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Adenovirus Methods and Protocols, Second Edition
V 2
OLUME
M E T H O D S I N M O L E C U L A R M E D I C I N E™
John M. Walker, SERIES EDITOR
138.Neutrophil Methods and Protocols, edited by 117.Fibrosis Research: Methods and Protocols,
Mark T. Quinn, Frank R. DeLeo, and Gary M. edited by John Varga, David A. Brenner,
Bokoch, 2007 and Sem H. Phan, 2005
137.Microtubule Protocols, edited by Jun Zhou, 2007 116.Inteferon Methods and Protocols, edited by
136.Arthritis Research: Methods and Protocols, Daniel J. J. Carr, 2005
Vol. 2, edited by Andrew P. Cope, 2007 115.Lymphoma: Methods and Protocols, edited by
135.Arthritis Research: Methods and Protocols, Timothy Illidge and Peter W. M. Johnson, 2005
Vol. 1, edited by Andrew P. Cope, 2007 114.Microarrays in Clinical Diagnostics, edited by
134.Bone Marrow and Stem Cell Transplantation, Thomas O. Joos and Paolo Fortina, 2005
edited by Meral Beksac, 2007 113.Multiple Myeloma: Methods and Protocols,
133.Cancer Radiotherapy, edited by Robert A. edited by Ross D. Brown and P. Joy Ho, 2005
Huddart and Vedang Murthy, 2007 112.Molecular Cardiology: Methods and Protocols,
132.Single Cell Diagnostics: Methods and edited by Zhongjie Sun, 2005
Protocols, edited by Alan Thornhill, 2007 111.Chemosensitivity: Volume 2, In Vivo Mod-
131. Adenovirus Methods and Protocols, Second els, Imaging, and Molecular Regulators, edited
Edition, Vol. 2: Ad Proteins, RNA, Lifecycle, by Rosalyn D. Blumethal, 2005
Host Interactions, and Phylogenetics, edited by 110.Chemosensitivity: Volume 1, In Vitro Assays,
William S. M. Wold and Ann E. Tollefson, edited by Rosalyn D. Blumethal, 2005
2007 109.Adoptive Immunotherapy: Methods and
130.Adenovirus Methods and Protocols, Second Protocols, edited by Burkhard Ludewig and
Edition, Vol. 1: Adenoviruses, Ad Vectors, Matthias W. Hoffman, 2005
Quantitation, and Animal Models, edited by 108.Hypertension: Methods and Protocols,
William S. M. Wold and Ann E. Tollefson, 2007 edited by Jérôme P. Fennell and Andrew
129. Cardiovascular Disease: Methods and Protocols, H. Baker, 2005
Volume 2: Molecular Medicine, edited by 107.Human Cell Culture Protocols, Second
Qing K.Wang, 2006 Edition, edited by Joanna Picot, 2005
128. Cardiovascular Disease: Methods and Protocols, 106.Antisense Therapeutics, Second Edition,
Volume 1: Genetics, edited by Qing K. Wang, edited by M. Ian Phillips, 2005
2006 105.Developmental Hematopoiesis: Methods and
127. DNA Vaccines: Methods and Protocols, Second Protocols, edited by Margaret H. Baron, 2005
Edition, edited by Mark W. Saltzman, Hong 104. Stroke Genomics: Methods and Reviews, edited
Shen, and Janet L. Brandsma, 2006 by Simon J. Read and David Virley, 2004
126. Congenital Heart Disease: Molecular Diagnostics, 103.Pancreatic Cancer: Methods and Protocols,
edited by Mary Kearns-Jonker, 2006 edited by Gloria H. Su, 2004
125.Myeloid Leukemia: Methods and Protocols, 102.Autoimmunity: Methods and Protocols,
edited by Harry Iland, Mark Hertzberg, edited by Andras Perl, 2004
and Paula Marlton, 2006 101.Cartilage and Osteoarthritis: Volume 2,
124.Magnetic Resonance Imaging: Methods and Structure and In Vivo Analysis, edited by
Biologic Applications, edited by Pottumarthi Frédéric De Ceuninck, Massimo Sabatini,
V. Prasad, 2006 and Philippe Pastoureau, 2004
123.Marijuana and Cannabinoid Research: 100.Cartilage and Osteoarthritis: Volume 1,
Methods and Protocols, edited by Emmanuel Cellular and Molecular Tools, edited by
S. Onaivi, 2006 Massimo Sabatini, Philippe Pastoureau,
122.Placenta Research Methods and Protocols: and Frédéric De Ceuninck, 2004
Volume 2, edited by Michael J. Soares and 99.Pain Research: Methods and Protocols,
Joan S. Hunt, 2006 edited by David Z. Luo, 2004
121.Placenta Research Methods and Protocols: 98.Tumor Necrosis Factor: Methods and
Volume 1, edited by Michael J. Soares and Protocols, edited by Angelo Corti and Pietro
Joan S. Hunt, 2006 Ghezzi, 2004
120.Breast Cancer Research Protocols, edited by 97.Molecular Diagnosis of Cancer: Methods
Susan A. Brooks and Adrian Harris, 2006 and Protocols, Second Edition, edited by
119.Human Papillomaviruses: Methods and Joseph E. Roulston and John M. S. Bartlett,
Protocols, edited by Clare Davy and John 2004
Doorbar, 2005 96.Hepatitis B and D Protocols: Volume 2,
118.Antifungal Agents: Methods and Protocols, Immunology, Model Systems, and Clinical
edited by Erika J. Ernst and P. David Rogers, Studies, edited by Robert K. Hamatake and
2005 Johnson Y. N. Lau, 2004
M E T H O D S I N M O L E C U L A R M E D I C I N E™
Adenovirus Methods
and Protocols
S E
ECOND DITION
Volume 2: Ad Proteins, RNA, Lifecycle,
Host Interactions, and Phylogenetics
Edited by
William S. M. Wold
Ann E. Tollefson
Department of Molecular Microbiology and Immunology
Saint Louis University School of Medicine
St. Louis, Missouri
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Adenovirus methods and protocols / edited by William S.M. Wold, Ann E. Tollefson. -- 2nd ed.
v. ; cm. -- (Methods in molecular medicine, ISSN 1543-1894 ; 130-131)
Includes bibliographical references and indexes.
Contents: v. 1. Adenoviruses, ad vectors, quantitation, and animal models -- v. 2. Ad proteins, RNA,
lifecycle, host interactions, and phylogenetics.
ISBN 1-58829-598-2 (v. 1 : alk. paper) -- ISBN 1-58829-901-5 (v. 2 : alk. paper)
1. Adenoviruses--Laboratory manuals. 2. Molecular biology--Laboratory manuals. I. Wold, William S. M.
II. Tollefson, Ann E. III. Series.
[DNLM: 1. Adenoviridae--Laboratory Manuals. W1 ME9616JM v.130-131 2007 / QW 25 A232 2007]
QR396.A336 2007
579.2'443--dc22
2006012284
Preface
Since their discovery in 1954, adenoviruses (Ads) have become a model for
studying virology, as well as molecular and cellular biology. Ads are easily
grown and manipulated, stable, and versatile. Ads replicate reproducibly, can
transform rodent cells to an oncogenic state, can induce tumors in certain ani-
mals, and have been instrumental in defining key cellular proteins and mecha-
nisms such as splicing, transcriptional regulation through transcription adaptor
proteins, and regulation of cell division and apoptosis. About half of the
approximately 35 Ad proteins physically interact with cellular proteins and
subvert them for use by the virus. In recent years, Ads have become premier
tools in vector technology and in experimental gene therapy research.
The Adenovirus Methods and Protocols volumes are designed to help new
researchers to conduct studies involving Ads and to help established researchers
to branch into new areas. The chapters, which are written by prominent investi-
gators, provide a brief general introduction to a topic, followed by tried and
true step-by-step methods pertinent to the subject. We thank returning con-
tributors for their updated and new chapters, and thank new contributors who
have expanded the content of this book.
Adenovirus Methods and Protocols, Second Edition, Volume 2: Ad Proteins,
RNA, Lifecycle, Host Interactions, and Phylogenetics, focuses on methods that
elucidate and quantitate the interactions of Ad with the host. This volume pro-
vides methods for analysis of transcription, splicing, RNA interference, sub-
cellular localization of proteins during infection, and cell cycle effects. Four
chapters are devoted to definition of interactions of viral and cellular proteins
(by co-immunoprecipitation or tandem mass spectrometry) or protein interac-
tions with viral DNA (by chromatin immunoprecipitation and electrophoretic
mobility shift assays). Other chapters provide thorough descriptions of the use
of microinjection procedures, transformation assays, and NK cell-mediated
cytolysis. Several chapters describe characterization of specific Ad proteins
(hexon, fiber, or protease/proteinase). Two chapters are devoted to defining
the phylogenetic relationships of Ads.
We thank contributors for sharing their secrets, John Walker for his patience,
and especially Dawn Schwartz, without whose expert assistance this work
would not have been possible.
William S. M. Wold
Ann E. Tollefson
v
Contents
Preface ..............................................................................................................v
Contents of the Companion Volume................................................................ix
Contributors .....................................................................................................xi
1 Analysis of the Efficiency of Adenovirus Transcription.........................1
Cristina Iftode and S. J. Flint
2 The Use of In Vitro Transcription to Probe Regulatory Functions
of Viral Protein Domains ................................................................15
Paul M. Loewenstein, Chao-Zhong Song, and Maurice Green
3 Preparation of Soluble Extracts From Adenovirus-Infected Cells
for Studies of RNA Splicing.............................................................33
Oliver Mühlemann and Göran Akusjärvi
4 In Vitro Methods to Study RNA Interference During
an Adenovirus Infection..................................................................47
Gunnar Andersson, Ning Xu, and Göran Akusjärvi
5 Simultaneous Detection of Adenovirus RNA and Cellular Proteins
by Fluorescent Labeling In Situ.......................................................63
Eileen Bridge
6 Study of Nucleolar Localization of Adenovirus Core Proteins............73
David A. Matthews
7 Analysis of Adenovirus Infections in Synchronized Cells ...................83
David A. Ornelles, Robin N. Broughton-Shepard,
and Felicia D. Goodrum
8 Co-Immunoprecipitation of Protein Complexes................................103
Peter Yaciuk
9 Chromatin Immunoprecipitation to Study the Binding of Proteins
to the Adenovirus Genome In Vivo ..............................................113
Jihong Yang and Patrick Hearing
10 Assaying Protein–DNA Interactions In Vivo and In Vitro Using
Chromatin Immunoprecipitation and Electrophoretic Mobility
Shift Assays ...................................................................................123
Pilar Perez-Romero and Michael J. Imperiale
11 Identifying Functional Adenovirus–Host Interactions Using
Tandem Mass Spectrometry..........................................................141
Anuj Gaggar, Dmitry Shayakhmetov, and André Lieber
vii
viii Contents
12 The Use of Cell Microinjection for the In Vivo Analysis of Viral
Transcriptional Regulatory Protein Domains................................157
Maurice Green, Andrew Thorburn, Robert Kern,
and Paul M. Loewenstein
13 Determination of the Transforming Activities
of Adenovirus Oncogenes.............................................................187
Michael Nevels and Thomas Dobner
14 Human Adenovirus Type 12: Crossing Species Barriers
to Immortalize the Viral Genome.................................................197
Walter Doerfler
15 Measurement of Natural-Killer Cell Lytic Activity of Adenovirus-
Infected or Adenovirus-Transformed Cells....................................213
John M. Routes
16 A Flow Cytometric Assay for Analysis of Natural-Killer
Cell-Mediated Cytolysis of Adenovirus-Transformed Cells...........221
Graham Bottley, Graham P. Cook, and G. Eric Blair
17 Large-Scale Purification and Crystallization of Adenovirus Hexon......231
John J. Rux and Roger M. Burnett
18 Synthesis and Assay of Recombinant Adenovirus Protease.............. 251
Joseph M. Weber
19 Assay for the Adenovirus Proteinase: Purification of the Enzyme
and Synthesis of a Fluorogenic Substrate......................................257
Walter F. Mangel and William J. McGrath
20 Cofactors of the Adenovirus Proteinase: Measuring Equilibrium
Dissociation Constants and Stoichiometries of Binding................269
Walter F. Mangel and William J. McGrath
21 Characterization of the Adenovirus Fiber Protein.............................281
Jeffrey A. Engler and Jeong Shin Hong
22 Phylogenetic Analysis of Adenovirus Sequences ..............................299
Balázs Harrach and Mária Benko"
23 Assessment of Genetic Variability Among Subspecies B1 Human
Adenoviruses for Molecular Epidemiology Studies.......................335
Adriana E. Kajon and Dean D. Erdman
Index ............................................................................................................357
C C V
ONTENTS OF THE OMPANION OLUME
Volume 1: Adenoviruses, Ad Vectors, Quantitation,
and Animal Models
1 Manipulation of Early Region 4
Julie Boyer and Gary Ketner
2 Isolation, Growth, and Purification of Defective Adenovirus
Deletion Mutants
Gary Ketner and Julie Boyer
3 Construction of Adenovirus Type 5 Early Region 1 (E1) and 4 (E4)
Virus Mutants
Peter Groitl and Thomas Dobner
4 Construction of Mouse Adenovirus Type 1 Mutants
Angela N. Cauthen, Amanda R. Welton, and Katherine R. Spindler
5 Generation of Recombinant Adenovirus Using Escherichia coli BJ5183
Recombination System
P. Seshidhar Reddy, Shanthi Ganesh, Lynda Hawkins,
and Neeraja Idamakanti
6 Production and Release Testing of Ovine Atadenovirus Vectors
Gerald W. Both, Fiona Cameron, Anne Collins, Linda J. Lockett,
and Jan Shaw
7 Construction of Capsid-Modified Recombinant Bovine Adenovirus
Type 3
Alexander N. Zakhartchouk, Qiaohua Wu, and Suresh K. Tikoo
8 Adenovirus Capsid Chimeras: Fiber Terminal Exon Insertions/Gene
Replacements in the MLTU
Jason Gall, John Schoggins, and Erik Falck-Pedersen
9 Temperature-Sensitive Replication-Competent Adenovirus shRNA
Vectors to Study Cellular Genes in Virus-Induced Apoptosis
Thirugnana Subramanian and Govindaswamy Chinnadurai
10 Evaluating Apoptosis in Tumor Cells Infected With Adenovirus
Expressing p53 Tumor Suppressor Gene
Elizabeth Perron, M. Behzad Zafar, Amanda Pister, Zhen-Guo Wang,
Jun Tian, and Prem Seth
11 Growth and Purification of Enteric Adenovirus Type 40
Vivien Mautner
ix
