Employing viruses to unravel the functional significance of the m5C epitranscriptome

利用病毒揭示 m5C 表观转录组的功能意义

• 批准号: 10638533
• 负责人: Charles M Rice
• 金额: $ 66万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-07 至 2028-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10638533
• 关键词: 5' Untranslated Regions; Acute; Address; Affect; Alphavirus; Animal Model; Attenuated; Binding; Binding Proteins; Biochemical; Biological Assay; Biology; Capsid Proteins; Cardiac; Cell physiology; Cells; Cellular biology; Chikungunya virus; Chronic; Clover; Code; Coxsackie Viruses; Data; Deposition; Excision; Exhibits; Family; Family member; Genetic Transcription; Genome; Goals; High-Throughput Nucleotide Sequencing; Homeostasis; Human Biology; Immune Evasion; Immunocompetent; Immunology; In Vitro; Infection; Innate Immune Response; Intervention; Knock-out; Knowledge; Learning; Life Cycle Stages; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Methylation; Methyltransferase; Modification; Molecular; Mutate; Mutation; Myocarditis; Outcome; Pathogenesis; Plant Leaves; Play; Post-Transcriptional RNA Processing; Process; Proteins; RNA; RNA Virus Infections; RNA Viruses; RNA methylation; Reader; Regulation; Research; Ribosomal RNA; Role; Sindbis Virus; Site; Specificity; Structure; System; Testing; Transfer RNA; Translation Initiation; Translations; Viral; Viral Genome; Viral Nonstructural Proteins; Viral Pathogenesis; Viral Physiology; Virion; Virus; Virus Diseases; Virus Replication; Work; antiviral drug development; bisulfite; cell type; developmental disease; epitranscriptome; human disease; immune activation; in vivo; innate immune sensing; insight; mRNA Translation; mouse model; mutant; neuron development; novel; overexpression; pathogenic virus; reconstitution; stem cell differentiation; stem cells; tRNA Methyltransferases; viral RNA; viral myocarditis; virus host interaction

5-methylcytosine (m5C) is an important RNA modification studied mostly for its role in tRNA biology. However, its roles in other aspects of RNA biology remain understudied. Our preliminary results, using bisulfite treatment of RNA followed by high-throughput sequencing, show that the genomes of many RNA viruses are m5C methylated in a site-specific manner, including Sindbis virus (SINV), chikungunya virus (CHIKV) and Coxsackievirus B3 (CVB3). The presence of m5C in diverse viruses, whose RNAs undergo many processes including translation, replication, transcription, and virion packaging, provides an attractive starting point for understanding the broader significance of this modification in regulating RNA function. A single dominant m5C site in SINV allowed us to generate an m5C-null mutant that exhibited cell-type dependent effects on virus replication. The host tRNA methyltransferase (MTase), NSUN2, which is important for host neuronal development and stem cell differentiation, appears to be the “writer” required for m5C modification of SINV. NSUN5, an MTase of ribosomal RNA is required for CVB3 methylation. We hypothesize that m5C plays a role in regulating viral RNA functions impacting virus-host interactions and viral life cycles. In three aims, using virologic, molecular, biochemical, high-throughput sequencing, and small animal model approaches: i) SINV will be exploited to learn how m5C is deposited and how it regulates RNA functions and viral infection and pathogenesis; studies of the related alphavirus CHIKV will allow conserved and virus-specific features to be uncovered, ii) how m5C regulates CVB3 RNA and what effects the modification has on virus replication and CVB3-associated myocarditis will be determined, and iii) SINV and CVB3 will be leveraged to characterize unknown functions of the NSUN2 and NSUN5 MTases, respectively, and as probes to discover novel m5C binding proteins that can exert their effect by direct binding (“readers”) or by removal of the m5C mark (“erasers”). This work will contribute to our understanding of human biology by revealing fundamental principles and functions of this widespread mark in the epitranscriptome with implications for its roles in maintaining cellular homeostasis. Since m5C methylation is a cellular process exploited by numerous viruses, this study could yield new targets for antiviral intervention.

5-甲基胞嘧啶（m5 C）是一种重要的RNA修饰，主要研究其在tRNA生物学中的作用。然而，它在RNA生物学的其他方面的作用仍然研究不足。我们的初步结果，使用亚硫酸氢盐处理RNA，然后进行高通量测序，表明许多RNA病毒的基因组以位点特异性方式被m5 C甲基化，包括辛德毕斯病毒（SINV），基孔肯雅病毒（CHIKV）和柯萨奇病毒B3（CVB 3）。m5 C在不同病毒中的存在，其RNA经历许多过程，包括翻译，复制，转录和病毒体包装，为理解这种修饰在调节RNA功能中的更广泛意义提供了一个有吸引力的起点。SINV中的单个显性m5 C位点使我们能够产生对病毒复制表现出细胞类型依赖性效应的m5 C无效突变体。宿主tRNA甲基转移酶（MTase），NSUN 2，其对于宿主神经元发育和干细胞分化是重要的，似乎是SINV的m5 C修饰所需的“作家”。NSUN 5是CVB 3甲基化所必需的核糖体RNA的MTase。我们假设m5 C在调节影响病毒-宿主相互作用和病毒生命周期的病毒RNA功能中起作用。在三个目标中，使用病毒学、分子、生物化学、高通量测序和小动物模型方法：i）将利用SINV来了解m5 C如何沉积以及它如何调节RNA功能和病毒感染和发病机制;相关甲病毒CHIKV的研究将允许揭示保守的和病毒特异性的特征，ii）将确定m5 C如何调节CVB 3 RNA以及该修饰对病毒复制和CVB 3相关心肌炎的影响，以及iii）将利用SINV和CVB 3分别表征NSUN 2和NSUN 5 MT酶的未知功能，以及作为探针发现新的m5 C结合蛋白，其可以通过直接结合（“读取器”）或通过去除m5 C标记（“擦除器”）来发挥其作用。这项工作将有助于我们了解人类生物学的基本原则和功能，这种广泛的标记在epitranscriptome与其在维持细胞内稳态的作用的影响。由于m5 C甲基化是许多病毒利用的细胞过程，因此这项研究可能会产生抗病毒干预的新靶点。