Genetic analysis of the HIV gp120-gp41 interface

描述(申请人提供)：HIV包膜糖蛋白复合体(gp120/gp41)通过与宿主细胞受体结合并促进病毒和细胞膜的融合来介导病毒进入。在生产能够引起有效的初级病毒中和抗体反应的包膜糖蛋白免疫原方面，一个主要的挑战被认为是保存该复合体的正确折叠构象。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持了gp41分子在游离病毒粒子上的亚稳态形式，并在gp120与CD4和辅受体结合时介导了gp41的激活。我们的研究目标是开发一个控制gp120-gp41界面的分子决定因素的工作模型。我们最近在gp41的六个螺旋核心中发现了专门影响gp120-gp41关联的突变。这些数据补充了以前的发现，即扩展的N-链和C-链以及二硫键结合环区对gp120-gp41的结合是重要的，并使我们假设gp41更大的中央外膜结构域可能在静止的前融合原包膜糖蛋白的稳定性和功能中起关键作用。在本提案中，我们寻求：(1)通过扫描突变来确定gp41中央胞外区内特异影响gp120-gp41界面的关键氨基酸侧链。(2)用特异性抑制剂和中和性单抗检测突变表型的基础，探讨突变效应与抑制或中和机制之间的相互作用。(3)分离补充gp41基因突变所致融合缺陷的gp120基因第二位点回复突变体。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体的结构与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的努力。

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.