Quantifying differential CD4 and CCR5 usage patterns amongst HIV-1/SIV strains

量化 HIV-1/SIV 毒株中 CD4 和 CCR5 使用模式的差异

• 批准号: 8026514
• 负责人: Benhur Lee
• 金额: $ 23.1万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8026514
• 关键词: 3-Dimensional; Accounting; Arts; Automobile Driving; Biological Assay; CCR5 gene; CD4 Antigens; CXCR4 gene; Cell Line; Cells; Characteristics; Chemokine (C-C Motif) Receptor 5; Clinical; Comparative Study; Complement; Complex; Computer Analysis; Data; Data Analyses; Dependence; Disease; Disease Progression; End Point Assay; Engineering; Grant; HIV; HIV-1; Infection; Kinetics; Luciferases; Mathematics; Measures; Methods; Metric; Molecular Virology; Monitor; Outcome; Pathogenesis; Pathogenicity; Patients; Pattern; Relative (related person); Reporter; Resolution; Resources; SIV; Staining method; Stains; Surface; System; Testing; Time; Tropism; Viral; Viral Pathogenesis; Virus; biomathematics; cohort; inhibitor/antagonist; interdisciplinary approach; multidisciplinary; novel; public health relevance; receptor; standardize measure; tool; vector; web based interface

DESCRIPTION (provided by applicant): HIV-1 enters cells by using the CD4 receptor, and one of two co-receptors, CCR5 or CXCR4. For those that harbor only CCR5-using viruses, there is evidence that the relative efficiency by which HIV-1 uses CD4 and CCR5 may correlate with the pathogenic potential of the virus. However, our ability to quantify the efficiency of CD4 and CCR5 usage has been limited by indirect and non-standardized measures for how well a virus uses CD4 and/or CCR5. Therefore, our driving hypothesis is that there are real underlying associations between strains of HIV-1 and their relative efficiencies of CD4 and CCR5 usage that account for specific aspects of viral pathogenesis, but that these associations have not been revealed due to the limitations of current methods. Our objective is to derive a comprehensive and quantitative way of characterizing the CD4/CCR5 usage efficiencies of any given viral isolate in order to facilitate comparative studies that will explore our hypothesis. We propose a multidisciplinary project that takes advantage of the unique expertise and resources of the PI (Dr. Benhur Lee), co-PI (Dr. Tom Chou, Biomathematics), and collaborators with access to precious cohorts of primary patient isolates with defined pathogenic characteristics. Dr. Lee has developed a dual-inducible cell line where CD4 and CCR5 can be simultaneously and independently regulated. For any given isolate, viral infectivity can be monitored at up to 48 distinct levels of CD4 and CCR5 expression. Dr. Chou has devised a method to transform the multi-dimensional data obtained into quantifiable metrics that describes the overall CD4/CCR5 usage efficiency of a particular isolate. This system for profiling the receptor usage efficiencies allows for large-scale intra- and inter-cohort comparisons of CD4 and CCR5 usage efficiencies. Thus, my Specific Aims are: (1) To develop higher throughput and more sophisticated methods for quantifying the relative CD4 and CCR5 usage efficiencies of various HIV isolates in our CD4/CCR5 dual-inducible cell line, We propose (a) engineering a tat/rev dependent Gaussia luciferase reporter vector that will allow for a higher throughput and kinetic analysis of the efficiency of CD4 and CCR5 usage of primary viral isolates, (b) optimizing an ultra-sensitive real-time fusion kinetics assay to complement the infection data, and (c) automating and optimizing the computational analysis via a web-based interface, and (2) To characterize the relative infectivity of primary HIV-1 strains, and to determine if viral pathogenicity or tropism correlates with the efficiency of CD4 and CCR5 usage. We will test the correlates of pathogenicity with viruses from various cohorts. Our underlying hypothesis is that the differential efficiency of CD4 and/or CCR5 usage are associated with specific aspects of viral pathogenesis, and that these differences may be better revealed by the system optimized in Aim 1. PUBLIC HEALTH RELEVANCE: Our dual inducible cells can directly measure and profile the CD4/CCR5 usage efficiency of any given viral isolate, and provide a quantitative metric that can be used for multiple comparisons. Our system is a valuable tool not only for better understanding the relationship between pathogenesis and the efficiency of CD4/CCR5 usage, but may also have clinical implications for guiding entry inhibitor use.

描述（由申请人提供）：HIV-1 通过使用 CD4 受体以及两个辅助受体 CCR5 或 CXCR4 之一进入细胞。对于那些仅携带使用 CCR5 的病毒，有证据表明 HIV-1 使用 CD4 和 CCR5 的相对效率可能与病毒的致病潜力相关。然而，我们量化 CD4 和 CCR5 使用效率的能力受到了对病毒使用 CD4 和/或 CCR5 的间接和非标准化测量的限制。因此，我们的驱动假设是，HIV-1 毒株与其 CD4 和 CCR5 使用的相对效率之间存在真正的潜在关联，这些关联解释了病毒发病机制的特定方面，但由于当前方法的局限性，这些关联尚未被揭示。我们的目标是得出一种全面、定量的方法来表征任何给定病毒分离株的 CD4/CCR5 使用效率，以促进探索我们假设的比较研究。我们提出了一个多学科项目，利用 PI（Benhur Lee 博士）、联合 PI（Tom Chou 博士，生物数学）和合作者的独特专业知识和资源，获得具有明确致病特征的珍贵原发患者分离群。 Lee 博士开发了一种双诱导细胞系，其中 CD4 和 CCR5 可以同时独立调节。对于任何给定的分离株，可以在多达 48 个不同的 CD4 和 CCR5 表达水平上监测病毒感染性。周博士设计了一种方法，将获得的多维数据转化为可量化的指标，描述特定分离株的整体 CD4/CCR5 使用效率。该用于分析受体使用效率的系统允许对 CD4 和 CCR5 使用效率进行大规模的队列内和队列间比较。因此，我的具体目标是：(1) 为了开发更高通量和更复杂的方法来量化我们的 CD4/CCR5 双诱导细胞系中各种 HIV 分离株的相对 CD4 和 CCR5 使用效率，我们建议 (a) 设计一个 tat/rev 依赖的 Gaussia 荧光素酶报告载体，该载体将允许对 CD4 和 CCR5 使用效率进行更高的通量和动力学分析 主要病毒分离株的分析，(b) 优化超灵敏实时融合动力学测定以补充感染数据，以及 (c) 通过基于网络的界面自动化和优化计算分析，以及 (2) 表征主要 HIV-1 毒株的相对感染性，并确定病毒致病性或向性是否与 CD4 和 CCR5 使用效率相关。我们将测试致病性与不同群体病毒的相关性。我们的基本假设是，CD4 和/或 CCR5 使用的差异效率与病毒发病机制的特定方面相关，并且目标 1 中优化的系统可能会更好地揭示这些差异。 公共卫生相关性：我们的双诱导细胞可以直接测量和分析任何给定病毒分离株的 CD4/CCR5 使用效率，并提供可用于多重比较的定量指标。我们的系统是一个有价值的工具，不仅可以更好地理解发病机制和 CD4/CCR5 使用效率之间的关系，而且还可能对指导进入抑制剂的使用具有临床意义。