IDENTIFICATION of a NOVEL CANDIDATE RECEPTOR for HUMAN RESPIRATORY SYNCYTL4L VIRUS SUBGROUP a by FARNOOSH TAYYARI M.Sc., The
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IDENTIFICATION OF A NOVEL CANDIDATE RECEPTOR FOR HUMAN RESPIRATORY SYNCYTL4L VIRUS SUBGROUP A by FARNOOSH TAYYARI M.Sc., The University of British Columbia, 2003 M.D., Mashhad University of Medical Sciences, Mashhad, Iran, 1997 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (PATHOLOGY AND LABORATORY MEDICINE) THE UNIVERSITY OF BRITISH COLUMBIA October, 2008 © Farnoosh Tayyari, 2008 Abstract Introduction: Respiratory syncytial virus (RSV) is an important pathogen, especially in children, the elderly, and immunocompromised individuals. Despite RSV being discovered decades ago, there is still no good treatment or prevention for RSV disease. The cell surface receptor for RSV is not known and identification of RSV receptor(s) will provide improved opportunities for understanding the pathogenesis of the viral disease and potential for discovering novel antiviral agents. Hypothesis: RSV infects cells via attachment to cell surface receptor(s) which can be identified by unbiased interrogation of cell membrane constituents and functionally characterized by blocking and competition experiments. Specific Aims: Chemical characterization of RSV receptor(s) by cell surface enzyme treatments, identifying candidate receptor(s), and confirming that any identified candidate has characteristics of a receptor were specific aims of the project. Methods: Chemical characteristics of RSV binding molecule(s) were investigated using enzyme digestion studies. Methods used for identification of candidate receptors included: co immunoprecipitation of candidate RSV receptors using whole virion; purification of RSV surface proteins (either by chromatography or by cloning), and virus overlay protein binding assay (VOPBA) combined with mass spectrometry (MS) and protein database searching. Neutralization experiments, in which cells were incubated with anti-candidate receptor antibodies prior to RSV exposure, and competition experiments, in which virus was pre incubated with purified candidate molecule prior to inoculation of cell cultures were performed. Results: The results of enzyme digestion studies showed that the RSV binding molecule is a non-glycosylated, non-glycosyiphosphatidylinositol-anchored protein. Experiments involving 11 co-immunoprecipitation of RSV receptor using whole virion, or purification of RSV surface proteins (either by chromatography or by cloning), were unsuccessful. By contrast, VOPBA combined with MS resulted in cell surface nucleolin being identified as a candidate RSV binding molecule, and was reproducible in several cell lines originating from different species. Neutralization and competition experiments showed decreased RSV infection in vitro. Conclusion: Nucleolin, expressed on the surface of multiple cell types from diverse species, was identified as a candidate receptor for RSV. Subsequent blocking and competition experiments showed evidence of nucleolin having characteristics of a functional receptor. These findings provide a basis for future work to investigate RSV-nucleolin interactions. 111 Table .of Contents Abstract ii Table of Contents iv List of Tables viii List of Figures ix List of Abbreviations x Acknowledgements xii Dedication xiii 1 II’ITRODUCTION 1 1.1 Cell Membranes 1 1.1.1 Membrane Architecture 2 1.1.1.1 The Lipid Bilayer 2 1.l.1.2LipidRafts 2 1.1.1.3 Glycolipids 3 1.1.1.4 Membrane Proteins 3 1.1.1.5 Glycoproteins 5 1.1.1.6 Cell Membrane Structure: Summary 5 1.2 Virus Receptors 5 1.3 Discovery of Virus Receptors 8 1.3.1 Identification of Candidate Receptors 8 1.3.1.1 Cell Surface Enzyme Treatments 9 1.3.1.2 Virus Overlay Protein Binding Assay (VOPBA) 9 1.3.1.3 Virus Overlay on Thin Layer Chromatography 10 1.3.1.4 Expression Cloning 10 1.3.1.5 Anti-receptor Antibodies 10 1.3.1.6 Immunoprecipitation (IP) of the Receptor Using Viral Proteins 11 1.3.1.7 A Clever Guess 11 1.3.2 Confirmation that the Candidate is the Receptor 12 1.3.3 Summary 13 1.4 Antivirals 14 1.4.1 Vaccines 14 1.4.2 Interferons 14 1.4.3 Chemical Compound Based Drugs 15 1.4.4 Biochemical Agents 15 1.4.5 Summary 18 1.5 RSV Overview 19 1.5.1 Historical Background 19 1.5.2 Classification 19 1.5.3 Structure 20 1.5.3.1 SHProtein 21 1.5.3.2 G Protein 21 1.5.3.3 F Protein 25 1.5.4 Replication 28 1.5.5 Attachment and Entry 30 1.5.5.1 Surfactant Proteins 31 1.5.5.2 GAGs 31 1.5.5.3ICAM-1 31 iv 1.5.5.4 Lipid Rafts. 32 1.5.5.5Actin 33 1.5.6 RSV Prevention and Management 33 1.5.6.1 Immunization 33 1.5.6.2 Immunoprophylaxis 34 15.6.3 Management 34 1.5.7 Summary 35 2 SCOPE OF THE THESIS 37 2.1 Hypothesis 37 2.2 Specific Aims 38 2.3 General Research Plan 38 3 METHODS 39 3.1 Cells 39 3.2 Trypan Blue Exclusion Test of Cell Viability 39 3.3 Preparation of RSV Stocks 41 3.4 RSV Plaque Assay 42 3.5 CVB3 Infection 42 3.6 CVB3 Plaque Assay 43 3.7 Cell Surface Enzyme Treatment 43 3.7.1 Trypsin 44 3.7.2 Glycosidases 45 3.7.3 P1-PLC 45 3.8 Protein Sample Preparation and Sodium Dodecyl Sulfate-Polyacralamide Gel Electrophoresis (SDS-PAGE) 46 3.8.1 Gel Electrophoresis of Native Proteins 47 3.8.2 Running Large Gels 47 3.9 Coomassie Blue Staining 49 3.10 Western Blot 49 3.11 Preparation of “Crude” Membrane Proteins 51 3.12 Preparation of “Enriched” Cell Surface Membrane Proteins 51 3.13 Preparation of Biotinylated Cell Surface Membrane Proteins 52 3.14 Caveolin-rich Light Membrane (CLM) Fractionation 54 3.15 Immunoprecipitation 55 3.15.1 Cell Lysis 55 3.15.2 Pre-Clearing 56 3.15.3 Coupling Antigen to Antibody 56 3.15.4 Precipitation of Immune Complexes 56 3.15.5 Dissociation and Analysis 57 3.16 Virus-Receptor Co-Immunoprecipitation 57 3.16.1 Co-immunoprecipitation of CAR Using CVB3 as Bait 57 3.16.2 Co-immunoprecipitation of an Unknown Receptor Using RSV as Bait 58 3.17 Chromatography for Purification of RSV F and G proteins 58 3.17.1 Cell Preparation 59 3.17.2 Column Preparation 59 3.17.2.1 HiTrapQ FF (GE Healthcare) 59 3.17.2.2 Lentil-lectin (GE Healthcare) 59 3.17.3 Processing of RSV-infected HEp-2 Cell Lysate 60 3.17.4 Running HiTrapQ FF Column 60 3.17.5 Running Lentil-Lectin Column 61 v 3.17.6 Concentrating and Spin-dialysing Purified RSV F Protein 62 3.18 RSV F Protein Cloning and Protein Expression 63 3.18.1.1 Growing and Purification of Plasmids 64 3.18.1.2 Restriction Enzyme Digestion 66 3.18.1.3 Agarose Gel Electrophoresis 66 3.18.1.4 Isolation of DNA from Agarose Gel 67 3.18.1.5 Polymerase Chain Reaction (PCR) 67 3.18.1.6 Insert-plasmid Vector Ligation 68 3.18.1.7 Transformation 69 3.18.1.8 Transient Transfection 69 3.18.2 Insertion of RSV F protein Excluding Its Signal Peptide or Extracellular Domain of RSV F Protein into pcDNA3. 1/V5-His-TOPO® 70 3.19 Pierce In-Gel® Chemiluminescent Detection 72 3.20 Virus Overlay Protein Binding Assay (VOPBA) 74 3.21 Sample Preparation for MS 75 3.22 MS Analysis 75 3.23 Enzyme Treatment of Membrane Proteins 76 3.23.1 Proteases 76 3.23.2 Glycosidases 77 3.24 Antibody Blocking Experiments 77 3.25 Competition Experiments 78 3.26 Flow Cytometry 78 3.27 Statistical Analysis 79 4 RESULTS 80 4.1 Cell Surface Enzyme Treatment 80 4.1.3 P1-PLC 86 4.2 Virus-Receptor Co-Immunoprecipitation 91 4.2.1 Immunoprecipitation of CVB3 Using Anti-enterovirus VP1 91 4.2.2 Co-immunoprecipitation of CAR Using CVB3 as Bait 91 4.2.3 Co-immunoprecipitation of an Unknown Receptor Using RSV as Bait 93 4.3 Purification of RSV F and G Proteins by Chromatography 93 4.4 Cloning RSV F Protein 97 4.5 VOPBA and In-Gel Chemiluminescent Detection 101 4.6 Membrane Protein Enzyme Treatment 108 4.7 Further Localization of the 100 kDa RSV VOPBA Signal 112 4.8 Antibody Blocking Experiments 112 4.9 Competiton Experiment: RSV Infection in the Presence of Purified Nucleolin 116 5 DISCUSSION 121 5.1 General Characteristics of RSV Receptor 121 5.1.1 Protein Digestion 122 5.1.2 Carbohydrate Digestion 123 5.1.3 Lipid Digestion 125 5.2 Feasibility of Using Proteomics for Identification of Candidate RSV Receptors 126 5.2.1 Immunoprecipitation Using RSV Virion 126 5.2.2 Immunoprecipitation Using Purified RSV Proteins 129 5.2.3 Production of Recombinant Epitope-tagged RSV F Protein 130 5.2.4 Summary: Immunoprecipitation Experiments 133 5.3 VOPBA 134 5.3.1. RSV VOPBA 138 vi . 5.4. Nucleolin Structure and Biology 139 5.5 Conclusion and Future Directions 146 References 151 Appendix A Maps of Vectors 161 Appendix B Sequences and Primers 162 Appendix C Sequencing Results 164 Appendix D MS Results 167 vii List of Tables Table 1- The Species of Origin and Description of Mammalian Cells Used in this Thesis 40 Table 2.- SDS-PAGE Details 48 Table 3- List and Source of Primary Antibodies Used for Western Blot 50 Table 4- HEp-2 Cell Viability after Enzyme Treatment 81 Table 5. Protein Identification, Score and Overall Coverage of Protein Hits Reported by MS and Highlighted in Appendix D 111 viii List of Figures . Figure 1. Map of RSV G protein 24 Figure 2 Map of RSV F0 Protein 26 Figure 3.