Induction of Apoptosis by HIV-1 vpr

描述（由申请人提供）：HIV-1辅助基因Vpr编码一种保守的96个氨基酸的蛋白质，可诱导细胞周期阻滞在62期。Vpr在CD 4+淋巴细胞中的表达也诱导凋亡。我们已经确定ATR激酶作为介导Vpr诱导的细胞周期阻滞和凋亡的细胞因子。我们还证明了G2期阻滞和凋亡的诱导需要ATR下游靶点的磷酸化，如Chk 1，BRCA 1和GADD 45 α。因此，ATR激酶代表了Vpr诱导的发病机制的关键决定因素。该提案的主要重点是阐明Vpr激活ATR的精确机制。与感染的活化T细胞相比，体内和体外感染HIV-1的巨噬细胞是长寿的。我们发现Vpr在巨噬细胞中的表达不能诱导细胞凋亡，我们推测其根本原因是Vpr不能激活这些细胞中的ATR。Vpr诱导活化T细胞与巨噬细胞凋亡的能力差异与ATR活化需要持续细胞DMA复制的新兴模型完全一致。Vpr对ATR的激活也导致病毒基因表达的增强，这解释了T细胞中归因于Vpr的中度反式激活活性。该提案的具体目标是：具体目标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叉。我们将研究Vpr是否通过影响DNA的完整性、通过阻碍宿主细胞复制或通过直接激活ATR信号复合物来激活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。我们已经观察到MDM对Vpr诱导的细胞凋亡是难治的，但对其他促凋亡刺激不是。我们推测，Vpr是无法激活ATR在MDM中，由于他们的非分裂状态。我们还假设，Vpr诱导的反式激活，这是依赖于ATR激活，不发生在MDM。目标3.评估ATR活化对HIV-1复制动力学的贡献。我们已经表明，ATR的抑制导致Vpr的反式激活病毒启动子的能力的完全抑制。因此，我们假设Vpr将能够诱导分裂细胞（活化的T细胞）中的反式激活，但不能诱导非分裂细胞（MDM）中的反式激活。我们还假设，在允许的情况下，Vpr激活ATR将导致病毒复制率提高。

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.