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

这项建议的总体目标是研究生物化学和 神经细胞中控制HIV-1生命周期的分子机制 以及HIV-1对神经细胞基因表达的影响 了解HIV-1神经趋向性及其在艾滋病中的作用的模型 脑病。建议的研究是基于我们最近的发现。 它确定了HIV-1生命周期的几个不同特征 神经细胞(胶质细胞、星形胶质细胞或神经元起源)与T 淋巴细胞或巨噬细胞。其中包括艾滋病毒-1的有效融合 带有CD4阴性神经细胞膜的包膜，有限病毒 复制不顾高效的融合和进入，瞬间的高水平 在表达CD4的神经细胞中产生病毒和有效的HIV-1DNA 部分独立于TAT/TAR的转录 转录系统。我们假设这些特征构成 HIV-1嗜神经性的基础是允许选择特定的 病毒变异体，在瞬间调节细胞基因表达 高水平的HIV-1DNA转录，并创造了一个可诱导的储存库 神经组织中的病毒。这些直接的(尽管不能溶解细胞) 巨噬细胞介导的神经毒性与其他效应的相互作用 HIV-1感染，将在本项目的其他组成部分项目中进行调查 计划项目。 项目1的具体目标是：1)确定艾滋病毒-1如何进入 决定HIV-1在体外神经细胞中感染的结局；2) 研究env、nef和vif在HIV-1神经趋向性中的作用 原发嗜神经性HIV-1‘S和嵌合神经型HIV-1构建；3) HIV-1 DNA在神经细胞中的瞬时高水平转录 体外细胞，包括新的TAT/TAR非依赖性转录 机制；4)识别在HIV-1感染过程中调节的细胞基因 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、其他试剂， 以及在神经细胞生物学和分子病毒学方面的经验 在过去的几年里，我们对艾滋病和HIV-1进行了研究 实验室。此外，密切合作、信息交流、 与计划项目中的其他成员一起使用试剂将是必不可少的 目标1和目标2(L.Epstein，项目3；B.Blumberg，核心A；目标3(H. Gendelman，项目2)和目标4(L.Sharer，项目4)。我们希望 拟议的研究将产生关于 HIV-1在人类神经细胞中感染和持续的机制， 补充补充HIV-1的全面调查 本计划项目中的神经致病机制。

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.