Mechanisms and functional implications of SARS-CoV-2 mRNA capping and modification.

SARS-CoV-2 mRNA 加帽和修饰的机制和功能意义。

• 批准号: 10185716
• 负责人: Brian Geiss
• 金额: $ 40.71万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10185716
• 关键词: 2019-nCoV; Address; Adenosine; Antiviral Agents; Area; Attention; Biochemical; Biological; Biology; Cell physiology; Cells; Cellular biology; Coronavirus; DNA Polymerase II; Data; Defense Mechanisms; Detection; Development; Ensure; Enzymes; Event; Family; Foundations; Future; Gene Expression; Generations; Genetic Transcription; Genetic Translation; Genome; Genomics; Glare; Guanosine; Guanosine Triphosphate; Immunologic Surveillance; Immunoprecipitation; Infection; Influentials; Knowledge; Label; Lysine; Mass Spectrum Analysis; Messenger RNA; Methylation; Modification; Molecular; Molecular Biology; Mutation Analysis; Open Reading Frames; Pathogenesis; Play; Process; Property; Proteins; RNA; RNA Caps; RNA Stability; RNA Virus Infections; RNA Viruses; RNA metabolism; RNA methylation; RNA replication; Regulation; Replicon; Ribose; Role; SARS coronavirus; Structure; System; Therapeutic; Transcript; Transfection; Translation Initiation; Untranslated RNA; Viral; Viral Genes; Viral Pathogenesis; Viral Proteins; Virus; Virus Diseases; Virus Replication; Work; base; drug development; guanylyltransferase; innate immune mechanisms; innate immune sensing; insight; mRNA Stability; mRNA capping; novel; novel therapeutics; novel vaccines; nucleotidyltransferase; success; therapeutic development; vaccine development; viral RNA; virus host interaction

SARS-CoV-2 must cap and methylate its mRNAs to ensure their stability, translatability, and avoid detection by host innate immune mechanism as non-self transcripts. The process of RNA capping, therefore, is pivotal to the success of a SARS-CoV-2 infection. It also represents a key contributor to the molecular mechanisms of pathogenesis as well as a very attractive target for the development of antiviral therapeutics. However, there are three key knowledge gaps that have slowed progress in our understanding of this important area of coronavirus molecular biology that will be addressed in this proposal. First, the identity of the guanylyltransferase (GTase), the centerpiece of the viral RNA capping machinery that transfers GTP to the 5' end of the nascent transcript, is unknown. We will use a two-pronged strategy of complementary molecular and biochemical approaches to address this glaring gap in our understanding of SARS-CoV-2 mRNA capping mechanisms, laying the foundation for the development of capping-targeted antivirals. Second, while RNA capping is a regulated process and uncapped RNAs play an influential role in the biology of other positive sense RNA viral infections, it is not known if RNA capping is a regulated or a default event in coronaviruses. We will determine if uncapped RNAs are produced by SARS-CoV-2 in order to establish the foundation for a role of regulated capping and non-coding viral transcripts in SARS-CoV-2 infections. Finally, every cellular mRNA that begins with a terminal adenosine has that residue 2'O methylated at the ribose ring as well as N6 methylated on the adenosine base (m6Am). The strong conservation of this m6Am modification indicates its importance in cell biology, an assertation recently confirmed with data suggesting that the modification increases translatability and facilitates recognition of the transcript as `self'. Interestingly, SARS-CoV-2 and other coronaviruses all initiate their transcripts with an A residue, but it is not known whether that A residue contains an m6A modification. In the final part of this project, we will determine the m6A modification status of the terminal 5' A residue of SARS-CoV-2 mRNAs and investigate the role that the modification (or lack thereof) plays in the biology of coronaviral transcripts. Collectively these studies will provide important new insights into the molecular biology of SARS-CoV-2 and open up avenues for the development of broad-spectrum anti- coronaviral therapeutics.

SARS-COV-2必须限制并甲基化其mRNA，以确保其稳定性，翻译和避免 通过宿主先天免疫机制作为非自我转录本的检测。因此，RNA封盖的过程， 对于SARS-COV-2感染的成功至关重要。它也代表了分子的关键因素 发病机理的机制以及抗病毒疗法发展的非常有吸引力的靶标。 但是，有三个关键知识差距在我们的理解中放缓了进步 冠状病毒分子生物学的重要领域将在本提案中解决。首先， Guanylyltransferase（GTase），病毒RNA封盖机械的核心，将GTP转移至5' 新生转录本的结尾是未知的。我们将使用互补分子的两种策略 和生化方法，以解决我们对SARS-COV-2 mRNA封盖的理解中的明显差距 机制，为开发定位的抗病毒药的发展奠定了基础。第二，而RNA 封盖是一个受调节的过程，未盖上的RNA在其他阳性的生物学中起着影响力 有义务RNA病毒感染，尚不清楚RNA限值是冠状病毒中的调节还是默认事件。 我们将确定SARS-COV-2是否生产未盖的RNA，以建立A的基础 SARS-COV-2感染中受调节的封盖和非编码病毒转录本的作用。最后，每个细胞 从末端腺苷开始的mRNA具有核糖环和N6的残基2'O甲基化 在腺苷碱（M6AM）上甲基化。该M6AM修改的强大保护表明其 在细胞生物学中的重要性，最近用数据证实了这一主张，表明修改 提高翻译性并促进对成绩单的认识为“自我”。有趣的是，SARS-COV-2和 其他冠状病毒均用A残留物启动其笔录，但尚不知道该残留物是否是 包含M6A修改。在该项目的最后部分，我们将确定M6A修改状态 末端5'SARS-COV-2 mRNA残留物，并研究了修饰（或缺乏修饰）的作用 发挥冠状病毒转录的生物学作用。这些研究集体将为重要的新见解 SARS-COV-2的分子生物学，并为开发广谱抗的途径开辟了道路 冠状病毒疗法。