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Noninvasive High-throughput Single-cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry

Overview
Journal Sensors (Basel)
Publisher MDPI
Specialty Biotechnology
Date 2013 Nov 30
PMID 24287545
Citations 4
Authors
Affiliations
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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.

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References
1.
Lu J, Yan S, Jamaluddin S, Weakley S, Liang Z, Siwak E . Ginkgolic acid inhibits HIV protease activity and HIV infection in vitro. Med Sci Monit. 2012; 18(8):BR293-298. PMC: 3560711. DOI: 10.12659/msm.883261. View

2.
Gershkovich A, Kholodovych V . Fluorogenic substrates for proteases based on intramolecular fluorescence energy transfer (IFETS). J Biochem Biophys Methods. 1996; 33(3):135-62. DOI: 10.1016/s0165-022x(96)00023-1. View

3.
Shaner N, Steinbach P, Tsien R . A guide to choosing fluorescent proteins. Nat Methods. 2005; 2(12):905-9. DOI: 10.1038/nmeth819. View

4.
Bulgheroni E, Citterio P, Croce F, Lo Cicero M, Vigano O, Soster F . Analysis of protease inhibitor combinations in vitro: activity of lopinavir, amprenavir and tipranavir against HIV type 1 wild-type and drug-resistant isolates. J Antimicrob Chemother. 2004; 53(3):464-8. DOI: 10.1093/jac/dkh103. View

5.
Mulato A, Cherrington J . Anti-HIV activity of adefovir (PMEA) and PMPA in combination with antiretroviral compounds: in vitro analyses. Antiviral Res. 1998; 36(2):91-7. DOI: 10.1016/s0166-3542(97)00043-0. View