Vero cells, in 6-well plates at 90% confluency, were co-transfected with 2 μg of different pCI-F constructs, 1 μg of the various pCI-H plasmids with 9 μl of TransIT-LT1 (Mirus). Vero cells in 24 wells were transfected with 1μg of the various pCI-H plasmids with 3 μl of TransIT-LT1 (Mirus). All transfections were performed according to the manufacturer’s protocol. In some experiments, phase contrast pictures were taken 24 h post-transfection with a confocal microscope (Olympus, Fluoroview, FV1000).
The quantitative fusion assay was performed as described previously [73 (link), 74 (link)] with subtle modification. Briefly, Vero cells were co-transfected with the F and H expression plasmids and 0.1 μg of pTM-Luc (kindly provided by Laurent Roux, University of Geneva). In parallel, separate 6-well plates of Vero-cSLAM cells at 30% confluency were infected with MVA-T7 at a multiplicity of infection (MOI) of 1. After overnight incubation, both cell populations were mixed and incubated at 37°C. 2.5 hours later, the cells were lysed using Bright Glo Lysis Buffer (Promega), and the luciferase activity was determined using a luminescence counter (PerkinElmer Life Sciences) and the Britelite reporter gene assay system (PerkinElmer Life Sciences).
The quantitative fusion assay was performed as described previously [73 (link), 74 (link)] with subtle modification. Briefly, Vero cells were co-transfected with the F and H expression plasmids and 0.1 μg of pTM-Luc (kindly provided by Laurent Roux, University of Geneva). In parallel, separate 6-well plates of Vero-cSLAM cells at 30% confluency were infected with MVA-T7 at a multiplicity of infection (MOI) of 1. After overnight incubation, both cell populations were mixed and incubated at 37°C. 2.5 hours later, the cells were lysed using Bright Glo Lysis Buffer (Promega), and the luciferase activity was determined using a luminescence counter (PerkinElmer Life Sciences) and the Britelite reporter gene assay system (PerkinElmer Life Sciences).