STEPHEN FULLER

Influenza haemagglutinin: illuminating fusionThe recently determined structure of influenza haemagglutinin at the pH of

membrane fusion gives new insights into the mechanism of viral fusion, and islikely to stimulate further work on this best understood of all fusion proteins.

Structure 15 October 1994, 2:903-906

Electron microscope images of influenza virus reveal afearsome-looking spiked virion resembling a nauticalmine. As with the mine, it is contact with the spikes[composed of the haemagglutinin (HA) protein] that doesthe damage. HA fulfils at least three functions for thevirus. Firstly, it attaches the virion to the host cell viainteractions with sialic acid residues on the cell surface.When the attached virion is endocytosed, HA is exposedto the low pH of the endosome and fulfils its second roleof mediating the fusion of the viral and endosomal mem-branes, enabling the viral genome to escape into the cyto-plasm. Finally, HA is also a site of antigenic variation,which enables the virus to evade the host's immunesystem. Considerable variation of the HA surface is possi-ble while preserving the framework necessary for attach-ment and membrane fusion. This poses considerableobstacles to the development of broad-spectrum vaccinesagainst influenza.

Incubation of HA with the protease bromelain releases asoluble fragment, and in 1981 the structure of this brome-lain fragment of HA (BHA) was determined [1]. Thisprompted many-functional studies of HA, so that it is nowthe best characterized viral fusion protein. The BHAstructure resolved several important questions but alsoraised others. The structure revealed that the' receptor-binding site is placed between modifiable surface regions[21 and that the conserved hydrophobic sequence believedto be essential for membrane fusion is buried. These find-ings explained how variation in antigenicity could beachieved without compromising function. Further work,informed by the BHA structure, has validated and refinedthis mechanism, which is now known to be common tomany viral systems.

The BHA structure was less illuminating about the mecha-nism by which haemagglutinin is able to mediate the fusionof two membranes [1]. The fact that the viral membranewas separated from the target membrane by the 135 Alength of the molecule was an obvious problem, as was thefact that the fusion sequence which presumably interactswith the target membrane is buried in the centre of thestructure. These features are so inappropriate for the workof membrane fusion that it was immediately proposed thata drastic conformational change must result from theexposure to low pH which triggers fusion [1]. In a recentpaper Bullough et al. [3] have determined the structure ofthis dramatically altered conformation. This new structureprovides striking insights into conformational changes inviral proteins and into the mechanism of fusion.

A flexible foeA number of groups have demonstrated that haemagglu-tinin is capable of enormous conformational flexibility.The development of batteries of conformation-sensitiveantibodies have allowed the mapping of some of theseconformational changes [4-6]. This work has shown thatHA is synthesized as a single polypeptide chain, HA0.This initially folds to form the epitopes in the HA1domain at the top of the molecule, and trimerizes toproduce (HA0)3 which is transported from the endoplas-mic reticulum to the Golgi [5] (Fig. 1). The cleavage ofthe HA0 to HA1 and HA2 occurs late in its passagethrough the secretory pathway and is accompanied byanother conformational change, seen as a change in anti-genicity. This cleavage and the conformational change arerequired for the fusion activity of haemagglutinin.Exposure of the mature haemagglutinin to low pH causesa series of antigenic changes which can be divided intotwo groups [7]: a rapid and relatively temperature-insensi-tive exposure of the stem region and a slower, more tem-perature-dependent change which dissociates the threeHA1 domains, exposing sites between them.

Fusion requires a pH between 5.0 and 6.0 and occurs inseveral distinguishable stages [8,9]. Kinetic experimentshave shown that the pH-induced conformational changeand the insertion of the fusion peptide into the targetmembrane occur within seconds although the mixing oflipids from the two membranes occurs on the timescale ofminutes [10]. The state which mediates fusion appears tobe relatively short lived so that low pH treatment of thevirus causes inactivation if a target membrane is not avail-able. Careful quantitative experiments have shown that thebinding and fusion activities are distinct and that efficientfusion requires several haemagglutinin trimers whichcombine to form a pore [10-12].

The effect of low pH on the overall properties of themolecule is dramatic [13]. The soluble BHA trimersaggregate upon prolonged exposure to low pH to formprotein micelles which are joined by their now exposedfusion sequences [13,14]. This micellar aggregate can besolubilized by digestion with a combination of trypsinwhich removes the HA1 residues 28 to 328, and ther-molysin which removes the HA2 residues 1 to 28, releas-ing a soluble trimeric fragment. containing residues38-175 of HA2 joined to residues 1-27 of HA1 by adisulphide bond (Fig. 1). This fragment (TBHA 2) issoluble and can be crystallized [15]. It retains its low pHconformation when returned to neutral pH. TBHA 2 is

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MINIREVIEW

904 Structure 1994, Vol 2 No 10

projects a full 100 A through the structure. This reversaland extension involves converting a loop region to helixand converting a portion of the previously helical regionto a turn. The resulting helical core of the TBHA2 mol-ecule looks enormously stable with an almost completelyconsistent pattern of heptad repeats forming the knobsinto holes interface between the three helices and moreregular than those in BHA (compare Fig. 2b with Fig. 2e).This rearrangement would place the amino-terminalregion of HA2 at the distal most region of the molecule sothat the fusion sequence could interact with the targetmembrane. The conformational change is so dramatic thatthe interaction of HA1 with HA2 seen in BHA would nolonger be possible even if the appropriate portions of HA1were still present. The fact that the secondary structurechanges are compensatory results in TBHA 2 and BHAhaving similar helix contents and explains the seeminglyparadoxical result of circular dichroism measurements [14],which showed that the conformational change did notalter the total amount of secondary structure.

Fig. 1. The forms of haemagglutinin. The haemagglutinin is syn-thesized as a single chain HAO which oligomerizes to form(HA0)3 in the endoplasmic reticulum (ER). After trimerization,the HAO passes to the Golgi. A further cleavage either by a cellu-lar protease in the late Golgi or by exogenous protease outsidethe cell converts (HAO)3 to the mature HA (HA1 HA2) 3.Treatment of the mature HA with bromelain cleaves away thetransmembrane region at the carboxyl terminus of HA2 leavingthe soluble trimeric bromelain fragment BHA3 whose structurewas solved previously [1]. Low pH treatment of BHA causesaggregation of the protein into micelles. Successive treatment ofthese micelles with trypsin (which removes HA1 residues28-328) and thermolysin (which removes HA2 residues 1-37)generates the TBHA 2 fragment which was crystallized 1141 andhas now been solved [3].

also stable toward thermal denaturation being unaffectedat 95C at low pH and stable until 760 C at neutral pHin comparison with BHA which is only stable to 65°Cat neutral pH.

Strange effects of acidThe structures of BHA and the fragment in low pH con-formation (TBHA2) are shown in Fig. 2. The structure ofBHA revealed a novel protein fold in which eachmonomer formed a loop extending more than 130 A,then folding back to the position of the carboxy-terminalmembrane anchor [16]. The trimeric molecule comprisestwo structurally distinct regions: a triple-stranded coiledcoil extending 76 A from the membrane forming the stemof the molecule, and a globular region of antiparallel13-sheet which sits on top of the coiled coil and containsthe receptor-binding site and a number of antigenic sites.Even a casual comparison of the corresponding regions ofthe two structures (Fig. 2c versus 2e) shows that low pHevokes a dramatic conformational change. Only 30residues have the same structure in BHA and TBHA2.The long ot-helical stem, which formed the core of theBHA structure, has been extended and reversed so that it

A number of mutations in the haemagglutinin whichincrease the fusion pH can now be understood in terms oftheir stabilizing the TBHA 2 form relative to the BHAform [17,18]. As pointed out by Bullough et al. [3] thesefall into four categories (Fig. 3a). Three of these representmutations of residues which stabilize the buried positionof the amino-terminal fusion sequence (orange in Fig. 3a),the interaction of the short helix and turn with the longhelix of BHA (yellow-green in Fig. 3a), and the inter-action of HA1 with the loop and helix of HA2 (red inFig. 3a). The TBHA2 structure shows that each of theseinteractions must be broken during the conformationalchange (Fig. 3b). The fourth class which stabilize inter-actions between the HA1 tops of the trimer (purple inFig. 3a) are not seen in the TBHA 2 structure, but asdescribed above, these interactions are lost in the secondstep of the pH-mediated rearrangement. The exposure ofthe sites recognized by peptide antibodies [7] is also neatlyexplained. The most rapidly exposed sites (orange andyellow-green in Fig. 3c) are in the areas of BHA whichmust be opened during the formation of the TBHA2structure. In the low pH conformation, the exposed sitesare either on the surface of TBHA 2 or in disorderedregions (Fig. 3d).

The comparison of BHA and TBHA2 indicates that theneutral form of HA is metastable. Carr and Kim [19]showed that the sequences which form the bend in BHAhave the potential to form a helix when isolated from therest of the structure, and Baker and Agard discuss the ener-getics of this transition more fully in a separate minireviewin this issue of Structure [20]. How is the metastable versionof the protein synthesized without prematurely convertingto the more stable form? A natural answer is that HAO,which is not capable of the pH transition, is actually themolecule which folds and that the activating cleavage toHA1 and HA2 leaves it in a metastable state. If so, theeffect of cleavage upon the structure must be dramatic.The timing of this cleavage at a late stage of transport tothe plasma membrane also protects the protein from

Illuminating fusion Fuller 905

Fig. 2. Comparison of the BHA andTBHA structures.The course of thechain from amino to carboxyl terminusis marked by shading between blue andpurple for the region of the chain whichis retained in TBHA 2 fragment. The rep-resentation was generated withWHATIF [23] using the algorithm ofThomas [241 to smoothly fit the sec-ondary structure. The structures of theBHA trimer (a) and monomer (b) withthe position of the the receptor-bindingsite marked by the ligand and the singledisulphide connecting HA1 and HA2displayed in yellow. The region of theBHA structure which is retained inTBHA2 is shown in (c). The monomer(d) and trimer (e) structures of TBHA2are shown in with the correspondingregions coloured as for BHA.

Fig. 3. The sites df mutations known toaffect fusion pH [17].(a) BHA and(b) TBHA2. The positions of mutationsin residues which are believed to stabi-lize the buried position of the amino-terminal fusion sequence (orange), theinteraction of the short helix and turnwith the long helix of BHA (yellow-green), the interaction of HA1 with theloop and helix of HA2 (red) and stabi-lize interactions between the HA1 topsof the trimer (purple) are all indicated.Some mutations do not appear in Bbecause the corresponding region hasbeen lost in the production of TBHA 2.The sites of regions which vary in theirreactivity to peptide antibodies uponexposure to low pH [7] are shown in(c) for BHA and (d) for TBHA2. Thesequences in the loop (orange) andfusion peptide (yellow-green) region areexposed much more rapidly than thosenear the carboxyl terminus of HA1, theinterface or hinge regions (purple).

exposure to low pH in the trans Golgi network. A similar The TBHA2 structure illustrates the dramatic conforma-activating cleavage is seen in alphaviruses such as Semliki tional change of the haemagglutinin upon exposure to lowForest virus and there it is known that this cleavage results pH. However only indirect evidence connects this struc-in a dramatic conformational change [21]. This metastable ture with the form of the molecule that is responsible forcharacter of mature fusion proteins may be quite general fusion. The fusion-mediating form of the protein iswhen the pH-mediated conformational change is irre- believed to be short-lived and indeed treating the virusversible [8,9]. For other systems, such as the rhabdoviruses, with low pH in the absence of target membranes - as isthe pH-induced change appears reversible in the absence done in the preparation of TBHA2 - causes inactivationof membranes [21]. [7-10,13]. Nevertheless, the stability of the TBHA 2 fold

906 Structure 1994, Vol 2 No 10

Fig. 4. The possible orientations of haemagglutinin upon themembrane. The orientation of HA at neutral pH is fixed by therelatively short length of the sequence between the carboxyl ter-minus of HA2 and the membrane-spanning region (left). The lossof 13 residues and the lack of secondary structure of an addi-tional 10 in TBHA 2 make the possible orientations much lessconstrained. three orientations of the 23 extended residues areindicated by the blue arrow. The position of the fusion sequencein BHA is shown in red. Much of this portion is missing fromTBHA 2, but the 30 residues that remain after cleavage are alsoshown in red.

and the likelihood that inactivation results from inappro-priate insertion of the fusion sequence into the viral mem-brane (rather thah a further rearrangement) both support acorrespondence between the TBHA 2 structure and thestate mediating fusion. The carboxyl terminus of HA2,which marks the closest approach of BHA to the mem-brane, is disordered in TBHA 2 so that any orientationof the molecule could be consistent with membraneassociation (Fig. 4).

Careful electron microscopic observations have establishedthe dimensions of the low pH form of the complete mol-ecule while it is still attached to the virus and of thevarious states of the solubilized bromelain fragment [16].The comparison of this data with the TBHA2 structuresupports the idea that the HA2 terminus does protrudefrom the distal end of the low pH form (Fig. 4). Trypsincleavage of virus which has been exposed to low pH(removing the HA2 carboxyl terminus) results in a struc-ture decorated with molecules which show a bulbous end(presumably the bottom of Fig. le) protruding away fromthe membrane. This indicates that the fusion sequence caninteract with its own membrane and supports the notionof the flexibility of the low pH form.

The TBHA 2 structure gives us a striking structure, somefirst answers about the mechanism of fusion, a new appre-ciation of conformational changes in viral proteins and ahost of new and interesting questions. The role of thetrimeric nature of TBHA 2 in stabilizing the structure isclear. The possible role of higher oligomers which arebelieved to play a role in the process, perhaps by forming apore [10,12], remains undefined. Similarly the orientationof the molecule during the fusion process needs to beclarified. It will probably be necessary to combine the well

defined structure of the TBHA2 fragment with obser-vations of the whole molecule during fusion in order tounderstand the process. As with BHA, TBHA2 willprovide the base upon which much future work will beconstructed.

Acknowledgements: The author gratefully thanks Dr Gert Vriendand Dr David Thomas (EMBL) for their help with the figures.

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Stephen Fuller, Biological Structures and BiocomputingProgramme, European Molecular Biology Laboratory,Postfach 10.2209, Meyerhofstrasse 1, 69012 Heidelberg,Germany.