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Adeno-associated virus: integration at a specific chromosomal locus
Adeno-associated virus: integration at a specific chromosomal locus Richard J. Samulski U n i v e r s i t y of Pittsburgh, Pittsburgh, Pennsylvania, USA Recent characterization of integration of the human parvovirus, adenoassociated virus, has uncovered the exciting occurrence of targeted integration. Thus far, such specific integration has been found to be unique among the eukaryotic viruses. The molecular details of the steps involved in virus integration are actively being pursued and should yield significant information for our understanding of the mechanisms of DNA transposition. Current Opinion in Genetics and Development 1993, 3:74-80
Introduction One of the most intriguing aspects of the adenoassociated virus (AA.V) life cycle is the abilit3, of the virus to integrate into the host genome in the absence of a helper virus. The integration step ensures that the genetic continuity of the virus is preserved until the host is exposed to appropriate conditions (e.g. lytic helper virus), whereupon the AAV genome is rescued and re-enters the lyric cycle (Fig. 1). Virus integration appears to have no apparent effect on cell growth or morphology [1]. In spite of its p r o p e n s w to integrate into the cellular genome as a rescuable provirus, there is no evidence of AAV functioning as a tumor vires. This host-viral interaction appears to be similar to symbiotic relationships often seen in the biological world, although here it exists at the molecular level. While much has been learned about AAV replication (see [2] and Fig. 2), relatively little is kalown about the events involved in virus integration. For these reasons, a number of laboratories have made a major effort to delineate the molecular steps involved in integration. Besides the opportunity to characterize this intriguing phenomenon, recent developments have suggested a potential use of this virus as a vector for gene delivery (see [2,3] for reviews). The incorporation of site-specific integration into AAV-vector schemes would make the system extremely attractive for human gene-therapy approaches.
The AAV life cycle AAV is the only known DNA animal virus that requires coinfection by a second unrelated virus in order to undergo productive infection. For this reason, AAV is classifted as a defective parvovirus. The DNA tumor viruses,
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Ad Latency [ ~
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Fig. 1. Schematic representation of the adeno-associated virus (AAV) life cycle, which is biphasic. The lytic phase requires the presence of a helper virus, for example, adenovirus (Ad), which results in complete replication of both AAV and the helper (lytic infection). During the alternative latent phase the AAV genome is integrated into the host's chromosome in the absence of the helper (latency). The latent virus genome is stable for many generations and can be 'rescued' to enter the lytic phase upon helpervirus superinfection.
adenovirus (Ad) or herpes simplex x4rus, provide the necessary helper flmctions required for e~cient AAV replication in vitro (Fig. 1). Although AAV is a human vires, its host range for lyric growth is unusually broad. Virtually every mammalian cell line evaluated (including a variety of human, simian, canine, bovine and rodent cell lines) can be productively infected with AA.V, provided that an appropriate helper virus is used (e.g. canine Ad in canine cells) [4]. Despite the wide range of susceptible cell types, no disease has been associated with AAV in either human or animal populations [5], even though
Abbreviations AAV--adeno-associated virus; Ad--adenovirus; HSV--herpes simplex virus; TR--terminal repeat; trs--terminal resolution site. 74
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Adeno-associated virus Samulski 75 exposure is commonplace. Anti-AAVantibodies have frequently been found in humans and monkeys. Estimates suggest that about 70--80% of infants acquire antibodies to AAV types 1, 2 and 3 within the first decade, and more than 50% of adults have been found to maintain detectable anti-AAV antibodies. AAV has been isolated from fecal, ocular and respiratory specimens during acute adenovirus infections, but not during other illnesses [6]. While AAV can grow in cultured cells utilizing any helper virus, no clinical isolates of herpes carrying wild-type AAV have been described. All these results point to an integral relationship between AAV, the host, and the helper virus.
In vivo integration of AAV AAVwas first documented as cryptic infections in primary African green monkey and human embryonic kidney cells in 1970 [5,7]. In these studies, 20% of the monkey and 2% of the human cells tested scored positive for the appearance of AAV antigens after Ad challenge [8]. This was the first indication of virus latency for this parvovirus in human and non-human primates. These results suggest that AAV infection in vivo is common. It is interesting to note that the high frequency of AAV in non-human primates is observed only in animals kept in captivity (RW Atchison, personal communication). While AAV transmission in humans is believed to occur horizontally, one study has documented the vertical germline transmission of avian AAV in chickens [9]. Since these early studies, relatively little information has accrued on the frequency and tissue tropism of AAV latent infection in man. However, with the advent of PCR technology and the potential relevance of AAV as a vector for human gene therapy, information concerning these important questions should be forthcoming. The first of such studies has been reported by Grossman et aL [10]. Using PCR analysis, they reported the detection of AAV-2 sequences in 2/55 healthy blood donors, and 2/16 hemophilic patients. While the sample size used in this study was too small to draw many conclusions, this work points to the type of information that will accumulate rapidly concerning AAV latency in vivo. A similar PCR analysis of airway epithelial cells from 36 cystic fibrosis and normal patient biopsies scored negative for AAV integration (RJ Samulski and M Welch, unpublished data). While the obvious interpretation is a lack of AAV latency in this tissue, a negative PCR result may be the result of other factors such as the turn-over rate of epithelial cells in the lung, or the relatively small portion of the organ that was subject to analysis. It is obvious that while positive results using PCR analysis will be informative, negative results remain nondefinitive and require follow-up experiments.
In vitro integration of AAV After AAV latent infection in human primary kidney cells was described [8], investigators focused on the question
of AAV latency in vitro. A human tissue-culture line, Detroit 6 (7374), was used to demonstrate the first in vitro AAV latent infection in 1972 [8]. In these experiments, infection at high multiplicity of AAV (250 tissue-culture infectious doses per cell) in the absence of helper virus resulted in 30% of the clones scoring positive for AAV after 39 passages. Further characterization of these latent cell lines demonstrated that the AAV genome existed as an integrated provirus [13,14]. This was the first evidence that AAV latency involved viral integration. Since this ame, some 80-100 independently-isolated proviral lines have been established and characterized in various cell types, including non-human primates [5,10,11-16,17-]. In fact, to date, no cell line has been isolated that is refractive to AAV integration. These observations suggest a very common receptor or mechanism of virus uptake (yet to be described), and also address the conservation of the integration mechanism in various cell types. In general, latenfly infected cells established in vitro appear to be stable, being able to maintain the viral DNA for greater than 150 200 passages [5,18"]. Overall, virus integration appears to have no apparent effect on cell growth or morphology [1]. A more recent study on AAV infection in either Epstein-Barr virus transformed B-cell line or an untransformed CD4 + T cell from a healthy donor demonstrated that many of the functional capabilities and growth properties of the T cells were not altered by virus integration. Parameters tested included DNA ploidy, lectin-dependent cytotoxic T-cell activity, anti-CD3 driven proliferation, and the analysis of surface-antigen expression using flow cytometry for CD2, CD5, CD7 and class 11 major histocompatibility products [17"]. While continued characterization of AAV latency in various cell lines is providing more information about the viral-host interaction [19,20], the definitive role of viral integration will require in vivo studies.
Proviral structure To date, no complete AAV provimses have been isolated, and therefore critical information on viral as well as cellular sequences is still lacking [18o,21]. The failure to isolate an intact AAV provirus is in part because of the recombinogenic potential of these sequences in traditional cloning vehicles. However, many studies (genomic blots) on the physical structure of integrated AAV genomes suggest that viral insertions are usually in a tandem head-to-tail orientation mediated by the AAV terminal repeats (TRs) [5,11,13-15]. Concatemers consisting of two to four tandem copies exist at the integration locus regardless of the initial multiplicity of infection [13,15]. These tandems appear to have at least one or two copies of the terminal-repeat sequences between two adjacent genomes [13,14]. The generation of low-copy tandem arrays using various multiplicities of infection suggest that these structures are not the result of end-to-end joining of input viral templates but rather the result of replicating a single AAV genome. Several immediate questions come to mind with regard to a pos-
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Viral genetics: e
sible mechanism involved in AAV integration. First, the infecting AAV genome is a unit-length, tsingle-stranded molecule with hairpin terminal repeats (Fig. 2), which must at some point convert'to duplex DNA. Whether this step is a prerequisite for integration, or occurs afterwards, remains to be seen. Second, the current model fcrr.AAV DNA replication predicts the formation of headto-head or tail-to-tail tandems, the exact opposite from that characterized for the integrated provirus. Third, productive AAV replication is completely dependent on the presence of the qiral rep genes, sequenees typically absence from AAV transducing vectors [13,16,22]. It is in.teresting to note that in one cell line where fragments of the" provirus have been isolated, some of the viral sequences exist in a tail-to-tail tandem arrangement [21]. It is elear that until a complete provirus is isolated and sequenced, ~at best we can only guess about the.mechanism for AAV integration. Data generated by restriction-enzyme digestion and Southern hybridization analysis of independently derived latent cell lines have revealed that the viral--cellular junction fragments migrate with different mobilities, suggesting that AAV integration occurs randomly [13-15]. These results, although interpreted correctly, ~ere misleading as a result of the deletion and substitution of sequences around the target site. Concurrent with these results, the fact that free AAV DNA can be found in some latent cell lines, suggests that the AAV sequences are in a dynamic state of flux in the latent (a)
3" A'C'CB'BA Flop
Targeting integration AAV-cellular junction probes used against a panel of independently derived AAV latent cell lines scored 68% positive for co-migration between AAVfragments and the junction sequences [23]. Using somatic cell hybrid mapping, and in situ hybridization, this flanking sequence was localized to the long arm (q) of human chromosome 19 [18°,24°]. These data represent the first evidence to support the suggestion that AAV integration prefers a target sequence present only on chromosome 19. The location of the other AAV proviruses (30%) has not been defined, but once obtained should add useful information concerning AAV targeting. A recent report characterizing three AAV latent cell lines suggested virus integration in chromosome 17 [20]. While these integrants may represent other target sites, the flanking sequences have not been isolated or mapped, a prerequisite for determining specificity. "Results supporting use of target sites
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form. This scenario adds another level of complexity to the characterization of AAV integrants. Only after analysis of latent cell lines using cellular flanking sequences was evidence gained that supports AAV specificity in integration [18°,23].
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Fig. 2. AAV replication scheme. (a) The single-stranded AAV genome has inverted terminal repeats, with the terms flop and flip designating the two potential arrangements of their DNA sequence. (b) These sequences can form a secondary T-shaped structure that is used as a self-primer (here shown in flop orientation) for DNA replication. (c) A specific parental-progeny (solid and broken lines, respectively) replicativeform (RF) intermediate is generated after the first round of replication. A virusspecific nicking activity that acts at the terminal resolution site (trs) is required for resolution of the RF structure. (d) This results in transfer of the parental hairpin sequences (solid line, ABB'CC'A'). (e) This duplex intermediate can then re-initiate DNA replication using the self-priming mechanism (T-structure shown with a broken line). (f) Finally, replication proceeds through strand displacement, generating a single-stranded hairpin molecule and a RF intermediate, each of which can re-enter the replication pathway.
Adeno-associated virus Samulski not identical to those characterized on chromosome 19 have been documented in diploid cells by A Srivastava a n d colleagues (personal communication). Utilizing a
protein-DNA-binding enrichment technique to isolate AAV proviral DNA from human cell lines, Samulski et aZ [18-] isolated and confirmed the initial results of Berns and his colleagues [23]. In the study, multiple latent cell lines were characterized using Southern-blot analysis, DNA sequencing, and in atu hybridization of latent chromosomes. Results from the analysis demonstrated viral targeting to within a 100 bp sequence on chromosome 19 [18-]. Even latent clones that by Southern analysis appeared as multiple copies of complex arrangement, when assayed by in situ, scored positive for integration at one locus' on chromosome 19. Sequence information from a cosmid pre-integration site has enabled PCR-based amplification for further characterization of viral-host junction sequences. These studies scored positive for targeted integration in latent aneuploid HeLa cell lines as well as latent diploid W138 [18 °] and have been extended to human colon, T cells, and monkey kidney cells (X Zhu, X Xiao and RJ Samulski, unpublished observations). In agreement with other studies [21], analysis of these viral-cellular sequences demonstrated patchy homology and rearranged sequences at the viral--cellular crossover points [18"]. Although these rearranged clones, isolated by PCR amplification, carried various amounts of the AAV terminal-repeat (TR) sequence, examples of both the flip and flop orientation (see Fig. 2) were isolated. This suggests that orientation of the TR sequence is not essential for virus targeting. To date, a complete viral TR-cellular junction has not been isolated. The inability to isolate an intact AAV TR-cellular junction may in part be because of the secondary structure of the AAV terminal sequences. The 145 bp TR of AAV can form a 125 bp palindromic T-shaped structure (see Fig. 3), which appears to be unstable in bacterial cells [25-28]. This instability is not unique to AAV but has also been observed with TRs from the autonomous parvoviruses MVM and JNC, and the pathogenic human parvovirus B19 [29-31]. We have modified a PCR amplification protocol to yield viral-junction sequences that pre-
serve the AAV terminal nucleotides (X Zhu and RJ Samul-
ski, unpublished data). Sequence analysis of virion DNA has demonstrated heterogeneity at the ends of the AAV TRs (at the 5' TR: 15% TTG; 50% TG; and 30% G). Junctions characterized by this procedure have so far only resulted in crossover points at 5' nucleotides TG and G. As described for other junctions, patchy homology of 3-5 bp was observed. The two break points for these junctions were separated by 30 bp (X Zhu and RJ Samulski, unpublished data). It is interesting to note that some of these AAV terminal nucleotides are similar to those of retroviruses (5' TG-CA 3' ).
Cis and trans sequences required for integration
To better define the target integration sequences, studies have been initiated to characterize the cellular preintegration site. Limited sequence information from the target site showed a 21 bp direct repeat flanking the viral-cellular break points [18.]. A 37bp element, repeated 10 times within 500 bp of the viral-cellular junction, has been identified [24.]. This repeat, a minisatellite sequence, was originally found in the third intron of the apolipoprotein C-U gene [32] and is uniquely located on 19q. Hybridization studies using the target sequence demonstrate conservation between humans and primates [18"]. Continued analysis of this region should further illuminate both the integration mechanism and the role AAV may play in the host cell. This host sequence may also facilitate the establishment of an in vitro integration system for the virus. It has been well documented that AAVvectors containing only the viral TR sequences integrate at high frequency [13,14,22,33-36]. in fact, in the absence of viral coding sequences, transduction of the AAV vector is even more efficient than that of wild-type virus (70% versus 30%) [13,14]. As the provirai structure of these recombinants are similar to wild type (low copy tandems and
T T..T co.G G..C G..C C' G..C C..G C..G C..G A G..C C. • G G C C T C A G T G A G C G A G C G A G C G C G C A G A G A G G G A G T G G C C A A A " ~ ~" : ' ' ' ' ' ' ' " : :'''''" :" ~'''.'''''''''''.'''." G. C C G G A G T C A C T C G C T C G C T C G C G C G T C T C T C C C T C A C C G G T T G C G G T C G T G C T C C C C A A G G A C. G
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secondary structure when presented as a single-stranded molecule. Two small palindromes, B-B' and C-C' are ernSedded in a larger palindrome, A-A'.
77
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Viral genetics
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Fig. 4. AAV rescue model. (a) Dimer AAV DNA molecule before integration. The typical terminal repeat CIR) of AAV is 145 bp with one terminal resolution sequence (black-grey boundary). A key feature in the dimer is a unique 165 bp TR shared by two AAV genomes that contains flanking 20 bp viral D sequences (black shading; nucleotides shown in Fig. 2). The presence of the flanking viral D sequences provides two terminal resolution sites in the unique TR. (b) Targeted integration of this substrate into the host chromosome with imprecise joining of the external viral TR sequences to cellular DNA. (c) Conservation of the AAV nicking sites (arrowed) allows rep-mediated nicking after helper virus induction. (d) Bidirectional replication (open arrows) from the internal unique TR (dotted here) displaces single-stranded AAV molecules with 145 bp TRs {hairpin structures). (e) Sequence homology between the D regions of the viral TRs forms a panhandleshaped replicative intermediate. (f} Gene conversion of the terminal sequences results in a full-length, single-stranded AAV molecule that can enter the AAV replication scheme shown in Fig. 2.
rescuable), it suggests that the AAV termini are efficiently recognized for limited amplification and integration. In preliminary studies, AAV vectors that contain only the vi ral TRs have been analyzed for targeted integration (X Zhu and RJ Sanmlski, unpublished data). These viruses,
while proficient for integration and subsequent rescue, do not target to 19q at high frequencT and (X Zhu, X Xiao and RJ Samulski, unpublished data) [37]. Efficient targeting appears to be lost once the AAV coding sequences are removed, suggesting that AAV trana:acting
Adeno-associated virus Samulski factors are required for site-specific integration. However, as AAV vectors containing only the TR can target to 19q, albeit at a low frequency, protein components of the virion may participate in viral targeting (X Xiao and RJ Samulski, unpublished data). AAV proteins that may be potential candidates for a role in virus integration are the AAV rep 68 and 78 kD proteins. These proteins, which are essential for viral-DNA replication in vivo, have been biochemically characterized in vitro (RO Snyder et aL, unpublished data) [38-42]. They interact with AAV terminal sequences only if the termini exist in the T-shaped secondary conformation [40,43], a conformation that is believed to exist in virion DNA. Both rep 68 and 78 contain ATP-dependent site-specific and strand-specific endonuclease acdvity that recognizes a sequence in the AAV TR that is referred to as the terminal resolution site (trs) [38,42]. A DNA helicase activity is also associated with these viral proteins [41]. These enzymatic activities are required in the first steps of AAV DNA replication, and may also be involved in virus integration.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest e• HAND^ H, SHIROKIK, SHIMOJOH: Establishment and Characterization of KB Cell Lines Latendy Infected with Adenoassociated Virus Type 1. Virology 1977, 82:84--88. MUZYC.ZKA N (ED): Use of Adeno-as$ociated Virus as a General Transduction Vector for Mammalian Cells. In Current Topics in Microbiology and Immunology, vol 158. Berlin/Heidelberg: Springer-Verlag; 1992:97-129. CARTER BJ: Adeno-associated Virus Vectors. Curr Opin Biotechnol 1992, 3:533-539. CUKOR G, BLACKLOWNR, HOGGAN D, BERNS KI: Biology of Adeno-Associated Virus. In The Parvovirusex Edited by Berns KI. New York: Plenum Press; 1984:33--66. BERNS KI, CHEUNGA" OSTROVEJ, LEWISM: Adeno-associated Virus Latent Infection. In Virus Persistence. Edited by Mahy BWJ, Minson AC, Darby GK. Cambridge: Cambridge University Press; 1982:249. DULBECCO R, GINSBERG HS: Virology. In Microbiology, 2nd edn. Edited by Davis BD et al. Hagerstown: Harper & Row; 1973:1236.
Conclusion and perspective Identification of AAV targeting into the human genome is an important milestone in the study of AAV integration. While current data suggest that AAV integration occurs through a deletion substitution mechanism, a primary objective in viral latency is to ensure the ability to rescue from chromosomal DNA and re-enter the replication cycle after helper virus superinfection. Therefore, at least one complete viral genome, or sequences sufficient to generate a complete AAV DNA molecule, must exist in the proviral state. A model for AAV integration and rescue that has been constructed in the light of available data is presented in Fig. 4. A key element in this model is the presence of a 160bp TR shared between two AAV molecules. This intermediate has been shown to be obligatory in the current AAV replication scheme (Fig. 2). PCR analysis of the TR sequences between tandem proviral molecules have shown that they contain this specific TR sequence (X Zhu and RJ Samulski, unpublished data; Zolotokhin and N Muzyczka, personal communication). A synthetic TR retaining these sequences is sufficient for virus rescue, replication, packaging and integration (X Xiao and RJ Sarnulski, unpublished data). The formation of a concatemer may be a mechanism for Ensuring the integrity of at least one copy of the AAV TR and a method for the rescue and replication of the viral genome. A combination of genetic and biochemical studies is now required to fully dissect the protein-DNA interactions involved in this process of recombination.
HOGGAN MD: Adeno-associated Viruses. Prog Med Viro11970, 12:211-239. HOGGAN MD, THOMAS GF, THOMAS FB, JOHNSON FB: Continuous Carriage of Adenovirus Associated Virus Genome in Cell Cultures in the Absence of Helper Adenoviruses.
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Acknowledgements Acknowledgements are especially in order for those people who shared their ideas and information freely with me; these include B Carter, N Muzyczka, A Srivastava, X Xiao and X Zhu. I aJso thank C Berliner for editorial and graphics work.
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•
25.
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LAUGHLINCA, TRATSCHINJ-D, COON H, CARTER BJ: Cloning of Infectious Adeno-associated Virus Genomes in Bacterial Plasmids. Gene 1983, 23:65-73.
29.
MERCHLINSKYMJ, TATTE~Ala. PJ, LEARY JJ, COTMORE SF, GARDINER EM, WARD IX:: Construction of an Infectious Molecular Clone of the Autonomous Parvovirus Minute Virus of Mice. J Virol 1983, 47:227-232.
RJ Samulski, 269 Crawford Hall, Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Introduction One of the most intriguing aspects of the adenoassociated virus (AA.V) life cycle is the abilit3, of the virus to integrate into the host genome in the absence of a helper virus. The integration step ensures that the genetic continuity of the virus is preserved until the host is exposed to appropriate conditions (e.g. lytic helper virus), whereupon the AAV genome is rescued and re-enters the lyric cycle (Fig. 1). Virus integration appears to have no apparent effect on cell growth or morphology [1]. In spite of its p r o p e n s w to integrate into the cellular genome as a rescuable provirus, there is no evidence of AAV functioning as a tumor vires. This host-viral interaction appears to be similar to symbiotic relationships often seen in the biological world, although here it exists at the molecular level. While much has been learned about AAV replication (see [2] and Fig. 2), relatively little is kalown about the events involved in virus integration. For these reasons, a number of laboratories have made a major effort to delineate the molecular steps involved in integration. Besides the opportunity to characterize this intriguing phenomenon, recent developments have suggested a potential use of this virus as a vector for gene delivery (see [2,3] for reviews). The incorporation of site-specific integration into AAV-vector schemes would make the system extremely attractive for human gene-therapy approaches.
The AAV life cycle AAV is the only known DNA animal virus that requires coinfection by a second unrelated virus in order to undergo productive infection. For this reason, AAV is classifted as a defective parvovirus. The DNA tumor viruses,
© Ad
Mv
Ad Latency [ ~
Lytic infecion
Fig. 1. Schematic representation of the adeno-associated virus (AAV) life cycle, which is biphasic. The lytic phase requires the presence of a helper virus, for example, adenovirus (Ad), which results in complete replication of both AAV and the helper (lytic infection). During the alternative latent phase the AAV genome is integrated into the host's chromosome in the absence of the helper (latency). The latent virus genome is stable for many generations and can be 'rescued' to enter the lytic phase upon helpervirus superinfection.
adenovirus (Ad) or herpes simplex x4rus, provide the necessary helper flmctions required for e~cient AAV replication in vitro (Fig. 1). Although AAV is a human vires, its host range for lyric growth is unusually broad. Virtually every mammalian cell line evaluated (including a variety of human, simian, canine, bovine and rodent cell lines) can be productively infected with AA.V, provided that an appropriate helper virus is used (e.g. canine Ad in canine cells) [4]. Despite the wide range of susceptible cell types, no disease has been associated with AAV in either human or animal populations [5], even though
Abbreviations AAV--adeno-associated virus; Ad--adenovirus; HSV--herpes simplex virus; TR--terminal repeat; trs--terminal resolution site. 74
(~) Current Biology Ltd ISSN 0959-437X
Adeno-associated virus Samulski 75 exposure is commonplace. Anti-AAVantibodies have frequently been found in humans and monkeys. Estimates suggest that about 70--80% of infants acquire antibodies to AAV types 1, 2 and 3 within the first decade, and more than 50% of adults have been found to maintain detectable anti-AAV antibodies. AAV has been isolated from fecal, ocular and respiratory specimens during acute adenovirus infections, but not during other illnesses [6]. While AAV can grow in cultured cells utilizing any helper virus, no clinical isolates of herpes carrying wild-type AAV have been described. All these results point to an integral relationship between AAV, the host, and the helper virus.
In vivo integration of AAV AAVwas first documented as cryptic infections in primary African green monkey and human embryonic kidney cells in 1970 [5,7]. In these studies, 20% of the monkey and 2% of the human cells tested scored positive for the appearance of AAV antigens after Ad challenge [8]. This was the first indication of virus latency for this parvovirus in human and non-human primates. These results suggest that AAV infection in vivo is common. It is interesting to note that the high frequency of AAV in non-human primates is observed only in animals kept in captivity (RW Atchison, personal communication). While AAV transmission in humans is believed to occur horizontally, one study has documented the vertical germline transmission of avian AAV in chickens [9]. Since these early studies, relatively little information has accrued on the frequency and tissue tropism of AAV latent infection in man. However, with the advent of PCR technology and the potential relevance of AAV as a vector for human gene therapy, information concerning these important questions should be forthcoming. The first of such studies has been reported by Grossman et aL [10]. Using PCR analysis, they reported the detection of AAV-2 sequences in 2/55 healthy blood donors, and 2/16 hemophilic patients. While the sample size used in this study was too small to draw many conclusions, this work points to the type of information that will accumulate rapidly concerning AAV latency in vivo. A similar PCR analysis of airway epithelial cells from 36 cystic fibrosis and normal patient biopsies scored negative for AAV integration (RJ Samulski and M Welch, unpublished data). While the obvious interpretation is a lack of AAV latency in this tissue, a negative PCR result may be the result of other factors such as the turn-over rate of epithelial cells in the lung, or the relatively small portion of the organ that was subject to analysis. It is obvious that while positive results using PCR analysis will be informative, negative results remain nondefinitive and require follow-up experiments.
In vitro integration of AAV After AAV latent infection in human primary kidney cells was described [8], investigators focused on the question
of AAV latency in vitro. A human tissue-culture line, Detroit 6 (7374), was used to demonstrate the first in vitro AAV latent infection in 1972 [8]. In these experiments, infection at high multiplicity of AAV (250 tissue-culture infectious doses per cell) in the absence of helper virus resulted in 30% of the clones scoring positive for AAV after 39 passages. Further characterization of these latent cell lines demonstrated that the AAV genome existed as an integrated provirus [13,14]. This was the first evidence that AAV latency involved viral integration. Since this ame, some 80-100 independently-isolated proviral lines have been established and characterized in various cell types, including non-human primates [5,10,11-16,17-]. In fact, to date, no cell line has been isolated that is refractive to AAV integration. These observations suggest a very common receptor or mechanism of virus uptake (yet to be described), and also address the conservation of the integration mechanism in various cell types. In general, latenfly infected cells established in vitro appear to be stable, being able to maintain the viral DNA for greater than 150 200 passages [5,18"]. Overall, virus integration appears to have no apparent effect on cell growth or morphology [1]. A more recent study on AAV infection in either Epstein-Barr virus transformed B-cell line or an untransformed CD4 + T cell from a healthy donor demonstrated that many of the functional capabilities and growth properties of the T cells were not altered by virus integration. Parameters tested included DNA ploidy, lectin-dependent cytotoxic T-cell activity, anti-CD3 driven proliferation, and the analysis of surface-antigen expression using flow cytometry for CD2, CD5, CD7 and class 11 major histocompatibility products [17"]. While continued characterization of AAV latency in various cell lines is providing more information about the viral-host interaction [19,20], the definitive role of viral integration will require in vivo studies.
Proviral structure To date, no complete AAV provimses have been isolated, and therefore critical information on viral as well as cellular sequences is still lacking [18o,21]. The failure to isolate an intact AAV provirus is in part because of the recombinogenic potential of these sequences in traditional cloning vehicles. However, many studies (genomic blots) on the physical structure of integrated AAV genomes suggest that viral insertions are usually in a tandem head-to-tail orientation mediated by the AAV terminal repeats (TRs) [5,11,13-15]. Concatemers consisting of two to four tandem copies exist at the integration locus regardless of the initial multiplicity of infection [13,15]. These tandems appear to have at least one or two copies of the terminal-repeat sequences between two adjacent genomes [13,14]. The generation of low-copy tandem arrays using various multiplicities of infection suggest that these structures are not the result of end-to-end joining of input viral templates but rather the result of replicating a single AAV genome. Several immediate questions come to mind with regard to a pos-
76
Viral genetics: e
sible mechanism involved in AAV integration. First, the infecting AAV genome is a unit-length, tsingle-stranded molecule with hairpin terminal repeats (Fig. 2), which must at some point convert'to duplex DNA. Whether this step is a prerequisite for integration, or occurs afterwards, remains to be seen. Second, the current model fcrr.AAV DNA replication predicts the formation of headto-head or tail-to-tail tandems, the exact opposite from that characterized for the integrated provirus. Third, productive AAV replication is completely dependent on the presence of the qiral rep genes, sequenees typically absence from AAV transducing vectors [13,16,22]. It is in.teresting to note that in one cell line where fragments of the" provirus have been isolated, some of the viral sequences exist in a tail-to-tail tandem arrangement [21]. It is elear that until a complete provirus is isolated and sequenced, ~at best we can only guess about the.mechanism for AAV integration. Data generated by restriction-enzyme digestion and Southern hybridization analysis of independently derived latent cell lines have revealed that the viral--cellular junction fragments migrate with different mobilities, suggesting that AAV integration occurs randomly [13-15]. These results, although interpreted correctly, ~ere misleading as a result of the deletion and substitution of sequences around the target site. Concurrent with these results, the fact that free AAV DNA can be found in some latent cell lines, suggests that the AAV sequences are in a dynamic state of flux in the latent (a)
3" A'C'CB'BA Flop
Targeting integration AAV-cellular junction probes used against a panel of independently derived AAV latent cell lines scored 68% positive for co-migration between AAVfragments and the junction sequences [23]. Using somatic cell hybrid mapping, and in situ hybridization, this flanking sequence was localized to the long arm (q) of human chromosome 19 [18°,24°]. These data represent the first evidence to support the suggestion that AAV integration prefers a target sequence present only on chromosome 19. The location of the other AAV proviruses (30%) has not been defined, but once obtained should add useful information concerning AAV targeting. A recent report characterizing three AAV latent cell lines suggested virus integration in chromosome 17 [20]. While these integrants may represent other target sites, the flanking sequences have not been isolated or mapped, a prerequisite for determining specificity. "Results supporting use of target sites
A'C'CB'BA 5" Flip
AAV genome
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form. This scenario adds another level of complexity to the characterization of AAV integrants. Only after analysis of latent cell lines using cellular flanking sequences was evidence gained that supports AAV specificity in integration [18°,23].
RF synthesis
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Fig. 2. AAV replication scheme. (a) The single-stranded AAV genome has inverted terminal repeats, with the terms flop and flip designating the two potential arrangements of their DNA sequence. (b) These sequences can form a secondary T-shaped structure that is used as a self-primer (here shown in flop orientation) for DNA replication. (c) A specific parental-progeny (solid and broken lines, respectively) replicativeform (RF) intermediate is generated after the first round of replication. A virusspecific nicking activity that acts at the terminal resolution site (trs) is required for resolution of the RF structure. (d) This results in transfer of the parental hairpin sequences (solid line, ABB'CC'A'). (e) This duplex intermediate can then re-initiate DNA replication using the self-priming mechanism (T-structure shown with a broken line). (f) Finally, replication proceeds through strand displacement, generating a single-stranded hairpin molecule and a RF intermediate, each of which can re-enter the replication pathway.
Adeno-associated virus Samulski not identical to those characterized on chromosome 19 have been documented in diploid cells by A Srivastava a n d colleagues (personal communication). Utilizing a
protein-DNA-binding enrichment technique to isolate AAV proviral DNA from human cell lines, Samulski et aZ [18-] isolated and confirmed the initial results of Berns and his colleagues [23]. In the study, multiple latent cell lines were characterized using Southern-blot analysis, DNA sequencing, and in atu hybridization of latent chromosomes. Results from the analysis demonstrated viral targeting to within a 100 bp sequence on chromosome 19 [18-]. Even latent clones that by Southern analysis appeared as multiple copies of complex arrangement, when assayed by in situ, scored positive for integration at one locus' on chromosome 19. Sequence information from a cosmid pre-integration site has enabled PCR-based amplification for further characterization of viral-host junction sequences. These studies scored positive for targeted integration in latent aneuploid HeLa cell lines as well as latent diploid W138 [18 °] and have been extended to human colon, T cells, and monkey kidney cells (X Zhu, X Xiao and RJ Samulski, unpublished observations). In agreement with other studies [21], analysis of these viral-cellular sequences demonstrated patchy homology and rearranged sequences at the viral--cellular crossover points [18"]. Although these rearranged clones, isolated by PCR amplification, carried various amounts of the AAV terminal-repeat (TR) sequence, examples of both the flip and flop orientation (see Fig. 2) were isolated. This suggests that orientation of the TR sequence is not essential for virus targeting. To date, a complete viral TR-cellular junction has not been isolated. The inability to isolate an intact AAV TR-cellular junction may in part be because of the secondary structure of the AAV terminal sequences. The 145 bp TR of AAV can form a 125 bp palindromic T-shaped structure (see Fig. 3), which appears to be unstable in bacterial cells [25-28]. This instability is not unique to AAV but has also been observed with TRs from the autonomous parvoviruses MVM and JNC, and the pathogenic human parvovirus B19 [29-31]. We have modified a PCR amplification protocol to yield viral-junction sequences that pre-
serve the AAV terminal nucleotides (X Zhu and RJ Samul-
ski, unpublished data). Sequence analysis of virion DNA has demonstrated heterogeneity at the ends of the AAV TRs (at the 5' TR: 15% TTG; 50% TG; and 30% G). Junctions characterized by this procedure have so far only resulted in crossover points at 5' nucleotides TG and G. As described for other junctions, patchy homology of 3-5 bp was observed. The two break points for these junctions were separated by 30 bp (X Zhu and RJ Samulski, unpublished data). It is interesting to note that some of these AAV terminal nucleotides are similar to those of retroviruses (5' TG-CA 3' ).
Cis and trans sequences required for integration
To better define the target integration sequences, studies have been initiated to characterize the cellular preintegration site. Limited sequence information from the target site showed a 21 bp direct repeat flanking the viral-cellular break points [18.]. A 37bp element, repeated 10 times within 500 bp of the viral-cellular junction, has been identified [24.]. This repeat, a minisatellite sequence, was originally found in the third intron of the apolipoprotein C-U gene [32] and is uniquely located on 19q. Hybridization studies using the target sequence demonstrate conservation between humans and primates [18"]. Continued analysis of this region should further illuminate both the integration mechanism and the role AAV may play in the host cell. This host sequence may also facilitate the establishment of an in vitro integration system for the virus. It has been well documented that AAVvectors containing only the viral TR sequences integrate at high frequency [13,14,22,33-36]. in fact, in the absence of viral coding sequences, transduction of the AAV vector is even more efficient than that of wild-type virus (70% versus 30%) [13,14]. As the provirai structure of these recombinants are similar to wild type (low copy tandems and
T T..T co.G G..C G..C C' G..C C..G C..G C..G A G..C C. • G G C C T C A G T G A G C G A G C G A G C G C G C A G A G A G G G A G T G G C C A A A " ~ ~" : ' ' ' ' ' ' ' " : :'''''" :" ~'''.'''''''''''.'''." G. C C G G A G T C A C T C G C T C G C T C G C G C G T C T C T C C C T C A C C G G T T G C G G T C G T G C T C C C C A A G G A C. G
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Fig. 3. The AVV 145bp terminal repeat sequence, illustrating the potential
secondary structure when presented as a single-stranded molecule. Two small palindromes, B-B' and C-C' are ernSedded in a larger palindrome, A-A'.
77
78
Viral genetics
IN
D
(a)
• Viral DNA 145 bp
165 bp 20 bp Cellular DNA
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Fig. 4. AAV rescue model. (a) Dimer AAV DNA molecule before integration. The typical terminal repeat CIR) of AAV is 145 bp with one terminal resolution sequence (black-grey boundary). A key feature in the dimer is a unique 165 bp TR shared by two AAV genomes that contains flanking 20 bp viral D sequences (black shading; nucleotides shown in Fig. 2). The presence of the flanking viral D sequences provides two terminal resolution sites in the unique TR. (b) Targeted integration of this substrate into the host chromosome with imprecise joining of the external viral TR sequences to cellular DNA. (c) Conservation of the AAV nicking sites (arrowed) allows rep-mediated nicking after helper virus induction. (d) Bidirectional replication (open arrows) from the internal unique TR (dotted here) displaces single-stranded AAV molecules with 145 bp TRs {hairpin structures). (e) Sequence homology between the D regions of the viral TRs forms a panhandleshaped replicative intermediate. (f} Gene conversion of the terminal sequences results in a full-length, single-stranded AAV molecule that can enter the AAV replication scheme shown in Fig. 2.
rescuable), it suggests that the AAV termini are efficiently recognized for limited amplification and integration. In preliminary studies, AAV vectors that contain only the vi ral TRs have been analyzed for targeted integration (X Zhu and RJ Sanmlski, unpublished data). These viruses,
while proficient for integration and subsequent rescue, do not target to 19q at high frequencT and (X Zhu, X Xiao and RJ Samulski, unpublished data) [37]. Efficient targeting appears to be lost once the AAV coding sequences are removed, suggesting that AAV trana:acting
Adeno-associated virus Samulski factors are required for site-specific integration. However, as AAV vectors containing only the TR can target to 19q, albeit at a low frequency, protein components of the virion may participate in viral targeting (X Xiao and RJ Samulski, unpublished data). AAV proteins that may be potential candidates for a role in virus integration are the AAV rep 68 and 78 kD proteins. These proteins, which are essential for viral-DNA replication in vivo, have been biochemically characterized in vitro (RO Snyder et aL, unpublished data) [38-42]. They interact with AAV terminal sequences only if the termini exist in the T-shaped secondary conformation [40,43], a conformation that is believed to exist in virion DNA. Both rep 68 and 78 contain ATP-dependent site-specific and strand-specific endonuclease acdvity that recognizes a sequence in the AAV TR that is referred to as the terminal resolution site (trs) [38,42]. A DNA helicase activity is also associated with these viral proteins [41]. These enzymatic activities are required in the first steps of AAV DNA replication, and may also be involved in virus integration.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest e• HAND^ H, SHIROKIK, SHIMOJOH: Establishment and Characterization of KB Cell Lines Latendy Infected with Adenoassociated Virus Type 1. Virology 1977, 82:84--88. MUZYC.ZKA N (ED): Use of Adeno-as$ociated Virus as a General Transduction Vector for Mammalian Cells. In Current Topics in Microbiology and Immunology, vol 158. Berlin/Heidelberg: Springer-Verlag; 1992:97-129. CARTER BJ: Adeno-associated Virus Vectors. Curr Opin Biotechnol 1992, 3:533-539. CUKOR G, BLACKLOWNR, HOGGAN D, BERNS KI: Biology of Adeno-Associated Virus. In The Parvovirusex Edited by Berns KI. New York: Plenum Press; 1984:33--66. BERNS KI, CHEUNGA" OSTROVEJ, LEWISM: Adeno-associated Virus Latent Infection. In Virus Persistence. Edited by Mahy BWJ, Minson AC, Darby GK. Cambridge: Cambridge University Press; 1982:249. DULBECCO R, GINSBERG HS: Virology. In Microbiology, 2nd edn. Edited by Davis BD et al. Hagerstown: Harper & Row; 1973:1236.
Conclusion and perspective Identification of AAV targeting into the human genome is an important milestone in the study of AAV integration. While current data suggest that AAV integration occurs through a deletion substitution mechanism, a primary objective in viral latency is to ensure the ability to rescue from chromosomal DNA and re-enter the replication cycle after helper virus superinfection. Therefore, at least one complete viral genome, or sequences sufficient to generate a complete AAV DNA molecule, must exist in the proviral state. A model for AAV integration and rescue that has been constructed in the light of available data is presented in Fig. 4. A key element in this model is the presence of a 160bp TR shared between two AAV molecules. This intermediate has been shown to be obligatory in the current AAV replication scheme (Fig. 2). PCR analysis of the TR sequences between tandem proviral molecules have shown that they contain this specific TR sequence (X Zhu and RJ Samulski, unpublished data; Zolotokhin and N Muzyczka, personal communication). A synthetic TR retaining these sequences is sufficient for virus rescue, replication, packaging and integration (X Xiao and RJ Sarnulski, unpublished data). The formation of a concatemer may be a mechanism for Ensuring the integrity of at least one copy of the AAV TR and a method for the rescue and replication of the viral genome. A combination of genetic and biochemical studies is now required to fully dissect the protein-DNA interactions involved in this process of recombination.
HOGGAN MD: Adeno-associated Viruses. Prog Med Viro11970, 12:211-239. HOGGAN MD, THOMAS GF, THOMAS FB, JOHNSON FB: Continuous Carriage of Adenovirus Associated Virus Genome in Cell Cultures in the Absence of Helper Adenoviruses.
In Proceedings of the Fourth Lepetit Colloquiur& Co2~ ocac, Mexico. Amsterdam: North-Holland Publishing Co.; 1972:243-249. DAWSON GJ, YATES VJ, CHANG PW, WATFANAVIJARINW: Egg Transmission of Avain Adeno-associated Virus and CELO Virus during Experimental Infections. Am J Vet Res 1981, 42:1833. 10.
GROSSMANZ, MENDELSONE, BROK-SIMON1F, I¢flLEGUmF, LErrNER Y, RECHAWG, RAMOTB: Detection of Adeno-associated Virus Type in Human Peripheral Blood Cells. J Gen Virol 1992, 73:961-966.
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12.
BERNS KI, PINKERTONTC, THOMAS GF, HOGGAN MID: Detection of Adeno-associated Virus (AAV)-specific Nucleotide Sequences in DNA Isolated from Latenfly Infected Detroit 6 Cells. Virology 1975, 68:556-560.
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McLAUGHtlNSK, COt-Us P, HERMONATPL, MUZYCZKAN: Adenoassociated Virus General Transduction Vectors: Analysis of Proviral Structures. J Virol 1988, 62:1963-1973.
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SAMULSKIRJ, CHANG LS, SHENK T: Helper-free Stocks of Recombinant Adeno-associated Viruses: Normal Integration does not Require Viral Gene Expression. J Virol 1989, 63:3822-3828.
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LAUGHLIN CA, CARDELLICHIOCB, COON HC: Latent Infection of KB Cells with Adeno-as$odated Virus Type 2. J Viro/ 1986, 60:515-524.
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MURO-CACHO C.~ SAMULSKIRJ, KAPLAND: Gene Transfer in Human Lymphocytes Using a Vector Baaed on Adeno-associated Virus. J lmmunother 1992, 11:231-237.
Acknowledgements Acknowledgements are especially in order for those people who shared their ideas and information freely with me; these include B Carter, N Muzyczka, A Srivastava, X Xiao and X Zhu. I aJso thank C Berliner for editorial and graphics work.
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30.
JOURDANM, JOUSSET FX, GERVAIS M, SKOrY S, BERGOIN M, DUMAS B: Cloning of the Genuine of a Densovirus and Rescue of Infectious Virions from Recombinant Plasmid in the Insect Host Spodoptera Littoralis. Virology 1990, 179:403-409.
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DEISSV, TRATSCHINJD, WEITZ M, SIEGL G: Cloning of the Human Parvovirus B19 Genuine and Structural Analysis of its Palindromic Termini. Virology 1990, 175:247-254.
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SRIVASTAVACH, SAMULSKIRJ, LU L, LARSENSH, SRIVASTAVAA: Construction of a Recombinant Human Parvovirus B19: Adeno-associated Virus 2 DNA Inverted Terminal Repeats are Functional in an AAV-BI9 Recombinant Virus. Proc Natl Acad Sci USA 1989, 86:8078-8082.
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WALSH CE, LIU JM, XIAO X, YOUNG NM, NIENHUIS AW, SAMULSKI RJ: Regulated, High Level Expression of a Human G-globin Gene Introduced into Erythroid Cells by an Adeno-associated Virus Vector. Proc Nail Acad Sci USA 1992, 89:7257-7261.
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SNYDERRO, IM D-S, MUZYCZKAN: Evidence for Covalent Attachment of the Adeno-associated rep Protein to the Ends of the AAV Genuine. J Virol 1990, 64:6204-6213.
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IM D-S, MUZYCZKAN: Factors that Bind to Adeno-associated Virus Terminal Repeats• J Virol 1989, 63:3095-3104.
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IM D-S, MUZYCZKAN: The AAV Origin Binding Protein Rep68 is an ATP-dependent Site-specific Endonuclease with DNA Hellcase Activity. Cell 1990, 61:447-457.
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1M D-S, MUZYCZKAN: Partial Purification of Adeno-associated Virus Rep78, Rep52, and Rep40 and Their Biochemical Characterization. J virol 1992, 66:1119-1128.
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SAMULSKIRJ, ZHU X, XlAO X, BROOK JD, HOUSMAN DE, EPSTEINN, HUNTER LA: Targeted Integration of Adeno-associated Virus (AAV) into Human Chromosome 19. EMBO J 1991, 10:3941-3950. This paper describes independent evidence that AAV integration into chromosome 19 occurs with high frequency. The location on the chromosome was confirmed by the in situ hybridization of DNA probes to metaphase.chromosomes, and multiple latent junctions were mapped to a 100bp sequenceusing PCR amplification. •
19.
BANTEL-SCHAALU, STOHR M: Influence of Adeno-associated
Virus on Adl~rence and Growth Properties of Normal Cells. J virol 1992, 66:773-779. 20•
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WALZ C, SCHLEHOFERJR: Modification of Some Biologi-
Ko'riN RM, SINISCAmO M, SAMULSKiRJ, ZHU X, HUNTER L LAUGHLINCA, McLAUGHLINS, MUZUCZKAN, ROCCHI M, BERNS KI: Site-specific Integration by Adeno-associated Virus. Proc Nail Acad Sci USA 1990, 87:2211-2215.
KOTINRM, MENNINGERJC, WARD De, BERNSK]: Mapping and Direct Visualization of a Region-specific Viral DNA Integration Site on Chromosome 19ql3-qter. Genomics 1991, 10:831-834. This paper uses in situ hybridization to directly visualize the regionspecific viral-integration site. See also [23]. 24.
•
25.
SAMULSKIRJ, BERNS KI, TAN M, MUZUCZKA N: Cloning of Adeno-associated Virus into pBR322: Rescue of Intact Virus from the Recombinant Plasmid in Human Cells. Proc Natl Acad Sci USA 1982, 79:2077-2081.
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RJ Samulski, 269 Crawford Hall, Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.