HIV Protocols 2nd Edition Kimdar Sherefa Kemal Phd

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Author(s): Kimdar Sherefa Kemal PhD, Milan Reinis, Barbara Weiser, Harold
Burger (auth.), Vinayaka R. Prasad PhD, GanjamV. Kalpana PhD (eds.)
ISBN(s): 9781597451703, 1597451703
Edition: 2
File Details: PDF, 6.97 MB
Year: 2009
Language: english

M E T H O D S I N M O L E C U L A R B I O L O G YTM
John M. Walker, SERIES EDITOR
489. Dynamic Brain Imaging: Methods and Protocols,
edited by Fahmeed Hyder, 2009
485. HIV Protocols: Methods and Protocols, edited by
Vinayaka R. Prasad and Ganjam V. Kalpana, 2009
484. Functional Proteomics: Methods and Protocols,
edited by Julie D. Thompson, Christine
Schaeffer-Reiss, and Marius Ueffing, 2008
483. Recombinant Proteins From Plants: Methods and
Protocols, edited by Loı̈c Faye and Veronique
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482. Stem Cells in Regenerative Medicine: Methods and
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481. Hepatocyte Transplantation: Methods and
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Hughes, 2008
480. Macromolecular Drug Delivery: Methods and
Protocols, edited by Mattias Belting, 2008
479. Plant Signal Transduction: Methods and Protocols,
edited by Thomas Pfannschmidt, 2008
478. Transgenic Wheat, Barley and Oats: Production
and Characterization Protocols, edited by Huw D.
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477. Advanced Protocols in Oxidative Stress I, edited by
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476. Redox-Mediated Signal Transduction: Methods
and Protocols, edited by John T. Hancock, 2008
475. Cell Fusion: Overviews and Methods, edited by
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474. Nanostructure Design: Methods and Protocols,
edited by Ehud Gazit and Ruth Nussinov, 2008
473. Clinical Epidemiology: Practice and Methods, edited
by Patrick Parfrey and Brendon Barrett, 2008
472. Cancer Epidemiology, Volume 2: Modifiable
Factors, edited by Mukesh Verma, 2008
471. Cancer Epidemiology, Volume 1: Host Susceptibility
Factors, edited by Mukesh Verma, 2008
470. Host-Pathogen Interactions: Methods and
Protocols, edited by Steffen Rupp and Kai Sohn, 2008
469. Wnt Signaling, Volume 2: Pathway Models, edited
by Elizabeth Vincan, 2008
468. Wnt Signaling, Volume 1: Pathway Methods and
Mammalian Models, edited by Elizabeth Vincan, 2008
467. Angiogenesis Protocols: Second Edition, edited by
Stewart Martin and Cliff Murray, 2008
466. Kidney Research: Experimental Protocols, edited by
Tim D. Hewitson and Gavin J. Becker, 2008.
465. Mycobacteria, Second Edition, edited by Tanya Parish
and Amanda Claire Brown, 2008
464. The Nucleus, Volume 2: Physical Properties and
Imaging Methods, edited by Ronald Hancock, 2008
463. The Nucleus, Volume 1: Nuclei and Subnuclear
Components, edited by Ronald Hancock, 2008
462. Lipid Signaling Protocols, edited by Banafshe
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2008
461. Molecular Embryology: Methods and Protocols,
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460. Essential Concepts in Toxicogenomics, edited by
Donna L. Mendrick and William B. Mattes, 2008
459. Prion Protein Protocols, edited by Andrew F. Hill,
2008
458. Artificial Neural Networks: Methods and
Applications, edited by David S. Livingstone, 2008
457. Membrane Trafficking, edited by Ales Vancura, 2008
456. Adipose Tissue Protocols, Second Edition, edited by
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455. Osteoporosis, edited by Jennifer J. Westendorf, 2008
454. SARS- and Other Coronaviruses: Laboratory
Protocols, edited by Dave Cavanagh, 2008
453. Bioinformatics, Volume 2: Structure, Function, and
Applications, edited by Jonathan M. Keith, 2008
452. Bioinformatics, Volume 1: Data, Sequence Analysis,
and Evolution, edited by Jonathan M. Keith, 2008
451. Plant Virology Protocols: From Viral Sequence to
Protein Function, edited by Gary Foster, Elisabeth
Johansen, Yiguo Hong, and Peter Nagy, 2008
450. Germline Stem Cells, edited by Steven X. Hou and
Shree Ram Singh, 2008
449. Mesenchymal Stem Cells: Methods and Protocols,
edited by Darwin J. Prockop, Douglas G. Phinney,
and Bruce A. Brunnell, 2008
448. Pharmacogenomics in Drug Discovery and
Development, edited by Qing Yan, 2008.
447. Alcohol: Methods and Protocols, edited by
Laura E. Nagy, 2008
446. Post-translational Modifications of Proteins: Tools
for Functional Proteomics, Second Edition, edited by
Christoph Kannicht, 2008.
445. Autophagosome and Phagosome, edited by Vojo
Deretic, 2008
444. Prenatal Diagnosis, edited by Sinhue Hahn and
Laird G. Jackson, 2008.
443. Molecular Modeling of Proteins, edited by Andreas
Kukol, 2008.
442. RNAi: Design and Application, edited by Sailen Barik,
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441. Tissue Proteomics: Pathways, Biomarkers, and Drug
Discovery, edited by Brian Liu, 2008
440. Exocytosis and Endocytosis, edited by Andrei I.
Ivanov, 2008
439. Genomics Protocols, Second Edition, edited by Mike
Starkey and Ramnanth Elaswarapu, 2008
438. Neural Stem Cells: Methods and Protocols, Second
Edition, edited by Leslie P. Weiner, 2008
437. Drug Delivery Systems, edited by Kewal K. Jain,
2008
436. Avian Influenza Virus, edited by Erica Spackman,
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435. Chromosomal Mutagenesis, edited by Greg Davis
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434. Gene Therapy Protocols: Volume 2: Design and
Characterization of Gene Transfer Vectors, edited by
Joseph M. Le Doux, 2008
433. Gene Therapy Protocols: Volume 1: Production and
In Vivo Applications of Gene Transfer Vectors, edited
by Joseph M. Le Doux, 2008
432. Organelle Proteomics, edited by Delphine Pflieger
and Jean Rossier, 2008
431. Bacterial Pathogenesis: Methods and Protocols,
edited by Frank DeLeo and Michael Otto, 2008
430. Hematopoietic Stem Cell Protocols, edited by
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429. Molecular Beacons: Signalling Nucleic Acid Probes,
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Oliver Seitz, 2008
428. Clinical Proteomics: Methods and Protocols, edited
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427. Plant Embryogenesis, edited by Maria Fernanda
Suarez and Peter Bozhkov, 2008
426. Structural Proteomics: High-Throughput Methods,
edited by Bostjan Kobe, Mitchell Guss, and Thomas
Huber, 2008
425. 2D PAGE: Sample Preparation and Fractionation,
Volume 2, edited by Anton Posch, 2008

ME T H O D S I N MO L E C U L A R BI O L O G Y TM
HIV Protocols
SecondEdition
Edited By
Vinayaka R. Prasad, PhD
and
Ganjam V. Kalpana, PhD
AlbertEinsteinCollegeofMedicine,Bronx,NY,USA

Editor
Vinayaka R. Prasad Ganjam V. Kalpana
Albert Einstein College of Medicine Albert Einstein College of Medicine
Bronx, NY Bronx, NY
USA USA
prasad@aecom.yu.edu kalpana@aecom.yu.edu
Series Editor
John M. Walker
University of Hertfordshire
Hatfield Herts
UK
ISBN: 978-1-58829-859-1 e-ISBN: 978-1-59745-170-3
ISSN: 1064-3745 e-ISSN: 1940-6029
DOI 10.1007/978-1-59745-170-3
Library of Congress Control Number: 2008938341
c
Humana Press, a part of Springer Science+Business Media, LLC 2009
All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the
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Preface
Why another book of HIV protocols? The question is sure to arise in the minds of
the readers. The AIDS epidemic continues unabated despite strong advances in thera-
peutics and an unprecedented level of efforts in vaccine development. Although some
of the reasons for the failure in AIDS control can be attributed to poor prevention
measures or paucity of antiretrovirals, a major drawback is the absence of efficacious,
potent antiretrovirals that can both suppress viral load completely and that do not
have toxic side effects. Toxicity can lead to nonadherence, which in turn results in
poor virus control, emergence of drug resistance and the eventual clinical drug fail-
ure. Development of novel drugs and vaccines require definition of new targets, bet-
ter definition of already known viral targets and understanding the interplay between
viral and host factors. Similarly, in order to develop an AIDS vaccine, we need to
be equipped with effective methods to measure immune response. More importantly,
these studies require the development of efficient and powerful in vitro and in vivo
systems to study viral replication and pathogenesis. Therefore, HIV researchers have
a real need for access to well-described, state-of-the-art methods to study HIV.
Approaches in HIV/AIDS investigation have continuously advanced in tune with
the evolution of modern experimental science. Development of new technologies in
investigating familiar aspects of HIV replication or immune response to it have led
to new insights that have improved our understanding of the biology of HIV. In
compiling this collection, our objective is threefold. First, we aim to document up-to-
date protocols available for select aspects of HIV biology. Second, we bring together
both virological and immunological approaches in a single volume. Third, we provide
a comprehensive account of techniques that are not already part of an existing HIV
protocol book.
HIV Protocols, Second Edition, is organized into five sections. Section I delin-
eates the methods to isolate full-length DNA clones of HIV-1 from patient samples,
isolation of HIV-1 particles free of contaminating cellular proteins and a method to
titer these virus particles. Section II delineates the study of early and late events. Early
events include entry, reverse transcription, nuclear transport, integration as well as
recombination, a process that occurs during reverse transcription. A thorough study
of early events would be incomplete without the analysis of complexes formed during
reverse transcription and prior to integration as well as the interaction between viral
and host proteins within these complexes. In the subsection on late events, we take
you through methods to study assembly and particle production within the producer
cells and the use of cell free systems to study the interaction of viral proteins and
nucleic acids including the cognate tRNALys,3.
No HIV-1 investigation is complete without the analysis of the dynamics of host-
virus interactions. HIV-1, being an intra-cellular parasite, not only invades the host,
but also subverts cellular antiviral mechanisms and hijacks host proteins for its own
purposes. Section III explores approaches to investigate the interplay between the
v

vi Preface
host and the virus by employing genetic, molecular and cellular techniques including
novel small animal models. Methods to investigate specific, immunological techniques
to understanding host-HIV-1 interplay are discussed in Section IV. Chapters in this
section delineate methods to study mucosal immunity, T-cell responses and antiviral
responses in cell culture and in Rhesus monkey models.
The last section of the book delves into the intense battle between the host and
the HIV-1. The virus continues to evade antiretrovirals owing to its ability to develop
drug resistance and its baffling ability to evolve and escape the immune system. The
chapters included should facilitate investigations of drug resistant viruses and virus
evolution.
We would like to draw the readers’ attention to the Notes sections in each chapter.
These notes come from the experts who have used these methods successfully many
times and contain many ‘tricks’ and little details that are rarely mentioned in standard
protocols. We find them to be a unique aspect of the Methods in Molecular Biology
series.
We would like to thank Humana Press for the opportunity to edit this book, the
series editor, Dr. John Walker, for his continuous support and guidance and David
Casey of Humana Press for his patience and support. We extend our sincerest gratitude
to all contributors for their submission of critical contributions to this collection. The
advice of Dr. Barbara Shacklett of the University of California, Davis in the selection
of immunological topics is specially acknowledged. The administrative assistance of
Ms. Emilia Ortiz in the production of the book went beyond the call of duty and we
are thankful to her. We also wish to express our gratitude to our graduate students,
post-doctoral fellows and other close colleagues at Einstein who helped with editing
the book for scientific content (Dhivya Ramalingam, Elizabeth Hanna Luke, Sonald
Duclair and Vasudev Rao) or for style (Andrea Provost, Aviva Joseph) and in the
beta testing and improving of the subject index (Chisanga Lwatula, James Gaudette,
Jennifer Cano, Melissa Smith, SeungJae Lee, Sheeba Mathew, Sohrab Khan, Supratik
Das).
We would like to thank Humana Press for the opportunity to edit this book as
well as the series editor, Dr. John Walker, for his continuous support and guidance.
We acknowledge the advice of Dr. Barbara Shacklett of the University of California,
Davis in the selection of immunological topics. The administrative assistance of Ms.
Emilia Ortiz often went beyond the call of duty. We also wish to express our grati-
tude to colleagues at Einstein who helped with editing for scientific content (Dhivya
Ramalingam, Elizabeth Hanna Luke, Sonald Duclair and Vasudev Rao) or for style
(Andrea Provost). Finally, we extend our sincerest gratitude to all contributors for
their submission of critical contributions to this collection.
Vinayaka R. Prasad, PhD
Ganjam V. Kalpana, PhD

Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
SECTION I. PREPARATION OF VIRUS PARTICLES AND THEIR
ANALYSIS
1 Methods for Viral RNA Isolation and PCR Amplification for Sequencing
of Near Full-Length HIV-1 Genomes
Kimdar Sherefa Kemal, Milan Reinis, Barbara Weiser, and Harold Burger . . . . . . . . 3
2 Purification of HIV-1 Virions by Subtilisin Digestion or CD45
Immunoaffinity Depletion for Biochemical Studies
David E. Ott . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3 Calculating HIV-1 Infectious Titre Using a Virtual TCID50 Method
Yong Gao, Immaculate Nankya, Awet Abraha, Ryan M. Troyer, Kenneth N. Nelson,
Andrea Rubio, and Eric J. Arts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
SECTION II. METHODS TO STUDY HIV-1 REPLICATION
SUBSECTION A. EARLY EVENTS
4 Cell-Free Assays for HIV-1 Uncoating
Christopher Aiken. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
5 Real-Time PCR Analysis of HIV-1 Replication Postentry Events
Jean L. Mbisa∗, Krista A. Delviks-Frankenberry∗, James A. Thomas,
Robert J. Gorelick, Vinay K. Pathak . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
6 Analysis of 2-LTR Circle Junctions of Viral DNA in Infected Cells
Dibyakanti Mandal and Vinayaka R. Prasad . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
7 HIV-1 Recombination: An Experimental Assay and a Phylogenetic
Approach
Michael D. Moore, Mario P.S. Chin, and Wei-Shau Hu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
8 Methods of Preparation and Analysis of Intracellular Reverse Transcription
Complexes
Ariberto Fassati. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .107
9 Analysis of Viral and Cellular Proteins in HIV-1 Reverse Transcription
Complexes by Co-immunoprecipitation
Sergey N. Iordanskiy and Michael I. Bukrinsky . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .121
10 Isolation and Analysis of HIV-1 Preintegration Complexes
Alan Engelman . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .135
11 Bisarsenical Labeling of HIV-1 for Real-Time Fluorescence Microscopy
Nathalie J. Arhel and Pierre Charneau . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .151
SUBSECTION B. LATE EVENTS
12 Methods for the Study of HIV-1 Assembly
Abdul A. Waheed, Akira Ono, and Eric O. Freed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .163
vii

viii Contents
13 Assembly of Immature HIV-1 Capsids
Using a Cell-Free System
Jaisri R. Lingappa and Beth K. Thielen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .185
14 Preparation of Recombinant Hiv-1 Gag Protein and Assembly
of Virus-Like Particles In Vitro
Siddhartha A.K. Datta and Alan Rein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .197
15 Methods for the Analysis of HIV-1 Nucleocapsid Protein Interactions
with Oligonucleotides
Andrew G. Stephen and Robert J. Fisher. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .209
16 Methods for Analysis of Incorporation and Annealing
of tRNALys in HIV-1
Shan Cen, Fei Guo, and Lawrence Kleiman. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .223
SECTION III. SPECIALIZED APPROACHES TO STUDY HIV-1 BIOLOGY
AND PATHOGENESIS
17 Somatic Cell Genetic Analyses to Identify HIV-1 Host Restriction Factors
Susana T. Valente and Stephen P. Goff . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .235
18 Rapid, Controlled and Intensive Lentiviral Vector-Based RNAi
Manuel Llano, Natassia Gaznick, and Eric M. Poeschla . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .257
19 Reverse Two-Hybrid Screening to Analyze Protein–Protein Interaction
of HIV-1 Viral and Cellular Proteins
Supratik Das and Ganjam V. Kalpana. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .271
20 Methods to Study Monocyte Migration Induced by HIV-Infected Cells
Vasudev R. Rao, Eliseo A. Eugenin, Joan W. Berman, and Vinayaka R. Prasad . . . .295
21 Novel Mouse Models for Understanding HIV-1 Pathogenesis
Aviva Joseph, Kaori Sango, and Harris Goldstein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .311
SECTION IV. IMMUNOLOGICAL STUDIES OF HIV
SUBSECTION A. MUCOSAL IMMUNOLOGY
22 Mucosal Antibody Responses to HIV
Zina Moldoveanu and Jiri Mestecky . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .333
23 Isolating Mucosal Lymphocytes from Biopsy Tissue for Cellular
Immunology Assays
Barbara L. Shacklett, J. William Critchfield, Donna Lemongello . . . . . . . . . . . . . . . . . . . . .347
SUBSECTIOM B. MEASURING T CELL RESPONSES VIA FLOW CYTOMETRY
24 Quantifying HIV-1-Specific CD8+ T-Cell Responses Using ELISPOT
and Cytokine Flow Cytometry
Barbara L. Shacklett, J. William Critchfield, and Donna Lemongello . . . . . . . . . . . . . . . .359
25 Multiparameter Flow Cytometry Monitoring of T Cell Responses
Holden T. Maecker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .375
SUBSECTION C. ANTIVIRAL RESPONSES
26 Measuring HIV Neutralization in a Luciferase Reporter Gene Assay
David C. Montefiori . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .395
27 Assessing the Antiviral Activity of HIV-1-Specific Cytotoxic
T Lymphocytes
Otto O. Yang . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .407

Contents ix
28 Methods for Quantitating Antigen-Specific T Cell Responses Using
Functional Assays in Rhesus Macaques
Rama Rao Amara. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .417
SECTION V. DRUG RESISTANT VIRUSES AND VIRAL EVOLUTION
29 Isolation of Drug-Resistant Mutant HIV Variants Using Tissue Culture
Drug Selection
Maureen Oliveira, Bluma G. Brenner, and Mark A. Wainberg . . . . . . . . . . . . . . . . . . . . . .427
30 Virus Evolution as a Tool to Study HIV-1 Biology
Ben Berkhout and Atze T. Das. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .435
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .453

Contributors
AWET ABRAHA, BA • Division of Infectious Diseases, Department of Medicine,
Case Western Reserve University, Cleveland, OH, USA
CHRISTOPHER AIKEN, PHD • Department of Microbiology and Immunology,
Vanderbilt University School of Medicine, Nashville, TN, USA
RAMA RAO AMARA, PHD • Department of Microbiology and Immunology, Emory
Vaccine Center, Yerkes National Primate Research Center, Emory University,
Atlanta, GA, USA
NATHALIE J. ARHEL, PHD • Institut Pasteur, Molecular Virology and Vectorology
Group, Virology Department, Paris, France
ERIC J ARTS, PHD • Division of Infectious Diseases, Department of Medicine;
Department of Molecular Biology and Microbiology, Case Western Reserve
University, Cleveland, OH, USA
BEN BERKHOUT, PHD • Laboratory of Experimental Virology, Academic Medical
Center, Amsterdam, The Netherlands
JOAN W. BERMAN, PHD • Department of Pathology, Albert Einstein College of
Medicine, Bronx, NY, USA
BLUMA G. BRENNER, PHD • McGill University AIDS Centre, Lady Davis Institute
for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada
MICHAEL I. BUKRINSKY, MD, PHD • The George Washington University,
Washington, DC, USA
HAROLD BURGER, MD, PHD • Wadsworth Center, New York State Department of
Health, Albany, New York Albany Medical College, Albany, NY, USA
SHAN CEN, PHD • Department of Medicine, McGill University, Lady Davis
Institute for Medical Research, Jewish General Hospital, Montreal, QC, Canada
PIERRE CHARNEAU, PHD • Institut Pasteur, Molecular Virology and Vectorology
Group, Virology Department, Paris, France
MARIO P.S. CHIN, PHD • HIV Drug Resistance Program, National Cancer
Institute, Frederick, MD, USA
J. WILLIAM CRITCHFIELD, PHD • Department of Medical Microbiology and
Immunology and School of Medicine, University of California, Davis, CA, USA
ATZE T. DAS, PHD • Laboratory of Experimental Virology, Academic Medical
Center, Amsterdam, The Netherlands
SUPRATIK DAS, PHD • Department of Molecular Genetics, Albert Einstein College
of Medicine, Bronx, NY, USA
SIDDHARTHA A.K. DATTA, PHD • HIV Drug Resistance Program, National
Cancer Institute, Frederick, MD, USA
KRISTA A. DELVIKS-FRANKENBERRY, PHD • HIV-Drug Resistance Program,
NCI-Frederick, Frederick, MD, USA
xi

xii Contributors
ALAN ENGELMAN, PHD • Department of Cancer Immunology and AIDS,
Dana-Farber Cancer Institute and Division of AIDS, Harvard Medical School,
Boston, MA, USA
ELISEO A. EUGENIN, PHD • Department of Pathology, Albert Einstein College of
Medicine, Bronx, NY, USA
ARIBERTO FASSATI, MD, PHD • Wohl Virion Centre and MRC Centre for Medical
Molecular Virology, Division of Infection and Immunity, University College
London, London, UK
ROBERT J. FISHER, PHD • Protein Chemistry Laboratory, Research Technology
Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, MD, USA
ERIC O. FREED, PHD • Virus-Cell Interaction Section, HIV Drug Resistance
Program, NCI-Frederick, National Institutes of Health, Frederick, MD, USA
YONG GAO, MD, PHD • Division of Infectious Diseases, Department of Medicine,
NATASSIA GAZNICK, BS • Molecular Medicine Program, Mayo Clinic College of
Medicine, Rochester, MN, USA
STEPHEN P. GOFF, PHD • Howard Hughes Medical Institute, Department of
Biochemistry and Molecular Biophysics, Columbia University, College of
Physicians and Surgeons, New York, NY, USA
HARRIS GOLDSTEIN, MD • Departments of Microbiology and Immunology and
Pediatrics, Albert Einstein College of Medicine, Bronx, NY, USA
ROBERT J. GORELICK, PHD • AIDS Vaccine Program, SAIC-Frederick, Inc.,
FEI GUO, PHD • Lady Davis Institute for Medical Research, Jewish General
Hospital Montreal, QC, Canada
WEI-SHAU HU, PHD • HIV Drug Resistance Program, National Cancer Institute,
Frederick, MD, USA
SERGEY N. IORDANSKIY, PHD • The D.I. Ivanovsky Institute of Virology, Moscow,
Russia
AVIVA JOSEPH, PHD • Department of Microbiology and Immunology, Albert
Einstein College of Medicine, Bronx, NY, USA
GANJAM V. KALPANA, PHD • Department of Molecular Genetics, Albert Einstein
College of Medicine, Bronx, NY, USA
KIMDAR SHEREFA KEMAL, PHD • Wadsworth Center, New York State Department
of Health, Albany, NY, USA
LAWRENCE KLEIMAN, PHD • Department of Medicine, McGill University, Lady
Davis Institute for Medical Research, Jewish General Hospital, Montreal,
QC, Canada
DONNA LEMONGELLO, BS • Department of Medical Microbiology and
Immunology and School of Medicine, University of California, Davis,
CA, USA
JAISRI R. LINGAPPA, MD, PHD • Departments of Pathobiology and Medicine,
University of Washington, Seattle, WA, USA
MANUEL LLANO, MD, PHD • Molecular Medicine Program, Mayo Clinic College
of Medicine, Rochester, MN, USA
HOLDEN T. MAECKER, PHD • Palo Alto, CA, USA

Contributors xiii
DIBYAKANTI MANDAL, PHD • Department of Microbiology and Immunology,
Albert Einstein College of Medicine, Bronx, NY, USA
JEAN L. MBISA, PHD • HIV-Drug Resistance Program, NCI-Frederick, Frederick,
MD, USA
JIRI MESTECKY, MD, PHD • University of Alabama, Birmingham, AL, USA
ZINA MOLDOVEANU, PHD • University of Alabama, Birmingham, AL, USA
DAVID C. MONTEFIORI, PHD • Department of Surgery, Laboratory for AIDS
Vaccine Research and Development, Duke University Medical Center, Durham,
NC, USA
MICHAEL D. MOORE, PHD • HIV Drug Resistance Program, National Cancer
Institute, Frederick, MD, USA
IMMACULATE NANKYA, MBCHB, PHD • Division of Infectious Diseases,
Department of Medicine, Department of Molecular Biology and Microbiology,
KENNETH N. NELSON, BA • Division of Infectious Diseases, Department of
Medicine, Case Western Reserve University, Cleveland, OH, USA
MAUREEN OLIVEIRA, BSC • McGill University AIDS Centre, Lady Davis Institute
AKIRA ONO, PHD • Department of Microbiology and Immunology, University of
Michigan Medical School, Ann Arbor, MI, USA
DAVID E. OTT, PHD • AIDS Vaccine Program, SAIC-Frederick, Inc.,
VINAY K. PATHAK, PHD • HIV-Drug Resistance Program and AIDS Vaccine
ERIC M. POESCHLA, MD • Molecular Medicine Program, Mayo Clinic College of
Medicine, Rochester, MN, USA
VINAYAKA R. PRASAD, PHD • Department of Microbiology and Immunology,
VASUDEV R. RAO, MBBS, MS • Department of Microbiology and Immunology,
ALAN REIN, PHD • HIV Drug Resistance Program, National Cancer Institute,
Frederick, MD, USA
MILAN REINIS, PHD • Institute of Molecular Genetics, Academy of Sciences of the
Czech Republic, Prague
ANDREA RUBIO, BSC • National Reference Center for AIDS, Department of
Microbiology, School of Medicine, University of Buenos Aires, Buenos Aires,
Argentina
KAORI SANGO, MS • Department of Microbiology and Immunology, Albert
Einstein College of Medicine, Bronx, NY, USA
BARBARA L. SHACKLETT, PHD • Department of Medical Microbiology and
Immunology and Division of Infectious Diseases, Department of Internal
Medicine, School of Medicine, University of California, Davis, CA, USA
ANDREW G. STEPHEN, PHD • Protein Chemistry Laboratory, Research Technology
BETH K. THIELEN, BS • Departments of Pathobiology and Medicine, University of
Washington, Seattle, WA, USA

xiv Contributors
JAMES A. THOMAS, PHD • AIDS Vaccine Program, SAIC-Frederick, Inc.,
RYAN M. TROYER, PHD • Department of Microbiology, Immunology and
Pathology, Colorado State University, Fort Collins, CO, USA
SUSANA T. VALENTE, PHD • Howard Hughes Medical Institute, Department of
Biochemistry and Molecular Biophysics, Columbia University, College of
Physicians and Surgeons, New York, NY, USA
ABDUL A. WAHEED, PHD • Virus-Cell Interaction Section, HIV Drug Resistance
Program, NCI-Frederick, National Institutes of Health, Frederick, MD, USA
MARK A. WAINBERG, PHD • McGill University AIDS Centre, Lady Davis Institute
BARBARA WEISER, MD • Wadsworth Center, New York State Department of
Health, and Albany Medical College, Albany, NY, USA
OTTO O. YANG, MD • Division of Infectious Diseases, Department of Medicine,
Department of Microbiology, Immunology, and Molecular Genetics and AIDS
Institute, Geffen School of Medicine, UCLA Medical Center, Los Angeles,
CA, USA

Section I
Preparation of Virus Particles and Their Analysis

Chapter 1
Methods for Viral RNA Isolation and PCR Amplification
for Sequencing of Near Full-Length HIV-1 Genomes
Kimdar Sherefa Kemal, Milan Reinis, Barbara Weiser, and Harold Burger
Abstract
HIV-1 in plasma represents the viral quasispecies replicating in the patient at any given time. Studies of
HIV-1 viral RNA from plasma or other body fluids therefore reflect the virus present in real time. To
obtain near full-length genomic sequences derived from virion RNA it is first necessary to carefully isolate
and amplify the RNA.
The procedure described below, involves viral RNA extraction, reverse transcription (RT) of the
extracted RNA to produce cDNA copies, and PCR amplification of long HIV-1 gene fragments using
site-specific, overlapping primers. The primers are based on subtype B HIV-1 strains, and plasma speci-
mens are used in the procedures. However, the protocol can easily be adapted to other HIV-1 subtypes
by modifying the primers to match the subtype of interest.
Key words: HIV-1, HIV-1 viral RNA, HIV-1 primers, Long RT-PCR amplification of HIV-1.
1. Introduction
HIV-1 infection is characterized by continuous replication of
∼ 9 kb RNA genomes resulting in a viral swarm of closely related
molecules called quasispecies (1, 2). Sequence variation is a hall-
mark of lentivirus infection; surviving viral species reflect replica-
tion and selection (3). When studying the relationship between
HIV-1 sequences and pathogenesis, it is highly desirable to ana-
lyze the complete HIV-1 genome because variability in multiple
regions of the genome may play a role in pathogenesis and virus–
host interactions (4–6). Full-length HIV-1 sequencing provides
essential data needed to address vaccine design and molecular
epidemiology (7, 8). Full-length sequence analysis also helps to
identify the presence of dual infections and recombination in vivo
Vinayaka R. Prasad, Ganjam V. Kalpana (eds.), HIV Protocols: Second Edition, vol. 485
C
2009 Humana Press, a part of Springer Science+Business Media
DOI 10.1007/978-1-59745-170-3 1 Springerprotocols.com
3

4 Kemal et al.
(9–11). Furthermore, knowledge of the complete genomic HIV-
1 RNA sequence and HLA type of the infected individual makes
it possible, with the use of an immunologic database (http://hiv-
web.lanl.gov/content/immunology/index), to predict cytotoxic
T-cell epitopes encoded by the virus (4,5).
HIV-1 in plasma represents the replicating virus population
at any given time; by contrast, proviral DNA, integrated in cel-
lular genomes, represents a repository of older sequences, the
majority of which have been shown to lack replication compe-
tence (12,13).
Amplification of a full-length HIV-1 genome requires careful
step-by-step procedures involving viral RNA extraction, synthesis
of cDNA copies of the viral RNA (RT-PCR), PCR amplification
of target gene fragments using specific primers, and the purifica-
tion of the amplified PCR products for further analyses. Successful
amplification of a near full-length HIV-1 viral RNA depends on
several factors, including viral RNA load in the specimen, quality
of specimen, viral RNA isolation methods used, cDNA synthesis
from RNA using RT-PCR, primer selection, and PCR amplifica-
tion conditions (6, 9, 14–17). The quality of the specimen, such
as plasma, is directly affected by factors such as storage temper-
ature, repeated freezing and thawing, and general handling of
the specimen from initial processing to the RNA extraction steps.
Although we cannot rule out the possibility of amplifying full-
length HIV-1 genomes from specimens with viral loads as low as
5,000 copies/mL, we recommend using samples with viral loads
of at least 10,000 copies/mL. Specimens should be aliquoted into
1 mL volume and stored at temperatures below −80 ◦C. Repeated
freeze and thaw steps need to be avoided (see Note 1 ).
2. Materials
2.1. HIV-1 Viral RNA
Isolation
1. RNAgentsR
Total RNA Isolation System kit (Promega). The
kit includes combined guanidine thiocyanate crystals and
the Citrate/Sarcosine/β-Mercaptoethanol (CSB) buffer in a
single bottle of denaturing solution. Phenol/chloroform/
isoamyl alcohol solution, isopropanol, 2 M sodium acetate
(pH 4.0), and nuclease free water are also included.
2. Yeast t-RNA (Sigma).
3. Glycogen (Sigma).
4. Siliconized Flat Top microfuge tubes (Fisher Scientific).
2.2. Reverse
Transcription and
cDNA Synthesis
SuperScript
TM
First-strand Synthesis System for RT-PCR kit
(Invitrogen).

PCR Amplification of Full-Length HIV-1 5
2.3. PCR
Amplification
1. GeneAmp XL PCR Kit (Applied Biosystems).
2. AmpliWax, PCR Gem 50 (Applied Biosystems).
3. 10 mM dNTP Mix with dTTP (Applied Biosystems).
4. TE buffer (pH 8.0).
2.4. PCR Primers All the PCR primers and PCR amplification programs specific for
each fragment are listed in Table 1.1.
2.5. Agarose Gel
Analysis
1. Agarose (Fisher Scientific).
2. 6X gel-loading buffer: 0.25% bromophenol blue, 0.25% xylene
cyanol, 30% glycerol, water up to desired volume. A stock
solution can be prepared and aliquots can be made in 1.5 mL
microfuge tubes and stored in a refrigerator. Once a tube is
taken out of the refrigerator, it can be stored at room temper-
ature and used.
3. Tris-Borate-EDTA (TBE) buffer: 0.45 M Tris-borate, 0.01 M
EDTA, pH 8.3.
4. Ethidium bromide tablets, 100 mg/tablet (Sigma). To have a
10 mg/mL stock solution, dissolve one tablet in 10 mL deion-
ized water and keep the solution at room temperature away
from direct light (see Note 2 ).
5. DNA ladder, 1 kb plus (Invitrogen).
6. Disposable scalpels.
7. QIAquick Gel Extraction Kit (Qiagen).
3. Methods
HIV-1 viral RNA isolation is one of the critical steps for the
success of the rest of the procedures. RNA should always be han-
dled with care; gloves should be worn at all times to help elim-
inate the introduction of endonucleases. It is important to work
in an RNase-free environment and use RNase-free reagents. Work
should be done in a Bio-safety level 2 (BSL-2) laminar flow hood;
RNA isolation areas should be separate from DNA or PCR ampli-
fication areas in the lab. It is also important to have dedicated
pipettes for RNA isolation. If this is not possible, always clean the
hood and the pipettes with RNase and DNA contaminant remov-
ing reagents and turn on the UV light, in the laminar flow hood,
for 20–30 min after use. To increase viral recovery, we suggest pel-
leting the plasma virions in siliconized microfuge tubes. We also
recommend using 0.5–1.0 mL sample volumes, particularly if the
viral load is low ( 10, 000 copies/mL). When available, 1 mL is
preferred. Using this method, RNA for full-length amplification
can be recovered from plasma samples with viral loads as little as
5,000 RNA copies/mL (see Note 3 ).

6 Kemal et al.
Table 1.1
PCR primers and amplification conditions used to amplify a 9-kb HIV-1 subtype
B genome as four overlapping fragments
Primer Primer sequences (5 − 3) HXB2 location
Fragment 1/partial 5 LTR and gag
517FA CTT-AAG-CCT-CAA-TAA-AGC-TTG-CCT-TGA 517–543B
2348RC TAC-TGT-ATC-ATC-TGC-TCC-TGT-ATC 2348–2325
548F TCA-AGT-AGT-GTG-TGC-CCG-TCT-G 549–570
2314R TCC-TTT-AGT-TGC-CCC-CCT-ATC 2314–2294
PCR conditions: 1X 94 ◦C: 5 min
5X 94 ◦C: 15 s, 45 ◦C: 45 s, 72 ◦C: 3 min
30X 94 ◦C: 15 s, 55 ◦C: 45 s, 72 ◦C: 3 min
1X 72 ◦C: 10 min
hold at 4 ◦C: until next step
Fragment 2/pol
1999F AAT-TGC-AGG-GCC-CCT-AGA-AAA-AAG-GGC-TGT 1999–2028
5304R TTC-TAT-GGA-GAC-TCC-CTG-ACC-CAA-ATG-CCA 5304–5275
2138F AGA-GCA-GAC-CAG-AGC-CAA-CAG 2138–2158
5202R TCC-CCT-AGT-GGG-ATG-TGT-ACT 5222–5202
5X 94 ◦C: 15 s, 45 ◦C: 45 s, 72 ◦C: 5 min;
30X 94 ◦C: 15 s, 55 ◦C: 60 s, 72 ◦C: 4 min;
1X 72 ◦C: 10 min
Fragment 3/env and accessory genes
4650F ATT-CCC-TAC-AAT-CCC-CAA-AGT-CAA-G 4650–4674
9626R CTT-GAA-GCA-CTC-AAG-GCA-AGC-TTT-ATT-G 9626–9599
4956F TGG-AAA-GGT-GAA-GGG-GCA-GTA-GTA-ATA-CAA-G 4956–5014
9173R TGG-TGT-GTA-GTT-CTG-CCA-ATC-AGG-GAA-G 9173–9146
5X 94 ◦C: 15 s, 55 ◦C: 45 s, 72 ◦C: 5 min;
30X 94 ◦C: 15 s, 60 ◦C: 5 min
1X 72 ◦C: 10 min
(continued)

PCR Amplification of Full-Length HIV-1 7
Table 1.1
(continued)
Fragment 4/nef and LTR
8081F TGG-AAT-AAC-ATG-ACC-TGG-AGG-G 8091–8112
9626R CTT-GAA-GCA-CTC-AAG-GCA-AGC-TTT-ATT-G 9626–9599
8296F CAT-AAT-GAT-AGT-AGG-AGG-CTT-GG 8279–8301
9608R TGA-AGC-ACT-CAA-GGC-AAG-C 9608–9590
35X 94 ◦C: 15 s; 55 ◦C: 45 s; 72 ◦C: 3 min
1X 72 ◦C: 10 min
AF: stands for forward primers, the numbers indicate the nucleotide positions of the for-
ward or reverse genome fragment.
BIndicates nucleotide positions in HIV-1 HXB2 strain.
CR: stands for reverse primers.
3.1. RNA Extractions
3.1.1. Beginning
1. Clarify plasma by spinning at 400 × g, at room temperature,
for 10 min (see Note 4 ).
2. Transfer the plasma to sterile, siliconized, 1.5 mL microfuge
tubes.
3. To pellet virions, centrifuge the plasma 18, 500 × g, at 10 ◦C,
for 90 min.
4. Remove most of the now virion-free plasma without disturb-
ing the pellet (see Note 5 ).
5. To extract virion-associated RNA, re-suspend the pellet in
300 μL pre-chilled denaturing solution.
6. Vortex the pellet until it is completely dissolved. This usually
takes 5–10 min (see Note 6 ).
7. Mix in order: 1 μL yeast tRNA (1 μg/μL), 1 μL glycogen
(2 μg/μL) and 30 μL of 2 M sodium acetate, pH 4.0 (see
Note 7 ).
8. Add 32 μL of the above mixture into each tube, and then add
0.5 mL phenol/chloroform/isoamyl alcohol solution, being
careful to remove only from the lower organic phase (see
Note 8 ).
9. Vortex for 10 s, then chill on ice for 20 min.
10. Centrifuge at 18, 500 × g for 20 min, at 4 ◦C.
11. Transfer the top phase, which contains the RNA, to fresh
microfuge tubes (see Note 9 ).
12. To precipitate the RNA, add an equal volume of isopropanol
(0.3–0.5 mL) and keep the tubes at −20 ◦C for at least 2 h
(see Note 10 ).

8 Kemal et al.
3.1.2. After 2 h or the
Next Day
1. Pellet the RNA by centrifugation at 18, 500 × g for 30 min at
4 ◦C. Discard the supernatant.
2. Re-suspend the RNA pellet in 200 μL denaturing solution.
Vortex until RNA is dissolved; usually 1 min is enough.
3. Add 300 μL isopropanol and precipitate the RNA at −20 ◦C
for at least 2 h.
4. After 2 h, pellet the RNA by centrifugation at 18, 500 × g for
25 min at 4 ◦C.
5. Wash the pellet, once with 1 mL and then with 0.5 mL of 75%
ice-cold ethanol, by centrifugation at 18, 500 × g for 20 min
at 4 ◦C each time. During each wash break up the pellet by
using RNase-free pipette tips before each centrifugation; do
not vortex (see Note 11 ).
6. Air-dry the pellet for 5–20 min, depending on the amount of
ethanol left in the tubes or the size of the pellet.
7. Dissolve the RNA in 25 μL nuclease-free water by using the
pipette tips. Do not vortex (see Note 12 ).
8. Store the RNA at −20 ◦C until use. For long-term use store at
−80 ◦C.
3.2. Reverse
Transcription PCR
(RT-PCR)
The procedure used for RT-PCR is essential for synthesis of a long
cDNA. The procedure is given below, with details of the reagents
given in Tables 1.2 and 1.3. There are a number of commer-
cial kits available for RT-PCR and each laboratory has its own
preferences. The method described here uses the SuperScript
TM
III First-Strand Synthesis System for RT-PCR kit (Invitrogen).
SuperScript
TM
III Reverse Transcriptase enzyme is used to syn-
thesize cDNA at the temperature range of 42–55 ◦C, providing
increased specificity, higher yields of cDNA, and more full-length
product than other reverse transcriptase enzymes (see Note 13 ).
We recommend using oligo(dT) primer, as it is more specific and
allows the synthesis of full-length cDNA fragments.
1. Prepare RNA/Primer master mix which includes oligo(dT)20
and the dNTP mix according to Table 1.2.
2. Dispense 10 μL of the master mix into each tube.
Table 1.2
RNA/primer master mix
Component Volume Final concentration
50 μM oligo(dT)20 1 μL 2. 5 μM
10 mM dNTP mix 1 μL 500 μM
Nuclease free water 5 μL –

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248.
249.
250.
251.
252.
Virtue, says Montesquieu, etc. Esprit des Lois, III. 6.
‘Honour dishonourable.’ Paradise Lost, IV. 314–15.
‘Of outward shew,’ etc. Cf. Ibid. VIII. 538–9.
‘To tread,’ etc. Hamlet, Act I. Sc. 3.
‘Nice customs,’ etc. Henry V. Act V. Sc. 2.
‘In form and motion,’ etc. Cf. Hamlet, Act II. Sc. 2.
‘Vice is undone,’ etc. Pope, Epilogue to the Satires, I. 142–9.
A Coronation-day. The coronation of George IV. had taken
place on July 19, 1821.
Prince Leopold. Prince Leopold of Saxe-Coburg (1790–1865),
who had married the Princess Charlotte, and afterwards
(1831) became King of the Belgians.
Castlereagh ... unstained, etc. Castlereagh committed suicide
on Aug. 12, 1822.
‘A present deity,’ etc. Dryden, Alexander’s Feast, 35–6.
‘Worth makes the man,’ etc. Pope, An Essay on Man, IV.
203–4.
‘The only amaranthine flower,’ etc. Cowper, The Task, III.
268–9.
‘A man may read,’ etc. Holy Dying, chap. i. § 2.

PAG
E
253.
253.
254.
255.
ON THE SCOTCH CHARACTER
Now republished for the first time. See Mr. W. C. Hazlitt’s
Memoirs, etc. (1867), I. xxvii.
‘Edina’s darling seat.’ ‘Edina! Scotia’s darling seat!’ Burns,
Address to Edinburgh.
Lismahago. In Humphry Clinker.
Lord Erskine. Lord Erskine was entertained at a banquet in
Edinburgh on Feb. 21, 1820. He had not been in Scotland
for more than fifty years.
Teres et [atque] rotundus. Horace, Satires, II. vii. 86.
A very learned man. (?) Sir David Brewster, editor of The
Edinburgh Encyclopædia. Cf. post, p. 316.
Mr. Macvey Napier. Macvey Napier (1776–1847), editor of a
supplement to the 4th, 5th, and 6th editions and of the 7th
edition of The Encyclopædia Britannica, and Jeffrey’s
successor as editor of The Edinburgh Review. Hazlitt had
contributed to the Supplement. See vol. IX. (Essays on the
Fine Arts), p. 377 and note. In A Selection from the
Correspondence of the late Macvey Napier, Esq. (1879), p.
21, there is the following letter from Hazlitt to Napier:—
‘Winterslow Hut, near Salisbury,
‘August 26, 1818.
‘My dear Sir,—I am sorry to be obliged, from want of health
and a number of other engagements, which I am little able
to perform, to decline the flattering offer you make me. I
have got to write, between this and the end of October, an
octavo volume or a set of lectures on the Comic Drama of
this country for the Surrey Institution, which I am anxious

256.
258.
not to slur over, and it will be as much as I can do to get it
ready in time. I am also afraid that I should not be able to
do the article in question, or yourself, justice, for I am not
only without books, but without knowledge of what books
are necessary to be consulted on the subject. To get up an
article in a Review on any subject of general literature is
quite as much as I can do without exposing myself. The
object of an Encyclopædia is, I take it, to condense and
combine all the facts relating to a subject, and all the
theories of any consequence already known or advanced.
Now, where the business of such a work ends, is just where
I begin, that is, I might perhaps throw in an idle
speculation or two of my own, not contained in former
accounts of the subject, and which would have very little
pretensions to rank as scientific. I know something about
Congreve, but nothing at all of Aristophanes, and yet I
conceive that the writer of an article on the Drama ought to
be as well acquainted with the one as the other. If you
should see Mr. Constable, will you tell him I am writing
nonsense for him as fast as I can?—Your very humble
servant,
W. HAZLITT.’
It is difficult to know what ‘nonsense’ Hazlitt was writing for
Constable.
‘Damnable iteration.’ 1 Henry IV., Act I. Sc. 2.
Not like La Fleur, etc. See Sterne, The Sentimental Journey,
The Passport, Paris.
Note 1. Cockney School of Poetry. See vol. VI. (Table-Talk), 99
and note.
Note 1. ‘Kernes and Gallowglasses.’ Macbeth, Act I. Sc. 2.
‘Sins,’ etc. Cf. Hebrews xii. 1.
A much-talked-of publication. Hazlitt no doubt refers to The
Beacon, which, like John Bull, was intended to counteract
the progress of Radical doctrine during the period of the

259.
Queen’s trial. For an account of it and of Scott’s connection
with it, see Lockhart’s Life of Scott, v. 152–3.
‘Leaning,’ etc. Cf. The Faerie Queene, I. vi. 14.
The editor. Theodore Hook, the editor of John Bull, was an
Englishman.
‘Entire affection,’ etc. Cf. The Faerie Queene, I. viii. 40.

PAG
E
259.
260.
262.
263.
264.
265.
266.
MY FIRST ACQUAINTANCE WITH POETS
Republished in Literary Remains and Winterslow. The germ of
the essay appeared in a short letter to The Examiner, reprinted in
Political Essays. See vol. III. pp. 152–3 and notes.
W——m. Wem.
‘Dreaded name,’ etc. Paradise Lost, II. 964–5.
‘Fluttering,’ etc. Cf. Coriolanus, Act V. Sc. 6.
‘High-born Hoel’s harp,’ etc. Gray, The Bard, 28.
‘Bound them,’ etc. Pope, Ode on St. Cecilia’s Day, 90–91.
The fires in the Agamemnon. Cf. ante, p. 240 and note.
It was in January, etc. This paragraph and the next are from
The Examiner. See the notes to vol. III. (Political Essays),
pp. 152–3.
‘As are the children,’ etc. Cf. Thomson, The Castle of
Indolence, II. xxxiii.
‘A certain tender bloom,’ etc. Cf. ante, p. 207 and note.
‘Somewhat fat and pursy.’ Cf. ‘He’s fat and scant of breath’
(Hamlet, Act V. Sc. 2), and ‘For in the fatness of these pursy
times,’ etc. (Ibid. Act III. Sc. 4).
‘No figures,’ etc. Julius Cæsar, Act II. Sc. 1.
Note 1. For an account of the Rev. William Hazlitt, see Mr. W.
C. Hazlitt’s Four Generations of a Literary Family, The
First Generation.
T. Wedgwood. A Life of Tom Wedgwood was published
recently (1903) by the late Mr. R. B. Litchfield.
‘Sounding on his way.’ See vol. IV. (The Spirit of the Age),
note to p. 214.
Credat Judæus Apella! Horace, Satires, I. v. 100.

267.
268.
269.
270.
271.
272.
273.
274.
‘Thus I refute him, Sir.’ See Boswell’s Life (ed. G. B. Hill), I.
471.
‘Kind and affable,’ etc. Cf. Paradise Lost, VIII. 648–50.
He has somewhere told himself. See Biographia Literaria,
chap. x.
That other Vision of Judgment. Byron’s, first published in
The Liberal, No. 1.
Bridge-street junto. Cf. vol. VI. (Table-Talk), p. 190 and note.
Tom Jones and the adventure of the muff. See Tom Jones,
Book X. chap. v. et seq.
At Tewkesbury. According to the essay ‘On Going a Journey,’
it was at Bridgwater. See vol. VI. (Table-Talk), p. 186.
A friend of the poet’s. This is a mistake. Wordsworth paid £23
a year for Alfoxden. The agreement is given in Mrs. Henry
Sandford’s Thomas Poole and his Friends, I. 225.
‘In spite of pride,’ etc. Pope, An Essay on Man, I. 293.
‘While yet,’ etc. Cf. Thomson, The Seasons, Spring, 18.
‘Of Providence,’ etc. Paradise Lost, II. 559–560.
Chantry’s bust. Sir Francis Chantrey’s bust, now at Coleorton.
Castle Spectre. Originally produced (at Drury Lane)
December 14, 1797.
‘His face,’ etc. Cf. Macbeth, Act I. Sc. 5.
Tom Poole. Thomas Poole (1765–1837), for an account of
whom see Mrs. Sandford’s Thomas Poole and his Friends.
‘Followed in the chase,’ etc. Cf. Othello, Act II. Sc. 3.
Sir Walter Scott’s, etc. Hazlitt probably refers to the banquet
given to George IV. by the Magistrates of Edinburgh, August
24, 1822.
The Death of Abel. Solomon Gessner’s Tod Abels (1758).

275.
‘Ribbed sea-sands.’ The Ancient Mariner, 227. This was one
of the lines for which Coleridge was indebted to
Wordsworth.
‘But there is matter,’ etc. Wordsworth, Hart-leap Well, 95–
96.

PAG
E
276.
277.
280.
281.
282.
PULPIT ORATORY, ETC.
Now reprinted for the first time. See Mr. W. C. Hazlitt’s Memoirs,
etc., I. xxvii. Cf. the essay on Edward Irving in The Spirit of the Age
(vol. IV. pp. 222–231). After Hazlitt’s essay there follows a savage
attack on Irving (? by T. J. Hogg), as to which the editor says: ‘The
following has also lost its way to us. We take it in as a foundling, but
without adopting all its sentiments.’
‘Got the start,’ etc. Cf. Julius Cæsar, Act I. Sc. 2.
‘Kingly Kensington.’ Swift’s Ballad, Duke Upon Duke, St. 14.
Lady Bluemount. Lady Beaumont presumably, the wife of
Wordsworth’s friend, Sir George Howland Beaumont.
Mr. Botherby.? William Sotheby (1757–1833), whose
persistent attempts as a dramatic author may explain the
nickname.
Mr. Theodore Flash. Theodore Hook, no doubt, who
afterwards denounced Irving as a humbug. See John Bull,
July 20, 1823.
Note. Mr. Dubois. Edward Dubois (1774–1850), wit and
journalist.
Note. ‘Rose,’ etc. Cf. Hamlet, Act III. Sc. 1.
‘His foot mercurial,’ etc. Cymbeline, Act IV. Sc. 2.
‘The iron,’ etc. The Psalter, Psalm CV. 18.
‘Come, let me clutch thee.’ Macbeth, Act II. Sc. 1.
‘Spins,’ etc. Cf. Love’s Labour’s Lost, Act V. Sc. 1.
‘Loop or peg,’ etc. Cf. Othello, Act III. Sc. 3.
‘Fire hot from Hell.’ Cf. Julius Cæsar, Act III. Sc. 1.
The swimmer. See this passage quoted by Hazlitt in vol. V.
(Lectures on the Age of Elizabeth), pp. 323–4.

283.
284.
285.
Mr. Croly. George Croly (1780–1860), a regular contributor
to Blackwood’s Magazine, had published Paris in 1815
(1817).
‘Best virtue.’ Cf. All’s Well That Ends Well, Act IV. Sc. 3.
‘We pause for a reply.’ Cf. Julius Cæsar, Act III. Sc. 2.
Daniel Wilson. Daniel Wilson (1778–1858), at this time
incumbent of St. John’s Chapel, Bedford Row, Bloomsbury,
afterwards Bishop of Calcutta.
‘Oh! for an eulogy,’ etc. Cf. ‘Oh, for a curse to kill with.’
Otway, Venice Preserved, Act II. Sc. 2.

PAG
E
285.
286.
287.
288.
289.
ARGUING IN A CIRCLE
Now reprinted for the first time. See Mr. W. C. Hazlitt’s Memoirs,
etc., I. xxvii.
‘Fancies and good-nights.’ Cf. 2 Henry IV., Act III. Sc. 2.
‘Base cullionly fellow.’ Cf. 2 Henry VI., Act I. Sc. 3.
‘Beggarly, unmannered corse.’ Cf. 1 Henry IV. Act I. Sc. 3.
‘The age of chivalry,’ etc. Cf. Burke, Reflections on the
Revolution in France (Select Works, ed. Payne, II. 89).
‘The melancholy Jacques,’ etc. As You Like It, Act II. Sc. 1.
The present Duke of Buckingham. Richard Temple Nugent
Brydges Chandos, created Duke of Buckingham and
Chandos, Feb. 1822.
‘New manners,’ etc. Thomas Warton, Sonnet, Written in a
Blank Leaf of Dugdale’s Monasticon.
‘Submits,’ etc. Burke, Reflections on the Revolution in France
(Select Works, ed. Payne, II. 90).
‘Long insulted,’ etc. Quoted elsewhere. See vol. III. (Political
Essays), pp. 13 and 100.
‘With jealous leer malign.’ Paradise Lost, IV. 503.
‘Cause was hearted.’ Cf. Othello, Act I. Sc. 3.
‘The open,’ etc. Cf. Paradise Lost, X. 112–113.
‘The shame,’ etc. Cf. 2 Samuel i. 16.
The Editor of the New Times. Dr. Stoddart.
‘Make the worse,’ etc. Paradise Lost, II. 114.
‘So musical,’ etc. A Midsummer Night’s Dream, IV. 1.

290.
291.
292.
293.
294.
295.
‘So well,’ etc. Cf. Paradise Lost, IX. 549.
Mr. Canning’s present ... situation. Canning had become
Foreign Secretary in 1822, and had shortly afterwards
acknowledged the independence of the Spanish American
Colonies.
‘Turnspit of the king’s kitchen.’ See Burke’s ‘Speech on
Economical Reform,’ (Works, Bohn, II. 85–86), and cf. vol.
I. (The Round Table), p. 427.
‘Undoing all,’ etc. 2 Henry VI., Act I. Sc. 1.
‘Though that their joy,’ etc. Cf. Othello, Act I. Sc. 1.
‘Like an exhalation,’ etc. Cf. Comus, 556.
‘Ride in the whirlwind,’ etc. Addison, The Campaign, and
Pope, The Dunciad, III. 264.
Noctes, etc. Horace, Satires, II. vi. 65.
‘The beautiful,’ etc. Coleridge, The Death of Wallenstein, Act
V. Sc. 1.
‘A thick scarf.’ See ante, note to p. 82.
‘Sweet smelling gums.’ Paradise Lost, XI. 327.
‘Dews of Castalie.’ Cf. Spenser, The Ruines of Time, 431.
The Six Acts. Passed by Lord Sidmouth in 1819 after the
Manchester reform meeting.

PAG
E
297.
QUERIES AND ANSWERS; OR THE RULE OF
CONTRARY
Now republished for the first time. See Mr. W. C. Hazlitt’s
Memoirs, etc. (1867), I. xxix.
Thimble. Cf. a passage, ante, at the foot of p. 39. The editors
have not been able to identify the person here referred to as
‘Thimble.’

298.
299.
300.
301.
ON KNOWLEDGE OF THE WORLD
This paper and the two following ones were republished in
Sketches and Essays.
‘Who shall go about,’ etc. Cf. The Merchant of Venice, Act II.
Sc. 9.
‘Subtle,’ etc. Cf. Cymbeline, Act III. Sc. 3.
‘The children,’ etc. Cf. S. Luke xvi. 8.
‘To see ourselves,’ etc. Burns, To a Louse, St. 8.
‘No figures,’ etc. Cf. Julius Cæsar, Act II. Sc. 1.
‘His soul,’ etc. Pope, An Essay on Man, I. 101–2.
‘What shall it profit,’ etc. S. Mark viii. 36.
Non ex quovis, etc. Erasmus, Adagiorum Chiliades, ‘Munus
aptum.’
‘No mark,’ etc. 1 Henry IV., Act III. Sc. 2.
‘The soul,’ etc. Cf. Othello, Act I. Sc. 3.

PAG
E
301.
302.
303.
304.
305.
THE SAME SUBJECT CONTINUED
Bub Doddington said, etc. Cf. vol. VI. (Table-Talk), p. 100 and
note.
Salus populi, etc. The Twelve Tables, De Officio Consulis.
The upstart, etc. This sentence was omitted in Sketches and
Essays.
Mr. Cobbett seemed disappointed, etc. The reference is
probably to The Weekly Political Register for Oct. 29, 1825,
where Cobbett deplores the fact that Baron Maseres (1731–
1824), who had visited him in prison, had left the bulk of
his large property to a ‘little Protestant parson.’
‘His patron’s ghost,’ etc. Cf. Thomson, The Castle of
Indolence, I. St. 51.
‘Never standing upright,’ etc. See Macklin’s The Man of the
World, II. 1.
‘In large heart enclosed.’ Cf. Paradise Lost, VII. 486.
‘The world,’ etc. Thomson, The Seasons, Autumn, 233.
‘The heart of man,’ etc. Cf. Jeremiah xvii. 9.
‘As the flesh,’ etc. Cf. Measure for Measure, Act II. Sc. 1.
‘Tread,’ etc. Cf. Hamlet, Act I. Sc. 3.
‘If thine eye,’ etc. Cf. S. Matthew v. 29.
‘The little chapel-bell,’ etc. Hazlitt refers to The Chapel Bell,
an early poem of Southey’s (1793), and The Book of the
Church, published by Southey in 1824.
Camille-Desmoulins, etc. Camille Desmoulins (1760–1794),
the well-known Revolutionary pamphleteer; Camille
Jordan (1771–1821), called ‘Jordan Carillon,’ from a speech
(July 4, 1797) in which he proposed to restore the use of
bells to the clergy. See Hazlitt’s Life of Napoleon, chap. 15.

‘His own miniature-picture,’ etc. ‘On my own Miniature
Picture’ (1796).

306.
307.
308.
309.
310.
THE SAME SUBJECT CONTINUED
‘Give us pause.’ Hamlet, Act III. Sc. 1.
‘Does somewhat smack.’ Cf. The Merchant of Venice, Act II.
Sc. 2.
Peter Finnerty. Peter Finnerty (1766?–1822) at one time on
the staff of The Morning Chronicle with Hazlitt.
J——. Jeffrey.
‘In some sort handled.’ Cf. Henry V. Act II. Sc. 3.
‘The high and palmy state.’ Cf. Hamlet, Act I. Sc. 1.
‘Keep this dreadful pudder,’ etc. King Lear, Act III. Sc. 2.
‘When a great wheel,’ etc. Cf. Ibid. Act II. Sc. 4.
‘Will be,’ etc. Dr. Johnson, Preface to Shakespeare (Works,
Oxford, 1825, vol. V., p. 118).

311.
312.
313.
314.
315.
ON PUBLIC OPINION
Published (together with the next essay) in Winterslow.
‘Scared,’ etc. Cf. Collins’s Ode, The Passions, 20.
‘The world rings,’ etc. Cowper, The Task, III. 129–30.
‘No man knoweth,’ etc. Cf. S. John iii. 8.
‘Casting,’ etc. Il Penseroso, 160.
‘Wink,’ etc. Cf. Marston, Antonio’s Revenge, Prologue.
‘Fed fat,’ etc. Cf. The Merchant of Venice, Act I. Sc. 3.

PAG
E
316.
317.
318.
320.
ON THE CAUSES OF POPULAR OPINION
Published (with preceding essay) in Winterslow.
The Editors of the Edinburgh Encyclopædia. The Edinburgh
Encyclopædia (18 vols., 1810–30) was edited by Sir David
Brewster.
‘Among the rocks,’ etc. Cf. Michael, 455–7.
‘A man of ten thousand.’ Cf. Hamlet, Act II. Sc. 2.
‘Who loved,’ etc. Othello, Act V. Sc. 2.
J——. Jeffrey.

PAG
E
321.
322.
323.
324.
325.
A FAREWELL TO ESSAY-WRITING
Republished in an imperfect form in Winterslow. In the Magazine
the essay is dated ‘Winterslow, Feb. 20, 1828.’
‘This life is best,’ etc. Cymbeline, Act III. Sc. 3.
‘A friend,’ etc. Cf. Cowper, Retirement, 741–2.
‘Done its spiriting gently.’ Cf. The Tempest, Act I. Sc. 2.
‘The spring,’ etc. Coleridge, Christabel, 22.
‘Fields are dank,’ etc. Milton’s Sonnet (XX.), ‘Lawrence, of
virtuous father virtuous son.’
‘Peep,’ etc. Cf. Macbeth, Act I. Sc. 5.
‘Open,’ etc. Cowper, The Task, VI. 11–12.
‘Of all the cities,’ etc. Dryden, Theodore and Honoria, 1–2.
‘Which when Honoria view’d,’ etc. Ibid. 342–3.
‘And made th’ insult,’ etc. Dryden, Sigismonda and
Guiscardo, 668–9.
I am much pleased, etc. This sentence (to the end of the
paragraph) was omitted in Winterslow.
‘Fall’n,’ etc. Scott, Glenfinlas, last stanza.
Mr. Gifford once said, etc. See vol. IV. (The Spirit of the Age)
p. 307.
I am rather disappointed, etc. This sentence was omitted in
Winterslow.
‘The admired,’ etc. Cf. Hamlet, Act III. Sc. 1.
What I have here stated, etc. This paragraph and the next two
were omitted in Winterslow.

326.
327.
328.
‘I know not seems.’ Hamlet, Act I. Sc. 2.
L——. Lamb, no doubt.
Antonio. Godwin’s Antonio was produced at Drury Lane and
damned Dec. 13, 1800.
‘Nor can I think,’ etc. Dryden, The Hind and the Panther, I.
315.
Chaucer’s Flower and Leaf. See vol. V. (Lectures on the
English Poets) p. 27 and note.
‘And ayen,’ etc. The Flower and the Leaf, St. 15.
Mr. and Miss L——. Charles and Mary Lamb.
‘And curtain close,’ etc. Cf. Collins’s Ode, On the Poetical
Character, 76.

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