Regulation of the influenza A virus polymerase activity by post-translational ubiquitin-modification

通过翻译后泛素修饰调节甲型流感病毒聚合酶活性

• 批准号: 386496948
• 负责人: Dr. Linda Brunotte
• 金额: --
• 依托单位: Abteilung für Molekulare Virologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/386496948?language=en
• 关键词: Regulation; influenza; virus; polymerase; activity

Influenza A viruses (IAV) are the causative agent of severe respiratory disease with high morbidity and mortality in humans. Today, only two classes of antiviral drugs are available for the treatment of acute influenza infections. Rapid generation of drug resistant viruses against these drugs is worrisome and new targets for antiviral approaches are urgently needed for the development of future antiviral drugs. During IAV infection replication of the viral genome as well as the synthesis of viral mRNA are accomplished by the viral RNA-dependent RNA polymerase in a complex and highly regulated processes. Therefore, the viral polymerase provides a vulnerable target for novel approaches to develop highly effective antiviral drugs. Recent evidence suggests that the cellular ubiquitin-proteasome system plays an important role in the regulation of viral replication and transcription processes by post-translational linkage of ubiquitin to lysine residues in the proteins of the viral polymerase. Using mass spectrometry we were able to identify 54 ubiquitylated lysines in the IAV polymerase in infected cells. Of these, 9 lysines are not only highly conserved in the polymerase of IAV but are also conserved in influenza B and C viruses, indicating, that these residues, and potentially their ubiquitin-modification, are essential for the enzymatic activity of the viral polymerase. Structure modeling on the basis of the published crystal structure of the IBV polymerase revealed that many of the modified lysines are located in functionally relevant regions of the polymerase proteins. This includes known regions for protein-interactions, RNA-promoter binding, cap-binding, nuclear localization signals and the active center of the PA-endonuclease. However, the functional relevance of these modifications for the regulation of the polymerase activity are yet unknown. The aim of this project is to determine which ubiquitylation sites are involved in the regulation of the activity of the viral polymerase and to unravel the underlying functional mechanisms. Using siRNA- as well as inhibitor studies in cell culture, we aim to identify the cellular E3 ligases responsible for the modifications with regulatory functions. Finally, we will perform inhibitor studies in a newly established human lung tissue system in our laboratory and lay the basis for novel antiviral approaches based on the target-specific inhibition of cellular E3 ligases in the human lung.

甲型流感病毒(IAV)是人类严重呼吸道疾病的病原体，具有较高的发病率和死亡率。今天，只有两类抗病毒药物可用于治疗急性流感感染。对这些药物迅速产生抗药性病毒令人担忧，未来抗病毒药物的开发迫切需要新的抗病毒方法的靶点。在IAV感染过程中，病毒基因组的复制以及病毒mRNA的合成是由依赖于病毒RNA的RNA聚合酶在一个复杂和高度调控的过程中完成的。因此，病毒聚合酶为开发高效抗病毒药物的新方法提供了一个脆弱的靶点。最近的证据表明，细胞泛素-蛋白酶体系统通过翻译后泛素与病毒聚合酶蛋白中赖氨酸残基的连接，在病毒复制和转录过程中发挥重要作用。利用质谱仪，我们能够在感染细胞中鉴定出IAV聚合酶中的54种泛素化赖氨酸。其中，9个赖氨酸不仅在IAV的聚合酶中高度保守，而且在B型和C型流感病毒中也保守，这表明这些残基及其潜在的泛素修饰对于病毒聚合酶的酶活性是必不可少的。根据已发表的IBV聚合酶的晶体结构进行的结构模拟显示，许多修饰的赖氨酸位于聚合酶蛋白的功能相关区域。这包括已知的蛋白质相互作用区域、RNA启动子结合区域、帽结合区域、核定位信号区域和PA-内切酶的活性中心。然而，这些修饰对聚合酶活性调节的功能相关性尚不清楚。这个项目的目的是确定哪些泛素化位点参与了病毒聚合酶活性的调节，并揭示了潜在的功能机制。利用siRNA-以及细胞培养中的抑制剂研究，我们的目标是确定负责具有调节功能的修饰的细胞E3连接酶。最后，我们将在我们实验室新建立的人肺组织系统中进行抑制剂研究，并为基于靶向抑制人肺细胞E3连接酶的新的抗病毒方法奠定基础。