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/E BP) (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 s hift assay. Transfection of NTera-2 cells with a HIV-1-LTR CAT constru ct (pC15CAT), together with C/EBP beta or C/EBP delta expression plasm ids showed that C/EBP proteins strongly activated the HIV-1 promoter. Deletions encompassing the C/EBP-binding site resulted in the enhancem ent of the LTR activation mediated by C/EBP proteins, suggesting that other sequences located 3' to -170 were indeed the target for C/EBP fa ctors, This possibility was confirmed by using the pCD54E9CAT plasmid, in which the NF-kappa B enhancer was inserted 5' to the HIV-1 LTR TAT A 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 ge nerated in tested cells and strongly activated the HIV-1 LTR by bindin g 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 gl utathione S-transferase fusion proteins. Moreover, p50 . C/EBP beta co mplexes were observed in vivo by using DNA affinity studies with bioti nylated 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 p 50 . C/EBP beta complexes required the DNA-binding domain of p50 and t he transcription activation domain of C/EBP beta.