Structural Plasticity of an HIV-1 Nef/Pak-2 Activity Surface

HIV-1 Nef/Pak-2 活性表面的结构可塑性

• 批准号: 7167782
• 负责人: J. Victor Garcia-Martinez
• 金额: $ 23.55万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7167782
• 关键词: gene mutation; human immunodeficiency virus; information systems; model; phenotype; protein kinase; proteins; recombinant virus; simian immunodeficiency virus

DESCRIPTION (provided by applicant): Nef is a major determinant of pathogenesis; both human and simian immunodeficiency viruses (HIV/SIV) show an attenuated phenotype when the accessory protein Nef is deleted. In vitro, both HIV and SIV Nef demonstrate several activities which include enhancement of infectivity, CD4 and MHCI downregulation and the activation of p21-activated protein kinase 2 (Pak-2) among others. Interestingly, Nef achieves all these activities in the face of high sequence divergence. It has been suggested that the complex biology of Nef is regulated through conformational changes induced by its cellular location and specific protein-protein interactions during cell traffic. In order to understand the importance of each of the Nef-mediated activities, in particular the activation of the host cell kinase Pak-2 to HIV/SIV pathogenesis, we have identified a surface on the subtype B Nef molecule that is specific for this activity. However, analysis of the amino acid sequence of subtype E Nef sequences indicated that dramatically different amino acid substitutions in this surface that completely abolish the ability of subtype B Nef to activate Pak-2 result in fully functional subtype E Nefs. Our biochemical analysis together with in silico modeling of Nef structure resulted in the identification of compensatory mutations that restore Nef activity. As part of this grant we first propose to identify these compensatory mutations in Nef from additional HIV-1 subtypes to fully define this surface and secondly to extend these observations to SIV Nef. We propose to identify alternative structures of this surface as it relates to variation between subtypes and even between strains. We will compile sequences in all available databases and translate our observations by generating mutated versions of the prototypical SF2 Nef that reflect naturally occurring sequences. The effect of these substitutions to the Nef-mediated activities, particularly the activation of Pak-2, will be correlated with changes in the tridimensional structure of Nef. The studies proposed here will help elucidate the structural requirements for the activation of Pak-2 by Nef and will serve as the basis for future in vivo experiments aimed at characterizing its functional significance.

描述（由申请人提供）：Nef 是发病机制的主要决定因素；当辅助蛋白 Nef 被删除时，人类和猿猴免疫缺陷病毒 (HIV/SIV) 均表现出减毒表型。在体外，HIV 和 SIV Nef 均表现出多种活性，包括增强感染性、下调 CD4 和 MHCI 以及激活 p21 激活蛋白激酶 2 (Pak-2) 等。有趣的是，Nef 在面对高序列分歧的情况下实现了所有这些活动。有人认为，Nef 的复杂生物学是通过其细胞位置和细胞运输过程中特定的蛋白质-蛋白质相互作用诱导的构象变化来调节的。为了了解每种 Nef 介导的活性的重要性，特别是宿主细胞激酶 Pak-2 的激活对 HIV/SIV 发病机制的重要性，我们鉴定了 B 亚型 Nef 分子上对该活性具有特异性的表面。然而，对 E 亚型 Nef 序列的氨基酸序列的分析表明，该表面中显着不同的氨基酸取代完全消除了 B 亚型 Nef 激活 Pak-2 的能力，从而产生了功能齐全的 E 亚型 Nef。我们的生化分析与 Nef 结构的计算机模型一起确定了恢复 Nef 活性的补偿突变。作为这笔资助的一部分，我们首先建议从其他 HIV-1 亚型中识别 Nef 的这些补偿性突变，以完全定义该表面，其次将这些观察结果扩展到 SIV Nef。我们建议确定该表面的替代结构，因为它与亚型之间甚至菌株之间的变异有关。我们将编译所有可用数据库中的序列，并通过生成反映自然发生序列的原型 SF2 Nef 的突变版本来翻译我们的观察结果。这些取代对 Nef 介导的活性的影响，特别是 Pak-2 的激活，将与 Nef 三维结构的变化相关。这里提出的研究将有助于阐明 Nef 激活 Pak-2 的结构要求，并将作为未来旨在表征其功能意义的体内实验的基础。