Fusion core structure of the severe acute respiratory syndrome coronavirus (SARS-CoV): in search of potent SARS-CoV entry inhibitors

Abstract

Severe acute respiratory coronavirus (SARS-CoV) spike (S) glycoprotein fusion core consists of a six-helix bundle with the three C-terminal heptad repeat (HR2) helices packed against a central coiled-coil of the other three N-terminal heptad repeat (HR1) helices. Each of the three peripheral HR2 helices shows prominent contacts with the hydrophobic surface of the central HR1 coiled-coil. The concerted protein-protein interactions among the HR helices are responsible for the fusion event that leads to the release of the SARS-CoV nucleocapsid into the target host-cell. In this investigation, we applied recombinant protein and synthetic peptide-based biophysical assays to characterize the biological activities of the HR helices. In a parallel experiment, we employed a HIV-luc/SARS pseudotyped virus entry inhibition assay to screen for potent inhibitory activities on HR peptides derived from the SARS-CoV S protein HR regions and a series of other small-molecule drugs. Three HR peptides and five small-molecule drugs were identified as potential inhibitors. ADS-J1, which has been used to interfere with the fusogenesis of HIV-1 onto CD4+ cells, demonstrated the highest HIV-luc/SARS pseudotyped virus-entry inhibition activity among the other small-molecule drugs. Molecular modeling analysis suggested that ADS-J1 may bind to the deep pocket of the hydrophobic groove on the surface of the central coiled-coil of SARS-CoV S HR protein and prevent the entrance of the SARS-CoV into the host cells.

Figure 1

Amino acid sequences of recombinant proteins and synthetic peptides used in this study. His₆‐HR1 (residues 899 iV 958) and GST‐removed HR2 (1,145 iV 1,192) are recombinant proteins, whereas HR1‐a (residues 899 iV 926), HR1‐b (residues 916 iV 950) and HR2 (residues 1,151 iV 1,185) are synthetic peptides. The conserved hydrophobic amino residues, a and d positions of the predicted heptad repeat patterns are shown above the amino acid sequences.

Figure 2

HR1‐b interacts with HR2 in vitro. HR1‐a or HR1‐b was incubated with HR2 at the indicated concentration before subjected to Native‐PAGE analysis. HR1‐a and HR1‐b are negatively charged in the running pH and do not migrate into the gel on their own (lanes 2–5). A high mobility species was found when HR2 was incubated with HR1‐b (lanes 6 and 7) but not with HR1‐a (lanes 8 and 9).

Figure 3

Folding and thermostability of the peptides and recombinant proteins. A: The CD spectra of HR1‐b (blue), HR2 (magenta), and HR1‐b/HR2 complex (yellow). B: Thermal denaturation curve of HR2. The Tm was 36.3°C. C: Thermal denaturation curve of HR1‐b/HR2 complex. The Tm was 59.5°C. D: The CD spectra of His₆‐HR1 (blue), GST‐removed HR2 (magenta) and the His₆‐HR1/GST‐removed HR2 complex (yellow). E: Thermal denaturation curve of His₆‐HR1. The Tm was 62.9°C. F: Thermal denaturation curve of His₆‐HR1/GST‐removed HR2 complex. The Tm was 86.2°C.

Figure 4

Laser light scattering analysis of the stoichiometry of the HR1/HR2 complex. Molar mass distribution plots of His₆‐HR1 (A), GST‐removed HR2 (B), His₆‐HR1/GST‐removed HR2 complex (C). The solid horizontal bar indicates the average molecular weights for each peak.

Figure 5

Characterization of HR‐2‐Helix. CD spectrum (A) and thermal denaturation curve (B) are shown. The Tm was 101.00°C. Laser light scattering analysis showed that HR‐2‐Helix forms a monodispersed species of 40,110 Da. The solid horizontal bar indicates the average molecular weight for the HR‐2‐Helix peak (C).

Figure 6

ADS‐J interferes with the interactions between HR1 and HR2 in vitro. His₆‐HR1 was preincubated in PBS, control (lanes 1–4, panel A and lanes 1–3, panel B), 1,500 µM ADS‐J1 (lanes 5–8, panel A) or 1,500 µM XXT (lanes 4–6, panel B) before loading into the glutathione resin captured GST‐HR2. The elution fractions were subjected to SDS–PAGE followed by Western blot analysis probed with anti‐His‐tag antibodies. Panel A, lanes 1 and 5: flow‐through; lanes 2 and 6: first elution; lanes 3 and 7: second elution; lanes 4 and 8: third elution. Panel B, lanes 1 and 4: flow‐through; lanes 2 and 5: first elution; lanes 3 and 6: second elution.

Figure 7

Interaction of HR2 with the deep pocket in the relative deep groove region of HR1 in the SARS‐CoV S protein fusion core. A: Hydrophobic residues (Leu1168 (light green), Val1171 (orange), Leu1175 (light blue) and Leu1179 (pink)) of the HR2 helix (yellow) penetrating deep into the deep pocket on the surface of HR1 (violet solid surface). B: Interaction of the hydrophobic residues (Phe909, Ile913, Ile916, and Leu920) (red) in the deep pocket formed by the HR1 central coiled‐coil (violet ribbon) with the hydrophobic residues of the HR2 helix (yellow ribbon). Note that there is a peripheral ionic HR1/HR2 interaction between Lys911 (orange) on the HR1 helix (violet ribbon) and Glu1177 (blue) on the HR2 helix (white ribbon).

Figure 8

Interactions of HR2 and ADS‐J1 with the deep pocket of the relatively deep groove region of HR1. The carbon, nitrogen, oxygen, and sulfur atoms of ADS‐J1 are shown in green, blue, pink, and yellow, respectively. ADS‐J1 was docked onto the deep pocket. The hydrophobic residues in deep pocket of the HR1 helix (violet ribbon) are shown in red and Lys911 is shown in orange with its amino group shown in blue. Hydrophobic interaction between Leu1168 (light green), Val1171 (orange), Leu1175 (light blue) and Leu1179 (pink) of HR2 (yellow ribbon) and the hydrophobic residues (red) in the deep pocket. The molecular weight of ADS‐J1 is 1079.01 Da, the calculated energy terms are −37.05 kcal/mol, −4.41 and −41.46 for van der Waals, electrostatic and total energy, respectively. The distance between Lys911 and SO₃H of ADJ‐S1 is 8.65 Å.