Structural Analysis of Adeno-Associated Virus Transduction Circular Intermediates

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Virology 261, 8–14 (1999) Article ID viro.1999.9821, available online at on

RAPID COMMUNICATION Structural Analysis of Adeno-Associated Virus Transduction Circular Intermediates Dongsheng Duan,* ,† Ziying Yan,* Yongping Yue,* and John F. Engelhardt* ,† ,‡ ,1 *Department of Anatomy and Cell Biology and ‡Department of Internal Medicine, †Center for Gene Therapy, College of Medicine, The University of Iowa, Iowa City, Iowa 52242 Received March 8, 1999; returned to author for revision April 27, 1999; accepted May 24, 1999 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. © 1999 Academic Press

Although the existence of circular AAV intermediates has been suspected for years, confirming evidence has remained elusive. Recently, using genetic approaches and Southern analysis of Hirt DNA, we have demonstrated the existence of head-to-tail circular intermediates in rAAVinfected muscle and cell lines (6, 7, 14). Importantly, the abundance of these rAAV circular intermediates correlates with the long-term persistence of transgenes in muscle (7). Integrated proviral genomes with similar head-to-tail conformations have also been reported for both wild-type and rAAV genomes in cell lines (4, 5, 10, 12). Furthermore, studies of wild-type AAV integration in an episomal Epstein–Barr viral vector harboring the AAVS1 locus (the wildtype AAV integration site in human chromosome 19) also demonstrate head-to-tail structures (11). Although it is clear that ITRs are directly involved in many aspects of the AAV life cycle, including viral packaging, integration, and rescue, the mechanism(s) responsible for ITR-mediated integration of recombinant viral genomes is poorly understood. Importantly, the head-totail structure typical of integrated genomes cannot be directly deduced from the replicative forms (Rf m and Rf d) associated with current models of lytic AAV replication. The recent identification of circular head-to-tail AAV genomes may clarify some features of the AAV latent life cycle, including proviral structure and genome persistence, as both episomes and integrated proviruses. Investigation regarding the potential role of circular AAV genomes as preintegration intermediates requires a de-

Recombinant adeno-associated virus (rAAV) has emerged as a very promising gene delivery vehicle in several organs including muscle and the central nervous system (7). Wild-type AAV is a 4.7-kb single-stranded DNA virus. In vitro production of rAAV is based on the rescue and subsequent replication of viral sequences from plasmids containing viral inverted terminal repeats (ITRs) as the only cis-acting component necessary for packaging of recombinant viral genomes. Additional functions required for the propagation of rAAV are provided by helper viruses, such as adenovirus or herpes virus, and the wild-type AAV viral gene products Cap and Rep (1). It is generally accepted that AAV replication in vivo occurs through self-priming, extension, and subsequent single strand displacement (13). Inherent in this model of replication is the need for the strand-specific endonuclease activity of the Rep protein, which produces free 39-OH groups necessary for subsequent rounds of extension (15). During repeated rounds of terminal resolution, reinitiation, and strand displacement, only head-to-head or tail-to-tail intermediates are generated (1). Without helper virus or genotoxic stimuli, rAAV will enter its latent cycle and persist as an integrated or episomal provirus in transduced mammalian cells. 1 To whom correspondence and reprint requests should be addressed at Department of Anatomy and Cell Biology, University of Iowa, College of Medicine, 51 Newton Road, Room 1-111 BSB, Iowa City, Iowa 52242. Fax: 319-335-7198. E-mail: [email protected]

0042-6822/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.




FIG. 1. Sequencing strategy for AAV circular intermediate ITR arrays. Seven head-to-tail circular intermediates isolated from Hela cells or primary fibroblasts were chemically sequenced. (A) The variable length of the ITR arrays seen in circular intermediates, as shown by SphI digestion in a 1.3% agarose gel. The majority of isolated head-to-tail fragments (80%) migrated at 300 bp, as illustrated for p81, p14f, and p1202. Since the predicted intact SphI head-to-tail ITR fragment is approximately 360 bp, we originally hypothesized that the frequently observed 300-bp fragment might reflect either anomalous migration due to the high secondary structure of the ITR or an internal deletion (6). A limited number of clones with SphI ITR fragments of 320 bp (p79) to 360 bp (p3f) were also observed. In addition, two clones (p82 and p140f) yielded fragments running at an apparent size indicative of less than one intact ITR. The Maxam-Gilbert chemical sequencing strategy is outlined in B. For presentation, nucleotide base pair numbers are according to the originally hypothesized circular intermediate structure, with two full-length inverted ITRs (6). To generate templates for sequencing from the 59 end of the junctional ITR region, cesium chloride-purified circular intermediates were digested with PstI to generate a 2.9-kb fragment. This fragment was gel isolated and end-labeled with g-ATP 32 in a 50-ml reaction containing 1 mg of the DNA fragment, 50 mCi g-ATP 32 (NEN, Dupont), and 1 unit T4 kinase (New England BioLabs, Inc.). The labeled DNA was subsequently digested to completion with HaeII to release a 600-bp ITR-containing segment labeled on the PstI end (371 bp). Up to 300 bases were successfully sequenced with this strategy. The 59 ITR sequencing results were subsequently confirmed by sequencing from the 39 end of the ITR array. To generate templates for sequencing from the 39 end, circular intermediates were first digested to completion with SacI, followed by partial digestion with SphI to yield an 800-bp fragment. This fragment was then gel purified and end-labeled with g-ATP 32. The end-labeled fragment was subjected to an additional digestion with SphI to release a 300-bp segment labeled at one end and gel purified once again. Chemical sequencing reactions were performed using a Maxam-Gilbert DNA sequencing kit from Sigma (Catalog No: SEQ-1), and the sequences of the ITR arrays from both directions were aligned and a consensus was generated.

tailed structural characterization of these circular genomes as well as knowledge of the mechanisms responsible for their formation. This information may in turn explain the structural diversity of the ITR sequences within integrated proviruses (5, 20), and may provide indications for the structural characteristics imparting long-term persistence. Double-D Structure Is the Predominant Type of ITR Array Contained within AAV Circular Intermediates. A central issue in the formation of AAV circular intermediates is the ligation event joining the 59 and 39 ITRs from single-stranded or double-stranded AAV genomes. Southern blot analysis of Hirt DNA and dideoxy sequencing of rescued genomes have indicated a head-to-tail conformation of the ITRs within circular AAV genomes (6). However, detailed sequence information within the central region of ITR arrays was not achieved by this sequencing strategy, reflecting the limitations of primer

extension methods for crossing through ITR sequences with high secondary structure. To more clearly define the exact position of ligation/recombination events involved in genome circularization at the two ITR ends, we have undertaken a rigorous chemical sequencing strategy to clarify the internal ITR junctional structures within AAV circular intermediates. The Maxam-Gilbert DNA sequencing method, based on the cleavage of specific chemically modified bases, represents an alternative approach less prone to problems associated with the secondary structure of DNA. Seven independent clones of rAAV circular intermediates (p79, p81, p82, p1202 from Hela cells and p3f, p14f, p140f from human primary fibroblasts) were isolated from AV.GFP3ori-infected Hela cells and human primary fibroblasts as previously described (6, 7, 14). Based on our previous control experiments using purified singlestranded or double-stranded linear viral genome for bac-



FIG. 2. Comparison of sequencing results in the ITR junctional region of circular AAV intermediates. Alignment of the junctional sequences within the ITR arrays of circular AAV intermediates is shown in A. Illustrated are results from seven circular AAV intermediates isolated from Hela cells (p82, p81, p1202, and p79) or from primary fibroblasts (p140f, p14f, and p3f). Palindromic ITR sequences in each of the clones are indicated by D, A9, B9, B, C9, C, A, D9 according to the standard nomenclature. SphI (GCATGC) and PstI (CTGCAG) sites used in the chemical sequencing reactions are indicated in the 39 and 59 ITR flanking regions. Regions flanking the D sequence of the 39 ITR included 40 bases from wild-type AAV (nucleotides 4535–4498; marked by a shaded box) which was retained during the cloning of the rAAV ITR from the original pSub201 plasmid. Flanking the D9 sequence of the 59 ITR was this same region from wild-type AAV-2 (nucleotides 4533–4498), in addition to part of the CMV promoter (marked by a crossed bar box). The only mutation noticed in all sequences was a point mutation of A to G transition in the B9 region of p81 (marked by †). In some clones, several regions of the ITR junctional arrays also contain incomplete ITR sequences. The deletions in the 39 end of any ITR regions are marked an asterisk, while the deletions in the 59 end of any ITR regions are marked by double asterisks. The most consistent motif observed in all clones was the conserved two intact D sequences adjoined by transgene sequences from both sides. Five of seven clones (p81, p1202, p14f, p79, p3f) contained additional conserved regions, including intact D and A9 sequences at the 59 end and the A and D9 sequences at the 39 end of the junctional ITR arrays. In addition, the terminal resolution sites (trs) (59-AGTTGG) were retained in both ends of the junctional sequences in all clones (boxed nucleotides). (B) A two-dimensional reconstitution of the ITR regions contained within all seven circular intermediates. Dashed lines illustrate DNA sequences that were deleted in the final plasmid form of the rescued circular intermediates. The potential sites of recombination are marked by double-headed arrows. The predicated flip or flop orientations of the ITR sequences are marked on each molecule. The majority of the head-to-tail fragments appeared to originate from at least one flop ITR. An exception was clone p3f, which may have originated from two flip ITRs. A panhandle or stem-loop configuration is consistent with a precursor form for all the circular intermediate clones.

terial rescue of circular intermediates, it was clear that replication-competent circular AAV genomes are not formed in bacteria (7). Furthermore, the characteristic digestion pattern for head-to-tail circular intermediates has been directly demonstrated on Hirt DNA Southern blots prior to bacteria transformation (6, 14). Taken together, these previous studies provided substantial evidence for the existence of circular AAV genomes in latent phase transduction. Similar to previous reports (6), the monomer circular intermediate clones selected for detailed structural evaluation exhibited identical head-totail ITR conformations, as determined by restriction en-

zyme analysis and Southern blotting with ITR and GFP probes (data not shown). These seven clones were chosen because they represented the heterogeneity found in the length of ITR arrays seen in circular intermediates (from ,1 to ;2 ITRs, Fig. 1A). Since the junctional ITR region (SphI-digested segment) in 80% of the circular intermediates migrated as a 300-bp fragment on nondenaturing agarose gels, we have randomly picked three independent clones from this group for our sequencing analysis. Based on further restriction mapping, a chemical sequencing strategy was generated (Fig. 1B). The templates for chemical sequencing were end-labeled at



FIG. 2—Continued

either the SphI (2 bp) or PstI (371 bp) site as described in Fig. 1. Complete sequence alignments of all seven clones are depicted in Fig. 2A. Interestingly, the D and A9 segments in the 39 ITR and the A and D9 regions in the 59 ITR were highly conserved in all of the clones analyzed. The existence of this consistent motif may reflect a uniform pathway in the formation of circular intermediates. The most predominant ITR array contained within circular genomes consists of one complete ITR flanked by two D-region elements (p81, p1202, and p14f). This structure closely resembles the configuration proposed for integrated AAV proviruses (2). Furthermore, it also shared strong homology with the ITR array in a previously described AAV double-D plasmid. Although the reported double-D plasmid was cloned by PCR-mediated approaches, it was shown to possess the minimal cis elements required for the AAV life cycle (19). The importance of the 20-nucleotide-long D sequence in AAV res-

cue and replication has also been confirmed by other studies (16, 17). Taken together, these findings suggest that double-D structures in circular intermediates are consistent with latent forms of the viral genomes. Results from the sequence analysis also confirmed the variable lengths of ITR arrays in SphI-digested head-to-tail ITR fragments. For example, the ITR array of p82 was composed of two complete inverted D sequences (labeled D and D9) and internally truncated A9 and A sequences. This head-to-tail ITR array was the shortest of the group analyzed. In contrast, additional partial B9A9 and C9CA9 sequences were inserted within the ITR arrays in p79 and p3f, respectively, leading to structures characterized by more than one complete ITR. Proposed Model for AAV Circular Intermediate Formation by ITR Recombination/Ligation Events within a Common Panhandle Single-Stranded Viral Genome. The formation of circular genomes in cell lines and tissues



FIG. 3. Model for the formation of AAV circular intermediates. Following infection with AAV and translocation of the viral DNA to the nucleus, single-stranded DNA can be maintained in different conformations (A). These forms may include linear molecules with hairpin ends or panhandle/ stem-loop structures. Additionally, the partial folding of the panhandle ends may produce intermediates by intermolecular hydrogen bonding at the ITR regions. According to our chemical sequencing results, the most likely precursor of head-to-tail circular intermediate monomers is the panhandle or stem-loop conformation. As shown in B, the formation of the double strand circular molecules might involve resolution at trs (terminal resolution site) followed by second strand synthesis by template strand switch and extension. Last, one must invoke a second nick at the trs following extension and a final ligation event. This model would be similar to a rolling circle mode of replication. It is also possible that religation of the free ends of the ITR happens first, followed by subsequent deletion and recombination in the B, B9, C, C9 region to create the double D molecule. Intramolecular or intermolecular recombination within the ITR sequences produces the substrate for circular monomer and dimer genome formation, respectively. We hypothesize that folding within the annealed panhandle ends of ITRs may in part determine the location of subsequent resolution and ligation sites required for head-to-tail circular intermediate formation. According to this model, a head-to-tail conformation will be the most abundant circular genome. However, head-to-head and tail-to-tail genomes could also occur through intermolecular recombinational events. This model is consistent with the frequency of head-to-tail, tail-to-tail, and head-to-head integration events reported for both wt and rAAV proviruses. The crossed double-headed arrows represent the potential recombination sites.

represents a new and potentially important aspect of the rAAV viral transduction process. A knowledge regarding the mechanisms responsible for circular intermediate formation is critical to our understanding of rAAV transduction. Models incorporating these molecular structures should be capable of explaining both integration and episomal persistence, which are features of both wt and rAAV latent infection. The presence of ITR structures with both less and more than one intact ITR sequence suggests three important mechanistic considerations. First, the original ITR array may contain two intact ITRs, which is reduced to a more stable double-D ITR structure either in vivo or by bacteria following amplification. Second, directed rearrangement or deletion of ITR arrays may occur following formation of a common intermediate. Third, the mechanism of circular intermediate forma-

tion may involve ITR recombination events from a panhandle or stem-loop conformation of single-stranded viral DNA. To address the potential for recombination/rearrangements within ITRs during bacteria amplification of rescued genomes, we performed the following experiment. The linear double-stranded viral genome (HindIII/PvuII fragment of pCisGFP3.ori) was blunted with T4 polymerase and religated at low concentrations to induce intramolecular circularization. This molecule should represent a circular proviral genome containing two full-length ITRs in a head-to-tail orientation. Consistent with our previous report (7), bacterial clones were obtained only following religation of blunted HindIII/PvuII fragments but not in controls lacking either T4 DNA polymerase or ligase. Importantly, all synthetically engineered circular


viral genomes isolated by this method revealed a consistent ITR array following SphI digestion of 400 bp, which is the expected length of the two intact ITRs in a head-to-tail orientation (data not shown). Hence, we concluded that recombination in bacteria might not have a significant influence on the findings in this study. The most appealing explanation for circular intermediate formation from our current results appears to involve ITR recombination/ligation processes from single-stranded self-annealed AAV DNA. Figure 2B schematically illustrates potential crossover/recombination sites within ITR segments that are contained within the head-to-tail circularized AAV genomes analyzed in this study. These sites might reflect a process by which circular form genomes are generated from single-stranded panhandle or stem-loop configurations of the AAV genome. According to this model, the panhandle or stem-loop intermediate structures could originate from either flip or flop ITR orientations, and the length of the stem could vary depending on the location of the recombination/ligation event. Furthermore, as shown in Fig. 2B, the secondary structure of BB9 and CC9 segments within this stem-loop region may influence the point of recombination/ligation by as yet unknown mechanisms. Previous findings regarding AAV genome structure have suggested that circular AAV molecules might be generated from a common single-stranded panhandle circular molecule. For example, direct electron microscopic visualization of purified single-stranded AAV DNA demonstrates a predominant structure consistent with self-annealed circular molecules closed by duplex hydrogen-bonded segments (3, 8). Although the predominant form of annealed genomes appears to be circular monomers, noncovalently linked circular dimers and linearized molecules were also detected, although with a reduced frequency (9). Based on these observations, a duplex, self-annealing panhandle model has been proposed (3). Formation of panhandle/stem-loop structures has also been observed in an in vitro AAV replication system (18). These observations of monomer and dimer panhandle structures formed from single-stranded AAV genomes are consistent with the fact that both monomer and dimer covalently closed circular genomes can be rescued from cells lines and muscle tissue. Taken together, we suggest a direct intramolecular ITR recombination/ligation model for the formation of head-to-tail monomer circular intermediates (Fig. 3). In contrast to previous models, we propose that intermolecular ITR recombinational events might be responsible for head-to-tail dimer and multimer circular intermediate formation. According to this model, if circular AAV genomes function as preintegration intermediates (16), the predominant forms of integrated AAV viral DNA should be in a head-to-tail conformation. Although this model cannot exclude the possible formation of head-to-head and tail-to-tail proviral structures by in-


termolecular ITR recombination, the frequency of this type of recombination will be lower than the frequency of head-to-tail dimer genomes produced by intramolecular annealing of genomes. In support of the hypothesis, head-to-head and tail-to-tail ITR sequences are only rarely found in integrated proviruses in the absence of adenovrius (2, 5). According to our proposed model, the first step in the formation of AAV transduction circular intermediates is the creation of a single-stranded circular genome. It remains to be determined what priming mechanism(s) may be involved in second strand DNA synthesis. This could involve some novel aspects of self-priming during the recombination process (Fig. 3B) or a secondary nucleic acid primer such as tRNA. In summary, detailed chemical sequencing of AAV circular genomes has raised the possibility of new molecular pathways fundamental to the formation of circular intermediates in latent phase transduction. Furthermore, this information provides further insight regarding the transduction biology of rAAV vectors developed for gene therapy. ACKNOWLEDGMENTS This work was supported by National Institutes of Health (NIDDK/ NHLBI) Grant DK/HL51887 (J.F.E.) and a pilot grant (D.D.) of the Gene Therapy Center for Cystic Fibrosis and Other Genetic Diseases from National Institute of Health and Cystic Fibrosis Foundation (ENGELH98S0, J.F.E.).

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