Interaction of HIV-1 gp120 (IIIB and 89.6 isolates) with glycosphingolipid monolayers. (A) The SDS-gel electrophoresis analysis of purified HIV-1 gp120s (from isolates IIIB and 89.6) is shown. Five micrograms of purified proteins was electrophoresed on a 10% polyacrylamide gel in the presence of SDS. The proteins were stained with Coomassie blue. A unique diffuse band typical of a glycoprotein with an apparent molecular mass of 120 kDa was observed in both cases. (B to D) Monolayers of Gb3, GM3, or GM1 purified from human erythrocytes were prepared at an initial surface pressure of 10 mN/m. The data show the variations of surface pressure induced by the addition of various concentrations of HIV-1 gp120 from isolate IIIB (C) or 89.6 (D). The kinetics of interaction between glycosphingolipid monolayers and cholera toxin (1 μg/ml) are also shown (B).

CD4-induced interaction of HIV-1 gp120 with glycosphingolipid monolayers. Recombinant CD4 (0.5 ng/ml) was added beneath a GM3 (A and B) or Gb3 (C and D) monolayer prepared at an initial surface pressure of 10 mN/m. The insertion of CD4 into the GM3 or Gb3 monolayer induced a mean increase in surface pressure of 3 to 5 mN/m (Fig. 2). After stabilization of the monolayer, gp120 from IIIB (A and C) or 89.6 (B and D) was added at a concentration of 1.85 nM. The data show the variations of surface pressure induced by gp120. For comparison, the surface pressure increase induced by gp120 (1.85 nM) alone at an initial surface pressure of 13 mN/m is indicated.

CD4-induced interaction of HIV-1(IIIB) gp120 with a reconstituted microdomain of Gb3. At time zero, recombinant CD4 (0.5 ng/ml) was added beneath a monolayer of Gb3 prepared at an initial surface pressure of 10 mN/m. The increase in surface pressure is due to the interaction of CD4 with the Gb3 monolayer. After the surface pressure reached a plateau value, HIV-1(IIIB) gp120 was added at a concentration of 1.85 nM. Secondary gp120-Gb3 interactions are evidenced by a second phase of surface pressure increase.

Plasma membrane glycosphingolipid microdomains as preferential sites of formation of the HIV-1 fusion complex. In the plasma membrane of CD4⁺ cells, CD4 is present in glycosphingolipid-enriched microdomains but is not associated with HIV-1 coreceptors. Once bound to CD4, the viral particle is conveyed to an appropriate coreceptor by the glycosphingolipid raft, which moves freely in the external leaflet of the plasma membrane. Ch, cholesterol; GSL, glycosphingolipid; PC, phosphatidylcholine.