The human dependovirus, adeno-associated virus (AAV) requires co-infection with a non-related DNA virus, e.g. adenovirus, to productively infect cells. AAV serotype 2 (AAV2) has been the prototype for this genus. Infection of cells in vitro with AAV2, in the absence of helper virus, may result in a integration of the virus genome. We have demonstrated that AAV2 DNA preferentially integrates into a specific region of the q-arm of human chromosome 19, locus AAVS1. Targeted integration is unique among eukaryotic viruses and requires cis-acting and trans-acting elements. The non-structural proteins of AAV, Rep, are required for targeted integration. The Rep proteins expressed from a promoter at map position 5, Rep 78 and Rep 68, are capable of binding both single stranded and dupex DNA and cleaving duplex and single-stranded DNA. The p5 Rep proteins multimerize and may form hexamers in the presence of a DNA substrate. Tyrosine 156 is required for the endonuclease activity and a covalent tyrosyl-phosphodiester is formed as an intermediate with the DNA. Through an as yet, uncharacterized reaction, we have shown that Rep 78 can then join the 5'-end of the nicked DNA to the 3'-end of another DNA substrate. Therefore the biochemical requirements for AAV targeted integration may be satisfied by the known activities of the p5 Rep proteins. Overexpression of the p5 Rep proteins has adverse effects on cells in vitro. Cell growth rate is retarded and there is increased percentage of dead cells. We have recently demonstrated that Rep 78 induces apoptposis. Cell death was found to occur in G1 and early S phases of the cell cycle. In an attempt to identify the activities of Rep that contribute to apoptosis induction, a set of Rep 78 derivatives was overexpressed in cells. The results were unexpected: there was no difference between wild-type Rep 78 and a endonuclease deficient Rep 78 mutation, Y156F. Rep 52, expressed from p19, lacks the amino terminus of the p5 Rep proteins which contains the DNA binding and enduclease functions, was nearly as cytotoxic. Rep 78 K340H has a mutation within the conserved ATPase motif is intermediate in cytotoxicity. Therefore, it appears that Rep mediated apoptosis results from several Rep encoded activities: helicase/ATPase activities and DNA binding, but not endonuclease activity. Additional experiments have implicated interactions between Rep proteins and cellular proteins as contributing to cytotoxicity.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Intramural Research (Z01)
Project #
1Z01HL002231-15
Application #
7594391
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
15
Fiscal Year
2007
Total Cost
$113,751
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Hickman, Alison Burgess; Ronning, Donald R; Perez, Zhanita N et al. (2004) The nuclease domain of adeno-associated virus rep coordinates replication initiation using two distinct DNA recognition interfaces. Mol Cell 13:403-14
Hickman, Alison Burgess; Ronning, Donald R; Kotin, Robert M et al. (2002) Structural unity among viral origin binding proteins: crystal structure of the nuclease domain of adeno-associated virus Rep. Mol Cell 10:327-37
Mastakov, Mihail Y; Baer, Kristin; Kotin, Robert M et al. (2002) Recombinant adeno-associated virus serotypes 2- and 5-mediated gene transfer in the mammalian brain: quantitative analysis of heparin co-infusion. Mol Ther 5:371-80
Ding, Chuantian; Urabe, Masashi; Bergoin, Max et al. (2002) Biochemical characterization of Junonia coenia densovirus nonstructural protein NS-1. J Virol 76:338-45
Schmidt, Michael; Chiorini, John A; Afione, Sandra et al. (2002) Adeno-associated virus type 2 Rep78 inhibition of PKA and PRKX: fine mapping and analysis of mechanism. J Virol 76:1033-42
Yang, Grace S; Schmidt, Michael; Yan, Ziying et al. (2002) Virus-mediated transduction of murine retina with adeno-associated virus: effects of viral capsid and genome size. J Virol 76:7651-60
Yamano, Seiichi; Huang, Li-Yun; Ding, Chuantian et al. (2002) Recombinant adeno-associated virus serotype 2 vectors mediate stable interleukin 10 secretion from salivary glands into the bloodstream. Hum Gene Ther 13:287-98
Urabe, Masashi; Ding, Chuantian; Kotin, Robert M (2002) Insect cells as a factory to produce adeno-associated virus type 2 vectors. Hum Gene Ther 13:1935-43
Zabner, J; Seiler, M; Walters, R et al. (2000) Adeno-associated virus type 5 (AAV5) but not AAV2 binds to the apical surfaces of airway epithelia and facilitates gene transfer. J Virol 74:3852-8
Davidson, B L; Stein, C S; Heth, J A et al. (2000) Recombinant adeno-associated virus type 2, 4, and 5 vectors: transduction of variant cell types and regions in the mammalian central nervous system. Proc Natl Acad Sci U S A 97:3428-32

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