肠道病毒A71型亚单位蛋白对RD细胞自噬活化的影响  

The Effects of Subunit Proteins of Enterovirus-A71 on Activation of Autophagy in Human RD Cells

作　　者：麻雯熠 孙萍萍 王佳慧 杨世昭 王蒋丽[2] 谢广成 MA Wenyi;SUN Pingping;WANG Jiahui;YANG Shizhao;WANG Jiangli;XIE Guangcheng(School of Basic Medicine,Chengde Medical University,Chengde 067000,China;Department of Microbiology Laboratory,Chengde Center forDisease Control and Prevention,Chengde 067000,China)

机构地区：[1]承德医学院基础医学院 [2]承德市疾病预防控制中心微生物检验室

出　　处：《病毒学报》2023年第3期720-728,共9页Chinese Journal of Virology

基　　金：国家自然科学基金(项目号:81702008),题目:EV71衣壳蛋白经TLR2/TLR4异源二聚体活化细胞因子反应的研究;承德医学院重大项目(项目号:KY2020002),题目:EV71 2A和2C亚单位蛋白互作P13K的分子机制研究;承德医学院国家级大学生创新创业训练计划项目(项目号:2021001),题目:EV71衣壳蛋白VP1与PI3K调节亚基p85互作的分子机制;河北省科技厅“技术创新引导专项-科技工作会商”项目。

摘　　要：肠道病毒A71型(Enterovirus-A71, EV-A71)能够活化宿主细胞的自噬并依赖自噬促进其复制,然而EV-A71的亚单位蛋白对自噬的活化目前仍不清楚。为探讨EV-A71亚单位蛋白对人横纹肌肉瘤(Human rhabdomyosarcoma, RD)细胞自噬活化的影响,将EV-A71的亚单位蛋白重组真核质粒转染至RD细胞,采用抑制剂MK-2206阻断PI3K/Akt途径,共聚焦显微镜和免疫印迹检测自噬活化。过表达EV-A71亚单位蛋白的RD细胞中PI3K/Akt途径、p38、JNK和ERK途径均呈现不同程度活化,同时RD细胞呈现出绿色荧光表明自噬发生活化,特别是EV-A71的VP2和2A。EV-A71亚单位蛋白使LC3-II/LC3-I的转化水平提升,EV-A71亚单位蛋白(VP2、VP3、VP4、2A、2B和2C)显著提升p62的表达水平,EV-A71 VP1显著下调p62的表达水平但显著上调LAMP-1和LAMP-2的表达水平。阻断PI3K/Akt途径后,过表达EV-A71亚单位蛋白的RD细胞绿色荧光强度显著减弱、自噬被阻断,同时LC3-II/LC3-I的转化水平显著降低而p62的表达水平提升,过表达EV-A71 VP1能提升p62表达水平但降低LAMP-1表达水平。因此,EV-A71亚单位蛋白能够不同程度依赖PI3K/Akt途径活化细胞自噬。Enterovirus-A71(EV-A71)can activate autophagy of host cell to promote replication of the virus.However,the roles of EV-A7lsubunit proteins in activation of autophagy are still unclear.In order to investigate the effects of EV-A71subunit proteins on activation of autophagy in human rhabdomyosarcoma(RD)cells,endotoxin-free recombinant eukaryotic plasmids of EV-A71subunit proteins were transfected into RD cells.Meanwhile,inhibitor MK-2206 was used to block PI3K/Akt pathway.The activation of autophagy was detected through confocal microscopy and immunoblotting.PI3K/Akt pathway,p38,JNK and ERK pathways were activated at different levels in RD cells which overexpressed EV-A7lsubunit proteins,and green fluorescence was also detected which indicated the activation of autophagy,especially for EV-A71subunit proteins VP2 and 2A.Conversion ratio of LC3-II/LC3-I was up-regulated by overexpression of EV-A71subunit proteins.Expression level of p62 was also up-regulated by EV-A7lsubunit proteins VP2,VP3,VP4,2A,2B and 2C.However,the level of p62 was down-regulated and the levels of LAMP-1 and LAMP-2 were significantly up-regulated by EV-A71subunit protein VP1.Intensities of green fluorescence were weakened and autophagy was blocked,at the same time,conversion ratio of LC3-II/LC3-I was significantly down-regulated and the level of p62 was up-regulated in RD cells which overexpressed EV-A71subunit proteins after PI3K/Akt pathway was blocked by inhibitor MK-2206.The level of p62was up-regulated and the level of LAMP-1 was down-regulated in RD cells which overexpressed EV-A71subunit protein VP1 after PI3K/Akt pathway was blocked.In conclusion,EV-A71subunit proteins can activate autophagy of host cell in a PI3K/Akt pathway dependent manner at different degrees.

关 键 词：肠道病毒A71型 自噬 亚单位蛋白 PI3K/AKT途径 

分 类 号：R373.24[医药卫生—病原生物学]