Identification of the Receptor-Binding Domain of the Spike Glycoprotein of Human Betacoronavirus HKU1

Coronavirus spike (S) glycoproteins mediate receptor binding, membrane fusion, and virus entry and determine host range. Murine betacoronavirus (beta-CoV) in group A uses the N-terminal domain (NTD) of S protein to bind to its receptor, whereas the beta-CoVs severe acute respiratory syndrome CoV in group B and Middle East respiratory syndrome CoV in group C and several alpha-CoVs use the downstream C domain in their S proteins to recognize their receptor proteins. To identify the receptor-binding domain in the spike of human beta-CoV HKU1 in group A, we generated and mapped a panel of monoclonal antibodies (MAbs) to the ectodomain of HKU1 spike protein. They did not cross-react with S proteins of any other CoV tested. Most of the HKU1 spike MAbs recognized epitopes in the C domain between amino acids 535 and 673, indicating that this region is immunodominant. Two of the MAbs blocked HKU1 virus infection of primary human tracheal-bronchial epithelial (HTBE) cells. Preincubation of HTBE cells with a truncated HKU1 S protein that includes the C domain blocked infection with HKU1 virus, but preincubation of cells with truncated S protein containing only the NTD did not block infection. These data suggest that the receptor-binding domain (RBD) of HKU1 spike protein is located in the C domain, where the spike proteins of alpha-CoVs and beta-CoVs in groups B and C bind to their specific receptor proteins. Thus, two beta-CoVs in group A, HKU1 and murine CoV, have evolved to use different regions of their spike glycoproteins to recognize their respective receptor proteins. IMPORTANCE Mouse hepatitis virus, a beta-CoV in group A, uses the galectin-like NTD in its spike protein to bind its receptor protein, while HCoV-OC43, another beta-CoV in group A, uses the NTD to bind to its sialic-acid containing receptor. In marked contrast, the NTD of the spike glycoprotein of human respiratory beta-CoV HKU1, which is also in group A, does not bind sugar. In this study, we showed that for the spike protein of HKU1, the purified C domain, downstream of the NTD, could block HKU1 virus infection of human respiratory epithelial cells, and that several monoclonal antibodies that mapped to the C domain neutralized virus infectivity. Thus, the receptor-binding domain of HKU1 spike glycoprotein is located in the C domain. Surprisingly, two beta-CoVs in group A, mouse hepatitis virus and HKU1, have evolved to use different regions of their spike glycoproteins to recognize their respective receptors.