Regulating Influenza Polymerase Structure and Function by Phosphorylation

蛋白质的功能是由翻译后事件动态控制的。一种常见的调节 其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， 在这个系统中，我们可以研究这些过程。监管的一个主要目标是流感病毒 包含病毒RNA、核蛋白（NP）和三聚体聚合酶的复制机制 由蛋白质PB1、PB2和PA组成。复制机器控制着 从基因表达到基因组复制的转变，这对生产性感染至关重要。在 此外，病毒聚合酶是流感病毒宿主范围内的关键决定因素， 流感病毒从禽类传播到人类。控制的过程 复制机制和聚合酶与宿主之间的相互作用知之甚少。 我们提出了一个综合的方法，利用生物化学，遗传学和结构研究，以确定 调节流感复制机制的宿主蛋白质和分子机制。 具体来说，目标1将确定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影响和宿主范围的确定。 AIM2将建立在最近的相互作用遗传图谱的基础上，以产生高分辨率的晶体 详细描述复制复合物的蛋白质界面的结构。第三，目标 将利用功能丧失筛选来识别调节流感聚合酶的细胞蛋白 并控制禽流感从鸟类向人类的传播。这些研究将提供重要的见解 控制流感复制的病毒和宿主因素，并将为 合理的策略来治疗和预防未来的人类流感爆发。

Protein function is dynamically controlled by post-translation events. A connmon 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 ofthe 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 build on recent genetic mapping of interactions to produce high-resolution crystal structures that characterize in detail the protein interfaces of the replication complex. 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. 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.