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List of Contributors xvii
Abbreviations xix
Extraction, purification, and 1 (14)
characterization of virus DNA
Ian W. Halliburton
Introduction 1 (1)
Tissue culture 2 (2)
Cell lines 2 (2)
Virus culture 4 (2)
Preparation of virus working stocks 4 (1)
Titration of virus stocks 5 (1)
Virus purification 6 (1)
Manipulation of virus DNA 6 (3)
Extraction of virus DNA 6 (3)
Restriction enzyme digestion of virus DNA 9 (1)
Mapping DNA virus genes 9 (6)
Marker rescue 10 (1)
Transcript mapping 11 (1)
Mapping by use of intertypic recombinants 12 (1)
Sequencing 12 (1)
References 12 (3)
Investigation of DNA virus genome structure 15 (32)
Michael A. Skinner
Stephen M. Laidlaw
Introduction 15 (1)
Scope 16 (1)
Genome mapping 16 (5)
Southern blotting 17 (2)
Pulsed-field agarose gel electrophoresis 19 (2)
Correlation of genotype with phenotype by 21 (1)
marker rescue
Design of marker rescue 21 (1)
Nucleotide sequencing 22 (9)
Primer walking 23 (1)
Library construction 23 (4)
Minipreparation of sequence grade DNA 27 (2)
Sequence determination 29 (1)
Data collection 30 (1)
Sequence assembly 31 (8)
The Staden package 31 (8)
Sequence analysis 39 (5)
Prediction of open reading frames 39 (2)
Homology searches 41 (1)
Sequence alignment 42 (1)
Phylogenetic analysis 43 (1)
Mutational analysis 44 (1)
Future directions 44 (3)
Acknowledgements 44 (1)
References 44 (3)
Mutagenesis of DNA virus genomes 47 (36)
Keith N. Leppard
Introduction 47 (4)
The diversity of DNA viruses 47 (1)
Alternative mutagenesis strategies 47 (1)
Suiting mutagenesis strategy to the virus 48 (3)
Preparation and titration of virus stocks 51 (5)
Cell culture techniques 51 (1)
Generating and titrating virus stocks 52 (4)
Chemical mutagenesis of viral DNA in vitro 56 (1)
Chemical mutagenesis of virus particles in 56 (3)
vitro
Mutagenesis through growth of virus in the 59 (1)
presence of nucleoside analogues
Site-directed mutagenesis in vitro 60 (4)
Reintroducing mutagenized sequences into 64 (11)
virus
Complementing cell lines 68 (2)
Polyomaviruses 70 (1)
Parvoviruses 71 (1)
Adenoviruses 72 (2)
Herpes viruses and pox viruses 74 (1)
Selection of mutant phenotypes in randomly 75 (1)
mutagenized stocks
Mapping mutations in isolates obtained by 76 (1)
random mutagenesis
Characterization of mutant viruses 77 (6)
Single step growth curve 77 (1)
DNA replication assay 77 (2)
Late protein expression 79 (1)
Virus assembly 80 (1)
References 80 (3)
Interactions between viral and cellular 83 (30)
proteins during DNA virus replication
Catherine H. Botting
Ronald T. Hay
Introduction 83 (1)
Identification and purification of proteins 84 (13)
involved in replication
Obtaining replication-active extracts 84 (2)
Assaying for activity 86 (6)
Purification of proteins required for 92 (5)
adenovirus DNA replication from HeLa cells
Overexpression of replication components 97 (4)
Overexpression and purification of pTP 97 (2)
and pol
Expression and purification of the 99 (2)
cellular factors
Mapping interactions 101 (8)
Protein-DNA interactions 101 (3)
Protein-protein interactions 104 (5)
Investigating the dynamics of the 109 (4)
replication process
Immobilized replication assay 109 (1)
Glycerol gradient centrifugation 109 (1)
References 110 (3)
Analysis of transcriptional control in DNA 113 (44)
virus infections
S. K. Thomas
D. S. Latchman
Introduction 113 (1)
Analysis of viral gene expression during 114 (14)
infection
Preparation of RNA samples 115 (3)
Analysis of transcripts by Northern 118 (4)
blotting
Analysis of transcripts by reverse 122 (2)
transcription-polymerase chain reaction
(RT-PCR)
Analysis of transcripts by in situ 124 (4)
hybridization (ISH)
Analysis of cloned promoter sequences using 128 (13)
reporter gene constructs
Methods of transfection 129 (1)
Analysis of promoter activity 130 (4)
Preparation of indicator viruses for the 134 (6)
analysis of promoter activity during
infection
Using transgenic animals to analyse the 140 (1)
tissue-specific expression of viral
promoters
Analysis of transcriptional control by 141 (7)
mutagenesis
Types of mutation 141 (1)
Linker scanning mutagenesis 142 (1)
Oligonucleotide insertion 142 (2)
Site-directed mutagenesis 144 (4)
Identification of cellular transcription 148 (9)
factors involved in the control of viral
transcription
The DNA mobility-shift assay 148 (5)
South-western blotting 153 (1)
Methods for isolating cloned 154 (1)
transcription factors
Acknowledgements 154 (1)
References 154 (3)
Identification and analysis of trans-acting 157 (20)
proteins involved in the regulation of DNA
virus gene expression
Adrian Whitehouse
David M. Meredith
Introduction 157 (1)
Identification of transactivating proteins 158 (1)
Transfection of mammalian cells 158 (1)
Analysis of the mechanism of transactivation 159 (5)
Characterization of RNA 160 (4)
Identification of cis-acting elements 164 (13)
Mobility shift assays 164 (5)
Purification of transcription factors 169 (4)
Conventional purification procedures 173 (1)
Acknowledgements 174 (1)
References 174 (3)
Interaction of DNA virus proteins with host 177 (32)
cytokines
Alshad S. Lalani
Piers Nash
Bruce T. Seet
Janine Robichaud
Grant McFadden
Introduction 177 (1)
Identification of novel virus soluble 178 (8)
cytokine-binding proteins
Generation of secreted virus proteins 178 (1)
from infected cells
Use of chemical cross-linking to detect 179 (1)
viral cytokine-binding proteins
Ligand blot overlays for detection of 180 (2)
viral cytokine-binding proteins
Immunoprecipitation of viral 182 (2)
cytokine-binding proteins
Use of plasmon resonance for analysis of 184 (2)
interactions of cytokines with viral
proteins
Synthesis and purification of 186 (11)
cytokine-binding proteins
Vaccinia virus expression system 187 (2)
Synthesis of cytokine binding proteins by 189 (1)
Baculovirus expression systems
Fc fusion protein production 189 (2)
Purification of secreted viral 191 (5)
cytokine-binding proteins by fast protein
liquid chromatography (FPLC)
Purification by affinity chromatography 196 (1)
Analysis of cytokine-binding partners 197 (12)
Solid phase binding 197 (2)
Scintillation proximity assay 199 (2)
The use of surface plasmon resonance 201 (1)
(SPR) for detailed kinetic studies
Inhibition of cell-surface binding 202 (2)
Inhibition of cytokine-induced cytolysis 204 (1)
Growth inhibition/proliferation assay 205 (1)
Measuring the effect of viral 206 (1)
chemokine-binding proteins on
chemokine-induced calcium flux
References 207 (2)
Analysis of DNA virus proteins involved in 209 (38)
neoplastic transformation
Kersten T. Hall
Maria E. Blair Zajdel
G. Eric Blair
Introduction 209 (7)
Human adenovirus oncoproteins 209 (2)
Simian virus 40 and polyomavirus 211 (3)
oncoproteins
Human papillomavirus oncoproteins 214 (2)
Cell systems utilized for study of DNA 216 (5)
tumour viruses
DNA transfection of virus oncogenes into 217 (1)
mammalian cells
Selection of virus-transformed cells 217 (2)
Cloning of transformed cells, and assay 219 (2)
of cell growth
Characterization of virus-transformed cells 221 (14)
Analysis of virus oncoproteins by 224 (6)
radiolabelling of transformed cells and
immunoprecipitation
Sub-cellular distribution of virus 230 (5)
oncoproteins
Biological activity of virus oncoproteins 235 (12)
Studies of interactions between virus 238 (2)
oncoproteins and cellular proteins
Use of the yeast two-hybrid screen to 240 (4)
identify interactions between virus
oncoproteins and cellular proteins
Acknowledgements 244 (1)
References 244 (3)
Chemotherapy of DNA virus infections 247 (20)
Patricia A. Cane
Deenan Pillay
Introduction 247 (1)
Antivirals effective against herpesviruses 247 (7)
Aciclovir 247 (2)
Penciclovir 249 (1)
Foscarnet 249 (1)
Cidofovir 250 (1)
Testing susceptibility of HSV to 250 (2)
antiviral drugs
Resistance assays for therapeutic 252 (2)
antivirals used for HSV
Resistance to antivirals of human 254 (6)
cytomegalovirus (HCMV)
Phenotypic CMV drug susceptibility assay 256 (1)
Genotypic assays for detection of 257 (3)
ganciclovir resistance-associated
mutations in CMV
Antivirals active against hepatitis B virus 260 (3)
Genotypic assay for resistance associated 261 (2)
mutations in HBV using nucleotide
sequencing
Real time PCR and fluorimetry for detection 263 (4)
of mutations
Acknowledgements 265 (1)
References 265 (2)
Herpes simplex virus and adenovirus vectors 267 (40)
Cinzia Scarpini
Jane Arthur
Stacey Efstathiou
Yvonne McGrath
Gavin Wilkinson
Introduction 267 (1)
Herpes simplex virus 267 (20)
Biological properties 267 (1)
Gene expression during lytic infection 268 (1)
The latent state 268 (1)
Basic techniques of virus handling 269 (4)
Construction of recombinant virus genomes 273 (7)
The use of wild-type and 280 (4)
replication-defective viruses as vectors
to deliver genes to the peripheral and
central nervous system
In vitro culture of neurones to study the 284 (3)
biology HSV
Adenovirus 287 (20)
Biological properties 287 (1)
Pattern of gene expression during lytic 288 (1)
adenovirus infection
Adenovirus vectors 288 (4)
The helper cell line 292 (1)
Basic adenovirus handling techniques 292 (3)
Quantification of adenovirus stocks 295 (1)
Construction of replication-deficient 296 (3)
adenovirus recombinants
Characterization of virus 299 (2)
Infection of cells with adenovirus vectors 301 (1)
Enhanced infection 301 (1)
References 302 (5)
Appendix 307 (6)
Index 313

DNA Viruses: A Practical Approach groups together the major experimental methods currently employed to study DNA viruses, from the fundamentals of virus culture to novel techniques such as surface plasmon resonance spectrometry and realtime PCR analysis of drug resistance mutations in clinicalisolates. Chapter 1 provides an overview of the extraction, purification and characterizations of virus DNA, but also covers the fundamentals of DNA virus culture. Chapters 2 and 3 describe approaches to the molecular investigation and mutagenesis of DNA virus genomes. Chapter 4 considers DNAvirus replication and then chapters 5 and 6 describe methods to study transcription control. Chapters 7 to 9 consider aspects of the pathogenesis of DNA virus infections. The final chapter describes the current technology being applied to the development of DNA virus vectors for gene delivery.This volume will therefore be of interest to all those working on DNA viruses whether in academia, industry or clinical research.