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

描述(申请人提供)：HIV-1通过使用CD4受体和两个共同受体CCR5或CXCR4之一进入细胞。对于那些只携带使用CCR5的病毒的人来说，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有关。然而，我们量化CD4和CCR5使用效率的能力受到了间接的和非标准化的衡量病毒使用CD4和/或CCR5的程度的限制。因此，我们的驱动假设是，HIV-1毒株与它们使用CD4和CCR5的相对效率之间存在真正的潜在关联，这解释了病毒致病的特定方面，但由于当前方法的限制，这些关联尚未被揭示。我们的目标是得出一种全面和定量的方法来表征任何给定病毒分离株的CD4/CCR5利用效率，以便促进将探索我们的假设的比较研究。我们提出了一个多学科项目，该项目利用PI(Benhur Lee博士)、共同PI(Tom Chou博士，生物数学)和合作者的独特专业知识和资源，可以获得具有明确致病特征的原始患者分离株的宝贵队列。李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，病毒的传染性可以在高达48个不同的CD4和CCR5表达水平上进行监测。周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，描述特定菌株的整体CD4/CCR5利用效率。这种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)开发更高的吞吐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的利用效率，我们建议(A)设计一种依赖于TAT/rev的Gaussia荧光素酶报告载体，该载体将允许对初级病毒分离株的CD4和CCR5的利用效率进行更高的吞吐量和动力学分析，(B)优化超灵敏的实时融合动力学分析以补充感染数据，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)确定主要HIV-1毒株的相对传染性，并确定病毒的致病性或趋向性是否与CD4和CCR5的使用效率相关。我们将测试致病性与来自不同队列的病毒的相关性。我们的基本假设是，CD4和/或CCR5使用的不同效率与病毒致病机制的特定方面有关，这些差异可能会通过AIM 1中优化的系统更好地揭示出来。 公共卫生相关性：我们的双重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利用效率，并提供可用于多次比较的定量指标。我们的系统不仅是一个有价值的工具，不仅可以更好地了解发病机制与CD4/CCR5使用效率之间的关系，而且可能对指导进入抑制剂的使用具有临床意义。

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.