Zika virus M protein as a viroporin drug target Emma Tabitha Brown Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds Faculty of Medicine and Health August 2020 The candidate confirms that the work submitted is their own and that appropriate credit has been given where reference has been made to the work of others. Chapter 4 Figure 4.4 Grids stained by myself and transmission electron microscopy (TEM) images generated by Dr Daniel Maskell and Dr Rebecca Thompson Figure 4.6 Images taken by Dr Daniel Maskell and Dr Rebecca Thompson, processing carried out by myself. Figure 4.17 In vivo assay carried out by Dr Daniella Lefteri and Dr Clive McKimmie, sample processing carried out by myself. Chapter 5 Figure 5.3 Identification of binding sites carried out by Dr Ravi Singh, Dr Stephen Griffin and by myself. Figure 5.4 Docking and images generated by Dr Ravi Singh using PyMOL and Maestro Table 5.2 & 5.3 High-throughput screen carried out by Ravi Singh using Maestro Figure 5.7- 5.12 Maestro interaction images generated by Dr Ravi Singh i Acknowledgements Thank you to my supervisor Dr Stephen Griffin for all his support throughout my PhD, encouraging and believing in me. Thank you to my co-supervisor Dr Antreas Kalli for joining my supervisory team, your MD training and endlessly answering my questions. I would also like to thank co-supervisors Dr Clive McKimmie and Dr Richard Foster for your support. Thank you to Dr Ravi Singh for your help using the modelling software and carrying out the in silico docking. Thank you to Dr Daniella Lefteri for carrying out the in vivo work. Thank you to Dr Daniel Maskell and Dr Rebecca Thompson for providing the TEM images. Thank you to Griffin group members Dr Matthew Bentham, Dr Abigail Bloy and Hannah Beaumont for your technical help throughout my first couple of months, answering my questions and ongoing moral support with lots of cups of tea and cake. Thank you also to newer group member Alex Kilvington, I wish you the best of luck in the future. Thank you to all the lovely people on level 5 who made my time enjoyable with endless support and baked goods! Thank you to Hyde Park Harriers for encouraging me to get out running on an evening and weekends, but most importantly for the amazing friendships and memories. I would like to thank all my friends and family for the support over the years. A special thank you to my parents Sally and Mick for your support and encouragement throughout my higher education. Last but not least, thank you to Brad for being understanding, cheering me up and ensuring that, outside of the lab and writing I got out for plenty of climbing adventures. ii Abstract Zika virus (ZIKV) an arbovirus that became widely known in 2015 due to the epidemic in Brazil, spreading across South and North America. Whilst previous Old World ZIKV outbreaks comprised largely mild, or even asymptomatic infections, the New World epidemic became notorious for its association with foetal microcephaly following maternal infection, and an increased incidence of various neurological symptoms, including Guillain-Barré syndrome. Mature, infectious ZIKV particles comprise three structural proteins, Capsid (C), small Membrane (M) and the envelope (E) glycoprotein; the latter is responsible for receptor binding and mediates membrane fusion upon encountering low pH within the acidifying endosome. However, the function of M within this context is unknown. Based upon its structural similarity to “viroporins”, a class of virus-coded ion channels mediating virus entry and uncoating, we investigated whether M could form alternative oligomeric forms to the dimeric structure seen within mature virions, and in so doing exhibit channel activity. Gratifyingly, M peptides adopted higher order structures within membrane-mimetic environments and displayed channel activity in vitro, sensitive to the prototypic viroporin inhibitor, Rimantadine. Accordingly, ZIKV entry was blocked in a dose-dependent fashion by the drug, which also prevented virus spread in mouse models of ZIKV infection. Molecular dynamics simulations supported that M protein is able to oligomerise into a hexameric viroporin channel, opening of which was within acidified environments via protonation of a conserved histidine residue. Rimantadine was predicted in silico to interact at a lumenal binding site, against which we derived improved inhibitors from a library of generic, FDA-approved and other bio-active small molecules, providing a basis for novel M protein targeted drug discovery. Significantly, due to its role during virus entry, M- targeted drugs might either prevent or reduce the severity of ZIKV infections, including those crossing the placenta, and may also show activity against closely related M proteins from other Flaviviruses. iii Table of Contents Acknowledgements ..................................................................................... ii Abstract ....................................................................................................... iii Table of Contents ........................................................................................ iv Table of Figures ........................................................................................ viii Abbreviations ............................................................................................. xii Chapter 1 Introduction ................................................................................ 1 1.1 Flaviviruses ...................................................................................... 1 1.2 Zika virus (ZIKV) ............................................................................... 1 1.2.1 Origin and Discovery ............................................................... 1 1.2.2 Transmission ........................................................................... 2 1.2.3 Symptoms ............................................................................... 5 1.3 Epidemiology .................................................................................... 6 1.4 Molecular biology of Zika virus (ZIKV) ............................................ 11 1.4.1 Zika virus genome structure .................................................. 11 1.4.2 Zika virus structural proteins ................................................. 11 1.4.3 Zika virus Non-structural proteins .......................................... 21 1.5 Zika virus life cycle ......................................................................... 29 1.5.1 Entry ...................................................................................... 29 1.5.2 Translation ............................................................................ 33 1.5.3 Polyprotein processing .......................................................... 34 1.5.4 Virus genome replication ....................................................... 35 1.5.5 Capping ................................................................................. 36 1.5.6 Virus assembly, budding, maturation and release ................. 36 1.6 ZIKV model systems....................................................................... 38 1.7 Vaccines ......................................................................................... 39 1.8 Antivirals and pharmacological disease management .................... 39 1.9 M protein as an antiviral target ....................................................... 41 1.9.1 M protein structure and function ............................................ 41 1.10 Viroporins ....................................................................................... 42 1.10.1 Influenza A Virus (IAV) M2 .................................................... 44 1.10.2 Hepatitis C Virus (HCV) p7.................................................... 46 1.10.3 Human immunodeficiency Virus type-1 (HIV-1) Vpu ............. 48 1.10.4 Human Respiratory Syncytial Virus (hRSV) SH .................... 49 iv 1.10.5 Human Papillomavirus type 16 (HPV-16) E5 ........................ 50 1.10.6 Enterovirus VP4 .................................................................... 51 1.11 Aims ............................................................................................... 52 Chapter 2 Materials and Methods ............................................................. 53 2.1.1 Zika Virus .............................................................................. 53 2.2 Mammalian Cell Culture ................................................................. 53 2.2.1 Recovery of frozen cells ........................................................ 53 2.2.2 Maintenance and Passage of Cells ....................................... 53 2.2.3 Freezing cells ........................................................................ 53 2.2.4 MTT Assay ............................................................................ 54 2.2.5 EGF Uptake Assay ................................................................ 54 2.2.6 Immunofluorescence ............................................................. 54 2.3 Virus production and storage .......................................................... 55 2.3.1 Virus stock propagation ......................................................... 55 2.3.2 Freezing virus ........................................................................ 55 2.3.3 Determination of Virus Titre................................................... 55 2.3.4 Virus assays .........................................................................