Influenza Fusion Mechanism Figure 4. Stalk evolution in response of removing the pore (top view on porated bilayer). (I,II,III) Sudden removal of the pore before completion of the p-HD reverses the stalk elongation process, and the stalk completely disappears (DOPC at 310 K, with 16 water molecules per lipid between the membranes). Hydrophobic lipid tails are colored grey, polar-headgroups (DOPC) tan. doi:10.1371/journal.pone.0038302.g004 strong propensity to self-associate into 2 nm-wide trans-membrane arranged bundles consisting of 4–6 peptides (Fig. 5 B & methods). These bundles were found to be stable in the course of a 10 m s- lasting simulation. Such a peptide bundle conceptually differs from the externally stabilized hydrophilic pore that we studied in our previous example. The internal rim of the peptide ‘pore’ is lined with the amphiphilic peptides that replace the lipid head groups and solvent. The bundle’s interior is mainly composed of the hydrophilic residues Glu11 and Asn12, which point toward the central axis of the bundle (Fig. 5 C). This particular region in the peptide, i.e. the kink region, has been shown to play a major role in the peptide’s fusogenicity [11,15,25]. Such bundle structure is, in fact, similar to the hexameric bundle formed by the related parainfluenza virus 5 (PIV5) fusion peptide as observed in recent atomistic simulations [9]. Such pore structure seems essentially non-leaky and therefore rather resembles the closed structure of the mechanosensitive channel protein MscL [9] than a hydrophilic toroidal pore formed by antimicrobial peptides [49]. Although the peptide bundle is a self-stabilized structure, this does not imply that the lipid rim that surrounds the bundle is characterized by a vanishing excess of free-energy [50,51]. Assigning this quantity to the bundle circumference, we define its line-tension. Whereas the line-tension of the hydrophilic pore in our previous example was counter-balanced by an externally applied potential, the line-tension of the rim of the peptide bundle stems from a balance between the remaining hydrophobic mismatch between peptides and lipids, and the short-range repulsions (excluded volume) between the densely packed peptides in the bundle. A non-vanishing positive line-tension is important: (i) It can both stabilize and promote the expansion of the stalk, and (ii) it favors minimization of the bundle’s perimeter and thereby exerts a constricting force on the peptide bundle that ensures formation of a closed, essentially non-leaky structure. If the peptide bundle were an in-vivo relevant fusion intermediate the latter would be important. Hence, like synaptic fusion, viral fusion is generally believed to proceed via a non-leaky process, because Figure 5. (A) Overlap between the coarse-grained model (backbone red and side-chains yellow) of the wild-type influenza fusion peptide and the NMR structure [71]. The two helices are joined by a linker region at a slightly bent angle (boomerang-shape). (B) The wild-type influenza fusion peptides (side-chains not shown) aggregate into a stable hexameric bundle. The bundle interior is depleted in solvent (colored blue) and lipid head groups. For sake of clarity, the first backbone residue (Gly1) is colored yellow. (C) Top view of the bundle. The bundle’s interior is mainly composed of the hydrophilic residues Glu11 (colored blue) and Asn12 (colored green) that are located in the kinked region of the peptide and which point toward the central axis of the bundle. doi:10.1371/journal.pone.0038302.g005 PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e38302