A cooperative interaction between NF‐kappa B and Sp1 is required for HIV‐1 enhancer activation

• Published in: The EMBO journal 1993-09, Vol.12 (9), p.3551-3558
• Main Authors: Perkins, N.D., Edwards, N.L., Duckett, C.S., Agranoff, A.B., Schmid, R.M., Nabel, G.J.
• Format: Article
• Language: English
• Subjects: Avian Sarcoma Viruses - genetics; Base Sequence; Binding Sites; Biological and medical sciences; Cell Line; Chloramphenicol O-Acetyltransferase - metabolism; DNA, Viral - metabolism; Enhancer Elements, Genetic; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Viral - drug effects; HIV Long Terminal Repeat; HIV-1 - genetics; HIV-1 - metabolism; human immunodeficiency virus 1; Humans; Macromolecular Substances; Microbiology; Molecular Sequence Data; Mutagenesis, Site-Directed; NF-kappa B - metabolism; Oligodeoxyribonucleotides; Recombinant Proteins - metabolism; Replicative cycle, interference, host-virus relations, pathogenicity, miscellaneous strains; Restriction Mapping; Sp1 Transcription Factor - metabolism; Tetradecanoylphorbol Acetate - pharmacology; Transcriptional Activation; Transfection; Tumor Cells, Cultured; Virology
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1002/j.1460-2075.1993.tb06029.x

The human immunodeficiency virus (HIV‐1) long terminal repeat (LTR) contains two binding sites for NF‐kappa B in close proximity to three binding sites for the constitutive transcription factor, Sp1. Previously, stimulation of the HIV enhancer in response to mitogens has been attributed to the binding of NF‐kappa B to the viral enhancer. In this report, we show that the binding of NF‐kappa B is not by itself sufficient to induce HIV gene expression. Instead, a protein‐protein interaction must occur between NF‐kappa B and Sp1 bound to an adjacent site. Cooperativity both in DNA binding and in transcriptional activation of NF‐kappa B and Sp1 was confirmed by electrophoretic mobility shift gel analysis, DNase footprinting, chemical cross‐linking and transfection studies in vivo. With a heterologous promoter, we find that the interaction of NF‐kappa B with Sp1 is dependent on orientation and position, and is not observed with other elements, including GATA, CCAAT or octamer. An increase in the spacing between the kappa B and Sp1 elements virtually abolishes this functional interaction, which is not restored when these sites are brought back into the same helical position. Several other promoters regulated by NF‐kappa B also contain kappa B in proximity to Sp1 binding sites. These findings suggest that an interaction between NF‐kappa B and Sp1 is required for inducible HIV‐1 gene expression and may serve as a regulatory mechanism to activate specific viral and cellular genes.