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o-<l12-4749lHl

DEFECTIVE CYTOTOXIC T LYMPHOCYTE FUNCTION IN HIV INFECTION

By

Shyamasundanm Kottilil, M.B; B.S.

A thesis submitted to tbe School ofCraduateStudies in partial fulfilmentorlbe requirements for tbe degree of Doctor of Philosophy

DivisioD of Basic Medical Sciences

Faculty of Medicine

Memorial Univenity of Newfoundland

October 1999

St. John's Newfoundland

TABLE OF CONTENTS

Page ABSTRACT

ACKNOWLEDGMENTS ABBREVIAnONS

CIIAPTER I: INTRODUCTION

iii

1.0 INTRODUCTION . ..1

I1.1 DISCOVERY OF THE AIDS VIRUS ... 1

I 1.2 HIV VIRION...

I 1.3 TRANSMISSION OF HJV

.... 2 ...J 1.IJ.1 TRANSMISSION BY BLOOD AND BLOOD PRODUcrs ..

I. 13.2SEXUAL TRANSMISSION

I.1.3.3TRANSMISSION FROM MOTHER TO CHILD I.1.4 CLINICAL SYNDROME OF ACUTE HIV INFECTION (. 1.5 HIV lNFECTION OF CELLS

I. 1.5.1cO'MOLECULE: VIRUSRECPTOR ON CELLS . 1.5.1.\CORECEPTORS IN H1V INFECTION

1.1.5.2 ROLE OF CHEMOKINES IN HlV PATHOOENESIS 10

L1.5.3. SUPPRESSTVE CHEMOKINES SECRETED BY

CD8+T CELL.... . 11

1.1.5.4 CHEMOK.INE·BASED lHERAPEUTlC APPROA.CHES... . 12 I.1.5.2POSTBINDING EVENTS IN VIRUS INFEcnON . 13

I. 1.5.1.1ENVELOPE SHEDDING.. [3

1.1.5.2.2ENVELOPE CLEAVAGE 14

I.5.3 VIRUS CELL FUSION... 14

I.5.4 DOWN·MODULAnON OF THE CD4 PROTEIN. IS

I.5.5 POSSlBILfT'Y OF A.NOTHER CELLULAR RECEPTOR:

INFECTION OF CD4- CELLS. 16

l.5.6 CELL·TQ.CELL TRANSFER OF VIRUS.. . 17

I. 1.6 OTHER MECHANISMS OF HIV ENTRY lNTO CELLS. . 18

1.1.7 HIV CYTOPATHOLOGY.. 19

I. U CONTROL OF HIV REPLICATION. . 20

I.U MECHANISMS OF HIV·INDUCED KlLLlNG 23

I.1.9.1HlV·INDUCED APOPTOSIS. 24

I.1.9.2INFLUENCE OF SUPERANTIGENS. 25

I.[.9.3OTHER CAUSESOFHIV·INDUCED CELL DEATH. 26

1.1.10HIV-INDUCEDIMMUNEDEFICIENCY.. 26

1.1.10.1 DIR£CTCYTOPATIflCITYOFTHEVIRUS.. 26

I.1.10.2 SIGNAL TRANSDUCTION ABNORMALITtES.. 27

L1.103BYSTANDER EFFECT.. 27

f.1.1004IMMUNECOMPLEXESOFVIRAL PROTEINS... 28

I.1.10.SCYTOTOXIC T CELLS AND CD8+ CELL SUPPRESSOR

FACTORS.. 28

I. 1.10.6 ANTI-L YMPHQCYlC ANTIBODIES. . 29

I. 1.10.7ROLE OF CYTOKlNES . . 29

I. 1.11HUMORALIMMUNERESPONSES TOHIV INFEcrION .... 29

l. 1.11.1NEUTRALIZING ANTIBODlES . . 29

l.1.11.2 ANTIBODY DEPENDENT CELL CYTOTOXICITY. . 31

I.1.11.3 ANTIBODY ENHANCEMENT. . . 31

I.1.12 CELL-MEDIATED lMMUNE RESPONSES TO HIV.. 32 L 1.12..1CYTOTOXIC NATURAL KILLER CELLS . 32

I.1.12.2 CD4+ T CELL RESPONSES. 32

I.1.12.3 CYTOTOXIC CD8+ TCELLS . ]]

I.1.12.3.1 THE GRANULE EXQCYTOSIS MECHANlSM. 34 I.1.12.3.2 GRANZVME-PERFORIN SYNERGY.. 35 1.1.12.3.3 PERfORlN-GRANZVMECOLLABORATtON. 37

1.1.12.3.4EFFECTSOFCELLCYCLECONlROL.. 37

I.1.12.3.5 SPECIFICSITEOF GRANZYME ACTION.. ]8

r.1.12..].6ASECOND MECHANISM:THEFASIFAS

LIGAND SYSTEM. . 38

I.1.l2.3.7THE NATURE OF THE FAS DEATH SIGNAL ... 39 1.1.12..].8 rMMUNOREGULATtON BY FASlFASL.. 42

L 1.12.3.9 FAS/FASL IN PATHOLOGY. 43

I.1.12.3.10 lMPUCATtONS OF FAS·MEDrATEDCELL

DEATH[NHIV INFECTION ...•... 44 I.1.12.3.11 ROLE OFCU IN VIRAL INFEcnONS.. 46

I.1.13 CTLS IN HIV: ARE THEY AUTOREACTIVE' . 50

I.1.1],1 EPIDEMIOLOOICAL EVIDENCE.. 50

I.1.132 PHENOTYPEQFCOS' TCELlS .... 51

I. 1.14 IS AIDS AN AUTOIMMUNE DISEASE' . 55

I.1.1.5COFACTORS INHIVINFECTION. . . • . . 56

I.1.16 FEATURES OF HlV PATHOGENESIS 58

1.1.17 FACTORS AFFECTING PROGNOSIS 60

J.1.18 GENERAL PURPOSEiHYPOTHESIS .. 61

1.1. 19 REFERENCES... .. . .. 6]

CHAPTERII

107 110 105 10J 104 THE DIFFERENTIAL SENSITMTY OF PSIS CELLS TO ANTI·FAS ANTIBODY AND FAS LIGAND ILLUSTRATES THE MECHANISM OF CYTOTOXICITY OF DrvERSE EFFECTOR CELLS

n.o nnE ..

u.

1 ABSTRACT. . .. . II. 2 INTRODUCTION.

n. ] MATI:RIALS AND METHODS U.4 RESUlTS ..

n.S DISCUSSION.. . _..

n.6 ACKNOWLEOOEMENTS II. 7 REFERENCES.

CHAP'fERIII

NON-APOPTOTIC ACTIVATION INDUCED-LYMPHOCYTE DEATH IN HIV INFECTION

113 117 118

11I.0 Ill. 1 111.2 lII.) 11I.4 11I.5 m.6

ill.7 TITLE...

SUMMARY...

INTRODUCTION ..

MATERIALS AND METHODS ..

RESULTS ..

DISCUSSION ..

ACKNOWLEDGMENTS ..

REFERENCES

128 129 IlO III 135 119 143 143

CHAPTERIV

THERELATIONSHIP BETWEEN CIRCUUTINGAUTOREACTIVECYTOTOXICT

LYMPHOCYTE ACTMTY AND STAGE OF DISEASE IN KJV lNFECTION

v.a TITLE... [64

V. I ABSTRACT 165

V.2 INTRODUCTION ...•...•...•... 166

V.) MATERlALSANOMEnlODS .. 169

v.,

RESULTS .. \74

V.5 DISCUSSION 180

V.• REFERENCES .. \85

CHAYfERV SUMMARY AND FUTURE DIREcrIONS

VI. 0 SUMMARY .. .. \99

VI. I FUTURE DIRECTIONS .. 204

VI. 2 REFERENCES. " 209

ABSTRACT

Theprimaryobjective arthis thesis is to determine whether cy1()(oxic T lymphocytes (CTl) detectable againstCD4"lymphocytes in vitro act in vivo to promote CD4- depletion and thereby contributing to immune dysfu.nc:tionand diseaseprogressionin HIV-Iinfectedindividuals.Duringthisstudy,the relarionship between the: level ofenactivity against CD4 lymphocytes and disease progression wasassased by carryingout a series of in vitro experiments in a HIV-posilive cohon of-70individuals.

£[is well established mateTLuse c1onotypic T cell~eplors(TCR) associated with the invariant CDJ signalling complex. torecognizeantigenic peprides bound to major histocomparibilitycompl~'l:(MHC) molecules on the wget cells. Since PSI S cells express an FeR and Fas wligen. IgG ant'-CD3 antibodies can trigger non-specific killing of PSIS cells by a variety of effector cells. Comparable inhibition of cellular cytotoxicityagainstPSIScellsby1~2or by cycloheximide, a protein synthesis inbibilorprevenling Fas ligand indLICtion.. confumed that the differenl levels of killing of10-2 treated and untrealtd. PSISs reflected the extent thatperforin and Fas ligand. rc:spectively.wereutilized in larget cell killing.

Abnonnallyhighnumbers ofTcells from HIV·infected individuals undergo spontaneous and aclivalion-induccd cell dead! (AleD), and also arc especially sensitive 10 Fas-mcdiakd apoplOSis. sUgge:sling thatFaslFas ligand (FasL) interactions might contribute to AlCDinHIV infection. We used treatmenl with PMA and ionomycin to investigate the possible role of FaslFasL interactions in AICD in HIV infection.

PMAlionomycin·induced AICD measured using Cr release, DNA analysis and electron microscopy.

demonstrated that PMA and ionomycin acted synergi:stically to induce up 10 70%~Icascof incorporated Cr fromfreshPBMCofHIV-infected individualsCOOlparedwith up to 26%rc:asebyhealthy volunteers.Cell delthrequiredceU-«1Icoatllct and extracellularcalcium.whileitdid not involveF~asLinteractions or

i i DNA fiagmenlation. bUI showed plasma membrane disruption with intact nuclear membranes ofdamaged cell.

We describe a novel fonn of AICO in T lymphocytes from HIV-infected individuals.

The presence. number and proportion of activated CDS" T lymphocytes in the peripheral blood of HIY-inf«ted individuals correlates with disease progression. We examined the associations between autoreactive CTL in the peripheral blood of HlY-infected individuals and disease progression. A significant percentage ofHlV-seropositive persons(>50%)in our study cohort, in contrast to healthy individuls showed cytolysis ofPHA-activated uninfected lymphocytes. These autoreactive CTL were found to be C02S· CDS' T cells which expanded with disease progression. A high proportion ofC028· CD8" T cells was seen in all HIV-infected individuals with demonstrable levels of circulating CTL.

We have shown direct association between dte autoreaetivity and other markers of disease progression such as plasma viral load. CD4- T-cell count, CDS' T cell count. and plasma levels OfPl microgloblilin. The datais in agreement with the proposed hypothesis that dtese CTL actually conDiblite to immunodeficiency and clinical progression to AIDS. Based on our data. CTL appe:1r to be a major conDibutor to disease progression.

Further studies based on longitudinal follow-up of these patients may help uncover the functional significance ofautoreactiveCTL.

i i i

ACKNOWLEDGMENTS

First and foremost. I would like to express my sincere gralilUdc co mysupervisorDr.Michael Grant for his valuable guidance during thelatterhalf of my PhD lraining and in preparation ofthismanuscripl. I would also like COe.xpresssincett gratitudecoDr. C. H..J. Ford who acceptedmeintothePhDprogramand trained me to develop research skills.Iamextremely graceful towardbothMike and Chris for their support.

confidence, and friendship, which enabled me to compJele this program successfully.Iwould like10express my gratitude10Dr.K. M.Kutty who encouraged me to apply10Memorial Ph.D program and helped me to achieve mygoal.Thanks to DB. Thomas Michalak. Vernon Richardson. Bodil Larsen. George Carayanniottis andGaryPaterno. who helped me dUring my entire PhD lraining by serving in my supervisory committee.

Thanks toDrsVema Skanes. Thomas Micl1a1ak. Ranjit Chandra and Karen Mearow for serving as members of the PhD comprehensive committee.talso wish to e.'C:lend my thanks to the School ofGraduate Studies.

Memorial University of Newfoundland for providing me with financial support during my training.

Iwould also likecoacknowledge with gratitude the support and guidance extended by Dept of Surgery. Faculty of Medicine. Memorial University of Newfoundland during this study.Iwould like to thank Dr.Maroun, Chainnan of Surgery for his encouragementandguidance. I would also like to e:<tend my gratitude to Ms.Sharon Wadden forsecretarialhelp and encouragement duringmyprogram..

My sincere gratitude goes CO Maureen Gallant for the technical assistance I received during my laboraJory work.Iwould also like to acknowledge the support and assistance of my colleagues in the lab, Jane andRod. I would also like to thank my close friends who supporttd me during stressful limesinmy graduate program, Bachar. Raghuram. Balaji, Lewis, Ptny and Arlene.

I would like to tbanJcDr.Vema Skanes, AssistantDeanof Re:search and Graduate Studies for her understanding and encouragement during myprngram.I would also like to acknowledge the encouragement

iv and supportsoownbymy landlords. Wynnan and John Cross, inspiteofthefact thattheyhadUl put upwith me for three years!twould also like to express my gratitude 10 Mrs andDr.K. M. Kuny, for their love, affection and guidance throughout my stay in Newfoundland. I am thankful to my aunt Dr. Sanda Subrahmanyam. who has been my menlor until she succumbed to ovarian cancerlast year.

Finally [ would like 10 thank my mother for her support. love and cncoul'tgcment whichcaniedme through the tough timesoflhis sNdy. I amindebted to her for everything.

ADCC:

ADE:

AICD:

AIDS:

APe:

ARC:

ARV:

As....

ASMase ATP:

CAD CAT:

CCRS CD:

CDC:

CDR:

CL:

CMY:

Can A:

CsA:

CTL, CXCR4:

ABBREVIAnONS

Antibody Dependent Cellular Cytotoxicity Antibody Dependent Enhancement Aetintion·induced CellDeath Acquired Immunodeficiency Syndrome Antigen Presenting Cell AIDS Related Complex AIDS associated retrovirus As,,,,",,, addic sphingomyelinase Adenosine Triphosphate caspase.-activatedDNasc Chloramphenicol Acetyl Transferase Cemokine Re<:eplor 5:

Cluster Designation Centre for Disease Control Complementarity Detennining Region Cytolytic Lymphocyte Cytomegalovirus Concanavalin A CyclosparinA CytOtoxic T Lymphocyte Chemoltinereceptor4

vi

DO: Death Domain

OED: Death Effector Domain

DISC: Deam.lnducing Signaling Complex

ON: Dominant Negative

DNA: Deoxyribonucleic acid

EBY: Epstein Barr Virus

EDTA: Ethylenediamine letra acetic acid

rADD: Fas-associated death domain

FAP-I: fa,s.-associatedphosphatase-I

FasL Fasligand

FeS: FetalCalfserum

FITC: Fluorescein Isothiocyanalc

gld: Generalizedlymphoproliferativedisease

HCJ: Hydrochloric acid

HHV-6: Human HerpesVirus-6

HlVo Human Immunodeficiency Virus

HLAo Human leukocyte Antigen

HUV: Human T Lymphottopic Virus

ICE: Incerleukin-l13 Converting Enzyme

lEt: Intra Epithelial Lymphocyte

tFN..-,: Interferon-Gamma

IgEo Immunoglobulin E

1000 rnununogiobulinG

'oMo UnmunogIobulinM

IL-I: lnterleukin-l

vii

lL-2: lnterleukin-2

lL-2R: Interleukin-2recepmr

lL-]: Interleukin-]

IL4: Interleukin4

IL-5: Interleukin-S

lL-6: lnterieukin-6

IL-7: Interleukin-7

IL-S: Inter\cukin·a

IL-9: Interleukin-9

IL-IO: lnterleukin-IO

IL-II: lnterlcukin-II

IL-12: Interieukin-12

IL-I]: Interleukin-I]

IL-14: Interleukin-14

IL-IS: Interleukin-IS

TL-16: Interleukin-16

LAK: Lymphokinc Activated Killer

LAS; Lymphadenopathy Syndrome

LAV; Lymphadenopathy Associated Virus

LCMV: Lymphocytic Choriomeningitis Virus

LFA-l: Lymphocyte function Antigen-I

Ipr. Lymphoprolifenation

LTOe Longterminal~

MA, M.mx

mAb: Monoclonal Antibodies

MHC:

MIP·la:

MIP·IIl:

NC:

N,r

NGF:

NK;

NSI:

PBl:

PBMe:

PHA:

PI:

PKC;

PMA:

RANTES:

RBC:

RBl:

Rev RNA:

... Ho RT:

sCD4:

SClDo SCID-hu SCID-huIPBl:

Major Histocmlpatibility Complex Macrophage inflammatory protein·la Macrophage inflammatory prolein·lp Nucleocapsid

Negativefaetor NerveGrowthFactor NaruralKiller Non-syncytium Inducing PeripheralBloodLeukocyte Peripheral Blood Mononuclear Cells Phytohaemagglutinin Propidium Iodide Protein KinascC Phorbol myristic acwte

Rtgulateduponactivation normal T cell expressed and secreted Red Blood Cell

Red Blood Leukemia Regulator of viral protein c.,<pression Ribonuclcicacid

RibonucleaseH ReVer5Clranscripwe SolubleCD4

SevereCombined immunodefICiency Severe Combined Immunodeficiency.auman

~Combined lmmunodefidency.auman lymphocyteS viii

SI:

SN_:

SN:

SLE:

TAT TCR:

THI:

TH2,

m,

TNF-R:

TRAIL:

TRAMP:

v-FLIP:

Syncytium Inducing

Simian Immunodeficiency virus-African Green Monkey Simian Immunodeficiency Virus

Systemic Lupus Erythematosus Transactivaringprotein TCeliReceptor THelperl THelper2 Transmembrane Tumor Necl'05is Factor-Alpha TumotNecl'05isFaclor- ra:eptor lNF-related apoptosis-inducing ligand TNF-receptor-related apoptosis-mediating protein viral FLICE-inhibitory protein

ix

CHAPTER!.

INTRODUCTION'

1.0 lNTROOUCTION

This chapter broadly outlines current concepts of the immunopathogenesis of human immunodeficiency virus(HIV)infection. This is followed by a more specific discussion ofcytoto:l:ic Tlymphocytes(CTL)and their possible roles in the progression ofHIV infection to the invariably falal immunodefICient state or acquired immune deficiency syndrome (AIDS).

1.1 DISCOVERY OF THE VIRUS CAUSING AIDS

HIV is a Ientivirus thaI has only recently beenrecognised asthe causalive agent of AIDS. The fll5t indication that AIDS could be caused by a retrovirus came in 198]. when Barrc-SinoussietaI.

(1983) atthe Pasteur Institute recovered a reverse transcripuse containing virusfromthe lymph node of a man with persistent lymphadenopathy syndrome(LAS). Aconcomitant publication by Gallo et al. (1983) reponed the isolation of human T<ellieukemia virus (HTLY.UI) from individualswith AIDSandarguedthattheca.usaJive viruswasthispreviouslyrcalgllisedhuman retrovirus. Further studies by Montagnier and coworkers clarified these issues regarding the LAS agent, indicating thac AlDs..assoc::iated hwnan retroviruswasdistinctfrom HTLY. Their virus. later called lymphadenopathy

&ocialedvirus(LAV)_gm¥10high literinCD4'cells and lOlled these cellular targets(Mootagnier etaLJ934). These observations on LAY supported the potential etiological role ofa mrovirusinAIDS.

Levy C1aI.(1984) also reported the identification of a retrovirus, they named the AIDS-associated

retrovirus (ARVl. All diese virusesw~~recovered from AIDS palients from diffeRnl known risk groups.,asw~11asfromother sympcomatic and asymplomatic people.HIVisolaleswe:Rsubsequently recoveredfromdie blood of many patients withAIDS. AIDSrelated complex (ARC), and neurological syndromes. as wellISfrom die peripheral blood mononuclear cells(PBMC)of scveral clinically healthy individuals (Levyet al.. 1985a; Salahuddin et al..1985~Soon after the discovery ofHJV-1.

a scparale virus. HIV·2.wasidentified in We:sr:e:m AtTica (Clavel et al. 1986). ItisIlOwcstablished that bolhvirusescanlead to AIDS, although the pathogenic course with HIV-2 appears 10 be longer.

I.lHIVVIRJON

Byelectron microscopy,HJV·IandHlV-2have cone shaped coreswhichare:biochemically conslilUte:d by the viral p25 Gas protein. Inside the capsid, ue two identical RNAstlandswith which

!he viral RNA dependent DNApolym~rase(Pol orreverseIl'anscripWC) and the nucleocapsid(NC) proteinsarec1ose[y associated. The inner portion of the viral membraneissurrounded by a myrisloylated pl7co~(Gag) pnKein that providesthematrix(MA)for me vinlSInlCILlteand is vital for me integrity of the virion (Gclderblom Cl al_ \988: Gc[derblom et al., 1989). Recenl studies have suggested matMAis required for incorporation orEnv proteins into mature virions (Yu et al., 1992).

The viral covelopc{cov)ischaracteristically made up oftrimcrsor tetrame:rsofglycoptoteins (EarlClaL. 1990;Gelderblomcut.., \98S; Oztlet aL [988; Pinteretal_ 1989: Wei$setaL. 1990). The mature Env proteins are derived from a 160,000 D precursor, which is cleaved inside thec~1linto a glycoprotein (gp) 120externalsurface (SU) envelope protein and a gp41 transmembrane (TM) protein (McCune et aL, 1988). Thescprnteinsart;transported10 Ibcccilsurface, wherepartJoftheccntral and N tenninal portions ofgp41 are also expre::sscd on me outside oflhc virion. The central region oftbe TM protein binds to the external viral gp[20 in a noncovaleot manner, most probably in the

hydrophobic regions in the amino andcarboxytermini ofgpl20 (Helseth et al., 1991). Generally. die virionhasabout 100 limes more p2S Gag proteinthanenvelope gpl20 (Layne etaL 1992; Moore et al., 1991) and 10 times more p2S dian Ihepolymaase prolein(Layneet al.• 1992).

The gpl20 sinwedonthe virus surfaceemlainsthe binding siteforcellular receptor(s) and die major neutralizing domains. Nevertheless, die external ponion of gp41 and perhapspanofpl7 have also been reported to be sensitive to neUlraJizingantibodies (Chanh et al.• 1986; Dalgleish elal..

1988; Naylorel al., 1987; Sarin etal..1986).

lJ TRANSMISSION OF HIV

Thereareessenlially Ihreemodesofrransmission oflhe virus fromaninf~tedindividual10 another person;exposure10blood or blood products., sexual transmission and vertical transmission.

IJ.1 TRANSMISSION BY BLOOD AND BLOOD PRODUCTS.

The polenlialriskof infection oftransfusion recipients depends on the virus load in the conlaminated bloodusedfortransfusion.which increases as an infmcd individual (as donor) advances to disease (PerkinseIat,1987). In hemophiliacs. infection could only occur through transmission of free virus andwasassociated with receipt ofmany vials of unheated coagulation faclOfS (Evan et aI., 1984; Eyster et aI., 1987; Goedert et al.. 1989; Koerper elaI.,1989).

IJ.2 SEXUAL TRANSMISSION

AIDSwasfltSlidentifiedas a sexually lrans.mitted disease in bomosexual men. However subsequent studies dernonsttatedheterosexua.l spreadofHJV. which accounts for the large majorilyof infectionswork:Iwide(Nkowaneetal_1991; Stonebumeretal, 1990). Transmission ofHIVingenital

fluids most probably occurs through virus-infected cells since they can be presenlinlarge numbers in the body fluids.Presenceofocherconcomitant sexually transmitted diseases can~IevelsofHIV in genital fluids and lhus make transmission more likely (Cameron et aI.. 1989; PlununeretaL1991).

Infection through anal inlercourse could occur following inlenlction of virus with cellular receptors.

especially those on the bowel mucosa Cfahi et ai. 1992) or the attachment of virus-antibody complexes toFe receptors onthemucosal cells (Hussain et aL 1991). Anolher possible means ofHIV entry eould bevia intestinal M cells present in the bowel epithelium (Amerongen el al.. 1991).

In the case of vaginal intercourse. the columnar and squamous cell epithelium oflhe vagina can be a barriu to virus infection. so thai ulcerations caused by venereal diseases might be required for infection at this site.

The insertive partner in sexual contact carries a relalively 'ower risk of infection, although (Winkelstein elal.. 1987) transmission could occur through infection of macrophages or lymphocytes in the foreskin or the urethral canal. Finally oral-genital contact could also potenlially lead to infection of eitherpartner.albeit at a 'ower frequency (WinkelsteinetaL 1987). Non tnIumaric oral exposure tocell-free srvwasshown to infect adult macaques{BabaCla.l~1996). These infected macaques later developed full blown AIDS indicating the possibility of an onll transmission of the virus and subsequent systemic disease in humans also (BIba et al, 1996). In all roules of sexual contact. an increased numberofvirus mfectedcellsinthe genital fluids andar ksionson the mucosal surfaces(eg....

resulting from venereal disease} would increase the risk of transmission oflhe virus.

1.J.JTRANSMISSION FROM MOTHER TO CHILD

Thetnmsmission of HIVfrommother" to child appe:m to occurinII to 60% of children born toHIV'"mothers (Ades elat,1991; Blancbeelat.,1989). It appears thai tnnsmission

can occur either in utero during or after delivery (Rossi, (992). Suppan for intrauterine transmission comes from the detet:tion ofHIVin placentas and fetuses by in situ hybridization. polymerase chain reaction (PCR) and immunobistochemisuy (CoorgnaudelaL,1991~HJVhasabobeenisolatedlTom con:!blood amniotic fluid. and placental and fetal tissues (Chandwani et aI., 1991).

Thefactors involved in transfer ofHIV from mother to chikl could be studied in relation10Ihe SIVinfection. Transmission of this primate virus appears to occur primarily when the animalsare sexually active and not during binh (McClute et al.. 1992). SlY transmission to one of the three offsprings was demonstrated in pigtailed macaques (Mac:oco nemesrrina)(Oehsetal.. 1991). The consistency of this result. which resembles lransmission in humans.needsfunhersrudy.Ho~erthis observation could suggest an HIV transmission model. since pigtailed macaques have been found 10 be sensitive toHJVinfection (Agy et al.. 1992). If the factors influencing maternal transmission ofHIV can be well defined. anliviral approaches could be better IargCled.

1.4 CLlNICAL SYNDROME OF ACUTE HIV lNFECTION

A recendy infected indiyidual can present wilhin I103 weeks with signs of acule virus infection. Symptomsincludeheadaches,reIrOOrbitalpain.muscle aches, sore throats. lowgrnor high gradefever. and swollen lymph nodes, IS well as a nonpnlritic.maculopapular erythematous rash involving theII'\lnkand lalerlheexttmlities (Cooper et aL (98S). In acutely infected indiyiduals.

pneumonilis, diarrhea, and other gastrointestinal complainES havealsobeen reponed (Tindall and Cooper.1991). These symptoms usually last for 1 10 3 weeks. although lymphadenopathy, lethargy, and malaise can persist for many months. In general, primary H1V infection is followed by an uymplonwic period of many months toyears..

In a groupof23 persons at riskofHJV infectionwhowen:followedevff'jsbc: months and who became infected. 11% I\adsymptomlessacute infection and 95% of these patients sought mcd.ical evaluation (Schacker et al., 1997). But only one in four patienlS in this study received die appropriate diagnosis of acUieHIVinfection at thefirstclinical visit, even though there should have been a high level of suspicion. Laboralory studies perfonncd during the initial infection may show lymphopenia and thrombocylopcnia, but atypical Iymphocylcsareinfrequent (Quinn, 1997).

Infected individualsareoften lymphopenic and thrombocylopenic during the first week followingHIVinfection. In thesecondWft:k,lite lola! number of lymphocyteS increases. primarily becauseofe:<pansion ofCOl'cells. CD4' cellsarereduced in number.Thus.during ltIis period, the C04'/C08'cell "llio is invened. Moreover, atypical lymphocylescanappear in Ihe blood (Cooperct al.. 19&8) but usually in smaller numbers in primary HIV infection Ihan in EBV, CMVorolher infections IItal elicit • similarresponse(Coopetet aI., 1988; Gaines erat.,1988). Overthc following months, number ofltle CD8- cells remains grealerthan that of the CD4' cells, which increases10a smaller extent andsothe ratio remains invcned..

Duringacute HJV infection, the infected individual dcmonstraIcs antigcnemia and viremia with high levels of infectious virus in the peripheml blood(Clarkel aI., 1991;Ouret aI., 1991).

Seroconvcrsion can occur days after infection, but antibodies to HlV generally appear after I to 4 weeks.InstudiesperformedbyCoopereraI.,(198S) and Gaines etal.. (1988),{gMantibodieswere:

fIrStdetected in some paticnlS IS early as 6dayspostinfection. IgO levels could usually be demonstrated by an indirect-immunofluorescent assay within two week!.

Some studies havereportedHIV specific: helper T cellresponsesshortly after acute infection before seroconversioa (Clerici etaI..1991). Conceivably other cellular immuneresponsesare evident

before humoral immunity is evident {Clerici et aI.• 1992a). Mackewiczand Levy, (1992) have reported COS' cell &nri-HIV activity in one individual before sernconvenion.

1.5 KJV INFECTION OF CELLS HIV infection of human cells involves a series of steps.

1.5.1 CIM MOLECULE: VIRUS RECEPTOR ON CELLS

An early breakthrough in the study ofHIV was the discovery of its major cellular receptor, the C04 molecule.P~fe~ntialgrowthof HlV in C04· lymphocytes was then e:<plaincdbyits anachment toIheC04 prn(ein on the cell surface {Dalgleishetat, 1984: Klaamann et al.. 19&4; Klaamann etaL [984a).

Withthe cryStal structure ofC04 now known (Ryu el al.• 1990; Wanget al.. 1990) the gpl20 binding site has been located on a protube1'1Ult ridge along one face of the VI domain. Recently using viral mutanlS and high resolution CD4a1omic:SttUCture.Moebiuser 81.•(1992) havedelinea!edthe viral attaclunentsitefurther.Thesestudiesindicate that Ihe classnMHC binding siteappearstoinclude the same CD4 regionasthe gp 12(l.binding sire. (Moebius ct al.. (992). Thus. this overlap might affect the useof inhibitof$ of the CD4-gp120 interaction.

FurtherSi1CS on CD4 could still be involved in HlV binding and (orfusionsuch asthe CoRJ domain of the VI region (Autieroerat.,1991; Corbeau etal., 1993). It wassuggestedthat this region playa role because CDRJ·related peptides block the CD4-gp120 interaction (LiMn et al., 1991) and mutationsinthis~giondecrease fusogenic activityofHIV (Camerini and Seed, 1990).

R.eml.tfindingsbyDeng et al (1996) andDragicetat.{I 996) haveindicated thatin on:Ierto infect a cell, not only does HIV have to bind to the CD4 receptor on the cell surface.itmUSt also

Cl\rol the help ofa secondrec~orknownas CC-CKR·5. This co-re«ptor isa binding site fortbe attractant molecules RANTC:S. MIP-Ia, and MIP-Ip,.Thesestudies show that RANTES. MTP-Ia. and MlP.\P, all inhibitHJV·\infection by blocking viral fusion and Cl\try, anddwexpressimofCC-CKR·

S chemokine-receptor genetogetherwith that for CD4 renders the cells susceptible to infectmby prinwy non-syncytium forming (NSl) strains ofHIV·\. It is not yet clear whethertherespective chemokines competitively block their receptors against HIV entry, or whether the chemokine binding results in down-regulation ofcell-surface expression of the receptor.

1.5.1.l CORECEPTORS IN HIV INFECfION

Thefirstidentified coreceplor. CXCR-4. is a receplor lor the a. (CXC) subclass ofchemokines and mediates Cl\try/fusion of T-cell tropic strains ofHIV (Fcng et aJ.. 1996). Another receptor forthc members of the p, (ee) chemokine subclass, eCR·S. medilUes entry/fusion of mac:rophage-tropic isolatcsofHIV (Alkhatibetal.• 1996). This moleculc SCTVes as a receptor for RANTES. Mlp·1a.and MIP-\P and thus provides the basis for chemokine-mcdiatedsupp~ionof HIV. Later. a CXC chemokinecalkd SDF·I. a ligand forCXCR-4wasshowntosuppteU replication of1-«1I tropjc HIV isolatcs(Bleuletal•• l996).

The selectivity for the specific cOre<:eptor is governed by the HIV envelope glycoprotein gp120.Thethirdhypervariable region (V3) of the molecule appears to be the major determinant (Cochi et al.• 1996). Paradoxically the V) loopis alsothe leastconservcd region ofgpl20 and contains highly strainspecific neutralizing epitopes. However, certain highly conserved residues are present, and they may contribute to a conserved structural motiftbat broadly facilitates chemokine receptor interactions.

Chemokinessupp~HI\'infectionbyblocking the viral entry process supported by chernokine

meptoTS. Following the binding of oligomeric vil'lll gpl20 10 CD4 on the host cell surface. the resulting CD4-gp 120 complex binds a coreceptor (Wu et aI.• 1996) which. intum.exposes the N- terminal fusogenic sequence ofgp41 (Chan eta1~1997). Che:rnokinesdisrupt thisprocessbymueing cOttCeptor internalization (Chan et al. 1997) into endosomes (Chan etaI~1997) which effectively prevents the fonnation of gpI201CD4/corecptor tricomplex.Mostof the evidence indicates !hat this inhibition does not require coreceptor activation. First. Ireatment of cells with pertussis toxin. which is a sele1:!ive inhibitor ofGproteins involved mainly in chemokine-induced intracellular signalling, rails10mfuce chemokine-mediated suppression orHIVreplication.Doranzetal..I~Second, mutanl chemokine receptorsareimpaired in their ability to transduce intracellular signal-cxhibited comeptor activity (Atchison et al.. 1996). Third. the chemokines modified at their N-tennini actas antagonists ortheir wild-type counterpartS. but do not trigger receptor activation. andare:activeasanli- HIV(Simmons etaL,1996).Finally,somemonoclonal antibodies matpossessanti-HJV activity bind tobind toCeR-SorCXCR-4without triggering intracellular signalling (Endres elaI.• 1996).

Another enigmatic reature orchemokine-mediated HJV suppression is thataligand specific ror one cote1:eplorcanbe effective againsl some viral strains even when other usable coreceptor species arepresent. Brain derived cell cultures expressing both CCR-] and CCR-S were protectedfrom intemonbyreponedpseudovirions containing NSI envelopes by treaImentwitheithereotaxinor MlP- I~alone (8ron et aI., 1997). This suggests that certain chemoreceptCltS might be downregulaled from the cell surface in a co-ordinated manner or that there may be cooperative use ofdifferent coreceptors by at[eastsomeHJVisolates.

10 1.5.1.2 ROLE OF CHEMOKINES IN HIV PATHOGENESIS

At the coreccptor level, ithasbeen established that specifIC allelesincoreceptor genes modulate expression and profoundly influenceHIV infection.The moststudied is aCCR·Sallele (.132) encoding a32base pair deletion. This geneproduces.truncatedand non-functional receptor formthat isnottransportedto the cell surface. Homozygosity ofrhisgene confers strong resistance to HlV infection although afew cases of HIV·infected 632 homozygous individuals have been reported (Michael etaL1997). HeterozygosityforIhe mutated allele is associated with a slowercour.;eofd~

progression (Endresetal. 1996). The effect in hClerozygoles appears tobea transdominant suppression o(wiJd..typeCCR.S coreceptor function due 10 an intracellular association ofdefectiveandnonnaI gene products (deRodaHusman et aI., 1997) leadingto retentioninthe endoplasmic reticulum. The net ft:Sult is decreased surfaceCeR·Sexp~ion relati~towil~typehomozygOCcs. However.lhep~

of .632 CCR·) is not associated with any known patliology in either the homozygoteOf"the heterozygote'.

In addition 10 the conuol al the il:nd:M: level, coreccptOrexp~ionisa function of~eptOf·

ligand inleractions that mediale surface down·regulalion. Givl:n the Significant effeclS of red.uced co~eplorexpression onHIVinfeclion, it is entirely plausible to expect that chemokine levels themselves axrelatewith disease.progression. "isalsopossibll: thaI certain get1d:ic alleles thatred~

~ptorexpressionwort inconcertwith chemokines10modulate infection.Severalgroups have attempted to determine whether there is an inverse COI'TCIa!ion with chemokine levels and disease progression.One approach was to measureplasmaorserum levelsofRANffiS,MIP·IQ.andMIP.lp inseveralcohorts.Overall, these studies havefailed10showan inverse comlation betweenchemok.ine levels and disease:sIaIUs(ZanlJSSietaL, 1996). A much moresuccesful approachhasbeen to measure

11 chemokine releasebyprimlllY PBMCs activated in vitro.Inearlystudies.twogroups failed 10 delect

• com:larionbetweenproduction ofchemokincs and disease progmsion (Lusteret al.. 1995: Bl.a2rlic etaL, 1996).. However. adlergroupssubsequently found a signirtcanc corT'elation between RANTES prodUC1ion andresistancetoinfectionandckcreased MIP-Ia. productioninsymptOmaticHIV'patients (Mackewicz etat1996). Thedispariti~between various studies likely arise from subtle differences in sample acquisition. storage and manipulation.

Taken together. these stUdies present an emerging picrut of HIV pathogenesis in which the production of suppressive chemokines controls disease progression. The results also suggest that in response10 HIVantigcns.CD4-etTCl:torTcells release antiviral chemokines at the site of viral production.Thiswillnot only protects localtargetcells. but will also protect the activated effcclOr cells by inducing autocrinc down-regulal:ion ofeCR-S.The induction ofthisresponse pmduc:cs an asymptomatic state for some period oftimeinallindividuals, but abroaderandmore robustresponse leads[0 non-progression or in~cases. pltlrection from infection.

1.5.1.3 SUPPRESSrvE CHEMOKINES SECRETED BY CDS'" T CELLS It is known thai CD8+ T cells fiom HIV seropositive individuals produce a soluble non·

cytolytic activity thai suppresses infection by HIV in"jrro(Walker et a!. \986). The production of suppressive activity was shown 10 correlate with immune stalUS and to steadily decline in parallel with disease progression (Walker et a!. 1986), indicating that the responsible factors may control infection.

Because of these propenies, the identification offactors responsible for HIV·l suppression has been a major objectiveinHIV·\ research.

Although SDF-\ is an obvious possiblity for the balance ofsoluble activity effective with SI isolates., recent studies have shown thalthiscytokine isnoc:involved(MoriuchietaJ., 1996).Therefore.

12 an alternate possibilty is that other c:hemokincs make up lhe balance of soluble suppressive activity.

When HTLV·! immortalized CDS" Tcells ftomHrV~1infected individuals werescreencd focHIV-SF.

using an acute infectivity assay with activattd.CDS' Tcell-depletedPBMesand virus HlV^lllll.a cell line exhibited suppressive activity against HlVWBas well as primary T.tropic and M.tropic isolates (Lacey etaI.•1991).The moleculewaslater identified as a~chemokine. macrophage-derived chemokine (MOC) (PaletaI. 1997). The ptIrifted chemokine suppressed a variety ofT-tropic and M·

tropic primary isolates and thus demonsQ"llted a broader panern ofsuppression. It has beenreponedthal MDC mRNA and protein are also expressedbyactivated CD4' and CDS" T cells from healthy individuals (Pal ctaI..1997: Godiskaet aJ.. 1997). The production byCDS' T cells further implicates MDCas lIle solublesup~ivefaclor.

1.5.1.4 CHEMOKINE·BASED THERAPEUTIC APPROACHES

Given the features of com:epior functions and novel insights into me pathogenesis of HIV infection. there are at least two different ways one can exploit the knowledge for therapeutic implications. First, molecules can be ciesiifled that can prevent HIV binding to the coreceptors. without aiggering intracellular signalling.Second,mokcules can also be designed thatdown~1alereceptor c:xpos~on dte cell surface.

An approach described recently uses a novel concept to induce receptor down-regulation.

Chemokine receptors arc coupled with an endoplasmic reticulum retention signal, thus causing the chemokine to be retained intracellularly in the endoplasmic reticulum ("intrakine"). Receptor expression on the membraneisdown~gulatedincells producine intrakincs. as the receptor binds 10 its ligand int:raeellularly and therefore its exposureisprevented (Ooranzet al.. 1991; Donzellaet al••

13 1998). As a result HN infectionis inhibited in cellsexp~ingintrakines. This approach couldbe useful as a gene: therapy protoeol 10 ptofectcells from infection.

Another recentstudyshowed that viral pseudotypes containing CD4 and appropriate chemokine recepfors were able 10 urget HfV- and SlY-infected cell lines and primary macrophages.

The targeting was shown 10be related 10 the spe<:ificity of the viral envelope for a given CD4·

chemokine receptor complex. This novel approach might contribute a new stralegy 10talXet specifically HJV·infecled ccl!s.lhus providing an imponanllool for genetherapyapproaches (Chen etal. 1997).

1.5.2 POST BINDING EVENTS IN VIRUS INFECTION

The post binding events ofHIV corry are also made up ofa sel ofsequenlial steps.

1.5.2.1 ENVELOPE SHEDDING

The HIV envelope proteins can~involvedinsteps other than binding to cell surface receptor. during virus infection ofa cell.Somereportshave suggestat that after attaehmenltothe CD4 molecule, the gp120 is displaced. uncovering the domains of gp41. which is needed for virus-ceJl fusion (Saneotau andMoore. (991).Recent analyses suggest that this displacement~ultsfrom the dissociation ofa knob and socket likestruetule involving the carboxylCmIinai~ofgpl20 and the cenrralponioo ofgp41 (Sc.hullZetal., 1992). Sheddingdoesnotappeartooccur(DimitrovetaL.1992) Orlo be necessaryaslongas thefusion domain on gp41 is exposed (Sanentauand Moore. 1991).tn this regani. the soluble CD4 (sCD4}-induced sheddingofgpl20from viruses,observed invitro.has not correlated well with virus entry or the viral envelope syncytial properties (Berger et al.• 1992).

14 1.5.2.2 ENVELOPE CLEAVAGE

Another event involving theHJVenvelope that influencesHJVentryinfOcells is the intracellular cleavage of gp120. Certain studies of gpl20 have revealed sites withintheV] loop that couldbe sensitive to selected cellular proleases (Hattori el a!., 1989). The proposed concept was that these e!ttymes, when present in the cell. cleave gpl20 in the V) region after binding. This m tum facilirates a conformational change in lhe envelope so that a vn! region like: gp41 can subsequently fuse with the cell membrane ($anentauandM~1991}. Thus, theCD4' cells dtatcannOlbeinfected by certain HlV strains mighllack lhenetessaryproteolytic enzymes recognizing a cleavage site oflhat particular viral V3 region.

Thehypothesis ofenvelope: cleavage has gainedsome:support f'rom evidence demonsrntini gpl20 digestionbyproteases(Clements elaI.,1991). Recent studies have shown thate.~~ofHIV tosCD4 leads 10cleavage ofgpl20 by proteolytic enzymes (Moore etat.199 [). This phenomenon can be blocked by monoclonal antibodies (rnAbs) to certain regions of the V3loop(McKuting el al..

199'2). However the importanCe arlhis event hastobe funher investigated.

1.5.3 VIRUS CE:LL FUSION

Envelopedvirusessuchas HIVentercellsfollowing fusion with the cellmembrane.The mechanism for this processinHN invasion is noc yet known.Thefusion step could follow a confonnarional change in !he CD4 molecule as well as. dissociation of the envelope gpl20 or~ ofitsV) loop (0 prolcolytic cleavage.

TheUnerics orlhis fusion reaction suggestS continued attaclunentofvirus 10 mcmbraneCD4 whilethefusion takes place (Dimitrov et al, 1992). Thus, a»npicte gpl20 shedding docs nOI occur

15 although some displacement. might be involved.. The V) loop as well as gp41 could be importantin thismembl1lllcfusionC'VCl\t(Berger Cl aI.•1m).Infectivity presumably results from the virus<ell fusion (Bergeron et at. 1992).

Some observations suggest thattheCD4molecule. in addition to binding couklbe required for the fusion afme viral envelopewiththe celJ surface.EliminationaCIhe proximal regionofCD4 through molecular techniques reduces viral infection. It also eliminates the ability ofcells tofusetothe infected cells (Poulin et at. 1991). Nevenheless. whether virus-cell fusion and cell-eell fusion involve the same processesis still unknown. Finally, the nature of putative cellular fusion receptor (s) is unknown although a glycolipid that mediates HIV infection has been identified on CD4' brain-derived and bowcl-derived cells (Harouseecal" 1991).

1..5.4 DOWN MODULAnON OF THE CD4 PROTEIN

Anomerearlyevenlse.=n with HIVinfed:ioninhuman Tc;cl1sislhedisappc:ar.lnct: oftheCD4 pmteinfrommeccll surface (Hoxieetal.1986). Thee.xte:ntandtimingaCmedown.;egulation depend on lbe level of virus production in the infected cells (Stevenson etaL, 1987; Zaguryet aI., 1986).

In viao. lossofC04e:~prt:SSiongtnerallyoccurs sevem days afterHI\'infectionofceUs when suffkiem progeny virions are produced. Thus. a reduction in chronicHI\' production ofa T-cell line with a Tal antagonist,restored CD4 expression (Shahabuddin e1aL. 1992). The mechanism for allered expression of this cell surface receptorisstill nOI dear. By using interviral recombinants,thisCD4 down-modulation has been linked to the envelope region (York-Higgins etaI.,1990). The CO' receptor does not internalile with HIV during infection, and CD4-related signal transduction events are not involved in virus entty (Orloffelat,1991a& b).

16 SomereportSindicalc that down-modulation involves an amst ofCD4 mRNA transcription (SalmonelaL. 1988), wbile others demonsttatecomplexing ofCD4 with the envelope gpl60 within (he cell (CriseCIaI., (990). Some studies have also suggested masking ofCD4bythe envelope gp 120 attached to the cell surface (McDougal el al.. 1986: Sanentau et al.. 1986). Finally. some researchers suggestthai theCD4moleculeis removed by budding virions (Meerloo et aL 1992). The relative effect of each of these processes on CD4 expression again most probably depends ontheparticular virus and the cell infected. The rtlevance 10 pathogenesis is unclear since some viruses do not modulale the expression ofC04. however the removal

armis

HIV binding site dots prevent superinfection of the cellswith other HIV wains.

1.5.5 POSSIBILITY OF ANOTHER CELLULAR RECEPTOR: lNFECf10N OFCJ).f"

CELLS

HIV can infeel manytypesof CD4- cells. These include human skin fibroblasts. (Tareoo et al..1989) muscle and bone derived fibroblastoid «lIlincs (Clapham et al.. 1989), humanaophoblast cells. follicular dendritic cells. brain derived glia.! cells (Cheng Meyer et al.. 1987), brain apillary endothelial cells fetal adrenal cells (Barboza et al.• 1992) and human liver carcinoma cell lines (Cao etaI.•1990). Evidence for another cellular re<:plor in virus entry into these cells comes from studies with Mabs 10 CD4, incubation of virus with sCD4, and lack of detectable CD4 mRNA in the vina- wgettedcells.

The rate of viral replication is generally law in the CD4' cells (Tateno etaI.,1989),andthe limited virus production is a consequence ofincfficient viralcnay,sinceusuallyfewer than l%ofCD4- cells become infected (Mellen etat,1990). To detect HIV productionintheseCD4- cells,co-- cultivation of

me

cells with other sensitive targets, such as PBMC, has been required (Tateno et aI.•

1989), Recent sNdiessu~tthat eytOkinc:s product<! by the PBMC can enhancemvproduction in CD4'cells. in partK:ular tbose of brain origin (Swingleret al.• 1992).

The nature of theeell surface molecule(s) responsible for viral entry into lheCD4' cells isnot known. but entry conceivably amid involve a fusion reeeplor (Taleno et al.. 1989). This route ofentry.

however. lIS noted above. is quile limited when compared to the CD4-mediared process. One conclusion could be thai forCD4' cells. !heanachmenl [() CD4 enhances the inlt:raction of the viral envelope with the cell surface fusion recepIOr. Cells lacking the CD4 molecule would use the same mode ofentry but it would be much less efficienl

Finally.workdemonstrating !he lymphocyte funcljon associated antigen' I (LFA.1) adhesion molecule as a participantinHIV infection offers alternative mechanism for viral entry. although its role appears primarily to be in cell-eell fusion (Hansen el al.. 1989).

1.5.6 CELL-TO-CELL TRANSFER OF VIRUS

Besides entering a cell as afreeinfeclioos particle. HIV mightbe passedduring cell to cell contacl Evidencehasbeen presetlted that HIV can spread rapidly from one cell10anolher without fonningmaturevirions (Sam et&I..1992). Transfer of nudeocapsids is probably involved. with subseqUeDl denovoreverse transcriplion(Liet aI.•1992), MOm)ver. HIV can be ttansmined from monocytes or lymphocytes10epi!helial cells during such close contact that neutralizing antibodies do nOI block !he transfer (Philips el al.. 1992). Thus. HlV spreadinIhe host could resultfromcell·to cell transfer (via cores or virions) as well as from circulating free virus. During cell-to-eell contact, neutralizing antibodies might not prevent this!ypeofinfeclion.

Insummary. the leading coocepl onearlyevenlS inHJVinfection is that .attachment of HIV [Qthe CD4 moleculemostprobably leads to someconfonn.ation.alchangesinvirus gpl20 and perhaps

18 CD4 moIKu[e. The initial attachment appears to be at one site on CD4 (the complementarity determining region (CDR) 2 domain). Subsequent displacement of gp 120 or cleavage afthe envelope proteinbycellular enzymes most likely causes changes in the viral envelope, permitting the interaction of gp41with the target cell membrane. This could possibly involve another cell surface receplor and su~uently.virus-celt fusion occurs (Sanentau and Moore. (991). Without CD4 expression. fusion of viral gp41 with the cell membrane might take place but the efficiency of this process mightbe greatly limited. Finally, the spread aflhe HIV in the host results ftom production of infectious progeny and also. most probably. by cell·!Q.Cell transfer of immature virions.

1.6 OTHER MECHANISMS OF "IV ENTRY INTO CELLS

HIVcan ioret!cells by mechanisms olher than the interaction ofits envelope proteins with surface receptors. Anlibody-dependent enhancemenl (ADE) ofHIV infei::tion involves binding of the Fab portion of non-neulralizing antibodies to the surface oflhe virion and transfer of the virus into cells through complement or Fc receplors (Homsy et aI., 1989: Horvat et aL. 1989). Fc-mediated infection by HIV highlights a polenlial role of herpes viruses as cofaclorsinl-DV infection. Since these viruses can induce Fc receptors on the surface of infccled cells (Keller et aI., 1976; Bauke and Spear, 1979) they can then serve as polential target cells for HIV. Further serological slUdies of individuals infected wilh HIV are needed to answer the question of the clinical relevance of ADE.

Another mechanism for HIV entry inlo cells is phenolypic mixing (Boettiger, (979). By this process.a viral genome canbe enclosed within the envelope ofa different virus and have the hOSlrange of that virus. Moreover, HIV pseudo-typeS have been produced in vitro willi herpes virus and rhabdoviruses (e.g., vesicular stomatitis virus) (Weiss et aI., 1986). Viruses lhat can undergo phenotypic mixing with HIV-I are HIV-2.HTI..V·I. vesicular stomatitis virus, herpes virus and murine

'9

xenotropic, ampholropic. andpolytropictypeC retroviruses. Whether fonnation of pseudo-lYpe virus particles occurs in natureis unknown.lflhis is the case, HIY·infetted individuals co-infected with herpes viruses or with Hn V-\ would haveviruspopulations representing phenotypic mi.'tlUreswith these two agents (landaue!al, 1991).

The tenn 'superinfeaion'isalsoIJSCdtodeno!t infection of an individual by more than one HIV strain. Inthiscase. individuals canyingboth HJV·JandHIV-2 are documenled (Evans etat, 1988; Rayfield et aI., 1988). although this event is most probably uncommon. Moreover. chimpantte:S canbesimultaneously infected by more than one HrV·l strain (Fultz et at, 1989). In none oflhese cases, has recovery of two dinincl viral strains from lhe same cell been documented.

1.7 HIV CYTOPATHOLOGY

Anot:herimportant biologic feature ofHJV infection is fonnation of multinucleated cellsin culture (syncytium). resulting from the fusion of infcc led cells with uninfected CD4' cells (Lirson el al.. 1986; Lifsonetal.. (988). Syncytium formation is often the fimsign of HI Vinf~tionin culture and can appear within 2·] days. Ba[[ooning of the cells accompaniesthiscytopathic effect most probably resultingfrommembrane permeability changes. This ce[k:cll fusion doesnotrequire DNA.

RNA orprotein synthesis (Hansen etaI.•[989: Tang and Levy.l990). Whether this process is directly relaled to virus<ell fusion isno(clear. but the cell fusion process certainly involves the CD4 molecule andboththe HlV gp120 and gp41 envelope proteins (Sodroski et ai., 1986).

The role of cellular membrane proteins. such as the adherin LFA·l, in cell-cell fusionhas recently been emphasized (Hildreth andOrenw,1989). Monoclonal antibodies (Mab) to this cell surfaceprotein block cell aggregation and syncytiwn fonnation, but not virus infection (Pantaleo etaL,

20 1991).Theprocessoreel!fusion has been linked 10viralcytolO:ticiry and cell death (LirsonetaL 1986).

Cytopalhologyand cell death during acute HIV infection invitrois often associatedwith accumulation ofvlral DNA inthec:y1Oplasmof the inf«tedceUs(Levyet aL 1986: ShawetaL 1984).

However,itis not known whether Ihis process occurs during virus infectioni"vivo. Similar observations have been madewith infectedTcellsarrestedindivision (Tanget al. 1992). These Observalionssupporttheconclusionthathigh levels of intracellular viralDNAcanbe!Oxietothecell and, in the early events of infection. contribute to cell killing (Levy et al.. 1986). Nevertheless, single cell killingisnot associatcd\Nittiaccumulation of viral DNA inthecytOplasm (BergeronandSodI'O$k.i 1992). Avarietyofpnxesses can be involvedinvirus-mediated celldeath.reflecting toxicity of vimI proteins.

U CONTROL OF HIV REPLICATION

Once the virus enters cells as a ribonucleocapsid. several intnu:cllular events lake place that lead to thcintegrationora proviral formintothe cellcluomosome. The viral RNA.stillassoc:ialerl with coreproteins.undergoes revene transcription, usingits RNAdependenlDNApolymerase and Rnase Hactivilies and eventually fonns doubled strandedDNA(reviewed in Greene,1991),TheseDNA copies of viralRNAthen migrate into the nucleus, whererheyinlegrate randomly to the cell chmmosome.lnlegmion ofl:he provirusappearsto be random andis essential for tbe cells10produce progeny virions. Recent observations on the early evenlS ofviraJ infection have revealed notewonhy featur~ofHIVreplication. In T cells arresttd in division. virus infection isabortive. wheteas in nondividingmacrophagesandepithelial cells, progenyproduction takes place.Inpennissive activated Tcells.,HIVundergoes integration and replication within 24 h(Kimetat,1990).Inmacrophages., the

21 processis similar but progeny production appears to require 48 h (Muniset&I.,1992). The earliest mRNAspecies madeinthe infected cell have low molecular weights, representing the vinllong terminal repealS (lTR) and the rcgulallxy genes, particularly^tQt.rnoand lief (Greene, 1991). (I appears thai Tal is madefirstand up regulates the production arReY. Presumably, the predominance ofany one oflhese gene products can determine whether HIV infection will lead to a productive or latent stale. The pr6ence of Tat at high levels will slimulate substantial virus production (Dayton et al..,1986).lntheIalestage$ofthevinas replicativecycle.,Rev would down-regulaJeitsown production andcause: decreased progeny formalion and perhaps latency. In cells nOI fully permissivetoHIV replication.therelative e.xpression oftheseregulatory pro(einscan differ. leadingtoabortive infection.

persistence of virus traces. or a talcOI state (Pomerantz el aI., 1990).

Cellular proteins thaI could innuence virus ll:plication are those reponed 10 increase rat binding 10 TAR (AlonsoetaI., 1992). Recent experimenlS have suggested thai two related cellular Tat- binding pl'Olt:ins might compete to up regulate (e.g.. MSSI) or down-regulate (e.g.. Tat binding p('O(ein I)Tat activilyandthereby affeclHIVproduction (Nclbock et aI.,1990:Shibuya etaI., 1992).Their mechanisms ofldionneedfurtherclucidation. The potential forusing these observationswithTat 10 develop more effective antiviral therapy meritsfurtherevaluation.

The processes involved inTcell activalion areIK)(fully defined but areknowntoaffectHIV replication viatheinlmlCtion ofinlnCellular regulatory factOB withregionsintheviral LTR(reviewed in Gaynor, 1992). This activationispart of a signal transduction process by which the binding of antigens or mitogenstothe surfaceTCRandCOO8moleculesaffectsgene expression within the cell.

Theactivationisreflectedbyanincrnscin the concentration ofintraeellularfrtccalciumanddepends onthe subsequent activarionofcakium-dependent protein kinase C (pKC)andodinphospkorylarion events(Kinteretal., 1990). FollowingtheT cell stimulation. cellular transeriprion factors are released

22 fi'om intracellular inhibitors (e.g., NF·dl&omitsinhibitor IKB) (Nolan et aI.. 1991). via phosphorylation by PKC. They enter the nucleus, where binding to other cellular proteins and. in the case ofHlV. attachmenttovir.L.I DNA sequences take place. The interaction of these transcriptional factors with viral LTR regions can up regulate viral replication. Identification of the cellular transcription factors involved isstill ongoing. bUI many have been rerognised (reviewed in Gaynor.

1992). CertaincytOk.ines..as well as rransactivaling proreinsencodcdbyotherviruses. can also increase HIV production vi. these intracellular events (Kinter clat.1990).

CytOkines and othere.'<temalstimuli often effect HIV expr$ion via interae:tion with the viral lTR. through their influence onintracellular factors. like NFJeB (Matsuyama et at. (991). For clGlIT1ple. rumor necrosis factor(TNF~)increases HIV production. Some viruses such as CMV and herpessimplex virus can enhance HlV productionthroughactivation oflhe viral LTRbyviral proteins.

Finally, a{atgene product. after interactingwithcellular factors binds to the TAR of tile viral lntinconjunction with cellular RNA bindingpmleins (Wu et aL 1991) that may be pbosphorylal:ed (Han eta.I.,1992)and up-regulalion of viral expression occurs. The induction oflNF-a production in T cellsby HIV Tar could also be involved in increased virus production (Buonagouro et II.. 1992).

Theseobservations reflect how bod! viral and cellular factOl'S intenlCt10affectHIVreplicatiolL Many viruses also enhance HlV production by induction ofcytokines. Co-infection of cells with viruses likeherpesvirus, papovavirus. hepatitis viruses,andretroviruses can enhance the production ofHIV·1. GenuallY,d!e lran5aetivating factol'S producedbydie infecting virus, usually early aene products, interact dmtly or indirectly via Ultr.lcellular faclol'S with the HIV·LTR, usually attheresponsived3region.For example, CMV. humanherpes virus6(HHV-6)andEBVactivate the HIV·LTR as measuredincell culnmbythechloramphenicol acetyl transferue (CA1)assay (reviewed

23 in Nelson et a!.. 1990). Some biological assays involving HTLV.! and other animal viruses have demonstrated an increased production ofHIV aftercoinfet:tion (Canivc[ et aI., 1990). Similarly. several B cell lines already tnlnsfonned byEeVappear!O replicate the virusbest.perhaps resulting from a postinfection prncess(DahIetaL.1987: Monroe:eta1~19118: Montagnieret a1.. 19&4).The mechanism (5)for this enhanced expression ofHIVin coinfected cells is unknown, bot again could reflect a cross- reaction orlhe HIV LTR.

1.9 MECHANISMS OF HIV lNDUC£D KILLING

Some understanding orlbe pathogenesis of HI V infection has come ftom studying the direct loxic effec:tofthe virus or its prtllcins on individual cells. Cenain strains ofHlV-I. particularly those recovered from individuals with advanced disease, have a grtater capacity for killing infected cells.

Seven.1observations associate celldeathwith directtoxic:ity of theViNSOfviral prol:cins. The relative quantities of viral envelope protein producedbythe cell can determine cytopathicity(Sodmski etaL. (986). Moreover.lhe cell fusion Ihatoften leadstocell death has been associated with gpl20 (Lifsone1aL 1981).(none study. doubling Ihe production ofgpl20 pcoducedcylopaIhic effects and cell death followingffiVinfection (Stevenson ct II.. 1988). Moreover, addition ofgp[20 to fBMe Ofcultured brain cells caused killing inadosedependent mann« (Dreyer etaJ..[990).The ,((gene hasbeen linked to cytopathic effects, probably by increasing infectious virus replication (Sakaieta.I.•

1991).

The mechanism of thisinductionofcdIdeathbythe viral envelope p""ein is not clearatthe moment.Disturbancesin the membtanc: permeability could be involved., as reflected by the balklon degeneration ofcellsobserved in vitro.HNbinding to andentryinto these cells produces membnne discontinuities andporesin associationwithballooning (Fermin and Garry, 1992). Hence, cells

2.

infectedbyandprnduc:ing cytopalhic HIVdemonstrate aninability 10 control the infllL,( o[monovalenl anddivaJentcarionslhataccumulateinthecell along with waIer(C1oyrlandlynn, 1991).Theresuhing lossinintracellular ionic strengthnoIonly leads 10 cell death,but also at relarively non.cytopathic levels. could change the electric potential afme cell so thai nonnal ccllliJnctioniscompromised.

1.9.1 HIV INDUCED APOPTOSIS

R«enlly. apoptosis was put forwani as a cause ofCD4' cell loss in HIV infection (Growe: cl al..1992;Laurent-Crawford cl aJ., 1991). This process has also been observed in T cells during other viral infections like EBV (Uehara cl aI., 1992). This phenomenon involves the reemergence of a programmedTcell death that is a normal physiological response during thymocyte maturation (Coffin.

1992; Ken' et al.. 1972). The process requires cell activation. protein synlhesis., and the action of a calcium dependent endogenous endonuclease that produce fragmentation ofcellular DNA. Apparently C04" cellsdo not undergo apoplosisinHIV-infecledchimpanzec:s(DeRienzi etaL.1992). This may help 10 explain

me

Ial:k of diseaseinthese animals. Cell proliferation inducedbyphytOhemagglutinin (PHA)alone.doesno[ lead[0apoplOSis. However. Slimulation with MHC-restricted classII recall anligens(e.g..letal1US 10xin) or with !hepokeweedmitogen can causedeathof up to40%in !he CD4- cells from asymptomalic HJV-infected individuals in two days (Groux et al.• 1992). Although most studiesfocus onCD4'cells undergoingapoptosis in HIV-infected individuals,someindicate thatmany C08" cells also diebythis process (Meyaard etaJ..1992). However,therole ofC08⁴T cell apoptosis in the pathogenesis ofHlV infection requires further investigation.

There is considerable speculation as 10 whether apoptosis results from direct effects ofHIVQ("

its viral proleins, antibodies to CD4, gp!20 antibody complexes., variationsincytokine production.Q("

fmally super.wigensfromotherinfecting parhogens (e.g.. srapbylococci, SIrepIOCOCCi, or myatplasma).

25 Some l'CSults suggest that gpl20 or virus-antibody complexes can elicit apoplosis(ferai et al.. 1991).

Recently, cross linking afthe gpl20 bound to human CD4- lymphocytes followedbyT celllCtival:ion byanti COJ antibodieswasshown toinduce apopl:osis (Banda etat,1992).Somec:ytOkines like Il4 canalsoincrease apoptosisinmacrophagesbycounteringmeprotective effects of other cytokines (TNF-a.and interferon-y) on lh6e cells (Manganetal.. 1992). Theserypesof interactions could be taking place in HIV infection.

Since unstimulated CD4" cells~movedfrom the infected individual do nol undergo apoplOSis., whether this phenomenon occurs to a substantial extentin vivois not clear. However, recent reports suggesting enhanced cell death from Ihis process even in PBMC taken directly from the blood of infected individuals (Groux et al" 1992) are especially relevant

1.9..1INFLUENCE OF SUPERANTIGENS

HIV may have a peptide thatac15 like asuperantigenbyattachingto CQ4-lymphocytesbyone ponion aCme T cell receptorandrriggering cell death by apopcosis (Coffin. (992). Support forthis concept comesfiomme observationthatthe individuals with AIDS showa disproportionateIos:sofT cells with a cenain TCR~chain V regions (Imbertietal.. 1991). Moreover, superantigens are responsible for the loss ofT cells in other retrovirus infet:tions, such as the murine mammary nunour virus and the murine model ofAIDS (Woodlandet at. 1991). If this process occurs in HIV infection the antigen involved has yet tobecharacterised. Conceivably,thismechanism for the eliminalion of CD4· cells maybecaused by other organisms or antigens present during HIV infet:tion.

26 l.9.3 OTHER CAUSES OF KIV·INDUCED CELL DEATH

Anumber ofoilier events in theviral cycle have also been believed[0beinvolvedwith cell death. The accumulation ofuninlcgrated viral DNAappears[0be toxic, and the viral Tat protein can kill brain cells (Sabatieretat,[991). Moreover, inleratlions ofcettain cytolcines.like TNF-a. with HIV-inrected lTagile cells might bring about additionaldamage(Matsuyama et aI., (991). Finally. anti cellular responses of immune cells also couldbeinvolved.

1.10 HIV lNDUCED [MMlJNE DEFICIENCY

The rnet.hanismbywhich HIV causes a loss ofimmunc responsiveness is a major mystery in AIDS research. Numerous studies have confirmed Ihal immune abnormalities canbeobserved in T cells., B cells. and macrophages early in the infection well before loss ofCD4" cells begins(Cleridet .11.,1992b).

1.10.1 DIRECT CYTOPATHICITY OF THE VIRUS

The prominent immunologic disorder~ognjsedin-patientswith AIDS is a loss ofCD4' T lymphocytes (Mildvan CI al.. [982). Whether tltis cell loss reflects direct cell destruclion by the virus or its proteins or a secondary effect of immune dysfunction is unclear.

Manyfeaturesordi:rC'c1 HIV·Iinfectionmay contribute to the reduction inCD4' cells and their function. First, despite Ihe inability 10 detect HIV-I in a large number orCD4' cells, even in healthy individuals, HN could be present in a latent or silent state and affect the function, long teno viability, and pmlifeBtion ofthesc cells (Bagasta etaI.,1992). Second, the virus could Wed or supPf'CSS the productionortheearty pm:urscxsorIheCD4·cells andreduce!hequantityoffresh lymphocytes added regularlyfromthebonemarrnwto theperipberal blood (FolksetaI.,1988). A loss ofmemo()' T cells

27 has been reported in asymptomatic HIV individuals (van Noesel el aI., 1990). Third. the HIV tot gene expressed in infected cells mighl reduce the responses ofCD4" cells to recall antigens (Viscidi etal..

(989) and contribule to immunodeficiency. Finally, even ifHIV does not replicateathigh levels,it might aller the membcane integrityofCD4' ccllssuJfK:icntly roaffect not only norma! function btJ[a!so increase their overaJl sensilivity to ccllutarfactCX$..

l.lOJ: SIGNAL TRANSDUCTION ABNORMALITIES

Besides the direct effects of HIV on CD4' lymphocytes and macrophages. infection o(these cells by HIV could interferewiththe nonnal(venlS in signal transduction. This may involve activation by an extracellular signal thai subsequently affects the activity of sequence specific transcription factors.Thisprocess oa:urs when natural ligands bind 10 CD4 or inlenactwithother membrane surface proteins activating T cells and elliei1ing immune response:inlIivo(reviewed in Greene. 1991). The HIV·I gpl20hasbeenfound to(onn an inrraccllularcomplexwithCD4 and pS6^1dl in the endoplasmic reticulum (Crise and Rose, 1992). The retention of this r:yrosine kinase in the cytoplasm couldbetoxic tothecell or affects its function. Funhennore. a possible gp 120-receptor interactionwithcellular proreins on CO' negative bnLin cellswithsubsequent activation of tyrosine phosphorylation of cenain cellular proteins mightbeinvolved inthepathogenesis of r»eurological manifestations ofHIV infectjon(Schneider.Schauliesetat, 1992).

t.lO.J BYSTANDER [FFECf

Another possible mcchanism ofCD4- cell loss is absorption ofsoluble gpl20 byuninfcctM cells carrying the CO' molecule. These celis canbethen~izcdas ."irus infected cells by NK effector cellsCH"CTLs(Lanzavechiaetat1988)anddestroyed, even though they~not infectedby

28 the virus. This hypothesis requires the detection ofcirculating gp120 in Iht: blood of individuals or on uninfected cells. Although some gpl20 released from cellshas beenfoundby in vi\/(}studies (Gilbert t:1 al.. 1991), this feature has nOI been well documented in vivo.

1.IO.~IMMUNECOMPLEXES OFVIRAL PROTEINS

Many investigators have showed that viral envelope proteins have immunosuppressive effects on the mitogenic responses ofT lymphocytes (Chanh et al., 1988) or NK cell activity (Cauda ct al..

1988). In the case of B cell function, gp120 could interferewith normal T cell help via a block in conlaCt-dependenl interactions (Chirmu!e t:1 a!.. 1992). Finally, the formation of anti-viral antigen complexes (Morrow et aI., (986) could tie up the rcticulo-endothelial system, affect cytokine production and influence immune function.

1.10.5 CYTOTOXIC T CELLS AND CDS· SUPPRESSOR CELL DERIVED FACTORS

Studies llSiflg lymphocytes frominfected individuals have suggested that cytOtoxic CDS' cells may kill nonnal CD4' cells as well as those infected with HlV (Pantaleo et al 1990; Zarlingetal., 1990). Some have found cytotoxic CD4' T cells against infected CD4' cells (Orenw el al.. 1990).

Production of immunosuppressive factoB by CDS' cells hasbeendescnbed(uurence.1990) and recently a factor producedbyCOS·cellswasfound10reduce the response ofCD4' cells10cenain recall antigens (Clmci et al.,1992). Production of Chis faclOfCOOki explainlheearly abnonna1itiesseen inbelperT cellfunction (Shearer and Clerici, (992).