An improved labeling strategy enables automated detection of single-virus fusion and assessment of HIV-1 protease activity in single virions
Abstract Enveloped viruses transfer their genomes into host cells by fusing their membrane to that of the cell. To visualize single-virus fusion in living cells, researchers take advantage of HIV-1's proteolytic maturation, which can generate free fluorescent proteins within the viral particle. Co-labeling viruses with a content marker and a fluorescently tagged Vpr (a viral core protein) enables detection of single-virus fusions, but a major limitation of this approach is that not all viral particles incorporate both markers. Here, we designed a labeling strategy based on the bifunctional mCherry-2xCL-YFP-Vpr construct, in which 2xCL denotes a tandem cleavage site for the viral protease. This bi-functional marker was efficiently cleaved during virus maturation, producing free mCherry and the core-associated YFP-Vpr. A nearly perfect colocalization of these two markers in virions and their fixed 1:1 ratio enabled automated detection of singleparticle fusion in both fixed and live cells based upon loss of the mCherry signal. Furthermore, a drop in FRET efficiency between YFP and mCherry due to cleavage of the bi-functional marker, which manifested as a marked shift in the normalized YFP/mCherry fluorescence ratio, reliably predicted viral protease activity in single virions. This feature could discriminate between the particles containing free mCherry, and therefore likely representing mature viruses, and immature particles whose fusion cannot be detected. In summary, our new labeling strategy offers several advantages compared with previous approaches, including increased reliability and throughput of detection of viral fusion. We anticipate that our method will have significant utility for studying viral fusion and maturation.