Role of Pur-alpha in Viral CNS Interaction

中枢神经系统的HIV-1感染诱导多种临床异常，包括 痴呆共济失调和记忆丧失进行性多灶性白质脑病（PML）是HIV-1感染最常见的神经系统并发症之一。PML是一种致命的脱髓鞘疾病，由人嗜神经性多瘤病毒JCV的再活化及其对少突胶质细胞和星形胶质细胞的感染引起。PML曾经是一种罕见的疾病，但艾滋病患者中PML的发病率较高，这表明HIV-1和JCV在大脑中存在交叉交流。分子生物学和病毒学研究的结果已经确定了达特增加JCV基因组的能力。这一事件至少部分需要一种细胞蛋白 Pur-alpha是一种单链DNA和RNA结合蛋白，其表达在大脑发育过程中受到控制。Pur-alpha还刺激HIV-1基因表达，并且其与达特的结合增强LTR的达特活化。此外，Pur-alpha通过与JCV早期蛋白T抗原相互作用来控制JCV DNA复制和神经胶质细胞中的基因表达。Pur-alpha有一个不寻常的结构特征， 除了核酸之外，还有各种重要的细胞蛋白质。动物模型中Pur-alpha的消融导致大脑发育不完全。我们的初步观察表明，Pur-alpha能够控制细胞周期进程，并延长DNA受损的细胞处于S期的时间。另一方面，Pur-alpha已经显示出与Rad 51（同源重组途径中的关键因子）相互作用的能力，降低Rad 51基因转录水平，并且干扰Ku 70（非同源重组途径的主要组分之一）的功能。 末端连接途径这些观察结果归因于Pur-alpha作为DNA修复的看门人的新角色，其通过调节细胞周期进程以及参与细胞DNA修复机制的因子的表达和活性水平，确保高效和适当的DNA修复具有高水平的保真度和准确性。支持这一观察结果源于我们的结果显示，大量的染色体异常与缺乏Pur-alpha的细胞中的功能失调修复相关。根据这些观察结果，可以设想一种模型，其中Pur-alpha与JCV调节蛋白、T抗原和免疫球蛋白的物理相互作用是相互作用的。 HIV-1反式激活蛋白达特在HIV-1感染和JCV再激活过程中对Pur-alpha在DNA修复中发挥作用的能力具有功能性影响。在这个项目中，实验拟描绘Pur-alpha对星形胶质细胞和少突胶质细胞中DNA修复机制的影响，并确定JCV和HIV-1对Pur-alpha在细胞周期调控和基因组稳定性中功能的影响。 这些研究的结果将提供重要的信息，可用于更好地了解两种不同的病毒通过细胞蛋白的间接通信及其在CNS疾病进展中的合作作用。

HIV-1 infection of the central nervous system induces a variety of clinical abnormalities including dementia, ataxia, and memory loss. Progressive multifocal leukoencephalopathy (PML) represents one of the most common neurological complications of HIV-1 infection. PML is a fatal demyelinating disease that results from the reactivation of the human neurotropic polyomavirus, JCV, and its infection of oligodendrocytes and astrocytes. Once a rare disorder, the higher incidence of PML among AIDS patients suggests cross-communication between HIV-1 and JCV in the brain. Results from molecular biology and virology studies have established the ability of Tat to augment the JCV genome. This event requires, at least in part, a cellular protein named Pur-alpha, a single stranded DNA and RNA binding protein whose expression is controlled during brain development. Pur-alpha also stimulates HIV-1 gene expression and its association with Tat augments Tat activation of the LTR. Furthermore, Pur-alpha controls JCV DNA replication and gene expression in glial cells by interacting with the JCV early protein, T-antigen. Pur-alpha has an unusual structural feature allowing the protein to interact with various important cellular proteins in addition to nucleic acids. Ablation of Pur-alpha in animal models causes incomplete brain development. Our preliminary observations have shown the ability of Pur-alpha to control cell cycle progression and prolong cells with damaged DNA in S-phase. On the other hand, Pur-alpha has shown the ability to interact with Rad51 (a key factor in the homologous recombination pathway), decrease the level of Rad51 gene transcription, and interfere with the function of Ku70, one of the major components of the non-homologous end-joining pathway. These observations ascribed a new role for Pur-alpha as a gatekeeper of DNA repair, which ensures the efficient and appropriate repair of DNA with high levels of fidelity and accuracy by modulating cell cycle progression and the level of expression and activity of factors involved in cellular DNA repair machinery. Support for this observation stems from our results showing substantial chromosomal abnormalities associated with dysfunctional repair in cells lacking Pur-alpha. In light of these observations one can envision a model in which the physical interaction of Pur-alpha with the JCV regulatory protein, T-antigen, and the HIV-1 transactivator protein, Tat, will have a functional consequence on the ability of Pur-alpha to execute its role in DNA repair during the course of HIV-1 infection and JCV reactivation. In this project, experiments are proposed to delineate the effect of Pur-alpha on DNA repair mechanisms seen in astrocytes and oligodendrocytes, and determine the impact of JCV and HIV-1 upon Pur-alpha functions in cell cycle regulation and genomic stability. The outcome of these studies will provide important information which can be utilized to better understand the indirect communication of two distinct viruses by cellular proteins and their cooperative role in the progression of diseases in the CNS.