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.

项目总结/文摘