IFITM3 blocks viral entry by sorting lipids and stabilizing hemifusion

Interferon-induced transmembrane protein 3 (IFITM3) is a broad-spectrum restriction factor of viral entry, yet the molecular mechanism is poorly understood. We found that IFITM3 localizes to late endosomes, preventing viral fusion. Atomistic molecular dynamics simulations and continuum membrane modeling predicted IFITM3-induced local lipid sorting, resulting in an increased concentration of lipids disfavoring viral fusion at the hemifusion site. This increases the energy barrier of fusion pore formation and the hemifusion dwell time, promoting viral degradation in lysosomes. In situ cryo-electron tomography verified this model by visualizing influenza A virus-induced arrested membrane fusion at molecular resolution. Accumulated hemifusion diaphragms between viral particles and late endosomal membranes confirmed hemifusion stabilization as the molecular mechanism of IFITM3. The presence of hemagglutinin in post-fusion conformation close to hemifusion sites further showed that IFITM3 does not interfere with the viral fusion machinery, which corroborates IFITM3-induced lipid sorting as the primary cause of hemifusion stabilization. HIGHLIGHTS IFITM3 is localized in late endosomes and blocks viral membrane fusion IFITM3-induced lipid sorting can increase the energy barrier of viral fusion In situ cryo-ET confirmed hemifusion-stabilization as IFITM3’s molecular mechanism IFITM3 does not interfere with conformational changes of the viral fusion machinery GRAPHICAL ABSTRACT