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CELLULAR ASPECTS OFHIV INFECTION

Edited by

ANDREA COSSARIZZASection of General PathologyDepartment of Biomedical SciencesUniversity of Modena and Reggio Emilia School of MedicineModena, Italy

DAVID KAPLANDepartment of PathologyCase Western Reserve University School of MedicineCleveland, Ohio

A JOHN WILEY & SONS, INC., PUBLICATION

Innodata0471459151.jpg

CYTOMETRIC CELLULAR ANALYSIS

Series Editors

J. Paul Robinson George F. BabcockPurdue University Cytometry

LaboratoriesDepartment of Surgery

Purdue UniversityUniversity of Cincinnati College of

MedicineWest Lafayette, Indiana Cincinnati, Ohio

Phagocyte Function: A Guide for Research and Clinical EvaluationJ. Paul Robinson and George F. Babcock, Volume Editors

ImmunophenotypingCarleton C. Stewart and Janet K. A. Nicholson, Volume Editors

Emerging Tools for Single Cell Analysis: Advances in Optical MeasurementTechnologiesGary Durack and J. Paul Robinson, Volume Editors

Cellular Aspects of HIV InfectionAndrea Cossarizza and David Kaplan, Volume Editors


Edited by

ANDREA COSSARIZZASection of General PathologyDepartment of Biomedical SciencesUniversity of Modena and Reggio Emilia School of MedicineModena, Italy

DAVID KAPLANDepartment of PathologyCase Western Reserve University School of MedicineCleveland, Ohio

A JOHN WILEY & SONS, INC., PUBLICATION

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CONTENTS

Preface ix

Contributors xi

Part I MOLECULES 1

1 HIV and Molecular Biology of the Virus-Host Interplay 3Massimo Clementi

2 Telomere Length, CD28C T Cells, and HIV Disease Pathogenesis 13Rita B. E¨ros

3 Immune Dysregulation and T-Cell Activation Antigens inHIV Infection 33Mara Biasin, Fulvia Colombo, Stefania Piconi, and Mario Clerici

4 Quantification of HIV/SIV Coreceptor Expression 53Benhur Lee and Robert W. Doms

Part II CELLS 67

5 B Cells in the Line of Sight of HIV-1 69Yolande Richard, Eric A. LefeÁvre, Roman Krzysiek, Christophe

Legendre, Dominique Dormont, Pierre Galanaud, and Gabriel Gras

6 Cytotoxic T-Cell (CTL) Function in HIV Infection 103M. L. Garba and J. A. Frelinger

7 Analysis of the a /b T-Cell Receptor Repertoire in HIV Infection 127Hugo Soudenys

v

8 Gamma-Delta (gd) T Cells and HIV-1 Infection 147Roxana E. Rojas and W. Henry Boom

9 Natural Killer Cells in HIV Infection and Role in the Pathogenesisof AIDS 183Benjamin Bonavida

10 Alveolar Macrophages 207Jianmin Zhang and Henry Koziel

11 Dendritic Cells Ferry HIV-1 from Periphery into Lymphoid Tissues 229Teunis B. H. Geijtenbeek and Yvette van Kooyk

Part III PROCESSES 249

12 Homeostasis and Restoration of the Immune System in HAART-Treated HIV-Infected Patients: Implications of Apoptosis 251Marie-Lise Gougeon, HerveÂ LeCoeur, Luzia Maria de Oliveira

Pinto, and Eric Ledru

13 Mitochondria Functionality During HIV Infection 269Andrea Cossarizza, Marcello Pinti, Milena Nasi, Maria Garcia

Fernandez, Laura Moretti, Cristina Mussini, and Leonarda Troiano

14 Multiple Roles of Cytokines in HIV Infection, Replication, andTherapy 293Massimo Alfano and Guido Poli

Part IV TECHNOLOGIES 313

15 The Use of Peptide/MHC Tetramers to Visualize, Track, andCharacterize Class I-Restricted Anti-HIV T-Cell Response 315Clive M. Gray and Thomas C. Merigan

16 Tagging of HIV with Green Fluorescent Protein 333Nadya I. Tarasova

17 Flow Cytometric Analysis of Cells from Persons with HIV-1 Diseaseby Enzymatic Amplification Staining 351David Kaplan

vi Contents

18 Antigen-Specific Cytokine Responses in HIV Disease 371Vernon C. Maino, Holden T. Maecker, and Louis J. Picker

Part V ORGANISMS 383

19 Chimeric Models of SCID Mice Transplanted with Human Cells:The Hu-PBL-SCID Mouse and Its Use in AIDS Research 385S. M. Santini, C. Lapenta, M. Logozzi, S. Parlato, M. Spada,

T. Di Pucchio, S. Fais, and F. Belardelli

20 Immune Reconstitution of the CD4 T-Cell Compartment inHIV Infection 399Guislaine Carcelain, Taisheng Li, Marc Renaud, Patrice DebreÂ,

and Brigitte Autran

21 New Perspectives in Antiretroviral Therapy of HIV Infection 423Stefano Vella

Index 439

viiContents

Preface

Infection with human immunode®ciency virus (HIV) has produced one of themost dramatic epidemics of the twentieth century. It has spread worldwide,leaving no region of the world unaëcted. Before eëctive therapies were de-veloped, infection with HIV meant an inexorable decline in health until deathwas a welcome relief. Now that the capability to decrease viral replicationhas been achieved, those who can aörd this expensive treatment survive bykeeping the infection dormant, not by eliminating the virus altogether. Un-fortunately, the antiviral reagents available come with serious side eëcts,and resistance to these agents develops readily for a consistent percentage ofpatients. At the same time that therapies with speci®c antiviral agents havedecreased morbidity and mortality, they have resulted in a relaxation of ap-propriate public and private health measures, which threatens a recrudescenceof epidemic infection. Of course, many if not most infected persons world-wide cannot even aörd treatment and perish quickly without any speci®cintervention.

Many biomedical scientists have investigated HIV and the disease syndromethat it produces in infected persons. These investigators have contributedgreatly to our understanding of the mechanisms that the virus uses to replicate,to infect new hosts, and to cause disease. These mechanisms have been de-scribed in molecular, cellular, organismal, and social terms.

At the cellular level, investigators ®rst identi®ed the cells that are infected byHIV or that act as reservoirs for the virus. Then the crucial mechanisms of theimmune response, including the importance of HIV-speci®c cytotoxic cells andhumoral responses, the way in which cells die after the infection, the death ofinnocent bystanders, and the role of costimulatory molecules and coreceptorswere described. These studies at the cellular level have relied on many diërenttechnologies, one of the most important being ¯ow cytometry.

Flow cytometry is a powerful technique for the analysis of multiple param-eters of single cells. It is capable of assessing six to ten parameters on 10,000cells in less than a minute. Moreover, cells with speci®ed characteristics can besorted live and cultured for additional investigation. Flow cytometry has beenused since the beginning of the HIV era as a key approach to study the cellular

ix

level in HIV infection. Millions of analyses have been performed on samplesfrom persons infected with HIV. These analyses have allowed us to follow thecourse of the infection, to observe the complex response of the immune sys-tem to the virus, and to help in deciding how to treat infected patients, and tounderstand the patients' cellular response to the therapy.

This book includes chapters by renowned experts on various aspects of HIVinvestigations. The main aim of this book is to present these descriptions andanalyses with particular attention to the role that ¯ow cytometric techniqueshave played in shaping our current conceptualizations. The book is divided into®ve partsÐmolecules, cells, pathophysiological processes, technologies, andorganisms. Each chapter emphasizes an intelligent, concise synthesis of thetopic without an attempt to provide an exhaustive review.

The book is intended for experts in the ®eld of HIV studies, including im-munologists, virologists, and clinicians, as well as for other researchers whoare primarily interested in the use of ¯ow cytometric techniques in biomedicalinvestigations.

Andrea Cossarizza and David Kaplan

x Preface

CONTRIBUTORS

Massimo AlfanoAIDS Immunopathogenesis UnitSan Ra¨aele Scienti®c InstituteMilano, Italy

Brigitte AutranLaboratoire d'Immunologie

Cellulaire et TissulaireHoÃpital PitieÂ-SalpeÃtrieÁreParis, France

Filippo BelardelliLaboratory of VirologyIstituto Superiore di SanitaÁ

Roma, Italy

Mara BiasinCattedra di ImmunologiaUniversitaÁ di MilanoMilano, Italy

W. Henry BoomDivision of Infectious DiseasesCase Western Reserve UniversityCleveland, Ohio

Benjamin BonavidaDepartment of Microbiology,

Immunology, and MolecularGenetics

UCLA School of MedicineUniversity of CaliforniaLos Angeles, California

Guislaine CarcelainLaboratoire d'Immunologie


Massimo ClementiDepartment of Biomedical SciencesUniversity of TriesteTrieste, Italy

Mario ClericiCattedra di ImmunologiaUniversitaÁ di MilanoMilano, Italy

Fulvia ColomboCattedra di ImmunologiaUniversitaÁ di MilanoMilano, Italy

Andrea CossarizzaDepartment of Biomedical SciencesUniversity of Modena and Reggio

Emilia School of MedicineModena, Italy

Patrice DebreÂ

Laboratoire d'ImmunologieCellulaire et Tissulaire

HoÃpital PitieÂ-SalpeÃtrieÁreParis, France

xi

Robert W. DomsUniversity of Pennsylvania Medical

CenterDepartment of Pathology and

Laboratory MedicinePhiladelphia, Pennsylvania

Dominique DormontINSERM U131Institut Paris-Sud sur les CytokinesClamart, France

Rita B. EffrosDepartment of Pathology and

Laboratory MedicineUCLA Medical CenterLos Angeles, California

Stephano FaisLaboratory of ImmunologyInstituto Superiore di SanitaÁ

Roma, Italy

J. A. FrelingerUniversity of North CarolinaChapel Hill, North Carolina

Pierre GalanaudINSERM U131Institut Paris-Sud sur les CytokinesClamart, France

M. L. GarbaUniversity of North CarolinaChapel Hill, North Carolina

Maria Garcia FernandezDepartment of Human PhysiologyUniversity of MalagaMalaga, Spain

Teunis B. H. GeijtenbeekDepartment of Tumor ImmunologyUniversity Medical Center St.

RadboudNijmegen, The Netherlands

Marie-Lise GougeonUnite d'Oncologie Virale and

CNRS URA 1930Department SIDA et RetrovirusInstitut PasteurParis, France

Gabriel GrasService de NeurovirologieCEA/CRSSAInstitut Paris-Sud sur les CytokinesFontenay aux Roses, France

Clive M. GrayCenter for AIDS Research at

StanfordDivision of Infectious Diseases and

Geographic MedicineStanford University Medical CenterPalo Alto, California

David KaplanDepartment of PathologyCase Western Reserve UniversityCleveland, Ohio

Henry KozielAssistant Professor of MedicineHarvard Medical SchoolBoston, Massachusetts

Roman KrzysiekINSERM U131Institut Paris-Sud sur les CytokinesClamart, France

C. LapentaLaboratory of VirologyIstituto Superiore di SanitaÁ

Roma, Italy

HerveÂ LecoeurUnite d'Oncologie Virale and

CNRS URA 1930

xii Contributors

Department SIDA et RetrovirusInstitut PasteurParis, France

Eric LedruUnite d'Oncologie Virale and


Benhur LeeUniversity of Pennsylvania Medical

CenterDepartment of Pathology and

Laboratory MedicinePhiladelphia, Pennsylvania

Eric A. LefeÁvreINSERM U131Institut Paris-Sud sur les CytokinesClamart, France

Christophe LegendreService de NeurovirologieCEA/CRSSAInstitut Paris-Sud sur les CytokinesFontenay aux Roses, France

Taisheng LiLaboratoire d'Immunologie


M. LogozziLaboratory of VirologyIstituto Superiore di SanitaÁ

Roma, Italy

Holden T. MaeckerBD BiosciencesSan Jose, California

Vernon C. MainoBD BiosciencesSan Jose, California

Thomas C. MeriganCenter for AIDS Research at

StanfordDivision of Infectious Diseases and

Geographic MedicineStanford University Medical CenterPalo Alto, California

Laura MorettiDepartment of Biomedical SciencesUniversity of Modena and Reggio


Cristina MussiniInfectious Diseases ClinicsUniversity of Modena and Reggio


Milena NasiDepartment of Biomedical SciencesUniversity of Modena and Reggio


Luzia Maria de Oliveira PintoUnite d'Oncologie Virale and


S. ParlatoLaboratory of VirologyIstituto Superiore di SanitaÁ

Roma, Italy

Louis J. PickerUniversity of Oregon Health

Sciences CenterBeaverton, Oregon

xiiiContributors

Stefania PiconiDivisione di Malattie InfettiveOspedale L. SaccoMilano, Italy

Marcello PintiDepartment of Biomedical SciencesUniversity of Modena and Reggio


Guido PoliAIDS Immunopathogenesis UnitSan Ra¨aele Scienti®c InstituteMilano, Italy

T. Di PucchioLaboratory of VirologyIstituto Superiore di SanitaÁ

Roma, Italy

Marc RenaudLaboratoire d'Immunologie


Yolande RichardINSERM U131Istitut Paris-Sud sur les CytokinesClamart, France

Roxana E. RojasDivision of Infectious DiseasesCase Western Reserve UniversityCleveland, Ohio

S. M. SantiniLaboratory of VirologyIstituto Superiore di SanitaÁ

Roma, Italy

M. SpadaLaboratory of VirologyIstituto Superiore di SanitaÁ

Roma, Italy

Hugo SoudeynsUnite d'immunopathologie viraleCentre de recherche de l'Hopital

Sainte-JustineDepartments de microbiologie and

immunologie et de peÂdiatrieFaculteÂ de meÂdicineUniversiteÂ de MontreÂal

Nadya I. TarasovaNational Cancer InstituteFrederick Cancer Research and

Development CenterFrederick, Maryland

Leonarda TroianoDepartment of Biomedical SciencesUniversity of Modena and Reggio


Yvette Van KooykDepartment of Tumor ImmunologyUniversity Medical Center St.

RadboudNijmegen, The Netherlands

Stefano VellaIstituto Superiore di SanitaÁ

Roma, Italy

Jianmin ZhangResearch FellowHarvard Medical SchoolCambridge, Massachusetts

xiv Contributors

P A R T I

MOLECULES

1HIV and Molecular Biology of theVirus-Host Interplay

Massimo ClementiDepartment of Biomedical Sciences, University of Trieste, Trieste, Italy

INTRODUCTION

The precise understanding of the molecular mechanisms in each step of thehuman immunode®ciency virus (HIV) life cycle has provided an essential basisfor designing antiviral compounds and strategies aimed at blocking viral repli-cation and preventing or delaying disease progression. As in other retroviralinfections, the replication cycle of HIV can be described as proceeding in twophases. The ®rst phase includes entry of the virion into the cell cytoplasm,synthesis of double-stranded DNA (provirus) using the single-stranded genomeRNA as a template, transfer of the proviral DNA to the nucleus, and inter-gration of the DNA into the host genome. The second phase includes synthesisof new copies of the viral genome, expression of viral genes, virion assembly byencapsidation of the genome by precursors of the HIV structural proteins,budding, and ®nal processing of the viral proteins. Whereas the former phaseis mediated by proteins that are present within the virion and occurs in theabsence of viral gene expression, the latter, leading to production of infectiousvirions, is a complex process requiring the interplay of viral and cellular factors.

The precise understanding of the molecular mechanisms of HIV replicationand the use of new technologies in virology has lead to exciting discoveries onmany aspects of the biology of this virus. In particular, a growing body of newdata on the HIV replication mechanisms together with the results from molec-

3

ular studies carried out directly in vivo have allowed researchers to address thevirus-host relationships, including the pathogenic role of this virus in diseaseprogression.

In this chapter, two virologic aspects that are regulated by the complexmechanisms of the HIV-host interplay and have crucial pathogenic implicationsare discussed: the dynamics of HIV replication and the intrahost evolution ofthe HIV population.

HIV GENOME AND CONTROL OF VIRUS EXPRESSION

The HIV genome encodes for precursor polypeptides of structural and func-tional virion proteins, regulatory proteins, and other proteins that are dispens-able for replication and are called accessory proteins (Table 1.1). As for other

T A B L E 1.1. Genes of HIV, Proteins, and Function

HIV

Gene Protein Function

Essential for

Replication

gag Pr55gag Polyprotein precursor for matrix protein

(p17), capsid protein (p24), nucleocapsid

protein p9, and p7

Yes

pol Pr160gag-pol Polyprotein precursor for virion enzymes

reverse transcriptase (RT)/RNAse-H (p51),

protease (PR) (p10), and integrase (IN)

(p32)

Yes

env gp160 Polyprotein precursor for envelope

glycoproteins gp120 and gp41 (receptor

binding and membrane fusion, respectively)

Yes

tat p14 Transcriptional transactivator (initiation and

elongation of viral transcripts)

Yes

rev p19 Regulates viral gene expression at post-

transcriptional levels (regulates splicing and

transport of viral RNAs from the nucleus

to the cytoplasm)

Yes

nef p27 Downregulates CD4 receptor, enhances virion

infectivity, in¯uences T-cell activation

No

vif p23 Viral infectivity factor (infectivity reduced

in vif-minus mutants)

No

vpr p15 Virion protein (associated with the

nucleocapsid) implicated in regulation of

viral and cellular gene expression

No

vpu p16 In¯uences virus release No

4 HIV and Virus-Host Interplay

retroviruses, the genomic HIV RNA is synthesized and processed by the cellu-lar mRNA handling machinery starting from the proviral HIV DNA. For thisreason, the viral genome contains a cap structure at the 5 0 end and a poly-A tailat the 3 0 end. Moreover, the diploid lentiviral genome has the additional featureof being rich in A residues (on average 38±39%) (Myers and Pavlakis, 1992). Asa direct consequence, the HIV codon usage di¨ers dramatically from that ofcellular genes (Berkhout and van Hemert, 1994; Kypr et al., 1989).

Control of HIV RNA synthesis is complex and requires the presence ofseveral cellular proteins as well as of viral transactivators and cis-acting viralelements. Indeed, retroviral long terminal repeats (LTRs) are divided into do-mains (designated U3, R, and U5) that have distinct functions in transcriptioneither in regulating basal levels or inducing high levels of HIV gene expression.The U3 domain of HIV contains basal promoter elements, including the TATAbox for initiation by RNA polymerase II and the site for binding the cellulartranscription factor SP1. Immediately upstream of the core promoter, the viruscontains one or more copies of a 10-bp sequence recognized by the enhancerfactor nuclear factor (NF)-kB. However, whereas in simple retroviruses regu-lation of viral transcription is passive (i.e., regulated by cellular factors), in HIVinfection, this process is more complex and products of the HIV genome arerequired to achieve high levels of expression. Initiation of HIV RNA occurs atthe U3/R level (cap site) of the 5 0 LTR, and the viral transactivator Tat func-tions through a cis-acting sequence (designated Tat-responsive element, TAR)an RNA encoded by a region located in R (+ 19 to + 43). R-U5 is the leadersequence of the full-length and spliced viral transcript, whereas the 3 0 ends ofmRNAs are de®ned by the R/U5 border in the 3 0 LTR. Finally, the accessorygenes of HIV (vif, vpr, vpu, and nef ) (Table 1.1) are generally de®ned as dis-pensable for viral replication based on studies in tissue culture systems. On theother hand, accessory genes are expressed in vivo and increasing data indicatethat they play important roles in the virus-host interplay.

MOLECULAR CORRELATES AND DYNAMICS OF HIV ACTIVITYIN VIVO

The relevant data on mechanisms of HIV replication have been coupled withthe results from in vivo studies, thus obtaining a precise understanding of thevirus-host relationships. Indeed, natural history and pathogenicity studies havesupplied a pro®le of HIV activity during the di¨erent phases of this infection,have contributed to a better understanding of virus-host interactions, haveallowed the application of mathematical models to evaluate the intrahost HIVdynamics, and, ®nally, have provided a theoretical basis for therapeutic anti-viral intervention.

In vivo, systemic HIV activity is a formal entity that consists of a sum ofdynamic processes, including productive infection of target cells, release ofvirions outside the infected cell and eventually in the blood compartment, and

5Molecular Correlates and Dynamics of HIV Activity in vivo

de novo infection of permissive cells. The virus variables in¯uencing the levelof systemic HIV activity and cell-free virus dynamics include degree of viralexpression and host cell range, whereas the host variables include the speci®c(humoral and cytotoxic) immune response and polymorphism of genes codingfor cell receptors of HIV.

The vast majority of quantitative studies carried out in vivo have highlightedthe role of cell-free viremia as a reliable index of mean viral activity in HIVinfection. Indeed, viremia-based studies have provided clear evidence thatchanges in HIV load during the di¨erent phases of this infection can be e½-ciently evaluated by measuring cell-free virus in plasma samples (Bagnarelliet al., 1994; Perelson et al., 1996), and that substantial increases in viral loadparallel or even predict (Mellors et al., 1996) the disease progression. These®ndings have greatly contributed in the last few years to a clearer understand-ing of the virologic correlates of disease progression, to driving new attemptsat understanding the pathogenic potential of HIV, and to designing e¨ectiveantiretroviral strategies. Although recent research has highlighted the diagnos-tic role of other quantitative parameters, including viral transcription patternand provirus copy numbers, and although in some cases virus compartmen-talization may in¯uence the exact correspondence between cell-free plasmaviremia and systemic viral activity, the analysis of viral genome molecules inplasma samples is still a major molecular correlate of systemic viral activity atthe level of the whole body in many human viral infections.

The evaluation of patients undergoing potent antiviral treatments has allowedthe dynamics of cell-free virus in plasma to be addressed in vivo (Ho et al.,1995; Perelson et al., 1996; Wei et al., 1995). Importantly, these studies havedocumented the dynamics of cell-free virions in plasma (half-life being approx-imately 5.7 h) and the turnover of infected cells. Furthermore, the sensitivityand speci®city performances of most quantitative molecular methods haveprovided in the last few years a simple approach to the evaluation of genetranscription in vivo and in vitro. In HIV infection, consistent evidence has in-dicated that progression of disease is driven by an increase in viral load eval-uated as cell-free plasma virus. To address whether this increase is contributedby the dysregulation of the molecular mechanisms governing virus gene ex-pression at the transcriptional or post-transcriptional levels, several quantitativevirologic parameters (including provirus transcriptional activity and splicingpattern) have been analyzed in subjects with nonprogressive HIV infection andcompared with those of matching groups of progressor patients. It was ob-served not only that high levels of unspliced (US) and multiply spliced (MS)viral transcripts in peripheral blood mononuclear cells (PBMCs) correlate withthe decrease in CD4 T cells (Bagnarelli et al., 1996; Furtado et al., 1995) fol-lowing the general trend of systemic HIV-1 activity, but also that MS mRNAlevels in PBMCs are closely associated with the number of productively infectedcells (Bagnarelli et al., 1996), because the half-life of this class of transcriptsafter administration of a potent protease inhibitor is very consistent with that ofproductively infected cells. The transcriptional pattern observed during in vitro


infections of T-cell lines, primary PBMCs, and monocytes/macrophages sup-ports these ®ndings.

INTER- AND INTRASUBJECT HIV VARIABILITY

Comparative analysis of the sequences of the HIV env gene from a greatnumber of viral isolates has revealed a pattern of ®ve hypervariable regions(designated V1 to V5) interspersed with more conserved sequences in the gp120.This sequence variation consists of mutations (resulting in amino acid sub-stitutions), insertions, and deletions (Leigh-Brown, 1991). Among HIV isolatesfrom geographically di¨erent locations, gp120 amino acid sequences may di-verge up to 20±25%, whereas other regions of the genome are relatively con-served. More recently, molecular epidemiology surveys based on env sequencesof numerous HIV isolates have revealed at least nine distinct HIV subtypes (orclades) in the acquired immunode®ciency syndrome (AIDS) pandemic (Myers,1994; Myers et al., 1994) (intersubject HIV variability).

Subsequent analysis has revealed that both linear and conformational de-terminants in¯uence the functional and antigenic structure of the gp120; this isa crucial pathogenic issue, inasmuch as all neutralizing antibodies are directedagainst env-encoded domains in HIV-infected hosts. Indeed, infections withretroviruses are also characterized by di¨erent (from moderate to high) levels ofintrahost viral genetic variation. This viral variability is dependent upon muta-tion, recombination, degree of viral replication, and the host's selective pressure(Dougherty and Temin, 1988; Hu and Temin, 1990; Pathak and Temin, 1990a,b; Temin, 1993). In HIV infection, the viral population is represented by re-lated, nonidentical genetic variants (Goodenow et al., 1989; Hahn et al., 1996;Meyerans et al., 1989; Pedroza Martins et al., 1992), designated quasispecies.The error-prone nature of the HIV reverse transcriptase (RT) and the absenceof a 3 0-exonuclease proofreading activity determine in vitro about 3 × 10ÿ5

mutations per nucleotide per replication cycle (Yu and Goodman, 1992).Although the mutation rate observed in vivo is lower than that predicted fromthe ®delity of puri®ed RT (because a number of newly generated variants areunable to replicate or are cleared by the host's immune system) (Mansky andTemin, 1995), the viral replication dynamics (Ho et al., 1995) and the host'sselective forces determine a continuous process of intrahost HIV evolution(Bagnarelli et al., 1999; Holmes et al., 1992; McNearney et al., 1992; Wolinskyet al., 1996). Overall, the data currently suggest that viral genetic variability isthe molecular counterpart of a continuous dynamic interplay between viral(i.e., HIV-1 replication dynamics and generation of variants by mutation andrecombination) and host factors (i.e., selective pressure). In this context, intra-host evolution of HIV-1 populations may be compatible with a Darwinianmodel system, as recently suggested (Bagnarelli et al., 1999; Ganeshan et al.,1997; Wolinsky et al., 1996).

The complete elucidation of the mechanisms driving intrahost HIV-1 evolu-

7Inter- and Intrasubject HIV Variability

tion is of crucial importance for understanding the natural history of this in-fection and developing eëctive anti-HIV strategies. In particular, the envelopeglycoproteins of HIV-1 interact with receptors of the target cells and mediatethe process of virus entry. This process is complex, including binding of theviral gp120 to CD4, conformational changes of the viral glycoprotein, andsubsequent use of a coreceptor before gp41-mediated fusion of the viral enve-lope and the cellular membrane (Kwong et al., 2000; Rizzuto and Sodroski,2000; Rizzuto et al., 1998; Wu et al., 1996; Wyatt et al., 1995; Zhang et al.,1999) (Table 1.2). The evolutionary changes characterizing the HIV-1 popula-tion during the natural history of infection strongly in¯uence crucial regionsof the viral env gene (Bagnarelli et al., 1999; Menzo et al., 1998; Salvatori etal., 1997; Scarlatti et al., 1997; Shankarappa et al., 1998, 1999; Wolinsky et al.,1996). Because diërent variable domains of the HIV-1 gp120 play a key role indriving the early steps of the viral infection cycle, including coreceptor usage(Isaka et al., 1999; Sato et al., 1999; Verrier et al., 1999; Xiao et al., 1998) andCD4 independence (LaBranche et al., 1999), careful analysis of the intrahostevolution of the HIV-1 env gene is strategic for addressing the relevant featuresof the virus-host relationships (Yamaguchi and Gojobori, 1997; Yamaguchi-Kabata and Gojobori, 2000). In addition, HIV entry is at present an attractivetarget for new classes of antiretroviral compounds (Sodroski, 1999); at present,these compounds include inhibitors of HIV binding to CCR5 and CXCR4coreceptors and fusion inhibitors (Murakami et al., 1999; Ono et al., 1997;Sakaida et al., 1998; Torre et al., 2000).

The V3 sequence is a variable domain in the HIV gp120 and contains 35amino acids arranged in a loop. This domain plays a crucial role in drivingimportant biological properties of the virus, including cell tropism. Generally,mutations in the V3 loop do not aëct the ability of gp120 to interact with theCD4 receptor, although several studies have unambiguously indicated that V3sequences play an important role in two correlated biological features withpathogenic implications, that is, syncytium formation (Willey et al., 1994) and

T A B L E 1.2. HIV Cell Receptors and Their Natural Ligands

CD4 CCR5 CXCR4 CCR3 CCR2b BOB BONZO

HIV

NSI ÿ SI ()

Natural ligands

MHC MIP-1a SDF-1 RANTES MCP-1 ? ?

Cl. II MIP-1b MCP-3 MCP-2

RANTES EOTAXIN MCP-3

MCP-4

NSI, nonsyncytium inducing; SI, syncytium inducing.


coreceptor usage (Isaka et al., 1999). Importantly, analysis of chimeric viruseshas revealed that changes in the V3 loop can convert a nonsyncytium inducing(NSI), slowly replicating virus into a syncytium inducing (SI), rapidly repli-cating virus (Shioda et al., 1992).

CONCLUSION

Expanded analysis of the molecular biology of HIV has been the key to under-standing the mechanisms by which this virus persists in the host and causesAIDS, and to developing eëctive antiretroviral strategies. Application of pow-erful molecular biology tools has allowed researchers to obtain fundamentalresults on many aspects of HIV biology in vitro (i.e., in cell-free and tissueculture systems) and in vivo (i.e., directly in samples from the susceptible host).Importantly, knowledge of the molecular mechanisms in each step of the viruslife cycle has provided an essential basis for discovering new antiviral com-pounds. Otherwise, a ®rm understanding of the relevant features of both theHIV turnover in vivo and the intrahost HIV evolution is crucial for developingeëctive anti-HIV strategies. Indeed, the HIV biology poses several challengesto the development of these strategies. In particular, sequence variation result-ing from errors of the viral RT and recombination renders HIV an elusive tar-get for both antiviral compounds and vaccines. In this context, novel diagnos-tic molecular tools to control development of viral resistance to the diërentclasses of antivirals and new eëctive therapeutic approaches, including geneticand immunologic strategies, could be the key to inhibiting HIV replication inthe future.

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2Telomere Length, CD28ÿ T Cells, and HIVDisease Pathogenesis

Rita B. EffrosDepartment of Pathology and Laboratory Medicine, UCLA Medical Center,Los Angeles, California, USA

INTRODUCTION

In human immunode®ciency virus (HIV) disease, as in all viral infections, CD8T cells constitute a critical component of the protective immune response(Borrow et al., 1994; Brodie et al., 1999; Koup et al., 1994). Loss of CD8 T-cellactivity coincides with the progression to acquired immunode®ciency syndrome(AIDS), and studies on long-term nonprogressors have underscored the impor-tance of cytotoxic T lymphocyte (CTL) function (Cao et al., 1995; Goulder etal., 1997; Harrer et al., 1996). One of the intriguing alterations in the peripheralT-cell pool of individuals infected with HIV is the progressive accumulationwithin the CD8 T-cell subset of a population of cells that lack expression of theCD28 costimulatory molecule. Indeed, in some HIV-infected persons, >65% ofthe CD8 T cells are CD28ÿ. A more complete characterization of this unusualcell population, therefore, is essential for understanding disease pathogenesis aswell as for the development of appropriate strategies for treatment. BecauseCD28ÿ T cells are poorly proliferative, do not contribute to production ofsoluble antiviral suppressive factors, and also show alterations in apoptosis andin cell-cell adhesion, the presence of large proportions of such cells will un-doubtedly have a profound in¯uence on the immune control over HIV infec-

This chapter is dedicated to my friend and colleague Janis V. Giorgi.

13

tion. Although there had been much speculation by AIDS researchers on theorigin of the CD28ÿ T cells, elucidation of the nature of this expanded popu-lation of cells in HIV disease has emerged from research in a totally di¨erentarena of scienti®c investigation, namely basic cell biology studies on the processof replicative senescence. This chapter will review the ®ndings that have led tothe unexpected convergence of these two seemingly unrelated ®elds.

REPLICATIVE SENESCENCE

Normal human somatic cells have an intrinsic natural barrier to unlimited celldivision. Following a fairly predictable number of cell divisions in culture,most, if not all, mitotically competent human cells reach an irreversible state ofgrowth arrest known as replicative senescence, a process ®rst identi®ed byHay¯ick in human fetal ®broblasts (Hay¯ick, 1965). Replicative senescence is astrict characteristic of human cells, and has, in fact, been proposed to constitutea tumor suppressive mechanism (Smith and Pereira-Smith, 1996). Interestingly,experimental cell fusion studies have demonstrated that the property of senes-cence is genetically dominant over immortality in a variety of human cell types,and spontaneous transformation of human cells in vitro rarely, if ever, occurs(Smith and Pereira-Smith, 1996). By contrast, most rodent cells have a highpropensity to bypass senescence and transform spontaneously in culture (Cam-pisi et al., 1996). The divergent behavior of human and mouse cells with respectto spontaneous immortalization in vitro suggests that conclusions regardingreplicative properties, telomeres, and telomerase drawn from murine studiesmay not be applicable to human cells.

The characteristics of replicative senescence, or the so-called Hay¯ick Limit,have been explored in a variety of human cell types for more than 30 years, butonly relatively recently has this model been applied to the immune system.Ironically, the Hay¯ick Limit may be particularly deleterious for immune cells,inasmuch as the ability to undergo rapid clonal expansion is absolutely essentialto their function.

During the past decade, human T cells have been extensively characterizedin cell culture models with respect to replicative senescence. A number of large-scale studies have shown that following multiple rounds of antigen, mitogen, oractivatory antibody-driven proliferation, T cells reach a state of growth arrestthat cannot be reversed by further exposure to antigen, growth factors, or anyother established T-cell stimuli (E¨ros and Pawelec, 1997). The occurrence ofreplicative senescence has been documented for both clonal and bulk culturesof CD4 and CD8 T cells (Adibzadeh et al., 1995; Grubeck-Loebenstein etal., 1994; McCarron et al., 1987). It has also been shown that the replicativepotential of memory CD4 T cells is reduced compared with naõÈve CD4 T cellsfrom the same individual, a ®nding that is consistent with the notion thatmemory cells are the progeny of antigen-stimulated naõÈve T cells (Weng et al.,1995). It is important to emphasize that although cell cycle arrest is the most

14 Telomere Length, CD28ÿ T Cells, and HIV Pathogenesis

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