ArticleViruses2025
A Highly Sensitive BRET-Based Reporter for Live-Cell Detection of HIV-1 Protease Activity and Inhibitor Screening.
Article in Viruses, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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4 authors.
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Abstract
Given their role in viral polyprotein processing, viral proteases (PRs) are excellent targets for antiviral therapy. Most assays developed for screening PR inhibitors are in vitro assays, and therefore have several limitations, including the inability to account for cell permeability, toxicity and the need for compounds activation within cells. The development of cellular reporters overcoming these limitations is therefore highly desirable. In this study, we developed two different Bioluminescence Resonance Energy Transfer (BRET)-based reporters for Human Immunodeficiency virus-1 (HIV-1) PR, allowing the simultaneous monitoring of cell viability and HIV-1 PR activity. The reporters employ two different BRET pairs as donor and acceptor moieties: Renilla luciferase (RLuc) with Yellow Fluorescent Protein (YFP), and Nano luciferase (NLuc) with mNeonGreen (mNG), both linked by the HIV-1 p2/p7 cleavage site. While both reporters specifically detected HIV-1 protease activity, mNG-p2/p7-NLuc exhibited higher sensitivity, increased energy transfer and better spectral separation between donor and acceptor emissions, resulting in a significantly higher BRET ratio. mNG-p2/p7-NLuc was used to quantify the effect of a panel of protease inhibitors in living cells, assessing simultaneously cell viability and HIV-1 PR activity. Additionally, it was employed to measure the potency of well-known HIV-1 PR inhibitors. Together, these findings demonstrate the utility of the mNG-p2/p7-NLuc reporter as a cell-based tool for the evaluation of HIV-1 PR activity and inhibitor efficacy. Its dual-readout capability provides a valuable platform for antiviral drug screening in physiologically relevant conditions.
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