Analysis of putative AP-1 binding sites in HIV-1 clade C and E promoters/enhancers. (A) Polymorphisms among transcription factor binding sites in the core promoter/enhancer regions of HIV-1 clades B, C, and E. The putative clade C and E noncanonical AP-1 binding sites have never before been analyzed experimentally. (B) 5′–3′ strands of the TRE, B, C, and E dsDNA used in gel-shift and competition analyses. The TRE of human collagenase 1, bearing a canonical AP-1 binding site (in bold), was used as positive control. C and E nucleotide sequences span the putative AP-1 binding sites (in bold) of the HIV-1 clade C and E enhancers, respectively. The clade B sequence spans the homologous sequence (underlined) in the enhancer region of the HIV-1 clade B LTR, used as negative control. TRE is flanked by AluI, and viral sequences, by XbaI (B, C, and E) restriction sites. (C) Gel-shift assays with HeLa nuclear extracts. Arrowhead and asterisk indicate AP-1–specific and nonspecific binding, respectively, to the indicated labeled dsDNA (0.5 ng), described in B. The presence of an 80-fold excess of unlabeled dsDNA TRE competitor (+) is indicated. (D and E) Gel-shift competition assays with HeLa nuclear extracts. Arrowhead and asterisks indicate AP-1–specific and nonspecific binding, respectively, to the labeled TRE (0.5 ng). Competition with increasing concentrations of the indicated unlabeled dsDNA competitors is shown on a gel (D) and was quantified by ImageQuant and plotted (E).