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。具体而言，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制细节，以帮助有效开发针对具有显著健康相关性的病原体的新型化合物。流感病毒就是这种病原体的一个最好的例子。流感病毒的新毒株每年通过在包括人类在内的许多动物宿主中感染和复制而发展。这些菌株引起疾病的相对能力或毒力由病毒和细胞蛋白之间的许多相互作用决定。虽然已知流感病毒非结构蛋白1（NS 1）在毒力中起关键作用，但我们对促进宿主细胞中归因于NS 1的多种菌株依赖性功能的遗传和结构决定因素的认识存在根本性差距。因此，我们的长期目标是了解NS 1的应变依赖性功能的分子机制。本申请的目的是从结构上表征与两种细胞蛋白（CPSF 30和RIG-1）的相互作用，这两种蛋白对先天免疫应答的激活至关重要。我们的中心假设是，结构和动力学特征独特的某些NS 1的变种帐户的各种各样的功能归因于NS 1。拟议研究的基本原理是，阐明NS 1与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特定目标进行测试：1）在结构和功能上表征NS 1和RIG-I之间的多重相互作用，2）确定微秒-毫秒（µs-ms）运动在NS 1效应结构域（NS 1 ED）正常功能中的作用，3）确定JJ 3297抑制流感复制的作用机制。在目标1中，NMR光谱和突变重组流感病毒将用于结构和功能表征NS 1和RIG-I之间的多重相互作用。在目标2中，松弛分散实验将用于确定蛋白质动力学在促进NS 1 ED和CPSF 30之间的相互作用和NS 1的细胞内定位中所起的作用。在目标3中，NMR光谱将用于确定JJ 3297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异性结构变异如何在病毒复制和致病性的背景下促进NS 1的适当功能。这也将是第一个确定多种流感病毒株之间NS 1功能变异的系统性研究。这项拟议中的研究意义重大，因为它将确定NS 1调节流感毒力功能的分子机制。通过定义这些分子机制，该提案将为开发针对NS 1的流感抗病毒药物提供信息，从而支持NIH的整体使命。

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.