Coronavirus Replication and Reverse Genetics (eBook)

Preface 5
List of Contents 8
List of Contributors 9
Coronavirus Genome Structure and Replication 10
1 Introduction 11
2 Common Features of Genome Structure Among Coronaviruses 12
3 Cis-Acting RNA Elements in Coronavirus Genome Replication 17
4 Packaging Signals 26
5 Minimum Sequence Requirements for ( Autonomous) Genome Replication 27
6 Importance of Gene Order for Genome Replication 28
7 Future Directions 30
References 31
Coronavirus Transcription: A Perspective 40
1 Introduction 41
2 Discontinuous Transcription by Coronaviruses 44
3 Kinetics of Plus- and Minus-Strand RNA Synthesis and Sensitivity to Translational Inhibition 50
4 The Kinetics of Synthesis of the Subgenomic Minus Strands 51
5 Subgenomic MHV RIs Exist in Infected Cells and Are Transcriptionally Active in mRNA 2–7 Synthesis 52
6 Characterization of Coronavirus Native RI/TIs and Native RF/TFs 53
7 Turnover of MHV Replicative/Transcriptive Intermediates 57
8 A Working Model 59
References 61
The Coronavirus Replicase 66
1 Introduction 67
2 Organization and Expression of the Replicase Gene 68
3 Replicase Polyproteins 70
4 Subcellular Localization of the Coronavirus Replicase 88
5 Concluding Remarks 92
References 92
Viral and Cellular Proteins Involved in Coronavirus Replication 104
1 Introduction 105
2 Viral Proteins in Coronavirus Replication 108
3 Cellular Proteins in Coronavirus Replication 118
4 Perspectives 127
References 128
Coronavirus Reverse Genetics by Targeted RNA Recombination 142
1 Introduction 143
2 Coronavirus RNA Recombination 143
3 Targeted RNA Recombination: Methodology and Technical Issues 146
4 Targeted RNA Recombination: Spectrum of Applications 154
5 Conclusions and Future Prospects 161
References 163
Coronavirus Reverse Genetics and Development of Vectors for Gene Expression 170
1 Introduction 171
2 Pathogenesis Induced by Group 1 Coronaviruses 173
3 Engineering Coronavirus Genome 174
4 Essential Genes Required for TGEV Replication 177
5 Transcription-Regulating Sequences 179
6 Expression Systems Based on Group 1 Coronaviruses 186
7 Coronavirus Vector Cloning Capacity 192
8 Insertion Site, Stability, and Expression Levels 193
9 Molecular Basis of Group 1 Coronavirus Tropism 195
10 Modulation of Coronavirus Vector Virulence 196
11 Biosafety in Coronavirus-Derived Vectors 197
12 Conclusions 198
References 199
Reverse Genetics of Coronaviruses Using Vaccinia Virus Vectors 208
1 Introduction 209
2 The Use of Vaccinia Virus as a Vector for Coronavirus cDNA 211
3 Recombinant Coronaviruses 220
4 Generation of Replicon RNAs 226
5 Development of Coronavirus-Based Multigene Vectors 230
6 Discussion 234
References 235
Development of Mouse Hepatitis Virus and SARS- CoV Infectious cDNA Constructs 238
1 Introduction 239
2 The Coronavirus Genome 239
3 Systematic Approaches to Assembling Coronavirus cDNAs from a Panel of Contiguous Subclones 240
4 Assembling MHV Infectious cDNAs 244
5 SARS-CoV Infectious Clone 254
6 Future Applications 255
References 257
Subject Index 262