13
HIV Protocols
HIV Protocols
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 TM
John M. Walker, SERIES EDITOR
31. Hemostasis and Thrombosis Protocols: Methods in Molecular Medicine, edited by David J. Perry and K. John Pasi, 1999
30. Vascular Disease: Molecular Biology and Gene Therapy Protocols, edited by Andrew H. Baker, 1999
29. DNA Vaccines: Methods and Protocols, edited by Douglas B. Lowrie and Robert Whalen, 1999
28. Cytotoxic Drug Resistance Mechanisms, edited by Robert Brown and Uta B6ger-Brown, 1999
27. Clinical Applications of Capillary Electrophoresis, edited by Stephen M. Palfrey, 1999
26. Quantitative PCR Protocols, edited by Bernd Kochanowski and Udo Reischl, 1999
25. Drug Targeting, edited by G. E. Francis and Cristina Delgado, 1999
24. Antiviral Methods and Protocols, edited by Derek Kinchington and Raymond F. Schinazi, 1999
23. Peptidomimetics Protocols, edited by Wieslaw M. Kazmierski, 1999
22. Neurodegeneration Methods and Protocols, edited by Jean Harry and Hugh A. Tilson, 1999
21. Adenovirus Methods and Protocols, edited by William S. M. Wold, 1998
20. Sexually Transmitted Diseases: Methods and Protocols, edited by Rosanna Peeling and P. Frederick Spatting, 1999
19. Hepatitis C Protocols, edited by Johnson Yiu-Nam Lau, 1998
18. Tissue Engineering, edited by Jeffrey R. Morgan and Martin L. Yarmush, 1999
17. HIV Protocols, edited by Nelson Michael and Jerome H. Kim, 1999
16. Clinical Applications of PCR, edited by E M. Dennis Lo, 1998
15. Molecular Bacteriology: Protocols and Clinical Applications, edited by Neil Woodford and Alan Johnson, 1998
14. Tumor Marker Protocols, edited by Margaret Hanausek and Zbigniew Walaszek, 1998
13. Molecular Diagnosis of Infectious Diseases, edited by Udo Reischl, 1998
12. Diagnostic Virology Protocols, edited by John R. Stephenson and Alan Warnes, 1998
11. Therapeutic Application of Ribozymes, edited by Kevin J. Scanlon, 1998
10. Herpes Simplex Virus Protocols, edited by S. Moira Brown and Alasdair MacLean, 1998
9. Leetin Methods and Protocols, edited by Jonathan M. Rhodes and Jeremy D. Milton, 1998
8. Helicobacter pylori Protocols, edited by Christopher L. Clayton and Harry L. T. Mobley, 1997
7. Gene Therapy Protocols, edited by Paul D. Robbins, 1997
6. Molecular Diagnosis of Cancer, edited by Finbarr Cotter, 1996
HIV Protocols
Edited by
Nelson L. Michael, MD, PhD Walter Reed Army Institute of Research, Rockville, MD
and
Jerome H. Kim, MD Henry M. Jackson Foundation, Rockville, MD
Humana Press ~ Totowa, New Jersey
© 1999 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512
All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular Medicine TM is a trademark of The Humana Press Inc.
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Cover illustration: Taken from The Sourcebook of Medical Illustrations, edited by Peter Cull. Copyright 1989 by the Medical College of St. Bartholomew's Hospital, London, UK. Published in the United States by The Parthenon Publishing Group, Inc., Park Ridge, NJ.
Cover design by Patricia F. Cleary.
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Library of Congress Cataloging in Publication Data
Main entry under title:
Methods in molecular medicine,s.
HIV protocols / edited by Nelson Michael and Jerome H. Kim. p. cm.--(Methods in molecular medicine ; vol. 17)
Includes index. ISBN 0-89603-369-4 (alk. paper) 1. AIDS (Disease)--Laboratory manuals. 2. HIV (Viruses)--Laboratory manuals. 3. HIV infections--Laboratory manuals. 1. Michael, Nelson. 1I. Kim Jerome H. III. Series. [DNLM: 1. HIV Infections---prevention & control. 2. Clinical Protocols. 3. Molecular Biology--methods. 4. Immunologic Techniques. 5. Virology--methods. WC 503.6H6765 1998] QR201.A37H56 1998 616.97'92--dc21 DNLM/DLC 98-27244 for Library of Congress C1P
Preface
The worldwide impact of infection with human immunodeficiency virus type 1 (HIV- 1) is reflected in the cumulative number ofHIV- 1 infections, which is now predicted to exceed 40 million by the year 2000---equivalent to the num- ber of humans who perished in World War II. The medical and scientific re- sponse to the HIV-1 pandemic has steadily grown since its recognition in 1981. The outlay by the United States alone for HIV research funded by the National Institutes of Health in 1997 was $1.4 billion. Laboratory-based HIV research has brought together academic clinicians, retrovirologists, molecular biologists, and immunologists in the formation of research teams attempting to dissect the viral and host factors contributing to disease pathogenesis. Increasing focus is being placed on those aspects of viral biology and host immune responses that bear on the development of vaccines to prevent HIV infection.
HIVProtocols reflects the state of HIV research in several ways. First, chapters are organized into four sections: Virology, Molecular Biology, Humoral Immunology, and Cellular Immunology. This organization is a natural consequence of the diverse scientific disciplines that have been attracted to HIV research. Second, the chapters reflect such diverse research directions as viral coreceptor usage, quantitation of viral genomes, HIV promoter function, B-cell epitope mapping, and measurements of T-cell function, each of which bears on the goal of understanding the viral and host immune responses that will be critical to the design of effective preventive vaccines. Third, HIV Protocols includes several chapters on the analysis of HIV-1 entry coreceptors only about two years after the first of these receptors was discovered, which is indicative of the explosion of research on these molecules.
All chapters in HIVProtocols open with an introduction to both a specific technique and its role in the field of HIV research. Following a thorough list of materials, a highly detailed methods section presents the technique in a clear, understandable fashion to ensure the sucessful execution of the protocol. This is followed by a Notes section that we believe many readers will perceive to be the core of these chapters. The Notes essentially go beyond the standard proto- col to unlock the specifics or "tricks" inherent in the sucessful execution of the technique. A brief series of references rounds off each chapter.
V
vi Preface
Thanks are due to all of the contributing authors for the efforts each of them made in writing both highly detailed and understandable chapters. The guidance, and forbearance, of Professor John M. Walker, editor of Methods in Molecular Medicine series, and Mr. Thomas Lanigan, President of Humana Press, were also instrumental in bringing HIYProtocols to fruition.
Nelson L. Michael
Jerome H. Kim
Contents
Preface ............................................................................................................. v Contributors ..................................................................................................... xi
PART I. VIROLOGY 1 Isolation and Expansion of HIV from Cells and Body Fluids
by Coculture James R. Lane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2 Quantitative HIV Culture James R. Lane ...................................................................................... 11
3 Quantitation of Cell-Free HIV by Reverse Transcriptase Activity James R. Lane ...................................................................................... 17
4 Determination of Syncytium-lnducing Phenotype of Primary HIV-1 Isolates Using MT-2 Cells
Mark K. Louder and John R. Mascola ............................................... 23
5 Determination of HIV-1 Chemokine Coreceptor Tropism Using Transduced Human Osteosarcoma (HOS) Cells
Mark K. Louder and John R. Mascola ............................................... 29
6 Use of Luciferase Reporter Viruses for Studying HIV Entry Rong Liu, Benjamin Chen, and Nathaniel R. Landau ..................... 35
7 A Cell-Cell Fusion Assay to Monitor HIV-1 Env Interactions with Chemokine Receptors
Aimee L. Edinger and Robert W. Doms ............................................ 41 8 Methods of Culturing HIV-1 from Semen
Bruce L. Gilliam ................................................................................... 51
PART II. MOLECULAR BIOLOGY
9 Detection of HIV-1 Nucleic Acids by Southern Blotting Richard A. McDonald .......................................................................... 61
10 Detection of HIV-1 Nucleic Acids by Northern Blotting Richard A. McDonald and Christopher A. D. Smith ........................ 71
viii Contents
11 Rapid Identification of Cloned HIV-1 Fragments Wei Dong, Anindita Kar Roy, and Yen Li .......................................... 83
12 PCR Amplification and Cloning of Virtually Full-Length HIV-1 Provirus
Mika Salminen ...................................................................................... 89
13 Quantitation of HIV-lgag DNA and RNA From Single Frozen Cell Pellets
Maryanne Vahey, Sandra C. Barrick, and Martin Nau ..................... 99
14 Quantitative PCR for HIV-1 Proviral DNA Davide Zella and Paola Secchiero ................................................... 119
15 Diagnosis and Direct Automated Sequencing of HIV-1 From Dried Blood Spots (DBS) Collected on Filter Paper
Sharon A. Cassol, Stanley Read, Bruce G. Weniger, Richard Pilon, Barbara Leung, and Theresa Mo ....................... 125
16 Quantification of HIV-1 RNA in Dried Plasma Spots (DPS): A Field Approach to Therapeutic Monitoring
Sharon A. Cassol, Francisco Diaz-Mitoma, Richard Pilon, Michelle Janes, and D. William Cameron ................................... 139
17 Quantitation of HIV-1 RNA in Genital Secretions Bruce L. Gill iam ................................................................................. 151
18 Detection of Nucleic Acids in Cells and Tissues by/n Situ Polymerase Chain Reaction
Omar Bagasra, Lisa E. Bobroski, Mohammad Amjad, Matthew Memo/i, and Maureen V. Abbey ................................... 165
19 Determination of the Promoter Activity of HIV-1 Using the Chloramphenicol Acetyltransferase Reporter Gene Assay
Loretta Tse and Nelson L. Michael .................................................. 185
20 In Vitro Techniques for Studies of HIV-1 Promoter Activity Fabio Romerio and David M. Margolis ........................................... 197
21 Detection of Polymorphisms in the HIV-1 Coreceptor CCR5 Using Single-Strand Conformation Polymorphism
Maureen P. Martin and Mary Carrington ......................................... 205
22 Sequence-Specific Priming as a Rapid Screen for Known Mutations Mary Carrington ................................................................................. 213
23 Quantitation of HIV-1 Entry Cofactor Expression James L. Riley and Richard G. Carrofl ............................................ 219
Contents ix
24 Retrovirus-Mediated Gene Transduction of SupT1 Cells Jen-Tsun J. Lin and Jerome H. Kim ................................................ 227
25 Cloning of HIV Single-Strand DNA Binding Protein from Human Lymphocyte Lambda gtl 1 Expression Library with [32P]-Oligomers
Sook-Jin Hur and Derhsing Lai ........................................................ 231
26 Telomeric Terminal Restriction Fragment (TRF) Sumesh Kaushal ................................................................................ 237
PART III. HUMORAL IMMUNOLOGY
27 Serologic Analysis by Enzyme-Linked Immunosorbent Assay (ELISA)
Melissa Krider and Lawrence D. Loomis-Price ............................ 245
28 Quantitative Immunoblotting with Fusion Proteins Margaret M. Mitchell and Lawrence D. Loomis-Price ................. 255
29 Analysis of Antibody Interactions with HIV-1 Envelope Expressed on the Surface of Acutely Infected H9 Cells
Thomas C. VanCott and Paul L. Hallberg ..................................... 265
30 Analysis of Antibody-Antigen Interactions Using Surface Plasmon Resonance
Thomas C. VanCott ......................................................................... 273
31 Measurement of HIV-1 Specific and Total Antibody Secreting Cells by ELISPOT
Frederik W. van Ginkel, John L. VanCott, Robert Kaminski, and Thomas C. VanCott .............................................................. 283
32 Linear Epitope Mapping by the PEPSCAN Method Lawrence D. Loomis-Price ............................................................. 293
33 Neutralization of HIV-1 Infection of Human Peripheral Blood Mononuclear Cells (PBMC): Antibody Dilution Method
John R. Mascola .............................................................................. 309
34 Neutralization of HIV-1 Infection of Human Peripheral Blood Mononuclear Cells (PBMC): Infectivity Reduction Method
John R. Mascola .............................................................................. 317
35 Collection and Processing of Mucosal Secretions from HIV-1 Infected Women
Andrew W. Artenstein and Thomas C. VanCott ........................... 323
x Contents
36 Collection and Processing of Mucosal Secretions from Mice Robert W. Karninski and Thomas C. VanCett .............................. 329
PART IV. CELLULAR IMMUNOLOGY
37 Lymphocyte Proliferation Assay Karl V. Sitz and Deborah L. Birx .................................................... 343
38 HIV-1 Specific Cytotoxic T-Cell Assays Jesephine H. Cox ............................................................................. 355
39 HIV-1-Specific Antibody-Dependent Cellular Cytotoxicity (ADCC) Josephine H. Cox ........................................................................... 373
40 Evaluation of Natural Killer Cell Activity Josephine H. Cox ........................................................................... 383
41 Generation and Expansion of HIV-1 CD4+ Antigen-Specific T Cell Lines
Silvia Ratte-Kirn ............................................................................. 391
42 Immunophenotyping and Assessment of Cell Function by Three-Color Flow Cytometry of Peripheral Blood Lymphocytes
Stephen P. Perfetto and Gilbert McCrary .................................... 397
43 Detection of Apoptosis in HIV-Infected Cell Populations using TUNEL
Patrick J. Blair and Stephen P. Perfetto ....................................... 407
Index ............................................................................................................ 413
Contributors
MAUREEN V. ABBEY " Division of Infectious Diseases, Thomas Jefferson University, Philadelphia, PA
MOHAMMAD AMJAD • Division of Infectious Diseases, Thomas Jefferson University, Philadelphia, PA
ANDREW W. ARTENSTEIN ° Division of Retrovirology, Walter Reed Army Institute of Research, Rockville, MD
OMAR BAGASRA ° Division of Infectious Diseases, Thomas Jefferson University, Philadelphia, PA
SANDRA C. BARRICK • Henry M. Jackson Foundation, Rockville, MD DEBORAH L. BIRX • Division of Retrovirology, Walter Reed Army Institute of
Research, Rockville, MD PATRICK J. BLAIR ° Immune Cell Biology Program, Naval Medical Research
Institute, Bethesda, MD LISA E. BOBROSKI ° Division of Infectious Diseases, Thomas Jefferson University,
Philadelphia, PA D. WILLIAM CAMERON ° Department of Laboratory Medicine, Children's
Hospital of Eastern Ontario, Ottawa, Ontario, Canada M A R Y CARRINGTON • NCI-FCRDC, Frederick, MD RICHARD G. CARROLL ° Henry M. Jackson Foundation, Rockville, MD SHARON A. CASSOL • Ottawa General Hospital Research Institute, Ottawa,
Ontario, Canada BENJAMIN CHEN ° Aaron Diamond AIDS Research Center, New York, NY JOSEPHINE H. Cox ° Henry M. Jackson Foundation, Rockville, MD FRANCISCO DIAZ-MITOMA ° Department of Microbiology and Immunology,
University of Ottawa, Ottawa, Canada ROBERT W. DOMS • Department of Pathology and Laboratory Medicine,
University of Pennsylvania, Philadelphia, PA WEI DONO • Department of Microbiology and Immunology, University of
Maryland School of Medicine, Baltimore, MD AIMEE L. EDINGER ° Department of Pathology and Laboratory Medicine,
University of Pennsylvania, Philadelphia, PA
xi
xii Contributors
BRUCE L. GILLIAM • Henry M. Jackson Foundation, Rockville, MD PAUL L. HALLBERG • Henry M. Jackson Foundation, Rockville, MD SOOK-JIN HUR • Department of Microbiology and Immunology, Thomas
Jefferson University, Philadelphia, PA MICHELLE JANES • Ottawa General Hospital Research Institute, Ottawa,
Ontario, Canada ROBERT KAMINSKI • Henry M. Jackson Foundation, Rockville, MD SUMESH I(AUSHAL ° Naval Medical Research Institute, Bethesda, MD JEROME H. KJM • Henry M. Jackson Foundation, Rockville, MD MELISSA KRIDER ° Henry M. Jackson Foundation, Rockville, MD DERHSING LAI • Department of Microbiology and Immunology, Thomas
Jefferson University, Philadelphia, PA NATHANIEL R. LANDAU • Aaron Diamond AIDS Research Center, New York, NY JAMES R. LANE • sea Technologies, Inc., Rockville, MD BARBARA LEUNG • Saint Paul's Hospital, Vancouver, British Columbia, Canada YEN LI • Department of Microbiology and Immunology, University of
Maryland School of Medicine, Baltimore, MD JEN-TSUN J. LIN • Henry M. Jackson Foundation, Rockville, MD RONG LIU • Aaron Diamond AIDS Research Center, New York, NY LAWRENCE D. LOOMIS-PRICE • Henry M. Jackson Foundation, Rockville, MD MARK K. LOUDER • Henry M. Jackson Foundation, Rockville, MD DAVID M. MARGOLIS ° Institute of Human Virology, Medical Biotechnology
Center, University of Maryland, Baltimore, MD MAUREEN P. MARTIN ° NCI-Frederick Cancer Research and Development
Center, Frederick, MD JOHN R. MASCOLA • Division of Retrovirology, Walter Reed Army Institute of
Research, Rockville, AID GILBERT MCCRARY • Division of Retrovirology, Walter Reed Army Institute
of Research, Rockville, MD RICHARD A. MCDONALD ° Division of Retrovirology, Walter Reed Army
Institute of Research, Rockville, MD MATHEW MEMOLI ° Division of Infectious Diseases, Thomas Jefferson University,
Philadelphia, PA NELSON L. MICHAEL • Division of Retrovirology, Walter Reed Army Institute
of Research, Rockville, MD MARGARET M. MITCHELL ° Henry M. Jackson Foundation, Rockville, MD THERESA Mo ° Saint Paul's Hospital, Vancouver, British Columbia, Canada MARTIN NAU ° Henry M. Jackson Foundation, Rockville, MD
Contributors xiii
STEPHEN P. PERFETTO • Henry M. Jackson Foundation, Rockville, MD RICHARD PILON ° Ottawa General Hospital Research Institute, Ottawa,
Ontario, Canada SILVIA RATTO-KIM • Henry M. Jackson Foundation, Rockville, MD STANLEY READ " Department of Pediatrics, Hospital for Sick Children,
Toronto, Ontario, Canada JAMES L. RILEY • Walter Reed Army Institute of Research, Rockville, MD FABIO ROMERIO • Institute of Human Virology, Medical Biotechnology
Center, University of Maryland, Baltimore, MD ANINDITA ROY ° Department of Microbiology and Immunology, University of
Maryland School of Medicine, Baltimore, MD MIKA SALMINEN • Department of Chronic Virus Diseases, National Public
Health Institute, Helsinki, Finland PAOLA SECCHIERO ° Institute of Human Virology, Medical Biotechnology
Center, University of Maryland, Baltimore, MD KARL V. SITZ ° Division of Retrovirology, Walter Reed Army Institute of
Research, Rockville, MD CHRISTOPHER A. D. SMITH ° Division of Retrovirology, Walter Reed Army
Institute of Research, Rockville, MD LORETTA TSE ° Henry M. Jackson Foundation, Rockville, MD MARYANNE VAHEY ° Division of Retrovirology, Walter Reed Army Institute of
Research, Rockville, MD JOHN L. VANCOTT ° Immunology Vaccine Center, The University of Alabama
at Birmingham, Birmingham, AL THOMAS C. VANCOTT ° Henry M. Jackson Foundation, Rockville, MD FREDERIK W. VAN GINKEL • Immunology Vaccine Center, The University of
Alabama at Birmingham, Birmingham, AL BRUCE G. WENIGER ° Division of HIV/AIDS, Centers for Disease Control and
Prevention, Atlanta, GA DAVlDE ZELLA ° Institute of Human Virology, Medical Biotechnology Center,
University of Maryland, Baltimore, MD
