Abstract
As an early event in the viral life cycle, the entry of enveloped viruses into target cells has received considerable attention. Viral fusion to cellular targets has been studied principally with fusion assays in which cells engineered to express the viral envelope are cultured with the target cells1,2. These assays yield valuable information but do not fully recapitulate all of the variables governing the fusion of actual virions to their cellular targets. The virion membrane and the plasma membrane, for example, differ strikingly in their lipid and protein compositions3,4,5. Two virion-based fusion assays have been described6,7,8. One is based on the redistribution of a self-quenching fluorophore6,7, whereas the second depends on photosensitized activation of a hydrophobic probe by a fluorescent lipid loaded into the target membrane8. These assays are complex and have not been adapted to study fusion in complex cell populations. We have developed a simple, rapid assay allowing the detection of HIV-1 virion fusion to biologically relevant target cells, including primary CD4+ T lymphocytes. It is based on the incorporation of β-lactamase–Vpr chimeric proteins (BlaM-Vpr) into HIV-1 virions and their subsequent delivery into the cytoplasm of target cells as a result of virion fusion. This transfer is then detected by enzymatic cleavage of the CCF2 dye, a fluorescent substrate of β-lactamase (BlaM), loaded in the target cells. BlaM cleaves the β-lactam ring in CCF2, changing its fluorescence emission spectrum from green (520 nm) to blue (447 nm) and thereby allowing fusion to be detected by fluorescence microscopy, flow cytometry, or UV photometry.
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Acknowledgements
We thank Dominique Schols for the gift of AMD3100, Michael Miller for informal discussions, Marty Bigos for expertise in the flow cytometry–based experiments, Stephen Ordway and Gary Howard for editorial assistance, and John C.W. Carroll for graphic arts. W.C.G. thanks the J. David Gladstone Institutes, National Institutes of Health (P01 HD40543), the University-wide AIDS Research Program (C99-SF-02), and the James B. Pendleton Charitable Trust for funding support.
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Cavrois, M., de Noronha, C. & Greene, W. A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes. Nat Biotechnol 20, 1151–1154 (2002). https://doi.org/10.1038/nbt745
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DOI: https://doi.org/10.1038/nbt745
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