Simian Varicella Virus Induces Apoptosis in Monkey Kidney Cells by the Intrinsic Pathway and Involves Downregulation of Bcl-2 Expression
Simian Varicella Virus Induces Apoptosis in Monkey Kidney Cells by the Intrinsic Pathway and Involves Downregulation of Bcl-2 Expression
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DOI：
10.1128/jvi.00768-09
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发表时间：
2009-09-15
影响因子：
5.4
通讯作者：
Mahalingam, Ravi
中科院分区：
文献类型：
--
作者：
Pugazhenthi, Subbiah;Gilden, Donald H.;Mahalingam, Ravi
猿猴水痘病毒 (SVV) 在灵长类动物中引起水痘，在神经节神经元中潜伏，然后重新激活产生带状疱疹。 SVV 在组织培养的猴肾细胞中产生细胞病变效应。为了研究 SVV 感染细胞死亡的机制，我们检查了感染后 24 至 64 小时 (hpi) 的细胞凋亡标记物。病毒感染细胞裂解物的蛋白质印迹分析显示，Caspase-3 裂解活性形式的水平显着增加，同时 40 至 64 hpi 时 caspase-3 活性也相应增加。 Caspase-9（内在途径标记物）在所有时间点的 SVV 感染细胞中均显着激活，而活性形式的 Caspase-8（外在途径标记物）的微量水平仅在 64 hpi 时检测到。在病毒感染过程中，Bcl-2 mRNA 和蛋白水平的表达下降了 50% 至 70%。 64 hpi 时，细胞色素 c（一种 caspase-9 激活剂）从线粒体释放到细胞质中的量增加了 200%。在 64 hpi 时对表达绿色荧光蛋白 (SVV-GFP) 的 SVV 感染的 Vero 细胞进行分析，揭示了 caspase-3 和 caspase-9 活性形式的共定位以及末端脱氧核苷酸转移酶介导的 dUTP-生物素缺口末端标记 (TUNEL) 与 GFP 的染色。在感染 SVV-GFP 的 Vero 细胞部分中，bcl-2 mRNA 水平显着降低，并且 SVV 基因 63、40 和 21 特异的 mRNA 丰富。总之，这些发现表明 SVV 通过 Bcl-2 下调的内在途径诱导培养的 Vero 细胞凋亡。
Simian varicella virus (SVV) causes varicella in primates, becomes latent in ganglionic neurons, and reactivates to produce zoster. SVV produces a cytopathic effect in monkey kidney cells in tissue culture. To study the mechanism by which SVV-infected cells die, we examined markers of apoptosis 24 to 64 h postinfection (hpi). Western blot analysis of virus-infected cell lysates revealed a significant increase in the levels of the cleaved active form of caspase-3, accompanied by a parallel increase in caspase-3 activity at 40 to 64 hpi. Caspase-9, a marker for the intrinsic pathway, was activated significantly in SVV-infected cells at all time points, whereas trace levels of the active form of caspase-8, an extrinsic pathway marker, was detected only at 64 hpi. Bcl-2 expression at the mRNA and protein levels was decreased by 50 to 70% throughout the course of virus infection. Release of cytochrome c, an activator of caspase-9, from mitochondria into the cytoplasm was increased by 200% at 64 hpi. Analysis of Vero cells infected with SVV expressing green fluorescent protein (SVV-GFP) at 64 hpi revealed colocalization of the active forms of caspase-3 and caspase-9 and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining with GFP. A significant decrease in the bcl-2 mRNA levels along with an abundance of mRNA specific for SVV genes 63, 40, and 21 was seen in the fraction of Vero cells that were infected with SVV-GFP. Together, these findings indicate that SVV induces apoptosis in cultured Vero cells through the intrinsic pathway in which Bcl-2 is downregulated.