Coronavirus capping and its impact on the host metabolism

SARS-CoV-2导致了新冠肺炎大流行，导致全球数百万人死亡。尽管疫苗的研发时间创下纪录，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。新药已经被提出作为抗病毒药物，然而，众所周知，病毒也会产生抗药性。因此，有必要寻找新的治疗靶点来应对SARS-CoV-2和新的人畜共患冠状病毒，以防止新的大流行和另一场全球卫生危机。在这方面，一个被证明未被研究的治疗靶点是抑制病毒封顶，这是一种通过修改病毒RNA的5‘非编码区来模仿哺乳动物RNA的过程。封顶可防止病毒RNA的降解，改善翻译，并防止检测到天然细胞免疫系统。病毒复制和封顶发生在宿主膜和病毒非结构蛋白(NSP)形成的受限双膜囊泡(DMV)中。由于大量使用三磷酸腺苷和S-腺苷蛋氨酸，这些过程导致宿主细胞的新陈代谢和生物能量学产生严重的应激和失衡。许多代谢途径通过形成蛋白质复合体来提高效率，这些复合体避免了产物的抑制，并将反应的平衡转移到了产物上。SARS-CoV-2的复制-转录(RTC)复合体已被描述，但对封闭酶(nsp14-nsp10，nsp16-nsp10)知之甚少。由于封闭酶是甲基转移酶(MTase)，被S-同型半胱氨酸(SAH)反应的产物强烈抑制，并且该产物只能被宿主SAH-水解酶(AHCY)水解，因此需要宿主代谢产物如三磷酸腺苷、GTP、SAM和SAH水解物，这表明可能存在未知的病毒-宿主混合代谢产物。这项提议的总体目标是确定DMV中是否存在一个混合的病毒-帽-宿主代谢池，以及这些变化对宿主生物能量学的影响。为了解决这些知识差距，我们将采取综合战略，使用计算、生化、结构和细胞生物学方法。该建议的目的是：1.确定病毒甲基转移酶-SAH水解酶代谢蛋白的存在。用AlphaFold 2多聚体软件预测甲基转移酶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下拉试验进行测试，使用纯化的蛋白质和结构生物学。目的2.建立冠状病毒甲基转移酶和S腺苷蛋氨酸水解酶在DMVS中的定位。封闭酶nsp14、nsp16和AHCY水解酶在病毒囊泡中的共存将通过使用肺-大鼠上皮细胞(L2)的MHV感染时程、随后的亚细胞分离、免疫共沉淀以及使用免疫荧光的共聚焦显微镜来评估。目的3.评估病毒复制和封端过程中宿主糖酵解和氧化磷酸化的变化。L2-MHV感染细胞的外部酸化速率(糖酵解-乳酸产生)和氧气消耗速率(线粒体活性)将使用海马分析仪进行测量。在DMV中，糖酵解酶和线粒体与SAM代谢酶和病毒蛋白的相互作用将按照目标2进行测试。

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.