title: Identification of the Receptor-Binding Domain of the Spike Glycoprotein of Human Betacoronavirus HKU1.
abstract: Coronavirus spike (S) glycoproteins mediate receptor binding, membrane fusion, and virus entry and determine host range. Murine betacoronavirus (β-CoV) in group A uses the N-terminal domain (NTD) of S protein to bind to its receptor, whereas the β-CoVs severe acute respiratory syndrome CoV in group B and Middle East respiratory syndrome CoV in group C and several α-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 β-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 α-CoVs and β-CoVs in groups B and C bind to their specific receptor proteins. Thus, two β-CoVs in group A, HKU1 and murine CoV, have evolved to use different regions of their spike glycoproteins to recognize their respective receptor proteins.
  
  
  OBJECTIVE
  Mouse hepatitis virus, a β-CoV in group A, uses the galectin-like NTD in its spike protein to bind its receptor protein, while HCoV-OC43, another β-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 β-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 β-CoVs in group A, mouse hepatitis virus and HKU1, have evolved to use different regions of their spike glycoproteins to recognize their respective receptors.
authors.affiliation: MOH Key Laboratory, Institute of Pathogen Biology, Chinese Academy of Medical Science, Beijing, China Department of Pediatrics, University of Colorado School of Medicine, Aurora, Colorado, USA zqian2013@sina.com Samuel.dominguez@ucdenver.edu.
authors.forename: Zhaohui
authors.initials: Z
authors.lastname: Qian
authors.affiliation: MOH Key Laboratory, Institute of Pathogen Biology, Chinese Academy of Medical Science, Beijing, China.
authors.forename: Xiuyuan
authors.initials: X
authors.lastname: Ou
authors.affiliation: Department of Pediatrics, University of Colorado School of Medicine, Aurora, Colorado, USA.
authors.forename: Luiz Gustavo Bentim
authors.initials: LG
authors.lastname: Góes
authors.affiliation: Department of Pediatrics, University of Colorado School of Medicine, Aurora, Colorado, USA.
authors.forename: Christina
authors.initials: C
authors.lastname: Osborne
authors.affiliation: Department of Microbiology, University of Colorado School of Medicine, Aurora, Colorado, USA.
authors.forename: Anna
authors.initials: A
authors.lastname: Castano
authors.affiliation: Department of Microbiology, University of Colorado School of Medicine, Aurora, Colorado, USA.
authors.forename: Kathryn V
authors.initials: KV
authors.lastname: Holmes
authors.affiliation: Department of Pediatrics, University of Colorado School of Medicine, Aurora, Colorado, USA zqian2013@sina.com Samuel.dominguez@ucdenver.edu.
authors.forename: Samuel R
authors.initials: SR
authors.lastname: Dominguez
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D017934:Coronavirus / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018352:Coronavirus Infections / Q000821:virology*; D004285:Dogs; D004847:Epithelial Cells / Q000821:virology; D057809:HEK293 Cells; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011991:Receptors, Virus / Q000235:genetics*; D020545:Respiratory Mucosa / Q000166:cytology / Q000821:virology; D016415:Sequence Alignment; D064370:Spike Glycoprotein, Coronavirus / Q000235:genetics* / Q000276:immunology; D014709:Vero Cells; D056189:Viral Tropism / Q000235:genetics*; D053586:Virus Internalization
chemical_list: D000911:Antibodies, Monoclonal; D011991:Receptors, Virus; D064370:Spike Glycoprotein, Coronavirus