Activation of a DNA damage pathway by HIV-1 vpr

描述（由申请人提供）：人类免疫缺陷病毒1型（HIV-1）编码4个辅助基因，调节宿主细胞生物学的各个方面。vpr基因编码一个96个氨基酸的蛋白质（Vpr），导致细胞周期停滞在G2期和受感染的淋巴细胞凋亡。导致Vpr诱导的细胞停滞和凋亡的细胞途径可以在大多数哺乳动物细胞类型中被激活，因此被认为是进化保守的。Vpr可能利用细胞信号传导途径来诱导其细胞抑制和细胞毒性作用。几项研究表明，Vpr对细胞的影响与遗传毒性药物和其他DNA损伤剂（如辐射）的影响相似。然而，我们以前证明，一个主要的细胞蛋白参与的遗传毒性剂，p53，介导Vpr的影响是不必要的。在本研究中，我们研究了一个最近确定的DNA损伤信号蛋白（ATM和Rad 3相关蛋白，ATR）的潜在作用，诱导G2期阻滞Vpr。我们发现，通过遗传手段或药理学抑制剂抑制ATR可消除哺乳动物细胞中Vpr诱导的细胞周期阻滞。ATR是一种丝氨酸-苏氨酸激酶，其响应于DNA损伤而被激活。ATR的活化通常导致其靶标Chkl激酶的直接磷酸化。我们检测到响应Vpr表达的Chkl的磷酸化。由于Chkl激活导致Cdc 2失活，我们的观察结果与Vpr诱导的细胞周期停滞涉及导致G2停滞的ATR依赖性应激反应途径的概念一致。通过抑制ATR，我们证明了vpr的另一种功能，即病毒启动子的反式激活的抑制。这些观察结果可能在抗HIV治疗领域产生重要影响。该提案的目的是阐明Vpr激活的DNA反应途径的具体步骤，并将这些步骤与生理激活剂DNA损伤进行比较。

DESCRIPTION (provided by applicant): The human immunodeficiency virus type-1 (HIV-1) encodes four accessory genes that regulate various aspects of the host cell biology. The vpr gene encodes a 96-amino acid protein (Vpr) that causes cell cycle arrest in G2 and apoptosis in the infected lymphocytes. The cellular pathways leading to Vpr-induced arrest and apoptosis can be activated in most mammalian cell types and are therefore, thought to be evolutionarily conserved. Vpr presumably utilizes cellular signaling pathways to induce its cytostatic and cytotoxic effects. Several studies have indicated that the effects of Vpr on the cell are similar to the effects of genotoxic drugs and other DNA damaging agents, such as radiation. However, we previously demonstrated that a major cellular protein involved in the response to genotoxic agents, p53, was not necessary for mediating the effects of Vpr. In the present study, we examined a recently identified DNA damage-signaling protein (the ATM- and Rad3-related protein, ATR) for its potential role in the induction of G2 arrest by Vpr. We show that inhibition of ATR by genetic means or with pharmacological inhibitors abrogates Vpr-induced cell cycle arrest in mammalian cells. ATR is a serine-threonine kinase that becomes activated in response to DNA damage. Activation of ATR typically results in direct phosphorylation of its target, the Chkl kinase. We detected phosphorylation of Chkl in response to Vpr expression. Since Chkl activation results in inactivation of Cdc2, our observations are consistent with the notion that the Vpr-induced cell cycle arrest involves an ATR-dependent stress-response pathway that leads to G2 arrest. By inhibiting ATR, we demonstrated suppression of another function of vpr, transactivation of the viral promoter. These observations may have important ramifications in the area of anti-HIV therapy. The goal of this proposal is to elucidate the specific steps of the DNA-response pathway that are activated by Vpr, and to compare such steps with the physiological activator, DNA damage.