Identification of a minimal peptide derived from heptad repeat (HR) 2 of spike protein of SARS-CoV and combination of HR1-derived peptides as fusion inhibitors

Abstract

The heptad repeats (HR1 and HR2) of the spike protein of SARS-CoV are highly conserved regions forming a critical 6-helix bundle during the fusion step of virus entry and are attractive targets of entry inhibitors. In this study, we report that a minimal HR2 peptide, P6 of 23-mer, can block the fusion of SARS-CoV with an IC(50) of 1.04+/-0.22 microM. This finding supports the structural prediction of the deep groove of HR1 trimer as a target for fusion inhibitors, and suggests P6 as a potential lead peptide for future drug development. Moreover, combination of an HR-1 peptide, N46, and its mutated version, N46eg, shows synergistic inhibition with an IC(50) of 1.39+/-0.05 microM and combination index of 0.75+/-0.15, suggesting a common strategy to achieve promising inhibition by HR1 peptide for other class I envelope viruses.

Fig. 1

Schematic drawing of S protein of SARS-CoV and the designed peptides. (A) The S2 subunit contains two heptad repeats (HR1, HR2). The amino acid sequences of peptides (P1–P7) in HR2 are shown. ss, signal sequence. TM, transmembrane domain. CY, cytoplasmic tail. The numbers of amino acid residues are indicated. (B) The amino acid sequences of the HR1 peptide (N46) and its derivative (N46eg), designed based on residues at the e and g positions of the interacting HR2 peptide (C35), are shown (Xu et al., 2004). The helical wheel representation of three HR1 and three HR2 helices in the 6-helix bundle is shown.

Fig. 2

Inhibition of the S protein-mediated cell fusion by HR2 and HR1 peptides. (A) The cell fusion assay and (B) syncytia formation. β-Galactosidase activities were measured for co-cultured HeLa cells #1, cells #2 and various controls with (+) or without (−) transfection and/or infection. Syncytia formation was observed under light microscope. (C) Inhibitory effects of HR2 peptides (P1–P7) at high concentrations (25 and 9 μM). (D, E) Inhibitory effects of HR2 peptides (P1, P4 and P6), HR1 peptides (N46, N46eg) and their combination (N46 plus N46eg, molar ratio 1:1) at various concentrations. Data are means ± standard errors of duplicate wells. One representative experiment of more than three is shown.

Fig. 3

Secondary structure of HR1, HR2 and HR1/HR2 complex by CD analysis. CD spectra of (A) P1, N46 and P1/N46 complex (B) P6, N46 and P6/N46 complex and (C) P1, N46eg and P1/N46eg complex in equimolar concentrations were analyzed at 25 °C by a spectropolarimeter.