SARS-CoV fusion peptides induce membrane surface ordering and curvature

Abstract

Viral membrane fusion is an orchestrated process triggered by membrane-anchored viral fusion glycoproteins. The S2 subunit of the spike glycoprotein from severe acute respiratory syndrome (SARS) coronavirus (CoV) contains internal domains called fusion peptides (FP) that play essential roles in virus entry. Although membrane fusion has been broadly studied, there are still major gaps in the molecular details of lipid rearrangements in the bilayer during fusion peptide-membrane interactions. Here we employed differential scanning calorimetry (DSC) and electron spin resonance (ESR) to gather information on the membrane fusion mechanism promoted by two putative SARS FPs. DSC data showed the peptides strongly perturb the structural integrity of anionic vesicles and support the hypothesis that the peptides generate opposing curvature stresses on phosphatidylethanolamine membranes. ESR showed that both FPs increase lipid packing and head group ordering as well as reduce the intramembrane water content for anionic membranes. Therefore, bending moment in the bilayer could be generated, promoting negative curvature. The significance of the ordering effect, membrane dehydration, changes in the curvature properties and the possible role of negatively charged phospholipids in helping to overcome the high kinetic barrier involved in the different stages of the SARS-CoV-mediated membrane fusion are discussed.

Figure 1. Perturbation of the thermodynamics of lipid phase transitions by the peptides.

Representative thermograms illustrating the temperature dependence of the excess molar heat capacity of DPPC, DPPG, DPPS, POPE, POPA, and POPS vesicles without (black) and with incorporation of 5 mol% (20:1 lipid/peptide molar ratio) of SARS_FP (red) and SARS_IFP (blue). Inset: Effects of the peptides on the pretransition of DPPC and DPPG and also on the higher-temperature transition found in the peptide-containing DPPS vesicles. Buffer used was 20 mM potassium phosphate, pH 7.4.

Figure 2. Curvature strain induced by SARS-CoV fusion peptides.

Representative DSC traces illustrating the effects of the peptides on the liquid crystalline (Lα) to inverted hexagonal (H_II) phase transition of DiPoPE multilamellar vesicles at the listed molar percentages and at low (A) and high (B) ionic strength. Buffer used was 20 mM sodium phosphate, pH 7.4, without or with 150 mM NaCl.

Figure 3. Changes in the ordering and mobility of different regions of DPPG bilayers in the gel and fluid phases.

Plots of the rotational diffusion rate, R_⊥ (left), and the variation of the order parameter, ΔS₀ (right), of (A) DPPTC, (B) 5-PCSL, and (C) 16-PCSL incorporated in DPPG MLVs without (white) and with 5 mol% of SARS_FP (gray) and SARS_IFP (light gray) at 25 °C, 37 °C, and 45 °C. ΔS₀ was calculated as the difference between the S₀ obtained from the peptide-containing liposome with that of the peptide-free liposome. The 16-PCSL ESR spectra at 37 °C presented two components with different ordering and dynamics.

Figure 4. Changes in the ordering and mobility of different regions of DPPS bilayers in the gel and fluid phases.

Plots of the R_⊥ (left) and ΔS₀ (right) of (A) DPPTC and (B) 16-PCSL incorporated in DPPS MLVs without (white) and with 5 mol% of SARS_FP (gray) and SARS_IFP (light gray) at 37 °C, 50 °C, and 60 °C. The 16-PCSL ESR spectra at 50 °C presented two components with different ordering and dynamics.

Figure 5. Changes in the ordering of different regions of POPA bilayers in the fluid phase.

Plots of ΔS₀ of (A) DPPTC, (B) 5-PCSL, and (C) 16-PCSL incorporated in POPA MLVs without (white) and with 5 mol% of SARS_FP (gray) and SARS_IFP (light gray) at 25 °C and 37 °C. The 16-PCSL ESR spectra at 25 °C presented two components with different ordering and dynamics.

Figure 6. Effect of membrane fusion promoter and inhibitor on the membrane surface ordering of lipid bilayers.

Variation of the order parameter S₀ of DPPTC in equimolar mixtures of DPPC/DPPG (white) and DPPC/POPA (gray) multilamellar vesicles upon incorporation of 5 mol% of SARS_FP and SARS_IFP or 10 mol% of LA and LPC relative to the pure bilayer.

Figure 7. Membrane dehydration induced by the SARS fusion peptides.

(A) Frequency-domain ESEEM spectra of DOPTC, 5-PCSL, and 16-PCSL embedded in peptide-free and peptide-bound POPC/POPG 7/3 (mol/mol) membranes. Insert: amplification of the low-frequency region corresponding to the deuterium signal. The intensity of the quadrupole interaction, Δ, defined as illustrated, is related to free D₂O content between 0.5 to 1.0 nm from the nitroxide. (B) Corresponding spectral densities at 2.09 MHz, I(²H), and quadrupole doublet intensities, Δ, of the signals shown in (A).