Molecular Mechanisms of ZAP/ISG20 mediated HBV RNA Decay

描述(申请人提供)：干扰素-a是一种被批准的治疗慢性乙型肝炎的药物。在干扰素刺激基因(ISG)的筛选中，我们发现了两种宿主蛋白，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，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的酶失活形式仍然通过干扰病毒前基因组RNA被包裹到核衣壳中而保持着抗病毒复制的活性。此外，ZAP和ISG20在肝细胞中都有基础水平的表达，并可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。在这项应用中，我们将进一步阐明ZAP/ISG20介导的HBVRNA衰退的分子机制，拟议的目标包括：(1)将确定HBVRNA与ZAP/ISG20之间的潜在物理作用；(2)将确定HBVRNA中负责ZAP和ISG20介导的RNA降解的序列元件(S)；(3)ZAP和ISG20在干扰素诱导的抗乙肝应答中的生理作用；(4)将通过蛋白质组方法鉴定ZAP和ISG20介导的HBVRNA衰退中的辅因子(S)，以及ZAP和ISG20之间潜在的合作作用。了解ZAP和ISG20介导的这种复杂的细胞抗病毒反应的分子机制将促进对病原体和宿主细胞在病毒致病过程中如何相互作用的理解，并有可能为利用这些宿主细胞内分子控制慢性乙肝病毒感染的新疗法的开发开辟新的平台。

DESCRIPTION (provided by applicant): Interferon-a 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 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 still retains antiviral activity against HBV replication by interfering vira pregenomic RNA encapsidation into nucleocapsid. In addition, both ZAP and ISG20 have basal level expressions in hepatocytes, and can be further induced upon interferon stimulation, indicating their potential roles in host restriction of HBV. In this application we will further elucidate the molecular mechanisms of ZAP/ISG20 mediated HBV RNA decay, the proposed Aims include: (1) The potential physical interaction between HBV RNA and ZAP/ISG20 will be determined; (2) The sequence element(s) within HBV RNA responsible for ZAP and ISG20 mediated RNA degradation will be identified; (3) The physiologic role of ZAP and ISG20 in IFN elicited anti-HBV response will be determined; (4) Co-factor(s) in ZAP and ISG20 mediated HBV RNA decay will be identified through proteome approach, and the potential cooperative role between ZAP and ISG20 will also be studied. Understanding the molecular mechanisms of such intricate cellular antiviral response mediated by ZAP and ISG20 will advance the understanding of how pathogen and host cell interact during viral pathogenesis, and potentially open a new venue for development of novel therapeutics that exploit such host intracellular molecules to control chronic HBV infection.