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通过研究计划的成功独立科学家，使用组织培养和感染的小鼠模型在分子水平上的病毒宿主相互作用，并特别感兴趣地发现病毒蛋白的功能。冠状病毒在哺乳动物物种中引起多种疾病。正如严重的急性呼吸综合征冠状病毒（SARS-COV）以及最近的中东呼吸道综合征冠状病毒（MERS-COV）的出现所强调的那样，它们有可能引起重大的人类发病率和死亡率。所有冠状病毒在非结构蛋白（NSP）3中编码一个高度保守的宏域，该结构域使其成为有用的治疗靶标。宏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磷酸酶活性，可将ADP-核糖1“ - 磷酸（ADRP）转化为ADP-核糖（ADR）。但是，这种活性在病毒复制或发病机理中的作用仍然是一个谜。使用小鼠适应的SARS-COV（MA15），一种宏观培养的型号，并以宏观的培养为单位，并以宏观的形式培养了型号，以培养型型号，以使其与内部培养的病毒培养物进行了复杂性，以培养型型号。但是，与野生型病毒相比，动力学完全无法在BALB/C小鼠中引起致命的肺炎，并引起特定的促炎细胞因子的增长。在野生型和突变病毒感染过程中，使用微阵列，QRT-PCR和ELISA在野生型和突变病毒感染中受到差异调节的细胞因子。目的2。为了确定感染突变病毒的细胞中促炎细胞因子上调的基础，将分析其激活途径中的各个步骤。此外，纯化的ADRP将用于测试ADRP可能是一种新型病原体相关的分子模式（PAMP）的有趣可能性，可激活先天免疫反应。训练完成后，申请人将精通研究冠状病毒遗传学，分子生物学和发病机理所需的工具。