Genetic Analysis of U5-PBS Role in HIV Neuropathogenesis

描述（由申请人提供）：与HIV-1感染中枢神经系统相关的疾病，HIV-1相关痴呆复合体（HAD），发生在约15%至25%的感染者中。与HAD相关的发病机制是由于巨噬细胞和小胶质细胞的感染，以及随后释放的细胞因子和神经毒素，这些细胞因子和神经毒素放大了疾病的病理和临床表现。虽然HIV-1感染单核细胞、巨噬细胞和小胶质细胞的许多特征是已知的，但我们的知识中仍然存在重要的空白。被忽视的一个最重要的领域是HIV-1选择启动逆转录所需的tRNA引物的过程。所有HIV-1只使用tRNA Lys，3来启动逆转录。HIV-1选择tRNA lys3的机制尚不完全清楚。在U5区的引物结合位点（PBS）上游发现了一个由RNA干环组成的遗传元件，它可以显著影响连续和原代t细胞中tRNA的选择和病毒复制。本课题的实验将集中在产生感染性HIV-1的关键步骤之一，即tRNA赖氨酸的选择过程。在这些研究中，我们将比较HIV-1与突变的U5-PBS在单核细胞、巨噬细胞和小胶质细胞中的复制情况，并确定这些细胞类型的病毒产生是否与已知神经免疫调节分子（tnf - α、il -1 β和癌他素- m (OSM)）和类二十烷酸（PGE2）的合成相关，这些分子已被证明具有神经毒性。具体目的1：确定U5-PBS对HIV-1复制和单核细胞、巨噬细胞和小胶质细胞中神经毒性蛋白的作用。将构建含有U5-PBS突变的病毒，并检测其在单核细胞、巨噬细胞和小胶质细胞中的复制。将比较野生型病毒和突变型病毒引发已知的神经毒性细胞因子和分子的能力。特异性目的2：确定单核细胞、巨噬细胞和小胶质细胞中tRNA Lys、3的细胞内可用性是否与HIV-1复制相关。尽管HIV-1依赖tRNA Lys，3作为逆转录引物，但HIV-1和细胞翻译需要tRNA Lys，3来合成蛋白质。了解HIV-1如何进化以平衡tRNA赖氨酸的双重需求，将为控制复制和翻译提供见解。我们将测定HIV-1感染前后单核细胞、巨噬细胞和小胶质细胞中游离和氨基酰化tRNA的水平。我们还将评估HIV-1蛋白Tat和Vpr是否可以调节细胞内tRNA Lys的表达和位置，3这将对感染性病毒的产生产生影响。我们的研究结果将为我们了解HIV-1的遗传元件（U5- PBS）如何进化到在单核细胞、巨噬细胞和小胶质细胞中有效地选择tRNA Lys，3，以及这与这些细胞中神经毒性分子的细化之间的关系提供基础，这些神经毒性分子是HAD严重程度的重要指示。

DESCRIPTION (provided by applicant): The disease associated with HIV-1 infection of the CNS, HIV-1 associated dementia complex (HAD), occurs in approximately 15% to 25% of infected individuals. The pathogenesis associated with HAD is due to infection of macrophage and microglial cells, and the subsequent release of cytokines and neurotoxins that serve to amplify the pathological and clinical manifestation of the disease. Although many of the features of HIV-1 infection of monocytes, macrophages and microglia are known, there are still important gaps in our knowledge. One of the most important areas that has been neglected is the process by which HIV-1 selects the tRNA primer required for initiation of reverse transcription. All HIV-1 exclusively use tRNA Lys,3 to initiate reverse transcription. The mechanism for the selection of tRNA Lys,3 by HIV-1 is not completely understood. A genetic element consisting of a RNA stem loop has been found upstream of the primer binding site (PBS) in the U5 region that can dramatically influence the selection of the tRNA and virus replication in continuous and primary T-cells. The experiments in this proposal will focus on one of the key steps for the production of infectious HIV-1, the selection process for tRNA Lys,3. For these studies, we will compare the replication in monocytes, macrophages and microglia of HIV-1 with mutated U5-PBS, and determine whether the production of virus from these cell types correlates with the elaboration of known neuroimmunomodulatory molecules (TNF-alpha, IL-1beta and oncostatin-M (OSM)) and eicosanoids (PGE2) that have been shown to be neurotoxic. The following Specific Aims are proposed: Specific Aim 1: To determine the importance of the U5-PBS for HIV-1 replication and elaboration of neurotoxic proteins from monocytes, macrophages and microglia. Viruses will be constructed that contain mutations in the U5-PBS and will be assayed for replication in monocytes, macrophages and microglia. The capacity of the wild type virus and mutants to elicit known cytokines and molecules that are neurotoxic will be compared. Specific Aim 2: To determine if intracellular availability of tRNA Lys,3 in monocytes, macrophages and microglia correlates with HIV-1 replication. Although HIV-1 is dependent upon tRNA Lys,3 as a primer for reverse transcription, HIV-1 and cellular translation require tRNA Lys,3 for protein synthesis. Understanding how HIV-1 has evolved to balance these dual needs for tRNA Lys,3 will provide insights in the control of replication and translation. We will determine the levels of free and aminoacylated tRNA in monocytes, macrophages and microglia pre and post infection with HIV-1. We will also assess whether HIV-1 proteins Tat and Vpr can modulate the intracellular expression and location of tRNA Lys,3 that will have impact on the production of infectious virus. The results of our studies will provide us with a foundation to understand how genetic elements of HIV-1 (U5- PBS) have evolved to effectively select tRNA Lys,3 in monocytes, macrophages and microglia, and how this correlates with the elaboration of neurotoxic molecules from these cells that are an important indication for the severity of HAD.