{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,26]],"date-time":"2025-11-26T16:40:30Z","timestamp":1764175230116,"version":"build-2065373602"},"reference-count":63,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2022,6,21]],"date-time":"2022-06-21T00:00:00Z","timestamp":1655769600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Membrane fusion constitutes an essential step in the replication cycle of numerous viral pathogens, hence it represents an important druggable target. In the present study, we established a virus-free, stable reporter fusion inhibition assay (SRFIA) specifically designed to identify compounds interfering with virus-induced membrane fusion. The dual reporter assay is based on two stable Vero cell lines harboring the third-generation tetracycline (Tet3G) transactivator and a bicistronic reporter gene cassette under the control of the tetracycline responsive element (TRE3G), respectively. Cell\u2013cell fusion by the transient transfection of viral fusogens in the presence of doxycycline results in the expression of the reporter enzyme secreted alkaline phosphatase (SEAP) and the fluorescent nuclear localization marker EYFPNuc. A constitutively expressed, secreted form of nanoluciferase (secNLuc) functioned as the internal control. The performance of the SRFIA was tested for the quantification of SARS-CoV-2- and HSV-1-induced cell\u2013cell fusion, respectively, showing high sensitivity and specificity, as well as the reliable identification of known fusion inhibitors. Parallel quantification of secNLuc enabled the detection of cytotoxic compounds or insufficient transfection efficacy. In conclusion, the SRFIA reported here is well suited for high-throughput screening for new antiviral agents and essentially will be applicable to all viral fusogens causing cell\u2013cell fusion in Vero cells.<\/jats:p>","DOI":"10.3390\/v14071354","type":"journal-article","created":{"date-parts":[[2022,6,23]],"date-time":"2022-06-23T01:59:47Z","timestamp":1655949587000},"page":"1354","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Broadly Applicable, Virus-Free Dual Reporter Assay to Identify Compounds Interfering with Membrane Fusion: Performance for HSV-1 and SARS-CoV-2"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2745-5748","authenticated-orcid":false,"given":"Nica","family":"Classen","sequence":"first","affiliation":[{"name":"Institute of Pharmaceutical Biology and Phytochemistry, University of M\u00fcnster, Corrensstr. 48, 48149 M\u00fcnster, Germany"},{"name":"Institute of Virology\u2014Clinical Virology, University of M\u00fcnster and University Hospital M\u00fcnster, Von-Stauffenberg-Str. 36, 48151 M\u00fcnster, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Diana","family":"Ulrich","sequence":"additional","affiliation":[{"name":"Institute of Pharmaceutical Biology and Phytochemistry, University of M\u00fcnster, Corrensstr. 48, 48149 M\u00fcnster, Germany"},{"name":"Institute of Virology\u2014Clinical Virology, University of M\u00fcnster and University Hospital M\u00fcnster, Von-Stauffenberg-Str. 36, 48151 M\u00fcnster, Germany"},{"name":"Vibalogics GmbH, Zeppelinstr. 2, 27472 Cuxhaven, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Arne","family":"Hofemeier","sequence":"additional","affiliation":[{"name":"Institute of Virology\u2014Clinical Virology, University of M\u00fcnster and University Hospital M\u00fcnster, Von-Stauffenberg-Str. 36, 48151 M\u00fcnster, Germany"},{"name":"Third Institute of Physics\u2014Biophysics, University of G\u00f6ttingen, Friedrich-Hund-Platz 1, 37077 G\u00f6ttingen, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marc Tim","family":"Hennies","sequence":"additional","affiliation":[{"name":"Institute of Virology\u2014Clinical Virology, University of M\u00fcnster and University Hospital M\u00fcnster, Von-Stauffenberg-Str. 36, 48151 M\u00fcnster, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wali","family":"Hafezi","sequence":"additional","affiliation":[{"name":"Institute of Virology\u2014Clinical Virology, University of M\u00fcnster and University Hospital M\u00fcnster, Von-Stauffenberg-Str. 36, 48151 M\u00fcnster, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aleksandra","family":"Pettke","sequence":"additional","affiliation":[{"name":"Institute of Virology\u2014Clinical Virology, University of M\u00fcnster and University Hospital M\u00fcnster, Von-Stauffenberg-Str. 36, 48151 M\u00fcnster, Germany"},{"name":"The Public Health Agency of Sweden, Nobels v\u00e4g 18, 171 82 Solna, Sweden"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marie-Luise","family":"Romberg","sequence":"additional","affiliation":[{"name":"Institute of Virology\u2014Clinical Virology, University of M\u00fcnster and University Hospital M\u00fcnster, Von-Stauffenberg-Str. 36, 48151 M\u00fcnster, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5570-1897","authenticated-orcid":false,"given":"Eva U.","family":"Lorentzen","sequence":"additional","affiliation":[{"name":"Institute of Virology\u2014Clinical Virology, University of M\u00fcnster and University Hospital M\u00fcnster, Von-Stauffenberg-Str. 36, 48151 M\u00fcnster, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0886-8384","authenticated-orcid":false,"given":"Andreas","family":"Hensel","sequence":"additional","affiliation":[{"name":"Institute of Pharmaceutical Biology and Phytochemistry, University of M\u00fcnster, Corrensstr. 48, 48149 M\u00fcnster, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joachim E.","family":"K\u00fchn","sequence":"additional","affiliation":[{"name":"Institute of Virology\u2014Clinical Virology, University of M\u00fcnster and University Hospital M\u00fcnster, Von-Stauffenberg-Str. 36, 48151 M\u00fcnster, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,6,21]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"690","DOI":"10.1038\/nsmb.1456","article-title":"Viral membrane fusion","volume":"15","author":"Harrison","year":"2008","journal-title":"Nat. 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