Host Cell Factors and AIDS Pathogenesis

宿主细胞因素与艾滋病发病机制

• 批准号: 6726154
• 负责人: GHALIB ABBAS ALKHATIB
• 金额: $ 33.53万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-15 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6726154
• 关键词: AIDS; cell line; cytokine receptors; frameshift mutation; genetic polymorphism; genetic susceptibility; human immunodeficiency virus 1; human tissue; immunoprecipitation; laboratory mouse; laboratory rabbit; lymphocyte; monocyte; open reading frames; pathologic process; receptor expression; virus genetics; virus infection mechanism; virus receptors; yeast two hybrid system

Human immunodeficiency virus type 1 (HIV-1) requires CD4 and a coreceptor for entry, into susceptible host cells. The chemokine receptors CXCR4 and CCR5 are the major coreceptors used by T cell line-tropic (X4) and macrophage-tropic (R5) HIV-1 isolates. A naturally occurring frame-shift mutation caused by a 32 base pair deletion (delta32) in the human CCR5 gene resulted in a truncated protein that does not function as a coreceptor. Relative to the general population, CCR5delta32 homozygotes (-/-) are rarely found among HIV-1 infected individuals. In addition, HIV-1 infected CCR5delta32 heterozygotes (+/-) progress more slowly to AIDS than individuals lacking this allele indicating that even one delta32 allele confers some resistance. We have found that expression of full-length delta32 protein in human cell lines or normal human peripheral blood mononuclear cells (PBMCs) can specifically downmodulate endogenous CXCR4 and inhibit X4 infection. We have demonstrated efficient co- localization of delta32 and either CXCR4 or CCR5. Truncation of the delta32 C-terminus resulted in the loss of CXCR4 downmodulation activity. Furthermore, we showed that PBMCs from several (-/-) individuals express lower CXCR4 levels in comparison to wild-type CCR5 (+/+) PBMCs. We hypothesize that one mechanism of resistance to HIV-1 is caused by the unique activity of the delta32 protein. Using delta32-specific antibodies, we have shown that native delta32 protein was expressed in (-/-) PBMCs isolated from exposed/uninfected [1]but was absent in an infected (-/-) individuals [2], indicating a critical role for the delta32 protein in resistance to HIV-1 infection. The overall purpose of this proposal is to determine the mechanism(s) that underlies the observed more broad protective effect by the naturally occurring delta32 protein. The following specific aims are based on preliminary data generated in my laboratory: 1. Analyze how delta32 impairs functional expression of CCR5 and CXCR4. 2. Analyze the mechanism of failure of delta32 protective effect in an infected delta32 homozygous individual. 3. Determine the structural determinants involved in delta32 activity.

1 型人类免疫缺陷病毒 (HIV-1) 需要 CD4 和辅助受体才能进入易感宿主细胞。 趋化因子受体 CXCR4 和 CCR5 是 T 细胞系嗜性 (X4) 和巨噬细胞嗜性 (R5) HIV-1 分离株使用的主要辅助受体。 由人类 CCR5 基因中的 32 个碱基对缺失 (delta32) 引起的自然发生的移码突变导致产生了不能作为辅助受体发挥功能的截短蛋白。相对于一般人群，CCR5delta32 纯合子 (-/-) 在 HIV-1 感染者中很少发现。 此外，感染 HIV-1 的 CCR5delta32 杂合子 (+/-) 比缺乏该等位基因的个体发展为艾滋病的速度更慢，这表明即使是一个 delta32 等位基因也能产生一定的抵抗力。 我们发现，全长delta32蛋白在人细胞系或正常人外周血单核细胞（PBMC）中的表达可以特异性下调内源性CXCR4并抑制X4感染。 我们已经证明了 delta32 与 CXCR4 或 CCR5 的有效共定位。 delta32 C 末端的截断导致 CXCR4 下调活性丧失。 此外，我们发现，与野生型 CCR5 (+/+) PBMC 相比，来自多个 (-/-) 个体的 PBMC 表达较低的 CXCR4 水平。 我们假设一种 HIV-1 抵抗机制是由 delta32 蛋白的独特活性引起的。 使用 delta32 特异性抗体，我们发现天然 delta32 蛋白在从暴露/未感染者中分离的 (-/-) PBMC 中表达 [1]，但在感染的 (-/-) 个体中不表达 [2]，这表明其发挥着关键作用Delta32 蛋白可抵抗 HIV-1 感染。 该提案的总体目的是确定天然存在的 delta32 蛋白所观察到的更广泛的保护作用背后的机制。以下具体目标基于我实验室生成的初步数据： 1. 分析 delta32 如何损害 CCR5 和 CXCR4 的功能表达。 2. 分析受感染的delta32纯合个体中delta32保护作用失效的机制。 3. 确定参与 delta32 活性的结构决定因素。