A Highly Sensitive BRET-Based Reporter for Live-Cell Detection of HIV-1 Protease Activity and Inhibitor Screening

Area of Science:

• Biochemistry
• Molecular Biology
• Virology

Background:

• Viral proteases (PRs) are crucial for viral replication and represent key targets for antiviral therapies.
• Current in vitro assays for PR inhibitors have limitations, including lack of cellular context, inability to assess cell permeability, toxicity, and intracellular compound activation.

Purpose of the Study:

• To develop novel, cell-based reporters for monitoring Human Immunodeficiency virus-1 (HIV-1) protease activity and cell viability concurrently.
• To establish a more physiologically relevant platform for screening antiviral compounds targeting HIV-1 PR.

Main Methods:

• Development of two Bioluminescence Resonance Energy Transfer (BRET)-based reporters utilizing different BRET pairs (RLuc/YFP and NLuc/mNG) linked by the HIV-1 p2/p7 cleavage site.
• Evaluation of reporter sensitivity, energy transfer efficiency, and spectral properties.
• Application of the most sensitive reporter (mNG-p2/p7-NLuc) to assess the effects of protease inhibitors on HIV-1 PR activity and cell viability in living cells.

Main Results:

• Both BRET reporters specifically detected HIV-1 PR activity.
• The mNG-p2/p7-NLuc reporter exhibited higher sensitivity, enhanced energy transfer, and superior spectral separation, leading to a significantly higher BRET ratio.
• The mNG-p2/p7-NLuc reporter successfully quantified the impact of protease inhibitors on HIV-1 PR activity and cell viability in cell-based assays.
• The potency of known HIV-1 PR inhibitors was accurately measured using this reporter system.

Conclusions:

• The mNG-p2/p7-NLuc reporter is a sensitive and effective cell-based tool for evaluating HIV-1 PR activity and inhibitor efficacy.
• This dual-readout reporter system offers a valuable platform for antiviral drug discovery and screening under physiologically relevant conditions.