GENE EXPRESSION STUDIES USING BRAIN DERIVED HIV1 LTRS

人类免疫缺陷病毒(HIV-1)侵袭中枢神经 系统(CNS)感染后早期，导致缓慢进展 痴呆，获得性免疫缺陷综合征痴呆复合体 (ADC)在相当数量的患者中。鉴于HIV-1的规模 感染，ADC是美国最常见的痴呆症原因之一， 尤其是在年轻人身上。大脑中的病毒载量与ADC相关 以及HIV-1脑炎的病理特征，但这些区域 病毒基因组与在中枢神经系统内复制的能力有关 还没有被完全定义。我们之前已经表明， 用HIV-1长末端重复序列构建转基因小鼠 JR-CSF和JR-FL菌株，来自于一例脑炎患者的中枢神经系统 ADC，在中枢神经系统表达一个报告基因，而小鼠用 而血源性IIB LTR则不能。这表明DNA序列 可能在一定程度上对组织特异性基因负有责任 HIV-1所显示的表达和复制。为了帮助定义这些 序列，我们已经分析了从人脑中分离出来的56个LTR克隆 四名HIV-1感染者。在这些样本中，我们发现至少有五个 LTRS的独立准种，具有显著的变异量 在大脑样本内部和之间都可以看到。绝大多数人 核因子-kappaB和Sp1转录上游发生变异 在核因子-AT和Lef-1结合内的LTR中的因子结合位点 以及转录因子结合之外的几个位置 网站。在许多这样的地方，我们的大脑衍生的LTR克隆共享 与中枢神经系统衍生的JR-CSF和JR-FL LTRS的独特替代，在 与血源性IIIB LTR的比较。我们的假设是，一个或 HIV-1 LTR上游DNA序列的更多替换 对组织特异的，可能还有细胞特异的基因有贡献 在中枢神经系统内表达。这项提案的目标是界定 与不同水平基因相关的特定LTR序列变异 转录因子在不同细胞类型中的表达和结合 与中枢神经系统有关。我们已经构建了表达 不同HIV-1基因调控下的报告基因β-半乳糖苷酶 LTRS，包括IIIB、JR-FL和OUT脑源性LTRS的子集 从这五个准种中的每一个。我们已经开始了短暂的 将这些载体导入培养的T淋巴细胞、巨噬细胞、 星形胶质细胞和神经元，寻找基因表达的差异 在不同细胞类型的不同克隆之间。最后，我们将表演 核糖核酸酶保护和凝胶迁移率变化分析 从上述细胞中提取，以确定LTR的特定区域 它们以不同的方式与它们的核蛋白结合。这些研究将 确定与细胞特异性基因表达相关的HIV-1 LTR区域 ，并有可能提供攻击HIV-1的新策略 中枢神经系统感染与青少年痴呆的重要原因 美国人。

The human immunodeficiency virus (HIV-1) invades the central nervous system (CNS) early after infection, causing a slowly progressive dementia, the acquired immunodeficiency syndrome dementia complex (ADC) in a significant number of patients. Given the magnitude of HIV-1 infection, ADC is one of the most common causes of dementia in the US, especially in the young. Viral load in the brain correlates with ADC and the pathologic hallmarks of HIV-1 encephalitis, but those regions of the viral genome associated with ability to replicate within the CNS have not been completely defined. We have shown previously that transgenic mice constructed with the HIV-1 long terminal repeat (LTR) of strains JR-CSF and JR-FL, derived from the CNS of a patient with ADC, express a reporter gene in the CNS, while mice constructed with the blood-derived IIB LTR do not. This suggests that DNA sequences within the LTR may be responsible, in part, for tissue-specific gene expression and replication displayed by HIV-1. To help define these sequences, we have analyzed 56 LTR clones isolated from the brains of four HIV-1-infected patients. In these samples, we found at least five separate quasispecies of LTRs, with a significant amount of variation seen both within and between brain samples. The vast majority of variation occurred upstream of the NF-kappaB and Sp1 transcription factor binding sites in the LTR, within the NF-AT and LEF-1 binding sites, and at several locations outside of transcription factor binding sites. At many of these locations, our brain-derived LTR clones shared unique substitutions with the CNS-derived JR-CSF and JR-FL LTRs, in comparison to blood-derived IIIB LTR. Our hypothesis is that one or more substitutions in the upstream DNA sequences of the HIV-1 LTR contribute to tissue-specific, and, possibly, cell-specific, gene expression within the CNS. The goal of this proposal is to define specific LTR sequence variations associated with varying levels of gene expression and transcription factor binding in different cell types relevant to the CNS. We have constructed vectors expressing the reporter gene beta-galactosidase under the control of various HIV-1 LTRs, including IIIB, JR-FL and a subset of out brain-derived LTRs from each of the five quasispecies. We have begun transiently transfecting these vectors into cultured T lymphocytes, macrophages, astrocytes and neurons, looking for differences in gene expression between various clones in different cell types. Finally, we will perform DNase protection and electrophoretic mobility shift assays using nuclear extracts from the above cells to define the specific regions of the LTR which differentially bind to their nuclear proteins. These studies will define HIV-1 LTR regions associated with cell-specific gene expression in the CNS, and potentially provide novel strategies to attack HIV-1 infection in the CNS, and important cause of dementia in young Americans.