Proc. Natl. Acad. Sci. USA Vol. 95, pp. 14435–14440, November 1998 Medical Sciences The neutral glycosphingolipid globotriaosylceramide promotes fusion mediated by a CD4-dependent CXCR4-utilizing HIV type 1 envelope glycoprotein ANU PURI*, PETER HUG*, KRISTINE JERNIGAN*, JOSEPH BARCHI†,HEE-YONG KIM‡,JILLON HAMILTON‡, i JOE¨LLE WIELS§,GARY J. MURRAY¶,ROSCOE O. BRADY¶, AND ROBERT BLUMENTHAL* *Section of Membrane Structure and Function, Laboratory of Experimental and Computational Biology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Frederick, MD 21702; †Laboratory of Medicinal Chemistry, Division of Basic Sciences, National Cancer Institute and ¶Developmental and Metabolic Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892; ‡Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD 20852; and §Centre National de la Recherche Scientifique Unite´Mixte de Recherche 1598, Institut Gustave Roussy, Villejuif Cedex 94805, France Contributed by Roscoe O. Brady, September 25, 1998 ABSTRACT Previously, we showed that the addition of enabling individual HIV strains to choose between alternate human erythrocyte glycosphingolipids (GSLs) to nonhuman modes of entry into cells. CD41 or GSL-depleted human CD41 cells rendered those cells The GSL hypothesis is based on a number of observations, susceptible to HIV-1 envelope glycoprotein-mediated cell fu- including recovery of fusion of nonsusceptible cells after sion. Individual components in the GSL mixture were isolated transfer of protease- and heat-resistant components from by fractionation on a silica-gel column and incorporated into human erythrocytes (12, 13), physicochemical studies on the the membranes of CD41 cells. GSL-supplemented target cells interactions of the gp120 V3 loop with defined GSLs (14–17), were then examined for their ability to fuse with TF228 cells and inhibition of HIV-1 infection in vitro after treatment of expressing HIV-1LAI envelope glycoprotein. We found that one cells with several inhibitors of GSL biosynthesis (18–20). GSL fraction, fraction 3, exhibited the highest recovery of Recently, we have shown that incorporation of GSLs fusion after incorporation into CD41 nonhuman and GSL- isolated from human (but not bovine) erythrocytes into the 1 depleted HeLa-CD4 cells and that fraction 3 contained a membranes of fusion-incompetent CD4 cells complements single GSL fraction. Fraction 3 was characterized by MS, subsequent HIV-1 fusion (20). In this study, we have fol- NMR spectroscopy, enzymatic analysis, and immunostaining lowed up on this observation by isolating individual compo- with an antiglobotriaosylceramide (Gb3) antibody and was nents from the human erythrocyte GSL mixture by using found to be Gal(a134)Gal(b134)Glc-Cer (Gb3). The addi- silica-gel column chromatography and testing the fractions tion of fraction 3 or Gb3 to GSL-depleted HeLa-CD4 cells for HIV-1LAI-gp120-gp41-mediated fusion activity after in- 1 recovered fusion, but the addition of galactosylceramide, corporation into CD4 nonhuman cells and GSL-depleted 2 glucosylceramide, the monosialoganglioside, GM3, lactosyl- HeLa-CD4 cells (HeLa-CD4yGSL ). We have identified a ceramide, globoside, the disialoganglioside, GD3, or a-galac- neutral GSL fraction, fraction 3 (Fr. 3), in the crude GSL tosidase A-digested fraction 3 had no effect. Our findings show mixture, which conferred the highest HIV-1 fusion activity that the neutral GSL, Gb3, is required for CD4yCXCR4- when incorporated into nonsusceptible cells. Structural anal- dependent HIV-1 fusion. ysis of this fusion-active fraction showed its chemical struc- ture to be Gal(a134)Gal(b134)Glc-Cer (Gb3). HIV-1 enters permissive cells by binding to the cellular receptor, CD4 (1), followed by gp120-gp41-mediated fusion of MATERIALS AND METHODS the viral and target cell membranes (2, 3). In addition to CD4, several members of the chemokine-receptor family have been Materials. Fluorescent probes were obtained from Molec- ular Probes, and tissue culture media were obtained from shown to act as necessary coreceptors for HIV-1 fusion and y infection (4, 5). Chemokine-receptor usage varies depending GIBCO BRL. Phospholipids were purchased from Avanti on the viral strain and the target cells used and is the primary Polar Lipids, and 1-phenyl-2-hexadecanoylamino-3-morpho- determinant of viral tropism (6–8). Although the discovery of lino-1-propanol (PPMP), GSLs, neutral GSL Qualmix stan- chemokine receptors has revolutionized our thinking about dards, and rabbit anti-asialo-GM2 antibody were from Ma- treya (Pleasant Gap, PA). Other reagents were from Sigma. the way HIV-1, HIV-2, and the simian immunodeficiency virus Mouse anti-galactosylceramide mAb was from Boehringer gain entry into the cell (9), some crucial pieces of the puzzle Mannheim. Rat anti-Gb3 mAb (38.13) was generated as are missing. Certain HIV-1 strains have been shown to infect 1 described (21). CD4 cells in the absence of known chemokine receptors (10, Fractionation of Human Erythrocyte GSLs. Total GSLs 11). Moreover, a panel of HIV-1 and HIV-2 strains, whose 1 1 were extracted from human erythrocytes as described (20). infection of CXCR4 CD4 RD rhabdomyosarcoma cells was The GSL mixture was fractionated further on a silica-gel inhibited by a mouse anti-CXCR4 monoclonal antibody column by using a CHCl yMeOH solvent mixture of increasing (12G5), resisted 12G5 inhibition on T cell lines. These obser- 3 vations suggest that CXCR4 could be processed or presented differently depending on the cell type, allowing some strains to Abbreviations: CMTMR, 5- and 6-{[(4-chloromethyl)benzoyl]- amino}tetramethylrhodamine; DiO, 3,39-dioctadecyloxacarbocyanine evade 12G5 inhibition. Therefore, it is conceivable that other perchlorate; Fr. 3, fraction 3; aGalA, a-galactosidase A; Gb2, lacto- cofactors, possibly glycosphingolipids (GSLs), are involved, sylceramide [Gal(b134)Glc-Cer]; Gb3, globotriaosylceramide [Gal(a134)Gal(b134)Glc-Cer]; Gb4, globoside [GalNAc(b133) a 3 b 3 The publication costs of this article were defrayed in part by page charge Gal( 1 4)Gal( 1 4)Glc-Cer]; Glc-Cer, glucosylceramide; GSL, glycosphingolipid; HeLa-CD4yGSL2, GSL-depleted HeLa-CD4 cells; payment. This article must therefore be hereby marked ‘‘advertisement’’ in HPTLC, high-performance thin layer chromatography, PPMP, 1-phe- accordance with 18 U.S.C. §1734 solely to indicate this fact. nyl-2-hexadecanoylamino-3-morpholino-1-propanol. i © 1998 by The National Academy of Sciences 0027-8424y98y9514435-6$2.00y0 To whom reprint requests should be addressed. e-mail: blumen@ PNAS is available online at www.pnas.org. helix.nih.gov. 14435 Downloaded by guest on October 1, 2021 14436 Medical Sciences: Puri et al. Proc. Natl. Acad. Sci. USA 95 (1998) polarity. The solvents were removed under vacuum, and the Addition of GSLs to CD41 Cells. GSLs were added to CD41 components of individual GSL fractions were detected by cells as described (20). Briefly, liposomes containing egg chromatography on silica-gel TLC. Bial’s reagent, which de- phosphatidylcholineyegg phosphatidylethanolamineyGSLs tects sugars, GSLs, sulfolipids, and gangliosides, was used to (3:1.5:1, wtywt; 0.9 mgyml total lipid) were prepared in PBS 1 1 detect the GSLs on the TLC plates. without Ca2 and Mg2 by extrusion through a 0.2-mm filter Acetylation of Fr. 3. The GSL was acetylated with acetic by using an extruder (Lipex Biomembranes, Vancouver, BC). anhydrideypyridine in a 1:2 ratio (volyvol) for 24 h at room HeLa-CD4 cells on microwells (5 3 104 per dish) were infected temperature. The reagents were evaporated under reduced with PRy8 influenza virus (AyPR8y34yH1N1) overnight at pressure. The residue was taken up in water and extracted 37°C. Target cells were treated with 5 mgyml trypsin for 5 min three times with ethyl acetate. The combined organic extracts at room temperature to convert the hemagglutinin precursor were washed with water and brine and dried by filtration to its fusogenic form. Liposomes (1 ml) were allowed to bind through a pad of sodium sulfate. The crude peracetate was to hemagglutinin-expressing cells. Liposome–cell fusion was purified on a silica-gel column with a gradient of CH3OH induced by brief exposure (60 s) of the cells to pH 5.1, followed (0–3%) CH Cl . by incubation in DMEM supplemented with 10% heat- 2 2 y Electrospray Liquid-ChromatographyyMS Analysis. A inactivated fetal bovine serum, 100 units ml penicillin, and 100 m y Hewlett–Packard 5989B mass spectrometer with an Analytica g ml streptomycin. The modified cells were then used as (Branford, CT) electrospray source was used to analyze GSL targets in the cell–cell fusion assays described below. samples with the same parameters described (22). Samples and HIV-1 Envelope Glycoprotein-Mediated Cell–Cell Fusion. known standards were injected onto a C-18 HPLC column To monitor cell–cell fusion, target cells were labeled with the (Phenomenex, Belmont, CA; 5 mm; 2.0 3 150 mm) and cytoplasmic dye 5- and 6-{[(4-chloromethyl)benzoyl]- y amino}tetramethylrhodamine (CMTMR; 10 mM, excitationy separated by using a mobile phase consisting of water MeOH y with 0.5% NaOHyhexane changing linearly from 12:88:0 to emission 541 565 nm) for 2–4 h at 37°C (13). Cells expressing y y gp120-gp41LAI (TF228 cells) were labeled either with the 0:98:2 (vol vol) in 7 min at the flow rate of 0.3 ml min, after 9 holding at the initial composition for 3 min. The final mobile- fluorescent membrane probe DiO (3,3 -dioctadecyloxacarbo- cyanine perchlorate; excitationyemission 484y501) or aqueous phase composition was maintained for 15 min. y y NMR Spectroscopy. NMR spectroscopy was performed probe calcein-AM (excitation emission 496 517 nm) as de- with a Bruker AMX spectrometer at 500 MHz (Billerica, MA). scribed (13). Fluorescently labeled TF228 cells were cocul- We collected 1H one-dimensional spectra at different temper- tured with CMTMR-labeled target cells for 2–4 h at 37°C.