FIG. 4.

DTT treatment of cells coexpressing H-Edm and F-Edm-452C/460C results in partial reactivation of F-Edm-452C/460C fusion activity. (A) (Top two rows) microphotographs of Vero cells cotransfected with 3 μg plasmid DNA each encoding H-Edm or F-Edm, or encoding H-Edm or F-Edm-452C/460C, or transfected with F-Edm encoding plasmids alone. Thirty hours posttransfection, cells were treated with DTT or left untreated (w/o), followed by an iodoacetamide wash and microscopic assessment of fusion activity 150 min posttreatment. F-Edm-452C/460C-expressing cells formed syncytia only after treatment with DTT. Prior to the time of DTT treatment, H-Edm/F-Edm-expressing cells were kept in the presence of fusion inhibitory peptide (Bachem) to prevent premature syncytia formation. (Bottom row) cells cotransfected with H-Edm and F-Edm-452C/460C as described above but treated with 6.25, 12.5, or 50 mM DTT for 150 min for comparison. The most substantial activation of F-Edm-452C/460C occurs when cells were treated with 12.5 or 25 mM DTT. All microphotographs were taken at a magnification of ×200. (B) Quantification of fusion activity of cells transfected as in panel A using the luciferase reporter assay outlined in Fig. 2C. While treatment with 25 mM DTT reduces fusion activity of F-Edm by approximately 40% compared to untreated (w/o) H-Edm/F-Edm-expressing cells, it restores activity of F-Edm-452C/460C to levels corresponding to 20% of untreated F-Edm. No fusion activity was detected in untreated cells expressing H-Edm and F-Edm-452C/460C. Luciferase activities were normalized for values obtained for untreated, H-Edm/F-Edm-expressing cells. Averages and standard error of the mean values are shown.