Figure 6

Inhibition of gene expression by CPX and DEF is promoter specific. A-C. Inhibition is independent of Tat. Total RNA was isolated 15 hr after transfection with pLTR-FF and pCMV-Ren in the absence or presence of Tat expression plasmid. Drugs were added as in Fig. 2D. RPA analysis was conducted by probing with antisense HIV-1 leader RNA probe complementary to LTR nt +83 to -117 (panel A). Protected fragments corresponding to promoter-proximal (Short) and promoter-distal (Long) transcripts were resolved (panel B) and quantified relative to Renilla RNA (panel C) analyzed as in Fig. 3. D. Stability of RNA transcribed from the HIV promoter in the presence of CPX. Actinomycin D (1 μg/ml) was added at 12 hr where indicated. RPA was carried out for FF mRNA as in Fig. 3. Upper panels: expression of FF RNA from the HIV promoter in control and CPX treated cells. Lower panel: FF mRNA decay rate in the presence or absence of CPX plotted relative to levels at 12 hr post-transfection (~50% less in the presence of CPX).