FIG. 3.

(A) Conditions determining whether HIV-1 mRNA was expressed in MCF7 cells. Cultures were grown for 2 days, reached 80% confluence, and were incubated at 37°C with 1 ml of conditioned medium alone (lane a) or containing 1,000 pg of HIV-1 IIIb p24 (lane b). Three other cultures were incubated in estrogen-free medium plus HIV-1 IIIb pretreated for 10 min with either buffer (lane c), 2 μg of cathepsin D per ml (lane d), or vaginal wash sample 22 (lane e). After a 2-h incubation, cells were washed three times with phosphate-buffered saline. Samples of 10⁶ cells were immediately harvested (time, 0 h) while the remaining cultures were grown for 24 h at 37°C and then collected with EDTA and centrifuged (time, 24 h). Cell pellets were treated with lysing buffer, and total mRNA was extracted and reverse transcribed. With the cDNA, a specific amplification of gp120 HIV-1 sequences was performed with primers described in the text. PCR products were run in a 2% agarose gel. MCF7 cells subcultured for several passages after HIV-1 inoculation were also tested (lane f). (B) Assay for receptor mRNA specific for CXCR4, CCR5, and CD4 in PBL (lane g) and in MCF7 cells (lane h).