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.

项目摘要/摘要 SARS-CoV、MERS-CoV和SARS-CoV-2属于贝塔冠状病毒属，编码一整套特异的 辅助性蛋白质。冠状病毒编码的辅助蛋白不是病毒生命周期所必需的，但 重要的调节因子，调节免疫逃逸，以实现病毒的最佳复制和繁殖。独一无二的 贝塔冠状病毒的特征是在冠状病毒科的其他属中没有看到的是存在一个小的 由+1开放阅读框(ORF)相对于ORF并在ORF内编码的辅助蛋白(I) 核衣壳(N)基因。SARS冠状病毒(ORF9b)、MERS冠状病毒(ORF8b)和SARS冠状病毒(SARS-CoV)的内部(I)蛋白- 2(ORF9b)还没有得到广泛的表征。然而，体外实验表明，病毒的I蛋白 这些病毒具有抑制干扰素-I表达的作用，这可能有助于发病。在……里面 在这个应用中，我们假设I蛋白是一个毒力因子，具有每种病毒特有的功能。这个 本项目的目的是研究i蛋白在发病机制中的作用，并确定i蛋白是否具有功能。 针对贝塔科纳病毒属内的病毒。我们获得了突变型MERS-CoV和SARS-CoV-2 通过反向遗传学在不改变N蛋白编码序列的情况下删除I蛋白表达 发现缺乏I蛋白表达的MERS-CoV对小鼠的毒力增加，而缺乏 SARS-CoV-2I蛋白引起细胞减毒。耐人寻味的是，i蛋白的缺失导致了 两个相关冠状病毒毒力的不同变化。在目标1中，我们将调查病毒特性 将MERS-和SARS-CoV-2的I蛋白插入小鼠肝炎病毒(MHV)的功能 另一种贝塔冠状病毒。此外，我们将询问参与调节病毒的i蛋白的作用。 通过干扰病毒复制所需的病毒/宿主机制进行生产。这将由以下人员执行 用质谱仪分析I蛋白相互作用组，检测病毒粒子中I蛋白的存在， 将与病毒粒子组装和释放中的角色一致，并比较突变和非突变的病毒生命周期。 亲代感染病毒的小鼠。在目标2中，我们将评估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。我们将询问干扰素-I信号的作用 在突变病毒的毒力变化中，AS I蛋白已被证明在体外抑制干扰素-I的诱导。在……里面 此外，将通过测量血液中的炎性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本病毒的小鼠的肺。免疫特征分析 将对感染突变病毒的小鼠进行检测，并与感染亲本病毒的小鼠进行比较 ScRNA-seq.这项职业发展奖中提出的培训和实验不仅将提供 对于我来说，获得发展自己独立所需的新技能和技能的机会是无价的 病毒免疫学和发病机制的研究，也解决了关于如何特异性的重要问题 病毒蛋白在冠状病毒感染的细胞中充当毒力因子。