A versatile reporter system to monitor virus infected cells and its application to1 dengue virus and SARS-CoV-22 3 Felix Pahmeiera, Christoper J Neufeldta, Berati Cerikana, Vibhu Prasada, Costantin4 Papeb,c, Vibor Laketad,, Alessia Ruggieria, Ralf Bartenschlagera,d,e,#, Mirko Cortesea,#5 6 aDepartment of Infectious Diseases, Molecular Virology, University of Heidelberg,7 Center for Integrative Infectious Disease Research, Heidelberg, Germany8 bHCI/IWR, Heidelberg University, Heidelberg, Germany9 cEuropean Molecular Biology Laboratory, Heidelberg, Germany10 dGermanCenter for Infection Research, Heidelberg partner site, Heidelberg, Germany11 eDivision<Virus-Associated Carcinogenesis=, German Cancer Research Center12 (DKFZ), Heidelberg, Germany13 14 #Address correspondence to:15 ralf.bartenschlager@med.uni-heidelberg.de16 mirko.cortese@med.uni-heidelberg.de17 18 Running title: A reporter system for SARS-CoV-2 and dengue viruses19 20 Keywords: SARS-CoV-2; dengue virus; live cell imaging; fluorescent reporter21 system; reporter cell lines; viral proteases22 23 Word count for the abstract: 19524 Word count for the text: 516925 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 1, 2020.;https://doi.org/10.1101/2020.08.31.276683doi:bioRxiv preprint
ABSTRACT26 Positive-strand RNA viruses have been the etiological agents in several major disease27 outbreaks over the last few decades. Examples of that are flaviviruses, such as dengue28 virus and Zika virus that cause millions of yearly infections and spread around the29 globe, and coronaviruses, such as SARS-CoV-2, which is the cause of the current30 pandemic. The severity of outbreaks caused by these viruses stresses the importance31 of virology research in determining mechanisms to limit virus spread and to curb32 diseaseseverity.Suchstudiesrequiremoleculartoolstodeciphervirus-host33 interactions and to develop effective interventions. Here, we describe the generation34 and characterization of a reporter system to visualize dengue virus and SARS-CoV-235 replication in live cells. The system is based on viral protease activity causing36 cleavageand nuclear translocation of anengineered fluorescentproteinthat is37 expressed in the infected cells. We show the suitability of the system for live cell38 imaging and visualization of single infected cells as well as for screening and testing39 of antiviral compounds. Given the modular building blocks, the system is easy to40 manipulate and can be adapted to any virus encoding a protease, thus offering a high41 degree of flexibility.42 IMPORTANCE43 Reporter systems are useful tools for fast and quantitative visualization of viral44 replication and spread within a host cell population. Here we describe a reporter45 system that takes advantage of virus-encoded proteases that are expressed in infected46 cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization47 sequence. Upon cleavage, the fluorescent protein translocates to the nucleus, allowing48 for rapid detection of the infected cells. Using this system, we demonstrate reliable49 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 1, 2020.;https://doi.org/10.1101/2020.08.31.276683doi:bioRxiv preprint
reporting activity for two major human pathogens from theFlaviviridaeand the50 Coronaviridaefamilies: dengue virus and SARS-CoV-2. We apply this reporter51 system to live cell imaging and use it for proof-of-concept to validate antiviral activity52 of a nucleoside analogue. This reporter system is not only an invaluable tool for the53 characterization of viral replication, but also for the discovery and development of54 antivirals that are urgently needed to halt the spread of these viruses.55 INTRODUCTION56 Positive sense single stranded RNA viruses constitute a major fraction of endemic and57 emerging human viruses (1). Among the positive-strand RNA viruses, flaviviruses58 such as dengue virus (DENV) and Zika virus (ZIKV) are some of the most prevalent59 arboviralpathogensandareconsideredamajorpublichealthproblem(2,3).60 Currently, there are no universal vaccines or specific antiviral drug approved for the61 preventionortreatmentofinfectionswiththeseviruses(4).Membersofthe62 Coronaviridaefamily also have a positive-strand RNA genome and have caused63 major outbreaks in the last two decades (5, 6). Currently, the world is facing the64 pandemic outbreak of SARS-CoV-2, the causative agent of coronavirus disease 201965 (COVID-19) (7, 8). As of August 2020, over 19 million confirmed cases and more66 than 700,000 confirmed deaths have been reported in 216 countries (9). Despite67 immense efforts by research teams around the world, there is still a dire need for68 effective and widely available treatment options and a prophylactic vaccine.69 Once released into the cell, the full genome of flaviviruses and the large open reading70 frame (ORF1ab) of coronaviruses are translated as polyproteins. Signal peptides and71 internaltransmembraneregionsdirectpolyproteinsynthesistotheendoplasmic72 reticulum (ER) membrane where co-translational cleavage generates the mature viral73 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 1, 2020.;https://doi.org/10.1101/2020.08.31.276683doi:bioRxiv preprint
proteins (10, 11). The flaviviral protease NS2B/3, together with host proteases,74 cleaves the flavivirus polyprotein into three structural and seven non-structural75 proteins(12,13).Inthecaseofcoronaviruses,ORF1abisexpressedastwo76 polyproteins, which are cleaved into sixteen non-structural proteins (nsp) by the viral77 papain-like protease (PLpro) residing in nsp3 and the 3C-like protease (3CLpro) of nsp578 (14–17). The replication of viral RNA of both virus groups was shown to occur on ER79 derived membranes, in specialized virus-induced membrane compartments termed80 replication organelles (10, 11, 18–20).81 Reporter systems for detection of virus infection are an invaluable tool for the82 characterization and quantification of virus infection kinetics, for the characterization83 of virus-host cell interactions and for the identification of antiviral compounds. One84 approach is the insertion of tags into the viral genome that, upon replication and85 translation, allow for visualization of the infected cells. However, this approach86 requires functional molecular clones of a given genome, which are not always87 available. In addition, insertion of atag frequently causesattenuationof viral88 replication competency and therefore, the search for adequate insertion sites is time-89 consuming or might fail.90 An alternative approach is the use of engineered fluorescent reporter proteins stably91 expressed in cells and altering their subcellular distribution upon viral infection (21–92 23). Building on this idea, here we established a reporter system based on an ER-93 anchored green fluorescent protein (GFP) that upon cleavage by a viral protease is94 released from the ER and translocated into the nucleus. Using this system, we95 demonstrate the reliable reporting activity of DENV and SARS-CoV-2 infected cells.96 Moreover, we apply this reporter cell system to live cell imaging and assessment of an97 antiviral compound.98 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 1, 2020.;https://doi.org/10.1101/2020.08.31.276683doi:bioRxiv preprint
MATERIALS AND METHODS99 Cell lines and virus strains.HEK-293T, A549 and VeroE6 cells were purchased100 from ATCC; Huh7 cells (24) were obtained from Heinz Schaller (Center for101 Molecular Biology, Heidelberg). Generation of the cell lines Huh7-Lunet and the102 derivative Huh7-Lunet-T7, stably expressing the RNA polymerase of bacteriophage103 T7, have been previously described (25, 26). All cells were cultured at 37°C and 5%104 CO2in Dulbecco’s modified Eagle medium (DMEM, Life Technologies) containing105 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin and 1% non-106 essential amino acids (complete medium). Huh7-Lunet-T7 cells were cultured in107 complete medium, supplemented with 5 µg/mL zeocin. A549-ACE2 were generated108 by transduction of A549 with lentiviruses encoding for the human Angiotensin-109 converting enzyme 2 (ACE2) gene as previously described (41).110 Wild-type (WT) DENV-2 was produced from an infectious molecular clone based on111 strain 16681 as described elsewhere (27). The DENV reporter virus genome encoding112 theTurbo farredfluorescentproteinFP635(DENV-faR)hasbeenpreviously113 described (28). SARS-CoV-2 (strain BavPat1) was kindly provided by Prof. Christian114 Drosten (Charité Berlin, Germany) through the European Virology Archive. Except115 for DENV-faR that was generated by electroporation of BHK-21 cells as previously116 described(28),allvirusstocksweregeneratedbyinfectionofVeroE6cells.117 Supernatants were harvested, filtered, and virus concentration was determined by118 plaque assay. For infection experiments, cells were inoculated as specified in the119 results section for 1 h at 37°C. Fresh complete medium was then added, and cells120 were incubated for the indicated time spans.121 122 123 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted September 1, 2020.;https://doi.org/10.1101/2020.08.31.276683doi:bioRxiv preprint
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