Influenza Fusion Mechanism Figure 7. Point mutations that are known to inhibit fusion destabilize the bundle. (top) Mutating a single residue, Gly1 to Val1 (colored blue), destabilizes the peptide bundle (mutant G1V). (bottom) Mutating a single residue, Trp14 to Ala14 (colored cyan), rapidly destabilizes the peptide bundle (mutant W14A). Notice the flexible kink that points out of the membrane [25]. doi:10.1371/journal.pone.0038302.g007 between the cis-leaflets, similar to the standard stalk-hemifusion pathway (cf. Fig. 1 A) because lipid head-groups are excluded from the interior of the preserved bundle structure and thereby hinder the occurrence of flip-flops from the trans- to cis-leaflets (Fig. 6B). (IV) The last point mutation of interest is the recently discovered mutant G13A [11]. Whereas the wild-type peptide has a steep kink-angle of 105 degrees, mutant G13A has a shallower kink- angle of about 150 degrees [11]. Fluorescence microscopy studies have observed that when red blood cells expressing such mutant merge, the content of the cells is released into the inter-cellular space rather than being transferred between the fusing cells [11]. Such massive leakage is readily observed more than 5 minutes before the occurrence of lipid mixing [11]. Leakage of the target membrane prior to lipid mixing, albeit less pronounced, has also been observed in model influenza fusion with wild-type peptides [6,7,12]. Notably, there seems to be a close relationship between a peptide’s ability to induce membrane lysis (leakage) and its ability to induce fusion. Antimicrobial peptides such as melittin, which are primarily pore-forming, have been shown capable to induce fusion [54,55], whereas fusion peptides, such as influenza hemagglutinin, have been shown capable to induce membrane lysis [56–59]. Furthermore, it has been observed that deletion of the N- or C-terminus of the influenza fusion peptide or its mutants, decrease its ability to induce both lysis and fusion [56,57]. With respect to the here-proposed stalk-bundle mechanism, such a relationship is intuitive because both fusion and lysis relate to the line-tension and the peptides are lineactants in both cases. We explored the behavior of the bundles formed by mutant G13A. To do so, we positioned 4 bundles, each consisting of six trans-membrane arranged peptides plus two ‘bystander’ peptides, in a 15615 nm POPC bilayer. We also duplicated this setup for the wild-type bundles. Fig. 9 shows both setups. In the course of the 20 ms simulation we observed a strong repulsive behavior between the separate wild-type bundles, i.e. the bundles tend to maximize their separation distance (Fig. 9, upper panel). Eventually, one of the bundles breaks up and the peptides redistribute among the remaining bundles. The size of these bundles ranges from tetrameters to hexamers, with pentamers being the most abundant (Fig. 9, lower panel). Notably, also the surface adhered peptides display an interaction with the bundle and likely play a role in its stability. In contrast to the wild-type bundles, the bundles formed by mutant G13A are ‘attractive’ and coalescence between the different bundles is observed (Fig. 9, central panel). Eventually, the latter results in one large bundle consisting of 10 trans-membrane arranged peptides and several associated bystander peptides. Obviously, the formation of such a ‘super’ bundle would be ominous for the integrity of the membrane (Fig. 10), especially in the presence of additional stress. Thus, the action of mutant G13A, i.e. massive leakage prior to lipid mixing, is very well explained by the formation of a corrupted peptide bundle. How can we rationalize this effect? We PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e38302