The S1 subunit of the S protein is composed of four domains, A through D (S1A through S1D) domains from the N-terminus [112]. By using OC43 or HKU1 S1A–Fc proteins in a direct binding assay, HCoV-OC43 and HCoV-HKU1 were shown to bind to the receptors via domain A (S1-NTD (Figure 5a), residues 15–302 based on the S protein of OC43 strain ATCC VR-759) [92]. However, binding of HKU1 S1A to its receptors on rat erythrocytes can be detected when HKU1 S1A–Fc proteins have been conjugated to nanoparticles but cannot be detected by using free HKU1 S1A–Fc proteins (the standard method), indicating the requirement of multivalency of HKU1 S1A–Fc proteins for binding to rat erythrocytes. Based on structural analysis, residues 28–34 (element 1) and/or residues 243–252 (element 2) in HKU1 S1A (Figure 5b) were thought to hamper the binding of HKU1 S1A. The mutant HKU1 S1A was generated by replacement of one or both of their elements with the corresponding element(s) from bovine coronavirus (BCoV), which is believed to be the ancestor of OC43. The free mutant HKU1 S1A–Fc proteins with only one replacement at element 2 were found to bind to rat erythrocytes. The free mutant HKU1 S1A–Fc proteins with replacement of both elements showed greater binding to rat erythrocytes. In comparison with binding of the wild-type HKU1 S1A conjugated with nanoparticles to rat erythrocytes, the mutant HKU1 S1A with removal of a glycosylation site at element 2 (N251Q) showed increased binding, and the mutant HKU1 S1A with removal of the glycosylation sites in both elements (N29Q in element 1 + N251Q in element 2) showed greater binding. These findings indicated that binding of HKU1 S1A to its receptors on rat erythrocytes is impeded by both the RBS architecture and N-glycans on the RBS [92]. Binding of free HKU1 S1 or free HKU1 S1A not only to rat erythrocytes but also to mouse erythrocytes and to BSM cannot be detected by the standard method unlike other 9-O-Ac-Sia-binding β1CoVs, including HCoV-OC43 for which their free S1 and S1A detectably bind to those erythrocytes and BSM [92,191]. The difference of HKU1 from other 9-O-Ac-Sia-binding β1CoVs was suggested to be due to receptor fine-specificity determined by elements 1 and 2. The effects of the internal part of the glycan structure, such as Siaα2,3/2,6Gal and LN repeats, on binding of HKU1 in comparison with other 9-O-Ac-Sia-binding β1CoVs should be further determined.