C-18 is the principal fusion-inhibiting component in STAg. (A) Inhibition of HIV-1 envelope–mediated cell fusion with STAg and Cyclophilin (C-18) from T gondii. PM1 cells were incubated with serial dilutions of STAg and C-18 for one hour at 37°C and then mixed (1:1, in triplicates) with 12E1 cells infected overnight with recombinant vaccinia (vCB28) expressing JR-FL envelope. Syncytia were scored between 3 and 4 hours of incubation. Calculated ID₅₀ values were as follows: STAg, 7 μg/mL; C-18, 2 μg/mL. Data represent 4 different experiments. No inhibition was observed with recombinant human cyclophilin or with P falciparum cyclophilin (data not shown). (B) Antibodies to C-18 inhibit STAg-mediated inhibition of HIV-1 fusion. PM1 cells were incubated with STAg (10 μg/mL) in the presence of increasing concentrations of an immunoglobulin G (IgG) fraction from rabbit antiserum raised against recombinant C-18 (1499). 12E1 cells expressing R5 envelope (JR-FL) were added after one hour at 37°C, and syncytia were scored after 3 to 4 hours. Control cultures (no inhibitor added) contained between 400 to 500 syncytia per well. IgG from a control antiserum prepared against an irrelevant peptide (1492) did not block STAg-mediated inhibition of HIV-1 fusion (not shown). The data are representative of 3 experiments performed. Error bars represent SD of the means of 3 replicates per data point.