Treatment of human osteosarcoma cells, expressing CD4 and various chemokine receptors, with the glucosylceramide synthase inhibitor 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP), blocked target membrane glycosphingolipid (GSL) biosynthesis and reduced the susceptibility of cells to infection and fusion mediated by envelope glycoproteins from a variety of human immunodeficiency virus type 1 (HIV-1) isolates that utilize CXCR4 and/or CCR5. PPMP treatment of the cell lines did not significantly change the cell surface expression of CD4, CXCR4, and/or CCR5, nor did it alter the chemokine receptor association with CD4. PPMP-treated cells exhibited no changes in chemokine-induced Ca 2؉ mobilization and chemotaxis. However, massive envelope glycoprotein conformational changes triggered by CD4 and the appropriate chemokine receptor on the target membrane were inhibited when the target cells were treated with PPMP. Addition of various purified GSLs to PPMP-treated target cells showed that for all isolates tested, globotriaosylceramide (Gb3) was the most potent GSL in restoring the fusion susceptibility of target cells with cells expressing HIV-1 envelope glycoproteins; addition of the monosialoganglioside GM3 yielded a slight enhancement of fusion susceptibility. Our data are consistent with the notion that a limited number of specific GSL species serve as crucial elements in organizing gp120-gp41, CD4, and an appropriate chemokine receptor into a membrane fusion complex.Human immunodeficiency virus type 1 (HIV-1) infects susceptible cells by fusion of the viral membrane with the cell plasma membrane. This process is mediated by the interactions of the HIV-1 envelope glycoprotein gp120-gp41 (11,45,46) with CD4 on the host cell surface (26) and requires additional coreceptors such as CXCR4 (X4) and CCR5 (R5) (4,5,14,28), which determine the tropism of different HIV-1 isolates. Several viral envelope glycoprotein oligomers then assemble into a viral fusion machine (18, 23), forming a molecular scaffold that brings the viral and target cell membranes into close apposition and allow the actual fusion event (29). Previously reported work suggests that glycosphingolipids (GSLs) may be involved in the assembly and functioning of the HIV-1 fusion machine. This is based on the demonstration that inhibition of target cell GSL biosynthesis affects HIV-1 envelope glycoprotein-mediated cell-cell fusion (37) and that fusion activity can be recovered following addition of human erythrocyte membrane components (15, 38) or purified GSLs to the impaired cells (35). Moreover, studies using reconstituted monolayers of purified GSLs at the air-water interface provide evidence for CD4-induced interactions between HIV-1 gp120 and the GSLs globotriaosylceramide (Gb3) and the monosialoganglioside GM3 (21). These observations have led to the hypothesis that plasma membrane GSL microdomains are preferential sites for assembly of the HIV-1 fusion machine (21,36).In this study, we have examined the effect of treating a variety of cell ...

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 GSLdepleted 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(␣134)Gal(␤134)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 ␣-galactosidase A-digested fraction 3 had no effect. Our findings show that the neutral GSL, Gb3, is required for CD4͞CXCR4-dependent HIV-1 fusion.