In vitro replication of adeno-associated virus DNA.

Abstract

An in vitro assay for adeno-aociated virus (AAV) DNA replication has been developed. The substrate is a plasmid containing the duplex form of AAV DNA in pBR322. The AAV insert is excised or rescued from the pas by extracts of uninfected cells. Replication was assayed by production of full-length excised AAV DNA resistant to Dpn I (ii) Only the excised AAV insert was replicated; pBR322 sequences were not. (iv) Replication was dependent on the presence of the AAV terminal repeat. (v) If the terminal 55 bases were deleted from both ends of the AAV insert, no rescue took place: replication occurred and both AAV and pBR322 sequences were replicated. We conclude that the AAV terminal repeat is essential for DNA replication but that under some conditions an initiation mechanism that does not involve hairpin priming may be used.The adeno-associated virus (AAV) genome has a palindromic inverted terminal repeat thought to serve as a "hairpin" primer to initiate DNA replication (1-3). In vivo AAV DNA replication appears to occur by a single-strand displacement mechanism leading to a duplex replicative intermediate covalently crosslinked at one end by the hairpin primer ( Fig. 1) (2, 3). To resolve the crosslink, the AAV-encoded rep 68/78 protein specifically nicks the hairpin at a point on the parental strand directly opposite the original 3' terminus (4). As a result of the nick, the 3'-terminal 125 bases are transferred from parental to progeny strand and are inverted in the process. The nick/transfer process leaves a 3'-OH at the end of the shortened parental strand, which then serves as a primer for synthesis to fill in the gapped region created by the transfer. In this reaction, the transferred hairpin sequence acts as the template.AAV DNA replication can be initiated from two very different substrates. In a permissive cell, in the presence of helper virus, the starting point for a productive infection is a small, linear, single-stranded DNA whose palindromic 3' terminus is free to form a hairpin and serve as a primer. Alternatively, in a latently infected cell the viral DNA is integrated as a duplex molecule into the host genome (5, 6). In the latter case, rescue or excision of the integrated genome is required for virion production. However, it is not known whether excision per se precedes the onset of free virion DNA synthesis or is a consequence of it. In vivo, the AAV rep gene is required in trans for rescue from the integrated state and for replication (7,8).A plasmid containing the duplex form of AAV DNA in pBR322 is fully infectious in adenovirus-infected HeLa cells (9,10 Plasmids. Plasmid pSM620 has been described (9). Plasmid pGM106 was created by insertion of the 4400-base-pair Bal I fragment of AAV into the Pst I site of pBR322 vector (14).Abbreviations: AAV, adeno-associated virus; SV40, simian virus 40;T antigen, large tumor antigen. 4673The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance w...

Inhibition of PrKX, a Novel Protein Kinase, and the Cyclic AMP-Dependent Protein Kinase PKA by the Regulatory Proteins of Adeno-Associated Virus Type 2

“…Some of these effects have been attributed to the viral nonstructural Rep proteins, which are central to the regulation of AAV DNA replication and transcription (2,18,36,44,46). Two of the Rep proteins (Rep68 and Rep78) have been associated with the preferential integration of AAV genomes into a region of the long or q arm of human chromosome 19 (27)(28)(29)48).…”

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

“…In wt AAV, Rep68/78 causes the rearrangement of the AAVS1 site and replication of the ITR cassette before site-specific integration. 22,[50][51][52] Thus, additional copies of the transgene cassette can be generated and integrated. Further, in AAV vectors, which lack rep sequences, the ITR cassette is often integrated into actively transcribed regions of the genome.…”

“…AAV ITRs and Rep68 or Rep78 have been shown to catalyze replication as well as site-specific integration of the ITR-flanked DNA into the genome. [22][23][24][25] Recent experiments have demonstrated that a 138 bp fragment of p5, the integration efficiency element (p5IEE) containing the p5 promoter, can also mediate site-specific integration with a 10-100-fold higher efficiency than the ITRs. 26 HSV/AAV hybrid vectors combine the HSV-1 elements ori S and pac with the AAV ITRs flanking the gene of interest and the rep-gene placed outside the ITR cassette.…”

Role of the terminal repeat GAGC trimer, the major Rep78 binding site, in adeno‐associated virus DNA replication

“…As a parvovirus AAV replicates its DNA by a 'rolling hairpin' mechanism [7][8][9]. The terminal repeats (TR) elements, of 145 base pairs, located at each terminus of the linear AAV DNA genome, are cis required for AAV DNA replication [10].…”