Single Cell Analysis of HIV-1 Latent reservoir establishment in Humanized Mice

摘要 人类免疫缺陷病毒(HIV)已感染全球7600万人，导致38人死亡 百万美元。目前的治疗方法可以减少患者的病毒载量，但无法治愈患者的感染。这是由于 病毒在抗逆转录病毒治疗期间经历潜伏的能力。潜伏感染的细胞几乎 无法与未感染的细胞区分开来，而且没有有效的治疗方法来消除这些细胞。我们的长- 术语目标是在单个细胞水平上描述水库早期播种发生的方式以及 建立驱动器延迟。这项资助中提议的实验是基于这样的假设：在 急性感染，一部分感染的CD4T细胞来自不同血统的T细胞，如静息的CD4 记忆、活化T细胞、幼稚T细胞、效应记忆T细胞经历早期潜伏期和转录 MTOR和eIF2信号通路以及与转录抑制位点整合的前病毒是 早期潜伏期建立的机制。这一假设是基于以下观察结果：i)尽管极端 早期抗逆转录病毒治疗，HIV潜伏储备库可能在暴露后72小时内建立II)多样化 T细胞亚群可能含有潜伏的HIV III)宿主和病毒之间复杂的相互作用会导致潜伏期。 陈实验室开发了第一代潜伏期标记技术，用于报告艾滋病毒的历史 通过不可逆的标记感染所有HIV感染的细胞称为HIV诱导谱系追踪(HILT)。在这 应用，我提出了第二代创新的遗传标记技术，增强型柄(EHILT)，以 进一步了解艾滋病毒潜伏期的复杂性。使用EHILT和验证的小动物模型 潜伏期，我将使用EHILT定义体外和体内急性和ART治疗感染的早期潜伏期动力学， 确定与体内潜伏期相关的转录特征和途径，并描述 具有即时潜伏表型的前病毒。

Abstract Human Immunodeficiency Virus (HIV) has infected 76 million people worldwide and caused the death of 38 million. Current treatments can reduce patient viral loads but fail to cure patients of infection. This is due to the ability of the virus to undergo latency during antiretroviral therapy. Latently infected cells are nearly indistinguishable from uninfected cells and there is no effective treatment to eliminate these cells. Our long- term goal is to describe at a single cell level, how early reservoir seeding occurs and the mechanisms that drive latency establishment. The proposed experiments in this grant are based on the hypothesis that during acute infection, a proportion of infected CD4+ T cells from diverse lineages of T cells such as resting CD4+ memory, activated T cells, naïve T cell, effector memory T cell undergo early latency and that transcriptional pathways such as mTOR and EIF2 signaling and provirus integration into transcriptionally repressive site are mechanisms of early latency establishment. This hypothesis is based on observations that: i) despite extremely early ART treatment, HIV latent reservoir may be established earlier than 72 hours after exposure ii) a diverse subset of T cells can harbor latent HIV iii) complex interactions between host and viral factors drive latency. First generation latency marking technology has been developed by the Chen lab to report the history of HIV infection through irreversible marking all HIV-infected cells called HIV-Induced Lineage Tracing (HILT). In this application, I propose a second-generation, innovative genetic marking technology, Enhanced HILT (EHILT), to further our knowledge the complexities of HIV latency. Using EHILT and validated small animal models of latency, I will define early latency kinetics in in vitro and in vivo acute and ART-treated infection using EHILT, identify transcriptional profiles and pathways associated with latency in vivo, and profile the integration sites of provirus with immediate latent phenotype.