NK killing of coronavirus-infected cells

摘要 自然杀伤(NK)细胞是先天的淋巴细胞，不依赖于多肽就能杀死肿瘤和病毒感染的细胞。 抗原。NK细胞通过激活和抑制NK受体与靶细胞相互作用识别靶细胞 靶细胞表面的特定配体。激活和抑制受体信号的平衡决定了 目标细胞是否被杀死。我们的实验室最近发现，表达最保守和普遍的NK 激活受体NKp46通过结合识别内质网应激的细胞 外化钙网织蛋白(ecto-CRT)，一种内质网伴侣蛋白，在下列条件下转运到细胞表面 急诊室压力。ECTO-CRT结合并激活NKp46，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感染过程中的内质网应激下调了主要组织相容性 NK抑制受体复合体(MHC)配体。冠状病毒(CoV)复制与ER密切相关。覆盖层 成熟是通过萌芽进入内质网高尔基体中间隔室(ERGIC)，在那里核衣壳基因组RNA 复合体被包裹在含有膜结合的结构病毒蛋白S、E和M的膜内。 这些蛋白质在内质网上表达。伴随着ERGIC的耗尽而大量产生病毒蛋白 在冠状病毒感染过程中，通过子代病毒粒子的萌发和外出而破坏ER的膜，如使用 以小鼠肝炎病毒为模型系统。鉴于我们之前的工作显示识别ZIKV感染和ER 通过NKp46/外钙网织蛋白应激细胞，我们想知道冠状病毒感染的细胞是否也可能被 NK通过相同的相互作用。大多数研究SARS-CoV-2的NK反应的论文都集中在抗体- 依赖的细胞细胞毒性(ADCC)，因此正在测量依赖于 适应性免疫反应。三篇论文研究了抗体非依赖性NK细胞与SARS的相互作用-- CoV-2体外感染靶细胞，提示NK细胞与感染靶细胞共培养可抑制病毒 通过测量病毒蛋白或RNA的减少来复制。然而，这些出版物都没有探讨如何 NK识别受感染的细胞。在初步实验中，我分析了内质网应激和CRT外化 被无毒人类冠状病毒OC43感染。我的初步数据证实OC43感染会导致 内质网应激基因上调，表明CRT外化发生在感染过程中。基于这些 数据，我假设冠状病毒复制导致内质网应激，CRT外化，以及NK下调 抑制受体配体，使冠状病毒感染的细胞被NKp46识别并被NK杀死。这就做 通过测量激活的ecto-CRT和抑制的MHC配体的表达来验证这一中心假设 在冠状病毒感染细胞表面(目标1)。然后我将测试原代和NK细胞株杀死CoV- 并确定感染细胞的杀伤是否由NKp46/ecto-CRT介导 互动(目标2)。我将使用强毒OC43和强毒SARS-CoV-2来研究这个系统。这个 这项提议将进一步加深我们对先天免疫系统如何防御冠状病毒感染的理解。

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.