Amino-Terminal Domain Exchange Redirects Origin-Specific Interactions of Adeno-Associated Virus Rep78 In Vitro

Abstract

The unique ability of adeno-associated virus type 2 (AAV) to site-specifically integrate its genome into a defined sequence on human chromosome 19 (AAVS1) makes it of particular interest for use in targeted gene delivery. The objective underlying this study is to provide evidence for the feasibility of retargeting site-specific integration into selected loci within the human genome. Current models postulate that AAV DNA integration is initiated through the interactions of the products of a single viral open reading frame, REP, with sequences present in AAVS1 that resemble the minimal origin for AAV DNA replication. Here, we present a cell-free system designed to dissect the Rep functions required to target site-specific integration using functional chimeric Rep proteins derived from AAV Rep78 and Rep1 of the closely related goose parvovirus. We show that amino-terminal domain exchange efficiently redirects the specificity of Rep to the minimal origin of DNA replication. Furthermore, we establish that the amino-terminal 208 amino acids of Rep78/68 constitute a catalytic domain of Rep sufficient to mediate site-specific endonuclease activity.Adeno-associated virus type 2 (AAV) has drawn much attention as a potential vector for human gene therapy. Advantages include its apparent lack of pathogenicity, its capacity to infect many cell types, and, notably, the ability of the wild-type (wt) virus to site-specifically integrate into a region on chromosome 19 known as AAVS1 (19q13.4) (14, 26-28, 35-37, 47). Integration, and thus the establishment of latency, is thought to occur preferentially over productive replication in the absence of coinfection of the host cell by a helper virus (3,4,8,18).The AAV genome is linear, single stranded, and flanked on either side by inverted terminal repeats (ITRs) (45, 53). Palindromic sequences within the ITRs allow for the formation of hairpin secondary structures, which contain the viral origins of replication (5,15,17,34,38,53). The minimal AAV DNA replication origin (AAVori) is organized into three segments: the Rep binding site (RBS), the terminal resolution site (TRS), and a spacer region separating the RBS from the TRS (Fig. 1A and 2B). The RBS coordinates the sequence-dependent recruitment of Rep to the origin (11), where it can then deliver a strand-specific nick at the TRS (23, 24). The spacer region has been implicated in favorably positioning the TRS so that it is efficiently nicked by the Rep protein (6). Interestingly, within AAVS1 is a region homologous to the minimal AAV origin in that it also contains a TRS and an RBS. Rep interaction with the minimal origin sequence in AAVS1 (57, 62) is required for site-specific integration (36, 63), thereby suggesting that Repmediated DNA replication may be linked to site-specific integration (Fig. 1B).Mechanistically, the initial steps of both AAV DNA replication and AAV site-specific integration are thought to be similar (25,36). In this model, Rep78/68 initiates replication at the viral origin by nicking the TRS in a site-and s...

Cross-Packaging of a Single Adeno-Associated Virus (AAV) Type 2 Vector Genome into Multiple AAV Serotypes Enables Transduction with Broad Specificity

“…Recent studies have demonstrated that the first 232 amino acids of AAV2 rep is required for origin specific binding when the AAV2/Goose parvovirus rep chimera is used (48). With respect to the type 5 hybrid construct, we were able to use as few as the first 250 amino acids of AAV2 rep to efficiently replicate and package rAAV2 ITR containing templates into type 5 virions.…”

The Expression Strategy of Goose Parvovirus Exhibits Features of both the Dependovirus and Parvovirus Genera

“…6A, lanes 3 and 4). GPV Rep1 shares a high degree of homology to the AAV2 Rep 78, although they do not complement for replication (29). For AAV2, a Rep binding site either in P5 or the AAV2 ITR is required for transactivation of the AAV2 P40 promoter by AAV2 Rep (17,18,21).…”

Residues within the B′ Motif Are Critical for DNA Binding by the Superfamily 3 Helicase Rep40 of Adeno-associated Virus Type 2

“…These activities are separated into three domains, all of which are present in the largest of the Rep proteins, Rep78. The Rep amino terminus possesses specific DNA binding and endonuclease activity (11)(12)(13), while the central motor domain bears motifs necessary for ATPase and helicase activity as well as the nuclear localization sequence (14 -16). The carboxyl-terminal zinc finger domain has been implicated in interaction with several cellular factors, although the biological significance of most of these interactions is not yet understood.…”