Royle, Jamie (2021) Discovery and characterisation of host-factors involved in Zika virus. PhD thesis. http://theses.gla.ac.uk/82306/ Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten: Theses https://theses.gla.ac.uk/ [email protected] Discovery and characterisation of host-factors involved in Zika virus infection Jamie Royle, BSc, MBiol Submitted in the fulfilment for the degree of Doctor of Philosophy in Virology. MRC-University of Glasgow Centre for Virus Research Institute of Infection, Immunity and Inflammation College of Medical, Veterinary and Life Sciences University of Glasgow 2 Abstract Zika virus (ZIKV, Flaviviridae), like other emerging arboviruses, poses a considerable threat to human health. It is estimated that approximately half of the world’s population is at risk from contracting a mosquito-borne arboviral infection, and this was exemplified during the 2015/16 ZIKV outbreak in the Americas. ZIKV infection is thought to be largely asymptomatic, although ZIKV disease has previously been characterised by mild symptoms such as a maculopapular rash, conjunctivitis, and fever. However, recent outbreaks have been associated with an increased incidence of Guillain-Barré syndrome, and a pattern of neurological and developmental symptoms in neonates which is now termed congenital Zika syndrome. Despite intense efforts, no therapeutic or vaccine has been developed. As such, it is vital that further fundamental research is conducted to discover novel host-virus interactions in both vector and mammalian host systems, which may allow development of targeted interventions. Here, multiple approaches were used to generate basic tools for ZIKV research, and siRNA screens and data from mass-spectrometry based proteomics were utilised to uncover important host interactors of ZIKV. A study investigating the Aedes aegypti immune response was conducted, and the classical RNAi effector Argonaute 2 (Ago2) was not found to be antiviral, whereas PIWI 4 was. Data from a previous proteomics experiment suggested that glucose-regulated protein 78 kDa (GRP78) may interact with ZIKV E. In this study, co-immunoprecipitation and immunofluorescence was used to verify that GRP78 interacts with ZIKV E in both mammalian and Aedes aegypti cell culture. GRP78 is a key modulator of the unfolded protein response (UPR), and while small-molecule inhibitors (EGCG and HNK) of the GRP78-mediated UPR did not inhibit ZIKV infection, EGCG was able to inhibit ZIKV entry independent of GRP78, likely through direct binding of the virion. Further study of GRP78 revealed that while it is not important for entry, replication, or egress of ZIKV, it did aid viral translation. Depletion of GRP78 with siRNA resulted in a loss of coordination of viral replication factories and relieved 3 a virus-specific inhibition of host translation. Furthermore, STRING analysis of GRP78 host-interactors followed by a targeted siRNA screen revealed that DnaJC1 is also a pro-viral factor. DnaJC1 has previously been shown to coordinate GRP78 localisation to ribosome exit tunnels, and so may contribute to ZIKV infection through GRP78, though this was not assessed in this study. Additionally, by using a circular polymerase extension reaction (CPER) system, a reverse genetics ZIKV was generated. This CPER ZIKV represents a genetically stable source of virus which can be easily modified and can support future research. Collectively, the data herein informs on important ZIKV interactions in both arthropod vector and mammalian systems, and highlights tools and techniques that can be used to conduct future fundamental ZIKV research. 4 Table of Contents Abstract ...................................................................................... 2 List of Tables ................................................................................ 8 List of Figures ............................................................................... 9 Abbreviations .............................................................................. 12 Publications ................................................................................ 15 Acknowledgements ........................................................................ 16 Authors Declaration ....................................................................... 18 Chapter 1. Introduction ................................................................. 19 1.1 Flaviviridae ....................................................................... 19 1.1.1 Taxonomy .................................................................... 19 1.1.2 Flavivirus Genomic RNA .................................................... 22 1.1.3 Flavivirus protein expression ............................................. 25 1.1.4 Virus structure .............................................................. 35 1.1.5 Geographical distribution and vectors ................................... 37 1.1.6 Economic and health burden .............................................. 40 1.1.7 Infection cycle .............................................................. 42 1.2 ZIKV: historical perspective and properties ................................. 45 1.2.1 Discovery and spread ....................................................... 45 1.2.2 Transmission ................................................................. 48 1.2.3 Health implications ......................................................... 52 1.2.4 Replicative cycle ........................................................... 54 1.2.5 Antivirals and vaccine development ..................................... 57 1.2.6 Interactions with the host cell ............................................ 58 1.3 GRP78 ............................................................................. 60 1.3.1 Zika virus E protein may interact with GRP78 .......................... 60 1.3.2 The structure and function of GRP78 .................................... 60 1.3.3 GRP78 and virus lifecycles ................................................ 64 Chapter 2. Aims .......................................................................... 68 Chapter 3. Materials and Methods ..................................................... 69 5 3.1 Materials .......................................................................... 69 3.1.1 Cell culture .................................................................. 69 3.1.2 Cell culture reagents ....................................................... 70 3.1.3 Viruses ........................................................................ 70 3.1.4 Plasmids ...................................................................... 71 3.1.5 Primers ....................................................................... 72 3.1.6 siRNA ......................................................................... 74 3.1.7 Antibodies ................................................................... 75 3.1.8 Commercial kits and general reagents .................................. 76 3.1.9 Buffers ....................................................................... 77 3.1.10 Enzymes ...................................................................... 78 3.1.11 Bacteria ...................................................................... 78 3.2 Methods ........................................................................... 79 3.2.1 Cell culture maintenance ................................................. 79 3.2.2 Cell culture transfection .................................................. 80 3.2.3 Lentivirus production and transduction of cells........................ 81 3.2.4 Virus rescue and amplification ........................................... 82 3.2.5 Circular polymerase extension reaction ................................. 83 3.2.6 Large scale siRNA screen .................................................. 85 3.2.7 Plaque assays ................................................................ 85 3.2.8 Luciferase assays ........................................................... 85 3.2.9 Freeze/thaw assay ......................................................... 86 3.2.10 Western blot analysis ...................................................... 87 3.2.11 Immunofluorescence ....................................................... 87 3.2.12 RNA extraction and RT-qPCR .............................................. 88 3.2.13 dsRNA synthesis ............................................................. 88 3.2.14 Co-Immunoprecipitation ................................................... 89 3.2.15 Mass spectrometry analysis ............................................... 89 3.2.16 Bacterial transformation .................................................. 90 3.2.17 Plasmid purification ........................................................ 91 6 Chapter 4. Investigating Aedes aegypti immune factors that regulate ZIKV infection .................................................................................... 92 4.1 Introduction .....................................................................