Figure 3.

Vpr effects on long DNA integration activity. (A) Phosphorimage of long DNA strand transfer assay. Reactions were performed by incubation for 1 h at 37°C of 2 pmol IN, 10 ng of U3U5 substrate and 40 ng of pSP70 target in the presence of increasing concentrations of Vpr and its subdomains: 0.4 µM (lanes 3, 6 and 9), 0.8 µM (lanes 4, 7 and 10), 1.6 µM (lanes 5, 8 and 11) of Vpr, (1–51)Vpr or (52–96)Vpr, respectively. Components were pre-incubated for 10 min on ice before incubation at 37°C, with the exception of the pSP70 target, which was added at the beginning of the reaction. The nature of the integration product is reported on the right: bold arrows indicate the main products corresponding to integration of one mini-viral DNA; fainter arrows indicate products corresponding to multiple integration events. (a) ³²P-labeled U3U5 substrate; (b) homologous integration products; (c) heterologous integration products. (B) Quantification of homologous integration products using ImageQuant software. Homologous integration in the presence of (52–96)Vpr (filled circles); (1–51)Vpr (filled inverted triangles) and Vpr (filled squares).