Properties and structures of the influenza and HIV fusion peptides on lipid membranes: implications for a role in fusion

Abstract

The fusion peptides of HIV and influenza virus are crucial for viral entry into a host cell. We report the membrane-perturbing and structural properties of fusion peptides from the HA fusion protein of influenza virus and the gp41 fusion protein of HIV. Our goals were to determine: 1), how fusion peptides alter structure within the bilayers of fusogenic and nonfusogenic lipid vesicles and 2), how fusion peptide structure is related to the ability to promote fusion. Fluorescent probes revealed that neither peptide had a significant effect on bilayer packing at the water-membrane interface, but both increased acyl chain order in both fusogenic and nonfusogenic vesicles. Both also reduced free volume within the bilayer as indicated by partitioning of a lipophilic fluorophore into membranes. These membrane ordering effects were smaller for the gp41 peptide than for the HA peptide at low peptide/lipid ratio, suggesting that the two peptides assume different structures on membranes. The influenza peptide was predominantly helical, and the gp41 peptide was predominantly antiparallel beta-sheet when membrane bound, however, the depths of penetration of Trps of both peptides into neutral membranes were similar and independent of membrane composition. We previously demonstrated: 1), the abilities of both peptides to promote fusion but not initial intermediate formation during PEG-mediated fusion and 2), the ability of hexadecane to compete with this effect of the fusion peptides. Taken together, our current and past results suggest a hypothesis for a common mechanism by which these two viral fusion peptides promote fusion.

FIGURE 1

Effects of fusion peptides on the surface properties of fusogenic and nonfusogenic vesicles. The fluorescence lifetime of C₆NBD-PC in the presence of peptide relative to the lifetime in the absence of peptide (Delta Lifetime) is presented as a function of gp41 (circles) and HA fusion (triangles) peptide/lipid ratios in panels A, B, and C. The mol fractions (X_mem) of C₆-NBD-PC partitioned into DOPC SUVs (nonfusogenic), DOPC/DOPE/SM/CH SUVs (fusogenic), and LUVs (nonfusogenic) are shown in panels D, E, and F. Open and solid symbols show data obtained at pH 7.5 and 5.5, respectively, and 23°C.

FIGURE 2

Effect of fusion peptides on acyl chain packing. DPH fluorescence emission anisotropy in DOPC SUVs, DOPC/DOPE/SM/CH (35:30:15:20) SUVs and DOPC/DOPE/SM/CH LUVs as a function of HA (left panels) and gp41 (right panels) peptide/lipid ratio. The experiment was done at 23°C.

FIGURE 3

Effect of fusion peptides on the interfacial region of vesicle membranes. The TMA-DPH fluorescence emission anisotropy in DOPC/DOPE/SM/CH (35:30:15:20) SUVs, LUVs, and DOPC SUVs is shown as a function of HA (▴) and gp41 (•) peptide/lipid ratio. All values represent the average of three measurements with representative error bars shown.

FIGURE 4

PATIR-FTIR amide I spectra of HA peptide (A) and gp41 peptide (B) on monolayers of POPC and DOPC/DOPE/SM/CH. Parallel-polarized spectra are shown; perpendicularly polarized spectra had similar shapes but slightly lower amplitudes.

FIGURE 5

Model of HA and gp41 peptides associated with lipid bilayers based on depth of penetration and FTIR results. The detailed arguments that led to these models are described in the Discussion. The models reflect our hypothesis that, even though the two peptides adopt somewhat different secondary structures, they interact with the bilayer with basically the same structural organization, and promote fusion by the same mechanism. This involves bilayer penetration by the hydrophobic N-terminus, thereby compensating for hydrophobic mismatch and stabilizing fusion intermediates leading to pore formation.