ArticleJournal of virological methods2005
A human immunodeficiency virus type 1 protease biosensor assay using bioluminescence resonance energy transfer.
Article in Journal of virological methods, 2005. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 20 papers.
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Who cites it
20 citing papers in PubMed, 37 citations in OpenAlex.
- A Highly Sensitive BRET-Based Reporter for Live-Cell Detection of HIV-1 Protease Activity and Inhibitor Screening.Viruses · 2025Article
- A highly sensitive NanoLuc-based protease biosensor for detecting apoptosis and SARS-CoV-2 infection.Scientific reports · 2023Article
- Luciferase-Based Biosensors in the Era of the COVID-19 Pandemic.ACS nanoscience Au · 2021Review
- Pan-retroviral Nucleocapsid-Mediated Phase Separation Regulates Genomic RNA Positioning and Trafficking.Cell reports · 2020Article
- Development of A Fission Yeast Cell-Based Platform for High Throughput Screening of HIV-1 Protease Inhibitors.Current HIV research · 2019Article
- Development of a Protease Biosensor Based on a Dimerization-Dependent Red Fluorescent Protein.ACS chemical biology · 2018Article
- Identification and characterization of loop7 motif and its role in regulating biological function of human APOBEC3G through molecular modeling and biological assay.Acta pharmaceutica Sinica. B · 2017Article
- A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors.Frontiers in microbiology · 2015Article
- A step beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL).Journal of visualized experiments : JoVE · 2014Article
- Noninvasive high-throughput single-cell analysis of HIV protease activity using ratiometric flow cytometry.Sensors (Basel, Switzerland) · 2013Article
- Novel two-round phenotypic assay for protease inhibitor susceptibility testing of recombinant and primary HIV-1 isolates.Journal of clinical microbiology · 2012Article
- Effects of HIV-1 protease on cellular functions and their potential applications in antiretroviral therapy.Cell & bioscience · 2012Article
- Setting Up a Bioluminescence Resonance Energy Transfer High throughput Screening Assay to Search for Protein/Protein Interaction Inhibitors in Mammalian Cells.Frontiers in endocrinology · 2012Article
- Visualization of human immunodeficiency virus protease inhibition using a novel Förster resonance energy transfer molecular probe.Biotechnology progress · 2011Article
- Bioluminescence technologies to detect calicivirus protease activity in cell-free system and in infected cells.Antiviral research · 2011Article
- An assay to monitor HIV-1 protease activity for the identification of novel inhibitors in T-cells.PloS one · 2010Article
- Small molecular compounds inhibit HIV-1 replication through specifically stabilizing APOBEC3G.The Journal of biological chemistry · 2010Article
- Biosensing and imaging based on bioluminescence resonance energy transfer.Current opinion in biotechnology · 2009Review
- Fluorescence resonance energy transfer-based assay for characterization of hepatitis C virus NS3-4A protease activity in live cells.Antimicrobial agents and chemotherapy · 2009Article
- Rational design of novel red-shifted BRET pairs: Platforms for real-time single-chain protease biosensors.Biotechnology progressArticle
Corrections and comments
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Authors and funding
7 authors at 4 institutions in 2 countries.
Funding
Abstract
A sensitive reporter assay to measure human immunodeficiency virus type 1 (HIV-1) protease (PR) activity is described in this manuscript. This assay measures PR activity as a function of the resonance energy transfer (RET) between a donor molecule [humanized sea pansy Renilla reniformis luciferase (hRLuc)] and an energy acceptor molecule, humanized green fluorescent protein (hGFP2) when expressed in mammalian cells. This is a naturally occurring phenomenon and is an emerging and powerful technology that has significant advantages over alternative in vitro PR assays. The HIV-1 Gag-p2/Gag-p7 (p2/p7) PR site was inserted between hGFP2 and hRLuc. The newly created vector, hRLuc-p2/p7-hGFP2 was co-expressed with an HIV-1 codon-optimized PR+ or PR- Gag/Pol expressor. Expression of the hRLuc-p2/p7-hGFP2 alone or with the PR- Gag-Pol expressor generated a BRET2 indicating that the PR cleavage site was not cleaved, whereas the inclusion of the PR+ Gag-Pol produced a significant reduction in the BRET2. The inclusion of PR inhibitors Saquinavir or Amprenavir, or the expression of a p2/p7 PR substrate mutant also blocked the cleavage to result in a stable BRET2 signal. Because the HIV-1 auxiliary protein Vif has been shown to modulate the HIV-1p2/p7 cleavage, this assay was then validated in studies in which Vif was expressed. When Vif was overexpressed along with hRLuc-p2/p7-hGFP2 and PR+ Gag-Pol, the decrease in BRET2 was abrogated in a dose-dependent manner, demonstrating that supraphysiologic levels of Vif block p2/p7 cleavage. An accumulation of a Gag processing intermediate was observed, indicating that p2/p7 cleavage was negatively affected. Overexpression of an RNA-binding-defective Staufen protein or a related dsRNA-binding protein TRBP had no effect on PR cleavage activity as shown by Western and BRET2 analyses. The p2/p7 processing data were confirmed by Western blot analyses. BRET is non-invasive and occurs within live cells, is measured in real time, and is not restricted to cellular compartments making it an especially attractive technology to identify small bioactive inhibitory molecules. This PR BRET2 biosensor assay can be adapted for high throughput screening of new HIV-1 PR inhibitors. It can be employed to screen for antiviral compounds that also target the proteases of other viruses.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.