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使用CD4受体和两个共受体CCR5或CXCR4进入细胞。对于仅使用CCR5病毒的人，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。但是，我们量化CD4和CCR5使用效率的能力受到间接和非标准化的措施的限制，即病毒使用CD4和/或CCR5。因此，我们的驾驶假设是，HIV-1菌株与CD4和CCR5使用的相对效率之间存在真正的潜在关联，这些用法涉及病毒发病机理的特定方面，但是由于当前方法的局限性，这些关联尚未得到揭示。我们的目标是得出一种表征任何给定病毒分离株的CD4/CCR5使用效率的全面和定量方法，以促进将探讨我们假设的比较研究。我们提出了一个多学科项目，该项目利用PI（Benhur Lee博士），Co-Pi（Tom Chou博士，生物护理学）的独特专业知识和资源，以及具有具有依赖病原特征的宝贵主要患者隔离的合作者。 Lee博士开发了一条双重诱导的细胞系，其中CD4和CCR5可以同时和独立调节。对于任何给定的分离株，可以在多达48个不同水平的CD4和CCR5表达的情况下监测病毒感染性。 Chou博士设计了一种方法，将获得的多维数据转换为可量化的指标，该指标描述了特定分离株的总体CD4/CCR5使用效率。该系统用于分析受体使用效率，允许对CD4和CCR5用法效率进行大规模内和循环间比较。因此，我的具体目的是：（1）开发较高的吞吐量和更复杂的方法，用于量化各种HIV隔离株的相对CD4和CCR5使用效率，我们建议（a）工程（a）允许使用CD和KIN的较高的cd和kin的cds andity（a），以允许使用CD/REV依赖的cD/rev依赖。原发性病毒分离株，（b）优化超敏感的实时融合动力学测定法以补充感染数据，以及（c）通过基于Web的界面自动化和优化计算分析，（2）以表征原代HIV-1菌株的相对感染性，并确定病毒性致病性或Tropism或Tropism cormage或CD4以及CD4以及CD4和ccrcr cd4及其cd4＆ccr cd4和ccrccr y。我们将测试与各种同类病毒的致病性相关性。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理的特定方面有关，并且在AIM 1中优化的系统可以更好地揭示这些差异。 公共卫生相关性：我们的双重诱导细胞可以直接测量和介绍任何给定病毒分离株的CD4/CCR5使用效率，并提供可用于多次比较的定量度量标准。我们的系统不仅是更好地理解发病机理与CD4/CCR5使用效率之间的关系的宝贵工具，而且还可能对指导进入抑制剂的使用具有临床意义。