ABSTRACT
SARS-CoV-2 viruses engage ACE2 as a functional receptor with their spike protein. The S1 domain of the spike protein contains a C-terminal receptor-binding domain (RBD) and an N-terminal domain (NTD). The NTD of other coronaviruses includes a glycan-binding cleft. However, for the SARS-CoV-2 NTD protein-glycan binding was only observed weakly for sialic acids with highly sensitive methods. Amino acid changes in the NTD of Variants of Concern (VoC) shows antigenic pressure, which can be an indication of NTD-mediated receptor binding. Trimeric NTD proteins of SARS-CoV-2, Alpha, Beta, Delta, and Omicron did not reveal a receptor binding capability. Unexpectedly, the SARS-CoV-2 Beta subvariant strain (501Y.V2-1) NTD binding to Vero E6 cells was sensitive to sialidase pretreatment. Glycan microarray analyses identified a putative 9-O-acetylated sialic acid as a ligand, which was confirmed by catch-and-release ESI-MS, STD-NMR analyses, and a graphene-based electrochemical sensor. The Beta (501Y.V2-1) variant attained an enhanced glycan binding modality in the NTD with specificity towards 9-O-acetylated structures, suggesting a dual-receptor functionality of the SARS-CoV-2 S1 domain, which was quickly selected against. These results indicate that SARS-CoV-2 can probe additional evolutionary space, allowing binding to glycan receptors on the surface of target cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=76 HEIGHT=200 SRC="FIGDIR/small/507904v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1f98a70org.highwire.dtl.DTLVardef@1efc119org.highwire.dtl.DTLVardef@16e8bc6org.highwire.dtl.DTLVardef@9a74e6_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisCoronaviruses utilize their N-terminal domain (NTD) for initial reversible low-affinity interaction to (sialylated) glycans. This initial low-affinity/high-avidity engagement enables viral surfing on the target membrane, potentially followed by a stronger secondary receptor interaction. Several coronaviruses, such as HKU1 and OC43, possess a hemagglutinin-esterase for viral release after sialic acid interaction, thus allowing viral dissemination. Other coronaviruses, such as MERS-CoV, do not possess a hemagglutinin-esterase, but interact reversibly to sialic acids allowing for viral surfing and dissemination. The early 501Y.V2-1 subvariant of the Beta SARS-CoV-2 Variant of Concern has attained a receptor-binding functionality towards 9-O-acetylated sialic acid using its NTD. This binding functionality was selected against rapidly, most likely due to poor dissemination. Ablation of sialic acid binding in more recent SARS-CoV-2 Variants of Concern suggests a fine balance of sialic acid interaction of SARS-CoV-2 is required for infection and/or transmission.
ABSTRACT
Emerging evidence suggests that host glycans influence infection by SARS-CoV-2. Here, we reveal that the receptor-binding domain (RBD) of the spike (S)-protein on SARS-CoV-2 recognizes oligosaccharides containing sialic acid (SA), with preference for the oligosaccharide of monosialylated gangliosides. Gangliosides embedded within an artificial membrane also bind the RBD. The monomeric affinities (Kd = 100-200 M) of gangliosides for the RBD are similar to heparan sulfate, another negatively charged glycan ligand of the RBD proposed as a viral coreceptor. RBD binding and infection of SARS-CoV-2 pseudotyped lentivirus to ACE2-expressing cells is decreased upon depleting cell surface SA level using three approaches: sialyltransferase inhibition, genetic knock-out of SA biosynthesis, or neuraminidase treatment. These effects on RBD binding and pseudotyped viral entry are recapitulated with pharmacological or genetic disruption of glycolipid biosynthesis. Together, these results suggest that sialylated glycans, specifically glycolipids, facilitate viral entry of SARS-CoV-2.
