Purification of the human immunodeficiency virus type 1 enhancer and TAR binding proteins EBP-1 and UBP-1

Abstract

Transcription of the human immunodeficiency virus type 1 (HIV-1) is regulated by viral proteins and cellular factors that bind to the viral long terminal repeat (LTR). At least five regions of the HIV LTR serve as binding sites for HeLa cellular proteins. One region containing two copies of the sequence GGGACTTTCC functions as an enhancer element for HIV transcriptional regulation. Another region between -17 and +44 known as the TAR region contains two copies of the sequence CTCTCTGG and is also important in tat-induced activation of the HIV LTR. HeLa cell extracts were used to purify cellular proteins binding to portions of the enhancer region (EBP-1) and the TAR region (UBP-1) by a combination of conventional and DNA affinity chromatography. Several species of proteins of between 55 and 60 kd were found to bind to specific sequences in the enhancer region and these proteins also bound to a portion of the NF-kappa B binding site in the immunoglobulin kappa enhancer. Two proteins of between 61 and 63 kd were the major species found to bind to specific sequences in the TAR region and fractions containing these proteins also bind to the TATA region. Both UBP-1 and EBP-1 exhibited specific binding as demonstrated by both UV cross-linking and DNase I footprinting. Mutations of either the enhancer or TAR regulatory regions prevented binding of these purified factors. These results demonstrate the binding of highly purified cellular proteins to important transcriptional regulatory regions in the HIV LTR.