RNA Viruses. A Practical Approac h. Alan J. Cann

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1 RNA Viruses A Practical Approac h Alan J. Cann

2

3 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 RNA extraction 1 2 Genome RNA extraction from virions 1 3 Genome RNA extraction without virion purification Fractionation of RNA, and analysis by Northern (RNA) blotting 1 6 Fractionation of RNA 1 6 Northern blotting and detection with labelled probes 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 : W.S. Barclay and J.W. Almon d 1 Introduction Generation ofrna virus mutants 26

4 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 Analysis of the frequency of RNA virus mutants 3 9 References 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 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 Analysis of RNA virus-encoded proteinases 69 M.D. Ryan,'M. Flint, M.L.L. Donnelly, E. Byrne and V. Cowton 1 Introduction Defining the proteinase type 7 0 Inhibitor studies 7 0 Sequence analysis 7 0 Sequence motifs 70 Sequence alignments 71

5 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 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 Transduction of virus oncogenes 99 Properties of transducing viruses 9 9 Biological methods of detection 100 Molecular methods of detection 10 1 References Analysis of RNA virus quasispecies 105 J.K. Ball 1 Virus quasispecies Choice of analytical method 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 Single-stranded conformation polymorphism analysis 122 Heteroduplex and quantitative heteroduplex tracking analyses 12 4 Length polymorphism analysis (LPA) 12 7 Point mutation assays Sequence analysis 132 Sequence manipulation and phylogenetic analyses 138 References 139

6 7 In vitro replication of RNA viruses 14 1 R. Banerjee, M. Igo, R. Izumi, U. Datta and A. Dasgupta 1 Introduction 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 Expression of virus proteins with enzymatic and RNA binding activities 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 Membrane binding of virus proteins 17 1 Indirect immunofluorescence 171 In vitro membrane binding assay 175 Acknowledgements 17 7 References 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 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 Introduction of site-specific mutations in the genome of negative-strand viruses Influenza virus genome packaging 190 References RNA virus reverse genetics 20 1 A. Bridgen and RM. Elliott 1 Introduction 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 Synthesis of RNA templates 208

7 4 Purification/synthesis of virus proteins required for replication Rescue of infectious virus Creation of mutant viruses Analysis of mutant phenotypes 220 Genomic studies of the virus mutants 220 Phenotypic studies Technical and ethical issues Perspectives 224 References Development of RNA virus vectors for gene delivery 229 B.A. Usmani, A. Fassati and G. Dickson 1 Introduction 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 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

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