Fig 2: BZLF1 disrupts the TRIM24/TRIM33/TRIM28 complex.A. AGS-BZLF1 cells before (-) or 24 hrs after dox-induction of BZLF1 were used to perform IPs with TRIM33 or IgG control antibodies, followed by Western blotting with antibodies against TRIM24, TRIM28 and TRIM33. Input lysates were also probed with BZLF1 and tubulin antibodies. B. 293T cells were transfected with a BZLF1 expression plasmid (+) or empty plasmid (-), then IPs were performed with TRIM33 or IgG negative-control antibodies, followed by Western blot on inputs and IPs with antibodies against TRIM24, TRIM28 and TRIM33. Input lysates were also probed with BZLF1 and actin antibodies. C. NPC43-Z cells before (-) or 24 hrs after dox-induction of BZLF1 were used to perform IPs with TRIM33 or IgG control antibodies, followed by Western blotting with antibodies against TRIM24, TRIM28 and TRIM33. Input lysates were also probed with BZLF1 and actin antibodies. D Akata-Z cells before (-) or 24 hrs after dox-induction of BZLF1 were used to perform IPs with TRIM33 or IgG control antibodies, followed by Western blotting as in C. E. AGS-BZLF1 cells before (-) or the indicated hours after dox-induction of BZLF1 were analyzed by Western blotting with antibodies against TRIM33, TRIM28, TRIM24, BZLF1 and actin. TRIM bands were quantified from three independent experiments and average values with standard deviation were plotted. * = 0.01Western blotting with TRIM33 and vinculin antibodies. G. AGS-BZLF1 cells were transfected with 6His-SUMO2 expression plasmid then treated with dox for 24 hrs or left untreated. 10% of cell lysates were analyzed as input samples. The remaining lysates were used to isolate His-SUMO-modified proteins on metal chelating resin. Samples were analyzed by Western blotting using TRIM33, BZLF1 and actin antibodies.

Fig 3: Model of the repression of the BZLF1 promoter by TRIM24/TRIM28/TRIM33 complexes in latent infection and their disruption by BZLF1 in lytic infection.The interaction of BZLF1 with TRIM24 and TRIM33 and/or the disruption of TRIM24/TRIM28/TRIM33 complexes by BZLF1 leads to degradation of TRIM24, and phosphorylation and SUMOylation of TRIM33 followed later by its degradation. Created with BioRender.com.

Fig 4: TRIM33 and TRIM24 suppress EBV reactivation.A. TRIM33 or TRIM24 was knocked out using CRISPR-Cas9 in pools of AGS-EBV, HONE-Akata and NPC43 cells with two different guide RNAs (E and F for TRIM33; B and C or D for TRIM24) and compared to a negative control guide RNA (CG) targeting the Adeno-Associated Virus Integration Site 1 (AAVS1). Western blots were performed on whole cell lysates with antibodies against TRIM33, TRIM24, four EBV proteins (BZLF1, BMRF1, BALF2, Vcap18) and actin. B and C. Total DNA and RNA were extracted from AGS-EBV cells in A and EBV genome amplification (B) and BZLF1 transcripts (C) were quantified by qPCR. D. AGS-EBV cells were transfected with plasmids expressing a negative control FLAG-tagged protein (DDX24) or FLAG-tagged TRIM33 or TRIM24, then stained for BZLF1 and FLAG and imaged by IF. For each of the transfected plasmids, the percentage of FLAG-positive cells expressing BZLF1 was determined in 100 cells in three independent experiments. E. AGS cells were co-transfected with pZp-luc (firefly luciferase) and pRL-promotorless (renilla) reporter plasmids along with FLAG-TRIM33 or FLAG-TRIM24 expression plasmid or empty plasmid control and harvested 24 hours post-transfection. Firefly and renilla luciferase levels were quantified and luciferase values were normalized to renilla for three independent experiments. F. WT and SUMO mutant (SUMOm) TRIM33 were expressed in 293T cells with or without 6His-SUMO1 and 10% of cell lysates were analysed as input samples. SUMO-modified proteins were isolated from the remaining samples on metal chelating resin (His pull down). Samples were analysed by Western blotting using anti-FLAG antibody. G. The same experiments as in D except that WT TRIM33 was compared to the TRIM33 SUMOm. All graphs show average values with standard deviation where * = 0.01

Fig 5: Verification of changes in SUMOylation in selected cellular and viral proteins.A and B. Whole cell lysates from AGS-EBV-Z-6His-SUMO1 and AGS-EBV-Z-6His-SUMO2 cells before (Un) or 12 or 24 hours after dox induction were analysed by Western blotting using the indicated antibodies. Examples of cellular proteins with altered SUMOylation after EBV reactivation (A) and EBV proteins with SUMO modifications (B) are shown. Stars indicate nonmodified proteins. Arrowheads and brackets indicate SUMO conjugated proteins. C. The indicated cell lines were treated with ML792 or left untreated then reactivated with dox for 24 hours or left untreated. Whole cell lysates were then analysed by Western blotting and probed for EBV lytic proteins (BORF2, BMRF1, Vcap18) and TRIM33.