Single-cell analysis of HIV-1 production and transmission

项目摘要 单细胞技术已经开始揭示HIV-1复制的新方面。但尚不清楚 细胞异质性如何影响病毒感染细胞的传播性。我们开发了定量的 单细胞检测HIV-1的产生和传播。我们的初步研究显示，大量的细胞对- 病毒产量和传播性的细胞变异。这种不同的感染表型结果是 这是一个出乎意料的发现，可能为HIV-1生物学提供新的范例。该提案的主要目标是获得 对HIV-1产生和传播中的单细胞异质性的定量和机制见解。在 此外，将利用一种简单快速的单细胞检测产病毒细胞的方法， 开发研究病毒潜伏期的新方法。在具体目标1中，我们将研究病毒如何诱导多样性， 细胞内在异质性导致个体生产力和传染性的细胞差异 病毒感染的细胞条形码病毒库将用于单细胞感染的高通量追踪 在体外和离体组织外植体模型中的事件。在具体目标2中，我们将研究病毒如何感染 细胞在潜伏期建立期间关闭病毒体产生，以了解细胞命运的时间变化， 延迟建立。病毒粒子产生的单细胞检测将用于定量病毒粒子的大小。 潜伏库和潜伏逆转试剂的效力评价。在具体目标3中，我们将分析单个- 细胞转录组谱，以阐明病毒感染细胞细胞间异质性的生物学基础， 将用于病毒产生和传播的定量单细胞测定与单细胞RNA相结合 测序

Project Summary Single-cell technologies have begun to reveal novel aspects of HIV-1 replication. However, it remains unclear how cellular heterogeneity influences transmissibility of virus-infected cells. We have developed quantitative single-cell assays for HIV-1 production and transmission. Our preliminary studies showed substantial cell-to- cell variations in both virus yield and transmissibility. Such diverse phenotypic outcomes of infection were unexpected and may offer new paradigms for HIV-1 biology. The major goal of this proposal is to gain quantitative and mechanistic insights into single-cell heterogeneity in HIV-1 production and transmission. In addition, a simple and rapid method for single-cell detection of virus-producing cells will be harnessed to develop novel assays for studying viral latency. In specific aim 1, we will study how virally induced diversity and cell-intrinsic heterogeneity contribute to cellular differences in productivity and infectiousness of individual virus-infected cells. A barcoded viral library will be utilized for high-throughput tracking of single-cell infection events in vitro and in an ex vivo tissue explant model. In specific aim 2, we will investigate how virus-infected cells shut off virion production during latency establishment to learn temporal changes in the cell fate during latency establishment. Single-cell detection of virion production will be used for quantification of the size of latent reservoirs and efficacy evaluation of latency reversal reagents. In specific aim 3, we will analyze single- cell transcriptome profiles to elucidate the biological basis for cell-to-cell heterogeneity of virus-infected cells by combining quantitative single-cell assays for virus production and transmission with single-cell RNA sequencing.