Membranotropic and biological activities of the membrane fusion peptides from SARS-CoV spike glycoprotein: The importance of the complete internal fusion peptide domain

Abstract

Fusion peptides (FP) are prominent hydrophobic segments of viral fusion proteins that play critical roles in viral entry. FPs interact with and insert into the host lipid membranes, triggering conformational changes in the viral protein that leads to the viral-cell fusion. Multiple membrane-active domains from the severe acute respiratory syndrome (SARS) coronavirus (CoV) spike protein have been reported to act as the functional fusion peptide such as the peptide sequence located between the S1/S2 and S2' cleavage sites (FP1), the S2'-adjacent fusion peptide domain (FP2), and the internal FP sequence (cIFP). Using a combined biophysical approach, we demonstrated that the α-helical coiled-coil-forming internal cIFP displayed the highest membrane fusion and permeabilizing activities along with membrane ordering effect in phosphatidylcholine (PC)/phosphatidylglycerol (PG) unilamellar vesicles compared to the other two N-proximal fusion peptide counterparts. While the FP1 sequence displayed intermediate membranotropic activities, the well-conserved FP2 peptide was substantially less effective in promoting fusion, leakage, and membrane ordering in PC/PG model membranes. Furthermore, Ca²⁺ did not enhance the FP2-induced lipid mixing activity in PC/phosphatidylserine/cholesterol lipid membranes, despite its strong erythrocyte membrane perturbation. Nonetheless, we found that the three putative SARS-CoV membrane-active fusion peptide sequences here studied altered the physical properties of model and erythrocyte membranes to different extents. The importance of the distinct membranotropic and biological activities of all SARS-CoV fusion peptide domains and the pronounced effect of the internal fusion peptide sequence to the whole spike-mediated membrane fusion process are discussed.

Keywords: COVID; Fusion peptide; Lipid-protein interaction; Membrane protein; SARS-CoV; SARS-CoV-2; Viral fusion.

Graphical abstract

Fig. 1

Schematic representation of the SARS-CoV spike glycoprotein containing the fusion peptide sequences. Top. The primary structure of the spike protein contains functionally relevant domains both in the N-terminal S1 subunit (gray), such as the receptor binding domain (RBD), and in the C-terminal S2 fusion subunit (white), such as the fusion peptide sequences (FP1, FP2, and cIFP), the heptad repeats 1 (HR1) and 2 (HR2), and the transmembrane domain (TM). The locations of the cleavage sites S1/S2 and S2’ are shown. Bottom. Sequence of the designed host-guest SARS-CoV fusion peptides used in the present study and sequence alignment with the SARS-CoV-2 fusion peptide domains (Accession numbers: AAP13441.1 for SARS-CoV-Urbani, and QHD43416.1 for SARS-CoV-2-Wuhan-Hu-1). Asterisks represent fully conserved residues, whereas colons and periods correspond to residues exhibiting strongly and weakly similar properties, respectively. Negatively (positively) charged residues are shown in red (blue). The host H7 sequence is underlined. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Membrane permeabilization induced by the SARS-CoV fusion peptides. Representative data on the kinetics of the leakage of calcein encapsulated in 50 μM of (A) POPC and (B) POPC/POPG 3/2 (mol/mol) at pH 7.4 and at different lipid-to-peptide molar ratios (L/P). (C, D) The extent of membrane permeabilization as a function of peptide-to-lipid molar ratio for POPC (panel C) and for POPC/POPG (panel D) lipid vesicles. The peptides were added to the vesicle solution at ~50 s. For each measurement, Triton X-100 (1%) was added to the sample to achieve 100% of calcein release.

Fig. 3

Membrane fusion elicited by the SARS-CoV fusion peptides. (A) Representative kinetics data of phospholipid mixing induced by addition of 2.5 mol% (L/P = 40) peptides measured by the dilution of the probes NBD-PE and Rho-PE from labeled vesicles (30 μM) into unlabeled POPC/POPG 3/2 (mol/mol) LUVs (120 μM) at pH 5.0. For each measurement, the peptides were added to the vesicle solution at the time indicated by the arrow. Triton X-100 (1%) was added to the sample to achieve 100% probe dilution. (B, C) Extent of lipid mixing as a function of peptide-to-lipid molar ratio for POPC/POPG 3/2 (mol/mol) lipid vesicles at pH 5.0 (panel B) and 7.4 (panel C).

Fig. 4

FP2H7 mediated lipid mixing is not enhanced in the presence of Ca²⁺. (A) Representative kinetics data of phospholipid mixing induced by addition of 5 mol% FP2H7 (L/P = 20) measured by the dilution of the probes NBD-PE and Rho-PE from labeled vesicles (30 μM) into unlabeled POPC/POPS/Chol 3/1/1 (molar ratio) LUVs (120 μM) at pH 5.0 in the absence and presence of Ca²⁺. Box. Extent of lipid mixing as a function of Ca²⁺ concentration. Buffer used: 10 mM MES/HEPES, 150 mM NaCl, pH 5.0. (B) Kinetics of lipid mixing induced by addition of 5 mol% FP2H7 (L/P = 20) into a mixture of labeled and unlabeled POPC/POPS/Chol 3/1/1 (molar ratio) LUVs at different NaCl concentrations. Inset. Extent of lipid mixing as a function of NaCl concentration.

Fig. 5

Peptide conformation in solution and bound to membrane mimetics. CD spectra of the peptides (A) FP1H7, (B) FP2H7, and (C) cIFPH7 in solution and incubated with 10 mM LPC or LPG micelles at pH 5.0 or 7.4. Peptide concentration was 10 μM. Spectra were recorded at 20 °C.

Fig. 6

SARS-CoV membrane peptides promote ordering on anionic lipid bilayers. Representative ESR spectra of DPPTC (A), 5-PCSL (B), and 14-PCSL (C) embedded in 5 mM DMPC/DMPG 3/2 (mol/mol) SUVs in the fluid phase (30 °C) and at neutral pH in the absence (black) and presence (red) of 5 mol% of cIFPH7. The parameters defined on the spectra were used for spectral analysis shown in the panels on the right. The spectra were normalized by the height of the central line (h₀). (D—I) Percentage of spectral change as a function of peptide-to-lipid molar ratio obtained from the analysis of the ESR spectra of DPPTC (D, G), 5-PCSL (E, H), and 14-PCSL (F, I) at pH 7.4 (D, E, F) and 5.0 (G, H, I). The plots are shown as the percentage of h₊₁/h₀ reduction of the indicated line amplitudes of both DPPTC (panel A) and 14-PCSL (panel C) spectra and the percentage of 2A_max increase of the 5-PCSL spectra (panel B) induced by the peptides. Positive values indicate membrane ordering, whereas negative values imply lipid disordering. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Biological activities of the SARS-CoV membrane fusion peptides. (A) Percentage of hemolysis as a function of peptide concentration. 100% hemolysis was achieved by adding 1% Triton X-100. (B) Representative DSC thermograms of erythrocyte ghosts in the absence and presence of 20 μM of FP1H7, FP2H7, and cIFPH7. The thermograms show four endothermic transitions corresponding to the melting of different erythrocyte ghost proteins. Besides peak IV, the other three transitions have been assigned to the denaturation of the following proteins : (I) bands 1 and 2 of the spectrin complex (~ 48 °C); (II) bands 2.1, 4.1, and 4.2 along with some glycophorins (~ 58 °C); and (III) the anion-transporting domain of the band 3 protein (~ 70 °C). (C) Hemagglutination assays with different concentrations of SARS peptides (1 to 50 μM). The red dots represent the deposition of the red blood cells onto the bottom of the wells, indicating a negative hemagglutination reaction. Absence of the red dot means positive reaction. PBS and ACN (acetonitrile) were used as negative controls. (D) Optical microscopy imaging of the SARS peptides hemagglutinating activity. Erythrocyte suspensions were incubated with FP1H7 (50 μM), FP2H7 (25 μM), and cIFPH7 (25 μM) and readily imaged. The data shown are representative of two independent experiments at the selected concentration. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)