Influenza Fusion Mechanism Figure 3. Evolution of a stalk in the absence and presence of a pore. For sake of clarity the size of the lipid headgroups is exaggerated (solvent is not shown). (A) Two apposed DOPC bilayers. A preformed stalk is not stable (see Fig. 4). (B) A stable ‘hour-glass shaped’ stalk structure formed between a DOPE and DOPC bilayer (4 ms). (C) Elongation of a stalk formed between two DOPE bilayers (4 ms). (I-III) Evolution of a stalk formed between two DOPC bilayers in the vicinity of a pore (stalk-pore complex). Elongation of the stalk, which circumvents the pore, results in the formation of a p-shaped hemifusion diaphragm (p-HD). doi:10.1371/journal.pone.0038302.g003 stable in these lamella-forming bilayers (DOPC at 310 & 350 K, with 16 water molecules per lipid between the membranes), in qualitative agreement with X-ray studies [42]. The latter process might partly explain why lipid mixing in electrofusion is observed after formation of membrane pores rather than the opposite [43]. Hence, stabilization of the stalk under unfavorable conditions, e.g., a non-optimal temperature, membrane curvature [16], hydration level, or lipid composition, will necessarily require the presence of the pore. We additionally note that the disappearance of the stalk progressed through a similar intermediate as the one that has been observed in simulations of stalk formation [30,44,44,45,45,46], i.e. a splayed lipid connecting the two adjacent leaflets (seeFig. S2). In summary, these stalk-pore simulations suggest that stalk stabilization and expansion do not necessarily require the presence of lipids with a negative spontaneous curvature but can alternatively be facilitated by an external pore, whose curvature stress gives rise to a positive line-tension. There is, however, one conceptual difference: In our first example (Fig. 3 B & 3 C), the stalk or elongated stalk was stabilized by the presence of the intrinsic negatively curved DOPE lipids. In such a case, the DOPE lipids were the ‘lineactants’ [47,48], i.e., molecules that favorably partition towards the stalk’s perimeter and thereby reduce its excess free-energy or line-tension. Likewise, also fusion peptides are often thought to be ‘lineactants’. In our second example, however, it was the stalk itself that was the lineactant, i.e. the stalk favorably partitioned toward the pore’s rim and reduced its line- tension. Could such inverted role, i.e. the stalk itself is the lineactant, explain influenza fusion? Influenza Fusion Peptides form a Trans-membrane Arranged Peptide Bundle that Drives Stalk Elongation Motivated by the experimental observation of a p-HD [7], and the hypothesis of the funnel structure [6,7,9], we next address the question of whether influenza peptides can, in principle, stabilize a ‘functional’ pre-fusion intermediate such as the pore shown in our previous example. To this aim, we included a near-atomistic coarse-grained model of the influenza hemagglutinin fusion peptide [23,24]. This model accurately reproduces the general structure of two helices joined by a linker region at a slightly bent angle (Fig. 5 A) [15,25]. In addition, it successfully mimics the amphiphilic nature of the influenza hemagglutinin fusion peptide. Intriguingly, we observed that these fusion peptides possess a PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e38302