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. 2004 Jun 18;319(1):283-8.
doi: 10.1016/j.bbrc.2004.04.141.

Following the rule: formation of the 6-helix bundle of the fusion core from severe acute respiratory syndrome coronavirus spike protein and identification of potent peptide inhibitors

Jieqing Zhu  1 , Gengfu Xiao, Yanhui Xu, Fang Yuan, Congyi Zheng, Yueyong Liu, Huimin Yan, David K Cole, John I Bell, Zihe Rao, Po Tien, George F Gao
Affiliations

Following the rule: formation of the 6-helix bundle of the fusion core from severe acute respiratory syndrome coronavirus spike protein and identification of potent peptide inhibitors

Jieqing Zhu et al. Biochem Biophys Res Commun. .

Abstract

Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is a newly identified member of Family Coronaviridae. Coronavirus envelope spike protein S is a class I viral fusion protein which is characterized by the existence of two heptad repeat regions (HR1 and HR2) (forming a complex called fusion core). Here we report that by using in vitro bio-engineering techniques, SARS-CoV HR1 and HR2 bind to each other and form a typical 6-helix bundle. The HR2, either as a synthetic peptide or as a GST-fusion polypeptide, is a potent inhibitor of virus entry. The results do show that SARS-CoV follows the general fusion mechanism of class I viruses and this lays the ground for identification of virus fusion/entry inhibitors for this devastating emerging virus.

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Figures

Fig. 1
Fig. 1
Prediction of the HR regions of SARS-CoV S protein. Schematic diagram of S protein (amino acids 1–1255 for the full-length) is shown in the upper panel. As the basic amino acid cluster required for S1/S2 cleavage is not present it is unlikely that SARS-CoV S protein would be processed further into S1 and S2. However, the positions of the putative S1 and S2 are indicated for the convenience to compare with other coronaviruses. “SS” represents signal sequence and “TM” for transmembrane domain. In the lower panel, likelihood of HR1 and HR2 predicted by LearnCoil-VMF program is shown.
Fig. 2
Fig. 2
Characterization of the HR1–HR2 6-helix bundle. (A) The 30a-HR1 bound to GST-removed HR2. On gel-filtration, the equal-molar mixture of HR1 and HR2 forms 6-helix bundle complex. The molecular weight of the complex on gel filtration is approximately 67 kDa and the peak shows the existence of both HR1 and HR2 in similar stoichiometry (inset picture). Inset picture is a Tris–Tricine SDS–PAGE gel. Lane M, protein standard marker in kDa; lane 1, purified GST-removed HR2; lane 2, purified his-HR1; and lane 3, peak from gel-filtration, clearly showing the co-existence of the HR1 and HR2 bands from a single peak. (B) Assembled HR1/HR2 forms typical α-helix coiled coil as shown by circular dichroism (CD) spectroscopy (double minima at 208 and 222 nm). (C) Thermodynamic stability of the complex. The melting temperature is over 85 °C, typical stable coiled coil bundle.
Fig. 3
Fig. 3
Peptide fusion–inhibition curve. (A) Typical CPE of SARS-CoV (Strain WHU) formed on Vero E6 cells. (B) CPE-inhibition curves of HR2 peptides. The IC50 of the different polypeptides were, respectively: synthetic HR2-38 (amino acids 1149–1186), 0.5–5 nM; GST-HR2-38 (amino acids 1149–1186), 66.2 nM; GST-HR2-44 (amino acids 1149–1192, 6-extra amino acids from the program prediction at the C-terminus), 500 nM; and GST protein itself was used as control and no inhibitions were observed.
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