A genome-wide screen of Epstein-Barr virus proteins that modulate host SUMOylation identifies a SUMO E3 ligase conserved in herpesviruses
A.293T cells were transfected with plasmids expressing ICP0, FLAG-BRLF1 or empty vector (EV) control, then treated with MG132 (+) or left untreated (-). 34 hours post-transfection, cell lysates were analysed by Western blotting using the antibodies against SUMO1, SUMO2, ICP0, FLAG (BRLF1) or actin. Samples for SUMO1 and SUMO2 blots were run on separate gels and the actin loading control is shown for each. B. Experiments in A (without MG132) were repeated in CNE2Z cells with (siRNF4) and without (Control) silencing of RNF4. C. 293T (left) or CNE2Z (right) cells were transfected with plasmids expressing either ICP0, FLAG-BRLF1, Strep-Rta or empty vector (EV) along with a plasmid expressing myc-his-ubiqutin. Cell lysates were Western blotted directly (input) with antibodies against myc (to detect ubiquitin conjugates), FLAG (for BRLF1), Strep (for Rta) or ICP0. Myc-his-ubiquitin containing proteins were isolated from the lysates on metal chelating resin (Pulldown) prior to blotting for myc.