Prikryl David, Zhang You, Melikyan Gregory B
Department of Pediatrics, Division of Infectious Diseases, Emory University School of Medicine, Atlanta, GA 30322, USA.
Children's Healthcare of Atlanta, Atlanta, GA 30329, USA.
bioRxiv. 2025 May 27:2025.05.27.656272. doi: 10.1101/2025.05.27.656272.
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.
干扰素诱导跨膜蛋白（IFITMs）可抑制多种包膜病毒的进入。IFITMs的抗病毒活性谱在很大程度上取决于它们的亚细胞定位。IFITM1定位于质膜并主要在质膜处阻断病毒融合，而IFITM3通过在晚期内体中积累来阻止晚期内体中的病毒融合。我们之前曾报道，环孢素A（CsA）处理可缓解甲型流感病毒的融合阻断，这可能是通过分别将IFITM1和IFITM3从质膜和内体重新定位到高尔基体区域来实现的。在此，我们报告在CsA处理的细胞中存在至少两个不同的IFITMs池。虽然使用温和的通透剂（如洋地黄皂苷）对CsA处理的细胞进行免疫染色表明IFITM优先定位于高尔基体，但更严格的通透方案揭示了晚期内体中大量以前未被识别的IFITMs池。值得注意的是，IFITM的重新分布与其降解无关。CsA处理后，与IFITMs的细胞外C末端相比，抗体进入细胞质N末端的比例失调，这与晚期内体内腔泡内N末端结构域的隔离一致。因此，超分辨率显微镜显示CsA诱导IFITM3从晚期内体的周边重新分布到内部。总之，我们的结果表明，在CsA存在的情况下，IFITMs会重新定位到晚期内体的内腔泡中，从而使病毒能够与这些区室的限制膜融合。我们的发现突出了IFITM转运在抗病毒防御中的关键作用，并提出了一种CsA调节细胞对病毒感染易感性的新机制。
