Department of Biological Science and Technology, National Pingtung University of Science and Technology, Pingtung, Taiwan.
General Research Service Center, Pingtung, Taiwan.
Intervirology. 2019;62(3-4):116-123. doi: 10.1159/000502047. Epub 2019 Aug 20.
Pseudorabies virus (PRV) infection induces apoptosis in swine cells both in vitro and in vivo; however, the mechanism associated with host-cell signaling has not been studied. This study investigated the role of free radicals caused by cellular oxidative stress after viral infection and examined whether the DNA damage response plays an important role in PRV-induced apoptosis.
Several apoptosis assays and western blotting confirmed PRV-induced apoptosis. PRV-mediated oxidative stress was evaluated by reactive oxygen species (ROS) assay.
Our results showed that PRV caused apoptosis in a porcine kidney cell line, PK15, and induced expressions of proapoptotic Bcl family proteins in a dose- and time-dependent manner. Expressions of specific DNA damage sensors and phosphorylation of histone H2AX were also significantly increased, which subsequently activated the expressions of checkpoint kinase 1/2 and proapoptotic p53. Caffeine, a known DNA damage inhibitor, was found to inhibit caspase-3 activation and protect cells from PRV-induced apoptosis. Additionally, the antioxidant N-acetyl-L-cysteine was shown to prevent the production of cellular ROS, protecting DNA from cleavage.
Our results confirmed that oxidative stress and free radicals arising from PRV infection cause DNA damage, which consequently triggers apoptosis.
伪狂犬病毒（PRV）感染可在体外和体内诱导猪细胞凋亡；然而，与宿主细胞信号相关的机制尚未得到研究。本研究探讨了病毒感染后细胞氧化应激引起的自由基的作用，并研究了 DNA 损伤反应是否在 PRV 诱导的细胞凋亡中发挥重要作用。
通过几种细胞凋亡检测和 Western blot 验证了 PRV 诱导的细胞凋亡。通过活性氧（ROS）测定评估了 PRV 介导的氧化应激。
我们的结果表明，PRV 可诱导猪肾细胞系 PK15 发生凋亡，并以剂量和时间依赖的方式诱导促凋亡 Bcl 家族蛋白的表达。特定的 DNA 损伤传感器的表达和组蛋白 H2AX 的磷酸化也显著增加，随后激活检查点激酶 1/2 和促凋亡 p53 的表达。咖啡因是一种已知的 DNA 损伤抑制剂，被发现可抑制 caspase-3 的激活并保护细胞免受 PRV 诱导的凋亡。此外，抗氧化剂 N-乙酰-L-半胱氨酸可防止细胞 ROS 的产生，从而防止 DNA 断裂。
我们的结果证实，PRV 感染引起的氧化应激和自由基会导致 DNA 损伤，从而引发细胞凋亡。
