EIAV Rev蛋白拮抗eqTRIM5α介导的AP-1信号通路的机制研究  

Mechanism of EIAV Rev protein antagonizing eqTRIM5α-mediated AP-1 signaling pathway

作　　者：宋丹丹 那雷[1] 王晓钧[1] SONG Dan-dan;NA Lei;WANG Xiao-jun(State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,ChineseAcademy ofAgricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2020年第1期12-17,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(31602034);中国博士后科学基金(2015M581223).

摘　　要：为探究马传染性贫血病毒(EIAV)附属蛋白Rev负调控Tripartite motif-containing protein 5α(TRIM5α)介导的AP-1信号通路的机制,本研究将pEIAV-Rev-HA和pcDNA3.1质粒分别与含TRIM5α基因的质粒及pGL3-AP-1-Luc(AP-1报告质粒)共转染HEK 293T细胞,采用荧光素酶试验检测Rev对TRIM5α激活的AP-1信号通路的影响;将pEIAV-Rev-HA和pcDNA3.1质粒分别与含TAK1、TAB2、P38和c-Jun基因的质粒及pGL3-AP-1-Luc共转染HEK 293T细胞,采用荧光素酶试验检测Rev对TRIM5α下游转导分子(TAK1、TAB2、P38、c-Jun)激活的AP-1信号通路的影响;将pEIAV-Rev-HA和pcDNA3.1质粒分别与含TAK1、TAB2、P38基因的质粒共转染HEK293T细胞,利用western blot试验分别检测TAK1、TAB2、P38的表达水平;将pEIAV-Rev-HA和pcDNA3.1质粒分别与含P38基因的质粒共转染HEK 293T细胞后加入蛋白酶体抑制剂MG132,利用western blot检测P38蛋白的表达情况。结果显示,共转染EIAV-Rev-HA实验组中TRIM5α对AP-1的激活倍数为0.4,而共转染pcDNA3.1对照组中相应的激活倍数为26.0;共转染pEIAV-Rev-HA实验组中,TAK1、TAB2、P38和c-Jun对AP-1信号通路的激活倍数分别为7.7、0.1、0.6、9.8,而共转染pcDNA3.1对照组中对AP-1信号通路的激活倍数分别为60.0、1.5、6.3、12.0;转染pEIAV-Rev-HA+pP38-Flag组与转染pcDNA3.1+pP38-Flag组相比,前者P38蛋白的表达量显著降低;加入蛋白酶体抑制剂组则恢复了P38蛋白的表达。上述结果表明,EIAV Rev显著下调eqTRIM5α及其下游转导分子TAK1、TAB2、P38激活的AP-1信号通路,但不显著下调c-Jun激活的AP-1信号通路;EIAV Rev通过蛋白酶体途径降解P38蛋白的表达而抑制eqTRIM5α激活的AP-1信号通路。本研究结果为理解EIAV与宿主蛋白相互作用提供参考依据。To investigate the mechanism of tripartite motif-containing protein 5α(TRIM5α)-mediated AP-1 signaling pathway negatively regulated by equine infectious anemia virus(EIAV)accessory protein Rev activated,pEIAV-Rev-HA or pcDNA3.1 empty vector was co-transfected with plasmids containing eqTRIM5α,TAK1,TAB2,P38 and c-Jun into HEK 293 T cells respectively,and pGL3-AP-1-Luc was also co-transfected.and the effect of AP-1 signaling pathway was then evaluated by luciferase reporter assay.p EIAV-Rev-HAor pcDNA3.1 empty vector was co-transfected with TAK1,TAB2,P38 plasmids into HEK 293 T cells respectively,and the expressions of TAK1,TAB2 and P38 were thenevaluated by western blot.pEIAV-Rev-HA or pcDNA3.1 was co-transfected with P38 into HEK 293 T cells for 24 h,the proteasome inhibitor MG132 was added and the expression of P38 protein was evaluated by western blot.The results showed that EIAV Rev protein significantly down-regulated the AP-1 signaling pathway activated by eqTRIM5 and its downstream transduction molecules TAK1,TAB2 and P38,but did not significantly down-regulate the AP-1 signaling pathway activated by c-jun.The EIAV Rev protein inhibited the eqTRIM5α-activated AP-1 signaling pathway by degrading the expression of P38 via the proteasome pathway.These results provide a reference for understanding the interaction between EIAV and host proteins.

关 键 词：EIAV TRIM5α REV P38 

分 类 号：S852.65[农业科学—基础兽医学]