NK killing of coronavirus-infected cells

摘要 自然杀伤（NK）细胞是一种先天性淋巴细胞，其杀伤肿瘤和病毒感染的细胞，不依赖于肽 抗原NK细胞通过激活和抑制NK受体识别靶细胞， 靶细胞表面的特异性配体。激活和抑制受体信号的平衡决定了 靶细胞是否被杀死。我们的实验室最近发现，最保守和普遍表达的NK 活化受体NKp 46通过结合到细胞膜上识别经历内质网（ER）应激的细胞。 外化钙网蛋白（ecto-CRT），一种ER分子伴侣，在细胞外基质形成的条件下易位至细胞表面。 急诊室压力。Ecto-CRT结合并激活NKp 46，导致杀死肿瘤、衰老和寨卡病毒（ZIKV）。 被感染的细胞我们还发现ZIKV感染期间的ER应激下调了主要组织相容性， NK抑制性受体的MHC复合物配体。冠状病毒（CoV）复制与ER紧密相关。盖 通过出芽进入ER-高尔基体中间室（ERGIC）而成熟，在那里核衣壳-基因组RNA 复合物被封闭在含有膜结合的结构病毒蛋白S、E和M的膜内。 这些蛋白质在ER上表达。病毒蛋白的大量产生沿着ERGIC的消耗 在CoV感染期间，通过子代病毒体的出芽和排出破坏ER，如使用 小鼠肝炎病毒作为模型系统。鉴于我们之前的工作显示ZIKV感染和ER 通过NKp 46/外钙网蛋白应激细胞，我们想知道CoV感染的细胞是否也可以被NKp 46/外钙网蛋白识别。 通过相同的互动。大多数研究NK对SARS-CoV-2反应的论文都集中在抗体上， 依赖性细胞毒性（ADCC），并因此测量NK细胞的功能，该功能取决于 适应性免疫反应三篇论文研究了非抗体依赖性NK细胞与SARS的相互作用- CoV-2感染的靶细胞，并表明NK细胞与感染的靶细胞共培养抑制了病毒 通过测量病毒蛋白或RNA的减少来检测病毒复制。然而，这些出版物都没有探讨如何 NK细胞识别感染细胞。在初步实验中，我分析了ER应激和CRT外化， 感染了无毒力的人CoV OC 43。我的初步数据证实OC 43感染 ER应激基因的上调，并显示CRT外化发生在感染期间。基于这些 数据，我假设CoV复制导致ER应激，CRT的外部化和NK的下调 抑制性受体配体，导致CoV感染的细胞被NKp 46识别并被NK杀死。我会 通过测量激活性外CRT和抑制性MHC配体的表达来研究这一中心假设 在CoV感染细胞的表面上（Aim 1）。然后我将测试原代和NK细胞系杀死CoV的能力- 感染的细胞，并确定感染的细胞的杀伤（如果存在）是否由NKp 46/外-CRT介导 相互作用（目标2）。我将使用无毒OC 43和毒性SARS-CoV-2来研究这个系统。的 这项提案将进一步加深我们对先天免疫系统如何抵御CoV感染的理解。

Abstract Natural killer (NK) cells are innate lymphocytes that kill tumor and virus-infected cells, independently of peptide antigens. NK cells recognize target cells through activating and inhibitory NK receptors, which interact with specific ligands on the target cell surface. The balance of activating and inhibitory receptor signaling determines whether target cells are killed. Our lab recently found that the most conserved and universally expressed NK activating receptor, NKp46, recognizes cells undergoing endoplasmic reticulum (ER) stress by binding to externalized calreticulin (ecto-CRT), an ER chaperone that translocates to the cell surface under conditions of ER stress. Ecto-CRT binds to and activates NKp46, leading to killing of tumor, senescent, and Zika virus (ZIKV)- infected cells. We also found that ER stress during ZIKV infection downregulates the major histocompatibility complex (MHC) ligands of NK inhibitory receptors. Coronavirus (CoV) replication is tightly linked to the ER. CoV mature by budding into the ER-Golgi intermediate compartment (ERGIC), where the nucleocapsid-genomic RNA complex is enclosed within a membrane containing the membrane-bound structural viral proteins, S, E, and M. These proteins are expressed on the ER. Massive production of viral proteins along with depletion of the ERGIC membrane by budding and egress of progeny virions disrupts the ER during CoV infection, as shown using murine hepatitis virus as a model system. Given our previous work showing recognition of ZIKV-infected and ER stressed cells via NKp46/ecto-calreticulin, we wondered whether CoV-infected cells may also be recognized by NK via the same interaction. Most papers studying NK responses to SARS-CoV-2 have focused on antibody- dependent cellular cytotoxicity (ADCC) and are thus measuring a function of NK cells that depends on an adaptive immune response. Three papers investigated antibody-independent NK cell interactions with SARS- CoV-2-infected target cells in vitro, and suggest that NK cell co-culture with infected targets suppresses viral replication by measuring a reduction in viral protein or RNA. However, none of these publications explored how NK recognize infected cells. In preliminary experiments I assayed for ER stress and CRT externalization during infection by the avirulent human CoV OC43. My preliminary data confirm that OC43 infection induces upregulation of ER stress genes and show that CRT externalization occurs during infection. Based on these data, I hypothesize that CoV replication causes ER stress, externalization of CRT, and downregulation of NK inhibitory receptor ligands, causing CoV-infected cells to be recognized by NKp46 and killed by NK. I will investigate this central hypothesis by measuring expression of activating ecto-CRT and inhibitory MHC ligands on the surface of CoV-infected cells (Aim 1). I will then test the ability of primary and NK cell lines to kill CoV- infected cells and determine whether the killing of infected cells, if present, is mediated by the NKp46/ecto-CRT interaction (Aim 2). I will investigate this system using both avirulent OC43 and virulent SARS-CoV-2. The proposal will further our understanding of how the innate immune system may defend against CoV infection.