Influenza Fusion Mechanism Figure 9. Interaction between multiple peptide bundles. (upper panel) Four wild-type bundles (top-view). The bundles strongly repel each other and maximize their separation distance in the course of the simulation. Eventually one of the bundles vanishes. (middle panel) Four G13A mutant bundles. The bundles are attractive and their coalescence results in a ‘super’ bundle consisting of 10 trans-membrane arranged peptides. (lower panel) Aggregation number of the largest bundle in the course of the simulation (Only the trans-membrane arranged peptide are counted). The brown line shows a separate simulation where the G13A mutation is reversed after 20 ms (G13A -. wild-type). The wild-type ‘super’ bundle readopts its usual size in the course of the simulation. doi:10.1371/journal.pone.0038302.g009 through the HD [30], and thereby actively open the fusion pore (see [39] for a recent review). A similar scenario may apply to influenza fusion. If the fusion peptide indeed facilitate the fusion process up to the formation of the HD, with the C-terminus of the TMD being chiefly involved in the subsequent pore opening, this would explain why the influenza fusion mechanism is relatively insensitive to overall changes in TMD sequence or length [62–65]. In such a case, the ability of hemagglutinin to complete fusion mainly relates to the ability of the C-terminus to perforate the membrane (HD). The latter would, e.g., be affected by altering the hydrophobicity, electrostatics or size of the TMD C-terminus and/ or cytoplasmic domain [66]. We predict that the trapped hemifusion state that is due to substantial shortening of the hemagglutinin TMD [62,63] or mutations herein [64–66], is a kinetically trapped p-shaped hemifusion diaphragm. Future cryo- tomography studies should be able to resolve these different hemifused structures. In this work we have mainly focused on how the peptides stabilize the stalk and drive its expansion into the hemifusion diaphragm. We have thus far not discussed the process of stalk formation itself. Molecular simulations suggest that stalk formation is facilitated by the formation of splayed lipid intermediates (i.e., the stalk barrier) which form a bridge between the apposed leaflets [30,44–46]. Recent coarse grained and atomistic simulations have revealed that the wild-type peptide induces a substantial disorder in the packing of the lipid tails (tail protrusions) and thereby substantially reduces the membrane thickness [22,46,67]. The non-fusogenic mutants, however, show a less pronounced distor- tion of the membrane integrity [22]. The absorbed wild-type peptide induces an approximately 5-fold increase in the normal tail protrusion frequency [46], i.e. a tail-end reaches the lipid PLoS ONE | www.plosone.org 10 June 2012 | Volume 7 | Issue 6 | e38302