The Role of Scavenger Receptor gp340 in Mucosal HIV-1 Transmission and Inhibition

描述（由申请人提供）：迫切需要了解粘膜组织中HIV-1感染的启动和传播，并开发针对早期感染的新方法，以预防粘膜HIV-1传播。HIV-1感染需要病毒和靶细胞膜的融合，这是一个由病毒包膜糖蛋白（Env）和宿主细胞受体介导的过程。人清道夫受体gp 340已被鉴定为人唾液中的分泌组分，其通过与Env刺突的表面gp 120亚基的特异性相互作用抑制HIV-1的经口传播。令人惊讶的是，女性生殖道中gp 120与膜结合gp 340的相互作用促进HIV-1通过屏障上皮细胞的转胞吞作用，从而感染CD 4+细胞。我们在这个项目中的目标是确定可溶性gp 340为基础的HIV-1进入抑制剂的分子靶点和作用机制。我们的发现工作将基于用gp 340的gp 120结合SRCR结构域获得的初步数据，该结构域以纳摩尔IC 50值有效地抑制HIV-1的体外感染。我们提出了一个全面的，跨学科的方法，结合蛋白质化学，高分辨率结构测定，热力学和动力学结合分析，蛋白质工程，分子病毒学和动物模型功效研究。在这个项目中，我们寻求发现新的HIV-1特异性干预策略，以抑制粘膜上皮-Env结合和病毒-细胞融合。具体目标是：1。了解gp 340与gp 120结合的结构基础，为新型SRCR结构域进入抑制剂的设计和优化提供信息。(a)阐明SRCR结构域片段的结构特性，包括游离和与V3区结合。(b)描述gp 120-gp 340相互作用的能量学特征。(c)使用突变分析和生物物理方法来剖析各自结合位点的结构决定因素。(d)定义一个“最小的”gp 120结合序列或一组这样的序列。(e)优化SRCR结构域变体的结合亲和力，并在单轮感染性试验中评估其抗HIV-1活性。2.探讨gp 120-gp 340相互作用对Env结构、功能和抗原性的影响机制。(a)确定目标1c中鉴定的V3突变如何影响Env的功能能力。(b)表征SRCR结构域对gp 120受体结合特性及其对中和和非中和单克隆抗体的反应性的影响。(c)确定选择SRCR结构域进入抑制剂对不同的主要HIV-1毒株的杀病毒活性。3.检测优化的SRCR结构域进入抑制剂对粘膜HIV-1感染的保护作用。(a)在人宫颈和阴道组织HIV-1感染的体外模型中表征选定SRCR进入抑制剂的特异性和效力。(b)使用人源化小鼠阴道传播模型评估优化的SRCR进入抑制剂单独以及与gp 41融合抑制剂C52 L和小分子CCR 5辅助受体抑制剂CMPD 167组合的体内效力和活性宽度。1

DESCRIPTION (provided by applicant): There is an urgent need to understand the initiation and dissemination of HIV-1 infection in mucosal tissues, and to develop new methods for targeting early infection to prevent mucosal HIV-1 transmission. HIV-1 infection requires fusion of the viral and target cell membranes, a process mediated by the viral envelope glycoprotein (Env) and host cell receptors. The human scavenger receptor gp340 has been identified as a secreted component in human saliva that inhibits oral transmission of HIV-1 through a specific interaction with the surface gp120 subunit of an Env spike. Surprisingly the gp120 interaction with the membrane-bound gp340 in the female reproductive tract promotes HIV-1 transcytosis through barrier epithelia and thus infection of CD4+ cells. Our goal in this program is to define the molecular target and mechanism of action of soluble gp340-based HIV-1 entry inhibitors. Our discovery effort will be based on preliminary data obtained with the gp120-binding SRCR domain of gp340 that potently inhibits in vitro infection by HIV-1 with nanomolar IC50 values. We propose a comprehensive, interdisciplinary approach that combines protein chemistry, high- resolution structural determination, thermodynamic and kinetic binding analyses, protein engineering, molecular virology, and animal model efficacy studies. In this project we seek to discover novel HIV-1-specific intervention strategies to inhibit mucosal epithelium-Env binding and virus-cell fusion. The Specific Aims are: 1. To understand the structural basis of gp120 binding by gp340 to inform the design and optimization of novel SRCR domain entry inhibitors. (a) Elucidate the structural properties of the SRCR domain fragment both free and bound to the V3 region. (b) Characterize the energetics of the gp120-gp340 interaction. (c) Use mutational analysis and biophysical methods to dissect the structural determinants of the respective binding sites. (d) Define a "minimal" gp120-binding sequence or set of such sequences. (e) Optimize binding affinity of variants of the SRCR domain, and evaluate their anti-HIV-1 activity in a single-round infectivity assay. 2. To determine the mechanisms by which the gp120-gp340 interaction influences Env structure, function and antigenicity. (a) Determine how V3 mutations identified in Aim 1c influence the functional competence of Env. (b) Characterize the effects of the SRCR domain on the receptor-binding properties of gp120 and its reactivities to neutralizing and non-neutralizing monoclonal antibodies. (c) Determine the virucidal activity of select SRCR domain entry inhibitors against diverse primary HIV-1 strains. 3. To test the effectiveness of optimized SRCR domain entry inhibitors to protect against mucosal HIV-1 infection. (a) Characterize the specificity and potency of select SRCR entry inhibitors in an in vitro model of HIV-1 infection of human cervical and vaginal tissue. (b) Use the humanized mouse vaginal transmission model to assess the in vivo potency and breadth of activity of optimized SRCR entry inhibitors alone and in combination with the gp41 fusion inhibitor C52L and the small-molecule CCR5 coreceptor inhibitor CMPD167. 1