NK killing of coronavirus-infected cells

NK 杀死冠状病毒感染的细胞

• 批准号: 10671834
• 负责人: Mercedes Lewandrowski
• 金额: $ 4.17万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10671834
• 关键词: 2019-nCoV; Affect; Antibodies; Antigens; Binding; Biological Assay; Biological Models; Blocking Antibodies; Cell Communication; Cell Line; Cell surface; Cells; Cellular Stress; Coculture Techniques; Complex; Coronavirus; Coronavirus Infections; Data; Down-Regulation; Endoplasmic Reticulum; Equilibrium; Flow Cytometry; Genes; Golgi Apparatus; Human; Immune system; Immunofluorescence Microscopy; In Vitro; Infection; Innate Immune System; Knock-out; Life Cycle Stages; Ligands; Link; Literature; Lymphocyte; Major Histocompatibility Complex; Measures; Mediating; Membrane; Messenger RNA; Molecular Chaperones; Murine hepatitis virus; Natural Killer Cells; Nucleocapsid; Paper; Pathway interactions; Peptides; Phosphorylation; Production; Productivity; Proteins; Publications; RNA Splicing; Receptor Signaling; SARS-CoV-2 infection; Signal Induction; Stress; Surface; System; Testing; Up-Regulation; Viral; Viral Proteins; Viral Structural Proteins; Virion; Virulent; Virus; Virus Diseases; Virus Replication; Work; XBP1 gene; ZIKV infection; Zika Virus; adaptive immune response; adaptive immunity; antibody-dependent cell cytotoxicity; betacoronavirus; calreticulin; cell killing; endoplasmic reticulum stress; experimental study; genomic RNA; human coronavirus; killer inhibitory receptor; neoplastic cell; receptor; response; senescence; tumor; viral RNA

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.

抽象的 自然杀伤 (NK) 细胞是先天性淋巴细胞，可以独立于肽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 受体来识别靶细胞，NK 受体与 靶细胞表面的特异性配体。激活和抑制受体信号传导的平衡决定 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 激活受体 NKp46 通过结合识别正在经历内质网 (ER) 应激的细胞 外化钙网蛋白 (ecto-CRT)，一种 ER 伴侣，在以下条件下易位至细胞表面 急诊室压力。 Ecto-CRT 结合并激活 NKp46，从而杀死肿瘤、衰老病毒和寨卡病毒 (ZIKV) - 被感染的细胞。我们还发现 ZIKV 感染期间的 ER 应激会下调主要组织相容性 NK 抑制性受体的复合体 (MHC) 配体。冠状病毒（CoV）的复制与内质网密切相关。冠状病毒 通过出芽进入内质网高尔基体中间室 (ERGIC) 成熟，核衣壳基因组 RNA 在此 复合物被包裹在含有膜结合结构病毒蛋白 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以及 ERGIC 的耗尽 如使用所示，在 CoV 感染期间，子代病毒粒子的出芽和排出会破坏膜 鼠肝炎病毒作为模型系统。鉴于我们之前的工作表明对 ZIKV 感染和 ER 的认识 通过 NKp46/ecto-calreticulin 应激细胞，我们想知道 CoV 感染的细胞是否也可以被识别 NK 通过相同的相互作用。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），因此测量 NK 细胞的功能，该功能依赖于 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 体外检测 CoV-2 感染的靶细胞，表明 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的减少来进行复制。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 ER 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 感染会诱发 ER 应激基因的上调并表明 CRT 外化发生在感染期间。基于这些 根据数据，我假设 CoV 复制导致 ER 应激、CRT 外化和 NK 下调 抑制性受体配体，导致 CoV 感染的细胞被 NKp46 识别并被 NK 杀死。我会 通过测量激活 ecto-CRT 和抑制性 MHC 配体的表达来研究这一中心假设 冠状病毒感染细胞的表面（目标 1）。然后我将测试原代细胞系和 NK 细胞系杀死 CoV 的能力- 感染细胞并确定对感染细胞的杀伤（如果存在）是否由 NKp46/ecto-CRT 介导 互动（目标 2）。我将使用无毒的 OC43 和剧毒的 SARS-CoV-2 来研究这个系统。这 该提案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。