mutant displays a similar secondary structure and structural dynamics and, in addition, also inserts similarly in the membrane [15]. It is difficult to conceive how the wild-type peptide, when adhering to the membrane outside of the virus or host cell [15], and when not being associated with the stalk structure [15,21–24], would open the fusion pore. Is it possible that the terminal- hemifusion mutant G1S rather inhibits the fusion step prior to pore opening, i.e. the expansion of the stalk into a p-HD? The aim of this work is to relate the three different experimentally observed phenomena in influenza fusion: the funnel-like pre-fusion structure, the p-HD and the observed effect of the point mutations. We will demonstrate that these three phenomena can be understood from a fundamental and general concept in membrane physics: the line-tension, i.e. the free-energy per unit length [26,27]. To this end, we use molecular simulation of a coarse-grained model [28,29], where computational efficiency is enhanced by representing several atoms by a single interaction site. This description captures the underlying driving forces and evolution of the fusion process in near-atomic detail. Recently, this model has been successfully applied to study membrane fusion mediated by SNARE proteins [30] and long surfactant protein B [31]. To rationalize the above mentioned phenomena, we (i) study the physical properties of an isolated stalk formed between two apposing bilayers, (ii) investigate how these stalk properties are modified by the presence of a small hydrophilic pore in one of the bilayers, (iii) explore the relation between wild-type influenza peptides and stalk expansion, and (iv) study the effects of point mutations that are experimentally known to alter the peptide’s fusogenity. Results and Discussion Large Negative Spontaneous Curvature Results in Linear Stalk Elongation Fusion is believed to be initiated by the formation of an energetically costly negatively curved stalk structure [1–4,13,16– 20]. To be able to rationalize the link between stalk and line- tension, we first study the expansion of an isolated stalk formed between two tension-less lipid membranes of area 20|20 nm2 (1152 lipids each) that are separated by a 2 nm-thick water layer, corresponding to approximately 16 H2O molecules per lipid. After stalk formation, fusion has been proposed to progress by either a radial, axially symmetric expansion [3,13,16,18] or, alternatively, by a linear elongation of the stalk [13,19,32,33]. Lipids that are characterized by a large negative spontaneous curvature, e.g. DOPE lipids, form inverse lipid phases, e.g. the inverted hexagonal phase, at sufficiently low hydration and high temper- ature. The inverted hexagonal phase consists of an hexagonally ordered array of cylindrical bilayer structures. As observed in both, coarse-grained and atomistic simulations [34,35], inverted hexag- onal structures are formed by linear elongation of stalks between multiple stacked bilayers. This universal type of stalk instability characterizes the transition from the lamellar to the inverted hexagonal phase [36]. Such a linear elongation of a stalk is shown in Fig. 3 C for a stalk formed between two DOPE bilayers at 350 K. We estimated the line-tension l, a measure for the free- energy per unit length, of such a linearly elongated DOPE stalk from the pressure tensor (see Fig. S1 for a detailed explanation) and obtained l = 260+15 pN or about 214 kBT per nm. A negative line-tension implies that it is thermodynamically favor- able to increase the negatively curved perimeter of the stalk, i.e. stalk elongation. When we replace one of the DOPE bilayers by a pure DOPC bilayer, however, stalk elongation is inhibited and the initially formed ‘hour glass’-shaped (rhombohedral) stalk remains Influenza Fusion Mechanism stable over the course of the 4 ms simulation (see Fig. 3 B). The latter effect can be explained by a positive value of l as a result of the increasing fraction of DOPC lipids, which have a more positive spontaneous curvature. Such asymmetric setup mimics the experimentally studied fusion between a pure DOPC membrane and the fusogenic viral envelope [7], where the occurrence of spontaneous stalk elongation presumably is not favorable [37,38]. In such a case, a radial, axially symmetric stalk expansion, stipulated by the standard hemifusion mechanism, becomes favored over a linear expansion [13,33]. This latter process, however, would face a substantial nucleation barrier that, depending on lipid polymorphism, has been estimated to be 15– 63 kBT [4] and, indeed, such a radial stalk expansion is not observed in our simulation. Thus, in the absence of a sufficiently large negative spontaneous curvature, stalk expansion, either radially or by elongation, does not occur spontaneously [4,13,16,18,33,39]. The Presence of a Pore Results in the Formation of a p-HD in Lamellar PC Bilayers Alternative fusion mechanisms, which involve pores in one or both of the apposing membranes have been observed in computer simulation [5,32,40] and have been studied by self-consistent field (SCF) theory [33]. Next, we study whether the presence of an externally stabilized pore might overcome the free-energy cost of stalk elongation even for more symmetric, lamella-forming lipids. Since we focus on the stalk to HD transition, we do not investigate the formation of a stalk in the vicinity of a pore or vice versa but we start from the energetically favorable stalk-pore complex (Fig. 3 I) [32]. To this end, we consider that a stalk has been formed between two pure DOPC bilayers in the vicinity of a pore in the lower bilayer. Panels I-III of Fig. 3 show that the presence of a small 3 nm–wide pore, which is stabilized by an external field (cf. methods), is indeed able to facilitate stalk elongation even between lamella-forming DOPC bilayers at 310 & 350 K, and that such a process leads to the formation of a p-HD. Note that this process does not necessarily involve bending of the host membrane. Thus, the presence of a rigid, shape-stabilizing protein-matrix on the trans-leaflet of the viral envelope [7] would not impede such formation of a p-HD. It is easy to rationalize that the observed effect has a simple essence. A pore generated in a lipid membrane, i.e. by membrane stretching or application of an electric field, has a rim covered by an extremely strongly curved lipid monolayer. The bending energy of this monolayer determines the rim’s line-tension. Sewing this rim to the host membrane replaces it with a circular three- bilayer junction, which is energetically favorable and leads to p- HD formation. We estimated the line-tension of the rim from the simulation of the corresponding bilayer edge (mimicking a pore with infinite radius) [41] and obtained lP~65+3 pN at 310 K. Indeed, the line-tension of a three-bilayer junction (the HD) was much lower, namely 23+10 pN (see Fig. S1). This value is in qualitative agreement with values derived from continuum elastic models (15–20 pN) [13]. Elongation is favorable, and the metastable p-HD is formed, provided that the line-tension of the three-bilayer junction is lower than that of the replaced pore edge. Obviously, the topology of the fusion site resulting from such pore fusion (a p-HD) is different from that generated by a dimple fusion (an H-HD) but the physical forces are the same. Finally, to demonstrate that the observed linear elongation of the stalk crucially depends on the presence of a pore, we remove the pore when the stalk has slightly elongated. In such a case, the elongation reverses, and the stalk completely disappears (Fig. 4 I, II, III). Thus, in the absence of a pore, stalk structures are not PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e38302