Pine P S, Weaver J L, Oravecz T, Pall M, Ussery M, Aszalos A
Division of Applied Pharmacology Research, CDER, Food and Drug Administration, Laurel, Maryland 20708, USA.
Exp Cell Res. 1998 Apr 10;240(1):49-57. doi: 10.1006/excr.1998.3939.
A novel fluorescence-based method was developed to measure HIV envelope glycoprotein (env)-CD4-mediated cell fusion. This method measures the spread of a fluorescent dye as the cytosolic compartments of adjacent cells become contiguous upon cell-to-cell fusion. Calcein-labeled CD4+ Sup-T1 cells were seeded onto a monolayer of unlabeled TF228.1.16 cells, which stably express env, the gp120-gp41 complex. Changes in the following parameters were measured using a stage-scanning laser microscope: total fluorescent area, average fluorescent area, and average shape factor. Anti-CD4 monoclonal antibodies, anti-Leu3a, and OKT4E were shown to block fusion in a dose-dependent manner, while OKT4 had no effect. Aurin tricarboxylic acid, a compound that interferes with the binding of anti-Leu3a mAb and gp120 to CD4+ human peripheral blood lymphocytes, T20, a peptide that interferes with gp41, and cytochalasin D, a microfilament disrupter, all blocked fusion in a dose-dependent manner. This semiautomated assay can be used to quickly assess the effectiveness of compounds acting at different sites to block CD4 and env initiated cell-to-cell fusion.
