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Invest Ophthalmol Vis Sci. 2020 Jun 3;61(6):39. doi: 10.1167/iovs.61.6.39.
Herpes simplex virus type I (HSV-1) infection of corneal epithelial cells activates ataxia telangiectasia mutated (ATM), an apical kinase in the host DNA damage response pathway, whose activity is necessary for the progression of lytic HSV-1 infection. The purpose of this study is to investigate the mechanism of ATM activation by HSV-1 in the corneal epithelium, as well as its functional significance.
Mechanistic studies were performed in cultured human corneal epithelial cell lines (hTCEpi, HCE), as well as in esophageal (EPC2) and oral (OKF6) cell lines. Transfection-based experiments were performed in HEK293 cells. HSV-1 infection was carried out using the wild-type KOS strain, various mutant strains (tsB7, d120, 7134, i13, n208), and bacterial artificial chromosomes (fHSVΔpac, pM24). Inhibitors of ATM (KU-55933), protein synthesis (cycloheximide), and viral DNA replication (phosphonoacetic acid) were used. Outcomes of infection were assayed using Western blotting, qRT-PCR, immunofluorescence, and comet assay.
This study demonstrates that HSV-1-mediated ATM activation in corneal epithelial cells relies on the viral immediate early gene product ICP4 and requires the presence of the viral genome in the host nucleus. We show that ATM activation is independent of viral genome replication, the ICP0 protein, and the presence of DNA lesions. Interestingly, ATM activity appears to be necessary at the onset of infection, but dispensable at the later stages.
This study expands our understanding of HSV-1 virus-host interactions in the corneal epithelium and identifies potential areas of future investigation and therapeutic intervention in herpes keratitis.
单纯疱疹病毒 I 型（HSV-1）感染角膜上皮细胞会激活共济失调毛细血管扩张突变（ATM），这是宿主 DNA 损伤反应途径中的顶端激酶，其活性对于裂解性 HSV-1 感染的进展是必要的。本研究旨在探讨 HSV-1 在角膜上皮细胞中激活 ATM 的机制及其功能意义。
在培养的人角膜上皮细胞系（hTCEpi、HCE）以及食管（EPC2）和口腔（OKF6）细胞系中进行了机制研究。在 HEK293 细胞中进行了基于转染的实验。使用野生型 KOS 株、各种突变株（tsB7、d120、7134、i13、n208）和细菌人工染色体（fHSVΔpac、pM24）进行 HSV-1 感染。使用 ATM 抑制剂（KU-55933）、蛋白质合成抑制剂（环已酰亚胺）和病毒 DNA 复制抑制剂（膦甲酸钠）。使用 Western blot、qRT-PCR、免疫荧光和彗星试验来检测感染的结果。
本研究表明，HSV-1 在角膜上皮细胞中诱导的 ATM 激活依赖于病毒早期基因产物 ICP4，并且需要病毒基因组存在于宿主核内。我们表明，ATM 激活不依赖于病毒基因组复制、ICP0 蛋白和 DNA 损伤的存在。有趣的是，ATM 活性似乎在感染开始时是必需的，但在后期是可有可无的。
本研究扩展了我们对 HSV-1 病毒-宿主相互作用在角膜上皮细胞中的理解，并确定了疱疹性角膜炎未来研究和治疗干预的潜在领域。
