TABLE 1.

Effects of specific inhibitors on AAV DNA replication in vitro

Inhibitor	Concn	Relative activity (%)
None		100
Aphidicolina	10 μg/ml	0
ddTTPa	500 μM	42
BuPdGTPb	40 nM	100
	25 μM	50
BuAdATPb	20 nM	100
	120 μM	50
Dimethyl sulfoxidea	0.1%	79
	10%	0

^aThe level of AAV DNA synthesis was measured by scanning the intensity of monomer and dimer duplex DNA that were resistant to DpnI digestion by a gas flow counter to calculate the amount of incorporation of [α-³²P]dAMP into NE DNA substrate in the standard in vitro AAV DNA replication assay using uninfected crude HeLa cell extract (255 μg/ml), crude baculovirus Rep78 (8 μg), and the indicated inhibitors in a 2-h reaction. The percent activity remaining was then calculated by comparison to the reaction that contained no inhibitor. 

^bThe level of [α-³²P]dAMP incorporation in a standard (2-h) in vitro replication assay using uninfected crude HeLa cell extract and crude baculovirus Rep78 as described above was measured by DE-81 filter assay in the presence of the following inhibitor concentrations: 0.04, 0.4, 1, 10, 100, and 200 μM for BuPdGTP and 0.02, 0.2, 2, 20, 70, 100, and 200 μM for BuAdATP. The percent activity remaining was then calculated by comparison to a reaction that contained no inhibitor, and the concentration of inhibitor required to achieve 50% inhibition was calculated. Also shown, is the percent remaining activity at the lowest concentration of inhibitor used.