FIGURE 4.

The nuclear export mutant HDAg-L-P205A cannot bind to TAP. A, GST pulldown assay with TAP and GST fusion proteins containing either HDAg-L(198–210) or the P205A mutant. The GST pulldown assay was performed with either GST-HDAg-L(198–210) or the mutant precoupled to glutathione-Sepharose beads and lysates were prepared from Huh7 cells. Following GST pulldown, Western blotting analysis was performed using antibodies against TAP and HDAg (top and bottom panels) or the pulled down proteins were detected by Coomassie Blue staining (middle panel). The positions of the molecular weight markers are shown on the left. B, Huh7 cells were transfected with plasmid pECE-d-BE or pECE-d-BE(P205A) containing cDNA encoding, respectively, HDAg-L and HDAg-L-P205A, then, at 2 days posttransfection, were harvested and the cell lysates were subjected to immunoprecipitation (IP) with anti-TAP antibodies, followed by Western blotting analysis with antibodies against TAP or HDAgs. NT, non-transfected.