Cyclosporine A rescues the influenza virus fusion with IFITM3-expressing cells by relocating the restriction factor to intraluminal vesicles of multivesicular bodies.

Interferon-induced transmembrane protein 3 (IFITM3) blocks fusion of many enveloped viruses with endosomes, likely by rigidifying the cell membrane. We and others have previously reported that cyclosporine A (CsA) treatment rescues the ability of the influenza A virus (IAV) to fuse with and infect IFITM3-expressing cells, but the mechanism of such rescue remained controversial. Here, we employed correlative light-electron microscopy (CLEM) and on-section electron tomography workflow to visualize the IAV fusion intermediates arrested by IFITM3. Most common intermediates were tight IAV contacts with the limiting membrane and intraluminal vesicles of late endosomes. A hemifusion intermediate was also detected. We further visualized the effect of CsA on IFITM3 localization by immunoelectron microscopy (immuno-EM), which revealed a marked relocation of IFITM3 from the limiting membrane to intraluminal vesicles of endosomes. This CsA-induced IFITM3 sequestration in intraluminal vesicles likely relieves the block for IAV fusion with the limiting membrane of endosomes that culminates in a release of the viral nucleoprotein complex into the cytoplasm.IMPORTANCEThe mechanism by which interferon-induced transmembrane protein 3 (IFITM3) inhibits the fusion of diverse enveloped viruses with endosomes is not fully understood. Using correlative light-electron microscopy (CLEM) and on-section electron tomography, we detected IFITM3-arrested influenza A virus fusion intermediates with the limiting membrane and intraluminal vesicles of late endosomes. The most common arrested intermediate was a tight contact between the viral membrane and the limiting membrane of late endosomes, which formed more frequently compared to tight contacts with intraluminal vesicles. Immunoelectron microscopy (immuno-EM) revealed that pretreatment of IFITM3-expressing cells with cyclosporine A induced relocation of IFITM3 from the limiting membrane to intraluminal vesicles of endosomes and rescued the influenza A virus fusion. Collectively, these findings imply that the influenza virus fuses with the limiting membrane of endosomes, but not with intraluminal vesicles, leading to productive infection, and that IFITM3 enrichment at these sites is critical for blocking viral fusion.