Molecular Biology of the SARS-Coronavirus (eBook)

Foreword 5
Preface 7
Contents 10
Part I: Viral Entry 13
Chapter 1: Cellular Entry of the SARS Coronavirus: Implications for Transmission, Pathogenicity and Antiviral Strategies 14
Introduction 14
The Spike Protein: Key to the Host Cell 15
The Attachment Factors DC-SIGN and DC-SIGNR: Enhancers or Inhibitors of SARS-CoV Infection? 17
The Two Faces of ACE2: SARS-CoV Receptor and Protector Against Lung Damage 18
The Structure of the Interface Between SARS-S and ACE2 19
Sequence Variations at the SARS-S/ACE2 Interface Might Impact Viral Transmission and Pathogenicity 20
The Human Coronavirus NL63 Uses ACE2 for Cellular Entry 21
SARS Versus NL63: A Correlation Between ACE2 Downregulation and Viral Pathogenicity? 21
Cleavage by Endosomal Cathepsin Proteases Activates SARS-S 23
Membrane Fusion is Driven by Conserved Elements Located in the S2 Subunit of the SARS Spike Protein 24
Conclusions 26
References 26
Chapter 2: The Cell Biology of the SARS Coronavirus Receptor, Angiotensin-Converting Enzyme 2 34
Introduction 34
Clues from Homologous Proteins 35
Regulation of ACE2 Expression on the Cell Surface 37
Proteolytic Cleavage Secretion 37
The Role of Membrane Microdomains 38
Conclusions and Future Perspectives 39
References 39
Chapter 3: Structural Molecular Insights into SARS Coronavirus Cellular Attachment, Entry and Morphogenesis 42
Structure of SARS Coronavirus (SARS-CoV) 43
Structure of the Coronavirus Spike 45
Viral Membrane Fusion in SARS-CoV 47
Cellular Attachment and Entry of SARS-CoV 47
References 52
Part II: Structures Involved in Viral Replication and Gene Expression 55
Chapter 4: RNA Higher-Order Structures Within the Coronavirus 5 and 3 Untranslated Regions and Their Roles in Viral Replica 56
Introduction 56
cis-Acting RNA Elements in Coronavirus Replication 57
The Transcription Regulatory Sequence 58
The 5 cis-Acting RNA Elements 59
The 3 cis-Acting RNA Elements 62
Proteins Binding to the 5 and 3 cis-Acting Elements 64
Future Directions 66
References 66
Chapter 5: Programmed -1 Ribosomal Frameshifting in SARS Coronavirus 71
Introduction 71
Programmed -1 Ribosomal Frameshifting 72
Programmed Frameshifting Rates and Virus Propagation 73
Different Models, Different Assay Systems, Different Results 74
The Biology of -1 PRF in SARS-CoV is Different 75
A Unique Feature of the SARS-CoV Frameshift Signal: A Three-Stemmed mRNA Pseudoknot 76
A Second PRF Signal in SARS-CoV? 77
Summary and Perspectives 78
References 78
Part III: Viral Proteins 81
Chapter 6: Expression and Functions of SARS Coronavirus Replicative Proteins 82
Introduction 82
Organization and Expression of the Coronavirus Replicase Gene 85
Functions and Activities of Replicase Gene-Encoded Nonstructural Proteins 86
ORF1a-Encoded Nonstructural Proteins 1-11 86
ORF1b-Encoded Nonstructural Proteins 12-16 93
Future Directions 97
References 99
Chapter 7: SARS Coronavirus Replicative Enzymes: Structures and Mechanisms 106
The ADP-Ribose-1-Phosphatase Domain 107
The nsp7-nsp8-nsp9-nsp10 Cistron 109
The nsp7 Protein 109
The nsp8 Protein 110
The nsp9 Protein 113
The nsp10 Protein 115
The Endoribonuclease, nsp15 116
Conclusion 118
References 119
Chapter 8: Quaternary Structure of the SARS Coronavirus Main Protease 122
Introduction 123
Molecular Biology of the SARS-CoV Polyproteins 123
Structure of the SARS-CoV Main Protease 124
Three-Dimensional Structure of the SARS-CoV Main Protease 124
Quaternary StructureMproQuaternary structure of the SARS-CoV Main Protease 126
Enzyme Activity-AssayMproactivity-assay for the SARS-CoV Main Protease 126
Catalytic MechanismMproCatalytic mechanism of the SARS-CoV Main Protease 129
Structure and Function of the SARS-CoV Papain-Like Protease 131
Conclusions 131
References 133
Chapter 9: The Nucleocapsid Protein of the SARS Coronavirus: Structure, Function and Therapeutic Potential 136
Introduction 136
N-Protein: Structure and Composition 137
Stability of the N-Protein 138
Posttranslational Modification 138
Localization of the N-Protein 140
Genome Encapsidation: Primary Function of a Viral Capsid Protein 140
Recognition and Binding with the Genomic RNA 141
Formation of the Capsid 141
Perturbation of Host Cellular Process by the N-Protein 142
Deregulation of Host Cell Cycle 142
Inhibition of Host Cell Cytokinesis 143
Inhibition of Host Cell Translation Machinery 144
Inhibition of Interferon Production 144
Modulation of TGFbeta Signaling Pathway 144
Upregulation of COX2 Production 145
Upregulation of AP1 Activity 146
Induction of Apoptosis 146
Upregulation of Prothrombinase (hfgl2) Gene Transcription 147
Association with Host Cell Proteins 147
N-Protein: An Efficient Diagnostic Tool 148
N-Protein: A Suitable Vaccine Candidate 150
Future Perspective 151
References 153
Chapter 10: SARS Coronavirus Accessory Gene Expression and Function 159
Introduction 159
SARS-CoV Accessory Gene Expression and Function 162
ORF3a and ORF3b 162
ORF6 163
ORF7a and ORF7b 164
ORF8a and ORF8b 166
ORF9b 168
Conclusions 168
References 169
Chapter 11: SARS Accessory Proteins ORF3a and 9b and Their Functional Analysis 173
The SARS-CoV ORF3a Protein 173
Functional Analysis of ORF3a 175
Ion-Channel Activity of the ORF3a Homologue in Other Human Coronavirus 176
The SARS-CoV ORF9b Protein 178
Functional Role of ORF9b 179
SARS-CoV Accessory Proteins: An Important Part of the Virus Genome 179
References 180
Chapter 12: Molecular and Biochemical Characterization of the SARS-CoV Accessory Proteins ORF8a, ORF8b and ORF8ab 182
Introduction 182
Genetic Variations in the ORF8 Region 184
Expression of ORF8a, ORF8b and ORF8ab During Infection 185
Expression During Infection In Vivo 185
Expression During Infection In Vitro 186
Cellular Localization and Posttranslational Modification of ORF8a, ORF8b and ORF8ab 187
Cellular Localization 187
Glycosylation 187
Ubiquitination 188
Participation of ORF8a, ORF8b and ORF8ab in Viral-Viral Interactions and Their Effects on Other SARS-CoV Proteins 188
Interaction of ORF8a, ORF8b and ORF8ab with Other Viral Proteins 188
Downregulation of the E Protein by ORF8b 189
Impact of ORF8a, ORF8b and ORF8ab on Viral Replication and/or Pathogenesis 190
Contribution to Viral Replication 190
Abilities to Modulate Cellular Events 191
Conclusion 192
References 192
Part IV: Viral Pathogenesis and Host Immune Response 197
Chapter 13: SARS Coronavirus Pathogenesis and Therapeutic Treatment Design 198
Introduction 199
Human SARS-CoV Pathogenesis 199
The Clinical Course of Human SARS-CoV Infection 199
The Human Adaptive Immune Response to SARS-CoV 202
The Human Innate Immune Response to SARS-CoV 203
Animal Models of SARS-CoV Pathogenesis 203
In Vitro Models of SARS-CoV Pahtogenesis 207
SARS-CoV and MyD88 208
SARS-CoV and the Renin-Angiotensin System 210
SARS-CoV Therapeutic Design 213
SARS-CoV Antigenic Variation and Therapy Efficacy 213
Animal Models to Assess Passive Immunization Therapy Efficacy Against Divergent SARS-CoV Antigens 214
Animal Models to Assess Vaccine Immunization Therapy Efficacy Against Divergent SARS-CoV Antigens 215
SARS-CoV Vaccine Efficacy in Immunosenescent Populations 216
SARS-CoV Vaccine Immunopotentiation 220
Conclusion 222
References 223
Chapter 14: Modulation of Host Cell Death by SARS Coronavirus Proteins 234
Cell Death During SARS-CoV Infection 234
Induction of Host Cell Death by SARS-CoV Structural Proteins 236
E Protein 237
M Protein 237
S Protein 238
N Protein 238
Accessory Proteins 239
Protein 3a 241
Protein 3b 241
Protein 6 242
Protein 7a 242
Proteins 8a/8b 243
Conclusion 244
References 244
Chapter 15: SARS Coronavirus and Lung Fibrosis 249
Introduction 249
SARS-CoV-Mediated Lung Fibrosis 251
TGF-beta and SARS-Induced Lung Fibrosis 252
TGF-beta Signal Transduction 252
TGF-beta and Lung Fibrosis 252
The Role of TGF-beta in SARS-Induced Lung Fibrosis 253
ACE2/Angiotensin II and SARS-Induced Lung Fibrosis 254
Other Mechanisms of SARS-Mediated Lung Fibrosis 255
Conclusion 256
References 256
Chapter 16: Host Immune Responses to SARS Coronavirus in Humans 261
Introduction 262
Immune Responses 265
Innate Immunity 265
Viral Evasive Strategies 267
T Cell Responses 268
Antibody and B-Cell Responses 271
Vaccines 272
Summary and Perspectives 273
References 274
Chapter 17: The Use of Retroviral Pseudotypes for the Measurement of Antibody Responses to SARS Coronavirus 281
Introduction 281
Construction of Retroviral Pseudotypes Bearing SARS-CoV (S) Glycoproteins 282
Neutralization Assay 284
Expanding the Repertoire of SARS-CoV (S) Pseudotypes 284
Pseudotype Titration and Target Cell Lines 286
Reporter Systems 286
Correlation with Other Immunological Assays 287
Conclusion 288
References 288
Chapter 18: SARS Coronavirus Spike Protein Expression in HL-CZ Human Promonocytic Cells: Monoclonal Antibody and Cellular Trans 291
SARS Coronavirus: A Lethal Zoonotic Virus with Future Potential for Reemergence 292
SARS-CoV Spike Glycoprotein Contributes to Virulence and Pathogenesis 292
Generation of Human Monoclonal Antibodies by Selection from Recombinant Single-Chain Antibody-Phage Libraries Constructed from 293
Expression of SARS-CoV Spike Protein in Mammalian Cell Lines and Specific Detection by Human Monoclonal Antibodies: Potential 294
Western Blot Characterization of HL-CZ Cells Transfected with Codon-Optimized SARS-CoV Spike Construct 294
Microscopic Analysis of SARS-CoV Spike Expression in HL-CZ Cells by Indirect Immunofluorescence Labeling 295
Transcriptomic Analysis Reveals Differentially Regulated Genes in HL-CZ Cells Following SARS-CoV Spike Transfection 297
Immune and Inflammatory Genes 298
Cell Cycle Genes 301
Cytoskeletal Genes 302
Trafficking and Transport Genes 303
Conclusions and Future Prospects 303
References 304
Chapter 19: Signaling Pathways of SARS-CoV In Vitro and In Vivo 307
Introduction 307
p38 MAPK Signaling Pathway in Viral Infection 309
p38 MAPK Signaling Pathway in SARS-CoV-Infected Cells 311
Downstream Signaling Pathways of p38 MAPK 312
ERK1/2 Activation by SARS-CoV Infection 313
JNK Activation by SARS-CoV 313
PI3K/Akt Activation by SARS-CoV 314
NF-kappaB Activation and Inhibition by SARS-CoV Proteins 315
Inhibitory Effects of Viral Proteins on the Cell Cycle 315
Apoptotic Signaling Pathway 316
Signaling Pathways in SARS Patients 317
Conclusion 318
References 319
Index 325