Chu Ling Hon Matthew. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 201-223). / Abstracts in English and Chinese. / Declaration --- p.i / Thesis/Assessment Committee --- p.ii / Abstract --- p.iii / 摘要 --- p.vi / Acknowledgements --- p.viii / General abbreviations --- p.xi / Abbreviations of chemicals --- p.xv / Table of Contents --- p.xvi / List of Figures --- p.xxiii / List of tables --- p.xxviii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Severe Acute Respiratory Syndrome (SARS) - Three Years in Review --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.1 / Chapter 1.1.2 --- Clinical presentation --- p.3 / Chapter 1.1.3 --- Diagnostic tests --- p.5 / Chapter 1.2 --- Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) --- p.7 / Chapter 1.2.1 --- SARS - Identification of the etiological agent --- p.7 / Chapter 1.2.2 --- The coronaviruses --- p.9 / Chapter 1.2.3 --- The genome organization of SARS-CoV --- p.11 / Chapter 1.2.4 --- The life cycle of SARS-CoV --- p.13 / Chapter 1.3 --- Spike Glycoprotein (S protein) of SARS-CoV --- p.15 / Chapter 1.3.1 --- SARS-CoV S protein --- p.15 / Chapter 1.3.2 --- S protein-driven infection --- p.17 / Chapter 1.4 --- SARS-CoV S Protein Fusion Core --- p.22 / Chapter 1.4.1 --- Heptad repeat and coiled coil --- p.22 / Chapter 1.4.2 --- The six-helix coiled coil bundle structure --- p.25 / Chapter 1.5 --- 3C-like Protease (3CLpro) of SARS-CoV --- p.28 / Chapter 1.5.1 --- Extensive proteolytic processing of replicase polyproteins --- p.28 / Chapter 1.5.2 --- SARS-CoV 3CLpro --- p.30 / Chapter 1.5.3 --- Substrate Specificity of SARS-CoV 3CLpro --- p.31 / Chapter 1.6 --- SARS Drug Development --- p.32 / Chapter 1.6.1 --- Drug targets of SARS-CoV --- p.32 / Chapter 1.6.2 --- Current anti-SARS drugs --- p.36 / Chapter 1.7 --- Project Objectives --- p.39 / Chapter 1.7.1 --- Characterization of SARS-CoV S protein fusion core --- p.39 / Chapter 1.7.2 --- Characterization of SARS-CoV 3CLpr0 substrate specificity --- p.40 / Chapter 2 --- Materials and Methods --- p.42 / Chapter 2.1 --- Characterization of SARS-CoV S Protein Fusion Core --- p.42 / Chapter 2.1.1 --- Bioinformatics analyses of heptad repeat regions of SARS- CoV S protein --- p.42 / Chapter 2.1.2 --- Recombinant protein approach --- p.43 / Chapter 2.1.2.1 --- Plasmids construction --- p.43 / Chapter 2.1.2.2 --- Protein expression and purification --- p.52 / Chapter 2.1.2.3 --- Amino acid analysis --- p.57 / Chapter 2.1.2.4 --- GST-pulldown experiment --- p.58 / Chapter 2.1.2.5 --- Laser light scattering --- p.61 / Chapter 2.1.2.6 --- Size-exclusion chromatography --- p.62 / Chapter 2.1.2.7 --- Circular dichroism spectroscopy --- p.62 / Chapter 2.1.3 --- Synthetic peptide approach --- p.64 / Chapter 2.1.3.1 --- Peptide synthesis --- p.64 / Chapter 2.1.3.2 --- Native polyacrylamide gel electrophoresis --- p.65 / Chapter 2.1.3.3 --- Size-exclusion high-performance liquid chromato-graphy --- p.66 / Chapter 2.1.3.4 --- Laser light scattering --- p.66 / Chapter 2.1.3.5 --- Circular dichroism spectroscopy --- p.67 / Chapter 2.2 --- Identification of SARS-CoV Entry Inhibitors --- p.70 / Chapter 2.2.1 --- HIV-luc/SARS pseudotyped virus entry inhibition assay --- p.70 / Chapter 2.2.2 --- Recombinant protein- and synthetic peptide-based biophysical assays --- p.74 / Chapter 2.2.3 --- Molecular modeling --- p.75 / Chapter 2.3 --- Characterization of SARS-CoV 3CLpro Substrate Specificity --- p.79 / Chapter 2.3.1 --- Protein expression and purification --- p.79 / Chapter 2.3.2 --- """Cartridge replacement"" solid-phase peptide synthesis" --- p.80 / Chapter 2.3.3 --- Peptide cleavage assay and mass spectrometric analysis --- p.83 / Chapter 3 --- Results --- p.84 / Chapter 3.1 --- Characterization of SARS-CoV S Protein Fusion Core --- p.84 / Chapter 3.1.1 --- Bioinformatics analyses of heptad repeat regions of SARS- CoV S protein --- p.84 / Chapter 3.1.2 --- Recombinant protein approach --- p.87 / Chapter 3.1.2.1 --- "Plasmids construction of pET-28a-His6-HRl, pGEX-6P-l-HR2 and pGEX-6P-l-2-Helix" --- p.87 / Chapter 3.1.2.2 --- Protein expression and purification --- p.92 / Chapter 3.1.2.3 --- GST-pulldown experiment --- p.101 / Chapter 3.1.2.4 --- Laser light scattering --- p.103 / Chapter 3.1.2.5 --- Size-exclusion chromatography --- p.105 / Chapter 3.1.2.6 --- Circular dichroism spectroscopy --- p.107 / Chapter 3.1.3 --- Synthetic peptide approach --- p.112 / Chapter 3.1.3.1 --- Peptide synthesis --- p.112 / Chapter 3.1.3.2 --- Native polyacrylamide gel electrophoresis --- p.116 / Chapter 3.1.3.3 --- Size-exclusion high-performance liquid chromatography --- p.117 / Chapter 3.1.3.4 --- Laser light scattering --- p.122 / Chapter 3.1.3.5 --- Circular dichroism spectroscopy --- p.124 / Chapter 3.2 --- Identification of SARS-CoV Entry Inhibitors --- p.129 / Chapter 3.2.1 --- HIV-luc/SARS pseudotyped virus entry inhibition assay --- p.129 / Chapter 3.2.2 --- Recombinant protein- and synthetic peptide-based biophysical assays --- p.131 / Chapter 3.2.3 --- Molecular modeling --- p.135 / Chapter 3.3 --- Characterization of SARS-CoV 3CLpro Substrate Specificity --- p.141 / Chapter 3.3.1 --- Protein expression and purification --- p.141 / Chapter 3.3.2 --- Substrate specificity preference of SARS-CoV 3CLpr0 --- p.142 / Chapter 3.3.3 --- "Primary and secondary screening using the ""cartridge replacement strategy""" --- p.142 / Chapter 4 --- Discussion --- p.149 / Chapter 4.1 --- Characterization of SARS-CoV S Protein Fusion Core --- p.149 / Chapter 4.1.1 --- Design of recombinant proteins and synthetic peptides of HR regions --- p.149 / Chapter 4.1.2 --- Recombinant protein approach --- p.151 / Chapter 4.1.3 --- Synthetic peptide approach --- p.153 / Chapter 4.1.4 --- Summary of the present and previous studies in the SARS-CoV S protein fusion core --- p.157 / Chapter 4.2 --- Identification of SARS-CoV Entry Inhibitors --- p.167 / Chapter 4.2.1 --- HIV-luc/SARS pseudotyped virus entry inhibition assay --- p.167 / Chapter 4.2.2 --- Identification of peptide inhibitors --- p.168 / Chapter 4.2.3 --- Identification of small molecule inhibitors --- p.172 / Chapter 4.3 --- Characterization of SARS-CoV 3CLpro Substrate Specificity --- p.183 / Chapter 4.3.1 --- A comprehensive overview of the substrate specificity of SARS-CoV 3CLpro --- p.184 / Chapter 4.3.2 --- The development of the rapid and high-throughput screening strategy for protease substrate specificity --- p.188 / Appendix --- p.191 / Chapter I. --- Nucleotide Sequence of S protein of SARS-CoV --- p.191 / Chapter II. --- Protein Sequence of S protein of SARS-CoV --- p.194 / Chapter III. --- Protein Sequence of 3CLpro of SARS-CoV --- p.195 / Chapter IV. --- Vector maps --- p.196 / Chapter 1. --- Vector map and MCS of pET-28a --- p.196 / Chapter 2. --- Vector map and MCS of pGEX-6P-l --- p.197 / Chapter V. --- Electrophoresis markers --- p.198 / Chapter 1. --- GeneRuler´ёØ 1 kb DNA Ladder --- p.198 / Chapter 2. --- GeneRuler´ёØ 100bp DNA Ladder --- p.198 / Chapter 3. --- High-range Rainbow Molecular Weight Markers --- p.199 / Chapter 4. --- Low-range Rainbow Molecular Weight Markers --- p.199 / Chapter VI. --- SDS-PAGE gel preparation protocol --- p.200 / References --- p.201