Influenza Fusion Mechanism Figure 6. Evolution of the stalk in the presence of the peptide bundle. For sake of clarity the size of the lipid headgroups is exaggerated (solvent is not shown). (A) The elongated stalk (wild-type peptides) after 0.4 ms. The bundle has opened up and the stalk and has partly surrounded the formed hole. Notice the readily adopted banana-shape. The stalk forces the peptides to the remaining rim portion. At this stage mixing occurs between both the cis-leaflets and the trans-leaflet of the target membrane (colored gray), while the cis-leaflet of the host cell (colored yellow) does not contribute to lipid mixing. (B) Mutating a single residue in the peptides, Gly1 to Ser1 (colored green), known as the terminal hemifusion mutant G1S [14,25], stabilizes both bundle and stalk but inhibits elongation of the stalk (10 ms). Consequentially, the fusion reaction becomes trapped. Note that lipid head-groups are excluded from the pore interior and the trans-leaflets (colored yellow) are hindered from participating in the lipid mixing. doi:10.1371/journal.pone.0038302.g006 and lines the interior pore (5 out of 5 simulations). This first observation suggests that the stresses imposed on the lipid rim of the preserved bundle are either too small and/or the affinity between the peptides in the bundle is too large. In other words such closed bundle structure seems too favorable/stable with respect to the elongated stalk. Since stalk elongation seems to be a nucleated event that requires an initial ‘opening’ of the bundle (Fig. 6A), the presence of a persistent bundle opposes stalk elongation. To further explore the stability of such bundle against stalk elongation, we took a corresponding wild-type simulation where nucleation of the elongation process had readily occurred, and ‘on the fly’ mutated the wild-type into the G1S mutant. Intriguingly, such mutation actually reversed the elongation process and recovered the bundle (Fig. 8). This reversibility indicates that the G1S mutation in fact makes stalk elongation a continuous energetically ‘uphill’ process rather than a nucleated event, and illustrates that the peptide and the stalk are competitive lineactants – a hydrophobic lipid rim is a prerequisite for the peptide/bundle, whereas a hydrophilic rim is a prerequisite for the elongated stalk (Fig. 8). Apparently, the G1S mutant is the better lineactant, i.e. it has the higher affinity for the lipid rim. The inability of the stalk to elongate and form a p-HD imparts a higher barrier onto the fusion process, which traps the membranes in an incomplete fusion state in our simulation as well as in experiments [14]. We note that such a trapped incomplete fusion state facilitates mixing predominantly PLoS ONE | www.plosone.org 7 June 2012 | Volume 7 | Issue 6 | e38302