Characterization of Adeno-Associated Virus Rep Protein Inhibition of Adenovirus E2a Gene Expression

Abstract

Adeno-associated virus (AAV) replication (Rep) proteins are pleiotropic effectors of viral DNA replication, RNA transcription, and site-specific integration into chromosome 19. In addition to regulating AAV gene expression, the Rep proteins modulate expression of a variety of cellular and viral genes. In this report we investigate Rep-mediated effects on expression of the adenovirus (Ad) E2a gene and the Ad major late promoter. We have found that all four Rep proteins repress E2a expression at the protein level, with Rep40 showing the weakest repression. Mutations in the purine nucleotide binding (PNB) site weakened each of the protein's abilities to repress expression. Analysis of steady-state E2a mRNA showed that Rep proteins decreased mRNA levels, but to a lesser extent than E2a protein levels. Analysis of mRNA stability demonstrated that neither Rep78 nor Rep52 affected E2a mRNA stability, suggesting that the decrease in mRNA is due to Rep-mediated inhibition of Ad E2a transcription. To determine if Rep68 proteins could directly inhibit RNA transcription, we performed in vitro transcription assays using HeLa nuclear extracts supplemented with Rep68 and Rep68PNB. We demonstrate that Rep68, but not mutant Rep68PNB, blocked in vitro transcription of a template containing the Ad major late promoter. These results provide insight into how AAV and its encoded Rep proteins interact with Ad and provide a model system for the study of AAV and host-cell interactions.