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A simple, high-throughput stabilization assay to test HIV-1 uncoating inhibitors

A. Dostálková, R. Hadravová, F. Kaufman, I. Křížová, K. Škach, M. Flegel, R. Hrabal, T. Ruml, M. Rumlová,

. 2019 ; 9 (1) : 17076. [pub] 20191119

Jazyk angličtina Země Velká Británie

Typ dokumentu časopisecké články, práce podpořená grantem

Perzistentní odkaz   https://www.medvik.cz/link/bmc20028749

Shortly after entering the cell, HIV-1 copies its genomic RNA into double-stranded DNA in a process known as reverse transcription. This process starts inside a core consisting of an enclosed lattice of capsid proteins that protect the viral RNA from cytosolic sensors and degradation pathways. To accomplish reverse transcription and integrate cDNA into the host cell genome, the capsid shell needs to be disassembled, or uncoated. Premature or delayed uncoating attenuates reverse transcription and blocks HIV-1 infectivity. Small molecules that bind to the capsid lattice of the HIV-1 core and either destabilize or stabilize its structure could thus function as effective HIV-1 inhibitors. To screen for such compounds, we modified our recently developed FAITH assay to allow direct assessment of the stability of in vitro preassembled HIV-1 capsid-nucleocapsid (CANC) tubular particles. This new assay is a high-throughput fluorescence method based on measuring the amount of nucleic acid released from CANC complexes under disassembly conditions. The amount of disassembled CANC particles and released nucleic acid is proportional to the fluorescence signal, from which the relative percentage of CANC stability can be calculated. We consider our assay a potentially powerful tool for in vitro screening for compounds that alter HIV disassembly.

Citace poskytuje Crossref.org

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$a Shortly after entering the cell, HIV-1 copies its genomic RNA into double-stranded DNA in a process known as reverse transcription. This process starts inside a core consisting of an enclosed lattice of capsid proteins that protect the viral RNA from cytosolic sensors and degradation pathways. To accomplish reverse transcription and integrate cDNA into the host cell genome, the capsid shell needs to be disassembled, or uncoated. Premature or delayed uncoating attenuates reverse transcription and blocks HIV-1 infectivity. Small molecules that bind to the capsid lattice of the HIV-1 core and either destabilize or stabilize its structure could thus function as effective HIV-1 inhibitors. To screen for such compounds, we modified our recently developed FAITH assay to allow direct assessment of the stability of in vitro preassembled HIV-1 capsid-nucleocapsid (CANC) tubular particles. This new assay is a high-throughput fluorescence method based on measuring the amount of nucleic acid released from CANC complexes under disassembly conditions. The amount of disassembled CANC particles and released nucleic acid is proportional to the fluorescence signal, from which the relative percentage of CANC stability can be calculated. We consider our assay a potentially powerful tool for in vitro screening for compounds that alter HIV disassembly.
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$a Hadravová, Romana $u Department of Biotechnology, University of Chemistry and Technology, Prague, 166 28, Czech Republic. Institute of Organic Chemistry and Biochemistry IOCB Research Centre & Gilead Sciences, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 10, Prague, Czech Republic.
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$a Kaufman, Filip $u Department of Biotechnology, University of Chemistry and Technology, Prague, 166 28, Czech Republic.
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$a Křížová, Ivana $u Department of Biotechnology, University of Chemistry and Technology, Prague, 166 28, Czech Republic.
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$a Škach, Kryštof $u Department of Chemistry of Natural Compounds University of Chemistry and Technology, Prague, 166 28, Prague, Czech Republic.
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$a Flegel, Martin, $u Department of Chemistry of Natural Compounds University of Chemistry and Technology, Prague, 166 28, Prague, Czech Republic. $d 1946-2021 $7 jk01031385
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$a Ruml, Tomáš $u Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague, 166 28, Prague, Czech Republic.
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$a Rumlová, Michaela $u Department of Biotechnology, University of Chemistry and Technology, Prague, 166 28, Czech Republic. michaela.rumlova@vscht.cz.
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