Identification of residues in the receptor-binding domain (RBD) of the spike protein of human coronavirus NL63 that are critical for the RBD–ACE2 receptor interaction

Abstract

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...

Interaction of severe acute respiratory syndrome-coronavirus and NL63 coronavirus spike proteins with angiotensin converting enzyme-2

“…However, this study demonstrated that the role of ACE-2 residue 353 was noticeably different. Substitution of the resident lysine for alanine reduced, but did not abolish, SARS-CoV S binding, while it effectively abolished binding by NL63 S. In addition, as the end points in our constructs differed somewhat from those published by Li et al (2007), our data reduced the carboxy-terminal boundary of the NL63 S RBD in an analogous Fc-fusion protein configuration from residue 749 to 739; however, this boundary is not reduced as far as residue 616 as recently described by Lin et al (2008). An affinity of interaction with ACE-2 by NL63 S of 10–100-fold less than that of SARS-CoV S would explain our inability to obtain a sound K d for the interaction by surface plasmon resonance.…”

“…However, while confirming that full-length SARS-CoV S appeared to bind to ACE-2-expressing cells more efficiently than NL63 S, the SARS-CoV RBD was less efficient at competing with NL63-mediated pseudotype entry than competition with SARS-CoV-mediated entry (IC 50 of ∼200 ng ml −1 for NL63 compared with ∼50 ng ml −1 for SARS-CoV) (Li et al , 2007). A recent report suggests that the minimum NL63 RBD lies between residues 476 and 616 and that, expressed alone, the binding affinity with ACE-2 reaches that observed for SARS-CoV S (Lin et al , 2008). Here, using a wholly recombinant system that precludes the involvement of any other cell surface factor present on mammalian cells, such as lectins [e.g.…”

“…In addition, we confirmed that ACE-2 residues key to SARS-CoV S binding are also involved in NL63 binding but that the contribution of individual residues, exemplified here by K353A, may differ. This will relate to the molecular contact between S protein and the receptor, which has been described for SARS-CoV but remains unknown in NL63 (Li et al , 2005a), despite the identification of residues critical for contact (Lin et al , 2008). It remains to be determined exactly what difference in ACE-2 signalling, if any, follows SARS-CoV and NL63 S protein binding and whether this relates to the pathology of infection.…”

Novel polymeric inhibitors of HCoV-NL63

“…One may assume that such binding will result in the efficient inactivation of the virus. Unfortunately, it was not possible to determine whether HTCC polymer hampers NL63-S/ACE2 interaction, as the affinity of NL63-S to ACE2 is very low (Lin et al, 2008;Mathewson et al, 2008) and it was not possible to visualize this process with accessible methods. Careful analysis of polymers suggests that the combination of particular polymeric chain and its proper substituent is indispensable for its activity.…”

Characterisation of human coronavirus-NL63 nucleocapsid protein

“…Unfortunately, the virus is not well studied at a molecular level with only the spike (Lin et al, 2008(Lin et al, , 2011Mathewson et al, 2008) and ORF3 proteins (Fielding and Suliman, 2009;Muller et al, 2010) previously characterized. In this work, the HCoV-NL63 N was expressed in mammalian and bacterial systems.…”