A COOPERATIVE INTERACTION BETWEEN NF-KAPPA-B AND SP1 IS REQUIRED FOR HIV-1 ENHANCER ACTIVATION

The human immunodeficiency virus (HIV-1) long terminal repeat (LTR) contains two binding sites for NF-chiB in close proximity to three binding sites for the constitutive transcription factor, Spl. Previously, stimulation of the HIV enhancer in response to mitogens has been attributed to the binding of NF-chiB to the viral enhancer. In this report, we show that the binding of NF-chiB is not by itself sufficient to induce HIV gene expression. Instead, a protein - protein interaction must occur between NF-chiB and Sp1 bound to an adjacent site. Cooperativity both in DNA binding and in transcriptional activation of NF-chiB 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-chiB 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 chiB 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-chiB also contain chiB in proximity to Spl binding sites. These findings suggest that an interaction between NF-chiB and Sp1 is required for inducible HIV-1 gene expression and may serve as a regulatory mechanism to activate specific viral and cellular genes.