Induction of Apoptosis by HIV-1 vpr

HIV-1 vpr 诱导细胞凋亡

• 批准号: 7545468
• 负责人: VICENTE PLANELLES
• 金额: $ 32.04万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-15 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7545468
• 关键词: Affect; Agreement; Amino Acids; Apoptosis; Apoptotic; BRCA1 gene; CD4 Positive T Lymphocytes; Cell Cycle; Cell Cycle Arrest; Cells; Complex; DNA; DNA biosynthesis; G2 Phase; Gene Expression; HIV-1; In Vitro; Induction of Apoptosis; Interphase Cell; Kinetics; Life; Mediating; Modeling; Pathogenesis; Phase; Phosphorylation; Phosphotransferases; Proteins; Refractory; Signal Transduction; Stimulus; Surveys; T-Lymphocyte; Transactivation; Viral; Viral Genes; in vivo; macrophage; monocyte; promoter; vpr Genes

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.

HIV-1辅助基因VPR编码诱导细胞块的保守的96-氨基酸蛋白 在62阶段的循环。 VPR在CD4+淋巴细胞中的表达也诱导凋亡。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。我们也有 证明G2停滞和凋亡的诱导需要ATR的下游靶标的磷酸化， 例如CHK1，BRCA1和GADD45A。因此，ATR，激酶代表VPR诱导的关键决定因素 发病。该提案的主要重点是阐明VPR的确切机制 激活ATR。 与感染，激活相比，感染HIV-1的巨噬细胞长期存在 T细胞。我们发现VPR在巨噬细胞中的表达无法诱导凋亡，我们假设 根本的原因是VPR无法激活这些单元中的ATR。 VPR诱导的差异能力 活化的T细胞与巨噬细胞的凋亡完全与新兴模型完全吻合。 ATR的激活需要持续的细胞DMA复制。 VPR激活ATR也会导致 增强病毒基因表达，解释了t-中VPR的中等反式激活活性 细胞。 该提案的具体目的是： 特定目的1。阐明VPR激活ATR的机制。 ATR的功能是调查 细胞DNA复制并检测停滞的复制叉。我们将检查VPR是否引起激活 通过影响DNA的完整性，阻碍宿主细胞复制或激活ATR信号，ATR 复合物直接。 具体目的2。探索VPR对单核细胞衍生的巨噬细胞（MDM）中ATR的影响。我们有 观察到MDM对VPR诱导的细胞凋亡是难治性的，但不是其他促凋亡刺激。我们 假设VPR由于其非个人状态而无法在MDM中激活ATR。我们也假设 MDM中不发生VPR诱导的反式激活，这取决于ATR激活。 目的3。评估ATR激活对HIV-1复制动力学的贡献。我们已经表明 抑制ATR会导致VPR反式激活病毒启动子的能力完全抑制。因此，我们 假设VPR将能够在分裂细胞（激活的T细胞）中诱导反式激活 分裂细胞（MDM）。我们还假设，允许VPR激活ATR将导致速率提高 病毒复制。