NEUROTROPISM AND MICROGLIAL INVASION

小胶质细胞是人类免疫缺陷病毒(HIV-1)在中枢神经系统(CNS)发生主要并发症的关键，因为它们是最常见的感染细胞，它们的感染代表了CNS中病毒载量的大部分。这项建议将以小胶质细胞中HIV-1的生物学为中心，以便更好地了解病毒在这种并发症发生中的作用，并与该计划的其他组成部分合作，确定潜在的、针对中枢神经系统的治疗策略。在第一个特定目标中，我们将继续我们的研究，利用成人和儿童的原代分离株，以及一株适应于小胶质细胞的分离株，通过连续传代来研究HIV-1分离株的小胶质细胞嗜性。我们将首先使用基于聚合酶链式反应的分析方法来分析小胶质细胞中HIV-1感染的顺序步骤。对于那些表现出快速进入表型的分离株(如小胶质细胞适应的HIV-1/BORI-15)，我们将从分子上克隆包膜，并使用分子和生化(结合)分析来确定增强细胞渗透的机制。如果小胶质细胞的嗜性与进入后的步骤有关，前病毒的其他部分，或整个前病毒基因组，将被克隆。然后，我们将确定不替换到小胶质细胞高水平的分离株是否仍能建立慢性感染。这些分离株随后将用于另一个项目的SCID-HU模型。在第二个特定目标中，我们将确定包膜蛋白，特别是来自HIV脑病分离株的gp120是否可以调节单核细胞来源的巨噬细胞(MDM)、小胶质细胞和其他神经细胞内游离钙浓度的变化。那些诱导细胞内信号的gp120将被测试其介导细胞凋亡的能力。在第三个特定目标中，我们将确定某些HIV分离株感染小胶质细胞是否会导致趋化因子的产生增加，这可能是导致细胞向中枢神经系统转运增加的原因，并可能导致慢性感染的放大。这些实验的结果将加强对HIV-1和小胶质细胞之间相互作用的了解。

Microglia are critical to the primary complications of the human immunodeficiency virus (HIV-1) in the central nervous system (CNS), since they are the most commonly infected cell and their infection represents the majority of the viral load in the CNS. This proposal will center on the biology of HIV-1 in microglial in order to develop a better understanding of the role of the virus in the development of this complication, and to identify potential, CNS-specific, treatment strategies in collaboration with the other components of this program. In the first specific aim we will continue our studies on microglial- tropism of HIV-1 isolates using primary isolates from adults and children, and an isolate adapted to microglia bu sequential passage. We will first use a PCR-based assay to analyze the sequential steps of HIV-1 infection in microglia. For those isolates (like the microglia-adapted HIV-1/BORI- 15) which demonstrate a rapid entry phenotype, we will molecularly clones the envelopes, and define the mechanism of enhanced cellular penetration using molecular and biochemical (binding) assays. Where tropism for microglial cells is related to post-entry steps, other portions of the provirus, or the entire proviral genome, will be cloned. We will then determine whether isolates that do not replace to high levels in microglia can nevertheless establish a chronic infection. These isolates will then be used in a SCID-hu model in another Project. In the second specific aim we will determine whether the envelope proteins, and specifically gp120 from isolates with HIV encephalopathy can mediate changes in intracellular free Ca2+ concentrations in monocyte-derived macrophages (MDM), microglia, and other neural cells. Those gp120s that induce intracellular signals will be tested for their ability to mediate apoptosis. In the third specific aim we will determine whether microglial infection by certain HIV isolates results in increased production of chemokines, which could be responsible for increased cellular trafficking into the CNS, and potential amplification of a chronic infection. The result from these experiments will strengthen knowledge about the interactions between HIV-1 and microglial cells.