Cells were fixed at 48 hpi and stained for double-stranded RNA (dsRNA) by IF. (F) Huh7-derived cells expressing reporter construct 1 (Huh7-RC) were infected with DENV serotypes 1 to 4 at an MOI of 5. Black arrowheads, uncleaved reporter red arrowheads, reporter cleavage products. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) served as a loading control. NS3 and GFP were detected with a specific antibody. At 48 hpi, cells were lysed and 10 μg of total protein for each sample was resolved by SDS-PAGE. (E) Cells expressing the reporter constructs 1 to 8 or an empty plasmid (empty) were infected with DENV2 (MOI = 5). For each construct, more than 60 cells were counted. The percentages of cells positive for NS3 and positive for both nuclear GFP and NS3 signals were quantified. (D) Quantification of images as in panel C. Magenta, DENV NS3 protein green, reporter GFP signal. Samples were analyzed by confocal microscopy. Cells were fixed at 48 hpi and NS3 was stained by immunofluorescence. (C) Huh7 cells were transduced as above for 24 h before being infected with DENV2 at an MOI of 5. For each construct, more than 70 cells were counted. The percentage of cells showing nuclear or cytosolic GFP localization is shown. (B) Quantification of images acquired as in panel A. Cells were fixed at 72 h postransduction and the subcellular distribution of GFP was analyzed by confocal microscopy. ![]() (A) Huh7 cells were transduced with lentiviruses encoding the different DENV GFP-based reporter constructs 1 to 8 (Table 1) at an MOI of 5. SARS-CoV-2 dengue virus live cell imaging reporter cell lines reporter system viral proteases.Ĭopyright © 2021 American Society for Microbiology.Įvaluation of DENV reporter constructs. 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. We apply this reporter system to live cell imaging and use it for proof-of-concept to validate antiviral activity of a nucleoside analogue. Using this system, we demonstrate reliable reporting activity for two major human pathogens from the Flaviviridae and the Coronaviridae families: dengue virus and SARS-CoV-2. ![]() Upon cleavage, the GFP moiety translocates to the nucleus, allowing for rapid detection of the infected cells. 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. IMPORTANCE Reporter systems are useful tools for fast and quantitative visualization of virus-infected cells within a host cell population. 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. 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. This system is based on viral protease activity that mediates cleavage and nuclear translocation of an engineered fluorescent protein stably expressed in cells. 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. Such studies require molecular tools to decipher virus-host interactions and to develop effective treatments. 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. 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. Positive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades.
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