Genetic analysis of the HIV gp120-gp41 interface

描述（由申请人提供）：HIV包膜糖蛋白复合物（gp120/gp41）通过结合宿主细胞受体和促进病毒与细胞膜融合介导病毒进入。生产包膜糖蛋白免疫原的一个主要挑战被认为是能够引发一种有效的初级病毒中和抗体反应，在于保持复合物的正确折叠构象。包膜糖蛋白的稳定性和功能的核心是gp120和gp41亚基之间的界面。这些分子相互作用保留了游离病毒粒子上gp41分子的亚稳态形式，并介导gp120与CD4和辅助受体结合时gp41的激活。我们的研究目标是建立一个控制gp120-gp41界面的分子决定因子的工作模型。我们最近在gp41的六个螺旋核心中发现了特异性影响gp120-gp41关联的突变。这些数据进一步证实了先前的发现，即在延长的N链和c链以及二硫脱键环区域内的位置对于gp120-gp41的结合是重要的，并使我们假设gp41较大的中央外结构域可能对静息前体细胞包膜糖蛋白的稳定性和功能至关重要。在本提案中，我们寻求：(1)通过扫描诱变确定gp41中央外域内特异性影响gp120-gp41界面的关键氨基酸侧链。(2)利用特异性抑制剂和中和性单克隆抗体研究突变表型的基础，探讨突变效应与抑制或中和机制之间的相互作用。(3)在gp120中分离出第二位点的复归物，以弥补gp41突变导致的融合缺陷。这些研究将产生gp120和gp41亚基之间相互作用的功能图谱，并将为理解天然包膜糖蛋白复合物的结构-功能关系提供独特的视角。这些知识将指导设计包膜糖蛋白免疫原和开发抗病毒抑制剂以防止病毒进入。

DESCRIPTION (provided by applicant): The HIV envelope glycoprotein complex (gp120/gp41) mediates viral entry by binding to host cell receptors and promoting fusion of the virus and cell membranes. A major challenge in producing an envelope glycoprotein immunogen capable of eliciting a potent primary virus neutralizing antibody response is thought to lie in preserving the correctly folded conformations of the complex. Central to the stability and functioning of the envelope glycoprotein is the interface between the gp120 and gp41 subunits. These molecular interactions retain the metastable form of the gp41 molecule on the free virion and mediate the activation of gp41 upon gp120 binding to CD4 and coreceptor. Our research objective is to develop a working model of the molecular determinants that control the gp120-gp41 interface. We have recently identified mutations within the six helix core of gp41 that specifically affect the gp120-gp41 association. These data add to previous findings that positions within the extended N- and C-chains and the disulfidebonded loop region are important for gp120-gp41 association, and lead us to hypothesize that the larger central ectodomain of gp41 may be critically involved in the stability and functioning of the resting prefusogenic envelope glycoprotein. In this proposal, we seek: (1) To determine, by scanning mutagenesis, key amino acid side-chains within the central ectodomain of gp41 that specifically affect the gp120-gp41 interface. (2) To examine the basis for the mutant phenotype by using specific inhibitory agents and neutralizing monoclonal antibodies to probe the interactions between the effect of the mutation and the mechanism of inhibition or neutralization. (3) To isolate second-site revertants in gp120 that complement the fusion deficiency engendered by the mutation in gp41. These studies will yield a functional map of the interactions between the gp120 and gp41 subunits, and will provide a unique perspective towards understanding the structure-function relationships of the native envelope glycoprotein complex. This knowledge will guide efforts to design envelope glycoprotein immunogens and to develop antiviral inhibitors to prevent viral entry.