Visualization of human immunodeficiency virus protease inhibition using a novel Förster resonance energy transfer molecular probe

Biotechnology Progress
Sha JinKaiming Ye

Abstract

The in vivo high-throughput screening (HTS) of human immunodeficiency virus (HIV) protease inhibitors is a significant challenge because of the lack of reliable assays that allow the visualization of HIV targets within living cells. In this study, we developed a new molecular probe that utilizes the principles of Förster resonance energy transfer (FRET) to visualize HIV-1 protease inhibition within living cells. The probe is constructed by linking two fluorescent proteins: AcGFP1 (a mutant green fluorescent protein) and mCherry (a red fluorescent protein) with an HIV-1 protease cleavable p2/p7 peptide. The cleavage of the linker peptide by HIV-1 protease leads to separation of AcGFP1 from mCherry, quenching FRET between AcGFP1 and mCherry. Conversely, the addition of a protease inhibitor prevents the cleavage of the linker peptide by the protease, allowing FRET from AcGFP1 to mCherry. Thus, HIV-1 protease inhibition can be determined by measuring the FRET signal's change generated from the probe. Both in vitro and in vivo studies demonstrated the feasibility of applying the probe for quantitative analyses of HIV-1 protease inhibition. By cotransfecting HIV-1 protease and the probe expression plasmids into 293T cells, we showed ...Continue Reading

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Citations

Oct 5, 2018·Current Protocols in Chemical Biology·Rachel A Coleman, Darci J Trader
Jan 13, 2017·Journal of Immunology Research·Han LeiKaiming Ye
Jul 3, 2021·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Bara CihlovaJan Silhan
Mar 20, 2018·ACS Combinatorial Science·Rachel A Coleman, Darci J Trader

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