Kedia Nandita, Banerjee Saptarshi, Mondal Arindam
School of Bioscience, Indian Institute of Technology Kharagpur, Kharagpur, India.
Front Microbiol. 2022 May 25;13:868367. doi: 10.3389/fmicb.2022.868367. eCollection 2022.
The mini-genome reporter assay is a key tool for conducting RNA virus research. However, procedural complications and the lack of adequate literature pose a major challenge in developing these assay systems. Here, we present a novel, yet generic and simple, cloning strategy for the construction of an influenza B virus reporter RNA template and describe an extensive standardization of the reporter RNP/polymerase activity assay for monitoring viral RNA synthesis in an infection-free setting. Using this assay system, we showed for the first time the effect of viral protein NS1 and host protein kinase C delta (PKCD) on influenza B virus RNA synthesis. In addition, the assay system showed promising results in evaluating the efficacy of antiviral drugs targeting viral RNA synthesis and virus propagation. Together, this work offers a detailed protocol for the standardization of the influenza virus minigenome assay and an excellent tool for screening of host factors and antivirals in a fast, user-friendly, and high-throughput manner.
微型基因组报告基因检测是进行RNA病毒研究的关键工具。然而，程序上的复杂性以及缺乏足够的文献在开发这些检测系统方面构成了重大挑战。在此，我们提出了一种新颖、通用且简单的克隆策略，用于构建乙型流感病毒报告RNA模板，并描述了报告核糖核蛋白/聚合酶活性检测的广泛标准化，以在无感染环境中监测病毒RNA合成。使用该检测系统，我们首次展示了病毒蛋白NS1和宿主蛋白激酶Cδ（PKCD）对乙型流感病毒RNA合成的影响。此外，该检测系统在评估靶向病毒RNA合成和病毒增殖的抗病毒药物疗效方面显示出有前景的结果。总之，这项工作提供了一份用于流感病毒微型基因组检测标准化的详细方案，以及一个以快速、用户友好且高通量的方式筛选宿主因子和抗病毒药物的优秀工具。
