Department of Molecular, Microbial and Structural Biology and Molecular Biology and Biochemistry Graduate Program, University of Connecticut Health Center, Farmington, Connecticut 06030, USA.
J Virol. 2010 Dec;84(23):12152-64. doi: 10.1128/JVI.01643-10. Epub 2010 Sep 22.
Although the herpes simplex virus type 1 (HSV-1) genome might be expected to induce a DNA damage response, the ATR kinase is not activated in infected cells. We previously proposed that spatial uncoupling of ATR from its interaction partner, ATRIP, could be the basis for inactivation of the ATR kinase in infected cells; however, we now show that ATR and ATRIP are in fact both recruited to HSV-1 replication compartments and can be coimmunoprecipitated from infected-cell lysates. ATRIP and replication protein A (RPA) are recruited to the earliest detectable prereplicative sites, stage II microfoci. In a normal cellular DNA damage response, ATR/ATRIP are recruited to stretches of RPA-coated single-stranded DNA in an RPA- and kinase-dependent manner, resulting in the phosphorylation of RPA by ATR in damage foci. In contrast, in HSV-1-infected cells, RPA is not phosphorylated, and endogenous phosphorylated RPA is excluded from stage II microfoci; in addition, the recruitment of ATR/ATRIP is independent of RPA and the kinase activity of ATR. Furthermore, we show that ATR/ATRIP play a beneficial role in viral gene expression and virus production. Although ICP0 has been shown to be important for partial inactivation of other cellular DNA repair pathways, we show that ICP0 is not responsible for the inactivation of ATR signaling and, furthermore, that neither ATR nor ATRIP is a target of ICP0 degradation. Thus, ATR and ATRIP may function outside the context of the canonical ATR damage signaling pathway during HSV-1 infection to participate in the viral life cycle.
尽管单纯疱疹病毒 1 型（HSV-1）基因组可能引发 DNA 损伤反应，但ATR 激酶在受感染细胞中并未被激活。我们之前曾提出，ATR 与其相互作用伙伴 ATRIP 的空间解偶联可能是ATR 激酶在受感染细胞中失活的基础；然而，我们现在表明，ATR 和 ATRIP 实际上都被招募到 HSV-1 复制隔间中，并且可以从受感染细胞裂解物中共同免疫沉淀。ATRIP 和复制蛋白 A（RPA）被招募到最早可检测到的复制前位点，即 II 期微焦点。在正常的细胞 DNA 损伤反应中，ATR/ATRIP 以 RPA 和激酶依赖性的方式被招募到 RPA 覆盖的单链 DNA 区域，导致ATR 在损伤焦点中对 RPA 的磷酸化。相比之下，在 HSV-1 感染的细胞中，RPA 未被磷酸化，内源性磷酸化的 RPA 被排除在 II 期微焦点之外；此外，ATR/ATRIP 的募集独立于 RPA 和 ATR 的激酶活性。此外，我们表明 ATR/ATRIP 在病毒基因表达和病毒产生中发挥有益作用。尽管 ICP0 已被证明对其他细胞 DNA 修复途径的部分失活很重要，但我们表明 ICP0 不负责 ATR 信号的失活，并且 ICP0 既不是 ATR 也不是 ATRIP 的降解靶标。因此，ATR 和 ATRIP 在 HSV-1 感染期间可能在经典 ATR 损伤信号通路的背景之外发挥作用，以参与病毒生命周期。
