Exploring viral infection with single cell transcriptomics

项目总结/摘要 BKV是一种普遍存在的多瘤病毒，由大多数人群携带， 无害的终身持续感染。BKV成为一种危险的病原体 免疫功能低下的个体，特别是那些经历肾脏、骨髓或多器官移植的个体， 器官移植在这些条件下，BKV可以进行猖獗的肾脏感染 病毒的产生主要发生在肾小管上皮细胞中。在生产性感染期间 BKV经历了一个基因表达程序，导致三个早期的合成， 称为大T抗原（LT）、小T抗原（ST）和truncT的蛋白质。这些早期蛋白质 改变细胞基因表达，捕获复制所需的细胞蛋白，并启动 病毒DNA复制从病毒晚期启动子的转录大约是协调的 与病毒DNA合成的开始，并导致表达衣壳基因和 新病毒粒子的后续组装。我们对BKV生物学的所有了解几乎都源于 来自细胞培养系统中的工作，其中同质克隆细胞群被感染 病毒粒子的高度多样性。这使得病毒转录和DNA复制等事件， 以及随着感染的进行而监测细胞转录的变化。但每 这些测量值是从大量培养物中获得的 发生在单个细胞中。此外，人类的BKV感染发生在复杂的组织中， 例如肾脏，由多种细胞类型紧密相连组成。知识很少 至于病毒感染是如何受到邻近的间充质或其他细胞类型的影响， 受感染的细胞，或受感染的细胞如何影响其邻居的生物学。本申请中 我们将开发单细胞转录组学与高通量液滴微流控技术相结合， 解决这些重要问题。个体中病毒和细胞转录的模式 用BKV感染的人肾近端小管上皮细胞将通过 将单个细胞包封在微滴中并提取RNA，微滴用作 反应容器从每个细胞分离的RNA将被条形码化，使得合并的感染的RNA将被标记。 细胞群可进行批量下一代测序（NGS）。转录 然后通过解码条形码可以计算地提取来自单个细胞的模式。 我们还将使用感染的肾脏上皮细胞与未感染的细胞混合进行这项实验。 血管细胞这将允许对病毒和细胞基因表达的协调检查， 混合细胞群。这一研究策略的成功执行将导致一部小说 该平台广泛适用于许多类型的病毒。

Project Summary/Abstract BKV is a ubiquitous polyomavirus that is carried by the majority of the human population as a harmless lifelong persistent infection. BKV becomes a dangerous pathogen in immunocompromised individuals, especially those undergoing kidney, bone marrow, or multi- organ transplants. Under these conditions BKV can undergo a rampant infection of the kidney with virus production occurring largely in the tubular epithelial cells. During productive infection BKV undergoes a program of gene expression that results in the synthesis of three early proteins called large T antigen (LT), small T antigen (ST), and truncT. These early proteins act to alter cellular gene expression, capture cellular proteins needed for replication, and to initiate viral DNA replication. Transcription from the viral late promoter is coordinated approximately with the onset of viral DNA synthesis, and results in expression of the capsid genes and the subsequent assembly of new virus particles. Nearly all our knowledge of BKV biology stems from work in cell culture systems in which a homogeneous clonal cell population is infected with a high multiplicity of virions. This allows events such as viral transcription and DNA replication, and changes in cellular transcription to be monitored as infection proceeds. However, each of these measurements is obtained from bulk culture and thus represents an average of the events occurring in individual cells. Furthermore, BKV infections of humans occur in complex tissues, such as the kidney, that consist of multiple cell types in close proximity. There is little knowledge as to how viral infection is influenced by mesenchymal or other cell types that are adjacent to infected cells, or of how infected cells influence the biology of their neighbors. In this application we will develop single-cell transcriptomics coupled with high-throughput droplet microfluidics to address these important questions. The patterns of viral and cellular transcription in individual human kidney proximal tubule epithelial cells infected with BKV will be measured by encapsulating individual cells in microdroplets and extracting RNA, with the droplets serving as reaction vessels. The RNA isolated from each cell will be barcoded such that pooled infected cell populations can be subjected to next generation sequencing (NGS) in bulk. Transcription patterns from individual cells can then be extracted computationally by decoding the barcodes. We will also perform this experiment using infected kidney epithelial cells mixed with uninfected vascular cells. This will allow the coordinate examination of viral and cellular gene expression in mixed cell populations. The successful execution of this research strategy will lead to a novel platform that is broadly applicable to many types of viruses.