Measuring Pseudovirus Fusion Using BlaM Assay

The pseudovirus fusion with target cells was measured using the BlaM assay, as described previously [10 (link), 83 (link)]. Briefly, cells cultured in 96-well black clear-bottom plates were pre-treated for 30 min, at 37 °C, 5 % CO₂ with growth medium containing 20 μM MG132. The viruses were bound to target cells by centrifugation at 4 °C for 30 min at 1550×g, in the absence of MG132. After the virus binding step, cells were washed once with cold PBS and incubated in live cell imaging buffer/2 % FBS/20 μM MG132 at 37 °C for 90 min. The fusion reaction was stopped by placing the plates on ice, and the media was replaced with the BlaM substrate, CCF4-AM (Invitrogen). Cells were incubated at 11 °C overnight, and the BlaM activity was determined from the ratio of coumarin (blue) and fluorescein (green) fluorescence signals, using a SpectraMaxi3 fluorescence plate reader (Molecular Devices, Sunnyvale, CA, USA).