Induction of Apoptosis by HIV-1 vpr

HIV-1辅助基因vpr编码一个保守的96个氨基酸的蛋白质，可诱导细胞阻断。 在62阶段循环。VPR在CD4+淋巴细胞中的表达也可诱导细胞凋亡。我们已经确定了 ATR激酶是介导VPR诱导的细胞周期停滞和凋亡的细胞因子。我们还有 证明了诱导G2停滞和凋亡需要ATR下游靶标的磷酸化， 如CHK1、BRCA1和GADD45a。因此，ATR，Kinase是VPR诱导的一个关键决定因素 发病机制。这项提案的主要焦点将是阐明vpr 激活ATR。 体内和体外感染HIV-1的巨噬细胞比感染、激活的巨噬细胞活得更长 T细胞。我们发现VPR在巨噬细胞中的表达不能诱导细胞凋亡，我们假设 其根本原因是VPR无法激活这些细胞中的ATR。VPR诱导分化能力的研究 活化的T细胞与巨噬细胞的凋亡完全符合一个新的模型，在这个模型中 ATR的激活需要持续的蜂窝DMA复制。VPR激活ATR也会导致 增强病毒基因表达，这解释了归因于VPR的适度反式激活活性。 细胞。 这项建议的具体目标是： 具体目的1.阐明VPR激活ATR的机制。ATR的功能是调查 细胞DNA复制和检测停滞的复制分叉。我们将研究VPR是否会导致激活 ATR通过影响DNA的完整性、通过阻碍宿主细胞复制或通过激活ATR信号来实现 直接复杂。 具体目的2.探讨VPR对单核细胞来源的巨噬细胞ATR的影响。我们有 观察到MDM对VPR诱导的细胞凋亡不敏感，但对其他促凋亡刺激不敏感。我们 假设VPR无法在MDM中激活ATR，因为它们处于非分割状态。我们还假设 这种依赖于ATR激活的VPR诱导的反式激活在MDM中不发生。 目的3.评价ATR激活在HIV-1复制动力学中的作用。我们已经证明了 抑制ATR导致完全抑制VPR反式激活病毒启动子的能力。因此，我们 假设VPR将能够诱导分裂细胞(激活的T细胞)的反式激活，但不能诱导非 细胞分裂(MDM)。我们还假设，当允许时，VPR激活ATR将导致更高的速率 病毒复制。

The HIV-1 accessory gene vpr encodes a conserved 96-amino acid protein that induces block of the cell cycle at the 62 phase. Expression of Vpr in CD4+ lymphocytes also induces apoptosis. We have identified the ATR kinase as the cellular factor that mediates Vpr-induced cell cycle arrest and apoptosis. We have also demonstrated that induction of G2 arrest and apoptosis require phosphorylation of downstream targets of ATR, such as Chk1, BRCA1 and GADD45a. Therefore, the ATR, kinase represents a key determinant of Vpr-induced pathogenesis. The major focus of this proposal will be to elucidate the precise mechanism by which Vpr activates ATR. Macrophages infected with HIV-1 in vivo and in vitro are long-lived, when compared to infected, activated T-cells. We find that expression of Vpr in macrophages is unable to induce apoptosis, and we hypothesize that the underlying cause is that Vpr is unable to activate ATR in these cells. The differential ability of Vpr to induce apoptosis in activated T-cells versus macrophages is in complete agreement with an emerging model in which activation of ATR requires ongoing cellular DMA replication. Activation of ATR by Vpr also leads to an enhancement viral gene expression, which explains the moderate transactivation activity ascribed to Vpr in T- cells. The specific Aims of this proposal are: Specific Aim 1. To elucidate the mechanism by which Vpr activates ATR. The function of ATR is to survey cellular DNA replication and to detect stalled replication forks. We will examine whether Vpr causes activation of ATR by affecting the integrity of DNA, by hindering host cell replication, or by activating the ATR signaling complex directly. Specific Aim 2. To explore the effect of Vpr on ATR in monocyte-derived macrophages (MDM). We have observed that MDM are refractory to Vpr-induced apoptosis, but not to other pro-apoptotic stimuli. We hypothesize that Vpr is unable to activate ATR in MDM due to their non-dividing status. We also hypothesize that Vpr-induced transactivation, which is dependent on ATR activation, does not occur in MDM. Aim 3. To evaluate the contribution of ATR activation to the kinetics of replication of HIV-1. We have shown that inhibition of ATR leads to complete supression of Vpr's ability to transactivate the viral promoter. Therefore, we hypothesize that Vpr will be able to induce transactivation in dividing cells (activated T-cells) but not innon- dividing cells (MDM). We also hypothesize that ATR activation by Vpr, when allowed, will result in enhanced rate of viral replication.