Shi. Ou et al., ROLE OF FLANKING E-BOX MOTIFS IN HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 TATA ELEMENT FUNCTION, Journal of virology, 68(11), 1994, pp. 7188-7199
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.