Figure 3.

AAV ssDNA-dependent in vitro interaction of AAV Rep and HSV ICP8. In vitro-translated, ³⁵S-labeled Rep78 was incubated with either GST–ICP8 or GST alone. Protein–protein interaction was studied either alone, or in the presence of varying amounts of gel-purified AAV template DNAs. The DNA was either in the double-stranded (ds) conformation, or was heat-denatured to adopt a single-stranded conformation, as indicated. (A and C) Autoradiographies of SDS–PAGE after separation of GST-pull-down products; +RBS, ssDNA flanked by AAV-ITRs with Rep-binding sites. –RBS, ssDNA without ITRs and deleted internal RBS homology sequences. Lanes 1 represent an aliquot of in vitro translated Rep78 alone. The faint high molecular weight band appears to represent multimerized Rep78. (B and D) Phosphoimager quantification of three separate experiments as displayed in (A) and (C).