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

描述(申请人提供)：迫切需要了解HIV-1感染在粘膜组织中的启动和传播，并开发针对早期感染的新方法，以防止粘膜传播HIV-1。HIV-1感染需要病毒和靶细胞膜的融合，这一过程由病毒包膜糖蛋白(Env)和宿主细胞受体介导。人类清道夫受体gp340已被确定为人类唾液中的一种分泌成分，通过与环境蛋白尖峰的表面gp120亚单位的特异性相互作用来抑制HIV-1的口服传播。令人惊讶的是，gp120与女性生殖道中结合在膜上的gp340相互作用，促进HIV-1通过屏障上皮细胞转运，从而感染CD4+细胞。我们在这个项目中的目标是确定基于gp340的可溶性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与gp120结合的gp340的SRCR结构域获得的初步数据，该结构域在体外有效地抑制具有纳摩尔IC50值的HIV-1的感染。我们提出了一种综合的、跨学科的方法，将蛋白质化学、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疗效研究结合起来。在这个项目中，我们试图发现新的HIV-1特异性干预策略来抑制粘膜上皮-环境结合和病毒-细胞融合。具体目的是：1.了解gp120与gp340结合的结构基础，为设计和优化新型SRCR结构域进入抑制剂提供依据。(A)阐明SRCR结构域片段的结构特性，包括游离的和与V3区结合的。(B)表征gp120-gp340相互作用的能量学。(C)使用突变分析和生物物理方法剖析各自结合部位的结构决定因素。(D)定义与gp120结合的“最小”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，并在单轮传染性试验中评估其抗HIV-1活性。2.确定gp120-gp340相互作用影响Env结构、功能和抗原性的机制。(A)确定Aim 1c中发现的V3突变如何影响Env的功能能力。(B)鉴定SRCR结构域对gp120受体结合特性的影响及其对中和性和非中和性单抗的反应性。(C)确定选定的SRCR结构域进入抑制剂对各种主要艾滋病毒-1毒株的杀灭病毒活性。3.检测优化的SRCR结构域进入抑制剂对黏膜HIV-1感染的保护作用。(A)在人类宫颈和阴道组织感染HIV-1的体外模型中，表征选定的SRCR进入抑制剂的特异性和有效性。(B)使用人源化的小鼠阴道传播模型，评估优化的SRCR进入抑制剂单独以及与gp41融合抑制剂C52L和小分子CCR5共受体抑制剂CMPD167联合使用的体内效力和活性广度。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