The AP-1 Binding Sites Located in the pol Gene Intragenic Regulatory Region of HIV-1 Are Important for Viral Replication
Figure 6
Binding and functional properties of intragenic AP-1 sites are independent of Tat protein expression.
(A) HeLa cells were transiently transfected with the expression vector pTat72 encoding the one-exon form of Tat (72 amino acids named Tat72), the expression vector pTat101 encoding the two-exon form of Tat (101 amino acids or Tat101), or with the corresponding empty vector pREP9 used as a control. Twenty-four hours post-transfection, cells were mock-treated (-) or treated with PMA (+) for one hour. Nuclear extracts from these transiently transfected cells were prepared and used in EMSA experiments. The AP-1#1, AP-1#2 and AP-1#3 probes incubated with 10 µg of nuclear extracts from HeLa cells (lanes 1 and 2), HeLa cells expressing Tat72 (lanes 3 and 4) or HeLa cells expressing Tat101 (lanes 5 and 6), which were mock-treated (lanes 1, 3 and 5) or treated with PMA (lanes 2, 4 and 6). Retarded bands of interest are shown and indicated by arrows. (B) HeLa cells were transiently cotransfected with 100 ng of the pTK, pTK-5103as-wt or pTK-5103as-totmut reporter construct and with 200 ng of an expression vector encoding either the one-exon form of Tat (pTat72) or the two-exon form of Tat (pTat101) or of the corresponding empty vector pREP9. All transfection mixtures contained 1 ng of pRL-TK as a control of transfection efficiency. 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 post-transfection. Results are expressed as LuciferaseFirefly/[Proteins]*LuciferaseRenilla and presented as histograms indicating the relative luciferase activities with respect to the activity of the control vector pTK in the absence of PMA, which was assigned an arbitrary value of 1. Means and standard errors of the means of one representative experiment from three independent transfection experiments each performed in triplicate are indicated. * indicates p<0.01 compared to the empty vector pTK.