MECHANISTIC UNDERSTANDING OF INFLUENZA-HOST INTERACTIONS FROM A ZOONOTIC PERSPECTIVE

项目摘要 甲型流感病毒是一种负链RNA病毒，根据抗原性分为亚型， 两种表面糖蛋白，即血凝素（HA）和神经氨酸酶（NA）的性质。有18 已知HA亚型（H1至H18）和11种已知NA亚型（N1至N11）。野生水鸟是主要的 甲型流感病毒的天然宿主。然而，天然水库的溢出可能会造成人类 流行病三种亚型（H1N1，H2 N2和H3 N2）引起了人类大流行，其中两种 (H1N1和H3 N2）目前正在人群中传播，导致季节性爆发。其他 在自然宿主中发现的亚型偶尔也会传播给人类，例如 H5 N1、H5 N6、H6 N1、H7 N7、H7N9、H9 N2和H10 N8病毒。这些人畜共患病亚型可以高度 致病性，并且当感染人类时具有高死亡率（>50%的住院个体）。病毒- 宿主蛋白质-蛋白质相互作用构成流感致病性的分子基础。许多“组学”研究 已经探索了流感与宿主的相互作用。然而，已知的机械细节还远没有 全面，特别是关于不同病毒亚型和宿主之间致病性的变化。 这项研究的目的是了解流感病毒的遗传变异和 寄主共同影响致病性。这项提案将集中在流感NS 1蛋白，这是一个主要的 致病性决定因子，并在宿主适应中发挥作用。已知NS 1与许多主机交互 proteins.其中一些相互作用已知是流感亚型依赖性的。在拟议的研究中， 将使用来自两种季节性亚型（H1N1和H3 N2）和一种人畜共患亚型（H5 N1）的NS 1。的 这三种NS 1在人类和禽类细胞中的功能限制的差异将通过以下方式来询问： 深度突变扫描和系统相互作用组分析（K99期）。随后，生物物理 将进行NS 1-TRIM 25相互作用的表征。NS 1与TRIM 25相互作用，TRIM 25是一种E3 泛素连接酶，以抑制先天免疫反应。NS 1-TRIM 25相互作用依赖于病毒亚型 和主机依赖性。潜在的生物物理机制将通过亲和力成熟和冷冻来探索。 电镜观察（K99/R 00相）。建议的研究将有助于详细了解流感 从分子生物学、遗传学、进化生物学和结构生物学等方面对病原性和嗜性进行了研究 视角更重要的是，本研究所建立的整合研究架构将适用于 其他流感病毒蛋白，甚至来自其他病毒的蛋白。

PROJECT SUMMARY Influenza A virus is a negative-strand RNA virus and is classified into subtypes based on the antigenic properties of the two surface glycoproteins, namely hemagglutinin (HA) and neuraminidase (NA). There are 18 known HA subtypes (H1 to H18) and 11 known NA subtypes (N1 to N11). Wild aquatic birds are the main natural reservoir for influenza A viruses. However, spillover from natural reservoirs can cause human pandemics. Three subtypes (H1N1, H2N2, and H3N2) have caused human pandemics, and two of them (H1N1 and H3N2) are presently circulating the in human population, causing seasonal outbreaks. Other subtypes found in natural reservoirs also occasionally transmit to the human population, as exemplified by H5N1, H5N6, H6N1, H7N7, H7N9, H9N2, and H10N8 viruses. These zoonotic subtypes can be highly pathogenic and have a high mortality rate (>50% of hospitalized individuals) when infecting humans. The virus- host protein-protein interaction constitutes the molecular basis of influenza pathogenicity. Many “omics” studies have explored influenza-host interactions. However, the known mechanistic details are far from comprehensive, especially regarding variation in pathogenicity across different virus subtypes and hosts. The objective of the proposed study is to understand how genetic variation in influenza virus and the host together influence pathogenicity. This proposal will focus on influenza NS1 protein, which is a major pathogenicity determinant and plays a role in host adaptation. NS1 is known to interact with many host proteins. Some of these interactions are known to be influenza subtype-dependent. In the proposed study, NS1 from two seasonal subtypes (H1N1 and H3N2) and one zoonotic subtype (H5N1) will be employed. The difference in the functional constraints of these three NS1 in human and avian cells will be interrogated by deep mutational scanning and systematic interactome analysis (K99 phase). Subsequently, biophysical characterization of NS1-TRIM25 interaction will be performed. NS1 interacts with TRIM25, which is an E3 ubiquitin ligase, to suppress the innate immune response. NS1-TRIM25 interaction is virus subtype-dependent and host-dependent. The underlying biophysical mechanism will be probed by affinity maturation and cryo- electron microscopy (K99/R00 phase). The proposed study will facilitate a detailed understanding of influenza pathogenicity and tropism from the molecular biology, genetics, evolutionary, and structural biology perspectives. More importantly, the integrative research framework developed in this study will be applicable to other influenza virus proteins, or even to proteins from other viruses.