Human Coronavirus HKU1 Spike Protein Uses O -Acetylated Sialic Acid as an Attachment Receptor Determinant and Employs Hemagglutinin-Esterase Protein as a Receptor-Destroying Enzyme

Abstract

Human coronavirus (hCoV) HKU1 is one of six hCoVs identified to date and the only one with an unidentified cellular receptor. hCoV-HKU1 encodes a hemagglutinin-esterase (HE) protein that is unique to the group a betacoronaviruses (group 2a). The function of HKU1-HE remains largely undetermined. In this study, we examined binding of the S1 domain of hCoV-HKU1 spike to a panel of cells and found that the S1 could specifically bind on the cell surface of a human rhabdomyosarcoma cell line, RD. Pretreatment of RD cells with neuraminidase (NA) and trypsin greatly reduced the binding, suggesting that the binding was mediated by sialic acids on glycoproteins. However, unlike other group 2a CoVs, e.g., hCoV-OC43, for which 9-O-acetylated sialic acid (9-O-Ac-Sia) serves as a receptor determinant, HKU1-S1 bound with neither 9-O-Ac-Sia-containing glycoprotein(s) nor rat and mouse erythrocytes. Nonetheless, the HKU1-HE was similar to OC43-HE, also possessed sialate-O-acetylesterase activity, and acted as a receptor-destroying enzyme (RDE) capable of eliminating the binding of HKU1-S1 to RD cells, whereas the O-acetylesterase-inactive HKU1-HE mutant lost this capacity. Using primary human ciliated airway epithelial (HAE) cell cultures, the only in vitro replication model for hCoV-HKU1 infection, we confirmed that pretreatment of HAE cells with HE but not the enzymatically inactive mutant blocked hCoV-HKU1 infection. These results demonstrate that hCoV-HKU1 exploits O-Ac-Sia as a cellular attachment receptor determinant to initiate the infection of host cells and that its HE protein possesses the corresponding sialate-O-acetylesterase RDE activity. IMPORTANCEHuman coronaviruses (hCoV) are important human respiratory pathogens. Among the six hCoVs identified to date, only hCoV-HKU1 has no defined cellular receptor. It is also unclear whether hemagglutinin-esterase (HE) protein plays a role in viral entry. In this study, we found that, similarly to other members of the group 2a CoVs, sialic acid moieties on glycoproteins are critical receptor determinants for the hCoV-HKU1 infection. Interestingly, the virus seems to employ a type of sialic acid different from those employed by other group 2a CoVs. In addition, we determined that the HKU1-HE protein is an O-acetylesterase and acts as a receptor-destroying enzyme (RDE) for hCoV-HKU1. This is the first study to demonstrate that hCoV-HKU1 uses certain types of O-acetylated sialic acid residues on glycoproteins to initiate the infection of host cells and that the HKU1-HE protein possesses sialate-O-acetylesterase RDE activity.H uman coronaviruses (hCoVs) are enveloped RNA viruses. They are usually associated with mild to moderate respiratory tract illnesses but can also cause severe and highly lethal disease, depending on the virus strain (1). Six hCoV strains have been identified to date and belong to four different groups, including hCoV-229E and hCoV-NL63 in the alphacoronaviruses (group 1); hCoV-OC43 and hCoV-HKU1 in the group a betacoronaviruses (group 2a); severe acut...

Crystal structure of the receptor binding domain of the spike glycoprotein of human betacoronavirus HKU1

“…Recently, Huang et al 16. found that o-acetylated sialic acid is an attachment receptor determinant for HKU1 virus infection.…”

Molecular Evolution of Human Coronavirus Genomes

“…Incidentally, the N-terminal domain of the CoV spike protein is also believed to derive from a cellular lectin [28]. Unlike the influenza virus C enzyme, CoV HEs lack membrane-fusion activity and are accessory to the spike protein by serving primarily as receptor-destroying enzymes (RDE)that is, they aid viral detachment from carbohydrates present on infected cells [29,30]. In fact, HEs are present only in the genome of lineage A beta-CoVs, most of which use sialic acids as coreceptors [1] ( Figure 1B).…”

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“…This receptor recognition is important for initiating virus entry into the host cells, thereby playing a major role in the tissue and host species tropism of viruses. The receptors used by all human CoV are known (see Table 1): Aminopeptidase N by HCoV-229E [32], 9-O-acetylated sialic acid by HCoV-OC43 and HCoV-HKU1 [33,34], angiotensin-converting enzyme 2 (ACE2) by SARS-CoV [35] and HCoV-NL63 [36,37] and dipeptidyl peptidase 4 (DPP4) by MERS-CoV [38].…”