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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

基本信息

批准号：
10686243
负责人：
Lok-Yin Roy Wong
金额：
$ 5.67万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-18 至 2023-11-03
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10686243
关键词：
2019-nCoV Ablation Acceleration Address Attenuated Binding Biological Assay Blocking Antibodies Blood Bronchoalveolar Lavage Cell physiology Cells Code Coronavirus Disease Disease Outbreaks Disparate Edema Electron Microscopy Exhibits Family Gene Expression Genes Genetic Goals Health system Homeostasis Human IFNAR1 gene Immune Immune Evasion Immune response Immunologics Immunology Immunoprecipitation In Vitro Infection Infiltration Inflammatory Interferons Intervention K-Series Research Career Programs Kinetics Knockout Mice 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 Open Reading Frames Parents Pathogenesis Pathogenicity Phenotype Play Population Production Property Protein Deficiency 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 Visualization attenuation betacoronavirus cytokine disorder prevention experimental study health economics human coronavirus immunoregulation in vivo interferon alpha receptor lung injury member mouse model mutant pandemic disease protein expression protein function recombinant virus reverse genetics single-cell RNA sequencing skills viral RNA

项目摘要

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