Zhang Li-Sheng, Zhang Yuexiu, Sharma Amit, He Chuan, Li Jianrong
Department of Chemistry, Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, University of Chicago, Chicago, IL, USA.
Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA.
Methods Mol Biol. 2025;2948:233-247. doi: 10.1007/978-1-0716-4666-3_15.
Viral epigenetic modification is an emerging field. As an obligate intracellular parasite, viral genetic material is exposed to host RNA-modifying enzymes. Currently, more than 180 types of RNA modifications have been identified. Like all non-segmented negative-sense RNA viruses, human respiratory syncytial virus (RSV) infection produces three types of RNA: genome and antigenome that are encapsidated by viral nucleocapsid and 10 mRNAs that are capped, methylated, and polyadenylated. Recently, the RSV genome, antigenome, and mRNAs have been shown to be modified by N6-methyladenosine (mA), which plays critical roles in replication, gene expression, and innate immune responses. However, whether RSV RNAs contain other types of modification remains unclear. Here, we describe an optimized bisulfite sequencing method and rigorous data analysis procedure to quantitatively map 5-methylcytosine (mC), one of the most prevalent RNA modifications, in RNA isolated from A549 cells infected by RSV and vesicular stomatitis virus (VSV) at single-base resolution. This method identified 20 mC sites in RSV genome but no mC sites were found in RSV antigenome, RSV mRNAs, or VSV RNAs. This protocol can be easily adapted for mC mapping of the RNAs of other viruses.
病毒表观遗传修饰是一个新兴领域。作为专性细胞内寄生虫，病毒遗传物质会接触宿主RNA修饰酶。目前，已鉴定出180多种RNA修饰类型。与所有非节段性负链RNA病毒一样，人类呼吸道合胞病毒（RSV）感染产生三种类型的RNA：由病毒核衣壳包裹的基因组和反基因组，以及经过加帽、甲基化和多聚腺苷酸化的10种mRNA。最近，RSV基因组、反基因组和mRNA已被证明会被N6-甲基腺苷（mA）修饰，其在复制、基因表达和先天免疫反应中起关键作用。然而，RSV RNA是否包含其他类型的修饰仍不清楚。在此，我们描述了一种优化的亚硫酸氢盐测序方法和严格的数据分析程序，以单碱基分辨率定量定位从感染RSV和水疱性口炎病毒（VSV）的A549细胞中分离的RNA中最普遍的RNA修饰之一5-甲基胞嘧啶（mC）。该方法在RSV基因组中鉴定出20个mC位点，但在RSV反基因组、RSV mRNA或VSV RNA中未发现mC位点。该方案可轻松适用于其他病毒RNA的mC定位。
