The AP-1 Binding Sites Located in the pol Gene Intragenic Regulatory Region of HIV-1 Are Important for Viral Replication
Figure 5
Overexpression of the dominant negative A-Fos mutant impairs the PMA-dependent enhancer activity of fragment 5103.
(A) HeLa cells were transiently cotransfected with 100 ng of the pTK, pTK-5103as-wt or pTK-5103as-totmut reporter construct and with increasing amounts (0, 50 or 100 ng) of the dominant negative construct pCG-AFos. To maintain the same amount of transfected DNA and to avoid squelching artifacts, the different quantities of A-Fos expression vector cotransfected were complemented to 100 ng of DNA by using the empty vector pCG. Cells were additionally cotransfected with 1 ng of pRL-TK. Twenty-four hours post-transfection, cells were mock-treated (-) or treated with PMA (+). Luciferase activities (Firefly and Renilla) were measured in cell lysates 48 h after transfection. Results are expressed as LuciferaseFirefly/LuciferaseRenilla and presented as histograms indicating the relative luciferase activity of each construct with respect to the activity of the same reporter construct in the absence of both PMA and A-Fos, which was assigned an arbitrary value of 1. Means and standard errors of the means from three independent transfection experiments each performed in triplicate are indicated, with p<0.01 compared with the empty vector pTK indicated by an asterisk. (B) A-Fos mutant expression is efficient and not affected by PMA-treatment of the cells. HeLa cells were transiently transfected with increasing amounts (0, 2 or 4 µg) of the dominant negative construct pCG-AFos. To maintain the same amount of transfected DNA, the different quantities of A-Fos expression vector transfected were complemented to 4 µg of DNA by using the empty vector pCG. Twenty-four hours post-transfection, cells were mock-treated (mock) or treated with PMA (PMA) for one hour. Nuclear extracts were then prepared and analysed by western blot with an antibody directed against the N-terminal FLAG epitope of the A-Fos mutant.