{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,4]],"date-time":"2025-11-04T16:15:33Z","timestamp":1762272933166,"version":"build-2065373602"},"reference-count":30,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2022,7,15]],"date-time":"2022-07-15T00:00:00Z","timestamp":1657843200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002261","name":"Russian Foundation for Basic Research","doi-asserted-by":"publisher","award":["20-04-60370"],"award-info":[{"award-number":["20-04-60370"]}],"id":[{"id":"10.13039\/501100002261","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>In the SARS-CoV-2 lifecycle, papain-like protease PLpro cuts off the non-structural proteins nsp1, nsp2, and nsp3 from a large polyprotein. This is the earliest viral enzymatic activity, which is crucial for all downstream steps. Here, we designed two genetically encoded fluorescent sensors for the real-time detection of PLpro activity in live cells. The first sensor was based on the F\u00f6rster resonance energy transfer (FRET) between the red fluorescent protein mScarlet as a donor and the biliverdin-binding near-infrared fluorescent protein miRFP670 as an acceptor. A linker with the PLpro recognition site LKGG in between made this FRET pair sensitive to PLpro cleavage. Upon the co-expression of mScarlet-LKGG-miRFP670 and PLpro in HeLa cells, we observed a gradual increase in the donor fluorescence intensity of about 1.5-fold. In the second sensor, both PLpro and its target\u2014green mNeonGreen and red mScarletI fluorescent proteins separated by an LKGG-containing linker\u2014were attached to the endoplasmic reticulum (ER) membrane. Upon cleavage by PLpro, mScarletI diffused from the ER throughout the cell. About a two-fold increase in the nucleus\/cytoplasm ratio was observed as a result of the PLpro action. We believe that the new PLpro sensors can potentially be used to detect the earliest stages of SARS-CoV-2 propagation in live cells as well as for the screening of PLpro inhibitors.<\/jats:p>","DOI":"10.3390\/ijms23147826","type":"journal-article","created":{"date-parts":[[2022,7,17]],"date-time":"2022-07-17T22:57:06Z","timestamp":1658098626000},"page":"7826","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Genetically Encoded Fluorescent Sensors for SARS-CoV-2 Papain-like Protease PLpro"],"prefix":"10.3390","volume":"23","author":[{"given":"Elena L.","family":"Sokolinskaya","sequence":"first","affiliation":[{"name":"Center for Molecular and Cellular Biology, Skolkovo Institute of Science and Technology, Bolshoy Blvd 30, bld. 1, 121205 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0684-5672","authenticated-orcid":false,"given":"Lidia V.","family":"Putlyaeva","sequence":"additional","affiliation":[{"name":"Center for Molecular and Cellular Biology, Skolkovo Institute of Science and Technology, Bolshoy Blvd 30, bld. 1, 121205 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vasilisa S.","family":"Polinovskaya","sequence":"additional","affiliation":[{"name":"Center for Molecular and Cellular Biology, Skolkovo Institute of Science and Technology, Bolshoy Blvd 30, bld. 1, 121205 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9845-2088","authenticated-orcid":false,"given":"Konstantin A.","family":"Lukyanov","sequence":"additional","affiliation":[{"name":"Center for Molecular and Cellular Biology, Skolkovo Institute of Science and Technology, Bolshoy Blvd 30, bld. 1, 121205 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,7,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"106727","DOI":"10.1016\/j.ijsu.2022.106727","article-title":"Emergence of novel omicron hybrid variants: BA(x), XE, XD, XF more than just alphabets","volume":"104","author":"Thakur","year":"2022","journal-title":"Int. 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