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...

The spike (S) protein of human coronavirus NL63 (HCoV-NL63) mediates both cell attachment by binding to its receptor hACE2 and membrane fusion during virus entry. We have previously identified the receptor-binding domain (RBD) and residues important for RBD-hACE2 association. Here, we further characterized the S protein by investigating the roles of the cytoplasmic tail and 19 residues located in the RBD in protein accumulation, receptor binding, and pseudotype virus entry. For these purposes, we first identified an entry-efficient S gene template from a pool of gene variants and used it as a backbone to generate a series of cytoplasmic tail deletion and single residue substitution mutants. Our results showed that: (i) deletion of 18aa from the C-terminus enhanced the S protein accumulation and virus entry, which might be due to the deletion of intracellular retention signals; (ii) further deletion to residue 29 also enhanced the amount of S protein on the cell surface and in virion, but reduced virus entry by 25%, suggesting that residues 19-29 contributes to membrane fusion; (iii) a 29aa-deletion mutant had a defect in anchoring on the plasma membrane, which led to a dramatic decrease of S protein in virion and virus entry; (iv) a total of 15 residues (Y498, V499, V531, G534, G537, D538, S540, G575, S576, E582, W585, Y590, T591, V593 and G594) within RBD were important for receptor binding and virus entry. They probably form three receptor binding motifs, and the third motif is conserved between NL63 and SARS-CoV.