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

甲型流感病毒（IAV）是人类严重呼吸道疾病的病原体，具有高发病率和死亡率。今天，只有两类抗病毒药物可用于治疗急性流感感染。针对这些药物的耐药病毒的快速产生是令人担忧的，并且对于未来抗病毒药物的开发，迫切需要抗病毒方法的新靶点。在IAV感染期间，病毒基因组的复制以及病毒mRNA的合成通过病毒RNA依赖性RNA聚合酶在复杂且高度调控的过程中完成。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标。最近的证据表明，细胞内的泛素-蛋白酶体系统通过泛素与病毒聚合酶蛋白中的赖氨酸残基的翻译后连接在病毒复制和转录过程中起着重要的调节作用。使用质谱分析，我们能够确定54泛素化赖氨酸在感染细胞的IAV聚合酶。其中，9个赖氨酸不仅在IAV的聚合酶中高度保守，而且在流感B和C病毒中也是保守的，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需的。IBV聚合酶的已发表的晶体结构的基础上的结构建模显示，许多修饰的赖氨酸位于聚合酶蛋白的功能相关区域。这包括蛋白质相互作用、RNA启动子结合、帽结合、核定位信号和PA内切核酸酶的活性中心的已知区域。然而，这些修饰对调节聚合酶活性的功能相关性尚不清楚。本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，并揭示其潜在的功能机制。使用siRNA-以及抑制剂的研究，在细胞培养中，我们的目标是确定细胞E3连接酶负责与调节功能的修改。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，并为基于靶向特异性抑制人肺中细胞E3连接酶的新型抗病毒方法奠定基础。

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.