The Influence of Early Integration Events on HIV Latency and Reactivation Potential

项目摘要 人类免疫缺陷病毒（HIV）持续存在于感染患者的长期潜伏宿主中， 使用高效抗逆转录病毒药物进行持续、长期治疗。治愈性治疗旨在 已知通过使用潜伏逆转剂（LRA）来重新激活和清除这些潜伏感染的细胞 随着“休克和杀死”的方法，但这些方法在很大程度上失败，由于不完全重新激活的 潜伏池虽然细胞环境和前病毒整合位点的差异被认为是负责 对于这种可变性，关于特定的主机因素如何影响延迟和LRA，实际上知之甚少 主要模型的潜力。早期整合和延迟建立过程中的事件是否最终影响 延迟维护和重新激活？大部分潜在储层被认为是由 静止的CD 4 + T细胞携带有复制能力但转录沉默的前病毒。这些细胞 传统上很难离体产生，甚至更难进行基因操作 用于研究特定的宿主因素。原代细胞潜伏期模型和基因组研究进展 然而，工程学首次使这些研究变得可行。在这份提案中，我打算测试 假设HIV整合、前病毒沉默和潜伏期建立的早期事件有助于决定 潜伏期维持和再激活潜力。为了验证这个假设，我将使用一个新的平台， 在原代T细胞中进行CRISPR/Cas9编辑以消除参与整合位点偏好的两种宿主因子， LEDGF和CPSF 6。这些细胞将被双荧光报告病毒感染，以监测前病毒 通过深度测序确定沉默和整合位点谱。这些细胞将被送回一个 静止状态，并用代表性LRA治疗，靶向不同的功能途径， 潜伏期维持，并确定再激活潜力是否与早期 感染的遗传背景（目的1）。相反，LRA最初的设计是为了改变 等待时间维护也可以在早期集成和等待时间建立期间改变事件 作为分子探针，用于鉴定参与这些过程的新宿主因子。为此， 原代T细胞将用一组LRA预处理，用双荧光报道病毒感染， 监测整合、前病毒沉默和潜伏期建立的差异（目标2）。直系同源 这些途径中的小分子和靶向基因敲除将用于验证这些发现， 确认新潜伏期宿主因子的身份。综合起来，这些数据将是第一个分析 宿主因素、HIV整合、潜伏期建立和再激活潜力之间的关系直接影响着 原代T细胞了解感染早期发生的宿主依赖性事件如何与 在慢性疾病期间的治疗效果将是开发新的个性化治疗药物的关键。 治疗和治愈艾滋病毒以及其他疾病的战略。

PROJECT SUMMARY Human Immunodeficiency Virus (HIV) persists in long-lived, latent reservoirs in infected patients despite continuous, long-term treatment with highly active antiretroviral drugs. Curative therapies designed to reactivate and clear these latently infected cells through the use of latency reversing agents (LRAs) are known as “shock and kill” approaches, but these approaches have largely failed due to incomplete reactivation of the latent pool. While differences in cellular environment and proviral integration site are thought to be responsible for this variability, very little is actually known about how specific host factors can influence latency and LRA potential in primary models. Do events during early integration and latency establishment ultimately influence latency maintenance and reactivation? A majority of the latent reservoir is thought to be composed of quiescent CD4+ T cells harboring replication competent, but transcriptionally silent proviruses. These cells have traditionally been very difficult to generate ex vivo and have been even harder to manipulate genetically for the study of specific host factors. Recent advancements in primary cell latency models and genome engineering, however, have made these studies feasible for the first time. In this proposal, I intend to test the hypothesis that early events in HIV integration, proviral silencing, and latency establishment help dictate latency maintenance and reactivation potential. To test this hypothesis, I will use a novel platform for CRISPR/Cas9 editing in primary T cells to ablate two host factors involved in integration site preference, LEDGF and CPSF6. These cells will be infected with a dual fluorescent reporter virus to monitor proviral silencing and integration site profiles determined by deep sequencing. These cells will be returned to a quiescent state and treated with representative LRAs targeting distinct functional pathways to interrogate latency maintenance and determine if reactivation potential correlates with differences observed during early infection in each of genetic background (Aim 1). Conversely, LRAs that were originally designed to alter latency maintenance may alter events during early integration and latency establishment as well and therefore serve as molecular probes for identifying novel host factors involved in these processes. Towards this end, primary T cells will be pre-treated with a panel of LRAs, infected with a dual fluorescent reporter virus, and monitored for differences in integration, proviral silencing, and latency establishment (Aim 2). Orthologous small molecules in these pathways and targeted genetic knock-outs will be used to validate these findings and confirm the identity of novel latency host factors. Taken together, these data will be the first to analyze the relationship between host factors, HIV integration, latency establishment, and reactivation potential directly in primary T cells. Understanding how host-dependent events that occur early in infection are linked to therapeutic efficacy during chronic disease will be critical to the development of new personalized therapeutic strategies for the treatment and cure of not only HIV, but other disease states as well.