Fig 2: SUMOylation and phosphorylation of TRIM33 and verification of TRIM24 loss in NPC43 and Akata cell infections.A. Upper panel: Schematic of TRIM33 showing domain structure and sites of SUMOylation and phosphorylation identified in the proteomics experiment. Lower Panel: Summary of MS peptide intensity data for the indicated TRIM33 modifications. Mono-SUMOylated peptides, di-SUMOylated peptides, and peptides co-modified by SUMO and phospho moieties are grouped. Intensity in each sample relative to the average intensity across all samples is indicated and coloured according to the scale shown below (values represent the average of three replicates per condition). Peptides not detected in a particular sample are shown in grey. Cell line and duration of EBV reactivation are shown in the headers. B. AGS-EBV-Z cells were treated with dox (or left untreated) with and without ML792 for 12 hours as indicated. Whole cell lysates were incubated with or without FastAP phosphatase, followed by Western blotting with TRIM33 and vinculin antibodies. C. NPC43-Z cells were treated with dox for 0, 24 or 48 hours, then whole cell lysates were analysed by Western blotting using TRIM33, TRIM24, BZLF1 and actin antibodies. The bracket marks the position of bands corresponding to SUMO-modified TRIM33. D. NPC43-Z cells were treated with dox for 12 hours (or left untreated) in the presence of DMSO (negative control), ML792 or MG132 as indicated, and the lysates were anlaysed as in C. E. Akata-Z cells were treated with dox for 0, 24 or 48 hours, then whole cell lysates were analysed by Western blotting using TRIM33, TRIM24, BZLF1 and actin antibodies. Two exposures are shown for TRIM33, with the phospho shift evident in the lighter (top) exposure and the SUMO-modified forms seen in darker exposure and marked by the bracket. F. Akata-Z cells were treated with dox for 24 hours (or left untreated) in the presence of DMSO (negative control) or ML792, and the lysates were anlaysed as in E.

Fig 3: Proteomics data summary.A + B. Charts indicating changes in abundance to SUMO1 (x-axis) and SUMO-2 (y-axis) GG-K peptides (left) and cellular proteins (right) derived from whole cell extracts from the 6His-SUMO1 cells (x-axis) and 6His-SUMO2 cells (y-axis) in response to 12 hours (A) or 24 hours (B) EBV reactivation. Data derived from viral proteins are indicated by red markers and cellular proteins are grey, with the shade indicating the statistical significance of the change (see key) derived from the sum of the two -log10 p-values for the x and y-axis data. Selected sites and proteins with large deviation from 0 combined with high significance are indicated. C. Tabular summary of the SUMO1 and SUMO2 site-level data and total protein level data for TRIM24, TRIM28 and TRIM33. Cells are coloured by fold change with a thick border indicating statistical significance (p<0.05). D. AGS-EBV-Z cells were treated with dox (or left untreated) with and without MG132 for 12 hours as indicated. Western blots were then performed using antibodies against TRIM24, BZLF1 and actin.

Fig 4: BZLF1 binds to TRIM24 and TRIM33.A. TRIM33-FLAG or empty FLAG (negative control) was expressed in AGS-EBV-Z cells followed by reactivation with dox for 24 hrs, and TRIM33 and associated proteins were isolated on anti-FLAG resin. Recovered proteins were identified by tandem MS. Total spectral counts are shown for the five most prevalent interactors seen in two independent experiments. B. TRIM33-FLAG was expressed in AGS-EBV followed by reactivation by sodium butyrate/TPA treatment and FLAG IPs. Western blots on IP samples and 10% of input samples are shown using the indicated antibodies. C. AGS cells were transfected with plasmids expressing BZLF1 and FLAG-tagged TRIM33, TRIM28, TRIM24 or empty FLAG plasmid (EP). 36 hours later, IPs were performed using FLAG antibody, followed by Western blotting with BZLF1 and FLAG antibodies. Input lysates were also probed with actin antibody. D. AGS cells were transfected with plasmids expressing FLAG-TRIM33 and BZLF1 WT or mutants (or empty plasmid) as indicated. IPs were performed using anti-FLAG M2 beads, followed by Western blotting as in C. The lower band in the BZLF1 blot of IP samples is from the IgG light chain.

Fig 5: 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.