The Adeno-Associated Virus Rep78 Major Regulatory Protein Binds the Cellular TATA-Binding Proteinin Vitroandin Vivo

Abstract

Rep78 is the major regulatory protein of adenoassociated virus (AAV). Rep78 is able to transcriptionally regulate all three of AAV's promoters, as well as a variety of heterologous promoters. In an attempt to understand the mechanism of action by which Rep78 is able to regulate gene expression, we are investigating Rep78's possible protein–protein interaction with basal transcription factors. One such critical basal transcription factor is the human TATA binding protein, TBP. TBP is a core factor required for the assemblage of the transcription initiation complex, TFIID. In this report anin vitrointeraction between Rep78 and TBP was demonstrated in three different assay systems, including West(far)–Western analysis, electrophoretic mobility shift assay–supershift, and coimmunoprecipitation. Furthermore, using the yeast GAL4 two-hybrid system, anin vivointeraction between Rep78 and TBP was also demonstrated. Further still, the amino half of Rep78 is shown to be needed for Rep78–TBP interaction. Mutations within this region of Rep78 are known to be defective for transcriptional regulatory ability, suggesting a biological role for this interaction. Thus, Rep78 may regulate transcription through binding and regulating TBP's numerous interactions. Furthermore, as Rep78 is known to bind at least one other transcription factor (Sp1) and likely others, Rep78 may function as a TBP-associated factor in an altered TFIID-like complex.