A semiautomated fluorescence-based cell-to-cell fusion assay for gp120-gp41 and CD4 expressing cells.
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.
Pine
P S
PS
Division of Applied Pharmacology Research, CDER, Food and Drug Administration, Laurel, Maryland 20708, USA. PINE@CDER.FDA.GOV
Weaver
J L
JL
Oravecz
T
T
Pall
M
M
Ussery
M
M
Aszalos
A
A
D000911:Antibodies, Monoclonal; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology / Q000821:virology*; D002459:Cell Fusion / Q000502:physiology; D002460:Cell Line; D015638:Cytochalasin D / Q000494:pharmacology; D005452:Fluoresceins / Q000493:pharmacokinetics; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D015726:Giant Cells / Q000502:physiology; D015699:HIV Envelope Protein gp120 / Q000032:analysis*; D015700:HIV Envelope Protein gp41 / Q000032:analysis*; D015497:HIV-1 / Q000254:growth & development / Q000302:isolation & purification*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D013997:Time Factors; D014779:Virus Replication / Q000187:drug effects
D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D005456:Fluorescent Dyes; D015699:HIV Envelope Protein gp120; D015700:HIV Envelope Protein gp41; D019384:Nucleic Acid Synthesis Inhibitors; D015638:Cytochalasin D; C007740:fluorexon