Characterization of the spike protein of human coronavirus NL63 in receptor binding and pseudotype virus entry

► We developed an efficient pseudotype virus system for HCoV-NL63. ► We identified the domains and critical residues involved in the NL63 S-mediated viral entry. ► This viral system is useful for investigation of the effect of NL63 S on ACE2 expression and shedding as well as the pathogenicity. ► Our study may advance our understanding of the molecular mechanism of NL63 S-ACE2 interaction. ► Our data may provide important information for the development of vaccines, small peptides, compounds, or neutralizing antibodies against NL63 infections.