characterization of gp120 C3 Variants that Influence HIV Neurotropism

描述(申请人提供)：人类免疫缺陷病毒(HIV)感染的CMS是一种日益流行的艾滋病并发症。在10-20%的艾滋病患者中，CMS中的HIV感染会导致HIV相关性痴呆(HAD)或较轻微的神经认知障碍。虽然高效抗逆转录病毒疗法(HAART)降低了血浆和脑脊液(CSF)中的HIV RNA水平，并改善了神经认知功能，但大多数抗病毒药物无法跨越血脑屏障。因此，CMS是病毒长期持续存在的蓄水池。这项提案的总体目标是了解艾滋病毒神经嗜性的基础机制。其具体目的是：1)鉴定gp120 C3区域的特定氨基酸变异，以增强HIV相关痴呆患者脑源性Env介导的膜融合；2)确定增强脑源性Env膜融合活性的特定C3变异是否也增加了HIV在巨噬细胞/小胶质细胞中复制的能力，并在原代脑培养中诱导神经元凋亡；以及3)表征增强HIV巨噬细胞/小胶质细胞趋向性的C3变异对膜融合发挥作用的机制。通过对匹配的脑源性和淋巴源性Env氨基酸序列的分析，确定了8个与脑分区相关的gp120 C3区氨基酸变体。这些C3变异体将被检测在CD4/CCR5表达降低的细胞中促进融合的能力以及在巨噬细胞/小胶质细胞中的融合动力学。将研究突变体在巨噬细胞/小胶质细胞复制和神经元凋亡中的作用，以发现融合和/或融合动力学增强的突变体。根据前两个特定目标的结果选择的C3变异可能有助于神经亲和性和/或神经毒力的潜在机制将被研究。利用亲本和突变的env，我们将研究HIV env的受体结合部位暴露和受体结合后的构象变化。这些研究有望有助于理解特定的HIV Env C3氨基酸变异对神经趋向性和神经毒力的影响。此外，这些研究将提供对HIV环境受体相互作用和受体后结合事件的洞察，这些事件决定了膜融合的效率，并代表了进入抑制剂的靶标。

DESCRIPTION (provided by applicant): Human immunodeficiency virus (HIV) infection of the CMS is an increasingly prevalent complication of AIDS. HIV infection in the CMS causes HIV-associated dementia (HAD) or milder forms of neurocognitive impairment in 10-20% of AIDS patients. Although highly active antiretroviral therapy (HAART) reduces HIV RNA levels in plasma and cerebrospinal fluid (CSF) and improves neurocognitive function, most antiviral drugs cannot cross the BBB. Thus, the CMS is a reservoir for long-term viral persistence. The overall goal of this proposal is to understand mechanisms that underlie HIV neurotropism. The specific aims are: 1) to identify specific amino acid variants in the gp120 C3 region that enhance membrane fusion mediated by brain-derived Envs from patients with HIV-associated dementia; 2) to determine whether specific C3 variants that enhance membrane fusion activity of brain-derived Envs also increase the capacity of HIV to replicate in macrophages/microglia and induce neuronal apoptosis in primary brain cultures; and 3) to characterize mechanisms by which C3 variants that enhance HIV macrophage/microglia tropism exert their effects on membrane fusion. Eight gp120 C3 region amino acid variants associated with brain compartmentalization were identified through analysis of matched brain- and lymphoid-derived Env amino acid sequences. These C3 variants will be examined for the ability to enhance fusion in cells with reduced CD4/CCR5 expression and fusion kinetics in macrophages/microglia. The role of variants in macrophage/microglia replication and neuronal apoptosis will be studied for variants that demonstrate enhancement of fusion and/or fusion kinetics. Potential mechanisms by which C3 variants might contribute to neurotropism and/or neurovirulence will be investigated for variants selected based on results of the first two specific aims. Using parental and mutant Envs, we will investigate receptor binding site exposure and post-receptor binding conformational changes in HIV Env. These studies are expected to contribute to the understanding of the influence of specific HIV Env C3 amino acid variants on neurotropism and neurovirulence. Furthermore, these studies will provide insight into HIV Env-receptor interactions and post- receptor binding events that determine the efficiency of membrane fusion and represent targets for entry inhibitors.