Apoptosis Hiv(1)

8/6/2019 Apoptosis Hiv(1)

1/14

Apoptosis 2001; 6: 103116

C 2001 Kluwer Academic Publishers

Apoptosis in AIDS

M. Roshal, Y. Zhu and V. Planelles

Departments of Medicine Microbiology & Immunology, University of RochesterCancer Center, 601 Elmwood Avenue, Rochester, NY 14642, USA

Infection with the human immunodeficiency virus type 1(HIV-1) leads to progressive immunodeficiency and on-set of opportunistic infections and neoplasms. The lossof immune competence is associated with declines inboth the functionality and the number of CD4+ lympho-cytes. Multiple mechanisms have been proposed to ex-

plain death and dysfunction of CD4+ T-cells. The mech-anisms of HIV-1-mediated cell death which are relevantin vivo are unclear at present. However, in vitro explo-rations on the cytopathic effects of HIV-1 have yieldeda wealth of potential triggering events, and signaling andeffector pathways leading to apoptosis. The types of pro-and anti-apoptotic stimuli that have been associated withHIV-1 are multiple and often appear overlapping or evencontradictory. This review focuses on the various molec-ular determinants from HIV-1 that play a role in inductionof apoptosis in T-lymphocytes. Special attention is de-voted to the viral genes, env, nef, tat and vpr, for which asignificant body of literature on apotosis-related effectsis available.

Keywords: Apoptosis; survival; HIV; vpr; net; tat; env; AIDS

Introduction

Infection with the human immunodeficiency virus type1 (HIV-1) usually leads to progressive immunodeficiencyand onset of opportunistic infections and neoplasms. Theloss of immune competence is associated with declines inboth the functionality and the number of CD4+ lym-phocytes. Work published in recent years has establishedthat an important component of the decline in num-bers of CD4+ T-cells in vivo is death due to direct viral

infection.13 The half-life of most HIV-1-infected T-cellsin vivo is estimated to be in the range of 12 to 36 hours.13

Key factors in the loss of immune competence are deteri-oration of CD4+ T-cell function and decrease in numbersof CD4+ T-cells.

Multiple mechanisms, including viral and immunolog-ical processes, have been proposed to explain death and

Corresponding to: Vicente Planelles, Departments of Medicineand Microbiology & Immunology, University of Rochester Can-cer Center, 601 Elmwood Avenue, Box #704, Rochester, NY14642; Tel: (716) 273-4474; Fax (716) 273-1221; e-mail: vicente

[email protected]

dysfunction of CD4+T-cells. Among the virus-mediatedmechanisms proposed are toxicity caused by accumula-tion of unintegrated viral DNA,4 membrane permeabilitychanges resulting from viral particles budding at the sur-face of theinfectedcell,5 and terminal differentiation caus-

ing a shortened life span of the CD4+ T-lymphocytes.

6

Among the immunological mechanisms that may con-tribute to death of CD4+ lymphocytes during HIV-1infection are killing by specific cytotoxic T-lymphocytes(CTL) and signaling through the CD4 molecule, lead-ing to apoptosis.7,8 Various groups have suggested thatdeleterious immunological interactions lead to the loss ofimmune cells by apoptosis.7,9,10 Fresh peripheral bloodlymphocytes (PBL) from HIV-1 infected patients have anincreased propensity to undergo apoptosis following stim-ulation in vitro compared to those from healthyindividuals.11,12 In addition, infection with HIV-1 in vitro

leads to enhanced expression of Fas ligand (Fas-L).13,14

Thus, apoptosis may be caused by inappropriate ligationof overexpressed Fas-L with its receptor, Fas, on the surfaceof CD4+ lymphocytes.

The genetic structure of HIV-1

HIV-1 encodes three structural genes, gag, pol and env,which are common to all replication-competent retro-viruses (Figure 1). The product of gag is translated fromunspliced mRNA as a precursor poly-protein. This pre-cursor is cleaved by the viral protease (PRO) into the

following subunits: matrix (MAp21), capsid (CAp24),nucleocapsid (NCp7), and several additional polypeptidesof small size and unknown function, such as p1, p2 andp6.

The pol gene also encodes a polyprotein, and it is ex-pressed as a fusion protein with Gag upon ribosomalframeshifting.15 The Gag-Pol polyprotein is also pro-cessed by the viral protease. The cleavage of Pol givesrise to three enzymes: PRO, reverse transcriptase (RT)and integrase (IN). RT contains three enzymatic activi-ties: RNA-dependent DNA polymerase, RNAase H andDNA-dependent DNA polymerase. IN is responsible for

integration of the viral DNA in the cellular chromosome.

Apoptosis Vol 6 No 1/2 2001 103


2/14

M. Roshalet al.

Figure 1. Genetic structure of HIV-1. HIV-1 encodes nine known genes. Structural genes are shown in black boxes, accessory genes

are shownin hatched boxes, andregulatory genes areshown in dottedboxes. The long terminal repeats (LTR; grey boxes) are cis-acting,

regulatory sequences flanking the coding region. Genes marked with an asterisk are documented to play a central role in the modulation

of apoptosis by HIV-1 infection (see text for details).

The env gene is essential for viral binding and entryinto the host cells. It encodes the precursor glycoprotein,gp160, which is cleaved into a surface moiety, gp120 (SU),and a transmembrane moiety, gp41 (TM). The surface

glycoprotein is required for binding to cellular receptors,whereas the transmembrane glycoprotein is responsiblefor the fusion with the cellular membrane.

In addition to the structural genes, HIV-1 also encodessix small open reading frames (Figure 1). Two of those areregulatory genes, tatand rev, which encode transactivatorproteins essential for viral replication. Tat is a transcrip-tional transactivator. Rev is a post-transcriptional transac-tivator and allows nuclear export of unspliced and singlyspliced mRNAs encoding viral structural proteins. Inthe absence of Rev the only mRNAs detected in the in-fected cells are doubly spliced ones, encoding regulatory

and accessory genes, but not structural genes.The remaining four small open reading frames(Figure 1) are also known as accessory genes and theyinclude vif, vpr, vpu and nef. These genes were named ac-cessory because they are non-essential for virus replicationin cell culture.15

Vif (virion infectivity factor) is not essential for HIV-1replication in permissive cells such as HeLa-CD4 orSupT1.16,17 However, it is necessary for production ofinfectious virions by cells that are natural targets for in-fection, including CD4-positive T-lymphocytes, macro-phages, and H9 cells.1820 A recent study has suggestedthat non-permissive cells contain an endogenous inhibitor

of HIV-1 production that is overcome by the virus-encoded Vif protein.21

The vpu gene encodes a cytoplasmic viral protein thatpromotes degradation of CD4 in the endoplasmic reticu-lum of target cells.2225 Degradation of CD4 allows moreefficient transport of the envelope glycoprotein to the cellsurface and its incorporation into virions. Vpu is able tostimulate the release of viral particles from certain typesof cells including T-lymphocytes, HeLa cells and coloniccarcinoma SW480 cells.26,27 Another function of Vpu isto downregulate the expression of MHC I molecules onthe surface of infected cells.28 This downregulation may

prevent recognition by cytotoxic T-cells.

General aspects ofenv, tat, vpr, and nef, as well as theirroles in apoptosis will be discussed for each individualgene, below.

HIV-1 induces apoptosis at differentstages of its replication cycle

HIV-1 can induce apoptosis from without when thesurface glycoprotein, gp120, cross-links CD4 (Figure 2).This interaction primes cells for apoptosis29,30 in the ab-sence ofde novo viral gene expression.

HIV-1 can also induce apoptosis from within, via

expression of certain viral genes, such as tat, nef and vpr(Figure 2).

Tat was shown to induce apoptosis when expressed in

infected cells.

31,32

The Tat protein, however, can be se-creted to the medium by infected cells33 and later betaken up at a different site by uninfected lymphocytes viaendocytosis.34 Through this route of distribution, Tat caninduce apoptosis in trans in bystander cells.35

This review focuses on the various molecular determi-nants from HIV-1 that play a role in inductionof apoptosisin T-lymphocytes. A wealth of scientific work in the lastten years suggests that the connections between HIV-1and apoptosis induction are highly complex, involvingmultiple viral genes and diverse signaling pathways. Aswe will discuss, the types of pro- and anti-apoptotic stim-uli that have been associated with HIV-1 are multiple

and often may appear overlapping or even contradictory.(See Table 1) This is partly a consequence of the fact thatsurvival and apoptosis pathways are far from being com-pletely understood at present.

General aspects of apoptosis

Apoptosis is a form of cell death that was initially char-acterized at a morphological level.36 Distinctive featuresof apoptosis include cell shrinkage, membrane blebbing,chromatin condensation and nuclear fragmentation. Re-

cently, apoptosis has been characterized at the genetic and

104 Apoptosis Vol 6 No 1/2 2001


3/14

Apoptosis in AIDS

Figure 2. HIV-induced apoptosis is triggered at multiple steps of the viral life cyle. A. Induction of apoptosis from without. Virion-

contained protein, such as Vpr and the envelope glycoprotien, may trigger apoptotic signals prior to full completion of the viral life cycle.

Envelope binding to CD4 and/or a co-receptor may trigger early signals. Immediately after virus entry, release of Vpr in the cytosol may

lead to activation of the apoptotic machinery. B. Induction of apoptosis from within. After completion of the entry, reverse trancription

and integration steps, expression of viral mRNAs is followed by de novo production of viral proteins in the target cell. These proteins

may display pro- and anti- apoptotic abilities. C. Induction of apoptosis in trans. Tat can be secreted into the medium and taken up by

endocytosis by bystander, uninfected cells. The biological effects of Tat have been demonstrated to occur in bystander cells.

biochemical levels, and a plethora of genes and proteinsinvolved in the positive and negative regulation of apop-tosis have become known.

Three major types of stimuli induce apoptosis. Eachof these stimuli triggers a specific signaling cascade, al-though a certain degree of overlap exists among differentcascades.

The first group of stimuli includes binding of certainligands (TNF-, Fas-L, TRAIL) to death-inducing re-

ceptors (DR) on the surface of cells. The second type

of stimulus is DNA damage. DNA damage is a natu-ral signal that may induce cell cycle arrest and apopto-sis. Failure to repair mutations will lead to activation ofthe apoptosis signaling cascade and commitment of thecell to death.37,38 The third type of stimulus that maylead to apoptosis is delivered by cytotoxic T-cells whenthey recognize a target cell. Dissection of apoptosis sig-naling pathways can be accomplished based on analysis ofthe presence, localization and activity of various proteins

(such as caspases, and cytochrome C), physical properties



4/14

M. Roshalet al.

Table 1. Apoptosis-related molecular determinants of HIV-1

Molecular determinant Pro-apoptotic (A) or pro-survival (S)/Mode of action References

tat (A) Activation of caspase 8 31

(A/S) Transcriptional activation of NFB 42, 43

(A) Upregulation of FasL expression 52, 14

(A)Enhancement of TNF-secretion and toxicity 56, 53, 57, 54, 55, 58

(A/S) Change concentrations of Bcl-2 family members 35, 63, 62, 60, 61

(S) Activation of Akt 69

vpr (A) Abnormal centrosome duplication 105, 96

(A) Induction of genomic instabilty 108, 109

(A) Induction of mitochondrial abnormalities 118, 97

(A/S) Suppression of NF b activity 119

(S) Increase concentration of Bcl-2 and decrease concentration of Bax 121

env (A) Upregulation of Fas 140, 141

(A) Fas-L 136, 138

(A) Downmodulation of caspase activation inhibitor 137 (A) Decrease in Bcl-2 and increase in Bax protein concentrations 134, 137

(A) Induction of TNF- and TNF Receptor-II expression 147, 141

nef (A) Upregulation of Fas-L 160163

(A) Activation of PAKs 167173

(A) Upregulation of Fas 163

(S) Downregulation of MHC-I presentation 151153

of the cells (such as depolarization of the mitochondria),and susceptibility to inhibition by cellular anti-apoptoticproteins (such as Bcl-2).

The Tat transactivator has pro-and anti-apoptotic properties

The viral protein Tat upregulates viral transcription atthe level of elongation via interaction with the Tat ac-tivation region (TAR) located at the 5 end of all viralmRNAs. In addition to the interaction withTAR, Tat alsointeracts with a cellular kinase, termed Tat-associated ki-nase (TAK), which is a complex between cyclin-T and thecyclin-dependent kinase-9, cdk-9.3941 Both of the aboveinteractions are required for the transactivation function

of Tat. In addition to transactivating the HIV-1 LTR, Tathas been shown to also transactivate a number of cellularpromoters (see below).

HIV-1 Tat can be secreted by virus-infected cells33 andcan be taken up by bystander cells via endocytosis.34 Be-cause of this property, Tats biological activities can beexerted in uninfected cells.

Tat transactivates cellular genes involvedin apoptosis signaling

Tat increases the activity of a number of cellular transcrip-

tion regulators including nuclear factor kappa-B

(NFB),42,43 nuclear factor of activated T-cells(NFAT)4446 and AP-1.47 Tat can also influence a num-ber of cellular signaling networks affecting, among oth-

ers, cytokine secretion profiles,4850 MAP/SAPK kinasecascades47,45 and tyrosine kinases of the Src superfamily.51

These interactions are thought to increase viral replicationin the infected cells.

Because of the variety of the interactions with cellulargenes, the effects of Tat on the survival/apoptosis path-ways are likely to be multiple. The activation of NFB,for example, was shown to increase the cellular survivalin certain paradigms, but to lead to apoptosis in others.Tat leads to transcriptional activation of both pro- andanti-apoptotic genes, as well as changes in inflammatorycytokine secretion profiles. Similarly, the effect Tat on

the MAP kinase family member, Jun N-terminal kinase(JNK) and consequent activation of the AP-1 transcrip-tion factor may have both pro- and anti-apoptotic effects.Little is known about he cross talk between the variouspathways affected by Tat. For the purposes of this review,we will focus on those effects of Tat that have been linkedto a well-described apoptosis pathway.

Effects of Tat in receptor-mediated apoptosis

It has been suggested that expression of Tat renders cellsmoresusceptible to apoptosis via deathreceptors.14,52 The

effect of Tat on Fas and tumor necrosis factor-



5/14

Apoptosis in AIDS

(TNF-)-induced cell death has been observed at severalsignaling steps. Exogenous Tat can upregulate the ex-pression of the Fas ligand (Fas-L) mRNA and increase thesusceptibility of the Tat-expressing cells to CD4 cross-linking-induced cell death.14 Tat-responsive sites in theFas ligand (Fas-L) promoter are identical to NFB bind-ing sites, which suggests that upregulation of Fas-L byTat is NFB-dependent.52

Tat may affect various pro-apoptotic factors that actdownstream of Fas-L/Fas interaction. Five membranereceptors (Fas, TNF R1, DR3, DR4 and DR5) can actas sensors for their respective ligands and trigger pro-apoptotic signaling via a shared pathway. This pathwayinvolves the formation of an active complex known asdeath-induced signaling complex (DISC; see Figure 3).Death receptors share intracytoplasmic domains that bindto the death domain of Fas-associated death domain

protein (FADD/MORT-I). FADD acts as an adaptor byrecruiting caspases 8 and 10 through its death domains.The recruitment of caspases 8 and 10 by FADD leads tothe generation of the DISC in which pro-caspases 8 and10 are cleaved to give rise to their activated forms.

Barz and Emerman31 showed that upon expression ofTat, the levels of caspase 8 mRNA, protein and enzymatic

Figure 3. The death receptor signaling pathway. Schematic rep-

resentation of the main signaling steps involving apoptisis signal-

ing viadeath receptors.

activity increased. The upregulation of caspase 8, in turn,renders cells susceptible to apoptosis via FAS signaling.The previous observations raise the possibility that theinfected cells would also be more susceptible to apoptosissignaling through other death receptors that utilize theDISC. Additionally tat can accentuate TNF- signalingby both increasing TNF- secretion5355 and shiftingthe cellular redox potential towards oxidation.56,57 Thismay be accomplished by decreasing transcription of themitochondrial enzyme, manganese-dependent superoxidedismutase (Mn-SOD).57 The pro-oxidative state of thecells expressing Tat may render them susceptible to TNFtoxicity.58

Effects of Tat on Bcl-2 and related proteins

In an early report, Zauli et al. suggested that lymphoblas-toid (Jurkat), epithelial (293) and neuronal (PC12) celllines stably expressing Tat were protected from apoptoticdeath induced by serum withdrawal.59 This protectionwas associated with increased levels of the anti-apoptoticprotein, Bcl-2. The increase in Bcl-2 was found to be dueto an increase in the bcl-2 promoter activity.60,61 Both en-dogenously expressed and recombinant Tat were shownto increase Bcl-2 promoter activity. However the mecha-nism for this transactivation has not yet been elucidated.

On the other hand, other groups have observed a Tat-dependent decrease in Bcl-2 protein levels in hematopoi-

etic cell lines

62

or have not observed any change.

35,63

Thus, the effects of Tat in Bcl-2 levels remain unresolved.Finally, a Bcl-2-related protein with pro-apoptotic

effects, Bax, was shown to increase in levels followingTat expression.62

Interactions of Tat with membersof the survival pathway

Cell survival cannot be explained solely on the basis offailure of apoptosis to be activated. An increasing body ofevidence supports the notion that survival, like apoptosis,

is an active process. The principal intracellular media-tors of survival (Figure 4) are Akt/protein kinase B64 andNFB/Rel.65

The apoptosis-survival pathway is particularly relevantto the biology of T-lymphocytes because T-cell activa-tion is accompanied by certain pro-apoptotic signals.66 Toprevent apoptosis, T-lymphocytes upregulate the activityof NFB (an anti-apoptotic factor) in response to activa-tion signals. Thus, activation-induced apoptosis of T-cellsappears to be dictated by the balance between pro- andanti-apoptotic forces. Constitutive activation of NFB incertain T-cell lymphomas causes resistance to activation-

induced apoptosis.67

Remarkably, the HIV-1 promoter,



6/14

M. Roshalet al.

Figure 4. The survival signaling pathway. Schematic representa-

tion of signaling steps which determine suppression of apoptosis.

the long terminal repeat (LTR), contains NFB bindingsites that confer high levels of transcriptional activity inactivated T-cells. A role for Tat in activation and translo-cation of NFB to the nucleus is well established.43,68

Tat and Akt

Low levels of extracellular Tat were shown to activatephosphatidylinositol-3-kinase (PI-3-K) and Akt/PKB ki-nases in CD4+ T-lumphoblastoid Jurkat cells, leadingto increased cell survival following serum starvation.69

Interestingly, when cells were pre-treated with PI-3-Kinhibitors, the percentage of apoptotic cells was dramati-

cally increasedin the presence of Tat.69 These observationssuggest that the activation of PI-3-K is an important anti-apoptotic modulator of the cellular response to exogenousTat. It was also found that protection from apoptosis in

Jurkat cells expressing Tat was, at least in part, due to anautocrineloop, which could be blocked by addinganti-Tatantibodies to the growth medium.50,70

Tat may play a pleiotropic role in cell survival. Bothincresed and decreased apoptosis were observed inresponse to Tat often under similar conditions. Rea-sons for these discrepancies are not clear. Because Tataffects numerous pathways involved in cell growth, ac-

tivation and survival, the points of interaction between

Tat and the apoptotic machinery are likely to be multi-ple. The type of cell, the of exposure to Tat (exogenousversus endogenous),71 its expression levels as well as thelevels of the polypeptide in the medium,32,35,59,70,72 thestate of activation of cells73 and the cytokine milieu mayall play a role in the response.4850 Further explorationof the effects of Tat on the HIV-1 infected as well as by-stander cells is bound to provide important clues to themechanism of cell depletion in HIV-1 pathogenesis.

The vprgene induces transactivation,cell cycle arrest and apoptosis

HIV-1 vpr encodes a 96-amino acid protein with multi-ple functions in the viral life cycle. The first reported rolefor vpr was a moderate transactivation effect on the vi-

ral promoter, the long terminal repeat (LTR).7476 HIV-1mutants with deletions in vpr replicate with slower ki-netics than wild-type viruses.77,78 Vpr is encapsidatedinto virions in significant amounts.74,79,80 The presenceof vpr in the viral particle facilitates efficient infection ofmacrophages and other non-dividing cells78,81,82 by me-diating active nuclear import of pre-integrationcomplexes.83,84 In addition, the presence of vpr enhancesthe transcriptional activity of the viral LTR in macro-phages and T-cells, allowing for the production of a largerviral progeny.8587

HIV-1 vprcontributes to the multiple cytopathic effects

iduced by HIV-1 by inducing cell cycle arrest in G28892

and apoptosis.9396 Interestingly, the vpr-induced growtharrest and cell enlargement phenotypes are observed inyeast, which suggest the involvement a rather conservedpathway.97,98

The apoptogenic and cytostatic properties of Vpr havebeen mapped to its basic C-terminal domain.99104 Themechanism of apoptosis induction by Vpr is not fully un-derstood. A recent report suggests that Vpr-expressingcells die during the M phase of the cell cycle, which theyenter after a prolonged G2 delay. The researchers hypoth-esized that the death was due to an abnormal multipo-lar mitosis due an aberrant centrosome duplication.96,105

Caspases have been shown to be involved becase bothcaspase inhibition with either pharmacological or viralinhibitors results in abrogation of Vpr-inducedapoptosis.94,106

Vpr and DNA repair pathways

It has been hypothesized that vprutilizes the DNA dam-age pathway, which can also arrest cells in G2 and causeapoptosis.107 Several lines of evidence support the previ-ous hypothesis. Vpr expression in certain cell lines leads

to genomic instability cromosome breaks, micronuclei



7/14

Apoptosis in AIDS

formation, aneuploidy and gene amplification.108,109 Hy-perphosphorylation of Cdc-2 observed in Vpr-arrestedcells is reminiscent of cells treated with a DNA-alkylatingagent, nitrogen mustard.107 Furthermore, the arrest byVpr can be reversed by methylxanthines that can also re-verse cell cycle arrest induced by DNA damage.107

Although a direct binding of Vpr to DNA has been repor-ted,110 the possibility that Vpr activates DNA damage-dependent cellular pathways by directly causing alter-ations in the structure or the integrity of DNA has notbeen demonstrated.

Vpr has been shown to physically interact two proteinspossibly involved in DNA repair: Uracil DNA glycosy-lase (UDG)111,112 and HHR23-A,113 a humanhomologueof the yeast rad 23 gene, involved in repair of radiation-induced damage.

Mutagenesis experiments demostrated that the interac-

tion of Vpr with UNG is dispensable for Vpr-induced cellcycle arrest and apoptosis.112 Rather, Vpr-UNG interac-tion appears to be involved in determination the HIV-1genomic integrity.114

Overexpression of HHR23-A was shown to partiallyabrogate Vpr-induced G2 arrest.

113,115 However it is notclear at present how the interaction with HHR23-A maybe involved in Vpr-induced G2 arrest.

Other lines of evidence argue against the DNA dam-age pathway as a primary pathway for Vprs cytostaticand cytotoxic action. Cell cycle arrest and apoptosis in-duced by genotoxic stress are often mediated through

p53 and ATM (mutated in ataxia-telangiectasia) geneproducts.116 Bartz and colleagues have shown that Vpr-induced G2 arrest is independent of p53 and ATMfunction.117 p53 is also not involved in vpr-mediatedapoptosis.94 Furthermore genetic studies in the yeast S.pombe have suggested that in this organism the machinerythat arrests cells in G2/M in response to DNA damage is,at least in part, dispensable for Vpr-induced G2 arrest.

98

Furthermore, the aberrant mitotic spindle formationthat has been observed in Vpr-expressing cells has notbeen observed in cells treated with DNA-damagingagents.96

Vpr and the mitochondria

Given the importance of mitochondria in most mecha-nisms of apoptosis, it would be logical to propose thatmitochondria are involved in Vpr-induced apoptosis. In-deed, several groups have suggested that mitochondriaare the primary site for Vprs apoptogenic and cytostaticactions.97,118 Results from Macreadie et al. indicate thatyeast cells constitutively expressing vpr show gross mi-tochondrial defects including loss of several respiratorychain complexes and inability to utilize unconventional

carbon sources such as ethanol or glycerol.97

This defect

Figure 5. Relationships among various functions of HIV-1 Vpr.

HIV-1 vprtriggers G2/M arrest as a priymary effect. Transactivation

of theviralLTR andinduction of apoptosis appear to be secondary

effects which are dependent on the induction of cell cycle arrest.

appears to be causedby thesame carboxy-terminal domainof Vpr that is involved in mammalian cell apoptosis.

A recent report has shown that exogenous Vpr can di-

rectlycause the loss of mitochondrial potential and triggerapoptotic changes in cell-free system as well as in intactcells.118 In a cell-free system, Vpr interacted with thepermeability trasition pore complex (PTPC) to cause in-creased ion permeability and swelling of mitochondria,leading to the rapid release of cytochrome C and induc-tion of apoptosis within several hours of Vpr exposure.118

A model that integrates the transactivation, G2 arrestand apoptosis induction properties of Vpr is presented inFigure 5. Inhibition of G2 arrest by caffeine also inhibitsthe downstream effects, transactivation and induction ofapoptosis. The higher transcriptional activity of the vi-

ral LTR during the abnormally long G2 phase leads to ahigh progeny output leading to the next cycle of infec-tion. The taget cell, then, undergoes apoptosis, a rela-tively non-immunogenic form of cell death. It has beenproposed that induction of apoptosis by Vpr may decreasethe immunogenicity of virus-infected cells.106

Vpr as a pro-survival protein

Several investigators have suggested that expression ofvprmay protect cells from apoptosis. Ayyavoo et al. havesuggested that Vpr may protect T-cells from apoptosis

in the presence of T-cell receptor (TCR) activation.119 Itwas hypothesized that Vpr acted as a general suppres-sor of NFB activity. However this observation was re-cently questioned.120 Other groups have found that cellsthat stably express low levels of Vpr are protected fromapoptosis.121,122 HIV-1 vpr protected stably transfectedcells from sorbitol-induced122 as well as Fas-L-, TNF--and serum starvation-induced apoptosis.121 A putativemechanism for the previous protection effects is the up-regulation of bcl-2and downregulation of bax.121 A recentreport by Conti et al. has suggested that in the course ofacute HIV infection Vpr plays a cytoprotective role early

on, and increases apoptosis at later stages of infection.123



8/14

M. Roshalet al.

Binding of the HIV-1 envelopeglycoprotein induces signalingevents in the target cells

In order to enter a target cell HIV-1 must first bind two

molecules on the cell surface. Depending on the cell typeand the tropism of the virus, gp120 interacts with its pri-mary receptor CD4 and an accessory receptor (typically,but not exclusively, CXCR4 or CCR5).124,125 The bind-ing of gp120 to the cell receptors can induce signalingevents.29,126128 These signals have been implicated inHIV-1-induced apoptosis of both lymphoid and nonlym-phoid cells.

Binding of gp120 and CD4

Both soluble gp120 as well gp120 on the surface of theinfected cells can induce apoptosis independently of syn-cytium formation.29,129132 Cross-linking of boundgp120 on human CD4+ T-cells followed by signaling inthrough the T-cell receptor was found to result in activa-tion-dependent apoptosis.29 Alternatively T-cells may beinduced to undergo apoptosis in the absence of TCR en-gagement when monocyte/macrophages are present.129

Themechanism forthe inductionof apoptosisby gp120has been extensively studied in recent years. Both Fas-dependent and Fas-independent pathways have beenreported.133139 Premature CD4 engagement results inthe increased susceptibility of the CD4+ cells to Fas-induced apoptosis via two mechanisms: (1) upregulationof Fas140,141 and Fas-ligand;136,138 and (2) downmodu-lation of a caspase inhibitor, the FLICE-like inhibitoryprotein (FLIP).137

Fas-independent mechanisms of gp120-induced apop-tosis have also been demonstrated. It appears that gp120binding to CD4 may induce downregulation of bcl-2134

and upregulation of the pro-apoptotic gene, bax,134,137

leading to mitochondria-dependent apoptosis. Both Fasand Bcl-2-dependent pathwaysare caspase-dependent andthereforeit is not surprising that caspase inhibition resultsin downmodulation of gp120-induced apoptosis.142,143

HIV-1 co-receptors and apoptosis

Fas-independent, gp120-induced apoptosis of CD4+

lymphocytes appears to be enhanced by the interaction ofgp120 with CXCR4.144146 The gp120 interaction withCXCR4 has also been implicated in the increased apop-tosis of CD8+ positive cells in the presence of macro-phages.147 The binding of gp120 to CXCR4 can signalthrough the receptor in both CD8+ cells and macro-phages, leading to the induction of TNF receptor-II

(TNFR-II) on the CD8+ cells and membrane-bound

TNF on the macrophages. The TNFR-II interaction withTNF then leads to increased apoptosis of the CD8+ pos-itive cells.

NefThe nef gene is essential for viral infectivity in vivo, butnot in vitro.148 Nef reduces interactions between Env andintracellular CD4 by inducing internalization and degra-dation of CD4.149,150 Nef also downregulates cell surfaceexpression of MHC I molecules and protects infected cellsfrom killing by cytotoxic T-lymphocytes.151153 Nef wasalso shown to enhance the infectivity of viral particles, in-dependently of the effects on CD4.154,155 Nef-dependentenhancement of infectivity occurs at the level of proviralDNA synthesis, early in the viral life cycle.156158 Nefhas been shown to interact with a number of cellular sig-

nal transduction molecules including members of the Srcfamily of tyrosine kinases; p21-activated kinases (PAKs);mitogen-activated kinases (MAPs); the G protein, Raf1;and a proto-oncogene and guanine-nucleotide exchangefactor, Vav, involved in activation of T-cells.159

Nef may play an important role in the immune dys-regulation and lymphoid tissue apoptosis in HIV-1 in-fected patients. Several mechanisms for Nef-dependentapoptosis have been postulated. Nef was shown to in-duce activation of lymphocytes leading to upregulationof Fas-L which, in turn, can induce apoptosis of bothCD4+ and CD8+ positive cells.160163 In order to acti-

vate lymphocytes, Nef interacts with and signals throughthe tyrosine-kinase activation motifs of the T-cell recep-tor zeta chain.161,164166 A downstream signal involvesactivation of p21-activated kinases 1 and 2167173 andformation of the Nef-activated kinase complex, NAK,between PAK and Nef. Interestingly PAK2 is cleavedand activated by caspases [Rudel, 1997 #331; Walter,1998 #328; Chan, 1999 #327] and plays an importantrole in the regulation of morphology and biochemistry ofapoptotic cells. Thus, it would be logical to suspect thatNef may induce certain pro-apoptotic events through theinteraction with PAK2.174,175 Another mechanism thatmay be involved in Nef-dependent apoptosis include up-

regulation of Fas.163Our understanding of the mechanisms involved in lym-

phocyte depletion in HIV has improved dramatically overthe past decade. In 1991 several groups proposed apopto-sis as a mechanism for cell depletion in HIV infection.7,10

An extensive body of literature since then has supportedthis hypothesis. Despite this large and ever-growing bodyof literature, many questions still need to be answered.Relatively little information is available about the spe-cific interactions of the HIV-encoded proteins and cellularones that modulate apoptosis pathways. In particular, thespecific mechanisms of HIV-induced cell death in vivo are

poorly understood.



9/14

Apoptosis in AIDS

It is perhaps no accident that much research on HIV-induced apoptosishas focused on theFas pathway. TheFaspathway is relatively well understood. Because the apop-totic machinery of the cell is intricately linked to othercellular programs, and HIV clearly modulates a numberof these programs, the triggers of apoptosis are likely tobe multiple.

Understanding of the apoptosis determinants in HIVinfection will have profound implications in anti-HIVvaccine design. For instance, knowing that expressionof certain viral genes in antigen-expressing cells will in-duce apoptosis will prompt investigators to re-design im-munogens so that such genes are removed or modified. Aclear example of this is the Nef-dependent upregulationof Fas-L, which results in decreased anti-HIV cytotoxicT-lymphocyte (CTL) activity.162 One may rationalize thatonly a modified version ofnefthat cannot trigger Fas-L ex-

pression should be used as part of an HIV vaccine. Otherdeterminants of apoptosis, such as vpr, may decrease thelife span of an antigen-presenting cell, perhaps hinderingthe production of an effective immune response.

Although a better understanding of apoptosis will helpus dissect AIDS progression at the cellular and molecularlevels, it should be noted that prevention of bystandercell killing could prove a rational strategy in slowing orpreventing immune deterioration.

Acknowledgments

This work was supported by a grant from the NationalInstitutes of Health (R29-AI41407) to VP.

References

1. Ho DD, Neumann AU, Perelson AS, Chen W, Leonard JM,Markowitz M. Rapid turnover of plasma virions and CD4lymphocytes in HIV-1 infection. Nature 1995; 373: 123126.

2. Perelson AS, Neumann AU, Markowitz M, Leonard JM, HoDD. HIV-1 dynamics in vivo: Virion clearance rate, infectedcell life-span, and viral generation time. Science 1996; 271:15821586.

3. Wei X, Ghosh SK, Taylor ME, et al. Viral dynamics in human

immunodeficiency virus type 1 infection. Nature 1995; 373:117122.4. Shaw GM, Hahn BH, Arya SK, Groopman JE, Gallo RC,

Wong-Staal F. Molecular characterization of human T-cellleukemia (lymphotropic) virus type III in the acquired im-mune deficiency syndrome. Science 1984; 226: 11651171.

5. Fauci AS. The human immunodeficiency virus: Infectivityand mechanisms of pathogenesis. Science 1988; 239: 617622.

6. Zagury D, Bernard J, Leonard R, et al. Long-term culturesof HTLV-IIIinfected T cells: A model of cytopathology ofT-cell depletion in AIDS. Science 1986; 231: 850853.

7. Ameisen JC, Capron A. Cell dysfunction and depletion inAIDS: The programmed cell death hypothesis. Immunol Today1991; 12: 102105.

8. Finkel TH, Banda NK. Indirect mechanisms of HIV patho-genesis: How does HIV kill T cells? Curr Opin Immunol1994;6: 605615.

9. Laurent-CrawfordAG, KrustB, MullerS, etal. The cytopathiceffect of HIV is associated with apoptosis. Virology 1991; 185:829839.

10. Terai C, Kornbluth RS, Pauza CD, Richman DD, CarsonDA. Apoptosis as a mechanism of cell death in cultured Tlymphoblastsacutely infected with HIV-1. J Clin Invest1991;87: 17101715.

11. GrouxH, TorpierG, Monte D, MoutonY, CapronA, AmeisenJC. Activation-induced death by apoptosis in CD4+ T cellsfrom human immunodeficiency virus-infected asymptomaticindividuals. J Exp Med1992; 175: 331340.

12. Meyaard L, Otto SA, Jonker RR, Mijnster MJ, Keet RP,Miedema F. Programmed death of T cells in HIV-1 infection.Science 1992; 257: 217219.

13. Badley AD, McElhinny JA, Leibson PJ, Lynch DH, Alder-son MR, Paya CV. Upregulation of Fas ligand expression byhuman immunodeficiency virus in human macrophages me-

diates apoptosis of uninfected T lymphocytes. J Virol 1996;70: 199206.14. Westendorp MO,Frank R, Ochsenbauer C, et al. Sensitization

of T cells to CD95-mediated apoptosis by HIV-1 Tat andgp120. Nature 1995; 375: 497500.

15. Luciw PA. Human immunodeficiency viruses and their repli-cation. In: Fields BN, Knippe DM, Howley PM, ed.Fields Virology. Philadelphia: Lippincott-Raven 1996; 18811975.

16. Gabuzda DH, Lawrence K, Langhoff E, et al. Role of vifin replication of human immunodeficiency virus type 1 inCD4+ T lymphocytes. J Virol1992; 66: 64896495.

17. Sakai H, Shibata R, Sakuragi J, Sakuragi S, Kawamura M,Adachi A. Cell-dependent requirement of human immun-odeficiency virus type 1 Vif protein for maturation of virus

particles. J Virol1993; 67: 16631666.18. Blanc D, Patience C, Schulz TF, Weiss R, Spire B. Transcom-plementation of VIF-HIV-1 mutants in CEM cells suggeststhat VIF affects late steps of the viral life cycle. Virology 1993;193: 186192.

19. Fouchier RA, Simon JH, Jaffe AB, Malim MH. Human im-munodeficiency virus type 1 Vif does not influence expressionor virion incorporation of gag-, pol-, and env-encoded pro-teins. J Virol1996; 70: 82638269.

20. von Schwedler U, Song J, Aiken C, Trono D. Vif is crucial forhuman immunodeficiency virus type 1 proviral DNA synthe-sis in infected cells. J Virol1993; 67: 49454955.

21. Madani N, Kabat D. An endogenous inhibitor of human im-munodeficiency virus in human lymphocytes is overcome bythe viral Vif protein. J Virol1998; 72: 1025110255.

22. Geleziunas R, Bour S, Wainberg MA. Human immunode-ficiency virus type 1-associated CD4 downmodulation. AdvVirus Res 1994; 44: 203266.

23. Vincent MJ,Raja NU,JabbarMA. Human immunodeficiencyvirus type 1 Vpu protein induces degradation of chimericenvelope glycoproteins bearing the cytoplasmic and anchordomains of CD4: Role of the cytoplasmic domain in Vpu-induced degradation in the endoplasmic reticulum. J Virol1993; 67: 55385549.

24. Willey RL, Maldarelli F, Martin MA, Strebel K. Human im-munodeficiency virus type 1 Vpu protein inducesrapid degra-dation of CD4. J Virol1992; 66: 71937200.

25. Yao XJ, Friborg J, Checroune F, et al. Degradation of CD4 in-duced by human immunodeficiency virus type 1 Vpu protein:A predicted alpha-helix structure in the proximal cytoplasmic



10/14

M. Roshalet al.

region of CD4 contributes to Vpu sensitivity. Virology 1995;209: 615623.

26. Gottlinger HG, Dorfman T, Cohen EA, Haseltine WA. Vpuprotein of human immunodeficiency virus type 1 enhancesthe release of capsids produced by gag gene constructs ofwidely divergent retroviruses. Proc Natl Acad Sci USA 1993;90: 73817385.

27. Klimkait T, Strebel K, Hoggan MD, Martin MA, OrensteinJM. The human immunodeficiency virus type 1-specific pro-tein vpu is required for efficient virus maturation and release.

J Virol1990; 64: 621629.28. Kerkau T, Bacik I, Bennink JR, et al. The human immun-

odeficiency virus type 1 (HIV-1) Vpu protein interferes withan early step in the biosynthesis of major histocompatibil-ity complex (MHC) class I molecules. J Exp Med 1997; 185:12951305.

29. Banda NK, Bernier J, Kurahara DK, et al. Crosslinking CD4by human immunodeficiency virus gp120 primes T cells foractivation-induced apoptosis. J Exp Med 1992; 176: 10991106.

30. Lu YY, Koga Y, Tanaka K, Sasaki M, Kimura G, Nomoto K.Apoptosis inducedin CD4+ cells expressinggp160of humanimmunodeficiency virus type 1. J Virol1994; 68: 390399.

31. Bartz SR, Emerman M. Human immunodeficiency virus type1 Tat induces apoptosis and increases sensitivity to apoptoticsignals by up-regulating FLICE/caspase-8. J Virol 1999; 73:19561963.

32. Purvis SF, Jacobberger JW, Sramkoski RM, Patki AH,Lederman MM. HIV type 1 Tat protein induces apoptosisand death in Jurkat cells. AIDS Res Hum Retroviruses 1995;11: 443450.

33. Ensoli B, Barillari G, Salahuddin SZ, Gallo RC, Wong-StaalF. Tat protein of HIV-1 stimulates growth of cells derivedfrom Kaposis sarcoma lesions of AIDS patients. Nature 1990;345: 8486.

34. Mann DA, Frankel AD. Endocytosis and targeting of exoge-nous HIV-1 Tat protein. Embo J1991; 10: 17331739.35. Li CJ, Friedman DJ, Wang C, Metelev V, Pardee AB. Induc-

tion of apoptosis in uninfected lymphocytes by HIV-1 Tatprotein. Science 1995; 268: 429431.

36. Kerr JF, Wyllie AH, Currie AR. Apoptosis: A basic biolog-ical phenomenon with wide-ranging implications in tissuekinetics. Br J Cancer1972; 26: 239257.

37. Hartwell LH, WeinertTA. Checkpoints: Controls that ensurethe order of cell cycle events. Science 1989; 246: 629634.

38. Paulovich AG, ToczyskiDP, Hartwell LH. When checkpointsfail. Cell1997; 88: 315321.

39. Ivanov D, Kwak YT, Nee E, Guo J, Garcia-Martinez LF,Gaynor RB. Cyclin T1 domains involved in complex forma-tion with Tat and TAR RNA are critical for tat- activation. J

Mol Biol1999; 288: 4156.40. Romano G, Kasten M, De Falco G, Micheli P, Khalili K,

Giordano A. Regulatory functions of Cdk9 and of cyclin T1in HIV tat transactivation pathway gene expression. J CellBiochem 1999; 75: 357368.

41. Wei P, Garber ME, Fang SM, FischerWH, Jones KA.A novelCDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding toTAR RNA. Cell1998; 92: 451462.

42. Conant K, Ma M, Nath A, Major EO. Extracellular humanimmunodeficiency virus type 1 Tat protein is associated withan increase in both NF-kappa B binding and protein kinaseC activity in primary human astrocytes. J Virol 1996; 70:13841389.

43. Demarchi F, dAdda di Fagagna F, Falaschi A, Giacca M.

Activation of transcription factor NF-kappaB by the Tat pro-tein of human immunodeficiency virus type 1. J Virol 1996;70: 44274437.

44. Kinoshita S, Su L, Amano M, Timmerman LA, KaneshimaH, Nolan GP. The T cell activation factor NF-ATc positivelyregulates HIV-1 replication and gene expression in T cells.Immunity 1997; 6: 235244.

45. Li X, Multon MC, Henin Y, et al. Grb3-3 is up-regulatedin HIV-1 infected T-cells and can potentiate cell activationthrough NFATc. J Biol Chem 2000; 275: 3092530933.

46. Macian F, Rao A. Reciprocal modulatory interaction betweenhuman immunodeficiency virus type 1 Tat and transcriptionfactor NFAT1. Mol Cell Biol1999; 19: 36453653.

47. Kumar A, Manna SK, Dhawan S, Aggarwal BB.HIV-Tat pro-tein activates c-Jun N-terminal kinase and activator protein-1. J Immunol1998; 161: 776781.

48. Ambrosino C, Ruocco MR, Chen X, et al. HIV-1 Tat inducesthe expression of the interleukin-6 (IL6) gene by binding tothe IL6 leader RNA and by interacting with CAAT enhancer-binding protein beta (NF-IL6) transcription factors. J Biol

Chem 1997;272

: 1488314892.49. Scala G, Ruocco MR, Ambrosino C, et al. The expression ofthe interleukin 6 gene is induced by the human immunodefi-ciency virus 1 TAT protein. J Exp Med1994; 179: 961971.

50. Zauli G, Gibellini D, Celeghini C, et al. Pleiotropic effectsof immobilized versus soluble recombinant HIV-1 Tat pro-tein on CD3-mediated activation, induction of apoptosis, andHIV-1 long terminal repeat transactivation in purified CD4+T lymphocytes. J Immunol1996; 157: 22162224.

51. Manna SK,Aggarwal BB.Differential requirement forp56lckin HIV-tat versus TNF- induced cellular responses: Effectson NF-kappa B, activator protein-1, c-Jun N-terminal kinase,and apoptosis. J Immunol2000; 164: 51565166.

52. Li-Weber M, Laur O, Dern K, KrammerPH. T cell activation-induced and HIV tat-enhanced CD95(APO-1/Fas) ligand

transcription involves NF-kappaB. Eur J Immunol 2000; 30:661670.53. Buonaguro L, Barillari G, Chang HK, et al. Effects of the

human immunodeficiency virus type 1 Tat protein on theexpression of inflammatorycytokines.J Virol1992; 66: 71597167.

54. Sastry KJ, Reddy HR, Pandita R, Totpal K, Aggarwal BB.HIV-1 tat gene induces tumor necrosis factor-beta (lympho-toxin) in a human B-lymphoblastoid cell line. J Biol Chem1990; 265: 2009120093.

55. Shatrov VA, Ratter F, Gruber A, Droge W, Lehmann V.HIV type 1 glycoprotein 120 amplifies tumor necrosis factor-inducedNF-kappa B activation in Jurkat cells.AIDS ResHumRetroviruses 1996; 12: 12091216.

56. Banki K, Hutter E, Gonchoroff NJ, Perl A. Molecular order-

ing in HIV-induced apoptosis. Oxidative stress, activation ofcaspases, and cell survival are regulated by transaldolase. JBiol Chem 1998; 273: 1194411953.

57. Flores SC, Marecki JC, Harper KP, Bose SK, Nelson SK,McCord JM. Tat protein of human immunodeficiency virustype 1 represses expression of manganese superoxide dismu-tase in HeLa cells. Proc Natl Acad Sci USA 1993; 90: 76327636.

58. Westendrop MO, Shatrov VA, Schulze-Osthoff K, et al.HIV-1 Tat potentiates TNF-induced NF-kappa B activationand cytotoxicity by altering the cellular redox state. Embo J1995; 14: 546554.

59. Zauli G, Gibellini D, Milani D, et al. Human immunodefi-ciency virus type 1 Tat protein protects lymphoid, epithelial,and neuronal cell lines from death by apoptosis. Cancer Res



11/14

Apoptosis in AIDS

1993; 53: 44814485.60. Wang Z, Morris GF, Reed JC, Kelly GD, Morris CB. Ac-

tivation of Bcl-2 promoter-directed gene expression by thehuman immunodeficiency virus type-1 Tat protein. Virology1999; 257: 502510.

61. Zauli G, Gibellini D, Caputo A, et al. The human immun-odeficiency virus type-1 Tat protein upregulates Bcl-2 geneexpression in Jurkat T-cell lines and primary peripheral bloodmononuclear cells. Blood1995; 86: 38233834.

62. Sastry KJ, Marin MC, Nehete PN, McConnell K, el-NaggarAK, McDonnell TJ. Expression of human immunodeficiencyvirus type I tat results in downregulation of bcl-2 and in-duction of apoptosis in hematopoietic cells. Oncogene 1996;13: 487493.

63. Macho A, Calzado MA, Jimenez-Reina L, Ceballos E, LeonJ, Munoz E. Susceptibility of HIV-1-TAT transfected cells toundergo apoptosis. Biochemical mechanisms. Oncogene 1999;18: 75437551.

64. Burgering BM, Coffer PJ. Protein kinase B (c-Akt) in phos-phatidylinositol-3-OH kinase signal transduction. Nature

1995;376

: 599602.65. Wang CY, Mayo MW, Korneluk RG, Goeddel DV, BaldwinAS, Jr. NF-kappaB antiapoptosis: Induction of TRAF1 andTRAF2 and c-IAP1 and c- IAP2 to suppress caspase-8 acti-vation. Science 1998; 281: 16801683.

66. Kolenko V, Bloom T, Rayman P, Bukowski R, Hsi E, Finke J.Inhibition of NF-kappa B activity in human T lymphocytesinduces caspase-dependent apoptosis without detectable acti-vation of caspase-1 and -3. J Immunol1999; 163: 590598.

67. Giri DK, Aggarwal BB. Constitutive activation of NF-kappaB causes resistance to apoptosis in human cutaneous Tcelllymphoma HuT-78cells.Autocrine role of tumor necrosisfactor and reactive oxygen intermediates. J Biol Chem 1998;273: 1400814014.

68. Demarchi F, Gutierrez MI, Giacca M. Human immunode-

ficiency virus type 1 tat protein activates transcription fac-tor NF-kappaB through the cellular interferon-inducible,double-stranded RNA-dependent protein kinase, PKR. J Vi-rol1999; 73: 70807086.

69. Borgatti P, Zauli G, Colamussi ML, et al. Extracellular HIV-1Tat protein activates phosphatidylinositol 3- and Akt/PKBkinases in CD4+ T lymphoblastoid Jurkat cells. Eur J Im-munol1997; 27: 28052811.

70. Zauli G, La Placa M, Vignoli M, et al. An autocrine loop ofHIV type-1 Tat protein responsible for the improved sur-vival/proliferation capacity of permanently Tat-transfectedcells and required for optimal HIV-1 LTR transactivatingactivity. J Acquir Immune Defic Syndr Hum Retrovirol1995; 10:306316.

71. McCloskey TW, Ott M, Tribble E, et al. Dual role of HIV Tat

in regulation of apoptosis in T cells. J Immunol 1997; 158:10141019.

72. Ranki A, Lagerstedt A, Ovod V, Aavik E, Krohn KJ. Expres-sion kinetics and subcellular localization of HIV-1 regulatoryproteins Nef, Tat and Rev in acutely and chronically infectedlymphoid cell lines. Arch Virol1994; 139: 365378.

73. Katsikis PD, Garcia-Ojeda ME, Torres-Roca JF, GreenwaldDR, Herzenberg LA. HIV type 1 Tat protein enhancesactivation-but not Fas (CD95)-induced peripheral blood Tcell apoptosis in healthy individuals. Int Immunol 1997; 9:835841.

74. CohenEA, Dehni G, Sodroski JG,Haseltine WA. Human im-munodeficiency virus vpr product is a virion-associated regu-latory protein. J Virol1990; 64: 30973099.

75. Ogawa K, ShibataR, Kiyomasu T, etal. Mutational analysis of

the human immunodeficiency virus vpr open reading frame.J Virol 1989; 63: 41104114.

76. Sawaya BE, Khalili K, Gordon J, Taube R, Amini S. Cooper-ative interaction between HIV-1 regulatory proteins Tat andVpr modulates transcription of the viral genome. J Biol Chem2000; 275: 3520935214.

77. Balliet JW, Kolson DL, Eiger G, et al. Distinct effects inprimary macrophages and lymphocytes of the human im-munodeficiency virus type 1 accessory genes vpr, vpu, andnef: mutational analysis of a primary HIV-1 isolate. Virology1994; 200: 623631.

78. Connor RI, Chen BK, Choe S, Landau NR.Vpr is required forefficient replication of human immunodeficiency virus type-1in mononuclear phagocytes. Virology 1995; 206: 935944.

79. Accola MA, Bukovsky AA, JonesMS, Gottlinger HG. A con-served dileucine-containing motif in p6(gag) governs the par-ticle association of Vpx and Vpr of simian immunodeficiencyviruses SIV(mac) and SIV(agm). J Virol 1999; 73: 99929999.

80. Yuan X, Matsuda Z, Matsuda M, Essex M, Lee TH.

Human immunodeficiency virus vpr gene encodes a virion-associated protein.AIDS Res Hum Retroviruses 1990; 6: 12651271.

81. Gallay P, Stitt V, Mundy C, Oettinger M, Trono D. Role ofthe karyopherin pathway in human immunodeficiency virustype 1 nuclear import. J Virol1996; 70: 10271032.

82. Heinzinger NK, Bukinsky MI, Haggerty SA, et al. The Vprprotein of human immunodeficiency virus type 1 influencesnuclear localization of viral nucleic acids in nondividing hostcells. Proc Natl Acad Sci USA 1994; 91: 73117315.

83. Fouchier RA, Meyer BE, Simon JH, et al. Interaction of thehuman immunodeficiency virus type 1 Vpr protein with thenuclear pore complex. J Virol1998; 72: 60046013.

84. Popov S,Rexach M,Zybarth G, etal. Viral protein R regulatesnuclear import of the HIV-1 pre-integration complex. Embo

J1998; 17: 909917.85. Goh WC, Rogel ME, Kinsey CM, et al. HIV-1 Vpr increasesviral expression by manipulation of the cell cycle: A mecha-nism for selection of Vpr in vivo. Nat Med1998; 4: 6571.

86. Gummuluru S, Emerman M. Cell cycle- and Vpr-mediatedregulation of human immunodeficiency virus type 1 expres-sion in primary and transformed T-cell lines.J Virol1999; 73:54225430.

87. Subbramanian RA, Yao XJ, Dilhuydy H, et al. Human im-munodeficiency virus type 1 Vpr localization: Nuclear trans-port of a viral protein modulated by a putative amphipathichelical structure and its relevance to biological activity. J MolBiol1998; 278: 1330.

88. He J, Choe S, Walker R, Di Marzio P, Morgan DO, LandauNR. Human immunodeficiency virus type 1 viral protein R

(Vpr) arrests cells in the G2 phase of the cell cycle by inhibit-ing p34cdc2 activity. J Virol1995; 69: 67056711.

89. Jowett JB, Planelles V, Poon B, Shah NP, Chen ML, Chen IS.The human immunodeficiency virus type 1 vpr gene arrestsinfected T cells in the G2 +M phase of the cell cycle. J Virol1995; 69: 63046313.

90. Planelles V, Jowett JB, Li QX, Xie Y, Hahn B, Chen IS.Vpr-induced cell cycle arrest is conserved among primatelentiviruses. J Virol1996; 70: 25162524.

91. Re F, Braaten D, Franke EK, Luban J. Human immunod-eficiency virus type 1 Vpr arrests the cell cycle in G2 byinhibiting the activation of p34cdc2-cyclin B. J Virol 1995;69: 68596864.

92. Rogel ME, Wu LI, Emerman M. The human immunodefi-ciency virus type 1 vpr gene prevents cell proliferation during



12/14

M. Roshalet al.

chronic infection. J Virol1995; 69: 882888.93. Chang L, Chen C, Urlacher V, Lee T. Differential apoptosis

effects of primate lentiviral vpr and vpx in mammalian cells. J Biomed Sci 2000; 7: 322333.

94. Shostak LD, Ludlow J, Fisk J, et al. Roles of p53 and caspasesin the induction of cell cycle arrest and apoptosis by HIV-1vpr. Exp Cell Res 1999; 251: 156165.

95. Stewart SA, Poon B, Jowett JB, Chen IS. Human immun-odeficiency virus type 1 Vpr induces apoptosis following cellcycle arrest. J Virol1997; 71: 55795592.

96. Watanabe N, Yamaguchi T, Akimoto Y, Rattner JB, HiranoH, Nakauchi H. Induction of M-phase arrest and apoptosisafter HIV-1 vpr expression through uncoupling of nuclearand centrosomal cycle in HeLa cells. Exp Cell Res 2000; 258:261269.

97. Macreadie IG, Thorburn DR, Kirby DM, Castelli LA, deRozario NL, Azad AA. HIV-1 protein Vpr causes gross mito-chondrial dysfunction in the yeast Saccharomyces cerevisiae.FEBS Lett1997; 410: 145149.

98. Masuda M, Nagai Y, Oshima N, et al. Genetic studies with

the fission yeast Schizosaccharomyces pombe suggest involve-ment of wee1, ppa2, and rad24 in induction of cell cy-cle arrest by human immunodeficiency virus type 1 Vpr.

J Virol2000; 74: 26362646.99. Berglez JM, Castelli LA, Sankovich SA, Smith SC, Curtain

CC, Macreadie IG. Residues within the HFRIGC sequence ofHIV-1 vpr involved in growth arrest activities. Biochem BiophysRes Commun 1999; 264: 287290.

100. Chen M, Elder RT, Yu M, et al. Mutational analysis of Vpr-induced G2 arrest, nuclear localization, and cell death in fis-sion yeast. J Virol1999; 73: 32363245.

101. Di Marzio P, Choe S, Ebright M, Knoblauch R, Landau NR.Mutational analysis of cell cycle arrest, nuclear localizationand virion packaging of human immunodeficiency virus type1 Vpr. J Virol1995; 69: 79097916.

102. Macreadie IG, Castelli LA, Hewish DR, Kirkpatrick A, WardAC, Azad AA. A domain of human immunodeficiency virustype 1 Vpr containing repeated H(S/F)RIG amino acid motifscauses cellgrowtharrest and structural defects.Proc Natl AcadSci USA 1995; 92: 27702774.

103. MahalingamS, Collman RG,PatelM, MonkenCE, SrinivasanA. Role of theconserveddipeptide Gly75 andCys76on HIV-1Vpr function. Virology 1995; 210: 495500.

104. Zhou Y, Lu Y, Ratner L. Arginine residues in the C-terminusof HIV-1 Vpr are important for nuclear localization and cellcycle arrest. Virology 1998; 242: 414424.

105. Minemoto Y, Shimura M, Ishizaka Y, Masamune Y, YamashitaK. Multiple centrosome formation induced by the expressionof Vpr gene of human immunodeficiency virus. Biochem Bio-phys Res Commun 1999; 258: 379384.

106. Stewart SA, Poon B, Song JY, Chen IS. Human immunod-eficiency virus type 1 vpr induces apoptosis through caspaseactivation. J Virol2000; 74: 31053111.

107. PoonB, Jowett JB,Stewart SA, Armstrong RW, Rishton GM,Chen IS. Human immunodeficiency virus type 1 vpr gene in-duces phenotypic effects similar to those of the DNA alkylat-ing agent, nitrogen mustard. J Virol1997; 71: 39613971.

108. Shimura M, Onozuka Y, Yamaguchi T, Hatake K, Takaku F,Ishizaka Y. Micronuclei formation with chromosome breaksand gene amplification caused by Vpr, an accessory gene ofhuman immunodeficiency virus. Cancer Res 1999; 59: 22592264.

109. Shimura M, Tanaka Y, Nakamura S, et al. Micronuclei for-mation and aneuploidy induced by Vpr, an accessory geneof human immunodeficiency virus type 1. Faseb J 1999; 13:

621637.110. Zhang S, PointerD, SingerG, FengY, Park K, Zhao LJ.Direct

binding to nucleic acids by Vpr of human immunodeficiencyvirus type 1. Gene 1998; 212: 157166.

111. Bouhamdan M, Benichou S, Rey F, et al. Human immunod-eficiency virus type 1 Vpr protein binds to the uracil DNAglycosylase DNA repair enzyme. J Virol1996; 70: 697704.

112. Selig L, Benichou S, Rogel ME, et al. Uracil DNA glycosylasespecifically interacts with Vpr of both human immunodefi-ciency virus type 1 and simian immunodeficiency virus ofscooty mangabeys, but binding does not correlate with cellcycle arrest. J Virol1997; 71: 48424846.

113. Withers-Ward ES, Jowett JB, Stewart SA, et al. Human im-munodeficiency virus type 1 Vpr interacts with HHR23A, acellular protein implicated in nucleotide excision DNA repair.

J Virol 1997; 71: 97329742.114. Mansky LM, Preveral S, Selig L, BenarousR, Benichou S. The

interaction of vpr with uracil DNA glycosylase modulates thehuman immunodeficiency virus type 1 In vivo mutation rate.

J Virol 2000; 74: 70397047.

115. Gragerov A, Kino T, Ilyina-Gragerova G, Chrousos GP,Pavlakis GN. HHR23A, the human homologue of the yeastrepair protein RAD23, interacts specifically with Vpr pro-tein and prevents cell cycle arrest but not the transcriptionaleffects of Vpr. Virology 1998; 245: 323330.

116. Morgan SE, Kastan MB. p53 and ATM: Cell cycle, cell death,and cancer. Adv Cancer Res 1997; 71: 125.

117. Bartz SR,RogelME, Emerman M. Human immunodeficiencyvirus type 1 cell cycle control: Vpr is cytostatic and medi-ates G2 accumulation by a mechanism which differs fromDNA damage checkpoint control. J Virol 1996; 70: 23242331.

118. Jacotot E, Ravagnan L, Loeffler M, et al. The HIV-1 viralprotein R induces apoptosis via a direct effect on the mito-chondrial permeability transition pore. J Exp Med2000; 191:

3346.119. Ayyavoo V, Mahboubi A, MahalingamS, etal. HIV-1 Vpr sup-presses immune activation and apoptosis through regulationof nuclear factor kappa B. Nat Med1997; 3: 11171123.

120. Roux P, Alfieri C, Hrimech M, Cohen EA, Tanner JE. Activa-tion of transcription factors NF-kappaB and NF-IL-6 by hu-man immunodeficiency virus type 1 protein R (Vpr) inducesinterleukin-8 expression. J Virol2000; 74: 46584665.

121. Conti L, Rainaldi G, Matarrese P, et al. The HIV-1 vpr pro-tein acts as a negative regulator of apoptosis in a human lym-phoblastoid T cell line: Possible implications for the patho-genesis of AIDS. J Exp Med1998; 187: 403413.

122. Fukumori T, Akari H, Iida S, et al. The HIV-1 Vpr displaysstrong anti-apoptotic activity. FEBS Lett1998; 432: 1720.

123. Conti L, Matarrese P, Varano B, et al. Dual role of the HIV-1

vpr protein in the modulation of the apoptotic response of Tcells. J Immunol2000; 165: 32933300.

124. Dittmar MT, McKnight A, Simmons G, Clapham PR, WeissRA, Simmonds P. HIV-1 tropism and co-receptor use. Nature1997; 385: 495496.

125. Moore JP. Coreceptors: Implications for HIV pathogenesisand therapy. Science 1997; 276: 5152.

126. Davis CB, Dikic I, Unutmaz D, et al. Signal transductiondue to HIV-1 envelope interactions with chemokine receptorsCXCR4 or CCR5. J Exp Med1997; 186: 17931798.

127. Neudorf SM, Jones MM, McCarthy BM, Harmony JA, ChoiEM. The CD4 molecule transmits biochemical informationimportant in the regulation of T lymphocyte activity. CellImmunol1990; 125: 301314.

128. Weissman D, Rabin RL, Arthos J, et al. Macrophage-tropic



13/14


14/14

M. Roshalet al.

cellular signal transducing proteins. Front Biosci 2000; 5:D268283.

160. Hodge S, Novembre FJ,Whetter L, Gelbard HA,DewhurstS.Induction of fas ligand expression by an acutely lethal simianimmunodeficiency virus, SIVsmmPBj14. Virology 1998; 252:354363.

161. Xu XN, Laffert B, Screaton GR, et al. Induction of Fas ligandexpression by HIV involves the interaction of Nef with the Tcell receptor zeta chain. J Exp Med1999; 189: 14891496.

162. Xu XN, Screaton GR, Gotch FM, et al. Evasion of cytotoxicT lymphocyte (CTL) responses by nef-dependent induction ofFas ligand (CD95L) expression on simian immunodeficiencyvirus-infected cells. J Exp Med1997; 186: 716.

163. Zauli G, Gibellini D, Secchiero P, et al. Human immunode-ficiency virus type 1 Nef protien sensitizes CD4(+) T lym-phoid cells to apoptosis via functional upregulation of theCD95/CD95 ligand pathway. Blood1999; 93: 10001010.

164. Bell I, AshmanC, Maughan J, HookerE, Cook F, Reinhart TA.Association of simian immunodeficiency virus Nef with theT-cell receptor (TCR) zeta chain leads to TCR down-

modulation. J Gen Virol1998;79

: 27172727.165. Howe AY, Jung JU, Desrosiers RC. Zeta chain of the T-cellreceptor interacts with nef of simian immunodeficiency virusand human immunodeficiency virus type 2. J Virol1998; 72:98279834.

166. Schaefer TM, Bell I, Fallert BA, Reinhart TA. The T-cellreceptor zeta chain contains two homologous domains withwhich simian immunodeficiency virus Nef interacts and me-diates down-modulation. J Virol2000; 74: 32733283.

167. Barber SA, Flaherty MT, Plafker SM, Clements JE. A novelkinase activity associated withNef derivedfrom neurovirulent

simian immunodeficiency virus. Virology 1998; 251: 165175.

168. Brown A, Wang X, Sawai E, Cheng-Mayer C. Activation ofthe PAK-related kinase by human immunodeficiency virustype 1 Nef in primary human peripheral blood lymphocytesand macrophagesleads to phosphorylation of a PIX-p95 com-plex. J Virol1999; 73: 98999907.

169. Facker OT, Lu X, Frost JA, et al. p21-activated kinase 1 playsa critical role in cellular activation by Nef.Mol Cell Biol2000;20: 26192627.

170. Manninen A, Hiipakka M, Vihinen M, Lu W, Mayer BJ,Saksela K. SH3-Domain binding function of HIV-1 Nef isrequired for association with a PAK-related kinase. Virology1998; 250: 273282.

171. Nunn MF, Marsh JW. Human immunodeficiency virus type1 Nef associates with a member of the p21-activated kinasefamily. J Virol1996; 70: 61576161.

172. Renkema GH, Manninen A, Mann DA, Harris M, Saksela K.Identification of the Nef-associated kinase as p21-activatedkinase 2. Curr Biol1999; 9: 14071410.

173. Sawai ET, Baur A, Struble H, Peterlin BM, Levy JA, Cheng-Mayer C. Human immunodeficieny virus type 1 Nefassociateswith a cellular serine kinase in T lymphocytes. Proc Natl AcadSci USA 1994; 91: 15391543.

174. Bokoch GM. Caspase-mediated activation of PAK2 duringapoptosis: Proteolytic kinase activation as a general mecha-nism of apoptotic signaltransduction? Cell Death Differ1998;5: 637645.

175. Rudel T, Bokoch GM. Membrane and morphological changesin apoptotic cells regulated by caspase-mediated activation ofPAK2. Science 1997; 276: 15711574.


Documents

Apoptosis Hiv(1)