MECHANISTIC UNDERSTANDING OF INFLUENZA-HOST INTERACTIONS FROM A ZOONOTIC PERSPECTIVE

项目总结 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个 已知的HA亚型(H1至H18)和11个已知的NA亚型(N1至N11)。野生水鸟是主要的 甲型流感病毒的天然蓄水池。然而，天然水库的溢出会导致人类 大流行。三种亚型(H1N1、H2N1和H3N2型)导致了人类大流行，其中两种 (H1N1和H3N2型)目前在人群中传播，造成季节性暴发。其他 在天然水库中发现的亚型有时也会传播给人类，例如 H5N1、H5N6、H6N1、H7N7、H7N9、H9N2和H10N8病毒。这些人畜共患病的亚型可以高度 在感染人类时具有致病性并有很高的死亡率(50%的住院患者)。病毒- 宿主蛋白之间的相互作用构成了流感致病的分子基础。许多“组学”研究 已经探索了流感与宿主的相互作用。然而，已知的机制细节远远不是 全面，特别是关于不同病毒亚型和宿主之间的致病性差异。 这项拟议研究的目的是了解流感病毒的基因变异和流感病毒 寄主共同影响致病力。这项建议将集中在流感NS1蛋白上，这是一种主要的 致病性决定因素，并在宿主适应中发挥作用。已知NS1与许多宿主相互作用 蛋白质。这些相互作用中的一些已知是流感亚型依赖的。在拟议的研究中， 将使用来自两个季节性亚型(H1N1和H3N1)和一个人畜共患亚型(H5N1)的NS1。这个 这三个NS1在人和禽类细胞中的功能限制的差异将被询问 深度突变扫描和系统交互作用组分析(K99期)。随后，生物物理 将对NS1-TRIM25相互作用进行表征。NS1与TRIM25交互，TRIM25是E3 泛素连接酶，抑制先天免疫反应。NS1-TRIM25相互作用依赖于病毒亚型 并且依赖于宿主。其潜在的生物物理机制将通过亲和力成熟和冷冻来探讨。 电子显微镜(K99/R00相)。建议的研究将有助加深对流感的详细认识。 来自分子生物学、遗传学、进化生物学和结构生物学的致病性和趋向性 透视。更重要的是，本研究开发的综合研究框架将适用于 其他流感病毒蛋白质，甚至是来自其他病毒的蛋白质。

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.