Tat and CDK9 on gene expression alterations coupled to HIV-1 Associated Dementia

描述(申请人提供)：痴呆症和其他较轻微形式的中枢神经系统认知疾病影响感染艾滋病毒-1的患者。虽然HAART已经减少了HIV-1相关痴呆(HAD)的发生率，但较轻微的神经系统疾病仍然存在。此外，HAART导致的存活率提高也导致了神经疾病患病率的增加。HIV诱导的神经变性可能至少部分是由HIV-1 Tat诱导的多种细胞类型的细胞基因表达的变化造成的，包括星形胶质细胞和神经元。TAT是一种重要的病毒反式激活因子，需要与一种由细胞周期蛋白T1调节亚基和CDK9催化亚基组成的细胞激酶结合。这个宿主复合体被TAT招募到TAR，TAR是新生的HIV-1转录本中的一种RNA元件，CDK9在那里被定位为磷酸化RNA聚合酶II(RNAPII)和负的延伸因子，强烈激活HIV-1转录。CDK9与T型细胞周期蛋白结合形成正转录延伸因子(P-TEFb)，被认为是细胞基因表达所必需的。我们研究了限制CDK9活性对人脑星形细胞瘤细胞系的影响。我们的基因图谱分析表明，CDK9的活性并不是细胞基因表达所必需的，而是抑制CDK9导致基因表达的选择性变化。在下调的基因中，我们鉴定了多种转录因子。我们的数据还显示，抑制CDK9导致包括应激和刺激反应基因(Gadd45A、Gadd45B、IL-8和IL-12)在内的一组基因上调，以及一些与痴呆相关的基因(GLS、SOD2、FGF2和CHMP2B)的解除调控。细胞CDK9被隔离在催化活性和活性复合体中，处于一种平衡状态，以限制基因表达。重要的是，TAT通过与特定转录激活物的竞争机制抑制巨噬细胞、树突状细胞和B细胞中依赖于细胞周期蛋白T1/CDK9的某些基因的表达。由于TAT在HIV-1感染的HeLa细胞和表达原代人类星形胶质细胞和星形细胞瘤的细胞中重新配置CDK9复合体池，因此很可能与其他转录因子在星形胶质细胞和神经元中发挥类似的竞争机制。因此，这个探索性的R21提案的目标是检验这样一种假设，即TAT在基因表达中可能导致HAD的影响至少部分是通过干扰宿主CDK9功能来介导的，并且恢复正常的CDK9功能应该可以改善TAT表达和HIV-1在中枢神经系统感染的有害影响。为了验证这一假说，我们提出：(目的1)确定细胞CDK9在HIV-1 TAT引发的星形胶质细胞和神经元基因表达变化中的作用；(目的2)确定TAT在基因表达方面的影响是否可以通过恢复表达TAT的星形胶质细胞和神经元的正常细胞CDK9功能来逆转。这些研究的短期好处是深入了解TAT重塑星形胶质细胞和神经元中导致神经退化的细胞基因表达的分子机制。这些研究的结果可能为后续的结构研究开辟道路，寻找能够结合细胞周期蛋白T1阻断TAT而不改变依赖CDK9的细胞基因表达的小分子化合物。在星形胶质细胞和神经元公共卫生方面：星形胶质细胞是大脑中含量最丰富的细胞类型，对神经元功能、存活和神经发生至关重要。星形胶质细胞被HIV-1感染，并产生包括Tat在内的病毒蛋白，但病毒不能有效复制。考虑到它们的丰富性和重要性，星形胶质细胞是一个非常重要的受感染细胞的储存库，这些细胞表达具有神经毒性的HIV-1蛋白。这些蛋白可能影响星形胶质细胞的动态平衡，进而影响神经元的功能，或者可能被神经元分泌和摄取，直接影响它们对HAD的贡献。HIV-1Tat是一种重要的病毒反式激活因子，它可以劫持细胞周期蛋白T1/CDK9转录延伸因子，促进HIV-1的复制。TAT还通过改变免疫细胞功能来改变细胞基因表达，从而参与HIV-1相关的发病机制。由于TAT的表达重排了人脑星形胶质细胞中的P-TEFb复合体，这些复合体被认为在转录中起着限速作用，因此假设TAT对P-TEFb的解除调控会导致星形胶质细胞和神经元中基因表达的选择性变化，从而导致HIV-1相关性痴呆。我们建议检验这样一种假设，即TAT对基因表达的影响至少部分是通过干扰宿主CDK9功能来介导的，并且恢复正常的CDK9功能应该可以改善TAT表达和HIV-1在中枢神经系统感染的不利影响。

DESCRIPTION (provided by applicant): Dementia and other milder forms of cognitive diseases of the CNS affect HIV-1 infected patients. While HAART has diminished the incidence of HIV-1-associated dementia (HAD), milder forms of neurological disease persist. Also, increased survival rates resulting from HAART have led to an increase in the prevalence of neurological disorders. HIV-induced neurodegeneration may result at least in part from changes in cellular gene expression induced by HIV-1 Tat in a variety of cell types including astrocytes and neurons. Tat is an essential viral transactivator that requires association with a cellular kinase composed of a regulatory subunit designated cyclin T1 and a catalytic subunit named CDK9. This host complex is recruited by Tat to TAR, an RNA element in the nascent HIV-1 transcript, where CDK9 is positioned to phosphorylate RNA polymerase II (RNAPII) and negative elongation factors, strongly activating HIV-1 transcription. CDK9 in association with T-type cyclins forms Positive Transcription Elongation Factors (P-TEFb) thought to be required for the expression of cellular genes. We have investigated the effect of limiting CDK9 activity in a human astrocytoma cell line. Our gene profiling analysis shows that CDK9 activity is not homogeneously required for the expression of cellular genes, but rather that inhibition of CDK9 leads to selective changes in gene expression. Among the genes downregulated we have identified various transcription factors. Our data also show that inhibition of CDK9 results in the upregulation of a subset of genes including stress and stimuli response genes (Gadd45A, Gadd45B, IL-8 and IL-12), as well as deregulation of a number of genes that have been associated with dementia (GLS, SOD2, FGF2 and CHMP2B). Cellular CDK9 is sequestered in catalytically inactive and active complexes in an equilibrium that is in place to restrict gene expression. Importantly, Tat inhibits cyclin T1/CDK9-dependent gene expression of certain genes in macrophages, dendritic cells and B cells via a competition mechanism with specific transcriptional activators. Since Tat reconfigures pools of CDK9 complexes in HIV-1 infected HeLa cells and Tat expressing primary human astrocytes and astrocytoma cells, it is likely that similar competition mechanisms operate in astrocytes and neurons with other transcription factors. Thus, the goal of this exploratory R21 proposal is to test the hypothesis that the effects of Tat in gene expression that may contribute to HAD are at least in part mediated through interference with the host CDK9 function and that restoration of normal CDK9 function should ameliorate the detrimental effects of Tat expression and HIV-1 infection in the CNS. To test this hypothesis we propose: (Aim 1) to determine the role of cellular CDK9 in the alterations on gene expression triggered by HIV-1 Tat in astrocytes and neurons; and (Aim 2) to determine whether the effects of Tat in gene expression can be reverted by restoring normal cellular CDK9 function in Tat expressing astrocytes and neurons. The short-term benefit of these studies is gaining insight into the molecular mechanisms by which Tat remodels cellular gene expression in astrocytes and neurons contributing to neurodegeneration. The results of these studies could set the path to subsequent structural studies to search for small compounds with the ability to bind cyclin T1 blocking Tat without altering CDK9 dependent cellular gene expression. in astrocytes and neurons PUBLIC HEALTH RELEVANCE: Astrocytes are the most abundant cell type in the brain and are critical for neuronal function, survival and neurogenesis. Astrocytes are infected by HIV-1, and viral proteins including Tat are made, but the virus does not productively replicate. Given their abundance and importance, astrocytes represent a very significant reservoir of infected cells that express HIV-1 proteins with neurotoxic properties. These proteins may affect astrocyte homeostasis, which in turn will influence neuronal function or might be secreted and taken up by neurons to directly affect them contributing to HAD. HIV-1 Tat is an essential viral transactivator that hijacks the cellular cyclin T1/CDK9 transcription elongation factor to promote HIV-1 replication. Tat also alters cellular gene expression, which contributes to HIV-1-associated pathogenesis by altering immune cell function. As Tat expression rearranges cellular P-TEFb complexes in human astrocytes, and these complexes are thought to play rate-limiting roles in transcription, it is hypothesized that P-TEFb deregulation by Tat induces selective changes in gene expression in astrocytes and neurons that contribute to HIV-1-associated dementia. We propose to test the hypothesis that the effects of Tat in gene expression are at least in part mediated through interference with the host CDK9 function and that restoration of normal CDK9 function should ameliorate the detrimental effects of Tat expression and HIV-1 infection in the CNS.