Comparison of transient and successful fusion pores connecting influenza hemagglutinin expressing cells to planar membranes

Time-resolved admittance measurements were used to investigate the evolution of fusion pores formed between cells expressing influenza virus hemagglutinin (HA) and planar bilayer membranes. The majority of fusion pores opened in a stepwise fashion to semistable conductance levels of several nS. About 20% of the pores had measurable rise times to nS conductances; some of these opened to conductances of approximately 500 pS where they briefly lingered before opening further to semistable conductances. The fall times of closing were statistically similar to the rise times of opening. All fusion pores exhibited semistable values of conductance, varying from approximately 2-20 nS; they would then either close or fully open to conductances on the order of 1 microS. The majority of pores closed; approximately 10% fully opened. Once within the semistable stage, all fusion pores, even those that eventually closed, tended to grow. Statistically, however, before closing, transient fusion pores ceased to grow and reversed their conductance pattern: conductances decreased with a measurable time course until a final drop to closure. In contrast, pore enlargement to the fully open state tended to occur from the largest conductance values attained during a pore's semistable stage. This final enlargement was characterized by a stepwise increase in conductance. The density of HA on the cell surface did not strongly affect pore dynamics. But increased proteolytic treatment of cell surfaces did lead to faster growth within the semistable range. Transient pores and pores that fully opened had indistinguishable initial conductances and statistically identical time courses of early growth, suggesting they were the same upon formation. We suggest that transient and fully open pores evolved from common structures with stochastic factors determining their fate.     

The fusion kinetics of influenza hemagglutinin expressing cells to planar bilayer membranes is affected by HA density and host cell surface

Time-resolved admittance measurements were used to follow formation of individual fusion pores connecting influenza virus hemagglutinin (HA)- expressing cells to planar bilayer membranes. By measuring in-phase, out-of-phase, and dc components of currents, pore conductances were resolved with millisecond time resolution. Fusion pores developed in stages, from small pores flickering open and closed, to small successful pores that remained open until enlarging their lumens to sizes greater than those of viral nucleocapsids. The kinetics of fusion and the properties of fusion pores were studied as functions of density of the fusion protein HA. The consequences of treating cell surfaces with proteases that do not affect HA were also investigated. Fusion kinetics were described by waiting time distributions from triggering fusion, by lowering pH, to the moment of pore formation. The kinetics of pore formation became faster as the density of active HA was made greater or when cell surface proteins were extensively cleaved with proteases. In accord with this faster kinetics, the intervals between transient pore openings within the flickering stage were shorter for higher HA density and more extensive cell surface treatment. Whereas the kinetics of fusion depended on HA density, the lifetimes of open fusion pores were independent of HA density. However, the lifetimes of open pores were affected by the proteolytic treatment of the cells. Faster fusion kinetics correlated with shorter pore openings. We conclude that the density of fusion protein strongly affects the kinetics of fusion pore formation, but that once formed, pore evolution is not under control of fusion proteins but rather under the influence of mechanical forces, such as membrane bending and tension.     

Hemifusion between cells expressing hemagglutinin of influenza virus and planar membranes can precede the formation of fusion pores that subsequently fully enlarge

The chronological relation between the establishment of lipid continuity and fusion pore formation has been investigated for fusion of cells expressing hemagglutinin (HA) of influenza virus to planar bilayer membranes. Self-quenching concentrations of lipid dye were placed in the planar membrane to monitor lipid mixing, and time-resolved admittance measurements were used to measure fusion pores. For rhodamine-PE, fusion pores always occurred before a detectable amount of dye moved into an HA-expressing cell. However, with DiI in the planar membrane, the relationship was reversed: the spread of dye preceded formation of small pores. In other words, by using DiI as probe, hemifusion was clearly observed to occur before pore formation. For hemifused cells, a small pore could form and subsequently fully enlarge. In contrast, for cells that express a glycosylphosphatidylinositol-anchored ectodomain of HA, hemifusion occurred, but no fully enlarged pores were observed. Therefore, the transmembrane domain of HA is required for the formation of fully enlarging pores. Thus, with the planar bilayer membranes as target, hemifusion can precede pore formation, and the occurrence of lipid dye spread does not preclude formation of pores that can enlarge fully.     

Membrane permeability changes at early stages of influenza hemagglutinin-mediated fusion

While biological membrane fusion is classically defined as the leak-free merger of membranes and contents, leakage is a finding in both experimental and theoretical studies. The fusion stages, if any, that allow membrane permeation are uncharted. In this study we monitored membrane ionic permeability at early stages of fusion mediated by the fusogenic protein influenza hemagglutinin (HA). HAb2 cells, expressing HA on their plasma membrane, fused with human red blood cells, cultured liver cells PLC/PRF/5, or planar phospholipid bilayer membranes. With a probability that depended upon the target membrane, an increase of the electrical conductance of the fusing membranes (leakage) by up to several nS was generally detected. This leakage was recorded at the initial stages of fusion, when fusion pores formed. This leakage usually accompanied the "flickering" stage of the early fusion pore development. As the pore widened, the leakage reduced; concomitantly, the lipid exchange between the fusing membranes accelerated. We conclude that during fusion pore formation, HA locally and temporarily increases the permeability of fusing membranes. Subsequent rearrangement in the fusion complex leads to the resealing of the leaky membranes and enlargement of the pore.     

Influenza virus hemagglutinin-induced cell-planar bilayer fusion: quantitative dissection of fusion pore kinetics into stages

Cells expressing the influenza virus hemagglutinin (HA) fuse to planar bilayer membranes under acidic conditions. After an electrically quiescent perfusion stage (Q), a fusion pore forms that enlarges in three subsequent stages. A repetitively flickering pore stage (R) develops into a securely open stage (S) that exhibits conductances ranging from a few to tens of nS. The pore then expands to a terminal stage (T) with a large conductance on the order of one microSiemens. We have studied how virus strain, HA receptors in the target bilayer membrane, and cytoskeleton affect the time a fusion pore remains in each stage. These intervals are referred to as waiting times. In the quiescent stage, waiting times were very sensitive to the virus strain and presence of gangliosides (HA receptors) in the bilayer. When bilayers contained gangliosides, the waiting times in the Q stage for cells infected with the PR/8/34 strain of virus were exponentially distributed, whereas waiting times for cells infected with the Japan/305/57 strain were not so distributed. Without gangliosides, the waiting time distribution for PR/8/34 infected cells was complex. The waiting times in the R and S stages of pore growth were exponentially distributed under all tested conditions. Within the R stage, we analyzed the kinetics of the flickering pore by fitting the open and closed time distributions with a sum of two exponentials. Neither the open and closed time distributions nor the flickering pore conductance distributions were appreciably affected by virus strain or gangliosides. Colchicine and cytochalasin B increased the flicker rates, without affecting the waiting time in the R stage. We conclude that variations in amino acid sequences of the HAs and the presence of gangliosides as receptors within the target membrane critically affect the kinetics of fusion pore formation, but do not affect subsequent stages.     

Effects of membrane potential and sphingolipid structures on fusion of Semliki Forest virus

Cells expressing the E1 and E2 envelope proteins of Semliki Forest virus (SFV) were fused to voltage-clamped planar lipid bilayer membranes at low pH. Formation and evolution of fusion pores were electrically monitored by capacitance measurements, and membrane continuity was tracked by video fluorescence microscopy by including rhodamine-phosphatidylethanolamine in the bilayer. Fusion occurred without leakage for a negative potential applied to the trans side of the planar membrane. When a positive potential was applied, leakage was severe, obscuring the observation of any fusion. E1-mediated cell-cell fusion occurred without leakage for negative intracellular potentials but with substantial leakage for zero membrane potential. Thus, negative membrane potentials are generally required for nonleaky fusion. With planar bilayers as the target, the first fusion pore that formed almost always enlarged; pore flickering was a rare event. Similar to other target membranes, fusion required cholesterol and sphingolipids in the planar membrane. Sphingosine did not support fusion, but both ceramide, with even a minimal acyl chain (C(2)-ceramide), and lysosphingomyelin (lyso-SM) promoted fusion with the same kinetics. Thus, unrelated modifications to different parts of sphingosine yielded sphingolipids that supported fusion to the same degree. Fusion studies of pyrene-labeled SFV with cholesterol-containing liposomes showed that C(2)-ceramide supported fusion while lyso-SM did not, apparently due to its positive curvature effects. A model is proposed in which the hydroxyls of C-1 and C-3 as well as N of C-2 of the sphingosine backbone must orient so as to form multiple hydrogen bonds to amino acids of SFV E1 for fusion to proceed.     

Multiple local contact sites are induced by GPI-linked influenza hemagglutinin during hemifusion and flickering pore formation

Membrane fusion intermediates induced by the glycosylphosphatidylinositol-linked ectodomain of influenza hemagglutinin (GPI-HA) were investigated by rapid freeze, freeze-substitution, thin section electron microscopy, and with simultaneous recordings of whole-cell admittance and fluorescence. Upon triggering, the previously separated membranes developed numerous hourglass shaped points of membrane contact (∼10–130 nm waist) when viewed by electron microscopy. Stereo pairs showed close membrane contact at peaks of complementary protrusions, arising from each membrane. With HA, there were fewer contacts, but wide fusion pores. Physiological measurements showed fast lipid dye mixing between cells after acidification, and either fusion pore formation or the lack thereof (true hemifusion). For the earliest pores, a similar conductance distribution and frequency of flickering pores were detected for both HA and GPI-HA. For GPI-HA, lipid mixing was detected prior to, during, or after pore opening, whereas for HA, lipid mixing is seen only after pore opening. Our findings are consistent with a pathway wherein conformational changes in the ectodomain of HA pull membranes towards each other to form a contact site, then hemifusion and pore formation initiate in a small percentage of these contact sites. Finally, the transmembrane domain of HA is needed to complete membrane fusion for macromolecular content mixing.     

Minimum Membrane Bending Energies of Fusion Pores

Membranes fuse by forming highly curved intermediates, culminating in structures described as fusion pores. These hourglass-like figures that join two fusing membranes have high bending energies, which can be estimated using continuum elasticity models. Fusion pore bending energies depend strongly on shape, and the present study developed a method for determining the shape that minimizes bending energy. This was first applied to a fusion pore modeled as a single surface and then extended to a more realistic model treating a bilayer as two monolayers. For the two-monolayer model, fusion pores were found to have metastable states with energy minima at particular values of the pore diameter and bilayer separation. Fusion pore energies were relatively insensitive to membrane thickness but highly sensitive to spontaneous curvature and membrane asymmetry. With symmetrical bilayers and monolayer spontaneous curvatures of ?0.1 nm^?1 (a typical value) separated by 6 nm (closest distance determined by repulsive hydration forces), fusion pore formation required 43–65 kT. The pore radius of ~2.25 nm fell within the range estimated from conductance measurements. With bilayer separation >6 nm, fusion pore formation required less energy, suggesting that protein scaffolds can promote fusion by bending membranes toward one another. With nonzero spontaneous monolayer curvature, the shape that minimized the energy change during fusion pore formation differed from the shape that minimized its energy after it formed. Thus, a nascent fusion pore will relax spontaneously to a new shape, consistent with the experimentally observed expansion of nascent fusion pores during viral fusion.     

Sterols and sphingolipids strongly affect the growth of fusion pores induced by the hemagglutinin of influenza virus

Cells expressing the hemagglutinin (HA) of influenza virus were fused to planar phospholipid bilayer membranes to evaluate the effects of sterols and sphingolipids in the target bilayer membranes on properties of fusion pores. Typically, in the absence of sterol, flickering pores are observed, followed by a successful pore (i.e., a pore that fully opens). The incorporation of cholesterol into the lipid bilayer had a marked effect: it greatly decreased the number of flickers, and the first pore formed was usually successful. Similar effects were produced by the sterols epicholesterol and 5beta-cholestanol. In contrast, the sterols cholesteryl acetate, coprostanol, and stanolone did not affect pore flickering, and a successful pore was observed to follow the typical number of flickers. 5alpha-cholestanol gave intermediate results. From these results, it follows that the 3-OH of cholesterol is essential to reduce flickering, but it does not matter if the 3-OH is in an alpha or beta configuration. The double bond is also not critical for the actions of cholesterol nor is the fact that it is a flat molecule. The sphingolipids sphingomyelin, lactosyl cerebroside, and glucosyl cerebroside tended to inhibit full pore enlargement, prolonging the stage of pore flickering. If a sphingolipid and a sterol that strongly interact were both included in the planar membrane, the pattern of flickering was the same as if neither had been included in the bilayer. However, if a sphingolipid and sterol that do not interact with each other were included in the bilayer, the reduced flickering characteristic of the sterol was observed.     

Large cation-selective pores from rat liver peroxisomal membranes incorporated to planar lipid bilayers

Summary Fusion of a highly purified fraction of rat liver peroxisomal membranes to planar lipid bilayers incorporates large, cation-selective voltage-dependent pores. TheP _K/P _Cl ratio of these pores, estimated in KCl gradients, is close to 4. The pores display several conductance states and spend most of the time open at voltages near 0 mV, closing at more positive and negative voltages. At voltages near 0 mV the most frequent open state has a conductance of 2.4 nS in 0.3m KCl. At voltages more positive and more negative than 10 mV the most frequent open state displays a conductance of 1.2 nS in 0.3m KCl. With these results pore diameters of 3 and 1.5 nm, respectively, can be estimated. We suggest that these pores might account for the unusually high permeability of peroxisomes to low molecular weight solutes. Fusion also incorporates a perfectly anion-selective, two-open states channel with conductances of 50 and 100 pS in 0.1m KCl.     

Short-chain alcohols promote an early stage of membrane hemifusion.

Hemifusion, the linkage of contacting lipid monolayers of two membranes before the opening of a fusion pore, is hypothesized to proceed through the formation of a stalk intermediate, a local and strongly bent connection between membranes. When the monolayers' propensity to bend does not support the stalk (e.g., as it is when lysophosphatidylcholine is added), hemifusion is inhibited. In contrast, short-chain alcohols, reported to affect monolayer bending in a manner similar to that of lysophosphatidylcholine, were here found to promote hemifusion between fluorescently labeled liposomes and planar lipid bilayers. Single hemifusion events were detected by fluorescence microscopy. Methanol or ethanol (1.2-1.6 w/w %) added to the same compartment of the planar bilayer chamber as liposomes caused a 5-50 times increase in the number of hemifusion events. Alcohol-induced hemifusion was inhibited by lysophosphatidylcholine. Promotion of membrane hemifusion by short-chain alcohol was also observed for cell-cell fusion mediated by influenza virus hemagglutinin (HA). Alcohol promoted a fusion stage subsequent to the low pH-dependent activation of HA. We propose that binding of short-chain alcohol to the surface of membranes promotes hemifusion by facilitating the transient breakage of the continuity of each of the contacting monolayers, which is required for their subsequent merger in the stalk intermediate.     

Interaction of influenza virus proteins with planar lipid bilayers: A model for virion assembly

Changes in conductance of oxidized cholesterol planar lipid bilayers were measured following the incorporation of isolated surface glycoproteins; hemagglutinin and neuraminidase (HA+NA) or matrix protein (M-protein) of influenza virus. The conductance dependence of the lipid bilayers on the HA+NA or M-protein concentrations indicates different mechanisms of interaction of these viral proteins with the lipid bilayer. Adsorption of M-protein molecules on one side of the lipid bilayer affects the character of the HA+NA interaction with the opposite side. Planar lipid bilayers can be a useful model for investigation of the assembly of influenza virions and other enveloped viruses.     

Relationship of membrane sidedness to the effects of the lipophosphoglycan of Leishmania donovani on the fusion of influenza virus.

Cells expressing the influenza hemagglutinin protein were fused to planar lipid bilayers containing the viral receptor GD1a at pH 5.0. An amphiphile known to alter membrane properties is lipophosphoglycan (LPG). This glycoconjugate was added from aqueous solution to either the cis or the trans monolayer to examine its effects on the fusion process. LPG markedly inhibited the formation of fusion pores when present in the cis monolayer but LPG in the trans monolayer had no effect on the parameters of pore formation or on the properties of the pores. The N-terminal segment of the HA2 subunit of the influenza hemagglutinin protein is important for membrane fusion. The effect of LPG on the conformation and membrane insertion of a synthetic 20-amino-acid peptide, corresponding to the influenza fusion peptide, was examined at pH 5.0 by attenuated total reflection Fourier transform infrared spectroscopy and by the fluorescence properties of the Trp residues of this peptide. It was found that cis LPG did not prevent insertion of the peptide into the membrane but it did alter the conformation of the membrane-inserted peptide from alpha-helix to beta-structure. The beta-structure was oriented along the bilayer normal. The effect of cis LPG on the conformation of the fusion peptide probably contributes to the observed inhibition of pore formation and lipid mixing. In contrast, trans LPG has no effect on the conformation or angle of membrane insertion of the peptide, nor does it affect pore formation by HA-expressing cells. The ineffectiveness of trans LPG, despite it having strong positive curvature-promoting properties, may be a consequence of the size of this amphiphile being too large to enter a fusion pore.     

Meta-stability of the hemifusion intermediate induced by glycosylphosphatidylinositol-anchored influenza hemagglutinin.

Fusion between influenza virus and target membranes is mediated by the viral glycoprotein hemagglutinin (HA). Replacement of the transmembrane domain of HA with a glycosylphosphatidylinositol (GPI) membrane anchor allows lipid mixing but not the establishment of cytoplasmic continuity. This observation led to the proposal that the fusion mechanism passes through an intermediate stage corresponding to hemifusion between outer monolayers. We have used confocal fluorescence microscopy to study the movement of probes for specific bilayer leaflets of erythrocytes fusing with HA-expressing cells. N-Rh-PE and NBD-PC were used for specific labeling of the outer and inner membrane leaflet, respectively. In the case of GPI-HA-induced fusion, different behaviors of lipid transfer were observed, which include 1) exclusive movement of N-Rh-PE (hemifusion), 2) preferential movement of N-Rh-PE relative to NBD-PC, and 3) equal movement of both lipid analogs. The relative population of these intermediate states was dependent on the time after application of a low pH trigger for fusion. At early time points, hemifusion was more common and full redistribution of both bilayers was rare, whereas later full redistribution of both probes was frequently observed. In contrast to wild-type HA, the latter was not accompanied by mixing of the cytoplasmic marker Lucifer Yellow. We conclude that 1) the GPI-HA-mediated hemifusion intermediate is meta-stable and 2) expansion of an aqueous fusion pore requires the transmembrane and/or cytoplasmic domain of HA.     

Restricted movement of lipid and aqueous dyes through pores formed by influenza hemagglutinin during cell fusion

The fusion of cells by influenza hemagglutinin (HA) is the best characterized example of protein-mediated membrane fusion. In simultaneous measurements of pairs of assays for fusion, we determined the order of detectable events during fusion. Fusion pore formation in HA-triggered cell-cell fusion was first detected by changes in cell membrane capacitance, next by a flux of fluorescent lipid, and finally by flux of aqueous fluorescent dye. Fusion pore conductance increased by small steps. A retardation of lipid and aqueous dyes occurred during fusion pore fluctuations. The flux of aqueous dye depended on the size of the molecule. The lack of movement of aqueous dyes while total fusion pore conductance increased suggests that initial HA-triggered fusion events are characterized by the opening of multiple small pores: the formation of a "sieve".     

Secondary structure, orientation, oligomerization, and lipid interactions of the transmembrane domain of influenza hemagglutinin

Influenza virus hemagglutinin (HA), the viral envelope glycoprotein that mediates fusion between the viral and cellular membranes, is a homotrimer of three subunits, each containing two disulfide-linked polypeptide chains, HA(1) and HA(2). Each HA(2) chain spans the viral membrane with a single putative transmembrane alpha-helix near its C-terminus. Fusion experiments with recombinant HAs suggest that this sequence is required for a late step of membrane fusion, as a glycosylphosphatidylinositol-anchored analogue of HA only mediates "hemifusion" of membranes, i.e., the merging of the proximal, but not distal, leaflets of the two juxtaposed lipid bilayers [Kemble et al. (1994) Cell 76, 383-391]. To find a structural explanation for the function of the transmembrane domain of HA(2) in membrane fusion, we have studied the secondary structure, orientation, oligomerization, and lipid interactions of a synthetic peptide representing the transmembrane segment of X:31 HA (TMX31) by circular dichroism and attenuated total reflection Fourier transform infrared spectroscopy and by gel electrophoresis. The peptide was predominantly alpha-helical in detergent micelles and in phospholipid bilayers. The helicity was increased in lipid bilayers composed of acidic lipids compared to pure phosphatidylcholine bilayers. In planar lipid bilayers, the helices were oriented close to the membrane normal. TMX31 aggregated into small heat-resistant oligomers composed of two to five subunits in SDS micelles. Amide hydrogen exchange experiments indicated that a large fraction of the helical residues were accessible to water, suggesting the possibility that TMX31 forms pores in lipid bilayers. Finally, the peptide increased the acyl chain order in lipid bilayers, which may be related to the preferential association of HA with lipid "rafts" in the cell surface and which may be an important prerequisite for complete membrane fusion.     

The initial fusion pore induced by baculovirus GP64 is large and forms quickly

The formation of the fusion pore is the first detectable event in membrane fusion (Zimmerberg, J., R. Blumenthal, D.P. Sarkar, M. Curran, and S.J. Morris. 1994. J. Cell Biol. 127:1885-1894). To date, fusion pores measured in exocytosis and viral fusion have shared features that include reversible closure (flickering), highly fluctuating semistable stages, and a lag time of at least several seconds between the triggering and the pore opening. We investigated baculovirus GP64- induced Sf9 cell-cell fusion, triggered by external acid solution, using two different electrophysiological techniques: double whole-cell recording (for high time resolution, model-independent measurements), and the more conventional time-resolved admittance recordings. Both methods gave essentially the same results, thus validating the use of the admittance measurements for fusion pore conductance calculations. Fusion was first detected by abrupt pore formation with a wide distribution of initial conductance, centered around 1 nS. Often the initial fusion pore conductance was stable for many seconds. Fluctuations in semistable conductances were much less than those of other fusion pores. The waiting time distribution, measured between pH onset and initial pore appearance, fits best to a model with many (approximately 19) independent elements. Thus, unlike previously measured fusion pores, GP64-mediated pores do not flicker, can have large, stable initial pore conductances lasting up to a minute, and have typical lag times of < 1 s. These findings are consistent with a barrel-shaped model of an initial fusion pore consisting of five to eight GP64 trimers that is lined with lipid.     

GPI-anchored influenza hemagglutinin induces hemifusion to both red blood cell and planar bilayer membranes

Under fusogenic conditions, fluorescent dye redistributed from the outer monolayer leaflet of red blood cells (RBCs) to cells expressing glycophosphatidylinositol-anchored influenza virus hemagglutinin (GPI- HA) without transfer of aqueous dye. This suggests that hemifusion, but not full fusion, occurred (Kemble, G. W., T. Danieli, and J. M. White. 1994. Cell. 76:383-391). We extended the evidence for hemifusion by labeling the inner monolayer leaflets of RBCs with FM4-64 and observing that these inner leaflets did not become continuous with GPI-HA- expressing cells. The region of hemifusion-separated aqueous contents, the hemifusion diaphragm, appeared to be extended and was long-lived. But when RBCs hemifused to GPI-HA-expressing cells were osmotically swollen, some diaphragms were disrupted, and spread of both inner leaflet and aqueous dyes was observed. This was characteristic of full fusion: inner leaflet and aqueous probes spread to cells expressing wild-type HA (wt-HA). By simultaneous video fluorescence microscopy and time-resolved electrical admittance measurements, we rigorously demonstrated that GPI-HA-expressing cells hemifuse to planar bilayer membranes: lipid continuity was established without formation of fusion pores. The hemifusion area became large. In contrast, for cells expressing wt-HA, before lipid dye spread, fusion pores were always observed, establishing that full fusion occurred. We present an elastic coupling model in which the ectodomain of wt-HA induces hemifusion and the transmembrane domain, absent in the GPI-HA-expressing cells, mediates full fusion.     

Influenza hemagglutinin assumes a tilted conformation during membrane fusion as determined by attenuated total reflection FTIR spectroscopy.

Fusion of influenza virus with target membranes is mediated by an acid-induced conformational change of the viral fusion protein hemagglutinin (HA) involving an extensive reorganization of the alpha-helices. A 'spring-loaded' displacement over at least 100 A provides a mechanism for the insertion of the fusion peptide into the target membrane, but does not explain how the two membranes are brought into fusion contact. Here we examine, by attenuated total reflection Fourier transform infrared spectroscopy, the secondary structure and orientation of HA reconstituted in planar membranes. At neutral pH, the orientation of the HA trimers in planar membranes is approximately perpendicular to the membrane. However, at the pH of fusion, the HA trimers are tilted 55-70 degrees from the membrane normal in the presence or absence of bound target membranes. In the absence of target membranes, the overall secondary structure of HA at the fusion pH is similar to that at neutral pH, but approximately 50-60 additional residues become alpha-helical upon the conformational change in the presence of bound target membranes. These results are discussed in terms of a structural model for the fusion intermediate of influenza HA.     

Membrane mechanics can account for fusion pore dilation in stages.

Once formed, fusion pores rapidly enlarge to semi-stable conductance values. The membranes lining the fusion pore are continuous bilayer structures, so variations of conductance in time reflect bending and stretching of membranes. We therefore modeled the evolution of fusion pores using the theory of the mechanics of deforming homogeneous membranes. We calculated the changes in length and width of theoretical fusion pores according to standard dynamical equations of motion. Theoretical fusion pores quickly achieve semi-stable dimensions, which correspond to energy minima located in a canyon between energy barriers. The height of the barrier preventing pore expansion diminishes along the dimensions of length and width. The bottom of the canyon slopes gently downward along increasing length. As a consequence, theoretical fusion pores slowly lengthen and widen as the dimensions migrate along the bottom of the canyon, until the barrier vanishes and the pore rapidly enlarges. The dynamics of growth is sensitive to tension, spontaneous curvature, bending elasticity, and mobilities. This sensitivity can account for the quantitative differences in pore evolution observed in two experimental systems: HA-expressing cells fusing to planar bilayer membranes and beige mouse mast cell degranulation. We conclude that the mechanics of membranes could cause the phenomenon of stagewise growth of fusion pores.