Abstract
The β-lactamase (BlaM) assay was first revealed in 1998 and was demonstrated to be a robust Förster resonance energy transfer (FRET)-based reporter system that was compatible with a range of commonly-used cell lines. Today, the BlaM assay is available commercially as a kit and can be utilised readily and inexpensively for an array of experimental procedures that require a fluorescence-based readout. One frequent application of the BlaM assay is the measurement of viral fusion—the moment at which the genetic material harboured within virus particles is released into the cytosol following successful entry. The flexibility of the system permits evaluation of not only total fusion levels, but also the kinetics of fusion. However, significant variation exists in the scientific literature regarding the methodology by which the assay is applied to viral fusion analysis, making comparison between results difficult. In this review we draw attention to the disparity of these methodologies and examine the advantages and disadvantages of each approach. Successful strategies shown to render viruses compatible with BlaM-based analyses are also discussed.
Highlights
The β-lactamase (BlaM) assay is a commercially available system that relies on the action of β-lactamase on a Förster resonance energy transfer (FRET)-based substrate termed CCF2-AM. β-lactamases represent a family of bacterially-derived enzymes that cleave the β-lactam ring structure found within certain families of antibiotic including cephalosporins and penicillins, thereby deactivating them [1]
The BlaM assay represents an attractive FRET-based assay for researchers investigating the characteristics and timing of the fusion event occurring between virus particles and their target cells
The assay has been integral to a multitude of fusion-centric discoveries including the importance of endocytosis and endosomes during Human immunodeficiency virus type-1 (HIV-1) entry in addition to the role of interferon-induced transmembrane (IFITM) proteins in inhibiting HIV-1 and IAV fusion
Summary
The β-lactamase (BlaM) assay is a commercially available system that relies on the action of β-lactamase on a Förster resonance energy transfer (FRET)-based substrate termed CCF2-AM (or the alternative substrate CCF4-AM). β-lactamases represent a family of bacterially-derived enzymes that cleave the β-lactam ring structure found within certain families of antibiotic including cephalosporins and penicillins, thereby deactivating them [1]. Β-lactamases represent a family of bacterially-derived enzymes that cleave the β-lactam ring structure found within certain families of antibiotic including cephalosporins and penicillins, thereby deactivating them [1]. Since this enzymatic function promotes the development of bacterial antibiotic resistance, β-lactamases have been the subject of thorough research efforts that have in turn provided a plethora of information regarding their structures, enzymatic mechanisms and substrate specificities. Exposure to β-lactamase promotes hydrolysis of the CCF2-AM β-lactam ring and separates the 3-fluorescein from the remainder of the substrate In this scenario, FRET is disrupted and excitation of hydroxycoumarin leads to direct emission at 447 nm [2]. Cleavage of the β-lactam ring by β-lactamase separates the two molecules, disrupting FRET and producing a fluorescence shift from 520 nm to 447 nm
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