Single-Cell Analysis of HIV Replication

HIV 复制的单细胞分析

• 批准号: 9518151
• 负责人: Masahiro Yamashita
• 金额: $ 49.48万
• 依托单位: AARON DIAMOND AIDS RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-14 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9518151
• 关键词: Address; Affect; Biological; Biological Assay; Biology; Cell Count; Cells; Cervical; Chronic; Event; Genetic Transcription; Goals; HIV; HIV-1; Heterogeneity; In Vitro; Individual; Infection; Kinetics; Libraries; Maintenance; Measurement; Monitor; Outcome; Pathogenesis; Phenotype; Physiological; Production; Proviruses; Reporter; Resolution; Reverse Transcriptase Polymerase Chain Reaction; Role; Source; Techniques; Time; Variant; Viral; Viral Proteins; Viral reservoir; Virion; Virus; Virus Integration; Virus Latency; Virus Replication; chromosomal location; gene product; humanized mouse; in vivo; insight; integration site; mouse model; novel; single cell analysis; single cell technology; transmission process; viral transmission

Project summary Recent advances in single-cell technologies uncovered a significant degree of functional and phenotypic heterogeneity between individual cells. How such cell-to-cell variation affects HIV-1 transmission by virus- producing cells is unclear. We have developed single-cell assays to quantify virus production and transmission by individual virus-infected cells in vitro. Our preliminary studies using both quantitative RT-PCR and single-cell viral transmission assays revealed substantial single-cell variations in both virus yield and transmissibility. Namely, a fraction of individual cells produced a large number of virions whereas there was a significant portion of single cells that barely release virions. Such immense cellular heterogeneity should have broad implications for HIV-1 biology, from viral transmission, dynamics to latency. The major goal of this proposal is to gain quantitative, mechanistic and biological insights into cell-to-cell variation in HIV-1 transmissibility at the single-cell level. In specific aim 1, we will determine cell fate upon HIV-1 infection with single-cell resolution by using reporter virus and the single-cell virion production assay. Longitudinal monitoring of chronically infected cells at the single cell level will allow us to characterize temporal changes of virus production, providing important information about how individual virus-infected cells shut off virion production before transitioning to a transcriptionally latent state. In specific aim 2, we will track individual cells to analyze their ability to produce and transmit HIV-1. Rates of virion production by single cell can be studied by time-course analysis. We will also estimate the extent of cell-to-cell variation in viral transmission by utilizing a barcoded viral library for high- throughput tracking of single cell infection events in vitro and also in vivo by using a humanized mice model. In specific aim 3, we will use single-cell assays to address how virally induced diversity and cell-intrinsic heterogeneity contribute to cellular differences in infectiousness.

项目概要 单细胞技术的最新进展揭示了显着程度的功能和表型 个体细胞之间的异质性。这种细胞间变异如何影响 HIV-1 病毒传播？ 产生细胞尚不清楚。我们开发了单细胞测定法来量化病毒的产生和传播 由单个病毒感染的细胞在体外进行。我们使用定量 RT-PCR 和单细胞进行的初步研究 病毒传播测定揭示了病毒产量和传播能力的显着单细胞差异。 也就是说，一小部分单个细胞产生大量病毒颗粒，而有大量病毒颗粒产生。 几乎不释放病毒颗粒的单细胞部分。如此巨大的细胞异质性应该具有广泛的 对 HIV-1 生物学的影响，从病毒传播、动态到潜伏期。该提案的主要目标是 获得对 HIV-1 传播性细胞间变异的定量、机制和生物学见解 单细胞水平。在具体目标 1 中，我们将通过单细胞分辨率确定 HIV-1 感染后的细胞命运： 使用报告病毒和单细胞病毒粒子生产测定。慢性感染者的纵向监测 单细胞水平的细胞将使我们能够表征病毒生产的时间变化，提供 关于单个病毒感染的细胞在转变为病毒之前如何关闭病毒粒子生产的重要信息 转录潜伏状态。在具体目标 2 中，我们将跟踪单个细胞以分析它们产生的能力 并传播 HIV-1。单细胞的病毒体产生率可以通过时间过程分析来研究。我们将 还通过利用条形码病毒库来估计病毒传播中细胞间变异的程度 通过使用人源化小鼠模型对体外和体内单细胞感染事件进行通量跟踪。在 具体目标 3，我们将使用单细胞测定来解决病毒如何诱导多样性和细胞内在 异质性导致细胞感染性的差异。