Basic Sciences and Computational Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
Division of Virology, Department of Pathology, University of Cambridge, Cambridge, United Kingdom.
J Virol. 2019 Jun 28;93(14). doi: 10.1128/JVI.00500-19. Print 2019 Jul 15.
Influenza virus-infected cells vary widely in their expression of viral genes and only occasionally activate innate immunity. Here, we develop a new method to assess how the genetic variation in viral populations contributes to this heterogeneity. We do this by determining the transcriptome and full-length sequences of all viral genes in single cells infected with a nominally "pure" stock of influenza virus. Most cells are infected by virions with defects, some of which increase the frequency of innate-immune activation. These immunostimulatory defects are diverse and include mutations that perturb the function of the viral polymerase protein PB1, large internal deletions in viral genes, and failure to express the virus's interferon antagonist NS1. However, immune activation remains stochastic in cells infected by virions with these defects and occasionally is triggered even by virions that express unmutated copies of all genes. Our work shows that the diverse spectrum of defects in influenza virus populations contributes to-but does not completely explain-the heterogeneity in viral gene expression and immune activation in single infected cells. Because influenza virus has a high mutation rate, many cells are infected by mutated virions. But so far, it has been impossible to fully characterize the sequence of the virion infecting any given cell, since conventional techniques such as flow cytometry and single-cell transcriptome sequencing (scRNA-seq) only detect if a protein or transcript is present, not its sequence. Here we develop a new approach that uses long-read PacBio sequencing to determine the sequences of virions infecting single cells. We show that viral genetic variation explains some but not all of the cell-to-cell variability in viral gene expression and innate immune induction. Overall, our study provides the first complete picture of how viral mutations affect the course of infection in single cells.
流感病毒感染的细胞在病毒基因的表达上差异很大，并且仅偶尔激活先天免疫。在这里，我们开发了一种新方法来评估病毒群体中的遗传变异如何导致这种异质性。我们通过确定感染名义上“纯”流感病毒株的单个细胞中的所有病毒基因的转录组和全长序列来做到这一点。大多数细胞被有缺陷的病毒粒子感染，其中一些增加了先天免疫激活的频率。这些免疫刺激性缺陷是多样的，包括扰乱病毒聚合酶蛋白 PB1 功能的突变、病毒基因中的大片段内部缺失以及干扰素拮抗剂 NS1 的表达失败。然而，在感染具有这些缺陷的病毒粒子的细胞中，免疫激活仍然是随机的，并且偶尔甚至由表达所有基因未突变副本的病毒粒子触发。我们的工作表明，流感病毒群体中缺陷的多样谱有助于但不能完全解释单个感染细胞中病毒基因表达和免疫激活的异质性。由于流感病毒具有很高的突变率，许多细胞被突变的病毒粒子感染。但是到目前为止，由于传统技术（如流式细胞术和单细胞转录组测序（scRNA-seq））只能检测蛋白质或转录本是否存在，而不能检测其序列，因此仍然不可能完全表征感染任何给定细胞的病毒粒子的序列。在这里，我们开发了一种新方法，该方法使用长读 PacBio 测序来确定感染单个细胞的病毒粒子的序列。我们表明，病毒遗传变异解释了病毒基因表达和先天免疫诱导的细胞间变异性的一部分，但不是全部。总体而言，我们的研究提供了病毒突变如何影响单个细胞感染过程的第一份完整图片。
