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: Rok Gaber, Andreja Majerle, Roman Jerala, Mojca Benčina
• Journal: Sensors (Basel, Switzerland) (Sensors (Basel)) Vol. 13 Issue 12 Pg. 16330-46 (Nov 28 2013) ISSN: 1424-8220 [Electronic] Switzerland
• PMID: 24287545 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• HIV Protease

Topics

• Cell Line
• Flow Cytometry (methods)
• HIV Infections (diagnosis, virology)
• HIV Protease (metabolism)
• High-Throughput Screening Assays (methods)
• Humans
• Single-Cell Analysis (methods)