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Viral membrane fusion

Viral membrane fusion review Me M brane Fusion Stephen C Harrison Infection by viruses having lipid-bilayer envelopes proceeds through fusion of the viral membrane with a membrane of the target cell. Viral ‘fusion proteins’ facilitate this process. They vary greatly in structure, but all seem to have a common mechanism of action, in which a ligand-triggered, large-scale conformational change in the fusion protein is coupled to apposition and merger of the two bilayers. We describe three examples—the influenza virus hemagglutinin, the flavivirus E protein and the vesicular stomatitis virus G protein—in some detail, to illustrate the ways in which different structures have evolved to implement this common mechanism. Fusion inhibitors can be effective antiviral agents. ‘Enveloped’ viruses—those with lipid bilayers as integral parts of their anchor that holds the protein in the viral membrane and a distinct structure—enter the cells they infect by fusion of viral and host-cell hydrophobic patch (‘fusion peptide’ or ‘fusion loop(s)’) that ultimately membranes. One or more viral membrane proteins facilitate the vari- interacts with the target membrane. Moreover, they all happen to be ous fusion steps. Several such fusion proteins have now been studied trimeric in their fusion-active state. In the initial step in the fusion in great detail, with crystal structures determined for both the form of reaction, the fusion protein, responding to binding of a ligand (protons the protein present on the viral surface before interaction with the cell in many cases, as the mechanism has evolved to respond to the low pH 3,4 (‘pre-fusion’ conformation) and the form of the protein after fusion is of an endosome , but cellular or viral protein ligands in other cases), complete (‘post-fusion’ conformation). The proteins show a variety of undergoes a conformational change that extends each subunit toward molecular architectures, but what we can infer from the various struc- the target membrane and yields a contact between that membrane and tures and from experiments both in solution and with cells suggests the fusion peptide or loop(s) (Fig. 1a to Fig. 1b). Many fusion proteins that all of them catalyze fusion in essentially the same way. We can even are C-terminal fragments of a larger precursor (for example, the HA2 draw a rough analogy to serine proteases, which can have very different fragment of influenza virus hemagglutinin or the gp41 fragment of HIV polypeptide chain folds but identical active-site mechanisms. Env), and initiating the fusion process requires that they must first shed Fusion of two bilayer membranes is thermodynamically favorable, but their N-terminal fragment, which often contains a receptor-binding 1,2 there is a very high kinetic barrier . Fusogens of all kinds lower that domain (for example, HA1 or gp120—see the more detailed description kinetic barrier; viral fusion proteins do so by using the free energy liber- of influenza virus hemagglutinin below). Although strong, the evidence ated during a protein conformational change to draw the membranes for an extended intermediate is indirect. The putative extended state is together. The general outlines of the pathway leading from two separate sometimes called a ‘pre-hairpin intermediate’, as the next step is collapse bilayers to a single one is relatively well understood (Fig. 1). A ‘hemifusion’ into a folded-back conformation. The intermediate may have a relatively state—in which the apposed, proximal leaflets of the two bilayers, but long half-life; for HIV-1 gp41, the half-life seems to be many minutes, but not yet the distal leaflets, have merged—is almost certainly an obligatory in other cases, it may only be a few seconds. Step 2: The bridge collapses intermediate. The structure of the hemifusion intermediate is probably (Fig. 1c) so that the two membrane-inserted elements (the fusion pep- stalk-like (Fig. 1d). Studies of fusion mediated by viral proteins provide tide or loop in the target membrane and the C-terminal transmembrane some of the best evidence for hemifusion as a required intermediate stage . anchor in the viral membrane) come together. This collapse distorts the There are probably substantial kinetic barriers both leading into this inter- two bilayers, probably into a nipple-like configuration, with a relatively mediate and leading away from it toward the product (Fig. 2). restricted area of close approach . Whether insertion of the fusion pep- The accumulated evidence suggests that viral fusion proteins lower tide potentiates this distortion in the target membrane, by perturbing the various kinetic barriers and, hence, catalyze the membrane fusion the bilayer and lowering the distortion energy, remains uncertain. process, as follows. Step 1: The protein opens up and forms a bridge Step 3: The distortion of the individual membranes lowers the energy between the two bilayers (Fig. 1b). All viral fusion proteins studied so far barrier (not necessarily symmetrically, as the anchoring of the fusion have two membrane-interacting elements: a C-terminal transmembrane protein is different at the two ends) between separated and hemifused bilayers so that a hemifusion stalk forms (Fig. 1d). Step 4: The hemifusion stalk opens to form a transient fusion pore. A final conformational step Jack and Eileen Connors Structural Biology Laboratory, Harvard Medical School, in the protein refolding renders the open state irreversible, and the pore Laboratory of Molecular Medicine, Children’s Hospital Boston, and Howard expands (Fig. 1e). With some fusion proteins, but not with others, the Hughes Medical Institute, 250 Longwood Avenue, Boston, Massachusetts 6–8 02115, USA. Correspondence should be addressed to S.C.H. pore may flicker open and closed . Whether flickering occurs may ([email protected]). depend on the rapidity of the final conformational step. In most cases, Published online 3 July 2008; doi:10.1038/nsmb.1456 steps 3 and 4 probably require concerted action of more than one 690 volume 15 number 7 j ul Y 2008 nture a structural & molecular biology © 2008 Nature Publishing Group http://www.nature.com/nsmb review a Pre-fusion b Extended c Collapse of d Hemifusion e Fusion pore intermediate intermediate (post-fusion) Cell Virus Figure 1 Sequence of events in membrane fusion promoted by a viral fusion protein. Ambiguities remain in some aspects of this scheme (see main text). (a) The protein in the pre-fusion conformation, with its fusion peptide or loop (light green) sequestered. The representation is purely schematic, and various features of specific proteins are not incorporated—for example, the displacement of the N-terminal fragment of proteins that are cleaved from a precursor or the dimer-to-trimer rearrangement on the surface of flaviviruses. (b) Extended intermediate. The protein opens up, extending the fusion peptide or loop to interact with the target bilayer. The part of the protein that bears the fusion peptide forms a trimer cluster. (c) Collapse of the extended intermediate: a C-terminal segment of the protein folds back along the outside of the trimer core. The segments from the three subunits fold back independently, so that at any point in the process they can extend to different distances along the trimer axis, and the entire trimer can bow outward, away from the deforming membrane. (d) Hemifusion. When collapse of the intermediate has proceeded far enough to bring the two bilayers into contact, the apposed, proximal leaflets merge into a hemifusion stalk. (e) Fusion pore formation. As the hemifused bilayers open into a fusion pore, the final zipping up of the C-terminal ectodomain segments snaps the refolded trimer into its fully symmetric, post-fusion conformation, preventing the pore from resealing. fusion-protein trimer (as symbolized by the two apposed trimers in Influenza virus hemagglutinin Fig. 1). The number of trimers that participate and the nature of the The influenza virus hemagglutinin is the best characterized of all interaction that couples them may vary from case to case, and these issues viral fusion proteins. The crystal structure of its ectodomain in a pre- are still matters of some debate. fusion conformation was determined in classic work by Wiley, Wilson To illustrate these generalizations, we describe the fusion proteins and Skehel in 1981 (refs. 18,19); the post-fusion conformation was of three viruses, each with known three-dimensional structures for finally visualized in 1994 (ref. 20), and the uncleaved precursor, HA0, both the pre-fusion conformation (corresponding to Fig. 1a) and in 1998 (ref. 21). The core of HA1 is a sialic acid–binding domain, the post-fusion conformation (corresponding to Fig. 1e). These borne on a stalk formed by HA2. The central feature of the stalk is a 9 10,11 three viral fusion proteins—from influenza , dengue and three-chain, α-helical coiled coil. HA0 and its cleaved product, pre- 12,13 vesicular stomatitis viruses —are representatives of what have fusion HA1–HA2, are essentially identical in overall structure. The come to be called class I, class II and class III viral fusion proteins, cleavage, which normally happens in the trans-Golgi network (TGN), but as this typology now obscures as much as it clarifies, we avoid but which can also occur after viral budding, leads to a modest local it here. Other viral fusion proteins for which structures in both rearrangement, in which the newly generated N terminus of HA2 conformational states are known include those from representative inserts into a pocket along the three-fold axis, burying the fusion 14,15 16,17 paramyxoviruses and alphaviruses . The principles illustrated peptide (the first 20–25 residues of HA2). The pocket is created by a by those important studies reinforce the conclusions derived from splaying apart from each other of the C termini of the HA2 coiled-coil the three examples chosen here. helices, so that the three helices diverge from the three-fold axis and from each other, rather like a narrow tripod (Fig. 3a). Sialic acid, on glycoproteins or glycolipids, is the influenza virus receptor; HA1 bears the binding site, a shallow pocket exposed on its outward-facing surface . As the plasma membrane recycles regularly through various forms of endocytosis, the virus-receptor complex may not require a specific endocytic signal in order to reach an endo- Hemifusion some. When the HA1:HA2 trimer encounters low pH, it undergoes a large-scale conformational rearrangement, in which HA1 separates Two bilayers from HA2 (Fig. 3a to Fig. 3b), except for a residual disulfide tether, 20,23,24 and the latter effectively turns inside out . The two key features of this HA2 refolding are a loop-to-helix transition in the region connecting the fusion peptide to the central coiled coil (Fig. 3b to Fig. 3c) and reorientation of the C-terminal part of the molecule so that it zips up alongside the extended coil (Fig. 3c to Fig. 3d). These correspond, respectively, to formation of the extended intermediate Fusion pore and to its collapse into a conformation that brings together the fusion peptide and the transmembrane anchor. The loop-to-helix transition Figure 2 Schematic diagram illustrating the (free) energy changes during in the N-terminal part of HA2 augments the central coiled-coil at its fusion of two bilayers. The relative heights of the various barriers are N-terminal end (Fig. 3c); reorientation of the C-terminal part of the arbitrary. Fusion proteins accelerate the process by coupling traversal of these barriers to energetically favorable conformational changes. protein breaks the central helices where they splay apart, at the site nture a structural & molecular biology volume 15 number 7 j ul Y 2008 691 © 2008 Nature Publishing Group http://www.nature.com/nsmb review ab c d Figure 3 Influenza virus hemagglutinin: proposed sequence of fusogenic conformational changes. (a) The pre-fusion conformation. Each subunit is shown in a different color. The binding site for the receptor, sialic acid, is at the top of each subunit, but contact with a receptor molecule is not shown. Red asterisk, the sequestered fusion peptide of the red subunit, at the N terminus of HA2. (b) HA1 dissociates from its tightly docked position in response to proton binding. Each HA1 remains flexibly tethered to the corresponding HA2 by a disulfide bond (near the bottom of the ectodomain, in the orientation shown here). (c) The extended intermediate. The loop between the shorter and longer helices in HA2 (for example, the two red helices and the loop connecting them, in b) becomes a helix, thereby translocating the fusion peptide toward the target membrane. The fusion peptides (asterisk) are shown interacting as amphipathic helices with the target bilayer. The loop-to-helix transition creates a long, three-chain coiled coil at the core of the trimer. (d) Collapse of the extended intermediate to generate the post-fusion conformation. The lower parts of the protein (as seen in the orientation in c) fold back along the outside of the three-chain coiled coil. The collapse is complete only when the two membranes have fused completely. The post-fusion conformation is shown in a ‘horizontal’ orientation, to correspond to the sequence in Figure 1. (e) Detail illustrating some features of the membrane-proximal region of influenza virus HA2 after fusion is complete. The N termini of the coiled-coil helices are capped by contacts with amino acid residues in the link between the fusion peptide and the coiled coil, as well as with residues near the C terminus of the ectodomain, proximal to the transmembrane helices . This cap locks into place all the membrane-proximal components of the structure. The fusion peptides at the N termini of three HA2 chains are shown as cylinders (possible amphipathic helices) lying partly immersed in the outer leaflet of the membrane bilayer, as suggested by NMR and EPR studies . The transmembrane segments, likely to be α-helices, are also shown as cylinders. The relationships in this drawing among the fusion peptides and the transmembrane helices, chosen to illustrate the scale of the structures and the approximate distances between them, are purely schematic, as there is no single structure yet determined experimentally that contains all the elements included here. Only the crystallographically determined components are in ribbon representation. from which the fusion peptide has withdrawn earlier in the fusion conformation of HA2 includes a substructure in which residues process (Fig. 3c to Fig. 3d). connecting the fusion peptides to the coiled-coil and residues just The specific sites at which protonation initiates these large-scale N-terminal to the transmembrane anchors cap the N termini of the three conformational changes may not be uniformly conserved. Mutations central helices (Fig. 3e). Formation of this cap-like substructure seems 28,29 at widely distributed locations affect the stability of trimer interfaces to be important for the transition from hemifusion to fusion . that break during the rearrangement and thereby alter the threshold As the extended intermediate collapses, it must bend outward, away pH for fusion . Conserved ionizable residues (two aspartates and a from the nascent hemifusion stalk, to allow the two membranes to come histidine) in the vicinity of the buried fusion peptide may contrib- together (Fig. 1c). The C-terminal segments of HA2 do not interact ute to the trigger; comparison of their interactions before and after laterally with each other in the final, post-fusion conformation, and there cleavage of HA0 (and hence before and after insertion of the fusion is no reason to suppose that their zipping up is cooperative. That is, each peptide into the cavity) does not, however, lead to an obvious explana- HA2 chain can complete its refolding independently of the other two, and tion for why HA0 does not undergo a proton-induced conformational the loss of overall three-fold symmetry during the transition (Fig. 1c,d) is a change . Redundant contributions to pH-regulated processes often natural consequence of this independence. Formation of the cap structure make identification of critical residues difficult (as illustrated by the (Fig. 3e) restores global three-fold symmetry; if the transmembrane helices history of investigations into the hemoglobin Bohr effect) because loss pass completely through the bilayer, this step probably requires the pres- of one contributing site may not lead to significant loss of fitness or to ence of an aqueous channel—that is, a committed fusion pore (Fig. 1e). pronounced change in pH dependence. The fusion peptide, presented to the target membrane by the loop-to- Flavivirus E helix transition, is thought to form an amphipathic helix, by inference The flaviviruses (a family that includes various mosquito- and tick-borne from its conformation on the surface of a detergent micelle and from pathogens, such as yellow fever virus, dengue virus, and tick- borne- spectroscopic data consistent with partial penetration of the outer leaflet encephalitis virus) have two envelope proteins, known as M and E. of the lipid bilayer . When fusion is complete, the polypeptide chain E has both receptor-binding and fusogenic activities; M is the proteolytic segment just C-terminal to the fusion peptide and the membrane- residuum of a precursor, prM, which is the form incorporated into proximal segment of HA2 interact. The completed post-fusion immature virions. The virus assembles by budding into the endoplasmic 692 volume 15 number 7 j ul Y 2008 nture a structural & molecular biology © 2008 Nature Publishing Group http://www.nature.com/nsmb review ac d Figure 4 Flavivirus E: proposed sequence of fusogenic conformational changes. (a) The packing of 180 E subunits (90 dimers) in an icosahedral array on the surface of a flavivirus particle . The red, yellow and blue parts of each subunit correspond respectively to domains I, II and III of the ectodomain. (b) ‘Side view’ of the pre-fusion, dimeric conformation of the E protein, based on the crystal structure of dengue E (residues 1–395) , supplemented by a representation of the ‘stem’ segment (two helices linked by a short loop, lying in the plane of the membrane head groups) and the transmembrane anchor (a helical hairpin), derived from a cryo-EM reconstruction of the virion . The domains in one of the two subunits are colored as in a; the other subunit is in gray. The fusion loop is at the tip of domain II, on the far right of the colored subunit, buried at the contact with domain III of the dimer partner. (c) Monomeric transition between the pre-fusion dimer and the trimeric extended intermediate. The three subunits that will associate into the extended intermediate in d are not yet in contact. The drawing embodies the suggestion that domains I and II have swung outward, while domain III and the stem remain oriented against the membrane roughly as in the pre-fusion state. The fusion loop is now at the top of the diagram and is shown already interacting with the target bilayer. (d) Extended intermediate. Domains I and II have associated into the trimeric core of the post-fusion conformation, but domain III has not yet flipped over (upper arrows) to dock against them . To indicate that the stem segment must then zip back along the trimer core (lower arrows), the stem is represented by loops ‘poised’ to reconfigure. (e) Post-fusion conformation. Domain III has reoriented, and the stem (dashed line, as there is no direct structural information on its conformation or exact position in the post- fusion trimer) connects it to the transmembrane anchor, now brought together with the fusion loop in the single, fused bilayer. The post-fusion conformation is shown in a ‘horizontal’ orientation, to correspond to the sequence in Figure 1. reticulum, and furin cleavage of prM in the TGN releases most of its allowing the individual subunits to swing outward . The now-exposed ectodomain. Immature virions do not fuse, even when triggered by fusion loops insert into the target membrane, facilitating reclustering of lowering the pH, as prM is essentially a chaperone that prevents the the subunits into trimers. Collapse of the extended, trimeric intermediate fusion-inducing conformational transition. Cleavage of prM to M is the that results from these events can then proceed, by rotation of domain processing step that primes the particle for low pH–induced membrane III in each subunit about the segment that links it to domain I and fusion and hence is not a modification of the fusion protein itself, but (presumably) by zipping up of the stem alongside the clustered domains rather of its companion . The closely related alphaviruses (for example, II. Protonation of one or two conserved histidine residues at the domain Sindbis and Semliki Forest viruses) have a different cellular maturation I–domain III interface probably contributes to initiating this process . pathway—they bud at the plasma membrane—but their fusion proteins Mutations around a hydrophobic pocket at the domain I–domain II (designated E1) are very similar in structure to those of the flaviviruses, interface also affect the pH threshold; this region undergoes a hinge- and priming is likewise by cleavage of a partner protein (E2). like change during the transition, and alterations in the bulk of the The mature flavivirus particle is icosahedrally symmetric and hydrophobic side chains that face the pocket could influence both the about 500 Å in diameter; its membrane bilayer has a mean diameter kinetic barrier and the net free energy change of the process . of about 390 Å (refs. 31,32). E covers the virion surface as an array The structures of E protein trimers from dengue and TBE viruses of 90 dimers (Fig. 4a) in the pre-fusion conformation (Fig. 4b). show the C terminus of the ectodomain projecting toward the fusion The soluble ectodomain (sE) dimer illustrated there lacks about loop; the stem segment has not yet been detected directly in a crystal 50 residues connecting its C terminus with the transmembrane structure, but as the transmembrane segment at its C terminus must anchor; cryo-EM reconstructions show this so-called ‘stem’ region to reside after fusion in the same membrane as the fusion loop, the lie in the outward-facing surface of the lipid bilayer, in a conformation stem must in some sense zip up alongside the clustered domains II that seems to be two amphipathic helices and an intervening loop . (refs. 10,35) (Fig. 4d,e). The absence of a structure that includes the full The sE structure itself contains a central β-barrel (domain I; red in stem also leaves open the question of whether a defined ‘cap’ completes Fig. 4), from which extend two long extensions forming a distinct the zipping up. The residues just N-terminal to the transmembrane 10,33 subdomain (domain II; yellow) . The fusion loop, a short stretch segment are mostly hydrophobic, and one can imagine that they form at the tip of one these extensions, is buried at the dimer interface some sort of tight interaction with the fusion loop or its rim and (Fig. 4a). At the C-terminal end of the sE polypeptide chain, perhaps also with the lipid bilayer itself. connecting to the stem, is an immunoglobulin-like domain (domain III; blue), which probably has viral-attachment functions. Vesicular stomatitis virus G The response to lowered pH in the presence of a membrane leads to Unlike the low pH–induced conformational change in the fusion pro- 11,34,35 the following sequence of molecular events . The dimers dissociate, teins just described, the shift between high- and low-pH forms of the nture a structural & molecular biology volume 15 number 7 j ul Y 2008 693 © 2008 Nature Publishing Group http://www.nature.com/nsmb review ab cd e Figure 5 VSV-G: proposed fusogenic conformational changes. (a) Pre-fusion trimer. The three subunits are in red, blue and green. The fusion loops (asterisk) are held away from the target membrane. The crystal structure does not include about 40 residues, represented here for each subunit by a slightly wavy line, that connect to the transmembrane anchor. (b) Pre-fusion conformation of one subunit (in the orientation of the red subunit in a). Core domain, red; α-helix at the trimer contact, light blue; two-part fusion apparatus, dark blue; C-terminal segment, dark green. Dashed line, the part of the C-terminal segment that is missing from the crystal structure; letter N, the N terminus. (c) Suggested extended intermediate conformation of one subunit, colored as in b. The fusion domains have reoriented (curved arrow in b), with the fusion loops (asterisk) now in contact with the target membrane; the reorientation seems to be driven in part by a loop-to-helix transition that elongates the helix at the trimer contact. The C-terminal segment still connects to the viral membrane (dashed arrow), but it must fold back along the outside of the trimer (curved arrow) to complete the transition to the post-fusion conformation. (d) Post-fusion conformation of one subunit, in the orientation and colors of the subunit in b and c. The C-terminal segment has folded back, and it now projects toward the fusion loops. (e) Post- fusion conformation of the trimer , with colors as in a. It is shown in a ‘horizontal’ orientation, to correspond to the sequence in Figure 1. glycoprotein (G) of rhabdoviruses (for example, vesicular stomatitis virus a relatively long α-helix (light blue in Figs. 5b–d), dominates the three- (VSV) and rabies virus) seems to be reversible . That is, virions inacti- fold contact in the high-pH (pre-fusion) conformation. This domain vated by prolonged incubation at pH < 6 can be reactivated by raising the contains residues from the N-terminal segment of the polypeptide pH to neutral or above, and both conformations of the trimeric protein chain and residues from near the C-terminal part of the chain: we can described here can be obtained from the same protein preparation. consider it a framework around which the rest of the molecule reorients. The structures of VSV-G in high-pH (pre-fusion) and low-pH The other two domains form a jointed, two-part fusion machinery. The (presumably post-fusion) conformations show that, despite these net effect of their rotations relative to each other and to the core domain distinctive properties, the fundamental characteristics of the protein is to translate the fusion loops, at the tip of the outermost domain, and of how it facilitates fusion are similar to those of influenza virus away from the viral membrane and toward the target membrane. In a hemagglutinin or flavivirus E (Fig. 5). G has two hydrophobic loops likely extended intermediate conformation (shown in Fig. 5c, but for that can cross-link to derivatized membrane lipids ; the structures which there are as yet no direct structural data), the C-terminal seg- show that these loops, each of which links a pair of antiparallel ment still connects ‘downward’, even as the fusion loops interact with β-strands, lie next to each other at the tip of an elongated domain the target. In the fully rearranged, low-pH conformation (Figs. 5d,e), (Fig. 5a). In the pre-fusion conformation, these domains face the the C-terminal segment has zipped up along the fusion domains, much viral membrane. In the post-fusion conformation, they cluster as in the flavivirus fusion transition. Aside from this zipping up of the around the three-fold axis, presenting the fusion loops to the target C terminus, the most pronounced conformational change is the loop-to- membrane (Fig. 5e). The connectivity of the strands joined by helix transition of the 12-residue segment between the fusion domains the fusion loops is different from the connectivity in domain II of and the core domain (Fig. 5b to Fig. 5c, blue helix), reminiscent of the flavivirus E and alphavirus E1 (that is, the domains themselves have even more notable loop-to-helix transition in influenza HA2. As in different folds), but the general picture is quite similar: hydrophobic hemagglutinin, the loop-to-helix transition in VSV-G creates a long, residues (including at least one tryptophan) are displayed on tightly three-chain coiled coil at the trimer axis and seems to propel the fusion structured loops at the end of an elongated domain. loops toward the target membrane. The rhabdovirus G protein has a more intricately folded structure The herpesvirus fusion protein, gB, is a ‘stretched’ version of VSV-G . than do flavivirus E and alphavirus E1. We can analyze the structure as This unexpected similarity between fusion proteins of a DNA virus three domains, each of roughly invariant fold, linked by segments that and a negative-strand RNA virus has led to some evolutionary specula- change conformation as the domains rotate with respect to one another tions, but the important result from the point of view of understanding (Fig. 5). A core domain, based on a β-sandwich (red in Figs. 5b–d) and fusion mechanisms is that information about one protein (for example, 694 volume 15 number 7 j ul Y 2008 nture a structural & molecular biology © 2008 Nature Publishing Group http://www.nature.com/nsmb review the identification of the rhabdovirus fusion loops) can be carried over surface catalyst in this context: by aligning the individual subunits in a to the other . Only the presumptive post-fusion structure of gB has preferred orientation (fusion-loop tip in the membrane), it lowers the been determined so far. The gB conformational transition is triggered barrier that separates the free monomer from the true minimum-energy not by changes in pH, but rather by receptor binding to another surface state represented by the post-fusion trimer. protein, gD. How a binding-induced conformational change in gD The case of VSV-G is puzzling. If the protein could undergo the com- leads to the reorganization of gB remains to be worked out. The role plete transition illustrated in Figure 5, then it should become inactivated of one further, conserved, herpesvirus surface protein, the gH–gL at low pH, by inverting during the zipping-up process so that its fusion heterodimer, is likewise still undetermined. loops ultimately insert into the viral membrane. The hemagglutinins of many influenza strains undergo just this kind of inactivation when the Activating and initiating pH is lowered in the absence of attachment to a target membrane. One For most fusion proteins, we can distinguish a priming step and an acti- possibility is that on the viral surface, the protein can shift reversibly vating or triggering step for the sequence of events that follows. Priming between the states represented by Figure 5b and Figure 5c, but insertion is usually the result of proteolysis—either of the fusion protein itself, of the fusion loops into a target membrane somehow favors the further as in the case of influenza hemagglutinin or retroviral Env, or of an (irreversible?) transition to the state in Figure 5d. accompanying protein, as in the case of flavi- and alphaviruses. For these viruses, priming occurs during transport of the immature glycoprotein The extended intermediate to the cell surface, either before assembly of the virus particle by bud- The postulated extended intermediate has been characterized func- ding at the cell surface or after a formation of an immature particle by tionally for HIV-1 gp41 and somewhat less extensively for flavi- and 47 48 budding through an internal membrane. The glycoproteins of other alphaviruses and paramyxoviruses . The post-fusion conformation viruses—Ebola virus and severe acute respiratory syndrome (SARS) of the gp41 ectodomain is particularly simple—just a trimer of hair- 49–51 coronavirus in particular—require cleavage by endosomal cathepsins pins, in which both prongs of the hairpin are α-helices (Fig. 6). 42,43 B or L during cell entry rather than during maturation . The SARS Approximately 50 residues immediately C-terminal to the fusion pep- coronavirus spike protein, S, is a trimer of uncleaved chains on the virion tide (designated HR1, where “HR” stands for “heptad repeats”) form surface, but receptor binding seems to make it susceptible to cathepsin L a central, three-chain coiled coil. A loop that contains a conserved (ref. 43). In addition to making the fusogenic conformational rearrange- disulfide bond connects the HR1 segment to a second heptad-repeat ment possible, cathepsin attack, by releasing a covalent constraint, may element, HR2, which forms an outer-layer α-helix. Peptides from this 52,53 also be sufficient to induce the rearrangement—that is, cleavage may be outer layer can inhibit the fusion process . The mechanism involves a triggering as well as a priming step, after a ‘pre-priming’ by the recep- association of the peptide with the inner core, preventing transition tor interaction. The Ebola virus glycoprotein, GP0, is cleaved by furin to the post-fusion conformation. The lifetime of the intermediate, to GP1 and GP2 before incorporation into virions. Degradation of GP1 as detected by the capacity of such peptides to inhibit fusion during by cathepsins may be part of the triggering step, to release GP2 from the the period after attachment and initiation of conformation change, constraints that prevent its fusogenic conformational rearrangement, is at least several minutes ; its magnitude is probably determined but some further endosomal activity seems to be required as well . by the resistance of the two membranes to being pulled toward each A primed fusion protein is metastable in its pre-fusion state. A covalent other. Without that resistance, the zipping up of the outer layer might peptide bond (either in the fusion protein itself, as in hemagglutinin, or be too rapid for peptide to intervene. HIV fusion occurs at the cell in the chaperone or guard protein, as in dengue prM-E) restrains the ini- surface, and one such inhibitory peptide (T-20, or enfuvirtide) is a tial, folded conformation of the precursor. Once that covalent restraint clinically useful drug . Mutations conferring resistance to T-20 can has gone (irreversibly), a high kinetic barrier still separates the primed occur at various positions in the envelope protein, including resi- from the post-fusion conformation. The trigger that lowers this barrier dues in gp120 (ref. 56). Some of the mutations in HR1 that reduce (or provides the required activation energy) can be binding of a proton, T-20 binding also retard fusion and enhance sensitivity to anti- for viruses that have evolved to detect a low-pH, endosomal environ- bodies targeting the membrane-proximal region of gp41 (ref. 57). ment, or binding of a co-receptor in some other cases (for example, There is also biochemical evidence that the extended intermediate is HIV-1); for herpesviruses and paramyxoviruses, the trigger is an altered the target for this set of neutralizing antibodies . lateral contact with another viral surface protein that has itself changed Fusion of flavi- and alphaviruses occurs within endosomes, but it can conformation because of binding with a cellular receptor. Whatever the be induced experimentally at the cell surface by exposing receptor-bound trigger, association with the ligand alters the free energy profile, so that virus to a brief pulse of low-pH medium. Soluble domain III of the fusion rearrangement to the post-fusion state is rapid. The free energy liber- protein, or domain III plus the stem, can inhibit fusion when present dur- ated by the rearrangement can then be used to overcome the barrier to ing acidification . The target of inhibition is presumably the extended merging two membrane bilayers. intermediate, in which domain III and the stem have yet to curl around The initial response to the trigger is probably formation of an extended and project back toward the fusion loops at the tip of domain II. intermediate. For influenza virus hemagglutinin, the first event must be loosening of the restraints on HA2 imposed by HA1. HA1 is linked to Four questions HA2 by a disulfide near the base of the trimer, so that it cannot dissociate How many fusion-protein trimers contribute to formation of a fusion completely, but it clearly must get out of the way of the loop-to-helix pore? Diagrams such as those in Figure 1 implicitly suggest participation transition and the subsequent zippering step (Fig. 3b). For flavivirus E of more than one fusion-protein trimer per fusion event. But there proteins, the initial dissociation of the dimer probably allows the mono- seems to be no fundamental reason why the fusion machinery needs to mer to flex outward, encounter the target membrane and associate into surround the hemifusion stalk or fusion pore. The energy barrier that trimers (Fig. 4b–d). Soluble E ectodomain dimers dissociate reversibly must be overcome en route to a hemifusion stalk is thought to be about –1 at low pH and redimerize if the pH is returned to neutral. If liposomes ~40–50 kcal mol (refs. 2,5). A free energy of roughly this magnitude are present in the low-pH step, however, the fusion loops insert into the could in principle be recovered from the collapse of just one or two lipid bilayer and the protein trimerizes irreversibly . The bilayer is a trimers, if the interactions driving refolding were strong enough. nture a structural & molecular biology volume 15 number 7 j ul Y 2008 695 © 2008 Nature Publishing Group http://www.nature.com/nsmb review will contribute even if the former does also, results simply from the response of the two membranes to the distortions necessary to pro- mote fusion. At least two properties of bilayer membranes will cause them to resist the collapse of the extended protein intermediates that bridge them. One is the energy of bending—for example, into the nipple-like configuration (Fig. 1c); the other is the so-called ‘hydration force’, which creates a substantial barrier when apposing membranes come closer than about 10–20 Å (ref. 61). Because all the proteins participating in a fusion event bridge the same pair of membranes, the behavior of one extended intermediate is not independent of the behavior of a neighboring one—they are coupled by the deformation energies of the two bilayers they connect. Does insertion of the fusion peptide or loop(s) into the target membrane perturb the bilayer in a way that lowers the kinetic barrier for hemifusion, or does collapse of the extended intermediate do most of the work? Deformation of a planar membrane into a nipple-like bud (Fig. 1c) creates a nearly hemispherical cap, with roughly uniform posi- tive curvature, and a flared region joining it to the planar membrane of the membrane. The flared region has positive curvature in one direction (around the axis of the nipple) and negative in the other (within the Figure 6 The transition between the trimeric extended intermediate and plane of the cross-section in Fig. 1c), and hence little net elastic distor- the post-fusion conformation of the HIV gp41 ectodomain. In the extended 5 tion. Using reasonable dimensions for the bud , one can estimate the intermediate (left), the HR1 segment of each of the three subunits is difference in area between the inner and outer leaflets of the positively shown as an α-helix, and the HR2 segment as an extended chain. The 4 2 curved cap as 2 × 10 Å . Available structural data show that fusion loops fusion peptides are imagined to be inserted into or against the target- and fusion peptides insert only partway into the outer leaflet of the target cell membrane (top) and the transmembrane anchors pass into the viral membrane, displacing lipid head groups laterally and therefore favoring membrane (bottom). In the post-fusion conformation (right), the HR2 11,17,27 segment has zipped up into a helix along the outside of the HR1 three-chain curvature of the leaflet . But their contribution is probably only a coiled-coil, creating a ‘trimer of hairpins’, and the two membranes have modest fraction of the total curvature in the cap. For example, the tip of fused. Courtesy Gaël McGill (see http://www.molecularmovies.org). one flavivirus trimer occupies about 800 Å . Hence, insertion of the tips of even five or six trimers will provide only 20%–25% of the required distortion. The direction of curvature promoted by the observed inser- Cell-cell fusion mediated by influenza virus hemagglutinin responds tion does suggest, however, that as the extended intermediates collapse, nonlinearly to the concentration of hemagglutinin on the cell surface, their fusion peptides or fusion loops may tend to concentrate in the in a manner consistent with cooperativity in fusion-pore formation . positively curved cap and migrate toward the developing hemifusion An estimate obtained from analysis of the lag time to fusion (after a drop stalk. At this stage, they may contribute substantially to the difference in pH) for red blood cells with hemagglutinin-expressing cells suggests between the areas of the inner and outer leaflets. that three or four hemagglutinin trimers might participate in a single fusion event. As the resistance of the two membranes to deformation Does the membrane-proximal segment of the ectodomain have a and the height of the kinetic barrier leading to hemifusion will probably mechanistic role? The membrane-proximal segments (10–15 residues) change with membrane composition, it is likely that the actual number of many fusion-protein ectodomains have characteristic hydrophobic of hemagglutinin trimers required will vary from cell type to cell type. and often relatively tryptophan-rich sequences. At the end of the fusion- Fusion of two cells is a complex event, potentially involving a num- inducing conformational change, these segments are apposed to the ber of distinct pores at different positions across the area of contact. merged membrane and could in principle interact with the membrane Experiments with HIV and HIV pseudotypes containing a mixture of or the fusion peptides/loops (or both). The membrane-proximal segments cleavable and uncleavable (and hence active and inactive) fusion pro- of influenza virus hemagglutinin might have a defined role in stabilizing teins suggest that only one active retroviral envelope trimer is sufficient the final, post-fusion structure (and hence potentially in stabilizing an for fusion, while confirming the requirement for several hemagglutinin open fusion pore) (Fig. 3e). The membrane- proximal region of HIV trimers . The fusion proteins of HIV and other retroviruses may have gp41 has been studied with particular attention, as it is the target of two evolved to manage with a single fusion protein, as the complement of well- characterized, broadly neutralizing antibodies, both of which seem trimers on the virion surface can be rather sparse: estimates of 15–20 trim- to target the extended intermediate rather than the pre-fusion envelope ers total have been made for HIV. Rough calculations based on the known trimer. Some recent NMR experiments suggest that it could form a bent (K ≈ 1 nM) affinity of an HIV gp41 outer-layer peptide for a trimeric amphipathic helix lying in the outer part of the membrane bilayer . inner core suggest that the free energy liberated during collapse of the extended intermediate to the folded-back trimer of hairpins might What structural rearrangements lower the kinetic barrier between –1 approach the estimate of ~40–50 kcal mol for the hemifusion barrier. hemifusion and fusion-pore formation? If influenza virus hemagglu- Cooperativity, if it occurs, could have two distinct mechanisms. One tinin is linked to a glycosyl phosphatidylinositol anchor or to a truncated requires lateral contacts between adjacent trimers in a ring around the protein anchor that does not completely traverse the membrane, the 63–65 fusion pore site. A specific structure for such a ring has been proposed fusion process halts at hemifusion and proceeds forward very slowly . for the alphavirus fusion protein . Lateral contacts would couple the It thus seems that collapse of the extended intermediate—the transition conformational change of one trimer to that in another, as in allosteric that induces hemifusion-stalk formation—is not sufficient to drive the regulation of multi-subunit enzymes. The other mechanism, which fusion process to completion. At the hemifusion stage, a hemagglutinin- 696 volume 15 number 7 j ul Y 2008 nture a structural & molecular biology © 2008 Nature Publishing Group http://www.nature.com/nsmb review induced fusion pore can flicker open and closed , and we may infer that One of several papers in which Helenius, Simons and their co-workers showed that the acidic pH of an endosome is a trigger for viral fusion. The demonstration that viruses some property of the fusion protein, connected with complete traversal have evolved to ‘sense’ the local proton concentration was a contribution both to our of the membrane, must accelerate the forward reaction. The capping of knowledge of viral entry mechanisms and to our understanding of the properties of endocytic pathways more generally. the central helix in influenza HA2 (Fig. 3e) shows that formation of a 4. White, J. & Helenius, A. pH-dependent fusion between Semliki Forest virus membrane well-defined structure, with tight hydrophobic and hydrogen-bonding and liposomes. Proc. Natl. Acad. Sci. USA 77, 3273–3277 (1980). interactions among the participating amino acid residues, brings about 5. Kuzmin, P.I., Zimmerberg, J., Chizmadzhev, Y.A. & Cohen, F.S. A quantitative model for membrane fusion based on low-energy intermediates. Proc. Natl. Acad. Sci. USA a concerted closure of the conformational transition, drawing the near 98, 7235–7240 (2001). ends of the transmembrane helices together and causing the cytoplasmic 6. Zimmerberg, J., Blumenthal, R., Sarkar, D.P., Curran, M. & Morris, S.J. Restricted segments on the opposite side of the membrane to pass into or through movement of lipid and aqueous dyes through pores formed by influenza hemagglutinin 24 during cell fusion. J. Cell Biol. 127, 1885–1894 (1994). the nascent fusion pore . Presence of these segments in the aqueous 7. Plonsky, I. & Zimmerberg, J. The initial fusion pore induced by baculovirus GP64 is channel of a fusion pore will prevent its resealing and may explain how large and forms quickly. J. Cell Biol. 135, 1831–1839 (1996). 8. Melikyan, G.B., Markosyan, R.M., Brener, S.A., Rozenberg, Y. & Cohen, F.S. Role of a cytoplasmic element at the far end of the transmembrane anchor can the cytoplasmic tail of ecotropic Moloney murine leukemia virus Env protein in fusion drive the fusion process forward . In other words, formation of a cap on pore formation. J. Virol. 74, 447–455 (2000). the HA2 helical bundle when zipping-up is complete becomes a mecha- 9. Skehel, J.J. & Wiley, D.C. Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu. Rev. Biochem. 69, 531–569 (2000). nism for making pore formation irreversible because cap formation 10. Modis, Y., Ogata, S., Clements, D. & Harrison, S.C. A ligand-binding pocket in the couples to a translocation, into or through the pore, of material from dengue virus envelope glycoprotein. Proc. Natl. Acad. Sci. USA 100, 6986–6991 the cytoplasmic face of the bilayer. (2003). 11. Modis, Y., Ogata, S., Clements, D. & Harrison, S.C. Structure of the dengue virus envelope protein after membrane fusion. Nature 427, 313–319 (2004). Fusion inhibitors 12. Roche, S., Bressanelli, S., Rey, F.A. & Gaudin, Y. Crystal structure of the low-pH form Ligands can retard or block viral entry if they bind selectively to any of the vesicular stomatitis virus glycoprotein G. Science 313, 187–191 (2006). 13. Roche, S., Rey, F.A., Gaudin, Y. & Bressanelli, S. Structure of the prefusion form of conformation of the fusion protein that precedes, in the fusion pathway, the vesicular stomatitis virus glycoprotein G. Science 315, 843–848 (2007). merger of the two bilayers. For example, inhibition of fusion is a mecha- 14. Yin, H.S., Paterson, R.G., Wen, X., Lamb, R.A. & Jardetzky, T.S. Structure of the uncleaved ectodomain of the paramyxovirus (hPIV3) fusion protein. Proc. Natl. Acad. nism by which some neutralizing antibodies block viral infection. T-20, Sci. USA 102, 9288–9293 (2005). the outer-layer peptide from HIV gp41 described above, demonstrates 15. 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Structure of the haemagglutinin membrane the inner core of an intermediate is a universal feature of the fusion- glycoprotein of influenza virus at 3 Å resolution. Nature 289, 366–373 (1981). The ground-breaking initial structural result of the Skehel-Wiley collaboration on inducing conformational changes illustrated in Figures 3–6. the influenza virus hemagglutinin. A milestone in structural biology and surface- Small molecules have also been found that bind HIV gp120 in its pre- glycoprotein biochemistry, this paper antedated by nearly 15 years the next report of a distinct viral fusion-protein structure. It helped shape the entire field of enveloped fusion conformation, thereby raising the barrier to the initial steps of the virus entry and viral antigenicity. fusion sequence . The site for these molecules is probably a pocket in 19. Wiley, D.C., Wilson, I.A. & Skehel, J.J. 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A trimeric subdomain of the simian immunodeficiency Nature 433, 834–841 (2005). 698 volume 15 number 7 j ul Y 2008 nture a structural & molecular biology © 2008 Nature Publishing Group http://www.nature.com/nsmb http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nature Structural & Molecular Biology Pubmed Central

Viral membrane fusion

Harrison, Stephen C
Nature Structural & Molecular Biology , Volume 15 (7) – Jul 3, 2008
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Abstract

review Me M brane Fusion Stephen C Harrison Infection by viruses having lipid-bilayer envelopes proceeds through fusion of the viral membrane with a membrane of the target cell. Viral ‘fusion proteins’ facilitate this process. They vary greatly in structure, but all seem to have a common mechanism of action, in which a ligand-triggered, large-scale conformational change in the fusion protein is coupled to apposition and merger of the two bilayers. We describe three examples—the influenza virus hemagglutinin, the flavivirus E protein and the vesicular stomatitis virus G protein—in some detail, to illustrate the ways in which different structures have evolved to implement this common mechanism. Fusion inhibitors can be effective antiviral agents. ‘Enveloped’ viruses—those with lipid bilayers as integral parts of their anchor that holds the protein in the viral membrane and a distinct structure—enter the cells they infect by fusion of viral and host-cell hydrophobic patch (‘fusion peptide’ or ‘fusion loop(s)’) that ultimately membranes. One or more viral membrane proteins facilitate the vari- interacts with the target membrane. Moreover, they all happen to be ous fusion steps. Several such fusion proteins have now been studied trimeric in their fusion-active state. In the initial step in the fusion in great detail, with crystal structures determined for both the form of reaction, the fusion protein, responding to binding of a ligand (protons the protein present on the viral surface before interaction with the cell in many cases, as the mechanism has evolved to respond to the low pH 3,4 (‘pre-fusion’ conformation) and the form of the protein after fusion is of an endosome , but cellular or viral protein ligands in other cases), complete (‘post-fusion’ conformation). The proteins show a variety of undergoes a conformational change that extends each subunit toward molecular architectures, but what we can infer from the various struc- the target membrane and yields a contact between that membrane and tures and from experiments both in solution and with cells suggests the fusion peptide or loop(s) (Fig. 1a to Fig. 1b). Many fusion proteins that all of them catalyze fusion in essentially the same way. We can even are C-terminal fragments of a larger precursor (for example, the HA2 draw a rough analogy to serine proteases, which can have very different fragment of influenza virus hemagglutinin or the gp41 fragment of HIV polypeptide chain folds but identical active-site mechanisms. Env), and initiating the fusion process requires that they must first shed Fusion of two bilayer membranes is thermodynamically favorable, but their N-terminal fragment, which often contains a receptor-binding 1,2 there is a very high kinetic barrier . Fusogens of all kinds lower that domain (for example, HA1 or gp120—see the more detailed description kinetic barrier; viral fusion proteins do so by using the free energy liber- of influenza virus hemagglutinin below). Although strong, the evidence ated during a protein conformational change to draw the membranes for an extended intermediate is indirect. The putative extended state is together. The general outlines of the pathway leading from two separate sometimes called a ‘pre-hairpin intermediate’, as the next step is collapse bilayers to a single one is relatively well understood (Fig. 1). A ‘hemifusion’ into a folded-back conformation. The intermediate may have a relatively state—in which the apposed, proximal leaflets of the two bilayers, but long half-life; for HIV-1 gp41, the half-life seems to be many minutes, but not yet the distal leaflets, have merged—is almost certainly an obligatory in other cases, it may only be a few seconds. Step 2: The bridge collapses intermediate. The structure of the hemifusion intermediate is probably (Fig. 1c) so that the two membrane-inserted elements (the fusion pep- stalk-like (Fig. 1d). Studies of fusion mediated by viral proteins provide tide or loop in the target membrane and the C-terminal transmembrane some of the best evidence for hemifusion as a required intermediate stage . anchor in the viral membrane) come together. This collapse distorts the There are probably substantial kinetic barriers both leading into this inter- two bilayers, probably into a nipple-like configuration, with a relatively mediate and leading away from it toward the product (Fig. 2). restricted area of close approach . Whether insertion of the fusion pep- The accumulated evidence suggests that viral fusion proteins lower tide potentiates this distortion in the target membrane, by perturbing the various kinetic barriers and, hence, catalyze the membrane fusion the bilayer and lowering the distortion energy, remains uncertain. process, as follows. Step 1: The protein opens up and forms a bridge Step 3: The distortion of the individual membranes lowers the energy between the two bilayers (Fig. 1b). All viral fusion proteins studied so far barrier (not necessarily symmetrically, as the anchoring of the fusion have two membrane-interacting elements: a C-terminal transmembrane protein is different at the two ends) between separated and hemifused bilayers so that a hemifusion stalk forms (Fig. 1d). Step 4: The hemifusion stalk opens to form a transient fusion pore. A final conformational step Jack and Eileen Connors Structural Biology Laboratory, Harvard Medical School, in the protein refolding renders the open state irreversible, and the pore Laboratory of Molecular Medicine, Children’s Hospital Boston, and Howard expands (Fig. 1e). With some fusion proteins, but not with others, the Hughes Medical Institute, 250 Longwood Avenue, Boston, Massachusetts 6–8 02115, USA. Correspondence should be addressed to S.C.H. pore may flicker open and closed . Whether flickering occurs may ([email protected]). depend on the rapidity of the final conformational step. In most cases, Published online 3 July 2008; doi:10.1038/nsmb.1456 steps 3 and 4 probably require concerted action of more than one 690 volume 15 number 7 j ul Y 2008 nture a structural & molecular biology © 2008 Nature Publishing Group http://www.nature.com/nsmb review a Pre-fusion b Extended c Collapse of d Hemifusion e Fusion pore intermediate intermediate (post-fusion) Cell Virus Figure 1 Sequence of events in membrane fusion promoted by a viral fusion protein. Ambiguities remain in some aspects of this scheme (see main text). (a) The protein in the pre-fusion conformation, with its fusion peptide or loop (light green) sequestered. The representation is purely schematic, and various features of specific proteins are not incorporated—for example, the displacement of the N-terminal fragment of proteins that are cleaved from a precursor or the dimer-to-trimer rearrangement on the surface of flaviviruses. (b) Extended intermediate. The protein opens up, extending the fusion peptide or loop to interact with the target bilayer. The part of the protein that bears the fusion peptide forms a trimer cluster. (c) Collapse of the extended intermediate: a C-terminal segment of the protein folds back along the outside of the trimer core. The segments from the three subunits fold back independently, so that at any point in the process they can extend to different distances along the trimer axis, and the entire trimer can bow outward, away from the deforming membrane. (d) Hemifusion. When collapse of the intermediate has proceeded far enough to bring the two bilayers into contact, the apposed, proximal leaflets merge into a hemifusion stalk. (e) Fusion pore formation. As the hemifused bilayers open into a fusion pore, the final zipping up of the C-terminal ectodomain segments snaps the refolded trimer into its fully symmetric, post-fusion conformation, preventing the pore from resealing. fusion-protein trimer (as symbolized by the two apposed trimers in Influenza virus hemagglutinin Fig. 1). The number of trimers that participate and the nature of the The influenza virus hemagglutinin is the best characterized of all interaction that couples them may vary from case to case, and these issues viral fusion proteins. The crystal structure of its ectodomain in a pre- are still matters of some debate. fusion conformation was determined in classic work by Wiley, Wilson To illustrate these generalizations, we describe the fusion proteins and Skehel in 1981 (refs. 18,19); the post-fusion conformation was of three viruses, each with known three-dimensional structures for finally visualized in 1994 (ref. 20), and the uncleaved precursor, HA0, both the pre-fusion conformation (corresponding to Fig. 1a) and in 1998 (ref. 21). The core of HA1 is a sialic acid–binding domain, the post-fusion conformation (corresponding to Fig. 1e). These borne on a stalk formed by HA2. The central feature of the stalk is a 9 10,11 three viral fusion proteins—from influenza , dengue and three-chain, α-helical coiled coil. HA0 and its cleaved product, pre- 12,13 vesicular stomatitis viruses —are representatives of what have fusion HA1–HA2, are essentially identical in overall structure. The come to be called class I, class II and class III viral fusion proteins, cleavage, which normally happens in the trans-Golgi network (TGN), but as this typology now obscures as much as it clarifies, we avoid but which can also occur after viral budding, leads to a modest local it here. Other viral fusion proteins for which structures in both rearrangement, in which the newly generated N terminus of HA2 conformational states are known include those from representative inserts into a pocket along the three-fold axis, burying the fusion 14,15 16,17 paramyxoviruses and alphaviruses . The principles illustrated peptide (the first 20–25 residues of HA2). The pocket is created by a by those important studies reinforce the conclusions derived from splaying apart from each other of the C termini of the HA2 coiled-coil the three examples chosen here. helices, so that the three helices diverge from the three-fold axis and from each other, rather like a narrow tripod (Fig. 3a). Sialic acid, on glycoproteins or glycolipids, is the influenza virus receptor; HA1 bears the binding site, a shallow pocket exposed on its outward-facing surface . As the plasma membrane recycles regularly through various forms of endocytosis, the virus-receptor complex may not require a specific endocytic signal in order to reach an endo- Hemifusion some. When the HA1:HA2 trimer encounters low pH, it undergoes a large-scale conformational rearrangement, in which HA1 separates Two bilayers from HA2 (Fig. 3a to Fig. 3b), except for a residual disulfide tether, 20,23,24 and the latter effectively turns inside out . The two key features of this HA2 refolding are a loop-to-helix transition in the region connecting the fusion peptide to the central coiled coil (Fig. 3b to Fig. 3c) and reorientation of the C-terminal part of the molecule so that it zips up alongside the extended coil (Fig. 3c to Fig. 3d). These correspond, respectively, to formation of the extended intermediate Fusion pore and to its collapse into a conformation that brings together the fusion peptide and the transmembrane anchor. The loop-to-helix transition Figure 2 Schematic diagram illustrating the (free) energy changes during in the N-terminal part of HA2 augments the central coiled-coil at its fusion of two bilayers. The relative heights of the various barriers are N-terminal end (Fig. 3c); reorientation of the C-terminal part of the arbitrary. Fusion proteins accelerate the process by coupling traversal of these barriers to energetically favorable conformational changes. protein breaks the central helices where they splay apart, at the site nture a structural & molecular biology volume 15 number 7 j ul Y 2008 691 © 2008 Nature Publishing Group http://www.nature.com/nsmb review ab c d Figure 3 Influenza virus hemagglutinin: proposed sequence of fusogenic conformational changes. (a) The pre-fusion conformation. Each subunit is shown in a different color. The binding site for the receptor, sialic acid, is at the top of each subunit, but contact with a receptor molecule is not shown. Red asterisk, the sequestered fusion peptide of the red subunit, at the N terminus of HA2. (b) HA1 dissociates from its tightly docked position in response to proton binding. Each HA1 remains flexibly tethered to the corresponding HA2 by a disulfide bond (near the bottom of the ectodomain, in the orientation shown here). (c) The extended intermediate. The loop between the shorter and longer helices in HA2 (for example, the two red helices and the loop connecting them, in b) becomes a helix, thereby translocating the fusion peptide toward the target membrane. The fusion peptides (asterisk) are shown interacting as amphipathic helices with the target bilayer. The loop-to-helix transition creates a long, three-chain coiled coil at the core of the trimer. (d) Collapse of the extended intermediate to generate the post-fusion conformation. The lower parts of the protein (as seen in the orientation in c) fold back along the outside of the three-chain coiled coil. The collapse is complete only when the two membranes have fused completely. The post-fusion conformation is shown in a ‘horizontal’ orientation, to correspond to the sequence in Figure 1. (e) Detail illustrating some features of the membrane-proximal region of influenza virus HA2 after fusion is complete. The N termini of the coiled-coil helices are capped by contacts with amino acid residues in the link between the fusion peptide and the coiled coil, as well as with residues near the C terminus of the ectodomain, proximal to the transmembrane helices . This cap locks into place all the membrane-proximal components of the structure. The fusion peptides at the N termini of three HA2 chains are shown as cylinders (possible amphipathic helices) lying partly immersed in the outer leaflet of the membrane bilayer, as suggested by NMR and EPR studies . The transmembrane segments, likely to be α-helices, are also shown as cylinders. The relationships in this drawing among the fusion peptides and the transmembrane helices, chosen to illustrate the scale of the structures and the approximate distances between them, are purely schematic, as there is no single structure yet determined experimentally that contains all the elements included here. Only the crystallographically determined components are in ribbon representation. from which the fusion peptide has withdrawn earlier in the fusion conformation of HA2 includes a substructure in which residues process (Fig. 3c to Fig. 3d). connecting the fusion peptides to the coiled-coil and residues just The specific sites at which protonation initiates these large-scale N-terminal to the transmembrane anchors cap the N termini of the three conformational changes may not be uniformly conserved. Mutations central helices (Fig. 3e). Formation of this cap-like substructure seems 28,29 at widely distributed locations affect the stability of trimer interfaces to be important for the transition from hemifusion to fusion . that break during the rearrangement and thereby alter the threshold As the extended intermediate collapses, it must bend outward, away pH for fusion . Conserved ionizable residues (two aspartates and a from the nascent hemifusion stalk, to allow the two membranes to come histidine) in the vicinity of the buried fusion peptide may contrib- together (Fig. 1c). The C-terminal segments of HA2 do not interact ute to the trigger; comparison of their interactions before and after laterally with each other in the final, post-fusion conformation, and there cleavage of HA0 (and hence before and after insertion of the fusion is no reason to suppose that their zipping up is cooperative. That is, each peptide into the cavity) does not, however, lead to an obvious explana- HA2 chain can complete its refolding independently of the other two, and tion for why HA0 does not undergo a proton-induced conformational the loss of overall three-fold symmetry during the transition (Fig. 1c,d) is a change . Redundant contributions to pH-regulated processes often natural consequence of this independence. Formation of the cap structure make identification of critical residues difficult (as illustrated by the (Fig. 3e) restores global three-fold symmetry; if the transmembrane helices history of investigations into the hemoglobin Bohr effect) because loss pass completely through the bilayer, this step probably requires the pres- of one contributing site may not lead to significant loss of fitness or to ence of an aqueous channel—that is, a committed fusion pore (Fig. 1e). pronounced change in pH dependence. The fusion peptide, presented to the target membrane by the loop-to- Flavivirus E helix transition, is thought to form an amphipathic helix, by inference The flaviviruses (a family that includes various mosquito- and tick-borne from its conformation on the surface of a detergent micelle and from pathogens, such as yellow fever virus, dengue virus, and tick- borne- spectroscopic data consistent with partial penetration of the outer leaflet encephalitis virus) have two envelope proteins, known as M and E. of the lipid bilayer . When fusion is complete, the polypeptide chain E has both receptor-binding and fusogenic activities; M is the proteolytic segment just C-terminal to the fusion peptide and the membrane- residuum of a precursor, prM, which is the form incorporated into proximal segment of HA2 interact. The completed post-fusion immature virions. The virus assembles by budding into the endoplasmic 692 volume 15 number 7 j ul Y 2008 nture a structural & molecular biology © 2008 Nature Publishing Group http://www.nature.com/nsmb review ac d Figure 4 Flavivirus E: proposed sequence of fusogenic conformational changes. (a) The packing of 180 E subunits (90 dimers) in an icosahedral array on the surface of a flavivirus particle . The red, yellow and blue parts of each subunit correspond respectively to domains I, II and III of the ectodomain. (b) ‘Side view’ of the pre-fusion, dimeric conformation of the E protein, based on the crystal structure of dengue E (residues 1–395) , supplemented by a representation of the ‘stem’ segment (two helices linked by a short loop, lying in the plane of the membrane head groups) and the transmembrane anchor (a helical hairpin), derived from a cryo-EM reconstruction of the virion . The domains in one of the two subunits are colored as in a; the other subunit is in gray. The fusion loop is at the tip of domain II, on the far right of the colored subunit, buried at the contact with domain III of the dimer partner. (c) Monomeric transition between the pre-fusion dimer and the trimeric extended intermediate. The three subunits that will associate into the extended intermediate in d are not yet in contact. The drawing embodies the suggestion that domains I and II have swung outward, while domain III and the stem remain oriented against the membrane roughly as in the pre-fusion state. The fusion loop is now at the top of the diagram and is shown already interacting with the target bilayer. (d) Extended intermediate. Domains I and II have associated into the trimeric core of the post-fusion conformation, but domain III has not yet flipped over (upper arrows) to dock against them . To indicate that the stem segment must then zip back along the trimer core (lower arrows), the stem is represented by loops ‘poised’ to reconfigure. (e) Post-fusion conformation. Domain III has reoriented, and the stem (dashed line, as there is no direct structural information on its conformation or exact position in the post- fusion trimer) connects it to the transmembrane anchor, now brought together with the fusion loop in the single, fused bilayer. The post-fusion conformation is shown in a ‘horizontal’ orientation, to correspond to the sequence in Figure 1. reticulum, and furin cleavage of prM in the TGN releases most of its allowing the individual subunits to swing outward . The now-exposed ectodomain. Immature virions do not fuse, even when triggered by fusion loops insert into the target membrane, facilitating reclustering of lowering the pH, as prM is essentially a chaperone that prevents the the subunits into trimers. Collapse of the extended, trimeric intermediate fusion-inducing conformational transition. Cleavage of prM to M is the that results from these events can then proceed, by rotation of domain processing step that primes the particle for low pH–induced membrane III in each subunit about the segment that links it to domain I and fusion and hence is not a modification of the fusion protein itself, but (presumably) by zipping up of the stem alongside the clustered domains rather of its companion . The closely related alphaviruses (for example, II. Protonation of one or two conserved histidine residues at the domain Sindbis and Semliki Forest viruses) have a different cellular maturation I–domain III interface probably contributes to initiating this process . pathway—they bud at the plasma membrane—but their fusion proteins Mutations around a hydrophobic pocket at the domain I–domain II (designated E1) are very similar in structure to those of the flaviviruses, interface also affect the pH threshold; this region undergoes a hinge- and priming is likewise by cleavage of a partner protein (E2). like change during the transition, and alterations in the bulk of the The mature flavivirus particle is icosahedrally symmetric and hydrophobic side chains that face the pocket could influence both the about 500 Å in diameter; its membrane bilayer has a mean diameter kinetic barrier and the net free energy change of the process . of about 390 Å (refs. 31,32). E covers the virion surface as an array The structures of E protein trimers from dengue and TBE viruses of 90 dimers (Fig. 4a) in the pre-fusion conformation (Fig. 4b). show the C terminus of the ectodomain projecting toward the fusion The soluble ectodomain (sE) dimer illustrated there lacks about loop; the stem segment has not yet been detected directly in a crystal 50 residues connecting its C terminus with the transmembrane structure, but as the transmembrane segment at its C terminus must anchor; cryo-EM reconstructions show this so-called ‘stem’ region to reside after fusion in the same membrane as the fusion loop, the lie in the outward-facing surface of the lipid bilayer, in a conformation stem must in some sense zip up alongside the clustered domains II that seems to be two amphipathic helices and an intervening loop . (refs. 10,35) (Fig. 4d,e). The absence of a structure that includes the full The sE structure itself contains a central β-barrel (domain I; red in stem also leaves open the question of whether a defined ‘cap’ completes Fig. 4), from which extend two long extensions forming a distinct the zipping up. The residues just N-terminal to the transmembrane 10,33 subdomain (domain II; yellow) . The fusion loop, a short stretch segment are mostly hydrophobic, and one can imagine that they form at the tip of one these extensions, is buried at the dimer interface some sort of tight interaction with the fusion loop or its rim and (Fig. 4a). At the C-terminal end of the sE polypeptide chain, perhaps also with the lipid bilayer itself. connecting to the stem, is an immunoglobulin-like domain (domain III; blue), which probably has viral-attachment functions. Vesicular stomatitis virus G The response to lowered pH in the presence of a membrane leads to Unlike the low pH–induced conformational change in the fusion pro- 11,34,35 the following sequence of molecular events . The dimers dissociate, teins just described, the shift between high- and low-pH forms of the nture a structural & molecular biology volume 15 number 7 j ul Y 2008 693 © 2008 Nature Publishing Group http://www.nature.com/nsmb review ab cd e Figure 5 VSV-G: proposed fusogenic conformational changes. (a) Pre-fusion trimer. The three subunits are in red, blue and green. The fusion loops (asterisk) are held away from the target membrane. The crystal structure does not include about 40 residues, represented here for each subunit by a slightly wavy line, that connect to the transmembrane anchor. (b) Pre-fusion conformation of one subunit (in the orientation of the red subunit in a). Core domain, red; α-helix at the trimer contact, light blue; two-part fusion apparatus, dark blue; C-terminal segment, dark green. Dashed line, the part of the C-terminal segment that is missing from the crystal structure; letter N, the N terminus. (c) Suggested extended intermediate conformation of one subunit, colored as in b. The fusion domains have reoriented (curved arrow in b), with the fusion loops (asterisk) now in contact with the target membrane; the reorientation seems to be driven in part by a loop-to-helix transition that elongates the helix at the trimer contact. The C-terminal segment still connects to the viral membrane (dashed arrow), but it must fold back along the outside of the trimer (curved arrow) to complete the transition to the post-fusion conformation. (d) Post-fusion conformation of one subunit, in the orientation and colors of the subunit in b and c. The C-terminal segment has folded back, and it now projects toward the fusion loops. (e) Post- fusion conformation of the trimer , with colors as in a. It is shown in a ‘horizontal’ orientation, to correspond to the sequence in Figure 1. glycoprotein (G) of rhabdoviruses (for example, vesicular stomatitis virus a relatively long α-helix (light blue in Figs. 5b–d), dominates the three- (VSV) and rabies virus) seems to be reversible . That is, virions inacti- fold contact in the high-pH (pre-fusion) conformation. This domain vated by prolonged incubation at pH < 6 can be reactivated by raising the contains residues from the N-terminal segment of the polypeptide pH to neutral or above, and both conformations of the trimeric protein chain and residues from near the C-terminal part of the chain: we can described here can be obtained from the same protein preparation. consider it a framework around which the rest of the molecule reorients. The structures of VSV-G in high-pH (pre-fusion) and low-pH The other two domains form a jointed, two-part fusion machinery. The (presumably post-fusion) conformations show that, despite these net effect of their rotations relative to each other and to the core domain distinctive properties, the fundamental characteristics of the protein is to translate the fusion loops, at the tip of the outermost domain, and of how it facilitates fusion are similar to those of influenza virus away from the viral membrane and toward the target membrane. In a hemagglutinin or flavivirus E (Fig. 5). G has two hydrophobic loops likely extended intermediate conformation (shown in Fig. 5c, but for that can cross-link to derivatized membrane lipids ; the structures which there are as yet no direct structural data), the C-terminal seg- show that these loops, each of which links a pair of antiparallel ment still connects ‘downward’, even as the fusion loops interact with β-strands, lie next to each other at the tip of an elongated domain the target. In the fully rearranged, low-pH conformation (Figs. 5d,e), (Fig. 5a). In the pre-fusion conformation, these domains face the the C-terminal segment has zipped up along the fusion domains, much viral membrane. In the post-fusion conformation, they cluster as in the flavivirus fusion transition. Aside from this zipping up of the around the three-fold axis, presenting the fusion loops to the target C terminus, the most pronounced conformational change is the loop-to- membrane (Fig. 5e). The connectivity of the strands joined by helix transition of the 12-residue segment between the fusion domains the fusion loops is different from the connectivity in domain II of and the core domain (Fig. 5b to Fig. 5c, blue helix), reminiscent of the flavivirus E and alphavirus E1 (that is, the domains themselves have even more notable loop-to-helix transition in influenza HA2. As in different folds), but the general picture is quite similar: hydrophobic hemagglutinin, the loop-to-helix transition in VSV-G creates a long, residues (including at least one tryptophan) are displayed on tightly three-chain coiled coil at the trimer axis and seems to propel the fusion structured loops at the end of an elongated domain. loops toward the target membrane. The rhabdovirus G protein has a more intricately folded structure The herpesvirus fusion protein, gB, is a ‘stretched’ version of VSV-G . than do flavivirus E and alphavirus E1. We can analyze the structure as This unexpected similarity between fusion proteins of a DNA virus three domains, each of roughly invariant fold, linked by segments that and a negative-strand RNA virus has led to some evolutionary specula- change conformation as the domains rotate with respect to one another tions, but the important result from the point of view of understanding (Fig. 5). A core domain, based on a β-sandwich (red in Figs. 5b–d) and fusion mechanisms is that information about one protein (for example, 694 volume 15 number 7 j ul Y 2008 nture a structural & molecular biology © 2008 Nature Publishing Group http://www.nature.com/nsmb review the identification of the rhabdovirus fusion loops) can be carried over surface catalyst in this context: by aligning the individual subunits in a to the other . Only the presumptive post-fusion structure of gB has preferred orientation (fusion-loop tip in the membrane), it lowers the been determined so far. The gB conformational transition is triggered barrier that separates the free monomer from the true minimum-energy not by changes in pH, but rather by receptor binding to another surface state represented by the post-fusion trimer. protein, gD. How a binding-induced conformational change in gD The case of VSV-G is puzzling. If the protein could undergo the com- leads to the reorganization of gB remains to be worked out. The role plete transition illustrated in Figure 5, then it should become inactivated of one further, conserved, herpesvirus surface protein, the gH–gL at low pH, by inverting during the zipping-up process so that its fusion heterodimer, is likewise still undetermined. loops ultimately insert into the viral membrane. The hemagglutinins of many influenza strains undergo just this kind of inactivation when the Activating and initiating pH is lowered in the absence of attachment to a target membrane. One For most fusion proteins, we can distinguish a priming step and an acti- possibility is that on the viral surface, the protein can shift reversibly vating or triggering step for the sequence of events that follows. Priming between the states represented by Figure 5b and Figure 5c, but insertion is usually the result of proteolysis—either of the fusion protein itself, of the fusion loops into a target membrane somehow favors the further as in the case of influenza hemagglutinin or retroviral Env, or of an (irreversible?) transition to the state in Figure 5d. accompanying protein, as in the case of flavi- and alphaviruses. For these viruses, priming occurs during transport of the immature glycoprotein The extended intermediate to the cell surface, either before assembly of the virus particle by bud- The postulated extended intermediate has been characterized func- ding at the cell surface or after a formation of an immature particle by tionally for HIV-1 gp41 and somewhat less extensively for flavi- and 47 48 budding through an internal membrane. The glycoproteins of other alphaviruses and paramyxoviruses . The post-fusion conformation viruses—Ebola virus and severe acute respiratory syndrome (SARS) of the gp41 ectodomain is particularly simple—just a trimer of hair- 49–51 coronavirus in particular—require cleavage by endosomal cathepsins pins, in which both prongs of the hairpin are α-helices (Fig. 6). 42,43 B or L during cell entry rather than during maturation . The SARS Approximately 50 residues immediately C-terminal to the fusion pep- coronavirus spike protein, S, is a trimer of uncleaved chains on the virion tide (designated HR1, where “HR” stands for “heptad repeats”) form surface, but receptor binding seems to make it susceptible to cathepsin L a central, three-chain coiled coil. A loop that contains a conserved (ref. 43). In addition to making the fusogenic conformational rearrange- disulfide bond connects the HR1 segment to a second heptad-repeat ment possible, cathepsin attack, by releasing a covalent constraint, may element, HR2, which forms an outer-layer α-helix. Peptides from this 52,53 also be sufficient to induce the rearrangement—that is, cleavage may be outer layer can inhibit the fusion process . The mechanism involves a triggering as well as a priming step, after a ‘pre-priming’ by the recep- association of the peptide with the inner core, preventing transition tor interaction. The Ebola virus glycoprotein, GP0, is cleaved by furin to the post-fusion conformation. The lifetime of the intermediate, to GP1 and GP2 before incorporation into virions. Degradation of GP1 as detected by the capacity of such peptides to inhibit fusion during by cathepsins may be part of the triggering step, to release GP2 from the the period after attachment and initiation of conformation change, constraints that prevent its fusogenic conformational rearrangement, is at least several minutes ; its magnitude is probably determined but some further endosomal activity seems to be required as well . by the resistance of the two membranes to being pulled toward each A primed fusion protein is metastable in its pre-fusion state. A covalent other. Without that resistance, the zipping up of the outer layer might peptide bond (either in the fusion protein itself, as in hemagglutinin, or be too rapid for peptide to intervene. HIV fusion occurs at the cell in the chaperone or guard protein, as in dengue prM-E) restrains the ini- surface, and one such inhibitory peptide (T-20, or enfuvirtide) is a tial, folded conformation of the precursor. Once that covalent restraint clinically useful drug . Mutations conferring resistance to T-20 can has gone (irreversibly), a high kinetic barrier still separates the primed occur at various positions in the envelope protein, including resi- from the post-fusion conformation. The trigger that lowers this barrier dues in gp120 (ref. 56). Some of the mutations in HR1 that reduce (or provides the required activation energy) can be binding of a proton, T-20 binding also retard fusion and enhance sensitivity to anti- for viruses that have evolved to detect a low-pH, endosomal environ- bodies targeting the membrane-proximal region of gp41 (ref. 57). ment, or binding of a co-receptor in some other cases (for example, There is also biochemical evidence that the extended intermediate is HIV-1); for herpesviruses and paramyxoviruses, the trigger is an altered the target for this set of neutralizing antibodies . lateral contact with another viral surface protein that has itself changed Fusion of flavi- and alphaviruses occurs within endosomes, but it can conformation because of binding with a cellular receptor. Whatever the be induced experimentally at the cell surface by exposing receptor-bound trigger, association with the ligand alters the free energy profile, so that virus to a brief pulse of low-pH medium. Soluble domain III of the fusion rearrangement to the post-fusion state is rapid. The free energy liber- protein, or domain III plus the stem, can inhibit fusion when present dur- ated by the rearrangement can then be used to overcome the barrier to ing acidification . The target of inhibition is presumably the extended merging two membrane bilayers. intermediate, in which domain III and the stem have yet to curl around The initial response to the trigger is probably formation of an extended and project back toward the fusion loops at the tip of domain II. intermediate. For influenza virus hemagglutinin, the first event must be loosening of the restraints on HA2 imposed by HA1. HA1 is linked to Four questions HA2 by a disulfide near the base of the trimer, so that it cannot dissociate How many fusion-protein trimers contribute to formation of a fusion completely, but it clearly must get out of the way of the loop-to-helix pore? Diagrams such as those in Figure 1 implicitly suggest participation transition and the subsequent zippering step (Fig. 3b). For flavivirus E of more than one fusion-protein trimer per fusion event. But there proteins, the initial dissociation of the dimer probably allows the mono- seems to be no fundamental reason why the fusion machinery needs to mer to flex outward, encounter the target membrane and associate into surround the hemifusion stalk or fusion pore. The energy barrier that trimers (Fig. 4b–d). Soluble E ectodomain dimers dissociate reversibly must be overcome en route to a hemifusion stalk is thought to be about –1 at low pH and redimerize if the pH is returned to neutral. If liposomes ~40–50 kcal mol (refs. 2,5). A free energy of roughly this magnitude are present in the low-pH step, however, the fusion loops insert into the could in principle be recovered from the collapse of just one or two lipid bilayer and the protein trimerizes irreversibly . The bilayer is a trimers, if the interactions driving refolding were strong enough. nture a structural & molecular biology volume 15 number 7 j ul Y 2008 695 © 2008 Nature Publishing Group http://www.nature.com/nsmb review will contribute even if the former does also, results simply from the response of the two membranes to the distortions necessary to pro- mote fusion. At least two properties of bilayer membranes will cause them to resist the collapse of the extended protein intermediates that bridge them. One is the energy of bending—for example, into the nipple-like configuration (Fig. 1c); the other is the so-called ‘hydration force’, which creates a substantial barrier when apposing membranes come closer than about 10–20 Å (ref. 61). Because all the proteins participating in a fusion event bridge the same pair of membranes, the behavior of one extended intermediate is not independent of the behavior of a neighboring one—they are coupled by the deformation energies of the two bilayers they connect. Does insertion of the fusion peptide or loop(s) into the target membrane perturb the bilayer in a way that lowers the kinetic barrier for hemifusion, or does collapse of the extended intermediate do most of the work? Deformation of a planar membrane into a nipple-like bud (Fig. 1c) creates a nearly hemispherical cap, with roughly uniform posi- tive curvature, and a flared region joining it to the planar membrane of the membrane. The flared region has positive curvature in one direction (around the axis of the nipple) and negative in the other (within the Figure 6 The transition between the trimeric extended intermediate and plane of the cross-section in Fig. 1c), and hence little net elastic distor- the post-fusion conformation of the HIV gp41 ectodomain. In the extended 5 tion. Using reasonable dimensions for the bud , one can estimate the intermediate (left), the HR1 segment of each of the three subunits is difference in area between the inner and outer leaflets of the positively shown as an α-helix, and the HR2 segment as an extended chain. The 4 2 curved cap as 2 × 10 Å . Available structural data show that fusion loops fusion peptides are imagined to be inserted into or against the target- and fusion peptides insert only partway into the outer leaflet of the target cell membrane (top) and the transmembrane anchors pass into the viral membrane, displacing lipid head groups laterally and therefore favoring membrane (bottom). In the post-fusion conformation (right), the HR2 11,17,27 segment has zipped up into a helix along the outside of the HR1 three-chain curvature of the leaflet . But their contribution is probably only a coiled-coil, creating a ‘trimer of hairpins’, and the two membranes have modest fraction of the total curvature in the cap. For example, the tip of fused. Courtesy Gaël McGill (see http://www.molecularmovies.org). one flavivirus trimer occupies about 800 Å . Hence, insertion of the tips of even five or six trimers will provide only 20%–25% of the required distortion. The direction of curvature promoted by the observed inser- Cell-cell fusion mediated by influenza virus hemagglutinin responds tion does suggest, however, that as the extended intermediates collapse, nonlinearly to the concentration of hemagglutinin on the cell surface, their fusion peptides or fusion loops may tend to concentrate in the in a manner consistent with cooperativity in fusion-pore formation . positively curved cap and migrate toward the developing hemifusion An estimate obtained from analysis of the lag time to fusion (after a drop stalk. At this stage, they may contribute substantially to the difference in pH) for red blood cells with hemagglutinin-expressing cells suggests between the areas of the inner and outer leaflets. that three or four hemagglutinin trimers might participate in a single fusion event. As the resistance of the two membranes to deformation Does the membrane-proximal segment of the ectodomain have a and the height of the kinetic barrier leading to hemifusion will probably mechanistic role? The membrane-proximal segments (10–15 residues) change with membrane composition, it is likely that the actual number of many fusion-protein ectodomains have characteristic hydrophobic of hemagglutinin trimers required will vary from cell type to cell type. and often relatively tryptophan-rich sequences. At the end of the fusion- Fusion of two cells is a complex event, potentially involving a num- inducing conformational change, these segments are apposed to the ber of distinct pores at different positions across the area of contact. merged membrane and could in principle interact with the membrane Experiments with HIV and HIV pseudotypes containing a mixture of or the fusion peptides/loops (or both). The membrane-proximal segments cleavable and uncleavable (and hence active and inactive) fusion pro- of influenza virus hemagglutinin might have a defined role in stabilizing teins suggest that only one active retroviral envelope trimer is sufficient the final, post-fusion structure (and hence potentially in stabilizing an for fusion, while confirming the requirement for several hemagglutinin open fusion pore) (Fig. 3e). The membrane- proximal region of HIV trimers . The fusion proteins of HIV and other retroviruses may have gp41 has been studied with particular attention, as it is the target of two evolved to manage with a single fusion protein, as the complement of well- characterized, broadly neutralizing antibodies, both of which seem trimers on the virion surface can be rather sparse: estimates of 15–20 trim- to target the extended intermediate rather than the pre-fusion envelope ers total have been made for HIV. Rough calculations based on the known trimer. Some recent NMR experiments suggest that it could form a bent (K ≈ 1 nM) affinity of an HIV gp41 outer-layer peptide for a trimeric amphipathic helix lying in the outer part of the membrane bilayer . inner core suggest that the free energy liberated during collapse of the extended intermediate to the folded-back trimer of hairpins might What structural rearrangements lower the kinetic barrier between –1 approach the estimate of ~40–50 kcal mol for the hemifusion barrier. hemifusion and fusion-pore formation? If influenza virus hemagglu- Cooperativity, if it occurs, could have two distinct mechanisms. One tinin is linked to a glycosyl phosphatidylinositol anchor or to a truncated requires lateral contacts between adjacent trimers in a ring around the protein anchor that does not completely traverse the membrane, the 63–65 fusion pore site. A specific structure for such a ring has been proposed fusion process halts at hemifusion and proceeds forward very slowly . for the alphavirus fusion protein . Lateral contacts would couple the It thus seems that collapse of the extended intermediate—the transition conformational change of one trimer to that in another, as in allosteric that induces hemifusion-stalk formation—is not sufficient to drive the regulation of multi-subunit enzymes. The other mechanism, which fusion process to completion. At the hemifusion stage, a hemagglutinin- 696 volume 15 number 7 j ul Y 2008 nture a structural & molecular biology © 2008 Nature Publishing Group http://www.nature.com/nsmb review induced fusion pore can flicker open and closed , and we may infer that One of several papers in which Helenius, Simons and their co-workers showed that the acidic pH of an endosome is a trigger for viral fusion. 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Structure of the haemagglutinin membrane the inner core of an intermediate is a universal feature of the fusion- glycoprotein of influenza virus at 3 Å resolution. Nature 289, 366–373 (1981). The ground-breaking initial structural result of the Skehel-Wiley collaboration on inducing conformational changes illustrated in Figures 3–6. the influenza virus hemagglutinin. A milestone in structural biology and surface- Small molecules have also been found that bind HIV gp120 in its pre- glycoprotein biochemistry, this paper antedated by nearly 15 years the next report of a distinct viral fusion-protein structure. It helped shape the entire field of enveloped fusion conformation, thereby raising the barrier to the initial steps of the virus entry and viral antigenicity. fusion sequence . The site for these molecules is probably a pocket in 19. Wiley, D.C., Wilson, I.A. & Skehel, J.J. 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A trimeric subdomain of the simian immunodeficiency Nature 433, 834–841 (2005). 698 volume 15 number 7 j ul Y 2008 nture a structural & molecular biology © 2008 Nature Publishing Group http://www.nature.com/nsmb

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