HIV Protocols METHODS in MOLECULAR MEDICINE TM John M

HIV Protocols METHODS IN MOLECULAR MEDICINE TM John M. Walker, SERIESEDITOR

31. Hemostasis and Thrombosis 19. Hepatitis C Protocols, edited by Protocols: Methods in Molecular Johnson Yiu-Nam Lau, 1998 Medicine, edited by David J. Perry 18. Tissue Engineering, edited by and K. John Pasi, 1999 Jeffrey R. Morgan and Martin L. 30. Vascular Disease: Molecular Biology Yarmush, 1999 and Gene Therapy Protocols, edited 17. HIV Protocols, edited by Nelson by Andrew H. Baker, 1999 Michael and Jerome H. Kim, 1999 29. DNA Vaccines: Methods and 16. Clinical Applications of PCR, edited Protocols, edited by Douglas B. by E M. Dennis Lo, 1998 Lowrie and Robert Whalen, 1999 15. Molecular Bacteriology: Protocols 28. Cytotoxic Drug Resistance and Clinical Applications, edited by Mechanisms, edited by Robert Brown Neil Woodford and Alan Johnson, 1998 and Uta B6ger-Brown, 1999 14. Tumor Marker Protocols, edited by 27. Clinical Applications of Capillary Margaret Hanausek and Zbigniew Electrophoresis, edited by Stephen M. Walaszek, 1998 Palfrey, 1999 13. Molecular Diagnosis of Infectious 26. Quantitative PCR Protocols, edited Diseases, edited by Udo Reischl, 1998 by Bernd Kochanowski and Udo 12. Diagnostic Virology Protocols, edited Reischl, 1999 by John R. Stephenson 25. Drug Targeting, edited by G. E. and Alan Warnes, 1998 Francis and Cristina Delgado, 1999 11. Therapeutic Application of Ribozymes, edited by Kevin J. 24. Antiviral Methods and Protocols, Scanlon, 1998 edited by Derek Kinchington and Raymond F. Schinazi, 1999 10. Herpes Simplex Virus Protocols, edited by S. Moira Brown 23. Peptidomimetics Protocols, edited by and Alasdair MacLean, 1998 Wieslaw M. Kazmierski, 1999 9. Leetin Methods and Protocols, edited 22. Neurodegeneration Methods and by Jonathan M. Rhodes Protocols, edited by Jean Harry and Jeremy D. Milton, 1998 and Hugh A. Tilson, 1999 8. Helicobacter pylori Protocols, edited 21. Adenovirus Methods and Protocols, by Christopher L. Clayton and Harry edited by William S. M. Wold, 1998 L. T. Mobley, 1997 20. Sexually Transmitted Diseases: 7. Gene Therapy Protocols, edited by Methods and Protocols, edited by Paul D. Robbins, 1997 Rosanna Peeling and P. Frederick 6. Molecular Diagnosis of Cancer, Spatting, 1999 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

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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 number 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 protocol 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. Gilliam ...... 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 MARY 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

NATHANIELR. 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, Centersfor Disease Control and Prevention, Atlanta, GA

DAVlDE ZELLA ° Institute of Human Virology, Medical Biotechnology Center, University of Maryland, Baltimore, MD