干扰素诱导型核糖核酸酶ISG20通过直接结合到病毒RNA的ε茎干

StephenW

PLoS Pathog. 2017 Apr 11;13(4):e1006296. doi: 10.1371/journal.ppat.1006296. eCollection 2017.
Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA.
Liu Y1, Nie H2, Mao R1, Mitra B1, Cai D1, Yan R1, Guo JT3, Block TM3, Mechti N4, Guo H1.
Author information

1    Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
2    Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States of America.
3    Baruch S. Blumberg Institute, Doylestown, Pennsylvania, United States of America.
4    CNRS, UMR5235, DIMNP, University of Montpellier 2, Montpellier, France.

Abstract

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.

PMID:
    28399146
DOI:
    10.1371/journal.ppat.1006296

StephenW

PLoS Pathog。 2017年4月11日; 13（4）：e1006296。 doi：10.1371 / journal.ppat.1006296。 eCollection 2017。
干扰素诱导型核糖核酸酶ISG20通过直接结合到病毒RNA的ε茎干环结构来抑制乙型肝炎病毒复制。
刘Y1，聂H2，毛泽东，米特拉B1，蔡D1，阎R1，郭吉泰，TM3，Mechti N4，郭H1。
作者信息

1美利坚合众国印第安纳州印第安纳州印第安纳波利斯印第安纳大学医学院微生物与免疫学系。
2美国宾夕法尼亚州费城德累斯顿大学医学院微生物与免疫学系。
3 Baruch S. Blumberg Institute，Doylestown，Pennsylvania，United States of America。
4 CNRS，UMR5235，DIMNP，蒙彼利埃大学2，蒙彼利埃，法国。

抽象

乙型肝炎病毒（HBV）通过病毒RNA pregenome的逆转录复制其DNA基因组。我们在这里报道，干扰素（IFN）刺激20KD（ISG20）的核糖核酸外切酶基因通过HBV RNA的降解来抑制HBV复制。在基础水平观察到ISG20表达，并且在肝细胞中IFN治疗后高度上调，并且ISG20的敲低导致HBV复制升高和IFN介导的抗病毒作用的减弱。赋予HBV RNA对ISG20介导的RNA降解的敏感性的序列元件被绘制在含有ε（ε）茎环的HBV RNA末端冗余区域。此外，ISG20诱导的HBV RNA降解依赖于其核糖核酸酶活性，因为酶活性形式ISG20D94G不能促进HBV RNA衰变。有趣的是，ISG20D94G通过阻止由于ISG20-ε相互作用的结果而导致的pgRNA包封，保留了对HBV DNA复制的抗病毒活性。这种相互作用的进一步特征在于体外电泳迁移率变动分析（EMSA），ISG20能够在没有任何其他细胞蛋白的情况下直接结合HBVε，表明ISG20的直接εRNA结合能力;然而，ISG20可能需要辅因子来有效地降解ε。此外，ε的下部部分是主要的ISG20结合位点，并且从ε的底部去除4个碱基对消除了HBV RNA对ISG20的敏感性，表明ISG20-ε相互作用的特异性依赖于RNA结构和序列。此外，ISG20的C末端核酸外切酶III（ExoIII）结构域被确定为负责与ε相互作用，因为ExoIII的缺失在体外ISG20-ε结合和细胞内HBV RNA降解中被消除。总之，我们的研究揭示了IFN介导的HBV抑制的基本机制和ISG20的抗病毒机制。

PMID：
    28399146
DOI：
    10.1371 / journal.ppat.1006296