H5亚型禽流感病毒HA蛋白糖基化位点修饰诱导天然免疫差异性表达的机制研究

Glycosylation of hemagglutinin (HA) protein of H5N1 subtype avian influenza virus (AIV) plays an important role in its evolution life. Previous study showed that loss of glycosylation site at residue 158 or residue 169 on HA protein of H5N1 AIV would enhance the viral virulence to mice and induced higher expression of cytokine, however, the mechanism for differential expression on innate immune induced by the mutants remain unclear. The following experiments will be performed using A549 cells and THP-1 cells as model cells. First, we will compare the resistance of H5N1 AIV mutants with glycosylation site modification to collectins, and test the role of lectin receptor in inducing innate immune by the mutants. Second, we will compare expression of cellular TLR induced by the mutants and deterimine the TLR and critical proteins which can recognize the glycosylation site pattern on HA protein. Third, we will determine the critical proteins in the signal pathway against interferon receptor by the mutants. Fourth, we will determine the critical protein in endoplasmic reticulum stress pathway for stronger cytokine expression by the mutants. Finally, we will compare the virulence of the mutants in the knockout mice without the identified protein, and elucidate the role of the identified protein in mutants’ pathogenicity. This study will discover the critical pathway for differential expression of innate immune induced by the mutants, and provide new targets for drug design.

H5N1亚型禽流感病毒（AIV）血凝素（HA）蛋白的糖基化在病毒进化过程中起重要作用，前期研究表明，HA蛋白158位或169位糖基化位点的缺失可显著增强H5N1亚型AIV对小鼠的致病性，诱导产生更高水平的细胞因子，但诱导天然免疫差异性表达的机制还不清楚。本研究主要以A549和THP-1细胞为模式细胞，内容包括：（1）比较H5N1 AIV HA糖基化位点缺失株对凝集素的耐受能力，确定凝集素受体在缺失株诱导细胞天然免疫中的作用；（2）比较缺失株诱导细胞TLR表达能力，确定识别HA糖基化位点的TLR受体及关键蛋白；（3）确定缺失株拮抗干扰素受体通路中的关键蛋白；（4）确定缺失株引起更强烈的细胞内质网应激的关键蛋白；（5）以关键蛋白基因敲除小鼠测定病毒的毒力，明确该蛋白对HA糖基化位点缺失株毒力的影响。研究结果将解析HA糖基化位点诱导天然免疫差异性表达的关键路径，为药物设计提供新的靶标。

高致病性H5N1亚型AIV可通过改变HA蛋白上的糖基化修饰增加对哺乳动物的致病性，而HA蛋白糖基化位点的改变在调控AIV感染过程中的作用机制尚不清楚。本研究拟通过测定野生株及HA 158位和169位糖基化位点缺失株在体内外诱导细胞因子表达的差异，并研究其调控通路，明确HA糖基化位点影响细胞因子表达的机制；测定凝集素受体对糖基化位点缺失病毒感染的影响；测定病毒感染对干扰素下游信号通路和内质网应激通路的调控。结果显示（1）发现H5亚型AIV HA糖基化位点缺失株通过激活IRE1α通路增强内质网应激介导更强烈的炎症反应；靶向IRE1α磷酸化的Kira6可作为降低禽流感病毒诱导炎症因子风暴的有效药物。（2）发现H5亚型AIV HA糖基化位点改变会影响C型凝集素受体的抗病毒感染作用。（3）发现H5亚型AIV HA糖基化位点的缺失可通过TLR3受体来激活下游通路（4）发现H5亚型AIV HA糖基化位点的缺失能更快地降解干扰素受体，抑制早期ISG基因的表达，利于病毒的早期复制。（5）发现H5亚型AIV通过激活HSP47和TFE3通路引起细胞高尔基体应激，TFE3可作为抗流感病毒感染的靶标。本研究阐明了HA糖基化位点缺失的H5亚型AIV对哺乳动物致性增强的致病机理，鉴定出2个重要宿主靶标，对高致病性禽流感病毒感染哺乳动物后的预后干预具有重要意义。