Single-Cell Analysis of HIV Replication

项目总结 单细胞技术的最新进展揭示了相当程度的功能和表型 单个细胞之间的异质性。这种细胞间的变异如何影响艾滋病毒-1通过病毒的传播- 生产细胞的情况尚不清楚。我们已经开发了单细胞分析来量化病毒的生产和传播。 由单个感染病毒的细胞在体外进行。我们用定量RT-PCR和单细胞同时进行的初步研究 病毒传播分析显示，病毒产量和传播力在单细胞中都有很大的差异。 也就是说，个别细胞的一小部分产生了大量的病毒粒子，而有显著的 几乎不会释放病毒粒子的单细胞的一部分。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。这项提议的主要目标是 为了获得对HIV-1传播率细胞间差异的定量、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来确定HIV-1感染后的细胞命运 采用报告病毒和单细胞病毒粒子产生试验。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产生的时间变化，提供 有关单个感染病毒的细胞如何在转变为 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的生产能力 并传播HIV-1病毒。单细胞产生病毒粒子的速率可以通过时间过程分析来研究。我们会 还通过利用条形码病毒库来估计病毒在细胞间传播的变异程度，以获得高 使用人源化小鼠模型在体外和体内追踪单细胞感染事件的吞吐量。在……里面 具体目标3，我们将使用单细胞分析来解决病毒诱导的多样性和细胞固有的 异质性导致了细胞感染性的差异。

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.