Selective Cleavage of AAVS1 Substrates by the Adeno-Associated Virus Type 2 Rep68 Protein Is Dependent on Topological and Sequence Constraints

Lamartina, Stefania; Ciliberto, Gennaro; Toniatti, Carlo

doi:10.1128/jvi.74.19.8831-8842.2000

Cited by 20 publications

(30 citation statements)

References 67 publications

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“…However, we were unable to detect an enhancement in the probability of hotspot localization to RBS bearing canonical minimal TRS (CAAC/GTTG) compared to RBS alone. This lack of TRS correlation with RBS is consistent with in vitro experimentation that has found that constraints on this sequence exist but are minimal and difficult to define (22,42,43). Additionally, the spacing between the TRS and RBS as well as secondary structure may contribute to the complexity of determining a TRS influence (19,44).…”

Section: Hotspots and Rep Binding Sites (Rbs)supporting

confidence: 54%

“…Since 60 percent of sites bearing six or more RBS possessed hotspots, without an identifiable TRS, the presence of an optimal TRS may not greatly influence the localization of hotspots but may rather enhance hotspot intensity. AAVS1, for example, possesses a perfect TRS that can be cleaved by Rep and may contribute to the extremely high frequency of integration at that locus (17,18,43).…”

Section: Discussionmentioning

confidence: 99%

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High-Throughput Sequencing Reveals Principles of Adeno-Associated Virus Serotype 2 Integration

Janovitz

Klein

Oliveira

et al. 2013

J Virol

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Viral integrations are important in human biology, yet genome-wide integration profiles have not been determined for many viruses. Adeno-associated virus (AAV) infects most of the human population and is a prevalent gene therapy vector. AAV integrates into the human genome with preference for a single locus, termed AAVS1. However, the genome-wide integration of AAV has not been defined, and the principles underlying this recombination remain unclear. Using a novel high-throughput approach, integrant capture sequencing, nearly 12 million AAV junctions were recovered from a human cell line, providing five orders of magnitude more data than were previously available. Forty-five percent of integrations occurred near AAVS1, and several thousand novel integration hotspots were identified computationally. Most of these occurred in genes, with dozens of hotspots targeting known oncogenes. Viral replication protein binding sites (RBS) and transcriptional activity were major factors favoring integration. In a first for eukaryotic viruses, the data reveal a unique asymmetric integration profile with distinctive directional orientation of viral genomes. These studies provide a new understanding of AAV integration biology through the use of unbiased high-throughput data acquisition and bioinformatics.

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Section: Hotspots and Rep Binding Sites (Rbs)supporting

confidence: 54%

Section: Discussionmentioning

confidence: 99%

High-Throughput Sequencing Reveals Principles of Adeno-Associated Virus Serotype 2 Integration

Janovitz

Klein

Oliveira

et al. 2013

J Virol

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“…Multiple lines of evidence suggest that both binding and nicking of AAVS1 by Rep68/78 are required for preferential integration (32,35,56,57,59,61,64). This has led to the current hypothesis that AAVS1 is the preferred integration locus for AAV-2 because it contains the best Rep68/78 nicking site within the human genome.…”

mentioning

confidence: 99%

“…1) (5,6,22,32,51,64). RRS/trs combinations have been identified in the inverted terminal repeat (ITR) origins of DNA replication for all AAV serotypes that have been sequenced (10,22,65), as well as AAVS1 (11,57,59).Multiple lines of evidence suggest that both binding and nicking of AAVS1 by Rep68/78 are required for preferential integration (32,35,56,57,59,61,64). This has led to the current hypothesis that AAVS1 is the preferred integration locus for AAV-2 because it contains the best Rep68/78 nicking site within the human genome.…”

mentioning

confidence: 99%

Stable Secondary Structure near the Nicking Site for Adeno-Associated Virus Type 2 Rep Proteins on Human Chromosome 19

Jang¹,

Yarborough²,

Conyers³

et al. 2005

J Virol

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Adeno-associated virus serotype 2 (AAV-2) can preferentially integrate its DNA into a 4-kb region of human chromosome 19, designated AAVS1. The nicking activity of AAV-2's Rep68 or Rep78 proteins is essential for preferential integration. These proteins nick at the viral origin of DNA replication and at a similar site within AAVS1. The current nicking model suggests that the strand containing the nicking site is separated from its complementary strand prior to nicking. In AAV serotypes 1 through 6, the nicking site is flanked by a sequence that is predicted to form a stem-loop with standard Watson-Crick base pairing. The region flanking the nicking site in AAVS1 (5-GGCGGCGGT/TGGGGCTCG-3 [the slash indicates the nicking site]) lacks extensive potential for Watson-Crick base pairing. We therefore performed an empirical search for a stable secondary structure. By comparing the migration of radiolabeled oligonucleotides containing wild-type or mutated sequences from the AAVS1 nicking site to appropriate standards, on native and denaturing polyacrylamide gels, we have found evidence that this region forms a stable secondary structure. Further confirmation was provided by circular dichroism analyses. We identified six bases that appear to be important in forming this putative secondary structure. Mutation of five of these bases, within the context of a double-stranded nicking substrate, reduces the ability of the substrate to be nicked by Rep78 in vitro. Four of these five bases are outside the previously recognized GTTGG nicking site motif and include parts of the CTC motif that has been demonstrated to be important for integration targeting.Adeno-associated virus serotype 2 (AAV-2) is a human parvovirus. AAV-2 normally requires a helper virus, such as an adenovirus or herpesvirus for productive infection (7). In the absence of helper virus, AAV-2 can establish a latent infection by integrating its genome into the host DNA. AAV-2 preferentially integrates its DNA into a 4-kb region of human chromosome 19 (19q13-qter), designated AAVS1 (27-30). Subsequent infection by helper virus leads to rescue and productive infection. Preferential integration into AAVS1 requires the Rep68 or Rep78 protein (Rep68/78), encoded by AAV-2, as well as specific DNA sequences within the virus genome and AAVS1 (3,11,35,42,47,55,57,59). Binding sites for Rep68/78 within both the viral DNA and AAVS1 appear to be required (35,42,55,59). These sites, called Rep recognition sequences (RRSs), are comprised of imperfect repeats of the sequence 5Ј-GCTC-3Ј or its complement, 5Ј-GAGC-3Ј (11,20,59). Rep68/78 can form a bridge between RRS-containing DNAs that may facilitate preferential integration (34, 59).Rep68 and Rep78 also have a nucleoside triphosphate-dependent, strand-specific, site-specific endonuclease (nicking) activity (22,23). A Rep68/78 nicking site is called a terminal resolution site (trs) because of the role of such nicking sites in AAV-2 replication (22, 50, 52). Nicking requires both a specific sequence flanking the trs and a nearby...

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“…Further, in AAV vectors, which lack rep sequences, the ITR cassette is often integrated into actively transcribed regions of the genome. 53,54 Since AH-gBGAL and AHr-gBGAL both contain the AAV-ITR, the difference in b-gal activity is presumably not a consequence of transgene integration into different sites in the genome, but could be caused by the combination of different copy numbers of the BGAL gene and integration into different sites of the genome, with the AAVS1 site presumably supporting higher levels of transgene expression. A similar effect could be seen with Ad/AAV vectors, with a b-globin locus control region (LCR)-CMV-GFP cassette, integrated into the AAVS1 site.…”

Section: Integration Of 100 Kb Gene Via Hsv/aav Amplicon Vector a Oehmentioning

confidence: 99%

Integration of active human β-galactosidase gene (100 kb) into genome using HSV/AAV amplicon vector

et al. 2007

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Vectors based on herpes simplex virus type-1 (HSV-1) permit delivery of transgenes of up to 150 kb, while the inverted terminal repeats and Rep of the adeno-associated virus (AAV) can confer site-specific integration into the AAVS1 site, which allows sustained expression of a transgene. In this study, combination of the viral elements in HSV/AAV hybrid vectors has been applied for the infectious transfer of the human lysosomal b-galactosidase (BGAL) gene of 100 kb. Temporary expression and functional activity of b-galactosidase (b-gal) could be detected in human b-gal-deficient patient and glioblastoma (Gli36) cells upon infection with the basic BGAL amplicon vector. Sustained expression of b-gal was achieved in Gli36 cells infected with rep-plus, but not rep-minus, HSV/AAV hybrid vectors. None of five clones isolated after rep-minus hybrid vector infection showed elevated b-gal activity or site-specific integration. In contrast, 80% of the rep-plus clones possessed b-gal activity at least twofold greater than normal levels for up to 4 months of continuous growth, and 33% of the clones exhibited AAVS1-specific integration of the ITR-flanked transgene. One of the rep-plus clones displayed integration of the ITR cassette only at the AAVS1 site, with no sequences outside the cassette detectable and b-gal activity fourfold above normal levels. These data demonstrate AAVS1-specific integration of an entire genomic locus and expression of the transgene from the endogenous promoter mediated by an HSV/AAV hybrid vector.

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Selective Cleavage of AAVS1 Substrates by the Adeno-Associated Virus Type 2 Rep68 Protein Is Dependent on Topological and Sequence Constraints

Cited by 20 publications

References 67 publications

High-Throughput Sequencing Reveals Principles of Adeno-Associated Virus Serotype 2 Integration

High-Throughput Sequencing Reveals Principles of Adeno-Associated Virus Serotype 2 Integration

Stable Secondary Structure near the Nicking Site for Adeno-Associated Virus Type 2 Rep Proteins on Human Chromosome 19

Integration of active human β-galactosidase gene (100 kb) into genome using HSV/AAV amplicon vector

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