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。然而，目前仍不清楚 细胞异质性如何影响病毒感染细胞的传播性。我们已经开发了定量的 用于HIV-1产生和传播的单细胞检测。我们的初步研究显示了大量的细胞对- 病毒产量和传播力的细胞变异。这种不同的感染表型结果是 这是意想不到的，可能为HIV-1生物学提供新的范式。这项提议的主要目的是为了 对HIV-1生产和传播中单细胞异质性的定量和机械洞察。在……里面 此外，将利用一种简单而快速的单细胞检测病毒产生细胞的方法来 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和 细胞固有的异质性导致个体在生产力和感染力方面的细胞差异 被病毒感染的细胞。条形码病毒库将用于高通量跟踪单细胞感染 在体外和体外组织外植体模型中的事件。在特定目标2中，我们将调查病毒是如何感染的 细胞在潜伏期建立期间切断病毒粒子的产生，以了解细胞命运的时间变化 延迟建立。单细胞检测病毒粒子的产生将用于量化病毒的大小 潜伏期反转剂的潜在储集层及效果评价。在具体目标3中，我们将分析单个- 细胞转录组图谱以阐明病毒感染细胞细胞间异质性的生物学基础 单细胞病毒生产和传播的定量检测与单细胞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.