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 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 MedicineTM is a trademark of The Humana Press Inc. All authored papers, comments, opinions, conclusions, or recommendations are those of the author(s), and do not necessarily reflect the views of the publisher. This publication is printed on acid-free paper. (~) ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials. 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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Printed in the United States of America. 10 9 8 7 6 5 4 3 2 I 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

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