DNA viruses : a practical approach / edited by Alan J. Cann.

Enhanced descriptions from Syndetics:

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 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.

Table of contents provided by Syndetics

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

Author notes provided by Syndetics