Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany.
HCI/IWR, Heidelberg University, Heidelberg, Germany.
J Virol. 2021 Jan 28;95(4). doi: 10.1128/JVI.01715-20.
Positive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of this include flaviviruses, such as dengue virus and Zika virus, which cause millions of yearly infections around the globe, and coronaviruses, such as SARS-CoV-2, the source of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of research aimed at determining methods to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective treatments. Here, we describe the generation and characterization of a reporter system that can be used to visualize and identify cells infected with dengue virus or SARS-CoV-2. This system is based on viral protease activity that mediates cleavage and nuclear translocation of an engineered fluorescent protein stably expressed in cells. We show the suitability of this system for live cell imaging, for visualization of single infected cells, and for screening and testing of antiviral compounds. With the integrated modular building blocks, this system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility. Reporter systems are useful tools for fast and quantitative visualization of virus-infected cells within a host cell population. Here, we describe a reporter system that takes advantage of virus-encoded proteases expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence. Upon cleavage, the GFP moiety translocates to the nucleus, allowing for rapid detection of the infected cells. Using this system, we demonstrate reliable reporting activity for two major human pathogens from the and the families: dengue virus and SARS-CoV-2. We apply this reporter system to live cell imaging and use it for proof-of-concept to validate antiviral activity of a nucleoside analogue. This reporter system is not only an invaluable tool for the characterization of viral replication, but also for the discovery and development of antivirals that are urgently needed to halt the spread of these viruses.
正链 RNA 病毒在过去几十年中的几次重大疾病爆发中是病原体。例如，黄病毒，如登革热病毒和寨卡病毒，每年在全球范围内导致数百万人感染；冠状病毒，如 SARS-CoV-2，是当前大流行的源头。这些病毒引起的疫情的严重程度强调了研究的重要性，旨在确定限制病毒传播和遏制疾病严重程度的方法。这些研究需要分子工具来破译病毒-宿主相互作用并开发有效的治疗方法。在这里，我们描述了一种报告系统的生成和表征，该系统可用于可视化和鉴定感染登革热病毒或 SARS-CoV-2 的细胞。该系统基于病毒蛋白酶活性，介导稳定表达在细胞中的工程荧光蛋白的切割和核易位。我们展示了该系统用于活细胞成像、单个感染细胞的可视化以及抗病毒化合物的筛选和测试的适用性。通过集成的模块化构建块，该系统易于操作并且可以适应任何编码蛋白酶的病毒，因此具有高度的灵活性。报告系统是在宿主细胞群体中快速定量可视化病毒感染细胞的有用工具。在这里，我们描述了一种报告系统，该系统利用感染细胞中表达的病毒编码蛋白酶来切割融合到核定位序列的 ER 锚定荧光蛋白。切割后，GFP 部分易位到细胞核，从而可以快速检测感染的细胞。使用该系统，我们证明了两种主要的人类病原体（ 和 科）的可靠报告活性：登革热病毒和 SARS-CoV-2。我们将该报告系统应用于活细胞成像，并将其用于验证核苷类似物抗病毒活性的概念验证。该报告系统不仅是表征病毒复制的宝贵工具，也是发现和开发急需的抗病毒药物的工具，以阻止这些病毒的传播。
