MECHANISTIC CHARACTERIZATION OF DENGUE VIRUS RNA DEPENDENT RNA POLYMERASE NON-NUCLEOSIDE INHIBITOR BINDING POCKET THROUGH IN VITRO BIOCHEMICAL ASSAYS AND REVERSE GENETICS ANALYSES Dorcas Adobea Larbi NATIONAL UNIVERSITY OF SINGAPORE 2012 MECHANISTIC CHARACTERIZATION OF DENGUE VIRUS RNA DEPENDENT RNA POLYMERASE NON-NUCLEOSIDE INHIBITOR BINDING POCKET THROUGH IN VITRO BIOCHEMICAL ASSAYS AND REVERSE GENETICS ANALYSES Dorcas Adobea Larbi B.Sc (Hons.), University of Cape Coast A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE IN INFECTIOUS DISEASES, VACCINOLOGY AND DRUG DISCOVERY DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2012 DECLARATION I hereby declare that the thesis entitled "Mechanistic Characterization of Dengue Virus RNA Dependent RNA Polymerase Non-Nucleoside Inhibitor Binding Pocket through In Vitro Biochemical Assays and Reverse Genetics Analyses" is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously Dorcas Adobea Larbi 27 November 2012 i ACKNOWLEDGEMENTS My sincere gratitude is expressed towards Dr Shi, Pei-Yong of Novartis Institute for Tropical Diseases, NITD and National University of Singapore, NUS for his innovative ideas and encouragement during this research work I am forever indebted to my supervisor Dr Lim, Siew Pheng of NITD, whom I closely worked with for the success of this research Dr Lim, I really value your concern, support and attention to details in this work at all times Your indispensable directions, expertise, meticulousness and ground-breaking encouraging ideas really inspired me and have undoubtedly led to the success of this study Again, I would like to express my gratitude to Prof Pascal Mäser of Swiss Tropical and Public Health Institute (Swiss TPH), Switzerland for accepting to co-supervise my research study His support and tutoring most especially during our studies in Basel cannot be underestimated I am most grateful the Swiss TPH for funding my studies and NITD for enabling me to use their facilities for my research study Special thanks to coordinators of this programme most especially Prof Marcel Tanner of Swiss TPH and Prof Markus Wenk of National University of Singapore (NUS) and also to our two ladies; Ms Christine Mensch and Ms Susie Soh for contributing to the success of our studies in Basel and Singapore My appreciation as well goes to all our tutors for generously sharing their knowledge with us and also, making time to answer all the questions we asked during lectures ii Special thanks goes to Seh, Cheah Chen; Chung, Ka Yan and Ghafar, Nahdiyah for providing me the time, brilliant ideas and assistance whenever I approached them Their work and dedication to this study is very much appreciated Not forgetting my Disease Biology friends including: Xie, Xuping, Dong, Hongping, Yip, Andy; Zou, Jing; Vasudevan, Dileep; Susila, Agatha; Lee, Le Tian; Chang, David; Chew, Kelly; Chao, Alex and Yeo, Kim Long for their friendship and willingness to share their knowledge with me I would like to express my profound thanks and love to my wonderful Husband, Patrick Kwasi Otoo who is always there for me and whose love, care, companionship and motivation propelled me to have a smooth sail in my MSc studies My family is also not left out knowing that they have been of great asset to me Thank you so much I also acknowledge my friends and course mates for spicing my social life both in Basel and Singapore Finally, I would like to express my sincere thanks to the Almighty God in heaven whose blessings, favour, strength and grace has been with me and has granted me the opportunity to begin this interesting research paving way to my career in drug discovery iii TABLE OF CONTENTS DECLARATION……………………………………………………… i ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iv SUMMARY ix LIST OF TABLES xi LIST OF FIGURES xii ABBREVIATIONS xiv CHAPTER 1: LITERATURE REVIEW 1.1 Evolution of Dengue Virus 1.2 Divergence from Non-infectious to an Infectious Pathogen 1.3 DENV Epidemiology and Global Consequence 1.4 Dengue Virus Pathogenesis and Host Immune Response 1.4.1 Host Immune Response 1.4.2 Dengue Virus Pathogenesis 1.4.3 Antibody Induced Enhancement of Dengue Virus 1.5 Clinical Signs and Symptom 1.6 Life Cycle of Dengue Virus 10 1.7 Virus Morphology 13 1.8 Dengue virus genome 15 1.9 Virus Structural Proteins 17 iv 1.9.1 Capsid Protein 17 1.9.2 Membrane Protein 18 1.9.3 Glycoprotein Envelope 18 1.10 Virus Non-structural Proteins 19 1.10.1 NS1 19 1.10.2 NS2A 20 1.10.3 NS2B 20 1.10.4 NS3 21 1.10.5 NS4A 22 1.10.6 NS4B 22 1.10.7 NS5 23 1.10.7.1 Role of NS5 in DENV Pathogenesis 24 1.10.7.2 NS5 Methyltransferase 25 1.10.7.3 NS5 RNA-dependent RNA Polymerase 26 1.10.7.4 Structure of NS5 RNA-dependent RNA Polymerase 27 1.11 Rationale 31 1.11.1 Objectives of Study 33 CHAPTER 2: MATERIALS AND METHODS 34 2.1 Cloning of pET28a-D4MY01-NS5-22713 NS5 Mutants using Site-directed Mutagenesis 34 2.2 Expression and Purification of DENV FL NS5 Mutant Protein Histidine-tagged 36 2.2.1 Expression of DENV FL NS5 36 2.2.2 Purification of DENV FL NS5 mutant protein 37 v 2.3 In-vitro Transcription of pUC19-D4-5´UTR-L-3´UTR Plasmid 38 2.4 Cell-Free Assay 39 2.4.1 Biochemical Enzymatic Assays 39 2.4.1.1 FAPA De novo Initiation 41 2.4.1.2 FAPA Elongation Assay 41 2.4.2 Differential Scanning Fluorimetry (Thermofluorescence Assay) 44 2.4.3 Measurement of Steady-state Kinetic Parameters 44 2.4.3.1 RNA Km Studies in De novo Assay 45 2.4.3.2 NTP Km Studies in De novo Assay 46 2.5 Cloning of DENV TSV01-F subclone mutants 46 2.6 Cloning of DENV TSV01-F Subclone K402A Mutant using Overlapping PCR 48 2.7 Construction of Recombinant Plasmids 50 2.8 Ligation of DENV pACYC-FL TSV01 with TSV01-F Subclone mutants 51 2.9 Production of Recombinant Viruses 53 2.9.1 Linearization of Plasmid 53 2.9.2 In-vitro Transcription of DENV FL pACYC-FL TSV01…………………………………… 54 2.10 Cell Culture and Cell Lines 54 2.11 Media for Cell Biological Studies 55 2.12 Growing and Maintaining of Cell Lines 56 2.13 RNA Transfection of Cells 58 vi 2.14 Cell-Based Assay 59 2.14.1 Indirect Immunofluorescence Assay 59 2.14.2 Plaque Assay 61 CHAPTER 3: RESULTS 63 3.1 Site-directed Mutagenesis 63 3.2 Expression and Purification of DENV FL NS5 Mutant Proteins 63 3.3 In-vitro transcription of RNA using DENV Template 67 3.4 Background of Biochemical Enzymatic Assays 68 3.4.1 FAPA De novo Initiation 70 3.4.2 FAPA Elongation Assay 71 3.5 Differential Scanning Fluorimetry 74 3.6 Measurement of Steady-state Kinetic Parameters 76 3.6.1 RNA Km Studies 77 3.6.2 NTP Km Studies 78 3.7 Production of Recombinant Viruses 79 3.7.1 Indirect Immunofluorescence Assay 80 3.7.2 Plaque Assay 83 CHAPTER 4: DISCUSSION AND CONCLUSION 84 4.1 DENV NS5 RdRp Characterization for In vitro Polymerase Activity 85 4.1.1 Effects of Mutations on NS5 RdRp De novo Initiation and Elongation Activities 86 4.1.1.1 F399A and K402A 88 vii 4.1.1.2 F486A and N493A 88 4.1.1.3 G605A, Y607A, and N610A 89 4.1.1.4 D664A 90 4.1.1.5 W796A 92 4.1.2 Stability of DENV NS5 RdRp mutants 93 4.1.3 Effects of NTP Km and RNA Km on DENV FL NS5 Mutants 94 4.2 Characterization of DENV TSV01 NS5 Mutants 95 4.2.1 Expression of Viral Proteins and RNA 96 4.2.2 Plaque Morphology 98 4.3 Summary of Discussion 99 4.4 Conclusion 101 BIBLIOGRAPHY 102 viii Discussion and conclusion W796A is unclear although it could imply that the residue might play a role in viral replication through a nonenzymatic mechanism Table 4.1 summarises results obtained from the biological, biophysical and biochemical properties of all amino acid residues in this study Table 4.1: Summary of Mutagenesis Analysis of Nine NS5 RdRp Mutants Protein % difference in activity between mutants and WT De novo Elongation WT FL NS5 100 100 F399A 67.22 K402A Change in Tm between mutants and WT Steady-state kinetic parameters RNA substrate NTP substrate IFA Plaque assay Vmax (nM/min) Km (nM) kcat (min-1) Vmax (uM/min) Km (uM) kcat (min-1) 37.4 15.65 53.66 0.16 15.99 2.17 0.16 ++++ Yes 59.43 1.7 3.68 67.59 0.04 7.78 2.44 0.08 N.D N.D 92.98 61.51 0.6 - - - - - - - - F486A 35.10 20.37 -0.4 - - - - - - - - N493A 86.66 70.44 -0.2 2.86 122.5 0.03 10.83 4.98 0.11 N.D N.D G605A 6.84 16.07 1.6 - - - - - - - - Y607A 78.29 26.48 0.6 2.61 204.1 0.03 - - - - N610A 99.72 94.33 0.2 - - - - - - N.D N.D D664A 100.38 92.33 1.6 - - - - - - N.D N.D W796A 25.24 24.13 1.6 - - - - - - ++ Tiny Mutants that were not used for specific assay are indicated hyphen (-) IFA and plaque assay results are categorised into: strong (++++), weak (++), non-determined (N.D.) While it is clear that conserved sequences and structures of DENV play an important role in regulating viral RNA synthesis, our understanding of the role of NS5 in RNA synthesis and pathogenesis might be rudimentary Further revertant analysis can be undertaken for DENV NS5 RdRp mutant proteins to check for possible mutations that 100 Discussion and conclusion may possibly revive genomic RNA replication Additional studies should as well be done to examine the biological relevance of K402, F486, G605 and Y607 replication fitness in the context of the DENV TSV01 infectious virus 4.4 Conclusion DENV NS5 RdRp is an attractive target for antiviral development This study represents the first observation in characterising the mechanism of action of residues F399, K402, F486, N493, G605, Y607, N610, D664 and W796 in the RdRp domain inhibitor binding pockets Of all the nine DENV NS5 RdRp amino acid residues tested, our results have shown that residues F399, K402, N493, Y607, N610 and D664 are 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through In Vitro Biochemical Assays and Reverse Genetics Analyses" ... the inhibitor binding pocket for in vitro polymerase activity and as well for replication fitness in context of the DENV TSV01 infectious virus For these studies, individual amino acids lining