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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 3230-3239 |
| Number of pages | 10 |
| Journal | Journal of Virology |
| Volume | 75 |
| Issue number | 7 |
| DOIs | |
| State | Published - 2001 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Immunology
Cite this
Amino-terminal domain exchange redirects origin-specific interactions of adeno-associated virus Rep78 in vitro. / Yoon, M.; Smith, D. H.; Ward, P.; Medrano, F. J.; Aggarwal, A. K.; Linden, R. M.
In: Journal of Virology, Vol. 75, No. 7, 2001, p. 3230-3239.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Amino-terminal domain exchange redirects origin-specific interactions of adeno-associated virus Rep78 in vitro
AU - Yoon, M.
AU - Smith, D. H.
AU - Ward, P.
AU - Medrano, F. J.
AU - Aggarwal, A. K.
AU - Linden, R. M.
PY - 2001
Y1 - 2001
N2 - 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.
AB - 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.
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U2 - 10.1128/JVI.75.7.3230-3239.2001
DO - 10.1128/JVI.75.7.3230-3239.2001
M3 - Article
VL - 75
SP - 3230
EP - 3239
JO - Journal of Virology
JF - Journal of Virology
SN - 0022-538X
IS - 7
ER -