Mechanisms by which the cellular protein, Pur-alpha, and HIV-1 Tat mediate transcription of viral genes

Lara Gayle Chepenik, Thomas Jefferson University

Abstract

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.

Subject Area

Biochemistry|Molecular biology

Recommended Citation

Chepenik, Lara Gayle, "Mechanisms by which the cellular protein, Pur-alpha, and HIV-1 Tat mediate transcription of viral genes" (1999). ETD Collection for Thomas Jefferson University. AAI9920883.
https://jdc.jefferson.edu/dissertations/AAI9920883

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