key: cord-273083-xrydkiu4 authors: Pahmeier, Felix; Neufeldt, Christoper J; Cerikan, Berati; Prasad, Vibhu; Pape, Costantin; Laketa, Vibor; Ruggieri, Alessia; Bartenschlager, Ralf; Cortese, Mirko title: A versatile reporter system to monitor virus infected cells and its application to dengue virus and SARS-CoV-2 date: 2020-09-01 journal: bioRxiv DOI: 10.1101/2020.08.31.276683 sha: doc_id: 273083 cord_uid: xrydkiu4 Positive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of that are flaviviruses, such as dengue virus and Zika virus that cause millions of yearly infections and spread around the globe, and coronaviruses, such as SARS-CoV-2, which is the cause of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of virology research in determining mechanisms to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective interventions. Here, we describe the generation and characterization of a reporter system to visualize dengue virus and SARS-CoV-2 replication in live cells. The system is based on viral protease activity causing cleavage and nuclear translocation of an engineered fluorescent protein that is expressed in the infected cells. We show the suitability of the system for live cell imaging and visualization of single infected cells as well as for screening and testing of antiviral compounds. Given the modular building blocks, the 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 viral replication and spread within a host cell population. Here we describe a reporter system that takes advantage of virus-encoded proteases that are expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence. Upon cleavage, the fluorescent protein 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. Antibodies. The antibodies used in this study are listed in Table 1 . 124 Oligonucleotides encoding the protease cleavage sites were designed to allow 129 insertion into the vector via MluI and BamHI restriction sites. The primer pairs (Table TABLE 2 Sequences of oligonucleotides used in this study. SARS-CoV-2 (MOI = 10) and 2 hpi the medium was exchanged for imaging medium. Lid was moved to the locked position and silicon was used to seal the dish in order to Bioinformatics analysis. Images were analyzed using the Fiji software (32, 33). Graph generation and statistical analysis was performed using the GraphPad Prism 8. Design and characterization of DENV reporter constructs. In order to generate a reporter system that can specifically indicate virus infection, we 219 designed a construct expressing a GFP fusion protein that could selectively be cleaved 220 by viral proteases. The reporter construct was engineered for viruses that produce ER which can be easily detected and quantified by light microscopy. The DENV polyprotein is cleaved into the individual viral proteins by either the host 231 signal peptidase of the viral NS2B/3 serine protease (12, 13). The ER-resident NS2B 232 protein acts as a co-factor of NS3 protease and anchors it to ER membranes (35, 36). To determine an optimal system for reporting DENV infection, several previously 234 described NS2B/3 specific cleavage sequences were inserted into the reporter 235 construct (Table 3) . Recently, a plasmid-based expression system for induction of DENV replication 299 organelles in transfected cells has been described (40). This system, designated 300 "plasmid-induced replication organelle -dengue (pIRO-D), encodes the viral 301 polyprotein that is translated from an RNA generated in the cytoplasm by a stably 302 expressed T7 RNA polymerase. In this way, the pIRO-D system allows the analysis of viral proteins in cells, independent of viral replication. However, since no fluorescent 304 protein coding sequence is incorporated into the construct, expression of the DENV 305 polyprotein cannot be followed by live cell imaging. To overcome this limitation, we determined whether our DENV reporter cell line (Figures 2 and 3) . Interestingly, for several DENV reporter constructs, 418 nuclear GFP localization was observed in absence of the viral protease (Figure 2A) Huh7-RC and Lunet-T7-RC, we had to establish single cell clones for the were observed ( Figure 4A ). This is likely due to differences in the ability of cell lines 441 to respond to high levels of GFP fusion proteins. In addition to sorting for cells with 442 lower reporter expression as done here, this problem might be overcome by 443 employing less active promoter or by using an alternative fluorescent protein. 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