title: Cell-based fluorescence assay for human immunodeficiency virus type 1 protease activity.
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.
authors.affiliation: Microbiology and Tumor Biology Center, Karolinska Institutet, S-171 77 Stockholm, Sweden.
authors.forename: K
authors.initials: K
authors.lastname: Lindsten
authors.forename: T
authors.initials: T
authors.lastname: Uhlíková
authors.forename: J
authors.initials: J
authors.lastname: Konvalinka
authors.forename: M G
authors.initials: MG
authors.lastname: Masucci
authors.forename: N P
authors.initials: NP
authors.lastname: Dantuma
mesh_terms: D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005453:Fluorescence; D049452:Green Fluorescent Proteins; D016333:HIV Protease / Q000032:analysis* / Q000378:metabolism; D017320:HIV Protease Inhibitors / Q000032:analysis / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017186:Titrimetry / Q000379:methods; D014162:Transfection
chemical_list: D017320:HIV Protease Inhibitors; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D016333:HIV Protease