CTGC motifs within the HIV core promoter specify Tat-responsive pre-initiation complexes

Abstract

BackgroundHIV latency is an obstacle for the eradication of HIV from infected individuals. Stable post-integration latency is controlled principally at the level of transcription. The HIV trans-activating protein, Tat, plays a key function in enhancing HIV transcriptional elongation. The HIV core promoter is specifically required for Tat-mediated trans-activation of HIV transcription. In addition, the HIV core promoter has been shown to be a potential anti-HIV drug target. Despite the pivotal role of the HIV core promoter in the control of HIV gene expression, the molecular mechanisms that couple Tat function specifically to the HIV core promoter remain unknown.ResultsUsing electrophoretic mobility shift assays (EMSAs), the TATA box and adjacent sequences of HIV essential for Tat trans-activation were shown to form specific complexes with nuclear extracts from peripheral blood mononuclear cells, as well as from HeLa cells. These complexes, termed pre-initiation complexes of HIV (PICH), were distinct in composition and DNA binding specificity from those of prototypical eukaryotic TATA box regions such as Adenovirus major late promoter (AdMLP) or the hsp70 promoter. PICH contained basal transcription factors including TATA-binding protein and TFIIA. A mutational analysis revealed that CTGC motifs flanking the HIV TATA box are required for Tat trans-activation in living cells and correct PICH formation in vitro. The binding of known core promoter binding proteins AP-4 and USF-1 was found to be dispensable for Tat function. TAR RNA prevented stable binding of PICH-2, a complex that contains the general transcription factor TFIIA, to the HIV core promoter. The impact of TAR on PICH-2 specifically required its bulge sequence that is also known to interact with Tat.ConclusionOur data reveal that CTGC DNA motifs flanking the HIV TATA box are required for correct formation of specific pre-initiation complexes in vitro and that these motifs are also required for Tat trans-activation in living cells. The impact of TAR RNA on PICH-2 stability provides a mechanistic link by which pre-initiation complex dynamics could be coupled to the formation of the nascent transcript by the elongating transcription complex. Together, these findings shed new light on the mechanisms by which the HIV core promoter specifically responds to Tat to activate HIV gene expression.