The Adeno-Associated Virus Type 2 Rep Protein Regulates RNA Processing via Interaction with the Transcription Template

Abstract

The adeno-associated virus type 2 (AAV) large Rep proteins can act to increase the ratio of spliced to unspliced AAV RNA when they are targeted to the transcription template via a Rep binding element. The required Rep binding site is both location and orientation independent; however, Rep enhancement decreases as the distance between the promoter and the intron of the affected transcription unit increases. Only the AAV intron and an extended polyadenylation site must remain for the AAV transcription unit to manifest responsiveness to Rep. A number of promoters, when driving the AAV capsid gene transcription unit, were responsive to targeted Rep, though to various degrees. Transactivation of transcription initiation is not sufficient for the enhancement of RNA processing, because activation of the P40 transcription unit by other activators targeted to this transcription template did not result in enhancement of the ratio of spliced to unspliced AAV RNA. These results suggest that Rep may act as a trans regulator of RNA processing by modulating such functions coupled to RNA polymerase II (RNA pol II) transcription, perhaps by affecting the composition of the transcription complex either prior to or during elongation. These results reveal another way in which gene expression can be regulated by trans-acting proteins and help explain an important feature of the parvovirus life cycle.

We thank R. Jude Samulski (University of North Carolina, Chapel Hill), Nick Muzyczka (University of Florida), Bryan Cullen (Duke University), Matthew Weitzman (Salk Institute), and Kevin Brown (NIH) for supplying plasmids, virus, and antibodies used in this work. We thank Greg Tullis (University of Missouri) for plasmids, helpful discussion, and critical reading of the manuscript. We are particularly indebted to Matthew Mouw, who initiated the AAV work in this lab and whose involvement helped form the initial direction of this project. We also thank Lisa Burger for excellent technical assistance.

Jianming Qiu was supported by a University of Missouri Molecular Biology Program fellowship. This work was supported by Public Health Service grant ROI A14658 and ROI A121302 from NIAID to D.J.P.