禽流感病毒M2蛋白抑制细胞自噬体降解的研究

摘要

为探究禽流感病毒(AIV)M2蛋白对细胞自噬的影响,本研究构建了pEGFP-LC3和pCAF-M2重组质粒,并采用脂质体将这两种重组表达质粒共转染293T细胞或Hela细胞中,同时以单独转染这两种重组质粒的细胞作为对照组,利用间接免疫荧光和western blot的方法检测细胞自噬体及自噬流情况.自噬体检测结果表明,AIV M2蛋白能够诱导转染细胞内自噬体积累.通过LC3-Ⅱturnover试验、mRFP-GFP-LC3双荧光指示系统以及pEGFP-LC3与LAMP1的共定位检测进一步证明,M2蛋白通过阻断自噬体与溶酶体融合,抑制溶酶体对自噬体的降解从而诱导自噬体积累.本研究揭示AIV M2蛋白对细胞自噬的发生具有重要作用,为深入研究自噬在A型流感病毒复制中的调节作用奠定基础.%Autophagy, as a self-protective mechanism of cells, plays an important role in host cells to resist virus infection. To explore the effects of avian influenza virus (AIV) M2 protein on autophagy, the pGFP-LC3 and pCAF-M2 recombinant plasmids were constructed and transfected into 293T or Hela cells by cationic liposome method, and the cells transfected with only plasmid were as negative control group, then the autophagosomes and autophagic flux were detected by indirect immunofluorescence assay and western blot. The results showed that M2 protein of AIV was able to induce the accumulation of autophagosomes. Moreover, the results also indicated that M2 protein mediated the accumulation of autophagosomes by blocking lysosomal degradation of autophagosomes detected by the LC3-Ⅱ turnover, tandem mRFP-GFP-LC3 fluorescence microscopy, pEGFP-LC3 and LAMP1 colocalization observation. These data suggest that M2 protein plays an important role for autophagy in AIV infected cells and lays the foundation for further study on the role of autophagy in the regulation of influenza A virus replication.