Herpes simplex virus type 1 single strand DNA binding protein and helicase/primase complex disable cellular ATR signaling.
Herpes simplex virus type 1 single strand DNA binding protein and helicase/primase complex disable cellular ATR signaling.
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DOI：
10.1371/journal.ppat.1003652
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发表时间：
2013
期刊：
影响因子：
6.7
通讯作者：
Weller SK
中科院分区：
文献类型：
--
作者：
Mohni KN;Smith S;Dee AR;Schumacher AJ;Weller SK
单纯疱疹病毒1型（HSV-1）已经进化到使细胞DNA损伤反应激酶（ATR）失效。我们之前已经证明，即使在复制分叉停止的情况下，hsv -1感染的细胞也不能磷酸化ATR底物Chk1。在这里，我们报道了HSV-1单链DNA结合蛋白（ICP8）和解旋酶/引物酶复合体（UL8/UL5/UL52）在转染细胞中形成核复合体，这是禁用ATR信号的必要和充分条件。该复合体定位于DNA损伤位点，并与ATR/ATRIP和RPA共定位，但在这些条件下，Rad9-Rad1-Hus1检查点箝位（9-1-1）不存在。ATR通常被含有与dsDNA相邻的ssDNA的底物激活，我们实验室之前的研究表明，ICP8和解旋酶/引物酶也能识别这种底物。我们认为这四种病毒蛋白通过结合DNA底物和阻碍9-1-1检查点箝位的加载来阻止ATR的激活。排除9-1-1会阻止ATR激酶激活剂TopBP1的募集，从而有效地禁用ATR信号传导。这些数据提供了病毒DNA复制蛋白掩盖DNA底物通路的第一个例子，而DNA底物通常会触发DNA损伤反应和检查点信号。HSV使用的这种不寻常的机制表明，它可能通过阻止9-1-1钳和TopBP1的募集来抑制ATR信号。在细胞核内复制的DNA病毒已被证明可以激活和灭活细胞DNA损伤反应（DDR）的各种成分。先前来自我们实验室和其他实验室的报告表明，单纯疱疹病毒（HSV）利用了DDR的某些方面，同时灭活了其他方面。矛盾的是，HSV利用DDR激酶ATR完成其生命周期，同时使激酶无法激活DDR信号。在本报告中，我们详细描述了ATR失活的机制。ATR通常在单链DNA （ssDNA）的作用下被激活，单链DNA作为一个支架来招募ATR完全激活所需的几种蛋白质。在本文中，我们提供了证据，证明HSV编码ssDNA结合蛋白和解旋酶/引物酶复合物的功能可以掩盖招募ATR激酶激活剂的DNA底物。这代表了病毒DNA复制蛋白掩盖DNA底物的第一个例子，该底物可以被细胞感知为受损的DNA并激活检查点信号。它还解释了ATR如何在没有检查点信号的情况下被招募到病毒DNA复制的位点。
Herpes Simplex Virus type 1 (HSV-1) has evolved to disable the cellular DNA damage response kinase, ATR. We have previously shown that HSV-1-infected cells are unable to phosphorylate the ATR substrate Chk1, even under conditions in which replication forks are stalled. Here we report that the HSV-1 single stranded DNA binding protein (ICP8), and the helicase/primase complex (UL8/UL5/UL52) form a nuclear complex in transfected cells that is necessary and sufficient to disable ATR signaling. This complex localizes to sites of DNA damage and colocalizes with ATR/ATRIP and RPA, but under these conditions, the Rad9-Rad1-Hus1 checkpoint clamp (9-1-1) do not. ATR is generally activated by substrates that contain ssDNA adjacent to dsDNA, and previous work from our laboratory has shown that ICP8 and helicase/primase also recognize this substrate. We suggest that these four viral proteins prevent ATR activation by binding to the DNA substrate and obstructing loading of the 9-1-1 checkpoint clamp. Exclusion of 9-1-1 prevents recruitment of TopBP1, the ATR kinase activator, and thus effectively disables ATR signaling. These data provide the first example of viral DNA replication proteins obscuring access to a DNA substrate that would normally trigger a DNA damage response and checkpoint signaling. This unusual mechanism used by HSV suggests that it may be possible to inhibit ATR signaling by preventing recruitment of the 9-1-1 clamp and TopBP1. DNA viruses that replicate in the nucleus have been shown to both activate and inactivate various components of the cellular DNA damage response (DDR). Previous reports from our laboratory and others have demonstrated that Herpes Simplex Virus (HSV) utilizes some aspects of the DDR while inactivating others. Paradoxically, HSV utilizes the DDR kinase ATR to complete its life cycle while at the same time disabling the kinase from activating DDR signaling. In this report we provide detail describing the mechanism of ATR inactivation. ATR is normally activated in response to single strand DNA (ssDNA), which serves as a scaffold to recruit several proteins required for complete ATR activation. In this paper we provide evidence that the HSV encoded ssDNA binding protein and helicase/primase complex function to mask the DNA substrate that recruits the ATR kinase activator. This represents the first example of viral DNA replication proteins masking a DNA substrate that could be sensed by the cell as damaged DNA and activate checkpoint signaling. It also explains how ATR can be recruited to sites of viral DNA replication in the absence of checkpoint signaling.
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