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β冠状病毒属，编码多组特异性的 辅助蛋白由冠状病毒编码的辅助蛋白对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繁殖的重要调节因子。一个唯一 在冠状病毒科的其他属中没有看到的β冠状病毒的特征是存在小的 辅助蛋白（I），其由相对于编码辅助蛋白的ORF且在编码辅助蛋白的ORF内的+1开放阅读框（ORF）编码， 核衣壳（N）基因。SARS-CoV（ORF 9 b）、MERS-CoV（ORF 8b）和SARS-CoV（ORF 8b）的内部（I）蛋白质 2（ORF 9 b）尚未被广泛表征。然而，体外实验表明， 这些病毒具有抑制IFN-1表达的作用，IFN-1表达可能潜在地导致发病。在 在本申请中，我们假设I蛋白是对每种病毒具有特异性功能的毒力因子。的 本课题的目的是研究I蛋白在发病机制中的作用，并确定I蛋白是否具有功能 特异于β冠状病毒属内的病毒。我们产生了突变的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在小鼠中显示出增加的毒力，而缺乏I蛋白表达的MERS-CoV在小鼠中显示出增加的毒力。 SARS-CoV-2 I蛋白导致减毒。有趣的是，I蛋白的缺失导致了这种 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）， 另一种β冠状病毒此外，我们还将探讨I蛋白在调控病毒中的作用 通过干扰病毒复制所需的病毒/宿主机制来生产。这将由以下人员执行 通过质谱分析I蛋白相互作用组，检测病毒体中I蛋白的存在， 将与病毒体组装和释放中的作用一致，并比较突变体与 亲代病毒感染小鼠。在目标2中，我们将通过以下方式评估I蛋白在调节免疫应答中的作用： 比较感染突变病毒或亲本病毒的小鼠。我们将研究干扰素I信号的作用 在突变病毒毒力改变中，As I蛋白已显示在体外抑制IFN-I诱导。在 此外，免疫应答的变化将通过测量血液中的炎性细胞因子来研究， 支气管肺泡灌洗（BAL）和用突变体或亲本病毒感染的小鼠的肺。免疫分析 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.在这个职业发展奖中提出的培训和实验不仅将提供 这是一个宝贵的机会，让我获得发展自己的独立所需的新技能和技术。 研究病毒免疫学和发病机制，但也解决了重要的问题， 病毒蛋白在冠状病毒感染的细胞中充当毒力因子。