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 天然杀手（NK）细胞是杀死肿瘤和感染病毒细胞的先天淋巴细胞，独立于肽 抗原。 NK细胞通过激活和抑制性NK受体识别靶细胞，它们与 靶细胞表面上的特定配体。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，最保守和普遍表达的NK 激活受体NKP46通过与 外部钙网蛋白（ECTO-CRT），一种ER伴侣，可在条件下转移到细胞表面 ER应力。 ECTO-CRT与NKP46结合并激活NKP46，导致肿瘤，衰老和Zika病毒（ZIKV）杀死 - 感染细胞。我们还发现，ZIKV感染期间的ER应力下调了主要的组织相容性 NK抑制受体的复合物（MHC）配体。冠状病毒（COV）复制与ER紧密相关。 COV 通过萌芽到Er-Golgi中间室（ERGIC），其中成熟 复合物被封闭在包含膜结构的结构病毒蛋白S，E和M的膜中。 这些蛋白质在ER上表达。大量生产病毒蛋白以及ERGIC的耗竭 后代病毒体的萌芽和出口膜在COV感染过程中破坏ER，如图所示 鼠肝炎病毒作为模型系统。鉴于我们以前的工作显示了对ZIKV感染和ER的认可 通过NKP46/ecto-calreticulin胁迫细胞，我们想知道是否也可以通过COV感染的细胞来识别 NK通过相同的相互作用。研究NK对SARS-COV-2响应的大多数论文都集中在抗体上 依赖性细胞细胞毒性（ADCC），因此正在测量NK细胞的功能，取决于 自适应免疫反应。三篇论文研究了与抗体无关的NK细胞相互作用与SARS- COV-2感染的靶细胞体外，并表明NK细胞与感染靶标共培养抑制病毒 通过测量病毒蛋白或RNA的降低来复制。但是，这些出版物都没有探讨 NK识别感染的细胞。在初步实验中，我分析了在 无毒的人类COV OC43感染。我的初步数据证实OC43感染会诱导 ER应激基因的上调，并表明CRT外部化发生在感染期间。基于这些 数据，我假设COV复制会导致ER压力，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配体的表达来研究该中心假设 在COV感染的细胞表面（AIM 1）。然后，我将测试主要和NK细胞系杀死COV-的能力 感染细胞并确定杀死感染细胞（如果存在）是否由NKP46/ECTO-CRT介导 互动（目标2）。我将使用Aviruity OC43和有毒的SARS-COV-2研究该系统。这 提案将进一步了解天生免疫系统如何防御COV感染。