Interaction of HIV nef with its receptor binding partners

描述（由申请人提供）：拟议研究的广泛长期目标是表征HIV网络与其细胞结合伙伴的相互作用，并了解这种相互作用对HIV感染和免疫的功能后果。拟开展的研究重点是nef与几种T细胞表面蛋白的相互作用：T细胞抗原受体（TCR）、MHC I类分子和相关CD1抗原、CD4共受体和CD28共刺激分子。这些蛋白质构成了目前已知的HIV nef细胞表面靶点的补充。这些蛋白质中的每一种都参与T细胞免疫反应的激活，并且已知每一种都通过与nef相互作用而下调。对于T细胞抗原受体，已知与nef的相互作用也会导致T细胞信号通路的调节。目前，人们对nef与这些靶点相互作用的结构基础知之甚少，本研究的主要目标是对这些相互作用进行结构表征。对于这些受体中的每一个，假定的网络结合伙伴被认为是一个非结构化或部分折叠的细胞质结构域，而拟议研究的一个重要组成部分是开发用于分析具有天然未折叠或松散折叠结构域的蛋白质之间相互作用的方法。提出的研究包括两个具体目标和一套共享分析工具的开发。第一个目的是表征HIV nef与TCR zeta亚基细胞质结构域的相互作用，包括亲和性、动力学和相互作用的化学计量学测量、HIV毒株和亚型依赖性、复合物三维结构的测定、nef和TCR zeta结构域结合诱导的结构变化的研究、nef调节TCR信号传导的机制。第二个目标是将这些研究扩展到其他细胞表面受体结合靶点，包括MHC、CD1、CD4和CD28。将开发一种共同的方法来克服在这种和其他涉及非结构化或部分结构化蛋白质结构域的系统中结构和功能表征的挑战。拟议的研究分为两个阶段。R21阶段（第1年和第2年）包括改进nef-靶标相互作用的结构表征工具和评估这些相互作用的功能结果，将这些方法应用于HIV-2/SIV nef与TCR zeta亚基细胞质结构域的相互作用，以及HIV nef与CD4、MHC I类、CD1a、CD1d和CD28相互作用的基本结合参数的表征。R33阶段（3-5年）包括将结构和功能工具应用于后一组目标，获得特定nef相互作用的明确证据，并详细评估这些相互作用的应变/亚型依赖性。Nef对HIV的毒力很重要，但是在感染细胞中Nef如何与其结合伙伴相互作用的分子细节尚不清楚。在拟议的研究中，这些相互作用将使用生化和生物物理方法进行研究。这一信息将有助于了解nef如何改变受感染细胞中的信号机制，并开发治疗方法来干扰HIV感染和发病机制的这一方面。

DESCRIPTION (provided by applicant): The broad long-term objectives of the proposed research are to characterize the interaction of HIV nef with its cellular binding partners and to understand the functional consequences of this interaction for HIV infection and immunity. The proposed research focuses on the interaction of nef with several T cell-surface proteins: the T cell antigen receptor (TCR), MHC class I molecules and the related CD1 antigen, the CD4 co-receptor, and the CD28 co- stimulatory molecule. These proteins comprise the currently known complement of cell- surface targets for HIV nef. Each of these proteins is involved in the activation of T cell immune responses, and each is known to be down-modulated by interaction with nef. For the T cell antigen receptor, interaction with nef is known also to lead to modulation of T cell signaling pathways. Currently, little is known about the structural basis for the interaction of nef with these targets, and a major goal of the proposed research is structural characterization of these interactions. For each of these receptors, the presumptive nef binding partner is a believed to be an unstructured or partially-folded cytoplasmic domain, and an important component of the proposed research is the development of methods for the analysis of interactions among proteins with natively unfolded or loosely-folded domains. The proposed research includes two specific aims and the development of a set of shared analysis tools. The first aim is to characterize the interaction of HIV nef with the TCR zeta subunit cytoplasmic domain, including measurements of the affinity, kinetics, and stoichiometry of the interaction, the HIV strain and subtype dependence, determination of the three-dimensional structure of the complex, investigation of binding-induced structural changes in both nef and the TCR zeta domain, characterization of the mechanism of TCR signaling modulation by nef. The second aim is to extend these studies to other cell surface receptor binding targets including MHC, CD1, CD4, and CD28. A common methodology will be developed to overcome challenges for structural and functional characterization in this and other systems involving unstructured for partially-structured protein domains. The proposed research is structured in two phases. The R21 phase (years 1 and 2) includes the refinement of tools for structural characterization of nef-target interactions and for evaluation of the functional outcome of these interactions, the application of these methods to the interaction of HIV-2/SIV nef with the TCR zeta subunit cytoplasmic domain, and characterization of basic binding parameters for the interaction of HIV nef with CD4, MHC class I, CD1a, CD1d, and CD28. The R33 phase (years 3-5) includes the application of the structural and functional tools to the targets in the latter set for which clear evidence of specific nef interaction is obtained, and the detailed evaluation of the strain/subtype dependence of these interactions. Nef is important for the virulence of HIV, but the molecular details of how nef interacts with its binding partners in an infected cell are not clear. In the proposed research, these interactions will be studied using biochemical and biophysical approaches. This information will promote efforts to understand how nef modifies the signaling machinery in an infected cell, and to develop therapeutic approaches to interfere with this aspect of HIV infection and pathogenesis.