Structural Analysis Of Hemagglutinin-Induced Hemifusion By Volta Phase-Plate Cryo-Electron Tomography

Enveloped viruses feature metastable fusion proteins, which upon their conformational change transfer configurative energy into membrane fusion. To overcome the initial and substantial energetic barrier due to hydration repulsion of phospholipids, membrane fusion is thought to proceed through a low energy intermediate called hemifusion; yet for viral fusion the hemifusion structure has never been characterized. Variations of membrane curvature theory predict hemifusion diaphragms of varying dimensions. We study the hemifusion intermediate catalyzed by wild-type and G1S hemifusion mutant influenza hemagglutinin (HA) incorporated into virus-like particles (VLP) using Volta phase-plate cryo-electron tomography. We found “Y” shaped lipidic junctions with liposomal membrane inserted into the VLP membrane as suggested previously (Lee K. K., EMBO, 2010). Lipidic junctions and hemifusion diaphragms (HD) were typical membrane structures in both mutated and wild-type HA mediated fusion. Dimensions of HD and lipidic junctions ranged widely with their variance controlled by the rigidity imposed by the matrix layer, in agreement with our continuum mechanics model. We observed HA in close proximity to free membrane edges of ruptured liposomal membrane and lipidic junctions, suggesting that HA is capable of rupture, stabilization and resealing of membrane compartments in a step-wise manner permitting HD formation by lateral merging of lipidic junctions. Unambiguous determination of a lipidic junction consisting of the edge of a liposomal bilayer merged in the outer leaflet of a VLP opens the possibility of an alternate HA-mediated membrane fusion pathway.Lee, K. K. Architecture of a nascent viral fusion pore. The EMBO journal 29, 1299-1311, http://dx.doi.org/10.1038/emboj.2010.13 (2010).