Membrane Interactions of the Tick-Borne Encephalitis Virus Fusion Protein E at Low pH

Membrane fusion of the flavivirus tick-borne encephalitis virusis triggered by the mildly acidic pH of the endosome and ismediated by envelope protein E, a class II viral fusion protein.The low-pH trigger induces an oligomeric rearrangement in whichthe subunits of the native E homodimers dissociate and the monomericsubunits then reassociate into homotrimers. Here we provideevidence that membrane binding is mediated by the intermediatemonomeric form of E, generated by low-pH-induced dissociationof the dimer. Liposome coflotation experiments revealed thatassociation with target membranes occurred only when liposomeswere present at the time of acidification, whereas pretreatingvirions at low pH in the absence of membranes resulted in theloss of their ability to stably attach to liposomes. With thecleavable cross-linker ethylene glycolbis(succinimidylsuccinate),it was shown that a truncated soluble form of the E protein(sE) could bind to membranes only when the dimers were freeto dissociate at low pH, and binding could be blocked by a monoclonalantibody that recognizes the fusion peptide, which is at thedistal tip of the E monomer but is buried in the native dimer.Surprisingly, analysis of the membrane-associated sE proteinsrevealed that they had formed trimers. This was unexpected becausethis protein lacks a sequence element in the C-terminal stem-anchorregion, which was shown to be essential for trimerization inthe absence of a target membrane. It can therefore be concludedthat the formation of a trimeric form of sE is facilitated bymembrane binding. Its stability is apparently maintained bycontacts between the ectodomains only and is not dependent onsequence elements in the stem-anchor region as previously assumed.

INTRODUCTION

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Introduction

Enveloped viruses have evolved different but conceptually relatedmechanisms to fuse their membranes with cellular membranes duringentry into cells. The process is controlled by viral surfaceglycoproteins that undergo triggered conformational changesrequired for mediating fusion (16, 30).

At least two different classes of viral fusion proteins canbe distinguished (21). Class I is represented by orthomyxo-,retro-, paramyxo-, and filoviruses. Their fusion proteins matureby proteolytic cleavage of a precursor protein, yielding a membrane-anchoredsubunit with an amino-terminal or amino-proximal fusion peptide.Application of the fusion trigger (receptor binding or low pH)results in the formation of a characteristic trimeric postfusionstructure with a triple-stranded coiled coil at its core (6,17, 27, 30). Class II fusion proteins are not proteolyticallycleaved and have internal rather than amino-terminal fusionpeptides. They are synthesized as a complex with a second membraneglycoprotein, and the activation of the fusogenic potentialinvolves the cleavage of this accessory protein (11, 12, 18).X-ray crystallography of two class II fusion proteins, the Eprotein of the flavivirus tick-borne encephalitis (TBE) virus(24) and the E1 protein of the alphavirus Semliki Forest virus(SFV) (21), has revealed a common overall fold for these proteins,which are structurally unrelated to class I viral fusion proteins,for which the influenza virus hemagglutinin is the prototype(5, 31).

The E protein of TBE virus forms flat head-to-tail homodimers(24) that are oriented parallel to the viral membrane and organizedin an icosahedral lattice (9), another characteristic that flaviviruseshave in common with alphaviruses (21, 23). At the mildly acidicpH at which membrane fusion occurs, the virion surface undergoesa concerted rearrangement involving a dissociation of the nativeE homodimers and a subsequent irreversible reorganization intoa trimeric form (2, 29).

Full-length E dimers isolated by treating virions with detergenthave been shown to undergo the low-pH-driven dimer-trimer transitionin solution, but truncated forms lacking the membrane anchorand connecting stem region undergo the dissociation step withouttrimerizing. It has been proposed, based on deletion mapping,that a predicted ${alpha}$ -helix extending from amino acids 401 to 413in the proximal stem region is involved in trimer formation(3, 29), but its exact role in this process is not yet clear.

Although the low-pH-induced rearrangements of the TBE virusE protein have been studied fairly extensively, most studiesso far have been carried out in the absence of membranes. Inthe present work, we used in vitro experiments to relate thesechanges to the first step of the fusion process, the bindingof the E protein to a target membrane. We provide evidence thatthe low-pH-induced dissociation of the E homodimer during thedimer-to-trimer transition is necessary for the initial interactionsof the virus with its target membrane and that, contrary toearlier predictions, membrane-bound E proteins can form stablehomotrimers even when the stem-anchor region has been removed.

MATERIALS AND METHODS

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Materials and Methods

Virus growth and purification. TBE virus strain Neudoerfl was grown in primary chicken embryocells, harvested 48 h after infection, and purified by 2 cyclesof sucrose density gradient centrifugation as described elsewhere(13).

Preparation of sE dimers. Truncated soluble E dimers (sE dimers) were generated by limitedtrypsin digestion of purified virions at 0°C. Residual particleswere removed by ultracentrifugation, and the purification ofthe sE dimers was performed by anion-exchange chromatographyas described previously (14).

Liposomes. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine(Avanti Polar Lipids, Inc., Birmingham, Ala.) and 1-cholesterol(Sigma) were mixed in a molar ratio of 1:1:1 from stock solutionsin chloroform. The mixture was dried to a thin film with a rotaryevaporator and then dried further in high vacuum for at least2 h. The lipid film was hydrated in 10 mM triethanolamine (pH8.0)-140 mM NaCl and subjected to 5 cycles of freeze-thawing,followed by 21 cycles of extrusion through two 200-nm-pore-sizepolycarbonate membranes with a Liposofast syringe-type extruder(Avestin, Ottawa, Canada).

Coflotation of virions with liposomes. Virions were mixed with liposomes in a ratio of 1 µg ofE protein to 300 nmol of lipid and incubated for 5 min at 37°C.The mixture was acidified with 150 mM morpholineethanesulfonicacid (MES), incubated for 10 min at pH 5.5 and 37°C, back-neutralizedwith 150 mM triethanolamine, adjusted to a final volume of 2ml of 20% (wt/wt) sucrose in TAN buffer (0.05 M triethanolamine[pH 8.0], 0.1 M NaCl), and layered onto a 1-ml 50% (wt/wt) sucrosecushion. The gradients were completed with a layer of 1 ml of5% (wt/wt) sucrose. Centrifugation was carried out for 2 h at50,000 rpm at 4°C in a Beckman SW55 rotor, and fractionsof 200 µl were collected by upward displacement with anISCO model 640 fraction collector. The amount of E protein ineach fraction was determined by a quantitative four-layer enzyme-linkedimmunosorbent assay after denaturation of the samples with 0.4%sodium dodecyl sulfate (SDS) (15).

Coflotation of sE dimers with liposomes. sE dimers were mixed with liposomes in a ratio of 1 µgof sE protein to 15 nmol of lipid and incubated for 5 min at37°C. The samples were acidified with 300 mM MES, incubatedfor 30 min at 37°C at pH 5.5, back-neutralized, and adjustedto a final volume of 0.4 ml of 20% (wt/wt) sucrose as describedabove. The 0.4-ml sE protein-liposome mixture was applied toa 50% cushion and overlaid with 1.6 ml of 15% (wt/wt) sucroseand 1 ml of 5% (wt/wt) sucrose. Acidic step gradients were madein the same way with MES buffer (0.05 M MES [pH 5.5], 0.1 MNaCl) instead of TAN buffer (pH 8.0). Centrifugation was carriedout as described above for coflotation of virus with liposomes.For the inhibition of coflotation by monoclonal antibodies (MAbs),the sE protein (final concentration, 50 µg/ml) was incubatedwith E protein-specific MAbs (final concentration, 100 µg/ml)for 30 min at pH 8.0 or 5.5 and room temperature before theaddition of liposomes.

Chemical cross-linking of sE dimers with EGS and cleavage of EGS with hydroxylamine. For coflotation experiments with cross-linked sE dimers, thesamples were incubated for 30 min at room temperature in thepresence of 1 mM ethylene glycolbis(succinimidylsuccinate) (EGS)(Pierce). The reaction was stopped by the addition of Tris-HCl(pH 7.5) to a final concentration of 62.5 mM Tris. Cleavageof the cross-linker was achieved by incubation of the samplesin the presence of 1 M hydroxylamine-HCl (Pierce) at pH 8.5for 3 h at 37°C. The efficiency of cross-linking and cleavagewas analyzed by SDS-polyacrylamide gel electrophoresis (PAGE)(22). Before the samples were used for coflotation with liposomes,hydroxylamine was washed out by membrane filtration (Ultrafree30K; Millipore Corp, Bedford, Mass.) to a final concentrationless than 0.3 mM.

Sedimentation analysis. To analyze the oligomeric structure of E proteins that coflotatedwith the liposomes, the corresponding fractions were solubilizedwith 2% Triton X-100 for 1 h at room temperature. Then the sampleswere applied to 7-to-20% (wt/wt) continuous sucrose gradientsin TAN buffer (pH 8.0) or MES buffer (pH 5.5) containing 0.1%Triton X-100, and centrifugation was carried out for 20 h at38,000 rpm at 15°C in a Beckman SW40 rotor. The amount ofE protein in the fractions was determined by a quantitativeenzyme-linked immunosorbent assay as described above. As controls,whole virions or sE dimers were subjected to the same low-pHtreatment as described for the coflotation assay, but in theabsence of liposomes.

Chemical cross-linking with DMS, SDS-PAGE, and immunoblotting. The E-protein-containing fractions from the sedimentation analyseswere subjected to cross-linking with dimethylsuberimidate (DMS)as described earlier (2). The cross-linked samples were separatedby electrophoresis on SDS-5% polyacrylamide gels with a phosphate-bufferedsystem (22), blotted onto polyvinylidene difluoride membranes(Bio-Rad) with a Bio-Rad Trans-Blot semidry transfer cell, anddetected and visualized immunoenzymatically as described previously(25).

RESULTS

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Results

Interactions of virions with liposomes. In order to investigate the interactions of TBE virus with targetmembranes at an acidic pH, we studied the association with liposomesby coflotation analysis. Native virions were exposed to pH 5.5in the presence of liposomes, back-neutralized, and subjectedto centrifugation in sucrose step gradients at pH 8.0. As acontrol, the virions were kept at pH 8.0 during the whole procedure.After centrifugation, the E proteins from native virions treatedat low pH in the presence of target membranes were found atthe top of the gradient together with the liposomes, whereasno association was observed in controls incubated at pH 8.0,indicating that this association was completely dependent onlow pH (Fig. 1).

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FIG. 1. Liposome coflotation assay with three different samples of TBE virus. Native virions were incubated with liposomes for 10 min at pH 8.0 ( ${circ}$ ), incubated with liposomes for 10 min at pH 5.5 and readjusted to pH 8.0 (•), or preincubated for 10 min at pH 5.5 without a target membrane, back-neutralized, incubated for 10 min at pH 5.5 in the presence of liposomes, and readjusted to pH 8.0 ( ${blacklozenge}$ ). These samples were then analyzed by sucrose step gradient centrifugation at pH 8.0. The positions of the sucrose layers are shown below the curves; the 20% layer contains the virus-liposome mixture before centrifugation.

When virions were preexposed for 10 min to pH 5.5 in the absenceof a target membrane, back-neutralized, mixed with liposomes,and then subjected to the same treatment as above, they wereunable to stably associate with liposomes and instead sedimentedto the 20-to-50% interface of the gradient, similar to the pH8.0 control (Fig. 1). This indicates that the target membranemust be present at the time of acidification for membrane associationto occur. The same results were obtained when samples were notback-neutralized and the coflotation analysis was done at pH5.5 (data not shown).

Binding of truncated soluble E proteins (sE) to liposomes. Coflotation experiments using virions whose membranes had beenmetabolically labeled with fluorescent lipids (8) revealed thatnot only had the virions in the top fractions attached to theliposomes but also, as expected, membrane fusion had occurred(data not shown). Therefore, to experimentally uncouple theattachment step from the viral membrane fusion event, we carriedout similar coflotation experiments with sE instead of wholevirions. The sE protein, which was used previously for structuredetermination by X-ray crystallography (24), is a homodimerthat lacks the approximately 100 C-terminal amino acids comprisingthe stem-anchor region (14). It was shown in an earlier studythat the sE dimers, when acidified in the absence of membranes,dissociate into monomers without forming trimers and that back-neutralizationrestores the original dimeric state (29).

sE dimers were incubated at pH 8.0 or 5.5 in the presence ofliposomes and subjected to centrifugation in step gradientsat the corresponding pH. As shown in Fig. 2A, the dimeric sEproteins were unable to bind to liposomes at pH 8.0, but atpH 5.5 about 70% of the isolated sE proteins were able to stablyattach to liposomes. When the centrifugation step was carriedout at pH 8.0 rather than pH 5.5, no difference in the coflotationof sE was observed (Fig. 2A), indicating that the binding wasirreversible.

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FIG. 2. Coflotation with liposomes of sE protein (A) and sE that had already undergone a cycle of pH-dependent dissociation and reassociation (B). (A) sE was incubated with liposomes at pH 8.0 for 30 min and analyzed by sucrose step gradient centrifugation at pH 8.0 ( ${circ}$ ), incubated with liposomes at pH 5.5 for 30 min and analyzed by sucrose step gradient centrifugation at pH 5.5 ( ${blacklozenge}$ ), or incubated with liposomes at pH 5.5 for 30 min, back-neutralized, and then subjected to centrifugation in sucrose step gradients at pH 8.0 (•). (B) sE was pretreated for 10 min at pH 5.5 without liposomes (dissociation), back-neutralized (reassociation), incubated for 30 min at pH 5.5 in the presence of liposomes, and analyzed by sucrose step gradient centrifugation at pH 8.0 ( ${square}$ ); alternatively, sE was incubated with liposomes at pH 5.5 for 30 min, back-neutralized, and then subjected to centrifugation in sucrose step gradients at pH 8.0 ( ${blacksquare}$ ). The positions of the sucrose layers are shown below the curves; the 20% layer contains the sE-liposome mixture before centrifugation.

In the case of whole virions, preincubation at low pH inducesan irreversible trimerization of E (2) and in the previous experimentsled to a complete loss of the capacity to bind to liposomes.The sE protein, on the other hand, reverts to the dimeric formwhen back-neutralized (29). To see whether this reassociationalso restores membrane binding activity, a set of experimentssimilar to those described above (Fig. 1) was carried out withsE dimers that had already been preexposed to low pH and thenback-neutralized. In striking contrast to what was observedwith whole virions, the preexposure of the sE dimer for 10 minto pH 5.5 did not destroy its membrane-binding potential. Whenthese samples were reacidified in the presence of liposomes,approximately the same percentage of membrane-associated sEproteins was observed as with samples that had not been pretreated(Fig. 2B).

Effect of cross-linking on liposome binding. The experiments described above suggest that the monomeric formof E is required for membrane interactions. To examine whethersubunit dissociation is indeed a prerequisite for the associationwith target membranes, we carried out coflotation experimentswith sE dimers that had been pretreated with the cleavable cross-linkerEGS to prevent dissociation.

The results of these experiments are shown in Fig. 3. Treatmentwith EGS led to intersubunit cross-linking, as revealed by astrong band corresponding to a dimer in SDS-PAGE (Fig. 3B, lane2). These cross-linked dimers were found to be strongly impairedin their ability to associate with liposomes (Fig. 3A). However,partial cleavage of the cross-linker with hydroxylamine, asdemonstrated by a weakening of the dimer band and a correspondingincrease in the intensity of the monomer band (Fig. 3B, lane3), restored the ability of the sE proteins to interact withliposomes at low pH to 40 to 45% of control levels. This demonstratesthat the ability of the dimers to dissociate is essential forforming stable interactions with the target membrane.

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FIG. 3. Effect of cross-linking with EGS on liposome binding. (A) Percentage of sE protein associated with liposomes after treatment with EGS and/or hydroxylamine compared to untreated controls. (B) SDS-PAGE of cross-linked samples. Lane 1, sE proteins not subjected to cross-linking but treated with the amount of hydroxylamine used to cleave the cross-linker (lane 3); lane 2, sE proteins cross-linked with EGS; lane 3, sE proteins cross-linked with EGS and then treated with hydroxylamine to cleave the cross-linker. The data are representative of four experiments.

Blocking of liposome binding by a fusion peptide-specific antibody. It is likely that the stable association of the sE proteinswith liposomes is the result of specific interactions betweenthe membrane and the internal fusion peptide, which would bepredicted to become exposed when the sE dimer dissociates (1).To obtain additional evidence as to whether the interactionsof sE with liposomes occur via the fusion peptide, sE dimerswere incubated with a MAb (A1) that specifically recognizesthis region (1). Liposomes were then added, and the sampleswere acidified, back-neutralized, and analyzed by coflotationas described above. As shown in Fig. 4, MAb A1 completely inhibitedthe binding of sE dimers to liposomes, in contrast to a controlMAb (C5) that binds to another region of the E protein (10).To exclude the possibility that A1 inhibits binding indirectlyby obstructing the dissociation of the dimer, we also preincubatedthe sE proteins at low pH to dissociate them before adding MAbA1. As shown in Fig. 4, the antibody was able to block low-pH-inducedliposome binding under these conditions as well.

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FIG. 4. Inhibition of liposome binding by a fusion peptide-specific MAb (A1). sE proteins were preincubated with MAb A1 at pH 8.0, acidified in the presence of liposomes, back-neutralized, and analyzed by step gradient centrifugation at pH 8.0 ( ${circ}$ ). Alternatively, sE proteins were pretreated at pH 5.5 and then incubated with MAb A1. After the addition of liposomes at the same pH, these samples were analyzed by step gradient centrifugation at pH 5.5 (•). As a control, sE proteins were incubated with MAb C5 at pH 8.0, acidified in the presence of liposomes, back-neutralized, and analyzed by step gradient centrifugation at pH 8.0 ( ${blacklozenge}$ ).

Oligomeric structure of liposome-associated E proteins. To determine the oligomeric state of full-length viral E ortruncated sE after coflotation, the liposome-bound fractionswere solubilized with Triton X-100 and subjected to sedimentationanalysis (2, 29). As predicted from earlier studies, the solubilizedfull-length E from virions sedimented entirely in the fractionscorresponding to a trimer, and this was confirmed by cross-linkingwith DMS (Fig. 5).

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FIG. 5. Oligomeric state of full-length E after attachment of TBE virus to liposomes. Whole TBE virions and liposomes were mixed at pH 8.0, exposed to pH 5.5, and subjected to coflotation as shown in Fig. 1. Bound virus was solubilized with 2% Triton X-100 and analyzed by sedimentation in 7 to 20% sucrose gradients containing 0.1% Triton X-100 (•). As a control, virions were incubated with liposomes at pH 8.0 and the E-protein-containing fractions were analyzed in the same way ( ${circ}$ ). The sedimentation direction is from left to right, and the positions of E monomer (M), dimer (D), and trimer (T) are indicated. (Insets) Cross-linking of the corresponding peak fractions with DMS.

When liposome-associated sE protein like that used for Fig.2A was analyzed in the same way, we were surprised to find thatthese proteins were also homotrimeric (Fig. 6A). This was unexpectedbecause earlier studies carried out in the absence of targetmembranes (and confirmed here in the control in Fig. 6B) hadshown that the sE dimer dissociates but does not form trimersat low pH (29) and that trimerization in solution is dependenton the presence of a portion of the stem region that is absentin the sE protein (2). The stem region is therefore not absolutelyrequired for trimer formation if the E ectodomains are boundto a target membrane.

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FIG. 6. Sedimentation analysis of sE protein after incubation at low pH in the presence (A) and absence (B) of liposomes. (A) Liposome-bound sE proteins (Fig. 2) were back-neutralized, solubilized with 2% Triton X-100, and centrifuged into 7-to-20% sucrose gradients in TAN buffer (pH 8.0) containing 0.1% Triton X-100 (•). As a control we used sE protein that had been incubated with liposomes at pH 8.0 ( ${circ}$ ). (Insets) SDS-PAGE of samples from the corresponding peak fractions cross-linked with DMS. (B) Samples were incubated at pH 5.5 (•) or pH 8.0 ( ${circ}$ ) in the absence of liposomes, solubilized with Triton X-100, and centrifuged into 7-to-20% sucrose gradients (containing 0.1% Triton X-100) at the corresponding pH. The sedimentation direction is from left to right, and the positions of E monomer (M), dimer (D), and trimer (T) are indicated.

DISCUSSION

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Discussion

The fusion of TBE virus with membranes is a process that requiresa low-pH-triggered reorganization of the proteins of the viralenvelope. In mature virions the E proteins form a metastable,presumably icosahedral network of laterally interacting homodimersthat are quantitatively and irreversibly converted to a homotrimericform when exposed to low pH (2). Earlier work (2, 28, 29) providedevidence that this is a two-step process involving a reversible,protonation-dependent dissociation of the dimers followed byan irreversible trimerization step.

The results of this study suggest that specific interactionswith the target membrane probably occur after the dissociationstep but before the trimerization step. This is based on theobservations that (i) preventing the dissociation of the dimerwith EGS blocked liposome binding, but binding activity couldbe restored by cleaving the cross-linker, and (ii) the finaltrimeric form of E on low-pH-treated virions was no longer ableto mediate liposome coflotation at low pH, suggesting that thelipid-binding state is transient.

It is probable that the low-pH-induced dissociation of the Edimer results in the exposure of a specific element in the Eprotein whose function it is to bind the target membrane. Recently,using site-directed mutagenesis, we showed that a conservedregion that includes Leu 107 and lies at the distal tip of eachE protein monomer appears to be directly involved in membraneinteractions during fusion (1). This "internal fusion peptide"is buried in the dimer interface (24) but would presumably becomeexposed during the low-pH-induced dissociation step. In thisstudy we show that a MAb recognizing this region is able toblock liposome coflotation with the sE protein, supporting thenotion that membrane binding is mediated specifically by theinternal fusion peptide. This MAb was also able to block fusionof whole virions with liposomes (data not shown). Interestingly,phase-partitioning experiments with Triton X-114 did not reveala dramatic increase in the overall hydrophobicity of sE at acidicpH (data not shown), suggesting that the exposure of apolarresidues in the monomeric low-pH form is rather limited andmay involve only specific functional sites.

A surprising result of this study was that sE proteins thathad bound to liposomes at low pH were no longer monomeric butinstead had been irreversibly converted to stable trimers. Thiswas unexpected because earlier in vitro experiments had shownthat the sE protein does not trimerize at low pH in the absenceof target membranes (29), and subsequent deletion mapping studiesrevealed that this was attributable to the lack of a putative ${alpha}$ -helix (amino acids 401 to 413) in the proximal stem region(3). It now appears that contacts between the ectodomains aresufficient to stabilize a homotrimeric form of the E proteinbut that "facilitators"—either the stem-anchor regionor target membranes—are required for the trimerizationprocess. Both could be involved in interactions that lead toan increase of the local concentration of correctly alignedmonomers and thereby facilitate trimer assembly. The inductionof oligomerization upon lipid binding has also been describedfor other membrane-interacting proteins such as the matrix proteinof Ebola virus (26) and several bacterial pore-forming toxins(reviewed in reference 4). Alternatively, the binding of E tothe target membrane might induce structural alterations thatlead to trimer formation, and these changes could be similarto ones induced by interactions involving the stem regions.

The TBE virus E protein has a number of functional propertiesthat are similar to those of the fusion protein E1 of SFV andother alphaviruses (family Togaviridae) (18). Very recentlyit was shown that these proteins share a similar overall foldand are probably descended from a common ancestor (21). Unlikethe TBE virus E protein, SFV E1 is not homodimeric in its nativestate but instead exists as a heterodimer with another glycoprotein,E2. The fusion peptide of SFV E1 is at a similar position atthe tip of the monomer and is apparently buried in the E1-E2interface (21). Exposure to low pH results in the dissociationof the heterodimer and irreversible formation of E1 homotrimers(reviewed in reference 18). It therefore appears likely thatthe fusion mechanism used by alphaviruses is very similar tothat used by flaviviruses.

It was shown earlier that low-pH-induced fusion, but not membranebinding, by SFV can be inhibited by zinc ions (7). Under theseconditions the E1-E2 heterodimer was shown to dissociate butnot go on to form trimers (7). Furthermore, a nonfusogenic mutantof SFV that was able to dissociate but not to form trimers retainedits ability to bind to liposomes at low pH (19). This wouldsuggest that the initial membrane binding step with both TBEvirus and SFV requires subunit dissociation but not trimerization.Also, similar to what was observed in this study with the TBEvirus sE protein, it has been shown that a C-terminally truncatedmonomeric form of the SFV E1 protein is able to trimerize atlow pH when bound to liposomes, but not when membranes are absent(20).

The class II fusion proteins of alphaviruses and flavivirusesshare properties that are quite distinct from those of classI viral fusion proteins, suggesting that these two classes usedifferent mechanisms to achieve membrane fusion. The findingthat soluble sE dimers can be converted into a trimeric formby acidification in the presence of liposomes should facilitatethe generation of trimeric proteins that might be suitable forstructural studies and thereby could provide a basis for investigatingthe three-dimensional structure of the low-pH form of a classII viral fusion protein.

ACKNOWLEDGMENTS

We thank Angela Dohnal and Walter Holzer for excellent technicalassistance.

FOOTNOTES

* Corresponding author. Mailing address: Institute of Virology, University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria. Phone: 43-1-40490, ext. 79505. Fax: 43-1-40490, ext. 9795. E-mail: karin.stiasny{at}univie.ac.at.

Present address: INTERCELL Biomedizinische Forschungs- und EntwicklungsGmbH, A-1030 Vienna, Austria.

REFERENCES

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