Phenotypic and functional characterization of Betacoronavirus Internal protein in relation to virulence

与毒力相关的β冠状病毒内部蛋白的表型和功能特征

• 批准号: 10506647
• 负责人: Lok-Yin Roy Wong
• 金额: $ 12.43万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-18 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10506647
• 关键词: 2019-nCoV; Address; Attenuated; Binding; Biological Assay; Blocking Antibodies; Blood; Bronchoalveolar Lavage; Cell physiology; Cells; Code; Coronavirus; Disease; Disease Outbreaks; Edema; Electron Microscopy; Exhibits; Family; Gene Expression; Gene Proteins; Genes; Genetic; Goals; Health system; Homeostasis; Human; IFNAR1 gene; Immune; Immune Evasion; Immune response; Immunologics; Immunology; Immunoprecipitation; In Vitro; Infection; Infiltration; Inflammatory; Interferon-alpha; Interferons; Intervention; K-Series Research Career Programs; Kinetics; Knockout Mice; Lead; Life Cycle Stages; Lung; Maps; Mass Spectrum Analysis; Measures; Mediating; Membrane; Middle East Respiratory Syndrome; Middle East Respiratory Syndrome Coronavirus; Molecular; Murine hepatitis virus; Mus; Nucleocapsid; Nucleocapsid Proteins; Open Reading Frames; Parents; Pathogenesis; Pathogenicity; Phenotype; Play; Population; Production; Property; Proteins; Public Health; Replicon; Reporting; Research; Role; SARS coronavirus; Signal Transduction; System; Techniques; Training; Up-Regulation; Viral; Viral Genes; Viral Proteins; Virion; Virulence; Virulence Factors; Virulent; Virus; Virus Replication; Virus-like particle; attenuation; betacoronavirus; cytokine; disorder prevention; experimental study; human coronavirus; immunoregulation; in vivo; lung injury; member; mouse model; mutant; pandemic disease; protein expression; protein function; receptor; recombinant virus; reverse genetics; single-cell RNA sequencing; skills; viral RNA

Project summary/Abstract SARS-CoV, MERS-CoV and SARS-CoV-2 belong to the genus Betacoronavirus and encode sets of specific accessory proteins. Accessory proteins encoded by coronaviruses are not essential for the viral life cycle but are important regulators that mediate immune evasion for optimal virus replication and propagation. One unique feature of Betacoronavirus that is not seen in other genera of the family Coronaviridae is the presence of a small accessory protein (I) encoded by the +1 open reading frame (ORF) relative to and within the ORF encoding the nucleocapsid (N) gene. The internal (I) proteins of SARS-CoV (ORF9b), MERS-CoV (ORF8b) and SARS-CoV- 2 (ORF9b) have not been extensively characterized. However, in vitro experiments suggest that the I proteins of these viruses have a role in suppressing IFN-I expression, which could potentially contribute to pathogenesis. In this application, we hypothesize that the I protein is a virulence factor with functions specific to each virus. The goal of this project is to study the roles of I proteins in pathogenesis and determine if I proteins possess functions specific to viruses within the genus Betacoronavirus. We generated mutant MERS-CoV and SARS-CoV-2 with deletions of I protein expression without altering the coding sequence of the N protein by reverse genetics and found that MERS-CoV lacking I protein expression showed increased virulence in mice, while the absence of the SARS-CoV-2 I protein resulted in attenuation. It is intriguing that the absence of the I protein resulted in such disparate changes in the virulence of the two related CoVs. In Aim 1, we will investigate the virus-specifc functions of the I proteins by inserting the I protein of MERS- and SARS-CoV-2 into mouse hepatitis virus (MHV), another betacoronavirus. In addition, we will interrogate the role of I proteins involved in regulating virus production by interfering with the virus/host machinery required for virus replication. This will be performed by analyzing the I protein interactome by mass spectrometry, detecting the presence of I protein in virions, which would be consistent with a role in virion assembly and release, and comparing the viral life cycle in mutant vs. parental virus-infected mice. In Aim 2, we will assess the role of the I protein in regulating immune responses by comparing mice infected with mutant viruses or parental viruses. We will interrogate the role of IFN-I signaling in the altered virulence of mutant viruses, as I proteins have been shown to suppress IFN-I induction in vitro. In addition, changes in immune responses will be investigated by measuring inflammatory cytokines in the blood, bronchoalveolar lavage (BAL) and the lungs of mice infected with mutant or parental viruses. Immune profiling of mice infected with mutant viruses will be performed and compared to mice infected with parental viruses by scRNA-seq. The training and experiments proposed in this career development award will not only offer invaluable opportunities for me to acquire new skills and techniques required for developing my own independent research in viral immunology and pathogenesis but also address important questions regarding how specific viral proteins act as virulence factors in coronavirus-infected cells.

项目概要/摘要 SARS-COV、MERS-COV 和 SARS-COV-2 属于 Betacoronavirus 属，编码一组特定的病毒 辅助蛋白。冠状病毒编码的辅助蛋白对于病毒生命周期不是必需的，但 介导免疫逃避以实现最佳病毒复制和传播的重要调节因子。一件独一无二的 β冠状病毒的一个特征是存在一个小病毒，这在冠状病毒科的其他属中是不存在的。 辅助蛋白 (I) 由 +1 开放阅读框 (ORF) 编码，相对于编码 核衣壳 (N) 基因。 SARS-CoV (ORF9b)、MERS-CoV (ORF8b) 和 SARS-CoV 的内部 (I) 蛋白 2 (ORF9b) 尚未得到广泛表征。然而，体外实验表明 I 蛋白 这些病毒具有抑制 IFN-I 表达的作用，这可能有助于发病机制。在 在此应用中，我们假设 I 蛋白是一种毒力因子，具有针对每种病毒的特定功能。这 该项目的目标是研究 I 蛋白在发病机制中的作用并确定 I 蛋白是否具有功能 特定于β冠状病毒属内的病毒。我们生成了突变型 MERS-CoV 和 SARS-CoV-2 通过反向遗传学删除 I 蛋白表达而不改变 N 蛋白的编码序列， 发现缺乏 I 蛋白表达的 MERS-CoV 在小鼠中表现出毒力增加，而缺乏 I 蛋白表达的 MERS-CoV 在小鼠中表现出毒力增加 SARS-CoV-2 I 蛋白导致衰减。有趣的是，I 蛋白的缺失导致了这样的结果。 两种相关冠状病毒毒力的不同变化。在目标 1 中，我们将研究病毒特异性 通过将 MERS-和 SARS-CoV-2 的 I 蛋白插入小鼠肝炎病毒 (MHV) 中来研究 I 蛋白的功能， 另一种β冠状病毒。此外，我们将探讨 I 蛋白在调节病毒中的作用 通过干扰病毒复制所需的病毒/宿主机器来生产。这将由以下人员执行 通过质谱分析 I 蛋白相互作用组，检测病毒粒子中 I 蛋白的存在，这 将与病毒粒子组装和释放中的作用一致，并比较突变体与非突变体中的病毒生命周期。 亲代病毒感染的小鼠。在目标 2 中，我们将通过以下方式评估 I 蛋白在调节免疫反应中的作用： 比较感染突变病毒或亲本病毒的小鼠。我们将探讨 IFN-I 信号传导的作用 突变病毒的毒力改变，因为 I 蛋白已被证明可以在体外抑制 IFN-I 诱导。在 此外，将通过测量血液中的炎症细胞因子来研究免疫反应的变化， 支气管肺泡灌洗（BAL）和感染突变病毒或亲代病毒的小鼠的肺部。免疫分析 将进行感染突变病毒的小鼠的实验，并与感染亲代病毒的小鼠进行比较 单链RNA测序。该职业发展奖中提出的培训和实验不仅提供 对我来说获得发展自己的独立能力所需的新技能和技术的宝贵机会 病毒免疫学和发病机制的研究，但也解决了有关特异性如何的重要问题 病毒蛋白在冠状病毒感染的细胞中充当毒力因子。