GENE EXPRESSION STUDIES USING BRAIN DERIVED HIV1 LTRS

使用脑源性 HIV1 LTRS 进行基因表达研究

• 批准号: 2431021
• 负责人: JOHN R CORBOY
• 金额: $ 7.55万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2431021
• 关键词: AIDS dementia complex; DNA binding protein; DNA footprinting; T lymphocyte; astrocytes; gel mobility shift assay; gene expression; human immunodeficiency virus 1; interleukin 1; macrophage; nervous system infection; neurons; nucleic acid repetitive sequence; pathologic process; tissue /cell culture; transcription factor; transfection; tumor necrosis factor alpha

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.

人免疫缺陷病毒（HIV-1）侵入中枢神经 感染后的系统（CNS），导致缓慢进行 痴呆症，获得的免疫缺陷综合征痴呆症复合物 （ADC）在大量患者中。考虑到HIV-1的大小 感染，ADC是美国最常见的痴呆症原因之一， 特别是在年轻人中。 大脑中的病毒负荷与ADC相关 以及HIV-1脑炎的病理标志，但 与CNS内复制能力相关的病毒基因组 尚未完全定义。我们之前已经表明 用HIV-1长末端重复（LTR）构建的转基因小鼠 菌株JR-CSF和JR-FL的菌株，源自患者的中枢神经系统 ADC，在中枢神经系统中表达一个记者基因，而小鼠则用 血液来源的IIB LTR没有。这表明DNA序列 在LTR内可能部分负责组织特异性基因 HIV-1显示的表达和复制。帮助定义这些 序列，我们分析了从大脑中分离出的56个LTR克隆 四名HIV-1感染患者。在这些样品中，我们至少发现了五个 LTR的单独的准菜单，有很大的变化 在大脑样品之间和之间都可以看到。绝大多数 变异发生在NF-kappab和SP1转录的上游 NF-AT和LEF-1结合中LTR中的因子结合位点 站点，在转录因子结合以外的几个位置 站点。在许多这些位置，我们的大脑衍生的LTR克隆共享 用CNS衍生的JR-CSF和JR-FL LTRS替换 与血液衍生的IIIB LTR进行比较。我们的假设是一个或 HIV-1 LTR的上游DNA序列中的更多取代 有助于组织特异性和可能的​​细胞特异性基因 中枢神经系统中的表达。该提议的目的是定义 特定的LTR序列变化与不同水平的基因相关的变化 不同细胞类型的表达和转录因子结合 与中枢神经系统有关。我们已经构建了表达的向量 在各种HIV-1的控制下，报告基因β-半乳糖苷酶 LTR，包括IIIB，JR-FL和脑衍生的LTR的子集 从五个准菜单中的每个。我们已经暂时开始 将这些向量转染到培养的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 在不同细胞类型的各种克隆之间。最后，我们将表演 DNase保护和电泳移动性转移测定法 从上述细胞中提取以定义LTR的特定区域 它与其核蛋白的差异结合。这些研究会 定义与细胞特异性基因表达相关的HIV-1 LTR区域 在中枢神经系统中，并有可能提供攻击HIV-1的新型策略 中枢神经系统的感染，以及年轻的痴呆症的重要原因 美国人。