Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of a viral RNA pregenome. We report herein that the interferon (IFN) stimulated exoribonuclease gene of 20 KD (ISG20) inhibits HBV replication through degradation of HBV RNA. ISG20 expression was observed at basal level and was highly upregulated upon IFN treatment in hepatocytes, and knock down of ISG20 resulted in elevation of HBV replication and attenuation of IFN-mediated antiviral effect. The sequence element conferring the susceptibility of HBV RNA to ISG20-mediated RNA degradation was mapped at the HBV RNA terminal redundant region containing epsilon (ε) stem-loop. Furthermore, ISG20-induced HBV RNA degradation relies on its ribonuclease activity, as the enzymatic inactive form ISG20D94G was unable to promote HBV RNA decay. Interestingly, ISG20D94G retained antiviral activity against HBV DNA replication by preventing pgRNA encapsidation, resulting from a consequence of ISG20-ε interaction. This interaction was further characterized by in vitro electrophoretic mobility shift assay (EMSA) and ISG20 was able to bind HBV ε directly in absence of any other cellular proteins, indicating a direct ε RNA binding capability of ISG20; however, cofactor(s) may be required for ISG20 to efficiently degrade ε. In addition, the lower stem portion of ε is the major ISG20 binding site, and the removal of 4 base pairs from the bottom portion of ε abrogated the sensitivity of HBV RNA to ISG20, suggesting that the specificity of ISG20-ε interaction relies on both RNA structure and sequence. Furthermore, the C-terminal Exonuclease III (ExoIII) domain of ISG20 was determined to be responsible for interacting with ε, as the deletion of ExoIII abolished in vitro ISG20-ε binding and intracellular HBV RNA degradation. Taken together, our study sheds light on the underlying mechanisms of IFN-mediated HBV inhibition and the antiviral mechanism of ISG20 in general.乙型肝炎病毒（Hepatitis B virus, HBV）通过病毒RNA前体基因组的逆转录过程复制其DNA基因组。本研究报道，20千道尔顿干扰素刺激外切核糖核酸酶基因（ISG20）可通过降解HBV RNA来抑制乙型肝炎病毒的复制。在肝细胞中，ISG20呈基础水平表达，且经干扰素（interferon, IFN）处理后表达显著上调；敲低ISG20会导致HBV复制水平升高，并削弱干扰素介导的抗病毒效应。介导HBV RNA对ISG20介导的RNA降解敏感的序列元件，被定位在HBV RNA的末端冗余区域，该区域包含ε茎环结构。此外，ISG20诱导的HBV RNA降解依赖于其核糖核酸酶活性，因为酶活性失活的突变体ISG20D94G无法促进HBV RNA降解。有趣的是，ISG20D94G仍可通过阻断前基因组RNA（pregenomic RNA, pgRNA）的衣壳化，保留针对HBV DNA复制的抗病毒活性，这一效应源于ISG20与ε的相互作用。我们进一步通过体外电泳迁移率变动分析（electrophoretic mobility shift assay, EMSA）表征了这一相互作用：在无其他细胞蛋白存在的情况下，ISG20可直接结合HBV ε RNA，表明ISG20具备直接结合ε RNA的能力；但ISG20要高效降解ε RNA，可能需要辅助因子的参与。此外，ε茎环的下部茎区是ISG20的主要结合位点；若从ε的底部区域移除4个碱基对，则会使HBV RNA丧失对ISG20的敏感性，这表明ISG20与ε的相互作用特异性同时依赖RNA结构与序列。进一步研究发现，ISG20的C端外切核酸酶III（Exonuclease III, ExoIII）结构域是其与ε相互作用的关键区域：缺失ExoIII结构域会消除体外ISG20与ε的结合能力，以及细胞内HBV RNA的降解效应。综上，本研究阐明了干扰素介导的乙型肝炎病毒抑制的潜在机制，同时也为ISG20的广谱抗病毒作用机制提供了新的理论见解。