A synthetic all D-amino acid peptide corresponding to the N-terminal sequence of HIV-1 gp41 recognizes the wild-type fusion peptide in the membrane and inhibits HIV-1 envelope glycoprotein-mediated cell fusion

Abstract

Recent studies demonstrated that a synthetic fusion peptide of HIV-1 self-associates in phospholipid membranes and inhibits HIV-1 envelope glycoprotein-mediated cell fusion, presumably by interacting with the N-terminal domain of gp41 and forming inactive heteroaggregates [Kliger, Y., Aharoni, A., Rapaport, D., Jones, P., Blumenthal, R. & Shai, Y. (1997) J. Biol. Chem. 272, 13496-13505]. Here, we show that a synthetic all D-amino acid peptide corresponding to the N-terminal sequence of HIV-1 gp41 (D-WT) of HIV-1 associates with its enantiomeric wild-type fusion (WT) peptide in the membrane and inhibits cell fusion mediated by the HIV-1 envelope glycoprotein. D-WT does not inhibit cell fusion mediated by the HIV-2 envelope glycoprotein. WT and D-WT are equally potent in inducing membrane fusion. D-WT peptide but not WT peptide is resistant to proteolytic digestion. Structural analysis showed that the CD spectra of D-WT in trifluoroethanol/water is a mirror image of that of WT, and attenuated total reflectance-fourier transform infrared spectroscopy revealed similar structures and orientation for the two enantiomers in the membrane. The results reveal that the chirality of the synthetic peptide corresponding to the HIV-1 gp41 N-terminal sequence does not play a role in liposome fusion and that the peptides' chirality is not necessarily required for peptide-peptide interaction within the membrane environment. Furthermore, studies along these lines may provide criteria to design protease-resistant therapeutic agents against HIV and other viruses.

Figure 1

Dose dependence of lipid mixing of POPG LUV induced by WT and WT-D peptides. Peptide aliquots were added to mixtures of LUV (22 μM phospholipid concentration) containing 0.6% (molar ratio) each of NBD-PE and Rho-PE and unlabeled LUV (88 μM phospholipid concentration) in PBS. The increase of the fluorescence intensity of NBD-PE was measured at 10 min after the addition of the peptide, and the percentage from maximum was plotted versus the peptide/lipid molar ratio. The fluorescence intensity on the addition of Triton X-100 (Triton Biosciences) (0.25% vol/vol) was referred to as 100%. Squares, WT; triangles, WT-D. (Inset) Time profile of the lipid mixing at peptide/lipid ratio of 0.075.

Figure 2

(A) CD spectra of WT (solid line) and WT-D (dashed line) peptides in 40% trifluoroethanol. (B) ATR-FTIR spectra of the peptides in POPG multibilayers. The samples were prepared as described in Material and Methods; peptide/lipid molar ratio was 1:80. The spectra were analyzed by using the curve fitting of the amide I band area assuming Voight line shapes for the IR peaks. Solid line, WT; dashed line, WT-D.

Figure 3

Proteolytic digestion of membrane-bound NBD-labeled peptides. The fluorescence emission spectra of the NBD-labeled peptide was monitored at 530 nm with the excitation set at 467 nm: (1) addition of 0.1 μM NBD-labeled peptides; (2) addition of 400 μM SUV; and (3) addition of Proteinase-K (50 μg/ml). In A and B, WT was used, and in C, WT-D was used.

Figure 4

Fluorescence energy transfer dependence on NBD-labeled peptide acceptor concentration. The spectra were obtained for the donor peptide alone or in the presence of various amounts of an acceptor peptide. Each spectrum was recorded in the presence of 400 μM POPG-SUV in PBS. The excitation wavelength was set at 467 nm; emission was scanned from 500 to 600 nm. Transfer efficiencies between donor- and acceptor-WT (squares), donor- and acceptor-WT-D (triangles), and donor-WT and acceptor-WT-D (circles) are plotted versus the bound-acceptor/lipid molar ratio. A theoretical plot showing energy transfer efficiency as a function of the surface density of the acceptors, assuming random distribution of donors and acceptors and assuming R₀ = 51Å, is given for comparison (dashed line).

Figure 5

Percentage fusion of TF228 cells to SupT1 cells versus peptide concentration for WT-D. The two cell types were incubated together for 2 hr before video fluorescence microscopy images were taken. Only TF228 cells in contact with SupT1 cells were counted. Empty circles, curve–fit to the lipid mixing data according to Equation 1, with ɛ = 0.81 μM and K_I = 0.4 μM. (Inset) Histograms of percentage fusion of HeLa cells expressing HIV-2 gp41 (filled columns) and HIV-1 gp41 (empty columns) versus WT-D concentration.