Attenuation of IFITM proteins’ antiviral activity through sequestration into intraluminal vesicles of late endosomes

Interferon-induced transmembrane proteins (IFITMs) inhibit the entry of diverse enveloped viruses. The spectrum of antiviral activity of IFITMs is largely determined by their subcellular localization. IFITM1 localizes to and primarily blocks viral fusion at the plasma membrane, while IFITM3 prevents viral fusion in late endosomes by accumulating in these compartments. We have previously reported that cyclosporine A (CsA) treatment relieves the fusion block for the Influenza A virus, likely by relocating IFITM1 and IFITM3 from the plasma membrane and endosomes, respectively, to the Golgi area. Here, we report the existence of at least two distinct pools of IFITMs in CsA treated cells. While immunostaining of CsA treated cells using mild permeabilization agents, such as digitonin, suggests preferential IFITM localization at the Golgi apparatus, a harsher permeabilization protocol reveals a large, previously unidentified pool of IFITMs in late endosomes. Notably, IFITM redistribution was not associated with its degradation. A disproportionate loss of antibody access to the cytoplasmic N-terminus compared to the extracellular C-terminus of IFITMs after CsA treatment is consistent with sequestration of the N-terminal domain inside intraluminal vesicles of late endosomes. Accordingly, super-resolution microscopy reveals that CsA induces IFITM3 redistribution from the periphery to the interior of late endosomes. Together, our results imply that IFITMs relocate to intraluminal vesicles of late endosomes in the presence of CsA, thereby enabling viral fusion with the limiting membrane of these compartments. Our findings highlight the critical role of IFITM trafficking in antiviral defense and suggest a novel mechanism through which CsA modulates the cell’s susceptibility to viral infections.