Genetic Analysis of U5-PBS Role in HIV Neuropathogenesis

描述(申请人提供)：与HIV-1感染中枢神经系统相关的疾病，HIV-1相关性痴呆综合征(HAD)，发生在大约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HAD的病理和临床表现。尽管单核细胞、巨噬细胞和小胶质细胞感染HIV-1的许多特征已为人所知，但我们的认识仍然存在着重要的空白。被忽视的最重要的领域之一是HIV-1选择启动反转录所需的tRNA引物的过程。所有HIV-1病毒都只使用tRNA Lys，3来启动逆转录。HIV-1选择tRNA Lys，3的机制还不完全清楚。在U5区的引物结合位点(PBS)上游发现了一个由RNA茎环组成的遗传元件，它可以显著影响tRNA的选择和病毒在连续和原代T细胞中的复制。这项建议中的实验将集中在生产传染性HIV-1的关键步骤之一，tRNA Lys的选择过程，3.在这些研究中，我们将比较HIV-1在单核细胞、巨噬细胞和小胶质细胞中的复制与突变的U5-PBS，并确定从这些细胞类型产生的病毒是否与已知的神经免疫调节分子(肿瘤坏死因子-α、IL-1β和肿瘤抑素-M(OSM))和二十烷类化合物(PGE2)的产生有关，这些分子已被证明具有神经毒性。提出了以下具体目标：具体目标1：确定U5-PBS对HIV-1复制和从单核细胞、巨噬细胞和小胶质细胞中提取神经毒性蛋白的重要性。将构建包含U5-PBS突变的病毒，并将检测其在单核细胞、巨噬细胞和小胶质细胞中的复制情况。将比较野生型病毒和突变体诱导已知的细胞因子和神经毒性分子的能力。具体目标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复制有关。虽然HIV-1依赖tRNA Lys，3作为逆转录的引物，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 Lys，3的双重需求将为控制复制和翻译提供洞察力。我们将测定感染HIV-1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tRNA Lys，3的细胞内表达和位置，这将对传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(U5-PBS)的遗传元件是如何进化到有效地选择单核细胞、巨噬细胞和小胶质细胞中的tRNA Lys，3，以及这如何与这些细胞中神经毒性分子的阐述有关，这些分子是HAD严重程度的重要指标。

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.