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1 Department of Pathology and Molecular Medicine, McMaster University and Department of Microbiology, St Joseph's Healthcare, Hamilton, ON L8N 4A6, Canada
2 Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA 02115, USA
3 National Microbiology Laboratory, Canadian Science Center for Human and Animal Health, Winnipeg, MB R3E 3R2, Canada
Correspondence
Chengsheng Zhang
zhangch{at}mcmaster.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). 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, 2006). 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 gastroenteritis virus (TGEV) (van der Hoek et al., 2004, 2006). The genome of NL63 contains 27553 nt and encodes the polyproteins 1a and 1b, spike (S), open reading frame 3 (ORF3), envelope (E), membrane (M) and nucleocapsid (N) proteins (van der Hoek et al., 2004) (Fig. 1). The S protein of NL63 is a 1356 aa, type I membrane glycoprotein, which contains a signal peptide (1–20 aa), an ectodomain (1–1302 aa), a transmembrane domain (1303–1325 aa) and a cytoplasmic tail (1326–1356 aa). The S protein can also be divided into S1 (21–717 aa) and S2 (718–1356 aa) domains based on its similarity to S proteins of other coronaviruses (Jackwood et al., 2001; Sturman & Holmes, 1984). The S1 domain of coronaviruses is responsible for binding to the cellular receptor, whilst the S2 domain is mainly involved in membrane fusion during viral entry into the host cell (Bonavia et al., 2003; Gallagher & Buchmeier, 2001). Human aminopeptidase N (hAPN or CD13) and human angiotensin-converting enzyme 2 (hACE2) have been identified as the functional receptors for 229E and SARS-CoV, respectively (Li et al., 2003; Yeager et al., 1992). As all previously known group I coronaviruses use the APN of their own host species as functional receptors (Tresnan & Holmes, 1998; Tresnan et al., 1996), and viral sequence analysis indicates that NL63 is most closely related to 229E (van der Hoek et al., 2004), it has been proposed that NL63 may also employ hAPN as its receptor. However, hACE2 is a functional receptor for NL63 (Hofmann et al., 2005). This finding was quite surprising given that the S protein of NL63 shows only limited sequence similarities to that of SARS-CoV.
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; Wong et al., 2004). Whilst the first 330 residues at the N terminus of the mouse hepatitis virus (MHV) S1 subunit are able to bind efficiently to carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), the RBDs of 229E and TGEV are all located at the C terminus of their S1 domains (Ballesteros et al., 1997; Bonavia et al., 2003; Godet et al., 1994; Laude et al., 1995). A recent study suggested that the central and C-terminal domains of the NL63 S protein are required for hACE2 association (Hofmann et al., 2006). However, the location of the NL63 RBD remains to be defined and the critical residues for receptor binding have not been characterized. In this study, we generated a series of S1-truncated variants, tested their association with the hACE2 receptor and subsequently mapped an RBD that consists of 141 aa (aa 476–616) towards the C terminus of the NL63 S1 domain. In addition, we identified 15 residues in the RBD that appear to be critical for receptor binding. Interestingly, these critical residues seemed to cluster into three separate regions (designated RI, RII and RIII) inside the RBD, suggesting that these sites might be associated with receptor binding. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In this study, the domain structures of the NL63 S protein were defined using computer programs from the websites of SOSUI (http://bp.nuap.nagoya-u.ac.jp/sosui/sosui_submit.html) and Pfam (http://www.sanger.ac.uk/Software/Pfam/). To construct the plasmids expressing S1 and its variants, we first synthesized a full-length, codon-optimized NL63 S1 gene (encoding aa 21–717) according to the method described by Babcock et al. (2004). All of the NL63 S1-truncated variants used in this study were generated based on this template. The RBD variants were generated by site-directed mutagenesis and/or random mutant library screening assays using a GeneMorph Random Mutagenesis kit (Stratagene) and overlapping PCR strategy. The cDNA inserts encoding the S1, RBD or a null (aa 12–327) fragment of SARS-CoV were amplified based on a codon-optimized S protein sequence of SARS-CoV described previously (Li et al., 2003). For expression and production of soluble S1 or RBD variants, the cDNA inserts were cloned into a mammalian soluble protein expression vector, pSecTag/Hygro (Invitrogen), respectively, thereby producing c-myc- and His-tagged fusion proteins. In addition, some constructs were cloned into a modified pSecTag/Hygro-Ig vector that contains the mouse Ig
-chain leader sequence and human IgG Fc fragment, producing hACE2–Ig, S1–Ig, RBD–Ig or other Ig fusion proteins. All expression plasmids used in this study were confirmed by DNA sequencing.
Expression and purification of proteins.
To make soluble hACE2, S1 or RBD proteins, 293T cells were transfected with the expression plasmids described above using a standard calcium phosphate transfection method. At 16–20 h after transfection, the cells were washed with PBS and cultured in serum-free medium (293 SFM II medium; Invitrogen). The protein-containing supernatants were collected on days 2, 3 and 4 post-transfection and pooled. An aliquot of the pooled protein supernatants was analysed by Western blotting (WB) to confirm the target protein expression. The supernatants were then passed through a 0.45 µm filter (Nalgene) and dialysed with binding buffer prior to affinity purification. The affinity column packed with charged nickel beads (Qiagen) was used to purify the 6xHis-tagged proteins. To purify the Ig fusion proteins (hACE2–Ig, S1–Ig or RBD–Ig), the supernatants were incubated with protein A/G–Sepharose beads at 4 °C for 16 h and then washed with 0.5 M NaCl in PBS. The samples were subsequently eluted with 50 mM sodium citrate/50 mM glycine (pH 2.0), neutralized with NaOH and dialysed against PBS as described previously (Sui et al., 2004). The purified proteins were concentrated with Centricon filters (Amicon) and dialysed against PBS. Protein concentration was measured with a protein assay kit (Bio-Rad) and aliquots of the proteins were stored at –80 °C.
WB.
To detect proteins by WB, purified proteins, cell culture supernatants or cell lysates were resolved by 10 % SDS-PAGE and transferred to a nitrocellulose membrane (Amersham Biosciences). The membrane was blocked for 1 h at room temperature with blocking buffer (5 % skimmed milk, 0.05 % Tween 20 in PBS) and incubated with primary antibody (anti-c-myc antibody at a 1 : 2000 dilution or NL63-positive human serum at a 1 : 500 dilution) overnight at 4 °C, followed by three washes with washing buffer (0.05 % Tween 20 in PBS). The blot was then incubated with secondary antibody (HRP-conjugated anti-mouse IgG or HRP-conjugated anti-human IgG at a 1 : 4000 dilution; Amersham Bioscience) for 1 h at room temperature and washed three times with washing buffer and once with PBS. The protein bands were detected using ECL reagents (Amersham Biosciences). The density of the protein band was quantified using Scion Image (National Institutes of Health, Bethesda, MD, USA).
Immunoprecipitation (IP) assay.
Cell lysate was prepared from hACE2-transfected 293T cells with lysis buffer consisting of 100 mM (NH4)2SO4, 20 mM Tris/HCl (pH 7.5), 10 % glycerol, 1 % Cymal 5 and protease inhibitor cocktail. To detect an S–ACE2 association by IP, purified S1 or RBD proteins (1 µM) were mixed with 200 µg hACE2–C9 cell lysate and 2.5 µl anti-His antibody (Amersham Biosciences). The mixture was incubated at 4 °C overnight, followed by the addition of 30 µl protein A/G beads, and incubation was continued at 4 °C for 2 h. The precipitate was washed three times with Triton X-100 in PBS and resuspended in 40 µl protein loading buffer for detection of proteins by WB. Additionally, we performed IP using soluble hACE2–Ig proteins that were incubated with supernatant containing S1–c-myc–His or RBD–c-myc–His protein and protein A/G beads, followed by the steps described above.
Flow cytometry.
To detect receptor expression and/or the binding interaction between S1 (or RBD) and the receptors on the cell surface, flow cytometry (or FACS) was performed as described previously (Zhang et al., 1996). In brief, the target cells were detached with 5 mM EDTA in PBS and washed twice with immunofluorescence (IF) buffer (1 % fetal bovine serum and 0.02 % NaN3 in PBS). The cells were incubated with anti-c-myc antibody (for the detection of receptor expression) or with S1–Ig or RBD–Ig protein (for the detection of binding between S1 or RBD and the receptor) on ice for 1 h, followed by three washes with IF buffer. They were further incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG or FITC-conjugated anti-human IgG on ice for 30 min. The cells were washed three times with IF buffer, fixed with 1 % formaldehyde and analysed using a Beckman Coulter FC5000 cytofluorometer.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). We then expressed and purified these S1 variants from human 293T cells. We demonstrated by WB that both anti-c-myc antibody (Fig. 2b, top panel) and NL63-positive human serum (Fig. 2b, middle panel) recognized these proteins, suggesting that they folded properly and retained their antigenicity. We also expressed and purified three S1 variants from SARS-CoV: full-length S1 (S12–670), RBD (S318–510) and a null fragment (S12–327), which were detected by anti-c-myc antibody but not the NL63-positive human serum (Fig. 2b, top and bottom panels). To examine S1–hACE2 associations, we performed an IP assay using anti-His antibody, followed by WB using anti-C9 antibody to detect precipitated ACE2 receptors. Fig. 2b (middle panel) shows that S1 and S21–616 of NL63 were able to bind to hACE2, although their binding efficiencies appeared to be much lower than the S1 and RBD of SARS-CoV. In contrast, we did not detect an association between hACE2 and the other S1 variants (S21–516, S21–460, S21–410, S21–360 and S21–260), suggesting that the region spanning aa 516–616 is required for receptor binding, whereas the region at the very end of the C terminus (aa 617–717) is dispensable for receptor binding. These results are consistent with previous findings reported by others (Hofmann et al., 2006).
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). Protein expression of three S1 variants (S311–616, S361–616 and S411–616) was not detected in the supernatants of transfected 293T cells (data not shown), suggesting that the deleted regions could be important for stability, folding and/or secretion of the proteins. In contrast, the other five S1 variants were expressed at high levels in the supernatants of transfected 293T cells. We then purified these S1 variants that were detected by WB using anti-c-myc antibody (Fig. 3b, top panel) and performed IP to detect an S1–hACE2 association. As expected, S1 and RBD of SARS-CoV bound efficiently to hACE2, whilst the null fragment (S12–327) did not (Fig. 3b, bottom panel). As for the NL63 S1 variants, we could detect an association between hACE2 and full-length S1 (S21–717), S210–717, S311–717 and S461–616, respectively, but not between hACE2 and S311–566 or S517–717. In particular, we demonstrated that the S461–616 variant was able to bind to hACE2 much more efficiently than full-length S1 and had a binding efficiency comparable to that of S1 or RBD of SARS-CoV (Fig. 3b, bottom panel).
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, top panel) for reciprocal IP assays in which either the S proteins (Fig. 3c, middle panel) or the hACE2 receptors (Fig. 3c, bottom panel) were precipitated and detected. In both IP assays, S476–616 bound to hACE2 as efficiently as S461–616, whilst S461–601 did not bind. We also measured the S1–hACE2 association by flow cytometry using similar amounts of S1–Ig proteins of three NL63 S variants (S1, the minimal RBD S476–616 and the null fragment S21–460) and three SARS-CoV S constructs (S1, RBD and null) (Fig. 3d and e). The quantitative data presented in Fig. 3(e) showed dramatic differences in binding efficiency between NL63 RBD–hACE2 and S1–hACE2. However, we did not observe such differences among NL63 RBD–hACE2, SARS-CoV RBD–hACE2 and SARS-CoV S1–hACE2. Therefore, the flow cytometry data were consistent with the IP data. Collectively, our study defined the fragment S476–616 as the minimal RBD of the S protein of NL63.
Identifying critical residues for RBD–hACE2 binding
A previous study on the RBD of SARS-CoV suggested that cysteine and some negatively charged residues appear to be important for receptor association (Wong et al., 2004). To identify the critical residues in NL63 RBD for hACE2 binding, we first generated 20 RBD variants (Table 1) in which a single cysteine (C) or a negatively or positively charged residue was replaced by alanine (A). These mutants comprised six cysteine (C497A, C500A, C516A, C550A, C567A and C577A), two aspartic acid (D484A and D538A), three arginine (R518A, R525A and R530A), three glutamic acid (E572A, E582A and E602A) and six lysine (K501A, K532A, K546A, K556A, K562A and K564A) point mutations. The expression plasmids encoding these RBD variants were transiently transfected into 293T cells and protein expression levels in the cell culture supernatants were detected by WB. Six of the mutants (C516A, C550A, C567A, C577A, R525A and K556A) either did not express or had very low expression levels in the supernatants of transfected 293T cells, whereas the remaining 14 RBD mutants exhibited relatively high expression levels (Table 1 and Fig. 4a, top panel). Subsequently, similar amounts of protein were used for IP to detect RBD–hACE2 associations. As illustrated in the bottom panel of Fig. 4(a), alanine substitution of seven residues (C497, C500, D538, R518, R530, E582 and K501), respectively, could either abolish or dramatically decrease the receptor binding efficiency.
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, top panels). Similar amounts of protein were used for the detection of RBD–hACE2 association by IP assay (Fig. 4b, bottom panels). Eleven RBD mutants (1-5, 1-22, 1-31, 1-36, 1-74, 2-10, 3-2, 3-7, 3-42, 3-46 and 3-55) were found to have undetectable or dramatically decreased binding activities to hACE2 receptor, whilst the other 14 mutants (1-10, 1-11, 1-19, 1-23, 1-30, 1-38, 1-66, 1-72, 1-77, 1-80, 2-1, 2-20, 3-15 and 3-40) could bind efficiently to hACE2. Sequence analysis revealed that three mutants carried a single amino acid substitution (1-22 with V531E, 3-42 with V499I and 3-46 with G534A), whereas the other eight mutants harboured two or more amino acid substitutions in RBD (Table 2). To pinpoint further the role of individual amino acid substitutions in these mutants, we generated a series of new variants with only one amino acid substitution in each construct. Consequently, we identified an additional five residues (Y498, G537, S540, W585 and T591) that appeared to be critical for RBD–hACE2 binding (bottom panels of Fig. 4c and Table 2). The protein expression levels of two mutants (S523P and V515E) were undetectable in the supernatants and thus were not included in the IP assay (data not shown). Furthermore, we noticed that substitutions of some residues with different amino acids, such as E582A and E582K; V499I and V499L; K501A, K501M and K501N; T591A and T591I; and Y498H and Y498F exhibited similar effects on receptor binding (Tables 1 and 2 and Fig. 4), reinforcing the importance of these residues. Taken together, we identified 15 residues in the NL63 RBD (C497, Y498, V499, C500, K501, R518, R530, V531, G534, G537, D538, S540, E582, W585 and T591) that appear to be important for the RBD–hACE2 association.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Godet et al., 1994; Laude et al., 1995). In contrast, one group II coronavirus, MHV, has the RBD located at the N terminus of its S1 subunit, and the SARS-CoV RBD is located near the central region of the S1 domain (Kubo et al., 1994; Wong et al., 2004) (Fig. 5). Therefore, the relative location of RBD in S1 of different coronaviruses seemingly correlates with their evolutionary and serological relationships. Whether or not this represents a conservative trait in the evolution of coronaviruses remains to be addressed.
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). Whilst two previous studies of SARS-CoV have also suggested that RBD could bind to hACE2 more efficiently than full-length S1, they only found differences of less than twofold (Babcock et al., 2004; Wong et al., 2004). In contrast, we observed that hACE2 binding efficiency by the NL63 RBD was similar to that of SARS-CoV S1 or RBD, but was about six times higher than that of NL63 S1 (based on the mean fluorescent value in flow cytometry assays, Fig. 3e). This sharp difference in receptor-binding efficiency might have important implications for the pathogenesis, tropism and transmission of NL63 and SARS-CoV. It has been shown that hACE2 expression in cell lines correlates with susceptibility to SARS-CoV S-driven infection (Esposito et al., 2006; Hofmann et al., 2004). We have previously demonstrated that the receptor-binding affinity of the S1 protein of GD03T0013, a SARS-CoV strain associated with mild disease, was significantly lower than the S1 of Tor2, a highly pathogenic strain isolated from a patient who died of SARS (Li et al., 2005b). Additional studies of the SARS-CoV-infected mouse model also strongly suggest that disease severity is correlated with S protein-mediated downregulation of ACE2 in vivo (Imai et al., 2005; Kuba et al., 2005). Therefore, the weak association between hACE2 and NL63 S1 might contribute to mild clinical symptoms in NL63-infected patients. Furthermore, the strong association of NL63 RBD with hACE2 suggests that the folding structure of RBD might be different from that of S1. It may also indicate that some efficient binding sites may be buried inside the structure of S1. This implies that NL63 possesses the potential to use hACE2 as efficiently as SARS-CoV. Mutations accumulate over time, some of which might alter the protein folding and expose the efficient binding sites on the surface. This may lead to the emergence of new NL63 strains that would be highly pathogenic to humans.
We have also conducted a series of mutagenesis studies on the NL63 RBD and identified 15 residues that appear to be critical for RBD–hACE2 interaction. We found that all of the six cysteine residues within the NL63 RBD are important for protein expression and/or hACE2 association (Table 1 and Fig. 4a), which is similar to the roles of cysteine residues in the RBD of SARS-CoV (Wong et al., 2004). This previous study by Wong et al. (2004) identified two acidic residues (E452 and D454) that appeared to be important for the interaction between hACE2 and the RBD of SARS-CoV. Interestingly, two acidic residues (D538 and E582) in the NL63 RBD that appeared to be critical for receptor binding were also identified in our studies (Table 1 and Fig. 4a). In addition, five basic residues in the NL63 RBD (R518, R525, R530, K501 and K556) were identified to be critical for RBD protein expression and/or hACE2 binding (Table 1 and Fig. 4a). Furthermore, we identified seven critical residues from the screening assay of the random mutant library of NL63 RBD: two valines (V499 and V531), two glycines (G534 and G537), one tyrosine (Y498), one serine (S540) and one trytophan (W585). Our studies also suggest that, in addition to site-directed mutagenesis, screening of a PCR-generated random mutant library may become a powerful strategy for the identification of critical residues that are important for RBD–receptor binding or other protein–protein interactions.
When we tried to understand how these critical residues may interact with the hACE2 receptor, it was interesting to find that, with the exception of R518, the other 14 critical residues clustered into three separated regions (designated RI, RII and RIII) in the RBD. As shown in Fig. 5, the RI region contains residues C497, Y498, V499, C500 and K501, and RII contains residues R530, V531, G534, G537, D538 and S540, whilst RIII contains residues E582, W585 and T591. These regions may represent three receptor-binding sites inside the NL63 RBD. Previous studies of the SARS-CoV RBD–hACE2 interaction and the crystal structure of the SARS-CoV RBD–hACE2 complex have identified a panel of residues in the RBD region that are critical for the RBD–hACE2 association, and demonstrated that some of these residues may play important roles in the cross-species transmission and pathogenesis of SARS-CoV (Li et al., 2005a, b). Therefore, the current study and further characterization of NL63 RBD–hACE2 interactions, including examination of the crystal structure of the NL63 RBD–hACE2 complex may not only help our understanding of the molecular interactions between the S protein of NL63 and the hACE2 receptor, but may also advance our understanding of the pathogenesis of NL63 and SARS-CoV.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 22 July 2007;
accepted 17 December 2007.
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) indicate the RBD variants that had no or dramatically decreased binding activity, respectively, to hACE2 receptor. In addition, the asterisk (*) indicates the RBD variants bearing only one amino acid substitution, whereas the arrow (