It is well established that the promoter region of the viral genome (promoter) contains regulatory sites that are responsive to host cellular proteins. The HIV-1 promoter has been demonstrated to be absolutely dependent on such factors. In these studies, we describe the ability of the HIV-1 transcription factor, Tat, to associate with the cellular factor, pur-α, and subsequently enhances transcription of the neurotropic viruses, JC Virus, and HIV-1. pur-α alone had a bi-phasic effect on transcription of the JCV promoter. Increasing amounts of pur-α initially stimulated transcription; however transcriptional activity was repressed as higher concentrations of Pur-α were introduced into cells. The Pur-α responsive site was localized to a Pur-α consensus binding site present in the JCV promoter that overlaps the origin of replication. In synchronized cell cultures, transcriptional activation of the JCV promoter was dramatically enhanced by the co-transfection of pur-α and HIV-1 Tat. Together, these proteins activated transcription, even under conditions in which neither protein stimulated transcription on its own. Since previous reports described the association of Pur-α and Tat in vitro and in vivo, we hypothesised that the observed synergistic activation was due to direct interaction of Pur-α and Tat. We next evaluated the contribution of this interaction on Tat's native target, HIV-1 in U87MG cells. Gel shift analysis using a native RNA riboprobe, corresponding to the Transactivation Response Element (TAR), identified that both bacterially synthesized human Pur-α and Pur-α immumoprecipitated from mouse brain bind to TAR in a specific manner. Multiple factors, including the sequence of the bulge and the stability of the secondary structure of TAR, contribute to binding by Pur-α. The use of various Pur-α deletion mutants in a gel retardation experiment demonstrated that the amino terminus of Pur-α is important for binding to TAR RNA. Functional studies identified that Pur-α transactivates the HIV-1 LTR in a TAR dependent manner. Binding studies using both Pur-α and Tat indicate that the proteins form a complex on TAR RNA, and that the Tat binding site was not needed for this association. Additional studies demonstrated that this association occurs through Pur-α contact with TAR, and that Pur-α serves as a linking protein between TAR and Tat. Transient transfection with selected constructs revealed that Pur-α and Tat function synergistically to activate transcription from the HIV-1 LTR. This transactivation was observed in the absence of consensus binding sites for Pur-α and Tat, suggesting they function through another mechanism, likely by contact with proteins assembled at the TATAA box of the HIV-1 promoter. Synergistic activation was not observed with the other TATAA containing minimal promoters. These data demonstrate that Pur-α and Tat may function to activate transcription of the HIV-1 promoter by two separate mechanisms, one of which requires binding to target nucleotide sequences in TAR RNA, and one which is mediated through protein/protein contact at the TATAA element.
