Substitutions of N27 having an H-bond with OA9 of the 9-O-acetyl carbonyl group, K81 forming H-bonds with O1 and N5 of Sia, or S83 containing an H-bond with O3 of Sia C1, with alanine and mutations of L80, L86 or W90 in hydrophobic pockets accommodating the 5-N-acyl moiety and the 9-O-acetyl-methyl moiety provided a mutant HCoV-OC43 S1A that had lost the ability to bind to 9-O-Ac-6SLN.