Line-Tension Controlled Mechanism for Influenza Fusion Herre Jelger Risselada1*, Giovanni Marelli2., Marc Fuhrmans2., Yuliya G. Smirnova2, Helmut Grubmu¨ ller1, Siewert Jan Marrink3, Marcus Mu¨ ller2 1 Theoretical Molecular Biophysics Group, Max-Planck-Institute for Biophysical Chemistry, Go¨ ttingen, Germany, 2 Institut fu¨ r Theoretische Physik, Georg-August- Universita¨t, Go¨ ttingen, Germany, 3 Groningen Biomolecular Sciences and Biotechnology Institute, Nijenborgh, AG Groningen, The Netherlands Abstract Our molecular simulations reveal that wild-type influenza fusion peptides are able to stabilize a highly fusogenic pre-fusion structure, i.e. a peptide bundle formed by four or more trans-membrane arranged fusion peptides. We rationalize that the lipid rim around such bundle has a non-vanishing rim energy (line-tension), which is essential to (i) stabilize the initial contact point between the fusing bilayers, i.e. the stalk, and (ii) drive its subsequent evolution. Such line-tension controlled fusion event does not proceed along the hypothesized standard stalk-hemifusion pathway. In modeled influenza fusion, single point mutations in the influenza fusion peptide either completely inhibit fusion (mutants G1V and W14A) or, intriguingly, specifically arrest fusion at a hemifusion state (mutant G1S). Our simulations demonstrate that, within a line- tension controlled fusion mechanism, these known point mutations either completely inhibit fusion by impairing the peptide’s ability to stabilize the required peptide bundle (G1V and W14A) or stabilize a persistent bundle that leads to a kinetically trapped hemifusion state (G1S). In addition, our results further suggest that the recently discovered leaky fusion mutant G13A, which is known to facilitate a pronounced leakage of the target membrane prior to lipid mixing, reduces the membrane integrity by forming a ‘super’ bundle. Our simulations offer a new interpretation for a number of experimentally observed features of the fusion reaction mediated by the prototypical fusion protein, influenza hemagglutinin, and might bring new insights into mechanisms of other viral fusion reactions. Citation: Risselada HJ, Marelli G, Fuhrmans M, Smirnova YG, Grubmu¨ ller H, et al. (2012) Line-Tension Controlled Mechanism for Influenza Fusion. PLoS ONE 7(6): e38302. doi:10.1371/journal.pone.0038302 Editor: Stefan Poehlmann, German Primate Center, Germany Received January 31, 2012; Accepted May 3, 2012; Published June 28, 2012 Copyright: 2012 Risselada et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: These authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: hrissel@gwdg.de . These authors contributed equally to this work. Introduction Membrane fusion is a fundamental process in biological cells, being involved in viral infection, endo- and exocytosis, and fertilization. The understanding of its molecular mechanism will open avenues for controlling a variety of collective biophysical processes that alter membrane topology. It is widely accepted that influenza hemagglutinin mediates a fusion mechanism that progresses through hemifusion [1,2]. In the standard stalk- hemifusion pathway [3–5], illustrated in Fig. 1, the initial contact point between the apposing cis-leaflets, i.e. the stalk, progresses via an axially symmetric radial expansion (stalk widening), which thins the stalk such that the two distal trans-leaflets eventually meet and form a single-bilayer-thick H-shaped diaphragm (H-HD). After rupture of the H-HD, the fusion is completed. Conductance measurements and fluorescence spectroscopy studies of fusion between the influenza envelope (host membrane) and lipid model membranes (target membrane) have reported that lipid mixing is preceded by the formation of an essential pre-fusion structure in the target membrane [6–11]. Formation of such a pre- fusion structure is believed to involve perforation of the target membrane [6,7], because leakage through the target membrane is detected prior to lipid mixing [6–8,11,12]. Intriguingly, the formation of a small (&5 nm wide) stable p- shaped HD (p-HD), illustrated in Fig. 2, which appears to be generated by a stalk that has partially encircled a formed membrane pore, has recently been observed by electron cryo- tomography of influenza fusion between a viral envelope and a pure DOPC vesicle [7]. It was reported that a leaky funnel-like structure was formed in the target membrane prior to lipid mixing. Moreover, in this example, the trans-leaflet of the viral membrane is completely covered by a rigid, shape-stabilizing protein matrix [7], that impedes bending of the trans-leaflets, which is required for the stalk to expand into an HD (Fig. 1 II) [4,13]. Such limitation should have substantially increased the already large barrier to form an H-HD within the standard hemi-fusion mechanism, ranging from 15–63 kBT in free membranes without protein matrix [4]. In model influenza fusion, single point mutations of the fusion peptide have been shown to either completely inhibit fusion, specifically arrest fusion at a hemifusion stage, or induce pronounced leakage of the target membrane prior to lipid mixing [11,14,15]. It is still puzzling why the fusion mechanism is so sensitive to minor changes in the fusion peptide. The important philosophy behind point mutations is that they alter specific and essential peptide-membrane interactions and thereby modify the ability to overcome free-energy barriers in the underlying fusion mechanism of the wild-type peptide. Point mutation studies therefore play a key role in unraveling the mechanism of influenza fusion. It is reasonable to assume that, to stabilize the initial stalk, fusion peptides should stabilize the negatively curved stalk structure [3,4,13,16–20]. Experiments, PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e38302