Epitranscriptomic cytidine methylation of the Hepatitis B viral RNA encapsidation signal ensure the reverse transcription of viral RNA [PA-m5C-seq 2]. Epitranscriptomic cytidine methylation of the Hepatitis B viral RNA encapsidation signal ensure the reverse transcription of viral RNA [PA-m5C-seq 2]

Epitranscriptomic RNA modifications have emerged as important regulators of the fate and function of both cellular and viral RNAs. One prominent modification, the cytidine methylation 5-methylcytidine (m5C), is found on the RNA of HIV-1, where m5C enhances the translation and splicing of HIV-1 RNA. However, whether m5C functionally enhances the RNA of other pathogenic viruses remain elusive. Here, we report that the RNA of hepatitis B virus (HBV) is enriched with a high level of m5C, mediated mainly through the cellular methyltransferase NSUN2. Intrigingly, the most prominent cluster of NSUN2-deposited m5C is found on the epsilon hairpin, an RNA element required for viral RNA encapsidation and reverse transcription. Loss of m5C from HBV RNA due to depletion of NSUN2 resulted in a modest decrease in viral capsid protein (HBc) translation, yet this is accompaneied by a near-complete loss of the reverse transcribed viral DNA. Similarly, mutations introduced to remove the methylated cytidines resulted in a translation decrease and block of reverse transcription. Furthermore, pharmacological disruption of m5C deposition with a nucleoside analogue led to a significant decrease in HBV replication. Thus, our data indicates m5C methylations is a critical enhancer of the epsilon element in HBV reverse transcription. Our study suggests the theraputic potential of targeting the m5C methyltransfer process on the HBV 5’epsilon as an alternative antiviral stratagy. Overall design: PA-m5C-seq: Photoactivated UV crosslinking of m5C antibody to m5C+ RNA, followed by immunoprecipitation and sequencing. RNA from HuH-7 cells, transfected with HBV replicon plasmid, pulsed with 6-thioguanosine (6SG).

表观转录组RNA修饰已成为调控细胞RNA与病毒RNA命运及功能的关键调控因子。其中一类代表性修饰——胞嘧啶甲基化产物5-甲基胞嘧啶（m5C），存在于人类免疫缺陷病毒1型（HIV-1）的RNA中，该修饰可增强HIV-1 RNA的翻译与剪接过程。然而，m5C是否对其他致病病毒的RNA发挥功能性增强作用，目前仍未明确。
本研究证实，乙型肝炎病毒（HBV）的RNA中富集高水平的m5C修饰，该修饰主要由细胞源性甲基转移酶NSUN2介导。有趣的是，NSUN2催化沉积的m5C最显著的簇集区域位于ε发夹结构——这一RNA元件是病毒RNA衣壳化与逆转录过程所必需的核心元件。通过敲低NSUN2以消除HBV RNA的m5C修饰，可导致病毒衣壳蛋白（HBc）的翻译水平小幅下降，却同时使逆转录生成的病毒DNA几乎完全丢失。类似地，对甲基化胞嘧啶位点进行突变以去除m5C修饰，同样会引发翻译水平下降并阻断逆转录过程。
此外，采用核苷类似物药理学阻断m5C修饰的沉积，可显著抑制HBV的复制水平。综上，本研究数据表明，m5C修饰是HBV ε元件逆转录过程的关键增强因子。本研究提示，靶向HBV 5’ε区域的m5C甲基化过程，有望成为新型抗病毒治疗策略。
整体实验设计：PA-m5C测序（PA-m5C-seq）：先将m5C抗体经光活化紫外交联结合至含m5C的RNA，随后进行免疫沉淀与高通量测序。实验所用RNA提取自转染了HBV复制子质粒、并用6-硫代鸟苷（6-thioguanosine, 6SG）处理的Huh-7细胞。