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.2005 Jun;79(11):7195-206.
doi: 10.1128/JVI.79.11.7195-7206.2005.

Identification and characterization of the putative fusion peptide of the severe acute respiratory syndrome-associated coronavirus spike protein

Affiliations

Identification and characterization of the putative fusion peptide of the severe acute respiratory syndrome-associated coronavirus spike protein

Bruno Sainz Jr et al. J Virol.2005 Jun.

Abstract

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is a newly identified member of the family Coronaviridae and poses a serious public health threat. Recent studies indicated that the SARS-CoV viral spike glycoprotein is a class I viral fusion protein. A fusion peptide present at the N-terminal region of class I viral fusion proteins is believed to initiate viral and cell membrane interactions and subsequent fusion. Although the SARS-CoV fusion protein heptad repeats have been well characterized, the fusion peptide has yet to be identified. Based on the conserved features of known viral fusion peptides and using Wimley and White interfacial hydrophobicity plots, we have identified two putative fusion peptides (SARS(WW-I) and SARS(WW-II)) at the N terminus of the SARS-CoV S2 subunit. Both peptides are hydrophobic and rich in alanine, glycine, and/or phenylalanine residues and contain a canonical fusion tripeptide along with a central proline residue. Only the SARS(WW-I) peptide strongly partitioned into the membranes of large unilamellar vesicles (LUV), adopting a beta-sheet structure. Likewise, only SARS(WW-I) induced the fusion of LUV and caused membrane leakage of vesicle contents at peptide/lipid ratios of 1:50 and 1:100, respectively. The activity of this synthetic peptide appeared to be dependent on its amino acid (aa) sequence, as scrambling the peptide rendered it unable to partition into LUV, assume a defined secondary structure, or induce both fusion and leakage of LUV. Based on the activity of SARS(WW-I), we propose that the hydrophobic stretch of 19 aa corresponding to residues 770 to 788 is a fusion peptide of the SARS-CoV S2 subunit.

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Figures

FIG. 1.
FIG. 1.
Interfacial hydrophobicity plots corresponding to sequences of HIV-1 gp41, EboV GP2, and SARS-CoV S2. Interfacial hydrophobicity plots (mean values for a window of 19 residues) were generated by using the WWIH scales for individual residues (94) of HIV-1 strain HXB2 gp41 (amino acids 502 to 600) (A), EboV strain Zaire GP2 (amino acids 520 to 590) (B), and the SARS-CoV strain Urbani S2 subunit (amino acids 763 to 900) (C). The residues corresponding to the known fusion peptides of the HIV (A) and EboV (B) fusion proteins are indicated by black bars and labeled FP. In addition, the two putative SARS-CoV fusion peptides (C) studied in this work are indicated by black bar and are labeled SARSWW-I and SARSWW-II.
FIG. 2.
FIG. 2.
Tryptophan fluorescence emission spectra of SARS-CoV fusion peptides. (A) SARSWW-I; (B) SARSWW-II; (C) SARSWW-I-SCR. The peptides were incubated in 5 mM HEPES buffer alone (solid lines) or after the addition of LUV (dashed lines) composed of POPC and PI (9:1) (1,000 μM). The P:L molar ratio was 1:400.
FIG. 3.
FIG. 3.
SARSWW-I partitions into membranes of LUV. The graph shows changes in the tryptophan fluorescence of SARSWW-I as a function of increasing concentrations of LUV composed of POPC (▪), POPC and PI (9:1) (•), or POPC, PI, and CHOL (6.5:1:2.5) (○). LUV were titrated at concentrations of 100, 250, 500, 750, and 1,000 μM lipid with 2.5 μM peptide. Tryptophan fluorescence values at each lipid titration (F) were normalized to tryptophan fluorescence values in 5 mM HEPES buffer alone (Fo).
FIG. 4.
FIG. 4.
SARSWW-I induces fusion of LUV. NBD fluorescence was detected by the FRET assay as a function of time. A DMSO control (a), SARSWW-I (b), SARSWW-II (c), or SARSWW-I-SCR (d) (P:L ratio of 1:10) was added to a 500 μM suspension of lipid composed of 25 μM POPC-PI-NBD-POPE-Rho-POPE LUV (8.8:1:0.1:0.1) and 475 μM POPC-PI LUV (9:1). The arrow indicates the time of addition of the peptides.
FIG. 5.
FIG. 5.
SARSWW-1 induces leakage of LUV contents. (A) Tb3+/DPA microwell assay. Each well contained 250 μl of 50 μM DPA and 500 μM Tb3+-encapsulated LUV composed of POPC and PI (9:1). The wells were treated with SARSWW-I (1), SARSWW-II (2), SARSWW-I-SCR (3) (P:L molar ratio of 1:500, 1:250, 1:100, 1:50, or 1:25), 20 μl of Triton X-100 (4), or 20 μl of DMSO (5) or were left untreated (6). The plates were incubated for 2 h at room temperature, and membrane permeabilization was determined by visual detection of Tb3+/DPA fluorescence. (B) Time trace analysis of Tb3+ fluorescence after theaddition of a DMSO control (a) or SARSWW-I at a P:L molar ratio of 1:100 (b), 1:50 (c), or 1:25 (d). The arrow indicates the time of addition of the peptides. (C) Extent of leakage from Tb3+-encapsulated LUV. SARSWW-I (•), SARSWW-II (♦), or SARSWW-I-SCR (○) was added to LUV composed of POPC and PI (9:1) at the indicated P:L molar ratios. The samples were incubated at room temperature for 2 h before Tb3+ fluorescence was measured. The percent leakage was determined by using equation 3.
FIG. 6.
FIG. 6.
SARSWW-1 adopts a β-sheet conformation. The graphs show CD spectra (mean residue ellipticities [Θ]) of the SARS-CoV fusion peptides (50 μM) SARSWW-I (A) and SARSWW-I-SCR (B) in 10 mM PO4 buffer, pH 7.0, alone (•) or with 1 mM LUV composed of POPC and PI (9:1) (○) at room temperature. Inset, CD spectra of SARSWW-I (5 μM) in 10 mM PO4 buffer, pH 7.0, alone (•) or with 1 mM LUV composed of POPC and PI (9:1) (○) at room temperature. Settings were adjusted to a 32-s response time and a scan speed of 5 nm/min.
FIG. 7.
FIG. 7.
Schematic of SARS-CoV S protein. The putative fusion peptide (red) is depicted at aa residues 770 to 788, 9 aa downstream of the minimum furin cleavage site (758R-N-T-R761). The two α-helical regions, N-helix (HR1, orange) and C-helix (HR2, yellow), are depicted at aa residues 902 to 1011 and 1131 to 1185, respectively. This is consistent with the HR predictions of Tripet et al. (84), Liu et al. (49), and Xu et al. (98). An interhelical domain of approximately 120 aa is depicted between the N- and C-helices. This region is extremely similar to the interhelical region of retrovirus TM proteins and EboV GP2 and has therefore been modeled as a similar disulfide-stabilized apex. Just prior to the transmembrane anchor (indigo) of S2, there is a region enriched in aromatic aa. This region, termed the aromatic domain (green), is highly conserved throughout theCoronaviridae and lies in an identical location to that of the aromatic domains of HIV and EboV. The S1 subunit, which includes the receptor-binding domain, is depicted schematically as a large ellipse, corresponding to the characteristic large globular head groups seen in electron micrographs of SARS-CoV.
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References

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