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

描述（由申请方提供）：Nef是发病机制的主要决定因素;当辅助蛋白Nef缺失时，人类和猿免疫缺陷病毒（HIV/SIV）均显示减毒表型。在体外，HIV和SIV Nef都表现出几种活性，其中包括增强感染性、CD 4和MHCI下调以及激活p21活化的蛋白激酶2（Pak-2）等。有趣的是，Nef在面对高序列差异时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特定的蛋白质-蛋白质相互作用在细胞运输过程中诱导的构象变化来调节的。为了理解每种Nef介导的活性的重要性，特别是宿主细胞激酶Pak-2对HIV/SIV发病机制的活化，我们已经鉴定了亚型B Nef分子上特异于该活性的表面。然而，亚型E Nef序列的氨基酸序列的分析表明，在该表面中完全消除亚型B Nef激活Pak-2的能力的显著不同的氨基酸取代导致完全功能的亚型E Nef。我们的生化分析与Nef结构的计算机模拟一起鉴定了恢复Nef活性的补偿突变。作为这项资助的一部分，我们首先建议从其他HIV-1亚型中识别Nef中的这些补偿性突变，以充分定义这种表面，其次将这些观察结果扩展到SIV Nef。我们建议确定替代结构的表面，因为它涉及到亚型之间的变化，甚至菌株之间。我们将在所有可用的数据库中编译序列，并通过生成反映自然发生序列的原型SF 2 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的活化，将与Nef的三维结构的变化相关。这里提出的研究将有助于阐明Pak-2激活Nef的结构要求，并将作为未来体内实验的基础，旨在表征其功能意义。

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.