Identification of the Membrane-Active Regions of the Severe Acute Respiratory Syndrome Coronavirus Spike Membrane Glycoprotein Using a 16/18-Mer Peptide Scan: Implications for the Viral Fusion Mechanism

We have identified the membrane-active regions of the severeacute respiratory syndrome coronavirus (SARS CoV) spike glycoproteinby determining the effect on model membrane integrity of a 16/18-merSARS CoV spike glycoprotein peptide library. By monitoring theeffect of this peptide library on membrane leakage in modelmembranes, we have identified three regions on the SARS CoVspike glycoprotein with membrane-interacting capabilities: region1, located immediately upstream of heptad repeat 1 (HR1) andsuggested to be the fusion peptide; region 2, located betweenHR1 and HR2, which would be analogous to the loop domain ofhuman immunodeficiency virus type 1; and region 3, which wouldcorrespond to the pretransmembrane region. The identificationof these membrane-active regions, which are capable of modifyingthe biophysical properties of phospholipid membranes, supportstheir direct role in SARS CoV-mediated membrane fusion, as wellas facilitating the future development of SARS CoV entry inhibitors.

INTRODUCTION

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Introduction

An infectious disease, designated severe acute respiratory syndrome(SARS), broke out in China in late 2002 and quickly spread toseveral countries. The infectious agent responsible for thisepidemic outbreak was identified as a previously unknown memberof the family of coronaviruses (CoV), SARS CoV (10, 21, 30,31). Its phylogenetic analysis showed that it was neither amutant nor a recombinant of previously characterized CoV (35).These viruses are a diverse group of enveloped, positive-strandRNA viruses, with three or four proteins embedded in the envelope,that cause respiratory and enteric diseases in humans and otheranimals (10, 21, 30, 31). CoV infection, similarly to otherenvelope viruses, is achieved through fusion of the lipid bilayerof the viral envelope with the host cell membrane.

The fusion of viral and cellular membranes, the critical earlyevents in viral infection, are mediated by envelope glycoproteinslocated on the outer surfaces of the viral membranes (11, 17).SARS CoV membrane fusion is mediated by the viral spike glycoproteinlocated on the viral envelope, which is synthesized as a 180-kDaprecursor and displayed in $~$ 200 copies on the viral membranein a trimeric or dimeric structure (10, 21, 30, 35, 47). Insome CoV strains, the spike glycoprotein is cleaved by a proteaseto yield two noncovalently associated subunits, S1 and S2 (Fig.1A), which have different functions (16, 40). However, cleavageis not an absolute requirement for the mechanism of fusion,and the available data suggest that the SARS CoV spike glycoproteinis not cleaved into two subunits (9, 18, 35). S1, which formsthe globular portion of the spike, contains the receptor-bindingsite and thus defines the host range of the virus (42), whileS2, more conserved than S1, forms the membrane-anchored stalkregion and mediates the fusion between the viral and cellularmembranes (35, 47).

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FIG. 1. (A) Scheme of the structure of SARS-CoV spike glycoprotein S (amino acid residues 1 to 1255 for the full length), as well as the S1 and S2 domains, according to literature consensus. Boundaries between the different domains cannot be accurately identified at present but are indicated for the convenience of comparison with other coronaviruses. The relevant functional regions are highlighted: the N-terminal signal peptide (SP), the transmembrane domain (TM) and the predicted heptad repeat regions pertaining to the S2 domain, HR1 and HR2. (B) Sequence of the 169 16/18-mer peptides used in this study with respect to the sequence of the whole spike glycoprotein S. Maximum overlap between adjacent peptides is 10 amino acids.

S2 contains two predicted ${alpha}$ -helical heptad repeat (HR) domains(HR1 and HR2) which form coiled-coil structures (5, 6, 20, 22,35, 43, 50). These regions, separated by a stretch of 140 aminoacid residues called the interhelical domain, are thought toplay important roles in defining the oligomeric structure ofthe spike protein in its native state and its fusogenic ability(23). The presence of the HR regions, in conjunction with recentstudies, indicates that CoV spike proteins can be classifiedas class 1 viral fusion proteins (5, 6, 20, 22, 43, 50). Inthe current paradigm of virus-host cell membrane fusion forclass 1 viral fusion proteins, the HR domains form a six-helixbundle, where three HR1 helices fold into a central paralleltriple-stranded ${alpha}$ -helical coiled coil, and wrapped antiparallelon the outside of this core is an outer layer of three antiparallelHR2 ${alpha}$ -helices, each HR1-HR2 pair connected by a loop that reversesthe polypeptide chain (5, 15, 17, 20, 22, 43, 50). The HR1 andHR2 regions are believed to be important domains in this processand show different conformations in different fusion states(11, 49). Under the current model, there are at least threeconformational states of the envelope fusion protein, the prefusionnative state, the prehairpin intermediate state, and the postfusionhairpin state (11, 49). This trimeric helical hairpin structureis thought to form at a late stage during the membrane fusionprocess (15, 17). Formation of the six-helix coiled-coil bundlebrings into close proximity the fusion peptide (FP) and thepretransmembrane (PTM) and transmembrane (TM) domains, therebydriving the viral and host cell membranes into close contact,making possible the formation of the fusion pore (11, 15, 17).In class 1 viral fusion proteins, the FP invariably occurs upstreamof the HR1 region; however, no FP has been experimentally identifiedin any CoV spike protein, although a hydrophobic region hasbeen predicted recently at the N terminus of the HR1 region(5).

Although much information has been gathered in recent years,we do not yet know the exact mechanism of membrane fusion andthe processes which are behind it. The mechanism by which proteinsfacilitate the formation of fusion intermediates is a complexprocess involving several segments of fusion proteins (15, 32).These regions, either directly or indirectly, might interactwith biological membranes, contributing to the viral envelopeand cell membrane merging. Even though the detailed structuresof different segments of the SARS CoV spike glycoprotein havebeen elucidated, there are still many questions to be answeredregarding its mode of action in accelerating membrane fusion.Moreover, SARS CoV entry is an attractive target for anti-SARStherapy. To investigate the structural basis of SARS CoV membranefusion and identify new fusion inhibitors, we carried out theanalysis of the different regions of the SARS CoV spike glycoproteinthat might interact with phospholipid membranes, using an approachsimilar to that used for studying the human immunodeficiencyvirus (HIV) gp41 ectodomain (25), i.e., the identification ofmembrane-active regions of SARS CoV spike glycoprotein by determiningthe effect on membrane integrity of a 16/18-mer spike glycoprotein-derivedpeptide library. By monitoring the effect of this peptide libraryon membrane integrity, i.e., leakage, we have identified differentregions on the SARS CoV spike glycoprotein with membrane-interactingcapabilities, which supports their direct role in membrane fusionand therefore might help in understanding the molecular mechanismof membrane merging, as well as making possible the future developmentof SARS entry inhibitors, which may lead to new vaccine strategies.

MATERIALS AND METHODS

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Materials and Methods

Materials and reagents. Egg L- ${alpha}$ -phosphatidylcholine (EPC), egg sphingomyelin (SM), andcholesterol (Chol), were obtained from Avanti Polar Lipids (Alabaster,Ala.). 5-Carboxyfluorescein (CF) (>95% by high-performanceliquid chromatography) was from Sigma-Aldrich (Madrid, Spain).A set of 169 peptides 16 or 18 amino acids in length derivedfrom the SARS spike glycoprotein, with 10-amino-acid overlapbetween sequential peptides, was obtained through the AIDS Researchand Reference Reagent Program (Division of AIDS, National Instituteof Allergy and Infectious Diseases, National Institutes of Health,Bethesda, Md.). Porcine lungs were obtained from a local slaughterhouse.Plasma membranes from lung tissue pneumocytes were obtainedaccording to the method of Müller et al. (26), and lipidextraction from porcine lungs was performed according to theprocedure of Bligh and Dyer using a ratio of 1:1:0.9 (vol/vol/vol)between chloroform-methanol and the corresponding aqueous sample(4). All other reagents used were of analytical grade and wereobtained from Merck (Darmstad, Germany). Water was deionized,distilled twice, and passed through a Milli-Q apparatus (MilliporeIbérica, Madrid, Spain) to a resistivity better than18 M ${Omega}$ /cm.

Sample preparation. Aliquots containing the appropriate amount of lipid in chloroform-methanol(2:1 [vol/vol]) were placed in a test tube, the solvents wereremoved by evaporation under a stream of O₂-free nitrogen, andfinally, traces of solvents were eliminated under vacuum inthe dark for >3 h. After that, 1 ml of buffer containing10 mM Tris, 20 mM NaCl, pH 7.4, and CF at a concentration of40 mM was added, and multilamellar vesicles were obtained. Largeunilamellar vesicles (LUV) with a mean diameter of 90 nm wereprepared from multilamellar vesicles by the extrusion method(19), using polycarbonate filters with a pore size of 0.1 µm(Nuclepore Corp., Cambridge, Calif.). Breakdown of the vesiclemembrane leads to leakage of the contents, i.e., CF fluorescence.Nonencapsulated CF was separated from the vesicle suspensionthrough a Sephadex G-75 filtration column (Pharmacia, Uppsala,Sweden) eluted with buffer containing 10 mM Tris-0.1 M NaCl-1mM EDTA, pH 7.4.

Leakage measurement. Leakage of intraliposomal CF was assayed by treating the probe-loadedliposomes (final lipid concentration, 0.125 mM) with the appropriateamounts of peptide on microtiter plates using a microplate reader(FLUOstar; BMG Labtech, Offenburg, Germany), each well containinga final volume of 170 µl stabilized at 25°C. The mediumin the microtiter plates was continuously stirred to allow therapid mixing of peptide and vesicles. Leakage was measured atapproximate peptide-to-lipid ratios of 1:15, 1:10, and 1:5.Changes in fluorescence intensity were recorded with excitationand emission wavelengths set at 492 and 517 nm, respectively.One hundred percent release was achieved by adding Triton X-100to the microtiter plates to a final concentration of 0.5% (wt/wt).Fluorescence measurements were made initially with probe-loadedliposomes, then by adding peptide solution, and finally by addingTriton X-100 to obtain 100% leakage. Leakage was quantifiedon a percentage basis according to the following equation: %release = (F_f – F₀)/(F₁₀₀ – F₀) x 100, where F_fis the equilibrium value of fluorescence after peptide addition,F₀ is the initial fluorescence of the vesicle suspension, andF₁₀₀ is the fluorescence value after the addition of TritonX-100. The phospholipid concentration was measured by methodsdescribed previously (7).

Hydrophobic moments, hydrophobicity, and interfaciality. The hydrophobic-moment calculations were carried out accordingto the method of Eisenberg et al. (12, 13), and the scale forcalculating hydrophobic moments was taken from Engelman et al.(14). Hydrophobicity and interfacial values, i.e., whole residuescales for the transfer of an amino acid of an unfolded chaininto the membrane hydrocarbon palisade and the membrane interface,respectively, were obtained from http://blanco.biomol.uci.edu/hydrophobicity_scales.html(45, 46). Two-dimensional plots of the hydrophobic moments,hydrophobicity, and interfaciality were obtained using a windowof seven amino acids, taking into consideration the arrangementof the amino acids in the space and assuming an ${alpha}$ -helical structure(see Fig. 5). Each specific value in the two-dimensional plotrepresents the mean of the values pertaining to the hydrophobicmoment, hydrophobicity, and interfaciality of the amino acidat that position and its neighbors. Positive values correspondto positive bilayer-to-water transfer free-energy values, andtherefore, the higher the value, the greater the probabilityto interact with the membrane surface and/or the hydrophobiccore (45, 46).

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FIG. 5. (Top) Helical-wheel diagram indicating the amino acids which lie on the surface of a helix and their representation on a two-dimensional array to identify an amino acid position as a surface-interacting point. Each value in the two-dimensional plot represents the mean of the values pertaining to the hydrophobic moment, hydrophobicity, and interfaciality of the amino acid at that position plus those of its neighbors (window, seven amino acids). (Bottom) Hydrophobic moment, hydrophobicity, interfaciality distribution, and relative positions of peptides 113, 119, 123, 147, 162, and 163 along the SARS CoV spike S2 domain, assuming it forms an ${alpha}$ -helical wheel, as shown above, and its correlation with the S2 different functional regions. Only positive bilayer-to-water transfer free-energy values are depicted (solid lines). The dashed lines identify three different domains (R1 to R3) with highly positive values along the helix. The suggested FP, heptad repeats HR1 and HR2, and the PTM and TM domains are indicated (see the text for details). Column numbers define amino acid positions as in the diagram above, upper row.

RESULTS

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Results

The SARS CoV spike glycoprotein consists of an extracellulardomain, a TM domain, and an intracellular domain (Fig. 1A),and it can be classified as a class I viral fusion protein (5,6, 20, 22, 43, 50). Although it was previously thought thatthe FP of class I proteins was the only factor responsible forthe cell membrane interaction leading to membrane fusion, ithas been shown recently that, apart from the FP, other regionsof viral fusion proteins bind and interact with membranes andexperience conformational changes which all combine to makepossible the fusion of the viral and cell membranes (8, 15,25, 32). To explore the structural basis of SARS CoV membranefusion, we have carried out the analysis of the different regionsof the full SARS CoV spike glycoprotein that might interactwith phospholipid membranes by using a peptide library derivedfrom the SARS CoV spike glycoprotein. This peptide library wascomposed of 169 peptides 16 and 18 amino acids in length, andby using an approach similar to that used for studying the HIVgp41 ectodomain (25), we studied their effects on the releaseof an encapsulated fluorophore, CF, using the experimental setupdescribed in Materials and Methods. The 169 peptides we usedin this study and their correlation with the SARS CoV spikeglycoprotein sequence are shown in Fig. 1B. As can be observed,the 16/18-mer peptide library encompasses the whole sequenceof the SARS CoV spike glycoprotein, i.e., from the signal peptideto the sequence that follows the TM domain, including the recentlyidentified HR1 and HR2 domains.

The S1 domain of the SARS CoV spike glycoprotein forms the globularportion of the spike and mediates binding to the host cells(16, 40, 42), being the receptor-binding domain localized inthe amino acid sequence 318 to 510 (9, 47). Although the proposedrole for the S1 domain is the attachment of the protein to itsfunctional receptor, angiotensin-converting enzyme 2, severalhydrophobic patches have been identified, which might be importantnot only for protein-protein binding but also for membrane interaction,since, as was mentioned above, S1 probably remains covalentlyattached to the S2 domain during the fusion process (9, 34,35, 47). Figure 2A and B shows the effect of the S1 domain-derived16/18-mer peptides on membrane integrity, i.e., leakage, atdifferent peptide-to-lipid ratios and two different liposomecompositions, namely, EPC-SM-Chol at a molar ratio of 70:15:15and EPC-Chol at a molar ratio of 5:1. For liposomes composedof EPC-SM-Chol (Fig. 2A), it is clearly evident that some peptidesexerted hardly any effect, but other peptides showed notableeffects on liposome leakage in comparison. The most notableeffects were observed for peptides 34 and 85, which producedleakage of $~$ 33 and 22%, respectively, at the highest peptide-to-lipidratio used. Similar to what was found for EPC-SM-Chol liposomes,the same peptides, i.e., peptides 34 and 85, were the ones whichinteracted with liposomes composed of EPC-Chol (Fig. 2B). ForEPC-Chol liposomes, the leakage values for peptides 34 and 85were $~$ 22 and 10%, i.e., slightly lower than those found for EPC-SM-Cholliposomes. The other peptides from the S1 domain, apart frompeptides 34 and 85, exerted no significant effect on liposomeleakage.

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FIG. 2. Effects of the S1 domain derived 16/18-mer peptides on the release of LUV contents for different lipid compositions. Leakage data for LUV composed of (A) EPC-SM-Chol at a phospholipid molar ratio of 70:15:15, (B) EPC-Chol at a phospholipid molar ratio of 5:1, and (C) a lipidic extract of lung tissue pneumocytes at different peptide (P)-to-lipid (L) ratios as indicated. Experimental conditions are described in the text. The error bars indicate standard deviations of the mean for quadruplicate samples.

In order to compare leakage values obtained in phospholipidmodel membranes with those obtained in biologically derivedmodel membranes, we also studied liposome leakage produced bypeptides derived from the S1 domain of the SARS CoV spike glycoproteinin model membranes whose lipids were obtained from a lipidicextract of lung tissue pneumocytes (Fig. 2C). It can be observedthat the extent of leakage was slightly reduced compared tothe EPC-SM-Chol and EPC-Chol liposome systems mentioned above.However, patterns of leakage similar to those described abovecan be discerned, since peptides 34 and 85 again showed relativelyhigh leakage values compared to the leakage values exhibitedby the other peptides pertaining to the S1 domain. It is worthnoting that, for this liposome composition, peptide 50 alsoshowed a similar leakage value.

We studied the interaction of the 16/18-mer S2 spike glycoproteinlibrary with LUV model membranes having variable Chol and SMcompositions, namely, EPC-SM-Chol at molar ratios of 70:15:15,52:18:30, and 37:18:45 (Fig. 3A, B, and C, respectively). Thesemodel membranes, showing the coexistence of liquid-ordered andliquid-disordered phases, have a high probability of raft formation(2); as has been documented, the presence of laterally segregatedmembrane microdomains or lipid rafts is important for membranefusion (1, 3, 28, 33, 37, 38, 44). For liposomes composed ofEPC-SM-Chol at a molar ratio of 70:15:15 (Fig. 3A), it can beclearly observed that, whereas the vast majority of peptidesexerted hardly any effect on membrane leakage, peptide 162 showeda dramatic effect, i.e., ca. 100% leakage for peptide (comparethe ordinate scales of Fig. 3A with those in Fig. 2). Otherpeptides that showed a significant effect, although not as highas peptide 162, were peptide 163 and, to a lesser extent, peptides113, 123, 147, and 148. It is worth noting that peptide 163did have a significant effect, not as great as that of peptide162 but higher than the leakage values observed for peptides113, 123, 147, and 148 (Fig. 3A). Similar to what was foundfor liposomes containing EPC-SM-Chol at a molar ratio of 70:15:15,some peptides interacted with liposomes composed of EPC-SM-Cholat molar ratios of 52:18:30 and 37:18:45 (Fig. 3B and C). Themost notable effects were again observed for peptides 162 and163, the former presenting the higher leakage values. However,the leakage values were reduced compared to those observed forliposomes containing EPC-SM-Chol at a molar ratio of 70:15:15.The major difference in the compositions of these liposomesis the PC-Chol ratio. In EPC-SM-Chol at molar ratios of 70:15:15,52:18:30,and 37:18:45, the proportion of SM remains the same,whereas the PC-Chol ratio decreases, i.e., leakage values decreaseas Chol contents increase. When liposomes composed of PC-SMat a molar ratio of 5:1 were studied, peptides 162 and 163 wereagain the ones which presented the most significant extentsof leakage (Fig. 3D). Similar results were obtained for liposomescomposed of PC-Chol at a molar ratio of 5:1 (Fig. 3E). Otherpeptides that showed positive leakage values, although not assignificant as peptides 162 and 163, were peptides 113, 123,147, and 148, similar to what was found earlier. However, itis interesting that EPC-Chol liposomes presented a lower extentof leakage than EPC-SM liposomes, stressing the fact that, asmentioned above, the Chol content might be responsible for thelower extent of leakage rather than the presence of both SMand Chol.

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FIG. 3. Effects of the S2 domain-derived 16/18-mer peptides on the release of LUV contents for different lipid compositions at a peptide-to-lipid ratio of 1:15. Leakage data for LUV composed of (A) EPC-SM-Chol at a molar ratio of 70:15:15, (B) EPC-SM-Chol at a molar ratio of 52:18:30, (C) EPC-SM-Chol at a molar ratio of 37:18:45, (D) EPC-SM at a molar ratio of 5:1, and (E) EPC-Chol at a molar ratio of 5:1. The error bars indicate standard deviations of the mean of quadruplicate samples.

We also studied the effects of different lipid-to-peptide ratioson leakage for liposomes composed of EPC-SM-Chol at a phospholipidmolar ratio of 70:15:15 (Fig. 4A). It is clearly evident thatpeptide 162 was again the one which showed a dramatic effect,since nearly complete rupture of the liposomes was observed(ca. 100% leakage for peptide) at all lipid-to-peptide ratiostested. It is worth noting that, even at the lowest lipid-to-peptideratio used, peptide 163 was the only one, apart from peptide162, that had a relatively significant effect, although notas great as that of peptide 162 (Fig. 4A). As described above,other peptides that showed positive leakage values, althoughnot as significant as peptide 162 or even peptide 163, werepeptides 113, 123, 147, and 148.

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FIG. 4. Effects of the S2 domain-derived 16/18-mer peptides on the release of LUV contents for (A) EPC-SM-Chol at a phospholipid-molar ratio of 70:15:15 and (B) lipidic extract of lung tissue pneumocytes at different peptide (P)-to-lipid (L) ratios as indicated. The error bars indicate standard deviations of the mean for quadruplicate samples.

In order to compare leakage values observed in model membraneswith those obtained in biologically derived model membranes,we studied liposome leakage produced by peptides derived fromthe S2 domain in model membranes whose lipids were obtainedfrom a lipidic extract of lung tissue pneumocytes at differentlipid-to-peptide ratios (Fig. 4B; note the increase in leakagescale compared with Fig. 4A). It can be observed that the extentof leakage was greatly reduced compared to the model systemsstudied before. However, two patterns of leakage can be discerned,i.e., regions defined by peptide 119 and by peptides 162 and163 (Fig. 4B).

DISCUSSION

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Discussion

Recent studies point out that CoV spike envelope glycoproteinscan be classified as class I viral fusion proteins, and functionaland biochemical analyses of the SARS CoV spike glycoproteinshow that SARS CoV uses a membrane fusion mechanism that issimilar to that of class I viral fusion proteins (5, 6, 20,22, 43, 50). The SARS CoV spike glycoprotein consists of anextracellular domain, a TM domain, and an intracellular domain(Fig. 1A). SARS CoV spike glycoprotein does not contain a typicalproteolytic cleavage site, and the boundary between the S1 andS2 domains cannot be accurately identified at present (22).In addition, the available data suggest that the SARS CoV spikeglycoprotein is not cleaved into two subunits, so most likelythe S1 and S2 domains remain covalently bonded during the fusionprocess (9, 35). A signal peptide at the amino terminus hasbeen recognized, and analogous to other class I viral fusionproteins, two ${alpha}$ -helical heptad repeat domains, HR1 and HR2, havebeen identified (Fig. 1A). Although no obvious fusogenic peptidesequence has been experimentally identified, it is clear thatviral and cellular membrane fusion is mediated by an internalFP in the SARS CoV spike glycoprotein. Nevertheless, a hydrophobicregion located upstream of the HR1 region has been recentlypredicted to be the FP (5).

Several lines of evidence indicate that, in addition to classicalFPs, different regions of viral envelope glycoproteins are essentialfor membrane fusion to occur. In the case of the HIV type 1(HIV-1) gp41 ectodomain, we have recently shown that, apartfrom the FP, three different regions of this protein, namelythe 15- to 20-residue peptide segment that follows the FP atthe N terminus, the immunodominant loop, and the 10- to 15-residuepeptide segment that precedes the TM domain at the C terminus,i.e., the PTM domain, are capable of modifying the biophysicalproperties of phospholipid membranes, suggesting a direct rolein membrane fusion (25). In the case of the SARS CoV spike glycoprotein,both the S1 and S2 subunits probably remain attached to eachother during the fusion process; however, the most probablerole for the S1 subunit is the attachment to its specific receptor,whereas the S2 subunit is responsible for the fusion betweenthe viral and cellular membranes (35, 47). As mentioned above,peptides 34, 50, and 85, which belong to the S1 domain, showedrelatively significant membranotropic activity (Fig. 2). Similarto the HIV gp120 inner-domain regions interacting with gp41(48), the regions of the S1 subunit where these peptides residecould associate with the S2 domain by specific hydrophobic interactionsbefore the S1 subunit itself interacts with its receptors. Asdescribed above, these hydrophobic patches might be importantnot only for protein-protein binding but also for membrane interaction,since S1 probably remains covalently attached to the S2 domainduring the fusion process. In fact, we have found that severalpeptides derived from the HIV gp120 region suggested to associatewith its receptors have a significant effect on membrane leakage(unpublished data).

Hydrophobic moments measure the periodicity of residue distributionalong a secondary-structure element (12, 13). The preferentialorientation of the hydrophobic moments toward one face of thatelement (Fig. 5, top) has been proposed to favor hydrophobicinteractions between proteins and/or between proteins and membranes.In order to detect surfaces along the S2 domain of the SARSCoV spike glycoprotein that might be identified as membrane-partitioningand/or membrane-interacting zones, we plotted the average surfacehydrophobic moments, hydrophobicity, and interfaciality versusthe S2 domain amino acid sequence, assuming it adopts an ${alpha}$ -helicalstructure along the whole sequence (Fig. 5, bottom). It is readilyevident that there are three different regions with high positivevalues covering the surface of the helix and along it: one ofthese regions is located immediately upstream of the HR1 region(region 1 [R1]), another one is located between the heptad repeatsHR1 and HR2 (R2), and the last is located at the end of theS2 domain sequence (R3). The last region partially matches theproposed TM domain of the spike glycoprotein. These regions,having positive bilayer-to-water transfer free-energy values,might show a tendency to partition into and interact with membranesurfaces or proteins. The adoption of a specific conformationcan generate rich hydrophobic surfaces along the structure andemphasize that the actual distribution of hydrophobicity andinterfaciality, i.e., structure-related factors, along the S2domain sequence might affect the biological function of thesesequences (36).

As mentioned above, peptides 113, 119, 123, 147, and 162-163were the peptides that exerted the most significant effect onmodel membranes, with peptide 162 the one that showed the mostdramatic effects, since nearly 100% leakage was observed inmany of the different compositions studied (Fig. 3 and 4). Theextents of leakage observed for the other peptides were notas high as for peptide 162, but they were significantly higherthan for the rest of the S2-derived peptides tested. Interestingly,peptide 119 showed the most important effect on pneumocyte-derivedmodel membranes. We do not know the lipid compositions of thesemodel membranes, but they must be very complex; nevertheless,two regions, defined by peptides 119 and 162-163, can be discerned(three if we count peptide 147 [Fig. 4B]). Actually, some peptidesthat are very active in model membrane systems are much lessactive in the biological membrane system, and the contrary isalso true. However, it should be noted that membrane systemshaving different lipid compositions have diverse propertieswhich make them behave in significantly different ways (24).What is outstanding is that peptides 113 (ARDLICAQKFNGLTVL^828-843)and 123 (VLYENQKQIANQFNKAI^897-913) are located at the boundariesof R1, peptide 119 (GAALQIPFAMQMAYRF^873-888) is located insideR1, peptide 147 (FVFNGTSWFITQRNFF^1077-1092) is located insideR2, and peptides 162 and 163 (LGKYEQYIKWPWYVWLGF^1185-1202 andKWPWYVWLGFIAGLIAIV^1193-1210) are located inside R3; all theseregions represent surfaces with high bilayer-to-water transferfree-energy values (Fig. 5, bottom).

As mentioned above, the FP, a stretch of hydrophobic amino acids,is an essential factor in viral fusion proteins; however, ithas not been identified, although it was recently predictedthat the sequence comprising residues 858 to 886 from the S2domain of the SARS CoV spike glycoprotein is indeed the FP domain(5). Peptide 119, which has a significant experimental membranotropiceffect, as shown here, belongs to R1, as depicted in Fig. 5,bottom, and comprises residues 873 to 888 of the S2 domain ofthe SARS CoV spike glycoprotein. Since this peptide overlapsthe theoretically predicted FP sequence 858 to 886 (5), thesequence is therefore a candidate to be the FP domain of theSARS CoV spike glycoprotein. Inspection of the sequence wherethis peptide is located reveals that it has a high content ofalanine and glycine, characteristic of viral fusion peptides(15), and a high degree of interfaciality and hydrophobicity,essential properties for membrane-interacting sequences in proteins(Fig. 6). Its location immediately upstream of HR1, as foundfor other class I viral fusion proteins, is appropriate, sincethe formation of the six-helix coiled-coil bundle would bringinto close proximity the FP and the PTM and TM domains (11,15, 17).

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FIG. 6. Analysis of the interfacial and hydrophobic distribution using a window of 15 amino acids according to the scales of Wimley and White (46) along the proposed FP, PTM, and TM sequences of the SARS CoV spike glycoprotein S2 domain. Values above the 0 level correspond to positive bilayer-to-water transfer free-energy values.

Another fundamental structural and functional sequence for aclass I viral fusion mechanism is the PTM, and one of the mostthoroughly studied PTM sequences is that belonging to HIV-1gp41 (27, 39, 41). The PTM is a region immediately adjacentto the membrane-spanning domain of class I fusion proteins containinghighly conserved hydrophobic residues and unusually rich intryptophan residues. In addition, the PTM domain of HIV-1 gp41shows a significant tendency to partition into membranes andis highly fusogenic (41). Peptides 162 and 163 from the SARSCoV S2 domain exert a dramatic effect on leakage for differentmodel membranes (Fig. 3 and 4), and a careful inspection ofthe sequences reveals that they have characteristics similarto those of the PTM domain of HIV-1 gp41, notably, high tryptophancontent and an interfacial stretch immediately following a hydrophobicone (Fig. 6). It is likely, then, that peptides 162 and 163comprise the PTM domain of the SARS CoV spike glycoprotein S2subunit and, similar to the PTM domain of HIV-1 gp41, this sequenceof the SARS CoV S2 subunit might be involved in the promotionof the membrane destabilization required for fusion, as wellas in fusion pore formation and enlargement (32, 36).

Not only are the FP and PTM domains of HIV-1 gp41 essentialfor the perturbation of the membrane, but also, other regionsof the protein interact and destabilize the viral and host membranes;this is what has been suggested for the HIV-1 gp41 immunodominantloop, which could play an essential role in the viral fusionprocess (8, 25, 29). One of the main characteristics of thegp41 loop domain, located between the HR1 and HR2 regions andinstrumental in the formation of the gp41 central parallel triple-stranded ${alpha}$ -helical coiled coil, is its tendency to partition into andinteract with membrane surfaces (8, 25, 29). We have found ahighly hydrophobic and interfacial domain flanked by SARS CoVHR1 and HR2 domains (R2) (Fig. 5, bottom), and remarkably, peptide147, which has shown a notable leakage effect, is located init. Therefore, R2 could play a role similar to that of the HIV-1gp41 loop domain, i.e., destabilization of both viral and cellularmembranes, favoring the formation of the fusion pore as wellas its stabilization and therefore facilitating, along withthe other regions, membrane fusion.

In conclusion, our results demonstrate that peptides originatingfrom three different regions of the SARS CoV spike glycoprotein(Fig. 5, bottom)—R1, which corresponds to the 15- to 20-residuepeptide segment immediately upstream of HR1; R2, which correspondsto a sequence between HR1 and HR2; and R3, which correspondsto the 10- to 15-residue peptide segment that immediately precedesthe TM domain at the C terminus—are capable of modifyingthe biophysical properties of phospholipid membranes, a propertywhich could provide an additional driving force for the mergingof the viral and target cell membranes, suggesting that theyhave a direct role in SARS CoV-mediated membrane fusion andtherefore might be necessary for assistance and enhancementof the viral and cell fusion process. These results should alsofacilitate the development of SARS CoV entry inhibitors, whichmight lead to new vaccine strategies, an important focus forclinical intervention.

ACKNOWLEDGMENTS

This work was supported by grant BMC2002-00158 from MCYT, Madrid,Spain (J.V.). M. R. Moreno and A. J. Pérez-Bernáare recipients of predoctoral fellowships from Ministerio deEducación y Ciencia and Generalidad Valenciana, Spain,respectively.

We are especially grateful to Maite Garzón for her excellentassistance, as well as to the National Institutes of HealthAIDS Research and Reference Reagent Program for making availablethe peptides used in this work.

FOOTNOTES

* Corresponding author. Mailing address: Instituto de Biología Molecular y Celular, Universidad "Miguel Hernández", E-03202 Elche-Alicante, Spain. Phone: 34 966 658 762. Fax: 34 966 658 758. E-mail: jvillalain{at}umh.es.

REFERENCES

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