DNA损伤反应通过细胞核到细胞质的核因子κB激活促进多瘤病毒JC感染。
The DNA damage response promotes polyomavirus JC infection by nucleus to cytoplasm NF- kappaB activation.
作者信息
White Martyn K, Bellizzi Anna, Ibba Gabriele, Pietropaolo Valeria, Palamara Anna T, Wollebo Hassen S
机构信息
Center for Neurovirology, Department of Neuroscience, Lewis Katz School of Medicine at Temple University, 3500 N. Broad Street, Philadelphia, PA, 19140, USA.
Department of Public Health and Infectious Diseases, Institute Pasteur Italia, Cenci-Bolognetti Foundation, Sapienza University of Rome, 5 P.le Aldo Moro, 00185, Rome, Italy.
出版信息
Virol J. 2017 Feb 15;14(1):31. doi: 10.1186/s12985-017-0707-7.
BACKGROUND
Infection of glial cells by human neurotropic polyomavirus JC (JCV), the causative agent of the CNS demyelinating disease progressive multifocal leukoencephalopathy (PML), rapidly inflicts damage to cellular DNA. This activates DNA damage response (DDR) signaling including induction of expression of DNA repair factor Rad51. We previously reported that Rad51 co-operates with the transcription factor NF-κB p65 to activate JCV early transcription. Thus Rad51 induction by JCV infection may provide positive feedback for viral activation early in JCV infection. DDR is also known to stimulate NF-κB activity, a phenomenon known as nucleus to cytoplasm or "inside-out" NF-κB signaling, which is initiated by Ataxia telangiectasia mutated (ATM) protein, a serine/threonine kinase recruited and activated by DNA double-strand breaks. Downstream of ATM, there occurs a series of post-translational modifications of NF-κB essential modulator (NEMO), the γ regulatory subunit of inhibitor of NF-κB (IκB) kinase (IKK), resulting in NF-κB activation.
METHODS
We analyzed the effects of downstream pathways in the DDR by phosphospecific Western blots and analysis of the subcellular distribution of NEMO by cell fractionation and immunocytochemistry. The role of DDR in JCV infection was analyzed using a small molecule inhibitor of ATM (KU-55933). NEMO sumoylation was investigated by Western and association of ATM and NEMO by immunoprecipitation/Western blots.
RESULTS
We show that JCV infection caused phosphorylation and activation of ATM while KU-55933 inhibited JCV replication. JCV infection caused a redistribution of NEMO from cytoplasm to nucleus. Co-expression of JCV large T-antigen and FLAG-tagged NEMO showed the occurrence of sumoylation of NEMO, while co-expression of ATM and FLAG-NEMO demonstrated physical association between ATM and NEMO.
CONCLUSIONS
We propose a model where JCV infection induces both overexpression of Rad51 protein and activation of the nucleus to cytoplasm NF-κB signaling pathway, which then act together to enhance JCV gene expression.
背景
人嗜神经多瘤病毒 JC（JCV）可感染神经胶质细胞，它是中枢神经系统脱髓鞘疾病进行性多灶性白质脑病（PML）的病原体，可迅速对细胞 DNA 造成损伤。这会激活 DNA 损伤反应（DDR）信号通路，包括诱导 DNA 修复因子 Rad51 的表达。我们之前报道过 Rad51 与转录因子 NF-κB p65 协同作用以激活 JCV 的早期转录。因此，JCV 感染诱导 Rad51 表达可能为 JCV 感染早期的病毒激活提供正反馈。DDR 也已知会刺激 NF-κB 活性，这种现象被称为细胞核到细胞质或“由内而外”的 NF-κB 信号传导，它由共济失调毛细血管扩张症突变（ATM）蛋白启动，ATM 是一种由 DNA 双链断裂招募并激活的丝氨酸/苏氨酸激酶。在 ATM 的下游，NF-κB 必需调节因子（NEMO）会发生一系列翻译后修饰，NEMO 是 NF-κB 抑制因子（IκB）激酶（IKK）的γ调节亚基，从而导致 NF-κB 激活。
方法
我们通过磷酸化特异性蛋白质免疫印迹分析了 DDR 下游通路的作用，并通过细胞分级分离和免疫细胞化学分析了 NEMO 的亚细胞分布。使用 ATM 的小分子抑制剂（KU-55933）分析了 DDR 在 JCV 感染中的作用。通过蛋白质免疫印迹研究了 NEMO 的类泛素化修饰，并通过免疫沉淀/蛋白质免疫印迹分析了 ATM 与 NEMO 的相互作用。
结果
我们发现 JCV 感染导致 ATM 磷酸化并激活，而 KU-55933 抑制 JCV 复制。JCV 感染导致 NEMO 从细胞质重新分布到细胞核。JCV 大 T 抗原与 FLAG 标记的 NEMO 共表达显示 NEMO 发生了类泛素化修饰，而 ATM 与 FLAG-NEMO 共表达证明 ATM 与 NEMO 存在物理相互作用。
结论
我们提出了一个模型，即 JCV 感染诱导 Rad51 蛋白过表达和细胞核到细胞质的 NF-κB 信号通路激活，然后它们共同作用增强 JCV 基因表达。
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