Fusion activity of HIV gp41 fusion domain is related to its secondary structure and depth of membrane insertion in a cholesterol-dependent fashion

Abstract

The human immunodeficiency virus (HIV) gp41 fusion domain plays a critical role in membrane fusion during viral entry. A thorough understanding of the relationship between the structure and the activity of the fusion domain in different lipid environments helps to formulate mechanistic models on how it might function in mediating membrane fusion. The secondary structure of the fusion domain in small liposomes composed of different lipid mixtures was investigated by circular dichroism spectroscopy. The fusion domain formed an α-helix in membranes containing less than 30 mol% cholesterol and formed β-sheet secondary structure in membranes containing ≥30 mol% cholesterol. EPR spectra of spin-labeled fusion domains also indicated different conformations in membranes with and without cholesterol. Power saturation EPR data were further used to determine the orientation and depth of α-helical fusion domains in lipid bilayers. Fusion and membrane perturbation activities of the gp41 fusion domain were measured by lipid mixing and contents leakage. The fusion domain fused membranes in both its helical form and its β-sheet form. High cholesterol, which induced β-sheets, promoted fusion; however, acidic lipids, which promoted relatively deep membrane insertion as an α-helix, also induced fusion. The results indicate that the structure of the HIV gp41 fusion domain is plastic and depends critically on the lipid environment. Provided that their membrane insertion is deep, α-helical and β-sheet conformations contribute to membrane fusion.

Fig. 1

Effect of cholesterol on HIV gp41 fusion domain-induced lipid mixing (A,B) and release of contents (C,D) between and from liposomes composed of POPC:POPG (4:1) (A,C) or POPC: POPS (4:1) (B,D) plus 0% (red), 10% (green), 20% (blue), and 30% (purple) cholesterol. 10% of the large unilamellar liposomes were labeled with 1% each of NBD-PE and Rhodamine-PE (A,B) or 16.7% were labeled with 15 mM ANTS and 45 mM DPX (C,D) and the rest were unlabeled. Fusion was induced by the addition of 5 µM HIV gp41 fusion domain at the first sharp rise of each curve. Triton X-100 to give a final concentration of 1% disrupting the liposomes was added at the second sharp rise of each curve.

Fig. 2

Effect of cholesterol on HIV gp41 fusion domain-induced lipid mixing (A,B) and release of contents (C,D) between and from liposomes composed POPC:POPS:PI (12:2:1) (A,C) or POPC:POPE:SM:POPS:PI (20:5:2:2:1) (B,D) without (red) and with 33% (purple) cholesterol. 10% of the large unilamellar liposomes were labeled with 1% each of NBD-PE and Rhodamine-PE (A,B) or 16.7% were labeled with 15 mM ANTS and 45 mM DPX (C,D) and the rest were unlabeled. Fusion was induced by the addition of 5 µM HIV gp41 fusion domain at the first sharp rise of each curve. Triton X-100 to give a final concentration of 1% disrupting the liposomes was added at the second sharp rise of each curve.

Fig. 3

Effect of cholesterol on secondary structure of HIV gp41 fusion domain in lipid bilayers of different lipid compositions. Far-UV circular dichroism spectra of HIV gp41 fusion domain bound to small unilamellar vesicles at a peptide:lipid ratio of 1:100 were recorded at room temperature. The lipid bilayers were composed of (A) POPC:POPG (4:1), (B) POPC:POPS (4:1), (C) POPC:POPS:PI (12:2:1), or (D) POPC:POPE:SM:POPS:PI (20:5:2:2:1) with 0% (red), 10% (green), 20% (blue), 30% (A and B, purple), or 33% (C and D, purple) cholesterol.

Fig. 4

Effect of lipids and cholesterol on EPR spectra of spin-labeled HIV gp41 fusion domain with the MTSL spin label attached at four positions that were mutated to cysteines as indicated. Fusion domains were solubilized in buffer or bound at a peptide:lipid ratio of (at most) 1:900 to 100 mM large unilamellar vesicles composed of POPC:POPG (4:1), POPC:POPG:cholesterol (6:2:2), or POPC:POPG:cholesterol (5:2:3). The gray lines in the spectra taken in the presence of cholesterol are spectra taken in POPC:POPG (4:1) for comparison.

Fig. 5

Model of the α-helical HIV gp41 fusion domain docked to a lipid bilayer based on power saturation experiments performed in POPC:POPG (4:1). (A) Docked fusion domain (PDB accession code 2PJV ) with four cysteines substituted in positions 4, 7, 12 and 15 and with nitroxide spin labels attached in all four positions. (B). Same as in (A), but with restored native HIV gp41 fusion domain sequence. The green lines represent the average positions of lipid phosphate groups relative to the fusion domains. The positions of spin label nitroxides are marked with red circles.

Fig. 6

Schematic representations of the modes of HIV gp41 fusion domain insertion into lipid bilayers containing different amounts of cholesterol based on the current work. Red, fusion domains in α-helical conformation; green, fusion domains in β-sheet conformation; gray, gp41 ectodomain.