%0 Journal Article
%T Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry
%A Rok Gaber
%A Andreja Majerle
%A Roman Jerala
%A Mojca Ben£¿ina
%J Sensors
%D 2013
%I MDPI AG
%R 10.3390/s131216330
%X 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£¿rster 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.
%K mCerulean-mCitrine FRET-HIV protease sensor
%K ratiometric flow cytometry
%K sensitize emission FRET
%U http://www.mdpi.com/1424-8220/13/12/16330