Employing viruses to unravel the functional significance of the m5C epitranscriptome

5-甲基胞嘧啶（m5C）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，其在RNA生物学其他方面的作用仍未得到充分研究。我们的初步结果，使用亚硫酸盐处理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的方式m5C甲基化，包括辛德比病毒（SINV），基孔肯雅病毒（CHIKV）和柯萨奇病毒B3 （CVB3）。m5C存在于多种病毒中，其RNA经历许多过程，包括翻译、复制、转录和病毒粒子包装，这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。在SINV中单个m5C显性位点允许我们产生m5C-null突变体，该突变体对病毒复制表现出细胞类型依赖效应。宿主tRNA甲基转移酶（MTase） NSUN2对宿主神经元发育和干细胞分化具有重要作用，似乎是m5C修饰SINV所需的“作者”。核糖体RNA的MTase NSUN5是CVB3甲基化所必需的。我们假设m5C在调节病毒RNA功能中发挥作用，影响病毒-宿主相互作用和病毒生命周期。利用病毒学、分子生物学、生物化学、高通量测序和小动物模型方法实现三个目标：1)利用SINV来了解m5C是如何沉积的，以及它如何调节RNA功能和病毒感染和发病机制；对相关甲病毒CHIKV的研究将使保守的和病毒特异性的特征得以揭示，ii) m5C如何调节CVB3 RNA，以及这种修饰对病毒复制和CVB3相关心肌炎的影响将被确定，iii) SINV和CVB3将分别被利用来表征NSUN2和NSUN5 mtase的未知功能。并作为探针来发现新的m5C结合蛋白，这些蛋白可以通过直接结合（“读取器”）或通过去除m5C标记（“擦除器”）来发挥其作用。这项工作将有助于我们对人类生物学的理解，揭示这种广泛存在于表转录组中的标记的基本原理和功能，以及它在维持细胞稳态中的作用。由于m5C甲基化是许多病毒利用的细胞过程，因此该研究可能为抗病毒干预提供新的靶点。

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.