RNA Viruses A Practical Approach

by Cann, Alan J.

• ISBN13: 9780199637171
• ISBN10: 0199637172
• Format: Hardcover
• Copyright: 2000-06-01
• Publisher: Oxford University Press

Summary

RNA Viruses: A Practical Approach is wide ranging in scope, from emerging technology such as reverse genetics and retrovirus vectors, to money saving tips - how to make your own silica particles for high efficiency RNA extraction and liposomes for cell transfection! Chapter one covers thefundamentals of investigating RNA virus genome structure at a molecular level. Chapters two and three describe techniques for mutagenesis of RNA genomes and analysis of transcription. Chapter four deals with RNA virus-encoded proteinases, an important aspect of the control of RNA virus geneexpression. Chapter five considers retrovirus oncogenesis and chapter six analysis of RNA virus quasispecies. Chapter seven describes systems for investigation of in vitro replication of positive-stranded viruses and chapter eight the packaging of RNA virus genomes. In addition to the technicalaspects of reverse genetics and retrovirus vectors, both of the final two chapters also consider ethical aspects of these new technologies.

Table of Contents

List of protocols	xiii
Abbreviations	xvii
Investigation of RNA virus genome structure	1	(22)
A.J. Easton
A.C. Marriott
C.R. Pringle
Introduction---the nature of the virus genome	1	(7)
Properties of the genomes of RNA viruses	3	(1)
Properties of the virions of RNA viruses	3	(5)
Paradigm for analysis of RNA virus genome structure	8	(1)
Growth, assay, and purification of RNA viruses	8	(4)
Source of virus: in vivo versus in vitro methods	8	(1)
Assay of virus yield	9	(1)
Harvesting and concentrating of virus	9	(1)
Purification of virus	10	(1)
Rate-zonal gradient centrifugation	10	(1)
Isopycnic gradient centrifugation	11	(1)
Radiolabelling	12	(1)
RNA extraction	12	(4)
Genome RNA extraction from virions	13	(2)
Genome RNA extraction without virion purification	15	(1)
Fractionation of RNA, and analysis by Northern (RNA) blotting	16	(3)
Fractionation of RNA	16	(1)
Northern blotting and detection with labelled probes	17	(2)
Further analysis	19	(4)
Characterization by restriction-endonuclease digest patterns	19	(1)
Characterization by ribonuclease protection	20	(2)
References	22	(1)
Mutagenesis of RNA virus genomes	23	(20)
W.S. Barclay
J.W. Almond
Introduction	23	(3)
Generation of RNA virus mutants	26	(11)
Selection of mutants from the RNA quasispecies	26	(2)
Recombination of RNA virus genomes	28	(1)
Reassortment of segmented RNA virus genomes	28	(3)
Generation of defective RNA virus populations by passage in culture	31	(1)
Recovery of RNA virus mutants from infectious cDNA	32	(1)
Site-directed mutagenesis of infectious clones of RNA virus genomes	32	(3)
Recovery of virus from infectious cDNA	35	(1)
Transfection of RNA replicons	36	(1)
Determining the genotype of RNA virus mutants	37	(2)
Analysis of the frequency of RNA virus mutants	39	(4)
References	41	(2)
Analysis of transcriptional control in RNA virus infections	43	(26)
S. Makino
Introduction	43	(1)
Analysis of virus mRNAs in virus-infected cells	43	(5)
General considerations	43	(1)
Radiolabelling and extraction of virus RNAs	44	(2)
Gel electrophoresis of RNAs	46	(1)
Glyoxal gel electrophoresis	46	(1)
Formaldehyde gel electrophoresis	47	(1)
Analysis of virus mRNA structure	48	(7)
General considerations	48	(1)
Separation of virus RNA in preparative agarose gels	49	(1)
Extraction of virus RNA from gel slices	50	(3)
One-dimensional oligonucleotide fingerprinting	53	(1)
Northern blot hybridization	54	(1)
The use of RNA reporter constructs for transcriptional assays	55	(14)
RNA transcription in vitro	57	(2)
RNA transfection of DI RNA construct and helper virus infection	59	(1)
DNA transfection and vaccinia virus infection	60	(2)
CAT reporter assay system	62	(1)
Detection and quantitation of minute amount of virus RNAs	63	(1)
Quantitative RT-PCR	63	(2)
RNase protection assay	65	(1)
References	66	(3)
Analysis of RNA virus-encoded proteinases	69	(16)
M.D. Ryan
M. Flint
M.L.L. Donnelly
E. Byrne
V. Cowton
Introduction	69	(1)
Defining the proteinase type	70	(1)
Inhibitor studies	70	(1)
Sequence analysis	70	(1)
Sequence motifs	70	(1)
Sequence alignments	71	(1)
Delimiting the proteinase (domain)	71	(9)
Deletion/truncation analysis	71	(3)
Screening for proteolytic activity using translation systems in vitro	74	(1)
Uncoupled translation systems	75	(2)
Coupled transcription/translation systems	77	(1)
Translation systems and interactions in trans	77	(2)
Rapid analysis of site-directed mutants	79	(1)
Bacterial expression	80	(2)
Expression of inactive proteinase	80	(1)
Affinity `tagging'	81	(1)
Insolubility of expressed proteinases	81	(1)
Substrate specificity	82	(1)
Artificial `reporter' polyproteins	82	(3)
References	84	(1)
Detection and analysis of host gene targets for oncogenic retroviruses	85	(20)
J.C. Neil
A. Terry
Introduction	85	(1)
Insertional mutagenesis	86	(13)
Analysis of virus integration patterns	87	(2)
Cloning of virus integration sites	89	(4)
Analysis of host DNA flanking the provirus integration sites	93	(3)
Location of genes at integration sites	96	(3)
Transduction of virus oncogenes	99	(6)
Properties of transducing viruses	99	(1)
Biological methods of detection	100	(1)
Molecular methods of detection	101	(1)
References	102	(3)
Analysis of RNA virus quasispecies	105	(36)
J.K. Ball
Virus quasispecies	105	(2)
Choice of analytical method	107	(1)
Nucleic acid extraction	107	(15)
Reverse transcription of virus RNA	110	(2)
The polymerase chain reaction as a tool for quasispecies analysis	112	(3)
Polyacrylamide gel electrophoresis	115	(4)
Methods for labelling and staining DNA	119	(3)
Single-stranded conformation polymorphism analysis	122	(10)
Heteroduplex and quantitative heteroduplex tracking analyses	124	(3)
Length polymorphism analysis (LPA)	127	(1)
Point mutation assays	128	(4)
Sequence analysis	132	(9)
Sequence manipulation and phylogenetic analyses	138	(1)
References	139	(2)
In vitro replication of RNA viruses	141	(38)
R. Banerjee
M. Igo
R. Izumi
U. Datta
A. Dasgupta
Introduction	141	(1)
De-novo synthesis of poliovirus	142	(5)
Isolation of poliovirus genomic RNA	142	(2)
HeLa S10 extract preparation	144	(1)
Translation initiation-factor preparation	145	(1)
Coupled transcription and translation	146	(1)
Poliovirus plaque assay	146	(1)
Expression of virus proteins with enzymatic and RNA binding activities	147	(5)
RNA binding assays	152	(19)
In vitro transcription for labelled probe preparation	152	(5)
Labelled nucleoprotein complex formation	157	(1)
Gel shift assay	158	(1)
UV-crosslink analysis	158	(9)
Northwestern analysis	167	(1)
Western analysis	168	(3)
Membrane binding of virus proteins	171	(8)
Indirect immunofluorescence	171	(4)
In vitro membrane binding assay	175	(2)
Acknowledgements	177	(1)
References	178	(1)
Packaging of segmented and non-segmented RNA virus genomes	179	(22)
J. Barr
J.W. McCauley
Introduction	179	(1)
Assembly and packaging defined	179	(1)
Genome selection	180	(1)
Genome packaging in non-segmented, negative-strand RNA viruses	180	(8)
Investigation of genome packaging: analysis of nucleocapsid RNA polarity before and after pakaging into virions	181	(7)
Introduction of site-specific mutations in the genome of negative-strand viruses	188	(2)
Influenza virus genome packaging	190	(11)
References	199	(2)
RNA virus reverse genetics	201	(28)
A. Bridgen
R.M. Elliott
Introduction	201	(2)
Model systems for the manipulation of RNA viruses	203	(5)
Transient expression system	204	(1)
Transfection and electroporation techniques	205	(2)
Choice of expression system	207	(1)
Generation of RNAs containing authentic 5 and 3 termini	208	(1)
Synthesis of RNA templates	208	(2)
Purification/synthesis of virus proteins required for replication	210	(3)
Rescue of infectious virus	213	(3)
Creation of mutant viruses	216	(4)
Analysis of mutant phenotypes	220	(4)
Genomic studies of the virus mutants	220	(3)
Phenotypic studies	223	(1)
Technical and ethical issues	224	(1)
Perspectives	224	(5)
References	225	(4)
Development of RNA virus vectors for gene delivery	229	(30)
B.A. Usmani
A. Fassati
G. Dickson
Introduction	229	(1)
Vectors based on murine retroviruses	230	(13)
Design and choice of MoMLV retrovirus vectors	231	(1)
Single-gene MoMLV vectors	231	(1)
MoMLV vectors expressing multiple genes	231	(1)
Tropism of MoMLV retrovirus vectors	232	(1)
MoMLV packaging cell lines	233	(1)
MoMLV vector production	234	(3)
Titration of MoMLV virus vector stocks	237	(2)
Concentration of MoMLV virus vector stocks	239	(1)
Detection of replication-competent MoMLV (RCR) vector contamination	239	(1)
Infection of targe cells in vitro with MoMLV vectors	240	(2)
Direct transduction of target cells in vivo with MoMLV vectors	242	(1)
Retrovirus gene transfer vectors based on lentiviruses	243	(16)
Lentivirus vector sources and design	245	(2)
Packaging and pseudotyping constructs for lentivirus vectors	247	(1)
Production and concentration of recombinant vector	248	(3)
Ex vivo and in vivo transduction	251	(1)
Safety considerations in the use of lentivirus vectors	252	(2)
References	254	(5)
A1 List of suppliers	259	(6)
Index	265