Many early studies of HA-mediated fusion kinetics relied on fusion of HA-expressing cells with red blood cells (Spruce et al., 1989; Zimmerberg et al., 1994) or planar bilayers (Melikyan et al., 1993a, 1993b). In the latter case, measurements of conductance and capacitance showed reversible opening and closing of flickering pores, following initial acidification and commitment to secure pore opening only after a number of seconds. Good evidence that several HAs participate in pore formation came from red-blood cell fusion experiments in which the surface concentration of HA in the expressing cells varied over a roughly thirteenfold range (Danieli et al., 1996). Hill-plot treatment of the fusion times led to an estimate of 3–4 HAs per fusion event. A limitation of any cell–cell fusion experiment is the large contact zone and hence some ambiguity concerning the number of independent, local fusion events that occur in any one observation. Single-virion fusion experiments confirmed the earlier estimate of 3–4 HAs and showed that lipid-mixing precedes content mixing, as expected for an obligatory hemifusion intermediate (Floyd et al., 2008). In that work, the single, rate-limiting step for content mixing had a rate constant of about 0.05/s.

Calculations based on continuum models and molecular simulations have suggested various structures for hemifusion intermediates. The pathway with the lowest energy barrier appears to be through a so-called ‘hemifusion stalk', derived from the apposed leaflets (Kuzmin et al., 2001). Some proposals for the subsequent transition to a fusion pore postulate expansion of the stalk into a ‘hemifusion diaphragm', in which the distal leaflets form a continuous bilayer between the two membrane-bounded compartments; others suggest that a stalk can open directly into a pore. Hemifusion diaphragms can be seen by electron microscopy (e.g., Hernandez et al., 2012); evidence for stalk-like structures comes from x-ray analysis of crystalline lipid phases (Yang and Huang, 2002). Both calculations and observations suggest that hemifusion diaphragms are stable, long-lived intermediates and that their formation might be a dead-end side reaction. Recent work on calcium-triggered SNARE/synaptotagmin/complexin-mediated fusion of lipid vesicles on a physiological time scale shows that the reaction proceeds too quickly (<100 ms) to detect even a hemifusion intermediate and that fusion from a hemifusion diaphragm is slow (tens of seconds) and inefficient (Diao et al., 2012). In influenza-virus fusion, tight packing of proteins on the viral surface probably makes a transition from stalk or other point-contact intermediate to diaphragm even less likely than in SNARE-mediated fusion, in which protein coverage of the fusing membranes is sparser.

The experimental results we describe above apply the previously developed single-virion experimental design (Floyd et al., 2008) to virus particles with site-directed mutations in the HA protein, enabling us to relate the kinetics of the lipid-mixing step to molecular events in the HA trimer inferred from the pre-fusion and post-fusion structures. The experiments report on conformational rearrangements in the catalyst (HA); their interpretation does not depend on the details of membrane organization in the substrates (the hemifusing membranes). They provide strong evidence for a long-lived, extended HA intermediate and for a rapid and cooperative transition to lipid mixing when a critical number of these intermediates has accumulated within a contact neighborhood. Thus, they begin to elucidate features of the ‘gray zone' in Figure 1A.

Figure 6 illustrates the model derived from our current experiments (Figures 1–4), as supported by our computer simulation (Figure 5). Binding to sialic-acid-containing glycans is weak, and local forces, such as flow, can displace the particle (either by ‘rolling', with dissociation at the rear edge accompanied by new attachments at the front, or by translation across the membrane surface together with any associated lipids). The contact region between a virus particle and the target membrane is relatively large (probably 50–150 HA trimers, depending on the particle morphology: see comments above and Figure 6 caption). Any fusion peptide released within this area of contact and inserted into the target membrane will contribute to resisting buffer flow; the probability of arrest is then the probability for fusion-peptide exposure and insertion from 3–4 trimers within the area of membrane contact. Because hemifusion requires that fusion peptides of 3–4 adjacent trimers insert into the target membrane, the apparent rate constant for hemifusion relates to a different probability—that of inserting fusion peptides from several neighboring HAs. The data suggest that HA trimers independently undergo the first part of their low-pH-induced conformational change and pause as extended intermediates with their fusion peptides inserted into the target membrane. Capture by the target membrane restrains a trimer from collapsing to induce hemifusion until 2–3 neighbors help generate sufficient pull to deform the membrane. This interpretation also argues that when these conditions have been met, the final HA rearrangements are fast and cooperative.

Cooperativity of the final HA conformational rearrangements can derive either from contacts between neighboring HAs or simply from the linkage that is present among all participating HAs by virtue of their common insertion in the two hemifusing membranes. We favor the latter explanation. There is no evidence for defined, lateral contacts between HA trimers, and any such contacts, were they present, could not persist during the substantial changes in HA conformation and orientation that accompany the progression to hemifusion. The accumulation of N extended HA 2 neighbors, each fluctuating toward a more stable, fully collapsed conformation while pulling against the elastic restoring force of the two membranes, will lead to an abrupt and cooperative event, when the critical Nth molecule joins the cluster (rather like the abrupt consequence of adding a new member to the team on one side of a previously balanced tug-of-war). Evidence for the rapidity of the final HA rearrangements comes from the results of our simulation and gamma distribution analysis and the sensitivity of the derived rate constant to the mutations we have introduced. Any additional steps, not directly sensitive to those mutations, must be faster than any one of the steps of fusion-peptide engagement with the target membrane.

Engagement of the fusion peptide with a target membrane requires formation of an extended intermediate to project it beyond the outer margin of adjacent HA 1 heads (Figure 6B). For an intermediate to form that resembles the inner core of the ultimate, low-pH triggered structure (as drawn for the extended structure in Figure 1A), the HA 1 heads must dissociate from their contact with an HA 2 loop (HA 2 residues 58–74: see Figure 3D) between the short first α-helix (HA 2 , residues 38–57) and the long central α-helix (HA 2 , residues 75–127) (Godley et al., 1992), and that loop must in turn reverse direction and become itself a helix. Extension of the central trimeric coiled-coil probably drives the latter process, as the amino-acid sequence of the loop strongly favors such a conformation (Carr and Kim, 1993). Further necessary structural changes, in addition to release of the fusion peptide from its pocket, are dissociation of two strands (HA 2 , residues 21–38) from the edge of the small β-sheet near the base of the HA trimer and displacement outward of this segment of HA 2 along with the short first helix, which could unfold and refold as it adds to the extending, central helical coiled-coil. The structure thus pictured would project the fusion peptide no more than about 25 Å beyond the palisade of un-triggered HAs (Figures 1A and 6), even if the HA 2 segment that will fold back against the central core remains roughly in its pre-fusion state (again, as drawn for the extended structure in Figure 1A). In short, the intermediate that mediates arrest and hemifusion must indeed be almost completely extended, at least for part of its lifetime.

The structures of the initial and final states do not dictate a unique ordering of the events just listed, but some must clearly precede others. Our results show that breaking the interactions that restrain the fusion peptide is rate-limiting both for virion arrest and for hemifusion at the pH of endosomes from which influenza virus penetrates (approximately 5 to 5.5); this interpretation is consistent with the dominance hierarchy of mutations that influence the threshold pH for fusion (Steinhauer et al., 1996). Mutations that affect the pH dependence of fusion map to a number of sites distributed across the HA 2 trimer interface (Daniels et al., 1985). Changes in the stability of this interface will influence fluctuations from the equilibrium structure and hence the ‘window of opportunity' for the fusion peptide to withdraw productively from its pocket. Emergence of the fusion peptide can in principle precede the other events, which then must follow rapidly (or the peptide will reinsert); perhaps more frequently, extension of the central coiled-coil by residues 20–74 may exert a ‘tug' to extract the fusion peptide. The fusion-peptide contacts, conserved in all field-isolated influenza HA virions, form upon cleavage of HA 0 (Chen et al., 1998; Russell et al., 2004). Their stability is critical for viral transmission, not only in the passage from one host or cell to another, but also during entry, as fusion will be relatively unproductive if it occurs before the pH of the surrounding compartment has dropped enough to allow M2-mediated acidification of the particle interior (Ivanovic et al., 2012), which induces dissociation of the eight ribonucleoproteins (RNPs) of the viral genome from the matrix protein, M1 (Martin and Helenius, 1991). Fusion too rapid for adequate acidification would release an inactive M1-RNP complex.

Evidence for extended intermediates for other viral fusion proteins is largely indirect. The most extensive data are for HIV gp41, in which binding of peptides such as C34 (Chan et al., 1998) and T-20 (Wild et al., 1994), mimics for the outer layer of the final, hairpin structure, indicates that the inner-core coiled-coil must be present before the outer layer folds back against it. Outer-layer peptides from paramyxovirus F proteins similarly inhibit fusion (Lambert et al., 1996). Electron microscopy of human parainfluenza virus 5 (PIV5) fusing with target vesicles shows a distance between viral and target membranes that corresponds to the gap expected for the putative extended intermediate of F, which (unlike the HA intermediate) should project much farther from the viral membrane than does the pre-fusion form (Kim et al., 2011).

The relative kinetics of HA-mediated hemifusion and arrest show that because fusion requires several neighboring HA trimers, the lifetime of an isolated extended intermediate is substantial. Release of the fusion peptide is rate limiting for generating an extended HA intermediate, but subsequent collapse is rapid only when three or four neighboring extended intermediates have appeared, and the mean lifetime of the ‘pioneer' extended HA in any patch can be as long as a minute or more, depending on pH. Thus, a relatively long-lived extended intermediate could prove to be a clinically useful target for an influenza-virus entry inhibitor: the inherent lower limit to the stability of the pre-fusion HA combined with the requirement for the coordinated action of multiple, independently-triggered HAs to induce fusion, curbs the ability of a virus to escape such treatment by mutations that accelerate fusion-inducing rearrangements.

The only clinically approved inhibitor of virus fusion targets the extended intermediate of the HIV fusion protein, gp41 (Wild et al., 1994; Kilby and Enron, 2003); analogous inhibitors have been designed for paramyxoviruses (Lambert et al., 1996), influenza virus (Lee et al., 2011) and flaviviruses (Schmidt et al., 2010a, 2010b). For HIV fusion, the results of timing-of-inhibitor-addition experiments (Gallo et al., 2001; Miyauchi et al., 2009), the low surface density of the HIV-envelope protein (Chertova et al., 2002; Zhu et al., 2003), and the indication, from experiments with virions bearing mixtures of active and inactive spikes, that one HIV-envelope trimer may be sufficient to mediate fusion (Yang et al., 2005), have led to the inference that a single, trimeric gp41 mediates fusion through a very slow fold-back transition. The data presented here for influenza HA show that there can be a long-lived species, analogous to the extended form of gp41, even when the fold-back transition itself is fast. We suggest that the long delay between HIV binding and membrane fusion may reflect a sequence of events similar to those in our model for influenza virus fusion. In other words, we propose that HIV fusion may require recruitment of (at least) a second envelope trimer, following attachment of the first. The delay time would then be a combination of the time needed to recruit one or more additional envelope proteins and the time needed to activate them with CD4 and a coreceptor. Extensions of the approaches taken here to HIV and other viruses will be necessary to probe the true similarities and differences among them.