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

描述（由申请人提供）：HIV-1通过CD4受体和两种共受体CCR5或CXCR4中的一种进入细胞。对于那些只携带使用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病毒如何使用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。我们的目标是推导出一种全面和定量的方法来表征任何给定病毒分离物的CD4/CCR5使用效率，以促进将探索我们假设的比较研究。我们提出一个多学科项目，利用PI （Benhur Lee博士），co-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原发性患者分离株的宝贵队列。他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物，可以在多达48种不同的CD4和CCR5表达水平下监测病毒感染性。Chou博士设计了一种方法，将获得的多维数据转换为描述特定分离物的整体CD4/CCR5使用效率的可量化指标。这个分析受体使用效率的系统允许对CD4和CCR5的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)为了开发更高的通量和更复杂的方法来量化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，我们提出(a)设计一个依赖于tat/rev的Gaussia荧光素酶报告载体，以便对初级病毒分离株的CD4和CCR5使用效率进行更高的通量和动力学分析。(b)优化一种超灵敏的实时融合动力学试验，以补充感染数据；(c)通过基于web的界面自动化和优化计算分析；(2)表征原发HIV-1株的相对感染性，并确定病毒致病性或趋向性是否与CD4和CCR5的使用效率相关。我们将测试来自不同群体的病毒与致病性的相关性。我们的基本假设是CD4和/或CCR5使用效率的差异与病毒发病机制的特定方面有关，这些差异可能通过Aim 1中优化的系统更好地揭示出来。

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.