Human coronavirus 229E: receptor binding domain and neutralization by soluble receptor at 37 degrees C

Abstract

Truncated human coronavirus HCoV-229E spike glycoproteins containing amino acids 407 to 547 bound to purified, soluble virus receptor, human aminopeptidase N (hAPN). Soluble hAPN neutralized the infectivity of HCoV-229E virions at 37 degrees C, but not 4 degrees C. Binding of hAPN may therefore trigger conformational changes in the viral spike protein at 37 degrees C that facilitate virus entry.

FIG. 1.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified recombinant soluble receptor hAPN and truncated spike glycoproteins of HCoV-229E. (A) Molecular weight standards (lane 1), 16 μg of shAPN stained with Coomassie brilliant blue (lane 2), and 4 μg of shAPN immunoblotted with a rabbit polyclonal anti-hAPN antibody (lane 3). (B and C) Truncated HCoV-229E spike proteins in supernatant media from Sf9 cells (lanes 1) and after purification (lanes 2) are shown in silver stains (B) and in immunoblots (C) with polyclonal rabbit antibody to HCoV-229E virions.

FIG. 2.

Identification of the receptor-binding domain on the S1 domain of the spike glycoprotein of HCoV-229E. Purified shAPN was immobilized on ELISA plates, and the S1 domain of the HCoV-229E spike protein (S547) and C- or N-terminally-truncated S1 proteins expressed in baculovirus vectors were bound at 4°C for 4 h. Binding of the S proteins to shAPN at 4°C was detected by binding of the indicated anti-S MAbs followed by peroxidase-conjugated antirabbit immunoglobulin. The y axis shows the difference between the absorbance values (A₄₀₅) of each sample incubated with anti-HCoV-229E MAb and a control MAb.

FIG. 3.

Neutralization of HCoV-229E infectivity by soluble human APN. HCoV-229E virions were incubated with dilutions of shAPN at 37 or 4°C for 1 h at pH 7.0 or with sCEACAM1a as a control. Virus infectivity was quantified by plaque assay. The percentage of virus neutralization relative to that in samples without receptor is plotted against the molar concentration of purified shAPN.