The neutral glycosphingolipid globotriaosylceramide promotes fusion mediated by a CD4-dependent CXCR4-utilizing HIV type 1 envelope glycoprotein

The neutral glycosphingolipid globotriaosylceramide promotes fusion mediated by a CD4-dependent CXCR4-utilizing HIV type 1 envelope glycoprotein

^*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 Unité Mixte de Recherche 1598, Institut Gustave Roussy, Villejuif Cedex 94805, France

Contributed by Roscoe O. Brady

Abstract

Previously, we showed that the addition of human erythrocyte glycosphingolipids (GSLs) to nonhuman CD4⁺ or GSL-depleted human CD4⁺ cells rendered those cells susceptible to HIV-1 envelope glycoprotein-mediated cell fusion. Individual components in the GSL mixture were isolated by fractionation on a silica-gel column and incorporated into the membranes of CD4⁺ cells. GSL-supplemented target cells were then examined for their ability to fuse with TF228 cells expressing HIV-1_LAI envelope glycoprotein. We found that one GSL fraction, fraction 3, exhibited the highest recovery of fusion after incorporation into CD4⁺ nonhuman and GSL-depleted HeLa-CD4 cells and that fraction 3 contained a single GSL fraction. Fraction 3 was characterized by MS, NMR spectroscopy, enzymatic analysis, and immunostaining with an antiglobotriaosylceramide (Gb3) antibody and was found to be Gal(α1→4)Gal(β1→4)Glc-Cer (Gb3). The addition of fraction 3 or Gb3 to GSL-depleted HeLa-CD4 cells recovered fusion, but the addition of galactosylceramide, glucosylceramide, the monosialoganglioside, GM3, lactosylceramide, globoside, the disialoganglioside, GD3, or α-galactosidase A-digested fraction 3 had no effect. Our findings show that the neutral GSL, Gb3, is required for CD4/CXCR4-dependent HIV-1 fusion.

Footnotes

↵ ‖ To whom reprint requests should be addressed. e-mail: blumen{at}helix.nih.gov.
ABBREVIATIONS:

CMTMR,

5- and 6-{[(4-chloromethyl)benzoyl]amino}tetramethylrhodamine;

DiO,

3,3′-dioctadecyloxacarbocyanine perchlorate;

Fr. 3,

fraction 3;

αGalA,

α-galactosidase A;

Gb2,

lactosylceramide [Gal(β1→4)Glc-Cer];

Gb3,

globotriaosylceramide [Gal(α1→4)Gal(β1→4)Glc-Cer];

Gb4,

globoside [GalNAc(β1→3)Gal(α1→4)Gal(β1→4)Glc-Cer];

Glc-Cer,

glucosylceramide;

GSL,

glycosphingolipid;

HeLa-CD4/GSL−,

GSL-depleted HeLa-CD4 cells;

HPTLC,

high-performance thin layer chromatography, PPMP, 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol