FIG. 6.

Binding to UAP56 is crucial for pUL69-mediated RNA export. (A) Schematic diagram illustrating FLAG-tagged UL69 proteins with either an intact or a mutated/deleted UAP56 binding domain. The subcellular localization of each mutant was analyzed via indirect immunofluorescence analysis. DAPI, 4′,6′-diamidino-2-phenylindole. (B) Expression of wild-type and mutant UL69 proteins after transfection of HeLa cells as detected by Western blotting. (C) Relative amount of CAT protein expressed in HeLa cells cotransfected with pDM128/CMV/RRE and plasmids expressing the proteins indicated in panel A. (D) Interspecies heterokaryon analysis to detect nucleocytoplasmic shuttling of a UL69 protein with mutated UAP56 binding site. Upper part: schematic representation of mutant UL69 mUAP and an NLS/β-Gal fusion protein (CFN-β-Gal) that was used as an internal nonshuttling control in the interspecies heterokaryon analysis. Lower part: immunodetection of UL69 protein and β-Gal in a heterokaryon consisting of HeLa and NIH 3T3 cells. Human and murine nuclei were differentiated via counterstaining with Hoechst 33258 dye (Hoechst panel; arrows indicate the mouse nucleus). Phase, phase-contrast image of the heterokaryon; the cytoplasmic edge is highlighted by a broken line.