KSHV RTA介导的宿主细胞Bcl-2表达上调促进病毒的溶解性感染

发布时间：2018-12-18 13:10

【摘要】：目的研究卡泼肉瘤病毒(kaposis's sarcoma-associated herpesvirus,KSHV)编码的复制转录激活因子(replication and transcription activator,RTA)调控宿主细胞Bcl-2表达的分子机制及其生物学意义。方法用实时聚合酶链反应(RT-PCR)、蛋白质印迹(western blotting)技术检测RTA转染的细胞、TPA诱导的KSHV阳性细胞Bcl-2 mRNA和蛋白质表达水平;采用荧光素酶报告基因技术检测RTA对Bcl-2基因启动子活性的调节。构建RNA干扰慢病毒载体,筛选稳定感染的BC3细胞,TPA诱导48 h后,分别用PCR、PI染色流式细胞术检测病毒子DNA和细胞凋亡率。结果 RTA转染的细胞、TPA诱导的KSHV阳性细胞Bcl-2 mRNA和蛋白质表达水平显著增高。RTA反式激活Bcl-2基因启动子,这种激活作用具有剂量依赖性。用RNA干扰技术抑制Bcl-2基因后,TPA诱导的KSHV阳性细胞与对照组细胞相比凋亡率增加,病毒子产生减少。结论KSHV RTA能够调控内源性细胞凋亡信号通路,延缓溶解性感染宿主细胞的凋亡,并促进病毒子产生。
[Abstract]:Objective to investigate the molecular mechanism and biological significance of kapperosarcoma virus (kaposis's sarcoma-associated herpesvirus,KSHV)-encoded replication-activator (replication and transcription activator,RTA (replication and transcription activator,RTA) in regulating the expression of Bcl-2 in host cells. Methods Real-time polymerase chain reaction (RT-PCR) and Western blot (western blotting) were used to detect the expression of Bcl-2 mRNA and protein in RTA transfected cells and KSHV positive cells induced by TPA. Luciferase reporter gene technique was used to detect the regulation of RTA on the activity of Bcl-2 gene promoter. RNA interference lentivirus vector was constructed to screen stable infected BC3 cells. After 48 h of TPA induction, the viral DNA and apoptosis rate were detected by PCR,PI staining flow cytometry. Results the expression of Bcl-2 mRNA and protein in KSHV positive cells induced by TPA was significantly increased in RTA transfected cells. RTA transactivated Bcl-2 gene promoter in a dose-dependent manner. After Bcl-2 gene was inhibited by RNA interference, the apoptosis rate of KSHV positive cells induced by TPA was higher than that of control cells, and the production of virus was decreased. Conclusion KSHV RTA can regulate endogenous apoptosis signaling pathway, delay apoptosis of host cells and promote viral production.
【作者单位】：三峡大学医学院病理生理学教研室;美国宾夕法尼亚大学医学院微生物学教研室;
【基金】：National Cancer Institute 5R01CA091792-08,5R01CA108461-05,1R01CA137894-01 and 1R01CA138434-01A209 National Institute of Allergy and Infectious Diseases 5R01AI067037-04 National Institute of Dental and Craniofacial Research 5R01DE017338-03(to ESR)
【分类号】：R373

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