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

['Jinlin Li', 'Department Of Cell', 'Molecular Biology', 'Karolinska Institutet', 'Stockholm', 'Noemi Nagy', 'Jiangnan Liu', 'Soham Gupta', 'Teresa Frisan', 'Department Of Molecular Biology']

Date: 2021-10

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.

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.

Ubiquitin-specific proteases, or deubiquitinating enzymes (DUBs), regulate protein turnover by disassembling poly-ubiquitin chains that target the substrate for proteasomal degradation [ 27 ]. Several human and animal viruses encode DUB homologs that play important roles in the virus life cycle by promoting viral genome replication and inhibiting the host antiviral response [ 28 – 31 ]. In this study, we report that TOP2 is a substrate of the DUB encoded in the N-terminal domain of the EBV large tegument protein BPLF1 and provide evidence for the capacity of the viral enzyme to promote the non-proteolytic TDP2-dependent resolution of TOP2ccs, which enhances cell survival and favors virus production.

Topoisomerases sustain DNA replication, recombination and transcription by inducing transient single or double-strand DNA breaks that allow the resolution of topological problems arising from strand separation [ 13 , 14 ]. TOP2 homodimers mediate DNA disentanglement by inducing transient double strand-breaks (DSBs) through the formation of enzyme-DNA adducts, known as TOP2 cleavage complexes (TOP2ccs), between catalytic tyrosine residues and the 5’ends of the DSBs [ 15 ]. Following the passage of the second DNA strand, TOP2 rejoins the DNA ends via reversion of the trans-esterification reaction. While TOP2-induced DSBs are relatively frequent in genomic DNA [ 16 ], failure to resolve TOP2ccs, as may occur upon endogenous or chemical stress that inhibits TOP2 activity, results in the formation of stable TOP2-DNA adducts that hinder DNA replication and transcription and trigger apoptotic cell death [ 17 ]. Thus, cellular defense mechanisms attempt to resolve the TOP2ccs via proteolytic or non-proteolytic pathways [ 18 ]. The proteolytic pathways involve the displacement of TOP2 via for example, ubiquitin [ 19 ] or SUMO and ubiquitin-dependent [ 20 ] proteasomal degradation, which, upon removal of residual peptide-DNA adducts by the Tyrosyl-DNA phosphodiesterase-2 (TDP2) resolving enzyme [ 21 , 22 ], unmasks the DNA breaks and promotes the activation of DNA damage responses (DDR) [ 22 ]. Alternatively, the SUMOylation of TOP2 may induce conformational changes in the TOP2 dimer that exposes the covalent TOP2-DNA bonds to the direct action of TDP2 [ 23 ] without need for TOP2 proteolysis, which allows the repair of DSBs by TOP2 itself or other ligases. Two TOP2 isozymes expressed in mammalian cells share ~70% sequence identity and have similar catalytic activities and structural features but are differentially regulated and play distinct roles in biological processes [ 15 ]. While TOP2α is preferentially expressed in dividing cells and is essential for the decatenation of intertwined sister chromatids during mitosis [ 24 ], TOP2β is the only topoisomerase expressed in non-proliferating cells and is indispensable for transcription [ 25 , 26 ].

EBV replication is triggered by the expression of immediate early genes, which transcriptionally activates a variety of viral and host cell factors required for subsequent phases of the productive cycle [ 6 – 8 ]. Among the cellular factors, DNA topoisomerase-I and -II (TOP1 and TOP2) were shown to be essential for herpesvirus DNA replication [ 9 – 11 ], raising the possibility that topoisomerase inhibitors may serve as antivirals. Indeed, non-toxic concentrations of TOP1 and TOP2 inhibitors were shown to suppress EBV-DNA replication [ 9 ], and different TOP1 inhibitors reduced the transcriptional activity of the EBV immediate-early protein BZLF1 and the assembly of viral replication complexes [ 12 ]. However, the mechanisms by which the virus harnesses the activity of these essential cellular enzymes remain largely unknown.

Like other herpesviruses, EBV establishes latent or productive infections in different cell types. In latency, few viral genes are expressed resulting in the production of proteins and non-coding RNAs that drive virus persistence and cell proliferation [ 2 ]. In contrast, productive infection requires the coordinated expression of a large number of immediate early, early and late viral genes, which leads to the assembly of progeny virus and death of the infected cells [ 3 ]. Although much of the EBV-induced pathology has been attributed to viral latency, the importance of lytic products in the induction of chronic inflammation and malignant transformation is increasingly recognized [ 4 , 5 ], pointing to inhibition of lytic gene products as a useful strategy for preventing EBV associated diseases.

Results

BPLF1 selectively inhibits the degradation of TOP2 in cells treated with topoisomerase poisons To investigate whether the EBV encoded DUB regulates the proteasomal degradation of poisoned topoisomerases, FLAG-tagged versions of the N-terminal catalytic domain of BPLF1 that is generated by caspase I cleavage of the large tegument protein during productive infection [32], and an inactive mutant where the catalytic Cys61 was substituted with Ala (BPLF1C61A) were stably expressed by lentivirus transduction in HEK-293T cells under the control of a Tet-on regulated promoter (HEK-rtTA-BPLF1/BPLF1C61A cell lines). Inducible expression was monitored by probing immunoblots of cells treated for 24 h with increasing concentration of doxycycline (Dox) with antibodies to the FLAG or V5 tags (S1A Fig). Although the steady-state levels of BPLF1C61A were occasionally lower, both versions of the enzyme were readily detected by anti-FLAG immunofluorescence in more than 50% of the induced cells (S1B Fig). To monitor ubiquitin-dependent proteasomal degradation, HEK-rtTA-BPLF1/BPLF1C61A cells cultured overnight in the presence or absence of Dox were treated with the TOP1 poison Camptothecin (Cpt) or the TOP2 poison Etoposide (Eto) in the presence or absence of the proteasome inhibitor MG132, and topoisomerase levels were assessed by western blot. Camptothecin and Etoposide trap TOP1-DNA and TOP2-DNA covalent adducts, respectively [33], while MG132 prevents the proteasomal degradation of stalled topoisomerase-DNA intermediates [19]. As expected, TOP1 was efficiently degraded in control Camptothecin treated cells (Fig 1A and 1C upper panels), while treatment with Etoposide promoted the degradation of both TOP2α and TOP2β (Fig 1B and 1C middle and lower panels). The degradation was inhibited by treatment with MG132, confirming the involvement of the proteasome in the clearance of poisoned topoisomerases. Expression of catalytically active or mutant BPLF1 following Doxycycline treatment did not affect the Camptothecin-induced degradation of TOP1. In contrast, expression of the active BPLF1 was accompanied by stabilization of both TOP2α and TOP2β in Etoposide-treated cells with effect comparable to that induced by treatment with MG132. The mutant BPLF1C61A had no effect (Fig 1B and 1C). The selective rescue of the DNA-trapped TOP2 isozymes indicates that the effect cannot be ascribed to a global deubiquitination of cellular substrates by the overexpressed viral enzyme. PPT PowerPoint slide

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TIFF original image Download: Fig 1. BPLF1 selectively binds to TOP2 and inhibits the degradation of TOP2 in cells treated with topoisomerase poisons. HEK-293T cell expressing inducible FLAG-BPLF1 or FLAG-BPLF1C61A were seeded into 6 well plates and treated with 1.5 μg/ml Dox for 24 h. After treatment for 3 h with 5 μM of the TOP1 poison Camptothecin (Cpt) or 6 h with 40 μM of the TOP2 poison Etoposide (Eto) with or without the addition of 10 μM MG132, protein expression was analyzed in western blots probed with the indicated antibodies. GAPDH was used as the loading control. (A) Representative western blots illustrating the expression of TOP1 in control and Cpt treated cells. The proteasome-dependent degradation of TOP1 induced by the treatment was not affected by the expression of BPLF1 or BPLF1C61A in Dox treated cells. (B) Representative western blots illustrating the expression of TOP2α and TOP2β in Etoposide treated cells. Expression of BPLF1 protected TOP2α and TOP2β from Etoposide-induced proteasomal degradation while BPLF1C61A had no appreciable effect. (C) The intensity of the TOP1, TOP2α and TOP2β specific bands in 5 (TOP1) or 6 (TOP2α and TOP2β) independent experiments was quantified using the ImageJ software. The data are presented as intensity of the bands in Cpt/Eto treated samples relative to untreated control after normalization to the GAPDH loading control. Statistical analysis was performed using Student’s t-test. **P≤ 0.01; ns, not significant. (D) HEK293T cells transfected with FLAG-BPLF1, FLAG-BPLF1C61A, or FLAG-empty vector were treated with 40 μM Etoposide for 30 min and cell lysates were either immunoprecipitated with anti-FLAG conjugated agarose beads or incubated for 3 h with anti-TOP2α or TOP2β antibodies followed by the capture of immunocomplexes with protein-G coated beads. Catalytically active and inactive BPLF1 co-immunoprecipitate with both TOP2α and TOP2β in untreated and Etoposide treated cells (upper panels). Conversely, TOP2α (middle panels) and TOP2β (lower panels) interact with both catalytically active and inactive BPLF1. Representative western blots from one of two independent experiments where all conditions were tested in parallel are shown. https://doi.org/10.1371/journal.ppat.1009954.g001

TOP2 is a BPLF1 substrate To directly test whether the TOP2 isozymes are substrates of BPLF1, we first investigated whether they interact in cells and in pull-down assays performed with recombinant proteins. Lysates of HEK-293T cells transiently transfected with FLAG-BPLF1 or FLAG-BPLF1C61A were immunoprecipitated with antibodies recognizing FLAG, TOP1, TOP2α or TOP2β. In line with the failure to rescue Camptothecin-induced degradation, BPLF1 did not interact with TOP1 (S2A Fig), whereas both TOP2α and TOP2β were readily detected in western blots of the FLAG immunoprecipitates and, conversely, BPLF1 was strongly enriched in the TOP2α and TOP2β immunoprecipitates indicating that the proteins interact in cells (Fig 1D). Notably, the failure to co-immunoprecipitate TOP1 and rescue TOP1 from Camptothecin-induced proteasomal degradation, supports the conclusion that the interaction of BPLF1 with TOP2 is not a mere artifact of overexpression. To gain insight on the nature of the interaction, equimolar concentration of yeast expressed FLAG-TOP2α, or a TOP2α mutant lacking the unique C-terminal domain that is not conserved in the TOP2β isozyme, FLAG-TOP2α-ΔCTD, were mixed with bacterially expressed His-BPLF1 and reciprocal pull-downs were performed with anti-FLAG (S2B Fig) or Ni-NTA coated beads (S2C Fig). A weak BPLF1 band was reproducibly detected in western blots of the FLAG-TOP2α pull-downs probed with a His-specific antibody and, conversely, a weak FLAG-TOP2α band was detected in the His pull-downs, confirming that the interaction is direct. The binding of BPLF1 to TOP2α was not affected by deletion of the TOP2α C-terminal domain (S2D Fig), pointing to the involvement of a domain shared by TOP2α and TOP2β in the direct binding of BPLF1 to the TOP2 isozymes. Notably, the weaker binding observed in the pull-down of bacterially expressed proteins compared to co-immunoprecipitation in cell lysates suggests that the interaction may be strengthened by factors, such as TOP2 post-translational modification or additional binding partners, that are only present in cells. The capacity of BPLF1 to rescue TOP2 from proteasomal degradation together with the stronger interaction of TOP2 with the catalytically mutant BPLF1C61A (Fig 1D) point to TOP2 as a bona fide substrate of the viral enzyme. To investigate this possibility, TOP2α and TOP2β were immunoprecipitated from lysates of control and Etoposide-treated HEK-293T cells transiently transfected with BPLF1 or BPLF1C61A and western blots were probed with a ubiquitin-specific antibody. The cell lysates were prepared under denaturing conditions to exclude non-covalent protein interactions and working concentrations of NEM and iodoacetamide were added to all buffers to inhibit DUB activity. Transfection of the catalytically active BPLF1 appreciably reduced the total amount of ubiquitinated proteins in both untreated and Etoposide treated cells, (Fig 2A lower panels), confirming that the viral enzyme can deubiquitinate a broad range of cellular substrates. In line with the capacity of Etoposide to induce proteasomal degradation, smears of high molecular weight species corresponding to ubiquitinated TOP2α and TOP2β were detected in the immunoprecipitates of Etoposide-treated cells compared to untreated cells (Fig 2A). The intensity of the smears was strongly decreased in cells expressing active BPLF1, while the mutant BPLF1C61A had no appreciable effect. This, together with the selective rescue of TOP2 from proteasomal degradation, supports the conclusion that TOP2 is a true BPLF1 substrate. PPT PowerPoint slide

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TIFF original image Download: Fig 2. BPLF1 deubiquitinates TOP2 and stabilizes TOP2ccs. (A) HEK293T cells were transiently transfected with plasmids expressing FLAG-BPLF1, FLAG-BPLF1-C61A, or the FLAG empty vector, and aliquots were treated with 40 μM Etoposide for 30 min. TOP2α and TOP2β were immunoprecipitated from cell lysates prepared under denaturing conditions in the presence of DUB inhibitors and western blots were probed with antibodies to TOP2α, TOP2β and ubiquitin. The expression of catalytically active BPLF1 inhibits the ubiquitination of TOP2α and TOP2β induced by Etoposide treatment. Western blots from one representative experiment out of three are shown in the figure. (B) HEK-rtTA-BPLF1 cells were treated with 1.5 μg/ml Dox for 24 h followed by treatment with 80 μM Etoposide for the indicated time with or without the addition of 10 μM MG132. RADAR assays were performed as described in Materials and Methods and TOP2 trapped in 10 μg DNA was detected in western blots using antibodies to TOP2α or TOP2β. Trapped TOP2 appears as a major band of the expected size and a smear of higher molecular weight species. The intensity of the trapped TOP2α and TOP2β smears decreased over time in control untreated cells due to proteasomal degradation, while the decrease was significantly reduced upon expression of BPLF1 in Dox treated cells. Western blots from one representative experiment out of two are shown in the figure. (C) The intensity of the TOP2 smears was quantified using the ImageJ software. Clearance was calculated as 1- (intensity of the smears after treatment for 4 h/intensity of the smears after treatment for 30 min) x100. Treatment with MG132 reduced the clearance of TOP2ccs in BPLF1 negative cells and a similar reduction was achieved by expression of BPLF1 in Dox treated cells. The mean ± SD of two independent experiments is shown in the figure. Statistical analysis was performed using Student’s t-test. *P≤ 0.05. https://doi.org/10.1371/journal.ppat.1009954.g002 The degradation of TOP2 by the proteasome plays an important role in the debulking of persistent TOP2ccs generated by topoisomerase poisons [19]. To investigate whether the viral DUB may interfere with this process, HEK-rtTA-BPLF1 cells cultured for 24 h in the presence or absence of Dox were treated for 30 min or 4 h with Etoposide with or without addition of MG132, and DNA-trapped TOP2α and TOP2β were detected by RADAR (rapid approach to DNA adduct recovery) assays [34, 35]. Neither TOP2α nor TOP2β were detected in control DMSO treated cells confirming that only covalently DNA-bound species are isolated by this method (Figs 2B and S3). In addition, preliminary experiments where control and Dox-treated cells were exposed to different concentration of Etoposide for 5–30 min showed that the expression of BPLF1 does not interfere with the formation of TOP2ccs (S3 Fig). In western blots of Etoposide treated samples, TOP2α and TOP2β appeared as major bands of the expected size and smears of high molecular weight species corresponding to various post-translational modifications. Despite minor experimental variations, comparable amounts of trapped TOP2α and TOP2β were detected in cells treated with Etoposide for 30 min, independently of BPLF1 expression or MG132 treatment (Fig 2B, compare 0.5 h Dox- versus Dox+), confirming that neither treatment, either alone or in combination, has significant effects on the formation of TOP2ccs. As expected, in the absence of BPLF1 (Fig 2B, Dox- samples) the intensity of the TOP2 smears decreased after Etoposide treatment for 4 h. This was inhibited by MG132, confirming the involvement of proteasome-dependent degradation in the debulking of Etoposide-induced TOP2ccs. At the 4 h time point, the degradation of both TOP2α and TOP2β was significantly decreased in Dox treated cells (Fig 2B Dox- and Dox+ samples), corresponding to levels of stabilization comparable to those achieved by treatment with MG132. Quantification of the intensity of the TOP2 smears in repeated experiments confirmed that the expression of BPLF1 reduced the clearance of TOP2ccs as efficiently as treatment with MG132 (Fig 2C), supporting the conclusion that BPLF1 can deubiquitinate and stabilize TOP2 trapped in covalent DNA adducts. This finding was independently confirmed in experiments where TOP2ccs were stabilized by alkaline lysis [36] (S4A Fig). In this assay, smears of high molecular weight species were readily detected above the main TOP2β band in Dox-induced Etoposide-treated HEK-rtTA-BPLF1 cells, whereas smears were not detected when the blots were probed with a TOP1 specific antibody, confirming that the high molecular weight species correspond to DNA-trapped TOP2 (S4A Fig). As expected, the intensity of the smears decreased over time in BPLF1 negative cells, and the decrease was inhibited by MG132. In cells expressing catalytically active BPLF1, the intensity of the smears remained virtually constant over the observation time, resulting in significantly higher amounts of residual TOP2ccs (S4B Fig). Similar results were obtained when the blots were probed with antibodies to TOP2α.

[END]

[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1009954

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