高致病性H5N8禽流感（AI）的爆发给养禽业带来巨大经济损失，对人类健康也存在威胁。前期研究表明PB2-I283M-K526R联合突变增强了其对小鼠的致病性，而PB2基因这种适应性突变的致病机理尚不清楚。本研究旨在通过挖掘病毒感染过程中与PB2蛋白相互作用的宿主蛋白，为新型药物的设计提供靶标。研究内容主要包括：（1）通过RNA sequencing分析比较模式病毒在小鼠肺脏中基因的表达差异；（2）稳定表达外源标签模式病毒的拯救与鉴定；（3）通过亲和层析结合液相色谱-串联质谱筛选与模式病毒PB2相关的差异蛋白；（4）使用GST-pull down, co-IP和激光共聚焦方法对PB2与宿主细胞蛋白相互作用验证；（5）通过过表达、小RNA干扰和CRISPR/Cas9系统探索选择的宿主因子或蛋白在流感病毒复制周期中的具体功能，是如何对流感病毒复制及其病毒聚合酶活性产生影响的。

Outbreaks of HPAI H5N8 virus have caused significant economic losses to the poultry industry, and more importantly, its potential threat to public health cannot be neglected. Our previous study demonstrated that the synergistic effect of 283M and 526R in PB2 may enhance replication of an avian influenza virus in mice through enhanced polymerase activity. Therefore, it is crucial to explore the molecular basis and host mechanism for the high virulence of H5N8 AIV in mammals. The following experiments will be performed.(1) we will analyze the differences of the expression of gene expression in mice lung by RNA sequencing. (2) we will construct and identify the recombinant AIVs with His tag insertion in its PB2-coding region based up a pair of AIVs that differ greatly in their virulence in mice. (3)We will identify cellular proteins interacting with PB2 by affinity chromatography and LC-MS/MS and bioinformatics. (4)We will confirm the interaction of the cellular protein and PB2 protein by GST-pull down, co-immunoprecipitation and confocal microscopy assays. (5) We will explore the specific functions of selected host factors or proteins in influenza virus replication cycle by over-expression, siRNA and CRISPR/Cas9 system, and how they affect influenza virus replication and its polymerase activity.