Human coronavirus NL63 (NL63), a member of the group I coronaviruses, may cause acute respiratory diseases in young children and immunocompromised adults. Like severe acute respiratory syndrome coronavirus (SARS-CoV), NL63 also employs the human angiotensinconverting enzyme 2 (hACE2) receptor for cellular entry. To identify residues in the spike protein of NL63 that are important for hACE2 binding, this study first generated a series of S1-truncated variants, examined their associations with the hACE2 receptor and subsequently mapped a minimal receptor-binding domain (RBD) that consisted of 141 residues (aa 476-616) towards the C terminus of the S1 domain. The data also demonstrated that the NL63 RBD bound to hACE2 more efficiently than its full-length counterpart and had a binding efficiency comparable to the S1 or RBD of SARS-CoV. A further series of RBD variants was generated using site-directed mutagenesis and random mutant library screening assays, and identified 15 residues (C497, Y498, V499, C500, K501, R518, R530, V531, G534, G537, D538, S540, E582, W585 and T591) that appeared to be critical for the RBD-hACE2 association. These critical residues clustered in three separate regions (designated RI, RII and RIII) inside the RBD, which may represent three receptor-binding sites. These results may help to delineate the molecular interactions between the S protein of NL63 and the hACE2 receptor, and may also enhance our understanding of the pathogenesis of NL63 and SARS-CoV. INTRODUCTIONCoronaviruses consist of a large and diverse family of enveloped, positive-sense, single-stranded RNA viruses. They can infect a broad range of mammalian and avian species, causing a variety of diseases in the respiratory, gastrointestinal, hepatic and central nervous systems (Holmes & Lai, 1996). The outbreak of severe acute respiratory syndrome (SARS) in [2002][2003], which was caused by a highly pathogenic virus named SARS coronavirus (SARS-CoV), posed significant challenges to medical communities worldwide (Ksiazek et al., 2003;Peiris et al., 2003). Since then, two additional human coronaviruses, HCoV-NL63 (NL63) and HCoV-HKU1, have been identified (van der Hoek et al., 2004;Woo et al., 2005). NL63 has been found to be associated with diseases in the upper and lower respiratory tracts, such as bronchiolitis, conjunctivitis, croup and pneumonia in young children and immunocompromised adults (Arden et al., 2005;Bastien et al., 2005;Ebihara et al., 2005;Gerna et al., 2006;Kaiser et al., 2005;van der Hoek et al., 2005. So far, NL63 infections have been reported in 12 countries across Europe, Asia and North America, indicating that it is circulating among the human population worldwide (Arden et al., 2005;Bastien et al., 2005;Han et al., 2007;Koetz et al., 2006;Lau et al., 2006;Suzuki et al., 2005;Vabret et al., 2005).NL63 is a member of the group I coronaviruses, which also includes HCoV-229E (229E), feline infectious peritonitis virus 79-1146, feline enteric coronavirus 79-1683, canine coronavirus and porcine transmissible ga...

Purpose: Severe acute respiratory syndrome (SARS) is an atypical type of contagious pneumonia with a high mortality rate, however no effective drugs and vaccine were established. To identify an entry inhibitor of severe acute respiratory syndrome coronavirus (SARS-CoV), we have examined the effects of medicinal herbs which were found to be effective

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