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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.IMPORTANCE 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 Flaviviridae and the Coronaviridae 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.

Unlabelled: Production of proinflammatory cytokines indicative of potent recognition by the host innate immune system has long been recognized as a hallmark of the acute phase of HIV-1 infection. The first components of the machinery by which primary HIV target cells sense infection have recently been described; however, the mechanistic dissection of innate immune recognition and viral evasion would be facilitated by an easily accessible cell line model. Here we describe that reconstituted expression of the innate signaling adaptor STING enhanced the ability of the well-established HIV reporter cell line Tzm-bl to sense HIV infection and to convert this information into nuclear translocation of IRF3 as well as expression of cytokine mRNA. STING-dependent immune sensing of HIV-1 required virus entry and reverse transcription but not genome integration. Particularly efficient recognition was observed for an HIV-1 variant lacking expression of the accessory protein Vpr, suggesting a role of the viral protein in circumventing STING-mediated immune signaling. Vpr as well as STING significantly impacted the magnitude and breadth of the cytokine mRNA expression profile induced upon HIV-1 infection. However, cytoplasmic DNA sensing did not result in detectable cytokine secretion in this cell system, and innate immune recognition did not affect infection rates. Despite these deficits in eliciting antiviral effector functions, these results establish Tzm-bl STING and Tzm-bl STING IRF3.GFP cells as useful tools for studies aimed at dissecting mechanisms and regulation of early innate immune recognition of HIV infection., Importance: Cell-autonomous immune recognition of HIV infection was recently established as an important aspect by which the host immune system attempts to fend off HIV-1 infection. Mechanistic studies on host cell recognition and viral evasion are hampered by the resistance of many primary HIV target cells to detailed experimental manipulation. We describe here that expression of the signaling adaptor STING renders the well-established HIV reporter cell line Tzm-bl competent for innate recognition of HIV infection. Key characteristics reflected in this cell model include nuclear translocation of IRF3, expression of a broad range of cytokine mRNAs, and an antagonistic activity of the HIV-1 protein Vpr. These results establish Tzm-bl STING and Tzm-bl STING IRF3.GFP cells as a useful tool for studies of innate recognition of HIV infection., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)