Single-cell transcriptional analysis of murine norovirus infection in a human intestinal cell line.

IF 4 2区 医学 Q2 VIROLOGY
Journal of Virology Pub Date : 2024-11-19 Epub Date: 2024-10-30 DOI:10.1128/jvi.01617-24
Yuki Matsushima, Eric A Levenson, Natthawan Chaimongkol, Loyall Harris, Yongmei Zhao, Sevilay Turan, Francisco Otaizo-Carrasquero, Sundar Ganesan, Katherine M Hornick, Craig Martens, Stanislav V Sosnovtsev, Kim Y Green
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引用次数: 0

Abstract

Noroviruses are a major agent of acute gastroenteritis in humans, but host cell requirements for efficient replication in vitro have not been established. We engineered a human intestinal cell line (designated mCD300lf-hCaco2) expressing the murine norovirus (MNV) receptor, mouse CD300lf to become fully permissive for MNV replication. To explore the replicative machinery and host response of these cells, we performed a single-cell RNA sequencing (scRNA-seq) transcriptomics analysis of an MNV infection over time. Marked similarities were observed between certain global features of MNV infection in human cells compared to those previously reported in mouse cells by whole population transcriptomics such as downregulation of ribosome biogenesis, mitochondrial dysfunction, and cell cycle preference for G1. Our scRNA-seq analysis allowed further resolution of an infected cell population into distinct clusters with varying levels of viral RNA and interferon-stimulated gene ISG15 transcripts. Cells with high viral replication displayed downregulated ribosomal protein small (RPS) and large (RPL) genes and mitochondrial complexes I, III, IV, and V genes during exponential viral propagation. Ferritin subunit genes FTL and FTH1 were also downregulated during active MNV replication, suggesting that inhibition of iron metabolism may increase replication efficiency. Consistent with this, transcriptional activation of these genes with ferric ammonium citrate and overexpression of FTL lowered virus yields. Comparative studies of cells that support varying levels of norovirus replication efficiency, as determined by scRNA-seq may lead to improved human cell-based culture systems and effective viral interventions.IMPORTANCEHuman noroviruses cause acute gastroenteritis in all age groups. Vaccines and antiviral drugs are not yet available, in part, because it is difficult to propagate the viruses causing human disease in standard laboratory cell culture systems. In contrast, a norovirus found in mice [murine norovirus (MNV)] replicates efficiently in murine-based cell culture and has served as a model system. In this study, we established a new human intestinal cell line that was genetically modified to express the murine norovirus receptor so that the human cells became permissive to murine norovirus infection. We then defined the host response to MNV infection in the engineered human cell line at a single-cell resolution and identified cellular genes associated with the highest levels of MNV replication. This study may lead to the improvement of the current human norovirus cell culture systems and help to identify norovirus-host interactions that could be targeted for antiviral drugs.

人肠道细胞系感染鼠诺如病毒的单细胞转录分析。
诺如病毒是人类急性肠胃炎的主要病原体,但宿主细胞在体外有效复制的要求尚未确定。我们设计了一种表达小鼠诺如病毒(MNV)受体--小鼠 CD300lf 的人肠道细胞系(命名为 mCD300lf-hCaco2),使其完全允许 MNV 复制。为了探索这些细胞的复制机制和宿主反应,我们对MNV感染的时间进行了单细胞RNA测序(scRNA-seq)转录组学分析。与之前在小鼠细胞中通过全群转录组学观察到的MNV感染的某些全局特征(如核糖体生物发生的下调、线粒体功能障碍和细胞周期对G1的偏好)相比,在人类细胞中观察到了明显的相似性。我们的 scRNA-seq 分析进一步将受感染的细胞群划分为不同的群组,这些群组的病毒 RNA 和干扰素刺激基因 ISG15 转录本水平各不相同。在病毒的指数传播过程中,病毒复制量大的细胞显示出核糖体蛋白小(RPS)和大(RPL)基因以及线粒体复合物 I、III、IV 和 V 基因的下调。铁蛋白亚基基因 FTL 和 FTH1 也在 MNV 复制活跃期下调,这表明抑制铁代谢可能会提高复制效率。与此相一致的是,柠檬酸铁铵对这些基因的转录激活以及 FTL 的过表达降低了病毒产量。通过 scRNA-seq 对支持不同水平诺如病毒复制效率的细胞进行比较研究,可能会改进基于人体细胞的培养系统和有效的病毒干预措施。目前还没有疫苗和抗病毒药物,部分原因是很难在标准的实验室细胞培养系统中繁殖导致人类疾病的病毒。与此相反,一种在小鼠体内发现的诺如病毒(鼠诺如病毒(MNV))却能在小鼠细胞培养中高效复制,并被用作模型系统。在本研究中,我们建立了一种新的人类肠道细胞系,并对其进行了基因改造,使其表达鼠诺如病毒受体,从而使人类细胞对鼠诺如病毒感染具有容许性。然后,我们以单细胞分辨率定义了宿主对工程人细胞系中 MNV 感染的反应,并确定了与最高水平 MNV 复制相关的细胞基因。这项研究可能会改进目前的人类诺如病毒细胞培养系统,并有助于确定诺如病毒与宿主之间的相互作用,从而成为抗病毒药物的靶标。
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来源期刊
Journal of Virology
Journal of Virology 医学-病毒学
CiteScore
10.10
自引率
7.40%
发文量
906
审稿时长
1 months
期刊介绍: Journal of Virology (JVI) explores the nature of the viruses of animals, archaea, bacteria, fungi, plants, and protozoa. We welcome papers on virion structure and assembly, viral genome replication and regulation of gene expression, genetic diversity and evolution, virus-cell interactions, cellular responses to infection, transformation and oncogenesis, gene delivery, viral pathogenesis and immunity, and vaccines and antiviral agents.
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