Effect of poly(ADP-ribose) polymerase 1 on integration of the adeno-associated viral vector genome

• Published in: The journal of gene medicine 2011-06, Vol.13 (6), p.342-352
• Main Authors: Romanova, Liudmila G., Zacharias, Jeana, Cannon, Mark L., Philpott, Nicola J.
• Format: Article
• Language: English
• Subjects: Adeno-associated virus; Adeno-associated virus 2; Blotting, Southern; Blotting, Western; Cancer; Dependovirus - genetics; DNA Primers - genetics; DNA repair; DNA-Binding Proteins - metabolism; DNA-Binding Proteins - physiology; Endonuclease; Enzymatic activity; Gene therapy; Genetic Vectors - genetics; Genome, Viral - genetics; Genomes; HeLa Cells; Humans; Immunoprecipitation; Integration; ligase III; nonhomologous end joining; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases - metabolism; Poly(ADP-ribose) Polymerases - physiology; Polymerase Chain Reaction; Rep; Rep protein; RNA; Viral Proteins - metabolism; Viral Proteins - physiology; Virus Integration - physiology; XRCC1 protein
• ISSN: 1099-498X; 1521-2254; 1521-2254
• DOI: 10.1002/jgm.1577

Background Adeno‐associated virus type 2 (AAV) has the ability to target integration of its DNA into a specific locus of the human genome. Site‐specific AAV integration is mediated by viral Rep proteins, although the role of cellular factors involved in this process is largely unknown. Recent studies provide evidence showing that cellular DNA repair proteins are involved in targeted integration of AAV, although their specific roles are not well defined. Methods In the present study, we investigated the interaction between Rep and proteins of the back‐up nonhomologous end‐joining pathway (B‐NHEJ). We then analyzed the effect of one of these proteins, poly(ADP‐ribose) polymerase 1 (PARP1) on AAV integration. Results We show that AAV Rep interacts with B‐NHEJ members DNA ligase III and PARP1 but does not associate with the scaffolding factor XRCC1. Moreover, PARP1 and Rep bind directly and not via DNA–protein interactions. We also found that Rep increases the enzymatic activity of PARP1 potentially through the endonuclease activity of Rep. Finally, we demonstrate that both chemical inhibition of PARP1 and PARP1 depletion using small hairpin RNA enhance integration of the AAV genome in HeLa cells. Conclusions The findings of the present study indicate that manipulation of PARP1 activity could be used as a tool for developing new, effective AAV‐based therapies for the treatment of genetic diseases and cancer. Copyright © 2011 John Wiley & Sons, Ltd.