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.The protease encoded by human immunodeficiency virus type 1 (HIV-1) plays an essential role in the retroviral life cycle by processing the viral p55Gag and p160 Gag-Pol polyprotein precursors into structural proteins and enzymes. The activity of the protease is required for conformational rearrangement of the immature virion and production of infectious virus particles, thus providing an attractive target for development of antiviral agents to treat AIDS and related disorders (29). Several potent HIV-1 protease inhibitors are widely used in clinics (7, 10). However, the constant emergence of resistant strains due to the additive effect of multiple amino acid substitutions within and outside the catalytic site motivates the continuous development of new protease inhibitors (26). The availability of reliable and convenient assays for protease activity is, in this context, of great importance.The majority of assays available today are based on trans-or autocatalytic cleavage of reporter proteins in bacteria, in yeasts, or in vitro (1,8,17,24) or on the in vitro hydrolysis of synthetic peptides encompassing the scissile bonds in p55 Gag and p160 14,23). However, none of these assays allows probing of all the native HIV-1 protease specificity sites under physiologic conditions, a situation for which a human cell environment would be required. An important reason for the lack of convenient mammalian cell-based assays is the cytotoxicity observed upon expression of the protease in cells. Thus, while this retroviral aspartic protease possesses unique structural and functional properties that distinguish it from its cellular counterparts (6), several cellular proteins are efficient substrates of the protease. Among those are cytoskeletal proteins such as vimentin, actin, troponin, and tropomyosin (20, 22), microtubule-associated proteins (30), bcl-2 (25), and precursors of NF-B (19) providing a likely explanation for the capacity of the protease to induce apoptosis.We report here on the dev...

A human immunodeficiency virus type 1 protease biosensor assay using bioluminescence resonance energy transfer

“…Because the expression of HIV-1 PR has been associated to apoptosis and cell cycle when expressed (Strack et al, 1996;Wallin et al, 1990;Shoeman et al, 1990), cell cycle and apoptosis Annexin 5 analyses were performed using FACS. These analyses did not reveal any effects of PR expression on cell cycle or apoptosis levels using the transient conditions described in the assays shown here perhaps because the PR is expressed from a precursor protein, a context which has already been shown to prevent the cytotoxicity of HIV-1 PR (Lindsten et al, 2001 and data not shown).…”

Fluorescent Biosensors of Intracellular Targets from Genetically Encoded Reporters to Modular Polypeptide Probes

“…In particular, the human immunodeficiency virus protease is a major target for therapeutic strategies to treat AIDS. However, given the continual emergence of inhibitor-resistant strains, a diagnostic strategy was devised to monitor treatment efficacy and progression of viral infection, based on a genetically encoded GFP-protease precursor of HIV-1 [171,172].…”

Non-infectious fluorimetric assay for phenotyping of drug-resistant HIV proteinase mutants

“…Previously, we reported a new PR assay based on transient expression of an artificial PR precursor harboring the green fluorescent protein (GFP-PR) (Lindsten et al, 2001). The precursor is activated in vivo by HIV PR autocatalytic cleavage.…”

“…Treatment with therapeutic doses of PIs results in a dose-dependent accumulation of the fluorescent precursor in transiently transfected cells that can be easily detected and quantified. This provides a convenient and non-infectious model for high-throughput screenings of substances that can interfere with the activity of the PR in living cells (Lindsten et al, 2001).…”