On day one, 293T target cells were transfected in a 6-well plate overnight with 0.5 μg plasmid encoding a Gal4 response element driven TurboGFP-luciferase reporter (Gal4-TurboGFP-Luciferase). The 293T effector cells were transfected in a 6-well plate overnight either with 1 µg empty vector pNCS-mNeonGreen or AX571 SFVmmu-AX585 DPZ9524_1_CMVie-YFP (ST1) or DPZ9524_1_ R289hybAGMenv_CMVie-YFP (ST2) and 0.5 μg VP16-Gal4 transactivator plasmid. On day two, 16 h after transfection, the medium on the cells was completely removed and exchanged with fresh medium. Twenty-four hours after transfection, the effector cells were trypsinized and seeded in 96-well plates at 50,000 cells/well. On day three, the target cells were trypsinized and added to the effector cells. After 48 h, the cells were lysed in 65 μL 1× Luciferase Cell culture lysis buffer (E1531, Promega, Madison, WI, USA) for 20 min at room temperature and centrifuged for 10 min at 4 °C. Fifty microliters of each cell lysate were used to measure luciferase activity using the Beetle-Juice Luciferase Assay (PJK Biotech, Kleinblittersdorf, Germany) according to manufacturer’s instructions on a Biotek Synergy 2 plate reader. Four independent experiments were performed. Each experiment was normalized to fusion signal of 293T effector cells transfected just with empty vector pNCS-mNeonGreen and VP16-Gal4 fused with 293T target cells. A paired t-test was performed to compare the results of the four experiments.
For testing cell-cell fusion activity of Env expressed from CMVie promoter-driven expression plasmids, A549 target cells were transduced with the lentiviral Gal4-driven TurboGFP-luciferase reporter construct as described previously [33 (link)], and were selected using 10 µg/mL of Blasticidin for three passages prior to the experiment. On 96-well, 293T cells (30,000/well) were seeded as the effector cells. After attachment, 31.25 ng of the transactivator (Gal4-VP16) plasmid were transfected together with 93.75 ng of the respective Env expression plasmid or empty vector per well. One day post transfection, A549 target cells (40,000/well) were added to 293T effector cells. The coculture was incubated for 48 h and then processed to measure luciferase activity as described above.
For testing cell-cell fusion activity of Env expressed from CMVie promoter-driven expression plasmids, A549 target cells were transduced with the lentiviral Gal4-driven TurboGFP-luciferase reporter construct as described previously [33 (link)], and were selected using 10 µg/mL of Blasticidin for three passages prior to the experiment. On 96-well, 293T cells (30,000/well) were seeded as the effector cells. After attachment, 31.25 ng of the transactivator (Gal4-VP16) plasmid were transfected together with 93.75 ng of the respective Env expression plasmid or empty vector per well. One day post transfection, A549 target cells (40,000/well) were added to 293T effector cells. The coculture was incubated for 48 h and then processed to measure luciferase activity as described above.
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