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

描述(申请人提供)：NEF是致病的主要决定因素；当辅助蛋白Nef缺失时，人类和猿类免疫缺陷病毒(HIV/SIV)都表现出减弱的表型。在体外，HIV和SIV Nef都表现出多种活性，包括增强感染性，下调CD4和MHCI，激活p21激活的蛋白激酶2(Pak-2)等。有趣的是，Nef在面对高度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-蛋白质相互作用引起的构象变化来调节的。为了了解Nef介导的每一种活性，特别是宿主细胞激酶Pak-2的激活对HIV/SIV致病机制的重要性，我们在B亚型Nef分子上鉴定了这一活性的特异性表面。然而，对E亚型Nef序列的氨基酸序列分析表明，该表面的氨基酸替换完全不同，完全取消了B亚型Nef激活Pak-2的能力，导致了E亚型Nef的完全功能。我们的生化分析和对Nef结构的电子模拟导致了恢复Nef活性的代偿性突变的鉴定。作为这项资助的一部分，我们首先建议从更多的HIV-1亚型中识别Nef中的这些补偿突变，以充分定义这一表面，其次将这些观察结果扩展到SIV Nef。我们建议确定这种表面的替代结构，因为它与亚型之间甚至菌株之间的变异有关。我们将汇编所有可用的数据库中的序列，并通过生成反映自然发生的序列的原型SF2Nef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，特别是对Pak-2的激活，将与Nef三维结构的变化相关。本文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。

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.