Sulfatide Inhibits HIV-1 Entry into CD42/CXCR41 Cells

Jacques Fantini,*,1 Djilali Hammache,* Olivier Delezay,* Gerard Pieroni,† Catherine Tamalet,† and Nouara Yahi‡

*Laboratoire de Biochimie et Biologie de la Nutrition, CNRS ESA 6033, Faculte des Sciences St. Jerome, 13397 Marseille Cedex 20, France;‡Laboratoire de Virologie, UF SIDA, CHRU de la Timone, 13005 Marseille, France; and †INSERM U130, 13009 Marseille, France

Received January 27, 1998; returned to author for revision February 26, 1998; accepted April 28, 1998

Sulfatide (39sulfogalactosylceramide) is the natural sulfated derivative of galactosylceramide (GalCer), a glycosphingolipidreceptor allowing HIV-1 infection of CD4-negative cells from neural and intestinal tissues. The incorporation of exogenoussulfatide into the plasma membrane of HT-29 (a CD42/GalCer1/CXCR41 human intestinal cell line) or RD (CD42/GalCer2/CXCR41 human rhabdomyosarcoma) resulted in a dose-dependent inhibition of HIV-1 infection. Experiments with luciferasereporter viruses pseudotyped with HIV-1 or amphotropic murine leukemia virus envelopes demonstrated that sulfatide actsat the level of viral entry. Paradoxically, the transfer of sulfatide in the plasma membrane of various CD42 cells resulted inincreased binding of HIV-1. Surface pressure measurements were conducted to study the interaction of gp120 withglycosphingolipid monolayers. The data showed that gp120 could penetrate into a monomolecular film of GalCer, confirmingthe role of this glycosphingolipid as a functional receptor for HIV-1. In contrast, the insertion of gp120 into a monolayer ofsulfatide was very limited. Moreover, the incorporation of sulfatide in a monomolecular film of GalCer specifically inhibitedthe penetration of gp120. In conclusion, these data show that sulfatide mediates gp120 binding but, in marked contrast withGalCer, is not able to initiate the fusion event. © 1998 Academic Press

Key Words: HIV-1 infection; coreceptor; CXCR4; GalCer; CD4; glycolipids.

INTRODUCTION

The interaction of HIV-1 surface envelope glycoproteingp120 with the plasma membrane of CD41 cells involvesat least two distinct binding sites: (i) CD4 and (ii) amember of the chemokine receptor family, among whichthe SDF-1 receptor CXCR4 and the RANTES receptorCCR5 have recenly been characterized as major HIV-1coreceptors. Following a primary interaction with CD4, aconformational change in gp120 renders the V3 domainof the viral glycoprotein available for secondary interac-tions with either CXCR4 or CCR5 (Alkhatib et al., 1996;Berson et al., 1996a; Deng et al., 1996; Dragic et al., 1996;Feng et al., 1996; Lapham et al., 1996; Wu et al., 1996). Inthe absence of CD4, the V3 loop is not correctly exposedto allow direct binding of gp120 to chemokine receptors.Yet HIV-1 can infect in vitro several CD42 cell types,including fibroblasts (Tateno et al., 1989) and neural(Harouse et al., 1989) and epithelial cells (Fantini et al.,1993). The relevance of these observations is under-scored by the increasing evidence that CD42 cells canbe targets for human and simian immunodeficiency vi-ruses in vivo (Basgara et al., 1996; Dean et al., 1996;Livingstone et al., 1996).

Galactosylceramide (GalCer), a glycosphingolipidabundantly expressed in neural and intestinal tissues,

can serve as an alternative receptor for HIV-1 (Berson etal., 1996b; Bhat et al., 1991; Fantini et al., 1997; Harouseet al., 1991; Long et al., 1994; Yahi et al., 1992). Thisglycolipid is the prototype of a new family of alternativeHIV-1 receptors including also seminolipid on spermato-zoa (Brogi et al., 1996) and lactosylsulfatide on vaginalepithelium (Furuta et al., 1994). Thus, studying the inter-actions between HIV-1 and glycolipids may help to im-prove the understanding of the sexual transmission ofHIV-1, as well as of the pathogenesis of this virus in thebrain (Albright et al., 1996; Kimura-Kuroda et al., 1996)and in the intestinal mucosa (Delezay et al., 1997a). Thedomain of gp120 involved in GalCer recognition hasbeen mapped to the V3 loop based on the inhibitoryactivity of anti-V3 mAbs and V3-derived synthetic pep-tides (Cook et al., 1994; Yahi et al., 1994a, 1995). Up-stream regions in the C2 domain may also be involved inthe folding of the V3 loop that is optimal for binding toGalCer (Bhat et al., 1993). In addition, infection assayswith chimeric viruses have identified the V3 loop as theprincipal determinant of HIV-1 tropism for various Gal-Cer1 cells (Harouse et al., 1995; Trujillo et al., 1996; Yahiet al., 1996). HIV-1 isolates able to infect the intestinalHT-29 cell line belong to a subclass of T-cell-line-adapted and primary HIV-1 isolates (Yahi et al., 1996) thatuse the coreceptor CXCR4 to fuse with CD41 cells, i.e.,X4 viruses according to the new classification for HIV-1(Berger et al., 1998). In a recent study, we reported thatGalCer and CXCR4 are coexpressed on the surface ofHT-29 cells and that HIV-1 entry into these cells can be

1 To whom reprint requests should be addressed. Fax: 133 491-288-440. E-mail: JACQUES.FANTINI@LBBN.u-3mrs.fr.

VIROLOGY 246, 211–220 (1998)ARTICLE NO. VY989216

0042-6822/98 $25.00Copyright © 1998 by Academic PressAll rights of reproduction in any form reserved.

211

blocked by either anti-GalCer or anti-CXCR4 mAbs(Delezay et al., 1997b). These data suggested that CXCR4can cooperate with GalCer during the fusion process.Moreover, HIV-1 entry into HT-29 cells is determined bydual recognition of GalCer and CXCR4 since isolates likeHIV-1(89.6) that can use CXCR4 (Doranz et al., 1996), butdo not bind to GalCer, do not enter the cells (Delezay etal., 1997b). This isolate also does not infect the neuralSKNMC cell line which expresses both GalCer and itssulfated derivative 39sulfo-GalCer (sulfatide) (Harouse etal., 1995). Based on the observation that gp120 recog-nized GalCer and sulfatide with equal affinity (Bhat et al.,1991), it has been assumed that sulfatide could, likeGalCer, function as an alternative receptor for HIV-1.Curiously, studies on the binding of gp120 to variouslipids gave contradictory results as to the ability of sul-fatide to recognize gp120. Using a liposome bindingassay, Long et al. (1994) could confirm the interaction ofgp120 with GalCer, but not with sulfatide. By ELISA,McAlarney et al. (1994) could, in contrast, detect bindingof gp120 to sulfatide, but not GalCer. Moreover, thetransfer of sulfatide into the plasma membrane of B-lymphocytes rendered the cells competent for gp120binding (McAlarney et al., 1994). Finally, Harouse et al.(1995) demonstrated that intact viral particles of HIV-1(IIIB) could bind to sulfatide immobilized on ELISAplates, whereas HIV-1(89.6) virions could not. These dataprovided a molecular basis for the mechanism of infec-tion of neural SKNMC cells and suggested that sulfatidecan behave as a potential HIV-1 receptor in these cells.However, the lack of gp120 binding to sulfatide in theliposome membrane remained unclear.

In this report, we present evidence that sulfatide, likeGalCer, promotes the binding of HIV-1 to the plasmamembrane of CD42 cells through an interaction with theV3 loop of gp120. However, gp120 makes a clear distinc-tion between these two glycosphingolipids, as demon-strated by surface pressure measurements of GalCerand sulfatide monolayers. These data give some insightinto the role of GalCer as a receptor for HIV-1 and explainwhy membrane-associated sulfatide behaves like a mo-lecular decoy preventing HIV-1 entry into CD42/CXCR41

cells.

RESULTS

Binding of HIV-1 gp120 to sulfatide

In a first series of experiments, increasing amounts ofsulfatide were adsorbed on ELISA plates and probedwith gp120. As shown in Fig. 1, gp120 binding was easilydetected, starting from less than 100 ng of sulfatide perwell. The specificity of the binding reaction was demon-strated by the lack of interaction with GluCer used as anegative glycosphingolipid control, whereas GalCer wasfully recognized by the viral glycoprotein. Moreover,gp120 binding to sulfatide immobilized on ELISA plates

was inhibited by suramin, a V3 loop-binding sulfonylurea(Yahi et al., 1994a), and by anti-V3 antibodies (data notshown). However, sulfatide did not bind to gp120 whenthe glycoprotein was first coated on ELISA plates. Cor-respondingly, preincubation of HIV-1 with a solution ofsulfatide up to 1 mg/ml, followed by ultracentrifugation ofthe virus, did not decrease HIV-1 infectivity. Taken to-gether, these data suggest that the V3 domain of gp120can bind to sulfatide adsorbed on a solid substratum, butnot in solution.

Transfer of sulfatide in CD42 cells results inincreased binding of HIV-1

Since exogenously added glycosphingolipids incorpo-rate spontaneously into the plasma membrane of recip-ient cells (Callies et al., 1977), these data prompted us tostudy the binding of HIV-1 to cells following sulfatidetransfer. In a previous study, we isolated clonal cellderivatives of the CD42 human colon epithelial cell lineCaco-2, which are not sensitive to HIV-1 infection andexpress neither GalCer nor CXCR4 (Delezay et al.,1997b). Incubation of Caco-2/Cl2 cells with various con-centrations of sulfatide resulted in the incorporation ofthe glycolipid in the plasma membrane. The sulfatide-treated cells were recognized by the anti-GalCer/sul-fatide R-mAb (Rantsch et al., 1982) and the binding of theantibody increased with the amount of sulfatide incorpo-rated (Fig. 2). Moreover, expression of sulfatide in theplasma membrane of these cells was sufficient to allowthe attachment of HIV-1 and the level of virus binding

FIG. 1. Binding of recombinant HIV-1 gp120 to GalCer and sulfatide.Sulfatide (h), GalCer (■), GluCer (E) or solvent alone (F) was coatedon ELISA plates as indicated under Materials and Methods. Bindingof recombinant gp120 (IIIB isolate) was revealed with a mouse anti-gp120 mAb.

212 FANTINI ET AL.

increased in proportion with the amount of sulfatide towhich the cells were exposed (Fig. 2). Taken together,these data show a good correlation between the detec-tion of sulfatide with the R-mAb and the binding of HIV-1to sulfatide-treated cells. Similar results were obtainedwith the CD42/GalCer1/CXCR41 cell line HT-29, whichshowed an increased binding of the R-mAb followingsulfatide incorporation (Fig. 3). Interestingly, sulfatide didnot affect the binding of an anti-HLA mAb, showing thatthe transfer procedure did not induce a general maskingof cell surface epitopes. Cell surface accessibility ofCXCR4 was even slightly increased in sulfatide-treatedHT-29 cells (Fig. 3). Finally, the transfer of sulfatide in theplasma membrane of HT-29 cells resulted in an in-creased binding of HIV-1 (data not shown), confirmingthe data obtained with Caco-2/Cl2 cells.

Sulfatide blocks HIV-1 entry into CD42/CXCR41 cells

Human epithelial intestinal HT-29 cells are CD42 butexpress both CXCR4 and GalCer (Delezay et al., 1997b).The cells were pretreated with various concentrations ofsulfatide and subsequently exposed to HIV-1(NDK), anisolate that infects these cells productively. The infectiv-ity of HT-29 cells was then analyzed by p24 concentrationin the culture supernatant at 7 days postinfection. Asshown in Fig. 4A, sulfatide induced a dose-dependent

inhibition of HIV-1 infection of HT-29 cells, with an IC50 of90 mg/ml. This concentration is clearly overestimated,since less than 5% of the total glycolipid in solution canbe incorporated into the plasma membrane of the targetcells under our experimental conditions (Callies et al.,1977). The specificity of the antiviral activity of sulfatidewas demonstrated by normal replication noted with theuse of other lipids: cholesterol and ganglioside GM1.Moreover, a concentration of sulfatide of 100 mg/ml wassufficient to prevent the infection of HT-29 cells by an-other isolate, i.e., HIV-1(LAI). Since this virus replicatesless efficiently in these cells, the level of infection wasanalyzed by measuring the p24 antigen in the superna-tant of human PBMC cocultivated with the HT-29 cells.The results in Fig. 4B show that PBMC efficiently rescuedthe virus from HT-29 cells exposed to HIV-1(LAI). Incontrast, the virus was not rescued from HT-29 cellstreated with sulfatide before HIV-1(LAI) exposure, indi-cating that these cells were not infected.

To test whether sulfatide blocks infection at the level ofviral entry, we used HIV-1-based luciferase reporter vi-ruses. The luciferase reporter viruses infect cells in asingle round but are not competent for further replication(Deng et al., 1996). Thus measurement of luciferase ac-tivity with pseudotypes of these viruses allows compar-ison of the relative efficiency of entry mediated by differ-ent viral envelopes. As shown in Fig. 5, HT-29 cells wereinfected by pseudotypes bearing the HXB2 Env, but notthe macrophage-tropic Env of JRFL, which does not rec-ognize the GalCer receptor (J. Fantini, unpublished data).Sulfatide inhibited infection with virus pseudotyped by

FIG. 3. Incorporation of sulfatide in HT-29 cells. HT-29 cells weretreated with the indicated concentration of sulfatide and assayed forbinding of the anti-CXCR4 mAb 12G5 (Œ), the anti-HLA mAb IOT2 (E),the anti-GalCer/sulfatide R-mAb (■), or an irrelevant mouse mAb (‚) byCELLISA. The data are expressed as the mean of 3 independentdeterminations (SD ,10%).

FIG. 2. Incorporation of sulfatide into GalCer2/CD42/CXCR42 cellspromotes HIV-1 binding. Caco-2/Cl2 cells grown in 96-well plates wereincubated with the indicated concentrations of sulfatide, rinsed, andfixed with paraformaldehyde. The presence of sulfatide was detectedby CELLISA with the R-mAb (h). The data are expressed as the meanof 10 independent experiments 6 SD. For HIV-1 binding, the cells weretreated with the indicated concentrations of sulfatide and subsequentlyincubated with HIV-1(LAI) for 2 h at 4°C. The bound virus was detectedby CELLISA using anti-gp120 and anti-gp41 mAbs as described underMaterials and Methods (■). The data are expressed as the mean of 5independent experiments 6 SD.

213SULFATIDE INHIBITS HIV-1 INFECTION

HXB2 Env, whereas the glycolipid had no effect on in-fection with virus bearing the A-MLV Env. Taken together,these data strongly suggest that sulfatide inhibition ofHT-29 infection by T-tropic HIV-1 is due to a block at thelevel of viral entry and not to postentry or nonspecificeffects.

A similar study was conducted with the human rhab-domyosarcoma cell line RD, which expresses CXCR4 butneither CD4 nor GalCer (data not shown). Exposure ofthese cells to undiluted stocks of HIV-1(NDK) (m.o.i. up to

1 TCID50 per cell) did not result in productive infection.Nevertheless, HIV-1(NDK) could infect the RD cell line, asdemonstrated by PCR amplification of HIV-1 pol se-quences (Fig. 6, lanes 1 and 2). Preincubation of thesecells with sulfatide (500 mg/ml) resulted in a total block-ade of infection (lanes 5 and 6), whereas at 100 mg/ml theglycosphingolipid inhibited HIV-1 entry in two of fourexperiments (PCR signal detected in lanes 3 and 10, butnot in lanes 4 and 9). Taken together, these data showedthat sulfatide could inhibit HIV-1 entry into RD cells, withan approximate IC50 of 100 mg/ml.

FIG. 4. Inhibition of HIV-1 infection of HT-29 cells by sulfatide. (A)HT-29 cells were treated with various concentrations of sulfatide (h),cholesterol (E), or GM1 (■) for 2 h at 37°C. After thorough washing, thecells were exposed to HIV-1(NDK) overnight at 37°C. The cells werethen trypsinated to remove excess inoculum and the state of infectionwas determined at day 7 postinfection by measuring the amount of p24in the cell-free supernatant. The results shown are representative of 4separate experiments. (B) HT-29 cells were either not treated (h) ortreated (■) with 100 mg/ml of sulfatide. The cells were then exposed toHIV-1(LAI) overnight at 37°C. The cells were trypsinated, cultured for 7days, and then cocultured with normal human PBMC as describedunder Materials and Methods. The rescued virus produced in cell-freesupernatants of cocultured PBMC was quantified by measuring theamount of p24.

FIG. 5. Sulfatide blocks infection at the level of viral entry. HT-29 cellswere either not treated (2) or treated (1) with sulfatide (1000 mg/ml)and then infected with luciferase reporter viruses pseudotyped by Envsof HIV-1 (JRFL or HXB2) or with A-MLV. Luciferase activity was mea-sured 5 days later and expressed in arbitrary units [counts per seconds(cps)]. B, background luminescence.

FIG. 6. Inhibition of HIV-1 infection of RD cells by sulfatide. RD cellswere either not preincubated (lanes 1, 2) or preincubated with sulfatideat 100 mg/ml (lanes 3, 4, 9, 10) or 500 mg/ml (lanes 5, 6). Forty-eighthours after infection, DNA was extracted and PCR-amplified as de-scribed under Materials and Methods. The arrow shows the amplifiedfragment of 805 bp. Positive controls (cultures of PBMC infected withprimary isolates of HIV-1) were analyzed in lanes 7 and 8. A negativecontrol for PCR (lane 11) and molecular weight markers (12) are alsoshown.

214 FANTINI ET AL.

Sulfatide and GalCer are distinguished by gp120

Surface pressure experiments were conducted tostudy the interaction of gp120 with glycosphingolipidmonolayers. In these experiments, the increase in sur-face pressure (DP) caused by penetration of the mono-layer by the viral glycoprotein was measured as a func-tion of initial surface pressure of the monolayer. Accord-ing to this model, lipid monolayers show decreasedcompressibility with increasing surface pressure, so thatDP is expected to decrease as the initial surface pres-sure of the monolayer increases (Lear and Rafalski,1993). Under these conditions, Fig. 7 shows that gp120interacts specifically with a monomolecular film of Gal-Cer and less efficiently with a monomolecular film ofsulfatide. Indeed, sulfatide was at best as active asGluCer, a control glycosphingolipid that is not recog-nized by gp120: for an initial surface pressure of 10mN/m, the viral glycoprotein induced a variation (DP) of8.2, 3.9, and 3.4 mN/m for GalCer, sulfatide, and GluCer,respectively.

To further study the interaction of gp120 with glyco-sphingolipids, a mixed monolayer was prepared with anequal amount of GalCer and sulfatide. As shown in Fig.7, gp120 did not significantly penetrate into this mono-layer. In contrast, the viral glycoprotein could efficientlyinteract with a monolayer containing equal amounts ofGalCer and GluCer. These data suggest that sulfatide,which is specifically recognized by gp120, impairs thefunctional interactions between the viral glycoproteinand GalCer.

DISCUSSION

The main result of the present study is the discoverythat sulfatide can promote the binding of HIV-1 gp120but, unlike GalCer, is unable to initiate the subsequentsteps leading to the fusion process. The binding of gp120to both GalCer and sulfatide can be evidenced by ELISA,which contrasts with a previous study reporting gp120binding to sulfatide alone (McAlarney et al., 1994). In ourassay, the protocol for glycosphingolipid adsorption onELISA plates is based on the solubilization of the lipidwith a mixture of chloroform, ethanol, and n-hexane,which, following addition of methanol and evaporation ofthe solvent, optimizes the orientation of glycosphingolip-ids with the hydrophobic moiety bound to the ELISA plateand the polar head accessible to the ligand (D. Hamma-che and J. Fantini, manuscript in preparation). Interest-ingly, gp120 did not recognize sulfatide in solution, whichmay suggest that several sulfatide molecules associatedby hydrogen bonds are necessary for an optimal inter-action, as previously suggested for GalCer (Long et al.,1994). Indeed, a threshold level of GalCer was requiredto allow gp120 binding to liposomes (Long et al., 1994)and, consistently, to confer sensitivity to HIV infection(Fantini et al., 1993). Thus, assuming that a lattice ofsulfatide molecules stabilized by hydrogen bonds is re-quired for gp120 binding, this supramolecular organiza-tion may develop only when the glycosphingolipid isadsorbed on a solid sustratum, which can be either aplastic support (ELISA plate) or a plasma membrane(e.g., after sulfatide transfer). This may explain why thepreincubation of HIV-1 with sulfatide in solution does notreduce viral infectivity.

One major outcome of this study is the evidence thatgp120 does make a difference between GalCer andsulfatide, as assessed by using monomolecular films ofglycosphingolipids at the air–water interface as a modelfor glycosphingolipid patches of the plasma membrane.Addition of gp120 in the aqueous phase underneath amonolayer of GalCer induced a marked increase of thesurface pressure, and the effect was gradually de-creased as the initial pressure of the monolayer in-creased. The influence of the initial surface pressure onthe compressibility of the monolayer induced by gp120demonstrates the high specificity of the interaction, aspreviously established for several other lipids and li-gands (Lear and Rafalski, 1993). From a physical point ofview, these data are interpretated as an evidence ofligand insertion into the lipid monolayer (Maggio, 1996).In the case of HIV-1 gp120, this may represent a complexnetwork of interactions between amino acid residues ofthe V3 loop and several domains of the GalCer molecule,including both the polar sugar head and the ceramidemoiety. The initial event is probably a primary interactionwith galactose, since gp120 does not bind to GluCer orceramides and does not affect the surface pressure of

FIG. 7. Interaction of gp120 with glycosphingolipid monolayers.Monomolecular films of GalCer (F), GluCer (h), sulfatide (■), or mixedfilms [GalCer/GluCer (E); GalCer/sulfatide (‚)] were prepared at vari-ous initial surface pressures. The variation of surface pressure inducedby recombinant gp120 (IIIB isolate) added in the aqueous phase wasmeasured. Results with mixed films are illustrated with a dotted line.

215SULFATIDE INHIBITS HIV-1 INFECTION

monolayers formed by these lipids. Then, secondaryinteractions with the ceramide moiety may lead to partialinsertion of gp120 inside the monomolecular film of Gal-Cer. This secondary binding step would significantlystrengthen viral adhesion and prepare the fusion event,as recently pointed out by Haywood (1994). In the case ofsulfatide, the sulfatation of galactose in position 39 maynot affect the first binding step, as evidenced by immu-noenzymatic techniques. However, according to surfacepressure measurements, gp120 does not penetrate intoa monomolecular film of sulfatide. Thus, the interactionbetween sulfatide and gp120 is completed at the firststep, suggesting that the sulfated group impairs acces-sibility to secondary binding sites in the hydrophobic partof the glycosphingolipid. Consequently, binding of gp120to sulfatide is less efficient than that to GalCer and maynot be tight enough to initiate the fusion process. Anillustration of the relative weakness of the gp120/sul-fatide association is given by Long et al. (1994), whoreported that the binding of gp120 to sulfatide in lipo-somes does not resist high-speed centrifugation. Similardata have been reported by Haywood and Boyer (1982)for the attachment of Sendai virus to liposomes bearingganglioside receptors.

The transfer of sulfatide into the plasma membrane ofCaco-2/Cl2, a CD42/GalCer2/CXCR42 human cell line,confers HIV-1 binding, but not entry (Delezay et al.,1997b). For HT-29, a CD42/GalCer1/CXCR41 cell line,the incorporation of sulfatide leads to increased HIV-1binding and decreased infection. The hypothesis thatsulfatide could inhibit HIV-1 infection though a generalantiviral activity is dismissed by the lack of effect of theglycosphingolipid on the infection of various CD41 cells,including PBMC and HT-29/CD41 cells (data not shown).Moreover, sulfatide inhibited infection of luciferase re-porter viruses pseudotyped by HXB2, but not A-MLVEnvs. These findings strongly suugest that the inhibitoryactivity of sulfatide is due to a block at the level of viralentry. Thus, one can reasonably hypothesize that sul-fatide acts as a decoy which prevents gp120 from estab-lishing functional interactions with GalCer. In this re-spect, it is worth noting that transfer of a synthetic gan-glioside analogue on the surface of receptor-negativecells resulted in binding of influenza virus, but not entry(Brossmer et al., 1993). A tentative model is proposed inFig. 8 to explain the anti-HIV-1 activity of sulfatide inCD42/CXCR41 cells. GalCer and CXCR4 are coex-pressed on the surface of most HT-29 cells, and onlyviruses able to interact with both receptors can enterthese cells (Delezay et al., 1997b). The simpliest expla-nation to account for the inhibition of HIV-1 entry intoHT-29 cells by either anti-GalCer or anti-CXCR4 mAbs isthat the virus interacts sequentially with GalCer andCXCR4 (Fig. 8, top left). Since the binding of gp120 toGalCer and CXCR4 involves the same domain, i.e., the V3loop (Cook et al., 1994; Speck et al., 1997), a given gp120

molecule cannot bind to both receptors. Thus, the attach-ment of HIV-1 to a GalCer patch on the surface of HT-29cells may involve several gp120, which may increase theprobability of a functional interaction between availablegp120 units and CXCR4 molecules. The oligomeric as-sociation of gp120 allows the same viral spike to interactwith both GalCer and CXCR4 through distinct gp120molecules. In the absence of GalCer, e.g., for RD cells,the probability of a direct interaction between gp120 andCXCR4 is very low, and only selected isolates like HIV-1(NDK) can infect these cells, yet at a high m.o.i. and witha poor efficiency (Fig. 8, bottom left). Indeed, RD cells are1000-fold less sensitive to HIV-1(NDK) infection thanHT-29 cells (data not shown). Since CXCR4 expression ishigher in RD than in HT-29 cells (not shown), one canreasonably conclude that it is GalCer which rendersHT-29 cells particularly sensitive to HIV-1 infection. Onceincorporated in the plasma membrane of HT-29 cells,sulfatide competes with GalCer for gp120 binding (Fig. 8,top right). This is demonstrated by the inability of gp120to penetrate into a mixed monolayer of GalCer and sul-fatide. At high concentrations, sulfatide may occupy mostof the available viral spikes, which therefore cannot bindto CXCR4. Alternatively, the virus attached to sulfatidecannot be delivered to CXCR4 due to the instability of thegp120–sulfatide complex. A similar mechanism may ex-plain the inhibitory activity of sulfatide incorporated in theplasma membrane of RD cells (Fig. 8, bottom right). Itshould be emphasized that the inhibitory effect of exog-enously added sulfatide on HIV-1 entry does not implythat cells expressing both GalCer and sulfatide are pro-tected from HIV-1 infection (Albright et al., 1996). Forinstance, HIV-1 can infect SKNMC cells (Harouse et al.,1995) as well as primary oligodendrocytes (Albright et al.,1996) which are GalCer1/sulfatide1. However, it can bepostulated that CD42/CXCR41 cells expressing highamounts of sulfatide and low amounts of GalCer may notbe infected. The identification (or the genetic engineer-ing) of such cells would help to validate this model.

In conclusion, the data reported here show that mem-brane-associated sulfatide can efficiently prevent infec-tion of CD42/CXCR41 cells, whether or not they expressthe GalCer receptor. These data shed some light on therespective roles of GalCer and CXCR4 in the mechanismof HIV-1 entry into intestinal epithelial HT-29 cells. Stud-ies are now in progress to determine whether othergalactolipids, such as lactosylsulfatide in vaginal cells(Furuta et al., 1994) and seminolipid in spermatozoa(Brogi et al., 1996), are involved in CD4-independentpathways of HIV-1 infection.

MATERIALS AND METHODS

Viruses

HIV-1(LAI) (Barre-Sinoussi et al., 1983) and HIV-1(NDK)(Ellrodt et al., 1984) were harvested from chronically

216 FANTINI ET AL.

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217SULFATIDE INHIBITS HIV-1 INFECTION

infected CEM cells. The macrophage-tropic HIV-1(89.6)isolate (Collman et al., 1992) was produced in peripheralblood mononuclear cells (PBMC) obtained from healthydonors.

Cell culture

The human colon epithelial cell lines HT-29 and Caco-2/Cl2 (Fantini et al., 1994) and the human rhabdomyosar-coma cell line RD (Delezay et al., 1997b) were routinelygrown in 25-cm2 flasks (Costar) in DMEM/F12 supple-mented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 mg/ml) as described previ-ously (Fantini et al., 1993).

gp120 binding to glycosphingolipids

A stock solution of the indicated lipid (1 mg/ml) wasprepared in hexane:chloroform:ethanol: (11:5:4, v:v:v) anddiluted in methanol immediately before use. The glyco-lipids (100 ml) were then coated on Greiner ELISA plates(Osi, Elancourt, France) by evaporation of the solventunder a chemical hood. The wells were saturated for 2 hat 37°C in phosphate-buffered saline (PBS) containing2% bovine serum albumin. Recombinant gp120 (10 mg/ml) was incubated 2 h at 37°C. The plates were washedwith PBS containing 0.05% Tween 20, incubated with ananti-gp120 mAb (Immunotech, Marseille, France), andrevealed with peroxidase-conjugated goat anti-mouseIgG (Sigma) at a dilution of 1:1000). Reaction productswere developed with o-phenylenediamine and the absor-bance was measured at 490 nm with a Biotek EL311ELISA reader (Osi).

ELISA on intact cells (CELLISA)

The cells grown in 96-well plates (Costar) were treatedas indicated, washed, fixed in 3.7% paraformaldehyde,and incubated with PBS containing 2% bovine serumalbumin (PBS/BSA) to reduce nonspecific binding. Theindicated primary antibody was added in PBS/BSA for1 h at room temperature. The cells were then washedand incubated with peroxidase-conjugated goat anti-mouse for 45 min. The labeling was revealed as de-scribed above. The primary antibodies used in this studywere the anti-HLA-ABC mAb IOT2 (Immunotech), theanti-CXCR4 mAb 12G5 (Endres et al., 1996), the anti-GalCer/sulfatide R-mAb (Rantsch et al., 1982), or theanti-gp120 RL 16-76 (Immunotech) as an irrelevantmouse IgG.

For HIV-1 binding, the cells were treated with theindicated amount of sulfatide in serum-free medium,washed with culture medium, and incubated with HIV-1(LAI) (100 ng of p24/ml) for 2 h at 4°C. After washing inPBS, the cells were fixed with 3.7% paraformaldehydeand incubated with PBS/BSA for 1 h at room temperature.The virus was detected with a mixture of mouse nonneu-tralizing anti-gp120 and anti-gp41 mAbs: 41-A (anti-gp41),

160-A (anti-gp41), 110-B (anti-gp120), 25 (anti-gp120) ob-tained from the Agence Nationale de Recherche sur leSIDA (ANRS), and RL 16-76 (anti-gp120), purchased fromImmunotech. The mAbs were detected as describedabove.

HIV-1 infection of CD42 cells

When indicated, the cells were treated with sulfatide(or other lipids) in serum-free medium for 2 h at 37°C(Delezay et al., 1997b). HT-29 cells were exposed tovarious isolates of HIV-1 as previously described (Fantiniet al., 1993; Yahi et al., 1994b). The infections were doneat a multiplicity of infection (m.o.i.) of 0.1 tissue cultureinfectious dose (TCID50) per cell. After 2 h of incubationwith the virus, the cells were trypsinized and subculturedat least twice before analysis of the production of theHIV-1 p24 antigen. The amount of p24 was measured inthe cell-free culture supernatant with an antigen captureELISA assay (DuPont). To detect low levels of HIV-1infection in HT-29 cells, an aliquot of the cells wascocultivated with indicator cells (normal human PBMCobtained from healthy donors), and HIV-1 p24 was mea-sured in the cell-free culture supernatant at the timeindicated after coculture. RD cells were exposed to HIV-1(NDK) at 1 m.o.i. and analyzed by PCR after 7 days ofculture. Briefly, genomic DNA was prepared by usingQiagen columns following the maufacturer’s protocol(Qiagen, France). The RT region of HIV-1 DNA was am-plified by two rounds of nested PCR with Taq DNApolymerase and supplied buffer (Boehringer-Mannheim).The first round was performed with primers MJ3 (59-AGTAGGACCTACACCTGTCAAC-39) and MJ4 (59-CTGT-TAGTGCTTTGGTTCCTCT-39) at 1 mM MgCl2 for an initial7-min denaturation at 94°C and then 35 cycles of dena-turing for 40 s at 94°C, 1 min of annealing at 55°C, anda 2-min 30-s extension at 72¡C. The second round usedprimers A35 (59-TTGGTTGCACTTTAAATTTCCCCATTAGT-CCTATT-39) and NE135 (59-CCTACTAACTTCTGTATGTCAT-TGACAGTCCAGCT-39) under the first round conditions.Amplified RT regions of 805 bp were then electropho-resed on 1% agarose gels and visualized by ethidiumbromide staining. Normal human PBMC were infectedand analyzed as previously reported (Fantini et al., 1997).

Pseudotyped virus infection assays

Luciferase reporter viruses pseudotyped by Envs ofHIV-1 macrophage-tropic JRFL or T-cell-line-adaptedHXB2 or with amphotropic murine leukemia virus (A-MLV) were generously provided by D. Littman. The HIVreporter proviruses are single round defective viruseswhich bear deletions in env, vif, and vpr and thus requirecoexpression of envelope to produce an infectious par-ticle. Target cells were plated in 24-well plates at 105

cells per well. Forty-eight hours later, the cells wereeither not treated or treated with 1000 mg/ml sulfatide for

218 FANTINI ET AL.

2 h at 37°C. After washing, the cells were infected with0.5 ml of luciferase reporter virus per well as describedby Hill et al. (1997). The culture medium was changed48 h later. At day 5 postinfection, the cells were lysed andassayed for luciferase activity using the Packard LucLiteluciferase reporter assay kit and a Packard Tri-Carb2100TR counter.

Surface pressure measurements

The surface pressure was measured with a Langmuirfilm balance (A&D Instruments, Oxford, UK) using theCollect software (Labtronics Inc., Guelph, Ontario, Can-ada). After being dissolved in a mixture of hexane:chlo-roform:ethanol 11:5:4 (v:v:v), lipids were spread inside aTeflon tank. In all experiments, the subphases were purewater obtained by filtration through a milli-Q water puri-fication system (Millipore, Saint-Quentin, France). Tomeasure the interaction of gp120 with monomolecularfilms of glycolipids, the lipids were spread inside theTeflon tank at various initial surface pressures.

ACKNOWLEDGMENTS

This work was supported by the Fondation pour la Recherche Medi-cale (SIDACTION). O.D. is recipient of a SIDACTION fellowship. D.H. isrecipient of a Region PACA fellowship. We thank F. Gonzalez-Scaranofor the design of sulfatide transfer experiments. We are very grateful toD. Littman and V. Kewalramani for the generous gift of luciferasereporter viruses and their help with the pseudotyped virus infectionassay. We also thank the M.R.C. for providing the CHO cell line pro-ducing recombinant gp120 and J. Hoxie for the 12G5 mAb.

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