ALTERED GENE PRODUCTS IN HIV-1 INFECTED ASTROCYTES

星形胶质细胞可能是艾滋病神经发病机制中的一个重要靶点。 以前的研究(项目1；沃尔斯基)表明，艾滋病毒-1感染 星形胶质细胞，暴露于gp120或表达Tat抑制摄取 神经递质谷氨酸。这一修改后的申请建议澄清 基因表达变化的结果是 HIV-1感染星形胶质细胞或暴露于纯化的gp120或Tat 并鉴定控制GLAST表达的调控序列 谷氨酸转运体(GT)。这些信息将提供重要的 HIV-1致艾滋病脑病机制的研究进展 儿童和成人的痴呆症。此外，一旦调节分子和 介导HIV-1诱导的星形胶质细胞功能障碍的途径已经确定，它 可能会利用这些信息来开发改进的 治疗艾滋病痴呆的干预策略。一个敏感而高效的 减影杂交法(酱&Fisher，1993)将用于 鉴定和克隆表达改变的cDNA，无论是诱导 抑制，作为感染星形胶质细胞的功能 嗜星形细胞HIV-1毒株(A-HIV-1)，即优先 感染星形胶质细胞，而不是T细胞或巨噬细胞(项目1；沃尔斯基)。 特定全长cDNA在HIV-1调控中的功能作用 对星形胶质细胞的毒性将用瞬变和稳定来研究 正义和反义表达载体及新型逆转录病毒载体 反义功能基因敲除策略、快速反义基因 灭绝(愤怒)。为了全面回应评论家的评论，我们的研究 计划已被广泛修订，包括增加了一个新的目标 研究GLAST的表达调控。具体来说，我们建议：(1) 构建临时间隔消减(TSS)文库 感染A-HIV-1的星形胶质细胞；(2)使用反向Northern杂交 识别表达增强和降低的cDNA的程序为 A-HIV-1感染星形胶质细胞的作用及其治疗 纯化的gp120或tat；(3)分离表达基因的全长cDNA A-HIV-1感染星形胶质细胞后基因表达的变化 并用纯化的gp120或tat处理；(4)检测 特异性A-HIV-1基因调控星形胶质细胞的生理功能 感染A-HIV-1或使用纯化的gp120或 以及(5)鉴定和鉴定GLAST GT启动子和顺式- 控制基因表达的作用和反式作用调控元件 这个基因。这项研究将导致确定潜在的 与HIV-1诱导的神经退行性变相关的重要基因 疾病。这些遗传试剂和获得的信息将证明 对本计划项目中研究特定项目的其他研究人员有用 HIV-1相关脑病的几个方面。

Astrocytes may represent an important target in AIDS neuropathogenesis. Previous studies (Project 1; Volsky) demonstrated that HIV-1 infection of astrocytes, exposure to gp120 or expression of Tat inhibit uptake of the neurotransmitter glutamate. This revised application proposes to elucidate the repertoire of gene expression changes induced as a consequence of infection of astrocytes with HIV-1 or exposure to purified gp120 or Tat and to identify the regulatory sequences governing expression of the GLAST glutamate transporter (GT). This information will provide important insights into the mechanism by which HIV-1 induces AIDS encephalopathy in children and dementia in adults. Moreover, once regulatory molecules and pathways mediating HIV-1-induced astrocyte dysfunction are identified, it may be possible to exploit this information to develop improved intervention strategies to treat AIDS dementia. A sensitive and efficient subtraction hybridization protocol (Jiang & Fisher, 1993) will be used to identify and clone cDNAs displaying altered expression, either induction of suppression, as a function of infection of astrocytes with astrocytotropic HIV-1 strains (A-HIV-1), i.e., viruses that preferentially infect astrocytes rather than T cells or macrophages (Project 1; Volsky). The functional role of specific full-length cDNAs in modulating HIV-1 toxicity in astrocytes will be investigated using transient and stable sense and antisense expression constructs and a novel retroviral-based antisense functional gene knock-out strategy, Rapid Antisense Gene Extinction (RAGE). To fully respond to Reviewer's comments, our research plan has been extensively revised, including addition of a new aim to study regulation of GLAST expression. Specifically, we propose to: (1) construct temporarily spaced subtracted (TSS) cDNA libraries from astrocytes infected with A-HIV-1; (2) use reverse Northern hybridization procedures to identify cDNAs displaying enhanced and reduced expression as a function of infection of astrocytes with A-HIV-1 and treatment with purified gp120 or Tat; (3) isolate full-length cDNAs of genes displaying altered expressions as a function of infection of astrocytes with A-HIV-1 and treatment with purified gp120 or Tat; (4) test the functionality of specific A-HIV-1 modulated genes in regulating astrocyte physiology as a consequence of infection with A-HIV-1 or treatment with purified gp120 or Tat; and (5) identify and characterize the GLAST GT promoter and cis- acting and trans-acting regulatory elements that control expression of this gene. This studies will result in the identification of potentially important genes that correlate with HIV-1 induced neurodegenerative diseases. These genetic reagents and the information obtained will prove useful to other investigators in this program project studying specific aspects of HIV-1 associated encephalopathy.