published_or_final_version / Microbiology / Master / Master of Philosophy

Ho Hei Ming. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 189-199). / Abstracts in English and Chinese. / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Epidemiology of the Severe Acute Respiratory Syndrome (SARS) Outbreak --- p.2 / Chapter 1.2 --- The SARS Coronavirus --- p.3 / Chapter 1.2.1 --- Genome organization --- p.7 / Chapter 1.2.2 --- Structural proteins --- p.9 / Chapter 1.2.3 --- Non-structural proteins --- p.11 / Chapter 1.3 --- Introduction to SARS-CoV nsp10 Protein --- p.14 / Chapter 1.4 --- Introduction to SARS-CoV RNA-dependent RNA Polymerase (RdRp) Protein --- p.17 / Chapter 1.5 --- Objectives of the Present Study --- p.25 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Construction of Glutathione S-Transferase (GST) Fusion/Green Fluorescence Protein (GFP) N1 and C1 Fusion nsplO --- p.26 / Chapter 2.1.1 --- Primer design --- p.26 / Chapter 2.1.2 --- Gene amplification by PCR --- p.28 / Chapter 2.1.3 --- Purification of PCR product --- p.30 / Chapter 2.1.4 --- Enzyme restriction --- p.31 / Chapter 2.1.5 --- Ligation --- p.33 / Chapter 2.1.6 --- Transformation --- p.34 / Chapter 2.1.6.1 --- Preparation of competent cell DH5α --- p.34 / Chapter 2.1.7 --- Mini scale plasmid preparation --- p.36 / Chapter 2.2 --- Subcellular Localization Study --- p.39 / Chapter 2.2.1 --- Midi scale plasmid preparation --- p.39 / Chapter 2.2.2 --- Transfection of GFP recombinant plasmids --- p.41 / Chapter 2.2.2.1 --- Cell culture of Vero E6 cell line --- p.41 / Chapter 2.2.2.2 --- Lipofectamine based transfection --- p.41 / Chapter 2.2.3 --- Fluorescent microscopic visualization --- p.42 / Chapter 2.2.4 --- Western blotting for GFP fusion protein expression --- p.43 / Chapter 2.2.4.1 --- Protein extraction --- p.43 / Chapter 2.2.4.2 --- Protein quantification --- p.44 / Chapter 2.2.3.4 --- SDS-PAGE analysis --- p.45 / Chapter 2.3 --- "Expression of GFP-nsp10 in Vero E6 cells, SARS-CoV Infected Vero E6 Cells and Convalescent Patients' Serum" --- p.47 / Chapter 2.3.1 --- Cell-based immunostaining of VeroE6 cells and SARS-CoV infected Vero E6 cells --- p.47 / Chapter 2.3.1.1 --- Immobilization of Vero E6 cells and SARS-CoV infected Vero E6 cells --- p.47 / Chapter 2.3.1.2 --- Preparation of monoclonal antibodies against SARS-CoV nsp10 --- p.48 / Chapter 2.3.1.3 --- Immunostaining of SARS-CoV nsp10 in Vero E6 cells and SARS-CoV VeroE6 cells --- p.48 / Chapter 2.3.1.4 --- Fluorescent microscopic visualization --- p.49 / Chapter 2.3.2 --- Detection of SARS-CoV nsplO expression in SARS-CoV infected convalescent patients' serum --- p.50 / Chapter 2.3.2.1 --- Western blotting of SARS-CoV nsp10 by SARS-CoV infected convalescent patients' serum --- p.50 / Chapter 2.4 --- Expression of GST fusion SARS-CoV nsp10 in E.coli --- p.51 / Chapter 2.4.1 --- Preparation of competent cells --- p.51 / Chapter 2.4.2 --- Small scale expression --- p.51 / Chapter 2.4.3 --- Large scale expression of GST-nsp10 in optimized conditions --- p.54 / Chapter 2.5 --- Purification of GST fusion SARS-CoV nsp10 --- p.55 / Chapter 2.5.1 --- Glutathione Sepharose 4B affinity chromatography --- p.55 / Chapter 2.5.2 --- Superdex 75 gel filtration chromatography --- p.56 / Chapter 2.6 --- "CD Measurement, NMR and Crystallization Study of SARS-CoV nsp10" --- p.57 / Chapter 2.6.1 --- CD measurement --- p.57 / Chapter 2.6.2 --- NMR spectroscopy --- p.58 / Chapter 2.6.3 --- Crystallization of nsp10 --- p.58 / Chapter 2.7 --- "Glutathione-S-Sepharose Pull-down assay, 2D Gel Electrophoresis and Mass Spectrometry" --- p.59 / Chapter 2.7.1 --- GST pull-down assay --- p.59 / Chapter 2.7.2 --- Two-dimension gel electrophoresis --- p.59 / Chapter 2.7.2.1 --- First dimensional isoelectric focusing (IEF) --- p.59 / Chapter 2.7.2.2 --- Second dimension SDS-PAGE --- p.60 / Chapter 2.7.2.3 --- Silver staining --- p.61 / Chapter 2.7.3 --- Protein identification by mass spectrometry --- p.63 / Chapter 2.7.3.1 --- Data acquisition --- p.65 / Chapter 2.8 --- Proliferative study of SARS-CoV nsp10 in VeroE6 Cell Line and Mouse Splenocytes --- p.66 / Chapter 2.8.1 --- Assay of mitogenic activity by 3H-thymidine incorporation --- p.66 / Chapter 2.9 --- "Cloning, Expression and Purification of GST fusion SARS-CoV RNA-dependent RNA Polymerase (RdRp) Full- length Protein" --- p.67 / Chapter 2.9.1 --- Construction of GST-RdRp-full length expression plasmid --- p.67 / Chapter 2.9.2 --- Expression and purification of GST-RdRp full-length protein --- p.68 / Chapter 2.10 --- "Cloning, Expression and Purification of GST Fusion SARS-CoV RNA-dependent RNA Polymerase (RdRp) Catalytic Domain" --- p.70 / Chapter 2.10.1 --- Construction of GST-RdRp Catalytic Domain (p64) and MBP-RdRp-p64 expression plasmids --- p.70 / Chapter 2.10.2 --- Expression and purification of GST fusion catalytic domain of SARS-CoV RdRp (GST-p64) --- p.71 / Chapter 2.10.3 --- Expression and purification of MBP fusion catalytic domain of SARS-CoV RdRp --- p.72 / Chapter 2.11 --- "Cloning, Expression and Purification of the His-thioredoxin Fusion N-terminal Domain of SARS-CoV RdRp (pET32h-pl2)" --- p.74 / Chapter 2.11.1 --- Construction of His-thioredoxin fusion N-terminal domain of SARS-CoV RdRp (pET32h-pl2) expression plasmid --- p.74 / Chapter 2.11.2 --- Expression and purification of His- thioredoxin fusion N-terminal domain of SARS-CoV RdRp (pET32h-pl2) --- p.74 / Chapter 2.12 --- Interaction Study of RdRp Catalytic Domain and N-terminal Domain --- p.76 / Chapter 2.13 --- Electrophoretic Mobility Shift Assay of SARS-CoV Genomic RNA Strands with RdRp Full-length sequence --- p.76 / Chapter 2.13.1 --- Preparation of RNA transcripts --- p.76 / Chapter 2.13.2 --- EMSA --- p.77 / Chapter 2.14 --- Non-radiometric and Radiometric RdRp Assays --- p.78 / Chapter 2.14.1 --- Non-radiometric RdRp assay--luciferase coupled enzyme assay --- p.78 / Chapter 2.14.2 --- Radiometric RdRp assay ´ؤ filter-binding enzyme assay --- p.79 / Chapter 2.15 --- Western Blot Analysis for Interaction Study --- p.80 / Chapter Chapter 3 --- Results and Discussion on SARS-CoV nsplO --- p.81 / Chapter 3.1 --- "Cloning, Expression and Purification of SARS-CoV nsp10 in Prokaryotic Expression System" --- p.81 / Chapter 3.1.1 --- Cloning and expression of SARS-CoV nsp 10 --- p.81 / Chapter 3.1.2 --- Purification of GST-nsp10 by GST affinity chromatography --- p.84 / Chapter 3.1.3 --- Purification of nsp10 by size exclusion chromatography --- p.85 / Chapter 3.1.4. --- "Yield, purity and stability of SARS-CoV nsp 10" --- p.88 / Chapter 3.2 --- SARS-CoV nsp10 Sequence Alignment and Protein Structure Prediction --- p.89 / Chapter 3.2.1. --- Sequence alignment of SAR-CoV nsp10 with known viral proteins --- p.91 / Chapter 3.2.2 --- Protein structure prediction - homology modeling --- p.93 / Chapter 3.3 --- Circular Dichroism Analysis of nsp10 --- p.96 / Chapter 3.3.1 --- CD spectrum of SARS-CoV nsp10 --- p.98 / Chapter 3.3.2. --- Effect of divalent metal ions on SARS-CoV nsp10 --- p.99 / Chapter 3.4 --- Nuclear Magnetic Resonance Analysis of nsp10 --- p.101 / Chapter 3.4.1 --- Sample preparation for NMR Experiment --- p.102 / Chapter 3.4.2 --- Protein structure determination by NMR --- p.103 / Chapter 3.5 --- Crystallization of SARS-CoV nsp10 --- p.105 / Chapter 3.5.1 --- Sample preparation of nsp10 for crystallization --- p.105 / Chapter 3.5.2 --- Screening conditions for crystallization --- p.106 / Chapter 3.6 --- "Antigenic, Immunofluorescene and Subcellular Localization Studies on the SARS-CoV nsp10" --- p.110 / Chapter 3.6.1 --- Antigenic and immunofluorescene studies on the SARS-CoV nsp10 --- p.110 / Chapter 3.6.2 --- Subcellular localization of SARS-CoV nsp10 --- p.115 / Chapter 3.7 --- Proliferative Study of nsp10 --- p.120 / Chapter 3.7.1. --- Influence of proliferative effect on the host cell --- p.121 / Chapter 3.8 --- A Proteomics Strategy for Interaction Study of nsp10 --- p.124 / Chapter 3.8.1 --- 2D SDS-PAGE analysis of proteins associating with the nsp10 bait --- p.125 / Chapter 3.8.2 --- Silver staining of proteins associating with the nsp10 bait and their identification by mass spectrometry --- p.127 / Chapter 3.9 --- Discussion on SARS-CoV nsp10 --- p.129 / Chapter Chapter 4 --- Results and Discussion on SARS-CoV RdRp / Chapter 4.1 --- "Cloning, Expression and Purification of SARS-CoV RdRp Full-length, Catalytic Domain and N-terminal Domain" --- p.139 / Chapter 4.2 --- Interaction Study of RdRp Catalytic Domain and its N-terminal Domain --- p.147 / Chapter 4.3 --- Functional Analysis of RNA Binding by the SARS-CoV RdRp --- p.149 / Chapter 4.4 --- Characterization of RdRp by Non-radioactive RdRp Assay ´ؤ Luciferase-coupled Enzyme Assay --- p.152 / Chapter 4.5 --- Characterization of RdRp by Radioactive RdRp Assay ´ؤ 32P Incorporation Assay --- p.157 / Chapter 4.6 --- Discussion on SARS-CoV RdRp --- p.161 / Chapter Chapter 5 --- General Discussion / General Discussion --- p.170 / Appendix --- p.172 / References --- p.189

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

Chong Lin-Tat. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 76-78). / Abstracts in English and Chinese. / Chapter Chapter 1 --- introduction / Chapter 1.1 --- Severe acute respiratory syndrome Coronavirus (SARS CoV) --- p.13 / Figure 1.1 Genome organization and putative functional ORFs of SARS CoV --- p.14 / Chapter 1.2 --- SARS main protease / Chapter 1.2.1 --- Three dimensional structure --- p.15 / Figure 1.2 Ribbon illustration of the SARS-coronavirus main protease --- p.17 / Figure 1.3 Surface representations of P1 and P2 substrate-binding pocket of main protease --- p.18 / Chapter 1.2.2 --- Substrate specificities --- p.19 / Table 1.1. Eleven predicted cleavage sites of SARS CoV main protease --- p.21 / Chapter 1.3 --- Protein-based FRET assay system --- p.22 / Figure 1.4. The principle of fluorescent resonance energy transfer (FRET) --- p.24 / Chapter 1.4 --- Objectives --- p.25 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- General Techniques / Chapter 2.1.1 --- Preparation and transformation of competent E. coli DH5a and23 BL21 (DE3)pLysS --- p.26 / Chapter 2.1.2 --- Minipreparation of plasmid DNA (Invitrogen) --- p.27 / Chapter 2.1.3 --- Spectrophotometric quantitation DNA --- p.28 / Chapter 2.1.4 --- Agarose gel electrophoresis / Chapter 2.1.5 --- Purification of DNA from agarose gel (Invitrogen) / Chapter 2.1.6 --- Restriction digestion of DNA fragments --- p.29 / Chapter 2.1.7 --- Ligation of DNA fragments into vector / Table 2.1. Standard recipe of ligation reaction --- p.30 / Chapter 2.1.8 --- SDS-PAGE electrophoresis --- p.31 / Table 2.2. Standard recipe of separating gel for SDS-PAGE --- p.32 / Table 2.3. Standard recipe of stacking gel for SDS-PAGE --- p.33 / Chapter 2.2 --- Sub-cloning and site-directed mutagenesis / Chapter 2.2.1 --- Sub-cloning of SARS Co V main protease --- p.34 / Chapter 2.2.2 --- Sub-cloning of Substrate / Chapter 2.2.3 --- Site-directed mutagenesis of substrate variant --- p.35 / Table 2.4 Primer sequence for generating substrate variants --- p.36 / Table 2.5. Standard recipe of Polymerase Chain Reaction (PCR) --- p.40 / Table 2.6. Polymerase Chain Reaction (PCR) profile --- p.41 / Chapter 2.3 --- Sample preparation / Chapter 2.3.1 --- Expression of recombinant proteins --- p.42 / SARS CoV main protease / Substrate and substrate variants / Chapter 2.3.2 --- Purification of recombinant proteins / SARS CoV main protease / Substrate and substrate variants / Chapter 2.4 --- Protein-based FRET kinetic analysis --- p.45 / Chapter 2.5 --- A model for substrate-enzyme binding by docking simulation --- p.46 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Preparation of SARS CoV main protease and substrate / Chapter 3.1.1 --- Expression and purification of SARS main protease --- p.48 / Figure 3.1. Purification profile of SARS CoV main protease --- p.49 / Chapter 3.1.2 --- Expression and purification of substrate and substrate variants --- p.50 / Figure 3.2. Purification profile of substrate and substrate variants --- p.51 / Chapter 3.2 --- A novel protein-based FRET assay system was established / Chapter 3.2.1 --- "With the cleavage of active main protease, absorbance at 528nm dropped while signal at 485nm were slightly increased" --- p.52 / Figure 3.3. Absorbance at 528nm dropped and 485nm increased with the substrate hydrolysis --- p.53 / Chapter 3.2.2 --- FRET efficiency ratio (528/485) decreased over time --- p.54 / Figure 3.4. FRET efficiency ratio (528/485) decreased over time --- p.55 / Chapter 3.2.3 --- Comparable kcat/Km value of SARS CoV main protease was obtained --- p.56 / Figure 3.5. Catalytic parameter (kcat/ Km) was determined from the slope of straight Line --- p.57 / Chapter 3.3 --- Main protease activity towards substrate variants at different substrate-binding sites (S2'-S2) --- p.58 / Table 3.1. Kinetic parameterrs of 76 substrate variants in descending order --- p.59 / Chapter 3.3.1 --- S2'substrate-binding site --- p.60 / Chapter 3.3.2 --- S1' substrate-b inding site / Chapter 3.3.3 --- S1 substrate-binding site / Chapter 3.3.4 --- S2 substrate-binding site / Figure 3.6. Kinetic analysis of some typical substrate variants against main protease --- p.62 / Figure 3.7. SDS-PAGE analysis of some typical substrate variants against main protease --- p.63 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Quantitative and high-throughput analysis by protein-based FRET assay system --- p.64 / Chapter 4.2 --- Substrate specificities of SARS CoV main protease at S2'-S2 subsites / Chapter 4.2.1 --- β-strand conformation was preferred at S2，subsite / Chapter 4.2.2 --- Residues with small aliphatic side chain were preferred at S1 ´ة subsite --- p.65 / Chapter 4.2.3 --- "Glutamine at S1 subsite was absolutely conserved, but alternatives were disclosed" --- p.66 / Figure 4.1. Glutamine was not absolutely conserved in S1 subsite --- p.67 / Chapter 4.2.4 --- Hydrophilic residues were tolerated at S2 subsite --- p.68 / Figure 4.2. Hydrophilic residues were tolerated at S2 subsite --- p.70 / Table 4.1. Summary of types of residues preferred at individual subsites --- p.71 / Chapter 4.3 --- Predicted conformation of substrate towards SARS CoV main protease at S2' and S1' subsites --- p.72 / Figure 4.3. Small residues were preferred at S1´ة subsite and Val at S2' subsite was more favoured than the native one --- p.73 / Chapter Chapter 5 --- Summary --- p.74 / Chapter Chapter 6 --- Future work --- p.75 / References --- p.76

The biological function of RNA is often linked to an ability to adopt one or more mutually exclusive conformational states or isomers, a characteristic that distinguishes this biomolecule from proteins. Two examples of conformationally inconverting RNAs were structurally investigated. The first is found in the 3' untranslated region (UTR) of the coronavirus mouse hepatitis virus (MHV). A proposed molecular switch between mutually exclusive stable stem loop and pseudoknot conformations was investigated using thermal unfolding methods, NMR spectroscopy, sedimentation velocity ultracentrifugation and fluorescence resonance energy transfer (FRET) spectroscopy. Utilizing a "divide and conquer" approach we establish that the independent subdomains are folded as predicted by the proposed model and that a pseudoknotted conformation is accessible. Using the subdomains as spectral markers for the investigation of the intact 3' UTR RNA, we show that the 3' UTR is indeed a superposition of a double stem conformation and a pseudoknotted conformation in the presence of KCl and MgCl2. In the absence of added salt however, the 3' UTR adopts exclusively the double stem conformation. Analysis of the pseudoknotted stem reveals only a marginally stable folded state (deltaG25 = 0.5 kcal mol-1, tm = 31 oC) which makes it likely that a viral or host encoded protein(s) is required to stabilize the pseudoknotted conformation. A second conformationally interconverting RNA system investigated is an RNA element that stimulates -1 programmed ribosomal frameshifting in the human Ma3 gene. Structural analysis of the frameshifting element reveals a dynamic equilibrium between a functionally inactive double stem loop conformation and the active pseudoknotted conformation. Thermal melting and NMR spectroscopy reveal that the double stem loop is the predominant conformation in the absence of added KCl or MgCl2. The addition of KCl and MgCl2 results in the formation of a pseudoknot conformation. This conformation is dominant in solution only when the competing double stem loop conformation is abrogated by mutation. Functional studies of the Ma3 pseudoknot reveal that abrogation of double stem conformation increases frameshift stimulation by 2-fold and indicates that the pseudoknot is the active conformation.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Tight junctions, as zones of close contact between epithelial and endothelial cells, form a physical barrier as one of the first host defense strategies that prevent the intrusion of pathogens across epithelia and endothelia. Recently, an interaction between the Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) envelope protein (E) and PALS1, a member of the CRB tight junction complex, was identified in the Virus-Host Interaction group at HKU-Pasteur Research Centre (Teoh et al, 2010). In this report, I present in vitro data which helps to better understand how this protein-protein interaction could interfere with the formation and maintenance of tight junctions at the apical domain of epithelial cells. In previous research, the interaction between E and PALS1 was identified through a yeast two-hybrid screen and confirmed in vitro. A PDZ-binding motif (PBM) was identified at the C-terminal end of E, which interacts with the PDZ domain of PALS1. The objective of my research was to further enhance the knowledge of this interaction by studying the effect of E expression on PALS1 localization and tight junction structure in epithelial cells. I have shown that expression of E is associated with a partial relocalization of PALS1 to the Golgi compartment. Also, I discovered that when wild-type E, E(wt), was expressed in the MDCKII cell model, the time required for tight junction formation was extended to 6-8 hours, while normal cells only required two hours. Interestingly, expression of the E protein with a deletion of the PBM, E(ΔPBM) did not affect the timing of tight junction formation. This finding indicates that the PBM plays a critical role in the process of alteration of tight junctions mediated by E, most likely through its interaction with PALS1. Furthermore, the localization pattern of E was altered when its PBM was deleted. In the MDCKII model, E(wt) located, as expected, at membranes of the Golgi compartment, whereas E(ΔPBM) had a diffused distribution in the cytosol. This observation suggests that the PBM acts as a localization signal for the E protein to the Golgi region, which is the assembly site of the virus. Finally, to examine the role of the PBM in the context of the whole virus, I participated in the production of SARS-CoV recombinant viruses, with mutations in the PBM of E. Though this work is still in progress, the use of these viruses should help to delineate the role of E PBM in SARS-CoV induced pathogenesis in vitro and ultimately in vivo. / published_or_final_version / Pathology / Master / Master of Philosophy

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 83-101) Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 83-101) Also available online.