Regulating Influenza Polymerase Structure and Function by Phosphorylation

描述（由申请人提供）：蛋白质功能受翻译后事件动态控制。一种常见的调节机制是靶蛋白的磷酸化和随后的构象重排。病毒蛋白质通常由靶向宿主编码的蛋白质的相同途径控制。甲型流感病毒的复制周期受宿主蛋白质的影响，并提供了一个独特的系统来研究这些过程。调控的主要目标是流感病毒复制机制，其包含病毒RNA、核蛋白（NP）和由蛋白质PB1、PB2和PA组成的三聚体聚合酶。复制机制控制着从基因表达到基因组复制的有序过渡，这对生产性感染至关重要。此外，病毒聚合酶是流感病毒宿主范围内的关键决定因素，限制了流感病毒从禽类向人类的传播。控制复制机制的过程以及聚合酶与宿主之间的相互作用还知之甚少。我们提出了一个综合的方法，利用生物化学，遗传学和结构的研究，以确定宿主蛋白质和分子机制，调节流感复制机制。具体来说，目标1将识别病毒蛋白质内的磷酸化位点，并检查磷酸化对病毒复制的功能后果以及宿主范围的确定。目标2将使用遗传作图和高分辨率晶体结构来详细描述复制复合物的蛋白质界面，并阐明伴随复合物组装的构象变化。最后，Aim 3将利用功能丧失筛选来鉴定调节流感聚合酶和控制流感从鸟类传播到人类的细胞蛋白。重要的是，每个目标都为培训应用程序独立阶段所必需的新技术提供了重要机会。这些研究将为控制流感复制的病毒和宿主因素提供重要的见解，并为治疗和预防未来人类流感暴发的合理策略提供基础。 相关性：流感病毒复制机制是确定感染和确定其致病性的关键因素。这些研究将深入了解流感复制机制的调节，它如何控制流感病毒从鸟类传播到人类，并可能最终确定治疗干预的新靶点。

DESCRIPTION (provided by applicant): Protein function is dynamically controlled by post-translation events. A common regulatory mechanism is the phosphorylation and subsequent conformational rearrangement of target proteins. Viral proteins are often controlled by the same pathways that target proteins encoded by the host. The replication cycle of influenza A virus is influenced by host proteins and provides a unique system in which to study these processes. A major target of regulation is the influenza virus replication machinery containing viral RNA, nucleoprotein (NP), and the trimeric polymerase composed of the proteins PB1, PB2, and PA. The replication machinery controls the ordered transition from gene expression to genome replication that is essential for a productive infection. In addition, the viral polymerase is a key determinant in the host range of influenza virus and restricts the transmission of influenza virus from avian to human populations. The processes controlling the replication machinery and the interplay between the polymerase and the host are poorly understood. We propose an integrative approach using biochemical, genetic, and structural studies to determine the host proteins and molecular mechanisms that regulate the influenza replication machinery. Specifically, Aim 1 will identify sites of phosphorylation within viral proteins and examine the functional consequences of phosphorylation on virus replication and the determination of host range. Aim 2 will use genetic mapping and high-resolution crystal structures to characterize in detail the protein interfaces of the replication complex and elucidate the conformational changes that accompany complex assembly. Finally, Aim 3 will exploit loss-of-function screens to identify cellular proteins that regulate the influenza polymerase and control transmission of influenza from birds to humans. Importantly, each Aim presents significant opportunities for training in new techniques essential for the Independent phase of the application. These studies will provide crucial insight into the viral and host factors controlling influenza replication and will provide the foundation for rational strategies to treat and prevent future influenza outbreaks in humans. Relevance: The influenza virus replication machinery is a key player in establishing infection and determining its pathogenicity. These studies will provide insight into the regulation of the influenza replication machinery, how it controls transmission of influenza virus from birds to humans, and may ultimately identify new targets for therapeutic intervention.