Artificial 64-Residue HIV-1 Enhancer-Binding Peptide Is a Potent Inhibitor of Viral Replication in HIV-1-Infected Cells

Figure 4

Gel electrophoretic analysis of the effect of R64 on in vitro transcription of OVEC-LTR and Sp1-OVEC in HeLa cell nuclear extract (OVEC-LTR: target plasmid containing the two enhancer sequences of HIV-1 LTR and a β-globin reporter gene; Sp1-OVEC: control plasmid lacking the enhancer sequences and the first 19 bp of the β-globin reporter gene). Transcription experiments containing the two plasmids, 100 ng each, and increasing amounts of R64 were performed as described in [14]. The total volume per experiment was 25 μL. Transcripts were hybridized with a labeled synthetic 93-mer DNA, corresponding to positions –18 to +75 of the β-globin reporter gene and then treated with nuclease S1. The resulting digests were submitted to gel electrophoresis in Tris-borate (89 mM, pH 7.5), EDTA (2 mM), urea (7 M). The gel shows the undigested RNA/DNA hybrids obtained. Lane 1: Sp1-OVEC; lanes 2 to 6: OVEC-LTR + Sp1-OVEC in the presence of 0, 16, 32, 65 and 97.5 pmol of R64, respectively.