ROLE OF FLANKING E-BOX MOTIFS IN HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 TATA ELEMENT FUNCTION

Human immunodeficiency virus type 1 (HIV-1) gene expression is depende nt on a number of cis-acting DNA elements present in the HIV-1 long te rminal repeat. Previous studies have demonstrated that the TATA elemen t is critical for basal and Tat-induced HIV-1 gene expression. The HIV -1 TATA region has an unusual structure in that the TATA sequence is f lanked by two palindromic sequence motifs (CANNTG) known as E boxes wh ich can serve as binding sites for the basic helix-loop-helix (bHLH) c lass of DNA-binding proteins. In this study, we performed site-directe d mutagenesis of both the TATA and the flanking E box sequences of HIV -1. We also substituted the sequences flanking the adenovirus E3 promo ter TATA sequence for those flanking the HIV-1 TATA sequence. Construc ts were assayed for their levels of basal and Tat-induced gene express ion by both in vitro transcription and transient expression assays. Bo th the TATA box and flanking sequences including the E box motifs were found to be important in modulating both basal gene expression and Ta t-induced HIV-1 gene expression. Gel retardation analysis demonstrated that binding of both the recombinant-TATA-binding protei (TBP) and th e TFIID fraction which contains both TBP and TBP-associated factors wa s dependent primarily on-the TATA element. However, competition analys is; suggested that the E boxes may play a role in stabilizing the bind ing of TFIID but not recombinant TBP. Two proteins representing differ ent classes of bHLH proteins, E47 and AP-4, were assayed for their abi lity to bind to the flanking E box motifs. We isolated a cDNA clone en coding the complete AP-4 protein and demonstrated that both AP-4 and E 47 bound specifically to the 3' E box motif, which contains sequences that correspond to the consensus binding site (CAGCTG). Gel retardatio n analysis indicated that the binding of AP-4 to the E boxes excluded the binding of TBP to the TATA box. These studies are consistent with a model in which different classes of cellular proteins may be involve d in regulating HIV-1 TATA element function by either inhibiting or pr omoting the assembly of different preinitiation transcriptional comple xes.