JOURNAL OF VIROLOGY,0022-538X/97/$04.0010

Apr. 1997, p. 3077–3082 Vol. 71, No. 4

Copyright q 1997, American Society for Microbiology

Adeno-Associated Virus Type 2 DNA Replication In Vivo:Mutation Analyses of the D Sequence in Viral

Inverted Terminal RepeatsXU-SHAN WANG,1,2 KEYUN QING,1,2 SELVARANGAN PONNAZHAGAN,1,2

AND ARUN SRIVASTAVA1,2,3*

Division of Hematology/Oncology, Department of Medicine,1 Department of Microbiology and Immunology,3

and Walther Oncology Center,2 Indiana University School of Medicine, Indianapolis, Indiana 46202

Received 27 November 1996/Accepted 16 January 1997

The adeno-associated virus type 2 (AAV) genome contains inverted terminal repeats (ITRs) of 145 nucleo-tides. The terminal 125 nucleotides of each ITR form palindromic hairpin (HP) structures that serve asprimers for AAV DNA replication. These HP structures also play an important role in integration as well asrescue of the proviral genome from latently infected cells or from recombinant AAV plasmids. Each ITR alsocontains a stretch of 20 nucleotides, designated the D sequence, that is not involved in HP structure formation.We have recently shown that the D sequence plays a crucial role in high-efficiency rescue, selective replication,and encapsidation of the AAV genome and that a host cell protein, designated the D sequence-binding protein(D-BP), specifically interacts with this sequence (X.-S. Wang, S. Ponnazhagan, and A. Srivastava, J. Virol.70:1668–1677, 1996). We have now performed mutational analyses of the D sequences to evaluate their preciserole in viral DNA rescue, replication, and packaging. We report here that 10 nucleotides proximal to the HPstructure in each of the D sequences are necessary and sufficient to mediate high-efficiency rescue, replication,and encapsidation of the viral genome in vivo. In in vitro studies, the same 10 nucleotides were found to berequired for specific interaction with D-BP, but viral Rep protein-mediated cleavage at the functional terminalresolution site is independent of these sequences. These data suggest that AAV replication and terminalresolution functions can be uncoupled and that the lack of efficient replication of AAV DNA may not be aconsequence of impaired resolution of the viral ITRs. These studies further illustrate that the D sequence–D-BP interaction plays an important role in the AAV life cycle and indicate that it may be possible to developthe next generation of AAV vectors capable of encapsidating larger pieces of DNA.

Adeno-associated virus type 2 (AAV) is a nonpathogenichuman parvovirus which contains a single-stranded DNA ge-nome of 4,680 nucleotides (37). Optimal replication of theAAV genome requires coinfection with a helper virus such asadenovirus or herpesvirus (2–4). In the absence of a helpervirus, the wild-type (wt) AAV establishes a latent infection inwhich the viral genome integrates into host chromosomalDNA in a site-specific manner (6, 17–20, 32). When a latentlyinfected cell is superinfected with a helper virus, the proviralAAV genome undergoes rescue and proceeds through a nor-mal lytic infection (2, 24, 25). The viral genome can also berescued from recombinant plasmids containing the wt AAVgenome by transfecting plasmid DNA into adenovirus-infectedhuman cells (29, 31). Thus, recombinant plasmids serve as auseful model for studying rescue and replication of the latentproviral AAV. Two AAV sequences are required for viralDNA replication. The first is the viral rep gene, which codes forfour nonstructural proteins that are synthesized from a singleopen reading frame by the use of alternate promoters andsplicing (37), and the second is the viral origin of DNA repli-cation, which consists of a 145-nucleotide (nt) inverted termi-nal repeat (ITR) sequence (9, 21). Two of the viral Rep pro-teins (Rep78 and Rep68) are site-specific and strand-specificendonucleases that specifically bind to and cleave at the ter-

minal resolution site (trs) within the AAV ITRs (1, 14–16, 35).AAV genomes with mutations in the rep genes are defectivefor viral DNA replication (10, 27, 38). The terminal 125 nt inthe ITR form a palindrome that can fold back on itself to forma T-shaped hairpin (HP) structure, and the remainder of theITR consists of a domain, designated the D sequence, which isnot involved in HP formation. The terminal HP structure isused as a primer for initiation of viral DNA replication (2, 21,23, 26, 36). ITRs are required in cis for AAV DNA replicationas well as for rescue, or excision, from prokaryotic plasmids (8,12, 13, 29, 31, 33). Following rescue of the AAV genome fromplasmid sequences, only the viral sequences undergo selectiveDNA replication (12, 29, 44). Our recent studies have demon-strated that the D sequence plays a crucial role in the efficientrescue, selective replication, and encapsidation of the AAVgenome (39, 40).Which of the 20 nucleotides in the D sequence mediate

these diverse functions is not known. In addition, the trs is atthe junction of the D sequence and HP sequence, and when asubstitute (S) sequence was used to replace the D sequence, itwas not clear whether a functional trs site had been restored. Inorder to address these questions, we constructed partial-sub-stitution mutations in the D sequence and studied in detail theeffects of these mutations on AAV DNA rescue, replication,and encapsidation in vivo and on D sequence-binding protein(D-BP) interaction and trs functions in vitro. In this report, wepresent evidence that the proximal 10 nt in the D sequence arenecessary and sufficient for the AAV DNA rescue, replication,and encapsidation functions in vivo. However, whereas in vitroD-BP interaction studies closely mimic these functions, the

* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, 635 Barnhill Dr., Medical Science Building,Room 231-B, Indiana University School of Medicine, Indianapolis, IN46202-5120. Phone: (317) 274-2194. Fax: (317) 274-4090. E-mail: arun_srivastava@iucc.iupui.edu.

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Rep-mediated cleavage at the trs appears to be independent ofthe D sequence. Thus, an altered trs function may not beconsequential for high-efficiency rescue and selective replica-tion of the AAV genome. These studies also suggest the pos-sibility of the development of the next generation of AAVvectors capable of encapsidating larger pieces of DNA.

MATERIALS AND METHODS

Cells, viruses and plasmids. Human nasopharyngeal carcinoma cell line KBwas obtained from A. C. Antony, Indiana University School of Medicine, Indi-anapolis, Ind., and maintained as monolayer cultures in Iscove’s modified Dul-becco’s medium supplemented with 10% fetal bovine serum, penicillin, andstreptomycin as previously described (36). AAV and Ad2 virus stocks wereobtained, respectively, from K. I. Berns, Cornell University Medical College,New York, N.Y., and K. H. Fife, Indiana University School of Medicine, andwere propagated as previously described (24, 25). The recombinant AAV plas-mid pSub201 (30) was supplied by R. J. Samulski, University of North Carolina,Chapel Hill, N.C.Construction of the recombinant AAV plasmids. Standard cloning techniques

were used for constructing all recombinant plasmids (28). The construction ofplasmid pXS-36 has been described recently (39, 40). To generate D sequencemutant plasmids containing five successive nucleotide substitutions, the syntheticoligonucleotides shown in Fig. 1 were inserted between the XbaI and BalI sitesof plasmid pXS-22 (39) to generate recombinant plasmids pXS-64D5, pXS-64D10, pXS-64D15, and pXS-64D20. Plasmids pD-5, pD-10, pD-15, and pD-20were generated by ligating the blunted ClaI-PvuII insert from plasmids pXS-64D5, pXS-64D10, pXS-64D15, and pXS-64D20 between the ClaI-XbaI sites ofplasmids pXS-64D5, pXS-64D10, pXS-64D15, and pXS-64D20, respectively.AAV DNA rescue and replication assays. DNA-mediated transfections were

carried out by the DEAE-dextran procedure (29, 31) with 4 mg of each plasmidper 100-mm-diameter dish of 50% confluent KB cells. The transfection mixturealso contained 10 PFU of Ad2. At various times posttransfection, low-Mr DNAsamples were isolated by the procedure described by Hirt (11), digested exten-sively with DpnI, and analyzed on Southern blots by using 32P-labeled AAVcoding sequence-specific DNA probes.AAV-packaging assays. DNA transfections were performed by the calcium

phosphate method (28). Ad2-infected cultures were incubated at 378C in a CO2incubator for 60 h, and cells were harvested. Cell pellets were subjected to threecycles of freezing and thawing, CsCl was added to a final density of 1.4 g/cm3, andthe mixture was centrifuged in an SW41Ti rotor at 35,000 rpm for 48 h at 208C.Fractions with refractive indexes between 1.3740 and 1.3710 were pooled anddialyzed in phosphate-buffered saline, pH 7.0, followed by exhaustive digestionwith DNase I. Clarified supernatants were heated at 568C for 30 min to inactivateAd2. Equivalent amounts were analyzed on quantitative DNA slot blots with a32P-labeled AAV DNA probe as previously described (40). Human 293 cellswere infected with these culture supernatants in the presence of Ad2, and thelow-Mr DNA samples were analyzed on Southern blots as described above.Preparation of WCE. Whole-cell extracts (WCE) were prepared from HeLa

cells in accordance with the method of Muller (22). Total protein concentrationswere determined, and the extracts were frozen in liquid N2 and stored at 2808C.EMSA. Electrophoretic mobility shift assays (EMSA) were performed in ac-

cordance with the method of Carthew et al. (5). 32P-labeled oligonucleotidescontaining the sequences specific for the S sequence (59-AAGTGATATCAGATCTAATA-39), the D-5 sequence (59-AAGTGATATCAGATCTGGAG-39),the D-10 sequence (59-AAGTGATATCAGTGATGGAG-39), the D-15 se-quence (59-AAGTGCCCCTAGTGATGGAG-39), and the D-20 sequence (59-AGGAACCCCTAGTGATGGAG-39) were used as probes. DNA-binding reac-tions were performed in a volume of 20 ml with 2 mg of poly(dI-dC) and 2 mg ofbovine serum albumin. The complexes were separated from the unbound probeon low-ionic-strength 4% polyacrylamide gels with recirculating Tris-acetate-EDTA buffer (pH 7.9) containing 6.72 mM Tris-HCl, 3.3 mM sodium acetate,and 1 mM EDTA. In competition experiments, increasing concentrations ofunlabeled oligonucleotides were added to the reaction mixtures 10 min prior tothe addition of the radiolabeled S sequence, D-5, D-10, D-15, and D-20 probes;this was followed by incubation with the labeled probes and separation as de-scribed above. When D-20 was used as a probe, increasing concentrations ofunlabeled S sequence, D-5, D-10, D-15, and D-20 were added to the reactionmixtures as described above.In vitro Rep-mediated cleavage assays. Appropriate DNA substrates for Rep-

mediated cleavage assays were obtained as follows. Plasmid pSub201 was di-gested with XbaI, and, following treatment with shrimp alkaline phosphatase,DNA was digested by PvuII. Plasmids pXS-36, pXS-64D5, pXS-64D10, pXS-64D15, and pXS-64D20 were digested with BsaI and treated with shrimp alkalinephosphatase, followed by digestion with PvuII. ITR fragments were separated on6% polyacrylamide gels, excised from gels, and eluted. The 59 termini of ITRswere labeled with [g-32P]ATP (3,000 Ci/mmol) by using T4 polynucleotide ki-nase. The labeled ITRs were boiled and quickly chilled, and the 59 overhangswere repaired with the Klenow fragment. DNA fragments were separated on 6%polyacrylamide–7 M urea denaturing gels and were eluted as described above. Invitro cleavage assays were carried out in a 20-ml reaction mixture containing 25

mM HEPES–KOH (pH 7.5), 5 mM MgCl2, 1 mM dithiothreitol, 0.5 mM ATP,0.2 mg of bovine serum albumin, 1 mg of poly(dI-dC), ;1 ng of the 32P-end-labeled ITRs (105 cpm), and 10 ng of purified Rep68 protein, generously sup-plied by N. Muzyczka, University of Florida, Gainesville, Fla. The reactions wereinitiated with the addition of MgCl2, and the reaction mixtures were incubatedfor 1 h at 378C. The reactions were terminated by the addition of 50 ml of phenoland 30 ml of Tris-EDTA buffer containing 10 mg of tRNA. Following extractionwith phenol, the reaction mixture was precipitated with ethanol. The precipitateswere dissolved in 90% formamide containing 0.1% bromophenol blue and 0.1%xylene cyanol and were electrophoresed on 6% polyacrylamide–8 M urea se-quencing gels, as previously described (15, 26, 34).

RESULTS

Mutations in the D sequence affect rescue, replication, andpackaging of AAV. The recombinant AAV genomes into whichvarious mutations were introduced within the D sequence aredepicted in Fig. 1. Plasmid pXS-36, in which the D sequenceshave been replaced by a 20-nt S sequence, has been describedrecently (39). In plasmid pD-5, nt 6 to 20 in the D sequencewere replaced by 15 nt from the S sequence, and in plasmidpD-10, nt 11 to 20 in the D sequence were replaced by 10 ntfrom the S sequence. Similarly, in plasmid pD-15, nt 16 to 20in the D sequence were replaced by 5 nt from the S sequence.Plasmid pD-20 contains 20 nt of the authentic D sequence andis similar to plasmid pSub201 except that nt 146 to 190 and nt4485 to 4530 have been deleted. These plasmids were trans-fected separately into Ad2-infected KB cells, and low-Mr DNAsamples were isolated at various times posttransfection, di-gested with DpnI (45) to degrade unreplicated input plasmidDNA, and analyzed on Southern blots by using 32P-labeledprobes specific for the AAV-coding sequences. The results ofrescue and replication are shown in Fig. 2. It is evident thatrescue and replication of the AAV genome from plasmidpXS-36 (lanes 1 to 3) were significantly less than those fromplasmid pSub201 (lanes 16 to 18), an observation consistentwith our previous studies (39). It is also evident that whereasinclusion of 5 nt of the D sequence augmented rescue andreplication of the AAV genome from plasmid pD-5 (lanes 4 to6), the extent of accumulation of AAV DNA replicative inter-mediates from plasmids pD-10 (lanes 7 to 9), pD-15 (lanes 10to 12), and pD-20 (lanes 13 to 15) was nearly the same as thatfrom plasmid pSub201. These results suggest that 10 nt of theD sequence proximal to the HP structures are necessary and

FIG. 1. Schematic representation of AAV genomes containing sequentialsubstitutions of the authentic D sequence (hatched boxes, boldface type) with theS sequence (open boxes, italics). Five nucleotides were replaced successively ineach of the recombinant plasmids.

3078 WANG ET AL. J. VIROL.

sufficient for optimal rescue and replication of the AAV ge-nome. These results also indicate that nt 46 to 191 present inplasmid pSub201 are not necessary for efficient replication ofthe AAV genome.Since we have recently documented that the D sequence

serves as a packaging signal for AAV (40), we wished to ex-amine whether the proximal 10 nt in the D sequence were alsosufficient for efficient encapsidation of the viral genomes inprogeny virions. Various recombinant plasmids were trans-fected into Ad2-infected 293 cells by the calcium phosphateprocedure, and progeny virions were purified on CsCl equilib-rium density gradients. Following digestion with DNase I todegrade any unencapsidated DNA, equivalent amounts of vi-rus stocks were deproteinized to release the AAV DNA andwere analyzed on quantitative DNA slot blots by using a 32P-labeled AAV DNA probe as previously described (40). Such ablot is presented in Fig. 3. It can be seen that viral genomesrescued from plasmid pXS-36 failed to undergo encapsidationinto viral particles (vXS-36) and that inclusion of the first 5 ntof the D sequence in plasmid pD-5 partially restored encapsi-dation of viral genomes in progeny virions (vD-5). Strong hy-bridization signals were detected with progeny virions vD-10,vD-15, and vD-20 produced, respectively, from plasmids pD-10, pD-15, and pD-20. These signals were comparable to thatdetected with vSub201 generated from plasmid pSub201.These results further suggest that the first 10 nt in the Dsequence are necessary and sufficient for optimal packaging ofthe AAV genome.We next examined whether, following encapsidation, the

progeny AAV were infectious. Human 293 cells were infectedat a multiplicity of infection of 1 in the presence of Ad2.Because of inefficient rescue, replication, and encapsidation ofthe AAV genome from plasmids pXS-36 and pD-5, approxi-mately 100-fold more vXS-36 and approximately 60-fold morevD-5 virus stocks were used in these experiments. Equivalentamounts of low-Mr DNA isolated 48 h postinfection were an-alyzed on Southern blots with a 32P-labeled AAV DNA probe.

These results are shown in Fig. 4. It is evident that the extentof replication of virions produced from plasmids pD-10, pD-15,and pD-20 was roughly the same as that from plasmid pSub201since monomeric and dimeric viral DNA replicative interme-diates could be readily observed. However, vD-5 viral DNAreplication efficiency was significantly less under identical con-ditions. These data provide strong support for the conclusionthat the first 10 nt of the D sequence are crucial for efficientrescue and replication of the AAV genome.

FIG. 2. Southern blot analysis of rescue and replication of AAV genomescontaining D sequence substitutions. Each of the D sequence-mutated plasmids,along with plasmids pSub201 and pXS-36 as appropriate controls, was trans-fected into Ad2-infected KB cells, and low-Mr DNA isolated at 24 (lanes 1, 4, 7,10, 13, and 16), 48 (lanes 2, 5, 8, 11, 14, and 17), and 72 (lanes 3, 6, 9, 12, 15, and18) h postinfection was digested with DpnI and analyzed on Southern blots witha 32P-labeled AAV DNA probe as described under Materials and Methods.d and m, dimeric and monomeric viral replicative DNA intermediates, respec-tively.

FIG. 3. DNA slot blot analysis for encapsidation of AAV genomes contain-ing various substitutions in the D sequence. Twofold serial dilutions of equivalentamounts of viral stocks were analyzed with a 32P-labeled AAV DNA probe asdescribed in Materials and Methods.

FIG. 4. Southern blot analysis of replication of progeny AAV containing Dsequence substitutions. Equivalent amounts of each of the virus stocks were usedto infect Ad2-infected human 293 cells, and replication assays were performed asdescribed in the legend for Fig. 2. Mock-infected or Ad2-infected cells were usedas appropriate controls. d and m, dimeric and monomeric viral replicative DNAintermediates, respectively; ss, single-stranded progeny viral DNA strands.

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Replication of the D sequence mutants in vivo correlateswith interactions with D-BP in vitro. We have recently dem-onstrated that cellular protein(s) D-BP interacts specificallywith the D sequence (40). Since the D sequence mutationanalysis indicated that the first 10 nt of the D sequence wereresponsible for the D sequence functions, we wished to exam-ine whether these mutations also correlated with D-BP inter-actions. WCE were prepared from HeLa cells, were incubatedwith a 32P-labeled D-20 oligonucleotide probe in the absenceor presence of unlabeled D-5, D-10, D-15, D-20, and S se-quence-specific synthetic oligonucleotides, and were analyzedby EMSA as previously described (1, 40). The results are pre-sented in Fig. 5. It is apparent that the D-20 probe formed aspecific complex with D-BP and that complex formation couldbe competitively inhibited by incubations with 10-, 50-, and200-fold molar excesses of unlabeled D-10 (lanes 7 to 9), D-15

(lanes 10 to 12), and D-20 (lanes 13 to 15) oligonucleotides butnot by incubations with the S sequence (lanes 1 to 3) or the D-5(lanes 4 to 6) oligonucleotides. These data suggest that the first10 nt in the D sequence play an important role in the interac-tion with D-BP.These results were further corroborated by performing

EMSA with 32P-labeled D-5, D-10, D-15, and D-20 oligonu-cleotides under self- and cross-competition conditions. Theresults are shown in Fig. 6. It is evident that whereas D-10,D-15, and D-20 oligonucleotides formed complexes withD-BP, complex formation with the D-5 oligonucleotide wasvery inefficient (panel A). Furthermore, complex formationwith D-10, D-15, and D-20 oligonucleotides was specific sincethe binding could be effectively competed with 200-fold molarexcess amounts of unlabeled D-10 (panel D), D-15 (panel E),and D-20 (panel F) but not with the S sequence (panel B) orthe D-5 oligonucleotide (panel C) under identical conditions.These results establish that the first 10 nt in the D sequence areindispensable for specific interaction with D-BP.The trs function is not affected by mutations in the D se-

quence. Because terminal resolution is a key step during AAVDNA replication and because it remained possible that theobserved lack of optimal rescue and replication of the AAVgenome from plasmid pD-5 was due to impaired trs function,we next examined whether mutations in the D sequence af-fected Rep-mediated cleavage at the trs. HP structure DNAsubstrates were synthesized from each of the recombinant plas-mids, were radiolabeled at the 59 ends with 32P, and wereincubated in an in vitro reaction with and without purifiedRep68 protein as previously described (15, 34). The data areshown in Fig. 7. Plasmid pSub201 was used as a positive con-trol, and as can be seen, DNA substrate prepared from thisplasmid could be effectively cleaved by Rep68 to generate aDNA product with a predicted size of 73 nt (lane 12). Inter-estingly, Rep68-mediated cleavage also occurred with DNAsubstrates prepared from plasmids pD-5 (lane 8), pD-10 (lane9), pD-15 (lane 10), and pD-20 (lane 11), and the efficiencies ofthese cleavages were not significantly different among differentD sequence mutations, nor were they significantly differentfrom that with the wt substrate. Furthermore, DNA substrateprepared from plasmid pXS-36, in which the D sequence hasbeen replaced by the S sequence, could also be cleaved byRep68, albeit less efficiently (lane 7). No cleavage occurred in

FIG. 5. EMSA with the D-20 oligonucleotide. Radiolabeled D-20 probe wasincubated with WCE in the presence of up to a 200-fold molar excess of theunlabeled S sequence, D-5, D-10, D-15, and D-20 as competitor oligonucleo-tides. The assays were carried out as described in Materials and Methods. Theupper arrow indicates D-BP.

FIG. 6. EMSA with the D sequence-substituted oligonucleotides. The D-BP reaction with each individual radiolabeled oligonucleotide was carried out either in theabsence of competitor oligonucleotides (panel A) or in the presence of a 200-fold molar excess of unlabeled S sequence (panel B), D-5 (panel C), D-10 (panel D), D-15(panel E), and D-20 (panel F) oligonucleotides as competitors. The assays were carried out as described in the legend for Fig. 5. The upper arrow indicates D-BP.

3080 WANG ET AL. J. VIROL.

the absence of the Rep68 protein (lanes 1 to 6). These data,though not quantitative, suggest that the lack of optimal rep-lication of the AAV genome may not be a consequence ofimpaired cleavage at the trs.

DISCUSSION

Recent studies from our laboratory have suggested that theD sequence in AAV ITRs is crucial for efficient rescue, selec-tive replication, and successful encapsidation of the AAV ge-nome (39, 40). In the present study, when 10 nt in the distalhalf of the D sequence were removed, the AAV genome couldstill undergo efficient rescue, replication, and encapsidation.However, when this deletion was extended to 15 nt in the Dsequence, the efficiency of AAV genome rescue, replication,and packaging was severely compromised. Thus, it would ap-pear that only 10 nt in the D sequence proximal to the HPstructures are sufficient to mediate these functions. Why mustthen the AAV genome carry 10 extra nt in each of its ITRs?Although we did not carry out DNA footprint analyses, on thebasis of the data shown in Fig. 6, it would seem reasonable topropose that the proximal 10 nt in the D sequence are requiredfor initiating contact with D-BP and that the remainder of thenucleotides might be involved in enhancing this interactionsince there appeared to be a progressive increase in the effi-ciency of D-BP binding with D-10, D-15, and D-20 probes,respectively. These studies, nonetheless, add to the current

understanding of the requirements for an optimally functionaltrs.Correlation between rescue and replication functions. Ter-

minal resolution is a key step during AAV DNA replication.Several studies have suggested that rescue without replicationor replication without rescue can occur (8, 12, 13, 25, 41, 44).However, it has been difficult to uncouple these two steps inthe virus life cycle. Previous studies have shown that both thesecondary structure element of the ITR and a specific se-quence at the trs are required for Rep-mediated cleavage at thetrs and that 5 to 6 nt at the trs are required for Rep recognition(34). However, since the trs is located between the A sequenceand the D sequence, i.e., between nt 124 and 125 in the ITR,it was conceivable that mutations in the D sequence led todestruction of the functional trs, resulting in impaired rescueand replication of the AAV genome. Interestingly, however,the experimental data did not appear to support this possibil-ity. For example, Rep-mediated cleavage assays in vitro docu-mented that none of the mutations significantly affected Repprotein recognition followed by cleavage at the trs, regardlessof the number of authentic nucleotides of the D sequence. Infact, Rep-mediated cleavage also occurred in the completeabsence of the D sequence, suggesting that, at least in vitro,these two steps can be uncoupled and that the lack of optimalreplication of the AAV genome is not a consequence of im-paired cleavage at the trs. However, it remains possible that theefficiency of Rep-mediated cleavage at the trs is enhanced byD-BP in vivo (39, 40). These data are, nonetheless, consistentwith previous studies in which the absence of the D sequencedid not prevent the initiation of AAV DNA replication in vitro(43) and in vivo (39, 40).Implications of the D sequence mutations in the develop-

ment of AAV vectors for gene therapy. AAV has gained atten-tion as a potentially useful vector for human gene therapy,primarily because of its nonpathogenic nature and its broadhost range (23). However, one of the limitations of the cur-rently available vectors is the size of DNA molecules that canbe successfully packaged at high efficiency. For example, the wtAAV DNA is 4.68 kb long, but the foreign DNA insert size islimited to about 4.3 kb since ITRs in pSub201-based vectorscontain 191 nt each (;0.4 kb). Consequently, some of thelarger inserts, such as the cystic fibrosis transmembrane con-ductance regulator cDNA, have been inserted into AAV vec-tors without authentic promoter sequences, and although low-level expression has been obtained (7), it would appear that theinclusion of a strong promoter element upstream of the trans-duced gene would be desirable. In pD-10-based vectors, on theother hand, we have engineered an EcoRV site in each ITR,and as a result, the ITRs contain only 138 nt. Thus, thesevectors should allow an increase in packaging capacity of atleast 106 nt. Indeed, it has been possible to include additionalpromoter sequences upstream of large cDNAs, such as themultidrug resistance-1 (MDR-1) and the retinoblastoma sus-ceptibility-1 (RB-1) cDNAs, in the next generation of AAVvectors (42, 46).

ACKNOWLEDGMENTS

We are grateful to Nicholas Muzyczka for his kind gift of the puri-fied Rep68 protein as well as for helpful suggestions. We also thankKenneth I. Berns for supplying the AAV stock and Richard J. Samul-ski for providing pSub201 plasmid.This research was supported in part by Public Health Service grants

(HL-48342, HL-53586, and DK-49218, Centers of Excellence in Mo-lecular Hematology) from the National Institutes of Health and by agrant from the Phi Beta Psi sorority. A.S. was supported by an Estab-lished Investigator Award from the American Heart Association.

FIG. 7. Rep68-mediated cleavage at the trs in AAV ITRs containing varioussubstitutions in the D sequences. HP DNA substrates prepared from each indi-cated plasmid were radiolabeled at the 59 ends and were incubated in the absence(lanes 1 to 6) or the presence (lanes 7 to 12) of purified Rep68 protein in in vitrocleavage assays as described in Materials and Methods. The predicted sizes ofDNA cleavage products from pSub201 (solid arrowhead), pD-5 through pD-20(arrow), and pXS-36 (open arrowhead) are indicated.

VOL. 71, 1997 AAV DNA RESCUE AND REPLICATION 3081

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