HIV gp41-induced apoptosis is mediated by caspase-3-dependent mitochondrial depolarization, which is...

HIV gp41-induced apoptosis is mediated by caspase-3-dependent mitochondrial depolarization, which isinhibited by HIV protease inhibitor nelfinavir

Himanshu Garg and Robert Blumenthal1

Center for Cancer Research Nanobiology Program, National Cancer Institute, National Institutesof Health, Frederick, Maryland

Abstract: Apoptotic loss of CD4� T cells hasbeen proposed as a mechanism of T cell depletionin human immunodeficiency virus (HIV) infectionsresulting in immunodeficiency. The Env glycopro-tein has been implicated in apoptosis of uninfectedbystander cells via gp120 binding to CD4/CXCchemokine receptor 4 as well as the fusion/hemi-fusion process mediated by gp41. Using an in vitromodel of coculture of Env-expressing cells as ef-fectors and CD4� T cells as targets, we find thatapoptosis mediated by Env glycoprotein in by-stander cells in fact correlates with gp41-inducedhemifusion. Further, the apoptotic pathway initi-ated by this interaction involves caspase-3-depen-dent mitochondrial depolarization and reactive ox-ygen species production. HIV gp41-induced mito-chondrial depolarization is inhibited by proteaseinhibitor nelfinavir but not by other HIV proteaseinhibitors or inhibitors of calpain and cathepsin.This “kiss of death” (hemifusion) signaling pathwayis independent of p38 mitogen-activated proteinkinase and p53, making it distinct from the apo-ptosis seen in syncytia. We also show that virion-induced apoptosis is gp41-dependent. Our findingsprovide new insights into the mechanism via whichHIV gp41 mediates apoptosis in bystander cells. J.Leukoc. Biol. 79: 351–362; 2006.

Key Words: hemifusion � Env glycoprotein � CXCR4

INTRODUCTION

Human immunodeficiency virus (HIV) infections cause a pro-gressive and irreversible depletion of CD4� T cells, leading toimmunodeficiency. The exact mechanism of HIV-mediated Tcell loss is not clear, although apoptosis has been suggested tobe a major mechanism [1]. HIV-mediated apoptosis remainscontroversial for several reasons. First, whether HIV inducesapoptosis in infected cells or in uninfected bystander cellsremains hotly debated. Further, the mechanism via which HIVmediates apoptosis in infected or uninfected cells is not clear.

Various viral proteins including Env, Vpr, and Tat [2–5]have been implicated in HIV-mediated apoptosis. The Envglycoprotein is composed of a gp120 surface unit, which in-

teracts with CD4 (receptor) and CXC chemokine receptor 4(CXCR4)/CC chemokine receptor 5 (CCR5; coreceptor) ontarget cells and a gp41 transmembrane protein, which is a classI fusion protein mediating fusion of membranes via a pH-independent mechanism [6]. As Env is expressed on the sur-face of infected cells and can interact with CD4 on uninfectedcells, the preferential depletion of CD4� T cells in HIVinfection suggests that Env may be the most likely candidatefor induction of apoptosis in bystander cells. In fact, HIV Env,expressed on the cell surface, has been shown to interact withCD4 and CXCR4 to trigger apoptosis in uninfected cells [5, 7].The function of Env glycoprotein is not restricted to receptor/coreceptor binding but, in fact, culminates in fusion of effectorand target membranes. In this context, pathogenecity of HIVhas been associated with the fusogenic potential of the Envglycoprotein [8–10]. In a bystander scenario, whether Envsignals apoptosis via binding to CD4 and CXCR4 or via thefusion process remains controversial. In this regard, a recentreport shows that Env-mediated apoptosis in bystander cellscorrelates with a gp41-dependent, hemifusion-like event andcan be inhibited by gp41-specific fusion inhibitory peptides[11]. Furthermore, the requirement of membrane fusion forinduction of apoptosis has also been reported for CCR5 tropicviruses [12, 13].

Although the interaction of HIV gp120 with receptor andcoreceptor is required for the induction of apoptosis, sig-naling via either of these receptors seems dispensable forthis process [14, 15]. Hence, the signaling mechanism viawhich HIV Env mediates apoptosis in bystander cells is notclear, although several reports indicate that caspase activa-tion [16, 17], mitochondrial dysfunction [18, 19], and reac-tive oxygen species (ROS) may be involved [20]. Neverthe-less, the sequence of these events in HIV Env-mediatedapoptosis remains unclear. In this study, we have tried todetermine whether Env-mediated apoptosis in bystandercells is a result of a gp41-dependent hemifusion-like eventand the signaling pathway activated.

1 Correspondence: Center for Cancer Research, NCI-Frederick, P.O. Box B,Building 469, Room 152, Miller Drive, Frederick, MD 21702-1201. E-mail:[email protected]

Received August 2, 2005; revised October 7, 2005; accepted October 17,2005; doi: 10.1189/jlb.0805430.

0741-5400/06/0079-351 © Society for Leukocyte Biology Journal of Leukocyte Biology Volume 79, February 2006 351

MATERIALS AND METHODS

Cells and reagents

Chinese hamster ovary (CHO), expressing HIV HXB2 Env glycoprotein [CHOwild-type (WT)], glycosylphosphatidylinositol (GPI)-anchored Env (CHO PI),or vector-transfected {CHO (EE) [21]}, were obtained from the AIDS Referenceand Reagent program. All CHO cell lines were maintained in glutamine-deficient minimum essential media supplemented with 10% fetal bovine serum(FBS), penicillin-streptomycin (5000 U/ml), and L-methionine sulfoxamine(400 �m). SupT1 cells were maintained in RPMI media supplemented with10% FBS and penicillin-streptomycin (5000 U/ml).

Isolation of CD4� lymphocytes from peripheralblood mononuclear cells (PBMC)

Whole blood was collected from healthy donors via the anonymous blood donorprogram at the National Cancer Institute (NCI)-Frederick (MD). PBMC wereisolated using Ficoll gradient (Amersham, Little Chalfont, UK). The purifiedPBMC were washed with phosphate-buffered saline and passed through a CD4T cell enrichment column (Cedarlane Labs, Ontario, Canada). The purifiedCD4� T cells were activated with 2 �g/ml phytohemagglutinin (PHA) andinterleukin (IL)-2, 10 U/ml, and cultured for 3 days prior to use. The purity ofCD4� T cells (�95%) was determined via staining with phycoerythrin-conjugated anti-CD4 antibody (BD PharMingen, San Diego, CA) followed byflow cytometry prior to use.

Reagents

CXCR4 antagonist AMD3100; peptides C34, T20, and N36; and proteaseinhibitors nelfinavir, saquinavir, and indinavir were obtained from the AIDSReference and Reagent program. Caspase inhibitors E64D, pepstatin,SB203580, and cyclic pifithrin were from Calbiochem (San Diego, CA). Anti-Fas antibody clone CH-11 was from Upstate Inc. (Lake Placid, NY). Allfluorescent dyes were from Molecular Probes (Eugene, OR). N36mut(e,g) peptidewas a kind gift from Dr. Marius Clore (NIH, Bethesda, MD), 2F5 antibody wasa kind gift from Dr. Hermann Katinger (University of Natural Resources,Vienna, Austria), and NC-1 antibody was a gift from Dr. Shibo Jiang (New YorkBlood Center, New York, NY).

Induction of apoptosis

CHO WT, GPI-anchored Env (CHO PI), or vector-transfected (CHO EE) wereseeded in 24-well plates at 105 cells per well and allowed to adhere overnight.Subsequent day media were removed, and 0.5–1.0 � 106 SupT1 cells wereadded per well for a final effector-to-target ratio of 1:5–1:10. Different inhib-itors were preincubated with SupT1 cells or added at the time of coculture. Thecells were cocultured for 24 h, following which the nonadherent target cellswere collected and used for various analyses.

Detection of apoptosis

Apoptosis was determined in target cells 24 h post-coculture, as phosphatidylserine exposure via Annexin V staining (Molecular Probes) or as DNA frag-mentation using the deoxyuridine triphosphate nick-end labeling (TUNEL)assay (Roche, Nutley, NJ), as per the manufacturers’ instructions, followed byflow cytometry (FACSCalibur, BD Biosciences, San Jose, CA). At least 10,000events were collected and analyzed using Cellquest software.

Detection of mitochondrial depolarization and ROS

Mitochondrial depolarization was determined by staining cells with 10 nMDiOC6 dye for 15–20 min at 37°C, followed by flow cytometry. ROS productionin cells was determined via dihydroethidium (DHE) staining, 2.5 �M at 37°Cfor 30 min, followed by flow cytometry.

Caspase activation

Caspase-3 activation was determined by staining cells with fluorescein iso-thiocyante (FITC)-labeled anti-active caspase-3 monoclonal antibody (mAb;BD PharMingen), as per the manufacturer’s instructions, followed by flowcytometry. Conversely, caspase-3/7 or caspase-8 activity was determined by

substrate-based luminescent Caspase-Glo assay (Promega, Madison, WI), asper the manufacturer’s instructions.

Dye transfer assay

CHO cells were labeled with nondiffusible cytoplasmic dye 4-chloromethylbenzoyl amino tetramethyl rhodamine (CMTMR; 10 �M) or diffusible cyto-plasmic dye Calcein Red (10 �M) or lipophilic membrane dye 1,1�-dioctade-cyl-3,3,3�,3�-tetramethylindocarbocyanine (DiI; Vybrant cell labeling kit, Mo-lecular Probes). The cells were then cocultured with unlabeled SupT1 cells for24 h. Subsequently, the nonadherent target cells were collected and stainedwith Annexin V for detection of apoptosis and analyzed by flow cytometry. Atransfer of dye (cytoplasmic or membrane) was acquired in the red channel,and the green channel was used for apoptosis detection via Annexin V-FITC.This assay was used to determine whether the apoptotic target cells seen in oursystem had taken up cytoplasmic or membrane dyes from the effector cells.

Fusion from without

High-titer HIV-1 MN virions were a kind gift from Jeff Lifson and Julian Bess[National Institutes of Health (NIH) AIDS vaccine program, NCI-Frederick].SupT1 cells were incubated with 200 ng/ml p24 equivalent of virus in thepresence of 1 �M zidovudine (AZT) to prevent reverse transcription and anyproductive infection. Inhibitors C34 or AMD3100 were added prior to additionof virus. Fusion from without was observed, and photomicrographs werecollected at 2 h. Caspase-3 activity was determined at 24 h post-virus addition.

RESULTS

Fusion-competent HIV Env mediates apoptosisin bystander cells

The role of membrane-expressed HIV Env in mediating apo-ptosis of bystander cells has been well-documented [5, 22].The mechanism involved in this process, however, remainscontroversial, specifically the role of gp120 binding to CD4 andCXCR4 versus fusion process mediated via gp41 [23]. Toaddress the issue of whether HIV Env mediates apoptosis inbystander cells and whether this process requires gp41 func-tion, an in vitro model similar to one used by others [11, 14]was established. CHO WT, CHO PI HIV Env, or CHO EE cellswere used as effectors. The CHO cells were cocultured withSupT1 cells as target cells for 24 h, followed by collection ofthe nonadherent target cells and analysis of apoptosis. We haveconfirmed that CHO WT cells are fusion-competent, whereasCHO PI cells express similar amounts of HIV-1 gp120 but lackfusion capability [21, 24] (data not shown). Fusion-competentHIV Env induced apoptosis in SupT1 cells determined asphosphatidlyserine (PS) exposure by Annexin V staining (Fig.1a) or DNA fragmentation by TUNEL staining (Fig. 1b), andGPI-anchored Env had no effect similar to control cells. Thissuggests that the apoptosis-inducing potential of HIV Env isrelated to its fusion competence.

HIV Env-mediated apoptosis is gp41-dependent

To further corroborate the above findings, we blocked HIV-induced fusion at the level of CXCR4 using the CXCR4antagonist AMD3100 or at the level of gp41 six helix bundleformation using C34 peptide [25]. Blocking Env function at thelevel of gp120 (AMD) or gp41 (C34) completely inhibitedHIV-induced apoptosis (Fig. 2a), suggesting that the phenom-enon requires gp41 function.

352 Journal of Leukocyte Biology Volume 79, February 2006 http://www.jleukbio.org

The gp41 protein of HIV consists of two heptad repeatregions, which interact with each other in a zipper-like mannerto pull the viral and cellular membrane in close apposition-mediating fusion [6]. Peptides specific to either of these regionsinteract with the corresponding region and are capable ofdisrupting the fusion process. To determine whether inhibitionof apoptosis via C34 was a specific phenomenon unique to thepeptide, other fusion-inhibitory peptides were tested undersimilar conditions. As seen in Figure 2b, all of the fusion-inhibitory peptides derived from the C (C34, T20)- or N-terminal (N36, N36mut(e,g) [26]) heptad repeat regions inhibitedapoptosis, suggesting that the effect was not unique to C34peptide. Further, the use of gp41-specific, broadly neutralizingantibody 2F5 [27, 28] also showed inhibition of apoptosis, andanother gp41-specific antibody lacking neutralizing effectsNC-1 [29] had no effect (Fig. 2c). The binding site of 2F5 islocated outside the coiled domains (heptad repeats) in a regionjust proximal to the transmembrane region [30]. Although theexact mechanism of fusion inhibition via 2F5 is not clear, it isreported to be distinct from peptide inhibitors [31]. Thesestudies indicate that gp41 plays a critical role in HIV Env-mediated apoptosis, and inhibition of gp41 function at varioussteps inhibits apoptosis.

HIV Env-mediated apoptosis correlates with ahemifusion-like event

Hemifusion is a phenomenon mediated via a variety of viralfusion proteins [32–34], whereby the outer leaflet of effectorand target membranes fuse without fusion of the inner leafletsor the formation of the fusion pore. Under hemifusion condi-tions, mixing of lipid components between the membranesoccurs, which can be demonstrated via transfer of fluorescentlylabeled lipid dyes, and full fusion results in syncytia formation,

which can be studied by intracytoplasmic labeling of effectorand target cells. To determine whether the apoptosis seenunder our coculture conditions was a result of full fusion ofeffector and target cells (as suggested by Ferri et al. [35]) ormixing of lipid contents between cells (as suggested by Blancoet al. [11]), a dye transfer assay was used, whereby CHO cellslabeled with nondiffusable CMTMR dye, diffusible calcein reddye, or the lipophilic DiI dye were cocultured with unlabeledSupT1 cells. Mixing of cellular or lipid contents could bedetected via transfer of the dye to the unlabeled target cells.Further, to detect apoptosis, Annexin V was used as themarker. As seen in Figure 3, apoptosis occurred primarily incells that had taken up the lipid dye DiI from the effector cells,and there was little transfer of the diffusible (calcein) ornondiffusible (CMTMR) cytoplasmic dyes. Apoptosis andDiI transfer was blocked by C34, indicating that it was anEnv-mediated phenomenon and not a passive transfer ofdye. This suggests that HIV Env-mediated apoptosis corre-lates with exchange of lipid components between effectorand target cells.

HIV Env-mediated apoptosis involvesmitochondrial depolarization and ROS production

Apoptosis mediated via various stimuli involves mitochon-drial depolarization and ROS production as early events inthe apoptotic pathway. In HIV infection, mitochondrial dys-function has been associated with T cell loss in vivo [18]. Todetermine the signaling mechanism involved in HIV Env-induced apoptosis, we first determined whether apoptosiswas mediated via the mitochondrial pathway. For this pur-pose, cells were stained with the mitochondrial potentialsensitive dye DiOC6 and analyzed by flow cytometry. A

Fig. 1. Fusion-competent Env glycoprotein medi-ates apoptosis in bystander cells. SupT1 cells werecocultured with CHO WT, CHO PI, or CHO EE cellsat a ratio of 1:10. Twenty-four hours post-coculture,the nonadherent target cells were collected and ana-lyzed for apoptosis via Annexin V (a) or TUNEL (b)staining.

Garg and Blumenthal HIV gp41-mediated apoptosis 353

reduction in fluorescent intensity of DiOC6 staining is indica-tive of loss of mitochondrial potential. As seen in Figure 4a,a loss in mitochondrial potential was observed following co-culture of target cells with HIV Env-expressing cells. Thisphenomenon was inhibited by AMD as well as C34, suggestinggp41 dependence.

Oxidative stress in lymphocytes as a result of HIV infectionhas been reported in vitro [16, 20] and in vivo [36]. Hence, weanalyzed ROS production in apoptotic cells using the ROSsensor DHE. Staining of cells with DHE revealed that theapoptosis seen in our model involves ROS production, whichwas also inhibited via C34 and AMD (Fig. 4b) and not observedin GPI-anchored protein (data not shown).

Caspase-3 but not caspase-8 is involved in HIVEnv-mediated apoptosis

Caspases are critical mediators of apoptosis and have beenshown to be involved in HIV Env-mediated apoptosis [14, 17,37]. To determine whether caspase-3 was activated under ourapoptosis-inducing conditions, a direct, intracytoplasmic stain-ing of cells with a mAb against active caspase-3 was used. Asseen in Figure 5a, Env-mediated apoptosis involved activa-tion of caspase-3, which was again inhibited by C34, suggest-ing the requirement of gp41. Caspase-3 activation has oftenbeen shown to be downstream of caspase-8 activation in re-ceptor (Fas, tumor necrosis factor receptor)-mediated apopto-

sis. To determine whether caspase-3 activation was down-stream of caspase-8, a substrate-based assay was used todetermine the activity of both of these enzymes under identicalculture conditions. As seen in Figure 5b, there was no signif-icant activation of caspase-8 under conditions where caspase-3activity was fourfold higher in Env-exposed cells. As a control,we induced apoptosis in CEM cells or SupT cells using anti-Fas antibody CH-11. As shown in Figure 5c, although anti-Fasinduced caspase-3 and caspase-8 activation in CEM cells, itdid not induce apoptosis in SupT cells, consisitent with pre-vious reports [38]. This suggests that caspase-3 activation andapoptosis, under our culture conditions, were most likely in-dependent of caspase-8 activity.

Caspase-3 activation is an early event upstreamof mitochondrial depolarization, ROS production,and PS exposure

Having determined that the apoptosis induced by HIV Env inour coculture model involves mitochondrial depolarization,ROS production, and caspase-3 activation, we wished to de-termine the sequence of these activations to determine thesignaling pathway. We used the broad-spectrum caspase in-hibitor Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) todetermine whether caspase activation was upstream or down-stream of mitochondrial depolarization. Interestingly, Z-VAD-FMK inhibited mitochondrial depolarization as deter-

Fig. 2. Env-mediated apoptosis can be inhibited by gp41-specific fusion in-hibitors. (a) CHO WT cells were cocultured with SupT1 cells in the presence orabsence of CXCR4 antagonist AMD3100 (2 �M) or gp41 fusion inhibitor C34 (2�M), and apoptosis was determined 24 h later via TUNEL staining. Gp41-specific fusion-inhibitory peptides C34, T20, N36, and N36mut(e,g) (4 �M; b) orHIV gp41-specific neutralizing 2F5 or non-neutralizing NC-1 antibodies (20�g/ml; c) were added to inhibit Env-mediated apoptosis, determined by AnnexinV staining. Data are mean � SD triplicate observations. All experiments wererepeated twice with similar results. Statistical significance was assessed byStudent’s t-test (*, P�0.001).


mined by DiOC6 staining (Fig. 6a), suggesting that caspaseactivation was in fact upstream of mitochondria. Inhibition ofmitochondrial depolarization via Z-VAD has been linked toinhibition of caspase-8 upstream of caspase-3; however, nosignificant caspase-8 activation was observed under our con-ditions. This suggests that mitochondrial depolarization trig-gered by HIV Env in target cells is most likely caspase-8-independent. In this context, recent studies demonstrate therole of early caspase-3 activation and amplification of apoptosisvia caspase-3-mediated mitochondrial depolarization [39].Consistent with this hypothesis, we found that the caspase-3-specific inhibitor Ac-DEVD-CHO also inhibited apoptosisprior to mitochondrial depolarization. Further, Z-VAD-FMKand Ac-DEVD-CHO inhibited ROS production and PS expo-sure (Fig. 6b). To further corroborate these findings, we used

other caspase inhibitors, including inhibitors of caspase-9 (Ac-LEHD-CHO) and caspase-8 (Ac-IETD-CHO), to see the effecton mitochondrial depolarization. As seen in Figure 6c, Z-VADand DEVD almost completely inhibited mitochondrial depo-larization, and LEHD and IETD were significantly less effec-tive. The partial inhibition via LEHD (approximately 50%)suggests that caspase-3 may also act via the caspase-9-depen-dent amplification loop, as has been seen in other models [39,40]. The slight inhibition via caspase-8 inhibitor IETD (ap-proximately 30%) is most likely a result of its nonspecific effectof caspase-3, as has been shown by others [41]. However,neither of the inhibitors were as effective as Z-VAD andDEVD, suggesting that caspase-3 is a key regulator of mito-chondrial depolarization as well as ROS production in HIVgp41-mediated apoptosis.

Fig. 3. HIV gp41-mediated apoptosis occurs pre-dominantly in cells after a hemifusion-like event.CHO WT cells were labeled with DiI, calcein red,or CMTMR and cocultured with unlabeled SupT1cells. Twenty-four hours post-coculture, the nonad-herent cells were stained with Annexin V-FITC andanalyzed for dye transfer (FL-2) or apoptosis (FL-1)via flow cytometry. C34 (2 �M) was added to inhibitfusion (right panels).


HIV gp41-mediated apoptosis of bystander cellsis independent of p53 and p38 mitogen-activated protein kinase (MAPK)

Extensive work by Perfettini and co-workers [42] identified thesignaling pathway involved in apoptosis of syncytia formed viaEnv glycoprotein-mediated fusion. This pathway is activated insyncytia after a latent period post-fusion and involves p38MAPK-mediated activation of p53 and subsequent p53-depen-dent apoptosis. To determine whether apoptosis, seen in oursystem, followed a similar pathway, we tried to inhibit apopto-sis using p53 inhibitor cyclic pifithrin (10 �M) or p38 MAPKinhibitor SB203580 (1 �M). Interestingly, neither of theseinhibitors showed any inhibition (Fig. 7) at concentrationsshown to inhibit syncytial apoptosis [42], suggesting that p38-mediated p53 activation was most likely not involved. This isconsistent with a recent report [43], whereby transmission ofcontagious apoptosis by Env glycoprotein in syncytia is alsop53-independent.

HIV gp41-mediated apoptosis is inhibitedby nelfinavir

Protease inhibitors used in HIV therapy have been shown tohave beneficial effects in infected individuals by inhibitingapoptosis, which is distinct from the antiviral activities of thesecompounds [44]. Nelfinavir is a HIV-specific protease inhibi-tor, which has been shown to have antiapoptotic activities viainhibition of mitochondrial depolarization [45]. As apoptosisinduced in our experiments was mitochondria-dependent, wewished to determine whether nelfinavir would inhibit this pro-cess. Interestingly, we found that nelfinavir inhibited HIVgp41-mediated apoptosis in a dose-dependent manner (Fig.8a). Surprisingly, other HIV protease inhibitors (indinavir andsaquinavir) were not effective nor were inhibitors of cathepsin(E64D) or calpain (pepstatin; Fig. 8b). Furthermore, nelfinavirseemed to act at an early step in the process, as it inhibited

mitochondrial depolarization as well as PS exposure and ROSproduction (Fig. 8c). However, the inhibitory activity on mito-chondrial depolarization was more prominent than the effect onROS or PS exposure (Fig. 8c) consistent with its activity onmitochondria permeability transition pore [46]. This suggeststhat HIV gp41-mediated apoptosis involves a mitochondrialamplification loop that can be inhibited by nelfinavir but not byother HIV-specific proteases.

HIV Env-mediated apoptosis in primary CD4� Tcells is gp41-dependent

The preceding experiments were conducted with SupT1 cells,which are well-documented for HIV studies. Nevertheless, topreclude the possibility that our results were specific to SupT1cells, we used CD4� T cells derived from peripheral blood ofhealthy donors (PBMC) and activated with PHA and IL-2. Asseen in Figure 9, a and b, activated primary CD4� T cellsalso underwent apoptosis when cocultured with CHO WT cellsvia a gp41-dependent mechanism, as apoptosis was inhibitedby C34 as efficiently as by AMD. CD4� PBMC also showedactivation of caspase-3, and no significant increase incaspase-8 activity was observed (Fig. 9c). This suggests thateven in primary cells, HIV Env-mediated apoptosis is gp41-dependent.

Virion-induced “fusion from without” results incaspase-3 activation, which is dependent ongp41 function

The induction of apoptosis via HIV-1 virion is unlikely atmultiplicities of infection commonly used for the study of HIVinfection in vitro. Nevertheless, high titers of virus can inducefusion from without, a phenomenon whereby virus particlesmediate fusion of cells in the absence of virus replication [47].Interestingly, some recent studies suggest that HIV Env pre-sented on infectious virions can mediate apoptosis via gp120-

Fig. 4. HIV gp41-mediated apoptosis involves mi-tochondrial depolarization and ROS production.SupT1 cells cocultured with CHO WT cells in thepresence or absence of C34 or AMD were analyzedfor mitochondrial depolarization via DiOC6 staining(a) or ROS production via DHE staining (b) followedby flow cytometry.


binding to CXCR4, which cannot be inhibited by gp41-specificfusion-inhibitory peptides [48]. Our preceding data suggest therequirement of gp41 in apoptosis-mediated via cell-expressedHIV Env. To address the issue that virion-expressed Envglycoprotein-induced apoptosis is also gp41-dependent, weincubated SupT1 cells with 200 ng/ml p24 equivalents of HIVvirions in the presence of reverse transcriptase inhibitor AZT(1 �M) to prevent productive infection. This concentration ofvirus induced rapid fusion from without within 2 h (Fig. 10a)and resulted in caspase-3 activation without any significantcaspase-8 activity (Fig. 10b). We were able to inhibit virion-induced caspase-3 activation via AMD and C34 to the samelevel as mock-infected cultures. This argues against the role ofCXCR4 signaling in virion-induced apoptosis, as has beensuggested by others [48]. Although the role of other virion-associated proteins such as Vpr and Tat cannot be ruled out,we demonstrate that gp41 function is critical for HIV Env-mediated apoptosis.

DISCUSSION

HIV-mediated T cell loss leading to immunodeficiency haslong been an area of intense research. Although a number ofHIV genes have been associated with T cell loss in HIVinfection, the role of HIV Env in mediating T cell loss appearsto be predominant (reviewed by Perfettini et al. [23]). However,the mechanism via which HIV Env mediates apoptosis is notclear, especially the role of gp120 binding to CD4 and CXCR4versus gp41-mediated fusion. In this study, we have attemptedto determine whether the apoptosis-inducing potential of HIVEnv is dependent on gp120 binding to receptor/coreceptor orgp41 function. Also, our objective was to identify the apoptoticpathway involved in this phenomenon.

Our results confirm that under in vitro culture conditions,whereby HIV receptor/coreceptor- expressing target cells areexposed to Env-expressing effector cells, apoptosis of targetcells is initiated. This process can be inhibited by the gp41-

Fig. 5. HIV gp41-mediated apoptosis involvescaspase-3 but not caspase-8 activation. (a) SupT1cells were cocultured with CHO WT in the pres-ence or absence of C34 or AMD for 24 h, followingwhich caspase-3 activation was determined via an-ti-caspase-3 antibody (Ab) staining. (b) CHO WTSupT1 cocultures were assayed for caspase-3 and-8 activity using a substrate-based Caspase-Gloassay. AMD and C34 were used as inhibitors ofEnv-mediated fusion. (c) CEM or SupT1 cells wereincubated with 2 �g/ml anti-Fas antibody CH-11for 24 h, following which caspase-3 and -8 activitywas determined. Data are mean � SD of triplicateobservations. All experiments were repeated twicewith similar results.


specific fusion-inhibitory peptides, indicating that it is gp41-dependent. We also show here that GPI-anchored Env lackingfusion capabilities [24] but having an intact gp120 [21] doesnot induce apoptosis. This argues against the role of 120binding to CD4 and CXCR4 as the apoptosis-inducing signal,which is consistent with findings by others [14, 15]. Theinhibition of Env-mediated apoptosis by the anti-gp41-neutral-izing antibody 2F5 further strengthens the hypothesis that thisphenomenon is dependent on gp41. Furthermore, we show herefor the first time that virion-induced fusion from without in-duces apoptosis in CD4� T cells. Using a dye redistributionassay, we confirm findings by Blanco et al. [11], who haveshown that apoptosis via membrane-expressed HIV Env occurspredominantly in cells after a hemifusion-like event.

We have extended these findings by looking at various intra-cellular events leading to HIV Env-induced apoptosis. We ob-serve that the apoptosis in our model was mediated via a mito-chondrial pathway, as has been seen by others in Env cocultures[49] and virus infection [18]. We also find that the cells under-going apoptosis show signs of high ROS, a marker of oxidativestress, and a phenomenon reported in HIV-infected cultures [16]

Fig. 6. Env-mediated apoptosis is dependent oncaspase-mediated activation of mitochondrial depo-larization. (a) SupT1 cells were cultured with CHOWT cells in the presence or absence of C34 (2 �M)or Z-VAD-FMK (40 �M). Mitochondrial depolar-ization was determined via DiOC6 staining. (b) Pancaspase inhibitor Z-VAD-FMK (40 �M) orcaspase-3 inhibitor Ac-Asp-Glu-Val-Asp (DEVD)-CHO (40 �M) was used to inhibit apoptosis. PSexposure via Annexin V staining, mitochondrialdepolarization by DiOC6 staining, and ROS byDHE (HE) staining was determined 24 h post-coculture. (c) Caspase-3 (DEVD), caspase-9 [Ac-Leu-Glu-His-Asp (LEHD)-CHO], caspase-8 [Ac-Ile-Glu-Thr-Asp (IETD)-CHO], or pan caspase in-hibitor (Z-VAD) was added to inhibit Env-mediatedapoptosis. Mitochondrial depolarization (mito de-pol.) was determined via DiOC6 staining and nor-malized to percent inhibition of C34 control. Dataare mean � SD of triplicate observations. All ex-periments were repeated twice with similar results.

Fig. 7. HIV gp41-mediated apoptosis is independent of p38 MAPK and p53.Coculture experiments were conducted in the presence or absence of p53inhibitor cyclic pifithrin (10 �M) or p38 MAPK inhibitor SB203580 (1 �M).Apoptosis was determined 24 h later via Annexin V staining. Data areaverage � SD of triplicate observations.


or in lymphocytes from infected patients ex vivo [20]. Also, ananalysis of the caspase activation suggests a role of caspase-3 butnot caspase-8 in Env-mediated apoptosis. Interestingly that inhi-bition of mitochondrial depolarization by the pan-caspase inhib-itor Z-VAD-FMK suggests that caspase activation was upstream ofmitochondrial depolarization. The extrinsic pathway for apoptosisinitiated via Fas ligation involves caspase-8 activation upstream ofmitochondrial depolarization. This pathway is unlikely a result ofthe absence of caspase-8 activation observed in PBMC and lackof Fas-induced apoptosis in SupT1 cells [38]. This is consistentwith previous reports showing that Env-mediated apoptosis isFas-independent [17, 49]. Interestingly, caspase-3 inhibitor Ac-DEVD-CHO also inhibited mitochondrial depolarization andapoptosis similar to pan caspase inhibitor Z-VAD, and otherinhibitors were not as effective. However, partial inhibition wasseen with the caspase-9 inhibitor, suggesting that HIV Env-mediated apoptosis may involve early caspase-3 activation fol-lowed by amplification of signal via a caspase-3-mediated mito-chondrial amplification loop initiated by caspase-9. There ismounting evidence in favor of a caspase-3-mediated mitochon-drial amplification loop in a variety of stimuli including genotoxicdrugs [40] and granzyme B [50]-mediated apoptosis. In fact,caspase-3 has been shown to cleave the proapoptotic protein biddirectly, which in turn mediates mitochondrial depolarization [39].

Furthermore, a recent report shows caspase-3 to be a componentof lipid rafts and is activated early after Fas ligation in type IIcells, which poorly recruit caspase-8 to the Fas-associated deathdomain [51]. This suggests that caspase-3 may act as an initiatorand effector caspase and may be a key regulator of HIV Env-mediated apoptosis.

We also show that the apoptosis seen in our system wasdistinct from syncytial apoptosis, as p53 or p38 MAPK inhib-itors, shown to inhibit syncytial apoptosis [42], failed to inhibitapoptosis in our system. Furthermore, we observe that mito-chondrial depolarization was caspase-dependent, contrary towhat has been reported in syncytial apoptosis [37]. This sug-gests that although gp41-mediated fusion and hemifusion canresult in apoptosis, the mechanism of apoptosis under theseconditions is most likely distinct. We believe that successfulfusion events resulting in syncytia formation might result inless membrane perturbation or a suboptimal signal, and thus,the apoptosis seen in syncytia is delayed and a result ofintrinsic signaling via p53. Consistent with this idea, in amodel of contagious apoptosis, suboptimal apoptotic signal ineffector/target cells can be transmitted to full apoptosis insyncytia post-fusion via a p53-independent mechanism andmight be similar to apoptosis seen in our system [43].

Fig. 8. HIV-specific protease inhibitor nelfinavir inhibits apoptosis viainhibition of mitochondrial depolarizatiuon. (a) SupT1 cells were cocul-tured with CHO WT cells in the presence of varying concentrations ofnelfinavir. Apoptosis was determined 24 h later via Annexin V staining. (b)SupT1 cells were pretreated for 30 min with HIV protease inhibitorsnelfinavir (Nel; 10 �M), inidnavir (Ind; 10 �M), saquinavir (Saq; 10 �M),or the cysteine protease inhibtor E64D (50 �M) or the aspartate proteaseinhibitor pepstatin (50 �M) prior to coculture with CHO WT cells. Apo-ptosis was determined via Annexin V staining. (c) Nelfinavir (10 �M)-mediated inhibition of apoptosis was determined by Annexin V, DiOC6, orDHE (ROS) staining. Data are average � SD.


We demonstrate here for the first time the ability of HIV-specific protease inhibitor nelfinavir to inhibit gp41-mediatedapoptosis. Although other HIV protease inhibitors failed to inhibitapoptosis, it is conceivable that nelfinavir is distinct from other

inhibitors, as it has been shown recently to inhibit mitochondrialdepolarization by acting on the adenosine nucleotide transportersubunit of the mitochondrial permeability transition pore complex[46]. This holds clinical relevance, as the use of nelfinavir has

Fig. 9. CD4� PBMC are susceptible to gp41-dependentapoptosis. Peripheral blood-derived CD4� T cells were ac-tivated with PHA, 2 �g/ml, � IL-2, 10 U/ml, for 4 days andsubsequently cocultured with CHO WT cells. Apoptosis wasdetermined 24 h post-coculture via TUNEL (a), Annexin V(b), or caspase-3 or -8 activities via a substrate-based assay(c).

Fig. 10. Virion-induced fusion from without activates caspase-3, which is inhibited by C34. SupT1 cells were incubated with 200 ng/ml p24 equivalents of HIVvirions in the presence of 1 �M AZT to prevent productive infection and in the presence or absence of C34 or AMD. (a) Photomicrographs collected at 2 hpost-infection show fusion from without in the absence of inhibitors AMD or C34. (b) Caspase-3 and -8 activities were determined 24 h later using Caspase-Gloassay. Data are average � SD.


been shown to reduce apoptosis in infected individuals beyondvirus suppression [52]. We believe that our findings have twofoldimplications. First, that the inhibition of apoptosis via nelfinavirseen in our system is also seen in vivo suggests that the mecha-nism of HIV-induced apoptosis in vivo may be via gp41-mediatedmembrane perturbation. Second, it also suggests that nelfinavirmay be a drug of choice for inhibiting HIV replication andEnv-mediated apoptosis.

Although we find that pathogenesis of HIV is associated withthe fusogenic property of the Env glycoprotein, how gp41-mediated hemifusion events lead to apoptotic signaling is amatter of further investigation. Based on our preliminary find-ings, we hypothesize that gp41-mediated membrane perturba-tion may activate membrane-associated caspase-3, followed byactivation of proapoptotic members of the bcl-2 family such asbid and/or bax. Although Fas or caspase-8 is not involved,gp41-mediated apoptosis has similarities to extrinsic pathwayof apoptosis initiated via type II Fas signaling as a result ofseveral facts. First, gp41-mediated hemifusion most likelygenerates signals at the membrane, making it similar to extrin-sic pathway. Second, caspase-3 activation was upstream ofmitochondrial depolarization, as has been shown recently intype II Fas signaling [51]. Finally, nelfinavir, which inhibitstype II but not type I Fas-mediated apoptosis [45], also inhibitsgp41-mediated apoptosis.

Overall, we demonstrate here an involvement of gp41 ininduction of CD4� T cell apoptosis by a process that isdependent on caspase-3 activation upstream of nelfinavir-inhibitable mitochondrial depolarization. The requirement ofan amplification pathway for apoptosis via HIV gp41 suggeststhat the signal generated via the interaction of gp41 withcellular membranes may be weak and hence may induceapoptosis in vitro but may only prime cells for apoptosis invivo. Further analysis of this pathway would be of interest tocorrelate in vitro with in vivo findings. Although we believethat the loss of T cells in HIV infection is multifaceted involv-ing several phenomenon, we do wish to emphasize that thepotential of Env glycoprotein to induce apoptosis is largelydependent on gp41 function and not on gp120 binding toreceptor/coreceptor on bystander cells.

ACKNOWLEDGMENTS

This research was supported in part by the Intramural Re-search Program of the NIH, NCI, Center for Cancer Research.We are grateful to the NIH AIDS Research and ReferenceReagent program for supplying HIV peptide inhibitors, pro-tease inhibitors, and CHO and Sup-T1 cells. We thank Dr.Marius Clore for N36mut(e,g) peptide, Dr. Hermann Katinger for2F5 antibody, and Dr. Shibo Jiang for NC-1 antibody. We alsothank Jeff Lifson and Julian Bess for high-titer HIV-1 viruspreparation and the members of the Blumenthal laboratory fortheir helpful suggestions.

REFERENCES

1. Gougeon, M. L., Montagnier, L. (1993) Apoptosis in AIDS. Science 260,1269–1270.

2. Stewart, S. A., Poon, B., Jowett, J. B., Chen, I. S. (1997) Human immu-nodeficiency virus type 1 Vpr induces apoptosis following cell cycle arrest.J. Virol. 71, 5579–5592.

3. Li, C. J., Friedman, D. J., Wang, C., Metelev, V., Pardee, A. B. (1995)Induction of apoptosis in uninfected lymphocytes by HIV-1 Tat protein.Science 268, 429–431.

4. Jacotot, E., Ravagnan, L., Loeffler, M., Ferri, K. F., Vieira, H. L.,Zamzami, N., Costantini, P., Druillennec, S., Hoebeke, J., Briand, J. P.,Irinopoulou, T., Daugas, E., Susin, S. A., Cointe, D., Xie, Z. H., Reed,J. C., Roques, B. P., Kroemer, G. (2000) The HIV-1 viral protein Rinduces apoptosis via a direct effect on the mitochondrial permeabilitytransition pore. J. Exp. Med. 191, 33–46.

5. Laurent-Crawford, A. G., Coccia, E., Krust, B., Hovanessian, A. G. (1995)Membrane-expressed HIV envelope glycoprotein heterodimer is a power-ful inducer of cell death in uninfected CD4� target cells. Res. Virol. 146,5–17.

6. Gallo, S. A., Finnegan, C. M., Viard, M., Raviv, Y., Dimitrov, A., Rawat,S. S., Puri, A., Durell, S., Blumenthal, R. (2003) The HIV Env-mediatedfusion reaction. Biochim. Biophys. Acta 1614, 36–50.

7. Biard-Piechaczyk, M., Robert-Hebmann, V., Roland, J., Coudronniere, N.,Devaux, C. (1999) Role of CXCR4 in HIV-1-induced apoptosis of cellswith a CD4�, CXCR4� phenotype. Immunol. Lett. 70, 1–3.

8. Etemad-Moghadam, B., Rhone, D., Steenbeke, T., Sun, Y., Manola, J.,Gelman, R., Fanton, J. W., Racz, P., Tenner-Racz, K., Axthelm, M. K.,Letvin, N. L., Sodroski, J. (2001) Membrane-fusing capacity of the humanimmunodeficiency virus envelope proteins determines the efficiency ofCD� T-cell depletion in macaques infected by a simian-human immuno-deficiency virus. J. Virol. 75, 5646–5655.

9. LaBonte, J. A., Patel, T., Hofmann, W., Sodroski, J. (2000) Importance ofmembrane fusion mediated by human immunodeficiency virus envelopeglycoproteins for lysis of primary CD4-positive T cells. J. Virol. 74,10690–10698.

10. Etemad-Moghadam, B., Sun, Y., Nicholson, E. K., Fernandes, M., Liou, K.,Gomila, R., Lee, J., Sodroski, J. (2000) Envelope glycoprotein determinants ofincreased fusogenicity in a pathogenic simian-human immunodeficiency virus(SHIV-KB9) passaged in vivo. J. Virol. 74, 4433–4440.

11. Blanco, J., Barretina, J., Ferri, K. F., Jacotot, E., Gutierrez, A., Armand-Ugon, M., Cabrera, C., Kroemer, G., Clotet, B., Este, J. A. (2003) Cell-surface-expressed HIV-1 envelope induces the death of CD4 T cellsduring GP41-mediated hemifusion-like events. Virology 305, 318–329.

12. Blanco, J., Barretina, J., Clotet, B., Este, J. A. (2004) R5 HIV gp120-mediated cellular contacts induce the death of single CCR5-expressingCD4 T cells by a gp41-dependent mechanism. J. Leukoc. Biol. 76,804–811.

13. LaBonte, J. A., Madani, N., Sodroski, J. (2003) Cytolysis by CCR5-usinghuman immunodeficiency virus type 1 envelope glycoproteins is depen-dent on membrane fusion and can be inhibited by high levels of CD4expression. J. Virol. 77, 6645–6659.

14. Biard-Piechaczyk, M., Robert-Hebmann, V., Richard, V., Roland, J.,Hipskind, R. A., Devaux, C. (2000) Caspase-dependent apoptosis of cellsexpressing the chemokine receptor CXCR4 is induced by cell membrane-associated human immunodeficiency virus type 1 envelope glycoprotein(gp120). Virology 268, 329–344.

15. Blanco, J., Jacotot, E., Cabrera, C., Cardona, A., Clotet, B., De, C. E., Este,J. A. (1999) The implication of the chemokine receptor CXCR4 in HIV-1envelope protein-induced apoptosis is independent of the G protein-mediated signaling. AIDS 13, 909–917.

16. Banki, K., Hutter, E., Gonchoroff, N. J., Perl, A. (1998) Molecular order-ing in HIV-induced apoptosis. Oxidative stress, activation of caspases,and cell survival are regulated by transaldolase. J. Biol. Chem. 273,11944–11953.

17. Ohnimus, H., Heinkelein, M., Jassoy, C. (1997) Apoptotic cell death uponcontact of CD4� T lymphocytes with HIV glycoprotein-expressing cells ismediated by caspases but bypasses CD95 (Fas/Apo-1) and TNF receptor1. J. Immunol. 159, 5246–5252.

18. Macho, A., Castedo, M., Marchetti, P., Aguilar, J. J., Decaudin, D.,Zamzami, N., Girard, P. M., Uriel, J., Kroemer, G. (1995) Mitochondrialdysfunctions in circulating T lymphocytes from human immunodeficiencyvirus-1 carriers. Blood 86, 2481–2487.

19. Ferri, K. F., Jacotot, E., Blanco, J., Este, J. A., Kroemer, G. (2000)Mitochondrial control of cell death induced by HIV-1-encoded proteins.Ann. N. Y. Acad. Sci. 926, 149–164.

20. Dobmeyer, T. S., Findhammer, S., Dobmeyer, J. M., Klein, S. A., Raffel,B., Hoelzer, D., Helm, E. B., Kabelitz, D., Rossol, R. (1997) Ex vivoinduction of apoptosis in lymphocytes is mediated by oxidative stress: rolefor lymphocyte loss in HIV infection. Free Radic. Biol. Med. 22, 775–785.

21. Weiss, C. D., White, J. M. (1993) Characterization of stable Chinesehamster ovary cells expressing wild-type, secreted, and glycosylphosphati-


dylinositol-anchored human immunodeficiency virus type 1 envelope gly-coprotein. J. Virol. 67, 7060–7066.

22. Ahr, B., Robert-Hebmann, V., Devaux, C., Biard-Piechaczyk, M. (2004)Apoptosis of uninfected cells induced by HIV envelope glycoproteins.Retrovirology. 1, 12.

23. Perfettini, J. L., Castedo, M., Roumier, T., Andreau, K., Nardacci, R.,Piacentini, M., Kroemer, G. (2005) Mechanisms of apoptosis induction bythe HIV-1 envelope. Cell Death Differ. 12 (Suppl. 1), 916–923.

24. Weiss, C. D., Barnett, S. W., Cacalano, N., Killeen, N., Littman, D. R.,White, J. M. (1996) Studies of HIV-1 envelope glycoprotein-mediatedfusion using a simple fluorescence assay. AIDS 10, 241–246.

25. Chan, D. C., Fass, D., Berger, J. M., Kim, P. S. (1997) Core structure ofgp41 from the HIV envelope glycoprotein. Cell 89, 263–273.

26. Bewley, C. A., Louis, J. M., Ghirlando, R., Clore, G. M. (2002) Design ofa novel peptide inhibitor of HIV fusion that disrupts the internal trimericcoiled-coil of gp41. J. Biol. Chem. 277, 14238–14245.

27. Muster, T., Steindl, F., Purtscher, M., Trkola, A., Klima, A., Himmler, G.,Ruker, F., Katinger, H. (1993) A conserved neutralizing epitope on gp41of human immunodeficiency virus type 1. J. Virol. 67, 6642–6647.

28. Purtscher, M., Trkola, A., Gruber, G., Buchacher, A., Predl, R., Steindl,F., Tauer, C., Berger, R., Barrett, N., Jungbauer, A. (1994) A broadlyneutralizing human monoclonal antibody against gp41 of human immu-nodeficiency virus type 1. AIDS Res. Hum. Retroviruses 10, 1651–1658.

29. Jiang, S., Lin, K., Lu, M. (1998) A conformation-specific monoclonalantibody reacting with fusion-active gp41 from the human immunodefi-ciency virus type 1 envelope glycoprotein. J. Virol. 72, 10213–10217.

30. Barbato, G., Bianchi, E., Ingallinella, P., Hurni, W. H., Miller, M. D.,Ciliberto, G., Cortese, R., Bazzo, R., Shiver, J. W., Pessi, A. (2003)Structural analysis of the epitope of the anti-HIV antibody 2F5 sheds lightinto its mechanism of neutralization and HIV fusion. J. Mol. Biol. 330,1101–1115.

31. de Rosny, E., Vassell, R., Jiang, S., Kunert, R., Weiss, C. D. (2004)Binding of the 2F5 monoclonal antibody to native and fusion-intermediateforms of human immunodeficiency virus type 1 gp41: implications forfusion-inducing conformational changes. J. Virol. 78, 2627–2631.

32. Kemble, G. W., Danieli, T., White, J. M. (1994) Lipid-anchored influenzahemagglutinin promotes hemifusion, not complete fusion. Cell 76, 383–391.

33. Zavorotinskaya, T., Qian, Z., Franks, J., Albritton, L. M. (2004) A pointmutation in the binding subunit of a retroviral envelope protein arrestsvirus entry at hemifusion. J. Virol. 78, 473–481.

34. Melikyan, G. B., Brener, S. A., Ok, D. C., Cohen, F. S. (1997) Inner butnot outer membrane leaflets control the transition from glycosylphosphati-dylinositol-anchored influenza hemagglutinin-induced hemifusion to fullfusion. J. Cell Biol. 136, 995–1005.

35. Ferri, K. F., Jacotot, E., Geuskens, M., Kroemer, G. (2000) Apoptosis andkaryogamy in syncytia induced by the HIV-1-envelope glycoprotein com-plex. Cell Death Differ. 7, 1137–1139.

36. Romero-Alvira, D., Roche, E. (1998) The keys of oxidative stress inacquired immune deficiency syndrome apoptosis. Med. Hypotheses 51,169–173.

37. Roumier, T., Castedo, M., Perfettini, J. L., Andreau, K., Metivier, D.,Zamzami, N., Kroemer, G. (2003) Mitochondrion-dependent caspase ac-tivation by the HIV-1 envelope. Biochem. Pharmacol. 66, 1321–1329.

38. Gandhi, R. T., Chen, B. K., Straus, S. E., Dale, J. K., Lenardo, M. J.,Baltimore, D. (1998) HIV-1 directly kills CD4� T cells by a Fas-independent mechanism. J. Exp. Med. 187, 1113–1122.

39. Slee, E. A., Keogh, S. A., Martin, S. J. (2000) Cleavage of BID duringcytotoxic drug and UV radiation-induced apoptosis occurs downstream ofthe point of Bcl-2 action and is catalyzed by caspase-3: a potentialfeedback loop for amplification of apoptosis-associated mitochondrial cy-tochrome c release. Cell Death Differ. 7, 556–565.

40. Chen, Q., Gong, B., Almasan, A. (2000) Distinct stages of cytochrome crelease from mitochondria: evidence for a feedback amplification looplinking caspase activation to mitochondrial dysfunction in genotoxic stressinduced apoptosis. Cell Death Differ. 7, 227–233.

41. Garcia-Calvo, M., Peterson, E. P., Leiting, B., Ruel, R., Nicholson, D. W.,Thornberry, N. A. (1998) Inhibition of human caspases by peptide-basedand macromolecular inhibitors. J. Biol. Chem. 273, 32608–32613.

42. Perfettini, J. L., Castedo, M., Nardacci, R., Ciccosanti, F., Boya, P.,Roumier, T., Larochette, N., Piacentini, M., Kroemer, G. (2005) Essentialrole of p53 phosphorylation by p38 MAPK in apoptosis induction by theHIV-1 envelope. J. Exp. Med. 201, 279–289.

43. Andreau, K., Perfettini, J. L., Castedo, M., Metivier, D., Scott, V., Pierron,G., Kroemer, G. (2004) Contagious apoptosis facilitated by the HIV-1envelope: fusion-induced cell-to-cell transmission of a lethal signal.J. Cell Sci. 117, 5643–5653.

44. Badley, A. D. (2005) In vitro and in vivo effects of HIV protease inhibitorson apoptosis. Cell Death Differ. 12 (Suppl. 1), 924–931.

45. Phenix, B. N., Lum, J. J., Nie, Z., Sanchez-Dardon, J., Badley, A. D.(2001) Antiapoptotic mechanism of HIV protease inhibitors: preventingmitochondrial transmembrane potential loss. Blood 98, 1078–1085.

46. Weaver, J. G., Tarze, A., Moffat, T. C., Lebras, M., Deniaud, A., Brenner,C., Bren, G. D., Morin, M. Y., Phenix, B. N., Dong, L., Jiang, S. X., Sim,V. L., Zurakowski, B., Lallier, J., Hardin, H., Wettstein, P., van Heeswijk,R. P., Douen, A., Kroemer, R. T., Hou, S. T., Bennett, S. A., Lynch, D. H.,Kroemer, G., Badley, A. D. (2005) Inhibition of adenine nucleotidetranslocator pore function and protection against apoptosis in vivo by anHIV protease inhibitor. J. Clin. Invest. 115, 1828–1838.

47. Clavel, F., Charneau, P. (1994) Fusion from without directed by humanimmunodeficiency virus particles. J. Virol. 68, 1179–1185.

48. Holm, G. H., Zhang, C., Gorry, P. R., Peden, K., Schols, D., De, C. E.,Gabuzda, D. (2004) Apoptosis of bystander T cells induced by humanimmunodeficiency virus type 1 with increased envelope/receptor affinityand coreceptor binding site exposure. J. Virol. 78, 4541–4551.

49. Roggero, R., Robert-Hebmann, V., Harrington, S., Roland, J., Vergne, L.,Jaleco, S., Devaux, C., Biard-Piechaczyk, M. (2001) Binding of humanimmunodeficiency virus type 1 gp120 to CXCR4 induces mitochondrialtransmembrane depolarization and cytochrome c-mediated apoptosis in-dependently of Fas signaling. J. Virol. 75, 7637–7650.

50. Metkar, S. S., Wang, B., Ebbs, M. L., Kim, J. H., Lee, Y. J., Raja, S. M.,Froelich, C. J. (2003) Granzyme B activates procaspase-3 which signals amitochondrial amplification loop for maximal apoptosis. J. Cell Biol. 160,875–885.

51. Aouad, S. M., Cohen, L. Y., Sharif-Askari, E., Haddad, E. K., Alam, A.,Sekaly, R. P. (2004) Caspase-3 is a component of Fas death-inducingsignaling complex in lipid rafts and its activity is required for completecaspase-8 activation during Fas-mediated cell death. J. Immunol. 172,2316–2323.

52. Phenix, B. N., Angel, J. B., Mandy, F., Kravcik, S., Parato, K., Chambers,K. A., Gallicano, K., Hawley-Foss, N., Cassol, S., Cameron, D. W., Badley,A. D. (2000) Decreased HIV-associated T cell apoptosis by HIV proteaseinhibitors. AIDS Res. Hum. Retroviruses 16, 559–567.


HIV gp41-induced apoptosis is mediated by caspase-3-dependent mitochondrial depolarization, which is...

Documents

Transcript of HIV gp41-induced apoptosis is mediated by caspase-3-dependent mitochondrial depolarization, which is...