Control of HIV-1 Gene Transcription in Brain Cells

在感染过程的不同阶段控制HIV-1基因的转录，需要各种宿主因子和病毒蛋白之间相互作用的微妙平衡。在中枢神经系统(CNS)中，星形胶质细胞等神经源性细胞和巨噬细胞/单核细胞系的外周细胞是HIV-1基因组表达的主要部位。然而，在CNS细胞感染周期中调节病毒基因组表达的机制尚不清楚，即病毒基因组的基础转录由宿主因子启动的早期阶段；宿主因子与病毒反式激活因子TAT协同作用的早期阶段；以及其他病毒调控蛋白如VPR参与病毒基因转录的晚期阶段。HIV-1感染中枢神经系统细胞伴随着维持宿主功能所需的几个关键通路的诱导/失调。Tat是一种在影响宿主功能方面具有既定作用的病毒蛋白，它能够与几种调节因子沟通，增强HIV-1 LTR的转录，并改变宿主基因组的表达水平。 我们最近的结果表明，在HIV-1脑病(HIVE)患者的脑细胞中，RAD51上调，这是一种与同源重组有关的细胞基因。此外，体外研究表明，TAT能够提高星形胶质细胞和神经元培养中RAD51的表达。由于RAD51的升高可能直接或间接地影响HIV-1基因的表达和复制，我们研究了在RAD51水平升高的情况下病毒启动子的转录，并证明了RAD51能够增加基础和 TAT诱导的HIV-1基因组表达。对我们的缺失突变体的初步观察显示，跨越-120到-80的LTR序列在人类星形胶质细胞中LTR的RAD51激活中具有重要意义，这表明C/EBP家族和NF-kappaB等一系列转录因子的参与，它们的活性至少部分是通过它们与DNA序列的相互作用来调节的。因此，我们假设RAD51与C/EBPbeta和NF-kappaB家族等转录激活子的交叉通讯以及TAT结果 在正反馈调节事件中，导致刺激中枢神经系统细胞中HIV-1基因的表达。为了验证我们的假设，我们将使用最先进的分子生物学技术来破译RAD51与各种细胞蛋白(如C/EBPbeta、CHOP、NF-kappaB和P53)以及病毒蛋白(如TAT和VPR)的合作相互作用，并评估它们在病毒基因表达的早期、早期和晚期对HIV-1基因组的影响。这些研究的结果将提供重要的信息，可用于目前艾滋病患者的治疗和设计更有效的分子策略来抑制中枢神经系统中的病毒基因表达。

Control of HIV-1 gene transcription at different stages during the course of infection requires a delicate balance of interaction among various host factors and viral proteins. In the central nervous system (CNS), cells of neural origin such as astrocytes and peripheral cells of macrophage/monocytic lineage are the primary sites for the expression of the HIV-1 genome. However, the mechanism which modulates expression of the viral genome during the infection cycle in CNS cells, i.e. the immediate early stage when basal transcription of the viral genome is initiated by host factors; at the early stage when host factors cooperate with the viral transactivator, Tat; and at the late stage when other viral regulatory proteins such as Vpr participate in viral gene transcription, remains unknown. HIV-1 infection of CNS cells is accompanied by induction/dysregulation of several critical pathways required for the maintenance of host function. One of the viral proteins that has an established role in affecting host function is Tat, which has the ability to communicate with several regulatory factors to augment transcription of the HIV-1 LTR and alters the level of expression from the host genome. Our recent results demonstrate upregulation of Rad51, a cellular gene which is implicated in homologous recombination, in brain cells of patients with HIV-1 encephalopathy (HIVE). Further, in vitro studies revealed the ability of Tat to elevate Rad51 expression in astrocytes and neuronal culture. As the elevation in Rad51 may affect, directly or indirectly, HIV-1 gene expression and replication, we examined transcription of the viral promoter in the presence of an elevated level of Rad51 and demonstrated the ability of Rad51 to increase basal-and Tat-induced expression of the HIV-1 genome. Preliminary observations from our deletion mutants revealed the importance of the LTR sequence spanning -120 to -80 in Rad51 activation of the LTR in human astrocytes suggesting the involvement of a series of transcription factors such as C/EBP family and NF-kappaB, whose activities are mediated, at least in part, through their interaction with the DNA sequence. Thus, we hypothesize that cross-communication of Rad51 with transcription activators such as C/EBPbeta and NF-kappaB families and Tat results in a positive feedback regulatory event that leads to stimulation of the HIV-1 gene expression in CNS cells. To test our hypothesis we will employ a comprehensive approach using state of the art techniques of molecular biology to decipher the cooperative interaction of Rad51 with various cellular proteins such as C/EBPbeta, CHOP, NF-kappaB, and p53, and viral proteins such as Tat and Vpr and assess their impact on the HIV-1 genome at the immediate early, early, and late phases of viral gene expression. The outcome of these studies will provide important information which can be utilized in the current treatment of AIDS patients and in devising more effective molecular strategies toward inhibition of viral gene expression in the CNS.