Heptad repeat-derived peptides block protease-mediated direct entry from the cell surface of severe acute respiratory syndrome coronavirus but not entry via the endosomal pathway

Abstract

The peptides derived from the heptad repeat (HRP) of severe acute respiratory syndrome coronavirus (SCoV) spike protein (sHRPs) are known to inhibit SCoV infection, yet their efficacies are fairly low. Recently our research showed that some proteases facilitated SCoV's direct entry from the cell surface, resulting in a more efficient infection than the previously known infection via endosomal entry. To compare the inhibitory effect of the sHRP in each pathway, we selected two sHRPs, which showed a strong inhibitory effect on the interaction of two heptad repeats in a rapid and virus-free in vitro assay system. We found that they efficiently inhibited SCoV infection of the protease-mediated cell surface pathway but had little effect on the endosomal pathway. This finding suggests that sHRPs may effectively prevent infection in the lungs, where SCoV infection could be enhanced by proteases produced in this organ. This is the first observation that HRP exhibits different effects on virus that takes the endosomal pathway and virus that enters directly from the cell surface.

FIG. 1.

(A) Schematic of SCoV S protein and sequences of native sHRP (SR9) and its EK substitution derivatives. The S protein contains two α-helical heptad repeats (HR1 and HR2), a putative fusion peptide (FP), a transmembrane domain (TMD), and a trypsin cleavage site (17). The expanded region shows the amino acid sequence of HR2 (SR9), which consists of two extended parts (1151 to 1160 and 1178 to 1185) and one α-helix part (1161 to 1177). Substituted EKs are shown with italic white letters. (B) In vitro binding inhibition assay of HRPs. GST-HR2-coated plates were incubated with MBP-HR1 in the presence of various concentrations (1 nM to 100 μM) of sHRP. Inhibitory potency of the peptide was assessed using the anti-MBP antibody-alkaline phosphatase conjugate and staining with 5-bromo-4-chloro-3-indolylphosphate.

FIG. 2.

Inhibitory effect of sHRPs on SCoV infections via the endosomal pathway (A) or protease-mediated cell-surface pathway (B). (A) VeroE6 cells were pretreated with 50 μM sHRPs at 37°C for 30 min, placed on ice for 10 min, and then inoculated with SCoV at a multiplicity of infection of 1.0 on ice for 30 min. After the removal of unbound virus, the cells were incubated in medium containing 50 μM sHRPs at 37°C for 6 h. (B) Cells pretreated with 1 μM Baf and sHRPs at the indicated concentrations were inoculated with SCoV as described above. After the removal of unbound virus, the cells were treated with 200 μg/ml l-1-tosylamide-2-phenylethyl chloromethyl ketone-treated trypsin at room temperature for 5 min and incubated at 37°C for 6 h. sHRP and Baf were present in the media in all steps at indicated concentrations. To measure amounts of viruses that entered cells, cells were infected with 10-fold-stepwise-diluted SARS-CoV from 10⁶ to 10² PFU without Baf and trypsin and the amounts of mRNA9 were quantified by real-time PCR. Amounts of viral entry in this study were calculated from a calibration line obtained as described above and are shown as relative mRNA levels (20). (C) EK1 has no sequential similarity to sHRP and showed no inhibitory effect in vitro. Cells were treated with 1 μM EK1 as a control peptide, and other procedures were performed as described for panel B.

FIG. 3.

Effective inhibition by HRPs of SCoV infection in the presence of exogenous trypsin. VeroE6 cells pretreated with sHRPs at the indicated concentrations were inoculated with SCoV as described in the legend to Fig. 2. After the removal of unbound virus, the cells were treated with 200 μg/ml l-1-tosylamide-2-phenylethyl chloromethyl ketone-treated trypsin at room temperature for 5 min and incubated at 37°C for 6 h. sHRPs were present in the media in all steps at the indicated concentrations. The relative viral mRNA9 was measured quantitatively by real-time PCR as described in the legend to Fig. 2. In this assay, cells were not treated with Baf throughout the experiment.