Molecular Mechanisms of ZAP/ISG20 mediated HBV RNA Decay

描述(申请人提供)：干扰素是一种获得批准的治疗慢性乙型肝炎的药物。在筛选干扰素刺激基因(ISG)以研究其潜在的抗病毒活性时，我们确定了两种宿主蛋白，特别是锌指抗病毒蛋白(ZAP)和ISG20，它们主要通过转录后下调病毒RNA来抑制乙肝病毒的复制。我们的数据还表明，ZAP和ISG20都与HBVRNA发生物理作用；虽然ZAP介导的HBVRNA降解需要四个CCCH型锌指基序的完整性，但ISG20对HBVRNA的降解依赖于其核糖核酸酶活性。有趣的是，ISG20的酶失活形式通过与病毒前基因组(PG)RNA相互作用，随后干扰pgRNA的包裹，保持了抗病毒复制的活性。此外，ZAP和ISG20都在肝细胞的基础水平表达，并在干扰素刺激下被进一步诱导，这表明它们在限制宿主对乙肝病毒的作用中具有潜在的作用。在这里，我们建议进一步阐明ZAP/ISG20介导的HBVRNA衰退的分子机制。建议的目标包括：(1)将确定负责ZAP和ISG20介导的RNA降解的HBVRNA序列元件(S)；(2)将确定ZAP和ISG20在天然免疫诱导的抗HBVRNA应答中的生理作用；(3)将通过蛋白质组学方法确定ZAP和ISG20介导的HBVRNA衰退中的辅因子(S)，并将研究ZAP和ISG20之间潜在的合作作用；(4)确定ZAP和ISG20靶向的潜在宿主RNA分子，包括信使核糖核酸和miRNA。阐明ZAP和ISG20介导的复杂细胞抗病毒反应的分子机制将有助于阐明病毒致病过程中的细胞生物学和病毒-宿主相互作用，并有可能导致 利用宿主分子控制病毒感染的新疗法的开发。

DESCRIPTION (provided by applicant): Interferon- ¿ is an approved medication for chronic hepatitis B. In the screening of interferon stimulated genes (ISG) for their potential antiviral activity against HBV replication, we identified two host proteins, specifically zinc finger antivirl protein (ZAP) and ISG20, which inhibit HBV replication primarily through posttranscriptional downregulation of viral RNA. Our data also demonstrated that both ZAP and ISG20 physically interact with HBV RNA; while the integrity of the four CCCH-type zinc finger motifs is required for ZAP-mediated HBV RNA degradation, the reduction of HBV RNA by ISG20 depends on its ribonuclease activity. Interestingly, the enzymatically inactive form of ISG20 retains antiviral activity against HBV replication by interacting with viral pregenomic (pg) RNA and subsequently interfering with pgRNA encapsidation. In addition, both ZAP and ISG20 are expressed at basal levels in hepatocytes, and can be further induced upon interferon stimulation, indicating their potential roles in host restriction of HBV. Here we propose to further elucidate the molecular mechanisms of ZAP/ISG20-mediated HBV RNA decay. The proposed Aims include: (1) The sequence element(s) of HBV RNA responsible for ZAP- and ISG20-mediated RNA degradation will be identified; (2) the physiologic role of ZAP and ISG20 in innate immunity-elicited anti-HBV responses will be determined; (3) co- factor(s) in ZAP- and ISG20-mediated HBV RNA decay will be identified through a proteomic approach, and the potential cooperative role between ZAP and ISG20 will also be investigated; (4) identification of the potential host RNA molecules targeted by ZAP and ISG20, including mRNA and miRNA. Elucidation of the molecular mechanisms of the intricate cellular antiviral response mediated by ZAP and ISG20 will shed light on cell biology and virus-host interaction during viral pathogenesis, and potentially lead to the development of novel therapeutics that exploit host molecules to control viral infection.