The Epstein-Barr virus deubiquitinating enzyme BPLF1 regulates the activity of topoisomerase II during productive infection

Topoisomerases are essential for the replication of herpesviruses but the mechanisms by which the viruses hijack the cellular enzymes are largely unknown. We found that topoisomerase-II (TOP2) is a substrate of the Epstein-Barr virus (EBV) ubiquitin deconjugase BPLF1. BPLF1 co-immunoprecipitated and deubiquitinated TOP2, and stabilized SUMOylated TOP2 trapped in cleavage complexes (TOP2ccs), which halted the DNA damage response to TOP2-induced double strand DNA breaks and promoted cell survival. Induction of the productive virus cycle in epithelial and lymphoid cell lines carrying recombinant EBV encoding the active enzyme was accompanied by TOP2 deubiquitination, accumulation of TOP2ccs and resistance to Etoposide toxicity. The protective effect of BPLF1 was dependent on the expression of tyrosyl-DNA phosphodiesterase 2 (TDP2) that releases DNA-trapped TOP2 and promotes error-free DNA repair. These findings highlight a previously unrecognized function of BPLF1 in supporting a non-proteolytic pathway for TOP2ccs debulking that favors cell survival and virus production.
Author summary The N-terminal domains of the herpesvirus large tegument proteins encode a conserved cysteine protease with ubiquitin- and NEDD8-specific deconjugase activity. Members of the viral enzyme family regulate different aspects of the virus life cycle including virus replication, the assembly of infectious virus particles and the host innate anti-viral response. However, only few substrates have been validated under physiological conditions of expression and very little is known on the mechanisms by which the enzymes contribute to the reprograming of cellular functions that are required for efficient infection and virus production. Cellular type I and type II topoisomerases (TOP1 and TOP2) resolve topological problems that arise during DNA replication and transcription and are therefore essential for herpesvirus replication. We report that the Epstein-Barr virus (EBV) ubiquitin deconjugase BPLF1 selectively regulates the activity of TOP2 in cells treated with the TOP2 poison Etoposide and during productive infection. Using transiently transfected and stable cell lines that express catalytically active or inactive BPLF1, we found that BPLF1 interacts with both TOP2α and TOP2β in co-immunoprecipitation and in vitro pull-down assays and the active enzyme stabilizes TOP2 trapped in TOP2ccs, promoting a shift towards TOP2 SUMOylation. This hinders the activation of DNA-damage responses and reduces the toxicity of Etoposide. The physiological relevance of this finding was validated using pairs of EBV carrying HEK-293T cells and EBV immortalized lymphoblastoid cell lines (LCLs) expressing the wild type or catalytic mutant enzyme. Using knockout LCLs we found that the capacity of BPLF1 to rescue of Etoposide toxicity is dependent on the expression of tyrosyl-DNA phosphodiesterase 2 (TDP2) that releases DNA-trapped TOP2 and promotes error-free DNA repair.