Asymmetric replication in vitro from a human sequence element is dependent on adeno-associated virus Rep protein

Abstract

The DNA of human parvovirus adeno-associated virus type 2 (AAV) integrates preferentially into a defined region of human chromosome 19. Southern blots of genomic DNA from latently infected cell lines revealed that the provirus was not simply inserted into the cellular DNA. Both the proviral and adjoining cellular DNA organization indicated that integration occurred by a complex, coordinated process involving limited DNA replication and rearrangements. However, the mechanism for targeted integration has remained obscure. The two larger nonstructural proteins (Rep68 and Rep78) of AAV bind to a sequence element that is present in both the integration locus (P1) and the AAV inverted terminal repeat. This binding may be important for targeted integration. To investigate the mechanism of targeted integration, we tested the cloned integration site subfragment in a cell-free replication assay in the presence or absence of recombinant Rep proteins. Extensive, asymmetric replication of linear or open-circular template DNA was dependent on the presence of P1 sequence and Rep protein. The activities of Rep on the cloned P1 element are analogous to activities on the AAV inverted terminal repeat. Replication apparently initiates from a 3-OH generated by the sequence-specific nicking activity of Rep. This results in a covalent attachment between Rep and the 5-thymidine of the nick. The complexity of proviral structures can be explained by the participation of limited DNA replication facilitated by Rep during integration.

Cloning of adeno-associated virus type 4 (AAV4) and generation of recombinant AAV4 particles

“…The left ORF encodes the nonstructural Rep proteins which are involved in the regulation of replication and transcription and are necessary for the production of single-stranded progeny genomes (13-16, 19, 23, 28, 30, 34, 38-40, 43, 54, 57, 60-62). Furthermore, two of the Rep proteins have been associated with the preferential integration of AAV genomes into a region of the q arm of human chromosome 19 (37,56,64). Rep68/78 have also been shown to possess nucleoside triphosphate binding activity as well as DNA and RNA helicase activities (31,32,36,68).…”

“…In addition to their role in replication, these two elements appear to be central to viral integration. Contained within the chromosome 19 integration locus is a Rep binding site with an adjacent TRS (64,66). These elements are functional and necessary for locus-specific integration (26).…”

Viral Integration and Consequences on Host Gene Expression

“…More particularly, AAV-2 integration starts with Rep68/Rep78 complex introducing a nick at the adjacent cellular TRS that may induce the non homologous end-joining pathway (NHEJ) repair machinery. Non homologous recombination between the viral ITRs and the host DNA results in the viral insertion of AAV-2 in the host genome and the partial duplication of the integration site (Henckaerts and Linden, 2010, Lamartina et al, 2000, Urcelay et al, 1995. In conclusion, AAV long persistence, the absence of pathogenicity and the site-specific integration at AAVS loci render AAV a very attractive candidate for gene therapy.…”

Effect of poly(ADP‐ribose) polymerase 1 on integration of the adeno‐associated viral vector genome

“…The endonuclease activity of Rep mediates a nicking event at the terminal resolution site within the AAVS1 [20,21]. A poorly understood recombination event then occurs, resulting in partial duplication of the AAVS1 site and integration of the AAV genome [12,[20][21][22][23][24][25][26]. Although the role of AAV proteins in targeted integration has been extensively studied, the detailed mechanism and particularly the role of cellular factors in integration remains unclear.…”