Regulating Influenza Polymerase Structure and Function by Phosphorylation

描述（由申请人提供）：蛋白质的功能是由翻译后事件动态控制的。常见的调控机制是靶蛋白的磷酸化和随后的构象重排。病毒蛋白通常与宿主编码的靶蛋白受相同途径的控制。甲型流感病毒的复制周期受到宿主蛋白的影响，并为研究这些过程提供了一个独特的系统。调控的一个主要目标是流感病毒复制机制，其中包含病毒RNA、核蛋白（NP）和由蛋白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，这是生产感染所必需的。此外，病毒聚合酶是流感病毒宿主范围的关键决定因素，并限制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尚不清楚。我们提出一种综合方法，利用生化、遗传和结构研究来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，Aim 1将确定病毒蛋白磷酸化位点，并检查磷酸化对病毒复制的功能后果和宿主范围的确定。目标2将使用遗传作图和高分辨率晶体结构来详细表征复制复合体的蛋白质界面，并阐明伴随复合体组装的构象变化。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向人类传播的细胞蛋白。重要的是，每个Aim都为应用程序独立阶段所必需的新技术培训提供了重要的机会。这些研究将对控制流感复制的病毒和宿主因素提供重要的见解，并将为治疗和预防未来人类流感暴发的合理策略提供基础。

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.