The involvement of cellular components in west nile virus replication cycle

THE INVOLVEMENT OF CELLULAR COMPONENTS IN WEST NILE VIRUS REPLICATION CYCLE CHU JANG HANN, JUSTIN B. Sc. (Hons), NUS A THESIS SUBMITTED FOR THE DEGREE OF PHILOSOPHY OF DOCTORATE DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2004 THE INVOLVEMENT OF CELLULAR COMPONENTS IN WEST NILE VIRUS REPLICATION CYCLE CHU JANG HANN, JUSTIN B. Sc. (Hons), NUS A THESIS SUBMITTED FOR THE DEGREE OF PHILOSOPHY OF DOCTORATE DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2004 PATENT, PUBLICATIONS AND PRESENTATTIONS PATENT, PUBLICATIONS AND PRESENTATIONS GENERATED FROM THIS STUDY Patent Filed: 1. Ng ML and Chu JJH (2004). Molecules, composition and kits for application associated with flaviviruses. International Publications: 1. Chu JJH and Ng ML (2002). Trafficking mechanism of West Nile (Sarafend) virus structural proteins. J Med Virol. 67, 127-136. 2. Ng ML and Chu JJH (2002). Interaction of West Nile and Kunjin viruses with cellular components during morphogenesis. Current Topics in Microbiology and Immunology. 267, 353-72. 3. Chu JJH and Ng ML. (2002). Infection of polarized epithelial cells with flavivirus West Nile: Polarized entry and egress of virus occur through apical surface. J Gen Virol. 83, 2427-2435. 4. Chu JJH and Ng ML. (2003). Characterization of a 105-kDa plasma membrane associated glycoprotein that is involved in West Nile virus binding and infection. Virology. 312, 458-469. 5. Chu JJH, Choo BGH, Lee JWM and Ng ML. (2003). Involvement of actin filaments in the budding of West Nile (Sarafend) virus. J Med Virol. 71, 463-472. 6. Chu JJH and Ng ML. (2003). The mechanism of cell death during West Nile virus infection is dependent on the initial infective dose. J Gen Virol. 84, 33053314. 7. Chu JJH and Ng ML. (2004). Infectious entry of West Nile virus occurs through a clathrin-mediated endocytic pathway. J Virol. 78, 10543-10555. 8. Chu JJH and Ng ML (2004). Interaction of West Nile virus with alpha v beta integrin mediates virus entry into cells. J Biol Chem. 279, 54533-54541. 9. Chu JJH, Rajamanomani R, Li, J, Bhuvanakanathan R, Lescar J and Ng ML. (2005). Inhibition of West Nile virus entry by using a recombinant domain III from the envelope glycoprotein. J Gen Virol. 86, 405-412. i PATENT, PUBLICATIONS AND PRESENTATTIONS Conference Presentations: 1. Chu JJH and Ng NL (2002). West Nile virus, a hitchhiker of host cell cytoskeleton. The 3rd ASEAN Microscopy Conference and the 19th Annual Conference of EMST, Chiang Mai, Thailand. 2. Choo BGH, Chu JJH and Ng ML (2002). Late consequences of flavivirus West Nile (Sarafend) infection in vitro. The 17th Australia Conference on Electron Microscopy, Australia. 3. Chu JJH and Ng ML (2002). The search for the cellular receptor for West Nile virus infection. The 10th International Congress on Infectious Diseases, Singapore. (Winner of the International Society Infectious Diseases New Investigator Award). 4. Chu JJH and Ng ML (2002). Detail Analysis of Flavivirus West Nile entry mechanism into host cells. The 15th international Congress on Electron Microscopy, Durban, South Africa. 5. Ng ML, Chu JJH and Choo BGH (2002). Tracking West Nile virus from entry to egression. The 15th international Congress on Electron Microscopy, Durban, South Africa. 6. Chu JJH and Ng ML (2002). Interaction of flavivirus with host cells: A polarized cell model for West Nile and Kunjin virus infection. The 4th APOCB Congress, Taipei, Taiwan. 7. Chu JJH and Ng ML (2002). Flavivirus West Nile Infection: An insight to the gateway for virus entry. The 6th NUS-NUH Annual Scientific Meeting, Singapore. (Winner of the National University of Singapore – National University Hospital Young Investigator Award) 8. Ng ML, Chu JJH and Choo BGH (2002). Kinectics of West Nile virus budding at the plasma membrane. The Annual Malaysia Electron Microscopy Conference, Malaysia. 9. Chu JJH and Ng ML (2003). West Nile virus–induced cell death and its implication on disease severity. The 4th combined Annual Scientific Meeting of SSMB, BRETSS and SSBMB, Singapore. (Winner for merit award for poster presentation) 10. Chu JJH and Ng ML (2003). West Nile virus induced cytopathology and its implication on disease severity and outcome. The 6th Asia Pacific Medical Virology Congress, Kuala Lumpur, Malaysia. 11. Leong PWH, Chu JJH and Ng ML (2003). Mapping the Entry Mechanism of West Nile virus into Mosquito Cell Line (C6/36). The 6th Asia Pacific Medical Virology Congress, Kuala Lumpur, Malaysia. ii PATENT, PUBLICATIONS AND PRESENTATTIONS 12. Chu JJH, Chye WSY and Ng ML (2004). Microscopic imaging of West Nile virus entry and assembly pathway. The 4th ASEAN Microscopy Conference, Hanoi, Vietnam. 13. Chu JJH. and Ng ML. (2004). Events of West Nile virus entry: A possible target for anti-flavivirus strategy. The 5th Combined Annual Scientific Meeting of BRETTS, SSMB and GSS, Singapore. (Winner of the overall best poster presentation). 14. Rajamanomani R , Chu JJH, Li, J, Bhuvanakanathan R, Lescar J and Ng ML. (2004). Structural and functional characterization of the putative receptor binding domain of West Nile virus. Norvartis Institute for Tropical Diseases, symposium on Dengue fever and Tuberculosis, 2004. iii ACKNOWLEDGEMENT ACKNOWLEDGEMENT I am deeply indebted to my supervisor, Prof. Mah-Lee Ng for providing a motivating, enthusiastic and conducive environment for the past few years. Her encouragement, guidance, unmatched concern and support were of great help at all times. She has given me countless opportunities to attend and make presentations in conferences worldwide. It was a great pleasure for me to conduct this thesis under her supervision. I wish to thank Dr Lu Jinhua (Department of Microbiology, NUS), Dr Tang Bor Luen (Department of Biochemistry, NUS), A/P Hanry Yu (Department of Physiology, NUS), A/P Julien Lescar (School of Biological Sciences, Nanyang Technological University, Singapore), Dr Alexandre Benmerah (Pasteur Insitute, France), Dr D. Cheresh (Scripps Research Institute, USA) for providing the reagents and technical advice throughout this study. I would also like to express my sincere gratitude to the following persons: Boon for being a wonderful teacher and her selfless assistance and advice has helped to overcome many technical difficulties that I encountered. Mr Low and Mdm Chew for their technical support and concern in times of need. The EM girls, Suat Hoon, Micky and Patricia for their expert advice on electron microscopy and I treasure the friendship that was fostered over the years. Sethoe, John, Ann Teck, Jasline, Min Yi for the many memorable moments that we shared. Dr Li Jun, Bhuvana, Paul, Jason (assistance in atomic force microscopy), Wee Lee, Kelvin, Benjamin, Huiwen, SiewPei, Justin Tan, Asad and Terence for standing by me and working together to overcome many obstacles in our path. We have emerged stronger than before. Michelle, Clarice, Samuel, Becky and Adrian, the new blood of our laboratory and for bringing diversification to our existing ecosystem. Especially, I would like to give my special thanks to my grandparents, parents, my wife and my sister whose patient love enabled me to complete this work. iv TABLE OF CONTENTS TABLE OF CONTENTS PAGE NO. PATENTS, PUBLICATIONS AND PRESENTATIONS GENERATED FROM THIS STUDY i ACKNOWLEDGEMENTS iv TABLE OF CONTENTS v LIST OF TABLES xvi LIST OF FIGURES xvii ABBREVIATIONS xxii SUMMARY xxiv CHAPTER 1.0 LITERATURE REVIEW 1.1 FLAVIVIRUS CLASSIFICATION 1.2 FLAVIVIRUS WEST NILE 1.3 VIRAL MORPHOLOGY 1.4 FLAVIVIRUS RNA GENOME ORGANISATION 1.5 FLAVIVIRUS STRUCTURAL PROTEINS 10 1.5.1 Capsid (C) Protein 10 1.5.2 Precusor Membrane/Membrane (prM/M) Protein 12 1.5.3 Envelope (E) Protein 13 1.6 1.7 FLAVIVIRus NON - STRUCTURAL (NS) PROTEINS 16 1.6.1 Flavivirus NS1 Protein 17 1.6.2 18 Flavivirus NS2A, NS2B, NS4A & NS4B Proteins 1.6.3 Flavivirus NS3 Protein 18 1.6.4 Flavivirus NS5 Protein 19 CELLULAR RECEPTOR MOLECULES AND VIRUS ENTRY PROCESSES 20 1.7.1 Virus Receptors 20 1.7.2 Virus Entry Processes 23 v TABLE OF CONTENTS 1.8 METHODS TO STUDY VIRUS RECEPTORS AND ENTRY MECHANISMS INTO CELLS 25 1.8.1 Virus Overlay Protein Blot Assay (VOPBA) 25 1.8.2 Anti-Idotypic Antibodies as Probes for Cell Surface Receptors 26 1.8.3 Methods to Decipher Virus Entry Mechanism and Pathway 26 1.9 FLAVIVIRUS RNA GENOME REPLICATION STRATEGY 30 1.10 MORPHOGENESIS OF FLAVIVIRUS 32 1.11 INVOLVEMENT OF CELLULAR CYTOSKELETON NETWORK IN VIRUS REPLICATION CYCLE 34 1.11.1 Cellular Cytoskeleton Networks 34 1.11.1.1 1.11.1.2 1.12 Microtubule Network and its Associated Motor 34 Proteins Actin Filaments and its Associated Motor Proteins 37 1.11.2 Viruses Associate with the Cytoskeleton 38 1.11.3 Flavivirus Interaction with Cytoskeleton Network 40 APPLICATION OF GENE SILENCING TECHNOLOGY (SMALL INTERFERING RNA, siRNA) TO STUDY VIRUS REPLICATION MECHANISM 1.13 1.14 41 WEST NILE VIRUS – INDUCED CELL DEATH DURING LATE INFECTION 42 OBJECTIVES 47 CHAPTER 49 2.0 MATERIALS AND METHODS 49 2.1 CELL CULTURE TECHNIQUES 49 2.1.1 Cell Lines 49 2.1.2 Media and Solution for Cell Culture 49 2.1.3 Cultivation and Propagation of the Cell Lines 50 2.1.3.1 Adherence Cell Lines 50 2.1.3.2 Suspension Cell Lines 51 vi TABLE OF CONTENTS 2.1.3.3 Polarized Cell Line 2.1.4 2.1.5 2.1.6 2.2 2.3 Cultivation of Cells in 6-Well, 24-Well and 96-Well Tissue Culture Tray 52 Cultivation of Cells on Glass Coverslips 52 2.1.5.1 Pretreatment of Coverslips for Cultivation of Cells 53 2.1.5.2 Culture of Cells on Coverslips 53 Storage of Cells 53 INFECTION OF CELLS 2.2.1 Viruses 54 2.2.2 Infection of Cell Monolayers 54 2.2.3 56 Preparation of Virus Pool 2.2.4 Concentration and Purification of Virus 56 2.2.5 57 Plaque Assay 2.2.6 Plaque Neutralization Assay 58 2.2.7 58 Extraction of Virus RNA Molecules PRODUCTION OF RADIOLABELLED-VIRUS 2.3.1 2.4 52 Production of [35S]-Methionine-Labelled Virus 59 59 MOLECULAR CLONING TECHNIQUES 60 2.4.1 Bacterial Strains 60 2.4.2 60 Cloning and Expression Vectors 2.4.3 Agarose Gel Electrophoresis 61 2.4.4 Complementary DNA (cDNA) Synthesis 62 2.4.5 Amplification of Specific Genes 62 2.4.5.1 Primers Sequences and Related Information 62 2.4.5.2 Polymerase Chain Reaction (PCR) 62 2.4.6 DNA Purification from Agarose Gel 64 2.4.7 Transformation of Competent E.coli Cells 65 2.4.8 Plasmid Isolation and Purification 65 2.4.9 Verification of Vector Containing Insert 66 2.4.10 Isolation of Recombinant Plasmid Using QIAGEN® Plasmid Midi Kit 67 vii TABLE OF CONTENTS 2.4.11 Expression of Recombinant ukinesin-GST Fusion Protein 68 2.4.11.1 Bacterial Strain and Plasmid 68 2.4.11.2 Analysis of Isolated Plasmids and Agarose Gel 68 Electrophoresis. 2.4.11.3 Expression of Recombinant Fusion Protein 2.5 2.6 69 TRANSFECTION OF CELL LINES 69 2.5.1 Transfection of Vector-Based DNA Molecules into Cells 69 2.5.2 Transfection of Antibodies and Protein Molecules into Cells 70 SODIUM-DODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE) 70 2.7 IMMUNOBLOTTING 72 2.8 IMMUNODOT BLOTTING 73 2.9 IMMUNOPRECIPITATION ASSAY 74 2.10 EXTRACTION OF CELLS 74 2.10.1 High Salt and Octyl-glucoside Cell Extraction Procedure 75 BIO-IMAGING 75 2.11.1 Indirect Immunofluorescence Microscopy 75 2.11 2.11.1.1 Purification of Polyclonal Antibodies 2.11.1.2 Preparation of Samples for Immunofluorescence with Antisera Against Specific Proteins 2.11.2 Real-Time Imaging Using Laser Scanning Confocal Microscopy 2.11.2.1 Cy5 Mono-Reactive Dye Labelling of Virus 2.11.2.2 Fluorescence Labelling of Cellular Organelles and Structures 76 77 78 78 78 2.11.3 Atomic Force Microscopy 79 2.11.4 Transmission Electron Microscopy 79 2.11.5 Carbon-Platnium Shadowing of Cytoskeleton 81 2.11.5.1 Immunogold-Labelling of the Cytoskeleton, its Associated Proteins and Specific Virus Proteins 82 2.11.6 Cryo-Immuno-Electron Microscopy 82 2.11.7 Negative Staining of WNV 84 viii APPENDICES (e) (f) The effects of EDTA on WNV and JEV binding to Vero cells EDTA WNV mM mM 7mM 10 mM 12 mM Exp Exp Exp Avg SD 5.34 6.75 4.68 5.11 5.81 5.68 8.33 5.01 5.98 5.94 5.12 7.9 5.66 5.43 5.9 5.38 7.66 5.1166667 5.5066667 5.8833333 1.3631874 1.5188603 0.4717379 0.3489842 0.0665833 JEV mM mM 7mM 10 mM 12 mM Exp 3.11 5.9 7.34 3.83 7.11 Exp 3.24 5.12 5.12 4.98 7.81 Exp 3.56 6.07 6.09 4.05 6.72 Avg 3.3033333 5.6966667 6.1833333 4.2866667 7.2133333 SD 1.3573946 0.818022 1.3129014 1.6854317 0.5522982 Gene silencing of human β3 integrin reduced WNV entry pSilencer-INTB3 pSilencer-GADPH pSilencer (g) Avg 59 SD 3.5118846 0.5773503 3.7634204 Specific interactions between soluble αVβ3 integrin and WNV prevent virus infection INTAVB3 10 µg/ml 7.5 µg/ml µg/ml 2.5 µg/ml µg/ml INTAVB5 10 µg/ml 7.5 µg/ml µg/ml 2.5 µg/ml µg/ml (h) % inhibition of virus entry Exp Exp Exp 63 60 56 5 11 3.8 5.5 Exp 85.6 77.23 68.56 35.28 26 Exp 23.33 9.87 7.98 8.37 5.56 Exp 83.2 72.49 61.08 38.45 30.12 Exp 26.12 9.17 5.88 8.44 6.68 Exp 87 74.08 63.34 41.87 28.48 Exp 25.07 11.48 6.98 8.11 4.1 AVG 85.266667 74.6 64.326667 38.533333 28.2 AVG 24.84 10.173333 6.9466667 8.3066667 5.4466667 SD 1.9218047 2.4124054 3.8363698 3.2957902 2.0742227 SD 1.4091487 1.1844971 1.0503968 0.1738774 1.2937285 Expression of αVβ3 integrin in CS-1 cells increased its permissiveness for WNV CS-1 cells substraction of background CS-1β3 cells substraction of background Exp 542 441 Exp 487 386 Exp 410 309 Avg SD 378.66667 66.304852 3325 3202 2911 2788 2874 2751 2913.6667 250.38837 Background 101 123 349 APPENDICES (i) Competitive inhibition of flaviviruses entry into Vero / C6/36 cells with soluble WNV E DIII protein Vero cell WNV+DIII 100ug/ml 50ug/ml 25ug/ml 10ug/ml 5ug/ml WNV+BSA 100ug/ml 50ug/ml 25ug/ml 10ug/ml 5ug/ml DV2 + DIII 100ug/ml 50ug/ml 25ug/ml 10ug/ml 5ug/ml DV2+BSA 100ug/ml 50ug/ml 25ug/ml 10ug/ml 5ug/ml C6/36 WNV+DIII 100ug/ml 50ug/ml 25ug/ml 10ug/ml 5ug/ml WNV+BSA 100ug/ml 50ug/ml 25ug/ml 10ug/ml 5ug/ml Exp 66.82 53.95 45.62 36.73 23.99 Exp 68.51 55.73 32.42 37.45 19.87 Exp 67.02 55.4 40.62 36.09 22.09 Avg 67.45 55.0267 39.5533 36.7567 21.9833 SD 0.9234176 0.9469072 6.6643329 0.680392 2.0620702 9.543 3.982 5.321 9.11 5.765 9.776 2.476 5.99 10.12 6.112 9.642 3.479 5.802 9.892 6.027 9.65367 3.31233 5.70433 9.70733 5.968 0.1169373 0.7667088 0.345028 0.5297182 0.1808674 32.1 22.99 11.34 2.19 2.67 33.67 21.95 11.76 2.76 1.12 32.45 22.82 11.56 2.052 1.924 32.74 22.5867 11.5533 2.334 1.90467 0.8241966 0.5578829 0.2100794 0.3753239 0.7751808 1.67 3.36 5.31 1.053 0.665 2.89 3.11 5.88 2.08 0.599 2.24 3.239 5.954 1.603 0.645 2.26667 3.23633 5.71467 1.57867 0.63633 0.610437 0.1250213 0.3523994 0.5139322 0.0338428 Exp 70.32 58.88 53.92 44.62 24.3 Exp 63.43 59.04 45.96 43.12 25.09 Exp 69.15 59.58 50.03 43.25 25.86 Avg 67.6333 59.1667 49.97 43.6633 25.0833 SD 3.6869002 0.3667879 3.9803392 0.8310435 0.7800214 2.55 2.71 5.33 1.34 3.65 3.97 2.9 6.17 -2.44 4.04 2.16 2.94 5.28 -3.18 4.16 2.89333 2.85 5.59333 -1.4267 3.95 0.952593 0.1228821 0.5000333 2.4244037 0.2666458 350 APPENDICES DV2 + DIII 100ug/ml 50ug/ml 25ug/ml 10ug/ml 5ug/ml DV2+BSA 100ug/ml 50ug/ml 25ug/ml 10ug/ml 5ug/ml (j) 65.34 53.63 47.52 40.65 35.19 64.39 56.77 50.87 42.25 30.65 63.58 55.33 48.48 41.2 31.18 64.4367 55.2433 48.9567 41.3667 32.34 0.8809275 1.571793 1.7251184 0.8129166 2.4823578 3.98 2.89 0.32 1.93 1.54 2.34 2.61 0.83 2.54 2.77 3.56 2.7 0.55 1.98 2.09 3.29333 2.73333 0.56667 2.15 2.13333 0.8518998 0.1429452 0.2554082 0.3386739 0.6161439 Plaque neutralization of WNV with murine polyclonal antibody against WNV E DIII protein West Nile virus Sample dilution 16 32 64 128 256 512 Positive Control Mouse anti-DIII protein 1.20E+02 1.22E+02 1.25E+02 2.95E+02 2.98E+02 3.00E+02 5.72E+02 5.80E+02 5.79E+02 1.23E+03 1.22E+03 1.22E+03 1.05E+03 1.20E+03 1.25E+03 2.19E+03 2.22E+03 2.21E+03 5.32E+03 5.34E+03 5.35E+03 8.00E+03 7.98E+03 7.99E+03 1.32E+04 1.31E+04 1.30E+04 % Inhibition 99.63 99.62 99.61 99.07 99.06 99.06 98.23 98.20 98.21 96.07 96.09 96.09 96.69 96.21 96.06 93.16 93.06 93.09 83.36 83.30 83.26 75.86 75.92 75.88 58.27 58.59 58.94 SD 1.25 Avg (% Inhibition) 100.00 2.36 99.07 1.25 98.21 5.26 96.09 2.32 95.65 1.23 93.10 0.13 80.25 0.90 77.26 0.15 60.25 Negative Control Pre-immunized sera 3.21E+04 3.30E+04 3.09E+04 3.12E+04 3.20E+04 3.24E+04 3.16E+04 3.22E+04 3.31E+04 3.23E+04 3.14E+04 2.99E+04 3.17E+04 3.06E+04 3.28E+04 3.20E+04 3.30E+04 3.10E+04 3.28E+04 3.25E+04 3.06E+04 3.29E+04 3.38E+04 3.27E+04 3.00E+04 3.23E+04 3.26E+04 Avg 3.20E+04 3.19E+04 3.23E+04 3.12E+04 3.17E+04 3.20E+04 3.20E+04 3.31E+04 3.16E+04 351 APPENDICES 1024 2048 4096 8192 Sample dilution 16 32 64 128 256 512 1024 1.94E+04 1.96E+04 1.91E+04 2.50E+04 2.49E+04 2.53E+04 2.98E+04 2.97E+04 3.00E+04 3.09E+04 3.02E+04 3.05E+04 Mouse anti-DIII protein 1.77E+04 1.78E+04 1.79E+04 1.81E+04 1.88E+04 1.83E+04 1.89E+04 1.81E+04 1.93E+04 2.00E+04 1.93E+04 1.91E+04 2.10E+04 2.55E+04 2.34E+04 2.55E+04 2.76E+04 2.45E+04 3.01E+04 2.99E+04 3.12E+04 3.23E+04 3.11E+04 3.19E+04 3.29E+04 3.26E+04 3.31E+04 3.42E+04 3.45E+04 3.51E+04 37.98 37.25 38.85 21.30 21.62 20.36 9.61 9.91 9.00 1.24 3.45 2.52 % Inhibition 51.81 51.54 51.27 48.29 46.29 47.71 46.05 48.33 44.91 41.46 43.51 44.10 37.75 24.41 30.63 27.69 21.74 30.53 15.21 15.77 12.11 3.96 7.53 5.15 6.71 7.56 6.14 5.26 4.43 2.77 0.80 38.03 0.66 21.09 1.00 12.03 3.26 2.40 Dengue virus Avg SD Avg (% Inhibition) 51.54 0.27 100.00 47.43 1.03 47.43 46.43 1.74 46.43 43.02 1.38 43.02 30.93 6.67 95.65 26.65 4.49 26.65 14.37 1.97 80.25 5.55 1.82 77.26 6.81 0.71 60.25 4.16 1.27 4.16 3.02E+04 3.23E+04 3.12E+04 3.16E+04 3.29E+04 3.08E+04 3.40E+04 3.28E+04 3.21E+04 3.15E+04 3.11E+04 3.13E+04 3.12E+04 Pre-immunized sera 3.54E+04 3.67E+04 3.81E+04 3.51E+04 3.34E+04 3.65E+04 3.51E+04 3.45E+04 3.55E+04 3.44E+04 3.12E+04 3.69E+04 3.01E+04 3.44E+04 3.67E+04 3.65E+04 3.59E+04 3.34E+04 3.45E+04 3.59E+04 3.61E+04 3.55E+04 3.27E+04 3.27E+04 3.60E+04 3.51E+04 3.47E+04 3.71E+04 3.57E+04 3.55E+04 Avg 3.18E+04 3.30E+04 3.13E+04 3.67E+04 3.50E+04 3.50E+04 3.42E+04 3.37E+04 3.53E+04 3.55E+04 3.36E+04 3.53E+04 3.61E+04 352 APPENDICES 2048 4096 8192 (k) 3.41E+04 3.38E+04 3.35E+04 3.44E+04 3.33E+04 3.43E+04 3.49E+04 3.34E+04 3.52E+04 0.29 1.17 2.05 0.86 4.03 1.15 1.51 5.74 0.66 1.17 0.88 1.17 2.02 1.75 12.03 2.63 2.72 2.63 3.34E+04 3.41E+04 3.51E+04 3.47E+04 3.33E+04 3.61E+04 3.44E+04 3.57E+04 3.62E+04 3.42E+04 3.47E+04 3.54E+04 Specific binding of WNV E DIII protein to αVβ3 integrin WNV+DIII 100 µg/ml 50 µg/ml 25 µg/ml Exp 3224.98 5563.12 7773.9 Exp 3312.34 5009.77 7688.9 Exp 3200.77 5398.1 7745.08 Avg 75.797569 60.306715 42.320608 SD 0.437579 2.1181455 0.3223059 WNV+BSA 100 µg/ml 50 µg/ml 25 µg/ml Exp 1886.54 1900.34 1944.3 Exp 1802.56 1899.67 1906.3 Exp 1844.6 1895.34 1913.5 Avg 7.6543877 4.9567951 3.8095047 SD 2.1021703 0.1358772 1.0105075 353 APPENDICES APPENDIX 16 DATA FOR FIGURES IN CHAPTER (a) Effects of clathrin-mediated endocytosis-disrupting drugs on WNV entry into Vero cells Monodanslycadervine Pre-1hr 10 30 hr hr Chlorpromazine Pre-1hr 10 30 hr hr Sucrose Pre-1hr 10 30 hr hr Filipin Pre-1hr 10 30 hr hr (b) Exp 200 210 678 910 943 Exp 189 189 753 930 935 Exp 178 232 699 896 912 Avg No. Virus Infected Cells 986 986 986 986 986 Avg 189 210.333 710 912 930 Exp 289 356 745 980 943 Exp 302 325 702 945 968 Exp 311 386 714 925 945 Avg No. Virus Infected Cells 986 986 986 986 986 Avg 300.67 355.67 720.33 950 952 Exp 168 300 758 856 943 Exp 189 358 753 930 902 Exp 178 389 789 896 912 Avg No. Virus Infected Cells 986 986 986 986 986 Avg 178.333 349 766.667 894 919 Exp 856 924 914 910 943 Exp 823 922 956 930 953 Exp 845 902 922 931 904 Avg No. Virus Infected Cells 986 986 986 986 986 Avg 841.333 916 930.667 923.667 933.333 797 775.667 276 74 56 % Reduction 80.831643 78.668019 27.991886 7.505071 5.6795132 SD 5.2 10 8.6 2.5 1.5 685.33 630.33 265.67 36 34 % Reduction 69.50642 63.92833 26.94388 3.651116 3.448276 SD 12 5.5 1.5 0.25 0.8 807.667 637 219.333 92 67 % Reduction 81.913455 64.604462 22.24476 9.3306288 6.7951318 SD 4.5 5.5 0.25 0.6 144.667 70 55.3333 62.3333 52.6667 % Reduction 14.672076 7.0993915 5.6118999 6.3218391 5.3414469 SD 1.5 2.5 0.5 0.89 1.8 Inhibition of WNV entry into Vero cells expressing Eps15-dominant negative mutant protein Cell Samples Vero cell (Control) Vero cell (EGFP-C2) Vero cell (GFPE∆95/295) % of fluorescent unit Exp Exp Exp 93 94 80 85 92 78 22 19 21 Avg 89 85 SD 7.81 20.67 1.53 354 APPENDICES (c) Effects of PKC inhibitor (BIS I) on WNV entry into Vero cells Time hr Pre hr Pre 0.5 hr hr hr hr hr hr (d) Virus yield (Log PFU/ml) Exp Exp Exp Avg 7.00E+03 6.80E+03 7.20E+03 7.00E+03 6.50E+03 6.80E+03 6.40E+03 6.57E+03 6.80E+03 6.30E+03 6.40E+03 6.50E+03 3.00E+04 3.80E+04 3.30E+04 3.37E+04 4.00E+05 4.20E+05 4.00E+05 4.07E+05 1.00E+08 1.50E+08 1.30E+08 1.27E+08 3.00E+09 3.40E+09 2.80E+09 3.07E+09 2.00E+09 2.30E+09 2.50E+09 2.27E+09 Effect of BIS I on WNV infectivity for cells – transfected with virus RNA Concentration µM 10 µM 20 µM 30 µM 40 µM 50 µM 60 µM (e) PFU/ml 5.00E+05 8.50E+05 5.60E+05 7.20E+05 8.30E+05 7.80E+05 9.80E+04 Effect of BIS I on WNV binding to cell surface of Vero cells No treatment BIS - treated (f) SD 200 208.1666 264.57513 4041.4519 11547.005 25166115 305505046 251661148 Exp 25000 25100 Radioactivity (CPM) Exp Exp Avg 24987 24956 24981 25002 24996 25032.667 SD 22.605309 58.389497 Sub-cellular fractionations of cellular homogenates from WNV-infected cells in 20 % Percoll gradients. Fraction No. 10 11 12 13 14 15 16 Untreated BIS I treated BIS I washout CPM 505 512 509 518 511 516 517 536 1000 608 534 509 506 508 511 588 CPM2 1490 1465 1491 1477 1601 1444 1510 3004 3997 3452 2647 1566 1573 1400 700 523 CPM3 456 461 458 477 476 455 469 572 645 678 455 432 433 451 623 711 355 APPENDICES 17 18 19 20 21 22 23 24 (g) 1123 913 503 109 23 22 27 21 132 56 59 64 60 61 54 55 877 745 433 209 33 32 21 20 Enzymatic activites of PKC and its isoforms PKC enzymatic assay PKC α PKC ζ PKCβI PKCβII PKC Control PKC enzymatic assay PKC α PKC ζ PKCβI PKCβII PKC Control (h) 10µg (Exp 1) 1891.22 1732.43 1800.59 1888.21 1727.15 3382.47 10µg (Exp 2) 1832.67 1771.07 1812.98 1809.48 1732.76 3614.66 Avg 253.895 143.7 198.735 240.795 121.905 1890.515 SD 41.401102 27.322606 8.761053 55.670517 3.966869 8.2345 µg (Exp 1) 2413.2 2588.38 2391.83 2098.41 2309.47 3413.08 µg (Exp 2) 2011.26 2431.03 2400.19 2001.56 2335.77 3491.29 Avg 604.18 901.655 787.96 441.935 714.57 1844.135 SD 284.2145 111.26325 5.9114127 68.483292 18.596908 55.302821 Inhibition of WNV infectivity in cells transfected with antibodies against PKC and its isoforms PKC α PKC ζ PKCβI PKCβII PKC FITC control Exp 6.00E+03 1.00E+04 3.00E+04 3.00E+04 1.00E+03 2.00E+06 2.00E+06 Exp 7.00E+03 3.00E+04 5.00E+04 1.00E+04 5.00E+02 5.00E+06 6.00E+06 Exp 1.00E+03 2.00E+04 8.00E+04 9.00E+03 9.00E+02 8.00E+06 3.00E+06 Avg 3.5410831 4.25938375 4.69306042 4.14378792 2.88440417 6.63436333 6.5187675 SD 0.46978576 0.24127159 0.21304585 0.28958033 0.16221224 0.30618552 0.24127159 356 APPENDICES (i) Low pH-dependent of WNV into Vero cells Bafilomycin A Concentration 1.5 µm µm 0.5 µm 0.1 µm 0.05 µm Chloroquine Concentration 30 µm 25 µm 20 µm 15 µm 10 µm Amantidine Concentration µm 1.5 µm µm 0.5 µm 0.1 µm (j) Exp 225 356 456 789 895 Exp 111 145 200 256 789 Exp 256 356 562 845 856 Exp 200 333 478 777 888 Exp 125 155 214 289 777 Exp 300 389 555 878 898 Exp 189 389 411 747 874 Avg No. Virus Infected Cells 986 986 986 986 986 Avg 204.67 359.33 448.33 771 885.67 Exp 156 156 235 299 745 Avg No. Virus Infected Cells 995 995 995 995 995 Avg 130.67 152 216.33 281.33 770.33 Exp 304 400 589 881 911 Avg No. Virus Infected Cells 995 995 995 995 995 Avg 286.67 381.67 568.67 868 888.33 781.333 626.667 537.667 215 100.333 % reduction 79.2427 63.5565 54.5301 21.8053 10.1758 SD 5.6 5.5 2.5 1.2 1.3 864.333 843 778.667 713.667 224.667 % reduction 86.8677 84.7236 78.258 71.7253 22.5796 SD 2.3 1.5 1.9 0.5 708.333 613.333 426.333 127 106.667 % reduction 71.1893 61.6415 42.8476 12.7638 10.7203 SD 4.2 2.3 5.6 1.5 Cytochalasin D and nacodazole pre-treated Vero cells inhibit entry of WNV Cytochalasin D Concentration 2.0 µg/ml 1.5 µg/ml 1.0 µg/ml 0.5 µg/ml 0.1 µg/ml Nacodazole Exp 89 152 189 356 758 Exp 58 155 193 389 777 Exp 52 180 195 400 745 Avg No. Virus Infected Cells 995 995 995 995 995 Avg 66.3333 162.333 192.333 381.667 760 Concentration 40 µm 30 µm 20 µm 10 µm µm Exp 156 256 356 589 689 Exp 189 289 389 510 666 Exp 200 245 399 571 689 Avg No. Virus Infected Cells 995 995 995 995 995 Avg 181.67 263.33 381.33 556.67 681.33 928.667 832.667 802.667 613.333 235 % reduction 93.333333 83.685092 80.670017 61.641541 23.61809 SD 2.3 1.5 0.56 4.8 1.2 813.33 731.67 613.67 438.33 313.67 % reduction 81.74204 73.53434 61.67504 44.0536 31.52429 SD 5.5 5.6 4.5 1.2 357 APPENDICES (k) Sub-cellular fractionations of cellular homogenates from WNV-infected cells in 20 % percoll gradients Markers Fraction 10 11 Untreated Density 1.03 g/ml Fraction 10 11 Radioactivity (CPM) 10 12 15 14 10 15 14 45 25 18 Fraction 10 11 Radioactivity (CPM) 11 15 10 10 14 89 68 55 Fraction 10 11 Radioactivity (CPM) 10 11 12 10 10 12 13 14 10 12 13 14 10 12 13 14 95 56 20 15 16 11 11 15 16 10 11 15 16 15 17 26 17 17 18 19 20 21 22 23 24 59 45 22 10 18 19 20 21 22 23 24 6 18 19 20 21 22 23 24 10 11 1.042 g/ml 12 13 14 1.056 g/ml 15 16 1.063 g/ml 1.079 g/ml 17 18 19 20 21 22 23 24 1.1 g/ml 1.15 g/ml (l) Cytochalasin D Nacodazole Infectivity of WNV with Cy5 labelling at different D/P ratio. (R/D) 0.5 1.5 Exp 6.00E+08 2.40E+08 3.30E+08 9.00E+07 Exp 5.00E+08 3.10E+08 4.50E+08 3.00E+07 Exp 1.00E+08 4.00E+08 2.33E+08 2.00E+07 Avg 4.00E+08 3.17E+08 3.38E+08 4.67E+07 SD 208166600 50147929 108522570 13471506 358 APPENDICES APPENDIX 17 DATA FOR FIGURES IN CHAPTER (a) Extracellular virus production of WNV-infected Vero cells treated with 10 µg/ml of vinblastine sulphate 10 12 10 12 10 12 (b) Extracellular virus yield (PFU/ml) / vinblastine sulphate treated Log Log Log Avg 4.00E+03 3.6021 3.00E+03 3.47712 9.00E+02 2.95424 3.34E+00 6.00E+04 4.7782 4.00E+05 5.60206 2.00E+05 5.30103 5.23E+00 2.00E+05 5.301 3.00E+05 5.47712 5.00E+05 5.69897 5.49E+00 5.00E+05 5.699 1.80E+06 6.25527 6.00E+05 5.77815 5.91E+00 2.00E+06 6.301 3.00E+06 6.47712 4.00E+06 6.60206 6.46E+00 Extracellular virus yield / untreated Log Log Log Avg 4.00E+04 4.6021 4.00E+04 4.60206 2.00E+05 5.30103 4.84E+00 8.00E+05 5.9031 3.00E+06 6.47712 4.00E+06 6.60206 6.33E+00 4.00E+07 7.6021 2.00E+07 7.30103 5.00E+07 7.69897 7.53E+00 3.00E+08 8.4771 2.00E+08 8.30103 5.00E+08 8.69897 8.49E+00 1.00E+09 1.20E+09 9.07918 3.00E+09 9.47712 9.19E+00 Viability of Infected Vero cells Log Log Log Avg 7.90E+05 5.8976 7.80E+05 5.89209 7.80E+05 5.89209 5.893939 7.60E+05 5.8808 7.00E+05 5.8451 7.80E+05 5.89209 5.872669 7.00E+05 5.8451 6.60E+05 5.81954 6.30E+05 5.79934 5.821328 6.30E+05 5.7993 6.50E+05 5.81291 7.00E+05 5.8451 5.819117 6.30E+05 5.7993 5.90E+05 5.77085 5.40E+05 5.73239 5.767529 SD 0.34368 0.4169 0.19941 0.30094 0.15124 SD 0.40355 0.37276 0.20751 0.19941 0.25569 SD 0.00319 0.02453 0.02293 0.0235 0.0336 Release of WNV occurs predominantly at the apical domain, while KUN virus release occurs bi-directionally in Vero C1008 cells. Time hr hr 12 hr 14 hr Time hr hr 12 hr 14 hr Time hr 12 hr 18 hr WNV apical release (Vero C1008 cells) Virus yield (Log PFU/ml) Exp Exp Exp Avg 5.00E+05 4.80E+05 5.00E+05 4.93E+05 6.00E+07 6.20E+07 6.40E+07 6.20E+07 4.50E+08 4.50E+08 5.00E+08 4.67E+08 8.00E+09 8.40E+09 8.30E+09 8.23E+09 WNV basolateral release (Vero C1008 cells) Virus yield (Log PFU/ml) Exp Exp Exp Avg 6.00E+03 6.30E+03 6.10E+03 6.13E+03 6.00E+03 6.00E+03 6.20E+03 6.07E+03 7.00E+03 7.50E+03 7.20E+03 7.23E+03 1.00E+04 1.50E+04 1.00E+04 1.17E+04 KUN virus apical release (Vero C1008 cells) Virus yield (Log PFU/ml) Exp Exp Exp Avg 2.00E+04 2.50E+04 2.40E+04 2.30E+04 6.00E+04 6.70E+04 6.30E+04 6.33E+04 8.90E+05 8.80E+05 8.60E+05 8.77E+05 SD 11547.005 2000000 28867513 208166600 SD 152.75252 115.47005 251.66115 2886.7513 SD 2645.7513 3511.8846 15275.252 359 APPENDICES 24 hr Time hr 12 hr 18 hr 24 hr Time hr hr 12 hr 14 hr Time hr hr 12 hr 14 hr Time hr 12 hr 18 hr 24 hr Time hr 12 hr 18 hr 24 hr Time hr hr 12 hr 14 hr Time hr hr 12 hr 14 hr 2.30E+07 2.50E+07 2.40E+07 2.40E+07 KUN virus basolateral release (Vero C1008 cells) Virus yield (Log PFU/ml) Exp Exp Exp Avg 8.60E+03 8.60E+03 8.30E+03 8.50E+03 9.60E+03 9.10E+03 9.50E+03 9.40E+03 7.60E+05 7.70E+05 7.60E+05 7.63E+05 9.30E+06 9.00E+06 9.00E+06 9.10E+06 WNV apical release (Vero cells) Virus yield (Log PFU/ml) Exp Exp Exp Avg 8.00E+04 8.50E+04 7.80E+04 8.10E+04 3.50E+06 2.90E+06 3.90E+06 3.43E+06 8.30E+08 8.00E+08 8.50E+08 8.27E+08 8.90E+09 8.70E+09 8.90E+09 8.83E+09 WNV basolateral release (Vero cells) Virus yield (Log PFU/ml) Exp Exp Exp Avg 8.20E+04 8.40E+04 8.60E+04 8.40E+04 3.70E+06 3.75E+06 3.60E+06 3.68E+06 8.20E+08 8.00E+08 8.00E+08 8.07E+08 8.40E+09 8.50E+09 7.90E+09 8.27E+09 KUN virus apical release (Vero cells) Virus yield (Log PFU/ml) Exp Exp Exp Avg 9.50E+03 9.50E+03 9.30E+03 9.43E+03 4.10E+04 4.00E+04 4.50E+04 4.20E+04 9.60E+05 9.90E+05 9.50E+05 9.67E+05 9.80E+06 9.80E+06 9.50E+06 9.70E+06 KUN virus basolateral release (Vero cells) Virus yield (Log PFU/ml) Exp Exp Exp Avg 9.40E+03 9.80E+03 9.80E+03 9.67E+03 1.10E+04 9.80E+03 9.55E+03 1.01E+04 8.60E+05 8.00E+05 8.50E+05 8.37E+05 8.00E+06 8.00E+06 8.10E+06 8.03E+06 Poliovirus apical release (Vero C1008 cells) Virus yield (Log PFU/ml) Exp Exp Exp Avg 6.70E+02 6.00E+02 6.50E+02 6.40E+02 6.40E+04 6.50E+04 6.60E+04 6.50E+04 5.60E+05 5.70E+05 5.80E+05 5.70E+05 9.70E+06 9.00E+06 9.50E+06 9.40E+06 Poliovirus basolateral release (Vero C1008 cells) Virus yield (Log PFU/ml) Exp Exp Exp Avg 5.50E+02 5.50E+02 5.40E+02 5.47E+02 1.00E+04 9.80E+03 9.80E+03 9.87E+03 1.00E+05 9.70E+04 1.30E+05 1.09E+05 8.10E+06 7.50E+06 7.90E+06 7.83E+06 1000000 SD 173.20508 264.57513 5773.5027 173205.08 SD 3605.5513 503322.3 25166115 115470054 SD 2000 76376.262 11547005 321455025 SD 115.47005 2645.7513 20816.66 173205.08 SD 230.94011 775.1344 32145.503 57735.027 SD 36.055513 1000 10000 360555.13 SD 5.7735027 115.47005 18248.288 305505.05 360 APPENDICES (c) Role of microtubules in the polarized release of WNV and KUN virus in polarized epithelial cells Apical Basolateral Apical Basolateral (d) Productive yields (PFU/ml) of WNV Exp Exp Exp Avg 2.00E+04 2.50E+04 2.20E+04 2.23E+04 4.00E+03 3.80E+03 4.40E+03 4.07E+03 Productive yields (PFU/ml) of KUN virus Exp Exp Exp Avg 5.00E+07 5.50E+07 6.00E+07 5.50E+07 2.00E+07 2.50E+07 2.80E+07 2.43E+07 SD 2516.611 305.505 SD 5000000 4041452 Effect of cytochalasin B on WNV egression from Vero cells Virus yield (Log PFU/ml) w/o treatment Exp Exp Exp Avg SD 4.50E+03 3.90E+03 4.70E+03 4.37E+03 416.3332 2.00E+04 2.00E+04 2.50E+04 2.17E+04 2886.7513 6.00E+05 5.80E+05 6.30E+05 6.03E+05 25166.115 7.00E+07 6.80E+07 7.20E+07 7.00E+07 2000000 3.00E+09 2.50E+09 3.00E+09 2.83E+09 288675135 Virus yield (Log PFU/ml) after Cytochalasin treatment Time Exp Exp Exp Avg SD hr 2.00E+02 2.50E+02 2.30E+02 2.27E+02 25.166115 hr 6.00E+03 5.30E+03 5.50E+03 5.60E+03 360.55513 hr 7.00E+03 7.50E+03 6.90E+03 7.13E+03 321.45503 10 hr 6.00E+03 5.80E+03 5.90E+03 5.90E+03 100 12 hr 2.00E+04 2.00E+04 1.00E+04 1.67E+04 5773.5027 Virus yield (Log PFU/ml) after removal of cytochalasin treatment Time Exp Exp Exp Avg SD hr 5.00E+03 5.20E+03 5.00E+03 5.07E+03 115.47005 hr 9.00E+04 8.50E+04 8.70E+04 8.73E+04 2516.6115 hr 8.00E+07 7.50E+07 8.45E+07 7.98E+07 4752192.5 Time hr hr hr 10 hr 12 hr 361 APPENDICES APPENDIX 18 DATA FOR FIGURES IN CHAPTER (a) The effect of different infectious doses of WNV-infected Vero cells. Extracellular virus production (PFU/ml) at different M.O.I. M.O.I. = 100 4h 8h 12 h 14 h Exp 2.00E+03 4.00E+07 8.00E+08 2.00E+09 Log 3.30103 7.60206 8.90309 9.30103 Exp 4.00E+03 6.00E+07 3.00E+09 5.50E+09 Log 3.60206 7.7781513 9.4771213 9.7403627 Exp 5.80E+03 2.50E+07 5.00E+09 6.00E+09 Log 3.763428 7.39794 9.69897 9.7781513 Avg 3.555506 7.5927171 9.3597271 9.6065146 SD 0.2346879 0.1902777 0.4107217 0.2652313 M.O.I. = 10 4h 8h 12 h 14 h 1.00E+03 3.00E+07 1.00E+09 3.00E+09 7.4771213 9.4771213 5.50E+03 4.00E+07 8.00E+08 1.00E+09 3.7403627 7.60206 8.90309 2.00E+03 1.00E+07 9.00E+08 8.00E+08 3.30103 8.9542425 8.90309 3.3471309 7.3597271 8.9524442 9.1267371 0.3723281 0.3177343 0.04848 0.307286 M.O.I. =1 8h 16 h 24 h 32 h 1.00E+04 2.00E+05 4.00E+06 2.00E+07 5.30103 6.60206 7.30103 9.00E+03 3.00E+05 8.00E+06 8.00E+06 3.9542425 5.4771213 6.90309 6.90309 8.70E+03 1.80E+05 7.00E+06 1.00E+07 3.9395193 5.2552725 6.845098 3.9645873 5.3444746 6.783416 7.06804 0.0315395 0.1171315 0.1597131 0.2075118 M.O.I. = 0.1 8h 16 h 24 h 32 h 2.00E+04 1.00E+05 5.00E+06 6.00E+06 4.30103 1.00E+04 1.80E+04 4.2552725 1.50E+05 5.1760913 8.70E+04 4.9395193 6.69897 6.00E+06 6.7781513 1.20E+06 6.0791812 6.7781513 9.00E+06 6.9542425 8.00E+06 6.90309 Cell viability (cells/ml) after infection with WNV 4.1854342 5.0385368 6.5187675 6.8784946 0.1622122 0.122904 0.382746 0.0905855 M.O.I. = 100 4h 8h 12 h 14 h Exp 5.00E+06 3.00E+05 2.00E+05 2.00E+02 Log 6.69897 5.4771213 5.30103 2.30103 Exp 4.80E+06 8.00E+05 3.50E+05 1.00E+02 Log 6.6812412 5.90309 5.544068 Exp 5.50E+06 4.00E+05 1.00E+05 4.50E+02 Log 6.7403627 5.60206 2.6532125 Avg 6.706858 5.6607571 5.2816993 2.3180808 SD 0.0303398 0.2189666 0.2725486 0.3269399 M.O.I. = 10 4h 8h 12 h 14 h 5.00E+06 4.00E+05 1.00E+05 1.00E+02 6.69897 5.60206 4.00E+06 3.00E+05 9.00E+04 4.00E+02 6.60206 5.4771213 4.9542425 2.60206 4.50E+06 1.00E+05 8.00E+04 8.00E+02 6.6532125 4.90309 2.90309 6.6514142 5.3597271 4.9524442 2.5017167 0.04848 0.3177343 0.04848 0.4598309 M.O.I. =1 8h 5.00E+06 6.69897 8.00E+06 6.90309 3.00E+06 6.4771213 6.6930604 0.2130458 362 APPENDICES 16 h 24 h 32 h 6.00E+06 1.00E+05 8.00E+03 6.7781513 3.90309 4.00E+06 2.00E+05 7.00E+03 6.60206 5.30103 3.845098 1.00E+06 3.00E+05 1.00E+04 5.4771213 6.4600704 5.2593838 3.9160627 0.4080449 0.2412716 0.0782616 M.O.I. = 0.1 8h 16 h 24 h 32 h 5.50E+06 6.00E+06 2.00E+05 2.00E+04 6.7403627 6.7781513 5.30103 4.30103 6.00E+06 3.00E+06 9.00E+04 7.00E+03 6.7781513 6.4771213 4.9542425 3.845098 5.00E+06 4.00E+06 3.00E+05 9.50E+03 6.69897 6.60206 5.4771213 3.9777236 6.7391613 6.6191108 5.2441313 4.0412839 0.0396043 0.1512376 0.2660426 0.2345174 (b) Measurement of LDH activity in WNV-infected Vero cell supernatants LDH assay M.O.I. = 10 hr hr 12 hr 14 hr M.O.I. = hr 16 hr 24 hr 32 hr Exp Exp Exp Avg SD 5.6 8.5 0.5 6.5 8.8 0.6 6.6 8.5 0.7 6.233333 8.433333 8.5 0.264575 0.550757 0.404145 0.5 1.2 2.2 0.8 0.5 1.6 6.5 0.5 1.8 5.8 0.766667 0.9 1.866667 5.766667 0.251661 0.360555 0.305505 0.750555 Exp 0.8 2.5 2.8 Exp 0.5 0.5 3.2 3.5 Exp 0.6 0.5 3.3 Avg 0.633333 0.666667 2.9 3.2 SD 0.152753 0.288675 0.360555 0.360555 Cytochalasin B treatment hr hr 12 hr 14 hr (d) Activation of caspases activities by WNV-infected cells Time 12 hr 18 hr 20 hr 26 hr Exp 1.2 2.1 7.8 8.9 12 hr 18 hr 20 hr 26 hr 1.2 3.3 12 hr 18 hr 20 hr 26 hr 32 hr 0.3 0.5 1.5 6.3 7.5 Caspase - Activation Exp Exp Avg 1.4 1.5 1.3666667 1.8 1.9666667 7.5 7.7666667 8.8 8.7 8.8 Caspase – Activation 1.2 1.1 1.1 1.5 1.6 1.4333333 2.5 2.8 2.4333333 2.8 2.9 Caspase – Activation 0.5 0.2 0.3333333 0.8 1.2 0.8333333 1.2 0.8 1.1666667 6.9 5.8 6.3333333 7.9 6.8 7.4 SD 0.1527525 0.1527525 0.2516611 0.1 0.1 0.2081666 0.4041452 0.2645751 0.1527525 0.3511885 0.3511885 0.5507571 0.5567764 363 APPENDICES 364 [...]... GRAND ENTRANCE OF WEST NILE VIRUS 160 5.1 INTRODUCTION 160 5.2 ENTRY ROUTE OF WEST NILE VIRUS AND CELLULAR COMPONENTS INVOLVED IN THE ENTRY PROCESS 5.3 ENTRY OF WEST NILE VIRUS OCCUR THROUGH A CLATHRINMEDIATED ENDOCYTIC PATHWAY 5.4 160 171 PROTEIN KINASE C (PKC) AND ITS ISOFORMS ARE INVOLVED IN WEST NILE VIRUS ENTRY PATHWAY 5.4.1 Inhibition of PKC Does Not Affect West Nile Virus Binding to Cell Surface... SOLUBLE INTEGRIN αVβ3 INHIBITS WEST NILE VIRUS ENTRY 142 4.7 EXPRESSION OF INTEGRIN αVβ3 INCREASES THE SUSCEPTIBILITY OF MELANOMA CELL (CS-1) TO WEST NILE VIRUS INFECTION 4.8 αVβ3 INTEGRIN EXPRESSION AND SUSCEPTIBILITY OF CELLS TO WEST NILE VIRUS INFECTION 4.9 142 144 ACTIVATION OF INTEGRIN - ASSOCIATED SIGNALLING PATHWAY BY WEST NILE VIRUS 146 xi TABLE OF CONTENTS 4.10 SOLUBLE RECOMBINANT ENVELOPE PROTEIN... NILE VIRUS ENTRY IN VERTEBRATE CELLS 129 4.1 INTRODUCTION 129 4.2 PEPTIDE SEQUENCING OF THE 105-KDA PLASMA MEMBRANE ASSOCIATED GLYCOPROTEIN 4.3 129 FUNCTIONAL BLOCKING ANTIBODIES AGAINST αVβ3 INTEGRIN INHIBIT WEST NILE VIRUS BINDING AND PENETRATION INTO CELLS 4.4 EFFECTS OF INTEGRIN LIGANDS ON WEST NILE VIRUS ENTRY INTO CELLS 4.5 130 136 GENE SILENCING OF HUMAN β3 INTEGRIN SUBUNITS INHIBITS WEST NILE VIRUS. .. PROTEIN DOMAIN III RECOGNIZED BY MONO-SPECIFIC ENVELOPE ANTIBODY 4.11 COMPETITIVE INHIBITION OF WEST NILE VIRUS ENTRY WITH SOLUBLE RECOMBINANT WEST NILE VIRUS- DIII PROTEIN 4.12 151 MURINE POLYCLONAL ANTIBODY TO RECOMBINANT WEST NILE VIRUS- DIII NEUTRALIZED WEST NILE VIRUS 4.13 150 154 RECOMBINANT WEST NILE VIRUS E DIII BINDS TO αVβ3 INTEGRIN AND PREVENTS WEST NILE VIRUS ENTRY 156 CHAPTER 5 160 5.0 THE GRAND... PENETRATION INHIBITION ASSAY USING FUNCTIONAL BLOCKING INTEGRIN ANTIBODIES AND LIGANDS 96 2.17.1 Inhibition of Virus Entry with Soluble Integrin 96 2.17.2 Gene Silencing of Integrin β3 97 ix TABLE OF CONTENTS 2.17.3 Inhibition of West Nile Virus Entry by Soluble WNV E DIII Protein 98 2.17.4 Generation of Murine Polyclonal Antibodies Against Soluble West Nile Virus E DIII Protein 2.18 ANALYSES OF VIRUS MATURATION... Trafficking Endosomes to Lysosomes Containing Virus Particles 5.4.3 180 PKC Isoforms are Responsible for West Nile Virus Entry 183 5.5 LOW pH-DEPENDENT ENTRY OF WEST NILE VIRUS 5.6 INVOLVEMENT OF ACTIN FILAMENTS AND MICROTUBULES NETWORK IN WEST NILE VIRUS ENTRY PATHWAY 5.7 185 188 REAL-TIME IMAGING OF THE ENDOCYTIC PATHWAY OF WEST NILE VIRUS USING LASER SCANNING CONFOCAL MICROSCOPY 194 xii TABLE OF CONTENTS... 6.5 VIRUS STRUCTURAL PROTEINS ASSOCIATE WITH ACTIN FILAMENTS AT CELL PLASMA MEMBRANE 6.5.1 6.5.2 219 Budding of West Nile Virus at the Plasma Membrane is Associated with Actin Filaments 226 Disruption of Actin Filaments Inhibits West Nile Virus Budding at the Plasma Membrane CHAPTER 7 7.0 218 227 233 INTERPLAY OF VIRUS AND CELLULAR COMPONENTS DURING WEST NILE VIRUS – INDUCED CELL DEATH 7.1 233 INTRODUCTION... 201 HOST CYTOSKELETON, THE FACILITATOR OF WEST NILE VIRUS MORPHOGENESIS 201 6.1 INTRODUCTION 201 6.2 TRAFFICKING OF NEWLY SYNTHESIZED VIRUS STRUCTURAL PROTEINS ALONG MICROTUBULE NETWORK 201 6.2.1 Type of Association of Virus E Proteins with Microtubules 205 6.2.2 Effect of Microtubule-Disrupting Drug on the Trafficking Mechanism of E and C Proteins of West Nile Virus 6.3 207 KINESIN, A PUTATIVE MICROTUBULE-BASED... MOTOR PROTEIN RESPONSIBLE FOR VIRUS ENVELOPE PROTEIN TRAFFICKING ALONG THE MICROTUBULES 6.4 210 APICAL RELEASE OF WEST NILE VIRUS IS DEPENDENT ON PROTEIN SORTING ALONG MICROTUBULE NETWORK IN POLARIZED CELLS 213 6.4.1 Apical Egression of West Nile Virus from Polarized C1008 Cells 213 6.4.2 Polarized Sorting of West Nile Virus E Protein to the Apical Domain of Vero C1008 Cells Is Dependent on Intact Microtubule... silencing of human β3 integrin reduced WNV entry 141 Figure 30 Specific interactions between soluble αVβ3 integrin and WNV prevent virus infection Figure 31 143 Expression of αVβ3 integrin in CS-1 cells increased its permissiveness for WNV 145 Figure 32 West Nile virus activates the integrin-dependent FAK 148 Figure 33 Activation of FAK auto-phosphorylation via the engagement of WNV with αVβ3 integrin . GENE SILENCING OF HUMAN β3 INTEGRIN SUBUNITS INHIBITS WEST NILE VIRUS ENTRY 138 4.6 SOLUBLE INTEGRIN αVβ3 INHIBITS WEST NILE VIRUS ENTRY 142 4.7 EXPRESSION OF INTEGRIN αVβ3 INCREASES THE SUSCEPTIBILITY. FUNCTIONAL BLOCKING ANTIBODIES AGAINST αVβ3 INTEGRIN INHIBIT WEST NILE VIRUS BINDING AND PENETRATION INTO CELLS 130 4.4 EFFECTS OF INTEGRIN LIGANDS ON WEST NILE VIRUS ENTRY INTO CELLS 136. Disruption of Actin Filaments Inhibits West Nile Virus Budding at the Plasma Membrane 227 CHAPTER 7 233 7.0 INTERPLAY OF VIRUS AND CELLULAR COMPONENTS DURING WEST NILE VIRUS – INDUCED CELL