Fig 6. Deletion of EBNA1^SIM significantly increases SUMO2-modified STUB1 and its association with EBNA1.

(A) Identification of cellular proteins that specifically associate with EBNA1^SIM. Whole cell lysates from HEK293 cells individually transfected with vector alone or vectors encoding WT EBNA1 with myc tag, or its SIM-deleted mutants (ΔSIM2, ΔSIM3, and ΔSIM2+3), were subjected to IP with anti-myc antibodies. Equal amounts of EBNA1-precipitated proteins were subjected to SDS-PAGE separation followed by Coomassie staining or directly MALDI-TOF-MS analysis. The heatmap and identified target protein are shown in S4 Fig and S4 Table, respectively. HC, heavy chain; LC, light chain. The EBNA1 and its SIM-associated target proteins (STUB1, KAP1, Cul1, and Cul4A) with more counts of peptide hits detected by MS are highlighted in the right panel. (B) The functional cluster of EBNA1-associated molecules upon deletion of EBNA1^SIM. The number of cellular proteins related to functional clusters of DNA/RNA binding, mRNA metabolism, transcription and translation process with significant change (≥ 2 fold) in the absence of EBNA1^SIM from panel A is shown in the green circles. The molecules related to the proteasome component are highlighted using asterisks. (C) Deletion of EBNA1^SIM significantly increases the association of STUB1 with EBNA1. HEK293 cells were transfected with plasmids expressing WT EBNA1 or its mutants (ΔSIM2, ΔSIM3, ΔSIM2+3, and K477R) with myc tag. At 48 h post-transfection, WCL were subjected to IB or IP with anti-myc antibody followed by immunoblotting as indicated. The asterisk denotes an uncharacterized protein band of KAP1. The modified protein bands of STUB1 and USP7 were highlighted. HC, heavy chain. (D) Deletion of EBNA1^SIM3 enhances EBNA1-mediated STUB1 modification switch from SUMO1 to SUMO2, SUMO1-modified KAP1 and SUMO2-mediated degradation of USP7. HEK293 cells were individually transfected with different amounts of vectors expressing WT EBNA1, its ΔSIM3 mutant or vector alone. At 48 h post-transfection, WCL were subjected to directly immunoblotting or de-IP with antibodies specific against SUMO1 or SUMO2, followed by IB as the indicated antibodies. The degraded KAP1 or USP7 with SUMO2 modification is highlighted in the figure. (E) Schematic presents the role of EBNA^SIM in the regulation of SUMO1 and SUMO2 -associated complex. The SUMO1- and SUMO2-associated complex (SC1 and SC2) is shown in gray and pink, respectively. The effect of EBNA^SIM deletion is highlighted by red arrows in the right panels.