Regulation of HIV-1 long terminal repeats by interaction of C/EBP(NF-IL6) and NF-kappaB/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/EBPbeta and C/EBPdelta factors in DNA band shift assay. Transfection of NTera-2 cells with a HIV-1-LTR CAT construct (pC15CAT), together with C/EBPbeta or C/EBPdelta 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-kappaB enhancer was inserted 5' to the HIV-1 LTR TATA box. A NF-kappaB1(p50) expression plasmid was also utilized to test for functional co-operation between NF-kappaB and C/EBP factors. We observed that p50 middle dotC/EBPbeta and p50 middle dotC/EBPdelta complexes were generated in tested cells and strongly activated the HIV-1 LTR by binding to the NF-kappaB sequences. The physical association of NF-kappaB1(p50) with C/EBP factors was assayed by direct interaction of in vitro translated p50 proteins with C/EBPbeta or C/EBPdelta produced as glutathione S-transferase fusion proteins. Moreover, p50 middle dotC/EBPbeta complexes were observed in vivo by using DNA affinity studies with biotinylated NF-kappaB oligonucleotides. By using mutant forms of p50 or C/EBPbeta proteins we found that the transactivation of HIV-1 LTR by p50 middle dotC/EBPbeta complexes required the DNA-binding domain of p50 and the transcription activation domain of C/EBPbeta.
Ruocco
M R
MR
Department of Clinical and Experimental Medicine, Medical School, University of Reggio Calabria, 88100 Catanzaro, Italy.
Chen
X
X
Ambrosino
C
C
Dragonetti
E
E
Liu
W
W
Mallardo
M
M
De Falco
G
G
Palmieri
C
C
Franzoso
G
G
Quinto
I
I
Venuta
S
S
Scala
G
G
D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016322:HIV Enhancer; D016325:HIV Long Terminal Repeat / Q000502:physiology*; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit; D052003:NF-kappa B p52 Subunit; D009687:Nuclear Proteins / Q000378:metabolism*; D020572:Oncogene Proteins v-rel; D011518:Proto-Oncogene Proteins; D015682:Retroviridae Proteins, Oncogenic / Q000378:metabolism; D051996:Transcription Factor RelA; D051841:Transcription Factor RelB; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D052003:NF-kappa B p52 Subunit; D009687:Nuclear Proteins; D020572:Oncogene Proteins v-rel; D011518:Proto-Oncogene Proteins; C497593:RELB protein, human; D015682:Retroviridae Proteins, Oncogenic; D051996:Transcription Factor RelA; D014157:Transcription Factors; D051841:Transcription Factor RelB