Strain Dependent Structure and Function of the Influenza NS1 Protein

开发有效的抗病毒药物需要详细了解靶向宿主-病原体相互作用的分子机制。具体地说，这些相互作用的精确结构模型可以提供具有原子分辨率的机械细节，以帮助高效开发抗重大健康相关病原体的新化合物。流感病毒就是这种病原体的一个典型例子。新的流感病毒株每年通过感染和在包括人类在内的许多动物宿主中复制而发展。这些毒株致病或致病的相对能力取决于病毒和细胞蛋白之间的一些相互作用。虽然已知流感非结构蛋白1(NS1)在毒力中发挥关键作用，但我们对促进NS1在宿主细胞中的多种毒株依赖功能的遗传和结构决定因素的了解存在根本差距。因此，了解NS1依赖于应变的功能的分子机制是我们的长期目标。这项应用的目的是从结构上表征与两种细胞蛋白(CPSF30和RIG-I)的相互作用，这两种蛋白对激活先天性免疫反应非常重要。我们的中心假设是，某些NS1变体特有的结构和动态特征解释了NS1所具有的不同功能。这项拟议研究的基本原理是，阐明NS1及其细胞相互作用伙伴之间的结构-功能关系将有助于开发针对这些已知调节毒力的关键相互作用的抗病毒药物。我们的中心假设将通过追求三个具体目标来验证：1)从结构和功能上表征NS1与RIG-I之间的多重相互作用；2)确定微秒级的S-ms运动在NS1效应域(NS1ED)的正常功能中的作用；以及3)确定JJ3297抑制流感复制的作用机制。在目标1中，核磁共振波谱和突变的重组流感病毒将被用来从结构和功能上表征NS1和RIG-I之间的多重相互作用。在目标2中，松弛弥散实验将被用来确定蛋白质动力学在促进NS1ED和CPSF30之间的相互作用和NS1的细胞内定位中所起的作用。在目标3中，核磁共振波谱将用于确定JJ3297抑制流感复制的作用机制。我们的创新方法将是第一次研究蛋白质动力学和菌株特定的结构变化如何在病毒复制和致病的背景下促进NS1的适当功能。这也将是确定多个流感病毒株之间NS1功能差异的第一项系统研究。这项拟议的研究意义重大，因为它将定义NS1调节流感毒力的潜在分子机制。通过定义这些分子机制，这项建议将为开发针对NS1的流感抗病毒药物的努力提供信息，从而支持NIH的总体任务。

Developing effective antiviral drugs requires a detailed understanding of the molecular mechanisms underlying the targeted host-pathogen interaction. Specifically, precise structural models of these interactions can provide mechanistic details with atomic resolution to assist in the efficient development of novel compounds against pathogens of significant health relevance. The influenza virus is a prime example of one such pathogen. Novel strains of the influenza virus develop annually via infection and replication in a number of animal hosts, including humans. The relative ability of these strains to cause disease, or virulence, is determined by a number of interactions between viral and cellular proteins. Although influenza non-structural protein 1 (NS1) is known to play a critical role in virulence, there is a fundamental gap in our knowledge of the genetic and structural determinants that facilitate the multiple strain-dependent functions attributed to NS1 in the host cell. It is therefore our long-term goal to understand the molecular mechanisms that underlie strain-dependent function of NS1. The objective of this application is to structurally characterize interactions with two cellular proteins (CPSF30 and RIG-I) that are important for the activation of the innate immune response. Our central hypothesis is that structural and dynamic features unique to certain NS1 variants account for the diverse array of functions attributed to NS1. The rationale that underlies the proposed research is that elucidating structure- function relationships between NS1 and its cellular interaction partners will aid in the development of antiviral drugs that target these critical interactions known to modulate virulence. Our central hypothesis will be tested by pursuing three specific aims: 1) structurally and functionally characterize the multiple interactions between NS1 and RIG-I, 2) determine the role of microsecond-millisecond (µs-ms) motions in proper function of the NS1 effector domain (NS1ED), and 3) determine the mechanism of action by which JJ3297 suppresses influenza replication. In Aim 1, NMR spectroscopy and mutant recombinant influenza viruses will be used to structurally and functionally characterize the multiple interactions between NS1 and RIG-I. In Aim 2, relaxation dispersion experiments will be used to determine the role that protein dynamics play in facilitating the interaction between the NS1ED and CPSF30 and intracellular localization of NS1. In Aim 3, NMR spectroscopy will be used to determine the mechanism of action by which JJ3297 suppresses influenza replication. Our innovative approach will be the first investigation into how protein dynamics and strain specific structural variations facilitate proper function of NS1 in the context of viral replication and pathogenicity. This will also be the first systematic study to determine functional variations in NS1 between multiple strains of influenza. The proposed research is significant because it will define the molecular mechanisms underlying NS1 functions shown to modulate influenza virulence. By defining these molecular mechanisms, this proposal will inform efforts in developing influenza antiviral drugs targeting NS1, thereby supporting the overall mission of the NIH.