FIGURE 6.
Induced A3G protein is stable and exerts an antiviral effect. A. The stability of induced A3G expression in CEMss cells was determined by treating cells with ionomycin (Iono) over a 14-day time course. The kinetics of A3G expression induction were examined by immunoblotting for expression on days 4, 6, 10, and 14. B, mock and ionomycin-stimulated (Stim) CEMss (CEMss++) cells were treated for 5 days prior to viral challenge with wild-type HIV-1 (filled symbols) or HIV-1Δvif (open symbols). Subsequent viral replication was monitored by HIV-1 p24Gag ELISA. C, CEMss cells were mock or ionomycin-stimulated for 5 days prior to infection. Ionomycin- treated cells then either continued to receive stimulation (++) or ionomycin was washed out of the medium after a 24-h incubation with HIV-1Δvif virus (+). Spreading infection was assayed by p24Gag ELISA. D, whole cell lysates were prepared from mock, transient ionomycin-, and chronic ionomycin-stimulated cultures on d1 of the replication curve, corresponding to day 6 of the stimulation time course. Additional whole cell lysates were prepared on day 10 of the replication curve, corresponding to day 16 of the stimulation protocol. Whole cell lysates from both days were probed for A3G and actin expression by Western blot. Unstim, unstimulated.