Enteric coronavirus nsp2 is a virulence determinant that recruits NBR1 for autophagic targeting of TBK1 to diminish the innate immune response

Non-structural protein 2 (nsp2) exists in all coronaviruses (CoVs), while its primary function in viral pathogenicity, is largely unclear. One such enteric CoV, porcine epidemic diarrhea virus (PEDV), causes high mortality in neonatal piglets worldwide. To determine the biological role of nsp2, we generated a PEDV mutant containing a complete <i>nsp2</i> deletion (rPEDV-Δnsp2) from a highly pathogenic strain by reverse genetics, showing that nsp2 was dispensable for PEDV infection, while its deficiency reduced viral replication <i>in vitro</i>. Intriguingly, rPEDV-Δnsp2 was entirely avirulent <i>in vivo</i>, with significantly increased productions of IFNB (interferon beta) and IFN-stimulated genes (ISGs) in various intestinal tissues of challenged newborn piglets. Notably, nsp2 targets and degrades TBK1 (TANK binding kinase 1), the critical kinase in the innate immune response. Mechanistically, nsp2 induced the macroautophagy/autophagy process and recruited a selective autophagic receptor, NBR1 (NBR1 autophagy cargo receptor). NBR1 subsequently facilitated the K48-linked ubiquitination of TBK1 and delivered it for autophagosome-mediated degradation. Accordingly, the replication of rPEDV-Δnsp2 CoV was restrained by reduced autophagy and excess productions of type I IFNs and ISGs. Our data collectively define enteric CoV nsp2 as a novel virulence determinant, propose a crucial role of nsp2 in diminishing innate antiviral immunity by targeting TBK1 for NBR1-mediated selective autophagy, and pave the way to develop a new type of nsp2-based attenuated PEDV vaccine. The study also provides new insights into the prevention and treatment of other pathogenic CoVs. <b>Abbreviations</b>: 3-MA: 3-methyladenine; Baf A1: bafilomycin A₁; CoV: coronavirus; CQ: chloroquine; dpi: days post-inoculation; DMVs: double-membrane vesicles; GABARAP: GABA type A receptor-associated protein; GFP: green fluorescent protein; GIGYF2: GRB10 interacting GYF protein 2; hpi: hours post-infection; IFA: immunofluorescence assay; IFIH1: interferon induced with helicase C domain 1; IFIT2: interferon induced protein with tetratricopeptide repeats 2; IFITM1: interferon induced transmembrane protein 1; IFNB: interferon beta; IRF3: interferon regulatory factor 3; ISGs: interferon-stimulated genes; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; NBR1: NBR1 autophagy cargo receptor; nsp2: non-structural protein 2; OAS1: 2’-5’-oligoadenylate synthetase 1; PEDV: porcine epidemic diarrhea virus; PRRs: pattern recognition receptors; RIGI: RNA sensor RIG-I; RT-qPCR: reverse transcription quantitative polymerase chain reaction; SQSTM1: sequestosome 1; TBK1: TANK binding kinase 1; TCID₅₀: 50% tissue culture infectious doses; VSV: vesicular stomatitis virus.

非结构蛋白2（non-structural protein 2, nsp2）广泛存在于所有冠状病毒（coronavirus, CoV）中，但其在病毒致病性中的核心功能仍未完全明确。猪流行性腹泻病毒（porcine epidemic diarrhea virus, PEDV）作为此类肠道冠状病毒之一，在全球范围内可导致新生仔猪出现高死亡率。为明确nsp2的生物学功能，本研究通过反向遗传学技术，从一株高致病性PEDV毒株中构建了完全缺失nsp2的PEDV突变株（rPEDV-Δnsp2）。实验结果显示，nsp2并非PEDV感染所必需，但其缺失会在体外（in vitro）环境中降低病毒的复制能力。有趣的是，rPEDV-Δnsp2在体内（in vivo）完全无致病性，攻毒后的新生仔猪各肠道组织中，干扰素β（interferon beta, IFNB）与干扰素刺激基因（interferon-stimulated genes, ISGs）的表达量均显著升高。值得注意的是，nsp2可靶向并降解固有免疫应答中的关键激酶——TANK结合激酶1（TANK binding kinase 1, TBK1）。机制研究显示，nsp2可诱导巨自噬/自噬过程，并招募选择性自噬受体NBR1（NBR1自噬货物受体）。随后，NBR1可促进TBK1发生K48位泛素化修饰，并将其递送至自噬体介导降解。因此，rPEDV-Δnsp2的复制受到自噬水平降低与I型干扰素及ISGs过度表达的双重抑制。综上，本研究明确肠道冠状病毒nsp2是一种新型毒力决定因子，提出nsp2可通过靶向TBK1并经NBR1介导的选择性自噬途径，抑制机体固有抗病毒免疫，为开发基于nsp2缺失的减毒PEDV新型疫苗奠定了基础。本研究同时为其他致病性冠状病毒的防控提供了新的思路。

**缩写说明**：3-MA：3-甲基腺嘌呤（3-methyladenine）；Baf A1：巴弗洛霉素A₁（bafilomycin A₁）；CoV：冠状病毒（coronavirus）；CQ：氯喹（chloroquine）；dpi：接种后天数（days post-inoculation）；DMVs：双膜囊泡（double-membrane vesicles）；GABARAP：GABA A型受体相关蛋白（GABA type A receptor-associated protein）；GFP：绿色荧光蛋白（green fluorescent protein）；GIGYF2：GRB10相互作用GYF蛋白2（GRB10 interacting GYF protein 2）；hpi：感染后小时数（hours post-infection）；IFA：免疫荧光试验（immunofluorescence assay）；IFIH1：含解旋酶C结构域1型干扰素诱导蛋白（interferon induced with helicase C domain 1）；IFIT2：四肽重复序列2型干扰素诱导蛋白（interferon induced protein with tetratricopeptide repeats 2）；IFITM1：干扰素诱导跨膜蛋白1（interferon induced transmembrane protein 1）；IFNB：干扰素β（interferon beta）；IRF3：干扰素调节因子3（interferon regulatory factor 3）；ISGs：干扰素刺激基因（interferon-stimulated genes）；mAb：单克隆抗体（monoclonal antibody）；MAP1LC3/LC3：微管相关蛋白1轻链3（microtubule associated protein 1 light chain 3）；MAVS：线粒体抗病毒信号蛋白（mitochondrial antiviral signaling protein）；NBR1：NBR1自噬货物受体（NBR1 autophagy cargo receptor）；nsp2：非结构蛋白2（non-structural protein 2）；OAS1：2’-5’-寡腺苷酸合成酶1（2’-5’-oligoadenylate synthetase 1）；PEDV：猪流行性腹泻病毒（porcine epidemic diarrhea virus）；PRRs：模式识别受体（pattern recognition receptors）；RIGI：RNA传感器RIG-I（RNA sensor RIG-I）；RT-qPCR：逆转录定量聚合酶链反应（reverse transcription quantitative polymerase chain reaction）；SQSTM1：隔离体1（sequestosome 1）；TBK1：TANK结合激酶1（TANK binding kinase 1）；TCID₅₀：50%组织培养感染剂量（50% tissue culture infectious doses）；VSV：水疱性口炎病毒（vesicular stomatitis virus）