Regulation of HIV-1 long terminal repeats by interaction of C/EBP(NF-IL6) and NF-kappa B/Rel transcription factors

We report the characterization of a CAAT enhancer-binding protein (C/EBP) (NF-IL6) element encompassing the region from -174 to -166 of the U3 long terminal repeat (LTR) region of HIV-1. This C/EBP cis sequence was found to bind to C/EBP beta and C/EBP delta factors in DNA band shift assay. Transfection of NTera-2 cells with a HIV-1-LTR CAT construct (pC15CAT), together with C/EBP beta or C/EBP delta expression plasmids showed that C/EBP proteins strongly activated the HIV-1 promoter. Deletions encompassing the C/EBP-binding site resulted in the enhancement of the LTR activation mediated by C/EBP proteins, suggesting that other sequences located 3' to -170 were indeed the target for C/EBP factors, This possibility was confirmed by using the pCD54E9CAT plasmid, in which the NF-kappa B enhancer was inserted 5' to the HIV-1 LTR TATA box. A NF-kappa B1(p50) expression plasmid was also utilized to test for functional co-operation between NF-kappa B and C/EBP factors. We observed that p50 . C/EBP beta and p50 . C/EBP delta complexes were generated in tested cells and strongly activated the HIV-1 LTR by binding to the NF-kappa B sequences, The physical association of NF-kappa B1(p50) with C/EBP factors was assayed by direct interaction of in vitro translated p50 proteins with C/EBP beta or C/EBP delta produced as glutathione S-transferase fusion proteins. Moreover, p50 . C/EBP beta complexes were observed in vivo by using DNA affinity studies with biotinylated NF-kappa B oligonucleotides. By using mutant forms of p50 or C/EBP beta proteins we found that the transactivation of HIV-1 LTR by p50 . C/EBP beta complexes required the DNA-binding domain of p50 and the transcription activation domain of C/EBP beta.