Antigenic modules in the N-terminal S1 region of the transmissible gastroenteritis virus spike...

Antigenic modules in the N-terminal S1 region ofthe transmissible gastroenteritis virus spike protein

Juan Reguera,13 Desiderio Ordono,1 Cesar Santiago,1 Luis Enjuanes2

and Jose M. Casasnovas1

Correspondence

Jose M. Casasnovas

[email protected]

Received 28 September 2010

Accepted 5 January 2011

1Department of Macromolecule Structure, Centro Nacional de Biotecnologia, CSIC, CampusUniversidad Autonoma, Darwin 3, 28049 Madrid, Spain

2Department of Molecular Biology, Centro Nacional de Biotecnologia, CSIC, Campus UniversidadAutonoma, Darwin 3, 28049 Madrid, Spain

The N-terminal S1 region of the transmissible gastroenteritis virus (TGEV) spike (S) glycoprotein

contains four antigenic sites (C, B, D and A, from the N- to the C-terminal end) and is engaged in

host-cell receptor recognition. The most N-terminal portion of the S1 region, which comprises

antigenic sites C and B, is needed for the enteric tropism of TGEV, whereas the major antigenic

site A at the C-terminal moiety is required for both respiratory and enteric cell tropism, and is

engaged in recognition of the aminopeptidase N (APN) receptor. This study determined the

kinetics for binding of a soluble S1 protein to the APN protein. Moreover, the S1 region of the

TGEV S protein was dissected, with the aim of identifying discrete modules displaying unique

antigenic sites and receptor-binding functions. Following protease treatments and mammalian cell

expression methods, four modules or domains (D1–D4) were defined at the S1 region. Papain

treatment identified an N-terminal domain (D1) resistant to proteolysis, whereas receptor binding

defined a soluble and functional APN receptor-binding domain (D3). This domain was recognized

by neutralizing antibodies belonging to the antigenic site A and therefore could be used as an

immunogen for the prevention of viral infection. The organization of the four modules in the S1

region of the TGEV S glycoprotein is discussed.

INTRODUCTION

Coronaviruses (CoVs) are enveloped, positive-sense,ssRNA viruses (de Groot et al., 2008; Enjuanes et al.,2008; Masters, 2006) involved in respiratory, enteric,hepatic and neuronal infectious diseases in animals andhumans that often lead to important economic losses(Perlman, 1998; Weiss & Navas-Martin, 2005). Since theoutbreak of severe acute respiratory syndrome (SARS) in2002, the interest in CoVs has increased dramatically aspotential generators of new, serious zoonotic infectiousdiseases (Drosten et al., 2003; Holmes, 2005; Lau, 2004;Rota et al., 2003), making evident the necessity for a deeperunderstanding of the determinants responsible for CoVreceptor recognition and tropism.

The CoV spike (S) glycoprotein is anchored to theenvelope and is specialized both in receptor recognitionand in virus–cell fusion (Bosch et al., 2003; Gallagher &Buchmeier, 2001). Receptor recognition appears to bespecies specific, and there is significant variability in

receptor usage among CoVs. Several cell-surface glycopro-teins have been identified as CoV receptors: members ofthe genus Alphacoronavirus such as transmissible gastro-enteritis virus (TGEV) and human (HCoV-229E), canineand feline CoV use the aminopeptidase N (APN) receptor(Delmas et al., 1992; Tresnan et al., 1996; Yeager et al.,1992). Members of the genus Betacoronavirus such ashuman SARS-CoV use the human angiotensin-convertingenzyme 2 (ACE2) (Li et al., 2003), whereas murinehepatitis virus (MHV) uses the cell adhesion moleculeCEACAM1a (Yokomori & Lai, 1992). Nevertheless, theexistence of alternative receptors that can confer anextended tropism has been revealed for several CoVs.SARS-CoV can use liver/lymph node-specific intercellularadhesion molecule-3-grabbing non-integrin (L-SIGN) forcell attachment and entry (Jeffers et al., 2004), whereas anMHV virus strain isolated from persistently infected cells(MHV/BHK) enters the cells in a heparan sulfate-dependent manner (de Haan et al., 2005; Miura et al.,2008). It has been suggested that binding of the TGEV Sprotein to sialic acids is responsible for its enteric tropism(Krempl et al., 1997).

The CoV S glycoproteins are type I membrane proteinsthat form trimers that protrude out of the virus envelope

3Present address: European Molecular Biology Laboratory, GrenobleOutstation, Grenoble Cedex 9, France.

Supplementary data are available with the online version of this paper.

Journal of General Virology (2011), 92, 1117–1126 DOI 10.1099/vir.0.027607-0

027607 G 2011 SGM Printed in Great Britain 1117

and give a crown-like appearance to the CoV particles(Beniac et al., 2006; Delmas & Laude, 1990). The trimericspikes have a globular region mostly formed by theN-terminal S1 region, and a protein stalk connectingthe globular region to the membrane, formed by theC-terminal S2 region (Beniac et al., 2006). The S1 regionbears the receptor-binding epitopes (Godet et al., 1994;Wong et al., 2004), whereas S2 is responsible for viral andcell membrane fusion, and adopts a helical conformationcharacteristic of class I fusion proteins (Bosch et al., 2003;Supekar et al., 2004). The CoV S protein is cleaved betweenthe S1 and S2 regions in some CoVs during virus particlematuration (Abraham et al., 1990; Cavanagh et al., 1986; deHaan et al., 2008), but no cleavage was observed inalphacoronaviruses such as TGEV and canine and felineCoVs.

TGEV is a porcine CoV with respiratory and enterictropism, whereas a naturally derived mutant of this virus,porcine respiratory CoV (PRCV) has only respiratorytropism (Laude et al., 1995; Sanchez et al., 1992). The S1region of the PRCV S protein has a 224 aa deletion withrespect to the TGEV glycoprotein at the N terminus,although both proteins are highly homologous and theybind to the porcine APN (pAPN) receptor. It has beensuggested that the N-terminal region of TGEV S proteinconfers enteric tropism (Ballesteros et al., 1997; Callebautet al., 1988; Sanchez et al., 1992), apparently related tosialic acid recognition (Krempl & Herrler, 2001; Kremplet al., 1997). The main antigenic profile of the S protein iswell characterized for TGEV and other porcine CoVs(Correa et al., 1988; Delmas et al., 1990; Sanchez et al.,1990). The TGEV S protein harbours four antigenic sites,named from the N- to the C-terminal end as C, B, D and A,whereas the PRCV S protein lacks the N-terminal C and Bantigenic sites (Callebaut et al., 1988; Sanchez et al., 1990).The position of each antigenic region has been mapped byanalysis of mAb-resistant mutant viruses that eitherovercome antibody neutralization or lack reactivity tospecific mAbs (Correa et al., 1990; Gebauer et al., 1991).Receptor-binding domains (RBDs) and neutralizing anti-genic sites have been characterized for several CoVs(Breslin et al., 2003; Corapi et al., 1995; Delmas et al.,1990; Sanchez et al., 1990; Tsai et al., 2003; van den Brinket al., 2005; Wong et al., 2004; Wu et al., 2009; Zhang et al.,2004). The RBD of porcine CoVs binding to pAPN hasbeen identified as a 150 aa fragment at the C-terminalmoiety of the S1 region and includes the antigenic A site(Godet et al., 1994). These data confirmed the reportedinhibition of TGEV binding to ST cells with site A-specificmAbs (Sune et al., 1990).

The present work shows the dissection of the S1 region ofTGEV by expression of soluble protein length variants,which defined four distinct modules or domains (D1–D4)in the N-terminal S1 region of the TGEV S glycoprotein.Proper folding of the domains and their antigenicproperties were proved with S1-specific mAbs and thepAPN receptor protein. A soluble and isolated domain

(D3) displayed receptor-binding activity similar to the fullS1 region. The kinetics for binding of the S1 protein topAPN were determined.

RESULTS

Dissection of the S1 region of the TGEV Sglycoprotein

Aiming to isolate folding modules displaying uniqueantigenic and functional properties linked to the S1 regionof the TGEV S protein, length variants were designedfollowing the antigenic map described with TGEV escapemutants (Correa et al., 1990; Gebauer et al., 1991; Sanchezet al., 1992) (Fig. 1a). We designed two TGEV-derivedvariants comprising all four antigenic sites: a longer variantcomprising the complete S1 region (S1) and a shorterprotein (S3) with its C-terminal end after the antigenic Asite. Similar variants were also engineered for the sameregion of the PRCV HOL87 strain (S1H and S3H) (Fig. 1a),which has 96 % sequence identity with the TGEV protein(see Supplementary Fig. S1, available in JGV Online). TGEVvariants S4 and S5 had sequential deletion of antigenic sitesA and D, respectively (Fig. 1a). An S4 protein derived fromthe PRCV HOL87 strain (S4H) and bearing only antigenicsite D was also engineered (Fig. 1a). A short proteinfragment including the RBD region and having justantigenic site A (SA) was produced (Fig. 1a).

Proteins were engineered fused to the Fc region of IgG1(bivalent proteins) and to Flag or haemagglutinin (HA)epitopes (monovalent proteins) at their C-terminal ends,and were produced in 293T and Chinese hamster ovary(CHO) cells (see Methods). We monitored expression formost of the designed protein variants in the supernatant oftransfected cells from which they were purified (Fig. 1b).Production of Fc fused to S4 (S4–Fc), S5 (S5–Fc) and SA(SA–Fc) was significantly higher than that of the HA- orFlag-tagged protein variants (not shown), but the longestvariants (1 and 3) were not expressed fused to Fc. Inaddition to the proteins presented in Fig. 1(b), the S3H andS4H proteins derived from PRCV HOL87 strain were alsosecreted into the supernatant of transfected cells (notshown). Therefore, soluble protein expression allowed usto produce full-length protein and fragments of the S1moiety of TGEV and PRCV containing unique antigenicsites.

An additional approach for the identification of proteindomains was carried out following protease treatments.The S1 variant prepared in CHO Lec cells was treated withtrypsin and papain proteases for the identification offragments resistant to proteolysis. A papain-resistantfragment of about 35 kDa (S35) was seen followingelectrophoresis of the S1 protein treated with a highpapain concentration (Fig. 2a), and a longer intermediateprotein (S60, 60 kDa) was observed at a low proteaseconcentration. Mass spectrometry determined that the

J. Reguera and others

1118 Journal of General Virology 92

mean mass of the papain-resistant fragment was 35 421 Da.The same fragments were observed following treatment ofthe TGEV S3 variant, but the papain-resistant-fragmentwas not seen with the S1H protein (not shown), whichlacks the N-terminal 224 aa of S1 (Fig. 1a); therefore, it

must include the N-terminal region. Sequencing of S1 andthe papain-resistant S35 and S60 fragments confirmed thatthey had the same N terminus (DNFP amino acidsequence). Therefore, they must have been derived fromproteolysis of the C-terminal region of the S1 protein.Based on the S60 size, the cleavage site that leads to thisintermediate fragment should be on the N-terminal side ofantigenic site A. Mass spectrometry of the S35 proteintreated overnight at 30 uC with N-glycosidase F (PNGase F;Biolabs), which hydrolyses N-linked glycans (not shown),gave a mass of 27 231 Da, suggesting that, most probably,its C-terminal residue is Ala240. The expected mass for thefragment comprising residues Asp1–Ala240 was 27 228 Da.The mass difference between the PNGase F-treated anduntreated protein (8190 Da) was also consistent with thepresence of six N-linked glycosylation sites with a mass ofabout 1343 Da (see Supplementary Fig. S1), attached toglycoproteins produced in CHO Lec cells (Stanley, 1989).

Circular dichroism (CD) of the purified S35 fragment gavean approximate secondary structure composition of 18 %a-helices, 30 % b-sheets and about 20 % b-turns (seeSupplementary Fig. S2, available in JGV Online, andMethods). These data, together with its protease resistanceproperties and its absence in homologous PRCV, indicated

Fig. 1. Design and production of soluble CoV S proteins. (a)Designed S1 length variants derived from the enteric TGEV PUR-MAD and respiratory PRCV HOL87 strains. The open rectanglerepresents the S1 region, with the approximate location of the C,B, D and A antigenic sites indicated. Antibodies used in this studyspecific for each site are indicated above. N-Linked glycosylationsites are indicated with an upside-down ‘Y’. The S2 region at the Cterminus of S1 is not shown. Solid lines show the soluble lengthvariants designed in this study, with the name on the left and the C-terminal residue of the mature proteins on the right. They are 16residues shorter than the immature S protein with the signalsequence. (b) 10 % SDS-PAGE under reducing conditions of thepartially purified protein variants produced in CHO Lec cells,except for the S4–Fc fusion protein, which was prepared in 293Tcells (see Methods). Coomassie blue staining is shown for the S1,S3, S1H, S4 and SA proteins, and an immunoblot (HA mAb) forthe HA-tagged S5 variant. Electrophoresis of the S4–Fc proteinuntreated (”) or treated (+) with thrombin for release of the Fcportion is shown. The fusion protein, S4 and Fc fragments andcontaminant immunoglobulins (Ig) from the serum are labelled. Thebroader band of the isolated S4 fragment reflects high glycosyla-tion heterogeneity because of its production in 293T cells. Thesizes (kDa) and migration of the molecular mass markers areindicated.

Fig. 2. Protease-resistant fragments in the TGEV S1 protein. (a)10 % SDS-PAGE of TGEV S1 protein incubated for 16 h at 37 6Cin the absence (”) or presence of papain at several enzyme : pro-tein ratios (w/w): 1 : 500 (1), 1 : 1000 (2), 1 : 2000 (3) and1 : 10 000 (4). The size (kDa) and migration of the molecular massmarkers are indicated. (b) Scheme showing the S1 fragmentsresulting from papain digestion shown in (a), with approximatesizes of 60 and 35 kDa. The arrows indicate the location of thecleavage sites. The length of the S35 fragment was determined bymass spectrometry to be 240 aa.

Domains in the TGEV spike protein

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that the N-terminal portion must be an independentfolding module in the TGEV S protein.

Folding of S1 length variants proved withconformational antibodies

We used a panel of mAbs (Sune et al., 1990) specific for thefour antigenic sites defined in the S1 region of TGEV andPRCV to analyse folding of the soluble protein variants(Fig. 1a). ELISAs were performed with purified S proteinsand the mAbs 6A.A6 (C site), 1D.B12 (B site), 1D.G3 (Dsite) and 6A.C3 (A site) (Fig. 3a). All mAbs recognized theTGEV S1 protein, which contains all four antigenic sites(Fig. 1a), whereas mAbs for the antigenic C and B sitesmapping to the N-terminal region failed to bind to thePRCV S1H protein. A mAb-binding profile similar to that ofthe S1 and S1H proteins was observed with the shorter S3and S3H variants, respectively (not shown). Furthermore,the mAbs recognizing conformational epitopes (1D.B12,1D.G3 and 6A.C3) (Correa et al., 1990; Gebauer et al., 1991;Sanchez et al., 1992) revealed that the antigenic B, D and Aregions were properly folded.

Antibody binding to the S4–Fc and S5–Fc proteins wasconsistent with the location of the antigenic sites (Fig. 3a andFig. 1), whereas S4H–Fc was just recognized by the site DmAb 1D.G3 (not shown). Unexpectedly, the S35 and S5proteins lacking the Fc region were not recognized by mAb1D.B12 (Fig. 3a) or the site B mAbs 1B.H11 and 8F.B3 (see

Supplementary Fig. S3, available in JGV Online), althoughsome binding to the S5 protein was recorded with the lattertwo mAbs. These results indicated a certain complexity forantigenic site B, as its recovery required the inclusion of anextended polypeptide chain at its C-terminal region, such ashas been reported for protein members of the immuno-globulin superfamily with certain mAbs (Casasnovas et al.,1998). Recognition of the SA variant by mAbs 6A.C3(Fig. 3a) and 1A.F10 (not shown) indicated that this virusprotein fragment comprising antigenic site A must be foldedcorrectly.

We observed that mAb 1D.G3 bound to the soluble S1H,but did not bind to PRCV particles (Fig. 3a, b) (Gebaueret al., 1991), indicating that the 1D.G3 epitope must behidden in the trimeric spike at the PRCV envelope.

Binding of S1 protein variants to soluble andmembrane-bound pAPN

Binding of the soluble pAPN receptor to plastic-boundTGEV S length variants was determined for a range ofprotein concentrations (Fig. 4a). The S1, S1H and SAproteins comprising the antigenic A site were recognized bythe soluble pAPN protein, whereas the S5 protein was not(Fig. 4a). The binding difference observed between the S1and S1H proteins at intermediate concentrations could berelated to differences in the accessibility of the receptor-binding region in the plastic-bound proteins, but pAPNbinding to both proteins at the highest concentration wassimilar. Binding of soluble pAPN to the S1 protein wasblocked specifically by the site A mAb 6A.C3 (Fig. 4b). Asimilar binding activity of the SA protein and thoseproteins including the complete S1 region (S1 and S1H)indicated that it should include all the essential epitopesrequired for receptor recognition.

The involvement of S protein domains in receptorrecognition was analysed further using permissive swinetesticle (ST) and baby hamster kidney (BHK) cells with(BHK-pAPN cells) or without the pAPN protein on the cellsurface. The SA–Fc and S5–Fc proteins were used for cell-binding analysis by flow cytometry (Fig. 4c). We observedspecific binding of the SA–Fc protein to the ST and BHKcells expressing pAPN, whereas the S5–Fc protein did notbind to any of the cell lines.

Kinetics of soluble S1 binding to the pAPNreceptor protein

Surface plasmon resonance was applied to characterizefurther S protein binding to the pAPN receptor protein(Fig. 5). About 800 resonance units (RU) of purified pAPNwith an HA epitope at the N-terminal end were capturedfirst onto a sensor chip with an immobilized HA-specificmAb (see Methods). The TGEV S1 protein was injected ontosurfaces with or without pAPN, and the specific increase inthe response related to S1 protein binding was monitoredfor surfaces on which pAPN was captured (Fig. 5).

Fig. 3. Antibody binding to soluble S1 length variants and virusparticles. Binding of the mAbs shown in the figure to the indicatedplastic-bound proteins (a) or virus particles (b) was carried out asdescribed in Methods. mAb binding to each sample was normal-ized to that determined for the S1 protein or the TGEV particles,which bear all the antigenic sites (Fig. 1a). Binding to PRCVpartides heated at 55 6C for 30 min is shown in (b). Means±SD

from three independent measurements are shown.



The soluble S1 protein was injected at concentrationsranging from 1 to 6 mM, which gave binding responses of50 to ~300 RU (Fig. 5, inset). The sensorgrams did notplateau, indicating that no steady-state binding wasreached at the flows used. The binding kinetics were

determined from analysis of the association and dissoci-ation phases (Table 1 and Methods). The associationkinetic rate (kass) was relatively low for protein–proteininteractions, similar to that described for binding of thepoliovirus receptor to poliovirus (Xing et al., 2000). The

Fig. 4. Binding of S1 protein variants to soluble and membrane-bound pAPN. (a) Absorbance measurements monitoring thebinding of soluble, biotin-labelled pAPN to the plastic-bound S1 protein variants indicated. The concentration of S1 proteinsused for plastic coating is shown (see Methods). (b) Normalized binding of biotin-labelled pAPN to S1 protein in the absence(”) or presence of the indicated mAb. Means±SD from three different experiments are plotted in (a) and (b). (c) Binding ofsoluble S1 protein to pAPN on the cell surface. FACS monitoring binding of SA–Fc (grey shading), S5–Fc (solid line) and 2 %BSA in PBS (dotted line) to cells lacking (BHK) or having (BHK-pAPN) the receptor protein on the cell surface, and topermissive ST cells.

Fig. 5. Binding of soluble S1 to pAPN using the BIAcore-X instrument. Sensorgrams monitoring the response (RU) for twocycles where a Flag-tagged S1 protein was run onto sensor chip surfaces with immobilized HA mAb after the injection ofsoluble HA-tagged pAPN (thick line) or buffer (thin line). Inset: overlay plot of sensorgrams recorded for association anddissociation of the S1 protein from captured pAPN at the indicated concentrations.



dissociation rate (kdis) was also low, indicating a strongbinding interaction. The TGEV S1 protein was prepared inthe lectin-resistant CHO Lec 3.2.8.1 cells and thereforecontained only high-mannose carbohydrate (Stanley,1989), which could be removed by endoglycosidase Htreatment. We also determined the affinity of theendoglycosidase H-treated S1 (S1eh) protein, and nosignificant differences were observed after removal of theglycans (Table 1), showing that they did not contribute torecognition of the pAPN receptor protein.

DISCUSSION

In this study, we dissected the S1 region of the TGEV Sprotein and identified four modules bearing uniqueantigenic sites, based on expression of soluble S1 lengthvariants and proteolysis experiments (Fig. 6a). Besides itsresistance to papain treatment shown here, the naturaldeletion of the D1 region in certain PRCVs indicated that itmust be a unique folding module. D3 could be expressedindependently of the remaining S1 fragment and its correctfolding was confirmed by both mAb and pAPN receptorbinding. We assigned D2 to the protein region between D1and D3 (Fig. 6a), which carries antigenic site D. Thisantigenic site was recovered when this region was expressedtogether with D1 (S4–Fc protein) or isolated (S4H–Fcprotein), showing that it is a distinct module in the S1region of the protein. The C-terminal moiety labelled D4could be considered a kind of S1 linker to the S2 region ofthe S protein, although we did not test whether it can foldindependently from the remaining S1 fragment.

D3 can fold independently and comprises both antigenicsite A and the pAPN receptor-binding epitopes. Thereceptor-binding activity of the SA protein that defined D3was similar to that of the complete S1 region (Fig. 4a), andthe protein could bind to both soluble and membrane-bound pAPN protein. Therefore, this domain shouldinclude all the critical residues required for recognitionof the pAPN protein. Sequence identity of the TGEV D3

domain with the crystallized receptor-binding domain ofSARS-CoV of the genus Betacoronavirus (Li et al., 2005)and NL63-CoV of the genus Alphacoronavirus (Wu et al.,2009) is around 10 and 24 %, respectively. Therefore, wewould expect certain structural similarities of the TGEVdomain with that of NL63-CoV, whose structure is quitedistinct from the SARS-CoV receptor-binding region (Wuet al., 2009), even though both viruses bind to the ACE2receptor protein.

Antigenic site A displays the major neutralization epitopesin TGEV, and antibodies clustered at this site can efficientlyprevent virus binding to the pAPN receptor and infection(Sune et al., 1990). The soluble SA protein fragmentdeveloped here could therefore be used as an immunogenicagent to elicit a neutralizing response. Its IgG fusionvariant should have increased stability and therefore couldenhance antigen-specific T-cell responses (Nam et al., 2010;Siberil et al., 2007).

We determined the affinity and kinetics for binding of theTGEV S1 region to its pAPN receptor (Table 1). Thedetermined KD of around 100 nM for monovalent bindingindicated a relatively high-affinity virus–receptor inter-action. Previously reported KD values for receptor bindingto viruses range from about 100 nM to 1 mM (Lea et al.,1998; Myszka et al., 2000; Santiago et al., 2002; Xing et al.,2000). The KD is close to the 50 nM value estimated forbinding of a bivalent SARS-CoV S1 fragment to its ACE2

Table 1. Affinity kinetic rate constants for binding of the TGEVS1 protein to pAPN

Affinity and kinetic rate constants were determined from sensorgrams

recorded at 25 uC during injection of the TGEV S1 protein untreated

or treated with endoglycosidase H (eh) through sensor chips having

pAPN captured (about 1000 RU) with an HA mAb (see Fig. 5). The

S1 protein was produced in CHO Lec cells (see Methods) and

therefore contained only high-mannose carbohydrates that can be

removed by endoglycosidase H treatment. Means (SD) from four

experiments carried out at two different flow rates are shown. KD was

determined from the kinetic rates (kdis/kass).

Protein kass (M”1 s”1) kdis�103 (s”1) KD (nM)

CoV S1 17 000 (5140) 2.28 (0.25) 135

CoV S1eh 17 400 (4580) 2.00 (0.64) 115

Fig. 6. Modules in the S1 region of TGEV and its arrangement inthe spike in the virus envelope. (a) Domains in the S1 protein ofTGEV. The open rectangle above represents the S1 region ofTGEV with the C, B, D and A antigenic sites indicated. DomainsD1–D4, defined in the S1 region, with the approximate amino acidnumber of their terminal residues, are shown below. (b)Arrangement of the S1 domains (ellipses) in the TGEV andPRCV spikes. The two heptad repeat regions of the S2 fragmentare shown as rectangles (Xu et al., 2004). The N-terminal region(NH2) of the S1 fragment is in close contact with S2, as proposedfor the MHV spike (Grosse & Siddell, 1994).



receptor (Wong et al., 2004). The kinetic rates reportedhere are similar to those described for receptor bindingto poliovirus (Xing et al., 2000). The low kass rate(17 000 M21 s21) indicated either a receptor–virus inter-face with low accessibility or certain conformationalchanges associated with the interaction. Nevertheless, asseen with poliovirus, TGEV-neutralizing antibodies canblock receptor binding, so that the receptor-binding site inthe virus must be accessible to antibody neutralization. Thekdis rate was also low and indicated a relatively strongbinding interaction, as has been described for other virus–receptor interactions (Santiago et al., 2002; Xing et al.,2000). The carbohydrates of the heavily glycosylated Sprotein did not have any effect on receptor-binding affinity(Table 1); therefore, the pAPN-binding region on the Sprotein must be free of glycans.

Antibody binding to soluble S length variants showed agood correlation with antibody recognition of the S proteinin TGEV, but some differences with the recognition ofPRCV particles (Fig. 3). Antibodies mapping to theantigenic C, B, D and A sites in TGEV recognized thesoluble proteins bearing those antibody epitopes.Nevertheless, mAb 1D.G3 bound efficiently to the solubleS1 and S1H proteins but poorly to the trimeric membrane-bound S protein in the HOL87 PRCV particles (Fig. 3).These data suggested that antigenic site D must be partiallyburied in the trimer formed by the S protein at the PRCVenvelope but exposed in TGEV spikes, indicating that thisregion (D2) must be differentially displayed in the tworelated viruses (Fig. 6b). According to our domainarrangement model presented in Fig. 6(b), the N-terminalD2 domain in the PRCV spike is mostly inside the trimer,with the N terminus close to the S2 region. This proximitybetween the N-terminal region of the S protein and S2 waspreviously proposed based on MHV mutants (Grosse &Siddell, 1994). Differing from PRCV, TGEV should haveD1 inside the trimer, with D2 being more exposed toantibody binding. In good agreement, the N-terminalantigenic C site at D1 was not exposed in freshly purifiedTGEV particles, although it became accessible to mAb6A.A6 upon virus binding to plastic or following partialvirus denaturation (Gebauer et al., 1991).

The dissection of the S1 region of the TGEV S protein hasprovided new insights into the modular architecture of theCoV S glycoprotein. We identified four modules ordomains in the S1 region, which displayed uniqueantigenic sites and functions. D3 is specialized in therecognition of the pAPN receptor recognition, but thefunctional relevance of the D1 domain in recognition ofadditional receptor molecules is unclear. Binding to anenteric receptor molecule by the N-terminal D1 of TGEVhas been suggested to confer its enteric tropism (Krempl &Herrler, 2001). However, the distinct domain arrangementin the S proteins of TGEV and PRCV proposed here couldprovide differential receptor-binding properties to thoseviruses in the enteric track. Our model opens up thepossibility that binding sites for enteric cellular receptors in

TGEV S1 may be allocated to D2, which is not accessible toligands such as site D mAbs in the PRCV virions. Furtherstructural studies are now required to determine theconformation of the modules defined here and theirarrangement in the CoV S glycoprotein.

METHODS

Cells. Hybridoma, 293T, BHK and ST cells were grown in Dulbecco’smodified Eagle’s medium (DMEM) supplemented with 10 %inactivated FCS. Stable transfected CHO Lec 3.2.8.1 (CHOLec) cells(Stanley, 1989) were grown with selective medium containing30–200 mM L-methionine sulfoximine, as described previously(Casasnovas & Springer, 1995; Ordono et al., 2006). BHK-pAPNcells have been described elsewhere (Delmas et al., 1993).

Preparation of TGEV S1 length variants and pAPN protein. Thecoding sequences for the S1 variants were prepared by PCR from thecDNA clones of virus strains SC11 (GenBank accession no. AJ271965)and HOL87 (Sanchez et al., 1990). The soluble region of the pAPN(residues 36–963 of reference sequence GenBank accession no.P15145) was amplified from the cDNA coding for the receptorprotein. The recombinant cDNAs of proteins requiring an exogenoussignal peptide for cellular secretion (SA and pAPN) were cloned inframe with the IgK leader sequence and an HA epitope in a vectorderived from pDisplay (Invitrogen). All recombinant cDNAs werecloned in a vector derived from the pEF-BOS (Mizushima & Nagata,1990) in frame with either a Flag (DYKDDDDK) or HA(YPYDVPDYA) epitope, or the human IgG1 Fc region at their 39

end. A thrombin recognition sequence (LVPRGS) was introducedbetween the protein and the C-terminal tags. The recombinantvectors were transfected into 293T cells using the calcium phosphatemethod for transient protein expression (Pear et al., 1993).Transfected 293T cells were maintained in DMEM with 10 % FCSor serum-free OPTI-MEM (Invitrogen) for up to 3–4 days post-transfection for protein production. The concentration of theproteins in the cell supernatants was determined by ELISA usingpurified proteins as reference.

The recombinant cDNAs with the Flag and Fc tags were cloned intothe unique SalI and NotI sites of the pBJ5-GS vector and used forpreparation of stable CHOLec cells using the glutamine synthetaseexpression system (Casasnovas & Springer, 1995; Ordono et al.,2006). Clones secreting the proteins into cell supernatants wereselected by ELISA.

Protein purification and papain digestion. Proteins were purifiedfrom cell supernatants by affinity chromatography. The TGEV Sproteins were purified with either mAb 6A.C3 or a Flag M2 mAb(Sigma) coupled to Sepharose, and the pAPN protein with the HAmAb 12AC5 (Roche). Fc fusion variants were purified using a proteinA column (GE Healthcare). After loading the cell supernatants, thecolumns were washed with Tris/saline (pH 8.0) and the proteinseluted with glycine buffer (pH 3.0). The eluted proteins at neutralpH were concentrated and in some cases treated with endoglycosidaseH at 30 uC for removal of N-linked glycans. Final size-exclusionchromatography with a Superdex 200 column was usually run withHBS buffer [20 mM HEPES (pH 7.5) and 100 mM NaCl].Concentration of purified proteins was determined by absorbanceat 280 nm, using the extinction coefficient given by its amino acidsequence.

The S1 protein in HBS buffer with 1 mM EDTA and 5 mM cysteinewas treated with papain from Carica papaya (Boehringer Mannheim)at different protein : papain ratios (w/w). The reaction was stopped bythe addition of E64 (Sigma) at a final concentration of 2 mM, and the



S35 fragment was purified by size-exclusion chromatography on a

Superdex 200 column (GE Healthcare).

Antibody- and pAPN-binding assays. The TGEV S mAbs were

purified from hybridoma supernatants using a Hi-Trap protein A

column (GE Healthcare). Binding of mAb and pAPN to TGEV S

protein variants was carried out in 96-well plates. Proteins (50 ml),

either purified or in serum-free 293T cell supernatant, were first

bound to wells by overnight incubation at 4 uC. The protein

concentration ranged from 10 to 0.1 mg ml21 for mAb binding or

at the concentrations indicated in Fig. 4 for pAPN binding. Wells

were blocked with 2 % BSA in PBS, and antibody binding was carried

out with the TGEV S mAb at a concentration of 5 mg ml21 in PBS

with 1 % BSA for 1 h at 37 uC. The bound antibody was detected with

a secondary biotin-labelled rabbit anti-mouse antibody, HRP–

streptavidin and o-phenylenediamine (Zymed). Absorbance at

492 nm was monitored for the wells having S protein and corrected

for the absorbance of wells without proteins. Receptor binding to the

plastic-bound proteins was carried out with biotin-labelled pAPN

protein (10 mg ml21) for 1 h at 37 uC and the bound protein was

monitored by absorbance determination as carried out for antibody

binding.

Antibody binding to the plastic-bound TGEV and PRCV particles was

performed as described elsewhere (Sanchez et al., 1990). Partially

purified viruses were titrated with mAb 6A.C3 and added in similar

amounts to the wells in 96-well plates for the antibody-binding

experiments.

Binding of Fc fusion proteins to cell-surface-expressed pAPN was

carried out by fluorescence activated cell sorting (FACS).

Approximately 26105 cells in 250 ml PBS with 2 % BSA were

incubated with 20 mg Fc protein overnight at 4 uC. The cells were

washed three times with the binding buffer and incubated for 1 h

with an Alexa Fluor 488-labelled goat anti-human Fc secondary

antibody (Invitrogen). The cells were washed three times and analysed

in a Beckman Coulter EPICS XL-MCL Laser 488 cytometer.

Kinetics for binding of soluble S1 to pAPN. Kinetic rate

determination by surface plasmon resonance was carried out on a

CM5 sensor chip using a BIAcore-X instrument. The HA mAb 12AC5

was covalently immobilized on the sensor chip. The HA-tagged pAPN

protein in HBS buffer was first captured on the sensor chip surface

with immobilized mAb and the S1 protein was run at flow rates of 10

and 20 ml min21 onto the surface with captured receptor. After each

cycle of S1 protein binding to the captured pAPN, the sensor chip was

regenerated by two injections of 50 mM citrate buffer (pH 3.0) for

1 min each. Successive binding cycles with a range of S1 protein

concentrations (1–6 mM) were carried out in each experiment (Fig.

5). The binding sensorgrams recorded with surfaces lacking pAPN

were subtracted from those recorded with captured pAPN receptor.

Association and dissociation phases of the corrected sensorgrams

were analysed using BIAevaluation software (BIAcore) and the kinetic

rates determined by adjusting the sensorgrams to a Langmuir 1 : 1

binding model.

CD. CD spectra (mean of four scans) were recorded in the far-UV

region using a J810 spectropolarimeter (Jasco) equipped with a

Peltier-type cell holder. Measurements were performed at 20 uC using

a scan rate of 20 nm min21, a response time of 4 s, a bandwidth of

1 nm and a protein concentration of 4.3 mM (1 mm path-length

quartz cells). The buffer contribution was subtracted from the protein

spectrum and the corrected curve was converted to mean residue

ellipticity with a mean molecular mass per residue of 113.45 Da.

Secondary structure content was estimated by deconvolution of the

normalized spectrum with the CDNN program (Bohm et al., 1992)

using a reference dataset with 33 proteins.

ACKNOWLEDGEMENTS

This work was supported by grants from MICINN of Spain to J. M. C.(BFU2005-05972 and BFU2008-00971) and L. E. (CIT-010000-2007-8and BIO2007-60978). L. E. acknowledges support from NIH (ARRA-W000151845) and European Commission FP7 (EC grant no. 245141).J. R. was supported by the Juan de la Cierva program. D. O. was arecipient of a Severo Ochoa fellowship from Fundacion Ferrer and 1-year JAE-CSIC fellowship. We acknowledge Nuria Cubells fortechnical assistance, the CNIO for providing a BIAcore instrumentand Margarita Menendez (IQRF-CSIC) for the CD experiments.

REFERENCES

Abraham, S., Kienzle, T. E., Lapps, W. & Brian, D. A. (1990). Deducedsequence of the bovine coronavirus spike protein and identification ofthe internal proteolytic cleavage site. Virology 176, 296–301.

Ballesteros, M. L., Sanchez, C. M. & Enjuanes, L. (1997). Two aminoacid changes at the N-terminus of transmissible gastroenteritiscoronavirus spike protein result in the loss of enteric tropism.Virology 227, 378–388.

Beniac, D. R., Andonov, A., Grudeski, E. & Booth, T. F. (2006).Architecture of the SARS coronavirus prefusion spike. Nat Struct MolBiol 13, 751–752.

Bohm, G., Muhr, R. & Jaenicke, R. (1992). Quantitative analysis ofprotein far UV circular dichroism spectra by neural networks. ProteinEng 5, 191–195.

Bosch, B. J., van der Zee, R., de Haan, C. A. & Rottier, P. J. M. (2003).The coronavirus spike protein is a class I virus fusion protein:structural and functional characterization of the fusion core complex.J Virol 77, 8801–8811.

Breslin, J. J., Mørk, I., Smith, M. K., Vogel, L. K., Hemmila, E. M.,Bonavia, A., Talbot, P. J., Sjostrom, H., Noren, O. & Holmes, K. V.(2003). Human coronavirus 229E: receptor binding domain andneutralization by soluble receptor at 37 uC. J Virol 77, 4435–4438.

Callebaut, P., Correa, I., Pensaert, M., Jimenez, G. & Enjuanes, L.(1988). Antigenic differentiation between transmissible gastroenteritisvirus of swine and a related porcine respiratory coronavirus. J GenVirol 69, 1725–1730.

Casasnovas, J. M. & Springer, T. A. (1995). Kinetics andthermodynamics of virus binding to receptor. Studies with rhino-virus, intercellular adhesion molecule-1 (ICAM-1), and surfaceplasmon resonance. J Biol Chem 270, 13216–13224.

Casasnovas, J. M., Bickford, J. K. & Springer, T. A. (1998). Thedomain structure of ICAM-1 and the kinetics of binding torhinovirus. J Virol 72, 6244–6246.

Cavanagh, D., Davis, P. J., Pappin, D. J., Binns, M. M., Boursnell, M. E.& Brown, T. D. (1986). Coronavirus IBV: partial amino terminalsequencing of spike polypeptide S2 identifies the sequence Arg-Arg-Phe-Arg-Arg at the cleavage site of the spike precursor propolypep-tide of IBV strains Beaudette and M41. Virus Res 4, 133–143.

Corapi, W. V., Darteil, R. J., Audonnet, J. C. & Chappuis, G. E. (1995).Localization of antigenic sites of the S glycoprotein of feline infectiousperitonitis virus involved in neutralization and antibody-dependentenhancement. J Virol 69, 2858–2862.

Correa, I., Jimenez, G., Sune, C., Bullido, M. J. & Enjuanes, L. (1988).Antigenic structure of the E2 glycoprotein from transmissiblegastroenteritis coronavirus. Virus Res 10, 77–93.

Correa, I., Gebauer, F., Bullido, M. J., Sune, C., Baay, M. F.,Zwaagstra, K. A., Posthumus, W. P., Lenstra, J. A. & Enjuanes, L.(1990). Localization of antigenic sites of the E2 glycoprotein oftransmissible gastroenteritis coronavirus. J Gen Virol 71, 271–279.



de Groot, R. J., Ziebuhr, J., Poon, L. L., Woo, P. C., Talbot, P., Rottier,P. J. M., Holmes, K. V., Baric, R., Perlman, S. & other authors. (2008).Revision of the family Coronaviridae. Taxonomic proposal of the

Coronavirus Study Group to the ICTV Executive Committee. http://talk.

ictvonline.org/media/p/1230.aspx.

de Haan, C. A. M., Li, Z., te Lintelo, E., Bosch, B. J., Haijema, B. J. &Rottier, P. J. M. (2005). Murine coronavirus with an extended host

range uses heparan sulfate as an entry receptor. J Virol 79, 14451–

14456.

de Haan, C. A., Haijema, B. J., Schellen, P., Schreur, P. W.,te Lintelo, E., Vennema, H. & Rottier, P. J. (2008). Cleavage of group

1 coronavirus spike proteins: how furin cleavage is traded off against

heparan sulfate binding upon cell culture adaptation. J Virol 82,

6078–6083.

Delmas, B. & Laude, H. (1990). Assembly of coronavirus spike

protein into trimers and its role in epitope expression. J Virol 64,

5367–5375.

Delmas, B., Rasschaert, D., Godet, M., Gelfi, J. & Laude, H. (1990).Four major antigenic sites of the coronavirus transmissible gastro-

enteritis virus are located on the amino-terminal half of spike

glycoprotein S. J Gen Virol 71, 1313–1323.

Delmas, B., Gelfi, J., L’Haridon, R., Vogel, L. K., Sjostrom, H.,Noren, O. & Laude, H. (1992). Aminopeptidase N is a major receptor

for the entero-pathogenic coronavirus TGEV. Nature 357, 417–420.

Delmas, B., Gelfi, J., Sjostrom, H., Noren, O. & Laude, H. (1993).Further characterization of aminopeptidase-N as a receptor for

coronaviruses. Adv Exp Med Biol 342, 293–298.

Drosten, C., Gunther, S., Preiser, W., van der Werf, S., Brodt, H.-R.,Becker, S., Rabenau, H., Panning, M., Kolesnikova, L. & otherauthors (2003). Identification of a novel coronavirus in patients with

severe acute respiratory syndrome. N Engl J Med 348, 1967–1976.

Enjuanes, L., Gorbalenya, A. E., de Groot, R. J., Cowley, J. A.,Ziebuhr, J. & Snijder, E. J. (2008). Nidovirales. In Encyclopedia

of Virology, 3rd edn, pp. 419–430. Edited by B. W. J. Mahy &

M. H. V. Van Regenmortel. Oxford: Elsevier.

Gallagher, T. M. & Buchmeier, M. J. (2001). Coronavirus spike

proteins in viral entry and pathogenesis. Virology 279, 371–374.

Gebauer, F., Posthumus, W. P. A., Correa, I., Sune, C., Smerdou, C.,Sanchez, C. M., Lenstra, J. A., Meloen, R. H. & Enjuanes, L. (1991).Residues involved in the antigenic sites of transmissible gastroenteritis

coronavirus S glycoprotein. Virology 183, 225–238.

Godet, M., Grosclaude, J., Delmas, B. & Laude, H. (1994). Major

receptor-binding and neutralization determinants are located within

the same domain of the transmissible gastroenteritis virus (corona-

virus) spike protein. J Virol 68, 8008–8016.

Grosse, B. & Siddell, S. G. (1994). Single amino acid changes in the

S2 subunit of the MHV surface glycoprotein confer resistance to

neutralization by S1 subunit-specific monoclonal antibody. Virology

202, 814–824.

Holmes, K. V. (2005). Structural biology. Adaptation of SARS

coronavirus to humans. Science 309, 1822–1823.

Jeffers, S. A., Tusell, S. M., Gillim-Ross, L., Hemmila, E. M.,Achenbach, J. E., Babcock, G. J., Thomas, W. D., Jr, Thackray, L. B.,Young, M. D. & other authors (2004). CD209L (L-SIGN) is a receptor

for severe acute respiratory syndrome coronavirus. Proc Natl Acad Sci

U S A 101, 15748–15753.

Krempl, C. & Herrler, G. (2001). Sialic acid binding activity of

transmissible gastroenteritis coronavirus affects sedimentation behav-

ior of virions and solubilized glycoproteins. J Virol 75, 844–849.

Krempl, C., Schultze, B., Laude, H. & Herrler, G. (1997). Point

mutations in the S protein connect the sialic acid binding activity

with the enteropathogenicity of transmissible gastroenteritis corona-

virus. J Virol 71, 3285–3287.

Lau, Y.-L. (2004). SARS: future research and vaccine. Paediatr Respir

Rev 5, 300–303.

Laude, H., Godet, M., Bernard, S., Gelfi, J., Duarte, M. & Delmas, B.(1995). Functional domains in the spike protein of transmissible

gastroenteritis virus. Adv Exp Med Biol 380, 299–304.

Lea, S. M., Powell, R. M., McKee, T., Evans, D. J., Brown, D., Stuart,D. I. & van der Merwe, P. A. (1998). Determination of the affinity andkinetic constants for the interaction between the human virus

echovirus 11 and its cellular receptor, CD55. J Biol Chem 273,

30443–30447.

Li, W., Moore, M. J., Vasilieva, N., Sui, J., Wong, S. K., Berne, M. A.,Somasundaran, M., Sullivan, J. L., Luzuriaga, K. & other authors(2003). Angiotensin-converting enzyme 2 is a functional receptor forthe SARS coronavirus. Nature 426, 450–454.

Li, F., Li, W., Farzan, M. & Harrison, S. C. (2005). Structure of SARScoronavirus spike receptor-binding domain complexed with receptor.

Science 309, 1864–1868.

Masters, P. S. (2006). The molecular biology of coronaviruses. AdvVirus Res 66, 193–292.

Miura, T. A., Travanty, E. A., Oko, L., Bielefeldt-Ohmann, H., Weiss, S. R.,Beauchemin, N. & Holmes, K. V. (2008). The spike glycoprotein of

murine coronavirus MHV-JHM mediates receptor-independent infec-

tion and spread in the central nervous systems of Ceacam1a2/2 mice.

J Virol 82, 755–763.

Mizushima, S. & Nagata, S. (1990). pEF-BOS, a powerful mammalian

expression vector. Nucleic Acids Res 18, 5322.

Myszka, D. G., Sweet, R. W., Hensley, P., Brigham-Burke, M., Kwong,P. D., Hendrickson, W. A., Wyatt, R., Sodroski, J. & Doyle, M. L.(2000). Energetics of the HIV gp120–CD4 binding reaction. Proc NatlAcad Sci U S A 97, 9026–9031.

Nam, H. J., Song, M.-Y., Choi, D.-H., Yang, S.-H., Jin, H.-T. & Sung, Y.-C.(2010). Marked enhancement of antigen-specific T-cell responses by

IL-7-fused nonlytic, but not lytic, Fc as a genetic adjuvant. Eur J

Immunol 40, 351–358.

Ordono, D., Enjuanes, L. & Casasnovas, J. M. (2006). Methods for

preparation of low abundance glycoproteins from mammalian cell

supernatants. Int J Biol Macromol 39, 151–156.

Pear, W. S., Nolan, G. P., Scott, M. L. & Baltimore, D. (1993).Production of high-titer helper-free retroviruses by transienttransfection. Proc Natl Acad Sci U S A 90, 8392–8396.

Perlman, S. (1998). Pathogenesis of coronavirus-induced infections.

Review of pathological and immunological aspects. Adv Exp Med Biol440, 503–513.

Rota, P. A., Oberste, M. S., Monroe, S. S., Nix, W. A., Campagnoli,R., Icenogle, J. P., Penaranda, S., Bankamp, B., Maher, K. & otherauthors (2003). Characterization of a novel coronavirus associated

with severe acute respiratory syndrome. Science 300, 1394–1399.

Sanchez, C. M., Jimenez, G., Laviada, M. D., Correa, I., Sune, C.,Bullido, M., Gebauer, F., Smerdou, C., Callebaut, P. & other authors(1990). Antigenic homology among coronaviruses related to trans-

missible gastroenteritis virus. Virology 174, 410–417.

Sanchez, C. M., Gebauer, F., Sune, C., Mendez, A., Dopazo, J. &Enjuanes, L. (1992). Genetic evolution and tropism of transmissible

gastroenteritis coronaviruses. Virology 190, 92–105.

Santiago, C., Bjorling, E., Stehle, T. & Casasnovas, J. M. (2002).Distinct kinetics for binding of the CD46 and SLAM receptors to

overlapping sites in the measles virus hemagglutinin protein. J BiolChem 277, 32294–32301.



Siberil, S., Dutertre, C.-A., Fridman, W.-H. & Teillaud, J.-L. (2007).FccR: the key to optimize therapeutic antibodies? Crit Rev OncolHematol 62, 26–33.

Stanley, P. (1989). Chinese hamster ovary cell mutants with multipleglycosylation defects for production of glycoproteins with minimalcarbohydrate heterogeneity. Mol Cell Biol 9, 377–383.

Sune, C., Jimenez, G., Correa, I., Bullido, M. J., Gebauer, F.,Smerdou, C. & Enjuanes, L. (1990). Mechanisms of transmissiblegastroenteritis coronavirus neutralization. Virology 177, 559–569.

Supekar, V. M., Bruckmann, C., Ingallinella, P., Bianchi, E., Pessi, A.& Carfı, A. (2004). Structure of a proteolytically resistant core fromthe severe acute respiratory syndrome coronavirus S2 fusion protein.Proc Natl Acad Sci U S A 101, 17958–17963.

Tresnan, D. B., Levis, R. & Holmes, K. V. (1996). Felineaminopeptidase N serves as a receptor for feline, canine, porcine,and human coronaviruses in serogroup I. J Virol 70, 8669–8674.

Tsai, J. C., Zelus, B. D., Holmes, K. V. & Weiss, S. R. (2003). The N-terminal domain of the murine coronavirus spike glycoproteindetermines the CEACAM1 receptor specificity of the virus strain.J Virol 77, 841–850.

van den Brink, E. N., Ter Meulen, J., Cox, F., Jongeneelen, M. A., Thijsse,A., Throsby, M., Marissen, W. E., Rood, P. M., Bakker, A. B. & otherauthors (2005). Molecular and biological characterization of humanmonoclonal antibodies binding to the spike and nucleocapsid proteins ofsevere acute respiratory syndrome coronavirus. J Virol 79, 1635–1644.

Weiss, S. R. & Navas-Martin, S. (2005). Coronavirus pathogenesisand the emerging pathogen severe acute respiratory syndromecoronavirus. Microbiol Mol Biol Rev 69, 635–664.

Wong, S. K., Li, W., Moore, M. J., Choe, H. & Farzan, M. (2004). A 193-amino acid fragment of the SARS coronavirus S protein efficientlybinds angiotensin-converting enzyme 2. J Biol Chem 279, 3197–3201.

Wu, K., Li, W., Peng, G. & Li, F. (2009). Crystal structure ofNL63 respiratory coronavirus receptor-binding domain complexedwith its human receptor. Proc Natl Acad Sci U S A 106, 19970–19974.

Xing, L., Tjarnlund, K., Lindqvist, B., Kaplan, G. G., Feigelstock, D.,Cheng, R. H. & Casasnovas, J. M. (2000). Distinct cellular receptorinteractions in poliovirus and rhinoviruses. EMBO J 19, 1207–1216.

Xu, Y., Liu, Y., Lou, Z., Qin, L., Li, X., Bai, Z., Pang, H., Tien, P., Gao,G. F. & Rao, Z. (2004). Structural basis for coronavirus-mediatedmembrane fusion. Crystal structure of mouse hepatitis virus spikeprotein fusion core. J Biol Chem 279, 30514–30522.

Yeager, C. L., Ashmun, R. A., Williams, R. K., Cardellichio, C. B.,Shapiro, L. H., Look, A. T. & Holmes, K. V. (1992). Humanaminopeptidase N is a receptor for human coronavirus 229E.Nature 357, 420–422.

Yokomori, K. & Lai, M. M. (1992). Mouse hepatitis virus utilizes twocarcinoembryonic antigens as alternative receptors. J Virol 66, 6194–6199.

Zhang, H., Wang, G., Li, J., Nie, Y., Shi, X., Lian, G., Wang, W., Yin, X.,Zhao, Y. & other authors (2004). Identification of an antigenicdeterminant on the S2 domain of the severe acute respiratory syndromecoronavirus spike glycoprotein capable of inducing neutralizingantibodies. J Virol 78, 6938–6945.



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