Adeno-associated virus type 2 preferentially integrates single genome copies with defined breakpoints
© Janovitz et al.; licensee BioMed Central Ltd. 2014
Received: 1 November 2013
Accepted: 22 January 2014
Published: 27 January 2014
Adeno-associated virus (AAV) serotype 2 prevalently infects humans and is the only described eukaryotic virus that integrates site-preferentially. In a recent high throughput study, the genome wide distribution of AAV-2 integrants was determined using Integrant Capture Sequencing (IC-Seq). Additional insight regarding the integration of AAV-2 into human genomic DNA could be gleaned by low-throughput sequencing of complete viral-chromosomal junctions.
In this study, 140 clones derived from Integrant-Capture Sequencing were sequenced. 100 met sequence inclusion criteria, and of these 39 contained validated junction sequences. These unique sequences were analyzed to investigate the structure and location of viral-chromosomal junctions.
Overall the low-throughput analysis confirmed the genome wide distribution profile gathered through the IC-Seq analysis. We found no unidentifiable sequence inserted at AAV-2 chromosomal junctions. Assessing both left and right ends of the AAV genome, viral breakpoints predominantly occurred in one hairpin of the inverted terminal repeat and AAV genomes were preferentially integrated as single copies.
Adeno-associated virus, a human Parvovirus in the genus Dependovirus, possesses a linear single-strand 4.7 Kb genome . AAV serotype 2 infects up to eighty percent of the human population [2, 3] and is the only described eukaryotic virus that integrates site-preferentially [4–6]. The dominant integration hotspot, AAVS1, is located in the first exon of protein phosphatase 1 regulatory subunit 12C (PPP1R12C) [1, 7]. Site-preferential integration requires two cell-extrinsic factors: the large AAV replication proteins, Rep68 or Rep78 [8–11], and DNA integration substrates containing Rep binding sites, which are GAGC repeats [12–14].
The genome-wide integration profile of AAV-2 has recently been revealed by a high-throughput sequencing approach coupled with bioinformatics . That study was the first high-throughput analysis of AAV integration and led to a number of discoveries, including the presence of several thousand novel genomic hotspots. However, paired-end sequencing generates short reads that do not sequence the entirety of viral-chromosomal junctions.
Integration junction sequences mapped to ten chromosomes, with chromosome 19 receiving 36% of all events (Figure 2B). Three genomic loci were represented by greater than one unique integrant (Figure 2C and E). AAVS1 was the most frequent site of viral genome insertion, accounting for one-third of all events, while the other two sites, PTH1R (chromosome 3) and LOC729862 (chromosome 5), each represented five percent of detected integrations. These were also the three largest hotspots identified via IC-Seq , and two of these hotspots were detected in a previous low-throughput analysis . The thirteen unique integrants identified in AAVS1 begin proximal to the AAV Rep binding site and span the first 15 Kb of PPP1R12C (Figure 2D). This distribution mirrors, on a diminutive scale, the peak-and-tail integration phenotype described in the high-throughput analysis .
Several previous studies, mostly involving AAV vectors, have identified the ITRs as frequent viral recombination points in the absence of Rep [20, 21, 26, 27]. Since the AAV genome is linear and flanked by ITRs, viral-cellular recombination would be expected to occur in this region. Additionally, the complex secondary structure of the ITRs is sufficient to induce a host DNA damage response [28–30]. Based on the data presented in this study, and considering the accumulated insight from previous work [20, 21, 26–30], the identification of the extreme targeting of one specific ITR hairpin as the primary recombination hotspot is an important observation.
Interestingly, the data provided in this study offer insight into the question of whether wild-type AAV genomes integrate as single copies or concatamers. Previous work using Southern blotting to characterize integrations from several cell lines suggested that AAV integrates as head-to-tail concatamers . The data analyzed in this study are one hundred unique sequences from a diverse cell population. Of the one hundred sequences that met our inclusion criteria, forty-six were intact viral sequence, thirty-six were direct viral-chromosomal events, fifteen were viral-viral recombinations and three sequences possessed both viral-viral and viral-chromosomal recombination. Therefore, 66.7% of all recombination events captured were between single viral genomes and human chromosomal DNA (Figure 3C). Additionally, we noted that 82% of all sequences were free of viral-viral recombinations (Figure 3D). Thus, analyzing both ends of integrated AAV-2 sequences, the data indicate viral genomes predominantly integrate into host DNA as single copies.
This study of complete viral-chromosomal junctions derived from cloning and sequencing IC-Seq DNA pools provides valuable insight into AAV integration. The structurally complex, repetitive, and GC-rich nature of these sequences may hinder capture of the entire junction-population. We have taken many steps to mitigate these effects. These steps included using: short sequences from random breaks, two primer sets, stringent sequence validation, robust polymerases, and high melting temperatures. Therefore, we believe that the junctions captured and analyzed in this study are not unduly influenced by sequence constraints, and present a valuable representation of the AAV-2 junction population. The insertion profile of AAV-2 maintained the same top three hotspots found using high-throughput technology and the distribution around AAVS1, the largest hotspot, was also quite similar. In the absence of Rep, the unique AAV-2 ITR structure is a target for cellular DNA repair and recombination pathways which can vary in a cell dependent manner [21, 30, 32, 33]. In the case of wild-type AAV-2, Rep binding to the RBE as well as the hairpin stem influences helicase activity . Therefore, Rep, in concert with cellular DNA repair complexes, may contribute to formation of the internal stem-loop ITR recombination hotspot identified in this study. We anticipate that cell-specific differences in DNA repair proteins and Rep interacting proteins may also influence the integration profile to some extent. However, direct Rep-DNA interactions appear to play the dominant role in defining the genome-wide targets for AAV-2 integration [15, 19]. Finally, based on the population of junctions captured, AAV-2 genomes were found to predominately integrate as single genome copies, and viral-viral recombination was modest. This study may impact Rep-mediated gene therapy approaches and highlights how long read length, even on a modest scale, may serve to significantly augment the understanding of high-throughput data sets.
T.J. was supported by a Medical Scientist Training Program grant from the National Institute of General Medical Sciences of the National Institutes of Health under award number T32GM07739 to the Weill Cornell/Rockefeller/Sloan-Kettering Tri-Institutional MD-PhD Program. E.F.P. received support from the WR Hearst Foundation and PHS grant RO1 AI094050. The content of this study is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
- Linden RM, Ward P, Giraud C, Winocour E, Berns KI: Site-specific integration by adeno-associated virus. Proc Natl Acad Sci U S A 1996, 93: 11288-11294. 10.1073/pnas.93.21.11288PubMedPubMed CentralView ArticleGoogle Scholar
- Halbert CL, Miller AD, McNamara S, Emerson J, Gibson RL, Ramsey B, Aitken ML: Prevalence of neutralizing antibodies against adeno-associated virus (AAV) types 2, 5, and 6 in cystic fibrosis and normal populations: implications for gene therapy using AAV vectors. Hum Gene Ther 2006, 17: 440-447. 10.1089/hum.2006.17.440PubMedPubMed CentralView ArticleGoogle Scholar
- Calcedo R, Vandenberghe LH, Gao G, Lin J, Wilson JM: Worldwide epidemiology of neutralizing antibodies to adeno-associated viruses. J Infect Dis 2009, 199: 381-390. 10.1086/595830PubMedView ArticleGoogle Scholar
- Kotin RM, Siniscalco M, Samulski RJ, Zhu XD, Hunter L, Laughlin CA, McLaughlin S, Muzyczka N, Rocchi M, Berns KI: Site-specific integration by adeno-associated virus. Proc Natl Acad Sci U S A 1990, 87: 2211-2215. 10.1073/pnas.87.6.2211PubMedPubMed CentralView ArticleGoogle Scholar
- Kotin RM, Linden RM, Berns KI: Characterization of a preferred site on human chromosome 19q for integration of adeno-associated virus DNA by non-homologous recombination. EMBO J 1992, 11: 5071-5078.PubMedPubMed CentralGoogle Scholar
- Samulski RJ, Zhu X, Xiao X, Brook JD, Housman DE, Epstein N, Hunter LA: Targeted integration of adeno-associated virus (AAV) into human chromosome 19. EMBO J 1991, 10: 3941-3950.PubMedPubMed CentralGoogle Scholar
- Linden RM, Winocour E, Berns KI: The recombination signals for adeno-associated virus site-specific integration. Proc Natl Acad Sci U S A 1996, 93: 7966-7972. 10.1073/pnas.93.15.7966PubMedPubMed CentralView ArticleGoogle Scholar
- Urcelay E, Ward P, Wiener SM, Safer B, Kotin RM: Asymmetric replication in vitro from a human sequence element is dependent on adeno-associated virus Rep protein. J Virol 1995, 69: 2038-2046.PubMedPubMed CentralGoogle Scholar
- Surosky RT, Urabe M, Godwin SG, McQuiston SA, Kurtzman GJ, Ozawa K, Natsoulis G: Adeno-associated virus Rep proteins target DNA sequences to a unique locus in the human genome. J Virol 1997, 71: 7951-7959.PubMedPubMed CentralGoogle Scholar
- Urabe M, Kogure K, Kume A, Sato Y, Tobita K, Ozawa K: Positive and negative effects of adeno-associated virus Rep on AAVS1-targeted integration. J Gen Virol 2003, 84: 2127-2132. 10.1099/vir.0.19193-0PubMedView ArticleGoogle Scholar
- Young SM, McCarty DM, Degtyareva N, Samulski RJ: Roles of adeno-associated virus Rep protein and human chromosome 19 in site-specific recombination. J Virol 2000, 74: 3953-3966. 10.1128/JVI.74.9.3953-3966.2000PubMedPubMed CentralView ArticleGoogle Scholar
- Young SM, Samulski RJ: Adeno-associated virus (AAV) site-specific recombination does not require a Rep-dependent origin of replication within the AAV terminal repeat. Proc Natl Acad Sci U S A 2001, 98: 13525. 10.1073/pnas.241508998PubMedPubMed CentralView ArticleGoogle Scholar
- Pieroni L, Fipaldini C, Monciotti A, Cimini D, Sgura A, Fattori E, Epifano O, Cortese R, Palombo F, La Monica N: Targeted integration of adeno-associated virus-derived plasmids in transfected human cells. Virology 1998, 249: 249-259. 10.1006/viro.1998.9332PubMedView ArticleGoogle Scholar
- Philpott NJ, Giraud-Wali C, Dupuis C, Gomos J, Hamilton H, Berns KI, Falck-Pedersen E: Efficient integration of recombinant adeno-associated virus DNA vectors requires a p5-rep sequence in cis. J Virol 2002, 76: 5411-5421. 10.1128/JVI.76.11.5411-5421.2002PubMedPubMed CentralView ArticleGoogle Scholar
- Janovitz T, Klein IA, Oliveira T, Mukherjee P, Nussenzweig MC, Sadelain M, Falck-Pedersen E: High-throughput sequencing reveals principles of adeno-associated virus serotype 2 integration. J Virol 2013, 87: 8559-8568. 10.1128/JVI.01135-13PubMedPubMed CentralView ArticleGoogle Scholar
- Hamilton H, Gomos J, Berns KI, Falck-Pedersen E: Adeno-associated virus site-specific integration and AAVS1 disruption. J Virol 2004, 78: 7874-7882. 10.1128/JVI.78.15.7874-7882.2004PubMedPubMed CentralView ArticleGoogle Scholar
- Klein IA, Resch W, Jankovic M, Oliveira T, Yamane A, Nakahashi H, Di Virgilio M, Bothmer A, Nussenzweig A, Robbiani DF, Casellas R, Nussenzweig MC: Translocation-capture sequencing reveals the extent and nature of chromosomal rearrangements in B lymphocytes. Cell 2011, 147: 95-106. 10.1016/j.cell.2011.07.048PubMedPubMed CentralView ArticleGoogle Scholar
- Oliveira TY, Resch W, Jankovic M, Casellas R, Nussenzweig MC, Klein IA: Translocation capture sequencing: a method for high throughput mapping of chromosomal rearrangements. J Immunol Methods 2012, 375: 176-181. 10.1016/j.jim.2011.10.007PubMedPubMed CentralView ArticleGoogle Scholar
- Hüser D, Gogol-Döring A, Lutter T, Weger S, Winter K, Hammer E-M, Cathomen T, Reinert K, Heilbronn R: Integration preferences of wildtype AAV-2 for consensus rep-binding sites at numerous loci in the human genome. PLoS Pathog 2010, 6: e1000985. 10.1371/journal.ppat.1000985PubMedPubMed CentralView ArticleGoogle Scholar
- Drew HR, Lockett LJ, Both GW: Increased complexity of wild-type adeno-associated virus-chromosomal junctions as determined by analysis of unselected cellular genomes. J Gen Virol 2007, 88: 1722-1732. 10.1099/vir.0.82880-0PubMedView ArticleGoogle Scholar
- Yang CC, Xiao X, Zhu X, Ansardi DC, Epstein ND, Frey MR, Matera AG, Samulski RJ: Cellular recombination pathways and viral terminal repeat hairpin structures are sufficient for adeno-associated virus integration in vivo and in vitro. J Virol 1997, 71: 9231-9247.PubMedPubMed CentralGoogle Scholar
- Im DS, Muzyczka N: The AAV origin binding protein Rep68 is an ATP-dependent site-specific endonuclease with DNA helicase activity. Cell 1990, 61: 447-457. 10.1016/0092-8674(90)90526-KPubMedView ArticleGoogle Scholar
- Wu JJ, Davis MDM, Owens RAR: Factors affecting the terminal resolution site endonuclease, helicase, and ATPase activities of adeno-associated virus type 2 Rep proteins. J Virol 1999, 73: 8235-8244.PubMedPubMed CentralGoogle Scholar
- Zhou X, Zolotukhin I, Im DS, Muzyczka N: Biochemical characterization of adeno-associated virus rep68 DNA helicase and ATPase activities. J Virol 1999, 73: 1580-1590.PubMedPubMed CentralGoogle Scholar
- Brister JR, Muzyczka N: Mechanism of rep-mediated adeno-associated virus origin nicking. J Virol 2000, 74: 7762-7771. 10.1128/JVI.74.17.7762-7771.2000PubMedPubMed CentralView ArticleGoogle Scholar
- Miller DG, Petek LM, Russell DW: Adeno-associated virus vectors integrate at chromosome breakage sites. Nat Genet 2004, 36: 767-773. 10.1038/ng1380PubMedView ArticleGoogle Scholar
- Miller DG, Trobridge GD, Petek LM, Jacobs MA, Kaul R, Russell DW: Large-scale analysis of adeno-associated virus vector integration sites in normal human cells. J Virol 2005, 79: 11434-11442. 10.1128/JVI.79.17.11434-11442.2005PubMedPubMed CentralView ArticleGoogle Scholar
- Schwartz RA, Carson CT, Schuberth C, Weitzman MD: Adeno-associated virus replication induces a DNA damage response coordinated by DNA-dependent protein kinase. J Virol 2009, 83: 6269-6278. 10.1128/JVI.00318-09PubMedPubMed CentralView ArticleGoogle Scholar
- Raj K, Ogston P, Beard P: Virus-mediated killing of cells that lack p53 activity. Nature 2001, 412: 914-917. 10.1038/35091082PubMedView ArticleGoogle Scholar
- Cataldi MP, McCarty DM: Hairpin-end conformation of adeno-associated virus genome determines interactions with DNA-repair pathways. Gene Ther 2013, 20: 686-693. 10.1038/gt.2012.86PubMedPubMed CentralView ArticleGoogle Scholar
- Cheung A, Hoggan M, Hauswirth W, Berns KI: Integration of the adeno-associated virus genome into cellular DNA in latently infected human Detroit 6 cells. J Virol 1980, 33: 739-4832.PubMedPubMed CentralGoogle Scholar
- Daya S, Cortez N, Berns KI: Adeno-associated virus site-specific integration is mediated by proteins of the nonhomologous end-joining pathway. J Virol 2009, 83: 11655-11664. 10.1128/JVI.01040-09PubMedPubMed CentralView ArticleGoogle Scholar
- Inagaki K, Ma C, Storm TA, Kay MA, Nakai H: The role of DNA-PKcs and artemis in opening viral DNA hairpin termini in various tissues in mice. J Virol 2007, 81: 11304-11321. 10.1128/JVI.01225-07PubMedPubMed CentralView ArticleGoogle Scholar
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