Journal article
Adeno-associated virus (AAV) site-specific recombination does not require a Rep-dependent origin of replication within the AAV terminal repeat
Proceedings of the National Academy of Sciences - PNAS, Vol.98(24), pp.13525-13530
11/20/2001
DOI: 10.1073/pnas.241508998
PMCID: PMC61074
PMID: 11707592
Abstract
Adeno-associated virus (AAV) is the only known eukaryotic virus capable of targeted integration in human cells. An AAV Rep binding element (RBE) and terminal resolution site (
trs
) identical to the viral terminal repeats required for AAV DNA replication are located on chromosome (ch) 19. Both ch-19 RBE and
trs
elements have been shown to be essential for viral targeting to this locus. To characterize the role of the AAV inverted terminal repeat (ITR) cis-acting sequences in targeted integration an AAV
trs
mutant incapable of supporting viral replication was tested. Wild-type and mutant substrates were assayed for targeted integration after cotransfection in the presence or absence of Rep. Our results demonstrated that, in the presence of Rep78, both ITR substrates targeted to ch-19 with similar frequency. Molecular characterization of the mutant ITR integrants confirmed the presence of the
trs
mutation in the majority of samples tested. Complementation analysis confirmed that the mutant targeted viral genomes were unable to rescue and replicate. In addition, Rep78 induced extensive rearrangement and amplification of ch-19 sequences independent of wild-type or mutant targeting substrate. These studies demonstrate that Rep-dependent nicking of the viral cis-acting
trs
sequence is not a prerequisite for site-specific recombination and suggests AAV targeting is mediated by Rep78/68-dependent replication from the ch-19 origin of replication (
ori
). These studies have significant impact toward the understanding of AAV site-specific recombination and the development of targeting vectors.
Details
- Title: Subtitle
- Adeno-associated virus (AAV) site-specific recombination does not require a Rep-dependent origin of replication within the AAV terminal repeat
- Creators
- Samuel M Young - Curriculum in Genetics and Molecular BiologyRichard Jude Samulski - Curriculum in Genetics and Molecular Biology
- Resource Type
- Journal article
- Publication Details
- Proceedings of the National Academy of Sciences - PNAS, Vol.98(24), pp.13525-13530
- DOI
- 10.1073/pnas.241508998
- PMID
- 11707592
- PMCID
- PMC61074
- NLM abbreviation
- Proc Natl Acad Sci U S A
- ISSN
- 0027-8424
- eISSN
- 1091-6490
- Publisher
- National Academy of Sciences
- Language
- English
- Date published
- 11/20/2001
- Academic Unit
- Anatomy and Cell Biology; Iowa Neuroscience Institute; Otolaryngology
- Record Identifier
- 9984025418402771
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