The HCoV-HKU1 N‑Terminal Domain Binds a Wide\nRange of 9‑<i>O</i>‑Acetylated Sialic Acids\nPresented on Different Glycan Cores

Coronaviruses (CoVs)\nrecognize a wide array of protein and glycan\nreceptors by using the S1 subunit of the spike (S) glycoprotein. The\nS1 subunit contains two functional domains: the N-terminal domain\n(S1-NTD) and the C-terminal domain (S1-CTD). The S1-NTD of SARS-CoV-2,\nMERS-CoV, and HCoV-HKU1 possesses an evolutionarily conserved glycan\nbinding cleft that facilitates weak interactions with sialic acids\non cell surfaces. HCoV-HKU1 employs 9-<i>O</i>-acetylated\nα2-8-linked disialylated structures for initial binding, followed\nby TMPRSS2 receptor binding and virus–cell fusion. Here, we\ndemonstrate that the HCoV-HKU1 NTD has a broader receptor binding\nrepertoire than previously recognized. We presented HCoV-HKU1 NTD\nFc chimeras on a nanoparticle system to mimic the densely decorated\nsurface of HCoV-HKU1. These proteins were expressed by HEK293S GnTI<sup>–</sup> cells, generating species carrying Man-5 structures,\noften observed near the receptor binding site of CoVs. This multivalent\npresentation of high mannose-containing NTD proteins revealed a much\nbroader receptor binding profile compared to that of its fully glycosylated\ncounterpart. Using glycan microarrays, we observed that 9-<i>O</i>-acetylated α2-3-linked sialylated LacNAc structures\nare also bound, comparable to OC43 NTD, suggesting an evolutionarily\nconserved glycan-binding modality. Further characterization of receptor\nspecificity indicated promiscuous binding toward 9-<i>O</i>-acetylated sialoglycans, independent of the glycan core (glycolipids, <i>N-</i> or <i>O</i>-glycans). We demonstrate that HCoV-HKU1\nmay employ additional sialoglycan receptors to trigger conformational\nchanges in the spike glycoprotein to expose the S1-CTD for proteinaceous\nreceptor binding.