Elucidating the roles of CMPK2 in mitochondrial homeostasis and antiviral immunity

项目总结 胞苷/尿苷单磷酸激酶2(CMPK2)是一种干扰素调节的酶，最初 据报道，在体外催化依赖于ATP的dCMP和DUMP磷酸化为二磷酸形式。到期 根据推测的线粒体靶向序列，CMPK2被认为在线粒体中起作用 线粒体DNA合成和维持所需的脱氧核苷酸抢救途径 (MtDNA)。然而，最近的数据显示，CMPK2更喜欢核糖核酸二磷酸底物 在体外，并在细胞检测中限制艾滋病毒和其他RNA病毒的功能。除了这些看似 与此形成鲜明对比的是，目前还没有其他研究涉及细胞定位和组织表达模式。 CMPK2或利用基因敲除来确定真正的生物活性。因此，这一总体目标是 建议是弥合这些知识差距，并机械地提高对CMPK2的理解 使用多种细胞和动物工具包的线粒体功能、组织动态平衡和抗病毒先天免疫 模特们。核心假设是CMPK2通过维持线粒体动态平衡，促进细胞内源性 先天免疫力，限制由线粒体应激源和病毒感染引发的炎症失控。在……里面 支持这一假说，正在进行的研究表明，CMPK2强烈定位于线粒体和 CMPK2的异位过表达足以保护细胞免受RNA病毒的感染。此外，在 在先天免疫激动剂的全身攻击下，CMPK2在肺和肝脏显著上调，并且 CMPK2基因敲除小鼠表现出促炎症细胞因子和I型干扰素(干扰素-I)的高表达 刺激Toll样受体(TLR)后的反应。要更深入地了解CMPK2在 线粒体和抗病毒免疫是两个相互关联但又相互独立的目标。目标1将阐明 CMPK2在安静和应激状态下维持线粒体动态平衡的分子机制。这里, CMPK2基因敲除细胞，由缺乏核苷酸激酶活性的突变CMPK2载体重组的新株系 或者线粒体靶向，全身CMPK2基因敲除小鼠将被利用。目标2将决定 CMPK2的线粒体活性抑制冠状病毒复制并维持线粒体功能 感染。在这里，模拟急性呼吸窘迫的鼻腔内小鼠肝炎病毒挑战方案 综合症和与人类冠状病毒肺炎密切相关的病毒将被使用。这项研究将 从根本上提高我们对CMPK2如何在线粒体稳态和抗病毒中发挥作用的理解 在细胞和组织层面上的先天免疫。此外，它可能会对公众产生积极的影响 通过揭示以CMPK2为中心的新策略来维持线粒体动态平衡，增强抗病毒作用 免疫，并限制在冠状病毒感染期间的破坏性炎症。

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.