{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,28]],"date-time":"2026-06-28T04:40:51Z","timestamp":1782621651015,"version":"3.54.5"},"reference-count":21,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2013,11,28]],"date-time":"2013-11-28T00:00:00Z","timestamp":1385596800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/3.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Sensors"],"abstract":"<jats:p>To effectively fight against the human immunodeficiency virus infection\/ acquired immunodeficiency syndrome (HIV\/AIDS) epidemic, ongoing development of novel HIV protease inhibitors is required. Inexpensive high-throughput screening assays are needed to quickly scan large sets of chemicals for potential inhibitors. We have developed a F\u00f6rster resonance energy transfer (FRET)-based, HIV protease-sensitive sensor using a combination of a fluorescent protein pair, namely mCerulean and mCitrine. Through extensive in vitro characterization, we show that the FRET-HIV sensor can be used in HIV protease screening assays. Furthermore, we have used the FRET-HIV sensor for intracellular quantitative detection of HIV protease activity in living cells, which more closely resembles an actual viral infection than an in vitro assay. We have developed a high-throughput method that employs a ratiometric flow cytometry for analyzing large populations of cells that express the FRET-HIV sensor. The method enables FRET measurement of single cells with high sensitivity and speed and should be used when subpopulation-specific intracellular activity of HIV protease needs to be estimated. In addition, we have used a confocal microscopy sensitized emission FRET technique to evaluate the usefulness of the FRET-HIV sensor for spatiotemporal detection of intracellular HIV protease activity.<\/jats:p>","DOI":"10.3390\/s131216330","type":"journal-article","created":{"date-parts":[[2013,11,28]],"date-time":"2013-11-28T12:06:47Z","timestamp":1385640407000},"page":"16330-16346","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry"],"prefix":"10.3390","volume":"13","author":[{"given":"Rok","family":"Gaber","sequence":"first","affiliation":[{"name":"Laboratory of Biotechnology, National Institute of Chemistry, Ljubljana 1000, Slovenia"},{"name":"Center of Excellence EN-FIST, Ljubljana 1000, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andreja","family":"Majerle","sequence":"additional","affiliation":[{"name":"Laboratory of Biotechnology, National Institute of Chemistry, Ljubljana 1000, Slovenia"},{"name":"Center of Excellence EN-FIST, Ljubljana 1000, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Roman","family":"Jerala","sequence":"additional","affiliation":[{"name":"Laboratory of Biotechnology, National Institute of Chemistry, Ljubljana 1000, Slovenia"},{"name":"Center of Excellence EN-FIST, Ljubljana 1000, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mojca","family":"Ben\u010dina","sequence":"additional","affiliation":[{"name":"Laboratory of Biotechnology, National Institute of Chemistry, Ljubljana 1000, Slovenia"},{"name":"Center of Excellence EN-FIST, Ljubljana 1000, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2013,11,28]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"250","DOI":"10.3389\/fimmu.2012.00250","article-title":"Controlling the HIV\/AIDS epidemic: Current status and global challenges","volume":"3","author":"Demberg","year":"2012","journal-title":"Front. Immunol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1016\/j.antiviral.2009.10.003","article-title":"Fifteen years of HIV Protease Inhibitors: Raising the barrier to resistance","volume":"85","author":"Wensing","year":"2010","journal-title":"Antivir. Res."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"413","DOI":"10.1086\/515642","article-title":"Relative potency of protease inhibitors in monocytes\/macrophages acutely and chronically infected with human immunodeficiency virus","volume":"178","author":"Perno","year":"1998","journal-title":"J. Infect. Dis."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"464","DOI":"10.1093\/jac\/dkh103","article-title":"Analysis of protease inhibitor combinations in vitro: Activity of lopinavir, amprenavir and tipranavir against HIV type 1 wild-type and drug-resistant isolates","volume":"53","author":"Bulgheroni","year":"2004","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2000","DOI":"10.1128\/jcm.32.8.2000-2002.1994","article-title":"A Comparison of cord blood and peripheral blood mononuclear cells as targets for viral isolation and drug sensitivity studies involving human immunodeficiency virus type 1","volume":"32","author":"Salomon","year":"1994","journal-title":"J. Clin. Microbiol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1016\/S0166-3542(97)00043-0","article-title":"Anti-HIV activity of adefovir (PMEA) and PMPA in combination with antiretroviral compounds: In vitro analyses","volume":"36","author":"Mulato","year":"1997","journal-title":"Antivir. Res."},{"key":"ref_7","first-page":"293","article-title":"Ginkgolic acid inhibits HIV protease activity and HIV infection in vitro","volume":"18","author":"Yan","year":"2012","journal-title":"Med. Sci. Monit. Int. Med. J. Exp. Clin. Res."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1016\/S0165-022X(96)00023-1","article-title":"Fluorogenic substrates for proteases based on intramolecular fluorescence energy transfer (IFETS)","volume":"33","author":"Gershkovich","year":"1996","journal-title":"J. Biochem. Biophys. Methods"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1016\/j.jviromet.2005.04.012","article-title":"A human immunodeficiency virus type 1 protease biosensor assay using bioluminescence resonance energy transfer","volume":"128","author":"Hu","year":"2005","journal-title":"J. Virol. Methods"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1107","DOI":"10.1002\/btpr.628","article-title":"Visualization of human immunodeficiency virus protease inhibition using a novel F\u00f6rster resonance energy transfer molecular probe","volume":"27","author":"Jin","year":"2011","journal-title":"Biotechnol. Prog."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"16759","DOI":"10.3390\/s121216759","article-title":"Enhancement of probe signal for screening of HIV-1 protease inhibitors in living cells","volume":"12","author":"Yao","year":"2012","journal-title":"Sensors"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"589","DOI":"10.1128\/JVI.75.2.589-594.2001","article-title":"Human immunodeficiency virus type 1 protease cleavage site mutations associated with protease inhibitor cross-resistance selected by indinavir, ritonavir, and\/or saquinavir","volume":"75","author":"Brumme","year":"2001","journal-title":"J. Virol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1186\/1754-1611-2-5","article-title":"Engineering BioBrick vectors from BioBrick parts","volume":"2","author":"Shetty","year":"2008","journal-title":"J. Biol. Eng."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1016\/j.bpc.2007.01.008","article-title":"High-precision FLIM-FRET in fixed and living cells reveals heterogeneity in a simple CFP-YFP fusion protein","volume":"127","author":"Millington","year":"2007","journal-title":"Biophys. Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"6705","DOI":"10.1128\/jvi.69.11.6705-6711.1995","article-title":"Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity","volume":"69","author":"He","year":"1995","journal-title":"J. Virol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"935","DOI":"10.1006\/viro.1995.1016","article-title":"Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes","volume":"206","author":"Connor","year":"1995","journal-title":"Virology"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1002\/jemt.20215","article-title":"PixFRET, an ImageJ plug-in for FRET calculation that can accommodate variations in spectral bleed-throughs","volume":"68","author":"Feige","year":"2005","journal-title":"Microsc. Res. Tech."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"477","DOI":"10.1002\/cyto.a.20300","article-title":"Measurement of two caspase activities simultaneously in living cells by a novel dual FRET fluorescent indicator probe","volume":"69","author":"Wu","year":"2006","journal-title":"Cytom. Part A J. Int. Soc. Anal. Cytol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"25935","DOI":"10.1074\/jbc.272.41.25935","article-title":"Thioltransferase (glutaredoxin) is detected within HIV-1 and can regulate the activity of glutathionylated HIV-1 protease in vitro","volume":"272","author":"Davis","year":"1997","journal-title":"J. Biol. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"905","DOI":"10.1038\/nmeth819","article-title":"A guide to choosing fluorescent proteins","volume":"2","author":"Shaner","year":"2005","journal-title":"Nat. Methods"},{"key":"ref_21","first-page":"413","article-title":"Quantification of F\u00f6rster resonance energy transfer by monitoring sensitized emission in living plant cells","volume":"4","author":"Galliardt","year":"2013","journal-title":"Front. Plant Sci."}],"container-title":["Sensors"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1424-8220\/13\/12\/16330\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T21:50:57Z","timestamp":1760219457000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1424-8220\/13\/12\/16330"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2013,11,28]]},"references-count":21,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2013,12]]}},"alternative-id":["s131216330"],"URL":"https:\/\/doi.org\/10.3390\/s131216330","relation":{},"ISSN":["1424-8220"],"issn-type":[{"value":"1424-8220","type":"electronic"}],"subject":[],"published":{"date-parts":[[2013,11,28]]}}}