Abstract
The HIV-1 fusion assay measures all steps in the HIV-1 life cycle up to and including viral fusion. It relies on the incorporation of a β-lactamase–Vpr (BlaM-Vpr) protein chimera into the virion and the subsequent transfer of this chimera into the target cell by fusion (Figure 1). The transfer is monitored by the enzymatic cleavage of CCF2, a fluorescent dye substrate of β-lactamase, loaded into the target cells. Cleavage of the β-lactam ring in CCF2 by β-lactamase changes the fluorescence emission spectrum of the dye from green (520 nm) to blue (447 nm). This change reflects virion fusion and can be detected by flow cytometry (Figure 2).
Materials and Reagents
Equipment
Software
Procedure
Representative data
Figure 1. Principle of the fusion assay Figure 2. Representative data. A. Gating strategy to measure HIV-1 fusion to resting PBL cultures. The population of cells was first gated on Forward Scatter area (FSC-A) and Side Scatter area (SSC-A). Single cells were then gated on a FACS plot with FSC-A and FSC-H. The population of CD4 T cells was gated on APC-Cy7 and PE-Cy7. The fusion gate was drawn on the non infected sample in a FACS plot representing cleaved CCF2 versus uncleaved CCF2. B. Representative fusion FACS plot obtained for non-infected sample (NI), the X4-tropic NL4-3, the R5-tropic 81A and a primary HIV-1 envelope (Env) subcloned into TN6-GFP (Cavrois et al., 2011; Cavrois et al., 2014).
Notes
Recipes
Acknowledgments
This assay was first published in (Cavrois et al., 2002) and the protocol described in detail in (Cavrois et al., 2004). The fusion assay also allows studies of fusion mediated by other viruses, including Ebola (Yonezawa et al., 2005) and henipavirus (Wolf et al., 2009). We would like to thank the team lead by Prof. Tsien for developing CCF2, the BlaM substrate (Zlokarnik et al., 1998).
References
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