DNA Viruses A Practical Approach

by Cann, Alan J.

• ISBN13: 9780199637195
• ISBN10: 0199637199
• Format: Hardcover
• Copyright: 2000-02-03
• Publisher: Oxford University Press

Summary

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 clinical isolates. 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 DNA virus replication and then chapters 5 & 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.

Table of Contents

List of Contributors	xvii
Abbreviations	xix
Extraction, purification, and characterization of virus DNA	1	(14)
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 marker rescue	21	(1)
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 vitro	56	(3)
Mutagenesis through growth of virus in the presence of nucleoside analogues	59	(1)
Site-directed mutagenesis in vitro	60	(4)
Reintroducing mutagenized sequences into virus	64	(11)
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 mutagenized stocks	75	(1)
Mapping mutations in isolates obtained by random mutagenesis	76	(1)
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 proteins during DNA virus replication	83	(30)
Catherine H. Botting
Ronald T. Hay
Introduction	83	(1)
Identification and purification of proteins involved in replication	84	(13)
Obtaining replication-active extracts	84	(2)
Assaying for activity	86	(6)
Purification of proteins required for adenovirus DNA replication from HeLa cells	92	(5)
Overexpression of replication components	97	(4)
Overexpression and purification of pTP and pol	97	(2)
Expression and purification of the cellular factors	99	(2)
Mapping interactions	101	(8)
Protein-DNA interactions	101	(3)
Protein-protein interactions	104	(5)
Investigating the dynamics of the replication process	109	(4)
Immobilized replication assay	109	(1)
Glycerol gradient centrifugation	109	(1)
References	110	(3)
Analysis of transcriptional control in DNA virus infections	113	(44)
S. K. Thomas
D. S. Latchman
Introduction	113	(1)
Analysis of viral gene expression during infection	114	(14)
Preparation of RNA samples	115	(3)
Analysis of transcripts by Northern blotting	118	(4)
Analysis of transcripts by reverse transcription-polymerase chain reaction (RT-PCR)	122	(2)
Analysis of transcripts by in situ hybridization (ISH)	124	(4)
Analysis of cloned promoter sequences using reporter gene constructs	128	(13)
Methods of transfection	129	(1)
Analysis of promoter activity	130	(4)
Preparation of indicator viruses for the analysis of promoter activity during infection	134	(6)
Using transgenic animals to analyse the tissue-specific expression of viral promoters	140	(1)
Analysis of transcriptional control by mutagenesis	141	(7)
Types of mutation	141	(1)
Linker scanning mutagenesis	142	(1)
Oligonucleotide insertion	142	(2)
Site-directed mutagenesis	144	(4)
Identification of cellular transcription factors involved in the control of viral transcription	148	(9)
The DNA mobility-shift assay	148	(5)
South-western blotting	153	(1)
Methods for isolating cloned transcription factors	154	(1)
Acknowledgements	154	(1)
References	154	(3)
Identification and analysis of trans-acting proteins involved in the regulation of DNA virus gene expression	157	(20)
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 cytokines	177	(32)
Alshad S. Lalani
Piers Nash
Bruce T. Seet
Janine Robichaud
Grant McFadden
Introduction	177	(1)
Identification of novel virus soluble cytokine-binding proteins	178	(8)
Generation of secreted virus proteins from infected cells	178	(1)
Use of chemical cross-linking to detect viral cytokine-binding proteins	179	(1)
Ligand blot overlays for detection of viral cytokine-binding proteins	180	(2)
Immunoprecipitation of viral cytokine-binding proteins	182	(2)
Use of plasmon resonance for analysis of interactions of cytokines with viral proteins	184	(2)
Synthesis and purification of cytokine-binding proteins	186	(11)
Vaccinia virus expression system	187	(2)
Synthesis of cytokine binding proteins by Baculovirus expression systems	189	(1)
Fc fusion protein production	189	(2)
Purification of secreted viral cytokine-binding proteins by fast protein liquid chromatography (FPLC)	191	(5)
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 (SPR) for detailed kinetic studies	201	(1)
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 chemokine-binding proteins on chemokine-induced calcium flux	206	(1)
References	207	(2)
Analysis of DNA virus proteins involved in neoplastic transformation	209	(38)
Kersten T. Hall
Maria E. Blair Zajdel
G. Eric Blair
Introduction	209	(7)
Human adenovirus oncoproteins	209	(2)
Simian virus 40 and polyomavirus oncoproteins	211	(3)
Human papillomavirus oncoproteins	214	(2)
Cell systems utilized for study of DNA tumour viruses	216	(5)
DNA transfection of virus oncogenes into mammalian cells	217	(1)
Selection of virus-transformed cells	217	(2)
Cloning of transformed cells, and assay of cell growth	219	(2)
Characterization of virus-transformed cells	221	(14)
Analysis of virus oncoproteins by radiolabelling of transformed cells and immunoprecipitation	224	(6)
Sub-cellular distribution of virus oncoproteins	230	(5)
Biological activity of virus oncoproteins	235	(12)
Studies of interactions between virus oncoproteins and cellular proteins	238	(2)
Use of the yeast two-hybrid screen to identify interactions between virus oncoproteins and cellular proteins	240	(4)
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 antiviral drugs	250	(2)
Resistance assays for therapeutic antivirals used for HSV	252	(2)
Resistance to antivirals of human cytomegalovirus (HCMV)	254	(6)
Phenotypic CMV drug susceptibility assay	256	(1)
Genotypic assays for detection of ganciclovir resistance-associated mutations in CMV	257	(3)
Antivirals active against hepatitis B virus	260	(3)
Genotypic assay for resistance associated mutations in HBV using nucleotide sequencing	261	(2)
Real time PCR and fluorimetry for detection of mutations	263	(4)
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 replication-defective viruses as vectors to deliver genes to the peripheral and central nervous system	280	(4)
In vitro culture of neurones to study the biology HSV	284	(3)
Adenovirus	287	(20)
Biological properties	287	(1)
Pattern of gene expression during lytic adenovirus infection	288	(1)
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 adenovirus recombinants	296	(3)
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