Four-dimensional visualization of the simultaneous activity of alternative adeno-associated virus replication origins

D L Glauser, O Saydam, N A Balsiger, I Heid, R M Linden, M Ackermann, C Fraefel

Research output: Contribution to journalArticlepeer-review

19 Citations (Scopus)

Abstract

The adeno-associated virus (AAV) inverted terminal repeats (ITRs) contain the AAV Rep protein-binding site (RBS) and the terminal resolution site (TRS), which together act as a minimal origin of DNA replication. The AAV p5 promoter also contains an RBS, which is involved in Rep-mediated regulation of promoter activity, as well as a functional TRS, and origin activity of these signals has in fact been demonstrated previously in the presence of adenovirus helper functions. Here, we show that in the presence of herpes simplex virus type 1 (HSV-1) and AAV Rep protein, p5 promoter-bearing plasmids are efficiently amplified to form large head-total concatemers, which are readily packaged in HSV-1 virions if an HSV-1 DNA-packaging/cleavage signal is provided in cis. We also demonstrate simultaneous and independent replication from the two alternative AAV replication origins, p5 and ITR, on the single-cell level using multicolor-fluorescence live imaging, a finding which raises the possibility that both origins may contribute to the AAV life cycle. Furthermore, we assess the differential affinities of Rep for the two different replication origins, p5 and ITR, both in vitro and in live cells and identify this as a potential mechanism to control the replicative and promoter activities
Original languageEnglish
Pages (from-to)12218 - 12230
Number of pages13
JournalJournal of Virology
Volume79
Issue number19
DOIs
Publication statusPublished - Oct 2005

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