FIGURE 3.

Time-course effects of siUPF1 and UPF1 overexpression on HIV-1 pr55^Gag and RNA levels. HeLa cells were transfected with siNS or siUPF1 for 24 h. They were then transfected with pNL4-3 (HIV-1) and Flag or UPF1^Rescue (2 μg) and again with siNS or siUPF1 (Time 0). At 6, 12, 24, and 30 h post-transfection (hr PT), cells were harvested for Western and Northern analyses. (A) A set of mock-transfected cells (no pNL4-3) with siNS or siUPF1 is shown on the left at 30 h post-transfection. Expression levels of UPF1, UPF2, pr55^Gag, and GAPDH (as loading control) were monitored by Western analyses. Expression levels of HIV-1 genomic RNA and gapdh mRNA (as loading control) were assayed by Northern blotting. Two exposures of the results for HIV-1 genomic RNA are shown. Flag and (lanes 1–4) siNS- or (lanes 5–8) siUPF1-transfected cells; UPF1^Rescue and (lanes 9–12) siNS- or (lanes 13–16) siUPF1-transfected cells. (B) Genomic RNA levels based on densitometric scanning of the lower exposure shown in A were expressed relative to those of gapdh mRNA at each time point post-transfection. (C) The levels of (gray bars) HIV-1 pr55^Gag and (black bars) RNA were expressed relative to the corresponding expression levels in the UPF1^Rescue-siNS treatment group that were considered maximal.