Structural Analysis of Adeno-Associated Virus Transduction Circular Intermediates
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Recombinant adeno-associated virus (rAAV) has recently been demonstrated to form circular intermediates following transduction in muscle tissue and cell lines. Although restriction enzyme and Southern blot analysis has revealed a consistent monomer and multimer head-to-tail conformation, detailed structural sequence analysis has been lacking due to the high secondary structure of the ITR arrays. To gain further insight into potential mechanisms by which AAV circular genomes are formed from linear single-stranded viral DNA, we have performed chemical sequencing of ITR arrays within seven circular intermediates independently isolated from primary fibroblasts and Hela cells. Results from these studies demonstrated several types of circular intermediates with mosaic ITR elements flanked by two D sequences. The most predominant form consisted of a structure similar to that of previously generated AAV double-D plasmids, with one complete ITR flanked by two D-region elements. However, intermediately deleted ITR arrays with more than one complete ITR were also seen. Based on this structural information, we have proposed a model for formation of AAV circular intermediates by recombination/ligation between ITR ends of panhandle single-stranded AAV genomes.
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During transduction, rAAV genomes form circular or linear (mostly) head-to-tail arrays of viral genomes.
“During transduction, rAAV genomes have been shown to form circular or linear (mostly) head-to-tail arrays of viral genomes, 14,50,51”
From Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids (2016) · cites this paper as [50]
The Abstract reports circular rAAV intermediates after transduction and describes monomer and multimer head-to-tail conformations. The paper does not establish that linear arrays formed during transduction are mostly head-to-tail; its statement about the most abundant head-to-tail form is limited to a model of circular genomes. (LLM, conf=0.85)
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“ITR then circularizes the double-stranded AAV genome or intermolecular genomic recombination, facilitating recombinant AAV (rAAV) genomes to form episomes [59], allowing their sustained and stable expression in postmitotic cells [60].”
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Yizhar, O., & Adamantidis, A. (2017) · Neuromethods · doi:10.1007/978-1-4939-7417-7_2 · Cited by 8 · see citing works
Sherpa, C., & Grice, S. F. J. L. (2020) · Current HIV Research · doi:10.2174/1570162x18666200210121339 · Cited by 8 · see citing works
Shirk, P., & Furlong, R. B. (2016) · Plasmid · doi:10.1016/j.plasmid.2016.01.001 · Cited by 7 · see citing works
“The mechanism that results in the integration of the AAV genome begins with the formation of circular intermediates (Duan et al., 1998; Duan et al., 1999).”
BackgroundWasala, N., Million, E. D., Watkins, T., Wasala, L., Han, J., Yue, Y., Lu, B., Chen, S.-J., Hakim, C. H., & Duan, D. (2022) · Human Gene Therapy · doi:10.1089/hum.2021.130 · Cited by 6 · see citing works
Self-citationAdachi, K., & Nakai, H. (2011) · InTech eBooks · doi:10.5772/24265 · Cited by 6 · see citing works
“The initial study of the structure of ITR-ITR junction sequences revealed that the majority of the recombination junctions in ds circular monomer genomes exhibited a 165-nt double-D ITR structure, the hallmark of HR (Duan et al., 1999; Xiao et al., 1997).”
Stabilization of a single-stranded DNA of adeno-associated virus by inverted terminal repeats
Yuan, Y.-Z., Higashiyama, K., Ito-Kudo, E., Masumi-Koizumi, K., Yusa, K., & Uchida, K. (2024) · Scientific Reports · doi:10.1038/s41598-024-78612-y · Cited by 5 · see citing works
Dean, J., Plante, J., Huggins, G., Snyder, R., & Aikawa, R. (2009) · Human Gene Therapy · doi:10.1089/hum.2009.054 · Cited by 5 · see citing works
“In vitro , the vector may exist in a circular episomal state (Duan et al. , 1999) or integrated into the host chromosome where it can be repressed, but reactivated with histone deacetylase inhibitors (Chen et al. , 1997; Chen and Townes, 2000).”
Wang, X.-S., Yang, Z., Pla, A., Ng, M., Zhuang, B., Ahuja, S., Tjoanda, C., Purde, V., Wenzel, S., Marshall, B., et al. (2025) · Molecular Therapy — Methods & Clinical Development · doi:10.1016/j.omtm.2025.101579 · Cited by 3 · see citing works
“This figure illustrates that upon lysis of the viruses, single-stranded DNA is released, and when these strands are annealed, different double-stranded variants may be created.”
Wasserman, S. R., Muron, S. J., Lee, H. R., Routh, M. L., Hepperla, A. J., Scoville, D. M., Huber, A., Umana, J. D., Pereira, N. E., Foley, C., et al. (2025) · Nucleic Acids Research · doi:10.1093/nar/gkaf345 · Cited by 3 · see citing works
“The formation of extragenomic circular episomes is a well-known hallmark of functional rAAV infection [ 4 , 53 , 54 ].”
Kubisch, H. M., Gagliardi, C., Romero, D. G., Bunnell, B. A., & Ratterree, M. S. (2008) · Molecular Reproduction and Development · doi:10.1002/mrd.20901 · Cited by 3 · see citing works
“Evidence further suggests that vector molecules are extensively processed prior to integration, sometimes forming circularized concatemers reminiscent of the fate of DNA injected into zygote pronuclei (Duan et al., 1998, 1999; VincentLacaze et al., 1999).”
Gene Delivery Technology: Adeno- Associated Virus
Carter, B. (2003) · Drugs and the pharmaceutical sciences · doi:10.1201/9780203912331.ch8 · Cited by 3 · see citing works
Ginn, S., Doroudian, F., Christina, S., Chan, O. P. Y., Lucas, C., Zhu, E.-H., Yang, S. F., Devanapalli, B., Klein, A. H., Scott, S., et al. (2026) · Molecular Therapy · doi:10.1016/j.ymthe.2026.06.044 · Cited by 2 · see citing works
“51,52 This would ensure correct orientation of therapeutic donor sequences in the target intron ready for transcription and mRNA splicing.”
Adeno-Associated Viral Vectors for Clinical Gene Therapy in the Brain
Samulski, R., & Giles, J. (2005) · KARGER eBooks · doi:10.1159/000084460 · Cited by 1 · see citing works
Anti-CRISPR Proteins: Applications in Genome Engineering
Lee, J. (2020) · doi:10.13028/xxj4-6a50 · Cited by 1
“The AAV genome undergoes circularization and concatemerization to stabilize its presence as episomal DNA, resulting in persistent expression in postmitotic cells (D. Duan et al., 1998, 1999).”
BackgroundMöller, C., Leal-Garza, L., Celauro, E., Nitsch, R., & Westerlund, F. (2025) · Applied Biochemistry and Biotechnology · doi:10.1007/s12010-025-05450-7
“…could identify the presence of multimers, reports have shown that the circularization and stabilization of transduced rAAV vectors can occur via recombination between the ITRs of panhandled fragments generating fractional combinations [42], which was not visible in the above-mentioned results.”
Lkharrazi, A., Tobler, K., Marti, S., Bratus-Neuenschwander, A., Vogt, B., & Fraefel, C. (2023) · bioRxiv · doi:10.1101/2023.11.13.566888
Farris, K. D. (2008) · doi:10.32469/10355/6687
“During wild-type AAV and adenovirus co-infection, the E4orf6 protein is responsible for the head-to-tail concatamerization of AAV genomes, a process required for AAV replication (35, 36).”
BackgroundNeurodegenerative Diseases: Gene therapy
Sarkis, C., & Mallet, J. (2005) · Cambridge University Press eBooks · doi:10.1017/cbo9780511544873.023
- Authors
- Duan, D., Yan, Z., Yue, Y., & Engelhardt, J. F.
- Affiliations
- University of Iowa (US)
- Corresponding author
- Engelhardt, John F.
- Venue
- Virology
- Publisher
- Elsevier BV
- Published
- 1999-08
- Volume
- 261
- Issue
- 1
- Pages
- 8-14
- ISSN
- 0042-6822 (ISSN-L)
- OpenAlex
- W1985285497
- Cited by
- 100
- References
- 22
- Citations by year
- 2026: 1 · 2025: 3 · 2024: 3 · 2023: 3 · 2022: 1 · 2021: 4 · 2020: 4 · 2019: 3 · 2018: 1 · 2017: 4 · 2016: 3 · 2015: 2 · 2014: 2 · 2013: 1
- FWCI
- 3.31
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Virology (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institutes of Health
- National Heart, Lung, and Blood Institute (HL51887)
- National Institute of Diabetes and Digestive and Kidney Diseases (DK/HL51887)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
- Keywords
- adeno-associated virus, inverted terminal repeats, transduction
- MeSH terms
- Dependovirus, Base Sequence, Cells, Cultured, DNA, Circular, DNA, Viral, HeLa Cells, Humans, Models, Genetic, Molecular Sequence Data, Nucleic Acid Conformation, Proviruses, Recombination, Genetic, Transduction, Genetic, Transfection, Genome, Viral, Terminal Repeat Sequences
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