Edwards Terri G, Bloom David C, Fisher Chris
Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, Florida, USA.
Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, Florida, USA
J Virol. 2018 Feb 26;92(6). doi: 10.1128/JVI.01884-17. Print 2018 Mar 15.
The ATM and Rad3-related (ATR) protein kinase and its downstream effector Chk1 are key sensors and organizers of the DNA damage response (DDR) to a variety of insults. Previous studies of herpes simplex virus 1 (HSV-1) showed no evidence for activation of the ATR pathway. Here we demonstrate that both Chk1 and ATR were phosphorylated by 3 h postinfection (h.p.i.). Activation of ATR and Chk1 was observed using 4 different HSV-1 strains in multiple cell types, while a specific ATR inhibitor blocked activation. Mechanistic studies point to early viral gene expression as a key trigger for ATR activation. Both pATR and pChk1 localized to the nucleus within viral replication centers, or associated with their periphery, by 3 h.p.i. Significant levels of pATR and pChk1 were also detected in the cytoplasm, where they colocalized with ICP4 and ICP0. Proximity ligation assays confirmed that pATR and pChk1 were closely and specifically associated with ICP4 and ICP0 in both the nucleus and cytoplasm by 3 h.p.i., but not with ICP8 or ICP27, presumably in a multiprotein complex. Chemically distinct ATR and Chk1 inhibitors blocked HSV-1 replication and infectious virion production, while inhibitors of ATM, Chk2, and DNA-dependent protein kinase (DNA-PK) did not. Together our data show that HSV-1 activates the ATR pathway at early stages of infection and that ATR and Chk1 kinase activities play important roles in HSV-1 replication fitness. These findings indicate that the ATR pathway may provide insight for therapeutic approaches. Viruses have evolved complex associations with cellular DNA damage response (DDR) pathways, which sense troublesome DNA structures formed during infection. The first evidence for activation of the ATR pathway by HSV-1 is presented. ATR is activated, and its downstream target Chk1 is robustly phosphorylated, during early stages of infection. Both activated proteins are found in the nucleus associated with viral replication compartments and in the cytoplasm associated with viral proteins. We also demonstrate that both ATR and Chk1 kinase activities are important for viral replication. The findings suggest that HSV-1 activates ATR and Chk1 during early stages of infection and utilizes the enzymes to promote its own replication. The observation may be exploitable for antiviral approaches.
ATM与Rad3相关（ATR）蛋白激酶及其下游效应分子Chk1是对多种损伤的DNA损伤反应（DDR）的关键传感器和组织者。先前对单纯疱疹病毒1型（HSV-1）的研究未显示ATR途径激活的证据。在此我们证明，感染后3小时（h.p.i.）时Chk1和ATR均被磷酸化。在多种细胞类型中使用4种不同的HSV-1毒株观察到了ATR和Chk1的激活，而一种特异性ATR抑制剂可阻断激活。机制研究表明早期病毒基因表达是ATR激活的关键触发因素。到感染后3小时，磷酸化的ATR（pATR）和磷酸化的Chk1（pChk1）均定位于病毒复制中心内的细胞核，或与其周边区域相关，在细胞质中也检测到了显著水平的pATR和pChk1，它们与ICP4和ICP0共定位。邻近连接分析证实，到感染后3小时，pATR和pChk1在细胞核和细胞质中均与ICP4和ICP0紧密且特异性相关，但不与ICP8或ICP27相关，推测是在一个多蛋白复合物中。化学性质不同的ATR和Chk1抑制剂可阻断HSV-1复制和感染性病毒粒子的产生，而ATM、Chk2和DNA依赖性蛋白激酶（DNA-PK）的抑制剂则无此作用。我们的数据共同表明，HSV-1在感染早期激活ATR途径，且ATR和Chk1激酶活性在HSV-1复制适应性中起重要作用。这些发现表明ATR途径可能为治疗方法提供思路。病毒已与细胞DNA损伤反应（DDR）途径形成复杂关联，DDR途径可感知感染期间形成的麻烦DNA结构。本文首次提供了HSV-1激活ATR途径的证据。在感染早期，ATR被激活，其下游靶点Chk1被强烈磷酸化。两种激活的蛋白均在与病毒复制区室相关的细胞核以及与病毒蛋白相关的细胞质中被发现。我们还证明ATR和Chk1激酶活性对病毒复制都很重要。这些发现表明HSV-1在感染早期激活ATR和Chk1，并利用这些酶促进自身复制。这一观察结果可能可用于抗病毒方法。
