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Abstract
We have produced a novel, simple and rapid method utilising genetically encodable FRET-based biosensors to permit the detection of HIV-1 virion fusion in living cells. These biosensors show high sensitivity both spatially and temporally, and allow the real-time recovery of HIV-1 fusion kinetics in both single cells and cell populations simultaneously.
Highlights
We have produced a novel, simple and rapid method utilising genetically encodable FRET-based biosensors to permit the detection of HIV-1 virion fusion in living cells
Various entry-specific assays have been developed over the last decade and include the redistribution of an unquenching fluorophore embedded in the virus envelope[6], the photosensitized activation of a hydrophobic dye loaded in the host membrane[7], or more recently, real-time single virus tracking (SVT) techniques[8]
The biosensor is comprised of a pair of fluorophores capable of undergoing FRET and held together by a short peptide sequence containing the Tobacco Etch Virus (TEV) protease (TEVp) cleavage site[15]
Summary
We have produced a novel, simple and rapid method utilising genetically encodable FRET-based biosensors to permit the detection of HIV-1 virion fusion in living cells. Our protease/substrate system (Fig. 1A), relies on the expression of a genetically-encodable fluorescent biosensor (termed HIV-Chameleon) within target cells. The HIV-Chameleon cell line was infected with both VSV- and HIV- (strain JR-FL) pseudotyped particles at MOIs of 10 and 2 (Fig. 2).
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