HIV & CORECEPTORS IN CNS: SINGLE CELL MOLECULAR ANALYSIS

艾滋病病毒

• 批准号: 6392723
• 负责人: Ronald G Collman
• 金额: $ 26.96万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2004-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6392723
• 关键词: AIDS dementia complex; HIV envelope protein; astrocytes; central nervous system; chemokine; clinical research; cytokine receptors; gene expression; human immunodeficiency virus 1; human subject; human tissue; immunocytochemistry; macrophage; microglia; molecular cloning; neurons; neuropathology; polymerase chain reaction; protein protein interaction; receptor expression; virus DNA; virus infection mechanism

DESCRIPTION (adapted from applicant's abstract): The discovery of chemokine receptors as co-receptors for HIV-1 fusion and entry has raised critical questions in the pathogenesis of HIV encepahlopathy. Such questions include the role of CXCR4, which is widely expressed on microglia, macrophages and neurons in the CNS and can mediate viral entry but also functional and neurotoxic effects of the HIV-1 envelope glycoprotein. Other receptors include CCR3, CX3CR1, the cytomegalovirus receptor US28 and APJ. All are highly expressed in mainly on sequences, clones or isolates from CSF or brain tissue. However, CSF variants may not reflect those in parenchyma, and viral species isolated from tissue may not reflect minority variants or can be distinguished from those contaminating the brain from blood cells. Such limitations are an important gap in the understanding of how viruses in vivo interact with chemokine viral co-receptors and how these interactions, suggested by in vitro or non-human animal models, contribute to pathogenesis. The hypothesis being tested is that HIV-1-co-receptor interactions are important in viral compartmentalization and in the pathogenesis in HIV-1 encephalitis (HIVE). In addition, an important role in viral neuropathogenesis may be played by viral variants that are minority species present in specific cells in brain or that that replicate poorly in culture. In this work a novel approach that enables the cloning of viral sequences from individual infected cells in brain using single-cell PCR will be employed. The goals are to define at the individual cell level chemokine receptor interactions of HIV-1 species in brain during HIVE. Specifically the investigators will: (1) Amplify HIV-1 env from individual cells in HIVE autopsy brain and construct functional clones for cell-specific viral analysis: (2) define the use of major, minor and CD4-independent co-receptors by these functional envs: (3) determine their target cell tropism and replication characteristics using pseudotype and recombinant infectious viruses, and (4) use single cell-derived cDNAs to define cell-specific patterns of altered cellular gene expression in infected versus uninfected microglia in brain tissue from HIVE

描述（改编自申请人摘要）：趋化因子的发现 作为HIV-1融合和进入的共受体， HIV脑病发病机制的问题。这些问题包括 CXCR4广泛表达于小胶质细胞、巨噬细胞和神经元， 在中枢神经系统，可以介导病毒进入，但也有功能和神经毒性 HIV-1包膜糖蛋白的作用。其它受体包括CCR 3， CX3CR1、巨细胞病毒受体US 28和APJ。所有这些都高度表达于 主要基于来自CSF或脑组织的序列、克隆或分离物。然而，CSF 变异体可能不反映薄壁组织中的变异体， 组织可能不反映少数变异体，或者可以与那些 血细胞污染了大脑这种限制是一个重要的差距 在理解体内病毒如何与趋化因子病毒相互作用方面， 辅助受体以及这些相互作用如何通过体外或非人类 动物模型，有助于发病机制。正在测试的假设是， HIV-1-共受体相互作用在病毒区室化中很重要， 在HIV-1脑炎（HIVE）的发病机制中。此外，重要的 在病毒神经发病机制中的作用可能由病毒变异体发挥， 存在于大脑中特定细胞或复制细胞中的少数物种 文化差。在这项工作中，一种新的方法可以克隆 使用单细胞PCR从脑中的单个感染细胞获得病毒序列 将被雇用。目标是在单个单元格级别定义 在HIVE期间脑中HIV-1种类的趋化因子受体相互作用。 具体而言，研究人员将：（1）扩增来自个体的HIV-1 env 细胞，并构建细胞特异性功能克隆 病毒分析：（2）定义使用的主要，次要和CD4-独立 这些功能性Env的共受体：（3）决定它们的靶细胞向性 使用假型和重组感染性 病毒，和（4）使用单细胞衍生的cDNA来定义细胞特异性模式 感染与未感染的小胶质细胞中细胞基因表达的改变， Hive的脑组织