Influenza Fusion Mechanism Figure 8. The G1S mutation reverses the stalk elongation process facilitated by the wild-type. Notice the removal of solvent (colored blue) and lipid head-groups (colored tan) from the membrane interior when the peptide bundle ‘reseals’ itself – the stalk and peptide are competitive lineactants. doi:10.1371/journal.pone.0038302.g008 hypothesize that such effect stems form altering the Janus-like structure of the peptide, i.e. their mutual interactions and their hydrophobic mismatch with the lipid membrane. The Janus-like structure of the peptides and the concomitant directionality of their mutual interaction is very important. If the peptides were merely axially symmetric cylinders, the balance between their mutual interaction and the hydrophobic mismatch (i.e. the interaction with the lipids) would likely dictate clustering or absence thereof [51]. In order to obtain a well-defined finite aggregation number one needs either a special geometry of packing or a special geometry of the interactions. The increased hydrophobicity of residue 13 (Gly13 to Ala13), which directly faces the lipid rim (Fig. 10), and the concomitant shallower kink-angle of the peptide most likely changes the optimal packing within the bundle and thereby drastically increases the aggregation number. In support of these arguments, we note that the ‘super’ bundle spontaneously reduces to its normal size after reversing the G13A mutation (Fig. 9, lower panel). For a lineactant, there appears to be a thin line between acting like a functional fusion peptide or antimicrobial peptide [60]. The G13A mutant, however, seems to behave more like an antimicrobial peptide. In summary, our molecular simulations and free-energy arguments in conjunction with experimental observations suggest that the influenza fusion mechanism might proceed through the formation of a trans-membrane arranged peptide bundle in the target membrane [6,7,9]. We have shown that the lipidic rim around a pore or peptide bundle is a critical factor in membrane fusion, equivalent to membrane curvature, the commonly cited determinant. In fact, curvature stress and line-tension are closely related quantities in membrane physics. For example, in the process of domain budding, minimization of the unfavorable perimeter of raft domains drives membrane bending (budding) and formation of vesicles (fission) [61]. Our simulations further suggest that ‘fusogenic’ peptides strike a balance: On one hand, they induce or stabilize a bundle in one of the membranes by gaining relatively favorable peptide-peptide and peptide-lipid interactions. On the other hand, the affinity of the peptides to pack into a bundle should be smaller than the stalk’s affinity for the lipid rim and the concomitant line-tension of the lipid rim has to be large enough in order to allow the stalk to surround it and form a p-HD. In addition, the peptide bundle should keep a low aggregation number. Such a complex and subtle balance might very well explain the experimentally observed sensitivity of the fusion process towards point mutations in the influenza fusion peptide. Based on our simulations, we predict that the puzzling trapped hemifusion state formed by point mutants G1S and E11A [14,25] is a kinetically trapped stalk that forms a stable complex with the peptide bundle. Notably, the transmembrane domain of influenza hemagglutinin (TMD) is also known to be involved in the formation of the fusion pore [62–65]. In SNARE-mediated membrane fusion such pore formation seems facilitated by the end residues (C-termini) of the TMD, which are being pulled PLoS ONE | www.plosone.org 9 June 2012 | Volume 7 | Issue 6 | e38302