Cell-based fluorescence assay for human immunodeficiency virus type 1 protease activity

Area of Science:

• Biochemistry
• Virology
• Drug Discovery

Background:

• Human immunodeficiency virus type 1 (HIV-1) protease is critical for viral replication and a key target for AIDS therapeutics.
• HIV-1 protease's cellular toxicity due to non-specific cleavage of host proteins hinders development of traditional cell-based assays.
• Existing assays often fail to accurately reflect drug activity within a living cellular environment.

Purpose of the Study:

• To develop a novel, convenient, and non-infectious cell-based assay for screening HIV-1 protease inhibitors.
• To leverage the inherent cytotoxicity of HIV-1 protease for assay development.
• To enable high-throughput screening (HTS) of potential therapeutic compounds against HIV-1 protease activity in vivo.

Main Methods:

• Transient expression of an artificial precursor protein, green fluorescent protein fused to HIV-1 protease (GFP-PR), in host cells.
• Autocatalytic activation of the GFP-PR precursor by the HIV-1 protease itself upon expression.
• Quantification of accumulated fluorescent precursor via flow cytometry and fluorimetry following inhibitor treatment.

Main Results:

• The assay demonstrated dose-dependent accumulation of the fluorescent GFP-PR precursor upon treatment with HIV-1 protease inhibitors.
• Inhibitor treatment prevented autocatalytic cleavage and subsequent cell elimination, leading to detectable fluorescence.
• The assay proved effective in detecting interference with HIV-1 protease activity in living cells.

Conclusions:

• The GFP-PR precursor assay provides a robust, non-infectious model for evaluating HIV-1 protease inhibitors.
• This novel assay overcomes limitations of previous methods by utilizing protease-induced cell death.
• The developed assay is suitable for high-throughput screening, accelerating the discovery of new anti-HIV-1 drugs.