Translational Regulation of SARS-CoV-2 in response to viral S protein-induced signaling

SARS-CoV-2 响应病毒 S 蛋白诱导信号传导的翻译调控

• 批准号: 10721101
• 负责人: BARSANJIT MAZUMDER
• 金额: $ 22.28万
• 依托单位: CLEVELAND STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-14 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10721101
• 关键词: 2019-nCoV; 3' Untranslated Regions; Adopted; Affinity Chromatography; Binding; Biochemical; Cells; Complement; Complex; Coronavirus; DAP kinase; Development; Elements; Foundations; Gastroenteritis; Gel Chromatography; Genes; Genome; Human; Infection; Inflammation; Inflammatory; Innate Immune Response; Interferon Type II; Intervention; Journals; Laboratories; Lentivirus; Lung; Macrophage; Mass Spectrum Analysis; Mediating; Messenger RNA; Modeling; Molecular Sieve Chromatography; Mutation; Myeloid Cells; Names; Nucleic Acids; Phosphorylation; Phosphotransferases; Physiological; Play; Process; Proteins; Publishing; RNA; RNA Binding; RNA Folding; RNA Viruses; RNA vaccine; RNA-Binding Proteins; Recombinants; Replicon; Respiratory syncytial virus; Ribonucleoproteins; Ribosomal Proteins; Ribosomes; Role; SARS-CoV-2 genome; SARS-CoV-2 infection; SARS-CoV-2 spike protein; Series; Signal Induction; Signal Transduction; Small Interfering RNA; System; Testing; Translational Regulation; Translational Repression; Translations; Viral Proteins; Virulence; Virus; Virus Diseases; Virus-like particle; Work; chemokine; chemokine receptor; complement system; genetic information; genomic RNA; inhibitor; insight; kinase inhibitor; member; monocyte; next generation; novel; respiratory pathogen; response; small molecule; small molecule inhibitor; upstream kinase; viral RNA; viral fitness; viral genomics; virology; virus host interaction

Adopted folding of the RNA elements within the genomes of RNA viruses allows binding of host and viral proteins and plays an essential role in host-virus interactions. However, the mechanistic insights on how these RNA elements could sense the transduced signals from host cells are lacking for SARS-CoV-2. This project is based on our recent discovery of a novel and structurally conserved RNA element within the ORF1a RNA and S mRNA of SARS-CoV-2. Our studies showed that this element regulates the translation of viral proteins in response to viral S protein-induced signaling in human lung cells. In prior studies, we identified a translational silencing-dependent mechanism of controlling inflammation in myeloid cells. These studies showed that the assembly of an L13a-dependent multi-protein RNA-binding complex (IFN-gamma-activated-inhibitor of translation) or GAIT complex on the GAIT elements found in the 3' untranslated region (UTR) of target mRNAs causing translational inhibition. Our studies also showed the physiological importance of this mechanism as an endogenous defense against inflammation. Importance of the GAIT-like elements in human responses to coronavirus and other respiratory pathogens is emerging from the published studies by us and others. Our preliminary work presented in this proposal shows that treatment of human lung cells with SARS- CoV-2 S protein activates a DAP kinase-dependent signaling mechanism causing the release of L13a from large ribosomal subunit followed by the formation of L13a-dependent RNA-binding complex on “GAIT-like” viral RNA elements of SARS-CoV-2, thus inhibiting translation. This led us to name these elements virus activated inhibitor of translation (VAIT) elements. Together, these studies lead us to hypothesize that ribosome-released form of L13a upon SARS-CoV-2 infection promotes the assembly of RNA- binding complex on VAIT element of ORF1a RNA and S mRNA, which is biochemically and functionally distinct from IFN-g-induced GAIT complex. The role of translational inhibition caused by the VAIT complex is testable. We will test this hypothesis by pursuing the following two aims. Aim 1. To elucidate the composition of the translation inhibitory VAIT RNP complex and identification of the upstream kinase in signaling. In this aim we will use the combined approach of Gel filtration, RNA-affinity chromatography and mass spectrometry to identify the subunits of the RNA-binding complex. Small molecule inhibitors and siRNA of the potential kinases will be used to dissect the signaling aspect. Aim 2. To test the virological significance of VAIT element mediated translational silencing. In this aim we will use extensive mutation in the VAIT element of the S gene within the viral genomic RNA of the trans-complementation system to test the role of VAIT element-mediated translational silencing in the intracellular expression of viral proteins and nucleic acid. Mechanistic insights on VAIT element mediated translational silencing of SARS-CoV-2 RNAs may provide novel targets of small molecule intervention and facilitate the development of next-generation mRNA vaccines.

RNA病毒基因组内RNA元件的采用折叠允许宿主与病毒的结合 蛋白质，在宿主与病毒的相互作用中起着至关重要的作用。然而，关于这些如何实现的机械论见解 RNA元件可以感知SARS-CoV-2缺乏的宿主细胞的转导信号。这个项目是 基于我们最近在ORF1a RNA和ORF1a RNA中发现的一个新的结构保守的RNA元件 SARS-CoV-2的S基因。我们的研究表明，该元件调节病毒蛋白的翻译。 病毒S蛋白诱导的人肺细胞信号转导反应。在之前的研究中，我们发现了一种翻译的 控制髓系细胞炎症的沉默依赖机制。这些研究表明， 依赖L13a的多蛋白RNA结合复合体(干扰素-γ激活抑制物)的组装 翻译)或在目标的3‘非翻译区(UTR)中发现的步态元素的步态复杂性 导致翻译抑制的mRNA。我们的研究也表明了这一点的生理重要性。 作为对抗炎症的内源性防御机制。步态元素在人体中的重要性 对冠状病毒和其他呼吸道病原体的反应正在从我们和 其他。我们在这项提案中介绍的初步工作表明，治疗患有SARS的人肺细胞- 冠状病毒2型S蛋白激活DAP激酶依赖的信号机制，导致L13a从 “步态”病毒大核糖体亚基形成依赖L13a的RNA结合复合体 SARS-CoV-2的RNA元件，从而抑制翻译。这让我们给这些 病毒激活的翻译抑制因子(VAIT)。总而言之，这些研究引导我们 假设SARS-CoV-2感染时核糖体释放形式的L13a促进RNA的组装- ORF1aRNA和S基因Vait元件的结合复合体，其生化和功能截然不同 来自干扰素-g诱导的步态复合体。VAIT复合体引起的翻译抑制作用是可以测试的。 我们将通过追求以下两个目标来检验这一假设。目的1.阐明药物的组成 翻译抑制因子Vit RNP复合体与信号转导中上游激酶的识别。为了实现这一目标，我们 将使用凝胶过滤、RNA亲和层析和质谱法相结合的方法 鉴定RNA结合复合体的亚基。小分子抑制剂与潜在激酶的siRNA 将被用来剖析信令方面。目的2.检测VAIT元件的病毒学意义 介导的翻译沉默。为此，我们将在S基因的Vait元件中进行广泛的突变 在病毒基因组RNA的反式互补系统中测试VAIT元件介导的作用 病毒蛋白质和核酸细胞内表达的翻译沉默。对机械的洞察 Vait元件介导的SARS-CoV-2 RNA的翻译沉默可能提供新的小分子靶点 分子干预和促进下一代信使核糖核酸疫苗的开发。