FLAG pulldown bioinformatics and proteomics programme comparison 78 3.6.3: Streptavidin pulldown bioinformatics 106 3.7: Validation 3.7.1: Validation targets 123 3.7.2: Validation experiments 124 4: Discussion 4:1: The importance of a direct comparison of NS5 interactors 126 4.2: ZNS5 damages cell health 126 4.3: The validity of the ZNS5-BirA cell line 127 4.4: Reliability of FLAG and streptavidin pulldowns based on bioinformatic analysis 127 4.5: Difference and similarities of ZNS5 and DENV-NS5 interactors 129 4.6: Streptavidin pulldown compared to FLAG pulldown 131 4.7: Cdk1 and UBR5 132 4.8: The limitations of proteomic data 4.8.1: The general concept of pulldowns/TMT-MS/MS 134 4.8.2: Programmes 135 4.9: Future directions 136 5: References 137 6: Appendix 6.1: Appendix A – Recipes 6.1.1: Cell culture 147 6.1.2: DNA/RNA techniques 147 6.1.3: Protein techniques 148 3 6.2: Appendix B – Sequences 6.2.1: ZNS5-FLAG gene 153 6.2.2: ZNS5-BirA gene with sequencing primers 155 6.2.3: ZNS5-BirA with RT-PCR primers 157 6.2.4: ZNS5-BirA RT-PCR sequencing results 159 6.3: Appendix C – CD 170 4 Table of figures and tables Figure 1 – The burden of dengue disease over time Figure 2 – The path that the recent ZIKV outbreak has taken along with details of its evolution Figure 3 – The scale of DENV infection symptoms as ranked by severity of disease Figure 4 – The structure of the flavivirus particle Figure 5 – Flavivirus genome arrangement and polyprotein processing Figure 6 – The transmission cycle of DENV Figure 7 – The transmission cycle of ZIKV Figure 8 – DENV internalisation Figure 9 – DENV uncoating Figure 10 – DENV replication Figure 11 – Uptake of DENV with protective antibodies during primary infection Figure 12 – Uptake of a second serotype of DENV with a non-protective, enhancing antibody response Figure 13 – Thermocycler programme Figure 14 – Wet transfer ‘sandwich’ Figure 15 – The 293 Flp-In™ system Figure 16 – Time lapse IFA of ZNS5-FLAG cell lines exposed to doxycycline and probed with FLAG antibody Figure 17 – Time lapse western blot of ZNS5-FLAG cell lines exposed to doxycycline and probed with FLAG antibody Figure 18 – IFA showing the regulation of ZNS5-FLAG expression with doxycycline Figure 19 – DNA electrophoresis of post-midi-prep ZNS5-BirA plasmids after double RE digest Figure 20 – Transient/Stable cell line generation flow diagram Figure 21 – IFA showing the specificity of the BirA antibody on ZNS5-BirA-expressing cells Figure 22 – IFA showing the specificity of the ZNS5 antibody on ZNS5-BirA-expressing cells Figure 23 – Western blot showing the efficacy of the ZNS5 antibody Figure 24 – Fluorescent WB on ZNS5-BirA cells probing for biotin 5 Figure 25 – RT-PCR products of C3/C4/C5 with/without 4 days doxycycline induction using ZNS5- BirA primers Figure 26 – Western blot showing pre- and post- FLAG pulldown samples Figure 27 – Fluorescent western blot showing pre- and post- Streptavidin pulldown samples Figure 28 – Sample preparation, labelling, and subsequent MS/MS in TMT-MS/MS proteomics Figure 29 – Histograms showing the distribution of protein concentrations of each FLAG pulldown sample from the 0.01 FDR MaxQuant-processed TMT-MS/MS data Figure 30 – Histograms showing the distribution of protein concentrations of each FLAG pulldown sample from the 0.05 FDR MaxQuant-processed TMT-MS/MS data Figure 31 – Histograms showing the distribution of protein concentrations of each FLAG pulldown sample from the Proteome Discoverer-processed TMT-MS/MS data Figure 32 – Scatterplot graphs showing the correlation between FLAG pulldown samples from the 0.01FDR MaxQuant-processed TMT-MS/MS data Figure 33 – Scatterplot graphs showing the correlation between FLAG pulldown samples from the 0.01FDR MaxQuant-processed TMT-MS/MS data Figure 34 – Scatterplot graphs showing the correlation between FLAG pulldown samples from the Proteome Discoverer-processed TMT-MS/MS data Figure 35 – STRING pathway analysis on ZNS5-FLAG interactors in MaxQuant 0.01FDR Figure 36 – STRING pathway analysis on ZNS5-FLAG interactors in MaxQuant 0.05FDR Figure 37 – STRING pathway analysis on ZNS5-FLAG interactors in Proteome Discoverer Figure 38 – STRING pathway analysis on D2NS5-FLAG interactors in MaxQuant 0.01FDR Figure 39 – STRING pathway analysis on D2NS5-FLAG interactors in MaxQuant 0.05FDR Figure 40 – STRING pathway analysis on D2NS5-FLAG
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