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

Abstract

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 (TOP2cc), 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 line 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 TOP2cc debulking that favors cell survival and virus production.

Suppression of cGAS- and RIG-I-mediated innate immune signaling by Epstein-Barr virus deubiquitinase BPLF1

“…Together with results from the infection experiments with enforced overexpression of BPLF1 (Fig 3), these data demonstrated the biological importance of BPLF1 DUB during EBV infection. Furthermore, the results from the infection experiments for wild type p2089 and mutant p2089C61A viruses has also demonstrated the biological significance of BPLF1 DUB during EBV infection (S4 Fig) . BPLF1 has also been shown to promote EBV replication and evade innate immunity through other mechanisms [35][36][37][38][39][40][41][42][43]. The IFN antagonism of BPLF1 demonstrated in our study is generally consistent with its role in viral replication and immune evasion.…”

“…It also targets TRIM25, TRAF6 and SQSTM1/p62 to inhibit innate immune response and selective autophagy [35][36][37]. Other known targets of BPLF1 include PCNA, Rad18, translesion polymerase η and TOP2 involved in cellular DNA repair and translesion synthesis [38][39][40][41], as well as EBV ribonucleotide reductase [42], which inhibits APOBEC3 to preserve viral genome integrity [43].…”

Remodeling of the Ribosomal Quality Control and Protein Translation by a Viral Ubiquitin Deconjugase

“…To investigate whether physiological levels of the vDUB may regulate the translation of EBNA1 and other viral proteins, the productive virus cycle was induced in a pair of lymphoblastoid cell lines (LCLs) established by immortalization of B lymphocytes with recombinant EBV encoding either wild-type (LCLwt) or mutant BPLF1 (LCLcm). Since the virus is tightly latent in LCL cells, stable sublines expressing the Dox-regulated EBV transactivator BZLF1 were produced to ensure efficient and synchronous induction (Li et al, 2021). Control and Dox-treated cells were cultured for 72 h before protein and mRNA expression analysis by western blot and qPCR.…”

The Central Role of the Ubiquitin–Proteasome System in EBV-Mediated Oncogenesis

“…A recent study demonstrated that BPLF1 could target topoisomerase II (TOP2) and stabilize sumoylated TOP2. This results in inhibition of the DNA damage response and etoposide-induced apoptosis [77]. The resistance of etoposide toxicity in EBV-transformed cells is mediated by the expression of tyrosyl-DNA phosphodiesterase 2 (TDP2) that promotes TOP2 releases and DNA repair [77].…”

“…This results in inhibition of the DNA damage response and etoposide-induced apoptosis [77]. The resistance of etoposide toxicity in EBV-transformed cells is mediated by the expression of tyrosyl-DNA phosphodiesterase 2 (TDP2) that promotes TOP2 releases and DNA repair [77]. BPLF1 can disturb the cellular DNA repair pathway through the deubiquitination of the DNA processivity factor PCNA [78].…”