Species‐specific ACE2 determinants of differential S‐protein association. (A) HEK293T cells transfected with plasmid encoding human or palm‐civet ACE2, with human ACE2 bearing the indicated palm‐civet residues, or with vector alone were analyzed by flow cytometry using S1‐Ig variants of the TOR2, GD, and SZ3 isolates. Error bars indicate the range of two or more experiments. (B) HEK293T cells transfected with plasmid encoding the ACE2 variants used in (A) were incubated with HIV‐1‐luciferase virus pseudotyped with the S proteins of TOR2, GD, or SZ3 viruses. Infection was assayed as in Figure 6C. (C) HEK293T cells transfected with plasmid encoding human ACE2, human ACE2 variants bearing the indicated palm‐civet residues, palm‐civet ACE2, or the palm‐civet ACE2 variant D354G were metabolically labeled and lysed. Cell lysates were immunoprecipitated with an anti‐tag antibody recognizing an amino‐terminal tag on these ACE2 variants (α‐myc), or with RBD‐Ig of TOR2 or SZ3, or with their variants with the indicated alterations of residues 479 and 487. The experiment is representative of at least two with similar results. (D) Amino‐acid content of critical regions of ACE2 from human, palm civet, and rat. Orange indicates human‐ACE2 residues whose alteration interferes with TOR2 S‐protein association. Red indicates rat‐ACE2 residues whose alteration to their human counterparts converts rat ACE2 to an efficient SARS‐CoV receptor. Yellow indicates residues of palm‐civet ACE2 that accommodate S‐protein lysine 479 of SARS‐CoV isolated from palm civets. Cyan indicates additional residues of palm‐civet ACE2 that, when introduced into human ACE2, result in more efficient association with all S proteins assayed. This effect may be due to the loss of glycosylation at asparagine 90 of human ACE2, shown in green. (E) Ribbon diagram of human ACE2 from the top of the protein. Brown on the ribbon identifies regions shown in (D). Residues highlighted in (D) are shown in the same colors. (F) Surface diagram of human ACE2, from the same orientation as in (E), and colored consistently with (D, E).
Binding of RBD chimeras to ACE2 chimeras
Figure 7C compares the ability of eight RBD variants to bind to human and civet ACE2, as well as to the chimeric molecules assayed in Figure 7A and to a point‐mutation variant of palm‐civet ACE2, in which aspartic acid 354 was altered to a glycine present in human ACE2. RBD variants were generated from that of TOR2 (left panels) or SZ3 (right panels), and altered at positions 479, 487, or both, as indicated. The panels of Figure 7C permit several conclusions. First, the rough equivalence between the left and right panels indicates that, as implied by Figure 6B and C, S‐protein residues 479 and 487 account for most of the differences between TOR2 and SZ3 RBD. Second, no consistent differences were observed between palm‐civet ACE2 and its variant with glycine at residue 354, indicating little or no contribution of this residue to S‐protein association. Third, consistent with infection data in Figure 6C, the presence of threonine at residue 487 enhanced the affinity of most RBDs for civet and human ACE2, and for variants of these receptors. (Compare, for example, K479/S487 RBD variants with K479/T487 variants for their ability to precipitate each ACE2 variant.) Fourth, and in contrast, substitution of lysine 479 for asparagine in most contexts increased the ability of each RBD variant to associate with human, but not with palm‐civet, ACE2. (Compare the binding of N479/T487 RBD with that of K479/T487 variants for binding to human ACE2 (lane 1) and palm‐civet ACE2 (lane 6); likewise for N479/S487 and K479/S487 variants.) This enhancement was also observed for ACE2 chimeras containing human α‐helix 1 residues (lanes 1 and 2), but not those of palm civet (lanes 3, 5, and 6), whereas residues 90–93 did not determine sensitivity to RBD residue 479. Fifth, consistent with infection data in Figure 7B and Supplementary Figure 3, all RBDs bound ACE2 variants bearing residues 90–93 of palm‐civet ACE2 substantially more efficiently than they bound equivalent variants with human ACE2 residues at these positions (compare lanes 1 and 2 and lanes 3 and 4 in each panel). Thus, the data of Figure 7C indicate that a lysine at S‐protein residue 479 interferes with RBD association with human, but not palm‐civet, ACE2. Supplementary Figure 4 shows data consistent with a steric interaction between lysine 31 of human ACE2 and lysine 479 of the SZ3 S protein. Our data also show that alteration of S‐protein serine 487 to threonine increases RBD affinity for both human and civet ACE2. Finally, they suggest that no S protein studied has fully adapted to human ACE2 residues 90–93, consistent with a recent zoonotic transmission of the virus.
Mapping determinants to the ACE2 surface
Figure 7D–F summarizes our findings. Figure 7D shows amino‐acid sequences of regions critical to S‐protein association for palm‐civet, rat, and human ACE2. Figure 7E shows ACE2 oriented with the C‐terminal membrane‐associated collectrin domain facing away from the viewer. Red indicates residues whose alteration transformed rat ACE2 to an efficient SARS‐CoV receptor. Orange indicates additional residues common to rat and human ACE2 whose alteration also interferes with S‐protein association. Yellow indicates residues along the α‐helix 1 ridge that are unique to palm‐civet ACE2, and which permit efficient association with RBD isolates from palm civet and likely interact with lysine 479 of the palm‐civet RBD. K31 of human ACE2, which interferes with palm‐civet RBD lysine 479, is labeled with white text in Figure 7E. Four residues at the beginning of α‐helix 4 that permit more efficient binding and infection by all S proteins assayed are shown in cyan, and the glycosylation site in this region, present in human but not palm‐civet ACE2, is shown in green.
ACE2 is a functional receptor for SARS‐CoV, and is likely to play a critical role in viral replication in an infected host (Li et al, 2003; Hamming et al, 2004; Nie et al, 2004). Here we describe the S‐protein‐binding domain of ACE2. In particular, residues along the first α‐helix, and lysine 353 and proximal residues at the N‐terminus of β‐sheet 5, participate in S‐protein binding and in infection. By altering histidine 353 in rat ACE2 and modifying a glyosylation site that may alter the shape of α‐helix 1, we converted rat ACE2 to an efficient receptor for SARS‐CoV. This S‐protein‐binding region of ACE2 remains intact in the presence of an inhibitor that dramatically alters the overall conformation of ACE2 (Dales et al, 2002; Towler et al, 2004), consistent with the inability of this inhibitor to block infection, and with the inability of the S protein to modulate ACE2 activity.
Although there can be multiple constraints on interspecies transmission of viruses (Webby et al, 2004), S‐protein alterations are sufficient to extend or alter the host range of a number of coronaviruses (Kuo et al, 2000; Casais et al, 2003; Haijema et al, 2003; Schickli et al, 2004). We have shown that entry is the primary barrier to SARS‐CoV infection of murine cells (Li et al, 2004). These observations suggest that S‐protein changes may be critical to or sufficient for the adaptation of SARS‐CoV to human cells. Accordingly, we compared the S proteins derived from the 2002–2003 outbreak (TOR2), from the less severe 2003–2004 outbreak (GD), and from apparently healthy palm civets (SZ3) (Guan et al, 2003; He et al, 2004). Strikingly, the receptor‐binding regions of each of these S proteins bound palm‐civet ACE2 efficiently, but only that from the 2002–2003 outbreak bound human ACE2 with comparable efficiency. These data are consistent with the absence of human‐to‐human transmission during the 2003–2004 outbreak, and with recent transmission of SARS‐CoV from palm civets to humans (Guan et al, 2003; Zhong, 2004; Song et al, 2005).
Differences among these S proteins permitted identification of key changes necessary for adaptation to the human receptor. In particular, changes at S‐protein residues 479 and 487 appear to be critical for high‐affinity association with human ACE2. The alteration at 479 to a small, uncharged residue is a consistent property of all described SARS‐CoV obtained from humans, whereas most civet‐derived viruses retain a basic residue at this position (Guan et al, 2003; Marra et al, 2003; Rota et al, 2003; He et al, 2004; Zhang et al, 2004; Song et al, 2005). Our data indicate that residue 479 interacts with residues along a ridge formed by ACE2 α‐helix 1, and in particular with lysine 31, which is present in human but not palm‐civet ACE2. Alteration of S‐protein residue 479 to the asparagine found in virus isolated from humans appears to accommodate this human ACE2 lysine.
Differences at S‐protein residue 487 are also of interest. A threonine at position 487 is absolutely conserved in all of the more than 100 S proteins isolated during the severe 2002–2003 outbreak. In contrast, the S proteins of viruses isolated during the 2003–2004 outbreak, and all 14 animal SARS‐CoV isolated, had a serine at this position (Guan et al, 2003; He et al, 2004; Zhang et al, 2004; Song et al, 2005). A threonine at position 487 increased affinity of most RBDs assayed for both human and palm‐civet ACE2, and all chimeras thereof, and substantially enhanced the efficiency with which palm‐civet‐derived S protein infected cells expressing human ACE2. These observations indicate that the additional methyl group of threonine 487 participates in the efficiency of infection of human and non‐human cells.
S‐protein alterations at residues 479 and 487 are important for high‐affinity association with human ACE2, and for efficient infection of cells expressing this receptor. Knowledge of these residues may be useful in assessing the risk posed by any new SARS‐CoV outbreak. Our data also show that, even with these and other changes outside the RBD, SARS‐CoV is imperfectly adapted to its human receptor. In particular, introduction of residues 90–93 of civet ACE2 into the human receptor increased binding of, and infection mediated by, all S proteins assayed. This effect may be due to removal of a glycosylation site at position 90 to which no SARS‐CoV has fully adapted. This observation raises the possibility that soluble human ACE2 lacking this glycosylation would more effectively inhibit SARS‐CoV replication than wild‐type human ACE2.
We have previously shown that replication of SARS‐CoV in a murine cell line is limited by the low affinity of the S protein for murine ACE2 (Li et al, 2004). Moreover, the affinity of S protein for the receptors of rats, mice, and humans correlates with the ability of virus to replicate in these animals. The lower affinity of palm‐civet‐derived S protein for the palm‐civet receptor is consistent with this pattern in that no overt disease was manifest in animals from which this virus was isolated (Guan et al, 2003), but disease was observed in palm civets challenged with isolates obtained during the 2002–2003 outbreak (Wu et al, 2005). Together, these observations suggest that the affinity of S protein for ACE2 is an important determinant in the overall rate of viral replication and in the severity of disease. If so, adaptations within the S protein that are critical for high‐affinity association with human ACE2 may have contributed to the unusual severity of SARS.
Construction of S‐protein and ACE2 variants
Plasmid encoding a codon‐optimized form of the SARS‐CoV S protein of the TOR2 isolate (accession number AY274119) has been previously described (Li et al, 2003; Moore et al, 2004). Plasmids encoding the corresponding S proteins of the GD03T0013 isolate, isolated during the mild 2003–2004 outbreak (accession number AY525636; denoted GD herein), and the SZ3 isolate, isolated from palm civets (accession number AY304486), were generated de novo by recursive PCR. Plasmids encoding the S1 domain (residues 12–672) and the RBD (residues 318–510) of the TOR2 S protein, fused to the Fc domain of human IgG1 (S1‐Ig and RBD‐Ig, respectively), have been previously described (Li et al, 2003; Wong et al, 2004). Corresponding S1‐Ig and RBD‐Ig variants of the GD and SZ3 isolates and variant ACE2 molecules were generated by mutagenesis using the QuikChange method (Invitrogen). Human, rat, and palm‐civet ACE2 molecules were amplified from cDNA of corresponding tissue by PCR, and cloned into a vector encoding previously described amino‐ and carboxy‐terminal tags (Li et al, 2004).
Association of S1‐Ig or RBD‐Ig with ACE2 variants was determined by flow cytometry and by immunoprecipitation. Flow cytometry using ACE2‐expressing cells has been previously described (Li et al, 2003; Wong et al, 2004). Briefly, HEK293T cells were transfected with a plasmid encoding ACE2 variants, or with vector alone. At 2 days post‐transfection, cells were detached in PBS/5 mM EDTA and washed with PBS/0.5% BSA. S1‐Ig or RBD‐Ig, or variants thereof, or the anti‐tag antibody 9E10, were added to 106 cells, and the mixture was incubated on ice for 1 h. Cells were washed three times with PBS/0.5% BSA, and then incubated for 30 min on ice with anti‐human IgG FITC conjugate (Sigma). Cells were again washed with PBS/0.5% BSA, and analyzed.
Immunoprecipitations were performed as previously described (Li et al, 2003; Wong et al, 2004). Briefly, HEK293T cells were transfected with plasmid encoding ACE2 variants and radiolabeled with [35S]cysteine and [35S]methionine. After 2 days, transfected cells were harvested and lysed in PBS buffer containing 1% CHAPSO. Cell lysates were incubated with Protein A–Sepharose beads together with 2 μg S1‐Ig or RBD‐Ig variants, or with the antibodies 1D4, recognizing a carboxy‐terminal C9 tag on ACE2, or 9E10, recognizing an amino‐terminal myc tag. Protein A–Sepharose beads were washed three times in PBS/0.5% CHAPSO, and analyzed by SDS–PAGE. Immunoprecipitated ACE2 variants were quantified by phosphorimaging.
TOR2 RBD‐Ig variants were also assayed by surface plasmon resonance using a Biacore 3000. A 200 nM portion of purified RBD‐Ig of TOR2 variants was bound to an anti‐human antibody (Sigma I‐2136) immobilized on a CM5 sensor chip. Soluble human ACE2 in HBS‐EP buffer (Biacore) was introduced at a flow rate of 20 μl/min at concentrations of 700, 200, 40, 8, 1.6, and 0 nM. Kinetic parameters were determined with BIA‐EVALUATION software (Biacore).
Infection with S‐protein‐pseudotyped retrovirus
MLV expressing GFP and pseudotyped with SARS‐CoV S‐protein variants has been previously described (Moore et al, 2004). Briefly, MLV virions were generated by cotransfecting plasmid encoding MLV gag and pol genes, the pQCXIX vector (BD Sciences) expressing GFP, and plasmid encoding S‐protein variants. At 48 h post‐transfection, cell supernatants were normalized for reverse transcriptase activity and incubated with HEK293T cells transfected with ACE2 variants. At 48 h postincubation, GFP fluorescence of infected cells was measured by flow cytometry. In some cases, cells were preincubated for 1 h with the ACE2 inhibitor MLN‐4760 or with NH4Cl before infection, and equivalent concentrations were maintained during infection.
Infection was also assayed with a lentivirus expressing a luciferase reporter gene and pseudotyped with S‐protein variants, as previously described (Sui et al, 2005). Briefly, 293T cells were cotransfected with plasmid encoding S‐protein variants, a plasmid (pCMVΔR8.2) encoding HIV‐1 Gag‐Pol, and a plasmid (pHIV‐Luc) encoding the firefly luciferase reporter gene under control of the HIV‐1 long terminal repeat. At 2 days post‐transfection, viral supernatants were harvested and 3 ìl of S‐protein‐pseudotyped virus was used for infection of 6000 ACE2‐expressing 293T cells in a 96‐well plate. Infection efficiency was quantitated by measuring the luciferase activity in the target cells with an EG&G Berthold Microplate Luminometer LB 96V.
The enzymatic activity of ACE2 was assayed using a fluorogenic substrate, 7‐methoxycoumarin‐YVADAPK(2,4‐dinitrophenyl)‐OH (R&D Systems). Cleavage of this peptide by ACE2 removes the 2,4‐dinitrophenyl moiety that quenches the fluorescence of the 7‐methoxycoumarin moiety. A 1 μg portion of a soluble form of ACE2 (Moore et al, 2004) was incubated in 100 mM Tris buffer with varying concentrations of the ACE2 inhibitor MLN‐4760 (Dales et al, 2002). Fluorescence was monitored at 5 min intervals using an excitation wavelength of 330 nm and emission wavelength of 450 nm.