Fig 7. TDP2 is required for resistance to Etoposide toxicity and virus production in induced LCLs.
The productive virus cycle was induced in TK6 and TK6-TDP2-/- LCLs expressing a tetracycline regulated BZLF1 transactivator by treatment with 1.5 μg/ml Dox. (A) TK6/TK6-TDP2-/- LCLs were harvested 72 h after induction. Five x104 live cells were seeded in triplicate wells of 96 well plates and treated overnight with the indicated concentration Etoposide before assessing cell viability by MTT assays. The rescue of Etoposide toxicity observed in induced TK6 was abolished in cells lacking TDP2. The mean ± SE of cell viability in three independent experiments is shown.**P≤0.01. (B) The amount of EBV-DNA in cell pellets and DNAse treated culture supernatants was quantified by qPCR 72 h after induction. The mean ± SE fold induction relative to untreated controls in three (supernatants) or four (cells) independent experiments is shown. *P≤0.05, **P≤0.01. (C) Model of TOP2 regulation by BPLF1. TOP2 (violet) trapped in TOP2ccs (yellow) is targeted for proteasomal degradation via SUMOylation (light blue) and ubiquitination (red) mediated by the SUMO ligase ZNF451, the SUMO-targeting ubiquitin ligase RNF4 and other cellular ubiquitin ligases, leading to the display of partially digested 5’-phosphotyrosyl-DNA adducts. Processing by the TDP2 resolvase generates protein-free DSBs that trigger the DDR. Imprecise repair leads to apoptosis and genomic instability. Ectopically expressed BPLF1 is recruited to DNA-trapped TOP2 and inhibits proteasomal degradation, which prevents the activation of the DDR. In the absence of ubiquitination, SUMOylation may alter the conformation of the TOP2 dimer allowing direct access of TDP2 to the 5’-phosphotyrosyl-DNA bonds, which promotes error-free repair. During productive EBV infection, the concomitant expression of BPLF1 and viral miRNA mediated downregulation of RNF4 favors the accumulation of SUMOylated TOP2β and the activation of non-proteolytic pathways for TOP2ccs debulking that are dependent on the resolution of TOP2-DNA adducts by TDP2. This promotes the error free repair of TOP2-induced DSBs and enhances cells survival and virus production.