Preferential Integration of Adeno-Associated Virus Type 2 into a Polypyrimidine/Polypurine-Rich Region within AAVS1

Abstract

Adeno-associated virus type 2 (AAV2) preferentially integrates its genome into the AAVS1 locus on human chromosome 19. Preferential integration requires the AAV2 Rep68 or Rep78 protein (Rep68/78), a Rep68/78 binding site (RBS), and a nicking site within AAVS1 and may also require an RBS within the virus genome. To obtain further information that might help to elucidate the mechanism and preferred substrate configurations of preferential integration, we amplified junctions between AAV2 DNA and AAVS1 from AAV2-infected HeLaJW cells and cells with defective Artemis or xeroderma pigmentosum group A genes. We sequenced 61 distinct junctions. The integration junction sequences show the three classical types of nonhomologous-endjoining joints: microhomology at junctions (57%), insertion of sequences that are not normally contiguous with either the AAV2 or the AAVS1 sequences at the junction (31%), and direct joining (11%). These junctions were spread over 750 bases and were all downstream of the Rep68/78 nicking site within AAVS1. Two-thirds of the junctions map to 350 bases of AAVS1 that are rich in polypyrimidine tracts on the nicked strand. The majority of AAV2 breakpoints were within the inverted terminal repeat (ITR) sequences, which contain RBSs. We never detected a complete ITR at a junction. Residual ITRs at junctions never contained more than one RBS, suggesting that the hairpin form, rather than the linear ITR, is the more frequent integration substrate. Our data are consistent with a model in which a cellular protein other than Artemis cleaves AAV2 hairpins to produce free ends for integration.Adeno-associated virus type 2 (AAV2) is a naturally defective human parvovirus that usually requires a helper virus, such as an adenovirus or a herpesvirus, for productive infection (41). In the absence of helper virus, AAV2 can establish a latent infection by episomal persistence (54) or by integrating its DNA into the host chromosomes, preferentially within a 4-kb region of human chromosome 19 designated AAVS1 (11). This is not site-specific integration in the classic sense, but approximately 70% of integration events occur somewhere within this locus (Fig. 1A) (20,26,51). The mechanism is unknown, but it does require the Rep68 or Rep78 (Rep68/78) protein encoded by AAV2 and a 33-bp region of AAVS1 (Fig. 1A) that includes a Rep68/78 binding site (RBS) and a nicking site for Rep68/78 that resembles the terminal resolution site (trs) within the 145-base inverted terminal repeats (ITRs) of the AAV2 genome (Fig. 2) (29,57,64,67). The trs got its name because of its role in AAV2 replication. AAV2 has a linear, single-stranded DNA genome (4,679 bases; GenBank accession no. AF043303), and the ITRs are essential for replication. The ITRs are palindromic and fold into T-shaped hairpin structures, one of which provides a 3Ј end that primes secondstrand synthesis by a cellular DNA polymerase (Fig. 2) (10, 65). The hairpin is then nicked by Rep68/78 at trs (Fig. 2) and unwound, and the end is replicated. As a result of this mo...

Site-specific integration of adeno-associated virus involves partial duplication of the target locus

“…1). Few of the previously identified left junctions were found closer to the TRS/RBS motifs (22,24,25), suggesting that the left cellular breakpoint might occur anywhere downstream of the TRS motif, most frequently at a considerable distance. In a recent study, Drew et al (26) identified 11 left viral-cellular junctions, of which the cellular breakpoints were scattered from 2.3 kb to Ͼ14 kb from the TRS motif (26).…”

“…Other hallmarks are the microhomology between the viral and cellular sequences, the presence of a short ''unknown'' sequence (23,24), and the close proximity of the right junction to the cellular TRS motif. It should be noted that most previous studies focused on the isolation of viral-cellular junctions based on direct PCR technologies that would only allow for the amplification of junctions located close to the AAVS1 TRS/RBS motifs (17,24).…”

Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas

“…They are defective viruses that require coinfection with adenovirus for productive infection 12,13 . Early studies showed that, during infection, the AAV2 DNA integrates into the human genome, where it remains quiescent until a new parvovirus infection occurs 12,14,15 . To investigate the functional consequences of viral integration, we generated a construct based on the pGL3 reporter vector, reproducing the exact AAV2 inserted sequence.…”

Impact of the MRN Complex on Adeno-Associated Virus Integration and Replication during Coinfection with Herpes Simplex Virus 1

“…Interestingly, the analysis of the AAVS1-AAV junctions also revealed that many integration junctions corresponded to short regions of homology between AAV and AAVS1 sequences (65,67,68). These findings strongly suggest that AAV integration might be the result of a DNA repair pathway involving end-joining via microhomologies.…”