Interferon-induced BST2 (bone marrow stromal cell antigen 2) inhibits viral replication by tethering enveloped virions to the cell surface to restrict viral release and by inducing the NFKB-dependent antiviral immune response. However, the mechanism by which BST2 uses the selective autophagy pathway to inhibit viral replication is poorly understood. In this study, we showed that BST2 expression was significantly increased during porcine epidemic diarrhea virus (PEDV) infection of Vero cells by IRF1 targeting its promoter. We also showed that BST2 suppressed PEDV replication by binding and degrading the PEDV-encoded nucleocapsid (N) protein. The downregulation of N protein was blocked by macroautophagy/autophagy inhibitors but not a proteasome inhibitor, implying that the N protein was degraded via the selective autophagy pathway. Both the BST2 and N protein interacted with the E3 ubiquitin ligase MARCHF8/MARCH8 and the cargo receptor CALCOCO2/NDP52, and the ubiquitination of N protein was necessary for the degradation of N mediated by the BST2-MARCHF8 axis. The knockdown of MARCHF8 or ATG5 with small interfering RNAs blocked the selective autophagy pathway, rescued the protein abundance of PEDV N in 293T cells, and prevented the inhibition of PEDV replication by BST2 in Vero cells. Together, our data demonstrate the novel mechanism of BST2-mediated virus restriction, in which BST2 recruits MARCHF8 to catalyze the ubiquitination of the PEDV N protein. The ubiquitinated N protein is then recognized by CALCOCO2/NDP52, which delivers it to autolysosome for degradation through the selective autophagy pathway.

Abbreviations: 3MA: 3-methyladenine; ATG: autophagy-related; Baf A1: bafilomycin A₁; BST2: bone marrow stromal cell antigen 2; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CC: coiled-coil; ChIP: chromatin immunoprecipitation; Co-IP: co-immunoprecipitation; CQ: chloroquine; CT: cytoplasmic tail; DAPI: 4′,6-diamidino-2-phenylindole; GPI: glycosyl-phosphatidylinositol; hpi: hours post infection; IRF1: interferon regulatory factor 1; ISG: IFN-stimulated gene; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MARCHF8/MARCH8: membrane-associated ring-CH-type finger 8; MOI: multiplicity of infection; N protein: nucleocapsid protein; PED: porcine epidemic diarrhea; PEDV: porcine epidemic diarrhea virus; RT: room temperature; siRNA: small interfering RNA; STAT: signal transducer and activator of transcription; TCID₅₀: 50% tissue culture infectious doses; TM: transmembrane.

干扰素诱导的 BST2（骨髓基质细胞抗原 2）通过将包膜病毒粒子束缚在细胞表面以限制病毒释放和诱导 NFKB 依赖性抗病毒免疫反应来抑制病毒复制。然而，BST2 使用选择性自噬途径抑制病毒复制的机制知之甚少。在这项研究中，我们发现在猪流行性腹泻病毒 (PEDV) 感染 Vero 细胞期间 BST2 表达显着增加，IRF1 靶向其启动子。我们还表明 BST2 通过结合和降解 PEDV 编码的核衣壳 (N) 蛋白来抑制 PEDV 复制。N蛋白的下调被巨自噬/自噬抑制剂而非蛋白酶体抑制剂阻断，这意味着N蛋白是通过选择性自噬途径降解的。BST2 和 N 蛋白都与 E3 泛素连接酶 MARCHF8/MARCH8 和货物受体 CALCOCO2/NDP52 相互作用，并且 N 蛋白的泛素化对于 BST2-MARCHF8 轴介导的 N 降解是必需的。用小干扰 RNA 敲低 MARCHF8 或 ATG5 可阻断选择性自噬途径，挽救 293T 细胞中 PEDV N 的蛋白质丰度，并阻止 BST2 在 Vero 细胞中抑制 PEDV 复制。总之，我们的数据证明了 BST2 介导的病毒限制的新机制，其中 BST2 招募 MARCHF8 来催化 PEDV N 蛋白的泛素化。然后泛素化的 N 蛋白被 CALCOCO2/NDP52 识别，通过选择性自噬途径将其递送至自溶酶体进行降解。

缩写：3MA：3-甲基腺嘌呤；ATG：自噬相关；Baf A1：巴弗洛霉素 A ₁; BST2：骨髓基质细胞抗原2；CALCOCO2/NDP52：钙结合和卷曲螺旋结构域 2；CC：盘绕线圈；ChIP：染色质免疫沉淀；Co-IP：共免疫沉淀；CQ：氯喹；CT：胞质尾；DAPI：4',6-二脒基-2-苯基吲哚；GPI：糖基-磷脂酰肌醇；hpi：感染后的小时数；IRF1：干扰素调节因子1；ISG：干扰素刺激基因；MAP1LC3/LC3：微管相关蛋白1轻链3；MARCHF8/MARCH8：膜相关环-CH 型指 8；MOI：感染复数；N蛋白：核衣壳蛋白；PED：猪流行性腹泻；PEDV：猪流行性腹泻病毒；RT：室温；siRNA：小干扰RNA；STAT：信号转导和转录激活因子；TCID ₅₀：50% 组织培养感染剂量；TM：跨膜。