非洲猪瘟病毒E423R蛋白诱导细胞自噬的功能
作者:
作者单位:

1.扬州大学,江苏省人兽共患病学重点实验室,江苏 扬州;2.扬州大学,江苏省高校动物重要疫病与人兽共患病防控协同创新中心,江苏 扬州;3.扬州大学,农业农村部农产品质量安全生物性危害因子(动物源)控制重点实验室,江苏 扬州;4.扬州大学,教育部农业与农产品安全国际合作联合实验室,江苏 扬州

作者简介:

黄静怡:实验执行,数据分析和论文撰写;康喜龙:实验设计,数据分析和论文修改;黄霞:实验执行;周懿:协助实验操作,数据分析;刘弘知:提供技术支持,参与论文讨论;焦新安:指导论文思路,论文修改;潘志明:研究构思和设计,论文修改。

通讯作者:

中图分类号:

基金项目:

江苏省重点研发计划(现代农业)项目(BE2021331);江苏省研究生科研与实践创新计划(KYCX24_3823);江苏高校优势学科建设工程项目(PAPD)


African swine fever virus protein E423R induces autophagy
Author:
Affiliation:

1.Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, Jiangsu, China;2.Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, Jiangsu, China;3.Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agrifood Safety and Quality, Ministry of Agriculture and Rural Affairs, Yangzhou University, Yangzhou, Jiangsu, China;4.Joint International Research Laboratory of Agriculture and Agri-product Safety of the Ministry of Education, Yangzhou University, Yangzhou, Jiangsu, China

Fund Project:

This work was supported by the Key Research and Development Program (Modern Agriculture) Project of Jiangsu Province (BE2021331), the Postgraduate Research and Practice Innovation Program of Jiangsu Province (KYCX24_3823), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

  • 摘要
  • |
  • 图/表
  • |
  • 访问统计
  • |
  • 参考文献
  • |
  • 相似文献
  • |
  • 引证文献
  • |
  • 资源附件
  • |
  • 文章评论
    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-DifluoTM 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-DifluoTM 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.

    参考文献
    相似文献
    引证文献
引用本文

黄静怡,康喜龙,黄霞,周懿,刘弘知,焦新安,潘志明. 非洲猪瘟病毒E423R蛋白诱导细胞自噬的功能[J]. 微生物学报, 2025, 65(7): 3195-3207

复制
相关视频

分享
文章指标
  • 点击次数:
  • 下载次数:
  • HTML阅读次数:
  • 引用次数:
历史
  • 收稿日期:2025-01-15
  • 最后修改日期:
  • 录用日期:
  • 在线发布日期: 2025-07-04
  • 出版日期:
文章二维码