Reshi Latif, Wu Horng-Cherng, Wu Jen-Leih, Wang Hao-Ven, Hong Jiann-Ruey
Laboratory of Molecular Virology and Biotechnology, Institute of Biotechnology, Department of Biotechnology and Bioindustry Sciences, National Cheng Kung University, No 1. University Road, Tainan City, 701, Taiwan, ROC.
Department of Life Sciences, College of Bioscience and Biotechnology, National Cheng Kung University, No. 1. University Road, Tainan City, 701, Taiwan, ROC.
Apoptosis. 2016 Apr;21(4):443-58. doi: 10.1007/s10495-016-1219-4.
Previous studies have shown that GSIV induces apoptotic cell death through upregulation of the pro-apoptotic genes Bax and Bak in Grouper fin cells (GF-1 cells). However, the role of viral genome-encoded protein(s) in this death process remains unknown. In this study, we demonstrated that the Giant seaperch iridovirus (GSIV) genome encoded a serine/threonine kinase (ST kinase) protein, and induced apoptotic cell death via a p53-mediated Bax upregulation approach and a downregulation of Bcl-2 in fish cells. The ST kinase expression profile was identified through Western blot analyses, which indicated that expression started at day 1 h post-infection (PI), increased up to day 3, and then decreased by day 5 PI. This profile indicated the role of ST kinase expression during the early and middle phases of viral replication. We then cloned the ST kinase gene and tested its function in fish cells. The ST kinase was transiently expressed and used to investigate possible novel protein functions. The transient expression of ST kinase in GF-1 cells resulted in apoptotic cell features, as revealed with Terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL) assays and Hoechst 33258 staining at 24 h (37 %) and 48 h post-transfection (PT) (49 %). Then, through studies on the mechanism of cell death, we found that ST kinase overexpression could upregulate the anti-stress gene p53 and the pro-apoptotic gene Bax at 48 h PT. Interestingly, this upregulation of p53 and Bax also correlated to alterations in the mitochondria function that induced loss of mitochondrial membrane potential (MMP) and activated the initiator caspase-9 and the effector caspase-3 in the downstream. Moreover, when the p53-dependent transcriptional downstream gene was blocked by a specific transcriptional inhibitor, it was found that pifithrin-α not only reduced Bax expression, but also averted cell death in GF-1 cells during the ST kinase overexpression. Taken altogether, these results suggested that aquatic GSIV ST kinase could induce apoptosis via upregulation of p53 and Bax expression, resulting in mitochondrial disruption, which activated a downstream caspases-mediated cell death pathway.
先前的研究表明，石斑鱼虹彩病毒（GSIV）通过上调石斑鱼鳍细胞（GF-1细胞）中促凋亡基因Bax和Bak来诱导凋亡性细胞死亡。然而，病毒基因组编码蛋白在这一死亡过程中的作用仍不清楚。在本研究中，我们证明了巨石斑鱼虹彩病毒（GSIV）基因组编码一种丝氨酸/苏氨酸激酶（ST激酶）蛋白，并通过p53介导的Bax上调途径和下调鱼类细胞中的Bcl-2来诱导凋亡性细胞死亡。通过蛋白质印迹分析确定了ST激酶的表达谱，结果表明其表达在感染后（PI）1小时开始，至第3天增加，然后在感染后第5天下降。这一表达谱表明了ST激酶表达在病毒复制早期和中期的作用。然后我们克隆了ST激酶基因并在鱼类细胞中测试了其功能。ST激酶被瞬时表达并用于研究可能的新蛋白功能。在GF-1细胞中瞬时表达ST激酶导致凋亡性细胞特征，在转染后（PT）24小时（37%）和48小时（49%）通过末端脱氧核苷酸转移酶生物素-dUTP缺口末端标记（TUNEL）分析和Hoechst 33258染色得以揭示。然后，通过对细胞死亡机制的研究，我们发现ST激酶过表达在PT 48小时可上调抗应激基因p53和促凋亡基因Bax。有趣的是，p53和Bax的这种上调也与线粒体功能改变相关，线粒体功能改变导致线粒体膜电位（MMP）丧失，并激活下游的起始半胱天冬酶-9和效应半胱天冬酶-3。此外，当p53依赖的转录下游基因被一种特异性转录抑制剂阻断时，发现pifithrin-α不仅降低了Bax表达，还避免了GF-1细胞在ST激酶过表达期间的细胞死亡。综上所述，这些结果表明水生GSIV ST激酶可通过上调p53和Bax表达诱导凋亡，导致线粒体破坏，进而激活下游半胱天冬酶介导的细胞死亡途径。
