excessive leakage might harm the host cell or virus. The stability condition of the peptide bundle/pore further implies that the effective, attractive medium-ranged peptide-peptide interactions, which are gained when the bundle is assembled, exceed the induced line-tension. We simulated the fusion reaction between the two DOPC membranes in the presence of the peptide bundle and explored if such bundle can, in principle, stabilize the stalk and drive its subsequent expansion. In 5 out of 5 simulations, stalk elongation occurred as a nucleated event [32,33] within 4 ms. Fig. 6 A depicts such elongation of the stalk in the presence of the peptide bundle (see Fig. S4, for additional simulations). It appears that the elongating stalk does not surround the bundle but rather ‘opens’ the closed bundle structure and pushes the peptides away. Such nucleated opening of the bundle indicates that a stalk formed near the intact bundle (Fig. 6 B) is under high stress, and vice versa. As the stalk elongates, the free rim portion of the resulting stalk-pore complex [33], which is not surrounded by the stalk, shrinks and the peptides crowd in this location (Fig. 6 A). The elongated stalk stabilizes a hydrophilic rim which is composed of lipid head groups (i.e, the p-HD). In contrast, the free portion of the rim which is stabilized by the peptides remains hydrophobic. Because the peptides are forced in close contact with the ends of the stalk, they might partly stabilize these ends by lowering their Gaussian curvature energy [23,24,33,38]. The replacement of the peptides by the stalk at the rim of such a pore further indicates (i) a non- vanishing positive line-tension, (ii) a pronounced difference of line- tension between its rim and the p-HD, and (iii) that the stalk is the better lineactant, i.e. it has a higher affinity for its rim than the peptides. To conclude, our simulations suggest that the wild-type influenza fusion peptide can in principle form a functional pre- fusion intermediate [6,7,9], i.e. a bundle consisting of multiple trans-membrane arranged fusion peptides, and that such an intermediate can facilitate the subsequent formation of the experimentally observed p-HD [7]. Point Mutations Affect Stability and Fusogenicity of the Peptide Bundle The relation between the peptide bundle and fusion is further corroborated by studies of the influence of peptide mutations. We discuss four specific examples: (I) A point mutation (G1V) where the first residue in the peptide, glycine, is replaced by valine, completely inhibits membrane fusion (lipid mixing) [14]. When adhered to the membrane surface, the fusogenic wild-type peptide adopts a boomerang shape (cf. Fig. 5 A), while the non-fusogenic G1V mutant remains linear, a-helical [15]. It has been demonstrated by atomistic simulations that such linear a-helical structure strongly reduces the peptides ability to penetrate the hydrophobic membrane core [15], i.e., the peptides remain parallel to the membrane surface. It is intuitive that the inability of the G1V mutant to adopt a trans-membrane orientation will also affect the formation/stabilization of the observed peptide bundle. We have performed simulations of wild-type peptides forming a stable bundle (see methods). Then, the first residue, glycine, was replaced with valine together with adapting the secondary structure to the linear a-helical structure [15]. Such point mutation rapidly destabilized the bundle in 5 out of 5 simulations (Fig. 7). Thus, our simulations indicate that the G1V mutation indeed impairs the ability of the peptides to form stable bundles. We found this effect, however, to be independent of secondary structure. Bundle destabilization also occurred when we conserved the secondary structure of the wild-type. In contrast, the bundle Influenza Fusion Mechanism remained stable in the presence of fully linear, a-helical wild-type peptides. We therefore relate the destabilization of the bundle to the increased hydrophobicity of the first residue rather than the concomitant change in secondary structure. This effect can be rationalized by the reduced ability of the hydrophobic Val1 residue to line-up with both the lipid/solvent interface and the hydrophilic center of the bundle (Fig. 5 B & C). However, the boomerang shape of the wild-type [15,25] might help the peptides to more easily adapt the trans-membrane orientation that is required to form such bundle. Although the G1V mutation will also affect other functions of these peptides related to fusion, e.g. the formation of the dimple [7,52], the lipid protrusion frequency [46], solvent dynamics [53], or membrane curvature/elasticity [38], the important role of the bundle in the viral fusion process indicated by our simulations suggests that the absence of a bundle substantially affects both the stability of the initial stalk and the pathway of the following fusion reaction. Aside from stabilizing the stalk structure, a bundle also enhances formation of the stalk by allowing a closer proximity between the adjacent fusion sites [39], e.g., by locally perturbing the membrane structure and reducing the inter-membrane repulsion (see Fig. S3). If viral fusion in model systems proceeded only after the formation of an initial ‘funnel’ structure in the target membrane [6,7,9], the non-fusogenicity of the point mutation could be rationalized by its inability to induce this early step of the fusion process. (II) Similar to mutant G1V, also mutant W14A has been shown to inhibit lipid mixing [25]. The structure of W14A determined by NMR and site-directed spin labeling features a flexible kink that points out of the membrane, in sharp contrast to the more ordered boomerang shape of the wild-type, which points into the membrane [25]. Although the flexible structure of mutant W14A rather differs from the conserved linear a-helical structure of mutant G1V, we expect that also mutant W14A destabilizes the bundle because it shares the inability of mutant G1V to induce fusion. To this end, we modeled mutant W14A based on its NMR derived structure (cf. methods) and studied its ability to stabilize the bundle. Figure 7 shows that mutant W14A destabilizes the bundle within 100 ns. Thus, the inability to stabilize the bundle seems common to both of these structurally different point mutations. (III) Another well known and intriguing point mutation of the peptide, Gly1 to Ser1 (mutant G1S), facilitates lipid mixing but selectively impairs its ability to complete fusion, i.e. content mixing [14]. Combined NMR and atomistic simulation studies have shown that this G1S mutant adopts a similar boomerang shape, displays similar structural dynamics, and inserts similarly in the hydrophobic bilayer core as the bundle-forming wild-peptide [15]. In contrast to mutant G1V, mutant G1S does not destabilize the bundle over the course of all 5 simulations lasting 10 ms. Thus, our simulations predict that also the G1S mutant is able to stabilize a peptide bundle. The latter might be explained by the fact that, unlike valine (mutant G1V), serine is even slightly more hydrophilic than glycine (wild-type), which makes lining up with both the hydrophilic center of the bundle and the lipid/solvent interface energetically favorable. Thus, we assume that the G1S mutant forms a stable bundle and, according to the here-proposed stalk-bundle mechanism, such a bundle will, in turn, facilitate the formation/stabilization of a stalk. This stalk-bundle formation is corroborated by the experimentally observed lipid mixing [14]. How can this situation be reconciled with the peptide’s inability to complete fusion? To this end, we investigated the behavior of a stalk-bundle complex formed between two DOPC bilayers. We observed that the presence of the G1S mutant bundle stabilizes the stalk in the course of the simulation of 10 ms but, in contrast to the bundle of wild-type peptides, the stalk does not ‘open’ the bundle PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e38302