Adenovirus induced adeno-associated virus gene expression is not dependent on AAV non-structural Rep protein

The normal lifecycle of Adeno-Associated Virus (AAV) alternates between a latent, repressed provirus and an actively transcribing viral genome. This switch in gene expression is the combination of cellular and viral factors to promote AAV replication. Previous studies of AAV have indicated regulatory pathways, which maintain the correct AAV transcription levels for productive infection. Autoregulation of the AAV genome is controlled by Rep protein interactions with cellular proteins. The addition of Adenovirus helper activities induce changes in both cellular and AAV transcriptional regulation. This combination of regulatory factors allows the dissection of a complex transcriptional regulation mechanism in a relatively simple genetic system. The dissection of the AAV transcriptional regulation was performed by a series of promoter constructs that would access the factors required for transcriptional activation of the AAV p19 promoter. Initially, the first promoter constructs containing a single AAV Rep protein binding site were used to determine if the nonstructural Rep protein would function as a transcriptional activator of AAV gene expression. The Rep protein was determined not to be a transcriptional activator but instead to function as a position-dependent inhibitor of p19 promoter activity. This position-dependent inhibition of the AAV p19 promoter activity suggested that the previously characterized Rep and Sp1 interaction might not be the direct cause of Rep-mediated transactivation. In order to prove that the previous characterized Rep and Sp1 interaction was not responsible for induction of the p19 promoter, a novel system of hybrid transcription factors was used to replace the Rep and Sp1 proteins. The novel hybrid transcription factors modified from the yeast two-hybrid system, consisted of two different proteins containing a GAL4-DNA binding domain fused to either an interaction domain from p53 or T-antigen. The replacement of the Rep and Sp1 binding sites with GAL4 binding sequences in the p19 promoter showed that Rep and Sp1 were not required for transactivation. We conclude that the Rep and Sp1 transcription factors function as architectural proteins facilitating a DNA loop to form between the Rep binding site in the p5 promoter and the Sp1 site within the p19 promoter.