High throughput analysis of latency/reactivation with barcoded proviruses

 描述：清除所有携带可能重新引发感染的前病毒的细胞可以为许多患者带来功能性治愈，并将减少病毒在人群中的传播。因此，更好地了解潜伏的前病毒宿主是关键的公共卫生优先事项。一些HIV-1整合事件会导致感染性后代或潜伏的前病毒，从而可以重新激活。在其他 在某些情况下，逆转录或表观遗传沉默过程中的突变使整合成为功能上的死胡同。潜伏期和再激活已经使用有限数量的克隆细胞系或集合细胞的聚集表型进行了广泛的研究。然而，更好地了解个体未表达前病毒的重新激活表型的全谱，应该会极大地促进减少钝化治疗以追求功能性治愈的长期目标。目前的应用将分子病毒学、T细胞发育和人类基因组学方法应用于一种新的高通量方法，以更好地确定潜伏感染细胞的类别及其重新激活的特征。为R21探索阶段提出的研究将开发一种新的条形码前病毒技术并验证其在培养细胞中的使用，建立在原代T细胞中启用该系统的技术，并在测试案例中开发用于高通量分析大量单个整合子的生物信息学，该测试案例解决了T细胞系中沉默在多大程度上由整合位置与随机效应决定。在R33阶段的概念验证研究中，条形码前病毒系统将被进一步改进和应用，以定义响应细胞分化或治疗性再激活的分子和基因组特征，包括细胞系和原代CD4+T细胞的再激活，研究潜伏期和CD4效应T细胞功能多样性之间的关系，以及评估一种新的分子遗传学方法跟踪残留病毒血症克隆的可能性，该方法未来可能应用于患者样本。

 DESCRIPTION: Purging all cells harboring proviruses that are likely to reinitiate infection could lead to a functional cure for many patients and would diminish viral spread in populations. Thus, better understanding of the latent proviral reservoir is a key public health priority. Some HIV-1 integration events lead to infectious progeny or to latent proviruses that can reactivate. In other cases, mutation during reverse transcription or epigenetic silencing makes integration a functional dead end. Latency and reactivation have been studied extensively using limited numbers of clonal cell lines or aggregate phenotypes of pooled cells. However, a better understanding of the full spectrum of reactivation phenotypes of individual unexpressed proviruses should greatly advance the long term goal of reducing blunt treatments in pursuit of a functional cure. The current application applies molecular virology, T cell development, and human genomics approaches to a novel high-throughput method for better defining classes of latently infected cells and their signatures of reactivation. Studies proposed for the R21 exploratory phase will develop a novel barcoded provirus technology and validate its use in cultured cells, establish technologies to enable this system in primary T-cells, and develop the bioinformatics for high throughput analysis of large numbers of individual integrants in a test case that addresses the extent to which silencing in a T cell line is determined by integration location vs. stochastic effects. In R33 phase proof-of- concept studies, the barcoded provirus system will be further refined and applied to defining molecular and genomic signatures of reactivation and persistence in response to cellular differentiation or therapeutic reactivation inT cell lines and primary CD4+ T-cells, to studying the relationship between latency and functional diversity of CD4 effector T cells, and to evaluating the potential of a novel molecular genetic approach for tracking the clonality of residual viremia, which may in the future be applied to patient samples.