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）中的主要并发症的关键，因为它们是最常见的感染细胞，它们的感染代表了CNS中的大部分病毒载量。该提案将以小胶质细胞中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展中的作用，并与该计划的其他组成部分合作确定潜在的CNS特异性治疗策略。在第一个具体目标中，我们将使用来自成人和儿童的原代分离株以及通过连续传代适应小胶质细胞的分离株继续我们对HIV-1分离株的小胶质细胞嗜性的研究。我们将首先使用基于PCR的测定来分析HIV-1感染在小胶质细胞中的顺序步骤。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），我们将分子克隆包膜，并使用分子和生物化学（结合）测定来确定增强细胞渗透的机制。当小胶质细胞的嗜性与进入后步骤有关时，将克隆前病毒的其他部分或整个前病毒基因组。然后，我们将确定在小胶质细胞中没有高水平取代的分离株是否仍然可以建立慢性感染。这些分离株将用于另一个项目的SCID-hu模型。在第二个具体的目标，我们将确定是否包膜蛋白，特别是gp 120分离与HIV脑病可以介导的单核细胞衍生的巨噬细胞（MDM），小胶质细胞和其他神经细胞的细胞内游离Ca 2+浓度的变化。将测试那些诱导细胞内信号的gp 120介导细胞凋亡的能力。在第三个具体目标中，我们将确定某些HIV分离株的小胶质细胞感染是否会导致趋化因子的产生增加，这可能是导致细胞向CNS的运输增加以及慢性感染的潜在扩增的原因。这些实验的结果将加强关于HIV-1和小胶质细胞之间相互作用的知识。

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.