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Structural & Functional Characterization of the Dengue Virus Non This document is downloaded from DR‑NTU (https://dr.ntu.edu.sg) Nanyang Technological University, Singapore. Structural & functional characterization of the dengue virus non‑structural protein 5 (NS5) Soh, Sherryl Tingjin 2018 Soh, S. T. (2018). Structural & functional characterization of the dengue virus non‑structural protein 5 (NS5). Doctoral thesis, Nanyang Technological University, Singapore. http://hdl.handle.net/10356/73218 https://doi.org/10.32657/10356/73218 Downloaded on 06 Oct 2021 23:07:19 SGT STRUCTURAL & FUNCTIONAL CHARACTERIZATION OF THE DENGUE VIRUS NON-STRUCTURAL PROTEIN 5 (NS5) SOH TINGJIN SHERRYL SCHOOL OF BIOLOGICAL SCIENCES 2017 STRUCTURAL & FUNCTIONAL CHARACTERIZATION OF THE DENGUE VIRUS NON-STRUCTURAL PROTEIN 5 (NS5) SOH TINGJIN SHERRYL School of Biological Sciences A thesis submitted to the Nanyang Technological University in partial fulfilment of the requirement for the degree of Doctor of Philosophy 2017 Acknowledgements Acknowledgements First and foremost, I would like to express my deepest gratitude to both my supervisors, Associate Professor Julien Lescar in NTU and Dr Lim Siew Pheng in Novartis Institute for Tropical Diseases (NITD), for their continuous guidance, encouragement and intense patience throughout my graduate education. Their expertise, meticulous attention and support had been greatly beneficial to me and to the success of my project. I am grateful to my colleagues in NITD, especially Ms Seh Cheah Chen and Dr Shan Chao who generously shared their knowledge and provided me with ample support and assistance. I would also like to thank my colleagues in NTU, especially Dr El Sahili Abbas and Ms Grace Chan Kar Yarn, who had rendered me with extensive help and advices for my crystallography experiments in NTU. My heartfelt gratitude also extends to my collaborators, Assistant Professor Luo Dahai, Dr Zhao Yongqian and Dr Subhash G. Vasudevan, for useful and critical discussions which led to the success of three publications. It was a privilege to work alongside with many brilliant and enthusiastic scientists and I truly enjoyed my stay in NITD and NTU for the past four years. It had been a fulfilling and enriching experience for me. Lastly, I thank Singapore Economic Development Board (EDB) for funding my research studies. i Table of Contents TABLE OF CONTENTS Acknowledgements.......................................................................................................................................... i List of Figures ....................................................................................................................... vi List of Tables ......................................................................................................................... viii List of Appendices .................................................................................................................... x Abbreviations .......................................................................................................................... xi Abstract ................................................................................................................................ xv CHAPTER 1: INTRODUCTION ........................................................................................... 1 1.1 Background & Classification of Flaviviruses ...................................................................... 1 1.2 Epidemiology of Dengue virus ............................................................................................ 1 1.3 The life cycle of Dengue virus ............................................................................................. 3 1.4 Dengue virus RNA genome ................................................................................................. 4 1.5 Translation and proteolytic processing ................................................................................ 8 1.6 Genome replication ............................................................................................................ 10 1.6.1 General introduction to polymerases ........................................................................ 10 1.6.2 Viral polymerases ..................................................................................................... 11 1.6.3 RNA-dependent RNA polymerases (RdRps) ........................................................... 15 1.6.3.1 Structural motifs of RdRps .................................................................................... 16 1.6.3.2 Two distinct initiation mechanisms adopted by plus-sense ssRNA viruses .......... 18 1.7 RNA capping process ........................................................................................................ 23 1.7.1 Conventional viral RNA capping mechanisms ......................................................... 24 1.7.2 Unconventional viral RNA capping pathways ......................................................... 26 1.7.3 Cap-snatching mechanism ........................................................................................ 28 1.8 DENV RNA replication ..................................................................................................... 29 1.9 NS5 protein ........................................................................................................................ 32 1.9.1 The methyltransferase domain .................................................................................. 33 1.9.2 The RNA-dependent RNA polymerase domain ....................................................... 36 1.9.3 NS5 inter-domain interactions .................................................................................. 39 1.10 Inhibitors of Flavivirus replication .................................................................................. 41 1.10.1 Inhibitors of RNA capping...................................................................................... 41 1.10.2 Inhibitors of RNA synthesis.................................................................................... 45 1.11 Next challenges in Flavivirus RNA capping and RNA replication research ................... 49 ii Table of Contents 1.12 Aims of this thesis ............................................................................................................ 52 CHAPTER 2: MATERIALS AND METHODS ................................................................. 56 2.1 Cells ................................................................................................................................ 56 2.2 Construction of DENV replicon cDNAs ........................................................................... 56 2.2.1 Generation of DENV-4 NS5 interface mutant replicons .......................................... 56 2.2.2 Generation of DENV-4 NS5 310-helix mutant replicons .......................................... 56 2.2.3 Generation of DENV-2 NS5 linker mutant replicons ............................................... 57 2.2.4 Generation of DENV-4 NS5 F1 motif mutant replicons .......................................... 58 2.2.5 Generation of DENV-4 NS5 N-pocket mutant replicons ......................................... 58 2.3 Construction of DENV infectious clones .......................................................................... 59 2.3.1 Generation of DENV-4 NS5 interface mutant infectious clones .............................. 59 2.3.2 Generation of DENV-2 NS5 E111 and G2 mutant infectious clones ....................... 59 2.4 RNA in vitro transcription and transfection....................................................................... 60 2.5 Cell viability assay ............................................................................................................. 61 2.6 Renilla luciferase assay ...................................................................................................... 61 2.7 Immunofluorescence assay (IFA) ...................................................................................... 62 2.8 Plaque assay ....................................................................................................................... 63 2.9 Intracellular and extracellular virus RNA extraction and quantification ........................... 63 2.10 Construction of DENV WT and mutant FL NS5 plasmids ............................................. 64 2.10.1 Generation of DENV-4 WT and interface mutant FL NS5 plasmids ..................... 64 2.10.2 Generation of DENV-4 WT and 310-helix mutant FL NS5 plasmids ..................... 65 2.10.3 Generation of DENV-2 WT and linker mutant FL NS5 plasmids .......................... 65 2.10.4 Generation of DENV-4 WT and E111 mutant FL NS5 plasmids .......................... 66 2.10.5 Generation of DENV-4 WT and F1 motif mutant FL NS5 plasmids ..................... 66 2.11 Construction of DENV WT and mutant MTase plasmids ............................................... 66 2.12 Expression and purification of DENV WT and mutant proteins ..................................... 67 2.12.1 Expression and purification of DENV-4 and DENV-2 WT and mutant FL NS5 proteins ................................................................................................................... 67 2.12.2 Expression and purification of DENV-4 WT and mutant MTase proteins ............ 68 m7 2.13 Construction of mutant G0ppp-DENV4 5’UTR nt-110 RNAs for 2’-O MTase assay... 69 2.14 Thermo-fluorescence assay .............................................................................................
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