Epitranscriptomic cytidine methylation of the Hepatitis B viral RNA encapsidation signal ensure the reverse transcription of viral RNA [PAR-CLIP]. Epitranscriptomic cytidine methylation of the Hepatitis B viral RNA encapsidation signal ensure the reverse transcription of viral RNA [PAR-CLIP]

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: PAR-CLIP: HuH-7 cells, transfected with HBV replicon plasmid along with FLAG-GFP or FLAG-NSUN2 expression plasmids, were lysed and subject to FLAG-antibody pulldown of proteins, the co-immunoprecipitated RNA isolated and sequenced.

表观转录组RNA修饰（epitranscriptomic RNA modifications）已成为调控细胞与病毒RNA命运及功能的重要机制。其中一类关键修饰为胞苷甲基化产物5-甲基胞苷（5-methylcytidine，m5C），该修饰存在于人类免疫缺陷病毒1型（HIV-1）的RNA中，可增强HIV-1 RNA的翻译与剪接过程。然而，m5C是否对其他致病性病毒的RNA同样具有功能增强作用仍不明确。本研究发现，乙型肝炎病毒（hepatitis B virus，HBV）的RNA中存在高水平的m5C修饰，该修饰主要由细胞内甲基转移酶NSUN2催化沉积。有趣的是，NSUN2介导的m5C最显著的富集簇位于ε发夹结构——一种对病毒RNA包装与逆转录至关重要的RNA元件。当通过敲低NSUN2消除HBV RNA的m5C修饰后，病毒衣壳蛋白（HBc）的翻译水平出现轻度下降，但同时逆转录生成的病毒DNA几乎完全消失。同样，通过定点突变去除甲基化胞苷位点后，也会导致翻译水平下降并阻断逆转录过程。此外，使用核苷类似物药物干扰m5C修饰的沉积，可显著降低HBV的复制水平。综上，本研究数据表明，m5C修饰是HBV逆转录过程中ε元件的关键增强因子。本研究提示，靶向HBV 5'ε区域的m5C甲基化过程可作为一种新型抗病毒治疗策略。实验设计：紫外交联核糖核苷增强交联免疫沉淀测序（Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation, PAR-CLIP）：将转染了HBV复制子质粒以及FLAG-GFP或FLAG-NSUN2表达质粒的HuH-7细胞裂解后，使用FLAG抗体进行蛋白下拉实验，分离并测序共免疫沉淀得到的RNA。