Coronavirus capping and its impact on the host metabolism

冠状病毒加帽及其对宿主代谢的影响

• 批准号: 10814128
• 负责人: Monica Rosas Lemus
• 金额: $ 23.16万
• 依托单位: UNIVERSITY OF NEW MEXICO HEALTH SCIS CTR
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10814128
• 关键词: 2019-nCoV; 5' Untranslated Regions; Address; Adenosylhomocysteinase; Antiviral Agents; Autophagocytosis; Biochemical; Bioenergetics; Biological Assay; COVID-19 pandemic; Cell Fractionation; Cells; Cellular biology; Cessation of life; Co-Immunoprecipitations; Complex; Computer software; Confocal Microscopy; Coronavirus; Detection; Development; Disease; Enzymes; Epithelial Cells; Equilibrium; Genetic Transcription; Genus Hippocampus; Glycolysis; Goals; Guanosine Triphosphate; Herd Immunity; Hybrids; Hydrolase; Hydrolysis; Immune system; Immunity; Immunofluorescence Immunologic; Infection; Inflammation; Knowledge; Lung; Measures; Membrane; Metabolic; Metabolic Pathway; Metabolism; Methyltransferase; Mitochondria; Oxidative Phosphorylation; Oxygen Consumption; Phase; Process; Production; RNA; Rattus; Reaction; S-Adenosylhomocysteine; S-Adenosylmethionine; Stress; Testing; Time; Transferase; Translations; Vaccines; Variant; Vesicle; Viral; Viral Nonstructural Proteins; Viral Proteins; Virus; Virus Replication; coping; drug resistance development; global health; improved; new pandemic; new therapeutic target; novel therapeutics; prevent; protein complex; protein purification; structural biology; therapeutic target; viral RNA; zoonotic coronavirus

SARS-CoV-2 caused the COVID-19 pandemic and millions of deaths worldwide. Although vaccines were developed in record time, the natural cycle of immunity is short and the rise of new variants complicates the development of herd immunity. New drugs have been proposed as antivirals however, it is known that viruses also develop drug resistance. Therefore, it is necessary to find new therapeutic targets to cope with SARS-CoV-2 and new zoonotic coronaviruses to prevent new pandemics and another global health crisis. In this regard, a proven therapeutic target that is understudied is the inhibition of viral capping, a process that modifies the 5’UTR of the viral RNA to mimic the mammalian RNA. Capping prevents the degradation of viral RNA, improves translation, and prevents the detection of the innate cell immune system. Viral replication and capping take place in confined double-membrane vesicles (DMV) formed by host membranes and viral non-structural proteins (nsps). These processes cause severe stress and imbalance in the metabolism and bioenergetics of the host cell since high amounts of ATP and S-adenosylmethionine are used. Many metabolic pathways improve their efficiency by forming protein complexes, which avoid product inhibition and move the equilibrium of the reaction to the product. The replication-transcriptional (RTC) complex of SARS-CoV-2 was previously described; however little is known about the capping enzymes (nsp14-nsp10, nsp16-nsp10). Since capping enzymes are methyl transferases (MTases), which are strongly inhibited by the product of the reaction S-adenosylhomocysteine (SAH), and this product can only be hydrolyzed by a host SAH-hydrolase (AHCY), the need for host metabolites such as ATP, GTP,SAM and SAH hydrolysis, indicates a possible viral-host hybrid metabolon which is unknown. The overall goal of this proposal is to determine the existence of a hybrid viral-capping-host metabolic pool within the DMVs and the impact of these changes on the bioenergetics of the host. To address these knowledge gaps, we will take an integrated strategy using computational, biochemical, structural, and cell biology approaches. The aims of the proposal are: 1. Determine the existence of a viral methyltransferases-SAH hydrolase metabolon. Using AlphaFold 2 multimer software as a computational approach to predict the interactions of the methyl transferases nsp14-nsp16-nsp10 and nsp14, nsp16, nsp10 with AHCY. In parallel, these interactions will be tested by pull-down assays, using purified proteins and structural biology. Aim 2. Establish the localization of the methyltransferases from coronaviruses and S-adenosylmethionine hydrolase within the DMVs. The co-localization of capping enzymes nsp14, nsp16, and AHCY hydrolase within viral vesicles will be assessed by a time-course of MHV infection using lung-rat epithelial cells (L2), followed by subcellular fractionation, Co-immunoprecipitation as well as confocal microscopy using immunofluorescence. Aim 3. Assess the changes in the glycolysis and oxidative phosphorylation of the host upon viral replication and capping. The rate of external acidification (glycolysis-lactate production) and the rate of oxygen consumption (mitochondrial activity) will be measured in L2-MHV-infected cells using a seahorse analyzer. And the interaction of the glycolytic enzymes and mitochondria with SAM-metabolic enzymes and viral proteins in the DMV, will be tested as in aim 2.

SARS-CoV-2 导致了 COVID-19 大流行并导致全球数百万人死亡，尽管疫苗的开发速度创历史新高，但免疫的自然周期很短，而且新变种的出现使群体免疫的开发变得更加复杂。然而，作为抗病毒药物，众所周知，病毒也会产生耐药性，因此，有必要寻找新的治疗靶点来应对 SARS-CoV-2 和新型人畜共患冠状病毒，以防止新的大流行和另一场全球健康危机。一个经过验证的治疗目标正在研究的是病毒加帽的抑制，这是一种修饰病毒 RNA 的 5'UTR 以模仿哺乳动物 RNA 的过程，可防止病毒 RNA 的降解、改善翻译并防止先天细胞免疫系统的检测。复制和加帽发生在由宿主膜和病毒非结构蛋白（nsps）形成的受限双膜囊泡（DMV）中，这些过程会导致代谢和生物能的严重应激和不平衡。由于使用了大量的 ATP 和 S-腺苷甲硫氨酸，许多代谢途径通过形成蛋白质复合物来提高其效率，从而避免产物抑制并将反应平衡转移到产物复制转录 (RTC) 复合物。先前描述了 SARS-CoV-2；然而，由于加帽酶是甲基转移酶，因此对加帽酶（n​​sp14-nsp10、nsp16-nsp10）知之甚少。 (MTases)，其被S-腺苷同型半胱氨酸(SAH)反应产物强烈抑制，且该产物只能被宿主SAH-水解酶(AHCY)水解，需要宿主代谢物如ATP、GTP ,SAM 和 SAH 水解表明可能存在未知的病毒-宿主混合代谢。该提案的总体目标是确定病毒-加帽-宿主混合代谢库的存在。 DMV 以及这些变化对宿主生物能量学的影响 为了解决这些知识差距，我们将采用计算、生物化学、结构和细胞生物学方法的综合策略 该提案的目标是： 1. 确定是否存在。使用 AlphaFold 2 多聚体软件作为计算方法来预测甲基转移酶 nsp14-nsp16-nsp10 和 nsp14-nsp16-nsp10 之间的相互作用。 nsp14、nsp16、nsp10 与 AHCY 同时，将使用纯化蛋白和结构生物学通过下拉分析来测试这些相互作用。目标 2. 确定冠状病毒和 S-腺苷甲硫氨酸水解酶在 DMV 中的定位。 -将评估病毒囊泡内封端酶 nsp14、nsp16 和 AHCY 水解酶的定位通过使用肺大鼠上皮细胞 (L2) 进行 MHV 感染的时间过程，然后进行亚细胞分级、免疫共沉淀以及使用免疫荧光的共聚焦显微镜观察。 目标 3. 评估宿主糖酵解和氧化磷酸化的变化。将测量外部酸化速率（糖酵解-乳酸产生）和耗氧速率（线粒体活性）。使用海马分析仪检测 L2-MHV 感染的细胞，并按照目标 2 测试 DMV 中的糖酵解酶和线粒体与 SAM 代谢酶和病毒蛋白的相互作用。