A SIRNA SCREEN TO PROBE FOR HYDROXYLASES THAT CAN MODULATE THE REPLICATION OF DENGUE VIRUS WONG PHUI YEW ANDREW B.SCI (HONS) A THESIS SUBMITTED FOR THE DEGREE OF MASTERS OF SCIENCE DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2010 Acknowledgements ACKNOWLEDGEMENTS First and foremost, I would like to express gratitude to my supervisor, Dr Chu Jang Hann, Justin and co-supervisor Professor Ng Mah Lee, Mary for their constant supervision and patience throughout the course of my project. Deep appreciation also goes out to the members of my lab mates, Ong Siew Pei, Chen Jin Cheng, June Low Su Yi, Karen Chen Cai Yun, Wu Kan Xing, for the valuable suggestions and for making my experience in the lab enjoyable. Lastly, I would like to dedicate this thesis to my wife Grace. I am extremely grateful for all the time which she stood by me throughout the course of my post-graduate studies. Thank you for always being a pillar of support and encouragement. i Contents CONTENTS ACKNOWLEDGEMENTS . i CONTENTS .ii TABLES AND FIGURES .iv ABBREVIATIONS .vii ABSTRACT xi 1. INTRODUCTION 1.1 DENGUE VIRUS 1.1.1 DENGUE VIRUS AND THE HOST INNATE IMMUNITY 1.1.2 DENGUE VIRUS AND THE HOST ADAPTIVE IMMUNITY 1.2 RNA INTERFERENCE (RNAI) 1.2.1 SMALL-INTERFERING RNA (SIRNA) . 10 1.2.2 MICRORNA (MIRNA) 12 1.3 GENETIC SCREENING W ITH RNAI 15 1.3.1 CONTROLS AND Z FACTOR . 17 1.3.2 SECONDARY ASSAYS . 18 2. AIM 21 3. MATERIALS AND METHODS 22 3.1 CELL CULTURE 22 3.2 VIRUS PROPAGATION . 22 3.3 VIRAL PLAQUE ASSAY 23 3.4 SMARTPOOL SIRNA . 23 3.5 TRANSFECTION OF SIRNA 24 3.6 CELL VIABILITY ASSAY . 24 3.7 SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS (SDSPAGE) AND W ESTERN BLOT 25 3.8 SIARRAY™ PROTEIN HYDROXYLASE SIRNA LIBRARY . 27 3.9 384-W ELL HIGH THROUGHPUT SIRNA SCREENING ASSAY . 28 3.10 IMMUNOFLUORESCENCE ASSAY 30 3.11 IMAGING AND DATA ANALYSIS BY IMAGEXPRESS™ . 31 3.12 SECONDARY ASSAY . 31 3.13 TOTAL CELLULAR RNA EXTRACTION . 35 3.14 REAL-TIME QUANTITATIVE REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION . 36 3.15 RT2 PROFILER™ PCR ARRAY SYSTEM 37 ii Contents 4. DEVELOPMENT OF 384-WELL SIRNA SCREENING ASSAY 40 4.1 DETERMINATION OF OPTIMAL SEEDING CELL DENSITY . 40 4.2 OPTIMIZATION OF DHARMAFECT®4 CONCENTRATION FOR EFFICIENT SIRNA TRANSFECTION 42 4.3 DETERMINATION OF OPTIMAL TIME POINT FOR VISUALIZATION OF DENV2INFECTED HUH-7 CELLS VIA IMMUNOFLUORESCENCE ASSAY 45 5. SIRNA SCREENING OF HUMAN PROTEIN HYDROXYLASE . 55 5.1 HYPOXIA-INDUCIBLE FACTOR 1, ALPHA SUBUNIT INHIBITOR MODULATES DENV2 REPLICATION IN HUH-7 CELLS 55 5.2 STATE OF CELLULAR HYPOXIA MODULATES REPLICATION OF DENV2 IN HUH-7 CELLS . 58 5.3 THE HYPOXIC-INDUCIBLE PATHW AY ACTIVATED VIA HIF2α/HIF1β IS PREDOMINANTLY RESPONSIBLE FOR THE MODULATION OF DENV2 REPLICATION IN HUH-7 CELLS 66 6. MODULATION OF DENV2 REPLICATION BY HIFS . 73 6.1 REPLICATION OF DENV2 IN HUH-7 CELLS DOES NOT RESULT IN INCREASED HIFS . 73 6.2 HYPOXIA-INDUCIBLE FACTORS COULD POSSIBLY MODULATE REPLICATION OF DENV2 BY ACTIVATION OF INTERFERON VIA THE NF-ΚB PATHW AY 77 7. DISCUSSION 86 8. CONCLUSION 102 REFERENCES 104 APPENDIX I: ACTIVATION AND REGULATION OF NF-KB PATHWAY 120 APPENDIX II: PRODUCTION AND SIGNALING OF TYPE I INTERFERON . 121 APPENDIX III: MATERIALS FOR CELL CULTURE . 122 APPENDIX IV: MATERIALS FOR WESTERN BLOT . 124 APPENDIX IV: FIRZAN ANG, ANDREW PHUI YEW WONG, MARY NG AND JUSTIN CHU, VIROLOGY JOURNAL (2010) VOL.7: 24 125 iii Tables and Figures TABLES AND FIGURES FIGURE 1.1: ARRANGEMENT OF THE DENGUE VIRUS RNA GENOME. . . . .3 FIGURE 1.2: THE PATHWAY OF RNA INTERFERENCE. 11 TABLE 1.1: FORMULA AND CHARACTERIZATION OF SCREENING ASSAY QUALITY BY THE VALUE OF ZFACTOR. . 19 TABLE 3.1: FORMULA OF STACKING GEL (5%) AND RESOLVING GEL (10%) FOR SDS-PAGE. 26 FIGURE 3.1: THE 384-WELL HIGH-THROUGHPUT ASSAY. . 29 FIGURE 3.2: IMAGEXPRESS MICRO™ AUTOMATED ACQUISITION AND ANALYSIS SYSTEM BY MOLECUAR DEVICES. 32 FIGURE 3.3: EXPERIMENT TIMELINE FOR SECONDARY ASSAYS USED TO VALIDATE “HITS” FROM SIRNA SCREENING ASSAY. . . . 34 TABLE 3.2: NUCLEOTIDE SEQUENCES OF REAL-TIME QRT-PCR PRIMERS. . 39 FIGURE 4.1: FLUORESCENCE IMAGE OF HUH-7 CELLS WITH NUCLEI STAINED WITH DAPI. FIGURE 4.2: CELL VIABILITY ASSAY OF HUH-7 CELLS. . . 41 44 ® FIGURE 4.3: EFFICIENT DELIVERY OF CLATHRIN-SIRNA INTO HUH-7 CELLS BY DHARMAFECT 4. .46 FIGURE 4.4: VISUALIZATION OF DENV2 PROTEINS IN HUH-7 CELLS. FIGURE 4.5: GROWTH CURVE OF DENV2 (NGC) IN HUH-7 CELLS. . 47 . 48 FIGURE 4.6: EFFECTS OF CLATHRIN KNOCK-DOWN ON REPLICATION OF DENV2. 51 FIGURE 4.7: EFFECTS OF VIMENTIN KNOCK-DOWN ON REPLICATION OF DENV2. 52 TABLE 4.1: Z-FACTOR OF THE 384-WELL HIGH-THROUGHPUT SIRNA SCREENING ASSAY. 54 FIGURE 5.1: 384-WELL HIGH-THROUGHPUT SIRNA SCREENING ASSAY OF HUMAN PROTEIN HYDROXLASES ON DENV2 IN HUH-7 CELLS. 56 TABLE 5.1: 384-WELL HIGH-THROUGHPUT SIRNA SCREENING ASSAY OF HUMAN PROTEIN HYDROXLASES ON DENV2 REPLICATION IN HUH-7 CELLS. FIGURE 5.2: THE TRANSLOCATION OF HYPOXIA-INDUCIBLE FACTORS INTO THE CELL NUCLEUS. 57 . 59 ® FIGURE 5.3: ALAMARBLUE CELL VIABILITY ASSAY OF HUH-7 CELLS TREATED WITH CO(II)CL2 AND FE(II)CL2. 60 iv Tables and Figures FIGURE 5.4: EFFECTS OF CO(II)CL2 AND FE(II)CL2 ON REPLICATION OF DENV2. 62 ® FIGURE 5.5: ALAMARBLUE CELL VIABILITY ASSAY OF HUH-7 CELLS TREATED WITH CHETOMIN. . 64 FIGURE 5.6: EFFECTS OF CHETOMIN ON REPLICATION OF DENV2 IN HUH-7 CELLS. 65 FIGURE 5.7: EFFECTS OF HIF1α KNOCK-DOWN ON REPLICATION OF DENV2 IN HUH-7 CELLS. . 68 FIGURE 5.8: EFFECTS OF HIF1β (ARNT) KNOCK-DOWN ON REPLICATION OF DENV2 IN HUH-7 CELLS. 69 FIGURE 5.9: EFFECTS OF HIF2α (EPAS1) KNOCK-DOWN ON REPLICATION OF DENV2 IN HUH-7 CELLS. 70 FIGURE 5.10: EFFECTS OF COMBINATION KNOCK-DOWN ON THE REPLICATION OF DENV2 IN HUH-7 CELLS. . . 71 FIGURE 5.11: A DIAGRAMMATIC VIEW OF HOW THE STATE OF HYPOXIA LIMITS DENV2 REPLICATION IN HUH-7 CELLS BY ACTIVATION OF THE HYPOXIA-INDUCIBLE PATHWAY PREDOMINANTELY VIA HIF2α AND HIF1β. . 72 TABLE 6.1: REAL-TIME QRT-PCR OF HIF1α AND HIF2α TRANSCRIPTS IN HUH-7 CELLS INFECTED WITH DENV2. 74 FIGURE 6.1: REAL-TIME QRT-PCR OF HUH-7 CELLS INFECTED WITH DENV2. . 75 FIGURE 6.2: A DIAGRAMMATIC REPRESENTATION OF THE EFFECT OF DENV2 INFECTION ON THE EXPRESSION OF HIFS IN HUH-7 CELLS. . 76 TABLE 6.3: A POSSIBLE MECHANISM THAT HYPOXIX FACTORS COULD MODULATE DENV2 REPLICATION VIA THE INFLAMMATORY RESPONSE PATHWAY. . 78 TABLE 6.2: QUALITY CONTROL FOR THE TEST SAMPLE (HYPOXIA) AND CONTROL SAMPLE (NORMOXIA) FOR THE NF-KB PROFILE PCR ARRAY. ™ . 81 TABLE 6.3: RESULTS OF THE RT PROFILER PCR ARRAY SYSTEM ON NF-KB. . 82 FIGURE 6.4: PCR ARRAY ANALYSIS OF NF-KB TRANSCRIPT LIBRARY . 85 . FIGURE 7.1: THE REGULATION OF HYPOXIA-INDUCIBLE FACTOR TRANSCRIPTION COMPLEX. . 87 FIGURE 7.2: THE PROTEASOMAL DEGRADATION OF HYPOXIA-INDUCIBLE FACTOR ALPHA SUBUNIT VIA UBIQUITINATION. . . 89 FIGURE 7.3: A COMPARISON ON THE EFFECTS OF DENV2 REPLICATION WHEN DIFFERENT SUBUNITS OF THE HIF GENES WERE KNOCKED-DOWN WITH SIRNA. . 91 v Tables and Figures FIGURE 7.4A: AN ILLUSTRATION OF HIF2α AND HIF1β ACTING INDEPENDENTLY IN THE HYPOXIAINDUCIBLE PATHWAY TO MODULATE DENV2 REPLICATION: THE EFFECTS OF HIF2α AND/OR HIF1β KNOCK-DOWN ON THE REGULATION OF DENV2. . . . 94 FIGURE 7.4B: AN ILLUSTRATION OF HIF2α ACTING SOLELY VIA HIF1β IN THE HYPOXIA-INDUCIBLE PATHWAY TO MODULATE DENV2 REPLICATION: THE EFFECTS OF HIF2α AND/OR HIF1β KNOCK- DOWN ON THE REGULATION OF DENV2. . 95 FIGURE 7.4C: AN ILLUSTRATION OF HIF2α ACTING VIA AN ALTERNATIVE PATHWAY OTHER THAN HIF1β IN THE HYPOXIA-INDUCIBLE PATHWAY TO MODULATE DENV2 REPLICATION: THE EFFECTS OF HIF2α AND/OR HIF1β KNOCK-DOWN ON THE REGULATION OF DENV2. . 96 FIGURE 7.5: AN OVERVIEW ON THE POSSIBLE RELATION BETWEEN THE STATE OF CELLULAR HYPOXIA AND THE REPLICATION OF DENV2. .98 vi Abbreviations ABBREVIATIONS ADE antibody dependent enhancement AGO Argonaute ARNT aryl hydrocarbon receptor nuclear translocator Asn asparagine ASPH aspartate beta-hydroxylase BHK baby hamster kidney BSA bovine serum albumin C capsid C-TAD C-terminal trans-activation domain CHK checkpoint kinase CPE cytopathic effect Ct cycle threshold DAPI 4’,6-diamidino-2-phenylindole DC dendritic cell DC-SIGN DC-specific ICAM-3-grabbing non-intergrin DENV dengue virus DF dengue fever DHF dengue haemorrhagic fever DNA deoxyribonucleic acid dNTP 2’deoxyribonucleoside-5’triphosphate dsRNA double stranded RNA DSS dengue shock syndrome DVHF dengue virus host factor E envelope ECL enhanced chemiluminescence EDTA ethyleneditrilo tetraacetic acid EPAS endothelial PAS domain-containing protein ER endoplasmic reticulum ETP epidithiodiketopiperazine vii Abbreviations FCS fetal calf serum FITC fluorescein isothiocyanate g gram HIF hypoxia/hypoxic-inducible factor HRE hypoxia/hypoxic response element Hsp heat shock protein HTS high-throughput screening IC50 inhibitory concentration of 50% ICAM-3 intercellular adhesion molecule IFA immunofluorescence assay IFN interferon IKK inhibitor of kappa light polypeptide gene enhancer in B cells kinase IL interleukin ISRE interferon-stimulated response element IRF interferon regulatory factor LEPRE leucine proline-enriched proteoglycan LNA locked nucleic acid M molar mg milligram minutes miRNA microRNA mL millilitre MLV-RT murine leukimia virus reverse transcriptase mM millimolar MOI multiplicity of infection mRNA messenger RNA NF-kB nuclear factor kappa-light-chain-enhancer of activated B cells nm nanometers nM nanomolar NO nitric oxide viii Abbreviations NS non-structural nt nucleotide ODDD oxygen-dependent degradation domain OH hydroxyl ORF open reading frame PBS phosphate buffered saline PCR polymerase chain reaction PFU plaque forming units PHD prolyl hydroxylase domain piRNA Piwi-interacting RNA PKR RNA-dependent protein kinase Pol polymerase PS phosphothioates prM pre-membrane Pro proline pVHL von Hippel-Lindau tumour suppressor rasiRNA repeat associated small interfering RNA RCL RISC loading complex RME receptor-mediated endocytosis RISC RNA-induced silencing complex RNA ribonucleic acid RNAi RNA interference RNase ribonuclease qRT-PCR quantitative reverse transcriptase-polymerase chain reaction SDS sodium dodecyl sulfate sec seconds siRNA small interfering RNA STAT signal transducer and activator of transcription TBK TANK binding kinase TNF tumor necrosis factor ix Appendix V: Firzan Ang, Andrew Phui Yew Wong, Mary Ng and Justin Chu, Virology Journal (2010) Vol.7:24 126 Appendix V: Firzan Ang, Andrew Phui Yew Wong, Mary Ng and Justin Chu, Virology Journal (2010) Vol.7:24 127 Appendix V: Firzan Ang, Andrew Phui Yew Wong, Mary Ng and Justin Chu, Virology Journal (2010) Vol.7:24 128 Appendix V: Firzan Ang, Andrew Phui Yew Wong, Mary Ng and Justin Chu, Virology Journal (2010) Vol.7:24 129 Appendix V: Firzan Ang, Andrew Phui Yew Wong, Mary Ng and Justin Chu, Virology Journal (2010) Vol.7:24 130 Appendix V: Firzan Ang, Andrew Phui Yew Wong, Mary Ng and Justin Chu, Virology Journal (2010) Vol.7:24 131 Appendix V: Firzan Ang, Andrew Phui Yew Wong, Mary Ng and Justin Chu, Virology Journal (2010) Vol.7:24 132 Appendix V: Firzan Ang, Andrew Phui Yew Wong, Mary Ng and Justin Chu, Virology Journal (2010) Vol.7:24 133 Appendix V: Firzan Ang, Andrew Phui Yew Wong, Mary Ng and Justin Chu, Virology Journal (2010) Vol.7:24 134 Appendix V: Firzan Ang, Andrew Phui Yew Wong, Mary Ng and Justin Chu, Virology Journal (2010) Vol.7:24 135 Appendix V: Firzan Ang, Andrew Phui Yew Wong, Mary Ng and Justin Chu, Virology Journal (2010) Vol.7:24 136 Appendix V: Firzan Ang, Andrew Phui Yew Wong, Mary Ng and Justin Chu, Virology Journal (2010) Vol.7:24 137 Appendix V: Firzan Ang, Andrew Phui Yew Wong, Mary Ng and Justin Chu, Virology Journal (2010) Vol.7:24 138 Appendix V: Firzan Ang, Andrew Phui Yew Wong, Mary Ng and Justin Chu, Virology Journal (2010) Vol.7:24 139 Appendix V: Firzan Ang, Andrew Phui Yew Wong, Mary Ng and Justin Chu, Virology Journal (2010) Vol.7:24 140 Appendix V: Firzan Ang, Andrew Phui Yew Wong, Mary Ng and Justin Chu, Virology Journal (2010) Vol.7:24 141 [...]... the mean signal and signal variation (standard deviation) of the positive and negative controls, it takes into account three aspects of a HTS assay: 1) the dynamic range of assay signal, 2) data variation associated with sample measurement and 3) data variation associated with reference control measurement Hence, not only can the Z-factor can be used as a tool for comparison and evaluation of the robustness... cytoplasm for replication [Kimura and Ohyama, 1988; Guirakhoo et al., 1989] Following the adsorption and entry of DENV is primary translation and early viral RNA replication The viral RNA not only serves as a template for translation of viral proteins but also for the replication of RNA genome for progeny virus Hence, the translation of the viral RNA must first be carried out to produce the viral RNA... modulate the replication of not only dengue virus but other RNA viruses as well • And to use the developed screening assay to screen a small library of human protein hydroxylases as the pilot screening The screening assay would be applied on the replication of dengue virus and possibly be able to identify candidate hydroxylases which could mediate the replication of dengue virus in-vitro Hence, with the. .. regulating the viral RNA synthesis, 5’ and 3’ UTR are also involved in the generation of newly synthesized positive-strand viral RNA by means of circularization of the viral RNA to form a more stable RNA replication complex [You and Padmanabhan, 1999; You et al., 2001] More recently, structural studies also suggested that the cis-trans activity of the viral protease (NS3) and its cofactor (NS2B) may play... where, p = sample/test and n = control/normal Z-Factor Value Structure of Assay Related to Screening 1 SD=0 (no variation), dynamic range → ∞ An ideal assay 1 > Z ≥ 0.5 Large separation band An excellent assay 0.5 > Z > 0 Small separation band A double assay 0 No separation band sample and control bands touch A “yes/no” type assay . A SIRNA SCREEN TO PROBE FOR HYDROXYLASES THAT CAN MODULATE THE REPLICATION OF DENGUE VIRUS WONG PHUI YEW ANDREW B.SCI (HONS) A THESIS SUBMITTED FOR THE DEGREE OF. dependent enhancement AGO Argonaute ARNT aryl hydrocarbon receptor nuclear translocator Asn asparagine ASPH aspartate beta-hydroxylase BHK baby hamster kidney BSA bovine serum albumin C capsid. RNAi-based screening platform was developed to screen genomic libraries for host factors that could modulate the replication of DENV in host cells. The application of the developed RNAi-based