Fluorescence resonance energy transfer-based HIV-1 virion fusion assay

Abstract

The fluorescence resonance energy transfer (FRET)-based HIV-1 virion fusion assay exploits the incorporation of beta-lactamase-Vpr chimeric proteins into HIV-1 virions and their subsequent delivery into the cytoplasm of target cells as a marker of fusion. This transfer can be monitored by the enzymatic cleavage of the CCF2-AM dye, a fluorescent substrate of beta-lactamase (BlaM), loaded into the target cells. BlaM cleavage of the beta-lactam ring in CCF2-AM prevents the FRET between the coumarin and fluorescein moieties of the dye. This cleavage changes the fluorescence emission spectrum of CCF2-AM from green (520 nm) to blue (447 nm), and thus permits detection of fusion by fluorescence microscopy, flow cytometry, or UV photometry. This assay is simple and rapid to perform, and exhibits high sensitivity and specificity. Importantly, it can be applied to study HIV-1 virion fusion in primary cells and can be combined with immunostaining for subset discrimination in heterogeneous target cell populations. Finally, the assay can also be adapted to study fusion mediated by the envelope proteins from other viruses through the construction of HIV-1 pseudotypes.