The AP-1 Binding Sites Located in the pol Gene Intragenic Regulatory Region of HIV-1 Are Important for Viral Replication
Figure 2
AP-1 transcription factors specifically bind in vitro to each of the three intragenic AP-1 sites of fragment 5103.
(A) The nucleotide sequence of the wild-type AP-1#1, AP-1#2 and AP-1#3 site oligonucleotides used as probes in our EMSAs are shown properly aligned with the AP-1 consensus sequence. The position of the AP-1 binding site is indicated by an arrow on each coding strand and mismatches in the AP-1 sites with respect to the AP-1 consensus sequence are designated by asterisks. The conservation of the intragenic AP-1 sites was assessed by comparing their sequences at the nucleotide level based on the full spectrum of HIV and SIV sequences compiled in the HIV compendium database (hiv-web.lanl.gov). Sequence logos that represent the frequency of the nucleotide present at each position in the intragenic AP-1 binding sites were generated based on these sequence analyses for each intragenic AP-1 site. (B) The AP-1#1, AP-1#2 and AP-1#3 oligonucleotide probes were incubated with nuclear extracts (10 µg) from mock-treated (lane 1) or PMA-treated (lanes 2 to 6) HeLa cells in the absence of competitor (lanes 1 and 2) or in the presence of a molar excess (5 fold) of a competitor corresponding to the homologous AP-1 site (lane 3), to the heterologous Sp1 consensus (lane 4; nucleotide sequence of the coding strand: 5′-ATTCGATCGGGGCGGGGCGAGC-3′), to the AP-1 consensus (lane 5; nucleotide sequence of the coding strand: 5′-CGCTTGATGACTCAGCCGGAA-3′) or to the mutated AP-1 consensus (lane 6; nucleotide sequence of the coding strand: 5′-CGCTTGATGACTTGGCCGGAA-3′, where mutations compared to the consensus are indicated in bold). The figure shows the specific retarded bands of interest, which are indicated by arrows. The terms C1, C2 and C3 refer to complexes 1, 2 and 3. (C) Nuclear extracts from PMA-treated HeLa cells (10 µg) were incubated, before the addition of the AP-1 probe, either with a purified rabbit IgG as a negative control (lane 1), or with an antibody directed against AP-1 family members including c-Fos (lane 2), FosB (lane 3), Fra-1 (lane 4), Fra-2 (lane 5), c-Jun (lane 6), JunB (lane 7) and JunD (lane 8), or with an antibody directed against other members of the B-ZIP family such as CREB (lane 9), CREM (lane 10), ATF-1 (lane 11), ATF-2 (lane 12), C/EBPα (lane 13), C/EBPβ (lane 14) and C/EBPδ (lane 15), or with an antibody directed against Ets-1 (lane 16). The figure shows the specific retarded bands of interest indicated by arrows. Supershifted complexes are indicated by asterisks. (D) Ten µg of nuclear extracts from PMA-treated HeLa cells were incubated with an antibody directed against c-Fos (lane 18), an antibody directed against JunB (lane 19) or a combination of both antibodies (lane 20). A purified rabbit IgG was used as a negative control (lane 17). The AP-1#1, AP-1#2 or AP-1#3 oligonucleotide probe was then added to the mixture. The figure shows the specific retarded bands of interest indicated by arrows. The supershifted complexes are indicated by asterisks.