The Untranslated 3'End of SARS-CoV-2 RNA as a Determinant of Obesity-Accelerated Infectivity

SARS-CoV-2 RNA 的非翻译 3 末端是肥胖加速感染的决定因素

• 批准号: 10318871
• 负责人: PAUL L FOX
• 金额: $ 40.25万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-14 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10318871
• 关键词: 2019-nCoV; 3' Untranslated Regions; ACE2; Acute; Adipocytes; Adipose tissue; Adult; Affect; Affinity; Antisense RNA; Antiviral Agents; Binding; Binding Proteins; Biological Assay; COVID-19; COVID-19 pandemic; COVID-19 therapeutics; Cell Surface Receptors; Cleaved cell; Code; Complex; Coronavirus; Effectiveness; Elements; Epidemic; Epitopes; Event; Foundations; GTP-Binding Protein alpha Subunits, Gs; Gait; Genetic Translation; Genome; Genomics; Hepatitis B; Hormones; Human; Hypersensitivity; Incidence; Inflammatory; Influenza B Virus; Insulin; Interferon Type II; Knowledge; Luciferases; Messenger RNA; Molecular; Mutate; Mutation; Obesity; Oligonucleotides; Pathologic; Pathway interactions; Patients; Polyproteins; Population; Proteins; Proteomics; RNA; RNA-Binding Proteins; RNA-Directed RNA Polymerase; Regulation; Regulatory Pathway; Reporter; Resistance; Ribosomal Frameshifting; Risk Factors; Role; SARS coronavirus; SARS-CoV-2 inhibitor; Series; Stimulus; Structural Protein; Structure; Syndrome; Testing; Therapeutic; Therapeutic Agents; Therapeutic Intervention; Translations; Untranslated RNA; Untranslated Regions; Vaccination; Vaccines; Viral; Viral Genome; Viral Proteins; Virus; Virus Assembly; Virus Replication; base; betacoronavirus; coronavirus disease; cytokine; demographics; experimental study; genomic RNA; glutamyl-prolyl-tRNA synthetase; improved; inhibitor/antagonist; mimetics; new therapeutic target; novel; obese patients; pandemic disease; particle; protein expression; receptor; targeted agent; therapeutic development; therapeutic target; viral RNA

Project Summary/Abstract Obesity is an epidemic-scale problem in the U.S. affecting about 35% of the adult population, and is a major risk factor for the ongoing pandemic, COVID-19. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the RNA betacoronavirus that is the causative agent of COVID-19. Despite rapid progress in developing effective vaccines, progress in devising improved therapeutics has been slow, and there is an urgent need for anti-viral therapeutics for treatment of infected patients not protected by vaccines. Certain demographics are variably resistant to vaccination, for example, obesity markedly reduces effectiveness of several vaccines. Therapeutics can also be critical in the eventuality that mutations in the virus render the vaccine ineffective. The molecular events responsible for expression of SARS-CoV-2 proteins are known from studies of other betacoronaviruses; however, the regulatory pathways and pathological conditions determining their expression are poorly understood. The translation of viral RNA utilizes the host mRNA translation machinery, primarily regulated by specific RNA-binding proteins or complexes that bind sequence or structural elements in terminal non-coding regions. Importantly, the coding regions of SARS-CoV-2 genomic RNA and the ten subgenomic mRNAs (sgmRNAs) are likewise bordered by non-coding upstream and downstream regions, termed the 5'- leader and 3'-end sequences, respectively. A critical feature of the genome and the sgmRNAs of SARS-CoV-2 is that identical 5'-leader and 3'-end sequences are present in all. Thus, the terminal sequences represent novel, unexplored targets for interference with virus assembly and function. We have discovered a 39-nt sequence in the 3'-end of SARS-CoV-2 bearing structural similarity to the GAIT (interferon-gamma-activated inhibitor of translation) RNA element previously described by us. We show that glutamyl-prolyl tRNA synthetase (EPRS) a protein that binds the human GAIT element also binds the vGLE. Moreover, IFN-γ, a pro-inflammatory cytokine, and insulin, an obesity-induced hormone, markedly increases expression of a luciferase reporter bearing the intact vGLE. These results are the first to show a functional consequence of an RNA element in the 3'-end sequence of SARS-CoV-2. We hypothesize that binding of EPRS to the vGLE stimulates sgmRNA translation required for expression of structural and other SARS-CoV-2 proteins, and for programmed ribosomal frameshifting required for genome replication. We will test this hypothesis by pursuing two Specific Aims: In Aim 1 we elucidate the role of EPRS binding to the SARS-CoV-2 3'UTR vGLE in regulating viral replication and sgmRNA translation. In Aim 2 we develop RNA inhibitors that target EPRS/vGLE interactions and block viral protein expression. We anticipate these fundamental studies will provide the first information on the function of the 3'-end of SARS-CoV-2, and will provide a foundation for development of therapeutic agents to be used in combination with mechanistically distinct anti-viral agents targeting the vGLE and possibly emerging betacoronaviruses.

项目概要/摘要 肥胖在美国是一个普遍存在的问题，影响着约 35% 的成年人口，并且是一个主要风险 造成持续流行病 COVID-19 的因素。严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 是 RNA β冠状病毒是 COVID-19 的病原体。尽管开发进展迅速 有效的疫苗，设计改进疗法的进展缓慢，迫切需要 用于治疗不受疫苗保护的感染患者的抗病毒疗法。某些人口统计数据是 对疫苗接种的抵抗力各不相同，例如，肥胖会显着降低几种疫苗的有效性。 如果病毒突变导致疫苗无效，治疗也可能至关重要。这 从其他研究中已知负责 SARS-CoV-2 蛋白表达的分子事件 β冠状病毒；然而，决定其表达的调控途径和病理条件 人们了解甚少。病毒RNA的翻译主要利用宿主mRNA翻译机制 受特定RNA结合蛋白或结合末端序列或结构元件的复合物调节 非编码区。重要的是，SARS-CoV-2基因组RNA的编码区和十个亚基因组 mRNA (sgmRNA) 同样以非编码上游和下游区域为界，称为 5'- 分别是前导序列和 3' 端序列。 SARS-CoV-2 基因组和 sgmRNA 的一个关键特征 是所有都存在相同的 5'-前导序列和 3'-末端序列。因此，末端序列代表 干扰病毒组装和功能的新颖的、未经探索的目标。我们发现了一个39-nt SARS-CoV-2 3'端的序列与步态（干扰素γ激活 翻译抑制剂）我们之前描述过的RNA元件。我们证明谷氨酰脯氨酰 tRNA 合成酶 (EPRS) 一种结合人类步态元件的蛋白质，也结合 vGLE。此外，IFN-γ是一种促炎剂 细胞因子和胰岛素（一种肥胖诱导的激素）显着增加荧光素酶报告基因的表达 承载完整的 vGLE。这些结果首次显示了 RNA 元件在 SARS-CoV-2 的 3' 端序列。我们假设 EPRS 与 vGLE 的结合刺激 sgmRNA 结构蛋白和其他 SARS-CoV-2 蛋白表达以及程序化核糖体蛋白表达所需的翻译 基因组复制所需的移码。我们将通过追求两个具体目标来检验这个假设： 1 我们阐明了 EPRS 与 SARS-CoV-2 3'UTR vGLE 结合在调节病毒复制和 sgmRNA翻译。在目标 2 中，我们开发了针对 EPRS/vGLE 相互作用并阻断病毒的 RNA 抑制剂 蛋白质表达。我们预计这些基础研究将提供有关功能的第一个信息 SARS-CoV-2 的 3' 末端，将为开发用于治疗的药物奠定基础 与针对 vGLE 的机制不同的抗病毒药物组合，并可能出现 β冠状病毒。