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: 10808399
• 负责人: Monica Rosas Lemus
• 金额: $ 22.13万
• 依托单位: UNIVERSITY OF NEW MEXICO HEALTH SCIS CTR
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10808399
• 关键词: Coronavirus; capping; its; impact; host

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大流行和全球数百万人死亡。尽管疫苗在创纪录的时间内被开发出来，但免疫的自然周期很短，新变种的出现使群体免疫的发展复杂化。人们提出了新的抗病毒药物，然而，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卫生危机。在这方面，一个被证实的治疗靶点是抑制病毒封顶，这是一个修饰病毒RNA的5'UTR以模仿哺乳动物RNA的过程。封顶阻止病毒RNA的降解，改善翻译，并阻止先天细胞免疫系统的检测。病毒复制和盖帽发生在宿主膜和病毒非结构蛋白（nsps）形成的受限双膜囊泡（DMV）中。由于大量的ATP和s -腺苷蛋氨酸被使用，这些过程引起宿主细胞代谢和生物能量学的严重压力和不平衡。许多代谢途径通过形成蛋白质复合物来提高效率，从而避免产物抑制并使反应平衡向产物转移。SARS-CoV-2的复制-转录（RTC）复合体先前已被描述；然而，对封盖酶（nsp14-nsp10, nsp16-nsp10）知之甚少。由于盖顶酶是甲基转移酶（mtase），受到s -腺苷型同型半胱氨酸（SAH）反应产物的强烈抑制，而该产物只能由宿主SAH水解酶（AHCY）水解，因此需要宿主代谢物，如ATP、GTP、SAM和SAH水解，表明可能存在未知的病毒-宿主杂交代谢。本提案的总体目标是确定dmv内存在一个混合病毒-帽盖-宿主代谢池，以及这些变化对宿主生物能量学的影响。为了解决这些知识空白，我们将采取综合策略，使用计算、生化、结构和细胞生物学方法。该提案的目的是：1。确定是否存在病毒甲基转移酶- sah水解酶代谢物。利用AlphaFold 2多定时器软件作为计算方法预测甲基转移酶nsp14-nsp16-nsp10和nsp14， nsp16， nsp10与AHCY的相互作用。同时，这些相互作用将通过拉下试验进行测试，使用纯化蛋白质和结构生物学。目标2。建立冠状病毒甲基转移酶和s -腺苷蛋氨酸水解酶在dmv内的定位。利用肺大鼠上皮细胞（L2）对MHV感染的时间过程进行评估，然后进行亚细胞分离、共免疫沉淀以及使用免疫荧光共聚焦显微镜。目标3。评估宿主在病毒复制和盖帽过程中糖酵解和氧化磷酸化的变化。使用海马分析仪测量l2 - mhv感染细胞的外部酸化率（糖酵解-乳酸生成）和耗氧量（线粒体活性）。糖酵解酶和线粒体与sam代谢酶和DMV中的病毒蛋白的相互作用将在目标2中进行测试。