title: Noninvasive high-throughput single-cell analysis of HIV protease activity using ratiometric flow cytometry.
abstract: 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 Förster 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.
authors.affiliation: Laboratory of Biotechnology, National Institute of Chemistry, Ljubljana 1000, Slovenia. mojca.bencina@ki.si.
authors.forename: Rok
authors.initials: R
authors.lastname: Gaber
authors.forename: Andreja
authors.initials: A
authors.lastname: Majerle
authors.forename: Roman
authors.initials: R
authors.lastname: Jerala
authors.forename: Mojca
authors.initials: M
authors.lastname: Benčina
mesh_terms: D002460:Cell Line; D005434:Flow Cytometry / Q000379:methods*; D015658:HIV Infections / Q000175:diagnosis* / Q000821:virology; D016333:HIV Protease / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D016333:HIV Protease