Characterization of a Membrane-Associated Trimeric Low-pH-Induced Form of the Class II Viral Fusion Protein E from Tick-Borne Encephalitis Virus and Its Crystallization

The interaction of a dimeric membrane anchor-free form of theenvelope protein E (sE dimer) from tick-borne encephalitis viruswith liposomes at acidic pH levels leads to its conversion intomembrane-inserted sE trimers. Electron microscopy shows thatthese trimers have their long dimensions along the threefoldmolecular axis, which is oriented perpendicularly to the planeof the membrane, where the protein inserts via the internalfusion peptide. Liposomes containing sE at their surface displayparacrystalline arrays of protein in a closely packing arrangementin which each trimer is surrounded by six others, suggestingcooperativity in the insertion process. sE trimers, solubilizedwith nonionic detergents, yielded three-dimensional crystalssuitable for X-ray diffraction analysis.

INTRODUCTION

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Introduction

Membrane fusion is a key step during entry of enveloped virusesinto cells. This function is mediated by viral surface glycoproteins(fusion proteins) that undergo extensive conformational changesupon receptor binding (fusion at the plasma membrane) or exposureto low pH (fusion at the endosomal membrane) (11, 23). In manycases, these triggers have been shown to release the fusionproteins from a metastable state and allow their conversionto a lower energy state. This structural change leads to theexposure of a previously buried functional element (fusion peptide)and is believed to provide the energy required for the mergerof the lipid bilayers (4, 5). So far, viral fusion proteinshave been shown to fall into two different structural classes,designated class I and class II (15). Class I fusion proteinsof orthomyxo-, paraymxo-, retro-, and filoviruses possess amino-terminalor amino-proximal fusion peptides and have a postfusion structurein which a triple-stranded alpha-helical coiled coil is a centralfeature (5, 19, 23). The atomic structures of protein fragmentscontaining the trimeric six-helix bundle have been determinedfor a number of class I fusion proteins (reviewed in reference5).

Class II viral fusion proteins, which have so far been foundin flavi- and alphaviruses, have a completely different structure.They are oriented parallel to the membrane, possess an internalfusion peptide, and form part of an icosahedral network in thevirion envelope. Fusion is triggered by acidic pH, which leadsto structural changes that convert the metastable fusion proteinspresent in mature virions into stable homotrimers (reviewedin references 8 and 13).

We are studying the structural basis of class II-mediated viralmembrane fusion using the flavivirus tick-borne encephalitisvirus (TBEV). As shown previously, exposure of the native, homodimericform of the fusion protein E to low pH leads to its irreversibleconversion into a homotrimer. In contrast, a truncated formof the E dimer (sE dimer) lacking the carboxy-terminal membraneanchor and the consecutive 40 amino acids (referred to as "stem")(3, 20) was shown to dissociate at low pH but not to form trimersin the absence of membranes (20). Trimerization did occur, however,when sE was acidified in the presence of liposomes, and thesetrimers remained bound to the target membrane (21). Similarto the lipid-induced trimerization of sE dimers of TBEV, ithas been shown that a C-terminally truncated form of the alphavirusclass II fusion protein is able to trimerize at low pH in thepresence of liposomes, but not when membranes are absent (14).

Here we show that sE trimers from TBEV form paracrystallinearrays on the surface of liposomes. Upon solubilization withnonionic detergents, these trimers yielded crystals suitablefor structure determination by X-ray diffraction analysis.

Liposome-associated sE trimers.

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Liposome-associated se trimers.

As starting material, native sE dimers were first prepared bylimited trypsin digestion of purified TBEV and purificationvia anion-exchange chromatography (9). To convert the dimersto trimers, we then exposed them to acidic pH in the presenceof large unilamellar liposomes, as described previously (21).Briefly, sE dimers were mixed with liposomes consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine(PE), and 1-cholesterol (CH) (molar ratio, 1:1:2) at a ratioof 1 µg of sE to 15 nmol of lipid (22). This mixture wasacidified with 300 mM morpholineethanesulfonic acid (MES), incubatedfor 30 min at 37°C at pH 5.4, back neutralized, adjustedto 20% (wt/wt) sucrose in 20 mM triethanolamine (TEA) and 130mM NaCl (pH 8.0) (TAN buffer), and was then applied to a 50%cushion, overlaid with 15% (wt/wt) sucrose and 5% (wt/wt) sucrose.Centrifugation was carried out for 1.5 h at 50,000 rpm at 4°Cin a Beckman SW 55 rotor, and the top fractions were collectedby upward displacement as described in reference 21. Under theseconditions, about 60 to 80% of the sE proteins were found tobind and float with the liposomes to the top of the gradient(Fig. 1A). Electron microscopy of these fractions stained withuranyl acetate revealed that the resulting sE trimers were distributedin patches of regular lattices on the surface of the liposomes,and structures with threefold rotational symmetry could be observedin the photomicrographs (Fig. 1B and C). Top views of the Etrimer display a donut appearance with inner and outer diametersof about 2 and 5 nm, respectively (Fig. 1B and D). The sideviews show conical shapes with mean width and height of about5 and 9 nm, respectively (Fig. 1C and D). The donut-shaped structuredisplaying threefold rotational symmetry is in agreement withthe biochemical data demonstrating that sE forms trimers duringits insertion into target membranes (21). All recognizable trimersinserted in the liposomes are closely packed, forming a latticein which each trimer is surrounded by six others. The arrangementof sE trimers at the liposome surface is different from thatobserved in the case of the Semliki Forest virus (SFV) E1* fragment,which displays a hexagonal lattice with a central hole (7) insteadof a central trimer, as observed here. The neutral pH form ofE is an elongated, brick-shaped dimeric molecule in which eachsubunit is contained within a cylinder having a diameter andheight of about 3 and 11 nm, respectively (17). In the dimer,the long dimension of the molecule is parallel to the viralmembrane. The fact that the long dimension of the trimer isnow along the threefold molecular axis, perpendicular to theliposome membrane, indicates that the E subunit has reorientedso that it lies perpendicular to its original orientation onthe virus. All these results are similar to those obtained withthe class II fusion protein E1 of the alphavirus Semliki Forestvirus, which also clusters in the plane of the membrane to formhexagonal lattices of E1 trimers (7). It can therefore be proposedthat the strong protein interactions leading to these arraysare an important parameter for the membrane fusion event inboth virus systems. Differences between flavi- and alphaviruses,however, exist in terms of the lipid dependence of membraneinsertion, trimerization, and fusion (6, 14, 22, 24). In contrastto alphaviruses, these processes in flaviviruses are not absolutelydependent on cholesterol and do not require sphingomyelin (6,22).

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FIG.1. (A) Coflotation of TBEV sE protein with liposomes at low pH. sE dimers were mixed with liposomes and acidified (pH 5.4), and sucrose was added to a final concentration of 20% (wt/wt). The mixture was applied to a 50% sucrose cushion (wt/wt), overlaid with 15% sucrose (wt/wt) and 5% sucrose (wt/wt), and subjected to ultracentrifugation as described previously (21). The amount of E protein in each fraction was determined by a quantitative four-layer ELISA after denaturation of the samples with 0.4% sodium dodecyl sulfate (10). The bracket indicates the top fractions, containing sE bound to liposomes. (B and C) Electron photomicrographs of liposome-bound sE trimers. The fraction containing sE inserted in liposome was negatively stained with a 2% uranyl acetate solution. sE forms clusters at the surface of the liposomes, displaying closely packed arrays. Two characteristic views, top and side, are shown. Panel B shows top views. The marked area (T) is enlarged in the inset. Note that the protein packing is much denser than in the case of E1 of SFV (7). Panel C shows mainly side views of the protein. The marked area (S) is enlarged in the inset. Top and side views have a donut and conical appearance, respectively. (D) Schematic of top (donut) and side (conical) views of sE trimers.

Isolated sE trimers.

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Isolated se trimers.

To isolate the sE trimers from liposomes and to remove the lipids,the membranes were solubilized with 1.5% n-octylglucoside (n-OG),followed by ultrafiltration using Vivaspin20 concentrators witha cutoff of 100 kDa (Vivascience AG, Hannover, Germany) andTAN buffer (pH 8.0) containing 0.8% n-OG. As revealed by rate-zonalcentrifugation in sucrose density gradients containing 0.8%n-OG (22), more than 90% of the material formed a defined peakat the position corresponding to an E trimer, and only a smallamount (5 to 10%) sedimented as a dimer (Fig. 2A). The trimericstate of the protein in the major peak was further confirmedby cross-linking with dimethylsuberimidate (DMS) (Fig. 2A, inset).

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FIG. 2. Sedimentation analysis of sE trimers in the presence (A) and absence (B) of detergent. The gradients were fractionated, and the amount of E protein in each fraction was determined by a quantitative four-layer ELISA after denaturation of the samples with 0.4% sodium dodecyl sulfate (10). The sedimentation direction is from left to right, and the arrows indicate the sedimentation positions of sE monomer (M), dimer (D) , and trimer (T) analyzed in the presence of detergent. P indicates sE recovered from the pellet. (A) sE trimers were subjected to sedimentation in 7 to 20% sucrose gradients in TAN buffer (pH 8.0) containing 0.8% n-OG as described in reference 22. The inset shows the Western blot analysis after chemical cross-linking of the trimer peak fraction with dimethylsuberimidate as described in reference 18. Panel B shows data for sE trimers (•) that were subjected to sedimentation in detergent-free 7 to 20% (wt/wt) sucrose gradients in TAN buffer (pH 8.0). As controls, sE dimers ( ${blacktriangleup}$ ) and sE monomers ( ${blacksquare}$ ) were used. The monomers were generated by incubation of sE dimers at pH 5.4, and sedimentation was carried out at pH 5.4 in MES buffer (50 mM MES, 100 mM NaCl) instead of pH 8.0.

To investigate whether the sE trimer remains soluble after removalof the detergent, we also analyzed its sedimentation behaviorin detergent-free sucrose gradients. sE monomers produced bydissociation of sE dimers at low pH (20) and sE dimers wereused as sedimentation standards; these behaved as expected forsoluble proteins in the absence of detergent (Fig. 2B). In thecase of sE trimers, however, most of the material was foundas a pellet at the bottom of the gradient, indicating that theremoval of the detergent had caused aggregation due to the exposureof hydrophobic sequence elements (Fig. 2B). The sE trimer thusappears to be more hydrophobic than either the sE monomer orsE dimer. Because the fusion peptide is presumably exposed inboth the monomer and the trimer (21), it is probable that otherstill-unidentified regions of the trimer contribute to thisincreased hydrophobicity. All further experiments with isolatedsE trimers were therefore carried out in the presence of detergents.

MAb reactivity of liposome-associated and isolated sE trimers.

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Mab reactivity of liposome...

Site-directed mutagenesis experiments have provided evidencethat the sequence element (designated the cd loop) at the tipof domain II of the E protein functions as an internal fusionpeptide and that it forms part of the epitopes recognized bythe monoclonal antibodies (MAbs) A1 and A2 (2) (Fig. 3B). Toobtain information about interactions of this sequence elementwith the liposomal membrane, we analyzed the reactivity of thesetwo MAbs with the membrane-bound and detergent-solubilized formof the sE trimer in a blocking enzyme-linked immunosorbent assay(ELISA) (12). In the course of this assay we investigated whetherthe binding of the MAbs to virions coated to the solid phasecould be inhibited by the two different trimer preparationsin solution. Although the fusion peptide loops and thus theepitopes recognized by A1 and A2 are normally buried in thenative state of E, they become accessible due to partial denaturationof the virions adsorbed to the plastic surface (12), allowingMAbs that are not bound to trimer preparations to be detectedby ELISA.

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FIG. 3. (A) Blocking ELISA with liposome-associated and solubilized sE trimers. A predetermined fixed dilution of the indicated MAb was mixed with decreasing concentrations of isolated detergent-solubilized trimers (solid lines) or liposome-bound trimers (dashed lines) as indicated. The mixture was added to microtiter plates that had been coated with purified virus at a concentration of 1 µg/ml. Free antibody that was not blocked by the antigen in solution was detected by using peroxidase-labeled rabbit anti-mouse immunoglobulin G as described in reference 12. Results are expressed as percentage of the absorbance value obtained with each MAb in the absence of a blocking antigen. (B) Top view of a single monomeric subunit of sE in its native conformation (16). The three domains of E are shown in different shades of grey. The MAbs reacting with each of the domains are indicated within the brackets.

As shown in Fig. 3A, the liposome-associated form of sE (shownin Fig. 1) did not react with MAbs A1 and A2. Their reactivity,however, was restored when the liposome-protein complex wasdispersed by detergent treatment (Fig. 3B). In contrast, thecontrol MAbs C4 (specific for domain I) and B4 (specific fordomain III) bound to their epitopes on the liposome-associatedform. These data provide evidence that the cd loop interactsdirectly with the target membrane and is inserted in the liposomalmembrane after the low-pH-induced conversion to a trimer, consistentwith its function as a fusion peptide (Fig. 3B). A similar resultwas obtained with the truncated form of SFV E1, which was shownto insert into liposomes at low pH via its fusion peptide (1).At present, however, it is still unknown how far domain II penetratesinto the membrane. MAb C4 consistently reacted better with theliposome-associated form than with the solubilized form, butso far we do not have a structural explanation for this finding.

Crystallization of sE trimers.

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Crystallization of se trimers.

Crystallization trials were carried out with isolated sE trimersin different detergents by using the hanging-drop method (16).The initial buffer (TAN buffer containing 0.8% n-OG) was exchangedfor 0.1 M ammonium acetate (pH 5.0) containing the correspondingdetergent at a concentration above the critical micellar concentration(1.5 mM undecyl maltoside, 1.0 mM dodecyl maltoside, 3 mM N,N-dimethyldodecylamine oxide [LDAO], or 15 mM N,N-dimethyl decylamineoxide [DDAO]) by using the following procedure. The solutionwas concentrated to 2 to 5 mg of trimer per ml by ultrafiltrationwith a Vivaspin500 concentrator (cutoff, 100 kDa). The trimersolution was then diluted 10-fold in the ammonium acetate bufferand reconcentrated to 2 to 5 mg/ml. The procedure was repeatedfour times before crystallization trays were set up. Crystalswere only obtained in the presence of DDAO. Several crystalforms were characterized (Table 1). They grew either at pH 7.8in 0.1 M Tris, 2 M ammonium sulfate, 2% polyethylene glycol(PEG) 400 or at pH 4.5 in 0.1 M sodium acetate with PEG as theprecipitant (20 to 30% PEG 2000, 15 to 20% PEG 4000, 10 to 20%PEG 10000, 10 to 20% PEG 20000). At pH 7.8, the crystals belongedeither to the cubic space group P2₁3 with two molecules perasymmetric unit (yielding eight trimers to make up the unitcell) or to the tetragonal space group P4₂22 with one trimerper asymmetric unit. These two forms were easily distinguishedby their appearance (cubic and rodlike, respectively) (Fig.4A). At pH 4.5, several orthorhombic crystal forms were grownthat could not be visually distinguished (Fig. 4B). The diffractionquality of these crystals is sufficient to allow structure determination(Table 1).

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TABLE 1. Growth conditions and characteristics of sE trimer crystals

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FIG. 4. Crystals of sE trimers grown in the presence of detergent (15 mM DDAO). (A) Cubic and tetragonal (rods) crystal forms that grow simultaneously in the same drops at pH 7.8. (B) Orthorhombic crystals grown at pH 4.5.

The described results represent a major step forward towarda more detailed understanding of the fusion process mediatedby class II viral fusion proteins. The formation of paracrystallinearrays of sE trimers upon membrane insertion suggests strongtrimer-trimer interactions that may be important for cooperativeeffects during the fusion process. Together with the alreadyknown structure of the native E dimer (17), the resolution ofthe low-pH structure will reveal details of the structural transitionsof E that are involved in fusion and thereby provide new insightsinto the fusion process mediated by class II proteins at a molecularlevel.

ACKNOWLEDGMENTS

We thank Steven Allison for helpful discussions and for criticalreading of the manuscript, Don Gibbons for technical discussions,and Walter Holzer, Silvia Röhnke, and Armelle Vigourouxfor technical assistance.

Stéphane Bressanelli has a long-term EMBO fellowship.

FOOTNOTES

* Corresponding author. Mailing address: Institute of Virology, University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria. Phone: 43-1-40490, ext. 79539. Fax: 43-1-40490, ext. 9795. E-mail: karin.stiasny{at}univie.ac.at.

REFERENCES

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