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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.
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