Deciphering the Role of the Coronavirus Macro Domain in SARS-CoV Infection

破译冠状病毒宏结构域在 SARS-CoV 感染中的作用

• 批准号: 8781200
• 负责人: Anthony R Fehr
• 金额: $ 5.33万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-25 至 2015-12-24
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8781200
• 关键词: Active Sites; Adenosine Diphosphate Ribose; Binding; Biological Assay; Biology; Brain; CXCL10 gene; Cell Line; Cells; Coronavirus; Coronavirus Infections; Cultured Cells; Disease; Engineering; Enzymes; Epidemic; Epithelial Cells; Eukaryota; Genetic; Genome; Goals; Hepatitis; Immune Cell Activation; Immune response; In Vitro; Inbred BALB C Mice; Individual; Infection; Inflammatory; Inflammatory Response; Interferons; Interleukin-6; Kinetics; Knowledge; Lead; Measures; Methods; Middle East; Modeling; Molecular; Molecular Biology; Molecular Virology; Murine hepatitis virus; Mus; Mutation; Natural Immunity; Pathogenesis; Pathology; Pathway interactions; Pattern; Phosphoric Monoester Hydrolases; Play; Pneumonia; Predisposition; Proteins; RNA Viruses; Reaction; Reporter; Research; Role; Scientist; Severe Acute Respiratory Syndrome; Shapes; Small Interfering RNA; Stream; Syndrome; System; Testing; Therapeutic Intervention; Training; Transcript; Up-Regulation; Viral; Viral Proteins; Virulence; Virus; Virus Diseases; attenuation; base; cell type; cytokine; experience; human morbidity; human mortality; in vivo; inhibitor/antagonist; interest; macrophage; mouse model; mutant; neutralizing antibody; new therapeutic target; novel; pathogen; positional cloning; prevent; programs; promoter; public health relevance; respiratory; ribose 1-phosphate; therapeutic target; therapeutic vaccine; tissue culture; tissue/cell culture; tool; transcription factor; vaccine development; virus host interaction

DESCRIPTION (provided by applicant): The long-term goal of the applicant is to be a successful independent scientist with a research program that focuses on virus-host interactions at the molecular level using tissue culture and mouse models of infection, with a particular interest in uncovering functions of viral proteins. Coronaviruses cause a variety of diseases in mammalian species. They have the potential to cause significant human morbidity and mortality as highlighted by the emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and more recently the Middle East Respiratory Syndrome Coronavirus (MERS-CoV). All Coronaviruses encode a highly conserved Macro domain within non-structural protein (nsp) 3 that makes it a useful therapeutic target. Macro domains are ADP-ribose (ADR) binding molecules conserved in eukaryotes and have diverse functions. The coronavirus Macro domain has phosphatase activity that converts ADP- ribose-1"-phosphate (ADRP) into ADP-ribose (ADR). However, the role of this activity in viral replication or pathogenesis has remained a mystery. Using a mouse-adapted SARS-CoV (MA15), a Macro domain mutant virus was engineered and shown to replicate in tissue culture cells with wild-type kinetics. However, it is completely unable to cause lethal pneumonia in BALB/c mice and induces a large increase in specific pro-inflammatory cytokines compared to wild-type virus. The overall goal of this project is to determine the role for the Macro domain during SARS-CoV and other Coronavirus infections and determine why these viruses universally encode for this protein. Aim 1. We will identify the complete spectrum of cytokines that are differentially regulated during wild type and mutant virus infection using a broad range of methods including microarrays, qRT-PCR, and ELISAs. Active site Macro domain mutants in Murine Hepatitis Virus (MHV, a mouse model for coronavirus biology) and MERS-CoV will be engineered and used to expand our analyses to multiple cell types and coronavirus infection models. Aim 2. To identify the basis of upregulation of pro-inflammatory cytokines in cells infected with mutant virus, individual steps in their activation pathways will be analyzed. Additionally, purified ADRP will be used to test the intriguing possibility that ADRP may be a novel pathogen-associated molecular pattern (PAMP) that activates an innate immune response. Following completion of training, the applicant will be versed in tools needed to study the genetics, molecular biology, and pathogenesis of coronaviruses.

描述（由申请人提供）：申请人的长期目标是成为一名成功的独立科学家，其研究计划侧重于使用组织培养和小鼠感染模型在分子水平上研究病毒-宿主相互作用，特别关注揭示病毒蛋白质的功能。冠状病毒在哺乳动物物种中引起多种疾病。它们有可能造成严重的人类发病率和死亡率，严重急性呼吸道综合征冠状病毒（SARS-CoV）和最近中东呼吸道综合征冠状病毒（MERS-CoV）的出现突出了这一点。所有冠状病毒都编码非结构蛋白（nsp）3内高度保守的宏结构域，使其成为有用的治疗靶点。大结构域是真核生物中保守的ADP-核糖（ADR）结合分子，具有多种功能。冠状病毒宏结构域具有将ADP-核糖-1”-磷酸（ADRP）转化为ADP-核糖（ADR）的磷酸酶活性。然而，这种活性在病毒复制或发病机制中的作用仍然是一个谜。使用小鼠适应的SARS-CoV（MA 15），对宏结构域突变病毒进行工程改造，并显示其在组织培养细胞中以野生型动力学复制。然而，与野生型病毒相比，它完全不能在BALB/c小鼠中引起致死性肺炎，并诱导特异性促炎细胞因子的大量增加。该项目的总体目标是确定SARS-CoV和其他冠状病毒感染过程中Macro结构域的作用，并确定为什么这些病毒普遍编码这种蛋白质。目标1.我们将使用广泛的方法，包括微阵列，qRT-PCR和ELISA，确定在野生型和突变型病毒感染期间差异调节的细胞因子的完整谱。小鼠肝炎病毒（MHV，冠状病毒生物学的小鼠模型）和MERS-CoV中的活性位点宏结构域突变体将被工程化并用于将我们的分析扩展到多种细胞类型和冠状病毒感染模型。目标2.为了鉴定突变病毒感染的细胞中促炎细胞因子上调的基础，将分析其活化途径中的各个步骤。此外，纯化的ADRP将用于测试ADRP可能是激活先天免疫应答的新型病原体相关分子模式（PAMP）的有趣可能性。完成培训后，申请人将精通研究冠状病毒遗传学，分子生物学和发病机制所需的工具。