LY6E blocks coronavirus fusion and confers immune control of viral disease

Zoonotic coronaviruses (CoVs) are significant threats to global health, as exemplified by the recent emergence of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Host immune responses to CoV are complex and regulated in part through antiviral interferons. However, the interferon-stimulated gene products that inhibit CoV are not well characterized. Here, we show that interferon-inducible lymphocyte antigen 6 complex, locus E (LY6E) potently restricts cellular infection by multiple CoVs, including SARS-CoV, SARS-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV). Mechanistic studies revealed that LY6E inhibits CoV entry into cells by interfering with spike protein-mediated membrane fusion. Importantly, mice lacking Ly6e in hematopoietic cells were highly susceptible to murine CoV infection. Exacerbated viral pathogenesis in Ly6e knockout mice was accompanied by loss of hepatic and splenic immune cells and reduction in global antiviral gene pathways. Accordingly, we found that Ly6e directly protects primary B cells and dendritic cells from murine CoV infection. Our results demonstrate that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis. These findings advance our understanding of immune-mediated control of CoV in vitro and in vivo, knowledge that could help inform strategies to combat infection by emerging CoV. Overall design: Ly6etm1a ES cells were obtained from the EUCOMM consortium and microinjected into C57BL/6J blastocysts by the UTSW Transgenic Technology Center. Chimeric mice with germline transmission were bred to obtain Ly6etm1a/+ offspring. Ly6etm1a/+ mice were crossed with FLPe-expressing mice (B6N.129S4-Gt(ROSA)26Sortm1(FLP1)Dyn/J, #16226, Jackson Laboratories) to obtain Ly6efl/+ offspring, which were bred to homozygosity. Conditional Ly6efl/fl mice were bred to Vav1-iCre transgenic mice (B6.Cg-Commd10Tg(Vav1-icre)A2Kio/J, #008610, Jackson Laboratories) to obtain Ly6e?HSC/+ (Ly6efl/+; Vav1-iCre) offspring. Ly6e?HSC/+ mice were bred to obtain Ly6e?HSC (Ly6efl/fl; Vav1-iCre) offspring, which harbor a deletion of exon 3 and 4 in hematopoietic stem cells (HSC). Experimental animals were obtained by crossing Ly6e?HSC and Ly6efl/fl mice. Genotype was confirmed by PCR of genomic DNA in-house or outsourced to Transnetyx. Ablation of Ly6e was confirmed by qPCR in immune cells from spleen, liver, or bone marrow. Animal studies were carried out in specific-pathogen-free barrier facilities managed and maintained by the UTSW Animal Resource Center. All procedures used in this study complied with federal and institutional guidelines enforced by the UTSW Institutional Animal Care and Use Committee (IACUC).

人畜共患冠状病毒（Zoonotic coronaviruses, CoVs）是威胁全球公共卫生的重大风险，近期出现的严重急性呼吸综合征冠状病毒2（Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2）便是典型案例。宿主针对冠状病毒的免疫应答极为复杂，其调控过程部分依赖于抗病毒干扰素。然而，目前对可抑制冠状病毒的干扰素刺激基因（interferon-stimulated gene, ISG）产物的认知仍较为匮乏。
本研究证实，干扰素诱导的淋巴细胞抗原6复合体E位点（interferon-inducible lymphocyte antigen 6 complex, locus E, LY6E）可强效限制多种冠状病毒对宿主细胞的感染，涵盖严重急性呼吸综合征冠状病毒（SARS-CoV）、SARS-CoV-2以及中东呼吸综合征冠状病毒（Middle East respiratory syndrome coronavirus, MERS-CoV）。机制研究显示，LY6E通过干扰刺突蛋白介导的膜融合过程，抑制冠状病毒侵入宿主细胞。
值得注意的是，造血细胞中缺失Ly6e的小鼠对鼠类冠状病毒感染呈现高度易感性。Ly6e基因敲除小鼠的病毒致病作用显著加剧，同时伴随肝脏与脾脏免疫细胞丢失以及全局抗病毒基因通路表达下调。据此，本研究发现Ly6e可直接保护原代B细胞与树突状细胞免受鼠类冠状病毒感染。
本研究结果证明，LY6E是关键的抗病毒免疫效应因子，可调控冠状病毒的感染与致病进程。上述发现加深了我们对冠状病毒体外与体内免疫调控机制的理解，可为应对新兴冠状病毒感染的防控策略提供理论支撑。

整体实验设计：研究人员从EUCOMM联盟获取Ly6etm1a胚胎干细胞（ES细胞），并由UTSW转基因技术中心将其显微注射至C57BL/6J小鼠囊胚中。将获得的种系传递嵌合小鼠与野生型小鼠交配，以获得Ly6etm1a/+子代。将Ly6etm1a/+小鼠与表达FLPe的小鼠（B6N.129S4-Gt(ROSA)26Sortm1(FLP1)Dyn/J，货号#16226，杰克逊实验室（Jackson Laboratories））交配，获得Ly6efl/+子代，随后将其繁育至纯合子状态。将条件性敲除小鼠Ly6efl/fl与Vav1-iCre转基因小鼠（B6.Cg-Commd10Tg(Vav1-icre)A2Kio/J，货号#008610，杰克逊实验室）交配，得到Ly6eΔHSC/+（Ly6efl/+; Vav1-iCre）子代。将Ly6eΔHSC/+小鼠互交，获得Ly6eΔHSC（Ly6efl/fl; Vav1-iCre）子代，该类小鼠的造血干细胞（hematopoietic stem cells, HSC）中存在第3、4外显子的缺失。实验动物通过交配Ly6eΔHSC与Ly6efl/fl小鼠获得。通过实验室内部PCR或委托Transnetyx公司完成基因组DNA的基因型鉴定。通过qPCR验证脾脏、肝脏或骨髓免疫细胞中Ly6e的敲除效果。动物实验在UTSW动物资源中心管理维护的无特定病原体（specific-pathogen-free, SPF）屏障设施中开展。本研究中所有实验操作均符合UTSW机构动物护理与使用委员会（Institutional Animal Care and Use Committee, IACUC）执行的联邦及机构指南要求。