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

Up-regulation of cellular Bcl-2 by the KSHV encoded RTA contributes to viral lytic replication

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。

[ Objective] To explore the molecular mechanism and biological significance of up-regulation of cellular Bcl-2 by the KSHV （Kaposis＇s Sarcoma-Associated Herpesvirus） encoded RTA （replication and transcription activator）. [Methods] The mRNA and protein levels of Bcl-2 in RTA-transfected 293, DG75 ceils and TPA-induced KSHV positive cells were analyzed by RT-PCR and Western blotting. Regulation of the Bcl-2 promoter activity by RTA was investigated by luciferase assay. A RNA interference lentiviral vector was generated, and stably infected BC3 cells were screened. At 48 h post-induction, cells were subjected to PCR, P1 staining flow cytometry for virion production and cellular apoptosis analysis. [Results] RTA up-regu- lates the mRNA and protein levels of Bcl-2 in RTA-expressing 293, DG75 cells, and TPA-induced KSHV positive ceils. Further analysis revealed that the up-regulation of cellular Bcl-2 promoter by RTA is dose-de- pendent. Furthermore, knockdown of cellular Bcl-2 using specific short hairpin RNA （shRNA） indicated that RTA plays an essential role in Bcl-2-mediated anti-apoptosis of the cell, prolonging the survival of lytically infected host cells to allow for maximum production of virus progeny. [Conclusion] These findings provide an insight into the mechanism by which KSHV utilizes the intrinsic apoptosis signaling pathways for promoting the survival of lyrically infected host cells to allow maximum production of virus progeny.