Induction of Apoptosis by HIV-1 vpr

描述（由申请人提供）：HIV-1附属基因Vpr编码一个保守的96个氨基酸的蛋白，诱导细胞周期在62期阻滞。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ATR激酶是介导vpr诱导的细胞周期阻滞和凋亡的细胞因子。我们还证明，诱导G2阻滞和细胞凋亡需要ATR下游靶点的磷酸化，如Chk1、BRCA1和GADD45alpha。因此，ATR激酶是vpr诱导发病机制的关键决定因素。本提案的主要重点将是阐明Vpr激活ATR的确切机制。体内和体外感染HIV-1的巨噬细胞比感染、活化的t细胞寿命更长。我们发现巨噬细胞中Vpr的表达不能诱导凋亡，我们推测其根本原因是Vpr不能激活这些细胞中的ATR。在活化的t细胞和巨噬细胞中，Vpr诱导凋亡的差异能力与一种新兴模型完全一致，该模型认为ATR的激活需要持续的细胞DMA复制。Vpr对ATR的激活也会导致病毒基因表达的增强，这就解释了Vpr在T细胞中具有适度的反激活活性。本建议的具体目标是：阐明Vpr激活ATR的机制。ATR的功能是调查细胞DNA复制并检测停止复制的分叉。我们将研究Vpr是否通过影响DNA完整性、阻碍宿主细胞复制或直接激活ATR信号复合体来激活ATR。具体目标2。探讨Vpr对单核细胞源性巨噬细胞（MDM） ATR的影响。我们观察到MDM对vpr诱导的细胞凋亡不耐受，但对其他促凋亡刺激不耐受。我们假设Vpr在MDM中由于它们的非分裂状态而无法激活ATR。我们还假设，依赖于ATR激活的vpr诱导的交易激活不会发生在MDM中。目标3。评估ATR活化对HIV-1复制动力学的贡献。我们已经证明ATR的抑制导致Vpr反激活病毒启动子的能力完全被抑制。因此，我们假设Vpr能够在分裂细胞（活化的t细胞）中诱导转激活，而在非分裂细胞（MDM）中则不能。我们还假设，在允许的情况下，Vpr激活ATR将导致病毒复制率的提高。

DESCRIPTION (provided by applicant): 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 GADD45alpha. 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 in non-dividing cells (MDM). We also hypothesize that ATR activation by Vpr, when allowed, will result in enhanced rate of viral replication.