MECHANISM OF HIV-1 ENTRY, REPLICATION, AND CYTOPATHOGENIC EFFECT IN NEURAL CELLS

本提案的总体目标是研究生物化学和 在神经细胞中控制HIV-1生命周期的分子机制 体外，以及HIV-1对神经细胞基因表达的影响，作为一种 理解HIV-1嗜神经性及其对艾滋病的贡献的模型 脑病 建议的研究是基于我们最近的发现 该研究确定了HIV-1生命周期的几个不同特征， 神经细胞（神经胶质细胞、星形胶质细胞或神经元来源）与T细胞相比 淋巴细胞或巨噬细胞。 其中包括HIV-1的有效融合 有CD 4阴性神经细胞膜的包膜，有限病毒 复制尽管有效的融合和进入，短暂的高水平 在表达CD 4的神经细胞中产生病毒， 部分独立于达特/TAR的转录 转录系统 我们假设这些特征构成了 HIV-1嗜神经性的基础是允许选择特定的 病毒变异体，在瞬时过程中调节细胞基因表达 高水平的HIV-1 DNA转录，并创造一个可诱导的 神经组织中的病毒 这些直接（尽管非细胞溶解） 巨噬细胞介导的神经元毒性和其他效应的相互作用 艾滋病毒-1感染，将在本项目的其他组成项目中进行调查 计划项目。 项目1的具体目标是：1）确定HIV-1如何进入 确定HIV-1感染在体外神经细胞中的结果; 2） 研究env、nef和vif在HIV-1嗜神经性中的作用， 原代亲神经HIV-1和嵌合神经HIV-1构建体; 3） 研究HIV-1 DNA在神经细胞中的瞬时高水平转录 包括新的达特/TAR非依赖性转录因子， 4）确定HIV-1感染过程中调控的细胞基因 在体外和体内。 拟议的研究将利用HIV-1毒株 以及分子克隆、重组载体、细胞系、其它试剂， 在神经细胞生物学和分子病毒学方面的经验 在过去几年的艾滋病和HIV-1研究中， 实验室 此外，密切合作，交流信息， 和试剂与其他成员的计划项目将是必不可少的 目标1和2（L. Epstein，Project 3; B. Blumberg，Core A; Aim 3（H. Gendelman，Project 2），and Aim 4（L. Sharer，项目4）。 我们希望 拟议的研究将产生重要的新信息， HIV-1感染的机制和在人类神经细胞中的持久性， 对互补性HIV-1的全面研究 神经病理机制在这个程序项目。

The general objective of this proposal is to study the biochemical and molecular mechanisms governing the HIV-1 life-cycle in neural cells in vitro, and the effects of HIV-1 on neural cell gene expression, as a model for understanding HIV-1 neurotropism and its contribution to AIDS encephalopathy. The proposed studies are based on our recent findings which identified several different features of the HIV-1 life cycle in neural cells (of glial, astroglial, or neuronal origin) compared to T lymphocytes or macrophages. These include efficient fusion of HIV-1 envelope with CD4-negative neural cell membranes, limited viral replication in spite of efficient fusion and entry, transient high-level viral production in CD4-expressing neural cells, and efficient HIV-1 DNA transcription that is partially independent of the Tat/TAR transcriptional system. We hypothesize that these features constitute the basis of HIV-1 neurotropism by allowing the selection of specific viral variants, modulating cellular gene expression during transient high-level HIV-1 DNA transcription, and creating a reservoir of inducible virus in the neural tissue. These direct (albeit non-cytolytic) interactions of macrophage-mediated neuronotoxicity and other effects of HIV-1 infection, to be investigated in other component projects of this Program Project. The Specific Aims of Project 1 are: 1) To determine how HIV-1 entry determines the outcome of HIV-1 infection in neural cells in vitro; 2) To study the role of env, nef, and vif in HIV-1 neurotropism using primary neurotropic HIV-1's and chimeric neural HIV-1 constructs; 3) To investigate the transient high-level transcription of HIV-1 DNA in neural cells in vitro, including the novel Tat/TAR-independent transcriptional mechanism; 4) To identify cellular genes modulated during HIV-1 infection in vitro and in vivo. The proposed studies will utilize HIV-1 strains and molecular clones, recombinant vectors, cell lines, other reagents, and experience in neural cell biology and molecular virology acquired during the past several years of research on AIDS and HIV-1 in this laboratory. In addition, close collaboration, exchange of information, and reagents with other members in the Program Project will be essential for Aims 1 and 2 (L. Epstein, Project 3; B. Blumberg, Core A; Aim 3 (H. Gendelman, Project 2), and Aim 4 (L. Sharer, Project 4). We hope that the proposed studies will yield significant new information regarding the mechanism of HIV-1 infection and persistence in human neural cells, adding to the comprehensive investigation of complementary HIV-1 neuropathogenic mechanisms in this Program Project.