GENE EXPRESSION STUDIES USING BRAIN DERIVED HIV1 LTRS

人类免疫缺陷病毒（HIV-1）侵入中枢神经系统， 系统（CNS）感染后早期，引起缓慢进行性 获得性免疫缺陷综合征痴呆综合征 (ADC)在很多病人身上。考虑到艾滋病病毒的严重性 感染，ADC是美国痴呆症最常见的原因之一， 尤其是年轻人 脑中的病毒载量与ADC相关 和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 还没有完全定义。我们之前已经证明， 用HIV-1长末端重复序列（LTR）构建的转基因小鼠 菌株JR-CSF和JR-FL，来源于患有 ADC在CNS中表达报告基因，而构建了ADC的小鼠在CNS中表达报告基因。 血源性IIB LTR则不然。这表明DNA序列 LTR内的基因可能部分负责组织特异性基因， HIV-1的表达和复制。为了帮助定义这些 序列，我们已经分析了56个LTR克隆分离自大脑， 四名HIV-1感染者在这些样本中，我们发现了至少五个 LTR的独立准种，具有显著的变异量 在大脑样本内部和之间都能看到。绝大多数 在NF-κ B和Sp1转录的上游发生变异 LTR中的因子结合位点，在NF-AT和LEF-1结合内 位点，以及转录因子结合外的几个位置 网站.在许多这些位置，我们的脑源性LTR克隆共享 CNS衍生的JR-CSF和JR-FL LTR的独特取代， 与血液来源的IIIB LTR相比。我们的假设是， 在HIV-1 LTR的上游DNA序列中有更多的取代 有助于组织特异性的，并且可能是细胞特异性的基因 在CNS内表达。该提案的目的是定义 与不同基因水平相关的特异性LTR序列变异 在不同细胞类型中的表达和转录因子结合 与CNS有关。我们构建了表达 报告基因β-半乳糖苷酶控制下的各种HIV-1 LTR，包括IIIB、JR-FL和外脑源性LTR的子集 五个准物种的基因我们已经开始短暂 将这些载体转染到培养的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 DNA酶保护和电泳迁移率变动分析，使用核 从上述细胞中提取以定义LTR的特定区域 它们与核蛋白的结合有差异。这些研究将 定义与细胞特异性基因表达相关的HIV-1 LTR区域 在中枢神经系统，并可能提供新的战略，攻击艾滋病毒-1 中枢神经系统感染，是年轻人痴呆的重要原因 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.