Elucidating the roles of CMPK2 in mitochondrial homeostasis and antiviral immunity

阐明 CMPK2 在线粒体稳态和抗病毒免疫中的作用

• 批准号: 10647630
• 负责人: Sylvia Torres-Odio
• 金额: $ 3.43万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2023-08-11
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10647630
• 关键词: Acute Respiratory Distress Syndrome; Address; Agonist; Animal Model; Biological; Biological Assay; Brain; Cell Culture Techniques; Cell model; Cells; Coronavirus; Coronavirus Infections; Cytidine; DNA Maintenance; DNA biosynthesis; Data; Deoxyribonucleotides; Diphosphates; Enzymes; Exhibits; Fibroblasts; Flavivirus; Generations; Genetic; Genetic Transcription; Goals; HIV; Health; Homeostasis; Human; Immune; Immunity; In Vitro; Infection; Inflammation; Inflammatory; Inflammatory Response; Innate Immune Response; Interferon Type I; Interferons; Knock-out; Knockout Mice; Knowledge; Ligands; Ligation; Liver; Lung; Maintenance; Mammalian Cell; Metabolism; Mitochondria; Mitochondrial DNA; Mitochondrial Proteins; Modeling; Molecular; Mouse Strains; Murine hepatitis virus; Mus; Natural Immunity; Nucleotides; Outcome; Pathology; Pathway interactions; Pattern; Phosphorylation; Phosphotransferases; Plaque Assay; Ploidies; Pneumonia; Predisposition; Proteins; Protocols documentation; Public Health; RNA Virus Infections; RNA Viruses; Reporting; Research; Rest; Ribonucleotides; Role; Runaway; Shapes; Signal Pathway; Stress; Time; Tissues; Toll-like receptors; Uridine Monophosphate; Viral; Viral Load result; Viral Proteins; Virion; Virus Diseases; antiviral immunity; base; combat; cytokine; experimental study; immune activation; improved; in vivo; inhibitor; insight; macrophage; mitochondrial metabolism; mouse model; mutant; novel; overexpression; pathogenic bacteria; pathogenic virus; reconstitution; response; stressor; tissue tropism; vector; viral RNA

PROJECT SUMMARY Cytidine/uridine monophosphate kinase 2 (CMPK2) is an interferon-regulated enzyme that was originally reported to catalyze the ATP-dependent phosphorylation of dCMP and dUMP to diphosphate forms in vitro. Due to a putative mitochondrial targeting sequence, CMPK2 was postulated to function in the mitochondrial deoxyribonucleotide salvage pathway necessary for the synthesis and maintenance of mitochondrial DNA (mtDNA). However, more recent data have revealed that CMPK2 prefers ribonucleotide diphosphate substrates in vitro and functions to restrict HIV and other RNA viruses in cell based assays. Beyond these seemingly contrasting findings, no other studies have addressed the cellular localization and tissue expression patterns of CMPK2 or utilized genetic knockouts to determine true biological activity. Therefore, the overall objective of this proposal is to close these knowledge gaps and mechanistically advance understanding of CMPK2 in mitochondrial function, tissue homeostasis, and antiviral innate immunity using a diverse toolkit of cell and animal models. The central hypothesis is that by maintaining mitochondrial homeostasis, CMPK2 boosts cell-intrinsic innate immunity and limits runaway inflammation triggered by mitochondrial stressors and viral infection. In support of this hypothesis, ongoing studies have revealed that CMPK2 localizes strongly to mitochondria and that ectopic overexpression of CMPK2 is sufficient to protect cells from RNA virus infection. Moreover, after systemic challenge with innate immune agonists, CMPK2 is markedly upregulated in the lungs and liver, and CMPK2 knockout mice exhibit elevated expression of proinflammatory cytokines and type I interferon (IFN-I) responses after Toll-like receptor (TLR) stimulation. To gain additional insight into how CMPK2 functions in mitochondria and antiviral immunity, two related, but independent aims are proposed. Aim 1 will elucidate the molecular mechanisms by which CMPK2 maintains mitochondrial homeostasis at rest and during stress. Here, CMPK2 knockout cells, novel lines reconstituted with mutant CMPK2 vectors lacking nucleotide kinase activity or mitochondrial targeting, and whole body CMPK2 knockout mice will be utilized. Aim 2 will determine that the mitochondrial activity of CMPK2 restricts coronavirus replication and maintains mitochondrial function during infection. Here, an intranasal mouse hepatitis virus challenge protocol that models acute respiratory distress syndrome and closely mirrors coronavirus pneumonia in humans will be employed. This research will fundamentally advance our understanding of how CMPK2 functions in mitochondrial homeostasis and antiviral innate immunity at both the cellular and organismal levels. Moreover, it may have a positive impact on public health by revealing novel CMPK2-centered strategies to maintain mitochondrial homeostasis, boost antiviral immunity, and limit damaging inflammation during coronavirus infection.

项目摘要 胞苷/尿苷单磷酸激酶2（CMPK 2）是一种干扰素调节的酶，最初是 据报道，在体外催化dCMP和dUMP的ATP依赖性磷酸化为二磷酸形式。由于 CMPK 2被假定在线粒体靶向序列中起作用， 线粒体DNA合成和维持所必需的脱氧核糖核苷酸补救途径 （mtDNA）。然而，最近的数据显示，CMPK 2更喜欢核糖核苷酸二磷酸底物， 并在基于细胞的测定中起限制HIV和其它RNA病毒的作用。除了这些看似 相比之下，没有其他研究解决了细胞定位和组织表达模式， CMPK 2或利用基因敲除来确定真实的生物活性。因此，这一总体目标 我们的建议是缩小这些知识差距，并机械地推进对CMPK 2的理解， 线粒体功能、组织稳态和抗病毒先天免疫 模型中心假设是通过维持线粒体稳态，CMPK 2促进细胞内分泌， 先天免疫和限制失控的炎症所引发的线粒体应激和病毒感染。在 为了支持这一假设，正在进行的研究表明，CMPK 2强烈定位于线粒体， CMPK 2的异位过表达足以保护细胞免受RNA病毒感染。而且经过 先天性免疫激动剂的全身性激发，CMPK 2在肺和肝中显著上调， CMPK 2敲除小鼠表现出促炎细胞因子和I型干扰素（IFN-I）的表达升高 Toll样受体（TLR）刺激后的反应。为了更深入地了解CMPK 2在 线粒体和抗病毒免疫，两个相关的，但独立的目标提出。目标1将阐明 CMPK 2在静息和应激期间维持线粒体稳态的分子机制。在这里， CMPK 2敲除细胞，用缺乏核苷酸激酶活性的突变体CMPK 2载体重建的新系 或线粒体靶向，并且将使用全身CMPK 2敲除小鼠。目标2将确定 CMPK 2的线粒体活性限制冠状病毒复制并在病毒复制期间维持线粒体功能。 感染在这里，鼻内小鼠肝炎病毒攻击协议，模型急性呼吸窘迫 综合征和密切反映冠状病毒肺炎在人类将被采用。这项研究将 从根本上推进我们对CMPK 2如何在线粒体内稳态和抗病毒中发挥作用的理解， 在细胞和器官水平上的先天免疫。此外，它可能会对公众产生积极的影响。 通过揭示以CMPK 2为中心的新策略来维持线粒体稳态，增强抗病毒 免疫力，并限制冠状病毒感染期间破坏性炎症。