Exploring viral infection with single cell transcriptomics

用单细胞转录组学探索病毒感染

• 批准号: 9167182
• 负责人: JAMES M PIPAS
• 金额: $ 25.58万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-03 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9167182
• 关键词: Address; Antiviral Agents; Antiviral Therapy; Biology; Blood Vessels; Bone Marrow; Capsid; Cell Culture System; Cell Culture Techniques; Cell Cycle; Cell Death; Cell Proliferation; Cells; Coculture Techniques; Complementary DNA; Complex; Complex Mixtures; Coupled; DNA Damage; DNA biosynthesis; Development; Disease; Encapsulated; Environment; Epithelial Cells; Epithelium; Event; Evolution; Foundations; Gene Expression; Gene Expression Profile; Genes; Genetic Recombination; Genetic Transcription; Global Change; Human; Immune system; Immunocompromised Host; Immunosuppression; Individual; Infection; Kidney; Knowledge; Laboratories; Large T Antigen; Late Promoters; Lead; Measurement; Measures; Mesenchymal; Messenger RNA; Microfluidics; Monitor; Natural Immunity; Organ Transplantation; Paper; Pattern; Peer Review; Physics; Polyomavirus; Population; Predisposition; Process; Production; Prognostic Marker; Proteins; Proximal Kidney Tubules; Publications; Publishing; RNA; Reaction; Research; Resolution; Reverse Transcription; Series; Small T Antigen; System; Therapeutic immunosuppression; Tissues; Transplant Recipients; Tubular formation; Urine; Viral; Viral Genes; Viral Tumor Antigens; Virion; Virus; Virus Diseases; Virus Replication; Work; abstracting; base; cell type; interest; kidney cell; kidney epithelial cell; kidney infection; kidney vascular structure; next generation sequencing; novel; pathogen; programs; research study; response; stem; transcriptomics; viral DNA; virology

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.

项目摘要/摘要 BKV是一种普遍存在的多瘤病毒，大多数人口作为一个 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，尤其是患有肾脏，骨髓或多种肾脏的人 器官移植。在这种情况下 病毒的产生主要发生在管状上皮细胞中。在生产感染期间 BKV经历了一个基因表达程序，该程序导致三个早期合成 蛋白质称为大T抗原（LT），小T抗原（ST）和三个。这些早期蛋白质作用 要改变细胞基因表达，请捕获复制所需的细胞蛋白，并启动 病毒DNA复制。病毒后期启动子的转录大约是协调的 随着病毒DNA合成的发作，并导致衣壳基因的表达和 随后组装新病毒颗粒。我们几乎所有对BKV生物学的了解 来自均质克隆细胞群感染的细胞培养系统的工作 高多种病毒体。这允许病毒转录和DNA复制等事件， 以及随着感染的进行监测的细胞转录变化。但是，每个 这些测量是从散装培养中获得的，因此代表了事件的平均值 发生在单个细胞中。此外，人类的BKV感染发生在复杂组织中， 例如肾脏，由多种细胞类型组成。知识很少 至于病毒感染如何受到间充质或其他与之相邻的细胞类型的影响 感染细胞，或感染细胞如何影响邻居的生物学。在此应用程序中 我们将开发单细胞转录组学，并加上高通量液滴微流体。 解决这些重要问题。个体中病毒和细胞转录的模式 人类肾脏近端小管上皮细胞感染了BKV 将单个细胞封装在微副细胞中并提取RNA，液滴用作 反应容器。从每个细胞中分离的RNA将进行条形码，以使被汇总感染 细胞种群可以进行大量的下一代测序（NGS）。转录 然后可以通过解码条形码来计算单个单元格的模式。 我们还将使用感染的肾上皮细胞与未感染的肾上皮细胞进行此实验 血管细胞。这将允许对病毒和细胞基因表达进行协调检查 混合细胞种群。该研究策略的成功执行将导致一本小说 广泛适用于多种类型病毒的平台。