UPA inhibits HIV expression in chronically infected U1 cells via binding of its ATF domain to uPAR. (A) U1 cells were preincubated with different concentrations of either uPA or peptides representing the low molecular weight (LMW), ATF, growth factor domain (GFD), or omega loop components of uPA. Cells were then stimulated with PMA and followed for virus expression in culture supernatants. All peptides significantly inhibited (P < .001) virus expression in the same range of concentrations, except for LMW, which was ineffective at all tested concentrations. (B) U1 cells were analyzed for uPAR expression 2 and 24 hours after incubation with PIPLC (10 U/mL). Down-modulation of uPAR expression was clearly detectable at both time points. Geometric mean fluorescence intensities (MFI) are shown: isotype, MFI: 4; Nil, unstimulated cells, MFI: 31; PIPLC, MFI: 14. (C) U1 cells were incubated with PIPLC for 2 hours and then stimulated with PMA in the presence or absence of uPA (10 nM). The RT activity levels were measured in the culture supernatants at the peak of virus expression (day 4 after stimulation). PIPLC abolished the inhibitory effect of uPA on virus expression without affecting the inductive effect of PMA (*P < .001). (D) U1 cells were stimulated with PMA in the presence or absence of suPAR and uPA, and RT activity was determined in the culture supernatants after 3 days of culture (*P < .001);a similar pattern of virus expression was observed even at days 2 and 4 of culture (data not shown). The error bars indicate the SD of duplicate samples. All the described experiments were performed in duplicate wells and repeated 3 times, and provided identical results. cpm indicates counts per minute.