PERMEABILIZATION AND FUSION OF UNCHARGED LIPID VESICLES INDUCED BY THE HIV-1 FUSION PEPTIDE ADOPTING AN EXTENDED CONFORMATION - DOSE AND SEQUENCE EFFECTS

The peptide HIVarg, corresponding to a sequence of 23 amino acid resid ues at the N-terminus of HIV-1 gp41 (LAV(1a) strain), has the capacity to destabilize negatively charged large unilamellar vesicles, As reve aled by infrared spectroscopy, the peptide associated with those vesic les showed conformational polymorphism: in the absence of cations the main structure was a pore-forming alpha-helix, whereas in the presence of Ca2+ the conformation switched to a fusogenic, predominantly exten ded beta-type structure, Here we show that an extended structure can a lso be involved in electrically neutral vesicle destabilization induce d by the HIV-I fusion peptide when it binds the vesicle from the aqueo us phase. In the absence of cations, neutral liposomes composed of pho sphatidylcholine, phosphatidylethanolamine, and cholesterol (molar rat io 1:1:1) selected for an extended structure that became fusogenic in a dose-dependent fashion. At subfusogenic doses this structure caused the release of trapped 8-aminonaphtalene-1,3,6-trisulfonic acid sodium salt/p-xylenebis(pyridinium)bromide from liposomes, indicating the ex istence of a peptide-mediated membrane destabilizing process before an d independent of the development of fusion. When compared to HIVarg, t he fusion activity of HIVala (bearing the R22 --> A substitution) was reduced by 70%. Fusogenicity was completely abolished when a second su bstitution (V2 --> E) was included to generate HIVala-E2, a sequence r epresenting the N-terminus of an inactive gp41. However, the three seq uences associated with vesicles to the same extent, and the three adop ted a similar extended structure in the membrane. Whereas 1-(4-trimeth ylaminophenyl)-6-phenyl-1 ,3,5-hexatriene emission anisotropy was unaf fected by the three peptides, DPH emission anisotropy in membranes was increased only by the fusogenic sequences. Taken together, our observ ations strongly argue that it is not an alpha-helical but an extended structure adopted by the HIV-I fusion peptide what actively destabiliz es cholesterol-containing, electrically neutral membranes. Moreover, m embrane destabilization is modulated by the amino acid sequence in the extended structure. The effect displayed by the aforementioned V2 --> E substitution suggests that the fusion process described here could be reflecting a physiologically relevant phenomenon.