细胞溶酶体降解A型流感病毒HA蛋白的机制研究

The degradation mechanism of influenza A virus hemagglutinin protein mediated by the lysosomal pathway

为研究A型流感病毒HA蛋白在宿主细胞内的降解通路及其机制,本研究首先将表达流感病毒H5亚型HA蛋白的重组质粒和内质网应激通路相关分子XBP1共转染293T细胞,通过检测荧光素酶活性分析HA蛋白引起细胞的内质网应激水平。结果显示HA蛋白在细胞内的表达能够引起内质网应激。进一步进行药物抑制试验,结果显示部分HA蛋白通过溶酶体进行降解。采用RNA干扰、ELISA、药物处理等相关试验对溶酶体降解的3条途径逐一分析,结果显示HA蛋白未通过分子伴侣介导的自噬途径、内吞途径以及巨自噬途径降解。利用表达细胞凝集结构标志蛋白与表达HA蛋白的重组质粒共转染293T细胞,通过观察并经荧光定量,发现HA蛋白在细胞内过表达时发生凝集,随后通过溶酶体降解。本研究结果表明HA蛋白在宿主细胞内错误折叠并诱发内质网应激,错误折叠的蛋白形成凝集结构后通过溶酶体降解。本研究结果初步阐明了HA蛋白通过溶酶体降解的机制,为开发新的抗病毒手段奠定了基础。

To study the degradation mechanism of influenza A virus hemagglutinin(HA) protein in host cells, the recombinant plasmid encoding H5 subtype HA of influenza virus and endoplasmic reticulumstress-related molecule XBP1 protein were co-transfected in 293 T cells, and then the luciferase activity of the cell lysates was determined as a measure of the level of ER stress. The results showed that expression of HA protein in 293 T cells induced ER stress;it was further found that HA protein was paritially degraded by lysosomes. To illuminate the HA protein degradation mechanism by lysosome, the RNA interference,ELISA, and drug treatment were respectively carried out to analyze the three classical lysosomal degradation pathways. Results indicated that HA protein degradation was not mediated by the pathway of chaperone-mediated autophagy(CMA), endocytosis,and macroautophagy. Finally, we found that the misfolding HA protein formed aggregate in 293 T cells co-transfected with GFP-250(protein aggregate marker) and HA, and then was degraded by lysosomes according to the analysis of the fluorescence signal observation and quantification. The above results in this study preliminarily elucidated the degradation mechanism of HA protein by lysosomes, which would provide scientific data for the development of antiviral approach.