Adeno-associated virus integrates site-specifically into human chromosome 19 in either orientation and with equal kinetics and frequency

Daniela Hüser and Regine Heilbronn

Department of Virology, Institute of Infectious Diseases, Free University of Berlin, Universitätsklinik Benjamin Franklin, Hindenburgdamm 27, 12203 Berlin, Germany

Correspondence
Regine Heilbronn
regine.heilbronn{at}ukbf.fu-berlin.de


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Adeno-associated virus type 2 (AAV-2) establishes latency by site-specific integration into a unique locus, AAVS1, on human chromosome 19 (chr19). To study the kinetics and frequency of chr19-specific integration, a rapid, sensitive and quantitative real-time PCR assay specific for AAV inverted terminal repeat (ITR)–chr19 junction sequences was developed. Since the assay only detected right-hand AAV ITR-specific integration events, the development of a complementary left-hand ITR-specific real-time PCR assay is described. The time-course of left-hand ITR-dependent AAV integration at AAVS1 of chr19 was determined in AAV-2-infected HeLa cells. Both the kinetics and frequencies of left-hand ITR-dependent integration were found to be similar to those of the right-hand ITR. In addition, left-hand ITR-specific fusion sequences and chromosomal breakpoints within AAVS1 were variable, yet were the same as those found in right-hand ITR–chr19 junction sequences. Thus, the AAV-2 genome integrates site-specifically into chr19 with similar efficiency in either orientation.


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Adeno-associated virus (AAV) has evolved a biphasic life cycle to ensure persistence in its primate host. It needs an unrelated helper virus, adenovirus or herpesvirus, for productive infection (Muzyczka & Berns, 2001). In the absence of a helper virus, AAV type 2 (AAV-2) establishes latency by preferential integration into a specific site (19q13.3-qter or AAVS1) on human chromosome 19 (chr19) (Kotin et al., 1990). The site-specificity of AAV integration is mediated by the AAV Rep78 protein or by its C-terminally spliced variant Rep68 (Berns, 1996; Linden et al., 1996a). During productive AAV replication, Rep78 or Rep68 is needed for AAV gene expression and DNA replication. The AAV origins of DNA replication reside in the 145 bp inverted terminal repeats (ITRs), which flank the 4.7 kb single-stranded AAV genome. Rep78 and/or Rep68 bind to the Rep-binding site (RBS) on the AAV ITR (Snyder et al., 1993), and nick and unwind the ITR at the terminal resolution site (trs) (Im & Muzyczka, 1990). AAV ITRs also serve as cis elements for chromosomal integration (Berns, 1996; Linden et al., 1996a). Binding of Rep to the ITR is required for site-specific integration (Weitzman et al., 1994), whereas nicking of the trs is not essential (Young & Samulski, 2001). DNA sequences homologous to RBS and trs sequences of the AAV ITR are found in the chr19 preintegration site (Kotin et al., 1992; Samulski et al., 1991). In vitro studies demonstrated ternary complex formation of Rep68 with the AAV ITR and chr19 AAVS1 (Weitzman et al., 1994). A 33 bp sequence spanning the chr19 RBS and the trs homology element is sufficient to mediate site-specific AAV integration in vivo. Either of these elements, and proper spacing between them, is essential (Linden et al., 1996b; Meneses et al., 2000). Mapped chr19 integration sites have been derived from cloned cell lines generated with and without drug selection (Kotin et al., 1992; Palombo et al., 1998; Pieroni et al., 1998; Samulski et al., 1991; Tsunoda et al., 2000; Yang et al., 1997). In addition, site-specific AAV integration into AAVS1 has been studied in Epstein–Barr virus shuttle vectors carried as episomal plasmids in human 293 cells (Giraud et al., 1994, 1995). The integration site sequences were highly variable within a range of a few 100 bp from the RBS of AAVS1. We have established recently a quantitative real-time PCR assay that allows detection and quantification of integration at the authentic chr19 integration site early after AAV infection (Hüser et al., 2002). Due to restrictions in primer design, the AAV-specific primer only detects integration events that involve the right-hand AAV ITR (nt 4529–4674). To evaluate and compare integration frequencies of AAV irrespective of genome orientation, a real-time PCR assay that detects left-hand ITR-specific integrations (nt 1–145) was developed and evaluated. Our results show that AAV integrates with equal efficiency in either orientation. Junction DNA sequences display left-hand ITR–chr19 fusions with similar variability of the chromosomal breakpoints, as described before for right-hand ITRs.

The real-time PCR assay described recently (Hüser et al., 2002) made use of an AAV ITR-specific primer that partially overlapped with unique sequences of the right-hand end of AAV-2, so that only one orientation of integration was detected. To evaluate the efficiency and kinetics of left-hand ITR-dependent chr19 integration, a left-hand ITR-specific primer (PITR left, 5'-CTCCAGGAACCCCTAGT-3') was designed and evaluated. The corresponding AAVS1-specific primer was PAAVS1b (5'-ATCCGCTCAGAGGACATCAC-3'). The internal primer set needed for PCR product detection by fluorescence resonance energy transfer (FRET) technology (Fig. 1A) has been described (Hüser et al., 2002). Real-time PCR conditions were optimized by the aid of a control plasmid, pAAVS1-LTR, which carries an artificial left-hand AAV ITR–chr19 junction sequence and which was constructed as described (Hüser et al., 2002). Real-time PCR conditions were adapted from the described protocol (Hüser et al., 2002) with the following modifications. Both the preamplification step and the real-time PCR were performed at 62 °C. In addition, real-time PCR was done in the presence of 3 mM MgCl2. Uninfected HeLa cell DNA (1 µg) was spiked with varying dilutions of pAAVS1-LTR (102–5 DNA copies per reaction). This led to a linear standard curve (Fig. 1). PCR products of varying length were expected due to the variability of integration sites within AAVS1. To ensure efficient amplification of all fragments, including long PCR fragments, the time for the PCR elongation step was set at 30 s. This is longer than the calculated time (25 s) for the efficient amplification of the 635 bp PCR product amplified from the control plasmid pAAVS1-LTR.



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Fig. 1. The kinetics of AAV-2 site-specific integration into chr19. (A) Schematic representation of AAV integration into AAVS1 of chr19 (wavy line). AAV sequence elements are represented as boxes shaded differentially in grey, with nucleotide position 1 at the right-hand end and nt 4647 at the left-hand end. Sequence elements within the AAV ITR are as outlined in Fig. 2(A). RBS, Rep-binding site; trs, terminal resolution site. Amplification, detection and quantification of site-specific integration were performed with the real-time PCR LightCycler system. Arrows mark the primers used for the amplification of AAV ITR–AAVS1 junction sequences. Primer PITR left hybridizes to AAV sequences at positions 133–149 (Srivastava et al., 1983) and primer PAAVS1b hybridizes to AAVS1 on chr19, 19q13.3-qter, at positions 1615–1596 (Kotin et al., 1992). Further sequence-specificity is provided by the hybridization probe assay format used for the detection of the PCR product. Fluorescent dye-labelled probes hybridize to the amplified PCR fragment (Donor probe, 1541–1560; acceptor probe, 1562–1583), thereby eliciting FRET. Fluorescence emission intensity is directly proportional to the amount of PCR product. (B) Raw data of the LightCycler analysis of HeLa cell DNA (1 µg) spiked with known copy numbers (102–5) of standard plasmid pAAVS1-LTR. (C) Kinetics of left-hand ITR-specific integration at AAVS1. HeLa cells were infected with AAV at an m.o.i. of 500. Total genomic DNA was isolated atdifferent times p.i. Purified DNA (1 µg) was amplified by LightCycler PCR as outlined in the text. The copy numbers of AAV ITR–chr19 integration site junctions per µg of AAV-infected HeLa cell DNA were quantified by comparison to values of the standard curve run in parallel (B). The columns represent the mean±SD of three independent cultures. The values at 96 h p.i. represent the mean of two samples.

 
To analyse the left-hand ITR-specific integration kinetics of AAV into AAVS1, HeLa cells were infected at an m.o.i. of 500, as described (Hüser et al., 2002). At the time-points indicated, total genomic DNAs of independent plates were extracted. Site-specific integration was analysed by left-hand ITR-specific real-time PCR as outlined in Fig. 1. Site-specific integration became detectable at 16 h post-infection (p.i.) and peaked at 96 h p.i. The overall kinetics and the junction copy numbers that reflect the integration frequencies per cell matched the results of the right-hand ITR-specific assay (Hüser et al., 2002). The specificity of the assay was documented by a series of negative controls which scored negative (Table 1). These included the mixing of the two plasmids carrying either one of the primer binding sites alone (pTAV-2 containing the AAV ITR and pRVK containing AAVS1). The negative PCR results excluded the possibility of polymerase-mediated template switches resulting in artefactual PCR products. Similarly, neither of the external primers alone detected left-hand ITR–AAVS1 junction sequences. Due to a partial sequence identity of the left- and right-hand AAV ITR-specific primers, amplification efficiency of either right- or left-hand ITR-specific PCRs for the other orientation of integration was tested. As seen in Table 1, either PCR detects the opposite orientation of integration very inefficiently. Although quantification of real-time PCR below a value of 200 copies per reaction is very inaccurate, the highest estimate for the detection of the opposite orientation of integration lies below 1 % of total junction copy numbers. Direct comparison of the results obtained with the right-hand ITR-specific assay with those of the left-hand ITR-specific assay showed that sample-to-sample variations were higher with the left-hand ITR-specific assay. The high degree of specificity achieved required a compromise with respect to sensitivity. Nearly identical frequencies and kinetics were obtained for the right- (Hüser et al., 2002) and the left-hand (this report) ITR-specific integration assays, in spite of separate control plasmids used as external standards, of specifically designed primer sets and of different assay conditions. In summary, the findings document clearly the independence of site-specific integration from the orientation of the AAV genome.


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Table 1. Real-time PCR analysis of AAV–AAVS1 junctions

 
To analyse junction structures, PCR fragments derived from the sample at 96 h p.i. were cloned into pCR4-TOPO (Invitrogen) by the use of an uracil-N-glycosidase (UNG)-deficient strain of Escherichia coli (TaKaRa Biomedicals). Plasmid DNAs from real-time PCR-positive colonies were subjected to DNA sequence analysis (Fig. 2). Variable crossover points and both ‘flip’ and ‘flop’ orientations of the AAV ITR were found. The breakpoints within AAVS1 of chr19 varied within a range of a few 100 bp, which is in line with the findings of right-hand ITR-specific integration (Hüser et al., 2002) (Fig. 3). In addition, there is good agreement of the chr19 integration sites detected with real-time PCR within hours after AAV infection with those described in latently infected cell lines (Palombo et al., 1998; Pieroni et al., 1998; Recchia et al., 1999; Rizzuto et al., 1999; Yang et al., 1997). The described integration sites also agree with those in AAVS1 transgenic mice or rats (Rizzuto et al., 1999). The quantitative evaluation of integration frequencies for either AAV orientation as described here confirms previous circumstantial evidence for comparable efficiencies (Giraud et al., 1995). Our results describe the events hours after AAV infection in the absence of selection. In addition, the results were independently generated for the right- (Hüser et al., 2002) and left-hand AAV ITRs. Together these data virtually exclude the possibility that early integration sites on chr19 differ from the ones detected after clonal selection and extensive chromosomal rearrangements.



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Fig. 2. Analysis of PCR-amplified left-hand ITR–AAVS1 junction sequences. PCR fragments of the sample at 96 h p.i. (see Fig. 1) were cloned into pCR4-TOPO and an UNG-deficient E. coli strain and subjected to DNA sequence analysis. (A) The hairpin structure of the left-hand AAV ITR is represented in both the ‘flip’ and the ‘flop’ orientation. Palindromic sequence elements of the left-hand ITR are indicated in lower case letters. (B) Structural maps deduced from DNA sequence analysis of cloned junctions. The black arrow indicates the hybridization site of primer PAAVS1b on chr19. The grey arrow indicates thebinding site of primer PITR left on the left-hand ITR. Positions of the last unambiguous cellular and/or viral nucleotide are indicated. Numbering is according to published sequence information (Kotin et al., 1992; Srivastava et al., 1983). Non-AAV, non-AAVS1 nucleotides in clone 3 are represented as lower case letters. (C) Schematic representation of left- and right-hand AAV ITR-specific integration sites within AAVS1. The bottom line represents AAVS1 of chr19. The nucleotide sequence and numbering correspond to those determined by Kotin et al. (1992). The trs and RBS homology sequences as determined by Linden et al. (1996a) are indicated as boxes, as is the described DNase hypersensitive site (Lamartina et al., 2000). The primers used for the detection of junction fragments are indicated below. Nucleotide positions in the 5'->3' direction are nt 1609–1593 (PAAVS1) and nt 1615–1596 (PAAVS1b). Integration sites determined for either AAV orientation are represented as characteristic symbols above. Each symbol represents one integration site.

 
The data from our previous study (Hüser et al., 2002), together with the data presented here, provide a solid basis for the calculation of site-specific integration frequencies as follows. The total of the right- and left-hand ITR-specific integration rates add up to a peak rate of 14 000 junction copies per µg of genomic DNA. Since HeLa cells have a hypertriploid (3n+) karyotype (Macville et al., 1999) with a calculated DNA content of 10 pg per cell, 14 % of HeLa cells are targeted. Assuming additional integration sites beyond the limits of the primers used in this study (Giraud et al., 1995; Tsunoda et al., 2000), and assuming that long or structurally complicated PCR products are difficult to obtain, our calculation represents a lower estimate. The similarity of right- and left-hand ITR-specific integration frequencies shown in this report leads to a more solid calculation basis for the natural site-specific integration frequency: at least 15 % of unselected HeLa cells will display targeted integration at 4 days p.i.

Our results show that a carefully controlled real-time PCR is not only sensitive but also highly specific for AAV ITR–AAVS1 junctions. In addition, copy numbers can be quantified reliably over a concentration range of several logs. Thus, the natural course of AAV-2 integration into chr19 (19q13.3-qter) can be quantified reliably within hours after AAV infection in an unselected cell population. This opens the possibility to rapidly monitor and quantify integration efficiencies of other AAV types and mutants thereof. Targeted integration analysed in cells infected with recombinant AAV (rAAV) vectors did not lead to site-specific integration, neither in the absence of selection at 72 h post rAAV infection nor in a pool of G418-selected cell clones at 14 days after infection with a neomycin-expressing rAAV vector (unpublished data). This confirms recent data on rare and non-AAVS1-specific rAAV integration (Miller et al., 2002). Real-time PCR screening for AAV–AAVS1 junction sequences will also be useful for the study of AAV integration in transgenic animals carrying AAVS1 (Rizzuto et al., 1999; Young et al., 2000).


   ACKNOWLEDGEMENTS
 
The authors wish to thank M. Alex for help with the analysis of cloned junctions, S. Weger who continuously supported this work by helpful discussions and critical reading of the manuscript and M. Linden and N. Muzyczka for plasmids. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 506).


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Received 23 July 2002; accepted 11 September 2002.