摘要
目的探讨早期生长反应基因1（EGR1）对成人T淋巴细胞白血病病毒1型（HTLV-1）感染后宿主细胞自噬的影响。方法获得病毒早期感染模型，免疫印迹检测病毒核心蛋白p19、宿主细胞EGRl的表达：荧光素酶报告基因检测EGRl的5’端调控序列转录活性；筛选有效的EGRlsiRNA，将EGR1 siRNA转染HeLa细胞，随后HTLV．1病毒阳性细胞系MT2与HeLa细胞进行共培养，免疫印迹检测病毒核心蛋白p19、EGR1、自噬相关蛋白LC3；real-timePCR检测病毒载量；免疫荧光检测自噬体数目。结果共培养后，宿主细胞EGR1蛋白表达和调控序列荧光素酶活性逐渐增强，且EGRl蛋白的表达与细胞自噬水平呈现正相关；筛选了有效的EGRlsiRNA，命名为siE2；共培养模型干扰EGRI的表达后，病毒核酸载量、核心蛋白p19的表达以及自噬相关蛋白LC3B／LC3A比例均下调，同时伴有胞内自噬体数目的减少（P〈0．01）。结论EGR1与HTILV-1感染后宿主细胞自噬水平和病毒复制密切相关。
Objective To investigate the influence of early growth response gene-1 （ EGR1 ） on the autophagy of host cells following infection with human T cell leukemia virus type 1 （ HTLV-1 ）. Methods A HTLV-1-positive cell line MT2 was co-cultured with HeLa cells for 24 h to construct the virus early infection model. Immunoblotting assay was used to detect the expression of HTLV-1 core protein p19 and EGR1. Luciferase reporter gene analysis was used to detect the transcriptional activity of 5＇-regulatory sequence of EGRI at different time points after co-culturing. An effective small interfering RNA （siRNA） targeting EGR1 was screened out and transfected into HeLa cells by Lipofectamine 2000. Then the transfected HeLa ceils were co-cultured with the HTLV-I-posltive cell line MT2 for 24 h. Immunoblotting assay was used to detect HTLV-1 core protein p19, EGR1 and autophagy-related protein LC3. Real-time PCR was performed to detect viral load. Autophagosome was analyzed by immunoflnorescence after co-culturing. Results The expression of EGR1 and the transcriptional activity of pEGRI-luc gradually increased after co-culturing HeLa cells with MT2 cells for 8 h （ P〈0.01 ）. The expression of EGR1 was positively correlated with host cell au- tophagy following HTLV-1 infection. The effective siRNA for silencing the expression of EGR1 was obtained and named as siE2. The viral load, the expression of HTLV-1 core protein p19 and the proportion of LC3B/ LC3A in the co-culture model were markedly down-regulated by RNA interference with siE2, which was con- comitant with a persistent decrease of intracellular autophagosome （P〈0.01）. Conclusion EGR1 is asso- ciated with host cell autophagy and viral replication in HTLV-1 infection.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2017年第7期492-496,共5页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金（81273241,81471559）
河南省高校科技创新团队（15IRTSTHN025）
河南省教育厅科学技术研究重点项目（13A310843）
国家大学生创新创业实践计划（201610472001）
