RNA Viruses A Practical Approac h Alan J. Cann
List of protocols page xiii Abbreviations xvii Investigation of RNA virus genome structure 1 A j. Easton, A.C. Marriott and C.R. Pringl e 1 Introduction-the nature of the virus genome 1 Properties of the genomes of RNA viruses 3 Properties of the virions of RNA viruses 3 Paradigm for analysis of RNA virus genome structure 8 2 Growth, assay, and purification of RNA viruses 8 Source of virus : in vivo versus in vitro methods 8 Assay of virus yield 9 Harvesting and concentrating of virus 9 Purification of virus 1 0 Rate-zonal gradient centrifugation 1 0 Isopycnic gradient centrifugation 1 1 Radiolabelling 1 2 3 RNA extraction 1 2 Genome RNA extraction from virions 1 3 Genome RNA extraction without virion purification 1 5 4 Fractionation of RNA, and analysis by Northern (RNA) blotting 1 6 Fractionation of RNA 1 6 Northern blotting and detection with labelled probes 1 7 5 Further analysis 1 9 Characterization by restriction-endonuclease digest patterns 1 9 Characterization by ribonuclease protection 20 References 22 2 Mutagenesis of RNA virus genomes 23 11 : W.S. Barclay and J.W. Almon d 1 Introduction 2 3 2 Generation ofrna virus mutants 26
Selection of mutants from the RNA quasispecies 2 6 Recombination of RNA virus genomes 2 8 Reassortment of segmented RNA virus genomes 28 Generation of defective RNA virus populations by passage in culture 3 1 Recovery of RNA virus mutants from infectious cdna 3 2 Site-directed mutagenesis of infectious clones of RNA virus genomes 32 Recovery of virus from infectious cdna 3 5 Transfection of RNA replicons 36 3 Determining the genotype of RNA virus mutants 3 7 4 Analysis of the frequency of RNA virus mutants 3 9 References 4 1 3 Analysis of transcriptional control in RNA virus infections 4 3 S. Makin o 1 Introduction 43 2 Analysis of virus mrnas in virus-infected cells 4 3 General considerations 4 3 Radiolabelling and extraction of virus RNAs 4 4 Gel electrophoresis of RNAs 4 6 Glyoxal gel electrophoresis 46 Formaldehyde gel electrophoresis 4 7 3 Analysis of virus mrna structure 4 8 General considerations 4 8 Separation of virus RNA in preparative agarose gels 4 9 Extraction of virus RNA from gel slices 5 0 One-dimensional oligonucleotide fingerprinting 5 3 Northern blot hybridization 54 4 The use of RNA reporter constructs for transcriptional assays 5 5 RNA transcription in vitro 5 7 RNA transfection of DI RNA construct and helper virus infection 5 9 DNA transfection and vaccinia virus infection 6 0 CAT reporter assay system 62 Detection and quantitation of minute amount of virus RNAs 6 3 Quantitative RT-PCR 63 RNase protection assay 65 References 6 6 4 Analysis of RNA virus-encoded proteinases 69 M.D. Ryan,'M. Flint, M.L.L. Donnelly, E. Byrne and V. Cowton 1 Introduction 6 9 2 Defining the proteinase type 7 0 Inhibitor studies 7 0 Sequence analysis 7 0 Sequence motifs 70 Sequence alignments 71
3 Delimiting the proteinase (domain) 7 1 Deletion/truncation analysis 7 1 Screening for proteolytic activity using translation systems in vitro 7 4 Uncoupled translation systems 75 Coupled transcription/translation systems 7 7 Translation systems and interactions in trans 77 Rapid analysis of site-directed mutants 79 4 Bacterial expression 80 Expression of inactive proteinase 8 0 Affinity `tagging' 8 1 Insolubility of expressed proteinases 8 1 5 Substrate specificity 82 6 Artificial `reporter' polyproteins 8 2 References 84 5 Detection and analysis of host gene targets for oncogeni c retroviruses 85 J.C. Neil and A. Terry 1 Introduction 85 2 Insertional mutagenesis 86 Analysis of virus integration patterns 87 Cloning of virus integration sites 89 Analysis of host DNA flanking the provirus integration sites 9 3 Location of genes at integration sites 9 6 3 Transduction of virus oncogenes 99 Properties of transducing viruses 9 9 Biological methods of detection 100 Molecular methods of detection 10 1 References 102 6 Analysis of RNA virus quasispecies 105 J.K. Ball 1 Virus quasispecies 10 5 2 Choice of analytical method 107 3 Nucleic acid extraction 10 7 Reverse transcription of virus RNA 11 0 The polymerase chain reaction as a tool for quasispecies analysis 11 2 Polyacrylamide gel electrophoresis 115 Methods for labelling and staining DNA 11 9 4 Single-stranded conformation polymorphism analysis 122 Heteroduplex and quantitative heteroduplex tracking analyses 12 4 Length polymorphism analysis (LPA) 12 7 Point mutation assays 12 8 5 Sequence analysis 132 Sequence manipulation and phylogenetic analyses 138 References 139
7 In vitro replication of RNA viruses 14 1 R. Banerjee, M. Igo, R. Izumi, U. Datta and A. Dasgupta 1 Introduction 14 1 2 De-novo synthesis of poliovirus 142 Isolation of poliovirus genomic RNA 142 HeLa S10 extract preparation 144 Translation initiation-factor preparation 145 Coupled transcription and translation 14 6 Poliovirus plaque assay 146 3 Expression of virus proteins with enzymatic and RNA binding activities 147 4 RNA binding assays 152 In vitro transcription for labelled probe preparation 152 Labelled nucleoprotein complex formation 157 Gel shift assay 158 UV-crosslink analysis 15 8 Northwestern analysis 167 Western analysis 16 8 5 Membrane binding of virus proteins 17 1 Indirect immunofluorescence 171 In vitro membrane binding assay 175 Acknowledgements 17 7 References 178 8 Packaging of segmented and non-segmented RNA virus genomes 179 J. Barr and J.W. McCauley 1 Introduction 179 Assembly and packaging defined 17 9 Genome selection 180 2 Genome packaging in non-segmented, negative-strand RNA viruses 180 Investigation of genome packaging : analysis of nucleocapsid RNA polarity befor e and after packaging into virions 18 1 3 Introduction of site-specific mutations in the genome of negative-strand viruses 188 4 Influenza virus genome packaging 190 References 19 9 9 RNA virus reverse genetics 20 1 A. Bridgen and RM. Elliott 1 Introduction 20 1 2 Model systems for the manipulation of RNA viruses 203 Transient expression system 204 Transfection and electroporation techniques 20 5 Choice of expression system 20 7 Generation of RNAs containing authentic 5' and 3' termini 20 8 3 Synthesis of RNA templates 208
4 Purification/synthesis of virus proteins required for replication 21 0 5 Rescue of infectious virus 21 3 6 Creation of mutant viruses 21 6 7 Analysis of mutant phenotypes 220 Genomic studies of the virus mutants 220 Phenotypic studies 22 3 8 Technical and ethical issues 224 9 Perspectives 224 References 225 10 Development of RNA virus vectors for gene delivery 229 B.A. Usmani, A. Fassati and G. Dickson 1 Introduction 229 2 Vectors based on murine retroviruses 230 Design and choice of MoMLV retrovirus vectors 231 Single-gene MoMLV vectors 231 MoMLV vectors expressing multiple genes 23 1 Tropism of MoMLV retrovirus vectors 232 MoMLV packaging cell lines 23 3 MoMLV vector production 234 Titration of MoMLV virus vector stocks 23 7 Concentration of MoMLV virus vector stocks 23 9 Detection of replication-competent MoMLV (RCR) vector contamination 23 9 Infection of target cells in vitro with MoMLV vectors 240 Direct transduction of target cells in vivo with MoMLV vectors 242 3 Retrovirus gene transfer vectors based on lentiviruses 243 Lentivirus vector sources and design 24 5 Packaging and pseudotyping constructs for lentivirus vectors 24 7 Production and concentration of recombinant vector 24 8 Ex vivo and in vivo transduction 25 1 Safety considerations in the use of lentivirus vectors 252 References 25 4 Al List of suppliers 259 Index 265