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Cell-Based Fluorescence Assay for Human Immunodeficiency Virus Type 1 Protease Activity

By Kristina Lindsten, Tat'ána Uhlíková, Jan Konvalinka, Maria G. Masucci and Nico P. Dantuma

Abstract

The human immunodeficiency virus type 1 (HIV-1) protease is essential for production of infectious virus and is therefore a major target for the development of drugs against AIDS. Cellular proteins are also cleaved by the protease, which explains its cytotoxic activity and the consequent failure to establish convenient cell-based protease assays. We have exploited this toxicity to develop a new protease assay that relies on transient expression of an artificial protease precursor harboring the green fluorescent protein (GFP-PR). The precursor is activated in vivo by autocatalytic cleavage, resulting in rapid elimination of protease-expressing cells. Treatment with therapeutic doses of HIV-1 protease inhibitors results in a dose-dependent accumulation of the fluorescent precursor that can be easily detected and quantified by flow cytometric and fluorimetric assays. The precursor provides a convenient and noninfectious model for high-throughput screenings of substances that can interfere with the activity of the protease in living cells

Topics: Antiviral Agents
Publisher: American Society for Microbiology
Year: 2001
DOI identifier: 10.1128/AAC.45.9.2616-2622.2001
OAI identifier: oai:pubmedcentral.nih.gov:90701
Provided by: PubMed Central
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