Secreted phospholipases A₂, a new class of HIV inhibitors that block virus entry into host cells

Mammalian and venom secreted phospholipases A₂ (sPLA₂s) have been associated with a variety of biological effects. Here we show that several sPLA₂s protect human primary blood leukocytes from the replication of various macrophage and T cell–tropic HIV-1 strains. Inhibition by sPLA₂s results neither from a virucidal effect nor from a cytotoxic effect on host cells, but it involves a more specific mechanism. sPLA₂s have no effect on virus binding to cells nor on syncytia formation, but they prevent the intracellular release of the viral capsid protein, suggesting that sPLA₂s block viral entry into cells before virion uncoating and independently of the coreceptor usage. Various inhibitors and catalytic products of sPLA₂ have no effect on HIV-1 infection, suggesting that sPLA₂ catalytic activity is not involved in the antiviral effect. Instead, the antiviral activity appears to involve a specific interaction of sPLA₂s to host cells. Indeed, of 11 sPLA₂s from venom and mammalian tissues assayed, 4 venom sPLA₂s were found to be very potent HIV-1 inhibitors (ID₅₀ < 1 nM) and also to bind specifically to host cells with high affinities (K_0.5 < 1 nM). Although mammalian pancreatic group IB and inflammatory-type group IIA sPLA₂s were inactive against HIV-1 replication, our results could be of physiological interest, as novel sPLA₂s are being characterized in humans.

HIV-1 infection is initiated by the interaction of the virion envelope complex (gp120/gp41) with at least 2 cellular receptors: the CD4 molecule (1, 2) and a member of the chemokine receptor family (3–6). Subsequent to binding with these cellular receptors, the gp120/gp41 complex undergoes conformational changes that mediate fusion of the viral membrane with the target-cell membrane (7–9). After virus-cell fusion, virion disassembly occurs (uncoating) to release the reverse transcription (RT) complex that dissociates from the plasma membrane and moves toward the cell nucleus (10). This complex contains all the viral functions necessary for the synthesis of the proviral DNA, its transport to the cell nucleus, and its integration into the host cell DNA (11–14). The molecular basis of viral tropism has now been well characterized and resides in the ability of gp120 to interact specifically with a chemokine receptor (3–9). Macrophage-tropic (M-tropic) strains of HIV-1 replicate in macrophages and CD4⁺ T cells and use the CC chemokine receptor CCR5 (R5 viruses). T-cell–tropic (T-tropic) isolates of HIV-1 replicate in primary CD4⁺ T cells and established CD4⁺ T cells and use the CXC chemokine receptor CXCR4 (X4 viruses). Usually, R5 viruses have a non–syncytium-inducing (NSI) phenotype, whereas X4 viruses have a syncytium-inducing (SI) phenotype (10).

Several HIV-1 inhibitors have been described to block HIV entry into cells by antagonizing the interaction between gp120 and the corresponding chemokine receptor. Such inhibitors have been derived from CC or CXC chemokines (3, 5, 15, 16) or are small-molecule inhibitors that bind to the coreceptor (17, 18). In addition, recent advances in AIDS research have focused on the development of new combination therapies that have led to a dramatic and sustained reduction of viral load (19–21). Although these therapies extend the life of patients, such approaches require rigorous compliance with complicated and expensive drug regimens that cause significant side effects. These factors, coupled with the emergence of resistant viruses that escape to treatment with time, argue for the continued development of new compounds capable of protecting cells from HIV replication.

Secreted phospholipases A₂ (sPLA₂s; 14 kDa) are found in mammalian tissues and animal venoms and catalyze the hydrolysis of glycerophospholipids to release FFAs and lysophospholipids (22–27). They have been classified into different groups on the basis of the number and position of the cysteine residues present in their sequences (24, 27). These sPLA₂s have a similar overall organization and the same catalytic mechanism but display very distinct pharmacological effects (22, 23, 27). So far, 6 mammalian sPLA₂s referred to as group IB, IIA, IIC, IID, V, and X have been cloned and associated with different physiological and pathological processes (25–29). Aside from their function as enzyme, sPLA₂s have been shown to associate with specific membrane receptors that participate to their biological activities (27). To date, 2 main types of sPLA₂ receptors have been identified. N-type receptors are expressed at high levels in brain, but they are also present in other tissues (30–32). These receptors bind with high affinities different venom sPLA₂s, such as bee venom sPLA₂ (bvPLA₂) (31). The 180-kDa M-type receptor is expressed in various tissues including lung, kidney, and liver and belongs to the C-type lectin superfamily (27). The M-type receptor has been proposed to be involved in a variety of biological effects of sPLA₂s including cell migration, eicosanoid release, and septic shock (33, 34), and recent data have indicated that this receptor is the physiological target for the mammalian endogenous group IB and group IIA sPLA₂s (35).

Mammalian sPLA₂s are likely to play important roles in host defense (25, 27, 36); sPLA₂ products have been shown to interfere with viral infection (37, 38); and venom sPLA₂s display a wide and intriguing array of biological effects. We therefore analyzed whether sPLA₂s have antiviral properties against HIV-1. Our results indicate that several, but not all, assayed sPLA₂s can protect efficiently various host cells from the replication of HIV-1 isolates and are likely to define a new class of HIV-1 inhibitors.

sPLA₂s have antiviral properties against T-tropic (X4 viruses) and M-tropic (R5 viruses) HIV-1 isolates. The effect of various sPLA₂s on HIV-1_BRU infectivity was first analyzed with a single-round infection assay using CD4⁺ HeLa cells (P4 cells). As shown in Figure 1, 4 venom sPLA₂s (bvPLA₂, Nmm_CMIII, taipoxin, and nigexine) appear as very potent inhibitors of HIV-1 infection. These 4 sPLA₂s reduce HIV-1_BRU replication with ID₅₀ values lower than 1 nM and completely prevent infection above 10 nM (Figure 1). In contrast, other venom sPLA₂s have weaker (OS₁, BaIV) or no (PA2, PA5) antiviral activity (Figure 1). The inhibitory effect observed with BaIV appears interesting, as BaIV is a naturally occurring catalytically inactive sPLA₂ (41). We also analyzed the effect of human inflammatory-type group IIA sPLA₂ (hGIIA) and human and porcine pancreatic-type group IB sPLA₂s (hGIB and pGIB, respectively) (25). As shown in Figure 1, hGIIA, hGIB, and pGIB are very weak inhibitors of HIV-1_BRU replication. Finally, we analyzed whether these sPLA₂s can interfere with the antiviral activity of bvPLA₂. In these experiments, P4 cells were infected with HIV-1_BRU in the simultaneous presence of bvPLA₂ (10 nM) and either hGIB or hGIIA (up to 1 μM). In all cases, bvPLA₂ was found to retain full antiviral activity (data not shown), indicating that the inactive mammalian sPLA₂s do not interfere with the inhibitory activity of bvPLA₂.

Figure 1

Inhibitory effects of sPLA₂s on HIV-1_BRU replication. P4 cells were infected with HIV-1_BRU in the presence of various concentrations of sPLA₂s for 8 hours. Cells were then washed, and AZT (50 μM) was added to prevent a second round of viral replication. Two days after infection, the level of viral replication was determined by β-gal assay. (a) Representative dose-response curves obtained with the venom sPLA₂s taipoxin, bvPLA₂, OS₁, BaIV, and hGIIA. (b) Concentration of sPLA₂s (ID₅₀) that inhibit 50% of HIV-1_BRU replication. Values are representative of results obtained with at least 2 or 3 independent experiments with SEM less than 10%.

Taipoxin, bvPLA₂, and Nmm_CMIII were then assayed for their ability to inhibit the replication of primary HIV-1 isolates and cell line–adapted HIV-1 strains that use either CXCR4 or CCR5 as a coreceptor. The replication of these HIV-1 isolates was measured in activated PBMCs maintained in the continuous presence or absence of sPLA₂s for 6 days (Table 1). HIV-1 replication, as monitored by the production of the HIV-1 Gag p24 protein, was dramatically reduced by sPLA₂s when PBMCs were infected either with a X4 virus (HIV-1_BRU), a R5 virus (HIV-1_ADA), or primary HIV-1 isolates of SI (91US054, CMU02) and NSI phenotype (91US056, 92US657). Similar antiviral activity of sPLA₂s was also observed when P4-CCR5 cells were infected with the HIV-1 R5 viruses HIV-1_ADA or HIV-1_YU2 (data not shown). Taken together, these data indicate that several, but not all, sPLA₂s protect efficiently different cellular models from the replication of HIV-1 isolates independently of the coreceptor usage.

Table 1

sPLA₂ inhibition of HIV-1 replication in human PBMCs^A,B

sPLA₂s do not exert virucidal or cytotoxic effects and block virus entry. The lipid composition of the HIV viral membrane is similar to that of cell membranes and mainly contains glycerophospholipids, which are sPLA₂ substrates (46). A first possible mechanism for the inhibition of virus infection would then be that sPLA₂s inactivate viral particles by hydrolyzing the lipids of the viral membrane. To address this possibility, HIV-1_BRU viral particles were treated with bvPLA₂ and then used to infect P4 cells (Table 2). Before infection, viral particles were centrifuged and the viral pellet was resuspended in the presence of specific bvPLA₂ antibodies (rbvAb) added to neutralize the residual bvPLA₂ that contaminates the viral pellet. As shown in Table 2, HIV-1_BRU viral particles treated with bvPLA₂ were still able to infect P4 cells, clearly indicating that the antiviral activity of bvPLA₂ does not result from a direct action on the viral particles. Furthermore, pretreatment of target cells with bvPLA₂ followed by extensive washes and infection with HIV-1 does not lead to inhibition of HIV-1 replication (Table 2). Another possibility would be that sPLA₂ exerts a cytotoxic action on the HIV-1 host cells. However, using the XTT cytotoxic assay, we found that the growth of the various HIV-1 host cells, including PBMCs (Table 1), was not affected by sPLA₂ treatment, indicating that the antiviral activity of sPLA₂s does not result from a cytotoxic effect (data not shown).

Table 2

Pretreatment of cells or viral particles with bvPLA₂ is not sufficient to inhibit HIV-1 replication

HIV-1 infection is initiated by the binding of gp120 to the CD4 receptor. This step was not blocked by sPLA₂s, as the binding of ¹²⁵I-gp120 to CD4 was not inhibited by sPLA₂s including bvPLA₂ (data not shown). This conclusion was confirmed by time-of-addition experiments showing that sPLA₂s are still able to inhibit HIV-1 replication when added to cells after virus binding (Figure 2a). In these experiments, P4 cells were incubated with HIV-1 at 4°C to allow virus binding, but not virus entry. Unbound viruses were then washed away, and cells were shifted to 37°C to trigger the virus-cell fusion process that initiates virus entry into host cells. At different times after virus addition to cells, bvPLA₂ was added until virus entry was fully achieved (8 hours). As shown in Figure 2a, a complete inhibition of HIV-1 infection was observed when bvPLA₂ was added before or after virus binding to cells. After the temperature shift, the sensitivity to bvPLA₂ was gradually lost with an inhibitory half-time effect of about 2 hours. The inhibitory effect of bvPLA₂ on virus entry occurs before the RT step, as the half-time effect of AZT, which is known to inhibit this step, was about 4 hours (Figure 2a). The half-time effects of taipoxin and Nmm_CMIII (data not shown) were found to be similar to those of bvPLA₂, indicating that all of these sPLA₂s are acting after virus binding to cells, but before the RT step. These results were confirmed by Southern blot analysis of the proviral DNA synthesis in HIV-1–infected cells (Figure 2b). For these experiments, MT4 cells were preferred because these cells are highly susceptible to HIV-1 infection and are also sensitive to sPLA₂ action (data not shown). Early after virus addition (8 hours), no unintegrated proviral DNA was detected in cells treated with bvPLA₂, Nmm_CMIII, or taipoxin (Figure 2b), whereas it was abundantly accumulated in untreated cells and in cells treated with the mammalian sPLA₂s pGIB (Figure 2b) and hGIIA (data not shown).

Figure 2

sPLA₂s block HIV-1 viral cycle before RT. (a) Time-of-addition experiments with bvPLA₂ and AZT on the replication of HIV-1_BRU. P4 cells were incubated with virus at 4°C for 90 minutes to allow virus binding. Unbound viruses were then removed by several washes and cells were shifted to 37°C to allow virus entry and infection. bvPLA₂ (100 nM) or AZT (50 μM) were added at different times after virus addition until 8 hours, after which the cell culture medium was replaced by fresh medium containing AZT. Two days later, the level of viral replication was measured by β-gal assay. (b) sPLA₂ effect on proviral DNA synthesis. MT4 cells (10⁷ cells) were infected with high doses of infectious HIV-1_BRU supernatant in the presence of AZT or 100 nM bvPLA₂, Nmm_CMIII, taipoxin, or pGIB. Early after infection (8 hours), HIV-1 proviral DNA was extracted and analyzed by Southern blot. Each lane was loaded with 10 μg of soluble DNA, and the blot was hybridized with a specific ³²P-labeled HIV-1 riboprobe. The arrow indicates the position of the unintegrated linear proviral HIV-1 DNA (9.2 kb).

The fusion between viral and cellular membranes is usually addressed indirectly by syncytium formation assays between CD4⁺ cells and cells expressing gp120. The possible effect of sPLA₂s on the formation of syncytia was thus analyzed by cocultivation of P4 cells with 293 cells expressing HIV-1_BRU proteins, including gp120. As shown in Figure 3a, bvPLA₂ was unable to prevent the formation of syncytia, whereas both the chemokine SDF-1 (known to block virus-cell fusion by interacting with CXCR4; ref. 16) and an mAb specific for CD4 (Leu3A) led to a dramatic decrease in the formation of syncytia. These results indicate that the formation of syncytia between cells is not inhibited by sPLA₂s and suggest that the virus-cell fusion process is probably not affected by sPLA₂s.

Figure 3

Effect of sPLA₂s on HIV-1 entry into host cells. (a) Effect of bvPLA₂ on the cell-cell fusion process. P4 cells and 293 cells expressing HIV-1 proteins, including gp120/gp41, were mixed and treated with or without bvPLA₂ (10 nM), SDF-1 (250 nM), or anti-CD4 mAb (Leu3A, 1/40). Levels of cell-cell fusion were estimated after 48 hours by β-gal assay. (b) Effect of sPLA₂s and SDF-1 on the intracellular release of Gag p24, used as an index of virus entry. CEM cells (10⁷ cells) were incubated at 4°C for 45 minutes with HIV-1_BRU, washed extensively, and then incubated for 1 hour at 37°C in the presence or absence of sPLA₂s (10 nM) or SDF-1 (250 nM). After pronase treatment, cells were lysed, ultracentrifuged, and analyzed for Gag p24 content in the cytosolic fraction. Results are representative of at least 2 (a) or 4 (b) independent experiments with SEM less than 10%.

Recently, Maréchal et al. have shown that the detection of Gag p24 in the cytosol of host cells can be used as a good index to monitor productive entry of HIV-1 into host cells (44). We therefore analyzed the effect of sPLA₂s on the level of cytosolic Gag p24 to determine whether sPLA₂s are inhibiting HIV-1 entry before or after virion dissociation from the cell membrane (Figure 3b). These experiments were carried out with different cell systems (P4, CEM, and MT4 cells), and suitable conditions were obtained with CEM cells. As shown in Figure 3b, CEM cells infected with HIV-1_BRU in the presence of SDF-1, bvPLA₂, taipoxin, or Nmm_CMIII contain much lower amounts of cytosolic Gag p24 compared with untreated cells or cells treated with hGIIA. Interestingly, sPLA₂s were as efficient as SDF-1 in preventing cytosolic Gag p24 accumulation (Figure 3b), indicating that these enzymes are potent inhibitors of virus entry into host cells.

Antiviral activity of sPLA₂s is linked to sPLA₂ binding to host cells, rather than sPLA₂ catalytic activity. The role of sPLA₂ catalytic activity in the HIV-1 antiviral action of sPLA₂s was addressed by using various sPLA₂ inhibitors that are known to inhibit sPLA₂ activity in vitro (23). Phenacyl bromide (0.1 mM), aristolochic acid (0.1 mM), and oleoyloxyethylphosphocholine (10 μM), which do not show antiviral activity by themselves, were unable to prevent the HIV-1 inhibitory effects of bvPLA₂ (data not shown). The cyclooxygenase blocker indomethacin (0.1 mM), which has no effect on HIV-1 infection, had also no effect on the antiviral activity of bvPLA₂ (data not shown). The effect of mepacrine (0.1 mM), a general PLA₂ inhibitor, and nordihydroguaiaretic acid (0.1 mM), a lipoxygenase inhibitor, was not addressed because these inhibitors were toxic to P4 cells. On the other hand, several catalytic products of sPLA₂s, such as arachidonic acid, lysophosphatidylethanolamine, lysophosphatidic acid, and oleoyl- and palmitoyl-lysophosphatidylcholine (up to 10 μM), were also found to be unable to block HIV-1 replication (data not shown). We also addressed the possible role of leukotriene B4 (LTB4), a downstream metabolite of PLA₂ activity whose receptor has recently been shown to act as a coreceptor for HIV-1 entry (47). Addition of LTB4 (up to 1 μM) to P4 cells did not block HIV-1_BRU replication (data not shown), making it unlikely that sPLA₂s mediate their inhibitory effect through production of LTB4. Finally, the relatively weak but significant ability of the catalytically inactive sPLA₂ BaIV (41) to block HIV-1 replication (Figure 1) suggests that the sPLA₂ catalytic activity is not involved in HIV-1 inhibition. Together, these results indicate that the catalytic activity of sPLA₂s would not play a crucial role in the sPLA₂ antiviral effect.

Because sPLA₂s have previously been shown to bind specifically to membrane receptors (27), we analyzed the presence of sPLA₂ binding sites in the different cell systems used in this study. A specific sPLA₂ binding was observed in P4 cells (Figure 4, inset), as well as in PBMCs and CEM and MT4 cells (data not shown). Competition binding experiments (Figure 4) indicate that K_0.5 values for bvPLA₂, taipoxin, nigexine, and Nmm_CMIII, which all block HIV-1 replication efficiently (Figure 1), are 0.08, 0.15, 0.06, and 0.17 nM, respectively (Figure 4 and data not shown). Conversely, K_0.5 values for sPLA₂s that have weak or no antiviral activity (Figure 1) are in the micromolar range (OS₁, BaIV) or higher (PA2, PA5, pGIB, hGIB, and hGIIA) (Figure 4 and data not shown). Taken together, the observed binding profile shows that the sPLA₂s that efficiently inhibit HIV-1 replication also display high affinities to P4 cell membranes, whereas the sPLA₂s that have no or weak inhibitory effect have much lower affinities.

Figure 4

Binding properties of sPLA₂s to P4 cell membranes. P4 cell membranes (20 μg/mL) were incubated with iodinated sPLA₂ (25 pM) and various concentrations of unlabeled sPLA₂s. Results are expressed as percentage of the maximal specific binding measured in the absence of sPLA₂ competitor. A total of 100% corresponded to 1.8 pM of specifically bound labeled sPLA₂. Nonspecific binding was measured with 100 nM unlabeled ligand and accounted for 10% of total binding.

Mammalian and venom sPLA₂s have been implicated in a variety of physiological and pathological effects including cell proliferation, cell contraction, hormone release, inflammation, cancer, and antibacterial defense and exert various types of toxicities (22, 25, 30, 36, 48). We show here that several, but not all, sPLA₂s are potent inhibitors of HIV-1 replication. These sPLA₂s were found to protect different types of host cells efficiently from the replication of various HIV-1 isolates independently of the viral phenotype. Furthermore, bvPLA₂ was also able to inhibit the replication of HIV-2_ROD10 in P4-CCR5 cells with ID₅₀ values close to 1 nM (data not shown), indicating that sPLA₂s can protect cells from infection with both HIV-1 and HIV-2 viruses.

Our results show that HIV inhibition by sPLA₂s takes place early during the viral life cycle. HIV enters the cell by fusion at the plasma membrane, a process that is triggered by the binding of gp120 to CD4 and chemokine receptors. Our data indicate that the binding of gp120 to these cellular receptors is not inhibited by sPLA₂s, because (a) binding of gp120 to CD4 is not inhibited by sPLA₂s; (b) sPLA₂s can block the entry of viruses that have been first bound to cells; (c) both X4 and R5 viruses that use different chemokine receptors are sensitive to sPLA₂s; and (d) syncytia formation between gp120 and CD4 expressing cells is not affected by the presence of sPLA₂s. The absence of sPLA₂ effect on syncytia formation also suggests that sPLA₂s do not act by blocking the virus-cell fusion process. However, this assumption must be tempered, as cell to cell fusion experiments do not always reflect the virus-cell fusion process (49).

Shortly after fusion, the disengagement of the RT complex from the cell membrane (often referred as uncoating) leads to the cytosolic accumulation of virion components. This step is believed to be a prerequisite for the synthesis of the proviral DNA that then moves toward the cell nucleus (10, 13, 14, 50). Our data show that sPLA₂s do not act directly on viral particles (Table 2), do not act on cells before infection (Table 2), do not block CD4-gp120 interaction, and have no antiviral activity when added a few hours after virus addition (Figure 2). We thus conclude that sPLA₂s display antiviral activity only when they are present during the first hours of virus entry that correspond to the critical period of sPLA₂ action. This is in agreement with our results showing that sPLA₂s prevent the release of Gag p24 and the synthesis of proviral DNA in the cytosol of cells infected with HIV-1 (Figures 2 and 3), and suggests that sPLA₂s block virus entry by preventing virion uncoating and dissociation of the RT complex from host cell membranes. This blockade of virion disassembly is likely to explain the lack of proviral DNA synthesis in the presence of sPLA₂s, as these enzymes were unable to inhibit the RT activity (data not shown), as measured in both exogenous and endogenous in vitro assays (51). Several previous reports have shown that postfusion steps can be modulated by different cellular and viral factors. The phosphorylation of RT complex components by a virion-associated kinase facilitates the disengagement of this complex from the cell membrane and its subsequent nuclear targeting (12, 14, 52, 53). Cyclophilin A, a host protein isomerase, is required for an early step in the HIV-1 life cycle, probably to favor virion uncoating by destabilizing the capsid structure (54, 55). Nef, a regulatory viral protein found associated to the virion (56), increases the efficiency of RT (51, 57) and may increase viral nuclear import (58). It thus appears that postfusion steps are tightly regulated, making it possible that sPLA₂s act on one of these steps through direct or indirect interaction with viral or cellular factors.

The sPLA₂ catalytic activity does not appear to be involved in HIV-1 antiviral effect. First, different sPLA₂ inhibitors were unable to prevent sPLA₂ antiviral activity. Second, sPLA₂ products, such as arachidonic acid and various lysophospholipids, do not prevent HIV-1 replication. Third, several sPLA₂s like PA2, PA5, pGIB, hGIB, and hGIIA are catalytically active enzymes (25, 27), but do not have antiviral activities (Figure 1). Finally, BaIV, a catalytically inactive sPLA₂-like protein (41), was found to have antiviral properties, although with a relatively low ID₅₀ value of 400 nM (Figure 1). On the other hand, the sPLA₂ antiviral activity appears to be associated to the ability of sPLA₂s to bind to host cells. Indeed, the K_0.5 values measured for the binding of the various sPLA₂s including BaIV to P4 cells are in good agreement with their ID₅₀ values to inhibit HIV-1 infection (Figures 1 and 4). So far, 2 main types of sPLA₂ receptors have been identified (27). N-type receptors display high affinities for bvPLA₂, taipoxin, and Nmm_CMIII sPLA₂, and low affinities for OS₁ and pGIB. Conversely, M-type receptors have high affinity for OS₁ and pGIB, and do not bind bvPLA₂ and Nmm_CMIII. Based on these binding profiles, the sPLA₂ binding sites detected in P4 cells appear most similar to N-type receptors. Furthermore, no M-type receptors were detected in P4 cells (data not shown). The molecular nature of the sPLA₂ binding sites found in P4 cells remains to be determined, but are likely to be related to the N-type like receptors that have been identified in different tissues and cells including immune cells (27, 31, 32).

In conclusion, our data indicate that several venom sPLA₂s can protect various types of host cells including PBMCs from the replication of primary HIV-1 isolates. Conversely, human group IB and human group IIA sPLA₂s were unable to block HIV-1 replication. However, because several other sPLA₂s are being identified in mammals (26, 28, 29), it is tempting to speculate that some of these novel human sPLA₂s may have clinical relevance in HIV infection. In individuals repeatedly exposed to HIV but who remain uninfected, several possible reasons for protection have been proposed but not clearly elucidated (59). In this respect, the putative antiviral activity of the novel group IID, V, and X sPLA₂s, which are expressed in immune tissues and cells such as macrophages and PBMCs (28, 29), will be particularly interesting to analyze in the future, when these enzymes will be available in high enough amounts to assay them, as we have assayed venom sPLA₂s.

We thank P. Rochet V. Sansoldi, and N. Gomez for expert technical assistance. The authors are grateful to P. Charneau, F. Clavel, K. Peden, N. Israel, and the NIH-AIDS Research and Reference Reagent Program for the generous gifts of the various plasmids and cell lines. We are also indebted to A. Ménez, J. Christophe, H. Verheij, J.M. Gutiérrez, and S. Lizano for providing the various venom sPLA₂. This work was supported by the Centre National de la Recherche Scientifique (CNRS), the BV Foundation Limited, the Association pour la Recherche sur le Cancer (ARC), and the Ministère de la Défense Nationale (Grant DGA-DRET 96/096). E. Valentin is a recipient of a grant from the region Provence Alpes Côte d’azur–CNRS program.

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