Department of Microbiology and Immunology, Hamamatsu University School of Medicine, Shizuoka 431-3192, Japan.
Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640, Japan.
Int J Mol Sci. 2022 Aug 5;23(15):8711. doi: 10.3390/ijms23158711.
In microbiological research, it is important to understand the time course of each step in a pathogen's lifecycle and changes in the host cell environment induced by infection. This study is the first to develop a real-time monitoring system that kinetically detects luminescence reporter activity over time without sampling cells or culture supernatants for analyzing the virus replication. Subgenomic replicon experiments with hepatitis C virus (HCV) showed that transient translation and genome replication can be detected separately, with the first peak of translation observed at 3-4 h and replication beginning around 20 h after viral RNA introduction into cells. From the bioluminescence data set measured every 30 min (48 measurements per day), the initial rates of translation and replication were calculated, and their capacity levels were expressed as the sums of the measured signals in each process, which correspond to the areas on the kinetics graphs. The comparison of various HuH-7-derived cell lines showed that the bioluminescence profile differs among cell lines, suggesting that both translation and replication capacities potentially influence differences in HCV susceptibility. The effects of RNA mutations within the 5' UTR of the replicon on viral translation and replication were further analyzed in the system developed, confirming that mutations to the miR-122 binding sites primarily reduce replication activity rather than translation. The newly developed real-time monitoring system should be applied to the studies of various viruses and contribute to the analysis of transitions and progression of each process of their life cycle.
在微生物研究中，了解病原体生命周期中每个步骤的时间进程以及感染引起的宿主细胞环境变化非常重要。本研究首次开发了一种实时监测系统，该系统无需采样细胞或培养上清液即可对病毒复制进行分析，从而对荧光报告活性进行动态检测。丙型肝炎病毒（HCV）的亚基因组复制子实验表明，可以分别检测瞬时翻译和基因组复制，翻译的第一个峰值出现在 3-4 小时，复制在病毒 RNA 导入细胞后约 20 小时开始。从每 30 分钟（每天 48 次测量）测量的生物发光数据集计算翻译和复制的初始速率，并将其容量水平表示为每个过程中测量信号的总和，这与动力学图上的面积相对应。对各种 HuH-7 衍生细胞系的比较表明，生物发光图谱在细胞系之间存在差异，这表明翻译和复制能力可能会影响 HCV 的易感性差异。在开发的系统中进一步分析了复制子 5'UTR 内 RNA 突变对病毒翻译和复制的影响，证实突变 miR-122 结合位点主要降低复制活性，而不是翻译。新开发的实时监测系统应该应用于各种病毒的研究，并有助于分析其生命周期中每个过程的转变和进展。
