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Journal of Virology, September 2007, p. 9718-9726, Vol. 81, No. 18
0022-538X/07/$08.00+0     doi:10.1128/JVI.00746-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Preferential Integration of Adeno-Associated Virus Type 2 into a Polypyrimidine/Polypurine-Rich Region within AAVS1{triangledown}

Victor J. McAlister and Roland A. Owens*

Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892

Received 5 April 2007/ Accepted 5 July 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adeno-associated virus type 2 (AAV2) preferentially integrates its genome into the AAVS1 locus on human chromosome 19. Preferential integration requires the AAV2 Rep68 or Rep78 protein (Rep68/78), a Rep68/78 binding site (RBS), and a nicking site within AAVS1 and may also require an RBS within the virus genome. To obtain further information that might help to elucidate the mechanism and preferred substrate configurations of preferential integration, we amplified junctions between AAV2 DNA and AAVS1 from AAV2-infected HeLaJW cells and cells with defective Artemis or xeroderma pigmentosum group A genes. We sequenced 61 distinct junctions. The integration junction sequences show the three classical types of nonhomologous-end-joining joints: microhomology at junctions (57%), insertion of sequences that are not normally contiguous with either the AAV2 or the AAVS1 sequences at the junction (31%), and direct joining (11%). These junctions were spread over 750 bases and were all downstream of the Rep68/78 nicking site within AAVS1. Two-thirds of the junctions map to 350 bases of AAVS1 that are rich in polypyrimidine tracts on the nicked strand. The majority of AAV2 breakpoints were within the inverted terminal repeat (ITR) sequences, which contain RBSs. We never detected a complete ITR at a junction. Residual ITRs at junctions never contained more than one RBS, suggesting that the hairpin form, rather than the linear ITR, is the more frequent integration substrate. Our data are consistent with a model in which a cellular protein other than Artemis cleaves AAV2 hairpins to produce free ends for integration.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adeno-associated virus type 2 (AAV2) is a naturally defective human parvovirus that usually requires a helper virus, such as an adenovirus or a herpesvirus, for productive infection (41). In the absence of helper virus, AAV2 can establish a latent infection by episomal persistence (54) or by integrating its DNA into the host chromosomes, preferentially within a 4-kb region of human chromosome 19 designated AAVS1 (11). This is not site-specific integration in the classic sense, but approximately 70% of integration events occur somewhere within this locus (Fig. 1A) (20, 26, 51). The mechanism is unknown, but it does require the Rep68 or Rep78 (Rep68/78) protein encoded by AAV2 and a 33-bp region of AAVS1 (Fig. 1A) that includes a Rep68/78 binding site (RBS) and a nicking site for Rep68/78 that resembles the terminal resolution site (trs) within the 145-base inverted terminal repeats (ITRs) of the AAV2 genome (Fig. 2) (29, 57, 64, 67). The trs got its name because of its role in AAV2 replication. AAV2 has a linear, single-stranded DNA genome (4,679 bases; GenBank accession no. AF043303), and the ITRs are essential for replication. The ITRs are palindromic and fold into T-shaped hairpin structures, one of which provides a 3' end that primes second-strand synthesis by a cellular DNA polymerase (Fig. 2) (10, 65). The hairpin is then nicked by Rep68/78 at trs (Fig. 2) and unwound, and the end is replicated. As a result of this mode of replication, the AAV2 ITRs can exist in two configurations, called flip and flop (Fig. 2) (10, 65).


Figure 1
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  		FIG. 1. AAVS1 locus. (A) Schematic of the AAVS1 locus. Positions of trs and the RBS and the range of reported AAV2 integration sites (integration zone) are indicated. (B) Expanded view of bases 1 to 2100, with both strands indicated. The two upper arrows represent the positions of the first-round PCR primers, and the lower arrow represents the positions of the two second-round primers (arrows not drawn to scale). (C) Nicking at trs can result in covalent attachment of Rep68/78 (R) to the 5' side of the nick and induce unidirectional DNA synthesis (dashed line).

 

Figure 2
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  		FIG. 2. AAV2 replication. (A) Schematic of packaged single-stranded AAV2 DNA (not drawn to scale). The ITRs are shown as hairpins, and the RBSs within the ITRs and the positions of the replication (rep) and capsid (cap) genes are indicated. For simplicity, only the minus strand is shown, even though the plus strand is packaged with the same efficiency. (B) Second-strand synthesis (dashed line) initiated from an ITR creates an additional RBS in the promoter for the rep gene (p5RBS). (C) Expanded views of the region boxed in panel B, shown in the flip or flop configuration. (D) Rep68/78 nicking at trs and replication of the end of the genome (lower dashed line) result in conversion from flip hairpin to flop linear ITR and vice versa (as indicated by the change in the distance from the SmaI site[s] to the end of the genome). RBS', second RBS created by replication of the ITR. (E) Schematic of the AAV2 genome, showing the positions of the PCR primers. The two upper arrows represent the positions of the first-round PCR primers, and the three lower arrows represent the positions of the second-round primers (arrows not drawn to scale).

 
Most published AAV2 breakpoints at integration junctions are near RBSs (11, 12, 46). Figure 2 shows that AAV2 has RBSs within its ITRs (bases 18 to 33, 93 to 108, 4572 to 4587, and 4647 to 4662) and near the promoter at map position 5 (p5) (bases 262 to 277) (33, 34, 42, 67). Resolution of the hairpin into a linear extended form results in a duplication of the hairpin sequence. The duplicated region includes the RBS within the ITR (Fig. 2D).

One curious feature is that all AAV2 junctions within the 4-kb AAVS1 locus are to one side of the nicking site (11). This side represents the direction in which unidirectional DNA synthesis (using the intact strand as a template) can be initiated by Rep68/78 nicking and a crude cell lysate in vitro (Fig. 1C) (64).

Preferential integration has been detected in human and nonhuman primate tissue culture cell lines (2, 12, 13, 24, 27, 43, 47, 49, 53, 55, 62), cells and tissues of transgenic rodents containing the human AAVS1 locus (3, 48, 70), and one patient sample (35). However, each publication described only a small number of junctions. The goal of the work presented here was to generate numbers of junctions sufficient for subtle patterns in the junction sites to be detected, which might shed light on the mechanism of integration. Since AAV2-AAVS1 junctions resemble those found with cellular nonhomologous DNA end joining, we also examined integration in two cell lines with known defects in DNA repair.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Human cell lines and virus. The HeLaJW cell line, a derivative of a HeLa cervical carcinoma cell line that has previously been selected for attachment to plastic dishes (36), was grown in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (FBS), penicillin, streptomycin, and neomycin (Gibco, Carlsbad, CA). A fibroblast line with a defective xeroderma pigmentosum group A (XPA) gene (repository identification no. GM04429) was obtained from Coriell Cell Repositories (Camden, NJ) and grown in minimal essential medium (Gibco) and 10% FBS. SC-3 cells, telomerase-immortalized skin fibroblasts from a patient that produces a defective Artemis protein (40), were a kind gift from Katheryn Meek of Michigan State University and were grown in Ham F-12 medium (Gibco), with antibiotics and 20% FBS.

Wild-type AAV2 was prepared as described previously (6), and titers were determined by DNA slot blot analysis. Cells were routinely infected at a multiplicity of infection of approximately 9 x 103 viral genomes per cell.

Revisions to the AAVS1 sequence. There are multiple differences between the initially reported AAVS1 sequence (GenBank accession no. S51329.1) (25) and the newer sequence generated as part of the sequencing of human chromosome 19 (GenBank accession no. AC010327.8). In the numbering system we use here, the complement base 11429 of the newer sequence (AC010327.8), which corresponds to base 1 of the old sequence (S51329.1), is designated base 1. The complement of base 7319 of the newer sequence (AC010327.8), which corresponds to base 4067 in the initially reported sequence (S51329.1) (25), is designated base 4111 in the numbering system used in this work. By our new numbering system, the Rep68/78 primary nicking site (64) is between T396 and T397. The RBS (67) runs from base 408 through base 423 (Fig. 1).

Integration assay. Cells were harvested 48 h after infection, and genomic DNA was isolated using a DNeasy tissue kit (QIAGEN, Valencia, CA). Primers 6-13-6-1 (5'-CAG GAC AGA GAT GTG TAC CTT CAG GG-3' [AAV2 bases 4021 to 4046]) and 6-13-6-4 (5'-AGG CAG ATA GAC CAG ACT GAG CTA TGG-3' [AAVS1 bases 1239 to 1213]) were used in the first round of PCR amplification, with 0.5 ng of genomic DNA as the substrate in a 50-µl reaction volume. After an initial incubation for 4 min at 94°C, the reaction mixture was subjected to 28 cycles of PCR amplification for 1 min at 94°C, 1 min of annealing at 63°C using FastStart DNA polymerase (Roche) or 60°C using Herculase Faststart polymerase (Stratagene), and 3 min at 72°C. One percent of the amplification product was diluted into a new reaction mixture containing a set of nested primers, 6-13-6-2 (5'-TGG ACA CTA ATG GCG TGT ATT CAG AGC-3' [AAV2 bases 4343 to 4369]) and 6-13-6-3 (5'-CAG GGA AGG AGA CAA AGT CCA GGA-3' [AAVS1 bases 1182 to 1159]). The PCR parameters were the same as those for the first amplification. For a subset of integration assays, nested AAV2 primers 6-13-6-5 (5'-CTC AAT CAG ATT CAG ATC CAT GTC AGA ATC TGG-3' [AAV2 bases 467 to 435]) and 6-13-6-6 (5'-TGG GGA CCT TAA TCA CAA TCT CGT AAA ACC-3' [AAV2 bases 357 to 328]) were used for the first and second PCRs, respectively. An additional subset of assays was performed using primer pairs 10-25-5-4 (5'-AGG ACA GAG ATG TGT ACC TTC AGG-3' [AAV2 bases 4022 to 4045]) and 8-15-5-13 (5'-CCC TGG AAG ATG CCA TGA C-3' [AAVS1 bases 2034 to 2016]) for the first PCR and 8-25-5-3 (5'-ACG TAG ATA AGT AGC ATG GCG GGT-3' [AAV2 bases 4496 to 4519]) and 8-15-5-7 (5'-CAG GGC AGG GAA GGA GAC [AAVS1 bases 1187 to 1170]) for the second PCR, using Herculase Hotstart polymerase and an annealing temperature of 59°C for 30 cycles of amplification for each round of PCR.

Sequence analysis. Topoisomerase-mediated ligation was used to insert PCR products into plasmid pCR4TOPO (Invitrogen). The ligation products were then transformed into Escherichia coli One Shot TOP10 competent cells (Invitrogen). Plasmids for sequencing were purified using a QIAGEN plasmid mini kit or a QIAprep spin miniprep kit. Sequencing was performed by Asthagen, Inc. (Gaithersburg, MD), or MWG Biotech, Inc. (High Point, NC), using either M13 universal primers or the same AAV2 primers used for the second round of the nested PCR.

Candidate cloned junction sequences were first compared directly to AAVS1 by use of the BLAST 2 sequence program (63), available at the website http://www.ncbi.nlm.nih.gov/BLAST/bl2seq/wblast2.cgi. The filters (for low-complexity DNA and human repeats) had to be turned off, since there are multiple sequences between AAVS1 bases 325 and 717 that are masked by these filters. The sequence was then compared to the GenBank nonredundant database by use of the BLASTn program (1), available at http://www.ncbi.nlm.nih.gov/BLAST/, to identify the AAV2 sequences. By convention, AAV2 sequences in databases have the left ITR in the flip configuration and the right ITR in the flop configuration, even though either conformation may exist at either end of the viral genome. The junction sequences were therefore checked manually against a flop left ITR and a flip right ITR sequence. Rare junction sequences with more than two ambiguously called bases within 15 bases of the junction were discarded, since this led to difficulty in identifying the precise junction point and determining whether or not the junction was distinct.

Putative binding motifs for cellular proteins were identified using the CONSENSUS algorithm (17), applying the web-based interface offered at http://rsat.ccb.sickkids.ca. The relative nucleotide frequencies in all selected upstream regions were used to estimate the prior frequencies, which enter into the algorithm estimation of information content and P values. The expected matrix length was left at the default value of 10.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Previously reported data are consistent with the hypothesis that Rep68/78-mediated preferential integration into AAVS1 employs elements of the cellular nonhomologous-end-joining DNA repair pathways. Since AAV2 integration is atypically specific, other cellular components may be involved. We set out to establish a baseline for junction characteristics by using HeLaJW cells (a derivative of a very commonly used human cell line) and then to look for qualitative differences in cell lines with known DNA repair defects, namely in the genes encoding the Artemis and XPA proteins. The Artemis protein is involved in locus-specific, nonhomologous end joining by cleaving hairpin DNA intermediates during immunoglobulin gene rearrangement (28, 30, 31, 40). Since fibroblasts from patients with Artemis gene defects are hypersensitive to ionizing radiation (40), the protein is suspected to have a broader role in DNA repair. The XPA protein is believed to be involved primarily in the recognition of bulky DNA adducts, as part of nucleotide excision repair (52). We hypothesized that the covalent attachment of Rep68/78 to the 5' side of the nick (Fig. 1C) might be perceived as a bulky adduct. In addition, a recent report shows that XPA can bind tightly to transition points between single-stranded and double-stranded DNA (69) similar to those found in hairpin ITRs (Fig. 2).

We also make use of the latest sequence data for AAVS1. Most reports of AAV2-AAVS1 integration junctions use an older and less accurate sequence (GenBank accession no. S51329.1) (25; see reference 11 for a review). By mapping published junctions onto a newer, more correct AAVS1 sequence (GenBank accession no. AC010327.8), we have determined that several of the sequences at junctions that were reported as being of unknown origin are actually from AAVS1 (data not shown). As a result, the frequency of junctions containing microhomologies has been underestimated and the frequency of junctions containing inserts has been overestimated.

Junction amplification. We used a nested-PCR system to amplify AAV2-AAVS1 junctions (see Fig. 1 and 2 for primer locations). One set of primers was within AAVS1 and was used in conjunction with a primer set within the capsid gene that was designed to detect junctions at the right end of the AAV2 genome or a primer set within the AAV2 rep gene that was designed to detect junctions at the left end of AAV2. AAV2-AAVS1 junction amplification products were ligated into a T/A cloning vector and sequenced. Although junctions could be defined with as little as 21 bases each of AAV2 and AAVS1 sequence, we typically obtained over 80 bases of sequence on the AAV2 side of the junction and over 100 bases on the AAVS1 side (data not shown). We found no differences in the AAVS1 sequences (other than those attributable to PCR replication errors) among the three cell lines.

Classes of AAV2-AAVS1 junctions. The junction sequences isolated from HeLaJW and XPA- and Artemis-defective cells are shown in Fig. 3, 4, and 5, respectively. Over 100 cloned PCR products were sequenced, yielding 61 distinct AAV2-AAVS1 junctions. Most of the junctions (57%) contained microhomologies ranging from 1 to 14 bases that could have come from either AAV2 or AAVS1 (Table 1). The second most prevalent class overall (31%) was that of junctions with DNA inserted that was not normally contiguous with either AAV2 or AAVS1 DNA. With the XPA-defective cells, these were as frequent as microhomology junctions (Table 1), but given the small number of junctions, the significance of this observation is not clear. Several of these inserts contained 1 to 6 bases that represented a repeat of AAV2 or AAVS1 DNA at or near the junction. For example, junction K08 (Fig. 3) contains a contiguous direct repeat of the last 2 AAVS1 bases (TT) within the insert. The insert of junction 8-9 (Fig. 3) contains a 3-base (TTT), noncontiguous direct repeat of AAV2 sequence near the junction and a 7-base (CAGCACA), noncontiguous direct repeat of AAVS1 sequence near the junction. Direct joining of AAV2 to AAVS1 DNA represented the smallest class of junctions (11% overall) in all three cell types. Although our PCR assay is not sufficiently quantitative to detect even significant drops in integration efficiency, we can say that there does not appear to be an absolute requirement for a fully functional Artemis or XPA protein in AAV2 preferential integration.


Figure 3
Figure 3
Figure 3
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  		FIG. 3. AAV2-AAVS1 junctions from HeLaJW cells. AAV2 sequences are shown in bold type. AAVS1 sequences are in italics. Other sequences are in lowercase. Regions of microhomology are underlined.

 

Figure 4
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  		FIG. 4. AAV2-AAVS1 junctions from XPA-defective cells. AAV2 sequences are shown in bold type. AAVS1 sequences are in italics. Other sequences are in lowercase. Regions of microhomology are underlined.

 

Figure 5
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  		FIG. 5. AAV2-AAVS1 junctions from Artemis-defective cells. AAV2 sequences are shown in bold type. AAVS1 sequences are in italics. Other sequences are in lowercase. Regions of microhomology are underlined. Additional homologous sequences are shown below the sequences for junctions 2-7 and 2-13 and are underlined.

 

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  		TABLE 1. Comparison of AAV2-AAVS1 junction type frequencies among cell lines

 
AAV2-AAVS1 junctions do not contain more than one ITR RBS. Since preferential integration can be seen with input of either packaged, single-stranded AAV2 DNA or a plasmid containing AAV2 DNA, there is a question as to whether the preferred substrate is single-stranded or double-stranded DNA. Since many AAV2 junction breakpoints are within the ITRs, there is also a question of whether the linear or hairpin form of the ITR (Fig. 2) is preferred. In addition, several reports show that significant numbers of AAV2 breakpoints occur in or near the p5 promoter (11). Our PCR primers within the AAV2 rep and cap genes were designed to amplify junctions up to about 180 bases away from the left ITR (which includes the p5 RBS) and up to about 160 bases away from the right ITR, respectively (Fig. 2). However, many of the reported breakpoints in p5 region-AAVS1 junctions result in a deletion of most of the rep gene (12), where our left-end PCR primers anneal. It was therefore not surprising that the majority of AAV2 breakpoints we identified at either end are within the 145-base ITRs (Table 1). Figure 6 shows the distribution of AAV2 breakpoints within the ITRs presented in their hairpin conformations. In their extended linear conformation, the complementary sequences that make up the RBS separate to form two RBSs (Fig. 2) (60). Although there are multiple breakpoints within what would be the RBS furthest from the end of the genome in the linear ITR and within the upper and lower stem loops, none were detected in the RBS that would be closest to the end of the genome in the linear ITR (Fig. 2 and 6). As seen in Table 1, the breakpoints are distributed approximately evenly between the flip and flop configurations in those junctions for which the configuration could be determined.


Figure 6
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  		FIG. 6. Mapping of AAV2-AAVS1 and AAV2-AAV2 junction breakpoints on the AAV2 ITR. For simplicity, only hairpin ITRs at the 3' end of the genome are shown. Narrow arrowheads indicate the breakpoints (estimated for microhomology junctions). Broad arrowheads indicate the positions of the trs and SmaI sites. Both the flip and flop hairpins are shown. Breakpoints that could not be unambiguously mapped to flip or flop are indicated on both hairpins.

 
AAV2-AAV2 junctions. Several of the junctions we sequenced contained AAV2-AAV2 junctions in addition to AAV2-AAVS1 junctions (Fig. 7). For example, AAV2-AAV2 junctions 9-02a and 9-02b (Fig. 7) were both upstream of junction 9-02c (Fig. 3), all within the same PCR product. The AAV2-AAV2 junctions shared several features with AAV2-AAVS1 junctions. Half of them (three of six) involved an ITR, although none involved two ITRs. They also showed the three classical nonhomologous-end-joining joints, with three microhomology junctions, one with an insertion at the junction, and two direct joinings.


Figure 7
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  		FIG. 7. AAV2-AAV2 junctions. AAV2 sequences upstream of the junction are shown in bold type. AAV2 sequences downstream of the junction are in italics. Other sequences are in lowercase. Regions of microhomology are underlined.

 
AAVS1 breakpoints cluster in a region rich in pPy tracts. Our AAVS1 breakpoints at junctions ranged from base 413, within the AAVS1 RBS (67), through base 1163 (Fig. 8). Due to the positions of our AAVS1 primers, we could not detect junctions further downstream. We detected a small cluster of integration junctions immediately downstream of the nicking site within AAVS1, near the region of homology with AAV2 that contains the RBS (Fig. 8), as had been seen in one previous study (12). Over two-thirds (69%) of our distinct sequenced junctions mapped to AAVS1 bases 813 to 1163. One obvious feature of this region is a high frequency of polypyrimidine (pPy) tracts on the strand that is nicked by Rep68/78. Figure 8 shows that there are eight pPy tracts of 9 bases or more between AAVS1 bases 800 and 1200. In contrast, AAVS1 bases 1 to 800 contain no pPy tracts of 9 bases or more. Further evidence for a correlation between pPy tracts and integration junctions is seen in the region between AAVS1 bases 430 and 550, which contains no integration junctions and no pPy tracts of greater than 3 bases on the nicked strand (Fig. 8).


Figure 8
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  		FIG. 8. Mapping of AAV2-AAVS1 junction breakpoints onto AAVS1. pPy tracts of greater than 8 bases are in bold type. The 2 bases flanking trs (T396 and T397) are in lowercase, and the RBS (bases 408 to 423) is in lowercase italics. Breakpoints found at one or more junctions are underlined. For microhomology junctions, the center of the microhomology is underlined.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Several patterns have emerged from our analysis of AAV2-AAVS1 junctions. All integration junctions are on the same side of the Rep68/78 nicking site of AAVS1, consistent with the hypothesis that unidirectional DNA replication, initiated at the nick (Fig. 1), is somehow involved (64). Our data also suggest that the majority of junctions are the result of microhomology-mediated end joining, which implies a strand invasion or single-strand annealing event as part of the mechanism.

In addition, about two-thirds of junctions were found in a 350-base region enriched for pPy tracts on the nicked strand of AAVS1. One trivial explanation for this clustering of junctions could be that the amplification reaction is more efficient over shorter distances (18). However, it should also be noted that there is not a perfect correlation between the distance from the AAVS1 primer and the frequency of integration junctions. For example, we detected six junctions between AAVS1 bases 851 and 900 but only two junctions between AAVS1 bases 951 and 1000 (Fig. 8). In addition, at least six different studies using proximal AAVS1 primers ranging from bases 1209 to 1621 have reported two or more junctions within the region from base 800 to base 1200 (18, 27, 43, 48, 49, 51, 53). This is one of the few regions in which all of the above-cited works reported AAV2-AAVS1 junctions. Due to the small number of sequences analyzed in each of these studies, the pattern was not detected.

Polypurine (pPu) tracts and their complementary pPy tracts are believed to stimulate recombination (4, 21-23). They can form triplex DNA, which can stabilize strand invasion events (21-23). In addition, pPy tracts as short as 18 bp have been demonstrated to promote oligonucleotide-directed gene correction at least 23 bp away in vivo and strand invasion (D-loop formation) at sites up to 4 kb away in vitro (4). As most of our junctions were outside the actual pPy tracts, we hypothesize that smaller pPy tracts may be able to stimulate recombination over shorter distances. A minimum of 9 bp was arbitrarily set for identifying a pPy tract because the longest pPu tract on the nicked strand from bases 1 through 1200 is 8 bases. It should also be noted that there is a 21-base pPy tract comprised of AAVS1 bases 1717 to 1737 (data not shown), which is in theory large enough to stimulate strand invasion throughout the entire AAVS1 locus. There are also 10-base pPy/pPu tracts within the AAV2 ITRs (bases 8 to 17, 109 to 118, 4562 to 4571, and 4663 to 4672), which may contribute to the ITRs’ role in recombination (50, 60).

In addition to the intrinsic properties of pPu/pPy tracts, there are cellular proteins that bind specifically to these sequences. For example, recent work shows that a complex of pPy tract binding protein-associated splicing factor with a 54-kDa nuclear RNA-binding protein may be involved in DNA double-strand-break rejoining (5). Also, the human purine-rich element binding protein alpha (PUR{alpha}) is a single-stranded DNA-binding protein that binds preferentially to the purine-rich element termed PUR, which is present at origins of replication and in gene-flanking regions in a variety of eukaryotes (14, 15, 32, 56, 61, 68). PUR{alpha} is believed to be involved in the control of both DNA replication and transcription and can promote the unwinding of double-stranded DNA containing pPu/pPy tracts (68). Computer-assisted analysis (17) of the junction sites revealed that several were near potential binding sites for the human PUR{alpha} protein (data not shown).

We also identified potential binding sites for transcription factors SP1 and MZF1 (38) near several of our AAV2-AAVS1 junctions (data not shown). SP1 has been reported to bind Rep68/78, and this interaction may allow the creation of DNA loops which might help to direct or restrict integration (16, 44, 45). SP1 is also a substrate for DNA-dependent protein kinase, a key protein in nonhomologous end joining (9, 19).

The pattern of AAV2 breakpoints at junctions is explained most easily by the ITRs being in the hairpin, as opposed to the linear conformation, prior to junction formation (Fig. 2 and 6). If the breakpoints were random or strictly dependent on the primary base sequence, we would expect to see at least a few breakpoints within the RBS nearest to the end of the genome if the linear ITR were the more common substrate. As with our data in Fig. 6, a recent large-scale study of rep– AAV2 vector "random" integration shows a similar lack of cellular junction breakpoints within the terminal 40 bases of the AAV2 ITR (37). This pattern is therefore Rep independent, indicating that it is determined by cellular proteins. Another recent study of the concatemerization and circularization of rep– AAV2 vectors also concluded that a forced hairpinned ITR is preferred, as a recombination substrate, to an ITR that could exist in either a hairpinned or an extended linear conformation (8).

We detected several complex junctions which involved one or two AAV2-AAV2 junctions in addition to an AAV2-AAVS1 junction. We hypothesize that such junctions result from circularization of AAV2 genomes by cellular proteins, followed by a Rep68/78-induced opening of the circles, prior to forming the junctions with AAVS1. Recent work has demonstrated that in the absence of Rep68/78, circularization efficiency can be greater than 90% within 24 h postinfection (7, 8). Work from other groups suggests that circularization is a dead end for integration (39, 58, 59). Since Rep68/78 is required for the efficient rescue of AAV2 ITR-containing DNA from plasmids (66), it is reasonable to speculate that Rep68/78 can resolve AAV2 circles, producing ends that can recombine with AAV2 or chromosomal DNA.

In theory, our assay should have detected so-called head-to-head and tail-to-tail fusions of AAV2 genomes. Such fusions are common replicative intermediates during productive infection (65) and have been detected previously in patient samples (54). Our results indicate that these are rare with a wild-type AAV2 infection in the absence of helper virus. This observation is also consistent with circularization being an efficient reaction compared to dimerization of the AAV2 genome. Circularization of AAV2 monomer DNA would result in a head-to-tail junction that would not be amplified by our system.

We originally hypothesized that Artemis might stimulate recombination by cleaving hairpins within the ITRs, but our data suggest that a protein other than Artemis can cleave the AAV2 hairpins. We cannot, however, rule out the possibility that Artemis is one of several proteins that cleaves the ITR.

We have also identified two junctions (Fig. 5) consistent with homeologous recombination (recombination between slightly mismatched sequences). Junctions 2-7 and 2-13 are at approximately the same sites, just upstream of an 11-base pPy tract in AAVS1. Although technically defined as a direct joining and a 2-base microhomology junction, respectively, each had 8 of 10 bases in common between AAVS1 and AAV2 (Fig. 5). We hypothesize that the recombination events were initiated by a strand invasion event, followed by the use of the partially mismatched sequence as a primer for DNA synthesis. This would have been followed by DNA mismatch repair that in some cases corrected the mismatch in favor of AAV2 and in other cases corrected in favor of AAVS1. Microhomeology junctions were detected only with the Artemis-defective cells. Since the Artemis protein is not known to be involved in homeologous recombination, we suspect that this is the result of a secondary defect.

Based on our observations, we propose a novel predominant mechanism for Rep68/78-mediated integration into AAVS1 in which a hairpinned ITR is first processed by a cellular endonuclease to produce a 3' overhang. This overhang then anneals to its complementary sequence within the intact strand of AAVS1, which has been exposed by DNA unwinding ahead of an active or stalled replication complex assembled at the Rep68/78 nicking site. This annealed 3' end is then extended by a DNA polymerase to create the first de facto joining of AAV2 to AAVS1 DNA sequences. Further analysis of AAV2 integration in cells with known defects in DNA damage repair/signaling should help to further constrain models of preferential integration.


   ACKNOWLEDGMENTS
 
We thank Peggy Hsieh, Anthony Furano, and Kevin Gardner for their critical reading of the manuscript as well as for other useful advice. We thank Sven Bilke for assistance with the sequence analysis. We thank Irving Miller for supplying much of the purified AAV2 used in this study and J. Rodney Brister and Dik van Gent for useful technical advice. We also thank Katheryn Meek for supplying the Artemis cell line.

R.A.O. is a coinventor on several patents related to recombinant AAV technology. To the extent that the work in the manuscript increases the value of this patent, he has a conflict of interest.

This research was supported by the Intramural Research Program of the NIH, National Institute of Diabetes and Digestive and Kidney Diseases.


   FOOTNOTES
 
* Corresponding author. Mailing address: Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bldg. 8, Rm. 307, National Institutes of Health, Department of Health and Human Services, 8 Center Drive MSC 0840, Bethesda, MD 20892-0840. Phone: (301) 496-3359. Fax: (301) 402-0053. E-mail: address: ro6n{at}nih.gov Back

{triangledown} Published ahead of print on 11 July 2007. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Journal of Virology, September 2007, p. 9718-9726, Vol. 81, No. 18
0022-538X/07/$08.00+0     doi:10.1128/JVI.00746-07
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