A 16bp Rep binding element is sufficient for mediating Rep-dependent integration into AAVS1

Journal of Molecular Biology
DengMin FengW Jia

Abstract

Adeno-associated virus (AAV) is a non-pathogenic virus and the only known eukaryotic virus capable of targeting human chromosome 19 for integration at a well-characterized AAVS1 site. Its site-specific integration is mediated by Rep68 and Rep78, viral proteins that bind to both the viral genome and AAVS1 site on ch19 through a specific Rep-binding element (RBE) located in both the viral genome and AAVS1. There are three RBEs in the AAV genome: two identical ones in both inverted terminal repeats (ITR) and another one in a recently discovered region termed the P5 integration efficiency element (P5IEE) that encompasses the viral P5 promoter. In order to identify the viral cis-acting sequence essential for Rep-mediated integration, we tested a series of constructs containing various lengths of P5IEE and compared the two RBEs from ITR (RBE(itr)) and P5IEE (RBE(p5)) in terms of their efficiency in Rep-dependent integration. Methods employed included a colony-forming assay, a PCR-based assay and Southern blotting analysis. We found that 16bp of the RBE cis-element was sufficient for mediating Rep-dependent site-specific integration. Furthermore, RBE(itr) was both more effective and specific than the RBE(p5) in Rep-dependent integrati...Continue Reading

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Citations

Mar 16, 2012·Applied Microbiology and Biotechnology·Yuan-Yuan XueJin-Zhong Chen
Apr 3, 2013·Trends in Biotechnology·Coral González-PrietoMatxalen Llosa
Oct 16, 2008·Clinical Microbiology Reviews·Shyam Daya, Kenneth I Berns
Dec 6, 2011·Biochemical and Biophysical Research Communications·Shuohao HuangMasamichi Kamihira
Oct 9, 2014·Journal of Cardiovascular Pharmacology·Zhengxin XuJinzhong Chen
Oct 5, 2012·Acta Biochimica Et Biophysica Sinica·Qiantong XiangDepei Liu
Sep 16, 2015·The Journal of Biological Chemistry·Faik N MusayevCarlos R Escalante

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