characterization of gp120 C3 Variants that Influence HIV Neurotropism

描述（由申请人提供）：CMS的人类免疫缺陷病毒（HIV）感染是艾滋病日益普遍的并发症。CMS中的HIV感染在10-20%的AIDS患者中引起HIV相关性痴呆（HAD）或较轻度形式的神经认知障碍。尽管高效抗逆转录病毒治疗（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不能穿过BBB。因此，CMS是长期病毒持久性的储存库。这项提案的总体目标是了解HIV嗜神经性的机制。具体目标是：1）鉴定gp 120 C3区中增强由来自HIV相关痴呆患者的脑源性Env介导的膜融合的特异性氨基酸变体; 2）确定增强脑源性Env的膜融合活性的特异性C3变体是否也增加HIV在巨噬细胞/小胶质细胞中复制的能力并诱导原代脑培养物中的神经元凋亡;和3）表征增强HIV巨噬细胞/小胶质细胞嗜性的C3变体对膜融合发挥作用的机制。通过分析匹配的脑和淋巴源性Env氨基酸序列，确定了8个与脑区室化相关的gp 120 C3区氨基酸变体。将检查这些C3变体在CD 4/CCR 5表达降低的细胞中增强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，以确定增强融合和/或融合动力学的变体。C3变体可能有助于神经嗜性和/或神经毒力的潜在机制将针对基于前两个特定目的的结果选择的变体进行研究。使用亲本和突变体Env，我们将研究HIV Env的受体结合位点暴露和受体结合后构象变化。这些研究有望有助于理解特定HIV Env C3氨基酸变异体对嗜神经性和神经毒力的影响。此外，这些研究将提供对HIV Env-受体相互作用和受体后结合事件的深入了解，这些事件决定膜融合的效率并代表进入抑制剂的靶标。

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.