Deletion of EBNA1^SIM reduces the co-localization of EBNA1 with SUMO2.

(A) K477R mutation but not SIM3 deletion results in punctate localization of EBNA1 on chromatin DNA. HEK293 cells transfected WT, SIM3-deleted (ΔSIM3) or the K477R mutant EBNA1 with myc tag were individually cultured on coverslips, fixed with 3% paraformaldehyde, and then stained with anti-myc antibody as indicated. Nuclei were counterstained with DAPI. Scale bars, 5 μm. Right panels show the magnified view and schematic of localization of EBNA1 (green) with host chromatin DNA (blue). (B) WT EBNA1, but not its SIM-deleted or K477R mutant, reduces the formation of SUMO1 punctate dots. HEK293 cells co-transfected Cherry-SUMO1 with vector alone, WT or SIM-deleted (ΔSIM3) or the K477R EBNA1 mutant with myc tag vectors, were individually subjected to immunofluorescence assays as described in panel A. The magnified view and schematic of localization of EBNA1 (green), SUMO1 (red) and host chromatin DNA (blue) and the percentage of co-localization from 30 counted cells are shown in the bottom panels. (C) WT EBNA1, but not its SIM-deleted or K477R mutant, co-localizes with SUMO2 as punctate dots. HEK293 cells co-transfected Cherry-SUMO2 with vector alone, WT or SIM-deleted (ΔSIM3) or the K477R EBNA1 mutant with myc tag vectors, was individually subjected to immunofluorescence assays as described in panel A. The magnified view and schematic of localization of EBNA1 (green), SUMO2 (red) and host chromatin DNA (blue) and the percentage of co-localization from 30 counted cells are shown in the bottom panels. The co-localization of WT (not K477R) EBNA1 with SUMO2 was highlighted by arrows.