Viral Fusion Assay Using BlaM-Vpr

Viral fusion was analyzed as described (57 (link)). Briefly, the assay relies on incorporation of a β-lactamase Vpr (BlaM-Vpr) protein chimera into the virion, and in target cells, upon fusion, the transfer is monitored by enzymatic cleavage of CCF2, a β-lactamase fluorescent dye substrate. The cleavage causes changes in fluorescence from green (520 nm) to blue (447 nm) that can be monitored by flow cytometry. Only cytoplasmic virions are detected (55 (link)). In our experiment, day 5 DCs were plated into a 96-well plate in triplicate (1.5 × 10⁵ cells/100 μl) in RPMI in the presence of 10 mM HEPES (Life Technologies) and 2 mg/ml DEAE-dextran (Sigma-Aldrich). Cells were exposed to the indicated concentrations of non-opsonized (HIV) or complement-opsonized (HIV-C) HIV-1 containing BlaM-Vpr. After 3 h, cells were washed twice in CO₂-independent medium (Life Technologies), resuspended in CO₂-independent medium containing 10% fetal calf serum (FCS), and loaded with the CCF2-AM substrate solution (LiveBLAzer FRET-B/G loading kit with CCF2-AM; Life Technologies). After 2 h of incubation at room temperature (dark), cells were washed twice in CO₂-independent medium and fixed in 4% paraformaldehyde for 30 min, and well contents were pooled for flow-cytometric analysis.