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Journal of Virology, October 2000, p. 9441-9450, Vol. 74, No. 20
0022-538X/00/$04.00+0

Adeno-Associated Virus Type 2 Rep78 Induces Apoptosis through Caspase Activation Independently of p53

Michael Schmidt, Sandra Afione, and Robert M. Kotin*

Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892

Received 16 May 2000/Accepted 25 July 2000

Adeno-associated virus (AAV) type 2 Rep78 is a multifunctional protein required for AAV DNA replication, integration, and gene regulation. The biochemical activities of Rep78 have been described, but the effects of Rep proteins on the cell have not been characterized. We have analyzed Rep-mediated cytotoxicity. We demonstrated that Rep78 expression is sufficient to induce cell death and disruption of the cell cycle. Cell death was found to be mediated by apoptosis. Rep78 expression resulted in the activation of caspase-3, a terminal caspase directly involved in the execution of cell death. A peptidic inhibitor of caspase-3, Z-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-FMK), abrogated Rep78-induced apoptosis, indicating that Rep78-mediated apoptosis is caspase-3 dependent. Rep78 induced apoptosis in wild-type p53-containing human embryonal carcinoma NT-2 cells and in p53-null promyelocytic human HL-60 cells, indicating that at least one pathway of Rep78-induced apoptosis is p53 independent. Apoptosis was shown to occur during the G1 and early S phases of the cell cycle. By analyzing the effects of Rep78 mutations on cell viability, the cause of cell death was attributed in part to two biochemical activities of Rep78, DNA binding and ATPase/helicase activity. The endonuclease activity of Rep78 did not contribute to apoptosis induction.

* Corresponding author. Mailing address: LBG, NHLBI, Bldg. 10, Rm. 7D05, Bethesda, MD 20892-1654. Phone: (301) 496-1594. Fax: (301) 496-9985. E-mail: KotinR{at}NHLBI.NIH.GOV.

Journal of Virology, October 2000, p. 9441-9450, Vol. 74, No. 20
0022-538X/00/$04.00+0


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