-
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
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CELLULAR ASPECTS OFHIV INFECTION
-
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
-
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
-
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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. Eros
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. Lefevre, 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
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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, Herve 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
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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
Debre,
and Brigitte Autran
21 New Perspectives in Antiretroviral Therapy of HIV Infection
423Stefano Vella
Index 439
viiContents
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Preface
Infection with human immunodeciency virus (HIV) has produced one
of themost dramatic epidemics of the twentieth century. It has
spread worldwide,leaving no region of the world unaected. Before
eective 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 aord this expensive treatment survive bykeeping the
infection dormant, not by eliminating the virus altogether.
Un-fortunately, the antiviral reagents available come with serious
side eects,and resistance to these agents develops readily for a
consistent percentage ofpatients. At the same time that therapies
with specic 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 aord treatment and perish quickly without any
specicintervention.
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 identied 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-specic 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 dierenttechnologies, 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 specied 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 intove
partsmolecules, 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
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CONTRIBUTORS
Massimo AlfanoAIDS Immunopathogenesis UnitSan Raaele Scientic
InstituteMilano, Italy
Brigitte AutranLaboratoire d'Immunologie
Cellulaire et TissulaireHopital Pitie-SalpetriereParis,
France
Filippo BelardelliLaboratory of VirologyIstituto Superiore di
Sanita
Roma, Italy
Mara BiasinCattedra di ImmunologiaUniversita 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
Cellulaire et TissulaireHopital Pitie-SalpetriereParis,
France
Massimo ClementiDepartment of Biomedical SciencesUniversity of
TriesteTrieste, Italy
Mario ClericiCattedra di ImmunologiaUniversita di MilanoMilano,
Italy
Fulvia ColomboCattedra di ImmunologiaUniversita di MilanoMilano,
Italy
Andrea CossarizzaDepartment of Biomedical SciencesUniversity of
Modena and Reggio
Emilia School of MedicineModena, Italy
Patrice Debre
Laboratoire d'ImmunologieCellulaire et Tissulaire
Hopital Pitie-SalpetriereParis, 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
Sanita
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 Sanita
Roma, Italy
Herve LecoeurUnite d'Oncologie Virale and
CNRS URA 1930
xii Contributors
-
Department SIDA et RetrovirusInstitut PasteurParis, France
Eric LedruUnite d'Oncologie Virale and
CNRS URA 1930Department SIDA et RetrovirusInstitut PasteurParis,
France
Benhur LeeUniversity of Pennsylvania Medical
CenterDepartment of Pathology and
Laboratory MedicinePhiladelphia, Pennsylvania
Eric A. LefevreINSERM U131Institut Paris-Sud sur les
CytokinesClamart, France
Christophe LegendreService de NeurovirologieCEA/CRSSAInstitut
Paris-Sud sur les CytokinesFontenay aux Roses, France
Taisheng LiLaboratoire d'Immunologie
Cellulaire et TissulaireHopital Pitie-SalpetriereParis,
France
M. LogozziLaboratory of VirologyIstituto Superiore di Sanita
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
Emilia School of MedicineModena, Italy
Cristina MussiniInfectious Diseases ClinicsUniversity of Modena
and Reggio
Emilia School of MedicineModena, Italy
Milena NasiDepartment of Biomedical SciencesUniversity of Modena
and Reggio
Emilia School of MedicineModena, Italy
Luzia Maria de Oliveira PintoUnite d'Oncologie Virale and
CNRS URA 1930Department SIDA et RetrovirusInstitut PasteurParis,
France
S. ParlatoLaboratory of VirologyIstituto Superiore di Sanita
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
Emilia School of MedicineModena, Italy
Guido PoliAIDS Immunopathogenesis UnitSan Raaele Scientic
InstituteMilano, Italy
T. Di PucchioLaboratory of VirologyIstituto Superiore di
Sanita
Roma, Italy
Marc RenaudLaboratoire d'Immunologie
Cellulaire et TissulaireHopital Pitie-SalpetriereParis,
France
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
Sanita
Roma, Italy
M. SpadaLaboratory of VirologyIstituto Superiore di Sanita
Roma, Italy
Hugo SoudeynsUnite d'immunopathologie viraleCentre de recherche
de l'Hopital
Sainte-JustineDepartments de microbiologie and
immunologie et de pediatrieFaculte de medicineUniversite de
Montreal
Nadya I. TarasovaNational Cancer InstituteFrederick Cancer
Research and
Development CenterFrederick, Maryland
Leonarda TroianoDepartment of Biomedical SciencesUniversity of
Modena and Reggio
Emilia School of MedicineModena, Italy
Yvette Van KooykDepartment of Tumor ImmunologyUniversity Medical
Center St.
RadboudNijmegen, The Netherlands
Stefano VellaIstituto Superiore di Sanita
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 immunodeciency 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, inuences 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 Inuences 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 3839%) (Myers and Pavlakis, 1992). Asa
direct consequence, the HIV codon usage diers 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 dened 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 dened 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 prole of
HIV activity during the dierent 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
inuencing the levelof systemic HIV activity and cell-free virus
dynamics include degree of viralexpression and host cell range,
whereas the host variables include the specic(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 dierent phases of this infection can be e-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. Thesendings 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
eectiveantiretroviral 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 inuence 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 specicity 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
6 HIV and Virus-Host Interplay
-
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 dierent locations, gp120 amino acid sequences may
di-verge up to 2025%, 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 immunodeciency syndrome (AIDS) pandemic (Myers,1994; Myers
et al., 1994) (intersubject HIV variability).
Subsequent analysis has revealed that both linear and
conformational de-terminants inuence 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 dierent (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 3 105
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 puried 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 eective 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
inuence 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 dierent 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 aect 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 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.
8 HIV and Virus-Host Interplay
-
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 eective
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 developingeective 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 dierentclasses of antivirals and new eective
therapeutic approaches, including geneticand immunologic
strategies, could be the key to inhibiting HIV replication inthe
future.
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12 HIV and Virus-Host Interplay
-
2Telomere Length, CD28 T Cells, and HIVDisease Pathogenesis
Rita B. EffrosDepartment of Pathology and Laboratory Medicine,
UCLA Medical Center,Los Angeles, California, USA
INTRODUCTION
In human immunodeciency 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 immunodeciency 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 CD28. 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. BecauseCD28 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 inuence
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 CD28 T cells, elucidation of the
nature of this expanded popu-lation of cells in HIV disease has
emerged from research in a totally dierentarena of scientic
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 identied byHayick in human
fetal broblasts (Hayick, 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
Hayick 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 Hayick 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 (Eros 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 nave CD4 T cellsfrom
the same individual, a nding that is consistent with the notion
thatmemory cells are the progeny of antigen-stimulated nave T cells
(Weng et al.,1995). It is important to emphasize that although cell
cycle arrest is the most
14 Telomere Length, CD28 T Cells, and HIV Pathogenesis