Epstein–Barr virus (EBV) can infect both B cells and epithelial cells (ECs), causing diseases such as mononucleosis and cancer. It enters ECs via Ephrin receptor A2 (EphA2). The function of interferon-induced transmembrane protein-1 (IFITM1) in EBV infection of ECs remains elusive. Here we report that IFITM1 inhibits EphA2-mediated EBV entry into ECs. RNA-sequencing and clinical sample analysis show reduced IFITM1 in EBV-positive ECs and a negative correlation between IFITM1 level and EBV copy number. IFITM1 depletion increases EBV infection and vice versa. Exogenous soluble IFITM1 effectively prevents EBV infection in vitro and in vivo. Furthermore, three-dimensional structure prediction and site-directed mutagenesis demonstrate that IFITM1 interacts with EphA2 via its two specific residues, competitively blocking EphA2 binding to EBV glycoproteins. Finally, YTHDF3, an m⁶A reader, suppresses IFITM1 via degradation-related DEAD-box protein 5 (DDX5). Thus, this study underscores IFITM1’s crucial role in blocking EphA2-mediated EBV entry into ECs, indicating its potential in preventing EBV infection.

EB 病毒 (EBV) 可感染 B 细胞和上皮细胞 (EC)，引起单核细胞增多症和癌症等疾病。它通过肝配蛋白受体 A2 (EphA2) 进入内皮细胞。干扰素诱导的跨膜蛋白 1 (IFITM1) 在 EC 的 EBV 感染中的功能仍不清楚。在这里，我们报告 IFITM1 抑制 EphA2 介导的 EBV 进入 EC。 RNA测序和临床样本分析显示，EBV阳性EC中的IFITM1降低，并且IFITM1水平与EBV拷贝数之间呈负相关。 IFITM1 缺失会增加 EBV 感染，反之亦然。外源性可溶性IFITM1在体外和体内均能有效预防EBV感染。此外，三维结构预测和定点诱变表明，IFITM1 通过其两个特定残基与 EphA2 相互作用，竞争性阻断 EphA2 与 EBV 糖蛋白的结合。最后，YTHDF3（一种 m ⁶ A 阅读器）通过降解相关的 DEAD-box 蛋白 5 (DDX5) 抑制 IFITM1。因此，这项研究强调了 IFITM1 在阻止 EphA2 介导的 EBV 进入 EC 中的关键作用，表明其在预防 EBV 感染方面的潜力。