非洲猪瘟病毒E423R蛋白诱导细胞自噬的功能

African swine fever virus protein E423R induces autophagy

摘要:

目的 探究非洲猪瘟病毒(African swine fever virus, ASFV)蛋白E423R诱导细胞自噬的功能。方法 通过高内涵分析系统量化分析pCMV-Myc-E423R转染自噬报告细胞HeLa-Difluo^TM hLC3后的RFP-LC3荧光斑点数，评估自噬通量；利用Western blotting分析HeLa细胞中自噬关键蛋白LC3-Ⅱ、SQSTM1/p62的表达水平；采用激光共聚焦技术观察自噬体与溶酶体的共定位情况；通过Western blotting检测E423R蛋白与LC3-Ⅱ表达的剂量依赖效应以及对AKT/mTOR/ULK1信号通路的调控作用。结果 高内涵细胞分析实验显示，表达E423R蛋白的报告细胞中RFP-LC3荧光斑点明显增多，表明E423R蛋白可以诱导自噬发生活化；Western blotting分析证实，E423R蛋白表达促使LC3-Ⅱ/β-actin显著升高，p62的表达水平显著下调；共聚焦结果显示E423R蛋白可增强GFP-LC3的表达，促使GFP-LC3与Lyso-Tracker Red荧光共定位明显增多，诱导自噬体与溶酶体融合；同时，E423R蛋白表达水平与HeLa细胞中的LC3-Ⅱ表达水平呈正相关，E423R蛋白能下调mTOR信号通路中p-AKT、p-mTOR、p-ULK1 (Ser757)的表达。结论 本研究揭示了ASFV E423R蛋白通过AKT/mTOR/ULK1信号通路诱导完整的细胞自噬过程，E423R与LC3-Ⅱ的表达呈一定程度的剂量依赖效应，为深入研究自噬在ASFV感染与致病机制中的功能奠定了基础。

Abstract:

Objective To explore the autophagy induced by the African swine fever virus (ASFV) protein E423R.Methods The RFP-LC3 fluorescent spots following the transfection of HeLa-Difluo^TM hLC3 cells with pCMV-Myc-E423R was quantified by the High-Content Analysis System, and the autophagic flux was assessed. The expression levels of key autophagy proteins, LC3-Ⅱ and SQSTM1/p62, in HeLa cells were analyzed via Western blotting. Co-localization of autophagosomes and lysosomes was examined by laser confocal microscopy. Additionally, Western blotting was employed to investigate the dose-dependent effect of E423R on LC3-Ⅱ expression and the regulatory effect of E423R on the AKT/mTOR/ULK1 signaling pathway.Results The High-Content Analysis System demonstrated a significant increase in RFP-LC3 fluorescence spots in the reporter cells expressing E423R, suggesting that E423R induced the activation of autophagy. Western blotting further confirmed that the expression of E423R significantly elevated the LC3-Ⅱ/β-actin ratio while decreasing the expression level of p62. Confocal microscopy results indicated that E423R enhanced the expression of GFP-LC3, promoted the co-localization of GFP-LC3 with Lyso-Tracker Red, and facilitated the fusion of autophagosomes and lysosomes. Additionally, the expression level of E423R exhibited a positive correlation with the expression level of LC3-Ⅱ in HeLa cells. Furthermore, E423R down-regulated the expression of p-AKT, p-mTOR, and p-ULK1 (Ser757) in the mTOR signaling pathway.Conclusion This study demonstrates that the ASFV E423R protein induces complete autophagy via the AKT/mTOR/ULK1 signaling pathway, exhibiting a dose-dependent effect on LC3-Ⅱ expression to a certain degree. These findings provide a foundation for further investigation into the infection and pathogenic mechanisms of ASFV.